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"T ‘.5 R u 7'“ t LI." 3. . . .‘ ,' ‘ . mm It , -' - ~ ~ c» fi—J .1! u‘ #rrrF-“té-"Z :wmmi Mfr—v This is to certify that the thesis entitled EVALUATION OF THE NEONATAL TOXICITY AND 'THYROTOXICITY OF PENTACHLOROPHENOL IN CATTLE presented by BR I AN JAMES HUGHES has been accepted towards fulfillment of the requirements for M.S. degree in Animal Science -.\ (\ 7’1 / , “\— \./ Y ' Major professor Date 5-21-82 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU BEIURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will returned after the date LIBRARIES M . _ be charged if book IS stamped below. ___ I T—I EVALUATION OF THE NEONATAL TOXICITY AND THYROTOXICITY OF PENTACHLOROPHENOL IN CATTLE. BY BRIAN JAMES HUGHES THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of IflfiEROFSUEME DEPARTMENT CF ANIMAL SCIENCE I982 4 '~ /;1 :‘l ,3 5 " ABSTRACT EVALUATION OF THE NEONATAL TOXICITY AND THYROTOXICITY 0F PENTACHLOROPHENOL IN CATTLE BY BRIAN JAMES HUGHES Pentachlorophenol (PCP) is an antimicrobial agent widely used as a wood preservative. Three studies were done to characterize its toxicity in cattle. Experiment I examined the effects of purified PCP on thyroid hormone levels in mature dairy cattle; the results indicated a decrease in T3 but not Th' Experiment ll examined the transfer of PCP to the newborn calf placentally and via the milk. Results indicated 33% of the maternal serum concentrations were present in calves at birth, and that substantial exposure occurs with Subsequent consumption of PCP-tainted milk. Experiment Ill examined the toxicity of analytical and technical (t)PCP to the bovine neonate. Experiment Ill demonstrated that high doses of tPCP resulted in decreases of both body weight gain and serum T3 and Th concentrations. The high dose tPCP group also elicited path- ologic lesions of the thymus and eyelid. IN MEMORY OF SARA PERRY ACKNOWLEDGMENTS I wish to express my deep appreciation to Dr. Lee R. Shull for his patience and wisdom through the years. His guidance, support, and en- couragement made this work possible. I am likewise grateful to Drs. James Ireland and Steven Aust for serving on my committee. My thanks to Dr. John Gill for his statistical expertise. I also extend my appreciation to James Forsell, John Kinzell, Don Kirsch, Marylee Lockwood, and Barb Olson for their technical expertise, friendship, encouragement and efforts on my behalf. Thanks is due to the personnel at the Michigan State University Dairy Center for their assistance with the animals. To Cindy Warrick and her excellent secre- tarial skills, my deep thanks. I also wish to express my indebtedness to my parents for their con- stant understanding and support. Last, but not least, I thank my Lord and God who has done all things exceedingly abundantly beyond all that I could imagine or ask. TABLE OF Introduction. . . . . . . . Literature Review . . . . . . Pentachlorophenol Chemistry . . . . . . Uses. . . . . . . . . Production. . . . . . Past and Current Problems Toxicokinetics - Absorption Tran5port and Distribution. Biotransformation and Excretion CONTENTS 9 Mechanism of Action and Toxicity. Dioxins Absorption, Distribution, Metabolism, and Excretion Mechanism of Action, Toxicity, Pathological and Biochemical Effects . Objectives. . . . . . . . . Materials and Methods . . . . Reagents. . . . . . . . . Experiment I . . . . . Animals and Diets . . Dose . . . . . . . . Sampling Procedures . Statistical Analysis. e s c L c c 0 e a page #FN omoomm _a 20 2i 2l 22 22 22 Experiment II . . . . . . . . . . . . . . . . . Animals and Diets . . . . . . . . . . . . . Cows. . . . . . . . . . . . . . . . . . Calves. . . . . . . . . . . . . . . . . Dose. . . . . . . . . . . . . . . . . . . . Sampling Procedures . . . . . . . . . . . . Experiment "l. e o o o c a o a I o l a I I e O Anim‘s and Diets I I O Q I I O O I Q I C O - Dose. . . . . . . . . . . . . . . . . . . . Sampling Procedures and Measurements. . . . Statistical Methods . . . . . . . . . . . . Analytical Procedures . . . . . . . . . . . . . Determination of PentachlorOphenol Residues Thyroxine Analysis of Serum . . . . . . . . Triiodothyronine Analysis of Serum. Re5ults . . . . . . . . . . . . . . . . . . . . . . Experiment I. . . . . . . . . . . . . . . . . . Experiment ll . . . . . . . . . . . . . . . . . Placental Transfer of PCP . . . . . . . . . PCP Levels in Calves During the Neonatal Period Milk PCP Concentrations . . . . . . . Average T3 and Th Concentration in Serum of Cows o o O in Tissues. and Calves. . . . . . . . . . . . . . . . . . . ExPeriment III. . . . . . . . . . . . . . . . . Blood Levels of PCP . . . . . . . . . . . Effect of Body Weight . . . . . . . . . . . T3 and T4 Levels During the Course of the Experiment. page 23 23 23 23 2h 25 25 25 26 27 28 29 29 . 29 30 BI 31 34 35 . 35 . 36 36 TRH Challenge . . . . . . . Organ Weights of Calves . . . . Tissue Concentrations of PCP. . HistoPathologic Examination of Tissues. Discussion. . . . . . . . . . . . . . . Toxicodynamics of Pentachlorophenol-General Appearance, Growth. . . . . . . . . Thyroid Hormone Concentrations. . . Pathologic Findings . . . . . . . . Toxicokinetics of PentachlorOphenol Milk PCP Concentrations . . . . . . Tissue PCP Concentrations . . . . . Summary . . . . . . . . . .-. . . . . . AppendixA............... Appendix B. . . . . . . . . . . . . . . Appendix C. . . . . . . . . . . . . . . List of References. 0 o a O O o o g t 9 List of Tables. . . . . . . . . . . . . List of Figures . . . . . . . . . . . . List of Abbreviations . . . . . . . . . vi Serum - o 0 Health, page 63 63 65 68 69- 72 72 73 7h 94 IIO Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Ia lb 2 3 5 6 7 8 9 IO II I2 l3 IA IS 16 LIST OF TABLES Physical Properties of Pentachlorophenol. . . . . Solubility of PentachlorOphenol . . . . . . . . . Dioxins in Bovine Tissue Samples. . . . . . . . . LD 0's of Some Animals Administered PC by Various Routes . . . . . . . . . . . . . . Chlorophenol and Chlorodibenzo-p-dioxin Content of PentachlorOphenol. . . . . . . . . . . Composition of Calf Starter Ration. . . . . . . . Extraction Methods for Determination of Total PentachlorOphenol Residues in Tissues . . . . . . Serum T Concentrations (ng/ml) of Cows EXposed SUbChronically to pPCP. . C O O Q . O O C . O C 0 Serum T Concentrations (ng/ml) of Cows During reatment. . . . . . . . . . . . . . . . . . Design of Experiment II EXposure of Calf via Dam. Comparison of Blood PCP Concentrations at Birth Between Dams and Calves EXposed In Utero. . . PCP Blood Concentrations at End of Postpartum Exposure of Neonate Compared to Dam . . . . . . . Comparison of PCP in Blood and Milk of Treated Cows Average PCP Concentration (ppb) for Four Weeks After Parturition . . . . .*. . . . . . . . Average Serum T and T4 Values of Cows and Calves Exposed for Four Weeks After Parturition . Intake of Grain Fed During Last Three Weeks of Experiment to PCP Treated Calves. . . . . . . . . Organ Weight of Calves Fed aPCP or tPCP . . . . . Concentrations of Total PCP in Various Tissues of Calves Fed aPCP or tPCP. . . . . . . . . . . . . vii I2 20 29 32 33 34 36 37 ’42 43 SI SI 56 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure N .p-w 0) LIST OF FIGURES PentachlorOphenol, Pentachlorophenate , . _ . . . . . . . Formation of Chlorodibenzodioxins and Chiorodibenzofurans. e l o o e o o o o o 0 Suggested Pathway for PCP Metabolism . . ACCumulation of PCP in Blood of Calves Exposed to Contaminated Milk from 0.l mg/kg tPCP Treated Cows for Four Weeks After Parturition. Accumulation of PCP in Blood of Calves Exposed to Contaminated Milk from 10.0 mg/kg pPCP and tPCP Treated Cows for Four Weeks After Par- turition G o O O o 0 O o O 0 O O o 0 Serum PCP Levels of Calves Maintained During the Course of the Experiment , , , , , , , , . Comparison of Weight Gains in the High Dose aPCP and tPCP Groups With Controls , , , . Average T and TA Values During the Course of the Experiment . . . . . . . . . . . . . . . . Average Resultant T and T Outputs After Administration of TRH for hree Haurs, . , , , Cross-section of the Thyroid Gland in Control and High Dose tPCP Groups After #2 Days of Exposure (Magnified HOOX) . . . . . . . . . . Cross-section of Meibomian Gland in Eyelid of Cantrol and High Dose tPCP Groups After #2 Days of Exposure (Magnified HOOX) . . . . . . . . . Cross-section of the Thymus in Control and High Dose tPCP Groups After #2 Days of Exposure (Magnified 400x) . . , , , viii 38-39 tic—Ln h7-h8 49-50 52-53 SB-SS 57-58 59-60 61-62 AHH coo cor DNP choo HpCDD ocno PCP aPCP pPCP tPCP ppm ppb TBG TCDD TCH T3 T4 TSH LIST OF ABBREVIATIONS Aryl Hydrocarbon Hydroxylase Chlorinated dibenzo-p-dioxin Chlorinated dibenzofurans Dinitrophenol Hexachlorodibenzo-p-dioxin Heptachlorodibenzo-p-dioxin Octachlorodibenzo-p-dioxin Pentachlorophenol Analytical Grade Pentachlorophenol Purified Grade Pentachlorophenol Technical Grade Pentachlorophenol Parts per million Parts per billion Thyroid Binding Globulin Tetrachlorodibenzo-p-dioxin Tetrachlorohydquuinone Triiodothyronine Tetraiodothyronine Thyrotropin Releasing Hormone Thyroid Stimulating Hormone INTRODUCTION PentachloroPhenol (PCP) is a broad spectrum biocidal agent prin- cipally used as a wood preservative. In I979, 40 million pounds of PCP were produced in the United States in which 90-95% was used in wood or wood products. Some of these products are used in agriculture, partic- ularly on farms where its highly effective antimicrobial properties inhibit fungal and bacterial rots of wood in high moisture areas. The use of PCP in farms has alerted the scientific community to the possible effects of enrivonmental exposure to livestock. In Michigan, where PCP-treated wood is used in barns and for feed bunks, cattle were exposed by the inhalation and ingestion of PCP. Such exposure was sus- pected of being responsible for low milk production and high calf mortal- ity in dairy cattle; commensurately, blood and tissue PCP levels in these cattle were abnormally high. Whether such exposure results in intoxi- cation is unclear and needs further investigation. LITERATURE REVIEW l. PENTACHLOROPHENOL A. Chemistry Pentachlorophenol (PCP) is fully chlorinated phenol, (Figure la). In its pure form, PCP is a white, needle-like crystalline solid (Bevenue and Beckman, 1967; anonymous, I979). It has a high solubility in non-polar solvents; when solubility in polar solvents is preferred, the sodium salt is used, (Figure lb). Physical properties and solubil- ity in various solvents is presented in Tables la and lb. Cl Cl Cl‘ Cl - + Cl 0H Cl 0 Na Cl \ CI Cl Cl Figure la Pentachlorophenol Figure lb Pentachlorophenate TABLE la Physical Properties of Pentachlorophenol (Kozak, I979) Empirical Formula 'c6Hc150 Molecular Weight 266.35 Melting Point, 0c I90 Boiling Point, °C 293 Density, g/cm3 l.98 Vapor Pressure, mm H9 at 100°C 0.12 Dissociation Constant, 25°C 1.2 x 10-5 Table lb Solubility of Pentachlorophenol, g/lOOg solvent, 20°C (Kozak, I979) Water 0.00ih Methanol 57 Diethyl Ether 53 s the nol l+7 Acetone ' 2i Xylene l4 Benzene ll Carbon Tetrachloride 2 Pentachlorophenol is relatively stable and will not decompose when heated to its boiling point for extended periods of time. It is not subject to easy oxidative coupling or electrophilic substitution reac- tions, although its hydroxyl group tends to facilitate biodegradation. Its vapor pressure is relatively low, but losses do occur from soil, vvater and PCP-treated items. Greater volatilization occurs at higher ‘temperature when PCP is in its protonated (phenolic) form.- The earliest method of quantitating pentachlorOphenol was by colorimetric determination of the oxidative products when reacted with riitric acid. However, newer methods of isolating and quantitating PCP €:Xploit the ionization of the compound. PCP is a weak acid with a pKa crf 4.7h (Anonymous, l980). It exists as a polar anion under basic con- Ciitions and as a nonpolar molecule under acidic conditions. This allows ‘F<>r easy partitioning and extraction from a biological matrix at an acid F>f1 into a nonpolar solvent. Quantitation is usually by gas-liquid chro- flnaatography equipped with an electron-capture detector. This method allows for quick, easy, sensitive, and ineXpensive separation and 3 IIIIIIIll-:::;_______________ L. quantitation of PCP in various matrices. 