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The effects of in ovo and early post-hatch DDT expourse on American robins from the Okanagan Valley,… Smith, Lori Kim 2004

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THE EFFECTS OF  INOVO  A N DE A R L Y POST-HATCH DDT EXPOSURE O N AMERICAN  ROBINS F R O M T H E O K A N A G A N V A L L E Y , BRITISH C O L U M B I A  by LORI K I M SMITH B . S c , T h e U n i v e r s i t y o f L e t h b r i d g e , 1994 M . Sc., T h e U n i v e r s i t y o f L e t h b r i d g e , 1997  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E  STUDIES  (Faculty o f A g r i c u l t u r a l Sciences, Department o f A n i m a l Science) W e accept this thesis as c o n f o r m i n g t o the r e q u i r e d standard  THE UNIVERSITY OF BRITISH C O L U M B I A A p r i l 23, 2004 © L o r i K i m Smith, 2004  11  ABSTRACT  American robin (Turdus migratorius) eggs from orchard areas of the Okanagan Valley, British Columbia contain high levels of dichlorodiphenyltrichloroethane (DDT) and its metabolites. These contaminants are present in the soil as a result of heavy historical use. DDTs accumulate in the earthworms that live in the soil and are passed to the robins through their preference for earthworms as a food source during the breeding season. These chemicals are then passed to the offspring via the egg yolk and in the diet. Despite the high residue levels found in these robins, no impairments in their reproductive success have been found. In order to assess more subtle and/or long term effects of DDTs on a variety of parameters, ten-day old nestlings were collected from the Okanagan, along with controls from the Lower Mainland of British Columbia, and raised and bred in captivity. Eggs were collected in order to measure contaminant levels. Total DDTs in the Okanagan eggs ranged from 5.7 to 277.6 pig/g (mean 49.3 pig/g), whereas in the Lower Mainland eggs they ranged from 0.4 to 3.4 pig/g (mean 1.5 pig/g). Eggs collected from the Lower Mainland and Okanagan were of similar weights, lengths, and widths. Okanagan chicks collected in 1997 had significantly shorter middle toes than the other birds, and appeared to lag in their tarsus growth. As adults, these birds laid smaller eggs than the Lower Mainland controls, but showed no differences in the timing of their reproductive activities, their laying, hatching, and fledging success, or their reproductive behaviors. They demonstrated an increased susceptibility to infectious disease, lower corticosterone levels during the early phases of a restraint test, and enlarged hearts, livers, and kidneys. Although robins in the Okanagan continue to thrive and reproduce despite their high levels of contamination, D D T likely has the potential to influence several aspects of their lives, as evidenced by the many significant correlations with in ovo exposure. However, it remains that genetic differences between the Lower Mainland and Okanagan birds may account for many of the effects seen.  iii  TABLE OF CONTENTS  Abstract Table of contents L i s t of Tables List of Figures  ii iii viii x  Acknowledgements  xii  Overview  xv  CHAPTER I INTRODUCTION  1  1.1. W h a t Is D D T ?  1  1.2. T h e T r o u b l e with D D T  3  1.3. Effects of D D T on Birds  4  1.3.1. Effects o n G r o w t h and S u r v i v a l  5  1.3.1.1. Adult Survival 1.3.1.2. Offspring Growth and Survival 1.3.1.3. Hormonal Influences  5  6 7  1.3.2. Effects o n the I m m u n e S y s t e m  10  1.3.3. Effects o n Stress R e s p o n s e  11  1.3.4. Effects o n R e p r o d u c t i o n  14  1.3.4.1 Hormonal Influences 1.3.4.2. Early Exposure 1.3.5. E f f e c t s o n B e h a v i o r  15 16 16  1.3.5.1. Reproductive Behaviors 1.3.5.2. Parental Behaviors 1.3.5.3. Other Behaviors 1.4 References  17 17 18 20  C H A P T E R II DDT, A M E R I C A N ROBINS, AND T H E O K A N A G A N V A L L E Y  30  2.1. Soil  30  2.2. E a r t h w o r m s  31  2.3. A m e r i c a n Robins  33  iv  2.4. This Study  36  2.5 References  39  C H A P T E R III G R O W T H AND S U R V I V A L OF DDT C O N T A M I N A T E D A M E R I C A N ROBINS  43  3.1. Introduction  43  3.2. Methods  44  3.2.1. E g g C o n t a m i n a n t s  44  3.2.2. E g g M e a s u r e m e n t s  45  3.2.3. R e a r i n g P r o t o c o l  45  3.2.4. C h i c k M e a s u r e m e n t s  46  3.2.5. G r o w t h M e a s u r e m e n t s  46  3.2.6. T h y r o i d H o r m o n e s  47  3.2.7. I m m u n e R e s p o n s e  47  3.2.7.1. Differential White Blood Cell Counts  4  3.2.7.2. Phytohemagglutinin Skin Test  4  3.2.8. M o r t a l i t y  48  3.2.9. T i s s u e W e i g h t s and B o d y M e a s u r e m e n t s  48  3.2.10. Statistics  48  3.3. Results  49  3.3.1. E g g C o n t a m i n a n t s  49  3.3.2. E g g M e a s u r e m e n t s  49  3.3.3. C h i c k M e a s u r e m e n t s  51  3.3.4. G r o w t h M e a s u r e m e n t s  51  3.3.5. T h y r o i d H o r m o n e s  51  3.3.6. I m m u n e R e s p o n s e  56  3.3.6.1. White Blood Cell Ratios  56  3.3.6.2. Phytohemagglutinin Skin Test 3.3.7. M o r t a l i t y 3.3.8. T i s s u e W e i g h t s a n d B o d y M e a s u r e m e n t s  3.4. Discussion  5 56 59  59  3.4.1. G r o w t h a n d D e v e l o p m e n t  64  3.4.2. I m m u n i t y a n d S u r v i v a l  66  3.5. Conclusions  68  3.6. References  69  CHAPTER IV REPRODUCTION AND BEHAVIOR IN AMERICAN ROBINS EXPOSED IN OVO AND EARLY POST-HATCH TO DDT AND ITS METABOLITES 75 4.1. Introduction  75  4.2. Methods  77  4 . 2 . 1 . E g g s and C h i c k s  77  4.2.2. R e p r o d u c t i o n  77  4.2.2.1. Nesting Success 4.2.2.2. Egg Measurements 4.2.2.3. Chick Measurements 4.2.2.4. Chick Mortality and Tissue Weights at Sacrifice  77 78 78  4.2.3. T h y r o i d H o r m o n e s  78  4.2.4. B e h a v i o r a l O b s e r v a t i o n s  79  4.2.4.1. Reproductive and Parental Care Behaviors 4.2.4.2. Aggressive Behaviors 4.2.4.3. Vocalizations 4.2.4.4. Maintenance Behaviors 4.2.5. Statistics  4.3. Results  7 80 80 81 81  82  4.3.1. E g g C o n t a m i n a n t s  82  4.3.2. R e p r o d u c t i o n  83  4.3.2.1. Nesting Success 4.3.2.2. Egg Measurements 4.3.2.3. Chick Measurements 4.3.2.4. Chick Mortality and Tissue Weights at Sacrifice  83 85 85  4.3.3. T h y r o i d H o r m o n e L e v e l s i n B r e e d i n g B i r d s  89  4.3.4. B e h a v i o r  89  4.4. Discussion  92  4.5. Conclusions  102  4.6. References  104  CHAPTER V INFECTIOUS DISEASE AND IMMUNE RESPONSE IN AMERICAN ROBINS EXPOSED IN OVO AND EARLY POST-HATCH TO DDT  109  5.1 Introduction  109  5.2 Methods  Ill  vi 5.2.1  E g g Contaminants  Ill  5.2.2 E g g M e a s u r e m e n t s  Ill  5.2.3 C h i c k s  Ill  5.2.4  C h i c k Measurements  112  5.2.5  Thyroid Hormones  112  5.2.6 I m m u n e R e s p o n s e  112  5.2.6.1 White Blood Cell Counts 5.2.6.2 Phytohemagglutinin Skin Test 5.2.6.3 Hematocrits 5.2.6.4 Positive Controls  112 11 112 113  5.2.7  Stress R e s p o n s e  113  5.2.8  Blood Lead  113  5.2.9 T i s s u e W e i g h t s  ,  113  5.2.10 Statistics  113  5.3 Results  114  5.3.1  E g g Contaminants  114  5.3.2  Thyroid Hormones  116  5.3.3  Immune Response  116  5.3.3.1 Hematocrits 5.3.3.2 White Blood Cell Ratios 5.3.3.3 Phytohemagglutinin Skin Test  116  116 11  5.3.4. Stress R e s p o n s e  119  5.3.5 B l o o d L e a d . . .  121  5.3.6 Infectious D i s e a s e s and Parasites  121  5.3.7 M o r t a l i t y  •  121  5.3.8 E g g M e a s u r e m e n t s  123  5.3.9 C h i c k M e a s u r e m e n t s  123  5.3.10 T i s s u e W e i g h t s  123  5.4 Discussion  125  5.5 References  129  C H A P T E R VI G E N E R A L DISCUSSION.... 6.1 Egg Contaminants  134 134  6.2 Similarities and Differences Between the Lower Mainland and Okanagan Robins. 137  vii 6.2.1 E g g and C h i c k M e a s u r e m e n t s  137  6.2.2 B e h a v i o r and R e p r o d u c t i o n  143  6.2.3 H o r m o n e s  143  6.2.4 I m m u n i t y  144  6.2.5 M o r t a l i t y and T i s s u e W e i g h t s  146  6.3 Problems  147  6.4 Implications  148  6.5 References  151  APPENDIX 1  158  A P P E N D I X II  160  Vlll  List of Tables  Table 2-1.  M e a n D D T l e v e l s ( m g / k g , w e t w e i g h t ) i n A m e r i c a n r o b i n eggs c o l l e c t e d f r o m  o r c h a r d s i n the O k a n a g a n V a l l e y a n d n o n - o r c h a r d areas o f the O k a n a g a n a n d L o w e r Mainland o f British Columbia Table 3-1:  35  M e a n s ( ± se) a n d ranges o f D D T s , m o i s t u r e content, a n d l i p i d content (u.g/g) i n  A m e r i c a n r o b i n eggs c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r M a i n l a n d T a b l e 3-2:  50  M e a n s (+ se) a n d ranges o f b o d y w e i g h t , a n d tarsus, w i n g c o r d , a n d m i d d l e toe  lengths o f ten day o l d A m e r i c a n r o b i n nestlings c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r Mainland Table 3-3:  ;  52  C a u s e s o f d e a t h f o r A m e r i c a n r o b i n s c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r  Mainland  58  T a b l e 3-4: A g e s at t i m e o f death for A m e r i c a n r o b i n s c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r Mainland T a b l e 3-5:  60  M e a n s (+ se) a n d ranges o f b o d y m e a s u r e m e n t s a n d tissue w e i g h t s ( c o r r e c t e d for  b o d y w e i g h t ) o f O k a n a g a n a n d L o w e r M a i n l a n d r o b i n s at sacrifice  61  T a b l e 4 - 1 : N u m b e r s o f L o w e r M a i n l a n d and O k a n a g a n A m e r i c a n r o b i n females n e s t i n g , l a y i n g eggs, h a t c h i n g eggs, and f l e d g i n g y o u n g i n 1998 a n d 1999 Table 4-2:  84  M e a n s (+ se) a n d ranges o f b o d y w e i g h t s a n d tarsus, w i n g , a n d toe lengths o f the  o f f s p r i n g o f L o w e r M a i n l a n d a n d O k a n a g a n A m e r i c a n r o b i n females w h e n five a n d ten days o l d  •.  87  T a b l e 4 - 3 : M e a n s ( ± se) a n d ranges o f b e h a v i o u r s p e r f o r m e d b y L o w e r M a i n l a n d a n d O k a n a g a n robins  93  T a b l e 4-4: S i g n i f i c a n t correlations between b e h a v i o r a n d egg D D T l e v e l s i n O k a n a g a n r o b i n s . 95 T a b l e 4 - 5 : S i g n i f i c a n t effects o f sex o n b e h a v i o u r i n L o w e r M a i n l a n d a n d O k a n a g a n r o b i n s . ...96 T a b l e 4 - 6 : S i g n i f i c a n t effects o f y e a r o n b e h a v i o u r i n L o w e r M a i n l a n d a n d O k a n a g a n r o b i n s . .97 Table 4-7:  S i g n i f i c a n t effects o f p e n - t y p e o n b e h a v i o u r i n L o w e r M a i n l a n d a n d O k a n a g a n  robins Table 5-1:  98  M e a n s (+ s t a n d a r d error) a n d r a n g e s o f D D T s , m o i s t u r e , a n d l i p i d s (u.g/g) i n  A m e r i c a n r o b i n eggs c o l l e c t e d f r o m the L o w e r M a i n l a n d and O k a n a g a n V a l l e y o f B r i t i s h Columbia  115  T a b l e 5-2: M e a n s (+ se) a n d ranges o f b o d y a n d tissue w e i g h t s (grams) o f 1998 O k a n a g a n a n d L o w e r M a i n l a n d broods at t i m e o f sacrifice  124  ix T a b l e 6-1: S i g n i f i c a n t differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n birds a n d eggs • •.  138  T a b l e 6-2: S i g n i f i c a n t correlations b e t w e e n O k a n a g a n egg contaminant l e v e l s and other parameters  140  List of Figures  F i g u r e 1-1: S c h e m a t i c d i a g r a m o f p , p ' - D D T and its t w o p r i m a r y metabolites, p , p ' - D D D a n d p,p'DDE Figure 2-1:  2 C o l l e c t i o n sites o f A m e r i c a n r o b i n e g g s a n d n e s t l i n g s . L o w e r M a i n l a n d  =  V a n c o u v e r , Delta, and Surrey, B r i t i s h C o l u m b i a ; Okanagan = Naramata and Pentiction, British Columbia Figure 3-1:  38  M e a n (+ se) b o d y w e i g h t s (grams) o f L o w e r M a i n l a n d a n d O k a n a g a n A m e r i c a n  r o b i n s at v a r i o u s ages  53  F i g u r e 3-2: M e a n (+ se) tarsus lengths ( m m ) o f L o w e r M a i n l a n d a n d O k a n a g a n A m e r i c a n r o b i n s at v a r i o u s ages  54  Figure 3-3: P l a s m a levels o f A)triiodothyronine (pg/ml) and B)thyroxine (ng/ml) i n Okanagan a n d L o w e r M a i n l a n d r o b i n s at v a r i o u s ages  55  F i g u r e 3-4: D i f f e r e n c e s (+ se) i n w h i t e b l o o d c e l l ratios ( e o s i n o p h i l s + heterophils / l y m p h o c y t e s + m o n o c y t e s ) for L o w e r M a i n l a n d a n d O k a n a g a n A m e r i c a n r o b i n s as 10 day o l d n e s t l i n g s and post-breeding adults  57  F i g u r e 3 - 5 : R e l a t i o n s h i p b e t w e e n g o n a d w e i g h t at s a c r i f i c e (grams, c o r r e c t e d for b o d y w e i g h t ) and egg p , p ' - D D E (p.g/g) levels  62  F i g u r e 3-6: R e l a t i o n s h i p s b e t w e e n egg o , p ' - D D T (p.g/g) a n d f e m a l e O k a n a g a n A m e r i c a n r o b i n o v i d u c t w e i g h t s (grams) at t i m e o f sacrifice Figure 4-1:  63  R e l a t i o n s h i p b e t w e e n f e m a l e O k a n a g a n A m e r i c a n r o b i n in ovo p , p ' - D D T (p:g/g)  exposure a n d the m e a n w e i g h t s o f their eggs  86  F i g u r e 4 - 2 : M e a n (+ se) b r a i n , k i d n e y , a n d l i v e r w e i g h t s (grams) for birds hatched at S a n R a f a e l to same type a n d different type parents. L M = L o w e r M a i n l a n d , O K = O k a n a g a n , m a l e x female parents Figure 4-3:  M e a n ( ± se) differences i n a) t h y r o x i n e a n d b) t r i i o d o t h y r o n i n e l e v e l s i n L o w e r  M a i n l a n d a n d O k a n a g a n r o b i n s at the v a r i o u s t i m e p e r i o d s tested Figure 5-1:  90  91  M e a n (+ se) t r i i o d o t h y r o n i n e a n d t h y r o x i n e l e v e l s ( n g / m l ) i n the b l o o d o f 1998  L o w e r M a i n l a n d a n d O k a n a g a n b i r d s at ten d a y s o f age  117  F i g u r e 5-2: M e a n (+ se) hematocrit values for 1998 L o w e r M a i n l a n d and O k a n a g a n r o b i n s w h e n 10 and 5 0 - 5 7 days o f age  118  xi F i g u r e 5-3:  M e a n (+ se) e o s i n o p h i l + h e t e r o p h i l / m o n o c y t e + l y m p h o c y t e ratios for 1998  O k a n a g a n a n d L o w e r M a i n l a n d b i r d s w h e n 10 a n d 50 - 57 d a y s o l d F i g u r e 5-4:  120  M e a n (+ se) p l a s m a c o r t i c o s t e r o n e l e v e l s ( n g / m l ) i n 1998 L o w e r M a i n l a n d a n d  O k a n a g a n j u v e n i l e r o b i n s d u r i n g a restraint stress test. O u t l i e r r e m o v e d  122  xu ACKNOWLDEGEMENTS  F i r s t they t e l l y o u y o u ' r e w r o n g , and they c a n p r o v e it. T h e n they t e l l y o u y o u ' r e right, but i t ' s not important T h e n they t e l l y o u i t ' s important, but t h e y ' v e k n o w n it for years. C.F. Kettering  T h i s w o r k c o u l d not h a v e b e e n a c c o m p l i s h e d w i t h o u t the h e l p o f a n u m b e r o f p e o p l e .  My  a p o l o g i e s i f I ' v e m i s s e d anyone. I a m d e e p l y indebted to y o u a l l . S p e c i a l thanks to: m y f a m i l y and friends, e s p e c i a l l y m y parents, K e n a n d B r i g i t t e S m i t h , a n d m y s i b l i n g s , T i n a a n d K e l l y S m i t h for their c o n t i n u e d support throughout m y s e e m i n g l y n e v e r - e n d i n g student career, a n d to Z e d N o e l , T a n y a B e h r i s c h , a n d e s p e c i a l l y S h a r i W e e c h for h e l p i n g m e retain w h a t little sanity I have left my  supervisory committee, D r . John Elliott, D r . Raja Rajamahendren,  Dr. Tony  W i l l i a m s , a n d e s p e c i a l l y D r . K i m C h e n g for their h e l p w i t h the e x e c u t i o n a n d w r i t e - u p o f the study m y internal e x a m i n e r s , D r . S t e l v i o B a n d i e r a and D r . G a i l B e l w a r d and external e x a m i n e r , D r . D i a n e H e n s c h e l for their h e l p f u l c o m m e n t s Laurie W i l s o n , Sandi L e e , Harpreet G i l l , Christy M o r r i s e y , Gabriella K a r d o s i , Terry Sullivan, Chris G i l l , Chris Coker, M a r i a Fronteddu, Jamie D e W i t t , Diane Henschel, and e v e r y o n e else that h e l p e d w i t h c o n s t r u c t i o n a n d m a i n t e n a n c e o f the pens, a n i m a l care, s a m p l e c o l l e c t i o n s , a n d m u c h appreciated a d v i c e Pam  M a r t i n , J o h n E l l i o t t , a n d the N a t u r a l S c i e n c e s a n d E n g i n e e r i n g C o u n c i l o f C a n a d a  for f u n d i n g support M o n i k a T o l g s d o r f f a n d the volunteers at M o n i k a ' s W i l d l i f e Shelter for r a i s i n g the b i r d s , n u r s i n g t h e m b a c k to health, a n d offering a d v i c e o n their care T a n y a Jaques for the use o f a m i c r o s c o p e for the W B C counts S y l v i a L e u n g , G i l l e s G a l z i , a n d S i v a C h e n n a r e d d y at the U n i v e r s i t y o f B r i t i s h C o l u m b i a for their help i n c o l l e c t i n g e q u i p m e n t and supplies and a d v i c e o n w h a t to do w i t h t h e m the l a n d o w n e r s that p e r m i t t e d us to c o l l e c t birds a n d eggs f r o m their properties N S E R C a n d the C a n a d i a n W i l d l i f e S e r v i c e for f u n d i n g and,  finally,  the r o b i n s w h o , a l t h o u g h not v o l u n t a r i l y , d o n a t e d t h e i r eggs, their b l o o d ,  their t i m e , and u l t i m a t e l y their l i v e s i n the name o f scientific progress  Xlll  C h a p t e r III R o b i n eggs a n d nestlings w e r e c o l l e c t e d and m e a s u r e d b y L a u r i e W i l s o n a n d t e a m ,  from  the C a n a d i a n W i l d l i f e S e r v i c e . E g g c o n t a m i n a n t l e v e l s w e r e a n a l y z e d b y M i c h a e l M u l v i h i l l at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r i n H u l l , Q u e b e c .  B o d y w e i g h t s a n d tarsus lengths w e r e  m e a s u r e d b y C h r i s G i l l , C h r i s t y M o r r i s e y , C h r i s C o k e r , L a u r i e W i l s o n , S a n d i L e e , Harpreet G i l l , a n d L o r i S m i t h . B i r d s w e r e s e x e d b y B r e t t V a n d e r k i s t at S i m o n F r a s e r U n i v e r s i t y , B u r n a b y , British Columbia.  B l o o d samples were collected by K a r e n Petit, L a u r i e W i l s o n , S a n d i L e e ,  G a b b y K a r d o s i , H a r p r e e t G i l l , a n d C h r i s t y M o r r i s e y , w i t h h e l p f r o m L o r i S m i t h . B l o o d smears were prepared b y S a n d i L e e a n d L o r i S m i t h , a n d w h i t e b l o o d c e l l counts w e r e c o n d u c t e d b y L o r i Smith.  T h y r o i d h o r m o n e l e v e l s w e r e a n a l y z e d b y T r a c y M a r c h a n t at the U n i v e r s i t y o f  Saskatchewan, Saskatoon, Saskatchewan.  T h e p h y t o h e m a g g l u t i n i n s k i n test w a s p e r f o r m e d b y  L o r i S m i t h a n d M a r i a F r o n t e d d u , w i t h h e l p from L a u r i e W i l s o n . C o c c i d i o s i s w a s d i a g n o s e d b y Ted  L e i g h t o n at the C a n a d i a n C o o p e r a t i v e W i l d l i f e H e a l t h C e n t r e at the U n i v e r s i t y o f  Saskatchewan, Saskatoon, Saskatchewan.  B i r d s w e r e h o u s e d at M o n i k a ' s W i l d l i f e Shelter,  S u r r e y , B r i t i s h C o l u m b i a p r i o r to s e x u a l m a t u r i t y a n d c a r e d for b y M o n i k a T o l g s d o r f a n d her staff.  B i r d s w e r e h o u s e d at the U n i v e r s i t y o f B r i t i s h C o l u m b i a S a n R a f a e l R e s e a r c h A v i a r y ,  S u r r e y , B r i t i s h C o l u m b i a after s e x u a l maturity. T h e S a n R a f a e l pens w e r e b u i l t b y C h r i s C o k e r , C h r i s G i l l , S a n d i L e e , L o r i S m i t h , L a u r i e W i l s o n , K i m C h e n g , T e r r y S u l l i v a n , a n d others.  Pens  w e r e m a i n t a i n e d b y L o r i S m i t h a n d T e r r y S u l l i v a n . B i r d s w e r e c a r e d for at S a n R a f a e l b y L o r i Smith.  B i r d s were sacrificed and dissected by K i m C h e n g and L o r i S m i t h .  A l l statistical  analyses were c o n d u c t e d b y L o r i S m i t h w i t h help from K i m C h e n g .  Chapter I V R o b i n eggs a n d n e s t l i n g s w e r e c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r M a i n l a n d a n d m e a s u r e d b y L a u r i e W i l s o n a n d t e a m , f r o m the C a n a d i a n W i l d l i f e S e r v i c e . E g g c o n t a m i n a n t l e v e l s w e r e a n a l y z e d b y M i c h a e l M u l v i h i l l at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r i n H u l l , Quebec.  T h y r o i d h o r m o n e l e v e l s w e r e a n a l y z e d b y T r a c y M a r c h a n t at the U n i v e r s i t y o f  Saskatchewan, Saskatoon, Saskatchewan.  E g g s and chicks from San Rafael were weighed and  m e a s u r e d b y L o r i S m i t h . T h e b r e e d i n g b i r d s w e r e s e x e d b y B r e t t V a n d e r k i s t at S i m o n F r a s e r U n i v e r s i t y , B u r n a b y , B r i t i s h C o l u m b i a . S a n R a f a e l c h i c k s w e r e s e x e d b y M a r i a F r o n t e d d u at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B r i t i s h C o l u m b i a . A l l b e h a v i o r a l observations w e r e conducted by L o r i Smith.  B i r d s w e r e h o u s e d at M o n i k a ' s W i l d l i f e Shelter, S u r r e y , B r i t i s h  xiv C o l u m b i a p r i o r to s e x u a l m a t u r i t y a n d c a r e d for b y M o n i k a T o l g s d o r f a n d her staff.  B i r d s were  h o u s e d at the U n i v e r s i t y o f B r i t i s h C o l u m b i a S a n R a f a e l R e s e a r c h A v i a r y , S u r r e y , B r i t i s h C o l u m b i a after s e x u a l m a t u r i t y .  T h e San Rafael pens were built by C h r i s C o k e r , C h r i s G i l l ,  S a n d i L e e , L o r i S m i t h , L a u r i e W i l s o n , K i m C h e n g , T e r r y S u l l i v a n , a n d others.  Pens were  m a i n t a i n e d b y L o r i S m i t h a n d T e r r y S u l l i v a n . B i r d s w e r e c a r e d for at S a n R a f a e l b y L o r i S m i t h . B i r d s w e r e s a c r i f i c e d a n d dissected b y K i m C h e n g a n d L o r i S m i t h . A l l statistical analyses w e r e c o n d u c t e d b y L o r i S m i t h w i t h h e l p from K i m C h e n g .  Chapter V R o b i n eggs a n d nestlings were c o l l e c t e d a n d m e a s u r e d b y L a u r i e W i l s o n a n d team, f r o m the C a n a d i a n W i l d l i f e S e r v i c e . E g g c o n t a m i n a n t l e v e l s w e r e a n a l y z e d b y M i c h a e l M u l v i h i l l at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r i n H u l l , Q u e b e c .  B o d y w e i g h t s a n d measures, b l o o d  s a m p l e s , b l o o d smears, h e m a t o c r i t s , stress response t e s t i n g , p h y t o h e m a g g l u t i n i n s k i n testing o b t a i n e d a n d c o n d u c t e d b y L a u r i e W i l s o n and others. W i l s o n a n d others.  B l o o d samples w e r e c o l l e c t e d b y L a u r i e  W h i t e b l o o d c e l l counts w e r e c o n d u c t e d b y L o r i S m i t h . T h y r o i d h o r m o n e  a n d corticosterone l e v e l s w e r e a n a l y z e d b y T r a c y M a r c h a n t at the U n i v e r s i t y o f S a s k a t c h e w a n , S a s k a t o o n , S a s k a t c h e w a n . B l o o d lead l e v e l s were a n a l y z e d b y E w a N e u g e b a u e r at the N a t i o n a l W i l d l i f e R e s e a r c h Center. M y c o p l a s m a d i a g n o s i s c o n d u c t e d b y the A n i m a l H e a l t h C e n t r e at the B r i t i s h C o l u m b i a M i n i s t r y o f A g r i c u l t u r e , F o o d , a n d F i s h e r i e s . B i r d s w e r e h o u s e d at M o n i k a ' s W i l d l i f e Shelter, S u r r e y , B r i t i s h C o l u m b i a p r i o r to s e x u a l m a t u r i t y a n d c a r e d for b y M o n i k a T o l g s d o r f and her staff. B i r d s were s a c r i f i c e d and dissected b y M a l c o l m M c A d i e . A l l statistical analyses were c o n d u c t e d b y L o r i S m i t h w i t h help f r o m K i m C h e n g .  XV  Overview  Chapter I T h i s chapter represents a r e v i e w o f some o f the a v a i l a b l e literature o n D D T a n d p r o v i d e s b a c k g r o u n d i n f o r m a t i o n o n D D T a n d its detrimental effects e s p e c i a l l y o n b i r d s . F o c u s is g i v e n to effects o n g r o w t h a n d s u r v i v a l , r e p r o d u c t i o n , b e h a v i o r , and the stress response.  C h a p t e r II T h i s chapter p r o v i d e s b a c k g r o u n d i n f o r m a t i o n o n the study area (the O k a n a g a n V a l l e y o f B r i t i s h C o l u m b i a ) a n d s p e c i e s (the A m e r i c a n r o b i n ) u t i l i z e d for this r e s e a r c h .  I n c l u d e d is  information on soil, earthworm, and A m e r i c a n robin D D T contamination.  C h a p t e r III T h i s chapter e x a m i n e s the effects o f early D D T exposure o n the g r o w t h a n d s u r v i v a l o f A m e r i c a n r o b i n s f r o m the O k a n a g a n V a l l e y , B r i t i s h C o l u m b i a .  E g g contaminants,  egg  measurements, c h i c k measurements, t h y r o i d h o r m o n e l e v e l s , i m m u n e response, m o r t a l i t y , a n d tissue w e i g h t s at sacrifice are i n c l u d e d . T h e g o a l o f this part o f the study w a s to d e t e r m i n e i f O k a n a g a n b i r d s are at a disadvantage i n terms o f g r o w t h a n d s u r v i v a l as c o m p a r e d to c o n t r o l b i r d s from the L o w e r M a i n l a n d .  Chapter I V T h i s chapter focuses o n the effects o f early D D T exposure o n r e p r o d u c t i o n a n d b e h a v i o r i n A m e r i c a n r o b i n s . B r e e d i n g pairs w e r e o b s e r v e d for nest b u i l d i n g , e g g l a y i n g , egg h a t c h i n g , a n d c h i c k f l e d g i n g . R e p r o d u c t i v e b e h a v i o r s i n c l u d i n g m a t i n g , nest b u i l d i n g , a n d parental care w e r e m o n i t o r e d , as w e l l as v o c a l b e h a v i o u r s (e.g., s i n g i n g , c h i r p i n g ) , m a i n t e n a n c e  behaviors  (e.g., eating, d r i n k i n g , p r e e n i n g ) , a n d aggressive b e h a v i o r s (e.g., c h a r g i n g , c h a s i n g , b i t i n g ) . I n a d d i t i o n , a subset o f b r e e d i n g pairs w e r e b l o o d s a m p l e d o n a regular basis i n order to m o n i t o r t h y r o i d h o r m o n e l e v e l s . T h e o f f s p r i n g o f these b i r d s w e r e a l s o w e i g h e d a n d m e a s u r e d , their s u r v i v a l m o n i t o r e d , a n d their tissues w e i g h e d u p o n s a c r i f i c e . T h e g o a l o f this part o f the study w a s to d e t e r m i n e i f b i r d s e x p o s e d to D D T e x p o s u r e e a r l y i n l i f e s u f f e r e d f r o m  decreased  r e p r o d u c t i v e s u c c e s s , as c o m p a r e d to L o w e r M a i n l a n d b i r d s , a n d i f they d e m o n s t r a t e d alterations i n their b e h a v i o r s w h i c h c o u l d i n f l u e n c e their s u r v i v a l a n d r e p r o d u c t i v e success.  any  xvi Chapter V T h i s chapter deals w i t h a s e c o n d set o f eggs a n d b i r d s c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r M a i n l a n d i n order to l o o k m o r e c l o s e l y at the effects o f e a r l y D D T e x p o s u r e o n i m m u n e response a n d other parameters that c a n i n f l u e n c e i m m u n e response.  E g g contaminants were  d e t e r m i n e d , eggs a n d c h i c k s w e r e w e i g h e d a n d m e a s u r e d , t h y r o i d h o r m o n e s , w h i t e b l o o d c e l l ratios, hematocrits, and T - c e l l mediated i m m u n e responses c o r t i c o s t e r o n e l e v e l s d u r i n g a restraint stress test.  w e r e e v a l u a t e d , as w e l l  as  T h e g o a l o f t h i s study w a s to d e t e r m i n e i f  e a r l y e x p o s u r e to D D T a n d its effects o n i m m u n i t y , t h y r o i d h o r m o n e s , a n d corticosterone release p l a y e d a r o l e i n the c o c c i d i o s i s i n f e c t i o n s a n d subsequent deaths o f a n u m b e r o f j u v e n i l e r o b i n s ( C h a p t e r III).  Chapter V I T h i s chapter serves as an o v e r v i e w o f the results f o u n d i n the p r e v i o u s chapters, as w e l l as p r o b l e m s e n c o u n t e r e d d u r i n g the c o u r s e o f the study, t h i n g s that c o u l d h a v e b e e n d o n e differently, other factors that m a y have p l a y e d a r o l e i n the results, i m p l i c a t i o n s o f these f i n d i n g s , a n d avenues for future research.  1  Chapter I Introduction  1.1. W h a t Is D D T ? D i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e , better k n o w n as D D T , i s a n o r g a n o c h l o r i n e p e s t i c i d e that w a s first p r o d u c e d i n 1873.  It w a s r e d i s c o v e r e d i n 1939, a n d at this p o i n t its i n s e c t i c i d a l  properties w e r e r e v e a l e d . T h i s d i s c o v e r y w a s h a i l e d as "the m o s t r e v o l u t i o n a r y d e v e l o p m e n t i n the h i s t o r y o f pest c o n t r o l " and earned P a u l M u l l e r the N o b e l P r i z e for M e d i c i n e a n d P h y s i o l o g y i n 1948 (p. 1, M e l l a n b y , 1992). D D T w a s touted as the i d e a l p e s t i c i d e . It w a s h i g h l y t o x i c to insects, safe for plants and w a r m - b l o o d e d a n i m a l s , n o n - i r r i t a t i n g a n d odorless, w i d e l y a p p l i c a b l e , cheap and easy to p r o d u c e , a n d l o n g - l a s t i n g . N o n e o f the pesticides i n use at the t i m e f u l f i l l e d a l l o f these c r i t e r i a ( M e l l a n b y , 1992). It c o m e s i n a v a r i e t y o f f o r m s , w i t h the para, para' (p,p') a n d o r t h o , para' (o,p') i s o m e r s b e i n g the m o s t c o m m o n .  C o m m e r c i a l or t e c h n i c a l grade D D T is  c o m p o s e d o f p r i m a r i l y p , p ' - D D T (65 - 9 0 % ) , w i t h 10 - 3 0 % o , p ' - D D T a n d a v a r i e t y o f metabolites a n d other products (Jefferies, 1975; M e l l a n b y , 1 9 9 2 ; W H O , 1989). T h e r e are t w o m a j o r routes o f m e t a b o l i s m o f D D T ( F i g u r e 1-1), w i t h the i n i t i a l c h a n g e b e i n g either to D D E ( d i c h l o r o d i p h e n y l d i c h l o r o e t h y l e n e ) or D D D ( d i c h l o r o d i p h e n y l d i c h l o r o e t h a n e , a l s o k n o w n as TDE).  T h e p r i m a r y D D T m e t a b o l i t e i n l i v i n g tissues is D D E (Jefferies, 1 9 7 5 ; S t i c k e l , 1973).  U n d e r anaerobic c o n d i t i o n s a n d p o s t - m o r t e m , D D T is m e t a b o l i z e d p r i m a r i l y to D D D ( S t i c k e l , 1973). D D T k i l l s insects b y w o r k i n g as a nerve p o i s o n . It m a y be ingested or absorbed t h r o u g h a n insect's integument ( M e l l a n b y , 1992). A l t h o u g h D D T ' s m o l e c u l a r target a n d m o d e o f a c t i o n h a v e yet to be c l e a r l y d e f i n e d , it l i k e l y exerts its effects o n the n e r v o u s s y s t e m b y b l o c k i n g p o t a s s i u m e f f l u x across the n e r v e a x o n m e m b r a n e r e s u l t i n g i n a n i n c r e a s e d n e g a t i v e potential.  after-  C o n s e q u e n t l y , it s l o w s d o w n the t u r n i n g - o f f o f s o d i u m c o n d u c t a n c e across  the  m e m b r a n e a n d i n h i b i t s the t u r n i n g - o n o f p o t a s s i u m c o n d u c t a n c e , p r o b a b l y b y i n t e r f e r i n g w i t h the energy m e t a b o l i s m r e q u i r e d for i o n transport across the m e m b r a n e s ( B u n y a n & S t a n l e y , 1982; M u r p h y , 1980; Y o u n i s et a l . , 2002).  CCl,  p,p'-DDT  Cl  CH-  CHC1  p,p'-DDD  Cl  CCh  3  p,p'-DDE  Figure 1-1: Schematic diagram of p,p*-DDT and its two primary metabolites, p,p'-DDD and p,p'-DDE.  3  D D T b e c a m e c o m m e r c i a l l y a v a i l a b l e i n 1 9 4 1 , a n d w a s first a v a i l a b l e as dusts a n d w e t t a b l e p o w d e r s for p l a n t a n d f a b r i c p r o t e c t i o n a n d p u b l i c h y g i e n e .  D u r i n g W o r l d W a r II,  D D T - c o n t a i n i n g products were u s e d e x t e n s i v e l y to c o m b a t m a l a r i a - c a r r y i n g m o s q u i t o e s , t y p h u s b e a r i n g l i c e , and b u b o n i c p l a g u e - i n f e s t e d rat fleas.  The number o f lives saved w o r l d w i d e by  D D T are n u m b e r e d i n the m i l l i o n s , a n d the i l l n e s s e s p r e v e n t e d i n the h u n d r e d s o f m i l l i o n s ( M u r p h y , 1980). F o l l o w i n g W o r l d W a r II, D D T w a s s t i l l u s e d to c o m b a t disease-transmitting insects i n areas o f outbreak, but its use shifted to p r i m a r i l y plant p r o t e c t i o n . F r u i t tree pests w e r e a m o n g the first to be c o n t r o l l e d b y D D T p r o d u c t s .  D D T also f o u n d success against insects  d a m a g i n g e v e r y t h i n g f r o m vegetables to forage crops, cotton to forests ( M e l l a n b y , 1992). 1.2. T h e T r o u b l e w i t h D D T M a n y o f the qualities that m a d e D D T s u c h a n efficient p e s t i c i d e also m a d e it a cause for concern.  A l t h o u g h not h i g h l y v o l a t i l e or water s o l u b l e , D D T s c a n adsorb to p a r t i c l e s a n d be  c a r r i e d b y w i n d a n d w a t e r t o a l l e c o s y s t e m s o f the w o r l d (Repetto & B a l i g a , 1996). T h e y c a n r e m a i n i n s o i l s for m o r e than 50 years ( C o l b o r n et a l . , 1993), w h e r e they m a y be c o n s u m e d b y a variety o f organisms.  