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FILE NAME: Talc (TALC) DATE: 1990 DOC#: TALC200 DOCUMENT DESCRIPTION: Journal Article - Asbestos-A Standard TEM Procedure for Identification and Quantitation of Asbestiform Minerals in Talc The Tenth-Meter In a recent (1) piece about the test diatom "N itzschia firthii Fuge" I quoted Spitta's comments (2) which includ ed the tenth-meter in reference to the wavelength of light (4700 tenth-meters) recommended to best resolve the lines (striae) on the diatom valve face. I feel an explanation of this term as used by Spitta is owed to readers of this column. In a footnote on p. 267 of his text 1 find "It should be mentioned here perhaps, although hardly in logical sequence, that even the p or micron is not small enough a unit for the physicist when dealing with the m easurem ent of the wavelengths of light. In this case the German savants employ what is called the double mu (w ritten up), which is the thou sandth part of the micron; but the English scientist adopts a smaller unit still, called the tenth-meter, which is the ten thousandth part of the micron, the raison d'etre of the term being that 1010 (10 at the tenth power) go to a meter". From this then we realize that the tenth-meter is equivalent to the Angstrom Unit, named after A.J. Angstrom (1814-74), a Swedish physicist, and defined as one-ten-thousandth of a micron. However in this age of enlightenment, after great strides in clarifying scientific term inology, we have dropped the micron in favor of the micrometer, and the millimicron in favor of the nanometer, and so since the Angstrom is one ten thousandth of a micron (oops...m icrom eter), Spitta was recommending light of 470 nanometers. Got that? References 1. M cLaughlin, R.B. "N itzsch ia firth ii Fuge, A Test Diatom". The M icroscope, 1990, 38. 2. Spitta, Edmund J. M icroscopy, The Construction, Theory and Use o f the M icroscope. E.P. Dutton and Company, New York (1920). 92 M-00000 l.91NrNr 456 MICROSCOPE(l 990)38 c hrysotile intosite,crocidolite inthophyllue,trem otile,a c linotile,chryso tile,am osite,crocidolite,anthophyllite.trem olite.actinolite,chrysolite,a m osite.croidoltAMt)uaktllji^senuii^^etiMoli^oluys^ii.em osite lite,anthophyllite,trem olite,actinolite,chrysotile,anthophyllite,trem olite,chrysolite,am osite,crocidolite,anthophyllite,trem olite,actin olite,ch A Standard TEM Procedure for Identification and Quantitation of Asbestiform Minerals in Talc Thomas Kremer* McCrone Associates and James R. Millette, Ph.D., Millette, Vander Wood Associates, Inc.** I Keywords: Asbestiform, asbestos, MSHA, OSHA, talc, TEM, amphiboles, mineral fibers, microscopy, electron microscopy. ABSTRACT A standard method for the preparation of powdered talc for transmission electron microscope (TEM) analysis has been developed to identify and quantify small quantities of asbestos and related minerals. The method utilizes an aqueous suspen sion treated with methyl cellulose. Particles are mounted by transferring a drop of the talc-water suspension to a TEM grid bearing a formvar film which has been coated with evaporated carbon. Once dry, two grids per sample are examined in the TEM at magnifications of 5,OOOX and 20,000X for elongate and *850 Pasquinelli Drive, Westmont, IL 60559 **5500 Oakbrook Pkwy., Suite 200, Norcross, GA 30093 457 fibrous particles. Morphology, crystal structure and elemental composition are used to identify and characterize particles of interest. Calculations are included to determine a detection limit and/or determine concentration in weight percent. INTRODUCTION The regulatory definition of asbestos was established in 1972 when the National Institute of Occupational Safety and Health (NlOSH) published its definitions and analysis methods for asbestos (1). Under their method, asbestos was defined as any fiber of chrysotile, crocidolite, amosite, anthophyllite, tremolite or actinolite. What has come to be known as a "federal fiber" was defined as a particle with a length to width (aspect ratio) of at least 3:1 and a length greater than 5 micrometers (pm) (1). Although NlOSH has set forth phase contrast microscopy (PCM) as the analytical tool for regulating airborne asbestos, it has acknowledged the need for and has used analytical electron microscopy (AEM) to identify asbestos in air samples and bulk talc samples (2,3). Both the Occupational Safety and Health Administration (OSHA) and the Mine Safety and Health Administration (MSHA) refer to NlOSH methods. As regulatory agencies, OSHA and MSHA are responsible for monitoring the safety of workers in the work and mine environments, respectively, which includes setting limits on the levels of hazardous materi als to which a worker may be