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UBC Theses and Dissertations

Effects of dieldrin on reproduction in the rat Virgo, Bruce Barton 1970

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THE EFFECTS OF DIELDRIN ON REPRODUCTION IN THE RAT by BRUCE BARTON VIRGO B.Sc. (Honors), Un i v e r s i t y of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA JULY 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbi Vancouver 8, Canada Depa rtment ERRATA Through-out the text. Through-out the text. Through-out the text. Through-out the text. Page kt l i n e 12. Page 13, l i n e 19. Page 18, l i n e 15 • Page 3^,line 9. Page kZ, l i n e 23. Page kkt l i n e 1. Page l i n e 15. For "homogenous" read "homogeneous". For "homogenity" read "homogeneity". For "oonsistant" read "consistent". For "oonsistantly" read "consistently". For " i n " read " i t " . For "was" read "were". For " i s " read "are". For "have" read "has". For " i s " read "are". For "dehydrogenoses" read "dehydrogen-ases". For " a f f e c t s " read " e f f e c t s " . ABSTRACT The e f f e c t s o f c h r o n i c i n g e s t i o n o f t e c h n i c a l d i e l d r i n on r e p r o d u c t i o n i n t h e r a t were s t u d i e d by d e t e r m i n i n g t h e number o f l i t t e r s , t h e number and t h e v i a b i l i t y o f young b o r n t o a n i m a l s e x p o s e d t o d i e t a r y c o n c e n t r a t i o n s o f 0, 13, 25, 37 and 53 ppm. D i e l d r i n had no e f f e c t on t h e r e p r o d u c t i v e s u c c e s s o f t h e m a l e s . I n t h e f e m a l e s d i e l d r i n had no e f f e c t on f e r t i l i t y and d i d n o t s i g n i f i c a n t l y r e d u c e l i t t e r s i z e s b u t e v i d e n c e i s p r e s e n t e d w h i c h s u g g e s t s t h e l i t t e r s i z e r e d u c t i o n i s r e a l . E x p o s u r e o f t h e dam d u r i n g l a c t a t i o n r e d u c e s t h e s u r v i v a l o f s u c k l i n g young. I t i s s u g g e s t e d t h a t t h e m a j o r cause o f t h i s m o r t a l i t y i s a c u t e p o i s o n i n g from d i e l d r i n s e c r e t e d i n t h e m i l k , a l t h o u g h t h e growth r e s p o n s e s o f t h e young i n d i c a t e t h a t s t a r v a t i o n r e s u l t i n g from d e c r e a s e d l a c t a t i o n may a l s o be i n v o l v e d . None o f t h e m o r t a l i t y i s c a u s e d by i n c r e a s e s i n m a t e r n a l a g g r e s s i o n o r n e g l e c t . E x p o s u r e d u r i n g g e s t a t i o n a l o n e a l s o r e d u c e s t h e p o s t - p a r t u m s u r v i v a l o f young. The p r o b a b l e p h y s i o l o g i c a l mechanisms i n v o l v e d i n t h e v a r i o u s e f f e c t s a r e d i s c u s s e d . (85% 1 , 2 , 3, 4, 10, 10 - h e x a c h l o r o - 6, 7 - epoxy - 1, 4, 4 a , 5, 6, 7, 8, 8a - o c t a h y d r o - 1, 4 - endo, exo - 5, 8 - d i m e t h a n o n a p h t h a l e n e and 15% r e l a t e d compounds.) i i TABLE OF CONTENTS PAGE ABSTRACT ; i TABLE OF CONTENTS i i LIST OF TABLES i i i LIST OF FIGURES i v ACKNOWLEDGEMENTS v I . INTRODUCTION 1 I I . METHODS 4 1. E x p o s u r e o f a n i m a l s t o d i e l d r i n 4 2. Measurement o f e x p o s u r e t o d i e l d r i n ( A d u l t s ) 4 3. Measurement o f r e p r o d u c t i v e s u c c e s s 5 4. Measurement o f e x p o s u r e t o d i e l d r i n (Young) 7 5. The f o s t e r n u r s i n g e x p e r i m e n t s 7 6. O b s e r v a t i o n s on m a t e r n a l b e h a v i o r 7 7. Measurement o f d i e l d r i n r e s i d u e s 8 I I I . RESULTS 11 1. E x p o s u r e t o d i e l d r i n and t h e h e a l t h o f t h e a n i m a l s 11 2. S t a t i s t i c a l a n a l y s e s o f t h e d a t a on r e p r o d u c t i v e p e r f o r m a n c e . 13 3. E f f e c t s on t h e r e p r o d u c t i v e c a p a c i t y o f t h e male 15 4. E f f e c t s on t h e r e p r o d u c t i v e c a p a c i t y o f t h e fe m a l e 17 5. E f f e c t s o f p r e and p o s t - p a r t u m m a t e r n a l e x p o s u r e on t h e v i a b i l i t y o f t h e young 25 6. B e h a v i o r o f exposed f e m a l e s d u r i n g t h e r a i s i n g o f young . 29 IV. DISCUSSION. 34 1. E x p o s u r e t o d i e l d r i n and t h e h e a l t h o f t h e a n i m a l s 34 2. E f f e c t s on r e p r o d u c t i v e c a p a c i t y 36 3. E f f e c t s on t h e r e p r o d u c t i v e c a p a c i t y o f t h e male 38 4. E f f e c t s on t h e r e p r o d u c t i v e c a p a c i t y o f t h e fe m a l e 39 (a) F e r t i l i t y 39 (b) S i z e and s e x c o m p o s i t i o n o f t h e l i t t e r 40 (c) V i a b i l i t y o f t h e young 41 V. SUMMARY 46 V I . LITERATURE CITED 48 V I I . APPENDIX I . B e h a v i o r p a t t e r n s o f female r a t s w i t h young .... 53 V I I I . APPENDIX I I . The measurement o f d i e l d r i n r e s i d u e s 56 IX. APPENDIX I I I . The r e l a t i o n s h i p between dose and r e s p o n s e ... 68 i i i LIST OF TABLES PAGE Table I. The t o t a l food intake of animals eating d i e l d r i n contaminated food f o r 44 weeks 11 Table I I . The reproductive success of unexposed females mated by males exposed to dietary d i e l d r i n 16 Table I I I . The e f f e c t of dietary d i e l d r i n on the estrous cycle of the r a t 19 Table IV. The reproductive success of females exposed to dietary d i e l d r i n 20 Table V. The age at death of those young dying before weaning whose dams were exposed to dietary d i e l d r i n 23 Table VI. The growth responses of the l i t t e r s of females exposed to'0 or 53 ppm dietary d i e l d r i n during l a c t a t i o n 31 Table VII.".'., The frequency of occurrence of behavior patterns shown by females with young and exposed to dietary d i e l d r i n .. 32 Table VIII. The p o s s i b l e causation of d i e l d r i n induced reproductive f a i l u r e i n the r a t 37 Table IX. A comparison of the usefulness of the three regression l i n e s f o r the standard curve data i n p r e d i c t i n g the amount of d i e l d r i n 60 Table X. The 95% confidence l i m i t s on an estimate of x from the regression l i n e s of O p t i c a l Density vs D i e l d r i n 62 i v L IST OF FIGURES . J PAGE F i g u r e 1 The c u m u l a t i v e e x p o s u r e o f t h e r a t s t o d i e t a r y d i e l d r i n 12 F i g u r e 2 The e f f e c t o f d i e t a r y d i e l d r i n on t h e p o s t - p u b e r t a l g r o w t h o f t h e r a t 14 F i g u r e 3 The e f f e c t o f d i e l d r i n i n t h e d i e t o f t h e dam on t h e s u r v i v a l and l i f e e x p e c t a n c y o f t h e young 21 F i g u r e 4 The g r o w t h r e s p o n s e o f young r a t s n u r s i n g on dams expo s e d t o d i e t a r y d i e l d r i n 24 F i g u r e 5 The e f f e c t o f a f o s t e r dam on t h e s u r v i v a l , l i f e e x p e c t a n c y o f t h e young 26 F i g u r e 6 The' s u r v i v a l o f young r a t s b o r n t o f e m a l e s e x p o s e d t o 0 o r 53 ppm d i e t a r y d i e l d r i n d u r i n g g e s t a t i o n and n u r s e d on dams exp o s e d t o t h e same doses d u r i n g l a c t a t i o n 27 F i g u r e 7 The l i f e e x p e c t a n c y o f young r a t s b o r n t o f e m a l e s exposed t o 0 o r 53 ppm d i e t a r y d i e l d r i n d u r i n g g e s t a t i o n and n u r s e d on dams exposed t o t h e same doses d u r i n g l a c t a t i o n 28 F i g u r e 8 The g r o w t h o f s u c k l i n g r a t s b o r n t o f e m a l e s e x p o s e d t o 0 o r 53 ppm d i e t a r y d i e l d r i n d u r i n g g e s t a t i o n and n u r s e d on f e m a l e s exposed t o t h e same doses d u r i n g g e s t a t i o n 30 F i g u r e 9 The r e l a t i o n s h i p between t h e O p t i c a l D e n s i t y o f t h e d i e l d r i n - d i p h e n y l a m i n e r e a c t i o n p r o d u c t s and t h e amount o f d i e l d r i n i n v o l v e d i n t h e r e a c t i o n 59 F i g u r e 10 The r e l a t i o n s h i p between dose and r e s p o n s e 69 ACKNOWLEDGEMENTS I should l i k e to thank Drs. H.C. Nordan, J . Mary Taylor, J.E. P h i l l i p s and J.F. Bendell f o r t h e i r advice and c r i t i c i s m during the study and the preparation of t h i s manuscript. I am p a r t i c u l a r l y g r a t e f u l to the S h e l l Chemical Corporation whose g i f t of d i e l d r i n made t h i s work p o s s i b l e . I extend s p e c i a l thanks to my parents, p a r t i c u l a r l y my mother who typed.'the draf t s of t h i s paper. Most of a l l I wish to thank my wife S h e i l a f o r her encouragement and support. 1 I. INTRODUCTION The organochlorine i n s e c t i c i d e s are now d i s t r i b u t e d throughout the ecosystems of the world such that dietary exposure to these compounds i s probably common f o r most species. I t i s becoming i n c r e a s i n g l y apparent that such exposure can r e s u l t i n mutagenesis, carcinogenesis, s y n e r g i s t i c responses to other p e s t i c i d e s and lowered rates of reproduction. Of these effects,impairment of the normal reproductive process i s l i k e l y to have the most serious consequences on a population of a w i l d species. Many in v e s t i g a t o r s have suggested that the organochlorines are responsible f o r the decreased rates of reproduction shown by raptores and other birds and the subsequent decline i n population l e v e l s (Hickey and Anderson, 1968). Recent laboratory work has confirmed that these compounds can decrease rates of egg production, f e r t i l i t y and h a t c h a b i l i t y and the s u r v i v a l rate of young (Bitman et al_. , 1969; Heath et a l . , 1969; McFarland and Lacy, 1969). Most of the organochlorine i n s e c t i c i d e s are u n l i k e l y to s i g n i f i c a n t l y a f f e c t mammalian reproduction rates. The hexachlorocyclohexanes (lindane and BHC) are not apt to cause problems since t h e i r high cost p r o h i b i t s large scale use. The p o l y - c h l o r i n a t e d biphenols (DDT and i t s analogs) are known to reduce the pregnancy rate of the house mouse and the s u r v i v a l rate of young mice and rats (Bernard and Gaertner, 1964; Ware and Good, 1967; Tayan and Kemeny, 1969) but only at exposure doses u n l i k e l y to be obtained i n natural systems. For s i m i l a r reasons the s i n g l e r i n g cyclodienes are n o t . l i k e l y to cause problems; chlordane i s known to reduce the pregnancy rate and s u r v i v a l rate of young i n house mice and r a t s but only at very high doses (Ambrose et a l . , 1953; Conney et a l . , 2 1 9 6 7 ) ; t e l o d r i n , at e c o l o g i c a l doses, had no e f f e c t on house mouse reproduction (Ware and Good, 1967). Endosulfan and h e p t a c h l o r , the others i n t h i s group, w i l l probably behave i n a s i m i l a r manner. The double r i n g cyclodienes ( a l d r i n , i s o d r i n , d i e l d r i n , endrin) do present a r e a l p o t e n t i a l hazard to mammalian p o p u l a t i o n s . T h e i r s t a b i l i t y (Nash and Wilson, 1967) and l a r g e s c a l e (Anon., 1963) use r e s u l t i n environmental l e v e l s (Saha, 1969), which i n c o n j u n c t i o n w i t h t h e i r ease of absorption (Lehman,. 1956), could r e s u l t in- doses s u f f i c i e n t to lower r e p r o d u c t i o n i n some s p e c i e s . Snyder (1963) has presented the only evidence t h a t these compounds can reduce numbers i n a p o p u l a t i o n of mammals when he demonstrated t h a t e n d r i n reduced the reproduction r a t e of a p o p u l a t i o n of meadow v o l e s . There are other reasons t o b e l i e v e t h a t the double r i n g cyclodienes are apt t o be the compounds most harmful to mammalian p o p u l a t i o n s . I t has been shown th a t low doses of these compounds adversely a f f e c t r e p roduction i n some mammals. Deichmann and K e p l i n g e r (1966) demonstrated w i t h the house mouse t h a t d i e l d r i n and a l d r i n at 25 ppm reduced the pregnancy r a t e , the l i t t e r s i z e and the s u r v i v a l r a t e of young; l a t e r work (Good and Ware, 1969) showed t h a t d i e l d r i n at 5 ppm had no e f f e c t on the pregnancy r a t e but reduced l i t t e r s i z e ( s u r v i v a l was not s t u d i e d ) . The house mouse i s s e n s i t i v e to these compounds as i s the dog; doses i n excess of 3.0 ppm reduced the s u r v i v a l of pups but had no e f f e c t on pregnancy ra t e s or l i t t e r s i z e s (Kitselman, 1953). Good and Ware a l s o showed t h a t e n d r i n at 5 ppm reduced l i t t e r s i z e s i n the house mouse whereas Mo r r i s (1968) demonstrated t h a t 7 ppm had no e f f e c t on l i t t e r s i z e i n the deer mouse (Peromyscus m a n i c u l a t u s ) ; t h e a l a t t e r species a l s o showed a decreased s u r v i v a l r a t e i n the young. The double r i n g cyclodienes c l e a r l y 3 a f f e c t mammalian r e p r o d u c t i o n b u t t h e r e a r e l a r g e d i f f e r e n c e s i n r e s p o n s e between s p e c i e s s u c h t h a t g e n e r a l i z a t i o n s c a n n o t be made. There i s a l a c k o f d a t a on t h e e f f e c t s o f t h e s e compounds i n t h e r a t . B a l l e t a l . ( 1 9 5 3 ) , u s i n g a v a g i n a l smear t e c h n i q u e , n o t e d t h a t a l d r i n a p p e a r e d t o cause t r a n s i t o r y changes i n t h e e s t r o u s c y c l e b u t t h e y c o n d u c t e d no b r e e d i n g t e s t s . T r e o n and C l e v e l a n d ( 1 9 5 5 ) , u s i n g t h e 3 g e n e r a t i o n b r e e d i n g , t e c h n i q u e o f F i t z h u g h (1949) , c l a i m t h a t a l d r i n and d i e l d r i n i n doses as low as 2.5 ppm d e c r e a s e d f e r t i l i t y i n t h e f i r s t g e n e r a t i o n and t h e s u r v i v a l o f young i n a l l g e n e r a t i o n s , w h i l e doses as h i g h as 25 ppm had no e f f e c t on t h e l i t t e r s i z e ; however, t h e y p r e s e n t no d a t a o r s t a t i s t i c s t o s u b s t a n t i a t e t h e i r c l a i m s . No o t h e r work has been done on t h e r a t and i n v i e w o f t h e a p p a r e n t l y extreme s u s c e p t i b i l i t y o f t h i s s p e c i e s t h e r e i s a need f o r d a t a on i t . T h i s s u s c e p t i b i l i t y , i f r e a l , w o u l d make t h e r a t an i d e a l a n i m a l f o r s t u d y i n g t h e p h y s i o l o g i c a l mechanisms o f o r g a n o c h l o r i n e i n d u c e d r e p r o d u c t i v e f a i l u r e . The p u r p o s e o f t h i s s t u d y was t o d e t e r m i n e i f c h r o n i c e x p o s u r e t o d i e t a r y d i e l d r i n w o u l d cause d e c r e a s e s i n f e r t i l i t y , l i t t e r s i z e and s u r v i v a l r a t e o f s u c k l i n g s and t o d e t e r m i n e t h e amount o f e x p o s u r e r e q u i r e d t o i n d u c e t h e s e e f f e c t s . 