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D . c: :iozivti..1y)0{v.{lxb¢1i "UV. rtvf Y4)Vu v‘olhtisvfi‘vilhfionln o liltifi‘ s . gfivl. c .rlvvol I:..30I§.0E§1g§§$p’i‘ it: VOA-0613A! .volvlt‘LthIE I; £ikl;§f!t¢ itgzlrvgft “Wu"??? ini‘tgtfitxu:tr£zt7rfrveéuzv. . . It"? in: V! rtzlvlvfiLVDIl.’ It'lgfavlggnhti hit '1’»! 9...}? 7.. \\ r .8 .. . Z 1:15... . E~ixfi§fl : if? £§1§£§E fit all: . .. £Lti§e.l..§. A4115 % L. . I! ~ ti 3‘ I ll? '% | big. 9...}.95555. filtlikh . . , \s tfbbllrllfftn. .11.: L . . 1 iiz:§\;\z iizg‘ggafiz. iiiSjl‘c-W \ii\$\l.‘)(yfil\ ,i.‘ ‘ \ v5 . l .. ... x . . . THESIS Illll Hill H l‘lllllllVlfllllllll'llfllllflll 3 1293 01094 2054 This is to certify that the thesis entitled Toxicity of Dicyclopentadiene and Diisopropyl Methylphosphonate to the Bobwhite Quail (Colinus virginianus) presented by Kathy S. Howell has been accepted towards fulfillment of the requirements for M.S. degree in Poultry Science (904ij fiv’ Major professor Date May 22, 1981 0-7639 LIBRARY Michigan State University 6:7 ' gig .4 -“T tifaugfifiux L Wis dull” .‘J' OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS : Place in book neturn to remve charge from circulation records Jim . ergo TOXICITY OF DICYCLOPENTADIENE AND DIISOPROPYL METHYLPHOSPHONATE TO THE BOBWHITE QUAIL (COLINUS VIRGINIANUS) by Kathy S. Howell A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Poultry Science 1981 ABSTRACT TOXICITY OF DICYCLOPENTADIENE AND DIISOPROPYL METHYLPHOSPHONATE TO THE BOBWHITE QUAIL (COLINUS VIRGINIANUS) by Kathy S. Howell Three experiments, an LD50, an LCSO’ and a chronic study, were conducted to determine the toxicity of dicyclopentadiene (DCPD) and diisopropyl methylphosphonate (DIMP) to the Bobwhite quail. The 14-day LD50 values for adult Bobwhites dosed per 05 with DCPD or DIMP were 1010 i 80.0 mg/kg and 1000 i 68.2 mg/kg, respectively. LC50 values, based on five day dietary treatment followed by three day withdrawal, could not be determined for either chemical because of insufficient mortality. Predicted zero feed intake levels of the quail were 73028 ppm DCPD and 77959 ppm DIMP. The chronic study was conducted over a single reproductive period. The dietary levels used were; 400, 1250, and 4000 ppm DCPD plus a control and 1200, 3800, and 12000 ppm DIMP plus a control. The parameters measured included adult mortality, feed consumption, body weight change, egg production, incubation para— meters, blood parameters, internal organ weights, and progeny livability. No consistent treatment-effect results were observed for either chemical. ACKNOWLEDGEMENTS The author would like to express her gratitude and appreciation to all those that lended their time and efforts toward the comple— tion of this research. Special thanks are given to Dr. R. K. Ringer, Advisor, for his continued guidance and consultation; to Dr. R. J. Aulerich, for his constant encouragement and advice; and to Dr. T. H. Coleman for his patience and interest. For their many hours of assistance throughout the study, the author would like to give special recognition and thanks to Paul Bowen, Ross E. Jones, Terrance J. Kavanagh, and B. Kay Trosko. This research was supported by a grant from the US Army Medical Research and Development Command, Washington, DC. ii TABLE OF CONTENTS Page Introduction . ...... .... ....................... . ........... 1 Statement of the problem ................. .............. . 1 Background .................................... ........ .. 1 Literature Review ....... ....... .... ...... .... .......... ... 3 Objectives .... ............................................ 9 Procedure ................ ...... . ....... .. ................. 10 Experiment 1 ............. ............................ ... 10 Testing .............. .................... . ..... . ...... 11 Statistical analysis ....... .......... . .............. .. 12 Experiment 2 ... ......................................... 13 Testing .... ........ .. ................................. 13 Statistical analysis .................................. 14 Experiment 3 ................................... ......... 15 Testing .............................. ................. 15 Egg collection,xstorage, and incubation ...... ......... 17 Hematological preparation ............................. 18 Statistical analysis .................................. 19 Results ................... ................................ 21 Experiment 1 .. ............................... . .......... 21 ‘ Rationale . ..... ........ ............. ...... ............ 21 Results ......................................... ...... 24 Experiment 2 .... ......................................... 32 Rationale . ............................................ 32 Results ............................................... 34 DCPD ........... ..................................... 34 DIMP ... ........ .. ............................. . ..... 43 Experiment 3 .. ..... ...... ............................... 53 Rationale ......... ..... . .............................. 53 Results ............................................... 56 DCPD . ............................................... 56 DIMP ................................................ 73 Discussion ....... ......................................... 9O Experiment 1 . ........................................... 9O DCPD ..... .............. . ............................ 9O DIMP ... ............................................. 94 Experiment 2 ............................................ 96 Experiment 3 ................................... . ........ 101 Conclusions .......... .. ................................... 111 iii Table Table Table Table Table Table Table Table Table Table Table Table Table 10. ll. 12. 13. LIST OF TABLES DCPD LD50 values from the literature ........... DIMP LD50 values from the literature ........... Mortality results of DCPD and DIMP LD50 range— finding trials ................................. Mortality of adult Bobwhites during a 14-day observational period following a single per os dosing with DCPD ............................ Mdrtality of adult Bobwhites during a 14-day observational period following a single per os dosing with DIMP ............................ Body weight changes of adult Bobwhites during 14-day observational period following a single per os dosing with DCPD or DIMP ................ Feed consumption of adult Bobwhites during a 14-day observational period following a single per os dosing with DCPD or DIMP ................ Calculated DCPD intake over five days and mortality over eight days for 14-day—old Bobwhite chicks on LC50 trial .................. Body weight change of Bobwhite chicks during the three-day period after withdrawal of DCPD— treated diet ................................... Calculated DIMP intake over five days and mortality over eight days for l4-day-old Bobwhite chicks on LC50 trial .................. Body weight change of Bobwhite chicks during three-day period after withdrawal of DIMP— treated diets .................................. Effect of feeding DCPD at various levels in the diet on body weight changes of Bobwhites for the 10 weeks prior to the onset of egg production ..................................... Effect of feeding DCPD at various levels in the diet for 28 weeks on body weight change of Bobwhites during the 10 week reproductive period ......................................... iv Page 5 7 23 25 26 30 31 40 49 58 59 Table Table Table Table Table Table Table Table Table Table Table Table 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Effect of feeding DCPD on mortality of Bobwhites during the 28 week chronic study .... Effect of feeding DCPD at various levels in the diet for 28 weeks on incubation parameters of Bobwhite quail eggs laid in March, April, and May, 1977 ................................. Effect of feeding DCPD at various levels in the feed for 28 weeks on shell thickness values of adult Bobwhite eggs ................. Effect of feeding DCPD at various levels in the diet for 28 weeks on liver and gonad weights of adult Bobwhites ...... .............. Effect of feeding DCPD at various levels in the diet for 28 weeks on organ weight in adult Bobwhites ............................... Effect of feeding DCPD at various levels in the diet for 28 weeks on hemoglobin and hematocrit values of adult Bobwhites .......... Effect of feeding DCPD at various levels in the diet for 28 weeks on mean corpuscular hemo- globin concentration of adult Bobwhites ....... Effect of feeding DCPD at various levels in the diet for 28 weeks on leukocyte counts of adult Bobwhites ............................... Effect of feeding DIMP at various levels in the diet on body weight changes of Bobwhites for the 10 weeks prior to the onset of egg production ....... .... ......................... Effect of feeding DIMP at various levels in the diet for 29 weeks on body weight changes of Bobwhites during the 10 week reproductive period ........................................ Effect of feeding DIMP on mortality of Bobwhites during the 29 week chronic study .... Effect of feeding DIMP an: various levels in the diet for 29 weeks on incubation parameters of Bobwhite quail eggs laid in March, April, and May, 1977 Page 60 63 64 67 68 69 7O 72 75 76 77 80 Table Table Table Table Table Table Table Table 26. 27. 28. 29. 30. 31. 32. 33. Effect of feeding DIMP at various levels in the diet for 29 weeks on shell thickness values of adult Bobwhite eggs ..... ............ ... ....... Effects of feeding DIMP at various levels in the diet for 29 weeks on gonad and liver weights of adult Bobwhites .... ................... Effect of feeding DIMP at various levels in the diet for 29 weeks on organ weight of adult Bobwhites ..... ............................. Effect of feeding DIMP at various levels in the diet for 29 weeks on hemoglobin and hematocrit values of adult Bobwhites ............. Effects of feeding DIMP at various levels in the diet for 29 weeks on mean corpuscular hemo- globin concentration of adult Bobwhites .......... Effect of feeding DIMP at various levels in the diet for 29 weeks on leukocyte counts of adult Bobwhites .................................. The LD50 values of twelve compounds for the Bobwhite quail at various ages ................... Median lethal concentrations (LC50's) of various pesticidal chemicals for Bobwhite quail chicks two to three weeks of age ........... vi Page 84 85 86 88 89 91 97 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. 11. 12. LIST OF FIGURES Page Percent mortality of adult Bobwhites, equal numbers of each sex, given a single per os dose of DCPD and observed for 14 days post treatment ....................................... 27 Percent mortality of adult Bobwhites, equal numbers of each sex, given a single per os dose of DIMP and observed for 14 days post treatment .... ...... ... ........... .. ............. 28 Effect of feeding various levels of DCPD in the diet for five days on feed consump- tion of l4-day-old Bobwhite chicks .............. 35 Regression equation of the data shown in Figure 3 ....... ............. ... ................. 36 Effect of feeding various levels of DCPD in the diet for five days on body weight change of l4-day-old Bobwhite chicks ... ......... 37 Regression equation of the data shown in Figure 5 ........................................ 38 Feed consumption of Bobwhite chicks fed untreated feed during three day post-treatment period after withdrawal of DCPD treated diets ... 41 Effect of feeding various levels of DIMP in the diet for five days on feed consumption of l4-day-old Bobwhite chicks ................... 44 Regression equation of the data shown in Figure 8 ........................................ 45 Effect of feeding various levels of DIMP in the diet for five days on body weight change of l4-day-old Bobwhite chicks ............ 46 Regression equation of the data shown in Figure 10 .. ........... . ......................... 47 Feed consumption of Bobwhite chicks fed untreated feed during three day post-treatment period after withdrawal of DIMP treated diets ... 50 Vii Figure 13. Effect of feeding various levels of DCPD in the diet for 28 weeks on feed consumption Of adult BObWhiteS O O O O ..... O O O 0-: ..... O O ......... 57 Figure 14. Effect of feeding various levels of DCPD in the diet for 28 weeks on egg production of adult Bobwhites in their first reproductive cycle ........................................... 61 Figure 15. Percent livability of offspring of adult Bobwhites fed various levels of DCPD in their diet for 28 weeks . ..... .... ............... 65 Figure 16. Effect of feeding various levels of DIMP in the diet for 29 weeks on feed consump- tion of adult Bobwhites ......................... 74 Figure 17. Effect of feeding various levels of DIMP in the diet for 29 weeks on egg production of adult Bobwhites in their first reproductive cycle ........................................... 79 Figure 18. Percent livability of offspring of adult Bobwhites fed various levels of DIMP in their diet for 29 weeks ............................... 82 viii LIST OF APPENDIX Page Appendix A: Chemical structures and alternate names for DIMP and DCPD .............................. 112 Appendix B: Analysis of feed ............................... 113 Appendix C: Diet Preparation ..... ....... ................... 115 Appendix D: Preparation of Wright's stain and buffer ...... 0......OOOOOOOOOOOOOO00.00.000.000. 118 Appendix E: Preparation of Drabkin's reagent . ..... ... ...... 119 Appendix F: Determination of hemoglobin concentration ...... 120 Literature Cited ....... . ...................... . .......... ... 121 General References .......................................... 126 ix INTRODUCTION Statement of the Problem Increasing pressure from environmental lobbying groups and recently passed environmental legislation, bOth due to continued interest in the preservation of the balance of ecology, have forced those involved in the use, storage, and/or disposal of potentially hazardous chemicals to exercise more caution in their activities. Army installations, often employed in the production, storage, and disposal of chemicals, are examples of potential ecology offenders working toward environmental clean-up. Recent activities of the U.S. Army have included the identification and toxicological and ecological hazard assessment of potential environmental pollutants discovered in the surface and subsurface water near and around military lands. Two compounds, dicyclopen- tadiene (DCPD) and diisopropyl methylphosphonate (DIMP), have been identified at the Rocky Mountain Arsenal, but not yet evaluated for toxicological prOperties. Background To reduce potential hazards and incidence of toxicological mishaps allegedly related to the Rocky Mountain Arsenal, toxicological studies are being conducted on several compounds identified at or around the arsenal. Two compounds, DCPD and DIMP, are being investigated to determine their toxicity to both mammals and birds. DCPD is employed as a starting material for the production of chlorinated insecticides, and is used, as is its monomer, cyclopentadiene (CPD), in the manufacturing of plastics, elasto— mers, cycloaliphatic epoxides in resin coatings, rubber hydro- carbons, and other products. Both chemicals have been used at the pesticide facility leased by Shell Chemical Company at Rocky Mountain Arsenal. DIMP is a by-product produced during the manufacture of GB (Isopropyl methylphosphonofluoridate), a nerve gas. There is no evidence that DIMP is either a metabolite or environmental product of GB. GB was manufactured in a plant at the Rocky Mountain Arsenal. Several reasons for the investigation of the toxicity of DCPD and DIMP exist. These reasons are: the paucity of toxi- cological information on either chemical, the presence of both chemicals in sampling wells and surface water both on and off the Rocky Mountain Arsenal, and the occurrence of unexplained mortalities of wild and domesticated animals on or near the arsenal. In addition, the Shell Chemical Company has stated that accidental spillages of chemicals have occurred at the arsenal and the Committee on Military Environmental Research on the Status of Research into Biological Effects of Environmental Contaminants at Rocky Mountain Arsenal has described various improper chemical waste disposal at the arsenal (citation unpublished). Bobwhite quail were selected as the test subject of this study because they are a representative species at the site of contamina- tion, they are readily available for toxicological testing, they represent an upland game bird, and the Environmental Protection Agency has accepted the Bobwhite as a suitable species for toxi- cological studies. LITERATURE REVIEW Dicyclopentadiene (DCPD) (Appendix A: Chemical Structure and Alternate Names for DCPD and DIMP) is a liquid at room temperature but becomes a waxy solid after cooling to approximately 0°C. DCPD melts at 32°C and has a bulk density of 0.982 g/cc at 20°C (Shashkina, 1965). The calculated distribution coefficient (oil/water) of DCPD is approximately 60,000 and so it is considered to be insoluble in water (Shashkina, 1965). Hart (1976) reported that DCPD had appreciable volatility. Humans can detect a slight odor of DCPD at 0.0004 mg/l, and the odor becomes more unpleasant and can cause headaches and nausea at 0.003 ppm (Shashkina, 1965). Lethal dose values, both acute and dietary, that have been determined for DCPD are listed in Table 1. Animal species utilized as test