Document M4Njn4E5gyOLrx4vkq1ygyopk

Pathology Staodards/Microorganisms & Occupational Dust PATHOLOGY CLASSIFICATION AND GRADING SCHEMATA FOR SILICOSIS JOHN E. CRAIGHEAD1 J. Kleinerman2 J. L. Abraham3 A. R. Gibbs4 F. H. Y. Green5 R. A. Harley6 J. R. Ruttner7 V. Vallyathan8 E. Juliano9 University of Vermont, Burlington, VT ^ase Western Reserve University, Cleveland, OH 3State University of New York, Syracuse, NY 4Llandough Hospital, Penarth, South Glamorgan, United Kingdom 5University of Calgary, Calgary, Alberta, Canada 6Medical University of So. Carolina, Charleston, SC 7Institut fur Pathologie, der Universitat Zurich, Zurich, Switzerland 8National Institute for Occupational Safety and Health, Morgantown, WV litigation Management, Inc., Cleveland, OH ABSTRACT In 1985, the National Institute for Occupational Safety and Health established a committee to review the pathology of silicosis and the silicate-associated lung diseases, and to develop a classification and pathology grading system for epidemiological studies. The committee considered a number of different schemata developed in die USA and abroad. However, we were unsuccessful in devising a satisfactory approach which would permit pathology grading for correlative studies. An outline of the various proposed grading systems will be presented, and the shortcoming and problems in their utilization and broad application will be discussed. No Paper provided. 711 Pathology Staodards/Microorganisms & Occupational Dust MICROBIAL CONTAMINANTS OF STORED TIMBER AS POTENTIAL RESPIRATORY HAZARDS FOR SAWMILL WORKERS JACHK DUTKIEWICZ, Ph.D. W. G. Sorenson, Ph.Di D. M. Lewis, Ph.D. S. A. Olenchock, Ph.D. Immunology Section, Division of Respiratory Disease Studies, NIOSH Morgantown, West Virginia 26505, USA INTRODUCTION Occupational exposure to wood dust may be a cause of respiratory diseases such as hypersensitivity pneumonitis (allergic alveolitis),7,12'23>26) asthma8 and chronic obstructive lung disease (COLD).2 The etiology of these diseases is not fully known and both the allergenic and/or toxic constituents of wood tissue itself and the substances produced by microorganisms developing in wood have been suggested as potential agents.8,10,22*27 Many species of allergenic and/or toxic molds developing on wood (Altemaria tenuis, Aspergillus fumigatus, Cryptostroma corticale, Mucor spp., Paecilomyces spp., PenicUlium spp., Rhizopus spp.) have been described as causative agents ofpulmonary diseases in woodworkers.6,7,8,12,23,24,25,26 The role of bacterial factors in wood-associated diseases was studied to a lesser ex tent.10,22 It has been reported that woodworkers may be ex posed to notable amounts of gram-negative bacteria and endotoxin.1,4,25 The aim of this study was to extend the knowledge of the potential respiratory risk ofwoodworkers to wood-inhabiting microorganisms by quantitative and qualitative determina tion of the microflora of stored timber logs scheduled for processing in a sawmill. MATERIAL AND METHODS Two series of microbiological wood samples were taken in August and October of the year 1987 from timber logs stored on die lumber yard at a sawmill in Kingwood, West Virginia. The logs had been stored for a period of 4-6 weeks and did not show any apparent signs ofdecay. At each sampling time, samples were taken from a log of each of the following species: American basswood (Tilia americana L.), black cherry (Prunus serotina Ehrh.), black locust (Robinia pseudoaccacia L.), red oak (Quercus coccinea Muenchh.), soft maple (Acer saccbarinum L.) and white poplar (Populus alba L.). From each log, one sample was taken from the heartwood (by boring from the transverse section), one from the sapwood (by boring from the transverse section) and one from the bark (by centripetal boring). The wood samples were collected with a novel * `drill and collect" device (model 02) for quantification of microorganisms in wood.5 This is a manually operated drill ing device in which a combined action of a twist boring bit and a spring-containing mobile ring collects the pulverized 712 wood into a sterile flask attached beneath the bit in a onestep sterile process. The wood surface to be sampled was first sterilized by wiping with 70% propanol and "Clorox" (a commercial 5.25% sodium hypochlorite solution) and then an average sample was taken by multiple boring (5-7 times) in a circle up to 3 cm in diameter. The concentrations of bacteria and fungi in die wood samples were determined by dilution plating. Aliquots of 200 mg of each sample were suspended in 20 ml of sterile phosphate buffered saline (Sigma Chemical Co., St. Louis, MO) con taining 0.1% (v/v) Tween 80 (Fisher Scientific Co., Fair Lawn, NJ) and, after vigorous shaking, serial 10-fold dilu tions were made up to Iff6. Die 0.1 ml aliquots of each dilution were spread on duplicate sets of the following agar media: (i) sheep blood agar for total aerobic bacteria, (ii) eosin methylene blue agar (EMB agar; Difco Lab., Detroit, MI) for gram-negative bacteria, (iii) half-strength tryptic soya agar (Difco) for thermophilic actinomycetes, (iv) rose bengal streptomycin agar (RBS) for total fungi, and (v) yeast malt agar for yeasts. The blood agar and EMB plates were in cubated for 48 hrs at 35 C, the tryptic soya plates for 120 hrs at 55C, and the RBS and yeast malt plates for 96 hrs at 28C. Following incubation, bacterial colonies were counted and differentiated on die basis of colony morphology, Gram reac tion, and biochemical reactions. The gram-positive isolates were identified according to Bergey's Manual.21 The gram negative isolates were identified with the APIR Systems 20 E (for enterobacteria) and NFT (for non-fermenting bacteria) (API Analytab Products, Plainview, NY), using supplemen tary biochemical tests selected according to Bergey's Manual14 and APIR Systems recommendations. Mold col onies were counted and differentiated on the basis of mor phological properties. Representative yeast colonies were isolated and differentiated on the basis of morphological and biochemical properties.13 Final results for microbial concen trations were reported in terms of the colony forming units (cfu) in one gram of ground wood. For endotoxin determination, 100 mg portions of the wood samples were extracted with 5 ml of sterile non-pyrogenic water (Travenol Laboratories Inc., Deerfield, IL) by rock ing for 60 min. at room temperature. The suspension was centrifuged at 1000 g for 10 minutes to remove particulate debris, and the supernatant fluid was separated for further analysis. Quantification of gram-negative bacterial endotoxin content was performed in duplicate by a quantitative chromogenic modification of the Limulus amebocyte lysate test (QCL-1000; Whittaker Bioproducts, Walkersville, MA). Results were reported in terms of Endotoxin Units (EU) in one gram of ground wood. The Students' t-test for matched pairs, test for linear regres sion and chi-square test were used for statistical evaluation of the results. RESULTS The concentration of microorganisms and endotoxin varied significantly with the kind of wood examined (P< 0.001). As shown in Figures 1-6, the highest levels ofbacteria, fungi and endotoxin were found in the wood samples from logs of American basswood and black locust (HP-108 cfu/gm, 10M07 cfu/gm and 10M06 EU/gm, respectively). The levels were lower in the logs of soft maple and black cherry (0-106 cfu/gm, 0-107 cfu/gm and lO^lO4 EU/gm, respec tively) and lowest in the logs of white poplar and red oak (0-104 cfu/gm, 0-105 cfu/gm and H^-IO5 EU/gm, respectively). Pathology Standards/Microorganisms A Occupational Dust dotoxin (P<0.05). No thermophilic actinomycetes were found in the examined wood samples. B Total Bacteria @ Total Fi^gi Gran (-) Bacteria fl| Yeaets A GndotCKin S 8> 1 a c s Figure 2. Concentrations of bacteria, fungi and endotoxin in the samples of heartwood collected in October, 1987. B Total Bacteria B Tninl Kml S Total Bacteria Gran (-) Bacteria Total Firigi ID Yeasts I Endotoxin c ff Na> 3 Lil Figure 1. Concentrations of bacteria, fungi and endotoxin in the samples of heartwood collected in August, 1987. High concentrations of bacteria, fungi and endotoxin were found in all the examined kinds of wood tissue: heartwood (Figures 1-2), sapwood (Figures 3-4) and bark (Figures 5-6). No significant differences were noted between the con tamination rates in August and October (P>0.05). In most of the samples of heartwood and sapwood, gram negative bacteria dominated the total bacteria flora. Except for two cases (Figure 5), this was not observed in the baric samples. In bark samples taken in October, viable gram negative bacteria were absent completely and die very high level of bacteria found in the bark of the black locust was due to the presence of large numbers of spore-forming bacilli (Figure 6). For each kind of wood tissue (heartwood, sapwood and bark) a significant correlation has been found be tween the concentrations of gram-negative bacteria and en ftnerian Black Soft Mcpie Black White Red Ock Basswood Locust Cherry Poplar Figure 3. Concentrations of bacteria, fungi and endotoxin in the samples of sapwood collected in August, 1987. Twelve species and/or genera of gram-negative bacteria and seven genera of gram-positive bacteria were found in the wood samples (Table I). The gram-negative flora comprised five fermentative species (belonging to the Enterobacteriaceae family) which, in most cases, were associated with the sapwood and seven non-fermentative species (mostly of the genus Pseudomonas) which were mostly associated with the heartwood. Among