Pulmonary response to Mount St. Helens' volcanic ash

ENVIRONMENTAL

RESEARCH

Pulmonary

30, 361-371 (1983)

Response

VAL VALLYATHAN,' Pathology

to Mount

St. Helens’

Volcanic

Ash

M. SHARON MENTNECH,JAMES H. TUCKER,AND FRANCIS H.Y. GREEN

and Immunology Sections, Appalachian Laboratory for Occupational and Health, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505

Safety

Received November 4. 1981 The pulmonary response to a sedimented sample of Mount St. Helens’ volcanic ash from the first eruption was studied at 1,7, 28,90, and 180 days postintratracheal administration of 1 or 10 mg of ash in specific-pathogen-free rats. One day after administration of volcanic ash all animals exhibited a marked inflammatory cell response centered on respiratory bronchioles in which polymorphonuclear leukocytes predominated. At 7 days the reaction was characterized by mononuclear cellular infiltrates. The macrophages within the respiratory bronchioles and alveoli contained intracytoplasmic ash particles. At 28 days the intraalveolar aggregates of mononuclear cells had condensed to form granulomas. Most of the granulomas contained foreign body-type giant cells and some showed central necrosis. The granulomas enlarged in size from 28 days until the termination of the experiment at 180 days with progressive increase in the amount of collagenous tissue. The results of these studies suggest that the volcanic ash may pose a risk for pneumoconiosis in heavily exposed human populations.

INTRODUCTION

On May 18, 1980, Mount St. Helens erupted releasing approximately 4 km3 of rock and ash into the atmosphere. Subsequent eruptions and the reaerosolization of ash by wind and man has resulted in exposure of over one million people in the northwestern United States. In addition to the low level of background exposure, certain occupational groups, such as loggers and farmers, may be exposed to high concentrations of ash. The health implications of these exposures are difficult to assess as there have been few precedents for this kind of disaster. Mineralogical analyses have shown the majority of the ash to be composed of silicate minerals of the plagioclase class (Fruchter et al., 1980; Green et al., 1982). The majority of the ash particles are within the respirable range, i.e., ~10 pm in diameter (Green et al., 1982). Particle circular area equivalent diameter showed an average value of 1.7 pm with a range from 0.11 to 10.7 pm. Although nonfibrous rock-forming silicates are generally supposed to be only mildly fibrogenic, they are known to cause pneumoconiosis in both humans and animals (Mohanty et al., 1953; Sherwin et al., 1979; Parkes, 1974; Brambilla et al., 1979). Free crystalline silica in its polymorphic forms-quartz, cristobalite, and tridymite-constitutes approximately 2- 10% of the ash (Fruchter et al., 1980; Morbidity and Mortality 1 To whom correspondence should be addressed: Pathology Section, ALOSH, Chestnut Ridge Rd, Morgantown, W. Va. 26505.

NIOSH,

944

361 0013-9351/831020361-11$03.0010 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

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Weekly Report, 1980). Free silica is highly fibrogenic and the predominant form of free silica in the ash (cristobalite) is more fibrogenic than quartz (King et al., 1953). National Institute for Occupational Safety and Health’s (NIOSH) industrial hygiene studies have shown that workers in the logging industry in the vicinity of Mount St. Helens are exposed to respirable dust concentrations of 0.8 to 5.3 mg per cubic meter of air (mg/m3). Assuming that the majority of volcanic ash in the vicinity of the logging industry is similar with an average free silica concentration of 5% and a respirable dust level of 1 mg/m3; the loggers would be exposed to a concentration of more than 50 pg free silica/m3 of air (Morbidity and Mortality Weekly Report, 1980; Baxter et al., 1981). This equals the NIOSH recommended standard which is based on the risk of developing pneumoconiosis over a working lifetime. Therefore, loggers exposed to concentrations of ash exceeding 1 mg/m3 over a period of time are theoretically at risk of developing pneumoconiosis from the free silica component of the ash alone. Moreover, the toxicity of other silicate minerals present in the ash are completely unknown, as are the possible interactions between the various components of the ash. In order to assess the toxicity of the ash, we have conducted a series of in vitro tests that reflect the fibrogenic and cytotoxic potential of a mineral dust (Green et al., 1982). In three tests considered indicative of the fibrogenic potential of a mineral dust, the ash had an activity slightly less than that of free crystalline silica. These were the sheep red cell hemolysis assay (Green et al., 1982); the ability of ash to cause proliferation in WI-38 fetal lung fibroblast cultures (Green et al., 1982); and the effect of ash on lysosomal membranes as assessed by release of lysosomal enzymes by ash-exposed alveolar macrophages (unpublished observations). In vitro studies are useful procedures for rapid determination of the potential toxicity of an unknown substance and for a better understanding of the mechanisms involved. It is necessary, however, to extend these observations into more complex systems if meaningful information concerning toxicity to human populations is to be gained. We report here the results of our in vivo studies on the Iibrogenic properties of volcanic ash. MATERIALS

