THE FATHGLGGY OF SILO GAS ‘EQXICQSES {N Ple Thesis fab {in 0mm cg: M. S. MECEEGM STETE UNWEESETY Wiiiiam Ems Giddens,. It. 1966 Jflisxs LIBRARY ABSTRACT THE PATROL“! W SILO GAS TGXICOSIS IN PIGS. by William Ellis Giddens, Jr. Research was conducted to determine the composition of silo gas, the effects of silo gas in pigs, and the similarity between the lesions of. silo gas toxicosis in pigs, nitrogen oxides toxicosis in rats, and silo filler's disease in man. The analysis of gas was made with the mass spectrometer. The con- centrations in 2 silos were: (a) carbon dioxide, 0.173 to 9.0542, (b) nitric oxide, 0 to 140 parts per million, and (c) nitrogen dioxide, 8 to 360 parts per million. Pigs were exposed for 3, 4, 5, and 10 minutes to silo gas. All pigs exposed for 10 minutes died within 21 to 72 minutes. All other pigs survived exposure, with the exception of l pig exposed for 4 minutes, which died in 10 hours and 27 minutes. Survivors were killed and examined at 15, 29, 30, 60, and 61 days. In pigs dying from exposure to silo gas, the earliest lesions noted were alveolar edema and hyperemia. The edema was eosinophilic and formed large quantities of fibrin in pigs that survived the longest. In addi- tion, there was hemorrhage, bronchiolar and bronchial epithelial desqua- nation, and perivascular, interlobuler, and subpleural edema. These lesions were similar to those produced in rats by exposure to high con- centrations of nitrogen oxides. Pigs surviving the acute effects of William Ellis Giddens, Jr. silo gas toxicosis did not develop bronchiolitis fibrosa obliterans, which has been described for silo filler‘s disease in men. THE PAEBOLOGY OF SILO GAS TOKICOSIS IN PIGS 37 William Ellis Giddens, Jr. A_TEESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of , MASTER OF SCIENCE Department of Pathology 1966 ACKNOWLEDGEMENTS I wish to express my deep appreciation for the help of Dr. C. K. Whitehair, my major professor. He provided me with complete freedom in the choice of a problem for investigation and was a constant source of moral support for the ups and downs of research. I am grateful to Dr. Robert P. Langham, who taught me most of what I know about pathology, and to Dre. C. C. Merrill and S. D. Sleight for their suggestions and guidance in conducting this research and writing this thesis. Acknowledgement is made to Mr. Robert A. Brooks, who performed the hematologic studies, Hr. Harold E. Harris, who performed the mass spec- trometer analyses, Dr. John F. Poss, who advised me on methods of collect- ing gas, Hrs. Mae Sunderlin, Hrs. Nina Miller, and Mrs. Frances Whipple, who prepared the tissues for microscopic examination, and Mr. Sam Tate and Mr. James Southern, who assisted in the care, handling, and exposure of the animals. I wish to thank the Department of Animal Husbandry of Michigan State University and Mr. Hiram Bickford and Mr. Norman Lind of Williamston, Mchigan, for allowing me to use their silos in these experiments. This work was made possible through Postdoctoral Fellowship Ho. l-l‘2-Qi-3l,299-01, awarded by the National Institutes of Health, Bethesda, Maryland. 11 To Huda who made it worthwhile 111 TABLE (F C(NTEN'I‘S monm 1m 0 O O O O O O O 0 O O O O O O O 0 O O O O O O O mlm a m anMM 0 O O O O O O O O O O O O O O O O 0 Chemistry of the Nitrogen Oxides. . . . . . . . . . .1. Sources of the Nitrogen Oxides and Their Effects in Man Experimental Studies of Nitrogen Oxides Toxicosis . . . Silo Gas onicosis in Man . . . . . . Silo Gas I'oxicosis in Smary....... MATERIALS AND METHODS. O EXPERIMENTS AND RESULTS. Eprrimnt 1e 0 e Experiment 2. Experiment 3. Experiment 4. DISCUSSION . . . . Silo Gas. . . Pathology . . SUMARY. . . . . . BIBLIOGIAPHY . . . VITA. O O O O O 0 Animals iv 0 Page 10 16 23 24 26 31 31 39 41 54 56 56 58 64 65 72 Table LIST OF TABLES Page Summary of data from exposures of rats to the nitrogen “16.. O O O O O O O O O O O O O O O O O O O O O O O O O O O - 3 2 Concentrations of carbon dioxide, nitric oxide, nitrogen dioxide (as measured by peaks 44. 30, and 46 on the mass spectrographs) and total nitrogen oxides in gas .m 1.. O O O O O O O O O O O O O O O O O O O O O O O O O O 62 Composition (in percent) of gas samples analyzed (B - before removal of air; A.- after removal). . . . . . . 43 Identification of pigs dying 0-12 hours after exposure to silo gas, survival times, organ‘weights, and histopatho- laic l..1m. O O O O O O O O O O O O O O O O O O O O O O O 48 Figure 10 11 12 LIST OF FIGURES Glass tube used for collecting gas samples. . . . . . . ... lass spectrographs showing peaks of masses 44 and 45 (car- bon dioxide) and 46 (nitrogen dioxide) in normal air and in .110 8‘. O O O O O O O O O O O O O O O O O O O O O O O O Caged rat and nitrogen oxides produced by the action of nitricacidoncopperwire. .. . ... . . . . . .. . . . Thoracic cavities of exposed rat (left) and control rat. Dark brownish discoloration of tissues, pulmonary edema, and congOltioninexpOIOGrat...........o....o Lung of a control rat. Hematoxylin and eosin. x 188. . . . Hyperemia, edema, and hemorrhage of the terminal bronchiole, alveolar ducts, and alveoli. Rat 2, which died 1-2 minutes after the beginning of exposure. Hematoxylin and eosin. x 188’. O O O O O O O C I O O O O O O O O O O O O O O O O O Rupture of bronchiolar arteriole. Rat l, which died 1-2 minutes after the beginning of exposure. Hematoxylin and .m1n0x75eeeeeeeeeseeeeeeeeeeeese Cellular debris in the bronchiolar lumen. Alveolar macro- phages and desquamated bronchiolar epithelium. Rat 9, which died 125 minutes after exposure. Bentoxylin and .“1n. : 188. O O O O O O O O O O O O O O O O O O O O O O 0 Formation of fibrin from proteinaceous edema. Rat 9, which died 125 minutes after exposure. Hematoxylin and eosin. :75e-ereve4eveeeeeeeeeseeeeeeeeseeee Pibrinous alveolitis. Rat 9, which died 125 minutes after exposure. Hematoxylin and eosin. x 750 . . . . . . . . . . Lung of a control pig. Hematoxylin and eosin. x 62 . . . . Hyperemia and edema. Pig 058852, which died 21 minutes after exposure. Helmtoxylin and eosin. x 150 . . . . . . . Hemorrhage, edema, and fibrin formation in the alveoli. Pig 058831, which died 39 minutes after exposure. Hema- toxylinandeosin.x150.................. vi Pass 29 29 33 33 35 35 37 37 38 38 50 $0 51 Figure Page 14 Desquametion of bronchiolar epithelium. Pig 058847, which died 72 minutes after exposure. Hematoxylin and gain. ‘ 150. O O O O O O O O O O O 0 O O O O O O O O O O O 51 15 Pibrinous hemorrhagic alveolitis and bronchiolitis. Pig 058842, which died 10 hours and 27 minutes after exposure. amtuylin‘ndCN1n9162eeeseesoesoeeeee 53 16 leukocytes, erythrocytes and fibrin in the alveoli and bronchioles. Pig 058842, which died 10 hours and 27 minutes after exposure. Hematoxylin and eosin. x 150 . . . . . . . 53 vii INTRODUCI‘ ION "At first glance the lungs may seem uncomplicated, but many wise men have gone astray in their laby- rinths. ... Disease commonly results in a profound but variable revision of their architecture. With each breath, also, the innermost recesses of the respiratory tract are brought very much into con- tact with a sometimes hostile environment. That we are not more often disabled we owe to their marvelous capacity to recover from injury and to their large reserve." Averill A. Liebow (1962) In our diverse and ever-changing environment, new stresses constantly appear. One of the most ubiquitous of these is the nitrogen oxides. As lay-products of the combustion of gasoline and of tobacco, they are present in small quantities as pollutants in the air of populated areas of the world. In much higher concentrations, they are also a formidable occupational hazard in certain industrial, military, and agricultural occupations. In the toxicoses that occur following exposure to concentrations of nitrogen oxides, there is a wide spectrum of lesions. In men one of~ the most interesting lesions is bronchiolitis fibrosa obliterans (Bro). Al- though it is a co-on lesion in silo filler's disease and in other forms of nitrogen oxides toxicosis, it has never been convincingly reproduced in animals by exposing them to pure gaseous nitrogen dioxide. Stokinger (1964) has advanced 2 theories to explain this enigma: 1. Nitrogen dioxide may not be the only toxic gas produced in silos. 