subjects include the rat, mouse, rabbit, and Mallard duck. No toxicological information on humans could be found. In an acute study conducted by Shashkina (1965), reactions of rats exposed to DCPD vapor were categorized as, "at the moment of exposure" and "in the following period". During the exposure period, narcosis, clonic toxicity, motor disturbance, and excitability were observed. During the following period excessive excitability upon contact or noise, spasms, agressive- ness, and hemorrhagic discharges from the eyes and nose were reported. Body temperature and blood pressure were lowered by 24 and 6.2 percent, respectively. Pathological changes in the rats, post exposure, were pronounced plethora of the internal organs, internal hemorrhage in the lungs and brain, emphysema of the lungs, dystrophic changes in the canalicules of the kidney, dystrophy of the liver, lipoid depletion of the adrenal cortex, and hyperfunction of the thyroid gland. All of the observations in the above paragraph were taken from results reported by Shashkina (1965). Over a six month chronic DCPD inhalation study involving rats, Shashkina (1965) observed a general decrease in the number of leukocytes, no consistent change in the amount of hemoglobin in the blood, a decrease in the number of erythrocytes, and a rise in blood pressure. Observations were made relative to a control group. Pathological changes of the rats at the termina- tion of the study were: proliferative and sclerotic changes in the trachea, bronchial tubes, and lungs plus a wrinkling of the cells in the cerebral cortex and the cerebrum. Kinkead 35 21. (1971) conducted several inhalation tests utilizing rats, guinea pigs, rabbits, mice, and dogs to determine the mammalian toxicity of DCPD. In acute studies, Kinkead gt 31. (1971) reported dogs, guinea pigs, and rabbits more resistant than rats which were more resistant than mice but responses of all species were generally dose related and followed a pattern of eye irritation, lack of normal coordination, convulsions (if death followed), and normal body weight gain. Chronic study results reported by Kinkead et 31. (1971) include kidney lesions in male rats inhaling 73.8 and 35.2 ppm DCPD and inconsistent changes in acid and alkaline phosphatase values of dogs inhaling 23.5 ppm DCPD. Hflm H\©E a coflumamraH n rcH m AmocouflummmuucH I mH N 4.1. .us soon mx\ma H Ahmad .mGCObV mHQMCHEumumpcD ammo pumaamz Iakaa ..mm mm ommxcflsv Has rcH uflnnmm Ammlw hmnflxnmmnmv «45.0 mk.ouaw.o acH mmsoz Aasma ..Hm um tmmxcaxv mas raH mmso: Immmm.hmcflxgmmrmc emm.H mo.aunm.a haH umm Aasma ..Hm.wm ommxcflmv mmm ncH umm Aasma ..Hm mm ommxmamv mmm ncH umm Aasma ..Hm pm ommxcfimc owe ham :mmm mrcH ham lmwmfl ..Hm mm zusEmo oaks ommsauomam smegma panama Amoma “MHMIum spasmc omvv omam Iowan Hmsumo pannmm Aasma ..Hm um ommxcflmc omom ommm uoaam Hmsumo panamm cm H on Ahhma .mmGObv oooovA Hmuo pumaamz lemma .uummc omm mom nona Hmuo o mmsoz ll II.AmhmH .uummv omH mom Imma Hugo o wmsoz Avnma ..Hm um cmmamumflunov oom mH mmsoz Aakmmllnwm mm wmmxcflmc cam 8H umm karma ..Hm um ammcmumaunoc oom NEH umm Amhma .uummv mum msv umom ammo o umm hwsmw .uummc omm mam Iowa Hugo o umm Imoma hhHMIum spasms oav omm loam Hmuo umm karma ..Hm um cmmxcamv mmm msv ummm ammo pom moonmummmm Homnq Hmmcmm bongo: HmEH:< omoo no name sufloflxoa .H manna Horace Gerarde, cited by Kinkead at 31. (1971), reported that the principal pathological findings in rats dosed with DCPD were generalized congestion, hyperemia, and focal hemorrhage in many tissues including the kidney, intestine, stomach, bladder, and particularly the lungs. Gage (1970) conducted inhalation studies with rats at levels of 100, 250, 1000, and 2500 ppm DCPD. Gage reported no adverse effects at 100 ppm with 15 6-hour exposures; weight loss, diffi- culty in breathing, tremors, hypersensitivity, one death and normal blood pressure at 250 ppm with 10 6-hour exposures; eye and nose irritation, dyspnea, incoordination, tremors, hyper— sensitivity, and no survivors at 1000 ppm with l 4-hour exposure; and the same symptoms as the 1000 ppm group but with only one death at 2500 ppm with l l—hour exposure. Jones (1977) conducted DCPD toxicity tests with Mallard ducks and reported an undeterminable LD50, an undeterminable LC50, and no adverse effects after feeding 32, 100, and 320 ppm DCPD rations for 22 weeks during a chronic study. Diisopropyl methylphosphonate (DIMP) (Appendix A: Chemical Structures and Alternative Names for DCPD and DIMP) is an organo— phosphorus compound, liquid at room temperature with a bulk density of 0.976 g/cc at25%2(Dacre, 1975) and water soluble at l—2 g/l (Ringer, 1978). Odor detection limits have not been reported. Thus, it is not known if DIMP could unknowingly be ingested. Lethal dose values that have been determined for DIMP are listed in Table 2. Table 2. Toxicity Data on DIMP. LD50 Animal Method Range mm3/kg References Rabbit IV1 179- 280 224 (Jacobson, 1953) Rabbit Pc2 >200 (Jacobson, 1953) Rabbit SC3 >100 <200 (Ford-Moore and Perry, 1948) Mouse IP4 >250 (Horton, 1948) Mouse (M) Oral 903-1201 1041 (Hart, 1976) Mouse (F) Oral 1165-1594 1363 (Hart, 1976) Rat SC >200 (Ford-Moore and Perry, 1948) Rat (M) Oral 1125 (Hart, 1976) Rat (F) Oral 747- 914 826 (Hart, 1976) Mallard Oral 1414—1566 1490 (Jones, 1977) 1 IV intravenous 2 PC percutaneous 3 SC subcutaneous 4 IP intraperitoneal Jones (1977) reported an undeterminable LC50 value in young Mallard ducks fed DIMP-treated diets. Results of a 24-week chronic study with adult Mallard ducks showed increased feed consumption at 3200 ppm and decreased egg production at 10000 ppm DIMP in their diets. Jacobson (1953) reported moderate corneal damages in rabbits dosed with 0.25 mm3/kg DIMP. Hart (1976) found that application of an unspecified amount of DIMP to the eyes of rabbits produced variable results ranging from slight redness to clouding of most or all of the cornea. The symptoms persisted for up to seven days with subsequent complete recovery. Skin applications of 200, 632, and 2000 mg/kg DIMP to rabbits caused some erythema in some rabbits with complete recovery in ten days if the rabbit survived the systemic effects (Hart, 1976). Rabbit mortalities in the skin application studies were 25 and 75 percent at 632 and 2000 mg/kg, respectively. OBJECTIVES To determine the single oral acute LD50 and dose-response curve for DCPD and DIMP to the adult Bobwhite. To determine the eight day subacute dietary LC50 for DCPD and DIMP to the young Bobwhite. To determine chronic toxicity, including long-range effects on reproduction parameters, blood parameters, and general body changes, from feeding DCPD or DIMP to adult Bobwhites over their first reproductive cycle. PROCEDURE The research consisted of the determination of the lethal dose for 50% of the test subjects (LD50), the determination of the lethal dietary concentration for 50% of the test subjects (LC50), and a long term chronic study. All experiments employed the Bobwhite (Colinus virginianus) as a research subject, and were conducted in a windowless house at the Michigan State University Poultry Science Research and Teaching Center. The Bobwhites were procured from the Poultry Science Department, Michigan State University, East Lansing, MI 48824. EXPERIMENT 1 This experiment was designed to determine the single, 14-day, oral dose LD50 of diisopropyl methylphosphonate (DIMP) and dicyclopentadiene (DCPD) to the Bobwhite. Dosages were reported in mg/kg, a standard unit of measure in toxicological studies. mg/kg is equivalent to ppm of body weight. Adult Bobwhites, approximately one year of age, in non—laying condition, were utilized. The birds were maintained indoors in cages measuring 85.1 cm (1) x 89 cm (w) x 24.1 cm (h), 20 birds per cage. Cage space per bird was 379 cm2. Body weights of all birds were recorded succeeding a one-week holding period. A two-week acclimatization period followed. Body weights were again recorded at the termination of acclimatiza- tion to note any significant weight loss before range finding was initiated. ‘ Preliminary range finding was conducted to establish the approximate lethal dose and a geometric scale of dosages was 10 11 employed for the test to give a mortality range of 10 to 90 percent. Testing Birds used for testing were maintained on quail breeder diet from Michigan State University (Appendix B1: Analysis of Feed). The feed was free of antibioticsand medication. Feed and water were provided 29.1121EEE throughout the testing period with the exception of a lS-hour minimum fasting period before oral admini- stration of test chemicals. Weekly feed consumption was determined for each group. The DIMP test and the initial DCPD test utilized twenty birds, ten of each sex, per dose level. The additional DCPD test utilized 10 birds, five of each sex. Weights were recorded immediately preceeding the dosing, and on the third, seventh, and fourteenth days of the succeeding two-week observational periOd. Post treatment behavior was observed for one hour immedi— ately following dosing, again at 4-5 hours, and daily thereafter for the duration of the observational period. Administration was by drenching per os from a syringe with a length of polyethylene tubing attached to a needle. The length of tubing corresponded with the distance from the back of the oral cavity to the esophageal opening of the proventriculus. This insured a uniform location for the introduction of the test substances. The syringe was 1 cc, the needle 22 ga, 2.54 cm long, and the tubing measured 0.762 mm ID and 1.29 mm OD. Necropsies were performed on all birds, including controls, at the time of death or termination of the observational period. 12 A general gross inspection was performed with special emphasis on the digestive tract, liver, kidneys, heart, and spleen. Statistical Analysis The LD50 was analyzed by the method of Litchfield and Wilcoxon (1949). Weight changes were analyzed by least squares analysis of covariance with log transformation and the two-sided Dunnett t-test with modification for unequal replication. Feed consump- tion data were not appropriate for meaningful statistical analysis. E XPE RIMEN T 2 This experiment followed the subacute toxicity test protocol as described by Heath and Stickel (1965) and Heath §E_a1. (1972), to determine the minimum repeated oral dosage (mg/kg/day) of DIMP or DCPD that was lethal to Bobwhite chicks. Range finding pilot studies were conducted with both chemicals to determine the effect or noneffect on mortality, feed consump- tion, and body weight. Since the mortality that did occur appeared unrelated to the dietary levels of DIMP or DCPD, a geometric scale of dosages was utilized to determine the point of feed refusal instead of 50 percent mortality. Testing Randomly selected day old Bobwhite chicks were housed indoors in a Petersime Brood Unit2 and maintained on a standard quail starter ration (Appendix B2: Analysis of Feed), free of anti— biotics and medication. Feed and water were provided ad libitum. At 14 days of age the chicks were segregated into groups of ten birds of undetermined sex. Each group of birds was randomly assigned to one of twenty dietary treatments (ten treatments per chemical). At the initiation of the experiment one bird from each of the control groups and the low level DIMP group escaped. The experiment was conducted with nine birds in the latter groups. During the eight day test period, treated feed was fed for the first five days and clean feed was fed for the remaining three days. Feed and.water were provided ag_1ibitum throughout the test period. 2 Petersime Incubator Company, Gettysburg, Ohio 45328. 13 14 The test diets were prepared by dissolving the chemical in corn oil, and hand mixing with quail starter to make a premix. The premix was then added to a standard quail ration to yield the appropriate dietary level (Appendix C: Ration Preparation). The DIMP treated diets' chemical-corn oil solution constant was greater than two percent of the diet. The DCPD treated diets‘ chemical-corn oil solution constant was two percent of the diet. Both control diets consisted of two parts corn oil to 98 parts feed by weight. The ten dietary treatments used for testing each chemical were as follows: DIMP (ppm): 0, 4000, 8000, 12000, 16000, 20000, 24000, 28000, 32000, and 36000. DCPD (ppm): 0, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, and 18000. Body weights were recorded on days zero, five, and eight of the test period. Feed was weighed on days zero and five (treated feed) and days six and eight (clean feed) to provide estimates of average feed consumption. Observations on bird wastage were taken into account in determining the estimated point of zero feed con- sumption. Any signs of intoxication or abnormal behavior during the test period were noted. All birds that died during the trial, and those that survived until the termination of the experiment, were necropsied. Statistical Analysis Slopes of feed consumption, body weight change, and predicted zero feed consumption were determined by regression analysis. EXPERIMENT 3 This experiment was designed to determine the effects of continuous long term exposure of DIMP or DCPD to the adult Bobwhite and their progeny over a single reproductive cycle. Two identical testing rooms were utilized to separate the dietary treatments by chemiéal. Each chemical test consisted of four dietary treatment groups, three treated levels plus one control. Each treatment group consisted of one female and one male housed in a single cage replicated by fifteen. The birds were allowed a two week acclimatization period before the initia- tion of the experiment. Initially dietary treatments were as follows: DIMP: 0, 1200, 3800, 12000 ppm DCPD: 0, 400, 1200, 4000 ppm Subsequent mortality at the 3800 and 12000 ppm DIMP levels caused the reduction to 380 and 0 ppm DIMP, respectively. This decision was reached by general concensus of the principal investi- gator and the project officer (U.S. Army). Testing Test diets were prepared by the addition of a premix to a standard quail breeder ration to attain the appropriate dietary levels. (Appendix C2: Ration Preparation). The control diets for both DIMP and DCPD consisted of two parts corn oil to 98 parts feed by weight. The diets were fed to the birds for a mini- mum of ten weeks before the initiation of egg production and a minimum of ten weeks after the attainment of 50 percent egg 15 16 production. Feed and water were provided ad libitum throughout the entire experiment. Feed consumption was measured biweekly for the full duration of the experiment. Body weights were measured at 0, 2, 4, 6, and 8 weeks and at the termination of the study. Body weights were not measured during egg production to avoid any adverse effects that handling may have had on egg production. During the pre egg production period (Nov. 1 to Jan. 8) both testing rooms were maintained at approximately 18°C with light provided six hours per day. To induce egg production the lighting schedule was increased to 16 hours of light per day. This schedule was maintained throughout the production period (Jan. 8 to May 28). Temperatures of the test rooms during the production period ranged from 15°C to 28°C. Egg production, mortality, morbidity, and any observable clinical signs of intoxication were recorded daily. All birds, including those that died during the study and those that survived the test, were subjected to gross necropsy, with the following organ weights measured; liver, kidneys, pancreas, proventriculus, gizzard, gonads, heart, and brain. Spleen weights were recorded for the DCPD treated birds but not for the DIMP treated birds, thus, no spleen weights were reported in the results of this experi- ment. Hemoglobin concentration, packed red cell volume (hematocrit value), and differential counts were determined for all surviving birds at the termination of the experiment. Egg Collection, Storage, and Incubation Each day, eggs were marked with the corresponding cage number and date, collected, and stored at 12.8 to 15.6°C until set. The storage time ranged from zero to six days. Eggs were set at weekly intervals in a Jamesway single stage Model 252 incubator3. The incubator was maintained at an average internal temperature of 37.5°C (range 36.9 to 38.1°C) and average relative humidity of 56 percent (range 52 to 65 percent). All eggs were candled on day 0 for shell cracks and again on day 14 to determine fertility and/or early embryonic death. Eggs that were cracked, infertile, or that contained early deads were removed and disposed of properly. Fertile, developing eggs were put in pedigree hatching baskets and were transferred to a hatching unit (Jamesway Model 252) on day 21. The average temperature and rela- tive humidity of the hatcher was 37.2°C (range 36.8 to 38.1°C) and 67 percent (range 65 to 70 percent) respectively. On day 24 the hatched chicks were removed from the hatcher, wing banded, and housed in a Petersime Brood unit for a two week observational period. Clean feed and water were provided 3g libitum during the two weeks. Mortality was recorded daily. Survivors were weighed and sacrificed at the termination of the two week observational period and liv- ability calculated. Eggs that did not hatch were broken open, examined, and placed in one of the following categories; pipped live, pipped dead, live in shell, or dead in shell. James Manufacturing Company, Inc. (a subsidiary of Butler Manu- facturing Co.), Fort Atkinson, Wisconsin 53538. 