Am. xcup. Hyf , Vol. « , No 2, pp 197-209, 1996
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A QUANTITATIVE METHOD USING A LIGHT MICROSCOPE
ON-LINE TO A MACINTOSH COMPUTER FOR THE
ANALYSIS OF TREMOLITE FIBRES IN DOLOMITE
Lennart Lundgren,* Sten Lundstrom.f Gunnel Sundstrom,* Gunnel Bergman*
and Staffan Krantz*
•Aerosol Division and tVentilation Division, National Institute of Occupational Health,
S-171 84 Solna, Sweden
{Received in final form 11 April 1995)
Abstract—A modern method for quantitative determination of tremolite fibres in dolomite has
been developed which uses light polarizing microscopy with phase-contrast equipment and
evaluation on-line to a Macintosh computer. The principle is mass calculation based on fibre
dimensions measured in the microscope with the help of a programmable multimedia program. The
sample is leached in acid, gently crushed and sieved before a small portion is deposited on a
cellulose nitrate membrane filter The filter is made transparent with cinnamaldehyde on a
microscope slide. The tremolite fibres will be coloured blue with a red to yellow halo when viewed
under phase-contrast and thus easy to identify. An ordinary Macintosh computer with special
video display cards and software, which enables the microscopist to perform on-line measurements
of the fibre dimensions on a display with the help of a mouse, is used. The method does not
differentiate between asbestiform and non-asbestifonn tremolite. AU particles/fibres of tremolite
with an aspect ratio of > 5:1 are measured. The method is very easy to use and the microscope as
well as the computer hardware and software are not unusual. Copyright © 1996 British
Occupational Hygiene Society. Published by Elsevier Science Ltd.
INTRODUCTION
The need to regulate the asbestos contaminant level in bulk materials is considered to
be of great importance. Sweden already has such a regulation. If a material contains
more than 1 % w/w of asbestos, special rules and precautions have to be applied
(AFS, 1992). The 1% quantitative limit needs reliable and extremely sensitive
methods. In the European Community the directive for classification and labelling of
hazardous materials has been extended with concentration limits for which mixtures
or contaminated products have to be labelled (EEC, 1967). This directive has not set
any limit for asbestos. This is owing to the absence of a suitable method to identify
and quantify low levels of asbestos. Therefore the EC has initiated a joint BCR
research project for the development of methods for the determination of low
concentrations of fibres in bulk materials (EEC, 1993) indicating the importance of
reliable methods for this kind of analysis.
For the analysis of tremolite asbestos contamination in dolomite or other
calcareous products no easy and reliable method has yet been published. Such an
analysis normally demands highly skilled operators and extremely expensive
analytical techniques such as electron microscopy or X-ray diffraction (Beamen
and File, 1976; Habermann and Tavakkoli, 1992; Ruud, 1978; Svingen, 1991). Each
of these techniques has limitations. X-ray diffraction is size dependent as well as
being unable to distinguish between fibrous forms and non-fibrous forms, and
197
198
L. Lundgren et at.
electron microscopy may have problems with the combined examining of extremely
large and small fibres simultaneously. These techniques are also extremely time
consuming and expensive.
The concentration of a fibre in weight per cent could be estimated by mass
calculation based on the measurement of fibre dimensions in a microscope, as was
done by Pooley and Clark (1979). If the number of fibres are to be evaluated or if the
fibres in a sample are extremely thin, electron microscopy might be the only choice
since the light microscope does not allow these smaller fibres to be detected.
However, if the concentration in weight per cent of asbestos is of main interest, light
microscopy can in some circumstances be used for a mixture/material where coarser
fibres do occur. This is possible since thin fibres only marginally contribute to the
total weight of fibres where coarser ones occur. If the analysis of such a sample could
be done with a conventional light microscope connected on-line to a personal
computer, it would be a very interesting alternative for the occupational chemists.
In this paper such an approach for the quantitative measurement of tremolite
fibres in dolomite is described (Lundgren et ai., 1993). The principle is mass
calculation based on fibre sizes measured in a light microscope connected on-line to a
Macintosh computer. The same type of equipment but with modification in the
software has recently been described for the measurements of airborne fibres on
membrane filters (Lundgren et al., 1995).
MATERIALS AND METHODS
Materials
Five dolomite samples of a Nordic origin with different levels of tremolite
contamination have been evaluated. They were all free from interfering fibres such as
anthophyllite.
