Document nmMNE5qEw67Z9xxOgEXYX5naR

v. ! I i : i EPA Report No. L~*-e " July 1984 METHODOLOGY FOR THE MEASUREMENT OF AIRBORNE ASBESTOS BY ELECTRON MICROSCOPY by George Yamate Satish C. Agarval Robert D. Gibbons IIT Research Institute Chicago, Illinois 60616 Contract No. 68-02-3266 Project Officer Michael E. Beard Quality Assurance Division Environmental Monitoring Systems Laboratory U.S. Environmental Protection Agency Research Triangle Park, North Carolina 27711 ENVIRONMENTAL MONITORING SYSTEMS LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711 HWBUI0007565 /' .* * , # DISCLAIMER This report has been reviewed by the Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, and approved for publica tion. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. ii HWBUI0007566 FOREWORD ill HWBUI0007567 I PREFACE i f! I I iv HWBUI0007568 ABSTRACT The provisional electron microscope methodology for measuring the concentration of airborne asbestos fibers was refined. The methodology is divided into separate protocols. The step-by-step procedures for each protocol are nearly identical, so that cumulative data can be obtained and uncertainties, especially in asbestos identification, can be clarified. The operational steps encompass (1) type of sample, (2) collection and transport, (3) sample preparation, (4) examination under the transmission electron microscope (TEM) and data collection, (5) data reduction and reporting of results, and (6) quality control-quality assurance. The TEM analytical protocol is subdivided into three levels of anal ysis: Level I, for screening many samples; Level II, for regulatory action; and Level III, for confirmatory analysis of controversial samples. Because identification of asbestos structures is critical, the level of analysis is directly related to the information sought: Level I--morphology and visual selected area electron diffraction (SAED) pattern recognition. Level II--morphology; visual SAED; and elemental analysis. Level III--morphology; visual SAED; a selected number of SAED micrographs of zone-axis patterns; and elemental analysis. This report was submitted in fulfillment of Contract No. 68-02-3266 by IIT Research Institute under the sponsorship of the U.S. Environmental Protection Agency. This report covers the period September 19, 1979, to June 19, 1981, and work was completed as of September 30, 1981. v HWBUI0007569 CONTENTS Page Disclaimer..................................................................................................................................................... ii Foreword.................................................... ......................................................................................... ..... .ill Preface............................................................................................................................................................ iv Abstract................................................................................................................................... ................... v Tables............................................................................................................................................................... ix Figures....................... ........................................................................................................... ..........................x List of Abbreviations............................................................................................................................ xi 1. Introduction.................................................................................................................... 2. Conclusions and Recommendations............... .3 3. Guidelines for Understanding the Methodology................................ ......4 Level of Analysis...............................................................................................................................5 Order of Analysis.................... 5 Collection and Reporting...............................................................................................................5 Costs................................................................................................................................ 5 Application to Nonairbo me Sources....................................................................................... 6 Geographical Considerations........................... 6 Laboratory Conditions............................................................................................................. ..6 4. Level I Analysis........................................................................... .........8 Summary of Protocol.......................................................................................................................... 8 Equipment, Facilities, and Supplies............................................................... ..................... 8 Description of Methodology.......................................................................................................... 9 1. Type of Samples--Source..................................................................... 9 2. Sample Collection and Transport............................................................................... 10 3. Sample Preparation for Analysis--Grid Transfer............................................ 13 4. TEM Examination and Data Collection...............................................................16 5. Data Reduction and Reporting of Results............................................................ 21 6. Quality Control/Quality Assurance...........................................................................22 5- Level II Analysis.............................. .`........................................................................................... 24 Summary of Protocol...................................................... .24 Equipment, Facilities, and Supplies.................................................................................... 25 Description of Methodology..................................... *25 1. Type of Samples--Source..................................................................................................25 2. Sample Collection and Transport............................................................................... 26 3. Sample Preparation for Analysis--Grid Transfer....................... ................... 29 vi 1 HWBUI0007570 CONTENTS (continued) Page 4. TEH Examination and Data Collection..................................................................... 33 5. Data Reduction and Reporting of Results............................................................ 39 6. Quality Control/Quality Assurance........................................................................ 42 6. Level III Asbestos Analysis........................................... ........................ ............................... 44 Discussion of Protocol............................................................................................................... 44 Summary of Protocol...................................................................................................................... 46 Equipment, Facilities , and Supplies..................................................................................4 7 Description of Methodology...................................................................................................... 48 1. Crystallography and Morphological Properties.................. ............................. 48 2. Chemical Properties--Elemental Analysis by EDS...........................................49 3. Selected Area Electron Diffraction (SAED)........................................................ 49 4. Use of Tilting to Acquire Exact Zone-Axis SAED Patterns........................50 5. Characteristics of SAED PatternsEncountered...................................................51 6. Determination of Camera Constant and SAED PatternAnalysis....................53 7. Determination of Camera Constant Using Gold Rings......................................54 8. Measurement of d-Spacings and Interplanar Angles........................................55 9. Identification of Unknown Fibers........................................................................... 58 7. Archival Samples.......................................... Discussion of Protocol............................ Description of Methodology................... 1. Samples with Adequate Loading 2. Samples with Heavy Loading... 60 60 61 61 62 8. Bulk-Sample Analysis..................................................................................................................... 65 Discussion of Protocol............................................................................. Description of Methodology...................................................................................... 65 65 1. Polarized Light Microscopy........................................................................................ 65 2. X-Ray Diffraction Analysis................................................................................. ....66 3. Electron Microscopy.......................................................................................................... 66 9. Numerical Relationships and Analytical Aids 67 Limits of Detection........................................................................................................................ 67 Statistical Methodology........................................................ 68 1. 952 Confidence Limits foraPoisson Variate.......................................................70 2. Comparison of Two PoissonVariates.......................................................................... 70 Magnification Calibration...................................................................................... 73 Preparation of Blanks....................................................................................................................74 Use of Computers............................................................................................................................... 74 References............................................................................................................................................. ....75 vii HWBUI0007571 r- CONTENTS (continued) Appendices A. Figures..........................................................................................................................................76 B. Computer Printout of Level I Analysis (Example).............................................. 96 C. Computer Printout of Level II Analysis (Example)............................................99 viii HWBUI0007572 TABLES Number Page X ProfHe Comparison of AsbesCos Standards*AO 2 Detertainatlon of Camera ConsCanC (Example 3 Dstermination of Spot Spacings (Examples)57 4 Comparison of d-spacings from SAED File and Powder Diffraction File (Example)....................... *..................................................... 59 3 Hypothetical Data69 6 95 Percent Confidence Limits71 7 Control and Test Sample Differences a*o*72! lx HWBUI0007573 FIGURES Number Page A1 Vacuum evaporator............................................................................................................................76 A2 Multiple coating arrangement in evaporator...................................................................77 A3 Close-up of multiple-coating arrangement................................................. '................... 78 A4 Modified Jaffe wick washer method (sketch)...................................................................79 A5 Modified Jaf fe wick washer....................................................................................................... 80 A6 Transmission electron microscope..........................................................................................80 A7 Morphology and counting guidelines used 1 in determining asbestos structure...................................................................................... 81 A8 Level I data sheet (example)...................................................................................................82 A9 Scanning of full-grid opening................................................................................................ 83 A10 Transmission electron microscope with energydispersive spectrometer...84 All Spectra profiles of asbestos standards........................................................................... 85 A12 Level II data sheet (example)................................................................................................ 86 A13 EM data report (example)........................................................................................................... 87 A14 Sample summary report (example)............................................................................................ 88 A15 Effects of tilting and alignment of fiber.....................................................................89 A16 Method of measuring two perpendicular diameters for each ring...................... 89 A17 Method of recognizing a horizontal row of spots...................................................... 90 A18 Relationship of dj , d2 , 01,2> and R................................................................................. 91 A19 Typical zone-axis SAED patterns from amosite standard specimen..................... 92 A20 Typical zone-axis patterns from crocidolite standard specimen....................... 93 A21 Typical zone-axis patterns from tremolite standard specimen............................ 94 A22 Typical SAED patterns and EDAX spectra from anthophyllite standard specimen................................ '.........................................................................................95 x HWBUI0007574 LIST OF ABBREVIATIONS AEM EDS EM JCPDS LTA NIOSH PLM QC/QA SAED SEM STEM TEM TSP UICC XRD XRF analytical electron microscope energy dispersive spectrometer electron microscope Joint Committee on Powder Diffraction Standards low-temperature ashing National Institute of Occupational Safety and Health polarized light microscopy quality control/quality assurance selected area electron diffraction scanning electron microscope scanning transmission electron microscope transmission electron microscope total suspended particulates Union Internationale Contre le Cancer x-ray diffraction x-ray fluorescence xi HWBUI0007575 SECTION 1 INTRODUCTION Asbestos is recognized as a health hazard, especially if inspired into the alveolar region of the respiratory tract. Asbestos may be present in air samples, water samples, biological or clinical samples, and other miscel laneous bulk samples, such as ores and food. These various types of samples require different collection methodologies and diverse preparation techniques. Asbestos analysis methodologies may be categorized as bulk-material analyses, or those providing concentration information, and single-fiber analyses, or those providing morphology, size distribution, and concentra tion. Bulk-material analysis techniques, which include infrared spectroscopy, differential thermal analysis, and x-ray diffraction analysis (XRD), are limited by an inability to analyze concentrations of less than 1 ug, and by an inability to differentiate between fibrous and nonfibrous forms of minerals. Single-fiber analysis techniques include optical microscopy and electron microscopy. Optical microscopy employing phase contrast has been promulgated into a monitoring method for the workplace environment (NIOSH-P&CAM 239). In addition, promulgation of a monitoring method for bulk-material asbestos samples (building insulation) using polarized light microscopy (PLM) is presently being considered. However, optical microscopic techniques cannot determine fibers of less than approximately I urn in diameter, and phase contrast cannot differentiate between asbestos and nonasbestos fibers. The electron microscope (EM) provides particle morphology and size, and a degree of identification. A comprehensive study of various EM procedures i I (Samudra et al., 1977) was conducted in development of a provisional method ology manual, Electron Microscope Measurement of Airborne Asbestos Concentra tions (Samudra, 1978). Three EM methods are available: the scanning electron microscope (SEM), the transmission electron microscope (TEM), and the analyti cal electron microscope (AEM). The SEM, with an x-ray energy-dispersive spectrometer (EDS), permits visual characterization (analogous to reflection optical microscopy) and fiber identification by elemental analysis. The TEM, providing an increased data-acquisition capability, permits visual characteri zation (in the transmitted mode) and fiber identification by crystal structure analysis. The AEM is a TEM with an EDS, and with the added capability of SEM/STEM (scanning transmission electron microscope) operation, which permits visual characterization (morphology and size) as well as fiber identification using both crystal structure by selected area electron diffraction (SAED) and elemental analysis by EDS. 1 HWBUI0007576 The original EM methodology was developed for Che U.S. Environmental Protection Agency (EPA) for measuring airborne asbestos concentrations, specifically for ambient air and for use as a "screening" cool. Development guidelines Included attainable precision and accuracy of results; relative rapidness in use; cost-effectiveness; applicability to a large number of laboratories possessing a TEM (at that time, very few laboratories had TEM's with x-ray analysis capability or an AEM); and procedural steps to be inde pendent of unique or exceptional in-house capabilities of a single laboratory (chat is, interlaboratory precision rather than intralaboratory). In usage, the EM method was successful within its prescribed limita tions--that is, the precision and accuracy of results between laboratories using the complete method was good. However, problems that had been recog nized in the study developing the methodology (Samudra et al., 1977) arose in the areas of (1) interpretation of airborne, (2) sample collection, (3) need for more exacting identification of asbestos, especially of amphibole type, and (4) use of only part of the methodology. The present study was undertaken to refine the methodology. The problem areas and related criticisms were addressed within the underlying goals and guidelines set for optimizing the methodology. Protocols similar to a cook book were not possible since basic knowledge or training was required regard ing (1) sample collection, (2) preparation of samples for EM, (3) use of the TEM-AEM, and (4) diffraction pattern analysis. The refined methodology is based on an assumption that each intended user of a particular level of analy sis has the necessary background and training to use it. 2 HWBUI0007577 SECTION 2 CONCLUSIONS AND RECOMMENDATIONS The EM methodology for measuring the concentration of airborne asbestos fibers has been refined and specified, and is recommended for field evalua tion. The methodology is based on a TEM analytical protocol that is divided into three levels of effort: Level I, for screening many samples; Level II, for regulatory action; and Level III, for confirmatory analysis of controver sial samples. The three-level analytical methodology is cost-effective, and will provide the required results for proper assessment of asbestos. 3 HWBUI0007578 SECTION 3 GUIDELINES FOR UNDERSTANDING THE METHODOLOGY The methodology is divided into separate protocols. The step-by-step procedures for each protocol are nearly identical, so that cumulative data can be obtained and uncertainties, especially in asbestos identification, can be clarified. These operational steps are: (1) Type of Sample--Source (2) Sample Collection and Transport (3) Sample Preparation for Analysis--Grid Transfer (4) TEM Examination and Data Collection (5) Data Reduction and Reporting of Results (6) Quality Control/Quality Assurance (QC/QA). The analytical protocol under the TEM examination and data collection procedure is subdivided into three levels of Increasing analytical effort in terms of requiring an Instrument of greater capability, an electron microscopist with greater expertise, and a longer analytical time. Level I, a monitoring or screening methodology, resembles the present EPA provisional methodology (Samudra et al., 1978; Anderson and Long, 1980). Level II is a regulatory method requiring additional analytical criteria to establish asbestos identification limits, and to provide guidance for Level I analyses by confirming or clarifying visual SAED patterns. Level III, the most sophisticated and the costliest of the methods, is intended for confirming asbestos identification, especially in judicial controversies and other special situations. In Sections 4, 5, and 6, the protocols for each of the three levels of analysis are presented independently of each other, and thus procedures common to each are repeated. All figures are presented in Appendix A. Section 7 describes modifications for using the methodology on archival samples, which are samples collected on nonprocedural filter substrates, or samples collected without regard to filter loading levels. Section 8 describes analysis of inorganic sources in bulk-air samples or in bulk form. Section 9 concludes the report with a discussion of analytical aids pertaining to the limits of detection, preparation of blanks, use of computers, magnification calibration, and statistical methodology. 