OX1GO AT MINML PROTECTION ------- 1OOR72008 TOXICOLOGY OF ATMOSPHERIC SULFUR DIOXIDE DECAY PRODUCTS Trent R. Lewis, Mary O. Amdur, Martin D. Fritzhand, and Kirby I. Campbell Division of Health Effects Research ENVIRONMENTAL PROTECTION AGENCY National Environmental Research Center Research Triangle Park, North Carolina July 1972 ------- The Aft series of reports is issued by the Environmental Protection Agency to report the results of scientific and engineering studies, and information of general interest in the field of air pollution. Information presented in this series includes coverage of intramural activities involving air pollution research and control technology and of coopera- tive programs and studies conducted in conjunction with state and local agencies, research institutes, and industrial organizations. Copies of AP reports are available free of charge to Federal employees, current contractors and grantees, and nonprofit organizations - as supplies permit - from the Air Pollution Technical Information Center, Environ- mental Protection Agency, Research Triangle Park, North Carolina 27711. Publication Number AP-111 ------- CONTENTS LIST OF FIGURES iv LIST OF TABLES iv ABSTRACT v INTRODUCTION 1 TOXICITY IN EXPERIMENTAL ANIMALS 3 STUDIES OF MORTALITY AND PATHOLOGY 3 Sulfuric Acid Mist Particle Size 3 Exposure Duration 5 Conclusions 6 STUDIES OF PULMONARY FUNCTION 7 Sulfuric Acid Mist Particle Size 7 Sulfur-Containing Particulate Aerosols 10 Particulates in Combination with Sulfur Dioxide 13 Hygroscopic Properties of Sulfuric Acid 15 Conclusions 16 TOXICITY IN MAN 19 STUDIES OF SUBJECTIVE AND PHYSIOLOGICAL RESPONSES TO IRRITATIVE PROPERTIES OF SULFURIC ACID MIST 19 Detection by Subjects 19 Effects of Humidity, Acidity, and Particle Size on Subject Response 20 Effects of Sulfuric Acid Mist on Respiratory Retention 21 Conclusions 23 STUDIES OF SENSORY AND CENTRAL NERVOUS SYSTEM RESPONSES TO SULFURIC ACID MIST 24 Optical Chronaxy 25 Dark Adaptation 26 Continuous Electroencephalography 28 Conditioned Reflexes 28 Conclusions 29 ill ------- SUMMARY 31 TOXICITY IN ANIMALS 31 TOXICITY IN MAN 33 REFERENCES 35 BIBLIOGRAPHY: SELECTED TOPICAL REFERENCES 39 LIST OF FIGURES Figure Page 1 Effect of Particle Size on Airway Response to 1 mg/m Zinc Ammonium Sulfate Aerosol 11 2 Dose-Response Curve of Zinc Ammonium Sulfate Aerosol for Different Particle Sizes 12 3 Comparison of Irritant Potency of Sulfur Dioxide and Sulfur-Containing Particulates 13 LIST OF TABLES Table Page 1 Optical Chronaxy on Ninth Minute of Exposure of One Subject to Test Atmospheres .......... 26 2 Eye Sensitivity to Light in Three Subjects During Twentieth Minute of Dark Adaptation ........ 27 Number of Trials with H^SO^. Plus Light Required for Appearance of Conditioned Reflex ........ 30 Threshold Aerosol Doses Required For Sensory Response . 30 IV ------- ABSTRACT Studies of atmospheric chemistry have shown that under certain conditions sulfur dioxide can be converted to sulfuric acid and particu- late sulfates. Additional aerometric and toxicologic research is needed to supplement what little is known of the actual atmospheric concentra- tions and chemical forms present under varying meteorological condi- tions and of the toxicologic significance of this group of compounds under different ecological conditions. Most available animal data, exclusive of data from mortality studies, relate to alterations of pul- monary function during and following exposure to sulfuric acid and particulate sulfates. Although results of pulmonary function studies in animals exposed to these substances cannot be extrapolated simply and directly to predict human responses, two significant toxicologic princi- ples related to the assessment of health hazards presented by such air- borne contaminants have been demonstrated. First, data on mass con- centration alone are an insufficient basis on which to predict irritant potency because particle size plays an important role in determining the irritant potency of sulfur oxide particles (submicron particles being more irritant). Second, particulate oxidation products of sulfur dioxide are generally much more potent irritants than the sulfur dioxide gas per se. Although sulfuric acid is known to be a much greater irritant than sulfur dioxide to man, the combined effect of particle size and concen- tration of sulfuric acid mist on exposed human subjects is still undeter- mined. Industrial- and episode-exposure studies have implicated long- and short-term exposure to sulfuric acid in the etiology of acute and/or chronic respiratory disease. Russian scientists have studied the thres- hold concentrations of oxides of sulfur that elicit certain sensory responses. ------- Other factors that affect the toxicity of particulate oxidation pro- ducts of sulfur dioxide, such as retention, site of deposition, hygro- scopic nature of the products, and the subject's breathing pattern are discussed. Key Words: toxicology, air pollution, sulfuric acid, sulfates, human, animal, respiratory, neurosensory Vi ------- TOXICOLOGY OF ATMOSPHERIC SULFUR DIOXIDE DECAY PRODUCTS INTRODUCTION Studies of atmospheric chemistry have shown that a portion of the sulfur dioxide (302) emitted into the atmosphere undergoes oxidation, leading, eventually, to the formation of sulfuric acid (H SO ) and sul- fate particulates. Studies in experimental toxicology demonstrate that some of these particulate oxidation products have a greater irritant potential than sulfur dioxide itself. Clearly then, air quality standards for sulfur dioxide should be set with due consideration for the potential sulfur dioxide has, under certain atmospheric conditions, for forming its more hazardous derivatives. The acute air pollution episodes that occurred in Donora, Penn- sylvania, and London, England, precipitated intensive research into the toxicological and physiological effects of sulfur dioxide and certain of its decay products. In spite of this research, most of which was conducted between,1950 and 1963, many questions regarding health problems related to atmospheric oxides of sulfur remain unanswered. To consolidate the information that is available, this report presents a review of the literature on the toxicology of some of the sulfur dioxide decay products, and, in one section, presents recent data on the com- parative toxicity of those decay products. ------- TOXICITY IN EXPERIMENTAL ANIMALS STUDIES OF MORTALITY AND PATHOLOGY Experimental studies that use mortality and pathology as the criteria of response by necessity involve the exposure of animals to pollutant concentrations far above those occurring in the atmosphere in order to produce measurable effects. Data from such studies cannot, without some misgivings, be extrapolated to give practical guidance in setting safe levels for those air pollutants. Such studies, however, are usually the initial step in investigating the toxicity of a substance. Considerable species differences have been observed in the response of small laboratory animals exposed to sulfuric acid mist. Treon et al. exposed small numbers of several species to H2SO4 mist, 95 percent of the particles of -which were smaller than 2 micrometers (jim) in diameter. Using concentrations ranging from 87 to 1600 milli- grams per cubic meter (mg/m3), they found increasing sensitivity as shown: rabbits < rats < mice < guinea pigs. Guinea pigs died of expo- sures not lethal for other species; when exposed to concentrations sub- lethal to them, they had proportionately greater respiratory irritation and lung pathology than the other species studied. The results of Mathur^ also corroborated that guinea pigs are less resistant than mice to sulfuric acid mist. Sulfuric Acid Mist Particle Size Questioning the influence of aerosol particle size on response, Amdur et al. ^ began by determining the 8-hour LCso* of solfuric acid particles with a mass median diameter (MMD) of 1 (im. The LC5Q was 18 mg/m3 for 1- to 2-month-old guinea pigs and 50 mg/m3 for 18-month- old animals. In subsequent studies, sulfuric acid mists of 2.7-(j.m-MMD particles proved more toxic than those of 0.8-fi.m-MMD particles and *LC50 is the concentration lethal for 50 percent of the experimental population. ------- the smaller material was more toxic at 0° C than at room temperature. * These data indicate that the age of the animal, particle size, and tem- perature all influence lethality. Other variables have been found to influence the toxicity of sulfuric acid: the presence of smoke^ resulted in increased mortality and the presence of aerosolized ammonium carbonate-* diminished the effects of the acid. The increase in mortality was attributed to the larger size of the toxic particles, which resulted from the adsorption of the acid, upon the smoke particles. The decrease in effects was attributed to chemical neutralization of the sulfuric acid. Amdur et al. and Pattle et al. both examined the gross and microscopic pathology of the lungs of exposed guinea pigs and agreed in their findings. Animals that died after exposures to H2SO4 of less than 2 hours had grossly distended and emphysematous lungs, but no other serious lesions. The cause of death appeared to be asphyxia- tion caused by bronchoconstriction and laryngeal spasm. The lungs of animals that died after longer exposures had gross pathological lesions, including capillary engorgement and hemorrhage. ' It has been sug- gested that these changes represent sequelae to the combined effects of anoxia and increased intrathoracic pressure caused by bronchoconstric- tion and laryngeal spasm. The lungs of animals that survived the longer exposures showed spotty areas of old hemorrhage and some areas of consolidation, especially around the hilar regions. Although such exposures do not give rise to death, during or afterwards, the damage is repaired only very slowly. Amdur et al. 3 reported that, when the H2SO4 concentration was 8 mg/m3, extending the exposure time to 72 hours did not increase mortality beyond the amount observed in 8-hour exposures. Pathologi- cal examination of the lungs, however, revealed that the longer expo- sures greatly increased the damage. In the 8-hour exposures, damage was limited to thickening of the alveolar walls and consolidation of lung tissue (CT*=64 mg/m3-hr). In animals exposed for 72 hours (CT=576 mg/m3-hr), damage was greatly enhanced. These workers postulated *CT is the product of time and concentration. 