I {ll Mill 4 ’ IWIHJ'WI THE EFFEC‘I‘S 3F mezum‘umem) F‘EITHALATE ON ADULT SINGLE COMB WHITE LEGEi‘éBN FEMALES m THEIR EGGS 145 049 THS “Wash gov 9510 Degree of M. 5. MECififiAR’ STATE UNEVERSITY Rage-r Dean Jacobs 1974 :1 LIBRARY ‘1 i Michigan State I L, University .7 ABSTRACT THE EFFECTS OF DI(2-ETHYLHEXYL) PHTHALATE ON ADULT SINGLE COMB WHITE LEGHORN FEMALES AND THEIR EGGS By Roger Dean Jacobs Forty-eight Single Comb White Leghorn hens were randomly divided into four groups and placed in cages. They were artificially inseminated twice weekly with pooled semen from Single Comb White Leghorn males. The eggs were set weekly and every seven days all infertile eggs and dead embryos were removed. The first two weeks (pretrial period) all hens were fed a layer breeder ration. The following four weeks (trial period) di(2-ethy1hexyl) phthalate (DEHP) was added to the feed at levels of 0, 1,000, 3,000 and 5,000 ppm. Parameters measured during the six weeks were: fertility, egg production, feed consumption, body weights and hatchability. Fertility and egg production were not significantly reduced when DEHP was added to the feed. DEHP signifi- cantly reduced body weights, feed consumption and hatch- ability at both the 3,000 and 5,000 ppm levels when compared to the 0 ppm level. There were no significant differences between the 0 ppm level and the 1,000 ppm level. 0{9 Roger Dean Jacobs Q7 Petechial hemorrhages were observed on many of the dead embryos from eggs produced by females fed the three levels of DEHP. No other teratogenic effects were observed. THE EFFECTS OF DI(2-ETHYLHEXYL) PHTHALATE ON ADULT SINGLE COMB WHITE LEGHORN FEMALES AND THEIR EGGS By Roger Dean Jacobs A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Poultry Science 1974 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. Robert K. Ringer for his help and guidance during this period of study and research. The author is grateful to Drs. T. H. Coleman, R. J. Aulerich,and H. C. Zindel of the Department of Poultry Science and to Dr. R. M. Cook of the Department of Dairy Science for their guidance and careful review of this manuscript. Sincere thanks are also extended to Drs. J. H. Wolford, D. Polin, L. R. Champion, T. S. Chang, 0. J. Flegal and C. C. Sheppard for their valuable criticisms and help in the preparation of this manuscript. To Dr. H. C. Zindel, Chairman of the Poultry Science Department, the author extends his appreciation for financial support provided. The author is also grateful to his parents and Mrs. Margaret Granger for their moral support. Finally, the author is indebted to his wife, Barbara, and two sons, John and Joseph, for their sacrifice and understanding during this period of study. ii TABLE OF CONTENTS Page LIST OF TABLES AND FIGURES . . . . . . . . . . . . . . iv INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . 2 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 3 Absorption, Distribution, Metabolism and Excretion O O O O O O O O O O O O O O O O 0 O O O 6 TOXiCOlOfl Of DEHP o o o o o o o o o o o o o o o o 8 Effects of DEHP on Reproduction and Teratogenicity . . . . . . . . . . . . . . . . . . 12 PROCEDURES . . . . . . . . . . . . . . . . . . . . . . 15 Preparation of Feed . . . . . . . . . . . . . . . 16 Artificial Insemination . . . . . . . . . . . . . 16 Storage and Incubation . . . . . . . . . . . . . . 17 Analyses of Data . . . . . . . . . . . . . . . . . 18 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 19 Fertility . . . . . . . . . . . . . . . . . . . . 19 Egg Production . . . . . . . . . . . . . . . . . . 22 Feed Consumption . . . . . . . . . . . . . . . . . 22 Body Weights . . . . . . . . . . . . . . . . . . . 28 Hatchability . . . . . . . . . . . . . . . . . . . 28 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . 36 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 37 iii LIST OF TABLES AND FIGURES Table Page i 1(- '0 .; Lethal dose levels for 50% of animals treated with di(2-ethylhexyl) phthalate . . . . . . . . . 1O 1.:- .3 . 2. Acute toxicity of metabolites of di(2-ethylhexyl) phthalate as measured by LD50 . . . . . . . . . . 11 3. Effects of feeding di(2-ethylhexyl) phthalate (DEHP) on fertility in chickens with data presented in arcsin conversions . . . . . . . . . 20 ' a. 1-‘ 4. Analysis of variance - fertility . . . . . . . . 21 5. Effects of feeding di(2—ethylhexyl) phthalate (DEHP) on egg production in chickens with data presented in arcsin conversions . . . . . . . . . 23 .. ._ I ‘.m-m--n‘n~m . . ' . v i ‘ I . Q _ 1;! mwrfipu-"¢z .-. . M- 6. Analysis of variance - egg production . . . . . . 24 Effects of feeding di(2-ethylhexyl) phthalate (DEHP) on feed consumption (gm) in chickens . . . 25 IT‘HT '1'. u?‘ '5‘"- L ‘ 2.5 C 8. Analysis of variance - feed consumption . . . . . 26 9. Effects of feeding di(2-ethylhexyl) phthalate (DEHP) on body weights (gm) in chickens . . . . . 29 .Em .. I ‘ . =_ . a i ’l 10. Analysis of variance - body weights . . . . . . . 