EPA/540/2-90/005a
OERR Directive 9285.5-02-1
February 1990
Environmental Asbestos Assessment Manual
Superfund Method for the Determination
of Asbestos in Ambient Air
Part 1: Method
INTERIM VERSION
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DISCLAIMER
!™ "7/J& report wa&'prepaYGd under contract tqtbg &£ Environmental
^(otecf/o/t !4g&noy, T/3e,(?7efj$d/jub? ?ffi!cfe or vjotptnefatef products \
fnot constitute U& Environmental Protection Agency endorsement or
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Contents
Figures vj
Tables '.'.'.'.'.'.'.'.','.'.'. vii
Acknowledgements vj(-j
1. Introduction 1
2. Background .....'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'. 3
2.1. Sensitivity 5
2.2. Sampled Air Volumes ' ' ' ' ' R
2.3. Filter Selection ',',', 7
2.4. Precision 9
2.5. Asbestos Characteristics '.'.'.'.'.''.'.'.'.'. 10
2.6. Cost Considerations 10
3. Overview of Method -12
3.1. Sample Collection 12
3.2. Sample Preparation 12
3.2.1 Indirect TEM Specimen Preparation ' ' 12
3.2.2 Direct TEM Specimen Preparation 13
3.3. Analysis 13
4. Scope and Field of Application 15
4.1. Substance Determined 15
4.2. Range 15
4.2.1 Upper Limit of Range 15
4.2.2 Lower Limit of Detection 1 g
4.3. Analytical Sensitivities _ 17
4.4. Dimensional Detection Limits 17
5. Definitions 1Q
6. Symbols and Abbreviations 22
6.1. Symbols 22
6.2. Abbreviations 22
7. Equipment and Apparatus 24
7.1. Air Sampling - Equipment and Consumable Supplies 24
7.1.1. Filter Cassette 24
7.1.2. Sampling Pump 24
7.1.3 Stand 24
7.1.4 Rotameter 24
7.2 Specimen Preparation Laboratory 25
7.3 Laboratory Equipment 25
7.3.1. Transmission Electron Microscope 25
7.3.2. Energy Dispersive X-ray Analyzer 27
7.3.3. Computer 28
7.3.4. Plasma Asher 28
7.3.5. Filtration Apparatus 28
7.3.6. Filtration Manifold 28
7.3.7. Vacuum Pump 29
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7.3.8. Vacuum Coating Unit 29
7.3.9. Sputter Coater 29
7.3.10. Solvent Washer (Jaffe Washer) 29
7.3.11. Condensation Washer 31
7.3.12. Slide Warmer or Oven 31
7.3.13. Ultrasonic Bath 31
7.3.14. Carbon Grating Replica 31
7.3.15. Calibration Grids for EDXA 31
7.3.16. Carbon Rod Sharpener 31
7.3.17. Disposable-Tip Micropipettes 31
7.4. Consumable Laboratory Supplies 33
7.4.1. Glass Beakers 33
7.4.2. Membrane Filters 33
7.4.3. Copper Electron Microscope Grids 33
7.4.4. Gold Electron Microscope Grids 33
7.4.5. Carbon Rod Electrodes 33
7.4.6. Disposable Tips for Micropipette 33
7.4.7. Routine Electron Microscopy Tools and Supplies 34
7.4.8. Reference Asbestos Samples 34
8 Reagents 35
8.1. Freshly-Distilled Water 35
8.2. Dimethyl Formamide, Analytical Grade 35
8.3 Glacial Acetic Acid, Analytical Grade 35
8.4. Acetone, Analytical Grade 35
8.5. Hydrochloric Acid, Analytical Grade 35
9. Air Sample Collection 38
9.1. Required Sensitivity 38
9.2. Air Volume 38
9.3. Row Rate 39
9.4. Sampling Procedures 39
10. Procedure for Analysis 41
10.1. Introduction 41
10.1.1. Indirect TEM Specimen Preparation Method 41
10.1.2. Direct TEM Specimen Preparation Method 42
10.2. Preparation of TEM Specimen Grids by the Indirect Method 42
10.2.1. Cleaning of Sample Cassettes 42
10.2.2. Ashing of MCE Filter 42
10.2.3. Re-dispersal of Ashed Residues 42
10.2.4. Rltration of the Aqueous Suspension 43
10.2.5 Selection of Area of Filter for Preparation 45
10.2.6. Preparation of Solution for Collapsing MCE Filters 45
10.2.7. Filter Collapsing Procedure 45
10.2.8. Carbon Coating of Filter Sectors 45
10.2.9 Preparation of the Jaffe Washer .• 46
10.2.10. Placing of Specimens Into the Jaffe Washer 46
10.2.11. Rapid Preparation of TEM Specimens from MCE Filters 46
IV
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10.3. Preparation of TEM Specimen Grids by the Direct Method 46
10.3.1. Cleaning of Sample Cassettes 46
i 0.3.2. Selection of Area of Filter for Preparation 47
10.3.3. Filter Collapsing Procedure 47
10.3.4. Plasma Etching of the Filter Surface 47
10.3.5. Carbon Coating of Filters 47
10.3.6. Preparation of the Jaffe Washer 47
10.3.7. Placing of Specimens into the Jaffe Washer 47
10.3.8. Rapid Preparation of TEM Speciimens from MCE Filters 47
10.4. Criteria for Acceptable TEM Specimen Grids 47
10.5. Procedure for Structure Counting by TEM 48
10.5.1 Introduction 48
10.5.2. Measurement of Mean Grid Opening Area 49
10.5.3. TEM Alignment and Calibration Procedures 49
10.5.4. Determination of Stopping Point 49
10.5.5. General Procedure for Structure Counting and Size Analysis 50
10.5.6. Measurement of Concentration for Asbestos Structures
Longer than 5 pm 54
10.6. Blank.and Quality Control Determinations 57
10.7. Calculation of Results 58
11. Performance Characteristics . . 59
11.1. Interferences and Limitations of Structure Identification 59
11.2. Precision and Accuracy 59
11.2.1. Precision 59
11.2.2. Accuracy 59
11.3. Analytical Sensitivity 60
11.4. Limit of Detection 61
12. Reporting Requirements 62
12.1. Sample Analysis Report 62
12.2. Sample Batch Report ' ' . 53
12.3. Data Review Report 64
Appendix A - Determination of Operating Conditions for Plasma Asher 71
Appendix B - Calibration Procedures 72
Appendix C - Structure Counting Criteria 75
Appendix D - Fiber Identification Procedure 84
Appendix E - Calculation of Results 99
Appendix F - Bibliography 107
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7.1 Calibration Markings on the TEM Viewing Screen
7.2 Design of a Solvent Washer (Jaffe Washer).
73 Design of a Condensation Washer
10.1 Structure Counting Form ;•
10.2 Scanning Procedure for a TEM Specimen Examination
12.1A Format for Reporting of Structure Counting Data, Page 1
12. IB Format for Reporting of Structure Counting Data, Page 2
12.1C Format for Reporting of Structure Counting Data, Page 3
12.2 Format for Summary Laboratory Report
12.3 Format for Data Review Summary Report
C.1 Fundamental Morphological Structure Types
C.2 Examples of Recording of Complex Asbestos Clusters
C.3 Examples of Recording of Complex Asbestos Matrices
C.4 Counting of Structures that Intersect Grid Bars
C.5 Counting of Structures that Extend Outside the field of View
D.I Measurement of Zone Axis SAED Patterns
D.2 Classification Chart for Fiber With Tubular Morphology
D.3 Chrysotile SAED Pattern
D.4 Classification Chart for Fibers Without Tubular Morphology
VI
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9.1
10.1
D.1
P.?
Examples of the Minimum Number of Grid Openings Required to Achieve a Particular
Analytical Sensitivity and Detection Limit
Specifications for Phase 1 and 2 Sampling and Analysis
Classification of Fibers With Tubular Morphology
Classification of Fibers Without Tubular Morphology
VII
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ACKNOWLEDGEMENTS
We would like to acknowledge the timely and critical support for this effort provided by Jean
Chesson, Chesson Consulting, Washington D.C. and Kenny Crump, ICF Clement, Huston Louisiana.
Kenny Crump also assisted directly with the preparation of the Sections of this document addressing
data manipulation. Acknowledgements are also extended to David Suder, ENVIRON Inc., Emeryville
California for his assistance during the preparation of the sampling sections of this document.
VIII
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1. INTRODUCTION
This is a sampling and analysis method for the determination of asbestos
in air. Samples are analyzed by transmission electron microscopy (TEM).
Although a small subset of samples are to be prepared using a direct
procedure, the majority of samples analyzed using this method will be prepared
using an indirect technique. The method allows for the determination of the
mineralogical type(s) of asbestos present and for distinguishing asbestos from
non-asbestos minerals. In the method, asbestos structures are characterized
as fibers, bundles, clusters, or matrices and the length and width of each
asbestos structure are measured. Although the method Is designed specifically
to provide results suitable for supporting risk assessments at Superfund
sites, it is applicable to a wide range of ambient air situations.
To support a risk assessment, this method addresses two objectives:
(a) to provide increased precision at the low concentrations of
asbestos typically found in the environment;
(b) to provide measurements that can be compared with risk factors
derived from existing epidemiology studies.
An additional consideration addressed in this method is the need to control
sampling and analysis costs.
The method focuses on sampling requirements for individual sampling
stations and the analysis of sample filters collected at such stations.
During a site investigation, sampling stations would be arranged in an array
designed to distinguish spatial trends in airborne asbestos concentrations.
Sampling schedules would be fashioned to establish temporal trends. Thus
proper design of a comprehensive sampling strategy, detailing the design of
the array of sampling locations and the schedule for sample collection, is
also critical to the success of an investigation. However, design of a
sampling strategy is necessarily site specific and site-specific
considerations are beyond the scope of this document.
Satisfying the two method objectives listed above requires innovations
that tax the limits of available technology. Consequently, several variations
were considered during development and this method represents a workable
compromise among several technical constraints. Although the method has not
been validated as a whole and the feasibility of a few procedures needs to be
better documented, many of the component procedures of this method have been
performed in the laboratory. Thus, the principle features of the method are
well enough established that the method can be profitably employed in current
field investigations. There is no better way to acquire the necessary
experience and data for completing method validation. At the same time, until
a validation study and appropriate pilot studies are completed, this should be
considered an interim method.
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Details of the Considerations addressed during the development of this
method are provided in a companion Technical Background Document, Part 2 of
this report, under separate cover. A summary is presented in Chapter 2
(Background) of this document.
NOTE
This document is intended to serve several audiences including site
proj ect managers, field sampling teams, data reviewers, and laboratory
analysts. The document may be separated into segments s6 that
individuals may focus on the sections of most interest to their
particular roles in a project.
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2. , BACKGROUND
During the development of this method, existing sampling and analysis
technologies were considered to determine an appropriate approach for
achieving defined method objectives while remaining within the known
constraints associated with the measurement of airborne asbestos. The method
is designed to provide the analytical sensitivity and precision necessary to
distinguish background from asbestos concentrations typical of environmental
contamination. The method is also designed to provide results recorded in a
format that allows comparison with published risk factors or other proposed
representations of the biological activity .of asbestos. :
Recent developments toward understanding the relationship between
asbestos exposure and risk indicate that, to assess risks, modern field
measurements should be tailored to quantify asbestos characteristics that best
relate to biological activity. This is true despite the fact that existing
risk factors are derived from epidemiology studies in which asbestos
concentrations were derived primarily by phase contrast microscopy (PCM). A
broader range of asbestos structures potentially contribute to biological
activity than can be detected by PCM.
To provide the needed capabilities for distinguishing the broadest range
of asbestos characteristics, transmission electron microscopy (TEM) was
selected as the analytical technique employed in this method. Although
considered, both PCM and scanning electron microscopy (SEM) were rejected for
use in this method due to their inherent limitations.
The method is designed to provide the best description possible of the
nature, numerical concentration, and sizes of asbestos-containing particles
found in an air sample. The method of data recording specified in the method
is designed to allow re-evaluation of the structure characterization and
counting data without the necessity for re-examination of the specimens.
Currently-recognized methods require collection of airborne particulate
on a membrane filter, followed by preparation of TEM specimens from the
filter. Airborne asbestos is collected either on mixed cellulose ester (MCE)
filters or polycarbonate filters. TEM specimens can be prepared from such
membrane filters either by a direct-transfer technique or by an indirect
method.
Direct-transfer TEM specimen preparation methods have the following
advantages:
(a) the particulate and fiber size distributions are undisturbed
during specimen preparation;
(b) the limited amount of specimen manipulation reduces the
possibility of fiber loss or introduction of extraneous
contamination.
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However, the direct-transfer TEM preparation methods also have some
significant disadvantages:
(a) the achievable detection limit is restricted by the particulate
density on the filter, which in turn is controlled by the sampled
air volume and the total suspended particulate concentration in
the atmosphere being sampled;
(b) the precision of the result is dependent on the uniformity of the
deposit of asbestos structures on the sample collection filter;
(c) air samples must be collected that have particulate and fiber
loadings within narrow ranges. If too high a particulate loading
occurs on the filter, it is not possible to prepare satisfactory
TEM specimens by a direct-transfer method. If too high a fiber
loading occurs on the filter, even if satisfactory TEM specimens
can be prepared, accurate fiber counting will not be possible.
Indirect TEM specimen preparation techniques permit some of the
disadvantages of direct-preparation to be overcome:
(a) air samples can be collected without regard to the amount of
deposit on the filter surface. The filter loading can be adjusted
in the laboratory to provide satisfactory TEM specimens;
(b) interfering particulate material can be completely or partially
removed through a combination of dissolution and oxidation
(ashing);
(c) the uniformity of distribution of asbestos structures on the
filters to be analyzed is improved.
Indirect TEM specimen preparation methods have the following disadvantages:
(a) the size distribution of asbestos structures is modified;
(b) there is increased opportunity for fiber loss or introduction of
extraneous contamination;
(c) when sample collection filters are ashed, any fiber contamination
in the filter medium is concentrated on the TEM specimen grid.
The question as to whether direct or indirect specimen preparation
yields the "correct" result (in terms of representing biological activity) is
currently unresolved. It can be argued that the direct methods yield an
under-estimate of the asbestos structure concentration because many of the
asbestos fibers present are concealed by other particulate material with which
they are associated. Conversely, the indirect methods can be considered to
yield an over-estimate because some types of complex asbestos structures
disintegrate during the preparation, resulting in an increase in the numbers
of structures counted.
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2.1. SENSITIVITY
To provide the required sensitivity at asbestos levels typically
found in the environment, it is necessary either to selectively
concentrate asbestos, or to count structures over a much greater area of
a TEM specimen grid than has traditionally been required. To the extent
that it can be employed, selective concentration is the least costly of
the alternatives for increasing sensitivity. The optimal asbestos
concentration on a filter requires filter loadings of total particulate
matter in excess of what can generally be tolerated for analysis using
direct-transfer methods for TEM specimen preparation. Consequently, an
indirect technique for TEM specimen preparation is employed in this
method, which incorporates steps allowing for the selective
concentration of asbestos.
In most ambient environments, a proportion of the suspended
particulate is organic, consisting of soot, spores, pollens and other
debris from vegetation. Organic materials such as these can be removed
from the analysis by low-temperature ashing.
It is common to find substantial numbers of calcium sulfate fibers
(gypsum) in airborne particulate collected in urban environments. These
can arise from various sources, and they can also be generated either in
the atmosphere or on the sample collection filter by reaction of
airborne calcite or dolomite particles with atmospheric sulfur dioxide.
Gypsum can be removed by dissolution in water or dilute acids.
Carbonates are another major component of most exterior
atmospheres. Calcite and dolomite are commonly found in urban
atmospheres; these originate from various sources, including erosion of
concrete and cement, local geology, and in some cases from industrial
operations. Such carbonates can be removed by extraction with
hydrochloric acid. If acid extraction procedures are carried out
correctly, no chemical or crystallographic degradation of asbestos can
be detected by routine methods of TEM analysis.
Removal of a large proportion of the suspended particulate by use
of these techniques permits the asbestos present in the sample to be
concentrated on to a smaller area of the TEM specimen. Consequently,
the area of the TEM specimen that must be examined to achieve a
particular analytical sensitivity is proportionately reduced. Also,
many of the non-asbestos fibrous structures normally found in a
directly-prepared TEM specimen, each of which must be identified and
rejected from the asbestos structure count, do not appear on the
indirectly-prepared TEM specimen.
For many environmental samples, a selective concentration of the
asbestos structures, incorporating low temperature ashing, re-suspension
in water, and acidification with HC1 is capable of removing substantial
amounts of interfering particulate from the analysis, and, for a
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particular analytical sensitivity, reducing the area of the TEM
specimens that must be examined.
The concentration steps included in this method, together with the
requirement to measure very low airborne asbestos concentrations, make
control of asbestos contamination more critical than for specimens
prepared by direct-transfer techniques. Accordingly, the procedures in
this method have been designed to minimize the effects of contamination.
2.2. SAMPLED AIR VOLUMES
This method incorporates an indirect preparation procedure to
provide flexibility in the amount of deposit that can be tolerated on
the sample filter and to allow for the selective concentration of
asbestos prior to analysis. To minimize contributions to background
contamination from asbestos present in the plastic matrices
(polycarbonate or mixed cellulose ester) of membrane filters while
allowing for sufficient quantities of asbestos to be collected, this
method also requires the collection,of a larger volume of air per unit
area of filter than has traditionally been collected for 'asbestos
analysis.
Due to the need to collect large volumes of air, higher sampling
flow rates are recommended in this method than have generally been
employed for asbestos sampling in the past. As an alternative, samples
may be collected over longer time intervals but this restricts the
flexibility required to allow samples to be collected while uniform
meteorological conditions prevail. Higher flow rates through a 25 mm
filter result in increased face velocities at the filter surface.
Potential problems associated with the higher flow rates and increased
face velocities include:
(a) destruction of the filter due to failure of its physical
support under force from the increased pressure drop;
(b) leakage of air around the filter mount so that the filter is
bypassed;
(c) damage to the asbestos structures due to increased impact
velocities.
The recommended flow rates and face velocities have been employed
in air sampling using membrane filters in the past. Based on such
studies, (c) is not likely to be a problem. Using the filters and flow-
rates specified, the first two concerns (a and b) are also unlikely to
impose limitations. However, documented experience at the increased
flow rates with the types of filter cassettes to be used in this method
is very limited. Consequently, a pilot study is recommended to confirm
that the structural integrity of a 25 mm filter cassette is not altered
at the increased flow rates recommended. There is also some question
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whether available pumps can maintain such flow rates throughout the
collection of large volumes of air while the filter loading and,
correspondingly, the resistance to flow steadily increase.
2.3. FILTER SELECTION
Selection of the type of filter to be used in this method was
determined by considering the performance of the filter during sample
collection, sample handling, and TEM specimen preparation (by an
indirect technique). Concerns include the degree of asbestos loss that
may occur during sample transport or handling, the efficiency with which
asbestos is transferred from the filter to the TEM specimen, and the
level of asbestos contamination potentially introduced to the TEM
specimen from the filter material itself. Both mixed cellulose ester
(MCE) and polycarbonate filters were evaluated.
It is generally accepted that deposited particles may move on the
surface of polycarbonate filters when these filters are heavily loaded.
Such movement may occur either during sample transport or handling in
the laboratory. Such movement may potentially lead to asbestos losses
between the time samples are collected and the time they are prepared
for analysis.
Unlike the smooth surface of a polycarbonate filter, MCE filters
exhibit sponge-like surfaces that trap filters within layers of porous
material. The potential for movement of deposits on the surface of this
type of filter is generally considered minimal. Correspondingly, the
potential for loss of asbestos during transport or handling is also
reduced. Thus, MCE filters are preferred over polycarbonate filters in
terms of ease of handling.
Two methods are available for removal of sampled particulate from
the sample collection filter in an indirect preparation: complete ashing
of the filter and the particulate deposit, or washing of the particulate
deposit from the filter surface followed by ashing of the wash-suspended
deposit only. A number of problems have been experienced with the
ashing of filters during indirect-transfer procedures. The efficiency
with which deposited asbestos is transferred by washing has not been
thoroughly investigated.
Complete ashing of a filter causes any asbestos contamination that
may be present within the polymer matrix of the filter to be transferred
to the final TEM preparation. Direct-transfer preparations from
polycarbonate filters are known to yield a measurable background of
asbestos contamination and current knowledge indicates that ashing of
polycarbonate filters will yield an even higher value. Direct-transfer
preparations of MCE filters generally yield low background asbestos
contamination. However, ashing of either filter may yield higher levels
of background asbestos contamination due to sources of contamination
associated with the preparation itself.
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Problems that have been experienced in association with low
temperature ashing include both potential sources of additional
contamination and filter behavior potentially leading to asbestos
losses. During filter ashing, background contamination can arise from
within the asher chamber, the containers in which the filters are ashed,
the distilled water supply used for re-dispersing the ash, the pipettes
used for transfer of dispersion for filtration, and the filtration
funnel.
During ashing, both MCE and polycarbonate filters have
occasionally been observed to curl up and give the appearance of being
completely ashed, yet asbestos is trapped in a few very large,
incompletely ashed particles that do not disperse. Consequently, such
asbestos is lost and does not appear on the final TEM preparation.
Procedures for avoiding this problem need to be refined.