the gram-positive bacteria, die most com mon organisms were endospore-forming bacteria ofthe genus Bacillus and coryneform bacteria belonging to the genera Artbrobacter, Brevibacterium, Corynebacterium and Microbacterium. In all kinds of wood samples examined, yeasts were the predominant fungi observed (Figures 1-6, Table II) and the 713 Pathology Standards/Microorganisms & Occupational Dust Table I Species of Bacteria Occurring in Wood Samples Mane of the species Heartwood Sapwood Bark Maximal concentration C X 10s cfu/graa) GRAM-NEGATIVE BACTERIA Fermentative Citrobacter freundii Enterobar.ter a** 1 omursn* Enterobacter cloacae Klebsiella sp. Serratia rubidaea on-fermentative + (B, K) + (B), + (M) (L) ++ (B), (M) +++ (B), (M) ++ (M) +++ (B), * (M) ++ (L) ++ CB, L) 0.10 CSapwood, B) 3.00 CSapwood. B) 0.38 (Sapwood, H) 1.45 (Sapwood, B) 0.41 (Sapwood, L) AMnefohnrter caleflareHrus Aernbacterium radiobacter Pseudomonas fluorescens Pseudomonas aaltoohilia Pseudomonas orvzihabitans Pseudomonas eutida Pseudomonas stutzerl ORAM-POSITIVE BACTERIA Bacillus spp. Corynaform bacteria* Staphylococcus spp. Streptomvces spp. (M) *+ (L) + (M) CM) +++ (B) ++ (B), ++ (M) ++ (B), + (H) *>* (L) + (B) + CM) *** CL) ++ (B), ++ (L). CM) +++ CB. L), * (P) + (H, 0, P) +++ CL), * CB, M) +++ CB, L) + CC, M. 0) +++ CL), + CO) +++ CL) +++ CL, M). ++ CB), CC, 0, P) +++ (L>, ++ CB, N), + CC, P) + CP) ++ CL), + CB) 30.50 (Bark, L) 2.64 (Sapwood, L) 0.07 (Heartwood, M) 0.02 (Heartwood, M) 15.10 (Heartwood, B) 3.84 (Heartwood, B) 0.45 (Heartwood, B) 154.00 (Bark, L) 20.30 (Heartwood, B) 5.00 (Sapwood, L) 2.51 (Sapwood, L) B American Basswood H = Soft Maple + : occurred in concentration below 1 x 10* cfu/grae C = Black Cherry 0 = Bed Oak ++ occurred in concentration 1 xlO* -lx 10^ cfu/graa i. Black Locust P = White Poplar +++ = occurred in concentration over 105 cfu/graa Comprise: Arthrobacter sop., BrevibS^teiHum gpp. Corynebacteriuis spp. , Microorganisms Ccfu/gram) Log n Endotoxin (EU/grom) Log n Q Total Bacterio 7 . iGran (-) Bacteria g Total Fingi 0 YeaBte Ertjotoxin 8* _i 6 u. 3 c s 8* i o Figure 4. Concentrations of bacteria, fungi and endotoxin in the samples of sapwood collected in October, 1987. Figure 5. Concentrations of bacteria, fungi and endotoxin in the samples of bark collected in August, 1987. 714 Pathology Standards/Microorganisms & Occupational Dust Table H Fungi Occurring in Wood Samples Organism Heartwood Sapwood Bark Maximal concentration ( x 105 cfu/gram) DBB- yeasts8 DBB+ yeastsb Acregionlum 2Oidiodendron so. Penicillium so. Trichoderma so. Nonsporulating 4-44 (B.L). 44 (M) 4 (C> 44 (B,L,M) 44 (L), 4 (B) 44 (L) 4 (B.C.M) 444 (B.C.L), 44(H) 444 (B.L.H), 44 (C.O), 4 (P> 444 (B), 44 (C.L) 44 (B.C.L,0) 44 (L) 444 (M), 4 (B.P) 44 (C,H) 44 (B,L) 44 (C) 44 (L,P), 4 (B.C.O) 44 (B.L) 44 (L,M). 4 (B.O.P) 78.35 (Sapwood, B) 1.45 (Sapwood, B) 14.00 (Bark, M) 0.79 (Bark, N) 0.72 (Bark, L) 0.49 (Bark, L) 0.26 (Bark, M) Negative reaction with Diazoniua Blue B (DBB); presumptive Aseomycetes and their anamorphs (includes Candida zeylanoides. other undetermined Candida app., and Hansenula silvicola. ^positive reaction with DBB; presumptive Baflidiomvcetes and their anamorphs (includes undetermined Candida spp., Cryptococcus laurentil. and Bhodotorula elutinls). B = American Basswood C = Black Cherry L = Black Locust M - Soft Maple 0 * Bed Oak P = White Poplar + * occurred in concentration below 1 x 10* cfu/gram 44 a occurred in concentration 1 x 10* -lx 10s cfu/gram ++ a occurred in concentration over 10^ cfu/gram Figure 6. Concentrations of bacteria, fungi and endotoxin in the samples of bark collected in October, 1987. most numerous among diem were presumptive Ascomycetes and their anamorphs, i.e., they gave a negative reaction with Diazonium Blue B (DBB).13 Yeast fungi tended to be found in die greatest numbers in the sapwood. Species of yeast isolated include undetermined Candida spp. (include both DBB+ and DBB-- species), Candida zeylanoides. Cryp tococcus laurentii, Hansenula silvicola and Rhodotorula glutinis. Molds found in these samples included Acremonium (Cqyhalosporium sp., A/ternaria sp., Aspergillus fumigatus, Aureobasidium pullulans, Bispora sp., Cladosporium sp., MortiereUa sp., and Trichoderma sp. as well a number of fungi that could not be identified because of their failure to sporulate. The molds that occurred in the greatest numbers were Acremonium sp., Oidiodendron sp., Penicillium sp., and Trichoderma sp.. The highest numbers of molds were found in the bark. DISCUSSION The levels of microorganisms and endotoxin in timber logs showed notable variation depending on die species ofthe tree. The concentrations of bacteria and fungi in the most con taminated wood species (basswood, locust) exceeded die level of 106 cfu/gm, and were comparable to the values reported for certain organic dusts related to harmful respiratory ef fects in workers.3 The concentration of endotoxin in the wood reached, in many cases, a level of 105--106 EU/gm which corresponds to the values found in organic materials (grain, silage, mushroom farm pre-flush) associated with the cases of respiratory disorders in exposed workers.17 This finding is in agree ment with the fact that some of the wood samples contained high concentrations of gram-negative bacteria. Among these bacteria were the species (Enterobacter agglomerans, Kleb siella spp., Pseudomonasputida) which are known producers of biologically active endotoxin that can cause pulmonary 715 Pathology Standards/Microorganisms & Occupational Dust injury through non-specific stimulation of alveolar macrophages.19 The occurrence of high concentrations of fungi in the wood presents another factor of potential respiratory risk for sawmill workers. The Penicillium species that were frequent ly isolated in this study have been reported as a source of die pathogenic respiratory allergens.6*24 Another potentially pathogenic species are Aspergillus fumigatus and Aureobasidium pullulans.*'10'22 The data conform to some earlier reports on the occurrence of bacteria and fungi in the wood9-15*16*18 The composition of the microflora of examined logs, characterized by the prevalence of yeasts and gram-negative bacteria indicates that it was in the stage of "pioneer colonization" which precedes the stage of wood decay by brown rot and white rot fungi.11,20 The main conclusion from this preliminary study is that some kinds of apparently not decayed timber stored for process ing in sawmill contain very high concentrations of ` `pioneer' ' microorganisms and their toxins. These organisms may cause respiratory disorders in the woodworkers if inhaled with the sawdust. Although not defined by the current study, the potential problems associated with microbiologically con taminated woods are intriguing and require further research. REFERENCES 1. A1 Zuhair, Y.S., Whitaker, C.J., Cinkotai, F.F.: Ventilatory Func tion in Workers Exposed To Tea and Wood Dust. Br. J. Iod. Med. 38:339-345 (1981). 2. Carosso, A.,Ruffino, C.,Bugiani, M.: Respiratory Diseases in Wood Workers. Br. J. lad. Med. 44:53-56 (1987). 3. Dutkiewicz, J: Exposure to Dust-borne Bacteria in Agriculture. I. En vironmental Studies, n. Immunological Survey. Arch. Environ. Health 33:250-270 (1978). 4. Dutkiewicz, J.: Bacteria, Fungi and Endotoxin in Stored Timber Logs and Airborne Sawdust in Poland. Abstracts ofthe Second Meeting of the Pan-American Biodeterioration Society. (Washington, D.C., July 28-31, 1981). 5. Dutkiewicz, J., Olenchock, S.A., Lewis, D.M., Ratajczak, Z., Kwapiszewski, C., Piech, T., Bilczuk, A.: Drill Samplers for Quan tification of Microorganisms in Wood. For. Prod. J. in press. 6. Dykewicz, M.S., Laufer, P., Patterson, R., Roberts, R.N., Sommers, H.M.: Woodman's Disease: Hypersensitivity Pneumonitis from Cut ting Live Trees. J. Allergy Clin. Immunol. 81:455-460 (1988). 7. Emanuel, D.A., Wenzel, F.J., Lawton, B.R.: Pneumonitis due to Cryp tostroma corticale (Maple Bark Disease). N. Engl. J. Mod. 274:1413-1418. 8. Goldsmith, D.F., Shy, C.M.: Respiratory Health Effects from Occupa tional Exposure to Wood Dusts. Scaod. J. WorkEnviron. Health 14:1-15 (1988). 9. Greaves, H, The Bacterial Factor in Wood Decay. Wood Sci. Techno/. 5:6-16 (1971). 10. Jagels, R.: Health Hazards of Natural and Introduced Chemical Com ponents of Boatbuilding Woods. Am. J. Ind. Med. 8:241-251 (1985). 11. Kaarik, A.: Succession of Microorganisms during Wood Decay. In: Biological Transformation ofWood by Microorganisms, pp. 39-51. W. Liese, Ed. Springer-Veriag, Berlin (1975). 12. Kolmodin-Hedman, B., Blomquist, G., Lofren, F.: Chipped Wood as a Source ofMould Exposure. Ear. J. Respir. Dis. 71 (Suppl. 154):44-51 (1987). 13. Kreger-van Rij, N.J.W., Ed.: The Yeasts. A Taxonomic Study, 3rd Ed. Elsevier Science Publishers, Amsterdam (1984). 14. Krieg, N.R., Holt, J.G., Eds.: Bergey's Manual of Systematic Bacteriology, Vol. 1. The Williams and Wilkins Co., Baltimore (1984). 15. Levy, J.F.: Colonization of Wood by Fungi. In: Biological Transfor mation ofWood byMicroorganisms, pp. 16-23. W. Liese, Ed. SpringerVeriag, Berlin (1975). 16. Murdoch, C.W., Campana, RJ.: Bacterial Species Associated with Wetwood of Elm. Phytopathology 73:1270-1273 (1983). 17. Olenchock, S.A.: Quantitation ofAirborne Endotoxin Levels in Various Occupational Environments. Scaod. J Work Environ Health 14 (Suppl. l):72-73 (1988). 18. Rossell, S.E., Abbot, E.G.M., Levy, J.F.: Bacteria and Wood. A Review of die Literature Relating to die Presence, Action and Interac tion of Bacteria in Wood. J. Inst. Wood. Sci. 6(2):28-35 (1973). 