Methods of monitoring and evaluating airborne man-made mineral fibres: report on a WHO consultation, Copenhagen, 29 April–1 May 1980

Tit~~ s - ;(.c,-6 c~ f 1:, ~ EURO Reports and Studies 48 ~~,, ,'\. /J'¼ ~ ht-,<..-..{, ~s / I"' / I '(. f-1. C ( <+ 11 - Methods of Monitoring and Evaluating Airborne Man-made Mineral Fibres Report on a WHO Consultation Copenhagen 29 April -1 May 1980 REGIONAL OFFICE FOR EUROPE World Health Organization COPENHAGEN 1981 IIIIIII IIIII IIIII IIIIIIIIIIIIIII IIIIIIIIIIIIIIIII .. 0 0 0 2 8 0 '-l 9 .. ICP/ WKH 014 ISBN 92 890 1214 5 © World Health Organization 1981 Publications of the World Health Organization enjoy copyright protection in accor- dance with the provisions of Protocol 2 of the Universal Copyright Convention. For rights of reproduction or translation, in part or in toto, of publications issued by the WHO Re- gional Office for Europe application should be made to the Regional Office for Europe, Scherfigsvej 8, DK-2100 Copenhagen 0, Denmark. The Regional Office welcomes such applications. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in pre- ference to others of a similar nature that are not mentioned. Errors and omissions ex- cepted , the names of proprietary products are distinguished by initial capital letters. The views expressed in this publication are those of the participants in the Consul- tation and do not necessarily represent the decisions or the stated policy of the World Health Organization. TYP ESET IN TH A IL A N D PRINT E D IN DENM A RK ISSN 0250-8710 Introduction Background . CONTENTS Methods currently used for monitoring man-made mineral fibres Measurement of total mass concentrations in air .. Measurement of fibre number concentrations in air Proposals for a reference method for monitoring man-made mineral fibres Organization of a central reference scheme for counting and size analysis of man-made mineral fibres . . . . . . . . . . . . . . . . . . Application of the reference scheme to epidemiological studies Conclusions . . . . Recommendations References . . . . Annex 1 A recommended standard method of monitoring airborne man-made mineral fibres . . . . . . . . . . . . . . . ... Annex 2 Organization of a central reference scheme for counting man-made mineral fibres . . . . . . . . . . . . . . . . . . Annex 3 A recommended reference method of monitoring the number concentration of airborne man-made mineral fibres Annex 4 List of participants . . . . . . . . . . . . . . . . . . . Page 2 3 3 5 6 8 9 11 13 30 33 52 INTRODUCTION A WHO Consultation on Monitoring and Evaluating Man-made Mineral Fibres met in Copenhagen from 29 April to 1 May 1980. Dr J.E. Asvall, Di- rector, Programme Management, opening the meeting on behalf of the Re- gional Director, said that it had been called in response to resolution WHA 30.47 of the Thirtieth World Health Assembly, which requested a study on health problems related to the growing use of chemicals. There has been a marked increase in the use of rr.an-made mineral fibres (MMMF) in the com- munity within recent years and a number of epidemiological studies, both national and international , have been carried out on their biological effects. It has been shown, however, that data from such studies must be comparable and, in particular , that research would be assisted if the methods now in use for monitoring and evaluating airborne MMMF were to be standardized. Accordingly, the aims of the meeting were : to review the methods used at present and to recommend reference methods for monitoring and evaluating airborne MMMF to ensure that accurate and consistent data are available for use in epidemiolo- gical studies to set up a central reference scheme for counting and size analysis of MMMF to establish a scheme for ensuring that the results of atmospheric monitoring are readily available and relevant to the needs of epidemio- logical studies. Mr J. I. Waddington , Director, Promotion of Environmental Health, WHO Regional Office for Europe, considered the meeting to be an important milestone in the Regional Office's activities concerning specific occupational health problems. It is encouraging that one section of the fibre industry is showing an appreciation of the need to harmonize approaches among Euro- pean countries. The report on the meeting should form a useful preparatory stage for the conference on man-made mineral fibres expected to be held in Copenhagen in 1982. BACKGROUND Following the demonstration that mesotheliomas can be induced in rats by intra pleural and intra peritoneal injection of MMMF (1, 2) , studies of the potential risk in man were initiated in the United States and Europe by the MMMF industries . The Joint European Medical Research Board was formed by the Comite international de la Rayonne et des Fibres synthetiques and the European Insulation Manufacturing Association in 1975 to initiate re- search into the health problems of MMMF or vitreous fibres . The policy of the Board is to support financially the work of research institutes of repute such as the United Kingdom Medical Research Council 's Pneumoconiosis Unit , the International Agency for Research on Cancer (IARC) , Lyon , and the Institute of Occupational Medicine , Edinburgh. The research is coordi- nated by a separate scientific and technical committee independent of in- dustry . The research programme was launched with a review of the current state of knowledge at a scientific meeting held under the auspices of WHO in Co- penhagen in 1976 (3- 7) . It included animal experiments at the MRC Pneu- moconiosis Unit , epidemiological studies throughout Western Europe by IARC , and a parallel assessment by the Institute of Occupational Medi- cine of environmental exposures at the factories included in the epidemio- logical studies. The work , which is summarized below , is now well advanced, and it is planned to present the final results at the conference in Copenhagen in 1982. The animal experiments repeat previous intrapleural injection experiments and include inhalation work with dust clouds of adequate intensities of respi- rable dust. They have recently been extended in France and Germany with studies of the lung deposition, clearance, and retention ofMMMF and research on the ability (or otherwise) of these fibres to migrate from the inner lung to the pleura. Following a feasibility study of 72 manufacturing plants in Europe (55 using glass and rockwool , I 7 continuous filament) , 13 plants were selected as suitable for a retrospective cohort study . In these factories the current levels of concentration and the size distribution of airborne fibres have been measured by the Institute of Occupational Medicine using optical and scanning electron microscopy respectively , and occupations can be grouped and categorized according to those levels . Some of the early results from the European studies have been published (8); the mass and fibre number concentrations were ge- nerally low - mainly < 3 mg/m3 and < 0.5 fibre/ml respectively , although a small number of higher exposures occurred. Similar exposures have been re- ported for MMMF workers in the United States (9). Workers are followed up by investigators at national institutes (e.g ., cancer registries and university de- partments) to determine the mortality and cancer incidence from first expo- 2 sure to MMMF (usually in the early 1950s) up to the present. A comparison of the mortality and cancer incidence will be made between exposed workers and the unexposed general population and between subgroups of workers differently exposed. A tentative estimate of the total man-years at risk is 130 000 , of which about 13 000 occur after 20 years from first exposure . It is proposed to extend the work to include morbidity studies , for example of bronchi tis. In this retrospective study , as in most studies of the same type in occupa- tional epidemiology , four main problems occur, which can only partially be overcome and limit interpretation of the results: (a) the absence of reliable measurements of past exposure ; (b) the absence of detailed information (as opposed to general data) on the job history of each worker ; (c) the Jack of information on potential confounding variables (typically smoking habits) ; and (d) difficulty in following up the medical history of past employees and ascertaining (if they are dead) the cause of death . This problem applies particularly in certain countries. A further difficulty is that interlaboratory comparisons of optional fibre counts have shown substantial differences ( up to about 5: I) , a difficulty also well known in the counting of asbestos fibres (J 0, 11 ). Differences of a smaller order of magnitude (up to 2 : I) have been observed between personal mass ex- posure measurements made by different laboratories during the studies of the Joint European Medical Research Board . These difficulties , together with the need for a large number of observa- tions to meet the requirements for the detection of rare tumours , indicate the need for a future policy of continuing surveillance and regular reporting. Such a policy , if carried out on an international scale , calls at a minimum for an agreed reference method of estimating occupational exposure and a system of interlaboratory calibration to support a programme of prospective epidemio- logy. METHODS CURRENTLY USED FOR MONITORING MAN-MADE MINERAL FIBRES Measurement of total mass concentrations in air The total airborne mass concentration of MMMF dusts is commonly measured in most countries by sampling a measured volume of air at a known 3 flow rate (usually 2 I/min) through a pre-weighted filter. The methods used, al- though similar in principle, differ appreciably in detail. A minimum sampling volume of 0.5 m3 (about 4 hours sampling at 2 I/min) is usually required to achieve a reasonably accurate measurement , though substantially larger volumes are sometimes measured either with a personal sampler or with a static instru- ment to maximize the precision of weighing. Either glass wool or cellulose ester membrane filters of a pore size of about 0 .6- 1.5 µm are used for this purpose together with an 'open ' filter holder of either 25 mm, 35 mm, or 37 mm diamete r. It was agreed by the participants that mass concentrations could be measured more precisely with glass fibre filters since they exhibit greater weight stability . Various methods of pre-conditioning the filter material to minimize the effect of changes in humidity affecting the precision of weighing are practised - constant humidity chambers, oven drying followed by de- siccation, or allowing the filters to reach equilibrium in the laboratory atmos- phere and re-weighing the sample filters against controls that have been treated in the same manner. National regulations in the Federal Republic of Germany and Czechoslovakia require total mass sampling to be carried out with a face velocity of 1.25 m/s or 1- 2 m/s , respectively. In the United Kingdom the Health and Safety Executive recommended the use of a 25-mm-diameter for- ward-facing filter holder for mass measurements only in which the glass fibre filter is protected by a front plate pierced by seven 4-mm-diameter orifices through which the air is drawn. The purpose is to prevent loss of fibrous ma- terial owing to the "balling" of the fibres - a problem that has been fairly widely observed by most workers during the sampling of MMMF, especially where the concentrations are high. In making environmental measurements for epidemiological purposes the Institute of Occupational Medicine , Edinburgh, has used a thin metal plate mounted 1 cm in front of the open filter holder (see Annex 1), both to pre- vent furnace sparks from damaging the filter and to safeguard it from acciden- tal damage from fingers. In Sweden, for the latter purpose, three 