17 18 One day's egg production was collected at biweekly intervals to be measured for egg shell thickness. The eggs collected for shell measurement were cracked open at the girth, the contents washed out, and the shells were air dried for a minimum of 48 hours. Measurements of the shell plus the membranes were taken at four points around the girth using a micrometer4 calibrated to 0.01 mm units. Hematological Preparation Determinations of differential counts, packed red cell volume, and hemoglobin concentration were completed on all birds that survived until the termination of the experiment. The dry, fixed smear method was used to determine blood differentials for each bird. This method involved air drying a thin smear of fresh blood on a clean glass slide, then flooding with avian Wright's stain (Appendix D: Preparation of Wright's stain and buffer). After a five minute period a few drops of buffer were added to allow maximum differential coloration of the blood cells. Following an additional five minute period the cells were rinsed with distilled water and blotted dry. Differential counts were determined by hand counting the first 100 white blood cells, repeating the count, then calculating the average of the two counts. Hematocrits were determined by collecting blood from a puncture of a vein in the wing into two heparinized capillary tubes, sealing one end of each tube and centrifuging at 4500 rpm for 7.5 minutes 4 Federal Products Corporation (a subsidiary of Esterline Corp.) 1144 Eddy Street, Providence, Rhode Island 02901. 19 in an International Microcapillary Centrifuge5. The packed red cell volume of each tube was measured using a microcapillary reader5 after centrifugatiOn. The cyan-methemoglobin method was used to determine the individual hemoglobin concentrations. In this method five ml of Drabkin's Reagent (Appendix E: Preparation of Drabkin's Reagent) were placed in a clean cuvette. Using a micropipetter5, 25 micro- liters of blood were pipetted into the reagent. The micropipetter was rinsed several times in the blood-reagent solution. The cuvette was stoppered and inverted several times to facilitate mixing. A ten minute period followed, allowing for maximum con- version of hemoglobin to cyan—methemoglobin. At the end of ten minutes the cuvette was wiped clean and placed in a Spectronic 20 Calorimeter-Spectrophotometer6. The percent absorbance of the sample at 540 nm, was recorded. The hemoglobin concentration was determined by the comparison of the sample percent absorbance against the percent absorbance of standards with human hemoglobin concentration7 (Appendix F: Determination of hemoglobin concen- tration). Statistical Analysis Data from the chronic study were treated statistically by analysis of variance; sample units with three exceptions, for the variables measured were the individual cages. The exceptions were for body weight change, organ weight, and hemotological parameters where the sample units were the individual birds. International Equipment Company, Boston, Mass. 6 Bausch and Lomb, Rochester, New York. 7 Cyanmethemoglobin certified standard, Hycel, Inc., Houston, Texas. 20 Dunnett's t-test (with modification for unequal replication where applicable) was used to compare all treatment groups against their respective controls for each variable except percent liv- ability of progeny. The latter was analyzed by the split-plot design (Gill, 1977) with arcsin transformation. Body weight change, egg production, and feed consumption were not analyzed by the split-plot design for three reasons: unequal replication, paral- lelism of the treatment, and control groups' trends and abnormal frequency distribution of the data. EXPERIMENT l Rationale The common method of expressing acute toxicity is the LD50 value; the single, oral dose of a compound that will produce death in 50% of the test subjects. It is this value that is the most rapid and convenient to determine when evaluating the hazard of a chemical, comparing inherent toxicity of chemicals, and pro- viding a basis for the determination of dose levels to be used in subacute and chronic toxicological studies. Caution, however, must be heeded when using the LD50 value because the severity of clinical signs, if any, are often excluded, species variation in susceptability can be considerable, and a maximum dose over pro- longed exposure may bear no relationship to the LDSO figure. The single oral median lethal dose (LD50) of DIMP and DCPD to the Bobwhite was determined to provide information that was deficient in the literature. Clinical signs noted during the test period, included mortality, morbidity, behavorial changes, and differences in clinical signs between the sexes. The infor- mation gathered was used to aid in the determination of the dose levels in the subacute and chronic studies. Range finding pilot studies were conducted to provide a practical dosage span to be used in the acute test. DIMP pilot tests began at 200 mg/kg body weight. The dose was repeatedly doubled until a level of 1600 mg/kg body weight was reached with deaths occurring at 800 mg/kg body weight and 1600 mg/kg body weight. Three additional trials were conducted to verify the information gathered from the initial trial. Dose 21 22 levels utilized in additional trials were between 200 and 1600 mg/kg body weight. Overall results are shown in Table 3. DCPD range finding began at 400 mg/kg body weight. The dose was repeatedly doubled until a level of 3200 mg/kg body weight was reached, with deaths occurring at 1600 mg/kg body weight and 3200 mg/kg body weight. As with DIMP range finding, three additional trials were conducted to provide more reliable data to use in the determination of the LD50 dose levels. Overall results are shown in Table 3. The first three range finding trials of each chemical employed one adult bird per dose level. The final range finding trial of each chemical utilized two adult birds per dose level. All birds were provided feed and water ad libitum with the exception of a minimum 15 hour fasting period before per os dosing. A two-week observational period followed dosing. Survivors were sacrificed at the termination of the trial. 23 Table 3. Results of DIMP and DCPD LD50 range finding trials. Number. . Level of DIMP of Mortality Chemical (mg/kg body weight) birds % DIMP 300 2 0 400 2 0 600 4 25 700 l 0 800 5 20 900 2 0 1000 2 50 1100 2 100 1200 2 50 1600 2 100 Number Level of DCPD of Mortality Chemical (mg/kg body weight) birds % DCPD 400 1 0 800 l 0 1000 1 0 1100 3 67 1200 3 100 1300 3 100 1400 3 100 1500 3 67 1600 4 100 3200 3 100 RESULTS Experiment 1 Mortality for the quail treated per os with DCPD and DIMP is listed in Tables 4 & 5. Determination of acute oral LD50 by the method of Litchfield and Wilcoxon (1949) for the compounds tested was: Compound Species LD50 mg/kg DCPD. Bobwhite 1010 i 80.0 DIMP Bobwhite 1000 i 68.2 Mortality curves of DCPD and DIMP for the Bobwhite are plotted in Figures 1 and 2, respectively. The curves may be considered parallel within experimental error (P< .05), and the compounds do not differ in potency (P < .05). Most deaths occurred within the first 24 hours after dosing with DIMP and from 48 to 96 hours after dosing with DCPD. There was no mortality nor clinical sign differences between the sexes among the treated groups. Clinical signs of reaction to DIMP per os dosing included an initial comatose state followed by death or recovery. During recovery, staggering, sitting still, and shallow breathing were noted. Recovery was usually complete within 24 hours. Responses of quail to DCPD dosing were noted after 24 hours when activity decreased and the birds became quiescent. Those that made efforts to walk were unsteady and lacked coordination. Recovery or coma and death followed by 96 hours post-treatment. During the 14-day post-treatment period, no further signs of intoxication nor significant weight changes of birds in treated groups from those of the control gorups were noted for birds 24 25 Table 4. Mortality of adult Bobwhite quail during a l4-day period following a single per os dosing with DCPD. Treatment Mortalityi level No. died/No. treated Combined (mg/kg) male female % 0 (control) 0/15 0/15 0 200 0/ 5 2/ 5 20 400 l/ 5 l/ 5* 10 600 l/ 5 0/ 5 10 800 5/15 4/15 30 900 4/10 1/10 25 1000 10/15 9/15 63 1100 2/10 4/10 30 1200 11/15 9/15 61 1400 5/ 5 4/ 5 90 1600 9/10 -- 9O Accidental death 26 Table 5. Mortality of adult Bobwhite quail during a l4-day period following a single per os dosing with DIMP. Treatment Mortality level No. died/No. treated Combined (mg/kg) male female % 0 (control) 0/10 0/10 0 800 0/10 2/10 10 900 . 3/10 4/10 35 1000 7/10 4/10 55 1100 7/10 4/10 55 1200 8/10 9/10 85 Figure l. 27 Percent mortality of adult Bobwhites, equal numbers of each sex, given a single per os dose of DCPD and observed for 14 days post treatment. In the regression equation x = dose of DCPD in mg/kg of body weight and y = percent mortality. 28 cov— L A.»> .m mozmfiv anon oonw coup oo: ooow 000 com CON. _ _ _ L _ _ L K0306 + No.mv-H> \ .H musmflm 000 [ON 0 M l r Humowv. O '0 l O 5 low '00 loop Figure 2. 29 Percent mortality of adult Bobwhites, equal numbers of each sex, given a single per os dose of DIMP and observed for 14 days post treatment. In the regression equation x = dose of DIMP in mg/kg of body weight and y = percent mortality. 3O cov— comp Agd mm\mEv onoo coup oop— Xhfio + NNTLH> Door 000 .m musmflm or ON on O? on 00 on cm 90 oo— ‘leow °/. 31 treated with either chemical (Table 6). Necropsies of all birds, i.e., those that died and those that were sacrificed at the end of the post treatment period, showed no gross pathological changes in the quail which had been administered either DIMP of DCPD. Feed consumption, for the 14—day post-treatment period, is listed in Table 7 for the quail dosed with DIMP or DCPD. With DIMP, feed consumption in the 900 and 1200 mg/kg dosed groups appeared to have been depressed during the first week. At the 100 mg/kg level, feed consumption may have been affected for the entire two-week period. Quail treated with DCPD at levels higher than 200 and 400 mg/kg showed depressed feed consumption with a marked decrease at the 1400 and 1600 mg/kg levels, during the first week. By the second week feed consumption had improved at all levels. Table 6. Quail body weight changes during post treatment for L050. Mean Level body weight (9) change mg/kg n Day 0 Day 14 (g/b/d) DIMP 1 0 20 202.95 207.05 +.293a 800 18 199.33 195.28 -.290a 900 13 190.23 195.23 +.357a 1000 9 203.22 197.33 --.421a 1100 9 193.67 195.22 +.1lla 1200 3 199.67 182.00 -1.262a DCPD o 19 205.74 198.74 -.soobl 200 8 188.75 196.38 +.545b 400 8 189.00 178.88 -.742b 600 9 186.44 182.11 —.309b 800 21 184.71 193.00 +.592b 900 15 201.80 198.73 —.219b 1000 11 207.45 191.18 -l.162b 1100 14 188.93 192.29 +.240b 1200 10 197.70 191.80 -.421b 1400 1 194.00 189.00 -.357b 1600 1 205.00 198.00 -.500b 1 Means having the same subscript are not significantly different from their respective control (P < .05). —. ..n—u—unr- ' __-——- Table 7. Quail feed consumption (g/b/d) during post-treatment for LD 5 0 Level First Second (mg/kg) n week week DIMP 0 20 13.04 14.96 800 18 12.63 13.75 900 13 8.34 15.31 1000 9 10.71 8.76 1100 10 12.43 12.49 1200 3 7.76 14.09 2932 0 19 10.26 13.60 200 8 11.04 11.17 400 9 9.42 9.04 600 9 6.29 11.35 800 21 6.25 10.14 900 15 7.65 13.19 1000 11 8.09 12.57 1100 14 7.65 15.21 1200 10 8.01 14.44 1400 1 3.36 13.57 1600 1 1.86 12.40 fl? EXPERIMENT 2 Rationale The subacute toxicity test is performed to eliminate error due to atypical results caused by a single oral dose of a chemical, thus providing a more accurate profile of a chemical's toxicity in the field. In this test, a chemical or test substance is incorporated into a standard ration which is subsequently fed ad libitum to the test subjects for a designated duration. Not only does this test better represent the repetitive dose exposure of wildlife to a chemical than the single dose test (LD50), but it also provides additional information by allowing for such variables as daily feed intake, absorption, and metabolism in the test design. Levels of both chemicals employed for the subacute test were partially determined by the LD50 value, the slope of the dosage- mortality curve, the variation within a group's response to the same dose, and the results of the range finding pilot studies. The pilot studies were performed to provide an estimate of the maximum dosage an animal will tolerate on repeated intake of a chemical, and to eliminate unnecessary or inappropriate dosages before initiating further, more costly testing. The eight-day range finding pilot test utilized six birds for each dietary treatment per chemical. Dietary treatments consisted of 4000, 8000, and 16,000 ppm of DIMP or DCPD. Treated feed was fed for the initial five days of the test and untreated feed for the remaining three days. The three-day clean feed period was included to avoid overestimation of the lethal dosage by calculating mortality before either compound had sufficient time to act. 34 35 Body weights were measured at the initiation of the test, the transition between feeding treated and untreated feed, and the termination of the test. Feed consumption was estimated by providing a known amount of treated or clean feed for the birds and weighing the remainder on days five and eight, of the test, respectively. Survivors were sacrificed at the end of the experiment. Results of the range—finding test were: Level in Mean change Mean feed Mortality diet in body wt. consumption (%) Chemical (ppm) (g/b/d) (S/b/d) DIMP 4000 +3.150 7.055 0 DIMP 8000 +0.185 5.650 33.3 DIMP 16000 +0.340 4.235 0 DCPD 4000 +2.315 7.085 0 DCPD - 8000 +0.965 3.740 66.7 DCPD 16000 +1.785 2.400 0 Since there was 33.3 percent mortality at the 8000 ppm DIMP level (the median level) during the range-finding study, the levels for the subacute study included four levels set above the maximum two percent level recommended in the Federal Register (1975). This was to hopefully result in at least 50 percent mortality or to establish a zero feed intake level if the mortality did not reach 50 percent at any level. Since the mortality on DCPD was greater than 50 percent (66.7 percent at the 8000 ppm level) in the range finding study, the subacute levels were set at below two percent of the diet. EXPERIMENT 2 Results DCPD. Compared to the control group, feed consumption of the chicks on diets that contained DCPD increased in six of the treated groups and decreased in three of the treated groups (Figure 3). The differences ranged from a 12.2 percent decline (0.16 g/b/d less than the control) for the birds that received the 10000. ppm diet to a 16.4 percent increase (1.82 g/b/d more than the control) for the birds that received the 4000 ppm diet, with a mean 1.4 percent (0.07 g/b/d) above that of the control. The equation describing the regression line depicting feed consumption ,is y = 5.343 - 0.00003x (Figure 4), with a correlation between feed consumption and level of DCPD in the diet of -0.4683; the predicted zero feed consumption was calculated from this line to be 73028 ppm DCPD in the diet. Body weight data (Figure 5) showed that all treatment groups gained weight. Total intake of the chemical ranged from 357.4 to 3051.3 mg/kg/day with the least amount of intake in the three lowest level groups (2000, 4000, and 6000 ppm). The lower level diets (2000 - 8000 ppm) showed a mean gain of 2.86 g/b/d, a 0.1 g/b/d decrease as compared to the control. Birds on higher levels (10000 - 18000 ppm) showed a mean gain of 2.38 g/b/d, a 0.58 g/b/d decrease as compared to the control. The slope of the regression line depicting body weight changes is -0.00004, (Figure 6 shows body weight changes, not body weight) with a correlation between the level of DCPD in the diet and weight gain of -0.6812. Predicted zero body weight gain was calculated to be 80108 ppm DCPD in the diet. 36 37 Figure 3. The effect of feeding DCPD at various levels in the diet for five days on feed consumption of 14-day old Bobwhite quail chicks. 38 Eon 11:023.: :3 _o>o_ ouoo .m musmflm POWIISIIO3 P331 p/q/s 39 Figure 4. The regression equation of the data shown in Figure 3. In the regression equation x = ppm of DCPD in the diet and y = feed consumption (g/b/d) . 40 — op Eng Antennaos. c: _u>o_ ouoo V« «w or 0 o v x mooood unfimfi n > .v mudmfim peas pamnsuo: 9/9/5 41 Figure 5. Effect of feeding various levels of DCPD in the diet for five days on body weight change of l4-day—old Bobwhite chicks. 42 o— o— .m musmflm Eon—51:02.0... c: .23. anon v— up on Q o v N O afiuoqa :qbgam Apoa P/q/B 43 Figure 6. Regression equation of the data shown in Figure 5. In the regression equation x = ppm DCPD and y = body weight change in g/b/d. 44 .o musmfim Eng Antennae—3 a: _o>o. unoo thoooodnvoodu> m p/q/B afiunq: “1619M .Apoa 45 There were no trends in mortality (Table 8). Both the 2000 and 10000 ppm groups had the highest mortality at 20 percent. The_18000 ppm group,which had the highest intake of DCPD in mg/kg/day, had a mortality of 10 percent. All other groups had no mortality even though levels of 6000 ppm and higher had DCPD intake (in mg/kg/day) levels above the LD50 value of 1010 mg/kg body weight. Correlation between mortality and mg DCPD/kg/day ingested is -0.0648. During the three day post-treatment period, all groups, except the 10000 ppm group, had increased feed consumption as compared to the control (Figure 7). There were no trends in feed consumption since the slope of the regression line is +.00001 and the correla- tion between the level of chemical in the treated diets and feed consumption is +0.1486. The increases in feed consumption ranged from 3 percent at the 4000 ppm level to 19.94 percent at 8000 ppm level, with a mean increase of 7.74 percent (5.16 g/b/d) as compared to the control. Body weight changes (Table 9) during the post-treatment period showed no trends. The slope of the regression line is +0.00004, and the correlation between the level of chemical in the treated diets and feed consumptionis +0.3730. All groups, with the exception of the 8000 ppm group, showed gains ranging from 4.83 g/b/d at 16000 ppm level to 3.37 g/b/d at 14000 ppm level, with a mean gain of 3.80 g/b/d. This was only 0.718 g/b/d greater than the control. At the 8000 ppm level the body weight gain was 2.6 g/b/d which was 0.44 g/b/d less than the control. 