VanOrden etal 60 2 p51 61 2012 1472588781
THE MICROSCOPE
•
Vol. 60:2, pp 51–61 (2012)
Effect of Sample Preparation on Observed Airborne
Fiber Characteristics
D.R. Van Orden, M. Sanchez and J.M. Wilmoth
RJ Lee Group, Inc.*
KEYWORDS
Asbestos, ASTM D7200, chrysotile, concentration,
crocidolite, grinding, NIST SRM 1866, phase contrast
microscopy (PCM), scanning electron microscopy
(SEM), sonication, transmission electron microscopy
(TEM), tremolite
ABSTRACT
Natural occurrences of asbestos and potential exposures to such materials have drawn significant public and regulatory interest in recent years. Proposed
revisions to a published analytical method (ASTM
D7200) that is designed to monitor airborne fiber concentrations are based on data developed using
inappropriate sample preparation strategies, including over-grinding the samples and the use of
ultrasonication. Tests demonstrating the effects of
grinding and preparation of asbestos minerals have
been conducted. Sample preparation procedures used
by some laboratories destroy the characteristics of
asbestos, thereby limiting the usefulness of ASTM
D7200 and similar analytical methods. The effects of
sample preparation on the morphology of particles
must be considered when creating or revising asbestos analytical procedures.
INTRODUCTION
When rocks that contain veins or pockets with
natural occurrences of asbestos are crushed, there is a
possibility that asbestos fibers will be released and
become airborne. If the airborne asbestos is of respirable size, these fibers pose a potential hazard to workers and bystanders who may breathe them.
Fortunately, minerals rarely grow as asbestos fibers, but more generally grow as (comparatively)
larger crystals that do not possess the characteristics
of asbestisform fibers. Unfortunately, when these nonasbestos minerals are fractured, many will fracture as
elongated particles that have dimensions that are encompassed by various regulatory analytical procedures. For example, OSHA (29 CFR §1910.1001) and
MSHA (30 CFR §56.5001) regulate airborne asbestos
fibers if they are at least 5 μm long, have a minimum
aspect ratio (length:width) of 3:1 and are visible in an
optical microscope. Fractured non-asbestos minerals
that frequently occur with asbestos are routinely observed with these dimensions even though they are
not asbestos.
Therefore, it is necessary to be able to analyze
samples to determine the amount of asbestos (independent of any non-asbestos mineral) in the samples.
ASTM D7200 (1) is an optical microscopy procedure
for analyzing airborne particles from mines, quarries
and construction activities to determine which
samples need additional analyses using electron microscopy to determine compliance with MSHA and
OSHA worker exposure regulations. ASTM D7200 provides an index used to estimate whether the population of counted fibers are potentially asbestos.
D7200 was originally developed in the late 1980s
and was round-robin tested at that time using filters
*350 Hochberg Road, Monroeville, PA 15146
51
collected at operating mines and quarries. The method
and the test results were produced during testimony
at an OSHA hearing in 1990 (2). The method was
adopted, with slight modification, by ASTM in 2006
after several years of consideration.
ASTM D7200 is an analytical method for airborne
particulate collected on filters. The method involves a
standard phase contrast microscopy (PCM) analysis
that then divides counted elongated particulate (minimum 3:1 aspect ratio, 5 μm or longer) into three bins:
one that includes obvious bundles and/or curved fibers, a second that includes long (> 10 μm), thin (high
aspect ratio) fibers, and a third bin for the remainder
of the counts. If the D7200 concentration of countable
fibers exceeds one half of the current regulatory limit
and the number of fibers reported in the first two bins
exceed 50% of the total count, the method strongly recommends the mine owner/operator to take additional
analytical measures to determine if the fibers are asbestos. If no further analyses are conducted, the fibers
should be considered to be asbestos. This trigger at
one-half the PEL and 50% of the counts in the first two
bins serves as a safety factor. Regardless of the distribution of counts in the bins, the reported concentration retains comparability to current regulatory levels and to historical PCM analyses. The binning process is a form of differential counting that is based on
published dimensional and crystal growth characteristics described in the literature (3–5).
Recently, Harper et al. published a paper (6) discussing the size characteristics of asbestos and nonasbestos amphibole particles that mischaracterizes
ASTM D7200 by suggesting that the method indicates
that particle dimensions (lengths and widths) are used
to positively identify asbestos fibers and to classify all
remaining particles as non-asbestos particles. It is important to understand, however, that D7200 is expressly
intended to be a screening tool designed to encourage
mine and quarry operators to be proactive in monitoring their employees for potential asbestos exposure. If
such a potential is indicated through the screening approach, additional analyses that are more complex, time
consuming and costly are required for positive identification of asbestos (see step 1.6 in the method).
