COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD 1^7^ HX64090213 QP981 .N5 C36 The physiological ac THE PHYSIOLOGICAL ACTION OF NITRO- BENZENE VAPOR ON ANIMALS RECAP A THESIS Presented to the Faculty of the Graduate School OF Cornell University, for the Degree op DOCTOR OF PHILOSOPHY BY WALLACE LARKIN CHANDLER i' ^ [Reprint from the Ck)mell University Experiment Station Memoir 20, March, 191 9] q^^ftN->^^ C3t intiieCttpofilfUjgork College of ^ftpsJicians; anb burgeons! Hihvavp Digitized by tine Internet Archive in 2010 witii funding from Columbia University Libraries http://www.archive.org/details/physiologicalactOOchan THE PHYSIOLOGICAL ACTION OF NITRO- BENZENE VAPOR ON ANIMALS A THESIS Presented to the Faculty of the Graduate School OF Cornell University, for the Degree of DOCTOR OF PHILOSOPHY BY WALLACE LARKIN CHANDLER [Reprint from the Cornell University Experiment Station Memoir 20, March, 1919] QCV'^flN^' CONTENTS PAGE Introduction 412 Review of literature ; . . 413 Clinical cases of poisoning 413 Clinical symptoms of poisoning 415 Seat of action of the drug; 416 Experimental cases of poisoning 417 Resume of the literature 426 Apparatus and technique of present experiments 427 Apparatus 427 Technique 428 Obtaining pure nitrobenzene 428 Aeration of the fumigation chamber 428 Maintaining constant temperature ' . . 429 Histological technique 429 Descriptions of experiments 430 Dog I (male dachshund) 430 Dog II (male) 433 Dog IV (female) 434 Dog V (female) 435 Dog VI (female) 436 Dog IX (male) 438 Dog X (female) 439 Dog XI (female) 441 Dog XVIII (female) 443 Rabbits I and II, and Guinea Pigs I and II 444 Rabbit III and Guinea Pig III 445 Gray rats (Af ms decumanus) 446 White rats 446 Cat X 446 Cat XVIII 447 Hen V 448 Hen VI 449 Hen VII 450 Hen VIII and Rooster III 451 Pigeons IV and V 451 Observations of the action of nitrobenzene on insects 452 Fleas 452 Biting lice 452 The follicular mite (Demodex folliculorum) 453 Observations of the action of nitrobenzene on internal parasites 454 Gapeworms (Syngamtis trachealis) 454 Intestinal worms 454 Coccidia 455 Experiment to determine the action of nitrobenzene on digestive functions 455 Experiments to determine the action of nitrobenzene on blood 455 Blood counts 455 Spectroscopic examination of the blood of animals poisoned by nitrobenzene 457 Histological examination of the tissues of animals poisoned by nitrobenzene 458 Dogs 458 Birds 461 407 408 Contents PAGE The symptom complex of nitrobenzene poisoning 462 Interpretation of the symptoms named 463 Disturbance of digestive functions 464 Cerebellar disturbances 464 Cause of the latent period 466 Conclusions 468 Literature cited 471 THE PHYSIOLOGICAL ACTION OF NITROBENZENE VAPOR ON ANIMALS THE PHYSIOLOGICAL ACTION OF NITROBENZENE VAPOR ON ANIMALSi Wallace Larkin Chandler^ The present rapid development in America of industries which utilize large quantities of benzene or its derivatives in the manufacture of their products has greatly augmented the importance of the question of industrial poisoning. This is especially true of those industries in which nitrobenzene is used in some particular stage of a manufacturing process. Because of the pleasant odor and the retarded physiological action of nitrobenzene its toxic properties are not generally recognized, with the result that many workmen are constantly endangered by being either in actual contact with the liquid or exposed to its poisonous fumes. Further- more, the physiological symptoms of nitrobenzene poisoning are not well understood, altho medical literature contains a large number of reports of such poisoning; and undoubtedly a large number of cases of industrial nitrobenzene-poisoning have been referred to other causes, the real cause having been obscured by the retarded and inconstant action of this chemical. In the hope of obtaining more specific data regarding the physiological action of nitrobenzene, the initial experiments conducted by Dr. M. Dresbach and the writer on the investigation of nitrobenzene as a parasiti- cide (Chandler, 1917)^ were continued. The present researches have resulted in findings which, it is hoped, may serve to make clear some of the factors regarding the action of nitrobenzene which hitherto have not been understood; for example, the cause of the "latent period," the rea.son for the inconstancy of certain symptoms, and the specific nervous centers involved. The work has also opened up new fields for investiga- tion along the lines of neurology, physiology, and biochemistry. The writer is indebted for advice and assistance to the following mem- bers of the faculty of Cornell University: Dr. M. Dresbach, of the Depart- ' From the Department of Physiology and Biochemistry, Cornell University Medical College, Ithaca, New York. 'Formerly Instructor in Parasitology, New York State College of Agriculture, Cornell University, Ithaca, New York. Research Assistant in Entomology, Michigan Agricultural Experiment Station, Ea-Ht Lansing, Michigan, since .July 1, 1918. ' Dates in parenthesis refer to Literature cited, page 471. 411 412 Wallace Larkin Chandler ment of Physiology and Biochemistiy ; Dr. William A. Riley, of the Department of Entomology; Dr. E. M. Chamot, of the Department of Sanitary Chemistry and Toxicology; Dr. H. M. Kingery, of the Depart- ment of Histology; and Professor S. H. Gage, of the Department of Histology. INTRODUCTION Nitrobenzene (mononitrobenzene, nitrobenzol, oil of mirbane, artificial oil of bitter almonds, and so forth), C6H5NO2, is a clear, straw-colored, oily liquid boiling at 210.9° C. and crystallizing in needles at 5.7° C.^ It has the odor of oil of bitter almonds, and when undiluted has a pungent, unpleasant taste. It is soluble in all parts of alcohol, in ether, chloro- form, benzene, oils, and liquid fats, and to some extent in lipoids. It is but slightly soluble in water. It has a vapor pressure of but 1 milli- meter of mercury at 53° C. It is combustible, burning in the open air with a sooty, yellowish flame, and is explosive when heated to high tem- peratures such as would be obtained by throwing it on red-hot iron. It was first made by Mitscherlich (1834). At present it is manufactured on a large scale by ''adding one part of benzene to three parts of a mixture of nitric acid(sp. gr. 1.40) and sulphuric acid (sp. gr. 1.84), this mixture being made up of 40 parts of the former to 60 parts of the latter " (Weaver, 1917). It is used in the manufacture of explosives (indurite); in the manufacture of anilin, which is used extensively in the making of dyes; in perfuming soaps, lotions, pomades, and other toilet articles; as a solvent in the manufacture of shoe polish, floor wax, and the like; in the manufacture of flavoring extracts and certam liqueurs; and for flavoring confections. It was recommended and used with friction as a parasiticide as early as 1863,^ and has recently been recommended as a fumigant for the extermination of the external parasites of domesticated animals (Moore, 1916). The poisoning effects of nitrobenzene were noted as early as 1856. Since that time, numerous cases of fatal poisoning in man have been reported, and several experiments on animals have been conducted for the purpose of studying the poisonous action of the chemical. * Determined experimentally. (See also Landolt, Bornstein, and Roth, 1912.) 5 This recommendation stimulated investigations by Ollivier and Bergeron C1863) and by Guttmann (1806). Physiological Action of Nitrobenzene Vapor on Animals 413 REVIEW OF LITERATURE According to Letheby (1865), the ancient Greeks were apparently familiar with a substance the physiological action of which is similar to that of nitrobenzene. He states as follows: It is said that Thrasyas, the father of botany, was so skilled in the preparation of drugs, that he knew how to compound a poison which would kill by a lingering illness. Theophrastus speaks of this poison, and says its force could be so modified as to occasion death in two, three, or six months, or even at the end of a year or two years; and the writings of Plutarch, Tacitus, Quintilian and Livy are full of instances of what seem to be the same kind of slow and occult poisoning. However, nitrobenzene itself was unknown to modern chemistry until its discovery by Mitscherhch in 1834. Most of the literature relating to the toxic properties of nitrobenzene consists of clinical reports of accidental cases of poisoning, and, from the standpoint of obtaining data on the physiological action of the drug, is unreHable for the reason that important information regarding the patient's "normal" condition is entirely lacking. Also, in the few experiments recorded dealing with its physiological action on animals, the drug was administered in the liquid form by either intravenous injections or intro- duction into the stomach by means of a tube, and very seldom by vapor inhalation. However, a review of some of these cases, both clinical and experimental, may serve to help interpret the results of the present experi- ments. Short abstracts of a few of these are therefore given. clinical cases of poisoning Probably the most interesting clinical record is the case reported by Grafe and Homberger (1914) of a man who worked in a nitrobenzene factory filling containers with nitrobenzene. After working in this capacity for a period of fourteen days the man began to show symptoms of poisoning. These symptoms were a blue-gray color of the skin, headache, backache, stomach-ache, and vomiting. He continued to work for a few days after the onset of the symptoms. On Friday, October 27, 1905, he filled a double number of containers (ordinarily he handled 1600 hters), and on Saturday it was with difficulty that he continued to work. On Sunday he felt better, went into a saloon, drank two glasses of beer, and started to play cards, when he suddenly became ill. He went to see his father, but felt so ill indoors that he went outside for fresh air; immediately on coming into the air, however, he fell to the ground unconscious and was 414 Wallace Larkin Chandler carried back into the house. A doctor who was called gave the following report of the symptoms: skin blue-gray; pupils dilated ; resphation retarded; pulse weak and irregular; patient unconscious. The man regained con- sciousness on the following day, and complamed of pains in his head, his stomach, and his back. On July 30, 1906, the patient was found to have a chronic gastritis; his hemoglobin was 125 per cent and his red-cell count was 6,500,000. His intelligence and sense of perception had become dimmed. By July, 1908, his muscles had become atrophied, and he was extremely emaciated. His blood examination showed the following: red-cell count, 5,600,000; hemoglobin, 80 per cent; lymphocytes, 32 per cent; polymorphonuclear leucocytes, 47 per cent; eosinophiles, 20 per cent. By October, 1909, his memory had failed; otherwise he was in about the same condition as in 1908. In 1911 he was asked the date; he looked for a calendar and said that he did not know whether it was , 1910 or 1911. His perception of distances had failed also. On October 4, 1912, he was visited by the doctor. Wlien asked whether he recog- nized the doctor, he said that he had seen him before, but where and when he could not recall; thus he showed loss of perception' of both tune and space. He gave correctly the names of his children but could not remem- ber which was the eldest. In March, 1913, his condition was about the same. . If he wandered some distance from his home he was unable to find his way back. Several other incidents revealed the loss of perception of time and space. Grafe and Homberger beheve that the type of psychosis shawn by this patient is identical with Korsakoff's syndrome, in which there is a loss of memory not only of things occurring before the accident but also of things occurring afterward. However, a careful analysis of the symp- toms as recorded brings out the probability that the loss of perception of time and space was the principal feature; and this, according to JeUiffe (1913), indicates cerebellar lesions. Taylor (quoted by Adams, 1912) reports the case of a young man who worked in a chemical laborator3^ The report states that the young man placed one or two drops of nitrobenzene on his tongue in order to remove the odor of a pipe he had been smoking. He repeated this action one and one-half hours later. In a few hours he was seized with convulsions and became unconscious. The coma lasted for about six hours, but the patient died in about fifteen hours after regaining conscioiJisness. Physiological Action of Nitrobenzene Vapok on Animals 415 Another case of- fatal poisoning; resulting; from a small amount of nitro- benzene is reported by Stone (1904). The report states that a strong man, weighing one hundred and sixty pounds, had stained the uppers of a pair of shoes with hquid shoe-blacking. He put the shoes on before they were dry and spent the evening in a cafe. About midnight he fell to the floor unconscious, and he died a few hours later. These two cases are interesting from the fact that small quantities of the drug were able to produce death^ Absorption in each case was prob- ably facilitated by the presence in the blood of some solvent for nitro- benzene. In the latter case, the ingestion of alcoholic liquor undoubtedly facihtated the absorption thru the skin. On the other hand, a number of cases have been reported in which the patient recovered after the ingestion of large quantities of nitrobenzene. Two interesting examples are as follows: Dodd (1891) reports the case of a man forty-seven years of age, who ingested two drams of nitrobenzene. The resulting symptoms were vomiting, extreme cyanosis, fixed jaws, contracted pupils. The patient eventually recovered. Schild (quoted by Adams, 1912) reports the cases of three gii'ls who took nitrobenzene as an abortifacient. The approximate amounts taken were as follows: one ingested 5 mils, and recovered; the second ingested 16 mils, and recovered; the third took l(i mils, and died. clinical symptoms of poisoning Regarding the characteristic clinical symptoms of nitrobenzene poison- ing, Tiirk (quoted by Roth, 1913) says:'' " The clinical picture consists on the one hand of a greater or less stimulation of the gastro-intestinal tract; secondly, of changes in the blood which consist of the formation of methemoglobin and destruction of erythrocytes, with associated phe- nomena of high-grade cyanosis, blue skin color, later icterus, dyspnoea, etc." Weisstein (1892) says that there is great variation in the symptoms. He says that those symptoms which might be called characteristic are: Great dyspnoea, livid, cyanotic color of the skin, and characteristic bitter-almond-oil odor of the breath; even the urine may have this odor. In addition we may have symptoms which are not exactly characteristic, as they are inconstant, such as incoordination, hesitating speech, drov/siness, numbness, vomitinj^, Cf)iivuIsioiis, coma, dilation or contraction of pupils, or unequal dilation, nystaKuius, irn^j^uhir pulse. Death is due to a failure of respiration and circulation. In animals and in man the symptoms may appear early or late, even days late." ■Translation from the original German. 