Identification of Fibrous Material in Two Public Water Supplies John Flickinger" and Jon Standridge Wisconsin Department of Natural Resources, Laboratory Services, 465 Henry Mall-U.W.,
An evaluation of three microscopic techniques for the enumeration of amphibole asbestos fibers in Lake Superior was undertaken to review existing methods, compare them for sensitivity, and modify the best method for achieving optimum results. Transmission electron microscopy with selected area electron diffraction capability can be used to identify amphibole asbestos fibers in water.
Industrial exposure to asbestos fibers has long been associated with cancer of the lung and peritoneum as well as asbestosis ( I ) . Recently, asbestos fibers have been identified in such diverse media as talc-coated rice ( 2 )and drinking water. Experimental studies in animals indicate that asbestos injected directly into the stomach is concentrated two days later in the omentum surrounding the large intestines. Asbestos was also found in the brain and the bloodstream ( 3 ) .A normally rare type of cancer in humans, mesothelioma, has been produced in animals by intrapleural inoculation of various types of asbestos ( 4 ) . While ingestion of these fibers in drinking water has not to date been established as a human health hazard ( 5 ) ,the possibility of a potential health hazard should not be ignored. The presence of amphibole mineral fibers in the waters of Lake Superior was established in 1969 (6).The major source of these mineral fibers is believed to be the taconite iron ore processing plant of the Reserve Mining Co., Silver Bay, Minn. (7). This company was established with the purpose of upgrading the low iron content taconite to high grade ore for shipment to the nation's blast furnaces. Lake Superior was a very desirable plant location since it supplies both the large volumes of water necessary and is a convenient location to dump the daily discharge of 67 OOO tons of waste material. The chemical and mineral composition of the waste tailings entering Lake Superior has been previously established (8). Among the various minerals present are several types of amphibole asbestos whose presence in water is suspected of being a potential health hazard. Based on the assumption that amphibole asbestos fibers present in water pose a potential danger to human health, this laboratory undertook a survey to evaluate the fiber content of Lake Superior water. Since comparisons of sensitivities of the various techniques for fiber identification have not been documented, the first part of this survey involved developing an adequate technique for amphibole asbestos fiber enumeration. The second part of this survey involved using this technique to determine whether slow sand filtration is a viable means of removing these fibers from a public water supply. Materials and M e t h o d s
Samples were collected from three sources serving two Wisconsin communities. Samples were taken before and after treatment (which included slow sand filtration) a t sources A and B. Source C was sampled before treatment only. Three basic techniques for the enumeration of asbestos fibers in water were attempted before an adequate method was developed. The first of these techniques to be tried was that of polarized light optical microscopy. The methodology used was as follows (9). 1028
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Madison, Wis. 53706
A known volume of water was agitated and passed through a 0.45-pm membrane filter which was then air dried overnight. After drying, the filter was placed on a glass slide. Three or four drops of diethyl oxalate-dimethyl phthalate mounting solution were then dropped on the filter paper. After the filter had cleared sufficiently, the slide was examined using polarized light microscopy. Those particles that were fibrous in appearance and could rotate the plane of polarized light were identified as crystalline fibers. This grouping included asbestos fibers as well as other mineral fibers. Further use of polarized light microscopy with determinations of refractive indices of individual fibers was abandoned in favor of electron microscopy because'fibers less than 1 ym long could not be resolved optically (Table I). A scanning electron microscope (SEM) with an x-ray energy spectrometer (XES) was chosen to facilitate the examination of ultrasmall fibers. Sample preparation was similar to that used by Maggiore and Rubin (IO).Water samples were filtered through 47-mm carbon-coated 0.4-ym polycarbonate membrane filters. To sufficiently wet the filter surface, a few tenths of a milliliter of 95% ethanol was filtered followed immediately by the water sample. Sample volumes varied from 100 to 500 ml depending on the sample source. Filtration was accomplished in a 250-ml membrane filter holder, and water samples were agitated by hand immediately prior to the withdrawal of the sample aliquot. After filtration the membrane filters were allowed to dry and coated a second time with carbon in a vacuum evaporator. This left the filtered particulates sandwiched between two carbon coats. The carbon film was then transferred to a locator transmission grid, and the filter dissolved with chloroform as described by Maggiore and Rubin (IO). Fiber counts and identifications were made with a 50-kV scanning electron microscope equipped with an x-ray energy spectrometry system. The entire grid was scanned a t 25 000-30 OOOX in the secondary electron mode using the TV monitor. When a fiber of interest was located, the operator switched to CRT viewing, and the fiber was centered at high magnification. An area slightly smaller than that of the fiber was scanned, and an x-ray spectrum of this area gathered. Appropriate background areas were also examined from time to time, and the spectra compared with those from the actual fibers. Each fiber that was counted was examined in this way
Table I. Fiber Counts: Optical Techniques Sample description
Source Source Source Source Source Source Source Source Source Source Source
finished raw finished raw raw C raw B finished B raw B finished B raw A raw
A A A A A
Date
Total fibers, I .
