EXPERIMENTAL LEAD TOXICOSIS m PONIES Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY KENNETH F. GALLAGHER 1975 5“.-. __ w-a—v g. ‘-,-~1AQ’ hé rll"til “Lit“ ‘1‘ vi“ ... «qr/a0“. “ -YAA ‘1‘. <6; LISA-21‘“ HY ABSTRACT EXPERIMENTAL LEAD TOXICOSIS IN PONIES BY Kenneth F. Gallagher Six adult pony mares were used to determine clinical signs, lesions, and tissue lead values during chronic lead toxicosis. Four ponies were fed lead oxide (Pb304) at the rate of l mg/lb of body weight/day mixed in a molasses-grain ration for 60 days. Two ponies were fed the same feed without the addition of lead. One month after the start of the experiment, 2 ponies had transitory clinical signs of hyperexcitability and diarrhea lasting 14 days. The other 2 ponies ingesting lead oxide had no observable clinical signs. Blood and urine samples, analyzed for lead by solvent extraction followed by the atomic absorption spectrophoto- metric method, had erratic but rising values throughout the study with final values markedly above those commonly accepted as indicative of lead toxicosis. There was a decrease in packed cell volumes, hemo- globins and erythrocyte counts as compared with baseline parameters. No specific gross lesions were observed at necropsy, but microscopi— cally early degenerative changes in hepatocytes and glomerular tufts and proximal tubular epithelium of the kidney were present. Kenneth F. Gallagher Liver and kidney tissue lead values, as determined by the dry ashing technique followed by the atomic absorption spectrophotometric method, were higher than normal. Results indicated that ponies do not always have classic signs of toxicosis even though their body burdens of lead are elevated above those levels generally accepted as indicative of lead intoxication. EXPERIMENTAL LEAD TOXICOSIS IN PONIES BY Kenneth F. Gallagher A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1975 ACKNOWLEDGEMENTS The author wishes to express his gratitude and appreciation to Dr. A. L. Trapp, major professor, and Drs. C. K. Whitehair, Kenneth K. Keahey and Gabel Conner, committee members, for their encouragement, advice, and guidance in fulfilling the research and thesis require- ments for this degree. Sincere thanks are expressed to other faculty members and technicians in the Department of Pathology who assisted in the histopathologic work related to this experiment. I also wish to acknowledge the veterinary students, and Mark Soper and Roberta Milar, Department of Large Animal Surgery and Medicine technicians, who provided much needed assistance in the conduct of this study. ii TABLE INTRODUCTION. . . . . . . . . . OBJECTI V38. 0 O O O O O I I O 0 LITERATURE REVIEW . . . . . . . Incidence. . . . . . . . Etiology . . . . . . . . source. . . . o 0 OF CONTENTS Absorption and Excretion. . . Dosage of Toxicant. . . . . . Interaction with Minerals . . Clinical Signs . . . . . Diagnostic Tests . . . . Gross and Microscopic Lesions. . . . Tissue Values. . . . . . MATERIALS AND METHODS . . . . . RESULTS Animals, Housing and Rations . . . Methods and Techniques . General Procedure Collection of Blood . . . . Collection of Urine . . . . . Necropsy Examination. . . . . General Observations . . Lead Levels in the Basal Blood Parameters . . . . Ration. . . Blood and Urine Lead Values. . . . iii Page O‘O‘U'IU'I U1 \1 10 11 11 11 ll 12 13 13 15 15 15 15 19 Page Liver, Kidney and Intestinal Contents . . . . . . . . . . l9 Postmortem Lesions. . . . . . . . . . . . . . . . . . . . 24 Gross Lesions. . . . . . . . . . . . . . . . . . . 24 Microscopic Lesions. . . . . . . . . . . . . . . . 24 Liver . . . . . . . . . . . . . . . . . . . 24 Kidney. . . . . . . . . . . . . . . . . . . 