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FIFFFF“-F’F““ I F'...’ “g FFFFFFFF' .. ‘ '.II‘lvxIIIFI'I “Fina“ IwipIIII.‘ Fr FFFFF‘I 'IF“1FF.F:FFIFF ‘F FFFFFwFIFII 1“ '11le “”2““ I .FFIF FF‘IIF“ 1'“ inufi 16%: “1““ “h“; F“ F'FFF“ “n“, ‘I F1FF"FFFFFFF Fl F 1 F“ F .FF.“ F )F .111...“ 1- - 'Flv...1;3I . 1-1121“ 1 ‘1 “FF“FFF . 1:“ 1... . 2 ' FFI,‘..1'IIII|II ”'1“: )1‘ F. In :JFpII .(F'F 3'1! II’III n WIN-11”?“ ’r“" '1; II“ 1. 11.1: '11 1 .1 1.1" 'F1 '1F'. 11‘ 'F'. '--.11- F ..1: 3' .1: .. . 'FF“ ' F “FFFFIIFF‘I F FrlF'F ' ' F n'F F F F “ F FF‘FF F F IHFIF’I “ 1F“. “‘.‘ .l “. 1“: IFu’a. '. III] ““5 . FF“ 11.11". "FIFFFF' 3.011 ,l ’ ,-F .1113 ‘, -- ,. .r. .- ' .F“ ’.'.Ff1F“ “FFFFFFR by“ “““FFKFIZIFFFFF‘FF FFI'. F'FFIIFIIFF II ““FFFIF F F “F‘F 'fi'). III III I 321 II II IIIIIIII IIIIIIIIIIII This is to certify that the thesis entitled TOXICITY 0F DIISOPROPYL METHYLPHOSPHONATE AND DICYCLOPENTADIENE TO THE MINK (MUSTELA VISON) presented by Terrance J. Kavanagh has been accepted towards fulfillment of the requirements for M.S. Physiology degree in @fi’m fig?) Major professor Date 1/4/80 - ' __.__L, Mflww OVERDUE FINES: 25¢ per du per item RETURNING LIBRARY MATERIALS: Place in book return to move charge from circulation records 9m: umvuun ILL-1“}. / ! J51” 0373290 TOXICITY 0F DIISOPROPYL METHYLPHOSPHONATE AND DICYCLOPENTADIENE TO THE MINK (MUSTELA VISON) By Terrance J. Kavanagh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1979 éHqVSE ABSTRACT TOXICITY OF DIISOPROPYL METHYLPHOSPHONATE AND DICYCLOPENTADIENE TO THE MINK (MUSTELA VISON) W Terrance J. Kavanagh Diisopropyl methylphosphonate (DIMP) and dicyclopentadiene (DCPD) were evaluated for their acute oral, sub-acute dietary, and chronic dietary toxicity to ranch—raised mink (Mustela vison). DIMP was shown to be moderately toxic to mink (LDSO = 503 mg/kg) by acute oral exposure. An acute oral LDSO for DCPD could not be determined, but was estimated to be greater than lOOO mg/kg. A 2l-day dietary LCSO to mink for DCPD was calculated to be 6800 ppm DCPD. Feed consumption and body weight were significantly reduced at the highest level of DCPD (l0,000 ppm). Gross pathologi- cal examination at necropsy revealed no consistent changes for any treatment. An LCSO for DIMP could not be determined since no mortal- ity occurred at the highest dietary concentration (l0,000 ppm). Post— mortem examination revealed no dose related pathologies. A reduction tion in body weight and feed consumption by mink fed DCPD or DIMP was attributable to decreased feed palatability. Chronic ingestion of dietary DIMP caused higher mortality in female mink. No adverse effects were noted for either DIMP or Terrance J. Kavanagh DCPD treatment regarding growth, feed consumption, blood para- meters, or reproduction. Body weight gain of mink kits during lac- tation was significantly less for 200, 400, and 800 ppm DCPD treat- ments. A reduction in testes weight for males on the 800 ppm DCPD treatment was noted. Histological examination revealed no consistent changes associated with toxicosis for any DIMP OR DCPD treatment. ACKNOWLEDGMENTS The author wishes to express his appreciation and thanks to all of those who have lended their time, consultation, and inter- est toward the completion of this research, especially Dr. R. K. Ringer, Dr. R. J. Aulerich, Dr. T. H. Coleman, and Dr. w. R. Duke- low. Special recognition and gratitude is expressed to Kathy S. Howell, Ross E. Jones, Angelo Napolitano, Kay Trosko, Mark Greenlee, and Ron Mehler for the many hours of assistance they provided. This research was supported by a grant from the U.S. Army Medical Research and Development Command, Washington, D.C. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES INTRODUCTION Statement of the Problem REVIEW OF LITERATURE . DIMP DCPD OBJECTIVES PART I TOXICITY 0F DIISOPROPYL METHYL PHOSPHONATE TO MINK . Test l--Acute LD50 Procedure . Results Discussion . . . Test 2--Subacute LCSO Procedure . . Results Discussion Test 3--Chronic Procedure . Results Discussion Conclusions . Page ix PART II TOXICITY OF DICYCLOPENTADIENE TO MINK Test l--Acute L050 Procedure . Results Discussion . . . Test 2--Subacute LC50 Procedure . . Results Discussion Test 3--Chronic Procedure . Results Discussion Conclusion APPENDICES LITERATURE CITED iv Page Table 10. ll. 12. T3. LIST OF TABLES Summary of toxicity data on DIMP for acute exposure . . . . . Summary of toxicity data on DCPD for acute exposure . . . . . Acute oral toxicity of DIMP to adult female mink Mortality associated with a subacute 21-day dietary administration of DIMP and a 7-day post-treatment recovery period . . . . . . . . . Change in body weight of mink on 2l-day dietary LCSO test and post-treatment recovery Effect of subacute dietary DIMP administration upon percent change in mink body weight taken at weekly intervals . . . . . . . . . . . Feed consumption of mink on 21-day dietary LC50 test and post-treatment recovery period Feed consumption, body weight, and amount of chemi- cal ingested by adult mink fed DIMP at various levels for 21 days . . . . . Effect of subacute dietary DIMP upon mink hematocrit values and differential leukocyte counts Effect of subacute dietary DIMP upon female mink organ weights . . . . Effect of subacute dietary DIMP upon male organ weights . . . . . . . . Mortality of mink fed DIMP at various levels for l2 months Effect of chronic dietary administration of DIMP to male and female mink upon body weight (g i S.E.) gain by date . . . . Page l8 T9 23 24 27 28 3O 31 38 39 Table Page 14. Effect of chronic dietary administration of DIMP to male and female mink upon mean percent change in body weight (:S.E.) by date . . . . . . . 42 15. Effect of chronic administration of DIMP to mink upon feed consumption (9 i S.E.) by date . . . . 45 T6. Calculation of estimated daily intake of DIMP by mink fed DIMP at various levels for l2 months . . 47 l7. Effect of chronic dietary administration of DIMP to male and female mink upon peripheral blood mean packed cell volume (hematocrit %) . . . . 48 18. Effect of chronic dietary administration of DIMP to male and female mink upon peripheral blood hemoglobin concentration . . . . . . . . . 49 l9. Effect of chronic dietary administration of DIMP to male and female mink upon peripheral blood mean corpuscular hemoglobin concentration (MCHC) . . . 50 20. Effect of chronic administration of DIMP to adult mink upon differential leukocyte count . . . . 5l 2l. Effect of DIMP on reproductive performance of mink . . . . . . . . . . . . . . . . 53 22. Performance of suckling offspring and dams fed DIMP . . . . . . . . . . . . . . . . 54 23. Effect of chronic administration of DIMP to mink on organ weights (9 i S.E.) at necropsy . . . . 55 24. Mortality associated with a subacute 2l-day dietary administration of DCPD and a 7-day post-treatment recovery period . . . . . . . . . . . . 65 25. Change in body weight of mink on 2l-day dietary LCSO test and post-treatment recovery . . . . . 68 26. Effect of subacute dietary DCPD administration upon percent change in mink body weights taken at weekly intervals . . . . . . . . . . . . 72 27. Feed consumption of mink on 2l-day dietary LCSO trial and post-treatment recovery period . . . . 73 vi Table 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. Feed consumption, body weight, and amount of chemi- cal ingested by adult mink fed DCPD at various levels for 2l days . . . . . . Effect of subacute dietary DCPD upon mink hemato- crit values and differential leukocyte counts Effect of subacute dietary DCPD upon male mink organ weights . . Effect of subacute dietary DCPD upon female mink organ weights . . . . . Mortality of mink fed DCPD at various levels for 12 months Effect of chronic dietary DCPD administration to male and female mink upon body weight (g + S. E. ) gain by date . . . . Effect of chronic dietary administration of DCPD to male and female mink upon percent change in body weight (i S.E.) by date . . . . . . Effect of chronic administration of DCPD to mink upon feed consumption Calculation of estimated daily intake of DCPD by mink fed DCPD at various levels for l2 months Effect of chronic dietary administration of DCPD to male and female mink upon peripheral blood mean packed cell volume (hematocrit %) Effect of chronic dietary administration of DCPD to male and female mink upon peripheral blood mean hemoglobin concentration . Effect of chronic dietary administration of DCPD to male and female mink upon mean corpuscular hemoglobin concentration (MHCH) . Effect of chronic administration of DCPD to adult mink upon differential leukocyte count Effect of DCPD on reproductive performance of mink . . . . . . . . . . . . vii Page 75 78 8O 81 88 89 92 95 96 98 99 100 lOl 102 Table Page 42. Performance of nursing offspring and dams fed DCPD . . . . . . . . . . . . . . . . l03 43. Effect of chronic administration of DCPD to mink on organ weights (9 i S.E.) at necropsy . . . . 