2 2. Animals may respond differently from.humans to nitrogen dioxide and, in fact, do not produce BFO as a response to this gas. This research was an attempt to determine more fully the pathology of silo filler's disease and of EEO. Two things were unique in this study: 1. Exposures were to naturally produced silo gas in freshly filled farm.silos, thus simulating the conditions of humen exposure. 2. The pig was selected for exposure to silo gas. This animal resembles the human in many aspects of pulmonary structure (McLaughlin ggpglp, 1961) and it has not been used in any of the previous research on nitrogen oxides toxicosis. REVIEW OF THE LITERATURE Von Oettingen (1941) published a thorough review of the early litera- ture of nitrogen oxides toxicosis. More recent reviews have been com- piled by Gray (1959) and Stokinger (1964). This review, while attempt- ing to give an over-all smary of the many ramifications of nitrogen oxides toxicosis, will be concerned particularly with the literature relating to the disease in man called silo filler's disease. Chemistgy of the Nitrggen Oxides Stokinger (1964) listed the nitrogen oxides that may contaminate the atmosphere as: nitrous oxide...............N20 nitric oxideOOOOOOOOOOOOOOOONO nitrogen dioxide............N02 nitrogen trioxide...........N203 nitrogen tetroxide..........N204 nitrogen pentoxide..........N205 Grayson (1957) reported that nitrous oxide is commonly used in inducing anesthesia and has no toxic effects, that nitrogen trioxide dis- sociates very rapidly into nitric oxide and nitrogen dioxide, and that nitrogen pentoxide reacts with air to form nitrogen dioxide and nitrogen tetroxide. Thus, the principal nitrogen oxides from.a toxicologic point of view are nitric oxide, nitrogen dioxide, and nitrogen tetroxide. Nitric oxide is produced in some reactions involving low concentra- tions of nitric acid (Jolly, 1964; Thienes and Haley, 1964) and in welding operations (Elkins, 1946). Von Oettingen (1941), Grayson (1957), and Thienes and Haley (1964) stated that nitric oxide is quickly 3 4 converted to nitrogen dioxide in the presence of air, whereas Elkins (1946) and Stokinger (1964) stated that conversion to nitrogen dioxide is dependent upon concentration of nitric oxide. Elkins (1946) described the kinetics of this conversion and stated that a concentration of 100 parts per million (p.p.m.) nitric oxide is converted to nitrogen dioxide at the rate of 2.8 p.p.m. per minute. This allows high concentrations of nitric oxide to persist long enough to exert toxic effects indepen- dently of nitrogen dioxide, and it may account for some of the variability of results in the literature. Pflesser (1936, cited by von Oettingen, 1941) reported that nitric oxide has no irritant properties, but it can induce a severe methemoglobinemia, which may cause death. von Oettingen (1941) mentioned that nitrogen dioxide is the most important oxide of nitrogen from the toxicologic point of view. It exists in equilibriumnwith its dimer, nitrogen tetroxide. Thienes and Haley (1964) reported that at 22 C all the gee is present as nitrogen tetroxide and at 140 C it is present as nitrogen dioxide. Von Oettingen (1941) stated that in.whatever molecular form nitrogen dioxide is inhaled it is at once altered to that equilibrium prevalent at body temperature, which is a mixture of approximately 301 nitrogen dioxide and 701 nitro- gen tetroxide. Test and Russell (1944, cited by Rounds and Bils, 1965) stated that the gas, which would reach the lung cells through a fihn of aqueous tissue fluid, combines with water in the fluid to form nitric and nitrous acids. Thienes and Haley (1964) stated that nitrogen dioxide is dark brown in color, while nitrogen tetroxide is yellow. ‘The mixture of these gnses may be any shade of brown, orange, or yellow. Because nitric oxide, nitrogen dioxide, and nitrogen tetroxide are all in equilibrium 5 with each other and because they are difficult to separate for purposes of measurement, they are usually collectively referred to as the oxides of nitrogen, nitrogen oxides, or simply as nitrogen dioxide. In this thesis the term nitrogen oxides is used to describe this mixture of gases. The most comonly used method for the measurement of the nitrogen oxides employs a specific reagent which changes color in their presence. This reagent, a mixture of sulfanilic acid, N- (l-naphthyl)-ethylenediamine dihydrochloride, and acetic acid, was first described by Saltzman (1954) and is referred to as the Saltznn reagent. The color change is measured in a spectrophotometer and calibrated with known concentrations of nitrogen dioxide. Gill (1960) compared this method with other earlier methods and described a simplified method for standardization. Peterson a; 3.1. (1958) used the-mass spectrometer to measure ,. the nitrogen oxides. They used masses of 30 and 46 to measure nitric oxide and nitrogen dioxide, respectively. Sources of the Nitrggen Oxides and Their Effects in Man The most ubiquitous source of the nitrogen oxides is the high tem- perature combustion of liquid fuels in gasoline motors and diesel engines. Hence, the chemical smogs today plaguing so many American cities are related directly to the density of their motor traffic. Stokinger (1964) has estimated that 200 to 300 tons of nitrogen oxides are emitted daily into the Los Angeles atmosphere, and this city has reported (Hills, 1962) levels of over 3 p.p.m. This approaches the Maximum Allowable Con- centration of 5 p.p.m. (Threshold Limit Values for 1960). Haagen-Smit _e_t_:_ $1. (1959) found concentrations of 145 to 655 p.p.m. nitrogen oxides in cigarette smoke and over 1100 p.p.m. in pipe and cigar 6 smoke. Stokinger (1964) confirmed these findings. ‘These levels of nitro- gen oxides in the atmosphere would be rapidly fatal to men. There is no evidence to indicate why the same levels in tobacco smoke are so innocuous. The nonagricultural sources of lethal concentrations of nitrogen oxides are usually related to the use of nitric acid, the detonation of explosives, the operation of carbon and electric arcs and acetylene torches, the incomplete combustion of celluloid and nitrocellulose films, and the combustion of rocket fuels. ' Exposures associated with nitric acid have been reported by'Wood (1912), Rosseno (1945), Adley (1946), Fail (1951), McAdams and Krop (1955), HbAdams (1955), and Darke and'Warrack (1958). In all these exposures nitric acid came into contact with some substance which it oxidized, yielding large amounts of nitrogen oxides. Cases reported by Wood (1912), Rossano (1945), and Adley (1946) terminated fatally in 26 hours to 10 days after exposure (AB) from puhmonary edema and/or broncho- pneumonia. MCAdams (1955) and Darke and Warrack (1958) described cases in man that terminated fatally l4 and 27 days AE with bronchiolitis fibrosa obliterans (BFO). Schultz-Brauns (1930, cited by von Oettingen, 1941) has reviewed other fatal cases associated with nitric acid. Becklake g£_gl, (1957) studied the long-term effects of 7 patients exposed for 5 to 75 minutes to blasting fumes from "asnonium dynamite". High concentrations of nitrogen dioxide‘were believed to’be present in the fumes, and pulmonary function studies on the patients for periods up to 64 months after exposure revealed changes consistent with fibrosis of the bronchi and bronchioli and narrowing of their lumina in 2 of the patients. They interpreted these findings as indicative of EEO. 7 Pflesser (1936, cited by von Oettingen, 1941) and Charleroy (1945, cited by Kooiker‘ggflg;., 1963) have reported fatalities resulting from explosions on naval vessels in which large quantities of nitrous fumes were produced. Williman (1935) and Lindqvist (1944) reported 5 deaths from 1 to 4 days AE to fumes produced by electric and acetylene‘welding in confined spaces. Coltman and MacPherson (1938) and Elkins (1946) described the high levels of nitrogen oxides that may be produced by these procedures. In several nonfatal cases which lindqvist reported, confluent areas of increased densitwaith a mottling effect that resembled miliary tubercu- los is were seen on radiographic examination. Large quantities of nitrogen oxides may be produced by the slow - combustion of celluloid and nitrocellulose film. The Cleveland Clinic disaster (Editorial, J.A.M;A., 1929), in'which roentgen film of nitro- cellulose type caught fire, resulted in the deaths of 125 people and the hospitalization of 80 to 100 others. Nichols (1930) described 3 of these fatalities. One patient died from