30 11. Effects of feeding di(2-ethylhexyl) phthalate (DEHP) on hatchability of eggs from chickens with data presented in arcsin conversions . . . . 31 if“ .9 "0-2. . - - m- " 't-‘x‘trvarxm ”Afw‘oq“ ’1' ' . ‘- 12. Analysis of variance - hatchability . . . . . . . 32 Figure 1. Linear regression curve of embryonic mortality of eggs from adult Single Comb White Leghorn hens fed di(2-ethylhexyl) phthalate . . . . . . . 35 iv INTRODUCTION Dr. Lloyd B. Tepper (1973), Associate Commissioner for Science, United States Food and Drug Administration, refers to the phthalic acid ester problem as an etiology in search of a disease. Since World War II the use of plastics and plasti- cizers has increased tremendously in building construc- tion, automobiles, home furnishings, clothing, food coverings and medical supplies. However, the increasing use of plasticizers contributes to environmental wastes and thus may affect the ecology. Although the toxic effects of phthalic acid esters have been documented in mammals and found to be of low order, there have been no reports of similar studies conducted in the class A122. This is a report of the effects of di(2-ethylhexyl) phthalate (DEHP) on fertility, hatchability, egg pro? duction, body weights and feed consumption in chickens. OBJECTIVES The specific objective of the research was to measure the effects of DEHP on adult Single Comb White Leghorn females and their eggs when this plasticizer was added to the diet at specific levels. The parameters measured were: 1. Fertility 2. Egg production 3. Feed consumption 4. Body weights 5. Hatchability REVIEW OF LITERATURE The first commercially marketable plasticizers were synthesized following the discovery of cellulose nitrate in 1846. In 1931, poly(vinyl chloride) was introduced and two years later, di(2-ethylhexyl) phthalate (DEHP) was discovered. The sales of poly(vinyl chlorides) and phthalates have shown a steady rise since the mid 1940's reaching about one billion pounds of plasticizers sold in 1972 (Graham, 1973). There are three forms of benzenedicarboxylic acids, the ggtg form (isophthalate esters), the para form (tere- phthalate esters) and the gathg form. The term "phthalate esters" involves only the gathg form and is the subject of this thesis. Twenty different phthalate ester compounds are produced commercially. They are normally synthesized from phthalic anhydride and the appropriate alcohol. Phthalic anhydrides are synthesized from either naphthalene or g—xylene. The type of plasticizer used in manufacturing depends upon the desired characteristic of the finished product. As much as 40% of the finished product may be plasticizer. 4 Plasticizers are volatile. The volatility of plasti- cizers in poly(vinyl chloride), under controlled conditions, is dependent upon the affinity for the material that is in contact with the poly(viny1 chloride) whether it be air, water, oil, lipids or blood (Quackenbos, 1954). The higher the temperature of the surrounding environment the greater the volatility. The rate at which a plasticizer is lost from a material is determined by Q/S = 2.26(Dt/L2)& where Q is equal to the weight in grams of plasticizer lost during 3 hours. 3 is the total weight of plasticizer in the film. D is the diffusion constant (sq cm/hr) and L is the film thickness in cm. Much of the present concern over phthalic acid esters began when Jaeger and Rubin (1970a) reported that phthalates were being lost from poly(viny1 chloride) tubing used in rat liver experiments. Later that same year they reported that blood stored for 21 days in plastic bags contained 5 to 7 mg of DEHP per 100 ml of blood. Tissues of spleen, liver, lung and abdominal fat obtained from two patients at Johns Hopkins University Hospital following blood trans- fusions contained DEHP ranging from 0.025 mg/g (dry weight) in spleen to 0.270 mg/g (dry weight) in abdominal fat. The blood used in transfusions had been stored in plastic bags (Jaeger and Rubin, 1970b). Milk has also been shown to contain phthalic acid esters after being pumped through poly(vinyl chloride) tubing (Wildbrett, 1973). Poly(viny1 chloride) tubing was found to be responsible for reduced 5 pulmonary vascular resistance during ventilation hypoxia in isolated perfused lungs of cats (Duke and Vane, 1968). Samples of soil (approximately 25 mm in depth) collected by Cifrulak (1969) from both highland and low- land areas near the Ohio River contained a black tar- like residue amounting to 0.08% by weight of the soil sample. Subsequent analysis indicated this residue to be a mixture of phthalic acid esters. Air samples in the vicinity of incinerators operating near Hamilton, Ontario have been reported to contain phthalic acid esters (Thomas, 1973). Water samples from the Charles River Basin near Boston showed DEHP concentrations to be about 1 part per billion (Hites and Biemann, 1972). Samples of water from the northeast part of the Gulf of Mexico, the Mississippi Delta and the Mississippi River indicated concentrations as high as 0.6 parts per