The testing reported by Harper, et al. (6) to address the reliability of these bins as a screening for
potential asbestos fibers (avoidance of false negatives
in particular) is also misleading, in part, due to the
inappropriate sample preparation procedures used to
generate the test fibers. In their test of D7200 to discriminate asbestos populations from non-asbestos
populations, Harper tested a number of samples that
52
could be classified as either asbestos or non-asbestos
on the basis of their appearance in hand samples. However, the purity of these specimens is unclear, and the
value of their work would be enhanced if the originating sample composition was fully characterized. Several standard reference materials for amphibole asbestos used in the Harper work are known to contain
considerable non-asbestiform fragments (7–9).
In the Harper paper, the mineral samples were
heavily processed to optimize the number of the 3:1
aspect ratio and particles longer than 5 μm, resulting
in powders whose particles had widths of generally
< 3 μm (10). Their general sample preparation procedure is shown in the flowchart in Figure 1. Part of
their sample preparation procedure used an ultrasonic
disruptor to further disaggregate the crushed particles. It is not clear from the publication what size
particles were subjected to ultrasonic treatment,
though Harper et al. (11) provides additional description of the sample preparation procedures in a second
paper. The mineral samples classified by Harper as
“asbestos” were not subjected to the initial crushing
step shown in Figure 1.
In regard to asbestos, the sample processing procedure used by Harper may well have destroyed the
asbestiform nature of the asbestos test minerals. Using ultrasonic energy to process asbestos fibers is
known to separate fiber bundles into the individual
fibers and to comminute the fibers into shorter fibers,
thus reducing the apparent aspect ratio of the asbestos fibers (12). This sonication effect is acknowledged
in other ASTM asbestos procedures that utilize
ultrasonication (13).
It is not known that particles prepared in the manner described by Harper will be equivalent to particles
observed in mining environments. Certainly there was
no attempt to collect airborne samples from operating
asbestos mines (either amphibole or chrysotile) to use
for comparison with the comminuted samples. It is
known that varying manufacturing processes produce
significant variations in fiber dimensions (14–17). In
particular, sonication has been shown to affect particle dimensions (12, 18). It has also been suggested
that the fibrosity index in bulk samples can be correlated with airborne data (19), although there are differences between bulk and airborne-size distributions
(20), and between different analytical procedures on
the same filter (21). The dimensions of asbestos fibers
have been shown to play an important role in exposure classifications (22, 23), hence the need to create an
appropriate analytical tool to screen for this potential.
The samples that Harper relied on as asbestos do
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
Figure 1. This flowchart of the sample preparation procedures was used by Harper et al. (6) in preparing amphibole samples for analyses.
not have the size characteristics of asbestos. The contract laboratory that produced the samples, reported
by Harper, evaluated some of the fiber samples using
transmission electron microscopy (TEM). A plot of
width versus aspect ratio (Figure 2) indicates that only
a small fraction of the fibers (16%) meet the minimum
aspect ratio of 20:1 recommended by the EPA (24). Of
the five minerals classified as asbestos, only the crocidolite sample contained more than 50% of fibers with
an aspect ratio greater than 20:1 (77%); the other
samples were 26% (amosite) or fewer than 3% (actinolite asbestos, tremolite asbestos and anthophyllite
asbestos).
Therefore, if the sample preparation procedures
affected the morphological and size characteristics of
the minerals used in his tests, Harper’s conclusions
about the efficacy of various test methods are not valid.
Analytical procedures need to be tested and modified
as new information about a program or analyte be-
comes known, and certainly the D7200 method should
be tested to confirm its performance. However, the tests
should be conducted with a clear understanding of the
effect of sample preparation on the reference test minerals. Only with this understanding can tests be properly designed and the results interpreted.
EXPERIMENTAL PLAN
Crocidolite from the NIST SRM 1866 standard was
examined in order to provide a direct comparison to
the samples used by Harper. Crocidolite from NIST
SRM 1866 was first placed in a beaker of distilled water and sonicated for three minutes using a 100 W ultrasonic bath. At the completion of the sonication, the
suspension was thoroughly mixed and allowed to settle
for 30 minutes. At that time, an aliquot of the remaining suspension was removed, deposited onto a polycarbonate filter (0.2 μm pore) and mounted on a car-
53
Figure 2. The graph shows the distribution of the width and aspect ratio of the amphibole asbestos samples from Harper et al. (6) Most of
the samples have aspect ratios below 20:1. The red box shows the area that Chatfield (7) has proposed for defining a particle as asbestos.
bon planchette for analysis by scanning electron microscopy (SEM).
In a second procedure, crocidolite from NIST SRM
1866 was placed in an elutriation chamber and aerosolized using compressed air. Air filters (MCE, 0.8 μm
pore) were placed at the top of the chamber and were
used to collect samples of the airborne dust at a sampling rate of 2 lpm. At the end of the sampling, a portion of the filter was excised and mounted on a carbon
planchette for analysis by SEM.