8. Uses Pentachlorophenol was initially prepared in I872 by chlorination of phenol at elevated temperatures in the presence of antimony tri-I chloride. Not until the I930‘s was pentachlorophenol used for preserv- ing wood (Anonymous, I979). In I979 nearly #0 million pounds of PCP was produced annually (Maloney and Pagliai, I980) with 90-95% of it used as a wood preservative. Because PCP has unique broad spectrum biocidal prOperties, it ex- cels in protecting wood from fungal rot, bacterial decay, and termite infestation. Not only is it a highly effective antifungal and anti- microbial agent, but it also is used as a preharvest plant desicant, al- gicide, slimicide, fungicide, herbicide, and mollusicide (Bevenue and Beckman, I967). C. Production PentachlorOphenol is produced commercially much the same way as vuhen it was first synthesized. Molten phenol undergoes direct chlorin- ation with chlorine in the presence of ferric or anmonium chloride in a ‘two-stage process. The first stage involves raising the reaction tem- t>erature to I05°C for the formation of tri- and tetrachlorinated phen- <>ls. The second step involves a gradual increase In temperature to ICI ° C‘ -HCI Cl 0 at c C] ‘3‘ A c1 lHo c: A >c, o c1 c1 CI Cl c C] CI c1 Cl Cl 0 Cl -Clz Cl 0 Cl C. -——-> 0 9 .. CICl Cl [3. CI CT CT C Cl ‘ Figure 2 Formation of Chlorodibenzodioxins and Chlorodibenzofurans In recent years various preparations are available with reduced concentrations of these impurities (Dougherty, I978). Technical penta- chlorophenol (tPCP) has the highest concentrations of dibenzodioxins Purified pentachlorophenol (pPCP) preparations and other impurities. such as DowicideDEC-7 Contain lower concentrations of dioxins. Compar- aatively, analytical grade pentachlorophenol (aPCP) is the ”cleanest” F>reparation (see Table 3, Materials and Methods). (3. Past and Current Problems Numerous and varied applications of PCP and consequently its highly ‘t<>xic contaminants suggest wide dispersal in the environment and inad- \I€ertent intrusion into the food chain of animals and humans. In Mich- ‘333an, for example, PCP was found at levels of 2 ppm to l2 ppm in the b lr overutilization of PCP-treated wood was noted on 39% of the farms in t1ichigan. This suggests overexposure of a potentially deleterious chemical to livestock. E . Toxicokinetics - Absorption Exposure to PCP can occur by three different routes; by inhalation I‘esulting from the volatilization of PCP off treated surfaces; dermally f’rom direct contact with PCP; or orally from direct ingestion of PCP itself or the treated products. Few large animal studies have been done regarding pulmonary ex- posure to PCP. In I978 Thompson evaluated the maximum expected air PCP level due to volatilization in barns constructed in part from PCP-treated poles and boards. He found concentrations of 0.02 mg/m3 PCP in air and estimated a dose of about 0.006 ppm to cattle. The hazards of the in- halation of PCP-laden air have yet to be scientifically evaluated. Dermal exposure has been observed more in swine than in cattle due to the use of farrowing crates and the direct contact of both sow and piglets to freshly treated wood. It has also been noted that young an- imals are more sensitive to PCP toxicosis and that tolerance is vauired with age (Walters, I952 and Schipper, l96l). Oral exposure of cattle and swine to PCP poses a serious problem. ShuII et aI (I98l) noted that PCP-treated wood was being used in feed bunks and bunk silos on many dairy farms in Michigan. This could poten- tially result in contamination of feeds and serve as a source of further exposure in addition to inhalation. Two herds in Michigan had to be destroyed because of elevated blood PCP and dioxin levels through environmental sources of contamination (Conklin and Fox, I978). In I979 Firestone et al determined that PCP is excreted through the milk of cows and thus is a source of contamination to their young. Furthermore, Kinzell (I982) determined that 5% of the PCP dose is eliminated via milk. In swine, exposure occurs by inhal- aation and ingestion when sows are housed in freshly treated farrowing <:rates and allow their young to suckle off teats contaminated with PCP can the surface. Piglets exposed in this manner exhibited burns on nos- ‘trils, face and tongue. Similarly, the skin of the udders and teats of 8 the sows were necrotic (Schipper, I96I). F. Transport and Distribution Whether PCP is taken up through the skin via the respiratory tract or through ingestion, it is bound to serum albumins and distributed to the various body compartments (Hoben, I976). Harrison (l959) showed that sheep force-fed PCP impregnated saw- dust reached peak serum levels of PCP in 3 to 6 hours. In I952 Walters looked at the tissue distribution of PCP after drenching with the chem- ical. Of the various biological matrices analyzed, urine was the highest followed by blood, kidney, and liver. Kinzell (I982) used Inc-PCP to determine the distribution of PCP in the cow. IhC -PCP ac- tivity was the greatest in serum followed by liver, kidney, gall bladder, and lung. A paucity of data exists in large animals to further define the distribution of PCP. It is assumed that data obtained from experi- mental animals is directly applicable to domestic and wild animals. Several studies have been done to detail the tissue distribution of PCP in experimental animals taking into account the various routes of administration. Jakobsen and Yllner (I97I) intraperitoneally in- jected mice with lb’C-PCP. Their findings were as follows: I) 63-79% of the total dose of PCP was excreted in 48 hours in the urine; 2) greatest concentrations of IAC were found in the gall bladder, in- testines, and stomach suggesting enterohepatic circulation and gastric secretion; 3) 89-98% of the IA C dose was accounted for in the urine, and h) liver and kidney contained substantial concentrations of 1III: indicating metabolism and excretion. Nearly all other work indicates similar findings. Braun et al, 9 (I977) and Braun and Sauerhoff (l976) established the half-life of PCP Inc-PCP half-life in rats and monkeys. In rats the orally administered was l3-I7 hours for males and females given I0 mg/kg and for males given I00 mg/kg. For females given l00 mg/kg, the half-life was 27 hours. PCP reached peak blood concentrations at four hours post-dose. Tissue concentrations of PCP were greatest in liver and kidney and the lowest in brain, spleen, and fat. In monkeys the half-life of PCP is approx- imately 83.0 hours for females and 72.0 hours for males. Peak concen- trations occurred at l2-2h hours after oral administration. Studies examining the placental transfer of PCP are conflicting and need to be further clarified. Hinkle (I973) observed that the maternal blood and the fetus have closely correlated concentrations of PCP when hamsters were given I.25 mg/kg or 20 mg/kg of PCP from day 5 to ID of gestation. Hinkle also showed greater resorptions in the treated groups. On the other hand Larsen (l975) administered PCP to rats and neither appreciable amounts of PCP in the fetus nor any evidence of re- sorptions were noted. In I979 Schwetz et al found the fetotoxic effects of purified PCP to be greater than the technical preparation when given to rats at 30 mg/kg/day and 50 mg/kg/day levels. Fetal resorptions were nearly I00% for the purified preparation at both levels. Skeletal de- fects were the main forms of anomaly in this group again purified PCP being more fetotoxic than technical PCP. G. Biotransformation and Excretion Information on the biotransformation of PCP is severely lacking in large animals. In experimental animals and humans, metabolites of penta- <:hlor0phenol include its conjugate, tetrachlorohydquuinone (TCH), and the TCH conjugate. In experimental animals the following information is available. In monkeys no metabolites have been formed from PCP (Braun and Sauerhoff, l976). Ahlborg et al (I97h) reported that Al% and 43% of PCP was ex- creted nonmetabolized thr0ugh the urine in mice and rats respectively. The other metabolites in urine are 29% TCH and 22% TCH conjugate for mice, and 5% TCH and 38% TCH conjugate for rats. The remainder is PCP conjugate. Jakobsen and Yllner (l97l) suggested the following pathway for PCP metabolism: 6R 0H 0H 0H Cl Cl CI Cl Cl Cl CI Cl <—— _a —> _ Cl Cl CI Cl Cl Cl CI Cl Cl I 0 0 TCH - conjugate CO0H PCP - conjugate PCP TCH ___O 0.... - 1< ”0 OH R- Figure 3 Suggested Pathway for PCP Metabolism H. Mechanism of Action and Toxicity PCP is a known uncoupler of oxidative phosphorylation similar to the activity of dinitrophenols (DNP) as shown by Weinbach (I9Sh). \Jeinbach (l957) Went on to show in vitro effects of PCP on the mito- chondria; these include uncoupling of oxidative phosphorylation, inhi- bition of mitochondrial and myosin adenosine triphosphate ATPase, II inhibition of glycolytic phosphorylation, inactivation of respiratory enzymes and overall gross damage to mitochondria. The binding of PCP to mitochondrial protein was later shown by Weinbach in I965. Hanstein and Hatifi (l97h) showed that DNP and PCP bind to the same site on mitochondrial protein. In still another binding study, this time of actinomyosin to PCP is was suggested that PCP bound to the amino acids lysine or arginine on proteins (Bowen et al, I965). In I977 Arrhenius et al proposed that PCP might cause malfunction of the detoxification of xenobiotics due to its high association with liver microsomes. Danner and Resnick (I980) showed that indeed penta- chlorOphenol does have a high affinity for biological membranes. Since PCP and DNP have similar activities, their signs of toxicosis are the same. The clinical signs caused by uncouplers are nausea, gas- tric upset, restlessness, sweating, rapid respiration, tachycardia, fever, cyanosis, thirst, loss of weight, finally collapse and death (Murphy, I980). Various studies have been done to determine LDSO's for various species. Table 3 gives some of the data. I2 TABLE 3 LDSO's of Some Animals Administered PCP by Various Routes Animal P5: Literature Sheep approx. I20 mg/kg Harrison I959 (oral) Calf approx. tho mg/kg Harrison 1959 (oral) Rat approx. ll.7 mg/kg Hoben I976 (aerosol) Rat approx. 3h mg/kg Hobenl976 (I?) Rat approx. 78 mg/kg Deichmann I9A2 (oral in I% olive oil) The LDSO's indicate PCP toxicosis is dependent on species, carrier, and route of administration. Kozak et al (I979) also stated that the toxicity of PCP may be influenced by three factors: I) ambient temper- ature, 2) renal competency, and 3) general‘health. Studies have been done to determine the effects of PCP on swine and cattle. Schipper (l96l) noticed feed refusal, increased neonatal mortality, and decreased body weights when holding sows in freshly treated farrowing crates. 0n necropsy lesions on the mucosal surface of the stomach, mild emphysema, congestion in lungs, enlarged lymph nodes, inflamation of small intestines, infarcted areas of liver and spleen, and hemorrhaged areas of the kidneys were evidenced. Recently, Greichus et al (I979) found enlarged livers in swine administered purified PCP at I0 mg/kg and IS mg/kg. The clinical chemistry showed elevated blood urea nitrogen (BUN) and white blood cell(WBC) count. In cattle, McConnell et aI (I980) examined the effect of analytical pentachlorOphenol (aPCP) versus technical pentachlorophenol (tPCP). l3 When heifers were treated with IS mg/kg/day aPCP, the treatment effects were decreased body weight, decreased feed efficiency, progressive an- emia, increases in liver and kidney weight and a decrease in thymus weight. Pathological findings in these cattle included villous hyper- plasia of urinary mucosa, hyperplasia of the gall bladder, and bile ducts. Other observations of the tPCP groups were thymic atrophy and keratin deposition in the Meibomian gland of the eyelid; these observations are characteristic of dioxin toxicosis. McConnell et al (I980) also report- ed various effects common to both tPCP and aPCP treated groups. For example, a decrease in thyroid hormone concentrations (T3, Tn) occurred with both grades of PCP. Hepatic mixed function oxidase activities show both types of PCP increased aryl hydrocarbon hydroxylase (AHH) al- though aPCP induced AHH to a lesser degree. AminOpyrine N-demethylase was induced by tPCP but not by aPCP. In vitro studies by Shull and McCarthy (I978) demonstrated that high concentrations of PCP inhibit cellulose utilization of rumen microorgan- isms, raising the questions of possible deleterious effects on rumen function. Other chrOnk studies treating cattle with lower levels of PCP showed no adverse effects. Herdt et al (l95l) gave calves 7.6 mg/kg/day of sodium pentachlorophenate in their drinking water for five weeks and observed no toxic effects in behavior, hematology or postqnortem exam- ination. Kinzell et al (I982) reported no adverse effects of milk production, feed intake, and body weight of cows administered 2 mg/kg/day PCP subchronically. However, he did note enlargement of the liver, lungs, kidney and adrenal on post-mortem examination. Renal function was also impaired in these cattle. In these same cattle, Forsell et al (I98l) I4 could not find any impairment of the immune system. Fleischer et al (I980), Goldstein et al (I977), Kimbrough and Linder (I975, I978) studied technical (tPCP), purified (pPCP) and ana- lytical (APCP) PCP to distinguish the toxicity of PCP from its contamin— ants. All of the above studies including McConnell's work in cattle are fairly consistent with each other. The following is a summary of the effects of tPCP which can occur in rats: vacuolation of hepatocytes, hepatic inclusions, singular hepatocellular necrosis, slight interstitial fibrosis, brown pigment in macrophages and Kupffer cells, hepatic por- phyria, increased liver weight, enlargement of hepatic central veins, bile duct proliferation, periportal fibrosis, and increases in AHH and glucuronyl transferase activity along with increases in cytochrome P-450 and microsomal heme. Knudsen et al (I97h) found that rats fed 50 ppm and 200 ppm PCP had decreased erythrocytes and hemoglobin levels in male rats. Kidneys appeared to have centrolobular vacuolization and calcium depositions. Purified and analytical PCP in the above experiments caused enlarged hepatocytes with cytOplasmic inclusions, slight increases in smooth endo- plasmic reticulum, atypical mitochondria, some lipid vacuoles, and in- I creased glucuronyl transferase activity in the high dose groups and are generally less toxic than the technical preparation. II. DIOXINS A. Absorption, Distribution; Metabolism, and Excretion Pentachlorophenol contains a variety of substances considered to be "inactive” from the aspect of antimicrobial efficacy. These 'hon- phenolics or ”neutral impurities” include chlorinated dibenzo-p-dioxins l5 (C005) and chlorinated dibenzofurans (CDFs). 