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I , I: III I I I.» ' ‘ THESIS LIBRARY Willi"I"!lllllllllllllll{WWW}!“W I 3 293 10410 9305 Michigan State University This is to certify that the thesis entitled A TOXICOLOGIC ASSESSMENT OF PENTACHLOROPHENOL IN LACTATING DAIRY CATTLE presented by JOHN HAROLD» KINZELL has been accepted towards fulfillment of the requirements for H: .DL degree in JnimaLScience (/M flit/7214? ”Z [/2 Major professor Date 6/2lI/E 2- 0-7639 I MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .—,_. your record. FINES will be charged if book is returned after the date stamped below. Til? t: t. CW 363 ("NA A.~M xix Dfihl 1flb=¥nv~~' an“ my 942 A TOXICOLOGIC ASSESSMENT OF PENTACHLOROPHENOL IN LACTATING DAIRY CATTLE By John Harold Kinzell A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1981 ABSTRACT A TOXICOLOGIC ASSESSMENT OF PENTACHLOROPHENOL IN LACTATING DAIRY CATTLE By John Harold Kinzell Pentachlorophenol (PCP) is a registered antimicrobial agent widely used for the preservation of wood. Two studies were conducted in.lactat- ing dairy cattle. The first study examined the effect of subchronic oral administration of technical PCP (penta) on performance and health in adult cattle. Eight lactating Holstein dairy cattle were allotted in pairs to a control or treatment group and were fed penta from 6 r l week pg§t_ partum_for about l35 days (0.2 mg/kg body wt/day for 75 - 84 days followed. by 2 mg/kg body wt/day for 56 - 60 days). Feed intake and milk production were measured daily. All cattle were weighed bimonthly and observed daily for health effects. Blood samples were collected twice monthly and subjected to standard hematologic and clinical chemistry measurements. At two weeks and one week prior to necropsy, urine was collected from each cow by urethral catheter and subjected to standard urinalysis. One week prior to necropsy all cattle were examined by a veterinarian. At necropsy all cattle were subjected to gross pathologic examination and selected tissues were examined histopathologically. lg_yitrg_kidney function tests were conducted on tissue slices collected at necropsy. Penta fed subchronically had no significant effect on performance as indicated by feed intake, body weight or milk production. Periodic somatic cell counts of milk samples indicated that treatment did not increase the incidence John Harold Kinzell of mastitis. Generally, all cattle appeared normal throughout the entire period of exposure. Periodically, two of the four treated cattle had an elevated body temperature, mild anorexia and decreased milk production. A physical examination revealed no abnormalities. There were no biolo- gically significant differences in hematologic measurements, clinical chemistry or urinalysis due to treatment. Organ to body weight ratios of liver, lungs, kidney and adrenals were significantly greater in the treated cattle. In three of the four treated cattle the kidneys had a pale white appearance. All other organs appeared normal. Microscopic examination of the discolored kidneys revealed a chronic, interstitial nephritis with mild hyperemia. Also the urinary bladders of all treated cattle had subacute urocystitis. The kidneys in the treated cattle were functionally impaired as demonstrated ifl_vj§rg_by significantly decreased uptake of para-aminohippurate, tetraethylammonium and amino isobutyrate. At the time of necropsy the kidneys may have been in a functional state of impairment, but not severe enough to cause significant changes in routine clinical laboratory tests, general health or performance of the animals. The interrelationship of the urocystitis and administration of penta is unclear. The results of PCP analysis of tissues from penta-fed cattle revealed that the liver and kidney had the highest PCP concentrations (13.6 and 9.6 ppm) and brain and thyroid the lowest (0.7 and 0.5 ppm). At the 0.2 and 2 mg/kg doses, average milk PCP (total) concentrations were 0.4 and 3.l ppm. The primary excretory route for PCP was urine, followed by feces and milk. Chlorodioxin (COO) analysis of liver tissue from penta-fed cattle showed average concentrations (ppm) of octa-CDD, hepta-CDD, hexa-CDD and tetra-COD to be 125, l4.8, 3.8 and non-detectable, respectively. John Harold Kinzell The pharmacokinetic profile of 14 C-PCP in a lactating dairy cow was studied in a second experiment. A mature Holstein cow fed 0.2 mg/kg body wt/day of technical pentachlorophenol for 95 days was given a gelatin capsule containing 14 C-PCP on alpha-cellulose. The subchronic oral admin- istration of penta was continued throughout the duration of the experiment. Concentrations of 14C were monitored in serum, urine, feces and milk through 76 hours post-administration. Maximum plasma concentrations were attained in about 10 hours. Both absorption into and elimination from serum followed first-order kinetics with corresponding half-lives of 4.28 and 42.8 hours. Approximately 75% of the dose was excreted in the urine with 5% each excreted in the feces and milk. Liver and kidney had the highest ‘4 C concentrations with brain, adipose tissue and spinal fluid having the lowest. Acid hydrolysis of the urine indicated that considerable quantities of PCP are excreted in a conjugated form. Fractionation of milk showed 62.2, 24.4 and l3.3% of the label in the whey, casein and fat, respectively. ACKNOWLEDGEMENTS I would especially like to express my gratitude to my wife Ann for her support and patience during the course of my Ph.D. program. Also, I thank my parents for initally encouraging me to seek a university education. Sincere appreciation is extended to Dr. Harold Hafs and the Department of Animal Science for their financial assistance during my program. I would also like to acknowledge the assistance of my graduate committee, Drs. Bill Thomas and Mel Yokoyama of Animal Science, Dr. Stuart Sleight of Pathology and Dr. Robert Roth of Pharmacology and Toxicology. The technical assistance of Barbara Olson and my fellow graduate students is also acknowledged and appreciated. To Dr. Peter Cheeke I feel a deep sense of appreciation for giving me the initial opportunity to pursue a graduate degree. Finally, I would like to thank Dr. Lee Shull for his friendship, support and guidance during my graduate training. TABLE OF CONTENTS List of Tables ....................... List of Figures ...................... Introduction ....... ' ................. Literature Review ..................... Historical Perspective ................ Chemical and Physical Properties of Pentachlorophenol ................. Synthesis of Pentachlorophenol ............ The Wood Preservation Process. ............ Chlorinated Dioxin and Furan Contaminants in Penta . . Toxicology of Chlorodioxins (COD) and Furans (CDF) in Penta ................. ,. . . . Analysis of Pentachlorophenol Separation .................... Detection .................... Toxicology of Pentachlorophenol ............ Exposure ..................... Toxicokinetics of Pentachlorophenol ........ Absorption .................. Distribution ................. Metabolism .................. Excretion ................... Toxicodynamics of Pentachlorophenol ........ Mechanism of Action .............. Studies in Domestic Animals .......... Studies in Other Species ........... Research Objectives .................... Research Methods and Procedures . . ............ Animals and Diets ................... Composition of Technical Pentachlorophenol ....... Preparation of the Pentachlorophenol Dose ....... Animal Care and Daily Observations . . ....... Collection of Blood and Urine for Clinical Analysis . . Collection and Analysis of Milk for Fat and Somatic Cells ....................... -11- -111- Necropsy Methods, Collection of Tissues and Histopathologic Techniques ............... Kidney Function Tests .................... Analysis of Pentachlorophenol ................ Extraction from Serum (for total PCP) ............ Extraction from Serum (for free PCP) ............ Extraction from Urine (for total PCP) ............ Extraction from Urine (for free PCP) ............ Extraction from Feces (for total PCP) ............ Extraction from Rumen Fluid (for total PCP) ......... Extraction from Tissues and Whole Milk (for total PCP) . . . Gas Chromatographic Analysis ................ Analysis of Chlorodioxins (C00) in Liver .......... Purification of l4C-Pentachlorophenol ............ Preparation of the 14C-Pentachlorophenol Dose ........ Preparation of the Lactating Dairy Cow for the 14C-PCP Radiotracer Study ............... Collection of Blood, Urine, Milk, Feces and Selected Tissues During the Radiotracer Study .......... Liquid Scintillation Counting Techniques .......... PCP Residue Analysis of Tissues and Fluids ......... Milk Fractionation Procedure ................ Analysis in Milk Fractions for PCP and 14C ......... Whey .......................... Casein ......................... Fat .......................... Quantitation of 14C in Tissues Using a Total Oxidation Method .................... Statistical Analysis .................... Results ............................. Experiment l: Subchronic Oral Administration of Penta to Lactating Dairy Cattle .................. Effect on Performance ................. Effect on Appearance and General Health ........ Effect on Clinical Chemistry, Hematology and Urinalyses ..................... Histologic Examination of Tissues ........... Effect on Kidney Function ............... Pentachlorophenol Residue Analysis ........... Serum PCP Concentrations ............. PCP Concentrations in Tissues and Fluids ..... Dioxin Residue Analysis of Liver ........... Experiment 2: The Fate of a Single Dose of 14C-Penta- chlorophenol in a Lactating Dairy Cow ......... l4 Absorption of C-PCP ............. 53 53 55 63 7O 7O 7O 77 77 77 8O 80 -iv- Distribution of 14C-PCP .............. 91 Metabolism of l4C-PCP ............... 9T Excretion of l4C-PCP ............... 94 Discussion .......................... l06 Toxicodynamics of Technical Pentachlorophenol ...... l06 Performance, Appearance, General Health, and Kidney Function .................... l06 Clinical Chemistry, Hematology and Urinalyses . . . 109 Pathologic Findings ................ llO Toxicokinetics of Technical Pentachlorophenol ...... ll2. Absorption and Serum Concentrations ........ ll2 Distribution .................... ll4 Metabolism ..................... ll7 Elimination .................... ll9 Excretion ..................... l20 Summary ........................... l23 Future Work ......................... l25 Bibliography ......................... 127 Appendix. . ......................... T35 Biographical Sketch of J.H. Kinzell ............. l48 Table 10. ll. l2. l3. 14. LIST OF TABLES Physical Characteristics of Pentachlorophenol . . . . Solubility of Pentachlorophenol in Various Organic Solvents ................... Pentachlorophenol and Dioxin Content of Dow's Improved Pentachlorophenol (Dowicide EC-7R). . . . Dioxin and Chlorophenol Analysis of Industrial Composite, Technical PCP and Dowicide EC-7R ...... Dioxin and Furan Analysis of an Industrial Composite of Penta by Monsanto, FDA and NIEHS ............ Organ Heights of Control and Penta-treated Cattle Expressed as Percent of Body Weight .......... lg_Vitro Renal Function in Penta-treated and Control Cattle ......................... Total Pentachlorophenol Concentration in Tissue (ng/g of wet tissue) or Fluid (ng/ml) Collected at Necropsy from Cattle Fed Penta ............ Total Pentachlorophenol Concentration in Urine, Serum, Rumen Fluid and Milk .............. 14C-Concentration and Corresponding Pentachlorophenol Concentration in Various Tissues and Fluids ...... Gas Chromatographic Analysis of Urine for Total and Free PentachlorOphenol ............... 14C-Concentration in Solvent xtracts of Urine Analyzed for Total and Free 1 c with Liquid Scintil- lation Counting ..................... Percentage Distribution of 14C-Concentration in Milk Fractions at 4-hour Intervals After Administration. . . Percentage Distribution of Conjugated and Unconjugated 14C and Pentachlorophenol (PCP) in Casein as Measured by Liquid Scintillation Counting (LSC) and Gas Chromatography (GC) at 4-hour Intervals After Administration ..................... Page 12 18 50 78 Bl 85 87 92 93 l02 l04 Table l5. T6. T7. l8a. 18b. l9a. T9b. 20. 21. 22. -VT- Percentage Distribution of Conjugated and Unconjugated 14C and Pentachlorophenol (PCP) in Milk Whey as Measured by Liquid Scintillation Counting (LSC) and Gas Chromatography (GC) at 4-Hour Intervals After Administration ..................... Values Generated by the "Curve Stripping" Method . . . . 14C-Concentration in Serum Over Time in a Cow Given a Single Dose of 14C-PCP ................. Results of Hematologic Examination From Control and Penta-fed Cattle (0.2 mg/kg/day Exposure Period) . . . . Results of Hematologic Examination From Control and Penta-fed Cattle (2 mg/kg/day Exposure Period) ..... Results of Clinical Chemistry Analyses From Control and Penta-fed Cattle (0.2 mg/kg/day Exposure Period) . . Results of Clinical Chemistry Analyses From Control and Penta-fed Cattle (2 mg/kg/day Exposure Period) . . . Results of Urinalyses from Control and Penta-fed Cattle ......................... Recovery of a 14C-PCP Spike in Various Tissues and Fluids of Cattle Fed Penta ............... Recovery of a 14C-PCP Spike From Urine, Serum Rumen Fluid and Milk .................. Page TOS T39 T40 T41 T42 T43 T44 T45 T46 T47 Figure boom TO. Tl. l2. T3. T4. LIST OF FIGURES Page The Phenol and Sodium Salt of Pentachlorophenol ..... 8 Synthesis of Pentachlorophenol ............. ll Common Dioxin Congeners ................. l6 Synthesis of Octachlorodibenzo-p-dioxin (OCDD) and Octachlorodibenzofuran (0CDF) .............. .l9 Metabolism of PCP in the Rat .............. 35 Effect of Penta on Body Weight ............. 7l Time Course Status of Daily Feed Intake of an As-Fed Basis (a), Ratio of Intake to Body Weight (b), and Ratio of Fat Corrected Milk Produced to Megacalories Consumed (c) in Cattle Fed 0.2 Mg Penta/Kg Body Weight/Day for 75 to 84 Days Followed by 2 Mg Penta/Kg Body Weight/Day for 56 to 60 Days. Each Graphed Point Represents the Mean of All Data Collected During the l4 Day Period Preceding the Specified Number of Days Indicated ........... 73 Time Course Status of Total Milk Production (a) and Milk Fat Production (b) in Dairy Cattle Fed 0.2mg Penta/Kg Body Weight/Day for 75 to 84 Days Followed by 2 mg Penta/Kg Body Weight/Day for 56 to 60 Days. Each Graphed Point Represents the Mean of all Data Collected During the T4 Day Period Preceding the Specified Number of Days Indicated. . 75 Concentrations of Total and Free Serum PCP in Cattle Fed Penta Subchronically ................ . 82 14C-Concentratiorlii in Serum Measured at Selective Intervals After C-PCP Administration .......... 89 14C-Concentrations in Urine and Cumulative Percentage of Dose Excreted into Urine Measured at 4-hour Intervals After Administration ............... 96 14C-Concentrations in Feces and Cumulative Percentages of Dose Excreted into Feces Measured at 4-hour Intervals After Administration .............. 97 14C-Concentrations in Milk and Cumulative Percentage of Dose Excreted into Milk Measured at 4-hour Intervals After Administration ............. l00 Use of "Curve Stripping" to Estimate the Rate Absorption Constant (ka). . . . ............ T37 -vii- 964:3 sure: tems "07 E i [no 3, 3 i v (1373\ a a, 9‘ a ll h‘~¢ “a '... Nifl . ‘Fn 1 JC ‘: I: . i.r39r‘i C I‘- ' “all! Vt 4.: 153‘: V. ~5:‘ INTRODUCTION Pentachlorophenol is an antimicrobial and fungicidal agent used in the preservation of wood and other materials. It has also occasionally been used as a herbicide, insecticide and molluscacide. As a clarification of terminology, pentachlorophenol or analytical pentachlorophenol (both abbreviated to PCP) refers to the chemically pure compound (99% purity). Penta and technical pentachlorphenol are . terms used for the commercially-produced pentachlorophenol which varies from 85-93% in purity and contains less than 1% of dibenzo-p-dioxins (no 2,3,7,8-TCDD) and dibenzofurans as contaminants. Penta's main use is in preservation of wood. According to Cirelli (l978) about 54 million pounds of penta were produced in 1974. Small quantities are converted to the alkali salts for use as broad-spectrum biocides, but more than 80% is used in the wood-preserving industry (Arsenault, 1976). Penta's popularity as a wood preservative stems from its superior ability to effectively control mold, termite infestation, powder post beetles and other wood-boring insects all of which decrease the structural longevity of wood (Monsanto, l958; Dow, l962; Carswell and Mason, l938). . Penta-treated wood has come into relatively widespread use in the construction of housing and feeding facilities on farms. A recent study has shown that 50% of Grade A dairy farms in Michigan have used penta- treated wood in the construction of animal housing and feeding facilities (Foss gt_gl,, 1980). As is the case with many uses of efficacious -2- pesticides, misuse often accompanies use. In the case of construction on dairy farms, penta treated wood has been improperly and unnecessarily used in locations where moisture is not a critical factor in the structural longevity of the wood. Examples include: rafters, door jambs, fencing and splash boards well above ground level; all locations in which con- tinual exposure to manure and moisture is not a significant problem. In recent years, numerous pesticides which have been in commerce for many years and have not been considered to pose significant health or environmental problems have come under close scrutiny of the regulatory agencies. Some have subsequently been withdrawn from the market permanently or until sufficient data were made available to answer questions regarding their safety. Penta is no exception, Until the late l960's,‘fiavquestions were raised concerning the safety of penta, other than it might pose a possible residue problem. However, during the l970's several situations occurred which created more concern about penta. As mentioned previously, most commercially-synthesized pentachlorophenol is contaminated to a level of about l% with several chlorinated dibenzo-p-dioxin and chlorinated diben- zofuran congeners. Although none of the much-publicized 2,3,7,8-tetra- Chlorodioxin (TCDD) has ever been detected in any commercial penta, the There presence of any dioxin congener was more than enough to bring penta into the political and scientific limelight. Early in l977 the state of Michigan was just recovering from the (nuisequences of a commercial fire retardant (polybrominated biphenyls) being accidentally mixed into livestock feed and the resulting animal health problems and contamination of the food chain. The burden on true dairy industry included removal of contaminated herds, decontamination of aninml facilities and purchase of replacement stock. People in the animal industry became keenly aware of the potential health and residue problems that can result from a chemical in an animal's environment. Consequently, in 1977 when herd health and production problems were observed on several Michigan dairy farms that had previously decontamin- ated their facilities of polybrominated biphenyls and replaced their stock, a chemical was the suspected cause. Penta was suspected as a cause because of the prevalence of penta-treated wood on several of . these farms. Attempts were made to quantitate penta in tissues of these cattle. Shortly thereafter, milk samples from one of the herds were found to contain both PCP and dibenzochlorodioxins. In these investigations, the blood of cattle in l3 herds was found to contain some PCP. These herds were subsequently quarantined by the Michigan Department of Agri- culture. Penta has since lost its anonymity and status as a safe chemical. Currently, regulatory agencies are concerned about penta's possible effects on the human population resulting from residues of dioxins, furans and PCP in the food chain. Moreover, questions concern- ing the effects of PCP and its contaminants on the health and productivity of domestic animals have also been raised. Prior to the initiation of the studies described herein, there were no reports which directly addressed the question of what effects subchronic exposure to penta had in lactating dairy cattle. Exposure to penta in the barn environment can occur through any one or all of three routes: oral, dermal or pulmonary. Clearly, an experiment examining all three routes would be a monumental task with considerable cost. Thus, to provide a starting point, these studies set out to address two questions. First, what are the effects of subchronic oral exposure to penta in lactat- -4- ing dairy cattle, and second, what is the fate of a single oral dose of 14C-PCP in a lactating dairy cow previously exposed to penta subchronically? LITERATURE REVIEW Historical Perspective The history of pentachlorophenol as a wood preservative dates back to l930 when some chlorinated phenols were produced for wood preserving experiments (EPA, 1980). Interestingly, the first experiments were carried out using stakes treated with tetrachlorophenol. The stakes were installed on Barro Colorado Island in the Panama Canal Zone in l93l as part of a study carried out by the U.S. Forest Products Laboratory and the U.S. Bureau of Entomology and Plant Quarantine. Similar studies carried out about two years thereafter with pentachlorophenol showed it to have superior wood preserving capabilities (EPA, 1980). Usage of penta increased to the point where in l947 about seven million pounds were reportedly used by commercial wood preservers (Hunt and Garratt, l953). Recently, the plant capacities of the four major U.S. manufacturers were listed as follows: Dow Chemical (17 million pounds), Monsanto Company (26 million pounds), Reichhold Chemical (20 million pounds) and Vulcan Materials Company (l6 million pounds) (EPA, l980). Pentachlorophenol has come to have many more uses than just as a wood preservative. These include use as a fungicide and/or bactericide in the processing of starches, adhesives, proteins, leather, oils, rubber and paints. It has also been incorporated into rug shampoos and textiles for mildew control. In food processing plants in pulp and paper mills PCP has been used for slime control (Bevenue and Beckman, -5- 1967). Agricultural uses have been as a herbicide for control of weeds and as a preharvest desiccant on pasture land (Grigsby and Farwell, 1950), on pineapple and sugarcane fields (Gordon, 1956; Hilton, 1966) and rice fields in Japan (Goto gt_al,, 1963). Sodium pentachlorophenate, the sodium salt of PCP has been used as a molluscacide for control of snails which serve as an intermediate host of human schistosomes (Barry §t_a1,, 1950). Agricultural applications of penta treated wood includes: treated posts and boards in the construction of fences, pens, feed troughs and bunk silos (Foss gt_al,, l980). Depending on the condition of the treated wood, its use in feed troughs presents a potential route for oral exposure to PCP. Similarly, applications such as on ceiling rafters and wall studs and boards provide a potential for inhalation of PCP. As with many other useful pesticides, PCP has also been misused. One such case was the inclusion of PCP in soy sauce as a preservative by a Japanese manufacturer (Narahu §t_al,, 1965). A pesticide with such wide and extensive use presents a potential risk to the environment. It was because of this suspected risk that pentachlorophenol was placed on notice of "Rebuttable Presumption Against Registration" (RPAR) by the Office of Pesticide Programs of the U.S. Environmental Protection Agency (Federal Register, 1978). Such a notice is issued when the EPA has determined there is evidence of sufficient risk in the use of a given pesticide to warrant a review of the advisabi- 1ity of its continued use and registration. Risk criteria are concerned with the following areas: acute toxicity; chronic toxicity (oncogenic and mutagenic); chronic effects such as reproductive (fetotoxicity, teratogenicity, spermatogenicity and testicular effects); significant reduction in wildlife, endangered species or nontarget species; and finally lack of an emergency antidote (Chemical Regulation Reporter, 1980). There are several RPAR categories: Pre-RPAR involves an investigational phase where all the toxicological information on a particular chemical is gathered and reviewed. This phase produces a preliminary regulatory position on the potential risks of a chemical. The document is referred to as a Position Document One (P01) and is published in the Federal Register with a formal Notice of Presumption Against Registration. The second stage or category involves issuance of the RPAR. This is a public process whereby interested parties are allowed to present rebuttals on the presumptions against registration. The period is usually about 105 days and if all risks are successfully rebutted, the chemical is returned to registration with the RPAR terminated for some or all of the uses. A second Position Document (P02) is published in the Federal Register and becomes the terminal document of the process. When the rebuttal is not successful, the rebuttal assessment, a risk-benefit analysis and a proposed regulatory position are published in a Position Document 2/3 (P02/3). Such a document has been released for penta (Federal Register, 1981). Review of the P02/3 document by various agencies and panels will produce a PD4 document which announces the final regulatory action to be taken on a given chemical. Chemical and Physical Properties of Pentachlorophenol Pure PCP is a white, solid, aromatic organic compound with needle- 1ike crystals (Bevenue and Beckman, 1967). Structurally it consists of five chlorine atoms (Figure 1). PCP is a weak acid with a pka of 4.74 (Table 1). As such, it reacts with strong alkali bases such as sodium or potassium Figure l. The Phenol and Sodium Salt of Pen tdchl orOphenol OH 0W5 CI 0 Cl Cl 0 Cl Cl Cl Cl Cl Cl ' Cl Phenol Sodium Salt TABLE 1 PHYSICAL CHARACTERISTICS OF PENTACHLOROPHENOL Molecular weight 266.36 Melting point 1900 C Boiling point 293° 0 Density 1.85 g/cm3 Vapor Pressure (20°-100° 0) 0.00011 - 0.12 mm Hg Solubility in water (20°-3o° c) 14 - 19 ppm pKa 4.74 (EPA, 1980) Bevenue and Beckman (1967) TABLE 2 SOLUBILITY OF PENTACHLOROPHENOL IN VARIOUS ORGANIC SOLVENTS Solvent g PCP/100g solvent (20-300 C) Methanol 57-65 Diethyl ether 53-60 Ethanol 47-52 Acetone 21433 Xylene 14-17 Benzene 11414 Carbon Tetrachloride . 2-3 Bevenue and Beckman (1967) -10- hydroxide to give the corresponding water-soluble salts (the sodium salt is shown in Figure l). The salts are highly water-soluble. At pH 8.0 the solubility is greater than 4,000 ppm (Myeling and Pitchford, 1966). As expected, the alkali salts are highly insoluble in most organic solvents. 0n the other hand, the phenol or protonated form is extremely soluble in most organic solvents (Table 2). Therefore,pH is a primary determinant of the dynamics and reactivity of PCP in a given environment. Moreover, pH is critical to the separation and analysis of PCP. Synthesis of Pentachlorophenol Pentachlorophenol is synthesized through the catalytic chlorination of phenol (Figure 2). Either ferric or aluminum chloride is the catalyst of choice. Analytical grade PCP (99% purity) can be produced in a chemistry laboratory. Synthesis of pentachlorophenol is a two-stage process which involves the catalytic chlorination of molten phenol. The temperature of the first stage is 1050 C and isomers of tri- and tetrachlorophenol are formed. The second stage involves an increase in reaction temperature and further chlorination of the tri-and tetrachlorophenols. This re- action is not quantitative. Tetrachlorophenols persist and are carried through into the end product (Cirelli. 1978). It is during this stage that the various dioxins and dibenzofuran congeners found in penta are formed from reaction intermediates. In an attempt to reduce the Chlorodioxin and dibenzofuran contamin- ants in commercial pentachlorophenol, Dow Chemical developed a process whereby significant amounts of the dioxin impurities are removed (Johnson gt_§l,, 1973). The process not only results in decreased contaminant levels, but also increased pentachlorophenol concentration (Table 3). -1]- Figure 2. Synthesis of Pentachlorophenol OH OH Cl Cl Cl Cl Cl Phenol PCP -12- TABLE 3 PENTACHLOROPHENOL AND DIOXIN CONTENT OF DON'S IMPROVED PENTACHLOROPHENOL (DOWICIDE EC-7R) Commercial PCP Improved Dow PCP Pentachlorophenol 85-90% 88-93% Tetrachlorophenol 4-8% 7-12% Trichlorophenol 0.1% 0.1% Higher Chlorophen01s 2-6% 0.1% Chlorinated dioxins Octa 575-2510 ppm 30 ppm (max) Hexa 9-27 ppm 1.0 ppm (max) Tetra 0 ppm 0 PPm Johnson gt a1. (1973). o I: v- \1) .-.A n,. .l d- -13- Depending on the manufacturer, penta is packaged in various physical forms including pellets, prills or one-half to one ton blocks. Penta is received at pressure-treating plants in bags, bulk and solid blocks The various forms are then dissolved in petroleum solvent to form a 5-7% solution which is then used in the pressure-treatment process (EPA, 1980). The Wood Preservation Process Some 38 million pounds or 78% of the penta produced in the U.S. is shipped to wood preservation plants. About 35% of this is used in treating lumber and fence posts. 62% is used on utility poles and cross arms and 3% used in treating miscellaneous types of wood products (Von Rumker _e__§_l_., 1975). Penta is purchased from the chemical manufacturer and formulated into a 5% solution with solvents such as kerosene, mineral spirits or Number 2 fuel oil (Cirelli, 1978). Methylene chloride has been used when the availability of other solvents was restricted. Wood to be treated with penta is debarked; cut into posts, poles or dimension lumber; conditioned, usually by kiln drying and then pressure- treated. Pressure treatment of hard wood species may be preceded by incising, a process in which the wood is pierced by knives to increase the penetration of the penta solution into the wood (EPA, 1980). Preservation methods can be categorized as either pressure or non- pressure processes. Briefly, the pressure significantly increases the penetration of the preservative into the wood, The non-pressure processes include diffusion, brush, dip, thermal and cold-soak methods. Approximately 95% of all treated wood is pressure treated (EPA, 1980). Two types of pressure-treatment systems are used, the "full cell" -14- Bethell Process and the "empty cell" Rueping or Lowry processes (EPA, 1980). The full cell process involves placing the dried wood in a cylinder in which it is subjected to a 22 inch vacuum for approximately 30 minutes. Then the penta solution is introduced under pressures of 50-259 psi and temperatures of 82-1040 C. After release of the pressure, the vacuum is applied to minimize bleeding or dripping. Bleeding may result when the low volatility solvents slowly migrate out and, in the. process, serve as a carrier for a certain amount of penta. The empty cell process differs only in that no vacuum is applied initially. Rather, two to four atmospheres of pressure are applied, followed by the release of a heated penta solution into the chamber from above so that there is no drop in pressure. Like the full cell process, a vacuum is applied as a final step to minimize bleeding (EPA, 1980). Additional treatments to reduce bleeding include an expansion bath and/or a steam flash. An alternative process is the Cellon method. It essentially involves replacement of the petroleum solvent carriers with liquefied petroleum gas (LPG). The advantage is that the LPG can be evaporated and recovered in a final step leaving only penta residue in the wood. As a result, the wood does not "bleed" because no solvent residue remains and the wood can be easily painted (EPA, 1980). Typical retentions achieved by the full cell process range from 20 to 30 pounds penta per cubic foot for most species, whereas the empty cell process results in retentions of 6 to 12 pounds penta per cubic foot (EPA, 1980). The Thermal Process is the most widely used of non-pressure processes. This method involves soaking the kiln-dried wood in a hot penta solution. In effect, a natural vacuum process is utilized which is created by the -15- expansion of the air in the wood cells in the hot penta solution and contraction when the wood is quickly transferred into a cold penta sol- ution, consequently, the cold penta solution is drawn into the wood (EPA, 1980). The pressure-type processes commonly utilize what are classified as low-volatility solvents: mineral spirits, kerosene or No. 2 fuel oil. Residues of the solvents remain in the wood at the conclusion of the treating cycle. A final cleanup step consisting of application of vacuum, expansion bath, or steam flash may be used. However, pressure processes often result in wood which may bleed considerably. Bleeding refers to the spontaneous migration of preservative onto the surface of the wood. Basically, it is a reversal of the pressure process. Consequently, when penta-treated wood is used in the construction of animal facilities on farms, the degree of exposure of animals by either the oral, dermal or pulmonary routes is directly related to the degree to which the surrounding wood is bleeding. Chlorinated Dioxin and Furan Contaminants in Penta A discussion of the various aspects of penta would not be complete without mention of its controversial contaminants. These contaminants are the primary reason penta is under scrutiny by the regulatory agencies. Much of the concern of the regulatory agencies stems from studies with 2,3,7,8-tetrach1orodibenzo-p-dioxin