The prepared crocidolite samples were examined
with a JEOL 6460 LV SEM at magnifications ranging
from 1,000x to 5,000x. Images of fields of particles were
recorded for offline measurements of approximately
650 particles. The counted particles were limited to
those longer than 5 μm with a minimum aspect ratio
of 3:1. Particles that were obvious bundles were la-
54
beled so their dimensions could be evaluated separately
from the other fibers.
Additional tests were conducted to demonstrate
the effect of grinding and sonication on the size and
morphology of amphibole minerals. An experiment
was designed to demonstrate the effect of sample
preparation procedures, specifically sonication, on
crushed rock samples. Three samples, shown in
Figure 3, were obtained and used in the testing. Each
sample was 50 mm–75 mm, which is a typical size of
railroad ballast or rock that would be used as base
material for road building. Each sample contained a
1
Byssolite is a fibrous form of amphibole minerals that has low
tensile strength and moderate fiber widths. A portion of the fibers
will show crystal faces. Byssolite fibers are not asbestiform fibers
and they are not asbestos.
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
3A
3B
3C
Figure 3. Rock samples used in the test program to demonstrate the effect of sample preparation procedures, specifically
sonication, on crushed rock: 3A, pieces of rock containing tremolite asbestos that were taken from a drill core; 3B, a
sample that contains actinolite (byssolite), which was also obtained from a drill core; and 3C, two pieces of chrysotile ore
from the mine in Lowell, Vermont.
vein or veins of visibly fibrous minerals that were
identified as the amphibole minerals tremolite asbestos, actinolite (byssolite1) or the serpentine mineral
chrysotile.
Each sample was stage ground so that the product
size was generally finer than 150 μm (100 mesh) (25).
The ground samples were riffled into equal parts.
The effect of sonication was examined by taking
one portion of each ground sample and sonicating it
using a Biologics Ultrasonic Homogenizer (Model 150
V/T) equipped with a titanium tip (0.5 inch). The pulverized material was suspended in approximately
100 ml of deionized water and then sonicated for 15
minutes, the same time used by Harper (6) and Beard
(10). Aliquots of the sonicated material were removed
from the suspension at the completion of the sonication (no settling) and filtered onto a new mixed cellulose ester filter (0.2 μm pore size).
The other portion of each sample was aerosolized
in a glove box using small fans; the airborne particles
were collected onto filters in general accordance with
NIOSH 7400 (26). While aerosolized, some of the
coarser particles did not remain airborne, therefore,
these aerosolized fractions represent a subset of the
entire sample.
Each collected filter was analyzed in general accordance with PCM (ASTM D7200 [1]) and SEM (ISO
14966 [27]). For the PCM analyses, the definition of
Bin 2 was modified to include fibers longer than 10
μm and thinner than 1 μm. The primary purpose of
the SEM analyses was to determine the size distribution of the counted fibers. The ISO procedure was
modified to count fibers 5 μm and longer with no minimum width.
ANALYTICAL RESULTS
The average dimensions of the crocidolite fibers
from these tests are shown in Table 1, compared to the
dimensions reported by Beard et al. (10). The fiber di55
mensions presented in Table 1 are limited to fibers and
do not include the dimensions of bundles. As can be
seen in Table 1, there are differences between the three
analyses primarily in the number of bundles counted.
In these tests, between 25% and 40% of the counts were
crocidolite bundles; none were reported by Beard et
al. (10). The dimensions of the fibers are relatively consistent between the three analyses.
Some of the fibers included in Table 1 are thinner
than 0.2 μm (the minimum width generally visible in
the PCM). Removing these very thin fibers from the
count slightly changes the dimensions as shown in
Table 2, which shows the dimensions for fibers, exclusive of bundles. The proportion of particles that were
bundles increased slightly in the sonication (25%–30%)
and aerosolization (42%–47%) data as a result of excluding the particles with widths less than 0.2 μm.
The lengths of the crocidolite fibers are similar in
the three data sets, but there is a slight difference in
the width of the fibers (resulting in a slight difference
in the aspect ratio). These dimensions indicate that the
sample preparation procedure used by Beard et al. (10)
resulted in fibers, with no bundles present in the
sample.
The presence of bundles of fibers is a hallmark
characteristic of asbestos. As an example, Figure 4
shows PCM photographs of crocidolite bundles and
curved crocidolite fibers observed in samples collected
at an operating cement plant where crocidolite was
56
used as a component of the cement product. Harper
instructed the laboratory not to differentiate between
bundles of fibers and single fibers during the TEM characterization of the fibers on the basis that identifying
bundles with PCM is difficult, subjective and judgmental. However, the characterization of particles as
bundles (Bin 1) is a key factor in the D7200 analysis
and should not be discarded if the method is to be properly evaluated. This error minimized the efficacy of
the method to identify populations of asbestiform fibers. Bundles of fibers are well known characteristics
of asbestiform minerals, and eliminating this hallmark
characteristic through both sample preparation and
analytical procedures will significantly bias the resulting data and conclusions.
