Txiclgical Studies with irex in Bbwhite Quail 1 RONALD J. KENDALL, RAYOND NOBLET, L. H. SENN, and J. R. HOLAN Department f Entmlgy and Ecnmic Zlgy, Clemsn University, Clemsn, Suth Carlina 29631 (Received fr publicatin April 27, 1978) ABSTRACT Bbwhite quail (Clinus virginianus) received dietary mirex in cncentratins f 1,, and ppm t investigate reprductive effects f lng term expsure t this chemical. Residue analyses f 0 generatin breeders indicated that male adipse tissue cntained apprximately 10 times the mirex level in the diet. Eliminatin f mirex in females prbably was facilitated by egg laying, which reduced mirex buildup in adipse tissue t five times the dietary level. Bth sexes were nted t cncentrate mirex in fat and breast tissue in direct prprtin t the intake f dietary mirex. Eggs cllected frm generatin breeders were nt affected deleteriusly by mirex as measured by embry survival t 3 weeks, and the number f eggs failing t hatch. Indeed, increased rates f egg fertility and hatchability were assciated with higher dietary cncentratins. Chick survival data was btained in 0 and [ generatin hatchlings frm hatching thrugh 2 weeks. N chick mrtality attributable t pesticide stress was detected in either grup f birds. Eggs cllected frm, generatin breeders that received 1 ppm were nt affected harmfully as measured by embrynatin, embry survival, and hatchability rates. Cmparisn f residues in wild bbwhites with residues in ur experimental findings indicates mirex is apparently nt affecting deleteriusly reprductive success f wild quail. INTRODUCTION The rganchlrine pesticide mirex (ddecachlrctahydr-1, 3,,-methen-2H-cyclbuta [cd] pentalene) has been used extensively in the Sutheastern United States fr cntrl f the imprted fire ant (Slenpsis invicta). Althugh mirex prvides effective cntrl f fire ants at very lw rates f applicatin (1.7 gm mirex/acre in 1.25 lb f crncb grits-sybean il bait) it is an extremely stable cmpund which allws it t becme incrprated int the fd webb (irex Reprt, 1972). Recent mnitring studies (Cllins et al, 197, arkin et al, 197, and Bevenue et al, 1975) fllwing mirex bait applicatins reprted widespread residues in terrestrial and aquatic bita. Brthwick et al. (1973) reprted bilgical cncentratin f mirex particularly in predacius birds. A few studies have been cnducted t determine effects f mirex in experimental birds. Heath et al (1972) reprted that mirex was less than ne-furth as txic as DDT t 2-week-ld bbwhites. In pen studies with Japanese quail (Cturnix cturnix japnica) receiving up t 80 ppm dietary mirex, Davisn et al. (1975) fund 'Technical cntributin n. 130 frm Suth Carlina Agricultural Experiment Statin. n reprductive effects. Hwever, Hyde et al (1973) determined mallard duckling {Anas platyrhynchs) survival t be reduced in a 100 ppm mirex treatment. Heath and Spann (1973) cncluded that shrt term mirex dsages f ppm in the feed f bbwhite quail induced n perceptible reprductive effects, but mrtality f dsed males averaged 75% cmpared t 17% f cntrl males. When chickens were given a diet cntaining 600 ppm mirex, hatching rate and chick survival were significantly reduced cmpared t cntrls (Naber and Ware, 196). The literature as a whle is variable as t cnclusive evidence f the effects f mirex in birds. Since the chlrinated hydrcarbn pesticides are such an integral part f the discussin f reprductive inhibitin in many avian species (Stickel and Rhdes, 1970), further studies with lw dietary levels f mirex in birds are needed. Bbwhite quail are imprtant game animals and their fd habits (Rsene, 1969) wuld allw frequent cntact with mirex; therefre, pen studies with bbwhites ver tw generatins were cnducted t investigate reprductive effects f lng term expsure t dietary mirex. At 1 ppm mirex treatment was designed t test the effects f a mirex dsage that bbwhites might cntact in the field (Kendall et al, 1977). Dietary levels f and ppm were used t determine effects f high levels f 1978 Pultry Sci 57:1539-155 1539