4 I I . METHODS 1. E x p o s u r e o f A n i m a l s t o D i e l d r i n F i v e g r o u p s o f p o s t - p u b e r t a l r a t s were f e d f o o d c o n t a i n i n g d i e l d r i n i n c o n c e n t r a t i o n s o f 0, 13, 25, 37 o r 53 ppm f o r 44 weeks. The a n i m a l s were o f t h e W i s t a r s t r a i n (UBC s t o c k , randomly b r e d f o r a p p r o x i m a t e l y 16 g e n e r a t i o n s ) and were 25-27 weeks o f age a t t h e o n s e t o f t h e e x p e r i m e n t s . There were 18 males and 18 f e m a l e s i n each group a t t h e s t a r t . The s e x e s were housed s e p a r a t e l y i n communal m e t a b o l i s m cages w i t h 9 a n i m a l s p e r cage. A l l t h e a n i m a l s were f e d t h e 0 ppm r a t i o n f o r 1 month a f t e r b e i n g p u t i n t h e cages t o a l l o w them t o a d j u s t t o t h e g round f e e d and t o combat a r e s p i r a t o r y d i s e a s e w h i c h a r o s e . Food and w a t e r were s u p p l i e d ad l i b i t u m . The f o o d was p r e p a r e d by g r i n d i n g c o m m e r c i a l l y p r e p a r e d UBC r a t r a t i o n i n a c r u s h e r m i l l and s p r a y i n g i n w i t h d i e l d r i n d i s s o l v e d i n 95% EtOH. To e n s u r e homogenity o f d i e l d r i n d i s t r i b u t i o n t h e s p r a y was a p p l i e d by a s m a l l h a n d - s p r a y e r t o 2 l b . p o r t i o n s o f f e e d ; s e v e r a l a p p l i c a t i o n s were made and t h e f e e d was t h o r o u g h l y m i x e d between a p p l i c a t i o n s . The 0 ppm r a t i o n was s p r a y e d w i t h e t h a n o l o n l y . The f o o d was f e d i n t h e g round s t a t e . 2. Measurement o f E x p o s u r e t o D i e l d r i n ( A d u l t s ) I t was d e s i r a b l e t o have 2 measures o f t h e amount o f e x p o s u r e t o d i e l d r i n . One i s t h e amount a c t u a l l y b e i n g a b s o r b e d f r o m t h e g u t , t h e s e c o n d i s t h e c o n c e n t r a t i o n i n t h e v a r i o u s o r g a n s . I t can be c a l c u l a t e d from t h e d a t a o f Heath and Vandekar (1964) t h a t o n l y 0.007% o f t h e f e c a l d i e l d r i n i s compound w h i c h has been a b s o r b e d and e x c r e t e d unchanged. T h e r e f o r e i f t h e amount o f d i e l d r i n i n g e s t e d , t h e amount o f f e c e s p r o d u c e d and t h e d i e l d r i n c o n c e n t r a t i o n i n t h e f e c e s a r e 5 known then the r a t e of absorption can b e ' c a l c u l a t e d . The animals were fed from s p i l l - p r o o f feeders which were weighed before and a f t e r f i l l i n g such t h a t the feed consumption could be determined by s u b t r a c t i o n . The amount of d i e l d r i n i n g e s t e d was c a l c u l a t e d by m u l t i p l y i n g the amount of feed consumed by the co n c e n t r a t i o n o f d i e l d r i n i n the feed. The animals were weighed once a week such t h a t exposure could be expressed as mgm. of ing e s t e d d i e l d r i n per kgm. of body weight. The feces were caught on screens below the cages and were c o l l e c t e d O once a week, d r i e d f o r 12 hours at 100 C, weighed and a sample s t o r e d f o r a n a l y s i s . The animals were sampled by s a c r i f i c i n g 2 males and 2 females at each l e v e l of exposure 1 day before the s t a r t of a breeding t e s t ; the carcasses were frozen and s t o r e d f o r a n a l y s i s . The method of a n a l y s i s i s presented below. 3. Measurement of Reproductive Success The animals were t e s t e d f o r r e p r o d u c t i v e c a p a b i l i t y a f t e r 10, 19, 28 and 39 weeks of exposure For the f i r s t 2 t e s t s a f a c t o r i a l mating system was used thus: 1. t r e a t e d male X t r e a t e d female 2. t r e a t e d male X untreated female 3. untreated male X t r e a t e d female P a i r s were made up by randomly s e l e c t i n g animals from the metabolism cages and p l a c i n g them i n s m a l l mating cages. Four p a i r s were used f o r each cross and each cross was made f o r the 5 l e v e l s of exposure. The males were removed a f t e r 2 weeks and the females l e f t i n the cages throughout pregnancy and l a c t a t i o n . The untreated animals were obtained from the UBC stock colony and were approximately the same age as the t r e a t e d animals. The r e s u l t s from the f i r s t 2 t e s t s i n d i c a t e d t h a t d i e l d r i n was 6 a f f e c t i n g the females but not the males. To enlarge the sample s i z e of treated females and yet s t i l l obtain data on the treated males a d i f f e r e n t mating system was used i n the l a s t 2 tests thus: 1. treated male X treated female 2. treated male X untreated female The females were not placed i n mating cages but were kept i n t h e i r metabolism cages and 2 males were placed i n each cage. The males were changed every day. Mating was allowed for 2 weeks and the animals were retained on t h e i r experimental d i e t s during t h i s time. The pregnant females were placed i n i n d i v i d u a l cages at about day 14 of gestation to give b i r t h and r a i s e t h e i r l i t t e r s . The data obtained from these tests were the number of pregnancies, the l i t t e r s i z e and sex r a t i o at b i r t h , and the s u r v i v a l of the young. The l a t t e r was determined by examining the l i t t e r s d a i l y and noting the number and sex of the young present. In ad d i t i o n , the weight of the young born to females i n the t h i r d and fourth tests was determined d a i l y . To determine i f d i e l d r i n had an e f f e c t on the estrous cycle of the female, vaginal smears were taken d a i l y from animals at each l e v e l of exposure for the periods of 4-8, 23-28 and 36-38 weeks i n c l u s i v e . Smears were obtained by a s p i r a t i n g the vagina with a drop of p h y s i o l o g i c a l s a l i n e and p l a c i n g the aspirant on a glass s l i d e . A f t e r the aspirant had d r i e d the c e l l s were f i x e d with absolute EtOH and stained f or 10 minutes with a 0.015% Giemsa s t a i n . The smears were examined and defined as estrus i f 80% of the c e l l s were c o r n i f i e d e p i t h e l i a l c e l l s , d i e s trus i f 80% were leucocytes and proestrus i f 80% were nucleated e p i t h e l i a l c e l l s . In the analysis of the r e s u l t s the data from the treated male X treated female and untreated male X treated female crosses were combined to t e s t female e f f e c t s i f there was no e f f e c t on the male. 7 4. Measurement o f Exposure to D i e l d r i n (Young) To c l a r i f y the cause of death of the young t h a t d i e d before weaning i t was d e s i r a b l e t o determine the amount of d i e l d r i n i n them. A l l young found dead were l a b e l l e d , f r o z en and s t o r e d f o r a n a l y s i s . In a d d i t i o n a l l young t h a t s u r v i v e d u n t i l weaning were s a c r i f i c e d , f r o z en and s t o r e d f o r a n a l y s i s . 5. The F o s t e r Nursing Experiments The data from the re p r o d u c t i o n t e s t s i n d i c a t e d t h a t d i e l d r i n i n the females d i e t caused a decreased s u r v i v a l of her young. To determine whether the cause was due to pre-partum or post-partum exposure o f the female, the young of 15 females exposed to 53 ppm i n t h e i r d i e t d u r ing g e s t a t i o n were f o s t e r nursed on females exposed only to the 0 ppm d i e t ; the reverse was a l s o f o l l o w e d . This was done by r e p l a c i n g h a l f the l i t t e r of a 53 ppm female w i t h h a l f the l i t t e r o f a 0 ppm female and v i c e versa. The t r a n s f e r s were made w i t h i n 6 hours of b i r t h but a f t e r the young had r e c e i v e d some colostrum from t h e i r own female. The young of these mixed l i t t e r s were t o e - c l i p p e d i n order to i d e n t i f y t h e i r r e a l dam. The s u r v i v a l and weight of the young were recorded d a i l y . To determine the e f f e c t s of a f o s t e r mother on the s u r v i v a l and growth of young an experiment was run previous t o t h a t d e s c r i b e d above but i d e n t i c a l i n every respect except a l l of the females (10) were on a 0 ppm d i e l d r i n d i e t . 6. Observations on Maternal Behavior The f o s t e r n u r s i n g experiment i n d i c a t e d t h a t there was both a pre-and post-partum component i n the causation of the decreased s u r v i v a l of young. To e l u c i d a t e the post-partum component of the cause(s) observations were made on the maternal behavior of females on the 0, 37 and 53 ppm 8 d i e t s . These o b s e r v a t i o n s were made on f e m a l e s u s e d i n t h e f o u r t h r e p r o d u c t i v e t e s t and t h e f o s t e r n u r s i n g e x p e r i m e n t . P r e v i o u s o b s e r v a t i o n s u g g e s t e d 12 b e h a v i o r p a t t e r n s were shown by f e m a l e s and i n a d d i t i o n 3 o t h e r s were added w h i c h <a p r i o r i c o u l d be d e f i n e d as a g g r e s s i v e (see A p p e n d i x I ) . • The 15 p a t t e r n s c o u l d be f i t t e d i n t o one o f 4 c a t e g o r i e s : 1. M a t e r n a l - c a r e o f young 2. A g g r e s s i o n - t o w a r d young 3. M a i n t e n a n c e - f e m a l e ' s c a r e o f h e r s e l f 4. O t h e r s - f i t t e d none o f above i . e . f u n c t i o n d e b a t a b l e Three 10 m i n u t e p e r i o d s o f o b s e r v a t i o n were made on each a n i m a l p e r day. One p e r i o d was chosen between 0800-1100 h o u r s , one between 1400-1700 and t h e t h i r d between 1900-2200. E v e r y 1.5 seconds t h e a n i m a l ' s b e h a v i o r was n o t e d . A metronome was u s e d f o r t i m i n g . 7. Measurement o f D i e l d r i n R e s i d u e s The method chosen t o measure t h e amount o f d i e l d r i n i n t h e f e c e s and a n i m a l t i s s u e s was t h a t o r i g i n a l l y d e s c r i b e d by Cueto (1960) . He a p p l i e d i t t o a n i m a l f a t and i t was l a t e r u s e d by Cueto and Hayes (1962) t o measure d i e l d r i n i n human u r i n e . The 3 s t e p s i n t h e e s t i m a t i o n o f t h e d i e l d r i n c o n t e n t o f a sample a r e : 1. e x t r a c t i o n o f t h e d i e l d r i n 2. r e m o v a l o f c o n t a m i n a n t s f r o m the e x t r a c t i . e . c l e a n - u p 3. c o l o u r development o f t h e e x t r a c t e d d i e l d r i n The c o l o u r r e a c t i o n i s t h e b a s i c p r i n c i p l e o f t h e t e c h n i q u e and depends on t h e f a c t t h a t d i e l d r i n forms a b l u e complex w i t h d i p h e n y l a m i n e i n t h e p r e s e n c e o f z i n c c h l o r i d e . The o p t i c a l d e n s i t y (OD) o f t h e r e a c t i o n p r o d u c t s i n a c e t i c a c i d s o l u t i o n s a t 650 mp f o l l o w s B e e r ' s Law such t h a t an unknown amount o f d i e l d r i n can be d e t e r m i n e d f r o m a s t a n d a r d c u r v e . 9 T h i s method was chosen because i t i s s e n s i t i v e t o m i c r o g r a m q u a n t i t i e s o f d i e l d r i n and i s v e r y i n e x p e n s i v e i n c o m p a r i s o n t o t h e gas c h r o m a t o g r a p h i c t e c h n i q u e s . Two m a j o r p r o b l e m s were e n c o u n t e r e d , one o f w h i c h was n o t s o l v e d and t h e t e c h n i q u e c o u l d n o t be use d . The c o l o u r r e a c t i o n as o r i g i n a l l y d e v e l o p e d was u s e d on d i e l d r i n w h i c h was a t l e a s t 99.5% p u r e . I n t h i s s t u d y t e c h n i c a l grade d i e l d r i n , w h i c h i s t h e b a s i s f o r c o m m e r c i a l i n s e c t i c i d e s , was u s e d and i t c o n t a i n s 85% d i e l d r i n and 15% r e l a t e d i n s e c t i c i d a l compounds. T e c h n i c a l g r a d e d i e l d r i n i s b i o l o g i c a l l y t h e more i m p o r t a n t p r e p a r a t i o n , p a r t i c u l a r l y s i n c e t h e r e i s e v i d e n c e t h a t t h e p r o p e r t i e s o f p u r i f i e d , r e c r y s t a l l i z e d compounds may d i f f e r c o n s i d e r a b l y f r o m t h e c o m m e r c i a l p r o d u c t s ( B a l l e t a l . , 1953; Treon e t a l . , 1955). M a j o r improvements were r e q u i r e d i n C u e t o ' s t e c h n i q u e b e f o r e i t h a d adequate s e n s i t i v i t y and p r e c i s i o n f o r use on t e c h n i c a l d i e l d r i n . The e x t r a c t i o n o f a p e s t i c i d e from b i o l o g i c a l m a t e r i a l s i n v a r i a b l y r e s u l t s i n t h e s i m u l t a n e o u s e x t r a c t i o n o f m a t e r i a l s w h i c h w i l l i n t e r f e r e w i t h t h e q u a n t i f i c a t i o n o f t h e p e s t i c i d e . An adequate method f o r r e m o v i n g c o n t a m i n a n t s f r o m an e x t r a c t i s e s s e n t i a l and i t i s t h i s p r o c e s s w h i c h i s u s u a l l y l i m i t i n g i n t h e e f f e c t i v e n e s s o f any measurement p r o c e d u r e . I n t h i s s t u d y m o d i f i c a t i o n s o f 3 c l e a n - u p p r o c e d u r e s ( t h o s e o f C u e t o , 1960; K u t n e y , 1969; de F a u b e r t Maunder e t a l _ . , 1964) were a p p l i e d t o e x t r a c t s o f b o t h f e c e s and t i s s u e s i n an u n s u c c e s s f u l a t t e m p t t o f i n d one w h i c h w o u l d a l l o w t h e use o f t h e c o l o u r r e a c t i o n . To use t h e c o l o u r r e a c t i o n i t became a p p a r e n t t h a t t h e r e must be a b s o l u t e l y no c o n t a m i n a n t s i n t h e f i n a l s o l u t i o n o f d i e l d r i n u n d e r g o i n g t h e r e a c t i o n b e c a u s e t h e i r p r e s e n c e r e s u l t e d i n t h e f o r m a t i o n o f r e d o r g r e e n m a t e r i a l s w h i c h were i n s o l u b l e i n t h e r e a c t i o n p r o d u c t s s o l v e n t and w h i c h c o m p l e t e l y masked t h e d i p h e n y l a m i n e - d i e l d r i n complex. The degree o f 10 p u r i t y required i n the f i n a l s o l u t i o n to be analyzed i s probably much higher than f o r gas chromatographic techniques. The i n t e r f e r i n g materials i n the feces were apparently carotenoid i n nature (Kutney, 1969) while those i n the tissues were l i p o i d a l ( i .e ether s o l u b l e ) . A f u l l d e s c r i p t i o n of the technique i s presented i n Appendix I I . I 11 I I I . RESULTS 1. E x p o s u r e t o D i e l d r i n and the He a l t h , o f t h e A n i m a l s The c u m u l a t i v e dose o f d i e l d r i n w h i c h t h e a n i m a l s i n g e s t e d i s p r e s e n t e d i n F i g . 