28 Special Stains . . . . . . . . . . . . . . . . . . 28 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 iv LIST OF TABLES Table Page 1 Blood and urine lead values (ppm) in experimental ponies . . . . . . . . . . . . . . . . . . . . . . . . . l6 2 Blood parameters in experimental ponies. . . . . . . . . l7 3 ' Averages of blood parameters of ponies (Pl-P4) ingesting lead oxide . . . . . . . . . . . . . . . . . . 18 4 Averages of blood and urine lead values of 4 ponies (Pl—P4) ingesting lead oxide . . . . . . . . . . . . . . 20 5 Lead values in liver, kidney and intestinal contents (ppm) from experimental ponies . . . . . . . . . . . . . 23 Figure 10 ll 12 LIST OF FIGURES Average blood lead and hemoglobin values of 4 ponies (Pl-P4) fed lead oxide . . . . . . . . . . . . . . . . Average blood lead and packed cell volume values of 4 ponies (Pl-P4) fed lead oxide. . . . . . . . . . . . Liver from a control pony. Normal hepatocytes . . . . Liver from a lead poisoned pony. There is cytoplasmic vacuolization and hydropic change in hepatocytes indicative of cellular degeneration. . . . . . . . . . Liver from a control pony. Normal lobular structure . Liver from a lead poisoned pony. Loss of normal lobular structure. Sinusoids and hepatic cords do not have a regular arrangement around the central vein 0 O O O O O O O O O I O O O C O O O O O O C C O 0 Liver from a lead poisoned pony. Basophilic stained pigment in cytoplasm of hepatocytes. . . . . . . . . . Kidney from a control pony. Normal proximal convo- luted tubule . . . . . . . . . . . . . . . . . . . . . Kidney from a lead poisoned pony. Hydropic degenera- tion of the epithelium of the proximal convoluted tubules. . . . . . . . . . . . . . . . . . . . . . . . Kidney from a control pony. Normal glomerular structure. . . . . . . . . . . . . . . . . . . . . . . Kidney from a lead poisoned pony. There is swelling of the glomerular tuft with reduction of Bowman's space and thickening of the endothelial lining of Bowman's capsule . . . . . . . . . . . . . . . . . . Kidney from a lead poisoned pony. Hypercellularity in glomerular tuft. Hyperemia of glomerular capillaries. . . . . . . . . . . . . . . . . . . . . . vi Page 21 22 25 25 26 26 27 29 29 30 3O 31 INT RODUCT I ON Lead has been a toxicant to man and animals at least as far back as biblical times. Yet even today with our present knowledge of the toxicity problems from this heavy metal, it is considered one of the most common causes of poisoning in domestic animals and children. The importance of lead as a toxic agent in our environment has increased because every year society utilizes more of this element in its quest for progress. The rising consumption of lead based fuels by vehicles is but one example. Much of the veterinary research to date has dealt with the ingestion, absorption, metabolism, and excretion of lead in different animals. Researchers have also measured lead values in blood, urine, and feces during lead toxicosis and the treatment of the condition. Little controlled research has been conducted on the equine species regarding lead poisoning. Results thus far have primarily come from clinical cases where the source of the toxicant has been suspected to be lead contaminated pastures and the amounts of lead ingested have been based on estimates of the average daily consump- tion of the forage. OBJECTIVES 1. To feed a known amount of inorganic lead to ponies to pro- duce a state of chronic intoxication. 2. To measure values of lead in blood and urine during the trial and attempt to correlate the values with any clinical signs of intoxication. 