19. Rylander, R., Snella, M-C.: Endotoxins and the Lung: Cellular Reac tions and Risk for Disease. Prog Allergy 33:332-344 (1983). 20. Shigo, A.L., Hills, W.E.: Heartwood, Discolored Wood, and Microorganisms in Living Trees. Ann. Rev. Phytopathol. 11:197-222 (1973). 21. Sneath, P.H.A., Mair, N., Sharpe, M.E., Holt, J.G., Eds.: Bergey's Manual of Systematic Bacteriology, Vol. 2. The Williams and Williams Co., Baltimore (1986). 22. Tatken, R.L., Ed.: Health Effects ofExposure to Wood Dust. A Sum mary ofthe Literature. National Institute for Occupational Safety and Health, Cincinnati (1987). 23. Terho, E.O., Husman, K., Kotimaa, M., Sjoblom, T.: Extrinsic Allergic Alveolitis in a Sawmill Worker: A Case Report. Scand. J. Work En viron. Health. 6:153-157 (1980). 24. Van Assendelft, A.H.W., Raitio, M., Turitia, V.: Fuel Chip-induced Hypersensitivity Pneumonitis Caused by Penicillium Species. Chest 87:394-396 (1985). 25. Wibelmsson, B., Jernud, Y., Ripe, E., Holmberg, K.: Nasal Hypersen sitivity in Wood Furniture Workers. An Allergological and Im munological Investigation with Special Reference to Mould and Wood. Allergy 39:586-595 (1984). 26. Wimander, K., BeHn, L.: Recognition ofAllergic Alveolitis in the Trim ming Department of a Swedish Mill. Eur. J. Respir. Dis. 61 (Suppl. 107): 163-167 (1980). 27. Whitehead, L.W.: Health Effects ofWood Dust--Relevance fbran Oc cupational Standard. Am. Ind. Hyg. Assoc. J. 43:674-678 (1982). ACKNOWLEDGEMENTS:This work was done when Jacek Dutkiewicz held the National Research Council--NIOSH research associateship. The authors thank Janet Simpson, Toni Bledsoe, Beverly Carter, Nanci Keenan, Mike Moore and Judith Mull for skillful technical assistance. We appreciate also the organizational support given by the Interstate Lumber Company in Kingwood, West Virginia. Mention of company names or products does not constitute endorsement by the National Institute for Occupational Safety and Health. 716 Pathology Standards/Microorganisms & Occupational Dust MICROBE EXPOSURE AND THE OCCURRENCE OF ANTIBODIES AGAINST THE EXPOSING MICROBES AMONG WOOD WORKERS IN CELLULOSE INDUSTRY M. KOTIMAA* P. Koskelat L. Saloranta$ Kuopio Regional Institute of Occupational Health, P.O.B. 93, SF-70701 Kuopio, Finland tNational Public Health Institute, P.O.B. 94, SF-70701 Kuopio, Finland $Metsa-Serla Ltd., SF-44100 Aanekoski, Finland ABSTRACT Exposure to airborne fungal spores and bacteria, and occurrence of antibodies against the most common fungal14 and bacterial3 species in sera of the 11 workers were studied in a cellulose factory. Air samples for microbiological analysis were taken by a six-stage Andersen impactor in barking department and on wood chip piles out-of-doors. Barking workers were exposed mainly to bacteria (geometric mean of bacterial concentration 46.3 xlO3 cfii/m3) and to lesser extent to fungal spores (5.9 xlO3 cfu/m3) in contrast to catepillar drivers on wood chip piles (1.5 xlO3 cfu/m3 and 45.5xlO3 cfii/m3 respectively). Rhodotorula glutinis was die dominating fungal species in the barking department and Aspergillus fumigatus and Penicillium brevicompactum on wood chip piles. Enzyme-linked immunosorbent assay (ELISA) found differences in IgG-andbody levels between different microbial species as also between different work environments. Highest antibody levels were found against Paecilomyces variotii, Sporobolomyces salmonicolor and Aspergillus niger while lowest levels were found against Rhizopus nigricans, Humicola grisea and Streptomyces albus. Generally, the levels of antibodies against fungal species were 2-5 times higher in the wood chip workers than those in the barking workers. Although the amount of the bacteria in the barking department was about 30 times higher than that on the wood chip piles, no differences in die levels of bacterial antibodies were found between the two groups. Probably the dry microbial material such as that in wood chip work penetrates into the lower parts of the respiratory tracts and initiates the formation of antibodies more easily than the moist aerosols. INTRODUCTION In pulp production wood used as raw material is cutted after barking into chips and stored outdoors in huge piles, where chips are transferred by caterpillars. Chips are stored ap proximately a couple of months before taking in to die pro duction. During the storage microbiological changes occur in the piles (Bergman and Nilsson 1979, Pellikka and Kotimaa 1983). Some cases of allergic alveolitis have been described among wood workers after exposure to fungal spores (van Assendelft et al. 1985, Lundgren and Rosenhall 1979, Jorgensen and Fjellheim 1982). Wood is barked in big barking drums, where water is used in the process. Water is circulated and becomes contaminated by bacteria and fungi. Process is mostly open and water becomes easily aerosolized. Microbial aerosols from humidifiers may cause so-called humidifier fever (Rylander et al. 1978, Marinkovich and Norey 1983). Because of respiratory symptoms among wood workers in a cellulose factory in central Finland, microbe exposure and the antibodies against 17 most common expos ing microbes were investigated. , MATERIALS AND METHODS Subjects and Serum Samples Serum samples from six catepillar drivers on wood chip piles and four workers in the barking department were taken within two weeks after air sampling. All the examined workers had work-related symptoms suggesting allergic background with microbial etiology (Table I). Air Sampling for Estimating the Microbe Exposure Air samples for microbiological analysis were collected by a six-stage fractionating impactor (model 10-800, Andersen Inc., Georgia, USA) (Andersen 1958). Three sets of media were used in successive samplings on each sampling site: Hagen-medium (incubated at 20C) was used for mesophilic fungi, die same medium incubated at 40C was used for tbermotolerant fungi and plate count agar was used for total count of mesophilic bacteria. 10 air samples for each microbe group were collected both on wood chip piles and in the barking 717 Pathology Standards/Microorganisms & Occupational Dust Table I Workers' Age, Type of Work, the Duration of Exposure, Symptoms, and the Clinical Findings ID..CODE AGE WORK (YRS) DUR.OF EXPOSURE (YRS) SYMPTOMS RE CFMD VA 1 SA 2 LA 3 LI 4 KU 5 TO 6 KA 7 KK 8 MA 9 HA 10 55 CD 25 38 CD 8 56 CD 14 39 CD 14 46 CD 26 36 WO/S 7/4 47 WO/B 4/3 37 B 17 37 B 15 47 B 17 _ _FM R C-- -- -- -- ---- C FM-- c F-- -- ---- R F-- -- ---- R D-- -- -- -- RE -FM-- R c-- -- - -- c-- -- ----- c-- """ CL. FINDINGS PEF ESR NORMAL NORMAL NORMAL NORMAL LOWERED NORMAL NORMAL NORMAL NORMAL NORMAL 12 2 12 5 17 4 7 2 2 5 CD - CATER PILLAR DRIVER WO - WOOD WORKER OUTDOORS S - SLASHER B - BARKING WORKER R - RHINITIS E * EYE IRRITATION C - COUGH F - FEVER M - MUSCLE PAIN D - DYSPNEA ESR - ERYTHRO CYTE SEDIMEN TATION RATE department. Alter incubation the number of colonies was counted, and the positive hole correction method was per formed to calculate the concentrations of viable airborne microbes. Antigens For antigen preparation 14 fungal end 3 bacterial strains were subcultured from original cultivation plates in nutrient broth containing 5 g/1 peptone (Difco Laboratories, Detroit, Mich., USA) and 3 g/1 beef extract (Difco) at optimal temperature for each species. Bacterial growth was haivested by centrifugation and fungal growth by filtering, and washed three times by distilled water. Microbial pellets were disrupted mechanically (Ultra Turrax, Janke and Kunkel, Staufen i Breisgan, FRG) and then by ultrasonic disintegrator (Soniprep ISO, MSE, Crawley, U.K.). The supernatants after a centrifugation at 40,000 g for 30 min were used as ELISA antigens. Antibody Determination IgG-antibodies were determinated by enzyme-linked im munosorbent assay (ELISA) carried out on disposable polystyrene microtiter plates (Immumoplate I, Nunc, Den mark). Microbial sonicates were used as antigen and alkaline 718 phosphatase-labelled swine anti-human IgG (Orion Diagnosdca, Espoo, Finland) was used as conjugate. An tibody levels were given as ELISA absorbance at a serum dilution of 1:100, read at 405 nm by a Titertek Multiskan (Eflab, Helsinki, Finland). RESULTS Microbe Exposure Marked qualitative differences were found in the microbial exposure of caterpillar drivers and barking workers. Bark ing workers were exposed mainly to bacteria (geometric mean of bacterial concentration 46.3 x 103 cfu/m3) and to lesser extent to fungal spores (5.9 x 103 cfu/m3) in contrast to caterpillar drivers on wood chip piles and 1.5X103 cfu/m3 and 4.5X103 cfu/m3 respectively) (Table II). Rhodotorula glutinis was the dominating fungal species in the barking department, and Aspergillus fumigatus and Penicillium brevicompactum on wood chip piles (Table HI). Antibodies Differences in antibody levels between different microbial species as also between different work environments were found by ELISA (Table IV). Highest antibody levels were found against Paecilomyces variotii, Sporobolomyces Pathology Standards/Microorganisms & Occupational Dust Table D Total Concentrations of Airborne Bacteria and Fungi (cfu/m3) in the Barking Department and on Wood Chip Piles Outdoors (x = geometric mean) MICROBE GROUP BARKING DEPARTMENT (n= 10) X RANGE ON WOOD CHIP PILES (n=10) x RANGE BACTERIA FUNGI 46000 5900 9200-230000 1400-70000 1500 770-35000 45000 1000-200000 Table HI Concentrations of Airborne Microbes (cfu/m3) in the Barking Department and on the Wood Chip Piles Outdoors (x = geometric mean) BARKING DEPARTMENT (n-10) X RANGE ON WOOD CHIP PILES (n-10) X RANGE Aspergillus fumigatus 28 Aspergillus