15 KENDALL ET AL. mirex n reprductive success; these levels were cnsidered t be sublethal t adults. ATERIALS AND ETHODS Pen Studies. ifty 1-day-ld bbwhite chicks ( 0 generatin) were placed in each f fur brders (0 chicks in 3 mirex treatments plus cntrl). Treatements cnsisted f mirex added t the diet as fllws: 0,1,, and ppm (jug f mirex/g f diet). The mirex was disslved in 25 ml f hexane, pured int the diet, and the feed was thrughly mixed fllwing evapratin f the hexane. The chicks received the mirex diets ad libitum beginning at 1-day f age and were cntinued n the diets thrugh the grw-ut and egg-laying perids. There was sme early mrtality in 0 chicks; hwever, this appeared t be natural die-ff and n antibitics were administered. The 35-day-ld 0 quail were transferred frm brders int tw quail battery breeding pens (GQ anufacturing Cmpany, Savannah, GA) fr grw-ut. The breeding pens were hused in a 2.1 x.3 m rm maintained under cnstant phtperid (12L:12D) and temperature (2 C). At 13 weeks 0 birds were paired ff and egg-laying was induced by increasing the light regimen 30 min/week until a 17 hr day was reached. A cnstant light duratin (17L:7D) was maintained in the study rm during the egg prductin perid. There were at least five replicates f pairs (1 male + 1 female/pen sectin) in each mirex treatment plus cntrls (cntrl, 7 pairs; 1 ppm, 5 pairs; ppm, 6 pairs; ppm, 6 pairs). Test birds began laying at 21 weeks and eggs were cllected daily, cded and stred at apprximately 19 C and 70% relative humidity. At fur biweekly intervals f laying, eggs were incubated and candled after 1 and 3 weeks f incubatin t measure embrynatin and embry survival rates. The eggs were incubated fr 2 days, and we determined hatchability as well as survival f the hatchlings t 2 weeks. After (36 weeks) the experiment terminated, adult birds ( 0 generatin) were sacrificed with chlrfrm vapr fr residue analysis. One replicate (3 mirex treatments plus cntrl) f i generatin hatchlings receiving dietary mirex was grwn ut fr investigatin f reprductive ptential f secnd generatin treatment birds. Ten-week-ld quail were transferred'accrding t mirex treatments (0, 1,, and ppm) int fur (.6 x 1.5 m) wire pens and placed in a pultry huse in the necessity f reducing maintenance csts. Ulcerative enteritis was detected in j birds and treated with ihjo sluble quail antibitic (Tylan, Elanc Chemicals). Birds were allwed t clny breed with sex ratis f five males t five females per pen. Tw treatments ( and ppm) and the cntrls were lst at 16 weeks due t predatin by dgs. One ppm treatment birds began laying at 21 weeks and the eggs were handled as previusly described. Analytical Prcedures. at (50-0 mg) and breast muscle (100-0 mg) were excised frm tw pairs f sacrificed generatin breeders/ treatment. The samples were weighed, freeze dried, reweighed, and grund with mrtar and pestle using granular sdium sulfate as an abrasive. irex was extracted frm breast muscles with hexane and frm fat samples using petrleum ether. After grinding, the samples were cllected in 125 ml flasks and fat samples were lightly biled n a steam bath fr 1 min. All samples were then shaken n a wrist-actin shaker fr 1 hr.. All the extracts were cleaned using standardized flrisil clumn chrmatgraphy. Ten centimeters f 60/100 mesh PR-grade flrisil tpped with 2 cm f anhydrus sdium sulfate was placed int a 1 cm