ASTM D6281-23

Standard Test Method for Airborne Asbestos Concentration in Ambient and Indoor Atmospheres as Determined by Transmission Electron Microscopy Direct Transfer (TEM)

SIGNIFICANCE AND USE
5.1 This test method is applicable to the measurement of airborne asbestos in a wide range of ambient air situations and for detailed evaluation of any atmosphere for asbestos structures. Most fibers in ambient atmospheres are not asbestos, and therefore, there is a requirement for fibers to be identified. Most of the airborne asbestos fibers in ambient atmospheres have diameters below the resolution limit of the light microscope. This test method is based on transmission electron microscopy, which has adequate resolution to allow detection of small thin fibers and is currently the only technique capable of unequivocal identification of the majority of individual fibers of asbestos. Asbestos is often found, not as single fibers, but as very complex, aggregated structures, which may or may not also be aggregated with other particles. The fibers found suspended in an ambient atmosphere can often be identified unequivocally if sufficient measurement effort is expended. However, if each fiber were to be identified in this way, the analysis would become prohibitively expensive. Because of instrumental deficiencies or because of the nature of the particulate matter, some fibers cannot be positively identified as asbestos even though the measurements all indicate that they could be asbestos. Therefore, subjective factors contribute to this measurement, and consequently, a very precise definition of the procedure for identification and enumeration of asbestos fibers is required. The method defined in this test method is designed to provide a description of the nature, numerical concentration, and sizes of asbestos-containing particles found in an air sample. The test method is necessarily complex because the structures observed are frequently very complex. The method of data recording specified in the test method is designed to allow reevaluation of the structure-counting data as new applications for measurements are developed. All of the feasible specimen preparation techn...
SCOPE
1.1 This test method2 is an analytical procedure using transmission electron microscopy (TEM) for the determination of the concentration of asbestos structures in ambient atmospheres and includes measurement of the dimension of structures and of the asbestos fibers found in the structures from which aspect ratios are calculated.  
1.1.1 This test method allows determination of the type(s) of asbestos fibers present.  
1.1.2 This test method cannot always discriminate between individual fibers of the asbestos and non-asbestos analogues of the same amphibole mineral.  
1.2 This test method is suitable for determination of asbestos in both ambient (outdoor) and building atmospheres.  
1.2.1 This test method is defined for polycarbonate capillary-pore filters or cellulose ester (either mixed esters of cellulose or cellulose nitrate) filters through which a known volume of air has been drawn and for blank filters.  
1.3 The upper range of concentrations that can be determined by this test method is 7000 s/mm2. The air concentration represented by this value is a function of the volume of air sampled.  
1.3.1 There is no lower limit to the dimensions of asbestos fibers that can be detected. In practice, microscopists vary in their ability to detect very small asbestos fibers. Therefore, a minimum length of 0.5 μm has been defined as the shortest fiber to be incorporated in the reported results.  
1.4 The direct analytical method cannot be used if the general particulate matter loading of the sample collection filter as analyzed exceeds approximately 10 % coverage of the collection filter by particulate matter.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropri...

General Information

• Status: Published
• Publication Date: 31-Aug-2023
• ICS: 13.040.01 - Air quality in general
• Technical Committee: D22 - Air Quality
• Drafting Committee: D22.07 - Sampling, Analysis, Management of Asbestos, and Other Microscopic Particles

Relations

• Refers: ASTM D1356-20a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Sep-2020
• Refers: ASTM D1356-20 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Mar-2020
• Refers: ASTM D1357-95(2019) - Standard Practice for Planning the Sampling of the Ambient Atmosphere
• Effective Date: 01-Aug-2019
• Refers: ASTM D6620-19 - Standard Practice for Asbestos Detection Limit Based on Counts
• Effective Date: 01-Jan-2019
• Refers: ASTM D7712-18 - Standard Terminology for Sampling and Analysis of Asbestos
• Effective Date: 15-Apr-2018
• Refers: ASTM D1356-15a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Oct-2015
• Refers: ASTM D1356-15 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Jul-2015
• Refers: ASTM D1356-14b - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Dec-2014
• Refers: ASTM D1356-14a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-May-2014
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM D1356-14 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Jan-2014
• Refers: ASTM E177-13 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2013
• Refers: ASTM E691-13 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-May-2013
• Refers: ASTM E691-11 - Standard Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
• Effective Date: 01-Nov-2011
• Refers: ASTM D1357-95(2011) - Standard Practice for Planning the Sampling of the Ambient Atmosphere
• Effective Date: 01-Oct-2011

Overview

ASTM D6281-23 is the authoritative ASTM standard for the measurement of airborne asbestos concentration in both ambient and indoor atmospheres using Transmission Electron Microscopy with direct transfer (TEM). This test method provides a highly sensitive means for identifying and quantifying asbestos structures in air samples, including detailed morphological and dimensional characterization of fibers. Given the health risks associated with asbestos exposure, accurate detection and identification in environments such as workplaces, schools, and public buildings are paramount for regulatory compliance and public safety.

Key Topics

• Transmission Electron Microscopy (TEM): The method relies on TEM analysis, which offers exceptional resolution and is currently the only technique capable of accurately identifying the majority of airborne asbestos fibers, including those too small for light microscopy.
• Fiber Identification: The method includes procedures for identifying fibers as asbestos or non-asbestos, recognizing that most fibers found in ambient air are not asbestos.
• Specimen Preparation: The standard provides detailed guidance on sample collection using polycarbonate or cellulose ester filters, preparation of specimens, and minimization of contamination to ensure data integrity.
• Quantification and Classification: The process involves counting and dimensionally measuring asbestos structures, classifying fiber type (e.g., chrysotile, amphibole varieties), and recording concentrations as structures per liter (s/L) of sampled air.
• Analytical Sensitivity: Analytical sensitivity is dictated by the air volume sampled and the number of microscope grid areas analyzed, with a lower reporting limit defined by fiber length (minimum 0.5 μm).
• Instrument Requirements: Specifics for TEM performance and supporting analytical equipment, such as energy-dispersive X-ray analysis (EDXA), are outlined to ensure precision and reliability.

Applications

Occupational and Environmental Health:
This standard is widely utilized in monitoring and assessment for asbestos in the air, especially:

• Workplace Monitoring: To ensure compliance with occupational exposure limits during abatement, demolition, or renovation activities that may disturb asbestos-containing materials.
• Building Inspections: For public safety in schools, offices, and residential buildings, especially under regulatory requirements (e.g., AHERA in the U.S.).
• Environmental Assessments: In evaluating ambient air in areas near former industrial sites or regions affected by natural events disturbing asbestos-bearing geology.
• Incident Response: Following accidents or natural disasters to assess the potential release and spread of airborne asbestos fibers.

Regulatory Compliance:

• Ensures data suitable for compliance with relevant air quality and worker safety regulations.
• Supports decision-making for remediation and protective controls in environments where asbestos may be present.

Related Standards

ASTM D6281-23 incorporates or references several related standards and international methods, enhancing coherence within regulatory and laboratory practices:

• ISO 10312: Ambient air - Determination of asbestos fibres - Direct-transfer transmission electron microscopy method
• ASTM D6620: Practice for Asbestos Detection Limit Based on Counts
• ASTM D1193: Specification for Reagent Water
• ASTM D1356/D7712: Terminology relating to sampling, analysis, and management of asbestos and particulates
• ASTM D1357: Practice for Planning the Sampling of the Ambient Atmosphere
• ASTM E691 & ASTM E177: Practices for precision and bias of test methods