General outline of the method
The sample preparation of the dolomite powder involves acid treatment, a gently
grinding and sieving practice before a small and known portion is deposited on a
pre-weighed cellulose nitrate membrane filter. The filter is made transparent with
cinnamaldehyde. When using phase-contrast in the polarizing microscope, fibres
with a refractive index of 1.62 will be seen as blue with a red to yellow halo (Berglund
and Lundgren, 1988). Anisotropy as well as other optical parameters for fibres can
be controlled during the examination (McCrone, 1985). By this it is possible to
selectively distinguish tremolite fibres from other fibres in the sample. Only fibres
longer than 5 /xm with an aspect ratio >5:1 are being measured. The measurement is
also performed at two different magnifications.
The microscope is connected to a Macintosh computer. The computer has special
video display cards and software which,makes it possible to on-line measure the
length and the diameter of a fibre.
Sample preparation, part I
Figure 1 describes schematically the preparation of a sample.
On-line analysis of trcmolite fibres
199
-lkg of powder
Sample splitting
1
50-100g
Acid leaching
Grinding
Sieving
I
Sample preparation, part II
Fig. 1. Sample preparation, part I
Approximately 1 kg of the bulk samples is truly representative, reduced to 50100 g with a sample splitter. The particle size of the original sample should be smaller
than 2 mm owing to great difficulties with the handhng of and the preparation with
coarse particles.
The split portion was dried (105°Q and weighed. The acid treatment is
performed with 2 M HC1 in a conical flask on a water bath at 95°C for 1 h or as long
as any CO2-formation could be seen. The flask was shaken vigorously on several
occasions.
The residue is filtered on a pre-weighed 47 mm membrane filter (pore size 0.8 fim,
regenerated cellulose) and is thoroughly washed with distilled water. The filter is
dried and weighed. The remaining quantity in per cent (Ca) gives the concentration
of the acid residue.
The residue is sieved through a 100 fim sieve. A small sieve (diameter less then
10 cm) is used to minimize losses in the sieve cloth. The coarse fraction is gently
reduced in size under acetone with a pestle in an agate mortar for only a few minutes.
This procedure is repeated up to 4 or 5 times. After each round of sieving, the weight
is determined to indicate how the grinding has been carried out.
For one of the samples, a simple test to determine the influence of grinding is
performed. The consequence of different forces on the pestle and the use of a
tungsten carbide ball micro-pulverizer has been investigated.
200
L Lundgren el at.
Sample preparation, part II
Figure 2 describes schematically the sample preparation for the microscopical
evaluation.
Pre-analysis. Before a quantitative determination of the tremolite fibre
concentration of this kind is done, a pre-analysis must be performed. This is carried
out on the acid-treated and sieved portion of a sample. Different available techniques
and analytical equipment like polarizing light microscopy (PLM), X-ray diffraction
(XRD) and electron microscopy (EM) could be used.
If the sample contains fibres of anthophyllite or fibres of wollastonite, then
polarizing filters are required during the fibre measurement.
It must be remembered that this pre-analysis is extremely important for the
following analysis. If there are any difficulties or problems in this qualitative analysis
for tremolite asbestos the suggested method might not be suitable. A clear
identification of the asbestos type of the sample must be performed prior to the
analysis. Any changes or other factors caused by the acid treatment or the grinding
of the fibres in each sample must be thoroughly investigated before the actual
analysis can be performed.
If all the tremolite fibres in a sample are very thin (thinner than 0.5-1 ^m) this
proposed technique is not suitable because of the limitations with light microscopy.
Filter and slide preparation. A number of 37-mm membrane filters of cellulose
nitrate (pore size 0.8 /un) was prepared for each sample. A pre-washing procedure is
necessary since the purpose is to determine the actual weight of the deposit on these
filters. The filters are dried and weighed on a micro weigh with an accuracy of 1 ^g.
Since cellulose nitrate filters are hygroscopic as well as easily charged, a consistent
procedure when weighing is necessary. A reading after exactly 20 s was quite
successful. Dissipating static charges with alpha particles during weighing is also
necessary.
Each of three different subsamples (up to 2000 pg can be used) is dispersed in a
mixture of ethanol and distilled water (10% ethanol). The subsamples are shaken
vigorously and filtered slowly. It is not advisable to rinse along the sides of the filter
holder. The remaining filters are used as blanks and filtered with the alcoholic
mixture only.