million, principally DEHP (Corcoran, 1973). He notes that if one third of this concentration or 0.2 ppm were found throughout the Mississippi River, this would equal the total production of DEHP in 1970-- 350 million pounds. Mayer, Stalling and Johnson (1972) detected phthalic acid ester residues in fish from many locations. Morris (1970) reported phthalic acids in the deep sea jellyfish Atolla at 0.01% of the total wet weight. He stated, "it is most unlikely that phthalic acid is present as a normal 6 metabolite and so it seems to have been assimilated and stored in the body." The proportion of man-made phthalic acid esters found in the environment is still unknown. They have been detected in poppies, cranberries, eucalyptus oil, olive oil, tobacco leaves, grapes, kewda and oxidized corn oil. They have also been found in shale and crude oil. Phthalic acid esters have been reported to be normal metabolites of the fungus Alternaria kikuchiana Tanaka (Graham, 1973). Absorption, Distribution._Metabolism and Excretion Phthalic acid esters can be absorbed through the gastro- intestinal tract, the peritoneal cavity, the lungs and the integument of experimental animals. Dillingham and Pesh- Imam (Autian, 1973) indicated that when 14c-diethyi 'phthalate was placed on the skin of rabbits, 9% of the activity was recovered in 24 hours and 14% in 48 hours. Shaffer §£_§l. (1945) placed 20 ml/kg of DEHP on the intact skin of rabbits for 24 hours. After 14 days, two of the six rabbits died. The LDSO for skin absorption was estimated to be about 25 ml/kg. This is similar to the reported LD50 for rabbits given DEHP orally. Jaeger and Rubin (1970b) found DEHP distributed in various tissues of patients following blood transfusions given from blood stored in plastic bags. Prior to this, Taborsky (1967) found di-gfoctyl phthalate and its isomer 7 DEHP in the bovine pineal gland. Nazir _e_t el- (1973) discovered DEHP and dimethyl phthalate in the mitochondria of beef hearts. Experiments by Dillingham and Pesh-Imam using 14C-DEHP showed that the highest activity was found in the lungs with lesser activity in the brain, fat, heart and blood of mice (Autian, 1973). There was no apparent preference by DEHP for fatty tissue. The mice were given both intraperitoneal and intravenous injections. Tissues were taken at 7 and 14 days following injections. Studies by Schulz and Rubin (1973) using a mixture of labeled and unlabeled DEHP in rats showed that DEHP was rapidly metabolized into water-soluble derivatives. Their results indicated that metabolism of DEHP is extensive and that the rate of metabolism is more rapid than the rate at which it is absorbed from the gastrointestinal tract. Jaeger and Rubin (1970a), using isolated perfused rat liver preparations, reported that butyl glycolylbutyl phthalates could be metabolized but DEHP could not. Furthermore, they stated that intracellular accumulation occurred. Urinary excretion studies by Shaffer 31 El- (1945) indicated that rabbits were unable to metabolize DEHP following a single oral dose of 2 gm/kg. Five male animals' average excretion was 41.9% of the phthalate ingested with a range of 26% to 65.4%. No quantitative data were reported for rats but the authors stated that considerable amounts of phthalic acids 8 were recovered. They concluded that the rat like the rabbit was largely unable to metabolize this acid. Further experiments by Dillingham and Pesh—Imam (Autian, 1973) measured urinary excretion of 14C-DEHP in mice following a pure phthalate ester intraperitoneal injection. In another group, labeled DEHP in a saturated saline solution was injected intravenously. The dose given intravenously was less than the dose given intra- peritoneally by a factor of 104. Seven days later the total amount excreted by intravenously injected mice paralleled the amount excreted by those receiving the intraperitoneal injection, 68% and 63% respectively. The intravenously injected group was characterized by a first order decline immediately following the injection. In the intraperitoneally injected group, excretion peaked at 3 days and then followed a first order decline. The author stated that the cumulative excretion rate ". . . shifts from zero order to first order at approximately 4 days at a time when there is approximately 20 times the amount of DEHP present required to saturate the aqueous compartment." Toxicology of DEHP The toxic effects of phthalates, especially DEHP, are considered to be of low order when given as single doses; no matter how the plasticizers are administered. In this section the chronic toxic effects of DEHP are discussed and for the sake of brevity acute toxicity of 9 DEHP and metabolites can be found in Table 1, page 10 and Table 2, page 11. One of the earliest experiments measuring the toxic effects of DEHP was performed by Hodge (1943). DEHP was given to rats via stomach tubes at levels between 1.1 g/kg and 34.5 g/kg. No deaths occurred at these levels but diarrhea was observed at 4.5 g/kg and