Summary

Adoption of ASTM D6281-23 ensures robust airborne asbestos monitoring and supports occupational, environmental, and public health initiatives. Laboratories, regulatory agencies, and field practitioners turn to this standard for its rigor, practical approach, and compatibility with international methodologies. Regular updates ensure alignment with current scientific consensus and regulatory needs, making it a cornerstone reference for airborne asbestos testing by TEM.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D6281 − 23
Standard Test Method for
Airborne Asbestos Concentration in Ambient and Indoor
Atmospheres as Determined by Transmission Electron
Microscopy Direct Transfer (TEM)
This standard is issued under the fixed designation D6281; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.5 The values stated in SI units are to be regarded as
2 standard. No other units of measurement are included in this
1.1 This test method is an analytical procedure using
standard.
transmission electron microscopy (TEM) for the determination
1.6 This standard does not purport to address all of the
of the concentration of asbestos structures in ambient atmo-
safety concerns, if any, associated with its use. It is the
spheres and includes measurement of the dimension of struc-
responsibility of the user of this standard to establish appro-
tures and of the asbestos fibers found in the structures from
priate safety, health, and environmental practices and deter-
which aspect ratios are calculated.
mine the applicability of regulatory limitations prior to use.
1.1.1 This test method allows determination of the type(s)
1.7 This international standard was developed in accor-
of asbestos fibers present.
dance with internationally recognized principles on standard-
1.1.2 This test method cannot always discriminate between
ization established in the Decision on Principles for the
individual fibers of the asbestos and non-asbestos analogues of
Development of International Standards, Guides and Recom-
the same amphibole mineral.
mendations issued by the World Trade Organization Technical
1.2 This test method is suitable for determination of asbes-
Barriers to Trade (TBT) Committee.
tos in both ambient (outdoor) and building atmospheres.
1.2.1 This test method is defined for polycarbonate
2. Referenced Documents
capillary-pore filters or cellulose ester (either mixed esters of
2.1 ASTM Standards:
cellulose or cellulose nitrate) filters through which a known
D1193 Specification for Reagent Water
volume of air has been drawn and for blank filters.
D1356 Terminology Relating to Sampling and Analysis of
1.3 The upper range of concentrations that can be deter-
Atmospheres
mined by this test method is 7000 s/mm . The air concentration
D1357 Practice for Planning the Sampling of the Ambient
represented by this value is a function of the volume of air
Atmosphere
sampled.
D4483 Practice for Evaluating Precision for Test Method
1.3.1 There is no lower limit to the dimensions of asbestos
Standards in the Rubber and Carbon Black Manufacturing
fibers that can be detected. In practice, microscopists vary in
Industries
their ability to detect very small asbestos fibers. Therefore, a
D6620 Practice for Asbestos Detection Limit Based on
minimum length of 0.5 μm has been defined as the shortest
Counts
fiber to be incorporated in the reported results.
D7712 Terminology for Sampling and Analysis of Asbestos
E177 Practice for Use of the Terms Precision and Bias in
1.4 The direct analytical method cannot be used if the
ASTM Test Methods
general particulate matter loading of the sample collection filter
E691 Practice for Conducting an Interlaboratory Study to
as analyzed exceeds approximately 10 % coverage of the
Determine the Precision of a Test Method
collection filter by particulate matter.
2.2 ISO Standard:
ISO 10312 Ambient air - Determination of asbestos fibres -
This test method is under the jurisdiction of ASTM Committee D22 on Air
Direct-transfer transmission electron microscopy method
Quality and is the direct responsibility of Subcommittee D22.07 on Sampling,
Analysis, Management of Asbestos, and Other Microscopic Particles.
Current edition approved Sept. 1, 2023. Published September 2023. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 1998. Last previous edition approved in 2015 as D6281 – 15. DOI: contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
10.1520/D6281-23. Standards volume information, refer to the standard’s Document Summary page on
This test method was adapted from International Standard ISO 10312 “Air the ASTM website.
quality—Determination of asbestos fibres—Direct transfer transmission electron Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
microscopy method.” 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6281 − 23
3. Terminology 3.2.7 asbestos structure, n—a term applied to isolated fibers
or to any connected or overlapping grouping of asbestos fibers
3.1 For definitions of general terms used in this test method,
or bundles, with or without other non-asbestos particles.
refer to Terminology D1356 or D7712 (see 2.1).
3.2.8 aspect ratio, n—the ratio of length to width of a
3.2 Definitions of Terms Specific to This Standard:
particle.
3.2.1 acicular, n—the shape shown by an extremely slender
3.2.9 blank, n—a structure count made on TEM specimens
crystal with cross-sectional dimensions that are small relative
to its length, that is, needle-like. prepared from an unused filter to determine the background
measurement.
3.2.2 amphibole, n—a group of more than 60 different
silicate minerals with similar crystal structures and complex 3.2.10 camera length, n—the equivalent projection length
compositions that conform to the nominal formula: between the specimen and its electron diffraction pattern, in the
absence of lens action.
A B C T O ~OH,F,Cl! (1)
021 2 5 8 22
3.2.11 chrysotile, n—a group of fibrous minerals of the
where:
serpentine group that have the nominal composition
A = K, Na, Ca,
Mg Si O (OH) and have the crystal structure of either
2+ 2 5 4
B = Fe , Mn, Mg, Ca, Na,
clinochrysotile, orthochrysotile, or parachrysotile; most natural
3+ 2+
C = Al, Cr, Ti, Fe , Mg, Fe , Mn, and
chrysotile deviates little from this nominal composition;
3+
T = Si, Al, Cr, Fe , Ti.
chrysotile may be partially dehydrated or magnesium-leached,
In some varieties of amphibole, these elements can be
both in nature and in building materials; in some varieties of
partially substituted by Li, Pb, Zn, Be, Ba, or Ni. Amphiboles
chrysotile, minor substitution of silicon by Al + may occur;
are characterized by a complex monoclinic or orthorhombic
chrysotile is the most prevalent type of asbestos.
structure that includes a double chain of T-O tetrahedra with a
3.2.12 cleavage, n—the breaking of a mineral along one of
T:O ratio of approximately 4:11; a variable morphology that
its crystallographic directions.
ranges from columnar to prismatic to acicular to fibrous; and
3.2.13 cleavage fragment, n—a fragment of a crystal that is
good prismatic cleavage at angles of about 56° and 124°. The
bounded in whole or in part by cleavage faces; some cleavage
cleavage may not be readily exhibited by small crystals that are
fragments would be included in the fiber definition used in this
bound by irregular growth and fracture surfaces (1).
method.
3.2.3 amphibole asbestos, n—amphibole in an asbestiform
3.2.14 cluster, n—a structure in which two or more fibers or
habit.
fiber bundles are randomly oriented in a connected grouping.
3.2.4 analytical sensitivity, n—the calculated airborne as-
bestos structure concentration in asbestos structures/L, equiva- 3.2.15 d-value or interplanar spacing, n—the perpendicular
lent to the counting of one asbestos structure in the analysis.
distance between identical adjacent and parallel planes of
atoms in a crystal.
3.2.5 asbestiform, n—a specific type of fibrous habit in
which the fibers are separable into thinner fibers and ultimately
3.2.16 electron diffraction, n—techniques in electron
into fibrils; this habit accounts for greater flexibility and higher
microscopy, including selected area electron diffraction
tensile strength than other habits of the same mineral.
(SAED) and microdiffraction, by which the crystal structure of
a specimen is examined.
3.2.6 asbestos, n—a collective term that describes a group
of naturally occurring, inorganic, highly-fibrous, silicate min-
3.2.17 electron scattering power, n—the extent to which a
erals that are easily separated into long, thin, flexible, strong
substance scatters electrons from their original courses.
fibers when crushed or processed.
3.2.18 energy dispersive X-ray analysis, n—measurement of
3.2.6.1 Discussion—Included in the definition are the asbes-
the energies and intensities of X-rays by use of a solid state
tiform varieties of serpentine (chrysotile); riebeckite (crocido-
detector and multichannel analyzer system.
lite); grunerite (grunerite asbestos [Amosite]); anthophyllite
3.2.19 eucentric, n—the condition when the area of interest
(anthophyllite asbestos); tremolite (tremolite asbestos); and
of an object is placed on a tilting axis at the intersection of the
actinolite (actinolite asbestos). The amphibole mineral compo-
electron beam with that axis and is in the plane of focus.
sitions are defined according to the nomenclature of the
International Mineralogical Association.
3.2.20 field blank, n—a filter cassette that has been taken to
the sampling site, opened, and then closed. Such a filter is used
Asbestos Chemical Abstracts Service Registry No.
Chrysotile 12001-29-5
to determine the background structure count for the measure-
Crocidolite 12001-28-4
ment.
Grunerite Asbestos [Amosite] 12172-73-5
Anthophyllite Asbestos 77536-67-5
3.2.21 fibril, n—a single fiber of chrysotile that cannot be
Tremolite Asbestos 77536-68-6
further separated longitudinally into smaller components with-
Actinolite Asbestos 77536-66-4
out losing its fibrous properties or appearances.
3.2.22 fiber, n—an elongated particle that has parallel or
The boldface numbers in parentheses refer to the list of references at the end of
stepped sides; for the purposes of this test method, a fiber is
this standard.
defined as having an aspect ratio equal to or greater than 5:1
The non-asbestiform variations of the minerals indicated in 5.2.6 have different
Chemical Abstracts Service (CAS) numbers. and a minimum length of 0.5 μm.
D6281 − 23
–6
3.2.23 fiber bundle, n—a structure composed of parallel,
μm = micrometer (10 m)
–9
smaller-diameter fibers attached along its length. A fiber bundle
nm = nanometer (10 m)
may exhibit diverging fibers at one or both ends.
W = watt
Pa = Pascals
3.2.24 fibrous structure, n—a fiber or connected grouping of
fibers with or without other particles. 3.4 Abbreviations:
3.2.25 habit, n—the characteristic crystal growth form or
DMF = dimethyl formamide
combination of these forms of a mineral, including character-
ED = electron diffraction
istic irregularities.
EDXA = energy dispersive X-ray analysis
FWHM = full width, half maximum
3.2.26 limit of detection, n—the mean count for a population
HEPA = high-efficiency particle absolute
of structures that has been determined, based on a measure-
MCE = mixed cellulose ester; also refers to pure cellulose
ment or average of measurements, to be different than the
nitrate filters
background population of structures (see Practice D6620); the
PC = polycarbonate
limit of detection may be restated in units of structures/L by
PCM = phase contrast optical microscopy
multiplying the mean count by analytical sensitivity (see
ED = selected area electron diffraction
3.2.4).
SEM = scanning electron microscope
3.2.27 matrix, n—a structure in which one or more fibers or
STEM = scanning transmission electron microscope
fiber bundles touch, are attached to, or partially concealed by a
TEM = transmission electron microscope
single particle or connected group of nonfibrous particles.
UICC = Union Internationale Contre le Cancer
3.2.28 miller index, n—a set of three integer numbers used
4. Summary of Test Method