41(3 Wallace Larkin Chandler Wei^stein states further that the lethal dose depends on the form m which the dose is taken, the condition of the stomach at the time of taking, and other factors. • SEAT OF ACTION OF THE DRUG Regarding the specific tissues acted upon by nitrobenzene, there has been much speculation. Roth (1913) writes as follows:^ " The action of nitrobenzene depends, as is known, upon blood changes. Hence hematological changes have excited the greatest interest, tho in differ- ent cases the findings differ greatly." Some writers report pronounced hemolysis accompanied by leucocytosis, and those who have subjected the blood of patients poisoned by nitrobenzene to spectroscopic analysis have for the most part found an absorption band occupying nearly the same position as the methemoglobin band. Some have regarded this band as the methemoglobin band and have concluded that methemoglobin is formed in the blood in cases of nitrobenzene poisoning. Others claim that the band is distinct from the methemoglobin band and is pecuhar to nitrobenzene alone. A discussion of these findings appears on a later page of this article. The following notes on the observations of blood changes in clinical cases may be of interest: Roth (1913) reports the case of a woman who drank nitrobenzene to produce abortion. Her blood became dark chocolate in color. The red- cell count dropped from 4,662,000 to 3,840,000; the hemoglobin dropped from 98 to 80 per cent; the number of leucocytes dropped from 16,840 to 9400; and the lymphocytes rose from 14.4 to 22.3 per cent. There. was no methemoglobin in the centrifugalized blood serum, but methe- moglobin appeared in the red corpuscles. Hence Roth concludes that methemoglobin is formed inside the red corpuscles. He thinks that the formation of methemoglobin is due to the action of p-aminophenol, formed from the nitrobenzene. Meyer (1905) reports the case of a patient who took about a teaspoonful of nitrobenzene, presumably to produce abortion, on June 15. On the 16th and 17th of the month the body temperature was 38° C. The red- cell count dropped to 2,180,000; the white-cell count dropped to 5200; the hemoglobin was 54 per cent. There was no morphological change in the red corpuscles. ' Translation from the original German. Physiological Action of Nitrobenzene Vapor on Animals 417 Massini (1910-11) cites two cases. The first is that of a man thirty years old, a worker in a chemical laboratory, who on November 30 drank by mistake 30 mils of nitrobenzene. Vomiting was at once induced by giving 1| grams of copper sulfate in 50 mils of water. The color of the blood was dark brown until December 3. The red-cell count had dropped to 1,800,000 by December 9, but returned to normal by January 11. The white-cell count was 22,400 on December 7, and 6800 on January 11. The lymphocytes rose from 22.5 per cent on December 2 to 41.7 per cent on December 26. The polymorphonuclears dropped from 76.8 per cent on December 11 to 54.7 per cent on December 26. The second case cited by Massini is one of chronic poisoning in a man thirty years old, who worked in a room with nitrobenzene. He was poorly nourished, the color of his skin was blue-gray, the spleen was enlarged, the urine was veiy dark, and urobilin was detected by spectroscopic analysis. The man was achnitted to the hospital on June 4. The red- cell count was then 2,500,000, but it had risen to 4,300,000 by June 29. The white-cell count was 6000 on June 4, had risen to 8400 by June 8, and had dropped to 3800 by June 24. A microscopic examination of the blood revealed embryonal forms. Bondi (1894) reports the case of a man twenty-five years old, who drank a "mandel liqueur" at nine o'clock in the evening. He was admitted to the hospital at four o'clock in the afternoon of the following day. The red-cell count was 6,340,000, the white-cell count 16,000; there was no methemoglobin ; the hemoglobin was 112 per cent. Only one examination was made. experimental cases of poisoning The literature dealing with experiments on animals is not very consider- able. Only twice have intensive experiments been carried on, by Letheby (1865) and by Filehne (1878). It may therefore be well to include here a short abstract of the literature dealing with the subject from the experi- mental side. The first experiment recorded was conducted by Jones (1857). He gave one dram of nitrobenzene to a rabbit, and reports that the rabbit was killefl instantly. One-half diani of nitrobenzene in two drams of water, given internally, killelve hours. This writer giv(!s no reference to earlier works, nor does he describe the symptoms 418 Wallace Larkin Chandler of nitrobenzene poisoning. He had been working on the toxic properties of a commercial oil of bitter aimonds which was known to contain hydro- cyanic acid. The experiments with nitrobenzene were conducted as a side issue and the purity of the nitrobenzene is doubtful. Casper (1859) was led to experiment with nitrobenzene because in post-mortem examinations of cases of poisoning, the cause of which had been diagnosed as hydrocyanic acid, he often detected the odor of nitro- benzene in the tissues. He mentioned the desirability of learning whether this drug was poisonous, since it was being used more or less extensively in the perfuming of soaps, pomades, and the like, and stated that so far as he knew there was no account of it as a poison. His article contains a short account of the chemical and physical properties of the drug. In his experimental work he gave an ounce of nitrobenzene (purity not stated) to a rabbit in four separate doses at intervals of fifteen minutes. Within a few minutes after the final dose, the animal fell suddenly on its left side. Its pupils were dilated. Convulsions occurred which involved the entire body, and within a few minutes the animal was dead. The body was allowed to remain untouched for a period of twenty-four hours in order to simulate forensic post-mortem cases. It was then opened. No odor of nitrobenzene was detected either externally or in the lower part of the digestive tract, but when the skull was opened a strong odor of nitrobenzene was given off. This odor was so pronounced that a new- comer, who was wholly unaware of the experiment, at once spoke of " almond oil." The odor was detected in the blood, in the brain, and in other tissues. The body was placed in a cellar for two weeks and at the end of that time had lost but very little of the odor. ^ F. Hoppe (reported by Casper, 1859) introduced 20 mils of nitro- benzene into the stomach of a medium-sized dog. After a few hours the dog appeared stupid, and at the end of twelve hours it was found in a deep coma. Respiration was slow and the skin temperature was lowered. The animal was killed by pithing without causing convulsions. Blood drawn from the subclavian vein was dark brown in color, and the odor of nitrobenzene was detected in it. The same odor was detected in the urine, which was dark brown, in the bile, and in all the organs. The stomach contained a few drops of nitrobenzene, and the contents of the stomach were strongly alkaline. The blood retained the odor of the drug for several days. Casper concludes that these experi- Physiological Action of Nitrobenzene Vapok on Animals 419 ments prove nitrobenzene to be a poison; and that a distinction between cases of poisoning by nitrobenzene and by hydrocyanic acid can be readily made, since the bitter-almond-oil odor due to hydrocyanic acid disappears within three or four days — the chemical (hydrocyanic acid) being destroyed by contact with the tissues — while the same odor due to nitro- benzene will persist for several days. Ollivier and Bergeron (1863) were led to study the action of nitrobenzene from the same standpoint as was Casper — that is, because the drug was being extensively used in perfuming toilet soaps and in making flavoring extracts. It had also been recommended afid used with friction for the cure of parasitic affections. These investigatois argued that since nitrobenzene is readily converted into anilin thru the action of nascent hydrogen, it is conceivable that it may be changed into anilin in the human body, and anihn is a poison. They gave a guinea pig ten or twelve drops of nitrobenzene. The animal, after the initial agitation and profuse salivation, remained motionless for some time and then began to run about again without showing the least signs of ill effects from the drug. To another guinea pig they gave approximately three grams. The experiment was begun at 2.12 o'clock, and at 2.40 the animal exhibited tremors without excessive convulsions. The heart beats were very faint and the respiration was decidedly labored. At 3.10 the animal attempted to turn around and fell on its right side. The animal died a few minutes before 4 o'clock. In another experiment Ollivier and Bergeron exposed the muscles of a frog's leg and placed on them a drop of nitrobenzene. The muscles remained sensitive to electric stimulations for a long time and did not show the slightest histological changes. The investigators then exposed the heart of a living frog and placed a few drops of nitrobenzene on it without obtaining the slightest modifications of the heart beat. In a fifth experiment OlUvier and Bergeron gave a large, healthy dog six drams of nitrobenzene, and in a half hour an additional dose of five drams. The experiment was begun at 12.40 o'clock. The animal appeared agitated and secreted saliva profusely, but did not vomit. Finally it lay down in a dark corner and remained motionless for some time. At 1.20 it started to howl, appeared excited, and moved its head convulsively. Its tongue was hanging out and its eyes were wide and animated. This condition lasted for about six minutes, when the dog again became motion- 420 Wallace Lakkin Chandler less. At 5 o'clock it showed tremors, and about an hour later its hind extremities were paralyzed. On the following morning it was found dead and rigid. A post-mortem examination showed the following: The blood was about normal, the cells being little altered, but the blood plasma contained fine, oily droplets which were recognized to be nitro- benzene. The meninges were congested, the veins turgid, the tongue and mucosae violet in color, and there was a stasis of the blood in the capillaries. The heart was dilated and filled with viscous blood, but there were no clots. The "authors claim to have found anilin in some of the organs and in the blood, as well as nitrobenzene. They state that they had tested the nitrobenzene for the presence of anilin just prior to the experiments and had not found a single trace. Hence they conclude that their original assumption was correct — that nitrobenzene may be con- verted into anilin in the living body of an animal. In another experiment these investigators caused a young dog to ingest a daily dose of from two to three grams of the drug for a period of sixteen days. They then killed the dog. They found no trace of anilin in any of the organs except the spleen and the liver. From these experiments Ollivier and Bergeron draw the following con- clusions: (1) that death due to nitrobenzene poisoning is delayed as compared with death due to an equal dose of anilin; (2) that nitrobenzene given in small daily doses is eliminated in part as such, and changed in part to anilin, which accumulates in the spleen and fiver; (3) that the drug is in time eliminated as nitrobenzene and anilin. and is not changed into picric acid ; (4) that animals poisoned by nitrobenzene die with symptoms of asphyxiation; (5) that the symptoms preceding death are similar to those in the case of anilin poisoning, except that the animal exhibits tremors, not convulsions of the whole body as in the case of anilin poison- ing; (6) that nitrobenzene does not appear to cause any direct alteration of the blood, the muscles, the heart, the nerves, or other organs.