Crystalline fibers, I.
3/18/74 3/18/74 3120174 3120174 3119/74 3119/74 3/19/74 311 9/74 3120174 3120174 3/22/74
28 44 600 300 1 a50 23 750 4 000 10 400 54 900 7 600 3 1 725 30 a75
2 1000 10 260 6 650 190 240 2 600 1187 2 375 4 275
and was classified as either an iron fiber, a silicate fiber, or a "tailing-like" fiber according to the elemental composition of its XES spectra. Fibers with large iron XES peaks and small silicon peaks were labeled iron fibers (possibly fractured iron bacteria). Those with large silicon peaks and no iron were la-
Iph of commercial asbestos fibers
heled silicate fihers which might he diatom fragments. "Tailing-like" fihers were those fihers with substantial iron and silicon peaks similar to those of the cummingtonitegrunerite series of minerals, a length-to-width ratio of 3 to 1, and approximately parallel sides (Figures la-10. The results
Figure Id. ES spectrum indicating major ell:m ent composition of min e tailing
,." -----
Figure lb. XES spectrum indicating major element composition of
Flgbre le. SEM micrograph of "tailing-like'' particle
Figure le. SEM micrograph of mine tailings
Figure 11. XES spectrum indicating major element composition of "tailing-like" particle from Lake Superior water Volume 10, Number 10. October 1976 1029
of the SEM fiber counts indicated tailing-like fiber concentrations of 1.95-7.80 X lo3 fibersfl. (Table 11). After several samples were counted, it became evident that when compared to previous EPA results (II), the SEM-XES method of asbestos detection was yielding unexplainably low results. To confirm this, a grid that had been counted by the SEM-XES technique was examined with a transmission electron microscope. Results indicated that SEM examination actually missed many “tailing-like” mineral fibers. These fibers were either too small to be resolved with SEM or were so thin that they became buried in the carbon coating. Next, the above-mentioned grid was examined using SEM modified to do scanning transmission electron microscopy (STEM). Using this technique the microscopist can penetrate the carbon film with the electron beam and see the outlines of fibers present. X-ray emission spectroscopy can be used to identify suspected “tailing-like’’fibers by their elemental composition. Although a SEM modified for transmission work affords the microscopist improved resolution over the scanning mode alone, it proved to be very cumbersome for routine fiber counting and was abandoned in favor of transmission microscopy. Transmitted electron microscopy (TEM) has three major advantages over the SEM or STEM that make it a desirable technique for mineral fiber counting. First of all, a better resolution, down to 5 A, is obtainable. Scanning microscopes generally have 50-100 A resolution in the scanning mode and down to 20 in the transmission mode. The increased resolution allows the microscopist to see smaller fibers. Secondly, T E M permits the analyst to see the internal features of thin fibers which is helpful for morphological identification of asbestiform amphibole fibers. Finally, selected area electron diffraction (SAED) patterns of individual fibers can be obtained with a transmission scope. Minerals have characteristic electron diffraction patterns, and this is a useful tool for assigning the fibers seen to the amphibole group. A new method of sample preparation and analysis was developed combining the best features of existing methods (IO, 12).A 25-ml membrane filter holder was used. The glass cylinder had parallel sides to ensure even distribution of particulate material on the filter. The filtration apparatus was rinsed in zero fiber water (double-distilled water that had been previously filtered through a 0.1-pm polycarbonate membrane filter). A 5-pm pore size membrane filter was used as a backup to ensure a good seal, and a 0.1-pm pore size polycarbonate membrane filter was placed on top of this. The filtration cylinder was then attached and clamped. This filtration cylinder was covered with a loose-fitting beaker to prevent contamination from dust. The original water bottle was then agitated
Table II. Fiber Counts: SEM Technique Sample description
Date
Source A raw Source A finished Source A raw Source A finished Source A raw Source A raw Source A raw Source C raw Source C raw Source C raw Source B finished Source B raw Source B finished Source B raw
3119/74 3120174 3120174 3/22/74 3/22/74 3/28/74 410 1/74 3119/74 3120174 312 1/74 3119/74 3/19/74 312 1/74 3/21/74
1030
Tailing-like Silicate fibers, I. fibers, I .
3900 1950 1950 7800 5850
Environmental Science & Technology
7 800 4 105 42 900 2 340 66300 15600 87 360 46 800 9 750 7800 2 925 29 250 7 800
Iron fibers, I.