niger 2 Humicola grisea 0 Paecilomyces variotii 2 PeniciIlium brevicompactum 500 Rhizopus nigricans 2 Streptomyces albus 2 Trichoderma viride 110 Aureobasidium pullulans 5 Cephalosporium curtipes 0 Cladosporium cladosporioides 52 Geotrichum candidum 2 Phialophora bubakii 0 Rhodotorula glutinis 3900 Sporobolomyces salmonicolor 1 Bacterium 1 930 Bacterium 2 33000 0-740 0-62 -- 0-41 41-3400 0-33 0-80 17-490 0-44 -- 0-180 0-21 -- 510-65000 0-10 180-4700 8300-210000 40000 9 9 6 6000 3 7 5 2 0 9 7 0 54 8 5 9 880-200000 0-190 0-48 0-110 12-88000 0-71 0-170 0-24 0-24 - 0-570 0-150 - 0-560 0-150 0-120 0-800 719 Pathology Standards/Microorganisms & Occupational Dust Table IV Antibody Levels (x + S.E.) Against the Microbes Found in the Working Environment in Barking Workers and in Wood Chip Workers MICROBE BARKING WOS (N-4) "7 + S.E. WOOD CHIP WOS (N-6) Y + S.E. ASP. FUMIGATUS ASP. NIGER HUM. GRISEA PAEC. VARIOTII PENIC. BREVICOMPACTUM RHIZ. NIGRICANS STR. ALBUS TRICH. VIRIDE AUR. PULLULANS CEPH. CURTIPES CLAD. CLADOSPORI01DES GEOTR. CANDIDUM PHIL. BUBAKII RHODOT. GLUTINIS SPOROB. SALMONICOLOR BACTERIUM 1 BACTERIUM 2 0.248 0.477 0.074 0.408 0.157 0.115 0.087 0.443 0.297 0.683 0.262 0.302 0.107 0.408 0.392 0.425 0.810 0.057 0.120 0.011 0.082 0.070 0.039 0.014 0.107 0.074 0.162 0.092 0.066 0.029 0.040 0.059 0.075 0.254 1.080 1.362 0.136 1.528 1.145 0.128 0.172 0.810 0.594 1.085 0.853 0.469 0.420 0.967 1.377 0.498 0.812 0.210 0.242 0.015 0.103 0.201 0.033 0.029 0.194 0.065 0.094 0.137 0.095 0.112 0.128 0.230 0.066 0.122 salmonicolor and Aspergillus niger while lowest were found against Rhizopus nigricans, Humicola grisea and Streptomyces albus. Generally, the levels of antibodies against fungal species were 2-5 times higher in the wood chip workers than those in the barking workers. No differences in the levels of bacterial antibodies were found between the two groups. DISCUSSION Ip the cellulose factory, the concentrations of airborne microbes except for bacteria were significantly higher on dus ty wood chip piles than in the barking department, and cor respondingly, the levels of antibodies against fungal species were higher in the wood chip workers than in die barking workers. Highest antibody levels were found against Paecilomyces variotii, Sporobolomyces salmonicolor, and Aspergillus niger and lowest antibody levels were found against Streptomyces albus, Humicola grisea and Rhizopus nigricans reflecting differences in capability of the species to stimulate a formation ofantibodies. Although the amount ofthe bacteria in the barking department was about 30 times higher because of aerosolized processing water than that on the wood chip piles, no differences in die levels ofantibodies against bacteria were found between the caterpillar drivers and toe barking workers. Probably the dry microbial material such as that in wood chip penetrates into the lower parts of toe respiratory tracts and initiates toe formation of antibodies more easily than toe moist aerosols. Fever and muscle pain as work-related symptoms in wood chip workers suggest the diagnosis of allergic alveolitis, which is supported also by high antibody levels in this group (Terho 1982), whereas rhinitis and cough found mostly in balking workers with low antibody levels seem not to be IgGmediated reactions. The comparison of antibody findings in toe cellulose factory with those in office workers gave a surprising result. The barking workers' antibody levels were not at all higher than those in bank clerks with minimal exposure to airborne microbes (unpubl. data). The dry microbial material occur ring in wood chip work and in office work penetrates prob ably easily into toe alveoli and initiates toe formation of an tibodies more effectively than toe moist aerosols irrespec tive of the amount of antigen. These results suggest that in addition to microbial concen tration toe physical nature of aerosols should be considered for evaluating toe health risks caused by airborne microbes. On toe other hand, toe immunization against occupationally exposing microbes could be diminished by controlled airhumidifying to prevent allergic respiratory diseases. 720 REFERENCES 1. Andersen, A.A., 1958. New sampler for collection, sizing and enumera tion of viable airborne particles. J Bact 76:471-484. 2. Bergman, 6. and Nilsson, T. 1979. An experiment on outdoor storage of whole-tree chips. Sveriges Lantbruksuniversitet. Institutionen for virkeslara. Rapport or 109:1-21, Uppsala Sverige. 3. van Assendelft, A.H.W., Raitio, M. and Turkia, V. 1985. Fuel chipinduced hypersensitivity pneumonitis caused by Penicillium species. Chest 87:394-396. 4. Jorgensen, H. and Fjellbeim, B. 1982. Allergisk alveolitt ved inhalasjon av soppsporer fra fiiktig treflis. Tidskx Nor Laegeform 102:737-739. Pathology Standards/Microorganisms & Occupational Dust 5. Lundgren, R. and Rosenhall, L. 1979. Fliseldarsjuka en ny variant av allergisk alveolit. Lakartidningen 76:4730-4731. 6. Marinkovicb, V.A. and Novey, H.S. 1983. Humidifier lung. Gin Rev Allergy 1:533-536. 7. PeOikka, M. and Kotimaa, M. 1983 The mould dust concentration caused by die handling of fuel chips and its modifying factors. Folia For 563. M8. 8. Rylander, R., Haglind, P., Lundholm, M., Mattsby, I. and Stenqvist, K. 1978. Humidifier fever and endotoxin exposure. Clin Allergy 8:511-516. 9. Terho, E.O. 1982. Extrinsic allergic alveolitis--The state of die art. Eur J Respir Dis 63:suppl 124,10-26. 721 Pathology Standards/Microorganisms & Occupational Dost ETIOLOGICAL INVESTIGATION OF FARMER'S LUNG --SEROLOGICAL STUDY SHEN YI-E* Shan Wei-Liang* Tao Bing-Gent Chen Guo-XIngf Wu Ai-Lianf Hong Zeng-Rongt Shanghai Medical University tHealth Agency of Jiangsu Province, Shanghai, P.R. China SUMMARY The reactions of precipitins in serum against the antigens from two strains of T. vulgaris were shown in 46.7 and 66.7% in 30 patients with farmer's lung, significantly higher than those in the control groups, while the reactions against M. faeni and A. fumigatus were low in the patients' group and not significantly higher than those in two control groups. The results indicated that the main etiological agents of farmer's lung were some strains of T. vulgaris in the patients. INTRODUCTION From 1980 through 1981, an epidemiological survey was conducted among 1054 hay grinders in Dafeng County, Jiangsu Province. 120 of them had history of farmer's lung disease. During follow-up study ofthese 120 grinders, acute episodes of farmer's lung after exposure to mouldy hay dust were seen in 67 of them.1 Meanwhile, a microbiological study of sputum of these patients and mouldy hay samples from their workplaces was performed. 80 strains of ther mophilic actinomycetes were isolated from these samples, 61.2% of them being T. vulgaris.2 In order to confirm whether T. vulgaris was die main etiological agent of former's lung in that county, we studied the precipitins in serum from the patients using serological method. MATERIALS AND METHODS Antigens Six strains of Thermoactinomycetes, including 4 strains of T. vulgaris called 801, 806, 816, 832 and 2 strains of Ther mophilic nocadia called 835 and 836, which were isolated from mouldy hay collected from the workplaces of the pa tients with former's lung, were selected and then, antigens were prepared by using a modified Salvaggio's method.3 Meanwhile, the strains of M. faeni 1., T. vulgaris 2. and T. candidus, one of each, provided by Dr. V. P. Kurup (Medical College ofWisconsin) were also selected to prepare antigens. The antigens were diluted to 30 or 40 mg per ml of normal saline, when they were used. In addition, other antigens including those form M. faeni 2., T. vulgaris 1. provided by Dr. J. H. Edwards (MRC Pneumoconiosis Unit) and A. fumigatus provided by Dr. J. Marx, JR (Marshfield Medical Foundation) were also used for detecting precipitins in serological test. Besides, the extracts from mouldy hay was prepared by using a modified Williams' method and diluted to 12 mg per ml of normal saline, also employed in die serological test. 722 Serum Samples Serum samples from 30 of these 67 patients were collected just one month after they ground mouldy hay. 30 serum samples from healthy people with no history of exposure to mouldy hay in the same area matched with the patients in sex and age were selected as control group A. Another 29 serum samples were collected from the healthy students in Shanghai Medical University as the control group B. Serological Test The presence of precipitins against the antigens was tested by using modified Ouchterlony's agar-gel double-diffusion assay.5 RESULTS It was shown that the reactions against two strains of T. vulgaris 1. and 2. were 46.7 and 66.7% in the patients' group, significantly higher than those in the control groups, whereas the reaction against T. candidus was 80% higher than that in group B, and it had not much difference with the control group A. Besides, the reactions against M. faeni 1. and 2. and A. fumigatus were rather low (16.7, 3.3 and 9.1 %) in the patients' group and not significantly higher than those in the two control groups. The reactions against six strains of thermophilic ac tinomycetes named T. vulgaris 801, 806, 816 and 832 and Thermophilic nocardia 835 and 836 ranged from 13.3 to 80.0% in the patients' group. The reactions against T. vulgaris 816 was 36.7% in the patients' group, significantly higher than that in the two control groups, and those against T. vulgaris 806 and 832 were 80.0 and 33.3% in the pa tients' group, significantly higher than those in the group B, but not in the group A. Besides, reactions