105 viii LIST OF FIGURES Figure Page 1. Regression equation of the data shown in Table 3 . 13 2. Mean body weights of mink on the 21-day subacute test fed DIMP at various levels . . . . . . . 20 3. Regression lines for the data presented in Table 7 . 25 4. Regression line for the data presented in Table 24 . 66 5. Mean body weights of mink on the 21 -day subacute test fed DCPD at various levels . . . . . 69 6. Regression lines for the data presented in Table 27 . . . . . . . . . . . . . . . 76 ix LIST OF APPENDICES Appendix Page A. Mink Feed constituents and Diet Preparation . . . 111 Determination of Hemoglobin Concentration . . . 114 C. Preparation of Wright's Stain and Buffer . . . . 116 D. Preparation of Drabkin's Reagent . . . . . . 118 INTRODUCTION Statement of the Problem In July of 1975 the U.S. Army Medical Research and Development Command was charged with the task of evaluating the toxicity of a number of chemicals polluting the ground and surface waters in the vicinity of the Rocky Mountain Arsenal (RMA). Two of these chemicals, dicyclopentadiene (DCPD) and diisopropyl methylphosphonate (DIMP), were included in a high priority list because of their detection in sampling wells both on and off the grounds of the RMA. DCPD is the spontaneously formed dimer of a compound (cyclopentadiene) used in the manufacture of cyclodiene pesticides. DIMP is a contaminant of demiliterized Sarin (a nerve gas) waste. A paucity of information on the plant and animal toxicity, and environmental disposition of these chemicals prompted a wildlife toxicology study at Michigan State University. Investigations were made regarding the acute, sub-acute, and chronic toxicity of these compounds in a terrestrial avian herbi- vore (Bobwhite quail), an aquatic avian herbivore (Mallard duck) and a semiaquatic mammalian carnivore (mink). This thesis is concerned with the latter species. REVIEW OF LITERATURE DIMP_ DIMP was produced in significant quantities during the demiliterization of the nerve gas Sarin (isopropyl methylphosphona- fluoridate) at the Rocky Mountain Arsenal. DIMP is a contaminant present in the principle demiliterization product IMP (isopropyl methylphosphonate). Limited toxicological data exists for DIMP. Table 1 summarizes the acute toxicity information on DIMP. Structurally, DIMP resembles the organophosphate insecticides (Matsumura, 1975), but McPhail and Adie (1960) reported that DIMP did not inhibit cholinesterase. DIMP has been shown to cause eye irritation in rabbits when applied directly to the corneal surface (Jacobson, 1953). Dacre and Hart (1977) demonstrated a similar effect, with corneal clouding the most severe form of ocular DIMP irritation in rabbits. Seven days following exposure, the clouding cleared, indicating the effect was reversible. As a part of the same study, rabbits were dermally painted with DIMP at levels as high as 2000 mg/kg Bw. Some death occurred at the higher levels with skin abrasions being the most common symptom of DIMP application. In addition, DIMP was shown to be a powerful inducer of liver microsomal enzymes, reducing the hexobarbitol-induced sleeping time in rats dietarily exposed to 3000 ppm DIMP for four days. 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Chronic dietary exposure of Mallard ducks (Jones, 1977) and Bob-white quail (Howell, 1979) to DCPD in concentrations as high as 320 ppm and 4000 ppm, respectively, caused no adverse changes in body weight, feed consumption, egg production, fertility, hatchability, eggshell thickness, or l4-day survival of offspring. Exposure of dogs to 40, 125, and 375 ppm DCPD for 14 days in the diet; of rats to 80, 250, and 750 ppm DCPD for 90 days in the diet; and of mice to 28, 91, and 273 ppm DCPD for 90 days, all revealed no evidence of toxicity (Hart and Dacre, 1977). In the same study it was shown that DCPD did not induce liver enzymes responsible for reduced hexobarbitol-induced sleeping time, in rats orally admin- istered 750 ppm DCPD for four days. Aquatic organisms are highly sensitive to DCPD contamination, as demonstrated by Bentley et a1. (1976). These workers found DCPD to be highly toxic to algae, invertebrates, and to fish in static and flow-through freshwater systems. The water flea (Daphnia magna) and the Channel catfish (Ictalurus punctatus) proved to be the most sensitive species' tested with a 48 Hr.'LC50 of 10.5 ppm, and a 96 Hr. LC50 of 15.7 ppm, for the water flea and the Channel catfish, respectively. DCPD was shown to bioconcentrate up to 53X in Bluegills (Lepomis macrochirus) exposed to 14C-DCPD contaminated water. Although no human toxicity data exists for DCDP, Shashkina (1965) reports the subjective reactions of volunteers exposed to DCPD vapors as being characterized by nausea, headache, and unpleas- ant sensations in the mouth at 0.023 ppm DCPD in air. Considering its irritating effects and odor theshold, a maximum permissible con- centration for occupational exposure was recommended at 1.0 ppm. The reproductive potential of mink has been shown to be extremely sensitive to selected compounds on a chronic exposure basis (Aulerich et al., 1971; Aulerich et al., 1974; Aulerich and Ringer, 1977; Gilbert, 1969). The reason for this reproductive sensi- tivity is probably related to the fact that these animals exhibit delayed implantation (Enders, 1952; Hansson, 1947). Delayed implanta- tion (or embryonic diapause) is influenced by photoperiod, and appears to be hormonally controlled (Duby and Travis, 1972; Dukelow, 1966; Enders, 1963; Hansson, 1947; Moller, 1973; Moller, 1974; Aulerich et al., 1963). The sensitivity of mink to Aroclor compounds (poly- chlorinated biphenyls) has been attributed to the increased metabolism of pregnancy maintenance hormones by Aroclor-induced hepatic micro- somal oxidases (Aulerich and Ringer, 1977). No information is available concerning the acute, subacute, or chronic toxicity of either DIMP or DCPD, to mink. OBJECTIVES To determine the acute oral toxicity of DIMP and DCPD to mink, and describe the symptoms of intoxication. To determine the sub-acute dietary toxicity of DIMP and DCPD to mink, and characterize the sequalae asso- ciated with that intoxication. To determine the effects of chronic ingestion of DIMP and DCPD by mink, on growth, survival, reproductive success, and neonate performance. PART I TOXICITY OF DIISOPROPYL METHYL PHOSPHONATE TO MINK TEST l--Acute L050 Procedure Testing.--To ascertain the effect of an acute oral exposure of DIMP to mink, 29 adult female mink were singly dosed intra- gastrically with the compound. The following progression of doses (and number of mink per dose) were used: 0.0 mg/kg (2); 75 mg/kg (2); 150 mg/kg (4); 300 mg/kg (4); 450 mg/kg (4); 500 mg/kg (6); 550 mg/kg (5); and 600 mg/kg (4). The larger doses (300 mg/kg and greater) were administered by gavage. This was accomplished by inserting a plexiglas rectangle (approximately 20 x 50 x 3 mm) with a 9 mm hole in the center, between the jaws of a restrained animal, and introducing the tube into the esophagus through the hole in the plexiglass. This con- sisted of a length of polythylene tubing (premeasured for average esophageal length) attached to a 3 ml syringe with an 18 gauge needle. Smaller doses were introduced into the stomach by gelatin capsule. The capsules were pushed down the eSOphagus by means of a length of polyethylene tubing to the level of the stomach. 10 11 Mortality and signs of intoxication were recorded during a 2 hour observation period after dosing and daily thereafter for 14 days. The mink were then killed by cervical dislocation, and exam- ined for gross pathomorphological changes. Statistical analysis.