4 TOXICOLOGY - S02 DECAY PRODUCTS ------- that sulfuric acid mist has two distinct toxic actions. It promotes laryngeal and bronchial spasms, causing immediate death by an action related primarily to concentration and individual sensitivity, and it also produces parenchymal lung damage, an effect that is more depend- ent upon total dosage. Pattle et al. 5 pointed out that the lung damage probably results from the direct irritant action of acid mist deposited on the lung tissue and that such deposition and irritation may be the mechanism by which the mist causes death in animals more resistant to bronchial-laryngeal spasm than the guinea pig. This mechanism may account, as well, for many of the marked differences in mortality observed by early investigators who used different exposure concen- trations. Exposure Duration Thomas et al. " exposed guinea pigs to sulfuric acid mist for periods of 18 to 140 days. They used three particle sizes (0. 6, 0. 9, and 9. 0 fim) in concentrations of 4 mg/m3 or less, except for occasional accidental higher exposures. ,The coarse particles produced lesions in the upper respiratory tract and slight edema in the larynx and trachea, with less mucus in the major bronchi. The mists composed of 0. 9-^m particles produced greater lung damage than either the 0.6- or 9. 0-|o.m particles. Exposure of young animals to 0. 9-nrn mists produced slightly edematous lungs and, rarely, capillary hemorrhage. Some increase in desquamated epithelial cells was observed in the minor but not in the major bronchi; the epithelium appeared intact. Major and minor bronchi both had less mucus than controls. The authors con- cluded that the guinea pig could tolerate continuous exposure to levels of about 2 mg/m sulfuric acid mist for more than 3 months with only minor effects. Bushtueva ' exposed guinea pigs to 2 mg/m sulfuric acid mist of unspecified particle size for 5 days. The animals developed edema and thickening of alveolar walls. Exposures to 1 mg/m3 for 1 to 2 •weeks produced slight catarrh in the mucous membranes of the trachea and bronchi, and slightly defined but widely distributed interstitial pro- liferation accompanied by round-lymphoid-cell infiltration around the Toxicity in Experimental Animals 5 ------- blood vessels and bronchi. These latter changes seemed to be progres- sive, for they were more highly developed 2 to 3 months after exposure. A concentration of 0. 5 mg/m produced only a slight "lung irritation. " Conclusions Sulfuric acid is a respiratory irritant, and, when present in sufficient concentration, it will cause the death of exposed animals. The guinea pig is the most sensitive species among the common small laboratory animals, followed in decreasing order by mice, rats, and rabbits. The guinea pig's greater sensitivity probably results from its greater susceptibility to bronchial and laryngeal spasm. Although the enhanced sensitivity makes this animal useful for some studies of the irritant potential of low concentrations of sulfuric acid, the results must be applied judiciously when extrapolation is made to the effects of sulfuric acid air pollution on human health. In all cases, con- centration has been shown to be a more important factor in sulfuric acid-produced death than duration of exposure. * Sulfuric acid also causes pathological lesions in the lungs after much less than lethal exposures. Effective, short exposures (up to 72 hours) produce a degree of lung damage related to the arithmetic product of concentration and time (CT) rather than to the concentration alone. Such damage is slow to repair. The fact that concentrations below 2 mg/m^ produce only minor lung damage after extended expo- sures (about 3 months) indicates that the CT-damage relationship observed in 8- and 72-hour exposures cannot be extrapolated directly to long-term effects of low concentrations. Data clearly indicate that the particle size of sulfuric acid mist is also an important factor in determining its irritant potency. When the criterion was mortality resulting from an 8-hour exposure, a mist of larger particles (2. 7 (im versus 0. 8 (im) was more toxic. When pathological lesions resulting from long-term exposures were the criterion of response, the effect of 0. 9-fj.m particles was greater than that of either 0. 6- or 9. 0-|j.m particles. The effect of particle size will be discussed further in the next section. TOXICOLOGY - S02 DECAY PRODUCTS ------- STUDIES OF PULMONARY FUNCTION Methods like the ones discussed in this section can be used to evaluate the effects of low concentrations of irritants that do not usually produce lung damage or cause death. In other words, they can be used to examine the response of experimental animals to air pollu- tants at concentrations known to occur in urban atmospheres. Sulfuric Acid Mist Particle Size Employing unanesthetized and spontaneously breathing guinea 9-11 pigs, Amdur measured the effect of sulfuric acid mist on pulmo- nary function by the following parameters: flow resistance of the lungs and airways; compliance (a measure of the elasticity of the lungs); tidal volume (the volume of each breath); respiratory frequency; and minute volume (tidal volume times respiratory rate). These data also per- mitted her to make rough estimates of elastic-resistive work and the total work of breathing. Amdur used concentrations ranging from 2 to 40 mg/m and particles of 0.8, 2.5, and 7.0 ^m (MMD). Experimental results indicated that the size of particles in the aerosol is critical to both the nature and degree of the irritant response elicited. Results of these experiments made clear that data obtained from exposure to high con- centrations could not have been extrapolated to indicate, in a valid manner, the reaction to lower concentrations , since irritant potency varied with both concentration and particle size of the sulfuric acid mist. This fact underlines the need for, and the importance of, methods that can directly assess responses to low concentrations of irritants. The 7. 0-fim-MMD particles, at a concentration of 30 mg/m , produced a statistically significant increase in flow resistance but no other detectable changes in respiration. Few particles of this size would be expected to penetrate beyond the nasal passages, so that the observed increase in flow resistance was probably restricted to the upper respiratory tract, especially since the other parameters measured were not altered. Toxicity in Experimental Animals ------- The 0. 8-nm-MMD particles produced an increase in resistance, accompanied by a proportionally lesser decrease in compliance, an increase in elastic and resistive work, and, hence, an increase in the total work of breathing. These changes were statistically significant at the lowest concentration tested (1. 9 mg/m^). Onset of response was prompt and resembled the pattern of changes observed after inhalation of sulfur dioxide. Changes in pulmonary function such as these are consistent with narrow air passages caused by bronchoconstriction, mucosal swelling, or increased mucosal secretion. The 2. 5-^m-MMD particles also increased flow resistance at all concentrations examined (2. 3 to 44 mg/m-). The relative toxicity of the 0. 8- and 2. 5-|j.m-MMD particles, evaluated by increased pulmonary flow resistance, was highly dependent upon concentration; however, at high concentrations, the larger particles elicited a much greater re- sponse, whereas at low concentrations the smaller particles gave the greater response, again demonstrating that data obtained from expo- sures to high concentrations could not be used to predict accurately the responses to low concentrations. The increased toxicity of 2.5-^m- as compared with 0. 8-(j.m-MM-D particles at high concentrations con- A firmed the earlier finding of Pattle et al. Further comparison of data for the two particle sizes suggested a difference in physiological action, presumably related to the site where the particles were deposited in the respiratory system. Response to the 2. 5-jim-MMD particles, but not to the 0. 