The distributions of particles from the three rock
samples aerosolized and analyzed using PCM are
shown in Table 3. The D7200 method was slightly
modified to distribute the PCM counts into the bins
by bundles or curved fibers (Bin 1), fibers longer than
10 μm and thinner than 1 μm (Bin 2) and all other
PCM counts (Bin 3). The data shown in Table 3 are the
percentage of fibers observed in each bin and are not
reflective of an air concentration or fiber loading density. Table 3 compares the distribution for samples
that were pulverized to the same degree but were subjected (or not) to sonication and shows that, with the
exception of chrysotile, that sonication reduces the
percentage of fibers that show characteristics of
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
Figure 4. Phase contrast microscopy images of airborne crocidolite asbestos fibers collected at an asbestos cement plant. Bundles of
crocidolite and curved fibers, components of the ASTM D7200 Bin 1, are visible in these photographs.
57
bundles or curvature (Bin 1).
What should be noted about Table 3 is that the
percentage of counts in Bins 1 and 2 for the asbestos
samples (tremolite and chrysotile) are approximately
50% or greater, regardless of the sample preparation
procedure. This suggests that the use of D7200 in mining environments to identify airborne particulate as
potential asbestos fibers is valid.
The size distributions of the samples for particles
5 μm and longer are shown in Tables 4–6. The sizes of
the particles are greatly affected by sonication, with a
reduction in length (less for the tremolite sample) and
width. There is some difference in the aspect ratios of
the minerals when comparing sonicated versus nonsonicated processes, but less of a difference than what
might have been expected when considering the length
and width distributions.
With the exception of the change in length for the
tremolite sample, all of the dimensions were statistically different (p < 0.05) for each sample when evaluated using both parametric (ANOVA) and nonparametric (Mann-Whitney test) analytical procedures.
These data confirm published observations that sonication adversely affects particle size (12).
DISCUSSION
The data from these experiments demonstrate that
the issues related to the pulverization and sonication
of asbestos minerals are complex. Liberation of particles is ongoing throughout all comminution processes,
including sonication. The energy input into a system,
whether as a mechanical force from the grinder or as
an effect of the sonic energy, affects each particle differently. Fibers and/or bundles can be released from complex (locked) particles; bundles can be split partially
or completely into the individual fiber components;
and fibers can be fractured into shorter, thinner, more
numerous particles.
Incorrect sample preparation will result in biased
analytical results. These studies demonstrate:
Sonicating samples destroys the observable asbestos characteristics. Fundamental asbestos characteristics, such as bundling of fibers or long, curved fibers,
are destroyed by sonication. As shown in Table 2, the
percentage of countable bundles or curved fibers (Bin
1) was significantly reduced solely due to sonication.
Sonicating asbestos samples biased the evaluation
of the D7200 analytical method. The effect of sonication has been extensively discussed in the asbestos
analytical literature (12, 28–31), consistently showing
that the use of ultrasonic energy disaggregates bound
58
particles from matrices, splits bundles of fibers longitudinally into separate fibrils and fractures fibers
transversely. Given the existing knowledge base of the
effects of sonication, it is inappropriate to use the sonication technique to prepare samples that would be
used to evaluate the ASTM D7200 procedure. The use
of sonication in preparation of the fiber samples (with
the intent of evaluating the D7200 method) significantly
biases the results.
Sonicating asbestos samples does not alter the
D7200 evaluation as to whether a sample contains a
population of asbestos or not. As shown in Table 2,
the percentage of counts that are used to suggest a
sample of airborne fibers may be asbestos found in
(Bin 1 + Bin 2) is 50% or greater. These tests support
the basic concept of the D7200 method that a population of asbestos fibers will be comprised of bundles
and/or long thin fibers.
This study demonstrates that sample preparation
procedures (grinding and sonication) do affect the asbestos fibers. It also suggests that these procedures can
be used inadvertently to skew results, reinforcing the
concept that standard but appropriate methods be
used for asbestos analyses. These data show that the
choice of material to test for evaluation of analytical
methods will affect the usefulness of the method evaluation. Any material chosen for method evaluation must
be fully and carefully characterized prior to use.
D7200 is a slight modification of standard PCM analytical procedures, requiring binning of the counts. The
observed concentrations are data that can be used for
regulatory compliance documentation. Because of the
binning of the counts, D7200 should provide information on the fraction of counted fibers that may be asbestos. Not all asbestos fibers are observable by PCM
(32), but the concentrations derived using D7200
should direct the user to additional (TEM) analyses and/
or personal protective measures for the employees.
Analytical test methods do need to be evaluated
and updated as new information on the analyte or procedure is obtained. However, these tests should be conducted using appropriately prepared reference materials in order to avoid inherent test bias.
ACKNOWLEDGMENTS
Funding for this study was provided by a grant
from the National Stone Sand & Gravel Association
(NSSGA). NSSGA did not participate in the study design, interpretation of results or in the decision to publish these findings. Samples used in this study were
provided by NSSGA member companies.