greater. At necropsy, the only observable changes were enlarged sto- machs. When DEHP was injected intraperitoneally at levels between 3.1 g/kg and 23.8 g/kg in rats, diarrhea was again observed at the higher levels. Enlarged livers were seen in rats killed 3 days after injection. In rats killed 7 days after injection the liver weights were normal, but the peritoneal cavity contained a milky watery emulsion. Calley 33 El- (1966) injected DEHP daily into the intraperitoneal cavity of mice for six weeks at levels of .125 g/kg to .5 g/kg. Body weights were the same as the controls although DEHP treated animals reached maxi- mum body weights later than the controls. Peritonitis, liver abscess, adhesions to the diaphragm and testicle abscesses were found in the treated mice. A slight decrease in hematocrit and hemoglobin concentration was noted. They reported that DEHP and dicapryl phthalate stimulated the central nervous system whereas all other phthalates tested were depressants. DEHP resulted in a marked inflammatory response when injected intradermally. 1O Table 1. Lethal dose levels for 50% of animals treated with di(2-ethylhexyl) phthalate “’50 Animal Route g/kg Reference Mouse Intraperitoneal 14.2 Calley 33 El- (1966) Rat Intraperitoneal 24.0 Hodge (1943) Rat Intraperitoneal 30.7 Shaffer 2£.§lo (1945) Rat Intraperitoneal 50.0aL Singh 21; 3;. (1972) Rat Intraperitoneal 30.6 Lefaux (1966) Rat Stomach Tube 34.0 Hodge (1943) Rat Stomach Tube 30.6 Shaffer 31 El. (1945) Rabbit Intraperitoneal 33.8 Lefaux (1966) Rabbit Stomach Tube 733.9 Shaffer J el- (1945) Rabbit Skin Absorption 25.0‘al Shaffer t _a_._l_. (1945) aml/kg 1 1 Amemev .mm mm soeaccm oe.e case sooaoem pom Hoaoxosaasemum Anamev omeom om.m case sonsopm pom Homewosaacemum Amsmev omoom mo.o HsoaoeasoaoessH ens Hosswosaasemum Amemfv smoom m>.o HmoqopanonmupsH mono: HoqsxonahanIm Amemev .mm.mm_sooeosm om.» spas consoem pom ease chansons Amemev .mm.mm omoom mm.o HooaoeasoanaeaH onsoz once chancesm oosouomom mx\w venom Hwawqd unconsoo owns cm as an nonsense no assassoaa «Haaosaaceonmvac mo noeaaoocpos mo messages oeaoa .m oases 12 Long term studies by Shaffer 33 al. (1945) indicated that after 90 days rats on diets of 0.375% to 3% DEHP had reduced growth at the highest levels with tubular atrophy and degeneration in the testes. They reported normal blood cell counts, hemoglobin concentrations and differential white cell counts. Carpenter 33 al. (1953) reported that rats fed DEHP for two years at levels of 0.04%, 0.13% and 0.4% of the diet had life expectancies the same as control rats. Only those fed 0.4% DEHP had less mean body weights, yet they consumed more feed. After two years, the 0.4% DEHP group had larger livers and kidneys. Similar results were found when guinea pigs were fed levels of 0.13% and 0.04% DEHP for one year. Dogs fed 0.03 ml/kg for 19 doses and then 0.06 ml/kg for 240 doses showed no effects other than slight kidney tubular degeneration. This work was confirmed by Harris 33 El. (1956) and Lefaux (1968) using rats as the experimental animal. Effects of DEHP on Reproduction and Teratogenicity One of the earliest studies mentioning the effects of DEHP on reproduction was by Carpenter 32 El- (1953). In this study DEHP was fed to male and female rats at levels of 0.4%, 0.13% and 0.04% of the diet for two years. They reported that at these levels DEHP had no effect on reproduction or fertility. The results of toxicity testing by the Cancer Insti- tute in France (Lefaux, 1968) indicated that DEHP fed in a 13 diet at 500 mg/kg to rats for four generations did not alter normal reproduction. No anomalies were observed during parturition or nursing. Further investigation by Singh 33 El- (1972) indi- cated that DEHP administered intraperitoneally hampered reproduction. Female rats were injected with 10 ml/kg and 5 ml/kg of DEHP on the fifth, tenth and fifteenth day of gestation. 0n the twentieth day of gestation the pregnant rats were killed and the fetuses were removed surgically. Resorption of fetuses was at 8.2% and 26.8% in the 5 ml/kg group and 10 ml/kg group respectively. Untreated controls had no resorptions. When injected at 10.0 ml/kg, distilled water caused 6.8% resorption, normal saline 11.5% resorp- tion and cottonseed oil 6.8% resorption. The authors reported neither dead fetuses nor skeletal abnormalities in the 5 ml/kg group. The weights of live fetuses were signi- ficantly less than those of untreated controls. The 10 ml/kg group had 22.0% more gross abnormalities than did the untreated controls. Peters and Cook (1973) investigated the effects of DEHP on reproduction in rats. In these experiments two groups of five females and one male were housed in large cages and given intraperitoneal injections with DEHP at levels of 2 and 4 ml/kg body weight at 3, 6 and 9 days of gestation. The results indicated DEHP prevented implanta- tion in 7 of 10 females (total of both levels). During 14 parturition, 2 of 3 rats where implantation occurred died. Excessive bleeding