46 Table 8. Calculated DCPD intake over 5 days and mortality over 8 days for l4-day-old Bobwhite chicks on LC50 trial. Level of DCPD Mean1 DCPD consumed/ DCPD in consumed/ body wt. kg bd. wt./ Mortality diet (ppm) day (mg) (gms) day (mg) (%) 0 0 28.3 0 0 2000 10.4 29.1 357.4 20 4000 23.3 30.9 754.0 0 6000 31.8 28.0 1135.7 0 8000 41.3 30.0 1376.7 0 10000 43.9 25.7 1708.2 20 12000 63.8 30.1 2119.6 0 14000 67.2 26.2 2564.9 0 16000 80.6 28.1 2868.3 0 18000 83.3 27.3 3051.3 10 1 Mean body weight of treatment group for five day interval. 47 Figure 7. Feed consumption of Bobwhite chicks fed untreated feed during three day post-treatment period after withdrawal of DCPD treated diets. 48 6 5 A. 3 11:0 10:23:00 100“— Dose level in (thousands) ppm Figure 7. 49 Table 9. Body weight change of Bobwhite chicks during the 3-day period after withdrawal of DCPD-treated diets. DCPD level in Weight change Feed consumed/ the diet (ppm) (g/b/d) weight change 0 3.04 2.19 2000 3.50 2.11 4000 3.70 2.04 6000 3.90 1.82 8000 2.60 3.08 10000 3.96 1.64 12000 3.67 2.09 14000 3.37 2.04 16000 4.83 1.53 18000 3.45 2.16 50 No gross pathological changes between the DCPD-treated groups and the control were observed during necropsies. EEMP. Feed consumption (Figure 8) of chicks on the three lowest levels (4000, 8000, and 12000 ppm) was increased compared to the control by 6.72 percent, 8.89 percent, and 7.8 percent, respectively (a mean increase of 7.80 percent or 0.43 g/b/d). Quail fed all other levels of DIMP showed decreased feed consumption as compared to the control, ranging from 15.97 percent at the 28000 ppm level to 43.01 percent at the 26000 ppm level. The slope of the regression line for feed consumption is -0.00008, with a correlation between feed consumption and level of DIMP in the diet of -0.8514 (Figure 9). The predicted zero feed consumption calculated from this line is 77959 ppm. Total intake of the chemical in mg/kg/day ranged from 755.60 at the 4000 ppm level to 4982.90 at the 36000 ppm level. With the exception of the 32000 ppm level, there was a continuous increase in the amount of DIMP ingested as the level of DIMP in the diet increased. Body weight data showed that all groups gained weight (Figure 10). Birds on lower DIMP levels (4000 to 16000 ppm) showed a mean gain of 2.06 g/b/d; a decrease of 1.03 g/b/d as compared to the control. Birds on the higher levels of DIMP, 20000 to 36000 ppm, showed a mean gain of 1.11 g/b/d; a decrease of 1.98 g/b/d as compared to the control. The slope of the regression line for body weight changes is -.00007 and the correlation between the level of DIMP in the diet and body weight change is -0.8540 (Figure 11). Predicted zero body weight gain ‘is 44547 ppm DIMP in the diet. l! 51 Figure 8. Effect of feeding various levels of DIMP in the diet for five days on feed consumption of 14-day-old Bobwhite chicks. 52 .canfinchnaos..:v_o>o_ oaoo .m musmflm Peas pawnsuo: p/q/B 53 Figure 9. Regression equation of the data shown in Figure 8. In the regression equation x = ppm of DIMP and y = feed consumption in g/b/d. 54. on an Eco 71:03.0... :3 .090. Once ON Va ON 0— up a x oaooooo - ooo u > .m musmflm P331 P/‘l/fi pawnsuo: 55 Figure 10. Effect of feeding various levels of DIMP in the diet for five days on body weight change of l4-day-old Bobwhite chicks. 56 page Au1:0u:O;—c_v_o>o_0noo .oH musmflm afiuoq: [qfigam Apoa p/q/s 57 Figure 11. Regression equation of the data shown in Figure 10. In the regression equation x = ppm of DIMP and y = body weight change in g/b/d. 58 page Anvcunaos. c_v_o>o_ ouoo x hooood - mo...m u > .HH musmflm afiuoqa qugaM Apog P/‘l/6 59 Mortality was limited and showed no trends (Table 10). The 28000 and 24000 ppm groups showed 10 and 60 percent mortality, respectively. The high mortality in the 24000 ppm group was attributed to cannibalism. No other groups showed any mortality even though the amount of DIMP ingested ranged from 755.6 to 4982.9 mg/kg/day which bracketed the LD50 value of 1000 mg/kg. Quail in all groups showing no mortality, except the 4000 ppm group, had values of DIMP ingested above the LD50 value. The correlation coefficient between mortality and mg DIMP ingested is +0.0905. During the three-day post-treatment period, feed consumption increased in all DIMP groups including the control, however, the greatest increase generally occurred hithose groups that had shown the greatest decrease in feed consumption during the five-day treat- ment period (Figure 12). Increases in feed consumption ranged from 105 percent (3.39 g/b/d) at 36000 ppm level to 22 percent (1.33 g/b/d) at 3000 ppm level with a mean of 58.33 percent. The control showed a 41 per- cent increase (2.27 g/b/d) in feed consumption. In total amount of feed consumed, the 4000 - 20000 ppm groups showed a mean feed consumption of 7.70 g/b/d; 0.08 g/b/d less than the control. Quail in groups fed 24000 - 36000 ppm had a mean feed consumption of 6.24 g/b/d; 1.54 g/b/d less than the control. Body weight changes, during the post treatment period showed no trends. The correlation between the level of chemical in the previous diet and feed consumption is -0.5222 (Table 11). All groups showed body weight gains ranging from 4.23 g/b/d at 20000 ppm level to 3.01 at 24000 ppm level, with a mean of 3.86 g/b/d. This was 1.47 g/b/d less than the control. 60 Table 10. Calculated DIMP intake over 5 days and mortality over 8 days for l4-day-old Bobwhite chicks on LC50 trial. Level of DIMP l DIMP DIMP consumed/ Mean consumed/kg in diet day body wt. bd. wt./ Mortality (ppm) (mg) (9) day (mg) (%) 0 0 30.8 0 0 4000 23.5 31.1 755.6 0 8000 _ 48.0 29.2 1643.8 0 12000 71.3 27.9 2555.6 0 16000 73.3 27.3 2685.0 0 20000 86.0 27.5 3127.3 0 24000 75.4 19.6 3846.9 60 28000 129.6 26.4 4909.1 10 32000 116.5 25.1 4641.4 0 36000 116.6 23.4 4982.9 0 1 Mean body weight of treatment group for five day interval. 61 Figure 12. Feed consumption of Bobwhite chicks fed untreated feed during three day post—treatment period after withdrawal of DIMP treated diets. 62 .caeA evennaeu—.V e. .o>e_ anon an 9... on o. «— .NH whomflm P/Q/Q pounsuo: pea, 63 Table 11. Body weight change of Bobwhite chicks during 3-day period after withdrawal of DIMP-treated diet. DIMP level Weight Feed in the change consumed/ diet (ppm) (g/b/d) weight change 0 5.33 1.46 4000 4.07 1.91 8000 3.80 1.93 12000 ' 3.93 2.00 16000 3.97 1.80 20000 4.23 1.99 24000 3.01 1.44 28000 3.93 1.86 32000 3.83 1.75 36000 3.93 1.69 64 No gross pathological changes between the DIMP—treated groups and the control were observed during necropsies. EXPERIMENT 3 Rationale The reproduction process is a complex phenomenon involving many physiological and behavioral changes governed by the action and interaction of chemicals and hormones. These carefully timed and coordinated changes in both parent and progeny can be very susceptable to a toxicant's effects even when a toxicant is fed continuously at exposure levels that exclude acute mortality and observable morbidity. Such effects in adults may include altera— tion in spermatogenesis, oogenesis, ovulation, and/or conception in birds, resulting indecreased production or fertility. Toxi- cants' effects at the progeny level may include embryonic death (embryogenicity) or alteration of embryonic organogenesis result— ing in nonlethal abnormalities or malfunctions of the fetus (teratogenicity). Single oral dose lethality and lethal dietary concentration values provide adequate information when used as an expression of potential hazard or as a comparison of relative toxicities of various substances. However, when compiling a complete data pro- file to evaluate the safety of a substance, more information of the chemical-biological properties of the substance is necessary. The chronic reproductive test was designed to produce such infor- mation through continuous long term exposure extended to repro- duction and teratology. The chronic test performed was a single generation reproductive test conducted over a single breeding season instead of a continuous year—long study. This is an accepted practice used by governmental agencies for toxicity studies 65 66 utilizing avian species. It is also a test situation applicable to field conditions of avian wildlife populations. The duration of the reproductive study of a representative avian wildlife species also allows for the determination of long term effects (or the absence of) due to the chronic feeding of a suspected toxicant. Such effects could include carcinogenecity or behav- ioral or physiological reactions previously unobserved. Dietary levels of the test substances were determined via the results of the LC50 experiment and consultations with the Project Officer (United States Army). Decreased feed consumption, as compared to the control, at levels above 12000 ppm DIMP coupled with a decrease in body weight gain, as compared to the control, at levels above 16000 ppm DIMP and absence of mortality at levels lower than 24000 ppm DIMP aided in the decision to place the reproductive study dietary levels of DIMP at 0, 1200, 3800, and 12000 ppm. Similarly, normal feed consumption and body weight gain at 4000 ppm DCPD coupled with mortality at the 10000, 18000 ppm DCPD levels aided in the determination of the reproductive study DCPD dietary levels of 0, 400, 1250, and 4000 ppml. Dietary intake of the test substances was expressed as ppm as opposed to mg/kg/day. Neither method is completely satisfactory. The use of ppm does not allow for reduced or increased feed con- sumption due to repellant or attractant properties of the test l The 3800 and 12000 ppm DIMP dietary levels were later reduced to 380 and 0 ppm, respectively. This action was taken after evalu- ating the mortality percentages occurring at the 3800 and 12000 ppm levels during the first four weeks of the study and consult- ing with the ProjeCt Officer (Unites States Army). 67 substance; however, the reaction by a laboratory bird to such a property of a test substance would undoubtedly be similar to the reaction of birds in the field. Mg/kg/day can be misleading when the length of time a toxicant is fed to a test subject and the excretion of a toxicant are not reported. The latter method would also require the continuance of biweekly body weight measurements throughout the reproductive period - an action highly undesirable due to possible adverse effects of handling on egg production. EXPERIMENT 3 Results 2922. Feed consumption data for adult Bobwhites fed DCPD treated diets or control diet are presented in Figure 13. Each point plotted is the mean of 15 cages, each housing one male and one female bird. There was no significant difference in feed consumption between birds fed DCPD diets and birds fed a control diet. A general increase over the entire experimental period in feed consumed on all dietary levels was noted. Body weight change data of Bobwhites fed DCPD treated or control diets for the initial 10 weeks of the test period are presented in Table 12. During the 10 week period no significant differences in body weight change were found between those birds fed a treated diet and those birds fed control diet. Body weight change was measured again at the commencement of egg production, and at the termination of the experiment. These data, catagorized according to sex, are given in Table 13. No significant differences between the mean body weight change of treated groups and the control group were found for either sex. Mortality data of Bobwhites fed DCPD or control diets are presented in Table 14. No diet related trends were noted. The majority of the deaths occurred during a 48-hour period at approximately the 62th day of the experiment. The cause of death of the twelve birds which died during the 48-hour period could not be determined. Other mortality was sporadic. Egg production data for the DCPD study Bobwhites are plotted in Figure 14. Each point plotted is the mean of 15 cages of one 68 Figure 13. 69 Effect of feeding various levels of DCPD in the diet for 28 weeks on feed consumption of adult Bobwhites. Each point represents the mean of fifteen cages, each containing one male and one female bird. g/b/d Feed consumpllee 70 PP.“ n cenlrel o 400 v 1250 #4000 Weeks Figure 13. 71 .Amo. A No Noaucoo m>flwoommmu Hflmnu Scum “cmHmMMHU waucmoHMHcmflm uoc mum pmfluomQSm mEmm may QDHS mHmQEsz N .QOHDMH>m© Guacamom H coma mm pmmuommn sumo 4 le.NHV INN.aHV Aaa.mflo Imm.oauv lam.muo omm.on boa.o+ omo.Nu oma.a+ mam.mn om oooa lam.NHo Amo.auo Aoa.mflo Aoa.aaflo AmN.aHV moo.an omk.o+ bao.ou boo.NH+ bom.mn om omNa ANH.NH3 Aaa.oHo ANo.aHv Ima.mwo loN.mHo mam.a- mmm.o+ boa.N- baN.NH+ oom.a- om ooa MmH.NHo Moo.NHo who.mflv Mom.awv Maa.mfio N as N- N oa m+ N om o+ N mo NH+ N oa on om o mxwm3 mxmwS mxmw3 mxmmB . mxmmz : AEQQV oH m o . a N Hm>ma afiwv mmcmno unmflms moon Naxmmzflm mnmumfla .gowuosoonm mom mo bongo map on HOHHQ mxmmz oa map Mow mmufl£3nom mo mmmcmno unmflm3 moon so pmflc map CH mam>ma mooflum> um omuo mcwomwm mo “ommmm .NH magma 72 Table 13. Effect of feeding DCPD at various levels in the diet for 28 weeks on body weight change of Bobwhites during the 10 week reproductive period. Mean body weight (gms) Level of Pre— Termina- Body DCPD in production tion weight 1 Sex diet (ppm) n (week 18) (week 28) change (%) Female 0 12 201.58 220.00 +9.21 : 6.7632 400 13 198.00 217.08 +9.64 i 13.85a 1250 14 205.43 215.21 +5.83 : 14.51a 4000 13 201.38 218.54 +8.69 : 6.11a Male 0 13 200.15 201.08 +0.42 : 6.53102 400 12 200.00 198.14 -0.91 : 2.89b 1250 13 201.85 222.08 +1.39 i 5.89b 4000 12 199.92 195.08 -2.31 : 5.10b 1 Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 73 Table 14. Effect of feeding DCPD on mortality of Bobwhites during the 28 week chronic study. Dietary Mortality levels days 61-63 Total Mortality (ppm) of the study mortality (%) 0 4 5/30 16.67 400 2 3/30 10.0 1250 2 3/30 10.0 4000 4 5/30 16.67 74 Figure 14. Effect of feeding various levels of DCPD in the diet for28 weeks on egg production of adult Bobwhites in their first repro— ductive cycle. Each point represents the mean of 15 females. Percents calculated from hen—day production. 75 00°? onu— 00v. .es.eeo Etc 0 .VH mnsoflm uollanposd 76 hen each. Percent production was based on hen-day production. Analysis of the data revealed that the egg production of the hens fed 400 ppm DCPD was significantly less than the egg produc- tion of the hens fed a control diet. No significant difference between the egg production of hens on 1250 ppm DCPD and 4000 ppm DCPD and the production of the control hens was found. However, production trends ofall dietary groups were similar. Analysis of incubation parameter data of Bobwhites fed DCPD treated diets or the control diet showed no significant difference between any treated group and the control in any category. The percentages of fertile eggs were based on the number of settable eggs (total eggs laid - [cracked eggs + eggs laid by unmated females + eggs used for egg shell thickness measurements]). Percent hatchability, early dead, dead in shell, live in shell, pipped lived, and pipped dead were based on the number of fertile eggs. Incubation parameter data are presented in Table 15. Eggshell thickness data for Bobwhites on the DCPD study are presented in Table 16. No significant difference was found between eggshell thickness from birds fed DCPD treated diets and birds fed a control diet. All eggs used for shell thickness measurements were included only in the calculations of total egg production percent. l4-day survivability of the progeny of Bobwhites fed DCPD or control diets is plotted in Figure 15. Each point plotted is the 14-day percent livability of all progeny of all Bobwhites in a particular dietary group for a particular hatch. No significant difference between the livability of the progeny of Bobwhites fed treated feed and progeny of Bobwhites fed control feed was found. '1' 7 Table 15. Effect of feeding DCPD at various levels in the diet for 28 weeks on incubation parameters of Bobwhite quail eggs laid in March, April, and May, 1977. Level in diet Month Parameter (PPm) March April May Combinedl .. .................... %_.__—.