3. To correlate blood parameters, i.e., packed cell volume (PCV), hemoglobin (Hb), red blood cell (RBC) counts, with the length of time exposed to a known amount of lead. 4. To determine if there are consistent gross and microscopic lesions in the equine from chronic lead poisoning. 5. To determine liver and kidney lead values after euthanasia. LITERATURE REVIEW Incidence Anonymous (1974) mentioned that lead is a serious environmental pollutant and that it has been incriminated more than any other substance as a cause of accidental poisoning in domesticated animals. Buck et a1. (1973) stated that even with our increased awareness of toxicities associated with lead, it is one of the leading causes of poisoning in large and small animals. Zook et a1. (1969) found lead to be the most common form of poisoning in dogs. Fenstermacher et a1. (1946) believed that many more cases of plumbism in cattle occurred than were diagnosed and that lead poisoning in cattle was infrequently reported. McIntosh (1956) noted that, although obvious forms of lead poisoning in animals have been recognized for a long time, more recent work has indicated that the toxic effects of lead contribute to illness and death of a much wider range of animals than previously realized. Zook et a1. (1972) diagnosed lead poisoning at postmortem examination in 34 simian primates, ll parrots, and 3 fruit bats at the National Zoological Park. Sanderson and Thomas (1961) found lead levels of 10 parts per million (ppm) or more in the livers of 23 of 101 wild raccoons in Illinois. Knight and Burau (1973) reported on incidences of lead toxicity on 2 horse ranches in California. Hammond and Aronson (1964) published a study of multiple 3 4 cases of lead intoxication of cattle and horses near a smelter in Minnesota. Priester and Hayes (1974) summarized case reports of lead poisoning in domestic animal species from 11 colleges of veteri- nary medicine in the United States for the period July 1968 to June 1972 but could find no regular pattern of geographic distribution of the problem. Reported Cases of Lead Poisoning by Species Bovine 102 Equine l7 Canine 37 Feline 2 Total I55 Allcroft and Blaxter (1950) contended that in dealing with farm ruminants the major, if not the only, danger of lead poisoning is from an acute standpoint and that chronic poisoning would not occur in cattle and sheep at least if the lead values involved were similar to those in human cases. Hammond and Aronson (1964) found horses to be somewhat more susceptible to chronic lead poisoning than cattle. Buck et a1. (1973) stated that young animals are more susceptible to lead toxicosis than mature animals and goats, chickens and pigs are more tolerant than horses and cattle. Schmitt et a1. (1971) suggested that horses may be more suscep- tible than cattle to chronic toxicity from lead because horses developed lead poisoning while grazing on pastures adjacent to a lead smelter while cattle grazing the same area appeared healthy. Etiology Source Radeleff (1964) wrote that the more common sources of lead that cause poisonings have been lead-based paints, pesticides containing lead, discarded wet cell batteries, and lead shot that accumulates around waterfowl hunting sites. Buck (1970) listed the most common sources of lead involved in cattle toxicities as lead-based paint, used motor oil, oil filters, storage batteries, and other petroleum products. Several articles (Egan et a1., 1970; Holm et a1., 1953; Aronson, 1972) incriminated industrial contamination of animals' environment through pollution of herbage by fumes or residual runoff from processing of lead or lead mining as a source of this toxicant. Absorption and Excretion Oehme (1973) stated that metallic lead is slowly