BOSTON PUBLIC LIBRARY 3 9999 06317 721 4 COTTONTAIL REPRODUCTION RELATED TO DIELDRIN EXPOSURE /// Bos. nient? Pm° 1 1 1974 DEPOSITORY UNITED STATES DEPARTMENT OF THE INTERIOR BUREAU OF SPORT FISHERIES AND WILDLIFE Special Scientific Report-Wildlife No. 177 UNITED STATES DEPARTMENT OF THE INTERIOR Bureau of Sport Fisheries and Wildlife COTTONTAIL REPRODUCTION RELATED TO DIELDRIN EXPOSURE By Richard A„ Malecki, Stephen H„ Allen, and John 0. Elliston U.S. Bureau of Sport Fisheries and Wildlife Missouri Cooperative Wildlife Research Unit Columbia, Missouri Kenneth C„ Sadler Missouri Department of Conservation Columbia, Missouri W„ Reid Goforth and Thomas S. Baskett U„S„ Bureau of Sport Fisheries and Wildlife Missouri Cooperative Wildlife Research Unit Columbia, Missouri Bureau of Sport Fisheries and Wildlife Special Scientific Report—Wildlife No. 177 Washington, D„C„ • 1974 Present addresses: Allen, North Dakota State Game and Fish Department, Bismarck; Elliston, Morgan Veterinary Clinic, Coos Bay, Oregon; Goforth, Northern Prairie Wildlife Research Center, James- town, North Dakota. CONTENTS ABSTRACT iii INTRODUCTION 1 MATERIALS AND METHODS 2 RESULTS 4 Onset and Synchrony of Breeding 4 Ovulation 5 Prenatal Mortality 6 Embryonic Litter Size 8 Testes 8 Lactation 9 Adult Mortality 10 Chemical Residue Determinations 11 Stress 15 CONCLUSIONS AND DISCUSSION 17 ACKNOWLEDGMENTS 18 REFERENCES 20 APPENDIX I 22 APPENDIX II 24 For sale by the Superintendent of Documents, U.S. Government Printing Office Washington. D.C. 20402- Price $1.05 Stock Number 2410-00384 1 1 ABSTRACT Wild-trapped young-of-the-year cottontails (Sylvilagus floridanus) , confined in 1-acre pens, were exposed to annual ground applications of 0.5 and 2.0 pounds/acre of granular dieldrin. Reproductive data from rabbits in treated pens were compared with control groups during six breeding seasons, 1966-1971. Breeding male rabbits in the pens showed no sign of reproductive abnormality. Testis weights were comparable at all dosage levels (P>0.05) and spermatozoa were evident in microscopic examinations of the seminiferous tubules. No measurable differences in onset and synchrony of breeding were found among females in treated and control groups. Conception dates for the penned rabbits corresponded closely with those of wild, unpenned cottontails collected nearby. Based on second pregnancies, ovulation rates, preimplantation losses, resorption of embryos, and embryonic litter size were not significantly affected (P>0.05) by the treatments . Residue levels in brain, liver, and muscle tissues of rabbits from the two treatment levels were significantly higher than those of control animals. A comparison of brain residue levels within the two treatment groups also indicated that accumulation of the pesticides was related to the amount applied. Lethal accumulations of dieldrin were found in brains of three cottontails. We recovered fewest breeding animals from pens with highest treatment levels, but differences in recovery rates were not significant (P>0.05). No adequate measures of postnatal mortality were obtained. Paired adrenal weight :body weight and spleen weight :body weight ratios, plus histological information about cortical lipid distribution in the adrenal glands showed no conclusive evidence of stress among penned animals, whether treated or not. Dieldrin persisted in soil from 1 year to the next, following the annual applications, which fell within the limits of recommended agricultural rates. Dieldrin residue levels in cottontail tissues (brain, liver, muscle) reflected application rates and soil residue levels . in INTRODUCTION Mechanisms by which organochlorine insecticides affect organisms are not completely understood. Toxic responses of wild animals, involving the peripheral and central nervous systems, are often observed following administration of lethal doses or heavy field applications. Subtle effects of non-lethal doses are far less distinguishable, but may be very important. Alterations of hormone and enzyme metabolism are possible consequences (Peakall, 1967; Hart et al., 1971). The influence of such changes on population dynamics of the species involved is not clear. Information about the effects of organochlorines on the reproductive performance of wild animals exposed to these insecticides is needed. The purpose of this investigation was to test the effects of dieldrin (1,2,3,4 ,10 ,10-hexachloro-6 ,7-epoxy-l , 4, 4a, 5, 6, 7,8, 8a- octahydro 1 :4 ,5 :8-dimethanonapthalene) on the reproductive processes of the cottontail (Sylvilagus f loridanus) . The cottontail was selected due to: (1) its prevalence on farmland where pest control practices make it a readily exposed nontarget species, and (2) the abundance of reproductive data on this species under both wild and semiwild conditions, facilitating evaluation of treatment effects. MATERIALS AND METHODS Twelve 1-acre pens were constructed at the University of Missouri's Ashland Wildlife Area in Boone County, Missouri. Pen walls made of 1-inch mesh poultry wire were 5 feet high with an additional 6 inches of wire buried in the ground. An electric wire was installed around the top of the fence and padded steel traps were placed on posts in and around the pens to minimize losses to predators . Cedar brush piles were provided to supplement natural cover. Commercial rabbit pellets were offered ad libitum in covered feeders. Food plots 20 x 150 feet were planted to milo in