8-|J.m-MMD particles, was slow to develop, occurring only after 40 to 45 minutes of exposure. This delay suggested the possibility that the different responses were mediated by different mechanisms, a possibility that was confirmed both by the nature of the changes in the mechanical properties and by the post-mortem appearance of the lungs. The alterations in mechanical properties that occurred indicated that large sulfuric acid particles produced a complete obstruction of some air passages,- whereas small particles caused a narrowing of the airways but not complete obstruction. The gross pathology of the lungs TOXICOLOGY - SO, DECAY PRODUCTS ------- was consistent with data derived from pulmonary function tests. Ani- mals exposed to the high concentration of 2. 5 -|J.m-MMD particles devel- oped extensive areas of atelectasis, frequently involving whole lobes. In addition, their lungs were edematous, as demonstrated by a signifi- cant increase in the ratio of lung weight to body weight. These findings suggested that high concentrations of larger particles, which probably are deposited in the vicinity of the major bronchi, produce severe local irritation of the larger air passages and cause swelling and increased exudation of fluid, which eventually lead to complete obstruction. The pulmonary flow-resistance values were slow to return to pre-exposure levels after the end of a 1-hour exposure. Animals exposed to 15 mg/m or more still showed elevated resistance 2 hours after exposure had ended. This slow return to normal has been found consistently after exposure to irritant aerosols or to the combination of irritant gas and inert particulate material. '•'•' ^' The reasonable explanation for the slower recovery from exposure to an aerosol as opposed to an irritant gas is apparently the fact that the particles, having been deposited on the respiratory tract, continue to exert their irritant effect. The irritant gases, on the other hand, are cleared from the lung when exposure ceases, and the respiratory system returns to normal functioning rather rapidly unless the exposure con- centration has been extremely high. The enhancement that results from combining inert aerosols and irritant gases appears to be mediated through the formation of an irritant aerosol, ' which could account for the prolonged response to such exposures. Lewis et al. ^> ^ studied the effects of chronic H2SO4-aerosol exposure on canine pulmonary function. Animals were exposed for 621 days to 0.5-(xm-MMD HzSO4 particles, at a concentration of 0. 8 mg/m^, and pulmonary function tests were performed 225 days and 621 days following the onset of exposure. At 225 days, diffusion capacity had decreased. After 621 days of exposure, average carbon monoxide diffusion capacity and residual lung volume were reduced and Toxicity in Experimental Animals ------- total pulmonary resistance was increased. Post-mortem examination showed that the net lung volume was reduced. Sulfur-Containing Particulate Aerosols Amdur and Corn'- studied the irritant potency of zinc ammonium sulfate, zinc sulfate, and ammonium sulfate, and found a difference in the potencies as judged by increases in flow resistance resulting from exposure to 1 mg/m concentrations having an MMD particle size of 0.29 him. Zinc sulfate exhibited about half the irritant potency of zinc ammonium sulfate. Ammonium sulfate was the least irritant of the three compounds, producing a response about one-third to one-fourth that observed from exposure to an equal concentration of zinc ammonium sulfate. In further studies of the effect of particle size on the irritant potency of zinc ammonium sulfate, four MMD sizes were used: 0.29, 0.51, 0.74, and 1. 40 (J,m. Concentrations ranged from 0. 25 to 3. 60 mg/m . Figure 1 shows the results obtained when the concentration of aerosol was held constant at about 1 mg/m and the particle size was varied. Particle size by weight is indicated in micrometers. This method for calculating mass-size is explained by Amdur and Corn. From the curve it is evident that the irritant potency, as judged by the increase in flow resistance produced by the 1-hour exposure, increased as the particle size decreased over the range of sizes examined. Amdur and Corn1" concluded that the greater irritant potency of the smaller particles could represent the reaction to an increased number of pinpoint stimuli produced by the greater number of particles present in the more finely dispersed material; to the differing depth of penetra- tion into the peripheral areas of the lung; or to a combination of these factors. From data available for several concentrations of zinc ammonium sulfate at various particle sizes, Amdur and Corn prepared a family of dose-response curves. As Figure 2 shows, not only was the response 10 TOXICOLOGY - S02 DECAY PRODUCTS ------- 200 100 — 20 J LJ 0.01 0.05 0.10 0.50 1.0 AEROSOL MEAN SIZE BY WEIGHT, micrometers 5.0 10.0 Figure 1. Effect of particle size on airway response to 1 mg/m^ zinc ammonium sulfate aerosol. Numerals beside each point indicate number of animals. Data and calculation of mean size by weight from Amdur and Corn.16 to a given concentration greater as the particle size decreased, but the slope of the dose-response curves was also steeper. This means that a small increase in mass concentration produced a larger increment in biological response as the particle size decreased, re-emphasizing the fact that if a compound is dispersed as particulate material a measure of mass concentration alone is not sufficient to permit useful assessment of its irritant potency. Nadel et al.l'' studied the effect of aerosols of histamine and of zinc ammonium sulfate on anesthetized, artificially ventilated cats. The aerosols were submicrometer in size and the concentrations of zinc ammonium sulfate were 40 to 50 mg/m . The sulfate aerosol produced a physiological response similar to that produced by hista- mine, but lesser in degree. The response to a 3-minute inhalation included increased pulmonary resistance, decreased pulmonary com- pliance, and increased end-expiratory transpulmonary pressure. As Toxicity in Experimental Animals 11 ------- a 60 0.4 1.2 1.6 2.0 2.4 ZINC AMMONIUM SULFATE, mg/m3 2.8 3.2 3.6 4.0 Figure 2. Dose-response curve of zinc ammonium sulfate aerosol for different particle sizes. Numerals beside each point indicate number of animals. Data from Amdur and Corn.16 correlation with the physiological studies, the authors made anatomical studies after rapidly freezing the lungs in the open chest. These showed that bronchioles up to 400 (im were narrower in exposed than in control animals and that the mucosa was longitudinally furrowed. Bronchi and bronchioles larger than 400 ^im were not significantly different from controls. The authors noted the similarity between the action of hista- mine and of zinc ammonium sulfate aerosol. In regard to extrapolating the results they concluded, "It remains to be demonstrated that acute changes reported here at aerosol concentrations of 40 to 50 mg/m are exaggerated but similar in nature to those produced, if any, at lower concentrations1'. Follow-up work is clearly needed in this field. In the course of examining the effect of aerosols on response to sulfur dioxide, Amdur and Underbill18 examined the irritant potency of several other sulfates. They reported that 1 mg/m3 ferric sulfate TOXICOLOGY - S02 DECAY PRODUCTS ------- [^62(304)3] produced a 77-percent increase in flow resistance during a 1-hour exposure, which would definitely indicate that this substance is an irritant. On the other hand, ferrous sulfate (FeSO4) and manganese sulfate (MnSO/j) at the same concentration produced no detectable alteration in resistance. Not all sulfates, then, are irritant in nature, and the sulfate ion per se is not known to have irritant potency. Particulates in Combination with Sulfur Dioxide Because data from atmospheric chemistry studies have indicated that sulfuric acid and particulate sulfates are formed from sulfur dioxide emitted to the atmosphere, it is essential that the comparative irritant potency of sulfur dioxide gas and its chemical reaction pro- ducts be examined. A long-term research program undertaken by Amdur et al. has provided data on the effects of sulfur dioxide gas and several particulate oxidation products on the guinea pig. The authors are using the same physiological techniques for all the pollutants, which permits direct comparison of the irritant potency of these compounds. Their data are summarized in Figure 3. 150 100 I ! 50 UJ », ^t 20 UJ 7 - 18 0.01 0,02 0.05 0.1 0.2 0.5 SULFUR EQUIVALENTS, i.o 2.0 5.0 1.0 0.01 0.1 SULFUR DIOXIDE, ppm 1.0 Figure 3 . Comparison of irritant potency of sulfur dioxide and sulfur-containing particulates. Numerals beside each point indicate number of animals. Data from Amdur.19 Toxicity in Experimental Animals 13 ------- Even a casual glance indicates clearly that an equivalent amount of sulfur present as sulfur dioxide gas produces a lesser irritant response than either the particulate sulfates or sulfuric acid. One part per million of SO is the equivalent of 1. 3 mg S/m3. When pre- sent as sulfur dioxide gas, this amount of sulfur increases flow resistance by about 15 percent. If, through reaction in the atmosphere, this sulfur equivalent were converted to 0. 