but constantly absorbed by most routes except the skin. Lead particles buried sub— cutaneously or intramuscularly are frequently absorbed in concentra- tions high enough to cause a toxicity. According to Goodman and Gilman (1965), lead is absorbed from all parts of the respiratory tract and, indeed, this absorption is more rapid and complete than by any other route. Blaxter (1950) pointed out that the absorption of lead from the alimentary tract of domestic animals proceeds slowly over a period of hours and that, due to solubility factors, most lead is absorbed in the small intestine. Kehoe et a1. (1940) stated that lead absorption from the gastrointestinal tract is slow and dependent upon species involved, and about 10% of ingested inorganic lead is absorbed. Buck et a1. (1973) wrote that only 1 to 2% of orally 6 administered lead is absorbed from the digestive tract and that a large portion of the absorbed lead is retained initially in soft tissues and eventually in the bone structure. Blaxter (1950) gave the distribution in body tissues of orally ingested lead as: 60% in bone, 25% in the liver, 4% in the kidney, 3% in the intestinal wall, 3% in the reticuloendothelial system, and 4% in the remaining tissues, including hair. Goodman and Gilman (1965) stated that nearly all circulating inorganic lead is associated with the erythrocytes, and only when lead is present in large amounts does the plasma contain significant amounts. Oehme (1973) stated that inorganic lead is excreted from the body chiefly in the feces and urine and that 90% of orally ingested lead is unabsorbed and passed in the feces. Blaxter (1950) found that the urinary excretion of lead in sheep is dependent upon the quantity of lead absorbed but does not exceed .8 mg/day. Dosage of Toxicant Hammond and Aronson (1964) discovered that horses died from lead toxicity in March following a winter intake in their hay of 2.4 mg lead/kg/day. Aronson (1972) reported that horses were poisoned when exposed to 1.7 mg/kg (approximately 80 ppm dry weight) of lead in their forage. A similar intake for cattle did not cause ill effects. Clinical signs did occur in cattle when their daily intake was 6 to 7 mg lead/kg of body weight/day. Interaction with Minerals Hsu et a1. (1975) found an interaction between lead, zinc, and calcium when fed to pigs. An elevated dietary zinc increased lead 7 toxicity, and a calcium deficiency increased the toxicity of both lead and zinc. Willoughby et al. (1972) could not reproduce results of previous studies (Shelling, 1932; Six and Goyer, 1970) which indicated animal diets low in calcium and phosphorus would increase the susceptibility to lead toxicity. Clinical Signs Zook et al. (1969, 1972a) reported that clinical signs of acute lead poisoning in the dog begin with anorexia and vomiting, colic, diarrhea or constipation, and then progress to nervous disorders such as excitability, hysteria, and convulsions. Radeleff (1964) reported that in acute lead toxicity more often seen in cattle and sheep, the symptoms can be walking in circles, apparent blindness, excessive salivation, grinding of teeth, muscular twitching, and convulsions. Weakness and prostration follow. In the acute phase horses are more prone to develop colic and diarrhea. In horses, chronic lead poisoning was manifested as anorexia, weight loss, muscular weakness, anemia, roaring and, terminally, inhalation pneumonia (Knight and Burau, 1973). Radeleff (1964) reported that chronic lead poisoning occurs more often in horses, cats, and dogs and that the condition might manifest these symptoms: anorexia, wasting, depression, muscular weakness, and prostration. The horse in particular might develop dyspnea due to laryngeal muscular paralysis. 