2.5-mm- diameter metal strips are mounted across the entry orifice of the 37 mm filter holder. It was generally accepted at the meeting that the best estimates of expo- sure are obtained by sampling within the breathing zone of the worker (i.e., within 30 cm of his nose or mouth). For this purpose a belt-mounted pump is used with sampling tubes connected to a filter holder attached to the lapel. A minimum sampling volume of 0 .5 m3 (about 4 hours sampling at 2 I/min) is generally recommended . Whereas international agreement has been reached on the definition of and methods for measuring the mass concentration of the fine respirable fraction of airborne dust (12) that penetrates to the lungs(< 7 µm aerodyna- mic diameter) , no similar agreement exists on measuring the mass of what until now has been called "total" dust (including fibres and other particles). It is now recognized that the only part of airborne dust that is of interest is 4 that which is taken into the body through the nose and mouth during the act of breathing. This is known as the "inhalable" fraction. All the fractions of dust deposited in the various regions of the respiratory sys- tem are described in terms of subfractions of inhalable dust. A commit- tee of the International Organization for Standardization (ISO) is cur- rently considering recommendations for defining tl1ese fractions , on the basis of work in several countries, and samplers that meet these criteria are being developed (J 3 - 16) . In the meantime , all the techniques in current use are arbitrary. The different methods that purport to sample inhalable dust all give different results, even for dust clouds that are sup- posed to be identical . This is hardly surprising, since the aerodynamic capture of the relatively coarse particles that make up inhalable dust is highly dependent on inertial effects , and so on ambient wind speed, sampler design, location , and orientation. The conclusion, therefore, is that a specified environmental dust standard is only of limited value so long as such a wide range of sampling devices is used. This situation will remain until the questions about the definition and sampling of inhalable dust are answered. Measurement of fibre number concentrations in air The measurement of fibre number concentrations is generally car- ried out with methods based on those used for evaluating asbestos fibres (J 7,18). For this purpose personal samples are taken on cellulose ester membrane filters (pore size 0 .5 - 1.5 µm) in a manner similar to that described in the previous section . Smaller volumes of air are sampled de- pending on the concentration, so that the fibre density is suitable for counting. Either the whole filter or a segment of it is then mounted on a microscope slide, the side with the dust upwards, and the filter made transparent with one of several clearing agents (e.g., triacetin, ace- tone, acetone/triacetin , or ethyleneglycol monomethyl ether). A cover slip is then placed over the cleared filter sample and the fibres are counted and classified, using phase contrast microscopy, according to the follow- ing criteria: Respirable fibres : Non-respirable fibres : (if counted) Length (I) ~ 5 µm Diameter ( d) < 3 µm Aspect ratio (I : d) ~ 3 : I Length (I) ~ 5 µm Diameter ( d) ~ 3 µm Aspect ratio (I : d) ~ 3 : I 5 To facilitate counting and sizing, the microscopes used are fitted with a graticule inscribed with dimensional areas appropriate to the analysis. The airborne fibre concentrations are determined from : An C = -- (fibres/ml) gaV where A n g a V area of filter exposed during sampling (mm2) number of fibres counted number of graticule areas counted area of graticule (mm2) sample volume (ml) Non-respirable fibres were not usually evaluated in the past, except for epidemiological studies. In Czechoslovakia measurements of glass fibres directly from the screen of a projection microscope were reported , the length and diameter of all fibres with an aspect ratio > 3 being determined. The volume and weight of the fibres were estimated from the number of fibres present, the length and diameter size distributions , and the fibre density (p = 2 .6 g/cm3 ) . Although the methods are apparently similar, differences in the sampling procedure , the filter size and type , the clearing agent, and the microscope equipment used and, particularly, statistical and subjective errors in count- ing all contribute to the evaluation error. Similar problems occur in evalu- ating asbestos samples , and a reference method for the determination of air- borne asbestos fibre concentrations has been recommended by the Asbestos International Association (J 9) so that results from different countries be- come more comparable . Attention was drawn in the meeting to an optical problem that may be of importance in identifying some MMMF materials. Certain ceramic fibres of refractive index of rather less than J .5 will be ·difficult to resolve by phase contrast microscopy using clearing agents that have a refractive index for the collapsed filter of approximately 1.5 . For example , when a filter of refractive index I .5 is collapsed with acetone the refractive in- dex falls to 1.49, and to 1 .46 if triacetin is used. To be visible under phase contrast microscopy the refractive index of the fibre must be at least 0 .03 higher than that of the collapsed filter. PROPOSALS FOR A REFERENCE METHOD FOR MONITORING MAN -MADE MINERAL FIBRES Epidemiological studies in industry require either that the same environ- mental sampling and evaluation procedures should be used throughout or that 6 the results obtained using different procedures should be referable to a com- mon basis. It was for this reason that the Institute of Occupational Medicine adopted the methods detailed in Annex I for measuring exposures to MMMF dusts at the 13 MMMF manufacturing plants included in the Joint European Medical Research Board study. The method was primarily intended to per- mit the measurement of fibre number concentrations but it also at the same time enabled any estimate of the total mass concentration to be made. Since there are differences in monitoring procedures for MMMF between the European countries , particularly in the measurement of total mass con- centrations , it was agreed by participants that a suitable reference method is necessary to permit national data to be compared in epidemiological studies covering MMM F workers in different countries. • -The participants discussed in detail the proposal for a reference method given in Annex I. As far as fibre counting is concerned, the method described is similar in principle to the methods used within the countries represented at the meeting. Detailed modifications were debated so that agreed amendments could be incorporated in a revised reference method for counting MMMF. The differences in the methods used for measuring the total mass con- centration of MMMF in the various countries precluded the ready acceptance of the proposals for this purpose contained in Annex I. It was generally agreed that mass concentration should be measured separately from fibre number concentration . This would enable a larger volume of air to be sampled in mass measurements, so that the greater weight of dust co!lected could be weighed more precisely . Even so , it was recognized , within the different countries the differences in filter holder design, face velocity used, and recommended orien- tation to wind direction are not in accordance with recent research on the sampling of inhalable dust. Further research is needed to establish suitable instruments for the sar,pling of MMMF in accordance with the concept of inhalable dust and to establish the relationship of measurements taken in this way with those currently in use. For these reasons it was decided that the reference method should concentrate on the method for measuring fibre numbers. A revised reference method for monitoring MMMF was prepared and circulated to the participants for comment and criticism, and the final pro- posals for the determination of the mean fibre number concentrations are given in Annex 3. It was agreed that the method should be reviewed in about three years. Further discussion is needed before agreement can be reached on a reference procedure for mass measurement. Such a discussion should take into account any new evidence that is available on the comparability of different methods. Since the results of the research on the sampling of inha- lable dust are unlikely to enable new techniques to be developed in the near future, an interim agreement on a reference procedure for mass measurement will be necessary before prospective epidemiological studies are started. 7 ORGANIZATION OF A CENTRAL REFERENCE SCHEME FOR COUNTING AND SIZE ANALYSIS OF MAN-MADE MINERAL FIBRES While the use of a reference method will enable environmental measure- ments of MMMF from different countries to be compared , variations in count- ing levels noted during retrospective epidemiological and other studies in counting asbestos fibres led the participants to recommend that a reference scheme should be established on an international scale to harmonize evaluation procedures. This will be essential if data from plants in different countries are to be used in prospective epidemiological studies. Reference schemes have already been or are being established within several countries (including Czechoslovakia , Denmark, France , Norway , Sweden , the United Kingdom, and the United States) for asbestos counting on a continuous basis so that counting levels in different laboratories can be monitored centrally by the appropriate national agency. The participants considered the proposals for a reference method outlined in Annex 2 but noted certain limitations in the method . Being based on opti- cal microscopy , the method was not considered suitable for the sizing of fibres, a task that should be performed by electron microscopy ; and its efficiency in the evaluation of new fibre types with a refractive index near 1.5 requires fur- ther investigation. In addition, the method most appropriate for gravimetric sampling needs to be developed. It was agreed that a reference scheme needs to include a systematic circulation of samples for the counting and sizing of fibres along the lines described in Annex 2, but it was considered that a for- mal method of controlling and developing the work is necessary. It was there- fore proposed that a technical committee of experts should be formed with the support of the Joint European Medical Research Board and the WHO European Office to direct the work through an appropriate central reference laboratory . The participants considered that the terms of reference of the technical committee should include one permitting it to investigate the tech- nical problems referred to earlier and other related matters . The scheme should be operated through appropriate national institutes (normally one per country) within the WHO European Region. - The participants recommended that the reference scheme should be ini- tiated as soon as possible so that the initial results of the work could be re- ported at the WHO conference on man-made mineral fibres in Copenhagen in 1982 and future needs assessed. 