AND METHODS

The volcanic ash used in these studies was a dry, sedimented sample-from the first volcanic eruption-collected at Spokane (a city 340 km away from Mt. St. Helens). The ash was collected within a few hours of sedimentation by an industrial hygienist; contamination was minimized by collecting the ash from hard surfaces. Mineralogic analysis and particle sizing was accomplished using scanning and transmission electron microscopy, energy dispersive X-ray analysis, X-ray powder diffraction, and automated image analysis, details of which have been reported elsewhere (Green et al., 1982). Before intratracheal administration (IT), the ash was suspended in 0.9% sodium chloride solution and sonicated to ensure even dispersion. Ten- to twelve-week-old (250-300 g), male and female, specific-pathogen-free (SPF) Fisher 344 rats (Charles River Laboratories, Wilmington, Mass.) were used

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in these experiments. The animals were maintained on a standard diet of rat chow and water ad fibitum. Animals receiving 10 mg volcanic ash IT were examined at intervals of l(8), 7(4), 28(5), 56(2), and 180(10) days. Animals receiving 1 mg volcanic ash IT were only examined at l(8), 56(8), and 180(8) days. The number of animals sacrificed is given in parentheses. Two groups of controls were used in these studies. The first group of 23 animals received 0.25 ml of 0.9% sodium chloride by IT, and the second group of 10 animals was not injected. Both groups of animals were maintained under the same conditions as the ash-exposed animals. The animals were anesthetized in a bell jar with ethyl ether and intratracheally instilled with either 0.25 ml of ash suspension or saline solutions using a curved 20-gauge needle with a 1.5-mm rounded tip. Instillations were made in phase with respiration in order to facilitate deposition in the lung periphery. Animals were anesthetized with ethyl ether and killed by exsanguination. The thorax was opened and the lungs were allowed to collapse. The trachea was cannulated and the lungs infiltrated to full inspirational volume with buffered Formalin. The trachea was tied and after 10 min of fixation in situ, the heart and lungs with trachea were removed and placed in a bath of Formalin for a minimum of 3 days. Tissue samples from other major organs were also obtained and fixed in Formalin. In each case a sagital block of tissue was taken from the midregion of the left and right lungs and a coronal block through the upper hilar region. The blocks were paraffin embedded and serial sections of 5 pm thick were cut and processed for light and scanning electron microscopic studies. The sections for light microscopy were stained with hematoxylin-eosin and examined under bright field and polarized light. In selected cases, 10 or more serial sections were made and sections 1, 2, 5, 6, 9, and 10 were stained with hematoxylin-eosin, Masson’s trichrome, elastin, reticulin, Perl’s iron, and Giesma stains, and evaluated by light microscopy. Sections 3, 4, 7, and 8 were mounted on spectroscopically pure carbon planchets, deparaffinized in xylene, and lightly coated with carbon in a vacuum evaporator. The specimens were studied in an ETEC Autoscan scanning electron microscope (SEM) equipped with a solid-state backscattered electron (BSE) detector and a Tracer Northern X-ray energy spectrometer (XES). Tissue lesions were identified and photographed in the backscattered electron mode (BSE) and particle inclusions in the lesions were analyzed by XES. Analyses were carried out in the point mode for 100 live-time set at a magnification of 3000 x with an accelerating voltage of 25 kV. Elemental peaks were identified and photographed. RESULTS