4 HWBUI0007579 General guidelines for understanding Che methodology are discussed in the following paragraphs. LEVEL OP ANALYSIS Knowledge of the history, source, and location of the sample, and Che purpose and objective of the analysis aids in selecting the correct level of analytical effort. Simply "grinding the samples out" neither is costeffective nor produces the best results, especially for Level II and Level III analyses. Instead of all Level I, all Level II, or all Level III, the majority of the analyses may be Level I, followed by some Level II. Level III could be used in its entirety or only at the analytical phase. If the source is known to contain no amphibole-type interference, or if chrysotile is of interest, gold-coating can be eliminated. If a legal proceeding Is anticipated, Level III analysis will be required where a chain-of-custody record is kept from collection, transport to the laboratory, preparation, analysis, data reduction, and reporting of results. EM finder grids must be used for grid transfer. In addition, for quality assurance, a second laboratory must be available for analyzing a portion of the sample using the same degree of custodial care. QC/QA protocols must be observed and records kept. Whenever possible, and especially for unknown source samples, 10 to 20Z of each set of samples should be analyzed by Level II analysis prior to using Level I as a screening procedure. Level I is a relatively rapid procedure, and can be used by many laboratories with access to a conventional TEM. However, Level I results should not be used in legal proceedings. If "positives" or "false positives" are found, especially in areas where asbestos is known to be absent, and the field blank and laboratory blank have been checked, Level II analysis, and possibly Level III analysis, should be performed. ORDER OF ANALYSIS The order of analysis is (1) field blanks, (2) laboratory blanks (if needed), and (3) field samples. COLLECTION AND REPORTING The counting rule, "minimum 100 fibrous structures per known area (complete grid opening) or 10 grid openings, whichever is first," is a minimum rule for cost limitation. For very low asbestos presence, or for asbestos contamination studies, where particulate loading is high and asbestos presence very low, counting 20 grid openings from each of 2 grids (10 per grid) is recommended. The EM magnification factor is very high or, conversely, the area of deposit examined is very small. Therefore, although the electron microscopist may report a zero count, the notation "Below Detectable Level" is more appro priate in the sample report. Along the same lines, the electron microscopist 5 HWBUI0007580 should report observations, measurements, and conclusions as objectively as possible, realizing the subjective nature of his decision-making, such as parallel-sided, 3:1 aspect ratio, number count, size measurements, recognition-discrimination of SAED patterns, and categorizing of asbestos structure. Data reduction and reporting of results must be consistent and stated. Dimensions of X-fibers (unknown length since complete fiber is not visible) may be doubled, not counted at all, or presented separately. Doubling of the visible portion is recommended, and should be so stated in the report. Mass or conversion of size measurements to an assumed shape-volumedensity relationship, is calculated, and thus is the least reliable of the data, especially for X-fibers, bundles, clusters, and matrices. Although morphology, SAED, and XRF either singly or in combination will provide identification of asbestos, not all structures will be identified. The nature of the asbestos structure prevents analysis of all structures by SAED and/or by elemental analysis with EDS. Such factors as specimen thick ness , orientation, and proximity to other particulates or to the grid wire will prevent attainment of good SAED patterns and limit the effectiveness of chemical analysis. COSTS Levels I, II, and III analyses are estimated to require 200, 400, and 1200 min per analysis, respectively. Additional costs will result from collection, preparation, and reporting of results. The equivalent monetary costs will depend on the laboratory rates of the personnel involved. APPLICATION TO NONAIRJBORNE SOORCES Although the methodology has been developed for airborne asbestos, other types of samples from different sources can be analyzed if the samples are finely divided and placed with proper loading and uniform distribution either on a polycarbonate membrane filter or on a carbon-coated EM grid. Of course, the limitations of the collection and preparation steps must be known and accounted for to prevent inaccuracies In comparing results. GEOGRAPHICAL CONSIDERATIONS In some parts of the country, such as the Upper Great Lakes area, the possibility of mis identification is much greater because some nonamphibole minerals have visual SAED patterns that closely resemble those of amphiboles. Gold-coating and Level II analysis will help in differentiating between these minerals. LABORATORY CONDITIONS Asbestos analysis involves sustained microscopy for periods of 3 to 7 hours with unscheduled rest breaks. Subjective decisions regarding such factors as morphology, size measurement, visual Identification, and possible 6 HWBUI0007581 EDS make it difficult to break a manipulative physical routine or rhythm. Therefore, a professional environment for the microscopist is essential for effective asbestos analysis. In particular, such factors as unnecessary or redundant procedural steps, lack of personal recognition, and unreasonable deadlines may contribute to poor precision. 7 HWBUI0007582 SECTION 4 LEVEL I ANALYSIS SUMMARY OF PROTOCOL Level I analysis is a monitoring or screening technique. It assesses the amount and type of asbestos structures in the atmosphere through the following steps: (1) A known volume of air is passed through a polycarbonate membrane filter (pore diameter, 0.4 pm; filter diameter, 37 or 47 mm) to obtain approximately 5 to 10 yg of particulates per cm2 of filter surface. (2) The particulate-laden filter is transported in its own filter holder. (3) The filter is carbon-coated in the holder. (4) The particulates are transferred to an EM grid using a refined Jaffe wick washer. (5) The EM grid, containing the particulates, is gold-coated lightly. (6) The EM grid is examined under low magnification (250X to 1000X) followed by high-magnification (16,000X on the fluorescent screen) search and analysis. (7) A known area (measured grid opening) is scanned, and the fibrous structures (fibers, bundles, clusters, and matrices) are counted, sized, and identified as to asbestos type (chrysotile, amphibole, ambiguous, or no identity) by morphology and by observing the SAED pattern. (8) The observations are recorded--a minimum of 100 fibrous structures or 10 grid openings, whichever is first. (9) The data are reduced and the results reported. EQUIPMENT, FACILITIES, AND SUPPLIES The following items are required for Level I analysis: (1) An 80 or 100-kV TEH with a fluorescent viewing screen inscribed with graduations for estimating the length and width of fibrous particulates. 8 HWBUI0007583 (2) A vacuum evaporator with a turntable for rotating specimens during coating, for such uses as carbon-coating polycarbonate filters, gold-coating EM grids, and preparing carbon-coated EM grids. (3) An EM preparation room adjacent to the room housing the EM. This room should either be a clean-room facility, or contain a laminar-flow class-100 clean bench to minimize contamination during EM grid preparation. Filter handling and transfer to EM grids should be performed in a clean atmosphere. Laboratory blanks should be prepared and analyzed weekly to ensure quality of work. (4) Several refined Jaffe wick washers for dissolving membrane filters. (5) Miscellaneous EM supplies and chemicals, including carboncoated 200-mesh copper grids, grid boxes, and chloroform. C6) Sample collection equipment, including 37-mm-diamecer or 47-mm-diameter filter holders, 0.4-ym (pore size) polycarbonate filters, 5.0-pm (pore size) cellulose ester membrane filters for back-up, a sampling pump with ancillary equipment, a tripod, critical orifices or flow meters, and a rain/wind shield. DESCRIPTION OF METHODOLOGY 1. Type of Samples--Source This protocol was originally developed for the EPA for measuring airborne asbestos (Samudra et al., 1977; Samudra et al., 1978). A broad interpretation of airborne has been to apply the term to samples obtained from ambient air (the original purpose), aerosolized source materials (such as Che asbestos workplace environment, and fugitive dust emissions), bulk-air material (such, as total suspended particulate (TSP) samples, dust, and powders) and any other type- of sample obtained by nonrestrictive use of (1) collection of a volume of air, (2) separation from the air, and (3) concentration of Che particulates onto a substrate. The airborne protocol has also been applied to samples collected in the regulatory areas of the EPA, as compared with, for example, the workplace environment (National Institute of Occupational Safety and Health), mining activities (U.S. Bureau of Mines), and shipboard atmosphere (Federal Maritime Administration). The present methodology has been optimized for application specifically to samples collected from a volume of air in which the asbestos concentration is considered a minor component of the total particulate loading (other analy tical methods are available for samples known to contain high concentrations of asbestos); and in which the particles are less chan 15 ym in diameter, since particles greater than 15 ym either are not Inhaled or are deposited In the upper respiratory tract and expelled, and preferably less chan 10 ym In diameter as recommended by the Clean Air Scientific Advisory Committee (Hileman, 1981), since particles up to 10 ym can be absorbed by the alveolar 9 HWBUI0007584 region of che lung. These concentration and size restrictions will preclude many air samples collected in an asbestos-processing environment and in bulkair material from the complete methodology. However, such samples can still be examined with the TEM, within the limitations of the instrument, through changes in preparation techniques--provided the effects on the final results, such as fractionation of size and representativeness of the sample, are carefully considered. 2. Sample Collection and Transport Sample Collection-- Sampling procedures vary depending on the nature of the sample, purpose of collection, analytical method to be used, sample substrate, and time and cost of sample collection relative to the total analytical effort. . Neverthe less, the primary objective of sample collection always is to obtain a repre sentative , unbiased sample. Impingers, impaction devices, electrostatic precipitators, and thermal precipitators have been used in sample collection, but each has limitations. Presently, the preferred substrates are membrane filters, which are manu factured from different polymeric materials, including polycarbonate, mixed esters of cellulose, polystyrene, cellulose acetate, and cellulose nitrate. Polycarbonate membrane filters differ from the others in being thin, strong, and smooth-surfaced, and in having sieve-like construction (circular pores from top surface to the bottom). The other membrane filters are thicker, have irregular-surfaces, and have depth-filter construction (tortuous paths from top surface to bottom). Consequently, polycarbonate filters have been selected for airborne asbestos analysis. The collection of small-sized particles (prefer less than 10 uo in diameter), the light loading of particulates, the uniform distribu tion of particulates attainable using a depth-type backing filter, the smooth surface and circular holes (which aid in determining size and instrument tilt axis), and the relative ease in grid transfer (chin and strong) minimize disadvantages of lack of retention and/or movement of large particles during handling. Other membrane materials, such as che cellulose ester type, are recommended for phase contrast and PLM, heavy particle loadings, and physical retention of large particles. In microscopical analysis, uniformity of particulate distribution and loading is critical to success. Air samples are taken on 37-ram-diameter or 47-mm-diamecer, 0.4-pm (pore size) polycarbonate membrane filters using the shiny, smooch side as the particle-capture surface. Cellulose ester-type membrane filters (pore size, 5.0 pm) are used Co support Che polycarbonate filter on che support pad (37-mm-diaraeter personal sampler) or on the support plate (47-mm-diamecer holder). Air monitoring cassettes (37-mm-diaraeter) of three-piece construction are available from several manufacturers. As with the 47-mm-diameter filters, loading che cassettes with che support pad, back-up filter, and 0.4 pm (pore size) polycarbonate filter should be carefully performed on a class-100 clean bench. Since the filters are held in place by pressure fit rather than by 10 HWBUI0007585 screw tightening, air muse not enter from the sides of the unit; a plastic band or tape (which can double as a label) should be used as a final seal. Collecting airborne samples with proper loading requires experience. Each of the following techniques is useful in collecting airborne samples for direct microscopy, preserving representative sizes, without diluting particulate deposits: (1) For long-term sampling at a site, test samples should be returned to the laboratory by express mail service, or air express service or by being hand-carried, and should then be analyzed by scanning electron microscopy. (2) The estimated particulate loading (deposit is barely visible to the naked eye) should be bracketed by varying the filtration rate and using the same time, or by varying the time and using the same filtration rate. (3) An automatic particle counter, such as a light-scattering instrument (0.3-ym detection) or a real-time mass monitor (0.1-pm detection), should be used to obtain an. approximate particulate-loading level of the area. Although any one of the three techniques will work, the suggested tech nique is to take the samples as a set, varying the sampling rates and using the same time so as to obtain filter samples with different particulate loadings. Each set is composed of a minimum of four 37-mm-diameter or 47-mmdiamecer filter units--three for different particulate loadings (low, medium, high), and the fourth for a field blank. Suggested sampling rates are 0 for the field blank, 2.48 L/min for the low loading, 7.45 L/min for the medium, and 17.62 L/min for the high, for a 30 min sampling period using a 47-mmdiameter filter holder. Simultaneous sampling will provide at least one sample with a particulate loading suitable for direct EM analysis. TSP's range from 10 yg/m3 in remote, nonurban areas, to 60 yg/m3 In nearurban areas, to 220 yg/m3 in urban areas. However, for heavily polluted areas, TSP levels may reach 2000 yg/m3. A loading of 5 to 10 yg per cm2 of filter is adequate for EM analysis; values beyond 20 to 25 yg per cm2 require a dilution treatment. As an example, for 47--mm-diameter filters at face velocities of 3.0 cm/s (2.48 L/min), 9.0 cm/s (7.45 L/min), and 21.2 cm/s (17.62 L/min), respectively, air volumes of 74.4 L, 223.5 L, and 528.6 L are sampled in 30 min. For a TSP level of 200 yg/m3, 14.88 yg (1.07 yg/cm2), 44.7 yg (3.23 yg/cm2), and 105.7 yg (7.63 yg/cra2), respectively, would be collected on 47-mm-diameter filters (which would have effective filtration areas of 13.85 cm2). The sampling time could be Increased to 60 min for areas having lower TSP levels, or reduced in a heavily polluted area (source emissions). Airborne samples from emission sources contain coarse particles (above the respirable size) of large matrix structures, binder materials, road dust, clay minerals, fillers, and other materials. For these samples, a fifth filter unit can be added that has a size-selective inlet (cyclone, impactor, or elutriator) attached prior to the filter unit. The flow pattern and flow 11 HWBUI0007586 races of Che candem sampling arrangement oust be checked before use. A satis factory, tested combination presently used In California is a cyclone-filter unit with a D$q cut-off of 2-5 urn at 21.7 L/min, and a D50 cut-off of 3.5 ura at 15.4 L/min (John and Reischl, 1980). Additional sampling devices, such as impingers (used in biological sampling), impactors, and other designated filter units (for TSP, XRJD, or x-ray fluorescence (XEF), for example) can be added to the system, to obtain supplementary as well as interrelated data. This expandable multifilter sampling unit, designated Hydra, offers the following advantages: (1) It is small, inexpensive, and compact, so that an adult can easily handle it. (2) It is efficiently designed, and includes a tripod, sampling pump, manifold, critical orifices, and a row of preloaded 37-mm-diameter or 47--mm-diameter filter holders. A rain/wind shield, size-selective cyclonefilter units, tubing, and other extras can be added as needed. (3) Its sample preparation steps and handling are minimized. (4) It allows complementary as well as supplementary analysis (TSP, size fractionation, bacteria, and XRF, for example), although additional air sampling capacity JLs required. (5) It accommodates ambient air and source emission samples, with or without a size-selective inlet. (6) It allows synchronous sampling in several places in the vicinity following the same sampling procedure, thereby accommodating particulate concentration fluctuations. (7) It includes filter holders that serve as transport and storage units. Hydra's disadvantages are a short sampling period, which may record an episode; a small sampling quantity or volume, which may not indicate the presence of asbestos fibers; and a detection limit of 2 x 10u fibers/s3 for sampling 1 m3 of air with the 47-mm-diameter filter. Using 8 inch x 10 inch, or 102-mm-diameter filter sizes, is not recom mended. The sampling units are designed for purposes other than microscopy. Interchanging the type of sample substrate filter (glass fiber or paper to polycarbonate) does not correct the inherent problems of filter size and sampling 'unit. Sample Storage and Transport-- Once the sample is acquired, its integrity must be assured, and contami' nation and loss of fibers prevented, until it is examined under the EM. The low cost and small size of the 37-mm-dlameter and 47-mm-dlameter filter holders enables them to be used as combination storage and transport con tainers . The filter holders should be maintained in a horizontal position 12 HWBUI0007587 during storage and transport to the laboratory so that the particulate-loaded filters can be removed under optimally controlled conditions in the laboratory. For 47-mm-dlameter holders (open-face) to be used in transport or stor age , the screw cap is carefully removed, and the shiny, waxy, stiff separator paper used to keep the polycarbonate filters apart is carefully placed on the retaining ring. The cap is then carefully screwed back on so that the sepa rator paper seals and protects the particulate-loaded filter without touching it. The 37-mra-diameter, three-piece filter holder (aerosol monitor) is used in its open-face position, and capped after usage for transport and storage. When the more expensive 47-mm-dtameter holder is to be re-used immedi ately, the particulate-loaded filter should be carefully removed and placed in a 47-mm-diaraeter Petri-slide (such as that manufactured by the Millipore Corp.*) this transfer takes place in the field rather than in the laboratory, so that the Petri-slide should be taken into the field. The 37-mm-diameter filter holder or the 47-mm-diameter holder/Petri-slide should be secured and all necessary sample identification marks and symbols applied to the holder. 3. Sample Preparation for Analysis--Grid Transfer Carbon-Coating the Filter-- The polycarbonate filter, with the sample deposit and suitable blanks, should be coated with carbon as soon as possible after sampling is completed. To begin this procedure, the particulate-loaded 47-mm-diameter polycarbonate filter is removed from the holder and transferred carefully to an open-faced 47-mm-diameter Petri-slide for carbon-coating in the vacuum evaporator (see Figure Al, Appendix A). If the 47-mm-diameter filter is already in the Petrislide , the cover is replaced with an open-face cover, minimizing filter disruption. The 37-mm-diameter filter is left in the holder, but the upper lid is removed to create an open-faced filter. The open-faced holders are placed on the rotating turntable in the vacuum evaporator for carbon-coating. Figure A2 shows the multiple-coating arrangement in the evaporator; Figure A3 shows a close-up of the 37-mm-diameter and the modified 47-ma-diameter holders for carbon-coating. For archival filters and those of larger sizes, portions of about 2.5 cm x 2.5 cm should be cut midway between the center and edge using a scalpel. The portions are then attached with cellophane tape to a clean glass micro scope slide and placed on the turntable in the vacuum evaporator for coating. Any high-vacuum carbon evaporator may be used to carbon-coat the filters (CAUTION: carbon sputtering devices should not be used). Typically, the electrodes are adjusted to a height of 10 cm above the level of the filters. A spectrographically pure carbon electrode sharpened to a neck of 0.1 cm x 0.5 cm Is used as the evaporating electrode. The sharpened electrode is * Millipore Corp., 80--T Ashby Rd., Bedford, Mass. 01730 13 HWBUI0007588 placed In its spring-loaded holder so that the neck rests against the flat surface of a second carbon electrode. The manufacturer's instructions should be followed to obtain a vacuum of about 1.33 x 10"3 Pa (1 x 10~5 torr) in the bell jar of the evaporator. With the turntable in motion, the neck of the carbon electrode is evaporated by increasing the electrode current to about 15 A in 10 s, followed by 20 to 25 A for 25 to 30 s. If the turntable is not used during carbon evaporation, the particulate matter may not be coated from all sides, resulting in an undesir able shadowing effect. The evaporation should proceed in a series of short bursts until the neck of the electrode is consumed. Continuous prolonged evaporation should be avoided, since overheating and consequent degradation of the polycarbonate filter may occur. Impeding the subsequent step of dissolving the filter. The evaporation process may be observed by viewing the arc through welders goggles (CAUTION: never look at the arc without appropriate eye protection). Preliminary calculations show that a carbon neck of 5 mm3 volume, when evaporated over a spherical surface 10 cm in radius, will yield a carbon layer that is 40 nm thick. Following carbon-coating, the vacuum chamber is slowly returned to ambient pressure, and the filters are removed and placed in their respective holders or in clean, marked Petri dishes for storage on a clean bench. Transfer of the Sample to the EM Grid-- Transferring the collected particulates from the carbon-coated polycar bonate filter to an EM grid is accomplished in a clean room or on a class-100 clean bench. The transfer is made in a Jaffe wick washer, which is usually a glass Petri dish containing a substrate to support the EM grid/carbon-coated membrane filter combination. Solvent is added to a level to just wet the combination and cause gentle dissolution of the membrane with minimum loss or dislocation of the particulates, resulting in a membrane-free EM grid with particles embedded in the carbon film coating. The substrate support can be stainless steel mesh bridges, filter papers, urethane foams, or combinations of these. The refined Jaffe wick washer is described as follows: (1) The glass Petri dish (diameter, 10 cm; height, 1.5 cm) is made airtight by grinding the top edge of the bottom dish with the bottom of the cover dish, with water and Carborundum* powder (80 mesh); this creates a groundglass seal (closer fit) and minimizes the need to refill the Petri dish with added solvent. (The usual glass Petri dish was found not to retain the solvent for long periods of time, and unless the wicking substrate is kept continuously wet, poor solubility of the membrane filter results, leading to a poor-quality EM grid). * Carborundum is a registered trademark of the Carborundum Co., Carborundum Center, Niagara Falls, N.Y. 14302. 