1. The f e m a l e s e x p e r i e n c e more e x p o s u r e t h a n t h e males a t e ach dose and t h i s i s a r e s u l t o f t h e i r e a t i n g more f o o d ( T a b l e I ) . T a b l e I . The t o t a l f o o d i n t a k e (kgm. feed/kgm. body w e i g h t ) o f a n i m a l s e a t i n g d i e l d r i n c o n t a m i n a t e d f o o d f o r 44 weeks. D i e l d r i n C o n c e n t r a t i o n (ppm) Sex 0 13 25 37 53 14.4 14.5 16.0 16.3 The d i f f e r e n c e i n f o o d i n t a k e i s n o t l a r g e b e i n g about 0.5 gm./day f o r an a v e r a g e s i z e d unexposed f e m a l e and about 1.5 gm./day f o r an ex p o s e d f e m a l e . The magnitude o f t h e d i f f e r e n c e and t h e ti m e a t w h i c h i t i s f i r s t d e t e c t a b l e a p p e a r t o be dose-dependent ( F i g . 1 ) . The a p p a r e n t e x c e p t i o n o f t h e 53 ppm a n i m a l s t o t h e t i m e e f f e c t i s due t o t h e males e a t i n g an a b n o r m a l l y l a r g e amount o f f o o d f o r t h e f i r s t 4 weeks. These d a t a s u g g e s t t h a t d i e l d r i n s t i m u l a t e s t h e female t o e a t more f o o d b u t has no e f f e c t on t h e male. The amount o f e x p o s u r e f o r f e m a l e s r a n g e d from 5.3 mgm./kgm. body weight/week a t 13 ppm t o 20.4 a t 53 ppm; t h i s r e p r e s e n t s 12% t o 47% o f th e a c u t e LD50 o f 43 mgm./kgm. f o r a d u l t f e m a l e s (Borgmann e t a l . , 1952b). The t o t a l e x p o s u r e f o r f e m a l e s o v e r t h e c o u r s e o f t h e e x p e r i m e n t r a n g e d ? 14.5 14.9 15.1 16.6 14.1 15.5 12 Weeks of Exposure 13 from 56.6 mgm./kgm. body weight at 13 ppm to 859.3 mgm. at 53 ppm. The amount of exposure f o r the males was s l i g h t l y less, which i n conjunction with the higher LD50 f o r males (47 mgm./kgm. Borgmann e_t aJ., 1952b)( r e s u l t e d i n a much lower " e f f e c t i v e dose" f o r t h i s sex. Males on the 53 ppm d i e t f o r example, ingested 18.2 mgm./kgm. body weight/week or only 39% of the l e t h a l dose. The growth of e i t h e r sex does not appear to be aff e c t e d by dietary d i e l d r i n (Fig. 2).. Each point i s the mean weight of 10-18 animals and range i s such that none of the apparent differences are r e a l . The sharp decline i n weight shown by the males during weeks 30-34 was caused by an epidemic of a re s p i r a t o r y disease. None of the animals were observed to show the c l a s s i c symptoms of • acute poisoning as described by Hodge e_t a l . (1967) . Females on the 37 and 53 ppm diet s were observed to show hyper-e x c i t a b i l i t y to external s t i m u l i , p a r t i c u l a r l y sound and to be nervous and i r r i t a b l e during weeks 7-10 of exposure. No other symptoms of chronic poisoning were noted. 2. S t a t i s t i c a l Analyses of the Data on Reproductive Performance The enumeration data (% pregnant, % s u r v i v a l , sex rati o s ) was tested f o r s i g n i f i c a n c e by use of the t e s t f o r independence i n r x c tables (Steele and T o r r i e , 1961) except f o r the % s u r v i v a l data from the f o s t e r nursing experiment which was tested by the G t e s t f o r r x c x t tables (Sokal and Rohlf, 1969). The l i t t e r s i z e , l i f e expectancy and age at death data were tested for s i g n i f i c a n c e by use of an analysis of variance (Steele and T o r r i e , 1961). . A l l p a i r e d comparisons between doses were made regardless of the s i g n i f i c a n c e of the o v e r a l l analysis of variance; the s i g n i f i c a n c e of these comparisons was tested by use of Scheffe's Fig. 2 The effect of dietary dieldrin on the post-pubertal growth of the rat. Weeks of Exposure 15 p r o c e d u r e f o r m u l t i p l e c o m p a r i s o n s w i t h u n e q u a l sample s i z e s ( S c h e f f e , 1959).. 3. E f f e c t s on t h e R e p r o d u c t i v e C a p a c i t y o f t h e Male The p r e g n a n c y r a t e o f u n t r e a t e d f e m a l e s mated w i t h t r e a t e d males i s 2 homogenous between doses (X = 8.182, d f = 4, P = 0.084) w h i c h s u g g e s t s t h a t t h e f e r t i l i t y o f t h e male i s u n i m p a i r e d by d i e l d r i n a t doses as h i g h as 53 ppm; n o t e however, t h a t t h e f e r t i l i t y r a t e o f males on 25 ppm i s much l o w e r t h a n t h o s e on t h e o t h e r doses ( T a b l e I I ) . S i m i l a r l y t h e s i z e o f l i t t e r a male s i r e s i s n o t a f f e c t e d by t h e dose o f d i e l d r i n he r e c e i v e s , ( o v e r a l l a n a l y s i s : F = 0.770, P>0.10; none o f t h e p a i r e d c o m p a r i s o n s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s ) . The s e x r a t i o o f young s i r e d by t r e a t e d males i s n o t homogenous between doses ( X 2 = 11.47, d f = 4, P = 0.022), b u t d i f f e r s a t t h e 0.05 l e v e l . However, a t what d o s e ( s ) t h i s a p p a r e n t e f f e c t o c c u r s i s . u n c l e a r , b e c a u s e r e m o v a l o f t h e d a t a f r o m any c e l l r e s u l t s i n an i n s i g n i f i c a n t X when i t i s r e - c a l c u l a t e d . T h i s e f f e c t i s v e r y d i f f i c u l t t o e x p l a i n and i s p r o b a b l y spurious.- The male d e t e r m i n e s s e x and i f t h e e f f e c t i s r e a l one w o u l d e x p e c t s i m i l a r r e s u l t s f r o m t h e young s i r e d by exposed males and b o r n t o e x p o s e d f e m a l e s and t h i s i s n o t t h e case ( T a b l e I V ) . I f t h e s e x r a t i o s f r o m b o t h T a b l e s I I and IV a r e combined t h e d a t a a r e homogenous ( X 2 = 3.82, d f = 4, P = 0.443). There c o u l d o f c o u r s e be some for m o f compensatory m o r t a l i t y o f embryos i n t h e t r e a t e d f e m a l e s w h i c h w o u l d mask t h e sex r a t i o e f f e c t b u t t h i s w o u l d p r o b a b l y be r e f l e c t e d by a d e c r e a s e i n t h e l i t t e r s i z e and t h i s does n o t o c c u r ( T a b l e I V ) . The s u r v i v a l r a t e o f p r e - w e a n i n g young s i r e d by t r e a t e d males i s n o t homogenous ( X 2 = 40.30, d f = 4, P<10~5) w h i c h s u g g e s t s t h a t d i e l d r i n i s a f f e c t i n g t h e sperm i n s u c h a way t h a t t h e young a r e r e l a t i v e l y i n v i a b l e . " S u r v i v i n g " i s d e f i n e d as b e i n g a l i v e a t w e a n i n g , 23 days o f age. However, s u r v i v a l i s a c r u d e i n d e x o f v i a b i l i t y and i t i s d i f f i c u l t t o d e t e r m i n e 16 Table I I . The reproductive success of unexposed females mated by males exposed to dietary d i e l d r i n . The number i n brackets i s the sample s i z e . Parametre $'s:100 9's Dietary Concentration (ppm) Control 13 25 37 53 % 83 83 50 83 67 Pregnant (18) (18) (18) (18) (18) L i t t e r Size 10.5±0.54 11.6+0.39 10.9±0.74 10.910.65 11.1±0.75 x±SE (12) (14)' (9) (13) (12) Sex Ratio 100 89 106 82 62 at B i r t h (136) (155) (87) (129) (133) % S u r v i v a l 58.1 63.9 39.1 66.7 36.1 to 23 Days (136) ' (155) (87) (129) (133) Expectancy of L i f e (days) j 16.011.1 17.511.0 12.311.2 18.910.9 14.9+1.2 at Day 1 of O (63) (66) (48) (57) (50) L i f e ? x+SE O 14.711.1 16.611.0 12.811.5 17.610.9 13.310.9 (64) (84) (34) (72) (81) 17 where the r e a l differences l i e using t h i s parametre because of the large e r r o r term associated•with binomial data. The l i f e expectancy of the young i s a much more s e n s i t i v e index of v i a b i l i t y and therefore was chosen as the parametre to compare between doses. The l i f e expectancy of the male o f f s p r i n g d i f f e r s s i g n i f i c a n t l y between doses (F = 5.36 P= 0.0004). B a r t l e t t ' s t e s t indicates that the variances are homogenous (P = 0.370) and therefore paired comparisons are v a l i d . Scheffe's t e s t i n d i c a t e s that none of the treatments d i f f e r s i g n i f i c a n t l y from the co n t r o l (P>0.05). As such,exposure of the male to d i e l d r i n does not a f f e c t the v i a b i l i t y of the male o f f s p r i n g he s i r e s . . The sources of variance which cause s i g n i f i c a n c e i n the o v e r a l l analysis are from the doses with the high and low extremes i n l i f e expectancy and t h i s can be a t t r i b u t e d to chance; 37 and 53 ppm d i f f e r at P<0.01 and 25 and 37 ppm d i f f e r at 0.01<P<0.05. S i m i l a r l y , the l i f e expectancy of the female young also d i f f e r s s i g n i f i c a n t l y between doses (F = 3.93, P = 0.0041). B a r t l e t t ' s t e s t i ndicates the variances are homogenous (P = 0.498) such that paired com-parisons are v a l i d . Scheffe's t e s t i n d i c a t e s that none of the treatments d i f f e r s i g n i f i c a n t l y from the co n t r o l (P>0.05).' As such exposure of the male to d i e l d r i n has no e f f e c t on the l i f e expectancy of the female o f f -spring he w i l l s i r e . The s i g n i f i c a n t variance arises from the doses with the two extremes of l i f e expectancy; 37 and 53 ppm d i f f e r at 0.01<P<0.05. The l i f e expectancy of the females i s lower than the male at a l l doses. However, Fisher's t t e s t i n d i c a t e s that the differences are not s i g n i f i c a n t (maximum t = 0.349, df = 00). 4. E f f e c t s on the Reproductive Capacity of the Female The e f f e c t of dietary d i e l d r i n on the length of the estrous cycle and the number of p o s i t i v e estrus' i s presented i n Table I I I . The data from 18 weeks 23-32 and 36-38 have been combined t o g i v e a sample s i z e comparable t o t h a t f o r weeks 4-8. The mean number o f p o s i t i v e e s t r u s ' has been c o r -r e c t e d f o r t h e t i m e d i f f e r e n c e i n t h e 2 p e r i o d s . There was a g r e a t d e a l o f v a r i a t i o n between i n d i v i d u a l s . A n i m a l s had c y c l e s r a n g i n g from 2-14 days i n l e n g t h ; many were a c y c l i c , b e i n g i n c o n s t a n t e s t r u s , d i e s t r u s o r p r o e s t r u s . There was no c o r r e l a t i o n between the t y p e o f c y c l e e x h i b i t e d by a f e m a l e and h e r f e r t i l i t y . M o r eover t h e r e i s no r e a s o n t o b e l i e v e t h a t e i t h e r t h e c y c l e l e n g t h o r t h e number o f p o s i t i v e e s t r u s ' d i f f e r between doees o r t i m e s . The f e r t i l i t y r a t e o f t h e t r e a t e d f e m a l e s and t h e s i z e and s e x c o m p o s i t i o n o f t h e i r l i t t e r s i s p r e s e n t e d i n T a b l e I V. The d a t a . f r o m a l l 4 b r e e d i n g t e s t s have been p o o l e d as t h e r e were no a p p a r e n t d i f f e r e n c e s between them. N e i t h e r t h e p r e g n a n c y r a t e ( X 2 = 5.97, d f = 4, P = 0.20) , th e l i t t e r s i z e ( o v e r a l l F = 0.74, P> 0.10 and a l l p a i r e d c o m p a r i s o n s a r e 2 n o t s i g n i f i c a n t ) , n o r t h e s e x r a t i o (X = 4.36, d f = 4, P = 0.360) d i f f e r between d o s e s . F o r f u t u r e r e f e r e n c e i t s h o u l d be n o t e d t h a t t h e l i t t e r s i z e o f t h e t r e a t e d f e m a l e s i s c o n s i s t a n t l y s m a l l e r t h a n t h o s e o f t h e c o n t r o l s . More i m p o r t a n t i s t h e f a c t t h a t t h e young o f t r e a t e d f e m a l e s were v e r y f r e q u e n t l y b o r n w i t h g r o s s hematomas i n t h e head and c a u d a l r e g i o n . The s u r v i v a l r a t e s o f t h e young b o r n t o t r e a t e d and c o n t r o l f e m a l e s ( F i g 3) a r e n o t homogenous ( X 2 = 127.99, d f = 4, P< 10~ 5) s u c h t h a t e x p o s u r e o f t h e female t o d i e l d r i n a f f e c t s t h e v i a b i l i t y o f h e r o f f s p r i n g . The d a t a i n F i g . 3 f i t l a t y p i c a l d o s e - m o r t a l i t y c u r v e (see A p p e n d i x I I I ) e x c e p t t h a t '•i t h e l o w e r p l a t e a u phase i s m i s s i n g . S i n c e s u r v i v a l i s s u c h a c r u d e i n d e x o f v i a b i l i t y / l i f e e x p e c t a n c y was use d as t h e i n d e x t o d e t e r m i n e where t h e s i g n i f i c a n t e f f e c t s l a y ( F i g 3 ) . C o n s i d e r i n g t h e males f i r s t , t h e a n a l y s i s o f v a r i a n c e i n d i c a t e s h i g h l y 19 Table I I I . The e f f e c t of dietary d i e l d r i n on the estrous cycle of the r a t . Parametre Weeks of Exposure Dietary Concentation (ppm) Control 13 25 37 53 Mean No. of P o s i t i v e Estrous' per Animal 4-8 -23-38 3.8 3.4 3.3 3.9 3.7 3.2 3.1 3.2 3.4 3.9 Mean Cycle" Length (days) 4-8 23-38 5.42 4.86 4.97 5.23 5.10 4.72 5. 20 6.13 5.40 5.21 Sample Size 4-8 23-38 16 16 16 17 17 17 17 17 17 17 20 Table IV. The reproductive success of females exposed to dietary d i e l d r i n . The number i n brackets r e f e r s to the sample s i z e . Dietary Concentration (ppm) Parametre Control 13 25 37 53 "o 36.8 44.7 23.7 31.6 44.7 • Pregnant (38) (38) (38) (38) (38) L i t t e r Size 11.311.0 9.310.9 10.110.9 10.111.3 9.111.0 xiSE (13) (16) (9) (12) (14) Sex Ratio 93 79 .65 93 90 at B i r t h (147) (143) (91) (104) (127) J'silOO 9 ' s Fig.3 The effect of dieldrin in the diet of the dam on the survival and life expectancy of the young. ro O 10 20 30 40 50 60 0 10 20 30 40 50 60 Concentration of Dieldrin in the Feed (ppm) 22 s i g n i f i c a n t d i f f e r e n c e s between doses (F = 20.57, P<10 b ) . Scheffe's t e s t shows t h a t : 0 ppm vs 13 ppm n s P > 0 . 0 5 0 ppm vs 25 or 37 or 53 ppm P<0.01 13 ppm vs 25 or 37 or 53 ppm * P<0.05 a l l comparisons between 25, 37 and 53 ppm ns. P>0.05 The r e s u l t s o f the a n a l y s i s on the l i f e expectancy of the females are i d e n t i c a l . The v a l i d i t y of Scheffe's t e s t may be questioned because B a r t l e t t ' s t e s t i n d i c a t e s t h a t the p r o b a b i l i t y of homogenous variances - f i i s l e s s than 10 . By F i s h e r ' s t t e s t there i s no d i f f e r e n c e between the l i f e expectancy of males and females (maximum t = 0.457, df = 0 0 ) . C o n s i d e r i n g these r e s u l t s i t i s c l e a r t h a t the dose r e c e i v e d f o r a s i g n i f i c a n t r e d u c t i o n i n the v i a b i l i t y of the o f f s p r i n g i s between 13 and 25 ppm but the Threshold Dose i s l e s s than 13 ppm. The age at death of those o f f s p r i n g dying.before weaning i s presented i n Table V where the sexes have been pooled. An a n a l y s i s of variance i n d i c a t e s t h a t there are s i g n i f i c a n t d i f f e r e n c e s at the 0.05 l e v e l (F = 3.49, P = 0.017). However Scheffe's t e s t f o r m u l t i p l e comparisons i n d i c a t e s t h a t none of the treatments d i f f e r from the c o n t r o l (F = 1.67 w i t h 4 and 476 df) and the s i g n i f i c a n t v ariance comes from the maximum and minimum doses (13 and 53 ppm) which d i f f e r a t the 0.01 l e v e l (F = 3.93). B a r t l e t t ' s t e s t i n d i c a t e s non-homogenity of v a r i a n c e at the 0.05 l e v e l (P= 0.038). The growth responses of the young are presented i n F i g . 