pulmonary edema shortly after exposure; another died several days AB from tracheobronchitis, pulmonary edema, and bronchOpneumonia; the 3rd died 25 days AE from subacute interstitial pneunonia. Convalescing patients had ntmnerous small nodular opaque areas in the lungs on radiographic examination. These resembled miliary tuberculosis and were diagnosed as exudative infiltration and inter- stitial fibrosis. The Opacities seemed to gradually fade as the patients recovered. The most recent source of air pollution from nitrogen oxides is the firing of ballistic missiles. Diamond and Johnson (1965) determdned the concentration of nitrogen oxides emitted by the test firing of the 8 Titan II missile and found 29 to 461 p.p.m. at 300 feet and 0 to 32 p.p.m. at 700 feet from.the firing point of the first stage. McLouth and Terry (1965) recorded 0 to 80 p.p.m. nitrogen dioxide when air was sampled 300 feet from the exhaust blast of the Titan missile. Flory (1930, cited by von Oettingen, 1941, and McAdams and Krop, 1955) has distinguished 4 clinical types of nitrogen oxides toxicosis: 1. The irritant gas type is characterized by severe irritation, choking in the throat and chest, and violent coughing, followed by a latent period of several hours when symptoms reside. Following this latent period the patient becomes more dyspneic, cyanotic, and dies 1 to 2 days later from pulmonary edema. MbAdams and KrOp (1955) stated that this is the phenomenon produced when nitrogen dioxide.is the main oxide present. 2. The reversible type is characterized by immediate dyspnea, cyanosis, vomiting, vertigo, loss of consciousness, and methemoglobinemis. This group of patients does not develop pulmonary edema and , if removed from the exposure early enough, may recover completely. Otherwise, the poisoning may rapidly end fatally. According to MbAdams and Krop (1955), this type is produced when nitric oxide is the main oxide present. 3. The shock type is characterized by severe symptoms of asphyxia- tion, convulsions and respiratory arrest, death presumably being-4 due to interference with the pulmonary circulation resulting in stasis in the blood vessels. This form is due to sudden inhalation of very high con- centrations of nitrogen oxides. 4. The combined type is characterized by symptoms of central nervous system.disorder, such as vertigo, somnolence, and staggering gait. There may be some cyanosis. After apparent recovery, this stage may be followed, 9 after some hours, by progressive dyspnea, marked cyanosis and pulmonary edema. Lowry and Schuman (1956b, cited by Stokinger, 1964) reviewed the litera- ture and correlated the clinical and pathologic processes with the concen- tration of gas. They estimated that acute pulmcnary edema, bronchopneumonia, and death in 2 to 10 days usually result from exposure to over 500 p.p.m. nitrogen dioxide. Exposure to 150 to 200 p.p.m. results in fatal BFO in 3 to 5 weeks. Bronchiolit is with focal pneumonitis results from exposure to 50 to 100 p.p.m. Spontaneous recovery usually occurs after 6 to 8 weeks. Blumgart and HacMahon (1929, Ehrlich and McIntosh (1932), La Due (1941), McAdam (1955), and Spencer (1962) have reviewed the literature and described the clinical and pathologic characteristics of BBC. This lesion was first described by lange in 1901. Fraenkel (1902, cited by La Due, 1941) was the first to describe it in association with nitrous fume poison- ing. Fraenkel divided the clinical course into 3 stages: (1) there was a short period imediately after exposure to the gas in which the patient was dyspneic, cyanotic, and coughing; (2) there was a latent period of variable length during which all symptoms vanished; (3) there was a final period during which symptans were markedly accentuated, the patient expec- torated bloody sputum, coarse rales wereheard, and there was increasing emphysema. Lowry and Schuman (1956a) list the causes as (1) inhalation of irritant gases, such as nitrogen dioxide, amonia, chloropicrin, chlorine, hydrogen chloride, sulfur dioxide, phosgene, and the war gases, and (2) a canplication of pneumonia, particularly influenza. Blumgart and MacMshon (1929) have described the pathogenesis of the lesion. Catarrhal bronchio- litis with necrosis and desquamation of bronchial and bronchiolar epithelium are first seen. The inflamatory process involves the basement membrane, 10 elastic and smooth muscle fibers, and peribronchiolar connective tissue. The bronchioli contain an inflamtory exudate with numerous desquamated epithelial cells, leukocytes, and large quantities of fibrin. Fibroblasts and some capillary endothelial cells proliferate into this exudate and attempt to organize it. The final result is a poly-paid mass of fibro- blastic granulation tissue, which may or may not be covered by epithelium, projecting into the lumen of the bronchiole. The lumen may be completely obliterated, or there may be a crescentic slit remaining. There is frequently emphysema in the alveoli distal to the obliteration. Grossly these fibroblastic masses appear as firm, irregular nodules 1 to 2 I'm. in diameter and scattered throughout the lungs. 0n radiographic examina- tion they have the appearance of miliary tuberculosis. The specific lesion of BFO is rare. Lowry and Schuman (1956a) reported that only 2 cases out of a total of 70,281 were diagnosed at the University of Minnesota lbdical School between 1919 and 1952. Experimental Studies of Nitrogen Oxides Toxicosis The Hygienic Guide Series (cited by McLouth and Terry, 1965) set the odor threshold for nitrogen dioxide as 5 p.p.m. McLouth and Terry reported that odor could be detected when less than 1 p.p.m. was the measured concentration. Because of their findings, the odor threshold at Cape Kennedy is considered to be 1 p.p.m. Henschler 9; 2;. (1960a, 1960b, cited by Stokinger, 1964) used nitrogen dioxide produced from the action of acid on sodium nitrate and reported that 0.1 p.p.m. could occasionally be detected and 0.4 p.p.m. could always be detected. Stokin- ger (1934) reported that the threshold of odor perception generally is l to 3 p.p.m. for the majority of individuals. 11 In a series of self-exposures, Lehmann and Hasegawa (1913, cited by von Oettingen, 1941) noted that exposures of 64 p.p.m. nitrogen dioxide caused moderate irritation of the larynx, 100 p.p.m. caused marked irri- tation, and 207 p.p.m. caused very marked irritation of the nose and larynx, with coughing, increased nasal secretion, and lacrimation. Huie (1962, as reported in personal communication to Stokinger, 1964) exposed volunteers to differing concentrations of nitrogen dioxide. Breathing 50 p.p.m. for 1 minute produced unfavorable reactions in 2 of 7 adult volunteers. About half the volunteers found that brief exposures to 25 p.p.m. were unpleasant but presunably not harmful. The Emergency Tolerance landts set by the technical manual, Oxides of NitrogenJToxicity (1963, cited by McLouth and Terry,l965) are 35 p.p.m. for 5 minutes, 25 p.p.m. for 15 minutes, 20 p.p.m. for 30 minutes, and 10 p.p.m. for 60 minutes. 'The Maximum Allowable Concentration (that concentration 'which can be tolerated 8 hours daily with no harmful effects) set by the Conference of Government and Industrial Hygienists is 5 p.p.m. nitro- gen dioxide (Threshold Limit Values for 1962). Vigdortschick 25.51, (1937) surveyed 127 men working in industries in which the concentration of nitrogen oxides averaged 2.6 p.p.m. These workers had higher rates of tooth decay, chronic bronchitis, pulmonary emphysema, and tuberculosis than did workers in nitrogen oxide-free environments. Le Towsky g; _a_l_. (1941), von Oettingen (1941), Gray (1959), Kooiker g§,g;, (1963), and Stokinger (1964) reviewed the early research on the toxicity and pathology of experimental nitrogen oxides toxicosis. Le Towsky _e_t_ 51... (1941), Gray gt. 3]... (1954a), Caastock and Rue (1961), and Kooiker g; 5;. (1963) described methods for exposing animals to measured concentrations of nitrogen oxides. 