B e c a u s e D D T s are v e r y l i p o p h i l l i c , they c a n b i o a c c u m u l a t e i n l i p i d - r i c h  tissues a n d b i o m a g n i f y i n f o o d w e b s .  T h e r e f o r e , they c a n be p a s s e d o n to o f f s p r i n g a n d  predators far r e m o v e d f r o n i the o r i g i n a l e x p o s u r e ( M u r p h y , 1 9 8 0 ; R e p e t t o & B a l i g a , 1 9 9 6 ) . D D T i s n o n - s e l e c t i v e a n d , therefore, d a m a g i n g not o n l y to detrimental insects, but also b e n e f i c i a l ones ( C a r s o n , 1962; M u r p h y , 1980). A s D D T w a s n o n - i r r i t a t i n g , w i d e l y a p p l i c a b l e , cheap, a n d easy to p r o d u c e , it tended to be o v e r - u s e d , a n d often p r o p e r care w a s not t a k e n to a v o i d o v e r s p r a y i n g , to l i m i t dosages, a n d to prevent c o n t a m i n a t i o n ( C a r s o n , 1962). It is this over-use o f D D T that l i k e l y c o n t r i b u t e d to the r a p i d d e v e l o p m e n t o f resistance i n a n u m b e r o f insects ( M e l l a n b y , 1992). T h e m e t a b o l i t e s , b y - p r o d u c t s , different i s o m e r s , a n d c o n t a m i n a n t s o f D D T m a y also h a v e different effects than the parent c o m p o u n d ( M u r p h y , 1980), a n d as D D T s are r a r e l y f o u n d a l o n e , p e s t i c i d e m i x t u r e s m a y h a v e a d d i t i v e or s y n e r g i s t i c effects ( F o s s i , 1 9 9 8 ; M c A r t h u r et a l . , 1983; T y l e r et a l . , 1998). A l t h o u g h m u c h less t o x i c than m a n y o f its predecessors (and successors), D D T has been l i n k e d to a n u m b e r o f a n i m a l deaths. N o n - t a r g e t species m a y be acutely or c h r o n i c a l l y p o i s o n e d , a l t h o u g h there is great v a r i a b i l i t y i n species s e n s i t i v i t y to the t o x i c effects o f D D T (Banerjee et a l . , 1996; C a r s o n , 1 9 6 2 ; M u r p h y , 1980).  F i s h , for e x a m p l e , are h i g h l y s u s c e p t i b l e to D D T  p o i s o n i n g , w i t h fish s u c h as the d w a r f p e r c h (Micrometrus minimus) d e m o n s t r a t i n g  LC50  levels  4 as l o w as, a n d e v e n l o w e r than, 0.3 p.g/liter o f water. larger fish o f the same species ( W H O , 1989).  S m a l l e r fish t e n d to be m o r e at r i s k than  M a m m a l s a n d b i r d s d o appear to be r e l a t i v e l y  tolerant to the t o x i c effects o f D D T s , but w i l l s u c c u m b to h i g h dosages a n d s o m e species are m o r e s e n s i t i v e t h a n o t h e r s are.  T h e L D 5 0 for A m e r i c a n b r o w n bats  (Eptesicus fuscus), for  instance, m a y be as l o w as 25 m g D D T / k g i n a s i n g l e dose ( W H O , 1989). T h e L D m a l e lab rats  5 0  (oral) for  (Rattus norvegicus), o n the other h a n d , is 2 1 7 m g / k g t e c h n i c a l grade D D T , a n d 8 8 0  m g / k g D D E ( M u r p h y , 1980). W h i l e the difference i n s u s c e p t i b i l i t y to D D T p o i s o n i n g b e t w e e n insects a n d other o r g a n i s m s m a y s i m p l y be due to differences i n scale ( M e l l a n b y , 1992), there are also l i k e l y to be differences i n their respective n e r v o u s systems.  F o r example, mammals do  n o t appear to h a v e a p a r t i c u l a r p r o t e i n that is part o f the s o d i u m / p o t a s s i u m p u m p i n n e r v e m e m b r a n e s ( Y o u n i s et a l . , 2 0 0 2 ) . A s i d e f r o m its p o t e n t i a l t o x i c i t y , D D T c a n have other d e t r i m e n t a l effects.  It has been  suggested that D D T s are t e r a t o g e n i c , d i s r u p t i n g the o r g a n i z a t i o n o f the b r a i n a n d b o d y o f d e v e l o p i n g e m b r y o s a n d l e a d i n g to a l t e r e d c o g n i t i o n a n d b e h a v i o r , as w e l l as p h y s i c a l d e f o r m i t i e s ( C o l b o r n et a l . , 1996). D D T a n d D D E have b e e n s h o w n to i n d u c e nerve c e l l death a n d suppress the differentiation o f these c e l l s i n rats a n d h a v e been l i n k e d to the deterioration o f n e u r o b e h a v i o r a l functions i n D D T - e x p o s e d w o r k e r s ( S h i n o m i y a & S h i n o m i y a , 2 0 0 3 ) . T h e r e i s a l s o e v i d e n c e that D D T d i s r u p t s s o d i u m c o n d u c t a n c e i n n e r v e c e l l s , s i m i l a r t o that s e e n i n insects.  S o d i u m c h a n n e l s are h e l d o p e n i n D D T p o i s o n e d a n i m a l s l e a d i n g to t r e m o r , h y p e r -  e x c i t a b i l i t y , a n d changes i n the l e v e l s o f s o m e n e u r o t r a n s m i t t e r s  ( H o n g et a l . , 1 9 8 6 ) .  The  c a r c i n o g e n i c i t y o f D D T is s t i l l under debate, but it has b e e n s h o w n to cause hepatic t u m o r s i n rodents a n d has b e e n l i n k e d to b o t h breast a n d t e s t i c u l a r cancers ( M u r p h y , 1 9 8 0 ; R e p e t t o & B a l i g a , 1996).  D D T s h a v e b e e n s h o w n to d i s r u p t the e n d o c r i n e s y s t e m b y m i m i c k i n g ,  a g o n i z i n g , or a n t a g o n i z i n g the functions o f e n d o g e n o u s h o r m o n e s ( C o l b o r n et a l . , 1996).  As  w e l l , they m a y have detrimental effects o n n o r m a l i m m u n e s y s t e m f u n c t i o n i n g (Banerjee et a l . , 1996; B a r n e t t & R o d g e r s , 1 9 9 4 ; R e p e t t o & B a l i g a , 1996; Street, 1 9 8 1 ; V o c c i a et a l . , 1999). B e c a u s e o f its p o t e n t i a l as an e n v i r o n m e n t a l c o n t a m i n a n t a n d h e a l t h r i s k , the use o f D D T w a s banned i n C a n a d a , the U n i t e d States, G r e a t B r i t a i n , a n d other i n d u s t r i a l i z e d countries i n the early 1 9 7 0 ' s ( M e l l a n b y , 1992).  1.3. Effects of DDT on Birds S o m e o f the first p u b l i c debates about D D T arose as a result o f t h i s c h e m i c a l ' s effects o n b i r d s ( C a r s o n , 1962).  "It w a s not u n t i l the A m e r i c a n p u b l i c b e c a m e a w a r e o f the p o s s i b l e  5 e x t i n c t i o n o f the A m e r i c a n R o b i n , because D D T causes p a r a l y s i s o f its central n e r v o u s s y s t e m , that s o m e t h i n g w a s f i n a l l y done to save it a n d other species....it w a s the A m e r i c a n R o b i n that b e c a m e the s y m b o l o f the fight to stop the use o f this d e a d l y c h e m i c a l . " (p. 80, W a u e r , 1999). B i r d s m a y be m o r e s e n s i t i v e to D D T s a n d other c h e m i c a l s t h a n m a m m a l s b e c a u s e o f t h e i r r e l a t i v e l y s m a l l e r l i v e r , r a p i d rates o f f o o d i n t a k e a s s o c i a t e d w i t h m a i n t e n a n c e o f h i g h b o d y temperature, a n d the p o t e n t i a l reabsorption o f u r i n a r y metabolites f r o m the c l o a c a (Rattner et a l . , 1984).  T h e y t e n d to r e s p o n d v e r y q u i c k l y to p e s t i c i d e use, but t h e i r a b i l i t y to a b s o r b a n d  metabolize chemicals may vary dramatically.  B i r d s that eat other b i r d s or f i s h u s u a l l y h a v e  h i g h e r residues than those that eat seeds, vegetation, or m a m m a l s ( S t i c k e l , 1973). also a c c u m u l a t e h i g h e r D D T burdens than g r a n i v o r e s ( K l e m e n s et a l . , 2 0 0 0 ) .  Insectivores  The half-life o f  (Larus argentatus) ( B r a u n e & N o r s t r o m , 1989), 2 2 9 days for c o m m o n g r a c k l e s (Quiscalus quiscalus), 2 5 0 d a y s for p i g e o n s {Columbia livia) ( S t i c k e l et a l . , 1984), and 129 days for Japanese q u a i l (Coturnix japonica) DDE w a s f o u n d to be b e t w e e n 2 0 0 and 3 0 0 days for h e r r i n g g u l l s  ( B r a u n e & N o r s t r o m , 1989; N o r s t r o m et a l . , 1986).  It c a n take u p to 9 8 8 d a y s for 9 5 % o f a  b i r d ' s b o d y b u r d e n o f D D T s to be depleted ( S t i c k e l , 1973).  1.3.1. Effects o h G r o w t h and S u r v i v a l  1.3.1.1.  Adult  Survival  A l t h o u g h c o n s i d e r e d o n l y m o d e r a t e l y t o x i c to b i r d s ( W H O , 1989), i n h i g h doses, D D T c a n result i n m o r t a l i t y . E x t r e m e l y h i g h m o r t a l i t y rates w e r e seen i n s o m e species d u r i n g the h e i g h t o f D D T use ( C a r s o n , 1962).  A n a p p l i c a t i o n o f 5.6 k g o f D D T per hectare resulted i n  i m m e d i a t e reductions i n p o p u l a t i o n s o f songbirds a n d invertebrates i n an u p l a n d h a r d w o o d forest i n P e n n s y l v a n i a ( B l u s , 1996). T h e use o f D D D to c o n t r o l gnats i n C l e a r L a k e , C a l i f o r n i a d u r i n g the 1 9 5 0 ' s d e c i m a t e d the p o p u l a t i o n o f w e s t e r n grebes  (Aechmophorus occidentalis).  Not on  d i d m a n y o f the adults d i e , the r e m a i n i n g o n e s f a i l e d to r e p r o d u c e ( C a r s o n , 1 9 6 2 ; F r y , 1 9 9 5 ; M e l l a n b y , 1992). T h i r t y pig/g o f D D T p l u s D D D i n the b r a i n has b e e n estimated as the l o w e r l e t h a l l i m i t i n b i r d s ( B l u s , 1996).  T h e r e i s , h o w e v e r , a great d e a l o f v a r i a t i o n i n the l e v e l s o f  D D T a n d its metabolites i n the brains o f different species that d i e d f r o m D D T . B r a i n l e v e l s o f DDT,  for i n s t a n c e , h a v e b e e n r e p o r t e d to range f r o m 15 p.g/g i n A m e r i c a n r o b i n s  (Turdus  migratorius) that d i e d i n tremors f o l l o w i n g s p r a y i n g to c o n t r o l D u t c h e l m disease, to 4 0 pig/g i n b r o w n - h e a d e d c o w b i r d s (Molothrus ater). L e t h a l D D D l e v e l s v a r i e d f r o m 2 p,g/g i n n o r t h e r n b o b w h i t e q u a i l (Colinus virginianus) to 99 p„g/g i n b r o w n - h e a d e d c o w b i r d s . B r a i n residues o f D D E v a r i e d f r o m less than 1 \xglg i n m o s t species studied to h i g h s o f 57 pig/g i n dead A m e r i c a n  6 robins.  T h i s suggests that there m a y be species differences i n the m e t a b o l i s m , storage, a n d  e x c r e t i o n o f D D T ( B l u s , 1996). Q u a i l t e n d to b u i l d u p h i g h e r concentrations before death than other species, a n d p i g e o n s are u n u s u a l l y susceptible to D D E p o i s o n i n g ( S t i c k e l , 1973).  1.3.1.2. Offspring Growth and Survival D D T s a c c u m u l a t e i n eggs i n p r o p o r t i o n t o the a m o u n t r e c e i v e d b y the m o t h e r ( O h l e n d o r f et a l . , 1978; S t i c k e l , 1973), w i t h the residue content o f e a c h e g g r e f l e c t i n g the r e l a t i v e l e v e l s i n the female at the t i m e o f y o l k l i p i d d e p o s i t i o n ( F o x et a l . , 1978). E m b r y o s are e x p o s e d not o n l y to the parent c o m p o u n d , b u t also its metabolites. C o n c e n t r a t i o n s o f D D T s m a y o r m a y n o t v a r y b e t w e e n eggs w i t h i n a c l u t c h as the mother's l i p i d stores are m o b i l i z e d ( O h l e n d o r f et a l . , 1 9 8 5 ; O t t i n g e r et a l . , 2 0 0 1 ) . C h i c k e n s  (Gallus)  m a y deposit up to 3 4 % o f their d a i l y p , p ' - D D T intake  into their eggs, a l o n g w i t h 4 2 % o f their p , p ' - D D E a n d 3 . 5 % o , p ' - D D T ( C e c i l et a l . , 1972).  As  they cannot be excreted f r o m the e g g , D D T s are present d u r i n g a l l c r i t i c a l p e r i o d s o f e m b r y o n i c d e v e l o p m e n t ( J i m e n e z , 1997). Effects o f D D T o n the e m b r y o or h a t c h l i n g m a y be m a n i f e s t e d i n a n e n t i r e l y different w a y , a n d w i t h p e r m a n e n t c o n s e q u e n c e s as c o m p a r e d to effects seen as a result o f exposure o n l y i n a d u l t h o o d . These effects m a y not b e c o m e apparent u n t i l the o f f s p r i n g r e a c h m a t u r i t y o r e v e n m i d d l e age ( C o l b o r n et a l . , 1 9 9 3 ) , a n d the e x t e n t o f p o t e n t i a l d e v e l o p m e n t a l a b n o r m a l i t i e s cannot be p r e d i c t e d f r o m c h e m i c a l exposures i n adults ( K e l c e et a l . , 1998).  In ovo e x p o s u r e s m a y r e s u l t i n m o r t a l i t y , r e d u c e d h a t c h a b i l i t y , w a s t i n g s y n d r o m e ,  s k e l e t a l a b n o r m a l i t i e s , a n d i m p a i r e d d i f f e r e n t i a t i o n o f the r e p r o d u c t i v e a n d n e r v o u s systems i n o f f s p r i n g ( F r y , 1995). D D T s m a y increase e m b r y o m o r t a l i t y , as seen i n b a r n o w l s  (Anas rubripes) gulls  ( B l u s , 1996), B e n g a l e s e finches  (Larus californicus)  et a l . , 1 9 7 2 ) , m a l l a r d s  (Tyto alba),  (Lonchura striata)  black ducks  (Jefferies, 1971), C a l i f o r n  ( F r y & T o o n e , 1981), Japanese q u a i l ( C h a n g & S t o k s t a d , 1975; L i l l i e  (Anas platyrhynchos)  ( L i l l i e et a l . , 1 9 7 2 ; W H O , 1 9 8 9 ) , a n d c h i c k e n s  ( B r i t t o n et a l . , 1974; L i l l i e et a l . , 1972; Sauter & Steele, 1972). It is p o s s i b l e for eggs to c a r r y e n o u g h D D T a n d metabolites to cause h i g h m o r t a l i t y a m o n g c h i c k s h a t c h e d f r o m t h e m w i t h o u t affecting t h e i r h a t c h a b i l i t y ( B l u s , 1 9 9 6 ; Jefferies, 1 9 7 1 ; J o n e s & S u m m e r s , 1 9 6 8 ; L i l l i e et a l . , 1972; W H O , 1989). F o r e x a m p l e , b l a c k d u c k s that w e r e fed 10 m g / k g D D E o v e r t w o b r e e d i n g seasons h a d r e d u c e d d u c k l i n g s u r v i v a l to three w e e k s o f age a n d this c o n t i n u e d e v e n t w o years after D D E d o s i n g h a d c e a s e d ( W H O , 1989). B e n g a l e s e f i n c h e s f e d p , p ' - D D T at b o t h l o w (1-50 p,g/day/bird) a n d h i g h ( 5 1 - 2 5 0 p g / d a y / b i r d ) doses for s i x w e e k s p r i o r to b r e e d i n g e x h i b i t e d not o n l y a decrease i n h a t c h i n g , but also c h i c k s u r v i v a l .  W h i l e 9 6 . 3 % o f the c h i c k s h a t c h e d to  7 c o n t r o l pairs s u r v i v e d to fledging, o n l y 7 5 . 8 % o f the c h i c k s f r o m l o w d o s e d b i r d s a n d 4 8 . 3 % o f the c h i c k s f r o m h i g h d o s e d birds s u r v i v e d that l o n g . M o s t o f the c h i c k s from treated p a i r s d i e d w i t h i n a d a y o f h a t c h i n g (Jefferies, 1971). Japanese q u a i l that c o n s u m e d a p p r o x i m a t e l y 28 m g o f p , p ' - D D T d u r i n g the first w e e k o f a study a n d 2 2 m g d u r i n g the s e c o n d w e e k , s h o w e d n o differences i n their h a t c h a b i l i t y , but 7 9 % o f the c h i c k deaths o c c u r r e d w i t h i n three d a y s o f h a t c h i n g , m o s t w i t h c l e a r s y m p t o m s o f D D T p o i s o n i n g (tremors, l o s s o f b a l a n c e , c o l l a p s e ) (Jones & S u m m e r s , 1968). A s n e w l y hatched c h i c k s r e l y o n stored y o l k for n u t r i t i o n for the first f e w days post-hatch, these early deaths c o u l d be attributed to the a b s o r p t i o n o f a large quantity o f p e s t i c i d e f r o m the y o l k ( B r i t t o n et a l . , 1974; Jefferies, 1 9 7 1 ; Jones & S u m m e r s , 1968).  Other  factors m a y i n c l u d e the stress o f h a t c h i n g , inadequate f e e d i n g or b r o o d i n g b y the parents, or r e d u c e d w e i g h t at h a t c h i n g due to s m a l l e r egg sizes (Jefferies, 1971). Three h u n d r e d m g / k g o f D D T has been suggested as the c r i t i c a l l e v e l i n the diet o f w h i t e l e g h o r n hens for n e g a t i v e effects o n p r o g e n y p e r f o r m a n c e , mortality and decreased c h i c k body weight.  as it results i n i n c r e a s e d c h i c k  T h e o f f s p r i n g o f h e n s fed 3 1 0 o r 6 2 0 m g / k g  t e c h n i c a l grade D D T for 133 d a y s h a d s i g n i f i c a n t l y l o w e r b o d y w e i g h t s at t w o w e e k s o f age c o m p a r e d to c o n t r o l s ( B r i t t o n at a l . , 1974).  C h i c k e n s , h o w e v e r , appear to be m o r e resistant to  the effects o f D D T o n g r o w t h d u r i n g the r e a r i n g p e r i o d t h a n w i l d b i r d s ( L i l l i e et a l . , 1972). I m p a i r e d g r o w t h has been reported i n the o f f s p r i n g o f b i r d s e x p o s e d to a v a r i e t y o f c h e m i c a l s , including D D T s .  G r e a t L a k e s h e r r i n g g u l l s , a n d other f i s h e a t i n g b i r d s , h a v e b e e n s h o w n to  e x h i b i t g r o w t h retardation a n d d e f o r m i t i e s , a s s o c i a t e d w i t h in ovo e x p o s u r e to  contaminants  ( F o x , 1992; T y l e r et a l „ 1998; V o s et a l . , 2 0 0 0 ) .  1.3.1.3. Hormonal  Influences  T h e t h y r o i d g l a n d h o r m o n e s , t r i i o d o t h y r o n i n e a n d t h r y o x i n e , p l a y an i m p o r t a n t r o l e i n p h y s i c a l g r o w t h a n d d e v e l o p m e n t ( S i n g h et a l . , 1968), b e h a v i o r a l , i n t e l l e c t u a l , a n d n e u r o l o g i c a l d e v e l o p m e n t ( H a u s e r et a l . , 1998), a n d feather g r o w t h and m o l t ( S i n g h et a l . , 1968; W e n t w o r t h & R i n g e r , 1986).  T h e s e h o r m o n e s not o n l y act to i n c r e a s e o x y g e n c o n s u m p t i o n , g l u c o s e  o x i d a t i o n , heat p r o d u c t i o n , a n d l i p o l y s i s , a n d regulate temperature (Jefferies, 1975), they also induce linear growth, protein synthesis, and skeletal maturation.  I n a d d i t i o n , they  have  p e r m i s s i v e effects o n g r o w t h h o r m o n e target c e l l s , they mediate the secretion o f g r o w t h h o r m o n e f r o m the p i t u i t a r y g l a n d ( B o l a n d e r , 1994; N e l s o n , 2 0 0 0 ) .  T h e t h y r o i d glands o f embryonic  c h i c k e n s b e c o m e f u n c t i o n a l a n d secrete t h y r o x i n e after t e n t o e l e v e n d a y s o f i n c u b a t i o n .  The  p i t u i t a r y g l a n d b e c o m e s sensitive to t h y r o t r o p i n r e l e a s i n g h o r m o n e f r o m the h y p o t h a l a m u s as  8 e a r l y as s i x to s e v e n days, w i t h the t h y r o i d g l a n d s b e c o m i n g s e n s i t i v e to t h y r o i d s t i m u l a t i n g h o r m o n e at the s a m e t i m e .  T h y r o x i n e concentrations increase during e m b r y o development,  w h e r e a s t r i i o d o t h y r o n i n e l e v e l s r e m a i n l o w . T h e concentrations o f b o t h h o r m o n e s p e a k o n the day o f p i p p i n g and then decrease after hatch u n t i l adult l e v e l s are reached ( W e n t w o r t h & R i n g e r , 1986). Jefferies and F r e n c h (1969) reported that the t h y r o i d g l a n d s o f feral p i g e o n s fed 3 to 36 m g D D T / k g / d a y for s i x w e e k s w e r e t w i c e as h e a v y as those o f c o n t r o l s . T h e t h y r o i d s o f d o s e d b i r d s h a d s m a l l e r f o l l i c l e s , less c o l l o i d , a n d h y p e r p l a s t i c e p i t h e l i a , regardless o f the dose l e v e l . S i m i l a r results were f o u n d i n birds d o s e d w i t h D D E (Jefferies, 1975). F e m a l e Japanese q u a i l fed 150 m g / k g D D E for 120 days h a d s i g n i f i c a n t l y e n l a r g e d t h y r o i d g l a n d s despite b e i n g o n a c l e a n diet for 85 days p r i o r to sacrifice ( R i c h e r t & P r a h l a d , 1972). In the same study, the t h y r o i d s o f b i r d s fed 100 m g / k g D D T or 2 0 0 m g / k g D D A ( 2 , 2 - b i s ( 4 - c h l o r o p h e n y l ) - a c e t i c a c i d ) w e r e not s i g n i f i c a n t l y h e a v i e r than those o f c o n t r o l s . enlarged thyroid gland follicles.  U n l i k e pigeons, D D T and D D E dosed quail had  T h e D D E treated g r o u p also demonstrated a r e d u c t i o n i n I  u p t a k e ( R i c h e r t & P r a h l a d , 1972).  1 2 5  B o b w h i t e q u a i l fed 5 0 0 m g / k g t e c h n i c a l grade D D T h a d  e n l a r g e d t h y r o i d glands after three m o n t h s o n treatment, a n d a n increase i n I  1 3 1  uptake b e t w e e n  one a n d three months o f treatment ( H u r s t et a l . , 1974). B e c a u s e t h y r o i d h o r m o n e s are i n s t r u m e n t a l i n the c o n t r o l o f m e t a b o l i c rate, they c a n i n f l u e n c e f u n c t i o n i n g o f the l i v e r a n d the heart.  B o b w h i t e quail fed 500 m g / k g D D T had  e n l a r g e d l i v e r s after t w o months o f treatment ( H u r s t et a l . , 1974). H o m i n g p i g e o n s fed 18, 3 6 , or 7 2 m g / k g p , p ' - D D T e v e r y s e c o n d d a y for 4 2 d a y s s h o w e d i n c r e a s i n g l i v e r w e i g h t s w i t h i n c r e a s i n g dose. T h e t h y r o i d g l a n d w e i g h t s o f these b i r d s also i n c r e a s e d as the c o n c e n t r a t i o n o f D D T i n the l i v e r increased. It has been suggested that this increase i n l i v e r size m a y h a v e b e e n due to i n c r e a s e d hepatic a c t i v i t y a n d m e t a b o l i s m o f c i r c u l a t i n g h o r m o n e s (Jefferies & F r e n c h , 1969).  D D T is k n o w n to i n d u c e e n z y m e b r e a k d o w n o f h o r m o n e s ( P e a k a l l , 1 9 6 7 ) .  Liver  h y p e r t r o p h y a n d l i v e r g l y c o g e n a c c u m u l a t i o n c a n be i n d u c e d b y h y p o t h y r o i d i s m i n c h i c k s ( W e n t w o r t h & R i n g e r , 1986). T h y r o x i n e c a n accelerate heart rate a n d increase heart w e i g h t i n d o m e s t i c f o w l s (Jefferies, 1975) and s i m i l a r effects w e r e seen w i t h D D T i n p i g e o n s . B i r d s fed a l o w dose o f D D T e x h i b i t e d increases i n a m p l i t u d e o f the v e n t r i c u l a r beat a n d heart w e i g h t , but b i r d s fed a h i g h dose s h o w e d heart beat a m p l i t u d e s l o w e r than that i n c o n t r o l s , decreased heart w e i g h t s , a n d t h i n , f l a c c i d heart m u s c u l a t u r e . hyperthyroidism  and  an  increase  L o w doses o f D D T i n pigeons  in metabolic  rate,  whereas  higher  doses  produce result  in  h y p o t h y r o i d i s m a n d a decrease i n m e t a b o l i c rate. B e n g a l e s e f i n c h e s , i n contrast, d o not appear  9 to d e v e l o p h y p o t h y r o i d i s m s y m p t o m s w i t h i n c r e a s i n g doses o f D D T , o n l y h y p e r t h y r o i d i s m . H e a r t rate, beat a m p l i t u d e , and w e i g h t c o n t i n u e to increase w i t h i n c r e a s i n g dose i n this species (Jefferies, 1975). Treatment w i t h t h y r o x i n e results i n a s i m i l a r trend i n c h i c k g r o w t h . I n s m a l l doses, t h y r o x i n e i m p r o v e s g r o w t h , doses b e y o n d p h y s i o l o g i c a l l e v e l s depress g r o w t h rate, a n d t o x i c doses accelerate c a t a b o l i c processes a n d reduce b o d y w e i g h t ( S i n g h et a l . , 1968). It has b e e n s u g g e s t e d that o r g a n o c h l o r i n e c o n t a m i n a n t s alter t h y r o i d f u n c t i o n i n g b y d i s r u p t i n g the transport o f t r i i o d o t h y r o n i n e a n d t h y r o x i n e b y p r e a l b u m i n (transthyretin) thyroid binding globulin (mammals).  and  H o w e v e r , o n l y the D D T m e t a b o l i t e D D O H (2,2-bis(4'-  c h l o r o p h e n y l ) e t h a n o l ) has b e e n s h o w n to b i n d transthyretin, a l b e i t w i t h l o w a f f i n i t y , a n d o,p'D D D a n d D D O H b i n d t h y r o i d b i n d i n g g l o b u l i n w i t h affinities 70 - 800 f o l d l o w e r than t h y r o x i n e ( C h e e k et a l . , 1999).  A n u m b e r o f researchers p u r p o r t that D D T s decrease t h y r o i d h o r m o n e  l e v e l s b y i n c r e a s i n g h e p a t i c e n z y m e a c t i v i t y ( C h e e k et a l . , 1 9 9 9 ; M c A r t h u r et a l . , 1 9 8 3 ; O h l e n d o r f et a l . , 1 9 7 8 ) .  P a r a , p a r a ' - D D T a n d o , p ' - D D T are k n o w n i n d u c e r s o f h e p a t i c  m i c r o s o m a l c y t o c h r o m e P - 4 5 0 m o n o o x y g e n a s e w h i c h c a t a l y z e s the m e t a b o l i s m o f n u m e r o u s x e n o b i o t i c s as w e l l as endogenous steroids ( K u p f e r & B u l g e r , 1980; R o b i s o n et a l . , 1984). T h i s , i n turn, c o u l d result i n e n l a r g e d l i v e r s ( B u n y a n & S t a n l e y , 1 9 8 2 ; O r b e r g & L u n d b e r g , 1974). D D T s m a y also affect the t h y r o i d glands i n d i r e c t l y v i a the h y p o t h a l a m i c - p i t u i t a r y - t h y r o i d a x i s . D i e t a r y l e v e l s as l o w as 2 m g . k g D D E c a n d r a m a t i c a l l y reduce b r a i n l e v e l s o f d o p a m i n e a n d norepinephrine i n ring doves  (Streptopelia risoria).  T h e n o r m a l f e e d b a c k l o o p s t h r o u g h the  h y p o t h a l a m u s m a y then be p a r t i a l l y b l o c k e d due to l o w l e v e l s o f neurotransmitters that w o u l d i n f l u e n c e the pituitary gland's release o f t h y r o i d s t i m u l a t i n g h o r m o n e and e v e n t u a l l y the release o f h o r m o n e s b y the t h y r o i d glands ( M c A r t h u r et a l . , 1983). G o n a d a l h o r m o n e s m a y also affect g r o w t h .  C h i c k s treated w i t h androgens s h o w e d a  r e d u c t i o n b o t h i n b o d y w e i g h t a n d skeletal g r o w t h ( F e n n e l l & Scanes, 1992). H o w e v e r , in ovo exposure to the anti-androgen, F l u t a m i d e , has also been s h o w n to suppress b o d y w e i g h t i n m a l e c h i c k s ( B u r k e , 1996). B u r k e (1996) has suggested that e m b r y o n i c androgens not o n l y i n f l u e n c e post-hatching g r o w t h i n m a l e c h i c k e n s , they also p l a y a role i n the s e x u a l a s y m m e t r y o b s e r v e d i n chicken body weights.  G r o w t h o f the n e u r o m u s c u l a r s y s t e m m a y a l s o b e e n h a n c e d  testosterone i n b o t h m a l e a n d f e m a l e b i r d s ( S c h w a b l , 1993).  by  E s t r o g e n s are k n o w n to p l a y a  c r u c i a l role i n bone d e v e l o p m e n t ( M i g l i a c c i o et a l . , 1995; O e s t r e i c h e r et a l . , 1971), as w e l l as the g r o w t h a n d f u n c t i o n o f m a n y organs i n c l u d i n g the b r a i n , breast, l i v e r , organs o f the r e p r o d u c t i v e s y s t e m , a n d the c a r d i o v a s c u l a r s y s t e m ( M c L a c h l a n & A r n o l d , 1 9 9 6 ) .  A v i a n embryos  e x p o s e d not o n l y to t h e i r o w n h o r m o n e s , but also to h o r m o n e s f r o m t h e i r m o t h e r s .  are  Both  10 e s t r a d i o l a n d testosterone h a v e b e e n s h o w n to be transferred i n t o the e g g ( S c h w a b l , 1 9 9 3 ; W i l l i a m s , 1999).  D D T s m a y i n f l u e n c e the l e v e l s o f g o n a d a l h o r m o n e s b y i n d u c i n g h e p a t i c  steroid h y d r o x y l a s e s w h i c h b r e a k d o w n g o n a d a l h o r m o n e s ( S t i c k e l , 1973; T h o m a s , 1998). T h e y m a y also i n h i b i t g o n a d o t r o p i n secretion (Rattner et a l . , 1984), l e a d i n g to a decrease i n the release o f g o n a d a l steroids. T h u s , there i s the potential for D D T s to h a v e s i g n i f i c a n t effects o n the l e v e l s o f g o n a d a l h o r m o n e s a n d therefore g r o w t h a n d d e v e l o p m e n t . f o u n d b e t w e e n p , p ' - D D T l e v e l s i n tree s w a l l o w  N o relationships, however, were  (Tachycineta bicolor)  eggs f r o m  southern  O n t a r i o orchards and e s t r a d i o l a n d testosterone l e v e l s i n c h i c k s ( B i s h o p , v a n der K r a a k , et a l . , 1998). 1.3.2. Effects o n the I m m u n e S y s t e m A variety o f pesticides, i n c l u d i n g D D T , are k n o w n to cause i m p a i r m e n t o f the vertebrate i m m u n e s y s t e m . T h e y m a y target the f u n c t i o n o f any o f the c e l l u l a r , s u b - c e l l u l a r , or m o l e c u l a r c o m p o n e n t s o f the i m m u n e s y s t e m (Banerjee, 1 9 9 9 ; R e p e t t o & B a l i g a , 1996).  A chemical's  effects o n the i m m u n e s y s t e m m a y be s u p p r e s s i v e a n d l e a d to a n i n c r e a s e d s u s c e p t i b i l i t y to i n f e c t i o u s diseases ( B a n e r j e e , 1 9 9 9 ; G r a s m a n et a l . , 1 9 9 6 ) , or it c a n be i m m u n o s t i m u l a t o r y l e a d i n g to h y p e r s e n s i t i v i t y , a u t o - i m m u n i t y , a n d a l l e r g y ( B i s h o p , B o e r m a n s et a l . , 1 9 9 8 ; S t i l l e r W i n k l e r et a l . , 1 9 9 9 ) .  B i r d s c o n t a m i n a t e d w i t h D D T s m a y be m o r e p r o n e to s u c c u m b to  infectious diseases a n d parasites.  C h i c k e n s fed 10, 3 0 , or 50 m g / k g D D T o n alternate d a y s for 8  to 38 d a y s d e m o n s t r a t e d a n i n c r e a s e d s u s c e p t i b i l i t y to the parasite  Histomonas meleagridis.  D u c k s fed 5 0 0 a n d 9 0 0 m g / k g D D T for 10 days h a d h i g h e r m o r t a l i t y rates t h a n c o n t r o l s w h e n e x p o s e d to a hepatitis v i r u s (Banerjee et a l . , 1996). I n contrast, c h i c k e n s g i v e n feed c o n t a i n i n g 5 0 0 m g / k g D D T for v a r y i n g p e r i o d s s h o w e d a n increase i n resistance to M a r e k ' s disease a n d  Mycoplasma gallisepticum.  T u r k e y s o n a s i m i l a r d i e t w e r e less s u s c e p t i b l e to h e m o r r h a g i c  enteritis v i r u s ( C o l m a n o & G r o s s , 1971). T h e n u m b e r a n d p r o p o r t i o n o f the v a r i o u s l e u k o c y t e s , or w h i t e b l o o d c e l l s , i n c l u d i n g l y m p h o c y t e s , h e t e r o p h i l s / n e u t r o p h i l s , m o n o c y t e s , b a s o p h i l s , a n d e o s i n o p h i l s , reflect the h e a l t h status o f i n d i v i d u a l s .  T h e t y p i c a l response to i n f e c t i o u s diseases i n b i r d s i s a n increase i n the  total l e u k o c y t e count, m a i n l y because o f increases i n h e t e r o p h i l s a n d l y m p h o c y t e s ( D u f v a & A l l a n d e r , 1995). Injecting 2 or 4 m g / k g p , p ' - D D E into Japanese q u a i l eggs resulted i n b i r d s w i t h h i g h e r l e u k o c y t e n u m b e r s ( Q u i n n et a l . , 2 0 0 2 ) .  C a s p i a n terns  (Sterna caspia)  f r o m the G r e a t  L a k e s s h o w e d i n c r e a s i n g h e t e r o p h i l : l y m p h o c y t e ratios w i t h i n c r e a s i n g D D E , a l t h o u g h h e r r i n g g u l l s f r o m the same areas d i d not e x h i b i t this pattern ( G r a s m a n et a l . , 1996). A n u m b e r o f other  11 h e m a t o l o g i c a l parameters m a y also be i n f l u e n c e d b y D D T c o n t a m i n a t i o n . F o u r i e a n d H a t t i n g h (1979) treated c r o w n e d g u i n e a - f o w l  (Numida meleagris)  w e i g h t v i a e s o p h a g e a l c a n u l a for s i x days.  w i t h 7 5 % pure D D T at 75 m g / k g b o d y  A n a l y s e s o f the p l a s m a f r o m these b i r d s r e v e a l e d  increased carbon d i o x i d e and cholesterol, decreased hematocrit and red b l o o d cell numbers, r e d u c e d h e m o g l o b i n , p o t a s s i u m , b l o o d sugar, a n d urea, as w e l l as e l e v a t e d a c t i v i t y o f the e n z y m e s a l k a l i n e phosphatase, l a c t i c dehydrogenase, a n d creatine p h o s p h o k i n a s e . B o t h h u m o r a l a n d c e l l - m e d i a t e d i m m u n i t y m a y be altered b y D D T exposure.  Japanese  q u a i l from eggs injected w i t h p , p ' - D D E h a d s i g n i f i c a n t l y h i g h e r b u r s a o f F a b r i c i u s w e i g h t s than c o n t r o l s , a l t h o u g h they s h o w e d n o differences i n h u m o r a l i m m u n e r e s p o n s e s ( Q u i n n et a l . , 2 0 0 2 ) . O n the other h a n d , D D T a d m i n i s t e r e d to c h i c k e n s at doses o f up to 800 m g / k g f r o m hatch to s i x w e e k s o f age, f a i l e d to affect b u r s a w e i g h t s a n d a n t i b o d y r e s p o n s e to b o v i n e s e r u m a l b u m i n , but r e d u c e d i m m u n o g l o b u l i n l e v e l s ( G l i c k , 1 9 7 4 ) .  pullorum  A n t i b o d y titers to  Salmonella  or b o v i n e s e r u m a l b u m i n antigen were s i g n i f i c a n t l y h i g h e r i n b i r d s r e c e i v i n g 50 or 125  m g / k g D D T for t w o w e e k s than i n c o n t r o l s and b i r d s r e c e i v i n g h i g h e r doses.  D D T at doses o f  100 or 5 0 0 m g / k g o v e r t w o w e e k s , d i d not i n f l u e n c e a g g l u t i n i n titers to sheep r e d b l o o d c e l l antigen i n c h i c k e n s , a l t h o u g h i m m u n o g l o b u l i n s w e r e r e d u c e d ( L a t i m e r & S i e g e l , 1974; R i s h i & G a r g , 1993).  G r a s m a n et a l . ( 1 9 9 6 ) , h o w e v e r , f o u n d n o e v i d e n c e for c o n t a m i n a n t a s s o c i a t e d  suppression o f total a n t i b o d y and i m m u n o g l o b u l i n G responses f o l l o w i n g i n o c u l a t i o n w i t h sheep r e d b l o o d c e l l s i n either h e r r i n g g u l l s or C a s p i a n terns.  T - l y m p h o c y t e s mature i n the t h y m u s ,  regulate i m m u n e responses, a n d attack v i r u s - i n f e c t e d a n d m a l i g n a n t c e l l s ( G r a s m a n et a l . , 1996). T h e p h y t o h e m a g g l u t i n i n s k i n test is often u s e d to m e a s u r e the p r o l i f e r a t i v e p o t e n t i a l o f T l y m p h o c y t e s i n response to a m i t o g e n ( S m i t s & W i l l i a m s , 1999).  