against T. vulgaris 801, Thermophilic nocardia 835 and 836 and the extracts of mouldy hay in the patients' group were not significantly higher than those in the control group. DISCUSSION The precipitin test against fanner's lung antigens has been widely used in clinical diagnosis and epidemiological survey of the farmer's lung disease. The positive reactions against these antigens always indicate that die people have die history of exposure to them. Based on these reactions, die etiological agents of farmer's lung could be determined.6 In our study, the precipitins against a variety of farmer's lung antigens in sera from the patients with farmer's lung in Dafeng Coun ty, Jiangsu Province were tested and it was found that the percentages of positive reaction against three strains of T. vulgaris 1., 2. and 816 were 46.7, 66.7 and 36.7% in the patients with farmer's lung, respectively, which were significantly higher than those in the two control groups. The results might indicate that the main causative agents were some strains of T. vulgaris. The microbiological study of the mouldy hay from that county and sputum from the pa tients had also indicated that T. vulgaris was the dominant thermophilic actinomyces in die samples, while M. faeni was not found in them2 So, the findings of our serological study and the microbiological study were consistent with each other. Pepys had reported that the percentage of positive reaction against M. faeni in the patients with farmer's lung was as high as 85 % in Britain.7 So, the main causative agent of die disease was M. faeni in Britain. But in Finland, Terho found that the main etiological antigen of farmer's lung was from T. vulgaris.8 Perhaps the difference might be referred to die different way of preparing and storing hay, perhaps also climatic differences and differences in crop types. In Dafeng County as well as other area in east part of China, hay before stocking would be sun-dried as much as it could be and then stocked outdoors. In this instance, the weather is rather humid and warm in these regions, but the time is not long enough for M. Faeni to grow in the stacks, which might be the reason why the percentage of positive serological reaction against M. faeni was very low in the patients in that county. It was reported that the reactions against different strains of T. vulgaris in the same group of patients with farmer's lung might be significandy different from each other, and similar results could be found from different strains of T. candidus.8,9,10 In our study, it was also found that the reactions against six strains of T. vulgaris in the patients with fanner's Pathology Standards/Microorganisms & Occupational Dust lung ranged widely from 13.3 to 80.0%. These findings may indicate that different strains of T. vulgaris could have dif ferent antigens. Therefore, a variety of strains of T. vulgaris should be used to test precipitins in serum from the patients with farmer's lung. In addition, the reactions against the extracts from Ther mophilic nocardia and mouldy hay in the patients was found not significant in this study. In conclusion, it may be said that the etiological agents of farmer's lung in Dafeng County were mainly from some strains of T. vulgaris, but not M. faeni, and different strains ofT. vulgaris should be applied to detect precipitins in serum diagnosis of farmer's lung. REFERENCES 1. Tao Bing-gen et al.: An Epidemiological Survey of Farmer's Lung in Dafeng County, Jiangsu Province. Chinese J. Industr. Hyg. Occup. Dis. 2:34-38 (1984). 2. Lu Yun-yu et al.: Studies of Pathogens of Fanner's Lung in Jiangsu-- Isolation and Classification of Thermophilic Actinomycetes. Acta Microbiologica Sinica 25:351-355 (1985). 3. Salvaggio J. et al.: Experimental Production of Granulomatous Pneumonitis--Comparison of Immunological and Morphological Se quelae with Particulate and Soluble Antigens Administered via the Respiratoiy Route. J. Allergy and Clin. Immunol. 56:364-380 (1975). 4. Williams, J.V.: Inhalation and Skin Test with Extracts ofHay and Fungi in Patients with Fanner's Lung. Thorax 18:182-196 (1963). 5. YuHeet al.: Clinical Immunologic Technique, 1st Ed., pp 39. Science and Technology Pub., Shanghai (1981). 6. Burrell, R. et al.: A Critical Review of the Role of Precipitins in Hypersensitivity Pneumonitis. Eur. J. Respir. Dis. 62:332-343 (1981). 7. Pepys, J. et al.: Precipitin (F.L.H.) Test in Farmer's Lung. Thorax 20:21-35 (1965). 8. Terho, E.O. et al.: Microbiological and Serological Studies of Farmer's Lung in Finland. Clin. Allergy 9:43-52 (1979). 9. Wenzel, F.J. et al.: Serologic Studies in Farmer's Lung--Precipitins to the Thermophilic Actinomycetes. Am. Rev. Respir. Dis. 109:464 (1974). 10. Kurup, V.P.: Serologic Diagnosis of Hypersensitivity Pneumonitis. Microbiology 180-182 (1985). ACKNOWLEDGEMENT: The writers thank Prof. Yu Qing-fu, Prof. Jiang Hui-hui, Prof. VP Kunip, Dr. JH Edwards and Dr. James J Marx, JR for their kindly advising and providing cultures of thermophilic actinomycetes related to farmer's lung and antigens. Dr. Yuan Jing-wei for his joining die field work. Miss Lu Yun-yu for her advising and identifying the strains of tbermoactinomycetes, Miss Wu Cui-e and Xin Ji-ling for their joining the laboratory work. Sincere thanks must also go to Prof. Gu Xue-qi and Lu Pei-lian for their advice. 723 Pathology--Human Studies J IN SITU QUANTITATION OF NON-FIBROUS INORGANIC PARTICLE BURDEN IN LUNG TISSUE USING SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY ANALYSIS J. L. ABRAHAM Department of Pathology, State University of New York Health Science Center, Syracuse, NY, USA ABSTRACT To investigate the interaction of inhaled inorganic particulates with the lung one needs quantitative informa tion on the particulate burden of lung tissue. The vast majority of tissue samples (biopsies and autopsies) are fixed with formaldehyde and embedded in paraffin wax. Over die past 16 years, I have attempted to obtain maximal analytical use of such tissue. Standard paraffin sections of tissue are analyzed in the scan ning electron microscope (SEM) using secondary electron and backscatter electron imaging (BEI). Inorganic material is detected in the BEI and is analyzed using Energy Dispersive X-ray Analysis (EDXA). Quantita tion ofdie tissue particulate burden is readily accomplished using a point-counting, morphometric approach. I have collected quantitative data from analyses of particulates in over 400 lung samples. The data includes numerous comparative digestion analyses. The results from this in situ method correlate well with other analytic methods, and with comparison of results from other laboratories using similar or other techniques-- with die exception of submicrometer metal particles which are better represented in the in situ analyses, as they may be lost during digestion and filtration. Currentiy, of over 30,000 particles in the data base, major non-fibrous particles are comprised of 13.8% silica, 47.7% silicates and 38.6% metals. These data, plus data on medical and occupational histories, smoking, age, sex, pathologic diagnoses, etc. are being queried regarding normal and diseased lungs. No Paper provided. 724 Pathology--Human Studies I PULMONARY FIBROSIS ASSOCIATED WITH SMOKING IN MEN RESIDING IN A CLEAN-AIR ENVIRONMENT J. E. CRAIGHEAD* A. M. Adeslna* V. Vallyathanf E. N. McQuillenf University of Vermont, Burlington, VT fNational Institute for Occupational Safety and Health, Morgantown, WV $Chief Medical Examiner, Burlington, VT, USA ABSTRACT The role ofcigarette smoking in the pathogenesis ofpulmonary fibrosis has not been defined. This question is important to pathologists concerned with pneumoconiosis since many inhalant-related lesions result in fibrosis in the smaller airways. Using Gough-Wentworth slices and microscope tissue sections, we analyzed lungs of Vermont males over a range of ages who died suddenly and unexpectedly and were autopsied. Gough sections were analyzed by the method of Thurlbeck and by planimetry. Microscopical tissue was evaluated by the method of Hogg, et al. (Med. J. Aust. 142:605, 1985). Postmortem interviews with nextof-kin were conducted by trained nurse epidemiologists to determine cigarette use and possible occupa tional exposures. Overall, enphysematous changes were not striking, but there was a gradual increase in scores with advanc ing age in both smokers and nonsmokers using both techniques of analysis. In microscopic sections, inflam mation reflected by cellular infiltration in the walls of bronchioles and the presence of intraluminal macrophages was most prominent in younger smokers, whereas fibrosis of the walls of the bronchioles increased with age among smokers. Inflammation and the lung fibrosis indices in smokers and nonsmokers differed significantly. This study provided an opportunity to evaluate pulmonary changes associated with smoking among men living in a clean air environment, and not employed in dusty trades. In addition, it excludes possible terminal effects on the tissue morphology. A significant association of smoking with fibrosis of die membranous and respiratory bronchioles was found. The data suggest that respiratory bronchiolitis may be a contributory pathogenetic factor. No Paper provided. Pathology--Human Studies I ACCUMULATION AND COMPOSITION OF INHALED PARTICULATES IN HUMAN LUNGS YUKIKO OHTA, DMSc. National Institute for Environmental Studies 16-2, Onogawa, Tsukuba 305, Japan SUMMARY The black particulate matters deposited and accumulated in the autopsied human lung of deceased residents of the Tokyo