0f the C005 found in PCP, the hexachlorodibenzo-p-dioxins (HxCDD), the heptachlorodibenzo-p-dioxins (HpCDD), and octachlorodibenzo-p-dioxin (OCDD) are the most prevalent. The most toxic and perhaps well known, 2,3,7,8-tetrachlorodibenzo-p-diox- in (TCDD), has not been detected in any sample in the United States al- though other TCDD isomers do exist in various commercial PCP preparations (Jensen and Renburg, I972; Firestone et al, I972; Villanueva et al, I973;. Kinzell et al, l98l). Since 2,3,7,8-TCDD has been more intensively stud- ied, it is generally believed that what we know about 2,3,7,8-TCDD can be applied to other CDD's as well. I Technical pentachlorophenol is a potential source of dioxin expos- ure. Firestone et al, 0979)reported the occurrence of PCP and dioxin residues in milk and blood of cows fed tPCP for 70 days. The findings indicated that PCP levels in the milk (h mg/kg) were Id% of the levels in the blood (#0 mg/kg). In the milk or body fat of treated cows, the levels of dioxins were about IOOO times those in blood. Average daily excretion of HxCDD, HpCDD and OCDD in the milk during days 90-70 was about 20, #0, and 23 micrograms, approximately 33%, 3%, and 0.6% res- pectively, of the daily intake of the dioxins. The feces of the treated cows had high levels of OCDD suggesting that a substantial portion of the ingested OCDD was not absorbed by the cow. Similarly, Norback et al (I975) reported that greater than 90% of the total radioactivity of OCDD administered to rats was recovered in the feces. At IOO days after PCP feeding was stapped, appreciable amounts of dioxin were still present in the fat of the cows. It is interesting to note that one of the cows calved after contamination; the calf showed lower levels of dioxins than the dam. The presence of dioxins in the calf is suggestive of placental l6 transfer. Norback et al (I975) found OCDD was poorly absorbed through the gastrointestinal lining. Of the dose that was absorbed, the greatest concentration of OCDD was in fat, skin, and liver. Parker et al (I98l) confirmed the accumulation of HxCDDs, HpCDDs, and OCDD in liver and fat of young cattle. More importantly he demon- strated that toxicity was proportional to the dioxin concentration in the tissues. 8 B. Mechanism OF Action, Toxicity, PathoIOgical and Biochemical Effects The mechanism of action of TCDD, the most studied COD, is poorly understood. Despite the wide Spectrum of deleterious effects, none have been fully understood with reSpect to the actual mechanism involved, nor is it known whether the toxicity of TCDD is the result of the action of the parent compound or some metabolite. In I973 Sinapol and Casida stated that no metabolites of TCDD have been found in mammalian systems either in vivo or in vitro. More recent studies have suggested the pre- sence of polar metabolites, possibly oxidation products (Poiger and Schlatter, I979). Furthermore, TCDD is a potent inducer of AHH, suggest- ing a metabolic relationship (Poland, I979). Clarification of COD metab- olism awaits further study. The toxicity of the various dioxins varies in relation to their chlorination. In general, the more chlorination, the less toxic the dioxin: TCDD is more toxic than HxCDD which is more toxic than HpCDD which is more toxic than OCDD. For example, in doses of I to 9 g/kg, OCDD failed to kill female rats and mice while the LD of HxCDD is 50 approximately IOO mg/kg in rats (Johnson, I973). Fortunately, acute I7 exposures are rarely seen in the environment. A more serious problem is posed by the accumulation of dioxins in the fatty compartments of chronically exposed animals to dioxins. Teratological studies with TCDD indicate a single dose of l-IO micrograms/kg is capable of triggering malformations in several species. The characteristic congenital defects are intestinal hemorrhage in rats, cleft palate in monkeys and mice, and kidney abnormalities in both spec+ ies (Schwetz et al, I973; Vos et al, I974; Zingesser, l979). Carcinogenesis is also indicative of COD toxicosis. A higher in- cidence of tumors was seen in female rats along with neoplastic lesions in the lungs and oral and nasal epithelia (Kociba et al, I978; I979; Van Miller et al, 1977). The effect on neonatal growth and development on the exposed fetus and mother is another important aspect of COD toxicosis. TCDD particu« Iarly suppresses the development of the immune system causing thymic atrophy in monkeys (McConnell et al, l978), in rats (Faith and Moore, I977) and in mice (Vos and Moore, I979). In addition to in utero ex- posure, offspring are exposed to significant amounts of these chemicals via their mothers milk. Dioxins have been found in the milk of cows long after exposure ceased (Firestone et al, l979). In the endocrine system, TCDD elicts a reduction of circulating levels of TA' Bastomsky (I977) noted in rats that biliary excretion of radiolabeled Th in the bile was increased fourfold but that biliary ex- cretion of radiolabeled T3 was not affected. Consequently, T4 levels in the blood were reduced while T3 levels were elevated. Thyroid weight was also increased due to TCDD treatment. Other hormonal aberrations include an increase in serum glucocorticoid levels (Neal et al, I979), and a l8 decrease in serum estradiol and progesterone concentrations with a re- sulting reproductive disfunction in monkeys (Barsotti et al, I979). The decreases of steroids are probably a result of increased metabolism and excretion. Urinary tract changes occur with TCDD eXposure probably due to ir- ritation of the epithelial lining by TCDD on excretion. The basic re- Sponse is hyperplasia of the transitional epithelium which may extend from the terminal portions of the collecting ducts of the renal medulla to the renal pelvis, ureter, and urinary bladder (McConnell and Moore, I979). It has been postulated that this hyperplastic response may be related to the species differences in metabolism and elimination of these chemicals from the body. It is known that monkeys secrete a higher pro- portion of a given dose of TCDD via the urine than rats which do not exhibit this lesion (Van Miller et al, l976). Various other toxic effects of TCDD include chloracne, hyperkera- tosis, keratin deposition in the Meibomian gland of the eyelid, hyper- trOphy of the liver, hepatocellular necrosis, and porphyria (McConnell et al, 1978; 1980; Poland, 1979). OBJECTIVES Three experiments were undertaken to examine the effects of various preparations of PCP in newborn calves and mature cows. These experi- ments were initiated for the following reasons: i) High calf mortality was noted on several farms in Michigan where PCP was in extensive use. 2) Several studies had previously established the toxicity of tPCP and aPCP in older cattle but no studies have been conducted in newborn calves. 3) Few studies have been done on the elimination of PCP in milk- but none has been done on its effects and extent of exposure in the offe- spring, and A) There is a paucity of research done on the inhibitory effect on thyroid hormones and the possible significance in dairy cattle. Therefore, it was of interest to conduct experiments with the fol9 lowing objectives: I) To determine the extent of exposure to the bovine neonate via pentachlorophenol treatment of the dam whether transferred placentally or by the milk. 2) To compare the subchronic toxicity of analytical and technical pentachlorophenol in newborn calves. 3) To evaluate the functional and pathologic effects of these chemicals on the thyroid gland. I9 MATERIALS AND METHODS I. Reagents The CD0 and chlorophenol content of available preparations of pentachlorOphenol (PCP) varies among preparations. The following experiments utilized three such preparations, namely, technical penta- chlorophenOI (tPCP), an industrial composite from Vulcan Materials Company, Wichita, Kansas; purified pentachlorophenol (pPCP) from The Dow Chemical Company, Midland, Michigan, lot 05l27D; and analytical grade pentachlorophenol (aPCP) from Aldrich Chemical Company, Milwaukee, Wisconsin, lot 032487. The chlorOphenol and dioxin content of all three of these preparations is listed in Table 4. TABLE A ChlorOphenol and Chlorodibenzo-p-dioxin Content of PentachlorOphenol Components Concentration Technicala Purifiedb Analyticalb Chlorophenols (%) Pentachlorophenol 85-90 88-92 99.02 TetrachlorOphenol h-8 8-l2 .98 Trichlorophenol 0.l -- -- Other 2-6 -- -- Chlorodibenzo-p-dioxin (ppm) Octa-CDD IOOO 2-4 I.2 Hepta-CCDs 378 2-3 I.8 Hexa-CDDs ' I73 0.2 0.2 Tetra-CCDs .035 -- -- a. Analysis as reported by Kinzell et al, I98l b. Analysis by The Dow Chemical Company, Midland, MI Personnel Communication, Dr. Robert L. Johnson 20 2l Trizma base, 8-anilino-l-naphthalene sulfonic acid (ANS), gamma- gIobulins ( Bovine, Cohn's Fraction II) were purchased from Sigma Chem- ical Company, St. Louis, Missouri. Sodium citrate, sodium chloride, sodium hydroxide, sodium phosphate monobasic, sulfuric acid, EDTA ((ethylenedinitrilo)-tetraacetic acid), and diethyl ether were purchased from Mallinckrodt, Inc., St. Louis, Missouri. Sodium phosphate dibasic (crystal), and hexane (Onmi-solve glass distilled) were purchased from MCB Chemists lnc., Norwood, Ohio. Benzene (Baker ResiqAnalyzed), and iso-prOpyl alcohol were purchased from J.T. Baker Chemical Company, Phillipsburg, New Jersey. B-2 barbital buffer was purchased from Beck- man instrument lnc., Fullerton, California. Dextran T-70 was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Charcoal (carbon de- colorizing neutral norit) was purchased from Fisher Scientific Company, New Lawn, New Jersey. Thyroxine [(Tu) 750 mCi/mé) and triiodothyronine ‘(fT3) 550 mCi/mg] tagged with 125! were purchased from New England Nucle- ar, Boston, Massachusetts. T3 standards (0 ng/IOO mg, l00 ng/l00 ml, 400 ng/lOO ml), T4 standards (0 micrograms/IOO mg, 5 micrograms/IOO ml, l5 micrograms/loo ml), T3 antibody, and Th antibody were purchased from Wien Laboratories Inc., Succassuna, New Jersey. Corn oil was purchased from Anderson Clayton Company, Dallas, Texas. ll. EXperiment l A. Animals and Diets Twelve dry Holstein cows three to five years of age were randomly assigned to the following treatment grOUps (three per treatment group): control, 0.I mg/kg body weight pPCP, l.0 mg/kg pPCP, and l0.0 mg/kg pPCP. All cows were fed a straight corn silage ration twice daily with trace 22 mineralized salt added to the p.m. feeding, and kept in comfort stalls in a warm enclosed barn. Cattle were weighed at two week intervals for the duration of the experiment. 8. Dose The pPCP dose was made by dissolving 2.lh, 2l.k and Zlk grams of pPCP per liter of corn oil for use in the 0.1, I.0 and l0.0 mg/kg treat- ment groups, respectively. Purified PCP dissolved easily in corn oil with the exception of the highest concentration which required prior solubilization in diethyl ether and mild heating of the mixture until ether vapors could no longer be detected by smell. The final volume of this mixture was one liter. The daily dose was calculated on the basis of body weight. One half of the daily dose was administered in the morning and the other half at night for a period of l# weeks. The apprOpriate aliquot was delivered directly into the rumen by inserting a 20 ml syringe through a surgically installed fistula (23 mm i.d.). The dose was readjusted every two weeks to account for changes in body weight. C. Sampling Procedures Blood samples were collected in 20 ml Vacutainer tubes from tail veins in two week intervals 0, 2, h, 6, 8, l0, l2, lh weeks into the experiment. The blood was allowed to clot, centrifuged, and serum was decanted into glass vials for later PCP, T3 and Th analysis. 0. Statistical Analysis The T3 and T data were statistically analyzed by split plot meth- b, ed with repeat measurements (Gill, I978). 23 III. EXPERIMENT II A. Animals and Diets l. Cows Five Holstein cows approximately three to five years of age and in the last trimester of pregnancy were assigned the following treatments: three cows received 0.I mg/kg body weight tPCP, one cow received l0.0 mg/kg body weight tPCP, and one cow received l0.0 mg/kg body weight pPCP. The treatments were administered the last three months of pregnancy and continued to four weeks after parturition. Controls were randomly selected from the MSU dairy herd for post- partum comparison with above treatments. Prior to parturition all cows were fed 20 to #5 pounds of corn silage and ID - l5 pounds of alfalfa hay per day. Within one month of calving, grain (up to 10 pounds) was added to the diet. After parturi- tion, high moisture corn, “2% protein supplement, and calcium carbonate were added to the diet. All cows were housed in a warm enclosed barn in stanchions equipped with vaccuum supply lines for bucket milkers. These animals were taken to box stalls at the time of parturition and later returned to the stan- chion barn for postpartum care. They were milked for the first 35 days of lactation and then dried off. All cows were weighed prior to the beginning of the experiment and once weekly during the course of the experiment. 2. Calves Three groups of calves were utilized for this experiment: I) five calves exposed in utero via contaminated dams fed milk contaminated via 24 the dam, 2) five calves not exposed in utero but fed the same contamin- ated milk as the previous five calves, and 3) five control calves not exposed to PCP. Two calves, one each in the 0.I mg/kg tPCP and l0.0 mg/ kg tPCP treatment groups, died at birth apparently from non-PCP related causes. (Refer to Appendix B) All calves were treated with naval dip and Vitamins A,D, and E at birth. Initially, all received colostrum, contaminated or 'tlean”, depending on the treatment group. Later milk was fed twice daily by nipple bottle for 28 days, the amount was equivalent to 8% of the calf's body weight at birth. The calves were housed in separate manure-pack pens in a warm en- closed barn for the duration of the experiment. 