By scanning electron microscopy, the majority of ash particles had smooth surfaces with sharp angular borders (Fig. 1). Occasional particles were rounded and had a more pitted surface resembling pumice. No fibrous minerals were identified in the ash preparations. The bulk of the ash sample, 99 and 81% by count and weight, respectively, was within the respirable range (i.e., less than 10 pm in diameter). Analysis of 1000 particles in 20 fields of view at 1000 x by SEM in combination with a LeMont B-10 automated image analysis system showed an average circular area equivalent diameter of 1.7 pm. Mass median diameter of 6.0

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FIG. 1. Scanning electron micrograph of volcanic ash sample. Majority of ash particles have sharp angular edges, x500.

pm for the ash was calculated from the analysis of 1216 particles in 10 fields of view at 400x. Mineralogical analysis showed that the majority of particles were plagioclase minerals such as albite, oligoclase, bytownite and labradorite consisting predominantly of silicon, aluminum, calcium, sodium with small quantities of potassium. Volcanic glass, labradorite, titano-magnetite, hornblende, and crystalline silica were present in smaller amounts. Total crystalline silica was 7.2% of which 4.2% was cristobalite and 3.0% quartz. All animals in the exposed and control groups survived the experimental period. Light microscopic evaluation of the lung sections from volcanic ash-instilled animals showed several significant morphologic changes at different periods of time. These changes were associated with the presence of volcanic ash identified by polarized light microscopy. At 1 day after the administration of 10 mg volcanic ash, the lungs of all the animals exhibited a marked acute inflammatory cell response which was centered on the respiratory bronchioles and alveolar ducts with involvement of adjacent alveoli. The inflammatory infiltrate was characterized by polymorphonuclear leukocytes (Fig. 2). Chronic inflammatory cells were few at this time. Focal alveolar edema was seen in areas of intense inflammation and greatest dust deposition. At 7 days, the inflammatory reaction was predominantly mononuclear in type with lesser numbers of polymorphonuclear cells evident. The macrophages formed granulomatous aggregates within the alveoli and contained numerous

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intracytoplasmic ash particles. Occasional true granulomata with central necrosis and ash particles were also seen (Fig. 3). At 28 days, well-formed granulomas composed of macrophages, lymphocytes, fibroblasts, and ash were seen scattered throughout the lung parenchyma. Foreign body-type giant cells were common and these usually contained brightly birefringent intracytoplasmic ash particles. In addition to the granulomata, mild thickening of the walls of some respiratory bronchioles and adjacent alveoli was observed. Ash was also seen in association with these lesions. The pulmonary lesions at 56 days were similar to those seen at 28 days. Giant cell granulomata and interstitial lesions were still evident. Necrosis was no longer present in the granulomata, and the number of lymphocytes and collagen fibers was increased. Nine of the ten animals studied at 180 days following 10 mg IT of volcanic ash showed widespread granulomata in both lungs. One animal in the group had histologically normal lungs, and ash could not be detected by polarized light microscopy. The granulomata ranged in size from 0.6 to 5 mm in diameter with a mean of 1.93 mm and contained more collagen, reticulin, and lymphocytes than were seen at earlier time periods. Occasional granulomata showed a rim of paracicatricial emphysema (Fig. 4). Although the lesions contained collagen, the amount was slight and none became hyalinized. Foreign body giant cells were also common at this time (Fig. 5). The distribution of the lesions at all time intervals was uneven. In most animals, lesions were seen throughout both lungs. Subjectively, the number of ash particles within the granulomata at 28, 90, and 180 days appeared to be similar as assessed by both polarized light microscopy and backscattered scanning electron microscopy. Animals that received 1 mg ash showed similar but less severe or extensive lesions. Inflammatory or granulomatous lesions were not seen in the lungs of control animals. Tissue sections on carbon planchets were examined in the SEM, and the lesions were identified and correlated with serial sections examined by light microscopy. At 24 hr, volcanic ash particles were identified by backscattered electron imaging in the respiratory bronchioles and adjacent alveoli. The ash particles at higher magnification were found to be lying both free on the airway surfaces and within the cytoplasm of phagocytic cells (Fig. 6). Granulomata examined from the 180day experimental group contained numerous evenly distributed ash particles (Fig. 7a-c). X-ray analysis of BSE-imaged particles within the lesions showed that the majority were silicates containing sodium, aluminum, silicon, calcium, and iron similar to the original ash sample (Fig. 7d). Histopathological evaluation of other major organs showed no abnormalities in either ash-exposed or control animals. A few particles of ash were seen within macrophages in the spleen, liver, and lymph nodes of animals intratracheally instilled with 10 mg volcanic ash. However, no cellular response was associated with the presence of ash in these organs. DISCUSSION