Due to the problems associated with ashing the filters, washing of
the particulate from the filter surface into ethanol is considered a
superior approach. It is considered that washing of particulate from
the surface of a polycarbonate filter can be accomplished with high
removal efficiency. However, contamination of polycarbonate filters by
asbestos has been measured by various laboratories, and currently
appears to be within the range of 50-200 asbestos structures/sq.mm. on
the active filter surface.1
During the washing of the filter surface, asbestos contamination
on the surface of a polycarbonate filter (observed on preparations from
direct-transfer procedures) may be detached along with the collected
particulate and contribute to the background against which the
concentrations of any collected asbestos must be measured. If the
filter is removed from the cassette and the washing of the filter is
performed ultrasonically in a beaker, contamination from both sides of
the filter may be detached, leading to higher background counts.
Therefore, potential but unquantified problems may be associated with
the washing of polycarbonate filters.
Currently, it is not known whether collected particulate can be
efficiently removed by washing the surfaces of 0.45 /zm or 0.8 /zm pore
size MCE filters. The MCE filter has a sponge-like texture, and many of
the fibers collected are embedded or entrapped within this structure.
It has been shown that any attempt to use ultrasonic techniques results
in disintegration of the filter surface and release of fragments of
Certain laboratories have consistently reported lower values for
polycarbonate filter contamination than other laboratories.
Although this is well known and the matter has been addressed by
numerous investigators, the source of the apparent discrepancy has
not been elucidated.
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filter material. It is possible that this disintegration of the filter
surface may also release most of the collected particulate, but this has
not been demonstrated. On the basis of current knowledge, it is
unlikely that the small amount of polymer removed from the surface of
the filter would contribute a significant level of contamination to the
analysis, because contamination levels on the surface of MCE filters
have been demonstrated to be very low.
Based on the above considerations, MCE filters were selected for
use in this method. Although available data suggest that the optimum
combination of filter and preparation technique would be for samples to
be collected on MCE filters and to re-disperse the particulate in water
by washing of the filters, little is known concerning the efficiency
with which asbestos can be removed from the surface of an MCE filter by
washing. It is known that some of the filter polymer is detached from
the filter surface during washing and particles of this polymer can
interfere with the analysis. Thus, ashing of the wash suspension would
be necessary in order to remove the filter polymer fragments. Because
the efficacy of the filter washing procedure has not been adequately
substantiated, the preparation procedure incorporated into this method
is ashing of the filter followed by re-dispersion of the ash in water.
2.4. PRECISION
To maximize precision, potential sources of variation within the
method must be controlled. To minimize systematic variation during
sampling and analysis, the method specifies detailed procedures. Random
variation is minimized by maximizing the number of structures to be
identified and counted, which includes increasing the probability of
encountering asbestos structures during analysis, as discussed in
Section 2.1. In addition, detailed and unambiguous counting rules and
rules for identification are specified in the method to minimize
variation due to subjective interpretation.
Airborne asbestos is often found, not as single fibers, but as
very complex structures that may or may not be aggregated with equant
(non-asbestos) particles. The mineralogy of structures found suspended
in an ambient atmosphere must be identified to confirm a structure as
asbestos. Such structures can often be identified unequivocally, if
sufficient measurement effort is expended. However, if each structure
were to be identified in this way, the analysis becomes prohibitively
expensive. Because of instrumental deficiencies or because of the
nature of the particulate, some structures cannot be positively
identified as asbestos, even though the measurements all indicate that
they could be asbestos. Subjective factors therefore contribute to this
measurement, and consequently a very precise procedure for
identification and enumeration of asbestos is incorporated in this
method.
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Despite the imposition of systematic counting and identification
criteria, there are large potential errors in characterizing asbestos in
ambient atmospheres, associated with the variability between filter
samples and the performance of individual microscopists. For this
reason, a replicate sampling and analysis scheme is also incorporated in
order to determine the accuracy and precision of the method.
2.5. ASBESTOS CHARACTERISTICS
To analyze asbestos samples so that results can be related to
potential risks, it is necessary to quantify the characteristics of
asbestos that relate to biological activity. This involves enumeration
of individual structures within certain size categories with particular
emphasis on the longest and thinnest structures. Although the range of
dimensions over which asbestos structures contribute to biological
activity has yet to be precisely defined, this method is designed to
provide a detailed characterization of structures encompassing the
entire range of potential importance.
When evaluating detailed asbestos size characterizations, it is
important to consider the effects of sample preparation. The appearance
of the size distribution representing a sample of asbestos may vary
depending on whether the sample was prepared by a direct or an indirect
technique. Existing risk factors are based largely on studies
incorporating direct transfer techniques.2 Because indirect preparation
is preferred to achieve the desired analytical sensitivity for
environmental analysis (see Section 2.1), a procedure for evaluating the
relationship between structure counts on samples prepared by an indirect
technique and by a direct transfer technique is included in the method.
2.6. COST CONSIDERATIONS
In environmental samples, the amount of airborne asbestos is
usually low relative to the total suspended particulate concentration.
Therefore, when using a direct-transfer preparation, large areas of the
TEM specimen grids must be examined to obtain a statistically valid
measurement for asbestos. Consequently, the analysis is expensive. It
is therefore not feasible to perform this type of analysis on all of the
filters collected as part of a site investigation.
A cost-effective compromise has been incorporated into this
method. A two phase sampling and analysis scheme has been adopted. In
Phase 1, the majority of samples collected at a site will be prepared by
PCM analyses are performed directly on sample filters where the
filter material has been rendered transparent by a suitable
solvent. Since the fibers are observed as originally deposited,
this corresponds closely to a direct transfer technique for TEM
analysis.
10
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the indirect-transfer technique (to maximize precision) and analysis
will be performed using an.efficient evaluation procedure. Air volumes
collected on Phase 1 samples may be maximized subject to the limitations
of sampling equipment.
In Phase 2 a small subset of samples (5 to 10%) will be collected
and analyzed using an extended TEM examination to derive a relationship
between counts from samples prepared by a direct technique and counts
from samples prepared by an indirect technique for each of the various
structure size fractions of interest that are observed in Phase 1 and
Phase 2 samples. Filters from Phase 2 samples will be split so that
half may be prepared by the direct-transfer procedure and half by the
indirect-transfer procedure. Because half of each filter to be analyzed
under Phase 2 will be prepared by the direct-transfer technique, the
volume of air collected for each sample is limited by the loading that
can be tolerated during analysis of a specimen prepared using a direct-
transfer technique. Phase 1 and 2 samples shall be collected simul-
taneously .
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3. OVERVIEW OF METHOD
Samples are collected and prepared for TEM examination by one of two
techniques depending on the purpose for the sample. The majority of air
samples (denoted Phase 1 samples) will be analyzed using an indirect procedure
for preparation of TEM specimens, optimized to provide low detection limits
and high precision. A small number of samples (denoted Phase 2 samples) will
be collected in such a way that they can be analyzed using both indirect and
direct procedures for preparation of TEM specimens, to allow comparisons to be
made between the results from the two specimen preparation procedures. TEM
examination procedures used for the two sets of samples also differ.
3.1. SAMPLE COLLECTION
A sample of airborne particulate is collected by drawing a
measured volume of air through a 25 mm diameter, 0.45 p,m pore size MCE
membrane filter by means of a pump. Air volumes collected on Phase 1
samples will be maximized. Air volumes collected on Phase 2 samples
will be limited to provide optimum loadings for filters to be prepared
by a direct-transfer procedure.
3.2. SAMPLE PREPARATION
TEM grids will be prepared according to either 3.2.1 or 3.2.2 below.
3.2.1. Indirect TEM Specimen Preparation
This preparation will be applied to all Phase 1 and Phase 2
samples. The filter is split and half of the filter is stored
for future use. The remaining half of the filter is ashed in a
low-temperature plasma asher. The residual ash is ultrasonically
dispersed in freshly-distilled water. The suspension is acidified
using hydrochloric acid, and immediately filtered through a 25 mm
diameter, 0.1 /jm pore size MCE filter. The filter is dried and
the filter structure is collapsed using a mixture of dimethyl
formamide, acetic acid and water. A thin film of carbon is
evaporated onto the collapsed filter surface and small areas are
cut from the filter. These areas of filter are supported on TEM
specimen grids and the filter medium is dissolved away by a
solvent extraction procedure.
NOTE
An alternate procedure for indirect preparation in which filter
deposits are washed off of MCE filters followed by the ashing of
the wash-suspended deposit may be substituted into this method
upon completion of validation in a pilot study.
12
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3.2.2. Direct TEM Specimen Preparation
The remaining one-half of the filters from all Phase 2
samples will be prepared by this procedure. One quarter of the
filter is collapsed using a mixture of dimethyl formamide, acetic
acid and water. The collapsed filter is etched for a short time
in a low temperature plasma asher to remove the surface layer of
. filter polymer which may have encapsulated asbestos structures
during the collapsing procedure. A thin film of carbon is
evaporated onto the collapsed filter surface and small areas are
cut from the filter. These areas of filter are supported on TEM
specimen grids and the filter medium is dissolved away by a
solvent extraction procedure.
3.3. ANALYSIS
The TEM specimen grids are examined at both low and high
magnifications to check that they are suitable for analysis before
carrying out a quantitative examination on randomly-selected grid
openings. In addition to isolated fibers, ambient air samples often
contain more complex aggregates of fibers, with or without equant
particles. Some particles are composites of asbestos fibers with other
materials. Individual fibers and these more complex structures are
referred to as "asbestos structures". A coding system is used to record
the type of fibrous structure, and to provide the optimum description of
each of these complex structures.
The method requires that separate examinations be made for
asbestos structures of all sizes (incorporating asbestos fibers with
lengths greater than 0.5 /im) and for long asbestos structures
(incorporating structures longer than 5 /*m). In both cases, asbestos
structures are defined as those exhibiting mean aspect ratios equal to
or greater than 5:1. This TEM examination procedure allows for
specification of a lower analytical sensitivity for the measurement of
the concentration of asbestos structures longer than 5 jum.
In the TEM analysis, electron diffraction (ED) is used to examine
the crystal structure of a fiber, or fibrous components of complex
structures, and the elemental composition is determined by energy
dispersive X-ray analysis (EDXA). For a number of reasons, it is not
possible to identify (determine the mineralogy of) each structure
unequivocally, and structures are classified according to the techniques
that have been used to identify them. A simple code is used to record
the manner in which each structure is classified.
The classification procedure is based on successive inspection of
the morphology, the electron diffraction pattern, and the energy
dispersive X-ray spectrum. Confirmation of the identification of
chrysotile is only by quantitative ED, and confirmation of amphibole is
only by quantitative EDXA and quantitative zone-axis ED.
13
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Several levels of analysis to confirm mineralogy are specified,
the higher levels providing a more rigorous approach to the
identification of structures. The procedure permits a minimum required
asbestos identification procedure to be defined on the basis of previous
knowledge, or lack of it, about the particular sample. Attempts are
then made to achieve this defined minimum procedure for each asbestos
structure, and the degree of success is recorded for each. The two
codes remove from the microscopist the requirement to interpret
observations made during the TEM examination, and allow this evaluation
to be made later without the requirement for re-examination of the TEM
specimens.
The lengths and widths of all classified asbestos structures are
recorded. The number of asbestos structures found on a known area of
the TEM specimen grids, together with the equivalent volume of air
filtered through this area, are used to calculate the airborne
concentration in asbestos structures/liter of air.
This method requires that the desired analytical sensitivities for
the measurements of:
(a) asbestos structures of all sizes (incorporating structures
longer than 0.5 /*m) ; and,
(b) asbestos structures longer than 5 pin,
shall be specified before air samples are collected. In both cases,
structures to be counted are defined as those exhibiting aspect ratios
equal to or greater than 5:1. It will not always be possible to achieve
these analytical sensitivities, because the volume of air that can be
sampled is dictated by the nature and concentration of the suspended
particulate in the atmosphere being sampled. To some degree, this
limitation can be overcome by selective concentration of asbestos
structures during the specimen preparation procedures and by examination
of a larger area of the TEM specimens. However, the ease and cost of
achieving a specific value for the analytical sensitivity will vary from
sample to sample.
14
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4. SCOPE AND FIELD OF APPLICATION
This method defines procedures for analyzing of airborne asbestos
collected by drawing a known volume of air through mixed cellulose ester (MCE)
filters. The method is suitable for the determination of asbestos in ambient
air.
4.1. SUBSTANCE DETERMINED
The method defined uses transmission electron microscopy for
determination of the concentration of asbestos in ambient atmospheres.
This method includes measurement of the lengths and widths of asbestos
structures and a calculation of their aspect ratios. In the method,
asbestos structures are characterized as fibers, bundles, clusters, or
matrices. The method allows for the determination of the mineralogical
type(s) of asbestos present. The method, however, cannot discriminate
between individual fibers of the asbestos and non-asbestos analogues of
the same amphibole mineral.
4.2. RANGE
The range of concentrations that can be determined on a TEM
specimen grid is approximately 50 to 7000 asbestos structures/mm2. The
lower limit is generally defined by the requirement that the
concentration of asbestos collected from the air be distinguishable from
background contamination contributed by the method and the filter. The
upper limit is defined by the concentration at which asbestos structures
will significantly overlap and interfere with proper detection.
4.2.1. Upper Limit of Range
The upper limit of the range for detecting asbestos on a
filter may be limited both by excessive overlap of asbestos
structures and by the presence of other interfering particles. In
practice, if obscuration of asbestos structures is not to be a
significant factor in a method's precision, the final TEM specimen
grid should not exhibit more than approximately a 10% coverage by
particulate. For the indirect TEM specimen preparation method,
this maximum permissible particulate loading is determined by the
concentration of that portion of the total suspended particulate
concentration that cannot be removed by procedures such as
oxidation or chemical dissolution.
Using the direct-transfer preparation method, it has been
found that if the total particulate loading of the sample
collection filter exceeds approximately 10 jig/cm2 of filter
surface, corresponding to approximately 10% coverage of the
collection filter by particulate, the TEM specimens are loaded to
a point that obscuration of asbestos structures by particulate
becomes a significant source of error. If the particulate loading
15
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exceeds approximately 25 /ig/cm2 of filter surface, the specimens
are seriously overloaded and it is often not possible to prepare
TEM specimens that have grid openings with intact carbon film.
The degree that non-asbestos particulates may interfere with
asbestos analysis is a function of the concentration of total
suspended particulate (TSP) present in the atmosphere in addition
to the asbestos being sampled. Concentrations of the TSP vary
over several orders of magnitude depending on location, time of
day, and weather. Urban and agricultural sites tend to have
significantly higher concentrations of the TSP than rural
locations. Consequently, higher volumes of air may be collected
at rural locations before interference by the TSP becomes a
limiting factor.
In addition to variation in overall concentration, the
composition of the TSP varies significantly as a function of
location. At urban sites and specific rural locations, the TSP
tends to be composed principally of organic matter that can be
ashed or inorganic substances that are soluble in acidified media.
Agricultural locations and other rural locations frequently
exhibit higher concentrations of refractory silicate particles.
Due to the wide spatial and temporal variation in the TSP
concentrations, a general rule for estimating levels can not be
provided. However, a review of available environmental studies
indicates that air volumes of 1 m3 per cm2 of filter area may
reasonably be collected for direct preparation samples in most
urban and rural environments. Subject to the types of the TSP
present, samples collected for indirect preparation can be loaded
to levels up to an order of magnitude higher. Data on the range
of local levels of TSP should be obtained and evaluated prior to
finalizing a sampling plan for a site.
4.2.2. Lower Limit of Detection
The detection limit theoretically can be lowered
indefinitely by:
(a) filtration of progressively larger volumes of air;
(b) use of intermediate specimen preparation procedures to
concentrate the collected particulate on to smaller
areas of the TEM specimen grids; and,
(c) examination of greater areas of the TEM specimens in
the electron microscope.
Although it might appear that the detection limit can be
lowered indefinitely by examination of a greater area of the TEM
16
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specimens, this is not actually the case if unused filters
(blanks), or the analytical procedure itself, contribute
background asbestos. Although it is considered that the back-
ground asbestos structure counts of TEM specimens prepared by the
collapsed MCE filter technique (direct preparation) are very low,
there are few data currently available concerning the asbestos
content of the MCE filter polymers themselves, which might
contribute to background during an indirect preparation.
In a technique that incorporates complete ashing of MCE
filter membranes, any asbestos contained in the filter polymer
will be concentrated on the final TEM specimens. Accordingly,
this effect should be minimized by collection of the maximum
volumes of air possible, and a continuous program of blank
measurements must form part of the quality assurance procedures.
4.3. ANALYTICAL SENSITIVITIES
Based on a review of data from past environmental asbestos
investigations and published studies of rural and urban background, the
lower end of the ranges of concentrations typical of these types of
studies center on 5 structures/liter (s/L) for asbestos structures of
all sizes and 0.2 s/L for structures longer than 5 /un. The significance
of the asbestos structures longer than 5 /im is discussed in Section 2.5.
To distinguish among such concentrations at acceptable levels of
statistical significance, it is assumed that a minimum of 10 asbestos
structures should be identified and counted (see Section 4.2 of the
Technical Background Document, Part 2 of this report). Thus, analytical
sensitivities (defined as the concentration represented by the detection
of 1 structure) required by this method are 0.5 s/L and 0.02 s/L for
asbestos structures of all sizes and asbestos structures longer than
5/im, respectively.
4.4. DIMENSIONAL DETECTION LIMITS
Microscopists vary in their ability to detect very small asbestos
structures. Therefore, a minimum length of 0.5 ^m has been defined as
the shortest fiber to be incorporated in the reported results.
The minimum detectable width for asbestos structures counted in
this method is determined by the ability of an operator to detect them
in a routine examination at the specified magnification of the method.
The minimum magnification specified in this method (10,000) is more than
sufficient to ensure visibility of the thinnest chrysotile asbestos
fibrils.
17
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5. DEFINITIONS
Acicular: The shape shown by an extremely slender crystal with cross-
sectional dimensions which are small relative to its length.
Amphibole: A group of rock-forming ferromagnesium silicate minerals, closely
related in crystal form and composition, and having the nominal formula:
where:
A - K, Na;
B - Fe2*, Mn, Mg, Ca, Na;
C - Al, Cr, Ti, Fe3+, Mg, Fe2*;
T - Si, Al, Cr, Fe3+, Ti.
In some varieties of amphibole, these elements can be partially substituted by
Li, Pb, or Zn. Amphibole is characterized by a cross-linked double chain of
Si-0 tetrahedra with a silicon:oxygen ratio of 4:11, by columnar or fibrous
prismatic crystals and by good prismatic cleavage in two directions parallel
to the crystal faces and intersecting at angles of about 56° and 124°.
Amphibole asbestos: Amphibole in an asbestiform habit.
Analytical sensitivity: The calculated airborne concentration, in asbestos
structures/liter, equivalent to counting of one asbestos structure in the
analysis.
Asbestiform: A specific type of mineral fibrosity in which the fibers and
fibrils possess high tensile strength and flexibility.
Asbestos: A term applied to a group of fibrous silicate minerals that readily
separate into thin, strong fibers that are flexible, heat resistant and
chemically inert.
Asbestos component: A term applied to any individually identifiable asbestos
sub-structure that is part of a larger asbestos structure.
Asbestos structure: A term applied to any contiguous grouping of asbestos
fibers, with or without equant particles.
Aspect ratio: The ratio of length to width of a particle.
18
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Blank: A fiber count made on TEM specimens prepared from an unused filter, to
determine the background measurement. Blanks consist of filter blanks, field
blanks and laboratory blanks.
Bragg Angle: The angle between a crystal surface and an incident ray of
incoming x-radiation.
Bundle: A fiber composed of parallel, smaller diameter fibers attached along
their lengths.
Camera length: The equivalent projection length between the specimen and its
electron diffraction pattern, in the absence of lens action.
Chrysotile: A mineral of the serpentine group which has the nominal composi-
tion:
Mg3Si205(OH)4
In some varieties of chrysotile, the silicon may be partially substituted by
Al or less commonly by Fe. The magnesium may be partially substituted by Fe,
Ni., Mn or Co. Some varieties contain Na, Cl or both. Chrysotile is a highly
fibrous, silky variety, and constitutes the most prevalent type of asbestos.
Cleavage: The breaking of a mineral along one of its crystallographic
directions.
Cleavage fragment: A fragment of a crystal that is bounded by cleavage faces.
Cluster: An assembly of randomly oriented fibers.
Component count: For any sample, a tally that includes the individually
identified components of complex asbestos structures and each single asbestos
structure with no identifiable components.
d-spacing: The distance between identical adjacent and parallel planes of
atoms in a crystal.
Detection limit: The calculated airborne fiber concentration in fibers/liter,
equivalent to counting of 3.69 fibers in the analysis.
Electron diffraction: A technique in electron microscopy by which the crystal
structure of a specimen is examined.
Electron scattering power: The extent to which a thin layer of substance
scatters electrons from their original directions.
Energy dispersive X-ray analysis: Measurement of the energies and intensities
of X-rays by use of a solid state detector and multi-channel analyzer system.
Eucentric: The condition when an object is placed with its center on a
rotation or tilting axis.
19
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Fiber: An elongated particle which has parallel or stepped sides. In this'
method, a fiber is defined to have an aspect ratio equal to or greater than
5:1.