to specify the orientation of a crystallographic plane in relation
to the crystal axes. 4.1 A sample of airborne particulate matter is collected by
drawing a measured volume of air through either a capillary-
3.2.29 PCM equivalent fiber, n—a particle of aspect ratio
pore polycarbonate membrane filter of maximum pore size
that is greater than or equal to 3:1, is longer than 5 μm, and that
0.4 μm or a cellulose ester (either mixed esters of cellulose or
has a diameter between 0.2 μm and 3.0 μm.
cellulose nitrate) membrane filter of maximum pore size
3.2.30 PCM equivalent structure, n—a fibrous structure of
0.45 μm by means of a battery-powered or mains-powered
aspect ratio that is greater than or equal to 3:1, is longer than
pump. TEM specimens are prepared from polycarbonate filters
5 μm, and has a diameter between 0.2 μm and 3.0 μm.
by applying a thin film of carbon to the filter surface by
3.2.31 primary structure, n—a fibrous structure that is a
vacuum evaporation. Small areas are cut from the carbon-
separate entity in the TEM image. coated filter, supported on TEM specimen grids, and the filter
medium is dissolved away by a solvent extraction procedure.
3.2.32 replication, n—a procedure in electron microscopy
This procedure leaves a thin film of carbon that bridges the
specimen preparation in which a thin copy, or replica, of a
openings in the TEM specimen grid and that supports each
surface is made.
particle from the original filter in its original position. Cellu-
3.2.33 residual structure, n—matrix or cluster material con-
lose ester filters are chemically treated to collapse the pore
taining asbestos fibers that remains after accounting for the
structure of the filter, and the surface of the collapsed filter is
prominent component fibers or bundles, or both.
then etched in an oxygen plasma to try to expose particles
3.2.34 serpentine, n—a group of common rock-forming
embedded in the collapsed filter. A thin film of carbon is
minerals having the nominal formula: Mg Si O (OH) .
3 2 5 4 evaporated onto the filter surface and small areas are cut from
the filter. These sections are supported on TEM specimen grids,
3.2.35 structure, n—a single fiber, fiber bundle, cluster, or
and the filter medium is dissolved by a solvent extraction
matrix.
procedure.
3.2.36 twinning, n—the occurrence of crystals of the same
4.2 The TEM specimen grids from either preparation
species joined together at a particular mutual orientation, and
method are examined at both low and high magnifications to
such that the relative orientations are related by a definite law.
check that they are suitable for analysis before carrying out a
3.2.37 unopened fiber bundle, n—a large-diameter asbestos
quantitative structure count on randomly-selected grid open-
fiber bundle that has not been separated into its constituent
ings. In the TEM analysis, electron diffraction (ED) is used to
fibrils or fibers.
examine the crystal structure of a fiber, and its elemental
3.2.38 zone-axis, n—the crystallographic direction parallel
composition is determined by energy dispersive X-ray analysis
to the intersection edges of the crystal faces defining the crystal
(EDXA). For a number of reasons, it is not possible to identify
zone.
each fiber unequivocally and fibers are classified according to
3.3 Symbols: the techniques that have been used to identify them. For each
fiber, a simple code is used to record the manner in which it
eV = electron volt
was classified. The fiber classification procedure is based on
kV = kilovolt
successive inspection of the morphology, the ED pattern, and
L/min = liters per minute
the qualitative and quantitative EDXA. Confirmation of the
–6
μg = micrograms (10 g)
identification of chrysotile is only by quantitative ED, and
D6281 − 23
confirmation of amphibole is only by quantitative EDXA and a two-dimensional filter surface can be considered a modifica-
quantitative zone axis ED. tion of the particulate matter, and some of the particles, in most
samples, are modified by the specimen preparation procedures.
4.3 In addition to isolated fibers, ambient air samples often
However, the procedures specified in this test method are
contain more complex aggregates of fibers, with or without
designed to minimize the disturbance of the collected particu-
other particles. Some particles are composites of asbestos
late material.
fibers with other materials. Individual fibers and these more
complex structures are referred to as asbestos structures. A 5.2 This test method applies to analysis of a single filter and
coding system is used to record the type of fibrous structure
describes the precision attributable to measurements for a
and to provide a description of each of these complex struc- single filter (see 13.1). Multiple air samples are usually
tures. Several levels of analysis are specified, the higher levels necessary to characterize airborne asbestos concentrations
providing a more rigorous approach to the identification of across time and space. The number of samples necessary for
fibers. The procedure permits a minimum required fiber iden- this purpose is proportional to the variation in measurement
tification criterion to be defined on the basis of previous across samples, which may be greater than the variation in a
knowledge, or lack of it, about the particular sample. Attempts measurement for a single sample.
are then made to achieve this minimum criterion for each fiber,
and the degree of success is recorded for each fiber. The lengths
6. Apparatus
and widths of all classified structures and fibers are recorded.
6.1 Air Sampling Equipment and Consumable Supplies:
The number of asbestos structures found on a known area of
6.1.1 Filter Cassette, 25 mm to 50 mm-diameter,
the microscope sample, together with the equivalent volume of
commercially-manufactured, nonreusable, three-piece
air filtered through this area, is used to calculate the airborne
cassettes, with cowls in front of the filter surface, used for
concentration in asbestos structures/L of air.
sample collection. Load the cassette with either a capillary pore
polycarbonate filter of maximum pore size 0.4 μm or an MCE
5. Significance and Use
of maximum pore size 0.45 μm. Back either type of filter with
5.1 This test method is applicable to the measurement of
a 5 μm pore size MCE, and support it by a cellulose back-up
airborne asbestos in a wide range of ambient air situations and
pad. Apply a shrink cellulose band or adhesive tape when the
for detailed evaluation of any atmosphere for asbestos struc-
filters are in position to prevent air leakage. Ensure that the
tures. Most fibers in ambient atmospheres are not asbestos, and
filters are tightly clamped in the assembly so that significant air
therefore, there is a requirement for fibers to be identified. Most
leakage around the filter cannot occur.
of the airborne asbestos fibers in ambient atmospheres have
6.1.1.1 It is recommended that representative filters from
diameters below the resolution limit of the light microscope.
the filter lot be analyzed as described in 10.7 for the presence
This test method is based on transmission electron microscopy,
of asbestos structures before any are used for air sample
which has adequate resolution to allow detection of small thin
collection.
fibers and is currently the only technique capable of unequivo-
6.1.2 Sampling Pump, capable of a flow-rate sufficient to
cal identification of the majority of individual fibers of asbes-
achieve the desired analytical sensitivity. The face velocity
tos. Asbestos is often found, not as single fibers, but as very
through the filter shall be between 4.0 cm ⁄s and 45.0 cm ⁄s. The
complex, aggregated structures, which may or may not also be
sampling pump used shall provide a stable air-flow through the
aggregated with other particles. The fibers found suspended in
filter. A constant flow or critical orifice-controlled pump meets
an ambient atmosphere can often be identified unequivocally if
these requirements. Use flexible tubing to connect the filter
sufficient measurement effort is expended. However, if each
cassette (see 6.1.1) to the sampling pump.
fiber were to be identified in this way, the analysis would
6.1.3 Stand, used to hold the filter cassette at the desired
become prohibitively expensive. Because of instrumental de-
height for sampling, and to isolate it from the pump vibrations.
ficiencies or because of the nature of the particulate matter,
6.1.4 Flow Meter, a calibrated flow meter with an appropri-
some fibers cannot be positively identified as asbestos even
ate range for the sampling flow rate used. The flow meter
though the measurements all indicate that they could be
should be calibrated to a primary standard.
asbestos. Therefore, subjective factors contribute to this
6.2 Equipment for Analysis:
measurement, and consequently, a very precise definition of the
procedure for identification and enumeration of asbestos fibers 6.2.1 Transmission Electron Microscope—A TEM operating
is required. The method defined in this test method is designed at an accelerating potential of 80 kV to 120 kV, with a
to provide a description of the nature, numerical concentration, resolution better than 1.0 nm, and a magnification range of
and sizes of asbestos-containing particles found in an air approximately 300 to 100 000 with the ability to obtain a direct
sample. The test method is necessarily complex because the screen magnification of about 100 000, shall be used for
structures observed are frequently very complex. The method inspection of fiber morphology. This magnification may be
of data recording specified in the test method is designed to obtained by supplementary optical enlargement of the screen
allow reevaluation of the structure-counting data as new image by use of a binocular. It is also required that the viewing
applications for measurements are developed. All of the screen of the microscope be calibrated such that the lengths and
feasible specimen preparation techniques result in some modi- widths of fiber images down to 1 mm width can be estimated
fication of the airborne particulate matter. Even the collection in increments of 1 mm regardless of fiber orientation. This
of particles from a three-dimensional airborne dispersion on to requirement is often fulfilled through use of a fluorescent
D6281 − 23
screen with calibrated gradations in the form of circles, such as specimen and acquisition of EDXA spectra without change of
the one shown in Fig. 1. specimen holder. If the goniometer does not permit eucentric
6.2.1.1 For Bragg angles less than 0.01 radians the TEM tilting, a gold or other metal film must be evaporated on the
shall be capable of performing ED from an area of 0.6 μm or sample so that ED patterns may be accurately calibrated.
less. This performance requirement defines the minimum
6.2.1.5 The TEM shall have an illumination and condenser
separation between particles at which independent ED patterns
lens system capable of forming an electron probe smaller than
can be obtained from each particle. If ED is used, the
250 nm in diameter. It is recommended that an anticontamina-
performance of a particular instrument normally may be
tion trap be used around the specimen.
calculated using the following relationship:
6.2.2 Energy Dispersive X-ray Analyzer—The TEM shall be
3 2
equipped with an energy dispersive X-ray analyzer capable of
A 5 0.7854 × D/M12 000 × C θ (2)
~ !
s
achieving a resolution better than 180 eV (FWHM) on the
where:
MnKα peak. Since the performance of individual combinations
A = effective ED area in μm ,
of TEM and EDXA equipment is dependent on a number of
D = diameter of the ED aperture in μm,
geometrical factors, the required performance of the combina-
M = magnification of the objective lens,
tion of the TEM and X-ray analyzer is specified in terms of the
C = spherical aberration coefficient of the objective lens in
s
measured X-ray intensity obtained from a fiber of small
mm, and
diameter, using a known diameter. Solid state X-ray detectors
θ = maximum required Bragg angle in radians.