ID glass clumn fitted with a fritted glass disc. The flrisil had been activated by ven heating at 150 C fr 3 hr. After the extract was passed thrugh Whatman- filter paper fr remval f tissue debris, it was placed n the clumn and allwed t perclate int the flrisil, at which time the mirex was eluted with ml f 5% ethyl ether in petrleum ether. The clumns were stripped with ml f a 15% ethyl ether in petrleum ether slutin befre intrductin f the next sample. The eluants were cllected in 80 ml glass beakers, evaprated t dryness n a steam bath, and transferred int aluminum-lined-cap sample vials that cntained 5 ml f nangrade hexane. Stck slutins were diluted when residues exceeded the linear respnse range f the electrn-capture detectr. irex residues were detected with a icr- Tek 2 gas chrmatgraph equipped with a 6 3 Ni electrn-capture detectr. Separatins and cnfirmatins were carried ut n 6 ft x.25 in OD U-shaped glass clumns packed with the fllwing materials: 10% DC-0 n Gas Chrm Q (80/100) and 1.5% OV-17, 1.95% Q- 1 n Gas Chrm Q (80/100). Nitrgen carrier gas flw rate was set at 100 cc/min and purge
IREX IN BOBWHITE QUAIL 151 flw at 10 cc/min. Injectin prt, clumn, and detectr temperatures were 2, 210, and 300 C, respectively. The limit f detectin f mirex in tissue samples was set at.01 ppm. Recvery rate fr samples spiked with mirex was apprximately 70%; residues are nt crrected fr recvery rates. All cncentratins f mirex are reprted n a dry weight basis. actrs fr cnverting mirex residue data frm dry-weight t wet-weight cncentratins are fat,.77 and breast,.29. Data were analyzed using analysis f variance with the cmputer-based statistical analysis system (SAS) (Barr and Gdnight, 1971). The experiment was set up as a cmpletely randm design (CRD) in assigning mirex treatments t the quail pairs. irex residue data fr breast and fat tissue f 0 generatin birds were analyzed as a 2 x factrial. A regressin analysis was perfrmed t test the crrelatin between mirex levels in breast and adipse tissue versus dietary levels. Analysis f egg prductivity data f 0 breeder quail was accmplished with a ne-way analysis f variance. RESULTS 0 ^ 3 2 2 160 X 80 = EALE QUAIL * =ALE QUAIL -2.0 6 1 22 30 IREX (PP) IN THE DIET IG. 1. irex residues in 0 generatin breeder quail (fat ppm, dry weight basis). All Revalues are significant (P<.05). Estimating equatins (Y = residues in ppm (at), Z = mirex cncentratin in the diet) ales, Y = 7.82 + 8.5Z-emales, Y =.1 +.8Z. Residues. irex residues were much higher in adipse tissue than breast muscle (Table 1), and males (P<.05) cntained mre mirex in fatty tissue (ig. 1) and breast muscle (ig. 2). There were psitive crrelatins (P<.05) between dietary levels f mirex and residues f mirex depsited in fat (ig. 1) and breast muscle (ig. 2). A significant interactin (P<.05) was fund between dietary levels f mirex X sex X mirex levels in fat, while the interactin was nt significant (P>.05) using mirex residues in breast muscle. At the highest treatment level TABLE 1. irex residues (ppm dry weight) in bbwbite quail 0 generatin breeders irex at ppm Breast ppm Sex.257.266.311.561 38 6.027 6.528 13.767.9.701.09.36.13 88.168 99.578 183.739 1.317 2.726 1.707 2.832 12.190 13.773 26.6 305.318 379.871 2.863 2.330 8.12 10.902