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D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
59
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•
Vol. 60:2, pp 51–61 (2012)
Effect of Sample Preparation on Observed Airborne
Fiber Characteristics
D.R. Van Orden, M. Sanchez and J.M. Wilmoth
RJ Lee Group, Inc.*
KEYWORDS
Asbestos, ASTM D7200, chrysotile, concentration,
crocidolite, grinding, NIST SRM 1866, phase contrast
microscopy (PCM), scanning electron microscopy
(SEM), sonication, transmission electron microscopy
(TEM), tremolite
ABSTRACT
Natural occurrences of asbestos and potential exposures to such materials have drawn significant public and regulatory interest in recent years. Proposed
revisions to a published analytical method (ASTM
D7200) that is designed to monitor airborne fiber concentrations are based on data developed using
inappropriate sample preparation strategies, including over-grinding the samples and the use of
ultrasonication. Tests demonstrating the effects of
grinding and preparation of asbestos minerals have
been conducted. Sample preparation procedures used
by some laboratories destroy the characteristics of
asbestos, thereby limiting the usefulness of ASTM
D7200 and similar analytical methods. The effects of
sample preparation on the morphology of particles
must be considered when creating or revising asbestos analytical procedures.
INTRODUCTION
When rocks that contain veins or pockets with
natural occurrences of asbestos are crushed, there is a
possibility that asbestos fibers will be released and
become airborne. If the airborne asbestos is of respirable size, these fibers pose a potential hazard to workers and bystanders who may breathe them.
Fortunately, minerals rarely grow as asbestos fibers, but more generally grow as (comparatively)
larger crystals that do not possess the characteristics
of asbestisform fibers. Unfortunately, when these nonasbestos minerals are fractured, many will fracture as
elongated particles that have dimensions that are encompassed by various regulatory analytical procedures. For example, OSHA (29 CFR §1910.1001) and
MSHA (30 CFR §56.5001) regulate airborne asbestos
fibers if they are at least 5 μm long, have a minimum
aspect ratio (length:width) of 3:1 and are visible in an
optical microscope. Fractured non-asbestos minerals
that frequently occur with asbestos are routinely observed with these dimensions even though they are
not asbestos.
Therefore, it is necessary to be able to analyze
samples to determine the amount of asbestos (independent of any non-asbestos mineral) in the samples.
ASTM D7200 (1) is an optical microscopy procedure
for analyzing airborne particles from mines, quarries
and construction activities to determine which
samples need additional analyses using electron microscopy to determine compliance with MSHA and
OSHA worker exposure regulations. ASTM D7200 provides an index used to estimate whether the population of counted fibers are potentially asbestos.
D7200 was originally developed in the late 1980s
and was round-robin tested at that time using filters
*350 Hochberg Road, Monroeville, PA 15146
51
collected at operating mines and quarries. The method
and the test results were produced during testimony
at an OSHA hearing in 1990 (2). The method was
adopted, with slight modification, by ASTM in 2006
after several years of consideration.
ASTM D7200 is an analytical method for airborne
particulate collected on filters. The method involves a
standard phase contrast microscopy (PCM) analysis
that then divides counted elongated particulate (minimum 3:1 aspect ratio, 5 μm or longer) into three bins:
one that includes obvious bundles and/or curved fibers, a second that includes long (> 10 μm), thin (high
aspect ratio) fibers, and a third bin for the remainder
of the counts. If the D7200 concentration of countable
fibers exceeds one half of the current regulatory limit
and the number of fibers reported in the first two bins
exceed 50% of the total count, the method strongly recommends the mine owner/operator to take additional
analytical measures to determine if the fibers are asbestos. If no further analyses are conducted, the fibers
should be considered to be asbestos. This trigger at
one-half the PEL and 50% of the counts in the first two
bins serves as a safety factor. Regardless of the distribution of counts in the bins, the reported concentration retains comparability to current regulatory levels and to historical PCM analyses. The binning process is a form of differential counting that is based on
published dimensional and crystal growth characteristics described in the literature (3–5).
Recently, Harper et al. published a paper (6) discussing the size characteristics of asbestos and nonasbestos amphibole particles that mischaracterizes
ASTM D7200 by suggesting that the method indicates
that particle dimensions (lengths and widths) are used
to positively identify asbestos fibers and to classify all
remaining particles as non-asbestos particles. It is important to understand, however, that D7200 is expressly
intended to be a screening tool designed to encourage
mine and quarry operators to be proactive in monitoring their employees for potential asbestos exposure. If
such a potential is indicated through the screening approach, additional analyses that are more complex, time
consuming and costly are required for positive identification of asbestos (see step 1.6 in the method).