14 HWBUI0007589  (2) A combination of foam and a single sheet of 9-cm filter paper is used as the substrate support. A 3-cm x 3-cm x 0.6-cm piece of polyurethane foam (the packing in Polaroid film boxes) is cut and placed in the bottom ( dish. A 0.5-inch V-shaped notch is cut into the filter paper; the notch Is oriented in line with the side of the foam, creating a well for adding solvent. Spectrographic-grade chloroform (solvent) is poured into the Petri dish through the notch until it is level with the top of the foam (also level with the paper). The foam will swell, and care is needed to avoid adding solvent above the filter paper. (3) On top of the filter paper, pieces of 100-mesh stainless steel screen (0.6 cm x 0.6 cm) are placed, usually in two rows, to make several grid transfers at one time (for I such uses as replicas), and to facilitate maintenance of proper identity of each transfer. (4) A 3-mm section (usually midway between the center and edge) of the carbon-coated polycarbonate filter is cut in a rocking motion with a scalpel. The section may be a square, rectangle, or triangle, and should just cover the 3--mm EM grid. (5) A section is laid carbon-side down on a 200-mesh carboncoated EM grid. (Alternatively, Formvar-coated* grids or uncoated EM grids may be used. Here, the carbon coating on the polycarbonate filter forms the grid substrate.) Minor overlap or underlap of the grid by the filter section can be tolerated, since only the central 2-mm portion of the grid is scanned in the microscope. The EM grid and filter combination is picked up at the edges with the tweezers and carefully laid on the damp 100-mesh stainless steel screen. The EM grid-filter combination will immediately "wet out" and remain on the screen. iI (6) Once all specimens are placed in the washer, more solvent is carefully added through the notch to maintain the liquid level so that it just touches the top of the paper I filter. Raising the solvent level any higher may float the EM grid off the mesh or displace the polycarbonate filter section. I (7) The cover is placed in the washer and oriented in place over the specimen, and a map of the filter/grid/screen arrangement is made on the glass cover and in the logbook. * Formvar is a registered trademark of the Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, Mo. 15 HWBUI0007590 (8) Solvent (chloroform) is added periodically to maintain Che level wichin the washer until the filter is completely dissolved by the wicking action (24 to 48 h). (9) The temperature in the room must remain relatively constant Co minimize condensation of solvent on the bottom of the cover and subsequent falling of solvent drops on the EM grid. Should day-night or other temperature differencials occur, solvent condensation on the under-surface of the cover can be minimized by placing the Jaffe washer at a slight tilt (three glass slides under one edge of the Petri dish parallel to the row of grids) to allow the condensation drops to flow toward the lower edge rather than fall on the EM grids. At temperatures lower than 20"C (68F), the complete filter solution may take longer than 72 h. (10) After the polymer is completely dissolved, the stainless steel mesh screen with the EM grid is picked up while wet and set on lens paper tacked to the bottom of a separate Petri dish. The EM grid is then lifted from and placed next to the screen to dry. When all traces of solvent have evaporated, the grid is stored in a grid box and identified by location and grid box in the logbook. Figure A4 illustrates the Jaffe wick washer method; Figure A3 shows the washer. The foam/filter combination is currently preferred, as is use of a closely fitted (by means of the ground-glass seal) Petri dish. i Gold Coating-- An additional step will aid in subjectively evaluating the SAED pattern. This step is required for specimens from Che upper Great Lakes area and for those of unknown origins. After the particulates on the filter are transferred to the EM grid, the grid is held to a glass slide with double i stick tape for gold-coating in the vacuum evaporator. Several EM grids may be taped to the glass slide for coating at one time. Approximately 10 mm of 0.015-cra-diameter (0.006-inch) pure gold wire is placed in a tungsten basket (10 cm from the rotating table holding the EM grids) and evaporated onto the grid. The thin gold-coating establishes an internal standard for SAED analysis. For some mineral species, an internal standard will clarify visual identifica tion of the pattern of a fibrous particulate as being or not being an amphibole species (for example, minnesotaite as opposed to amosite). With exper ience , differentiation in SAED patterns can be observed. For samples of known geographic origins, gold-coating is optional, since the additional coating hinders observation and identification of small-diameter chrysotile fibers. 4. TP! Examination and Data Collection Low Magnification Examination of Grids-- Figure A6 shows a modern TEM. The grid is observed in the TEM at magnifications of 250X and 1000X to determine its suitability for detailed 16 HWBUI0007591 study at higher magnification. The grid is rejected and a new grid used if: (1) the carbon film over a majority of the grid openings is damaged and not intact; (2) the specimen is 'dark due to incomplete dissolution of the polycarbonate filter; or (3) the particulate loading is coo light (unless a blank) or too heavy with particle-particle interactions or overlaps. TEM Analysis (Morphology and SAD)-- The following guidelines are observed for consistency in the analytical protocol: (1) Magnification at the fluorescent screen is determined by calibration with a diffraction-grating replica in the specimen holder. (2) A field of view or "gate" is defined. On some microscopes, the central rectangular portion of the fluorescent screen, which is lifted for photographic purposes, is convenient to use. On others, a scribed circle or the entire circular screen may be used as the field of view. The area of the field of view must be accurately measurable. (3) The grid opening is selected on a random basis. (4) The analysis, morphology, and SAED are performed at a tilt angle of 0*. (5) The recommended instrument settings are: accelerating voltage, 100 kV; beam current, 100 pA; film magnifi cation, 20.000X (which is equivalent to 16,000X on the fluorescent screen for this instrument); and concentric circles of radii 1, 2, 3, and 4 cm on the fluorescent screen. (6) The grid opening is measured at 1000X. (7) Since asbestos fibers are found isolated as well as with each other or with other particles in varying arrange ments , the fibrous particulates are characterized as asbestos structures: Fiber (F) is a particle with an aspect ratio of 3:1 or greater, with substantially parallel sides. Bundle (B) is a particulate composed of fibers in a parallel arrangement, with each fiber closer than the diameter of one fiber. Cluster (Cl) is a particulate with fibers in a random arrangement such that all fibers are intermixed and no single fiber is isolated from the group. 17 HWBUI0007592 Matrix (M) is a fiber or fibers with one end free and the other end embedded or hidden by a particulate. Combinations of structures, such as matrix and cluster, matrix and bundle, or bundle and cluster, are categorized by the dominant fiber quality--cluster, bundle, or matrix. (8) Counting rules for single fibers are: (a) Particulates meeting the definition of fiber are isolated by themselves. With this definition, edge view of flakes, fragments from cleavage planes, and scrolls, for example, may be counted as fibers. (b) Count as single entities if separation is equal to or greater than the diameter of a single fiber. (c Count as single entities if three ends can be seen. (d) Count as single entities if four ends can be seen. (e) In general, fibers that touch or cross are counted separately. (f) Two or more fibers are counted as a bundle if the distances between fibers are less than the diameter of a single fiber, or if the ends cannot be resolved. (g) Fibrils attached longitudinally to a fiber are counted as part of the fiber and the size (width) is estimated based on the fiber-to-fibril relationship. (h) A fiber partially hidden by grid wires (one or two ends) is counted, but labeled as an X-fiber. If the number of X-fibers is more than 20Z of the fibers identified as asbestos, a larger-mesh EM grid should be used, such as 100 mesh (about 200 ym wide). (9) Sizing rules for asbestos structures are: (a) For fibers, widths and lengths are obtained by orienting the fibers to the inscribed circles on the fluorescent screen. Since estimates are within 1 mm, small-diameter fibers have greater margins of error. Fibers less than 1 mm at the fluorescent screen magnification level are characterized as being 1 mm. A cylindrical shape is assumed for fibers. X-fibers are sized by measuring their entire visible portions in the grid opening. (b) Bundles and clusters are sized by estimating their widths and lengths. The sum of individual diameters is used to obtain the total width, and an average length for the total length. A laminar-sheet shape 18 HWBUI0007593 is assuaed, with the average diameter of the individual fiber as the thickness. ri (c) Matrices are sized by adding the best estimates of individual fiber components. A laminar or sheet ' ! structure is assumed for volume calculation. I (10) The method of sizing is as follows: (a) An asbestos structure is recognized, and its location in the rectangular "gate" relative to the sides, inscribed circles, and other particulates, is memorized. (b) The structure is moved to the center for SAED observation and sizing. I (c) Sizing is performed using the inscribed circles. If the structure, such as a fiber, extends beyond the rectangular "gate" (field of view), it is super imposed across the series of concentric circles (several times, if necessary) until the entire structure is measured. (d) The structure is returned to its original location by recall of the location, and scanning is continued. Figure A7 illustrates some of the counting and morphology guidelines used in determining asbestos structures. TEH Procedure-- The TEM procedure is as follows: ! (1) EM grid quality is assessed at 250X. (2) Particulate loading is assessed at 1000X. (3) A grid opening is selected at random, examined at 1000X, and sized. (4) A series of parallel traverses is made across the grid opening at the film magnification of 20,OOOX. Starting at one corner, and using the tilting section of the fluorescent screen as a "gate" or "chute," the grid opening is traversed. Movement through the "gate" is not continuous, but rather is a stop/go motion. On reaching Che end of one traverse, the image Is moved the width of one "gate," and the traverse is reversed. These parallel traverses are made until the entire grid opening has been scanned. ! (5) Asbestos structures are Identified morphologically and counted as they enter the "gate." 19 HWBUI0007594 (6) The asbestos structure is categorized as fiber (with or without X--) bundle, cluster, or matrix, and sized through use of the inscribed circles. (7) The structure (individual fiber portion) is centered and focused, and the SAED pattern is obtained through use of the field-limiting aperture. (a) SAED patterns from single fibers of asbestos minerals fall Into distinct groups. The chrysotile asbestos pattern has characteristic streaks on layer lines other than the central line, and some streaking also on the central line. Spots of normal sharpness are present on the central layer line and on alternate lines (that is, 2nd, 4th etc.) The repeat distance between layer lines is about 0.53 no. (b) Amphibole asbestos fiber patterns show layer lines formed by very closely spaced dots, and have repeat distances between layer lines also of about 0.53 mu. Streaking in layer lines is occasionally present due to crystal structure defects. (c) Transmission electron micrographs and SAED patterns obtained with asbestos standard samples should be used as guides to fiber identification. An example is the "Asbestos Fiber Atlas" (Mueller et al., 1975). (8) From visual examination of the SAED pattern, the structure is classified as belonging to one of four categories: (1) chrysotile, (2) amphibole group (includes amosite, crocidolite, anthophyllite, tremolite, and actinolite), (3) ambiguous (incomplete spot patterns), or (4) no identification. SAED patterns cannot be inspected for some fibers. Reasons for the absence of a recognizable diffraction pattern include contamination of the fiber, interference from nearby particles, fibers that are too small or too thick, and nonsuitable orientation of the fiber. Some chrysotile fibers are destroyed in the electron beam, resulting in patterns that fade away within seconds of being formed. Some patterns are very faint and can be seen only under the binocular microscope. In general, the shortest available camera length must be used, and the objective lens current may need to be adjusted to give optimum pattern visibility for correct identification. A 20-cm camera length and a 10X binocular are recommended for inspecting the SAED pattern on the tilted screen. HWBUI0007595 (9) Additional grid openings are selected, scanned, and counted until either the total number of structures counted exceeds 100 per known area or a minimum of 10 grid openings has been scanned, whichever is first. (10) The TEM data should be recorded in a systematic form so that it can be processed rapidly. Sample information, instrument parameters, and the sequence of operations should be tabulated for ease in data reduction and subsequent reporting of results. Figure A8 shows an example of a data sheet used in Level I analysis. Figure A9 illustrates the method of scanning a full-grid opening. The "field of view" method of counting previously included in the provisional methodology, which is based on randomly selected fields of view, has been discontinued. Originally, the method was recommended for medium loading level on the filter (50 to 300 fibers per grid opening). However, if samples are collected at three different loading levels and the optimum is selected, this medium loading on the filter will not be used. Samples with grid openings containing 50 to 300 fibers may be used as laboratory fiber preparations or selected source samples, but in field samples the particulate loading is usually of much higher concentration than the fiber. Filter loading is characterized by the particulate concentration, not by fiber concentration. 5. Data Reduction and Reporting of Results Data Seduction-- From the data sheet, size measurements are converted to microns (16.000X screen magnification), mass of asbestos structure is calculated, and other characterizing parameters are calculated through use of a hand calculator or computer. (Appendix B, an example of a computer printout from Level I analysis, shows reduced data--that is, what was found on the specified number of grid openings or area examined.) These measurements are summarized and related to the volume of air sampled and the total effective filtration area (area of deposit). Size measurements of X-fibers may be doubled and noted, or kept as a separate category. Fiber number concentration is calculated from the equation Fibers/m3 Total no. of fibers No. of EM fields Total effective filter area, era* Area of an EM field, cm2 _______________ I Volume of air sampled, ra3 The number of X-fibers, bundles, clusters, and matrices are calculated in a similar manner. X-fibers may be Included with fibers if they are few in 21 HWBUI0007596 number. Similarly, their corresponding mass (from their size measurements) may be included. Fiber mass for each type of asbestos (chrysotile or amphibole) in the sample is calculated by assuming chat both chrysotiles and amphiboles have circular cross-sections (cylindrical shape) and chat the width measurements are one diameter. The density of chrysotiLe is assumed to be 2.6 g/cm3, and of amphiboles to be 3.0 g/cm3. The individual mass is calculated from the equation Mass, pg -- x (length, pm) x (diameter, pm)2 x (density, g/cm3) x 10 6 The total mass concentration of fibers for each type of asbestos is then calculated from the total mass of all the individual fibers of that type. The individual masses of bundles, clusters, and matrices are calculated by assuming a laminar or sheet-like structure with an average thickness of the fiber make-up of the structure. Again, the density of chrysotile is assumed to be 2.6 g/cm3, and of amphiboles to be 3.0 g/cm3. The individual masses are calculated from the equation Mass, pg - (length, pm) x (width, pm) x (thickness, pm) x (density, g/cm3) x 10 6 The total mass for each type of structure for each type of asbestos is the sum of all the individual masses. Other characterizing parameters of the asbestos structures are: (1) length and width distribution of fibers, (2) aspect ratio distribution of fibers, and (3) relationships of fibers, bundles, clusters, and matrices. Reporting of Results-- The data in their acquired and reduced forms are reported as summarized, or, depending on the purpose of the analysis, are further reduced to present the interrelationships of Che various characterizing parameters. Again, the Level I methodology is a monitoring or screening technique, and its limita tions, such as the possibility of "false positives" and misldentification, should be noted. 6. Quality Control/Quality Assurance Sampling procedures will vary depending on the type of sample, objectives of the sampling, and time/cost factors. The primary goals of sampling are to obtain a representative sample at the location and time of sampling, and to maintain sample integrity. The sampling team will have written sampling 22 HWBUI0007597 procedures, and che field chief and/or designated individual will be responsible far all record-keeping (including sample Identification, labeling, logging of data, site description, and meteorological conditions), pre- and post-collection checks, and continuous sample custody and sign-outs until the sample is delivered to the laboratory and transferred to the appropriate quality assurance officer (QAO). Verification of sampling times, flow rates, equipment calibration, and taking of field blanks will be checked and recorded in the field logbook. Samples are turned over to the QAO for logging into a project logbook. Each sample is carefully examined for gross features, such as tears, breaks, and overall condition of container. The QAO registers the as-received sample number and other designated information, and assigns a simple internal code number that will accompany the sample through the preparation stage, grid transfer, grid analysis, data reduction, and reporting of results. After being logged into the project logbook, the sample is transferred to the custody of the electron microscopy staff, where every precaution is taken to maintain sample integrity and to prevent contamination and loss of collected particulates. During storage and transport, the filters in their respective holders are maintained in a horizontal position at all times. The sample logging, handling, and storing procedures ensure that all samples can be readily located and identified throughout the course of a program. The QAO has divisional responsibility for QC/QA activities, and must see that the laboratory maintains high standards. He must be aware of current standards of analysis, and must ensure that internal quality control standards, instrument calibration, and records of samples and completed analyses are kept for ease of later retrieval and use. For quality control, internal laboratory blanks are analyzed at least once a week, which may or may not coincide with a sample batch blank. In addition, a magnification calibration of the EM using a carbon grating replica (2,160 lines per mm) is performed once a week. The results are recorded in an EM instrument log, along with other routine instrumental performance checks. All photographs, TEM, SEM, and STEM Images are recorded in a photo log. These QC results are documented for inspection by the QAO. 23 HWBUI0007598 SECTION 5 LEVEL II ANALYSIS SUMMARY OF PROTOCOL Level II analysis is a regulatory technique consisting of Level I analysis plus chemical elemental analysis. Morphology, size, SAED pattern, and chemical analysis are obtained sequentially. By a process of elimination, mineral fibers are identified as chrysotile, amphlbole, ambiguous, or "no identity" by morphology and SAED pattern. X-ray elemental analysis is used to categorize the amphlbole fibers, identify the ambiguous fibers, and confirm or validate chrysotile fibers. Level II analysis is summarized as follows: (1) A known volume of air is passed through a polycarbonate membrane filter (pore diameter, 0.4 ym; filter diameter 9 37 or 47 mm) to obtain approximately 5 to 10 yg of particulates per cm2 of filter surface. (2) The particulate-laden filter is transported in its own filter holder. (3) The filter is carbon-coated in the holder. (4) The particulates are transferred to an EM grid using a refined Jaffe wick washer. (5) The EM grid, containing the particulates, is gold-coated lightly. (6) The EM grid is examined under low magnification (250X to 1000X) followed by high-magnification (16.000X on the fluorescent screen) search and analysis. (7) A known area (measured grid opening) is scanned, and the fibrous structures (fibers, bundles, clusters, and matrices) are counted, sized, and identified as to asbestos type (chrysotile, amphlbole, ambiguous, or no identity) by morphology and by observing the SAED pattern; and finally by elemental analysis using EDS. (8) The observations are recorded--a minimum of 100 fibrous structures or 10 grid openings, whichever is first. (9) The data are reduced and the results reported. 24 HWBUI0007599 EQUIPMENT, FACILITIES, AND SUPPLIES The following items are required for Level I analysis: (1) A modem 100-k.V TEM equipped with an EDS. A scanning accessory as found in a STEM will Increase Che versatility and analytical capability for very small fibers and for fibers adjacent to other particulate matter. The microscope should be equipped with the fluorescent viewing screen inscribed with graduation of known radii to estimate the length and width of fibrous particulates. (2) A vacuum evaporator with a turntable for rotating specimens during coating, for such uses as carbon-coating .polycarbonate filters, gold-coating EM grids, and preparing carbon-coated EM grids. (3)' An EM preparation room adjacent to the room housing the EM. This room should either be a clean-room facility, or contain a laminar-flow class-100 clean bench to minimize contamination duing EM grid preparation. Filter handling and transfer to EM grids should be performed in a clean atmosphere. Laboratory blanks should be prepared and analyzed weekly to ensure quality of work. (4) Several refined Jaffe wick washers for dissolving membrane filters. (5) Miscellaneous EM supplies and chemicals, including carboncoated 200-mesh copper grids, grid boxes, and chloroform. (6) Sample collection equipment, including 37-mm-diameter or 47-mm-diameter filter holders, 0.4-um (pore size) polycarbonate filters, 5.0-ym (pore size) cellulose ester membrane filters for back-up, a sampling pump with ancillary equipment, a tripod, critical orifices or flow meters, and a rain/wind shield. DESCRIPTION OF METHODOLOGY 1. Type of Samples--Source This protocol is an expansion of the method originally developed for the EPA for measuring airborne asbestos (Samudra et al., 1977; Samudra et al., 1978). A broad interpretation of airborne has been to apply the term to samples obtained from ambient air (the original purpose), aerosolized source materials (such as the asbestos workplace environment, and fugitive dust emissions), bulk-air material (such as total suspended particulate (TSP) samples, dust, and powders) and any ocher type of sample obtained by nonrestrictive use of (1) collection of a volume of air, (2) separation from the air, and (3) concentration of the particulates onCo a substrate. The airborne protocol has also been applied to samples collected in the regulatory areas of the EPA, as compared with, for example, the workplace environment (National 25 HWBUI0007600 Institute of Occupational Safety and Health), mining activities (U.S. Bureau of Mines), and shipboard atmosphere (Federal Maritime Administration). The present methodology has been optimized for application specifically to samples collected from a volume of air in which the asbestos concentration is considered a minor component of the total particulate loading (other analy tical methods are available for samples known to contain high concentrations of asbestos); and in which the particles are less than 15 urn in diameter, since particles greater than 15 yo either are not inhaled or are deposited in the upper respiratory tract and expelled, and preferably less than 10 ym in diameter as recommended by the Clean Air Scientific Advisory Committee (Hileman, 1981), since particles up to 10 ym can be absorbed by the alveolar region of the lung. These concentration and size restrictions will preclude many air samples collected in an asbestos-processing environment and in bulkair material from the complete methodology. However, such samples can still be examined with the TEM, within the limitations of the instrument by changes in preparation techniques--provided the effects on the final results, such as fractionation of size and representativeness of the sample, are carefully considered. 