each enclosure . Soil samples were obtained annually from all pens beginning in November 1965 (before initial application of dieldrin) and ending in April 1971. Twenty- five samples (minimum of 25 grams each) were randomly collected from approximately the top 3 inches of soil in each pen during 1965-69. These were then combined to give one aggregate sample for each pen. Prior to 1965, none of the 12 pens had been treated with pesticides, and all were relatively free of them. Four pens served as controls and received no treatment. From 1965 to 1971, annual treatments of 5% granular dieldrin were applied to the remaining pens during December and January, after the pens were stocked with cottontails. Four received 0.5 pound/acre of active dieldrin and the other four, 2.0 pounds/acre. Applications were made by a tractor-mounted broadcast seeder. Application levels were within ranges recommended (before inception of the experiments) for white- fringed beetle control in cottonfields or for control of several species of insects in ungrazed turf areas (United States Department of Agriculture, 1965) . Our application levels were well below the 3.0 pounds/acre used by Federal and State agencies in an intensive campaign against Japanese beetles near Sheldon, Illinois, in 1954-55 (see Scott et al., 1959). Test animals were live trapped within a 20-mile radius of the research site. Ear-tagged cottontails were stocked annually in a ratio of 1 male:2 females (except 1971) in each of the 12 pens. In 1966 through 1968, each pen received 2 males and 4 females; in 1969, 4 males and 8 females; and in 1971, 5 males and 12 females. Only young-of-the-year were used to assure some uniformity in reproductive performance (Conaway et al., 1963). Animals trapped in the wild prior to December, and weighing less than 900 grams, were considered to be juveniles of that year. This criterion has been substantiated under comparable conditions by lens weight comparisons (Sadler, unpublished data) . Rabbits were left in the treated and control pens until the females were approximately 18 days pregnant with the second litter. At this time all penned rabbits were collected, sacrificed, and weighed. Reproductive organs, spleens, and adrenals were removed and fixed in either 10% formalin or a solution of alcohol, formalin, and acetic acid (AFA) . Samples of brain, liver, and muscle were immediately frozen for dieldrin analysis . In 1970, rabbits introduced into the pens the previous fall were allowed to breed for a full season, then removed from the pens. Young-of-the-year progeny of these rabbits were then kept in the pens until the following spring breeding season. The purpose of this procedure was to evaluate the effects of dieldrin on first season reproduction of rabbits born to exposed parents in the pens . In the spring of 1968, heavy predation occurred in the pens and no meaningful reproductive data are available for that year. However, data on dieldrin residues and spleen and adrenal condition from that year are included in this presentation. Chemical analyses were made by WARF Institute, Inc.,-/ using electron capture gas chromatography. Methodology is described in Appendix I. Captive females were trapped each spring to determine breeding condition, using Haugen's (1942) procedure involving palpation of the abdominal walls between the thumb, index, and middle fingers to locate uterine swellings at the sites of embryo implantation. These swellings were classified by approximate size: peanut (age of embryo=10 days) ; acorn (age=15 days) ; or walnut (age=20 days) . The time of the year and the fact that abdominal hair had not been pulled to line a previous nest served as indicators that the female was pregnant with her first litter of the year. Wild, free-ranging cottontails were periodically collected in the vicinity of the pens, palpated, then autopsied to corroborate breeding condition assessments made by abdominal palpation of penned rabbits. We assumed that penned and wild rabbits were in similar stages of pregnancy, as was true in other central Missouri studies (Conaway and Wight, 1962) . The limited collections outside our pens reinforced this assumption. -f Companies referred to in this publication do not imply endorsement of the service by the Government. When females were approximately 18 days pregnant with their second litter, corpora lutea resulting from the second pregnancy were easily visible and the first-litter young had been weaned. Also, the critical stage of resorption was past, permitting a more accurate determination of litter size (Brambell and Mills, 1948) . Ovaries from all females were sectioned at 10 microns and mounted as interrupted serials (every tenth section mounted) . Sections were stained with a modified Schorr stain and examined microscopically. Particular attention was given to the following: 1) primary follicles in the ovarian cortex and sinusoids in the corpora lutea (di Fiore, 1961) ; 2) necrosis and degeneration of germinal epithelium (Albert, 1962) ; 3) cell size and nucleus size; and 4) character of the ovarian connective tissue. The ovulation rate was then determined by counting the number of corpora lutea in the sectioned ovaries. A wedge was removed from each testis, and slides were prepared using the same staining and mounting procedures as described for the ovaries. Sections were examined for the following indications of pathological conditions: 1) degeneration and disappearance