7-|j.m-MMD sulfuric acid mist, it could produce an increase in resistance of about 60 percent, a fourfold potentiation of irritant response. If, on the other hand, that same amount of sulfur were converted to zinc ammonium sulfate parti- cles of 0. 3-|j.m MMD, resistance would increase about 300 percent, a twentyfold potentiation. Such complete conversion in the atmosphere is of course highly unlikely, but an oxidation of 10 percent can occur readily. Ten percent of 1 ppm sulfur dioxide has an equivalent sulfur content of 0. 13 mg/mj, and if this were present as an irritant sulfate with a particle size of 0. 3 |am, it would increase flow resistance about 45 percent. If the response to the particulate sulfate and the response to the remaining sulfur dioxide (which increased resistance about 15 percent) were purely additive, one could estimate a total increase in pulmonary flow resistance of approximately 60 percent, an important fourfold potentiation in irritant response. The response to such com- binations is more than additive, 1">'-(J however, so that the overall irri- tant effect •would be even greater than these figures indicate. These data clearly suggest that a toxicological response to sulfur dioxide gas alone is not a sufficient basis for assessing its role in air pollution. The overall problem of the effect of inert aerosols on physiolog- ical responses to irritant gases will not be discussed in detail here. It has been reviewed in the air quality criteria documents for both sul- fur oxides and particulate material, and is discussed in a recent 23 18 review by Amdur. Amdur and Underhill reported the results of a study on the effects of various aerosols on the response to sulfur dioxide gas. Their findings indicated that the degree to which the response to sulfur dioxide was potentiated by an aerosol was determined by the solubility of sulfur dioxide in the liquid droplet and by the extent to 14 TOXICOLOGY - S02 DECAY PRODUCTS ------- which it was oxidized to sulfuric acid. The importance of solubility was suggested by the fact that the potentiating ability of aerosols of sodium chloride, potassium chloride, and ammonium thiocyanate could be related, in a reasonable manner, to the solubility of sulfur dioxide in solutions of these salts. Further evidence for the role of solubility came from the observation that none of the various solid, dry aeroools that were tested potentiated the response to sulfur dioxide. These included spectrographic carbon, activated coconut charcoal, iron oxide fume, triphenyl phosphate, fly ash, and manganese dioxide. None of these substances, at concentrations of 8 mg/m or above, produced a detectable effect on pulmonary flow resistance or potentiated response to levels of sulfur dioxide ranging from 1 to 100 ppm. In contrast, aerosols of soluble salts of ferrous iron, manganese, or vanadium at concentrations of 0. 7 to 1 mg/m potentiated response to sulfur dioxide about threefold. At significant humidity levels like those in the respi- ratory tract, these substances would not only form droplets, but would also absorb sulfur dioxide; they are known to be agents that favor the oxidation of sulfur dioxide to the more potent sulfuric acid. The pro- perties of these and other sulfur compounds have been studied thoroughly by many researchers, who have found that the toxicity of the compounds is directly related to the metal ion itself rather than to the attached sulfur radical. These compounds will not be reviewed here, but a relevant bibliography is appended. Hygroscopic Properties of Sulfuric Acid Another toxicologic aspect of sulfuric acid aerosol inhalation is related to its hygroscopic nature. Even when sulfuric acid particles are inhaled in relatively dry air, they will expand within the respiratory system by water absorption because inspired ambient air becomes satu- rated with water drawn through the nose, mouth, and pharynx. Since large particles are retained with greater efficiency, more hygroscopic particles, such as sulfuric acid and water-soluble sulfate aerosols, are deposited than non-hygroscopic materials of the same size. Depending on other factors, physiologic response may, but need not, be enhanced Toxicity in Experimental Animals 15 ------- in proportion to the increase in percentage retained because of expan- sion in situ. In the case of hygroscopic aerosols, the original size of the parent particle will also determine the subsequent size of the hydrated particle, and, hence, the site of its retention along the respiratory system. Larger particles will have a tendency to be trapped along the upper respiratory airways (nasopharnygeal), where their relative toxicity is usually lessened and from which removal is more rapid. Increases in particulate size from the nares to the alveoli are difficult to quantitate in vivo; therefore, studies of the effects of hygroscopic particles have been limited largely to empirical models, in which alterations in, or potentiation of, physiologic responses are related to variations in physicochemical properties of the particulate matter; for example, size, chemical composition, solubility, catalytic converti- bility, and moisture absorbability. When interpreting physiologic responses to aerosols of a sulfurous nature, assessing their toxic potential, and relating aerosols to models of respiratory deposition, one must not lose sight of the fact that these phenomena are influenced by the hygroscopic nature of the particles. Conclusions The studies discussed above cannot be extrapolated simply and directly to predict the human response to particular substances. They do provide useful information regarding the nature of mechanisms by which effects are produced; however, they may lend more insight into responses of the sensitive segment rather than of the robust healthy segment of the population. For practical considerations in using these experiments as indi- rect guidance in air pollution research, two critical points emerge. The first is the extreme importance of the particle size to the irritant potency of particulate products of sulfur dioxide. The most important size is in the submicron range. Data on mass concentration alone pro- vide an insufficient basis for predicting irritant potency of particulate products of sulfur dioxide. 16 TOXICOLOGY - S02 DECAY PRODUCTS ------- The second important point is that the particulate oxidation pro- ducts of sulfur dioxide generally have much greater irritant potency than the parent sulfur dioxide gas, as indicated by the comparative data in Figure 3 as well as by increased response to sulfur dioxide in the presence of particulate material known to dissolve the gas and/ or catalyze its conversion to sulfuric acid or sulfates. Particle sizes of oxidative products of sulfur dioxide are, in part, affected by the hygroscopic properties of the products; consequently, physiologic responses and pulmonary deposition of inhaled particulates may be significantly altered by moisture in the atmosphere and within the respiratory tract. Atmospheric levels of sulfur dioxide and their health implications should therefore be assessed in terms of the potential for formation of more irritant substances. To do otherwise is to miss the toxicological point and seriously becloud important Toxicity in Experimental Animals 17 ------- TOXICITY IN MAN Only limited research has been done on the effects of sulfuric acid on human pulmonary function, and none on the effects on man of the various sulfates that have irritant potency for the guinea pig. Studies that regard both physiological methodology and characterization of the aerosol particle size with meticulous attention are badly needed. The exposure of human volunteers to low concentrations of H2SC>4 mist, to define its toxicological effects, has involved two divergent, though complementary, branches of research. One branch has concerned itself with the effects of H^SCJj aerosols upon the normal physiological responses of the central nervous system: the role of H2SO4 mist and its interrelationship with sensory modalities and cerebral cortical function. These studies are reported exclusively in the Russian litera- ture. The other branch of research has been concerned primarily with the irritative effects of H2SO4 aerosols and the changes in respiratory function that occur subsequent to exposure to concentrations that approximate the allowable industrial standards set by the governments of the United States24'25 and the Union of Soviet Socialist Republics. 26 STUDIES OF SUBJECTIVE AND PHYSIOLOGICAL RESPONSES TO IRRITATIVE PROPERTIES OF SULFURIC ACID MIST Detection by Subjects 27 In 1913, Dorsch studied workers in the battery room of a German telephone exchange. He found that H2SO4 mist was only rarely detected by the employees when the concentration was below 0.5 mg/m3. They complained of slight nose and throat irritation at 2. 0 mg/m3, distinct discomfort at 3 to 4 mg/m3, and marked discom- ? Q fort when the concentration rose to 6. 8 mg H2SO4/m3 . Bushtueva exposed ten volunteers to H2SO4 aerosols with the following results. When exposed to concentrations less than 1. 1 mg/m3, the volunteers compained of ticklish, scratchy throats; when exposed to levels rang- ing from 1. 1 to 2. 4 mg/m3, all subjects noticed considerable 19 ------- irritation at the base of the esophagus and 40 percent of them experi- enced some irritation of the eye mucosa. When the aerosol level was raised to 2. 