^ In a further experiment with nitrobenzene, these authors placed guinea pigs, cats, and other small animals under a bell jar and introduced air saturated with the vapor of nitrobenzene, allowing a small opening for ventilation. Under these conditions they were able to produce death in from two to five hours, death being preceded by characteristic symptoms such as staggering, tremors, and paratysis of the hind legs. 8 The staining of nerve cells by the Nissl method was not developed until 1885. Physiolouical Action of Nitkobenzenk Vai'oii on Animals 421 Letheby (1865) carried out a number of experiments for the purpose of studjdng the effects of nitrobenzene on dogs and cats. The drug was invariably administered by introducing it into the stomach. The results obtained in his various experiments were fairly similar; therefore the following report (page 49 of reference cited), which is quoted verbatim, may be taken as characteristic of the results obtained by him: Experiment 2.^ January 16th, 1862, at half-past three p. m., I gave half a drachm of nitro-benzole to a small terrier dog. The poison was poured into the animal's mouth; it caused discomfort, as if from the unpleasant taste, and produced a copious flow of frothy saliva. This, however, soon subsided, and for an hour there was no perceptible effect beyond a little heaviness of look. At the end of an hour the animal was sick, and after that it became sleepy. In another hour it was again sick, and again in a quarter of an hour. For four hours the animal lay on its side asleep, and then some water was given to it, which it took freely; from that time till midnight nothing appeared to be the matter with it, and the next morning it seemed to be quite well, and ate its food heartily. It remained thus all day, and was left at night apparently well, but the next morning at half-past six o'clock it was found upon its side insensible. The b-^s were in constant motion, as if the animal was running. The head was drawn back, and the muscles of the neck were rigid, as if in spasm; the eyes were open, the pupils were widely dilated, and the conjunctiva was insensible to the touch. The animal lay in this state for sixty-six hours, that is, nearly three days, and then it died as if from exhaustion. During the whole of this time the legs were in constant motion; there were occasional spasms, and then a sort of struggle for breath. The heart beat in an irregular, tumultuous manner, and the breathing was somewhat laborious. The total time which elapsed from the taking of the poison to the death was one hundred and four and a half hours. The bod}^ was opened twelve hours after death. The brain and its membranes were very vascular; there was no odour of the poison in any part of the body; the lungs were slightly congested; the heart was full of blood on the right side, and there was a little on the left; the liver was of a deep purple colour; the gall-bladder was full of bile; the stomach was nearly empty, it only contained a little fluid and mucus; there was no sign of irritation. On analysis it yielded a trace of aniline, but no nitro-benzole; and nothing was found in the brain. Letheby divided the action of nitrobenzene into two classes, character- ized respectively by rapidly developing coma and by slow paralysis and coma after a considerable period of inaction. He summarizes the symp- toms as follows (page 42 of reference) : ■^'hen the effects were speedily fatal, the animals were soon seized with giddine,ss and an inability to walk. The weakness of the limbs first appeared in the hind extremities, and was manifested by a difficulty in standing; but very soon it extended to the fore legs, and then to the head and neck. There was complete loss of voluntary power; the animals lay upon the side with the head drawn a little back, and with the limbs in constant motion, as if in the act of trotting or running. The muscles of the back were occasionally fixed in spasm, and every now and then the animals had a sort of epileptic fit. They looked distressed, and howled as if in pain, and struggled violently; after which they always seemed exhausted, and lay powerless. The pupils were widely dilated, the action of the heart was tumultuous and irreg- ular, and the breathing was somewhat difficult. For some time, however, the animals retain«'d their consciousness, and gave signs of intelligence when spoken to; but suddenly, and nfU'u at the close of a fit, th(!y became comato.se, the eyes remaining open, although the Cf)njunctivii was insensible to touch, and the movements of the limbs would m-arly cease, the breathing became slow and ,sadily; 428 Wallace Larkin Chandler paraffin is used for effecting an air-tight seal. It was thru this opening that animals were introduced into the tank, thus obviating the necessity of removing the entire cover each time. A small glass window was also built into one side of the tank, thru which observations of the temperature inside could be made. The tank rests on two runners, one of which is lower than the other in order to provide a slant to facilitate drainage of urine thru a small hole in one corner of the bottom of the tank. A false removable bottom 1 inch in height, made of strong wire of No. 2 mesh and well supported, was constructed in three pieces, for ease in handling. This false bottom serves to keep the animal from contact with its excretions. Two parallel steel supports are placed across the width of the tank 1| feet above the bottom, and on the middle of these supports rests a wire cage 8 X 8 X 10 inches. This cage serves to protect a small fan, which is connected to a motor on the outside by a shaft passing thru a tightly fitting collar in one side of the tank. This cage also protected a triangular strip of cheesecloth from which the nitrobenzene was evaporated. The container into which the cheesecloth dipped rested on an aluminum tray in the bottom of the cage. The inside of the tank and all its internal accessories were coated with paraffin in order to prevent rusting. TECHNIQUE Obtaining pure nitrobenzene Practically pure nitrobenzene was obtained by redistilling the commercial liquid, at the temperature of the boiling point of nitrobenzene, until a product was obtained which proved experimentally to have a boiling point and a freezing point corresponding to pure nitrobenzene (page 412). Aeration of the fumigation chamber The tank was aerated by passing air saturated with nitrobenzene into it at a rate determined by the weight of the animal being fumigated. The air was saturated by passing it, after dehydration, thru a flask con- taining nitrobenzene and kept at a temperature of 50° C, and condensing it by passing it thru a series of U-tubes containing nitrobenzene. The final condensing tubes were placed inside the tank, so that the final con- densing temperature equaled the temperature of the tank. Physiological Action of Nitrobenzene Vapor on Animals 429 That this method for saturating air with nitrobenzene is practical was shown experimentally in the following way: A glass-stoppered U-tube containing a little nitrobenzene was dehydrated and weighed to constant weight. This U-tube was then placed in a constant temperature chamber, which was also a desiccator, with the final condensing tube, and air from this condensing tube was passed thru the U-tube for a given period of time. The U-tube was then reweighed, and the fact that it had neither lost nor gained in weight indicated that the air coming from the final condenser was saturated. Maintaining constant temperature A constant temperature ±1 degree centigrade was maintained in the fumigation chamber by regulating the temperature of the room, it being found that a direct relation existed between these two temperatures. Histological technique In preparing sections for histological studies the following technique was employed. The animal was quickly and painlessly killed by piercing the heart with a scalpel, since it was important that death should be produced without the use of drugs and in a manner which would produce a minimum shock. The body was opened immediately, a cannula was connected with the aorta, and a warm (normal body temperature) isotonic saline solution was transfused until all the blood was washed out. The saline solution was immediately followed by the warm fixing fluid, which consisted of 4- per-cent formaldehyde in a saturated aqueous solution of corrosive sub- limate. The brain and the cord were then quickly dissected out, small pieces from each being placed directly in the fixing fluid and allowed to remain for twenty-four hours. The pieces were then washed in run- ning water for twenty-four hours, after which they were carried thru 50-, 60-, 70-, and 82-per-cent alcohol. They were allowed to remain in 82-per-cent alcohol, to which was added a few drops of 5-per-cent alcoholic iodine, until the excess corrosive sublimate had been removed. The alcohol was changed twice a day for a time, and th(Mi as frequently as it became? decolorized. The tissues were then dehydrated, cleared, emljcflded, and sectioned. The sections were cut 4 and 5 microns thick 430 Wallace Larkin Chandler and were fixed on slides by the usual methods. They werei then cleared and carried down thru the various grades of alcohol to water, and then stained for ten minutes in hot (70° C.) 10-per-cent methylene blue in saturated anilin oil water (Rasmussen and Myers, 1916). When taken from the staining fluid they were hurriedly rinsed in a large volume of water, and were then placed directly in 95-per-cent alcohol where they were allowed to remain until they were sufficiently destained. They were then dehydrated, cleared in xylene, and mounted in balsam. Corre- sponding tissues from each of the poisoned animals and from the controls were carried thru the same fluids and stained on the same shdes. DESCRIPTIONS OF EXPERIMENTS A large number of experiments were carried out. Since, however, the symptoms for each group of animals were in general fairly similar, so far as locahzing the action is concerned, only two or three experiments from each group in which the animals showed typical symptoms are included in the descriptions. The other experiments included are those in which the symptoms were different in essential details. DOG I (male dachshund) Weight of dog, 16.4 kilograms. August 30, 1916 — Dog fumigated at 26° C. for a period of five and one- half hours. ^ Time when fumigation was begun, 11.15 a. m. Time when fumigation was finished, 4.45 p. m. Observations : After having become accustomed to the strangeness of the fumigation chamber, the animal lay down and became quiet. At 2 p. m. it was observed that the dog had vomited, urinated, and defecated. At 3 p. m. the animal was seen to stagg'er when attempting to walk from one end of the tank to the other. At 4 p. m. the animal was found lying on its side; it was unable to hft its head; its respiration was labored. At 4.30 the condition was about the same. At 4.45 the animal was removed from the tank. The respiration was slow and regular, except for intermittent long, deep inhalations; the animal was unconscious; ' The chamber was thoroly dehydrated before the experiment was begun, but in this instance no time was allowed for saturation. Ordinarily sufficient time was given to insure saturation of tlie chamber before the animal was introduced. Physiological Actiox of Nitrobenzene Vapor on Animals 431 the spinal reflexes were apparently gone; the conjunctival reflexes were present; there was profuse salivation. The muscles of the entire body were wholly relaxed when the animal was removed from the tank, but soon all of the legs became extended and rigid. This condition gradually passed off and the animal showed signs of recovery. By 11.30 p. m. the dog had regained consciousness and was able to stand on its feet. It walked slowly, with a trembling, staggering, uncertain gait and without aim. August 31 — The anim.al was found to have greatly improved. It refused food, however, and howled when the back of its head was touched. It would press its head against the attendant's legs or other objects, and remain thus for hours. Seytemher 1 — The dog was found normally active, eating meat and drinking water freely. No other effects followed. September 22, 1916 — Dog fumigated a second time, this time at 23° C. for a period of six hours. Time when fumigation was begun, 11.10 a. m. Time when fumigation was finished, 5.10 p. m. Observations: At 11.15 a. m. the dog was observed licking its chops. At 11.30 it was panting at intervals as if short of breath. At 12 m. it was slightly drowsy. At 12.05 p. m. it was unsteady, sitting on its haunches but keeping this position apparently with difficulty. At 12.10 it lay down in a natural position and closed its eyes; it would open its eyes when one rapped at the tank, but would immediately close them again. At 12.20 the dog's head was raised and the breathing was slow and labored. At 12.30 the animal appeared slightly confused, and uncertain as to the direction of the sound when one tapped on the tank. At 12.50 the animal stood up when called and walked across the chamber, but immediately went back and lay down again. From that time until 5.10, when it was removed from the tank, the animal lay quiet but alert as if sensing some danger. After the dog was removed from the fumigation chamber fresh vomit was found in the tank, which had probably been (iinitted while th(; chamber was being opened. A copious secretion of diva was ol)serv(.d and the animal was slightly unsteady on its feet. 11 refused water. 