Anal. Chem. 1984, 56, 1429-1431
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Comparison of Preparation Techniques for Analysis of Asbestos Samples by Transmission Electron Microscopy Mark B. Finn, William H. Hallenbeck,* and Edwin H. Chen University of Illinois a t Chicago, School of Public Health, P.O. Box 6998, Chicago, Illinois 60680
Four techniques were evaluated for the preparation of asbestos samples by transmission electron microscopy utilizing a chrysotile suspension of known mass concentration. The acetone droplet technique was compared to three direct transfer techniques: EPA interim method on ashed and unashed samples and the condensation washer technique on unashed samples. Recording of the analytical results on a fiber vs. a fibril basis was also evaluated. Asbestos recovery on the basis of number and mass concentration was significantly less for the acetone droplet technique than the recovery for the three direct transfer techniques. The three direct transfer techniques were not significantly different regarding number and mass concentrations. The acetone droplet technique is not recommended for the quantitative analysis of asbestos samples. Recording observations on a fiber basis was selected as the most appropriate system for reporting results.
Preparation of asbestos samples for transmission electron microscopy (TEM) requires the transfer of sample material from the collection medium to an electron microscope grid. Transfer procedures are critical as asbestos fiber loss must be minimized. Several grid preparation techniques are available which vary in technical difficulty and time requirements. The U.S. EPA has published an interim method for the analysis of asbestos in water ( I ) . This technique employs a modified Jaffe wick procedure to transfer the sample by solvent dissolution of a piece of carbon-coated filter placed on an electron microscope grid. A similar direct transfer technique uses a condensation washer (reflux apparatus) to dissolve the filter on which the sample has been deposited (2). Transfer of the sample material in a liquid suspension directly to a carbon-coated grid was done by Patel-Mandlik et al. (3) using an acetone droplet. Certain sample materials (i.e., blood, urine, and tissue) contain debris which can interfere with the observation of asbestos fibers. Low-temperature plasma ashing is one technique which has been used successfully to eliminate organic debris in samples ( 4 ) . Studies which compared preparation techniques reported that the direct transfer method using the modified Jaffe wick procedure was most effective in minimizing asbestos fiber loss (4, 5). However, they did not include an evaluation of the acetone droplet technique. The acetone droplet technique requires less time to complete and is simpler than the other techniques, but recovery studies have not been reported. In order to determine if the fairly simple acetone droplet technique provides reliable results, it was compared to the EPA interim method for grid preparation of ashed samples collected on polycarbonate filters. Application of the EPA interim method on unashed samples was included in the comparison to investigate any effects which ashing might have on the sample such as breakage of fibrils, bundles, or clusters. The condensation washer technique was also included in order to provide additional data regarding unashed samples and to 0003-2700/84/0356-1429$01.50/0
evaluate a technique which could be completed in less time than the modified Jaffe wick procedure. Microscopic observations were recorded on both fiber and fibril bases in order to evaluate the relative merits of each classification system.
EXPERIMENTAL SECTION The four sample preparation techniques will be referred to as follows: EPA interim method on unashed samples-EPAunashed; condensation washer method on unashed samplescondensation washer; EPA interim method on ashed samplesEPA-ashed; acetone droplet method on ashed samples-droplet. A chrysotile asbestos suspension was used to prepare sample filters in a target range of 15-25 asbestos fibers per grid opening. The target range was selected in order to make TEM scanning and counting less time consuming while maintaining asbestos counts well above a low mean level of background contamination. An additional objective was to create a homogeneous suspension of individual fibrils with as few bundles and clusters as possible. The chrysotile asbestos suspension of known asbestos mass concentration was prepared by using a procedure similar to that developed by R. S. Feldman of the U.S.Health Effects Research Laboratory, Cincinnati, OH. Chrysotile asbestos (978 pg) was dispersed in 1900 mL of 0.1% aqueous Aerosol OT (dioctyl sodium sulfosuccinate). A series of dilutions and sonications were used during suspension preparation. The final suspension provided acceptable grid opening counts and had a theoretical mass concentration of 7 x 10-~pg/mL. All of the sample fiiters, along with the appropriate blank filters, were prepared on the same day when the chrysotile asbestos suspension was formulated in an effort to have the greatest possible homogeneity among sample aliquots. The filtration assembly employed a fritted glass support, a 5 pm pore size 47 mm diameter mixed cellulose ester backup filter, and a 0.1 pm pore size 47 mm diameter polycarbonate filter. Sample and blank filters were cut into sectors which were randomly assigned to a preparation technique. Samples requiring low-temperature ashing were placed in &mL vials and ashed in an International plasma ashing chamber. The open vials were placed in the quartz rack of the ashing chamber and ashed for 6-7 h at approximately 1torr, 90 W, and 100 cm3 of oxygen/min. Care was taken to reduce air movement in the chamber by evacuating and purging slowly. Sample and blank filters were ashed simultaneously. For those samples to be analyzed by the EPA-ashed technique, a 7-mL volume of filtered deionized water was added to the vials, and the vials were sonicated for 5 min in a low energy ultrasonic water bath (50-60 Hz). The contents of the vials and two rinses were vacuum filtered, secured in a plastic petri dish by double stick tape, and set aside for modified Jaffe washer preparation. Ashed samples to be prepared by the droplet technique were resuspended in 3 mL of filtered acetone and sonicated for 5 min. A 5-pL aliquot was placed on a carbon-Formvar-coated grid. The grid was supported in anticapillary tweezers and placed under a 250-W heat lamp. Grid quality was carefully monitored and grids were discarded if any of the following conditions were observed: (1)sample liquid remained in the pipet tip, (2) sample liquid adhered to the tweezers, (3) uneven liquid evaporation, and (4) violent liquid evaporation as characterized by formation and bursting of a central bubble within the droplet. All samples to be prepared by direct transfer techniques (EPA ashed, EPA unashed, and condensation washer) were first carbon coated (20-40 nm thickness) with a vacuum evaporator unit. 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table I. Number Concentration of Chrysotile Asbestos Suspension (Blank Adjusted) number concn, 105/mL no. Of fiber fibril grid preparation openings std std technique scanned mean dev mean dev EPA-unashed condensation washer (unashed) EPA-ashed droplet (ashed)