Preanalysis
XRD PLM EM
Filter preparation
i,
Slide preparation
Microscopical evaluation
Fig. 2. Sample preparation, part II.
On-line analysis of tremolite fibres
201
The filters are dried and weighed together with blank filters. In this way, it is
possible to achieve a sufficient precision of the actual amount on the filter (mp). The
two weighing procedures are performed during the same day.
A quarter of each membrane filter is carefully cut with a scalpel blade. The filter
piece is placed, particles uppermost, on five drops of cinnamaldehyde (nD = 1.623)
on a clean microscope slide and covered with a clean cover slip. The filter piece is
completely transparent in 1 h and ready for examination. The slides are evaluated
immediately or at least during the same day.
Which of the three slides to use depends on the density of the deposit, on the
number of tremolite fibres present and on the homogeneity of the slide. One or two
tremolite fibres in one viewing field is a good choice. If there is any visible disorder of
particles/fibres, the slide is rejected.
Microscopical evaluation
Figures 3 and 4 describe and show the microscope and the Macintosh set-up.
Microscope and camera. A polarizing light microscope (Leitz Ortholux II) with
phase-contrast equipments is used. The microscope has two objectives on a revolver
with magnification of x 10 and x40 and has an internal magnification lens in the
tube of x 1.25. The numerical aperture of the objectives is 0.75 (x40) and 0.25
(xlO). The total magnification on the colour display is x l l 6 0 and x415,
respectively. If halogen illumination is used, the colour of the fibres could be slightly
enhanced with a colour compensating filter beneath the condenser, for instance a
Kodak CC10M filter.
The microscopic area viewed on the colour display is almost quadratic and
0.015 mm 2 ( x 40) and 0.25 mm 2 (x 10) in size.
Since the samples in this study are free of interfering fibres, such as anthophyllite
or wollastonite, no polarizing filters have been used. If these fibres were expected to
be present in a sample, one or two polarizing filters (one in the tube and one beneath
or in the condenser) must be used during the examination. In this way, the angle of
Printer
Network
Camera
Polarizing
microscope
with
phase contrast
equipment
Objective
10X
40X
Graphic card
DVA-4000
MicroMind
Director
Microsoft EXCEL
Other programmes
Macintosh computer
Fig. 3. Microscope and computer set-up.
Colour display
17"
202
L. Lundgren et at.
extinction and the sign of elongation of an anisotropic fibre can be checked and
determined. This requires a rotating microscopic stage and a compensator.
The camera is a Panasonic 2/3' 1-chip CCD camera, type WV-CD130/G with a
composite PAL video output signal.
Computer hard- and software. A Macintosh Quadra 700 with 8 MB primary
(RAM) memory and 150 MB secondary (hard disk) memory is used. The computer
is equipped with a MIC System II multimedia system (VideoLogic Ltd). This system
consists of two cards, a DVA-4000 and a 8-bit Graphic card. The cards are internally
connected and are working together. The system has a picture resolution of
640 x 480 pixels with an 8 bit per pixel colour resolution (256 colours).
The display is a multimode 17" colour display from SuperMatch, model CDS423V.
The application software used is MacroMind Director Version 3.0, an
inexpensive multimedia program. MacroMind Director is easily programmed with
script files (text instructions) and has two layers of graphics on the display, the
microscope picture live in the background and the computer graphics in the
foreground. The microscope picture is substituted against one of the different
graphics colours, which is set in the script. The size and the colour for up to 24
rectangular sprites ('movable markers') have been defined in the script. These sprites
can be placed anywhere on the screen with a mouse. When four sprites are placed,
the program calculates the distance between each two pair of sprites, for example the
length and the diameter of the fibre is being measured. One sprite is left to mark the
fibre. Data for fibres/particles with an aspect ratio less than 5:1 are not stored and
the remark "This is not a fibre!" is shown on the screen.
The data (the number offibres,the number offieldsexamined, the length and the
diameter of each fibre, the aspect ratio and comments on certain fibres) stored with
the application software are copied and transferred to a pre-programmed EXCELdocument (Microsoft EXCEL Version 4.0), where all the final calculations are
performed. This document could be used for other calculations further on.