8. Dose A tPCP premix was made for the 0.1 mg/kg treatment group dose. In- itially, 66.67 grams of tPCP was dissolved in three liters of acetone. This solution was incorporated in 20 kg of finely ground corn in a Hobart mixer and agitated until the acetone was driven off. This 'bremix'fi #74 grams, was mixed with 37.8 kg of finely ground corn containing 5% molasses to give a #I.8 mg PCP/kg corn mixture. This final mix was given to each cow with their daily morning and night ration so that half the daily dose was administered during each feeding. I The cow receiving l0 mg/kg tPCP was given the entire dose orally via gelatin capsule during the morning feeding only. The cow receiving l0 mg/kg pPCP was given her dose in the same form and method as described for the high dose group in Experiment I. The calves in this experiment were administered PCP via the milk 25 which was collected from PCP-exposed dams as previously mentioned (See Animals and Diets, EXperiment II). C. Sampling Procedures All blood samples were collected with a 20 ml Vacutainer via the jugular vein for both cows and calves. The blood was allowed to clot, centrifuged, and the serum was decanted into glass vials, frozen, and stored for later PCP, T3, and T4 analysis. Blood samples from treated cows were taken prior to initial dose, for two day intervals for the first two weeks of administration, and then weekly until parturition. After parturition, the blood sampling regime was 0, l, 2, 3, S, 7, ll, L5, I9, 23, and 28 days for PCP contaminated cows and their controls. At birth all calves were put on the following blood sampling regime 0 hours, 3 hours, 6 hours, l2 hours, and days i, 2, 3, 5, 7, ll, IS, 19, 23 and 28 into neonatad life. Cows were milked on a l2:l2 hour schedule by bucket milker. The milk was collected a.m. and p.m., weighed, and sampled into 200 ml bottles for freezing and storage for later PCP analysis. The milk was collected on days I, 2, 3, h, S, 6, 7, II, is, 19, 23 and 28. IV. EXPERIMENT III A. Animals and Diets Fifteen Holstein bull calves seven (plus or minus four) days of age were randomly assigned to the following treatment groups: control, l.0 mg/kg aPCP, l0.0 mg/kg aPCP, l.0 mg/kg tPCP, 10.0 mg/kg tPCP per day 26 for a period of #3 days with the exception of the first five-day inter- val when calves received 20.0 mg/kg PCP per day. The dose was lowered when two calves succumbed apparently from acute pentachlorophenol toxi- cosis, one in the high aPCP group and one in the high tPCP. These calves were replaced only to have the sec0nd calf receiving l0 mg/kg aPCP succumb with the same clinical signs. These calves were fed an amount of milk equivalent to 8% body weight throughout the experiment. At 20 days into the experiment, the calves were fed a standard calf starter ration ad libitum (Table 5) along with the milk ration. Water was provided at all times. TABLE 5 Composition of Calf Starter Ration Component Concentration Ground Shelled Corn 37.75% Ground Corn 28.0 % Soybean Meal (h8% protein) 20.0 % Alfalfa Meal 6.0 % Dicalcium Phosphate l.0 % Limestone 0.85% Trace Mineralized Salt 0.70% Magnesium Oxide 0.20% Vitamin A, D, and E Premix 0.50% Cane Molasses 5.00% Calves were individually housed in pens in a warm enclosed barn and weighed at five-day intervals. B. Dose For each treatment group, PCP solutions were made up as follows: the control group received corn oil, the l.0 mg/kg tPCP group had a 2l.h3 g tPCP/liter corn oil dose, the l0.0 mg/kg tPCP group had a 2I4.3 g tPCP/liter corn oil dose, the 1.0 mg/kg aPCP group had an l8.2 gram 27 aPCP/liter corn oil dose, the 10.0 mg/kg aPCP group had a I82 gram aPCP/liter corn oil dose. Since tPCP is 85% PCP and aPCP is 99% PCP, the remainder being impurities, the doses were adjusted to deliver equiv- alent amounts of PCP per mg/ body weight. PCP-containing corn oil was mixed with the milk twice daily for six weeks. At five-day intervals the dose was readjusted subsequent to weighing during the experiment. C. Sampling Procedures and Measurements Blood was taken by venipuncture with 20 ml Vacutainers, once prior to dosing and at five-day intervals during the experiment. The blood was allowed to clot, centrifuged, and serum decanted into glass vials and frozen for later PCP, T3 and Th analysis. At the end of 35 days, the calves were challenged with thyrotropin releasing hormone (TRH)(Abbott Labs, North Chicago, Illinois) to examine the response of the thyroid to a stimulus. Jugular cannula approximately 7.1 cm in length (SLV-lOS#-2h9, PVC cannula, ICD Rolly Company, Palo Alto, California) were installed through a 5.9l cm I4 gauge thin-walled needle (Abbott Labs, North Chicago, Illinois). Prior to TRH dose, three blood samples were taken to establish basal T3 and Th levels. Post- dose samples were taken at IO, 20, 30, #5, 60, 90, I20, l50, I80, 2l0, and 2h0 minutes. Approximately l0 mls of blood were drawn through the cannula and replaced by 2 mls of 3.9% sodium citrate solution to prevent clotting in the cannula. These samples were again allowed to clot, centrifuged, and serum decanted into glass vials then frozen for later T3 and Th analysis- Body weights were taken at five-day intervals during the course of the experiment to determine the effect of PCP on body weight. 28 On day #3 all calves were sacrificed by electrocution followed by exsanguination. A complete post-mortem and histopathol09ic examination was performed by Dr. S.D. Sleight, Department of Pathology, Michigan State University. Liver, lung, kidney, spleen, thyroid, thymus, and brain were removed intact, trimmed of excess fat and connective tissue, and weighed. Representative tissue samples selected for histopathologic examination included lung, liver, kidney, heart, spleen, adrenal, mesen- teric lymph node, reticulum, rumen, omasum, abomasum, small intestine, large intestine, gall bladder, urinary bladder, ureter, skeletal muscle, thyroid, thymus, skin, eyelid, and brain. All tissue samples were fixed in l0% buffered fonmalin. Parafin embedded sections of 6 micrometer thickness were routinely prepared and stained with hematoxylin and eosin for histologic examination under light microsc0pe. Samples of liver, lung, kidney, spleen, thyroid, thymus, brain, bone marrow, lymph, fat, and muscle were taken and frozen for later PCP analysis. D. Statistical Methods anferroni analysis (Gill, I978) was done to determine the signifi- cant differences of the following contrasts: l) Control vs. l.0 mg/kg aPCP 2) Control vs. l0.0 mg/kg aPCP 3) Control vs. l.0 mg/kg tPCP h) Control vs. l0.0 mg/kg tPCP 5) 10.0 mg/kg aPCP and tPCP vs. 1.0 mg/kg aPCP and tPCP 6) l.0 mg/kg and l0.0 mg/kg aPCP vs. l.0 mg/kg and l0.0 mg/kg tPCP 7) Interaction of grade and dose. 29 V. ANALYTICAL PROCEDURES A. Determination of Pentachlorophenol Residues in Tissues Quantitation of total PCP (Total 8 acid hydrolyzable and benzene extractable PCP) in tissues was by methods reported by Kinzell (l982). The extraction methods are briefly described in Table 6. All extracts were analyzed using a Varian 3700 gas chromatograph by electron capture detection with a column I.8 m long and 2 mm i.d. packed with l%.SP i200 DA on l00/l20 Supelcoport (Supelco Inc., Bellefonte, Pennsylvania). TABLE 6 Extraction Methods for Determination of Total Pentachlorophenol Residues in Tissues Tissue Method Serum Acid hydrolysis with heating and extraction with benzene. Liver, Kidney, Spleen Homogenized, hexane extraction Lung. Thymus, Thyroid, with NaOH, reSidue acidified and Lymph, Muscle, Milk heated then extracted with benzene. Bone Marrow, Fat, Brain Homogenized, hexane-isopropanol extraction with H2504, extract dried and re-extracted with hexane and NaOH, extracted again with benzene and H2504. PCP recoveries and the standard error (N36) of the various biological matrices were as follows: serum 993; .l+%, liver 9l.8_4_- i.l%, kidney 90.8: 2.2%, lung 96.0: l.'+%, thyroid 79.0: 2.0%, thymus 90.23: 2.2% brain 90.0; 0.2%. (Kinzell, 1982). B. Thyroxine Analysis of Serum Serum thyroxine was analyzed using commercial radioimmuniassay reagents (Vien Laboratories, Succassuna, New Jersey). Standard points of 0.0, 6.25, l0.0, l2.5, 20.0, 25.0, 37.5, 50.0, 75.0 and lOO mg/ml 30 were made using standards and dilutions thereof with 0.0 standard as the diluent. Standards and samples were diluted using 30 microliters of sample in 600 microliters of 0.I M phOSphosaline buffer with 0.0I M EDTA (pH 7.6). This diluent also contained 2.5 mg/ml protein, l.0 mg/ml 8-anilino-l-naphthalene sulfonic acid, and the radiolabeled hormone. Then l00 microliters of anti-Th was added. This mixture was then vor- texed, incubated at 37°C for 20 minutes, and stored at h°C for approx- imately 20 hours. A 2.5% Charcoal, 0.25% Dextran solution (0.5 mls) was added to each tube and centrifuged at 2800 rpm for IS minutes. An 0.2 ml aliquot of supernatant was counted by a gamma counter (Model ll85, Searle Analytic Inc., Des Plaines, illinois). C.’ Triiodothyronine Analysis of Serum Serum thyroxine was assayed using commercial radioimmunoassay reagents (Vein Laboratories, Succassuna, New Jersey). Standard points of 0.0, 0.25, 0.5, l.0, 2.0, 3.0, and h.0 mg/mi were made using stan- dards and dilutions thereof with 0.0 standard as the diluent. Standards and samples were diluted using l00 microliters of sample in 600 micro- liters of commercially available barbital buffer with 0.5% gamma-globu- lins (pH 8.6). This diluent also contained 0.75 mg/mg 8-anilino-l- naphthalene sulfonic acid and the radiolabeled hormone. Then l00 micro- liters of anti-T3 serum was added. The assay was then vortexed, incu- bated at 37°C for 30 minutes, and stored at h°C for approximately 20 hours. A 2.5% Charcoal, 0.25% Dextran solution (0.5 mls) was added to each tube and centrifuged at 2800 rpm for IS minutes. A 0.4 ml aliquot of supernatant was counted by a gamma counter (model ll85, Searle Anal- ytic Inc., Des Plaines, Illinois). RESULTS Experiment I Twelve Holstein cows were randomly assigned to the following treat- ment groups: 1) control, 2), 0.1 mg/kg pPCP, 3) l.0 mg/kg pPCP, and h) 10.0 mg/kg pPCP chronically dosed for a period of IA weeks. Blood samples were drawn via tail vein every 2 weeks for a period of lh»weeks. Average blood PCP concentrations during the sampling period were: i) controls 3 300 ppb, 2) 0.1 mg/kg - 3.1 DP”. 3) 1.0 mg/kg 3 19.5 ppm and h) 10.0 mg/kg 3 100 ppm. All the data from EXperiment i is report- ed in Appendix A. At ten days prior to administration of pPCP, thyroxine (T4) concen- trations in serum averaged over all groups was 35.9 ng/ml. Thyroxine in serum of cows after various durations of pPCP exposure (Table 7) indi- cates little evidence of altered Th concentrations due to treatments (P<:0.25) although differences between sampling periods were significant (P<0.01). At ten days prior to administration of pPCP, T3 concentrations in serum averaged over all groups was 0.77 ng/ml. T3 in serum of cows after various durations of pPCP exposure (Table 8) provides some evidence of altered T3 levels due to treatments (P<=0.10), however differences be- tween sampling periods were of greater significance (P<0.05). 31 32 TABLE 7 Serum T Concentrations (ng/ml) of Cows Exposed Subchronically to pPCP pPCP mg/kg/day Duration of Treatment (weeks) 0* ‘ 2 4 6 8 10 12 14 Ave. 0 34.0 42.0 53.7 45.7 53.5 47.0 54.8 70.7 50.2 0.1 32.3 33.2 42.4 40.8 44.2 45.0 46.8 51.5 42.0 1.0 34.3 33.7** 35.6 36.5 38.4 37.7 38.2 49.3 38.0 10.0 45.1 42.4 48.2 48.3 48.1 46.9 47.2 51.2 47.2 Ave. 36.4 37.8 45.0 42.8 46.1 44.2 46.8 55.7 44.4 STANDARD ERROR DUE TO TREATMENTS: :_ 3.91 STANDARD ERROR DUE TO TIME 2.16 1+ STANDARD ERROR DUE TO EACH CELL : i- 4.32 * Values at time of initiation of chronic dose. ** N : z 33 TABLE 8 Serum T3 Concentrations (ng/ml) of Cows During Treatment pPCP mg/kg/day Duration of Treatment (weeks) 0* 2 4 6 8 10 12 14 Ave. 0 0:74** 1.08 1.40 1.0 1.14 1.27 1.167 1.36 1.14 0.1 0.71** 0.74 1.10 1.03 1.08 1.14 1.05 1.09 0.99 1.0 0.93** 0.63 0.74 0.70 0.73 0.78 0.66 0.91 0.76 10.0 0.73 0.67 0.78 0.86 0.84 0.89 0.68 0.86 0.79 Ave. 0.78 0.78 1.01 0.90 0.95 1.02 0.89 1.06 0.92 STANDARD ERROR DUE TO TREATMENTS: i 0.12 STANDARD ERROR DUE 1'0 TIME f 0.05 STANDARD ERROR DUE TO EACH CELL : t. 0.10 * Values at time of initiation of chronic dose ** N 3 2 34 Experiment 11 This experiment was viewed as a pilot study to assess the extent of exposure in calves 1) via placental transfer and 2) via the milk (Table 9). Calves from two contaminated cows died at birth. Necropsy showed no unusual findings and therefore the deaths were not diagnosed as a result of PCP poisoning. (Necropsy reports and all data pertinent to Experiment II are reported in Appendix 8). Due to the preliminary nature of this' experiment and few subjects per group, no statistical analysis was per- formed on this data. However, best fit polynomial equations were gener- ated for serum concentrations of PCP in calves during postpartum expos- ure. These equations were used to plot the curves shown in Figures 4 and 5. TABLE 9 Design of Experiment II Exposure of Calf via Dam* Treatment Exposure N of Dam of Calves In Utero and Postpartum 2** 0.1 mg/kg tPCP Postpartum 3 n Utero and Postpartum i 10.0 mg/kg pPCP Postpartum I In Utero and Postpartum 0** l0.0 mg/kg tPCP Postpartum ' l * Exposure of calf by placental transfer (in utero) during the last trimester and by milk (postpartum). ** One animal died from each group at birth. 