-_—_—-———————————-2-——— Cracked 0 5.06 8.21 6.21 6.49 : 1-59a 400 4.90 8.85 13.75 9.17 : 4.43a 1250 4.14 6.41 5.00 5.18 : 1.14a 4000 4.17 10.56 7.14 7.29 : 3.20a Fertile 0 91.72 97.21 89.71 92.88 : 3.88b2 400 72.79 68.93 79.41 73.71 : 5.30b 1250 91.88 84.03 66.32 80.74 : 13.09b 4000 82.50 91.61 84.44 86.18 : 4.80b Hatched 0 80.00 75.29 81.15 78.81 i 3.1002 400 79.80 73.24 75.93 76.32 + 3.30G 1250 78.23 61.98 84.13 74.79 I 11.42C 4000 78.79 65.65 90.79 78.41 : 12.57C Early 0 1.94 6.32 7.38 5.21 : 2.88d2 dead 400 6.06 8.45 16.67 10.39 : 5.57d 1250 2.72 7.44 3.17 4.44 + 2.6ld 4000 3.79 9.17 2.63 5.20 I 3.49d Dead in 0 4.52 3.45 3.28 3.75 : 0.67e2 shell 400 5.05 7.04 0.00 4.03 + 3.63e 1250 4.76 4.96 1.59 3.77 I 1.898 4000 6.82 3.05 0.00 3.29 : 3.42e Live in 0 0.65 2.30 0.82 1.26 + 0.9lf2 shell 400 0.00 4.26 0.00 1.42 I 2.46f 1250 1.36 6.61 0.00 2.66 I 3.49f 4000 1.52 2.29 0.00 1.27 I 1 17f Pipped 0 12.26 12.64 7.38 10.76 i 2.9392 live 400 9.09 7.04 7.41 7.85 : 1.099 1250 12.24 18.18 11.11 13.84 i 3 8og 4000 7.58 19.85 5.26 10.90 i 7 84g Pipped 0 0.65 0.00 0.00 0.22 + 0 37h2 dead 400 0.00 0.00 0.00 » 0.00 I 0.00h 1250 0.68 0.83 0.00 0.50 I 0.44h 4000 0.76 0.00 1.32 0.69 i 0 66h Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 78 Table 16. Effect of feeding DCPD at various levels in the feed for 28 weeks on shell thickness values of adult Bob- white eggs. Dietary Shell level thicknessl (ppm) 11 (mm x 10-2) 0 41 21.95 i 1.83,,2 400 25 21.66 : 1.82a 1250 _ 37 22.40 : 2.06a 4000 37 22.25 " 1.69a 1 Data reported as mean : standard deviation. 2 Numbers with the same subscript are not significantly different from their respective control (P > .05). 79 Figure 15. Percent livability of offspring of adult bobwhites fed various levels of DCPD in their diets for 28 weeks. 80 III a cenlre o 400 4 1250 000 i 10 1'1 12 13 14 15. 81 Organ weight data for DCPD-treated Bobwhites and control INdbwhites are listed in Tables 17 and 18. The liver and gonad(s) (weights (absolute) showed differences attributed to sex and so *were separated into male and female categories. Liver weights of male Bobwhites fed 4000 ppm DCPD feed were significantly less than liver weights of male Bobwhites fed control feed. No other organ weights of DCPD-fed Bobwhites were significantly different from the respective organ weights of Bobwhites fed control feed. Hemoglobin values for the DCPD study Bobwhites are presented in Table 19. There were no significant differences found between the hemoglobin values of DCPD-fed males and control males, or the hemoglobin values of DCPD-fed females and control females. However, the mean hemoglobin value of all males was significantly greater than the mean hemoglobin value of all females. Hematocrit values are presented in Table 19. The results of the analysis of the data showed no significant difference between the hematocrit values of Bobwhites (male or female) fed DCPD diets and Bobwhites (male or female) fed control diets. However, as with the hemoglobin data analysis, the mean hematocrit of all the male Bobwhites was significantly greater than the mean hematocrit of all the female Bobwhites. The mean corpuscular hemoglobin concentration data of Bobwhites on the DCPD study is shown in Table 20. No significant difference between the mean corpuscular hemoglobin concentrations of male and female birds fed DCPD treated diets and their respective controls was found. Also no significant difference between mean corpuscular hemoglobin concentration of males and females was found. 82 Table 17. Effect of feeding DCPD at various levels in the diet for 28 weeks on liver and gonad weights of adult Bobwhites. Level Mean of DCPD organ Organ weight as % of in diet wt. Body Brain Sex Organ (ppm) 11 (gms) wt. wt.1 Female Ovary 0 12 6.357 2.84 560.86 : 174.16a2 400 13 7.602 3.29 655.55 : 181.52a 1250 14 6.193 2.79 560.46 : 142.62a 4000 12 7.175 3.33 622.77 i 209.50a Liver 0 12 6.746 3.06 605.64 : 190.72b2 400 13 7.211 2.88 554.35 i 256.50b 1250 14 6.689 3.03 607.07 : 225.88b 4000 12 6.263 2.71 509.79 : 98.05b Male Testes 0 12 1.35 0.68 118.60 : 15.20,:2 400 14 1.14 0.57 97.74 + 36.970 1250 13 0.98 0.49 85.71 I 39.57C 4000 13 1.28 0.65 113.34 i 54.49C Liver 0 13 3.80 1.91 333.50 i 65.41,,2 400 14 4.13 2.09 355.90 + 63.15d 1250 13 3.72 1.89 323.50 I 60.54d 4000 13 3.40 1.73 263.20 1 81.819 Data reported as mean : standard deviation. Means with the same subscript are not significantly different from their respective controls (P > .05). 83 Table 18. Effect of feeding DCPD at various levels in the diet for 28 weeks on organ weight in adult Bobwhites. Level Mean of DCPD organ in diet wt. Organ weight as % of 3 Organ (ppm) 11 (gms) Body wt. Brain wt.‘ Spleen 0 16 .064 0.030 5.61 i 2.538,2 400 15 .062 0.029 5.46 i 3.073 1250 11 .053 0.025 4.64 i, 2.25a 4000 20 .403 0.027 5.34 : 1.65a Kidneys 0 25 1.49 0.71 133.03 : 37.60,,2 400 27 1.33 3.30 113.61 : 31.37b 1250 27 1.50 1.08 132.82 i 43.71b 4000 25 1.44 0.70 124.93 : 31.04b pancreas 0 25 .524 0.221 41.06 i 12.00,,2 400 27 .527 0.257 43.37 : 20.51C 1250 27 .467 0.224 41.47 : 11.97C 4000 25 .485 0.227 40.87 : 12.54C Proventri- 0 25 .912 0.43 81.83 1 18.76.512 culus 400 27 .930 0.45 71.92 : 22.55d 1250 27 .971 0.47 86.37 : 17°89d 4000 25 .886 0.44 79.17 : 16.41d Gizzard 0 25 4.12 1.97 364.53 : 61.8492 400 27 3.99 1.92 342.12 : 83.44e 1250 27 4.24 2.06 376.07 : 68.978 4000 25 4.24 2.04 369.55 : 87.068 Heart 0 25 0.96 0.49 89.24 : 14.31,-2 400 27 1.02 0.50 87.44 : 19.27f 1250 27 1.08 0.52 95.69 i 22.03f 4000 25 0.96 0.47 83.55 : 18.53f Brain 0 25 1.14 i 0.0992 —-— -—- 400 27 1.17 : 0.11g --- --- 1250 27 1.13 i 0.109 -—- —-- 4000 25 1.17 : 0.10g -—— —-— Data reported as mean : standard deviation. Means with the same subscript are not significantly different from their respective controls (P > .05). 84 Table 19. Effect of feeding DCPD at various levels in the diet for 28 weeks on hemoglobin and hematocrit values of adult Bobwhites. Level of DCPD in Hemoglobin Hematocrit Sex diet (ppm) n (gm/d1. ) 1 n (%) 1 Female 0 8 9.61 : 1.22a2 12 33.2 i 5.96,,2 400 10 10.27 : 0.78a 13 32.9 i 4.39b 1250 11 10.72 : 0.91a 14 35.8 i 3.17b 4000 11 10.55 : 1.20a 12 37.0 : 3.39b Overall 40 10.34 i 1.073 51 34.75: 4.53 Male 0 10 12.28 i 0.9302 12 40.8 : 3.38d2 400 13 12.10 : 0.96C 14 40.4 : 3.97d 1250 11 11.79 : 0.79C 13 39.1 i 3.74d 4000 12 12.33 : 0.85C 13 43.2 i 3.72d Overall 46 12.12 i 0.881 52 40.87+ 3.91 Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 85 .Amo. A my maonpsoo m>Huommmmn Hflmnp Scum psmHmMMHp SHDCMOHmesmHm p0: mum pmHMUmQSm 08mm ocp mcfl>ma mono: m .GOHpcwpmp osmosmpm H some psmfipmmnu mm Uwpuommu mumo m .aa dance ca 6066 Foam coucasoaco a oNN.H H Na.oN mN am.mN NH MN.oN as coca oom.N H oo.oN oN am.mN as Hm.aN Na omNH oNH.N H Ha.aN mN om.aN ma HN.aN ca ooa NomN.a H am.aN V NN mo.om NH mo.oN ca o omanEoo pmcfinfioo mmHmE mmHmE mmamfimm mmHmEmm Afimmv umflp NANV omoz c lev omos s Nlmo omos c as neon . mo Hm>wq .mmuHQSQom “data mo HQOHHmuusmUsoo GHQonofims Hmasomsmuoo some so mxmmz mm How poet map as mam>ma mDOHHm> pm anon msflpmmm mo pomwmm .om canoe 86 Enumeration of leukocytes of the DCPD study Bobwhites is presented in Table 21. The number of eosinophils of the Bobwhites fed 1250 ppm DCPD was significantly greater than the number of eosinophils of the control Bobwhites. No other significant difference between leukocyte counts of DCPD—treated Bobwhites and control Bobwhites was found. 87 Table 21. Effect of feeding DCPD at various levels in the diet for 28 weeks on leukocyte counts of adult Bobwhites. Level DCPD in diet Cell (ppm) n Mean1 Range Basophil 0 25 2.60 : 2.04a2 0- 8 400 27 2.30 i 1.64a 0- 5 1250 26 2.81 i 1.77a 0- 7 4000 23 3.57 : 2.82a 0-12 Eosinophil 0 25 2.76 : 2.57b2 0-10 400 27 3.33 + 2.25b 0-11 1250 26 4.96 E 4.500 1-14 4000 23 3.70 : 2.98b 0-12 Heterophil 0 25 30.32 : 14.85d2 0-62 400 27 27.67 : 16.24d 2-69 1250 26 24.92 1 15-75d 5-67 4000 23 25.83 : 14.365 6-50 Lymphocyte 0 25 56.52 : 18.14e1 19-88 400 27 57.85 : 16.60e 20-89 1250 26 58.69 + 20.27e 2-88 4000 23 58.91 E 15.109 30-85 Monocyte 0 25 7.32 : 4.68fl 1-17 400 27 7.52 : 3.69f 1-15 1250 26 9.50 : 4.60f 2-17 4000 23 8.00 : 4.17f 0—15 1 Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). EXPERIMENT 3 Results Bl§§° Feed consumption data for adult Bobwhites fed DIMP treated rations or control rations are presented in Figure 16. Each point plotted is the mean of 15 cages, each housing one male and one female bird. The dietary levels of DIMP for the initial time interval plotted were: 0, 1200, 3800, and 12000 ppm. Due to con— siderable mortality occurring in the 3800 and 12000 ppm groups, the dietary levels were reduced to 380 and 0 ppm, respectively. Thus, the first time interval of the feed consumption data was analyzed separately from the remaining time intervals. The feed consumption of the birds receiving the 12000 ppm diet was found to be signifi- cantly less than the feed consumption of the control birds. No other significant differences were found between the feed consump- tion of the birds fed treated diets and the control birds. The body weight change data of Bobwhites fed DIMP treated diets or control feed for the initial ten weeks, are presented in Table 22. No significant differences in body weight change of treated birds and control birds were found. For reasons stated in the above paragraph, the initial time interval was analyzed separately from the remaining time intervals. Body weight change data over a ten—week reproductive period are given in Table 23. The data were analyzed separately for males and females. No significant differences between body weight changes of treated quail and control, of either sex, were found. During the initial four weeks of the DIMP study, considerable mortality occurred in the 3800 and 12000 ppm diet groups (Table 24). 88 Figure 16. 89 Effect of feeding various levels of DIMP in the diet for 29 weeks on feed consumption of adult Bobwhites. Each point represents the mean feed consumption of fifteen cages, each containing one male and one female bird. Food consul-9H0. g/b/d 90 Hm I conflrol 0 12000] 0’ A ‘3300/380’ #1200 Wool" Figure 16. .mmmw ma Ho0mm Ema 0 0p woospmn Ema oooma v .mwmw mm Hopmm Ema omm Op pwonpmu 8mm comm m .Amo. A mv Houuqoo m>auommmou Hflmnu Eoum pswuomwflo maustHMHsmHm won mum uQHHUmQSm meow map nuHB mnmnfisz m .coflumfl>mp pumpcmum H some mm wmuuomon mama a m .. .. .. - .. on.m+v Amo.m+v Amm.m+v Amm.ma+v mam.m+v wna.m- cvm.aa+ on.N- a¢6.ma+ me.HH- mm vo\oooma Aom.muc AHm.mHv wam.mwv 16H.» My Amm.mwc www.mu 6H¢.w + ah.o+ QNF.OH+ mvm.m I 6N momm\oomm www.mwc as.mHv Asm.mwc Ama.m He Avm.mflc om.m- mm.m + 0mm.o+ ama.> + waH.m . om coma Amm.~Hc «n.5Hc wan.eflv Amo.mHHv Awm.mHo www.mu oo.m +. oa.o- nee.» + mmo.m . om o m m m m mxomz mxmmz mxmm3 ‘mxmw3 mxomz fiH Afimmv OH m w v N Ho>ma Hva mmsmno unmwm3 moon maxmoSHm humumfla .COHQODUOHQ mom mo #mmco mnu ou uoflum mxme OH map How mmpHSSQom mo mmmcmao pamfloz moon so uoflw may CH maw>ma msOHHm> “m mzHQ mcflcmmm mo pommmm .Nm manna 92 Table 23. Effect of feeding DIMP at various levels in the diet for 29 weeks on body weight change of Bobwhites during the 10 week reproductive period. Level of Mean body weight (gms) DIMP in Pre- Body weight Sex diet (ppm) I1 production Termination change (%)1 Female 0 13 192.33 199.38 +3.29 i 8.69a2 1200 14 196.80 196.30 -0.91 : 9.69a 3800/380 13 197.15 207.15 +5.09 : 10.10a 12000/0 13 194.62 207.85 +6.88 : 7.94a Male 0 13 199.50 200.54 +0.53 : 5.79b2 1200 14 196.27 191.50 -2.06 : 8.43b 3800/380 12 193.85 202.17 +3.94 i 5.15b 12000/0 13 201.15 201.46 +0.17 : 4.79b 1 Data reported as mean : standard deviation. 2 Means having the same subscript are not significantly different from their respective controls (P > .05). 93 Table 24. Effects of feeding DIMP on mortality of Bobwhites during the 29 week chronic study. Dietary Mortality Total Mortality level (ppm) wks. 1-4 mortality (%) 0 0 4/30 13.33 1200 0 2/30 6.67 3800/380l 4 5/30 16.67 12000/02 ‘ 4 4/30 13.33 l 3800 ppm reduced to 380 ppm after 26 days. 12000 ppm reduced to 0 ppm after 18 days. 94 The cause of the deaths was attributed to dietary levels of DIMP but could not be verified by gross necropsy. Mortality, other than that previously mentioned, was sporadic with no diet related trends. Egg production data for the DIMP-study Bobwhites are plotted in Figure 17. Each point plotted is the mean of approximately 15 cages of one hen each. Percent production was based on hen-day production. Analysis of the data revealed that the egg production of hens fed 1200 ppm was significantly less than the production of hens fed control feed. No other significant difference in egg production between treated hens and control hens was found. Produc- tion trends of all dietary groups and the control were similar. Analysis of data on incubation parameters of Bobwhites fed DIMP treated diets or control diets showed no significant differences between any treated group and the control group in any category. The percentages of fertile eggs were based on the number of settable eggs (total eggs laid — [cracked eggs + eggs laid by unmated females + eggs used for eggshell thickness measurements]). Percent hatch— ability, early dead, dead in shell, live in shell, pipped live, and pipped dead were based on the number of fertile eggs. Table 25 contains the incubation parameter data. Eggshell thickness data for the Bobwhites on the DIMP study are given in Table 26. No significant difference was found between the shell thickness of eggs from hens fed DIMP treated diets and the hens fed a control diet. All eggs used for shell thickness measure- ments were only included in the percent egg production calculations. Fourteen-day survivability of the progeny of DIMP-study Bob— whites is plotted in Figure 18. Each point plotted is the l4-day Figure 17. 95 Effect of feeding various levels of DIMP in the diet for 29 weeks on egg production of adult Bobwhites in their first reproductive cycle. Each point represents the mean egg production of fifteen females. Percents calculated from hen—day production. 96 oo«—* Oon\ooon w o\oooup c .0330» u E...— .nH mnsmflm c': n nouanpon 97 Table 25. Effect of feeding DIMP at various levels in the diet for 29 weeks on incubation parameters of Bobwhite quail eggs laid in March, April, and May, 1977. Level in diet Month Parameter (ppm) March April May Combined1 _____________________ %_-_..__._-_—_________--___ Cracked 0 16.55 11.11 1.68 9.78 i 7.52,,2 1200 11.48 8.96 0.00 '6.81 : 6.03a 3800/380 5.62 5.48 10.48 7.19 : 2.85a 12000/0 5.66 3.47 7.79 5.64 i 2.16a Fertile 0‘ 89.66 90.44 87.07 89.06 : 1.76b2 1200 62.26 83.61 56.86 67.58 i 14.14b 3800/380 82.93 94.78 93.26 90.32 : 6.45b 12000/0 76.00 76.05 68.09 73.38 : 4.58b Hatched 0 83.65 73.98 78.22 78.62 : 4.85c2 1200 69.70 64.71 72.41 68.94 i 3.91c 3800/380 79.41 70.87 83.13 77.80 : 6.29C 12000/0 85.53 69.29 83.33 79.38 : 8.81C Early 0 5.77 4.88 6.93 5.86 :, 1.03d2 dead 1200 9.09 13.73 3.45 8.76 : 5.15d (3800/380 5.88 5.51 3.61 5.00 i 1.22d 12000/0 2.63 5.51 8.33 5.49 i 2.85d Dead in 0 3.85 2.44 2.97 3.09 i 0.7182 shell 1200 3.03 7.84 13.79 8.22 : 5.39e 3800/380 0.00 5.51 6.02 3.84 + 3.34 12000/0 5.26 5.51 3.13 4.63 E 1.31e Live in 0 0.96 4.07 3.96 3.00 : 1.76f2 shell 1200 0.00 7.84 3.45 3.76 : 3.93f 3800/380 0.00 3.15 2.41 1.85 i 1 65f 12000/0 0.00 6.30 4.17 3.49 i 3.20f Pipped 0 5.77 13.82 7.92 9.17 : 4.17g2 live 1200 15.15 5.88 3.45 8.16 : 6.17g 3800/380 13.24 13.39 3.61 10.08 : 5.60g 12000/0 2.63 13.39 0.00 5.34 i 7.099 Pipped 0 0.00 0.81 0.00 0.27 : 0.47h2 dead 1200 3.03 0.00 3.45 2.16 + 1.88 3800/380 1.47 1.57 0.00 1.01 f 0.88h 12000/0 ‘ 3.95 0.00 1.04 1.66 : 2.03h Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 98 Table 26. Effect of feeding DIMP at various levels in the feed for 29 weeks on shell thickness values of Bobwhite eggs. Dietary Shell thickness1 level (PPm) n (mm X 10’2) 0 38 21.17 :2.08a2 1200 18 21.83 : 2.19a 3800/3803 35 22.13 : 2.11a 12000/04 26 22.17 : 1.38a 1 Data reported as mean i standard deviation. 2 Numbers with the same subscript are not significantly different from their respective control (P > .05). 3800 ppm reduced to 380 ppm after 26 days. 12000 ppm reduced to 0 ppm after 18 days. 99 Figure 18. Percent livability of offspring of adult ' Bobwhites fed various levels of DIMP in their diet for 29 weeks. 100 ”— eobssz norm: N. __ o. a m a a a . cow—i can \ coon a. o\ OOONwo .0523»: E a a .ma musmflm ass Auuqu1 % r... -9:— lOl livability of the progeny of Bobwhites fed a particular diet for a particular hatch. Treated birds' progeny-percent-livability was not significantly different from control birds' progeny-percent- livability. Mean organ weight data are presented in Tables 27 and 28. Due to weight differences attributed to sex differences, the liver and gonad(s) weights were categorized according to sex (Table 27). Liver and gonad weights of females were further separated into "producing" and "non-producing" categories. Males were not differentiated by reproductive capacities. Significant increases in mean proventriculus and gizzard weights, were found in the 12000 ppm DIMP group, as compared to the controls. No other significant differences between organ weights of DIMP—treated birds and their respective controls were found. Hemoglobin values for DIMP—treated or control Bobwhites are given in Table 29. There was no significant difference found between the hemoglobin values of DIMP-fed Bobwhites and control Bobwhites of either sex. However, the hemoglobin values of the males were significantly greater than the hemoglobin values of the females. Hematocrit values for Bobwhites on the DIMP study are presented in Table 29. The results of the analysis of the data showed no significant difference between the hematocrit values of DIMP-fed females and control females, and no significant difference between DIMP-fed males and control males. The hematocrits of the male birds were significantly higher than the hematocrits of the female birds. 