--The determination of the acute oral L050 was made by the method of Litchfield and Wilcoxon (1949). Results The dose related mortality of mink to a single acute oral- exposure of DIMP is presented in Table 3. The acute oral L050 as determined by the method of Litchfield and Wilcoxon (1949) was 503 mg/kg with a 95% confidence interval of 379-668 mg/kg. A least- squares regression line of the probit analysis data shown in Table 3 is presented in Figure l. The clinical signs of acute intoxication with DIMP included. salivation, lethargy, myasthenia, immobilization, vomiting, and death. The mink exposed to 300-550 mg/kg that did not die, were immobilized to varying degrees, but eventually recovered. Recovery was complete within several hours of dosing. Discussion Acute oral administration of DIMP to female mink resulted in an L050 determination somewhat higher than that reported for rats (Kinkead et al., 1971; Dacre and Hart, 1977) and mice (Dacre and Hart, 1977) but less than that reported for Mallard ducks (Jones, 1977) and for Bobwhite quail (Howell, 1979). These data suggest an TABLE 3.--Acute oral toxicity of DIMP to adult female mink 12 Dose (mg/kg)a No. died/No. tested Mortality (%)b ProbitsC 0 0/3 0 -- 75 0/2 0 -- 150 0/4 0 -- 300 1/4 25 3.55d 450 1/4 25 4.33 500 4/6 66.7 5.45 550 2/5 40 4.75 600 4/4 100 -- aAdministered by gavage b Taken at 24 hours post-dosing cDetermined by method of Litchfield and Wilcoxon (1949) dRepresents adjusted value (Litchfield and Wilcoxon, 1949) 13 Figure l.--Regression equation of the data shown in Table 3. In the regression equation x = log dose DIMP in mg/kg body weight, y = probits. l4 Probl I: l 1! 7 2.5 2.0 2.7 ‘I '0 27.1 22 23 2.4 log Dose (mg/‘9 body WI.) 1.0 0.0 Y=0.12.+2.09 ° I, ruOJS / 5.0 / / , / , / / / . 4.0 I, / / ° / / I, 3.0 / / 2.8 l 2.9 “J 15 intermediate sensitivity for mink with respect to acute DIMP poison- ing. The clinical signs of acute oral toxicity of mink dosed with DIMP were consistent with those reported for Mallards (Jones, 1977), and for Bobwhite quail (Howell, 1979), as general depressive effects until death occurred. Test 2--Subacute L050 Procedure Testing.--The subacute dietary LC50 trial consisted of a 7-day quarantine and acclimation period, a 21-day dosing period, and a 7-day recovery period. Sixty juvenile pastel mink were separated into 6 groups of 10 mink each. Each group consisted of 5 males and 5 females randomly chosen from healthly stock, and was approximately 8 months of age. One group was assigned to each of the following logarithmically scaled dietary concentrations (Ppm) of DIMP: 0 (control),l, 10, 100, 1000, and 10,000. Diet constituents and preparation procedures are given in Appendix A. All animals in the subacute trial were housed indoors in an environmentally controlled cage room, at the Poultry Science Research and Teaching Center, Michigan State University. Each mink was housed individually in a 51 x 36 x 30 cm (length x width x height) cage equipped with water cup and feed container. Feed was provided in removable containers attached to the inside of the cage on a swinging door such that feed consumption 16 could be.ascertained from measurement of unconsumed feed. Water was provided ag_libitum. During the 7-day predosing acclimation period, all mink were provided with a control diet. Body weights were recorded at the beginning of the dosing period and on days 7, 14, and 21 of dosing, and on day 7 of the recovery period (termination of the test). Feed consumption was estimated by daily recovery of the unconsumed portion of a preweighed allotment of feed, and collectively weighed for each treatment level on days 7, 14, and 21 of dosing, and on day 7 of recovery. Mortality, signs of intoxication, and behavioral changes were noted throughout both the dosing and recovery periods. Blood for packed cell volume (hematocrit) and differential leukocyte counts were procured by toe-clip at the termination of the test. Blood was collected in heparinized microcapillary tubes (100ul) and centrifuged for 7 minutes at 4500 rpm on an International I for hematocrit determination. Blood Microcapillary Centrifuge smears were allowed to air dry and were then fixed and stained in Wright's stain (see Appendix C: Preparation of Wright's Stain and Buffer). After staining, slides were first rinsed with phosphate buffer, for differentiation, and then with distilled water. They were then blotted and air dried. Differential leukocyte counts were 1International Equipment Company, Boston, MA. 17 made under oil immersion (930-x) and any abnormalities in cells were recorded. At the end of the experiment animals were terminated by cervical dislocation, and necropsied. Gross pathomorphological observations were made, and the following organs were excised, weighed, and prepared for histopathological observation according to routine laboratory procedures: brain, heart, lungs, kidneys, spleen, and liver. Statistical analysis.--Differences in body weight changes, feed consumption, hematocrit values, differential leukocyte counts and organ weights were analyzed by a one-way analysis of variance and Dunnett's t-test. Zero predicted feed consumption was estimated by regression analysis. Results The determination of a subacute mean lethal dietary concen- tration of DIMP to mink was not possible since there was no signifi- cant mortality related to DIMP concentration in the diet (see Table 4). Only two animals died during the experiment. One was a female fed the control diet and the other was a female on the 1000 ppm diet. Both deaths resulted from wounds inflicted by neighboring mink which were able to squeeze under the partition between cages. The mean of body weights recorded weekly throughout the experiment are shown in Table 5 and Figure 2. There were signifi- cantly lower mean body weights for the 10,000 ppm DIMP treatment 18 o oF OF op op oF oooo_ op m m m m o_ coop o op op o_ o_ op oop o op op op op o_ op o op op o_ o_ op F mmxmm o_ m m m o_ o_ o mzHo nmceneou o m m m m m oooop om a e a w m coop o m m m m m oo_ o m m m m m op o m m m m m P om e v v m m o azfio mFmemd o m m m m m oooo_ o m m m m m coo— o m m m m m oop o m m m m m op o m m m m m _ o m m m m m o azHo mpmz Axv mp\~ m\~ mN\F NN\P m_\~ apppmugoz Agaav . ucosummculpmoa ucmEpmmLp xmm m=w>w>gsm xcws mo .oz ucmEpmmLu newest mcm>w>czm xcps mo .oz vowcma xem>oomc acmEummLuuumoa Xmuux m use mzmo mo :owumcumwcwscw xgmummv xmuupm monumnam m saw: empmwuommm aprmpLozuu.e m4m

mp mo.o A a pm Fogucou soc» gcmcmeewu prchwmwcmvm mcmmz n .mFochoo mzu seem ucmgwoewv xpucmuwmwcmwm yo: mew uawcomcmazm mamm mgu cow: mcmmzm QFm H mmpp amc h “cop nee h coop new A ampp mmm h NFNF oooop mmmp n mom, mpcp h poop memp h vamp wepp h «mmp m¢.wm h mmpp ooo_ «map A moop mwmp h New? momp n mmmp mmmp h mmop mmep H mm¢_ oo_ mwmp h mo¢_ mmmp h owe~ «amp n omep mmmp h mmep mmw— h Pomp OF mpmp n m~¢~ avoF n omcp mmmp h mmep ammF A one. mmmp h PoNP F mpmp h mom? mmmp n comp camp h msmp comp h mmmp mom_ h camp 0 azHo ucmEpmmLp ugmwmz lumen mxmu n mace Fm mace up mace n —mwuw:H Asaav pcmsummcp Amy agave; xcon cam: Acm>oomc acmEummLunumoa use Swap emu; staomwu sau-pm co secs Lo “cues; soon Cc mo:agu--.m msmqh 20 Figure 2.--Mean body weights of mink on the 21-day subacute test fed DIMP at various levels. 21 0000' occ— cow 0— — .o..coo 00.... 