Herring gulls and Caspian  terns f r o m the G r e a t L a k e s demonstrated a decrease i n p h y t o h e m a g g l u t i n i n response as D D E l e v e l s i n c r e a s e d ( G r a s m a n et a l . , 1996). A decrease i n D N A synthesis i n l y m p h o c y t e s e x p o s e d to p h y t o h e m a g g l u t i n i n w a s also f o u n d i n rabbits treated w i t h p , p ' - D D T ( K a n n a n & S h a r m a , 1979) a n d h u m a n s c o n t a m i n a t e d w i t h o , p ' - D D T ( L e e & P a r k , 1979).  (Carreta carreta)  L o g g e r h e a d sea turtles  w i t h h i g h o r g a n o c h l o r i n e l e v e l s , h o w e v e r , s h o w e d a n increase i n m i t o g e n -  i n d u c e d l y m p h o c y t e p r o l i f e r a t i o n ( K e l l e r et a l . , 2 0 0 2 ) . 1.3.3. Effects o n Stress R e s p o n s e O f a l l the e n d o c r i n e tissues, c h e m i c a l l y i n d u c e d l e s i o n s are m o s t frequent i n the adrenal g l a n d s o f b i r d s e x p o s e d to e n v i r o n m e n t a l c o n t a m i n a n t s .  T h e h i g h l i p i d content o f the a d r e n a l  c o r t e x m a k e s it p a r t i c u l a r l y susceptible ( L o r e n z e n et a l . , 1999), e s p e c i a l l y to the effects o f l i p i d  12 soluble organochlorine compounds like D D T . Significant accumulations o f D D T s have been f o u n d i n the adrenal glands o f b o t h m a m m a l s a n d b i r d s ( B i e s m a n n & v o n F a b e r , 1 9 8 1 ; L a t i m e r & S i e g e l , 1974). S o m e metabolites o f D D T h a v e been s h o w n to be a d r e n o t o x i c i n b i r d s .  Five  w e e k o l d c h i c k e n s treated w i t h as little as 5 m g / k g o f t e c h n i c a l grade D D T o v e r several w e e k s e x h i b i t e d a s i g n i f i c a n t decrease i n c o r t i c o s t e r o n e c o n c e n t r a t i o n s ( L a t i m e r & S i e g e l ,  1974;  L o r e n z e n et a l . , 1999). D D D has b e e n s h o w n to cause atrophy i n the adrenal cortex o f m a m m a l s ( B i e s m a n & v o n F a b e r , 1 9 8 1 ; J o n s s o n et a l . , 1994). O r t h o , p a r a ' - D D D (mitotane) i s so effective at d e c r e a s i n g l e v e l s o f g l u c o c o r t i c o i d s it has e v e n b e e n u s e d to treat patients s u f f e r i n g f r o m C u s h i n g ' s s y n d r o m e a n d a d r e n a l c a r c i n o m a , a n d to p e r f o r m c h e m i c a l a d r e n a l e c t o m i e s i n the treatment o f breast and prostate cancers ( P e a k a l l , 1967; T h o m a s , 1998). I n birds, h o w e v e r , D D T treatments are m o r e l i k e l y to result i n increased adrenal g l a n d w e i g h t s a n d a h i g h e r percentage o f c o r t i c a l c e l l s ( B i e s m a n n & v o n F a b e r , 1981).  P i g e o n s d o s e d w i t h p , p ' - D D E or p , p ' - D D T for  eight w e e k s e x h i b i t e d a decrease i n adrenal g l a n d w e i g h t at 6 m g / k g / d a y , but then a s i g n i f i c a n t increase w i t h i n c r e a s i n g dosage (Jefferies, 1975). T h e net effect o f the stress response is a n i n c r e a s e d a v a i l a b i l i t y o f e n e r g y , i n c r e a s e d o x y g e n intake, decreased b l o o d f l o w to b o d y areas not necessary for s u r v i v a l a n d m o v e m e n t , inhibition o f digestion, reproduction, immune function, growth, and pain perception, and enhancement o f m e m o r y a n d sensory f u n c t i o n ( N e l s o n , 2 0 0 0 ) . A n increase i n corticosterone is u s u a l l y the first s i g n i f i c a n t r e s p o n s e to stress ( P u v a d o l p i r o d & T h a x t o n , 2 0 0 0 ) .  Plasma  c o r t i c o s t e r o n e l e v e l s i n c h i c k e n s restrained b y h a n d , for e x a m p l e , b e g a n to increase w i t h i n 4 5 seconds a n d w e r e s i x - f o l d h i g h e r than b a s a l c o n c e n t r a t i o n s w i t h i n eight m i n u t e s o f h a n d l i n g ( S i e g e l , 1980).  T h y r o i d h o r m o n e s are also released i n t i m e s o f stress, as they are i m p o r t a n t  regulators o f m e t a b o l i s m ( N e l s o n , 2 0 0 0 ; S t i c k e l , 1 9 7 3 ) .  E n l a r g e d adrenal glands have been  f o u n d i n b i r d s a n d m a m m a l s w i t h h y p o f u n c t i o n i n g t h y r o i d glands, as w e l l as i n c r e a s e d l e v e l s o f adrenocorticotropic  (Jefferies,  1975).  Administration o f thyroxine may  decrease  the  c o n c e n t r a t i o n o f corticosterone b i n d i n g g l o b u l i n s , thus i n f l u e n c i n g the a c t i v i t y a n d m e t a b o l i s m o f this stress h o r m o n e ( H a r v e y et a l . , 1986). C h i c k e n e m b r y o s are capable o f secreting g l u c o c o r t i c o i d s b y day s i x o f i n c u b a t i o n . T h e y e x p e r i e n c e a surge i n corticosterone p r o d u c t i o n a r o u n d day fifteen o f i n c u b a t i o n ( H a r v e y et a l . , 1986).  T h e h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l a x i s o f d e v e l o p i n g c h i c k e m b r y o s is s e n s i t i v e to  stressors s u c h as c h a n g e s i n e n v i r o n m e n t a l t e m p e r a t u r e ( L o r e n z e n et a l . , 1 9 9 9 ) .  Levels of  corticosterone b e g i n to rise at the t i m e o f h a t c h i n g but d e c l i n e post-hatch, a n d for a b r i e f p e r i o d f o l l o w i n g h a t c h i n g , n e o n a t a l b i r d s m a y be r e l a t i v e l y i n s e n s i t i v e to e n v i r o n m e n t a l stressors  13 ( H a r v e y et a l . , 1986). S i m i l a r patterns are seen i n rats w h e r e corticosterone is secreted b y the fetus i n response to stressors d u r i n g the late fetal stage o f gestation, b u t f o r the first t w o w e e k s after b i r t h , rat p u p s h a v e e x t r e m e l y l o w l e v e l s o f a d r e n a l steroids a n d are h y p o r e s p o n s i v e to stress.  T h i s s u p p r e s s i o n o f the stress r e s p o n s e m a y be n e c e s s a r y f o r n o r m a l g r o w t h a n d  d e v e l o p m e n t ( K a w a t a , 1 9 9 5 ) , as i n c r e a s e d c o r t i c o s t e r o n e l e v e l s i n h i b i t g r o w t h a n d s k e l e t a l d e v e l o p m e n t ( S i e g e l , 1980). E l e v a t e d l e v e l s o f a d r e n o c o r t i c o t r o p i c or g l u c o c o r t i c o i d s h a v e b e e n s h o w n to result i n i n v o l u t i o n o f the a v i a n bursa and t h y m u s ( H a r v e y et a l . , 1986; S i e g e l , 1980), as a result o f B - and T - c e l l a p o p t o s i s ( L e c h n e r et a l . , 2 0 0 1 ) .  S e v e r a l studies h a v e r e p o r t e d d e p r e s s e d a n t i b o d y  p r o d u c t i o n i n stressed b i r d s ( B o l a n d e r , 1994; H a r v e y et a l . , 1 9 8 6 ; L a t i m e r & S i e g e l , 1 9 7 4 ; S i e g e l , 1980).  G l u c o c o r t i c o i d s serve as a n t i - i n f l a m m a t o r y agents, s t a b i l i z i n g l y s o s o m e s ,  lowering antibody levels, inhibiting leukocyte migration, and destroying lymphocytes (Bolander, 1994). W h i l e corticosterone l o w e r s the n u m b e r o f c i r c u l a t i n g l y m p h o c y t e s i n a v i a n b l o o d , the p o p u l a t i o n s o f heterophils a n d other granulocytes are often elevated ( H a r v e y et a l . , 1986; S i e g e l , 1980). T h e r e are a variety o f theories o n h o w D D T s exert their effects o n the adrenal glands a n d hormones.  A s D D T i s a n i n d u c e r o f m i x e d f u n c t i o n o x i d a s e e n z y m e s , it m a y d i m i n i s h the  b i o l o g i c a l a c t i v i t y o f estrogens, androgens, a n d g l u c o c o r t i c o i d s b y p r o m o t i n g their m e t a b o l i s m i n the l i v e r ( B u n y a n & S t a n l e y , 1982; K u p f e r & B u l g e r , 1976). D D T m a y i n f l u e n c e the adrenal g l a n d s i n d i r e c t l y v i a effects o n the t h y r o i d or p i t u i t a r y g l a n d s ( B i e s m a n n & v o n F a b e r , 1 9 8 1 ; Jefferies, 1975). It has e v e n b e e n suggested that D D T m a y act as a c o r t i c o s t e r o n e m i m i c a n d b i n d to corticosterone receptors, at least i n the S e n e g a l w a l k i n g frog  (Kassina senegalensis) a  potentially other a m p h i b i a n s ( H a y e s et a l . , 1997). DDT  e x p o s u r e i t s e l f m a y serve as a stressor.  S u b - l e t h a l doses o f D D T , a n d other  contaminants, have been s h o w n to p r o v o k e stress responses i n rats ( S t i c k e l , 1973). A s w e l l , the stress response m a y potentiate the t o x i c effects o f D D T . i m m u n e s u p p r e s s i o n m a y be due p r i m a r i l y to stress.  For example, in mice, D D T - i n d u c e d  Short-term, sub-lethal D D T exposure i n  c o n j u n c t i o n w i t h restraint stress y i e l d e d the same decrease i n a n t i b o d y titers as l o n g t e r m D D T exposure alone (Banerjee, 1999). D D T is stored i n a n a n i m a l ' s adipose tissue, w h i c h sequesters it a w a y from the n e r v o u s s y s t e m a n d v i t a l organs. W h e n these fat reserves are u t i l i z e d , the D D T is m o b i l i z e d .  A n y events that speed up this m o b i l i z a t i o n m a y k i l l apparently h e a l t h y a n i m a l s  l o n g after e x p o s u r e and at dosages that w o u l d not n o r m a l l y k i l l t h e m ( S t i c k e l , 1973). I n b i r d s , stressors associated w i t h m o l t i n g , m i g r a t i o n , a n d r e p r o d u c t i o n c o u l d m o b i l i z e l e t h a l l e v e l s o f  14 D D T s ( B u n y a n & S t a n l e y , 1 9 8 2 ; G i s h & C h u r a , 1 9 7 0 ; O h l e n d o r f et a l . , 1978; S t i c k e l , 1973). J a p a n e s e q u a i l p a r t i a l l y s t a r v e d p r i o r to p , p ' - D D T d o s a g e w e r e m o r e s u s c e p t i b l e to D D T i n t o x i c a t i o n than w e r e those not h u n g e r stressed.  M o r t a l i t y o f A m e r i c a n r o b i n s i n D D T treated  areas w a s h i g h e s t d u r i n g the b r e e d i n g season ( G i s h & C h u r a , 1 9 7 0 ; O h l e n d o r f et a l . , 1 9 7 8 ; Stickel, 1973).  T h e b i r d s w o u l d h a v e l o s t a c o n s i d e r a b l e a m o u n t o f w e i g h t d u r i n g the s p r i n g  m i g r a t i o n , a n d thus m o b i l i z e d a n y D D T that w a s stored i n t h e i r b o d i e s .  M a l e s were more  susceptible than females, as they c o n t i n u e d to lose w e i g h t w h i l e e s t a b l i s h i n g a n d d e f e n d i n g t h e i r b r e e d i n g territories. O n l y t w o o f 50 b r o w n - h e a d e d c o w b i r d s fed 40 m g / k g D D T for eight w e e k s d i e d d u r i n g treatment. S i x b i r d s , h o w e v e r , d i e d after b e i n g put o n a c l e a n diet, as a result o f the stress i n d u c e d b y w o r k e r s entering the cage. N o q u a i l d i e d d u r i n g s i x m o n t h s o f d o s i n g w i t h 25 m g / k g D D T , but eight d i e d w h e n the b i r d s were f o r c e d to m o l t ( S t i c k e l , 1973). It is l i k e l y that steroid homeostatic m e c h a n i s m s c a n deal w i t h m o s t sub-lethal c o n t a m i n a n t residues ( B u n y a n & Stanley, 1982), but w h e n homeostasis is disrupted b y stress, the r e s u l t i n g h o r m o n a l disturbances m a y be e n o u g h to h a v e p r o f o u n d effects o n a v a r i e t y o f systems.  E x p o s u r e to t o x i c a n t s i s o f  p a r t i c u l a r c o n c e r n d u r i n g d e v e l o p m e n t because m a n y o f the f e e d b a c k m e c h a n i s m s n e e d e d to m a i n t a i n homeostasis are not yet f u n c t i o n a l ( C r i s p et a l . , 1998).  1.3.4. Effects o n R e p r o d u c t i o n D D T e x p o s u r e has b e e n l i n k e d to r e d u c e d f e r t i l i t y , s u p p r e s s i o n o f e g g f o r m a t i o n , e g g s h e l l t h i n n i n g , i m p a i r e d i n c u b a t i o n a n d c h i c k r e a r i n g b e h a v i o r , a n d other r e p r o d u c t i v e p r o b l e m s i n b i r d s ( F r y , 1995). T h e r e i s a great d e a l o f species d i v e r s i t y i n the effects o f D D T a n d its m e t a b o l i t e s o n r e p r o d u c t i o n .  B r o w n pelicans  (Pelecanus occidentalis) are a m o n g the  most sensitive, s h o w i n g depressed reproduction w h e n egg D D E levels reach 3 p g / g and total r e p r o d u c t i v e failure w h e n l e v e l s e x c e e d 3.7 p g / g ( B l u s , 1996). D o m e s t i c c h i c k e n s , o n the other h a n d , appear to be v e r y t o l e r a n t o f D D T e x p o s u r e , a l t h o u g h reports are c o n f l i c t i n g .  For  e x a m p l e , one study reports that hens fed 25 m g / k g o f p , p ' - D D T , o , p ' - D D T or p , p ' - D D T for 28 w e e k s , f o l l o w e d b y doses o f 3 0 0 m g / k g for a n a d d i t i o n a l 12 w e e k s s h o w e d n o decrease i n their fertility, whereas another study f o u n d decreased e g g p r o d u c t i o n after t w o m o n t h s o f f e e d i n g as little as 10 m g / k g t e c h n i c a l grade D D T ( L i l l i e et a l . , 1972). F e r t i l i t y w a s r e d u c e d i n B e n g a l e s e finches fed l o w (1 - 50 pig/day/bird) or h i g h (51 - 2 5 0 p g / d a y / b i r d ) doses o f p , p ' - D D T a n d p,p'D D E for s i x w e e k s (Jefferies, 1971) a n d Japanese q u a i l fed 4 0 0 m g / k g D D T for as little as three days ( L i l l i e et a l . , 1972). Parental m o r t a l i t y i n these q u a i l w a s also increased ( L i l l i e et a l . , 1972).  15 D D E c o n t a m i n a t i o n has l o n g b e e n a s s o c i a t e d , i n a n u m b e r o f s p e c i e s , w i t h r e d u c e d e g g s h e l l q u a l i t y a n d t h i n n i n g . T h i n eggshells l e a d to a n i n c r e a s e d i n c i d e n c e o f b r o k e n eggs a n d i n e v i t a b l y , l o w e r e d r e p r o d u c t i v e success ( C e c i l et a l . , 1 9 7 1 ; S t i c k e l , 1 9 7 3 ; W H O , 1989). I n w i l d b i r d p o p u l a t i o n s , average e g g s h e l l t h i n n i n g greater than or e q u a l to 1 8 % , o v e r several years, has been l i n k e d to p o p u l a t i o n d e c l i n e s ( B l u s & H e n n y , 1997). W h i l e D D E adversely affects e g g s h e l l q u a l i t y , it does not seem to affect the size or shape o f the eggs. T h e effects o f D D E o n e g g s h e l l t h i c k n e s s are species s p e c i f i c , w i t h G a l l i f o r m s a n d L a r i d s s h o w i n g l i t t l e s e n s i t i v i t y ( B l u s et a l , 1997). D D T s m a y also i n d u c e c h a n g e s i n the t i m i n g o f r e p r o d u c t i v e events. d o v e s , Japanese q u a i l , a n d B e n g a l e s e  finches  Mallards, ring  h a v e d e m o n s t r a t e d d e l a y s i n e g g l a y i n g after  d o s i n g w i t h D D E or D D T ( C e c i l et a l . , 1 9 7 1 ; Jefferies, 1 9 7 1 ; W H O , 1989).  I n the  finch,  this  d e l a y b e t w e e n p a i r i n g a n d o v u l a t i o n w a s s t i l l present i n the s e c o n d generation (Jefferies, 1971). It is p o s s i b l e that the delay i n the r i n g d o v e s w a s due to l o n g e r retention o f the egg i n the o v i d u c t i n the absence o f the proper l a y i n g stimulus, a n adequate nest ( W H O , 1989).  1.3.4.1 Hormonal  Influences  M a n y o f the d e t r i m e n t a l effects o f D D T s o n r e p r o d u c t i o n are related to their e n d o c r i n e d i s r u p t i n g c a p a b i l i t i e s . T h e s e substances c a n act b y :  1) m i m i c k i n g e n d o g e n o u s h o r m o n e s , 2)  a n t a g o n i z i n g n o r m a l endogenous h o r m o n e s , 3) a l t e r i n g the pattern o f synthesis a n d m e t a b o l i s m o f n a t u r a l h o r m o n e s , a n d 4) m o d i f y i n g h o r m o n e receptor l e v e l s ( J i m e n e z , 1 9 9 7 ; K e l c e et a l . , 1998; Sonnenschein & Soto, 1998).  T h e o,p' i s o m e r o f D D T i s k n o w n to b i n d d i r e c t l y to  e s t r o g e n receptors, as d o e s o , p ' - D D D ( G a i d o et a l . , 1 9 9 7 ) ; the p , p ' i s o m e r o f D D T d o e s n o t ( R o b i s o n et a l . , 1984). O r t h o , p a r a ' - D D T c a n compete w i t h estradiol for b i n d i n g to the estrogen r e c e p t o r a n d d e m o n s t r a t e s e s t r o g e n - l i k e a c t i v i t y in vitro a n d in  vivo ( G a i d o et a l . , 1 9 9 7 ) .  P a r a , p a r a ' - D D E is an a n d r o g e n antagonist that b i n d s d i r e c t l y to a n d r o g e n receptors ( G a i d o et a l . , 1 9 9 7 ; K e l c e et a l . , 1 9 9 5 ; 1 9 9 8 ) .  T h e c o n c e n t r a t i o n o f p , p ' - D D E n e e d e d to i n h i b i t a n d r o g e n  receptor t r a n s c r i p t i o n a l a c t i v i t y i n c e l l culture is 63.6 parts per b i l l i o n , c o n s i d e r a b l y less t h a n l e v e l s that a c c u m u l a t e i n the e n v i r o n m e n t ( K e l c e et a l . , 1995). P a r a , p a r a ' - D D E has b e e n s h o w n to have v a r y i n g degrees o f a n d r o g e n i c , anti-androgenic, estrogenic, a n d anit-estrogenic a c t i v i t y ( H u t z , 1999; S o h o n i & S u m p t e r , 1998; S o n n e n s c h e i n & S o t o , 1998). T h e a n d r o g e n receptor is less s p e c i f i c than the estrogen receptor, so o , p ' - D D T a n d e n d o g e n o u s estrogens c a n also h a v e a n t i - a n d r o g e n i c capacities ( K e l c e et a l . , 1 9 9 5 ; S o h o n i & S u m p t e r , 1998). T h e presence o f a n t i androgens c a n create a n estrogenic e n v i r o n m e n t r e s u l t i n g i n s y m p t o m s i n d i c a t i v e o f estrogen  16 exposure ( S o h o n i & S u m p t e r , 1998). D D T s m a y i n f l u e n c e the l e v e l s o f g o n a d a l h o r m o n e s b y i n d u c i n g hepatic steroid h y d r o x y l a s e s ( S t i c k e l , 1973; T h o m a s , 1998) a n d i n h i b i t i n g g o n a d o t r o p i n secretion (Rattner et a l . , 1984).  O t h e r h o r m o n e s , i n c l u d i n g t h y r o i d h o r m o n e s , also i n f l u e n c e  r e p r o d u c t i o n a n d m a y be altered b y D D T e x p o s u r e ( C o o k e , 1996; H u r s t et a l . , 1974; N e l s o n , 2 0 0 0 ; W i l s o n & D o n h a m , 1988; W e n t w o r t h & R i n g e r , 1986).  1.3.4.2. Early Exposure In ovo  a n d e a r l y p o s t - h a t c h e x p o s u r e to D D T s m a y h a v e p r o f o u n d effects  d e v e l o p m e n t a n d s e x u a l d i f f e r e n t i a t i o n o f the r e p r o d u c t i v e systems o f b i r d s .  o n the  Male California  g u l l s f r o m eggs injected w i t h as little as 2 m g / k g o , p ' - D D T h a d f e m i n i z e d gonads. F i v e m g / k g or h i g h e r o , p ' - D D T resulted i n the d e v e l o p m e n t o f b o t h left a n d right o v i d u c t s i n female g u l l s ( F r y & T o o n e , 1981). I n c h i c k e n s , p , p ' - D D T injected into the y o l k p r i o r to i n c u b a t i o n altered g o n a d a l m o r p h o l o g y i n b o t h sexes ( S t i c k e l , 1973). F o u r w e e k o l d w h i t e l e g h o r n c o c k e r e l s fed D D T 12.5 to 37.5 m g / k g b o d y w e i g h t for 2 4 w e e k s , h a d a t r o p h i e d testes, a n d e x h i b i t e d a d i s r u p t i o n a n d n e c r o s i s o f s p e r m a t o g o n i a l c e l l s . T h e s e effects persisted e v e n after treatment w a s d i s c o n t i n u e d ( B a l a s u b r a m a n i a m & Sundararaj, 1993).  C o c k e r e l s injected w i t h i n c r e a s i n g a m o u n t s o f D D T  f r o m eight days after hatch for u p to 89 days h a d c o n s i d e r a b l y s m a l l e r testes than c o n t r o l s .  The  d e v e l o p m e n t o f c o m b s a n d wattles i n these birds w a s also severely i m p a i r e d , b e g i n n i n g 25 days after the start o f treatment ( B u r l i n g t o n & L i n d e m a n , 1950). 1.3.5. Effects o n B e h a v i o r A n organism's b e h a v i o r represents the final integrated result o f a v a r i e t y o f p h y s i o l o g i c a l a n d b i o c h e m i c a l processes ( P e a k a l l , 1996; S p y k e r , 1975).  A s b e h a v i o r patterns c a n be h i g h l y  sensitive to changes i n the steady state o f a n o r g a n i s m , changes i n b e h a v i o r c a n serve as e a r l y i n d i c a t o r s o f p o t e n t i a l p r o b l e m s ( P e a k a l l , 1996; S p y k e r , 1975). N e r v o u s tissue, e s p e c i a l l y the b r a i n , is e x t r e m e l y sensitive to the effects o f a v a r i e t y o f f o r e i g n substances, s u c h as pollutants. C h a n g e s i n b e h a v i o r m a y i n d i c a t e t o x i c a c t i o n s o f a c h e m i c a l l o n g before a n y c l a s s i c a l s y m p t o m s o f p o i s o n i n g o c c u r a n d i n the absence o f gross f u n c t i o n a l or structural defects.  Toxic  effects i n the n e r v o u s s y s t e m m a y m a n i f e s t as d e f i c i t s i n s e n s o r y f u n c t i o n , m o t o r c o n t r o l , intellectual processes, or e m o t i o n a l responses ( S p y k e r , 1975). O r g a n i s m s are m o r e v u l n e r a b l e to m a n y o f the adverse effects o f e x o g e n o u s c h e m i c a l s during development,  w h e n the c e n t r a l n e r v o u s  system is u n d e r g o i n g r a p i d changes  p r o t e c t i v e m e c h a n i s m s h a v e not yet d e v e l o p e d ( P a r m i g i a n i et a l . , 1998; S p y k e r , 1975).  and  Some  p o l l u t a n t s , at e n v i r o n m e n t a l l y r e l e v a n t l e v e l s , c a n l e a d to i r r e v e r s i b l e a l t e r a t i o n s i n b r a i n  17 d e v e l o p m e n t at exposure concentrations that m i g h t p r o d u c e l i t t l e effect i n a n adult ( P a r m i g i a n i et a l . , 1998).  T h e effects o f these c o m p o u n d s m a y not b e c o m e apparent u n t i l m u c h later i n a n  i n d i v i d u a l ' s life ( S p y k e r , 1975). A s b e h a v i o r represents a n integrated response o f a n o r g a n i s m , it m a y also be i n f l u e n c e d b y d e f i c i t s i n the f u n c t i o n o f systems other t h a n the n e r v o u s s y s t e m . A l t h o u g h not a l l b e h a v i o r s m a y be affected, defects i n those r e l a t i n g to s u r v i v a l a n d r e p r o d u c t i o n m a y p r o v e to be e x t r e m e l y disadvantageous ( S p y k e r , 1975). Subtle b e h a v i o r a l changes i n w i l d p o p u l a t i o n s c o u l d e v e n cause serious effects at l o w l e v e l s o f pollutants w h i c h d o not have overt effects o n m o r t a l i t y or r e p r o d u c t i o n ( P e a k a l l , 1996).  1.3.5.1. Reproductive Behaviors A n u m b e r o f b e h a v i o r s i n v o l v e d i n r e p r o d u c t i o n m a y be d e t r i m e n t a l l y affected b y D D T exposure. F o r e x a m p l e , d u r i n g the 1970s, m a l e g u l l s i n h i g h l y c o n t a m i n a t e d areas o f C a l i f o r n i a a n d i n the G r e a t L a k e s e x h i b i t e d f e m i n i z e d r e p r o d u c t i v e systems.  These birds showed reduced  s e x u a l b e h a v i o r a n d m a n y o f t h e m f a i l e d to e v e n migrate to the b r e e d i n g grounds. T h i s resulted i n s k e w e d sex ratios a n d h o m o s e x u a l p a i r i n g s b e t w e e n the females ( F o x , 1992; F r y & T o o n e , 1981). H e r r i n g g u l l s e x p o s e d to a m i x t u r e o f o r g a n o c h l o r i n e s s h o w e d a r e d u c t i o n i n defense o f their territories.  A s a g g r e s s i o n is r e l a t e d to the c o n c e n t r a t i o n o f c i r c u l a t i n g testosterone,  e n d o c r i n e d i s r u p t i n g c h e m i c a l s l i k e p , p ' - D D E that act i n a n anti-androgenic m a n n e r c o u l d cause aggressive b e h a v i o r to d i m i n i s h ( C r a i n & G u i l e t t e , 1997). Japanese q u a i l treated w i t h o , p ' - D D T s h o w e d an attenuation o f m a t i n g b e h a v i o r s i n b o t h sexes ( B r y a n et a l . , 1989). R i n g d o v e s fed a m i x t u r e o f o r g a n o c h l o r i n e s , i n c l u d i n g D D E d i s p l a y e d a series o f b e h a v i o r a l a b n o r m a l i t i e s . B e c a u s e the b e h a v i o r s o f the t w o sexes i n this species are so c l o s e l y l i n k e d , alterations i n the b e h a v i o r o f one c a u s e d changes i n the other.  T h e b i r d s i n this study s h o w e d a h increase i n the  courtship p e r i o d due to the females' failure to r e s p o n d to the m a l e s ' petitions for c o p u l a t i o n . T h i s forced the m a l e s to increase a n d p r o l o n g the use o f these s o l i c i t a t i o n b e h a v i o r s ( M c A r t h u r et a l . , 1983).  T h e s e b i r d s also s h o w e d a r e d u c t i o n or d e l a y i n the b e h a v i o r a l l y i n d u c e d increase i n  progesterone w h i c h l e a d to a d e l a y i n nest b u i l d i n g , o v u l a t i o n , a n d i n c u b a t i o n ( M c A r t h u r et a l . , 1983).  1.3.5.2. Parental Behaviors P a r e n t a l care b e h a v i o r s appear to be m o s t i n f l u e n c e d b y D D T c o n t a m i n a t i o n . M e r l i n s  (Falco columbarius) w h o s e eggs c o n t a i n e d h i g h l e v e l s o f o r g a n o c h l o r i n e p o l l u t a n t s , e s p e c i a l l y p , p ' - D D E , h a v e b e e n s h o w n to desert their c l u t c h e s m o r e r e a d i l y a n d d e f e n d their nests less a c t i v e l y than b i r d s w i t h less c o n t a m i n a t e d eggs.  T h e r e w a s a n i n v e r s e r e l a t i o n s h i p b e t w e e n the  18 D D E burdens i n the eggs a n d the intensity o f nest site defense ( F o x & D o n a l d , 1980). H e r r i n g g u l l s e x p o s e d to a n u m b e r o f c h e m i c a l c o n t a m i n a n t s h a v e a l s o d e m o n s t r a t e d d e c r e a s e d nest attentiveness.  T h e y a p p l i e d less heat to their eggs, w e r e absent f r o m the nest m o r e frequently  and for l o n g e r p e r i o d s , a n d m o r e r e a d i l y left their nests than b i r d s f r o m less c o n t a m i n a t e d areas. T h e degree o f nest inattentiveness w a s correlated w i t h the female's o r g a n o c h l o r i n e burdens ( F o x et a l . , 1978; Rattner et a l . , 1984). P r o l a c t i n l e v e l s w e r e affected b y o r g a n o c h l o r i n e diets i n r i n g d o v e s , d e c r e a s i n g the a m o u n t o f t i m e spent i n c u b a t i n g the eggs a n d b r o o d i n g the y o u n g .  The  squabs o f c o n t a m i n a t e d parents w e r e fed less a n d thus h a d decreased w e i g h t s a n d s u r v i v a l rates ( M c A r t h u r e t a l . , 1983). H y p e r - a g g r e s s i v e parents i n j u r e d s o m e o f the squabs i n M c A r t h u r et al.'s ( 1 9 8 3 ) study. Increased parental a g g r e s s i o n w a s also o b s e r v e d i n B e n g a l e s e finches d o s e d w i t h p , p ' - D D T a n d p , p ' - D D E (Jefferies, 1971).  Parent b i r d s c o n t a m i n a t e d w i t h D D T s a n d other c o m p o u n d s m a y  destroy their o w n eggs b y eating, b r e a k i n g , or ejecting t h e m ( S t i c k e l , 1973). G r a y herons (Ardea cinerea) h a v e b e e n o b s e r v e d b r e a k i n g t h e i r o w n eggs a n d d r o p p i n g l i v e y o u n g f r o m the nest ( O h l e n d o r f et a l . , 1 9 7 8 ) .  T h i s increase i n a g g r e s s i v e b e h a v i o r s e e m s c o n t r a r y to the a n t i -  a n d r o g e n i c , a g g r e s s i o n decreasing effects o f D D E . H o w e v e r , g o n a d a l h o r m o n e s are not the o n l y aspects o f the n e r v o u s a n d e n d o c r i n e systems i n f l u e n c e d b y D D T c o n t a m i n a t i o n . M c A r t h u r et al.'s ( 1 9 8 3 ) p i g e o n s also d e m o n s t r a t e d e l e v a t e d t h y r o i d h o r m o n e l e v e l s a n d d e c r e a s e d b r a i n l e v e l s o f the b i o g e n i c a m i n e s , d o p a m i n e , a n d n o r e p i n e p h r i n e , w h i c h i n f l u e n c e h y p o t h a l a m i c a n d pituitary hormones.  H i g h levels o f corticosterone, can also redirect behaviors away f r o m  r e p r o d u c t i o n a n d parental care a c t i v i t i e s ( S i l v e r i n , 1990).  T h u s , the increase i n a g g r e s s i o n i n  D D T c o n t a m i n a t e d parents m a y be related to one or m o r e o f these other factors.  O v e r a l l , birds  that e x h i b i t a b n o r m a l c o u r t s h i p a n d nest c o n s t r u c t i o n , altered b r e e d i n g s y n c h r o n y , d e c r e a s e d i n c u b a t i o n attentiveness  a n d p a r e n t a l care m a y alter t h e i r r e p r o d u c t i v e s u c c e s s ,  increase  energetic costs, a n d decrease r e p r o d u c t i v e fitness (Rattner et a l . , 1984). 1.3.5.3.  Other Behaviors  O t h e r , n o n - r e p r o d u c t i v e , b e h a v i o r s m a y also be i n f l u e n c e d b y D D T s .  F o r e x a m p l e , the  d u c k l i n g s o f m a l l a r d s fed D D E w e r e h y p e r - r e s p o n s i v e to m a t e r n a l c a l l s . T h e y a p p r o a c h e d the source o f the c a l l m o r e a n d stayed near it m o r e t h a n c o n t r o l d u c k l i n g s . shorter d i s t a n c e s f r o m f r i g h t e n i n g s t i m u l i ( O h l e n d o r f et a l . , 1 9 7 8 ) .  T h e y also traveled  Hyper-excitability and  h y p e r s e n s i t i v i t y to s t i m u l i are c o m m o n l y l i s t e d a m o n g the s y m p t o m s o f D D T p o i s o n i n g i n m a m m a l s , a n d s i m i l a r results are l i k e l y i n b i r d s (Jefferies, 1 9 7 5 ) .  H y p e r a c t i v i t y has b e e n  19 suggested as a p o s s i b l e r e a s o n for the nest inattentiveness o f o r g a h o c h l o r i n e f e d r i n g d o v e s ( M c A r t h u r et a l . , 1983). P e s t i c i d e use m a y reduce the f o o d s u p p l i e s o f s o m e b i r d s . T h i s w o u l d result i n a n increase i n t i m e spent f o r a g i n g ( B l u s & H e n n y , 1997; F o x & D o n a l d , 1980). A l a c k o f appetite has b e e n n o t e d i n p i g e o n s o r a l l y d o s e d w i t h p , p ' - D D E , p , p ' - D D T , o r o , p ' - D D T (Jefferies, 1 9 7 5 ; Jefferies & F r e n c h 1971). T h e a m o u n t o f c o v e r m a y also be r e d u c e d , affecting nest b u i l d i n g a n d nest p r e d a t i o n ( B l u s & H e n n y , 1997). T h u s , D D T s h a v e b e e n s h o w n to h a v e a n u m b e r o f d e t r i m e n t a l effects o n b i r d s .  The  f o l l o w i n g chapters w i l l focus o n a particular e x a m p l e o f D D T exposure i n a b i r d species and its effects o n s u r v i v a l , g r o w t h , r e p r o d u c t i o n , b e h a v i o r , i m m u n i t y , t h y r o i d h o r m o n e s , a n d the stress response.  20  1.4 References A n d r e w s , J. E . , Smith, C . A . , & Sinclair, A . H . (1997). aromatase e x p r e s s i o n i n the c h i c k e n e m b r y o .  Sites o f estrogen receptor a n d  General and Comparative Endocrinology, 108  182-190. B a l a s u b r a m a n i a m , G . A . , Sundararaj, A . (1993). 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A s the O k a n a g a n V a l l e y o f southern  B r i t i s h C o l u m b i a i s a m a j o r fruit g r o w i n g area, it w a s e x p o s e d to large q u a n t i t i e s o f t h i s p e s t i c i d e . P r i o r to the r e s t r i c t i o n o f D D T use i n the early 1970's, O k a n a g a n o r c h a r d s r e c e i v e d t w o to f o u r a p p l i c a t i o n s o f about 13.5 k g t e c h n i c a l grade D D T / h a / y e a r (3.4 - 6.8 k g a c t i v e ingredient/ha/year) ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ) .  2.1. Soil H a r r i s et a l . (2000) reported that s o i l i n the O k a n a g a n consists o f a t h i n s u p e r f i c i a l l a y e r o f about 10 c m above a dense sand/gravel m i x t u r e . W i t h i n O k a n a g a n orchards, this top 10 c m o f s o i l c o n t a i n e d 3 . 8 % o r g a n i c matter a n d a p H o f 6.4.  D e s p i t e the fact that D D T h a d not b e e n  a p p l i e d to these orchards i n n e a r l y thirty years, they f o u n d 0.25 m g / k g D D D (o,p' p l u s p,p', d r y w e i g h t ) , 4.9 m g / k g D D E , a n d 9.3 m g / k g D D T i n the t o p l a y e r o f s o i l .  I n contrast, at a n  O k a n a g a n n o n - o r c h a r d reference site D D D , D D E , a n d D D T l e v e l s w e r e less t h a n 0.05 m g / k g . S o i l p H at this site ranged f r o m 7.8 to 8.0 and the average o r g a n i c matter w a s 4 . 7 % . T h e ratio o f D D E to D D T i n the o r c h a r d s w a s o n l y 1.10 ( H a r r i s et a l . , 2 0 0 0 ) .  It has b e e n suggested that  fifteen t o t w e n t y years after D D T use has stopped, t h i s r a t i o s h o u l d e x c e e d 20:1 ( E l l i o t t et a l . , 1994).  T h u s , i n the O k a n a g a n , d e g r a d a t i o n o f D D T to D D E appears to be i m p e d e d .  DDT  r e s i d u e s w e r e o n l y f o u n d i n the s o i l s o f p r e v i o u s l y s p r a y e d areas, w h i c h s u g g e s t s that a t m o s p h e r i c d e p o s i t i o n does not contribute s i g n i f i c a n t l y to the D D T l e v e l s f o u n d ( H a r r i s et a l . , 2000). A n u m b e r o f factors i n f l u e n c e the extent to w h i c h p e s t i c i d e s w i l l r e m a i n i n the s o i l , i n c l u d i n g : s o i l type, temperature, m o i s t u r e , p H , d e g r a d a b i l i t y o f the p e s t i c i d e , m i c r o o r g a n i s m content, c o v e r c r o p s , a n d c u l t i v a t i o n ( M u r p h y , 1980).  W h e n o r c h a r d trees are s p r a y e d , m o r e  than 8 0 % o f the p e s t i c i d e m a y e n d u p o n the g r o u n d ( S t r i n g e r et a l . , 1974). D D T m a y be lost s o o n after a p p l i c a t i o n v i a v o l a t i l i z a t i o n ( B l u s et a l . , 1987; W H O , 1989), h o w e v e r , due to D D T ' s l o w v a p o r pressure, v o l a t i l i z a t i o n is l i m i t e d ( N a i r et a l . , 1 9 9 2 ) .  