4 to 6. 0 mg/m3, all subjects experienced acute irritation of the mucous membranes, a pronounced reflex cough, and irritation of the eye mucosa. The threshold concentration was approximately 0. 7 mg/m3 (0. 60 to 0. 85 mg/m3). In that study, the particle size of the acid mist was not stated. Amdur2° was unable to detect responses in subjects exposed to aerosol levels less than 1 mg/m3, using odor, taste, or irritancy as parameters, although all 15 of her subjects detected the aerosol when the level was raised to 3 mg/m , noting that it "felt like breathing dusty air. " Some subjects found 5 mg/m3 very objectionable, but others less so, and, for all subjects, a deep breath of this concentration usually produced coughing. In another study, Bushtueva3^ found that the presence of 0. 4 mg H2SO4/m3. even when combined with 1 mg SO2/m , was below the sensitivity threshold of all subjects. The fact that the results reported in each of these papers are remarkably similar to Dorsch's^^ original description of H2SO4 mist irritancy, indicates that the dose-response curve for human beings has remained quite stable during the past 60 years of experimentation and, in contrast to animal data, appears to be physiologically predictable in almost all instances. Effects of Humidity, Acidity, and Particle Size on Subject Response Sim studied the response of healthy males, aged 18 to 46, to r^SO,^ mist. He noted that at a given concentration a mist of larger particle size and lower acidity, produced under conditions of high humidity, was more irritant than a mist produced under lower humid- ity at the same concentration, but with smaller particles and higher acidity. Pulmonary air-flow resistance was 35 to 100 percent greater than normal when the volunteers were exposed to a ION H2SC>4 aerosol of 1-p.m-MMD particles at a concentration of 39 mg H2SC>4/m , a temperature of 18.4° C, and humidity of 61 percent. Altering the aerosol to 25° C, 91 percent humidity, acidity of 4N H2SO4, and particle size of 1. 5 (xm evoked intense coughing, lacrimation, and 20 TOXICOLOGY - SO, DECAY PRODUCTS ------- rhinorrhea, which continued throughout the exposure. Coughing decreased somewhat after the first 10 minutes of exposure and airway resistance ranged from 43 to 150 percent greater than normal. Adding magnesium oxide particulate to the exposure air did not alter the irri- tancy, although addition of ammonia toward the end of the H2SC>4 exposure resulted in rapid and complete disappearance of all symptoms, thus corroborating Pattle's5 observations on guinea pigs. Toyama33 also correlated airway resistance with particle size. In an aerosol of 4. 6-(im particles, a. mist concentration of 0. 8 to 1.4 mg/m3 increased airway resistance 36. 5 percent in 24 healthy males; as an aerosol of 1. 8-|_im particles, a mist concentration of 0. 01 to 0. 1 mg/m3 increased airway resistance 17. 9 percent. The latter concentrations appear questionable, since 0.01 to 0. 1 mg H^SO^/m are concentrations far below the reported thresholds for any physiological response. Hamilton32 stated in 1948 that the site of action for H2SO4 fumes is the upper respiratory tract, "that this action is not severe, and that no instance of bronchitis [is]. . .traceable to such fumes. " Sim3-! described, however, how he, himself, became increasingly sensitive to the mist during 10 months of exposure, developing a moderately severe but extremely persistent bronchitis, which immediately exacerbated into uncomfortable periods of coughing and wheezing that occurred whenever he was exposed to H2SC>4 mist. Effects of Sulfuric Acid Mist on Respiratory Retention Apparent increases in the respiratory rate of human subjects exposed to H2SO4 irritant were described in reports by Bushtueva, 2° Sim, 31 and Amdur. The increase is not suprising, for Wilson34 in 1948 showed that the retention of inhaled aerosol particles decreased as the respiratory rate increased. Morando3^ briefly (for 15 minutes) exposed normal human subjects not accustomed to inhaling HzSO4 fumes to concentrations varying from 0. 35 to 5. 0 mg/m . Even at concentrations not subjectively detected by the volunteers (less than 0.5 to 0. 7 mg/m3), the respiratory rate increased rapidly and remained Toxicity in Man 21 ------- elevated for several minutes after exposure. Amdur^9 exposed fifteen subjects to 0.35 to 0. 5 mg/m3 and noted that the respiratory rate increased about 30 percent above control values; the maximum inspira- tory and expiratory flow rates decreased by about 20 percent; and tidal volume increased by about 28 percent. These changes occurred during the first 3 minutes of exposure and were maintained throughout the 15- minute exposure period. Values returned to pre -exposure levels within 15 minutes after the exposure ended. During the first minute after expo- sure, the tidal volume rose above and then declined to control levels. These experiments do not provide the physiological evidence necessary to indicate conclusively that bronchoconstriction was the response to H2SC>4 mist, although, the data suggest that this was the case. 2R Bushtueva found clear-cut reflex changes in respiratory rhythm and amplitude when she gave volunteers multiple exposures to an of 1.8 to 2. 0 mg/m3. At concentrations of 1. 0 to 1. 1 mg/m , changes in respiration were also noted, though rhythm and amplitude were decidely lower. She was unable to demonstrate respiratory changes when the mist concentration was below 1. 0 mg/m3, but this inability may have reflected differences in the degree of "aerosol dispersion. " As the respiratory rate increases, the percentage of H2SO4 mist retained by the respiratory tract is definitely reduced. The change in respiratory rhythm and amplitude may be a reflex protective mechanism that prevents the retention of the irritant, ^9, 34 thereby decreasing its toxic effects. By measuring the total volume of air breathed, and the concentration of the H2SO4 aerosol in the mixing chamber and in ex- haled air, Amdur^° was able to show that average retention of inhaled H2S<-)4 varied from 50 to 87 percent with exposure concentrations of 0.4 to 1.0 mg/m . She gave two possible reasons for such relatively high retention. First, the aerosol levels used in the experiment in- creased the respiratory rate by only 35 percent (5 to 7 breaths per minute), certainly not enough to reduce retention far below baseline values; and, second, the strong affinity of H2SC>4 droplets for moist surfaces, such as the mucous membranes of the respiratory tract, may have enhanced retention. 22 TOXICOLOGY - SO? DECAY PRODUCTS ------- As noted earlier, the respiratory rate and pulmonary air-flow resistance of human volunteers rose rapidly as exposure concentrations of H2SO4 aerosol were increased. Symptomatically, bronchospasm was also evident in the healthy subjects, presenting a kaleidoscopic picture of wheezing, increased secretions, dyspnea, spasmodic cough- ing, and occasional chest pains. The intensity and combinations of the symptoms depended upon the aerosol concentration and length of exposure. Two observer-subjects reported occurrence of chronic bronchitis during long-term intermittent exposure to H2SC>4 aerosol. Bronchospasm, increased upper-respiratory-tract secretions, and a rapid respiratory rate were consistently found, along with eleva- ted pulmonary air-flow resistance, in volunteers exposed to mist levels greater than 0. 35 to 0. 5 mg/m . All of the subjects involved in pre- vious experiments were young healthy adults who could easily compen- sate for the increased resistance imposed on their breathing. Elevated airway resistance severely embarrasses breathing in individuals already burdened by cardiorespiratory deficiencies, however, making it more difficult to obtain needed oxygen. Moreover, bronchospasms and increases in secretory and respiratory rates impair gaseous exchange in the lungs, and carbon dioxide accumulates, which causes respiratory acidosis. An acidotic condition superimposed on a restricted oxygen intake explains many of the effects seen in pulmonary cripples when they are exposed to rapidly rising concentrations of t^SO^ aerosols, above threshold levels. Conclusions Research data concerning the effects of aerosol particle size, temperature, or humidity on the human response to H2SC>4 mist exposure are scarce. The few long-term human-exposure studies have been limited, essentially, to epidemiological surveys. Ethical con- siderations preclude achieving the requirements of controlled long- term, experimental, human exposure as such. Intensive industrial- exposure investigations, rather than controlled laboratory experiments, may then be the only acceptable source of needed information. Chronic Toxicity in Man 23 ------- bronchitis did develop over the course of at least one long-term study, however, and various case histories have reported the development of acute and/or chronic respiratory disease after patients endured short- or long-term exposure to H2SO4 aeros.ols. ^°> ' Definitive data are lacking, but exposure to low concentrations of sulfuric acid appears to increase pulmonary flow resistance in human subjects. Determining the minimum concentration at which such changes might be expected to occur, which is possible for SO2. is impossible for H2SO4- Unfortunately, most experiments on the general complex of sulfur -containing pollutants have dealt with the less toxic sulfur dioxide. Additional work, to be most meaningful, must be con- ducted by a design that incorporates the refinements of both physiolog- ical methodology and aerosol technology. A failure in either of these areas would severely limit the value of the data. STUDIES OF SENSORY AND CENTRAL NERVOUS SYSTEM RESPONSES TO SULFURIC ACID MIST In the U. S. S. R. , air pollutant limits have been rigidly set by the standard, "that the ambient air should not contain odors to be imposed on the population against its wishes". ..." With this standard as a guideline, the Russians in the middle fifties conducted studies to limit SC>2 and H2SC>4 in the air to acceptable levels. The limits finally selected were based on the subjective reactions of human volunteers exposed to either SC>2 or H2SO4- The perception threshold for odor and mucosal irritation was about 1. 