The testing reported by Harper, et al. (6) to address the reliability of these bins as a screening for
potential asbestos fibers (avoidance of false negatives
in particular) is also misleading, in part, due to the
inappropriate sample preparation procedures used to
generate the test fibers. In their test of D7200 to discriminate asbestos populations from non-asbestos
populations, Harper tested a number of samples that
52
could be classified as either asbestos or non-asbestos
on the basis of their appearance in hand samples. However, the purity of these specimens is unclear, and the
value of their work would be enhanced if the originating sample composition was fully characterized. Several standard reference materials for amphibole asbestos used in the Harper work are known to contain
considerable non-asbestiform fragments (7–9).
In the Harper paper, the mineral samples were
heavily processed to optimize the number of the 3:1
aspect ratio and particles longer than 5 μm, resulting
in powders whose particles had widths of generally
< 3 μm (10). Their general sample preparation procedure is shown in the flowchart in Figure 1. Part of
their sample preparation procedure used an ultrasonic
disruptor to further disaggregate the crushed particles. It is not clear from the publication what size
particles were subjected to ultrasonic treatment,
though Harper et al. (11) provides additional description of the sample preparation procedures in a second
paper. The mineral samples classified by Harper as
“asbestos” were not subjected to the initial crushing
step shown in Figure 1.
In regard to asbestos, the sample processing procedure used by Harper may well have destroyed the
asbestiform nature of the asbestos test minerals. Using ultrasonic energy to process asbestos fibers is
known to separate fiber bundles into the individual
fibers and to comminute the fibers into shorter fibers,
thus reducing the apparent aspect ratio of the asbestos fibers (12). This sonication effect is acknowledged
in other ASTM asbestos procedures that utilize
ultrasonication (13).
It is not known that particles prepared in the manner described by Harper will be equivalent to particles
observed in mining environments. Certainly there was
no attempt to collect airborne samples from operating
asbestos mines (either amphibole or chrysotile) to use
for comparison with the comminuted samples. It is
known that varying manufacturing processes produce
significant variations in fiber dimensions (14–17). In
particular, sonication has been shown to affect particle dimensions (12, 18). It has also been suggested
that the fibrosity index in bulk samples can be correlated with airborne data (19), although there are differences between bulk and airborne-size distributions
(20), and between different analytical procedures on
the same filter (21). The dimensions of asbestos fibers
have been shown to play an important role in exposure classifications (22, 23), hence the need to create an
appropriate analytical tool to screen for this potential.
The samples that Harper relied on as asbestos do
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
Figure 1. This flowchart of the sample preparation procedures was used by Harper et al. (6) in preparing amphibole samples for analyses.
not have the size characteristics of asbestos. The contract laboratory that produced the samples, reported
by Harper, evaluated some of the fiber samples using
transmission electron microscopy (TEM). A plot of
width versus aspect ratio (Figure 2) indicates that only
a small fraction of the fibers (16%) meet the minimum
aspect ratio of 20:1 recommended by the EPA (24). Of
the five minerals classified as asbestos, only the crocidolite sample contained more than 50% of fibers with
an aspect ratio greater than 20:1 (77%); the other
samples were 26% (amosite) or fewer than 3% (actinolite asbestos, tremolite asbestos and anthophyllite
asbestos).
Therefore, if the sample preparation procedures
affected the morphological and size characteristics of
the minerals used in his tests, Harper’s conclusions
about the efficacy of various test methods are not valid.
Analytical procedures need to be tested and modified
as new information about a program or analyte be-
comes known, and certainly the D7200 method should
be tested to confirm its performance. However, the tests
should be conducted with a clear understanding of the
effect of sample preparation on the reference test minerals. Only with this understanding can tests be properly designed and the results interpreted.
EXPERIMENTAL PLAN
Crocidolite from the NIST SRM 1866 standard was
examined in order to provide a direct comparison to
the samples used by Harper. Crocidolite from NIST
SRM 1866 was first placed in a beaker of distilled water and sonicated for three minutes using a 100 W ultrasonic bath. At the completion of the sonication, the
suspension was thoroughly mixed and allowed to settle
for 30 minutes. At that time, an aliquot of the remaining suspension was removed, deposited onto a polycarbonate filter (0.2 μm pore) and mounted on a car-
53
Figure 2. The graph shows the distribution of the width and aspect ratio of the amphibole asbestos samples from Harper et al. (6) Most of
the samples have aspect ratios below 20:1. The red box shows the area that Chatfield (7) has proposed for defining a particle as asbestos.
bon planchette for analysis by scanning electron microscopy (SEM).
In a second procedure, crocidolite from NIST SRM
1866 was placed in an elutriation chamber and aerosolized using compressed air. Air filters (MCE, 0.8 μm
pore) were placed at the top of the chamber and were
used to collect samples of the airborne dust at a sampling rate of 2 lpm. At the end of the sampling, a portion of the filter was excised and mounted on a carbon
planchette for analysis by SEM.
The prepared crocidolite samples were examined
with a JEOL 6460 LV SEM at magnifications ranging
from 1,000x to 5,000x. Images of fields of particles were
recorded for offline measurements of approximately
650 particles. The counted particles were limited to
those longer than 5 μm with a minimum aspect ratio
of 3:1. Particles that were obvious bundles were la-
54
beled so their dimensions could be evaluated separately
from the other fibers.