8 Diagnostic Tests Willoughby and Brown (1971), using the dithizone method followed by spectrophotometric analysis, determined that the normal whole blood lead value in the horse was 10.7 :_8.51 ug Pb/100 gm. The study involved 193 blood samples from 158 normal horses of various ages. The highest blood lead values were found in mares at parturition. Hammond and Aronson (1964) reported the following blood lead averages: 16 normal horses, 0.04 ppm; 6 exposed to lead but remaining asymptoma— tic, 0.40 ppm; 2 clinically involved, .29 ppm; and 5 fatally affected, 0.49 ppm. Holm et al. (1953) found blood lead values of 0.50, 0.30, 0.40, and 0.30 ppm in 4 poisoned horses. Buck et al. (1973) stated that in large domestic animals a blood value of 0.35 ppm lead or above is evidence of unusual exposure to lead. Normal values are in the range of 0 to 0.15 ppm. Knight and Burau (1973), using atomic absorption spectropho- tometry, reported urine lead values in 2 horses exposed to lead at 0.1 ppm and 1.2 ppm. Three control horses had urine lead values of 0.05 ppm, 0.05 ppm, and 0.04 ppm. Holm et al. (1953) performed hematologic examinations on lead poisoned horses and they did not have abnormal blood cells, baso- philic stippling, or anemia. McSherry et al. (1971) reported a correlation between the urinary aminolevulinic acid (ALA) test and clinical signs of lead poisoning in the cow, dog, and cat. Gross and Microscopic Lesions Smith and Jones (1966) stated that lesions of lead poisoning resolve themselves into those of gastroenteritis, destruction of 9 erythrocytes, and damage to nervous tissue. Radeleff (1964) wrote that in peracute cases a mild degree of gastritis may be the principal lesion, and that generally there is a gastroenteritis often with hemorrhages. The liver may be pale and the kidney may contain hemor— rhages. Buck et al. (1973) wrote that histologically the glomerular capsule is thickened and hyalinized. Degeneration and necrosis of proximal convoluted and descending tubular epithelium is a prominent lesion along with acid-fast intranuclear inclusion bodies. Hemor- rhages in the meninges are seen along with increased cerebrospinal fluid and edema throughout the brain. Radeleff (1964) wrote that in the chronic form of lead toxicity the gastroenteritis of acute poison- ing is usually absent. The liver tends to be yellowish. Hemorrhages are most regularly found on the heart and in the kidneys. Goyer (1968) reported intranuclear inclusion bodies and swelling of mitochondria in proximal convoluted tubules in rats. Thompson (1972) assessed the reliability of the presence of acid-fast inclusion bodies in renal epithelium in diagnosing lead poisoning. Twenty— seven percent of 55 cases of lead poisoning in cattle were negative for acid-fast inclusion bodies in renal cells. There was considerable variation between lead levels in the kidney and the number and size of inclusions. Zook et a1. (1972) based their diagnosis of lead toxi- city in monkeys on the findings of multiple acid-fast intranuclear bodies in renal tubular epithelial cells or hepatocytes. Sauer et al. (1970) observed that lead poisoning caused prolifera- tive and degenerative vascular changes, edema, laminar necrosis, and demyelinization in the central nervous system of 3 primates. Christian and Tryphonas (1971) described cerebrocortical malacia, status 10 spongiosus, astrocytic swelling, nerve cell degeneration, severe cavitation, and vascular proliferation in cattle supposedly suffering from prolonged lead poisoning. Tissue Values Fenstermacher et al. (1946), using the dithizone method of analysis, contended that 