2. Sample Collection and Transport Sample Collection-- Sampling procedures vary depending on the nature of the sample, purpose of collection, analytical method to be used, sample substrate, and time and cost of sample collection relative to the total analytical effort. Neverthe less , the primary objective of sample collection always is to obtain a representative, unbiased sample. Impingers, impaction devices, electrostatic precipitators, and thermal precipitators have been used in sample collection, but each has limitations. Presently, the preferred substrates are membrane filters, which are manufac tured from different polymeric materials, including polycarbonate, mixed esters of cellulose, polystyrene, cellulose acetate, and cellulose nitrate. Polycarbonate membrane filters differ from the others in being thin, strong, and smooth-surfaced, and in having sieve-like construction (circular pores from top surface to the bottom). The other membrane filters are thicker, have irregular-surfaces, and have depth-filter construction (tortuous paths from top surface to bottom). Consequently, polycarbonate filters have been selected for airborne asbestos analysis. The collection of small-sized particles (prefer less than 10 ym in diameter), the light loading of particulates, the uniform distribu tion of particulates attainable using a depth-type backing filter, the smooth surface and circular holes (which aid in determining size and instrument tilt axis), and the relative ease in grid transfer (thin and strong) minimize disadvantages of lack of retention and/or movement of large particles during handling. Other membrane materials, such as the cellulose ester type, are recommended for phase contrast and PLM, heavy particle loadings, and physical retention of large particles. 26 HWBUI0007601 In microscopical analysis, uniformity of particulate distribution and loading is critical Co success- Air samples are taken on 37-mm-dlaraecer or 47-mm-diameter, 0.4-pm (pore size) polycarbonate membrane filters using Che shiny, smooch side as the particle-capture surface. Cellulose ester-type membrane filters (pore size, 5.0 pm) are used to support the polycarbonate filter on the support pad (37-mm-diameter personal sampler) or on the support plate (47-mm-diatnecer holder). Air monitoring cassettes (37-mm-diameter) of three-piece construction are available from several manufacturers. As with the 47-mm-diameter filters, loading the cassettes with Che support pad, back-up filter, and 0.4 0m (pore size) polycarbonate filter should be carefully performed on a class-100 clean bench. Since the filters are held in place by pressure fit rather than by screw tightening, air must not enter from the sides of the unit; a plastic band or tape (which can double as a label) should be used as a final seal. Collecting airborne samples with proper loading requires experience. Each of the following techniques is useful in collecting airborne samples for direct microscopy, preserving representative sizes, without diluting particulate deposits: (1) For long-term sampling at a site, test samples should be returned to the laboratory by express mail service, or air express service or by being hand-carried, and should then be analyzed by scanning electron microscopy. (2) The estimated particulate loading (deposit is barely visible to the naked eye) should be bracketed by varying the filtration rate and using the same time, or by varying the time and using the same filtration rate. (3) An automatic particle counter, such as a light-scattering instrument (0.3-pm detection) or a real-time mass monitor (0.1--u detection), should be used to obtain an approximate particulate-loading level of the area. Although any one of the three techniques will work, the suggested technique is to take the samples as a set, varying the sampling rates and using the same time so as to obtain filter samples with different particulate loadings. Each sec is composed of a minimum of four 37-mm-diameter or 47-mmdiameter filter units--three for different particulate loadings (low, medium, high), and the fourth for a field blank. Suggested sampling rates are 0 for the field blank, 2.48 L/min for the low loading, 7.45 L/min for the medium, and 17.62 L/min for the high, for a 30 min sampling period using a 47-mmdiameter filter holder. Simultaneous sampling will provide at least one sample with a particulate loading suitable for direct EM analysis. TSP's range from 10 pg/m3 in remote, nonurban areas, to 60 pg/m3 in nearurban areas, to 220 pg/m3 in urban areas. However, for heavily polluted areas, TSP levels may reach 2000 pg/3 A loading of 5 to 10 pg per cm2 of filter is adequate for EM analysis; values beyond 20 to 25 pg per cm2 require a dilution treatment. As an example, for 47-mm-diameter filters at face velocities of 3.0 cm/s (2.48 L/min), 9.0 cm/s (7.45 L/min), and 21.2 cm/s 27 HWBUI0007602 (17.62 L/min), respectively, air volumes of 74.4 L, 223.5 L, and 528.6 L are sampled in 30 min. For a TSP level of 200 jig/m3 > 14.88 yg (1.07 ug/cm2), 44.7 ug (3.23 ug/cm2), and 105.7 ug (7.63 ug/c2), respectively, would be collected on 47-ram-diamecer filters (which would have effective filtration areas of 13.85 cm2). The sampling time could be increased to 60 min for areas having lower TSP levels, or reduced in a heavily polluted area (source emissions). Airborne samples from emission sources contain coarse particles (above the respirable size) of large matrix structures, binder materials, road dust, clay minerals, fillers, and other materials. For these samples, a fifth filter unit can be added that has a size-selective inlet (cyclone, impactor, or elutriator) attached prior to the filter unit. The flow pattern and flow rates of the tandem sampling arrangement must be checked before use. A satisfactory, tested combination presently used in California is a cyclonefilter unit wich a D50 cut-off of 2.5 pm at 21.7 L/min, and a D50 cut-off of 3.5 pm at 15.4 L/min (John and Reischi, 1980). Additional sampling devices, such as impingers (used in biological sampling), lmpactors, and other designated filter units (for TSP, XRD, or x-ray fluorescence (XRF), for example) can be added to the system to obtain supplementary as well as inter related data. This expandable multifilter sampling unit, designated Hydra, offers the following advantages: (1) It is small, inexpensive, and compact, so chat an adult can easily handle it. (2) It is efficiently designed, and includes a tripod, sampling pump, manifold, critical orifices, and a row of preloaded 37-mm-diaoeter or 47-mm-diaoeter filter holders. A rain/wind shield, size-selective cyclonefilter units, tubing, and other extras can be added as needed. (3) Its sample preparation steps and handling are minimized. (4) It allows complementary as well as supplementary analysis (TSP, size fractionation, bacteria, and XRF, for example), although additional air sampling capacity is required. (5) It accommodates ambient air and source emission samples, wich or without a size-selective inlet. (6) It allows synchronous sampling in several places in the vicinity following the same sampling procedure, thereby accommodating particulate concentration fluctuations. (7) It includes filter holders that serve as transport and storage units. Hydra's disadvantages are a short sampling period, which may catch an episode; a small sampling quantity or volume, which may not indicate the presence of asbestos fibers; and a detection limit of 2 x 10" fibers/m3 for sampling 1 m3 of air with the 47-mm-diameter filter. 28 HWBUI0007603 Using 8 inch x 10 inch, or 10 2-mra-d lame ter filter sizes, is nor recom mended. The sampling units are designed for purposes other than microscopy. Interchanging the type of sample substrate filter (glass fiber or paper to polycarbonate) does not correct the inherent problems of filter size and sampling unit. Sample Storage and Transport-- Once the sample is acquired, its integrity must be assured, and contami nation and loss of fibers prevented, until it is examined under the EM. The low cost and small size of the 37-mm-diameter and 47-mm-diameter filter holders enables them to be used as combination storage and transport con tainers. The filter holders should be maintained in a horizontal position during storage and transport to the laboratory so that the particulate-loaded filters can be removed under optimally controlled conditions in the labora tory. For 47-mm-diameter holders (open-face) to be used in transport or storage, the screw cap is carefully removed, and the shiny, waxy, stiff separator paper used to keep the polycarbonate filters apart is carefully placed on the retaining ring. The cap is then carefully screwed back on so that the separator paper seals and protects the particulate-loaded filter without touching it. The 37-mm-diameter, three-piece filter holder (aerosol monitor) is used in its open-face position, and capped after usage for transport and storage. When the more expensive 47-mm-diameter holder is to be reused immedi ately, the particulate-loaded filter should be carefully removed and placed in a 47-mm-diameter Petri-slide (such as that manufactured by the Millipore Corp.*). This transfer takes place in the field rather than in the labora tory, so that the Petri-slide should be taken into the field. The 37-mmdiameter filter holder or the 47--mm--diameter holder/Petri-slide should be secured and all necessary sample identification marks and symbols applied Co the holder. 3. Sample Preparation for Analysis--Grid Transfer Carbon-Coating the Filter-- The polycarbonate filter, with the sample deposit and suitable blanks, should be coated with carbon as soon as possible after sampling is completed. To begin this procedure, the particulate-loaded 47-mm-diameter polycarbonate filter is removed from the holder and transferred carefully to an open-faced 47-mm-diameter Petri-slide for carbon--coating in the vacuum evaporator (see Figure A1, Appendix A). If the 47-mm-diameter filter is already in the Petri-slide, the cover is replaced with an open-face cover, minimizing filter disruption. The 37-mm-diameter filter is left In Che holder, but the upper lid is removed to create an open-faced filter. The open-faced holders are placed on the rotating turntable in the vacuum evaporator for carbon-coating. Figure A2 shows the multiple-coating * Millipore Corp., 80-T Ashby Rd., Bedford, Mass. 01730 HWBUI0007604 arrangement In Che evaporator; Figure A3 shows a close-up of the 37-mradiameter and the modified 47-mm-diameter holders for carbon-coacing. For archival filters and chose of larger sizes, portions of about 2.5 era x 2.5 era should be cue midway between the center and edge using a scalpel. The portions are Chen attached with cellophane tape to a clean glass microscope slide and placed on che turntable in the vacuum evaporator for coating. Any high-vacuum carbon evaporator may be used to carbon-coat the filters ' (CAUTION: carbon sputtering devices should not be used). Typically, che electrodes are adjusted to a height of 10 cm above the level of che filters. A speccrographically pure carbon electrode sharpened to a neck of 0.1 cm x 0.5 cm is used as the evaporating electrode. The sharpened electrode is placed in its spring-loaded holder so that the neck rests against the flat surface of a second carbon electrode. The manufacturer's instructions should be followed to obtain a vacuum of about 1.33 x 10~3 Pa (1 x 10_s torr) in the bell jar of the evaporator. With the turntable in motion, che neck of the carbon electrode Is evaporated by increasing the electrode current to about 15 A in 10 s, followed by 20 to 25 A for 25 to 30 s. If the turntable is not used during carbon evaporation, the particulate matter may not be coated from all sides, resulting in an undesir able shadowing effect. The evaporation should proceed in a series of short bursts until the neck of the electrode is consumed. Continuous prolonged evaporation should be avoided, since overheating and consequent degradation of the polycarbonate filter may occur, impeding the subsequent step of dissolving the filter. The evaporation process may be observed by viewing the arc through welders goggles (CAUTION: never look at the arc without appropriate eye protection). Preliminary calculations show that a carbon neck of 5 mm3 volume, when evaporated over a spherical surface 10 cm in radius, will yield a carbon layer that is 40 nm thick. Following carbon-coating, the vacuum chamber is slowly returned to ambient pressure, and the filters are removed and placed in their respective holders or in clean, marked Petri dishes for storage on a clean bench. Transfer of che Sample to Che EM Grid-- Transferring the collected particulates from the carbon-coated polycar bonate filter to an EM grid is accomplished in a clean room or on a class-100 clean bench. The transfer is made in a Jaffe wick washer, which is usually a glass Petri dish containing a substrate to support the EM grid/carbon-coated membrane filter combination. Solvent, is added to a level to just wet the combination and cause gentle dissolution of the membrane with minimum loss or dislocation of the particulates, resulting In a membrane--free EM grid with particles embedded in the carbon film coating. The substrate support can be stainless steel mesh bridges, filter papers, urethane foams, or combinations of these. 30 HWBUI0007605 The refined Jaffe wick washer Is described as follows: (1) The glass Petri dish (diameter, 10 cm; height, 1.5 cm) is made airtight by grinding the top edge of the bottom dish with the bottom of the cover dish, with water and Carborundum* powder (80 mesh); this creates a groundglass seal (closer fit) and minimizes the need to refill the Petri dish with added solvent. (The usual glass Petri dish was found not to retain the solvent for long periods of time, and unless the wicking substrate is kept continuously wet, poor solubility of the membrane filter results, leading to a poor-quality EM grid). (2) . A combination of foam and a single sheet of 9-cm filter ' paper is used as the substrate support. A 3-cm x 3-cra x 0.6-cm piece of polyurethane foam (the packing in Polaroid film boxes) is cut and placed in the bottom dish. A 0.5-inch V-shaped notch is cut Into the filter paper; the notch is oriented in line with the side of the foam, creating a well for adding solvent. Spectrographic-grade chloroform (solvent) is poured into the Petri dish through the notch until it is level with the top of the foam (also level with the paper). The foam will swell, and care is needed to avoid adding solvent above the filter paper. (3) On top of the filter paper, pieces of 100-mesh stainless steel screen (0.6 cm x 0.6 cm) are placed, usually in two rows, to make several grid transfers at one time (for such uses as replicas), and to facilitate maintenance of proper identity of each transfer. (4) A 3-mm section (usually midway between the center and edge) of the carbon-coated polycarbonate filter is cut in a rocking motion with a scalpel. The section may be a square, rectangle, or triangle, and should just cover the 3-mm EM grid. (5) A section is laid carbon-side down on a 200-mesh carboncoated EM grid. (Alternatively, Formvar-coatedf grids or uncoated EM grids may be used. Here, the carbon coating on the polycarbonate filter forms the grid substrate.) Minor overlap or underlap of the grid by the filter section can be tolerated, since only the central 2-otm portion of the grid is scanned in the microscope. The EM grid and filter combination is picked up at the edges with the tweezers and carefully laid on the damp 100-mesh * Carborundum is a registered trademark of the Carborundum Co., Carborundum Center, Niagara Falls, N.Y. 14302. T Formvar is a registered trademark of the Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, Mo. 31 HWBUI0007606 stainless steel screen. The EM grid-filter combination will immediately "wet out" and remain on the screen. (6) Once all specimens are placed in the washer, more solvent is carefully added through the notch to maintain the liquid level so that it just touches the top of the paper filter. Raising Che solvent level any higher may float the EM grid off the mesh or displace the polycarbonate filter section. (7) The cover is placed in the washer and oriented in place over the specimen, and a map of the filter/grid/screen arrangement is made on the glass cover and in the logbook. (8) Solvent (chloroform) is added periodically to maintain the level within the washer until the filter is completely dissolved by the wicking action (24 to 48 h). (9) The temperature in the room must remain relatively constant Co minimize condensation of solvent on the bottom of the cover and subsequent falling of solvent drops on the EM grid. Should day-night or other temperature differentials occur, solvent condensation on the under-surface of the cover can be minimized by placing the Jaffe washer at a slight tilt (three glass slides under one edge of the Petri dish parallel to the row of grids) to allow the condensation drops to flow toward the lower edge rather than fall on the EM grids. At temperatures lower than 20C (68F), the complete filter solution may take longer than 72 h. (10) After the polymer is completely dissolved, the stainless steel mesh screen with the EM grid is picked up while wet and set on lens paper tacked to the bottom of a separate Petri dish. The EM grid is then lifted from and placed next to the screen to dry. When all traces of solvent have evaporated, the grid is scored in a grid box and identified by location and grid box in the logbook. Figure A4 illustrates the Jaffe wick washer method; Figure A5 shows the washer. The foam/filter combination is currently preferred, as is use of a closely fitted (by means of the ground-glass seal) Petri dish. Gold Coating-- An additional step will aid in subjectively evaluating the SAED pattern. This step is required for specimens from the upper Great Lakes area and for chose of unknown origins. After the particulates on the filter are trans ferred to the EM grid, the grid is held to a glass slide with double-stick tape for gold-coating in the vacuum evaporator. Several EM grids may be taped to the glass slide with double-stick tape for gold-coating in the vacuum evap orator. For comparison, one-half of the EM grids may be coated and the other one-half not coated; recognition of the gold-coating is helpful in searching and x-ray analysis. Several EM grids may be taped to the glass slide for coacing ac one time. Approximately 10 mm of 0.015-cm-diaraeter (0-006-inch) pure gold wire is placed in a tungsten basket (10 cm from the rotating table holding the EM grids) and evaporated onto the grid. The thin gold-coating establishes an internal standard for SAED analysis. For some mineral species, an internal standard will clarify visual identifi cation of the pattern of a fibrous particulate as being or not being an amphlbole species (for example, minnesotaite as opposed to amosite). With experience, differentiation in SAED patterns can be observed. For samples of known geographic origins, gold-coating is optional, since the additional coacing hinders observation and identification of small-diameter chrysotile fibers. 4. TEH Examination and Data Collection Figure A10 shows a modern TEM with capabilities for elemental analysis with an EDS. The grid is observed in the TEM at magnifications of 250X and 1000X to determine its suitability for detailed study at higher magnifica tion. The grid is rejected and a new grid used if: (1) the carbon film over a majority of Che grid openings is damaged and not intact; (2) the specimen is dark due to incomplete dissolution of the polycarbonate filter; or (3) the particulate loading is too light (unless a blank) or too heavy with particleparticle interactions or overlaps. TEM Analysis (Morphology, SAED, and X-Ray Analysis)-- The following guidelines are observed for consistency in the analytical protocol: (1) Magnification at the fluorescent screen is determined by calibration with a diffraction-grating replica in the specimen holder. (2) A field of view or "gate" is defined. On some microscopes, the central rectangular portion of the fluorescent screen, which is lifted for photographic purposes, is convenient to use. On others, a scribed circle or the entire circular screen may be used as the field of view. The area of the field of view must be accurately measurable. (3) The grid opening is selected on a random basis. (4) The analysis, morphology, and SAED are performed at a tilt angle of 0. (5) The recommended instrument settings are: accelerating voltage, 100 kV; beam current, 100 yA; film magnifi cation , 20.000X (which is equivalent to 16,000X on the fluorescent screen for this instrument); and concentric circles of radii 1, 2, 3, and 4 cm on the fluorescent screen. 33 HWBUI0007608 (6) The grid opening Is measured at low magnification (about 1000X). (7) Since asbestos fibers are found isolated as well as with each ocher or with other particles in varying arrange ments , the fibrous particulates are characterized as asbestos structures: Fiber (F) is a particle with an aspect ratio of 3:1 or greater with substantially parallel sides. Bundle (B) is a particulate composed of fibers in a parallel arrangement, with each fiber closer than the diameter of one fiber. Cluscer (Cl) is a particulate with fibers in a random arrangement such that all fibers are intermixed and no single fiber is isolated from the group. Matrix is a fiber or fibers with one end free and the other end embedded or hidden by a particulate. Combinations of structures, such as matrix and cluster, matrix and bundle, or bundle and cluster, are categorized by the dominant fiber quality--cluster, bundle, and matrix. (8) Counting rules for single fibers, which are illustrated in Figure A7 are as follows: (a) Particulates meeting the definition of fiber are Isolated by themselves. With this definition, edge view of flakes, fragments from cleavage planes, and scrolls, for example, may be counted as fibers. (b) Count as single entities if separation is equal to or greater than the diameter of a single fiber. (c) Count as single entities if three ends can be seen. (d) Count as single entities if four ends can be seen. (e) In general, fibers that touch or cross are counted separately. (f) Two or more fibers are counted as a bundle if the distances between fibers are less than the diameter of a single fiber, or if the ends cannot be resolved. (g) Fibrils attached longitudinally to a fiber are counted as part of the fiber and the size (width) is estimated based on the fiber-to-fibril relationship. (h) A fiber partially hidden by grid wires (one or two sides of the grid opening) is counted, but labeled as an X-fiber (X-F) in the structure column. If the number of X-fibers is high enough to affect the size distribution (mass, etc.), a large-mesh EM grid should be used, such as 100 mesh (about 200 ym wide). (9) Sizing rules for asbestos structures are: (a) For fibers, widths and lengths are obtained by orienting the fibers to the inscribed circles on the fluorescent screen. Since estimates are within 1 mm, small-diameter fibers have greater margins of error. Fibers less than 1 mm at the fluorescent screen magnification level are characterized as being 1 mm. A cylindrical shape is assumed for fibers. X-fibers are sized by measuring their entire visible portions in the grid opening. (b) Bundles and clusters are sized by estimating their widths and lengths. The sum of individual diameters is used to obtain the total width, and an average length for the total length. A laminar-sheet shape is assumed, with the average diameter of the individual fiber as the thickness. (c) Matrices are sized by adding the best estimates of individual fiber components. A laminar or sheet structure is assumed for volume calculation. (10) The method of sizing is as follows: (a) An asbestos structure is recognized, and its location in the rectangular "gate" relative to the sides, inscribed circles, and other particulates is memorized. (b) The structure is moved to the center for SAEO observation and sizing. (c) Sizing is performed using the inscribed circles. If the structure, such as a fiber, extends beyond the rectangular gate (field of view), it is superimposed across the series of concentric circles (several times, if necessary) until the entire structure is measured. (d) The structure is returned to its original location by recall of the location, and scanning is continued. 