or 2,3,7,8-TCDD (Arsenault, 1976). However, 2,3,7,8-TCDD has never been found in any commercial PCP in the U.S. (Plimmer gt_al,, 1973; Firestone, 1973; Villanueva gt_al,, 1973). Therefore, the task is to evaluate the hazard of the higher- cthrinated dioxins found in penta which are hexa-, hepta- and octachlorodioxin (OCDD) as shown in Figure 3. -15- Figure 3. Common Dioxin Congeners Cl Cl Cl 0 Cl Cl 0 Cl 2,3,7,8 TCDD l,2,3,7,8,9 HCDD Cl Cl Cl Cl 0 \ 0 Cl Cl 0 Cl Cl Cl Cl Cl l,2,3,4,7,8,9 Hepto CDD OCDD -17- The amounts of the various dioxin congeners in a given sample of penta vary significantly with the analytical technique. A case in point is the industrial composite used in the current study which was analyzed by two separate laboratories for dioxins (Table 4). There are marked differences in the quantities of the various dioxin congeners. One reason for the difference in content of the congeners in a given sample can be found in the analysis itself. A critical step in the analysis of penta for dioxins involves an ion-exchange cleanup of the dioxin extract of the penta sample. Arsenault (1976) reported that the initial analysis of a sample using gas chromatography showed it to contain 2800 ppm OCDD. However, after using an ion-exchange cleanup step, which removes such minor contaminants as hydroxychlorodiphenyl ethers, octachlorodibenzofurans and hexachlorocyclohexanedione which co- chromatograph with OCDD, 1100 ppm OCDD was measured. Another aspect of dioxin analysis is the actual generation of dioxins during gas chromatography. The same high temperature conditions which generated dioxins during the synthesis of the penta are similar to those found in the injection port and column of the GC. Typically, both the injection port and the column oven operate at temperatures greater than 2000 C. Jensen and Renberg (1972) demonstrated that two PCP molecules can react in a two-step reaction under alkaline conditions t0'fin110CDD (Figure 4). The intermediate product, hydroxynonachloro- diphenyl, is termed a "predioxin". These authors developed a procedure to block conversion of the predioxin to the dioxin and in so doing, found that a sample which would have normally analyzed at 1100 ppm OCDD contained only 50 ppm OCDD. Others have also shown that the temperature -13- TABLE 4 DIOXIN AND CHLOROPHENOL ANALYSIS OF INDUSTRIAL COMPOSITE, TECHNICAL PCP AND DOWICIDE EC-7R Dioxin Dowicidea Technicala Industriala Industrialb Congener EC-7 PCP Composite Composite (ppm) (ppm) (ppm) (ppm) Octa 16.7 340 2450 1000 Hepta 1.26 37.1 340 378 Hexa 0.08 1.8 16.7 173 Tetra - - - 0.035C Chlorophenolsd (%) Pentachlorophenol 85-90 Tetrachlorophenols 4-8 Trichlorophenols 0.1 Other 2.6 a Analysis courtesy of Agriculture Canada, Ottawa, Ontario b Analysis courtesy of Pesticide Research Center, Michigan State University. Determined by HPLC with confirmation by GC/MS. C Does not include 2,3,7,8-TCDD d Reported by supplier (Roman, 1978). -19- Figure 4. Synthesis of Octachlorodibenzo- p- dioxin (OCDD) and Octachlorodibenzofuran (0CDF) OH 0 Cl Cl Cl Cl 0 + 0 Cl Cl Cl Cl Cl Cl c1 c1 A/Cl Cl Cl 0 O 0 Cl Cl 0 O 0 Cl Cl Cl H0 Cl Cl Cl Cl Cl Cl Cl Cl 0 O 0 Cl Cl _ Cl Cl Cl Cl Cl Cl \ '0 Cl Cl Cl OCDD OCDF -20- in the heated injection port of the gas chromatograph can contribute significantly to the overestimation of OCDD in penta. Therefore, it is important to know the method by which a sample of penta was analyzed for dioxins before judging its quality, especially if the concentration of OCDD is suspiciously high. Analytical data available on dibenzofurans in penta are limited. However, it is apparent that similar quantitation problems like those found with dioxins also exist. In other words, the same composite analyzed by different laboratories may differ markedly in concentration of the various dibenzofuran congeners. Toxicology of Chlorodioxins (COD) and Furans (CDF) in Penta The toxicity of COD and CDF found in penta is probably best brought into perspective by comparing them to the toxicity of penta- chlorophenol itself. The acute oral LD50 of pentachlorophenol in rats is reported to be 27-80 mg/kg depending on the solvent used (Dow, 1965). Hexachlorodioxin (HCDD) is somewhat less toxic with an acute oral L050 of 100 mg/kg (Schwetz gt_al,, 1971). There is n0' established LD50 for octachlorodioxin (OCDD). However, oral doses of 1000 mg/kg in male mice did not result in death (Schwetz gt_al,, 1971). These data on OCDD are reasonable since it is poorly absorbed. In rats fed OCDD at the rate of 100 mg/kg, 95% of the compound was accounted for in the feces, 4% in the urine and 1% each in the fat, liver and skin (Norback §t_al,, 1973). No acute toxicity data are available on the heptachlorodioxins, however, one would expect this group to be less toxic than HCDD. Therefore, on the basis of acute oral L050, HCDD is the most toxic dioxin in penta. HCDD has been shown to be acnegenic (i.e. causes acne-like -21- dermatitis) and teratogenic (Schwetz gt_al,, 1971). Teratogenicity was noted in pregnant female rats when fed from days 6 through 15 of gestation at the rate of 100 mg/kg/day. For a pregnant female rat weighing 0.2 kg this would amount to 20 mg of HCDD. If a value of 200 mg/kg is used as the level of HCDD in penta, the female would have to consume 100 mg of penta or 500 mg/kg body weight to produce terata in her offspring. This level of exposure is highly unlikely. Therefore, unless substantial subchronic preconception exposure to dioxins in penta occurred, the most likely outcome would be PCP intoxication. There is the possibility that some HCDD congeners may be carcinogenic. The National Cancer Institute recently reported the results of an oral dosing study in rats and mice and a dermal study in mice (Chemical Regulation Reporter, 1980). Both studies were conducted over a period of 104 weeks. HCDD caused increased incidence of hepatocellular carcinomas in female rats and increased hepatocellular carcinomas and adenomas in both male and female mice. However, HCDD was not demonstrated to be carcinogenic in male rats. Similarly, HCDD was not carcinogenic in dermal bioassays in either female or male mice. An additional concern regarding COD and CDF is the translocation of residues into produce from domestic animals which are exposed to penta. Firestone gt_§l, (1979) found three CDD isomers in milk, blood and adipose tissue of lactating cattle fed penta; these were l,2,3,6,7,8-hexa-CDD; 1,2,3,4,6,7,8-hepta COD and octa-CDD. In another study, yearling Holstein heifers were fed various mixtures of PCP and penta ranging from 100% analytical or pure PCP to 100% technical PCP at a dose level of 15 mg/kg body wt. per day (Parker gt_al,, 1980). These authors showed that COD and CDF concentrations in liver and adipose tissue were correlated with the -22- concentration of technical PCP in the diet. Moreover, the liver was shown to contain much higher levels of CDD's than adipose tissue. Some caution should probably be used when comparing the risks of dioxin and the dibenzofuran contaminants in penta. However the concentrations of the octa- and heptachlorinated dibenzofurans are similar to the concentrations of the octa- and hepta-chlorodioxins. Moreover, they appear to cause toxic effects similar to Chlorodioxins, but with a somewhat lower relative potency (WHO/IARC, 1978). Analysis of Pentachlorophenol Separation There are several key considerations when determining an acceptable method for analysis of a given compound. One of these is the method by which the compound of interest is removed from a biological matrix such as blood, urine or tissue. Clearly, the optimal separation method is uncomplicated, rapid and recovers the maximum amount of the compound. Because pentachlorophenol is a weak acid (pKa 4.74) manipulation of pH provides a valuable tool for separating pentachlorophenol from a bio- logical tissue or fluid. For instance, at pH 8.0 the solubility of the sodium salt of PCP is greater than 4,000 ppm in water (Myeling and Pitchford, 1966) and it is essentially insoluble in an organic solvent such as benzene or hexane. Conversely, at pH 2 essentially all of the PCP will be in the phenolic form and, as such, is virtually insoluble in any aqueous phase. Therefore, in extracting PCP from serum, the pH of an appropriate volume (1-2 ml) is adjusted to pH 2 and then the acidified aqueous phase can be extracted with an organic solvent. Because PCP is essentially insoluble in the aqueous phase at this pH, it will very efficiently partition into the benzene (upper) phase. A portion of the benzene -23- phase can then be analyzed directly by gas chromatography. If the concentrations of PCP are high enough, the PCP can be partitioned back into an aqueous phase which can then be analyzed by high performance liquid chromatography. Manipulation of the partitioning characteristics of PCP through changing the pH becomes even more useful in analyses of lipid-containing tissues. Few chromatography systems are compatible with analysis of . extracts containing lipid. Therefore, an efficient method to remove the lipid prior to the final analysis of the solvent extract of the tissue is necessary. Traditionally, some kind of stationary phase has been used to trap the lipid, leaving the solvent extract lipid-free and ready for analysis. The alternative to this is to make a homogenate of the matrix of interest, then alkalinize it with weak base solution, such as NaOH, to a pH of approximately 10. The lipid in the alkalinized homogenate can then be extracted with a solvent such as hexane. Once the lipid is extracted, the aqueous phase can be acidified and the PCP extracted back into an organic solvent which can then be analyzed on the gas chromatograph. The main advantage of such a clean up method is that it is rapid and economical because both the clean up step and final extraction are carried out in the same test tube. Detection Once pentachlorophenol has been extracted into an organic solvent, the problem becomes one of how to determine the concentration using the appropriate detection method or analytical instrument. (Bevenue and Beckman (1967) reviewed the various methods for detection of PCP; -24- colorimetry, spectrophotometry, and gas chromatography (GC). Basically, the colorimetric methods lack the sensitivity of the gas chromatographic methods and, as such, require large samples for analysis. Moreover, other chlorinated phenols and substances inherent to the various matrices cause significant interference problems. GC methods, however, do not have these problems. For samples that have been sufficiently cleaned up, current column packing (solid phase) technology provides effective separation of PCP from other phenols (Supelco, 1978). 63Nickel electron capture (ECD). This detector The detector of choice is has a high sensitivity to halogenated aromatics with detection limits of 3-10 ppb. Cheng and Kilgore (1966) reported a GC method in which PCP was converted to the O-methylated derivative (pentachloroanisole) using diazomethane. The pentachloro- anisole was then detected using electron capture, with a lower detection limit of 10 ppb. Using the same derivatization method and detector, but with a different column packing Bevenue gt_al, (1966) reported an even lower limit of 3 ppb. Other detectors have been used such as flame ionization (Barry et_al,, 1962; Koloff gt_al,, 1963; Smith gt_al,, 1964),hot wire (Kanazawa, 1963), microcoulo- meter (Yip, 1964) and thermal conductivity (Narahu, 1965). Until recently, derivatization of PCP with diazomethane has been the most desirable method to prepare PCP for gas chromatographic analysis. Derivatization of PCP as described by Bevenue gt 11. (1966) and Cheng and Kilgore (1966) using diazomethane prepared by the method of Aldrich Chemical Co. (1954) is still routinely used in the preparation of PCP extracts for GC-ECD analysis. However, this derivatization step has several drawbacks. First, both preparation and use of the diazomethane carry with it the risk of explosion. Second, the glass diazomethane generation kit is relatively expensive and requires -25- extensive care when being handled and cleaned. Dust particles or minor surface scratches provide sites for cystallization of diazomethane and the resulting potential for explosion. Finally, the derivatization requires a second set of glassware in which to carry out the methylation reaction. Because of the problems associated with derivatization using diazo- methane, successful efforts have been made toward developing other derivatives. Chau and Colburn (1974) have reported a method where PCP was extracted from sewage water into benzene. The PCP was then extracted from the benzene into potassium carbonate and subsequently acetylated with acetic anhydride. The PCP acetate was then extracted from the potassium carbonate solution into hexane and analyzed using GC- ECD. The authors reported a minimum detection limit of 0.177 ppb. More recently, a nonderivatized method has been reported (Supelco, 1978). The column packing used has had the “active sites" on the column support deactivated. Most column packings used in the separation of pesticides by gas chromatography are composed of a support, which is often silica coated with an organic compound. The coating process is not complete and as such, leaves some silica exposed. Because PCP readily binds to silica, the result is some~loss of the sample of the column before it reaches the detector. The new packing developed by Supelco (Bellfonte, PA) is the result of a process whereby the exposed silica or "active sites" have been deactivated chemically. Consequently, PCP can be efficiently separated using this column packing with a minimal I'on column loss" from binding to the silica Support. -26- Toxicology of Pentachlorophenol Exposure PCP from penta-treated wood can enter animals through one or all of the following routes: oral, dermal of pulmonary. Unfortunately, relatively little is known about exposure of domestic animals to PCP through any of the routes. Studies on pulmonary exposure are essentially non-existent. However, a recent study examined the effect of the type of solvent used to treat wood with penta on subsequent air concentrations (Thompson, 1978). Wood treated with penta dissolved in either heavy oil, liquefied petroleum gas (LPG) or methylene chloride was placed on a 30 liter test chamber at 300 C with an air flow rate of one liter per minute. The air levels of PCP associated with the various solvents were 0.02, 0.048, 0.76 mg/m3 for heavy oil, LPG and methylene chloride, respectively. The vapor density for PCP at 200 C is 0.00011 mm Hg (Bevenue and Beckman, 1967) which trans- lates to an equilibrium vapor density of 0.0017 mg/liter or 1.7 4 mg/m3. Although it must be kept in mind that this value is for analytical PCP and not penta which is 85-94% PCP, both the solvent and the wood appear to have a significant retarding influence on the volatilization of PCP. Most wood is treated with solvents such as kerosene, mineral spirits or No. 2 fuel oil, which are similar to heavy oil in terms of solvent polarity. Although no information exists on pulmonary exposure in domestic animals, one can estimate the pulmonary exposure of a cow. In a barn partially constructed from wood treated with penta in methylene chloride, the expected air level of PCP would be about 0.076 mg/m3 (Thompson, 1978). If a 514 kg cow breathes at the rate of 109 liters per minute, (the average of 104 L/min lying down and 114 L/m standing)(Respiration and -27- Circulation, 1971), in 24 hours she would have inhaled 150,960 liters of air or 156.9 m3. This translates to a dose of 0.006 mg/kg. Factors such as ambient temperature, barn .ventilation, proximity of the animal to the wood and extent of bleeding of the wood could significantly affect pulmonary exposure. Exposure to dioxins or furans in penta via this route would be insignificant due to the fact their vapor pressures are quite low (Anonymous, 1978). Dermal exposure is probably morecrfa problem in swine than in cattle because of lack of a substantial hair coat and usage of penta-treated wood for flooring which swine lie on. Documented cases of health problems in swine have occurred when sows were confined in farrowing crates construct- ed of wood recently treated with penta (Walters, 1952; Schipper, 1961). The new-born piglets were found to be especially sensitive, but when the treated wood was covered with plywood or bedding, the problem disappeared. Gastric exposure is a potential route of entry for both cattle and swine. Feed bunks and bunk silos, especially when they are constructed of recently treated or bleeding penta lumber, present an opportune site for oral exposure for cattle. Exposure can occur by either eating con- taminated food from the bunk silo or directly by licking the penta lumber in the feed bunk. Swine, on the other