40 40
1.30 1.26
0.44 0.55
3.39 4.03
4.17 5.73
56 80
1.51 0.40
0.84 0.61
3.33 0.86
1.61 1.42
Table 11. Mass Concentration of Chrysotile Asbestos Suspensiona (Blank Adjusted)
preparation technique EPA-mashed condensation washer (unashed) EPA-ashed droplet (ashed)
mass concn, no. of fiber grid opefiings std scanned mean dev 40 40 28 40
10.4 24.6 6.7 1.1
,ug/mL fibril std mean dev
46.5 101.0
2.6 3.1
8.3 7.4
19.4 3.3
5.4 0.7
18.8 1.8
a The theoretical mass concentration of the suspension pg/mL. was 7 x
Randomly selected squares (approximately3 mm x 3 mm) of the carbon-coatedfiiter were placed particle side down on an uncoated London 200 mesh finder grid. A 5-llL drop of filtered chloroform was placed on the filter to start dissolution and affix the filter to the grid. Grids were positioned on a copper supporting screen to facilitate transfer to the modified Jaffe washer or the condensation washer. Grids were washed in the modified Jaffe washer for 6-7 days or in the condensation washer for 6-7 h. Grids prepared by direct transfer techniques were discarded if less than 90% of the grid opening films were intact. The filters also had to be dissolved enough to allow easy observation of the grid opening surface. These techniques were developed to a point where at least 95% of the samples resulted in satisfactory grid preparations on the first attempt. All grids were scanned at approximately 26 OOOX on a Philips 300 transmission electron microscope at 100 kV. Grid openings to be scanned were selected from a computer-generated table of randomly selected grid openings. Fibers were defined as having parallel sides and a minimum 31 length-to-diameterratio. Asbesto fibers were further classified as either an individual fibril, a bundle, or a cluster. The number of fibrils forming each bundle and cluster was also recorded. The total number of fibrils recorded was equal to the summation of individual fibrils and the fibrils which were incorporated into bundles and clusters. Asbestos fibers were identified as chrysotile primarily by morphology (central canal) and selected area electron diffraction patterns (6). Length and diameter measurements were made at magnifications of 26000 to 10OOOOx on all fibers and fibrils.
RESULTS AND DISCUSSION Fiber and fibril number concentrations were calculated for each grid opening (see Appendix). Grid opening fiber and fibril mass concentrations were obtained by summation of the calculated individual fiber or fibril mass values (see Appendix). Fiber and fibril mass values were calculated by employing the fiber or fibril length and diameter measurements and assuming cylindrical shape and uniform density (2.56 X lo4 ~ g / , u m ~ ) . Number and mass concentrations were adjusted by the technique-specific blank values for fiber number, fibril number, fiber mass, and fibril mass per cm2 of grid area. Calculated mean concentrations per milliliter of the chrysotile asbestos suspension (blank adjusted) are shown in Tables I and 11. The percentage of each fiber type was quite similar among the techniques: 67% individual fibrils; 32% bundles; and 1% clusters. The large standard deviation values in Tables I and I1 were due to the mass data not being normally distributed. All fibers were included in the data no matter how large in size. Therefore a large bundle or cluster (e.g., 100 or more fibrils) in one grid opening could have a greater mass (Table 11) than the combined mass of several grid openings not containing a large bundle or cluster. The same effect was present in the fibril number concentration standard deviation values (Table I). Statistical evaluations employed each grid opening concentration as an independent estimate of the suspension concentration. Calculation of the Kolomgorov-Smirnov D statistic (7) demonstrated that some data were random samples from normal distributions and others from nonnormal distributions. Therefore, nonparametric techniques were selected for further statistical analyses of the data: the Mann-Whitney rank sum and Kruskal-Wallis tests (8). The four techniques were compared by the Kruskal-Wallis test regarding fiber and fibril number and mass concentrations. Concentrations were significantly different 0, C 0.0001) among the techniques. By employing a multiple comparison technique (9),the droplet technique mean concentration was shown to be significantly different (p 2 0.005) from the mean concentrations of the other three techniques (Table 111). Further statistical analyses demonstrated that there were no significant differences among direct transfer techniques regarding mass and number concentrations. The droplet technique underestimated the suspension mass concentration due to sample loss or uneven distribution of the sample material on the grid surface. For all techniques, the concentrations were not significantly different (a = 0.05) between grids prepared from the same suspension filter. It was further determined that the differences in concentrations between the droplet and direct transfer techniques were not due to differences between suspension filters analyzed by the same technique. Therefore, the concentration differences between the droplet and direct transfer techniques were not due to variation within each technique. The data were next evaluated on the bases of the following six characteristics: fiber and fibril diameter, length, and mass.
Table 111. Multiple Comparisons of Number and Mass Concentrations p values
preparation techniques compared EPA-unashed vs. condensation washer EPA-unashed vs. EPA-ashed EPA-unashed vs. droplet condensation washer vs. EPA-ashed condensation washer vs. droplet EPA-ashed vs. droplet a
fiber no.
fibril no.
basis
basis
fiber mass basis
fibril mass basis
0.21 0.16 < 0.00001 0.83