Fibril: A single fiber of asbestos, which cannot be further separated
longitudinally into smaller components without losing its fibrous properties
or appearances.
Fibrous structure: A contiguous grouping of fibers, with or without equant
particles.
Field blank: A filter cassette which has been taken to the sampling site,
opened, and then closed. Such a filter is analyzed to determine the back-
ground asbestos structure count for the measurement.
Filter Blank: An unused filter which is analyzed to determine the background
asbestos structure count.
Habit: The characteristic crystal form or combination of forms of a mineral,
including characteristic irregularities.
Identify: During analysis, the use of a sequential set of procedures to
determine and confirm the mineralogy of a structure.
Laboratory Blank: An unused filter which is analyzed along with sample
filters to determine the background asbestos structure count.
Matrix: A connected assembly of asbestos fibers with particles of another
species (non-asbestos).
Miller index: A set of either three or four integer numbers used to specify
the orientation of a crystallographic plane in relation to the crystal axes.
PCM equivalent structure: A structure of aspect ratio greater than or equal
to 3:1, longer than 5 pm, and which has a mean diameter between 0.2 pm and
3.0 pm for a part of its length greater than 5 pm. In this method, PCME
structures also must contain at least one asbestos component.
Replication: A procedure in electron microscopy specimen preparation in which
a thin copy, or replica, of a surface is made.
Selected area electron diffraction: A technique in electron microscopy in
which the crystal structure of a small area of a sample is examined.
Serpentine: A group of common rock-forming minerals having the nominal
formula:
Mg3Si205(OH)A
20
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Structure Count: For any sample, a tally of each individually identified
asbestos structure regardless of whether the structure contains identifiable
components. This is equivalent to a count of the total number of separate
asbestos entities encountered on the sample.
Twinning: The occurrence of crystals of the same species joined together at a
particular mutual orientation, and such that the relative orientations are
related by a definite law.
Unopened fiber: A large diameter asbestos fiber bundle which has not been
separated into its constituent fibrils or fibers.
Zone-axis: The line or crystallographic direction through the center of a
crystal which is parallel to the intersection edges of the crystal faces
defining the crystal zone.
21
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6. SYMBOLS AND ABBREVIATIONS
6.1. SYMBOLS
A
eV
kV
L/min
run
Pa
s/L
W
6.2.
DMF
ED
EDXA
FWHM
HEPA
MCE
PCM
PCME
SAED
SEM
Angstrom unit (10~10 meter)
electron volt
kilovolt
liters per minute
microgram (10~6 grams)
micrometer (10~6 meter)
nanometer (10"9 meter)
Pascal
- structures per liter
watt
ABBREVIATIONS
Dimethyl formamide
Electron diffraction
Energy dispersive X-ray analysis
Full width, half maximum
High efficiency particle absolute
Mixed cellulose ester
Phase contrast optical microscopy
Phase contrast microscopy equivalent
Selected area electron diffraction
Scanning electron microscope
22
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STEM
TEM
TSP
UICC
Scanning transmission electron microscope
Transmission electron microscope
Total suspended particulate
Union Internationale Centre le Cancer
23
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7. EQUIPMENT AND APPARATUS
7.1. AIR SAMPLING - EQUIPMENT AND CONSUMABLE SUPPLIES
7.1.1. Filter Cassette
Commercially available field monitors, comprising 25 mm
diameter three-piece cassettes, with conductive extension cowls
shall be used for sample collection. The cassette must be new and
not previously used. The cassette shall be loaded with an MCE
filter of pore size 0.45 fjaa., and supplied from a lot number which
has been qualified as low background for asbestos determination.
The filter shall be backed by a 5 pm pore size MCE filter, and
supported adequately so that distortion of the filter by the
differential pressure across it does not occur during sampling.
The cassettes shall be purchased with the required filters in
position, or shall be assembled in a laminar flow hood or clean
area such that the filter blank determinations meet the criteria
of Section 10.6. When the filters are in position, a shrink
cellulose band or adhesive tape should be applied to cassette
joints to prevent air leakage. Suitable precautions shall be
taken to ensure that the filters are tightly clamped in the
assembly so that significant air leakage around the filter cannot
occur.
7.1.2. Sampling Pump
The sampling pump shall be capable of a flow-rate and
pumping time sufficient to achieve the desired volume of air
sampled. The face velocity through the filter shall be between
4.0 cm/s and 65.0 cm/s. The sampling pump used shall provide a
non-fluctuating air-flow through the filter, and shall maintain
the initial volume flow-rate to within ±10% throughout the
sampling period. A constant flow or critical orifice controlled
pump meets these requirements. Flexible tubing shall be used to
connect the filter cassette to the sampling pump. A means for
calibration of the flow-rate of each pump is also required.
7.1.3. Stand
A stand shall be used to hold the filter cassette at the
desired height for sampling and the filter cassette shall be
isolated from the vibrations of the pump.
7.1.4. Rotameter
A rotameter or other flow measuring device calibrated such
that the operator can measure flow rates to ± 5% accuracy at the
expected sampling flow rate.
24
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7.2. SPECIMEN PREPARATION LABORATORY
Asbestos, particularly chrysotile, is present in varying
quantities in many laboratory reagents. Many building materials also
contain significant amounts of asbestos or other mineral fibers that may
interfere with the analysis if they are inadvertently introduced during
preparation of specimens. It is most important to ensure that during
preparation, contamination of TEM specimens by any extraneous asbestos
fibers is minimized. All specimen preparation steps shall therefore be
performed in an environment where contamination of the sample is
minimized. The primary requirement of the sample preparation laboratory
is that a blank determination shall yield a result which will meet the
specifications in 10.6. A minimum facility considered suitable for
preparation of TEM specimens is a laminar flow hood. However, it has
been established that work practices in specimen preparation appear to
be more important than the type of clean handling facilities in use.
Preparation of samples shall be carried out only after acceptable blank
values have been demonstrated.
NOTE
Activities involving manipulation of bulk asbestos shall not be
performed in the same area as TEM specimen preparation, because of the
possibilities of contaminating the TEM specimens.
7.3. LABORATORY EQUIPMENT
7.3.1. Transmission Electron Microscope
A TEM operating at an accelerating potential of 80-120 kV,
with a resolution better than 1.0 nm, and a magnification range of
approximately 300 to 100,000 shall be used. The ability to obtain
a direct screen magnification of about 100,000 is necessary for
inspection of fiber morphology; this magnification may be obtained
by supplementary optical enlargement of the screen image by use of
a binocular if it cannot be obtained directly. It is also requir-
ed that the viewing screen be calibrated (as shown in Figure 7.1)
with concentric circles and a millimeter scale such that the
lengths and widths of fiber images down to 1 mm width can be
measured in increments of 1 mm.
For Bragg angles less than 0.01 radians, the TEM shall be
capable of performing ED from an area of 0.6 /zm2 or less, selected
from an in-focus image at a screen magnification of 20,000. This
performance requirement defines the minimum separation between
particles at which independent ED patterns can be obtained from
25
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Figure 7.1: Calibration Markings on the TEM Viewing Screen
26
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each particle. If SAED is used, the performance of a particular
instrument may normally be calculated using the following
relationship:
A - 0.7854 x (D/M + 2000 x CS93)2
Where: A - the effective SAED area in fan2
D - the diameter of the SAED aperture in fun
M - the magnification of the objective lens
C, - the spherical aberration coefficient of the
objective lens in mm
9 - maximum required Bragg angle in radians
It is not possible to reduce the effective SAED area
indefinitely by the use of progressively smaller SAED apertures,
because there is a fundamental limitation imposed by the spherical
aberration coefficient of the objective lens.
If zone-axis ED analyses are to be performed, the TEM shall
incorporate a goniometer stage which permits the TEM specimen to
be either:
(a) rotated through 360°, combined with tilting through at
least +30" to -30° about an axis in the plane of the
specimen; or,
(b) tilted through at least +30" to -30° about two
perpendicular axes in the plane of the specimen.
The analysis is greatly facilitated if the goniometer
permits eucentric tilting, although this is not essential. If
EDXA and zone-axis ED are required on the same fiber, the
goniometer shall be of a type which permits tilting of the
specimen and acquisition of EDXA spectra without change of
specimen holder.
The TEM shall have an illumination and condenser lens system
capable of forming an electron probe smaller than 250 nm in
diameter.
Use of an anti-contamination trap around the specimen is
recommended if the required instrumental performance is to be
maintained.
7.3.2. Energy Dispersive X-ray Analyzer
The TEM shall be equipped with an energy dispersive X-ray
analyzer capable of achieving a resolution better than 175 eV
(FWHM) on the Mnl^ peak. Since the performance of individual
combinations of TEM and EDXA equipment is dependent on a number of
27
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geometrical factors, the required performance of the combination
of the TEM and X-ray analyzer is specified in terms of the
measured X-ray intensity obtained from a fibre of small diameter,
using a known electron beam diameter. Solid state X-ray detectors
are least sensitive in the low energy region, and so measurement
of sodium in crocidolite shall be the performance criterion. The
combination of electron microscope and X-ray analyzer shall yield,
under routine analytical conditions, a background-subtracted NaKa
integrated peak count rate of more than 1 count per second (cps)
from a 50 nm diameter fiber of UICC crocidolite irradiated by a
250 nm diameter electron probe at an accelerating potential of
80 kV. The peak/background ratio for this performance test shall
exceed 1.0.
The EDXA unit shall provide the means for subtraction of the
background, identification of elemental peaks, and calculation of
background-subtracted peak areas.
7.3.3. Computer
Many repetitive numerical calculations are necessary, and
these may be performed conveniently by relatively simple computer
programs. For analyses of zone-axis ED pattern measurements, a
computer with adequate memory is required to accommodate the more
complex programs involved.
7.3.4. Plasma Asher
For ashing of filters or particulate deposits washed from
filters, a plasma asher, with a radio frequency power rating of
100 W or higher, shall be used. The asher shall be supplied with
a controlled oxygen flow, and shall be modified, if necessary, to
provide a valve to control the speed of air admission so that
rapid air admission does not disturb the ashed particulate. It is
recommended that filters be fitted to the oxygen supply and the
air admission line.
7.3.5. Filtration Apparatus
After re-dispersal of the ashed particulate in water,
the particulate suspension is filtered through a membrane filter
of 25 mm diameter. A glass frit support is required in order to
obtain a uniform deposit on the filter. The reservoir must be
easily cleaned in order to prevent sample cross-contamination. A
25 mm analytical filter holder (Millipore Corporation, Cat. No.
XX10 025 00) or equivalent has been found to be suitable.
7.3.6. Filtration Manifold
When a number of samples are to be filtered, several
28
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filtration units can be operated simultaneously from a single
vacuum source by using a multiple port filtration manifold
(Millipore Corporation, Cat. No. XX26 047 35 or equivalent has
been found to be suitable). The manifold should include valves to
permit each port to be opened or closed independently.
7.3.7. Vacuum Pump
A pump is required to provide a vacuum of approximately 20
kPa for the filtration of water samples. A water jet pump
(Edwards High Vacuum Inc., Grand Island, NY 14072, Cat. No.
01-C046-01-000-female connection or 01-C039-01-000-male connection
or equivalent) has been found to provide sufficient vacuum for a
3-port filtration mani-fold and also incorporates a non-return
valve to prevent back-streaming.
7.3.8. Vacuum Coating Unit
A vacuum coating unit capable of producing a vacuum better
than 0.013 Pa shall be used for vacuum deposition of carbon on the
membrane filters. A sample holder is required which will allow a
glass microscope slide to be continuously rotated during the
coating procedure. A mechanism which allows the rotating slide
also to be tilted through an angle of approximately 45° during the
coating procedure is recommended. A liquid nitrogen cold trap
above the diffusion pump may be used to minimize the possibility
of contamination of the filter surfaces by oil from the pumping
system. The vacuum coating unit may also be used for deposition
of the thin film of gold, or other calibration material, when it
is required on TEM specimens as an internal calibration of ED
patterns.
7.3.9. Sputter Coater
A sputter coater with a gold target may be used for
deposition of gold on to TEM specimens as an internal calibration
of ED patterns. Experience has shown that a sputter coater allows
better control of the thickness of the calibration material.
7.3.10. Solvent Washer (Jaffe Washer)
The purpose of the Jaffe washer is to allow dissolution of
the filter polymer while leaving an intact evaporated carbon film
supporting the fibers and other particles from the filter surface.
One design of a washer that has been found satisfactory for
various solvents and filter media is shown in Figure 7.2. Several
acceptable variations have been described in the scientific
literature. When specimens are not being inserted or removed, the
petri dish lid should be in place. The washer should be cleaned
before it is used for each batch of specimens.
29
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GLASS PETRI DISH
( lOOmm x 15mm )
ELECTRON MICROSCOPE
SPECIMENS
ST. STEEL MESH
BRIDGE ( SOmesh)
LENS
TISSUE
Figure 7.2: Design of a Solvent Washer (Jaffe Washer).
Solvent is added until the meniscus contacts the underside of
the stainless steel mesh
30
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7.3.11. Condensation Washer
For more rapid dissolution of the filter polymer, a
condensation washer, consisting of a flask, condenser and cold
finger assembly, with a heating mantle and means for controlling
the temperature may be used. A suitable assembly is shown in
Figure 7.3, and acetone should be used as the solvent for MCE
filters.
7.3.12. Slide Warmer or Oven
Either a slide warmer or an oven shall be used for heating
slides during the preparation of TEM specimens. It is required to
maintain a temperature of 65-70°C.
7.3.13. Ultrasonic Bath
An ultrasonic bath is required for cleaning of apparatus
used for TEM specimen preparation, and for re-dispersal of ashed
residues from MCE filters.
7.3.14. Carbon Grating Replica
A carbon grating replica with about 2000 parallel lines
per mm shall be used to calibrate the magnification of the
TEM.
7.3.15. Calibration Grids for EDXA
For calibration of the efficiency of the EDXA system,
reference silicate standard material must be used. The National
Institute for Standards and Technology (NIST) Standard Reference
Material (SRM) 2063 is a sputtered thin film containing known
concentrations of magnesium, silicon, calcium and iron. Other
suitable calibration materials include riebeckite, chrysotile,
halloysite, phlogopite, wollastonite and bustamite. The mineral
used for calibration of the EDXA system for sodium must be
prepared using a gold TEM grid.
7.3.16. Carbon Rod Sharpener
The use of necked carbon rods, or equivalent, allows the
carbon to be evaporated on to the filters without creating
sufficient heat to damage the filters.
7.3.17. Disposable-Tip Micropipettes
A disposable tip micropipette, capable of transferring a
volume of approximately 30 /^L, is required for the preparation of
TEM specimen grids.
31
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ADAPTER
CONDENSER
SPECIMEN
•4
COLD WATER
SOURCE
COLD FINGER
a c
WATER
DRAIN
THERMOSTATICALLY
CONTROLLED
HEATING MANTLE
Figure 7.3: Design of a Condensation Washer
. 32
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7.4. CONSUMABLE LABORATORY SUPPLIES
7.4.1. Glass Beakers
Borosilicate glass beakers of 100 mL capacity are required
for ashing of filters and re-dispersal of ashed residues.
7.4.2. Membrane Filters
For filtration of the particulate suspensions, two types of
filter are required:
(a) MCE filters of 0.1 pm pore size (Millipore Corporation
VCWP 025 00 or equivalent) are used to collect the
particles from the liquid suspension;
(b) MCE filters of 5 pm pore size (Millipore Corporation
SMWP 025 00 of equivalent). One of these filters is
placed between the glass frit of the filtration
apparatus and the 0.1 /tm pore size filter, in order to
ensure a uniform particulate deposit.
7.4.3. Copper Electron Microscope Grids
Two-hundred mesh TEM grids are required. Grids which have
grid openings of uniform size such that they meet the requirement
of 10.4.2 should be chosen.
7.4.4. Gold Electron Microscope Grids
Two-hundred mesh gold TEM grids are required to mount TEM
specimens when sodium measurements are required in the fiber
identification procedure. Grids that have grid openings of
uniform size such that they meet the requirement of 10.4.2 should
be chosen.
7.4.5. Carbon Rod Electrodes
Spectrochemically pure carbon rods are required for use in
the vacuum evaporator during carbon coating of filters.
7.4.6. Disposable Tips for Micropipette
Disposable tips for a micropipette are required for
measurement of 30 /zL volumes of the filter collapsing solution.
33
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7.4.7. Routine Electron Microscopy Tools and Supplies
Fine-point tweezers, scalpel holders and blades, microscope
slides, double-coated adhesive tape, lens tissue, gold wire,
tungsten filaments, liquid nitrogen, and other routine supplies
are required.
7.4.8. Reference Asbestos Samples
Asbestos samples are required for preparation of reference
TEH specimens of the primary asbestos minerals. The NIST SRM 1866
contains well-characterized samples of chryostile, crocidolite,
and amosite, although compositional data are not available for
these standards. The UICC set of minerals may be used as
additional reference materials.
34
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8. REAGENTS
8.1. FRESHLY-DISTILLED WATER
A supply of freshly-distilled water must be available. The water
must be produced using an all-glass still. Any water used for
re-dispersal of ash, or for final rinsing of glassware must have been
distilled no longer than 24 hours prior to use.
8.2. DIMETHYL FORMAMIDE, ANALYTICAL GRADE
8.3. GLACIAL ACETIC ACID, ANALYTICAL GRADE
8.4. ACETONE, ANALYTICAL GRADE
8.5. HYDROCHLORIC ACID, ANALYTICAL GRADE
WARNING - Use the reagents in accordance with the
appropriate health and safety regulations.
35
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9. AIR SAMPLE COLLECTION
Sampling procedures for this method were developed in consideration of
and designed to be compatible with the analytical requirements of this method
including, primarily, the need to achieve the desired analytical sensitivity.
Analytical sensitivity is a function of the volume of air sampled, the
active area of the collection filter, the dilution or concentration factor
introduced during specimen preparation, and the area of the TEM specimen over
which asbestos structures are counted. If total airborne dust levels are
high, it may be necessary to terminate sampling before the required volume has
been sampled. If this happens, the analytical sensitivity required can be
achieved only by counting structures over a greater area of the TEM specimen,
or by use of a specimen preparation technique that incorporates some selective
concentration of asbestos structures relative to other particulate species.
For example, depending on the nature of the suspended particulate, a con-
centration factor up to about 2 may be possible for 25 mm filters, if the
entire filter is used in the analysis. Larger concentration factors are
possible for larger sample filters. In either case, the number of grid
openings to be examined will be proportionately reduced.
The number of grid openings that must be examined in order to achieve a
particular analytical sensitivity, without any selective concentration of the
asbestos structures, is shown in Table 9.1. An average grid opening size of
0.0081 mm2 is assumed in this table. However, grid opening sizes vary
depending on the manufacturer of the grid. When using this table to derive
the number of grid openings to be counted to achieve a defined analytical
sensitivity, the number of grid openings to be counted must be adjusted to
preserve the total areas represented. For example, if a volume of 5000 liters
of air is collected, the table indicates that 96 grid openings must be counted
to achieve an analytical sensitivity of 0.1 s/L. This corresponds to an area
of 0.78 mm2 (96 x 0.0081). If the average grid opening size on another grid
is 80 pm square (resulting in an average grid opening area of 0.0064 mm2),
than a total of 122 grid openings will have to be counted, to cover the same
area and achieve the same analytical sensitivity. Under no circumstances
should fewer than 4 grid openings be counted.
The volume in liters required to achieve a specified analytical
sensitivity is calculated from the formula:
where:
V - Af/(N x Ag x S x F)
Af - Active area of sample collection filter in mm2
N - Number of grid openings to be examined. N shall be
rounded upwards to the next highest integer
Ag — Mean area of grid openings in mm2
S - Analytical sensitivity in structures/liter
F - Concentration factor.
36
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Table 9.1 - Examples of the Minimum Number of Grid Openings Required to Achieve
a Particular Analytical Sensitivity
Analytical
sensitivity
s/L
2000
Volume of air sampled (liters)
5000
7000
10000
15000
20000
0.01
0.02
0.03
0.04
0.05
0.07
0,
0,
0,
0.4
0.5
0.7
1.0
2.0
3.0
4.0
5.0
7.0
10.0
2377
1189
793
595
476
340
238
119
80
60
48
34
24
12
8
6
5
4
4
951
476
317
238
191
136
96
48
32
24
20
14
10
5
4
4
4
4
4
680
340
227
170
136
98
68
34
23
17
14
10
7
4
4
4
4
4
4
476
238
159
119
96
68
48
24
16
12
10
7
5
4
4
4
4
4
4
317
159
106
80
64
46
32
16
11
8
7
5
4
4
4
4
4
4
4
238
119
80
60
48
34
24
12
8
6
5
4
4
4
4
4
4
4
4
NOTES
In this table, it is assumed that the collection filter area is 385 mm2, there
is no concentration or dilution introduced during specimen preparation, and the
TEM grid openings are 90 jum square.
For grid openings with dimensions other than 90/jm square, the minimum number of
openings to be counted should be adjusted by the ratios of the squares of the
grid opening dimensions, such that the area of the TEM specimen examined remains
the same.