35 HWBUI0007610 Analytical Procedure-- The analytical procedure is as follows: (1) EM grid quality is assessed at 250K. (2) Particulate loading is assessed at 1000X. (3) A grid opening is selected at random, examined at 1000X, and sized, (4) A series of parallel traverses is made across Che grid opening at the film magnification of 2Q,000X. Starting at one corner, and using the tilting section of the fluorescent screen as a "gate" or "chute," the grid opening is traversed. Movement through the "gate" is not continuous, but rather is a stop/go motion. On reaching the end of one traverse, the image is moved the width of one "gate," and the traverse is reversed. These parallel traverses are made until the entire grid opening has been scanned. (5) Asbestos structures are identified morphologically and counted as they enter the "gate." (6) The asbestos structure is categorized as fiber (with or without X-) bundle, cluster, or matrix, and sized through use of the inscribed circles. (7) The structure (individual fiber portion) is centered and focused, and the SAED pattern is obtained through use of the field-limiting aperture. (a) SAED patterns from single fibers of asbestos minerals fall into distinct groups. The chrysotile asbestos pattern has characteristic streaks on layer lines other than the central line, and some streaking also on the central line. Spots of normal sharpness are present on the central layer line and on alternate lines (that is, 2nd, 4th etc.) The repeat distance between layer lines is about 0.53 nm. (b) Amphibole asbestos fiber patterns show layer lines . formed by very closely spaced dots, and have repeat distances between layer lines also of about 0.53 nn. Streaking in layer lines is occasionally present due Co crystal structure defects. (c) Transmission electron micrographs and SAED patterns obtained with asbestos standard samples should be used as guides to fiber identification. An example is the "Asbestos Fiber Atlas" (Mueller et al., 1975). (8) From visual examination of the SAED pattern, the struc ture is classified as belonging to one of four cate gories: (1) chrysotile, (2) amphibole group (includes amosice, crocidolite, anthophyllite, tremolite, and actinolite), (3) ambiguous (incomplete spot patterns), or (4) no identification. SAED patterns cannot be inspected for some fibers. Reasons for the absence of a recog nizable diffraction pattern include contamination of the fiber, interference from nearby particles, fibers that are too small or too thick, and nonsuitable orientation of the fiber. Some chrysotile fibers are destroyed in the electron beam, resulting in patterns that fade away within seconds of being formed. Some patterns are very faint and can be seen only under the binocular micro scope . In general, the shortest available camera length must be used, and the objective lens current may need to be adjusted to give optimum pattern visibility for correct identification. A 20-cm camera length and a 10X binocular are recommended for inspecting the SAED pattern on the tilted screen. (9) The specimen holder is tilted for optimum x-ray detection (40 tilt for the JEOL* 100C instrument's Tracor Northernf NS 880 analyzer and Kevex* detector). The categorized asbestos structure is maintained in its centered position for x-ray analysis by means of the Zcontrol. (10) The spot size of the electron beam is reduced and stigmated to overlap the fiber. As an option for STEM instruments, the electron beam may be used in the spot mode and the x-ray analysis performed on a small area of the structure. (11) The EDS is used to obtain a spectrum of the x-rays generated by the asbestos structure. (12) The profile of the spectrum is compared with profiles obtained from asbestos standards; the best (closest) match Identifies and categorizes the structure. The image of the spectrum may be photographed, or the peak heights (Na, Mg, Si, Ca, Fe) recorded for normalizing at a later time. No background spectra or constant acquisi tion time is required since the shape of the spectrum (profile) is the criteria. Acquisition of x-ray counts may be to a constant time; to a constant peak height for a selected element, such as silicon (1.74 keV); or just * JEOL (U.S.A.) Inc., 11 Dearborn Road, Peabody, Mass. 01960 t Tracor Northern Inc., 2551-T.W. Beltway Hvy., Middleton,.Wis. 53562 * Kevex Corp., Chess Dr., Foster City, Calif. 94404 37 HWBUI0007612 long enough to gee an adequate idea of the profile of Che spectra, and then aborted. Figure All illustrates spectra obtained from various asbestos standards and used as referenced profiles. (13) The specimen holder is returned to 0" tilt to examine other asbestos structures. (14) Scanning is continued until all structures are identified, measured, analyzed, and categorized in the I grid opening. (15) Additional grid openings are selected, scanned, and counted until either the total number of structures I counted exceeds 100 per known area, or a minimum of 10 grid openings has been scanned, whichever is first. (16) The TEM data should be recorded in a systematic form so that they can be processed rapidly. Sample information, instrument parameters, and the sequence of operations should be tabulated for ease in data reduction and subsequent reporting of results. Figure A12 shows an example of a data sheet used in Level II analysis. Figure A9 illustrates the method of scanning a full-grid opening. The "field of view" method of counting, which is based on randomly selected fields of view, has been discontinued. Originally, the method was recommended for medium loading level on the filter (50 to 300 fibers per grid opening). How ever, if samples are collected at three different loading levels and Che opti mum is selected, this medium loading on Che filter will not be used. Samples with grid openings containing 50 to 300 fibers may be used as laboratory fiber preparations or selected source samples, but in field samples, the particulate loading is usually of much higher concentration chan the fiber. Filter load ing is characterized by particulate concentration, not by fiber concentration. EDS is relatively time-consuming, and becomes redundant if used as repetitive analysis for a confirmatory check on chrysotile fibers. Chrysotile identity by morphology and visual SAED analysis is not as controversial as I amphibole identification and categorization. The following rules are recommended for EDS analysis (Level II) : (1) For chrysotile structure identification, the first five are analyzed by EDS, then one out of every 10. (2) For amphibole structure identification, Che first 10 are analyzed by EDS, then one out of every 10. (3) For amphibole structure identification and categorization, all confirmed amphiboles are analyzed by EDS. (4) For ambiguous structure identification and categorization, all are analyzed by EDS. 38 HWBUI0007613 -j Energy dispersive x-ray analysis as used in asbestos analysis is seraiquanticacive at best. X-ray analyzer manufacturers may claim quantitative .I i I results based on calibration standards and sophisticated computer software, but such claims are based on stoichiometric materials and extension of work with XRF instrumentation. Asbestos has a varying elemental composition. The electron beam in an EM is of varying size, and not all instruments are equipped to measure the beam current hitting the specimen. The size of the specimen has an effect on the x-ray output, and nearby materials may fluoresce and add to the overall x-ray signals being generated. Moreover, specimen tilting results in a loss of x-ray acquisition from particles hidden by grid wires or by other particles. The only consistency in x-ray analysis is that the intensity of the output, within restrictions, is proportional to the mass, therefore providing the semiquantitative analytical possibility. Asbestos minerals have been found to have a characteristic profile, although not an exact duplicate of each other. For example, the Mg:Si ratio of chrysotile may vary from 5:10 to 10:10, averaging about 7:10. The ratio can be used to confirm the morphology and visual SAED analysis. Table 1 illustrates the phenomena of variability with resemblance for some of the amphibole fibers. Peak heights and profile measurements were taken. To aid in the visual perspective of the spectrum profile, the peak heights were normalized to a silicon value of 10, resulting in a five-number series that is relatively easy to visualize--as in the following examples: chrysotile tremolite crocidolite anthophyllite amoslte ~ 0-7-10-0-0 ~ 0-4-10-3-<l ~ 1-1-10-0-6 ~ 0-3-10-0-1 ~ 0-2-10-0-7 These relationships are approximate, since chrysotile can vary from 0-5-10-0-0 to 0-10-10-0-0. However, for the others, the variation is only about one point, such that the profile (shape) of the five elements (Na, Mg, Si, Ca, Fe) is recognizable. 5. Data Reduction and Reporting of Results Data Reduction-- From the data sheet, size measurements are converted to microns (16,Q00X screen magnification), mass of asbestos structure Is calculated, and ocher characterizing parameters are calculated through use of a hand calculator or computer. (Appendix C, an example of a computer printout from Level II analysis, shows reduced data--that Is, what was found on the specified number of grid openings or area examined.) These measurements are summarized and related to the volume of air sampled and the total effective filtration area 39 HWBUI0007614 TABLE 1. PROFILE COMPARISON OF ASBESTOS STANDARDS Asbestos Tjrp* Anoslte (CF-38A) Sice U 0.19 c 1.44 (stignate) 0.19 X 0.75 (STEM) 0.19 x 1.25 0.19 x 0.88 (100 a) 0.25 x 1.81 (100 a) 0.12 x 1.56 0.31 x 2.38 0.19 x 1.56 Repeat Its m Si Ca Fe Profile 182 497 186 526 181 . 352 226 870 576 4207 253 2049 256 2127 276 1696 477 2945 386 387 289 674 3338 1515 1613 1116 ' 1959 0--3--1o-n-a n-i-in-n-a n-i-in-n-7 O-i-in-o-R 0-2-10-0-7 0-2-10-0-7 Anrhophylllte (AF-45) 0.56 x 2.38 (stignate) 0.31 x 2.38 (stignate) 0.31 x 5.19 (stignate) 0.19 x 1.56 (stignate) 0.19 x l.Bfl (stignate) 631 640 1064 507 787 2577 1670 3610 2191 2286 349 n-2-lo-o-i 71 n-4-io-o-o 466 0-3-1O-0-1 309 n-2-io-o-i 257 0** 3" t J Crseidoii.ee (CR-37) 0.19 x 0.81 (stignate) 0.06 x 0.50 (stignate) 0.06 x 0.69 (stignate) 0.12 x 1.00 (stignate) Repeat (STEM) 0.12 x 0.62 (stignate) 0.12 x 1.12 (stignate) 0.19 x 1.56 (stignate) 0.06 x 1.69 (stignate) Repeat (STEM) Repeat (STEM) Repeat (STEM) 131 ion 885 28 28 205 37 35 171 44 53 379 70 64 612 56 65 479 53 56 326 78 83 735 45 4R 290 72 85 892 35 42 373 16 22 166 501 2-1-10-0-4 115 l-l-jn-fwf. 96 204 i-i-lo-o-5 333 1-1-10-0-5 260 l-i-io-o-s 166 2-2-io-o-5 421 )-;_!0-0-6 159 2-2-jO-o-ft 463 l-l-to-0-5 237 1-1-10-0-6 104 1-1-10-0-6 Treoollte (T-79) 0.38 x 2.19 (stignate) 0.38 x 2.19 (spot) 0.25 x 1.75 (stignate) 0.25 x 1.75 (spot) Repeat (stignate). (STSM-ino *) (STEM-tno ,) (STEM-1 on ,,) (stem-ioo a> (STEM-IOO a) (STEM-40 *) (STEM-40 ) 138 114 80 95 70 376 135 1454 64 1072 46 123 368 327 197 252 211 1118 364 4Rin ii 3114 113 313 3 80 65 62 51 24 5 72 1235 48 8*2 27 qa 1-3-10-2-'' 0-5-10.3-0 jv-3-I 2 0-3-10-3-0 0-4-10-2-0 n-t-to-s-o 40 HWBUI0007615 (area of deposit). Size measurements of X-fibers may be doubled and noted, or kept as a separate category. Fiber number concentration is calculated from the equation Fibers/m3 Total no. of fibers No. of EM fields Total effective filter area, cm2 Area of an EM field, cm2 1 Volume of air sampled, m3 The number of X-fibers, bundles, clusters, and matrices are calculated in a similar manner. X-fibers may be Included with fibers if they are few in number. Similarly, their corresponding mass (from their size measurements) may be included. Fiber mass for each type of asbestos (chrysotile or amphibole) in the sample is calculated by assuming that both chrysotiles and amphiboles have circular cross-sections (cylindrical shape) and that the width measurements are one diameter. The density of chrysotile is assumed to be 2.6 g/cm3, and of amphiboles to be 3.0 g/cm3. The individual mass is calculated from the equation Mass, jxg = x (length, ym) x (diameter, ym)2 x (density, g/cm3) x 10 6 The total mass concentration of fibers for each type of asbestos is then calculated from the total mass of all the individual fibers of that type. The individual masses of bundles, clusters, and matrices are calculated by assuming a laminar or sheet-like structure with an average thickness of the fiber make-up of the structure. Again, the density of chrysotile is assumed to be 2.6 g/cm3, and of amphiboles to be 3.0 g/cm3. The Individual masses are calculated from the equation Mass, yg - (length, ym) x (width, ym) x (thickness, yo) x (density, g/cm3) x 10 6 The total mass for each type of structure for each type of asbestos is the sum of all the individual masses. 41 HWBUI0007616 Other characterizing parameters of the asbestos structures are: (1) length and width distribution of fibers, (2) aspect ratio distribution of fibers, and (3) relationships of fibers, bundles, clusters, and matrices. Reporting of Results-- The data and their subsequent reduction are reported as summarized, or can be further reduced to present the interrelationships of the various characterizing parameters. Figure A13 is an example of the EM data report; Figure A14 is an example of the sample summary report. The methodology can establish the limits of identity for unknown samples, act as a QC/QA method for Level I analysis, and satisfy most of the identification criteria for asbestos. 6 Quality Control/Quality Assurance Sampling procedures will vary depending on the type of sample, objectives of the sampling, and time/cost factors. The primary goals of sampling are to obtain a representative sample at the location and time of sampling, and to maintain sample integrity. The sampling team will have written sampling procedures, and the field chief and/or designated individual will be respon sible for all record-keeping (including sample identification, labeling, logging of data, site description, and meteorological conditions), pre- and post-collection checks, and continuous sample custody and sign-outs until the sample is delivered to the laboratory and transferred to the appropriate quality assurance officer (QAO). Verification of sampling times, flow rates, equipment calibration, and taking of field blanks will be checked and recorded in the field logbook. Samples are turned over to the QAO for logging into a project logbook. Each sample is carefully examined for gross features, such as tears, breaks, and overall condition of container. The QAO registers the as-received sample number and ocher designated information, and assigns a simple internal code number chat will accompany the sample through the preparation stage, grid transfer, grid analysis, data reduction, and reporting of results. After being logged into the project logbook, the sample is transferred to the custody of the electron microscopy staff, where every precaution is taken to maintain sample integrity and to prevent contamination and loss of collected particulates. During storage and transport, the filters In their respective holders are maintained in a horizontal position at all times. The sample logging, handling, and storing procedures ensure that all samples can be readily located and identified throughout the course of a program. The QAO has divisional responsibility for QC/QA activities, and must see that the laboratory maintains high standards. He must be aware of current standards of analysis, and must ensure that Internal quality control standards, instrument calibration, and records of samples and completed analyses are kept for ease of later retrieval and use. 42 HWBUI0007617 For quality control, internal laboratory blanks are analyzed at least once a week, which may or may not coincide with a sample batch blank. In addition, a magnification calibration of the EM using a carbon gracing replica (2,160 lines per mm) is performed once a week. The results are recorded in an EM instrument log, along with other routine instrumental performance checks. All photographs, TEM, SEH, and STEM images are recorded in a photo log. These QC results are documented for inspection by the QAO. 43 HWBUI0007618 SECTION 6 LEVEL III ASBESTOS ANALYSIS DISCUSSION OF PROTOCOL The Level III protocol is an extension of the Level II analysis proce dures described in Section 5- This extension may be necessitated by the need for positive identification of the specific amphibole species in situations where (1) fundamental disagreements between parties Involved in a litigation require further clarification; (2) for identification purposes, e.g., as causative agents in medical diagnosis or studies; (3) for quality control of Level II analysis in special situations, and/or; (4) for source samples whether as bulk material or bulk-air type where a legal judgment is antici pated. Since an SAED pattern may be considered as a signature of the crystal structure of the diffracting crystal (mineral fiber or particulate), the mineral giving the pattern can be identified by comparison of measured and standard sets of d-spacings and interplanar angles () from SAED patterns obtained in near-exact zone axis orientations. Such identification, however, may not be absolute without the provision of SAED patterns from more than one zone-axis orientation. The Level III analysis is an objective, confirmatory-type analysis and consists of Level II analysis plus quantitative SAED analysis from two different near-exact zone-axis orientations on a selected number of fibers identified for detailed SAED analysis during the course of Level II analysis. The Level III analytical procedure consists of locating the selected fibers contained in gold-coated grid openings (for internal calibration); photographing the fibers under bright-feld illumination; obtaining (by tilting) and recording two zone-axis SAED patterns from each selected fiber; and obtaining (recording and photographing) representative EDS spectra from the subject fiber. The present Level III protocol is based on the following guidelines: (1) Maintenance of procedural continuity so that results of Level II analytical effort will aid in conducting the Level III effort. (2) Since detailed SAED analysis on all the fibers measured in Level II analysis is not possible due to time and cost restraints, a selection criterion is needed to assure representative analyses. 44 HWBUI0007619 (3) The primary emphasis in Level III analysis is on the positive identification of the amphibole type. (4) The present protocol is designed to allow greater flexibil ity and freedom of decision for the microscopist in deter mining the selection criteria since, due to practical constraints (position, orientation, contamination, etc.) , all fibrous particulates may not be suitable for detailed SAED work. (5) It is recommended that approximately 20Z (at least 10Z) of the fibers examined in Level II analysis be selected for Level III SAED analysis. Fibers which would be classified as "amphiboles" or "ambiguous" in Level II analysis should be more often included for Level III analysis as compared to those fibers which could be readily identified as "not asbestos." In cases where the majority of the fibers in Level II belong to a single, easily-identifiable species (e.g., chrysotile), fibers that are different should be more often selected for detailed Level III analysis. This flexibility in selection criteria will maximize the gain (meaningful information) from Level III effort beyond what would be achieved from the analysis of 10-20Z randomly selected fibrous particulates. (6) The electron microscope grids used in Level III analysis (also Level II if Level III is anticipated) should be finder grids so that location of fibers examined could be referenced for quantitative SAED and for future rechecking. (7) Level III analysis should always be conducted by or under the close supervision of a professional electron micro scopist knowledgeable in crystallography, SAED analysis, mineralogy, plus Level I and Level II asbestos analyses. If such expertise is not available in-house, an outside consultant should be retained. (8) If enforcement proceedings and possible legal involvement may be part of the analytical procedure, the sample collec tion procedure entails additional record-keeping to maintain sample integrity. The field crew chief or a designated individual initiates, in addition to normal QC/QA activities, a chain-of-custody record. The sample is collected by the field team or by a representative of the adversary party in the presence of each other, and Is sealed and signed for with the date and time. The desig nated individual acknowledges receipt of the collected sample. In transferring the sample, the designate signs a release of the sample in the presence of the new recipient, who notes the date and time, and signs for acceptance in the designate's presence. The chain of custody ensures that only responsible personnel have access to and control of the sample, thereby avoiding the possibility of 45 HWBUI0007620 concamination before and after transport to the labora tory. Ac the laboracory, the QAO has first access to Che sealed sample container, and signs for it after obtaining a signed release by the hand-carrier. SUMMARY OF PROTOCOL (1) An EM grid is prepared as directed in Level II analysis using finder or locator grids instead of regular 200-mesh grids. (2) The particulate-loaded grid is then one-half or completely coated with a thin layer of gold. (3) The gold-coated grid is placed in a tilt-rotation or a double-tilt specimen holder, and examined in the AEM or STEM. (4) At low magnification the specimen grid is examined, and a grid opening is selected and identified for reference. (5) Fibers identified for detailed Level III SAED work during Level II analysis, employing the selection criteria described under Level III guidelines, are now examined one at a time. (6) A bright-field image of the fiber is taken at 0 tilt and at the magnification of analysis (20.000X). (7) With the tilt-rotation or double-tilt combination, welldefined SAED patterns of two different zone--axis orienta tions are observed and photographed. The fiber location with respect to the edges of the grid opening or to other particulates may prevent more than one zone-axis orienta tion from being obtained for some fibers. (8) X-ray elemental analysis is taken of the fiber after the SAED patterns. The EDS analysis also may be affected by proximity of the fiber to the edge of the grid opening or to other particles if tilting of the specimen is required for efficient use of the EDS. An image of the spectra is taken along with a record of the peak heights (the presence of grid peaks, such as Cu or Ni, as well as goldcoating may serve as markers). (9) As explained earlier, due to time and cost considerations, at least 10Z (preferably 20Z) of the fibers examined in Level II are analyzed in Level III work. (10) Those fibers whose EDS elemental analysis points to a possible amphibole identification are selected for SAED pattern indexing. 