are least sensitive in the low energy region, so measurement of
6.2.1.2 It is not possible to reduce the effective ED area
sodium in crocidolite shall be the performance criterion. The
indefinitely by the use of progressively smaller ED apertures
combination of electron microscope and X-ray analyzer shall
because there is a fundamental limitation imposed by the
yield, under routine analytical conditions, a background-
spherical aberration coefficient of the objective lens.
subtracted NaKα integrated peak count rate of more than
6.2.1.3 If zone-axis ED analyses of amphiboles are to be
1 count per second (cps) from a fiber of UICC crocidolite
performed, the TEM shall incorporate a goniometer stage that
50 nm in diameter or smaller when irradiated by an electron
permits the TEM specimen to be either:
probe of 250 nm diameter or smaller. The peak/background
(a) rotated through 360°, combined with tilting through at
ratio for this performance test shall exceed 1.0.
least +30° to –30° about an axis in the plane of the specimen;
6.2.2.1 The EDXA unit shall provide the means for subtrac-
or
tion of the background, identification of elemental peaks, and
(b) tilted through at least +30° to –30° about two perpen-
calculation of background-subtracted peak areas.
dicular axes in the plane of the specimen.
6.2.3 Carbon Rod Sharpener, to neck the carbon rods that
6.2.1.4 The analysis is greatly facilitated if the goniometer
allow the carbon to be evaporated on to the filters with a
permits eucentric tilting, although tilting is not essential. If
minimum of heating.
EDXA and zone-axis ED are required on the same fiber, the
6.2.4 Plasma Asher, for preparation of TEM specimens from
goniometer shall be of a type that permits tilting of the
MCE filters. The plasma asher shall have a radio frequency
power rating of 50 W or higher and be provided with a
controlled, filtered oxygen flow. Admission of filtered air shall
be through a valve to control the speed of air admission so that
rapid air admission does not disturb particulate matter from the
surface of the filter after the etching step.
6.2.5 Vacuum Coating Unit, a vacuum coating unit capable
of producing a vacuum better than 0.013 Pa, used for vacuum
deposition of carbon on the membrane filters. A sample holder
is required that will allow a glass microscope slide to be tilted
and continuously rotated during the coating procedure.
6.2.5.1 Equip the vacuum coating unit with a mechanism
that allows the rotating slide to be tilted also through an angle
of approximately 45° during the coating procedure. A liquid
nitrogen trap 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 calibra-
tion of ED patterns.
6.2.6 Sputter Coater, with a gold target used for deposition
of gold onto TEM specimens as an internal calibration of ED
patterns. Other calibration materials are acceptable. Experience
has shown that a sputter coater allows better control of the
FIG. 1 Example of Calibration Markings on TEM Viewing Screen thickness of the calibration material.
D6281 − 23
shown in Fig. 3. Use either acetone or chloroform as the
solvent, depending on the type of filter.
6.2.9 Slide Warmer or Oven, for heating slides during the
preparation of TEM specimens from MCE or cellulose nitrate
filters, capable of maintaining a temperature of 65 °C to 70 °C.
6.2.10 Ultrasonic Bath, for cleaning of apparatus used for
TEM specimen preparation.
6.2.11 Carbon Grating, with approximately 2000 parallel
lines per mm, used to calibrate the magnification of the TEM
(see 6.2.1).
6.2.12 Calibration Specimen Grids for EDXA, TEM speci-
men grids prepared from dispersions of calibration minerals
NOTE 1—Solvent is added until the meniscus contacts the underside of
the stainless steel mesh. required for calibration of the EDXA system: crocidolite
FIG. 2 Example of Design of Solvent Washer (Jaffe Washer)
asbestos (NIST SRM 1866) and chrysotile asbestos.
6.3 Reference Asbestos Samples, for preparation of refer-
ence TEM specimens of the primary asbestos minerals. The
UICC or NIST set of minerals are suitable for this purpose.
7. Reagents and Materials
7.1 Reagents: Warning—Use the reagents in accordance
with the appropriate health and safety regulations. Review their
Material Safety Data Sheets before use.
7.1.1 Purity of Water—Water shall be reagent water as
defined by Type II of Specification D1193.
7.1.2 Chloroform, analytical grade, distilled in glass (pre-
served with 1 % (v/v) ethanol).
7.1.3 1-Methyl-2-Pyrrolidinone, analytical grade.
7.1.4 Dimethyl Formamide, analytical grade.
FIG. 3 Design of Condensation Washer
7.1.5 Glacial Acetic Acid, analytical grade.
7.1.6 Acetone, analytical grade.
7.1.7 1-2-Diaminoethane, analytical grade.
7.2 Materials:
6.2.7 Solvent Washer (Jaffe washer (2)), allows for dissolu-
tion of the filter polymer while leaving an intact evaporated
7.2.1 Copper Electron Microscope Grids, 200-mesh TEM
carbon film supporting the fibers and other particles from the
grids with grid openings of uniform size such that they meet
filter surface. One design of a washer that has been found
the requirement of 10.6.3. Use grids with numerical or alpha-
satisfactory for various solvents and filter media is shown in
betical indexing of individual grid openings to facilitate the
Fig. 2. Use either chloroform or 1-methyl-2-pyrrolidinone for
relocation of individual grid openings for quality assurance
dissolving polycarbonate filters, and use dimethyl formamide
purposes.
or acetone for dissolving MCE or cellulose nitrate filters. A
7.2.2 Gold Electron Microscope Grids, 200 mesh gold to
mixture of 20 % 1-2-diaminoethane and 80 % 1-methyl-2-
mount TEM specimens when sodium measurements are re-
pyrrolidinone may also be used to dissolve polycarbonate
quired in the fiber identification procedure. Use grids that have
filters (3). The higher evaporation rates of chloroform and
grid openings of uniform size such that they meet the require-
acetone require that a reservoir of 10 mL to 50 mL of solvent
ment of 10.6.3. Use grids with numerical or alphabetical
be used, which may need replenishment during the procedure.
indexing of individual grid openings to facilitate the relocation
Because dimethyl formamide and 1-methyl-2-pyrrolidinone
of individual grid openings for quality assurance purposes.
have lower vapor pressures, much smaller volumes of solvent
7.2.3 Carbon Rod Electrodes, spectrochemically pure for
may be used. Use the washer in a fume hood, and keep the petri
use in the vacuum evaporator during carbon coating of filters.
dishes covered with their lids when specimens are not being
7.2.4 Disposable Tip Micropipettes, 30 μL.
inserted or removed during the solvent dissolution. Clean the
7.2.5 Core Borer, 7 mm.
washer before it is used for each batch of specimens.
6.2.8 Condensation Washer, used for more rapid dissolution 7.2.6 Routine Electron Microscopy Tools and Supplies, such
of the filter polymer or for dissolving the filter polymer if as fine-point tweezers, scalpel holders and blades, microscope
difficulties are experienced. The washer consists of a flask, slides, double-coated adhesive tape, gummed paper reinforce-
condenser, and cold finger assembly with a heating mantle and ment rings, lens tissue, gold wire, tungsten filaments, and other
means for controlling the temperature. A suitable assembly is routine supplies.
D6281 − 23
8. Specimen Preparation Laboratory Hold the cassette facing downwards vertically at a height of
approximately 1.5 m to 2.0 m above ground floor level, and
8.1 Asbestos, particularly chrysotile, may be present in
connect it to the pump with a flexible tube.
varying quantities in laboratory reagents. Many building ma-
9.2.2 Measure the sampling flow-rate at the front end of the
terials also contain significant amounts of asbestos or other
cassette, both at the beginning and end of the sampling period,
mineral fibers that may interfere with the analysis if they are
using a calibrated flow meter (6.1.4) temporarily attached to
inadvertently introduced during preparation of specimens. It is
the inlet of the cassette (see 6.1.1). Use the mean value of these
most important to ensure that during preparation, contamina-
two measurements to calculate the total air volume sampled. If
tion of TEM specimens by any extraneous asbestos fibers is
the difference in flow rate at the beginning and end of the
minimized. Perform all specimen preparation steps in an
sampling period is greater than 20 %, the result should be
environment where contamination of the sample is minimized.
labeled as suspect or void due to sampling errors.
The primary requirement of the sample preparation laboratory
9.2.2.1 If flow-meter contamination is suspected, clean and
is that a blank determination yields results that will meet the
requirements specified in 10.7. A minimum facility considered recalibrate the flow meter before use to avoid transfer of
asbestos contamination from the flow meter to the sample
suitable for preparation of TEM specimens is a positive-
pressure, laminar flow hood. However, it has been established being collected.
that work practices in specimen preparation appear to be more
9.2.3 Monitor sampling pumps on a periodic basis during
important than the type of clean handling facilities in use.
the entire sampling time. Place a cap over the open end of the
Carry out preparation of samples only after acceptable blank
cassette (6.1.1) after sampling, and store the cassette with the
values have been demonstrated.
filter face-upwards for return to the laboratory. Include blank
field filters, as described in 10.7, and process them through the
8.2 Do not perform activities involving manipulation of
remaining analytical procedures along with the samples.
bulk asbestos samples in the same area as TEM specimen
preparation because of the possibilities of contaminating the 9.2.4 Determine the analytical sensitivity S in structures/L
as follows:
TEM specimens.
S 5 A / A × V × K (3)
~ !
f g
9. Sampling
where:
9.1 See Terminology D1356 and Practice D1357 for general
A = area of sample filter exposed to the passage of air,
information on sampling and EPA Documents on AHERA (4)
f
mm ,
and Superfund (5) for information about sampling for asbestos.
A = mean area of TEM specimen grid openings, mm ,
g
9.2 Establish the desired analytical sensitivity for the analy-
V = volume of air sampled, L, and
sis prior to sample collection. It is defined as that structure
K = number of grid openings to be examined.
concentration corresponding to the detection of one structure in
9.2.5 To achieve a particular analytical sensitivity when the
the analysis. For direct transfer methods of TEM specimen
total airborne dust levels are high, it may be necessary to
preparation the analytical sensitivity is a function of the
collect low volumes of air and examine many grid openings.
volume of air sampled, the active area of the collection filter,
and the area of the TEM specimen over which structures are
10. Analysis
counted. Select the sampling rate and the period of sampling to
yield the required analytical sensitivity, as detailed in Table 1.
10.1 Introduction:
9.2.1 Collect air samples using cassettes as qualified in 10.7.
10.1.1 The techniques used to prepare TEM specimens are
Support the filter cassette on a stand (see 6.1.3) that is isolated
different for polycarbonate and cellulose ester filters. The
from the vibrations of the pump (see 6.1.2) during sampling.
preparation method to be used shall be either as described in
10.3 or 10.4, depending on the type of membrane filter used for
TABLE 1 Examples of the Minimum Number of Grid Openings
air sampling. Cleaning of the sample cassettes before they are
Required to Achieve a Particular Analytical Sensitivity for a
opened, preparation of the carbon evaporator, criteria for
Collection Filter Area of 385 mm and
acceptable specimen grids, and the requirement for blank
TEM Grid Openings of 85 μms (0.0072 mm )
determinations are identical for the two preparation techniques.
Analytical
Volume of Air Sampled, L
Sensitivity TEM examination, structure counting, fiber identification, and