A l l soils show a strong  a d s o r p t i v e c a p a c i t y for D D T ( W H O , 1989), but h i g h l y o r g a n i c s o i l s t e n d to h a v e the highest r e t e n t i o n t i m e s ( J o h n s o n et a l . , 1976; S t r i n g e r et a l . , 1974; W H O , 1 9 8 9 ) .  E v e n several years  31 after D D T a p p l i c a t i o n , the majority o f D D T residues f o u n d i n s o i l is located w i t h i n the top 10 c m ( C o o k e & S t r i n g e r , 1 9 8 2 ; S t r i n g e r et a l . , 1974).  T h e l o w water solubility o f D D T prevents  s i g n i f i c a n t p e r c o l a t i o n a n d l e a c h i n g ( S t r i n g e r et a l . , 1974), so d o w n w a r d m o v e m e n t o f D D T o c c u r s as litter accumulates at the s o i l surface ( D i m o n d et a l . , 1970). It has been suggested that the b r e a k d o w n o f D D T to D D E is the o n l y s i g n i f i c a n t change this p e s t i c i d e undergoes i n s o i l , as a d s o r p t i o n to the top 10 c m o f s o i l i m p e d e s v o l a t i l i z a t i o n a n d losses t h r o u g h l e a c h i n g are n e g l i g i b l e ( C o o k e & Stringer, 1982). S o m e m i c r o o r g a n i s m s are capable o f d e g r a d i n g D D T , but there is little m e t a b o l i s m i n situ ( W H O , 1989). B e c a u s e o f the v a r i o u s factors that i n f l u e n c e the l o s s a n d b r e a k d o w n o f D D T i n s o i l , a w i d e range o f h a l f - l i f e v a l u e s h a v e b e e n p r o p o s e d , f r o m 16 d a y s ( W H O , 1989) to 57.5 y e a r s ( B l u s et a l . , 1987).  C o n c e n t r a t e d D D T breaks d o w n m o r e s l o w l y ( H a r r i s et a l . , 2 0 0 0 ) , so the  h a l f - l i f e o f D D T increases w i t h i n c r e a s i n g c o n c e n t r a t i o n s ( B l u s et a l . , 1987).  T h u s , it is not  s u r p r i s i n g g i v e n the h e a v y a p p l i c a t i o n s o f D D T O k a n a g a n orchards r e c e i v e d that D D T l e v e l s i n this area r e m a i n h i g h t h i r t y years after D D T use w a s d i s c o n t i n u e d .  The lack o f regular soil  disturbances i n orchards m i n i m i z e s losses f r o m v o l a t i l i z a t i o n a n d e r o s i o n . O k a n a g a n m a y also contribute to the s l o w e l i m i n a t i o n o f D D T .  T h e c l i m a t e i n the  A s the O k a n a g a n is temperate,  l o w temperatures d u r i n g the w i n t e r m o n t h s m a y be i m p e d i n g D D T b r e a k d o w n ( H a r r i s et a l . , 2 0 0 0 ) . D D T breaks d o w n m o r e r e a d i l y i n w a r m e r , t r o p i c a l c l i m a t e s ( M e l l a n b y , 1992) a n d i s also subject to greater v o l a t i l i z a t i o n ( N a i r et a l . , 1992). A l t h o u g h O k a n a g a n orchards are i r r i g a t e d i n the s u m m e r , l o w s o i l m o i s t u r e l e v e l s i n the w i n t e r reduce the b r e a k d o w n o f D D T a c c u m u l a t i n g o r g a n i c matter and s l o w the m i c r o b i a l degradation o f D D T ( H a r r i s et a l . , 2 0 0 0 ) .  2.2. Earthworms A n o t h e r factor that i n f l u e n c e s the retention o f D D T i n s o i l s is the presence a n d species composition o f earthworms.  E a r t h w o r m s p l a y a n i m p o r t a n t r o l e i n the m a i n t e n a n c e o f s o i l  fertility b y i n c o r p o r a t i n g l e a f litter, a n d i m p r o v i n g water h o l d i n g c a p a c i t y , nutrient e n r i c h m e n t , a e r a t i o n , d r a i n a g e , r o o t p e n e t r a t i o n , a n d m i c r o o r g a n i s m p o p u l a t i o n s ( C o o k e et a l . , 1 9 8 0 ) . E a r t h w o r m s are also i n d i c a t o r s o f s o i l p o l l u t i o n b y o r g a n o c h l o r i n e pesticides ( S e n t h i l k u m a r et a l . , 2 0 0 1 ) . D D T m a y k i l l e a r t h w o r m s ( J o h n s o n et a l . , 1 9 7 6 ; T o m l i n , 1992) a n d w o r m s f o r c e d to l i v e i n D D T c o n t a m i n a t e d o r c h a r d s o i l h a v e b e e n k n o w n to e x h i b i t h i g h m o r t a l i t y rates a n d w e i g h t loss ( J o h n s o n et a l . , 1976). H o w e v e r , m o s t reports suggest that e a r t h w o r m s are r e l a t i v e l y i n s e n s i t i v e to the t o x i c effects o f D D T ( G i s h & H u g h e s , 1 9 8 2 ; W H O , 1 9 8 9 ) , a n d m a y e v e n d e v e l o p p h y s i o l o g i c a l , b i o c h e m i c a l , or b e h a v i o r a l resistance to e c o t o x i c a n t s ( T o m l i n , 1992).  32 T h e b e h a v i o r o f a n e a r t h w o r m m a y greatly affect its s u s c e p t i b i l i t y to D D T . F o r e x a m p l e , the surface m a t i n g a n d f e e d i n g o f  Lumbricus terrestris m a k e s it v u l n e r a b l e to c h e m i c a l s a p p l i e d  to v e g e t a t i o n or the s o i l surface.  T h i s species also has s l o w g r o w t h a n d r e p r o d u c t i o n rates.  habit o f b u r r o w i n g v e r t i c a l l y a n d c a s t i n g o n the surface a l l o w s it to translocate p o l l u t a n t s the s o i l surface to l o w e r l a y e r s a n d v i c e v e r s a ( T o m l i n , 1 9 9 2 ) .  Its from  E a r t h w o r m species that h a v e  permanent b u r r o w s take up D D T from the s o i l surface m o r e r e a d i l y than residues m i x e d into the s o i l , w h e r e a s species that t u n n e l h a p h a z a r d l y t h r o u g h the s o i l c a n take u p m o r e residues that h a v e b e e n m i x e d i n t o the s o i l ( E d w a r d s & Jeffs, 1974).  T h u s , p l o u g h i n g or r o t o v a t i o n after  s p r a y i n g i n c o r p o r a t e s D D T into the s o i l , d e c r e a s i n g the e x p o s u r e o f w o r m s that feed at the surface but i n c r e a s i n g exposure for subterranean species ( C o o k e et a l . , 1992). T h e uptake o f D D T b y e a r t h w o r m s is related not o n l y to its c o n c e n t r a t i o n i n the s o i l , but also to the a c t i v i t y o f the w o r m s ( W H O , 1989). D D T residues i n e a r t h w o r m s tend to be c y c l i c , w i t h h i g h e r l e v e l s b e t w e e n late s p r i n g a n d e a r l y a u t u m n a n d l o w e r l e v e l s f r o m late a u t u m n to early spring.  R e s i d u e l e v e l s p e a k i n M a y a n d are at t h e i r l o w e s t l e v e l s i n J a n u a r y .  This  c o i n c i d e s w i t h seasonal differences i n a c t i v i t y ( G i s h & H u g h e s , 1982; W H O , 1989). W h e n the w o r m s are m o r e a c t i v e they p r o c e s s m o r e s o i l t h r o u g h the gut a n d r e t a i n m o r e D D T ( W H O , 1989).  G i s h a n d H u g h e s ( 1 9 8 2 ) f o u n d that e a r t h w o r m s d i d n o t s h o w m a x i m u m r e s i d u e s  i m m e d i a t e l y after a p p l i c a t i o n , but rather t w o m o n t h s later. D D T c a n be c o n v e r t e d to b o t h D D D a n d D D E i n w o r m s ( E d w a r d s & Jeffs, 1974; G i s h & H u g h e s , 1 9 8 2 ) .  E a r t h w o r m s m a y , thus,  contribute s i g n i f i c a n t l y to the i n i t i a l degradation o f D D T ( E d w a r d s & Jeffs, 1974). H a r r i s et a l . (2000) f o u n d that e a r t h w o r m c o m m u n i t i e s i n the O k a n a g a n w e r e d o m i n a t e d  by Aporrectodea turgida, but Eisenia rosea, Lumbricus rubellus, and Octolasion tyrtaeum w also present. T h e total e a r t h w o r m b i o m a s s i n o r c h a r d areas w a s 16 g / m , w h e r e a s i n the n o n 2  o r c h a r d reference site it w a s 87 g / m . O v e r a l l , e a r t h w o r m s i n the O k a n a g a n orchards c o n t a i n e d 2  2.2 m g / k g D D D , 43.5 m g / k g D D E , 17.2 m g / k g D D T , w i t h a D D E : D D T ratio o f 2.56. D D D , D D E , a n d D D T l e v e l s i n the w o r m s f r o m the O k a n a g a n reference site w e r e b e l o w 0.6 m g / k g . E a r t h w o r m D D E v a l u e s increased w i t h i n c r e a s i n g s o i l o r g a n i c matter ( H a r r i s et a l . , 2 0 0 0 ) .  The  m a j o r i t y o f e a r t h w o r m s i n the O k a n a g a n l i v e a n d feed near the s o i l surface, thus h e l p i n g to m a i n t a i n the h i g h D D T l e v e l s i n the top l a y e r o f s o i l . O t h e r species that b u r r o w d e e p l y into the s o i l facilitate the d i l u t i o n o f pesticides throughout the s o i l p r o f i l e ( E l l i o t t et a l . , 1994). B e c a u s e o f the h i g h D D T residues earthworms c a n tolerate, they pose a s i g n i f i c a n t h a z a r d to predators ( W H O , 1989). R o b e r t s o n a n d A l e x a n d e r ( 1 9 9 8 ) f o u n d that the b i o a v a i l a b i l i t y o f D D T to flies a n d c o c k r o a c h e s decreased w i t h t i m e as the c h e m i c a l adsorbed to the s o i l , h o w e v e r ,  33 this l i k e l y does not a p p l y to a n i m a l s l i k e earthworms that l i v e i n a n d c o n s u m e s o i l .  Earthworms  i n c o n t a m i n a t e d s o i l s c o n t i n u e to a c c u m u l a t e D D T s n e a r l y t h i r t y y e a r s after its use d i s c o n t i n u e d ( H a r r i s et a l . , 2 0 0 0 ) .  D D T contamination o f earthworms can adversely  was affect  vertebrate predators either d i r e c t l y t h r o u g h secondary p o i s o n i n g or i n d i r e c t l y b y r e d u c i n g f o o d s u p p l i e s ( C o o k e et a l . , 1 9 9 2 ) . a c c u m u l a t e residues.  W o r m s that d o not die f r o m D D T p o i s o n i n g h a v e l o n g e r to  S u b - l e t h a l l e v e l s m a y also alter a n e a r t h w o r m ' s b e h a v i o u r . A l t h o u g h any  b e h a v i o u r s that result i n the w o r m s s p e n d i n g m o r e t i m e o n the surface m a y increase predator v u l n e r a b i l i t y ( C o o k e et a l . , 1992), D D T m a y e l i c i t a n a v o i d a n c e r e a c t i o n i n e a r t h w o r m s ( T o m l i n , 1992). B o t h surface c a s t i n g and the t u r n o v e r o f l e a f litter w e r e r e d u c e d i n D D T c o n t a m i n a t e d areas ( C o o k e et a l . , 1980; T o m l i n , 1992). T h i s a v o i d a n c e o f the s o i l surface m a y also l i m i t their a v a i l a b i l i t y as a f o o d source. B i r d predators m a y p i c k u p s i g n i f i c a n t l e v e l s o f D D T f r o m e a t i n g e a r t h w o r m s .  It has  b e e n suggested that 8 m g / k g D D T is a h a z a r d o u s t h r e s h o l d f o r b i r d s a n d 3 0 to 4 0 m g / k g represents a l e t h a l short-term h a z a r d .  T o t a l D D T concentrations o f 32 m g / k g i n w o r m s c o u l d  p o s e a h a z a r d t o the r e p r o d u c t i o n o f s o m e b i r d s p e c i e s ( H a r r i s et a l . , 2 0 0 0 ) .  Residues i n  e a r t h w o r m s f r o m the O k a n a g a n m a y e x c e e d this l e v e l . B i r d deaths h a v e b e e n attributed to the m o b i l i z a t i o n o f D D T i n fat reservoirs d u r i n g t i m e s o f r e p r o d u c t i v e stress, but the fact that f o o d intake is also increased d u r i n g the b r e e d i n g season m a y p l a y a role ( C o o k e et a l . , 1992).  2.3. American Robins A n i m p o r t a n t c o n s u m e r o f e a r t h w o r m s is the A m e r i c a n r o b i n . T h i s u b i q u i t o u s s o n g b i r d has adapted w e l l to h u m a n a c t i v i t i e s a n d is quite at h o m e i n o r c h a r d habitats ( C a n n i n g s et a l . , 1987). R o b i n s are o p p o r t u n i s t i c o m n i v o r e s w h o subsist p r i m a r i l y o n fruit t h r o u g h the s u m m e r and fall.  D u r i n g the b r e e d i n g season, h o w e v e r , they eat a n d feed their y o u n g p r i m a r i l y insects  a n d e a r t h w o r m s ( E h r l i c h et a l . , 1988; S a l l a b a n k s & J a m e s , 1 9 9 9 ; W a u e r , 1999). M o n t g o m e r i e a n d W e a t h e r h e a d (1997) reported that e a r t h w o r m s m a y c o m p r i s e up to 2 0 % o f a r o b i n ' s diet d u r i n g the b r e e d i n g season, a n d capture rates as h i g h as 2 0 w o r m s per h o u r h a v e b e e n r e c o r d e d . B e c a u s e o f their penchant for e a r t h w o r m s , r o b i n s are susceptible to secondary D D T p o i s o n i n g . A r o b i n that c o n s u m e s 100 earthworms m a y ingest m o r e than 3 m g o f D D T ( C o o k e et a l . , 1992). F o r a 77 g b i r d ( S a l l a b a n k s & James, 1999), this m a y be a n acutely lethal dose. E v e n sub-lethal l e v e l s o f D D T c a n be h a z a r d o u s , as D D T s a c c u m u l a t e i n the b o d y .  T u r k e y s fed D D T  a c c u m u l a t e d it i n their fat at concentrations four to eight t i m e s the l e v e l i n the diet ( P o l a n d et a l . , 1972). D D T s tend to be lost r e l a t i v e l y s l o w l y f r o m the b o d y . K a n et a l . (1978) f o u n d that the  34 loss o f total D D T i n feces as a p r o p o r t i o n o f d a i l y intake for c h i c k e n s w a s o n l y 7 % . M e t a b o l i s m a n d e x c r e t i o n o f D D T s depends o n species, diet ( C l a r k et a l . , 1987), l i p i d p o o l sizes ( N o r s t r o m et a l . , 1989) a n d the p a r t i c u l a r i s o m e r b e i n g m e a s u r e d .  P a r a , p a r a ' - D D E is c o n s i d e r e d the f i n a l  b r e a k d o w n product o f D D T i n l i v i n g birds ( P o l a n d et a l . , 1972). T h i s metabolite tends to be lost quite s l o w l y . blackbirds  S t i c k e l et a l . ( 1 9 8 4 ) f o u n d that i n g r a c k l e s  Agelaius phoeniceus),  (Molothrus ater),  starlings  (Quiscalus quiscula),  (Sturnus vulgaris),  the loss rate o f D D E w a s o n l y 0 . 3 0 % per day.  red-winged  and brown-headed  cowbir  O r t h o , p a r a ' - D D T tends to be  stored less r e a d i l y a n d e l i m i n a t e d m o r e q u i c k l y than p , p ' - D D T ( S t i c k e l , 1973).  D i m o n d et a l .  (1970) f o u n d that D D T concentrations i n r o b i n s w e r e one order o f m a g n i t u d e h i g h e r than those i n w o r m s . These authors also f o u n d traces o f o , p ' - D D T i n the e a r t h w o r m s but n o n e i n the r o b i n s s u g g e s t i n g differential a b s o r p t i o n and/or b r e a k d o w n b e t w e e n predator a n d p r e y .  A d u l t robins  c o n t a i n e d about 2.6 t i m e s h i g h e r residue l e v e l s than i m m a t u r e s , as w e l l ( D i m o n d et a l . , 1970). E l l i o t t et a l . (1994) e x a m i n e d o r g a n o c h l o r i n e residues i n the eggs o f O k a n a g a n o r c h a r d n e s t i n g California quail magpies  (Callipepla californica),  (Pica pica),  house wrens  tree s w a l l o w s  (Tachycineta bicolor),  (Troglodytes aedon),  and A m e r i c a n robins.  black-bille  R o b i n eggs  c o n t a i n e d 18 to 3,500 t i m e s m o r e D D T a n d 4 to 100 t i m e s m o r e D D E t h a n those o f the other species tested.  T h e y also h a d the l o w e s t m e a n D D E : D D T ratios (9.5:1).  T h r e e recent studies  ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s et a l . , 2 0 0 0 ) e x a m i n e d the l e v e l s o f D D T s i n r o b i n eggs f r o m the O k a n a g a n orchards and v a r i o u s c o n t r o l sites. T h e s e data are s u m m a r i z e d i n T a b l e 2 - 1 . R e s i d u e l e v e l s i n eggs reflect the a m o u n t o f D D T i n the l a y i n g female at the t i m e o f y o l k l i p i d d e p o s i t i o n ( F o x et a l . , 1978). T h e h i g h levels o f D D T s f o u n d i n O k a n a g a n r o b i n eggs c o u l d be attributed to a v a r i e t y o f sources.  L o w D D E : D D T ratios suggest recent a p p l i c a t i o n s o f D D T ( E l l i o t t et a l . , 1994; M o r a ,  1997). D D T has b e e n f o u n d as a c o n t a m i n a n t i n recent use p e s t i c i d e s s u c h as d i c o f o l ( B l u s et a l . , 1987; E l l i o t t et a l . , 1994; R i s e b r o u g h et a l . , 1986) a n d kelthane ( B u n c k et a l . , 1987), a n d it m a y s t i l l be used i l l e g a l l y ( B l u s et a l . , 1987; B u n c k et a l . , 1987; E l l i o t t et a l . , 1994). N e i t h e r o f these scenarios, h o w e v e r , i s l i k e l y to result i n the h i g h residue l e v e l s seen i n these b i r d s ( E l l i o t t et a l . , 1994). D D T b u r d e n s m a y be a c q u i r e d b y m i g r a t o r y b i r d s o n w i n t e r i n g g r o u n d s i n L a t i n A m e r i c a w h e r e D D T m a y still be used l e g a l l y ( E l l i o t t et a l . , 1994; K l e m e n s et a l . , 2 0 0 0 ; M o r a ,  35 Table 2-1. Mean DDT levels (mg/kg, wet weight) in American robin eggs collected from orchards in the Okanagan Valley and non-orchard areas of the Okanagan and Lower Mainland of British Columbia.  Period  1990-1991*  1993 -1995**  1993 -1998***  Orchard DDT  16.7  13.0  10.1  DDE  83.3  85.1  54.8  DDD  2.4  1.1  0.7  DDT:DDE  5.0  17.2  5.2  DDT  0.08  0.4  0.4  DDE  1.5  8.2  8.6  DDD  0.001  0.06  0.05  18.1  18.9  102.8  Non-orchard  DDT:DDE  * E l l i o t t et a l . , 1994 ** H a r r i s et a l . , 2 0 0 0 ; para,para' i s o m e r s o n l y *** G i l l e t a l , 2003  36 1997) .  A m e r i c a n r o b i n s i n the O k a n a g a n , h o w e v e r , d o not u s u a l l y m i g r a t e as far as L a t i n  A m e r i c a but rather t r a v e l to W a s h i n g t o n , O r e g o n , a n d C a l i f o r n i a ( C a m p b e l l et a l . , 1997).  Many  O k a n a g a n r o b i n s (up to 1,000) m a y e v e n o v e r - w i n t e r i n the o r c h a r d s a n d v i n e y a r d s o f the O k a n a g a n v a l l e y ( C a m p b e l l et a l . , 1997; C a n n i n g s et a l . , 1987) because o f the m i l d c l i m a t e a n d abundance o f food.  T h e r o b i n s i n E l l i o t t et al.'s ( 1 9 9 4 ) study h a d c o n s i d e r a b l y h i g h e r D D T  l e v e l s than b i r d s that w i n t e r i n L a t i n A m e r i c a (tree s w a l l o w s , b a r n s w a l l o w s (Hirundo a n d house w r e n s ) .  rustled),  T h u s , r o b i n s i n the O k a n a g a n are m o s t l i k e l y a c q u i r i n g their D D T burdens  f r o m the earthworms they eat, w h i c h , i n t u r n accumulate their residues f r o m h i s t o r i c a l l y a p p l i e d , s l o w l y d e g r a d i n g D D T i n the s o i l ( E l l i o t t et a l . , 1994). D e s p i t e their h i g h D D T burdens, A m e r i c a n r o b i n s c o n t i n u e to t h r i v e , a n d f i e l d studies h a v e f o u n d n o decreases i n their r e p r o d u c t i v e success ( E l l i o t t et a l . , 1994; G i l l et a l . 2 0 0 3 ) .  It  has b e e n i m p l i e d that aside f r o m the m a s s die-offs seen d u r i n g the peak o f D D T use ( C a r s o n , 1962), r o b i n s are r e l a t i v e l y unaffected b y the h i g h l e v e l s o f D D T they are e x p o s e d to.  It is  p o s s i b l e that the residues they ingest are d i l u t e d w h e n they eat " c l e a n " fruit a n d other less c o n t a m i n a t e d i n s e c t s ( J o h n s o n et a l . , 1 9 7 6 ) .  R o b i n s m a y also a v o i d foraging i n h i g h l y  c o n t a m i n a t e d areas ( D i m o n d et a l . , 1970). T h e e x t r e m e l y h i g h concentrations o f D D T s f o u n d i n these b i r d s a n d their eggs m a y , therefore, o n l y be f o u n d d u r i n g the b r e e d i n g season w h e n they are c o n s u m i n g p r i m a r i l y e a r t h w o r m s a n d i n s e c t s .  A g e d D D T , w h i l e s t i l l b i o a v a i l a b l e to  e a r t h w o r m s , m a y be m u c h less t o x i c t h a n w h e n it w a s first a p p l i e d ( R o b e r t s o n & A l e x a n d e r , 1998) .  It is p o s s i b l e that after decades o f e x p o s u r e , r o b i n s h a v e d e v e l o p e d a t o l e r a n c e o r  resistance to the detrimental effects o f D D T s as a result o f genetic selection. A v a r i e t y o f insects became  resistant  Procambarus  to D D T s o o n after  clarkii,  its i n t r o d u c t i o n ( M e l l a n b y ,  1992).  The crayfish,  a v a r i e t y o f other aquatic invertebrates, m o s q u i t o f i s h (Gambusia  a n d t w o s p e c i e s o f c r i c k e t f r o g (Acris  crepitans  and Acris  gryllus)  h a v e also  affinis),  demonstrated  tolerances to D D T s after l o n g - t e r m exposure ( W H O , 1989). A n i m a l s often d e v e l o p tolerances to the r e p r o d u c t i o n suppressing effects o f phytoestrogens ( C r a i n & G u i l e t t e , 1997; S o n n e n s c h e i n & S o t o , 1998), a n d resistance to the e n d o c r i n e - d i s r u p t i n g effects o f D D T s m a y have e v o l v e d i n a similar w a y i n robins. 2.4. T h i s S t u d y T h e o b j e c t i v e o f this study w a s to assess l o n g - t e r m , d e l a y e d , or subtle effects o f D D T c o n t a m i n a t i o n o n A m e r i c a n r o b i n s f r o m the O k a n a g a n .  T h e focus w a s o n t w o m a i n areas:  1)  g r o w t h a n d s u r v i v a l ( C h a p t e r III) a n d 2) r e p r o d u c t i o n a n d b e h a v i o u r ( C h a p t e r I V ) . I n c l u d e d i n  37 g r o w t h a n d s u r v i v a l are e g g m e a s u r e m e n t s ,  c h i c k measurements,  i m m u n e response, t h y r o i d  h o r m o n e l e v e l s , m o r t a l i t y , a n d tissue w e i g h t s . R e p r o d u c t i o n and b e h a v i o u r i n c l u d e the t i m i n g o f r e p r o d u c t i v e events, egg l a y i n g , e g g h a t c h i n g , c h i c k f l e d g i n g , a n d t h y r o i d h o r m o n e l e v e l s i n breeding birds, along w i t h frequencies o f reproductive, maintenance, aggressive, and v o c a l b e h a v i o u r s . T h e w o r k o f E l l i o t t et a l . (1994) a n d G i l l et a l . (2003) suggest that there s h o u l d be n o o v e r a l l d e t r i m e n t a l effects o f e a r l y D D T e x p o s u r e o n s u r v i v a l o r r e p r o d u c t i o n . these studies o n l y o b s e r v e d the r o b i n s u n t i l the y o u n g h a d  fledged.  However,  It m a y be that d e t r i m e n t a l  effects i n the y o u n g w e r e not e v i d e n t u n t i l after f l e d g i n g ( C o l b o r n et a l . , 1 9 9 3 ; S p y k e r , 1975), that the stable p o p u l a t i o n s i n the O k a n a g a n are due to the recruitment o f b i r d s from other areas, not f r o m the return (or r e s i d e n c y ) o f s u c c e s s f u l b r e e d i n g pairs a n d t h e i r p r o g e n y ( C o o k e et a l . , 1992), or that m o r e subtle detriments m a y w o r k at the i n d i v i d u a l rather than the p o p u l a t i o n l e v e l . O t h e r studies, o n a v a r i e t y o f s p e c i e s , i n d i c a t e that D D T c a n , i n fact, h a v e a n u m b e r o f deleterious effects (see C h a p t e r I for r e v i e w ) , w h i c h m a y s i m p l y not h a v e b e e n o b s e r v e d i n the field. I n order to study these potential l o n g - t e r m effects o f early D D T exposure, nestlings f r o m O k a n a g a n o r c h a r d s , e x p o s e d in ovo a n d for ten d a y s p o s t - h a t c h to D D T s , w e r e c a p t u r e d a n d r a i s e d i n c a p t i v i t y . These b i r d s , a l o n g w i t h controls f r o m the B r i t i s h C o l u m b i a L o w e r M a i n l a n d , w e r e m a i n t a i n e d i n c a p t i v i t y for three y e a r s .  R a i s i n g the b i r d s i n c a p t i v i t y e n s u r e d that a n y  differences b e t w e e n the t w o g r o u p s o f b i r d s c o u l d be r e l a t e d to t h e i r e a r l y e x p e r i e n c e s a n d c o n t a m i n a n t exposures.  T h i s e l i m i n a t e d any extraneous v a r i a b l e s due to differences i n habitat,  diet, a n d c l i m a t e . It w a s a s s u m e d that the c a p t i v e e n v i r o n m e n t w o u l d also reduce the n u m b e r o f p r e d a t i o n losses. E g g s w e r e c o l l e c t e d f r o m the same nests as the 10-day o l d c h i c k s i n order to evaluate differences b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n r e l a t i o n to the l e v e l s o f D D T c o n t a m i n a t i o n . T h e O k a n a g a n eggs a n d c h i c k s w e r e c o l l e c t e d from orchards near P e n t i c t o n a n d N a r a m a t a , w h e r e a s the L o w e r M a i n l a n d eggs a n d c h i c k s w e r e c o l l e c t e d from p a r k areas w i t h i n V a n c o u v e r , Surrey, and D e l t a , B r i t i s h C o l u m b i a (Figure 2-1).  A c o m p l e t e l i s t i n g o f the  o r g a n o c h l o r i n e s a n d p o l y c h l o r i n a t e d b i p h e n y l s a n a l y z e d i n these eggs is a v a i l a b l e i n A p p e n d i x I. B e c a u s e a n u m b e r o f r o b i n s d i e d after  fledging  but p r i o r to r e p r o d u c t i v e m a t u r i t y , a  s e c o n d study w a s c a r r i e d out i n order t o evaluate i m m u n e response i n m o r e d e t a i l . A s e c o n d c o h o r t o f eggs a n d b i r d s w e r e c o l l e c t e d ( C h a p t e r V ) a n d i m m u n e response, t h y r o i d h o r m o n e levels, a n d stress responses were e x a m i n e d .  38  Figure 2-1: Collection sites of American robin eggs and nestlings. Lower mainland = Vancouver, Delta, and Surrey, British Columbia; Okanagan = Naramata and Pentiction, British Columbia  39  2.5 References B l u s , L . J . , H e n n y , C . J., Stafford, C . J., G r o v e , R . A . (1987). Persistence o f D D T and metabolites i n wildlife  from  W a s h i n g t o n State o r c h a r d s .  Archives of Environmental  Contamination and Toxicology, 16, 461 Al6. B u n c k , C . M . , P r o u t y , R . M . , K r y n i t s k y , A . J . 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( 1 9 9 7 ) . H o w r o b i n s find w o r m s .  Animal Behaviour,  54, 143-151. M o r a , M . A . ( 1 9 9 7 ) . T r a n s b o u n d a r y p o l l u t i o n : Persistent o r g a n o c h l o r i n e p e s t i c i d e s i n m i g r a n t b i r d s o f the S o u t h w e s t e r n U n i t e d States a n d M e x i c o .  Environmental Toxicology and  Chemistry, 16(1), 3-11. M u r p h y , S. D . ( 1 9 8 0 ) . P e s t i c i d e s . I n J . D o u l l , C . D . K l a a s s e n , a n d M . O . A m d u r ( E d s . ) ,  Casarett and Doull's Toxicology: The Basic Science of Poisons (pp. 3 5 7 - 4 0 8 ) . N e w Y o r k : M a c M i l l a n Publishing. N a i r , A . , S a m u e l , T . , P i l l a i , M . K . ( 1 9 9 2 ) . B e h a v i o u r o f D D T i n three s o i l s e x p o s e d to solar r a d i a t i o n under different c o n d i t i o n s .  Pesticide Science, 34(4), 3 3 3 - 3 4 0 .  N o r s t r o m , R . J . , C l a r k , T . P . , Jefrey, D . A . , W o n , H . T . , G i l m a n , A . P . ( 1 9 8 6 ) . D y n a m i c s o f organochlorine compounds i n herring gulls o f [14C] D D E i n free-living herring gulls  (Larus argentatus): I. D i s t r i b u t i o n a n d clearance  (Larus argentatus). Environmental Toxicology and  Chemistry, 5, 4 1 - 4 8 . P o l a n d , J . J . , K i s s a m , J . B . , R e e d , J . K . , Barnett, B . D . ( 1 9 7 2 ) . T h e uptake o f D D T b y d o m e s t i c turkeys ranged o n treated c l a y l o a m s o i l .  Poultry Science, 51, 1 0 2 4 - 1 0 2 6 .  R i s e b r o u g h , R . W . , J a r m a n , W . M . , S p r i n g e r , A . M . , W a l k e r , W . , H u n t , W . G . (1986). A m e t a b o l i c d e r i v a t i o n o f D D E f r o m kelthane.  Environmental Toxicology and Chemistry, 5, 13-19.  Robertson, B . K . , A l e x a n d e r , M . (1998). Sequestration o f D D T and d i e l d r i n i n soil: Disappearance  o f a c u t e t o x i c i t y but n o t the c o m p o u n d s .  Environmental Toxicology and  Chemistry, 17(6), 1034-1038. S a l l a b a n k s , R . , James, F . C . ( 1 9 9 9 ) .  American Robin ( T h e B i r d s o f N o r t h A m e r i c a N o .  4 6 2 ) . C o r n e l l L a b o r a t o r y o f O r n i t h o l o g y a n d the A c a d e m y o f N a t u r a l S c i e n c e s . S e n t h i l k u m a r , K . , K a n n a n , K . , S u b r a m a n i a n , A . , T a n a b e , S. ( 2 0 0 1 ) . A c c u m u l a t i o n o f o r g a n o c h l o r i n e pesticides a n d p o l y c h l o r i n a t e d b i p h e n y l s i n sediments, aquatic o r g a n i s m s , b i r d s , b i r d eggs, a n d bat c o l l e c t e d f r o m S o u t h I n d i a .  Environmental Science and Pollution Research,  8(1), 3 5 - 4 7 . S o n n e n s c h e i n , C , Soto A . M . (1998). A n updated r e v i e w o f e n v i r o n m e n t a l estrogen a n d a n d r o g e n m i m i c s a n d antagonists.  Journal of Steroid Biochemistry and Molecular Biology, 65(1-  6), 143-150. S p y k e r , J . M . ( 1 9 7 5 ) . A s s e s s i n g the i m p a c t o f l o w l e v e l c h e m i c a l s o n d e v e l o p m e n t : B e h a v i o r a l a n d latent effects.  Federation Proceedings, 34, 1835-1844.  42 S t r i n g e r , A . , P i c k a r d , J . A . , L y o n s , C . H . ( 1 9 7 4 ) . T h e a c c u m u l a t i o n and d i s t r i b u t i o n o f p , p ' - D D T and related c o m p o u n d s i n an apple o r c h a r d . I. R e s i d u e s i n the s o i l .  Pesticide Science,  5, 5 8 7 - 5 9 8 . S t i c k e l , L . F . ( 1 9 7 3 ) . P e s t i c i d e residues i n b i r d s a n d m a m m a l s . I n C . A . E d w a r d s ( E d . ) ,  Environmental Pollution by Pesticides  (pp. 2 5 4 - 3 1 1 ) . L o n d o n : P l e n u m Press.  S t i c k e l , W . H . , S t i c k e l , L . F . , D y r l a n d , R . A . , H u g h e s , D . L . (1984). D D E i n b i r d s : L e t h a l residues and loss rates.  Archives of Environmental Contamination and Toxicology, 13,  1-6.  T o m l i n , A . D . (1992). B e h a v i o u r as a source o f e a r t h w o r m s u s c e p t i b i l i t y to e c o t o x i c a n t s . I n P . W . G r e i g - S m i t h , H . B e c k e r , P . J . E d w a r d s , and F . H e i m b a c h ( E d s . ) ,  Ecotoxicology of  Earthworms: Society of Environmental Toxicology and Chemistry's First European and International Workshop on Earthworm Ecotoxicology  (pp. 1 1 5 - 1 2 5 ) . A n d o v e r , E n g l  Intercept. W a u e r , R . H . (1999). W H O . (1989).  The American Robin.  A u s t i n , T X : U n i v e r s i t y o f T e x a s Press.  DDT and its derivatives - Environmental Aspects  C r i t e r i a N o . 83). W o r l d H e a l t h O r g a n i z a t i o n .  (Environmental Healt  43  Chapter III Growth and Survival of DDT Contaminated American Robins  3.1. Introduction D i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e ( D D T ) a n d its metabolites, i n h i g h doses, c a n be t o x i c to b i r d s , i n d u c i n g r e p r o d u c t i v e disturbances a n d e v e n m o r t a l i t y ( B l u s , 1996; C a r s o n , 1 9 6 2 ; F r y , 1995; W H O , 1989). M o t h e r b i r d s c a n pass these c h e m i c a l s to their eggs d u r i n g y o l k f o r m a t i o n ( B r a n d t et a l . , 1978; F o x et a l . , 1978; F r y , 1995; O h l e n d o r f et a l . , 1978; O t t i n g e r et a l . , 2 0 0 1 ; S t i c k e l , 1973). In ovo D D T e x p o s u r e m a y result i n r e d u c e d h a t c h a b i l i t y , e m b r y o or h a t c h l i n g mortality,  wasting  syndrome,  skeletal  abnormalities,  retarded  growth,  and  impaired  differentiation o f the r e p r o d u c t i v e and nervous systems ( B l u s , 1996; B r i t t o n et a l . , 1974; C h a n g & S t o k s t a d , 1 9 7 5 ; F r y , 1 9 9 5 ; F r y & T o o n e , 1 9 8 1 ; Jefferies, 1 9 7 1 ; J o n e s & S u m m e r s , 1 9 6 8 ; L i l l i e et a l . , 1972; Sauter & Steele, 1972; T y l e r et a l . , 1998; V o s et a l . , 2 0 0 0 ; W H O , 1989). F r u i t - g r o w i n g areas, s u c h as the O k a n a g a n V a l l e y o f southern B r i t i s h C o l u m b i a , w e r e h i s t o r i c a l l y treated w i t h vast a m o u n t s o f D D T ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s et a l . , 2000). 1973;  T h i s c h e m i c a l a n d its p r i m a r y m e t a b o l i t e s , D D E a n d D D D (Jefferies, 1 9 7 5 ; S t i c k e l , 1  W H O , 1989), adsorb to s o i l p a r t i c l e s ( C o l b o r n et a l . , 1 9 9 3 ; H a r r i s et a l . , 2 0 0 0 ; W H O ,  1989) a n d m a y be c o n s u m e d b y e a r t h w o r m s ( G i s h & H u g h e s , 1982; H a r r i s et a l . , 2 0 0 0 ; J o h n s o n et a l . , 1 9 7 6 ; S e n t h i l k u m a r et a l . , 2 0 0 1 ; T o m l i n ,  1992; W H O , 1989).  The  earthworms  b i o a c c u m u l a t e these residues i n their l i p i d r i c h tissues a n d t h e n pass t h e m o n to their predators ( C o o k e et a l . , 1992; H a r r i s et a l . , 2 0 0 0 ) .  T h e A m e r i c a n r o b i n (Turdus migratorius)  is a major  e a r t h w o r m predator ( E h r l i c h et a l . , 1988; M o n t g o m e r i e & W e a t h e r h e a d , 1 9 9 7 ; S a l l a b a n k s & James, 1999; W a u e r , 1999). P r e v i o u s studies have f o u n d v e r y h i g h l e v e l s o f D D T a n d D D E i n O k a n a g a n r o b i n eggs ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s et a l . , 2 0 0 0 ) . G i l l et a l . (2003), for e x a m p l e , reported D D T l e v e l s as h i g h as 60.7 m g / k g a n d D D E l e v e l s u p to 3 0 2 m g / k g i n r o b i n eggs f r o m O k a n a g a n orchards, c o m p a r e d w i t h D D T l e v e l s o f 0.5 m g / k g a n d D D E l e v e l s o f 12.0 m g / k g at r e l a t i v e l y u n c o n t a m i n a t e d c o n t r o l sites.  