432 Wallace Larkin Chandler September 26 — The animal had apparently recovered and no symptoms of the action of the drug appeared until on this day (four days after exposure to the vapor) when the attendant observed that the dog had difficulty in using its hind legs. An examination was made and the animal appeared normal. This was about 9 a. m. By 11 a. m., however, incoordination of the muscles of the hind legs was observed and the animal walked with a peculiar sprawhng gait. This condition became more pronounced as the day advanced, and by 3 p. m. it was almost impossible to induce the animal to walk at all. Its tendency was to crawl into dark corners and hide. Finally the animal refused to remain on its feet, and when placed on its feet it would stumble and fall down again. However, at 4.30 p. m., after a considerable period of rest, the dog walked into its kennel with a slow, staggering, sprawling gait. September 27 — There was evidence that the dog had thrashed about a good deal during the night and it was found lying prone on its side in the kennel. The animal appeared conscious, but was very irritable and thrashed about considerably. At 3 p. m. pronounced nystagmus was observed; the left pupil was dilated, the right pupil was contracted, and the jaws were set. At 4 p. m. the animal showed a tendency to remain on its left side; when turned on its right side, it executed a right-to-left rotation, finally coming to rest on the left side. The animal seemed to be conscious and wagged its tail when spoken to. The flexor muscles of the hind legs were in a state of tense tonic contraction, drawing the legs up against the body. The animal would neither eat nor drink. The anal temperature was 37.8° C. September 28 — The general condition of the animal was about the same. The trunk muscles were tremulous, the extensor leg muscles contracted. The legs were withdrawn once or twice when touched, but finally they failed to react even to the prick of a pin, so tense was the muscular, con- traction. The animal made swallowing movements and could work its jaws to some extent, swallowing water when placed in its mouth. Nystagmus was not so pronounced. The anal temperature was 38.3° C, September 29 — The animal appeared to be somewhat improved. Nystagmus was decreasing. The dog swallowed milk when placed in its mouth. The flexor muscles were relaxed. Clonic movements of the Physiological Action of Nitrobenzene Vapor on Animals 433 hind legs were observed. The anal tonipcraturc was 39° C. The heart rate was increased only when the animal struggled, but was a trifle irregular. September 30 — The animal was very much impi'oved. It ate chopped meat and drank milk, and could raise itself a little. Its head waved about in an uncontrollable manner. The anal temperature was 38.5 C. October 1 — Still more improvement was observed. The dog could almost regain its feet. It refused water, but drank milk without urging. In the afternoon the animal was able to stand on its feet; it walked eight or ten steps, and then staggered and fell. The anal temperature was 38.7° C. October 2 — The animal walked fairly well, but staggered a great deal. It ate greedily. October 3 — The animal had regained nearly the normal use of its legs and was found running about with other dogs. This dog finally recovered entirely and never developed any further symptoms. It is interesting to note that this animal exhibited symptoms just the reverse of those descril^ed by Filehne as following a retarded action of the drug (page 424). DOG II (male) Weight of dog, 11.5 kilograms. September 1, 1916 — Dog fumigated at 25° C. for a period of two hours and fifty-nine minutes. Time when fumigation was begun, 12.40 p. m. Time when fumigation was finished, 3.39 p. m. Observations: Soon after being introduced into the chamber, the animal was observed to lick its chops; it panted at intervals; respiration was accelerated. At 1.45 the animal appeared restless, howling a good deal; it appeared to stagger. At 2.30 the animal was unable to remain on its feet; it lay on its side, with the extensors of all legs in tonic con- vulsions. At 2.50 the condition was about the same as at 2.30. At 3 p. m. the dog made sounds as if it was becoming anesthetized ; there were clonic convulsions of the extensor muscles of the fore legs, and occasional clonic convulsions of the extensors of the hind legs followed by general abdominal 434 Wallace Larkin Chandler muscle tremors; respiration was quickened, with periodic long, deep inhalations; the dog was apparently unconscious, and could not be aroused; the eyes were open and winking; there was nothing abnormal about the pupils, and no nystagmus. At 3.04 the condition was about the same; the dog moaned at intervals. At 3.24 the respiration was 40, and increased in depth with periodic long, deep inhalations as before. At 3.25 there was opisthotonos, the convulsions lasting for about one-half minute and being followed by accelerated respiration. At 3.27 the respiration was 52. At 3.35 the respiration was shallow. At 3.39 the animal was removed from the tank; the muscles of the entire body were relaxed, but soon the leg muscles stiffened; the tongue and the Ups were cyanotic. At 3.40 the respiration was irregular, gasping; the animal was given artificial respiration and oxygen, but it died at 3.50. The body was opened immediately. The heart blood was of a chocolate color; the lungs were a dark gray; the intestines were hyperemic; the liver and the spleen were coffee-colored. In this case the type of symptoms described by Filehne as following a rapid action of the drug were undoubtedly shown. DOG IV (female) Weight of dog, about 12 kilograms. September 27 , 1916 — -Dog fumigated at 21.5° C. for a period of ten hours. Time when fumigation was begun, 8.30 a. m. Time when fumigation was finished, 6.30 p. m. Observations: Immediately after being introduced into the fumigation chamber the animal lay down and went to sleep. It scarcely moved from this position during the entire ten hours; when the observer tapped on the tank the animal would open its eyes; when the tapping was loud it raised its head but seemed confused and could not follow the sound. At 4.30 p. m. the respiration was observed to be decidedly increased. At 5 p. m. the observer tapped loudly on the tank, and the animal opened its eyes but did not raise its head, tho it appeared normal. When removed from the tafik at 6.30, the animal v/as lively, eating and drinking freely, and depositing a great quantity of apparently normal urine. This animal developed no symptoms of poisoning aftoi-ward. Physiological Action of Nitrobexzene Vapor on Animals 435 DO(i V (female) Weight of dog, 11.2 kilograms. October 26, 1916 — Dog fumigated at 20° C. for a period of twelve hours. Time when fumigation was begun, 8.30 a. m. Time when fumigation was finished, 8.30 p. m. Observations: The animal rested quietly during the entire course of the experiment, and was removed from the tank apparently unharmed. It ate heartily of roast beef and showed no symptoms of poisoning during the next three days. October 29 — The animal vomited when it was taken out of the kennel, but no other sjanptoms were especially noted during the day. October 30 — The animal was found on its side and was unable to stand. The following symptoms were observed : lack of coordination of the muscles of the extremities; neck muscles rigid and head drawn back on the body; ventroflexion of back; fore legs drawn up to the body; one or both hind legs involved in clonic convulsions; nystagmus. October 31 — The condition was slightly improved. The fore legs were drawn up as before; the hind legs were extended, but were flexed on the body. The right pupil was widely dilated and the left pupil was con- tracted; this is just the reverse of the pupillary reactions observed in Dog I. Nystagmus was still in evidence. The neck muscles were not so rigid. The dog was very restless all day, but quieted down toward evening. It swallowed milk when placed in its mouth. November 1 — There were no signs of nystagmus. The general condition was very much improved. The dog swallowed milk when placed in its mouth. The leg muscles were not particularly involved. Both pupils were widely dilated. The animal appeared to be conscious but did not howl. November 2 — The dog's condition was very much improved. It noticed the ob.server as soon as he entered the room. The animal took milk freel3^ Nystagmus was observed at times. The pupils were somewhat dilated. The animal was al)le to raise its head. November 3 — The condition was still more improved. The animal drank milk and water readily. Its head was raised. Its fore legs were f(jld(,'d beneath the body, but were stiff. . 436 Wallace Larkin Chandler November 4 — The animal was able to stand but was unsteady on its feet. It ate meat and drank milk and water. November 5 — The condition was about normal. The dog's appetite was good. The heart and the respiration were apparently not ajffected in this case. January 20, 1917 — Dog fumigated a second time, this time at 20° C. for a period of five hours. Time when fumigation was begun, 2.45 p. m. Time when fumigation was finished, 7.45 p. m. Observations: As before, the animal remained quiet during the entire course of the experiment, and was taken from the tank apparently unharmed, and eating and drinking heartily. January 21 — No symptoms had appeared by morning. At 8 p. m., however, the animal exhibited an apparent stiffness in the hind legs. This passed off dining the night, and no further symptoms were developed until on the morning of January 24, when a loss of coordination of the muscles of the hind legs was observed and the animal walked with a sprawling gait very similar to that shown by Dog I. Toward evening this condition was much more pronounced, and it persisted during the course of two days altho the animal developed no further symptoms. January 27 — All lameness was apparently gone, and recovery was complete. DOG VI (female) Weight of dog, 10.7 kilograms. October 27, 1916 — Dog fumigated at 25° C. for a period of three hours. Time when fumigation was begun, 3.05 p. m. Time when fumigation was finished, 6.05 p. m. Observations: The animal remained quiet during the experiment and was removed from the tank in a perfectly normal condition. It ate heartily of roast beef. It had developed no symptoms by November 3. November 3, 1916 — Dog fumigated a second time, this time at 22° C. for a period of four hours and forty -five minutes. Time when fumigation was begun, 3 p. m. Time when fumigation was finished, 7.45 p. m. Physiological Action of Nitrobenzene Vapor on Animals 437 Obsei-vations : -The animal was removed from the tank apparently unharmed, and developed no symptoms during the night. November 4 — At 8 a. m. the dog was running about in a lively condition and was apparently normal. At 2 p. m., however, the animal was found in convulsions; the legs, particularly the hind legs, exhibited tetany- and the muscles of the abdomen quivered violently. By 6 p. m. the convulsions had become even more pronounced, and the animal refused food and drink. November 5 — At 10 a. m. the animal was found with its body flexed to the left, and rigid; all the legs were rigidly extended; when an attempt was made to straighten the animal out, the head would crash violently against the floor and the animal would immediately return to its former position. At 2 p. m. the animal's condition had not changed and it was decided to attempt to relieve its condition b}^ a blood transfusion. At 4 p. m. this operation was undertaken; the animal became anesthetized with ether very readily, and did not struggle on coming out of the anesthesia; approximately 200 mils of dark coffee-colored blood was drawn from the carotid artery of the poisoned animal, and 500 mils of defibrinated blood from a healthy dog was transferred thru the femoral vein. At 7 p. m. the animal was found in a stupor; the res- piration was fairly regular. At 9 p. m. the respiration was very irreg- ular and labored; at intervals of about one and one-half minutes there appeared incoordinated movements of the muscles of the diaphragm and the chest, each set working alternately with the result that no air was inhaled; these spasms lasted for from one-half to three-fourths of a minute, and at their height all the legs would move as if the animal were swimming, and would then become extended and rigid; the muscles of the abdomen would quiver, then the animal would give one or two deep gasps and regular respiration was resumed for a time but gradually became lessened in depth again until the incoordinated movements reap- pciared; an attempt was made to obtain a kymographic record of the respiration, but the animal became so active that this was impossible. At 10 p. m. the animal's condition was about the same, tho a shght improve- ment in the respiration was observed; the heart rate was 52, the res- piration 30-40, 438 Wallace Larkin Chandler November 6 — The animal seemed not to be greatly improved; the respira- tion was irregular, with a tendency to return to the type observed the day before, but it never reached that type again sufficiently to give a good record At 12 m. the anal temperature was 26.8° C.;the animal urinated and passed very dark soft feces; the external anal sphincter was relaxed, but the internal sphincter was about normal; the respira- tion was regular but very weak; the animal made swallowing move- ments and was given a very little water, which was swallowed with difficulty. At 12.30 p. m. the animal was placed m a warm room (30° C)- the respiration was fairly regular but weak, and the animal gave occasional gasps. At 2.30 p. m. the external temperature was rather low and the room temperature was therefore increased; the dogs respiration was 48, and was regular but very shallow; the heart rate was 64 and was very regular. At 3 p. m. 1 milligram of strychnin sulfate was injected; the anal temperature was 30.5° C, the external anal sphincter was still relaxed. At 4 p. m. the