11 In a series of self-exposures, lehmann and Hasegawa (1913, cited by von Oettingen, 1941) noted that exposures of 64 p.p.m. nitrogen dioxide caused moderate irritation of the larynx, 100 p.p.m. caused marked irri- tation, and 207 p.p.m. caused very marked irritation of the nose and larynx, with coughing, increased nasal secretion, and lacrimation. Huie (1962, as reported in personal communication to Stokinger, 1964) exposed volunteers to differing concentrations of nitrogen dioxide. Breathing 50 p.p.m. for 1 minute produced unfavorable reactions in 2 of 7 adult volunteers. About half the volunteers found that brief exposures to 25 p.p.m. were unpleasant but presunably not harmful. The Emergency Tolerance Limits set by the technical manual, Oxides of NitrogenJToxicity (1963, cited by McLouth and Terry,1965) are 35 p.p.m. for 5 minutes, 25 p.p.m. for 15 minutes, 20 p.p.m. for 30 minutes, and 10 p.p.m. for 60 minutes. "The Haximm Allowable Concentration (that concentration ‘which can be tolerated 8 hours daily with no harmful effects) set by the Conference of Government and Industrial Hygienists is 5 p.p.m. nitro- gen dioxide (Threshold Limit Values for 1962). Vigdortschick gt _a_1_. (1937) surveyed 127 men working in industries in which the concentration of nitrogen oxides averaged 2.6 p.p.m. These workers had higher rates of tooth decay, chronic bronchitis, pulmonary emphysema, and tuberculosis than did workers in nitrogen oxide-free environments. Le Towsky gt filo (1941), von Oettingen (1941), Gray (1959), Kooiker g 5-1. (1963), and Stokinger (1964) reviewed the early research on the toxicity and pathology of experimental nitrogen oxides toxicosis. Le Towsky _e_§_ 9_1,. (1941), Gray £_t_ g1. (1954a), Comstock and Rue (1961), and Kooiker 25.31, (1963) described methods for exposing animals to measured concentrations of nitrogen oxides. 12 La Towsky Lt $1. (1941) exposed 112 animals (cats, rabbits, mice, guinea pigs, and rats) to concentrations of nitrogen oxides varying from 2.6 to 1000 p.p.m. Continuous exposure to 100 p.p.m. produced death in an average of approximately 5 hours, 400 p.p.m. in 1 hour, 800 p.p.m. in 30 minutes, and 1000 p.p.m. in 19 minutes. In a given concentration, length of survival depended upon length of exposure. Animals survived an average of 21 hours when exposed to 100 p.p.m. for 10 minutes. Het- hemoglobin was seen frequently in the blood during and_after exposure, but it disappeared rapidly when the animal was removed from.the exposure chamber. MacQuiddy 25,31, (1941) reported severe hemoconcentration (due to pulmonary edema), lower blood pressure (due to the effect of nitrite on the smooth muscle of the blood vessels), methemoglobinemia, and simul- taneous failure of both heart and respiration in these same animals. Tollman g; _a_l. (1941) described the lesions in these animals. Of the fatalities, 62 of those which were exposed to the higher concentration died from immediate asphyxiation due to severe methemogldbinemia. All the vessels were filled with brown blood, and there were no histologic lesions. Death due to pulmonary edema during or several hours after exposure accounted for 882 of the fatalities. Most of these animals died with dyspneic convulsions. Histologically, there was severe pul- monary edema and necrosis of bronchiolar epithelium. Lung'weights, expressed as percentage of bodywweight, increased 2.6 to 4.7 times over controls due to the pulmonary edema. Pneumonitis due to secondary bac- terial infection accounted for 62 of the fatalities. These animals died within 1 week after exposure. There were no lesions.in the animals that survived the acute and subacute effects of the gas. 13 Tollman‘gg 9_1_. (1941) produced similar lesions in laboratory animals by exposing them to carbon arc fumes. They recorded concentrations of _ 72 to 140 p.p.m. nitrogen dioxide in the fuses. Gray gt. 3;. (1952) demonstrated that exposure of rats to 9 to 14 p.p.m. nitrogen oxides at 4-hour periods for a total of 40 to 96 hours produced rhinitis, tracheitis, and pneumonitis. In a later report (19541:) no harmful effects were noted after exposure to 4 p.p.m. for 4 hours daily, 5 days per week, for 6 months. They reconnended on the basis of these data that the Maximum Allowable Concentration of 25 p.p.m. be lowered to 5 p.p.m. The median lethal concentration for male albino rats was 138 p.p.m. for 30 minutes and 67 p.p.m. for 4 hours (Gray 33; _ , 31., 1954b). Gray and his associates were the first to use pure sources of nitrogen dioxide and noted that nitrogen dioxide was the primary toxic substance in red fuming nitric acid, but that the presence of acid vapors increased the toxicity 257.. Ripperton and Johnson (1959) exposed rats to 0.5 p.p.m. nitrogen dioxide for 2 to 6 weeks and noted no histologic changes in the lung or liver. The levels of urinary aspartic acid were significantly increased in the exposed rats. . Carson g; 9;. (1962) exposed rats, rabbits, and dogs to concentra- tions of 10 to 1000 p.p.m. nitrogen dioxide. They reported that lung weight/body weight ratios provided the quickest and most objective indi- cation of nitrogen dioxide exposure. The threshold concentrations for toxic effects in rats were 104 p.p.m. for 5 minutes, 65 p.p.m. for 15 minutes, and 28 p.p.m. for 60 minutes. In animals that survived exposure there were no significant differences when compared with controls. 14 Kleinerman and'Wright (1961) exposed rats, rabbits, and guinea pigs to nitrogen dioxide for 2-hour periods. Exposures to 150 to 200 p.p.m. killed all rats and 1/3 :of the rabbits. With exposures of 15 to 75 p.p.m. they all survived. Imediately after exposure they had acute pneumonitis and pulmonary edema. By the 4th day the acute inflammatory changes had sub- sided and macrophages and epithelial regeneration were present, especially in the terminal bronchioles. Healing*was practically complete in 2‘weeks. Freeman and Haydon (1964) reported that rats exposed to 100 p.pEm. nitrogen dioxide began to die within 24 hours from pulmonary edema. _'l'wo- thirds of the rats exposed continuously to 50 p.p.m. died in 48 to 68 days. All rats survived 25 p.p.m. for periods up to 157 days, with hyper- plasia of epithelial and connective tissue cells of the bronchioles, accumulation of desquamated cells, and partial airway obstruction'causing some emphysema. Buckley and Balchum (1965) studied oxygen consumption rats, lactic dehydrogenase levels, and aldolase levels in guinea pigs exposed to short- and long-term dosage regimens of nitrogen dioxide. The short-term.exposures were to 40 p.p.m. at half-hour periods for a total of 4% hours. There was a 2-hour rest period between exposures. Thelong-term exposures were to 15 p.p.ms‘continuously for 10 weeks. They observed significant increases in oxygen consumption values in splenic and renal tissues of animals on short- and long-term exposures. Hepatic tissue values were increased in animals on short-term.exposures only, while lung tissue values were not significantly different in experimental and control groups. Aldolase and lactic dehydrogenase activities were increased by both exposure regimens. The relative change in activity varied widely in different organs, indicating that tissues may respond to nitrogen 15 dioxide through different mechanisms, or that the effects may not be the same in different organs. Balchum 9;; 5.1. (1965) demonstrated increased levels of antibodies against lung tissue in the serum of guinea pigs inhaling'nitrogen dioxide in concentrations of 5 p.p.m. and 15 p.p.m. The antibodies were detected in dilutions of serm greater than 106 by agglutinating latex particles coated with normal lung protein. Wagner _e_t_:_ _a_1_. (1965) exposed various species of laboratory animals (dogs, rabbits, guinea pigs, rats, hamsters, and mice) daily to 1, 5, and 25 p.p.m. nitrogen dioxide for periods up to 18 months. At no expo- sure level did body weights, hematologic values, biochemical indices, and histologic observations differ significantly in experimental and control groups. The only effect of nitrogen dioxide exposure was a slightly increased rate of tumorigenesis in the lungs of a pulm0nary tumor-susceptible strain of mice. Pace 3; 93.... (1961) found HeLa cells in tissue culture to survive daily 8-hour exposure to 5 to 10 p.p.m. for 8 days, but 30 minutes' exposure to 100 p.p.m. was quite toxic. Serum added to the tissue cul- ture median had a profound protective influence. Some cells survived a cmcentration of 8600 p.p.m. Rounds and Bile (1965) treated tissue cul- tures of pulmonary epithelial cells with soditnn nitrite, solutions and studied the chemical and morphological changes. Utilizing oxygen con- eruption values, they reported a partial reversible inhibition in oxida- tive activity during treatment with sodium nitrite. They demonstrated similar nuclear and mitochondrial changes in alveolar epithelial cells in tissue cultures treated with sodium nitrite and in animals exposed to L5 p.p.m. nitrogen dioxide in the atmosphere. 