37
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The concentration factor F arises from the fact that, when using an
indirect specimen preparation technique, the fibers on a particular area of
the TEM specimen have usually been transferred from a different area of the
original sample collection filter. The concentration factor may correspond to
a concentration or a dilution of the original fiber density on the collection
filter. It is calculated from the ratio between the areas of the original and
analytical filters, the volume of water used to re-disperse the ashed particu-
late, and the volume of the final dispersion used to prepare the analytical
filter. For the purposes of calculating the required volume of air to be
sampled in order to achieve the specified analytical sensitivity when a direct
transfer preparation is to be used, the concentration factor (F) is assumed to
be 1.
9.1. REQUIRED SENSITIVITY
The required analytical sensitivities for characterizing environ-
mental samples is established in section 4.3 as 0.5 s/L and 0.02 s/L for
asbestos structures of all sizes and asbestos structures longer than 5 /jm,
respectively. For other applications of this method, different analytical
sensitivities should be established prior to sample collection. Air
volumes to be collected in this method are selected to provide the desired
analytical sensitivity when coupled with the counting of a manageable
number of grid openings.
9.2. AIR VOLUME
The sampling rate and the period of sampling shall be selected to
yield as high a sampled volume as possible, which will minimize the
influence of filter contamination. Wherever possible, a volume of 15
cubic meters shall be sampled for those samples intended for analysis only
by the indirect TEM preparation method (Phase 1 samples). For those
samples to be prepared by both the indirect and the direct specimen
preparation methods (Phase 2 samples), the volumes must be adjusted so as
to provide a suitably-loaded filter for the direct TEM preparation method
(See Section 4.2.1). It has been found that the volume cannot normally
exceed 5 cubic meters in an urban or agricultural area, and 10 cubic
meters in a rural area for samples collected on a 25 mm filter and
prepared by a direct transfer technique.
Prior to planning a sampling program, air volumes to be collected
should be adjusted based on available information characterizing typical
local TSP concentrations (see Section 4.2.1). It has not proven possible
to judge the loading of filters in real time during sampling so that
filters may easily become overloaded to the point were analysis by the
direct method becomes impossible. To minimize the potential that filters
will have to be rejected for analysis due to overloading, conservative
estimates of the loading rate based on typical TSP concentrations have
been developed so that an "optimum" volume of air per unit area of filter
may be collected. However, because such estimates tend to be conservative
to avoid overloading, a fair percentage of the filters analyzed from such
38
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a sampling effort tend to be underloaded forcing a corresponding increase
in analysis costs.
NOTE
One option is to collect filters at several loadings to bracket the
estimated optimum loading for a particular site. Such filters can
be screened in the laboratory so that only those filters closest to
optimal loading are analyzed.
9.3. FLOW RATE
To remain within known constraints on filter face velocities as
reported in Section 2.2, an upper limit to the range of acceptable flow
rates for this method is 15 L/min. In practice, available pumps that can
be used for environmental sampling at remote locations operate under load
at a maximum of approximately 12 L/min. Consequently, the recommended
15 m3 of air for Phase 1 samples requires approximately 20 hours to
collect. Such pumps typically draw 6 amps at full power so that 2
lead/acid batteries should provide sufficient power to collect a full
sample. The lower volumes required for Phase 2 samples can be collected
in shorter periods with a corresponding reduction in stored energy
requirements. The use of line voltage, where available, eliminates the
difficulties associated with transporting stored electrical energy.
At many locations, wind patterns exhibit strong diurnal variations.
Therefore, intermittent sampling (sampling over a fixed time interval
repeated over several days) may be necessary to accumulate 20 hours of
sampling time over constant wind conditions. Other sampling objectives
also may necessitate intermittent sampling. The objective is to design a
sampling schedule so that samples are collected under uniform conditions
throughout the sampling interval. This method provides for such options.
9.4. SAMPLING PROCEDURES
Before air samples are collected, unused filters shall be analyzed
as described in Section 10.6 to determine the mean background asbestos
structure count for the analytical procedure.
Air samples shall be collected using cassettes as described in
7.1.1. The cassettes used for sampling shall be new and unused. Sampling
shall be conducted with the cassette open-face. During sampling, the
filter cassette shall be supported on a stand so that it is isolated from
the vibrations of the pump. The cassette shall be held facing vertically
downwards at a height of approximately 1.5-2.0 m above ground/floor level
and connected to the pump with a flexible tube. In many cases it will
likely be sufficient to collect samples with a short cassette. If
conditions dictate the need for additional protection, however, extension
cowls may be affixed to the front of the cassette. Such cowls must be
39
-------
constructed of electrically conducting material to minimize electrostatic
effects.
The sampling flow-rate should be measured at the cassette at the
beginning and at the end of the sampling period. Sampling equipment shall
be selected such that either the sampling flow rate can be maintained to
within ± 10%, using a flow controller or critical orifice, or flow.rates
shall be measured at least every 4 hours. If at any time the measurement
indicates that the flow-rate has decreased by more than 30%, the sampling
shall be terminated. The mean value of these flow-rate measurements shall
be used to calculate the total air volume sampled. After sampling, a cap
shall be placed over the open end of the cassette, and the cassette packed
in a clean plastic bag for return to the laboratory. Field blank filters
shall also be included, as described in Section 10.6, and processed
through the remaining analytical procedures along with the samples.
Should intermittent sampling be required, sampling cassettes must be
covered between active periods of sampling. To cover the sampler: turn
the cassette to face upward, attach a rotameter to measure the flow rate,
turn off the sampling pump, and place the cap. To resume sampling: remove
the cap, turn on the sampling pump, attach a rotameter to measure the flow
rate, and invert the cassette to face downward.
40
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10. PROCEDURE FOR ANALYSIS
10.1. INTRODUCTION
' Fi.lters representing Phase 1 samples will be prepared by the
indirect method. Filters representing Phase 2 samples will be split.
Half of the Phase 2 filters will be prepared by the indirect method and a
quarter sector of the filter will be prepared by the direct method.
10.1.1. Indirect TEM Specimen Preparation Method
The indirect method requires collection of particulate from a
large volume of air on a 0.45 /HB pore size MCE filter. In this
procedure, half of the filter is ashed by treatment in a low
temperature plasma asher, and the ashed residue is re-dispersed in
distilled water. Immediately before filtration, the suspension of
particles is acidified using hydrochloric acid to remove any
acid-soluble species. The suspension is filtered through a 0.1 pra.
pore'size MCE filter. The filter is collapsed using a mixture of
dimethyl formamide, acetic acid and water. A sector of the
collapsed filter is carbon coated, arid TEM specimen grids are
prepared from the carbon coated filter by the Jaffe washer
technique, using dimethyl formamide as the solvent.
Use of the 0.1 pm pore size membrane filter eliminates the
requirement for the plasma etching step incorporated into both NIOSH
Method 7402 and the method of Burdett and Rood. In previous work by
Chatfield, Dillon and Stott, no statistically-significant fiber
losses could be detected when TEM grids were prepared from 0.1 /zm
pore size MCE filters without including the etching step.
Indirect preparation of TEM specimens for determination of
asbestos is particularly sensitive to problems of contamination by
extraneous asbestos. This method requires that very low detection
limits be achieved so that control of extraneous asbestos
contamination becomes even more important. The ability to meet the
blank sample criteria is critically dependent on the cleanliness of
equipment and supplies. All supplies such as microscope slides and
glassware should be considered as potential sources of asbestos
contamination. All solvents used should be filtered through a
0.2 ^m pore size filter or they should be distilled in a glass
still. It is necessary to wash all glassware before it is used and
the final washing of glassware should be performed using
freshly-distilled water. Any tools or glassware that come into
contact with the air sampling filters or TEM specimen preparations
should be washed both before use and between handling of individual
samples. Where possible, disposable supplies should be used.
41
-------
10.1.2. Direct TEM Specimen Preparation Method
The direct TEM specimen preparation method requires that the
air volume sampled must not result in a filter that is over-loaded
by particulate. Particulate is collected on a 0.45 /zm pore size MCE
filter. A quarter sector of the MCE filter is collapsed using a
mixture of dimethyl formamide, acetic acid and water. The collapsed
filter is etched for a short time in a low temperature plasma asher.
The collapsed and etched filter is carbon coated, and TEM specimen
grids are prepared from the carbon coated filter by the Jaffe washer
technique, using dimethyl formamide as the solvent.
10.2. PREPARATION OF TEM SPECIMEN GRIDS BY THE INDIRECT METHOD
10.2.1. Cleaning of Sample Cassettes
Asbestos can adhere to the exterior surfaces of air sampling
cassettes and this asbestos can be inadvertently transferred to the
sample during handling. To prevent this possibility of
contamination, and after ensuring that the cassette is tightly
sealed, wipe the exterior surfaces of each sampling cassette with a
clean, wet paper towel before it is taken into the clean facility or
laminar flow hood.
10.2.2. Ashing of MCE Filter
Open the filter cassette and remove the MCE filter. Place the
filter on a clean microscope slide and cut it in half, using a
curved scalpel blade. Return one half of the filter to the
cassette. Place the other half filter in the bottom of a 100 mL
glass beaker and cover the top of the beaker with a piece of
aluminum foil. Make approximately 8 1-2 mm perforations in the
aluminum foil. Completely ash the half filter in the low
temperature plasma asher, using operating conditions as defined in
Appendix A.
10.2.3. Re-dispersal of Ashed Residues
After the filter has been completely ashed, remove the beaker
from the asher, add 40 mL of distilled water, and place the beaker
in the ultrasonic bath for a period of 15 minutes. Using a
disposable plastic pipette, add 0.2 mL of concentrated hydrochloric
acid and stir the suspension. After addition of the acid, place the
beaker in the ultrasonic bath for a period of 5 minutes. Filtration
of the suspension must be performed immediately after the ultrasonic
treatment. Pov,er in the ultrasonic bath should be maintained below
0.1 watt/mL.
42
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10.2.4. Filtration of the Aqueous Suspension
The separation of suspended particulate by filtration of the
aqueous suspension through a membrane filter is a critical step in
the analytical procedure. The objective is to produce an analytical
filter on which the suspended solids from the sample are distributed
uniformly, and at an acceptable loading for the microscopical
examination.
The volume to be filtered generally depends on either the
total suspended solids content or the asbestos structure
concentration of the sample, and the intention is to prepare a
filter that has a loading not exceeding about 50 asbestos structures
per grid opening and with a particulate coverage not exceeding 10%
of the filter area. Some judgment is required to achieve the
optimum loading and, if the asbestos concentration is very low, it
will be found that the suspended solids concentration will limit the
volume that can be filtered. The maximum particulate loading on the
filter that can be tolerated is about 10 fig/cm2.
In practice, the best procedure for optimizing the loading on
the final filter is to prepare several filters using different
volumes of the suspension. Any fraction of the 40 mL suspension
(prepared as per Section 10.2.3) may be selected as a unit for
filtration. For example, aliquots containing 1 mL, 4 mL, 8 mL, and
20 mL fractions from the original suspension may be used to prepare
a series of increasingly loaded filters. However, if the con-
centration of suspended solids dictates that a very small volume of
the suspension be filtered, small volume aliquots need to be further
diluted prior to filtering. Do not attempt to filter a volume of
less than 10 mL.
If aliquots smaller than 10 mL from the original suspension
are to be filtered, it is difficult to ensure that a uniform deposit
of particulate is obtained on the filter unless the aliquot is first
diluted. Samples of high solids content, or of high asbestos
concentration, may require filtration of volumes less than 10 mL.
Such aliquots shall be diluted with freshly-distilled water so that
the filtered volumes exceed the minimum of 10 mL. These dilutions
shall be made by transferring a known volume of the sample (such as
an aliquot from the original suspension) to a disposable plastic
beaker and making the volume up to a known volume with
freshly-distilled water. The mixture shall be stirred vigorously
before sub-samples are taken for filtration.
The following instructions for filtration must be followed
precisely:
(a) Assemble the filtration base and turn on the vacuum.
The upper surface of the filtration base (both the glass
43
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(b)
(c)
frit and the ground mating surface) must be dry before
the membrane filters are installed. Place a 5 pm pore
size MCE filter on the glass frit. If the filter
appears to become wet by capillary action on residual
water in the glass frit it must be discarded and
replaced by another filter. Place a 0.1 pm pore size
MCE filter, smooth side facing up, on top of the 5 pm
filter. The mating'surface of the reservoir component
of the filtration apparatus should be dried by shaking
off any surplus water and draining on paper towel or
tissue. Position the reservoir on the filters and
firmly clamp it, taking care not to disturb the filters.
The vacuum should not be released until the filtration
has been completed.
It is necessary to comment- on the 'use of filtration
equipment that is still wet after washing, -since
improper procedures at this -point can seriously
compromise the results. If the glass frit is wet when
the first filter is applied to it, capillary action will
result in some areas of the filter structure being
filled by water. When the second, smaller pore size
filter is added and the vacuum is applied, the
differential pressure across the 5 yum pore size filter
will be insufficient to overcome the surface tension of
the water in the filled areas. Thus no filtration will
take place through the corresponding areas of the 0.1 pm
pore size'filter, and a grossly non-uniform deposit of
particulate will be obtained.
Add the required volume of sample to the filtration
funnel. Disposable plastic beakers and pipettes provide
a means of measuring the sample volumes without
introducing problems of sample'cross-contamination. The
reservoir may not be sufficiently large to accommodate
the total volume of liquid to be filtered. In this
case, more of the sample may be added during the
filtration, but this should be done carefully and only
when the reservoir is more than half full. In this way
the addition of'liquid will riot disturb or affect the
uniformity of particulate already deposited on the
filter. Do not rinse the sides of the reservoir, and
avoid other manipulations which may disturb the
particulate deposit on the filter.
Disassemble the filtration apparatus, and transfer the
0.1 pro pore size filter to" a labelled, clean plastic
petri dish. Place the cover loosely over the top of the
dish to limit any deposition of material on the filter,
and allow the filter to dry. Discard the 5 pm pore size
filter. -....-•...•
44
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10.2.5. Selection of Area of Filter for Preparation
Using clean tweezers, and a clean, curved scalpel blade, cut
out a 90 degree sector of the filter while it is still in the
plastic petri dish.
10.2.6. Preparation of Solution for Collapsing MCE Filters
Mix 35 mL of dimethyl formamide, 15 mL of glacial acetic acid
with 50 mL of freshly-distilled water. Store this mixture in a
clean bottle.
10.2.7. Filter Collapsing Procedure
Using a micropipette with a disposable tip, place
approximately 30 pL of the collapsing solution on a cleaned
microscope slide, and using the end of the pipette tip spread the
liquid over the area to be occupied by the filter sector. Place the
filter sector, active surface upwards, on top of the solution,
lowering the edge of the filter at an angle of about 20 degrees so
that air bubbles are not created. Remove any solution not absorbed
by the filter by allowing a paper tissue to contact the liquid at
the edge of the filter. More than one filter sector may be placed
on one slide, and a laboratory blank filter sector shall also be
prepared on each slide. Place the slide either on a
thermostatically-controlled slide warmer at a temperature of
approximately 65-70°C, or in an oven at the same temperature, for
about 10 minutes. The filter collapses slowly to about 15% of its
original thickness. The procedure leaves a thin, transparent
plastic film adhering to the microscope slide, with particles and
fibers embedded in the upper surface.
10.2.8. Carbon Coating of Filter Sectors
Place the glass slide holding the filter sectors on the
rotation-tilting device, approximately 10-12 cm from the evaporation
source, and evacuate the evaporator chamber to a vacuum better than
0.013 Pa. The evaporation of carbon shall be performed in very
short bursts, separated by some seconds to allow the electrodes to
cool. If evaporation of carbon is too rapid, the surface of the
filter may be damaged. The thickness of carbon required is
dependent on the size of particles on the filter, and approximately
30-50 run has been found to be satisfactory. If the carbon film is
too thin, large particles will break out of the film during the
later stages of preparation, and there will be few complete and
undamaged grid openings.on the specimen. Too thick a carbon film
will lead to a TEM image which is lacking in contrast, and the
ability to obtain SAED patterns will be compromised. The carbon
film thickness, should be the minimum possible, while retaining most
of the grid openings of the TEM specimen intact.
45
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10.2.9. Preparation of the Jaffe Washer
Place several pieces of lens tissue on the stainless steel
bridge, and fill the washer with dimethyl formamide to a level where
the meniscus contacts the underside of the mesh, resulting in
saturation of the lens tissue. If it is intended to complete the
washing of the specimens in the condensation washer, the pieces of
lens tissue should be of such a size that they will fit on to the
stainless steel mesh of the condensation washer cold finger. Three
TEM specimen grids shall be prepared from each of the coated
analytical filters, and for each of the analytical filters, three
new specimen grids should be placed on to one of the pieces of lens
tissue.
10.2.10. Placing of Specimens Into the Jaffe Washer
Using a curved scalpel blade, cut along the radius of the
coated filter sector, approximately 1 mm from the edge. Discard the
thin strip of coated filter, which usually exhibits signs of edge
effects introduced during the collapsing procedure. Starting from
the freshly-cut edge, cut three approximately 3 mm square pieces of
the coated filter. Select three squares to represent the center and
the outer periphery of the active surface of the filter. Place each
square of filter, carbon side up, on one of the TEM specimen grids
already on the lens tissue in the Jaffe washer. Cover the Jaffe
washer with the lid, and allow the washer to stand. Specimens are
normally cleared after approximately 4 hours.
10.2.11. Rapid Preparation of TEM Specimens From MCE Filters
An alternative washing procedure may be used to prepare TEM
specimens from MCE filters more rapidly than can be achieved by the
Jaffe washing procedure. After the specimens have been washed in a
Jaffe washer for approximately 1 hour, transfer the piece of lens
tissue supporting the specimens to the cold finger of a condensation
washer operating with acetone as the solvent. Operate the
condensation washer for approximately 30 minutes. This treatment
removes all remaining filter polymer.
10.3. PREPARATION OF TEM SPECIMEN GRIDS BY THE DIRECT METHOD
10.3.1.
Cleaning of Sample Cassettes
After ensuring that the cassette is tightly sealed, wipe the
exterior surfaces of each sampling cassette with a clean, wet paper
towel as described in 10.2.1 before it is taken into the clean
facility or laminar flow hood.
46
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10.3.2. Selection of Area of Filter for Preparation
Using clean tweezers, remove the MCE filter from the filter
cassette, and cut a sector as described in 10.2.5.
10.3.3. Filter Collapsing Procedure
Collapse the filter sector as described in 10.2.7.
10.3.4. Plasma Etching of the Filter Surface
The conditions required in a particular plasma asher shall
first be established using the procedure defined in Appendix A.
Place the microscope slide holding the collapsed filter sectors in
the plasma asher, and etch for the time and under the conditions
determined. Care should be taken to ensure that the correct
conditions are used. After etching, admit air slowly to the chamber
and remove the microscope slide.
10.3.5. Carbon Coating of Filters
Carbon coat the microscope slide holding the collapsed filter
portions as described in 10.2.8.
10.3.6. Preparation of the Jaffe Washer
Prepare the Jaffe washer as described in 10.2.9.
10.3.7. Placing of Specimens into the Jaffe Washer
Place specimens in the Jaffe washer as described in 10.2.10.
Specimens are normally cleared after approximately 4 hours.
10.3.8 . Rapid Preparation of TEM Specimens from MCE Filters
The alternative rapid washing procedure, described in 10.2.11,
may be used to prepare TEM specimens from MCE filters more rapidly
than can be achieved by the Jaffe washing procedure.
10.4. CRITERIA FOR ACCEPTABLE TEM SPECIMEN GRIDS
Examine the TEM specimen grid in the TEM at a magnification
sufficiently low (300-1000) so that complete grid openings can be
inspected. Reject the grid if:
(a) the TEM specimen has not been cleared of filter medium by the
filter dissolution step. If the TEM specimen exhibits areas
of undissolved filter medium and, if at least two of the three
specimen grids are not cleared, either additional solvent
washing shall be carried out or new specimens shall be
prepared from the filter;
47
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(b) the sample is over-loaded with particulate. If the specimen
grid exhibits more than approximately 10% obscuration on the
majority of the grid openings, the specimen will be designated
as over-loaded. This filter cannot be analyzed satisfactorily
because the grid is too heavily loaded with debris to allow
separate examination of individual particles by ED and EDXA
and obscuration of structures by other particulate would lead
to under-estimation of the structure count;
(c) the particulate deposits on the specimen are not uniformly
distributed from one grid opening to the next. If the
particulate deposits on the specimen are obviously not uniform
from one grid opening to the next, the specimen will be
designated as non-uniform. This condition is caused either by
use of an unsatisfactory procedure for the water filtration or
it is a consequence of the fundamental nature of the airborne
particulate. Such a filter cannot be analyzed satisfactorily;
(d) the TEM grid is too heavily loaded with asbestos structures to
make an accurate count. Accurate counts cannot be made if the
grid has more than approximately 7000 asbestos structures/mm2;
or,
(e) less than approximately 75% of the grid openings have unbroken
carbon film over the whole grid opening. Since the breakage
of carbon film is usually more frequent in areas of heavy
deposit, counting of the intact openings can lead to an
underestimate of the structure count. An additional carbon
coating may be applied to the carbon coated filter and new
specimen grids prepared. The larger particles can often be
supported by using a thicker carbon film. If this action does
not produce acceptable specimen grids, this filter cannot be
analyzed and a filter prepared with a lighter loading of
particulate should be selected.
If any one or more of the conditions described in (b), (c), (d) or
(e) exists, the specimen grids cannot be analyzed.