5 mg SO2/m3 and about 0. 5 mg . Subsequent to these studies, in 1957, the Russian govern- ment established the allowable single concentrations of SC>2 and at 0. 5 and 0. 3 mg/m3, respectively levels one-half to one-third the threshold values. *26, 40 #The term threshold concentration applies to that level of H SO aerosol or SC>2 gas that is just perceptible as either odor or irritation. This concentration lies between 0. 6 to 0. 85 mg/m for the aerosol, and between 1. 6 to 2. 8 mg/m3 for the gas. 26> 28> 30> 40> 41 Subthres- hold implies that the level of exposure cannot be detected either by odor or by irritation. 24 TOXICOLOGY - S02 DECAY PRODUCTS ------- Between 1956 and 1962, Russian scientists attempted to define that level of SO2 and/or H2SO4 that could trigger responses in specific receptors of the respiratory system (including the nose). They exposed subjects simultaneously to subthreshold concentrations of SO2 and H2SC>4 in an effort to detect responses in the central nervous system (brain). The respiratory receptors, particularly those in the olfactory area, have a well-recognized sensitivity to the action of minimal concentra- tions of chemical substances that is partially explained by the fact that they are located where the outside air initially comes in direct contact with the internal organism. The varied responses to H2SC>4 exposure discussed in this section, although not strictly within the realm of classical toxicology, are included because the data are closely related to the biological considera- tions of this review. The practical ramifications and toxicological implications of the subsequent neurophysiological responses have not been adequately explored. In addition, one must exercise caution in interpreting such data, since no replicative studies have been made to confirm the reported neurophysiological responses, and informative descriptions of experimental conditions have been less than adequate. Optical Chronaxy Alterations in optical chronaxy* following stimulation of the respiratory receptors were examined extensively in hopes of finding the allowable limits of atmospheric pollutants. No change in optical chronaxy was noted when the legal maximum concentrations of SO2 and/or H2SC>4 aerosol were administered to volunteers (for H2SO4, *Weak electrical current applied to the eyeball that produces the sensa- tion of a flash of light. Each subject has a maximum intensity of stim- ulation below which he does not perceive light. The intensity thres- hold, expressed in units of electrical potential (i. e. , volts) is called the rheobase. Chronaxy is the electrical current times twice the intensity that produces the rheobase. A stimulating current of two rheobases causes light sensation only when it is sufficiently prolonged. This value, that is, the time threshold necessary for the appearance of light sensation, is called optical chronaxy.2° Toxicity in Man 25 ------- 0.3 mg/m ; for SC>2, 0.5 mg/m3); nor when subthreshold concentra- tions were administered (for t^SC^, 0. 6 mg/m3; for SC>2, 1.2 mg/m3) (Table 1). 30 Exposure to levels of SC>2 or H2SO4 beyond threshold consistently elicited increases in the optical chronaxy. When the two irritants were combined, such that the concentration of both was equal to or greater than the subthreshold concentration, the optical chronaxy value indicated an additive or synergistic depression of central nervous system excitation. These data were not sufficient to determine whether the primary response came from the optic cortex itself, or whether a response in the olfactory zone caused a secondary inhibitory effect in the visual cortex, for no changes were detected until concentrations exceeded the odor threshold. Ryazanov has postulated the latter mechanism as a possible explanation for the increased optical chronaxy effect. Table 1. OPTICAL CHRONAXY ON NINTH MINUTE OF EXPOSURE OF ONE SUBJECT TO TEST ATMOSPHERES3' 3° Test gas Fresh air so2 so2 H2S04 H2S04 S02 and H2S04 SO- and H2S04 S02 and H2S04 S09 and H0SO,, 2 24 Concentration, mg/m - 1.5 to 1.8 1.0 to 1.2 0.73 to 0.75 0.60 to 0.63 1.2 and 0.6 0.8 and 0.6 1.2 and 0.4 1.5 and 0.75 Average optical chronaxy, yF 0.61 ± 0.0037 0.80 t 0.0015 0.62 ± 0.0070 0.70 + 0.0090 0.62 + 0.0058 0.71 ± 0.0120 0.71 1 0.0120 (sic) 0.61 t 0.0100 0.98 + 0.0280 Results of tests on one subject, Madame B. Dark Adaptation Information based on optical chronaxy was substantiated by studies of dark adaptation. The Hagel adaptometer, an instrument that meas- ures changes in light sensitivity of the visual organ while the subject is in darkness, was used, and test subjects were exposed to several con- centrations of SC>2 and/or H2SO4 aerosol. The average sensitivity of subjects to light was computed from a curve illustrating changes in dark 26 TOXICOLOGY - SO? DECAY PRODUCTS ------- adaptation with time. The raw data (in relative units) in Table 23° show a marked similarity to the data obtained with optical chronaxy techniques. Subthreshold concentrations had no effect on dark adapta- tion, but levels exceeding threshold reduced adaptation activity in the visual cortex. ' ' According to Ryazanov, 2 this effect can also be explained as the result of a primary reflexive response in the olfactory zone that leads to a short period of optical cortex inhibition, which is followed by another, prolonged period of depressed activity in the optical cortex secondary to the influence of reticular formation.26'43 The second component of this diphasic response was noted in some subjects when their light sensitivity was found markedly depressed 50 to 60 minutes after the dark-adaptation curves had shown post-exposure return toward normal. Table 2. EYE SENSITIVITY TO LIGHT IN THREE SUBJECTS DURING TWENTIETH MINUTE OF DARK ADAPTATION30 Test no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Concentration of gas added, mg/m3 S02 H2S04 Madame D 0 0.65 3.0 0 3.0 0 0.3 0 0.7 0.7 Madame P 0 0.65 3.0 0 3.0 0 0.3 0 0.7 0.7 Madame V 0 0.65 3.0 0 3.0 0 0.3 0 0.7 0.7 Average light relative 33,400 + 1, 33,200 ± 1, 41,100 + 1, 42,400 t 54,600 t 1, 54,300 1 2, 55,500 ± 1, 69,300 + 5, 62,850 + 3, 82,750 t 1, 42,133 + 41,767 + 56,200 t 3, 54,700 t 1, 68,967 t 3, sensitivity, units 153 841 570 0 (sic) 200 857 385 100 268 432 972 546 377 570 050 Toxicity in Man 27 ------- Continuous Electroencephalography The Russian scientists next turned to continuous monitoring of the cerebral cortex by electroencephalography (EEC), directly record- ing the state of central nervous system function during exposure to SO2 40 43 44 and/or H2SO4 aerosols. ' ' Mucous membrane irritation, secondary to the sulfur irritation, innervates the trigeminal nerve, while odor perception results from direct excitation of the olfactory receptors. Stimulation of any receptor organs is followed by alpha- rhythm (low-frequency, high-amplitude EEC signal) suppression or desynchronization. Individuals with well-defined alpha rhythms were selected as subjects and the criterion of response was defined as the onset of electrical desynchronization, or alpha-rhythm suppression. Exposing subjects to subthreshold concentrations elicited no electro- encephalographic changes, nor did application of both SC>2 and H2SO4 at maximal allowable concentrations elicit alpha-rhythm desynchroni- zation, at either beginning or end of exposure. Desynchronization was observed only when threshold doses of H2SC>4 mist and/or SC>2 were used; then it lasted for 1 to 2 seconds. The H2SO4 aerosol changed alpha rhythms only when the concentration was sufficient to be noted subjectively by the volunteers. Subthreshold concentrations, not subjectively perceived, did not appear to alter the central nervous system signals, and, at higher concentrations, the crude subjective indices - odor perception and mucosal irritation - apparently correlated well with the onset of alterations in the central nervous system. Sub- jective evidence of cortical response was found only when the concen- trations were sufficient to affect the course of the other reflex effects (i. e. , optical chronaxy and dark adaptation), so that subjective responses apparently were valid as objective indices of the cortical effects of the irritants. Conditioned Reflexes An additional physiological concept is referred to in the Russian