Structures/L 500 1000 1200 2000 3000 4000 5000
reporting of results are independent of the type of filter or
0.1 1066 533 444 267 178 134 107
preparation technique used.
0.2 533 267 223 134 89 67 54
0.3 356 178 148 89 60 45 36
10.1.2 The ability to meet the blank sample criteria is
0.4 267 134 112 67 45 34 27
dependent on the cleanliness of equipment and supplies.
0.5 214 107 89 54 36 27 22
0.7 153 77 64 39 26 20 16 Consider all supplies, such as microscope slides and glassware,
1.0 107 54 45 27 18 14 11
as potential sources of asbestos contamination. Wash all
2.0 54 27 23 14 9 7 6
glassware before it is used. Wash any tools or glassware that
3.0 36 18 15 9 6 5 4
4.0 27 14 14 7 5 4 4 come into contact with the air sampling filters or TEM
5.0 22 11 13 6 4 4 4
specimen preparations, both before use and between handling
7.0 16 8 7 4 4 4 4
of individual samples. Use disposable supplies whenever
10.0 11 6 5 4 4 4 4
possible.
D6281 − 23
10.2 Cleaning of Sample Cassette—Asbestos fibers can 10.3.4 Use of the Jaffe Washer with Chloroform—Cut three
adhere to the exterior surfaces of air sampling cassettes (see 3 mm square pieces of carbon-coated polycarbonate filter from
the carbon-coated filter portion, using a curved scalpel blade.
6.1.1), and these fibers can inadvertently be transferred to the
sample during handling. To prevent this possibility of Select three squares to represent the center and the outer
contamination, and after ensuring that the cassette is tightly periphery of the active surface of the filter. Place each square
of filter, carbon side up, on a TEM specimen grid, and place the
sealed, wipe the exterior surfaces of each sampling cassette
before the cassette is taken into the clean facility or laminar grid and filter onto the saturated lens tissue in the Jaffe washer.
Place the three specimen grids from one sample on the same
flow hood.
piece of lens tissue. Any number of separate pieces of lens
10.3 Direct Preparation of TEM Specimens from Polycar-
tissue may be placed in the same Jaffe washer. Cover the Jaffe
bonate Filters:
washer with the lid, and allow the washer to stand for at least
10.3.1 Selection of Filter Area for Carbon Coating—Use a
8 h. It has been found that some lots of polycarbonate filters
cleaned microscope slide to support representative portions of
will not completely dissolve in the Jaffe washer, even after
polycarbonate filter during the carbon evaporation. Use
exposure to chloroform for as long as three days. This problem
double-coated adhesive tape to hold the filter portions to the
also occurs if the surface of the filter was overheated during the
glass slide. Take care not to stretch the polycarbonate filters
carbon evaporation.
during handling. Remove the polycarbonate filter from the
10.3.4.1 Condensation Washing—Prepare TEM specimens
sampling cassette (see 6.1.1), using freshly-cleaned tweezers,
by washing for approximately 1 h in a Jaffe washer (see 6.2.7),
and place it on to a second cleaned glass microscope slide that
transfer the piece of lens tissue supporting the specimen grids
is used as a cutting surface. Cut the filter by rocking the blade
to the cold finger of the condensation washer (see 6.2.8), which
from the point, using a freshly-cleaned curved scalpel blade,
has achieved stable operating conditions using chloroform (see
pressing it into contact with the filter. Repeat the process as
7.1.2) as the solvent. Operate the washer for approximately
necessary. Several such portions may be mounted on the same
30 min after inserting the grids.
microscope slide. Wash and dry the scalpel blade and tweezers
10.3.5 Use of the Jaffe Washer with 1-Methyl-2-
between the handling of each filter. Identify the filter portions
Pyrrolidinone—Cut three 3 mm square pieces of carbon-coated
by writing on the glass slide.
polycarbonate filter from the carbon-coated filter portion, using
10.3.2 Carbon Coating of Filter Portions—Place the slide
a curved scalpel blade. Select three squares to represent the
holding the filter portions on the rotation-tilting device, ap-
center and the outer periphery of the active surface of the filter.
proximately 100 mm to 120 mm from the evaporation source,
Place each square of filter, carbon side up, on a TEM specimen
and evacuate the evaporator chamber to a vacuum better than
grid, and place the grid and filter on the stainless steel mesh in
0.013 Pa. Perform the evaporation of carbon in very short
the Jaffe washer. Any number of separate grids may be placed
bursts, separated by a few seconds to allow the electrodes to
in the same Jaffe washer. Cover the Jaffe washer with the lid,
cool.
and allow the washer to stand for 2 h to 6 h. After dissolution
10.3.2.1 If evaporation of carbon is too rapid, the strips of
is complete, remove the stainless steel mesh from the Jaffe
polycarbonate filter will begin to curl, and cross-linking of the
washer and allow the grids to dry. 1-methyl-2-pyrrolidinone
surface will occur. This cross-linking produces a layer of
evaporates very slowly. If it is required to dry the grids more
polymer that is relatively insoluble in organic solvents, and it
rapidly, transfer the stainless steel bridge into another petri
will not be possible to prepare satisfactory TEM specimens.
dish, and add distilled water until the meniscus contacts the
The thickness of carbon required is dependent on the size of
underside of the mesh. After approximately 15 min, remove the
particles on the filter, and approximately 30 nm to 50 nm has
mesh and allow the grids to dry. If it is desirable to retain the
been found to be satisfactory. If the carbon film is too thin,
water-soluble particle species on the TEM grids, ethanol may
large particles will break out of the film during the later stages
be used instead of distilled water for the second wash.
of preparation, and there will be few complete and undamaged
10.3.6 Use of the Jaffe Washer with a Mixture of 20 %
grid openings on the specimen. Too thick a carbon film will
1,2-Diaminoethane and 80 % 1-Methyl-2-Pyrrolidinone—Cut
lead to a TEM image that is lacking in contrast, and the ability
three 3 mm square pieces of carbon-coated polycarbonate filter
to obtain ED patterns will be compromised. Ensure that the
from the carbon-coated filter portion, using a curved scalpel
carbon film thickness is the minimum possible while retaining
blade. Select three squares to represent the center and the outer
most of the grid openings of the TEM specimen intact.
periphery of the active surface of the filter. Place each square
10.3.3 Preparation of the Jaffe Washer—Place several
of filter, carbon side up, on a TEM specimen grid, and place the
pieces of lens tissue, as shown in Fig. 2, on the stainless steel
grid and filter on the stainless steel mesh in the Jaffe washer.
bridge, and fill the washer (see 6.2.7) with chloroform (see
Any number of separate grids may be placed in the same Jaffe
7.1.2) to a level where the meniscus contacts the underside of washer. Cover the Jaffe washer with the lid, and allow the
the mesh, resulting in saturation of the lens tissue.
washer to stand for 15 min. After dissolution is complete
Alternatively, without using lens paper, fill the washer with (15 min), remove the stainless steel mesh from the Jaffe washer
1-methyl-2-pyrrolidone (see 7.1.3) or a mixture of 20 %
and transfer the stainless steel bridge into another petri dish,
1,2-diaminoethane (see 7.1.7) and 80 % 1-methyl-2- and add distilled water until the meniscus contacts the under-
pyrrolidinone to a level where the meniscus contacts the side of the mesh. After approximately 15 min, remove the mesh
underside of the mesh. and allow the grids to dry. If it is desirable to retain the
D6281 − 23
water-soluble particle species on the TEM grids, ethanol may 10.4.4.1 Adjust the air admission valve of the plasma asher
be used instead of distilled water for the seco
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D6281 − 15 D6281 − 23
Standard Test Method for
Airborne Asbestos Concentration in Ambient and Indoor
Atmospheres as Determined by Transmission Electron
Microscopy Direct Transfer (TEM)
This standard is issued under the fixed designation D6281; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method is an analytical procedure using transmission electron microscopy (TEM) for the determination of the
concentration of asbestos structures in ambient atmospheres and includes measurement of the dimension of structures and of the
asbestos fibers found in the structures from which aspect ratios are calculated.
1.1.1 This test method allows determination of the type(s) of asbestos fibers present.
1.1.2 This test method cannot always discriminate between individual fibers of the asbestos and non-asbestos analogues of the
same amphibole mineral.
1.2 This test method is suitable for determination of asbestos in both ambient (outdoor) and building atmospheres.
1.2.1 This test method is defined for polycarbonate capillary-pore filters or cellulose ester (either mixed esters of cellulose or
cellulose nitrate) filters through which a known volume of air has been drawn and for blank filters.
1.3 The upper range of concentrations that can be determined by this test method is 7000 s/mm . The air concentration represented
by this value is a function of the volume of air sampled.
1.3.1 There is no lower limit to the dimensions of asbestos fibers that can be detected. In practice, microscopists vary in their
ability to detect very small asbestos fibers. Therefore, a minimum length of 0.5 μm has been defined as the shortest fiber to be
incorporated in the reported results.
1.4 The direct analytical method cannot be used if the general particulate matter loading of the sample collection filter as analyzed
exceeds approximately 10 % coverage of the collection filter by particulate matter.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and healthsafety, health, and environmental practices and determine
the applicability of regulatory limitations prior to use.
This test method is under the jurisdiction of ASTM Committee D22 on Air Quality and is the direct responsibility of Subcommittee D22.07 on Sampling and Analysis
of AsbestosSampling, Analysis, Management of Asbestos, and Other Microscopic Particles.
Current edition approved Dec. 1, 2015Sept. 1, 2023. Published January 2016September 2023. Originally approved in 1998. Last previous edition approved in 20092015
as D6281 – 09.D6281 – 15. DOI: 10.1520/D6281-16.10.1520/D6281-23.
This test method was adapted from International Standard ISO 10312 “Air quality—Determination of asbestos fibres—Direct transfer transmission electron microscopy
method.”
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6281 − 23
1.7 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D1356 Terminology Relating to Sampling and Analysis of Atmospheres
D1357 Practice for Planning the Sampling of the Ambient Atmosphere
D4483 Practice for Evaluating Precision for Test Method Standards in the Rubber and Carbon Black Manufacturing Industries
D6620 Practice for Asbestos Detection Limit Based on Counts
D7712 Terminology for Sampling and Analysis of Asbestos
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E691 Practice for Conducting an Interlaboratory Study to Determine the Precision of a Test Method
2.2 ISO Standard:
ISO 10312 Ambient air - Determination of asbestos fibres - Direct-transfer transmission electron microscopy method
3. Terminology
3.1 For definitions of general terms used in this test method, refer to Terminology D1356 or D7712 (see 2.1).
3.2 Definitions of Terms Specific to This Standard:
3.2.1 acicular—acicular, n—the shape shown by an extremely slender crystal with cross-sectional dimensions that are small
relative to its length, that is, needle-like.
3.2.2 amphibole—amphibole, n—a group of more than 60 different silicate minerals with similar crystal structures and complex
compositions that conform to the nominal formula:
A B C T O ~OH,F,Cl! (1)
021 2 5 8 22
where:
A = K, Na, Ca,
2+
B = Fe , Mn, Mg, Ca, Na,
3+ 2+
C = Al, Cr, Ti, Fe , Mg, Fe , Mn, and
3+
T = Si, Al, Cr, Fe , Ti.