1000 loan loom ucoo looow nocwp looup noonp 1°09— noonw scoop noos— uoay' ‘P°fl (I) lqlgo- 22 group than for the control group on days 7, l4, and 21 of dosing. Although the 7-day post-treatment period showed a weight gain for these animals (DIMP 10,00 ppm) the mean body weight was still sig- nificantly depressed compared to the control. Since mink have a high degree of variability in body weights, especially between sexes, the data in Table 5 may tend to obscure changes in body weight that might prove significant for one sex. Table 6 lists the mean percent change in body weight by sex over four weekly intervals. Highly significant losses (P < 0.01) in percent of body weight were recorded for both males and females fed 10,000 ppm DIMP during the first 7 days of dosing. A highly significant (P < 0.01) percent loss of body weight loss continued during the second week of dosing for these males. Females fed 10,000 ppm DIMP continued to lose weight (P < 0.01) only during the third week of dosing. It was also noted that the females fed the 10 ppm DIMP diet gained weight significantly over the controls during the second week of the test. During the 7-day post-treatment period, males on the 10,000 ppm DIMP diet gained weight significantly (P < 0.01) over the con- trols. Feed consumption during the DIMP subacute trial is reported in Table 7. The mean feed consumption for the 21 days on treatment was significantly less for the mink on the 10,000 ppm DIMP treatment than for the control. Feed consumption was greater for this group than for the controls during the 7 day post-treatment period. Figure 3 predicts the extrapolated dose (in ppm) required for zero feed .NNo.o v NV Nogpcou EogN NamwaNNc NNNcNonNcoNN Ncmmzu .Nmo.o v av Nogucoo Eogm acmemwwwu xNucmonNcmNN Ncmmzn .Nmo.o A NV Nocucou seem NemeNNNu NNpcmuNywcmNN No: NNN NNNLNNNNN mENN NNN: NcmmzN 23 NNNN.N N N.N NN NNN.N N N.NN NN NNNN N N N.NN NN NN.N N N.NN NN NNNNN NNN.N N N.NN N NNN. N N N.N N NNN. N N N.N N NNN. N N N.NN NN NNNN N NN.N N N.NN NN N.NN N N N.N NN NNN. N N N.NN NN N.NN N N N.NN NN NNN NNN.N N N.N NN MN5 N N N.N NN N NN. N N N.N NN ”NN. N N N.NN NN NN NNN.N N N.N NN NNN. N N N.N NN NNN.N N N.NN NN NNN. N N N.NN NN N NNNNN NNN N N N.N N NNNN. N N N.NN N ”NNN.N N N.NN N UNNN. N N N.NN NN N NzNN NNNNNNNN NNN.N N N.N N UNNN.N N N.NN N NNNN. N N N.NN N NN. N N N.NN N NNNNN NNN.N N N.N N N NN.N N N.NN N NNN5 N N N.NN N NN. N N N.N N NNNN N NN.N N N.NN N NNN. N N N.N N NNN. N N N.NN N NNN. N N N.NN N NNN NN.N N N.N N NN9 N N N.N N N NN. N N N.N N NNN. N N N.N N NN “NN.N N N.N N NNN. N N N.N N NNNN. N N N.NN N NNN.N N N.NN N N NNN.N N N.N N NNN. N N N.N N MMNN. N N N.NN N NNN.N N N.NN N N NzNN NNNENN NNNN.N N N.N N MNN. N N N.N N NN. N N N.NNN N N NN.N N N.NN N NNNNN NNNN N N N.NN N NN9 N N N.N N NNN. N N N.N N NNN.N N N.NN N NNNN NNNN.N N N.NN N NNN. N N N.N N NNNN. N N N.NN N NNN.N N N.NN N NNN NNNN.N N N.NN N NNN. N N N.N N NNNN. N N N.NN N NNN.N N N.NN N NN NNNN.N N N.NN N NNN. N N N.N N NNNN. N N N.NN N NNN.N N N.NN N N NNNN.N N N.NN N ”NNN. N N N.NN N NNNN. N N N.NN N NNN.N N N.NN N N NzNN NNN: .Nz NNNN NN .Nz NNNN NN N3 NNNN NN .Nz NNNN NN NNNoNV cNNw N z Nmmopv :wa N z NmNoNv :ch N z NNNoNV cNNw N z NENNV acme xmm NNNN-NNN NNN-NNNN NNNN-NNNN NNNN-NN\N -NNNNN Newsammcplumoa Newsammcp NNN>NNN2N NNxmmz um cmxmu pcmNmz Neon News :N wmcmgu ucmocma cog: :NNNNNNNNNNENN NzNo NNNNmNc NNNUNNNN No NNNNNN--.N mNmoumg ucmsummgpupmoa New uNmN ONUN Ngmumwu NNN-NN co ch2 No :oNNNENNcou vmmNNI.N m4m<~ 25 Figure 3.--Regression lines for the data presented in Table 7. In the regression equations x = log dose DIMP in ppm, y = mean feed consumption for 21 days, in g/mink/day. 26 «£33 umcdn .. «don n a m2_o no.— III." oou uoudwnsuoa p033 one” log a PIN/5 27 consumption. Based on regression analysis of data in Table 7, zero feed consumption would have occurred at.aconcentration greater than 100 percent DIMP, according to this analysis. Table 8 shows the calculated average amount of DIMP ingested/kg body weight by the animal over the 21-day treatment TABLE 8.--Feed consumption, body weight, and amount of chemical ingested by adult mink fed DIMP at various levels for 21 days DIMP in diet Feed consumed DIMP consumed Mean body DIMP consumed (ppm) (g/mink/day) (mg/mink/day) wt. (9) (mg/kg/day) 0 291.7 0 1561.7 0 291.7 0.292 1461.3 0.200 10 272.3 2.723 1449.3 1.879 100 279.0 27.9 1626.6 17.159 1000 273.5 273.5 1359.6 201.16 10000 201.3 201.3 1087.0 1851.9 period, based on mean feed consumption and mean body weight for the period. The animals on the 10,000 ppm DIMP treatment were calcu- lated to have received a daily dose of DIMP more than 3 times the acute, oral L050 as determined in Test 1. The hematological parameters measured at the termination of the test are given in Table 9. Hematocrit (packed cell volume) was found to be significantly depressed (P < 0.05) for the animals on the 10,000 ppm DIMP treatment. Differential leukocyte counts revealed a significantly lower percentage of lymphocytes in the peripheral 28 .NNN.N v NV .NNN.N v NV some Nocucoo Eogm ucmgmmNNu NNNcNuNNNcmNN cmme NamENNmNNNN some Nogucou EoNN ucmgmNNNu NNucNuNNNcmNN cams acmENNmNNN .m.m N :Nmzn NNNENNNNN NzNN NNN NNNN NNN N N “N N.N.m + NV max“ NNwo muzuoxamg ww.oNnm.m «NNN.NN N.NN No.mNum.mm mo.oNHN.o NN.oNum.o mN.oN”~.o mo.NNNN.~m oN ooooN NN.NNNNN «NN.—NN.NN NN.NNm.wm mmdNed NN.ONm.N wodN To mm.NNN.Nm m oooN $6de NNN.NNNdm $.NN 7mm .36de odeoN ONded $6258 ON ON: NN.NNNNN 3mm.NNw.NN 8.: N.NN 2:: To ~m.oNN.N o~.o.Nm.o 3.35% N: N: No.0NN.N No.mN~.o~ mN.mNN.mm oo.oNo.o om.oN~.m NN.NNNNNN 8.353 N: N omdN N.N NNN.NN N.NN. Nm.MN N.NNN. oodN o.o 8.: _.m NN.N: m.o NNN.N: N.NN m o NEE Nmpxuocoz NmuzuoggszN -omwwmm -owwwwu nowwwnm NNNzaoNNm NNNNuoNNEm: z Nwmmw umucmsmwm nucmm . nummgh NNNpcmNmmmNu ucw NNNNN> NNNuouNEm; News con: Npcaoo mpzooxsz NzNN NNNNNNN NNNNNNNN No NNNNNN--.N NNNNN 29 blood of mink on the 10, 1000, and 10,000 ppm treatments. No con- sistent signs of intoxication were recorded for any treatment group on the DIMP subacute trial. However, the mink fed 10,000 DIMP behaved much more aggressively than animals on other treatments. There were no consistent macroscopic lesions associated with a particular DIMP treatment at necropsy. No significant differences in organ weights of females were noted in any treatment group for brain, heart, lungs, or liver weights (see Table 10). However, there was a significant reduction in kidney weights for females on the 1 ppm DIMP diet. Male mink on the 1000 ppm DIMP treatment showed a significant decrease in lung weight (see Table 11). The male mink fed 10,000 ppm DIMP showed a significant decrease in heart, lung, kidney, and liver weights. Discussion Since no determination of lethal concentration of dietary DIMP to mink could be made at the concentrations and length of exposure used, DIMP was considered to be nontoxic to mink by inges- tion in the 21-day test. Although weight loss was noted for the animals receiving the highest dietary concentration (10,000 ppm), the reduced feed consumption by these animals while on the test diet may have been the attributing factor in weight loss. The increase in feed consumption and body weight displayed by these animals during the post-treatment period (when they were placed on the control diet), suggests a palatability problem with DIMP in high dietary concentrations. The marked aggressiveness of the 30 .Nmo.o v NV Nogucou Eocm ucmgmmmmu NpucmuwmmcmNN some pcmsamwgh k. .NNmu on» mo :oNuchENwN op LoNNN cmNu NmNNEmN ozpn .NNNENNNNN NzNN NNN NNNN NNN N N ZN m¢.© H ¢.Pm mm.o H N.m mp.o H «.9 mm.o H N.N wo.o H ¢.w am oooop ¢F.N H m.mm mm.o H N.m mp.o H m.m Pm.o H ~.m m¢.o H N.@ m coop Fm.m H m.mm om.o H m.m vw.o H w.o m¢.o H m.© mm.o H u.w m oo— Nm.P H p.mm mm.o H ¢.m mN.o H m.© ~m.o H m.© mm.o H 0.0 m op mm.~ H m.Nm «mp.o H N.m mo.o H ¢.N F¢.o H ¢.N . mm.o H w.m m P mm.N H m.©m FN.o H N.¢ mm.p H m.© —m.o H o.“ N_.o H m.w m o mzHo NNm>NN Nxmchx mmczN exam: :chm Nfimmp z -meNN N.N.m N av szNmz :mmgc Nuzmwmz cmmco xcws «Noam» con: NzNo agmuwwu ouaumnzm No «ummwmuu.op MNmNN :mmNam Nxmchx macs; Ngmmz :ngm NENNV 2 News N.N.m N my pgmNmz cam: -wagh Nugmwmz cmmgo mpms cog: NzNo NNmNmNc muaumnam we pummmm::.- m4m Nogpcoo o>NNoooNoN NNogp sogm ucmgowNNo NNpoNoNchmNN No: ogo uoNNoNooN osoN ocp gpNz :ENNoo oENN on“ oN NcoozN m m m w m NN NNNNN NN NN NNNNN NN NN NNNNN NN NNN NNNNN NN NN NNNNN NN NN NNNNN NN NNN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNN NNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NN NNN NNNN NN .NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN NNN NNNNN NN N NzNN NNNMMHMW NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN NNN NNNN NN N NzNN NNNENN NNN NNNNN N NNN NNNNN N NNN NNNNN N NNN NNNNN N NNN NNNNN N NNN NNNNN N NNN NNN NNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNN NNNNN N NNN NNN NNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNN NNNNN N NNN NNNNN N NN NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N NNNNNNNNN N N NzNN NNN: Agony NNNNNNN z NNNNNN z NNNNNNN z NNNNNN z NNNNNNN z NNNNNN z -Nwmwfi NNN vmocwucounu.mp m4m Fogucou m>NuumNng mecu eon» uNNLNNNNN zpucNuNwwcmNN No: mLN HQNLUNNNN mENN msp saw; caspou meNN ms“ cw NcNmz_ 44 NNN.N N N.N NN NANN.N N N.NN NN NNN.N N N.N NN NNN NNN.N N N.N NN NANP.N N N.NN NN NAN_.. N N.NN NN NN_ NNN.N N N.N KN NANN.N N N.NN RN NA_N.N N N.NN RN NN N N N memm NN.. N N.N NN NNN.N N N.NN NN ANF._ N N.NN NN N NzNN NNNNNNNN m . . m . . m . I . NN N N N N NN ANN N N N NN NN ANN N + F NN NN NNN NNN.N N N.N NN NAN~.F N N.NN NN NNN.N N N.N NN NN_ NNN.N N N.N NN NANN.N N N.NN NN NANN.N N N.NN NN NN NNN._ N N.N NN NANN.N N N.NN NN NANN._ N N.NN NN N NzNN N_NENN m . . m . . m . . F“ N N N N N ANN . N N NV N NN N N N N N NNN NNN.N N N.N N NANN.