A l t h o u g h these studies o n l y o b s e r v e d  r o b i n s u n t i l the o f f s p r i n g h a d f l e d g e d , they r e p o r t e d n o s i g n i f i c a n t d i f f e r e n c e s i n s u r v i v a l b e t w e e n birds from the O k a n a g a n and controls. T h i s study a i m e d to e x a m i n e m o r e l o n g - t e r m or d e l a y e d effects o f e a r l y D D T exposure, w i t h a focus o n g r o w t h a n d s u r v i v a l . A s m a n y parameters are d i f f i c u l t to study i n the w i l d , ten-  44 d a y o l d n e s t l i n g s f r o m o r c h a r d s i n the O k a n a g a n a n d r e l a t i v e l y c l e a n areas o f the B r i t i s h C o l u m b i a L o w e r M a i n l a n d w e r e c o l l e c t e d a n d r a i s e d i n c a p t i v i t y . T h e o b j e c t i v e s o f the study w e r e to: 1) D e t e r m i n e i f in ovo  and early post-hatch D D T exposure influenced g r o w t h and  d e v e l o p m e n t as m e a s u r e d b y tarsus length, b o d y w e i g h t , a n d tissue w e i g h t s .  Thyroid  h o r m o n e l e v e l s w e r e a l s o m e a s u r e d , as they p l a y a n i m p o r t a n t r o l e i n g r o w t h ( B o l a n d e r , 1994; H a u s e r et a l . , 1998; N e l s o n , 2 0 0 0 ; S i n g h et a l . , 1968). C o r r e l a t i o n s w e r e c o n d u c t e d to d i s c e r n i f the l e v e l o f D D T e x p o s u r e resulted i n differences w i t h i n the O k a n a g a n b i r d s . 2)  D e t e r m i n e i f in ovo a n d early post-hatch D D T exposure i n f l u e n c e d m o r t a l i t y . B i r d s w e r e m o n i t o r e d f r o m ten d a y s o f age u n t i l three years o f age, a n d age a n d cause o f death w e r e d i s c e r n e d w h e n e v e r p o s s i b l e . I m m u n e response w a s also measured, as it is essential for s u r v i v a l (Banerjee, 1 9 9 9 ; G r a s m a n et a l . , 1996; R e p e t t o & B a l i g a , 1996).  3.2. M e t h o d s 3.2.1. E g g C o n t a m i n a n t s  y  B e t w e e n June 2 a n d J u l y 3, 1997, 70 eggs w e r e c o l l e c t e d f r o m r o b i n nests i n o r c h a r d areas s u r r o u n d i n g P e n t i c t o n a n d N a r a m a t a i n the O k a n a g a n V a l l e y o f B r i t i s h C o l u m b i a .  An  a d d i t i o n a l 3 6 eggs w e r e c o l l e c t e d f r o m c o n t r o l sites i n V a n c o u v e r a n d D e l t a , i n the L o w e r M a i n l a n d o f B r i t i s h C o l u m b i a , b e t w e e n A p r i l 10 a n d J u n e 18, 1 9 9 7 .  T h i r t y - o n e o f these  O k a n a g a n e g g s a n d 16 o f the L o w e r M a i n l a n d e g g s w e r e a n a l y z e d f o r m o i s t u r e , o r g a n o c h l o r i n e , a n d p o l y c h l o r i n a t e d b i p h e n y l content.  lipid,  T h e O k a n a g a n eggs w e r e a n a l y z e d  i n d i v i d u a l l y , a n d the L o w e r M a i n l a n d eggs were p o o l e d (n = 3; one p o o l o f 6 eggs, one p o o l o f 9 eggs, a n d one i n d i v i d u a l egg) i n order to save costs a n d because the e x p e c t e d v a l u e s w o u l d be low. A l l o f the eggs w e r e a n a l y z e d at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r ( H u l l , Q u e b e c ) . A n a l y s e s were c o n d u c t e d u s i n g gas c h r o m a t o g r a p h y a n d m a s s spectrophotometry, f o l l o w i n g the m e t h o d s o u t l i n e d i n W o n et a l . ( 2 0 0 1 ) .  R e s u l t s w e r e e x p r e s s e d i n pig/g a n d o n a w e t w e i g h t  basis. A l l eggs w e r e c o l l e c t e d d u r i n g t i m e s o f n o current-use p e s t i c i d e a p p l i c a t i o n ( L . W i l s o n , Canadian W i l d l i f e Service, personal communication).  A l t h o u g h it has b e e n s u g g e s t e d that  contaminant loads m a y v a r y b e t w e e n eggs w i t h i n a c l u t c h ( O h l e n d o r f et a l . , 1985; O t t i n g e r et a l . ,  1  DDE = dichlordiphenyldichloroethylene, DDD = dichlorodiphenyldichloroethane  45 2 0 0 1 ) , for the p u r p o s e s o f t h i s study i t w a s a s s u m e d , as i n p r e v i o u s studies ( H a r r i s et a l . , 2 0 0 0 ; G i l l , 2 0 0 3 ) , that a l l eggs w i t h i n a c l u t c h c o n t a i n e d s i m i l a r c o n t a m i n a n t loads. 3.2.2. E g g M e a s u r e m e n t s A total o f 106 eggs (36 L o w e r M a i n l a n d , 70 O k a n a g a n ) w e r e w e i g h e d a n d m e a s u r e d . W h e r e measurements w e r e a v a i l a b l e for m o r e t h a n one e g g per nest, nest means w e r e u s e d for the analyses.  E g g l e n g t h refers to the distance f r o m end-to-end, w h i l e e g g w i d t h refers to the  measurement at the w i d e s t part o f the e g g . 3.2.3. R e a r i n g P r o t o c o l B e t w e e n A p r i l 28 a n d J u l y 16, 1997, 1 0 - d a y - o l d r o b i n nestlings w e r e c o l l e c t e d f r o m the same nests as the eggs. N i n e t y - o n e c h i c k s w e r e c o l l e c t e d f r o m the O k a n a g a n a n d 5 9 f r o m the Lower Mainland.  A l l nestlings were collected d u r i n g periods o f no current-use pesticide  treatment ( L . W i l s o n , C a n a d i a n W i l d l i f e S e r v i c e , p e r s o n a l c o m m u n i c a t i o n ) . T h e b i r d s w e r e h a n d reared at M o n i k a ' s W i l d l i f e S h e l t e r ( S u r r e y , B r i t i s h C o l u m b i a ) w h e r e they w e r e fed a m i x t u r e o f d r y cat f o o d , peanut butter, h a r d - b o i l e d c h i c k e n eggs, c o m m e r c i a l c h i c k starter, a n d v i t a m i n s u n t i l they were self-feeding ( M . T o l g s d o r f , M o n i k a ' s W i l d l i f e Shelter, personal c o m m u n i c a t i o n ) . A t this t i m e they w e r e p r o v i d e d w i t h d r y cat f o o d a n d c l e a n water for d r i n k i n g and bathing  ad libitum.  The birds were housed i n c o m m u n a l outdoor pens measuring  a p p r o x i m a t e l y 3.7 m l o n g x 3.7 m w i d e x 3.7 m h i g h a n d t h e n transferred to 3.7 m x 2.4 m x 3.7 m pens w i t h g r a v e l f l o o r s w h e n s e l f - f e e d i n g . E a c h b i r d w a s outfitted w i t h b o t h a n u m b e r e d m e t a l l e g b a n d a n d p l a s t i c c o l o u r e d l e g bands for i n d i v i d u a l i d e n t i f i c a t i o n . U p o n r e a c h i n g s e x u a l m a t u r i t y , the b i r d s w e r e t r a n s f e r r e d t o b r e e d i n g p e n s at the University o f B r i t i s h C o l u m b i a ' s San Rafael Research A v i a r y (Surrey, B r i t i s h Columbia). p a i r w a s h o u s e d per p e n a n d three p e n types w e r e e m p l o y e d .  One  T w e l v e pairs were housed i n  i n d o o r pens constructed o f w o o d frame a n d c h i c k e n w i r e , w i t h t a r p a u l i n u s e d for v i s u a l i s o l a t i o n . T h e s e pens h a d cement floors a n d m e a s u r e d 3.8 m x 1.9 m x 3 m . S i x w i n d o w s p r o v i d e d natural l i g h t i n g , a n d a d d i t i o n a l a r t i f i c i a l l i g h t i n g w a s t i m e d to m i m i c the n a t u r a l l i g h t / d a r k c y c l e . S i x t e e n p a i r s w e r e k e p t i n o u t d o o r h o l d i n g p e n s c o n s t r u c t e d o f m e t a l - p i p e frame w i t h n y l o n netting or c h i c k e n w i r e over c h a i n - l i n k fencing.  S e v e r a l c e n t i m e t e r s o f g r a v e l c o v e r e d the  g r o u n d a n d t a r p a u l i n w a s u s e d for v i s u a l i s o l a t i o n . T h e s e pens m e a s u r e d a p p r o x i m a t e l y 3.0 m w i d e x 3.6 m l o n g x 1.9 m h i g h . T w e l v e pairs w e r e h o u s e d i n o u t d o o r s i d e - b y - s i d e p i e - s h a p e d p e n s c o n s t r u c t e d w i t h m e t a l p i p e s , w o o d fence posts, a n d c h i c k e n w i r e .  Pens were visually  i s o l a t e d b y t a r p a u l i n , a n d netting w a s u s e d as r o o f i n g . T h e s e pens m e a s u r e d a p p r o x i m a t e l y 1.5  46 m w i d e (at the w i d e s t end) x 4.8 m l o n g x 2.1 m h i g h .  D r y cat f o o d ( K i r k l a n d S i g n a t u r e ,  B u r n a b y , B r i t i s h C o l u m b i a ) a n d water w e r e p r o v i d e d ad libitum a n d s u p p l e m e n t e d w i t h l i q u i d c a l c i u m (Stanley P h a r m a c e u t i c a l s L t d . , N o r t h V a n c o u v e r , B r i t i s h C o l u m b i a ) , v i t a m i n s (8 i n 1 Pet Products Inc., Hauppauge, N e w Y o r k ) , and m e a l w o r m s ( M . Tolgsdorf, M o n i k a ' s  Wildlife  Shelter, p e r s o n a l c o m m u n i c a t i o n ) . T h e b i r d s , e s p e c i a l l y those h o u s e d outdoors, also h a d access to any insect and/or plant m a t e r i a l they c o u l d p r o c u r e t h e m s e l v e s .  E a c h p a i r w a s also p r o v i d e d  w i t h a c o v e r e d feeder, t w o nesting platforms, paper b o w l s (to serve as a nest foundation), dishes o f m u d , and a variety o f nesting materials, including:  straw, shredded paper, s t r i n g , strips o f  c l o t h , w o o l , a n d assorted grasses c o l l e c t e d from a r o u n d the a v i a r y . C e d a r branches a n d t a r p a u l i n w e r e h u n g o v e r the n e s t i n g p l a t f o r m s for p r o t e c t i o n against i n c l e m e n t weather.  A l l treatment  a n d h o u s i n g p r o t o c o l s w e r e a p p r o v e d b y the U n i v e r s i t y o f B r i t i s h C o l u m b i a A n i m a l C a r e C o m m i t t e e (Certificate # A 9 7 - 0 0 4 3 ) . 3.2.4. C h i c k M e a s u r e m e n t s B l o o d samples w e r e sent to the C e n t r e for W i l d l i f e E c o l o g y , S i m o n F r a s e r U n i v e r s i t y , B u r n a b y , B r i t i s h C o l u m b i a , for D N A s e x i n g ( b y B r e t t V a n d e r k i s t ) , f o l l o w i n g the p r o t o c o l s o u t l i n e d i n G r i f f i t h s et a l . ( 1 9 8 8 ) .  S e x w a s later c o n f i r m e d w h e n the b i r d s w e r e d i s s e c t e d at  s a c r i f i c e at the e n d o f the study.  W e i g h t s a n d m e a s u r e m e n t s w e r e a v a i l a b l e for 55 L o w e r  M a i n l a n d ( f r o m 33 nests) a n d 91 O k a n a g a n ( f r o m 41 nests) 10-day o l d c h i c k s . C h i c k s w e r e w e i g h e d a n d b o d y m e a s u r e m e n t s t a k e n a p p r o x i m a t e l y ten days post-hatch, u s i n g a n e l e c t r o n i c scale a n d c a l i p e r s . T a r s u s l e n g t h refers to the distance from the j u n c t i o n o f the tibiotarsus a n d the tarsometatarsus to the j u n c t i o n w i t h the m i d d l e toe.  W i n g l e n g t h w a s m e a s u r e d f r o m the  b e n d o f the f o l d e d w i n g to the t i p o f the longest p r i m a r y . T o e l e n g t h refers to the distance f r o m the j u n c t i o n o f the distal e n d o f the tarsometatarsus to the p r o x i m a l e n d o f the c l a w o f the m i d d l e toe ( A l d r i c h & James, 1991). A s m o r e than one c h i c k w a s c o l l e c t e d from m o s t nests, m e a n s per b r o o d w e r e u s e d for the analyses to m i n i m i z e p s u e d o r e p l i c a t i o n . 3.2.5. G r o w t h M e a s u r e m e n t s B o d y w e i g h t s a n d tarsus lengths w e r e r e c o r d e d w h e n the c h i c k s w e r e a p p r o x i m a t e l y 10 days ( M a y - J u l y , 1997), 2 m o n t h s ( J u l y - S e p t e m b e r , 1997), 5 m o n t h s ( O c t o b e r - D e c e m b e r , 1997), a n d 7-9 m o n t h s (February, 1998) o f age. B o d y w e i g h t s w e r e also m e a s u r e d f o l l o w i n g their first b r e e d i n g season ( A u g u s t - S e p t e m b e r , 1998). M e a n s per b r o o d were u s e d for the analyses.  47 3.2.6. T h y r o i d H o r m o n e s B l o o d s a m p l e s w e r e c o l l e c t e d f r o m the b i r d s i n J u l y , A u g u s t , or S e p t e m b e r 1 9 9 7 ) , O c t o b e r , N o v e m b e r , or D e c e m b e r ( F a l l 1 9 9 7 ) , F e b r u a r y  (Summer  1998, and A u g u s t  1998.  S a m p l e s f r o m 11 L o w e r M a i n l a n d a n d 11 O k a n a g a n b i r d s ( a l l f r o m different nests) w e r e a n a l y z e d for p l a s m a t r i i o d o t h y r o n i n e a n d t h y r o x i n e l e v e l s . A l l b l o o d s a m p l e s w e r e c o l l e c t e d f r o m the j u g u l a r v e i n u s i n g a 2 7 g a u g e h e p a r i n i z e d n e e d l e a n d 1 m l s y r i n g e ( L . W i l s o n , Canadian W i l d l i f e Service, personal communication).  A p p r o x i m a t e l y one m l o f w h o l e b l o o d  w a s c o l l e c t e d f r o m e a c h b i r d , centrifuged at 3 3 0 0 r p m for 5 m i n u t e s , a n d the p l a s m a separated f r o m the b l o o d c e l l s . T o t a l p l a s m a t r i i o d o t h y r o n i n e a n d t h y r o x i n e concentrations w e r e a n a l y z e d b y T r a c y M a r c h a n t at the U n i v e r s i t y o f S a s k a t c h e w a n , S a s k a t o o n , S a s k a t c h e w a n . A n a l y s e s w e r e conducted using unextracted serum and a radioimmuno-assay f o l l o w i n g protocols outlined by C h o p r a (1972).  3.2.7. I m m u n e R e s p o n s e  3.2.7.1. Differential White Blood Cell Counts B l o o d smears w e r e m a d e w h e n the b i r d s w e r e 1 0 - d a y s - o l d (99 L o w e r M a i n l a n d b i r d s from  3 2 b r o o d s , a n d 159 O k a n a g a n b i r d s f r o m 41 b r o o d s ) a n d p o s t - b r e e d i n g adults i n A u g u s t  1998 (33 L o w e r M a i n l a n d b i r d s f r o m 24 broods, 45 O k a n a g a n b i r d s f r o m 31 broods). B l o o d for the smears w a s c o l l e c t e d f r o m the j u g u l a r v e i n u s i n g a 2 7 gauge h e p a r i n i z e d needle a n d 1 c c syringe.  S m e a r s w e r e a l l o w e d to d r y a n d t h e n s t a i n e d u s i n g a H e m a c o l o r ( E M D i a g n o s t i c  Systems, G i b b s t o w n , N e w Jersey) staining kit.  D i f f e r e n t i a l white b l o o d c e l l counts  were  c o n d u c t e d u s i n g lOOOx o i l i m m e r s i o n m i c r o s c o p y . O n e h u n d r e d w h i t e b l o o d c e l l s w e r e c o u n t e d a n d ratios o f heterophils + e o s i n o p h i l s to l y m p h o c y t e s + m o n o c y t e s w e r e d e t e r m i n e d . C e l l types w e r e c o m b i n e d due to c o u n t i n g d i f f i c u l t i e s ( H o d g e s , 1979), but as n u m b e r s o f e o s i n o p h i l s a n d m o n o c y t e s are n o r m a l l y l o w i n b i r d s ( D u f v a & A l l a n d e r , 1995), these values p r i m a r i l y represent h e t e r o p h i l a n d l y m p h o c y t e n u m b e r s a n d the ratio c a n be treated as h e t e r o p h i l . l y m p h o c y t e ratio.  3.2.7.2. Phytohemagglutinin Skin Test T w e l v e L o w e r M a i n l a n d and  13 O k a n a g a n a d u l t m a l e r o b i n s w e r e u s e d f o r  the  p h y t o h e m a g g l u t i n i n tests i n M a y 2 0 0 0 . F o r e a c h b i r d a s m a l l ( a p p r o x i m a t e l y 1 c m ) p a t c h o f s k i n o n e a c h w i n g w e b (patagium) w a s p l u c k e d or t r i m m e d c l e a n o f feathers a n d d o w n . T h i s spot was then cleaned w i t h a l c o h o l and measured using a calibrated M i t u t o y o digital pressuresensitive m i c r o m e t e r ( D y e r , L a n c a s t e r , P e n n s y l v a n i a ) . T h e left w i n g (control) o f e a c h b i r d w a s then injected sub-cutaneously w i t h 50 u l o f sterile D u l b e c c o ' s phosphate buffered saline ( S i g m a ,  48 St. L o u i s , M i s s o u r i ) u s i n g a 27 gauge needle. C a r e w a s t a k e n to ensure that the needle d i d not go t h r o u g h the p a t a g i u m a n d that the saline d i d not l e a k out o f the i n j e c t i o n site. T h e r i g h t w i n g s o f the same b i r d s w e r e injected w i t h 1 m g / m l p h y t o h e m a g g l u t i n i n ( S i g m a , St. L o u i s , M i s s o u r i ) d i s s o l v e d i n 50 p i s a l i n e . T w e n t y - f o u r hours later, the i n j e c t i o n sites o n b o t h w i n g s w e r e remeasured.  P a t a g i u m t h i c k n e s s w a s m e a s u r e d three t i m e s b o t h p r i o r to a n d 24 hours after the  injections a n d m e a n s were u s e d for the analyses. A w i n g i n d e x w a s c a l c u l a t e d for e a c h b i r d b y s u b t r a c t i n g the d i f f e r e n c e  i n w i n g w e b t h i c k n e s s f o r the s a l i n e w i n g f r o m that o f the  p h y t o h e m a g g l u t i n i n w i n g ( [ p o s t - p h y t o h e m a g g l u t i n i n - p r e - p h y t o h e m a g g l u t i n i n ] - [post-saline pre-saline]) ( K e a n & L a m o n t , 1994; S m i t s et a l . , 1999; S m i t s & W i l l i a m s , 1999).  3.2.8. M o r t a l i t y A g e at t i m e o f death or disappearance a n d cause o f death w e r e r e c o r d e d w h e n k n o w n . B i r d s that d i e d p r i o r to f l e d g i n g age ( a p p r o x i m a t e l y 14 d a y s p o s t - h a t c h ) w e r e c o n s i d e r e d n e s t l i n g s , w h e r e a s those that d i e d b e t w e e n 14 a n d 30 d a y s o f age w e r e l a b e l e d as f l e d g l i n g s . R o b i n s that d i e d after 30 days but p r i o r to their first b r e e d i n g season a n d the m o v e to S a n R a f a e l , at a p p r o x i m a t e l y 7 to 10 m o n t h s o f age, w e r e c o n s i d e r e d j u v e n i l e s . A n y b i r d s that s u r v i v e d to F e b r u a r y 1998 w e r e c o n s i d e r e d adults a n d s e x u a l l y m a t u r e . undetermined, accidental, escape/disappearance, starvation/coccidiosis.  C a u s e o f death w a s c l a s s e d as  d e p r e d a t i o n , e u t h a n a s i a (due to i n j u r y ) , or  H e a l t h y b i r d s that w e r e s a c r i f i c e d at the e n d o f the study w e r e not  i n c l u d e d here.  3.2.9. T i s s u e W e i g h t s a n d B o d y M e a s u r e m e n t s F o l l o w i n g c o m p l e t i o n o f the study 23 L o w e r M a i n l a n d a n d 31 O k a n a g a n b i r d s w e r e s a c r i f i c e d b y d e c a p i t a t i o n a n d dissected. C o l l e c t e d tissues (heart, spleen, l i v e r , k i d n e y s , gonads, t h y r o i d glands, t h y m u s , bursa, o v i d u c t , a n d b r a i n ) w e r e w e i g h e d w h e n fresh a n d then f r o z e n or stored i n f o r m a l i n for future analyses.  T h e same b o d y m e a s u r e m e n t s w e r e t a k e n as at 10 days  (tarsus, w i n g c o r d , m i d d l e toe, b o d y w e i g h t ) .  T i s s u e w e i g h t s a n d b o d y m e a s u r e m e n t s for  individual birds were analyzed. 3.2.10. Statistics D i f f e r e n c e s b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n samples i n e g g c o n t a m i n a n t s , e g g measurements,  c h i c k measurements,  t h y r o i d hormone levels, and i m m u n e response  a n a l y z e d u s i n g o n e - w a y a n a l y s e s o f v a r i a n c e ( A N O V A s ) a n d nest m e a n s . a n a l y z e d u s i n g n o m i n a l l o g i s t i c s (x ), 2  as were age a n d cause o f death.  were  S e x ratios w e r e  T i s s u e w e i g h t s at t i m e o f  sacrifice w e r e corrected for b o d y w e i g h t b y d i v i d i n g the tissue w e i g h t b y the b o d y w e i g h t m i n u s  49 the tissue w e i g h t . D i f f e r e n c e s i n tissue a n d b o d y measurements at sacrifice were a n a l y z e d u s i n g o n e - w a y A N O V A s a n d data f r o m i n d i v i d u a l b i r d s . D a t a w e r e c o m m o n l o g transformed, w h e r e n e c e s s a r y , i n o r d e r to i n c r e a s e n o r m a l i t y .  M a h a l a n o b i s o u t l i e r tests w e r e u s e d to i d e n t i f y  outliers. P a i r - w i s e c o r r e l a t i o n s w e r e c o n d u c t e d to determine the effects o f e g g p , p ' - D D T , o,p'D D T , p , p ' - D D D , o , p ' - D D D , p , p ' - D D E , a n d o , p ' - D D E i n the O k a n a g a n birds o n l y . C o n t a m i n a n t non-detects a n d zeros w e r e r e p l a c e d b y 0.00005 ( h a l f the d e t e c t i o n l i m i t ) for the c o r r e l a t i o n s . P v a l u e s less than or e q u a l to 0.05 were c o n s i d e r e d significant. A l l statistics w e r e c o n d u c t e d u s i n g J M P v e r s i o n 3.2.1 software ( S A S Institute, C a r y , N o r t h C a r o l i n a ) . V a l u e s presented represent m e a n s + standard errors, and ranges. 3 . 3 . Results 3.3.1. E g g C o n t a m i n a n t s T a b l e 3-1 illustrates the m e a n s , standard errors, a n d ranges for m o i s t u r e content, l i p i d content, p , p ' - D D T , p , p ' - D D D , p , p ' - D D E , o,p'- D D T , o , p ' - D D D , o , p ' - D D E , a n d the ratios o f D D E to D D T for the e g g s c o l l e c t e d i n 1 9 9 7 .  The complete  listing o f organochlorine  and  p o l y c h l o r i n a t e d b i p h e n y l s a n a l y z e d is a v a i l a b l e i n A p p e n d i x I. A s expected, O k a n a g a n eggs h a d significantly higher levels o f p , p ' - D D T ( F \  3 2  = 6 6 . 0 , p < 0 . 0 0 0 1 ) , p , p ' - D D D ( F i , 32 = 2 4 . 3 , p <  0.0001), a n d p , p ' - D D E ( F i , 32 = 4 1 . 8 , p < 0.0001) than L o w e r M a i n l a n d eggs. W h i l e p,p' i s o m e r s w e r e f o u n d i n a l l the s a m p l e s , o,p' i s o m e r s w e r e o n l y f o u n d i n the O k a n a g a n eggs.  0,p'-DDT  w a s detected i n 7 4 . 2 % , o , p ' - D D D i n 4 8 . 4 % , and o , p ' - D D E i n 4 5 . 2 % o f the O k a n a g a n eggs.  The  D D E : D D T ratio w a s not s i g n i f i c a n t l y different b e t w e e n the t w o g r o u p s , a l t h o u g h there w a s a trend (p = 0.06) t o w a r d s h i g h e r ratios o c c u r r i n g i n the L o w e r M a i n l a n d eggs than the O k a n a g a n eggs. L o w e r M a i n l a n d eggs c o n t a i n e d a s i g n i f i c a n t l y h i g h e r percentage o f l i p i d than O k a n a g a n eggs (F\j2 = 5.7, p = 0.02), but there were n o differences i n m o i s t u r e content. L i p i d content w a s n e g a t i v e l y correlated w i t h o , p ' - D D D (r = - 0.4, n = 3 1 , p = 0.02) i n the O k a n a g a n eggs, but there were n o other significant correlations.  3.3.2. E g g M e a s u r e m e n t s E g g s c o l l e c t e d f r o m the L o w e r M a i n l a n d a v e r a g e d 6.7 g r a m s ( ± 0 . 1 , range = 5.6 g r a m s ) i n w e i g h t , as d i d eggs c o l l e c t e d f r o m the O k a n a g a n (+ 0 . 1 , range = 5.1 - 8.4).  8.3 Egg  lengths r a n g e d f r o m 27.5 to 32.2 m m (29.7 + 0.2 m m ) i n the L o w e r M a i n l a n d samples a n d 26.3 to 32.6 m m (29.2 + 0.2 m m ) i n the O k a n a g a n samples. E g g w i d t h s w e r e v e r y s i m i l a r b e t w e e n  Table 3-1: Means (± se) and ranges of DDTs, moisture content, and lipid content (p:g/g) American robin eggs collected from the Okanagan and Lower Mainland. Lower Mainland n=3  Okanagan n = 31  % Moisture  82.0 ( ± 0.2)  82.8 ( ± 0.3)  81.6-82.3  80.2 - 85.3  % Lipid  5.7 ( ± 0.2)  4.3 ( ± 0.2)*  5.4-6.1  1.5-5.8  p,p'-DDT  0.1 ( ± 0 . 0 1 ) 0.1-0.2  12.1 ( ± 1.6)*  o,p-DDT  ND  p,p'-DDD  0.009 ( ± 0.004)  1.0 ( ± 0 . 3 ) *  0.003 - 0.02  0.06 - 8.7  o,p'-DDD  ND  p,p'-DDE  1.9 ( ± 0 . 7 )  51.7 ( ± 8 . 7 ) *  0.9-3.2  10.0-245.0  o,p'-DDE  ND  DDE:DDT N D = not detected, *p < 0.05  0.9 - 30.5  0.07 ( ± 0 . 0 1 ) N D - 0.3  0.005 ( ± 0 . 0 0 1 ) N D - 0.02  0.005 ( ± 0.0009) N D - 0.01  12.8 ( ± 3 . 7 )  6.8 ( ± 2.0)  8.0-20.1  0.7-63.1  51 the t w o groups ( L o w e r M a i n l a n d 2 1 . 0 ± 0 . 1 , range 19.3 - 2 2 . 6 m m ; O k a n a g a n 21.0 + 0.1, range 19.3 - 23.0 m m ) . T h e r e w e r e n o s i g n i f i c a n t differences i n w e i g h t , length, or w i d t h o f the L o w e r M a i n l a n d a n d O k a n a g a n eggs. T h e O k a n a g a n p a i r - w i s e correlations b e t w e e n e g g measurements a n d D D T contaminant l e v e l s w e r e also not significant. 3.3.3. C h i c k M e a s u r e m e n t s C h i c k m e a s u r e m e n t s are l i s t e d i n T a b l e 3-2. L o w e r M a i n l a n d c h i c k s h a d s i g n i f i c a n t l y l o n g e r m i d d l e toes than their O k a n a g a n counterparts (Fi,68 = 4 7 5 . 9 , p < 0.0001). T h e r e w e r e n o s i g n i f i c a n t type differences i n the other b o d y measurements.  F o r the O k a n a g a n c h i c k s , b o d y  w e i g h t w a s p o s i t i v e l y c o r r e l a t e d w i t h p , p ' - D D E (r = 0.5, n = 3 1 , p = 0.009), but there w e r e n o other s i g n i f i c a n t correlations. S e x w a s d e t e r m i n e d for 104 b i r d s . T h e r e w e r e 2 2 female a n d 21 m a l e c h i c k s c o l l e c t e d f r o m the L o w e r M a i n l a n d a n d 2 7 females a n d 34 m a l e s c o l l e c t e d f r o m the O k a n a g a n . T h e r e w e r e n o significant differences i n sex ratio b e t w e e n the t w o groups. 3.3.4. G r o w t h M e a s u r e m e n t s F i g u r e 3-1 demonstrates b o d y w e i g h t s for the b i r d s at the v a r i o u s ages m e a s u r e d , a n d F i g u r e 3-2 illustrates changes i n tarsus l e n g t h w i t h t i m e . T h e r e were n o s i g n i f i c a n t differences i n w e i g h t at a n y o f the ages tested.  H o w e v e r , w h e n o u t l i e r s w e r e r e m o v e d f r o m the a n a l y s e s ,  O k a n a g a n b i r d s w e r e s h o w n to w e i g h m o r e than L o w e r M a i n l a n d b i r d s at 2 m o n t h s o f age, but L o w e r M a i n l a n d b i r d s w e i g h e d m o r e at 7 - 9 m o n t h s o f age. W i t h i n the O k a n a g a n b i r d s , o n l y 10- d a y w e i g h t w a s p o s i t i v e l y c o r r e l a t e d w i t h p , p ' - D D E (r = 0.4, n = 2 5 , p = 0.04).  Tarsus  l e n g t h s w e r e s i g n i f i c a n t l y l a r g e r i n the L o w e r M a i n l a n d than i n the O k a n a g a n b r o o d s at 2 months ( F i ,  6 5  = 15.8, p = 0.0002), 5 m o n t h s ( F ,  j 6 2  = 6.2. p = 0.02), a n d 7 to 9 m o n t h s ( F i  > 6 ]  = 5.8,  p = 0.02) o f age. T h e difference i n tarsus lengths at 7 to 9 m o n t h s , h o w e v e r , w a s n o n - s i g n i f i c a n t w h e n a n o u t l i e r w a s r e m o v e d . T h e r e w e r e n o s i g n i f i c a n t correlations b e t w e e n egg c o n t a m i n a n t l e v e l s a n d tarsus length.  3.3.5. T h y r o i d H o r m o n e s T h e r e w e r e n o s i g n i f i c a n t differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n t r i i o d o t h y r o n i n e o r t h y r o x i n e l e v e l s , at any o f the ages tested ( F i g u r e 3-3). correlations b e t w e e n t h y r o i d h o r m o n e l e v e l s and e g g D D T residues w e r e f o u n d .  N o significant  52  Table 3-2: Means (+ se) and ranges of body weight, and tarsus, wing cord, and middle toe lengths of ten day old American robin nestlings collected from the Okanagan and Lower Mainland.  Weight (g)  Tarsus (mm)  Wing (mm)  Lower  58.4 ( + 1 . 5 )  37.3 ( ± 0.3)  63.4 ( ± 1.7)  Mainland  34.0 - 76.0  32.8-41.5  46.0 - 7 9 . 0  19.0-26.0  60.4 ( ± 0.8)  36.8 ( ± 0.2)  64.6 ( ± 0.9)  10.9 ( ± 0 . 3 ) *  50.1 - 7 5 . 1  33.9 - 40.6  50.7-74.3  6.7-20.0  Okanagan  *p < 0.05  Toe (mm)  21.6  (±0.3)  ;ure 3-1: Mean (+ se) body weights (grams) of Lower Mainland and Okanagan American robins at various ages.  39.5  n  Figure 3-2: Mean (+ se) tarsus lengths (mm) of Lower Mainland and Okanagan American robins at various ages. * p <  55  A ) T r i iodothyronine  Summer 1997  Summer 1997  Fall 1997  February 1998  Fall 1997  February 1998  August 1998  August 1998  Time  Figure 3 - 3 : Plasma levels of A)triiodothyronine (pg/ml) and B)thyroxine (ng/ml) in Okanagan and Lower Mainland robins at various ages.  56 3.3.6. I m m u n e R e s p o n s e  3.3.6.1. White Blood Cell Ratios L o w e r M a i n l a n d b i r d s h a d s i g n i f i c a n t l y h i g h e r h e t e r o p h i l t o l y m p h o c y t e ratios t h a n O k a n a g a n b i r d s at t e n d a y s o f age ( F i j i = 1 1 . 1 , p = 0 . 0 0 1 ; F i g u r e 3-4), b u t there w e r e n o s i g n i f i c a n t differences b e t w e e n the groups w h e n they w e r e p o s t - b r e e d i n g adults. W h i t e b l o o d c e l l ratios i n t h e O k a n a g a n b i r d s , h o w e v e r , w e r e s i g n i f i c a n t l y c o r r e l a t e d w i t h p , p ' - D D T w h e n they w e r e post-breeding adults (r = 0.4, n = 2 5 , p = 0.03). R a t i o s w e r e c o n s i d e r a b l y h i g h e r w h e n the b i r d s w e r e 1 0 - d a y - o l d n e s t l i n g s as c o m p a r e d t o p o s t - b r e e d i n g adults (F1J26  =  244.0, p <  0 . 0 0 0 1 ; F i g u r e 3-4).  3.3.6.2. Phytohemagglutinin Skin Test T h e r e w e r e n o s i g n i f i c a n t differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n m a l e s i n their response to the p h y t o h e m a g g l u t i n i n s k i n test, n o r w e r e there a n y s i g n i f i c a n t c o r r e l a t i o n s w i t h e g g D D T l e v e l s for the O k a n a g a n b i r d s . W i n g w e b i n d i c e s i n the L o w e r M a i n l a n d m a l e s r a n g e d f r o m - 0.04 t o 1.0 (0.5 ± 0 . 1 ) , w h i l e O k a n a g a n m a l e s ' r a n g e d f r o m 0.1 t o 1.1 (0.4 ± 0.08). 3.3.7. M o r t a l i t y T a b l e 3-3 illustrates cause o f death f o r the r o b i n s i n this study. cause o f death w a s n o t d e t e r m i n e d .  I n a n u m b e r o f cases,  F i v e o f the O k a n a g a n b i r d s w e r e d i a g n o s e d as h a v i n g  intestinal c o c c i d i o s i s (parasites Eimeria a n d Isospora), a n d it w a s suspected that a l l 13 o f the O k a n a g a n b i r d s that a p p e a r e d t o starve t o death ( w a s t i n g ) w e r e i n f e c t e d . T w o o f these b i r d s w e r e also suffering f r o m l e u c o c y t o z o o n o s i s ( p r o t o z o a n b l o o d parasite, Leucocytozoon). N o n e o f the L o w e r M a i n l a n d b i r d s h o u s e d u n d e r the same c o n d i t i o n s w e r e affected. euthanized f o l l o w i n g debilitating injuries.  Seven birds were  A c c i d e n t a l deaths i n c l u d e d those i n c u r r e d d u r i n g  b l o o d s a m p l i n g , d r o w n i n g ( i n w a t e r d i s h e s ) , a n d a v a r i e t y o f other i n c i d e n t s . d i s a p p e a r e d o r e s c a p e d w e r e p r e s u m e d d e a d a n d w e r e l i k e l y depredated.  B i r d s that  Several birds died  f o l l o w i n g t r a u m a as a result o f h o u s e c a t (Felis domesticus) a n d h a w k attack attempts f r o m outside the cages, b u t exact cause o f death w a s n o t d e t e r m i n e d . R a t s (Rattus norvegicus) w e r e c o m m o n i n the b r e e d i n g pens a n d w e r e c o n s i d e r e d r e s p o n s i b l e f o r the p r e d a t i o n deaths.  There  w a s a s i g n i f i c a n t difference b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n cause o f death (X - 14.2, p = 0.01), m o s t l i k e l y due to the starvation deaths i n the O k a n a g a n b i r d s . 2  57  Figure 3-4: Differences (+ se) in white blood cell ratios (eosinophils + heterophils / lymphocytes + monocytes) for Lower Mainland and Okanagan American robins as 10 day old nestlings and post-breeding adults. *p < 0.05  58  Table 3-3: Causes of death for American robins collected from the Okanagan and Lower Mainland.  Lower Mainland n = 33 undetermined  accidental  disappeared/ escaped  depredated  euthanized  starved/coccidiosis  Okanagan n = 60  Total n = 93  19  22  41  57.6%  36.7%  44.1%  3  10  13  9.1%  16.7%  14.0%  5  5  10  15.2%  8.3%  10.8%  3  9.1%  6 10.0%  9 9.7%  3  4  7  9.1%  6.7%  7.5%  0  13  13  21.7%  14.0%  59 C o n t a m i n a n t l e v e l s i n the e g g d i d not s i g n i f i c a n t l y i n f l u e n c e the cause o f death for O k a n a g a n b i r d s . T h e O k a n a g a n b i r d s that d i e d as j u v e n i l e s f r o m s t a r v a t i o n / c o c c i d i o s i s , for instance, c a m e f r o m nests w i t h a w i d e range o f c o n t a m i n a n t l e v e l s . T o t a l e g g D D T l e v e l s for these b i r d s r a n g e d f r o m 13.5 to 104.3 p g / g . A l t h o u g h , m o s t o f the b i r d s s u r v i v e d to a d u l t h o o d ( 1 0 7 / 1 4 7 , 7 2 . 8 % ) , 4 ( 2 . 7 % ) b i r d s d i e d w h e n s t i l l i n the n e s t l i n g stage (< 14 days o f age), a n d 5 (3.4%) c h i c k s d i e d d u r i n g w h a t w o u l d be c o n s i d e r e d the f l e d g l i n g (> 14 days but < 3 0 days) stage. A n a d d i t i o n a l 31 (21.1%>) b i r d s d i e d p r i o r to s e x u a l m a t u r i t y a n d transfer to the b r e e d i n g cages ( F e b r u a r y 1998). T h e r e w a s a s i g n i f i c a n t difference b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n t i m e o f death (k  2  = 14.6, p = 0 . 0 0 2 ; T a b l e 3-4).  A l t h o u g h the m a j o r i t y o f b o t h L o w e r M a i n l a n d a n d  O k a n a g a n d i e d as adults, a larger n u m b e r o f O k a n a g a n b i r d s d i e d as j u v e n i l e s , l i k e l y as a result o f c o c c i d i o s i s infections.  3.3.8. T i s s u e W e i g h t s a n d B o d y M e a s u r e m e n t s T i s s u e w e i g h t s a n d b o d y m e a s u r e m e n t s at s a c r i f i c e are l i s t e d i n T a b l e 3 - 5 . significant differences  The only  i n o r g a n w e i g h t s ( c o r r e c t e d f o r b o d y w e i g h t ) b e t w e e n the  Lower  M a i n l a n d a n d O k a n a g a n b i r d s w e r e i n l i v e r w e i g h t ( F i ^ = 4 . 3 , p = 0.04), a n d heart w e i g h t (Fi 36 ;  = 9.6, p = 0.004). B o t h o f these tissues w e r e h e a v i e r i n O k a n a g a n t h a n L o w e r M a i n l a n d b i r d s . T h e r e m o v a l o f an o u t l i e r rendered the difference i n l i v e r w e i g h t n o n - s i g n i f i c a n t . D i f f e r e n c e s i n b r a i n , t h y r o i d glands, t h y m u s , spleen, bursa, g o n a d , a n d o v i d u c t w e i g h t s w e r e not s i g n i f i c a n t , n o r w e r e the differences i n b o d y measurements.  W i t h i n the O k a n a g a n b i r d s , s i g n i f i c a n t c o r r e l a t i o n s  w e r e f o u n d b e t w e e n g o n a d w e i g h t a n d p , p ' - D D E (r = - 0.5, n = 18, p = 0.04; F i g u r e 3-5), a n d o v i d u c t w e i g h t a n d o , p ' - D D T (r = 0.8, n = 9, p = 0 . 0 0 9 ; F i g u r e 3-6).  E g g D D T l e v e l s d i d not  s i g n i f i c a n t l y correlate w i t h the other tissue w e i g h t s o r w i t h the b o d y m e a s u r e m e n t s t a k e n at sacrifice.  