46 HWBUI0007621 (11) Parameters of interest obtained form zone-axis SAED patterns are: the camera constant, CC (obtained from the gold ring); the diffraction spot spacing dj (along the slant vector), d2 (along a row); the inter-row spacing, R; and the interplanar angle elf2. See Figure A18 for details. (12) The reciprocal lattice values of the d-spacings dj and d2 and the inter-row spacing (R) are converted into direct lattice spacings and then , d2, R, and 0*,2 are compared to those of standard amphibole species listed in JCPDS Powder Diffraction Files, values computed from lattice parameters and crystal structures, or SAED Standard Pattern File developed internally from known amphibole minerals regulated by EPA. EQUIPMENT, FACILITIES, AND SUPPLIES Essential items required for a Level III analysis are: A 100-kV AEM equipped with the fluorescent viewing screen inscribed with graduations of known radii to estimate the lengths and widths of fibrous particulates; or a modem 100-kV TEM equipped with an EDS. A scanning accessory as found in an STEM will increase the versatility and analytical capability for very small fibers or for fibers adjacent to other particulate matter. This microscope should also be equipped with the fluorescent viewing screen inscribed with graduations of known radii to estimate the lengths and widths of fibrous particulates. A specimen holder with tilt-rotation or double-tilt capability to obtain diffraction patterns at different zone-axis orientations. Darkroom facilities for developing negatives, making enlarged prints of patterns, and facilitating measurement of distances, spots, lines, and circles. A vacuum evaporator with a turntable for rotating specimens during coating, for such uses as carbon-coating polycarbonate filters, gold-coating EM grids, and preparing carbon-coated EM grids. 9 An EM preparation room adjacent to the room housing the EM. This room should either be a clean-room facility, or contain a laminar-flow class-100 clean bench to minimize contamination during EM grid preparation. Filter handling and transfer to EM grids should be performed in a clean atmosphere. Laboratory blanks should be prepared and analyzed weekly to ensure quality of the work. In addi tion, a sample preparation room with a laminar-flow class100 clean bench should be available for handling bulk-air 47 HWBUI0007622 samples, ashing procedures, sedimentation, ultrasonification, filtration, and ocher prefilcer accivities. Several modifed Jaffe wick washers for dissolving membrane filters. Miscellaneous supplies and chemicals, such as membrane filters, EM grids, films, gold wire, chloroform, and carbon rods. Sample collection equipment, such as filter holders, sampling pumps, critical orifices, and tripods. DESCRIPTION OF HETHODOLOCT A detailed discusson of the morphology, crystallography and chemistry of asbestos minerals, electron microscopy, and SAED analysis is outside the scope of the present protocol. Basic knowledge In these areas and an adequate level of comprehensive knowledge of TEM and SAED are prerequisites for the microscopists participating in asbestos analysis, especially at Level III stage. Since Level III analysis is an extension of Level II analysis, common methodological details dealing with type of samples (source), sample collec tion and transport, sample preparation, TEM examination and data collection, data reduction and reporting of results, and quality control/quality assurance (QC/QA) program, which were discussed in detail in Section 5 (Level II Asbestos Analysis) will not be repeated here and Che users are advised to refer to Section 5 for details in these areas. Differences, if any, between Level II and Level III protocols in common areas have been dealt with earlier under "Guidelines" and "Summary of Protocol." The following provides brief descriptions of some of the essential areas of the Level III protocol that were not covered under Level II protocol. 1. Crystallography and Morphological Properties Both crystallographic and morphological characteristics of asbestos minerals can help considerably in asbestos indentification and analysis. Chrysotile displays a unique narrow tubular morphology. The amphibole asbestos minerals have very similar morphologies--they are elongated along the 2-axis (the chain direction) and generally lie with (100) planes approximately perpendicular to the electron beam. All varieties of amphlboles exhibit these Wadsley faults parallel to the length of the fiber. Chrysotile possesses a cylindrical lattice which produces a unique SAED pattern. All the amphlboles, except anthophyllite, which is orthorhombic, have a monoclinic crystal structure. The amphlboles are double-chain silicates in which the fiber axis, z, has a repeat of 0.53 nm (inter-row spacing 'R' in real space, Figure A18). Since the other lattice parameters are also very similar, detailed zone-axis SAED analysis in more than one orientation is needed for positive identification. The non-asbestos forms of amphlboles have properties very similar to their asbestos counterparts, thus they must be distlnquished from asbestos on the basis of morphology alone. 48 HWBUI0007623 2. Chemical Properties--Elemental Analysis by EDS 'I Amphiboles are nonscochiometrie minerals and often contain substitutional cations in varying amounts. Therefore, precise determination of their chem istry is difficult and positive identification based on chemistry alone is not reliable. This may be particularly pertinent when dealing with asbestos minerals present as minor constituents in mineral samples. Elemental ratios, which are sometimes used to distinquish between asbestos types, often vary over wide ranges even in standard samples. The presence of gold coating, which would tend to preferentially absorb x-rays from lighter elements more than heavier elements, may make the situation even worse. In view of these ambiguities, and due to inherent practical difficul ties in obtaining representative quantitative EDS elemental analyses from subraicroscopic fibers, the present Level II and Level III protocols specify the use of only qualitative EDS spectra, which are often very valuable for screening purposes in the identification procedure. For example, in distin guishing between tremoLite and actinolite type of amphibole, actinolite usually contains Fe, but tremolite does not. 3. Selected Area Electron Diffraction (SAED) The method of obtaining an SAED pattern of a randomly oriented specimen is usually described in the EM instruction manual. The general directions for using the instrument to obtain and photograph SAED patterns are: (1) Select the image magnification for the selected area. (2) Bring the desired field of view to the center of the screen. (3) Insert the appropriate field-limiting aperture (according to the desired field of view) into the beam path. (4) Obtain the sharpest field-limiting aperture shadow. (5) Confirm that the desired field of view is in the field- limiting aperture. (6) Focus the specimen image; a photograph of the selected area image can be taken. (7) Obtain the SAED pattern, remembering to retract the objective lens aperture from the beam path. The SAED pattern will be observed on the fluorescent screen. (8) Select the desired camera length (the shorter the length, the better for SAED patterns of asbestos taken at high magnification). (9) Focus the SAED pattern sharply. The beam stopper is used to intercept the bright center spot. I 49 HWBUI0007624 (10) For photography, the Illumination is expanded (condenser reduced) after focusing the pattern, so that the pattern becomes barely visible (indistinct). A manual time exposure of approximately 20 to 30 s (maybe more depending on such factors as specimen and film) is required. The beam stopper can be left in place or removed from the beam path 1 to 2 s before closing the shutter. A double exposure of the specimen image and the SAED pattern can be taken if particle-to-partide spacing is adequate. 4. Dse of Tilting to Acquire Exact Zone-Axis SAED Patterns Determination of the Tilt Axis-- In the side-entry type electron microscopes, the instrument tilt axis is always fixed. However, the position of the tilt axis on the viewing screen shifts with magnification. Also, there is always an angular rotation between the image and the SAED pattern. It is highly desirable to know the location of the tilt axis on the viewing screen and its relationship vis-a--vis SAED pattern under the operating conditions to make effective use of specimen tilting for obtaining exact zone-axis orientations. The following steps can be used to locate the position of the tilt axis: (1) A gold-coated EM grid with a standard asbestos mineral specimen on a polycarbonate replica film is placed in a tilt-rotation or double-tilt holder and inserted at 0 tilt into an aligned TEH set at 100 kV, 100 yA, 20.000X magnification, and 20-ym camera length operation. (2) The image is focused on the fluorescent screen, which is at approximately 16,000X magnification. (3) A circular hole in the polycarbonate replica is positioned in the center of the field of view. (4) On tilting, the circular feature changes to an ellipse with the major axis unchanged, and indicates the position (direction) of tilt axis at that magnification. The minor axis shows the perpendicular direction to the tilt axis. A high tilt angle defines the tilt axis more accurately than a small tilt angle. Figure A13 illustrates the effect of tilt. (5) A double-exposure photograph at 0 tilt and at some high tilt angle, such as 30, is taken of the focused circular hole for reference. Tilting--for zone-axis SAED Patterns-- Quantitative SAED requires knowledge of crystallography to obtain useful zone axis diffraction patterns from which precise measurements can be made for comparison with known asbestos standards on file. Thus the method of obtain ing the visual SAED pattern of randomly oriented specimens, as in Level I and II analysis, is modified for quantitative SAED pattern analysis. It requires tilting of the specimen to align major crystallographic directions with the 50 HWBUI0007625 electron beam. The zone axis Is a line parallel to a set of Intersecting crystal planes and nearly parallel to the electron beam. A zone-axis pattern thus gives regular repeat distances and even Intensities of spots throughout the pattern. Either a double-tilt or a tilt-rotation type specimen holder can be used for obtaining zone-axis patterns. A double-tilt holder is often preferred because tilt-rotation combination involves translational movement of the fiber during tilting, necessitating constant adjustment of the specimen-positioning controls to keep the specimen centered in the SAED aperture. On the other hand, it is much easier to obtain an accurate measure of the degree of tilt and perform systematic tilting with the tilt-rotation specimen holder. It is only necessary to rotate the specimen (fiber) until the tilt axis (as deter mined earlier) coincides with a major row of spots and then tilt until a major zone axis is parallel to the incident electron beam. Alternately, fiber axis of the fiber can be oriented either parallel or perpendicular to the tilt axis and then further tilting is used to obtain exact zone-axis orientations. In order to avoid flip-flopping between Image and diffraction modes while tilting, a recommended procedure Is to defocus the diffraction pattern (the aperature becomes visible and the specimen/fiber can be seen in it) so that a double image of fiber in aperture can be seen with a poorly focused diffrac tion pattern. The movement of the fiber can then be tracked in relation to the spot pattern during tilting and kept centered in the SAED aperture by use of the specimen-positioning controls (knobs) of the microscope. Sometimes a larger aperture aids in the tracking-pattern recognition process. An experienced electron mlcroscoplst can readily recognize the geometri cal features like Kikuchi lines or Laue zones in the SAED pattern and use these to obtain the exact zone-axis SAED patterns. A detailed discussion of Kikuchi patterns and Laue zones and their utility In tilting experiments may be found in any standard text book on electron microscopy. Use of the double tilt specimen holder is very helpful and less tedious in tilting experiments. However, all laboratories may not have both types of specimen holders avail able . A skilled mlcroscoplst can use either specimen holder without much difficulty. Experience and skill are more important factors in SAED analysis than the type of specimen holder used. 5. Characteristics of SAED Patterns Encountered In Asbestos Analysis Successful application and exploitation of SAED analysis in asbestos analysis needs prior knowledge of the general appearance and distinguishing characteristics of other SAED patterns which are often encountered. The following discussion summarizes some of the observed SAED features of asbestos and.other related minerals. This discussion is by no means comprehensive and assumes that the reader is familiar with general crystallography and the nomenclature pertaining to varous aspects of SAED patterns. Minnesotaite and Stilpnomelane-- These iron-rich non-asbestos layer minerals are often encountered in asbestos analysis of specimens from certain geographic locations. Particu lates of these minerals lie near their basal (001) planes. Stilpnomelane and 51 HWBUI0007626 minnesotaite boch possess large superlattices and their commonly observed SAED patterns are easily distinguishable from amphibole patterns. The spacing (in reciprocal space) is about half (for minnesotaite) or less than that for most amphiboles. These minerals can be readily distinguished in Level I or Level II analyses if a gold coating (optional) is applied to the specimen grids. A visual inspection of the number of rows of spots inside the (111) gold ring is sufficient to distinguish minnesotaite and stilpnomelane from amphiboles. Chrysotile-- Due to the cylindrical lattice of chrysotile the SAED pattern is unique. The SAED pattern observed is symmetrical about the cylinder axis, x, and the spacing of the rows of spots is proportional to 1/a, where Is 0.53 no. The most distinguishing features of the pattern are Che flared spots of the type (130) which occur in the firt layer line. The flaring is due to the cylin drical lattice. A typical EDS spectra shows the presence of only Mg and Si (Figure All). Amphiboles--Systematic Absences, Twinning, and Double Diffraction-- The most commonly observed row of diffraction shots found in SAED patterns in amphiboles is in the y* or b* direction, representing the shortest reciprocal spacing between the spots (18.4 A In real space). There are many strong zone axis orientations containing the y* row of spots. The lattice of amosice, crocxdolite, tremolite, and actinoliCe is c-centered, and for such a lattice the h + k odd spots are absent along the y* or b* row. In practice, however, weak spots may be present in forbidden positions due to the presence of thin multiple twinning on (100), which cause streaking parallel to a*. Often, reciprocal nets from both twins are present in the same SAED pattern. In a twinned crystal, the number of important diffraction nets containing b* is doubled, leading to the observation chat the diffraction patterns appear insensitive to tilt. In some cases SAED patterns can contain spots from both twin individuals which overlap. However, not all the spots present in the composite SAED patterns are generated by the overlapping nets; some spots may be present because of double diffraction where a diffracted beam from one twin becomes the transmitted beam when it enters the other twin. The purpose of the above discussion is to point out that although many complications exist in the analysis of SAED patterns, these can be overcome; in a good goniometric tilting stage most amphiboles can be identified by SAED analysis. Aaosite-- The nearest reciprocal lattice section to the (100) direct lattice plane in amosice is (301)* and it is also the most commonly observed section. Due to the presence of the thin (100) twins, this section closely resembles (100)*. Typical EDS spectra from amosice fibers (Figure All) show mainly Si and Fe with smaller amounts of Mg and Mn. Mn is frequently observed as a substi tutional cation in amosice. 52 HWBUI0007627 CrocidoliCe-- Most of Che commonly observed patterns are asymmetrical and cannot be indexed easily. However, they all show rows of spots separated by a reciprocal repeat (R) corresponding to the fiber axis (0.53 nm). The main elements observed in typical EDS analysis are Mg, Si, Ca, and Fe. Na, which is usually present in crocidolite, may not be detected in goldcoated specimens because of absorption, or because of overlapping secondary peaks from the copper grid. Tremolite--Actinolite-- Teraolite and actinolite show a variety of SAED patterns which have very similar appearances. In actinolite some of the Mg is replaced by Fe, with the result chat interplanar d-spacings of actinolite are slightly larger than tremolite. In both tremolite and actinolite, the main elemental constituents are Mg, Si, and Ca. Actinolite also contains some Fe. An thophyllite-- Even though anthophyllite has an orthorhombic crystal structure, its commonly observed patterns are similar to the monoclinic amphiboles. Antho phyllite fibers dehydrate more easily in an electron beam and are, therefore, more difficult to study. EDS elemental analysis shows the main constituents to be Si and Mg with a small amount of Fe. ? 6. Determination of Camera Constant and SAED Pattern Analysis As mentioned earlier, a thin film of gold is evaporated on the specimen EM grid to obtain zone-axis SAED patterns superimposed with a ring pattern from the polycrystalline gold film. Since d-spacings corresponding to identi fiable gold rings are known, these can be used as an internal standard in measuring unknown d-spacings on an SAED pattern from a fiber. The precision of measurement is as good as the quality of the photograph (or negative) and usually the measurements should be in the order of 0.1-0.2 mm with an angular tolerance of 0.5-1.5 degrees. The measurements can be made by several methods: manually with a ruler, with a mechanical aid, or a densitometer, etc. The patterns can be read directly on the developed negative or on an enlarged non-glossy print. In practice, it is desirable to optimize the thickness of the gold film so that only one or two sharp rings are obtained on the superimposed SAED pattern. Thicker gold film would normally give multiple gold rings, but it will tend to mask weaker diffraction' spots from the unknown fibrous particu lates . Since the unknown d-spacings of most interst in asbestos analysis are those which lie closest to the transmitted beam, multiple gold rings are unnecessary on zone-axis SAED patterns. 7. Determination of Camera Constant Oslng Gold Rings An average camera constant using multiple gold rings can be determined as explained below. However, in practice, in most cases determination of the 53 HWBUI0007628 average camera constant is not necessary and thicker gold films are not desirable. The camera constant, CC, is 1/2 the diameter, D, of the rings times the interplanar spacing, d, of the ring being measured and is express ed as: CC(mm-A) - ^IXd(4) The value of d for each ring can be obtained from the JCPDS file. (a) Measure the diameters (two perpendicular locations) of the gold rings in mm as precisely as possible (see Figure A16). (b) Measure as many distinct rings as possible to minimize systematic errors. (c) Example: if the measured values in ran are Dj , Dj, D3 , D4, and D5, these will represent, respectively, d-spacings of 4.079 4.079 4.079 4.079 2 ' T" ' 11 and MZa '12 (d) The camera constants will be: D1 4.079 D1 ,, CCj _ x ----------- vr- x 2.355 /3 1 2 4.079 D2 cc2 _ x --^-- " ~2~ x 2-04 3 4.079 3 cc3 _ x ----------- x 1.442 /8 l CQ, 04 4.079 D" , ,, x ------------- - y- X 1.23 /II L 5 4.079 S cc5 -5-- x --------- - x 1.178 /12 (e) The camera constant for the SAED pattern is the average of CCi , CC2 , CC3, CG*, and CC5. Table 2 presents an example of camera-constant determination. 54 HWBUI0007629 TABLE 2. DETERMINATION OF CAMERA CONSTANT (EXAMPLE) Ring No. 1 2 3 4 DjL readings (mm) 23.0, 22.0 27.4, 27.6 37.8, 38.2 44.6, 45.4 Mean Value of Camera Constant ^i n Mean (mm) 22.5 27.5 38.0 45.0 d-spacing, d. (A) 2.355 2.04 1.44 1.23 Camera constant Ct - Dt/2 x dt 26.5 28.0 27.4 27.7 26.5 + 28.0 + 4 27.4 + 27.7" * 27.4 (mm-A) 8. Measurement of d--Spactags and Interplanar Angles The go Id film, because of its small, randomly oriented crystallites, produces a ring pattern superimposed on the SAED pattern from the fibers. The diameters of the gold rings correspond to known values of d-spacings, and this provides an internal standard to correct for inherent uncertainties present due to variations in instrumental and/or operating conditions. Since the dspacings of interest on SAED patterns are usually the ones that lie closest to the center spot (transmitted beam), a camera constant measured from the first gold ring in the direction of measurement of d-spacings will usually give better accuracy in computed spacings than the use of an average camera constant. This method will account for any distortions in the symmetry of the gold ring pattern. The zone-axis SAED pattern usually has several rows of spots within the circular pattern of the gold rings. These rows of spots contain information about the two sets of planes in the crystal structure and the angle between them. The following procedure outlines the steps necessary to obtain the distances between planes (d-spacings) and the corresponding interplanar angle, 0 (see Figure A17): (1) From the spot pattern, determine the row with spots most closely spaced, and designate this as a horizontal row. Draw a fine line to show the row through the origin, and designate this the zeroeth row. Draw fine lines to show the first and succeeding horizontal rows. For a few horizontal rows, measure the mean spacing between adjacent spots (or the minimum vector): Distance between spots m units apart A1* " m" where m is chosen as an optimum number to minimize measurement errors. The mean horizontal spot distance, X, 55 HWBUI0007630 equals Che summation of divided by the number, n, of rows measured. The d-spacing in 4 corresponding co this vector is the camera constant divided by X, and is labeled d2 Table 3 presents an example of spot spacing measurement within a horizontal row. (2) The perpendicular distance between two adjacent horizontal rows is similarly measured. This interrow spacing, Z, is the mean separation between horiziontal rows, and equals the distance between a number of rows divided by the number of spaces. This distance is an additional veccor for comparison that coincides with the slant vector, di~ spacing, when angle 0i ,2 is 90. The row-spacing (_R) equals the camera constant divided by Z. Table 3 presents an example of perpendicular spacing between horizontal rows; Figure A17 illustrates spot and row spacing. (3) To obtain the dj-spacing and corresponding angle 81,2, a perpendicular is drawn to the zeroeth horizontal row through the origin. A line is drawn to the first spot to the right of the perpendicular in the first row and extended through the succeeding rows. This line, called the slant vector, forms the acute angle 81,2* The mean spacing, Y, between spots on the slant vector can be measured by dividing the maximum distance between spots by the number of spaces between them, or by calculating from the incerrow spacing: Y sin 2 The d-spacing in A corresponding to this vector is the camera constant, CC, divided by Y and labeled dj. dj(A) CC x sin 8j,2 cc R " 7" Figure A18 illustrates the relationship of dj, d2, 81,2 and R. In some cases, the lnterplanar angle 81,2 ma-7 be more than 90 degrees (not shown in Figure A18). Summary of Data from Each SAED Pattern: (a) The camera constant, CC, as determined from the gold rings, normalizes the distances on the SAED pattern regardless of such factors as magnification and tilting. 