~ N N.NNN N NANN.~ N N.NN N NN_ NNN.. N N.N N NANN.N N N.NN N NANN.F N N._N N NN NNN.. N N.N N NANN.. N N.NN N NANN.N N N.NN N N NzNN NNN: NN\N~\NF NN\N\NF NN\NN\FN NNN NNN xm -NN\N\N_ z -NN\NN\__ z -NN\N_\PP z N N N N vmzcwpcouuu.¢~ m4m 0.05). 46 only two instances. In one case the depressed feed consumption did not appear to be dose re1ated (50 ppm DIMP treatment on September 1, 1977); in the other case, depressed feed consumption may have been dose re1ated (450 ppm DIMP treatment on November 15, 1977) but was not trend oriented when compared to feed consumption of other treat- ments on the same date. An estimated dai1y ingested does of DIMP (as ca1cu1ated from body weight and feed consumption) by mink on each treatment is shown in Tab1e 16. Ana1ysis of the data co11ected on hemato1ogica1 parameters at the termination of the test revea1ed increased hematocrit va1ues for ma1es on the 150 ppm and 450 ppm DIMP treatments (see Tab1e 17). Significant differences in hemog1obin content or mean corpuscu1ar hemog1obin concentration were not found in any treatment groups with respect to contro1 va1ues (Tab1es 18 and 19). Differentia1 1eukocyte counts revea1ed no differences among DIMP treatments consistent with toxicosis (Tab1e 20). Reproductive success of mink on the various DIMP treatments is shown in Tab1e 21. No adverse effects upon whe1ping rates, ges- tation 1ength, fecundity, kit weight at birth, or secondary sex ratios were noted for the DIMP-treated anima1s. Kit weight at birth was significant1y greater for 50 ppm DIMP treatment animals than contro1s. Ma1e ferti1ity, as estimated by presence of sperm in post- coita1 vagina1 aspirations, was not adverse1y affected by chronic DIMP administration. 47 .Nzucoe NP cm>o cmeu NucmeLNNNmE mp com “sawmz anon :Nme NucmchNmmN .Ngucos N gm>o :mep NpcwEmcamNme m com cowunezmcou NNN; :Nms Npcwmmcamm F NN.NN NNNN NN.NN_ NNN NNN N_.NN NNN_ NN.NN NNN NN_ NN.NN NNN_ NN.NN NNN NN N FNNN N NNN N ANEN ANNN\mx\msv wNoN NANV .pz NNN\NmuNmch ”ANN :oNpstmcou AENNV prN NNNNNNNN N.NNN NNNN NNN: NzNN NNNN N_NNN NNN: NN _N>NN NzNN Ncucoa NP com NNN>N_ NNNNNN> NN NzNN NNN NNNE NN NzNN co NNNNNN N_NNN NNNNNNNNN NN NNNNNFNN_NN--.NF NNNNN 48 .Amo.o A NV Nm:_N> Pocpcoo m>Nuomach NNNNFNNN :N NNNNNUNN cones: NNN NmLNNNmE muNo LNNNN ENLN ucmchmNN apchuNwwcmNN No: NNN NNNLUNNNN wENN NNN NNN: caspoo NENN NNN :N NNN.N:F N . . N . . N . . N . - . NN N N N NN NN NN N N N NN NN NN _ N N NN NN NN N + _ NN NN NNN NNN.N N N.NN NN NNN.N N N.NN NN NNN.N N N.NN NN NNN.N N N.NN NN NN. 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NNN.N N N.N m.mp mm NN.NN N NNN NN.NN N NNN N.NNN NN.N N N.NN_ NNN.N N N.N w.om Nm NN.NN N NNN NN.NN N NNN N.NNN NN.N N N.NNN NNN.N N N.N m.om mm NN.NN N NNN NN.NN N NNN N.NNN m m.N N o.nm— w NoN.o N N N m.om mm N.N.m N av ENNLNN NNNN Nxmmz N N.N NNNNNNUNN No pgmwmz .m>< N.N.m N av NENN chNPmsz No ugmwmz mmNgm>< NNNNNNNN NNN N.NN N 3 NNNNN N NN NNNN mo Ncmez .m>< .N.N NNNNNNNNN «\NNNN .Nz NNN NNNNz N NN NNNNNNNNN NNN ANV Nxmmz N NN mNNNNNoNF N.” NNNNNNN: omN omp om AENNV NNNENNNLN NzHo azHo cum NENN NNN NNNNNNNNo mcwpxozm mo mucNELowLmN--.~N m4m 0.05)/ 56 greater than this natural mortality, preliminary evidence exists for a chronic toxic effect of DIMP ingestions to mink (especia11y females), at moderately high doses. The body weight changes which resulted in mink on either the control or the DIMP-treated diets are in agreement with the growth of mink reported elsewhere (Aulerich and Schaible, 1965; Kumeno, et al., 1970; Oldfield et a1., 1968; Seier et a1., 1970; Travis and Schaible, 1961). Feed consumption was not differentially affected in a trend consistent with dose. Sporadic differences in the consumption of test diets, as compared to the control diet, suggested no demonstra- ble pattern of differences in palatability, and were most likely attributable to chance or sampling error. Since daily ingestion of approximately one—fifth of the calcu- lated L050 by mink on the 450 ppm diet caused no growth impairment or radical change in appetite, it is unlikely that metabolic effi- ciency of food conversion was significantly altered by this chemical, at the concentrations used. Hematological parameters were not appreciably different in value from those reported by other workers. Hematocrit values (packed cell volume) similar to control values were reported by Asher et al. (1976), Fletch and Karstad (1972), Kubin and Mason (1948), and Rotenberg and Jorgensen (1971). Hemoconcentration was reported as a normal occurrence in ranch mink during the winter months and was attributed to decreased water consumption (Asher et a1., 1976; 57 Skrede, 1970). The increase in hematocrit values recorded at the termination of this test for males on the 150 and 450 ppm DIMP diets may have been related to a decrease in water intake with resultant hemoconcentration. Hemoglobin values and mean corpuscular hemoglobin concentra- tions were in good agreement with values published elsewhere (Fletch and Karstad, 1972; Kubin and Mason, 1948). Differential leukocyte counts of blood taken from mink at the termination of the chronic test differed slightly from counts made by Fletch and Karstad (1972). These workers showed approximately equal percentages of mature (e.g., segmented) neutrophils and lymphocytes (43% each), and an appreciably greater number of mono- cytes (9%) than found in the animals in this study. However, Asher et al. (1976) have shown a seasonal and age dependent variation in white cell percentages in mink. Mature (segmented) neutrophils were shown to comprise as high as 75% of all leukocytes during the repro- ductive season, with lymphocytes comprising as little as 15% during the same period. Monocytes were also shown to undergo seasonal shifts, but in concurrence with Fletch and Karstad (1972), monocytes remained in the 6-8% range throughout the year. Except for the depressed numbers of monocytes, the overall leukocyte percentages found in mink at the termination of this study are well correlated with values for that time of the year reported by Asher et a1. (1976). Both Gilbert (1969) and Kennedy (1935) reported monocyte numbers in the 1-2% range in adult mink, but as in the counts recorded by Fletch 58 and Karstad (1972), neutrophils and lymphocytes were nearly equally represented. Hence, thelower monocyte numbers reported in this study are in concurrence with two previous studies, whereas the values obtained for the remaining leucocyte types are in agreement with a number of other previously completed studies. DIMP was not shown to seriously alter the reproductive capacity of mink when chronically ingested. DIMP chronica11y admin- istered to Mallard ducks and (Jones, 1977) and to Bobwhite quai1 (Howell, 1979) did not have any adverse effect upon ferti1ity, hatchability, eggshell thickness, or hatchling survival at dietary levels of 10,000 ppm and 1200 ppm, respectively. However, egg pro- duction in both species was reduced at these dietary levels. Hardesty et a1. (1977) failed to demonstrate any chronic adverse effect upon reproduction in rats given 10 or 1000 ppm DIMP in their drinking water for 13 and 19 weeks (males and females, respectively). The increase in kit weight and aberrant secondary sex ratio observed on the 50 ppm DIMP diet was probably associated with chance varia- tion and/or sampling error, since a similar effect was not recorded at higher doses. Other reproductive indices (spermatogenesis, ges- tation length, whe1ping rate, litter size, and number of stillborn kits) were paralleled by data reported in other studies (Aulerich et al., 1963; Aulerich and Ringer, 1977; Enders, 1952; Hansson, 1947; Schaible and Travis, 1958). Performance of mink kits for DIMP-treatment groups was like- wise unaffected by chronic ingestion of DIMP by lactating fema1es. PART II 59 Kit mortality and growth data for all groups were similarly in agree- ment with data reported by Aulerich, et a1.