3.4. Discussion T h e o b j e c t i v e o f this study w a s to e x a m i n e whether e a r l y exposure to D D T has any d e l a y e d o r l o n g - t e r m effects o n the g r o w t h a n d s u r v i v a l o f A m e r i c a n r o b i n s .  T h i s w a s d o n e b y : 1)  c o m p a r i n g c o n t a m i n a t e d O k a n a g a n b i r d s w i t h u n c o n t a m i n a t e d L o w e r M a i n l a n d c o n t r o l s , a n d 2) c o r r e l a t i n g c o n t a m i n a t i o n l e v e l s o f O k a n a g a n eggs w i t h the g r o w t h parameters o f t h e i r c l u t c h mate(s).  T h e d e s i g n o f the study w a s b a s e d o n three a s s u m p t i o n s :  a) that the O k a n a g a n a n d  L o w e r M a i n l a n d b i r d s w e r e o f s i m i l a r genetic b a c k g r o u n d , b) the l e v e l o f c o n t a m i n a t i o n i n a l l  60  Table 3-4: Ages at time of death for American robins collected from the Okanagan and Lower Mainland.  adult  juvenile  fledgling  nestling  Lower Mainland  Okanagan  Total  n = 33  n = 60  n = 93  24  29  53  72.7%  48.3%  57.0%  4  27  31  12.1%  45.0%  33.3%  4  1  5  12.1%  1.7%  5.4%  1  3  4  3.0%  5.0%  4.3%  61 Table 3-5: Means (+ se) and ranges of body measurements and tissue weights (corrected for body weight) of Okanagan and Lower Mainland robins at sacrifice.  body weight (grams) wing (cm) toe (cm) tarsus (cm) brain (g) thyroids (g)  Lower Mainland n = 15  Okanagan n = 23  79.9 ( ± 1.1)  81.4 ( ± 1 . 0 )  72.1 - 8 5 . 4  66.6-86.6  12.8 ( ± 0.2)  12.9 ( ± 0 . 1 )  11.5-14.1  11.5-13.3  1.7 ( ± 0 . 3 )  1.7 ( ± 0 . 3 )  1.5-1.9  1.5-1.9  3.7 ( ± 0 . 3 )  3.7 ( ± 0 . 3 )  3.4-3.9  3.5-4.0  0.021 ( ± 0 . 0 0 0 4 3 ) 0.018-0.023  0.020 ( ± 0.00040) 0.018-0.024  0.00025 ( ± 0 . 0 0 0 0 1 8 )  0.00031 ( ± 0 . 0 0 0 0 2 5 )  0.00013-0.00038  0.00013-0.00062  0.0013 ( ± 0 . 0 0 0 1 5 ) 0.00039 - 0.0023  0.0011 ( ± 0 . 0 0 0 1 1 ) 0.00036 - 0.0025  heart (g)  0.012 ( ± 0 . 0 0 0 2 5 )  0.013 ( ± 0 . 0 0 0 2 9 ) *  0.011-0.014  0.011-0.016  spleen (g)  0.002 ( ± 0.00043) 0.00054 - 0.0074  0.0023 ( ± 0 . 0 0 0 1 9 ) 0.0010-0.0041  gonads (g)  0.00052 ( ± 0.000047)  0.00041 ( ± 0 . 0 0 0 0 3 9 )  0.00021 - 0 . 0 0 0 7 9  0.00016-0.00079  bursa (g)  0.00086 ( ± 0.000082)  0 . 0 0 0 9 2 ( ± 0.000072)  0.00042-0.0014  0.00052-0.0018  0.011 ( ± 0 . 0 0 0 5 3 )  0.011 ( ± 0 . 0 0 0 2 3 )  0.0082-0.015  0.0090-0.014  0.00073 ( ± 0.000072)  0.00070 ( ± 0.000096)  0.00044-0.0012  0.00031 - 0 . 0 0 1 5  0.024 ( ± 0.00093)  0.026 ( ± 0 . 0 0 0 6 0 ) * 0.022 - 0.035  thymus (g)  kidneys (g) oviduct (g) liver (g)  *p < 0.05  0.018-0.031  ;ure 3-5: Relationship between gonad weight at sacrifice (grams, corrected for body weight) and egg p,p'-DDE  (ng/g) levels.  vo O.OOI8-1  0.0016 4 0.0014 4  0.0002 0  -J  1  1  1  1  1  1  0  0.01  0.02  0.03  0.04  0.05  0.06  r-  0.07  1  0.08  i  0.09  o,p'-DDT (ppm)  Figure 3-6: Relationships between egg o,p'-DDT ((-ig/g) and female Okanagan American robin oviduct weights (grams) at time of sacrifice.  64 the eggs i n a c l u t c h w a s s i m i l a r , a n d c) a m o n g O k a n a g a n b i r d s , parental care a n d p o s t - h a t c h D D T exposure ( f r o m h a t c h i n g to about ten-days o f age) w a s n o t a s i g n i f i c a n t v a r i a b l e . 3.4.1. G r o w t h a n d D e v e l o p m e n t A s expected, O k a n a g a n eggs c o n t a i n e d c o n s i d e r a b l y h i g h e r l e v e l s o f D D T s t h a n L o w e r M a i n l a n d e g g s (See E l l i o t t et a l . , 1 9 9 4 ; G i l l et a l . , 2 0 0 3 ; H a r r i s et a l . , 2 0 0 0 ) . It w a s a l s o f o u n d that o,p' i s o m e r s w e r e o n l y present i n O k a n a g a n eggs and not L o w e r M a i n l a n d eggs. eggs also h a d h i g h e r l i p i d contents.  Okanagan  C h i c k s c o l l e c t e d from the O k a n a g a n h a d shorter m i d d l e toes  a n d at certain points i n d e v e l o p m e n t also had shorter tarsi. A f t e r r e a c h i n g a d u l t h o o d , their hearts a n d l i v e r s (adjusted for b o d y w e i g h t ) w e r e h e a v i e r than those o f L o w e r M a i n l a n d b i r d s . O f the parameters e x a m i n e d , o n l y b o d y w e i g h t at ten days o f age w a s c o r r e l a t e d w i t h p , p ' - D D E l e v e l s i n the eggs.  C h i c k s f r o m m o r e c o n t a m i n a t e d nests w e r e h e a v i e r at ten-days than those f r o m  nests w i t h l o w e r D D E l e v e l s .  T h e s e results suggest that in ovo c o n t a m i n a t i o n m a y h a v e  i n f l u e n c e d w e i g h t g a i n i n these c h i c k s s h o r t l y after h a t c h i n g .  T h e m a j o r i t y o f studies suggest  that D D T s suppress rather than p r o m o t e g r o w t h ( B r i t t o n et a l . , 1974; F o x , 1997; T y l e r et a l . , 1998; V o s et a l . , 2 0 0 0 ) . I n B e n g a l e s e finches  (Lonchura striata),  for instance, b o t h egg w e i g h t  a n d c h i c k w e i g h t decreased w i t h i n c r e a s i n g p , p ' - D D T a n d p , p ' - D D E l e v e l s (Jefferies, 1971). A s parental care measures a n d i m m e d i a t e post-hatch D D T exposure are not k n o w n for the r o b i n s i n this study, they c o u l d serve as c o n f o u n d i n g factors. O k a n a g a n b i r d s h a d shorter m i d d l e toes t h a n L o w e r M a i n l a n d c h i c k s , a n d they h a d shorter tarsi t h a n L o w e r M a i n l a n d b i r d s at t w o , f i v e , a n d s e v e n to n i n e m o n t h s o f age.  Lower  M a i n l a n d b i r d s s h o w e d the m o s t d r a m a t i c increase i n tarsus l e n g t h b e t w e e n ten d a y s a n d t w o m o n t h s o f age. T h e O k a n a g a n b i r d s , i n contrast, d i d not e x h i b i t this g r o w t h spurt u n t i l b e t w e e n t w o a n d five m o n t h s o f age. T a r s u s g r o w t h i s often u s e d as a n i n d i c a t o r o f b o d y g r o w t h ( A l d r i c h & James, 1991). These results suggest that g r o w t h i n O k a n a g a n b i r d s w a s d e l a y e d relative to the L o w e r M a i n l a n d controls.  T h i s is a n effect w h i c h m a y be attributed to in ovo D D T e x p o s u r e  ( B r i t t o n et a l . , 1974; F o x , 1997; T y l e r et a l . , 1 9 9 8 ; V o s et a l . , 2 0 0 0 ) .  While Okanagan chicks  w e i g h e d m o r e than L o w e r M a i n l a n d c h i c k s at t w o m o n t h s o f age, b y the t i m e they w e r e seven to n i n e m o n t h s o l d they w e i g h e d less. D D T s c o u l d p o t e n t i a l l y i n f l u e n c e b o d y w e i g h t a n d w e i g h t g a i n t h r o u g h their effects o n the t h y r o i d glands, as t h y r o i d h o r m o n e s i n f l u e n c e b o t h b o d y a n d o r g a n g r o w t h (Jefferies, 1975; K i n g & M c L e l l a n d , 1984). T h e steroid h o r m o n e effects o f D D T s m a y also p l a y a r o l e i n b o d y w e i g h t . T h e anti-androgen,  flutamide,  depressed the b o d y w e i g h t s o f three a n d s e v e n w e e k o l d  65 b r o i l e r c h i c k s , s u g g e s t i n g that interference w i t h the a c t i o n s o f e n d o g e n o u s a n d r o g e n s d u r i n g e m b r y o n i c l i f e c a n suppress p o s t - h a t c h i n g g r o w t h ( B u r k e , 1 9 9 6 ) .  A s p , p ' - D D E is an anti-  a n d r o g e n ( G a i d o et a l . , 1 9 9 7 ; K e l c e et a l . , 1995, 1998), it m a y h a v e s i m i l a r effects, a l t h o u g h this c a n n o t be c l e a r l y d e m o n s t r a t e d i n this study.  T h e r e w e r e n o s i g n i f i c a n t differences  between  L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n either p l a s m a t r i i o d o t h y r o n i n e or t h y r o x i n e l e v e l s at any o f the ages tested a n d there w e r e n o s i g n i f i c a n t correlations b e t w e e n the D D T s m e a s u r e d i n the eggs a n d t h y r o i d h o r m o n e s at any age. B o t h l i v e r s a n d hearts w e r e h e a v i e r i n O k a n a g a n than L o w e r M a i n l a n d b i r d s . P r e v i o u s studies have l i n k e d b o t h enlarged hearts a n d l i v e r s to t h y r o i d h o r m o n e l e v e l s . F o r e x a m p l e , l i v e r h y p e r t r o p h y a n d l i v e r g l y c o g e n a c c u m u l a t i o n c a n be i n d u c e d b y h y p o t h y r o i d i s m i n c h i c k s ( W e n t w o r t h & R i n g e r , 1986), a n d t h y r o x i n e c a n accelerate heart rate a n d increase heart w e i g h t i n d o m e s t i c f o w l s (Jefferies, 1975). S i m i l a r effects h a v e been seen w i t h D D T . B o b w h i t e q u a i l  (Colinus virginianus;  H u r s t et a l . , 1974) a n d p i g e o n s  (Columba livia;  Jefferies & F r e n c h , 1969  s h o w e d i n c r e a s i n g l i v e r w e i g h t s w i t h i n c r e a s i n g D D T dose. T h e increase i n l i v e r size m a y h a v e b e e n due to increased hepatic a c t i v i t y and m e t a b o l i s m o f c i r c u l a t i n g h o r m o n e s , as D D T is k n o w n to i n d u c e e n z y m e b r e a k d o w n o f h o r m o n e s ( P e a k a l l , 1 9 6 7 ) .  P i g e o n s f e d a l o w dose o f D D T  e x h i b i t e d increases i n a m p l i t u d e o f the v e n t r i c u l a r beat a n d heart w e i g h t , but b i r d s fed a h i g h dose s h o w e d heart beat a m p l i t u d e s l o w e r than that i n c o n t r o l s , decreased heart w e i g h t s , a n d t h i n , f l a c c i d heart m u s c u l a t u r e .  H e a r t rate, beat a m p l i t u d e , a n d w e i g h t c o n t i n u e d to increase w i t h  i n c r e a s i n g dose, h o w e v e r , i n B e n g a l e s e f i n c h (Jefferies, 1975). A l t h o u g h the r o b i n s i n this study d i d not demonstrate any s i g n i f i c a n t differences i n t h y r o i d h o r m o n e l e v e l s , little is k n o w n about the effects o f in ovo exposure to D D T o n these organs. W h i l e there w a s n o s i g n i f i c a n t d i f f e r e n c e i n g o n a d a n d o v i d u c t w e i g h t s  between  O k a n a g a n a n d L o w e r M a i n l a n d b i r d s , g o n a d w e i g h t s i n the O k a n a g a n b i r d s w e r e n e g a t i v e l y correlated w i t h p , p ' - D D E .  P a r a , p a r a ' - D D E has b e e n s h o w n to act as a n a n d r o g e n  antagonist  ( G a i d o et a l . , 1997; K e l c e et a l . , 1995, 1998), thus its effects o n testes g r o w t h a n d d e v e l o p m e n t i n p a r t i c u l a r c o u l d be p r o f o u n d . exposure.  O v i d u c t weights were positively correlated w i t h o , p ' - D D T  T h i s f o r m o f D D T is a k n o w n e s t r o g e n a g o n i s t that has b e e n s h o w n to increase  o v i d u c t w e i g h t i n other species ( S t i c k e l , 1973). T h i s study demonstrates that e a r l y exposures to these D D T isomers m a y also have l o n g t e r m effects o n these r e p r o d u c t i v e organs. It is therefore o f interest to e x a m i n e the r e p r o d u c t i v e p e r f o r m a n c e a n d b e h a v i o r o f these O k a n a g a n b i r d s (See Chapter I V ) .  66 3.4.2. Imrnunity a n d S u r v i v a l A t ten-days o f age, L o w e r M a i n l a n d c h i c k s h a d h i g h e r percentages o f h e t e r o p h i l s a n d eosinophils than Okanagan chicks, whereas O k a n a g a n birds had higher levels o f lymphocytes and monocytes.  T h e r e are several p o s s i b l e e x p l a n a t i o n s for this. N o r m a l stress response a n d  c o r t i c o s t e r o n e release i n c r e a s e h e t e r o p h i l l e v e l s a n d decrease l y m p h o c y t e l e v e l s ( D u f v a  &  A l l a n d e r , 1995; G r a s m a n et a l . , 1996; S i e g e l , 1980; S m i t s & W i l l i a m s , 1999), a n d the h e t e r o p h i l to l y m p h o c y t e ratio has b e e n u s e d e x t e n s i v e l y as a r e l i a b l e i n d i c a t o r o f p h y s i o l o g i c a l a n d s o c i a l stress ( G r o s s & S i e g e l , 1 9 8 3 ; E h r i c h & G r o s s , 1986; G r o s s , 1990; de J o n g et a l , 2 0 0 2 ) .  While  D D T m a y i n h i b i t the stress response i n O k a n a g a n c h i c k s (see C h a p t e r V ) thus i n f l u e n c i n g the w h i t e b l o o d c e l l ratios ( B i e s m a n n & v o n F a b e r , 1 9 8 1 ; L a t i m e r & S i e g e l , 1 9 7 4 ) , it has b e e n s h o w n i n c h i c k e n s that h e t e r o p h i l / l y m p h o c y t e ratios c a n take up to t w o days to reflect the effects o f a stressor ( P u v a d o l p i r o d & T h a x t o n , 2 0 0 0 ) . A s adults these b i r d s w o u l d l i k e l y be less p r o n e to stress-related effects o n w h i t e b l o o d c e l l ratios, as they h a d b e e n c a p t u r e d a n d h a n d l e d o n n u m e r o u s o c c a s i o n s a n d were l i k e l y s o m e w h a t habituated to it. T h u s , stress o f h a n d l i n g l i k e l y d i d not p l a y m u c h o f a r o l e i n the different w h i t e b l o o d c e l l ratios seen i n the r o b i n c h i c k s , as b l o o d samples were o b t a i n e d i m m e d i a t e l y u p o n capture.  Glucocorticoid levels and white b l o o d  c e l l n u m b e r s fluctuate t h r o u g h o u t the day, w i t h v a r i a t i o n s as h i g h as 5 0 % ( C r i s p et a l . , 1998). Thus,  simply  sampling  the  h e t e r o p h i l / l y m p h o c y t e ratios.  birds  at  different  times  of  the  day  may  influence  A s l y m p h o c y t e l e v e l s t e n d to r i s e i n response to v i r u s e s , a n d  h e t e r o p h i l l e v e l s tend to rise i n response to bacteria ( S i e g e l , 1980), it is also p o s s i b l e that one or b o t h groups w e r e b a t t l i n g l o w l e v e l s o f i n f e c t i o n . A l t e r n a t i v e l y , these differet w h i t e b l o o d c e l l ratios m a y n o r m a l l y be different b e t w e e n these t w o g r o u p s due to g e n e t i c d i f f e r e n c e s below).  (see  D D T exposure m a y have t r i g g e r e d i m m u n e responses i n the O k a n a g a n b i r d s o r m a d e  t h e m m o r e susceptible to i n f e c t i o n . H o w e v e r , the c e l l ratios w e r e not s i g n i f i c a n t l y c o r r e l a t e d w i t h in ovo D D T exposure. R e g a r d l e s s o f the the reason b e h i n d the difference i n w h i t e b l o o d c e l l ratios i n nestlings, as adults, L o w e r M a i n l a n d a n d O k a n a g a n b i r d s d i d not s h o w differences.  A s adults, the b i r d s  w o u l d have been less p r o n e to stress i n d u c e d changes as they w e r e m o r e u s e d to b e i n g h a n d l e d a n d the adrenal response to a d r e n i c o r t i c o t r o p i c h o r m o n e is l o w e r i n adults than i n y o u n g b i r d s , m a k i n g t h e m less r e s p o n s i v e to stressful s t i m u l i ( H a r v e y et a l . , 1 9 8 6 ) .  Differences i n white  b l o o d c e l l ratios at v a r i o u s life stages m a y s i m p l y reflect n o r m a l age and/or seasonal changes ( S t u r k i e & G r i m i n g e r , 1986). S m i t s and W i l l i a m s ( 1 9 9 9 ) f o u n d a n increase i n l y m p h o c y t e s and a decrease i n heterophils a n d e o s i n o p h i l s b e t w e e n 11 a n d 21 d a y s i n z e b r a finches  (Taeniopygia  67  guttata), but  D u f v a and A l l a n d e r (1995) f o u n d n o differences i n w h i t e b l o o d c e l l counts b e t w e e n  different age classes o f G r e a t tits  (Parus major).  S e a s o n a l changes, e v e n w i t h i n a p e r i o d o f a  f e w w e e k s w e r e associated w i t h changes i n the i m m u n e systems o f tree s w a l l o w s  bicolor)  (Tachycineta  ( B i s h o p et a l . , 1998). H o w e v e r , w i t h i n the O k a n a g a n b i r d s , p , p ' - D D T w a s p o s i t i v e l y  c o r r e l a t e d w i t h w h i t e b l o o d c e l l ratios.  T h u s , b i r d s f r o m m o r e c o n t a m i n a t e d nests h a d h i g h e r  h e t e r o p h i l l e v e l s as adults. It is not k n o w n for sure h o w m a n y c h i c k s d i e d p r i o r to ten-days o f age w h e n they w e r e c o l l e c t e d . G i l l et a l . (2003) reported n o s i g n i f i c a n t differences i n hatch rate, b r o o d size, or fledge rate b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n nests f r o m w h i c h the b i r d s w e r e c o l l e c t e d . U n d e r c a p t i v i t y , h o w e v e r , the O k a n a g a n b i r d s appeared to be m o r e s u s c e p t i b l e to i n f e c t i o u s disease t h a n L o w e r M a i n l a n d b i r d s . T h i r t e e n ( 2 1 . 7 % ) o f the O k a n a g a n c h i c k s d i e d o f starvation, l i k e l y d u e to c o c c i d i o s i s , w h e r e a s  no L o w e r M a i n l a n d birds appeared  to be  affected.  S i g n i f i c a n t l y h i g h e r j u v e n i l e m o r t a l i t y o c c u r r e d i n O k a n a g a n b i r d s due to p r e d a t i o n , accidents, a n d disease.  T h e s e three factors are p r o b a b l y related as s i c k b i r d s are m o r e e a s i l y depredated  a n d are m o r e p r o n e to accidents.  A s b i r d s f r o m the m o s t c o n t a m i n a t e d nests w e r e n o m o r e or  less l i k e l y to s u c c u m b to c o c c i d i o s i s t h a n those f r o m less c o n t a m i n a t e d nests, D D T e x p o s u r e m a y n o t be the f a c t o r i n v o l v e d .  A l t h o u g h G r a s m a n et a l . , ( 1 9 9 6 ) f o u n d a d e c r e a s e i n  p h y t o h e m a g g l u t i n i n response w i t h i n c r e a s i n g D D E l e v e l s i n g u l l s a n d terns, the r o b i n s i n this study s h o w e d n o differences i n response related to their l e v e l s o f i n o v o D D E exposure. al. ( 2 0 0 2 ) a l s o d i d not find that b a l d eagles  (Haliaeetus leucocephalus)  R o e et  from contaminated  coastal sites o n the G r e a t L a k e s were m o r e susceptible to b l o o d borne parasites than b i r d s f r o m c l e a n e r i n t e r i o r sites.  P a r a s i t e e x p o s u r e c a n suppress a n d / o r s t i m u l a t e i m m u n e r e s p o n s e s  d e p e n d i n g o n the life stage o f the parasite a n d the host ( B i s h o p et a l . , 1998). It i s p o s s i b l e that there are i n h e r e n t d i f f e r e n c e s b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s w h i c h affect their s u s c e p t i b i l i t y to disease. B i r d s f r o m the L o w e r M a i n l a n d m a y be g e n e t i c a l l y p r e d i s p o s e d to have greater resistance to the p a r t i c u l a r c o c c i d i a parasites f o u n d i n these b i r d s . T h e L o w e r M a i n l a n d a n d O k a n a g a n b i r d s m a y e v e n represent different subspecies ( A l d r i c h & James, 1991).  Turdus migratorius caurinus,  R o b i n s f r o m the L o w e r M a i n l a n d l i k e l y b e l o n g to the whereas O k a n a g a n r o b i n s are p r o b a b l y  ( A l d r i c h & James, 1 9 9 1 ; C a n n i n g s , 1998).  T. migratorius  subspecies  propinquus  I f the b i r d s h a d b e e n r a i s e d i n the O k a n a g a n , as  o p p o s e d to the L o w e r M a i n l a n d , L o w e r M a i n l a n d b i r d s m a y h a v e b e e n m o r e p r o n e to i n f e c t i o n . G e n e t i c differences b e t w e e n the b i r d s m a y also be exacerbated b y early D D T exposure.  68  3.5. Conclusions T h i s study demonstrated that in ovo D D T exposure i n f l u e n c e d g r o w t h a n d o r g a n w e i g h t s i n A m e r i c a n r o b i n s f r o m the O k a n a g a n V a l l e y . It appears that m o s t o f the detrimental effects o f in ovo c o n t a m i n a n t e x p o s u r e i n r o b i n s o c c u r r e l a t i v e l y e a r l y i n the b i r d s ' l i v e s .  W h e t h e r the  difference i n s u s c e p t i b i l i t y to c o c c i d i o s i s i n f e c t i o n b e t w e e n the O k a n a g a n a n d L o w e r M a i n l a n d b i r d s w a s D D T related cannot be established, a n d requires further e x a m i n a t i o n (see C h a p t e r V . ) . It is p o s s i b l e that genetic differences b e t w e e n the t w o g r o u p s o f b i r d s m a y h a v e i n f l u e n c e d the results.  69  3.6. References  A l d r i c h , J . W . , J a m e s , F . C . ( 1 9 9 1 ) . E c o g e o g r a p h i c v a r i a t i o n i n the A m e r i c a n R o b i n  (Turdus migratorius). The Auk, 108,  230-249.  B i e s m a n n , A . , v o n F a b e r , H . (1981). 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T e c h n i c a l R e p o r t S e r i e s N o . 3 3 5 E . C a n a d i a n W i l d l i f e S e r v i c e , Headquarters, H u l l , Q u e b e c , C a n a d a . W H O . (1989).  DDT and its derivatives - Environmental Aspects  C r i t e r i a N o . 83). W o r l d H e a l t h O r g a n i z a t i o n .  (Environmental Healt  75 Chapter I V Reproduction and Behavior in A m e r i c a n Robins Exposed In Ovo and E a r l y Post-Hatch to D D T and its Metabolites  4.1. Introduction  Dichlorodiphenyltrichloroethane  (DDT),  its  primary  metabolites  dichlorodiphenyldichloroethylene ( D D E ) and dichlorodiphenyldichloroethane ( D D D ) , and a v a r i e t y o f other o r g a n o c h l o r i n e c h e m i c a l s have l o n g b e e n l i n k e d to r e p r o d u c t i v e a b n o r m a l i t i e s a n d f a i l u r e i n b i r d s ( M u r p h y , 1980).  A s these c h e m i c a l s are d e p o s i t e d into eggs d u r i n g y o l k  f o r m a t i o n ( B r a n d t et a l . , 1 9 7 8 ; F r y , 1 9 9 5 ; O t t i n g e r et a l . , 2 0 0 1 ) , t h e y c a n i n f l u e n c e the r e p r o d u c t i o n n o t o n l y o f the e x p o s e d generation, b u t also their o f f s p r i n g . In ovo a n d early posthatch  exposure  to D D T s  m a y have  profound  effects  o n the d e v e l o p m e n t  and sexual  d i f f e r e n t i a t i o n o f the r e p r o d u c t i v e systems o f b i r d s ( B a l a s u b r a m a n i a m & S u n d a r a r a j , 1 9 9 3 ; B u r l i n g t o n & L i n d e m a n , 1950; F r y & T o o n e , 1 9 8 1 ; Stickel, 1973).  F o r example, male  C a l i f o r n i a g u l l s {Larus californicus) f r o m eggs injected w i t h as little as t w o m g / k g o , p ' - D D T h a d f e m i n i z e d gonads, w h i l e f i v e m g / k g o r h i g h e r o , p ' - D D T resulted i n the d e v e l o p m e n t o f b o t h left a n d right o v i d u c t s i n female g u l l s ( F r y & T o o n e , 1981). D D T c o n t a m i n a t i o n c a n also l e a d to alterations i n p a r e n t a l a n d other b e h a v i o u r s . F o r e x a m p l e , m e r l i n s (Falco columbarius) w h o s e eggs c o n t a i n e d h i g h l e v e l s o f p , p ' - D D E , a n d other o r g a n o c h l o r i n e s , h a v e b e e n s h o w n t o desert t h e i r c l u t c h e s m o r e r e a d i l y a n d d e f e n d t h e i r nests less a c t i v e l y than birds w i t h less c o n t a m i n a t e d eggs ( F o x & D o n a l d , 1980). H e r r i n g g u l l s (Larus argentatus) e x p o s e d to a n u m b e r o f c h e m i c a l c o n t a m i n a n t s h a v e a l s o d e m o n s t r a t e d  decreased  nest attentiveness ( F o x et a l . , 1978; Rattner et a l . , 1984). H y p e r - a g g r e s s i v e parents that injured their o f f s p r i n g w e r e f o u n d i n d o s i n g studies o f r i n g d o v e s (Streptopelia risoria) ( M c A r t h u r et a l . , 1983) a n d B e n g a l e s e f i n c h e s (Lonchura striata) (Jefferies, 1971). Parent b i r d s c o n t a m i n a t e d w i t h D D T s a n d other c o m p o u n d s m a y destroy their o w n e g g s b y e a t i n g , b r e a k i n g , o r e j e c t i n g t h e m ( O h l e n d o r f et a l . , 1 9 7 8 ; S t i c k e l ,  1973).  H y p e r e x c i t a b i l i t y a n d h y p e r s e n s i t i v i t y to s t i m u l i are c o m m o n l y l i s t e d a m o n g the s y m p t o m s o f DDT  p o i s o n i n g i n m a m m a l s , a n d s i m i l a r results  H y p e r a c t i v i t y has been  suggested  are l i k e l y i n b i r d s (Jefferies,  as a p o s s i b l e r e a s o n  o r g a n o c h l o r i n e f e d r i n g d o v e s ( M c A r t h u r et a l . , 1983).  1975).  f o r the nest inattentiveness o f  76 A m e r i c a n robins  (Turdus migratorius)  f r o m the O k a n a g a n V a l l e y o f B r i t i s h C o l u m b i a  are k n o w n to be h i g h l y c o n t a m i n a t e d w i t h D D T a n d D D E w h i c h they a c q u i r e f r o m e a t i n g e a r t h w o r m s that have c o n s u m e d c o n t a m i n a t e d s o i l ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s et al., 2000).  D e s p i t e the v e r y h i g h l e v e l s o f D D T f o u n d i n these b i r d s , r e p r o d u c t i o n does not  appear to be adversely affected. A study o f r o b i n s nesting i n o r g a n i c (non-pesticide sprayed) and c o n v e n t i o n a l l y (pesticide sprayed) m a n a g e d o r c h a r d s i n the O k a n a g a n r e v e a l e d n o s i g n i f i c a n t differences i n c l u t c h s i z e , nest success, h a t c h i n g success, or f l e d g i n g success b e t w e e n the t w o o r c h a r d types ( E l l i o t t et a l . , 1994).  G i l l et a l . ( 2 0 0 3 ) a l s o f o u n d n o s i g n i f i c a n t differences i n  h a t c h rate a n d fledge rate b e t w e e n r o b i n s f r o m O k a n a g a n orchards a n d c o n t r o l s from p a r k areas w i t h i n the B r i t i s h C o l u m b i a L o w e r M a i n l a n d . T h e s e authors, h o w e v e r , reported that c l u t c h size and b r o o d size were s i g n i f i c a n t l y h i g h e r i n o r c h a r d than n o n - o r c h a r d c o n t r o l sites. T h e purpose o f this study w a s to e x a m i n e latent effects o f in ovo a n d e a r l y p o s t - h a t c h DDT  exposure o n A m e r i c a n r o b i n reproduction.  T o do this, ten-day-old nestlings f r o m  Okanagan orchards, along w i t h L o w e r M a i n l a n d controls were raised i n captivity and then transferred as p a i r s to b r e e d i n g pens w h e r e they w e r e m o n i t o r e d o v e r t w o seasons.  Nesting  success w a s m o n i t o r e d , eggs a n d c h i c k s w e i g h e d a n d measured, a n d tissues c o l l e c t e d at the t i m e o f sacrifice. T h y r o i d h o r m o n e l e v e l s were also m e a s u r e d i n a subset o f b r e e d i n g pairs, every t w o w e e k s d u r i n g one b r e e d i n g season, as these h o r m o n e s c a n p l a y a s i g n i f i c a n t r o l e i n r e p r o d u c t i o n ( C h e e k et a l . , 1999; M c A r t h u r et a l . , 1983; N e l s o n , 2 0 0 0 ; W e n t w o r t h & R i n g e r , 1986; W i l s o n & D o n h a m , 1988).  A number o f behaviours were observed, i n c l u d i n g aggressive, v o c a l i z a t i o n ,  maintenance, and reproductive behaviours.  A s n o s i g n i f i c a n t d i f f e r e n c e s w e r e f o u n d i n the  r e p r o d u c t i o n o f B r i t i s h C o l u m b i a r o b i n s i n the w i l d , it w a s e x p e c t e d that c a p t i v e b i r d s f r o m the O k a n a g a n and L o w e r M a i n l a n d w o u l d demonstrate similar levels o f reproductive  success.  H o w e v e r , it i s p o s s i b l e that e a r l y D D T e x p o s u r e has m o r e subtle, l o n g - t e r m effects o n the r e p r o d u c t i o n o f these b i r d s that have heretofore gone u n n o t i c e d .  T h u s , the g o a l s o f this study  were to: 1) D e t e r m i n e i f e a r l y D D T e x p o s u r e h a d a d e t r i m e n t a l effect o n the r e p r o d u c t i v e success o f O k a n a g a n r o b i n s , as m e a s u r e d b y egg l a y i n g , e g g h a t c h i n g , a n d c h i c k fledging, as w e l l as egg a n d c h i c k measurements. 2 ) D e t e r m i n e i f e a r l y D D T e x p o s u r e affected r e p r o d u c t i v e  b e h a v i o u r s (e.g., nest b u i l d i n g ,  m a t i n g , c h i c k f e e d i n g , nest c l e a n i n g ) i n O k a n a g a n r o b i n s .  I n a d d i t i o n , a g g r e s s i v e (e.g.,  c h a r g i n g , c h a s i n g , a n d f i g h t i n g ) , v o c a l i z a t i o n (e.g., s i n g i n g , c h i r p i n g , l a u g h i n g ) , a n d m a i n t e n a n c e (e.g., eating, d r i n k i n g , p r e e n i n g ) b e h a v i o u r s w e r e also e x a m i n e d .  Aggressive  77 a n d v o c a l i z a t i o n b e h a v i o u r s p l a y a n i m p o r t a n t r o l e i n r e p r o d u c t i o n i n terms o f territory a n d mate a c q u i s i t i o n a n d defense ( S i l v e r et a l . , 1979).  4.2. Methods 4 . 2 . 1 . E g g s and C h i c k s T e n - d a y o l d A m e r i c a n r o b i n nestlings w e r e c o l l e c t e d f r o m nests i n the O k a n a g a n V a l l e y a n d the L o w e r M a i n l a n d o f B r i t i s h C o l u m b i a .  C h i c k s were marked w i t h numbered metal leg  b a n d s a n d c o l o u r p l a s t i c l e g b a n d s for i n d i v i d u a l i d e n t i f i c a t i o n . E g g s w e r e also c o l l e c t e d for c o n t a m i n a n t analyses. analyses protocols.  See C h a p t e r III for details o n eggs, n e s t l i n g s , h o u s i n g , a n d c o n t a m i n a n t  B i r d s w e r e s e x e d b y e x t e r n a l m o r p h o l o g y a n d later c o n f i r m e d b y D N A  s e x i n g b y B r e t t V a n d e r k i s t at S i m o n Fraser U n i v e r s i t y , B u r n a b y , B r i t i s h C o l u m b i a ( G r i f f i t h s et a l . , 1998). I n 1998, 4 0 b r e e d i n g pairs were established i n b o t h i n d o o r a n d o u t d o o r pens, one p a i r p e r p e n at the U n i v e r s i t y o f B r i t i s h C o l u m b i a S a n R a f a e l r e s e a r c h a v i a r y ( S u r r e y , B r i t i s h Columbia).  T h e b i r d s w e r e o v e r - w i n t e r e d at M o n i k a ' s W i l d l i f e S h e l t e r ( S u r r e y , B r i t i s h  C o l u m b i a ) , and i n 1999, 56 b i r d s w e r e brought b a c k to S a n R a f a e l a n d h o u s e d as b r e e d i n g pairs i n the outdoor pens o n l y . B o t h same type ( O k a n a g a n x O k a n a g a n a n d L o w e r M a i n l a n d x L o w e r M a i n l a n d ) a n d different type ( O k a n a g a n x L o w e r M a i n l a n d a n d L o w e r M a i n l a n d x O k a n a g a n ) p a i r s w e r e i n c l u d e d . A total o f 15 L o w e r M a i n l a n d x L o w e r M a i n l a n d ( m a l e x f e m a l e ) , 18 L o w e r M a i n l a n d x O k a n a g a n , 2 2 O k a n a g a n x L o w e r M a i n l a n d , a n d 23 O k a n a g a n x O k a n a g a n pairs w e r e studied. C a r e w a s taken to ensure that s i b l i n g s a n d c l o s e n e i g h b o r s w e r e not p a i r e d . I n d i v i d u a l s that d i e d or e s c a p e d w e r e r e p l a c e d w i t h another b i r d o f the s a m e type ( L o w e r M a i n l a n d or O k a n a g a n ) w h e n e v e r p o s s i b l e . T h u s , i n 1998, 4 6 females ( L o w e r M a i n l a n d = 2 2 , O k a n a g a n = 2 4 ) w e r e i n t r o d u c e d i n t o the b r e e d i n g pens, a n d i n 1 9 9 9 3 2 f e m a l e s ( L o w e r M a i n l a n d = 1 5 , O k a n a g a n = 1 7 ) w e r e studied.  4.2.2. R e p r o d u c t i o n  4.2.2.1. Nesting Success T h e pairs w e r e m o n i t o r e d d a i l y for e v i d e n c e o f nest b u i l d i n g , e g g l a y i n g , egg h a t c h i n g , a n d c h i c k f l e d g i n g f r o m the e n d o f F e b r u a r y u n t i l September.  T h e observer w a s b l i n d to the type  a n d l e v e l o f c o n t a m i n a t i o n o f a l l b i r d s . A s i n c u b a t i o n u s u a l l y b e g i n s after the last e g g is l a i d ( H o w e l l , 1 9 4 2 ; K e m p e r , 1 9 7 1 , but see S a l l a b a n k s & J a m e s , 1 9 9 9 ; W a u e r , 1 9 9 9 ) , i n c u b a t i o n p e r i o d w a s d e f i n e d as the t i m e b e t w e e n the l a y i n g o f the last e g g a n d the h a t c h i n g o f the last chick.  T h e n e s t l i n g p e r i o d e n c o m p a s s e d the t i m e b e t w e e n the first e g g h a t c h i n g a n d the last  78 chick  fledging.  H a t c h i n g success refers to the n u m b e r o f females w i t h eggs i n nests that h a t c h e d  at least one c h i c k . fledged  F l e d g i n g success refers to the n u m b e r o f f e m a l e s w i t h eggs i n nests that  at least one c h i c k . C l u t c h size a n d n u m b e r o f clutches l a i d w e r e also noted. O n l y eggs  that w e r e i n the nests w e r e i n c l u d e d i n c l u t c h s i z e a n d n u m b e r c a l c u l a t i o n s , as  females  s o m e t i m e s l a i d eggs o u t s i d e o f the nest a n d eggs c o u l d be r e m o v e d f r o m the nest b y the b i r d s themselves and/or predators.  4.2.2.2. Egg Measurements T h e first t w o eggs f r o m e a c h c l u t c h w e r e w e i g h e d a n d m e a s u r e d after the s e c o n d e g g w a s l a i d , u s i n g a n e l e c t r o n i c scale a n d c a l i p e r s . A n u m b e r o f a b a n d o n e d eggs w e r e also m e a s u r e d . E g g l e n g t h refers to the distance f r o m end-to-end, w h i l e e g g w i d t h refers to the m e a s u r e m e n t at the w i d e s t part o f the e g g .  4.2.2.3. Chick Measurements P r o g e n y w e r e w e i g h e d a n d b o d y m e a s u r e m e n t s t a k e n f i v e - a n d ten-days p o s t - h a t c h , u s i n g a n e l e c t r o n i c scale a n d c a l i p e r s . T a r s u s , w i n g , a n d m i d d l e toe lengths w e r e m e a s u r e d as d e s c r i b e d i n A l d r i c h a n d J a m e s ( 1 9 9 1 ) (see C h a p t e r III).  D N A s e x i n g o f the c h i c k s w a s  c o n d u c t e d at the U n i v e r s i t y o f B r i t i s h C o l u m b i a ( F r o n t e d d u , 2 0 0 1 ) u s i n g a d i f f e r e n t set o f p r i m e r s ( G r i f f i t h s et a l . 1998) than the s e x i n g o f the adults. T h e s e n e w p r i m e r s p r o d u c e d m o r e consistent a n d observable results.  