also occurred in all three rats at parturition. In a second experiment DEHP was injected at a level of 2 ml/kg body weight. Injection was on the first or third, sixth or ninth day of gestation or combinations thereof. Results indicated the failure to implant occurred only when injections were administered prior to 6 days of gestation. The authors noted that implantation normally occurs at 6 days of gestation. At parturition excessive bleeding was observed. DEHP had no effects on reproduction in female pups from dams used in the second experiment. The developing chick embryo is a very good experimen- tal system for observing teratogenic effects of drugs. McLaughlin gt 2;. (1963) reported that when 0.5 ml of DEHP was injected into the yolks of fertile eggs prior to incubation, 95% hatchability was observed with no observable teratogenic effects. Haberman 22.21- (1968) injected 0.1 m1 of various phthalates into the air cell and onto the choriollantoic membrane of nine day old incubated eggs. When dioctyl phthalate was used, 66 of 125 eggs hatched. Of these, 5 exhibited neurological abnormalities. One hatched with one eye missing and a malformed beak. PROCEDURES Forty-eight, 45 week old Single Comb White Leghorn hens were placed in single cages measuring 20.32 x 40.64 x 40.64 centimeters. They were randomly divided into four groups of twelve birds per group. The control group (P0) was treated like all others except that DEHP was not added to the feed. Three treat- ment groups received feed with varying amounts of DEHP added. The amounts were as follows: treatment group I (P1), 1,000 ppm; treatment group II (P3), 3,000 ppm; treat- ment group III (P5), 5,000 ppm. Previous experiments by this author indicated that DEHP did not significantly lower hatchability when added up to 1,000 ppm. Seven adult Single Comb White Leghorn males were selected to provide the semen used in this experiment and they were placed in separate cages. Throughout the experi- ment the males were fed Michigan State Layer-Breeder Ration LB-72 without DEHP. The experimental period lasted six weeks. During the first two weeks all chickens were fed LB-72 without DEHP. Body weights were taken weekly and feed consumption was measured daily during this period and the following four weeks. Feed and water were given ad libitum. 15 16 Preparation of Feed The feed for all groups was prepared by straining LB-72 through a no. 20 gauge screen. Five hundred grams of strained feed was placed into a 1,000 ml beaker. DEHP (P1 = 25 g, P3 = 75 g, P5 = 125 g) was diluted in one liter of hexane. It was then added to the screened feed which was then stirred and placed into a chemical exhaust hood until the hexane had evaporated. This was added to addi- tional LB-72 to make premixes of 5,000 gram lots each. One thousand grams of premix was added to LB-72 to make 5,000 grams of ration. Artificial Insemination Semen was collected into a common pool on Mondays and Fridays at 4:30 p.m. after most ovipositions had occurred. The average volume of pooled semen was 5 ml to which was added Ringer isotonic saline to make 7 ml. The semen was allowed to diffuse throughout. Approximately 0.1 ml of semen in a 1 ml glass disposable syringe was placed about 2 centimeters into the left oviduct of the hens. The contents in the syringe were then pushed out. The number of sperm per ml from Single Comb White Leghorn males per ejaculate averages 3.5 billion. The number of spermatozoa per insemination needed for optimum fertility is 100 million (Sturkie, 1965). In this experiment approximately 250 million spermatozoa were used 17 per insemination. The sperm were observed for motility under a microscope after all hens had been artificially inseminated. Storage and Incubation Eggs were marked and collected daily. They were put in a cooler which maintained a temperature of 50-60° F. On each Friday the eggs were removed from the cooler at 9:00 a.m. and allowed to warm to room temperature prior to incubation. When available, one egg from each hen was removed and the yolk and albumen were separated and placed in 16 oz bottles and frozen for future analysis. Four eggs from each hen were placed into a Jamesway 252 incubator at 2:00 p.m. For the first 18 days these eggs were turned every four hours. They were held at a temperature of 99- 100° F and a wet bulb temperature of 86-88° F. At seven days of incubation the eggs were removed from the incubator and all infertile and dead embryos were removed and observed for any possible teratogenic effects caused by DEHP. This procedure was repeated at 14 days of incubation. At 18 days of incubation the eggs were transferred to hatching baskets and placed into another Jamesway 252 incu- bator where the temperature was maintained at 99° F with a wet bulb temperature of 88-90° F. 0n the twenty-second day of incubation all chicks and unhatched eggs were removed. The live chicks were 18 killed via cervical dislocation. Again, all embryos were inspected for teratogenic effects. Analyses