8 APPLICATION OF THE REFERENC E SCHEME TO EPIDEMIOLOGICAL STlJDIES In view of the limitations of the data available for a retrospective cohort study, the participants supported the proposal for prospective epidemiological studies including large numbers of workers from a wide range of plants co- vering the full range of fibres and manufacturing processes. A properly planned study over a sufficiently long period would considerably improve precision in estimating the incidence of mortality and morbidity from cancer, and mor- bidity studies should be included. Concurrent measurements of exposure to total inhalable dust mass and fibre numbers should also be included so that reliance on estimates of past exposure, as in the Joint European Medical Re- search Board Studies, can be dispensed with . These measurements need to be linked to the reference scheme . The participants agreed that the full benefit of a prospective study can only be attained if arrangements are made to coordinate regular recording of the job history of each worker with environmental measurement of the mass and fibre number concentration. Good estimates of exposure will be obtained in this way and the results can be related to any medical effects found during follow-up studies . An appropriate dust sampling strategy needs to be developed at each plant. The Joint European Medical Research Board studies were limited to a short one-or-two-week survey at each plant. The data suggest that an occupational group structure may be evolved whose dust exposure and work history during a prospective study may both be recorded. No measurements of long-term variability of dust concentrations were possible in the Joint European Medical Research Board study and any future work must take this into account. The participants proposed that the feasibility of the procedures should be tested in a pilot study. It is clear that a prospective study could only be based on sampling carried out regularly at each plant by local personnel. For this to be effective in epi- demiological studies it is essential that there should be regular calibration of the measurement procedures using the reference scheme . If more than one plant from any given country is included in the study, national coordination could best be carried out internally through an appropriate national agency. Arrangements for this procedure and assessment of the results should be coor- dinated through the technical committee and central laboratory referred to in the section on the organization of a central reference scheme . One of the major problems in international epidemiological studies is obtaining access to the medical histo ry of past employees and ascertaining the cause of death, a problem that varies considerably between countries. The participants recognized the need for confidentiality . WHO might use its good offices to help obtain access in Member States to the information in death certificates, subject to appropnate safeguards of confidentiality, so that em- 9 ployment history could be linked to cause of death for medical research pur- poses . CONCLUSIONS The meeting recognized the value of the retrospective epidemiological and associated environmental studies conducted under the auspices of the Join t European Medical Reseach Board in assessing the health risks of expo- sure to MMMF. The studies nevertheless contain a number of limitations and shortcomings, which will affect the interpretation of the final results , parti- cularly as current knowledge suggests that the dose-related disease response to MMMF, if any , is likely to be long-term in its manifestation . Accordingly , the participants made a number of recommendations on the action needed to extend the work and improve the reliability and comparability of the results. RECOMMENDATIONS (1) Prospective epidemiological studies should be carried out at a large number of plants , both to increase the number of workers under surveillance and to cover the full range of fibres and manufacturing processes. The feasi- bility of the procedures employed should be tested in a pilot study. (2) In addition to assessing the feasibility of collecting and recording data on both previous and ongoing work, the prospective epidemiological studies should follow up the adverse effects of MMMF. For this purpose adequate facilities will be required. The effects studied should include the incidence of mortality and cancer , and measurements should be made on a continuing basis of total inhalable mass and of fibre numbers . (3) Since it would be difficult or impracticable to provide follow-up fa- cilities in some countries , there should be access to information in death certi- ficates for research purposes , subject to safeguards of confidentiality . WHO should actively encourage the linkage of occupational information with causes of death in Member States. (4) Methods of monitoring and evaluating mass and fibre number con- centrations, which differ in countries , should be brought into conformity. While differences persist , it is necessary to use a reference method enabling results 10 from different plants and countries to be compared for epidemiological pur- poses. (5) The monitoring method adopted for use in the retrospective studies has provided comparable data on plants over a short period of time. For theo- retical and practical reasons ce rtain modifications should be made, and the re- vised reference method should be reviewed in about three years. The revised method has certain limitations : being based on optical microscopy, it is not suitable for sizing fibres, an operation that should be performed by electron microscopy; and it may not be suitable for dealing with new fibre types with a refractive index less than 1.5. These and related technical matters (including the possible effects of electrostatics on sampling) should be investigated by the technical committee referred to in recommendation (6) . (6) Account must be taken of the substantial differences observed during the retrospective studies between laboratories in evaluating airborne concen- trations of MMMF, especially fibre number concentrations. A central refe- rence scheme should be developed for counting and sizing MMMF, aimed at harmonizing the levels and providing a basis for prospective epidemiology . The details of implementing the scheme should be the responsibility of a technical committee set up with the support of the Joint European Medical Research Board . The scheme should include research to solve the technical problems raised during this meeting. It should be operated through appro- priate institutes (normally one per country) within the WHO European Region. (7) Data on the long-term variability of exposure to MMMF are lacking. To assist the development of an experimental protocol for the prospective epidemiological studies in I 982, pilot studies should be undertaken of occu- pational exposure involving the measurement of variability at some (or all) of the four plants selected for the feasibility studies on surveillance and reporting. The agreed reference method should be used for this purpose. This would enable long-term sampling strategies to be assessed in terms of sampling fre- quency and cost. REFERENCES 1. Stanton, M. F. & Wrench , C. Journal of the National Cancer Institute, 48: 797- 821 (1972). 2. Pott , F. & Friedrichs, K. A. Naturwissenschaften, 59: 318 (1972). 3. Cameron, J. D. Annals of occupational hygiene, 20: 149-152 (1977). 4. Klingholtz , R .Annals of occupational hygiene, 20: 153- 159 (1977). 11 5. Hill, J. W. Annals of occupational hygiene, 20: 161 - 173 (I 977). 6. Gilson, J.C. Annals of occupational hygiene, 20: 175- 178 (1977). 7. Rossiter, C. E. Annals of occupational hygiene, 20: 179- 187 (1977). 8. Dod~on, J. et al. In: Proceedings of a Symposium on the Biological Ef- fects of Mineral Fibres, Lyon, 19 79. Lyon, International Agency for Research on Cancer, 1980 (Scientific Publication No . 30). 9. Esmen, N. et al . Environmental research, 15: 262- 277 (1978). I 0. Beckett, S. T.Annals of occupational hygiene, 16: 405-408 (1973). 11. Walton, W. H. et al.Annals of occupational hygiene, 19: 215 - 224 (1976). 12. Orenstein, A. J. ed.Proceedings of thePneumoconiosis Conference,Johan- nesburg, 1959. London , Churchill , 1960. 13. Ogden, T. L. & Birkett, J. L.Annals of occupational hygiene, 21: 41 - 50 (1978). 14. Wood, J. D. & Birkett, J. L. Annals of occupational hygiene, 22: 299- 310 (1979) . 15. Vincent, J. H. & Mark, D. In: Proceedings of the Fifth International Symposium on Inhaled Particles, Cardiff, 1980. In press. 16. Armbruster, L. & Breuer, H. In: Proceedings of the Fifth International Symposium on Inhaled Particles, Cardiff, 1980. In press. 17. Technical Note 1. Rochdale, Asbestosis Research Council , 1971. 18. Edwards, G. H. & Lynch, J. R.Annals of occupational hygiene, 11: 1- 6 (1968). 19. Recommended technical method No. 1. London, Asbestos International Association. 12 Annex 1 A RECOMMENDED ST AND ARD METHOD OF MONITORING AIRBORNE MAN-MADE MINERAL FIBRES J. Dodgson° SCOPE The techniques described in this Annex were developed to assess the full-shift dust exposure of workers in the man-made mineral fibre (MMMF) manufacturing industry . The methods described enable the mean fibre number concentration and the "total" mass concentration to be measured over an 8-hour period. Since the prime purpose was to measure the fibre exposures, the techniques used are based on those commonly adopted for asbestos mo- nitoring. Whereas international agreement has been reached on the measure- ment of respirable dust , such agreement has not been reached on a method for measuring total dust. Consequently, all techniques in common use , including that described here , are empirical. The methods described are not necessarily suitable for measuring peak exposures and may not be di- rectly applicable to the checking of compliance with statutory regulations or the monitoring of levels in the MMMF user industry. Full-shift measurements are , however , a better indication of the on-site exposure of personnel and are applicable for determining threshold limit values (i .e., the concentration to which a person may be exposed for 40 hours per week) and for epidemiologi- cal studies. SUMMARY OF METHOD Initially , membrane filters are pre-treated to remove any electrostatic charge before being stabilized and weighed . Monitoring is then carried out with the filter placed in the breathing zone of the subject. The sample is collected by drawing a measured quantity of air through the filter by means of a battery- powered sampling pump. The filter is again stabilized and reweighed to obtain the mass of deposited particulate matter. After the filter is made optically 0 Institute of Occupational Medicine , Edinburgh , United Kingdom. 