96600 10920 42 900
by hand for 5 min to ensure an even distribution of particulate matter. Immediately after shaking, a 50-ml wide-mouth plastic bottle was washed three times with the water sample, and approximately 40 ml of the sample was transferred to the bottle which was then capped and weighed on an analytical balance. To ensure rapid filtration, 0.5 ml of fiber-free ethanol was filtered first. When the alcohol was almost gone, approximately 25 ml of water sample was added from the weighed 50-ml bottle. Care was taken to ensure that the filter holder was covered with a loose-fitting beaker to prevent contamination from the surrounding work area. The vacuum was released as soon as the water sample had been completely filtered. The filter was then labeled and transferred to a labeled petri dish. The wide-mouth bottle was reweighed, and the filtered volume of water calculated by taking the mass difference of the two weighings. The filter with its particulate sample was then carbon coated in a vacuum evaporator. A transmission grid was prepared from this sample by the same method used for the scanning technique (10). This procedure yields particulate matter on a single carbon film that can be examined for mineral fibers in a transmission microscope. Fiber counts were made with a 100-kV transmission electron microscope. First, the grid was examined at a very low magnification to confirm an even particulate distribution. When a suitable grid space was located, it was scanned at 25 OOOX. When a suspected mineral fiber was found, an electron diffraction pattern was taken and compared with a pattern taken under identical conditions of the amphibole mineral amosite. Amosite was chosen as a standard material because it is readily available in pure form and because it is an asbestiform amphibole in the cummingtonite-grunerite series. Since taconite tailings contain cummingtonite (8),the authors believed that the water samples examined would contain amphibole fibers that had an identical structure (shown by SAED) to amosite. Fibers were indeed found that had the SAED pattern of amosite. (See Figures 2a-2d). All fibers that were counted had to meet the conditions of a t least a 3 to 1 aspect ratio, parallel sides, and the electron diffraction pattern of the standard. In addition, the mineral fibers counted had a characteristic appearance that was useful for identifying them. In some instances, only the first two of the above conditions could be met, because of unfavorable electron diffraction conditions. These fibers were considered questionable and were not used in calculating the results. The water samples were filtered without any extensive treatment since it was felt that this was the best way to prevent any fibers present from breaking up. Treatments such as the addition of a dispersing agent and sonication to ensure even distribution would tend to break up mineral fibers present into smaller pieces, thus increasing the actual fiber count. Counting time was another critical part of the TEM method. Because of the large amount of time required to examine even a small number of grid spaces, the number of fibers counted was 25 or 6-12 grid spaces on a 200 mesh grid. This generally involved 1-2 h of actual TEM counting, depending on how much interferring debris was present. However, since fibers were not found in negative controls, we feel that the 25 fibers counted represent only those actually present in the water sample.
Results By use of optical microscopy techniques, counts were inaccurate because of two major problems. The first and most obvious problem was that the majority of the tailing fibers present in the lake water were substantially smaller in size than 10 pm. Therefore, most of the fibers present were a t the
very limits of resolving power and were very easily missed. The second problem was not being able to implicate fibers ohserved as truly being amphibole asbestos. Both the SEM and STEM methodology exhihited a problem of resolution. Both short fibers (those approaching the instruments' resolution) and thin fibers (those whose surface irregularity approaching the resolution limit) could escape recognition if SEM was used. If STEM was used, short fibers became a resolution problem. Also, the use of x-ray emission spectroscopy as an identification tool could be questioned. Since a number of amphibole minerals have similar or identical element compositions (Mg, Si, Fe) to minerals in other groups, XES is not an absolute method of fiber identification. For these reasons, TEM with SAED analysis was selected as the method of choice. Transmission microscopy as applied to mineral fiber analysis was not without problems also. First, i t is not possible to get an electron pattern of thick fibers with a 100-kV transmission microscope. This was not a handicap in most cases, since the fibers identified were generally thin enough to yield patterns. However, those fibers too thick for SAED analysis were not counted. Secondly, a mineral fiber may assume any one of a number of orientations which have an effect on the appearance of the electron diffraction patterns (13).Visual comparison of standard Datterns with those from actual fibers isolated 1. -.....l__ ...&ult
.._-
Figure 28. TEM micrograph of commercial amosite fiber
Figure 2b. SAED pattern from commercial amosite fiber
a t times because of the orientation assumed by the unknown fiber. However, it is unlikely that a nonamphihole fiber would he mistakenly identified as an amphibole hy its SAED pattern. Other fibrous minerals with amphihole-like diffraction patterns are not generally found in Lake Superior water (14). All of the fibers observed in the finished water samples appeared to fall in a size range of 0.2-2.0 pm. Fibers counted in both of the raw water samples examined fell in a similar size range. The TEM-SAED data (Table 111) indicated that amphibole mineral fibers were present in all samples examined and ranged in concentration from 0.2 to 4.2 X 106 fibersh. of water. From the limited data presented here, it appears that slow sand filtration cannot remove small ashestiform amphiboles from water. The T E M results (Tahle 1111,when compared to those obtained by the optical and SEM techniques (Tables I and II), demonstrate a large difference in the number of fibers connted. Although the same samples were not carried through each procedure, the large and consistent count differences hetween optical and,SEM on one hand and " E M on the other hand illustrate the importance of using the TEM-SAED method for an analysis where small fibers are expected. It is also significant that the classification of fibers counted in the TEM method, amphibole fibers, is much narrower than the classifications used in the other methods described. The TEM
Figure wafer
2c. TEM
micrograpn of ampniDOle mer from LaKe superior
Figure 26. SAED pattern from amphibole fiber from Lake Superior water Volume 10, Number 10, October 1976 1031
Table Ill. Fiber Counts: TEM Technique Sample description
Data
Amphibole fibers, I.