of Data The data were statistically analyzed according to Duncan's multiple range test (1955) and by two-way analysis of variance. Because of the large variation between indivi- duals, each laying hen was compared weekly during the trial period to its average performance during the pretrial period. RESULTS AND DISCUSSION Fertility The effects that DEHP had on fertility are presented in Tables 3 and 4, pages 20 and 21. The analysis of variance computations in Table 4 indicate that there were no signifi- cant differences due to the amount of DEHP added to the feed (treatment level effects) or to the length of time that DEHP was added to the feed (weekly effects). This table also indicates that there was no interaction effect (level x weeks). During the six weeks of this experiment 3 hens of the total 47 hens had infertile eggs in more than one week. The maximum number of infertile eggs for any hen was two. Four hens had this many, one from each treatment level. The total numbers of infertile eggs for the pretrial period were 1, 2, 2 and 3 for the P0, P1, P3 and P5 levels respectively. The total numbers of infertile eggs during the trial period were 2, 4, 6 and 2. It should be noted that those levels with the highest fertility during the pretrial period had the lowest mean index number for the trial period. This is because an infertile egg during the pretrial period increased the index number in all four weeks of the trial period whereas 19 20 r some on» no soaps cnmvnwpm H Acownon Hmwnponmv mPHHHpnom name no: an cmcH>Hc Axes: Huanpv MPHHHpHoH m.onap come no use: u hoped: NoocH mac.” ewe.“ mecca mac.” mmo.e mmo.. cmo.e omo.e rec.” emo.u ewe.“ smog“ mace“ oeo.e emo.e cmo.e cmo.e eeo.e mm.em we 000m m 5 ..+. m8.“ m3.“ who.H moo.” seo.e 000.2 mmo.e mmo.e omm.o sm.om me coon 4N0.” .mo.u mmo.u amen“ 040.” 240.? seo.e 4mm.o seo.e mso.e mm.sm me 0002 m5...” mac..." So.” So...” Eon“. 040.? mmm.o emo.e smo.e sao.e me.mm e, o eoaaom e m N e coasom noaam Asaav Hanna Hoaseosa mo no .oz mama mo soc: apafiaesom mxmm3 HGHHB gm: msoamuo>soo swoops a“ oopsomonm name new: msoxoflno an apaaaeaoe so “gamma cesacroaa Aaawocaanpoumvao maaooou mo message .m canoe 21 Table 4. Analysis of variancea - fertility m “—1-! Level of Source d.f. M.S. F-Value Significance Treatment Level (L) 3 0.011 0.478 N.S. Week (W) 3 0.008 0.348 N.S. L x W 9 0.011 0.478 N.S. Within 172 0.023 aCalculated in arcsin conversions 22 an infertile egg during the trial period decreased the index number only for that week. Egg Production Tables 5 and 6, pages 23 and 24 show that there were no significant differences in egg production when DEHP was added to the feed. Only the PO level had a steady decline in egg production during the trial period although all four levels declined in egg production during the last week of the trial period. Because of the method of analysis, all three levels with DEHP added to the feed showed an increase in egg production. When comparing the actual differences in hen day egg production, only the P3 level showed an increase in egg production. Both the P1 and P5 levels' egg pro- duction decreased. This variation is reflected in the standard errors of the different index means. Feed Consumptign The analysis of variance for feed consumption shown in Tables 7 and 8, pages 25 and 26 indicate that DEHP, when added to the feed, reduced feed consumption significantly. Table 7 shows that there was a significant reduction in feed consumption after the first week of the trial period. This table also shows that there were significant differences due to the treatment level. The overall level effect indicates the P level had 0 significantly higher feed consumption than either the P3 or I gm Min‘n‘n‘: v..— A . ‘ . 23 name one no nouns cascades + Aboanom awauponav sofiposuong wwo name no: mp wooH>Hc Axum: Hafinpv nofiposvonm wMo m.cpap nose no use: a Renaud Hoan e neon“ .mo.+ Pmo.u mecca Nmm.o oeo.. coo.e Foo.e 2.0 .+. N9.” 63.” Re.” Re.” moo e mom.o omo.e mom.o Nmo.e mmm.ee N. 000m 30 H No...” 30..." or e .N. 2.0.... moo.e eem.o Neo.e omo.P 040.? FN>.ee NP coon nmo .+ $0.... of .H m8...” coo...” moo.e omm.o sso.e ono.. enm.o eme.mm Ne coo. N8... boo.“ N8.“ 30.... m3.” st.o on.o sea.o erm.o FEma omN.No .. o ceases e n N e coasom ocean Assay Hosea Hoasoosm no No .oz mama No one: .N.m soc: mxooz Hague unofimnobnoo canons an copsomoam dead sea: msoxoano as soaeoaeosa was so Ammmnv oeoHosesa Aaawosaaseoquao msaeoom No neooeem .m canoe 24 F4” Table 6. Analysis of variance8 - egg production Level of Source d.f. M.S. F-Value Significance Treatment Level (L) 3 0.048 0.358 N.S. Week (W) 3 0.055 0.410 N.S. L x W 9 0.040 0.299 N.S. Within 172 0.134 aCalculated in arcsin conversions 25 apaaanoposa so Hosea am one go economman mapsmoflwwsmam ohm panama ananomnoasm psmummmfiu m msfl>mn mammzm some map mo House unsccmpm.fl Acoaama Haaspmnav :ofipmasmnoo boom name no: as cooH>Ho Axes: HmHHpv soapmssmcoo noon n.6qu come no ado: u posses soanP Nee.“ oeo.u_ oeo.u. ocean ammm.o absm.o aNmm.o Unomad moo.H MNo.H NNo.H eNo.H 2N0.