(1975), Aulerich and Ringer (1977), and Oldfield et a1. (1968). At the termination of the experiment no gross or histopatho- logical abnormalities were found to be consistent with any particular DIMP treatment. Organ weights were not appreciably different from weights given in other studies (Aulerich and Ringer, 1977; Wood et a1., 1965). Kidney and lung weights for mink in this study were slightly lighterthan the weights reported for those organs by Wood et al. (1965). Conversely heart weights of mink in this study were found to be greater than reported by Wood et al. (1965). The lethal agent used in terminating animals was shown to affect the individual organ weights by these same workers. Since the method employed in this study to terminate the animals (cervical dislocation) was dif- ferent from that employed by Wood and co-workers (electrocution), the differences found in comparison of organ weights may be due to the different euthanatization techniques. Conclusions 1. The acute oral L050 of DIMP for mink was 503 mg/kg Bw with a 95% confidence interval of 379-668 mg/kg BN. 2. A 21 day subacute dietary LCSO of DIMP for mink was estimated to be greater than 10,000 ppm. 3. Chronic ingestion of dietary DIMP had no effect upon growth, reproductive success or neonate performance. A slightly higher mortality occurred in females fed all DIMP treatments than those fed the control diet. TOXICITY 0F DICYCLOPENTADIENE T0 MINK Test 1--Acute L050 Procedure Testing.--Twenty-four adult female mink were singly dosed intragastrically with DCPD in order to determine its acute oral toxicity to mink. The following progression of doses (and number of mink per dose) were employed: 0.0 mg/kg (2); 30 mg/kg (3); 60 mg/kg (2); 120 mglkg (2); 240 mg/kg (4); 480 mQ/kg (4); 600 mg/kg (4); 720 mg/kg (3); and 960 mg/kg (2). The larger doses (240 mg/kg and greater) were administered by gavage as described for DIMP. The smaller doses were introduced into the stomach by geletin capsule. In addition, five adult female mink were injected intra- peritoneally with DCPD according to the following regime: 960, 1200, 1440, 1680, and 1920 mg/kg (1 mink per dose). Mortality and signs of intoxication were recorded during a 2 hour observation period following dosing, and daily thereafter for 14 days. The mink were then terminated by cervical dislocation, and examined for gross pathomorphological changes. Results Calculation of an actue oral L050 for DCPD in mink was not possible since 100% of the animals survived the highest dosage (960 mg/kg). However, intraperitoneal injections of DCPD at 960, 60 61 1200, 1440, 1680, and 1920 mg/kg resulted in death for those animals. The clinical signs of intoxication following oral exposure to DCPD included hyperactivity, high-pitched vocalizations, dyspnea, diarrhea, opisthotonus, convulsions, vomiting, and paresis of the hind limbs. Recovery was generally rapid with resumption of normal appearance and behavior within an hour to an hour and a half of dosing. The mink exposed to the high doses of DCPD by I.P. injection all died within minutes of administration of the compound. Discussion The acute oral L050 of DCPD to mink was above the maximum dose of DCPD given in this test (> 1000 mg/kg). The acute oral toxicity of DCPD to Mallard ducks, (L050 > 40,000 mg/kg) (Jones, 1977) and to Bobwhite quai1 (1010 mg/kg) (Howell, 1979) as previously reported, and the L050 to mice (1041-1363 mg/kg) and rats (866-1125 mg/kg) reported by Hart and Dacre (1977), support the evidence that DCPD is slightly toxic, to practically nontoxic for species tested. Pharmacologically, DCPD seemed to act as a general excitant to mink, causing increased activity and convulsions as the most pronounced clinical signs. These observations are consistent with those for Mallard ducks (Jones, 1977). The acute intraperitoneal dosing of DCPD to mink caused mor- tality in all mink in doses of 960 mg/kg and above. 62 These data suggest a lower L050 for intraperitoneal adminis- tration than by the oral route, of DIMP to mink. Data of intra- peritoneal LDSO's for other species is lacking except for the mouse which was stated to be greater than 250 mg/kg (Horton, 1948). Test 2--Subacute L050 Procedure Testing.--The subacute dietary LC50 test consisted of a 7-day quarantine and acclimation period, a 21-day dosing period, and a 7—day recovery period. Sixty juvenile pastel mink were separated into six groups of 10 mink each. Each group consisted of five males and five females randomly chosen from healthy stock, and were approximately 8 months of age. One group was assigned to each of the following dietary concentrations of DCPD: 0 (control),1, 10, 100, 1000, and 10,000 ppm. Dietary constituents and preparation procedures are given in Appendix A. All animals in the subacute trial were housed indoors in an environmentally controlled cage room, at the Poultry Science Research and Teaching Center, Michigan State University. Each mink was housed individually in a 51 x 36 x 30 cm (length x width x height) cage equipped with a water cup and feed container. Feed was provided in removable feeders attached to the inside of the cage on a swinging door such that feed consumption could be ascertained from measurement of unconsumed feed. Water was provided ad 1ibitum. 63 During the 7-day predosing acclimation period, all mink were provided with a control diet. Body weights were recorded at the beginning of the dosing period and on days 7, 14, and 21 of dosing, and on day 7 of the recovery period (termination of test). Feed consumption was estimated by daily recovery of the uncon- sumed portion of a preweighed allotment of feed, and collectively weighed for each treatment level on days 7, l4, and 21 of dosing, and on day 7 of recovery. Mortality, signs of intoxication, and behavioral changes were noted throughout both the dosing and recovery periods. Blood for packed cell volume (hematocrit) and differential 1eukocyte counts was procured by toe-clip at the termination of the test. Blood was collected in heparinized microcapillary tubes (100 pl) and centrifuged for 7 minutes at 4500 rpm on an International Microcapillary Centrifuge1 for hematocrit determination. Blood smears were allowed to air dry and were then fixed and stained in Wright's stain (see Appendix C: Preparation of Wright's Stain and Buffer). After staining, slides were first rinsed with phosphate buffer, for differentiation, and then with distilled water. They were then blotted and air dried. Differentia1 1eukocyte counts were made under oil immersion (930-x), and any abnormalities in cells were recorded. 1International Equipment Company, Boston, MA. 64 At the end of the experiment animals were terminated by cervical dislocation, and necropsied. Gross pathomorphological observations were made, and the following organs were excised, weighed, and prepared for histopathological observation according to routine laboratory procedures: brain, heart, lungs, kidneys, spleen, and liver. Statistical analysis.--Differences in body weight changes, feed consumption, hematocrit values, differential leukocyte counts and organ weights were analyzed by a one-way analysis of variance and Dunnett's t-test. Zero predicted feed consumption was estimated by regression analysis. Determination of approximate LC50 was made by regression analysis. Results The mortality associated with feeding DCPD in the diet at various levels for 21 days, followed by a 7 day post-treatment period, is given in Table 24. Mortality occurred in only the highest dietary concentration of DCPD (10,000 ppm). Mortality of mink on the 10,000 ppm DCPD diet was greater for males than for females. A mean lethal concentration (LC50) of 6800 ppm was calculated from the regression line shown in Figure 4. The mean of body weights recorded weekly throughout the test are shown in Table 25 and Figure 5. There were significantly lower body weights for mink on the 10,000 ppm DCPD treatment on days 14 and 21 of the dosing period. Although the 10,000 ppm group showed a 65 on N N N m oF coco— o op op op op op coop o op op op op op cop o op op op op o_ op o op op op op o_ P mewm o o_ op op op o_ 0 came NmNNnEoo ON m m m N N scoop o N N N N N coop o N N N N N cop o N N N N N o_ o N N N N N N o N N N N N o aqua NNNEmN ow P m m N N scoop o N N N N N ooo_ o N N N N N cop o N N N N N op o N N N N N N o N N N N N o ammo NNN: ANN m_\~ N\~ m~\— NN\P N_\_ AENNV xmm NuNprcoz NcmENNmLH NNNENNNNN1NNNN NNN>N>LNN News mo .oz NNNENNNLN NNNNNN NNN>N>LNN xcws No .oz NNNLNN acm>oomg acmeNngu-NNoN NNN-N N NNN NNNN NN NNNNNNNNNNNENN NNNNNNN NNN-_~ NNNNNNNN N NNN: NNNNNNNNNN NNNNNNNNN--.N~ NNNNN 66 Figure 4.