10.5. PROCEDURE FOR STRUCTURE COUNTING BY TEM
10.5.1. Introduction
The TEM examination consists of a count of asbestos
structures, which are present on a specified number of grid
openings. Asbestos structures are classified into groups on the
basis of morphological observations, ED patterns and EDXA spectra.
The total number of asbestos structures to be counted depends on the
statistical precision desired. In the absence of asbestos
structures, the area of the TEM specimen grids which must be
examined depends on the analytical sensitivity required. The
48
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precision of the asbestos structure count depends not only on the
total number of asbestos structures counted, but also on their
uniformity from one grid opening to the next. Additional asbestos
structure counting will be necessary if greater precision is
required.
In order that the estimate of the asbestos structure density
on the sampling filter shall not be based on the asbestos structure
deposits found within the small area represented by one specimen
grid, grid openings shall be examined on at least two of the three
specimen grids prepared. The results shall then be combined in the
calculation of the asbestos structure density. Structure counts
shall be made at a magnification of approximately 20000,, and will be
terminated at the number of asbestos structures as defined below,
except that the count shall be continued until a minimum of 4 grid
openings have been examined. Otherwise, the asbestos structure
count shall continue to that number of grid openings at which the
specified analytical sensitivity has been achieved.
10.5.2. Measurement of Mean Grid Opening Area
The grid opening area shall be measured for the type of TEM
specimen grids in use. This may be performed either on an optical
microscope at a calibrated magnification of about 400, or at a
calibrated magnification in the TEM. A mean value for the grid
opening dimensions may be used if this value can be shown to be
sufficiently precise. If a mean value is used, the standard
deviation of the mean of 10 openings selected randomly from each of
10 grids shall be less than 5%. Alternately, the dimensions of one
grid opening on each of the grids examined shall be measured and the
mean of these used in the calculations for the particular analysis.
Values for the number of grid openings to be counted to
achieve a desired level of sensitivity (Table 9.1) will be adjusted
based on the average grid opening size derived in this section. The
procedure for performing the conversion is presented in a note at
the bottom of Table 9.1.
10.5.3. TEM Alignment and Calibration Procedures
Before structure counting is performed, align the TEM
according to instrumental specifications. Calibrate the TEM and
EDXA system according to the procedures described in Appendix B.
10.5.4. Determination of Stopping Point
Before structure counting is commenced, the area of specimen
to be examined in order to achieve the required analytical
49
-------
sensitivity shall be calculated. The maximum number of grid
openings to be examined shall be calculated from the formula:
N
Af/(Ag x V x S x F)
where:
N - Number of grid openings to be examined. N shall
be rounded upwards to the next highest integer
Af - Area of collection filter in mm2
A. — Area of TEM specimen grid opening in mm
V — Volume of air sampled in liters
S = Required analytical sensitivity in
asbestos structures/liter
F — Concentration factor.
The concentration factor F arises from the fact that the
asbestos structures on a particular area of the TEM specimen have
usually been transferred from a different area of the original
sample collection filter. The concentration factor may correspond
to a concentration or a dilution of the original asbestos structure
density on the collection filter. It is calculated from the ratio
between the areas of the original and analytical filters, the volume
of water used to re-disperse the ash, and the volume of the final
dispersion used to prepare the analytical filter. F is calculated
from the following formula:
F - A. x Vf/(Af x Vr)
where:
Aa - Area of filter ashed
Af - Area of analytical filter
VE — Volume of water used to re-disperse ashed particulate
Vf - Volume of dispersion filtered through analytical filter
10.5.5. General Procedure for Structure Counting and Size Analysis
Use at least two specimen grids prepared from the filter in
the structure count. Select at random several grid openings from
each grid, and combine the data in the calculation of the results.
Use a form similar to that shown in Figure 10.1 to record the
structure counting data. Insert the first specimen grid into the
TEM. Select a typical grid opening and set the screen magnification
to the calibrated value (approximately 20,000). Adjust the sample
50
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TEH ASBESTOS COUNT
Page of
Preparation Date:
Analysis Date:
Report Number:
Filename:
By:_
By:_
Sample Description:
Sample Number:
Sample Filter Area: sq.mm.
Area of Filter Taken: sq.mm.
Analytical Filter Area: sq.mm.
Redispersal Volume: mL
Liquid Volume Filtered: mL
Magnification:
Air Volume:
Grid Opening Dimension:
..Liters
urn
Grid
Opening
Structure
Number
Class
Structure
Type
Length
(mm)
Width
(mm)
Comments
Figure 10.1: Counting Form
51
-------
height until the features in the center of the TEM viewing screen
are at the eucentric point. Set the goniometer tilt angle to zero.
In column 1 of the structure counting form, record the
sequential number of the grid opening. Position the specimen so
that the grid opening is positioned with one corner visible on the
screen. Move the image by adjustment of only one translation
control, carefully examining the sample for asbestos structures,
until the opposite side of the grid opening is encountered. Move
the image by a pre-determined distance less than one screen
diameter, using the other translation control, and scan the image in
the reverse direction. Continue the procedure in this manner until
the entire grid opening has been inspected in a pattern similar to
that shown in Figure 10.2.
When a fibrous structure is detected, assign a sequential
number in column 2, perform the identification procedures required
as detailed in Appendix D, and enter the appropriate compositional
classification on the fiber counting form in column 3. Assign a
morphological classification to the structure according to the
procedures in Appendix C, and record this in column 4. Measure on
the TEM viewing screen the length and width of the image of each of
the components of the fibrous structure in mm and record these
measurements in columns 5 and 6 of the structure counting form.
If fibrous structures are present that are of obvious
biological origin or that are determined to be non-asbestos, record
data for a minimum of the first 10 such structures; further
recording of data from non-asbestos structures is optional. After a
fibrous structure has been examined and measured, re-locate the
original field of view accurately before continuing scanning of the
specimen. Failure to do this may cause asbestos structures to be
overlooked or counted twice. The point at which the examination is
to be terminated depends on the specimen, and is defined by either
(a), (b) or (c) below:
(a) for Phase 1 samples, which have been prepared by the indirect
TEM specimen preparation method only, complete the examination
at the end of the grid opening on which the 50th asbestos
structure is counted;
(b) for Phase 2 samples, which have been prepared both by the
direct and indirect TEM specimen preparation methods, complete
the examination at the end of the grid opening on which the
100th asbestos structure is counted; or
(c) for either of the sample types, until the number of grid
openings required to achieve the specified analytical
sensitivity for asbestos structures of all lengths, calculated
according to 10.5.4, have been inspected, whichever occurs
52
-------
TEM field of viei
Grid opening
First pass
Second pass
\
XX"
Figure 10.2: Scanning Procedure for TEM Specimen Examination
53
-------
first. The data shall be drawn approximately equally from the
two grids. Regardless of the value calculated according to
10.5.4, asbestos structures shall be counted on a minimum of 4
grid openings. Table 10.1 presents a summary of the counting
requirements for Phase 1 and Phase 2 samples.
(d) Only those structures that are identified as, or are suspected
to be, either chrysotile or one of the amphibole minerals,
will be included in either the original or the extended
structure count. Other materials, such as gypsum, cellulose
fibers, and filter artifacts such as undissolyed filter
strands, will not be included in the structure count. This
restriction is intended to ensure that the best statistical
validity is obtained for the materials of interest.
10.5.6. Measurement of Concentration for Asbestos Structures
Longer than 5 fj.ro.
To increase the statistical validity of the measurement, this
method requires that an extended count be made of those asbestos
structures with aspect ratios equal to or greater than 5:1 and
longer than 5 pm. This separate structure count takes account only
of the asbestos structures longer than 5 /zm, and scanning of the TEM
specimen shall be performed using a magnification of approximately
10,000.
Measurement of the asbestos structure lengths shall be
performed at a magnification of 10,000, but asbestos structure
diameters shall be measured at a magnification of a minimum of
20,000. For Phase 1 samples, which have been prepared by indirect
transfer only, the count is continued until either 50 asbestos
structures have been recorded or an area of specimen has been
examined that achieves the specified analytical sensitivity for
asbestos structures longer than 5 /wn. For Phase 2 samples, which
have been prepared both by indirect and direct transfer, the count
is continued until either 100 asbestos structures have been counted
or an area of specimen has been examined that achieves the specified
analytical sensitivity for asbestos structures longer than 5 fan.
Table 10.1 presents a summary of the counting requirements for
Phase 1 and Phase 2 samples.
Only those structures that are identified as, or are suspected
to be, either chrysotile or one of the amphibole minerals, will be
reported in either the original or the extended structure count.
Other materials, such as gypsum, cellulose fibers, and filter
artifacts such as undissolved filter strands, will not be included
in the structure count. This restriction is intended to ensure that
the best statistical validity is obtained for the materials of
interest.
54
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TABLE 10.1: SPECIFICATIONS FOR PHASE 1 AND 2 SAMPLING AND ANALYSIS
WORKING DRAFT - DO NOT COPY Oft QUOTE
Setting Definitions
Ul
Ul
Ambient:
Rural:
Urban
and
Agri-
cult
ural:
Activity
Specific:
Structure sizes to be counted
Number of structures to be counted
Volume of air to be collected is 15 m3
Maximum area to be scanned
Maximum number of grid openings
to be counted
Minimum number of grid openings
to be counted
Volume of air to be collected is 10 m
Maximum area to be scanned
Maximum number of grid openings
to be counted
Minimum number of grid openings
to be counted
Volume of air to be collected is 5 m3
Maximum area to be scanned
Maximum number of grid openings
to be counted
Minimum number of grid openings
to be counted
Volume of air to be determined in the field
Maximum area to be scanned
Maximum number of grid openings
to be counted
Minimum number of grid openings
to be counted
Phase 1 Samoles:
High Magnifi-
cation Scan
total
50
0.05 mm2
7
4
0.08 mm2
10
4
0.15mm2
20
4
TBD
TBD
4
Low Magnifi-
cation Scan
length > 5 im
50
1.3 mn2
160
4
2.0 mm2
240
4
3.9 mm2
480
4
TBD
TBD
4
Phase 2 Samoles:
High Magnifi-
cation Scan
total
100
0.11 mm2
13
4
0.16 rm2
20
4
0.32 mm2
40
4
TBD
TBD
4
Low Magnifi-
cation Scan
length > 5 jun
100
25 mm2
307
4
3.7mm2
460
4
7.5 irm2
920
4
TBD
TBD
4
-------
TABLE 10.1
(continued)
NOTES:
Phase 1 samples will be prepared by the indirect technique. Filters from
Phase 2 samples will be split. Half will be prepared by the indirect technique
and half will be prepared by the direct technique. Note that the required
sensitivity for the Phase 2 samples is higher than that necessary for the Phase
1 samples. Consequently, it is assumed that 20 structures need to be counted
for phase 2 samples at a minimum.
The information presented in this table is derived from the equation presen-
ted in the Section 10.5.4 assuming that Af - 385 mm2, Ag - 0.0081 mm2, F - 1, and
that 10 structures will have to be counted for statistical significance. Based
on a review of reported background concentrations and typical concentrations
encountered in previous studies, it is also assumed that the critical
concentration for total structures is 5.0 structures/liter and the critical
concentration for structures longer than 5 micrometers is 0.2 structures/liter.
During any site investigation, it will likely be necessary to characterize
both ambient air concentrations and concentrations of asbestos associated with
specific human activities. Although the method is designed for ambient
conditions, it will be possible to adapt it to activity specific sampling by
accounting with some adjustment for expected differences in conditions between
the two types of samples. The principle difference likely is that a much higher
concentration of non-asbestos particulates will likely be contained in air
sampled in association with specific, dust-generating activities. As a
consequence, the estimated values for maximum tolerable air volumes presented in
section 4.2.1 of the text can not be applied to activity specific sampling and
some measure of TSP will have to be provided to derive alternate sampling
volumes. Thus, "TBD" means: to be determined.
It must be emphasized that the volume of air recommended for collection in
each of the environmental settings listed represents a conservative estimate
(based on published data) of the maximum that can be collected and still allow
analysis without overloading the filter. If additional information regarding
the local average concentration of ambient TSP, air volumes to be collected in
any of these settings should be optimized accordingly.
To achieve the desired analytical sensitivity, the numbers of grid openings
required for counting (as presented in this Table) may either be distributed
over a set of specimen grids prepared from a single sample or a larger set of
specimen grids prepared from several samples collected in a manner assuring that
the samples are appropriately homogeneous (see Section 4.3 of the Technical
Background Document, Part 2 of this report).
56
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10.6. BLANK AND QUALITY CONTROL DETERMINATIONS
Before air samples are collected, a minimum of 2 unused 0.45 jum pore size
MCE filters from each lot of 100 filters shall be sectioned and analyzed
both by the indirect and the direct transfer procedure to determine the mean
background asbestos structure concentration. If the mean concentration for
all types of asbestos structures, expressed as the concentration per unit
area of the sample collection filter, is found to be more than
10 structures/mm2, the reasons for the high blank values shall be determined
and the situation corrected before air samples are collected.
To ensure that contamination by extraneous asbestos structures during
specimen preparation is insignificant compared with the results reported on
samples, it is necessary to establish a continuous program of blank measure-
ments . The number of field blanks incorporated into the program shall be at
least 10% of the total number of samples, and all of these shall be
analyzed. In addition, one unused 0.1 /im pore size filter shall be included
with every group of samples prepared on one microscope slide.
It is further recommended that laboratory blanks be collected intermit-
tently at all critical phases of the laboratory program. For example, in
addition to the filter blanks and field blanks, suspension blanks should be
prepared by subjecting a clean ashing tube to the asher, rinsing the tube
and completing the standard indirect preparation on the "clean" rinse.
Other blanks may be constructed to test for the potential introduction of
contamination at other phases of the process. Such blanks need not be
analyzed but may be stored to provide a chain for trouble-shooting should a
problem with contamination occur.
Initially, and also included at random points in the sampling program, it
is necessary to ensure that samples of known asbestos structure
concentrations can be analyzed satisfactorily by this procedure. There are
many opportunities in the procedure for a low recovery to be obtained, and
the recovery must be measured frequently, particularly if more than one
laboratory performs the analysis. Reference filters of known concentration
will be incorporated, and shall constitute a minimum of 5% of the total
analyses. These reference filters shall not be identified to the analytical
laboratory prior to the analysis of any group of samples. The results of
these reference analyses shall not differ at the 5% significance level from
the mean value obtained by a selected group of experienced laboratories.
Since there is a subjective component in the structure counting procedure,
it is necessary that re-counts of some specimens be made by different
microscopists, in order to minimize the subjective effects. Such recounts
provide a means of maintaining comparability between counts made by
different microscopists. Variability between and within microscopists and
between laboratories shall be characterized. These quality assurance
57
-------
measurements shall constitute a minimum of approximately 10% of the
analyses. Repeat results should not differ at the 5% significance level.
10.7. CALCULATION OF RESULTS
Prior
Calculate the results using the procedures detailed in Appendix E.
to the TEM examination of the specimens, the level of analysis was
specified. Before the results are calculated, the compositional and
morphological classifications to be included in the result shall be
specified. The chi-squared uniformity test shall be conducted using the
number of isolated asbestos structures (individual entities) found on each
grid opening, prior to the application of the cluster and matrix counting
criteria. The concentration result shall be calculated using the numbers of
asbestos structures reported after the application of the cluster and matrix
counting criteria.
58
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11. PERFORMANCE CHARACTERISTICS
11.1. INTERFERENCES AND LIMITATIONS OF STRUCTURE IDENTIFICATION
Unequivocal identification of every chrysotile structure is not possible,
due to both instrumental limitations and the nature of some of the component
fibers. The requirement for a calibrated ED pattern eliminates the pos-
sibility of an incorrect identification of the structure selected. However,
there is a possibility of misidentification of other chrysotile structures
for which both a morphology and ED pattern are reported on the basis of
visual inspection only. The only significant possibilities of
misidentification occur with halloysite, vermiculite scrolls or
palygorskite, all of which can be discriminated from chrysotile by the use
of EDXA and by observation of the 0.73 run (002) reflection of chrysotile in
the ED pattern.
As in the case of chrysotile structures, complete identification of every
amphibole structure is not possible due to instrumental limitations and the
nature of some of the component fibers. Moreover, complete identification
of every amphibole structure is not practical due to the limitations of both
time and cost. Particles of a number of other minerals having compositions
similar to those of some amphiboles could be erroneously classified as
amphibole when the classification criteria do not include zone-axis ED
techniques. However, the requirement for quantitative EDXA measurements on
all structures as support for the random orientation ED technique makes
misidentification very unlikely, particularly when other similar structures
in the same sample have been identified as amphibole by zone-axis methods.
The possibility of misidentification is further reduced with increasing
aspect ratio, since many of the minerals with which amphibole may be
confused do not display its prominent cleavage parallel to the c-axis.
11.2. PRECISION AND ACCURACY
11.2.1. Precision
The analytical precision that can be obtained is dependent upon the
number of structures counted, and also on the uniformity of the
particulate deposit on the original filter. Assuming that the structures
are randomly deposited on the filter, if 100 structures are counted and
the loading is at least 3.5 fibers/grid opening, computer modeling of the
counting procedure shows that a coefficient of variation of about 10% can
be expected. As the number of structures counted decreases, the precision
will also decrease approximately as ,/N, where N is the number of
structures counted. In practice, particulate deposits obtained by
filtration are rarely ideally distributed, and it is found that the
precision is correspondingly reduced from that predicted by the Poisson
distribution. This degradation is a consequence of:
59
-------
(a) non-uniformity of the filtered particulate deposit;
(b) distortion of the structure distribution by application of the
structure counting criteria;
(c) variation between microscopists in their interpretation of the
fibrous structures; and,
(d) variation between microscopists and instruments in their
ability to detect and identify structures.
The 95% confidence interval about the mean for a single structure
concentration measurement using this analytical method should be about
±25% when about 100 structures are counted.
11.2.2. Accuracy
There is no independent method available to determine accuracy.
11.3. ANALYTICAL SENSITIVITY
Analytical sensitivity is the concentration corresponding to observation
of one asbestos structure in an analysis. The analytical sensitivity of the
method can be varied by choice of the area of the collection filter, the
volume of air sampled, the dilution or concentration factor used in the
specimen preparation and the area of the specimen examined in the TEM. The
analytical sensitivity shall be quoted for each sample analysis.
NOTE
In practice, the lowest achievable value of analytical sensitivity
is frequently determined by the total suspended particulate
concentration in the air sample, since each particle on the TEM
specimen must be separated from adjacent ones by a sufficient
distance that the particle can be identified without interference.
Particulate loadings on filters greater than about 25 jig/cm2 usually
preclude preparation of TEM specimens.
If the analysis is to be performed with an acceptable expenditure of
time, the area of the specimen examined in the TEM must also be
limited. The analytical sensitivity that can be achieved for any
particular sample depends on the air volume, the area of TEM
specimen examined, and the dilution or concentration factor used in
the analysis, which in turn is controlled by the amount of
particulate that is not removed by the selective concentration
procedures.
60
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11.4, LIMIT OF DETECTION
Should asbestos be observed during analysis, it is generally important to
distinguish,whether such asbestos originated in the sampled medium or if it
was introduced as contamination during analysis. Asbestos that can be
attributed to the sampled medium is generally considered to have been
"detected". Thus, a detection limit is defined as the smallest measurement
that is unlikely (probability less than a specified value) to be due
entirely to contamination from sources other than the air being sampled.
Detection limits are generally quantified by considering the magnitude and
frequency of occurrence of the analytical background associated with a
particular method. However, detection limits for asbestos methods are
difficult to quantify because the distribution of analytical background
associated with asbestos analysis tends to be poorly behaved.
To avoid the problems typically associated with defining a detection limit
for asbestos analysis, an alternate procedure for distinguishing sampled
asbestos from contamination due to analytical background has been
incorporated into this method3. A statistical test is performed to compare
structure concentrations observed on sample filters with structure
concentrations observed on appropriate blanks to determine whether
differences between the concentrations observed on the two sets of filters
are statistically significant.
NOTE
Due to the difficulty in characterizing contributions to observed asbestos
on individual samples from contamination introduced as analytical
background, tests to distinguish "detected" asbestos from such
contamination are better applied to groups of samples. Adjustments to
account for analytical background are not recommended for individual
samples.
A detection limit is more appropriate for analytical methods that
employ wet-chemical or spectroscopic techniques rather than
structure counts (as in the case of asbestos) because the former
are invariably associated with a lower limit to the amount (number
of molecules) that can be detected. For asbestos, however,
analysis by TEM is sufficiently sensitive to detect the smallest,
single asbestos structure. Thus the issues involved in setting
detection limits for TEM analyses of asbestos structures are quite
different than from those involved with detecting populations of
molecules such as in gas chromatograph-mass spectrometry (GC-MS)
analyses, for example.
61
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12. REPORTING REQUIREMENTS
Reporting requirements for the laboratory include the individual documen-
tation for each sample (sample analysis report) and the cumulative report for
any batch of samples from a particular study. The structure counting form to
be used by the analyst was presented in Figure 10.1. Required data reduction
by the site data reviewer is presented at the end of this chapter, Section
12.3. It is critical that the following documentation requirements be
followed exactly to preserve the ability to interpret results without the need
to re-examine samples.