hand, could be exposed orally by eating out of penta-treated troughs, chewing on pen boards treated with penta, or rooting on pen floors treated with penta. Schipper (1961) reported high piglet mortality when sows and their litters were exposed to farrowing pens constructed of penta lumber. In this case, there was a strong indication that the piglets refused to nurse. This is in agreement with Deichmann §t_al,, (1942) who reported that when PCP was -28- included in the diet of rats their feed intake was reduced. Similarly, cats refused to eat salmon when it contained PCP (Deichmann gt 31., 1942). Also, cattle refused to graze pasture which had been sprayed with an emulsion of diesel fuel, water and penta (Grigsby and Farwell, 1950). However, it was not determined in this study whether the diesel fuel or the PCP was the reason for the avoidance. In summary, most studies have examined oral exposure of domestic animals to penta. Therefore, much remains to be done regarding the pulmonary and dermal routes of exposure and the toxic effects of the various solvents. Toxicokinetics of Pentachlorophenol Absorption Radiotracer studies, which have examined the kinetics of carbon- 14 pentachlorophenol (l4C-PCP), demonstrated that absorption of PCP, regardless of the route, is both rapid and extensive. Jakobsen and Yllner (1971) injected mice either subcutaneously or intraperitoneally 14 with C-PCP and were able to account 03085-89% of the dose in the urine, feces and various tissues. In another study, male and female rats were given a single oral dose of either 10 or 1000 mg/kg (Braun gt_al,, 1977). Again, by analysis of urine, feces and tissues these authors accounted for 99.8 and 97.6% of the dose in rats given 10 and l4 14 100 mg/kg C-PCP, respectively. Furthermore, plasma levels of C increased rapidly after ingestion and peaked within the first six hours. A sex difference was noted in that female rats absorbed PCP at a slower 14 rate, but had higher C concentrations in the plasma and almost every tissue at any given time, than did male rats. In a similar study with 14 male and female Rhesus monkeys given a single oral dose of C-PCP, the -29- 14C concentrations in the plasma also increased rapidly but required 12 to 24 hours to reach maximum concentration (Braun and Sauerhoff, 1976). A sex difference was also observed, however, in contrast to rats, the female monkeys absorbed PCP at a faster rate, but the 14C concentrations in the plasma peaked at a lower concentration compared to males. Male monkeys absorbed PCP at a somewhat slower rate and the peak 14C concentrations were higher than in the females. The rapid appearance of 14C in the blood and the high degree of absorption of pentachlorophenol are understandable in terms of gastric absorption. Most compounds cross both membranes through simple diffusion (Casarett and Doull, 1975). The current concept (Cohn, 1971) is that the cell membrane is a bimolecular layer of lipid molecules coated on each side with a protein layer. Therefore, movement across this structure is largely determined by the lipid-water partition coefficient of a given compound and secondly by the concentration gradient across the membrane (Casarett and Doull, 1975). Furthermore, many compounds are classified as weak acids or bases and as such can exist in solution in ionic and nonionic forms. For instance, the amount of a weak acid in the un- ionized form is dependent on its disassociation constant ( Ka) which is the negative logarithm of the acidic disassociation constant (Ka). Pentachlorophenol is classified as a weak acid with a pKa 4.74 (EPA, 1980). Consequently, at a low pH PCP is very lipid soluble (Table 2). Assuming that the pH of a Rhesus monkey's stomach is close to that of a human (pH 2) and applying the Henderson-Hasselbalch equation (pH = pKa - log unionized/ionized), one can calculate that the ratio of the unionized -30- or protonated form of PCP to the ionized form is 501 to 1. Conversely, if the pH of the intestine is approximately pH 6, the ratio of the' ionized to unionized forms is 20 to 1. Thus, under the latter condition much less PCP will be absorbed. Clearly, the absorption of PCP is favored in the more acidic environment of the stomach vs the environment of the intestine. The gastrointestinal tract of the ruminant presents a somewhat different situation in terms of absorption of PCP. Unlike the monogastric or simple-stomach animal, the ruminant has four stomachs or compartments prior to the intestine: the rumen, reticulum, omasum and abomasum. All differ in pH, the most basic being the rumen at pH 6.5 and the most acidic being the abomasum at pH 3 (Phillipson, 1977). Thus, the abomasum would be the most favorable environment in which PCP could be absorbed and the rumen the least favorable. _Distribution There are two important aspects associated with the distribution of PCP: its affinity for binding to serum proteins and the pattern of distribution in the various tissues of the body. Blood is the means of distribution for most compounds absorbed from the stomach or intestines. Depending on its chemical characterisitcs, a compound may become associated with one or several components of the blood. The proteins in the blood, particularly albumin, can act as a storage depot for many foreign compounds (Casarett and Doull, 1977). The first study to examine the binding relationship of PCP and plasma proteins utilized an ultrafiltration membrane system (Braun gt _l., 1977). The procedure involved adding. 5 ml of rat plasma to the untrafiltration membrane, which retained the protein components, plus -3]- PCP at l or 100 mg/ml. When the loaded system was washed with phosphate buffer, the ultrafiltrate contained no 14C. The authors concluded that the 140430 on the membrane was most likely due to PCP bind- l4 retention of the ing to plasma proteins. At 900 mg PCP/m1 94% of the C-PCP was retained, indicating that even at very high concentrations of PCP its binding is extensive. Through similar types of experiments these workers also charac- terized the binding to be of a heterogeneous nature and the molar binding affinity constants in bovine serum albumin (BSA) were found to be 0.66 x 106 per mole and 2.1 x 106 per mole for K1 and K2, respectively. It has been demonstrated, in studies examining the effects of the molar binding affinities on the properties of drugs, that protein binding becomes a significant property of a given drug when its affinity constant(s) is 104 or greater (Keen, 1971). The effects of temperature and pH on PCP binding to BSA have also been studied (Hoben §t_al,, 1976a). When a dialysis bag system was used in'conjunction with increasing concentrations of PCP against a fixed amount of BSA, these authors found that a linear relationship exists between the PCP concentration and the amount of PCP bound per milli-l gram of BSA. Moreover, by varying the BSA concentration with a fixed concentration of PCP they found that as the protein concentration increased the amount of PCP binding decreased. The authors also suggested these results indicate that PCP binding to BSA is of a heterogeneous type. The number of binding sites for PCP on BSA was determined to be thirteen. An inverse relationship between pH and binding of PCP to BSA was demon- strated. Two explanations were suggested, both supporting the idea that the unionized species is the one involved in binding. First, as the pH approaches the pKa of PCP (4.74) the proportion of unionized -32- species increases, as does binding. Similarly, as the pH increases so does the net negative charge of the protein and the amount of ionized PCP, resulting in a repulsion by the negative charges of each other and a concomitant decrease in binding. Moreover, binding was directly related to temperature, i.e. as the temperature was increased from 5°C- 40°c so did the amount of PCP bound. The amount of PCP bound to albumin is nearly the same in the cow, rat and man (Hoben et_al,, 1976a). However, in a comparison of rat and human plasma, both of which can be about 50% albumin, the amount of PCP bound in human plasma was twice that in the rat. The authors suggested that other factors, in addition to albumin, affect retention of PCP in plasma. These results agree with earlier observations that at the same dose, humans had significantly higher blood PCP concentrations than did rats (Casarett gt _l., 1969; Hoben gt_al,, 1976a). Studies examining the distribution of PCP in the various body tissues and fluids of different species generally show the same pattern. In a study carried out with mice, the highest concentrations of 14C activity were found in the liver and the intestines, followed by the kidney, heart and lungs. Whole body autoradiograms taken four hours after injection showed the greatest 14C concentration in the fundus wall of the stomach and in the intestinal contents (Jakobsen and Yllner, 1971). The high 140 content in the wall of the stomach would tend to suggest that this is a major site of absorp- tion for PCP. However, these workers suggested that this is also evidence of secretion of PCP in the gastric juices. Larsen gt_al, (1972) dosed rats -33- 14 with C-PCP and found the highest concentrations of 14C in the liver followed by the kidney, blood, stomach, adrenal and lung. Adipose tissue and muscle had the lowest, and nearly equal, concentrations of 14C. Similar results were obtained when male and female rats were 14 dosed with C-PCP (Braun g _a_1_., 1977). They found ”c primarily distributed, in decreasing magnitude, in the liver, kidney and plasma, with brain and adipose tissue having the least. The preferential re- 14 tention of C in the plasma was suggested to be primarily related to the strong tendency of PCP to bind to plasma protein. Almost every tissue, as well as the plasma, in female rats had a greater concentration 14 of C than did the male rats. In contrast, Larsen gt_al, (1972) reported no observable sex differences, in terms of tissue or fluid 14C, in rats. In'a study in which male and female 14 concentrations of Rhesus monkeys were given a single oral dose of 10 mg/kg C-PCP, the percentage distribution of 14C was: liver, 1.1%; small and large intestine, 7.6%; and the remainder of the tissues, 2.76% (Braun and Sauerhoff, 1976). Carbon-14 in the urine and feces accounted for 70 and 18% of the dose, respectively. The only study examining PCP residues in milk, to date, involved lactating dairy cattle which were fed 10 mg penta per kg body weight per day for 60 days (Firestone et 11., 1979). The PCP concentration measured in the milk, at steady state, was 4 ug/ml (ppm). Metabolism The metabolism of PCP has been relatively well defined in several mammalian species. With the exception of the mouse, most studies show PCP is excreted largely unmetabolized in the urine, with the metabolites -34- being tetrachlorohydroquinone (TCH) and the glucuronide conjugate of TCH and PCP. 14C-PCP metabolism in mice, about 3 % of the In a study examining dose was excreted unchanged in the urine as PCP (Jakobsen and Yllner, 1971). Upon acid hydrolysis, this value increased by about 8% indicating the presence of some type of conjugate. Approximately 21% of the 14C in the urine was identified as tetrachlorohydroquinone (TCH). An accurate estimate of the amount of conjugated TCH was not available, due to lack of sample. After an acid hydrolysis step, they found the 14c-TCH and 14C-PCP accounted for all of the 14C present. Similarly, Braun §3_ 14 91, (1977) reported that most of the 14 ‘ C in the plasma of rats administered C-PCP was unchanged PCP, with only a small amount present as the glucuro- nide conjugate. These authors further determined that 14C-PCP and 14C- tetrachlorohydroquinone accounted for 75% and 15% of the administered 140 in the urine 14 dose in the urine, respectively. The remainder of the (9.4%) after treatment with glucuronidase was shown to be C-PCP glucuronide (Figure 5). These authors reported if the urine were not extracted to remove both PCP and TCH, they would inhibit the glucuronidase hydrolysis of the conjugate. Ahlborg gt_al, (1974) also reported that TCH inhibited hydrolysis of the PCP-glucuronide by glucuronidase. Braun and Sauerhoff (1976) found that in male and f14 14 . female monkeys given a single oral dose 0 C-PCP, all of the C 1n the urine was PCP. Ahlborg gt_al, (1978) further characterized the metabolism of 14C-PCP and its metabolites in urine. They reported the percentage 14 distribution of the C-PCP dose in the urine to be 60% PCP; 9-16% PCP-glucuronide and 16-22% TCH-glucuronide. The -35- Figure 5. Metabolism of PCP in the Rat _1 OH ‘ C1 C1 > C1 Cl —*>PCP-glucuronide-*> Cl Pentachlorophenol (PCP) OH Cl Cl 5 (:1 Cl -—>TCH-glucuronid_e -1> OH Tetrachloro-p-hydroquinone (TCH) -I>Exc retion -35- liver enzymes involved in metabolism of PCP to TCH are inducible with phenobarbital (Ahlborg gt_al,, 1978). These workers characterized the effects of phenobarbital,a.known inducer of hepatic microsomal enzymes (Conney, 1967), on the metabolism of PCP. The effects of SKF 525-A, a known inhibitor of several microsomal enzymes (Anders, 1971), were also examined. Rats pretreated with phenobarbital showed a 6-fold increase in conversion of PCP to TCH in the first 24 hours, whereas rats pre- treated with SKF 525-A once or twice showed 3- and 2-fold increases, respectively. Rats treated repeatedly every 6 hours for 24 hours with SKF 525-A did not show an increased conversion of PCP to TCH. Further jg_yjtgg_studies using liver microsomes from phenobarbital-pretreated rats showed an approximate 3-fold increase in conversion of PCP to TCH. 14c-PCP was incubated with microsomes from SKF 525-A Conversely, when pretreated rats, there was a marked inhibition of conversion of PCP to TCH. Penta contains up to 12% 2,3,4,6 tetrachlorophenol (Johnson gt_ ‘11., 1973). Ahlborg and Larsson (1978) have shown that this isomer is excreted essentially unchanged in rats. Excretion The main route of excretion of pentachlorophenol appears to be urinary, with the remainder being fecal. Jakobson and Yllner (1971) 14 reported results of an experiment where C-PCP was administered sub- cutaneously and intraperitoneally to mice. They found that 73 to 83% of the dose was excreted in the urine within 4 days and 8 to 21% in the feces. Only traces (0.05%) were expired in the air. In another study, f 14 female mice were given oral doses 0 C-PCP (37 to 41 mg/kg); 68% and -37- 9-14% were recovered in the urine and feces, respectively, after ten days (Larsen §3__l,, 1972). Again, less than 0.04% of the dose was detected in the respired air. They concluded that the urinary excretion of pentachlorophenol is biphasic, the first phase having a half-life (ti) of 10 hours and the second, a t% of 102 days. These values were obtained by plotting body-burden over time. The body-burden was deter- mined by subtracting the cumulative amount excreted in the urine from the 14C dose initially administered. Apparently, there are problems associated with this method in that it may cause errors in estimation of elimination rate unless one can account for nearly all of the dose originally administered. Thus, the 102 day half-life for the second component of elimination reported by these authors is suspect. Ahlborg gt_al, (1974) found approximately 80% and 50% of the 14C activity in the urine of rats, 24 hours following intraperitoneal and oral administration, respectively. The metabolism and excretion of 14C-PCP were evaluated in female and male rats using single oral doses of either 10 mg/kg and 100 mg/kg (Braun et_al,, 1977). Males given 10 or 100 mg/kg and females given 10 mg/kg excreted about the same percentage in the urine (72-80%) and feces (l9-24%). However, females given 100 mg/kg excreted significantly less in the urine (54%) and more in the feces (43%). These authors proposed a two-compartment 14 open model to describe the elimination of C-PCP in rats. The model is comprised of a central, or fast-exchange, compartment from which there is rapid elimination. The half life of elimination (ti) in males given 10 mg/kg 14 C-PCP was 17 hours, and 13 hours in females given 10 mg/kg, or males given 100 mg/kg. A slow-exchange compartment has a slow rate of elimination, with a t% of 40 hours for males given 10 or 100 mg/kg - 38 - 14C-PCP, 121 hours for females given 10 mg/kg and 33 hours for females given 100 mg/kg. They suggested two possible reasons for the existence of the second, or slow-exchange compartment: 1) radioactivity is retained in the liver, where most of the radioactivity in the body remains at later times after administration, and 2) binding of PCP to plasma proteins. These two factors, in combination, may also be the cause of the long half life of PCP in urine, where small amounts are excreted over long periods. The influence of plasma-protein binding on the elimination rate is evidenced by a renal clearance rate