4. The mean body weight per s u r v i v i n g young was determined by weighing the l i t t e r o f a female, d i v i d i n g by the number of young i n the l i t t e r and then averaging these weights f o r a l l females. There would appear to be no d i f f e r e n c e s i n growth. The sample, however, i s very s m a l l and there i s normally l i t t l e growth i n the f i r s t 5 days by which time the o f f s p r i n g of females on 25, 23 Table V. The age at death of those young dying before weaning whose dams were exposed to dietary d i e l d r i n . Dietary Concentration (ppm) Control 13 25 37 53 Age at Death 5.0210.58 6.4410.63 4.35+0.64 4.6410.44 • 3.98+0.43 x+SE 24 Fig. 4 The growth response of young rats nursing on dams exposed to dietary dieldrin. (7) (7) Oppm I3ppm (2) \ 25ppm (5) (6) 37ppm 53ppm | litters increasing from birth weight until all litter members died 0 litters maintaining birth weight until all litter members died 13 litters decreasing from birth weight until all litter members died 25 37 and 53 ppm d i e l d r i n are dead. Moreover the growth of the young i s apparently a f u n c t i o n of the female such t h a t growth e f f e c t s are masked by averaging. The body weight of s u r v i v i n g young of any female c o n s i s t a n t l y increases., decreases, or remains constant and the p r o p o r t i o n of females whose' young grow decreases w i t h i n c r e a s i n g exposure to d i e l d r i n ( F i g . 4). The young t h a t d i d not grow c o n s i s t a n t l y had l i t t l e or.no m i l k i n the gut. 5. V i a b i l i t y of Young - The F o s t e r Nursing Experiment: E f f e c t s of Pre  and Post-partum Maternal Exposure The most e f f i c i e n t way of p a r t i t i o n i n g the causation of the decreased v i a b i l i t y of young i s by f o s t e r n u r s i n g as long as the e f f e c t of a f o s t e r dam per se on v i a b i l i t y i s known. The % s u r v i v a l , l i f e expectancy and growth of f o s t e r young does not d i f f e r from those r a i s e d by t h e i r own dam (Fig 5 ). Since post-partum maternal f a c t o r s do not i n f l u e n c e v i a b i l i t y a l l p o s s i b l e comparisons can be made between the c o n t r o l s (0-0) and the treatments (0-53, 53-0 and 53-53) - the f i r s t number r e f e r s to dose the female was r e c e i v i n g w h i le the young were i n utero and the second number r e f e r s to the dose r e c e i v e d by the female on which the young were nu r s i n g . Despite the apparent marked d i f f e r e n c e i n s u r v i v a l ( F i g . 6) between groups there i s no s t a t i s t i c a l s i g n i f i c a n c e to the d i f f e r e n c e s (G = 7.82, df = 4, P = 0.063). S i m i l a r l y the G t e s t s f o r p a i r e d comparisons i n d i c a t e no d i f f e r e n c e s . However the l i f e expectancy index does r e v e a l s i g n i f i c a n t d i f f e r e n c e s i n v i a b i l i t y ( F i g . 7). The o v e r a l l a n a l y s i s of variance i s s i g n i f i c a n t (F = 156.75, P<10~6). Scheffe's t e s t r e v e a l s t h a t the l i f e expectancy i s d i f f e r e n t between a l l groups (minimum F = 7.81 which w i t h 3 and 285 df r e s u l t s i n P<0.01) except f o r the 0-53 and 53-53 comparison (F =0.40, P>0.10) B a r t l e t t ' s t e s t casts some doubt on the v a l i d i t y of the comparisons as the p r o b a b i l i t y of homogenous variances i s l e s s than 10~6. Fig 5 The effect of a foster dam on the survival, life expectancy, and growth of young rats. 50 I. 40 g> a> * 30 >» TJ O m o 20 10 • 8 * e • 8 12 16 A g e (days) 2 0 Fig.6 The survival of young rats born to females exposed to 0 or 53ppm dietary dieldrin during gestation (I'st number) and nursed on dams exposed to the same doses during lactation (2'nd number). A g e ( d a y s ) Fig. 7 The life expectancy of young rats born to females exposed to 0 or 53ppm dietary dieldrin during gestation (I'st number) and nursed on dams exposed to the same doses during lactation (2'nd number) 20 a> at +i |5 cn" >» o TJ u o a> Q. X LU 10 ro ^^^^ 23-dtUI J o-o 53-0 0-53 53-53 29 The growth of the young i n the four groups i s presented i n F i g . 8; the body weights were c a l c u l a t e d as before. Fisher's t t e s t i n d i c a t e s that on no day does the weight d i f f e r between groups despite the f a c t that the young of the treatment groups are co n s i s t a n t l y smaller than the controls (for comparisons between a l l four groups maximum t was found to be t = 1.23 df.= 15 - f o r groups 0-0 and 53-53 on day 4 - and f o r groups 0-0 and 53-0 a f t e r day 7 maximum t i s t = 1.11, df = 23 on day 14). There are apparently no e f f e c t s on the growth of young as was the case i n the other experiment (Fig. 4). However, as before there appeared to be diff e r e n c e s i n the growth responses of e n t i r e l i t t e r s and i t i s c l e a r the young of some l i t t e r s grew while others d i d not (Table VI). The female then a f f e c t s the growth of her l i t t e r . The growth response of the young i s not dependent upon the exposure of the dam during gestation but i s greatly a f f e c t e d by the dam's exposure during l a c t a t i o n . C l a s s i f y i n g the data i n t o a 2 x 2 contingency table 2 y i e l d s a X =00 because of the 0 i n one c e l l . Moreover i f the body weight data i n F i g . 8 are pooled depending upon the post-partum exposure ( i e . 0-0 with 53-0 and 0-53 with 53-53) and t tests conducted the 2 groups d i f f e r from days 2 to 4 (minimum t = 5.26, df = 52, P 0.01); the weight does not d i f f e r at b i r t h or on days 5 or 6 but t h i s l a t t e r e f f e c t i s probably due to the small sample s i z e of 53 ppm post-partum young because of mor t a l i t y . The young being r a i s e d by females exposed to 53 ppm had l i t t l e or no milk i n t h e i r gut. 6. Behavior of Exposed Females during the Raising of Young The % frequency with which the 15 behavior patterns (see Methods p 8 and Appendix I) were shown by exposed and unexposed females i s presented i n Table VII. There i s no reason to believe that d i e l d r i n caused the females F i g 8 The growth of suckling rats born to females exposed to 0 or 5 3 ppm dietary dieldrin during gestation (I'st number) and nursed on females exposed to the some doses during gestation (2'nd number). 31 T a b l e V I . The growth r e s p o n s e s o f t h e l i t t e r s o f f e m a l e s exposed t o 0 o r 53 ppm d i e t a r y d i e l d r i n d u r i n g l a c t a t i o n . D i e t a r y C o n c e n t r a t i o n d u r i n g L a c t a t i o n - 0 53 D i e t a r y C o n c e n t r a t i o n d u r i n g G e s t a t i o n 0 53 0 53 Net Growth o f L i t t e r Mates 15 15 6 6 L i t t e r Mates M a i n t a i n e d B i r t h W e i g h t 0 0 4 4 Net D e c l i n e i n Body 0 0 5 5 W eight o f L i t t e r Mates 32 T a b l e VI1^ ' The f r e q u e n c y o f o c c u r r e n c e o f b e h a v i o r p a t t e r n s shown by f e m a l e s w i t h young and exposed t o d i e t a r y d i e l d r i n . % F r e q u e n c y B e h a v i o r P a t t e r n Unexposed Exposed Females Females A g g r e s s i v e B i t i n g o f Young 0.0 ) 0.0 ) K i c k i n g o f Young 0.0 ) 0. 0 0.0 ) 0.0 H i t t i n g o f Young 0.0 ) 0.0 ) M a i n t e n a n c e o f S e l f Grooming o f S e l f D r i n k i n g F e e d i n g 9.6 ) 1.7 ) 4.6 ) 15. 9 2.8 ) 0.0 ) 5.5 ) 8.3 M a t e r n a l N u r s i n g S t a n d i n g : o v e r Young 69.0 ) 3.8 ) 76.3 ) 3.0 ) L i c k i n g Anus' o f Young 1.8 ) 76. 4 2.4 ) 83.2 H o l d i n g o f Young 0.2 ) 0.3 ) S m e l l i n g : Young o r N e s t 1.6 ) 1.2 ) O t h e r s P l a y i n g 3.0 ) 1.6 ) D i g g i n g L o c o m o t i o n 1.7 ) 0.1 ) 7. 5 1.2 ) 0.0 ) 8.5 S t a n d i n g away f r o m Young 2.8 ) 5.7 ) 3 3 t o become a g g r e s s i v e towards t h e i r l i t t e r s o r t o d e c r e a s e t h e amount o f m a t e r n a l c a r e . I n p a r t i c u l a r t h e r e i s no d i f f e r e n c e i n t h e amount o f N u r s i n g . I t i s i m p o r t a n t t o r e a l i z e , however, t h a t N u r s i n g does n o t i m p l y l a c t a t i o n o r s u c k l i n g i t m e r e l y means t h a t t h e female was i n t h e n o r m a l n u r s i n g p o s i t i o n o v e r t h e young. 34 IV. DISCUSSION 1. Exposure to D i e l d r i n and Health of the Animals The rats i n t h i s study were exposed to 0, 13, 25, 37 or 53 ppm dietary d i e l d r i n f o r 44 weeks with no apparent i l l e f f e c t s . Minor and t r a n s i t o r y changes i n the behavior of females on the two highest doses were noticed and the r e s u l t s suggest that the d i e l d r i n may have stimulated an increase i n food consumption i n the female. Gross body weight i s a crude index of health commonly used i n tox-i c o l o g i c a l studies (Loomis, 1968) and the r e s u l t s of t h i s study i n d i c a t e that d i e l d r i n i n doses up to 53 ppm have no e f f e c t on the growth of e i t h e r sex i n the r a t . Treon and Cleveland, (1955) , Coulson and McCarthy, (1963) , and Walker e_t al_. , (1969) , employing s i m i l a r doses of d i e l d r i n and/or a l d r i n had i d e n t i c a l r e s u l t s and Borgmann -fet; a l . , (1952b) , have shown that the r a t i s not aff e c t e d by these compounds i n doses as high as 150 ppm. However, B a l l et a l . , (1953) reported the p e c u l i a r e f f e c t on an increased rate of growth i n the female r a t caused by 20 ppm a l d r i n . A s i m i l a r e f f e c t was noted by Treon et a l . , (1955) who claim that growth i n the male r a t was decreased by 5 and 25 ppm endrin (the endo-endo stereoisomer of d i e l d r i n ) but unaffected by doses of 1 or 50 ppm; the female was unaffected at a l l doses. Contradictory e f f e c t s of 25 ppm a l d r i n or d i e l d r i n on the dog have been reported by Kitselman'(1951) and Kitselman and Borgmann(1952). Only r a r e l y has the e f f e c t of an organochlorine i n s e c t i c i d e on food intake been studied, probably because of the d i f f i c u l t y of obtaining v a l i d data. Food intake i n mammals i s generally thought to be unaffected by low doses and decreased by high doses of organochlorines; f or example, the r a t does not decrease food intake u n t i l concentrations of d i e l d r i n are over 100 ppm (Borgmann e_t a l . , 1952b) and the cow withstands dietary d i e l d r i n at 35 75 ppm quite s u c c e s s f u l l y (Gannon et a l . , 1959). However, the r e s u l t s suggest that d i e l d r i n stimulates the female r a t to increase food con-sumption i n a dose-dependent manner but has no e f f e c t on the male: Increased food intake i s unusual and has only been reported by B a l l e_t a l . , (195 3) i n conjunction with increased growth. Moreover a sex d i f f e r e n c e has not been reported before although the growth e f f e c t s described by Treon e_t a l . , (1955) can be explained by a s i m i l a r mechanism Dietary d i e l d r i n at concentrations of up to 53 ppm d i d not induce . symptoms of acute poisoning. This i s consistant with the work of Borgmann et a l . , (1952a,b) who demonstrated that 100 ppm i s the minimum dose which w i l l cause acute poisoning. S i m i l a r l y endrin does not increase mortality at doses below 50 ppm (Treon et a l . , 1955). The r a t i s apparently the species most r e s i s t a n t to dietary double r i n g cyclodienes as the other species tested show acute poisoning i n the concentration range of 5-25 ppm (monkey, mouse, dog, r a b b i t , - Hodge et a l . , 1967; deer mouse - Morris, 1968). The females on the d i e t s containing 37 or 53 ppm d i e l d r i n showed an increase i n e x c i t a b i l i t y and a c t i v i t y during the week 7-10 of exposure; the males were unaffected. This sex d i f f e r e n c e i s probably r e l a t e d to the greater a b i l i t y of the male to detoxify d i e l d r i n as evidenced by the d i f f e r e n c e s i n the acute LD50. The period of changed behavior i s s l i g h t l y l a t e r than the periods of peak t i s s u e storage of d i e l d r i n (Coulson and McCarthy, 1963) and probably r e f l e c t s a temporary increase i n the amount of d i e l d r i n i n the metabolic pool. S i m i l a r but permanent changes i n behavior were caused i n the CFW r a t by 3 months exposure to 10 ppm d i e l d r i n (Walker et a l . , 1969) and i n the Carworth r a t by 50 ppm endrin (Treon et a l . , 1955). Both these authors noted occasional convulsions i n t h e i r animals which were not observed i n t h i s study. 36 In.summary, exposure to d i e t a r y d i e l d r i n i n concentrations of up to 53 ppm d i d not appear to have caused any i l l e f f e c t s on the male and had minimal e f f e c t s on the female, causing minor changes i n n u t r i t i o n a l physiology and temporary changes i n behavior. 