35 l. Placental Transfer of PCP Blood samples were taken within 10 minutes after birth from both the calves and exposed dams of the 0.1 mg/kg tPCP group. The analysis showed serum levels of PCP in the calves to be approximately 34% of that found in the dams (Table 10). Similarly the calves from cows contamin- ated with 10.0 mg/kg tPCP and 10.0 mg/kg pPCP had 32% and 38%, respec- tively, of the levels in the dam. 2. PCP Levels in Calves During the Neonatal Period Another group of calves were paired with those that were exposed to PCP in utero. Those calves that received their dose of PCP via the tainted milk from the 0.1 mg/kg tPCP treated cows achieved serum PCP concentrations as shown in Figure 4. In this group, calves exposed postpartum slowly accumulated PCP in the blood until at the end of four week‘s exposure, the serum levels were 98% of those that were exposed in utero. At the end of four weeks both groups of calves had serum levels similar to (92%) the treated dams. Those calves that received milk from cows administered 10.0 mg/kg pPCP had PCP blood levels which were 65% of the dams after four weeks eXposure. That calf which re- ceived milk from the cow administered 10.0 mg/kg tPCP had PCP blood levels 20% of the dam after four weeks exposure (Table 11). Concentrations of PCP in the blood of calves fed only tainted milk from the 10.0 mg/kg tPCP and pPCP cows without previous eXposure in utero were dissimilar. The calf fed tainted milk from the tPCP treated cow had approximately 25% to 33% the serum PCP concentration compared to the calf fed milk from the cow treated with pPCP (Figure 5). The data from two calves, one exposed both in utero and postpartum and the other eXposed only postpartum via the milk of the 10.0 mg/kg treated 36 cow showed that the latter accumulated 25% higher serum PCP levels than the former (Figure 5). 3. Milk PCP Concentrations. Concentrations of PCP in the milk are shown in Table 12. The ex- cretion of PCP in milk is about 7-IO% of the concentration found in the dam's serum. Also note that the milk concentrations of PCP in pPCP vs. tPCP treated cows are slightly different (3,900 ppb vs. 4,600 ppb). 4. Average T3 and T4 Concentration in Serum of Cows and Calves The averagevalues of T3 and Tg measured four weeks after partu- rition are shown in Table 13. There were no trends observed in this data to indicate alteration of thyroid hormones. TABLE 10 Comparison of Blood PCP Concentrations at Birth Between Dams and Calves Exposed In Utero % of Dam’s Blood Total PCP** PCP Animals Treatment N .in Blood (ppm) Concentration Calves (0.1 mg/kg tPCP) 3* 1.4 34 Cows (0.1 mg/kg tPCP) 3 4.1 Calf (10.0 mg/kg tPCP) 1* 17.9 32 Cow (10.0 mg/kg tPCP) 1 55.4 Calf ,(l0.0 mg/kg pPCP) 1 22.9 38 Cow (10.0 mg/kg pPCP) I 60.7 * One calf died at birth; blood sample was taken by surgical removal of heart and emptying of chambers ** Total PCP 8.Acid hydrolyzable and benzene extractable PCP 37 TABLE 11 PCP Blood Concentrations at End of Postpartum Exposure of Neonate Compared to Dam PCP in Blood ‘% of Dam's Blood (ppm) 28 days PCP Animals Treatment N After Calving Concentration Calves (0.l tPCP) 5 2.2* 92 Cows (0.1 tPCP) 3 2.4 Calves (10.0 pPCP) 2 34.2* 65 Cow (10.0 chP) 1 52.2- Calf (10.0 tPCP) 1 9.7 20 Cow (10.0 tPCP) 1 48.0 * The value for calves is the combined values of those eXposed in utero and postpartum and those exposed only postpartum. mm .comu.c:u 3 =Om o 300 h 0 60 m __ . can Loam a < mxoo x z to mc_EmwcoU ob oomoaww on ”ammo 600 m c m _ coflmoc mama mx\me mo co.uo c _:E:oo< : ocsmmu Oman ONNNONMNVNMNNN KONG—a— : 0.9 3 n— N— —— 9o 0 h o m e n N— u 41 1 1 1 1 d 1 J 4 1 1 333 mi..- . <1 4 ‘1 1 id 4 d .1 d 1 1111.. c o\ \m \\\° \\\ Amnzu Eaten «ace iiio \\ o \\0 awuzv Eaten Zoe new 9.3: c. x o \\\\ \ Eon: 9.32 2: $58me “.0 we? .\ \\ \\ \\ \ \\\ \ \ \\ O \ \ \\o x xx \ O \\ \ \ \ \ \ \ \ \\ x x \\ \\ x x 8.- E??? (qdd) suogmnuaouog pooia dOd o.» .oo- .o—— .ONP comp ...—. .omp ..op .o~— comp .oap .OON ..p~ ,.ONN .omu Figure 5 Accumulation of PCP in Blood of Calves Exposed to Contaminated Milk from 10.0 mg/kg pPCP and tPCP Treated Cows for Four Weeks After Partu- rition. 40,000] 30,000~f 36,0 001 i 34.0 00. 32.0001 I 30.00qu zeoom g D , 2¢0004 J zzooe d 20,0 0 04 18.0001 4' 1 16.0001 4 b O O O 4.... 9 12.0004 x BLOOD PCP CONCENTRATIONS (ppb) - ..‘ D 28.0 001://"/// - ... D TYPE OF EXPOSUR E (10 mo/kgPCRN-ll 1 CI x ...... postpartunflpPCP) o- .. .. - postpartum (tPCP) in utero and postpartum (pPCP) ‘ —.-7- -7 T ’T 111 “5'10 20 22 24' TIME (DAYS) 42 TABLE 12 Comparison of PCP in Blood and Milk of Treated Cows Average PCP Concentration (ppb) for. Four Weeks After Parturition % of Dam's Blood . PCP Treatment N In Whole Milk* In Serum** 'Concentration 0.1 mg/kg tPCP 3 200 2,800 7 10.0 mg/kg tPCP 1 3,700 48,400 8 10.0 mg/kg pPCP 1 4,400 46,200 10 * Data represents average of 24 milk samples taken a.m. and p.m. on days 0, I, 2, 3, 4, 5, 7, ll, 15, I9, 23, 28 after parturition. ** Data represents average of 11 blood samples taken on days 0, l, 2: 39 5t 79 'IO 159 '9, 23’ 28. 1+3 TABLE 13 Average Serum T and T1, Values of Cows and Calves Exposed for Eour Weeks After Parturition.* Exposure T3 (ng/ml) T4 (ng/ml) 2}** Controls *** 3.6 i 2.0 104.5 j; 60.6 68 C A In Utero and . L Postpartum 3.5 i 2.3 113.6 3; 70.6 27 v (0.1 tPCP) (n=2) E S Postpartum 3.2 :1; 1.7 94.3 i 55.9 40 (0.1 tPCP) (n=3) in Utero and Postpartum 3.7 t 1.9 95.3 i 44.3 14 (10.0 pPCP) (u=1) Postpartum (10.0 pPCP) (11:1) 2.8-: 1.9 73.5 348.8 14 Postpartum (10.0 tPCP) (11:1) 3.2 11.7 108.0 368.3 1L1 ‘Controls 161* 1.3 1 0.11 36.30 :1; 13.2 62 C B 0.1 mg/kg 1.1 :_0.3 25.88-:_ 6.7 33 w (tPCP) (N=3) S 10.0 mg/kg 0.7 t 0.2 26.75 i 9.5 11 (tPCP) (N-l) 10.0 mg/kg 0.7 t 0.2 19.61 1‘ 5.8 11 (PFC?) (N'l) * All calves had blood samples drawn at 0, 3, 6, 12 hours, and days 1, 2, 3, S, 7, 11, 15, 19, 23, and 28 after birth. A11 Cows had blood samples drawn on days 1, 2, 3, 5, 7, ll, 15, 19, 23, and 28 after parturition. **28- Number of samples that were averaged into the T3 and T4 Values 191* Controls - - Calves were MSU dairy herd animals born to cows not treated with PCP, also calves were not fed contaminated milk. Cows were the dams of the control calves. Experiment III Initially this experiment was carried out with dosages of 2.0 and 20.0 mg/kg of aPCP and tPCP. During the first five-day interval, two calves in the high dose groups died, one from each of the groups. (Nec- ropsy reports and all data pertinent to Experiment III are reported in Appendix C.) One of the replacement calves died five days after treat- ment with 10.0 mg/kg aPCP. After these deaths all calves were lowered to one half their original dose from 2 mg/kg to 1 mg/kg and from 20 mg/kg to 10 mg/kg five days after the initiation of the experiment. None of the calves that succumbed were included in the statistical analysis. Statistical analysis of data was done by Bonferroni t method with the following contrasts: treatments vs. control, low dose vs. high dose, analytical vs. technical PCP, and the interaction of grade and dose. 1. Blood Levels of PCP During the course of the experiment, the concentration of total PCP in serum at steady state for the 1.0 mg/kg and 10.0 mg/kg aPCP and tPCP groups averaged 32 ppm and 92 ppm respectively. There was no significant difference between aPCP and tPCP, but the latter tended to be slightly lower throughout the experiment. There was a slight rise in blood PCP levels in the controls due to high potential PCP volatilization from urine and subsequent pulmonary exposure. Figure 6 shows that steady state levels of calves are reached within five days. 2. Effect on Body Weight There was no statistically significant difference in body weight on either the 1.0 mg/kg tPCP or aPCP treatment groups when compared to con- trols. Figure 7 shows there was a significant effect on body weight (P‘1.05) with the high level of technical preparation; this was commen- 45 surate with a decrease in feed intake. The high dose analytical group initially showed some decrease in growth, but at day 20 apparent compen- satory growth occurred in these calves. All other contrasts show no statistical significance (P:>.10). Feed intake data is on Table 14. 3. T3 and T4 Levels During the Course of the Experiment. There was little evidence that 1.0 mg/kg treatment of either aPCP or tPCP had any effect on circulating thyroid hormone concentrations when compared to controls. Figure 8 shows that both serum T3 and Tp levels were decreased significantly (P0 CONTROL IO mo/ka aPCP 10 mg/kq tPCP v1 1 :1: 2': 011 16 DAYS ON EXPERIMENT Compared with control (11:, P 2'0 3'0 35 .1; H. P<.05; m. , P<.01) Figure 9 Average Resultant T and Th Outputs for Three I-Iours After Administration of TRI-l. Three Subjécts per Treatment Group Except for ID. 0 mg/kg aPCP where N82. 51+ T4 ( no/ml) T3 (no/ml) 90‘ 80‘ 20‘- O CONTROL N 11 T3 and T4 OUTPUT AFTER TRH CHALLENGE IN CALVES FED aPCP or tPCP T4 /Cr———_—’O A. 10 ma/kq aPCP A 10 mo/kg tPCP 0 2'0 4'0 60 do 1‘20 150 who 2'40 MINUTES AFTER TRH INJECTION 56 TABLE 16 Concentrations of Total PCP* In Various Tissues 0f Calves Fed aPCP or tPCP (ppm, N=3) Treatment Liver Lung Kidney Thyroid Thymus Brain Fat Control .h .3 .5 .3 2.5 ‘<.l .5 1 mg/kg: aPCP 6.8 5.8 8.7 2.9 3.3 ‘<.l 2.3 tPCP 6.2 h.8 7.2 2.5 3.5 <.l 3A l0 mg/kg: aPCP** 22.h l6.8 29.3 7.0 10.2 .2 l7.8 tPCP 2503 18.2 26.6 903 21.6 02 ‘- * Total PCP =.Acid Hydrolyzable and Benzene Extractable PCP ** N I 2 57 Figure 10 Cross-section of the Thyroid Gland in Control and High Dose tPCP Groups After Liz Days of Exposure. (Magnified 400x) A, A “a" ‘- ' .Ir‘. I . .- . UM‘ .1 ’94 \e d _.--.-';2 . a- ,4. X ..- a... T'. ”'Ku."u '4’]; ,.‘f (1.. c"\ - ~\;‘..:. ”6 .r\' . ".‘5‘IIE‘f-ft'F .11.“: 4\ . a z“, '0- D. ‘ 3“ '9‘ c (4‘ a 3. 59 Figure II Cross-section of Meibomian Gland in Eyelid of Control and High Dose tPCP Groups After #2 Days of Exposure. Note the filling of the interstitial space with keratin. (Magnified h00x) ‘wafilv' .1 ' - "hr-“0,515.13;- . w‘.‘ .4“; 'I . 3 ' i .. 5m *1” V "{p . o o 0, fl ‘ , o I W .. In L 'l - " “ \ . a . ?lltrv~nl‘t . a.» ' . w I“ 1‘ "1411‘ 11 .‘T'T'l‘e. 51“ it in i‘» 1 1 Pm‘ \: 11‘1'1011. 1“ 15;;0 ‘. . i ‘0. -( a“ ‘\y. g}.\v I' *7 ‘ ,‘ . ‘1‘].31- 5‘ ,‘l g ‘ I ‘ u” . . ’ , 61 Figure 12 Cross-section of the Thymus in Control and High Dose tPCP Groups After 42 Days of Exposure. Note the absence of darker stained cortical material in the treatment group indicative of absent t-lymphocytes. (Magnified #00x) I ‘ ‘ ' ‘ V. ' "5‘ . ‘ w . \ 9 ‘ ‘ 1 i - $9 ~ 9 9 'I ’9'1‘97'5‘ "L; I ' _,; ~ 1' 11,511.11" "."."Z;:‘ M- :14 ’7‘ t}; . fl“; ., :r r V I‘ )9- 12:? ‘99 " «t9 915.1(1‘93 0,955,131 9 It‘ll; .1," ”it: 191’ 7,93“; V11? £391“: :3 .u T .‘1’ 1 o ‘ _. I 5" fi‘rasi. J !/ ?)£.":’J”.1; i5} :{71 q! I", 31“ 3‘1“.» «11).! %1g,'§fl~7‘ <9 9 9 99 .19 29 gm: 9 ' ...' 9? "m; '-,-’{.7'°9'3’.3.’" ’ N‘ 739.379.999.9999799213' '19. 9.1, $41» \ flat-ll 3‘1”." H 1| “1‘ "1 DISCUSSION . High calf mortality on several Michigan dairy farms where penta- chlorophenol (PCP)-treated wood was in extensive use (Thomas, 1977) raised concerns among dairy farmers as to whether PCP would be detrimen- tal to the health of young stock. To determine such effects, toxico- logic evaluations concerning all three modes of exposure (dermal, respir- atory, and oral) should be conducted. Most studies have been conducted involving oral eXposure. But there is a great need for pulmonary and dermal exposures to be investigated, especially since poor ventilation high ambient temperatures, over-use and improper use of PCP in feed bunks and bunker silos increase the potential for exposure in livestock barns. Equally lacking are those studies investigating the effects of PCP subchronically administered to young cattle. Furthermore, calves may risk higher exposures due to the transfer of PCP from dam to neonate via the milk (Firestone et al, 1979). In view of the paucity of data invol- ving the toxicity of PCP orally administered subchronically to calves, the purpose of this study was to evaluate the toxicity of different pre- parations of PCP to cattle particularly the bovine neonate. Special em- phasis was placed on the thyroid gland due to an earlier study indicating alteration of serum thyroid hormone levels by PCP (McConnell et al, 1980). Toxicodynamics of Pentachlorgphenol - General Health,,Appearance, Growth There were no overt adverse health effects in any of the cattle treated with purified (p) PCP or analytical (a) PCP. The only toxic effects with associated clinical signs were in calves directly fed 10.0 mg/kg tPCP subchronically. The main effect.was on body weight which was significantly decreased (P‘<}05). These calves were also lethargic, anorexic and generally weak. The decrease in weight gain was due to feed 63 52. refusal commonly encountered when cattle were exposed to PCP contamin- ated feed. Deichmann (19h2), Grisby and Farwell (1950) and McConnell et a1 (1980) all reported feed refusal in cattle exposed to high levels of PCP. McConnell et a1 attributed the decrease in body weight gains to the dioxin or dibenzofuran impurities rather than PCP itself. Moreover, decreased body weight is characteristic of dioxin toxicosis in other species as well (McConnell, 1980). In 1981, Ball and Chhabra found that TCDD causes malabsorption of nutrients from the intestinal tract of rats.' Malabsorption, therefore, may account for some of the decreased growth rate. It was also observed that tPCP-treated calves tend to have a greater frequency of scours of longer duration. However, since scours also occurred in controls there is not sufficient evidence to state de- finitively that scouring was PCP-related. In addition, no irritation of gut lining was found during histological examination. Calves born to cows treated with various doses and preparations of PCP during the last trimester of pregnancy had no overt adverse health effects at birth or during the first 9 weeks of postnatal life. Two calves died at birth but subsequent necropsy could not determine any indication of PCP toxicosis. Teratogenesis could not be determined in this experiment because susceptability to teratogenic agents varies with the developmental stage at the time of exposure. In general, there is relatively a short critical period Of sensitivity to teratogens, when early organ09enesis is in progress. This period extends from the time of implantation to the end of the embryonic period (Harbison, 1980). Since the process of organogenesis was essentially completed before the last trimester of gestation, PCP would not be expected to produce ab- normulities in this eXperiment. 