The formation of granulomata with areas of interstitial fibrosis in exposed rats shows that volcanic ash is fibrogenic in viva, and this finding correlates well with

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the in vitro data on cytotoxicity previously reported (Green et al., 1982). It has been shown that the volcanic ash varies in both particle size and mineralogical composition depending on the distance from the volcano (Fruchter et al., 1980). The findings reported here are based on a single volcanic ash sample, therefore it should be borne in mind that other ash samples may produce a different type of pulmonary response. The initial tissue response to volcanic ash involved polymorphonuclear leukocytes. This finding is consistent with studies using other mineral dusts (Dodson et al., 1980; Morgan et al., 1980). The polymorphonuclear leukocyte response to volcanic ash and other mineral dusts is transient and could result from a variety of mechanisms, including release of chemotactic factors (Hunninghake et al., 1978) and direct activation of complement (Hasselbacher, 1979). The central necrosis seen in the ash granulomata was reminiscent of the early stages of silicosis. However, these lesions did not develop the whorled and hyalinized collagenous centers typical of classical silicosis. Giant cells, which were common in the ash-induced lesions, are also not a feature of silicosis. The latter are more commonly seen in the other forms of silicate pneumoconiosis such as talcosis (Vallyathan and Craighead, 1981). Giant cells were a common feature of the response to volcanic ash at all time periods studied. It is possible that the giant cell reaction was related more to the size of the particles than to their chemical composition. This question could be resolved by studying the cellular response to size-defined fractions of the ash in a similar experimental study. Overall, the tissue response to volcanic ash more closely resembled the nodular lesions associated with mixed-mineral dust exposure (Parkes, 1974). This type of reaction, in which the collagen is arranged in a haphazard pattern rather than in the concentric pattern of a mature silicotic nodule, is thought to represent a reaction to silica that is modified by other minerals, notably silicates and oxides of iron (Sherwin et al., 1979; Parkes, 1974; Brambilla et al., 1979). Experiments using pure minerals present in the ash, individually and in mixed combinations, would be necessary to define the fibrogenicity of the ash to a particular component or to a group of minerals. The risk of developing pneumoconiosis, if any, from volcanic ash in human populations has yet to be elucidated. However, our preliminary studies on lung tissues from two loggers who were heavily exposed to volcanic ash showed a reaction in their lungs similar in some respects to that seen in exposed rats (Green

FIG. 2. Photomicrograph of a rat lung examined 1 day after 10 mg volcanic ash IT. The cellular infiltrate is characterized by polymorphonuclear leukocytes. Hematoxylin-eosin, (X 145.) FIG. 3. Photomicrograph of the rat lung 7 days after IT of 10 mg of volcanic ash. Granuloma illustrated to show the central necrosis with cellular debris and ash particles. Hematoxylin-eosin, (x225.) FIG. 4. Photomicrograph of rat lung 180 days after IT of 10 mg of volcanic ash. The granuloma is composed of macrophages and lymphocytes and a few collagen fibers. A rim of paracicatricial emphysema is seen. Hematoxylin-eosin, (X 145.) FIG. 5. Photomicrograph of rat lung 180 days after IT of 10 mg of volcanic ash. The granuloma contains multinucleated giant cells and ash particles. Hematoxylin-eosin, (X 145.)