2. E f f e c t s on Reproductive Capacity Reproductive c a p a b i l i t y can be considered as 3 e m p i r i c a l l y d i s t i n c t phenomena: (a) f e r t i l i t y , or the a b i l i t y to produce young, (b) fecundity, or the number of young produced, (c) v i a b i l i t y of the young produced. The e f f e c t s of chronic exposure to d i e l d r i n on reproduction i s best con-sidered i n terms of these phenomena because such an approach delineates the area(s) where reproduction i s a f f e c t e d and allows inferences to be made as to the mechanisms involved. The various mechanisms which can be the cause of decreased reproductive a b i l i t y i n any of the three areas are presented i n Table VIII. C l e a r l y , there are mechanisms which are very general and may cause f a i l u r e i n any or a l l of the three phenomena, for example, genetic damage or lowered l e v e l s of the sex hormones. On the other hand there are mechanisms which are very s p e c i f i c to one phenomenon, induced maternal aggression for example. Judicious i n t e r p r e t a t i o n of the combinations of phenomena which are a f f e c t e d and the s e v e r i t y of the e f f e c t ( s ) can suggest the mechanisms involved. Let us consider f o r a moment the two mechanisms which may cause a l l of the phenomena. There i s l i t t l e reason to b e l i e v e that d i e l d r i n causes genetic damage. I t has been claimed to cause some questionable changes i n the DNA and RNA of rats and chicks (Daugherty et a l . , 1962) but these r e s u l t s have not been substantiated; s i m i l a r l y there i s no evidence i n the 37 Table VIII. The possible causation of d i e l d r i n induced reproductive f a i l u r e i n rats (1) Genetic damage (2) Germ c e l l or oocyte m o r t a l i t y (3) Decreased sex hormone l e v e l s (a) Decreased production (b) Increased metabolism ^ As f o r ^ (1) Decreased ovulation (a) Germ c e l l or oocyte mortality (b) Decreased sex hormone l e v e l s (c) Gonadotroph imbalance (2) Increased i n t r a - u t e r i n e m o r tality (a) Genetic damage (b) Decreased sex hormone l e v e l s (c) Acute poisoning of embryos (1) Genetic damage Decreased F e r t i l i t y ? Decreased Fecundity ? Decreased V i a b i l i t y of Pre-Weaning Young f (1) Genetic damage (2) Starvation (a) Maternal neglect (b) Decreased l a c t a t i o n (i) Decreased sex hormone l e v e l s ( i i ) "Blockage" of lactogenic hormone or oxytocin (3) I n f a n t i c i d e i e . increased maternal aggression (4) Teratogenic defects cf (1) Genetic damage 38 l i t e r a t u r e which suggests that t h i s compound induces chromosomal breakage or i n t e r f e r e s with chromosome mechanics. However, on the other hand i t i s very possible that d i e l d r i n causes decreased hormone l e v e l s . Decreased hormonal l e v e l s can r e s u l t from a decrease i n production and/or an increase i n metabolism. Decreased production would be a complex phenomenon i n v o l v i n g a d i r e c t attack on the hormone producing c e l l s or an imbalance i n gonadotroph l e v e l s ; there i s no evidence i n the l i t e r a t u r e to suggest that e i t h e r of these i s occurring. This leaves increased metabolism. I t has long been known that drugs stimulate t h e i r own metabolism by inducing hepatic enzymes. Hart and Fouts (1963) suggested that the organochlorines would induce s i m i l a r enzymes and t h i s was subsequently shown to be true by Hart and Fouts (1965) and others. Trivus (1965) and Peakall (1967) demonstrated that the normal substrates f o r these enzymes were the sex s t e r o i d s which suggested that small l e v e l s of the organochlorines could cause reproductive f a i l u r e . This was confirmed i n 1967 by Conney et a l . , who showed that chlordane caused a simultaneous reduction i n l i t t e r s i z e and an increase i n s t e r o i d hydroxylase a c t i v i t y . The threshold l e v e l s which cause enzyme induction have been determined f o r most of the common organochlorines and the l e v e l s f o r the double r i n g cyclodienes are the lowest (Street e t a l . , 1969). D i e l d r i n has the lowest threshold of a l l and i n the r a t i t i s approximately 1.0 ppm ( G i l l e t and Chan, 1968). There can be l i t t l e doubt that the animals i n t h i s study were experiencing enzyme induction. 3. E f f e c t s on the Reproductive Capacity of the Male The r e s u l t s i n d i c a t e that exposing male rats to dietary d i e l d r i n i n concentrations up to 53 ppm does not a f f e c t t h e i r f e r t i l i t y , the s i z e or sex composition of the l i t t e r s they s i r e or the v i a b i l i t y of the young they s i r e . These r e s u l t s suggest that d i e l d r i n does not cause genetic damage, 39 net lowering of androgen l e v e l s , or germ c e l l or sperm m o r t a l i t y . No work has been done on the e f f e c t s of the double r i n g cyclodienes on reproductive capacity of the male of any species and l i t t l e has been done on other organochlorines. The data of Ambrose e_t a l . , (1953) suggest: that 320 ppm chlordane does not a f f e c t f e r t i l i t y i n the male r a t . Huber ( 1 9 6 5 ) , studying the e f f e c t of Kepone on the house mouse, found a decrease i n the l i t t e r s i z e of unexposed females bred by treated males but h i s t o -l o g i c a l examination of t h e i r testes revealed normal spermatogenesis and i n t e r s t i t i a l c e l l content. McFarland and Lacy (1969) using Kepone on the Japanese q u a i l found that i t greatly enlarged the testes although the increase was i n s i g n i f i c a n t s t a t i s t i c a l l y ; h i s t o l o g y revealed normal spermatogenesis and a s l i g h t increase i n the number of i n t e r s t i t i a l c e l l s . A l b e r t (1962) showed that 1000 ppm DDT caused no h i s t o l o g i c a l changes i n the testes of the chicken despite the f a c t that t h i s dose precludes reproduction i n t h i s species (Ruben et al_. , 1947) . The present evidence suggests that the organochlorines do not disrupt the male reproductive system and therefore the disturbances these compounds are known to cause are mediated through the female. 4. E f f e c t s on the Reproductive Capacity of the Female (a) F e r t i l i t y The r e s u l t s show that dietary d i e l d r i n i n concentrations of up to 53 ppm has no e f f e c t on the f e r t i l i t y or estrous cycle of the female; my data suggest that the r a t i s i n s e n s i t i v e to the double r i n g cyclodienes. This i s i n d i r e c t opposition to the r e s u l t s of Treon and Cleveland (1955) who, using a 3 generation breeding t r i a l , found that d i e l d r i n at 2.5 ppm had a " s l i g h t e f f e c t " on the pregnancy rate which was "more pronounced" at 12.5 and 25.0 ppm. The v a l i d i t y of t h e i r r e s u l t s , p a r t i c u l a r l y at the low doses, i s questionable,however, since no s t a t i s t i c a l analyses were performed 40 and since the e f f e c t at 2.5 and 12.5 ppm "tended to disappear a f t e r the f i r s t generation". Assuming that t h e i r e f f e c t s are r e a l , the dichotomy between our r e s u l t s probably i s caused by a diffe r e n c e i n the age of the animal at the time of exposure. Treon and Cleveland exposed t h e i r animals from week 3-28 of age and mine were exposed from week 25-69; s i m i l a r l y the breeding t e s t s were conducted at d i f f e r e n t ages. The s i g n i f i c a n c e of a pos s i b l e age e f f e c t i s be t t e r appreciated when i t i s r e a l i z e d that young animals have a much lower threshold l e v e l f o r enzyme induction than do older animals (Kuntzman e_t a l . , 1964) . B a l l et_ a l . , (1953) found an a l d r i n caused t r a n s i t o r y decrease i n the number of p o s i t i v e estrus' which can also be explained as an age e f f e c t . The s i g n i f i c a n c e of these various r e s u l t s i s that even high doses of d i e l d r i n and probably the other double r i n g cyclodienes w i l l not reduce the pregnancy rate of a r a t population to zero. My r e s u l t s also suggest that d i e l d r i n does not cause genetic damage, germ c e l l or ova m o r t a l i t y or a net decrease i n the hormone l e v e l s before gestation. (b) Size and Sex Composition of the L i t t e r There i s no s t a t i s t i c a l evidence that d i e l d r i n i n doses up to 53 ppm a l t e r s the sex r a t i o or reduces the s i z e of a l i t t e r a female w i l l bear. This i s i n agreement with Treon and Cleveland (1955) who found that doses up to the maximum they used (25 ppm) had.no e f f e c t on l i t t e r s i z e . These data suggest that d i e l d r i n does not increase i n t r a - u t e r i n e mortality or decrease ovulation i n the r a t . Despite the lack of s i g n i f i c a n t r e s u l t s there i s reason to believe that exposed females had lowered gestagen t i t r e s . The young of treated females were often born with gross hematomas i n the head and caudal region, 41 the frequency and s e v e r i t y of which increased with in c r e a s i n g dose. Such a condition i s known to r e s u l t from hormonal d e f i c i e n c i e s during gestation (Kroc et a l . , 1959)! and i s associated with a decrease i n l i t t e r s i z e ; the l i t t e r s i z e of the exposed females was c o n s i s t a n t l y smaller than that of the c o n t r o l females. In view of t h i s ^ h e e f f e c t of d i e l d r i n and the other double r i n g cyclodienes on l i t t e r s i z e i n the r a t should be re-examined. Even i f d i e l d r i n does reduce the l i t t e r size,the\.:.effeet i s c e r t a i n l y small, even at high doses,and at the residue l e v e l s l i k e l y to be found i n the w i l d the reduction would be i n s i g n i f i c a n t . As such the fecundity of a r e a l population i s u n l i k e l y to be a f f e c t e d . On the other hand the mouse shows a d e f i n i t e dose dependent decrease i n the l i t t e r s i z e caused by d i e l d r i n and other organochlorines (Deichmann and Keplinger, 1966; Good and Ware, 1969). However, the mechanism i s probably d i f f e r e n t from that of the r a t . The mouse i s known to have a poor induction response to the.organo-chlorines i n comparison to the r a t (Kuntzmann et_ a_l. , 1964)- such that i t w i l l have r e l a t i v e l y high concentrations of blood organochlorines when exposed to the same dose. Backstrom et a l . , (1965) have demonstrated that d i e l d r i n r e a d i l y crosses the mouse placenta such that i_n utero f e t a l poisoning could e a s i l y and probably does occur. The r a t on the other hand with i t s greater enzyme systems probably d e t o x i f i e s enough of the compound to prevent acute poisoning of the embryos but i n doing so reduces the l e v e l s of progesterone and estrogen. (c) V i a b i l i t y of the Young The r e s u l t s i n d i c a t e that the % s u r v i v a l of pre-weaning young born to exposed females i s reduced i n a dose-dependent manner, although t h i s reduction i s s t a t i s t i c a l l y i n s i g n i f i c a n t . However, the l i f e expectancy of these young 42 does d e m o n s t r a t e t h a t t h e r e d u c t i o n i n v i a b i l i t y i s s t a t i s t i c a l l y s i g n i f i c a n t . The t h r e s h o l d dose f o r r e d u c e d v i a b i l i t y i s l e s s t h a n 10 ppm, t h e "LD50" i s a p p r o x i m a t e l y 17 ppm and v i a b i l i t y i s e f f e c t i v e l y 0 a t doses >25 ppm. The mean age a t d e a t h i s a p p r o x i m a t e l y 5 days and t h i s does n o t o c c u r a t a t i m e d i f f e r e n t f r o m n o r m a l p r e - w e a n i n g m o r t a l i t y . M oreover t h e mean age a t d e a t h i s much l e s s t h a n 14 days w h i c h i s when t h e young s t a r t e a t i n g s o l i d f o o d s u c h t h a t t h e y a r e n o t d y i n g o f a c u t e p o i s o n i n g f r o m t h e f e e d . I t i s c l e a r t h a t e x p o s u r e of t h e f e m a l e t o d i e l d r i n d u r i n g g e s t a t i o n and l a c t a t i o n r e d u c e s t h e v i a b i l i t y o f h e r o f f s p r i n g b u t i t i s n o t c l e a r w h e t h e r t h i s i s c a u s e d by t h e e x p o s u r e d u r i n g g e s t a t i o n ( h e r e a f t e r c a l l e d " p r e - p a r t u m " ) , l a c t a t i o n ("post-partum") o r an i n t e r a c t i o n o f t h e two. T h i s d e c r e a s e d v i a b i l i t y o f p r e - w e a n i n g young has been c o n s i s t a n t l y r e p o r t e d by t h e few i n v e s t i g a t o r s who have l o o k e d f o r i t . E v e r y s p e c i e s examined r e g a r d l e s s o f t h e compound u s e d and r e g a r d l e s s o f i t s e f f e c t s on f e r t i l i t y o r f e c u n d i t y has shown t h i s r e s p o n s e . However, some o f t h e o r g a n o c h l o r i n e s r e q u i r e doses u n l i k e l y t o be o b t a i n e d even i n t h e d i e t s o f c a r n i v o r e s (eg. DDT a t 250 ppm and c h l o r d a n e a t 100 ppm a r e r e q u i r e d f o r m i c e ; Deichmann and K e p l i n g e r , 1966) b u t t h i s i s n o t t r u e o f t h e d o u b l e r i n g c y c l o d i e n e s . T r e o n and C l e v e l a n d (1955) r e p o r t e d t h a t 2.5 ppm d i e l d r i n (and a l d r i n ) had "moderate e f f e c t s " on t h e s u r v i v a l o f young w h i l e 12.5 and 25.0 ppm had " s e v e r e e f f e c t s " . S i m i l a r l y , Deichmann and K e p l i n g e r (1966) c l a i m e d t h a t 25 ppm d i e l d r i n (and a l d r i n ) had " v e r y s e v e r e e f f e c t s " w h i l e 10 ppm had "some e f f e c t " . My d a t a and t h e d a t a o f t h e s e a u t h o r s i s c o m p a r a b l e , s u g g e s t i n g l i t t l e d i f f e r e n c e i n r e s p o n s e between t h e two s p e c i e s . The r a t and t h e mouse appear t o be more t o l e r a n t t o t h e s e compounds t h a n t h e dog where 3.0 ppm a l d r i n r e d u c e d s u r v i v a l by 60% ( K i t s e l m a n , 1953). The d e e r mouse has been shown t o s u f f e r a s i g n i f i c a n t 30% d e c r e a s e i n s u r v i v a l o f young c a u s e d by 4.0 ppm e n d r i n ( M o r r i s , 1968); w h e t h e r t h e a p p a r e n t 43 d i f f e r e n c e between the other two rodents and the deer mouse i s a species dif f e r e n c e or a chemical d i f f e r e n c e i s not c l e a r . I t i s widely known that a l l of the organochlorines are secreted i n the milk i n qua n t i t i e s proportional to the amount ingested and i t has been assumed that the decreased s u r v i v a l of pre-weaning young i s caused by acute poisoning from the compound i n the milk. There are however, other possible post-partum causes and pos s i b l e pre-partum causes as w e l l (Table V I I I ) . This f a c t has also been recognized by Harris et al_. , (1965) whose unpublished data has been summarized obscurely i n Appendix 10 of Hodge ejt al_. , (1967) . Harris et a l . found that lambs born to unexposed ewes died when nursed on ewes re c e i v i n g 25 ppm dietary d i e l d r i n and conversely lambs born to exposed ewes s t i l l died when nursed on dams r e c e i v i n g no d i e l d r i n . The e f f e c t s of pre and post-partum exposure of "the female to d i e l d r i n on the v i a b i l i t y of her o f f s p r i n g were in v e s t i g a t e d by the f o s t e r nursing experiment. Since the 0-0 and 53-0 groups d i f f e r there i s a pre-partum component i n the causation. Since 0-53 and 53-53 do not d i f f e r the post-partum component swamps the pre-partum and i s therefore the more important. The pre-partum causation i s