of spermatogenic cells; 2) dividing Sertoli cells; 3) giant multi-nucleated developing sperm cells; and 4) abnormally large numbers of fibroblasts in interstitial areas (Maximow and Bloom, 1957) . During autopsies, embryo age was obtained by visually estimating the size of the uterine swellings. From this information, the time of second litter conception was determined. Females having no visible embryos, but showing other signs of an earlier pregnancy (i.e. having little or no fat, possessing well-developed mammary glands which exuded milk when sliced, or having abdominal hair pulled) were considered as being in preimplantation postpartum pregnancy. This judgment was based on the findings of Wight and Conaway (1962) who noted that almost all female cottontails breed postpartum after first pregnancies. Approximate dates for the onset of breeding were established by backdating 28 days (average gestation) from second litter conception dates. RESULTS Onset and Synchrony of Breeding In each year, the majority of females from the control pens and those of both treatment levels could be divided into two main groups with respect to time of onset of breeding. For the entire study, dates of onset of breeding were as follows: March 1 and March 8-9, 1966; March 2 and March 10, 1967; March 16 and March 23, 1969; March 1 and March 12, 1971. In each instance breeding periodicity was substantiated by comparable data from wild cottontails in the region. Although cottontails are generally synchronous breeders, a few individuals conceived between the breeding dates established for most years, as shown by ages of embryos collected on a single day (Appendix II, Table A) . Such findings are not unusual, however, since unfavorable weather occurring at this time of the year has been shown to upset this synchrony (Marsden and Conaway, 1963; Sadler and Conaway, 1971) . Ovulation Each year, the ovulation rate of female cottontails was numerically higher for rabbits at the 2.0 pounds/acre treatment level than for those at the other levels (Table 1) . These differences, however, were not significant (analysis of variance; 0.5

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(0 vo vo vo r-- (0 vo vo vc r- (0 0) 0\ G\ G\ H rH rH rH H >H rH rH rH rH In our dieldrin-treated pens (both levels) preimplantation losses of ova were generally higher than in control pens, and ranged upward to 20.4% (Table 2). However, there was great variability, and differences between controls and treated pens were not significant (P>0.05; chi-square test). Litter Resorption. — Data from females with partially resorbed litters are shown in Appendix II, Table A. Table 3 presents a summary of those data. For partially resorbed litters, a chi-square test showed no significant differences (P>0.05) in embryo resorption between rabbits in either of the two dieldrin treatments and the controls Similarly, the number of females with partially resorbed litters was not related to the treatment level. In 1966, two instances of total litter resorption were noted; both occurred in control females. One female resorbed a litter of five and the other a litter of undetermined size. Three additional cases of total litter resorption were observed in subsequent years . All involved females were from the pens treated with 0.5 pound/acre of dieldrin; litter sizes were four and six (1967) and four (1971) . Embryonic Litter Size Complete data on numbers of embryos per female are shown in Appendix II, Table A and summarized in Table 4. Table 4. Mean embryonic litter sizes for female cottontails in control and treated pens, 1966-71 (viable embryos only) . No treatment Dieldrin treatment level _ (control^ 0.5 lb/acre 2.0 lbs/acre Year x Sx x Sx x Sx 1966 5.20 + 0.425 5.57 + 0.508 5.40 + 0.601 1967 6.00 + 0.950 5.17 + 0.548 6.33 + 0.776 1969 5.67 + 0.548 5.14 + 0.508 6.57 + 0.359 1971 5.32 + 0.269 4.89 ± 0.254 5.47 ± 0.308 Analysis of variance showed that litter size was not significantly different (P>0.05) between controls and treated groups or among years within treatments. Testes Testes were removed, fixed in AFA solution and stored for 2 months. After epididymis and connective tissue were removed, one testis (fixed) from each animal was weighed. Testis weights are presented in Appendix II, Table A, and are summarized in Table 5. Table 5. Mean testis weights in grams for breeding males (1966-71) . No treatment (control) Dieldrin trea tment level 0.5 lb/acre 2.0 lbs/acre Year x Sx x Sx x Sx 1966 9.44 + 0.691 8.63 + 0.691 9.06 + 0.640 1967 10.69 + 1.197 8.25 + 0.000 8.91 + 0.977 1969 9.34 + 0.452 9.59 + 0.535 9.63 + 0.691 1971 9.42 + 0.535 10.20 + 0.510 9.79 + 0.510 "" Analysis of variance comparing testis weights between treatment levels and between years revealed no significant differences (P>0.05). No signs of testis degeneration were evident through histological examination, and spermatozoa were found in the seminiferous tubules of all males. Lactation We assumed that increased proportions of nonlactating females in treated pens would reflect increased juvenile mortality related to dieldrin treatment levels, or possibly some delay in conceptions. Detailed lactation data are presented in Appendix II, Table A, and summarized in Table 6. Table 6. Recovery rates and lactation data for females collected between 1966 and 1971. No treatment (control) Dieldrin treatment level 0.5 lb/acre 2.0 lbs/acre 1966 Number of females: Recovered Lactating Percentage lactating 