152 KENDALL ET AL. 13.0 10.5 ;a. _ 5.5 3.01 EALE QUAIL x-ale QUAIL -2.0 6 1 22 30 38 IREX (PP) IN THE DIET IG. 2. irex residues in 0 generatin breeder quail (breast ppm dry weight basis). All R* values are significant (P<.05). Estimating equatins (Y = residues L \ ppm (breast), Z = mirex-cncentratin in the diet) ales, Y.53 +.25Z-emales, Y =.32 +.06Z. ( ppm) the relative difference in residues in adipse tissue and breast muscle f male vs. females was larger than at the lwer levels f dietary mirex (ig. 1 and 2). Prductivity. irex did nt significantly (P>.05) affect the percentage f quail embrys surviving t 3 weeks nr the percentage f eggs (P>.05) failing t hatch (Table 2). A difference was detected (P<.05) in the percentage f eggs which failed t embrynate. Eggs f 1 ppm treatment birds had a mean infertility f 30% cmpared t 17.5% fr eggs f the ppm treatment (Table 2). The cntrls had a 28% mean infertility f eggs which was apprximately midway between the 1 and ppm treatments; therefre, n fertility prblems attributable t pesticide stress were indicated. In cmparisn t a 2% hatchability rate in cntrls, the and ppm mirex treatment birds prduced eggs with higher hatchability rates f 58% and 52%, respectively (Table 2). Chick Survival. A similar pattern f chick survival ccurred during the first tw weeks f life in the mirex treatments as cmpared t the cntrl grup. The similarity in the slpes f the lines n the graph frm days 1 t 6 indicated that mirex was nt a factr in early chick die-ff. rtality prblems invlving dietary mirex were nt encuntered thrugh grw-ut and egg laying in 0 generatin quail. A 2-week survival check (hatching t 2 weeks) was accmplished with i generatin birds (ig. ). A plt f the means f tw replicates f j chick data (ig. ) revealed a similar rate as the 0 generatin (ig. 3). j generatin treatment chicks shwed apprximately the same mrtality pattern as cntrls between 1 t 6 days f age as nted by the slpes f the lines n the graph. The rate f die-ff, hwever, was smewhat sharper in the j than in the 0 generatin birds. Ulcerative enteritis was detected in the t quail, and this factr prbably influenced the livability f the chicks. The number f j quail chicks surviving stabilized at day 7 and remained stable thrugh the rest f the tw-week check (ig. ). N mrtality prblems ccurred thrugh grw-ut in j generatin quail. Only the 1 ppm j generatin quail were available fr the egg prductin phase f the experiment. Eggs cllected frm i generatin 1 ppm breeders were incubated, and n prblems in embrynatin, embry survival, and hatchability rates were detected (Table 2). Higher percentages f eggs failing t hatch and eggs embrynating were detected in 1 ppm t vs. 0 breeders. Due t the small number f eggs as well as nly ne treatment available, the authrs suggest that extraplatin frm the data be limited. Hwever, it was particularly nted that 1 ppm j breeders had the ability t lay fertile eggs. DISCUSSION Previus wrk n the txicity f mirex in birds has demnstrated that this cmpund is f lw subacute txicity and at best f mderate chrnic txicity (Galbreath 1965; Heath et al, 1972; Hyde et al, 1973; Stickel et al, 1973). Data presented in this paper cncerning the effects f dietary mirex n bbwhite quail d nt alter these cnclusins. Davisn et al. (1975) in studies with Japanese quail reprted that residues f mirex frm whle bdy hmgenate samples were apprximately five times the mirex cncentratin in the diet. These results were similar t the wrk reprted here; hwever care must be taken in interpretatin because nly fat and breast tissue were analyzed. Our results indicate that fatty tissue f female quail cntained apprxi-