12.1. SAMPLE ANALYSIS REPORT
The sample analysis report for each sample shall include at least the
following information:
(a) reference to this analytical method;
(b) identification of the air sample and the batch number of the
collection filter used;
(c) the dates and times of the air sample collection period;
(d) the volume of air passed through the sample collection filter, the
area of filter used for the sample preparation, the volume of water
used for re-dispersal, the batch number of the filters used for
filtration of the aqueous suspension, and, for the set of TEM
specimen grids examined, the volume of the aliquot used in the
aqueous suspension filtered;
(e) a complete listing of the structure counting data. The dimensions
of the TEM grid openings shall be specified, and for each asbestos
structure (or structure component) the following data shall be
included: grid opening number, structure number, identification
category (morphological classification), structure type /composi-
tional classification), length and width of the structure in /^m, and
any comments concerning the structure;
(f) a statement of the minimum acceptable identification category and
the maximum identification category attempted;
(g) a statement specifying which identification and structure categories
have been used to calculate the concentration values;
62
-------
(h)
(i)
(j)
(k)
(1)
(m)
separate concentration values for chrysotile and amphibole struc-
tures, expressed in asbestos structures/liter4;
separate concentration values for chrysotile and amphibole structure
components, expressed in asbestos structures/liter*;
the 95% confidence limits for the concentration values, expressed in
asbestos structures/liter;
the analytical sensitivity, expressed in asbestos structures/liter;
compositional data for the principal varieties of amphibole if
present; '
items d - 1 for the separate count of asbestos structures longer
than 5 fim;
_ An example of a suitable format for the structure counting data is shown
•*•**• £ IgUjTG J_^ . J_ *
NOTE
As improved definitions of the dimensions of asbestos structures critical
to the determination of risk become available, the reporting requirements
for this method may be modified accordingly. At the same time, sufficient
information is preserved in these reporting requirements to allow later
re-evaluation of existing analysis results without the need to re-examine
the original sample.
12.2. SAMPLE BATCH REPORT
In addition to the sample analysis report for each sample, a summary page
should be provided for each batch of samples representing an entire project
The summary sheet should include the following information-
(a)
(b)
(c)
project title;
date samples received and data results reported;
a summary listing of sample results with chrysotile and amphibole
reported separately including:
the sample number;
If asbestos is not detected during a particular analysis the
resulting mean concentration for that sample should be reported as
"NF" for "not found".
63
-------
the analytical sensitivity for each size range characterized
(structures of all sizes and structures longer than 5 mm)
the total number of structures counted;
the concentration of asbestos structures of all sizes in
structures per liter with appropriate 95% confidence limits ;
the concentration of asbestos structure components in struc-
tures per liter with appropriate 95% confidence limits;
the concentration of asbestos structures longer than 5 urn in
structures per liter with appropriate 95% confidence limits;
the concentration of asbestos structure components longer than
5 (im in structures per liter with 95% confidence limits;
A sample summary sheet is presented in figure 12.2.
12.3. DATA REVIEW REPORT
Once the Sample Batch Report is received from the laboratory, the
following reporting procedure will be performed by the project data
reviewer. The data reviewer shall provide a summary sheet reflecting
specific steps in data reduction. The summary data sheet shall include the
following information:
(a)
(b)
(c)
(d)
(a)
(f)
(g)
(h)
(i)
the sample number;
the type of sample (lab blank, field blank, sampling station
identifier) ;
the analytical sensitivity for each size fraction reported
(structures of all sizes and structures longer than 5 ^m) ;
the number of total structures counted;
the concentration of asbestos structures of all sizes in structures
per liter with 95% confidence limits;
the concentration of asbestos structure components in structures per
liter with 95% confidence limits;
the concentration of asbestos structures longer than 5
structures per liter with 95% confidence limits;
in
the concentration of asbestos structure components longer than 5 /«n
in structures per liter with 95% confidence limits;
the ratio of free asbestos fibers to total asbestos structures;
64
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SAMPLE ANALYSIS INFORMATION (Page 1)
Laboratory name
Report number
Date
SAMPLE: 456 Queen Street
San Diego
Downwind sample 1988-09-25
Collected: 8 a.m. to 4 p.m.
ANALYSIS: Name of method
Reference to method
Air volume:
Area of collection filter:
Area of filter ashed:
Volume of water used for re-dispersal:
Volume of suspension filtered:
Area of analytical filter:
Magnification used for fiber counting:
Mean dimension of grid openings:
12500.0 liters
385.0 mm2
96.3 mm2
40.0 mL
35.0 mL
199.0 mm2
20500 (high magnification)
10200 (low magnification)
90.0 /im
High Magnification Scan
Number of grid openings examined:
Analytical Sensitivity:
Number of chrysotile structures counted:
Number of chrysotile structure components counted:
Number of amphibole structures counted:
Number of amphibole structure components counted:
38
0.5 s/L
63 (including F, M, C and B)
91
12 (F only)
12
Low Magnification Scan
Number of grid openings examined:
Analytical sensitivity:
Number of long chrysotile structures counted:
Number of long chrysotile structure components counted:
Number of long amphibole structures counted:
Number of .long amphibole structure components counted:
357
0.02 s/L
3 (B and C)
14
0
NOTES: Only structures with components longer than Spin counted during
the low magnification scan.
Figure 12.1A: Format for Reporting of Counting Data, Page 1
65
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SAMPLE ANALYSIS INFORMATION (Page 2)
Laboratory name
Report number
Date
SAMPLE: 456 Queen Street
San Diego
Downwind sample 1988-09-25
Collected: 8 a.m. to 4 p.m.
ANALYSIS:
Name of method
Reference to method
Concentration of
Concentration of
Concentration of
Concentration of
Concentration of
Concentration of
Concentration of
Concentration of
chrysotile structures:
chrysotile structure components:
long chrysotile structures:
long chrysotile structure components;
amphibole structures:
amphibole structure components:
long amphibole structures:
long amphibole structure components:
Mean
(s/L)
31.5
45.5
0.06
0.28
6
6
NF
NF
95%
Confidence
Limits
Range
(s/L)
24
37
0 -
0.16
3.1
3.1
0 -
0 -
- 38
- 54
0.12
- 0.4
- 8.9
- 8.9
0.07
0.07
NOTES: NF means Not Found
Long structures and structure components are those greater than 5/*m in
length.
Figure 12.IB: Format for Reporting of Counting Data, Page 2
66
-------
TEM ASBESTOS COUMT - RAW DATA (Page 3 and higher)
Grid "Structure Classif- Structure Length
Opening Number ication* Type pm
1
1 '
1
1
2
3
' 3
3
3
3
3
> 4
5
5
6
7 ,
8
8
y
1
2
:/ 3-
4
5 "
6
" 7
8
9
10
11
12
13
14
is
•v \ %
16
17
,
'CD
CD
CDQ
'AD
CDQ
, CMQ
CMQ
CMQ
CD
ADQ
ADQ
CD
CD
CD
AQZZ
v , •.
CMQ
CD
F
MS002
MF002
MMQ02
B
CS006
CF006
CBQ06
DF006
F
F
M
"MS013
, MB013
v ,f ;
'"No Fibers
F
F'
1,7
5.5
2.6
2,0
8.7
8.5
5.41
8.1
3.6
9.0
4.5
3,0
6.1
4.1
3.2
1.3
1.1
Width Comments
'•••
0.045
4.3
0.045
0.53
0.21
4.3 :
i.'o
1.2
0.75
0.045 Crocidolite
0.60 Amosite
2.0
1.0
0.50
0.10 Amosite
0.030
0.045
* Classification codes listed in Tables Dl and D2
Figure 12.1C: Format for Reporting Counting Data, Page 3 and Subsequent Pages
67
-------
Batch tf:
Project:
Lab:
Date:
Sample Medium:
Analysis Type:
Preparation Technique:
Sample I.D.
Date Received:
Structures counted:
Structure concentration (s/L):
Mean
Range
Low
High
Analytical sensitivity:
Components counted:
Component concentration (s/L):
Mean
Range
Low
High
Analytical sensitivity:
Long structures
counted:
Long structure concentration (s/L):
Mean
Range
Low
High
Analytical sensitivity:
Long components counted:
Long component concentration (s/L):
Mean
Range
Low
High
Analytical sensitivity:
"~"~ ~""
••••-''• "~-
„,,,,*„,,,,.,,
I
;: ' ' i ' ' . :
! f
•:
$ '$
\ 1
r^~rr
—
:
i '
;••
i • -
—
,.,V.M..,V.'.V.'.'.V.V
NOTES:
NF = not found
Long structures or components are those longer than 5 fan.
The range presented represents the 95% confidence limits constructed as discussed in Section E.4.
Provide separate sheets for chrysotile and amphiboles or total asbestos, as necessary.
Figure 12.2: Format for the Summary Batch Report
68
-------
the ratio of free asbestos fibers to total asbestos structures and
components;
the ratio of matrices to total asbestos structures;
the ratio of bundles to total asbestos structures;
the ratio of clusters to total asbestos structures;
items i-m for asbestos structures longer than 5 urn;
a complete listing of the structure counting data for each sample
(item "e" of section 12.1).
An example of the summary form for data review is provided in Figure 12.3.
(j)
(k)
(1)
(m)
(n)
(o)
69
-------
Case No.:
Site:
Lab:
Reviewer:
Date:
ANALYTICAL RESULTS
Concentration of Asbestos (Chrysolite unless otherwise noted)
Analysis Type: Air for TEM asbestos
Indirect Preparation
Sample ID. (number and type)
Analysis/Preparation
Parameter
Number of liters sampled
HIGH MAGNIFICATION
Number of grids examined
Structures/area viewed
Fibers/area viewed
Bundles/area viewed
Clusters/area viewed
Matrices/area viewed
Structures/L
Components/L
Long structures/L
Long components/L
Analytical sensitivity
LOW MAGNIFICATION
Number of grids examined
Structures/area viewed
Fibers/area viewed
Bundles/area viewed
Clusters/area viewed
Matrices/area viewed
Long structures/L
Long components/L
Analytical sensitivity
Mean
95%
Low
,, .
CL
High
Mean
95%
Low
CL
High
1
Mean
95%CL
Low 1 High
!
Mean
95%
Low
CL
High
Mean
95%
Low
|
1
CL
High
-J
o
NOTES:
NF = not found
95% CL = 95% confidence limit
The range presented represents the 95% upper or lower confidence limit constructed as discussed in Section E.4.
Figure 12.3: Format for Data Review Summary Report
-------
APPENDIX A - DETERMINATION OF OPERATING CONDITIONS FOR PLASMA ASHER
A.I INTRODUCTION
It is important to establish standard conditions for operation of the
plasma asher, so that:
(a) ashing conditions for MCE filters and particulate are sufficient and
remain constant; and,
(b) so that a known amount of etching of MCE filters can be achieved on
the surface of filters being prepared by the direct-transfer method.
Therefore, the plasma asher shall be calibrated so that a known material
is completely ashed in a constant time. This is carried out by adjusting
the radio-frequency power output and the oxygen flow rate, and measuring the
time taken to completely oxidize an unused 25 mm diameter 0.45 um pore size
MCE filter.
A. 2 PROCEDURE
Place an unused, 25 mm diameter, 0.45 pm pore size MCE filter in the
center of a glass microscope slide. Position the slide approximately in the
center of the asher chamber. Close the chamber and evacuate to a pressure
of approximately 40 Pa, while admitting oxygen to the chamber at a rate of
8-20 cc/min. Adjust the tuning of the system so that the intensity of the
plasma is maximized. Measure the time required for complete oxidation of
the filter. Determine the power setting that result in complete oxidation
of the filter in a period of approximately 15 minutes.
NOTE
Plasma oxidation at high radio-frequency powers will cause the filter to
shrink and curl, followed by sudden violent ignition. At lower powers the
filter will remain in position, and will become slowly thinner until it
becomes nearly transparent. It is recommended that the radio- frequency
power be used is selected such that violent ignition does not occur. If
powers are used such that the particulate ignites violently, fibers will
possibly be disturbed and lost from the analysis.
71
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APPENDIX B - CALIBRATION PROCEDURES
B.I CALIBRATION OF THE TEM
B.I.I Calibration of TEM Screen Magnification
The electron microscope should be aligned according to the
specifications of the manufacturer. Initially, and at regular intervals,
calibrate the magnifications used for the analysis using a diffraction
grating replica. Adjust the specimen height to the eucentric position
before carrying out the calibration. Measure the distance on the
fluorescent viewing screen occupied by a convenient number of repeat
distances of the grating image, and calculate the magnification. Always
repeat the calibration after any instrumental maintenance or change of
operating conditions. The magnification of the image on the viewing
screen is not the same as that obtained on photographic plates or film.
The ratio between these is a constant value for the particular model of
TEM.
B.I.2 Calibration of ED Camera Length
Calibrate the camera length of the TEM when used in ED mode. Use a
specimen grid supporting a carbon film on which a thin film of gold has
been evaporated or sputtered. Form an image of the gold film and select
ED conditions. Adjust the objective lens current to optimize the pattern
obtained, and measure on the fluorescent viewing screen the diameters of
the innermost two rings. Calculate the camera constant, A x L, from the
relationship:
a x D
A x L -
+ k2
where:
A x L
a
D
the wavelength of the incident electrons
the effective camera length in mm
the unit cell dimensions of gold in Angstr6m units
the diameter of the (hkl) diffraction ring in mm
Using gold as the calibration material, the camera constant is given by;
A x L - 2.3548 x D (smallest ring)
A x L - 2.0393 x D (second ring)
72
-------
B.2 CALIBRATION OF THE EDXA SYSTEM
Energy calibration of the EDXA system for a low energy and high energy
peak shall be performed regularly. Calibration of the intensity scale of
the EDXA system permits quantitative composition data, at an accuracy of
about 10% of the elemental concentration, to be obtained from EDXA spectra
of silicate minerals involving the elements Na, Mg_, Al, Si, K, Ca, Mn, and
Fe. SRM 2063 should also be used to calibrate the EDXA system for Mg[ Ca,
and Fe relative to Si. If quantitative determinations are required for
minerals containing other elements, reference standards other than those
referred to below will be required. Well-characterized mineral standards
permit calibration of any TEM-EDXA combination that meets the instrumental
specifications of 6.3.1 and 6.3.2, so that EDXA data from different
instruments can be compared. Reference minerals are required for the
calibration; the criteria for selection being that they should be silicate
minerals with matrices as close as possible to those of the amphiboles or
serpentine, and that individual small fragments of the minerals are
homogeneous in composition within a few percent.
Determine the compositions of these standards by electron microprobe
analysis or chemical methods, and prepare TEM grids from aqueous dispersions
of crushed fragments of the same selected mineral specimens. These TEM
specimens can then be used to calibrate any TEM-EDXA system so that
comparable compositional results can be obtained from different instruments.
NOTE
The microprobe analyses of the mineral standards are made by conventional
techniques, which can be found in the bibliography. The mineral is first
embedded in a mount of polymethyl methacrylate or epoxy resin. The mount
is then ground and polished to achieve a flat, polished surface of the
mineral fragment. This surface is then analyzed, using suitable reference
standards, preferably oxide standards of the individual elements wherever
these are available. It is necessary to take account of the water
concentration in the minerals, which in the case of chrysotile amounts to
13% by weight.
Express the results of the electron microprobe analyses as atomic ratios
relative to silicon. X-ray peak ratios of the same elements relative to
silicon, obtained from the EDXA system, can then be used to calculate the
relationship between peak area ratio and atomic ratio. The technique was
described by Cliff and Lorimer (See Bibliography).
The X-rays generated in a thin specimen by an incident electron beam have
a low probability of interacting with the specimen. Thus mass absorption
and fluorescence effects are negligible. In a specimen composed of
elements i and j, the following relationship can be used to perform
quantitative analyses in the TEM.
73
-------
where :
A£ - the elemental integrated peak areas for element i
Aj - the elemental integrated peak area for element j
C± — the concentration or atomic proportion of element i
Cj - the concentration or atomic proportion of element j
k — a constant
To incorporate correction for the particle size effect on peak area ratios
(See Small et al. in Bibliography), extend the Cliff and Lorimer technique
by obtaining separate values of the constant k for different ranges of fiber
diameter. It is recommended that about 20 EDXA measurements be made for
each range of fiber diameter. Suitable ranges of fiber diameter are
<0.25 [an, 0.25-0.5 /jm, 0.5-1.0 /«n and >1.0 >m.
Insert the TEM grid into the TEM, obtain an image at the calibrated higher
magnification of about 20,000, and adjust the specimen height to the
eucentric point. If the x-ray detector is a side-entry variety, tilt the
specimen towards the x-ray detector. Select an isolated fiber or particle
less than 0.5 pm in width, and accumulate an EDXA spectrum using an electron
probe of suitable diameter. When a well-defined spectrum has been obtained,
perform a background subtraction and calculate the background- corrected peak
areas for each element listed, using energy windows centered on the peaks.
Calculate the ratio of the peak area for each specified element relative to
the peak area for silicon. All background- subtracted peak areas used for
calibration shall exceed 400 counts.
Repeat this procedure for 20 particles of each mineral standard.
Reject analyses of any obviously foreign particles. Calculate the
arithmetic mean peak area to concentration ratio, k, for each specified
element of each mineral standard and for each of the fiber diameter
ranges. Routine checks to ensure that there has been no degradation of
the detector performance shall be carried out. These k-values are used to
calculate the elemental concentrations of unknown fibers, using the Cliff
and Lorimer relationship.
74
-------
APPENDIX C - STRUCTURE COUNTING CRITERIA
G.1 INTRODUCTION
In addition to isolated fibers, other assemblages of particles and fibers
frequently occur in air samples. Groupings of asbestos fibers and
particles, referred to as "asbestos structures", are defined as fiber
bundles, clusters and matrices. The numerical result of a structure count
depends strongly on whether the analyst assigns each such assemblage of
fibers as a single entity, or as the estimated number of individual fibers
which form the assemblage. It is therefore important that a logical system
of counting criteria be defined, so that the interpretation of specific
structures is the same for all analysts, and so that the numerical result is
meaningful. Imposition of specific structure counting, criteria generally
requires^that some interpretation, partially based on uncertain health
effects information, be made of each asbestos structure found. It is not
the intention of this method to make interpretations based on health
effects. Rather, it is intended that a clear separation shall be made
between recording of structure counting data and later interpretation of
those data. The system of coding specified in this method permits a clear
morphological description of the structures to be recorded in a concise
manner suitable for later interpretation, if necessary, by a range of
different criteria, without the necessity for re-examination of the
specimens. The system requires each asbestos structure to be assigned a
predominant classification as fiber, bundle, cluster or matrix and in some
cases individual components of a structure are separately enumerated.
Examples of the various types of morphological structures and the manner in
which these shall be recorded are shown in Figure Cl..
C.2. STRUCTURE DEFINITIONS AND TREATMENT
C.2.1 Fiber
Any particle with parallel or stepped sides, with an aspect ratio of
5:1 or greater, shall be defined as a fiber. For chrysotile asbestos, the
single fibril shall be defined as a fiber. A fiber with stepped sides
shall be assigned a width equal to the average of the minimum and maximum
widths. This average shall be used as the width in determination of the
aspect ratio. '
C.2.2 Bundle
A grouping composed of apparently attached parallel fibers shall be
counted as a bundle of a width equal to an estimate of the mean bundle
width, and a length equal to the maximum length of the structure. The
overall aspect ratio of the bundle may be any value, provided that the
individual constituent fibers have aspect ratios equal to or greater than
J • -L •
75
-------
C.2.3 Cluster
An aggregate of randomly oriented fibers, with or without bundles,
shall be defined as a cluster. Clusters occur as two varieties:
Cluster type A; a disperse and open network, in which both ends of
individual fibers and bundles can be separately identified and their
dimensions measured. If the cluster consists of up to 5 such fibers or
bundles, the individual fibers and bundles comprising the cluster shall be
separately counted, measured, and recorded as individual fibers and
bundles that are components of the overall cluster. If the cluster
consists of more than 5 fibers or bundles it shall be noted as such on the
count sheet. In addition to characterizing and measuring individual
components, the overall outer dimensions of the cluster will be recorded
as prescribed for type B clusters.
Cluster type B: a complex and tightly bound network, in which one or both
ends of each individual fiber or bundle are obscured, such that the
dimensions of individual fibers cannot be measured. In this case the
cluster shall be recorded as a single cluster with no components, and the
overall dimensions in the two perpendicular directions defining the
maximum and minimum dimensions shall be recorded.
The recording of clusters shall be based on the predominant charac-
teristics of the structures. For example, a cluster consisting
predominantly of 4 fibers, but with smaller regions of attached material
containing fibers, shall be considered as a type A cluster of 4 fibers to
be recorded separately as fibers that are components of the overall
cluster.
The procedure for treatment of clusters is illustrated by examples
in Figure C2.
C.2.4 Matrix
A fiber, fibers, or bundles, may be attached to, or partially
concealed by, a single particle or group of overlapping non-fibrous
particles. This structure shall be defined as a matrix. The TEM image
does not discriminate between particles that are attached to fibers, and
those that, by chance, overlap in the TEM image. It is not known,
therefore, whether such a structure is actually a complex particle, or
whether it has arisen by a simple overlapping of particles and fibers on
the filter.