Additional tests were conducted to demonstrate
the effect of grinding and sonication on the size and
morphology of amphibole minerals. An experiment
was designed to demonstrate the effect of sample
preparation procedures, specifically sonication, on
crushed rock samples. Three samples, shown in
Figure 3, were obtained and used in the testing. Each
sample was 50 mm–75 mm, which is a typical size of
railroad ballast or rock that would be used as base
material for road building. Each sample contained a
1
Byssolite is a fibrous form of amphibole minerals that has low
tensile strength and moderate fiber widths. A portion of the fibers
will show crystal faces. Byssolite fibers are not asbestiform fibers
and they are not asbestos.
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
3A
3B
3C
Figure 3. Rock samples used in the test program to demonstrate the effect of sample preparation procedures, specifically
sonication, on crushed rock: 3A, pieces of rock containing tremolite asbestos that were taken from a drill core; 3B, a
sample that contains actinolite (byssolite), which was also obtained from a drill core; and 3C, two pieces of chrysotile ore
from the mine in Lowell, Vermont.
vein or veins of visibly fibrous minerals that were
identified as the amphibole minerals tremolite asbestos, actinolite (byssolite1) or the serpentine mineral
chrysotile.
Each sample was stage ground so that the product
size was generally finer than 150 μm (100 mesh) (25).
The ground samples were riffled into equal parts.
The effect of sonication was examined by taking
one portion of each ground sample and sonicating it
using a Biologics Ultrasonic Homogenizer (Model 150
V/T) equipped with a titanium tip (0.5 inch). The pulverized material was suspended in approximately
100 ml of deionized water and then sonicated for 15
minutes, the same time used by Harper (6) and Beard
(10). Aliquots of the sonicated material were removed
from the suspension at the completion of the sonication (no settling) and filtered onto a new mixed cellulose ester filter (0.2 μm pore size).
The other portion of each sample was aerosolized
in a glove box using small fans; the airborne particles
were collected onto filters in general accordance with
NIOSH 7400 (26). While aerosolized, some of the
coarser particles did not remain airborne, therefore,
these aerosolized fractions represent a subset of the
entire sample.
Each collected filter was analyzed in general accordance with PCM (ASTM D7200 [1]) and SEM (ISO
14966 [27]). For the PCM analyses, the definition of
Bin 2 was modified to include fibers longer than 10
μm and thinner than 1 μm. The primary purpose of
the SEM analyses was to determine the size distribution of the counted fibers. The ISO procedure was
modified to count fibers 5 μm and longer with no minimum width.
ANALYTICAL RESULTS
The average dimensions of the crocidolite fibers
from these tests are shown in Table 1, compared to the
dimensions reported by Beard et al. (10). The fiber di55
mensions presented in Table 1 are limited to fibers and
do not include the dimensions of bundles. As can be
seen in Table 1, there are differences between the three
analyses primarily in the number of bundles counted.
In these tests, between 25% and 40% of the counts were
crocidolite bundles; none were reported by Beard et
al. (10). The dimensions of the fibers are relatively consistent between the three analyses.
Some of the fibers included in Table 1 are thinner
than 0.2 μm (the minimum width generally visible in
the PCM). Removing these very thin fibers from the
count slightly changes the dimensions as shown in
Table 2, which shows the dimensions for fibers, exclusive of bundles. The proportion of particles that were
bundles increased slightly in the sonication (25%–30%)
and aerosolization (42%–47%) data as a result of excluding the particles with widths less than 0.2 μm.
The lengths of the crocidolite fibers are similar in
the three data sets, but there is a slight difference in
the width of the fibers (resulting in a slight difference
in the aspect ratio). These dimensions indicate that the
sample preparation procedure used by Beard et al. (10)
resulted in fibers, with no bundles present in the
sample.
The presence of bundles of fibers is a hallmark
characteristic of asbestos. As an example, Figure 4
shows PCM photographs of crocidolite bundles and
curved crocidolite fibers observed in samples collected
at an operating cement plant where crocidolite was
56
used as a component of the cement product. Harper
instructed the laboratory not to differentiate between
bundles of fibers and single fibers during the TEM characterization of the fibers on the basis that identifying
bundles with PCM is difficult, subjective and judgmental. However, the characterization of particles as
bundles (Bin 1) is a key factor in the D7200 analysis
and should not be discarded if the method is to be properly evaluated. This error minimized the efficacy of
the method to identify populations of asbestiform fibers. Bundles of fibers are well known characteristics
of asbestiform minerals, and eliminating this hallmark
characteristic through both sample preparation and
analytical procedures will significantly bias the resulting data and conclusions.