of 0.138 ml per minute (3% of the glomerular filtration rate in a 2009 rat) (Renken and Gilmore, 1973). They suggested this indicates that either the 14C-PCP is extensively reabsorbed or a large fraction is strongly bound to plasma protein and not filtered. In another study, 3 male and 3 female Rhesus monkeys were each given a single oral dose of 10 mg/kg 14C-PCP (Braun and Sauerhoff, 1976). Male monkeys excreted 75% in the urine and 12% in the feces whereas, in female monkeys, urinary excretion was 70% and fecal excretion was 18 percent. These authors suggested that although there was no direct evidence of biliary elimination of the 14C, that the 14C in the feces most likely represented unchanged PCP or metabolites secreted into the bile. Moreover, they also proposed that biliary elimination is supported by a slow, but steady elimination of 14C in the feces. In contrast, unabsorbed PCP would have been eliminated as a bolus in the first 24 hours. These authors also demonstrated that excretion of PCP by the kidneys is a first-order process, i.e. the rate of excretion is Prtuaortional to the concentration of PCP in the blood. Renal clearance was shown to be 14.5 ml/min which according to the authors suggests that -39- PCP is not actively transported into the tubular filtrate, nor reabsorbed at the same site, but is filtered by the glomerulus. Further- more, they could not find any evidence of saturation of the excretion mechanism. Toxicodynamics of Pentachlorophenol Mechanism of Action Pentachlorophenol's toxicity to a wide variety of organisms ranging from bacteria to mammals is based on its ability to interfere with the biochemical energetics of the cell. Experiments aimed at uncovering the enzymatic basis for PCP's molluscacidal activity led to the demonstration that minute concentrations of PCP completely inhibit the coupling of oxidation to phosphorylation in both rat and snail mitochondrial preparations (Weinbach, 1954, 1956). Substrate-level phosphorylation (glycolysis) was shown not be be interrupted by the same PCP concentra- tions which uncoupled oxidative phosphorylation. This author was the first to suggest that PCP's effect on oxidative phosphorylation in_ yjtrg_was the basis for the increased respiration and glycolysis observed in living snails. Subsequent in_yitrg_studies on rat liver mitochondria demonstrated that PCP is tightly bound to mitochondrial proteins (Weinbach and Garbus, 1965). These authors proposed that the protein binding was a major factor in the uncoupling phenomenon. They showed that oxidative phosphorylation, when interrupted by PCP in mitochondrial iareparations could be effectively restored by addition of albumin to the irn:ubation medium. The ability of PCP to strongly bind to albumin has beer: documented by other workers (Hoben gt_al,, 1976a ; Braun et_al,, 1977). -40- PCP has also been shown to be a selective inhibitor of P-450, the terminal oxygenation enzyme in liver microsomes (Arrhenius gt_al,, 1977). These authors suggest that the inhibition is due either to a specific attack on the P-450 enzyme itself or to a disturbance of the transfer of electrons from the flavin enzyme (NAD) to the P-450 enzyme. Studies on Domestic Animals The first attempt to examine the toxicity of pentachlorophenol in livestock involved the spraying of a diesel fuel-water emulsion containing penta on pasture (Grigsby and Farwell, 1950). They found that cattle avoided the sprayed areas. However, since the penta was applied at 2- 4 times the recommended amount and in diesel fuel, it is difficult to conclude whether diesel fuel or penta caused the avoidance behavior. In another study, swine were dosed with 35 grams of a solution containing 5% PCP, 5% diacetone alcohol and 95% mineral spirits (Walters, 1952). This single dose resulted in blood levels of 42 ppm. PCP-concentrations in urine and fecal samples were 184, 6.0 ppm (24-h) and 212, 30.6 ppm (48-h), respectively. Tissue samples, taken at necropsy had the following PCP concentrations: 0.5 ppm (fat), 6 ppm (kidney), 5.8 ppm (liver) and 0.5 ppm (muscle). No observable deleterious effects were noted in these animals. In two other experiments, the sows and their piglets were con- fined to either farrowing houses treated with PCP on the outside or, in a second experiment, farrowing houses in which both the outside and inside were treated with a 5% solution of PCP in mineral spirits (Walters, 1952). The newborn piglets were in the farrowing house from 2-8 weeks of age. None of the animals showed any detrimental effects, with respect to health or growth. However, there was no mention of the amount of ventilation and no blood PCP measurements were made. In contrast, Schipper (1961) observed increased mortality of fetuses and newborn piglets and birth -4]- of weak piglets when pregnant sows were confined in penta-treated farrowing pens. Moreover, nursing was decreased, possibly because of contamination of the udder with preservative, resulting in undernourished piglets. This agrees with the finding of Deichmann gt_al,, (1942) and others who have observed that both the odor and taste of penta dis- couraged eating. Schipper (1961) also found that, in some cases, piglets consumed enough PCP to result in severe toxicosis. Lesions in the liver, spleen, stomach, intestinal and respiratory tracts, kidneys and urinary bladder were seen at necropsy. In general, toxicosis was most severe with insufficiently dried lumber treated with penta. Also, the mortality rate of piglets at birth closely coincided with the length of time the sow was confined in the penta-treated farrowing pen prior to farrowing. Moreover, the age of the piglets, at the time of exposure was also inversely related to the degree of toxicity (i.e. the older the animal, the less severe the effects). Inclusion of bedding in the farrowing pen prevented the problem. Unfortunately PCP concentrations in the serum were not reported. In a clinical investigation reported by Blevins (1965), the owners of a newly-constructed farrowing house had overtreated the floor with a solution of PCP in used engine oil. Exposure to the preservative, in combination with poor ventilation and no bedding resulted in the death of nine newborn piglets. The author suggested that the piglets were potentially exposed via all three routes: oral, dermal and pulmonary. Harrison (1959) measured PCP concentrations in the blood of sheep after they were force-fed sawdust impregnated with pentachlorophenol . Results indicated that PCP was rapidly absorbed from the gut, since max- imum PCP concentrations blood were attained within 3-6 hours after dosing. This author observed a wide variability in blood concentrations -42- and individual animal responses resulting from various doses. When two animals were given a dose of 139 mg/kg, one lived for 24 days while the other died in 12 hours. Several studies examining the effects of penta and analytical pentachlorophenol on mixed cultures of rumen microbes indicate that both preparations are effective inhibitors of cellulose digestion (Shull gt_al., 1977; Shull and McCarthy, 1978). These authors also demonstrated that the j_n_ v_i_t_rg inhibitory effect was due to pentachlorophenol and. not the impurities in penta. The significance of this 12.!i39. is not yet clear. However, during the course of the subchronic study reported by this author, the digestibility jg_yiyg_of several feedstuffs was measured using an intraruminal nylon bag technique. McConnell gt_al, (1980) fed four groups of yearling female Holstein cattle either analytical PCP, penta (technical PCP) or various mixtures of the two for 160 days. The objectives were to determineiflwetoxic effects of long term exposure to analytical PCP and the influence of the dioxin contaminants in penta. The initial dose regime of 20 mg/kg body weight per day was decreased to 15 mg/kg per day after 42 days because of body weight loss in all cattle fed pentachlorophenol. The treatments were: 100% analytical PCP, 10% technical PCP (90% analytical), 35% technical PCP (65% analytical), and 100% technical PCP (penta). These authors observed a dose-related decrease in body weight, decreased feed efficiency and progressive anemia. These same cattle had dose-related increases in liver and lung weights and decreased thymus weights. Pathological findings included a marked villous hyperplasia of the urinary bladder mucosa in animals exposed to 100% technical PCP and hyperplasia of the gall bladder -43- and bile duct in some animals. The group exposed to 100% analytical PCP was essentially comparable to control animals. In Summary, there is little information regarding effects due to low-1eve1 exposure to PCP, chronically or subchronically, in domestic animals where accurate daily dosing was maintained and blood and urine PCP concentrations measured. Also, the effects of such exposure on production and health have not been documented. McConnell gt_al, (1980) also reported a decrease in triiodothyroxine and thyroxin (T3, T4) concentration in all animals exposed to both analytical and technical PCP. They proposed no definitive explanation; however, the results suggest that PCP itself is the causative agent. Hepatic mixed funtion oxidase activities were measured and included: aryl hydrocarbon hydroxylase (AHH), aminopyrine N-demethylase and cytochrome P-450. AHH activity was increased 3-fold in the analytical PCP-fed group over the control group whereas, the group given technical PCP had an AHH activity 5-fold greater than the controls. Only the group fed 100% technical PCP had a small, but significant increase in aminopyrine-N- demethylase activity over that of the control group. Immunologically, there was a demonstrable dose-related enhancement in the lymphoproliferative response, an ig_vitro correlate for cell-mediated immunity. Furthermore, a hyperkeratotic lesion of the lining of the Meibomian glands of the eyelids was also found to be dose related. The results of this study led the authors to propose that the toxic effects associated with chronic exposure to technical PCP are primarily associated with the dioxin or dibenzofuran contaminants. A noteworthy aspect of this study was the method used for administering the penta dose. A premix containing penta -44- was mixed with the basic diet so that each animal received 1 kg of feed per 32 kg body weight. The weight of the feed not consumed was recorded daily. However, there was no mention in the report as to whether there were rejections. Consequently, whether the animals were consistently exposed to 15 mg/kg per day of pentachlorophenol is questionable. Studies in Other Species Information in the literature on the effects of low dose, chronic or subchronic exposure via any of the three routes of exposure in experi- mental animals is limited. In the case of oral exposure,the reason is that PCP contaminated food for most animals is very repellent (Deichmann gt 31,, 1942). Consequently, the feed intake decreases with a concom- itant decrease in body weight. Kehoe gt_al,(l939) reported that dermal application of PCP, at levels low enough to avoid gross skin damage pro- duced no chronic systemic effects in rabbits. However, the information available on low dose, chronic exposure through any or all of the possible routes of exposure indicates there are no readily definable health effects. PlakhOVa (1966) reported several toxic responses of animals (species not stated) exposed via the pulmonary route to PCP (23 mg/m3 of air). for 4 hours per day for four months. The observed responses included: decreased body weight, hemoglobin content, and erythrocyte count. Also, an increased number of eosinophils and leucocytes was noted. However, a concentration of 7.44 mg PCP/m3 resulted in unstable, but insignificant changes in these same hematologic measurements. The results of an acute inhalation study with PCP in rats showed the LD50 to be 11.7 mg/kg body weight (Hoben gt_al,, 1976b). This is considerably less than -45- the acute oral LD50 in rats of 150 mg/kg (Schwetz gt_gl,, 1973). Knudsen (1974) administered PCP to female and male rats for a period of 12 weeks at doses of 9, 25, 50 and 200 mg/kg in their feed. Histo- pathologic examination of the kidneys indicated a dose-related decrease in calcium deposits. This author suggested that this was possibly due to a lowering of blood calcium through an indirect action of PCP on calcium metabolism. Laborers in Japan chronically exposed to PCP had elevated levels of total bilirubin and creatinine phosphokinase (Takahashi _t._l,, 1975). However, the authors noted that the values were within the normal limits. An epidemiological survey of occupationally-exposed workers showed them to have higher than normal serum levels of the liver enzymes, aspartate aminotransterase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LH) (Klemmer, 1972). However, the author did not speculate as to the clinical significance of these findings. Arsenault (1976) reviewed the results of an epidemiological study conducted by the University of Hawaii's Biomedical Research Center at Monoa which found no readily apparent long term chronic effects from exposure to pentachlorophenol. Considering the wide variety of uses of pentachlorophenol, one would expect some degree of chronic exposure in the general population. Survey data accumulated by Cranmer and Freal (1970) tend to support this expectation. They found urinary levels in the general population ranging from 6 to 11 parts per billion. Casarett gt 31. (1969) suggested that pulmonary exposure is a reasonable explanation for the existence of measurable PCP levels in the general population. Much of the survey per- formed by this group was conducted in Hawaii where penta-treated wood is commonly used to prevent termite infestation. ~46- Goldstein et_al,, (1977) examined the hepatic effects of technical grade and analytical grade PCP in rats. Technical PCP fed at 20 ppm in the diet produced hepatic porphyria and increased the following parameters: hepatic aryl hydrocarbon hydroxylase activity, liver weight, cytochrome P-450 activity, and microsomal heme content. These workers suggested that these effects were due to the contaminants. Analytical PCP fed at 550 ppm in the diet resulted in decreased body weight gains, a slight increase in liver glucuronyl transferase activity and pigmentation of the liver. RESEARCH OBJECTIVES Two experiments were undertaken to examine the effects of technical pentachloroohenol in lactating dairy cattle for the follow- ing reasons: 1) Information from the dairy industry indicated that production and health problems in several dairy herds were related to exposure to penta-treated wood. 2) Milk samples from some of - these herds had measurable PCP concentrations. 3) A perusal of the literature indicated that little was known about the effects of sub- chronic penta exposure in lactating dairy cattle. In an effort to provide information with regards to possible production and health problems, in addition to tissue and milk residues of PCP, two experiments were undertaken with the following objectives in mind: Experiment 1: To determine the effects on performance and health in lactating dairy cattle fed technical PCP subchronically. 