Since a matrix structure may involve more than one fiber, it is
important to define in detail how matrices shall be counted. Matrices
exhibit different characteristics, and three types can be defined:
Matrix type A: a disperse grouping of overlapping fibers and/or bundles
and associated equant particles or groups of particles in which some
76
-------
fibers have less than one third of their lengths obscured. If the matrix
consists of up to 5 such fibers or bundles, the fibers or bundles shall be
recorded as separate fibers or bundles that are components of the overall
matrix, and their lengths and widths shall also be recorded. If the
matrix consists of more than 5 fibers or bundles, it shall denoted as such
on the count sheet. In addition to characterizing and measuring
individual components, the overall outer dimensions of the matrix will be
recorded as prescribed for type B matrices.
Matrix type B: a structure consisting of an equant particle or linked
group of particles, in which the ends of fibers or bundles project from
the particles, but the other ends of the fibers or bundles are obscured.
Fibers and bundles shall be treated differently, depending on whether the
obscured length could not possibly be more than one third of the total
length. If the matrix exhibits up to 5 fibers or bundles, those fibers
for which the obscured length could not be more than one third of the
total length shall be counted as separate fibers or bundles that are
components of the overall matrix. The assigned length for each partially-
obscured fiber or bundle shall be equal to the visible length plus the
maximum possible contribution from the obscured portion. Fibers or
bundles which appear to cross the matrix, and for which both ends can be
located approximately, shall be included in the maximum of 5 and counted
as separate fibers or bundles that are components of the overall matrix.
If more than 5 such fibers or bundles can be individually identified, this
will be denoted on the count sheet. The residual matrix, if it exhibits
additional fiber terminations that cannot be separately counted because
the unobscured lengths are too short, shall be recorded as one matrix.
All other matrices of type B shall each be recorded as one matrix with no
components. The overall dimensions of each matrix in the two
perpendicular directions defining the maximum and minimum dimensions shall
be recorded.
Matrix type C: a structure in which fibers can be seen and identified in
the interior, but which incorporates no fibers which project from the
outside edges. This type of matrix can originate as a result of partial
dissolution of binders during specimen preparation, when the original
particle was a composite material containing asbestos. This type of
matrix shall be recorded as a single matrix with no components. The
overall dimensions of the matrix in the two perpendicular directions
defining the maximum and minimum dimensions shall be recorded.
In practice, structures can occur in which different areas exhibit
features of the three types of matrix, and more than one variety of
asbestos may be incorporated in the same structure. In this case, the
predominant characteristic of the structure should be determined,'and then
a logical procedure should be followed, in which predominant fibers and
bundles are enumerated first up to a maximum of 5 followed by assignment
of the remaining asbestos structures according to the counting rules.
Complex structures shall be assigned no more than 6 separate components.
Examples of the procedure which shall be followed are shown in Figure C3.
77
-------
0.3 OTHER STRUCTURE COUNTING CRITERIA
C.3.1 Structures that Intersect Grid Bars
Structures that intersect grid bar shall be counted only for two
adjacent sides of the grid opening, as illustrated in Figure C4. The
length of the structure shall be recorded as twice the unobscured length.
Structures intersecting either of the other two sides shall not be
included in the count. This procedure ensures that the numerical count
will be accurate, and that the best average estimate of length has been
made.
C.3.2 Structures That Extend Outside the Field of View
During scanning of a grid opening, systematically count structures
that extend outside of the field of view, so as to avoid double-counting.
In general, a rule should be established so that structures extending
outside the field of view in only two quadrants are counted. Structures
without terminations within the field of view shall not be counted. The
procedure is illustrated by Figure C5. Measure the length of each such
structure by moving the specimen to locate the other end of the structure,
and then return to the original field of view before continuing to scan
the specimen.
C.4 PROCEDURE FOR DATA RECORDING
When a fibrous structure is detected during the structure count, the com-
ponents of the structure are first identified according to the procedures in
Appendix D. The procedure for recording the morphological description
starts by classifying the predominant characteristic of the structure as a
fiber, bundle, cluster, or matrix. The code F, B, C or M shall be the first
component of the morphological descriptor. Depending on the nature of the
individual structure, additional coded descriptions will be added to
classify separate components of the structure.
C.4.1 Fibers
On the counting form, isolated fibers as defined in C.2.1
shall be recorded by the simple designation "F". If the fiber is a
separately-counted part of a cluster or matrix structure, the
sequential structure number shall be attached as a suffix, to
identify all such constituent components of the particular cluster
or matrix structure. For example, CF004 shall be used to denote a
fiber forming part of structure number 004, the overall and predomi-
nant characteristic of which is a cluster.
C.4.2 Bundles
On the counting form, isolated bundles as defined in C.2.1
shall be recorded by the designation "B". If the bundle is a
78
-------
separately-counted part of a cluster or matrix structure, the
sequential structure number shall be attached as a suffix, to
identify all such constituent components of the cluster or matrix
structure. For example, MB004 shall be used to denote a bundle
forming part of structure number 004, the overall and predominant
characteristic of which is a matrix.
C.4.3 Clusters
On the fiber counting form, isolated clusters, as defined in
C.I.4, shall be recorded by the designation "C". The dimensions of
the overall structure are recorded. If some components of the
cluster have been separately counted, the cluster shall be recorded
by the designation CS, followed by the sequential structure number
"n". For example, the code CS004 indicates that the primary
structure number 004 was a cluster, for which the overall dimensions
are specified. Thus if a localized cluster is attached to a group
of fibers or bundles, and the fibers and bundles have been counted
separately because this procedure was defined by the predominant
characteristics of the structure, the sequential structure number
shall be attached as a suffix, to identify all constituent
components of the structure. For example, CF004 shall be used to
denote a fiber forming part of structure number 004, the overall and
predominant characteristic of which is a cluster. When more than
5 components can be individually identified, the cluster is still
recorded by the designation CS and the overall dimensions of the
structure are recorded, but the sequential structure number is
excluded from the code and components are not recorded separately.
C.4.4 Matrices
On the fiber counting form, isolated matrices of type B or C,
as defined in C.I.5, shall be recorded by the designation "M". The
dimensions of the overall structure are recorded. If the type B or
C matrix is attached to a group of fibers or bundles, and the fibers
and bundles have been counted separately because this procedure was
defined by the predominant characteristics of the structure, the
sequential structure number shall be attached as a suffix, to
identify all constituent components of the structure. For example,
the primary matrix structure shall be designated as MS004, and the'
overall structure dimensions recorded. All separately-counted
components of the structure shall carry the suffix 004. Thus the
code MB004 shall be used to denote a bundle forming part of
structure number 004, the overall and predominant characteristic of
which is a matrix. When more than 5 components can be individually
identified, the matrix is still recorded by the designation MS and
the overall dimensions of the structure are recorded, but the
sequential counting number is excluded from the code and components
are not recorded separately.
79
-------
FIBERS
\
BUNDLES
CLUSTER TYPE A
CLUSTER TYPE B
MATRIX TYPE A
MATRIX TYPE B
MATRIX TYPE C
Figure C.I: Fundamental Morphological Structure Types
80
-------
Record primary structure as one cluster
designated C.
Record primary structure as 1 cluster
designated as CSn. Record 5 fibers as
components. Use code CFn for the component
fibers.
Record primary structure as 1 cluster
designated CSn. Record 3 fibers and 2 bundles
as components. Use code CFn for component
fibers and code CBn for component bundles.
Record primary structure as 1 cluster
designated as CSn. Record 4 fibers and 2 sub-
clusters as components. Use code CFn for
component fibers and code CCn for component
clusters.
Notes:
"n" is a three digit number identifying the structure number of the
primary structure that each component belongs to.
Dimensions assigned to each component represent the best estimate of the
maximum length and mean width of that particular component. Dimensions
recorded for a primary structure represent the best estimate of the
overall outside dimensions of that structure.
Figure C.2: Examples of Recording of Complex Asbestos Clusters
81
-------
Record primary structure as 1 matrix
designated as MSn. Record 1 fiber as a
component. Use code MFn for component fiber.
Record primary structure as 1 matrix
designated as MSn. Record 5 fibers as
components. Use code MFn for component
fibers.
Record primary structure as 1 matrix
designated as MSn. Record 3 fibers and 1 sub-
matrix as components. Use code MFn for
component fibers and code MMn for component
matrix.
Record as 1 matrix designated as M.
Notes:
"n" is a three digit number identifying the structure number of the
primary structure that each component belongs to.
Dimensions assigned to each component represent the best estimate of the
maximum length and mean width of that particular component. Dimensions
recorded for a primary structure represent the best estimate of the
overall outside dimensions of that structure.
Submatrices are only recorded when they incorporate asbestos fibers not
accounted for after separation of fiber, bundle, and cluster components
from the primary structure.
Figure C.3: Examples of Recording of Complex Asbestos Matrices
82
-------
-*-Grid opening
Figure C.4: Counting of Structures That Intersect Grid Bars
Grid opening
TEM field of view
Figure C.5: Counting of Structures That Extend Outside the Field of View
83
-------
APPENDIX D - FTBER IDENTIFICATION PROCEDURE
D.1 INTRODUCTION
Fibrous structures are classified as asbestos structures by identifying
one or more of the constituent fibers or bundles as asbestos. Accordingly,
this section refers to identification of fibers rather than structures.
The criteria which shall be used for identification of asbestos fibers are
selected, depending on the intended use of the fiber counting results. In
some circumstances, there can be a requirement that fibers shall be unequi-
vocally identified as a specific mineral species. In other circumstances
there can be sufficient knowledge about the sample that rigorous identifica-
tion of each fiber need not be carried out. The time required to perform
the analysis, and therefore the cost of analysis, can vary widely depending
on the identification criteria which are considered to be sufficiently
definitive. The combination of criteria considered definitive for
Identification of fibers in a particular analysis shall be specified before.
the analysis is made, and this combination of criteria shall be referred to
as the "Level" of analysis. Various factors related to instrumental
limitations and the character of the sample may prevent satisfaction of all
of the specified fiber identification criteria for a particular fiber.
Therefore, a record shall be made of the identification criteria which were
satisfied for each suspected asbestos fiber included in the analysis. For
example, if both ED and EDXA were specified to be attempted for definitive
identification of each fiber, fibers with chrysotile morphology which, for
some reason, do not give an ED pattern but which do yield an EDXA spectrum
corresponding to chrysotile, are categorized in a way that conveys the level
of confidence to be placed in the identification.
D.2- ED AND EDXA TECHNIQUES
D.2.1 General
Initially, classify fibers into two categories on the basis of
morphology: those fibers with tubular morphology, and those fibers without
tubular morphology. Conduct further analysis of each fiber using ED and
EDXA methods. The following procedures should be used when fibers are
examined by ED and EDXA.
The crystal structures of some mineral fibers, such as chrysotile,
are easily damaged by the high current densities required for EDXA
examination. Therefore, investigation of these sensitive fibers by ED
shall be completed before attempts are made to obtain EDXA spectra from
the fibers. When more stable fibers, such as the amphiboles, are
examined, EDXA and ED may be used in either order.
84
-------
D.2.2 ED Techniques
The ED technique can be either qualitative or quantitative.
Qualitative ED consists of visual examination, without detailed
measurement, of the general characteristics of the ED pattern obtained on
the TEH viewing screen from a randomly orientated fiber. ED patterns
obtained from fibers with cylindrical symmetry, such as chrysotile, do not
change when the fibers are tilted about their axes, and patterns from
randomly oriented fibers of these minerals can be interpreted
quantitatively. For fibers that do not have cylindrical symmetry, only
those ED patterns obtained when the fiber is oriented with a principal
crystallographic axis closely parallel with the incident electron beam
direction can be interpreted quantitatively. This type of ED pattern
shall be referred to as a zone-axis ED pattern. In order to interpret a
zone-axis ED pattern quantitatively, it shall be photographed and its
consistency with known mineral structures shall be checked. A computer
program may be used to compare measurements of the zone-axis ED pattern
with corresponding data calculated from known mineral structures. The
zone-axis ED pattern obtained by examination of a fiber in a particular
orientation can be insufficiently specific to permit unequivocal
identification of the mineral fiber, but it is often possible to tilt the
fiber to another angle and to photograph a different ED pattern
corresponding to another zone-axis. The angle between the two zone-axes
can also be checked for consistency with the structure of a suspected
mineral.
For visual examination of the ED pattern, the camera length of the
TEM should be set to a low value of approximately 250 mm and the ED
pattern then should be viewed through the binoculars. This procedure
minimizes the possible degradation of the fiber by the electron
irradiation. However, the pattern is distorted by the tilt angle of the
viewing screen. A camera length of at least 2 m should be used when the
ED pattern is photographed, if accurate measurement of the pattern is to
be possible. It is necessary that, when obtaining an ED pattern to be
evaluated visually or to be photographed, the sample height shall be
properly adjusted to the eucentric point and the image shall be focussed
in the plane of the selected area aperture. If this is not done there may
be some components of the ED pattern which do not originate from the
selected area. In general, it will be necessary to use the smallest
available ED aperture.
For routine sample analysis, calibration films of evaporated gold or
other materials shall not be applied to the TEM grids. Such films
seriously degrade the visibility of fine chrysotile fibers so that they
may not be detected by the EM operator. Moreover, the visibility of ED
patterns from chrysotile fibers are also seriously degraded resulting in
failure to identify them positively. Both effects lead to significant
reduction of the asbestos structure concentrations reported.
85
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To form an ED pattern, move the image of the fiber to the center of
the viewing screen and insert a suitable selected area aperture into the
electron beam so that the fiber, or a portion of it, occupies a large
proportion of the illuminated area. The size of the aperture and the
portion of the fiber shall be such that particles other than the one to be
examined are excluded from the selected area. Observe the ED pattern
through the binoculars. During the observation, the objective lens
current should be adjusted to the point where the most complete ED pattern
is obtained. If an incomplete ED pattern is still obtained, move the
particle around within the selected area to attempt to optimize the ED
pattern, or to eliminate possible interferences from neighboring
particles.
If a zone-axis ED analysis is to be attempted on the fiber, the
sample shall be mounted in the appropriate holder. The most convenient
holder allows complete rotation of the specimen grid and tilting of the
grid about a single axis. Rotate the sample until the fiber image
indicates that the fiber is oriented with its length coincident with the
tilt axis of the goniometer, and adjust the sample height until the fiber
is at the eucentric position. Tilt the fiber until an ED pattern appears,
which is a symmetrical, two dimensional array of spots. The recognition
of zone-axis alignment conditions requires some experience on the part of
the operator. During tilting of the fiber to obtain zone-axis conditions,
the manner in which the intensities of the spots vary should be observed.
If weak reflections occur at some points on a matrix of strong
reflections, the possibility of multiple diffraction exists, and some
caution should be exercised in the selection of diffraction spots for
measurement and interpretation. A full discussion of electron diffraction
and multiple diffraction can be found in the references by J. A. Card,
P.B. Hirsch et al, and H. R. Wenk, included in the Bibliography. Not all
zone-axis patterns that can be obtained are definitive. Only those which
have closely-spaced reflections corresponding to low indices in at least
one direction should be recorded. Patterns in which all d-spacings are
less than about 0.3 nm are not definitive. A useful guideline is that the
lowest angle reflections should be within the radius of the first gold
diffraction ring (111), and that patterns with smaller distances between
reflections are usually the most definitive.
Five spots, closest to the center spot, along two intersecting lines
of the zone-axis pattern shall be selected for measurement, as illustrated
in Figure Dl. The distances of these spots from the center spot and the
four angles shown provide the required data for analysis. Since the
center spot is usually very over-exposed, it does not provide a
well-defined origin for these measurements. The required distances shall
therefore be obtained by measuring between pairs of spots symmetrically
disposed about the center spot, preferably separated by several repeat
distances. The distances must be measured with a precision of better than
0.3 mm, and the angles to a precision of better than 2.5°. The diameter
of the first or second ring of the calibration pattern (111 and 200) must
also be measured with a precision of better than 0.3 mm.
86
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Using gold as the calibration material, the camera constant is given
A x L = 2.3548 x D (first ring)
A x L - 2.0393 x D (second ring)
D.2.3 EDXA Measurements
Interpretation of the EDXA spectrum may be either qualitative or
quantitative. For qualitative interpretation of a spectrum, the elements
originating from the fiber are recorded. For quantitative interpretation,
the net peak areas, after background subtraction, are obtained for the
elements originating from the fiber. This method provides for
quantitative interpretation for those minerals which contain silicon To
obtain an EDXA spectrum, move the image of the fiber to the center of the
screen and remove the objective aperture. Select an appropriate electron
beam diameter and deflect the beam so that it impinges on the fiber.
Depending on the instrumentation, it may be necessary to tilt the specimen
towards the X-ray detector, and in some instruments, to use Scanning
Transmission Electron Microscopy (STEM) mode of operation.
The time for acquisition of a suitable spectrum varies with the
fiber, diameter, and also with instrumental factors. For quantitative
interpretation, spectra should have statistically valid number of counts
in each peak. Analyses of small diameter fibers which contain sodium are
the most critical, since it is in the low energy range that the X-ray
detector is least sensitive. Accordingly, it is necessary to acquire a
spectrum for a sufficiently long period that the presence of sodium can be
detected in such fibers. It has been found that satisfactory quantitative
analyses can be obtained if acquisition is continued until the background
subtracted silicon Ka peak integral exceeds 10,000 counts. The spectrum
should then be manipulated to subtract the background and to obtain the
net areas of the elemental peaks.
After quantitative EDXA classification of some fibers by computer
analysis of the net peak areas, it may be possible to classify further
fibers in the same sample in the basis of comparison of spectra at the
instrument. Frequently, visual comparisons can be made after somewhat
shorter acquisition times.
D.3 INTERPRETATION OF FIBER ANALYSIS DATA
D.3.1 Chrysotile
The morphological structure of chrysotile is characteristic and
with experience, can be recognized readily. However, a few other minerals
have similar appearance, and morphological observation by itself is
inadequate for most samples. The ED pattern obtained from chrysotile is
quite specific for this mineral if the specified characteristics of the
87
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pattern correspond to those from reference chrysotile. However, depending
on the past history of the fiber, and on a number of other factors, the
crystal structure of a particular fiber may be damaged, and it may not
yield an ED pattern. In this case, the EDXA spectrum may be the only data
available to supplement the morphological observations.
D.3.2 Amphiboles
Since the fiber identification procedure for asbestos fibers other
than chrysotile can be involved and time-consuming, computer program such
as developed by Rhoades (see Bibliography) can be used for interpretation
of zone-axis ED patterns. The published literature contains composition
and crystallographic data for all of the fibrous minerals likely to be
encountered in TEH analysis of air samples, and the compositional and
structural data from the unknown fiber should be compared with the
published data. Demonstration that the measurements are consistent with
the data for a particular test mineral does not uniquely identify the
unknown, since the possibility exists that data from other minerals may
also be consistent. It is, however, unlikely that a mineral of another
structural class could yield data consistent with that from an amphibole
fiber identified by quantitative EDXA and two zone axis ED patterns.
Suspected amphibole fibers should be classified initially on the
basis of chemical composition. Either qualitative or quantitative EDXA
information may be used as the basis for this classification. From the
published data on mineral compositions, a list of minerals which are
consistent in composition with that measured for the unknown fiber should
be compiled. To proceed further, it is necessary to obtain the first zone
axis ED pattern, according to the instructions in D.2.1.
It is possible to specify a particular zone-axis pattern for
identification of amphibole, since a few patterns are often considered to
be characteristic. Unfortunately, for a fiber with random orientation on
a TEM grid, no specimen holder and goniometer currently available will
permit convenient and rapid location of two pre-selected zone-axes. The
most practical approach has been adopted, which is to accept those low
index patterns which are easily obtained, and then to test their
consistency with the structures of the minerals already pre-selected on
the basis of the EDXA data. Even the structures of non-amphibole minerals
in this pre-selected list must be tested against the zone-axis data
obtained for the unknown fiber, since non-amphibole minerals in some
orientations may yield similar patterns consistent with amphibole struc-
tures .
88
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SPOT I.
SPOT 3
SPOT 2.
SPOT 4
SPOT 5
Figure D.I: Measurement of Zone Axis SAED Patterns
89
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The zone-axis ED interpretation must include all minerals previously
selected from the mineral data file as being chemically compatible with
the EDXA data. This procedure will usually shorten the list of minerals
for which solutions have been found. A second set of zone-axis data from
another pattern obtained on the same fiber can then be processed, either
as further confirmation of the identification, or to attempt elimination
of an ambiguity. In addition, the angle measured between the orientations
of the two zone-axes can be checked for consistency with the structures of
the minerals. Caution should be exercised in rationalizing the inter-zone
axis angle, since if the fiber contains c-axis twinning the two zone-axis
ED patterns may originate from the separate twin crystals. In practice,
the full identification procedure will normally be applied to very few
fibers, unless for a particular reason precise identification of all
fibers is required.
D.4 FIBER CLASSIFICATION CATEGORIES
It is not always possible to proceed to a definitive identification of a
fiber; this may be due to instrumental limitations or to the actual nature
of the fiber. In many analyses a definitive identification of each fiber
may not actually be necessary if there is other knowledge available about
the sample, or if the concentration is below a level of interest. The
analytical procedure must therefore take account of both instrumental
limitations and varied analytical requirements. Accordingly, a system for
fiber classification is used to permit accurate .recording of data. The
classifications are shown in Tables Dl and D2, and are directed towards
identification of chrysotile and amphibole respectively. Fibers shall be
reported in these categories.