In some varieties of amphibole, these elements can be partially substituted by Li, Pb, Zn, Be, Ba, or Ni. Amphiboles are
characterized by a complex monoclinic or orthorhombic structure that includes a double chain of T-O tetrahedra with a T:O ratio
of approximately 4:11; a variable morphology that ranges from columnar to prismatic to acicular to fibrous; and good prismatic
cleavage at angles of about 5656° and 124°. The cleavage may not be readily exhibited by small crystals that are bound by irregular
growth and fracture surfaces (1).
3.2.3 amphibole asbestos—asbestos, n—amphibole in an asbestiform habit.
3.2.4 analytical sensitivity—sensitivity, n—the calculated airborne asbestos structure concentration in asbestos structures/L,
equivalent to the counting of one asbestos structure in the analysis.
3.2.5 asbestiform—asbestiform, n—a specific type of fibrous habit in which the fibers are separable into thinner fibers and
ultimately into fibrils. Thisfibrils; this habit accounts for greater flexibility and higher tensile strength than other habits of the same
mineral.
3.2.6 asbestos—asbestos, n—a collective term that describes a group of naturally occurring, inorganic, highly-fibrous, silicate
minerals that are easily separated into long, thin, flexible, strong fibers when crushed or processed.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
D6281 − 23
3.2.6.1 Discussion—
Included in the definition are the asbestiform varieties of serpentine (chrysotile); riebeckite (crocidolite); grunerite (grunerite
asbestos [Amosite]); anthophyllite (anthophyllite asbestos); tremolite (tremolite asbestos); and actinolite (actinolite asbestos). The
amphibole mineral compositions are defined according to the nomenclature of the International Mineralogical Association.
Asbestos Chemical Abstracts Service Registry No.
Chrysotile 12001-29-5
Crocidolite 12001-28-4
Grunerite Asbestos [Amosite] 12172-73-5
Anthophyllite Asbestos 77536-67-5
Tremolite Asbestos 77536-68-6
Actinolite Asbestos 77536-66-4
3.2.7 asbestos structure—structure, n—a term applied to isolated fibers or to any connected or overlapping grouping of asbestos
fibers or bundles, with or without other nonasbestosnon-asbestos particles.
3.2.8 aspect ratio—ratio, n—the ratio of length to width of a particle.
3.2.9 blank—blank, n—a structure count made on TEM specimens prepared from an unused filter to determine the background
measurement.
3.2.10 camera length—length, n—the equivalent projection length between the specimen and its electron diffraction pattern, in the
absence of lens action.
3.2.11 chrysotile—chrysotile, n—a group of fibrous minerals of the serpentine group that have the nominal composition
Mg Si O (OH) and have the crystal structure of either clinochrysotile, orthochrysotile, or parachrysotile. Mostparachrysotile;
2 5 4
most natural chrysotile deviates little from this nominal composition. Chrysotilecomposition; chrysotile may be partially
dehydrated or magnesium-leached, both in nature and in building materials. Inmaterials; in some varieties of chrysotile, minor
substitution of silicon by Al + may occur. Chrysotileoccur; chrysotile is the most prevalent type of asbestos.
3.2.12 cleavage—cleavage, n—the breaking of a mineral along one of its crystallographic directions.
3.2.13 cleavage fragment—fragment, n—a fragment of a crystal that is bounded in whole or in part by cleavage faces. Somefaces;
some cleavage fragments would be included in the fiber definition used in this method.
3.2.14 cluster—cluster, n—a structure in which two or more fibers or fiber bundles are randomly oriented in a connected grouping.
3.2.15 d-value or interplanar spacing—spacing, n—the perpendicular distance between identical adjacent and parallel planes of
atoms in a crystal.
3.2.16 decision value, n—the structure count that must be exceeded to claim that a measurement represents a population of
airborne structures that is different than the background population, which is established by analyzing blanks (see 3.2.9 and
Practice D6620).
3.2.16 electron diffraction—diffraction, n—techniques in electron microscopy, including selected area electron diffraction (SAED)
and microdiffraction, by which the crystal structure of a specimen is examined.
3.2.17 electron scattering power—power, n—the extent to which a substance scatters electrons from their original courses.
3.2.18 energy dispersive X-ray analysis—analysis, n—measurement of the energies and intensities of X-rays by use of a solid state
detector and multichannel analyzer system.
3.2.19 eucentric—eucentric, n—the condition when the area of interest of an object is placed on a tilting axis at the intersection
of the electron beam with that axis and is in the plane of focus.
The non-asbestiform variations of the minerals indicated in 5.2.6 have different Chemical Abstracts Service (CAS) numbers.
D6281 − 23
3.2.20 field blank—blank, n—a filter cassette that has been taken to the sampling site, opened, and then closed. Such a filter is used
to determine the background structure count for the measurement.
3.2.21 fibril—fibril, n—a single fiber of chrysotile that cannot be further separated longitudinally into smaller components without
losing its fibrous properties or appearances.
3.2.22 fiber—fiber, n—an elongated particle that has parallel or stepped sides. Forsides; for the purposes of this test method, a fiber
is defined as having an aspect ratio equal to or greater than 5:1 and a minimum length of 0.5 μm.
3.2.23 fiber bundle—bundle, n—a structure composed of parallel, smaller-diameter fibers attached along its length. A fiber bundle
may exhibit diverging fibers at one or both ends.
3.2.24 fibrous structure—structure, n—a fiber or connected grouping of fibers with or without other particles.
3.2.25 habit—habit, n—the characteristic crystal growth form or combination of these forms of a mineral, including characteristic
irregularities.
3.2.26 limit of detection—detection, n—the mean count for a population of structures that has been determined, based on a
measurement or average of measurements, to be different than the background population of structures (see 3.2.16 and Practice
D6620). The); the limit of detection may be restated in units of structures/L by multiplying the mean count by analytical sensitivity
(see 3.2.4).
3.2.27 matrix—matrix, n—a structure in which one or more fibers or fiber bundles touch, are attached to, or partially concealed
by a single particle or connected group of nonfibrous particles.
3.2.28 miller index—index, n—a set of three integer numbers used to specify the orientation of a crystallographic plane in relation
to the crystal axes.
3.2.29 PCM equivalent fiber—fiber, n—a particle of aspect ratio that is greater than or equal to 3:1, is longer than 5 μm, and that
has a diameter between 0.2 and 3.0 μm0.2 μm and 3.0 μm.
3.2.30 PCM equivalent structure—structure, n—a fibrous structure of aspect ratio that is greater than or equal to 3:1, is longer than
5 μm, 5 μm, and has a diameter between 0.2 and 3.0 μm.0.2 μm and 3.0 μm.
3.2.31 primary structure—structure, n—a fibrous structure that is a separate entity in the TEM image.
3.2.32 replication—replication, n—a procedure in electron microscopy specimen preparation in which a thin copy, or replica, of
a surface is made.
3.2.33 residual structure—structure, n—matrix or cluster material containing asbestos fibers that remains after accounting for the
prominent component fibers or bundles, or both.
3.2.34 serpentine—serpentine, n—a group of common rock-forming minerals having the nominal formula: Mg Si O (OH) .
3 2 5 4
3.2.35 structure—structure, n—a single fiber, fiber bundle, cluster, or matrix.
3.2.36 twinning—twinning, n—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.
3.2.37 unopened fiber bundle—bundle, n—a large-diameter asbestos fiber bundle that has not been separated into its constituent
fibrils or fibers.
D6281 − 23
3.2.38 zone-axis—zone-axis, n—the crystallographic direction parallel to the intersection edges of the crystal faces defining the
crystal zone.
3.3 Symbols:
eV = electron volt
kV = kilovolt
L/min = liters per minute
–6
μg = micrograms (10 g)
–6
μm = micrometer (10 m)
–9
nm = nanometer (10 m)
W = watt
Pa = Pascals
3.4 Abbreviations:
DMF = dimethyl formamide
ED = electron diffraction
EDXA = energy dispersive X-ray analysis
FWHM = full width, half maximum
HEPA = high-efficiency particle absolute
MCE = mixed cellulose ester; also refers to pure cellulose nitrate filters
PC = polycarbonate
PCM = phase contrast optical microscopy
ED = selected area electron diffraction
SEM = scanning electron microscope
STEM = scanning transmission electron microscope
TEM = transmission electron microscope
UICC = Union Internationale Contre le Cancer
4. Summary of Test Method
4.1 A sample of airborne particulate matter is collected by drawing a measured volume of air through either a capillary-pore
polycarbonate membrane filter of maximum pore size 0.4 μm 0.4 μm or a cellulose ester (either mixed esters of cellulose or
cellulose nitrate) membrane filter of maximum pore size 0.45 μm 0.45 μm by means of a battery-powered or mains-powered pump.
TEM specimens are prepared from polycarbonate filters by applying a thin film of carbon to the filter surface by vacuum
evaporation. Small areas are cut from the carbon-coated filter, supported on TEM specimen grids, and the filter medium is
dissolved away by a solvent extraction procedure. This procedure leaves a thin film of carbon that bridges the openings in the TEM
specimen grid and that supports each particle from the original filter in its original position. Cellulose ester filters are chemically
treated to collapse the pore structure of the filter, and the surface of the collapsed filter is then etched in an oxygen plasma to try
to expose particles embedded in the collapsed filter. A thin film of carbon is evaporated onto the filter surface and small areas are
cut from the filter. These sections are supported on TEM specimen grids, and the filter medium is dissolved by a solvent extraction
procedure.
4.2 The TEM specimen grids from either preparation method are examined at both low and high magnifications to check that they
are suitable for analysis before carrying out a quantitative structure count on randomly-selected grid openings. In the TEM analysis,
electron diffraction (ED) is used to examine the crystal structure of a fiber, and its elemental composition is determined by energy
dispersive X-ray analysis (EDXA). For a number of reasons, it is not possible to identify each fiber unequivocally and fibers are
classified according to the techniques that have been used to identify them. For each fiber, a simple code is used to record the
manner in which it was classified. The fiber classification procedure is based on successive inspection of the morphology, the ED
pattern, and the qualitative and quantitative EDXA. 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.
4.3 In addition to isolated fibers, ambient air samples often contain more complex aggregates of fibers, with or without other
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 a description
of each of these complex structures. Several levels of analysis are specified, the higher levels providing a more rigorous approach
to the identification of fibers. The procedure permits a minimum required fiber identification criterion to be defined on the basis
D6281 − 23
of previous knowledge, or lack of it, about the particular sample. Attempts are then made to achieve this minimum criterion for
each fiber, and the degree of success is recorded for each fiber. The lengths and widths of all classified structures and fibers are
recorded. The number of asbestos structures found on a known area of the microscope sample, together with the equivalent volume
of air filtered through this area, is used to calculate the airborne concentration in asbestos structures/L of air.
5. Significance and Use
5.1 This test method is applicable to the measurement of airborne asbestos in a wide range of ambient air situations and for