13 transparent , the fibres present within random areas are counted optically using a transmission phase contrast or interference microscope at a magnification of approximately S00X. The total number of fibres on the filter is then estimated. The main aim of this procedure is to evaluate the airborne dust concentration in terms of the fibre number. However , the method also permits simultaneous measurement of the mass concentration . If only the fibre number concentra- tion is required , then it is unnecessary to weigh the filters. DEFINITION OF A FIBRE AND A FRAGMENT For the purpose of optical counting, a "fibre" is defined as having a length ;;;i, 5 µm and a length/diameter (aspect) ratio ;;;i, 3: 1. Particles of MMMF with length/diameter ratio < 3 : 1 will be termed "fragments". Fibres of dia- meter < 3 µm are considered to be "respirable", whereas those of diameter ;;;i, 3 µm are "non-respirable". "Respirable" fragments are those with a mean projected diameter< 5 µm . Fibre 1/d ;;;i, 3:1 Fragment 1/d < 3:1 1 length of particle d diameter of particle Respirable d<3µm D<Sµm Non-respirable d;;;i,3µm n;;;i,sµm D = mean projected diameter (diameter of circle of equal area) In the absence of other convincing information ( e.g., morphology , re- fractive index) , fibres should be considered to be MMMF and counted as such. Fragments should only be evaluated when some form of compositional iden- tification is available (e.g., refractive index or dispersion colours). APPARATUS AND REAGENTS Sampling equipment Pumps should be sufficiently light to be worn for an entire shift without discomfort and be battery-powered and capable of functioning continuously for eight hours at the selected flow rate without recharging. The flow should also be rendered pulsation-free by means of an external smoothing unit if ne- cessary . Charging units should be capable of fully recharging the pump battery with- in 16 hours to allow the use of the pump for one shift every day. It should al- 14 so be safe to leave the batteries charging for longer than the recharge time without damage to the battery or charging unit. An indicator lamp that re- gisters when the battery is actually charging as opposed to when the charging unit is switched on is also preferable. Filters (see following section) Filter holders must be light, capable of securely holding the type of filter in use, and unobtrusive. Open-type filter holders are preferred , providing that a protective shield is employed to prevent filter damage . Filter holders with fitted covers may be used , providing that the covers have sufficient holes in them to prevent blockage of the airways by fibre agglomerates . On no account should a filter holder employ any form of tubing or similar restrictions in front of the filter. The most commonly used holders are the 25 mm Gelman holder and the 35 mm Millipore design (see Annex 3, Appendix A). Filter holder shields should be light and cover the whole filter surface without restricting the air flow through the filter. They should also displace easily to facilitate flow rate readings taken at the front of the filter holder. One possible design for use with the Gelman or Millipore type holder is shown in Fig. 1. Fig. 1. Filter holder with shield swivel jo int 15 Tubing should not kink readily and must be capable of maintaining leak- proof connexions. Flow meters must be able to measure the flow rate at the filter surface. Flow rate measurements taken at any other position in the sampling train are subject to errors due to leaks . Errors in flow rate measurements will also occur if the flow is not pulsation-free. Lapel fixings. Locating the filter in the breathing zone of the worker in- evitably means fixing the filter holder to a lapel or shoulder-strap. Spring clips are preferable to safety-pins, etc. for this purpose as they do not pierce or damage workers' clothing. Belts should be supplied to support the pumps when sampling, to im- prove workers' comfort, and to standardize the position in which the pumps are worn. Filter holder covers. Where these are not part of the sampling head pro• vided by the manufacturer , covers should be obtained to protect the filter during transportation. Plastic covers for Gelman sampling heads can be ob· tained from Airoquip UK Ltd (see Annex 3, Appendix A). Optical equipment Microscope. The microscope used should have a magnification of between S00X and 600X, with an objective lens of magnification of approximately 40X. Lower-power objectives with high-power eyepieces or immersion ob- jectives with low-magnification eyepieces must not be used . The microscope should be one of the following types: (a) interference; obtained by the shearing method , based on the prin- ciple of polarization (Normarski and separated objective types are not recommended). This type is preferred at the Institute of Occupational Medicine and should be used if an estimation is being made of the num- ber of MMMF fragments. (b) phase contrast; either positive or negative phase may be used. Ahigh- absorption (95%) phase ring in the objective is preferred . This type is not recommended for estimating fragments. In order to reduce eyestrain, binocular microscopes should be used. For phase contrast work it is recommended that a green or blue filter be incor- porated in the illumination system, particularly where low-absorption (60%) positive-phase rings are used. 16 Eyepiece graticule (see the section on microscopical and counting pro- cedures). Filter mounting equipment Microscope slides. Best-quality , 25 x 76 (0 .8- 1.0 mm thick) glass slides are commonly used. Cover slips. Cover slips are a necessary part of the slide mount and optical system. They should be of a size marginally greater than that of the filter being mounted and matched in thickness to the calibration of the objective . Scalpel. If filter sections are to be cut, a straight-edged blade surgeon's scal- pel is satisfactory. Scissors are unsuitable as their use may disturb the sample. Tweezers. A range of high-quality broad-tipped and pointed-tipped twee- zers is essential for filter manipulation . Lens tissue. Use of a lint-free lens tissue is recommended for cleaning cover slips. An industrial paper cleaning tissue is suitable for slides. Organic tissue fibre contamination can be readily identified and rejected. Heating device. A water bath and one-litre flask are recommended for heating acetone when this method of mounting is used . Reagents Acetone. Laboratory-grade acetone is satisfactory for mounting filters . Glycerol triacetate. The laboratory grade is sufficiently pure but it should not be used if an odour of acetic acid is present . Methanol. Laboratory-grade methanol may be used to clean slides and cover glasses, etc . Hyamine 2389 (see Annex 3, Appendix A; normally available in a 50% aqueous solution). FILTER PREPARATION Type of filter White cellulose ester membrane· filters of 0.8 or 1.2 µm pore size are re- commended, preferably with printed grids. Where mass concentrations only 17 are required, glass-fibre filters (GF A) may be substituted. The diameter of fil- ter used should be chosen taking into account the following three factors. Pressure drop across filters. Where the pressure drop is near or above the recommended limit for the pump being used , the filter diameter or pore size should be increased. This is particularly important for full-shift sampling . Expected fibre density on filter. Only a limited number of fields of view of the microscope are evaluated . With an increase in filter size there is a pro- portionate decrease in the fraction of filter examined. For low concentrations this can result in larger counting errors. Conversely, at high concentrations the use of a larger filter will reduce the degree of overlap. Filter mounting method. It may not be possible to mount the whole of a filter on one microscope slide. Dividing the filter into sections, however , in- creases the chances of sample loss . In addition, large differences may be found between sectors of the same filter (J). This may not be apparent if only one sector is evaluated, and where possible the size should be chosen so that the complete ftlter can be mounted on one slide. For sampling in the relatively low concentrations occurring in most parts of the MMMF manufacturing in- dustry , 0.8 µm pore size , 25 mm diameter filters are preferred . Removal of electrostatic charge from the filters The electrostatic charge on a filter can affect both the accuracy of its weight measurement and its sampling characteristics. The weighing error can be overcome by discharging the filter before or during weighing using a radioactive source. This may involve extra handling, which is undesirable, and not permanently remove the charge. A more satisfactory method was developed by Mark (2) , which can reduce the electrical resistance of the fil. ters by a factor of up to 106 . Hyamine 2389 is diluted to form a 0.1 % solution (care is needed: higher concentrations will result in a greatly increased pressure drop across the filter during sampling) . The solution is poured into a dish or tray to a depth of approximately 40 mm. One litre is sufficient for about 50 filters . The filters are placed on top of the solution and allowed to sink . Any remaining on the surface after 6 hours are prodded gently to the bottom of the container. After 24 hours the filters are taken out and dried between sheets of blotting paper to prevent localized globules of detergent from blocking up part of the filter. This can occur when filters are dried on a nonabsorbent material. Once dried, the filters can be stabilized ready for weighing. 18 Pre-weighing Filters must always be manipulated with blunt-nosed forceps gripping the filter edge. Occasionally within a batch of filters a filter is found that will increase weight dramatically with increased humidity . It is therefore necessary to ensure that the variation of filter weights with humidity is standard for the batch. Filters should be allowed to stabilize in individual tins with the tin lids slightly open in a balance room overnight before being weighed . A second weighing, preceded by a further 24 hours of stabilization, will reveal a standard of variation against which defective filters can be identified. Weighed filters should be kept in dust-proof tins or mounted in securely covered filter holders until use. A minimum of four filters per batch must be retained as controls. These should not be used for sampling, but are reweighed whenever any of the samples are being evaluated. This enables any changes in weight of the sampling filter due to humidity to be determined. SAMPLING General In order to estimate the mean shift exposure of an operative, samples must be taken in the breathing zone (i.e., the hemisphere of 300 mm radius extending in front of the face and centred on the nose). The filter surface may be at any angle between directly downwards and facing forward, and should have a guard placed in front of the surface to prevent accidental or de- liberate contamination (see section on equipment). This is particularly impor- tant in a furnace area , where high-velocity particles of molten glass can burn holes in the filter. The 37 mm Millipore holder should normally be used with the face section removed, and with a protective cover placed about 5 mm from the end of the holder. Where this cover is not available, six additional 4 mm holes should be drilled in some face sections (J). These are then used du- ring sampling and replaced by unadapted ones for transportation. Flow rate selection The sampling flow rate should be between 0.5 and 2 I/min. Lower flow rates are to be avoided as particle elutriation at these velocities may reduce the measured mass concentration and contamination effects are more im- portant. The sampling time and flow rate should be adjusted to obtain between 50 and 1000 fibres/mm2 on the filter to minimize counting errors . In high 19 particle/fibre concentrations a consecutive series of short-term samples may be necessary rather than one long-term one. Calibration of sampling pumps Pumps should be tested before use to ensure that they are operating at the manufacturer 's specification. The flow should be pre-set with the full sampling train (i.e ., smoothers , filters , etc .) in position . Some types of pumps include an air bleed to enable the flow through the filter to be changed . These bleeds should always be closed and the flow altered by adjusting the piston stroke. If the bleed is open more ai r will be drawn through it as the pressure drop across the filter increases due to particulate build-up on its surface. A large decrease in sampling flow is therefore liable to take place. No pump should be operated without a filter on the input , otherwise da- mage can occur. All pumps should be allowed to run for a few minutes before setting the flow . Some types are also susceptible to changes in temperature ; in these cases a warming-up period of at least half an hour should be allowed before calibration . Sampling procedure Starting routine. The pump should be attached to the worker by means of a belt or harness and the filter holder fastened to his or her clothing in the breathing zone. The connecting tubing may be secured to clothing by adhe- sive tape. At this stage any covering or cap over the filter should be removed , and the filter checked for damage . The switch-on time and any other time or volume reading on the pump is then noted. Progress checks. The flow rate through the filter should be checked and recorded during the sampling period, together with the time; if possible, this should be carried out every hour during long-term sampling. If a change in flow rate has occurred , the pump may be re-set to its previous value where practicable (see section on microscopical and counting procedures). Sampling should be terminated for any sample where the flow rate falls by more than 30%. Damaged filters should be withdrawn and the sample abandoned . Dense samples should be replaced by an unused filter ; as a rough guide, a filter should be changed when the printed grid lines are no longer easily visible . Completion of sampling. When the sampling period is completed, switch off the pump and immediately replace the filter holder cap, noting the time. Where possible, the filter should remain in the filter holder until it is ready for analysis. 