--Regression line for the data presented in Table 24. In the regression equation x = log concentration of DCPD in ppm; y = percent mortality. X Mon-1m 100 90 10 60 40 20 67 Lo. 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N awn.“ E3 NNNENNNL» ANN NNNNNN NNNN NNN: Ncm>oumg NNNENNNLN1NNNN NNN NNNN emu; NLNNNNN NNN-NN :o ch5 mo NNNNN: anon :N mchcu--.N~ NNNcmN=N NENH N.N>LNNNN N.NNN: NN :meu NNNNNwz anon News N. mchgo ucmocma cog: NN.NNNNNNNNENN NNua NLNNNNN NNNNNNNN mo uommwu--.NN NNNOOwN NONENNmNpuuNOO OON .NNNN ONUN NNNNNNO NNN-.N :O NONE NO :ONNNENNOOO ONNN--.NN m4mm_ maowcm> um smug now save “Faun An voummmcw FmUwswgo we uczosm can .ucmwmz xcoa .zowuasamcoo comm--.wm m4m uwcuoumem; xcws con: anon accumwc muaumnzm wo pomwwm--.mm m4mw4 mcmmwam mchvwx amass; Head: :wmcm Asaav A.m.m H av Hemwmz cam: -HWMWH mpcmwwz cameo xcwe opus cog: oauo Hempmwu muzumaam wo Humwwu--.om m4mw4 mammwam mxmcuwx mmcag Hcmm: :wmcm Asaav z Heme a.m.m H my pcmwmz cameo -Hmmcw mpgmwmz :mmco xcwe wwwemw cog: onus acmpmwu muzomnsm wo Humwwm--._m m4m «ocucou «>«Huwamwc «wmgu Eocw «Omcmww«c ««H:«o«wwcm«m Ho: «LO HO«LOOO=O oEOO we» ;H«3 3o; «EOO ecu :« Ocmmz« 91 ««OH««« «N «OOHO«O« «N «NOH«OO« «N «««HO«« «N «««HOO« «N «O«HN«« «N OO« «««HONO« «N «OOHOO«« O« ««OH«O«« O« «OOH««O« O« «OOH««O« O« «NOH«O«« O« OO« ««OHOO« ON ««OH«OO« «N «NOH««O« «N «««H«OO« «N «NOH«NO« «N «OOH«OO« «N OON ««OHOO« ON «OOHONO« «N «OOHN«O« «N ««OH««« «N ««OH«OO« «N «OOH««O« «N OO« OOxOO «O«HO«O« ON «OOH««O« «N OOOH««O« «N «OOHOOO« «N «OOH«OO« O« «««H««O« O« O «2«O «OO«OEOO «ONH««« «N «N«HOO« NN «N«H««« NN «ONH««« NN ««NH«O« «N «O«HO«« «N OO« «ONH««« «N ««NH««« «N ««NH««« «N «ONHO«« «N ««NHO«« «N «ONH«O« «N OO« ««NH««« «N ««NH««« NN «N«H«N« NN «O«HO«« «N ««NH««« «N «N«HOO« «N OON «ONHO«« «N «ONH««« «N «ONH««« «N «ONHO«« «N «ONH««« «N ««NHO«« «N OO« «ONH««« «« «NNH««« «N ««NHO«« «N «ONH«O« «N «NNH««« «N ««NH«N« «N O «2«O OO««EO« «««H««O« O «O«H««O« O «O«H«O«« O O««H«««« O «NOH«O«« O «OOH«««« O OO« «O«HNN«« O ««O«H«O«« O ««O«H«««« O «OO«H«O«« O «««H«««« O «««H«N«« O OO« «N«H«OO« O «OOH«NO« O «OOH«OO« O «O«HOO«« O «««HOOO« O «««H«NO« O OON ««OH««O« O «««HO«O« O «««H««O« O «O«H««O« O «««H««O« O «««HN«O« O OO« «NOHO«O« O ««OHN«O« O ««OH«OO« O ««OH««O« O ««OH«OO« O ««OHO«O« O O «2«O OO««z AEanv «««O««O z ««««\« z ««««««N z «««««N z ««««««« z «««««« z -meww XOO OO«:«OOOO--.«« «O«<« 92 «««.O H «.« O« OO«.O H «.« O« «OO.O H «.O O« «««.« H «.«« O« OOO «O«.« H «.« O« HO«.O H «.« O« «««.O H O.O O« «O«.« H «.O« O« OO« «««.O H «.« O« OO«.O H «.N O« «N«.O H N.« O« H««.« H «.«« O« OON «N«.« H «.N «N «O«.« H «.O O« «««.O H «.O O« ««O.« H «.«« O« OO« HHXOO HO«.O H O.O O« «NO.« H N.N O« «N«.O H «.« O« «O«.O H «.«« O« O OOOO OO««OEOO «N«.O H O.« «N ««N«.O H «.O« «N «OO.O H «.O «N HO«.« H «.O« «N OO« «O«.« H «.« «N H««.O H N.O «N ««O.« H O.« «N «OO.« H N.«« «N OO« HO«.O H «.« «N «««.O H «.« «N «««.O H O.« «N ««O.« H N.O« «N OON «««.« H «.N «N «««N.« H N.NO «N «««.O H «.« «N «««.« H «.«« «N OO« ««O.« H O.O «N H««.O H «.O «N «««.O H O.O «N ««O.O H «.«« «N O OOOO OO««EO« «««.« H «.« O ««N.« H O.« O «««.« H O.« O «O«.N H O.«« O OO« «OO.« H «.N O «««.O H «.« O «O«.« H «.« O H.««.O H «.NN O OO« ««O.« H «.O O «««.« H «.« O ««N.O H «.« O «NN.« H N.NN O OON «««N.« H «.O« O «««.« H «.«« O «««.N H «.« O ««O.« H «.O« O OO« «««.O H «.« O «««.« H «.O« O «««.N H O.O« O M««.N H O.ON O O O«OO OO««z «««O««« z ««\««« z ««««««« z «««««« z «E«O« «HO -«««N«« -««««««« -«««««« -««-«N«« «OOOHH«O« 3% «3 Add H v 33¢: «won ««.« mmcwzu ucwugma con: sews m«msww ucm mpms op ammo wo cowumgumwcwEnm «gmpmmu uwcoggu wo Humwwm--.¢m “4m «ogpcou m>wuumaOmg «Own» Eocw pcmgmwwwc ««HcmowwwcmOO Ho: mam qugoOOOO mEOO ;H«3 cs=«ou HEOO asp cw «cam: p HN«.« H O.« «N H«««.O H «.«« «N HO«.O H «.« O« OO« «««.O H «.O O« H«««.O H «.«« O« H««.O H «.« O« OO« H««.O H «.« O« H«O«.N H «.O« O« «N«.O H «.O O« OON H««.O H «.« «N H«««.O H «.«« «N H«O«.O H 0.00 «N OO« OHHHO HO«.O H «.O O« H«««.O H N.«« O« H«O«.O H O.«« O« O O«OO OHO«OOOO HON.« H «.« NN H«O«.O H «.«« NN HO«.« H N.N «N OO« HO«.« H 0.0 «N H«««.O H «.«« «N «««.O H O.N «N OO« ««O.« H O.« «N H«O«.O H O.«« «N H««.O H «.O «N OON HO«.« H N.O «N H«««.O H N.«« «N H«O«.O H «.O« «N OO« HO«.O H «.O «N H«««.O H «.«« «N H««O.« H «.O« «N O OHOO OH«HEH« ««O.N H O.« O H«««O H «.«« O ««««.O H O.OO O OO« HO«.« H «.« O «««N.« H O.OO O H««.O H «.« O OO« HOO.« H «.« O ««OO.« H «.O« O H««.« H O.O O OON HO«.O H O.« O H««O.« H «.«« O H««.« H «.O O OO« H««.« H N.N O H««N.N H «.O« O H«««.O H O.NO O O O«OO OH«Hz ««««N«N« z «««««N« z «««NN««« 2 «OO«« «HO -«««««N« -««««N««« -«««««««« HOHOHHHH« umzcwpcouuu.¢m m4mo :meH OHcHEHLOOOHE m« «ow usmwmz «won :«me OHcmOmgammN .Oswcos « gm>o cmxmp «Hewsmcsmmms m «ow cowuassmcou ummw came OpcmOmgamm« a.mmp mwm a.mm— O«N com o.mm «mop w.ow N«N oov e.m¢ «cop e.¢¢ NNN com o.m~ mmop m.¢~ mew cop o «mop o omm o ««mmwmwemwm may a”: \«Hmwfimnza «wwwcmmmuzwfi «Msflwhfie Ocucos N_ «ow O«H>H« OOO«HH> HH O«OO «HO «O«s «O O«OO «O HxHHO« ««««O OHHHO«HOH «O OO«HH«OH«HO--.O« «O««« 97 Hematological parameters showed no consistent changes asso- ciated with chronic DCPD administration. Hematocrit va1ues (packed cell volume) showed no significant differences in treated animals as compared to controls, with the exception of 100 ppm DCPD-fed animals on the second blood collection date (Table 37). The depres- sion in hematocrit values shown by these animals (combined sexes) was not apparent when separated by sex. At no time during the chronic study was hemoglobin concentra- tion of the treated animals shown to be significantly different from that of the control (Table 38). Mean corpuscu1ar hemoglobin concen- tration (derived by the division of hemoglobin concentration by the hematocrit value x 100) was not significantly different for treatment animals with respect to controls, except for an initial deviation for animals fed the 100 ppm DCPD diet. When analyzed on a sex- dependent basis, this difference failed to appear (Table 39). Differentia1 leukocyte counts failed to establish a dose related difference in treatment groups with respect to control (Table 40). The effect of chronic dietary exposure to DCPD upon reproduc- tive performance is shown in Table 31. Mhelping rates, gestation length, fecundity, kit weight at birth, and secondary sex ratios were not adversely affected by DCPD administration. No differences in male fertility, as determined by presence of sperm in post-coital vaginal aspirations was noted among any of the treatment groups. Performance of kits and whe1ping dams during 1acatation is shown in Table 42. 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N«O O + «« O ««O O + N« O ««O O + O« O «OO O + «« O ««O O + O« O O«HOHHHH H««.O H «.« H««.O H «.O H««.O H O.« HO«.O H O.« HN«.O H «.« O«OOO H«N.O H «.« H«N.O H «.« H««.O H «.« HNN.O H O.« HNN.O H «.« «HOO«¥ H«N.O H O.N HO«.O H «.N H«N O H O.N HON.O H O.N H«N O H «.« OHH«OO HO.N H N« HO.« H «N HO.« H ON H«.« H «N MO.« H «N «H««O OO« OO« OON OO« O III OOH«HO «zany HOHEHHHLH OOOO «angum: «H «.0.0 H «« «HO««Hz OHOHO OO «««O OH O«OO «O OO«HHHHO«O«OHH O«OOHOO «O HOHOHOII.