Source C rawa ‘Source B finisheda Source B finished Source B finished Source B finished Source B raw Source A finished Source A rawb
312 1/74 3/20/74 4/15/74 410 1/74 4/08/74 4/08/74 3/24/74 3/24/74
2.0 x l o 6 0.2 x l o 6 2.4 X l o 6 2.8 x 106 4.0 x l o 6 4.2 x l o 6 1.9 x 1os 0.7 X l o 6
a O n l y five fibers per grid were counted. b Most o f t h e particulate matter was clumped together. Some fibers m a y have been trapped and could n o t be counted, resulting i n an erroneously l o w count.
area would allow limitations to be placed on the size of fibers to be counted. Although the counts made using TEM-SAED must be considered in light of the restrictions of the method, the authors feel it is a promising analytical method for evaluating fiber removal processes. Acknowledgment
The authors thank Everet Glover, University of Wisconsin, Department of Geology, for his help in developing a scanning electron microscopic technique for fiber examination and Richard Dodd, Walter Yang, and Richard Casper, University of Wisconsin, Department of Material Science, for their help in developing a transmission electron microscopy technique for fiber examination. Literature Cited (1) Brodeau, P., “Asbestos and Enzymes”, Balantine, New York,
method is capable of detecting smaller fibers, but it is also looking a t a more limited definition of a fiber. This is additional evidence that a TEM method is necessary since evidently there are large numbers of fibers present that fall into the specific catagory of amphibole. Conclusions
Research and development in the following areas would greatly facilitate future fiber counting in water. First, background counts are needed to allow significance to be assigned to any counting data acquired to date. There is a definite need for more studying of mineral fibers in ground and surface waters. Secondly, there is no standard method of microscopic analysis for mineral fibers in water. The most promising technique to date is one combining the best features of SEM-XES systems and TEM-SAED systems. New transmission microscopes can be equipped with both selected area x-ray diffraction and x-ray energy spectrometry. The operator can acquire electron diffraction patterns of thin specimens and use XES spectra for those fibers too thick for SAED. Finally, it has not been possible to define a size range of fibers that are hazardous to health. Successful research in this
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Environmental Science & Technology
N.Y., 1972. (2) Cunningham, H. M., Ponterfract, R. D., J . Assoc. Off. Anal. Chem., 56 (41,976 (July 1973). (3) Blejer, H. P., Arlon, R., J . Occup. Med., 15,92 (Feb. 1973). (4) Wagner, J. C., Berry, G., Brit. J . Cancer, 23 (3), 567 (Sept. 1969). (5) Brit. J. Znd. Med., 30 (2), 180 (April 1973). (6) Andrew, R. W., Proceedings, Conference of Lake Superior and Its Tributary Basin Wisconsin, Minnesota, Michigan, FWPCA, p 15, 1969. (7) Cook. P. M.. Glass. G. E.. Tucker. J. H.. Science. 185 (4154). 853 (Sept. 6, 1974). (8) Plumb, R. H., Jr., PhD thesis, University of Wisconsin, Madison, Wis.. 1973. (9) Holmes, S., The Measurement of Airborne Asbestos Dust by the Membrane Filter Method, Asbestos Research Council, Technical Note 1, Lancashire, Great Britain, June 1969. (10) Maggiore, C. 3., Rubin, I. B., Proc. 6th Ann. Scan. Elect. Micros. Symp., ITT Res. Inst., Chicago, Ill., April 1973. (11) Fairless, B., Region V Central Regional Laboratory, USEPA, private communication. (12) Analytical Procedures for the Analysis of Asbestos Fibers, USEPA, Region V Central Regional Laboratory, April 1974. (13) Zvyagin, B. B., “Electron Diffraction Analysis of Clay Mineral Structures”, pp 70-75, Plenum, New York, N.Y., 1967. (14) Cook, P., National Water Quality Laboratory, Duluth, Minn., private communication.
Received for review December 24, 1975. Accepted May 3,1976.