exposed. The primary route of asbestos exposure is inhalation, therefore air concentration lim its were established (4). A primary source of airborne dust is fine particle bulk materials. Consequently, manufacturers have been concerned with the potential presence of these minerals in their products in any measurable quantity. In February, 1990 OSHA promulgated a Hazard Communication Standard (HCS) (5). The HCS requires producers to label as a carcinogen, bulk materials and bulk minerals and products which contain 0.1% or more of asbestos and, pending supplemental rulemaking to be completed November, 1990, non-asbestiform anthophyllite, tremolite and actinolite. To meet this standard, a more sensitive method to detect asbestos is needed. Such low levels are not quantifiable by polarized light microscopy and the morphology of fibers is not discernible by x-ray diffractometry. Transmission 458 MICROSCOPEil 990)38 THOMAS KREMER, et al. electron microscopy is capable of detecting these low levels and has been recognized by both government and industry for sev eral years (3,6). Given the NIOSH definition of asbestos and the low levels to which it must be detected, the need arose for quantitative TEM analysis of powdered talc. To meet this need, McCrone Environmental Services developed the following method, which has been in use since 1985. Standard Operating Procedure Analysis of Powdered Talc for Asbestiform Minerals by Transmission Electron Microscopy 1. Scope and Purpose This method is applicable to the identification and quantitation of small (typically 1-20 micrometer) elongate minerals in pow dered talc, and was designed for chrysotile and the elongate forms of tremolite, actinolite and anthophyllite. Samples may be previously screened by light microscopy or x-ray diffraction techniques. 2. Principle of Method The combined techniques of transmission electron microscopy (TEM), selected area electron diffraction (SAED) and energy dis persive x-ray spectrometry (EDS) permit the detection of asbestiform minerals based on morphological characteristics, followed by a definitive mineralogical identification of each fiber. These techniques are currently the best analytical tools for the determination of fine asbestos minerals in a talc matrix. 3. Interferences Interferences caused by other fibrous particles must be distin guished from positively identifiable asbestos. Large particle or particle aggregates may obscure fibers. Positively identified non-asbestos fibers include enrolled talc, ribbon talc (Figures 1A and B), antigorite (Figures 2A,B and C), talc fragments (Figures 3A and B), silica and iron oxide fibers. Organic additives such as perfumes may crystallize as fibers or needle-shaped crystals in 459 Figure la . Talc platelets and attached ribbon. Figure lb. SAED pattern o f a talc ribbon with its (001) face oriented approximately parallel with the electron beam. 460 MICROSCOPE(1990)38 THOMAS KREMER, et al. Figure Zb. SAED pattern with a diagnostic 35 to 40 A spacing in the a-crystallographic direction. 461 Figure 2c. The elemental composition, determ ined by EDXRA, is characteristic of a serpentine mineral. Figure 3a. SAED pattern of talc, showing the typical [0011 zone axis of a phyllosilicate mineral, with the (001) face oriented perpendicular to the electron beam. 462 MICROSCOPE(1990)38 r THOMAS KREMER, et al. C ursor: 1.240K*V 409 ggf tl ! ---------- 1j . j j ! fit u <j j i ! H S| i t ^ HUES' i - -- 1- t ! j u AJ I E * i ...................... ! sj. Figure 3 k EDXRA spectrum, characteristic o f talc. finished cosmetic products. In the absence of positive identifica tion, all other fibers must be classified as unidentified. 4. Instrumental Conditions The talc specimen grids are examined in the TEM at an acceler ating voltage of 120 kV and at magnifications of 5,000X and 20,OOOX. 5. Sensitivity This method can detect a single fiber as small as 1 micrometer (pm) long by 0.075 pm wide in the entire TEM field, which results in a theoretical detection limit of 10-5 weight percent. Such fibers usually can be identified readily by SAED and EDS. The mass of a fiber with the above dimensions is about 1.1 x 10- g for chrysotile and about 1.9 x 10- g for amphiboles. 6. Limit of Quantifiable Detection The detection of five or more asbestiform minerals of one vari ety in an analysis constitutes a quantifiable level of detection. When no asbestiform minerals are detected a representative fiber size is used to calculate a detection limit. A representative fiber size is 3 pm long by 0.2 pm wide by 0.06 pm thick, which is 463 considerably larger than the smallest fiber than can be detected (see Section 5, Sensitivity), but it is more typical of small asbestos fibers that are detected in talc analyses. The mass of five such fibers is calculated as follows: 3 pm x 0.2 pm x 0.06 pm = 0.036 pm3per fiber x 3.3 x 10-12 g/pm 3= 1.2 x 10-13g per fiber x 5 fibers = 6 x 10 grams The limit of quantifiable detection for most talc analyses is approximately 6 x 1CMweight percent. The theoretical and quan tifiable detection limits assume homogeneity of the material being sampled. 