20 Blanks Some control filters (a minimum of 2%) should be processed in the same way as for normal samples , but without air being drawn through them. If there are more than 15 fibres per mm2 on these filters , the entire sampling/analysing procedure should be examined to determine the cause of the contamination. Transporting filters Filters may be transported in their sampling holders , provided a top cap or cover is fitted to prevent contamination , or in a box or tin . In the latter case, the ftlter should be removed from the holder in a dust-free area with for- ceps , care being taken only to grasp its unexposed edge. This edge should be used to attach the filter to the base , using sticky tape. After transportation the filter can be easily removed from the container by means of a scalpel. Plastic containers made of highly insulating material should be avoided to mi- nimize electrostatic effects, which may result in fibre loss . The filter holders or containers should be packed into a rigid case with sufficient foam packing to prevent crushing and minimize vibrational effects. Care should be taken to avoid undue banging of the case. It is desirable to mount a proportion of the filters ( or sectors from them) before transportation, so that any fibre losses can be estimated. Experience during some surveys has shown fibre loss during transport to be unimportant. The use of fixatives , either cytological or perspex-based, is not recommended. FILTER WEIGHING AND MOUNTING Weighing the sample Extreme care must be taken when dismantling the filter holder to avoid disturbing the sample or damaging the edge of the filter. The filters should be stabilized and re-weighed following the procedure outline above . It is consi- dered that a five-place analytical balance is suitable for this purpose. At least four control filters must be weighed each time any batch of filters is evaluated. The mean change from their original weights provides the correction, which is then applied to all the sampling filter weights. Mounting the filter The mounting method must render the filter transparent without intro- ducing flaws that could confuse or hamper counting. A permanent method is prefe rred as this enables the sample to be re-assessed at a later date if required. The method used at the Institute of Occupational Medicine is based upon the 21 use of acetone vapour° (4) , as detailed below. However , non-permanent clear- ing methods may be used provided the samples are counted within a few weeks of mounting. Two acceptable methods currently in use are the triacetin method (5) and the dimethyl phthalate/diethyl oxalate method (3). These methods, however, may not be suitable for all makes of filters, e.g., triacetin cannot be used with some Gelman filters. Before they are mounted, all samples should be inspected to ensure that the deposit is not too dense for optical evaluation . Where there is some doubt as to the density of the collected ma- terial, the filter may be divided into two, one half being mounted using the standard technique. The dust on the remainder of the filter should be washed off and re-deposited on to a clean filter, which can then be analysed by the standard technique . The above procedure requires careful manipulation and is therefore not recommended for routine use . Dense samples should be avoided by restricting the sampling time or volume . Acetone/triacetin mounting method. The filter is placed, dust side upper- most, on a clean and warm microscope slide, then plunged into hot acetone vapour. The acetone vapour is produced in a glass flask heated by a water bath until the acetone is gently boiling (Fig. 2). If the acetone is not boiling suffi- ciently the filter may twist and buckle before clearing. After approximately two seconds in the vapour , the filter will clear and should be removed from the flask to allow excess acetone to evaporate. The filter is then covered with a clean cover slip, using a drop of triacetin to give a good optical contact be- tween it and the sample. MICROSCOPICAL AND COUNTING PROCEDURES Microscope adjustment The manufacturer's instructions for setting up the microscope must be followed carefully. The optical alignment of both phase and interference systems should be checked periodically, and always be checked following the changing of any lenses or transport of the microscope. Eyepiece graticule Any graticule may be used for MMMF evaluation providing that it satisfies three criteria : a Acetone mounting should be carried out only in a fume cupboard or well ventilated room, using a water bath for heating. On no account should a naked flame be allowed in the region of acetone vapour. • 22 (a) it has markings exactly 5 µm apart on it; (b) it has markings exactly 3 µm apart on it (this is particularly im- portant where separate counts are being made of respirable and non- respirable fibres) ; (c) it has a clearly defined central counting area occupying not more than one fifth of the full field of view of the microscope . This can be achieved by inserting one graticule to delineate the counting area into one eyepiece, while using a "sizing" graticule (e.g., Graticules Ltd - type E17) in the other . Alternatively , a single Walton-Beckett graticule (6) (Graticules Ltd - type E22) may be used. These were designed specifically for fibre counting, but must be manufactured individually for each combination of eyepiece and objective. The graticule must always be in focus , and the eyepiece used should pre- ferably have adjustable focusing. In addition, before use , and following any change or adjustment to the microscope lenses , the sizes of the marking on the graticule must be calibrated by means of a stage micrometer (note that ca- libration changes marginally with alternation of interocular distance on a bi- nocular microscope). Use of graticule The cent ral defined area alone is used for counting fibres and any fibre lying totally within it is evaluated. It is also necessary to define rules for de- termining which of the fibres crossing the boundary of this area are to be counted . Normally two adjacent sides (or one hemisphere for a circular gra- ti cule) are initially chosen. Fibres crossing these are counted , whereas any cutting the other two sides (or other hemisphere) are omitted for that grati- cule count. All fibres must have at least one end within the counting area to be evaluated. Fibres passing st raight through are not counted (Fig. 3) . The full field of view of the microscope should not be evaluated as this can lead to a greatly reduced fibre count (7). Counting. The aim of the counting procedure is to estimate the total number of fibres on the filter surface so that the original airborne concen- tration can then be calculate<l from the volume of the air sampled. The num- ber of respirable and non-respirable fib res is counted in 100 graticule areas. unless more than 200 fibres (either respirable or non-respirable) are observed. A minimum of 10 graticule areas should be evaluated , even if more than 200 fibres are recorded. One hundred Walton-Beckett graticule areas constitute approximately only 0 .2% of the area of a 25-mm-diameter filter and the method assumes 23 Fig. 2. Acetone mounting equipment acetone water bath 24 that the fibres are homogeneously distributed in the filter. It is necessary to ensure that : (a) filters with uneven deposits are rejected; (b) the randomly selected graticule areas are representative of the whole area of the mounted sample and do not overlap (the method used is to traverse the filter , starting at the top , evaluating randomly selected graticule areas along the traverse ; the process is then repeated on randomly selected traverses covering the whole filter area until the requisite num- ber of graticule areas have been evaluated); and (c) once selected, a graticule should always be evaluated except where over half the area is obscured. Fig. 3. Proposed criteria for evaluating fibres overlapping the edge of the counting area Fibre counted c==::::::::i Fibre not counted (a) Rectangular counting area " " " " " " " " " " " u (b) Circular counting area Counting criteria : top and left sides not evaluated bottom and right sides evaluated Counting criteria : upper hemisphere evaluated lower hemisphere not evaluated 25 Precision of counting. Differences in the counts of fibres may arise from two sources : (a) sy stematic differences or effects attributable to differences in technique (sampling, mounting, counting rules , etc.) or personal factors (subjective errors). The evaluation and discussion of systematic errors constitute a subject in themselves and as such are outside the scope of this document. (b) random errors, i.e ., those which are individually unpredictable and variable in magnitude. They arise because the counter examines only a small area of the membrane filter sample , typically< 0.2%. This implies that repeat counts on the same sample are likely to cover different areas . If a sufficiently large number of measurements are taken in a controlled way , differences arising from random errors may be unimportant and can , in principle , be made negligible . On the other hand , differences arising from systematic effects ( differences in technique or subjective errors) cannot be eliminated by increasing the area of the filter examined. Both the magnitude and the source of these systematic differences are difficult to distinguish if the random error is also high. Tests using MMMF data from environmental surveys have confirmed that the distribution of fibres on membrane filters may be reasonably described by the Poisson distribution. On this basis, when fibres are distributed ran- domly over a surface , the probability (P) that x fibres are observed in a spe- cific field is given by: µx exp(- µ) Px=----- x! The 95% confidence limits for the Poisson distribution are shown in Table 1. For example, when 4 fibres are observed in a specific area, then 95 times out of 100 the true fibre density lies between I and 10 fibres . It can be seen from Fig. 4 that the limits are markedly asymmetrical and proportionately very large where fewer than 20 fibres are observed . The limits tend to be much smaller symmetrical values as the number of fibres observed approaches 100. Where the evaluation involves samples of similar volumes (e.g., approxi- mately 500 litres) and all counts refer to the same proportion of the filter area (e .g. , approximately 0 .2%) it is possible to relate approximately the number of fibres observed on the filter to the density of fibres in the air: 26 1 fibre observed 10 fibres observed I 00 fib res observed 0 .001 fibres / ml 0.01 fibres/ml 0.1 fibres /ml . + 200% , ~ al t ~ .0 0 . .c E _g +. 100% . u C . . ii [ 0 1 IOO'li. Table 1. 