responsible f o r 30% of the t o t a l reduction i n s u r v i v a l at the 53 ppm dose used and as such the pre-partum e f f e c t i s l i k e l y to be experienced even at low doses. The proximate cause of the pre-partum induced., mortality must involve e i t h e r genetic damage, which i s u n l i k e l y f o r the reasons previously discussed, or a teratogenic defect (Table V I I I ) . The l a t t e r probably involves subtle disruptions of metabolism since no gross defects were observed, the weight at b i r t h i s not affe c t e d and the induced deaths occur at the same time as the normal deaths. The nature of the defect i s com-p l e t e l y unknown but could involve any of the following e f f e c t s known to be caused by d i e l d r i n : a l t e r e d N metabolism (Annau et_ al_. , 1952); a l t e r e d 44 a c t i v i t y of carbonic anhydrase, several dehydrogenoses and several esterases (Verrett and Desmond, 1959; Hosein and Proulx, I 9 6 0 ; Gowdey and Stavraky, 1955) ; decreased consumption (Crevier e_t al_. , 1954) ; decreased transport i n RBC's (Weikel et a l . , 1 9 5 8 ) ; or abnormal transport systems (Daugherty et a l . , 1962) . The proximate cause(s) of the post-partum component of mo r t a l i t y could involve any combination of acute poisoning v i a the milk, s t a r v a t i o n r e s u l t i n g from decreased l a c t a t i o n or maternal neglect, or i n f a n t i c i d e from increased maternal aggression. While die l d r i n - i n d u c e d changes i n the maternal behavior of the r a t can be postulated to occur,since the compound i s known to disrupt the function of the CNS (Gowdey et a l . , 1954) and has been shown to change the behavior of s h a r p t a i l grouse (McEwan and Brown, 1966) and sheep (Van Gelder e_t a l . , 1969), the r e s u l t s of t h i s study i n d i c a t e that such changes do not occur. Morris (1968) claims, without data, a s i m i l a r s i t u a t i o n e x i s t s i n the deer mouse. The r e s u l t s suggest that both acute poisoning from d i e l d r i n i n the milk and s t a r v a t i o n from decreased l a c t a t i o n are operating. At the higher doses a l l the o f f s p r i n g die but they may or may not grow u n t i l death. This growth response i s female dependent i n the sense that the growth of littermates i s consistant and the number of females whose l i t t e r s grow decreases with increasing dose. Moreover,the young that do not grow show l i t t l e or no milk i n the gut. The death of the young who grow can only be explained by acute poisoning while decreased l a c t a t i o n probably plays at l e a s t a part i n the mortality of the young who do not grow. Deichmann and Keplinger (1966) have claimed that 10 ppm d i e l d r i n r e s u l t e d i n a decrease i n an undefined l a c t a t i o n index i n the house mouse. DecreasedLlactation' i s ..also consistant with decreased sex hormone l e v e l s for which, as discussed previously, there i s some evidence of occurrence. 45 I n t h e r a t , e s t r o g e n and p r o g e s t e r o n e a r e n e c e s s a r y f o r mammary g l a n d development and t h e i n i t i a t i o n o f m i l k s e c r e t i o n f r o m t h e a l v e o l a r c e l l s (Cowie and F o l l e y , 1961). As such d i e l d r i n i n d u c e d d e c r e a s e s i n t h e s e hormones and c o u l d m a n i f e s t i t s e l f i n d e c r e a s e d m i l k p r o d u c t i o n . There i s however t h e p o s s i b i l i t y t h a t d e c r e a s e d l a c t a t i o n r e s u l t s f r o m d i e l d r i n d i r e c t l y a f f e c t i n g ' t h e b r a i n . I n t h e r a t l a c t o g e n i c hormone i s n e c e s s a r y f o r mammary g l a n d development and t h e i n i t i a t i o n and maintenance o f m i l k s e c r e t i o n ; o x y t o c i n I s n e c e s s a r y t o i n d u c e r e l e a s e o f t h e m i l k f rom t h e g l a n d and may a l s o cause i n c r e a s e d m i l k s e c r e t i o n by s t i m u l a t i n g t h e r e l e a s e o f t h e l a r g e amount o f l a c t o g e n i c hormone e f f e c t e d a t t h e o n s e t o f s u c k l i n g (Cowie and F o l l e y , 1961). The r e l e a s e o f t h e s e p i t u i t a r y f a c t o r s i s a p p a r e n t l y u n d e r t h e c o n t r o l o f complex CNS r e f l e x e s c o n t a i n i n g both. . a d r e n e r g i c and c h o l i n e r g i c pathways and can be a l t e r e d by f o r e i g n compounds a f f e c t i n g t h e s e pathways ( G r o s v e n e r and T u r n e r , 1957 and '58; M e i t e s , 1959). D i e l d r i n i s c h o l i n e r g i c and m e d i a t e s i t s a f f e c t s t h r o u g h t h e b r a i n (Gowdey e t a l . , 1954). The i m p l i c a t i o n i s o b v i o u s . I n summary, t h e e f f e c t o f s m a l l doses o f d i e t a r y d i e l d r i n on t h e r e p r o d u c t i v e c a p a c i t y o f t h e f e m a l e r a t i s s e r i o u s . The f e r t i l i t y and f e c u n d i t y a r e u n a f f e c t e d by doses as h i g h as 53 ppm b u t t h e v i a b i l i t y o f th e young i s r e d u c e d by a t h r e s h o l d dose o f l e s s t h a n 13 ppm. 46 V. SUMMARY (1) Groups of post-pubertal rats were exposed to 0 , 13, 25 , 37 or 53 ppm dietary d i e l d r i n for 44 weeks. In the female t h i s r e s u l t e d i n a dose dependent increase i n food intake and a t r a n s i t o r y h y p e r e x c i t a b i l i t y i n those r e c e i v i n g 37 and 53 ppm; t h i s l a t t e r e f f e c t coincided with the s t a r t of the decrease i n the storage of d i e l d r i n i n the t i s s u e s . No other e f f e c t s of chronic t o x i c i t y were observed i n e i t h e r sex. The animals were tested for reproductive success at 4 i n t e r v a l s during exposure. Other experiments were performed to elucidate the mechanisms of reproductive f a i l u r e . ( 2 ) D i e l d r i n had no e f f e c t on the reproductive success of the male. The number of l i t t e r s s i r e d by exposed males and the number, sex and v i a b i l i of young i n these l i t t e r s d i d not d i f f e r from the controls. This suggests that d i e l d r i n does not cause genetic damage, lowered androgen t i t r e s , or m o r t a l i t y of the sperm or germ c e l l s . (3) D i e l d r i n had no e f f e c t on the exposed females' estrous cycles, f e r t i l i t y or sex of o f f s p r i n g . This suggests that d i e l d r i n does not cause genetic damage, mortality of the eggs or germ c e l l s or decreases i n the pre-gestational hormone t i t r e s . (4) D i e l d r i n d i d not cause s i g n i f i c a n t decreases i n the si z e of l i t t e r s born to exposed females.. This suggests that d i e l d r i n was not causing i n utero poisoning of the embryos or d e f i c i e n c i e s of the g e s t a t i o n a l hormones. However the treatment l i t t e r s were co n s i s t a n t l y smaller than the controls and the young had defects known to be associated with hormone def i c i e n c y such that the e f f e c t of t h i s compound on l i t t e r s i z e i n the r a t should be re-examined. 47 (5) D i e l d r i n decreases the v i a b i l i t y of o f f s p r i n g born to exposed females i n proportion to the dose received by the dam; the threshold dose i s less than 10 ppm and the LD50 i s 17 ppm. The mor t a l i t y induced by d i e l d r i n occurs at the same time as normal pre-weaning m o r t a l i t y . (6) Exposure of the dam to d i e l d r i n during gestation increases the mort a l i t y of the young a f t e r b i r t h . This suggests that d i e l d r i n causes some manner of defect and p o s s i b i l i t i e s are discussed. (7) Exposure of the dam to d i e l d r i n during l a c t a t i o n increases the mort a l i t y of "the young; the causation of t h i s e f f e c t does not i n t e r a c t with the pre-partum defect. D i e l d r i n does not change the dams' behavior such that increased maternal aggression or neglect are not the causes of t h i s e f f e c t . The growth responses of the young i n d i c a t e that i n at l e a s t some females d i e l d r i n decreases l a c t a t i o n and st a r v a t i o n causes part of the mortal i t y . I t i s suggested that acute poisoning from d i e l d r i n secreted i n the milk i s the major cause of mortality.. (8) The probable p h y s i o l o g i c a l mechanisms of these e f f e c t s are discussed. 48 VI. LITERATURE CITED Anon. 1963. The Use of P e s t i c i d e s . Presidents' S c i . Adv. Committee. U.S. Government P r i n t i n g O f f i c e . Washington D.C. Al b e r t , T.F. 1962. E f f e c t of DDT on the sperm production of the domestic fowl. Auk 79: 104-107. Annau, E., H. Konst, and P.J.G. Plummer. 1952. Biochemical and h i s t o l o g i c a l changes i n the l i v e r of mice following feeding of the i n s e c t i c i d e a l d r i n . Can. J . Med. S c i . 30: 463-470. Ambrose, A.M., H.E. Christenson, D.J. Robbins, and L.J. Rather. 1953. T o x i c o l o g i c a l and pharmacological studies on chlordane. Arch. Ind. Hyg. Occupational Med. 7: 197-210. Backstrom, J.> E. Hansson, and S. Ullb e r g . 1965. D i s t r i b u t i o n of C!4-DDT and C l 4 - d i e l d r i n i n pregnant mice determined by wholebody autoradio-graphy. T o x i c o l . Appl. Pharmacol. 7: 90-96. B a l l , W.L., K. Kingsley, and J.W. S i n c l a i r . 1953. Observations on the t o x i c i t y of a l d r i n : 1.. Growth and estrus i n r a t s . Arch. Ind. Hyg. Occupational Medicine 7: 292-300. Bernard, R.F., and R.A. Gaertner. 1964. DDT and reproduction i n house mice. J . Mammal. 45: 272-276. Bitman, J . , H.C. C e c i l , S.J. Harris and G.F. F r i e s . 1969. DDT induces a decrease i n eggshell calcium. Nature 224: 44-46. Borgmann, A.R., C.H. Kitselmann, P.A. Dahm, J.E. Panaskie, and F.R. Dutra. 1952a. T o x i c o l o g i c a l studies of a l d r i n on small laboratory animals. Unpubl. rept. Kansas State College. c i t e d i n Hodge,. e_t al_. , 1967. Borgmann, A.R., C.H. Kitselmann, P.A. Dahm, J.E. Panaskie, and F.R. Dutra. 1952b. T o x i c o l o g i c a l studies of d i e l d r i n on small laboratory animals. Unpubl. rept. Kansas State College. c i t e d i n Hodge, et a l . , 1967. Conney, A.H., R.M. Welch, R. Kuntzman, and J . J . Burns. 1967. E f f e c t s of pe s t i c i d e s on drug and s t e r o i d metabolism. C l i n . Pharmacol. Therap. 8: 2-10. Coulson, D.M., and E.M. McCarthy. 1963. E f f e c t s of p e s t i c i d e s on animals and human beings. Report No. 13, Technical Report No. VI, Stanford Research I n s t i t u t e . 37 pp. Cowie, A.T. and S.J. F o l l e y . 1961. The mammary gland. In: Sex and  Internal Secretions. W.C. Young Ed. Williams and Wilkins, Co. Bai t . pp. 590-642. Crevi e r , M., W.L. B a l l , and K. Kingsley. 1954. Observations on the t o x i c i t y of a l d r i n . I I . Serum esterase changes i n rats following administration of a l d r i n and other chlorinated hydrocarbon i n s e c t i c i d e s . Arch. Ind. Hyg. Occupational Med. 9: 306-314. 49 C u e t o , C. 1960. C o l o r i m e t r i c d e t e r m i n a t i o n o f d i e l d r i n and i t s a p p l i c a t i o n t o a n i m a l f a t . J . A g r . Food Chem. 8: 273-276. C u e t o , C. and W.J. Hayes, J r . 1962. The d e t e c t i o n o f d i e l d r i n m e t a b o l i t e s i n human u r i n e . J . A g r . Food Chem. 10: 366-369. D a u g h e r t y , J.W., D.E. L a c y , and P. K o r t y . Some b i o c h e m i c a l e f f e c t s o f l i n d a n e and d i e l d r i n on v e r t e b r a t e s . A e r o s p a c e Med. 33: 1171-1176. de F a u b e r t Maunder, M.J., H. Egan, E.W. G o d l y , E.W. Hammond, J . Roburn, and J . Thompson. 1964. C l e a n - u p o f a n i m a l f a t s and d a i r y p r o d u c t s f o r the a n a l y s i s o f c h l o r i n a t e d p e s t i c i d e r e s i d u e s . A n a l y s t 89: 168-174. Deichmann, W.B. and M.L. K e p l i n g e r . 1966. E f f e c t s o f c o m b i n a t i o n s o f p e s t i c i d e s on r e p r o d u c t i o n i n m i c e . T o x i c o l . A p p l . P h a r m a c o l . 8: 337-338. F i t z h u g h , O. 1949. T o x i c i t y o f c h e m i c a l s i n f o o d s : L ong-term s t u d i e s . Food, D r u g , C o s m e t i c Law Q u a r t . 4: 423-425. Gannon, N., R.P. L i n k , and G.C. D e c k e r . 1959. I n s e c t i c i d e r e s i d u e s i n t h e m i l k o f d a i r y cows f e d i n s e c t i c i d e s i n t h e i r d a i l y r a t i o n . J . A g r . Food Chem. 7: 829-832. G i l l e t t , J.W. and T.M. Chan. 1968. C y c l o d i e n e i n s e c t i c i d e s as i n d u c e r s , s u b s t r a t e s and i n h i b i t o r s o f m i c r o s o m a l e p o x i d a t i o n . J . A g r . Food Chem. 16: 590-593. Good, E.E. and G.W. Ware. 1969. E f f e c t s o f i n s e c t i c i d e s on r e p r o d u c t i o n i n t h e l a b o r a t o r y mouse. IV: e n d r i n and d i e l d r i n . T o x i c o l . A p p l . P h a r m a c o l . 14: 201-203. Gowdey, C.W., A.R. Graham, J . J . S e g u i n , and G.W. S t a v r a k y . 1954. The p h a r m a c o l o g i c a l p r o p e r t i e s o f t h e i n s e c t i c i d e d i e l d r i n . Can. J . Biochem. P h y s i o l . 32: 498-503. Gowdey, C.W. and G.W. S t a v r a k y . 1955. A s t u d y o f t h e autonomic m a n i f e s t a t i o n s seen i n a c u t e a l d r i n and d i e l d r i n p o i s o n i n g . Can. J . Biochem. P h y s i o l . 33: 272-282. G r o s v e n e r , C.E. and C.W. T u r n e r . 1957. E v i d e n c e f o r a d r e n e r g i c and c h o l i n e r g i c components i n m i l k l e t - d o w n r e f l e x i n l a c t a t i n g r a t s . P r o c . Soc. E x p e r . B i o l , and Med. 95: 719-722. G r o s v e n e r , C.E. and C.W. T u r n e r . 1958. E f f e c t s o f o x y t o c i n and b l o c k i n g a gents on p i t u i t a r y l a c t o g e n d i s c h a r g e i n l a c t a t i n g r a t s . P r o c . Soc. E x p e r . B i o l , and Med. 97: 463-465. H a r r i s , L.E. , D.A. Greenwood, J . E . B u t c h e r , J.C. S t r e e t , J . L . Shupe, and C. Biddulph.- 1966. D i e l d r i n s t o r a g e i n sheep d u r i n g e x t e n d e d o r a l a d m i n i s t r a t i o n . P r e s e n t e d a t t h e Amer. Chem. Soc. M e e t i n g , S e p t . c i t e d i n Hodge, et_ a l . , 1967. H a r t , L.G. and J.R. F o u t s . 1963. DDT s t i m u l a t i o n o f d r u g m e t a b o l i s m . P r o c . Soc. Exp. B i o l , and Med. 114: 388-392. 50 Hart, L.G. and J.R. Fouts. 1965. Stimulation of hepatic microsomal drug metabolizing enzymes by DDT and i t s analogs. Arch. Exper. Pathol. Pharmacol. 249: 486-500. Heath, D.F. and M. Vandekar. 