65 Thyroid Hormone Concentrations Exposing mature cows to levels of pPCP up to 10.0 mg/kg body weight per day for 1h weeks (Experiment 1) resulted in no statistically signi- ficant effects on Th but did provide some evidence of decreased T3 levels due to treatment (P9<.lO). The pPCP in this experiment produced effects on T3 similar to what McConnell et a1 (1980), observed in heifers exposed to either aPCP or tPCP. However, in contrast to our data, McConnell et al also reported a decrease in Th levels caused by both preparations. The dissimilarities may be explained by noting: l) Cattle in the McConnell et al experiment were younger; therefore, an age-related sus- ceptability in the thyroid may be involved, and 2) The dosage of PCP was 15 mg/kg body weight of aPCP and tPCP for 160 days compared to 10.0 mg/kg body weight pPCP for 98 days in this study. Therefore, the difference between the two experiments may be dose related. Due to lack of subjects no statistical differences in circulating T3 and Th concentrations could be found in calves fed contaminated milk collected from exposed dams. However, when calves were administered PCP by direct incorporation into the milk prior to feeding, decreases in body weight and in serum T3 and Th levels were observed in the 10.0 mg/kg tPCP treatment group (P9<.05) during the course of exposure. in the same experiment, where incorporation of PCP directly into milk was done prior to feeding, all treatment groups were challenged with TRH after 5 weeks exposure and subsequent blood samples taken and analyzed for T3 and Th in order to assess the responsiveness of the thyroid. The rate of the increase in thyroid hormones did not differ significantly when comparing treatments with controls. Therefore the 66 functional response of the thyroid was not impaired. This suggests sev- eral possibilities yet to be investigated. First, the elimination of T3 and Th might be enhanced, thereby lowering blood levels. Bastomsky (1977) noted that biliary excretion of Th was increased four fold in rats exposed to 2,3,7,84TCDD. This suggests that induction of metabolism by tPCP and its contaminants might lower serum T3 and TL levels by in- creasing excretion of thyroid hormones. Although an assay was never con- ducted to assess glucuronidation in these calves, in a separate report Shull et al (1982) showed induction of some drug metabolizing enzymes. However, McConnell et al (1980) did not Observe an increase in glucuron- yltransferase in his cattle. Nevertheless, their p-nitrophenol conju- gation assay might not be a suitable indicator of the particular glucu- ronyltransferase responsible for T3 and Th conjugation, as in the case of rabbit estrone and p-nitrOphenol UDP-glucuronyltransferases in which only one of the substrates can be conjugated by the enzyme and not the other (Tukey and Tephly, 1981). A second explanation is that the treated calves had decreased cir- culating thyroid binding globulin (TBG). It is usually agreed that 99% of the thyroid hormones in blood are bound to globulins and 1%.are free (Guyton, 1976). it is this free 1% that reacts in a negative feedback fashion on the hypothalamus and pituitary to regulate T3 and Th levels. In the case of a chemical insult to the liver that causes a decrease in protein output (eg.depressed TBG), the result would be a lower total hormone concentration with a higher free fraction compared to bound fraction, therefore lower serum levels would result after the elimination of excess thyroid hormone. It remains to be studied whether PCP or its contaminants cause a decrease in binding globulins. However, a histological 67 examination of tissues showed no apparent hepatic damage. Another action that might mimick the effects brought about a depression in binding glob- ulins is the displacement of thyroid-binding hormones from serum-binding proteins. For example, PCBs displace Th from its binding proteins re- ported by Bastomsky (197h). Third, PCP and its contaminants may somehow inhibit TSH synthesis or impair its release at the level of the pituitary gland. This explan- ation seems unlikely for two reasons: 1) Analysis of various tissues for PCP residues indicates that the brain is not a-compartment into which PCP is distributed to any significant degree in the newborn calf, and 2) The resultant output of T3 and T4 after TRH challenge was not different with respect to amplitude of response when tPCP and aPCP were compared to controls. Quantitation of TSH in response to TRH would determine if de- pression of TSH was the Cause ofthe lowered thyroid secretions of T3 and T4, hence lower blood levels. However, TSH was not measured in this study. Fourth, PCP and its contaminants may inhibit the release or synthe- sis of T3 and T4 at the level of the thyroid gland. Because responses of treated groups are comparable with controls when challenged with TRH. it seems unlikely that PCP is acting at this level. The results are in contrast to those of McConnell et al (1980) where both tPCP and aPCP low- ered T3 and Th levels in blood. Again the differences in response be- tween the two studies might be due to the differences in age, sex, or dosage discussed previously. Fifth, the lowering of thyroid hormones may be nutritionally re- lated. Blum et a1 (1980) observed decreases in thyroid hormones in ruminants during weight loss. Since tPCP treated calves demonstrated signs of feed refusal and decreased weight gain, the resultant decrease , 0f circulating T3 and Th may be due to the poor nutritional state of 68 the animals. However, such effects need further investigation in light of the confounding effects of PCP and its associated highly toxic contam- inants. Pathologic Findings Exposure to tPCP had more profound effects on organ weights than aPCP. Pronounced decreases were evidenced on the thymus and spleen with an increase in liver weight. Calves administered aPCP showed less thymic involution than those fed the technical preparation. The 1.0 and 10.0 mg/kg tPCP treatment groups had thymic weights 61% to 18 % of controls indicating that the non-phenolic contaminants are mainly responsible for the effect and that it is dose-related. Thymic atrOphy also occurs in malnourished animals and is probably responsible for part of the atrophy. Van Logten (1981) reported that 2,3,7,8-TCDD potentiated thymic involu- tion in the starved rat. Therefore, the interaction of starvation and toxicosis might explain the overall effect of thymic involution observed in our calves. The effects on the liver and spleen are also contaminant related and are in agreement with those seen by McConnell et al (1980). The histologic examinations were not remarkable, with the exception of changes on the thymus and eyelid. The thyroid had no observable ab- normalities with respect to follicle size or the nature of the surround- ing epithelial cells. The only morphologic changes found in the tissues collected were: 1) atrOphy of the thymus due to cortical depletion of t-lymphocytes, and 2) keratin depostion in the Meibomian gland of the eyelid. Both of these lesions were found in the high tPCP groups and are characteristic of dioxin toxicosis (McConnell, 1980). This is in agreement with McConnell et al (1980) who reported similar lesions in 69 heifers treated with 15 mg/kg tPCP. Gross examination of the lungs revealed consolidation (2-h029 of pulmonary tissues in all calves of Experiment III. It was concluded that the effect was probably not treatment-related on the basis that it was seen with equal frequency in both control and treated cattle. Toxicokinetics of Pentachlorophenol Serum Concentrations When PCP was fed directly to calves, serum concentrations reached steady state within five days. This is in good agreement with Kinzell (1982) in which mature cows reached steady state concentrations in three days. However, when the exposure of PCP was by contaminated milk via exposed cows only (no intrauterine exposure) steady state levels were not established until the fourth week; some calves never clearly reached steady state at the experiment's end (28 days). A possible explanation to the slower establishment of steady state when PCP-containing milk was fed may be that PCP in milk is either bound to milk proteins or conjugated possibly to a glucuronide (Kinzell, 1982). The conjugated PCP must be converted to a free form before it could be absorbed by the calf. Therefore, the calf would require functional pro- teases or an established gut microflora for deconjugation (eg. B-glucu- ronidase). As the activities of proteases and gut microflora develp with age, (Huber et al 1961; Hill, 1970) so would the serum PCP concen- trations. Calves from PCP-exposed cows having received exposure during the last trimester of gestation were born with significant serum levels of PCP. This indicates placental transfer of PCP from dam to offspring. Few experiments have been conducted to show that PCP does pass into the fetus. Hinkle (1973) demonstrated placental transfer of PCP in rats, and 70 that concentrations of PCP in the fetus and dam were proportional. Con- versely, Larsen et al (1975) administered PCP on day 15 of gestation to pregnant guinea pigs and noted no placental transfer of PCP nor any evi- dence of fetotoxicity (or embryotoxicity). A possible explanation for the occurance of PCP in the blood of new- born calves is the longer exposure time (approximately 120 days) of the cows in the experiment as opposed to one day in Larsen‘s. This would allow sufficient time for steady state concentrations to be established between the dam and fetus.. it is interesting to note the morphological differences among the species involved in placental transfer studies. Anatomically the placental barrier is the result of a number of layers of cells interposed between the fetal and maternal circulations. The number of layers varies with the species and state of gestation and this probably affects the permeability of the placenta. This would seem to suggest that the rat not the cow would have the greatest capability of placental transfer since the rat has the fewest layers of cells interpos- ed between the maternal and fetal circulation. Nevertheless, the rela- tionship of the number of layers of the placenta to its permeability has not been thoroughly established. Since this study shows substantial levels in the calf, but, according to Larsen, not in the rat, length of exposure and other factors must be responsible. Another group of calves having no previous PCP exposure were paired with those exposed in utero for exposure via the milk. Calves fed milk collected from the 0.1 mg/kg treated cows for h-weeks into post-natal life achieved blood levels similar to those having in utero exposure. Together both groups had 99% of the serum PCP levels of the dam. How- ever, calves paired in a similar manner and exposed to milk collected 71 from the 10.0 mg/kg pPCP-treated cow had serum PCP levels 25% higher than the calf with in utero exposure at the end of 9 weeks; representing serum levels that were 65% of the dams. Calves exposed in utero had slowly rising PCP serum levels during the four weeks postpartum period. This represents that milk is a sub- stantial source of contamination for the neonate. The exception to this is the calf receiving milk from the 10.0 mg/kg tPCP-treated cow where, at the end of four weeks, PCP levels were 20%.which is a percentage well below the intrauterine-exposed calves at birth. Differences in the var- ious PCP preparations could account for this occurrence. The greater exposure to the non-phenolic contaminants in the tPCP preparation may increase glucuronidation and excretion of PCP. The metabolites, there- fore, would be preferentially eliminated via the kidney rather than the mammary gland thereby reducing exposure to the calf. For this particular cow it is true that the total PCP levels in the milk are lower. The same non-phenolic contaminants can be passed on to the calf via the milk (Firestone et al, 1979) giving rise to the possibility of in- creased metabolism (i.e. conjugation) and excretion in the calf. There- fore, the result of this would be lower blood PCP levels, although this increase in glucuronidation holds true in rats (Goldstein, 1977). McConnel et al (1980) found no such inerease in glucuronyl transferase in heifers. However, PCP conjugation in cattle due to non-phenolic con- taminants is still in question and needs to be further investigated. Nevertheless, increased metabolism may explain why the 10.0 mg/kg tPCP- treated calf had blood levels of PCP 25%.to 33% that of the pPCP-treated calf. 72 Milk PCP Concentrations All cows had PCP concentrations in milk around 