literature, although apparently not discussed at all in American toxico- 45 4.1 logical studies. According to Gershuni and Dubrovskaya, cortical 28 TOXICOLOGY - SO? DECAY PRODUCTS ------- reactions may occur upon exposure to certain subthreshold (subsensory) excitations. This may play an essential role in toxicological and health effects. If cortical reactions to subthreshold levels do occur, one must conclude that to be acceptable ambient pollutant levels must not only be below the level of odor perception and irritancy but also below the threshold of subconscious reflex reactions, particularly those capable of altering the functional state of the cerebrum. The electrocortical conditioned reflex is a central nervous system phenom- enon elicited only after a succession of repeated, conditioned reflex trials. As indicated previously, temporary desynchronization of the alpha rhythm occurs each time a subject is given a strong light signal. When light is excluded, the alpha rhythm is restored. Experimentally, a. system is established by which a subject inhales, for 10 seconds, a known subthreshold concentration of SO2 gas and/or H2SO4 aerosol that by itself does not cause the alpha rhythm to desynchronize. He then receives a light over a 5- to 10-second interval. After repeating this combination (irritant plus light) several or many times, desynchro- nization begins to appear before the light; that is, apparently in response to the unperceived odor. The unperceived odor thus appears to become the conditioning stimulus, generating the so-called condi- tioned electrocortical reflex. " Through this reflex, EEG alpha-wave desynchronization occurred when individuals were exposed to an H£SO4 aerosol concentration of 0. 4 mg/m , an SC>2 concentration of 0. 6 mg/m , or to a combination of either 0. 15 mg H2SC>4/m plus 0. 5 mg SO2/m3, or 0. 3 mg H2SO4/m3 plus 0. 25 mg SC>2/m3. 40' 43> 44 All of these concentrations are below the subjective sensory threshold. With respect to H2SO4, the conditioned reflex was usually developed in the course of 19 to 23 trials with H2SO4 aerosol plus light (Table 3). Remarkable as it may seem, the complex neurological process result- ing in disruption of cortical rhythm was initiated by a subjectively non- perceivable odor. ° Conclusions The Russian data are concerned wholly with the effects of H2SO4 aerosols upon the interrelationship between sensory modalities and Toxicity in Man 29 ------- Table 3. NUMBER OF TRIALS WITH ^04 PLUS LIGHT REQUIRED FOR APPEARANCE OF CONDITIONED REFLEX40 Subject NN GV SN ML H2S04 concentration, mg/m3 0.6 to 0.75 8 16 12 6 0.4 23 - 21 19 cerebral cortical function. Those concentrations of H2SO4 mist that produce subjective sensations (odor and mucous membrane irritation) were invariably associated with objective evidence of central nervous system depression. Subjective, subthreshold, acid mist concentra- tions were not associated with depression of either optical chronaxy, dark adaptation, or electroencephalographic patterns. Only when the electrocortical conditioned reflex technique was employed did sub- threshold levels result in detectable central nervous system changes. This is evidence that a chemical stimulator, unable to initiate an electroencephalographic response when used in subthreshold doses by itself, can stimulate the central nervous system and change the irri- tability of cortical cells of the optical and other analytic or cognitive 40 centers when it is used as a conditioning stimulus. Table 4 sum- marizes the sensory-neurologic dose-response data just described. Table 4. THRESHOLD AEROSOL DOSES REQUIRED FOR SENSORY RESPONSE44 (mg/m3) 44 Threshold dose Response Perception of odor and irritation of mucosa Suppression of dark adaptation Elevation of optical chronaxy Disruption of alpha rhythms Conditioning of electro- cortical reflex H2S04 0.6 to 0.85 0.63 to 0.73 0.73 0.63 0.4 S02 1.6 to 2.8 0.92 1.5 0.9 0.6 H2S04 + S02 0.3 + 0.5 0.3 + 0.5 0.6 + 1.2 0.3 + 0.5 0.15 + 0.5 or 0.3 + 0.25 30 TOXICOLOGY - S02 DECAY PRODUCTS ------- SUMMARY Studies of atmospheric chemistry have shown that under certain conditions sulfur dioxide can be converted to sulfuric acid and particu- late sulfates. Air quality criteria and standards for sulfur dioxide should, therefore, take into account the irritant potency of these oxidation products, recognizing that both sulfur dioxide and its degradation products can react further with other air pollutant consti- tuents. Additional aerometric and toxicologic research is needed to supplement what little is known of the actual existence in the atmosphere of this group of compounds and of their toxicology. TOXICITY IN EXPERIMENTAL ANIMALS Sulfuric acid is a respiratory irritant that in sufficient concentra- tion will cause the death of exposed animals. The guinea pig is the most sensitive species among the common small laboratory animals, followed in decreasing order by mice, rats, and rabbits. The greater sensitivity of the guinea pig results from its greater susceptibility to bronchial and laryngeal spasm, a characteristic that makes the animal useful for some studies of the irritant potential of low concentrations of sulfuric acid, but that makes extrapolation of the results obtained of questionable value in predicting the health effects of sulfuric acid air pollution in man. Experiments to date have shown that concentration is a more important factor than duration of exposure in producing death in animals. Sulfuric acid also produces pathological changes in lungs. The degree of lung damage observed after effective, short exposures (up to 72 hours) is related to the mathematical product of concentration and time (CT) rather than to concentration alone. Sulfuric acid damage is repaired slowly. Concentrations below 2 mg/m3 produce only minor luhg damage after extended exposures (about 3 months), which indicates 31 ------- that the CT relationship observed in 8- and 72-hour exposures cannot be extrapolated directly to long-term effects of low concentrations. The particle size of sulfuric acid mist seems to be another impor- tant factor in determining its irritant potency. When the" potency crite- rion was mortality resulting from an 8-hour exposure, a mist of larger particles (2. 7 |J.m versus 0. 8 |J.m) was more toxic. When pathological lung damage resulting from long-term exposures was the criterion, the effect of 0. 9-(J.m particles was greater than that of either 0. 6- or 9-|J.m particles. Studies of pulmonary function in animals exposed to these sub- stances cannot be extrapolated simply and directly to predict the human response. They are, however, providing useful information regarding the nature of mechanisms by which effects are produced, and may yet lend insight into responses that are more analogous to the responses of sensitive individuals in the population than.of healthy. For practical considerations, these experiments present two principles for indirect guidance in the area of air pollution research. First, because particle size plays an important role in determining irritant potency of sulfur oxide particulates (submicron particles being more irritant), data on mass concentration alone are insufficient for predicting irritant potency. The second important point is that the particulate oxidation products of sulfur dioxide have a generally much greater irritant potency than sulfur dioxide gas per se. This is indicated by comparative data as well as by increases in response to sulfur dioxide in the presence of particulate materials that are known to dissolve the gas and/or to cat- alyze its conversion to sulfuric acid or sulfates. Along with factors such as the original particle size, presence of other materials in the inhaled air, the subject's breathing pattern, and the site of particle deposition, the hygroscopic nature of sulfuric acid and certain sulfate aerosols affects physiologic responses to their inhalation. Atmospheric levels of sulfur dioxide and the implication .of these levels for health should thus be assessed in terms of their potential for forming more irritant substances. To do otherwise would be to miss the toxicological point and seriously becloud important issues. 