respiration had improved to some extent, but it became shallow again at 4.30; the rate was about, 50 a minute; the heart rate was 75. At 5 p. m. the annnal was found gasping weakly; the heart rate was above 100. The animal died at 5.10, apparently as the result of respiratory failure; the sound of the heart indicated that that organ was in excellent condition. At 6 p. m the body was opened; the general condition of the organs was found to be good; the spleen and pancreas were of a dark blue color; the peritoneum was shghtly hyperemic; the stomach and the intestines were slightly hyperemic, with occasional hemorrhagic areas possibly due to roundworms which were present in large numbers; the rectum contained a small amount of soft, brown feces. There was no nystagmus observed in this case, nor did the pupils appear to be involved. , DOG IX (male) Weight of dog, ?. (The dog, which was a very small one, was not weighed. Its weight was probably about 3 kilograms, and it was completely free of excess fat. This dog had a severe Demodex infection.) December 4, 1916 - Dog fumigated at 22° C. for a period of five hours. Time when fumigation was begun, 12.30 p. m. Time when fumigation was finished, 5.30 p. m. Physiological Action of Nitrobenzene Vapor on Animals 439 Observations: The dog appeared restless during most of the time it was in the fumigation chamber. At 1.30 p. m. it had vomited. At 5 p. m. it was observed to be unsteady on its feet; it staggered and fell, regained its feet, and fell again. At 5.30, when it was removed from the tank, the animal was able to walk but staggered about veiy much as if it had been intoxicated with alcohol; it ate cooked meat. December o — The animal was found lying on its side in a helpless con- dition; the tongue and the lips were cyanotic; the skin temperature was very low; the heart rate was 70, but was regular; the respiration was irregular, as if from disorganization of the respiratory center, and was difficult to count; the conjunctival reflex was good; the dog was unable to move its legs; tremors were observed in the leg muscles, the abdominal muscles, and the lips; the jaws moved incessantly, as if the animal was gasping for breath; the dog was placed in a warm room on a piece of cotton. The animal's condition remained unchanged during the remainder of the day; its respiration was always shallow and irregular. At 7 p. m. it was found dead. A post-mortem examination showed the following: heart distended and all the chambers filled with ante-mortem clots; these clots also appeared in the larger blood vessels; the stomach was very distended and was filled with gas and undigested food ; the duodenum was filled with a sticky, bloody mucus; the jejunum contained a dark brown mucous substance; the blood was a trifle darker than normal. In this case asthenia appeared to be the principal symptom. The action of the drug was rapid, but did not cause the type of convulsions described by Filehne as following a rapid action of the drug. DOG X (female) Weight of dog, ? ^medium-sized) . December 7, 1916 — Dog fumigated at 20° C. for a period of seven hours and fifty minutes. Time when fumigation was begun, 2.10 p. m. Time when fumigation was finished, 10 p. m. ' Obs(M-vations: ^i'h(^ animal remaiiuid quiet during the fumigation, and when n^moved from the tank at 10 p. m. it appeared entirely normal. No symptoms of poisoning appeared until two days later. 440 Wallace Larkin Chandler December 9 — At 9 a. m. the animal was apparently normal. At 3 p. m. it exhibited a weakening of the hind legs, and walked with a staggering, sprawling gait, showing a lack of coordination of the muscles of the hind legs; it had recently vomited. At 5 p. m. the animal was no longer able to walk, and the extensors of the fore legs were in tetany. At 6 p. m. the animal was no longer able to stand; nystagmus had appeared, and both pupils were dilated, the left more widely than the right; the dog drank a little milk. December 10 — The general condition of the animal was about the same as on the preceding night. Nystagmus was slight. The dog drank milk and water in the morning, but refused both food and drink later in the day. The legs were extended; there was nothing definite about the extension of the legs, one or both of the hind legs sometimes being extended and rigid, and the fore legs sometimes being thus affected; at times the tetany would last for a long period, and again it would be of short duration. At times the head was drawn strongly backward, with the muscles of the neck rigid. The pupils reacted sHghtly to light. There was an odor of nitrobenzene on the animal's breath. December 11 — At 8 a. m. the general condition of the animal was worse, but it was still conscious; all the legs were rigidly extended for minutes at a time, and the head was drawn backward; when this condition passed off the animal was left prostrated ; the pupils were contracted unequally. At about 1 p. m. the animal passed about 100 mils of dark urine (the first passed since the fumigation). At 10 p. m. the pupils were about normal; the animal swallowed a very little milk and water when these were placed in its mouth; when disturbed, the animal would attempt to use its legs, and this resulted in a tetanic convulsion involving the muscles of the legs and the neck. December 12 — During the night the animal passed about 100 mils of very dark urine (almost like black coffee). The animal had regained the ability to move its legs a httle, tho an attempt to do so usually threw them into tetany of the type described above. At 11 a. m. the animal was found with all four of its legs in constant motion; these movements were fairly well coordinated and rapid, as in the act of running or swim- ming; they would increase in rapidity and violence until the animal was thrown into a convulsion which apparently involved every muscle of the Physiological Action of Nitrobenzene Vapor o.\ Animals 441 body ; the legs were straight and the head was drawn down under the body between the hind legs ; these spasms lasted for a few seconds, during which respiration ceased entirely; as the spasms passed off, the animal would give a short, hollow ciy and resume the running movements, tho with evidence of exhaustion; both the heart rate and the respiration were rapid; if the animal was lifted up or its legs were held for a moment, the swimming or running movements would cease for a time; the excite- ment was very similar to postanesthetic excitement, except for the convul- sive periods. At 3.30 p. m. the animal was found quiet and relaxed; the respiration was 35. At 5 p. m. the animal swallowed a little milk and water which were placed in its mouth. At 10 p. m. convulsions were observed which were of tho opisthotonos type except that the muscles of the hind legs were relaxed; these convulsions appeared at intervals, and were induced if the animal was disturbed. December 13 — The animal was able to swallow but a very little liquid food when this was placed in its mouth, and so had had little or no nourishment. It lay on its left side, with its head diawn back on its body, its fore legs extended and ligid, and one or both of its hind legs drawn up to its body. The rcspiiation was slow, 16-20, and was deeper than normal; the heart was irregular, the rate being aliout 160; the tongue and the conjunctiva were slightly cyanotic. Toward evening the heart seemed weaker; the animal swallowed a little milk; it had passed very littk^ in-ine, and the little that was passed was of a dark color. The cause of the; color of this urine has not been determined; it was not hematoporphyrin, l)y the spectroscopic test. December I4. — The animal was found dead. It died sometime after 11 p. m. of the preceding day. A post-mortem examination showed the following: lungs, hypostatic congestion of the left lobe; liver, congested and dark brown in color; esophagus and stomach, containing a little clear mucous substance: all other organs normal. DOG XI (female) Weight of dog, ? (smull, about 8 kilograms). ./(inimry o, W17 — Dog fumigated at 20° C foi' a pcjriod of three hours. 'I'inic when funiigali(ji! was begun, 2.2o p. ni. Time when fumigation was finished, 5.25 p. ni. 442 Wallace Larkin Chandler Observations: The animal remained fairly quiet duririg the fumigation and was removed from the tank at 5.25 p. m. in an apparently normal condition. It was lively, ate heartily, and showed no symptoms of poison- ing. No symptoms developed during the next three days. January 8 — ^ When seen at 11 a. m. the animal was apparently well and normal. At 5 p. m., however, it exhibited a loss of coordination of the muscles of the hind legs, and had vomited at some time previously. At 8 p. m. it was unable to stand. , January 9 — The animal was found lying on its left side in a helpless condition; when placed on its right side, it struggled violently until it regained the left side; it refused cooked meat and water, but drank a little milk. At 12 m. nystagmus was observed; the pupils were normal; the animal ate a Httle cooked liver. By 3 p. m. nystagmus was very marked; the pupils were normal; knee-jerk reflex was good in both legs. When seen at 7 p. m. the animal tried to stand on its feet, but its hind legs were apparently paralyzed; when it did not succeed, it at once began to howl. January 10 — The condition of the animal was about the same. It lay continually on its left side and was unable to move its body. There was a certain amount of rigidity of the leg muscles at times, but this was not well marked. Knee-jerk reflex was good. The animal ate cooked meat, and drank milk but no water. Nystagmus was not notice- able. January 11 — The condition of the animal was slightly improved. It could move its legs a little. It ate meat and drank milk, but refused water (it was given meat and milk twice). The pupils were normal. January 12 — The condition of the animal was much improved. It was able to lift its body on its fore legs and crawl about the cage, but the fore legs weakened quickly. The animal passed urine which was very dark. It ate meat greedily and drank some water. January 13 — The condition was much improved. The animal was able to walk about very well, but its legs seemed weak and gave way at times. January I4 — ■ The condition was still further improved. Physiological Actiox of Nitrobenzene Vapor on Animals 443 January 15 — The condition was apparently normal. The dog was turned loose with the other dogs. In this case no definite convulsions were observed, the dominant symptom being paralysis such as was reported by Filehne as following a slow action of the drug. dog xviii (female) Weight of dog, ? (small, rather thin; heavy Demodex infection). April 23, 1917 — Bog fumigated at 23° C. for a period of four hours. Time when fumigation was begun, 2 p. m. Time when fumigated was finished, 6 p. m. Observations: The animal was I'cstless for a time after being placed in the fumigation chamber, but soon became quiet. It was removed apparently unharmed, and never developed any symptoms of poisoning as the result of this fumigation. Maij 16, 1917 — Dog fumigated a second time, this time at 20° C. for a period of five and one-half hours. (The dog was slightly fatter than when first fumigated.) Time when fumigation was begun, 1.10 p. m. Time when fumigation was finished, 6.40 p. m. Observations: As before, the animal was a bit restless when first placed in the tank, but it soon became accustomed to its new environment and became quiet. It was removed from the tank apparently unharmed, showing no signs of nitrobenzene poisoning and drinking water freely. May 17 — No symptoms had developed. May 18 — The animal was found with its hind legs paralyzed, and there were evidences of its having thrashed about during the night. It drank milk and water freely and ate meat. It was placed in a padded cage. May 19 — The general condition of the animal was worse. It could rai.se its head and wag its tail, but its legs were useless. It ate and drank. This condition remained al)Out the same until May 22, when some improve- ment was noticed. 444 Wallace Larkin Chandler May 23 — The animal was found with its body raised on its fore legs, swaying from side to side, apparently making efforts to stand up but its hind legs were useless. The animal had not defecated since being placed in the cage, altho it was taken out several times for this purpose. May 21 — The animal was very much improved.. It could use its fore legs very well and had some use of its hind legs, but when it attempted to walk it staggered and fell, or rather tumbled to the floor, striking its jaws against the floor with considerable force. It appeared very nervous, and was always moving and fidgeting about, apparently unable to remain quiet at all. The animal defecated for the first time since the beginning of the experiment. May 26 — The general condition of the animal was about the same. It was taken out on the lawn for exercise. In standing, its hind legs were spread far apart. It was unable to walk or to run, but it actually tumbled along, jumping high into the air and coming down on its head or its back, turning somersaults, or tumbling over sidewise. This dog was by nature playful and it had lost none of its playfulness as the result of the fumigation; its efforts to play always resulted in its throwing itself violently about. An interesting observation was the attempt of the dog to go toward any one when called; it made better progress in attempting to go in the opposite direction. It apparently was con- fused as to distances, and was wholly unable to make progress in a straight line.