detailed evaluation of any atmosphere for asbestos structures. Most fibers in ambient atmospheres are not asbestos, and therefore,
there is a requirement for fibers to be identified. Most of the airborne asbestos fibers in ambient atmospheres have diameters below
the resolution limit of the light microscope. This test method is based on transmission electron microscopy, which has adequate
resolution to allow detection of small thin fibers and is currently the only technique capable of unequivocal identification of the
majority of individual fibers of asbestos. Asbestos is often found, not as single fibers, but as very complex, aggregated structures,
which may or may not also be aggregated with other particles. The fibers found suspended in an ambient atmosphere can often
be identified unequivocally if sufficient measurement effort is expended. However, if each fiber were to be identified in this way,
the analysis would become prohibitively expensive. Because of instrumental deficiencies or because of the nature of the particulate
matter, some fibers cannot be positively identified as asbestos even though the measurements all indicate that they could be
asbestos. Therefore, subjective factors contribute to this measurement, and consequently, a very precise definition of the procedure
for identification and enumeration of asbestos fibers is required. The method defined in this test method is designed to provide a
description of the nature, numerical concentration, and sizes of asbestos-containing particles found in an air sample. The test
method is necessarily complex because the structures observed are frequently very complex. The method of data recording
specified in the test method is designed to allow reevaluation of the structure-counting data as new applications for measurements
are developed. All of the feasible specimen preparation techniques result in some modification of the airborne particulate matter.
Even the collection of particles from a three-dimensional airborne dispersion on to a two-dimensional filter surface can be
considered a modification of the particulate matter, and some of the particles, in most samples, are modified by the specimen
preparation procedures. However, the procedures specified in this test method are designed to minimize the disturbance of the
collected particulate material.
5.2 This test method applies to analysis of a single filter and describes the precision attributable to measurements for a single filter
(see 13.1). Multiple air samples are usually necessary to characterize airborne asbestos concentrations across time and space. The
number of samples necessary for this purpose is proportional to the variation in measurement across samples, which may be greater
than the variation in a measurement for a single sample.
6. Apparatus
6.1 Air Sampling Equipment and Consumable Supplies:
6.1.1 Filter Cassette, 25 to 50-mm-diameter,25 mm to 50 mm-diameter, commercially-manufactured, nonreusable, three-piece
cassettes, with cowls in front of the filter surface, used for sample collection. Load the cassette with either a capillary pore
polycarbonate filter of maximum pore size 0.4 μm or an MCE of maximum pore size 0.45 μm. Back either type of filter with a
5 μm pore size MCE, and support it by a cellulose back-up pad. Apply a shrink cellulose band or adhesive tape when the filters
are in position to prevent air leakage. Ensure that the filters are tightly clamped in the assembly so that significant air leakage
around the filter cannot occur.
6.1.1.1 It is recommended that representative filters from the filter lot be analyzed as described in 10.7 for the presence of asbestos
structures before any are used for air sample collection.
6.1.2 Sampling Pump, capable of a flow-rate sufficient to achieve the desired analytical sensitivity. The face velocity through the
filter shall be between 4.04.0 cm ⁄s and 45.045.0 cm cm/s. ⁄s. The sampling pump used shall provide a stable air-flow through the
filter. A constant flow or critical orifice-controlled pump meets these requirements. Use flexible tubing to connect the filter cassette
(see 6.1.1) to the sampling pump.
6.1.3 Stand, used to hold the filter cassette at the desired height for sampling, and to isolate it from the pump vibrations.
6.1.4 Flow Meter, a calibrated flow meter with an appropriate range for the sampling flow rate used. The flow meter should be
calibrated to a primary standard.
6.2 Equipment for Analysis:
D6281 − 23
6.2.1 Transmission Electron Microscope—A TEM operating at an accelerating potential of 80–120 kV, 80 kV to 120 kV, with a
resolution better than 1.0 nm, and a magnification range of approximately 300 to 100 000 with the ability to obtain a direct screen
magnification of about 100 000, shall be used for inspection of fiber morphology. This magnification may be obtained by
supplementary optical enlargement of the screen image by use of a binocular. It is also required that the viewing screen of the
microscope be calibrated such that the lengths and widths of fiber images down to 1 mm 1 mm width can be estimated in
increments of 1 mm 1 mm regardless of fiber orientation. This requirement is often fulfilled through use of a fluorescent screen
with calibrated gradations in the form of circles, such as the one shown in Fig. 1.
6.2.1.1 For Bragg angles less than 0.01 radians the TEM shall be capable of performing ED from an area of 0.6 μm or less. This
performance requirement defines the minimum separation between particles at which independent ED patterns can be obtained
from each particle. If ED is used, the performance of a particular instrument normally may be calculated using the following
relationship:
3 2
A 5 0.7854 × D/M12000 ×C θ (2)
~ !
s
where:
A = effective ED area in μm ,
D = diameter of the ED aperture in μm,
M = magnification of the objective lens,
C = spherical aberration coefficient of the objective lens in mm, and
s
θ = maximum required Bragg angle in radians.
6.2.1.2 It is not possible to reduce the effective ED area indefinitely by the use of progressively smaller ED apertures because there
is a fundamental limitation imposed by the spherical aberration coefficient of the objective lens.
6.2.1.3 If zone-axis ED analyses of amphiboles are to be performed, the TEM shall incorporate a goniometer stage that permits
the TEM specimen to be either:
(a) rotated through 360°, combined with tilting through at least +30+30° to –30° about an axis in the plane of the specimen;
or
(b) tilted through at least +30+30° to –30° about two perpendicular axes in the plane of the specimen.
6.2.1.4 The analysis is greatly facilitated if the goniometer permits eucentric tilting, although tilting is not essential. If EDXA and
zone-axis ED are required on the same fiber, the goniometer shall be of a type that permits tilting of the specimen and acquisition
FIG. 1 Example of Calibration Markings on TEM Viewing Screen
D6281 − 23
of EDXA spectra without change of specimen holder. If the goniometer does not permit eucentric tilting, a gold or other metal film
must be evaporated on the sample so that ED patterns may be accurately calibrated.
6.2.1.5 The TEM shall have an illumination and condenser lens system capable of forming an electron probe smaller than 250 nm
in diameter. It is recommended that an anticontamination trap be used around the specimen.
6.2.2 Energy Dispersive X-ray Analyzer—The TEM shall be equipped with an energy dispersive X-ray analyzer capable of
achieving a resolution better than 180 eV (FWHM) on the MnKα peak. Since the performance of individual combinations of TEM
and EDXA equipment is dependent on a number of 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 fiber of small diameter, using a known
diameter. Solid state X-ray detectors are least sensitive in the low energy region, 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 NaKα integrated peak count rate of more than 1 count 1 count per second (cps) from a fiber
of UICC crocidolite 50 nm 50 nm in diameter or smaller when irradiated by an electron probe of 250-nm250 nm diameter or
smaller. The peak/background ratio for this performance test shall exceed 1.0.
6.2.2.1 The EDXA unit shall provide the means for subtraction of the background, identification of elemental peaks, and
calculation of background-subtracted peak areas.
6.2.3 Carbon Rod Sharpener, to neck the carbon rods that allow the carbon to be evaporated on to the filters with a minimum of
heating.
6.2.4 Plasma Asher, for preparation of TEM specimens from MCE filters. The plasma asher shall have a radio frequency power
rating of 50 W or higher and be provided with a controlled, filtered oxygen flow. Admission of filtered air shall be through a valve
to control the speed of air admission so that rapid air admission does not disturb particulate matter from the surface of the filter
after the etching step.
6.2.5 Vacuum Coating Unit, a vacuum coating unit capable of producing a vacuum better than 0.013 Pa, used for vacuum
deposition of carbon on the membrane filters. A sample holder is required that will allow a glass microscope slide to be tilted and
continuously rotated during the coating procedure.
6.2.5.1 Equip the vacuum coating unit with a mechanism that allows the rotating slide to be tilted also through an angle of
approximately 45° during the coating procedure. A liquid nitrogen trap 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.
6.2.6 Sputter Coater, with a gold target used for deposition of gold onto TEM specimens as an internal calibration of ED patterns.
Other calibration materials are acceptable. Experience has shown that a sputter coater allows better control of the thickness of the
calibration material.
6.2.7 Solvent Washer (Jaffe washer (2)), allows for 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 Fig. 2. Use either chloroform or 1-methyl-2-pyrrolidinone for dissolving polycarbonate filters,
NOTE 1—Solvent is added until the meniscus contacts the underside of the stainless steel mesh.
FIG. 2 Example of Design of Solvent Washer (Jaffe Washer)
D6281 − 23
FIG. 3 Design of Condensation Washer
and use dimethyl formamide or acetone for dissolving MCE or cellulose nitrate filters. A mixture of 20 % 1-2-diaminoethane and
80 % 1-methyl-2-pyrrolidinone may also be used to dissolve polycarbonate filters (3). The higher evaporation rates of chloroform
and acetone require that a reservoir of 1010 mL to 50 mL of solvent be used, which may need replenishment during the procedure.
Because dimethyl formamide and 1-methyl-2-pyrrolidinone have lower vapor pressures, much smaller volumes of solvent may be
used. Use the washer in a fume hood, and keep the petri dishes covered with their lids when specimens are not being inserted or
removed during the solvent dissolution. Clean the washer before it is used for each batch of specimens.
6.2.8 Condensation Washer, used for more rapid dissolution of the filter polymer or for dissolving the filter polymer if difficulties
are experienced. The washer consists of a flask, condenser, and cold finger assembly with a heating mantle and means for
controlling the temperature. A suitable assembly is shown in Fig. 3. Use either acetone or chloroform as the solvent, depending
on the type of filter.
6.2.9 Slide Warmer or Oven, for heating slides during the preparation of TEM specimens from MCE or cellulose nitrate filters,
capable of maintaining a temperature of 65 to 70°C.65 °C to 70 °C.
6.2.10 Ultrasonic Bath, for cleaning of apparatus used for TEM specimen preparation.
6.2.11 Carbon Grating, with approximately 2000 parallel lines per mm, used to calibrate the magnification of the TEM (see 6.2.1).
6.2.12 Calibration Specimen Grids for EDXA, TEM specimen grids prepared from dispersions of calibration minerals required for
calibration of the EDXA system: crocidolite asbestos (NIST SRM 1866) and chrysotile asbestos.
6.3 Reference Asbestos Samples, for preparation of reference TEM specimens of the primary asbestos minerals. The UICC or
NIST set of minerals are suitable for this purpose.
7. Reagents and Materials