IREX IN BOBWHITE QUAIL 153 TABLE 2. Prductivity data f 0 and -, generatin bbwhite quail Treatment N a Eggsb % ail c % Died d % Hatch e % Hatch f 0 generatin C 1 50.8 39.3.5 7.5 28. 38.9 17.5 22.2 6.6 1.3 5.3 8.7 2.0 39.9 58.1 52.7 22.5 19.8 19.1 16.3, generatin 1 2 28.0g N = ttal number f replicates per treatment. 11.5 Eggs = mean number f eggs cllected fr incubatin ver the replicates f each treatment pairs f quail/treatment = C-7, 1 ppm-5, ppm-6, ppm-6. ean percentage f eggs which failed t embrynate when checked at 1 week. ean percentage f embrys which had died when checked at 3 weeks. ean percentage f eggs hatching. ean percentage f eggs failing t hatch. *Eggs cllected frm 1 ppm l generatin breeders hused in a clny breeding pen with a sex rati f five males t five females. mately five times the dietary mirex cncentratin. Hwever, fatty tissue f males had up t 10 times the level f mirex fund in the diet. This difference f mirex levels in male vs.female quail agree with data reprted by Heath and Spann (1973); they nted that the egg can serve as a majr eliminatin rute fr mirex in females. The ability f the female birds in eliminating mirex als shwed up when residues in 55 3 I -I z> O I 50 5 10 QUAIL AGE IN DAYS CONTROL IG. 3. Tw week survival check n 0 generatin birds (beginning n hatching date). 6.1 8.3 3.1 breast muscle were determined. A trend f higher mirex levels in breast muscle f males was bvius; hwever, differences were nt clearly defined except in the ppm treatment. Cntrl birds shwed lw mirex residues in fat > t X u < a: UJ m s => z 30 25 15 10 X 5 10, QUAIL AGE IN DAYS ppm Ippm ppm C0NTR0L IG.. Tw week survival check n, generatin birds (beginning n hatching date) (X f 2 replicates used fr plt f data). 15
15 KENDALL ET AL. tissue but nne was detected in breast muscle. This agreed with Putnam et al. (197) wh shwed that pultry huse dust n walls and grund litter can serve as a surce f cntaminatin f cntrls in pesticide studies. Cntrary t the results f Heath and Spann (1973), n mrtality prblems were encuntered with dsed male bbwhites even at the ppm mirex level. Cnflicting results may be due t differences in envirnmental cnditins experienced by quail in the tw experiments. Our birds were placed under little r n stress except frm the pesticide whereas Heath and Spann (1973) cnducted an utdr clny breeding experiment. Reprductive studies reprted here with bbwhite quail indicated that survival f chick embrys as well as the number f eggs failing t hatch were nt deleteriusly affected by mirex. These data agreed with results reprted by Heath and Spann (1973) and Davisn et al. (1975). In additin, ur results shw a lwer percentage f infertile eggs and increased hatchability rates in the higher mirex treatments. We have n explanatin fr this ther than the bservatin f large fat depsits in all f the experimental birds, which pssibly enabled partitining f ingested mirex int adipse tissue, thus allwing treatment birds t cmpete reprductively with cntrls even with high levels f mirex expsure. Sme prblems with egg fertility were nted, and we suggest that because f the small number f quail pairs available, extraplatin frm the data shuld be limited. ertility, survival f embrys t 3 weeks, and hatchability rates were determined fr eggs cllected frm 1 ppm t generatin breeders. N reprductive prblems attributable t pesticide stress were nted in these i breeder quail. Due t the fact that nly ne treatment (1 ppm) f j breeders was available, cnclusins were limited in this phase f the experiment. Hwever, the data shw that V x generatin mirex treatment birds laid fertile eggs. Beginning n the day f hatching, 2-week chick survival checks were made n 0 and j generatin quail. During this early critical perid (Rsene, 1969), mrtality f bbwhite chicks was nt related t expsure t mirex. Heath et al. (1972) als shwed bbwhite chicks were nt unusually sensitive t mirex; they listed an extremely high LC 50 f 2511 ppm mirex in 5-day diets f 2-week-ld birds. In relating the research reprted here t bbwhite quail fund in mirex treated areas, Kendall et al. (1977) fund mirex residues in wild quail that were