95% confidence limits for the Poisson distribution Observed number Lower limit Upper limit of fibres fibres fibres 1 0 6 2 0 7 3 9 4 10 5 2 12 10 5 18 20 12 31 30 20 42 40 28 54 50 37 66 100 82 122 200 174 230 Fig. 4 . 95% confidence limits for the unknown fibre density (assuming Poisson distribution) No of 1000 fibres observed It is evident from both Table I and Fig. 4 that large random variations in the number of fibres observed may occur at the lower densities; neverthe- less , expressed in terms of airborne fibre concentration , the variation is rela- tively unimportant. 27 Size distribution Owing to the present unce rtainty concerning the particular size parame- ters that are most biologically ac tive, it is desirable that data should be obtained concerning the length and diameter distribution of the various airborne fibres. Limits of resolution and technical convenience generally restrict the use of optical microscopy to length distributions > 5 µm. Shorter fibres are best evaluated by electron microscopy ,a and this is considered to be the only satis- factory system for determining diameter distributions in the respirable size range(< 3 µm). Length distribution may be measured using the preceding optical technique and the following criteria: (a) the criteria given above; (b) the rules used for counting fibres that overlap the graticule edge must be applied when sizing to make the result independent of fibre length; and (c) at least 200 fibres should be sized. Calculations Volume of air sampled. The volume of air samples is calculated by mul- tiplying the duration of sampling by the measured flow rate. If the flow rate changes during sampling, the air volume should be estimated assuming that a linear change occurred between flow rate measurements. Airborne fibre concentration. The airborne fibre concentration (C) is de- termined by dividing the number of fibres estimated to be on the filter (N) by the volume of air samples (V) . N An C= - ,and N= - where V ga A a g n area of sample deposit (not area of filter) (mm2) area of graticule (mm2) in the object plane number of graticule areas evaluated number of fibres counted in g graticule areas. a Details of the electron microscope methods used at the Institute of Occupational Medicine , Edinburgh, may be obtained on application to the Institute. 28 Combining these equations gives: An C = - - fibres /ml where V is measured in ml. gaV REFERENCES I . Bartosiewicz, L. American Industrial Hygiene Association journal, 34(6): 252 - 259 (I 973). 2 . Mark , D. Annals of occupational hygiene, 17: 35-40 (I 974) . 3. Edwards, G. H. & Lynch, J. R. Annals of occupational hygiene, 11 : 1-6 (1968). 4. Membrane filter method for estimating airborne asbestos dust. Canberra, National Health and Medical Research Council, 1976. 5. Technical Note I. Rochdale , Asbestosis Research Council, 1971 . 6. Walton, W. H. & Beckett, S. T . Annals of occupational hygiene, 20: 19- 23 (1977). 7. Beckett , S. T . et aJ . Annals of occupational hygiene, 19 : 69- 76 (I 976). 29 Annex 2 ORGANIZATION OF A CENTRAL REFERENCE SCHEME FOR COUNTING MAN-MADE MINERAL FIBRES (MMMF) J. Dr,dgson° Purpose To establish standard reference counts of MMMF samples and provide a service whereby the counting performance of different laboratories can be checked against optical or scanning electron microscope standards. Outline of method The standard of reference will be the average of counts made by a group of experienced "master" laboratories (e.g., one per country) working according to a method agreed with the appropriate agencies (the accepted method) . The average will be established by circulating "permanent" mounted samples to all the master laboratories for evaluation. These samples will subsequently be available for counting by other laboratories whose performance is to be as- sessed. Reasonable consistency between the members of the master group will be essential to the success of the scheme ; meetings and discussions be- tween representatives may be needed to achieve this. The distribution of sam- ples among the master laboratories will be repeated at suitable intervals (e.g., every six months). On each occasion new samples will be introduced so as to maintain or ir,crease the reference stock; in addition , some previously evaluated samples will be recirculated to check the long-term stability of the master group . Separate distributions will be needed for the optical and scanning elec- tron microscope standard samples. The results of other laboratories in a given country may be compared with the mean count of the master laboratories ( or with the count obtained by that country's master laboratory). This can be done by requesting those labo- ratories to evaluate a number of the standard samples. alnstitute of Occupational Medicine, Edinburgh, United Kingdom. 30 When a laboratory employs more than one person on MMMF counting, one nominated representative might evaluate the standard samples, while the other counters would be related to this representative by in-house check counting of local samples. The checking scheme and the count results would be submitted to the central reference agency for assessment. The purpose is to restrict the demand for standard samples, the supply of which will be li- mited. The achievable level of agreement between laboratories , acceptable limits of variation , the number of samples, frequency of checking, etc., can only be reliably assessed by statistical analysis of the results once the scheme has started. These matters will require discussion and agreement with the agencies concerned. Master laboratories The master laboratories will be established laboratories of experience and repute that have been prominent in the development of measurement proce- dures for MMMF , deal with the full variety of samples likely to be encountered, and represent different interests. Accepted method of counting The method of evaluating MMMF described in Annex III will form the basis of the method, subject to discussions with the laboratories and other agencies concerned. Work plan Initially each of, for example, 10 master laboratories will provide, say, 10 membrane filter samples of an agreed type (to give good overall represen- tation), mounted by the acetone triacetin method for permanency and for optical evaluation. These 100 samples will then be evaluated independently by all the laboratories. Further similar distributions will be undertaken at six-month intervals , using 50 new slides and 20 from earlier occasions. Addi- tionally, stub-mounted and gold-coated Nuclepore filter samples will be re- quired from the participating master laboratories for evaluation by scanning electron microscope. It is envisaged that about one-third of the above num- ber of samples could be conveniently dealt with. All of these will be used for count comparisons, but only some (say , one-quarter) will be included in the comparison of size distributions because of the time-consuming nature and cost of the technique. It is anticipated , on the basis of experience gained by 31 the Central Reference Laboratory for Asbestos Counting, that special inves- tigative exer,;ises will additionally be required in order to harmonize counting levels and procedures. These will be determined on the basis of the results of the counts themselves and the discussions between the representatives of the master laboratories. To be effective, the programme of work will need to be arranged on a continuing basis. It is proposed that the Institute of Occupational Medicine , Edinburgh, United Kingdom , should have two quite separate functions : (I) of serving as a member of the group of master laboratories and (2) of operating the service. The latter function would involve planning the master exchange ; receiving, coding and despatching slides , and evaluating the master counts ; keeping quality control charts for the master laboratories , showing scatter , trends , long-term stability, etc. ; organizing discussion meetings when necessary ; dispatching samples , analysing data , and maintaining records. Those concerned should have competence in actual fibre counting, as they would need to scru- tinize samples for damage , etc., to guide discussions between members of the master counters group , and to translate agreed procedures into written codes . 32 Annex 3 A RECOMMENDED REFERENCE METHOD OF MONITORING THE NUMBER CONCENTRATION OF AIRBORNE MAN-MADE MINERAL FIBRES The techniques described below are designed to assess the full-shift dust exposure of workers to man-made mineral fibres (MMMF). The methods described enable the mean fibre number concentration to be measured over an 8-hour period. Full-shift measurements are a better indication of the on-site exposure of personnel and can be used for determining thresh- old limit values (i.e ., the concentration to which a person may be exposed for 40 hours per week) and for epidemiological studies. The techniques used are based on those commonly adopted for asbestos monitoring (1, 2). SUMMARY OF METHOD Initially , membrane filters are pre-treated to remove any electrostatic charge . Monitoring is then carried out with the filter placed in the breathing zone of the subject. The sample is collected by drawing a measured quantity of air through the filter by means of a battery-powered sampling pump. The filter is made optically transparent, and the fibres present within random areas are optically counted using a transmission phase contrast microscope at a magnification of approximately S0OX. The total number of fibres on the filter is then estimated, and hence the airborne dust concentration in terms of the fibre number. DEFINITION OF A FIBRE For the purpose of optical counting, a "fibre" is defined as having a length~ 5 µm and a length/diameter (aspect) ratio~ 3: 1. Fibres of diameter < 3 µm are considered to be "respirable", whereas those of diameter~ 3 µm are "non-respirable". Fibre 1/d ~ 3: 1, 1 ~ 5 µm I = length of particle d = diameter of particle Respirable d<3µm Non-respirable d~3µm 33 In the absence of other convincing information (e.g. morphology) fibres should be considered to be MMMF and counted as such . APPARATUS AND REAGENTS Sampling equipment A list of suppliers of suitable equipment is given in Appendix A as an example . It does not imply any recognition by WHO of the quality of the goods they supply or of any preference for those goods. Fig. 1. Filter holder and protective cowl 34 Pumps must be sufficientl y light(< 1 kg) to be worn for an entire shift without discomfort , be battery-powered . :ir.d be capab le of funct ioning con- tinuously for at least eight hours at the selected flow rate without recharging. The flow must also be rendered pulsation-damped by means of an external smoothing unit if necessary. Charging units must be capable of fully recharging the pump battery within 16 hours to permit the use of the pump for one shift every day. It must also be safe to leave the batteries charging for longer than the recharge time with- out damage to the battery or charging unit. An indicator lamp that registers when the battery is actually charging, as opposed to when the charging unit is switched on . is also preferable . Filters. White gridded cellulose ester membrane filters of 1.2 µm pore size and 25 mm diameter must be used. Filter hulders should be light. capable of securely holding the type of fil. ter in use. and unobtrusive. An open-type filter holder must be used which is fitted with a protective metallic cowl (Fig. 1) to help protect the filter from accidental damage. Tubing should not kink readily and must be capab le of maintaining leak - proof connexions. Flow meters must be ab le to measure the flow rate at the filter surface : measurements taken at any other position in the sampling train are subject to errors due to leaks . The flow meter must be able to measure to an accuracy of at least I 0% at the selected flow rate. Calibration of flow meters against a suitable primary standard (i.e., wet gas meter , soap-film meter) should be undertaken annually. Lapel fixings. Spring clips should be used in preference to safety-pins. etc., since they do not pierce or damage workers ' clothing. Belts should be supplied to support the pumps when sam pling. to im- prove workers ' comfort. and to standardize the position in which the pumps are worn. Filter holder covers. Where these are not part of the sampling head pro- vided by the manufacturer , covers must be obtained to protect the filter du- ring transportation. 