0.3 mg% lead in liver tissue on a wet basis was not significant, 0.5 mg% of lead was suspicious, and 1.0 mg% of lead was considered lethal. Hatch and Funnell (1969) reported lead poisoning often results in values of 10 ppm for the liver and values of 15 ppm or greater for the renal cortex. Control values in liver range from 0.3 to 1.5 ppm. Control values in the kidney range from 1 to 3 ppm. Hammond et al. (1956), using the dithizone method of analysis, reported a lead value range of 0.2 to 1.9 ppm in livers from normal cattle. MATERIALS AND METHODS Animals, Housing and Rations The 6 experimental animals used were mixed breed adult pony mares ranging from 5 years to 15 years of age and weighing from 290 to 475 pounds. Prior to the study the ponies were physically examined to be sure they were in good health. They were dewormed via stomach tube with Equizolea at the dosage of 4 gm/100 lb body weight to elimi— nate as many internal parasites as possible so they would not affect the blood parameters. The ponies were housed at the Michigan State University Veteri- nary Clinic in individual stalls with aluminum pipe fronts and concrete walls. The walls were painted with non-lead based paint. The ration consisted of Omalene,b mixed grass and alfalfa hay, and water from the East Lansing, Michigan, city water supply. Methods and Techniques General Procedure Samples of the Omalene, hay and water were submitted for analysis of lead values. The Omalene and hay were dry ashedc and analyzed by a . Equizole, Merck and Company, Rahway, NJ. Omalene, Ralston Purina Company, St. Louis, MO. CMethods of analysis of the Association of Official Analytical Chemists. 1975. Vol. 12, 25.077. 11 12 an atomic absorption spectrophotometerd and the water was solvent extracted for lead and analyzed by atomic absorption spectropho- tometry.e All ponies were fed the ration for one week prior to the study to rule out palatability problems. Two ponies (P5 and P6) were used as controls. The other 4 ponies (Pl-P4) were fed lead oxidef for 60 days at the rate of 1 mg/lb of body weight/day mixed with the sweet grain mixture (Omalene). The hay and water were provided ad libitum. The ponies were observed daily for any clinical signs of lead toxicosis. Collection of Blood 1. Blood samples were drawn before and during the study on a g vials with EDTA weekly basis from the jugular vein using Vacutainer as an anticoagulant. The following procedures were performed on the samples: A. Packed cell volumes (PCV) were determined using the Micro-Hematocrit Capillary Tubeh and centrifuge method. dA. A. Spectrophotometer, Perkin-Elmer Model 403 with deuterium arc background corrector. eThe precise determination of lead by solvent extraction-atomic absorption spectrometry. 1972. Atomic Absorption Newsletter. Vol. 11, NO. 6' 120-1210 fLead oxide (Pb O ) (purified), Matheson Coleman and Bell, 3 4 Norwood, OH. gVacutainer, Becton-Dickinson, Rutherford, NJ. hBlu-Tip Capillary Tube, Sherwood Medical Industries, St. Louis, MO. 13 B. Hemoglobins (Hb) were determined using the Hycel Cyan- methemoglobin Standard.i C. Red blood cells (RBC count) were counted using the Spencer Hemacytometer.j D. Blood smears were examined for abnormal cells and baso— philic stippling after applying Wright's stain. 2. Blood samples were also drawn before and at 4-day intervals during the study from the jugular vein using 20 cc plastic heparinized syringes. These samples were used for lead determination by the solvent extraction method followed by analysis with an atomic absorp- . k tion spectrophotometer. Collection of Urine Urine was collected before and at 4—day intervals during the study using lead—free polyethylene containers. These samples were analyzed for lead by the solvent extraction method followed with an . . 