The distributions of particles from the three rock
samples aerosolized and analyzed using PCM are
shown in Table 3. The D7200 method was slightly
modified to distribute the PCM counts into the bins
by bundles or curved fibers (Bin 1), fibers longer than
10 μm and thinner than 1 μm (Bin 2) and all other
PCM counts (Bin 3). The data shown in Table 3 are the
percentage of fibers observed in each bin and are not
reflective of an air concentration or fiber loading density. Table 3 compares the distribution for samples
that were pulverized to the same degree but were subjected (or not) to sonication and shows that, with the
exception of chrysotile, that sonication reduces the
percentage of fibers that show characteristics of
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
Figure 4. Phase contrast microscopy images of airborne crocidolite asbestos fibers collected at an asbestos cement plant. Bundles of
crocidolite and curved fibers, components of the ASTM D7200 Bin 1, are visible in these photographs.
57
bundles or curvature (Bin 1).
What should be noted about Table 3 is that the
percentage of counts in Bins 1 and 2 for the asbestos
samples (tremolite and chrysotile) are approximately
50% or greater, regardless of the sample preparation
procedure. This suggests that the use of D7200 in mining environments to identify airborne particulate as
potential asbestos fibers is valid.
The size distributions of the samples for particles
5 μm and longer are shown in Tables 4–6. The sizes of
the particles are greatly affected by sonication, with a
reduction in length (less for the tremolite sample) and
width. There is some difference in the aspect ratios of
the minerals when comparing sonicated versus nonsonicated processes, but less of a difference than what
might have been expected when considering the length
and width distributions.
With the exception of the change in length for the
tremolite sample, all of the dimensions were statistically different (p < 0.05) for each sample when evaluated using both parametric (ANOVA) and nonparametric (Mann-Whitney test) analytical procedures.
These data confirm published observations that sonication adversely affects particle size (12).
DISCUSSION
The data from these experiments demonstrate that
the issues related to the pulverization and sonication
of asbestos minerals are complex. Liberation of particles is ongoing throughout all comminution processes,
including sonication. The energy input into a system,
whether as a mechanical force from the grinder or as
an effect of the sonic energy, affects each particle differently. Fibers and/or bundles can be released from complex (locked) particles; bundles can be split partially
or completely into the individual fiber components;
and fibers can be fractured into shorter, thinner, more
numerous particles.
Incorrect sample preparation will result in biased
analytical results. These studies demonstrate:
Sonicating samples destroys the observable asbestos characteristics. Fundamental asbestos characteristics, such as bundling of fibers or long, curved fibers,
are destroyed by sonication. As shown in Table 2, the
percentage of countable bundles or curved fibers (Bin
1) was significantly reduced solely due to sonication.
Sonicating asbestos samples biased the evaluation
of the D7200 analytical method. The effect of sonication has been extensively discussed in the asbestos
analytical literature (12, 28–31), consistently showing
that the use of ultrasonic energy disaggregates bound
58
particles from matrices, splits bundles of fibers longitudinally into separate fibrils and fractures fibers
transversely. Given the existing knowledge base of the
effects of sonication, it is inappropriate to use the sonication technique to prepare samples that would be
used to evaluate the ASTM D7200 procedure. The use
of sonication in preparation of the fiber samples (with
the intent of evaluating the D7200 method) significantly
biases the results.
Sonicating asbestos samples does not alter the
D7200 evaluation as to whether a sample contains a
population of asbestos or not. As shown in Table 2,
the percentage of counts that are used to suggest a
sample of airborne fibers may be asbestos found in
(Bin 1 + Bin 2) is 50% or greater. These tests support
the basic concept of the D7200 method that a population of asbestos fibers will be comprised of bundles
and/or long thin fibers.
This study demonstrates that sample preparation
procedures (grinding and sonication) do affect the asbestos fibers. It also suggests that these procedures can
be used inadvertently to skew results, reinforcing the
concept that standard but appropriate methods be
used for asbestos analyses. These data show that the
choice of material to test for evaluation of analytical
methods will affect the usefulness of the method evaluation. Any material chosen for method evaluation must
be fully and carefully characterized prior to use.
D7200 is a slight modification of standard PCM analytical procedures, requiring binning of the counts. The
observed concentrations are data that can be used for
regulatory compliance documentation. Because of the
binning of the counts, D7200 should provide information on the fraction of counted fibers that may be asbestos. Not all asbestos fibers are observable by PCM
(32), but the concentrations derived using D7200
should direct the user to additional (TEM) analyses and/
or personal protective measures for the employees.
Analytical test methods do need to be evaluated
and updated as new information on the analyte or procedure is obtained. However, these tests should be conducted using appropriately prepared reference materials in order to avoid inherent test bias.
ACKNOWLEDGMENTS
Funding for this study was provided by a grant
from the National Stone Sand & Gravel Association
(NSSGA). NSSGA did not participate in the study design, interpretation of results or in the decision to publish these findings. Samples used in this study were
provided by NSSGA member companies.
THE MICROSCOPE 60 (2012)
D.R. VAN ORDEN, M. SANCHEZ and J.M. WILMOTH
59
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