Fibre measurements. Two separate microscopical examinations are used. With the
objective (x 10), only fibres longer than 50 fim are measured. With the objective
(x 40), all fibres are measured and stored but only fibres between 5 and 50 fim in
length are used in the calculations. By doing this it is also possible to evaluate the
result for the x 40 magnification alone.
With the objective (x 10) almost the whole quarter of the filter is scanned and
evaluated. More than 300 fibres are often needed. At the higher magnification
[objective (x 40)], a practical limit of at least 200 has been used. The necessary
number of fibres depends on the homogeneity of tremolite fibres in the sample but
should be large. Occasionally up to 500 fibres have been measured.
It is also important that any remarks of the morphology of the fibres observed is
recorded. Notes on the aspect ratio, whether the edges of the fibres are parallel,
whether there are fibre bundles present, and so on will give valuable information
about the sample.
Calculations. The mass of the measured tremolite fibre (mf) is calculated using the
relationship
m{=ld2p
(1)
On-line analysis of tremolite fibres
Fig. 4. The microscope and computer set-up.
.183:
On-line analysis of tremobte
fibres
205
where / is the length, d is the diameter of the fibre and p the specific gravity (we have
used p = 2.9 g cm" 3 ) for tremolite. With this, we assume that the height of the fibre is
the same as the diameter we observe in the microscope. The weight (mtf) of all
tremolite fibres on the membrane filter are calculated from
where n is the number of fields examined, and A{ and Am are the exposed area of the
filter and the area of the microscope field, respectively.
The total weight of all tremolite fibres on the filter is the sum from the two
separate examinations at different magnifications (mt).
The concentration of tremolite fibres in the original sample (Ct) expressed in
per cent are calculated from
^ - ^ ,
(3)
where mp is the total weight of the sample on the filter and C a is the concentration of
the acid residue.
Since the data (the number of fibres, the number of fields examined, the length
and the diameter of each fibre, the aspect ratio and comments on some fibres) are
stored as a database in an EXCEL-document, they could be used for further
calculations. For instance, the concentration of respirable tremolite fibres (fibres
with a diameter of less than 3 Aim), and also fibres with other aspect ratios >5 :1,
could easily be obtained and calculated. In this study this has been done for the
aspect ratio >10:l. The number of fibres (observations) are, of course, less if only a
part of the original data are used. The mean fibre sizes are also calculated with a
statistical program.
An important feature in this kind of analysis is a graphical presentation of the
result—which shows how the weight of tremolite fibres on the filter may alter
depending on the number of fibres being measured. In this study this has been done
after the actual examinations and not while actually measuring the fibres.
RESULTS
The results from the analysis of the different dolomite samples are shown in
Table 1. The concentration of tremolite fibres is given in weight per cent. The
arithmetic mean, the deviation as the coefficient of variation (CV) and the number of
investigations on each sample can be seen. The corresponding data if only one
magnification ( x 40) is used are also shown.
A graphic presentation of how the weight of tremolite fibres varies with the
number of fibres for sample No. 5 is shown in Fig. 5. The plot to the left describes the
weight of fibres longer than 50 /im ( x 10) and the plot to the right fibres of length
between 5 and 50 pan ( x 40). Each dot represents a measured fibre.
The influence of grinding on the tremolite concentration has been studied for
sample No. 1 (Table 2). The sample has been reduced in size prior to sieving in
206
L Lundgren et a!.
Table 1. The concentration of fibrous tremolite in dolomite (aspect ratio >5 1)
Mean
Objective magnification
N
0.25
0.25
0.059
0.083
0.021
0 023
0.012
0013
0 70
0.72
x40
x 10/x40
x40
x 10/x40
x40
x 10/x40
x40
x 10/x40
x40
x 10/x 40
0
CV
Sample No.
50 100 150 200 250 300 350
0
34
84
51
1
18
17
47
15
53
29
50
Number of fibres
100 150 200 250
Number of fibres
Fig. 5. The weight of tremolite fibres on a filter, sample No. 5.
Table 2. The tremolite concentration with different grinding procedures, sample
No. 1
Mean
CV
Grinding condition
Gentle grinding*
Normal grinding*
Aggressive grinding*
Very aggressive grinding*
5 min in a ball pulverizerf
30 min in a ball pulverizerf
N
0.24
0.25
0.15
0.097
0.063
0.023
33
44
—
—
7.9
—
3
3
1
1
2
1
*In an agate mortar.
fin a tungsten carbide ball micro-pulverizer.
different ways. Four different conditions (different force on the pestle) in an agate
mortar and two different settings of a ball micro-pulverizer have been evaluated.