56 HWBUI0007631 TABLE 3. DETERMINATION OF SPOT SPACINCS (EXAMPLES) '" r Reading Separation (mm) Units Mean spacing, \ (1) Spot spacing within a horizontal row, dj: 1 49 16 3.006 2 42.7 14 3.05 3 -- -- -- 3.028 * Mean , d-spacing - 27.4 n nc . " 9,05 4 Perpendicular spacing between horizontal rows, R: 43 8 5.0375 5-0375 " Mean d-spacing, R 27.4 5.0375 5.44 4 i Note: It is preferable that the camera constant values used in computing d-spacings are measured from the first one or two gold ring diameters in the direction of d-spacing measurement. 57 HWBUI0007632 (b) The parameters of interest are: d-spacing of spots in a hoririontal row: CC/X - di d-spacing of spots in the slant vector: CC/Y - d; angle 61,2 formed between a horizontal row and slant vector d-spacing corresponding to row separation as an additional parameter of interest: CC/Z ** R. It should be noted that the use of camera constant in the form used here in calculating dj, d2, and R, which are measured in reciprocal space on SARD patterns, automatically converts the calculated numbers into real space spacings, which are then compared to those from a suitable standard file. 9. Identification of Unknown Fibers Unknown d-spacings (d* and d2), interrow spacing (R). and interplanar angles (0) measured from zone-axis SARD patterns of unknown fibers are compared with corresponding known values tabulated in JCPDS powder diffraction files. or those computed using lattice parameters and crystal structures of candidate asbestos minerals, or with the values contained in an internally developed file from standard specimens of candidate minerals. Table 4 is an example of the IITRI standards file (Jones et al., 1981). Figures A19 to A22 are examples of zone-axis SARD patterns. Unknowns are matched as closely as possible to the file parameters for positive identification. However, considerable care and competent judgment are required in Level III confirmatory analysis. For example, amphiboles are usually nonstochiometric minerals, and thus a perfect match may not be possi ble between the d-spacings and interplanar angles determined from unknown fibers and those available from standard minerals. JCPDS Powder Diffraction files do not list interplanar angles. Since amphlboles have low-symmetry crystal structures, tabulated values of d-spacings and interplanar angles would be extensive and very expensive to generate, and to get an accurate match may not be possible because these tables are derived assuming certain lattice parameters which may not be the same as those of the unknown fibers being analyzed. Given these inherent uncertainties, it would seem that use of internally developed SARD files consisting of several readily accessible orientations (by virtue of natural habit of amphibole fibers) from standard amphibole species could eliminate a lot of tedious unnecessary work and yet provide reliable data for comparison and identification of unknown fibers. In practice, SARD analysis combined with qualitative EDS analysis may help resolve certain cases where a close match in d-spacings and interplanar angles is not possible. For difficult specimens or SARD patterns of contro versial nature, a second opinion may be necessary, especially if a legal case is involved. 58 HWBUI0007633 Amphibole type Amosite TABLE 4. COMPAK.IS ION OF d-SPACINGS FROM SAED FILE AND POWDER DIFFRACTION FILE (EXAMPLE) Zone axis [100] [30T] [101] [To i] ["310] Internal Standard File Data Interrow dl d2 0 spacing. R (A) (A) (deg) (A) Powder Diffraction File Data ( 1973) File d i d2 index (A) (A) no. 5.3 1.79 4.88 4.14 5.22 9.14 9.26 9.23 9.11 5.13 90.0 84.0 74.0 78.0 95.0 .5.3 -- 5.17 4.21 -- 5.22 1.76 4.84 4.10 5.22 9.20 9.20 9.20 9.20 5.12 17-725 17-725 17-725 17-725 17-725 Crocidolice [100] [101] [HO] [307] [310] 5.22 4.94 4.79 1.75 5.12 8.97 ,9.05 8.19 8.97 5.12 90.0 75.0 79.0 83.5 96.0 5.22 5.19 5.23 -- -- 5.20 5.89 4.89 1.76 -- 9.02 9.02 8.40 9.02 -- 19-1061 19-1061 19-1061 19-1061 19-1061 Tremolite [100] UOI] [207] [307] 5.04 . 4.83 2.59 1.72 9.03 9.03 8.97 8.98 90.0 75.0 80.5 83.5 -- 5.07 8.98 13-437 -- 4.87 8.98 13-437 -- 2.59 8.98 13-437 -- 1.69 8.98 13-437 Anthophyllite U00] [742] -- 4.56 *T 4.56 90.0 60.0 ' 5.24 -- 5.28 4.50 8.90 4.50 9-455 9-455 59 HWBUI0007634 SECTION 7 ARCHIVAL SAMPLES DISCUSSION OP PROTOCOL Samples Chat have been collected on filter substrates ocher than poly carbonate, or that have been collected without regard to filter loading levels, are referred to as archival samples. These samples were usually collected for other analytical objectives, such as for opcical microscopy or gravimetric analysis, for defined sampling periods without regard to concen tration levels in the air, or for collection of particles larger than 10 um in diameter. Such samples were historically collected, and are of value and interest in determining the presence of asbestos fibers and/or structures. Filter substrates designated as archival samples include glass fiber filters; cellulose or modified paper filters; cellulose ester filters; other organic polymeric membranes, such as polystyrene, nylon, and polyvinyl chloride; and all overloaded organic polymeric membrane filters. The purpose of the preparation step is to transfer particles from a filter surface to an EM grid with a minimum of distortion in morphology and size distribution. The nature of non-polycarbonate filter substrates or particle loading makes it sometimes necessary to transfer a satisfactory quantity of particles to a polycarbonate filter prior to transfer to the EM grid. At present, only transfer to an EM grid from a polycarbonate filter has been standardized. A modified preparation technique is recommended for archival samples, followed by the analytical methodology using Level I, Level II, or Level III effort--with the understanding that these samples will indicate the presence of asbestos, and secondarily the number, size, distribution, and morphology. The results from sample to sample are less precise due to problems in standardizing the preparation procedures used for archival samples. The archival filter samples are prepared for analysis based on the information sought, type of filter material, and particle loading on the filter. The various preparation techniques for these filters include: (1) Individual particle picking and/or reverse washing of the filter, with subsequent filtration of the filtrate using a polycarbonate filter. (2) Collapsing the membrane filter structure by exposure to solvent vapor (surface fusion), to produce a more uniform substrate for replication and grid transfer. 60 HWBUI0007635 (35 Solubilizing filter material in selected solvents, followed by separation of particulates. (4) Low temperature ashing (LTA). Two preparation methods are recommended based on filter loading and type of filter material: surface fusion, and LTA. i DESCRIPTION OP METHODOLOGY Because the greatest number of archival samples have cellulose ester substrates, this type of filter material is used in examples describing the methodology. i 1. Samples with Adequate Loading Discussion-- As an example, samples collected on cellulose ester filters have been received by a laboratory. Direct transfer of the particulates to the EM grid is possible using acetone as the solvent in a modified Jaffe wick washer. However, a question arises concerning indeterminate particle loss in the transfer. Carbon-coating the cellulose ester filter prior to grid transfer minimizes particle loss. However, this improvement in particle count is offset by difficulty in visually observing and counting the fibrous particles against a replica background of the uneven surface topography of the cellulose ester filter, and by indeterminate loss of very small particles hidden in the crevices of the uneven filter surface. The NIOSH method of surface fusion (Zunwalde and Dement, 1977) is relatively reliable, and produces a more consistent result, although the question of loss of Che very small particles has not been resolved. LTA, described later in this section, may also be used for these samples. Procedure-- The NIOSH technique, a modification of a developed at Los Alamos Scientific Laboratory described as follows: particle-transfer technique (Ortiz and Isom, 1974), is (1) A section of the membrane filter is cut with a scalpel, and placed on a clean microscope slide with the sampled side facing up. (2) The cut section is fastened on all sides to the slide with narrow scrips of transparent tape. (3) The slide, with the cut section, is exposed to acetone vapor (not liquid) for approximately 10 min. The acetone vapor collapses the structure of the filter and produces a fused, relatively smooth-surfaced film. The size of the acetone vapor bach and time of filter response to the vapors are critical in obtaining the desired smooth, fused surface; each laboratory must determine its own optimum conditions. (4) The fused filter section is placed on the rotating stage of the vacuum evaporator for carbon-coating. 61 HWBUI0007636 (5) A 3-ram-diaraeter portion of Che carbon-coated filter Is transferred to a carbon--coated EH grid in the modified Jaffe wick washer. (6) Acetone is used in dissolving the fused membrane filter. (7) Transfer to the grid and options for analytical efforts were described previously. 2. Samples with Heavy Loading Discussion-- As an example, samples collected with a heavy deposit of particulates have been received by a laboratory. These particulates may be organic in nature (for example, pollen or soot), or of mineral matter. LTA is used to remove the organic material (filter as well as particulates), leaving the inorganic residue. The residue is gently resuspended and dispersed in filtered distilled water by low-wattage, short-time ultrasonification. The resuspension is then filtered onto a 0.l~um (pore size) polycarbonate filter. The dry, particulate-loaded polycarbonate filter is then carboncoated and transferred to EM grids for analysis as described previously. Low temperature ashers are available with one, two, or four chambers. The following modifications minimize contamination in using these units: ( 1) A single chamber is dedicated for ashing samples for EM analysis. (2) An in-line filter is placed in the oxygen supply between the regulator and entry into the asher. (3) For models with direct access to ambient laboratory air on completion of ashing and return to ambient pressure, a filter is placed in the inlet line to prevent laboratory air from being sucked into the chamber. In using the single-chamber method, a blank test tube and the sample tubes (up to four, for a total of five in a 10-cm-diameter chamber) are placed in the chamber lengthwise, with the opening facing the door. The filtration step is also used in diluting the initial heavy particulate loading. Filtration of aliquots is not recommended to obtain different levels of loading on the new filters; a representative sample from each aliquot in the filtration of suspensions is difficult to obtain. Instead, for heavy loadings, different known areas of filter segments (oneeighth, one-fourth, or one-half of the filter) should be ashed so that the entire contents of the resuspension tube can be filtered onto either a 25-ram--, 37-mm-, or 47-mm-dlameter polycarbonate filter for the desired dilution. Distilled water is filtered through a 0.1-um (pore size) polycarbonate filter prior to use. All glassware is washed wich soap and water, rinsed with acid, and then rinsed with particle-free distilled water. The dedicated asher chamber Is carefully wiped with damp lens paper. 62 HWBUI0007637 LTA Procedure-- The LTA manufacturer's instructions are followed since the power required for one, two, or four chambers, the mass (glassware plus sample) placed In the chamber, and the desired rate of ashing all vary. In general, the following steps are performed: (1) Each filter segment with a known deposit area is carefully placed in a clean test tube (13 mm x 80 mm) using a clean tweezer. (2) With forceps, the cubes containing the sample, and one lab blank (unused filter segment of the same size and type of filter material as the sample) are placed lengthwise, side by side in the chamber, with the mouths of the tubes facing Che open end (door) of the asher chamber. The tubes are laid in the center of the chamber within the region of the coils surrounding the chamber. Up to four sample tubes and one blank can be laid like logs inside the chamber. (3) The power is slowly and carefully increased to prevent "flashing" of the filter, which would result in loss of sample. (4) The filter membrane vanishes in about 30 min; ashing is continued for another 2 to 3 h to ensure complete ashing. The chamber is slowly allowed to reach ambient pressure. (5) The test tubes are carefully removed and placed in a beaker, covered, and stored on a class-100 clean bench for resuspension. Resuspension (Sonification) Procedure-- U1trasonificacion is used in resuspending and redispersing the ash resulting from the LTA. The superiority of a probe-type ultrasonic device over a bath-type device has not been demonstrated. However, the criteria of low energy and minimum sonification time appear valid. The probe-type instrument is more readily calibrated (desired reproducibility), but requires a larger volume of suspension to work with. The bath-type instrument is more difficult to calibrate, and is usually of fixed wattage. A generalization is that probes are used for dispersing, baths for cleaning. Recently, a bathtype ultrasonic unit (Ladd Research Industries, Inc., Burlington, Vermont) with a variable power source and timer has become available that appears to have the advantages of both types of ultrasonic devices. The resuspension procedure is as follows: (1) 10 mL of filtered distilled water is added to each test tube. (2) Each tube is placed in a 100-mL beaker containing 50 mL of water. (3) The beaker, wich the tube, is placed in the low--energy ultrasonic bach. 63 HWBU10007638 (4) Ultrasonic energy of about 50 to 60 W (607.) is applied for 3 min. Filtration Procedure-- Liquid filtration of suspensions for EM examination using polycarbonate filters is one of the more difficult procedures to standardize. Variations in the nature of the filter material, geometry and distribution of pores, and method of manufacturing make it difficult to obtain a uniform deposit of particulates on the filter. The following procedure is used for consistency in the filtration procedure: (L) A filtering apparatus having a filter size adequate for the desired dilution--preferably 25-mm or 47-mm diameter--is assembled. A polycarbonate filter (0.1-ym pore size) is used shiny side up for the deposit, with a cellulose ester filter (5--ym pore size) as a backing filter on the glass frit. (2) While dry, the filters are centered and suction is applied. The filter funnel is mounted on the centered, perfectly flat filters with the vacuum on. (3) The vacuum Is then turned off. A 2 mL amount of particlefree distilled water is added to the filter funnel, followed by careful addition of all the water in the test tube containing Che dispersed ash. The test Cube is rinsed twice with particle-free distilled water, and the contents are carefully added to the filter funnel. (4) Suction is Chen applied; neither rinsing the filter funnel nor adding extra liquid is permitted during the entire fil tration process. (5) At the end of filtration, suction is stopped. (6) If possible, the filter is dried on a glass slide or holder that can be placed directly in the vacuum evaporator for carbon-coating. (7) The dry filter is stored in a disposable Petri dish (taped on a glass slide), or in the special holder, until ready for carbon-coating, grid transfer, and EM analysis. (8) The effective area of the redispersion filter and the area of original filter deposit cut for ashing must be recorded (ashing factor) for inclusion in analytical data reduction and reporting. 64 HWBUI0007639 SECTION 8 BOTX-SAMFLE ANALYSIS DISCUSSION OF PROTOCOL Bulk samples may be air samples collected in large volumes using electro static precipitators, bag collectors, or high-volume samplers, for example; or they may be original pieces of source material containing asbestos, such as insulation, asbestos paper products, and asbestos cement products. Efficient usage of the three levels of analysis requires effective com munication between those requesting an analysis and those responsible for con ducting the analysis. Personnel requesting an analysis must understand the limitations of each level of analysis by EM. For example, requesting EM analysis of a bulk-material (solid) sample, where there is marked disagreement regarding the presence of asbestos (amphibole), and using Level I (screening) analysis, are incompatible. Bulk-material samples require grinding for analy sis ; grinding requires care to minimize such problems as contamination, change in size of the asbestos fiber, increase in fragments that morphologically meet the criteria of a fiber, possible change in the relationship of asbestos to nonasbestos components, and possible destruction of asbestos fiber crystal linity. Following grinding, bulk-material samples should first be analyzed by PLM, followed by XRD, if necessary. XRD provides information on samples having asbestos concentration levels of at least 2Z. PLM provides information on asbestos and nonasbestos components, as well as on the size of the asbestos fibers in the solid-bulk phase. The additional information aids in EM analysis of these samples at the selected level of analysis. DESCRIPTION OF METHODOLOGY 1. Polarized Light Microscopy Analysis of bulk samples, such as insulation material, for component identification and for determination of the type and concentration of asbestos present is best accomplished by PLM. With the polarized light microscope, particle properties--such as color, morphology, refractive index, bire fringence (which indicates a crystalline substance rather than an amorphous substance), surface texture, reflectivity, and magnetism--can be observed and determined. Determination of such a large number of particle properties allows identification of specific particle types, in most cases. For example, amorphous slags and crystalline minerals are common nonfibrous filler compon ents of insulation materials that can be easily distinguished by PLM. 65 HWBU10007640 2. X--Ray Diffracttoo Analysis XRD has been successfully used co measure asbestos content In both aerosol samples and bulk samples. The reported values for sensitivity and accuracy vary depending on the exact technique, but recent reports quote sensitivity values of about 12 chrysotile, whereas 5% was more common when the technique was first demonstrated. Much of the improved sensitivity derives from sample preparation techniques, which are important in XRD, but digital data collection and use of such accessories as x-ray monochromators to reduce background are also important. While IZ sensitivity has not been demonstrated for asbestos materials ocher chan chrysotile, the same sample preparation procedures are applicable to ocher mineral forms, with comparable sensitivities expected in cases with serious interfering lines. Interferences would hinder the analysis for chrysotile as well as for other asbestos minerals. Considering that the highsensitivity procedures have been only partially demonstrated at the 12 level, a sensitivity of, say, 22 is probably a more realistic expectation for asbestos minerals in general. 3. Electron Microscopy For bulk-air samples, asbestos analysis by EM entails an addition to the sample preparation procedure to attain a representative powder sample at a suitable concentration level to be placed on the EM grid. This additional step Is similar to the method used in preparing standards of known asbestos. The finely divided powder samples are split into representative fractions, and a small, weighed portion is suspended in a known volume of filtered distilled water containing 0.12 Aerosol OT.* A mild ultrasonic treatment is used to disperse the particles. Different known volumes of suspension are filtered through a 0.1-ym (pore size), 25-mm-diaraeter Nucleporet membrane filter. The dried Nuclepore filter is then carbon-coated and transferred to an EM grid using the refined Jaffe wick technique described previously. Bulk-solid samples are gently and slowly ground to a powder for EM analysis to minimize localized heating; the powder is then prepared for the EM grid by the method described for bulk-air samples. For EM analysis of bulk-air samples, a weighed portion is suspended in filtered distilled water, deagglomerated In an ultrasonic bath, transferred to a volumetric flask, and brought to volume with filtered distilled water. An aliquot is then filtered onto a 0.1-pra (pore size) polycarbonate filter using a 5.0-urn (pore size) cellulose ester filter as a back-up filter on the filtration apparatus. The dried polycarbonate filter Is then carbon-coated. A 3-mm x 3-mm portion of the carbon-coated filter Is then directly transferred to a 200-mesh carbon-coated copper EM grid using the refined Jaffe wick washer technique. EM analysis based on Level I, II, or III effort is then performed. * Fisher Scientific Co. (Cat. no. 50-A-292), 711 Forbes Ave., Pittsburgh, Pa. t Nuclepore Corporation, 7035 Commerce Circle, Pleasanton, Calif. 66 HWBUI0007641 SECTION 9 NUMERICAL RELATIONSHIPS AND ANALYTICAL AIDS The fibrous structures (fibers, bundles, clusters, and matrices) in an air sample are to be counted, sized, and identified as asbestos or nonasbestos. An air sample ranging from 1 to 5 ra3, depending on its total suspended' particulates (TSP) content (in ambient air, the average TSP is between 30 and 300 pg/m3) is drawn through a 37-mm filter (effective filtration area of 8.6 cm2) or a 47-mm filter (effective filtration area of 9.6 cm2). The asbestos content, unlike a prepared laboratory standard or sample, is a very small percentage (less than 12) of the particulate loading (TSP content) collected on the filter surface. Two small circular sections of the filter of approximately 3-mm diameter are transferred to EM grids for transmission electron microscopy. Either one or both EM grids are examined for asbestos content; 10 random grid openings are examined for each EM grid. Each grid opening measures approximately 85 urn x 85 pm. At 20,000X magnification, a field of view of 4.5 pm x 5.0 pm is used in the examination. This approximates to about 300 fields per grid opening or 3000 fields of view per grid examined (6000 fields for two EM grids) if less than 100 asbestos structures had been found. Unlike a field blank or laboratory blank, the statistical significance of obtaining a low asbestos count in the midst of atmospheric clutter needs to be recognized. LIMITS OF DETECTION The minimum detection limit of the EM method for counting airborne asbestos fibers varies depending on the amount of total extraneous particulate matter in the sample, and on the contamination level in Che laboratory environment. This limit also depends on the air sampling parameters, loading level, and EM parameters used. For example, assuming that a fiber count has an accuracy of 1 fiber, when 10 full-grid openings are scanned, each grid opening having an average area of 0.72 x 10"" cm2, the detection limit is determined from the equation Detection limit --1 x -A---r-e---a-.