32 TOXICOLOGY - S02 DECAY PRODUCTS ------- TOXICITY IN MAN The combined effects of particle size and concentration of mist on exposed human subjects have still not been determined. The effects of temperature and humidity on the response in humans toH2SO4 have not been satisfactorily examined either. Long-term human expo- sure information is, by necessity, limited to epidemiological surveys. Since controlled long-term experimental exposure of humans cannot ethically be performed, industrial and episodic exposures are the only sources of needed information and should be investigated. One long- term study and various case histories have reported the development of acute and/or chronic respiratory disease following short- and long-term exposure to 1^230^ aerosol. Although definitive data are lacking, animal studies indicate that sulfuric acid should increase pulmonary flow resistance in human subjects. The concentration at which such changes might be expected to occur is, however, impossible to determine. Regrettably, the bulk of the experiments relating to the general complex of sulfur-containing pollutants has dealt with the less irritating sulfur dioxide. More research should now be directed to the other components of the complex. Russian scientists have been almost entirely concerned with the effects of H2SO4 aerosols upon sensory modalities, cerebral cortical function, and their interrelationships. Concentrations of H2SO4 mist that resulted in subjective sensory stimulation (odor and mucous mem- brane irritation) were found to be associated invariably with objective evidence of central nervous system depression. Subjectively deter- mined subthreshold concentrations of acid mist were not associated with depression of either optical chronaxy, dark adaptation, or electro- encephalographic alterations. Only when the electrocortical conditioned reflex was used did sensory subthreshold levels produce central nervous system stimulation. This has been taken as evidence that, when used as a conditioning stimulus, a chemical stimulator that is unable to initiate an electroencephalographic response when used in subthreshold doses by itself can stimulate the central nervous system and change the irritability of cortical cells of the optical and other analytic or cognitive centers. Summary 33 ------- REFERENCES 1. Treon, J. F. et al. Toxicity of Sulfuric Acid Mist. A.M. A. Arch. Ind. Hyg. Occup. Med. 2^:716-734, July-December 1950. 2. Mathur, K. and P. Olmstread. Inhalation Toxicity of Sulfuric Acid Mist: Animal Experiment Study of the Problem. 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Acad. of Science, U.S.S.R. 2:98, 1945. 38 TOXICOLOGY - S02 DECAY PRODUCTS ------- BIBLIOGRAPHY: SELECTED TOPICAL REFERENCES BARIUM SULFATE 1. Cember, H. Bronchogenic Carcinoma from Radioactive Barium. AM A Arch. Ind. Health. r7_:230-235, March 1958. 2. Cember, H. e_t al. Elimination of Radioactive Barium Sulfate Particles from the Lung. AM A Arch. Ind. Health. 13:170-177. February 1956. 3. Cember, H. , J. A. Watson, and M. E. Novak. The Influence of Radioactivity and Lung Burden on the Pulmonary Clearance Rate of Barium Sulfate. Am. Ind. Hyg. Assoc. J. 22:27-32, February 1961. 4. Cember, H. et al. Pulmonary Effects from Radioactive Barium Sulfate Dust. AMA Arch. Ind. Health. j_2_:628-634, December 1955. 5. Clement, J. G. Inhalation Bronchography Method and Results in the Experimental Animal. J. Canad. Assoc. Radiol. 20; 106- 113, June 1969. 6. Johnson, T. H. , Jr. , and W. J. Howland. Aerosol Bronchography. A Preliminary Report. Amer. J. Roentgen. 104:787-791, December 1968. 7. LaBelle, C. W. and H. Brieger. Synergistic Effects of Aerosols. II. Effects on Rate of Clearance from the Lung. AMA Arch. Ind. Health. 2£: 100-105, August 1959. 8. Lacroix, Giuseppe. Fourfold Mortality from Commercial Barium Sulfate Intoxication. Sammlung v. Vergiftungsf. 13:63-64, July 1943. 9. Nice, C. M. , Jr. , and W. W. Waring. Bronchography in Infants and Children; Barium Sulfate as a Contrast Agent. Am. J. Roentgen. 2^:564-570, March 1964. 10. Pendergrass, E. P. , and R. R. Greening. Baritosis; Report of a Case. AMA Arch. Ind. Hyg. Occup. Med. 7^44-48, January- June 1953. BARIUM SULFONATE 1. Dooley, A. E. Toxicity of Petroleum Product Additive. Arch. Environ. Health. 6:324-328, March 1963. 39 ------- MAGNESIUM SULFATE 1. Sutton, W. R. and V. E. Nelson. Blood-Sugar Changes in the Rat Produced by Salts of Beryllium, Magnesium and Zinc, with Some Observations on Haemoglobin and Red Blood Corpuscles. Proc. Iowa Acad. Sci. 45:115-121, 1938. MANGANESE SULFATE 1. Shelley, W. B. e_t §L Intradermal Tests with Metals and Other Inorganic Elements in Sarcoidosis and Anthraco-Silicosis. J. Invest. Dermatol. 3^:301-303, July 1958. 2. Voigt, G. E. and T. Saldeen. On the Protective Effects of Zinc Against Magnesium Sulfate or Carbon Tetrachloride Poisoning. Frankf. z. Path. 74:572, 1965. (Abstract) In: Food and Cosmetics Toxicol~4:541-542, No. 1100, October 1966. SULFATES 1. Chien, L. , H. Robertson, and J. W. Gerrard. Infantile Gastroen- teritis Due to Water with High Sulfate Content. Canad. Med. Assoc. J. 92:102-104, July 1968. 2. Colucci, J. M. and C. R. Begeman. Carcinogenic Air Pollutants in Relation to Automotive Traffic in New York. Air Pollution Control Assoc. 61 st Annual Meeting, St. Paul, Minnesota, June 1968. Paper No. 68-71. 3. Commins, B. T. and R. E. Waller. Observations from a Ten- Year Study of Pollution at a Site in the City of London. Atmospheric Environment. l_:49-68, January 1967. 4. Domzalska, E. and K. Grabowska. The Influence of Chemical Factors on the Oral Cavity in Workers of the Szczecin Plant of Phosphorous Fertilizers, in the Light of Our Own Investigations. Czas. Stomat. (Poland), 21_:1081-1087, September 1968. 5. Forck, G. Frequency and Persistence of Persulfate Allergies. Berufsdermatosen. 1_6: 84-92, April 1968. 6. Gaultier, M. , P. Gervais, and F. Mellerio. Two Causes of Occupational Asthma in Hairdressers: Persulfate and Silk. Arch. Mai. Prof. (France). £7:809-813, October-November 1966. 7. Gottschalk, R. G. and H. N. Beers. Selective Toxicity of Radio- active Sulfate for Mouse Cartilage and Bone Marrow. AMA Arch. Pathol. 65:298-311, March 1958. 8. Gotz, H. and N. Kluken. On the Effect of Air Pollution on the Skin. Studies with Emission Substances in Industrial Areas. Med. Wschr. (Munchen). 111:1021-1027, May 1969. 40 TOXICOLOGY - SO, DECAY PRODUCTS ------- 9. Greenblatt, M. , C. Raha, and C. Roe. Bimethylnitrosamine and Hydrazine Sulfate. An Analysis of Combined Toxicity and Pathology in Mice. Arch. Environ. Health. 17:315-320, September 1968. 10. Malpartida, M. and F. Moncloa. Radiosulfate Space in Humans at High Altitude. Proc. Soc. Exp. Biol. Med. 125:1328-1330, August-September 1967. 11. Meindl, K. and R. Meyer. Asthma and Urticaria in Hairdresser Occupation Due to Persulfate-Containing Bleaching Agents. Zbl. Arbeitsmed. 19:15-79, March 1969. 12. Muto, J. Ozena and Environmental Factors. HNO (Germany). ljx71-73, March 1968. (sic) 13. Perin, G. and G. Rausa. Atmospheric Pollution in the City of Bolzano. I. Results of the First Year of Observations. Ann. Sanit. Pubblica (Italy). ^:627-678, May-June 1967. 14. Schutte, K. H. Esophageal Tumors in Sheep: Some Ecological Observations. J. Nat. Cancer Inst. 41_:821-824, September 1968. 15. Slonim, A. R. et al. Potable Water Standards for Aerospace Systems 1967. Aerospace Med. _38:789-799, August 1967. 16. Thiess, A. M. and P. J. Goldmann. Industrial Medical Problems in Connection with Dimethlsulfate Poisoning. Observations over 30 years in the Basf. Zbl. Arbeitsmed. J_8:195-204, July 1968. 17. Voropaev, A. A. Major Problems of Industrial Hygiene in the Production of Construction Gypsum (Hygienic Clinical and Experi- mental). Gig. Tr. Prof. Zabol. (Russia). _H_:23-26, October 1967. SULHTES 1. Dwornicka, B. and A. Jasienska. Changes in the Upper Respira- tory Tract in Tanning Worker s. Med. Pracy. (Poland). 17(4):357- 359, 1966. 2. Fitzhugh, G. O. , L. F. Knudsen, and A. A. Nelson. The Chronic Toxicity of Sulfites. J. Pharmacol. 86_:37-48, January 1946. 3. Locked;, M. F. and I. L. Natoff. A Study of the Toxicity of Sulphite. I. J. Pharm. Pharmacol. l_2:488-496, August I960. 4. Nater, J. P Allergic Contact Dermatitis Caused by Potassium Metabisulfite. Dermatologica (Basel). ^36_(6):477-478, 1968. 5. Recent Developments in the Sulphite Field. Food and Cosmetics Toxicol. 4_: 187-189, April 1966. 6. Recommended Methods for the Analysis of Trade Effluents. Methods for the Determination of Residual Chlorine, Cyanides and Thiocyanate, Fluoride, Formaldehyde, and Sulphite and Thiosul- phate. Analyst 83_: 230-241, April 1958. Bibliography 41 ------- SULFON1C ACIDS 1. Armeli, G. Occupational Diseases of Workers Employed in the Production of Aminoaphthodisulfonic Acid. Med. Lavoro. (Italy). 52:366-369, May 1968. 2. Fregly, M. J. and L. B. Kier. Effect of Some Substituted Sulfamic Acid Compounds on Development of Renal Hypertension in Rats. Toxicol. Appl. Pharmacol. 2:124-138> July 1966. 3. Oba, K. and J. Tamura. Acute Toxicity of n-x-Olefin Sulfonates. Agr. Biol. Chem. (Tokyo). 3^:1509-1510, December 1967. SULFUR 1. Disney, G. W. and J. T. Smith. Sulfur Metabolism and Malathion Toxicity. Fed. Proc. 26_:427, No. 957, March-April 1967. 2. Horton, A. W. et §1. Carcinogenesis of the Skin. III. The Contri- bution of Elemental Sulfur and of Organic Sulfur Compounds. Cancer Research. 25:1759, November 1965. SULFUR COMPOUNDS 1. Feiner, B. , W. J. Burke, and S. Moskowitz. Estimation of Sul- fur Compounds in Air. New York State Dept. Labor, Monthly Rev. 2_6_(2), February 1947. 2. Imai, M. , H. Oshima, and Y. Takatsuka. On the Yokkaichi- Asthma. Jap. J. Hyg. 2_2_:323-335, June 1967. 3. Johnstone, H. F. Technical Aspects of the Los Angeles Smog Problem. J. Ind. Hyg. Toxicol. 3^:358-369, November 1948. 4. Lodge, J. P. , Jr. , ed. Atmospheric Chemistry of Chlorine and Sulfur Compounds; Proceedings of a Symposium, Cincinnati, Ohio. Geophysical Monograph No. 3; NAS-NRC Publication No. 652. 1959. 5. Pirila, V. Skin Allergy to Simple Gaseous Sulphur Compounds. Acta Allergol. 7_(4): 397-402, 1954. 6. Stokinger, Herbert E. Protective Mechanisms and Tolerance Development Among Air Pollutants. Proc. Thirteenth International Congress on Occupational Health. New York, Book Craftsman Associates, Inc., 1961. 7. Young, L. and G. A. Maw. 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