7. Quality A ssurance Blank suspensions are routinely prepared and tested in order to monitor potential residual contamination from the sample jars. Blank carbon-coated grids are routinely tested to monitor the ambient fiber count. If greater than 4 fibers per grid are present, the jars are pre-cleaned or new carbon-coated grids are pre pared, respective of the test. For each analysis, one grid opening is examined by another ana lyst as a quality control check. 8. Background Correction As of the time of this writing, background correction has not been necessary. Blank contamination is very rare and corrective steps are taken before the testing of material proceeds. 9. Preparation and Analysis Time Preparation time per sample (including preparation of related materials) is one hour. Analysis search time per sample is a maximum o f two hours. 10. Apparatus A. Analytical balance with 0.0001 gram sensitivity B. Weighing boats C. Narrow spatula 464 MICROSCOPE(1990)38 THOMAS KREMER, et al. D. Wide mouth polyethylene jars (125 mL) E. Mild ultrasonic bath, 50-60 watts F. Micropipette (5-10 pL range) with disposable tips G. Standard 3 mm diameter, 200 mesh, copper TTsM grids, cov ered with a carbon-coated formvar film H. Transmission electron microscope (TEM) with an 80-120 kV accelerating voltage and energy dispersive x-ray analyzer 11. Reagents A. Methyl cellulose, powder, USP 4000 cps - Fisher Certified Reagent #M-352 or equivalent B. Water: deionized, particle free (<0.2 pm filtered) C. Methyl cellulose solution: 0.002% (w t/vl) (20 ppm). Dissolve 20 0 5 mg of methyl cellulose in 500 mL of deionized particlefree water to make a 0.004% stock solution. Dilute 1:1 to make a working solution. Note: Methyl cellulose acts as a wetting agent to aid in maintain ing a uniform particle distribution as the sample dries. 12. Sample Preparation 12-1. Transfer 30 to 50 mg of talc powder to a clean 125 mL polyethylene jar. 12-2. Add 80 mL of 20 ppm methyl cellulose solution, cap and shake vigorously for one minute. 12-3. After shaking, loosen cap and ultrasonicate for 10 minutes in order to disperse the finer particles. Then shake again for one minute to produce a uniform suspension. 12-4. Immediately after shaking, uncap and remove 9.2 pL by micropipette. 12-5. Transfer 9 pL drop to a carbon film covered TEM grid. (Grid is first lightly anchored by 2 parallel strips of double-stick tape mounted about 2 5 mm apart on a clean glass microscope slide.) Repeat to make two sample grids per talc sample. Note: Do not expel the remaining 0.2 pL suspension from the 465 micropipette tip. It tends to sputter and frequently destroys the stability of the sample drop. 12- 6. Transfer slide with grids to a desiccator. (Drying time is 23 hours.) Do not leave the grids on the slide for more than one day as the double-stick tape may adhere too tightly. Note: The talc:water ratio may need to be varied for some sam ples. Preparation of talc samples with significantly finer or coarser particles results in large differences in particle coverage on the TEM grid. 13. TEM Analysis 13- 1. Definition of fiber: an elongated particle with parallel sides and an aspect ratio >3:1. When this definition is employed, fibers which fit the OSHA (4), EPA (7) or client's definition can be selectively extracted from the total fiber data for each analy sis. 13-2. Scan sample at 120-150X magnification to check for even dispersion of particles and to locate grid squares with optimum particle density. (Optimum particle density is particle coverage over 15-35% of the field of view.) 13-3. Scan three grid squares on each grid at 20,000X magnifica tion and seven grid squares on each grid at 5,000X for asbestiform minerals. Each asbestiform mineral is recorded as to type (chrysotile, tremolite, anthophyllite, etc.), structure (bundle, dump, fiber) and dimensions (length x width). 13-4. Questionable fibers are examined first by SAED. The chrysotile SAED pattern is unique and diagnostic. Amphibole SAED patterns are variable but usually characteristic. Additional analysis and measurement of amphibole SAED pat terns are done if warranted. 13-5. Ten percent of chrysotile fibers are checked by EDS for further confirmation. If the SAED pattern is not clearly diagnos tic, or if it is consistent with an amphibole SAED pattern, then it is examined by EDS to confirm the identification or to identify 466 MICROSCOPEO990)38 THOMAS KREMER, et al. the type of amphibole. 