7.1 Reagents: Warning—Use the reagents in accordance with the appropriate health and safety regulations. Review their Material
Safety Data Sheets before use.
7.1.1 Purity of Water—Water shall be reagent water as defined by Type II of Specification D1193.
7.1.2 Chloroform, analytical grade, distilled in glass (preserved with 1 % (v/v) ethanol).
7.1.3 1-Methyl-2-Pyrrolidinone, analytical grade.
7.1.4 Dimethyl Formamide, analytical grade.
7.1.5 Glacial Acetic Acid, analytical grade.
7.1.6 Acetone, analytical grade.
D6281 − 23
7.1.7 1-2-Diaminoethane, analytical grade.
7.2 Materials:
7.2.1 Copper Electron Microscope Grids, 200-mesh TEM grids with grid openings of uniform size such that they meet the
requirement of 10.6.3. Use grids with numerical or alphabetical indexing of individual grid openings to facilitate the relocation of
individual grid openings for quality assurance purposes.
7.2.2 Gold Electron Microscope Grids, 200 mesh gold to mount TEM specimens when sodium measurements are required in the
fiber identification procedure. Use grids that have grid openings of uniform size such that they meet the requirement of 10.6.3. Use
grids with numerical or alphabetical indexing of individual grid openings to facilitate the relocation of individual grid openings
for quality assurance purposes.
7.2.3 Carbon Rod Electrodes, spectrochemically pure for use in the vacuum evaporator during carbon coating of filters.
7.2.4 Disposable Tip Micropipettes, 30 μL.
7.2.5 Core Borer, 7 mm.
7.2.6 Routine Electron Microscopy Tools and Supplies, such as fine-point tweezers, scalpel holders and blades, microscope slides,
double-coated adhesive tape, gummed paper reinforcement rings, lens tissue, gold wire, tungsten filaments, and other routine
supplies.
8. Specimen Preparation Laboratory
8.1 Asbestos, particularly chrysotile, may be present in varying quantities in 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. Perform all specimen preparation steps in an environment where
contamination of the sample is minimized. The primary requirement of the sample preparation laboratory is that a blank
determination yields results that will meet the requirements specified in 10.7. A minimum facility considered suitable for
preparation of TEM specimens is a positive-pressure, 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. Carry out preparation of samples
only after acceptable blank values have been demonstrated.
8.2 Do not perform activities involving manipulation of bulk asbestos samples in the same area as TEM specimen preparation
because of the possibilities of contaminating the TEM specimens.
9. Sampling
9.1 See Terminology D1356 and Practice D1357 for general information on sampling and EPA Documents on AHERA (4) and
Superfund (5) for information about sampling for asbestos.
9.2 Establish the desired analytical sensitivity for the analysis prior to sample collection. It is defined as that structure
concentration corresponding to the detection of one structure in the analysis. For direct transfer methods of TEM specimen
preparation the analytical sensitivity is a function of the volume of air sampled, the active area of the collection filter, and the area
of the TEM specimen over which structures are counted. Select the sampling rate and the period of sampling to yield the required
analytical sensitivity, as detailed in Table 1.
9.2.1 Collect air samples using cassettes as qualified in 10.7. Support the filter cassette on a stand (see 6.1.3) that is isolated from
the vibrations of the pump (see 6.1.2) during sampling. Hold the cassette facing downwards vertically at a height of approximately
1.51.5 m to 2.0 m above ground floor level, and connect it to the pump with a flexible tube.
9.2.2 Measure the sampling flow-rate at the front end of the cassette, both at the beginning and end of the sampling period, using
a calibrated flow meter (6.1.4) temporarily attached to the inlet of the cassette (see 6.1.1). Use the mean value of these two
measurements to calculate the total air volume sampled. If the difference in flow rate at the beginning and end of the sampling
period is greater than 20 %, the result should be labeled as suspect or void due to sampling errors.
D6281 − 23
TABLE 1 Examples of the Minimum Number of Grid Openings
Required to Achieve a Particular Analytical Sensitivity for a
Collection Filter Area of 385 mm and
TEM Grid Openings of 85 μms (0.0072 mm )
Analytical
Volume of Air Sampled, L
Sensitivity
Structures/L 500 1000 1200 2000 3000 4000 5000
0.1 1066 533 444 267 178 134 107
0.2 533 267 223 134 89 67 54
0.3 356 178 148 89 60 45 36
0.4 267 134 112 67 45 34 27
0.5 214 107 89 54 36 27 22
0.7 153 77 64 39 26 20 16
1.0 107 54 45 27 18 14 11
2.0 54 27 23 14 9 7 6
3.0 36 18 15 9 6 5 4
4.0 27 14 14 7 5 4 4
5.0 22 11 13 6 4 4 4
7.0 16 8 7 4 4 4 4
10.0 11 6 5 4 4 4 4
9.2.2.1 If flow-meter contamination is suspected, clean and recalibrate the flow meter before use to avoid transfer of asbestos
contamination from the flow meter to the sample being collected.
9.2.3 Monitor sampling pumps on a periodic basis during the entire sampling time. Place a cap over the open end of the cassette
(6.1.1) after sampling, and store the cassette with the filter face-upwards for return to the laboratory. Include blank field filters, as
described in 10.7, and process them through the remaining analytical procedures along with the samples.
9.2.4 Determine the analytical sensitivity S in structures/L as follows:
S 5 A / A ×V ×K (3)
~ !
f g
where:
A = area of sample filter exposed to the passage of air, mm ,
f
A = mean area of TEM specimen grid openings, mm ,
g
V = volume of air sampled, L, and
K = number of grid openings to be examined.
9.2.5 To achieve a particular analytical sensitivity when the total airborne dust levels are high, it may be necessary to collect low
volumes of air and examine many grid openings.
10. Analysis
10.1 Introduction:
10.1.1 The techniques used to prepare TEM specimens are different for polycarbonate and cellulose ester filters. The preparation
method to be used shall be either as described in 10.3 or 10.4, depending on the type of membrane filter used for air sampling.
Cleaning of the sample cassettes before they are opened, preparation of the carbon evaporator, criteria for acceptable specimen
grids, and the requirement for blank determinations are identical for the two preparation techniques. TEM examination, structure
counting, fiber identification, and reporting of results are independent of the type of filter or preparation technique used.
10.1.2 The ability to meet the blank sample criteria is dependent on the cleanliness of equipment and supplies. Consider all
supplies, such as microscope slides and glassware, as potential sources of asbestos contamination. Wash all glassware before it is
used. Wash any tools or glassware that come into contact with the air sampling filters or TEM specimen preparations, both before
use and between handling of individual samples. Use disposable supplies whenever possible.
10.2 Cleaning of Sample Cassette—Asbestos fibers can adhere to the exterior surfaces of air sampling cassettes (see 6.1.1), and
these fibers can inadvertently be 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 before the cassette is taken into
the clean facility or laminar flow hood.
D6281 − 23
10.3 Direct Preparation of TEM Specimens from Polycarbonate Filters:
10.3.1 Selection of Filter Area for Carbon Coating—Use a cleaned microscope slide to support representative portions of
polycarbonate filter during the carbon evaporation. Use double-coated adhesive tape to hold the filter portions to the glass slide.
Take care not to stretch the polycarbonate filters during handling. Remove the polycarbonate filter from the sampling cassette (see
6.1.1), using freshly-cleaned tweezers, and place it on to a second cleaned glass microscope slide that is used as a cutting surface.
Cut the filter by rocking the blade from the point, using a freshly-cleaned curved scalpel blade, pressing it into contact with the
filter. Repeat the process as necessary. Several such portions may be mounted on the same microscope slide. Wash and dry the
scalpel blade and tweezers between the handling of each filter. Identify the filter portions by writing on the glass slide.
10.3.2 Carbon Coating of Filter Portions—Place the slide holding the filter portions on the rotation-tilting device, approximately
100100 mm to 120 mm from the evaporation source, and evacuate the evaporator chamber to a vacuum better than 0.013 Pa.
Perform the evaporation of carbon in very short bursts, separated by a few seconds to allow the electrodes to cool.
10.3.2.1 If evaporation of carbon is too rapid, the strips of polycarbonate filter will begin to curl, and cross-linking of the surface
will occur. This cross-linking produces a layer of polymer that is relatively insoluble in organic solvents, and it will not be possible
to prepare satisfactory TEM specimens. The thickness of carbon required is dependent on the size of particles on the filter, and
approximately 3030 nm to 50 nm 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 that is lacking in contrast, and the ability to obtain ED patterns will be compromised.
Ensure that the carbon film thickness is the minimum possible while retaining most of the grid openings of the TEM specimen
intact.
10.3.3 Preparation of the Jaffe Washer—Place several pieces of lens tissue, as shown in Fig. 2, on the stainless steel bridge, and
fill the washer (see 6.2.7) with chloroform (see 7.1.2) to a level where the meniscus contacts the underside of the mesh, resulting
in saturation of the lens tissue. Alternatively, without using lens paper, fill the washer with 1-methyl-2-pyrrolidone (see 7.1.3) or
a mixture of 20 % 1,2-diaminoethane (see 7.1.7) and 80 % 1-methyl-2-pyrrolidinone to a level where the meniscus contacts the
underside of the mesh.
10.3.4 Use of the Jaffe Washer with Chloroform—Cut three 3-mm3 mm square pieces of carbon-coated polycarbonate filter from
the carbon-coated filter portion, using a curved scalpel blade. 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 a TEM specimen grid, and place the grid and filter
onto the saturated lens tissue in the Jaffe washer. Place the three specimen grids from one sample on the same piece of lens tissue.
Any number of separate pieces of lens tissue may be placed in the same Jaffe washer. Cover the Jaffe washer with the lid, and allow
the washer to stand for at least 8 h. It has been found that some lots of polycarbonate filters will not completely dissolve in the
Jaffe washer, even after exposure to chloroform for as long as three days. This problem also occurs if the surface of the filter was
overheated during the carbon evaporation.
10.3.4.1 Condensation Washing—Prepare TEM specimens by washing for approximately 1 h in a Jaffe washer (see 6.2.7), transfer
the piece of lens tissue supporting the specimen grids to the cold finger of the condensation washer (see 6.2.8), which has achieved
stable operating conditions using chloroform (see 7.1.2) as the solvent. Operate the washer for approximately 30 min 30 min after
inserting the grids.
10.3.5 Use of the Jaffe Washer with 1-Methyl-2-Pyrrolidinone—Cut three 3-mm3 mm square pieces of carbon-coated polycar-
bonate filter from the carbon-coated filter portion, using a curved scalpel blade. 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 a TEM specimen grid, and place
the grid and filter on the stainless steel mesh in the Jaffe washer. Any number of separate grids may be placed in the same Jaffe
washer. Cover the Jaffe washer with the lid, and allow the washer to stand for 22 h to 6 h. After dissolution is complete, remove
the stainless steel mesh from the Jaffe washer and allow the grids to dry. 1-methyl-2-pyrrolidinone evaporates very slowly. If it
is required to dry the grids more rapidly, transfer the stainless steel bridge into another petri dish, and add distilled water until the
meniscus contacts the underside of the mesh. After approximately 15 min, remove the mesh and allow the grids to dry. If it is
desirable to retain the water-soluble particle
...