12 6 50.0 7 4 57.1 5 2 40.0 1967 Number of females: Recovered Lactating Percentage lactating 2 2 100.0 8 7 87.5 4 3 75.0 1969 Number of females: Recovered Lactating Percentage lactating 16 13 81.3 14 10 71.4 16 14 87.5 1971 Number of females: Recovered Lactating Percentage lactating 29 19 65.5 30 21 70.0 19 11 57.9 No significant differences were found between the percentage of lactating females in the control pens and those of the two treatment levels (chi-square test: P >0.05). Inspection of lactation data and all reproductive parameters previously considered shows no consistent differences in reproductive performance between wild-trapped rabbits stocked annually in the pens, 1966-69, and the rabbits breeding in 1971, born of captive parents (Tables 1-6) . Adult Mortality Mortality estimates of breeding adults in the treated pens and control pens were obtained for 1966, 1969, and 1971 by a comparison of the numbers originally stocked with those recovered at the time of collection (Table 7) . Because of heavy losses to predation, similar data were not available for 1967 and 1968; in 1970, cottontails were left in the pens as breeders. Table 7. Adult cottontails recovered in spring from pens stocked the previous fall. Dieldrin treatment level No treatment 0.5 2.0 (control) lb/acre lbs/acre 1966 F M F M F M Number of rabbits: Stocked 16 8 16 8 16 8 Recovered 12 6 8 6 5 7 Percentage recovered 75.0 58.3 50.0 1969 Number of rabbits: Stocked 32 16 32 16 32 16 Recovered 19 14 20 10 17 6 Percentage recovered 68.8 62.5 47.9 1971 Number of rabbits: Stocked 48 20 48 20 48 20 Recovered 29 10 31 11 19 11 Percentage recovered 57.4 61.8 44.1 Although fewer rabbits were recovered from the treated pens than from controls, the difference was not statistically significant (chi-square test, P> 0.05). No systematic attempt was made to determine the fate of individual rabbits in the pens prior to collection. However, in the dieldrin-treated pens, some mortality was observed which appeared to be related to the pesticide. Rabbits found dead in these pens often showed no external injuries, were very emaciated, and in some instances, contained frothy intestinal contents. Rabbit mortality was most noticeable soon after application of dieldrin to the soil. 10 Raptorial birds preyed on some rabbits in most pens, but we could not determine whether raptors preferentially fed on rabbits in pens treated with dieldrin. Chemical Residue Determinations Dieldrin in Cottontail Tissues . — Residue data for 1966 through 1971 are presented in Appendix II, Table B, and summarized in Table 8. In 1966 and 1968, residue determinations were made separately on tissue samples from individual animals. In the other years, tissue samples from various numbers of individuals were pooled; numbers of samples comprising the pools are shown in Table 8 . In 1966, three separate sets of dieldrin residue analyses were made. One set consisted of analyses of tissue samples from individual adult rabbits; this set is alluded to above, and tallied in Appendix II, Table B. A second set was made on pooled tissues collected from different combinations of adult animals recovered at each of the three treatment levels as a guide for subsequent pooling procedure. The third set consisted of analyses performed on pooled tissues from the progeny (first litter of the year) of these adults. The latter two sets were not considered in the statistical analyses of the data, but are placed on record in Appendix II, Tables C and D. Large variances in the data and small sample size necessitated using the Mann-Whitney U test for statistical testing. This nonparametric test provides the same function as the "t" test, but does not require such restrictive assumptions. Comparisons of dieldrin tissue residue data are made by combining the sample results from any two groups and ranking these while still maintaining their individual identity. Differences in the sums of these ranks are then responsible for the different significance levels. In testing the null hypothesis that observations from two populations are identical, differences between controls and treated groups are expressed at the following significance levels : ** - implies a significant difference at the 10% level * - implies significance at the 25% level and is used whenever the sample size is so small that 10% significance cannot be achieved by use of the tables (from Siegel, 1956) . In 1967, too few males were collected for the data to be analyzed statistically (see column n, Table 8) . 11 Table 8. Dieldrin determinations (ppm wet weight) for adult cottontails in experimental and control pens (1966-71) No treatment Dieldrin treatment level (control) 0.5 lb/acre 2.0 lbs/acre k Sample X Sx nt x Sx nt x Sx nt 1966+ Females Brain 0.036 + 0.028 2 0.219 + 0.093 4 **0.420 + 0.078 4 Liver 0.083 + 0.007 2 **4.125 + 1.138 4 **7 .825 + 1.616 4 Muscle 0.01 — 2 **0.054 + 0.018 Males 3 **0.088 + 0.023 4 Brain 0.01 - 2 *0.183 + 0.034 4 *0.390 + 0.104 3 Liver 0.061 + 0.002 2 *5.300 + 1.520 4 *6.350 + 1.333 4 Muscle 0.01 - 2 *0.036 ± 0.003 3 *0.149 + 0.084 4 1967 + Females Brain 0.01 - 3(1) *0.049 + 0.008 9(5) *0.186 + 0.044 5(3) Liver 0.049 + 0.0 3(1) *0.997 + 0.142 6(3) *5.017 + 1.749 5(3) Muscle 0.01 — 3(1) 0.013 + 0.004 Males 8(4) 0.052 + 0.030 4(2) Brain 0.01 - 3(1) 0.069 + 0.0 KD 0.113 + 0.042 3(2) Liver 0.11 + 0.0 2(1) No data 0 3.500 + 0.639 3(2) Muscle 0.01 - 3(1) 0.01 KD 0.022 + 0.003 3(2) 1968 + Females Brain 0.01 - 5 **0.262 + 0.101 5 **0.661 + 0.251 7 Liver 0.052 + 0.013 5 **3.026 + 0.841 5 **4.330 + 1.455 7 Muscle 0.011 + 0.002 5 **0.043 + 0.015 Males 5 **0.149 + 0.094 7 Brain 0.01 - 4 **0.110 + 0.0 2 *0.36 + 0.0 1 Liver 0.071 + 0.011 4 **2.725 + 0.885 2 *6.69 + 0.0 1 Muscle 0.01 - 4 **0.112 + 0.019 2 *0.062 + 0.0 1 1969++ Females Brain 0.005 - 19(8) **0.094 + 0.012 20(9) **0.339 + 0.042 17(7) Liver 0.348 + 0.135 19(8) **2.619 + 0.433 20(9) **5.903 + 0.634 17(7) Muscle 0.005 — 19(8) **0.027 + 0.004 Males 20(9) **0.050 + 0.009 17(7) Brain 0.006 + 0.002 14(6) *0.148 + 0.024 10(5) *0.523 + 0.112 6(3) Liver 0.180 + 0.035 14(6) *5.100 + 0.634 10(5) *7.750 + 2 .653 6(3) Muscle 0.005 - 14(6) *0.028 + 0.006 10(5) *0.187 + 0.047 6(3) 1971 ++ Females Brain 0.005 - 30(7) **0.104 + 0.010 31(7) **0.328 + 0.057 19(5) Liver 0.033 + 0.003 30(7) **1.474 + 0.153 31(7) **3.448 + 0.564 19(5) Muscle 0.005 — 30(7) **0.020 + 0.003 Males 30(7) **0.064 + 0.012 19(5) Brain 0.005 - 9(3) **0.120 + 0.018 11(3) **0.375 + 0.068 11(2) Liver 0.067 + 0.013 9(3) **3.307 + 0.565 11(3) **6.185 + 1.172 11(2) Muscle 0.005 - 9(3) *0.045 + 0.008 11(3) 0.057 + 0.012 11(2) + Sensitivity = 0.01 ++ Sensitivity = 0.005 ** Significantly diffe * Significantly diffe t Individuals; number ppm (Sx values ppm one-half rent from contro rent from contro of pooled sampl involving trace amounts based on the sensitivity values) Is (P<0.10; Mann-Whitney U test) Is (P<0.25; Mann-Whitney U test) es in parentheses. 12 Residue levels in brain, liver, and muscle tissues of rabbits from the two dieldrin-treatments were significantly higher than those of control animals (see Table 8) . A comparison of brain residue levels by treatment groups also indicated that accumulation of the pesticide was related to the amount applied in the pens . Stickel et al. (1969:197) have shown that the brain is a useful tissue in appraising the probability of death from dieldrin poisoning. They cautioned that "...1 p. p.m. in the brain cannot be regarded lightly, for when brain residues are that high, body residues are many times as great." Some dieldrin levels in rabbit brains reported for this study thus strongly suggest a potential lethal condition for these animals. Three rabbits found dead in the pens during 1966 had brain residue levels of 26, 31, and 31 ppm of dieldrin. Dieldrin in the Soil .--Chemical analyses showed general agreement between annual application rates of dieldrin and residue levels in soils, with a minor exception in the samples collected in November 1969 (Table 9 and Appendix II, Table E) . Dieldrin persisted in the soil from one annual treatment to the next. After 1966, residue levels in soils of treated p§m exceeded amounts applied annually, based on computations described by Korschgen (1970:189-190). Residue levels in soil before the fifth treatment provided additional evidence of persistence (Table 9) . Variability in the data over the 6-year period precluded firm conclusions about rates of accumulation of dieldrin in the soil. This variability may be attributable to: 1) precipitation causing the mechanical movement of dieldrin into low areas; 2) nonuniform application of the pesticide because of vegetation; 3) time of year samples were collected; and 4) inconsistency in the soil collection technique (the 1970 and 1971 collections were taken at somewhat shallower depths than the intended 3 inches) . Dieldrin in Plants .--Three plant species in the pens were collected in June 1970 and analyzed for dieldrin (Table 10) by the Bureau of Sport Fisheries and Wildlife Fish-Pesticide Research Laboratory, Columbia, Missouri (see Appendix I for methodology) . Plants analyzed were among the preferred natural food sources in the pens (orchard grass, Dactylis glomerata; broomsedge, Andropogon virginicus; and panic grass, Panicum sp.) . In the treated pens, all plant samples tested had measurable amounts of dieldrin. Thus, plant food was one of the sources of dieldrin accumulation in the rabbits. 13 Table 9. Dieldrin residues in soil in experimental and control pens . ppm dieldrin Rate of dieldrin recovered Sx Range application (wet wt.)t November 1965 Control 0.5 lb/acre 2.0 lbs/acre + (before treatment) 0.01 0.01 0.01 May 1966 (after first treatment) Control 0.01 0.5 lb/acre 0.383 2.0 lbs/acre *0.765 0.112 0.211 0.15 - 0.56 0.25 - 1.22 1967 - No analysis made between second and third treatments May 1968++ (after third treatment) Control 0.015 0.5 lb/acre *3.050 2.0 lbs/acre *6.268 0.003 - - .02 0.668 2.22 - 4.62 1.640 4.50 - 10.10 ++ June 1969 Control 0.5 lb/acre 2.0 lbs/acre (after fourth treatment) 0.005 November 1969 Control 0.5 lb/acre 2.0 lbs/acre July 1970++ I Control 0.5 lb/acre 2.0 lbs/acre April 1971++ Control 0.5 lb/acre 2.0 lbs/acre ++ *2.720 0.878 0.72 - 5.00 *3.750 1.171 2.19 - 7.19 (before fifth treatment) 0,296 0.282 - - 1.14 *2.913 2.247 0.12 - 9.61 *2.408 0.410 1.72 - 3.53 er fifth treatment) 0.005 0.001 — - 0.007 *2.593 0.459 1.78 - 3.91 *8.230 1.471 5.31 - 10.0 (after sixth treatment) 0.005 *2.915 *10.333 0.448 2.845 1.89 - 3.91 5.39 - 17.7 + Sensitivity = 0.01 ppm. ++ Sensitivity = 0.005 ppm. t 1968 samples were dried before analyses, resulting in disproportionately high residue values. * Significantly different from controls (P<0.10) - "t" test (Sx values involving trace amounts based on one-half the sensitivity values.) 