^" RABBITS I AND II, AND GUINEA PIGS I AND II November 2, 1916 — Animals fumigated at 22° C. for a period of nine hours. Time when fumigation was begun, 10.30 a. m. Time when fumigation was finished, 7.30 p. m. Observations: These animals were apparently normal when removed from the tank, and never developed any symptoms afterward. '"This animal was killed in February, 1918, and a histological examination of the cerebellum revealed a striking absence of Purkinje cells. Only from 5 to 10 per cent of the number found in a normal dog were present. The cont.-^ur of t'-e cerebellum was aoparently normal, and those Purkin.ie cells which were present were scaifere! f'\lr! ,- uniformly thruoit t^ie cerebellum. The condition of the animal had never improved very markedly; and while it had learned many riew tricks regarding locomotion, its actions were always typically those of a cerebellar animal. Physiolocical Action of Nitrobenzene Vapok ox Animals 445 November IS, 1910 — Animals fumigated a second time, tiiis time at 24° C. for a period of nine hours and thirty minutes. Time when fumigation was begun, 10 a. m. Time when fumigation was finished, 7.30 p. m. Observations: As before, these animals showed no effects of the drug, either during the fumigation or afterward. rabbit III AND GUINEA PIG III Dec. 22, 1916 — These animals were placed together in the fumigation chamber at 5.15 p. m., and were allowed to remain there until 4 p. m. on December 23. The temperature of the chamber remained constant at 20° C. during the first seven hours, then it gradually dropped until at the end of the next seven hours it was 15° C, and then it rapidly rose to 20° C. again. During the second seven hours no air was intro- duced into the tank. Observations: The animals nestled together and remained quiet dur- ing the entire experiment. A string was tied to the rabbit's leg, and every hour or so the anunal's reflexes were tested. They remained good. Both animals were a trifle stupid when they were removed from the tank. They were offered food and water, but would not drink and barety nibbled at the food. Suddenly the guinea pig fell on its right side and was unable to regain its feet. When placed on its left side, it immediately turned again to the right side. Violent tremors were observed in all its muscles, and presently it was seized with convulsions; all the legs were rigid and the head was drawn back on the body. This spasm lasted but a few seconds, and when it ended the animal shook itself violently and then executed running movements similar to those described in dogs. These movements were extremely rapid and lasted until another convulsion appeared. At 8 p. m. the guinea pig seemed to be recovering and was able to raise itself on its fore legs. It remained quiet for some time, and when observed the next moi-ning it was dead. A post-mortem examination showed the lungs to be distended and the air passages were filled with blood; the blood. was dark brown; the liver was congested; the other organs were normal. At the end of two hours the rabbit had developed no symptoms, and at about G p. m. it was again placed in the fumigation (^hambei-. The animal reacted to the jerk of the string until about 9 p. m. It was then removed 446 Wallace Larkin Chandler from the tank and was found to be in a stupor; it was wholly relaxed and perfectly reactionless. It remained in this condition for a few hours, and then died, without any signs of convulsions. GRAY RATS {Mus decumanus) Five young rats were placed together in a large wooden box having a capacity of 10 cubic feet. Fifteen drops of nitrobenzene were placed on a strip of cheesecloth and the cloth was suspended in the box, which was then closed for twelve hours. At the end of that time the rats were removed. The animals were all perfectly anesthetized, and three of them were reactionless. One died five hours later, and another was killed for the purpose of examining its blood; both of these had dark brown blood. All the other three exhibited either right or left rotatory (pinwheel) movements; one of them was seen to roll over and over for several feet before becoming exhausted. Two of these remaining three died without showing other symptoms, and one recovered (at least tem- porarily) and escaped. Two adult rats of the same species were fumigated together m the regular fumigation chamber for three and one-half hours at a temperature of 23.5° C. When removed from the tank they were apparently unharmed. Both of these died two days later, probably from lack of nourishment since it was impossible to induce them to eat while in captivity. WHITE RATS It was found that white rats could not stand a fumigation at ordinary temperatures for longer than from one and one-half to two hours. How- ever, the rats used in these experiments were infected with trypanosomes and 'spirochetes, and this fact may have had something to do with hastening the action of the drug. The rats that were still alive when removed from the tank showed only paralysis and usually died very quickly. CAT X September 26, 1916 — Cat fumigated at 17° C. for a period of five hours. Time when fumigation was begun, 11.50 a. m. Time when fumigation was finished, 4.50 p. m. Physiological Action of Nitrobenzenp: Vapor on Animals 447 Observations: -The animal lay down and remained quiet foi- about three hours, and then became restless for a time but soon became quiet again. When first removed from the tank the animal appeared well and started to walk away, but lost control of its hind legs and tumbled about for a moment, then became excited. It vomited (chunks of meat which had not started to digest), and then lay prostrate for about one-half hour. The lips and the tongue were cyanotic; the pupils were dilated and did not react to hght. The animal's condition improved shortly and it again appeared well. On the following morning a quantity of sticky, l)loody fec(>s was found in the animars cage; no further symptoms of poisoning had developed, however, nor did any symptoms appeal- during the next four days. At the end of this tinu^ the cat was found dead. CAT XVIII October 5, 1917 — Cat fumigated at 22.5° C. for a period of three hours. Time when fumigation was begun, 9 a. m. Time when fumigation was finished, 12 m. Observations: The animal remained quiet during the fumigation and was removed from the tank apparently unharmed, but became sUghtly stupid toward evening. A sample of blood was taken and examined. It was dark brown and showed methemoglobin by spectroscopic analysis. October 6 — The animal was found with well-advanced symptoms of nitro- benzene poisoning — nystagmus, paralysis of the muscles of the hind legs, and periods of excitation followed l)y prostration. The cat re-fused milk, nor was it possible to place any in its mouth. A sample of blood drawn from the ear was coffee-colored and showed the same sixictrum as on the preceding day. October 7 — The condition of the animal remained about the same. It lay on its side entirely helpless and apparently unconscious. October 8 — TJie (x>ndition gradually grew worse and the animal died toward evening. A post-mortem examination showed a pronounced congestion of lungs a'nd viscera, and the blood vessels of the neck were turgid with blood. 448 Wallace Larkin Chandler HEN V March 21, 1917 — Hen fumigated at 30° C. for a period of six and one- half hours. Time when fumigation was begun, 1 p. m. Time when fumigation was finished, 7.30 p. m. Observations : The bird was removed from the tank apparently normal except that it appeared a Uttle stupid. The feces were formed when the bird was first placed in the fumigation chamber, but those deposited immediately after the bird was removed from the tank were soft, stringy, and slightly bloody. March 22 — The bird appeared normal except that the neck feathers were constantly ruffled — a condition which induced such a belligerent attitude on the part of the other birds in the cage that this hen had to be placed by itself. No further symptoms developed until March 27, on which day the bird was found lying prone on its side and apparently unable to stand. When the bird was lifted it exhibited a circular rotation of head and neck; and when it was placed on its feet, the legs stiffened, throwing the bird backward. March 28 — The bird's general condition was about the same. Nothing new was especially noted. March 29 — The bird's condition had grown worse. The skin was cold, and the bird was placed in a warm room. There appeared frequent periods of excitation, during which the bird thrashed about a good deal. March 30 — The bird was found lying on its left side, with its left leg ex- tended and rigid and its right leg exhibiting violent tremors. The respiration was normal. The head was moving by jerks sidewise. When placed on its feet the bu-d lunged forward instead of backward. At 2 p. m. the bird swallowed some water when its head was placed in water and then released; it also swallowed a little moistened bread when this was placed in its mouth. Each attempt at swallowing was followed by excitation, during which the head was drawn back rigidly between the wings or revolved slowly, describing broad circles. Wlien placed on its feet the bird lunged backward. March 31 — The l^ird was again fed as described. Physiological Action of Nitrobenzene Vapor on Animals 449 April 1 — The eondition was about the same, altho the bird appeared stronger. When placed on its feet it attempted to walk, but the legs stiffened and the bird was thrown foi'wai'd. This bird never fully recovered. It was able to squat on its feet after a time, but refused to walk; wh(>n urged, it took two or three rapid steps and then tumbled over forward. It was unable to remain on a perch, faUing either forward or backwai'd. The wing movements were well coordinated. Foi' a long time the bird was unable to eat without assist- ance, but it was finally taught to do so. The bird was killed on June 11, in order to make an examination of \\\v biain tissues. HEN VI March ,12, 1917 — Hen fumigated at 27-28° C. for a pei'iod of eleven hours. Time when fumigation was begun, 10 a. m. Time when fumigation was finished, 9 p. m. Ob.servations: The bird was removed fiom the tank ajiparently unharmed. The feces were as described for Hen V. March '23 — The bird was apparently well, but not very active. It ate cracked corn and di"ank water. The neck feathers were ruffled. March 24 — Wlien first seen on this day (at 8 a. m.) th(> bird appeared normal. It was taken from the cage and placed on the floor. It was able to walk and run very well, but suddenly showed a tendency to give way to the left side; it flew to a perch 18 inches high, but was unable to retain its position; it fatigued very easily. At 10 a. m. the attendant reported that the bird had been in convulsions; it appeared normal except that it was a little dull. At 3 p. m. the bird appeared to be drowsy; it was removed from the cage and placed on its feet; it stood swaying from side to side; when it attempted to walk, it staggered and then swayed, backward, taking several steps in an effort to catch itself; after this excite- ment its head was bent back between its wings. At 4 p. m. the bird was found lying on its side and was unable to stand; when it was [)icked up, its head rotated in a ciicle, sweeping the back and the wings. At 4.15 about an ounce of clear fluid came from the bird's mouth. At ") p. m. periods of excitation were observed; the legs wei'e in violent, motion as in the act c)f nuining, and ihe head shook viok'iitly as if the bird were 450 Wallace Larkin Chandler trying to dislodge something in it; these periods of excitation were of short duration and were followed by periods of prostration lasting for several minutes, during which the muscles were wholly relaxed and the neck was limp; then with a sudden jerk, the period of excitation would appear again; this excitation could always be elicited by disturbing the bird; when the bird was placed on its feet, the legs stiffened and threw the body backward; the legs were bluish (they were pink normally); frequent movements of the gullet were observed, these movements often involving the mouth. At 5.15 another ounce of clear fluid came from the mouth. At 6 p. m. the bird made a violent effort to regain its feet; the wing movements were well coordinated but weak; this effort was followed by excitation during which the legs moved violently as described above. March 25 — At 9 a. m. the bird's condition was worse; there was constant rotation of head and neck; convulsions occurred as before; when placed on its feet, the bird squatted quietly for a moment and discharged a large quantity of watery fluid from the anus, and then went into convulsions again. At 5 p. m. the bird was apparently better, and was resting quietly in an upright position. At 7 p. m. the bird was found in convulsions; the head was bent firmly on the venter (almost between the legs); the bird had no control of any muscle. March 26 — In the morning the bird was found with all muscles relaxed except the neck muscles, which were rigid and held the head firmly against the venter. At 6 p. m. the bird was found dead. A post-mortem exami- nation revealed a strong odor of nitrobenzene in all the organs; the crop and the gizzard were filled with cracked corn, which had not started to digest altho the bird had had nothing to eat during the preceding sixty hours : the other organs were about normal. HEN VII March 25, 1917 — Ren fumigated at 25° C. for a period of eight hours. Time when fumigation was begun, 2.15 p. m. Time when fumigation was finished, 10.15 p. m. Observations: When removed from the tank the bird was a trifle stupid but was otherwise normal. It developed no symptoms of poisoning until March 