1964. T o x i c i t y and metabolism of d i e l d r i n i n ra t s . B r i t . J.. Industr. Med. 21: 269-279. Heath, R.G., J.W. Spann, and J.F. K r e i t z e r . 1969. Marked DDE impairment of Mallard reproduction i n c o n t r o l l e d studies. Nature 224: 47-49. Hickey, J . J . and D.W. Anderson. 1968. The chlorinated hydrocarbons and eggshell changes i n r a p t o r i a l and f i s h - e a t i n g b i r d s . Science 162: 271-273. Hodge, H.C. , A.M. Boyce, W.B. Deichmann, and H.E. K r a y b i l l . 1967. Toxicology, and no-effect l e v e l s of a l d r i n and d i e l d r i n . T o x i c o l . Appl. Pharmacol. 10: 613-675. Hosein, E.A. and P. Proulx. 1960. Chemical and b i o l o g i c a l analyses of brain t i s s u e preparation during the e p i l e p t i f o r m - l i k e a c t i v i t y of d i e l d r i n and other cerebral convulsants. J . Agr. Food Chem. 8: 428-431. Huber, J . J . 1965. Some p h y s i o l o g i c a l e f f e c t s of Kepone i n the laboratory mouse. T o x i c o l . Appl. Pharmacol. 7: 516-524. Kitselman, C.H. 1951. A comparative study of dogs as a susceptible species to sublethal doses of a l d r i n and d i e l d r i n . Report, Kansas State College Agr. Exper. Sta. Dec. Kitselman, C.H. 1953. Long term studies on dogs fed a l d r i n and d i e l d r i n i n sublethal dosages, with reference to the h i s t o p a t h o l o g i c a l findings and reproduction. J . Am. Vet. Med. Assoc. 123: 28-30. Kitselman, C.H. and A.R. Borgmann. 1952. A comparative study of the reaction of dogs as a susceptible species to sublethal doses of a l d r i n and d i e l d r i n . J . Am. Vet. Med. Assoc. 121: 383-385. Kroc, R.L., B.G. Steinetz and V.L. Beach. 1959. The e f f e c t of estrogens, progestagens, and r e l a x i n i n pregnant and non-pregnant laboratory rodents. Annals N.Y. Acad. S c i . 75: 942-980. Kuntzman, R., M. Jacobson, K. Schneidman, and A.H. Conney. 1964. S i m i l a r i t i e s between oxidative drug metabolizing enzymes and s t e r o i d hydroxylases i n l i v e r microsomes. J . Pharmacol. Exper. Therap. 146:-280-285. Kutney, J.P. 1969. Personal communication. Dept. of Chem. Univ. of B.C. Lehman, A.J. 1956. The minute residue problem. Q u a r t . B u l l . Assoc. Food and Drug O f f i c i a l s U.S.A. 20: 95-99. L i t c h f i e l d , J.R., J r . and F. Wilcoxon. 1949. A s i m p l i f i e d method of evaluating dose-effect experiments. J . Pharmacol. Exper. Therap. 92: 99-113. .51 Loomis, T.A. 1968. E s s e n t i a l s of Toxicology. Lea and Febiger Co. P h i l a d e l p h i a . 162 pp. McEwan, L.C. and R.L. Brown. 1966. Acute t o x i c i t y o f d i e l d r i n and malathion to wild s h a r p - t a i l e d grouse. J . W i l d l . Mgt. 30: 604-611. McFarland, L.Z. and P.B. Lacy. 1969. P h y s i o l o g i c and endocrinologic e f f e c t s of the i n s e c t i c i d e Kepone i n Japanese Q u a i l . T o x i c o l . Appl Pharmacol. 15: 441-450. Meites, J . 1959. Induction and maintenance of mammary growth and l a c t a t i o n i n rats with acetylcholine or epinephrine. Proc. Soc. Exper. B i o l , and Med. 100: 750-754. M o r r i s , R.D. 1968. E f f e c t s of endrin feeding on s u r v i v a l and reproduction i n the deer mouse. Can J . Zool. 46: 951-958. Nash, R.G. and E.A. Wilson. 1967. Persistence of chlorinated hydrocarbons i n the s o i l . Science 157: 924-926. P e a k a l l , D.B. 1967. P e s t i c i d e induced enzyme breakdown of steroids i n b i r d s . Nature 216: 505-506. Ruben, M., H.R. B i r d , N. Green, and R.H. Carter. 1947. T o x i c i t y of DDT to l a y i n g hens. Poult. S c i . 26: 410-413. Saha, J.G. 1969. S i g n i f i c a n c e of organochlorine i n s e c t i c i d e residues i n fresh plants as possible contaminants of milk and beef products. P e s t i c i d e Rev.' 26: 89-126. Scheffe, H. 1959. The A n a l y s i s of Variance. Wiley. N.Y.C. 477 pp. Snyder, D.B. 1963. The e f f e c t s of endrin on vole reproduction i n bluegrass meadows. Ph.D. t h e s i s . Ohio State Univ. Sokal, R.R. and F.J. Rohlf. 1969. Biometry. W.H. Freeman and Co. San Francisco. 776 pp. Steele, R.G.D. and J.H. T o r r i e . 1960. P r i n c i p l e s and procedures of s t a t i s t i c s . McGraw-Hill Co. Toronto. 481 pp. Street, J.C. , F.L. Mayer, and D.J. Wagstaff. 1969. E c o l o g i c a l s i g n i f i c a n c e of p e s t i c i d e i n t e r a c t i o n s . Ind. Med. Surg. 38: 91=100. Tayan, R. and T. Kemeny. 1969. Multigeneration studies on DDT i n mice. ^Food and Cosmetic T o x i c o l . 7: 215-222. Treon, J.F. and F.P. Cleveland. 1955. T o x i c i t y of c e r t a i n chlorinated hydrocarbon i n s e c t i c i d e s f o r laboratory animals with s p e c i a l reference to a l d r i n and d i e l d r i n . J . Agr. Food Chem. 3: 402-408. Treon, J.F. , F.P7 Cleveland and J . Cappel. 1955. T o x i c i t y of endrin f o r laboratory animals. J . Agr. Food Chem. 3: 842-848. T r i v u s , R.H. 1965. K i n e t i c s of drug and s t e r o i d oxidations by hepatic microsomes. Pharmacologist 7: p. 149. 52 Van Gelder, G.A., B.E. Sandler, W.B. Buck, J.B. Maland, and G.G. Karas. 1969. Behavioral and e l e c t r o p h y s i o l o g i c a l e f f e c t s of d i e l d r i n i n sheep. Ind. Med. Surg. 38: 111-114. V e r r e t t , M.J. and A.H. Desmond. 1959. I n h i b i t i o n of carbonic anhydrase by chlorinated i n s e c t i c i d e s . Pharmacologist 1: p 72. Walker, A.I.T., D.E. Stevenson, J . Robinson, E. Thorpe, and M. Roberts. 1969. The toxicology and pharmacology of d i e l d r i n - 2 years o r a l exposure of rats and dogs. T o x i c o l . Appl. Pharmacol. 15: 345-373. Ware, G.W. and E.G. Good. 1967. E f f e c t s of i n s e c t i c i d e s on reproduction i n the laboratory mouse I I : Mirex, Te l o d r i n and DDT. T o x i c o l . Appl. Pharmacol. 10: 54-61. . Weikel, J.H., J r . , E.P. Lang and R. Tomchick. 1958. Ion movement across the r a b b i t erythrocyte membrane as affe c t e d by chlorinated i n s e c t i c i d e s . Arch. Intern. Pharmacodyn. 113: 261-272. 53 VII. APPENDIX I. Behavior Patterns of Female Rats with Young B i t i n g of Young ) ) Kicking of Younq ) ) H i t t i n g of Young ) Grooming of S e l f Drinking These were the patterns which were not seen but added to the l i s t as being a p r i o r i aggressive. They were also not seen during the experiment which since t h e i r meaning i s obvious anyway makes furthe r discussion unnecessary. The cleaning of the pelage, t a i l , limbs etc. with the paws and/or mouth. Also .includes scratching with the paws. Li c k i n g the spout of the water b o t t l e to obtain water. Feeding Nursing Eating from the j a r of food. This r e f e r s to the posture and p o s i t i o n of the female r e l a t i v e to the young and implies nothing about l a c t a t i o n or suckling. The t y p i c a l posture of the female was i n a loosely curled f o e t a l p o s i t i o n but l y i n g more on the v e n t r a l surface than on the l a t e r a l surface. Two v a r i a t i o n s of t h i s were noted. In one the female was i n a "sprawled" p o s i t i o n , l y i n g completely stretched out on the v e n t r a l surface; i n the other she was l y i n g on the l a t e r a l surface and almost completely stretched out. Regardless of the posture the female was t y p i c a l l y asleep. In the former two p o s i t i o n s 54 Standing over the Young Anus' L i c k i n g of Young Holding of Young Smelling Nest or Young Playing the young were hidden from s i g h t underneath the female while i n the l a t t e r p o s i t i o n the young could be seen along her v e n t r a l surface. The female would be standing s t r a d d l i n g the nest and the young. The young could be seen to be suckling on occasion. This p o s i t i o n was t y p i c a l l y observed at the end of a bout of Nursing. The female would hold the young, head down, i n her fore-paws and l i c k the anus of the young. The l i c k i n g probably induces defecation i n the young. This includes 3 patterns. The f i r s t was a f r i g h t response to external s t i m u l i where the female picked the young up i n her mouth and e i t h e r ran from the nest or stood nervously by the nest. The second was a maternal response where the female picked up a young who had strayed from the nest and returned i t to the nest by use of her mouth. The t h i r d pattern i s of unknown function and merely involved a female p i c k i n g up the young with her fore-paws, handling i t , and p u t t i n g i t down. The female, when standing about the nest was frequently observed to lower her head and^'smell" the nest and young. This i s a loose category for apparently functionless patterns. The most frequent pattern was holding the food dish tray i n the mouth and dragging i t 55 about the cage; a s i m i l a r pattern involved b a t t i n g the tray about the cage with the fore-paws. Chasing the t a i l was the other most frequent pattern. Digging This includes 2 patterns. The f i r s t involves the female standing i n one p o s i t i o n , usually at/or near.the nest, and p u l l i n g the l i t t e r away from one spot by use of the forepaws. In the second the female runs r a p i d l y about the cage with the head lowered and often ploughing i n the l i t t e r and with the forepaws " f l i c k s " l a t e r a l l y the l i t t e r ahead of her. Locomotion Walking or running about the cage. Standing Away from Usually an i n t e r r u p t i o n on a bout of locomotion, the Young 56 VIII. APPENDIX II The Measurement of D i e l d r i n Residues 1. The Colour Reaction and the Preparation of a Standard Curve The colour reaction was o r i g i n a l l y applied to d i e l d r i n which was at le a s t 99.5% pure (Cueto, 1960). In t h i s work t e c h n i c a l grade, d i e l d r i n , which i s the basis f o r commercial -insecticides arid i s b i o l o g i c a l l y the more important, was used. Technical d i e l d r i n contains 85% d i e l d r i n and 15% r e l a t e d i n s e c t i c i d a l compounds ( i e . a l d r i n , endrin, i s o d r i n ) . I t was necessary to make many refinements to improve the p r e c i s i o n of the colour reaction on t h i s material before a reasonable standard curve could be produced. The r e f i n e d technique i s discussed below. The reagents and t h e i r conditions required f o r the colour reaction are: D i e t h y l ether, C e r t i f i e d ACS. Ether containing peroxides i s unsa t i s f a c t o r y and cannot be used. The presence of free iodine as detected by the starch t e s t a f t e r shaking a small quantity of ether with aq. KI i s i n d i c a t i v e of peroxides. Hexane, C e r t i f i e d ACS. This solvent must be r e - d i s t i l l e d at l e a s t twice before use. Diphenylamine reagent. 0.25% diphenylamine i n hexane. The diphenyl-amine must be C e r t i f i e d ACS and r e - c r y s t a l l i z e d at l e a s t twice from hexane. This reagent should be used within 2 hours of preparation. Zinc chloride reagent. 0.25% zinc chloride i n ether. The zinc chloride must be c e r t i f i e d ACS and must be stored and measured under  conditions of absolute d e s s i c a t i o n . This reagent should be used within 0.5 hours of preparation and cannot be used a f t e r 4 hours. Solvent f o r reaction products. 10% C e r t i f i e d ACS a c e t i c anhydride by volume i n reagent grade g l a c i a l a c e t i c a c i d . 57 The procedure for the colour development i s ou t l i n e d below. The hexane containing the d i e l d r i n i s placed i n a 22 x 175 mm t e s t tube and taken j u s t to dryness i n a water bath at 68°C.' The evaporation rate under these conditions i s about 0.33 ml/hour. Allowing the tube to stay i n the water bath a f t e r a l l the hexane has evaporated r e s u l t s i n loss of d i e l d r i n . Two ml of the diphenylamine and zi n c chloride reagents are added to the tube and taken to dryness undear the same conditions as before. This w i l l take about 2 hours. A c o l o u r l e s s , dry r e s i d u a l f i l m should appear i n the tube. This f i l m should be l e f t i n the undeveloped condition f or the minimum amount of time for best r e s u l t s . To develop the colour the tube i s placed i n a t e s t tube heating block f o r exactly 3.0 minutes at 205°C. The tubes are then cooled f o r exactly 5.0 minutes i n running tap water. The white r e s i d u a l f i l m w i l l become cle a r where d i e l d r i n i s not present and a deep blue where i t i s . The reaction products are apparently unstable even i n the dry condition and they should be dissolved and the OD determined immediately. The re a c t i o n products are disso l v e d i n 3.0 mis of the g l a c i a l a c e t i c a c i d solvent and placed i n a 4 ml capacity, 10 mm l i g h t path absorption c e l l and OD determined at 650 mpi using the solvent as a reference. The OD must be determined within 10 minutes of the addition of solvent before the colour begins to fade. The spectrophotometer used was a Unicam SP 500. The diphenylamine and zinc chloride reagents have ODs which vary tremendously between batches and within batches between times such that i t i s absolutely necessary to run a reagent blank through the e n t i r e process above and correct the ODs of the d i e l d r i n samples with i t . The standard curve was prepared by determining the OD of known amounts of d i e l d r i n . Standard solutions of 2.5, 5.0, 10.0, 20.0, 50.0, and 100.0,y/ml 58 of d i e l d r i n ( r e - c r y s t a l l i z e d twice from hexane) i n t r i p l e - d i s t i l l e d C e r t i f i e d ACS hexane were prepared and s u i t a b l e aliquots were subjected to the colour reaction process. The range of the standard curve i s from 5.0 to 150 y. Attempts to increase the s e n s i t i v i t y i n the range of 0 to 20 y by d i s s o l v i n g the reaction products i n 1.0 mis of solvent and using a 0.8 ml capacity, 20 mm l i g h t path absorption c e l l were unsuccessful. The standard curve of gm of d i e l d r i n vs o p t i c a l density of the reaction products i s presented i n F i g . 9; the OD's have been corrected f o r the o p t i c a l density of the reagents. Since there i s no good l i n e a r r e l a t i o n s h i p over the e n t i r e range of observations two regression l i n e s have been f i t t e d , one f o r 0-70 7" d i e l d r i n and the other f o r 70-150. The l i n e f o r 70-1507 has been f i t t e d by the l e a s t squares method and the equation i s Y = 10-3 ( 3 .80 + 2.59 X). There are however two possible l i n e s f o r the 0-70 y range. One, which i s not shown on the f i g u r e , was f i t t e d by the l e a s t squares method and has the equation Y = 1 0 - 3 ( 1 . 