7-lO%.of those found in blood. This is in agreement with the data of Firestone et al (1979). This distribution seems reasonable in view of the pKa of PCP (pKa.a 9.79 Anonymous, 1980). Since organic xenobiotics are generally excreted into milk by simple diffusion, and since milk is more acidic (pH about 6.5) than plasma, PCP would be expected to attain a lower concentration in milk than in plasma water at steady state. Tissue PCP Concentrations Liver, kidney, and lung contained the greatest concentrations of PCP in calves fed PCP subchronically. This is logical for two reasons: I) These organs are highly perfused and therefore have much more expos- ure to PCP in the serum, and 2) Since PCP has the ability to associate with proteins, the high protein content of these organs make them com- partments which will sequester PCP. Brain contained the lowest concen- trations. There are three possible reasons for this result: 1) The high lipid content of the brain would prevent PCP accumulation. Since PCP is mainly in the ionized form it would seek out a polar environment instead of non-polar, 2) most compounds are unable to pass the capill- ary endotheiium, and 3) Low protein concentration of the interstitial fluid does not allow for binding of PCP. Therefore, there is little ability for PCP to concentrate in the brain (Klassen, 1980). The thyroid and thymus did show significant levels of PCP concentra- tions, but the subsequent effects do not appear to be PCP related. SUMMARY In the experiment involving transfer of PCP from dam to offspring via the milk it has been noted that substantial PCP levels are present in milk. No adverse effects were noted among calves in this experiment. However, this poses a needless and increased exposure not only to PCP but to the non-phenolic contaminants that may also be passed along with the milk to the calf. The toxic effects observed in Experiment III where both aPCP and tPCP were compared implicated the contaminants (dioxins, dibenzofurans, etc.) in tPCP as the toxic entities. In particular, pathologic lesions were seen in the thymus and eyelid which are unique to dioxin toxicosis. Therefore, it is believed that more purified preparations of PCP should be used for agricultural purposes, although the tPCP dose in this exper- iment far exceeds levels in the typical farm situation. Nevertheless, it is the highly toxic non-phenolic contaminants which accumulate over longer periods of time that should prompt the lowering of farm exposure to tPCP in order to avoid herd health problems. Lastly, the thyroid does not appear to be a site of PCP toxicosis. Only technical preparations high in non-phenolic contaminants induced any changes in serum T3 and T4 levels during the course of exposure. Again, this suggests that PCP in its purified form is not responsible for the adverse effects seen in this experiment and is an unlikely cause of serious herd health effects seen on some Michigan dairy farms. 73 APPENDICES APPENDIX A APPENDIX A The data on the following experiment concerns cows on the following treatment groups: Cows 1-3 - Controls 4-5 - 0.1 mg/kg pPCP 7-9 - 1.0 mg/kg pPCP 10-12 -10.0 mg/kg pPCP The following is a key for all the following data: Column 1 - T-Total PentachlorOphenol in blood Column 2-9 - Date assayed (ex: March 10, 1980 or 03/10/80 = 31080) Column 10-14 - Days into experiment Column 15-20 - Time of day (Military Time) Column 21-23 - Individual Cow Identification from 1-12 Column 24-31 - Tetrachlorophenol (Replicate l) cone. in blood (ppb) Column 32-38 - Pentachlorophenol (Replicate i) ” ” ” ” Column 39-45 - Tetrachlorophenol (Replicate 2) ” ” ” ” Column 46-53 - Pentachlorophenol (Replicate 2) ” “ ” ” These cows were given one initial dose on 10/13/79 and started on chronic dosing on 10/24/79. Blood sample taken on 10/29/79 prior to chronic dose was used as 0 time on Tables 7 and 8. Later, during chronic dosing, samples were taken every 2 weeks for 1% weeks to determine the effect on thyroid hormone levels. 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TTVTTTTTTTTTTTTTTTTTTTTTTTTTTT 00.0 00.1 00.0 01.1 00.0 00.0 01.1 01.1 00.0 00.1 00.1 OH.H 10.0 00.1 10.0 00.0 00.0 10.0 00.0 00.0 00.0 00.0 00.0 10.0 01.1 01.1 00.0 01.1 00.1 11.1 00.1 00.1 00.1 10.1 01.1 10.1 2 01 0003 00 x003 my Xmm 00.1 00.0 00.0 NO.H 00.0 00.0 00.1 00.1 00.0 00.1 00.0 10.0 010000. 00.1 00.0 00.0 00.1 00.0 1N0.10 00.1 00.0 00.0 01.1 00.1 00.0 0.0 00 .000 0c co_um.um_c_Eu< omcohnu 0o 00000 00.1 00.0 00.0 00.1 00.0 00.0 00.1 mm.o 00.0 00.0 00.0 00.0 0 0003 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 10.1 01.1 00.0 0 0002 00.1 00.0 00.0 00.0 .*ome cowpmwaficH 00.1 @m.~ 00.0 01.1 00.0 00.0 00.0 00.0 mO.H 00.0 00.0 00001000 mamv 01 AHE\mCV Lo mlmmmmmmmm,0mmmemHzH20< 0300 20 000000020 0002 N B< mzoh900021 0003 0 00 02010000200300 .0 APPENDIX B ,REBORT OF’ LABORATORY gamma new 0000.00, .2006 ANlMAL HEALTH DlAGNOSTlC LABORATORY P.0.Box30076 ' Lansing, MI 48909 Phone (517) 353-1683 ClinicNo. Dairy acct. 71-6111 Date Shipped " " . Date Received December 3, 1979 AMWLEGED MWKNWWAYWWN NOT FOR PUBLICATION FinaiReport December 26! 1979 Veterinarian Owner MSU Dairy Science full Specimen 1 calf Breed HOIStej-n Age _______tem Sex ____F Preliminary reports “one '- HISTORY: This calf was from a research project involving PCP (pentachlorophenol). There were no signs of illness in the dam. GROSS LESIONS : This was a stillbirth, born between 4 and 6 a.m. It weighed 96 pounds and f\ was in normal flesh. It was not dehydrated, and there was very little postmortem autolysis. There was a 25 cm ventral midline incision present over the thorax and through the sternum. The right ear had been removed. The calf was still wet. The lungs were dark purple throughout and sank in formalin (indicating the animal had not breathed). The liver appeared slightly yellowish over its entire surface, except at the margins. On cut surface it had an orange-gold color throughout. The small intestine contained a yellowish viscous liquid throughout. The large intestine con- tained green foreign material (meconium). Attached to the inside of the placenta on the opposite side from the caruncle was a yellowish-brown plaque. This measured 7 cm in diameter and 2 cm thick and was easily peeled from the placenta. EBOR'ATORY FINDINGS : Virologic examination: Fluorescent antibody tests on frozen sections were negative for IBR and BVD. Microbiologic examination: Darkfield examination of stomach contents did not reveal any Vibrio or LeptOSpira. The placenta had no growth at 72 hours. Stomach contents had an alpha hemolytic Streptococcus and a Pasteurella— like organism recovered from enrichment media. HiStOPathologic examinationi A section of liver had periportal aggregates Of lymphocytes. This was a very mild but yet diffuse type of change. No \ Other significant lesions were seen in tissues examined, including heart, 0WNER~ ‘ Y?“ are advised to consult your veterinarian for his analysis of this report and for any treatment that might be indicated. Fees for services of the lab will follow under separate cover and will not include Professmnal service fees of the veterinarian. 041192 \ New” Sun Unum hinmg I.-. v... -3 e...“ a... »- ...--. ’ANIMAL HEALTH DIAGNOSTIC LABORATORY P.0.Box30076 Lansing, MI 43909 95 APhone (517) 353.1683 Pam EGED Mom/1'. 1' ION NOT FOR PUBLICA TION Veterinarian Owner Specimen Breed rxoCCSSkNiIVO. -d'~ivvv tx)ut. Clinic NO. Date Shipped Date Received Final Report Age __________ Sex Preliminary reports - brain (multiple sections), lung, liver, spleen, mesenteric lymph node, thymus, adrenal, thyroid, parathyroid, kidney, pancreas, and small intestine. CONCLUS IONS : Stillbirth, etiology undetermined. [Whether or not the PCP initiated this problem was not clear. We did not see any significant morphologic changes in tissues, and there were no infectious agents of any significance isolated.] R. F. Taylor jf copies, to: Ambulatory, Dr. Erickson (Dairy), Dr. Shull (Dairy), Jim Miles (sr. vet. student), AHDL A 7%; o . «Dr-7'0 V. 0 OWNER: You are advised to consult your veterinarian for his analysis of this report and for any treatment that might be indicated. Fees for services of the lab will follow under separate cover and will not include professional service. fees of the veterinarian. 041192 Maegan Sm: Urinary hump . \ "‘ , REPORT OF LABORATOR I EXAMINA; lON Accession NOT_,20_2836 ‘ 96 d‘quulm‘” Dairy KCCE. ‘ANIF/IAL HEALTH DlAGNOSTiC LABORATORY g, cm: No 71—6111 P.0.Box3007s ‘ ' A Lansmg, Ml 48909 Date Shipped Phone (517) 353-188: Date Received W9 PR/V/LEGED INFORMA TION NOT FOR PUBL/CAT/ON January 15, 1980 Final Report Veterinarian Owner MSU Dairy Science SUPPLEL‘IENTAL REPORT l calf Holstein Breed Specimen Original report - December 26, 1979 . ?reiiminary reports Mycologic examination: Cultures made from the placenta submitted produced a white budding yeast and Penicillium sp. The organisms are probably not significant except if they are also found by histology. A R.F. Taylor CW . i .1 O ' copies to: Ambulatory, Dr. Erickson (Dairy), Dr. Shull (Dairy), Jim Miles (Sr. Vet. Stud.) , AHDL OWNER: You are advised to consult your veterinarian for his analysis of this report and for any treatment that might be indicated. Fees for services Of the lab will follow under separate cover and will not include professional service fees of the veterinarian. 041192 Michigan Sure Unmfl'v A-inn'ng " REPORT OF- LABORATORY exAMiNA'non ANlMAL HEALTH DIAGNOSTIC LABORATORY P.0. Box 30076 Lansing. Ml 48900 ?hono (517) 353-1883 RMMMEGEIJHMHOHnidTKWV . 204107 Accessmn No. Act. QTEBOSXOIY casein; Date Shipped DateReceived December 26: 1979 NOT FOR PUBLICATION ‘ Final Report January 7, 1980 Veterinarian Dr. Martcniuk Owner MSU Dairy MSU Veterinary Clinics (Dr. Lee Shuil) l calf Breed Holstein Age Newborn M Sex Preliminary report; ...... - None 7 HIS'l‘O :11: This calf was pulled by Dr. Marteniuk at approximately 12:00 p.m. on December 24, l979. ' GROSS LESIONS: This calf weighed ll6 pounds and was in good condition. Externally the animal had a yellowish—green staining throughout due to being covered with meconium. Internally the kidneys had considerable perirenal hemorrhage present. The lungs were not inflated and sank in formalin. No other significant gross lesions were seen. LABORATORY FINDINGS: Virologic examination: Fluorescent antibody examination on frozen tissue sections was negative for IBR and BVD. Microbioloqic examination: Darkfield examination on stomach contents was negative for Ecptospira sp. The liver and spleen had no bacterial growth. The lung had a very light growth of Acinetobacter sp. The thoracic fluid had a very light mixed growth of alpha-hemolytic Streptococcus sp., Micro- coccus sp., and Acinetobacter sp.. HistOpathologic examination: There was considerable meningeal hemorrhage present around the cerebellum. The fetal lung was markedly congested with blood. A section of adrenal had considerable hemorrhage present around the periphery or capsule of the adrenal. Similar findings were seen around the kidney.’ The thymus was well developed and no significant lesions were seen. CONCLUSIONS: Perirenal hemorrhage. [There was no evidence of infectious disease. [The perirenal hemorrhage was most likely a result of trauma of some sort.) R. E. Taylor js/copies to: Dr..Marteniuk, Dr. Erickson, Dr. Shull, AHDL, Ambulatory ‘7/0‘0w- afr~OJ7 OWNER: You are advised to consult your veterinarian for his analysis of this report and for any treatment that might be indicated. Fees for services of the lab will follow under separate cover and will not include professional service fees of the veterinarian. O. I 1192 Melvyn Sun Um firming - ———.— - r-v --_.._ v 'RéPUHl Ur" LHD'Ui‘tAI on I ANIMAL HEALTH DIAGNOSTIC LABORATORY P.O. Box 30076 Lansing, MI 48909 “Phone (517)353-1683 98 PRIVILEGED INFORMAl I‘ION _ NOT FOR PUBLICATION Dr. Marteniuk MSU Veterinary Clinical Center Veterinarian ilxé-unilUH I ILA .' “Haw“, Owner Accession No: " ' 204107 Ambulatory Clinic No. ACCt. 2 1'2954 Date Shipped Date Received December 26. 1979 Final Report January 21, 1980 MSU Dairy (Dr. Lee Shull) SUPPLEMENTAL REPORT Specimen 1 calf Breed Preliminaryrepons Oriqinal Report l-7-RO CLINICAL PATHOLOGY LABORATORY Veteninany Clinical Ce. mien Hnlcfnin Age u . k .a Sex —JI1—— Michigan State Universitysg, 30/ EAST LANSING. MICHIGAN 48824 )ST NT L Cage/SlallNO. TOTAL CHARGES / ? WILE— ,Wd/almu S /”/.C I “95““ 105/ 0 l— /.L Io sIONAL OIAcugsIs _ NO _~ ~ ,w 05% I)...“ ZZQ - ..."e-‘II- I I: ”'ICHEMISTRY~~ ~ '» :I» M / ,36 ?9=wv-I-w I] PROFILE : sepr lU/l M324, 961W /3 M mg/dl I'"'SGOT lU/l ; ..UCOSE mg/dl {-'ISDH__ lU/l :BlLl-TOTAL mg/dl {LALK PHOS lUI’l HM: DIRECT mg/dl IT‘CPK lU/l ‘ INDIRECT mg/dl L‘ILDH lU/I 26/;0 TIIME -TOTAL PROTEIN gm/dl j HBD_ lU/l L//" A/dgk'“ ALBUMIN gm/dl '_‘AMYLASE I .SESBULIN gmldl V'UPASE ELECTROPRORESIS: PROTEIN I % ! V T. uO/dl > - , ; 'psp—_____—__% .. 'CHOLESTEROL mg/dl ; [EP " ' ISOENZYMES (SPECIFYL ! ISOOIUM mEq/I 1" :TRIGLYCERIDES mg/dl OE‘Y‘OLAL“ ’- . ; POTASSIUM mEq/l ;_‘_LACTATE mg/dl 'SERUM Osm/kg _ URINE—fi—mOW/kg CHLORIDE mEq/l [)conTISOL COMMENTS. l CALCIUM___ mg/dl _ ‘IRON ug/dl Z; PHOSPHORUS mg/dl TIBC ug/dl ' ”VOOIS/ 3 5‘ N51" MAONESIUM mg/dl {‘ :____AMMONIA __ug/dl l TOT. co: mmol/l OTHE SPECIFY) at.) 3‘0!" a" | CREATININE _-_.__.mg/cII fizfimum dX'ILlL I ‘1‘ I GLUCOSE TOLERANCE ? “XYLOSE TOLERANCE ACTH TOLERANCE __ I. ;TSI-I TOLERANCE _____. l ‘PEARANCE OF SAMPLE: NORMAL __ LIPEMIC ' TIME .. HEMOLYZED ICTERIC UNSATISFACTORY TECH' 6%» OUT‘ li/f/ I \ Jim-I‘d III/«Iriuuliun - Nul jhr [Ill/Ill'('lllillll '/ CHEMISTRY—CHART COPY R. F. Taylor cm A'" O ‘N copies to: Dr. Martcniuk, Dr. Erickson, Dr. Shall, Ambulatory, AHDL OWNER: You are advised to consult your veterinarian for his analysis of this report and for any treatment that might be indicated. Fees for services of the lab will follow under separate cover and will not include professional service fees of the veterinarian. 