102 Table 27. Effects of feeding DIMP at various levels in the diet for 29 weeks on gonad and liver weights of adult Bobwhites. Level Mean of DIMP organ Organ weight as % of in diet wt. Body Brain Sex Organ (ppm) n (gms) wt. wt.l Female2 Ovary 0 7 1.51 0.74 125.98 : 11.00a4 1200 3 2.33 1.07 202.99 : 13.43a 3800/308 7 2.93 1.33 269.68 i 24.51a 12000/0 9 2.59 1.21 229.80 : 14.54a Liver 0 7 7.44 3.58 628.96 : 21.10C4 1200 3 6.59 3.02 560.65 : 11.77C 3800/380 7 8.05 3.74 736.60 : 21.91C 12000/0 10 7.67 3.88 682.02 : 20.49C Female3 Ovary 0 5 0.46 0.353 23.28 : 3.19104 1200 10 0.38 0.194 33.09 : 1.79b 3800/380 5 0.38 0.281 32.63 : 2.75b 12000/0 3 0.67 0.353 23.28 : 3.19b Liver 0 5 5.11 2.58' 464.24 1 10.30,;4 1200 10 4.27 2.28 379.85 : 9.50d 3800/380 5 5.89 2.99 532.02 : 11.55d 12000/0 3 5.96 3.13 542.50 : 364.16d Male Testes 0 14 1.05 0.51 87.01 1 15.8984 1200 14 0.86 0.44 80.29 : 20.82e 3800/380 13 1.08 0.54 96.72 : 12.91e 12000/0 13 0.97 0.48 81.25 : 13.08e Liver 0 14 3.81 2.58 320.01 1 21.67f4 1200 14 3.40 2.28 320.14 : 14.42f 3800/380 13 4.03 2.99 362.99 i 10.30f 12000/0 13 4.42 3.13 367.65 : 14.07f 1 Data reported as mean : standard deviation. 2 Females producing eggs. 3 Females not producing eggs. 4 Means with the same subscript are not significantly different from their respective controls (P > .05). 103 Table 28. Effect of feeding DIMP at various levels in the diet for 29 weeks on organ weight of adult Bobwhites. Level Mean of DIMP organ Organ weight as % of in diet wt. Body Brain Organ (ppm) 11 (gms) wt. wt.l Kidneys 0 26 1.47 0.75 125.74 : 2.26a2 1200 28 1.34 0.70 120.74 : 2.60a 3800/380 25 1.37 0.63 127.95 : 3.10a 12000/0 26 1.49 0.73 129.04 : 3.94a pancreas 0 26 0.52 0.27 44.31 i 1.88b2 1200 28 0.51 0.26 44.45 : 1.26b 3800/380 25 0.47 0.23 42.61 : 1.22b 12000/0 26 0.50 0.24 46.23 : 1-93b Proventri- 0 26 0.92 0.46 78.98 i 1.4502 culus 1200 28 1.11 0.57 98.39 : 3.64d 3800/380 25 0.96 0.47 87.64 : 1.98C 12000/0 26 0.96 0.47 83.25 : 1.92C Gizzard o 26 4.21 2.15 360.51 + 6.0492 1200 28 4.93 2.49 448.70 E 15.79f . 3800/380 25 4.48 2.23 410.75 + 10.499 12000/0 26 4.48 2.20 389.15 E 6.09e Heart 0 26 0.99 0.51 81.74 i 2.2792 1200 28 0.93 0.48 83.70 i 3.199 3800/380 25 1.01 0.50 91.62 i 1.759 12000/0 26 1.03 0.50 89.55 : 2.40g Brain 0 25 1.17 i 0.01h2 -- -- 1200 28 1.15 : 0.03h -- —- 3800/380 25 1.11 : 0.02h —- -- 12000/0 26 1.16 : 0.01h -— -— Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 104 Table 29. Effect of feeding DIMP at various levels in the diet for 29 weeks on hemoglobin and hematocrit values of adult Bobwhites. Level of DIMP in diet Hemoglobinl Hematocrit Sex (ppm) n (gm/d1.) n (%) Female 0 10 10.0 i 1.37 12 37.4 : 4.40102 1200 14 10.0 i 1.45 14 36.4 : 4.76b 3800/380 11 10.6 i 0.57 12 37.2 : 2.77b 12000/0 10 10.2 i 0.71 13 37.2 : 3.52b Overall1 45 10.2 i 1.12 51 37.0 i 3.87 Male 0 13 11.3 i 0.94 14 41.5 : 3.35d2 1200 14 10.9 i 1.00 14 40.7 : 2.79d 3800/380 12 11.7 i 1.02 13 42.3 : 4.46d 12000/0 12 11.2 i 1.19 13 40.9 : 3.91d 0vera111 51 11.3 i 1.05 54 41.4 i 3.61 1 Data reported as mean : standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). 105 Table 30 lists mean corpuscular hemoglobin concentration data for the DIMP-study Bobwhites. No significant difference was found between the mean corpuscular hemoglobin concentrations of the treated birds and the mean corpuscular hemoglobin concentrations of the control birds. Also, no significant difference was found between the mean corpuscular hemoglobin concentration of the males and the females. Enumeration of leukocytes of Bobwhites on the DIMP study is given in Table 31. No significant difference in leukocyte counts was found between the treated birds and their respective control. 106 .Amo. A do maouusoo m>fluommmmu Hflonp Eoum usmuwwmflp mapcmOHMHsmHm pom mum pmfluomnsm mEmm map msfl>m£ mammz m .coflumfl>mv Unmccmum H cmmfi mm Umpuommu mama N .em magma ca mums some commenceco a mma.H H mm.em mm am-em NH we.em OH cxcccma 6mm.a H m~.mm mm am.sm NH ec.mm as omm\comm 6cm.m H mm.em mm om.cm ea cm.em ea come mamm.e H ma.em . mm ca.em mH Hm.cm ea c wosflnfioo .omcflQEOo (ymmdmfi _. memE mmamamm mmHmEmw Afimmv pomp mxmv omoz . s Nxmv omoz a Name omoz a ma oaoo . mo Hw>®q . .mmpfln3nom pHDUm Ho ACOflumHucmosoo GHQOHmofimn Hmasomsmuoo cmmE so mxmmB mm How “map mnu CH waw>ma mSOHHm> um mzHQ mcficwmm mo muomwmm .om manme Table 31. Effect of feeding DIMP at various levels in the diet for 29 weeks on leukocyte counts of adult Bobwhites. Level of DCPD in 1 Cell diet (ppm) 11 Mean Range Basophil 0 26 2.57 : 1.28a2 0- 6 1200 28 2.63 i 1.78a 0- 8 3800/380 25 2.76 : 2.09a 1-10 12000/0 26 3.65 i 2.47a 0— 9 Eosinophil 0 26 4.27 i 2.63 2 0- 9 1200 28 2.85 : 2.91b 0-10 3800/380 25 3.44 i 2.26 0-11 12000/0 26 2.73 i 2 34b 0- 9 Heterophil 0 26 19.23 1 10.4702 4-46 1200 28 23.04 i 12.95C 2-56 3800/380 25 21.04 : 14.36C 1-63 12000/0 26 22.19 i 11.26C 2-43 Lymphocyte 0 26 65.77 1 13.01 2 39-93 1200 28 62.56 : 14.85 40-84 3800/380 25 63.24 i 18.55d 14-89 12000/0 26 61.88 : 15.45d 32-88 Monocyte 0 26 7.69 i 4.2482 1-21 1200 28 9.00 i 4.08e 1-20 3800/380 25 9.40 : 5.63e 4-24 12000/0 26 9.50 : 5.62e 2-21 Data reported as mean i standard deviation. Means having the same subscript are not significantly different from their respective controls (P > .05). DISCUSSION Experiment 1 The LD50 is the most common expression of acute toxicity. Though other values such as the LD5 or LD95 can be calculated, the dosage required to cause death of one half of the test subjects (LD50) is the easiest to determine and more precise statistically. This median lethal dosage is also used as a standard of comparison of the relatiVe toxicities of compounds to various species of animals. DEED. The LD50 of DCPD for rats and mice (Hart, 1975) were smaller than the LD50 of DCPD for Bobwhites. Quail were roughly twice as resistant to DCPD as were rats and approximately five times as resistant to DCPD as were mice. Bobwhites were, however, less resistant to DCPD than were Mallard ducks (Jones, 1977) and mink (Kavanagh, personal communication). LD50 values for the Mallard could not be determined because no deaths occurred at dosages up to and including 40000 mg/kg bwt (Jones, 1977). Mink suffered no effects other than regurgitation and/or suffocation due to the per 05 treatment with liquid DCPD at dosages up to and including 960 mg/kg bwt (Kavanagh, personal communication). Table 32 lists several compounds and their LD50 values of DCPD for the Bobwhite (Tucker and Crabtree, 1970). The LD50 value of DCPD for the Bobwhite is included in the table for comparison of relative toxicities. Table 1 lists the LD50 of DCPD for several species. Based upon the following chart (Loomis, 1968), DCPD is slightly toxic to the Bobwhite. 108 Table 109 32. .The LD50 values of thirteen compounds for the Bobwhite quail at various ages. Primary LD50 mg/kg Compound use Sex Age (95% conf. limits) Azodrin I1 Male 1-2 yr. 0.944 (0.749-1.19) Furadan I Female 3 mo 5.040 (3.64—6.99) Aldrin I Female 3—4 mo. 6.59 Dieldrin I Both 2—3 mo. 12—14 Accothion® I Male 2—3 mo. 27.4 (19.0-37.1) DDT oil sol. I Both --—- 60-85 Toxaphene I Male 3 mo. 85.4 (59.2-123) Lindane I Male 2-3 mo. 120-130 DDT crystalline I Both ———- 300 SD 15418 H2 Female 3—5 mo. 400-500 DCPD —3 Both Adult 1010 (930-1090) DIMP 1-3 Both Adult 1000 (931.8-1068.2) Ceresan L F4 Male 2-3 mo. 1060 (841-1330) 1 I = insecticide 2 H = herbicide : See Literature Review F: fungicide 110 1. Extremely Toxic (1 mg/kg or less) 2. Highly Toxic (1 to 50 mg/kg) 3. Moderately Toxic (50 to 500 mg/kg) 4. Slightly Toxic (0.5 to 5 g/kg) 5. Practically Nontoxic (5 to 15 g/kg) 6. Relatively Harmless (more than 15 g/kg) Two trials were conducted to determine the LDSO of DCPD for the Bobwhite. The results of the first trial were considered inconsistent and thus a second trial was performed. The additional test included three dosages of DCPD from the initial trial plus five other dosages. The results of the second trial were similar to the first trial. Coburn and Treichler (1946) in determining the acute toxicity of DDT to the Bobwhite reported the inconsistent data they collected as indicating the toxic action of DDT to be irregular due to low and variable absorption. The slope of the dose-response curve is an estimate of the margin of safety of a compound (the magnitude of the range of doses, and thus responses, between a no effect dose and a lethal dose). A steep curve limits the range of doses. A flat curve encompasses a large range of doses. The dose-response curve of DCPD for the Bobwhite is somewhat flat (Slope = .096) indicating variable response to the compound. When not lethal, DCPD did not produce lasting effects on feed consumption and/or body weight during the l4-day observation period. The feed consumption of Bobwhites post DCPD treatment in this study followed the same feed consumption pattern as Mallard ducks dosed with DCPD (Jones, 1977). Generally, a decrease in feed consumption the first week post—treatment was succeeded by an increase in feed consumption, up to control levels, the second 92 111 week post-treatment. Reduced feed consumption post chemical treatment is not an uncommon result in the reported literature. Coburn and Treichler (1946), and Dehlen and Haugen (1954) reported reduced feed consumption of the Bobwhite following treatment with DDT, aldrin, dieldrin, toxaphene, and lindane, respectively. During the l4-day post-treatment period, the body weight change of treated birds showed no significant difference from the body weight change of the control birds; a maximum of 0.5 percent change. Results from other investigators varied. Dahlen and Haugen (1954) reported an average weight loss of 15 to 25 percent in Bobwhites dosed with aldrin, dieldrin, toxaphene, or lindane. Bergstrand and Klimstra (1962) reported a mean weight gain of 3.5 percent in Bobwhites dosed with fenuron. Kinkead 33 31. (1971) conducted mammalian studies with DCPD and found normal weight gains in the treated animals. Jones (1977) reported a six percent mean increase in the body weight of Mallard ducks dosed with DCPD. The similarity in response to DCPD administration of male and female Bobwhites is consistent with findings on other compounds by Coburn and Treichler (1946); Dahlen and Haugen (1954); and Tucker and Haegele (1971). Hart (1975) reported no difference in response, attributable to sex, in either rats or mice dosed with DCPD. The majority of the deaths of Bobwhites dosed with DCPD occurred within 48 hours of the treatment. Of that majority, half occurred on day l and the other half occurred on day 2. The remaining deaths occurred sporadically. One to two day mortality time was reported by Coburn and Treichler (1954) after dosing quail with DDT. 112 DIME. The LD50 of DIMP for Bobwhites is similar to the LD50 of DIMP for rats and mice reported by Hart (1976), but approxi- mately 30 percent less than the LDSO of DIMP for Mallard ducks reported by Jones (1977). Based on the chart in the DCPD discus- sion, DIMP is slightly toxic to the Bobwhite. Table 32 lists the LD50's of several compounds for the Bobwhite. The DIMP LD50 for the Bobwhite is included for comparison purposes. Table 2 lists the LDSO'S of DIMP for several species. The slope of the dose-response curve of DIMP for Bobwhites can be considered flat. As mentioned in the DCPD discussion, a steep curve limits the range of dosages between the no-effect dose and the lethal dose. A flat curve provides more variability of dosages (thus responses) between the no-effect dose and the lethal dose. Male and female Bobwhites responded similarly to DIMP admini- stration. Lack of response difference due to sex is not uncommon. Hart (1976) and Jones (1977) found no difference in response to treatment with DIMP by sex for mice and Mallard ducks, respectively. Tucker and Haegele (1971) reported no difference in response by sex to 108 different compounds in 22 species of birds. Feed consumption of Bobwhites post DIMP treatment did not show a typical dose-response relationship. The 900 mg/kg and 1200 mg/kg groups showed reduced feed consumption compared to the control group during the first week with an increase to control levels the second week post-treatment. The 1000 mg/kg group showed a reduction in feed consumption the second week post-treatment. Other dietary levels showed little change during the observation 113 period. Jones (1977) reported similar inconsistent data with Mallard ducks dosed with DIMP. Body weight change of DIMP treated birds showed no signifi- cant difference from the control birds during the l4-day observa- tion period. This is consistent with findings by Jones (1977) who found no significant difference in body weight change of Mallard ducks dosed with DIMP with the exception of the ducks treated with the highest level of DIMP. Dahlen and Haugen (1954) reported weight losses in Bobwhites treated with either of four insecticides. Bergstrand and Klimstra (1962) found the percent weight gain of birds treated with fenuron greater than the weight gain of control birds. The vast majority of deaths of Bobwhites treated with DIMP occurred between one and three hours post-treatment. The lethal effects of per os dosing with DIMP appeared much more rapidly for Bobwhites than for rats, mice, or Mallard ducks. Jones (1977) reported Mallard duck deaths occurring within 24 hours after dosing with DIMP. Hart (1975) reported most deaths occurring during the first 24 hours and no deaths occurring more than 48 hours after administration of DIMP to rats and/or mice. EXPERIMENT 2 Toxicity can be expressed not only as a single lethal dose but also as a lethal dietary concentration. Bein (1963), Worden and Harper (1963), and Stickel gt gt. (1965) have reported that the toxicity of the compound administered via gavage can be considerably different from the toxicity resulting from incorporation of the compound into the diet. The results of this study exemplify such difference in toxicity due to method of administration. LCSO values of DCPD or DIMP could not be determined for the Bobwhite due to insufficient mortality, even though the average mg of compound consumed per bird per day of either chemical was greater than the respective LDSO value. The mortality occurring in the DCPD fed birds reached a maximum of only 20% and was not dose related. The mortality of the DIMP fed birds occurred in only one dietary level group and was attributed to cannibalism. The predicted points of zero feed consumption of both chemicals were above 70000 ppm. These results are in agreement with Jones (1977) who reported undeterminable LC50 values for the Mallard duck fed DCPD or DIMP treated diets and predicted points of zero feed consumption of 77,300 and 23,222 ppm of DCPD and DIMP, respectively. Several values taken from extensive LC5O determinations (Heath gt gt., 1972) of 89 pesticidal chemicals are listed in Table 33. The LC5O value of DDT in Table 33 was taken from results by Heath and Stickel (1965). In general, comparison of the susceptability of the Bobwhite to various compounds by Heath gt gt. (1972) has shown polychlorinated 114 115 Table 33. Median lethal concentrations (LCSO's) of various pesticidal chemicals for Bobwhite quail chicks two to three weeks of age, listed alphabetically. Primary LC5O 95% confidence Chemical use (ppm) limits Aldrin ll 37 33- 41 Aroclor 1254 (PCB) 1d2 604 410— 840 Ceresan M F3 57 42— 74 Chlordane I 331 197— 479 DDE Dp4 825 697- 976 DDT I 611 514— 724 Dieldrin I 39 37— 41 Diquat H5 2932 1811—5256 Endrin I 14 ll— 24 Fenitrothion I 157 135- 183 Fenuron H 5000 no mortality Heptachlor I 92 76- 113 Lindane I,A6 882 755-1041 Malathion I 3497 2959-4117 Paraquat dichl. H 981 784—1213 Toxaphene I 828 619-1102 1 I = insecticide 2 Id: industrial 3 F = fungicide 4 Dp= degradation product 2 H = herbicide A = acaricide 116 biphenyls (PCB) to be the most toxic, organochlorines the least toxic, and organophosphates as intermediately toxic. Heath gt gt. (1972) also reported aldrin and dieldrin to be more toxic than 20 of the 23 organophosphates tested. It was suggested in this same study that gradual exposure to a compound via the diet can allow the test subject's detoxification system sufficient time to degrade the relatively unstable compounds, such as the organophosphates but insufficient time to degrade the relatively stable compounds such as organochlorines. Obviously this was not the case for the Bobwhite in this investigation with the compounds under considera- tion. Results of studies by Hill (1971) and Heath gt gt. (1972) utilizing several species of aves have shown considerable variation in species sensitivity to various compounds. Hill (1971) suggested that avian sensitivities to DDT and organophosphates was associated with body size and species type, i.e., wild or farm raised. Hill found true wild Bobwhites were approximately 1.4 times as sensitive as farm raised Bobwhites. Heath gt gt. (1972) compared the sensi— tivity of the Bobwhite, Japanese quail, Mallard duck, and pheasant to various compounds. The conclusions of that comparison study are as follows: 1. Bobwhites, Japanese quail, and pheasants are similar in sensitivity to organochlorines. 2. Bobwhites and Japanese quail are similar in sensitivity to all the compounds tested with the exception of polychlorin— ated biphenyl; the Bobwhites were the most sensitive and the Japanese quail the least sensitive of the four species. 3. Bobwhites and Japanese quail are more sensitive to organo- phosphates than pheasants or Mallard ducks. 