35 Optical equipment Microscope. The microscope must conform to the following specifications : Illumination Substage assembly Objective Eyepiece Graticule Accessories Filter mounting equipment Koehler illumination , built in. Phase contrast condenser in centring focusing mount. The phase annulus centration should be independent of the condenser centring mechanism . X40 parfocal phase contrast achromatic objec- tive with numerical aperture at least 0.65. The phase rings should have an absorption coeffi- cient of between 70 and 90%. Positive or nega- tive phase is suitable . Compensating binocular. Total magnification between 500 and 600 X. One eyepiece must permit the insertion of a graticule and be of fo- cusing type . The Walton-Beckett graticule must be used (Fig. "2). Centring telescope or Bertrand lens. Green filter. Stage micrometer, I mm long with 2 µm divi- sions. A Health and Safety Executive/National Physical Laboratory (HSE/NPL) test slide to check the detection limit of the microscope- observer system. Microscope slides. Best-quality 25 X 76 (0.8- 1.0 mm thick) glass slides should be used. Cover slips. Cover slips are a necessary part of the slide mount and optical system. They must be of a size marginally greater than that of the filter being mounted and matched in thickness with the calibration of the objective . Tweezers. A range of high-quality broad-tipped and pointed-tipped tweezers must be available for filter manipulation . 36 - ----- - - --------------------- Fig. 2. The Walt on-Beckett grat icule 3 10µm ~ 3 _L T 5 ......... Lens tissue. Lint-free lens tissue must be used for cleaning cover slips . An industrial paper cleaning tissue is suitable for slides. Heating device. An electrically heated water bath with a two-neck flask and water condenser system must be used for heating acetone (see Fig. 3). 37 Fig. 3. Acetone mounting equipment condensing column cooling water---- water bath (electrically heated) Reagents (filtered free of fibres) orifice (1 cm diameter) Acetone. Laboratory-grade acetone should be used for mounting filters. Glycerol triacetate (triacetin). The laboratory grade is sufficiently pure but it should not be used if an odour of acetic acid is present. Ethanol. Laboratory-grade ethanol may be used to clean slides, cover glasses, etc. Hyamine 2389, normally available in a 50% aqueous solution. 38 FILTER PREPARATION Removal of electrostatic charge from the filters The electrostatic charge on a filter can affect its sampling characteristics. A method was developed by Mark (3), which can reduce the electrical resis- tance of the filters by a factor of up to l 06 . Hyamine 2389 is diluted to form a 0.1 % solution (care is needed: higher concentrations result in a greatly increased pressure drop across the filter du- ring sampling). The solution is poured into a dish or tray to a depth of appro- ximately 40 mm. One litre is sufficient for about 50 filters. The filters are placed on top of the solution and allowed to sink. Any remaining on the sur- face afte r 6 hours are prodded gently to the bottom of the container. After 24 hours the filters are taken out and dried between sheets of blotting paper to prevent localized globules of detergent from blocking up part of the filter , as can occur when filters are dried on a non-absorbent material. SAMPLING General To estimate the mean shift exposure of an operative, samples must be taken in the breathing zone (i.e. the hemisphere of 300 mm radius extending in front of the face and centred on the nose). The cowled filter holder should be attached to the lapel or shoulder so that the filter surface points downwards at an angle of about 45°. Flow rate selection The sampling flow rate must be between 0 .5 and 2 I/min. Lower flow rates are to be avoided as particle elutriation takes place and contamination effects are more important. The sampling time and flow rate should be adjusted to obtain between 50 and 1000 fibres/mm2 on the filter to minimize counting errors. In high particle/fibre concentrations a consecutive series of short-term samples may be necessary rather than one long-term one. Calibration of sampling pumps Pumps should be tested before use to ensure that they are operating at the manufacturer's specification. The flow must be pre-set with the full sam- pling train (i.e., smoothers, filters , etc.) in position . Some types of pumps in- clude an air bleed to enable the flow through the filter to be changed. These 39 bleeds must always be closed and the flow altered by adjusting the piston stroke. If the bleed is open more air will be drawn through it as the pressure drop across the filter increases owing to particulate build-up on its surface . A large decrease in the sampling flow is therefore liable to take place. No pump should be operated without a filter on the input, otherwise damage can occur. All pumps must be allowed to run for a few minutes be- fore setting the flow. Some types are also susceptible to changes in the tem- perature; in these cases a warming-up period of at least half an hour must be allowed before measurement of the flow rate. The mechanical timekeepers built into some types of pump should not be relied upon as they are prone to inaccuracy. The actual time must be used to determine the duration of sampling. Sampling procedure Starting routine. The pump should be attached to the worker by means of a belt or harness and the filter holder fastened to his or her clothing in the breathing zone. The connecting tubing should be secured to clothing by spring clips. At this stage any covering or cap over the filter must be removed and the filter checked for damage. The switch-on time and any other time or vo- lume reading on the pump is then noted. Progress checks. The flow rate through the filter must be checked and re- corded during the sampling period, together with the time; if possible, this should be carried out every two hours during long-term sampling. If a change in the flow rate has occurred, the pump may be re-set to its previous value where practicable (see section on microscopical and counting procedures). Sampling should be terminated for any sample where the flow rate falls by more than 20%. Damaged filters must be withdrawn and the sample abandoned. Dense samples should be replace by an unused filter; as a rough guide , a filter should be changed when the printed grid lines are no longer easily visible. Where filters have been changed during a shift, the average concentration should be calculated on a time-weighted basis . Breaks. If an employee takes a lunch or other break away from the work environment , the following procedure should be followed. The sampling equip- ment should be switched off and temporarily removed if the break is unpaid; if the break is paid the equipment should be left on and running. Completion of sampling. When the sampling period is completed , replace the filter holder cap and then immediately switch off the pump , noting the time. The filter must remain in the filter holder until it is ready for analysis. 40 Transporting filters Filters must be transported in their sampling holders, with a top cap or cover fitted to prevent contamination. After sampling the filter should be re- moved from the holder with forceps , care being taken to grasp the filter's un- exposed edge only. This operation must be carried out in a dust-free area. The filter holders or containers should be packed into a rigid case with sufficient foam packing to prevent crushing and to minimize vibrational ef- fects . Care should be taken to avoid undue banging of the case . Fixatives, either cytological or perspex-based , must not be used . Blanks Some control filters (a minimum of 2%) must be transported and pro- cessed in the same way as for normal samples but without air being drawn through them or without their being attached to an employee, to check the level of adventitious contamination. If there are more than 15 fibres per mm2 on these filters , the entire sampling/analysing procedure must be examined to determine the cause of the contamination . FILTER MOUNTING The whole of the 25 mm filter must be mounted on one microscope slide. Dividing the filter into sections increases the changes of sample loss. In addi- tion , large differences may be found between sectors of the same filter (4) and may not be apparent if only one sector is evaluated. The filter mounting method is based upon the use of acetone vapour1 (5), as detailed below. Before they are mounted, all samples should be inspected to ensure that the deposit is not too dense for optical evaluation. Where there is some doubt as to the density of the collected material, further samples should be collected using a restricted sampling time or volume. The filter is placed, dust side uppermost, on a clean and warm microscope slide, then plunged into hot acetone vapour. The acetone vapour is produced in the double-neck glass flask using an electrically heated water bath until the acetone is gently boiling (Fig. 3). The condenser outlet operates continuously but the jet outlet is kept stoppered until the filter is ready for clearing. The filter is placed under the jet. After approximately two seconds in the vapour 0 Acetone mounting should be carried out only in a fume cupboard or well venti- lated room, with a water bath used for heating. On no account should a naked flame be allowed in the region of acetone vapour. 41 it will clear and should be removed to allow excess acetone to evaporate. The stopper is then replaced and the filter covered with a clean cover slip using a drop of triacetin (approximately 2 µl /cm 2 of filter) to provide a good optical contact between it and the sample. If the acetone is not boiling sufficiently the filter may twist and buckle before clearing. It should be noted that the use of the acetone-triacetone method is un- suitable for materials with refractive indices of between 1.43 and 1 .49 . MICROSCOPICAL AND COUNTING PROCEDURES The microscopist must have good vision and be trained by someone ex- perienced in fibre counting. Microscope adjustment The manufacturer's instructions for setting up the microscope must be followed carefully. The optical alignment of the phase contrast microscope should be checked regularly, and always be checked following the changing of any lenses or transport of the microscope . An HSE/NPL test slide should be used to check that the microscope/observer performance is of an acceptable standard . The slide consists of seven sets of parallel Jines of varying width, and an observer should be able to see the sixth (0.36 µm) or the seventh (0 .25 µm) set. Eyepiece graticule The graticule used for MMMF evaluation must satisfy the following cri- teria : (a) it shol!ld have markings exactly 5 µm apart on it; (b) it should have markings exactly 3 µm apart on it (this is particularly important when separate counts are being made of respirable and non- respirable fibres); and (c) it should have a clearly defined central counting area occupying not more than one fifth of the full field of view of the microscope. The Walton-Beckett graticule (6) (see Appendix A), meets these require- ments and is shown in Fig. 2. It was