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FIG. 6. Rat lung 24 hr following IT injection of 10 mg volcanic ash. (a) SEM image of lesion centered on respiratory bronchiole. x70. (b) Higher magnification of area shown in inset of a. Inflammatory cells and volcanic ash particles are present in the respiratory bronchiole and adjacent alveoli. x400. (c) Higher magnification of inset of b showing polymorphonuclear cells (PMN) and alveolar macrophages (AM). x 1100. (d) Backscattered electron image of b showing volcanic ash particles (black) lying free (arrows) and within phagocytic cells (arrowheads). x400.

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FIG. 7. Rat lung 180 days following IT of 10 mg volcanic ash. (a) Low power SEM showing granuloma (inset). x42. (b) Higher magnification of granuloma shown in inset. x70. (c) BSE image of granuloma showing the distribution of volcanic ash particles (black). x70. (d) X-ray energy elemental spectrum of Particle No. 1. The major elemental peaks identified in the spectrum are similar to those obtained on the original ash sample. Particles Nos. 2, 3, and 4 identified by backscattering electron imaging in the granuloma showed similar elemental composition.

ef al., 1981). It is pertinent also to note that a severe form of pneumoconiosis was recently described in California agricultural workers, in which the dust in their lungs was similar, mineralogically, to volcanic ash (Sherwin ef al., 1979). Massive pulmonary fibrosis resulting from exposure to feldspar free of crystalline silica has also been reported (Barrie and Gosselin, 1960). In addition experimental studies have shown the development of simple pneumoconiosis in rats (Mohanty et al., 1953). Although we present evidence that the ash is fibrogenic in uivo and that the fibrosis appears to progress with time, it is difficult to extrapolate from these studies or to draw conclusions concerning ash exposures in human populations.

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First, the amount of ash intratracheally instilled in the rats was far greater than that likely to be inhaled by the general population or even by high-exposure occupational groups such as loggers and farmers. Second, the route of exposure (IT) is not comparable with the human situation where exposure is by inhalation. Finally, apart from a few individuals who had massive exposure after the fust eruption, human exposures are likely to be low doses over long periods of time. This may produce a totally different effect than that seen in animals receiving large doses over a short period of time. Despite these considerations and based on our knowledge of the toxicology of some of the ash constituents, volcanic ash should be considered to constitute some risk for pneumoconiosis in heavily exposed populations. Although it has been suggested that volcanic ash is relatively inert biologically (Fruchter et al., 1980), it would seem prudent to minimize ash exposure in the light of these experimental studies. ACKNOWLEDGMENTS The authors thank Dr. Donald D. Dollberg and Dr. Lloyd E. Stettler for mineralogical and particle size analyses; Ms. Marilyn Gamble for technical assistance; and Dr. Robert Bernstein and Mr. Michael McCawley for ash samples. We are also grateful to Dr. Vincent Castranova, Dr. William Chang, Dr. Mark Colson, Dr. David H. Groth, and Dr. Yusunosuke Suzuki for manuscript review; Ms. Molly Pickett Fredlock for editorial assistance; and Ms. Catherine M. Weydert and Ms. Kay Kennedy for manuscript preparation.