117 4. Mallard ducks were generally the least sensitive of the four species tested. In general, the feeding of DCPD treated diets to the Bobwhite had little effect on their feed consumption, though small decreases in feed consumption at the higher DCPD dietary levels were noted. Diets containing 16000 ppm or more of DIMP caused a pronounced reduction of feed.inUfl«awith the least feed intake at the highest DIMP dietary level. Both of the observations on feed consumption were made during the first five days of the eight day test period. This reduction of feed intake of birds fed either chemical was undoubtedly due to a repellant effect of the compounds rather than a toxic effect since the birds increased their feed consumption when fed clean feed during the three day post-treatment period. Voluntary feed restriction of treated diets is not uncommon; Nestler and Bailey (1941) observed discriminatory feeding habits of quail given a choice of toxic Crotalaria seeds or clean feed. Ernst (1966) reported that quail voluntarily restricted their feed intake when sufficient levels of some pesticides were added to their diets. Frings and Boyd (1952) reported olfactory discrimina- tion by the Bobwhite. Jones (1977) reported that incorporation of DCPD into the diet greatly reduced feed consumption in twelve-day- old Mallard ducklings but had little effect on the feed consumption of 23-week-old Mallard ducks. In this same investigation Jones reported a great decrease in feed intake of Mallard ducks fed DIMP treated diets at levels of 12000 ppm and above. Linduska and Springer (1951) reported various feed consumption results when feeding six different insecticides to Bobwhites for 32 days. Body weight gains were generally reduced in Bobwhites fed DCPD or DIMP diets; the least weight gains occurring in birds fed 118 the highest dietary levels of the compounds. However, while the reduced body weight gain of the birds fed DIMP diets was fairly consistent with the decrease in feed consumption, the decreased weight gain of the birds fed DCPD treated diets was more pronounced than the reduction in feed consumption. Jones (1977) suggested an "unknown mechanism causing decreased uptake of nutrients" as explanation of body weight loss coupled with unchanging feed consumption when feeding DCPD treated diets to 23-week-old Mallard ducks. Another possible explanation of noncorresponding body weight and feed consumption data is a low estimate of feed waste. Jones (1977) also reported decreased body weight gain for Mallard ducks fed DIMP treated diets. Feed efficiency of Bobwhites fed DCPD or DIMP treated diets during the three day post treatment period showed no trends. All dietary concentrations of DCPD or DIMP were greater than their respective LD50 values and all birds consumed a greater amount of chemical (mg/kg/day) than the respective LD50 values, with the exceptions of the birds fed the lowest dietary levels. Fitshugh and Schouboe (1965) reported that animals tolerating an amount of chemical in their diet that was greater than the LDSO value was uncommon. A possible explanation of the phenomena investigated by Fitshugh and Schouboe (1965) is an observation by Stickel gt gt. (1965) who reported that absorption of some compounds through the gastrointestinal wall can be more efficient if the compound is incorporated into the diet than when given as a single dose. EXPERIMENT 3 The chronic test is conducted to determine the effects of feeding a chemical in the diet over an extended period of time to a test subject. This test differs from the subacute test in duration and parameters measured. Feed consumption was unaffected in Bobwhites fed either DCPD or DIMP-treated diets with one exception: the 12000 ppm DIMP birds showed a reduced feed intake for the first two-week period of the experiment. As noted previously the 12000 ppm DIMP ration was reduced to 0 ppm at approximately the fourth week of the experiment. During the ten-week pre-production period all groups of birds fed either chemical followed the same feed consumption pattern. During production, feed consumption of all groups of birds again followed a general pattern, with feed consumption steadily increas— ing to a peak followed by a gradual decline. The feed consumption pattern just described is typical of normal, untreated birds during their reproductive period. Scott gt gt.(l969) reported that feed intake increases to accomodate for the increased energy expenditure of egg production then decreases as egg production declines. The chronic study feed consumption results are in agreement with the findings by Jones (1977) who reported that feed consumption patterns of Mallard ducks fed DCPD (32,100 or 320 ppm) or DIMP (1000, 32000, or 10000) ppm) treated diets remained unchanged. Uneffected feed consumption of several other avian species fed various "treated" diets has been reported. Genelly and Rudd (1965) reported that 300 ppm toxaphene and 25 and 50 ppm dieldrin reduced the feed 119 120 intake of laying pheasants while 100 and 400 ppm DDT did not. Lillie gt gt. (1975) reported unaltered feed consumption in producing Leghorn hens after feeding various levels of PCB and PBB in the diets for eight weeks. Polin and Ringer (1978) reported 126, 625, and 3125 ppm PBB reduced feed intake of producing Leghorn hens. Pre-production body weight change of Bobwhites fed either DCPD or DIMP treated diets coincided with their respective feed consumption results and showed no treatment effects. These results are consistent with results reported by Jones (1977). Jones found no treatment effect on the body weight changes of pre—productive Mallard ducks fed either DCPD or DIMP diets (for dietary levels see preceding paragraph). In other studies conducted, Japanese quail fed Dybar (fenuron) or DDT gained more weight than their respective controls but consumed less feed than the controls (Bergstrand and Klimstra, 1962; Ernst, 1966), respectively. During the reproductive period, both weight change of Bobwhites fed DCPD or DIMP-treated diets showed no treatment effects. Female birds did show a greater weight gain than male birds in all groups. This type of weight difference between the Bobwhite sexes is consis- tent with the findings of many other investigators; Stoddard (1931), Aldrich (1946), Nestler (1949), Baldini (1951), Ripley (1960), Mahmoud (1966), and Georgis (1970). Again, Jones (1977) reported similar body weight change results in producing Mallard ducks fed DCPD or DIMP diets. Lillie gt gt. (1975) reported no effect on body weight changes due to the levels of PCB or PBB in diets fed to Leghorn laying hens. 121 The egg production of the DCPD fed Bobwhites showed no treatment-egg production relationship. Percent productions of birds fed the higher levels of DCPD (1250 and 4000 ppm) or control feed were near the standard value of 68.2 percent reported by Coleman (1930) cited from Hutt (1949). Percent production of the low level group (400 ppm) was reduced but generally the egg produc- tion pattern followed the same pattern as that of the control birds. Egg production of DIMP fed Bobwhites was reduced in the 1200 ppm group, the highest level of DIMP fed. However, the egg produc- tion pattern was similar to the egg production pattern of the control birds except that it was consistently lower. Overall, egg production of the control birds, the 12000/0 ppm birds and the 3800/380 ppm birds was less than the standard reported by Colemen (1930) cited from Hutt (1949) but well within the ranges reported by Nestler (1943), Nestler gt gt. (1944), DeWitt gt gt. (1949), Baldini gt gt. (1952, 1954), Kirkpatrick (1964), and Wilson gt gt. 1973). No effect on the incubation parameters measured occurred in either the DCPD or the DIMP experiments. The percentages of cracked eggs for each of the dietary groups and the control groups were above the normal range reported in the Federal Register (1975) but no percentages differed significantly from their respective control. The percent fertility and percent hatchability of eggs produced by birds of each dietary group, including the control group, were well within the normal range reported in the Federal Register (1975). Three week embryo viability was not measured for the following reasons: the difficulty of its determination, the possible adverse effects on the developing embryos from removing 122 the eggs from the incubator for parameter measurement, and the difficulty in stabilizing the temperature and the humidity of the incubators during parameter measurement. Jones (1977) reported normal values for egg production fer- tility, hatchability, and number of cracked eggs of Mallard ducks fed either DCPD or DIMP treated diets, results which are in agree- ment with the results of this quail study. Many investigators have found adverse effects on egg production of various avian species treated with different compounds. Genelly and Rudd (1965) found unaffected egg production, fertility, and hatchability of pheasants fed two levels of DDT but reported depressed egg production in pheasants fed toxaphene (300 ppm) or dieldrin (25 and 50 ppm). In this same study, the investigators reported reduced hatchability at 300 ppm toxaphene and reduced fertility at 50 ppm dieldrin. Ernst (1966) reported variable egg production results and unaf— fected fertility and hatchability of Japanese quail fed DDT (100, 300, and 500 ppm), Zectran® (100 and 300 ppm), and Zytron® (500, 700, 2000, and 4000 ppm). Ernst (1966) also reported that ® reduced egg production. Polin and Ringer (1978) 300 ppm Zectran reported reduced egg production of chickens and reduced hatch- ability of their progeny, when the chickens were fed PBB at 45 ppm and 30 to 45 ppm, respectively. Dewitt (1955, 1956), Shellenberger and Newell (1965), and Walkergt gt. (1969) have reported adverse effects on egg production, fertility, and hatchability of quail fed various levels of dieldrin. The levels of dieldrin necessary to cause the adverse effects just mentioned are not in agreement among the workers cited. Cross gt gt. (1962) and Gish and Chura (1970) found reduced egg production in quail fed DDT treated diets. Sauter 123 and Steele (1972) reported reduced egg production of laying hens fed various levels of malathion, DDT, lindane, or diazinon. Schom gt_gt, (1972) found severely reduced egg production in quail fed azodrin (25 ppm)- Two week livability of Bobwhite hatchlings from parents treated with DCPD or DIMP showed no treatment effect. Percentages of chick survival of all groups including the control group, were within the normal range reported in the Federal Register (1975). These results are in agreement with Jones (1977) who reported no effect on two week livability of progeny of Mallard ducks fed DCPD or DIMP treated diets. Progeny livability of several other avian toxicological studies have shown no effect and/or adverse effects. Ernst (1966) found a reduction in the fourteen day livability of progeny of quail fed DDT-treated diets. Genelly and Rudd (1956) reported unaffected fourteen day progeny survival of pheasants fed various levels of DDT, toxaphene, or dieldrin. Jones and Summers (1968) reported 200 ppm DDT fed to Japanese quail reduced their progeny survival. Eggshell thickenss data from either DCPD or DIMP treated Bobwhites were consistent with normal values reported in the Federal Register (1975). These results were not unexpected since the Bobwhite is not susceptible to eggshell thinning (U.S. Army, 1975). Jones (1977) reported no effect on shell thickness of eggs produced by Mallard ducks fed either DCPD or DIMP—treated diets. In general, the feeding of DCPD or DIMP—treated diets to Bobwhites had no effect on the appearance and/or weights of their various internal organs. Exceptions to the preceding generality 124 were the proventriculus and gizzard weights of the Bobwhites fed 1200 ppm DIMP and the liver weights of the male Bobwhites fed 4000 ppm DCPD. The proventriculi and gizzards of Bobwhites fed 1200 ppm DIMP weighed more than did the same organs of the control birds. The livers of male Bobwhites, fed 4000 ppm DCPD, weighed less than the livers of the control birds but no lesions were ob- served. Jones (1977); however, reported unaffected organ weights of Mallard duCks fed DCPD or DIMP treated diets. Other various organ aberrations have been reported in avian toxicity studies. Gish and Chura (1970) found the number of males with testes exceeding 1000 mg (full spermatogenic activity) was inversely pro— portional to the amount of DDT in the diet. Burlington and Lindeman (1950) observed a decrease in the development and size of the testes of Leghorn cockerels injected with DDT. Genelly and Rudd (1955, 1956) reported liver degeneration in pheasants poisoned with DDT. Two blood parameters, hemoglobin concentration and hematocrit (packed cell volume), plus the calculated mean corpuscular hemo- globin concentration, were measured in the DCPD or DIMP treated Bobwhites and found to be unaffected by treatment. Male Bobwhites, in all groups fed either chemical, had both a higher level of hemoglobin and higher number of erythocytes than the respective female Bobwhites. There was no difference in the mean corpuscular hemoglobin concentration between male and female birds. The greater hemoglobin concentration and hematocrit values of the males compared to the females is consistent with findings by numerous investigators. The relationship of a greater level of hemoglobin and maleness is correlated with the increased numbers of erythro- cytes in the male due to testosterone. Androgen increases the 125 numbers of erythrocytes whereas estrogen tends to decrease the number of red cells (Sturkie, 1976). Nirmalin and Robinson (1972) reported that estrogen decreases erythrocyte numbers in Japanese quail by initially decreasing erythrOpoietic activity. The mean hemoglobin values determined for Japanese quail by Nirmalin and Robinson (1972) were slightly higher than the hemoglobin values determined for the Bobwhite in this study. The mean hematocrit values, male or female, were within the normal ranges (Spiers, personal communication) and very near the values reported by Bond and Gilbert (1958) and Ernst gt gt. (1971). Lucas and Jamroz (1961) reported a five percent variation in hematocrit values in chickens as normal. Ernst gt gt. (1971) reported no significant variation in the mean packed cell volume by sex, which was not in agreement with results of this study. Nice gt gt, (1935) reported a mean crythocyte count of 3.5 mill./ mm3 for three female Bobwhites. In agreement with the unaffected blood parameters of quail in this study, Jones (1977) reported normal hemoglobin concentrations, hematocrit, and mean corpuscular hemoglobin concentration values of Mallard ducks fed DCPD or DIMP—treated diets. Different effects on blood parameters of other avian species treated with various compounds have been reported. Rehfeld gt gt. (1972) found decreased hemoglobin and hematocrit values in chicks treated with PCB's. Ernst (1966) studied the effects of several pesticides on Japanese quail and found that DDT, Zeetran, and Zytron when incorporated into the diets of male birds caused a decrease in packed cell volume but not the mean corpuscular volume "indicating normocytic and normoblastic anemia". Burlington and Lindeman (1950) observed a 126 reduction in erythocytes coincident with normal hemoglobin concentrations in male chickens chronically injected with DDT. Van Thiel (1977) reported a decrease in hematocrit and hemoglobin concentration in Leghorn cockerels fed 150 ppm PBB. Leukocyte differentials of Bobwhites fed DCPD or DIMP were generally unaffected. Only the birds fed 1250 ppm DCPD showed any aberration. In the 1250 ppm DCPD group the mean number of eosinophils was significantly greater than the mean number of eosinophils of the control birds. However, the control birds of the DIMP experiment had a similar mean number of eosinophils. Sturkie (1976) and Lucas and Jamroz (1961) have reported a great variability in differential counts due to the methods used for counting, age of the subject, sex of the subject, physical con- dition of the subject, environment, diet, hormones, drugs, and other factors. Lucas and Denington (unpublished data cited in Lucas and Jamroz, 1961) found that "if the coefficient of vari- ability for the 95 percent fiducial interval were to be held to some fixed values,for example, i 10 percent of the group mean, then the following number of birds would be needed for a study on each of the components of the blood: Blood component Number of chickens Lymphocyte/mm3 130 Monocyte/mm3 352 Heterophil/mm3 240 Eosinophil/mm3 ‘ 3615 Basophil/mm3 551 The normal leucocyte differentials of Bobwhites are in agreement with Jones (1977). In Jones' investigation, he reported no 127 significant differences between the leucocyte differentials of Mallard ducks fed DCPD or DIMP treated diets and their respective controls. Again different results were obtained in studies utilizing other avian species and compounds. Ernst (1966) reported that Japanese quail fed DDT or Zectran showed no consistent changes in leukocyte differentials. In this same study, Ernst reported that the feeding of Zytron to Japanese quail caused a con- sistent increaSe in the lymphocyte percentage of blood of the male quail coincident with a decrease in the percentage of heterophils of the blood. A decrease in the blood percentage of heterophils of female quail was observed by Ernst (1966). Ernst (1971) reported the following leucocyte values for the Bobwhite: % of blood* Cell type Male Female Lymphocyte 63 50 Monocyte 2 2 Heterophil 30 42 Eosinophil 3 3 Basophil 2 2 * Values estimated from graphs During the chronic study the Bobwhites