1 atomic absorption spectrophotometer. Necropsy Examination The following procedures were performed after all the ponies were euthanatized with succinyl choline and electrocution: leanmethemoglobin Certified Standard, Hycel, Inc., Houston, TX. JHemacytometer, American Optical Company, Buffalo, NY. kThe precise determination of lead by solvent extraction-atomic absorption spectrometry. 1972. Atomic Absorption Newsletter. Vol. 11, No. 6, 120-121. 1Same as "k" (above). l4 1. A necropsy examination was performed to record gross lesions. 2. Samples of liver, kidney, and contents of the large intestine were taken for determination of lead values using the dry ash techniquem followed by analysis with an atomic absorption spectrophotometer.n 3. Tissues were taken for histopathologic study from most organs, including skin, fat, skeletal muscle, cardiac muscle, aorta, diaphragm, esophagus, stomach, small intestine, large intestine, trachea, lung, lymph node, spleen, liver, kidney, adrenal gland, urinary bladder, thyroid gland, brain, peripheral nerve, and costochondral junction of the rib. The tissues were fixed in 10% buffered formalin, trimmed and processed in an AutotechniconO and embedded in Tissue Prep.p Sections were cut at a 6 micron thickness and stained with hematoxylin and eosin. Liver and kidney sections were also stained with: Ziehl— Neelsen's acid-fast stain for intranuclear inclusion bodies, Prussian blue stain for iron containing pigments, and oil red O stain for lipids. mSame as footnote "c". n I! N Same as footnote d . OAutotechnicon, Technicon Company, Chauncey, NY. pTissue Prep, Fisher Scientific Company, Fairlawn, NJ. RESULTS General Observations The ponies usually ate the lead-sweet feed mixture readily. If there was any hesitancy, more sweet feed was added to mask the taste of the lead so that all the feed was consumed. All ponies gained some weight during the study. The average gain was 11 pounds. Clinical signs were observed in 2 of the 4 ponies (P2 and P4). Signs manifested were hyperexcitability and mild diarrhea. The ponies' signs began approximately 30 days into the study (Table l), lasted about 14 days, and were not observed at the termination of the experi- ment. Pony 3 could only be sampled twice in the beginning and at the end of the study because she became fractious and unsafe to handle. Lead Levels in the Basal Ration The lead levels in the hay-grain mixture, and water, before the lead oxide was added were all Hummmz «« .mcoflm HMUNGHHU mo coeumcflsuwu paw acaccflmmm k mcHH . . . . . . . . -mmmn um>o NN m be N OO N NN N O ON NO N mN N NO N mmmmuocH m0 uouomm NN. OO. ON. Hm. OO. N4. ON. ON. OO Nan NO. NN. OH. HO. OH. Ne. Om Nae ON. NN. OO.H ON. NO. NO. NN moo NN. « Na. OH. E HN. NN. NO. ON Nan NO. ON. OH. NN. NO. NN. av Ame NO. ON. OH. NN. NO. ON. ON NmO NH. O4. OH. . NN. HH. NO. ON Nan NO. 4 «N. NO. OH. NO. NN. NN Nan NH. NH. NO. ON. HH. ON. NN Nan ON. Ha. NO. ON. OH. NN. ON Nmo 4H. ON. OH. OH. OH. Ha. ON Nan ON. NN. OH. 4N. OH. 44. OH Nan .Omc NO. HO. NO. NO. ON. NO. ON. NO. NN. NH Nan .Omc 4O. .Omc «O. HH. NO. 4N. ON. NN. HN. m sea .Omc 4O. .Omc.. NO. OH. 4N. NH. NN. NN. NN. NH. ON. a sac NO. NH. NO. NH. NO. OH. NO. NN. NO. NH. 40. OH. mcHHmmmm imcHuOVAOOOHQO HOOHHOOAOOOHHO imcHHOVIOooHnO AmcHusVHOooan AmcHuaVAOOOHnO imcHusvloooan iHouucoov Om HHouucooO mm «N Nm Nm Hm mmflcom HmucwEHquXm :N Hemmv mosam> Gama mcflus Ucm UOOHm .H magma .uoquHHHHE oafldo\mcoflaafle ca ucsoo Hamo pooan pmu Hummv .HS ooa\mEmum CH CNQOHmOEm: u Anmv .ucmouom CH mESHo> Haoo voxomm u A>Omv o.ov o.hN m.hm m.mm @.ma w.mH ®.mm m.ma m.om mcHH Immmn Eonm h.av «.mm ©.mm mmmmuomv 7 unmoumm l Ob.m m.OH Hm mh.