4.2.2.4. Chick Mortality and Tissue Weights at Sacrifice A g e o f c h i c k death o r disappearance as w e l l as cause o f death, w h e n k n o w n , w e r e n o t e d . A t the e n d o f the study ( A u g u s t , 2 0 0 0 ) , the b i r d s w e r e s a c r i f i c e d b y d e c a p i t a t i o n a n d tissues i m m e d i a t e l y d i s s e c t e d out a n d w e i g h e d .  T a r s u s , toe, a n d w i n g m e a s u r e m e n t s  were also  r e c o r d e d . T i s s u e s c o l l e c t e d i n c l u d e d : heart, b r a i n , k i d n e y s , l i v e r , spleen, t h y r o i d g l a n d s , b u r s a , thymus, oviduct, and gonads.  T i s s u e w e i g h t s w e r e adjusted for b o d y w e i g h t b y d i v i d i n g the  tissue w e i g h t f r o m the total b o d y w e i g h t m i n u s the tissue w e i g h t . 4.2.3. T h y r o i d H o r m o n e s T e n p a i r s w e r e b l e d e v e r y t w o w e e k s d u r i n g the 1 9 9 9 b r e e d i n g s e a s o n ( 1 2 t e s t i n g p e r i o d s , F e b r u a r y to J u l y ) .  A l l b l o o d samples w e r e c o l l e c t e d f r o m the j u g u l a r v e i n u s i n g a 2 7  gauge h e p a r i n i z e d needle a n d 1 c c s y r i n g e . A p p r o x i m a t e l y one m i l l i l i t r e o f w h o l e b l o o d w a s c o l l e c t e d f r o m e a c h b i r d , c e n t r i f u g e d at 3 3 0 0 r p m f o r five m i n u t e s , a n d the p l a s m a separated f r o m the b l o o d c e l l s . T o t a l p l a s m a t r i i o d o t h y r o n i n e a n d t h y r o x i n e concentrations w e r e a n a l y z e d  79 b y T r a c y M a r c h a n t at the U n i v e r s i t y o f S a s k a t c h e w a n .  Analyses were conducted using  unextracted s e r u m a n d a r a d i o i m m u n o assay f o l l o w i n g p r o t o c o l s o u t l i n e d b y C h o p r a ( 1 9 7 2 ) . 4.2.4. B e h a v i o r a l O b s e r v a t i o n s A s u b - s a m p l e o f t w e l v e p a i r s , e n c o m p a s s i n g b o t h same a n d different type p a i r s w i t h v a r i o u s degrees o f c o n t a m i n a t i o n , were o b s e r v e d for thirty minutes a day (per p a i r ) , three t i m e s a w e e k , f r o m early M a r c h u n t i l early A u g u s t (1998 a n d 1999) to e x a m i n e aggressive, r e p r o d u c t i v e , v o c a l i z a t i o n , a n d m a i n t e n a n c e b e h a v i o r s , t h r o u g h o u t the b r e e d i n g season.  I n 1998 s i x o f the  o b s e r v e d pairs w e r e i n the i n d o o r pens a n d s i x i n the o u t d o o r p i e - s h a p e d pens. A l l 12 o b s e r v e d pairs w e r e i n the outdoor pie-shaped pens i n 1999.  4.2.4.1. Reproductive and Parental Care Behaviors R e p r o d u c t i v e b e h a v i o r s ( H o w e l l , 1942; S a l l a b a n k s & James, 1999; Y o u n g , 1955; W a u e r , 1999) i n c l u d e d : c o l l e c t i n g - b i r d p i c k s up a n d carries nesting m a t e r i a l ( m u d , h a y , string, etc.) b u i l d i n g - b i r d engages i n characteristic b o d y m o v e m e n t s i n the nest b o w l o r o n nest p l a t f o r m , b i r d u s u a l l y c r o u c h e s l o w , w i t h w i n g s spread, a n d stamps and b a c k - p e d a l s feet to m o v e nest m a t e r i a l a r o u n d , m o v e m e n t s m a y also be seen w h e n b i r d is o n p e r c h or b a t h i n g i n water d i s h , but these were not r e c o r d e d m o u n t i n g - m a l e m o u n t s o r attempts to m o u n t f e m a l e o r i n a n i m a t e object, n o t k n o w n i f i n t r o m i s s i o n and/or ejaculation a c h i e v e d sitting - f e m a l e sits o n nest i n c u b a t i n g eggs or b r o o d i n g c h i c k s , f e m a l e s l e e p i n g i n nest b o w l w h e n n o c h i c k s or eggs present w a s not r e c o r d e d f e e d i n g - b i r d c a r r i e s f o o d to nest a n d p l a c e s i n c h i c k ' s m o u t h , m a y r e m o v e f o o d f r o m one c h i c k ' s m o u t h a n d p l a c e i n another c l e a n i n g - b i r d r e m o v e s f e c a l sacs a n d other wastes f r o m nest, m a y be c a r r i e d a w a y f r o m nest and d i s c a r d e d or eaten 'other' parental care - b i r d is active at nest but observer unable to d i s c e r n b e h a v i o r , m a y i n c l u d e f e e d i n g o f c h i c k s , c l e a n i n g o f nest, and m o v i n g o f eggs or c h i c k s p a r e n t a l care - a l l b e h a v i o r s c o n c e r n i n g eggs a n d / o r c h i c k s i n the nest, i n c l u d e s f e e d i n g , c l e a n i n g , sitting, a n d other behaviors. M o u n t i n g w a s r e c o r d e d o n l y for m a l e s a n d s i t t i n g w a s r e c o r d e d o n l y for females.  As  m a l e s r a r e l y engaged i n nest m a t e r i a l c o l l e c t i n g a n d nest b u i l d i n g , o n l y the females' c o l l e c t i n g a n d b u i l d i n g w e r e i n c l u d e d here.  A l l the b e h a v i o r s e x c e p t m o u n t i n g w e r e r e c o r d e d  as  80 p r o p o r t i o n s (the n u m b e r o f m i n u t e s , out o f 3 0 , i n w h i c h the b e h a v i o u r w a s o b s e r v e d at least once). M o u n t i n g w a s r e c o r d e d as frequency per 3 0 - m i n u t e o b s e r v a t i o n p e r i o d .  4.2.4.2. Aggressive Behaviors Aggressive behaviors were performed by both males and females, and were directed t o w a r d s the mates, n e i g h b o r s , c h i c k s , or other b i r d s that m a y l a n d o n or near the pens. o f b i t i n g a n d f i g h t i n g w e r e rare a n d so w e r e not i n c l u d e d i n the a n a l y s e s . b e h a v i o r s were r e c o r d e d as frequencies.  Instances  A l l aggressive  T h e b e h a v i o r s are defined ( H o w e l l , 1942; S a l l a b a n k s &  James, 1999; Y o u n g , 1955; W a u e r , 1999) as: c h a r g i n g - b i r d rushes towards a c o n s p e c i f i c , m a y be flying or r u n n i n g c h a s i n g - one b i r d chases its mate, m a y be r u n n i n g or  flying  s n a p p i n g - s o u n d m a d e b y q u i c k l y b r i n g i n g m a n d i b l e s together, c o n s i d e r e d a threat d i s p l a y g a p i n g - b i r d h o l d s its m o u t h o p e n w h i l e f a c i n g other b i r d , m a y be c o n s i d e r e d a threat d i s p l a y , or signify intention to bite o v e r a l l aggression - frequency o f a l l aggressive b e h a v i o r s p e r f o r m e d b y a b i r d , s u m o f c h a r g i n g , chasing, snapping, and gaping  4.2.4.3. Vocalizations V o c a l i z a t i o n s were d e f i n e d ( H o w e l l , 1942; S a l l a b a n k s & James, 1999; W a u e r , 1999) as: c h i r p i n g - u s u a l l y o n l y one note, s i g n i f i c a n c e not k n o w n , used i n a variety o f contexts c h u k k i n g - series o f notes s i m i l a r to " c l u c k i n g " o f c h i c k e n s , a l s o d e s c r i b e d as " c u c k " , often s l o w e r and softer v e r s i o n o f l a u g h i n g , s i g n i f i c a n c e not k n o w n l a u g h i n g - series o f often l o u d a n d fast notes; d e s c r i b e d as " h a - h a - h i - h i - h i - h a - h a " , often u s e d f o l l o w i n g aggressive b e h a v i o r but m a y be u s e d i n other contexts, has b e e n d e s c r i b e d as b e i n g associated w i t h s o c i a b i l i t y a n d a sense o f w e l l b e i n g s i n g i n g - characteristic s o n g or parts thereof, often d e s c r i b e d as " c h e e r i l y - c h e e r u p " , m a y be u s e d for mate attraction and/or territorial d i s p l a y s i n g i n g p r o p o r t i o n - the p r o p o r t i o n o f minutes d u r i n g the o b s e r v a t i o n that the b i r d s e n g a g e d i n at least one s i n g i n g bout, out o f 30 minutes 'other' v o c a l i z a t i o n s - any other v o c a l i z a t i o n s that do not fit into the p r e v i o u s categories, m a y include song-like vocalizations performed by females  and c o m b i n a t i o n s o f other  categories o f v o c a l i z a t i o n s o v e r a l l v o c a l i z a t i o n s - s u m o f c h i r p i n g , c h u k k i n g , l a u g h i n g , and 'other' v o c a l i z a t i o n s  81 V o c a l i z a t i o n s , other than s i n g i n g p r o p o r t i o n , w e r e r e c o r d e d as frequencies.  B o t h males  and females e n g a g e d i n c h i r p i n g , c h u k k i n g , l a u g h i n g , a n d other v o c a l i z a t i o n s . A l t h o u g h s o m e females w e r e w i t n e s s e d s i n g i n g , it w a s r e l a t i v e l y rare, so s i n g i n g a n d s i n g i n g p r o p o r t i o n w e r e o n l y r e c o r d e d for males.  4.2.4.4. Maintenance  Behaviors  M a i n t e n a n c e b e h a v i o r s i n c l u d e d those a c t i v i t i e s related to d a i l y r o u t i n e s a n d w e r e not directly associated w i t h reproduction or aggression. B o t h males and females performed a l l o f these b e h a v i o r s .  W i t h the e x c e p t i o n o f e a t i n g , d r i n k i n g , a n d f l y i n g , w h i c h w e r e r e c o r d e d as  f r e q u e n c i e s , these b e h a v i o r s w e r e r e c o r d e d as p r o p o r t i o n s .  These behaviors were defined  ( S a l l a b a n k s & J a m e s , 1 9 9 9 ; W a u e r , 1999) as: b a t h i n g - b i r d sits o r stands i n water d i s h a n d proceeds to d u n k and s p l a s h to m o v e water o v e r its feathers p e c k i n g - b i r d p e c k s at v a r i o u s objects i n the p e n (weeds, cedar branches, insects, etc.), b i r d m a y be eating things it p e c k s at p r e e n i n g - b i r d uses b i l l to c l e a n a n d s m o o t h feathers eating - b i r d p i c k s u p a n d s w a l l o w s cat f o o d o r m e a l w o r m f r o m feeder o r other dishes d r i n k i n g - b i r d d r i n k s f r o m water or m u d d i s h f l y i n g - b i r d flies f r o m one p o s i t i o n (perch, feeder, g r o u n d , etc.) to another f l y i n g p r o p o r t i o n - the p r o p o r t i o n o f t i m e spent f l y i n g , out o f 3 0 m i n u t e s 4.2.5. Statistics A l l statistical a n a l y s e s w e r e c o n d u c t e d u s i n g J M P v e r s i o n 3.2.1 ( S A S Institute, C a r y , N o r t h C a r o l i n a ) software.  P v a l u e s less t h a n o r e q u a l to 0.05 w e r e c o n s i d e r e d s i g n i f i c a n t .  V a l u e s p r e s e n t e d represent m e a n s + s t a n d a r d errors, a n d ranges.  Data were c o m m o n log  transformed to increase n o r m a l i t y w h e r e necessary. F o u r females w e r e s t u d i e d i n b o t h b r e e d i n g seasons, but as they w e r e m a t e d w i t h different m a l e s a n d h o u s e d i n different p e n s i n the t w o y e a r s , t h e y w e r e treated as different s a m p l e s .  Outliers were determined using Mahalanobis  outlier tests. N o m i n a l l o g i s t i c ( / ) tests w e r e u s e d to determine differences i n w h e t h e r o r not a 2  female b u i l t a nest, l a i d eggs, h a t c h e d c h i c k s , f l e d g e d c h i c k s , i n c u b a t e d eggs, o r d u m p e d eggs. N e s t l i n g p e r i o d s , i n c u b a t i o n p e r i o d s , n u m b e r o f eggs per c l u t c h , a n d n u m b e r o f c l u t c h e s w e r e tested u s i n g standard least squares analyses o f v a r i a n c e ( A N O V A ) . u s i n g the m o d e l : Yijkim = u. + Mk + Fi + ( M F ) + U  Analyses were conducted  82 T h e dependent v a r i a b l e b e i n g m e a s u r e d is represented b y Y , M is m a l e type ( L o w e r M a i n l a n d vs. O k a n a g a n ) , F is female type ( L o w e r M a i n l a n d v s . O k a n a g a n ) , M F is the t w o - w a y i n t e r a c t i o n b e t w e e n m a l e type a n d f e m a l e t y p e , a n d E i s the error t e r m . r e m o v e d f r o m the m o d e l a n d the data w e r e r e - a n a l y z e d .  Non-significant variables were  T h e n u m b e r o f eggs l a i d per f e m a l e ,  e g g w e i g h t s a n d measurements, the n u m b e r s o f c h i c k s h a t c h e d a n d f l e d g e d , c h i c k w e i g h t s a n d measurements, a n d c h i c k sex ratios w e r e also a n a l y z e d u s i n g the same standard least squares ANOVA.  M e a n s per female per y e a r were used to a n a l y z e e g g a n d c h i c k measurements i n order  to a v o i d p s e u d o - r e p l i c a t i o n .  F e m a l e type and age at c h i c k death w e r e a n a l y z e d u s i n g /  2  tests.  P a i r - w i s e correlations w e r e c o n d u c t e d to determine the effects o f the O k a n a g a n mothers' in ovo p , p ' - D D T , o , p ' - D D T , p , p ' - D D D , o , p ' - D D D , p , p ' - D D E , a n d o , p ' - D D E (see C h a p t e r III a n d A p p e n d i x I for e g g c o n t a m i n a n t l e v e l s ) e x p o s u r e o n the m o r p h o l o g i c a l a n d p h y s i o l o g i c a l m e a s u r e m e n t s o f their eggs a n d c h i c k s . C o n t a m i n a n t non-detects a n d zeros w e r e r e p l a c e d b y 0 . 0 0 0 0 5 ( h a l f the d e t e c t i o n l i m i t ) for the c o r r e l a t i o n s .  A g e a n d cause o f death, as w e l l as the  presence o f bruises a n d w o u n d s w e r e a n a l y z e d u s i n g n o m i n a l l o g i s t i c s . B o d y measurements a n d tissue w e i g h t s at t i m e o f sacrifice were also a n a l y z e d u s i n g the same A N O V A m o d e l as a b o v e . H e r e data for i n d i v i d u a l b i r d s w e r e used, as at the t i m e o f sacrifice a l l o f the b i r d s hatched at S a n R a f a e l h a d b e e n f u l l - g r o w n a n d i n d e p e n d e n t for at least a year.  Differences between L o w e r  M a i n l a n d and Okanagan birds i n thyroid hormone levels were analyzed using one-way A N O V A s and log-transformed data, as w e r e sex differences. B e h a v i o r data w e r e c o m m o n l o g t r a n s f o r m e d to i n c r e a s e n o r m a l i t y a n d  differences  between L o w e r M a i n l a n d and Okanagan birds were analyzed using one-way A N O V A s .  Pair-  w i s e c o r r e l a t i o n s w e r e c o n d u c t e d to d e t e r m i n e the effects o f in ovo p , p ' - D D T , p , p ' - D D D , p,p'DDE,  o , p ' - D D T , o , p ' - D D D , a n d o , p ' - D D E ( C h a p t e r III a n d A p p e n d i x I) e x p o s u r e o n the  b e h a v i o r s o f O k a n a g a n b i r d s . T h e untransformed data w e r e u s e d for the correlations. A l t h o u g h not the focus o f this study, other factors w e r e also i n v e s t i g a t e d .  T h e effects o f y e a r (1998 v s .  1999), sex (male v s . female), p e n type ( i n d o o r v s . outdoor), a n d mate type (same v s . different) o n b e h a v i o u r were a n a l y z e d u s i n g o n e - w a y A N O V A s .  4.3. Results 4.3.1. E g g Contaminants See C h a p t e r III for details o n the differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n eggs i n terms o f contaminant l e v e l s .  83 4.3.2. R e p r o d u c t i o n  4.3.2.1. Nesting Success In 1998, the first e g g w a s l a i d o n A p r i l 7, a n d the last c h i c k w a s f l e d g e d o n J u l y 24, thus d e f i n i n g the nesting p e r i o d . I n 1999 the nesting p e r i o d w a s f r o m A p r i l 10 to A u g u s t 5. U s u a l l y one e g g w a s l a i d p e r d a y d u r i n g the l a y i n g p e r i o d , h o w e v e r s e v e r a l females s k i p p e d a d a y b e t w e e n eggs i n a c l u t c h , a n d o c c a s i o n a l l y m o r e than o n e d a y w o u l d be s k i p p e d ( Y o u n g , 1955). I n c u b a t i o n p e r i o d s ranged from 8 to 14 days ( m e d i a n = 1 3 days). C h i c k s w i t h i n a b r o o d t o o k 1 to 3 d a y s to hatch.  N e s t l i n g p e r i o d s r a n g e d f r o m 13 to 17 d a y s ( m e d i a n = 1 5 d a y s ) , w i t h a l l  c h i c k s l e a v i n g the nest w i t h i n a d a y o r t w o o f e a c h other a n d u s u a l l y o n the s a m e d a y . N e i t h e r i n c u b a t i o n p e r i o d n o r n e s t l i n g p e r i o d w a s s i g n i f i c a n t l y i n f l u e n c e d b y the m a l e t y p e o r f e m a l e type. O v e r a l l , 21 o f 67 (31.3%) pairs nested d u r i n g the t w o - y e a r study. T h i r t y - t h r e e (49.3%) pairs l a i d at least one e g g , 15 (22.4%) hatched at least one e g g , a n d 11 (16.4%) f l e d g e d one o r m o r e c h i c k s . T a b l e 4-1 illustrates the n u m b e r s o f L o w e r M a i n l a n d a n d O k a n a g a n females that s u c c e s s f u l l y nested, l a i d eggs, h a t c h e d eggs, a n d f l e d g e d y o u n g .  O k a n a g a n females w e r e n o  m o r e o r less l i k e l y to b u i l d a nest, l a y eggs, hatch c h i c k s , o r fledge c h i c k s t h a n L o w e r M a i n l a n d females. males ( /  L o w e r M a i n l a n d m a l e s , h o w e v e r , f l e d g e d p r o p o r t i o n a l l y m o r e c h i c k s than O k a n a g a n 2  = 4.9, p = 0.03). I n total, 19 L o w e r M a i n l a n d females l a i d 150 eggs a n d 14 O k a n a g a n  females l a i d 62 eggs. O f the females that l a i d eggs, L o w e r M a i n l a n d females l a i d s i g n i f i c a n t l y (Fi,3i = 5.1, p = 0.03) m o r e eggs (7.9 ± 1.1) t h a n their O k a n a g a n (4.4 + 0.8) counterparts. H o w e v e r , one L o w e r M a i n l a n d female l a i d 16 eggs i n b o t h years a n d r e m o v a l o f this b i r d f r o m the analyses r e s u l t e d i n n o n - s i g n i f i c a n c e .  O f the eggs l a i d b y L o w e r M a i n l a n d f e m a l e s , 53  (35.3%) hatched, 53 (35.3%) b r o k e , 19 (12.7%) w e r e m i s s i n g o r their fate w a s u n k n o w n , a n d 25 (16.7%) w e r e u n h a t c h e d .  O k a n a g a n females h a d 28 (45.2%) eggs that h a t c h e d , 24 (38.7%)  b r o k e , 5 (8.1%) h a d u n k n o w n fates, a n d 5 (8.1%) w e r e u n h a t c h e d .  S e v e n o f the 11 (63.6%)  L o w e r M a i n l a n d females w i t h nests p r o d u c e d at least one f l e d g l i n g , whereas 4/10 (40.0%) o f the O k a n a g a n females w i t h nests w e r e successful.  O f the 20 females that l a i d eggs i n a nest, n i n e  (45.0%o) l a i d one c l u t c h , f i v e (25.0%) l a i d t w o c l u t c h e s , three (15.0%) l a i d three c l u t c h e s , a n d three (15.0%) l a i d four clutches. T h e r e w e r e n o s i g n i f i c a n t m a l e type effects o n the n u m b e r o f c l u t c h e s l a i d b y a female, n o r o n the n u m b e r o f eggs l a i d p e r c l u t c h . L o w e r M a i n l a n d females l a i d s i g n i f i c a n t l y ( F j ^ s = 4.3, p = 0.05) m o r e clutches (2.3 ± 0.3) than O k a n a g a n females (1.6 + 0.2). H o w e v e r , the r e m o v a l o f one h i g h l y p r o d u c t i v e female from the analysis rendered the  84  Table 4-1: Numbers of Lower Mainland and Okanagan American robin females nesting, laying eggs, hatching eggs, and fledging young in 1998 and 1999.  Nested  Laid  Lower Mainland  5/19 26.4%  Okanagan  6/19 31.4%  Lower Mainland  Okanagan  Hatched  Fledged  11/19  3/5  57.9%  60.0%  2/5 40.0%  9/19  4/6  1/6  47.4%  66.7%  16.7%  6/15 40.0%  8/15 53.3%  5/5 100%  5/5 100%  4/14  3/4  3/4  28.6%  5/14 35.7%  75.0%  75.0%  11/34 32.4%  19/34  8/11  7/11  55.9%  72.7%  63.6%  14/33 42.4%  7/10  4/10  70.0%  40.0%  1998:  1999:  Total: Lower Mainland  Okanagan  10/33 30.3%  85 result n o n - s i g n i f i c a n t . F o r t y clutches w e r e l a i d i n total b y these b i r d s : 12 ( 3 0 . 0 % ) w e r e o f three eggs, 2 7 (67.5%>) were o f four eggs, a n d one (2.5%) w a s o f f i v e eggs. T h e r e w e r e no differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n females i n the n u m b e r o f eggs l a i d per c l u t c h . C l u t c h e s w e r e not d e t e r m i n e d w h e n eggs w e r e f o u n d outside o f a nest. T h e s e eggs m a y h a v e b e e n l a i d outside o f the nest or r e m o v e d f r o m the nest b y the b i r d s or predators. O v e r the t w o seasons, 81 c h i c k s w e r e h a t c h e d b y 15 f e m a l e s ( O k a n a g a n = 7, L o w e r M a i n l a n d = 8). L o w e r M a i n l a n d females that l a i d eggs hatched a m e a n o f 2.7 + 0.9 c h i c k s , a n d f l e d g e d 0.9 + 0.3 o f t h e m . O k a n a g a n females hatched a m e a n o f 2.1 + 0 . 7 c h i c k s a n d f l e d g e d a m e a n o f 0.9 + 0.4 c h i c k s . These differences w e r e not statistically significant.  4.3.2.2. Egg  Measurements  W e i g h t s a n d m e a s u r e m e n t s w e r e o b t a i n e d for 102 eggs, l a i d b y 14 L o w e r M a i n l a n d females a n d 11 O k a n a g a n females.  E g g s l a i d b y L o w e r M a i n l a n d females a v e r a g e d 7.0 g (+  0.07, range = 5.5 - 8.5 g) i n w e i g h t , 28.8 m m (+ 0.2 m m , range = 25.1 - 33.5 m m ) i n length, a n d 21.2 m m ( + 0.2 m m , range = 19.1 - 30.2 m m ) i n w i d t h . E g g s from O k a n a g a n females averaged 6.5 g (± 0.08, range = 5.8 - 7.3 g) i n w e i g h t , 2 8 . 9 6 ( ± 0.2, range = 27.1 - 30.9 m m ) i n l e n g t h , a n d 2 0 . 3 m m (± 0 . 1 , range = 19.1 - 21.5 m m ) i n w i d t h .  L o w e r M a i n l a n d females laid  s i g n i f i c a n t l y h e a v i e r ( F 1 2 3 = 7.6, p = 0.01) a n d w i d e r (Fi,23 = 1 2 . 1 , p = 0 . 0 0 2 ) eggs t h a n O k a n a g a n females.  E g g w e i g h t , l e n g t h , a n d w i d t h w e r e not s i g n i f i c a n t l y i n f l u e n c e d b y m a l e  t y p e nor w e r e there s i g n i f i c a n t m a l e b y f e m a l e i n t e r a c t i o n s . M e a n e g g w e i g h t w a s p o s i t i v e l y c o r r e l a t e d w i t h the O k a n a g a n females' in ovo p , p ' - D D T (r = 0.7, n = 9, p = 0 . 0 3 ; F i g u r e 4-1) exposure, e g g l e n g t h w a s p o s i t i v e l y correlated w i t h p , p ' - D D D (r = 0.7, n = 9, p = 0.03), a n d p,p'D D E (r = 0.9, n = 9, p = 0.0009) l e v e l s . , a n d egg w i d t h w a s p o s i t i v e l y correlated w i t h p , p ' - D D T (r = 0.7, n = 9, p = 0.04) a n d o , p ' - D D T (r = 0.8, n = 9, p = 0.02) burdens.  4.3.2.3. Chick  Measurements  W e i g h t s and m o r p h o m e t r i c measurements were obtained for the o f f s p r i n g o f eight L o w e r M a i n l a n d a n d s i x O k a n a g a n females. F i f t y - e i g h t c h i c k s w e r e m e a s u r e d at f i v e d a y s o f age a n d 4 0 c h i c k s at ten days o f age. T h e means (+ se) a n d ranges are s u m m a r i z e d i n T a b l e 4-2.  00 7.2  y = 0.0351 x + 5.9508 R = 0.4983  6.8  2  6.6  M  6A  6.2  5.8 10  15  20  25  p^-DDTCppm)  Figure 4-1: Relationship between female Okanagan American robin in ovo p,p'-DDT (p-g/g) exposure and the mean weights of their eggs.  87 Table 4-2: Means (+ se) and ranges of body weights and tarsus, wing, and toe lengths of the offspring of Lower Mainland and Okanagan American robin females when five and ten days old.  5 day weight (g)  Lower Mainland  Okanagan  n = 37  n = 19  23.5 ( ± 1 . 3 )  28.3 ( ± 1 . 9 ) 14.2-39.1  10.0-37.3  n = 39  n= 19  5 day tarsus (mm) *  20.7 ( ± 0.7) 12.0-35.0  23.3 ( ± 0 . 9 ) 16.0-30.0  n = 39  n = 19  5 day wing (mm) *  21.3 ( ± 0 . 1 )  25.2 ( ± 0 . 2 )  11.0-32.0  12.0-38.0  n = 39  n=19  15.3 ( ± 0 . 0 5 )  15.5 ( ± 0 . 0 6 )  9.0 - 22.0  10.0-19.0  5 day toe (mm)  10 day weight (g)  10 day tarsus (mm)  10 day wing (mm)  10 day toe (mm)  *p < 0.05  n = 23  n = 16  48.2 ( + 2 . 4 ) 25.6 - 69.0  46.8 ( ± 3 . 4 ) 14.9-61.7  n = 24  n = 16  33.9 ( ± 0 . 9 )  33.9 ( ± 0 . 9 )  20.0 - 39.0  25.0-38.0  n = 24  n = 16  54.2 ( ± 0 . 2 )  55.3 ( ± 0 . 3 )  27.0 - 68.0  30.0 - 73.0  n = 24 19.8 ( ± 0 . 0 4 )  n = 16 19.2 ( ± 0 . 0 4 )  15.0-23.0  16.0-21.0  88 O k a n a g a n females h a d c h i c k s w i t h l o n g e r tarsus (Fi i2 = 5.9, p = 0.03) ;  0.04)  a n d w i n g (Fi,i2 = 5.6, p =  measurements at five days o f age t h a n those o f L o w e r M a i n l a n d females. Parent type d i d  not s i g n i f i c a n t l y influence the c h i c k s ' ten-day measurements. S e x w a s d e t e r m i n e d for 69 o f the 81 c h i c k s hatched. I n total, L o w e r M a i n l a n d females hatched  21 (44.7%)  (63.6%) m a l e  male chicks and  chicks and  26 (55.3%)  8 (36.4%) female  female chicks.  O k a n a g a n females had  14  c h i c k s . T h e ratio o f m a l e to female c h i c k s w a s not  s i g n i f i c a n t l y i n f l u e n c e d b y parent type.  4.3.2.4. Chick Mortality and Tissue Weights at Sacrifice A l l eight o f the L o w e r M a i n l a n d females that h a t c h e d c h i c k s h a d at least one c h i c k die o r d i s a p p e a r w i t h i n the first t w o w e e k s p o s t - h a t c h , w h e r e a s o n l y f o u r o f the s e v e n O k a n a g a n females  (57.1%)  h a d c h i c k s die o r disappear d u r i n g this n e s t l i n g stage.  c h i c k s h a t c h e d to L o w e r M a i n l a n d females d i e d d u r i n g the n e s t l i n g stage,  Thirty-seven  2 (3.9%)  (71.2%)  d u r i n g the  1 (1.9%) d u r i n g the j u v e n i l e stage, a n d 12 (23.1%) as adults ( i n c l u d i n g the 11 that w e r e s a c r i f i c e d at the e n d o f the s t u d y ) . O k a n a g a n f e m a l e s h a d 15 (51.7%) c h i c k s d i e as nestlings, 4 (13.8%) as fledglings, 1 (3.5%) as a j u v e n i l e , a n d 9 (31.0%) as adults ( s a c r i f i c e d at f l e d g i n g stage,  the e n d o f the study). C h i - s q u a r e tests r e v e a l e d that female t y p e i n f l u e n c e d the n u m b e r o f c h i c k s d y i n g d u r i n g the n e s t l i n g stage, w i t h m o r e L o w e r M a i n l a n d females h a v i n g c h i c k s d i e at this early age t h a n O k a n a g a n females  (x = 5.5, p 2  =  0.02).  C a u s e o f death w a s u n k n o w n for m o s t o f  the c h i c k s , a n d therefore, w a s not statistically a n a l y z e d . T h e c h i c k s that s u r v i v e d to the e n d o f the study i n A u g u s t , m o s t l i k e l y b y rats. offspring.  2000 w e r e  sacrificed.  C h i c k s that d i s a p p e a r e d w e r e p r e s u m e d depredated,  A L o w e r M a i n l a n d male was witnessed shaking and biting two o f his  S i x c h i c k s w e r e d i s c o v e r e d c o l d i n their nests, suggesting abandonment, b u t it is not  k n o w n i f they w e r e a b a n d o n e d p r i o r to or after death. O f those c h i c k s for w h i c h cause o f death w a s not d e t e r m i n e d , 12 h a d w o u n d s and/or b r u i s i n g , but i t i s n o t k n o w n i f these w e r e i n c u r r e d p r i o r to o r after death o r h o w they w e r e i n f l i c t e d .  F e m a l e t y p e d i d not s i g n i f i c a n t l y i n f l u e n c e  whether o r not a c h i c k demonstrated bruises or w o u n d s . O f the 20 c h i c k s that w e r e s a c r i f i c e d at the end o f the study, tissue w e i g h t s w e r e a v a i l a b l e for 18 o f t h e m , s i x f r o m L o w e r M a i n l a n d x L o w e r M a i n l a n d pairs, s e v e n f r o m L o w e r M a i n l a n d x O k a n a g a n p a i r s , three f r o m O k a n a g a n x L o w e r M a i n l a n d p a i r s , a n d t w o f r o m O k a n a g a n x O k a n a g a n pairs (male x female). T h e o f f s p r i n g o f O k a n a g a n m a l e s h a d h e a v i e r hearts  0.0006 g) t h a n those fathered b y L o w e r M a i n l a n d (0.012 ± 0.0003 g) m a l e s ( F 0.05), a l t h o u g h the r e m o v a l o f a n o u t l i e r c a n c e l s t h i s effect. S i g n i f i c a n t m a l e  U  6  (0.013 + = 4.6, p =  b y female  89 interactions were f o u n d for b o d y w e i g h t (F] i6  =  ;  6.3, p = 0.02) a n d b r a i n w e i g h t (Fjj6 ~ 10.3, p =  0.006). Parents o f the same type ( L o w e r M a i n l a n d x L o w e r M a i n l a n d , 8 2 . 9 + 2.8 g ; O k a n a g a n x O k a n a g a n , 8 5 . 7 + 4 . 2 g) h a d y o u n g w i t h h i g h e r b o d y w e i g h t s at s a c r i f i c e t h a n parents o f different types ( O k a n a g a n x L o w e r M a i n l a n d , 75.5 + 0.8 g ; L o w e r M a i n l a n d x O k a n a g a n , 7 9 . 0 ± 1.1 g ) . Parents o f different types, h o w e v e r , h a d y o u n g w i t h h e a v i e r b r a i n s t h a n parents o f the same type ( F i g u r e 4-2). A significant m a l e b y female interaction affected k i d n e y (Fij6= 6.1, p = 0.03) a n d l i v e r (Fi i6 (  =  9 . 1 , p = 0.009) w e i g h t s ( F i g u r e 4-2). T h e o f f s p r i n g o f O k a n a g a n parents  h a d h e a v i e r k i d n e y s and l i v e r s . T h e r e m o v a l o f a n outlier l e d to n o n - s i g n i f i c a n t effects o n l i v e r w e i g h t . C a r e must be t a k e n w h e n interpreting these results, as the sample size is quite s m a l l a n d i n c l u d e s s i b l i n g s . T h e t w o O k a n a g a n x O k a n a g a n b i r d s , f o r e x a m p l e , c o m e f r o m the s a m e b r o o d . S e x w a s also c o n f i r m e d at this t i m e .  4.3.3. T h y r o i d H o r m o n e L e v e l s i n B r e e d i n g B i r d s There were n o s i g n i f i c a n t differences b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n p l a s m a t r i i o d o t h y r o n i n e o r t h y r o x i n e l e v e l s at a n y o f the ages tested. T r i i o d o t h y r o n i n e l e v e l s i n the O k a n a g a n b i r d s , h o w e v e r , w e r e n e g a t i v e l y c o r r e l a t e d w i t h p , p ' - D D T o n J u l y 2 0 , 1 9 9 9 ( p e r i o d 12) (r = - 0.8, n = 8, p = 0.01). T h y r o x i n e l e v e l s w e r e p o s i t i v e l y c o r r e l a t e d w i t h b o t h o , p ' - D D T (r = 0.7, n = 8, p = 0.05) a n d o , p ' - D D D (r - 0.7, n = 8, p = 0.05) o n M a r c h 3 0 , 1 9 9 9 ( p e r i o d 4 ) , but n e g a t i v e l y correlated w i t h p , p ' - D D T (r = - 0.9, n = 8, p = 0.003) o n A p r i l 2 7 , 1999 ( p e r i o d 6 ) . There w e r e s i g n i f i c a n t t i m e effects for t r i i o d o t h y r o n i n e ( F i 1,208 = 3 3 . 0 , p < 0 . 0 0 0 1 ; F i g u r e 4 - 3 ) a n d t h y r o x i n e (Fu,2i9 = 8.1, p < 0 . 0 0 0 1 ; F i g u r e 4 - 3 ) . M a l e s h a d h i g h e r t h y r o x i n e levels than females d u r i n g testing periods 1 ( F 1 J 7 = 52.2, p < 0.0001), 7 ( F i j 8 8 (F1.17 = 9.6, p - 0.007), 9 ( F  U  7  = 8.1, p = 0.01), 10 ( F  U  7  =  10.5, p = 0.005),  = 17.8, p = 0.0006), a n d 11 ( F  u  8  =  17.3, p = 0.0006). F e m a l e s h a d h i g h e r t r i i o d o t h y r o n i n e l e v e l s than m a l e s d u r i n g p e r i o d s 1 ( F i n ;  = 6 0 . 5 , p < 0.0001) a n d 9 ( F  U  7  = 8.8, p = 0.009).  4.3.4. B e h a v i o r There were f e w behavioral differences between L o w e r M a i n l a n d a n d O k a n a g a n birds ( T a b l e 4-3). O k a n a g a n b i r d s drank ( F i ^ = 6.9, p = 0.01) m o r e frequently than L o w e r M a i n l a n d birds, a n d L o w e r M a i n l a n d b i r d s engaged i n m o r e 'other' parental care b e h a v i o r s than O k a n a g a n b i r d s (F1.10 = 5.3, p = 0.04). T h i s latter difference, h o w e v e r , c a n be attributed p r i m a r i l y to one bird.  Same type pairs (Okanagan x O k a n a g a n a n d L o w e r M a i n l a n d x L o w e r M a i n l a n d )  e x h i b i t e d preening b e h a v i o u r s s i g n i f i c a n t l y (Fi 48 = 4 . 1 , p = 0.05) m o r e frequently (0.2 + 0.01) ;  0.035 b  0.03  X  i  0 LM x LM Q LM x OK • OK x LM • OK x OK  0.025  0.02  s  0.015  H  0.01  0.005  Brain  Kidneys  Liver-  Tissue  Figure 4-2: Mean (+ se) brain, kidney, and liver weights (grams) for birds hatched at San Rafael to same type and different type parents. L M = Lower Mainland, OK = Okanagan, male x female parents. For each tissue, columns with different letters are significantly different.  91 14  n  A) Thyroxine  1400 -|  B) Triiodothyronine  1200  ~Cu, 5b  1000 800  8 •B o -u _o  600  £  400  t  200 0 A  , 1  , 2  , 3  , 4  , 5  , 6 7 Test Period  ,  , 8  , 9  , 10  , 11  12  Figure 4-3: Mean (+ se) differences in a) thyroxine and b) triiodothyronine levels in Lower Mainland and Okanagan robins at the various time periods tested.  92  t h a n different t y p e ( O k a n a g a n x L o w e r M a i n l a n d , L o w e r M a i n l a n d x O k a n a g a n ) p a i r s (0.2 + 0.02).  D i f f e r e n t t y p e p a i r s , o n the other h a n d , e x h i b i t e d c h u k k i n g v o c a l i z a t i o n s m o r e ( F i ^ s =  3.9, p = 0.05) frequently (3.0 + 0.7) than same t y p e pairs (1.8 + 0.3). A n u m b e r o f b e h a v i o r s w e r e s i g n i f i c a n t l y correlated w i t h the b i r d s ' in ovo D D T exposures; these are listed i n T a b l e 4-4. C o r r e l a t i o n s that b e c a m e n o n - s i g n i f i c a n t w h e n outliers w e r e r e m o v e d are i n d i c a t e d . S i g n i f i c a n t differences b e t w e e n years, p e n types, a n d the sexes are o u t l i n e d i n T a b l e s 4 - 5 , 4 - 6 , a n d 4 - 7 .  4.4. Discussion  A l t h o u g h A m e r i c a n r o b i n s w e r e once r a i s e d as pets ( H o w e l l , 1942), this study is l i k e l y the first report o f large-scale successful b r e e d i n g o f these b i r d s i n c a p t i v i t y , d e m o n s t r a t i n g that the A m e r i c a n r o b i n c a n be u t i l i z e d as a m o d e l for t o x i c o l o g y studies. O r t e g a et a l . , ( 1 9 9 7 ) reported that e v e n i n the w i l d , A m e r i c a n r o b i n s are v e r y t o l e r a n t o f r e s e a r c h a c t i v i t i e s a n d r a r e l y a b a n d o n e d their nests after a h u m a n d i s t u r b a n c e .  T h e onset o f b r e e d i n g , a n d the t i m i n g f o r  i n c u b a t i o n a n d n e s t l i n g p e r i o d s for the r o b i n s i n this study w e r e s i m i l a r to those r e p o r t e d b y other authors ( C a m p b e l l et a l . , 1 9 9 7 ; C a n n i n g s et a l . , 1 9 8 7 ; H o w e l l , 1 9 4 2 ; K e m p e r , 1 9 7 1 ; W a u e r , 1999; Y o u n g , 1955) for w i l d r o b i n s , a n d there w e r e n o major differences b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s . T h e o v e r a l l success rates o f the c a p t i v e r o b i n s i n this study ( L o w e r M a i n l a n d 6 3 . 