16 Interrelationships between the nitrogen oxides and other factors have been studied by several workers. Fairchild _e_t _a_l_. (1959) found that com- pounds bearing the sulfhydryl group were capable of antagonizing the acute toxic effects of nitrogen dioxide. Antagonism was demonstrated with mercaptans, disulfides, and thio-urea derivatives administered by inhala- tion or by injection. Wagner _e_t_:. 2.}.- (1961) demonstrated similar but less drantic protection with oil mists. They hypothesized that the oil coated the alveoli, producing a film which protected animals against otherwise lethal exposures to nitrogen dioxide. Purvis and Ehrlich (1963) enhanced the virulence of Klebs iell_a_ mgmnonige infections in mice by exposing them to 25 p.p.m. nitrogen dioxide for 2 hours. Gray (1959), Kooiker g_t_ 3;. (1963), Stokinger (1964), and Wagner g2 $1. (1965) have consented on the confusing and conflicting nature of existing data regarding the concentrations, types of exposure, and responses to the nitrogen oxides. Kooiker g; g. (1963) stated: "The literature reveals inadequate data concerning the hazard of such poisoning. Experimental studies are numerous, but the data are conflicting, and comparison of results is difficult. Few reports include sufficient information to permit evaluation of the validity of the reported findings.” Silo Gas Taicosis in M The literature describing silo gas toxicosis is scattered throughout both medical and agricultural journals. Hayhurst and Scott (1914) recorded the first known deaths due to silo gas. Four men ascended a 40-foot metal silo, which was in the process of being filled. Within 5 minutes all 4 men appeared to be dead. They were removed inedistely and were given artificial respiration, but they all died. Lesions observed at necropsy were a dark appearance of the blood, failure of the blood to 17 clot, cyanosis of the lungs, trachea, bronchi, liver, and kidneys, and capillary injection of the brain. When a lantern was lowered into the silo it went out about 18 inches above the silage. Death occurred in 10 and 42 minutes, respectively, in a guinea pig and a rabbit when they were lowered into the silo. When a dog was placed in the silo it held its nose up in the air and was not affected by the gas. Samples of the gas were found to contain 38% carbon dioxide, 13.5% oxygen, and 48.5% nitro- gen, these being the only 3 gases tested for. Attempts were made with animals and with the lantern to detect the presence of poisonous gas in ‘2 other silos, without avail. It was concluded that the men had become asphyxiated with carbon dioxide. In 1926, Fabian warned of the dangers from poisonous gases in silo filling in an agricultural bulletin. He reported that large concentra- tions of carbon dioxide were formed during fermentation and that each year cases of asphyxiation and, in some instances, death occurred. 1e Rossignol (1932), in a letter to the editors of the Journal of the American Medical Association, described a patient with symptoms of coughing, headache, aching joints, and subjective sensations of fever within-4 to 8 hours after working in a silo. Symptcmls disappeared within 1 to 2 days. The editors suggested that these synptmas may have been caused by (1) volatile organic acids given off during fermentation, (2) allergic phenomena, (3) a secondary bacterial infection, or (4) a combi- nation of these. Price 33; 51. (1937) described several fatalities in silos and grain elevators: (l) a 9-year-old child was overcome by the gas present in a pit silo. In an attempt to rescue the child, the mother and a sister were also overcou. All 3 died; (2) a farmer was overcome while trying 18 to clean out his pit silo. Four of his children attempted to rescue him; all 5 died. (3) Two men entered a silo which had been partially filled the day before; both were overcome' and died. (4) Two men were fatally asphyxi- ated while working in a bin filled with damp corn. (5) A workman was fatally asphyxiated after 15 minutes' exposure in a bin filled with deep barley. (6) A workman was fatally asphyxiated while working-(in a bin filled with damp oats; another worker went in to rescue him and was also fatally asphyxiated. ‘ Analyses of air samples of 2 of the above bins showed that the air contained 10.2 to 12:657.. carbon dioxide. We test was conducted for any “1'... gas. Three fatalities were reported in a partly-filled silo (Deadly Gases Sometimes Accmaulate in Silos, 1939). Other agricultural publi- cations warned of dangers due to high levels of carbon dioxide in the gas from freshly filled silos (Silos, 1939; Briggs £51., 1956, cited by Grayson, 1956). Credit for detecting the presence and possible hazard of the nitro- gen oxides in silo gas must be given to Peterson and his associates (1949), who observed a heavy, yellow-brown gas with a distinct odor on the floor of a silo roan. A sample of the gas revealed the equivalent of 151 p.p.m. nitrogen dioxide. In agricultural bulletins (Peterson _e_t_ $1., 1952: Take Frecautions Against Deadly Silo Cases, 1957) farmers were urged to take precautions against nitrogen dioxide poisoning. In 1 study (Peterson, 1958), 5 silos were examined. The gas, visible in 2, was heavy, yellow, irritating, and smelled like bleach. It stained the silage a yellow color, which persisted for days after the gas was gone. An air seaple taken near the opening of a pipe draining the silo had 58,500 p.p.m. nitrogen dioxide. 19 Peterson 55.31, (1958) studied nitrogen oxides production in arti- ficial silos. They found that nitric oxide was the principle nitrogen oxide produced, that it could occupy up to 14.81 of the air (148,000 p.prm.), and that the peak point of production‘was 23 hours after filling. By 60 hours, the concentration of nitric oxide was down to 6% (60,000 p.p.m.). nitrogen dioxide was present in concentrations up to 101 (100,000 p.p.m.). Ten times as much nitrogen oxides were produced when nitrogen was added as nitrates to the artificial silo, compared to nitrogen added as amino acids. They believed the process for conversion ' of nitrate to nitrogen oxides to be: nitrate-onitrite--nitrous acid-- nitric oxide-~nitrogen dioxide. When silage was autoclaved, little or no nitrogen oxides were produced, indicating the importance of bacteria in the formation of the gases. Mbst of Peterson's early work appeared in agricultural publications and escaped the early attention of the medical profession. Fostvedt (1951, cited by Grayson, 1956) reported a case of nonfatal asphyxiation attributed to carbon dioxide in a man'who entered a silo. Schroeppel (1953), in a letter to the editors of the Journal of the American Medical Association, asked for advice concerning a man found dead in.a silo filled 24 hours previously with corn silage. There was a definite slope and the body was found lying in the lowest part of theslope. The editors replied that high levels of carbon dioxide in the silo‘were probably responsible for the fatality. The first published study of silo gas toxicosis in which nitrogen dioxide was incriminated was that of Delaney 25. l. (1956). In their 1st case, a man died 5 days after the beginning of exposure to silo gas. At autOpsy, there was extensive congestion and edema of the lungs. In 20 a 2nd case, a man working in a silo had vague symptms of constriction in the chest, coughing, and weakness. He remained in bed for a week and then recovered. During this tine deaths occurred in some pigs penned near the silo. Two weeks after exposure he became progressively more dyspneic. He was admitted 1 month after exposure with a clinical diagnosis of suba- cute bronchopneumonia and eventually recovered with supportive therapy. Radiographic examination revealed extensive miliary mottling throughout the lungs, especially in the upper lobes. Delaney and his associates, citing the work of Peterson 3; 51. (1949), attributed the symptoms and lesions in their patients to the possible presence of nitrogen dioxide in the silo gas, and named this new condition silo filler's disease. Lowry and Schuman (1956) described 4 cases of silo filler's disease. In 2 of their cases 2 men attempted to climb the chute of a silo 1 day after it had been filled with corn silage. Both were exposed to what