REFERENCES Barrie, J., and Gossehn, L. (1960). Massive pneumoconiosis from a rock dust containing no free silica. Arch. Environ. Healrh 1, 109-117. Baxter, P. J., Ing, R., Falk, H., French, J., Stein, G. F., Bernstein, R. S., Merchant, J. A., and Allard, J. (1981). Mount St. Helens eruptions, May 18 to June 12, 1980. An overview of the acute health impact. J. Amer. Med. Assoc. 246, 2585-2589. Brambilla, C., Abraham, J., Brambilla, E., Berinschke, K., and Bloor, C. (1979). Comparative pathology of silicate pneumoconiosis. Amer. J. Pathol. 96, 149- 170. Dodson, R. F., Hurst, G. A., and Williams, M. G. (1980). Short-term response to lung parenchyma following “amosite” asbestos exposure. Amer. Rev. Respir. Dis. 121, 232. Fruchter, J. S., Robertson, D. E., Evans, J. C., Olsen, K. B., Lepel, E. A., Laul, J. C., Abel, K. H., Sanders, R. W., Jackson, P. O., Wagman, N. S., Perkins, R. W., Van Tuyl, R. H., Beauchamp, R. H., Shade, J. W., Daniel, J. L., Erikson, R. L., Sehmel, G. A., Lee, R. N., Robinson, A. V., Moss, 0. R., Brian& J. K., and Cannon, W. C. (1980). Mount St. Helens ash from the May 18, 1980 eruption: Chemical, physical, mineralogical and biological properties. Science 209, 1116-1125. Green, F. H. Y., Bowman, L., Castranova, V., Dollberg, D. D., Elliot, J. A., Fedan, J. S., Hahon, N., Judy, D. J., Major, P. C., Mentnech, M. S., Miles, P. R., Mull, J., Olenchock, S., Ong, T., Pailes, W. M., Resnick, H., Stettler, L. E., Tucker, J. G., Vallyathan, V., and Wong, W. (1982). Health implications of the Mount St. Helen’s eruption: Laboratory investigations. Ann. Occup. Hyg. 26, 921-933. Green, F. H. Y., Vallyathan, V., Mentnech, M. S., Tucker, J. H., Kiessling, P. J., Antonius, J. A., Parshley, P., and Merchant, J. A. (1981). Is volcanic ash a pneumoconiosis risk? Nature (London) 293, 216-217. Hasselbacher, P. (1979). Binding of immunoglobulin and activation of complement by asbestos fibers. .I. Allergy

Hunninghake,

Clin.

Immunol.

64, 294-298.

G. W., Gallin, J. I., and Fauci, A. S. (1978). Immunological

reactivity of the lung. The

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in vivo and in vitro generation of a neutrophil chemotactic factor by alveolar macrophages. Amer. Rev. Respir. Dis. 117, 15-23. Hunninghake, G. W., Kawanami, O., Ferrans, V. J., Young, R. C., Roberts, W. C., and Crystal, R. G. (1981). Characterization of the inflammatory and immune effector cells in the lung parenchyma of patients with interstitial lung disease. Amer. Rev. Respir. Dis. 123, 407-412. King, E. J., Mohanty, G. P., Harrison, C. V., and Nagelschmidt, G. (1953). The action of different forms of pure silicates on the lungs of rats. Brit. J. Znd. Med. 10, 9- 17. Mohanty, G. P., Roberts, D. C., King, E. J., and Harison, C. V. (1953). The effect of feldspar, slate and quartz on the lungs of rats. J. Pathol. Bacterial. 65, 501-511. Morbidity and Mortality Weekly Report (1980). Vol. 29. No. 28, p. 338. Center for Disease Control, Atlanta. Morgan, A., Moore, S. R., Holmes, A., Evans, J. C., Evans, N. H., and Black, A. (1980). The effect of quartz, administered by intratracheal instillation, on the rat lung. I. The cellular response. Environ. Res. 22, 1-12. Parkes, R. W. (1974). “Occupational Lung Disorders.” pp. 194- 197. Butterworths, London. Sherwin, R. P., Barman, M. L., and Abraham, J. L. (1979). Silicate pneumoconiosis in farm workers. Lab Invest.

40, 576-582.

Vallyathan, V., and Craighead, J. E. (1981). Pulmonary asbestiform talc. Human Pathol. 12, 28-35.

pathology of workers exposed to non-