041x92 Marl-gm Sm: Umm nanny W.. mm m.mm m.~q m.om «.mm m.ss m.mm m.om <.ae «.55 a.moH m.om~ n.0sfi S.Hm~ m.¢m wees . OOH: moan mx\ws o.oa mzpmoaxe zaee semcaxm sneeem emom 92< ozeezzh 9me is“ a.mq mom 0:3 «flow a.mq p.mm mg»: 0.3; «is? ad? mdmm Wm? ammo OOH: leap mx\ms o.oH ammoaxm zzeeeaemoa S.m¢ c.9m H.mm m.sm a.mm ¢.>m N.~m o.se 4.0me H.sma «.msa o.mHm a.mom III: mmoo N.ne III- a.me e.m< a.mm o.wm 0.4“ a.me m.em N.Ho~ a.mmfi a.maa a.mml a.mm emoe m.Mm $.0m m.am m.oo a.ee w.Sm a.mm c.ms m.mo m.o;H m.e0m m emm «.mma a.mm some OOH: mega H.o mx\ma O.o EH> ammoexm zsee acme mxxxe c.o# mgzmoaxe :segeaemoe 5 EN 3 a.m. em 9m 3. ma. 3 «a cs E 3t 2 $2. 34H: an acme mx\ms c.c# oemcaxa zae: amucaxe z:%: ammoaxe zzezqa ems; ~.m s.a m.~ III o.~ c.m e.e ¢.m c.I o.m c.“ m.m m.e s._ Hmow S.m e.m m.m H.H q.a m.w s.m s.e m.m s.c m.c a.» m.o m.m #moc Ede: 2H mom; mxxma H.c agnoaxe zsem e 23:”. m (.g/ml) of ed up we T Time (days) SERUM T. VA 23 O/ «L 15 mi 52.4 #2.? 3A.1 Al.3 56.7 58.2 35-h L3«3 32.5 2L.8 30.1 52.9 22.3 35.0 IS-L .2 1 33.0 19.7 1678 I) P mu n/~ 18.8 30.5 ALI? lOl 1560 I. UIV. .‘fiJLI O .14 6) Av 5 1; 64 Q) 6 r) fi/ 03 n7, I4 fié max 54 flu O 8 0V AU 02 AI/ )4 né 2 .2 AU AU a) 2 any mg. L 8 04 1L th mg «I . .ZW no no 5 a... 04 4I. /9 a0 no 00 0g 1. HI «2 Q/ I.» a; 1L 4L hm, d3, 0; n/~ a; ¢I U 0 O 41, 1. fié 1. 9. G4 Pa 8. Dr /_.J 0 I14 GD J. k. .K I/II If. co w. mu. «1.. nu Ad 0 in _v.. 4J1.) A'VYT‘T‘ [L V ’2 'r‘ l ‘. s. \ I! QT)“ U CONTROL 20 4.; n; pw/ In. 1L ‘. \/ O 'K. 4 3. ‘_. 0 \J pl 0) x? 1' Cu 0.. AL 1.. 1.2 1.705 '\ 0.]. :ng/kg tPCP a... "‘I O \I a.) 1. l02 1 .O rag/kg tPCP v? o' v w- m I- - «fl “7‘“ "Fl 1" Dt- . .vtg'r I'm..".'.'~ I-ID h-'. .- I.* - ...-“N..- I|-4-Jc.i ‘- . Jt . ...- J"tl I "ms m - «so V ‘Trr'r-x 'r - ~ " - ("YA‘T / ‘. ‘.- " J... ' ...".. .’ .'.I. ‘. .- . .“.;.'.‘~-.I ' -u- ‘ \ I 7";“.'-" ..‘."..;.s..- 1 . "F '1‘ I t: ”I '7 . ‘I t ‘ ' 1': "v.3 :, / I; / x : _ I_ ‘ I I I +pno r' ' "\ I ‘ 08 C‘- ‘. pum v.11 b l b l F l COW “II/VIN. I/ C ."“ \ ‘F'j A ' " '3 : 'r‘ >- _' E L") O r.” '— ‘I’I "I """ " r‘" I '5 v’ I " . . | l v " .‘l I‘ ' . ,J,L, L‘J.) 'l-Ff'“ J .U L’9.L:> t-‘k5.C' 4:95? 3"f.,) j). l-. O: ‘Igl );‘.‘\- ,)I_) L. I 1"‘1 "" " A A 7' "' "I 1'" V.“ ' h —“ ' [E IW ’ ’\ ‘fi ‘3 ' f" (‘ -_|f I" ‘. I " . ‘_ ' \"< " '\ \ I ..‘ / JCJv‘v ' ()2. ’l .1 ‘5. I "" ‘1 ‘l' p' .r .- ' “'K’ ' " - ‘ '- " . a V F. I — ~ .- -’ ’l ‘ - ~ ... o r1 (firm .0 'T 71> 2‘ 3 7'7 r" I’- -. ' .Q I ‘ " " z- ," ’ VC'II ’_I )f'); HA) 0% 4-~ /-- o I. , . . J" o I . Z I I. I _ I . — . . . x . . ‘ pm L: r. '3 .4 \ O O E Q\ \1 \h ' - “ I“ I /\ n ’\ I p. ,- ,.,. n ‘ 8 1"“, 32+'O 30.5 4‘18.” [ll—O." )90‘. "500.) ill.) ill. ‘.:o(.l “50C 3 ,_‘.'O I a .I ..-.'.- “:Il-'_ >‘vbof0\. ._ . i -' . . ‘i‘..\J _ . v . _' :‘. |‘ 103 IOh Best Fit Polynomial Equations for Experiment ll Calf PCP Serum Concen- trations as in Figures 4 and 5. For For For For For All calves contaminated in utero and postpartum with 0.l mg/kg tPCP y = l616.236+-26.303x calves contaminated postpartum via dam's milk with 0.1 mg/kg tPCP y = l62.68h+-69.3l3x calf contaminated postpartum via dam's milk with 10.0 mg/kg pPCP y : o.3oI+ lLI6l.969x + 109.1on2 - 5.055x3 + 0.036xu calf contaminated in utero and postpartum with 10.0 mg/kg pPCP y 26816.269+ 80,155.. calf contaminated postpartum via dam's milk with l0.0 mg/kg tPCP y = -o.ooe+ 729.370x a 78.526x2 + l.l87x - 0.005x postpartum exposure was via milk collected from the contaminated dam. POP IN MILK*OF PCF CONTAMINATED cows DURING FIRST 22 DAYS INTO LACTATION (ppb) DAYS 1 2 3 h 5 6 7 11 15 19 23 28 Dose 0.1 ppm tPCP Cow 566A the 300 330 330 ' 260 29c 350 zuo 330 410 360 370 Cow 6681 870 590 580 550 600 550 #80 620 590 LOO 570 650 Cow 6682 773 360 230 35q 460 500 430 380 280 500 290 390 Dose 10.0 ppm tPCP - Cow 5758 5370 L37+ touo 3900 3830 3730 3600- 3512 2850 2700 2410 3930 Dose 10.0 ppm pPCP Cow 1560 7150 t3t- 6590 7230 4890 ;3790 3&50 30701 4280 3730 3A1o 2720 * BKG Of 200 ppb not subtracted from values. lOS 106 PCP BLOOD CONCENTRATIONS (ppb) OF A POST-NATALLY EXPOSED CALF VIA MILK OF A 10 mg/kg pPCP TREATED cow Calf TIME (Days) 7045 0.0 146 0.125 150 0.250 495 0.50 1039 1.0 1986 2.0 3243 3.0 4929 5.0 8990 7.0 12165 11.0 24930 15.0 31969 19.0 37365 23.0 37965 28.0 38325 107 PCP BLOOD CONCENTRATIONS (ppb) 0F CALF RECEIVING POST-PARTUN EXPOSURE To PCP VIA MILK CF 10.0 mg/kg tPCP DOSED 00w AND ALSO OF THAT cow Calf Cow Time (Dgyg) 6929 5857 0 16h 55430 0.125 868 -— 0.250 962 -- 0.50 1127 ... 1.0 1505 65h60 2.0 2796 46520 3.0 h438 A9370 5.0 6983 53980 7.0 8082 49820 11.0 12270 #8000 15.0 11322 28880 19.0 115h0 h7850 23.0 11840 39370 28.0 9672 47980 PCP BLOOD CONCENTRATIONS (ppb) OF IN UTERO & POST-NATALLY EXPOSED CALVES VIA NILK CF 10.0 mg/kg pPCP TREATED cow AND OF THE CORRESPONDING DAN. Calf Cow Calf Time (Days) 7044 1560 Time(Days) 7045 0.0 22933 60700 0'0 1A6 0.125 30168 -— 0.125 150 0.250 30252 ~- 0.25 495 0.50 28035 -- 0.50 1039 1.0 26414 61340 1.0 1986 2.0 26327 64450 2.0 3243 3.0 25802 59550 3.0 4929 5.0 26207 50890 5.0 8990 7.0 23627 40820 7.0 12165 11.0 30172 50240 11.0 24930 15.0 29296 51440 15.0 31969 19.0 28653 46160 19.0 37365 23.0 26736 43220 23.0 37965 28.0 28.0 38325 300h2 52200 PCP BLOOD CONCENTRATIONS (ppb) OF 108 POST NATALLY EXPOSED CALVES*' Calves Time (Days); 6909 6930 6932 0.0 <100 171.0 -—- 0.125 (100 (100 (100 0.250 177.0 {100 187.0 0.500 4100 168.0 178.0 1.0 131.0 148.0 301.0 2.0 236.0 266.0 521.0 3-0 257.0 358.0 683.0 5.0 371.0 606.0 1000.0 7.0 610.0 688.0 863.0 11.0 649.0 889.0 876.0 15.0 795.0 2035.0 1308.0 19.0 817.0 1958.0 1768.0 23.0 792.0 .... 2130.0 28.0 1051.0 2869.0 2507.0 * Fed milk collected from 0.] tPCP treated cows. 109 PCP BLOOD CONCENTRATIONS (ppb) OF IN UTERALLY AND POST NATALLY EXPOSED CALVES AND THEIR DAMS Calf No. VINE (DAYS) 6921 6931 0 1579 1367 0.125 1634 1417 0.250 1924 1412 0.500 1605 1433 1.0 1555 1452 2.0 1661 1705 3.0 1800 1886 5.0 1919 1807 7.0 2015 2291 11.0 1919 2146 15.0 1513 -— 19.0 1768 2258 23.0 2084 2379 28.0 2169 2589 Cow No. TIME (DAYS) *6664 6681 6682 0 3755 4219 4444 1.0 3394 3486 2993 2.0 3455 3202 2776 3.0 3418 2935 2311 5.0 3012 2898 2667 7.0 2934 2269 2711 11.0 2464 2560 3106 15.0 2092 2462 2238 19.0 1965 2496 2158 23.0 2069 2779 2027 28.0 2116 2586 2398 *Calf of 6664 died at birth post- mortem PCP blood level analysis indicated 1167 ppb APPENDIX C 7. U“ a US ht oU‘-- -.. . . .-. o. coo-u: . v. t [fichSSIOn l‘uu. "’ ANIMAL HEALTH DIAGNOSTIC LABORATORY P.O.Box30076 Lansing.Ml 48900 110 Phone (517) 353-1683 Clinic No.05” rY SCi- 71‘6111 Date Shipped DateReceived September 23, 1980 A PR/VIL EGED INFORMA TION . NOT FOR PUBLICATION Final Report October 7, 1980 Veterinarian . Owner Msu Dairy (Lee Skull) Specimen 2 calves #7181, #7176 Breed Hostein 134963.351... Sex M __ Preliminary reports “one HISTORY: 'These 2 calves had temperatures of 107-110F and had signs of weakness, rapid respiration, and scouring. They had been treated with chloramphen- icol, gentamicin, BO—SE, vitamins A and D, 3 cc of penicillin and oral electrolytes. Calf #7181 was given lactated Ringer's IV. The animals were housed in pens enclosed in an insulated barn. They were fed pen- tachlorophenol (20 mg/kg body wt/day) in the milk as part of an experi- mental protocol. However, these animals died. GROSS LESIONS: The carcasses were in fair nutritional condition but appeared 5—68 de— hydrated. There was moderate postmortem autolysis of the tissues. Ex- ternally, there was yellow, watery fecal material in the hair around the anal orifice. The eyes were sunken into the orbits. Internally, the trachea contained white foam extending from the mid-cervical region throughout the terminal bronchioles. There were multifocal areas of _. atelectasis involving the lungs of both animals. Calf (221907) had ‘ a large pus-filled abscess involving the diaphragmatic lobe of the lung. There was a large area of emphysema measuring 3 inches in diameter and lying adjacent to this area of abscessation. LABORATORY FINDINGS: Microbiologic examination: Please refer to the attached report. Histopathologic examination: The most outstanding histopathological findings were in the lungs. In sections of lung from both animals, there was diffuse congestion of blood vessels. In 221908, there were areas of lobular atelectasis with infiltration of inflammatory cells consisting of many neutrophils and a few lymphocytes. The bronchioles in these areas had narrow lumens. There were adjacent areas of a] veolur .\ interstitial emphysema. In 221907, there were many neutrophils within conti . OWNER: You are adciggg °tE>°consult your veterinarian for his analysis of this report and for any treatment that mlSlht be indicated. Fees for services of the lab will follow under separate cover and will not include Professmnal service fees of the veterinarian. OrlllDZ MSU ,3 . an Affirmative A ' Cflon/Equal O ' n maria/7 Mot-Mm Sm. Unwom'ry Pmrmg . Pportumry I s . . 7 m... pk , __- 112.‘Un‘ Ur m ~-0 4 C.‘ -.4 ..‘_.."¢onii“é ‘l a. i ' o-‘~ ' ' .' an, e w h. l 0 iv I"\L:CGSS‘OH N0. Och—AJKJ / JUU LUHLo ' ex ANIMAL HEALTH DIAGNOSTIC LABORATORY ‘ Clinic No P.0.Box30076 ' V - . Lansing,Ml 48909 m Phone (517) 353-1683 Date Shipped Date Received l 1.- PRIV/LEGED INFOR 94A TION NOT FOR PUBLICATION Final Report i Veterinarian Owner Breed Age ...__._.__. Sex .... Specimen Preliminary reports \. the bronchiolar lumens. In other areas, these inflammatory cells were within alveoli and interstitium. There were areas of hemorrhage in .random locations. Interstitial edema and emphysema was observed as well. In both animals, there was vascular congestion in the brain with dark purple bodies in the vessel lumens. These bodies were thought to be hemoconcentrated precipitated protein. There was 1 focus of subcapsular hemorrhage in the adrenal gland of calf 221908. Changes in other organs were thought to be insignificant. CONCLUS IONS : Acute purulent pneumonia. [The isolated highly resistant Escherichia coli was thought to be a significant problem, especially in the presence of scouring in these animals.) P. E. Tippett/R. F. Taylor:knw copies to: Dr. Hughes (Dairy Science), Dr. Tippett, senior student Forbes, AH Dr. Erickson (Dairy) , Ambulatory 5 607’ ‘fm‘ °' OWNE . . . ’ R. You are advrsed to consult your veterinarian for his analysis of this report and for any treatment that might be Indicated. Fees for services of the lab will follow under separate cover and will not include Drafessional service fees of the veterinarian. MSU’k.nA”’-’MOW.A \ _k' 0- l l 192 Michigan State Unruly” Raining anion/Equal Opportunity Institution DL Nausea '. 2.. ‘ “1., . : "’ "717711.14: ' ilfNTIMIcaoaI ' .-.'.':. 7“," '. Animal Health Diagnostic LabOratory "it“? AL o ca 0 - .."N’ o. " .... Igggfgengy 321907-908 42:2...- PO.~Box"30076~-~--~—- 8:42:21.-- -..;L ‘ . my,“ Lansing. Michigan «43909 .. 3.~ V’sjfp... grggm‘gofiat. 1980 4“.“ _fPhqne (517) 353-1 5:13 ‘ " ~ .'.‘ .17 .' I - . apt . I. "..~ . . I '.. ‘ "1' .' . ; .2. r . 'J‘WW. ‘C‘ «“1 "~ 1 a .. ' V - ‘ . 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'- u ’ l~3nl2 K4]. . \/.~.J/ o" 3o 1'9 . ’3 - *L—J CLntrols ' {‘1 I‘L-6 o“ d- UP CIRCULATING T1 CCNCEHTRATICIS (mg/ml) AFTER TRH CHALLENGE 1v 4 * TIME (Minutes) Predose—semnles POST—TRH SAMPLES Calves ~60 -30 0 10 20 3: 45 ’60 90 120 180 1 86.3 84.1 85.8 85.6 86 7 87.2 78.0 84.C 87.9 109.7 115.8 2 47.3 49.7 49.2 50.0 47 5 52 49 7 53-8 57.6 60.9 59.4 3 50.2 49.7 54.6 50.6 54.3 5 47.4 52.0 58.8 58.0 70.8 4 64.9 58.0 58.8 69.9 63.1 62 1 64.0 72.7 76.4 80 3 89.9 5 64.4 61.6 63.1 65.9 63.0 60.6 65.3 64.2 68.5 70.0 73.7 6 65.1 62.3 62.5 65 7 58 8 66.8 55.3 73.8 75 1 81 5 81.5 7 42.6 40.4 41.1 41.4 42.4 43 5 38.7 48.3 52 7 59.9 67.5 8 32.3 29.4 30.5 32.6 33.5 33.9 32.5 41.2 45 O 45.6 54.9 9 64.0 61.9 62.8 65.0 62.6 58.5 58.8 65.9 72.0 83.2 97.9 10 NO DATA AVAILABLE 11 48.6 45.4 45.5 45.9 L7.9 49 O 48.9 A? 5 L9 8 53.5 57.6 12 51.8 49.9 1’ 3 3; u “1.4 5- 7 5? 0 54 3 53 h 57.4 67.9 13 13.4 13.9 *4 9 ‘5 3 LS.O 16.4 17 3 18 7 23.7 27.5 36.1 14 34.7 32.0 29 9 30.0 3-.3 9” 4 33.4 3: b 39.4 49.5 52.9 15 19.1 19.8 13 3 9 - 1839 20 4 21 4 2* 3 25 3 32 7 35 2 1-3 COHTROLS h-6 1.0 mg/kg aPCP 7—9 1.0 mg/kg tPCP 10-12 10.0 mg/Ag aPCP 13-15 10.0 mg/kg tPCP 119 ORGAN WEIGHTS on CALVES (GRAMS) Liver Lung Thyroid 513393 Spleen Thymus 1200 880 10.3 300 1&5 139.8 2 1050 970 8.7 320 205 128.8 3 1300 11h5 11.3 280 200 188.9 A 900 640 8.5 195 107 99.6 5 1200 1170 6.8 300 135 128.8 6 1325 1110 8.3 370 180 247.A 7 1290 820' .5 335 140 50.1 8 15A0 850 1&.8 350 280 102.2 9 1520 1020 12.4 360 160 126 9L5 522 8.5 290 80 22.5 1L90 8&0 10.7 LOO 170 120.2 1110 775 15.6 370 95. 29.9 1230 800 11.3 320 80 21.8 Controls 1.0 mg/kg aPCP 1.0 mg/kg tPCP A mg/kg aPCP 1.9..) 10.0 mv/kg tPCP .m— oqom -11-: o¢wq~ 4.8qefifi ¢.mflm ans: 11:: qm¢m ~ m.oqmmH um¢fi oqm~m ouomfi zflmo NJQFN omoofl oaflwm mfi om.“ a.mwwm ommm_ o.~_- :.w~_ o~:o_ o:ow Nmom «mmNm o_mm_ mm::~ N. :11: oam.cm Omomy o.mmmm o.uqm ammo 05mm mmflc oo~m~ cocoa cmmom #8 a.mqmg c.0cqm wa¢ n.0mm o.mm mJJm qwmm mfimm «New Nmmm omfic a 0.8288 o.Hwom mo¢m a.mmo #.mm mmmm wflofi comfi mm>¢ mflmm ¢¢Hm m 1...: o.~>¢< .mufim 0.0mqm q.mc mmwm OQmoq ocwm omwm OMmHH ooflm mmym m 1.1:: o.«<m a.mmqfi m.om o.mmqm c.qpm o.omo~ m.¢>< q.omm o.cmm m aaaaa ~.mMm c.0mflm o.om~ 5.8m c.8ocm c.5mm o.moq~ a.mmq m.mqm u.mmmm n.0mm m.mq> ¢.50m a.mqm q.mmm a m Fonds ii £953 28me 9.8.5 mzzqfit. 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