14. Calculation of Results 14-1-A. where: Mass of chrysotile fibers: M{f) M(f) = n r2l x d r = fiber radius 1= fiber length d = density of chrysotile = 2.55 x 10-12g/pm* n = 3.14159 14-1-B. Mass of asbestifonm amphibole particles: M(a) M(a) = 1 x w x th x d 1= length w = width th = thickness = 0.3 width (approximation) d = density of amphiboles = 3.3 x 10-'2g /p m 3 14-2-A. Mass of talc deposited on each TEM grid (step 12-5) M(s) = T x (V/H) T = amount of talc sampled (step 12-1) V = volume of aliquot transferred to TEM grid (step 12-5) H = volume of methyl cellulose solution (step 12-2) 14-2-B. Total estimated talc mass examined: M(t) M(t) = M(s) x (N x A(s))/A(g) N = number of grid squares examined A(s) = area of a single TEM grid square A(g) = area of an entire TEM grid (effective area over which a 9 pL drop of suspension dries) 14-3. Weight percent: sum total of Mff) or M(a) x 100 M(t) 15. Calculation of a Detection Limit 15-1. M(dl) = A minimum quantifiable mass of asbestos fibers, based on the detection of 5 fibers (approximately 6 x 10-'3grams, from Section 6) 15-2. Detection-Limit (Weight Percent) = M(dl) x 100 M(t) 467 REFERENCES 1. National Institute for Occupational Safety and Health: USPHA/NIOSH Membrane Filter Method for Evaluating Airborne Asbestos Fibers by N.A. Leidel, S.G. Bayer, R.D. Zumwalde, and K.A. Busch (Technical Report No. 79-127). Cincinnati, OH, 1979. 2. National Institute of Occupational Safety and Health, "Method 7402" NIOSH Manual of Analytical Methods, Carter, J.W., Baron, P.A. and Taylor, D.G., pp. 7402-1-7402-7,1986. 3. Dement, J.M., Asbestiform Minerals in Industrial Talcs: Commercial Definitions versus Industrial Hygiene Reality. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods (National Bureau of Standards Special Publication 506). Washington, D.C.: Government Printing Office, pp. 313-323 (1978). 4. Occupational Safety and Health Administration, 29 CFR Part 1910 Occupational Safety and Health Standard, Federal Register, Special Publication. Washington, D.C.: Government Printing Office, 1989. 5. Occupational Safety and Health Administration, 29 CFR Parts 1910 and 1926 "Occupational Exposure to Asbestos, Tremolite, Anthophyllite, and Actinolite; Notice of Proposed Rulemaking", Federal Register, pp. 4939 ff., Vol. 55, No. 29,12 February 1990. 6. Stanley, H.D., The Detection and Identification as Asbestos and Asbestiform Minerals in Talc. Proceedings of the Workshop on Asbestos: Definitions and Measurement Methods (National Bureau of Standards Special Publication 506). Washington, D.C.: Government Printing Office, pp. 325-337 (1978). 7. Environmental Protection Agency, "Asbestos-Containing Materials in Schools Final Rule and Notice", Federal Register, Vol. 52, No. 210,30 October 1987. JNJNL61 i 468 MICROSCOPE(1990)38 V (dBook Reviews "GOD BLESS THE M ICRO SCO PE", A H istory o f the Royal Microscopical Society over 150 years, Gerald L'e Turner, published by the Society, 116 pages, 48 figures. On 3 September 1839,17 microscopists met at No. 50 Wellclose Square at the invitation o f Edwin Quekett, the owner. They met to consider "forming a society for the promotion of microscopi cal investigation, and for the introduction and improvement of the microscope as a scientific instrument". The timing was right, because during that decade Joseph Jackson Lister had published his classic paper on the achromatization of microscope lenses. Only then was the compound microscope able to out-perform the simple (Leeuwenhoek-type) microscope. (Note: Brian Ford tells us that Antony only followed the directions of Hooke, a Britisher who had earlier produced "Leeuwenhoek-type" micro scopes.) In any case, the Society was formed by Quekett and his guests among whom were Joseph Jackson Lister, Jam es S. Bowerbank and Nathaniel Ward. These men had met informally over the previous several years and this culminated in the deci sion taken on 3 September 1839 to form "The Microscopical Society of London". Philologists will be interested to learn that Joseph B. Read, one of the 17, advised the use of microscopical rather than microscopic to prevent "the possibility of ourselves being mistaken for microscopic objects". Dr. Turner's book covers little microscopical science but there is much biographical data and a complete and very read able account of the history of the (after 1867) Royal Microscopi cal Society. It is a great success story with many trials and tribu lations, all overcome with renewed and enhanced status. The book is well written, well illustrated and should be required reading for microscopists. Walter C. McCrone