14 0.127 0.250 0.028 0.075 0.024 0.139 0.012 0.093 Table 10. Dieldrin content of some selected plant species (ppm) . No treatment June 1970 (controls) 0.5 lb/acre 2.0 lbs/acre Orchard grass (Dactylis) 0.0 (blades) Orchard grass (Dactylis) 0.004 (seed heads) Broomsedge (Andropogon) 0.0 (seed heads) Panic grass (Panicum) 0.009 (blades and stalks) Stress Adrenal glands were fixed in 10% formalin at the time of removal from the rabbits. Gland capsules were then stripped of extraneous connective tissue and weighed . One adrenal from each rabbit was sectioned at 20 microns with a freezing microtome, collected in water, and stained with oil-red-0 lipid specific stain according to the method described by Fickess (1963) . Following microscopic examination of the sections, adrenals were classified according to content and distribution of cortical lipid material as described by Fickess (1963) . This classification system has six types of lipid distribution in adrenals, which can be related to stress. In our study, no differences in lipid distribution were noted in adrenals from control rabbits compared with those of the dieldrin-treated pens . Mean paired adrenal weight (mg) :body weight (g) ratios and mean spleen weight (mg) :body weight (g) ratios were also used to determine if any major physiologic responses were apparent as a result of the dieldrin treatments. We assume that if the pesticide had acted as a constant source of stress, some deviation in these ratios would be observed . This response would be in accord with the rationale of Selye's General Adaptation Syndrome (Selye, 1946) , in which thymo- lymphatic atrophy and adrenal cortical hypertrophy are recognized as two of the first responses observed in animals under stress. Both adrenal and spleen weight :body weight ratios differed by sex, year, and treatment, but differences showed no consistent patterns (Tables 11 and 12) . Thus, these ratios provide no indication of differential physiological stress as a result of treatment. 15 Table 11. Mean adrenal weioht (mg) :body weight (g) ratios of adult cottontails treated with dieldrin and of controls (1966-71) . No treatment (control) Dieldrin treatment level 0.5 lb/acre 2.0 lbs/acre X Sx n X Sx n X Sx n 1966 Females 0.20 0.015 12 0.18 0.021 6 0.16 0.023 5 Males 0.18 0.025 4 0.20 0.021 6 0.22 0.019 7 1967 Females 0.13 0.029 3 0.14 0.018 8 0.15 0.021 6 Males 0.20 0.029 3 0.17 - 1 0.16 0.029 3 1968 Females 0.35 0.025 4 0.22 0.025 4 0.24 0.234 5 Males 0.37 0.025 4 0.30 0.036 2 0.37 - 1 1969 Females 0.15 0.013 15 0.15 0.012 17 0.16 0.013 16 Males 0.21 0.015 12 0.18 0.016 10 0.20 0.021 6 1971 Females 0.11 0.012 18 0.12 0.012 19 0.13 0.019 7 Males 0.18 0.016 10 0.16 0.019 7 0.17 0.036 2 Table 12. Mean spleen weight (mg) :body weight (g) ratios of adult cottontails treated with dieldrin and of controls (1966-71) . 1 ' " ' No treatment (control) x Sx n Dieldrin treatment level 0.5 X lb/acre Sx n 2.0 It X is/acre Sx n 1966 Females Males 0.29 0.54 0.160 0.277 12 4 0.28 0.41 0.209 0.209 7 7 0.32 0.26 0.248 0.209 5 7 1967 Females Males 0.83 1.46 0.320 0.320 3 3 0.47 1.28 0.196 8 1 0.90 0.75 0.248 0.320 5 3 1968 Females Males 1.11 1.23 0.277 0.277 4 4 0.70 1.29 0.320 0.392 3 2 0.79 1.21 0.248 5 1 1969 Females Males 1.09 1.38 0.134 0.154 17 13 1.15 1.02 0.124 0.175 20 10 1.18 0.56 0.134 0.226 17 6 1971 Females Males 0.55 0.75 0.109 0.161 26 10 0.32 0.40 0.105 0.185 28 9 0.45 0.44 0.127 0.167 19 11 16 It should be noted that density of cottontails may affect adrenal and spleen weight :body weight ratios (Conaway and Wight, 1962) . In our experiments, the stocking rates were equal in all pens each year. Recovery rates for rabbits at the end of the breeding seasons were generally higher in the control than in the experimental pens, but these differences were not statistically significant (see Table 7). From the available data, we doubt that density affected the measures of stress that we used; however, we cannot completely rule out this possibility. CONCLUSIONS AND DISCUSSION The following principal points emerged from this study: (1) Dieldrin behavior in soil — Dieldrin levels in soil samples taken a few months after application generally reflected the application levels. Dieldrin persisted from 1 year to the next in the soils of the pens treated annually. However, variable residue readings, attributable at least in part to experimental procedure, permitted no assessment of long-term accumuation of dieldrin in the treated pens. Annual carryover in the soil of organochlorine pesticides, including aldrin and dieldrin, has been demonstrated in other studies (reviewed by Korschgen, 1970) . Persistence over periods of a few years has also been demonstrated previously, as has been the perplexing variability in soil residue levels long after application of dieldrin (see Caro and Taylor, 1971) . (2) Dieldrin residues in cottontail tissues — Residue levels in brain, liver, and muscle tissues of rabbits from pens subjected to dieldrin treatment were significantly higher than those of control animals. Tissue residue levels reflected residue levels in the soils of the treated pens. (3) Mortality of adults — At the end of each year's experiment, lower percentages of stocked cottontails were recovered from the pens treated with dieldrin than from the control pens. These differences were not statistically significant (P > 0.05), but they may have been real: three animals found dead in treated pens had lethal brain residue levels, and a few others found dead showed some symptoms of dieldrin poisoning. (4) Reproductive performance — No major differences were apparent in the reproductive performance of either female or male cottontails according to treatment. 