they described as an irritating, oppressive gas in the chute. They immediately suffered from coughing, dyspnea, and shortness of breath. Their distress never did subside, an! they were eventually hospitalized. Radiographic examination revealed scattered focal opacities resembling miliary tuberculosis. They died 27 and 30 days after exposure. Grossly, the lungs contained numerous uniformly distributed lesions which were visible and palpable as firm, discrete nodules of miliary size. Micro- scopically there was BFO. The bronchioles were filled with a cellular fibrinous exudate, and organisation of this adherent plug of fibrin by ingrowth of fibroblasts from the bronchial wall tended eventually to occlude the lumen. In the other 2 cases there was innediate respiratory distress, then rapid recovery. latent periods in which no distress was felt were 21 followed by relapses 10 and 19 days after exposure, with symptoms of pro- gressively increasing respiratory distress and coughing. Radiographic examination revealed the presence of scattered, diffuse, nodular infiltra- tions. Both patients were treated with prednisolone, which caused a -dramatic reversal in symptoms, and they recovered with no ill effects. All 4 cases were diagnosed as BFO resulting from the inhalation of nitro- gen dioxide in the silo gas. Lowry and Schuman (1956) stated that the potential concentration of nitrogen dioxide was roughly preportional to the nitrate and nitrite concentration in the silage. Factors known to ‘increase the nitrate concentration were: (1) highly nitrated soil, either naturally occurring or due to heavy nitrate fertiliser applica- tion, (2) drought, and (3) immaturity of the plant'when ensiled. Grayson (1956) described 2 cases of silo filler's disease, which he called ”nitrogen dioxide pneumonia". In a later article (Grayson, 1957) he defended this term against the more commonly used tenm, silo filler's disease. In Grayson's 1st case a man entered a silo and was rendered unconscious by a yellow-brown gas present above the silage. He died from fulminating pulm0nary edema 29 hours after exposure. In the 2nd case, exposure for 2 to 3 minutes was followed by dyspnea. This patient eventually recovered with no ill effects, but on radiographic exmmination there were patchy diffuse confluent infiltrations throughout the middle 2/3 of both lungs. The radiologic diagnosis was "chemical pneumonitis". Dickie (1957, cited by Grayson, 1957) described 2 cases of silo filler's disease, resulting in subacute bronchOpneumonia with BBC. Gailitis g5_§l, (1958) reported a case in which a patient had dyspnea and hyperpnea 2 to 3 weeks after exposure. There were no lesions upon radiographic examination. Treatment with cortisones brought dramatic relief and recovery. 22 Leib 9.; 3;... (1958) described a case of chronic pulmonary insufficiency secondary to silo filler's disease. A man was exposed to high concentra- tions of silo gas which caused burning of the eyes and nose and a choking feeling. He was bedridden for 3 weeks and retained shortness of breath for l to 2 years. On radiographic exandnation there was an accentuation of the bronchovascular markings. 0n pulmonary function examinations, there 'was an increase in residual volume, a decrease in the maximum breathing “capacity, and a lowered pulmonary compliance value, the latter'indicating that there may have been decreased elasticity of the lungs. The signs and lesions were largely irreversible, and were believed to be due to the exposure to silo gas. Schell (1958) described a similar case in‘which there was a diffuse, finely nodular infiltration of the lungs on radiographic examination. Treatment for several months with corticosteroids caused symptoms to disappear, but the radiographic lesions did not regress. Cornelius and Betlach (1960) reported 2 cases of silo filler's disease. In 1 case a mannwas treated for pneumonia 10 days after exposure to silo gas. He was hospitalized 34 days later with a fine nodulation in the upper 2/3 of both’lungs on radiographic examination. Four mdnths after treatment he had no symptoms. His lungs had no lesions on radio- graphic examination. In a 2nd case a man had severe pulmonary edema after exposure. He improved rapidly after treatmenthith corticosteronds. There was a patchy infiltration through most of the lungs on radiographic examination, but the lesions were gone 15 days after treatment. Rafil and Godwin (1961) have written a detailed description of the' pathology of BPO in a case of silo filler's disease. A patient admitted with severe dyspnea and hemoptysis had scattered nodular infiltration of 23 the lungs on radiographic examination. The patient had worked in a silo for 5 hours some 25 days before. He died from cardio-respiratory failure 3 days after admission, despite supportive and corticosteroid therapy. At necropsy the lungs were firm, voluminous, mottled, grayish-red, and equally involved throughout. There were numerous small nodules and hemor- rhagic blotches scattered throughout the lung parenchyma. 0n histopatho- logic examination there was severe congestion, hemorrhage, edema, fibrin formation, and numerous alveolar macrophages in the alveoli. Bronchiolar ‘walls‘were edematous and infiltrated with chronic inflammatory cells. The basement membrane was prominent, hyalinised, and thickened. The bronchioles were filled with fibrin and a cellular exudate. In many places this exudate was undergoing organisation, the fibrous stroma come pletely filling the terminal bronchioles and alveolar ducts. There were attempts by the bronchial epithelium to grow over the fibrous protrusions. Some terminal bronchioles, alveolar sacs, and alveoli were completely lined by a hyaline membrane composed of dense fibrinoid material. There was alveolar emphysema near the areas of bronchiolar occlusion. Silo Gas Toxicosis in Animals The nitrogen oxides have been suspected in the etiology of pulmonary adenomatosis, a specific disease of cattle. This condition, first de- scribed by Honlux‘g§,g;, (1953, 1955) and Seaton (1957b), is an acute atypical interstitial pneumonia of unknown cause. Seaton (1957a) repro- duced the typical lesions seen in field cases by exposing a cow to fumes produced by the action of nitric acid on copper wire. He postulated that nitrogen dioxide was produced in the rumen by the fermentation of forage containing high levels of nitrates and was then eructated and inhaled. 24 Dougherty gt 9_l_._. (1962) provided a physiologic basis for this by demon- strating that rtmninants do inhale part of the gases they eructate. Grayson (1957) reported, on the basis of Seaton's work (1957), that nitrogen dioxide pneumonia does exist in cattle. Seaton was unable (1957a), however, to reproduce the condition by repeatedly pumping the rumen full of pure nitrogen dioxide gas, and Houlton _e_t_ g}... (1963) were unable to find nitro- gen oxides on examination of rmen air samples of field cases. Haynes (1963) described a respiratory. condition in cattle which he called "silo filler's disease". The condition was associated with the feeding of corn silage and was characterised by coughing, hyperpnea, and dyspnea. The condition was observed for several months after the silage was processed. A silage sample analysed for nitrite content was within the normal limits . 8mg It is evident from this literature review that the nitrogen oxides and silo gas are public health problems. Silo filler‘s disease is an occupational hazard in farmers and has been recognised and described by a number of workers. Exposures usually occur 1 to 2 days after the silo is filled or partially filled. The farmer is exposed either in the chute or within the silo. The length of exposure may vary from several minutes to several hours. Initially there is respiratory eabarrassment and pul- monary edema. Distress may continue throughout the course of the disease, but frequently there is temporary recovery, followed by the return of respiratory distress 2 to 5 weeks after exposure. Death may result frma pulmonary edema within 1 to 2 days after exposure or free BFO 2 to 5 weeks after exposure. Recovery is usually complete but may be partial, with permanent debilitating impairment of respiration. 