7 1 + 3 . 3 1 x); the other, which i s shown, was also f i t t e d by the l e a s t squares method but was forced through the o r i g i n and has the equation Y = 3.34 x 10" 3 x. The reason for p u t t i n g the l i n e through 0, 0 i s t h e o r e t i c a l : i f there i s no d i e l d r i n the o p t i c a l density must be zero. In theory the lower l i m i t of s e n s i t i v i t y should be extended by t h i s l i n e . The l a t t e r two l i n e s d i f f e r i n t h e i r slope (F = 664, df = 1 and 156, P<0.01) J such that they cannot be considered estimates of a common l i n e . This implies that only one or the other i s the "true" l i n e and that only one can be used. In r e a l i t y there i s l i t t l e d i f f e r e n c e i n the estimates of the amount of d i e l d r i n (x) c a l c u l a t e d from the two l i n e s (Table IX). Moreover the confidence l i m i t s on x are wide such that i t i s r e a l l y inconsequential which l i n e i s used (Table IX). For computational ease the f i x e d l i n e i s preferable. Fig. 9 The relationship between the Optical Density of the dieldrin - diphenylamine reaction products and the amount of dieldrin involved in the reaction. 0.501-Dieldrin (jjgm) 60 Table IX. A comparison of the usefulness of the 3 r e g r e s s i o n l i n e s i n p r e d i c t i n g the amount of d i e l d r i n . 95% Confidence L i m i t s on x OD D i e l d r i n Line (Y) (x) T % of X Y = 10~ 3 (1.71+3. 31X) 0.050 14. 59 12. 84 53. 02 -12 - +263 0.100 29. 69 22. 81 - 33. 73 -23 - + 14 Y = 3.34 x 10" 3X 0.050 14. 95 12. 97 — 53. 13 -13 - +255 0.100 29. 95 23. 18 - 34. 22 -23 - + 14 Y = 10" 3 (3.80+2. 59X) 0. 300 101. 10 4. 01 _ 162. 01 -60 - + 60 61 The equations f o r c a l c u l a t i n g the 95% confidence l i m i t s on an estimate of the amount of d i e l d r i n c a l c u l a t e d from e i t h e r of the three l i n e s i s presented i n Table X; they have been derived from Sokal and Rohlf (1969). The CL are not symmetrical about x because i n c a l c u l a t i n g x from Y]_f a l l of the assumptions made i n o r i g i n a l l y regressing Y on X are v i o l a t e d and the necessary mathematical corrections d i s t o r t the CL. As i s t y p i c a l the CL are widest at the ends of the l i n e s (Table X). In general the CL are narrowest when Y^=Y. Therefore the most accurate estimates are obtained when the amount of d i e l d r i n present i s about 30"? and the lower l i n e s i s used. The upper l i n e i s v i r t u a l l y useless since the CL are so wide even when Y ^ Y . For p r a c t i c a l use the colour reaction should be performed on a sample containing about 30y d i e l d r i n even though t h i s necessitates the use of a p i l o t sample. The minimum quantity of t e c h n i c a l d i e l d r i n which can be measured i s about 2 . 5 7 . 2. The Ext r a c t i o n of D i e l d r i n from Feces and Animal Tissues Three d i f f e r e n t e x t r a c t i o n processes corresponding to the three clean-procedures were used on the tissues but only one, a modification of Zweig ( 1 9 6 4 ) , was used on the feces. This was to avoid the p o s s i b i l i t y of converting f e c a l d i e l d r i n metabolites i n t o d i e l d r i n . The d e t a i l s of the extraction and clean-up procedures are presented below. A l l of the t r i a l s on feces were performed on 50 g. (dry weight) sample from unexposed animals. The samples were ground to a f l o u r i n a Waring blendor before e x t r a c t i o n . The feces were extracted by placing the 50 g sample i n a 500 ml fl a s k with 200 ml of hexane and shaking vigorously f o r 5 minutes. Approximately Table X. The 95% confidence l i m i t s on an estimate of x from the regression l i n e s of O p t i c a l Density vs D i e l d r i n . Regression Line Confidence Limits Y = 1.71 x 10" 3 + 3.31 x 10 3X CL = 36. 88 + 231{Y± - 0. 125) + 97.5\f ).00376 + (Y x - 0.125) 2 for 0>X>70 V (floating) Y = 3.34 x 10 _ 3X CL = 36. 88 + 327(Y^ " 0. 125) + 98.5\f0.00385 + CY-L - 0.125) 2 for 0>X>70 (fixed through 0) Y = 3.80 x 10~ 3 + 2.59 x HT3X CL =110. 0 + 1038 (Y^ ^ - 0. 336) + 529^0.0227 + ( Y 1 " 0.326) 2 for 70>X>150 63 90 ml of hexane could be recovered and a 75 ml a l i q u o t of t h i s was centrifuged f o r 20 minutes at 2500 rpm to remove p a r t i c u l a t e matter. The 75 ml of extract was then concentrated to 5 ml by use of a water bath at O 68 C. The concentrate was deep green i n colour and subjected to the column chromatography of the clean-up procedures discussed below. The t r i a l s on animals' tissues were performed on whole body homogenates of unexposed pre-weaning young. The carcasses were homogenized by grinding them, while s t i l l frozen, several times i n a hand grinder. A modification of the technique of Cueto (1960) was t r i e d f i r s t . (a) A 50 g sample of t i s s u e was refluxed f o r 2 hours i n 75 ml of 95% ETOH and 15 ml of 50% aq. KOH and the mixture allowed to cool. This process hydrolyzes organochlorine i n s e c t i c i d e s other than d i e l d r i n , cleaves the peptide bond of proteins and the ester bond of l i p i d s . (b) F i f t e e n ml of the r e f l u x mixture and 25 ml of hexane were mixed i n a Waring blendor f o r 1 minute and the mixture was then t r a n s f e r -red to a 1000 ml separatory funnel. This was repeated u n t i l a l l of the r e f l u x mixture had been extracted. (c) The contents of the separatory funnel were mixed by vigorous shaking and the layers allowed to separate. This was repeated 3 times and a f t e r the t h i r d time the f l a s k was allowed to s i t for 30 minutes and the polar layer was then drawn to waste. (d) The extract was n e u t r a l i z e d with 1 N HCl and washed once with 75 ml d i s t i l l e d HOH and twice with 20% ag. NACl. The washings were performed by shaking the mixture vigorously and allowing the layers to separate; t h i s was repeated three times. The polar layers were discarded. (e) The f i n a l traces of HOH were removed from the hexane extract by 64 adding, anhydrous Na2S0^ and allowing i t to s i t overnight. (f) A 75 ml a l i q u o t of extract was concentrated to 2 ml i n a water O bath at 68 C and subjected to column chromatography. (g) The chromatography columns were prepared by adding small q u a n t i t i e s of unactivated dry alumina (80-200 mesh) and tapping the column (10 x 330 mm) to ensure packing u n t i l the column of absorbant i s 10 cm high. (h) The concentrated extract i s tr a n s f e r r e d to the column with the a i d of a few ml of 10% ether i n hexane (the same c r i t e r i a of p u r i t y as i n the colour reaction) and the column i s eluted with 10% ether i n hexane at a flow rate of 2 ml/minute. (i) One hundred ml of elutant are c o l l e c t e d i n 10 ml f r a c t i o n s and each f r a c t i o n i s subjected to the colour procedure. To determine which f r a c t i o n s would contain the d i e l d r i n 100 7" were placed on a column and eluted. There was an average recovery of approximately 90% with the f r a c t i o n s 2, 3, 4, 5, 6, and 7 containing 15, 20, 25, 25 and 5 7 d i d l d r i n . A l l ten f r a c t i o n s of the elutant from a f e c a l blank contained i n t e r -f e r i n g materials. The green contaminants apparently consisted of two major f r a c t i o n s ; one with a very low Rf which was picked up i n f r a c t i o n s 1-7 and another which was picked up i n 8-10. S i m i l a r l y a l l ten elutant f r a c t i o n s from a tis s u e blank contained i n t e r f e r i n g materials; the peak was i n f r a c t i o n s 4-7. The inadequacy of t h i s clean-up procedure precluded i t s use with the colour reaction'.' The second extraction-clean-up procedure which was t r i e d was that of Kutney (1969). (a) A 50 g sample of t i s s u e was placed i n a 250 ml f l a s k and 100 ml 65 of hexane was added. The f l a s k was then shaken vigorously f o r 5 minutes. A 75 ml a l i q u o t was then concentrated to 5 ml by use O of a water bath at 68 C. Feces were extracted as previously described. (b) Chromatography columns were prepared by f i l l i n g the column h a l f f u l l of hexane and pouring a s l u r r y of unactivated, 80-200 mesh alumina i n t o i t . The excess hexane was drained o f f and the pro-cedure repeated u n t i l the desired height of alumina was obtained. The column si z e s used were 360 x 25 mm, 300 x 25 mm, 240 x 25 cm and 180 x 25 mm. The extracts were tr a n s f e r r e d to the column by use of a few ml of hexane and eluted with hexane at a flow rate of 2 ml/min. The various s i z e columns were evaluated f o r t h e i r e f f i c t i v e n e s s i n cleaning up the concentrated extracts. To determine i f the 360 x 25 mm column could be used 2000 T of d i e l d r i n were placed on i t and eluted with 600 ml of hexane; the elutant was c o l l e c t e d i n 10 ml f r a c t i o n s . These f r a c t i o n s were subjected to the colour develop-ment process. The compound was found i n f r a c t i o n s 40-51 with the majority of i t i n f r a c t i o n s 41-46. To t a l recovery was about 20%. The elutants of f e c a l extracts showed no contaminants i n the f r a c t i o n s 40-51. Therefore a concentrated f e c a l extract plus 100T of d i e l d r i n were added to a column i n order to estimate recovery at r e l a t i v e l y low concentrations. Under these conditions i t was found that recovery was 0% even when the f r a c t i o n s 40-51 were pooled. Subsequent t r i a l s with various amounts of d i e l d r i n showed that 10007 i s the minimum l e v e l which r e s u l t s i n detectable l e v e l s i n the elutant. As such t h i s s i z e column i s not useful f o r detecting microgram q u a n t i t i e s i n b i o l o g i c a l materials. The 180 x 25 mm column was the next tested by methods i d e n t i c a l to those above and i t was found that contaminants were found i n the elutant 66 f r a c t i o n s containing the d i e l d r i n . S i milar work with the 2 intermediate s i z e d columns showed that they were also unsuitable because of poor recovery or clean-up. The f i n a l method which was t r i e d was that of de Faubert Maunder ejt a l . , (1964) . This i s the mos-t e f f e c t i v e e x t r a c t i o n and clean-up procedure which has been devised f o r p e s t i c i d e residue analysis using the gas chromatograph. (a) A 25 g sample of homogenzied tis s u e was ground i n a mortar with 25 g of quartz and 100 g of anhydrous Na2SC>4 u n t i l a free flowing powder resulted. (b) This was t r a n s f e r r e d to 250 ml f l a s k and 100 ml of hot hexane was added and the f l a s k shaken vigorously for 5 minutes. (c) The hexane was decanted o f f and allowed to c o o l . (d) A '25 ml a l i q u o t of hexane and 10 ml of dimethylformamide (DMF) saturated with hexane were shaken vigorously f o r 5 minutes i n a 100 ml separatory funnel. The funnel was allowed to s i t for 10 minutes and the polar DMF layer was run i n t o another funnel; any emulsion was l e f t i n the f i r s t funnel. The e x t r a c t i o n of the hexane with 10 ml DMF s o l n . was repeated twice more. The DMF extracts were combined and washed with 10 ml of hexane saturated with DMF to remove the l a s t traces of f a t . The 10 ml of hexane were separated and washed with 10 ml of DMF which was added to the o r i g i n a l 30 ml of DMF extract. This procedure was repeated on another 2 aliquots of hexane and a l l the DMF extracts were' combined. (e) The combined DMF extracts were shaken b r i s k l y f o r 2 minutes with 600 ml of 2% aq. Na^O^ i n a 1000 ml separatory funnel. The funnel was allowed to s i t for 30 minutes while the hexane, previously held i n s o l u t i o n , separated out. The polar layer was then run to waste 67 and the hexane was c o l l e c t e d . (f) The hexane was concentrated to 2 ml and subjected to the column chromatography. (g) The chromatography columns were prepared by h a l f - f i l l i n g a 10 x 330 mm column with hexane and pouring a hexane s l u r r y of O lOg of 80-200 mesh alumina (activated by heating to 200 C f o r 4 hours) i n t o i t . The hexane was drained o f f u n t i l the alumina was j u s t covered. A 5 cm- layer of anhydrous Na^SO^ was added on top of the alumina and t h i s layer was j u s t covered with hexane. (h) The extract was applied to the top of the column and washed on with three successive 2 ml portions of hexane. The column was eluted with 100 ml of hexane and the elutant was c o l l e c t e d i n 10 ml f r a c t i o n s . To determine which f r a c t i o n s would contain the d i e l d r i n 1007 were placed on a column and eluted. There was an average recovery of 90% with the f r a c t i o n s 2, 3, 4, 5, 6, and 7 containing 15, 20, 25, 25 and 5 7 d i e l d r i n . As with the chromatography of Cueto (1960) a l l ten f r a c t i o n s of f e c a l and t i s s u e blanks contained materials which i n t e r f e r e d with the colour reaction although the amount was reduced several f o l d . I t was concluded a f t e r t r y i n g these three techniques that Cueto's spectrophotometry method of d i e l d r i n q u a n t i f i c a t i o n i s not applicable to e i t h e r feces or animal t i s s u e s . 68 IX. APPENDIX III The Relationship between Dose and Response An i d e a l dose response curve i s presented i n F i g . 10'."-'! Frequently, empirical curves are l a c k i n g the top and/or bottom plateau phase. The AB segment of the curve represents the doses where a l l members of the population, can detoxify a l l the compound they absorb. These animals may or may not d i f f e r i n the amount they absorb. The BC segment represents the doses where the highly susceptible members of the population die; these animals have very poor mechanisms of d e t o x i f i c a t i o n and/or very "good" mechanisms of absorption. Dose B i s defined as the Threshold Dose (i e . minimum dose producing e f f e c t ) and the s o - c a l l e d No-Effect Dose i s s l i g h t l y l e s s than B. The CD p o r t i o n of the curve includes the doses where the "normal" members of the population undergo mort a l i t y . The DE portion of the curve represents the doses to which only a very few, highly r e s i s t a n t members of the populations w i l l not respond; these animals have very good mechanisms of d e t o x i f i c a t i o n or very poor mechanisms of absorption. I t i s possible to c a l c u l a t e an LD50 from t h i s curve or portions of i t ( L i t c h f i e l d and Wilcoxon, 1949). 69 FiglO The relationship between dose and response. 

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