17 (However, the data for females pertained only to second pregnancies; it is possible that effects would have been detected in subsequent litters following longer exposure.) These findings are concordant with other studies of the effects of dieldrin on mammals. For example, Murphy and Korschgen (1970) studied effects on reproduction in white-tailed deer (Odocoileus virginianus) fed 0, 5, and 25 ppm dieldrin. They found no consistent differences in conception rates and in mortality in utero among treatment groups. Fertility of male progeny was not affected. However, they showed a greater postpartum mortality of fawns from dieldrin-treated does. In the present study, there were no consistent differences in reproductive performance between wild-trapped rabbits stocked annually in the pens, 1966-69, and the rabbits breeding in 1971, born of captive parents. Several studies besides that of Murphy and Korschgen (1970) have shown increased postnatal mortality of mammals born to females treated with organochlorines . For example, Morris (1968) studied the effects of feeding endrin (0-7 ppm) on survival and reproduction in the deer mouse (Peromyscus maniculatus) . Frequency of litter production and mean litter size were similar for each group before and during experimental feeding. Increased postnatal mortality before weaning was observed among the treated animals. He concluded that the postnatal period may be the crucial one for survival of young mammals subjected to pesticides . Harr et al. (1970) reported reproductive data for 220 female Wistar rats fed a semipurified ration to which dieldrin was added. The maximal dietary exposure level of dieldrin consistent with reproductive values of normal rats was 0.24 ppm (0.014 ug/g of body weight per day) . Exposure levels in excess of 0.24 ppm resulted in a lowered percentage of females that conceived, an increased concentration of dieldrin in the stomach milk curd of pups as compared with the ration fed the dam, and death of nursing pups. In the present study, there was no adequate measure of postnatal mortality. Thus, a possibility remains that dieldrin application suppressed productivity of the cottontails, even though reproductive rates themselves, at least during second pregnancies, were apparently not affected. ACKNOWLEDGMENTS The authors acknowledge the valuable advice and assistance of Clinton H. Conaway, Director, Caribbean Primate Research Center, Puerto Rico, whose effort was considerable in the early phases of this study. We appreciate the efforts of Frank Sapp 18 and Charles Shaiffer, managers of the research area on which the study was conducted. We are grateful to the graduate students who gave their time . We are indebted to Nicholas R. Holler, Jerry R. Longcore, and James R. Palmer, Bureau of Sport Fisheries and Wildlife, Washington, D. C, for editorial assistance and criticism. E. H. Dustman, Lucille F. Stickel, and William H. Stickel, Patuxent Wildlife Research Center, U. S. Bureau of Sport Fisheries and Wildlife, provided valued advice throughout the study. Sandra Clark deserves special thanks for manuscript work. This paper is a contribution from the Missouri Cooperative Wildlife Research Unit: U. S. Bureau of Sport Fisheries and Wildlife, Missouri Department of Conservation, Wildlife Management Institute, and University of Missouri-Columbia cooperating. Support was provided to Richard Malecki through an Edward K. Love Fellowship and to John Elliston through a Paul K. Wehmiller Fellowship. All research was supported by research contract number USDI 17-14-0008-703 with the U. S. Bureau of Sport Fisheries and Wildlife, Patuxent Wildlife Research Center, Laurel, Maryland. 19 REFERENCES Albert, T. F. 1962. The effect of DDT on sperm production in domestic fowl. Auk, vol. 79, p. 104-107. Brambell, F. W. R., and I. H. Mills. 1948. Studies on sterility and prenatal mortality in wild rabbits. Journal of Experimental Biology, vol. 25, p. 241-269. Caro, J. H., and A. W. Taylor. 1971. Pathways of loss of dieldrin from soils under field conditions. Journal of Agricultural and Food Chemistry, vol. 19, p. 379-384. Conaway, C. H., and H. M. Wight. 1962. Onset of reproductive season and first pregnancy of the season in cottontails. Journal of Wildlife Management, vol. 26, p. 278-290. , , and K. C. Sadler. 1963. Annual production by a cottontail population. Journal of Wildlife Management, vol. 27, p. 171-175. di Fiore, M. S. H. 1961. An atlas of human histology. Lea and Febiger, Philadelphia. 224 p. Fickess, D. R. 1963. Seasonal sudanophilic variation in the cottontail (Sylvilagus floridanus) adrenal cortex. Ph.D. Thesis, University of Missouri, Columbia. 106 p. Harr, J. R., R. R. Claeys, J. F. Bone, and T. W. McCoveie . 1970. Dieldrin toxicosis: rat reproduction. American Journal of Veterinary Research, vol. 31, p. 181-189. Hart, M. M., R. H. Adamson, and S. Fabro . 1971. Prematurity and intrauterine growth retardation induced by DDT in the rabbit. Archives International Pharmaco-dynamics 192, p. 286-290. Haugen, A. O. 1942. Life history studies of the cottontail rabbit in southwestern Michigan. American Midland Naturalist, vol. 38, p. 204-244. Korschgen, L. J. 1970. Soil- food-chain-pesticide wildlife relationships in aldrin-treated fields. Journal of Wildlife Management, vol. 34, p. 186-199. Marsden, H. M., and C. H. Conaway. 