25 Wagner and his co-vworkers (1965) made the following comments in their report: "Despite all the efforts made in this and past studies, one large question remains. Can nitrogen dioxide per se produce BFO? ... BFO is the cannon endpoint in many human exposures to silage gases. When generalised fibrosing reactions were found in animals, they occurred when nitro- gen dioxide was associated with other contaminants, notably nitric acid vapors. It may be that animals cannot repro- duce the htmmn disease from nitrogen dioxide, or it is equally likely that nitrogen dioxide is not the sole exposure in silage gas. Whatever the answer, it would seem that some future research effort should be made to determine the effects of combinations of agents with nitrogen dioxide in animals to attempt simulation of the hunan response." MATERIALS AND METHODS Objectives The objectives of this research were: (a) to study the pathology of silo gas toxicosis in pigs, (b) to compare the lesions of. silo gas toxicosis in pigs with those of reported cases of silo filler's disease in man, (c) to compare the lesions of silo gas toxicosis in pigs with nitrogen oxides toxicosis in rats, and (d) to determine the presence and amounts of the toxic components of silo gas. Sources, Housing, and Care of .. Animals Rats for Experiment 1 came from 2 groups which were raised on a standard laboratory animal diet. Rats! 1 through 4 and 1 control weighed an average of 300 Gm. Rats 5 through 9 and the 2nd control weighed an average of 332 Gm. The pig used in Experiment 2 was a lZ-week-old Yorkshire male weighing 27.3 kg. and was raised on a conventional grower ration. Pigs for Experiments 3 and 4 were from a Specific Pathogen Free herd in which there was no history of respiratory disease. They were purchased when 46 to 49 days old and were exposed to silo gas when 65 to 69 days old, at which time the average weight was 10.6 kg. Before and after exposures, the pigs were pastured and fed standard rations, free choice. The pigs were given an anthelmint ic, piperasine hexahydrate, in their drinking water approximately once a month. Hematolgic and Fecal Examinations Blood samples were collected periodically before and after exposures. Packed cell volume and hemoglobin (cyanmethemoglobin method) values and 26 27 total and differential white cell counts were determined according to methods described by Benjamin (1961). Fecal samples from the herd were collected periodically and examined for parasites according to the flota- tion method described by Benjamin (1961). Gross and Microscgpic Egg-nation of Tissues Control rats were killed by intraperitoneal injection of sodium pentobarbital. Pigs not killed by the gas were killed by electrocution. At necropsy of the pigs, bacteriologic examinations were made of lung, kidney, spleen, and intestine using blood agar and MacConkey's agar. Tissues were fixed in 10% buffered formalin. Rat lungs were fixed by ligation of the trachea and slow intratracheal injection of fixative until the lungs expanded to fill the thoracic cavity. The lungs and ligated trachea were removed and placed in a container of fixative until trim-ed. Pig lungs were fixed by intratracheal or intrabronchial infusion of fixative at a continuous pressure of 15 to 30 cm. of fixative for 48 to 72 hours, using an apparatus described by Heard (1960). Sections were cut and labeled from the dorsal cranial, middle, and caudal and ventral cranial, middle, and caudal parts of either the right or the left lung fraa each pig. All tissues were washed and dehydrated in ethyl alcohol, cleared in xylene, and embedded in paraffin. Sections were cut at 7‘ I" and stained with hematoxylin and eosin for general observations, Gaaori's trichrome stain for collagen and smooth muscle, Wilder's reticulum stain for reticular fibers, Mallory's Phosphotungstic-Acid Hematoxylin for fibrin, and Verhoeff's elastic stain for elastin, using procedures described in the Manual of Hiatolggic and Special Stainigg Technics of the Armed Forces Institute of Pathology, Washington, D. C. (1957). 28 Gas Anglzsis Gas samples were collected in specially made glass tubes (Figure 1). 0n 1 side of the tubes #10-30 joints were attached so that they could be mounted to the inlet of the mass spectrometer. Tubeswere evacuated with a vacuum pump* for 5 minutes and sealed. Samples were collected in the silos and the exposure chamber by opening 1 of the stopcocks, thus allowing the air to be pushed into the tube. Air sampleswere analysed in a mass spectrometer.” All gases, with a molecular weight between 12 and 60 were measured. Each sample was analysed once at room tempera- ture, then cooled with liquid nitrogen, thus liquifying most gases except nitrogen and oxygen, which were then vacuum removed. The sample was allowed to return to room temperature, at which point a 2nd analysis was made. By comparing peak heights in the 2nd sample with that of carbon dioxide (mass 44) which appears in both analyses, a more accurate measurement of the gases in lower concentrations could be made (Figure 2). Masses 30, 44, and 46 were used to estimate the concentrations of nitric oxide, carbon dioxide, and nitrogen dioxide. Values obtained by this method were compatible with the data of previous reports and with the lesions observed in the animals exposed. Estimates of nitric oxide, nitrogen dioxide, total oxides, and carbon dioxide are listed in TABLE 2. The complete data from all analyses are presented in TABLE 3. *Model 1400' Duo-Seal Pmnp, Welch Company, Chicago, Illinois. *fliodel 21-1030, Consolidated Electrodynamics Corporation, Pasadena, California. 29 Figure 1. Glass tube used for collecting gas samples. Figure 2. ‘Bass spectrographs showing peaks of masses 44 and 45 (carbon dioxide) and 46 (nitrogen dioxide) in normal air and in silo gas. 30 Queral A rubber mask* covering the face was worn to protect against the inhalation of noxious gases. It was connected to a 50-foot rubber hose, through which inhaled air cams frma outside the silos. Exhaled air was evacuated directly out through a valve built into the mask. These experiments were conducted in the fall of 1965. lili'odel 4104-P, Davis Emergency Equipment Company, Newark, New Jersey. EXPERIMENTS AND RESUIIS .m..._._._E as... 1 Emriggntal plan. Nine rats were exposed to nitrogen oxides according to the schedule in TABLE 1. Two rats were randomly selected as controls. Exposures were made inside polyethylene plastic chambers measuring approximately 2 x 2 x 4 feet. The rats were caged inside the chamber. Nitrogen oxides were generated by placing measured concentra- tions of copper wire into a beaker of concentrated nitric acid, using arm-length rubber gloves built into the side of the plastic chamber. The analyses of the gases produced are presented in TABLES 2 and 3. Exposure times were measured beginning at the time the gas reached the caged rats. Obsegations and clinical sigs. When the copper wire was dropped into the nitric acid, a heavy, brown gas poured over the edges of the beaker. The gas was heavier than air and filled the chamber free the bottom up (Figure 3). As soon as it reached the caged rats, they scurried rapidly about in their cages, rubbing their faces frequently with their front paws and gasping. Within 1 minute after the nitrogen oxides had reached Rats 1 through 4, the concentration, was so high that the rats were no longer visible. When the cage was removed after 2 minutes of exposure, all the rats were dead. Rate 5 through 9 were exposed to a lower concentration of nitrogen oxides. They scurried frantically around the cage, often standing on 31 32 chosen a +++ someones u_++ usoeewm n + Hashes no undone n.c O o o o O O --- ..as . --- .-- --- Ofimo he snows o o o o + o It... a!- .::.. 