© v.HH mm mm.® N.HH Hm mm.m m.m mm m x003 oa.h m.HH mm 00.0 v.HH mm on.h m.NH mm b xmmz oo.h v.NH mm mo.® H.NH mm ov.h H.ma mm o xmwz om.h m.ma mm mm.h o.NH mm Oh.h v.ma mm m xwm3 om.h N.vH 0v mm.o o.NH Hm ov.h m.ma om v xmmB mm.w m.ma mm mm.© H.NH «m Om.b v.NH mm m xme om.m m.va 0v ov.b m.HH mm mh.h N.ma mm N xmmz mh.h m.ma mm ov.h m.ma mm mv.m o.mH Nv mv.h o.mH Vm mm.h ®.HH mm m>.m ©.MH hm H xmwB mm.m ©.ma mm Om.h m.mH om Om.m m.vH mv mh.m m.ma mm mh.m m.MH mm oo.m m.mH mv mcwammmm Hummv Harv A>Omv Hummv Harv A>Omv Hommv Harv H>omv Aummv Anmv H>Umv Hummv Anmv A>Umv Aummv Hazy A>Umv AHOHDCOOV mm AHOHDCOOV mm vm mm mm Hm mmwcom Hmucmeflummxm :N mumumemumm poon .N magma 18 Table 3. Averages of blood parameters of ponies (Pl-P4) ingesting lead oxide PCV (%) Hb (gm/100 ml) RBC (mil/mm3) Baseline 41.3 14.4 9.00 Week 1 36.5 13.1 8.15 Week 2 36.0 13.0 8.02 Week 3 36.0 12.8 7.53 Week 4 35.7 13.2 7.28 Week 5 35.3 13.0 7.58 Week 6 34.0 12.5 7.02 Week 7 33.3 11.9 7.13 Week 8 30.8 10.8 5.99 % decrease from base— 25.4 25.0 33.4 line (PCV) = packed cell volume (Hb) = hemoglobin (RBC) = red blood cell count 19 Mean corpuscular volumes (MCV) were determined using the data from Table 3. The baseline MCV was 44.8 U3 and the MCV at the end of the experiment was 51.3 U3- Evidence of abnormal cells or basophilic stippling was not seen on Wright's—stained blood smears. Blood and Urine Lead Values The blood and urine lead values of the ponies fed lead oxide increased in an erratic manner throughout the study. The final values were elevated markedly above the controls and also their individual baseline values (Table l). Averaging the final values for the 4 ponies gave an increase by a factor of 2.88 for the blood and a factor increase of 7.75 for the urine over the baseline averages (Table 4). The graphs (Figures 1 and 2) depict the rela- tionship between the blood lead values and both the packed cell volumes and hemoglobins. There did not appear to be a good rela- tionship between the blood and urine lead values and the onset and severity of clinical signs. Liver, Kidney and Intestinal Contents The lead levels of samples from the ponies exposed to the toxicant were all elevated over the control pony values. Ponies P3 and P4 had higher values in the kidney tissues compared to the liver samples. There was not consistency of results from the intestinal contents between the poisoned ponies (Table 5). 20 Table 4. Averages of blood and urine lead values of 4 ponies (Pl-P4) ingesting lead oxide Blood lead (ppm) Urine lead (ppm) Baseline .18 .04 Day 4 .30 .20 Day 8 .38 .27 Day 12 .23 .08 Day 16 .40 .18 Day 20 .33 .14 Day 24 .32 .14 Day 28 .24 .10 Day 32 .25 .02 Day 36 .47 .13 Day 40 .27 .08 Day 44 .25 .09 Day 48 .39 .20 Day 52 .72 1.17 Day 56 .46 .10 Day 60 .55 .31 Factor of increase 2.88 7.75 over baseline 21 AHE OOH\EOO 3H: :NQOHO Ioaom NH MH vH ma 0H 00 .mpflxo Umma pom AvaHmv moflcom v mo mwsHm> canoamofimn can Umoa vooHn mommm>4 mm om mv 0v mm mwmp Om mm ON ma OH .H onsmflm Umwd UOOHm CNQOHOOEmm H.o m.o m.o m.o 0.0 o.H €53 Ummq vooam 22 .wpflxo omoa pom Avmuamv moflcom v mo mmsHm> mesao> Hamo onomm Ucm pme pooHQ wmmum>¢ .m wusmflm mxmp 00 mm om mv ov TMM om mm om ma 0H m o m H.o 0H omen poon N.o ma m.o om ¢.o va A>Omv mfisao> mm m .0 can: HHmO Ummq pwxomm GOOHm Om 0.0 mm h.o oEDHo> HHwO pmxomm 0v m.o mv m.o 0m o.H 23 Table 5. Lead values in liver, kidney and intestinal contents (ppm) from experimental ponies Intestinal con- Liver Kidney tents (lg. colon) P1 27 16 123 P2 19 9 850 P3 13 21 44 P4 21 29 27 P5 (control) *neg. neg. neg. P6 (control) neg. neg. neg. * Negative =