14 Experiment 2: To determine the fate of a single dose of C-PCP in a lactating dairy cow previously fed technical PCP subchronically. -47- RESEARCH METHODS AND PROCEDURES Animals and Diets Eight mature Holstein dairy cattle were randomly allotted as pairs to either a control or treatment group. Cows were paired on the basis of stage of lactation. Rumen cannulas (10.2 cm, i.d.) were surgically installed hitwo of the four pairs, 5-6 weeks pre-partum. Each pair of animals was started on experiment, 6 t 1 weeks postpartum. All cattle were housed in portable tie stalls located in the toxicology wing of the Michigan State University Dairy Cattle Teaching and Research Center. In addition, each animal had water available on an ag_1ibitum basis. The basal diet consisted of alfalfa haylage and high moisture corn, supplemented with 38% protein concentrate added to a rate of one pound per three pounds of milk production. The total ration was maintained at 14.7% protein, 47% dry matter, 1.85 Mcal net energy/lb dry matter, 0.5% Ca and 0.39% P. Composition of Technical Pentachlorophenol The penta was a composite sample from three manufacturers of technical pentachlorophenol. The dioxin content of the composite penta was determined by Dr. M.J. Zabik using high-performance liquid chromatography (HPLC) according to the method of Pfeiffer (1976) and is given in Table 4. The method involved dissolving 4 grams of penta in 50 ml of 1N NaOH which was then combined with 10 m1 of benzene in a 100 ml separatory funnel and the contents shaken for 20 minutes. The aqueous phase was re-extracted with another 10 ml of benzene. The extracts -48- -49- were combined and placed on an anion-exchange column (Bio-Rad AG-21R, hydroxyl form; 17.8 mm x 16 cm glass column) and eluted with 150 m1 of benzene-methanol (1:1). The column effluent was evaporated to 10 ml and washed 3 times in a separatory funnel using 1N NaOH. The 10 ml of solution was then brought to 100 ml with hexane. The dioxin congeners in the solution were quantitated using a high-pressure liquid chromatograph (Altex pumps, Rheodyne valve, Hitachi variable wave length UV detector and Altex microprocessor). Congeners were separated by using two Du Pont RP-18 columns in series. The solvent system was absolute methanol with a flow rate of 1 ml/min. Chlorodioxin standards included 100 mg/liter solutions of Octa-CDD; 1,2,3,4,6,7,8-hepta-CDD; 1,2,3,4,6, 7,9-hepta-CDD; l,2,3,6,7,9-hexa-CDD; l,2,3,6,7,8-hexa-CDD; 1,2,3,4,6, 8-hexa-CDD, and 2,3,7,8-tetra CDD. Except for tetra-COD, quantitative results were based on peak areas for each CDD congener in comparison with standard curves for that congener. The quantitation of tetra-COD peaks was based on the assumption that the relationship of detector response to concentration for tetra-COD congeners is the same as that .05). All cattle in the treatment group readily consumed the penta con- centrate. Although treated cattle in this study consumed less total feed than did control cattle (Figure 7a), when the data were expressed on a body weight basis the amount of feed consumed per day by each group was equivalent (Figure 7b). I The cattle fed penta tended toward a more efficient conversion of feed to milk, i.e. they produced more fat-corrected milk (FCM) per Mcal (NEL) of feed consumed (Figure 7c). However, this effect was signifi- cant (P < .05) only through 14-28 days in the 2 mg/kg dose period. Less total milk was produced by treated cattle, but the difference was not significant (P > .05) (Figure 8a). Results of somatic cell measure- ments on milk collected during the last week of the second treatment period indicated that the penta-fed cattle did not have cell counts (number of cells/m1 t SE) different from that of control cattle (control, 180,917 i 8,374; treated 20,042 : 121,784). There was no significant difference (P > .05) in milk fat production between control and penta fed cattle -70- -71- Figure 6. Time Course Status of Body Weight of Cattle Fed 0.2 Mg Penta/Kg Body Weight/Day for 75 to 84 Days Followed By 2 Mg Penta/Kg Body Weight/Day for 56 to 60 Days. 700 r ’6 600 - 35 E l g 5001’ 9‘ A/é f\é——Q/ A 1c? 0—0 Control a: A—A Treated 400 '- <—O.2rng/kg/ddy —>+02mg/kg/doy>1 /L l l L L l 1 l 1 J 0 I4 28 42 56 7075-8414 28 42 56-62 Days Fed Pento Figure 7. -73- Time Course Status of Daily Feed Intake of an As-Fed Basis (a), Ratio of Intake to Body Weight (0), and Ratio of Fat Corrected Milk Produced to Megacalories Consumed (c) in Cattle Fed 0.2 Mg Penta/Kg Body Weight/Day for 75 to 84 Days Followed by 2 Mg Penta/Kg Body Weight/Day for 56 to 60 Days. Each Graphed Point Represents the Mean of All Data Collected During the 14 Day Period Preceding the Specified Number of Days Indicated. 4:. (3 Kg Feed Consumed/Day on o a m— -74- ALI \\/I’I. ’ I o—-—o Control A—‘ Treated . L I l l \ > 080 B 8 E ,\_ .070 a 5. 5'3; 060 0 3 81>; 050 0 o ”- an 040 O—-0 Control fg‘ a ' A—ATreaied :2 l? L, L, 1 1 1 1 1 .1 l c. ‘8’. 1.6]- 2 1.2 2 g '0 I o——0 Control 5 .90 44—4 Treated m . .70 - \ /z’;\I-I Q 50 i ' j:- 0. 2 mg/kg/day —>-1—2 Omg/kg/dayu O 14 28 42 56 7075‘8414 28 42 56 62 Days Fed Penta Figure 8. -75- I Time Course Status of Total Milk Production (a) and Milk Fat Production (b) in Dairy Cattle Fed 0.2 mg Penta/kg Body Weight/Day For 75 to 84 Days Followed by 2 mg Penta/kg Body Weight/Day for 56 to 60 Days. Each Graphed Point Represents the Mean of all Data Collected During the 14 Day Period Preceding the Specified Number of Days Indicated. Kg Milk Fat Produced/Day Kg Milk Produced/ Day -75- ! l l l L 14 28 42 56 70 758414 a. 30 l. H COl‘l‘erl A—‘Treated 25% 201’- r1” 1 5 - \I-‘g/ l O - <—o.2 mg/ kg/day —el<- 2.0 mg/kg/dayDl 1 1 1 1 1 1 1 1 1 l b. HC’ontrol A—‘Treaied L4 " - 1 1.21 1.0 I .80 \I\ g \ l L l I 28 42 566 Days Fed Penta -77- (Figure 8b). EffectscniAppearance and General Health In general, all cattle appeared normal throughout the entire period of exposure. However, some treated cattle had problems, not observed in control cattle, but they were not believed by the investigators to be treatment related. One treated cow contracted mastitis early in the experiment and eventually milk production ceased in two quarters even though antibiotic therapy was administered. Also, two treated cattle periodically contracted a health condition characterized by elevated body temperature, mild anorexia and decreased milk production for one or two days. The cause of the condition was never diagnosed. The physical examination conducted one week prior to necrOpsy revealed no abnormalities in any cattle, other than that the papillae in the buccal caVity of two of the treated cattle were noticeably eroded. Effectscn1C1inical Chemistry, Hematology and Urinalyses There were no biologically significant differences in the results of hematologic and blood chemical measurements that could be attributed to the subchronic administration of penta (Appendix,pg 140. Also, no stat- istically significant differences between groups were found in the urin- alyses obtained on subsamples from samples collected one or two weeks before necr0psy (Appendix, pg 145). Histologic Examination of Tissues The liver, lungs, kidneys and adrenals represented a significantly greater percentage of the body weight in penta-fed cattle than in control cattle (Table 6). Macroscopic examination of these and other organs _ ~78- Table 6. Organ Weights of Control and Penta-Treated Cattle Expressed as a Percent of Body Weight . Qgggn_ Control Treated '7 ::SE 7 i:SE liver 0.63 0.03 0.86 0.07b lungs 0.31 0.01 0.40 0.07b kidneys 0.106 0.004 0.140 0.009b spleen 0.082 0.005 0.094 0.004 heart 0.196 0.011 0.219 0.007 thyroid 0.003 0.001 0.003 0.001 adrenals 0.003 0.0004 0.004 0.0001b aOrgans were trimed of extraneous body fat before weighing b Different from control mean (P< .05), n = 4 -79- revealed certain distinct changes in the treated cattle. The wall of the urinary bladder was thickened in all four treated cattle. The kidneys in three of the treated cattle were pale in appearance. The discoloration was localized primarily in the cortical areas but occasionally, extended into the medullary areas. All other organs in the treated cattle appeared normal. No abnormalities were observed in any organs of control cattle, except for multiple nodular abscesses in the liver of one control cow. Histologic examination of this 1iver resulted in a diagnosis of chronic focal suppurative hepatitis. Microsc0pic examination revealed pathologic changes in several organs of treated cattle, particularly in the kidneys and urinary bladder. The kidneys of three treated cattle had a chronic, diffuse, interstitial nephritis and werenfildly'hyperemic. In all four treated cattle, some glomeruli were swollen, while others were atr0phied or had disappeared and been replaced by connective tissue. Also, the Bowman's capsules were noticeably thickened. In one treated cow, the subcapsular space of many capsules was obliterated and the parenchyma replaced by connective tissue. Some of the tubules hithree treated cattle were dilated and connective tissue had proliferated into the interstitial areas. In two treated cattle, there were foci of lymphocytic infiltration in the medulla and cortex. The basement membrane of some renal tubules in one treated cow had undergone hyaline degeneration and deposits of a homogeneous eosinOphilic hyalinoid substance were seen in the tubular lumenS'hitwo treated cattle. The urinary bladder of all four treated cattle had a subacute uro- cystitis, characterized by edema and diffuse infiltration of inflammatory cells, primarily lymphocytes. ~In one treated cow, the bladder had undergone epithelial desquamation and there was a prominent neutr0philic infiltration -80- into the transitional epithelial lining. The ureters of one treated cow had a mild ureteritis with some infiltration of inflammatory cells. The kidneys, urinary bladder, and ureters in all control cattle were considered to be histologically normal. Organ enlargement was the only significant effect noted in the liver. However, a relationship of enlargement to treatment is not conclusive. Furthermore, there was a general enlargement, although not as dramatic, in several organs. The adrenal glands of one treated cow were abnormal. The capsule was thickened by connective tissue, foci of neutrophils were seen in the zona fasciculata, and small hemorrhages were evident. Except for the one instance of suppurative hepatitis described above, all other tissues of control cattle examined were considered histologically normal. Effect of Kidney Function The kidneys of treated cattle were functionally impaired (Table 7). This was demonstrated by the significantly decreased uptake of para- aminohippurate (PAH), tetraethylammonium (TEA) and amino isobutyrate (AIB) in renal cortical slices (P < .05). Conversely, the rate of ammonia- genesis was not significantly different (P > .05) from that of the control cattle. Pentachlorophenol Residue Analysis Serum PCP-Concentrations Analysis of serum from the penta-fed cattle for PCP during the low dose period showed concentrations to have essentially reached a steady state by three days and averaged 2802 ng/ml (ppb) PCP during the next 65 days (Figure 9). For reasons which cannot be fully explained, there was a slight decline in serum PCP-concentration at the 2, 4 and 6 week sampling times. However, this trend -8] - Table 7. In Vitro Renal Function in Penta-Treated and Control Cattle. Renal Function Control Treated ‘x t SE 7' t SE PAH S/Ma 5.32 0.05 3.47e 0.27 TEA S/M° 3.01 0.16 2.35e 0.07 AIB S/MC 1.90 0.10 ‘1.579 0.03 Ammoniagenesisd 121.00 22.00 153.00 22.00 ap-Aminohippurate, slice to medium ratio bTetraethylammonium, slice to medium ratio C Amino isobutyrate slice to medium ratio d Mole NH4 produced per gram dry tissue per hr eDifferent from control mean (P < .05), n = 4 -82- Figure 9. Concentrations of Total and Free Serum PCP in Cattle Fed Penta Subchronically. -83- 20,000 1-1 1 Po J J 1 71 .___,l l .000 j \l/ E l/ B. 10 000 — I , c ’ - \ ‘5 : I\!/ .2 _ g, - B ?\i\i/i"i4 S L) CL C) CL O—O Free Serum PCP (ng/mI) I—l Total Serum PCP (ng/ml) ;-6——0.2 mg/ kg——-) (——2.0 mg/kg-—) 100 L I l l i l l l l l J 0 I4 28 42 56 70 73214 28 42 56-65 Days Fed Penta -84- was reversed at the 8 and 10 week sampling periods. The PCP extracted under very mild acidic conditions is classified as free or unconjugated PCP. Such PCP is not covalently bound to any blood component, but is bound electrostatically or hydr0phobica11y to serum proteins or is nOt associated with any component of the blood. Total PCP is the amount that is extracted after rigorous acid hydrolysis and consists of both the free PCP and any PCP that is not acid extractable such as conjugated PCP. Therefore, the difference between total and free PCP is expressed as a percent of the total and probably consists of conjugated PCP ( (total - free/total) x 100 = % conjugated PCP). Further, 100% - % conjugated should approximately represent that percentage of PCP which is not associated with any serum component other than water or that which is protein bound. The average free serum PCP concentration during the low dose period was 1714 ng/ml. The corresponding values for unconjugated and conjugated PCP are 61% (1714/ 2082 x 100) and 39% (100% - 61%). Similarly, the average free PCP concentration during the high dose period was 9364 ng/ml and the average total PCP concentration was 14,726 ng/ml. The calculated values for the amounts of unconjugated and conjugated PCP are 64% and 34%. In a manner similar to that observed during the low dose period there was a slight decline in serum PCP concentrations after the fourth week. PCP Concentrations in Tissues and Fluids The body tissues and fluids analyzed for total PCP are shown in Table 8, in order of decreasing concentration. 0f the tissues analyzed, liver and kidney contained the highest concentration of PCP, with brain and thyroid having the lowest concentration. -35- Table 8. Total Pentachlorophenol Concentration in Tissue (ng/gm of wet tissue) or Fluid (ng/ml) Collected at Necr0psy from Cattle Fed Penta. Tissue PCP Concentration Number of Samples Analyzed 7' t SE 1iver 13,587 1867 4 kidney 9,579 706 4 spleen 3,437 265 4 lung 3,376 955 4 bile 2,829 734 4 thymus 2,218 166 4 muscle 2,725 168 4 mammary 2,058 247 4 adrenal 1,886 0 l thyroid 754 78 3 brain 504 0 1 Serum 14,520 2289 4 -86- Total PCP concentrations were also measured in rumen fluid, milk, urine and feces after 10 weeks of feeding 0.2 mg penta/kg and after four weeks of feeding 2 mg/kg (Table 9). Total PCP analysis of serum also collected at these times is included for purposes of comparison. At both sampling times, feces and milk had significantly lower PCP concen- trations than did urine. After four weeks of being fed 2 mg penta/kg in their diet, the four penta-fed cows had average total PCP concentrations of 14,520 ng/ml in serum and 3,141 ng/ml in milk. As expected, rumen fluid contained significant quantities of PCP. Interestingly, the initial attempts to analyze a sample of rumen fluid known to contain PCP proved fruitless. Only after fractionating the rumen fluid and analysis of the supernatant and pellet was it determined that the PCP was associated with the particulate portion. As a result of this finding, subsequent analyses were carried out on a homogenate of the whole rumen fluid with the final PCP concentration expressed on a dry matter basis. Dioxin Residue Analysis of Liver The'livers.O5). McConnell et_al, (1980) reported that heifers fed 15 mg/kg body weight per day of penta had reduced body weight -106- -107- gains. However, this effect was due to the dioxin or dibenzofuran impurities in penta, rather than PCP itself. Moreover, these same animals also had a significantly poorer feed conversion ratio in conjunction with a decreased rate of gain. In contrast, the penta-fed cattle in this study maintained their body weight throughout both treatment periods. In studies reported by McConnell et_al, (1978b) the toxicity of several dioxins was compared in several species. Weight changes were observed which could not be entirely attributed to decreased feed intake. The fact that all cattle in the treatment group in our study readily consumed the penta-containing concentrate suggests that the levels of penta were below the taste threshold or the molasses added to the ration effect- ively masked the reported repellant properties of penta (Deichmann et_al,, 1942). McConnell 33 fl. (1980) successfully fed 15 mg/kg body weight of both analytical and technical PCP. In our study, when feed intake was related to body weight over the test period, the amount of feed consumed by the penta fed-cattle was not different (P> .05) from the control cattle (Figure 7b). This suggests that penta had no effect on metabolic processes. The observation that the penta-fed cattle tended to be more efficient in the conversion of feed to milk during the later part of the high-dose period is not readily explainable. This tends not to agree with studies that showed PCP to interfere with cellulose digestion when it was added to mixed cultures of rumen microorganisms (Shull et_al,, l977; Shull and McCarthy, 1978). However, the lack of an effect of penta on body weight, milk production and efficiency suggests that penta does not have an in_ yiyg_effect on cellulose digestion. This may, in part, be explained by the fact that the rumen is a dynamic system where PCP is removed from the rumen -108- rapidly, whereas the jg_yitrg_digestion method involves a static system. The lack of an adverse effect on milk production does not coincide with the field observations of Thomas gt_al, (1977) which included decreased milk production in penta exposed animals. However, there is the suggestion that expression of the toxic effects of penta may be a time-dependent phenomenon. Firestone gt_al, (1979) found that dioxins accumulate in the bodies of cattle fed penta chronically, but require a significant period of time to reach a steady-stateconcentration. In Firestone's cattle, the dioxin content had not yet reached a steady-state concentration after 70 days of continuous exposure. This may be, in part, due to the fact that dioxins are poorly absorbed from the gastrointestinal tract (Schwetz gt_al., 1971). Accordingly, MCconnell §t_al, (1980) showed that growth suppression, in heifers fed penta is both dose and time-dependent. Therefore, the performance of the penta-fed cattle in this study may have been impaired had penta administration continued for a longer period of time. Somatic cell measurement in milk provides