Metropolitan area with no history of lung disease were separated, and their composing elements and substances were identified using several analytical techniques. The origin of lung contamination was examined. The carbon free radicals detected from human lungs were an original finding. Alpha-quartz was identified, carbon element, minerals and trace elements were determined, asbestos fibers were found and a result of the mutagenicity test on deposits was positive. The results observed in most cases were almost identical to the composition of an urban atmosphere. As for the exogenous factors related to the formation of pulmonary lesions, the effect of tobacco smoking cannot be ignored. In our pathohistological study, the observations of pulmonary lesions were found to be related to smoking. In view of the high concentration ofelement observed therein, it is considered that multiple factors participate in the development of exogenous pulmonary disease due to substances in the environment. These findings may be important in elucidating factors involved in the development of the lung disease due to particle deposition. INTRODUCTION The pulmonary anthracosis which has generally been as sumed to have little pathological significance has been used as a simple indicator for estimating exogenous lung contami nation. In studing the effects of suspended particulate matters in the atmospheric environment, it should be noted that the amount ofblack dusts deposited in a life time in human lungs depend upon various factors such as age, place of residence, smoking habits, and occupation. Published research reports pertain to anthracosis in the lung of such animals as dog, monkey, pigeon and autopsied human tissues.1*2*3 These studies were conducted from the pathological standpoint. However, there has been a need to chemically analyze the composition of black deposits only, because it has been thought that black deposits are mainly composed of inhaled suspended particulate matters in the atmosphere. In a report published4 in which Ohta was a co-author, multiple elements analysis was first conducted on anthracosis using spark source mass spectrometry. The ongoing studies5*6*7 have concentrated on the establishment of a relatively large base ofdata obtained by analyzing particulate matters isolated from autopsied lungs not only for element but also for ac cumulated toxic materials using several analytical techniques. MATERIALS AND METHODS Lung Specimens and Pathohistological Findings The autopsied lung samples used for this study were taken exclusively from people living in the Tokyo Metropolitan area with no history of lung disease. The age of 108 cases ranged from toe second decade to toe ninth decade. A defined 726 site of tire left upper autopsied lung lobes of these cases was employed, and pathohistological observations were ex amined. Separation of Black Deposits from the Lung Tissue The lung tissue was dissolved in alkaline solution. First, a test was made to determine whether 0.5N NaOH or 0.5N KOH would be satisfactory. Results by these solutions were not so different in regard to dissolve toe lung tissue. After weighing the lung tissue which was kept in room temperature after removal from storage at -- 80C, they were cut into small pieces and placed in polyethylene bottles with demineralized water to eliminate blood. After repeating this procedure for a few times using high speed centrifugation, 0.5N NaOH which was used for many samples were poured into the bottles. By repeated ultrahigh speed centrifugations at 12,000 rpm and 30,000 rpm, toe solid residue was re tained. The final residue was then washed using water, ethanol, acetone and finally dried. These black powders were used as samples for analysis. Deposition Rate and Observation of Particulates Using Scanning Electron Microanalyzer (SEM-EDAX) Elementary Analysis 1. Determination of elementary content using SEM-EDAX for obtaining general survey of toe particulate components 2. Quantitative analysis by neutron activation The analysis of Mn, V, A1 and Ti in 92 samples was completed, and other selected 13 samples were analyzed into trace elements. The samples were irradiated for 30 sec. for short halflife nuclides and for 5 hours for long half life nuclides at 1.5 x 1012 n/cm2 sec. 3. Determination of carbon content using CHO Elemen tal Analyzer In the analysis of total carbon content, CHO Elemental Analyzer was used. A sample measured precisely to 0.3 mg or 1.2 mg was placed in a sample container. Elemental carbon content was measured by combustion at 300C for 30 min. The volatilized carbon was calculated by subtracting the weight of residue carbon from the total carbon. Detection of Free Radicals in the Black Particulate Deposited in Lungs Using Electron Spin Resonance (ESR) Soot, tobacco, other kinds of smoke and products of com bustion are serious sources of harmful particulates. Samples of black deposits from the lung which were removed with tweezers without any chemical treatment were lyophilized and approximately 20 mg of each sample was subjected to ESR analysis at room temperature. Solid DPPH was used as standard for the g factor and its benezene solutions was used for the estimation of radical concentration. Mutagenicity Test for Black Deposits in the Lung A mutagenicity test for black deposits isolated directly from the lung tissue was examined by the Ames Test. The strain used for this test was Salmonella typhimurium TA98 and TA100. Identification of Crystallized Materials in Deposited Dust The crystallized material in the black dust which was treated with alkaline solution was identified using X-ray diffraction for 50 cases. Detection of Asbestos The asbestos fibers were detected and identified using a transmission electron microscope (TEM) coupled with X-ray microanalyzer for selected samples. RESULTS AND DISCUSSION Pathohistological Findings Some of 108 cases were detected to have pathohistological findings. The main observed findings were chronic bron chiolitis, emphysema and pavement epithelium metaplasia. These cases were found in relating to smoking considerable amount of cigarettes. This is especially true of9 cases found in this study. Deposition Rate and Observation of Deposited Particulates The deposition rate of inhaled dust was positively correlated with age. Correlation factor (r) was 0.65 (n=95, p<0.001). The particle size and shape of collected dust particles from human lungs were observed that an individual particulate was Pathology--Human Studies I approximately 0.1 micron in diameter, and many particles had aggregated into clumps. Elementary Composition of Deposited Particulates and their Accumulation in the Lung Usually Mg, Al, Si, P, S, K, Ca, Ti and Fe were detected in almost all samples, while Cl and Zn were detected in many samples, those contents were represented in weight percent (wt%). The concentration of Hg, Cr, Fe, Zn, Co, Ag, Sb, Cd and As in 13 specimen's samples were determined by neutron activation analysis. In case of chromium worker, Cr concentration was very high because of exposure to hexavalent chromium. V and Mn in die particulate are considered to originate from artificial sources, such as fuel or combus tion, Al and Ti are assumed coming from soil or sand in the natural environmental sources. The concentration of these elements were determined. We attempted to correlate the concentration of element to age. The correlation factor (r) of Al was r=0.48 (n=92, ***), that of V, r=0.40 (n--91, ***), that of Si, r=0.46 (n=95, ***), that of Fe, r=0.34 (n=95, ***). These elements showed a positive correlation to age, that is, they were accumulated in the lung according to increase in age. However, Mn and Ti were not correlated to age. And furthermore, Ca concentration showed a negative correlation to age (r= --0.56, n=72,***). The average total carbon content was 55 wt% (n=77). The data comparing the total carbon content between smokers and nonsmokers were not discriminating. The average content of elemental carbon was 39 wt% (n=39). The volatilized carbon was con sidered to be organic carbon. Determination of Free Radicals in Black Deposits Carbon-centered free radicals were detected in all 21 specimens. Figure 1-a shows an ESR spectrum from specimen A who was 81 year-old woman, where a narrow singlet is seen with a width of 2.7G and a factor of 2.0025. This spectral component is designated R]. This is more evi dent in the spectrum obtained with a wider field sweep in Figure 1-b. Such a broad signal apparently arises from in organic magnetic species in the black deposits. Carboncentered free radicals are not among those substances com monly expected to be contained in the air dust. Thermolysis or combustion of hydrocarbons is essentially a free radical process accompanying bond cleavage. As a simple com parison, tar and ash from Japanese cigarettes were collected and measured. The observed spectra were the same as that shown in Figure 1. The intensity of Ri component in each specimen measured by the height ofdie derivative peaks was obtained. Mutagenicity Test for Black Deposits A mutagenicity test which is called the Ames Test was used to determine the black deposits, which were isolated direct ly from lung tissues, and a small amount of them was used in this test. They were set in the center of an agar plate, the so-called spot test. The strains used for mutagenesis testing were salmonella typhimurium TA98 and TA100, S-9(--) and S-9(+). some inhibition was observed, however, His+ revertants were not more than the numbers of spontaneous revertants both in S-9(--) and