30. Physiological Action of Nitrobenzene Vapor on Animals 451 March 31 — The' bird showed symptoms very similar to those described for Hen VI. April 1 — The bird was found dead. On March 27 (two days after the fumigation) this hen laid an egg- The egg was opened on March 29 ; a strong odor and a very characteristic taste of nitrobenzene were detected in the yolk, but the white did not contain more than a trace of the chemical. This phenomenon can easily be explained by the fact that nitrobenzene is soluble in fats, but is scarcely soluble at all in the white of eggs. HEN VIII AND ROOSTER III May 10, /5/ 7 — Birds fumigated together, at 23° C, for a period of eight hours. Time when fumigation was begun, 2 p. m. Time when fumigation was finished, 10 p. m. Observations: The rooster was found dead at the end of the fumigation period. The hen appeared slightly stupid when removed from the tank and became easil}^ fatigued, but was otherwise normal. No further symptoms developed in the hen until on May 14, when it was seen to stagger on attempting to run. During the next few da3^s the bird was very stupid. It did not eat much, staggered in attempting to walk, and fatigued easily. May 25 — The condition of the bird was apparently normal again. No further symptoms ever developed in this bird. PIGEONS IV AND V June 11, 1917 — Birds fumigated at 24° C. for a period of six hours. Time when fumigation was begun, 1.10 p. m. Time when fumigation was finished, 7.10 p. m. Observations: The l)irds wen> apparently normal when removed from the tank. They could fly and run easily. June 12 — One of the birds showed the following symptoms: it was unable to fly, tho the wing movements were fairly well coordinated; in attempting to walk, it lunged forward and tumbled on its head; there was rotation of head and neck. 452 Wallace Larkin Chandler June 13 — The bird showing on June 12 the symptoms described, flew the length of the room along the floor, its head touching the floor. It probably had the use of its wings but could not direct the flying move- ments. The other bird showed symptoms of poisoning on this day. Both the birds were killed about noon, and the nervous tissues were fixed as described earlier (page 429). OBSERVATIONS OF THE ACTION OF NITROBENZENE ON INSECTS During the fumigation of the animals a number of external parasites dropped from the hosts. These were collected at the end of the fumigation period and observations were recorded regarding the action of the drug on them. Some of these observations were as follows: fleas Eighty-three fleas of the genus Ctenocephalus were recovered from the bottom of the tank after the fumigation of Cat X (fumigated for five hours at 17° C). Most of these began to show signs of life in about one and one-half hours; they were put into a glass tube and placed in a warm room, and in about twelve hours all the fleas had recovered with the exception of about one-half dozen, which were stuck fast to the tube. Nineteen fleas of the same genus were recovered in a stupefied con- dition from the bottom of the tank after the fumigation of Dog I (second fumigation, six hours at 23° C). Three of these .showed signs of life, and most of them recovered during the next twelve hours. Seventy-six fleas of the same genus were recovered from the tank after the fumigation of three kittens for a period of four hours at 22° C. Some of these fleas showed signs of life when removed from the tank, and twenty of them recovered and lived for several days. BITING lice A large number of biting lice (Trichodectes suhrostratus) were recovered in a stupefied condition from the bottom of the tank after the fumigation of Cat X (fumigated for five hours at 17° C.) . Nearly all of these recovered during the next twelve hours. A large number of the biting lice of poultry {Menopon gallinae, Menopon stramineum, Lipeurus heterographus, and Goniocotes gigas) were recovered Physiological Action of Nitrobenzene Vapor on Animals 453 from the tank after the fumigation of Hen V (fumigated for six and one- half hours at 30° C). The hce were all apparently dead, showing no signs of life, at the end of twelve hours. At the end of eighteen hours, however, some of them were seen to be moving their legs. In Goniocotes these movements were lapid, and were very similar in character to the movements of the legs of poisoned guinea pigs. Approximately three hundred biting lice, representing six species, were recovered from th(^ tank after the fumigation of a chicken for a period of one and one-half hours at 20° C. Some of these insects showed signs of life when removed from the tank, and nearly all of them recovered entirely during the next few hours. Nineteen specimens of biting lice were recovered from the bottom of the tank after the fumigation of Hen VI (fumigated for eleven hours at 27-28° C). None of these showed signs of Hfe when taken from the tank, and none recovered. THE FOLLICULAR MITE (Demodex folUculorum) Two or three cases of mild infection of demodecic scabies in dogs apparently cleared up after fumigations for long periods at low tem- peratures. One of these cases was Dog V, which was fumigated twice — once for twelve hours at 20° C, and once for five hours at 20° C. These observations led to the following experiments for the purpose of determining the value of nitrobenzene in controlling demodecic scabies. A special fumigation chamber was constructed in such a way that the animal's nose passed thru a rubber collar and remained on the outside of the chamber, and an attempt was made to fumigate the body of the animal without permitting it to inhale a large amount of the vapor. Morphine and chloral hydrate were given in order to cause the animal to remain quiet during the fumigation. A six-hour fumigation under these special conditions had no effect on the mites, nor was it possible to induce dogs to remain quiet without giving them large doses of the narcotics. The method was therefore abandoned. Small pieces of skin heavily infected with D. folUculorum var. cards were placed in a petri dish in which there was a drop or so of nitrobenzene. The dish was then kept at a temperature of 30° C. for six hours. At the end of that time the mites weie still alive. 454 Wallace Larkin Chandler It was observed that a 33-per-cent solution of nitrobenzene in olive oil would have no apparent effect on dogs if applied externally and if the animal was allowed to remain in the open air, in spite of the fact that an external application of a solution of this strength invariably killed cats and rabbits. Hence a small, short-haired dog ^^ having a heavy infection of Dernodex folliculorum was bathed thoroly and fre- quently with the 33-per-cent solution ; but no improvement in the condition of the animal could be noticed. OBSERVATIONS OF THE ACTION OF NITROBENZENE ON INTERNAL PARASITES GAPEWORMS {Syngamus trachealis) Three chicks showing symptoms of gapes were fumigated together for two hours at 25-26° C. on June 23, 1917. Two of the chicks developed symptoms of nitrobenzene poisoning on June 24. One of these died on June 25 and the other on June 26. Two nearly mature pairs of Syngamus trachealis were recovered alive from the trachea of the former, and three living pairs were taken from the trachea of the latter. The third chick developed typical symptoms of nitrobenzene poisoning on June 26. By July 8, however, this chick had fully recovered from the effects of the drug, and it had also recovered from the gapes by July 8. The recovery from the gape worms, however, cannot be attributed to the "action of the drug on the worms, since with its improved environment it would in all probability have recovered anyway, INTESTINAL WORMS A number of roundworms {Belascaris marginata) were found in the feces of Dog III on the^ morning following a fumigation for five hours at 18° C. These worms were obsei^ved to be dead and their death was attributed to the action of the drug. However, roundworms of the same species were recovered, very much alive, from the intestines of nearly all the dogs examined, even in cases following long periods of fumigation. " The solution was not tried on long-haired dogs, nor was a stronger solution tried. The animal did HOC lick the solution off, presumably because of the burning taste of nitrobenzene. Physiological Action ok Nitrobenzene Vapor on Animals 455 coccidia Specimens of Eimeria avium in the oocyst stage were recovered from the feces of Hen VII. These oocysts appeared normal, and the develop- ment of sporocysts and sporozoites occurred as usual. EXPERIMENT TO DETERMINE THE ACTION OF NITROBENZENE ON DIGESTIVE FUNCTIONS Post-mortem examinations of animals poisoned by nitrobenzene usually revealed the fact that food which had been in the stomach for some days had not started to digest. A special experiment was therefore conducted in order to check these observations. At 11 a. m. on October 3, 1916, four kittens, all from the same litter, were each given an equally large portion of boiled hamburg steak. At 11.10 three of these kittens were placed in the fumigation chamber, where they were fumigated for four hours at a temperature of 22° C. At 2.20 p. m. (three hours and ten minutes after the fumigation was begun) one of the animals died. The other two were removed from the tank at 3.10 and showed well-advanced symptoms of poisoning. One of them died at about 4 p. m. and the other at about 5 p. m. At about 5.30 p. m. the control kitten was killed with chloroform and a post-mortem examination was made of all four animals. The stomachs of the animals that had been fumigated contained the full amount of the hamburg steak eaten, and in no case had it even started to digest. The digestion of the hamburg steak in the stomach of the control animal was well advanced. EXPERIMENTS TO DETERMINE THE ACTION OF NITROBENZENE ON BLOOD BLOOD COUNTS Two pups, from the same litter and about three months old, were kept under similar conditions,^^ ^ji^j blood counts were made on each for several days in order to determine the normal counts. Each animal was then fumigated, on different days and for different periods of time, and the blood counts were continued. Always, of course, as soon as an animal developed pronounced symptoms of poisoning it refused food, and this interfered with exact compaiisons. The lesults of these experiments are indicated in the following tables: '2 These animals were fwi twire a day, each reoeiving at a meal 40 grarns of cookesis is, jierliaps, the best interpretation of the type of convulsions observed. This is a term originally used by Babinski to describe a pecuhar type of incoordination in patients (Jelhffe, 1913). It is seen in the absence of paralysis, muscle palsies, and sensibility disturbances, and is characterized by a loss of ability to carry out rapidly alternating movements, such as flexion and extension of the forearm on the arm. In dogs, })irds, rats, and other animals poisoned by nitro- benzene, it was observed that at times one set of muscles would.be con- tracted and another set relaxt^d, and at other times just the reverse happened. For example, if motor impulses passed to the extensors of the hind legs, the animal was unable to stop these impulses or to use the antagonistic muscles, and the leg remained for a time rigidly extended ; again, if the impulses passed to the flexors, then the animal was unable to extend the leg. This condition, according to Babinski, indicates involvement of the cerebellar paths. Nystagmus. — According to JelHffe (1913), Holmes states that " nystagmus .is a common and very valuable localizing sign of local cerebellar lesions. It is almost certainly a true cerebellar symptom." Nystagmus does not always appear, ]mt when present it is invariably a cerebellar type, distinguishable from vestibular nystagmus by the jerky movements of the eyeball. The shifting is in a lateral plane, and is equally rapi'd in either direction. Asthenia. — Asthenia is one of the first symptoms to appear and is almost invariably present. The animal is usually so weak that when it is placed on its feet, its legs literally double up beneath it, and its head sways about as if- the neck were disjointed. This symptom, according to Jelliffe (1913), indicates "disorder of the tractus cerebellovestibularis spinalis, or rubro-spinalis." Hypotonus. — Hypotonus, as revealed by palpation of the muscles and Vjy special tests of the tendon reflexes, was observed in the more advanced cases, especially, of nitrobenzene poisoning. In some cases this symptom was accompanifid by poor tendon reflex reactions, while in other cases the tendon reflexes were noiinul oi' even exaggerated. These conditions indicate cerebellar hypotonus. 