a means of monitoring the presence of mastitis in lactating dairy cattle. Somatic cell measurements in milk from control and penta-fed cattle showed treated cattle to have cell counts not significantly different (P>.05) from those of control cattle. These results do not conform to the findings of Thomas gt a1. (1977) where lactating cattle housed in penta-treated barns had an increased incidence of mastitis. The lack of an effect of feeding penta on milk fat production coin- cides well with other measurements of performance such as feed intake, milk production and feed efficiency. The generally normal appearance Of the treated cattle throughout the -109- entire period of exposure tends to support other findings with respect to the performance of these cattle. As for the health problem which periodically plagued two of the treated cattle, the inconsistency of its appearance and lack of correlation with dose suggest that it is not treat- ment related. The findings of the physical examination generally support the lack of any treatment effects on performance. In fact, there were no abnormal observations and a generally normal appearance in all cattle. The presence of eroded papillae in two of the four treated cattle may have been a local effect of penta. However, because the papillae were not evaluated prior to exposure, a cause-effect relationship cannot be established. Clinical Chemistry, Hematology and Urinalyses There were no biologically significant differences in the results of hematologic examinations, blood chemistry evaluations or urinalyses which could be attributed to the subchronic administration of penta. Minor variations in blood measurements, routinely performed throughout both treatment periods were inconsistent between sampling periods and failed to follow any definitive trends or relationships with dose. In contrast, McConnell gt_al, (1980) found a dose-related decrease in packed cell volume (PCV), accompanied by a comparable decrease in the hemoglobin concentration. It should be remembered that these findings were in heifers fed 15 mg penta/kg per day. In our study, no statistically significant differences (P > .05) were found between groups in urinalyses obtained one or two weeks before necropsy. McConnell et_g1, (1980) also found no statistically significant differences in the results of urinalyses in heifers fed penta subchronically for 160 days. -110- Pathologic Findings The microscopic findings in the kidneys coincide with the gross pathologic findings. However, the observed lesions were apparently not severe enough to be reflected by significant changes in routine clinical laboratory tests. In general, more than half of the original renal parenchyma must be destroyed before hematologic, clinical chemistry and urinalysis values are altered (Kaneko and Cornelius, 1971). Thus, at the time of necropsy the kidneys may have been in an early stage of- functional impairment. Even though there was no similar kidney damage in control cattle, there is not sufficient evidence to state definitively that the observed effects were in fact caused by penta exposure. A similar situation was encountered by McConnell _t._l. (1980). They observed inter- stitial nephritis in heifers fed penta chronically, but concluded that the effect was not treatment related on the basis that it was seen with equal frequency in both control and treated cattle. The pathologic condition of the urinary bladders in the treated cattle in our study was classified as urocystitis. McConnell gt 31. (1980) des- cribed a thickening of the bladder wall in heifers fed 15 mg penta per kg body weight. These authors classified the thickening of the bladder as epithelial hyperplasia rather than urocystitis. Moreover, in their study the effect was directly related to the amount of penta fed, which totalled more than ten times the amount fed in the present study. Also, they observed concurrent hyperplasia of renal papillae, which was not detected in the penta-fed cattle in our study. The nature of the inter- relationship among bladder wall hyperplasia, urocystitis and the admin- istration of penta are unclear. The effect could be related to the use of urethral catheters for urine collections, i.e., catheterization may have been responsible for the direct introduction of an infectious microorganism -111- into the urinary bladder which caused the urocystitis. However, control cattle were also catheterized using an identical procedure and equivalent time frame, without ill effect. Because urine is the primary excretory route of PCP in cattle, an increased susceptibility of the bladder to infection may have developed. However, the status of the immune system in these cattle was not impaired by the penta treatment. (Forsell et_gl,, 1980). Organ enlargement coupled with liver monooxygenase induction was reported by McConnell gt_gl, (1980) in heifers fed either penta or purified PCP. Microsc0pic examination showed mild vacuolation of hepatocytes in addition to the presence of foci of lymphocytic infiltration and capillary dilation in the lobules. Hepatocytic vacuolization had also been observed in cattle environmentally contaminated with penta (Thomas gt 11., 1977). However, in the absence of specific serum chemistry changes this histologic abnormality is not a sufficient basis for establishing a cause-and-effect relationship with treatment. In view of the fact that the liver is a primary site of dioxin accumulation, the lack of severe hepatic injury is somewhat surprising. McConnell §t_gl, (1980) reported other pathologic changes associated with the liver, namely bile duct hyperplasia and dila- tion of bile canaliculi. Neither of these effects was observed in the present study. The relative lung weights, like those of the liver were significantly increased, an effect also noted by McConnell et_gl, (1980) in heifers fed penta. Enlargement was accompanied by interstitial pneumonia in two of four treated cattle. Microscopic examination showed mild proliferation of connective tissue and infiltration of lymphocytes. The relationship of this lesion to treatment is not clear, as a similar effect was not observed -112- in heifers fed 15 mg/kg per day penta for approximately the same duration (McConnell gt_gl,, 1980). The results of the jn_yitrg_kidney function tests tend to support the histologic findings in the kidneys. The significantly decreased uptake of para-aminohippurate (PAH), tetraethylammonium (TEA), and amino- isobutyrate (AIB) in renal cortical slices indicate some degree of renal impairment, but not to such a degree that the animal's general health was overtly impaired. One possible explanation for the decreased uptake of these prototype substances is decreased active transport, which is an ATP-requiring process, in the proximal tubule. PCP is known to interfere with the synthesis of ATP. Toxicokinetics of Technical Pentachlorophenol Absorption and Serum Concentrations In the interest of integrating the information on residue analysis from the lactating cattle fed penta subchronically (Experiment 1) and the one lactating cow given a single dose of 14C-PCP (Experiment 2) the results of both will be discussed in this section. A relatively short period of timeimasrequired for PCP concentrations in serum to reach steady state in the cattle fed penta subchronically. By the third day, steady state was clearly attained. In the cow dosed with 14 14 C-PCP the serum C-concentration peaked in approximately 10 hours. This is a somewhat longer time than in rats, but is a shorter time than in 14C-PCP. Braun et_gl (1977) gave single 14 monkeys given a single dose of f ‘4 C-concentrations peaked 14 doses 0 C-PCP to rats and observed that plasma in about four hours, whereas Rhesus monkeys given a single dose of C-PCP -113— attained maximum plasma 14C-concentrations in 12-24 hours (Braun and Sauerhoff, 1976). Apparently the female monkeys absorbed the PCP more slowly than did the males, but reached a greater 14C-concentration in the blood. The difference in the rate of absorption between the bovine 14 vs monogastric species (rat, monkey) may be because the C-PCP was administered in a gelatin capsule with alpha-cellulose as a carrier. The -1 rate absorption constant (ka) of 0.162 hr in our study is similar to the ka of 0.215 hr ‘1 found in male Rhesus monkeys dosed orally with 14C-PCP (Braun and Sauerhoff, 1976). The absorption t1 of 4.28 hours in the cow is also similar to the absorption t; of 3.64 hours in male monkeys. Both male and female rats have significantly larger absorption rate constants of 1.95 and 1.52 hr ‘1 respectively (Braun et_gl,, 1977). These convert to absorption ti of 0.355 and 0.456 hours, respectively. The deviations in linearity in the serum data in Figure 10 may be partially explained by the possible enterohepatic recirculation of PCP. This would involve elimination of PCP in the bile and storage of bile containing PCP in the gall bladder. Then, upon ingestion of food, biliary secretion into the small intestine would result, followed by the sub- sequent reabsorption of PCP. The amounts of PCP classified as unconjugated (i.e., bound to serum proteins) and conjugated, in cattle fed penta subchronically, appears to remain relatively constant regardless of dose. Interestingly, the serum analysis data from these cattle suggest that a considerable amount of PCP is present in a conjugated form. In contrast, Braun gt_gl, (1977) 14C found in the serum of rats given 14C-PCP showed that the majority of was unchanged PCP, with only a small amount present as the glucuronide conjugate. However, since these rats were not previously exposed sub- -114- chronically to penta, it is possible the conjugating enzymes, such as glucuronyl transferase were not induced by previous exposure to dioxins and furans. Distribution Liver and kidney contained the greatest concentrations of total PCP in cattle fed penta subchronically. Analysis of liver and kidney 14 14 tissue, for total C, from the cow given a single dose of C-PCP produced similar results. In terms of compartments, these data are reasonable, as both tissues represent rapidly equilibrating compartments because of their profuse blood supply. In the 14 14 C study, brain and adipose tissue contained the lowest C-concentrations. Adipose tissue represents a slowly equilibrating compartment. It is likely in the case of the brain that although it is highly vascularized, it has a high phos- pholipid content into which PCP does not readily partition at physiologic 14 pH. The low levels of C in the spinal fluid, in addition to the low concentration in the brain clearly demonstrate that PCP or its metabolites have a limited distribution to the central nervous system. Braun et_gl, (1977) 14 also found the highest concentrations of C, in decreasing magnitude, 14 in the liver, kidney and plasma of rats dosed with C-PCP. Brain and adipose tissue contained the lowest concentrations of 14C. Similarly, 1. (1972) found that after dosing rats with 14C-PCP, the Larsen §t_ liver contained the highest 14C-concentration followed by the kidney, blood, stomach, adrenals and lungs. These authors found adipose tissue and muscle to have the lowest, and nearly equal concentrations of 14 C. In the present subchronic study the spleen, lungs and muscle had relative- ly high concentration of PCP. One explanation for this may be the presence of residual blood in these tissues. -115- A significant 14C-concentration was found in the gall bladder and lymph nodes. This may be partially explained by the fact that they were both in contact with fluids which contained high concentrations of 14 14C-concentration in the kidney, lung and liver may C. Considerable be due to their extensive vascularization in conjunction with their high protein content. Barbiturates behave in the blood in a manner not unlike PCP, i.e., they are highly serum protein bound (Harvey, 1975). It appears that the ability of tissues to concentrate barbiturates is largely dependent on their protein binding capacity for barbiturates. Further, the tissue binding capacity of the various barbiturates is closely related to their plasma protein binding capacity. Thus, both liver and kidney tend to have higher concentrations of barbiturates than do other tissues. In contrast, several of the short acting barbiturates should be able to concentrate easily in the body fat, but because of the plasma protein binding, slow rate of blood flow and low surface-to-volume ratio of adipocytes, uptake is slow. This may also explain the significant concentrations of 14C measured in the endocrine organs. Rumen contents contained no 14C-activity, which suggests that no PCP or its metabolites are secreted back into the rumen or in the saliva. Note, however, that GC analysis of rumen fluid shows it to have about 800 ppb PCP, on a dry matter basis. Unfortunately, neither samples of abomasal tissue ("true stomach") nor its contents were collected at necropsy. Although the stomach in monogastrics is probably the major site of PCP absorption because of its low pH, there is one report which suggests that PCP may be secreted back into this compartment (Jakobson and Yllner, 1971). In contrast to rumen fluid, duodenal contents contain a considerable concentration of 14C. Given that the duodenum preceded the area of the small intestine into which bile is released, this may be -116- indirect evidence that PCP was secreted into the abomasum. However, it is also possible that the glands of Brunner, which are the major contributor to secretion in the upper duodenum (Knobel, 1971) are the source of the 14C appearing in the duodenal contents. This is reasonable, given that the cow had been given half of her 0.2 mg/kg daily dose of penta five hours prior to necropsy. The rumen fluid samples measured for total PCP from the cows fed penta subchronically averaged about 4 ppb PCP during the 0.2 mg/kg dose period. The marked difference in PCP concentrations can be partially explained by the fact that rumen fluid sampling in the cattle fed penta subchronically was done about one hour after dosing. The sig- nificance of PCP being associated with rumen particulates and the resulting effect on digestion of feed by rumen bacteria is unknown. Serum and lymph had the highest 14C-concentrations of the fluids examined. This is understandable in that they share common proteins (Knobel, 1971). Lymph contains 1-4 times less protein than serum. How- ever, albumin is present in significant quantities in lymph, due to its small size and ability to cross capillary walls. Braun gt El- (1977) showed that PCP can bind both tenaciously and in extremely large quantities to serum albumin. These authors suggested that this relationship is the 14 primary reason for the preferential retention of C in the plasma. 