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Dark brown discoloration of tissues, pulmonary edema, and congestion in exposed rat. 34 their hind legs in order to breathe the less concentrated gas. higher off the floor of the chamber. They rubbed their eyes and nostrils fre- quently. After 2 to 3 minutes of exposure their activity lessened and they became lethargic. Rat 5 died in the chamber after 12 minutes of exposure. The survivors were dyspneic, hyperpneic, and lethargic after removal from the chamber. They gradually became comatose before death. Survival times and histopathology are summarised in TABLE 1. Gross pathology._ The hair and skin of all exposed rats were stained a light yellow color by the gas. The blood and viscera of Rats 1 through 5 were brownish-red. The viscera were congested. The lungs were brown, edematous, hyperemic, and did not collapse (Figure 4). Ratm6phad a slight brownish discoloration of the tissues and blood. Rats 7 through 9 had no discoloration but did have congested viscera and edematous, hyperemic, uncollapsed lungs. There was much blood-tinged froth in the trachea and bronchi. Histgpatholggy. Rats 1 through 4 were exposed to approximately“ 13,220 p.p.m. nitrogen oxides (TABLE 2) and died within 1 to 2 minutes after the beginning of exposure with massive pulmonary edema. In some sections alveoli, alveolar ducts, respiratory and terminal bronchioles were filled with an eosinophilic proteinaceous fluid (Figure 6). The edema was more severe near the peripheral alveoli, especially near the ventral surface of the lungs. The lungs were severely hyperemic, and some alveoli contained erythrocytes. In Rats l and 4 there was rupture of some bronchiolar arteries with perivascular hemorrhage (Figure 7). Strands of fibrin could be seen in some of the alveoli which were not so edematous. In all exposed rats there was a brownish discoloration of 35 Figure 5. Lung of a control rat. Hematoxylin and eosin. x 188. ~ Figure 6. Hypersmia, edema, and hemorrhage of the terminal bronchiole, alveolar ducts, and alveoli. Rat 2, which died 1-2 minutes after the beginning of exposure. Hustoxylin and eosin. x 188. 36 the erythrocytes in areas surrounding the bronchioles. This was more pro- nounced in Rats 1 through 4. There was slight peribronchiolar, perivascular, and interlobular edema. There was no evidence of necrosis or desquamation ‘of alveolar, bronchiolar, bronchial, or tracheal epithelium. Rats 5 through 9 were exposed to approximately 6,670 p.p.m. nitrogen oxides (TABLE 2) and died 12 to 125 minutes after the beginning of exposure. The degrees of alveolar edema and hyperemia generally decreased in comparison to Rats 1 through 4. Fibrin formation in the alveoli, the presence of interlobuler, perivascular and peribronchiolar edema, and the degree of epithelial necrosis were increased in comparison to Rats 1 through 4 (Figure 8). All the changes became more pronounced as the rats survived exposure for longer periods of time. Rats 7,98 and 9 had extensive fibrin formation in the alveoli. The fibrin strands occurred either singly or in loose bundles (Figures 9 and 10). Epithelial necrosis was manifested, particularly in Rat 9, by sloughing of the bronchiolar epithelima, with the acctmmlation of almost normal appearing cells in the lumina of the alveolar ducts, bronchioles, and bronchi. In the kidneys of Rats 5 through 9, and in 1 control rat (these rats ‘were from 1 group), there was increased cellularity and swelling of the glomeruli with adhesions between the parietal and visceral layers of Bowman's capsule. There was dilation of collecting tubules and eosino- philic, homogeneous casts were present in some of them. In Rate 5 through 9, especially in Rats 7 and 8, there was swelling of the convoluted tubules around some glomeruli to such an extent that no lumina were visible. 'There was marked swelling and congestion of the glomeruli so, that no Bowman's space could be seen. These changes appeared to be 37 Figure 7. Rupture of bronchiolar arteriole. Rat l, which died 1-2 minutes after the beginning of exposure. Hematoxylin and eosin. x 75. Figure 8. Cellular debris in the bronchiolar lumen. Alveolar macrophages and desquamated bron- chiolar epithelium. Rat 9, which died 125 minutes after exposure. Hematoxylin and eosin. x 188. 38 Figure 9. Formation of fibrin from proteinaceous edema. Rat 9, which died 125 minutes after exposure. Hematoxylin and eosin. x 75. Figure 10. Fibrinous alveolitis. Rat 9, which died 125 minutes after exposure. Bematoxylin and eosin. x 750. ' 39 confined to certain areas in the renal cortex. Between and around these swollen areas the renal architecture appeared relatively normal. In all exposed rats there was congestion of the kidneys, liver, spleen, and intestines. Centrolobular congestion of the liver was especially marked in an. .6 through 9. Emr iment 2 Emrigntal plan. A concrete silo 50 feet high and 12 feet in diameter was filled approximately 1/8 full of freshly chopped corn silage, so that at its peak it was 12 feet high and at its edges it was 2 to 6 feet high. Approximately 24 hours after filling, a lZ-week-old male Yorkshire pig weighing 27.3 kg. was placed in a wire cage located on the edge of the silage. The objectives were to observe the formation of the silo gas and its effect upcm the pig. Obserntions gnd eligicgl sins. Approximately 44 hours after the silage was placed in the silo a heavy, brownish-orange gas was observed. It appeared to extend upwards approximately 30 cm. above the silage at the edge of the peak. The caged pig was moved down into the gas, but the cage was large enough to permit the pig to sit with his head up. In this position the gas came up only halfway to the height of his nose, and after 20 minutes there were no signs of respiratory distress. he was removed from the cage and held in a horisontal position'in the gas. I-sdiately he began to display hyperpnea, dyspnea, and coughing. lie was held in this position for 15 minutes, by which time he exhibited open-mouthed breathing. He was then placed back in the cage, where he sat up and breathed with difficulty. Eighty-five minutes later he was dead . 40 gross patholgn. There was yellow discoloration of the skin due to the silo gas. Some hyperelaia of the skin over the abdomen was observed. an the abdominal viscera were congested. The turbinates, trachea, and major bronchi were severely inflamed, and blood-tinged froth was present in the trachea. The lungs were very hyperemic and slightly edenatous, particularly in the ventral portions. Histgpgtholggz. There was severe rhinitis and tracheitis with desquamation of epithelium and acctnaulation of epithelial cells in a fibrinonucous exudate. The desquanated epithelial cells were colt-mar, and most of then appeared to be morphologically normal. Within the lungs the desquanation of epithelium continued down to the respiratory bron- chioles. A cellular exudate consisting of bronchiolar and bronchial epitheliun, alveolar amcrophages, erythrocytes, and fibrin was found in the bronchi, bronchioles, and occasionally in the alveoli. There was severe general hyperemia, and in the ventral parts of the lung there was extensive alveolar and interlobulsr edema with fibrin forming in many of the alveoli. Some lobules appeared atelectatic and very hyperemic in comparison to surrounding lobules. The bronchioles in these lobules were lined by intact epithelial cells. Throughout the lungs there were many eosinophils scattered diffusely and in clusters around vessels and bronchioles and in the interlobular connective tissue. There was congestion of the viscera and the meningeal vessels. There was a focal interlobular eosinophilic hepatitis. This lesion and the eosinophils in the hmgs resembled the lesions produced by larval migra- tion of sue porcine parasites (Jubb and Kennedy, 1963). 41 Eaeriment ' 3 Exggriantgl plan. A concrete silo measuring 10 x 50 feet was filled 2/3 full of freshly chopped corn silage 36 to 42 hours before exposure. The silo was connected to a dairy barn by a small room which enclosed the bottom of the silo chute. Two plastic sheets were taped in an overlapping fashion to the bottom of the chute so that they hung down to the floor. The sheets were put into place 5 hours before exposure. Twelve pigs were exposed in 4 groups of 3 pigs each for 10, 5, 4, and 3 minutes by lifting up the plastic sheets and pushing the caged pigs under them. Air samples were taken before and after the 10-minute exposure and during the 5- and 4-minute exposures (TABLES 2 and 3). Survivors were killed at 15, 29, 30, 60 and 61 days after exposure. Four litter-mate pigs were killed at 6 hours and at 30, 60, and 61‘days after exposure of the experimental pigs to obtain tissues for control data. Observations and clinical siggg. Before. exposure it was noted that the spilled silage near the bottom of the chute was stained orange-yellow. An orange haze could be seen in the silo chute. The odor of the nitrogen oxides was strong, and the concentration seemed to increase after the plastic sheets were put into place. Within 1 to 3 minutes after the beginning of exposure, the pigs began coughing, sneezing, and struggling to get out of the cage. 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