in S-9(+). The result was that one sample was positive in both of TA98 and TA100 to 727 Pathology--Human Studies / b Figure 1-a. ESR spectrum of carbon free radical signal of specimen A who lived in die center of Tokyo for 60 years. Age: 81, Tobacco (--). The signal consists primary of Rj type radical case. Gain = 4 x 104. Modulation width = 1G at g = 2.00. Figure 1-b. The whole ESR spectrum of die same specimen taken with a wide magnetic field sweep. Besides Rt radical (the sharp signal in the center), a very broad absorption due to inorganic magnetic species is observed. Gain = 1 x 104. Modula tion width = 10G. S-9(--) and S-9(+), 3 samples were probably positive in TA98 only. Identification of Crystal Structure in the Deposited Particulates Alpha quartz was detected in 55 samples and crystallized stearate calcium was also detected. In a few samples, talc and ferric hydroxide were detected. Alpha quartz (a--SiO^) is a natural mineral, originating from soil and rock. They were blown up in die atmosphere. The stearate calcium detected in the lung deposit was produced by chemical pro cedures with alkaline solution at 40C for several days. Detection of Asbestos Fibers in the Black Deposits Asbestos fibers were detected in three cases among the 10 samples. We studied them using a TEM-XMA to identify asbestos fibers in die lung deposits. Chrysotile fibers were found in specimen B who was 78 year-old medical doctor, tremolite fibers and crocidolite fibers were detected in specimen C who was 65 year-old man. These fibers were qualitatively examined. However, other samples were not detected asbestos fibers. CONCLUSION The black dust deposited and accumulated in the human lung were separated and through die identification of their com posed elements, crystallized materials, carbon free radicals, asbestos fibers and the mutagenicity test, the origin of lung contamination was examined. The results observed in most 728 cases were identical to the composition of an urban at mosphere except for several cases, which were depended on their profession. The one case was a hexavalent chromium worker, and others were laborers worked at a industrial fac tory, public engineering works, construction industry etc. As for the exogenous factors related to the formation of pulmonary lesions, die effect of smoking cannot be ignored. We have detected free radicals in die human lung deposits as an original finding related to smoking and soot. A mutagenicity test for black deposits also was examined and a few cases were positive. Some of these data provide a large information base for future work and will be useful for mak ing a risk evaluation for lung contamination by low exposure to toxic substances. REFERENCES 1. Shibata, E.: The relation between human lung deposits and air pollu tion observed to autopsied lungs. Kyobu Shittan. 7(10):52-57 (1963). 2. Tateishi, R., Morimura, Y. and Hattori, M.: Multiple reactions ofsmok ing and air pollution on the human lung (pathological study). Nippon Rinsho. 25:184-192 (1967). 3. Shima, S. and Kato, Y.: The index ofliterature ofpneumoconiosis. pp. 158-161, 168-170. Fujita Gakuen University Publication, Japan (1982). 4. Tpyohara, K., Shigematsu, A., Hajikano, H., Iwai, K., Aoyama, T., Watanabe, E., (Rita, Y., and Yoshimura, S.: Elementary analysis of dust particulates deposited in human lungs. Proc. 8th Conference on Radioisotopes. 13-15 November.Tokyo. 226-228 (1967). 5. Ohta, Y., Inui, M., Shiraishi, H., Tabata, Y. and Waldsaka, I.: In vestigation ofcarbon black dust deposited in human lungs with special reference to influence ofthe environment. Proc. Vltb World Congress on Air Quality. 2:181-188 (1983). 6. Ohta, Y., Inui, M., Shiraishi, H., Matsumoto, M.: Air quality ofTokyo Metropolis evaluated by analysis of human lung deposits. Internal Medicine Today and Tomorrow. T. Oda et al. Editors. 106-112. Elsevier Science Pub.(1986). 7. Ohta, Y., Shiraishi, H. and Tabata, Y.: An electron spin resonance Pathology--Human Studies I study offree radicals in black dust deposited in human lungs. Archives ofEnvironmental Health. 40:279-282 (1985). The author thanks Mrs. Michi Matsumoto, B.Sc., Junior Scientist of our Department, for her helpful cooperation on the Ames Test study. 729 Pathology--Human Studies I CARCINOMA OF THE LUNG AND SILICOSIS: PATHOLOGICAL STUDY ISAMU EBIHARA Masaki Kawami Institute of Environmental Epidemiology, Faculty of Medicine Chiba University. (Chiba Japan) ' INTRODUCTION The relationship between silicosis and lung carcinoma can be approached from both epidemiologic and morphologic viewpoints. The majority of epidemiologic studies indicate that lung carcinoma occurs less frequently in coal miners than in comparable populations. However, excess lung carcinoma has been reported among metal miners, pottery workers, foundry workers and silicotic patients. To our knowledge, detailed morphologic studies of lung car cinoma in silicotic patients have not been reported. This is a report on pathologic evaluation of lung carcinoma associated with silicosis which was reviewed between 1960 and 1986 at our Laboratory. MATERIALS AND METHODS Between 1960 and 1986, the authors evaluated about 450 autopsies ofsilicosis. Ofthese, 140 were our own consecutive autopsies in our Laboratory and remaining 310 were kindly provided to us from other hospitals in Japan. Carcinoma of die lung was seen in 48 of the autopsies. Pathological studies, including cell types, cancer sites, severi ty of silicosis, were performed in the 48 cases of lung car cinoma associated with silicosis. Severity of silicosis was determined by extent ofprogressive massive fibrosis as follows; (1) mild silicosis; silicosis without PMF (simple silicosis); (2) medium silicosis: silicosis with small PMF that were formed within lung segment; (3) severe silicosis: silicosis with large PMF including some segments. RESULTS Carcinoma ofthe lung was seen in 25 ofour own consecutive autopsies, an incidence of 19.9%. The incidence of lung car cinoma was definitely elevated among mild silicosis and lowered among severe silicosis (Table I). Table II shows the distribution of lung carcinomas by histologic cell type in all 48 lung carcinomas. Over all, the predominant cancer was squamous cell car cinoma (54.2%) followed by small-cell carcinoma (22.9%) and adenocarcinoma (14.6%). There was a clear trend of squamous cell carcinomas arising in the larger airways, whereas the adenocarcinoma was found only in peripheral lung tissue. More tumors were observed in the right lung, but the dif ference was not observed between upper and lower lobes (Table m). In case of mild silicosis, die majority of tumors arose in the right, upper and larger airways. On the other hand, in case of medium and severe silicosis, more tumors arose in the left, lower and peripheral lung tissues. The distribution of primary focus in the large bronchi are illustrated in Figure 1. In case of mild silicosis, many tumors arose in stem and lobar bronchi, whereas many tumors arose in segmental bronchi in case of medium and severe silicosis. In case of silicosis with PMF, the majority of tumors arose in the segmental bronchi leading to PMF. (Figure 2 shows the typical case of such cases.) The primary foci of tumors arising in peripheral lung tissues were illustrated in Figure 3. Almost all the tumors in periph- Table I Incidence of Lung Carcinomoma Among Our Autopsy Cases Severity of Pn. Hi Id Med iua Severe Total Nuobcr of Cases 40 51 49 1 40 Lung Carcinoia 13 ]0 2 25 % 3 2.5 19. 6 4. 1 1 7. 9 730 Pathology--Human Studies I Table H Distribution of Lung Carcinoma Associated with Pneumoconiosis Cases by Histologic Type Squaaous Cell Ca. Adenocarcinoaa Siall-Cell Ca. Large*Cell Ca. Total Central Type Peripheral Type 15 10 06 64 21 23 21 Unknown 1 1 1 1 4 Total 26 7 1 4 48 % 5 4. 2 1 4. 6 2 2.9 0. 3 10 0. 0 Table HI Location in Lung of Tumors by Site of Origin Hi Id Pn. Hcdiue Pn. Severe Pn. Hediui + Sevc r RiSh t:Left 16t7 Upper : ff i d<11 c: Lower 14: 0: 9 7: 7 5 : 1:8 4: 3 4:0:3 11: 10 D : 1 : 11 Cen tral:Periphera 1 14:9 7: 7 Total 23 14 __________________ 1______ . 2: 5 7 9 : 12 21 J Total 2 7; 17 2 3: 1 : 20 2 3:21 44 ........... . .. eral lung tissues arose in S2, S3, S6, S9 where PMF were usually formed. The majority of the tumors in peripheral lung tissues were centered on closely adjacent to PMF or originated on the basis ofpathologic course to PMF. In these cases, there were three cases of scar cancer arising in scar tissues of PMF (Figure 4 and Figure 5). Diffuse interstitial fibrosis of the lung was often associated with silicosis. Five cases of carcinoma of lung were found in these cases (Figure 6). SUMMARY The data indicate the close relationship between pathological, changes of lung tissues by dust exposure and carcinoma of the lung. 731 Pathology--Human Studies I Upper Lobe Bronchus Lower Lobe Bronchus B2 C 8i * z ) B4 Be B7 B B* Total Right Lung 4 1 4 1 2 1 1 2 16 Left Lung 3 2 2 0 0 0 0 0 7 Figure 1. Location in bronchial trees of central type of tumors by site of origin. 732 Pathology--Human Studies I Figure 2. Squamous cell carcinoma originated from bronchus leading to progressive massive fibrosis. 733 Pathology--Human Studies I Figure 3. Location in lung of peripheral type of tumors by site of origin. 734 Pathology--Human Studies I Figure 4. Scar cancer originated from anterior portion of progressive massive fibrosis. Pathology--Human Studies I Figure 5. Scar cancer originated on the basis of pathologic course to progressive massive fibrosis. 736 Pathology--Human Studies I Figure 6. Adenocarcinoma associated with mild pneumoconiosis and diffuse interstitial fibrosis. 