1963. Behavior and the reproductive cycle in the cottontail. Journal of Wildlife Management, vol. 27, p. 161-170. Maximow, A. A., and W. Bloom. 1957. A textbook of histology. W. B. Saunders Company, Philadelphia. 628 p. 20 Morris, R. D. 1968. Effects of endrin feeding on survival and reproduction in the deer mouse (Peromyscus maniculatus) . Canadian Journal of Zoology, vol. 46, p. 951-958. Murphy, D. A., and L. J. Korschgen. 1970. Reproduction, growth, and tissue residues of deer fed dieldrin. Journal of Wildlife Management, vol. 34, p. 887-903. Peakall, D. B. 1967. Pesticide-induced enzyme breakdown of steroids in birds. Nature, vol. 216, p. 505-506. Sadler, K. C, and C. H. Conaway. 1971. Cold temperatures, snow and ice as reproductive inhibitors in cottontail rabbits. In Proceedings of the Snow and Ice in Relation to Wildlife and Recreation Symposium. Iowa State University Press, Ames, p. 197-202. Scott, T. G., Y. L. Willis, and J. A. Ellis. 1959. Some effects of a field application of dieldrin on wildlife. Journal of Wildlife Management, vol. 23, p. 409-427. Selye, H. 1946. The general adaptation syndrome and diseases of adaptation. Journal of Clinical Endocrinology, vol. 6, p. 117-230. Siegel, S. 1956. Nonparametric statistics for the behavioral sciences. McGraw-Hill Book Company, New York. 312 p. Stickel, W. H., L. F. Stickel, and J. W. Spann. 1969. Tissue residues of dieldrin in relation to mortality in birds and mammals. Chemical Fallout; Current Research on Persistent Pesticides. In Proceedings First Rochester Conference on Toxicity, p. 174-204. United States Department of Agriculture. 1965. Suggested guide for the use of insecticides to control insects affecting crops, livestock, and households. Agriculture Handbook No. 290. Agricultural Research Service, Entomology Research Division and Federal Extension Service. 200 p. Wight, H. M., and C. H. Conaway. 1962. Determination of pregnancy rates of cottontail rabbits. Journal of Wildlife Management, vol. 26, p. 93-95. 21 APPENDIX I ANALYTICAL METHODOLOGY Rabbit Tissue Analyses for dieldrin were made at the WARF Institute, Inc., Madison, Wisconsin. All samples were stored frozen until time for analysis. Tissues analyzed included brain, muscle, liver, testes, and fat. Samples from individual animals weighed approximately 5-10 grams, except for fat, which was 2-4 grams. Fat samples were taken from the supra-scapular region of the body; muscle samples were from the thigh. In some years, tissue samples from more than one animal from the same pen were combined and homogenized before analysis. Samples were then proportionately larger. The numbers of individuals represented in each sample are shown in the tables in Appendix II. Samples were dried to constant weight in a 40 C oven for 72-96 hours, then ground with sodium sulfate and extracted with a mixture of ethyl and petroleum ether (70 ml: 170 ml) for 8 hours in a Soxhlet apparatus. The extract was cleaned and separated into two fractions by elution through a florisil column with mixtures of ethyl and petroleum ether (5:95 and 15:85). Analysis was by electron capture gas chromatography on a Barber-Coleman Pesticide Analyzer Model 5360. Instrument conditions were: Column, 1.2 m x 4 mm glass, packed with 5% DC-200 on 80/100 mesh Gas Chrom Q? injector temperature 230 C, column 200 C, and detector 240 C; carrier gas, nitrogen; flow such that dieldrin had a retention time of 4-5 minutes. No corrections were made for recovery, which was 85% or better. Lipids were determined on an aliquot of the extract reduced to dryness on a steambath and placed in a 40 C oven for 204 hours. Soil Analyses for dieldrin were made at the WARF Institute, Inc., Madison, Wisconsin. Samples were stored frozen until analysis. The soil sample (approximately 800 grams) was passed through a mesh screen to remove stones and other foreign materials. A 20-gram portion was taken for analysis. The sample was extracted in a 1-quart Waring Blender with 200 ml acetonitrile, filtered through glass wool into a separatory funnel containing 600 ml of tap water, partitioned from acetonitrile into petroleum ether, dried with sodium sulfate, and passed through a florisil column as described for rabbit tissue. Subsequent steps and instrument conditions are also as described for rabbit tissue. 22 Soil moisture was determined for a separate 10-gram aliquot by heating in a vacuum oven at 100 C for 5 hours, then reweighing for moisture calculation. Organic matter was measured in the same sample used for moisture determination; the dry sample was heated in a muffle furnace at 500 C for 4 hours, then reweighed for organic matter calculation. pH was determined for a separate 5-gram sample and read to the nearest 0.1 pH unit on a Beckman Zeromatic II pH meter. Plant Tissue Analyses for dieldrin were made at the Bureau of Sport Fisheries and Wildlife, Fish-Pesticide Research Laboratory, Columbia, Missouri. Tissues analyzed included seed heads, blades, or blades plus stalks. All samples were stored frozen until time for analysis. Two-gram samples were ground and dried with anhydrous sodium sulfate; extracted in a 1 cm ID glass column, with reservoir, with 100 ml of 5% diethyl ether in hexane; cleaned by eluting with 75 ml of diethyl ether and petroleum ether (5:95) through a 1 cm ID florisil column topped with sodium sulfate. Analysis was by electron capture gas chromatography on a Packard 804. 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