«« OO«<« 106 The growth of mink on the DCPD diets and the control was similar to growth patterns reported by other workers (Aulerich and Schaible, 1965; Kumeno et a1., 1970; Oldfield et al., 1968; Seier et al., 1970; Travis and Schaible, 1961). Feed consumption was depressed in several instances for mink on the 800 ppm DCPD treatment. This may have been due to a decreased palatability associated with the odor of DCPD at higher concentra- tions. However, this depression in feed consumption was transitory in nature, and the significantly greater feed consumption of mink fed DCPD over the control value on one occasion tends to contra- dict any supposed palatability problem at high dietary concentra- tions. In general, feed consumption for all groups was somewhat higher than reported for adult female mink by Schiable, 1970. Based upon the amount of DCPD ingested daily by each treatment group, it is unlikely that DCPD, when ingested chronically in moderately high concentrations, adversely affects the feed-conversion effi- ciency of mink. The analysis of hematological indices revealed no indica- tions of hemopoietic disturbances caused by chronic DCPD adminis- tration. Hematocrit (packed cell volume) hemoglobin and mean corpuscular hemoglobin concentrations values were in accordance with values reported by other workers for normal adult mink (Asher et al., 1976; Fletch and Karstad, 1972; Kubin and Mason, 1948; and Roten- berg and Jorgensen, 1971). Even though there was no difference between treatment groups and controls, differential 1eukocyte counts made at the termination 107 of the study, on blood collected from all treatment groups, differed in several respects from the values reported by other researchers. Mature (segmented) neutrophils and lymphocyte numbers differed from the results of counts made by Fletch and Karstad (1972), Gilbert (1969), and Kennedy (1935), who all reported nearly equal percent- ages of these leukocyte types at about 45-47% each. However, Asher et al. (1976) have shown seasonal and age dependent variations in white cell percentages in mink; a consideration not given by pre- vious workers. When compared to values given by Asher et al. for an equivalent time of the year, the neutrophil and lymphocyte percentages of mink in this study were well correlated. Monocyte percentages were less in the counts made in the animals in this test, when compared to the 6-9% values from several studies (Fletch and Karstad, 1972; Asher et al., 1976). However, counts made by Gilbert (1969) and by Kennedy (1935) place monocytes in the 1-2% range, as was found for the animals in this test. The reproductive potential of mink chronically exposed to DCPD was not adversely affected. Indices of reproductive performance were not markedly different from those present in the literature (Aulerich et al., 1975; Aulerich and Ringer, 1977; Enders, 1952, Hansson, 1947; Schaible and Travis, 1958). Performance of kits nursed by females on the 200, 400, and 800 ppm DCPD diets was poorer than that of control kits. The decreased weight gain of these kits over a four week nursing period is suggestive of mammary excretion of the chemical, especially since 108 it is highly lipid soluble. However, disturbances in maternal meta- bolism such as lactogenic capability, fat metabolism and excretion, calcium metabolism, or a myriad of other problems may be responsible for the reduced kit growth. Weight gain of control kits during this period was well-correlated with data supplied by other workers (Aulerich et al., 1975; Aulerich and Ringer, 1977; Oldfield et al., 1968). No gross or histopathological abnormalities were found to be consistent for any DCPD treatment, at the conclusion of the test. Sp1een weights were substantially heavier in the 400 ppm diet than in the control, but this difference was not seen in the 800 ppm DCPD-treated animals. Since individuals on a higher dosage treat- ment failed to show a similar effect, the difference in spleen weights is probably associated with chance variation or sampling error. Organ weights were not far removed from values reported by other workers (Aulerich and Ringer, 1977; Wood et al., 1965). Kidney and lung weights for mink in this test were slightly lighter than the weights reported for these organs by Wood et al. (1965). Conversely, heart weights were found to be greater than heart weights reported in the same study. According to Wood et al. (1965), the method of euthanatization can affect organ weights. Since the method of euthanatization employed by Wood and co-workers (electrocution) was different from the technique used in this study (cervical dislocation), the difference in these organ weights is more easily reconciled. 109 The reduction in testes weight exhibited by the males fed 800 ppm DCPD may have been due to an acceleration of the normal seasonal reduction which occurs in this species (Bostrom et al., 1968). However, histological examination revealed no differences in the state of seasonal regression. Conclusion l. The acute oral toxicity of DCPD for mink was estimated to be greater than 1000 mg/kg BW. 2. The 21-day subacute dietary L050 of DCPD for mink was estimated to be 6800 ppm. 3. The chronic ingestion of DCPD in the diet by mink had no effect on growth, survival, or reproductive performance. Neonate weight gain was significantly reduced by the ingestion of 200, 400, and 800 ppm DCPD by lactating dams. Testes weight of males fed 800 ppm DCPD was significantly less than the controls. APPENDICES 110 APPENDIX A MINK FEED CONSTITUENTS AND DIET PREPARATION 111 APPENDIX A MINK FEED CONSTITUENTS AND DIET PREPARATION Mink Feed Constituents Mink feed used in these experiments consisted of the follow- ing constituents: Commercial cereal (XK-40)1 25% Whole chicken 20% Ocean fish (cod, haddock & flounde trimmings) A 20% Beef tripe 15% Beef lung 7.5% Beef liver 5% Beef trimmings 5% Corn oil (during lactation) 1% Powdered milk 0.1% Vitamin E (March 1 to weaning) 55,000 units/100kg finished feed The chicken, fish, and beef by-products were ground in a 6 inch commercial feed grinder,2 and added to the remaining constituents 1XK Sales and Development Co., Thiensville, WI. 2Weiler and Co., Whitewater, WI. 112 113 in a commercial three-quarter ton feed mixer.3 Feed was allowed to mix for 15 minutes, and was then unloaded from the mixer for further diet preparation. Preparation of Diets For each diet, the amount of chemical (DIMP or DCPD) required for the proper final dietary concentration (dilution to 100 kg feed) was preweighed, and added to 500 ml corn oil as a vehicle. The chemical-vehicle mixture was then added to a mixing can contianing one kg of ground cereal, and mixed until absorbed. This premix was then added to 98.5 kg of feed (described above) in a one-quarter ton commercial feed mixer and allowed to mix thoroughly. The fin- ished diet was then unloaded into premarked color coded cans and frozen for future use. Ibid. APPENDIX B DETERMINATION OF HEMOGLOBIN CONCENTRATION 114 APPENDIX B DETERMINATION OF HEMOGLOBIN CONCENTRATION Twenty microliters of blood were added to 5 m1 of Drabkin's Reagent (see Appendix 0), mixed, and allowed to stand for 10 minutes for maximum conversion of hemoglobin to cyanmethemoglobin. This mixture was then placed in a quartz cuvette and optical density determined at 540 nm in a Spectronic 20 calorimeter- spectrophotometer.‘ The optical density of the sampel was then compared to a standard curve. The standard curve was constructed from values of optical density and hemoglobin concentration which were previously determined with human hemoglobin standards.2 1Bausch and Lomb, Rochester, N.Y. 2Cyanmethemoglobin certified standard, Hycel, Inc., Houston, Texas. 115 APPENDIX C PREPARATION OF WRIGHT'S STAIN AND BUFFER 116 APPENDIX C PREPARATION OF WRIGHT'S STAIN AND BUFFER Wright's Stain 3.3 grams Wright's powder was added to 500cc fresh, pure methyl alcohol. The stain was ripened for several months to room temperature in a stoppered brown bottle. Buffer 3.80 gm NazHPO4 5.47 gm KH2P04 Dissolve in 500 ml distilled water and bring total volume to 1000 ml. Set pH at 6.4. 117 APPENDIX D PREPARATION OF DRABKIN'S REAGENT 118 APPENDIX D PREPARATION OF DRABKIN'S REAGENT 1000 mg Soidum bicarbonate (NaHC03) 50 mg Potassium cyanide (KCN) 200 mg Potassium ferricyanide [K3Fe(CN)6] Mix to dissolve and dilute to 1 liter. The solution was stored in a sealed amber bottle and kept refriger- ated. 119 LITERATURE CITED 120 LITERATURE CITED Asher, S. J., R. J. Aulerich, R. K. Ringer, and H. Kitchen: Seasonal and age variations which occur in the blood parameters of ranch mink. U.S. Fur Rancher 56, 4, 6, 9 (1976). Aulerich, R. J., L. Holcomb, R. K. Ringer, and P. J. Schaible: Influence of photoperiod on reproduction in mink. Mich. Agr. Expt. Sta. Quart. Bul. 46, 132-138 (1963). Aulerich, R. J., and R. K. Ringer: Current status of PCB toxicity to mink, and effect on their reproduction. Arch. Environ. Contam. Toxicol. 6, 279-292 (1977). Aulerich, R. J., R. K. Ringer, and G. R. 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