737 Pathology--Human Studies I STUDY ON DUST PARTICLE SIZE IN AUTOPSIED LONGS OF UNDERGROUND COALMINERS XING GCIO-CHANG Fu Mu-Sen et al. Institute for Occupational Medicine of Ministry of Coal Industry Beijing, China INTRODUCTION Some scholars suggested that particle less than 5pm was most harmful. Others thought that particle less than 5-7 pm had the selective effect on die lungs. Some countries adopted the respirable dust concentration as dust standard.1 But both the measurement of particle size distribution of dust after death from the lungs of coalminers and the experimental study on dust retained in die respirable organ ofanimals failed to reach a consensus about hygienic evaluation of different fractions of particle sizes. Dust 40-60 pm in diameter were found at autopsy.2 Professor Chen Hongquan3 observed particle size distribution of dust at autopsy, using biological microscope and scanning electron microscope and suggested that the particles with dia. below 2, 5 and 10 pm made up 65.2, 88.4 and 95% of total number. Author4 thought that dust more than 5-7 pm must be considered when working out limit standard of dust and monitoring dust in production environment. In this paper, our results of study on particle size distribu tion ofdust in underground coalminers' lungs were reported. MATERIALS AND METHODS Subjects 120 histological sections were at random sampled from the lung sections of 60 autopsies (2 sections per case), who had been exposed to coal dust or with Coal Workers' Pneumoconiosis for this study, most of them had been coal mining workers and die few had been rock drifting workers. In view of the difference of physical and chemical proper ties of coal mineral dusts, 120 sections were divided into two dust groups which were treated with digestion and microin cineration, respectively. Hydrogen Peroxide Digestion The specimen were digested in 70 5 centigrade temperature H2C>2 for 3 hrs and treated with concentrated HC1 and observed by polarizing microscope. Microincineration Thin sections with their waxembedding material were moved by washing in xylene, dried and microincinerated in a muf fle furnace at 540 centigrade temperature for 4 hrs, treated with concentrated HC1. Size-Groups by Particle Size Dusts were divided into down to 2, 2-5,5-7,7-10 and over 10 pm size-groups by geometric projection diameter. Frac tions of particle numbers and masses of dust were calculated. RESULTS Number Distribution of Particle Sizes of Dust in lungs The observations ofdie treated specimens which were divided into many parts equally were performed using tire X 400 light microscope and Polarizing microscope. 500 particles were measured per section. Results were in Table I. The % of particle numbers was similar in the small particles of two dust-groups, significantly different in two dust-groups of par ticle 7-10 pm and over 10 pm in dia.(t >to.oi P <0.01). In mineral dust-group, numbers of particle 7-10 pm in dia. made up 7.7 % of all mineral particle numbers. In coal dustgroup, number of particle in 7-10 pm dia. only constituted 4.4 % of all coal particle numbers, number of dust > 10 pm covered 0.4% and 0.2% respectively in mineral dust-group and in coal dust-group. It was clear that large particle mineral dust predominated over that of coal dust. To account of different definitions of the respirable dust in die inspirable dust curve recommended by some countries and organisms5 Table I was changed into Table II. Grain size distribution >7 pm fraction had significant difference in mineral dust-group and coal dust group (t >tooi* P <0.01). Mass Distribution of Dust Particle Size in Lungs Accumulative distribution derived from number distribution of particle size was plotted on logarithmic normal logprobability graph paper so as to attain number distribution N(D) which was necessary to account and more minute than die measuring of groups by means of geometric projection using microscope. Total mass of particles with dia. ranging from Di to D2 is given by fD2 9 1)3 N (d) dD 738 Pathology--Human Studies I Table I Number Distribution of Particle Sizes of Two Dust-Groups & of Number Distributioni of Particle Types of Number of Sizes (/*)____________________ Dusts Samples <2 2-5 >5 7-10 >10 Mineral Coal 53 57 55.6 57.6 25. 26.5 10.5 11.2 7.7 4.4 0.4 0.2 Types of Dusts Mineral Coal Mean Table n Numbers of Particle Sizes of Two Dust-Groups Number of Samples 53 57 % of Particle Sizes (Fm) <5 5-7 7-.10 >10 72.9 79.1 76.0 18.6 15.9 17.2 8.1 4.7 6.4 0.4 0.3 0.4 Here: p: Particle Density Oy,: Volume Shape Factor of Particle D: Geometrical Projective Diameter of Particle. If composition ofparticles and mechanism of producing par ticle are same, p and cty are not related to particle size, so the formula above is changed into: "1,2 =^"vfD2 d3 n (D) do J Di But relation between N(D) and D measured really showed that distributions of N(D) was different within the range of particle sizes considered. For the sake of convenience, in tegral method of numerical value was used. So: Flf2 rol,2 rat P v D. D N (Di) =Dl Dmax t> v X! N (Di) Di DissDmin D2 2Z ul N (Di) Di*Di Within the given range of Particle size (D1-D2), the per cent of particle mass in total particle mass is: 2Z Dj N (Di) DisD-4n Where Dmin and D^ are the smallest and largest particle diameters. Mt is total mass. The calculated results were shown in Table m and Figure 1. It was seen in Table TTT that masses of Particle greater than 5, 7 or 10 pm in size made up respectively 83.7 3.3,66.3 6.1 and 14.2 7.7 739 Pathology--Human Studies I Table in Mass Percent of Particle Sizes in Two Dust-Groups Types of Coal Mineral Coal Mean Number of Samples 53 57 % Mass of Particle Sizes <5 5-7 7-10 16.3 22.3 iy.4 17.4 11.3 14.3 52.1 55.5 53.8 ) >io 14.2 12.5 12.5 Gerometrical Projective Diameter( pm ) Figure 1. Mass percent of particle sizes of two types of dusts. percent of the total mass in mineral dust in autopsic lung tissue and in coal dust in lung, masses ofparticle with more than 5, 7 or 10 pm in dia. accounted for 77.6 7.2, 45.3 10.4 and 10.8 8.4 % of total mass, respectively. Significance tests showed that particle fractions over 5 pm and 7 pm of two types of dusts were significantly different (t >to.oi, P< 0.05)and that fractions > 10 pm of two types of dust had statistical significance^ >t 0.05 , P < 0.05). Relationship between the Mass and the Number Distribution of Dust Particle Sizes in Autopsic Lungs The mass and the number distributions ofdust particle sizes in the lung tissue were studied. (Figure 2). Figure 2 illustrated that number of dust < 5 pm amounted to 76.0% of total number, but its mass was only 19.4%, of total mass; that number of dust >5 pm made up only 24.0% , but its mass accounted for 80.6% of total mass and that number of dust t 7 pm was 6.8% total number, its mass was 66.3% total mass and that number of dust > lO pm was 0.4% of total number, its mass constituted 12.5%, total mass. Some scholars had observed 37297 airborne particles of samples from the gold mine and come to the conclusion that number of particle < 1 pm made up 92/% of total number and its mass only 10.5% weight of sample, which was cor respondence with our results. 740 Pathology--Human Studies I (0 u O Ch O 4J c 0) o (4 9 a, Figure 2. Comparison of percent content of particle sizes of dusts in autopsic lungs. DISCUSSION Some scholars2 had injected the same weight of quartz 0.8-2.Opm and 5-10 pm in dia. into two groups of rats (A group and B group), respectively and observed 19 small dust focuses and 3 large dust focuses in A group and 47 small dust focuses and 15 large dust focuses in B group. It may be seen that quartz 5-7 pm.in dia. caused the more and the larger dust focus than quartz 0.8-2.0 pm in dia.. Hence, the respirable dust concentration was only part of dust concentration. Other articles6*7,8 and our study confirmed that the level of pathological change and categories by X-ray were closely relative to mass and content of dust retained in lungs. Such research has shown that CWP is related to exposure to respirable dust, partially dust 2 pm or larger in size, is the most important factor associated with CWP. So we think that when drawing up the dust hygiene standard and monitoring dust concentration, we considered not only die respirable dust concentration but also the total dust concentration. REFERENCES 1. Morgan, W.K.C. and Seaton, A.: Occupational Lung Diseases, 2nd Ed. pp.440. W.B. Saunders Co., New York (1975). 2. Tatsuo Sano: Japanese Pneumoconiosis and Environmental Pollution of Dust. Associate Number of Institute for Science of Labour, pp. 155-178. Tokyo (1977). 3. Chen Hong-Quan: Study on Etiology of Miner's lung Cancer and Pneumoconiosis of Yun Xi. Journal of China Medical University, p. 7 (1985). 4. Beeckmans, J.M.: The deposition ofAerosols in The Respiratory Tract I. Mathematical Analysis and Comparison with Experimental Data. Can. J. Physiol-Pharmacol. 43:157-172 (1965). 5. Xing Guo-Chang: Coal Water's Pneumoconiosis. Institute for Occupa tional Medicine of Ministry of Coal Industr, pp. 40-60, Beijing (1986). 6. IAEA: Particle Size Analysis in Estimating the Significance ofAirborne Contamination, Technical Reports Series No. 179. Vienna (1978). 7. Rivers, D., Wise, M.E., King, E.J. and Nagelschmidt, G.: Dust Con tent, Radiology and Pathology in Simple Pneumoconiosis of Coal Workers. Brit. J. Med. 17:87-108 (1960). 8. Nagelshmidt, G.: Dust and Collagen Content of Lungs ofCoal Workers with Progressive Massive Fibrosis. Brit. J. Ind. Med. 20:3, 181-191 (1963). 741