--o--f----f--i--l-t--e---r---- (--c--m---2- )- x 1 10 0.72 x 10~" (cm2) Volume of air (m^) The minimum detection limit, then, is lower for very dilute samples. Examining full-grid openings leads to a lower value of the minimum detection limit because of the large area scanned, as compared with the field of view method. With a given sample, the detection limit can be lowered considerably, but the required experimental effort increases. The guideline of using 10 67 HWBUI0007642 fall-grid openings represents a judicious compromise between a reasonable experimental effort and a fairly low value of the detection limit. However, using two or more TEM grids reduces the detection limit further and improves the precision of the estimates. STATISTICAL METHODOLOGY Several statistical strategies have been used to characterize airborne asbestos distributions and estimate the abundance of asbestos fibers in a given sample. These methods range from simple tabulation of observed frequen cies of fibers across grid opening samples to the fitting of statistical prob ability distribution such as the Poisson. This statistical section outlines a general methodology for fitting observed data to a statistical probability distribution (either Poisson or normal depending on the fit of the former). The mean and 95% confidence interval are then estimated and used for the purpose of sample description and drawing inference regarding the abundance of airborne asbestos fibers in the environment in which the samples were obtained. As an illustration, consider the hypothetical data in Table 5. The expected number of grid openings with no fibers is: Ne"u - (98) (e-3*0204) - 4.7806 The expected member of grid openings with 1, 2, 3, ... fibers are found by multiplying Neu by u/r , l.e., 4.7806 x 3.0204, 4.7806 x 3.0204/2, 4.7806 x 3 .0204/3, successively. To test the fit of the Poisson distribution to the observed data we compute a chi-square statistic, *2 = r (observed - expected)2/expected - 8.26. Since there are nine different observed frequencies (l.e., numbers of fibers) there are 9-2*7 degrees of freedom (since we estimate one parameter). The probability of xa2 - 8.26 is p - 0.4; therefore, we conclude that the Poisson distribution fits the observed data. In certain cases, the observed frequencies will not have a Poisson distribution (as determined by the previously described chi-square statistic). In this case we estimate the mean (X), variance S2, and 95% confidence limits 68 HWBUI0007643 TABLE 5. HYPOTHETICAL DATA No. of fibers on grid 1 opening (r) 0 1 2 3 4 5 6 7 8 9 10 11 or more Total Observed* frequency (f) 3 17 26 16 18 9 3 5 0 It 0 0 98 Expected frequency 4.78 14.44 21.81 21.96 16.58 10.03 5.04 2.181 0.82 0.27 1.20 0.08 0.03, 98.0 CO e 1 Observed - expected +2.56 +4.19 -5.96 + 1.42 -1.02 -2.04 +2.84 -0.20 Probability (r) .049 .149 .224 .224 .168 .101 .050 .022 .003 * The number of grid openings showing that number of fibers. t We combine adjacent frequencies to get a minimum of 1 fiber per group. Assuming a Poisson distribution, the mean is u r fr/lf 296/98 = 3.0204. That is, the sum of the product of the observed frequencies and number of fibers divided by the sum of the frequencies. ! 69 HWBUI0007644 assuming normality. The 95Z confidence limits are computed as a function of the sample variance and C distribution. kn n X2 - [z X ]2 S2 - JLiL, ____ i-f . n(n - 1) where k is the number of grid openings, n is the total number of fibers found, and X is the number of fibers found in grid opening i. The 95Z confidence limits are given by X ---- /K where t is the value of the two-tailed t distribution for probability p <.025 and n - 1 degrees of freedom. 1. 95Z Confidence Limits for a Poisson Variate To generate a level of confidence regarding our estimate of the number of asbestos fibers per grid opening, a 95Z confidence limit can be derived. For counts of 0 through 20, Table 6 may be used. For example, if out of 20 grid openings 15 fibers are found, the 95Z confidence limits are obtained by taking the lower and upper bounds from Table 6 as 8.40 and 24.74. Then per grid opening, the 95Z confidence limit is 8.40/20 to 24.74/20, or 0.42 to 1.237 fibers per grid opening. For counts greater than 20, a simple normal approximation is computa tionally convenient. The normal approximation is X - L x * (S^) where L is Che observed count, is /IT , and is 1.96 or 2.58 for the 95Z or 99Z confidence limits, respectively. For example, if 35 fibers were observed from inspection of 20 grid openings, L - 35, Sr - /33 - 5.91, 1-35 1.96 (5.91) - 23.4 to 46.6 or 23.4/20 - 1.38 to 46.6/20 - 2.74 fibers per grid opening. 2. Comparison of Two Poisson Variates In certain cases, a new test sample is compared to a "blank" or "control" sample. Table 7 gives those differences between control and test samples chat are significant at the 5Z level. Inspection of Table 7 reveals that the minimal detectable difference between test and control samples is 5 fibers in the test sample and 0 fibers in the blanks. Typically, inspection of 20 grid openings for a blank control reveals between 0 and 5 fibers. Ac the upper bound (i.e., 5 fibers in a 70 HWBUI0007645 TABLE 6. 95 PERCENT CONFIDENCE LIMITS r No. of Fibers 95Z Limits Lower Upper 0 0.000 3.69 1 0-0253 5.57 2 0.242 7.22 3 0.619 8.77 4 1.09 10.24 5 1.62 11.67 6 2.20 13.06 i 7 2.81 14.42 8 3.45 15.76 9 4.12 17.08 10 4.80 18.39 11 5.49 19.68 12 6.20 20.96 13 6.92 22.23 14 7.65 23.49 15 8.40 24.74 16 9.15 25.98 17 9.90 27.22 18 10.67 28.45 19 11.44 29.67 20 12.22 30.89 71 HWBUI0007646 TABLE 7. CONTROL AND TEST SAMPLE DIFFERENCES Fiber Count Control Test sample 1 2 3 4 05 06 07 18 l9 1 10 2 11 2 12 3 13 3 14 3 15 4 16 4 17 5 18 5 19 5 20 Fiber Count Control Test sample 6 21 6 22 7 23 7 24 7 25 8 26 8 27 9 28 9 29 10 30 10 31 10 32 11 33 11 34 12 35 12 36 13 37 13 38 13 39 14 40 72 HWBU10007647 control sample), 18 fibers in Che test sample are required for statistical significance. This estimate of 18 fibers may prove useful for establishing an asbestos detection limit criterion. MAGNIFICATION CALIBRATION The following steps should be performed to calibrate the magnification of the EM: (1) Align the EM using the manufacturer's instructions. (2) Insert mag-calibration gracing replica* (with 54,864 lines per inch, or 2160 lines per mm) in the specimen holder. (3) Switch on the beam, obtain the image of the replica gracing at " 20.000X magnification (or at the magnification at which the asbestos samples will be analyzed), and focus. (4) If the fluorescent screen has scribed circles of known diameters, align one line tangentially to the circumference of one circle using stage control. Count the number of lines in a diameter perpendicular to the lines. In most cases, the other end of the diameter will be between the nc^ and nc^ + 1 line. The fractional spacing can be estimated by eye. Alternatively, the separation between lines can be estimated using the scribed circles. (5) If X line spacings span Y mm on the fluorescent screen using this gracing replica, the true magnification, M, is given by ,, Y x 2160 The readings should be repeated at different locations on the replica, and the average of about six readings should be taken as the representative or true magnification for chat setting of the EM, as in the following example: * For example, Cat. no. 1002, E. F. Fullam Co., Schenectady, N.Y. 73 HWBUI0007648 Line Spacings, X 9.5 9.3 7.0 8.8 9.0 9.0 mm on Screen, Y 83 80 60 80 80 80 Magnification, _____ M 18871 18580 18514 19636 19200 19200 Average: 19000 On most EM's with large (18-cm diameter) fluorescent screens, the magnification is substantially constant only with the central 8- to 10-cradiameter region. Therefore, calibration measurements should be made within this small region and not over the entire screen. PREPARATION OF BLANKS Even after taking the utmost precautions to avoid asbestos contamination the possibility of some contamination cannot be ruled out. Contamination should be checked periodically by running field blank samples in addition to laboratory blanks. Field blanks should be analyzed prior to laboratory blanks. A blank sample may consist of a clean filter subjected to all the processing conducted for an actual air sample. This processing may include ashing, resuspension, redeposition, carbon-coating, transfer to a TEM grid, and TEM examination. When analyses of blank samples show significant background levels of asbestos, these should be subtracted from the values obtained for field samples. Also, the minimum detection limit may be calculated as twice or three times the standard deviation of the blank or background value. USE OF COMPUTERS Data reduction is facilitated by computers. Computer printouts can be used in reports. Each laboratory should develop software suitable for its needs as well as to maintain basic information, such as fiber, areas examined, volume/mass of sample, and size distribution, for possible interlaboratory comparison. Appendixes B and C present sample printouts from Level I and Level II analyses, respectively. 74 HWBUI0007649 / APPENDIX A FIGURES Figure Al. Vacuum evaporator. 76 HWBUI0007650 I 77 HWBUI0007651 F ig u re A2. M u ltip le c o a tin g arrengem ent in e va p o ra to r. HWBUI0007652 79 HWBUI0007653 F ig u re A 4. M o d ifie d J a ffe w ic k washer m ethod (s k e tc h ). F ig u re A8. L e ve l 1 d a ta s h e e t (e x a m p le ). Struc .I \ * u<* \ \ o n Sce < N t_t>. % \\ \ eo CV r -J r- c c\ - -- *5 \s es .J tr r O' N C 3 vr c tr N e | O 5 N \ s\ > \ \ r; -tasj ir rr a t > i3 2 U U. 2 a fC 2 2 2 UL 2 a CC a u u 2 a a a a a r CN a tv o <N f efsr cr ? $ * c *sl ff V \r . -r V ao V v 5 to e o e e* * <>u J6s & s o *vr>' Jas UC*i u \ 'i c c e 3* c & 3o tcs r cr - c cs l e .** r \ \\\ s >> \ \ \ a i2* lr CO r- ts a < o- n vr n r~ 2 to Struct. u. U a a 1 t a a a 2 2 2 2 2 2 u 2 2 u 2 2 a 2 3 -- N eO 10 r C o> c - cr 2 tr J r- oC S *sJ CO OJ rsj vn cs -- <s ft" 82 HWBUI0007654 r ~1 83 HWBUI0007655 F ig u re A 9. S canning o f f u ll- g r id o p e n in g . Figure AlO. Transmission electron microscope with energy dispersive spectrometer. 8i HWBUI0007656 t 1.1-10-0-6 0-2-10-C-7 Figure All. Spe a profiles of asbestos standards. 85 HWBUI0007657 86 HWBUI0007658 I IF ig u r e A12. L e v e l d a ta s h e e t ( e x a m p le ). EH DATA REPORT Sample Number: R09-2865_______ OaLc Analyzed:___ 3/26/81 IITRI Sample No.: CO 10-1859_______ Date Sample Received: 1/30/81 Sample Type: Bulk, Air. Water, Misc. (circle one) Filter Type: Nurlgonre________ Area of Filter Ocposit (cm2): 8.6 Volume of Fluid Sampled: nA Mass Deposited:NA 1. Total Number of Structures:10J 2. Total Number of Asbestos Structures:93 2.1 Chrysotlie 88 2.* Amphibole 5 Crocidoli te 5 Anthophyllite Tremolite Actinolite Amosite Non-Identity 2.3 Non-Identity 8 Asbestos Structure Description 3.1 Total Number of Fibers: 85 3. 1.1 Chrysotile 80 Fiber Length; Ranqe (urn) .31 - 2.25 Fiber Diameter; Range (um) .06 - ?5 Aspect Ratio; Ranqe (um) 3.5 - 31,0 3.2.2 Amphibole 5 Fiber Lenqth; Ranqe (um) .31 - 2.19 Fiber Diameter; Range (um) .06 - .31 Aspect Ratio; Ranqe (um) 5.0 - 10.0 3.2 Total Number of Bundles: 5 3.3 Total Number of Clusters/Clumps: 2 3.4 Total Number of Matrix/Debris: 1 Mass (nq) Mass (ng) Mean (um) Mean (um) Mean (um) Mass (ng) Mean (um) Mean (um) Mean (um) .Hass (ng) .Hass (ng) .Mass (ng) .0016 .0010 0.78 0.07 11.00 .0006 1.00 0.15 6.40 .0007 .0002 neqliqible Area of Filter Sample Analyzed, (cm2): 0007225 Total1 Mass of Asbestos Analyzed (ng): 0025 6. Number of Pictures Attached:2 7. ( Qualitative Description of Non-Asbestos Particles Few, small oarticles-non-descriotive 8. Comments: Particulate loading OK.______________________________ _ Figure A13. EM data report (example). 87 HWBUI0007659 sample suMMAur mrtmr Sample Number: R09-2865__________ Date of Report: 4/1/81 IITRI Sample No.: C010-1S59_______ Date Sample Received: 1/30/81 Sample Type: Bulk, Water, Mi sc. (circle one) Filter Type: Nucleoore_________ Area of Filter Deposit (cmJ) Volume of Fluid Sampled: NA Mass Deposited: Nfl 8.6 1. Total Number of Structures:1 ,202 ,21S 2. Total Number of Asbestos Structures: 1,106,990 2.1 Chrysotile 1 ,047,474 2.2 Amphibole 59.516 Crocidolite 59,516 Anthophyl1ite Tremolite Actino!ite Amosite Non-Identity 2.3 Non-Identity 95,225 Asbestos Structure Description 3.3 Total Number of Fibers: 1 .011.765 Mass (ng) 19.00 3. 1.1 Chrysotile 952 .249 Mass (ng) 11.90 Fiber Length; Range (pm) .31 - 2.25 Mean (um) 0.78 Fiber Diameter; Range (urn) .06 - .25 Mean (pm) 0.07 Aspect Ratio; Range (urn) 3.5 - 31.0 Mean (um) 11.0.0 3.2.2 Amphibole 59.516 Fiber Length; Range (pm) .31 - 2.19 Mass (ng) Mean (u) 7 14 1.00 Fiber Diameter; Range (pm) .06 - .31 Mean (pm) 0.15 Aspect Ratio; Range (pm) 5.0 - 10.0 Mean (w) 6.40 i !I 3.2 Total Number of Bundles: 59,516 .Mass (ng) 8.30 3.3 Total Number of Clusters/Clumps: 23 .806 .Mass (ng) 2.10 3.4 Total Number of Matrix/Debris: 11,,903 .Mass (ng) 0.10 Area of Filter Sample Analyzed, (cm*): .0007225 Total Mass of Asbestos Analyzed {no)-. 29.5 6. Number of Pictures Attached:2 7. Qualitative Description of Non-Asbestos Particles Few, small particles-- Non-descripti ve; 8. Comments: Particulate loading OK. Figure A14. Sample summary report (example). 88 HWBUI0007660 (a) Effects of tilting. (b) Fiber alignment. Figure AL5. Effects of tilting and alignment of fiber. HWBUI0007661 gO Perpend Ic u la r F ig u r e A18. R e la tio n s h ip o f. d i , d z , 8 2 , and^ se& (w0 C oN o T3 XC 4) - w X L. K 0) * 4) W- c > X X? c o fO CD o<=. 4 2 4J o o *" Vu CT> 4} C X C. w <3 X 2o 3 uo u JH--- X to 4-* cu o N 3 UU o 4) w W 4) 3 UX U <o u- c 43 4? MU X X w 2 uO 4) U o TM & <u> 3 3 X U 4< c to o X *Q c > "3 43 C E 4) X w a? a. w 4) X u a O 4* o 0) U 0) o. o 41 k. 4> O. 4) EO X (0 X 4) C u* $ 2m w o u o C o X u~ 4) X & c -- -3 O <w """ 91 HWBUI0007663 I (c) Zone axis [101]. (d) Zone axis [101]. Figure Al9. Typical Zone-axis SAED patterns from amosite standard specimen. (Jones et al., 1981) 92 HWBUI0007664 (c) Zone axis [110]. (d) Zone axis [301]. Figure A20. Typical zone-axis patterns front crocidolite standard specimen. (Jones et al., 1981) 93 HWBUI0007665 (c) Zone axis [201]. (d) Zone axis [30l]. Figure A21 Typical zone-axis patterns iron treaolice standard specimen. (Jones et al., 1981) 94 HWBUI0007666 I \ (a) Zone axis [TOO]. (b) Zone axis [ 1423 - ! i I Figure A22 . Typical SAED patterns and EDAX spectra from ant'nophyllite standard specimen. (Jones et al., 1981) HWBUI0007667 w J fioj iI c X H w s < 25 WM *2 pa s> 63 3S3 IX. ft 6 K C c X tl X UJ 1- u u u <* u < C- 3X u 8 tt 8 8 a a & 8 8 a a s n a a ii t! n s a s it 8 a 0 tt. 8 C I! Q ti > it n tt -* R U 2 II * 1 n u U -J i t8i B a n <r ft tt ua a < Ut n 1- H" u 8<C li 2 rt KX a uc s tii -- u -5 rt a A< a &: n < tt s UJ a 3 9: n (A M m a ue a O LJ n ^4 w* tt tt ^ 2 <C R * u Q U Ut w X O <9 z . tt l ** X i xx * X X - XX XX X X X - X - X -XX -XXXX 0W1 z : P! x1 X1 e1 <c i 1 &i 1 <-- i 1 G! 1XI i **" ! 1 JC 1 ** l 0. I I ? a1I I i 6i CI U s **-! ! V* ! 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C3 *" UXTK O r-t _, a w 3^ O & n u" T-i IN 0 0tt m 0 JN tN 0 0 < M * n f-4 -^ N> O rt CM 0 rt --' -- G rt O 0 it* < c* rt 5^ O w r: rt O O * a?ni rt a *: ^0 *3 'Si -- ~ N. ^0 w 0 0 m IN ^sr O U*5 M U*l IN n tOn fN trn; < IN r-t - O ti*S r tft*-i a ar-tt rt S"i CM "C W m c 0 'st r-t X a -* ii rt ^ MN --s rt -- IN rt c ?< rt rt r-t *43 O .N w ft >5 m N &> O N c r-t O 0 00000 c O O 0 0 c 0 oocoo&ooeo O - O O ooooooooooooooooooooooooooooo oooooooc ocooccooooococoocoooc OOOOpOOGOOOOOGOOCOOOOOQwOOOO oooooooccoooooocoooooooocoooo ^ fi rj v ifi n a > o- ^<? w n s<* *i f-'i nn n*i "j n "^* fr-4 ft r>- - fi f-J ft n W N N K 2 a M W ?l !*) n M V v ^ bn* < ^ li u It II InI a a n It II nu nI! n II H ii ts li li ii u ii ii u u B it 0 s n II ii II a tt ft 0 ii n it H n u it ii i$ y u ii r. u n U a u n y u s y u it u 8 8 a ii a tt 8y n u 8 n u u II r. 3 a un 96 HWBU10007668 I ! tt n Q 1 O M n1 lIaI 1 8! 8tt s s COO c rj tfs -* -- r-i 0" u R i 01 i tn X 3 C if5 -- rs. a im -3 o r. a r 8 i s. 3 * ! Hi c in n o ti i > & 3 a u i HX 8t U> i c o <; ni C U3 > fi !i s ui **> -- n 8 a. a 8 i Qj i 5 5 -y -r ** l y j XS y c o O' u j & c^ rj c -c u. 18 cU i > tt x <J~ - C X M w 1 ii a fi u ii i --?s. w R ut n -> liJ J! f- ii Cj X ii 2 t1t! -j sn &. &c r. 9 aX ~ . y at " S S3 .-n C i! >--< 1! & & 3 '<"4 > -X ** a H* n u cs a ss Cc: 0 e. n 38 -- sr L. u c UJ o a X aX fc. a 3 - U"3 `-5 a Cl. i\ us 0 Cw b 85 US Q w O' reU si a US -* -- si T 3 i. 3 Oi u XU 11T RESEARCH IN S T IT U T E STRUCTURE ANALYSIS HATA ii K it u ii H u ii u a n 1! u u & ` - * n X , < a3 S'* > f rj n c u c r &u t r: r-i 3 n "9 X u *>* r>* & si 3 - ? s sr X X w *-- u. ? a n sn f8i uj u -j a Btt }-- utt ii >- u u X si X 31 2 S3 C5 U y JUJ 8 tt-J -- n u. UJ X x tt o tt tn u tt B u u 31 Q 3 U. 2X M 21 R U a 8 3 u # m UJ *3 c r c 3 G Ut J u & X c o 0 0 t. 8! ; ; ! 1 i *4 w ! S Q t nn : -t a j m T3 (J t Q i s 1 c CU 8 1 i& 1 -- S3 i^ 0 i cX ! US 6 13X0 4' 4 3i 1 e -> i UC s. &' X u. < k 3 w^ ; C O i &3 i o "X u : i X; c cn ! U I CJ 9 ca f: rs. c oc c o o r-i fi 13 3 ES 2" *T>* in a c Xa> X C *4 Oi 4& CL 0 X % > *3* o yn U X 9 ft:i i 6 6 X n~ss &h. *34 C 3 2 m uX . i h W>) & X e B -<4 C X u. si J* 1 s1 Xi i i Ci ^4 i r 93 i. j r CJ U C3 e sfi. ! 3 5 & i cn x X s. 1 4. ; o i c o i * Ca 4 H 4 i ci 9 n nn H 4 i { 16 Ci 3 1 i fit B s. & K> l Qt s- e c l a * CJ 8 3 u H | mm u? 6: s 0 i r> fi x> 4 4S oo D i 4 X "3 S *** & u* 3b cs f X 2 .99- HWBUI0007669 I IT RESEARCH IN S TITU TE STRUCTURE ANALYSIS DATA IN D IV ID U A L ORJECT DATA TADLE ( F = F I6ERi D=DUNDLF.i C.=C,LUSTF.R H =hA TR IX ) TADLE PREPARATION DATE 1 21-A P R -Q 1 n f! 8 n a K a UJ Ct Q CJ UJ &. X < CD i i I t i i ii fi X \ ! re ; i ii Xi *3 i *> C ft i X u. { - ft I 1 O ft i 2c \ XXX - o CM r%s 33 S JQ 1 2 -C * ** o ! ft ! - O 1 *- ! O 1 0 i ii*5 *"* iX i I -- i m - *o iX i -* 5 ft i t t QIC \ 6- 1 33 i Gift ! O 1 f* \1 *< 1 * 11 i +* i1 --! 0 [ * * - I ft . ft 1 3* ,i ft i s- !l a tX i 1U ! o o S3 fs n r: P4 o o c o c- G I[ CMNOJ1! 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(MS Mh ** 83 w Li Si ts u a si :t u a is a a a a a 39 ! 83 y3 r-i ^ Q <8 z i oOMC rvv> 3 o n w n <W -* <M> S3 rs ww i i c O c 3 e a uj x .; Z cj t-- z u z u X L. is n 63 ^ w" C3 ZU 83 2 C X US si u u u Si ti u it a si na a 0 (8 S s Z i. s I? ii il Ra U s a 0} i > 2 | a O w: i a z <r XXX X X X - M U a u u aa a a w; u ss a <C a u a s eI <c i U ii is sn <8s u ra a a %z a ua frCft 3 nu n J a SJ R an wa 3 fS f-i O fS O* fs O'* "0 - 83 O S ns T r; ri a. ; e! i - * o in 3 -r r-j a r< :s cs Cl a rs rs n a ss c rs ss a ts n ;s a a a n u on a oa a c il W S* ^u 2 3 s a a A oi &a a r. a Ms o 08 0t9f 3ut SC X Z CJ u u ss ts a a a cn >* 3 U C c u. a a a & ft o c o* n. 6s3 a & S3 n * 3 n - nc 63 m 'C * 83 83 83 n n 83 c n O* e S3 U u ii ts Si aII s-- 38 n UJL.SE 0a i I } Ce: ~ i n 3 CfT a - u. 8 a us a D j i a . a -< a h II cn *- a ~a li <c u b *- K 8 30 hw u96 I- K 2 B (OUCH 2 'J *- n h> a ft < A !X 1^ } 3? i i& a i --: ci o sX t ^ Of I 33 ** 1 **' z 13 t 1X 6: I ** N t it E Qi m 1 n fS H S3 mn s** ' * U3 8a3 rj 63 <1 f-i 63 fS O 63 63 >0 n r-i oo o o o oo > o o 83 ro rs rs S3 Q * o o o o rs e* S S3 * -< in m it. ps CJ *4 n _ <M 9> w coo ooo ooo ooo " * ** ooc m*o u3 sn ^ u ^ is o o n 63 w ' tOfSt *6 ^ f4 i\ z e -- S3 * c S" to ta l Count it B H Uii ii a a zU: a & aa f: Ju 0 ecus UJ a to * L, L. L. L. u. u. u t. S_ ti. L L. L. a a a c I US 3 we a (A w & 1 UJ B CC u B H* *&-* &j s8 u "3 W4 r-i n X a n ss rs B O -v c mm n n 63 83 S3 fS fS 83 ^ is * w4 S> R II a a a a m ZC 8 m -) *- S U 4. C2 n 99 HWBUI0007671  s 8 a o** ^ y n C a f8i U lIiI 3 &X ^SS X T3 ?3 W O i cr f*3 f -* &>. m m rx n Ua a s. B 3 *< >ft fCt O f*t > fv a _J - fi Le3 S 05 Rna u n ^a 23 H I8 ad u. II O O 'O o '! in ~ J o -- -* ft c 8a1 s0 ** -H * L ^ ft N 'S*' > ft X <C J ^ fl M bnl CM P* o * o o n fl M B APPENDIX C (C o n tin u e d ) --i aa cm an -- ti u}--i a& is n o o r* ri w CM ft O m 2n n e *-- si c. ft * u . ft ft T1 > X 3 0 0 onn o n& o * o l! cr n a L c Ui u Q ft ft r U e-- ft X fc. rj on CT 8 cW 3 . fi 65 ft 0 w it h~ U a ft ft ft ft ft ft c - u u X m o u. ft 'fl W ft 5 r*> fact. z &* c i c ft e N** "--D C *8 --<f*t* CU in o * cm o w T4 -- *- c -- r- ft w 094S; x* {BOH ft ^ *o '8 -> ft e -- *8 e -- a t C3 O * 2 1ft &H Xu U en *- 8 B fut tn n ~3 JTM, s i <\ X s. o 4 0 in ft ftB u c ft r c i tn 8 U s3^ n i n 2: s T? ft 8 ft c 3> u 3 ** n o 2 ft wfl 1 M i UI 3 ft z u w u. i C R ft ft > c. w04 ffil ft ft fffttt-L 3 {pi. o 03 3 UI -J H* St 03 fr- UI >* 3a j-- X *-" X 01 3 x 03 M Ui X J u cc u. X Ui **. U9 03 U 0? UI UI UI -J 3 U. XX M H 03 03 8 B B 8 B U a B s B B 8 a B B fl S S B S B 8 8 8 a 8 8 a 3 ft ft U 8 C C 2? Q * (j ft "J C "* 0 ft 0 t. ft +3 m ft D ft U TM3 ? ft > O O3 rv 4 ! 1 ft -* ft (> *** ^fl 3 X O > R . u. ? X ft ft . ft X ^4 3* X * u O ft fc. Z* *> ft ft 4 ft 3 1 ft n- 1 X x > 1 Wl O 01 u. u roc ZJ ft fi. u U3 6 03 C 03 061 L o o o C 4 <N "( >*4 c98oC n B R 8 gffMoottt ft f4ftt- ft ffUiti U8 afnt ,t >e a* ft &ft " #4 ft 6fi. ft tn u4 Offit. Xfftt tff?tt C 100 HWBU10007672 1. EPO"T NO. TECHNICAL REPORT DATA (Please read Instructions on the reverse before completing} 3. RECIPIENT'S ACCESSION no. 4. TITLE AND SUBTITLE Methodology for the Measurement of Airborne Asbestos by Electron Microscopy 7. AUTHOR(SI George Yamate, Satish C. Agarval, Robert D. Gibbons s. report DATE July 1984 I. performing organization CODE 8. PERFORMING organization REPORT no C06470 9. PERFORMING ORGANIZATION name ANO address IIT Research Institute 10 West 35th Streeet Chicago, Illinois 60616 10. PROGRAM ELEMENT NO. 1 1. CONTRACT/GRANT NO. 12. SPONSORING AGENCT NAME ANO ADDRESS Environmental Monitoring Systems Laboratory Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, North Carolina is. supplementary notes 27711 13. TYPE OF REPORT ANO PERlOO COVERED 14. SPONSORING AGENCT CODE 16. abstract The provisional electron microscope methodology for measuring the concentration of airborne asbestos fibers was refined. The methodology is divided into separate protocols. The step-by-step procuedures for each protocol are nearly identical, so that cumulative data can be obtained and uncertainties, especially in asbestos iden tification, can be clarified. The operational steps encompass (1) type of sample, (2) collection and transport, (3) sample preparation, (4) examination under the transmission electron microscope (TEM) and data collection, (5) data reduction and reporting of results, and (6) quality control-quality assurance. The TEM analytical protocol is subdidvided into three levels of analysis: Level I, for screening many samples; Level II, for regulatory action; and Level III, for confirmatory analysis of controversial samples. Because identification of asbestos structures is critical, the level of analysis Is directly related to the information sought: Level I--morphology and visual selected area electron diffraction (SAED) pattern recognition. Level II--morphology; visual SAED; and elemental analysis. Level III--morpholgy; visual SAED, a selected number of SAED micrographs of zone-axis patterns; and elemental analysis._______ _________ ________________ DESCRIPTORS KEY WORDS ANO DOCUMENT ANALYSIS b.tOENTI PIERS/OPEN ENDED TERMS c. cosati t-irld.'Gioup 8. DISTRIBUTION STATEMENT RELEASE TO PUBLIC 19 SECURITY CLASS iThis Report) UNCLASSIFIED 30 SECURITY CLASS I Tim ptftl UNCLASSIFIED EPa 2220-1 (R... 4-57) IRC *Ou taitlON i A p ftfrOk. C T t 101 21 NO O*?aG5 112 22. PRICE HWBUI0007673