The general principle to be followed in this analytical procedure is first
to define the most specific fiber classification which is to be attempted,
or the "level" of analysis to be conducted. Then, for each fiber examined,
record the classification which is actually achieved. Depending on the
intended use of the results, criteria for acceptance of fibers as "iden-
tified" can then be established at any time after completion of the
analysis.
In an unknown sample, chrysotile will be regarded as confirmed only if a
recorded, calibrated ED pattern from one fiber in the CD categories is
obtained, or if measurements of the ED pattern are recorded at the
instrument. Amphibole will be regarded as confirmed only by obtaining
recorded data which yields exclusively amphibole solutions for fibers
classified in the AQZ, AZZ or AQZZ categories.
D.4.1 Procedure for Classification of Fibers with Tubular Morphology,
Suspected to be Chrysotile
Occasionally, fibers are encountered which have tubular morphology
similar to that of chrysotile, but which cannot be characterized further
either by ED or EDXA. They may be non-crystalline, in which case ED
90
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techniques are not useful, or they may be in a position on the grid which
does not permit an EDXA spectrum to be obtained. Alternatively, the fiber
may be of organic origin, but not sufficiently definitive that it can be
disregarded. Accordingly, there is a requirement to record the fiber, and
to specify how confidently each fiber can be identified. Classification
of fibers will meet with various degrees of success. Figure D2 shows the
classification procedure to be used for fibers which display any tubular
morphology. The chart is self explanatory, and every fiber is either
rejected as a non-asbestos mineral (NAM), or classified in some way which
by some later criterion could still contribute to the chrysotile fiber
count.
Morphology is the first consideration, and if this is not similar to
that usually seen in chrysotile standard samples, designate the initial
classification as TM. Regardless of the doubtful morphology, examine the
fiber by ED and EDXA methods according to Figure D2. Where the morphology
is more definitive, it may be possible to classify the fiber as having
chrysotile morphology (CM).
are:
For classification as CM, the morphological characteristics required
(a) the individual fibrils should have high aspect ratios
exceeding 10:1, and be about 20 to 40 nm in diameter;
(b) the electron scattering power of the fiber at 60 to 100 kV
accelerating potential should be sufficiently low for internal
structure to be visible; and,
(c) there should be some evidence of internal structure suggesting
a tubular appearance similar to that shown by reference UICC
chrysotile, which may degrade in the electron beam.
Examine every fiber having these morphological characteristics by
the ED technique, and classify as chrysotile by ED (CD) only those which
give diffraction patterns with the precise characteristics shown in Figure
D3. The relevant features in this pattern for identification of
chrysotile are as follows:
(a) the (002) reflections should be examined to determine that
they correspond approximately to a spacing of 0.73 nm;
(b) the layer line repeat distance should correspond to 0.53 nm;
and,
(c) there should be "streaking" of the (110) and (130)
reflections.
91
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FIBER WITH TUBULAR-MORPHOLOGY
Is fiber morphology characteristic
of that displayed by reference chrysotile?
NO
Examine by SAED
Pattern not
chrysotile
Chrysotile
pattern
Pattern not present
or indistinct
Examine by quantitative EDXA
Composition not
that of chrysotile
Chrysotile
composition
No Spectrum
TM
YES
Examine by SAED
Chrysotile
pattern
Pattern not
chrysotile
Pattern not present
or indistinct
Examine by quantitative EDXA
Chrysotile
composition
No S
CMQ
Composition not
that of chrysoti
Dectrum
CM
le
NAM
Examine by quantitative EDXA
Composition not
that of chrysotile
Chrysotile
composition
No Spectrum
{CDQ j
Figure D.2: Classification Chart for Fiber With Tubular Morphology
92
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/ass
nm
X
0002,
Figure D.3: Chrysotile SAED Pattern
93
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Using the millimeter calibrations on the TEM viewing screen, these
observations can readily be made at the instrument. If documentary proof
of fiber identification is required, record a TEM micrograph of at least
one representative fiber, and record its ED pattern on a separate film or
plate. This film or plate shall also carry calibration rings from a known
polycrystalline substance such as gold. This calibrated pattern is the
only documentary proof that the particular fiber is chrysotile, and not
some other tubular or scrolled species such as halloysite, palygorskite,
talc or vermiculite. The proportion of fibers which can be successfully
identified as chrysotile by ED is variable, and to some extent dependent
on both the instrument and the procedures of the operator. The fibers
that fail to yield an identifiable ED pattern will remain in the TM or CM
categories unless they are examined by EDXA.
In the EDXA analysis of chrysotile there are only two elements which
are relevant. For fiber classification, the EDXA analysis must be
quantitative. If the spectrum displays prominent peaks from magnesium and
silicon, with their area in the appropriate ratio, and with only minor
peaks from other elements, classify the fiber as chrysotile by
quantitative EDXA, in the categories CQ, CMQ, or CDQ, as appropriate.
D.4.2 Procedure for classification of Fibers Without Tubular
Morphology, Suspected to be Amphibole
Every particle without tubular morphology and which is not obviously
of biological origin, with an aspect ratio of 5:1 or greater, and having
parallel or stepped sides, shall be considered as a suspected amphibole
fiber. Further examination of the fiber by ED and EDXA techniques will
meet with a variable degree of success, depending on the nature of the
fiber and on a number of instrumental limitations. It will not be
possible to identify every fiber completely, even if time and cost were of
no concern. Moreover, confirmation of the presence of amphibole can be
achieved only by quantitative interpretation of zone-axis ED patterns, a
very time-consuming procedure. Accordingly, for routine samples from
unknown sources, this analytical procedure limits the requirement for
zone-axis ED work to a minimum of one fiber representative of each
compositional class reported. In some samples, it may be necessary to
identify more fibers by the zone-axis technique. When analyzing samples
from well-characterized sources, the cost of identification by zone-axis
methods may not be justified.
The 0.53 nm layer spacing of the random orientation ED pattern is
not by itself diagnostic for amphibole. However, the presence of c-axis
twinning in many fibers leads to contributions to the layers in the
patterns by several individual parallel crystals of different axial
orientations. This apparently random positioning of the spots along the
layer lines, if also associated with a high fiber aspect ratio, is a
characteristic of araphibole asbestos, and thus has some limited diagnostic
value. If a pattern of this type is not obtained, the identity of the
94
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fiber is still ambiguous, since the absence of a recognizable pattern may
be a consequence of an unsuitable orientation relative to the electron
beam, or the fiber may be some other mineral species.
Figure D4 shows the fiber classification chart to be used for
suspected amphibole fibers. This chart shows all the classification paths
possible in analysis of a suspected'amphibole fiber, when examined
systematically by ED and EDXA. Two routes are possible, depending on
whether an attempt to obtain an EDXA spectrum or a random orientation ED
pattern is made first. The normal procedure for analysis of a sample of
unknown origin will be to examine the fiber by random orientation ED,
qualitative EDXA, quantitative EDXA, and zone-axis ED, in this sequence.
The final fiber classification assigned will be defined either by
successful analysis at the maximum required level, or by the instrumental
limitations. Record the maximum classification achieved for each fiber on
the counting sheet in the appropriate column. The various classification
categories can then be combined later in any desired way for calculation
of the fiber concentration, and a complete record of the results obtained
when attempting to identify each fiber is maintained for reassessment of
the data if necessary.
In the unknown sample, zone-axis analysis will be required if the
presence of amphibole is to be unequivocally confirmed. For this level of
analysis, attempt to raise the classification of every suspected amphibole
fiber to the ADQ category by inspection of the random orientation ED
pattern and the EDXA spectrum. In addition, examine at least one fiber
from each type of suspected amphibole found by zone-axis methods to
confirm their identification. In most cases, some ambiguity of
identification can be accepted, because information exists about possible
sources of asbestos in close proximity to the air sampling location.
Lower levels of analysis can therefore be accepted for these situations.
95
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[ FIBER WITHOUT TUBULAR MORPHOLOGY
Ootk
-------
Table Dl
Classification of fibers with tubular morphology
TM
CM
CD
CQ
CMQ
CDQ
NAM
Tubular Morphology, not sufficiently characteristic for
classification as chrysotile
Characteristic Chrysotile Morphology
Chrysotile SAED pattern
Chrysotile composition by Quantitative EDXA
Chrysotile Morphology and composition by Quantitative
EDXA
Chrysotile SAED pattern and composition by Quantitative
EDXA
Non-asbestos Mineral
97
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Table D2
Classification of fibers without tubular morphology
UF
AD
AX
ADX
AQ
AZ
ADQ
AQZ
AZZ
AQZZ
NAM
Unidentified Fiber
Amphibole by random orientation SAED (shows layer
pattern of 0.53 nm spacing)
Amphibole by qualitative EDXA. Spectrum has elemental
components consistent with amphibole
Amphibole by random orientation SAED and qualitative
EDXA
Amphibole by quantitative EDXA
Amphibole by one Zone-Axis SAED pattern
Amphibole by random orientation SAED and Quantitative
EDXA
Amphibole by Quantitative EDXA and one Zone-Axis SAED
pattern
Amphibole by two Zone-Axis SAED patterns with consistent
inter-axial angle
Amphibole by Quantitative EDXA, two Zone-Axis SAFD
patterns, and consistent inter-axial angle
Non-asbestos Mineral
98
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APPENDIX E - CALCULATION OF RESULTS
E.1 INTRODUCTION
Calculate the results using the procedures described below. The results
can be conveniently calculated using a computer program.
E.2 TEST FOR UNIFORMITY OF FIBER DEPOSITS ON TEM GRIDS
A check shall be made, using the chi-square test, to determine whether the
asbestos structures found on individual grid openings are randomly and
uniformly distributed/among the grid openings. If• the total number found in
k grid openings is n, and the areas of the k individual grid openings are
designated At to Ak, then the total area of TEM specimen examined is:
A =
The fraction of the .total area examined' which is represented by the
individual grid opening area, pi( is given by A±/A. If the structures are
randomly and uniformly dispersed over the k grid openings examined, the
expected number of structures falling in one grid opening with area ^ is
np^ If the observed number of structures; found on that grid opening is n,,
then:
x2
Z
(nt -
This value shall be compared with significance points of the x2 distribu-
tion, having (k - 1) degrees of freedom. Significance levels lower than
0.1% may be cause for the sample analysis to be rejected, since this
corresponds to a very inhomogeneous deposit. If the structure count fails
this test, the precision of the result will be uncertain, and new analytical
filters should be prepared from the original collection filter.
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E.3 CALCULATION OF THE ANALYTICAL SENSITIVITY
Calculate the analytical sensitivity S, in structures/liter, using the
formula:
where:
Af
N
AS
V
F
S - Af/(N x Ag x V x F)
- Active area of sample collection filter in mm2
— Number of grid openings examined
- Mean area of grid openings
- Volume of air sampled in liters
— Concentration factor
E.4 CALCULATION OF THE MEAN AND CONFIDENCE LIMITS FOR A REPORTED
ASBESTOS STRUCTURE CONCENTRATION
In the asbestos structure count made according to this method, a number of
grid openings have been sampled from a population of grid openings and it is
required to determine the mean grid opening asbestos structure count for the
population on the basis of this small sample. The upper and lower 95%
confidence limits are also to be reported with every mean. When such limits
are constructed as indicated in Section E.4.2, the interval between the two
limits should contain the true mean 90% of the time (95% of the time if the
lower confidence limit is zero).
E.4.1 Calculation of the Mean Asbestos Structure Concentration
Calculate the mean concentration from a particular measurement, C,
in structures/liter:
C - S x N x n
where:
S - Analytical sensitivity in structures/liter
N - Number of grid openings examined
n - mean number of asbestos structures found per grid opening
E.4.2 Calculation of Upper and Lower 95% Confidence Limits
If the total structures counted from a filter is 30 or less, the
Poisson distribution is recommended to be used for constructing confidence
limits. Let P(x;m) represent the cumulative probability function from a
Poisson distribution with mean m. That is, P(x;m) is the probability of
observing x or fewer counts given by
P(x;m) - e'm(l
m
m2/2!
+ mx/x!).
100
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The upper 95% confidence limit, xa, on the mean count, based on an
observed count of x, is the value that satisfies:
P(X)XU) = 0.05,
and the lower 95% confidence limit, XL, on the mean count, based on an
observed count of x, is the value that satisfies
1 - P(x-l,xL) - 0.05
(e.g., Miller and Freund 1965)5. Tables of P(x;m) for values of m less
than 26 may be found in Miller and Freund. A computer program can be
easily written for calculating P(x;m) for larger values of m. If such a
program is not available, confidence limits may alternately be based on
the normal distribution approximation to the Poisson distribution (see
Miller and Freund 1965). Corresponding approximations to the upper and
lower 95% confidence limits are
— X
1.65*x1/2
and
=• x - 1.65*x1/2.
The Poisson distribution is correct whenever structures are randomly
distributed in the sampled air and on the filter. However, as a hedge
Confidence limits derived as described in this section are one-
tailed limits meaning that 95% of the distribution lies below the
upper 95% confidence limit and, similarly, 95% of the distribution
lies above the lower 95% confidence limit. If the two limits are
combined to create a confidence interval, the interval between the
two limits represents 90% of the distribution (which is different
than a 95% confidence interval).
Construction of confidence limits in this manner, rather than the
more traditional approach of constructing confidence intervals
(where, for example, the 95% confidence interval corresponds to
upper and lower 97.5% confidence limits), is recommended to avoid
confusion when referring to an asymmetric distribution or when the
lower confidence limit falls to zero. When the lower confidence
limit falls to zero, for example, the upper 95% confidence limit now
corresponds to the upper 95% confidence interval (rather than the
90% confidence interval, as indicated in the last paragraph) because
the lower confidence limit (zero) removes nothing from the
distribution. Thus, we have chosen to refer to one-tailed
confidence limits throughout and avoid emphasis on confidence
intervals.
101
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against possible clumping of structures, it is recommended that, whenever
the total structure count on a filter is greater than 30, confidence
limits also be calculated using the following procedure and the more
extreme limits (i.e., larger Xu and smaller XL) be reported.
Suppose k grid openings are examined and let xt be the structure
count for the ith grid opening. Note that a minimum of four grid openings
(k>4) should be examined and they should be of equal size. Then 95% upper
and lower confidence limits are calculated according to the formulas
xu - 1.65s
XT - 1.65s
Where
s2 - tk/(k-l)
k
E
- x/k)2,
and 1.65 is the cumulative 95th percentage point of the standard normal
distribution.
Upper or lower confidence limits, for airborne concentrations derived
from a particular measurement are computed by multiplying the
corresponding upper or lower confidence limits derived as described above
for the structure count by the analytical sensitivity of the measurement.
E.4.3 Reporting the Mean and Confidence Limits for a Measured
Concentration
Where a result is to be interpreted as a single isolated
measurement, the following procedures shall be used for reporting:
No structures detected - the concentration shall be reported as "NF"
meaning "not found" and it should be accompanied by the upper and lower
95% confidence limits derived as described in Section E.4.2. Note that
the lower 95% confidence limit is zero in this case.
At least 1 structure detected - report the mean concentration derived as
described in Section E.4.1 along with the upper and lower 95% confidence
limits derived as described in Section E.4.2.
Rules for combining the results of individual measurements as part
of a site evaluation are presented in the Technical Background Document,
Part 2 of this report.
102
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E.5 DISTINGUISHING DETECTED ASBESTOS FROM ANALYTICAL BACKGROUND
non-parametric Wilcoxon Test (Hollander and Wolfe 1973) is employed in
this method to test for significant differences between asbestos
concentrations (in units of s/cm2) observed on sample filters and
concentrations observed on blanks. It must be emphasized, however, that the
than indf^/1? atSi0n °f ^ teSt iS tO ^°^S °f samPle resu^ "ther
than individual results. This should not be a severe limitation because
conclusions based on the totality of data from an entire study tend to be
more important than the results from individual analyses.
The Wilcoxon test performs better on multiple samples. A test comparing
the concentration from an individual filter to those from a group of blanks
0^ ' ^ SUCh a teSt 1S Ukely t0 be of low P°w*r ansensiSve
di!fe"nces). In fact, data from at least 20 blanks are likely
Hr~ teS? beC°meS CSpable °f exhibiting a statistically
dlfference (p < -0.05) between blank concentrations and the
n ? °bSTed °n S Slngle Sample filter- ^ P°wer of the test
a SS £ ^ d ^^ comParing multiple sample filter analyses to
a limited number of blanks. For example, at least seven sample filters
b~lankl ^ ^ C°mbined t0 find S siSnific-nt difference from two
WU1 *enerally be available from two sources:
are i hlf°rical- Within a particular study, two filter blanks
be fSS M lyvanalred fr°m eaCh lot °f filt^s. Typically, there will also
be field blanks Depending on the length of time that a particular
ac^af0? ^6n USlng thlS meth°d' the laboratory should also be
accumulating a historical database of blank analyses! Either set of blank
data may be combined with care to provide the needed data to perform the
Wilcoxin Test and distinguish sampled asbestos from analytical
levels of"* bTk H°Ter> lf different lots of alter, exhibit different
levels of background contamination or if results from a laboratory chanee
over time (due possibly to changes in methodology or analys^s'then i* Xy
er0lae ? !jist°rical blank d^a in analysis . On the
l data are representative, use of this data may
the power of the analysis for detecting airborne
this methi hist°rical blank da*a are used in «%tatl.tlc.l test in
tnis method, this is to be noted and results of corresponding tests based
only on concurrent blanks shall also be reported. ponainS Cests based
Pr°cedur,e can be used f°r applying the Wilcoxon test to
results' More detailed discussions of the procedure are
113 S°UrCeS ^^luding Hollander and Wolfe 1973).
M blank and N non-blank samples are available. Let X, X« be
the measured filter concentrations from the blank samples and Y" " %! the
" COcnoCebtrat:irSMfMr0m ^ n°n-blank -"»Pl-
-------
exhibiting the same concentration) among the M+N observations, assign the
average rank to each group of tied observations. (E.g., if the observations
are 0, 0, 0, 0, 0.001, 0.002, then the corresponding ranks would be 2.5,
2.5, 2.5, 2.5, 5, 6.) Let W be the sum of the ranks assigned to the
concentrations from the non-blank samples. Large values of W are evidence
that concentrations from non-blank samples are larger than those from blank
samples, and the hypothesis that the blank and non-blank samples have equal
concentrations of fibers is rejected if W is large enough.
Tables are available for determining when W is large enough to be
statistically significant in cases in which there are no ties and M and N
are small (see e.g., Table A.5 in Hollander and Wolfe 1973). As an example
of the application of these tables, if N - 4 and M - 5, then W, the sum of
the ranks of the concentrations from the non-blank samples, would have to be
28 or larger before it could be concluded at the 0.05 level of significance
that the concentrations from the non-blanks are higher than those from the
blanks (see Table A.8 in Hollander and Wolfe 1973, page 276).
If ties are present in the data or if the number of samples is so large
that tables are not available, a normal approximation to the distribution of
W may be used. Define
W* - W - rN(M+N+l)/21
[Var(W)]1/2
where
Var(W) - M
12
M+N+1 -
S tj(tj2-l)
j-1
(M+N) (M+N-1) J
where g is the number of groups of tied concentrations (included among the
combined set of blank and nonblank samples) and tj is the size of tied group
j. The hypothesis test is based on an assumed normal distribution for W*.
Thus one would reject the hypothesis of equal concentrations on blanks and
non-blanks at the 0.05 level of significance if W*>1.65. (Note: This
normal approximation may not work well if the number of samples is small.
Consequently, if there are ties in the data and M or N is less than about 4,
consideration should be given to consulting with a statistician regarding a
more accurate approach to computing the statistical significance of W.)
E.6 CALCULATION OF ASBESTOS STRUCTURE LENGTH, WIDTH, AND ASPECT RATIO
DISTRIBUTIONS
The distributions all approximate to logarithmic-normal, and so the size
range intervals for calculation of the distribution shall be spaced
logarithmically. The other characteristics required for the choice of size
intervals are that they should allow for a sufficient number of size
classes, while still retaining a statistically-valid number of asbestos
104
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structures in each class. Interpretation is also facilitated if each size
class repeats at decade intervals. A ratio from one class to the next of
1.47 satisfies all of these requirements and this value shall be used The
distributions, being approximately logarithmic-normal, when presented
graphically, shall be plotted using a logarithmic ordinate scale and a
Gaussian abscissa.
E.6.1
Calculation of Asbestos Structure Length Cumulative Number
Distribution
This distribution allows the fraction of the total number of
asbestos structures either shorter or longer than a given length to be
determined. It is calculated using the relationship:
i=k
E
i-N
x 100
where:
C(N)k =
nl
N
Cumulative fraction asbestos structures (expressed in
percent) which have lengths less than the upper bound of
the k'th class
Number of asbestos structures in the i'th length class
Total number of length classes.
E.6.2
Calculation of Asbestos Structure Width Cumulative Number
Distribution
This distribution allows the fraction of the total number of
asbestos structures either narrower or wider than a given width to be
determined. It is calculated in a similar way to that used in E.6.1, but
using the asbestos structure widths.
105
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E.6.3 Calculation of Asbestos Structure Aspect Ratio Cumulative
Number Distribution
This distribution allows the fraction of the total number of
asbestos structures which have aspect ratios either smaller or larger than
a given aspect ratio to be determined. It is calculated in a similar way
to that used in E.6.1, but using the asbestos structure aspect ratios.
106
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