14C concentrations in Probably a more obvious reason for the similar serum and lymph is that lymph is derived from serum. Examination of the distribution of 14C in milk from the cow given a 14C-PCP showed that the majority of 14C in milk is associated single dose of with the whey fraction (62.2%) with lesser amounts associated with the casein (24.4%) and fat (13.3%) fractions. This can be partially explained -117- by considering the gross composition of milk which is about 82% whey, 15% casein and 3.0% fat. Although the '4 C-concentration is higher in the casein than in the whey, the latter still represents the largest fraction in milk. Analysis of the casein and whey fractions of the 140 milk suggested that significant quantities of conjugated PCP are associated with these fractions. Metabolism The urine from the lactating cow given a single dose of 14C-PCP was analyzed using two methods to determine the extent of metabolism of PCP. The first involved extraction of urine under mildly acidic conditions with benzene. The benzene extract was subsequently analyzed using LSC and GC to quantify '4 C and PCP respectively. This method was assumed to extract PCP and/or its metabolites which were not conjugated. The second method involved treatment of urine with strong acid and heat followed by extraction with benzene and subsequent analysis of the benzene extract using LSC and 00. Use of strong acid and heat constitute conditions which are known to hydrolyze the chemical bond between PCP or tetrachlorohydroquinone and glucuronic acid (Ahlborg et_gl,, 1978). The additional 14C released by this method and analyzed by liquid scintillation counting was believed 14 to be C-PCP and/or its metabolites which are conjugated. Further, the additional PCP released by hydrolysis and measured by gas chromatography of the benzene extract was conjugated PCP. The percentage of conjugated 14 PCP or C was determined by the calculation:((tota1 - free)/total)x 100. Similarly, the percentage of unconjugated PCP was determined by the calculation: 100% - % conjugated. It should be noted that the values 14 produced by LSC analysis of the benzene extracts of the C urine most -118- likely represent 14C-PCP and its metabolites. In contrast, GC analysis of the benzene extracts represents PCP only. The results (Table 14, 15) 14 strongly suggest through indirect evidence, that about 85% of the C-PCP dose was excreted as a glucuronide conjugate. In contrast, Rhesus monkeys 14C-PCP in the urine as unchanged PCP excreted all of the administered (Braun and Sauerhoff, 1976). However, rats metabolize PCP to a moderate degree. When rats were 14 '40 in the urine was characterized as PCP (60%), given C-PCP orally, the PCP-glucuronide (9-16%), tetrachlorohydroquinone (TCH) (7%) and TCH-glu- curonide (16-22%) (Ahlborg gt_gl,, 1978). Similar results were obtained 14 by Braun gt 21, (1977) when rats were given 100 mg/kg C-PCP. In this 14C was excreted in the urine as PCP (75%), TCH (15%) and as PCP- study glucuronide (9%). However, the rats and monkeys in these studies had not been previously exposed to PCP orally unlike the present study. The rationale for the previous subchronic exposure of the experimental cow is that cattle are typically exposed chronically to penta on farms. There- fore, a single dose pharmacokinetic study with an animal having had no previous exposure may not give an accurate assessment of the fate of PCP in animals exposed to penta under typical environmental conditions. It is likely that with previous exposure, the degree of conjugation and metabolism would be somewhat different, particularly in light of the fact that dioxin contaminants are potent inducers (Goldstein gt 91,, 1977). PCP analysis Of serum from cattle fed 0.2 mg/kg/day suggests that a significant amount of the PCP in the serum was not conjugated, whereas 39% was in a conjugated form. This appears reasonable since one would expect that a glucuronide conjugate produced in liver would be transported in the blood and subsequently excreted via the kidney. -119- Elimination The terms elimination and excretion are pharmacokinetic terms which are often used in a manner which would suggest they are interchangeable. Elimination typically describes the clearance of a chemical from a compartment, such as serum or plasma, whereas excretion is the process whereby the chemical is actually discharged from the body, such as by way of the urine, milk, feces or lungs. In Experiment 2, a single dose of 14 14 C-PCP was given to a lactating cow and the concentration of C in serum was subsequently monitored over the next 76 hours. Applying the methods described by Gehring gt 31. (1976) which are shown in the Appendix (pgl35), the rate constant of elimination (ke) and its associated half-life (t1) were calculated to be 0.016 hr " and 42.8 hrs, respectively. Because studies with orally administered 14C-PCP in other species were done in animals which were not previously exposed to PCP, a direct comparison of values for ke and t1 may not be valid. However, some general comparisons can be made. The plot of serum 14C-concentrations in the cow shows that absorption and clearance follows 14am also followed first-order kinetics. Absorption and elimination of first-order kinetics in Rhesus monkeys (Braun and Sauerhoff, 1976). How- ever, this is not the case in rats. Although absorption appears to follow first-order kinetics, elimination is biphasic and requires description with two elimination constants and their corresponding half-lives (Braun. gt_§1,, 1977). These authors suggested that the secondary, or slow elimination phase was related to heterogeneous plasma protein binding and a small, but strongly protein-bound PCP in the liver and kidneys, which they categorized as ”deep" (slowly-equilibrating) compartments in the rat. -120- Excretion The results of the analysis of urine collected from the cow given a 14 single dose of C-PCP demonstrate that urine provides the main excretory route of PCP. When mice were administered 14C-PCP subcutaneously (s.cl) or intraperitoneally(i.p.),73-83% of the dose was excreted into the urine 14 (Jakobson and Yllner, 1971). Similarly, when mice were given C-PCP orally, about 86% of the label was recovered in the urine (Larsen gt 21,, 1972). Rats dosed with 14C-PCP either orally Or 141.excreted 50 and 80% of the dose, respectively, in the urine (Ahlborg gt 31., 1974). Male Rhesus monkeys 14 given C-PCP orally excreted 75% of the dose in the urine whereas female Rhesus monkeys similarly exposed excreted 70% of the dose in the urine (Braun and Sauerhoff, 1976). 14 In contrast, the results of analysis of feces from the C-PCP cow (Experiment 2) and cattle fed penta subchronically (Experiment 1) showed that feces provides a minor excretory route. Similar findings have been reported in rats and monkeys. Braun §t_al,(l977) found that male rats 14 given a single oral dose of C-PCP excreted 19-24% of the dose in the feces. Female rats given the same dose excreted significantly more in the 14 feces (43%). Further, male Rhesus monkeys given C-PCP orally excreted about 12% of the dose in the feces (Braun and Sauerhoff, 1976). Female 14 Rhesus monkeys excreted about 18% of an oral dose of C-PCP in the feces. Milk also provides a minor route for excretion of PCP. In the cow dosed with '4 C-PCP, only 5% of the dose was accounted for in the milk. This is also shown in a comparison of milk and urine PCP levels from the cattle fed penta subchronically (Figure 11). The serum and milk PCP levels in these cattle when fed 2 mg penta/kg per day were 14,520 and 3,141 ng/ml, respectively. Interestingly, when Firestone gt_al, (1979) fed cattle 10 mg -121- technical pentachlorophenol per kg per day, they found 40,000 ng PCP/m1 in serum and 4,000 ng PCP/ml in milk. However, when heifers were fed 15 mg penta per kg, blood PCP concentrations averaged 32,800 ng/ml (Parker §t_§l,, 1980). Development of the serum analysis used in the present study clearly showed that acid hydrolysis (acid & heat) of serum for a period of three hours, significantly increased the PCP yield. In contrast, extraction of the serum after mild acidification followed by solvent extraction resulted in significantly lower PCP concentrations. The acid hydrolysis step was subsequently included in the milk PCP analysis. Since Firestone gt El: (1979) did include a hydrolysis step (acid 8 heat) in their blood analysis, the PCP levels reported are probably accurate. Whereas, a partial explanation for the lower blood PCP concentration in the study reported by Parker gt_gl, (1980) is the lack of a hydrolysis step in their blood PCP analysis procedure. Our serum analyses suggest approximately 38% of the PCP in the blood of penta- fed cattle is conjugated. Thus, applying such a factor to the Parker gt_gl, data (i.e. 32,800 ng/m1/.62) would result in a blood PCP level of 54,903 ng/ml which is more reasonable considering the cattle were receiving 5 mg more penta per kg than cattle in the Firestone gt 31. study. Firestone et_gl,(l979) did use concentrated acid in their PCP milk analysis, but did not expose the milk & acid mixture to heat as was done in their blood PCP analysis. Lack of additional heat for an extended period of time appears to have resulted in only partial release of the bound PCP in the milk samples. Such an explanation would account for the average milk PCP concentration of 4,000 ng/ml in cattle fed 10 mg -122- penta/kg per day. In contrast the cattle in our study fed 2 mg penta/ kg per day had an average milk PCP concentration of 3141 ng/ml. SUMMARY Experiment 1 indicated that technical PCP or penta fed subchronically to lactating dairy cattle does not significantly affect feed intake, milk production or body weight gains at the levels administered. If there is any subchronic effect from oral exposure, it is possibly an effect on the kidney. Histopathological changes were present in the kidneys of the treated animals, but not in the control cattle. Furthermore, kidney function tests tended to support the histopathologic observations. The impaired jg_yitrg_kidney function may have manifested itself in other ways, had the exposure continued for a longer time. Generally, the results of this study do not support the findings of a fact-finding task force, in that penta exposure does not seem to result in decreased milk production or increased incidence of mastitis. It should be pointed out that the cattle in our study would be considered low to medium producers with respect to their level Of milk production. It is possible that cattle producing 80 to over 100 lbs of milk per day, under a high metabolic stress and also exposed subchronically to penta may have exhibited some or all of the production and health problems observed by the fact-finding task force in penta-exposed dairy herds. The pharmacokinetic profile of PCP in lactating cattle shows that PCP is both rapidly and extensively absorbed. In addition, the results of tissue residue analyses agree with the findings of other studies in that the‘liver and kidneys are major sites of PCP deposition, whereas brain and adipose tissue are minor ones. In terms of metabolism, hydrolysis Of both urine and serum samples indicated that PCP is extensively conjugated. -123- -124- Analysis of fecal, urine and milk samples showed that urine is the major excretory route for PCP in cattle. In contrast, both milk and feces represent only minor routes by which PCP is eliminated from the body. FUTURE WORK A review of the current toxicological data base on pentachlorophenol, as it relates to penta, suggests several possible studies which would significantly increase our knowledge of the effects of this chemical on domestic animals. One essentially unknown component is the contribution that inhalation of PCP makes to an animal's overall exposure. As compared to the assessment of dermal exposure, the assessment of inhalation of xenobiotics is a difficult task. However, practicality suggests that some of the most meaningful information might easily be obtained by field studies. Such studies might involve monitoring serum, urine and milk PCP residues in cattle, previously unexposed to penta, before and after being moved into a facility containing penta-treated wood. Clearly, one requirement would be that oral exposure be at a minimum or nonexistent. A study of this kind would eliminate the costs and intricate considerations associated with inhalation chambers and the need to extrapolate to actual conditions. To date, the studies in cattle have involved limited numbers of year- ling heifers (McConnell gt_§l,, 1980) and the lactating cattle in the current study. Thus, it would seem that the definitive study yet to be done is one whereby heifers exposed to penta-treated wood are bred, carried through one complete lactation and then bred once more. Such a study would involve making manywyfthe same kinds of measurements made in the current study, plus it would provide data on the effects of penta on such aspects as fertility, gestation and teratogenicity, which are of critical interest in dairy production. Clearly, to provide both statistically and biologically meaningful information, this study would have to involve sufficient numbers -125- -126- of cattle, and more importantly, it would be very costly. However, until such a study is conducted, significant questions will remain as to the effects of chronic exposure to penta on lactating dairy cattle. BIBLIOGRAPHY Ahlborg, U.G., J.E. Lundgren and M. Mercier. 1974. Metabolism of pentachlorophenol. Archives of Toxicology 32:271-281. Ahlborg, U.G., K. Larsson and T. Lundberg. 1978. 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Journal of the Association of Official Agricultural Chemists 47:1116-1120. APPENDIX Estimation of the.Rate Constants of Absorption (ka) and Elimination (ke) and Their Associated Half-Lives (t%) The rate constant for elimination (ke) can be estimated by taking a straight edge and extrapolating back to the point where the line inter- cepts the y-axis (Figure 10). The line is described by the equation: lnC=inCO-k_e where: C0 - concentration at the point where the extrapolated line intercepts the y-axis C = concentration at time t ke = rate constant for elimination Solving for ke, the equation becomes: lnC " 1“ Co ke 3 t Using the following data: C - 9300 dpm/ml, Co = dpm/ml, and t 3 76 hrs, then: 1n 32,000 - 1n 9,300 76 ke 0?“, 1 ke 0.0162 hr- -135- -136- Thus, the half-life for elimination is: 0.693 = 0.693 ke 0.0162 = 42.8 hours t% 3 The rate constant for absorption (ka) is obtained by a method called "curve stripping" or "feathering". This involves determining the differences between the experimental values from the absorption phase of the curve and the corresponding values on the line which was previously extrapolated back to an intercept on the y-axis (Figure 14). These values are then plotted vs their respective times. The values for C, CO, and t can then be used in an identical manner as shown above, to determine ka as follows: 1nC-lnCQ t ka using the following data: 30,408 dpm/ml, Co = 7,029 dpm/ml, and t - 9 hr, then: 1n 30,408 - 1n 7,029 ka - 9 OY‘, ka = 0.162 hr '1 Thus, the half-life for elimination is: t, . 9292; . 0.693 _ 4.28 hr. ka 0.162 -137- Figure 14. 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N.o. w._ N.m _a\=_ Azomv mmmcmuoguxcmu _ou_ngom Apm_ummm= n c .& as oo— no u m .& as an no u m .mucgu .m u mch msmguxm .N n mNo; wumgmcos .p u «No: uzm__m .o u mepu .m u gmzuo .e u zeppm» xgcv .m n zappmx .m u zeppmx m_ma ._ u :mLum .o n Lam—um mama m>?unwzo:c go: _mu_:._u mmv9>ogg noguma m_;~ .Aummu cum; u:c-a_vv x_umpu—:z an umago$gma mmmxpccvgap II QG — .m>w9mmm: n o MQ'LDKDN «.0 ©.— N.c w.c N.o N.— N.o m.o Oemwgmuucm o.c o.o c.c o.c o.o o.c c.o o.o op.wmummu N.o m.c c.o c.— «.0 o.— v.0 o.— am——mu paw—wzu_au m.m ©.m c.— —.N N.m ¢.N— m.o _.m— wm——mu too—a umz m.o v.c— —.v m.m N.c— o.¢m —.—— a.pp mm—pmu woopn Mews: N.c N.c c.o c.c m.c m.o N.o N.c mace—n u—zuuo N.o w.o N.o w.o mo.o mo.o mc.o mo.o ficwmocwppnogz c.o o.o o.o 0.0 c.o o.c N.o N.o mcwnagp—wm o.c c.O o.o o.c o.c c.o N.o N.o amateumx O.o o.c c.o o.o c.c o.o c.o c.o mmmouzpo m.o m.o o.o o.c c.c o.o o.o c.o vcwmuogm v.c c.m N.o N.w N.o N.m e.c 0.x :Q Nco.c —NO.— —oo.o mmo.— mco.o —No.~ mco.o mmo.— xuw>mgm uwwwumam c.o o.c o.o O.o m.c w.o o.o o.o mxuwvwngah 0.0 O.N 0.0 o.m o.o c.m c.o o.m NLO—ou mm w com: mm H cam: mm H com: um fl cam: mmu_n:_ umucmgh _ogucou mwummgh _ogucou- xmzogumz-mmm xmmzup . xmmmuumznmga xwwz-m w_uucu nmu-mu:ma use _ogucou sag» mgmx—m:_L: yo mu_:mwm .cm «paw» — -l46- Table 2l. Recovery of a 14C-PCP Spike in Various Tissues and Fluids of Cattle Fed Penta Tissue Percentage Recovery of 14C-PCP Spikea i’ t SE liver 91'8 1'] kidney 90-8 2'2 spleen 89-8 1'6 lung 96.0 1-4 bile 86.6 0.6 thymus 90°2 2'2 muscle 93'2 1'9 mammary 95.0 .1-5 adrenal 98°3 2'5 thyroid - 79.0 2.4 brain 90.0 0-2 a l4 average percentage recovery C-PCP from replicate spiked samples of tissue homogenate per animal -l47- Table 22. Recoveryirfa 14C-PCP Spike from Urine, Serum, Rumen Fluid and Milk Sample Percentage Recovery of a l4c-Pcp Spikea E' i SE Rumen Fluid 98.4 0.64 rfilk 90.2 l.46 Urine 99.0 - Serum 99.0 0.38 Feces 94.0 1.20 a average percentage recovery of 14 spiked samples C-PCP from replicate BIOGRAPHICAL SKETCH OF J.H. KINZELL The author of this thesis was born in Calgary, Alberta on April l4, 1950. Shortly thereafter, he moved with his family to a mixed farm 30 miles south of Calgary at High River. He attended school at High River and in his free time, assisted his father in managing their beef cattle operation and participated in school athletics and 4H programs. After graduating from high school in l969, he attended junior college in Everett, Washington and thereafter, obtained an Associate Science Degree in Biology. He subsequently transferred to Oregon State University and two years later received a Bachelor's of Science Degree in Zoology. His farm background led him to choose a graduate program in animal nutrition at Oregon State University under Dr. Peter R. Cheeke. Thesis research for this degree involved experiments which examined the nutritional and toxicological aspects of novel protein sources for monogastric animals. In the fall of 1976, he began his doctoral studies and research at Michigan State University in the Department of Animal Science with Dr. Lee Shull. As Dr. Shull's first graduate student, the author assisted his supervisor in developing what is now the Food Animal Toxicology program as an integral part of the Center for Environmental Toxicology at Michigan State University. At the present time the author is employed as a toxicologist in the Toxicological Evaluation Division of the Health Protection Branch (Health and Welfare Canada) in Ottawa, Ontario. His duties as a toxicologist -l48- 449- include examining toxicological data supporting the safety-in-use of agricultural chemicals and supplying consultative services regarding these chemicals, to other governmental agencies.