suffered two periods of high mortality. The initial period occurred during the first four weeks of the experiment when four birds from each of the highest levels of DIMP‘(3800 and 12000 ppm) died. The cause of death could not be determined by gross examination of the birds at necropsy. Upon consultation with the Project Officer (U.S. Army) the decision was made to reduce the DIMP dietary levels of 3800 and 12000 ppm to 380 and 0 ppm, respectively. The second period of high mortality occurred during a three day span in the eleventh week of the experiment. In these three days, mortality was independent of DCPD dietary level. At necropsy, hemorrhagic 128 lungs were observed in all the birds that expired during the three day period. No other abnormalities were observed. The cause of death could not be determined. Mortality other than the two cases of high mortality mentioned was sporadic and not treatment related. CONCLUSIONS LD50:. Observations on mortality, feed consumption, and body weight change of Bobwhite quail show both DCPD and DIMP to be slightly toxic to that species. LC50= Generally the lack of treatment effect on feed consump- tion, mortality, and body weight gain of young Bobwhites fed DCPD treated diets suggests that the quail could not consume enough chemical to produce sufficient mortality to calculate a LC50 value. Bobwhites fed DIMP treated rations showed de— creasing feed consumption coincident with decreasing body weight gain as dietary level of DIMP increased. These results coupled with no mortality trends suggest that the quail voluntarily restricted their feed intake and thus did not consume a suffi- cient amount of chemical to cause enough deaths to calculate a LC50 value. Chronic: Of the various parameters measured very few of those of the treated quail were significantly different from the respective parameters of the control quail. The parameters that did show significant aberrations showed no consistent treatment- effect results. Thus, the results suggest that DCPD and DIMP have little effect on Bobwhite survival and reproduction at the levels tested. However, before the 3800 and 12000 ppm DIMP dietary levels were reduced, unexpected mortality occurred at those levels. There— fore, additional studies should be conducted, perhaps using different criterias of toxicity, different animal species, and/or different parameters to provide a better toxicity profile of DIMP. 129 APPENDICES APPENDIX A CHEMICAL STRUCTURES AND ALTERNATE NAMES FOR DIMP AND DCPD DIMP Structural formula ((CH3)2CHO)2(CH3)P Alternative names C M3 \C / +5 0 \ //0 G3 \ / \ o ’fifia”c> CH 3 cu, diisopropyl methylphosphonate; DIMP; phosphonic acid, bis- (l-methylethyl) ester (Chem._Abstr. after 1971); phOSphonic acid, methyl-, diispropyl ester (1947-1971); methanephosphonic acid, diisopropyl ester. DCPD Structural formula AlternatiVe names CH 3‘ EC.“ ‘0,“ Q“ CH3 dicyclopentadiene; bicyclopentadiene; biscyclopentadiene; 3a,4,7,7a-tetrahydro-4, 7—methanoindene. 130 APPENDIX B ANALYSIS OF FEED Quail Breeder QB 72 Ingredients Corn Soybean meal, 49% Meat scrap, 50% Alfalfa meal, dehy. Animal fat, stabl. Limestone Dicalcium phosphate Choline chloride, 50% Methionine hydroxy analogue Salt, iodized Mineral mix A Vitamin mix A Antioxidant 131 kg 408.41 296.65 45.36 40.82 51.71 45.36 6.35 2.72 0.91 907.29 132 Quail Starter Q8 72 Ingredients Corn Soybean meal, 49% Fish meal Meat scraps, 50% Alfalfa meal, dehy. Animal fat, stabl. Dicalcium phosphate Choline chloride, 50% Methionine hydroxy analogue Salt, iodized Vitamin mix A Mineral mix A Antioxidant ethoxyquin/or BHT k9 348.64 384.55 28.18 31.82 40.91 53.64 11.82 2.73 .91 3.18 2.73 2.73 0.11 911.95 APPENDIX C DIET PREPARATION C1: LC50 diets. A premix of DCPD or DIMP was prepared by adding the pure chemical to corn oil and mixing by hand with quail starter diet. The final individual diets were prepared in one kilogram quan- tities by combining a quantity of premix with quail starter diet (Table C1). VAll final diet mixing was completed by tumbling the mixture for 15 minutes in a seven kilogram capacity feed can in a Paul G. Abbe mixerl. The total amount of chemical-corn oil solution was not more than two percent of the diets containing DCPD. Table C1. Premix 7 Amount of Amount of Total Chemical chemical (gms) feed (gms) (gms) ppm DCPD 90' 4410 4500 20,000 DIMP 180 4320 4500 40,000 1 Paul G. Abbe, Inc., Little Falls, New Jersey 07424. 133 134 Table C1 con't. Diets Amount of Amount of Total Chemical premix (gms) feed (gms) , (gms) ppm DCPD 100 900 1000 2000 200 800 1000 4000 300 700 1000 6000 400 600 1000 8000 500 500 1000 10000 600 400 1000 12000 700 300 1000 14000 800 200 1000 16000 900 100 1000 18000 DIMP 100 900 1000 4000 200 800 1000 8000 300 700 1000 12000 400 600 1000 16000 500 500 1000 20000 600 400 1000 24000 700 300 1000 28000 800 200 1000 32000 900 100 1000 36000 C2: Chronic diets A premix of DCPD or DIMP was prepared by the same method as employed in the premix preparation of the LC50 experiment. Final individual diets were prepared by the addition of an appropriate amount of premix to quail breeder diet. All mixing was completed by handmixing for ten minutes. Table C2. Premix Amount of Amount of Amount of Total oil (gms) chemical (gms) feed (gms) (gms) ppm DCPD 60 120.0 2820.0 3000 40000 60. 37.5 2902.5 3000 12500 60 12.0 2928.0 3000 4000 DIMP 60 360 2580 3000 120000 60 114 2826 3000 38000 60 36 2904 3000 12000 60 11.4 2928.6 3000 3800 Diets Premix (gms) Feed (gms) Total (gms) ppm DCPD 100 900 1000 4000 100 900 1000 1250 100 900 1000 400 DIMP 100 900 1000 12000 100 900 1000 3800 100 900 1000 1200 100 900 1000 380 135 APPENDIX D PREPARATION OF WRIGHT'S STAIN AND BUFFER Wright's Stain 3.3 grams Wright's powder is added to 500 cc fresh, pure methyl alcohol. The stain is ripened for several months to room temp— erature in a stoppered brown bottle. Buffer 3.80 gm Na2HP04 5.47 gm KH2P04 Dissolve in 500 ml distilled water and bring total volume to 1000 m1. Set pH at 6.4. 136 APPENDIX E PREPARATION OF DRABKIN'S REAGENT 1000 mg Sodium Bicarbonate NaHCO3 50 mg Potassium Cyanide KCN 200 mg Potassium Ferricyanide K3Fe(CN)6 1250 mg Mix to dissolve and dilute to 1 liter. The solution should be stored in a sealed amber bottle and kept refrigerated. 137 APPENDIX F DETERMINATION OF HEMOGLOBIN CONCENTRATION 138 Figure F1. Sample hemoglobin concentration calculation. The line is constructed by plotting the % absorbance of each standard against its known hemoglobin concentration. Absorbance 139 0.5 Standard 0.4 0.3 ———-S—aT-P'—3-————; Absorbance l l | 0.2 l l Calculated I Hemoglobm 1 Concentration 0.1 . l . l l | \l) O 4 8 12 16 Hemoglobin gm/dl Figure F1. CITED REFERENCES Aldrich, J. W., 1946. The United States races of the Bobwhite. AUK 63:493-507. Baldini, J. T., 1951. A study of the nutritive requirements of the captive Bobwhite quail. Ph.D. Thesis, Purdue Univ., Lafayette, IN. Baldini, J. T., R. E. Roberts, and C. M. Kirkpatrick, 1952. Studies of the reproduction cycle of the Bobwhite quail. J. Wildl. Man. 16:91-93. Baldini, J. T-, R. E. Roberts, and C. M. Kirkpatrick, 1954. The reproductive capacity of Bobwhite quail under light stimula- tion. Poultry Sci. 33:1282-1283. Bein, J. H., 1963. Rational and irrational numbers in toxicology. Proc. of the European Soc. for the Study of Drug Toxicity. 2:15-26. Bergstrand, J. and W. D. Klimstra, 1962. Toxicity of "Dybar" to Bobwhite quail. J. Wildl. Man. 26:325—327. Bond, C. F. and P. W. Gilbert, 1958. Comparative study of blood volume in representative aquatic and nonaquatic birds. Am. J. Physiol. 194:519-521. Burlington, H. and U. F. Lindeman, 1950. Effect of DDT on testes and secondary sex characters of White Leghorn cockerels. Proc. Soc. Exp. Biol. and Med. 74:48-51. Christensen, H. E., T. T. Lugenbyth, and B. S. Carroll, 1974. The Toxic Substance List. U.S. Dept. of Health, Education, and Welfare. Coburn, D. R. and R. Treichler, 1946. Experiments on toxicity of DDT to wildlife. J. of Wildl. Man. 10:208-216. Cross, D. L., H. L. King, and D. L. Haynes, 1962. The effects of DDT in the diet of the Japanese quail. Quart. Bull. Mich. Agr. Exp. Sta. 44:688-696. Dacre, J., 1975. Fact Sheet-DIMP Toxicity. U.S. Army Chemical Center, MD. Dahlen, J. H. and A. O. Hougen, 1954. Acute toxicity of certain insecticides to the Bobwhite quail and Mourning dove. J. Wildl. Man. 18:477-481. DeWitt, J. B., 1955. Effects of chlorinated hydrocarbon insecti- cides upon quail and pheasants. J. Agr. Food Chem. 3:672-676. 140 141 DeWitt, J. B., 1956. Chronic toxicity to quail and pheasants of some chlorinated insecticides. J. Agr. Food Chem. 4:863-866. DeWitt, J. B., R. B. Nestler, and J. V. Derby, Jr., 1949. Calcium and phosphorus requirements of breeding Bobwhite quail. J. Nutr. 39:567-577. Ernst, R. A., 1966. The physiological effects of selected pesti— cides on the Japanese quail (Coturnix coturnix japonica) and embryogenesis of the domestic fowl. Ph.D. Thesis, Michigan State Univ., E. Lansing, MI. Ernst, R. A., T. H. Coleman, A. W. Kulenkamp, R. K. Ringer, and S. Pangborn, 1971. The packed cell volume and differential leucocyte count of Bobwhite quail (Colinus virginianus). Poultry Sci. 50:389-392. Federal Register, Wednesday, June 25, 1975. Environmental Protec- tion Agency; Pesticide program, guidelines for registering pesticides in the United States. Vol. 40, No. 123, Part II. Fitshugh, O. G. and P. J. Schouboe, 1965. Subacute toxicity. In: Appraisal gt the Safety of Chemicals in Foods, Drugs, and Cosmetics. The AssociatIEn of Food afid Drug Officials of the United States, P.O. Box 1494, Topeka, KS. Ford-Moore, A. H. and B. J. Perry, 1948. The chemistry of the alkane fluorophosphorates: Part VI. The dialkanepyrophosphonates, Summary of Portor Technical Paper No. 68. U.S. Army Chemical Center, MD. Frings, H. and W. A. Boyd, 1952. Evidence of Olfactory Discrimina- tion by the Bobwhite quail. Am. Midl. Nat. 48(1):181-184. Gage, J. C., 1970. The subacute inhalation toxicity of 109 industrial chemicals. Brit. J. Industr. Med. 27:1-18. Genelly, R. E. and R. L. Rudd, 19561. Chronic toxicity of DDT, Toxophene, and dieldrin to ring—necked pheasants. Calif. Fish and Game 42:5-14. Genelly, R. E. and R. L. Rudd, 19562. Effects of DDT, Toxophene, and dieldrin on pheasant reproduction. AUK 73:529-539. Georgis, H. D., 1970. Heritability estimates, correlation coefficients and other parameters in Bobwhite quail. Ph.D. Thesis, Michigan State Univ., E. Lansing, MI. Gill, J. L., 1978. Design and Analysis gt Experiments tg the Animal and Medical Sciences. Vol. 1-3. The Iowa State University Press, Ames, IA. 142 Gish, C. D. and N. J. Chura, 1970. Toxicity of DDT to the Japanese quail as influenced by body weight, breeding condition, and sex. Toxicol. and Appl. Pharmaco., 17:740—751. Hart, E. R., 1975. Unpublished data. Hart, E. R., 1976. Unpublished data. Heath, R. G., J. W. Spann, E. F. Hill, and J. F. Kreitzer, 1972. Comparative Dietary Toxicities of Pesticides tg Birds. U.S. Fish and Wildlife Service Spec._Sci. Rept.—Wildlife. No. 152, 57 pp. Heath, R. G. and L. F. Stickel, 1965. Protocol for testing the acute and relative toxicity of pesticides to penned birds. IN: The Effects of Pesticides on Fish and Wildlife. U.S. Fish and Wildlife Circular 226. Hill, E. F., 1971. Toxicity of selected mosquito larvicides to some common avian species. J. Wildl. Man. 35:757—762. Horton, R. G., 1948. Screening candidate chemical agents. U.S. Army Chemical Center, MD. Hutt, F. B., 1949. Genetics gt the Fowl. McGraw—Hill Book Co., NY. pp. 288. Jacobson, K. H., 1953. Report No. 17. The Acute Toxicity of Some Intermediates tg Gt Manufacture, U.S. Army Chemical Center, MD. Jones, F. J. S. and D. D. B. Summers, 1968. The relation between DDT in diets of laying birds and viability of their eggs. Nature 217:1162-1163. Jones, R. E. Jr., 1977. Toxicity of diisopropyl methylphosphonate and dicyclopentadiene on the Mallard. M.S. Thesis, Mich. State Univ., E. Lansing, MI. Kavanagh, T., 1978. Personal communication. Kinkead, E. R., U. C. Pozzani, D. L. Geary, and C. P. Carpenter, 1971. The mammalian toxicity of dicyclopentadiene. Toxicol. and Appl. Pharmaco., 20:552—561. Kirkpatrick, C. M., 1964. Age versus environment as conditions for reproduction in caged Bobwhites. J. Wildl. Man. 28:240-243. Lillie, R. J., H. C. Cecil, T. Bitman, and G. F. Fries, 1975. Toxicity of certain polychlorinated and polybrominated biphenyls on reproduction efficiency of caged chickens. Poultry Sci. 54:1550. Linduska, J. P. and P. F. Springer, 1951. Chronic toxicity of some new insecticides to Bobwhite quail. U.S. Dept. Interior, Spec. Sci. Rept., Wildlife No. 9, Mimeo, 11 pp. 143 Lucas, A. M. and C. Jamroz, 1961. Atlas gt Avian Hematology. Agric. Monogr. 25, U.S. Dept. Agric., Washington, D.C. Mahmoud, T. H. H., 1966. Growth and reproduction of Bobwhite quail raised in confinement. Ph.D. Thesis, Michigan State Univ., E. Lansing, MI. Nestler, R. B., 1943. Effect of large quantities of common salt in the diet of Bobwhite quail. J. Wildl. Man. 7:418-419. Nestler, R. B., 1949. Nutrition of the Bobwhite quail. J. Wildl. Man. 13:342-358. Nestler, R. B. and W. W. Bailey, 1941. The toxicity of Crotularia specabilis seeds for quail. J. Wildl. Man. 5:309-313. Nestler, R. B., L. M. Llewellyn, and M. Y. Benner, 1944. War-time diets for growing Bobwhite quail. J. Wildl. Man. 8:221-228. Nice, L. B., M. M. Nice, and R. M. Kraft, 1935. Erythrocytes and hemoglobin in the blood of some American birds. Wilson Bull. 47:120-124. Nirmalin, G. P. and G. A. Robinson, 1972. Hematology of Japanese quail treated with exogenous stilbestrol dipropionate and testosterone proprionate. Poultry Sci. 51:920. Polin, D. and R. K. Ringer, 1978. PBB fed to adult female chickens: Its effect on egg production, reproduction, viability of off- spring, and residues in tissues and eggs. Environmental Health Perspectives 23:283-290. Rehfeld, B. M., R. L. Bradley, Jr., and M. L. Sunde, 1972. Toxicity studies on PCB's in the chick. Poultry Sci. 51:488-492. Ringer, R. K., 1978. Personal communication. Ripley, T. H., 1960. Weights of Massachusetts quail and comparisons with other geographic samples for taxonomic significance. AUK 77:445. Sauter, E. A. and E. E. Steele, 1972. The effect of low level pesticide feeding on fertility and hatchability of chicken eggs. Poultry Sci. 51:71-76. Schom, C. B., N. E. Walker, and U. K. Abbott, 1972. Azodrin and its effects on four avian species. Poultry Sci. 51:1860. Scott, M. L., M. C. Nesheim, and R. J. Young, 1969. Nutrition gt the Chicken. Scott and Associates, Ithaca, NY. Shashkina, L. F., 1965. Materials for substantiation of maximum permissible concentration of cyclopentadiene and of its dimer - dicyclopentadiene - in the atmosphere of industrial premises. Gigiena Truda: Prof. Zabolevaniya, 9:13-19. 144 Shell Chemical Company, restricted information. Shellenberger, T. E. and G. W. Newell, 1965. Toxicological evalua- tions of agricultural chemicals with Japanese quail (Coturnix cotrunix japonica). Lab. Anim. Care 15:119-130. Smyth, H. F., C. P. Carpenter, C. S. Weil, U. C. Pozzani, and J. A. Striegel, 1962. Range—finding toxicity data: List VI. Am. Indust. Spiers, D., 1978. Personal communication. Stickel, W. H., W. E. Dodge, W. G. Sheldon, J. B. DeWitt, and L. F. Stickel, 1965. Body condition and response to pesticides in woodcocks. J. Wildl. Man. 29:147—155. Stoddard, H. L., 1931. The Bobwhite quail - Its habits, preserva- tion, and increase. Charles Scribner's Sons, NY. Sturkie, P. D., 1976. Avian Physiology. 3rd ed. Springer Verlag, New York, NY. Tucker, R. K. and D. G. Crabtree, 1970. Handbook of toxicity of pesticides to wildlife. U.S. Bureau of Sport Fish and Wildl. Res. Publ. 84. Tucker, R. K. and M. A. Haegele, 1971. Comparative acute oral toxicity of pesticides to six species of birds. Toxicol. and Appl. Pharmacol. 20:57-65. U.S. Army, 1975. Supplement to general protocol for evaluating the effects of possible toxicants on wildlife with specific material pertaining to the Rocky Mountain Arsenal. Medical Research and Development Command. Van Thiel, L. R., 1977. The effects of polybrominated biphenyls on the hematology and plasma erythropoietin levels of the Single Comp White Leghorn Cockerel. M.S. Thesis, Mich. State Univ., E. Lansing, MI. Walker, A. I. 1., C. H. Neill, D. E. Stevenson, and J. Robinson, 1969. The toxicity of dieldrin (HEOD) to Japanese quail (Coturnix coturnix japonica). Toxicol. and Appl. Pharmacol. 15:69-73. Wilson, H. R., M. W. Holland, Jr., and R. L. Renner, Jr., 1973. Egg laying cycle characteristics of the Bobwhite (Colinus virginianus). Poultry Sci. 52:1571-1573. Worden, A. N. and Harper, K. H., 1963. Oral toxicity as influenced by method of administration. Proc. of the European Soc. for the Study of Drug Toxicity 2:15-26. GENERAL REFERENCES Blood, F. R., 1969. Essays tg Toxicology, 2 volumes. Academic Press, Inc., New York, NY. 7 Buck, W. B. and G. D. Osweiler, 1976. Clinical and Diagnostic Toxicology. 2nd ed., Kendall/Hunt Publishing Co., Dubuque, IA. Clyne, R. M. and C. B. Shaffer. Toxicological Information Cyanamid Organophosphate Pesticides. 3rd ed., Am. Cyanamid Co., Princeton, NJ. DuBois, K. P. and E. M. K. Geiling, 1959. Textbook gt Toxicology. Oxford University Press, Inc., New York, NY. Jones, L. M., N. H. Booth, and L. E. McDonald, 1977. veterinary Pharmacology and.Therapeutics. The Iowa State Univ. Press, Ames, IA. . Loomis, T. A., 1968. ‘Essentials gt Toxicology. Lea and Febiger, Philadelphia, PA. 145 MICHIGAN ST ATE UNIV. 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