“ oeeN.o eem.o sem.o nem.o mmN.o a.mae Ne 000m Nee.” 6N0.“ NNo.H_ eNo.H mNo.H pomm.o sem.o mom.o mmN.o smm.o m.mee Ne coon rec.” mNo.u mNo.u. NNo.H_ 0N0.“ poONm.o mNm.o mam.o mmN.o sbm.o a.moe Ne 0002 No.” No.” No.“ No.“ 30.... seem.o on.o Nmm.o Nem.o emmm.o e.moe F? o Noonwom e n N e eoaaom madam Asaav Hesse Heaaeosa No No .oz ammo no nos: .o.a soc: egos: Hoses msoxofino a“ Asmv soaeassnsoo once so Aammmv oesacresa Afiaxocaareoquae Naaeooe No neoomem .s oases Table 8. 26 Analysis of variance - feed consumption Level of Source d.f. M.S. F-Value Significance Treatment Level (L) 3 0.780 65.000 0.05 Week (W) 3 0.056 4.667 0.05 L x W 9 0.003 0.250 N.S. Within 172 0.012 27 P5 levels, and that both the P1 and P3 levels had signifi- cantly greater feed consumption than the P5 level. No significant differences existed between the PO and P1 levels or the P1 and P3 levels. During the trial period more feed wastage was noticed than during the pretrial period although no changes in eating habits were seen. The amount of feed wasted was not r“ 4 ‘ul.. measured. Feed consumption for each individual hen remained com- paratively the same as its previous weeks average until the third day of the first week of the trial period. At that {L time the P5 level showed a marked decrease in feed consump- tion. 0n the fourth day of the first week all levels except the PO level had significantly reduced feed consump- tion. At the end of the first week of the trial period 5 hens out of 11 from the PO level, 7 hens out of 12 from the P1 level, 9 hens out of 12 from the P3 level and 10 hens out of 12 from the PS level consumed less feed than they had the previous week. Feed consumption did not decrease until 3 to 4 days after the introduction of DEHP into the feed. This indi- cates the possibility that the reduction in feed consump- tion may have been due more to the physiological effects of DEHP on the eating centers in the hypothalamus than to palatability. 28 Body Weights The effects that DEHP had on body weights of hens in this experiment are presented in Tables 9 and 10, pages 29 and 30. Table 10 shows that DEHP had a significant effect at the treatment level. Both the PO and P1 levels had mean gains at the end of the trial period. The P level and the P level showed 3 5 re steady decreases in body weights during the trial period. J't‘ “Ll. The superscripts indicate that the PO and the P1 levels had significantly higher body weight changes than the PS level, but only the PO level was significantly greater than . the P3 level. The mean net changes in body weights are closely associated with the combination of egg production and feed consumption. During the pretrial period both the P3 and P5 levels showed much greater average gains in body weights. They also consumed approximately 10 grams more feed per day and produced approximately 6% more eggs than did the P0 or P1 levels. With the addition of DEHP to the feed a signifi- cant decrease in feed consumption was observed although there was not a significant change in egg production. Hatchability The effects that DEHP had on hatchability are shown in Tables 11 and 12, pages 31 and 32. Table 12 shows that hatchability was affected at the treatment level only. The P level had significantly higher hatchability than either 0 29 a..H.ponosa 0o Hose. *0 on» as ecoummmwc mapcsowm.cmflm one Hopped pQHHOmaomzm #:0ANMMN0 w wsa>wn mnsozm uses on» no nouns cascades H_A00HH0Q Hmfinponmv vnmflo3 hoop name no: as 000.».0 Axes: Haunpv pnmfioz heap n.0an £000 Ho :00: u Hopadc chsH. 000 .H 000.... 000.“. 000.“. m00.o mmm.0 000.0 000.. 000.H 0.0.” N.0.u_ 000.“ 000.H osm0.0 0.0.0 000.0 .00.0 000.0 N0.N N. 0000 000.“ N.0.H N.0.H 000.“ N00.“ opmm0.0 ss0.0 0.0.0 400.0 .00.. 0.NN N. 0000 000.“ 4.0.“ 0.0.” N.0.H 000.“ poN00.. 000.0 e00.. 000.. .00.. 0N.N N. 000. 400.H 0.0.”. 000.H. 000.“ .00.” 0000.. 000.. 000.. 000.0 .e00.. 00.N .. 0 NooHNoN e n N . coasoa nopam Asaav Heaps Ho.a.os0 No No .oz mama No ado: .3.m aces neon: adage .smv nermHoz hoop so .Nmmnv opoaorsra .Hawocaaseoqu.o waaooom No neoommm mqoxofino ca .m manna 3O Table 10. Analysis of variance - body weights Level of Source d.f. M.S. F-Value Significance Treatment Level (L) 3 0.0037 2.85 0.05 Week (W) 3 0.0017 1.31 N.S. L x W 9 0.0008 0.62 N.S. Within 172 0.0013 31 0008 90: an omcabflc 0x003 H0anpv thHHQ0nop0n 0.0909 2000 no 000: u amped: chsH . . r...“ 3:9? .0. n_. 00.4.000000 0o asses 00 0:» p0 900nmmmflo zapq0ofimasmflw 090 nmppma paanomammsm pqmnwmmwu 0 wcfl>0g 0:002m 0008 one no Hohhm 0H0cn0pm H.0000nom H0anmnmv hpwawp0sop0s 0 040.0 N00.. 000.“ 040.0 000.0 000.0 0N0.0 N00.0 040.“. 400.H N...H. 0...” 000.“ 0000.0 000.0 000.0 00N.0 004.0 04..0 N. 0000 040.“ 4.0.0 000.” 04..H_ 000.“ 0400.0 0.0.0 040.0 0N0.0 N00.0 0..00 N. 0000 000.H N4..H .4..H 00..” 0...” 00000.0 000.0 000.0 400.0 0.0.0 04.00 N. 000. 000.” 0...” N40.H 0...” 00..” 