similar t thse receiving 1 ppm mirex in this study. Baetcke et al. (1972) reprted that adipse tissue f bbwhite quail cntained as much as 3.18 ppm mirex 1 year after mirex bait applicatin. Cllins et al. (197) reprted that mirex residues in bbwhites cllected frm treated areas were similar t the cncentratins discussed here. Cmparisn f mirex residues in ur experimental quail, that exhibited n treatment effects in regard t mrtality r reprductivity, t residues in wild bbwhites indicates n demnstrable threat f mirex t wild bbwhite quail ppulatins. ACKNOWLEDGENTS The authrs express their gratitude t Lynn Luszcz wh assisted in the pesticide analyses and Jay D. Hair fr helpful suggestins during the curse f the experiment. This research was supprted by a grant frm the Suth Carlina Plant Pest Regulatry Service. REERENCES Baetcke, K. P., J. D. Cain, and W. E. Pe, 1972. irex and DDT residues in wildlife and miscellaneus samples in ississippi 1970. Pestic. nit. J. 6: 1-22. Barr, A. J., and J. H. Gdnight, 1971. Statistical analysis system. Nrth Carlina State University Press, Raleigh, NC. Bevenue, A., J. N. Ogata, L. S. Tengan, and J. W. Hylin, 1975. irex residues in wildlife and sils, Hawaiian Pineapple Grwing Areas 1972 197. Pestic. nit. J. 9:11-19. Brthwick, P. W., T. W. Duke, A. J. Wilsn, Jr., J. I. Lwe, J.. Patrick, Jr. and J. C. Oberheu, 1973. Accumulatin and mvement f mirex in selected estuaries f Suth Carlina, 1969-71. Pestic. nit. J. 7:6-26. Cllins, H. L., G. P. arkin, and J. Davis, 197. Residue accumulatin in selected vertebrates fllwing a single aerial applicatin f mirex bait, Luisiana-1971-72. Pestic. nit. J. 8:125-130. Davisn, K. L., J. H. Cx, and C. K. Graham, 1975. The effect f mirex n reprductin f Japanese quail and n characteristics f eggs frm Japanese quail and chickens. Arch. Envirn. Cntam. Txicl. 3:8-95. Galbreath, E. H., 1965. Txicity f mirex bait t certain species f wildlife..s. Thesis, University f Gergia. Heath, R. G., E.. Hill, and J.. Kreitzer, 1972. Cmparative dietary txicities f pesticides t birds. N. 152. U. S. Bur. Sprt ish, and Wildl. Heath, R. G., and J. W. Spann, 1973. Reprductin and related residues in birds fed mirex. Page 21 3 in Pestic. Symp., 8th Inter-Amer. Cnf. Txicl. Occup. ed.
IREX IN BOBWHITE QUAIL 155 Hyde, K.., J. B. Graves, A. B. Watts, and. L. Bnner, 1973. Reprductive success f mallard ducks fed mirex. J. Wildl. anage. 37:79-8. Kendall, R. J., R. Nblet, J. D. Hair and H. B. Jacksn, 1977. irex residues in bbwhite quail after aerial applicatin f bait fr fire ant cntrl, Suth Carlina-1975-76. Pestic. nit. J. 11:6-68. arkin, G. P., H. L. Cllins, and J. Davis, 197. Residues f the insecticide mirex in terrestrial and aquatic invertebrates fllwing a single aerial applicatin f mirex bait, Luisiana 1971 1972. Pestic. nit. J. 8:131-13. irex reprt, 1972. Reprt f the irex Advisry Cmmittee, Envirnmental Prtectin Agency, Washingtn, DC. Naber, E. C, and G. W. Ware, 1965. Effects f kepne and mirex n reprductive perfrmance in the laying hen. Pultry Sci. :875-880. Putnam, E.., R. N. Brewer, and G. J. Cttier, 197. Lw level pesticide cntaminatin f sil and feed and its effect n briler tissue residue. Pultry Sci. 53:1695-1698. Rsene, W., 1969. The bbwhite quail, its life and management. Rutgers University Press, New Brunswick, NJ. Stickel, L.., and L. I. Rhdes, 1970. The thin eggshell prblem. Page 31 35 in Prc. Symp., The Bilgical Impact f Pesticides in the Envirnmental Health Sciences Series N. 1, Oregn State University, Crvallis, OR. Stickel, W. H., J. A. Gaylen, R. A. Dyrland, and D. L. Hughes, 1973. Txicity and persistence f mirex in birds. Page 37 66 in Pestic. Symp. 8th Inter- Amer. Cnf. Txicl. Occup. ed.