Some other calculations can be seen in Tables 3 and 4. In Table 3, the
concentration of tremolite fibres with the aspect ratio of > 10:1 is shown. In Table 4
the average geometric length and average geometric diameter together with the
standard deviations are listed.
On-line analysis of tremolite
fibres
207
Table 3. The concentration of fibrous tremolite in dolomite (aspect
ratio >10:l)
Sample No.
Mean
(%)
CV
(%)
N
1
2
3
4
5
0.07
0.03
0.004
0.006
0.1
11
23
15
52
49
5
2
3
4
7
Table 4. Fibre statistics (geometric values)
Sample No.
1
2
3
4
5
Mean length
(/an)
SD
Mean diameter
(/an)
SD
13.9
13.6
14.3
19.1
16.1
1.7
17
1.9
1.6
1.8
1.7
1.4
1 6
23
2.0
1.7
1.7
1.7
1.6
1.8
DISCUSSION
A modern method for quantitative determination of tremolite fibres in dolomite
has been developed which uses light polarizing microscopy with phase-contrast
equipment and evaluation on-line to a Macintosh computer. The sample is treated
with acid, gently crushed and sieved before a small portion of the sample is deposited
on a membrane filter of cellulose nitrate. This filter is made transparent with
cinnamaldehyde. The mass of the blue tremolite fibres are calculated by measuring
the length and width and knowing the specific gravity of the fibre.
All five samples in this study contained measurable amounts of tremolite fibres.
The concentration varied between 0.013 and 0.72 weight per cent (Table 1).
Working with two magnifications (with objective magnification of x 10 and
x 40) increases the precision considerably, see Table 1.
The sieving and grinding practice might cause problems if the grinding process is
too aggressive, as can be seen in Table 2. It is therefore essential that a gentle and
stepwise grinding-sieving procedure in a mortar, as suggested, is used. There has
been no indication that this gentle grinding procedure could to any greater extent
reduce the fibres below the detection limit of the light microscope. Of course, with a
more aggressive procedure this might be the case.
The computer used is an ordinary Macintosh computer. The Macintosh concept
makes this technique very user-friendly and does not demand an extremely skilled
programmer or operator. Since the actual measurement is done with the help of a
mouse on a large screen instead of in the microscope, the technique makes tedious
microscopic investigation much easier: The application software (Director 3.0) is
also easy to use and to program.
A graphical presentation of the result is a useful tool when determining how
many fibres orfieldswill be necessary to examine. At present this is not done on-line
but hopefully might be implemented in future programs.
208
L. Lundgren et at.
The method does not differentiate between asbestiform or non-asbestiform
tremolite. All particles/fibres of tremolite with an aspect ratio of greater than 5 are
measured. As can be seen from this study, there is quite a difference in result between
the aspect ratios >5 :1 and >10:l (see Tables 1 and 3). The fibre statistics (the length
and the diameter of the tremolite fibres) also verifies that the fibres are quite short
and coarse (Table 4).
This method does not give an absolute estimate of the number concentration of
tremolite asbestos in a sample. The only fibres counted are those visible in the light
microscope. Thin fibres under the detection limit will not be measured or counted.
This means that this technique is not suitable for an overall analysis of the number
concentration of asbestos in a bulk material. If the number of asbestos fibres will
be the only measured dimension for the level of asbestos contamination, then
electron microscopy is the only choice. But for many samples with moderate levels
of contamination and a high degree of large fibres this technique can be very useful
for the estimation of mass concentration of tremolite fibres. The pre-analysis
procedure described is extremely important in determining when this technique is
suitable. Under these circumstances the proposed method is not just a screening
technique.
Finally the method is very easy to use and the microscope as well as the computer
hardware and software are commercially available and not extremely expensive. This
makes this technique an excellent choice for tremolite analysis in dolomite.
Acknowledgements—The authors would like to thank all participating members in the inter Nordic
reference group working with the project "Development of a Nordic reference method for the
determination of tremolite fibres in dolomite". The authors would also like to thank Mrs Gun-Britt
Berglund and Mrs Birgit Paulsson for their suggestions, helpful comments on the manuscript and
photographic assistance.
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On-line analysis of tremolite
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