designed specifically for fibre counting, but must be manufactured individually for each combination of eyepiece and objective so that the effective diameter is 100 µm ± 2 µm . The details re- quired when ordering such a graticule are given in Appendix B. 42 The graticule must always be in focus, and the eyepiece used should pre- ferably have adjustable focusing . In addition , before use, and following any change or adjustment to the microscope lenses, the sizes of the markings on the graticule must be calibrated with a stage micrometer. The exact diameter should be measured so that the graticule area can be calculated. (Note. The calibration may change marginally on a binocular microscope if changes in the intraocular distance alter the tube length.) Use of graticule The central defined area alone is used for counting fibres. The procedure is to count the total number of fibre ends within the graticule area and to di- vide by two to determine the number of fibres. Counting. The aim of the counting procedure is to estimate the total number of fibres on the filter surface so that the original airborne concen- tration can then be calculated from the volume of the air sampled. The num- ber of respirable and non-respirable fibres is counted in 100 graticule areas, unless more than 100 fibres (either respirable or non-respirable) are observed. A minimum of 20 graticule areas should be evaluated , even if more than 100 fibres are recorded. One hundred Walton-Beckett graticule areas only constitute approximately 0.2% of the area of a filter with a 22 mm diameter , and the method assumes that the fibres are homogeneously distributed on the filter. It is necessary to ensure that : (a) filters with uneven deposits or evidence of leakage around the filter edge are rejected; (b) the randomly selected graticule areas are representative of the whole area of the mounted sample and do not overlap {the method used is to traverse the filter, starting at the top, evaluating randomly selected graticule areas along the traverse ; the process is then repeated on randomly selected traverses covering the whole filter area until the requisite num- ber of graticule areas has been evaluated); (c) once selected, a field is always evaluated except where over one eighth of the area is obscured by particulate material ; and (d) fibres conforming to the size criteria given in section 3 are counted in each graticule according to the "end in" rule defined above. 43 The following conventions should be used: ( I) Single fibres (2) Split fibres (3) Grouped fibres ( 4) Fibres attached to particles count according to their geometric di- mensions occur infrequently with MMMF but count as one fibre according to their geometric dimensions when several fibres cross or intersect and the individual component fibres can be easily distinguished , count each fibre se- parately according to its geometric di- mension (when crossed fibres form clumps that cannot be readily identified into se- parate fibres, do not count the clumps as fibres) count as fibres according to their geo- metric dimensions . Precision of counting. Differences in the counts of fibres may arise from two sources : (a) Systematic differences or effects owing to differences in techniques (sampling, mounting, counting rules, etc.) or personal factors (subjective errors). The evaluation and discussion of systematic errors constitute a subject in themselves and as such are outside the scope of this document. (b) Random e"ors, i.e., those which are individually unpredictable and variable in magnitude. They arise because the counter examines only a small area of the membrane filter sample, typically< 0.2%. This implies that repeat counts on the same sample are likely to cover different areas. If a sufficiently large number of measurements are taken in a controlled way , differences arising from random errors may be unimportant and can , in principle , be made negligible . On the other hand , differences arising from systematic effects cannot be eliminated by increasing the area of the filter examined. Both the magnitude and the source of these systema- tic differences are difficult to distinguish if the random error is also high. Tests using MMMF data from environmental surveys have confirmed that the distribution of fibres of membrane filters may reasonably be described by the Poisson distribution . On this basis, when fibres are distributed randomly over a surface , the probability that x fibres are observed in a specific field is given by: 44 µx exp(- µ) Px =----- x! where µ is the mean number of fibres observed in a large number of fields. The 95 per cent confidence limits for the Poisson distribution are shown in Table 1. For example , when the true average fibre density is 4 fibres in a specific area, then the observed number in 95 such areas out of 100 will be between 1 and 10 fibres. Table 1. 95% confidence limits for the Poisson distribution True number Lower limit Upper limit of fibres fibres fibres 0 6 2 0 7 3 9 4 10 5 2 12 10 5 18 20 12 31 30 20 42 40 28 54 50 37 66 100 82 122 200 174 230 Where the evaluation involves samples of similar volumes (e.g., approxi- mately 500 litres) and all counts refer to the same proportion of the filter area (e.g., approximately 0.2%), it is possible to relate approximately the number of fibres observed on the filter to the density of fibres in the air : 1 fibre observed 10 fibres observed 100 fibres observed 0 .001 fibres/ml 0.01 fibres /ml 0.1 fibres/ml It is evident from Table 1 that large random variations in the number of fibres observed may occur at the lower densities ; nevertheless , expressed in terms of airborne fibre concentration, the variation is relatively unimportant. 45 Calculation Volume of air sampled. The volume of air sampled is calculated by mul- tiplying the duration of sampling by the measured flow rate. If the flow rate cnanges during sampling, the air volume should be estimated assuming that a linear change occurred between flow rate measurements. Airborne fibre concentration. The airborne fibre concentration (C) is de- termined by dividing the number of fibres estimated to be on the ftlter (N) by the volume of air samples (V). N An C= - ,and N= - V ga where A = area of sample deposit (not area of filter) (mm2) a = area of graticule (mm2) in the object plane g number of graticule areas evaluated n number of fibres counted in g graticule areas. Combining these equations gives: An C = -- fibres/ml where V is measured in ml. gaV REFERENCES 1. Technical Note 1. Rochdale, Asbestosis Research Council, I 971. 2. Reference method for the determination of airborne asbestos fibre concentrations at workplaces by light microscopy. London, Asbestos International Association, 1979. 3. Mark,D.Annals ofoccupationalhygiene, 17 : 35-40(1974). 4. Bartosie~icz, L. American Industrial Hygiene Association journal, 34(6) : 252 - 259 (I 973). 5. Membrane filter method for estimating airborne asbestos dust. Canberra, National Health and Medical Research Council, 1976. 6. Walton, W.H. & Beckett, S. T. Annals of occupational hygiene, 20 : 19- 23 (1977). 46 Pumps Appendix A Type Tl3051/2 or TI3055 C. F . Casella & Co . Ltd , Regent House, Britannia Walk, London, NI 7ND Type C2000 or L25 F Rotheroe & Mitchell Ltd, 14 Aintree Road, Perivale , Middlesex, UB6 7l.J , United Kingdom MSA : pump type G Mine Safety Appliances Company 400 Penn Centre Boulevard , Pittsburgh , Pennsylvania , USA Birger Carlsson & Co. AB, Kaptensgatan 6, 114 57 Stockholm, Sweden Scan Agent AB, Box 15006, 161 15 Bromma, Norway Ramstri:im AB, Fack, 16 I 20 Bromma, Norway - High flow sampler model P2500 and P4000 E.I. du Pont de Nemours & Co. (Inc), Fabrics and Finishes Department , Applied Technology Division , Brandywine Building 4300, Wilmington , Delaware 19898, USA RAC 209016 (American) Oleico AB, Sandhamnsgatan 25, 115 28 Stockholm, Sweden Scan Agent AB , Box 15006, 161 15 Bromma, Norway 47 Spectrex PAS-3000 (American) Wartsila 8077DS (Finnish) Birger Carlsson & Co. AB , Kaptensgatan 6 , 114 57 Stockholm, Sweden also Birger Carlsson & Co. AB Filter holder 25 mm diameter open delrin filter holder Gelman Hawksley Ltd , 12 Peter Road , Lancing, Sussex , United Kingdom Filters Gridded membrane filters (mixed ester cellulose) 25 mm diameter , 1 .2 µm pore size. Millipore UK Ltd , Millipore House , Abbey Road , London, NWl0 7SP, V.A. Howe & Co. Ltd, 88 Peterborough Road , London SW6 Interlab (Gelman Type TCM-1200) Millipore AB, Box 17094, 402 61 Goteborg 17, Sweden Interlab , Fack, 161 20 Bromma, Norway Filter cowl 48 The cowl is not available from a commercial manufacturer and should be manufactured from the following diagram . T _L T 36 mm 10mm 38mm l T I ---------------------------- 1 I· 88mm • I Filter holder covers Plastic covers for the 25 mm diameter Gelman filter holder are available from : Spring clips Airoquip UK Ltd , 165 Factory Centre, Redditch, Birmingham, United Kingdom Plastic strips with bulldog type clips, 5 cm between studs Morane Plastic Co . Ltd, Gresham Road , Staines, Middlesex , United Kingdom 49 Eyepiece graticule Type G22 Walton-Beckett (I 977) Graticules Ltd , Sovereign Way , Botany Trading Estate , Tonbridge , Kent , TN91RN , United Kingdom Test slide HSE/NPL test slide PTR Optics Ltd , 46 Church Road , Teddington , Middlesex , United Kingdom Hyamine 2389 50 Robin & Haas Company , Philadelphia, USA Supplied by Hopkin & Williams, Chadwell Heath, Essex, United Kingdom Appendix B ORDERING A WALTON-BECKETT GRATICULE Manufacturer : Graticules Ltd , Sovereign Way , Botany Trading Estate , Tonbridge , Kent TN9 I RN, United Kingdom When ordering please specify: Walton-Beckett graticule, Type G22, and the diameter of the circle in millimetres that corresponds to I 00 µm object size . This can easily be determined by taking any available graticule , measuring the actual grid dimensions (to three significant figures) and then measuring the corresponding object length with a stage micrometer. It is then a simple matter to calculate the graticule length corresponding to I 00 µm in the object plane. 51 Annex 4 LIST OF PARTICIPANTS Temporary advisers Mr B. Carton , Institut national de Recherche et de Securite pour la Pre- vention des Accidents de Travail et des Maladies professionnelles , Paris , France Mr J. Dodgson, Institute of Occupational Medicine , Edinburgh , United Kingdom (Rapporteur) Dr J . Gilson , Hembury Hill Farm, Honiton , Devon , United Kingdom Dr J . Goscicki, Institute of Occupational Medicine , Lodz , Poland Dr J . M. Le Guen , Occupational Medicine and Hygiene Laboratories, Health and Safety Executive , Cricklewood, London , United Kingdom Mr B. Gylseth , Institute of Occupational Health , Oslo, Norway Dr M. Navez, Isover Saint-Gobain , Neuilly-sur-Seine , France Mr I. Oehberg, Rockwool AB, Skovde , Sweden Dr A. van der Meulen, Physical Laboratory , National Institute of Public Health , Bilthoven , Netherlands Dr T. Schneider, National Institute for Occupational Health , Hellerup , Denmark Dr A. Schi.itz , Dust Research Institute , Bonn , Federal Republic of Ger- many Dr Y. Shimecec, Institute of Epidemiology and Public Health, Prague, Czechoslovakia Dr C. Tillman , National Board of Occupational Safety and Health , Solna , Sweden Representatives of other organizations International Agency for Research on Cancer Dr R. Saracci, Lyon , France 52 International Labour Office Dr N. Gavrilescu , Geneva, Switzerland Joint European Medical Research Board Dr T. Guthe , Elkem-Spigerverket A/S, Oslo, Norway Dr J. W. Hill , Pilkington Brothers Ltd Medical Centre , St Helens , United Kingdom World Health Organization R egional Office for Europe Dr G. Lamm, Regional Officer for Chronic Diseases Dr M. I. Mikheev, Regional Officer for Workers' Health . Mr J . I. Waddington , Director , Promotion of Environmental Health 53