0000.0 000.0 000.0 0.0.. .000.0 00.00 .. o Neo.000 4 0 N . soascm 000.0 .saav 00.00 00.00000 we 00 .oz 0000 no 000: 00.000000000 0x003 H0099 s00: mnoflmnobqoo :000H0 :0 copcmmonm 0900 spa: mnmxofiso Bonn 0000 Ho 00.H.000000s so Ammmnv 0.0400000 .0000040000uN000 00.0000 no 0.00000 ... earns 32 Table 12. Analysis of variancea - hatchability A { i Level of 7 Source d.f. M.S. F-Value Significance Treatment Level (L) 3 3.426 23.785 0.05 Week (W) 3 0.026 0.181 N.S. L x W 9 0.052 0.361 N.S. Within 170 0.144 aCalculated in arcsin conversions 33 the P3 or P5 levels and both the P1 and P3 levels were higher than the P5 level. Birds in all four levels had a 'decrease in hatchability during the trial period as com- pared to the pretrial period. In the case of the PO level, all 8 eggs placed in the incubator from one hen hatched during the pretrial period, but only 2 out of 16 eggs from this bird hatched during the F, .4. all ‘ trial period. The hatchability of this hen had the overall effect of reducing the mean hatchability of the trial period index number by 0.084. This hen was left in the A analysis because it was impossible to tell if hens in other ; levels may have had a similar type hatchability response. Dead embryos from eggs whose dams had consumed DEHP had small petechial hemorrhages on the integument of both the head and body regions. No other gross abnormalities were observed. The cause of the hemorrhages is unknown. Griminger (1964) observed hemorrhagic areas on dead embryos from hens whose diets were deficient in vitamin K. This suggests the possibility that somehow vitamin K was inter- fered with by DEHP or a metabolite of DEHP. There are other possible reasons for the observed hemorrhages. Peters and Cook (1973) reported that when DEHP was injected into female rats excessive bleeding at parturition was observed. It was also noted that less than 45% of all dead embryos during the treatment period in the P0, P1, and P3 34 levels died in the first fourteen days of incubation whereas 61% of the dead embryos died during this period in the P5 level. Figure 1 on page 35 shows the linear regression curve on hatchability to be S" = 14.930 + 0.168x where 3? equals the estimated embryonic mortality in arcsin conversions and x equals the amount of DEHP consumed in milligrams per kilogram body weight. The calculated correlation coeffi- cient (r = 0.571) has been calculated by the F test to have a P value less than 0.05. ' ”Trim-l" - . 35 ammo no 000H0pnos canohupam H0 0>nso scammmnwon H0osaq FIG—w} >oOm .8. «mm OmxbszU stub 62 0.0400000 .H000000000anae 009 0:05 sycamon 090:3 psoo mawsam 9H000 Bonn con CON 3N Ova CNN 8N cup 00.. 0: ON. 000 agave“ .— . U 05““ Illlllll. o O D— On on 0? ch 00 .. 0000.0 (SNOISHANOD NISDUV) Ainvmow amouawa SUMMARY The purpose of this experiment was to measure five parameters when di(2-ethylhexyl) phthalate (DEHP) was added to the feed of 45 week old Single Comb White Leghorn hens at levels of 0, 1,000, 3,000 and 5,000 ppm. The five parameters investigated were fertility and hatchability of eggs, egg production, body weights and feed consumption of v. . x. u I r 1 I m f. a r: I . the hens. The analysis of variance indicated that no significant differences existed in two of these parameters, fertility of eggs and egg production. The analysis also showed that feed consumption and body weights of the hens plus hatch- ability of their eggs were significantly decreased at levels greater than 1,000 ppm of DEHP in the feed. It was noticed that the dead embryos, from eggs where the hens were fed DEHP, had petechial hemorrhages on the integument of both the head and body regions. No other teratogenic effects were observed. 36 BIBLIOGRAPHY BIBLIOGRAPHY Autian, J., 1973. Toxicity and health threats of phthalate esters: review of the literature. Environ. Health Perspect. 4: 3-26. Calley, D. J., J. Autian and W. L. Guess, 1966. Toxicology of a series of phthalate esters. J. Pharm. Sci. 55: 158. Carpenter, C. P., C. S. Well and H. F. Smyth, Jr., 1953. Chronic oral toxicity of di(2-ethylhexyl) phthalate for rats, guinea pigs, and dogs. Arch. Ind. Hyg. 8: 219. Ins-u. Cifrulak, S. D., 1969. Spectroscopic evidence of phthalates in soil organic matter. Soil Sci. 107: 63. Corcoran, E. P., 1973. 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Hyg. Toxicol. 27: 130. Singh, A. R., W. H. Lawrence and J. Autian, 1972. Teratogenicity of phthalate esters in rats. J. Pharm. Sci. 61: 51. Sturkie, P. D., 1965. Avian Ph siolo . Second edition. Cornell University Press, Itfiaca, New York. 765 pp. Taborsky, R. G., 1967. Isolation studies on a lipoidal portion of the bovine pineal gland. J. Agr. Food Chem. 15: 1073. Tepper, L. B., 1973. Phthalic acid esters-~an overview. Environ. Health Perspect. 3: 179-182. Thomas, G. H., 1973. Quantitative determination and confir- mation of identity of trace amounts of dialkyl phthalates in environmental samples. Environ. Health Perspect. 3: 23-28. Wildbrett, G., 1973. Diffusion of phthalic acid esters from PVC tubing. Environ. Health Perspect. 3: 29-35. MICHIGAN STATE UNIV 1 m | I. IIIIIIIIIIIJIIIIIIIIM 3 1293 30