466 Wallace Larkin Chandler Disturbance of respiration. — In moribund animals poisoned by nitro- benzene, one almost invariably observes a disturbance of respiration, and in some instances a slowing of the pulse rate. Musser (1904) states that cerebellar tumors often cause symptoms of this type. Headache and tenderness of the occiput. — Headache in animals is very difficult, if not entirely impossible, to determine. However, certain actions exhibited by animals poisoned by nitrobenzene might be interpreted to indicate headache; for instance, the animal's desire to press its head against some object. Dog I was observed to stand for long intervals with its head pressed against the attendant's legs or against some solid object (page 431). And certainly the fact that the animal exhibited some dis- comfort if the back of its head was touched, indicated a tenderness in the vicinity of the occiput. These symptoms have been observed in cases of cerebellar tumors. Circus movements. — Rotation of the head and the neck was always observed in the case of birds poisoned by nitrobenzene. This is a character- istic symptom observed in cerebellar pigeons. Rotation of the body about its longitudinal axis, observed especially in gray rats and sometimes in dogs, is a symptom exhibited in cerebellar mammals. CAUSE OF THE LATENT PERIOD The fact that there often exists a long latent period, the period of time elapsing between the administration of the drug and the onset of the symptoms, has led to much theorizing as to its cause. The following two theories have been the most popular: the theory advanced by Olhvier and Bergeron and held to by Letheby, that nitrobenzene is converted into anilin in the body and that time is required for this transformation;, and the theory investigated first by Filehne and accepted by most recent writers, that the drug is so lightly soluble in the tissues of the body that time is required for the absorption of it in sufficient amounts to produce the poisonous effects. Filehne has ably shown that the action of the drug does not depend on its conversion into other chemicals. Furthermore, he failed to find any trace of anilin in any of the organs of animals poisoned by nitrobenzene even by the hypochlorite test. His criticism of the isophenylcyanide test (used by Letheby) is justified, since in conducting this test nitrobenzene Physiological Action of Nitrobenzene Vapor on Animals 407 is itself convci'tcd into auilin. Filehne has shown also that the theor}- of slow absorption does not account for the rapid action of the ch'ug observed in certain cases. In all probability the rate of absorption of nitrobenzene by the body tissues does have something to do with the cause of the latent period; but the explanation must be primarily based on the readiness with which the drug is absorbed by certain tissues as compared with other tissues, and not alone on the rate of absorption by the tissues in general. It will be recalled that nitrobenzene is readily soluble in oils and the liquid fats; it is soluble also to a certain extent in lipoids, but probably to a less extent in certain lipoids than in others, and even the same lipoid may absorb the drug more readily under certain conditions than under other con- ditions. When the drug is administered by vapor inhalation, the amount absorbed by the blood (at a given vapor pressure) depends, undoubtedly, on the amount and the condition of the fats or the lipoids in the blood. Also, the amount absorbed from the blood and held in solution by the body fats is, in all probability, directly proportional to the absorption power of the body fats over the absorption power of the fats in the blood. Since nitrobenzene is more readily absorbed by the liquid body-fats than by the lipoids (of the lirain), large amounts of it may be stored in the liquid fats of the body without the animal's showing any immediate symptoms of poisoning. Moreover, since the action of the drug on the cells of the brain probably depends on its concentration in the vicinity of these cells — as in the case of chloroform, ether, and other drugs that act directly on the cells of the brain — niti'obenzcne, depending on the amount and the condition of the lipoids and the fats held' in suspension in the blood, may be picked up from the body fats by the blood in such small amounts as to be in time entirely eliminated from the body without ever giving a sufficient concentration in the vicinity of Pui'kinje's cells to cause .symptoms of poisoning; so that large amounts of the drug may be stored in the body without the animal's ever showing any symptom of poisoning. On the other hand, depending on the concentration of nitrobenzene in the blood supply to the cerc^bellum, a sufficient concentration of the drug in the vicinity of I he Purkinje cells might be reached, even after miniite 468 Wallace Larkin Chandler doses, to affect these cells and produce the typical symptoms of poisoning, or even death, within a very short time after the administering of the poison. If the above assumptions are correct — and they can be so proved only after a long series of experiments dealing more exhaustively with the physics, chemistry, and physiology of the subject — - then it is not sur- prising that, as was found, the latent period, as well as the intensity of the action of the drug, should vary in different individual animals, or even in the same individual at different periods of time; for neither the amount nor the kind of fats in the blood or the nervous tissues is abso- lutely constant. CONCLUSIONS The present work has confirmed the findings of previous investigators regarding all six of the points listed in the first paragraph following the review of the literature. In addition the following conclusions have been deduced : 1. Aside from the possible disturbance of digestive functions and a possible asphyxia due to a direct action of nitrobenzene on the blood, most, if not all, of the observed symptoms of nitrobenzene poisoning may be explained on the basis of disturbances in the cerebellum or the cerebellar paths. 2. Toxic doses of nitrobenzene, when administered by vapor inhalation, exert a direct action on the Purkinje cells in the cerebellum, causing chromatolytic degeneration of these cells. 3. Histological examinations have failed to reveal any definite changes in any of the organs of the body except the blood (presence of methemo- globin and morphological alterations of erythrocytes) and the cerebellum (chromatolytic degeneration of the Purkinje cells) .^^ 4. The size of the lethal dose probably depends on conditions such as the amount and the kind of fats in the blood, which favor or disfavor a concentration of the drug in the vicinity of the nerve cells. 5. The latent period (the time elapsing between the administration of the poison and the onset of the symptoms) is undoubtedly due to the " The writer does not mean to assert that histological changes do not occur in other tissues, especially in other parts of the central nervous system. Indeea, a further study of tlie present sections may yet reveal such changes. It would be strange if the action of nitrobenzene on the central nervous system were confined to a single type of cells only. Probably in cases of fatal poisoning other nerve cells are irtvolved also, but it will be difficult to determine whether such changes are due to a direct action of the poison or are the result of a complication of changes attending death by poisoning. Further investigations are being made along this line. Physiological Action of Nitrobenzene ^apok on Animals 469 absorption of the nitrobenzene from the blood, and its retention by the liquid fats of the bod}^ in which it is easily soluble. As the concentration of the poison in the blood lipoids and fats diminishes, in "relation to its concentration in the body fats, the nitrobenzene is given up again to the blood; and in the course of time, a sufficient concent ratioji of the poison in the lipoids of the cerebellar or other brain cells is reached to produce an onset of the motor symptoms. The time requircnl to bring this about (the latent period) depends on the same factors as those on which the size of the lethal dose depends, and is undoubtedly hastened by the ingestion of solvents of nitrobenzene, such as alcohols, fats, oils, milk, and the like. Possibly, also, the condition of the brain lipoids at a given time may be an important factor in hastening or retarding the al)sorption of nitroben- zene by these lipoids. 6. Nitrobenzene cannot be used, with au}^ degree of safety, for the fumigation of animals to destroy their external parasites. The lethal dose for })irds and manunals is i-ather variable but it may be very small; and from these experiments it will be seen that apparently a shoi'ter period of fumigation at a given temperature is required in older to kill a domestic animal than to kill even a fair proportion of eith(M- fleas or biting lice. However, as has been pointed out by the writer in a previous paper (Chandler, 1917), the drug may be used, with the exercise of caution, for collecting external parasites from animals, by fumigating the animals at low temperatures and for shoit periods of time — at a temperature not over 20° C, and for a period not longer than one and one-half hours. Under these conditions the i)arasites may be stupefied without any appre- ciable damage being done to t he host ; but the drug is dangerous to handle under any conditions. 7. Because of the extreme toxic properties and the su])tle action of nitrobenzene, the following uses of this drug should be prohibited by legislation : for perfuming soaps, lotions, and pomades ; as a solvent in shoe polish, floor wax, and th(> like; and especially .as an ingredient of flavoring extracts, confections, and liqueurs. The drug should be regarded as one of the most dangerous of poisons, and its sale and use should be regulated by law just as in th(> cas(; of any other deadly poison. 8. Nitrobenzene should be given serious consideration as an industrial poison. Munition plants, dye works, and other factories which handle 470 Wallace Larkin Chandler nitrobenzene, should be inspected with the view of instalHng devices to prevent workmen from inhahng the vapor or coming into contact with the hquid. 9. In view of the evident relation existing between the lethal dose of nitrobenzene and the amount and kind of fats and other solvents of the drug present in the blood, it would appear highly desirable that further investigations should be undertaken with the view of working out rational therapeutics for cases of poisoning. 10. Since the findings in these experiments indicate that the symptoms of nitrobenzene poisoning are caused by a direct action of the drug on the Purkinje cells, causing these cells to degenerate, and since different sets of muscles appear to be involved at different times, it would appear that further investigations might be of value from the standpoint of obtaining more exact data regarding the localization of functions in the cerebellum. 1 Physiological Action of Nitrobenzene Vapor on Animals 471 LITERATURE CITED Adams, S. S. Nitrobenzol poisoning. Assn. Amer. Physicians. Trans. 27:503-513. 1912. BoNDi, ]\'Iaximilian. Ein casuistischer Beitrag zur Lehre von der Nitro- benzolvergiftung. Prager Med. Wochensch. 19:129-143. 1894. Casper, Johann Ludwig. Ein neiies Gift. Vrtljschr. gerichtl. u. offentl. Med. i6:l . 1859. Chandler, W. L. Investigations of the vakie of nitrobenzol as a para- siticide, with notes on its use in collecting external parasites. Journ. par. 4:27-32. 1917. DoDD, A. H. Poisoning by nitro-benzole ; recovery. Brit. med. journ. i:849 . 1891. Dresbach, M., and Chandler, W. L. Some physiological disturbances induced in animals by nitrobenzol fumigation. Amer. journ. physiol. 42:604-605. 1917. Eulenberg, H. Die aromatischen Korper. In Handbuch der Gewerbe- hygiene, p. 606-640. 1876. Filehne, Wilhelm. Ueber die Giftwirkimgen des Nitrobenzols. Arch. exp. Path. u. Pharmakol. 9:329-379. 1878. Grafe, E., and Homberger, A. Gewerbliche Nitro-benzolvergiftung mit Korssakowschem Svndrom und Ausgang in geistigen Schwilchezu- stand. Ztschr. Gesell. Neurol, u. Psychiat. 25:343-352. 1914. Guttmann, Paul. Ueber die giftigen Eigenschaften des Nitrobenzin. Arch. Anat., Physiol., u. wiss. Med. i866: 214-223. 1866. Howell, William H. A text-book of i:)hvsiology for medical students and physicians. 8th ed., p. 1-1059. 19l"8. Jelliffe, Smith Ely. Disorders of the cerebellum. In A reference handbook of the medical sciences 2:725-737. 1913. Jones, W. Price. Poisoning by essential oil of bitter almonds. The lancet [London] 1857': 45-46. 1857. Landolt, II., B()rnstein, Richard, and Roth, Walther A. Phys- ikalisch-chemische Tabellen, p. 1-1313. (Reference on p. 243.) 1912. Leiiieby, H. On the poisonous properties of essence of mirbane, or, artificial oil of l)itt(M- almonds (nitro-benzole). Clinical Lectures and Repts., London Hospital, 2:34-57. 1865. Massini, Rudolph. tJber Nitrobenzolvergiftung, Blutbefund, und Ver- halten des Reizens bei derselbe. Deut. Arch. klin. Med. ioi:72 — . 1910-11. Meyer, Erich, t'ber das Verbal ten des Nitrobenzols und einigcr anderer aromatischer Nitrokorper iiii Organismus. Ztschr. physiol. Chem. 46:497-509. 1905. 472 Wallace Laekin Chandler MiTSCHERLicH, E. Ueber die Zusammensetzimg des Nitrobenzids iind Sulfobenzids. Ann. Phys. u. Chem. 31: 625-631. 1834. Moore, William. Fumigation of animals to destroy their external parasites. Journ. econ. ent. 9:71-80. 1916. MussER, John H. A practical treatise on medical diagnosis, p. 17-1213. (Reference on p. 1173.) 1904. Ollivier, Augusts, and Bergeron, Georges. L'action physiologique de la nitrobenzine. Journ. physiol. 6:455-459. 1863. Rasmussen, a. T., and Myers, J. A. Absence of chromatolytic change in the central nervous system of the woodchuck (Marmota monax) dur- ing hibernation. Journ. compar. neurol. 26:391-401. 1916. Roth, O. Zur Kenntnis der Nitrobenzolvergiftung. Zentbl. inn. Med. 34:417 . 1913. Stone, Willard J. Fatal poisoning due to skin absorption of liquid shoe blacking (nitrobenzol). Amer. Med. Assn. Journ. 43:977-980. 1904. Weaver, Erasmus M. Notes on military explosives, p. 1-382. (Refer- ence on p. 68-69.) 1917. Weisstein, Karl. Beitrage zur Kenntnis der Wirkung des Nitrobenzols auf Blut. Inaug. Diss., Wlirzburg. 1892. Zieger, Jos. Studien iiber die Wirkung von Nitrobenzol, etc. 1903. Memoir 18, A Study of Bacteria in Ice Cream during Storarje, the second preceding number in this series of pubhcations, was mailed on February 12, 1919. Date Due 1 f) QP981,N5 cze Chandler Physiological action of nitroben- zene vapor on animals ■T-itwr-'i, .' >:"r.'^-ifi\'!'-,^?9ss^.'^)'