6 % , O k a n a g a n 4 0 . 0 % ) w e r e l o w e r t h a n those reported for f r e e - l i v i n g w i l d b i r d s ( C a n n i n g s et a l . , 1987; E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ) . H o w e v e r , the b i r d s i n this study w e r e first t i m e breeders i n 1998 a n d their p e r f o r m a n c e w a s e x p e c t e d to be substandard ( E m l e n , 1984; L a w t o n & G u i d o n , 1981). I n 1 9 9 9 , w h e n the b i r d s w e n t t h r o u g h t h e i r s e c o n d b r e e d i n g season, h a t c h i n g a n d  fledging  rates ( L o w e r M a i n l a n d 1 0 0 % , O k a n a g a n 7 5 . 0 % ) w e r e  greatly i m p r o v e d a n d w e r e c o m p a r a b l e to the p e r f o r m a n c e o f w i l d b i r d s . C h i c k f e e d i n g , nest c l e a n i n g , a n d o v e r a l l parental care w e r e p e r f o r m e d s i g n i f i c a n t l y m o r e i n 1999 than i n 1998.  93  Table 4-3: Means (+ se) and ranges of behaviors performed by Lower Mainland and Okanagan robins.  Total parental care Collect (nest material) (proportion) Building (nest) (proportion) Mounting (frequency) Sitting (on nest) (proportion) Feeding (chicks) (proportion) Cleaning (nest) (proportion) Other parental care * (proportion) Aggression (frequency) Charging (frequency) Chasing (frequency) Snapping (frequency) Gaping (frequency) Overall vocalizations (frequency) Chirping (frequency) Chukking (frequency) Laughing (frequency)  Lower Mainland  Okanagan  n = 26  n = 24  0.07 ( ± 0.04)  0.01 ( ± 0.02)  0-0.8 0.03 ( ± 0.006)  0-0.4 0.04 ( ± 0 . 0 1 )  0-0.1 0.03 ( ± 0.007)  0-0.3 0.04 ( ± 0.01)  0-0.1 0.03 ( ± 0 . 0 1 )  0-0.3 0.1 ( ± 0 . 0 6 )  0-0.3 0.08 ( ± 0.03)  0-1.4 0.02 ( ± 0 . 0 1 )  0-0.5 0.06 ( ± 0.04)  0-0.3 0.02 ( ± 0.02)  0-0.8 0.002 ( ± 0.0008) 0 - 0.02 0.02 ( ± 0.004)  0-0.4 0.001  (±0.0007)  0 - 0.02 0.001  (±0.001)  0 - 0.07  0 - 0.03  3.0 ( ± 0 . 5 )  2.9 ( ± 0.5)  0.6-10.5  0.3-7.1  1.9 ( ± 0 . 3 6 )  2.2 ( ± 0.4)  0.2-9.0  0.06-6.3  0.3 ( ± 0 . 1 )  0.3 ( ± 0.08)  0-3.2 0.3 ( ± 0 . 1 ) 0-2.2 0.5 ( ± 0.1) 0-3.4  0-1.2 0.2 ( ± 0.04) 0-0.8 0.3 ( ± 0.04) 0-0.7  10.8 ( ± 1 . 5 )  8.9 ( ± 1 . 3 )  2.7-35.1  1.3-23.4  4.5 ( ± 0.8)  3.1 ( ± 0 . 5 )  0.6-17.2  0.4-9.9  2.7 ( ± 0.5)  2.0 ( ± 0.6)  0.2-10.6  0.3-13.5  2.4 ( ± 0 . 5 )  2.6 ( ± 0.5)  0.02-9.5  0.05-10.9  Singing (frequency) Singing (proportion) Other vocalizations Eating (frequency) Drinking (frequency) * Bathing (proportion) Preening (proportion) Pecking (proportion) Flying (frequency) Flying (proportion)  *p < 0.05  11.4 ( ± 3 . 2 )  20.7 ( ± 6 . 1 )  0-59.0  0-108.8  0.1 ( ± 0 . 0 2 )  0.1 ( ± 0 . 0 3 )  0-0.3  0-0.5  1.2 ( ± 0 . 2 )  1.3 ( ± 0 . 3 )  0.3-4.9  0.1-8.6  3.0 ( ± 0 . 2 )  3.6 ( ± 0 . 3 )  1.3-6.3  1.5-6.7  3.2 ( ± 0 . 2 )  4.0 ( ± 0.2)  2.1-5.8  2.5-6.1  0.004 ( ± 0.0007)  0.005 ( ± 0 . 0 0 1 )  0-0.01  0 - 0.02  0.2 ( ± 0 . 0 1 )  0.2 ( ± 0.02)  0.09-0.4  0.08-0.4  0.2 ( ± 0 . 0 1 )  0.2 ( ± 0 . 0 1 )  0.1-0.4  0.1-0.4  50.4 ( ± 5.9)  44.4 (+ 4.8)  17.5-166.1  2.4-85.6  0.5 ( ± 0.02)  0.4 ( ± 0.03)  0.3-0.7  0.2-0.7  95  Table 4-4: Significant correlations between behavior and egg DDT levels in Okanagan robins.  Behavior  Contaminant  r  P*P - D D E  0.25  0.03"  -DDT  0.25  0.04"  P»P- D D E  0.25  0.02  Snapping  P>P- D D E  0.49  0.002  Overall vocalizations  P»P'- D D D  0.25  0.03  Laughing  P»P'- D D T  -0.26  0.02  Other vocalizations  P»P'- D D E  0.64  0.0002  o,p' - D D T  0.25  0.04  o,p' - D D D  0.36  0.004  o,p' - D D E  0.49  0.002  Eating  P»P'- D D E  0.25  0.03  Flying (frequency)  P,P' - D D T  0.36  0.01  P»P'- D D E  0.25  0.05  P,P' - D D D  0.25  0.04  P>P'- D D E  0.49  0.002  P»P'- D D T  0.25  0.02  8  Building (nest) Collecting (nest material)  fly (proportion)  S a m p l e size = 18 b i r d s ;  2  P  b  b  b  r e m o v a l o f outliers renders c o r r e l a t i o n n o n - s i g n i f i c a n t  96  Table 4 - 5 : Significant effects of sex on behaviour in Lower Mainland and Okanagan robins.  Behavior  Female  Male  F  P  2.57 (0.35)  9.3  0.004  0.084 (0.022)  0.51 (0.13)  25.5  < 0.0001  aggression  2.08 (0.44)  3.85 (0.46)  13.5  0.0006  laugh  1.34 (0.29)  3.65 (0.55)  12.4  0.0009  overall vocalizations  7.97(1.18)  11.85 (1.53)  5.6  0.02  preen  0.18(0.013)  0.21 (0.016)  4.2  0.05  other parental care  0.011 (0.0038)  0.00039 (0.00022)  24.5  0.0006  o v e r a l l parental care  0.088 (0.044)  0.0045 (0.0020)  12.1  0.004  Mean + se  Mean + se  charging  1.51 (0.40)  chasing  97  Table 4-6: Significant effects of year on behaviour in Lower Mainland and Okanagan robins.  1998  1999  Mean + se  Mean ± se  snap  0.44 (0.11)  laugh  Behavior  F  P  0.055 (0.012)  22.9  < 0.0001  1.57 (0.11)  3.36 (0.45)  14.6  0.0004  sing  18.39(5.60)  13.55 (4.01)  5.6  0.02  s i n g (proportion)  0.13 (0.033)  0.10(0.024)  6.3  0.02  eat  3.99 (0.27)  2.63 (0.19)  16.7  0.0002  preen  0.23 (0.017)  0.16(0.0079)  17.4  0.0001  peck  0.29 (0.013)  0.20 (0.0084)  32.3  < 0.0001  fly (proportion)  0.50 (0.029)  0.41 (0.018)  4.6  0.04  feed ( c h i c k s )  0.0029 (0.0012)  0.072 (0.040)  15.2  0.003  c l e a n (nest)  0.00078 (0.00037)  0.0021 (0.00097)  7.5  0.03  0.0778 (0.043)  7.9  0.01  o v e r a l l parental care  0.012 (0.0046)  98 Table 4-7: Significant effects of pen-type on behaviour in Lower Mainland and Okanagan robins.  Indoor  Outdoor  Mean + se  Mean ± se  snap  0.63 (0.19)  gape  Behavior  F  P  0.11 (0.024)  16.6  0.0002  0.87 (0.28)  0.24 (0.031)  11.2  0.002  aggression  4.88 (0.88)  2.36 (0.29)  9.5  0.003  laugh  0.94 (0.25)  2.99 (0.42)  8.7  0.005  other v o c a l i z a t i o n s  2.04 (0.69)  0.99 (0.11)  5.4  0.02  eat  4.02 (0.36)  3.04 (0.21)  5.6  0.02  preen  0.28 (0.025)  0.17(0.0065)  30.3  < 0.0001  peck  0.32 (0.019)  0.22 (0.0083)  23.5  < 0.0001  f l y (proportion)  0.55 (0.043)  0.42 (0.017)  8.0  0.007  99  W h i l e there i s a b u n d a n t  l i t e r a t u r e o n the effects o f i m m e d i a t e D D T e x p o s u r e  on  r e p r o d u c t i o n i n b i r d s ( B l u s et a l . , 1997; B r y a n et a l . , 1989; C e c i l et a l . , 1 9 7 1 ; C h a n g & Stokstad, 1975; F o x & D o n a l d , 1980; F r y , 1995; G i s h & C h u r a , 1970; Jefferies, 1 9 7 1 , 1975; L i l l i e et a l . , 1972), there i s less k n o w n about the effects o f in ovo exposure o r the l o n g - t e r m effects c a r r i e d to the next generation. caused Japanese q u a i l  G i l d e r s l e e v e et a l . ( 1 9 8 5 ) f o u n d that in ovo exposure to d i e t h y l s t i l b e s t r o l  (Coturnix japonica)  females to have s i g n i f i c a n t l y r e d u c e d o v i d u c t w e i g h t s  a n d decreased egg p r o d u c t i o n . T h e r e p r o d u c t i v e b e h a v i o r s a n d s o c i a l - d o m i n a n c e b e h a v i o r s o f m a l e s were also m a r k e d l y attenuated. W i l l i a m s (1999) investigated parental and effects  first-generation  o f exogenous estrogens (17(j-estradiol) o n female reproduction i n zebra  (Taeniopygia guttata)  finches  a n d f o u n d that the m e a n e g g m a s s o f d a u g h t e r s o f e s t r a d i o l - t r e a t e d  females w a s larger than that o f c o n t r o l o f f s p r i n g . T h e r e w e r e n o treatment effects o n o f f s p r i n g c l u t c h size or l a y i n g i n t e r v a l . B e r g et a l . (2001) also f o u n d in ovo exposure o f Japanese q u a i l to e t h y n y l e s t r a d i o l (a s y n t h e t i c estrogen)  resulted i n various oviduct malformations.  They  c o n c l u d e d that these o v i d u c t a b n o r m a l i t i e s c o u l d b e u s e d as b i o m a r k e r s o f x e n o e s t r o g e n exposure i n w i l d bird populations.  T h e o,p' i s o m e r s o f D D T a n d its m e t a b o l i t e s are k n o w n to  b i n d d i r e c t l y to e s t r o g e n receptors ( R o b i n s o n et a l . , 1984) a n d d e m o n s t r a t e e s t r o g e n - l i k e activities ( K u p f e r & B u l g e r , 1980; G a i d o et a l . , 1997). M a l e C a l i f o r n i n i a g u l l s e x p o s e d in ovo to 2 m g / k g o , p ' - D D T h a d f e m i n i z e d g o n a d s , w h i l e 5 m g / k g o r h i g h e r d o s e s r e s u l t e d i n the d e v e l o p m e n t o f b o t h left a n d r i g h t o v i d u c t s i n female g u l l s ( F r y & T o o n e , 1981). T h i s i s s i m i l a r to B e r g et a l . ' s ( 2 0 0 1 ) f i n d i n g w i t h in ovo exposure o f q u a i l to e t h y n y l e s t r a d i o l . I n the r o b i n s , e g g w e i g h t a n d s i z e w e r e c o r r e l a t e d w i t h the in ovo D D T l e v e l s o f the mothers.  T h e s e results  support W i l l i a m s ' (1999) f i n d i n g s that egg m a s s for daughters o f estradiol-treated z e b r a finches w a s larger than c o n t r o l s . H o w e v e r , the O k a n a g a n r o b i n s here w e r e not o n l y e x p o s e d in ovo to D D T , but they w e r e l i k e l y the o f f s p r i n g o f b i r d s that w e r e also e x p o s e d to D D T in ovo.  Despite  the p o s i t i v e c o r r e l a t i o n s b e t w e e n egg s i z e a n d in ovo D D T l e v e l s , the u n c o n t a m i n a t e d L o w e r M a i n l a n d f e m a l e s l a i d s i g n i f i c a n t l y larger eggs a n d m o r e eggs t h a n the O k a n a g a n f e m a l e s . G e n e t i c differences b e t w e e n the t w o types o f females l i k e l y p l a y e d a s i g n i f i c a n t r o l e i n egg size ( S t y r s k y et a l . , 2 0 0 2 ) , and there c a n be a great deal o f i n d i v i d u a l v a r i a t i o n ( S t y r s k y et a l . , 2 0 0 2 ; W i l l i a m s , 1999). T h e s e factors m a y h a v e c o n f o u n d e d the effects o f D D T exposure. It w a s f o u n d that the p o s t - b r e e d i n g g o n a d w e i g h t s o f O k a n a g a n r o b i n s w e r e n e g a t i v e l y c o r r e l a t e d w i t h in ovo p , p ' - D D E l e v e l s w h i l e o v i d u c t w e i g h t w a s p o s i t i v e l y c o r r e l a t e d w i t h in ovo o , p ' - D D T l e v e l s (see C h a p t e r III). T h e r e w a s also m o r e v a r i a t i o n i n g o n a d w e i g h t s i n b i r d s  100 f r o m l o w l e v e l exposures c o m p a r e d to h i g h l e v e l exposures (see F i g u r e 3-6).  The majority o f  studies reported i n the literature m e a s u r e d g o n a d w e i g h t s w h e n the b i r d s w e r e i n f u l l b r e e d i n g c o n d i t i o n , h o w e v e r , K e m p e r ( 1 9 7 1 ) f o u n d that m a l e r o b i n testes w e i g h e d f r o m 8.6 to 84.5 m g i n A u g u s t a n d September a n d that female ovaries ranged f r o m 10.8 to 36.0 m g . M e a n testes w e i g h t i n this study w a s 24.7 m g (range 21.4 - 30.8 m g ) for L o w e r M a i n l a n d m a l e s and 31.1 m g (16.1 49.5 m g ) for O k a n a g a n m a l e s , so they are s i m i l a r to the b i r d s K e m p e r studied. O v a r y w e i g h t s i n the L o w e r M a i n l a n d females r a n g e d f r o m 9.0 to 20.3 m g ( m e a n 13.2 m g ) a n d 7.7 to 22.8 m g ( m e a n 12.2 m g ) i n the O k a n a g a n females.  A g a i n , these v a l u e s are w i t h i n the ranges f o u n d i n  K e m p e r ' s b i r d s d u r i n g the same t i m e p e r i o d . U n l i k e p r e v i o u s studies w i t h laboratory b i r d s (e.g., c h i c k e n s , q u a i l , z e b r a finches) ( F r y & T o o n e , 1 9 8 1 ; G i l d e r s l e e v e et a l . , 1985; B e r g et a l . , 2 0 0 1 ; W i l l i a m s , 1 9 9 9 ) , this study m a y be c o n f o u n d e d b y the effects o f the f e m a l e s ' o w n in exposure a n d the in ovo exposure o f the p r e v i o u s generation.  ovo  Further c o m p l i c a t i o n comes w i t h  the fact that i n m a n y laboratory studies, birds are e x p o s e d to a s i n g l e c h e m i c a l or i s o m e r w h i l e the r o b i n s w e r e e x p o s e d to a c o m b i n a t i o n o f i s o m e r s .  W h i l e this represents a m o r e r e a l i s t i c  situation, the i n t e r a c t i o n o f these i s o m e r s cannot be teased apart w i t h the g i v e n s a m p l e s i z e . E v e n t h o u g h the O k a n a g a n females l a i d s m a l l e r eggs than L o w e r M a i n l a n d females, their c h i c k s at 5 days o f age w e r e s i g n i f i c a n t l y larger (as i n d i c a t e d b y tarsus a n d w i n g length). T h i s is interesting as h a t c h w e i g h t a n d e g g w e i g h t s h o u l d be h i g h l y c o r r e l a t e d ( S t y r s k y et a l . , 2 0 0 2 ) . T h i s finding is consistent w i t h the f i n d i n g i n C h a p t e r III that O k a n a g a n c h i c k s at this early age w e r e h e a v i e r t h a n L o w e r M a i n l a n d c h i c k s o f the s a m e age.  H o w e v e r , s i n c e the O k a n a g a n  females i n this study w e r e p a i r e d w i t h b o t h types o f m a l e s , the difference i n c h i c k size or w e i g h t c a n n o t be attributed to genetics b u t to the p a r e n t i n g a b i l i t y o f the females.  T h i s h y p o t h e s i s is  further supported b y the f i n d i n g that c h i c k s hatched b y L o w e r M a i n l a n d females (regardless o f w h a t type o f m a l e s they w e r e p a i r e d w i t h ) suffered s i g n i f i c a n t l y h i g h e r m o r t a l i t y at this age than c h i c k s hatched b y O k a n a g a n females. A g g r e y & C h e n g (1993) f o u n d that b o d y w e i g h t i n p i g e o n (Columba livid) squabs w e r e m o s t l y affected b y the parents' p a r e n t i n g a b i l i t y d u r i n g the first t w o w e e k s after h a t c h i n g . T h e squabs' o w n genetic p o t e n t i a l to g r o w o n l y e x p r e s s e d i t s e l f after the first t w o w e e k s o f b r o o d i n g . I n A m e r i c a n r o b i n s , the i m p o r t a n c e o f the f e m a l e s ' a n d the m a l e s ' parenting roles seem to be at different stages o f the r e a r i n g p e r i o d . I n the early stage, m a l e s w e r e n e v e r o b s e r v e d b r o o d i n g the c h i c k s , a l t h o u g h they w o u l d o c c a s i o n a l l y p e r c h o n the edge o f the nest a n d stand g u a r d w h i l e the female w a s a w a y and b o t h parents fed the c h i c k s a n d r e m o v e d f e c a l sacs. A f t e r f l e d g i n g , the m a l e m a y take o v e r care o f the y o u n g w h i l e the f e m a l e b e g i n s to prepare for and l a y the next c l u t c h o f eggs ( H o w e l l , 1942, S a l l a b a n k s & James, 1 9 9 9 ; W a u e r ,  101 1999). L o w e r M a i n l a n d m a l e s , regardless o f the type o f female they w e r e p a i r e d w i t h , f l e d g e d s i g n i f i c a n t l y m o r e ( p r o p o r t i o n a l l y ) c h i c k s than O k a n a g a n m a l e s . It is interesting that the r o l e o f the m a l e is s t i l l i m p o r t a n t e v e n w h e n f o o d w a s p r o v i d e d they d i d not have to p h y s i c a l l y fight o f f intruders.  ad libitum  a n d territories w e r e set a n d  N e v e r t h e l e s s , the c o - o r d i n a t i o n o f the m a l e  a n d female parents w a s a n i m p o r t a n t factor i n affecting the c h i c k s ' g r o w t h . I n this study, same type parents h a d h e a v i e r f l e d g l i n g s t h a n different t y p e parents.  O n e w o u l d e x p e c t that c h i c k s  f r o m different t y p e parents s h o u l d h a v e a n advantage because o f h y b r i d v i g o u r .  Same type  parents preened m o r e than different type parents whereas different type parents v o c a l i z e d ( c h u k ) more.  A l t h o u g h the s i g n i f i c a n c e o f these t w o b e h a v i o r s is not clear, the differences b e t w e e n  parent types suggest that there m a y also be differences i n c o m p a t a b i l i t y . T h i s , i n turn, suggests that there are differences b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s a n d that the r o b i n s c a n differentiate their o w n type f r o m the other e v e n w h e n r a i s e d together f r o m an early age. L i k e t h e i r p a r e n t s (see C h a p t e r III), the f u l l - g r o w n r o b i n c h i c k s f r o m O k a n a g a n p a r e n t s h a d s i g n i f i c a n t l y h e a v i e r organs (standardized w i t h b o d y w e i g h t ) than those f r o m L o w e r M a i n l a n d parents. T h e O k a n a g a n b i r d s s e e m to be m o r e c o m p a c t c o m p a r e d to L o w e r M a i n l a n d b i r d s . O u r f i n d i n g s s u p p o r t the n o t i o n that the O k a n a g a n a n d L o w e r M a i n l a n d r o b i n s b e l o n g to t w o different sub-species ( A l d r i c h & James, 1 9 9 1 ; C a n n i n g s , 1998). I m m e d i a t e exposure to D D T has b e e n s h o w n to affect b e h a v i o u r s i n b i r d s . I n Japanese q u a i l , h e r r i n g g u l l s , r i n g d o v e s , a n d m e r l i n s , D D T e x p o s u r e has b e e n s h o w n to attenuate r e p r o d u c t i v e a n d aggressive b e h a v i o r s a n d l e a d to r e p r o d u c t i v e f a i l u r e ( F o x & D o n a l d , 1 9 8 0 ; M a A r t h u r et a l . , 1983; F r y & T o o n e , 1 9 8 1 ; F o x , 1997; B r y a n et a l . , 1989). I n B e n g a l e s e finches and gray herons  (Ardea cinerea),  o n the other h a n d , D D T e x p o s u r e l e d t o h e i g h t e n e d  m i s d i r e c t e d a g g r e s s i o n (Jefferies, 1 9 7 1 ; O h l e n d o r f et a l . , 1978) w h i c h a l s o l e d to r e p r o d u c t i v e failure.  I n the r o b i n s , in ovo D D T l e v e l s w e r e c o r r e l a t e d w i t h several r e p r o d u c t i v e a n d related  b e h a v i o u r s , but the results w e r e c o m p r o m i s e d b y the s m a l l s a m p l e s i z e . A s noted, m a n y o f the c o r r e l a t i o n s b e c a m e n o n - s i g n i f i c a n t w h e n o u t l i e r s w e r e r e m o v e d f r o m the a n a l y s e s .  These  " o u t l i e r s " , h o w e v e r , w e r e important observations. I n m o s t cases, they w e r e i n d i v i d u a l s e x p o s e d to the highest l e v e l s o f in ovo D D T c o n t a m i n a t i o n , and they expressed exaggerated b e h a v i o u r . F e m a l e O K 9 7 - 2 1 C 2 , for e x a m p l e , e n g a g e d i n a b n o r m a l l y h i g h l e v e l s o f nest m a t e r i a l c o l l e c t i n g a n d nest b u i l d i n g but f a i l e d to c o m p l e t e a nest or l a y any eggs.  H e r mate, m a l e O K 9 7 - 3 0 C 3 ,  another h i g h l y c o n t a m i n a t e d o u t l i e r , w a s o b s e r v e d m o u n t i n g h i s m a t e i n a b n o r m a l l y h i g h frequency.  W h i l e it w o u l d be d i f f i c u l t to d r a w c o n c l u s i o n s b a s e d o n one or t w o i n d i v i d u a l s , it  w o u l d be reasonable to p r o p o s e the hypothesis that in ovo D D T exposure a d v e r s e l y affects o n l y  and  102 h i g h l y c o n t a m i n a t e d b i r d s , a n d there m a y be a t h r e s h o l d l e v e l b e l o w w h i c h A m e r i c a n r o b i n b e h a v i o u r is not affected. P l a s m a t h y r o i d h o r m o n e l e v e l s i n the O k a n a g a n a n d L o w e r M a i n l a n d b r e e d i n g b i r d s w e r e not s i g n i f i c a n t l y different d u r i n g d e v e l o p m e n t , n o r w e r e there s i g n i f i c a n t c o r r e l a t i o n s w i t h in ovo D D T l e v e l s ( C h a p t e r III).  D u r i n g the b r e e d i n g season, there w e r e also n o s i g n i f i c a n t  differences except i n p e r i o d 7, w h e n L o w e r M a i n l a n d b i r d s h a d h i g h e r p l a s m a t r i i o d o t h y r o n i n e levels than Okanagan birds.  W h i l e plasma triiodothyronine levels were negatively correlated  w i t h in ovo p , p ' - D D T d u r i n g the p o s t - b r e e d i n g p e r i o d ( p e r i o d 12), a n d t h y r o x i n e l e v e l s w e r e p o s i t i v e l y c o r r e l a t e d w i t h p , p ' - D D T a n d o , p ' - D D D d u r i n g p e r i o d 4 , f e w c o n c l u s i o n s c a n be d r a w n f r o m these data.  I n this study, t h y r o x i n e l e v e l s p e a k e d i n late A p r i l ( p e r i o d 6 ) .  As a  n u m b e r o f pairs l a i d eggs i n early M a y , this peak i n t h y r o x i n e m a y be related to increases i n nest b u i l d i n g a n d m a t i n g a n d the increased energy d e m a n d s o f these b e h a v i o r s . It is important to note that r o b i n s o f t e n r a i s e m o r e t h a n o n e b r o o d i n a s e a s o n ( H o w e l l , 1 9 4 2 ; K e m p e r , 1 9 7 1 ; Sallabanks & James, 1999), and their gonadal hormones, and presumably t h y r o i d hormones, w o u l d be expected to rise a n d fall a c c o r d i n g l y w i t h each b r o o d . T h y r o i d h o r m o n e l e v e l s are also l i n k e d to m o l t i n g a c t i v i t y , w i t h m a x i m u m t h y r o i d a c t i v i t y p r e c e d i n g or c o i n c i d e n t w i t h the onset o f m o l t ( W e n t w o r t h & R i n g e r , 1986). B y early J u l y , m a n y b i r d s have f l e d g e d their last b r o o d o f c h i c k s a n d are p r e p a r i n g to m o l t a n d t h e n m i g r a t e ( C a m p b e l l et a l . , 1 9 9 7 ; K e m p e r , 1 9 7 1 ; S a l l a b a n k s & James, 1999; W a u e r , 1999). T h e b i r d s i n this study d i d i n d e e d e x h i b i t a p e a k i n t h y r o x i n e l e v e l s i n J u l y ( p e r i o d 11) w h i c h is w h e n they w o u l d b e g i n m o l t i n g ( C a m p b e l l et a l . , 1997; H o w e l l , 1942; K e m p e r , 1 9 7 1 ; S a l l a b a n k s & James, 1999).  4 . 5 . Conclusions E x c e p t for a c o u p l e o f h i g h l y c o n t a m i n a t e d i n d i v i d u a l s , b i r d s e x p o s e d in ovo a n d e a r l y p o s t - h a t c h to D D T e x p o s u r e d i d not e x h i b i t detriments i n t h e i r r e p r o d u c t i v e success.  They  demonstrated n o differences i n the t i m i n g o f their r e p r o d u c t i v e b e h a v i o r s n o r i n their h a t c h i n g a n d f l e d g i n g success, as c o m p a r e d to b i r d s f r o m r e l a t i v e l y u n c o n t a m i n a t e d areas. E g g w e i g h t , l e n g t h a n d w i d t h w e r e p o s i t i v e l y correlated w i t h the f e m a l e s ' in ovo D D T c o n t a m i n a t i o n , but it is not certain whether this is a n in ovo exposure effect or effects c a r r i e d o v e r f r o m the p r e v i o u s generation.  T h e results a l s o s u g g e s t that the O k a n a g a n a n d L o w e r M a i n l a n d b i r d s w e r e  g e n e t i c a l l y different a n d m a y b e l o n g to different sub-species.  Some h i g h l y contaminated birds  d e m o n s t r a t e d h y p e r - a c t i v i t y a n d l e d to the h y p o t h e s i s that in ovo e x p o s u r e to D D T a d v e r s e l y affects o n l y h i g h l y c o n t a m i n a t e d b i r d s , a n d there m a y be a t h r e s h o l d l e v e l b e l o w w h i c h  103 A m e r i c a n r o b i n b e h a v i o r is not affected.  It is also p o s s i b l e that after e x p o s u r e to D D T f o r  several decades, r o b i n s have d e v e l o p e d a tolerance to it ( G i l l et a l . , 2 0 0 3 ) or at least a n a b i l i t y to compensate for any detrimental effects.  104  4.6. References A g g r e y , S. E . , C h e n g , K . M . ( 1 9 9 3 ) . G e n e t i c a n d p o s t - h a t c h p a r e n t a l i n f l u e n c e s o n g r o w t h o f p i g e o n squabs. Journal of Heredity, 84, 184-187. A l d r i c h , J . W . , J a m e s , F . C . ( 1 9 9 1 ) . 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D a y l e n g t h a n d c o n t r o l o f seasonal r e p r o d u c t i o n i n m a l e b i r d s . I n M . H . Stetson ( E d . ) ,  Processing of Environmental Information in Vertebrate  101-119). N e w Y o r k : S p r i n g e r - V e r l a g . Y o u n g , H . ( 1 9 5 5 ) . B r e e d i n g b e h a v i o r a n d n e s t i n g o f the E a s t e r n R o b i n . T h e  Midland Naturalist, 53(2),  329-352.  American  109 Chapter V Infectious Disease and Immune Response in American Robins Exposed In Ovo and Early Post-Hatch to DDT  5.1 Introduction T h e O k a n a g a n V a l l e y o f B r i t i s h C o l u m b i a is an important f r u i t - g r o w i n g r e g i o n . O r c h a r d s i n this area w e r e h e a v i l y treated w i t h the i n s e c t i c i d e d i c h l o r o d i p h e n y l - t r i c h l o r o e t h a n e ( D D T ) p r i o r to its b a n n i n g i n the early 1 9 7 0 ' s ( E l l i o t t et a l , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s et a l , 2000).  T w o t o f o u r treatments o f 13.5 k g o f t e c h n i c a l grade D D T  a p p l i e d ( E l l i o t t et a l . , 1994).  2  p e r hectare p e r y e a r w e r e  T h i s o r g a n o c h l o r i n e has b e e n l i n k e d to r e d u c t i o n s i n s u r v i v a l ,  r e p r o d u c t i o n , a n d i m m u n e response, as w e l l as d i s r u p t i o n s i n n o r m a l e n d o c r i n e f u n c t i o n i n g , i n a v a r i e t y o f a v i a n species (e.g., Banerjee, 1999; B l u s , 1996; C a r s o n , 1962; C o l b o r n et a l . , 1996; F r y & T o o n e , 1 9 8 1 ; Jefferies, 1 9 7 1 ; K e l c e et a l . , 1995; M u r p h y , 1980; S o n n e n s c h e i n & S o t o , 1998; S t i c k e l et a l . , 1984; W H O , 1989). A l t h o u g h not u s e d i n the O k a n a g a n V a l l e y for a p p r o x i m a t e l y 30 years, D D T s t i l l persists i n the s o i l w h e r e it i s c o n s u m e d a n d a c c u m u l a t e d b y e a r t h w o r m s ( H a r r i s et a l . , 2 0 0 0 ) . earthworms are a f a v o r e d p r e y o f the A m e r i c a n r o b i n  These  (Turdus migratorius), e s p e c i a l l y d u r i n g the  b r e e d i n g season ( E h r l i c h et a l . , 1 9 8 8 ; S a l l a b a n k s & James, 1 9 9 9 ; W a u e r , 1999). T h e o f f s p r i n g o f these b i r d s a c q u i r e s i g n i f i c a n t D D T residues not o n l y f r o m eating c o n t a m i n a t e d e a r t h w o r m s offered b y the parents, but also v i a m a t e r n a l transfer o f D D T into the e g g y o l k ( B r a n d t et a l , 1978; C e c i l et a l . , 1972; F o x et a l . , 1978; O h l e n d o r f et a l . , 1978; O t t i n g e r et a l . , 2 0 0 1 ; S t i c k e l , 1973). R o b i n eggs i n O k a n a g a n orchards have been reported to c o n t a i n total D D T  3  l e v e l s up to  3 0 0 m g / k g ( G i l l et a l . , 2 0 0 3 ) . D D T s have been s h o w n to i n f l u e n c e several aspects o f the i m m u n e s y s t e m i n a variety o f species, i n c l u d i n g b i r d s (See C h a p t e r I). B i r d s c o n t a m i n a t e d w i t h D D T s m a y be m o r e p r o n e to s u c c u m b to infectious diseases a n d parasites (Banerjee et a l . , 1996). I n 1997, t e n - d a y - o l d r o b i n n e s t l i n g s f r o m O k a n a g a n o r c h a r d s , a l o n g w i t h c o n t r o l s f r o m the B r i t i s h C o l u m b i a L o w e r M a i n l a n d , were c o l l e c t e d a n d r a i s e d i n c a p t i v i t y as part o f a study o n l o n g - t e r m effects o f early D D T exposure (see C h a p t e r s III a n d I V ) . W h e n a p p r o x i m a t e l y 50 to 57 days o f age 13 o f the  Technical grade DDT is composed of 65 to 90% p,p'-DDT, with 10 to 30% o.p'-DDT and a variety of metabolites and other products (Jefferies, 1975; Mellanby, 1992; WHO, 1989). Total DDT includes both ortho.para' and para, para' isomers of DDT and its two primary metabolites, dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD). 2  3  110 c h i c k s c o l l e c t e d f r o m the O k a n a g a n d i e d o f apparent starvation ( w a s t i n g s y n d r o m e ) . (parasites  Eimeria  and  Isospora)  Coccidiosis  w a s c o n f i r m e d i n f i v e o f these c h i c k s . N o n e o f the c h i c k s  c o l l e c t e d f r o m the L o w e r M a i n l a n d s u c c u m b e d t o t h i s disease, s u g g e s t i n g that there m a y be differences i n disease resistance a n d i m m u n e response b e t w e e n these t w o groups o f b i r d s . These differences m a y b e related to the O k a n a g a n b i r d s ' in ovo a n d e a r l y p o s t - h a t c h exposure to D D T a n d other contaminants (Banerjee, 1999; C o l m a n o & G r o s s , 1 9 7 1 ; G r a s m a n et a l . , 1996; Repetto & B a l i g a , 1996).  H o w e v e r , w h i l e r o b i n s f r o m the L o w e r M a i n l a n d h a d s i g n i f i c a n t l y h i g h e r  h e t e r o p h i l to l y m p h o c y t e ratios at ten days o f age c o m p a r e d to O k a n a g a n r o b i n s o f the same age, p h y t o h e m a g g l u t i n i n tests r e v e a l e d n o d i f f e r e n c e s i n i m m u n o c o m p e t a n c e b e t w e e n the t w o groups. T h u s , the objective o f this study was to e x a m i n e i n m o r e d e t a i l , a n d w i t h a larger sample s i z e , w h e t h e r e a r l y D D T e x p o s u r e s u p p r e s s e d the i m m u n e response o f r o b i n c h i c k s f r o m the O k a n a g a n V a l l e y . T h e goals w e r e : 1) T o replicate the 1997 e x p e r i m e n t to see i f L o w e r M a i n l a n d a n d O k a n g a n r o b i n s w e r e different i n t h e i r response to the p h y t o h e m a g g l u t i n i n s k i n test a n d d i f f e r e n t i a l w h i t e b l o o d c e l l counts. 2) T o determine i f there w e r e differences i n egg s i z e , c h i c k s i z e , p a c k e d r e d b l o o d c e l l v o l u m e s , t h y r o i d h o r m o n e l e v e l s , a n d corticosterone l e v e l s i n response to stress, b e t w e e n the L o w e r M a i n l a n d and O k a n a g a n c h i c k s . These factors have the p o t e n t i a l to i n f l u e n c e the i m m u n e s y s t e m a n d s u r v i v a l a n d / o r serve as i n d i c a t o r s o f d i s r u p t i o n s i n n o r m a l i m m u n e responses ( H a r v e y et a l . , 1986; Jefferies, 1 9 7 5 ; L e c h n e r et a l . , 2 0 0 1 ; M a i e r & W a t k i n s , 1999; S i e g e l , 1980; S t y r s k y et a l . , 2 0 0 2 ) . 3) T o determine i f the l e v e l s o f D D T and D D T metabolites the c h i c k s w e r e e x p o s e d to in ovo c o r r e l a t e d w i t h differences i n i m m u n e response a n d h o r m o n e l e v e l s .  B l o o d lead  l e v e l s w e r e a l s o assessed, as O k a n a g a n o r c h a r d s w e r e h i s t o r i c a l l y treated w i t h l e a d arsenate ( L . W i l s o n , C a n a d i a n W i l d l i f e S e r v i c e , p e r s o n a l c o m m u n i c a t i o n ) .  L e a d has  b e e n s h o w n to increase s u s c e p t i b i l i t y to i n f e c t i o u s agents a n d i m p a i r b o t h c e l l - m e d i a t e d a n d h u m o r a l i m m u n i t y at d o s e s l o w e r t h a n t h o s e r e q u i r e d f o r other t o x i c ( G r a s m a n & S c a n l o n , 1995; L e e et a l . , 2 0 0 2 ) .  effects  Ill 5.2 Methods 5.2.1 E g g C o n t a m i n a n t s B e t w e e n June 14 a n d J u l y 2 0 , 1998, 53 A m e r i c a n r o b i n eggs w e r e c o l l e c t e d f r o m nests i n o r c h a r d areas near N a r a m a t a , B r i t i s h C o l u m b i a i n the O k a n a g a n V a l l e y . A l l eggs were c o l l e c t e d d u r i n g t i m e s o f n o current-use p e s t i c i d e a p p l i c a t i o n ( L . W i l s o n , C a n a d i a n W i l d l i f e S e r v i c e , p e r s o n a l c o m m u n i c a t i o n ) . A n a d d i t i o n a l 4 0 eggs (controls) w e r e c o l l e c t e d f r o m p a r k areas i n the L o w e r M a i n l a n d ; Stanley Park, V a n c o u v e r , Tinehead Park, Surrey, U n i v e r s i t y o f B r i t i s h C o l u m b i a B o t a n i c a l G a r d e n s , V a n c o u v e r , a n d the g r o u n d s s u r r o u n d i n g the C a n a d i a n W i l d l i f e S e r v i c e o f f i c e , D e l t a b e t w e e n A p r i l 15 a n d June 2 9 , 1998. T w e n t y - t w o o f the O k a n a g a n eggs w e r e a n a l y z e d i n d i v i d u a l l y a n d the L o w e r M a i n l a n d eggs w e r e p o o l e d (one p o o l o f f i v e eggs, t w o p o o l s o f t w o eggs, a n d one i n d i v i d u a l egg). A l l o f the eggs w e r e a n a l y z e d at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r ( H u l l , Q u e b e c ) for contaminant l e v e l s u s i n g the same p r o t o c o l as those for the eggs c o l l e c t e d i n 1997 (See C h a p t e r III). R e s u l t s w e r e expressed i n p g / g a n d o n a w e t w e i g h t basis.  5.2.2 E g g M e a s u r e m e n t s A t o t a l o f 83 eggs (50 O k a n a g a n , 33 L o w e r M a i n l a n d ) w e r e w e i g h e d a n d m e a s u r e d , i n c l u d i n g those u s e d for c o n t a m i n a n t analyses. W h e r e m e a s u r e m e n t s w e r e a v a i l a b l e for m o r e than one e g g per nest, nest m e a n s w e r e u s e d for the analyses.  E g g l e n g t h refers to the distance  f r o m end-to-end, w h i l e egg w i d t h refers to the m e a s u r e m e n t at the w i d e s t part o f the e g g (See C h a p t e r III).  5.2.3 C h i c k s S e v e n t y - s e v e n t e n - d a y - o l d A m e r i c a n r o b i n nestlings w e r e c o l l e c t e d b e t w e e n J u n e 14 a n d J u l y 2 0 , 1 9 9 8 , f r o m the same O k a n a g a n nests f r o m w h i c h eggs w e r e c o l l e c t e d .  Forty-eight  nestlings w e r e c o l l e c t e d f r o m the L o w e r M a i n l a n d nests, b e t w e e n M a y 1 a n d J u l y 2 8 , 1998. A l l nestlings were c o l l e c t e d d u r i n g periods o f no current-use pesticide treatment ( L . W i l s o n , Canadian W i l d l i f e Service, personal communication).  T h e b i r d s w e r e r e a r e d at M o n i k a ' s  W i l d l i f e Shelter (Surrey, B r i t i s h C o l u m b i a ) under the same c o n d i t i o n s as those c o l l e c t e d i n 1997 (See C h a p t e r III). A l l b i r d s w e r e e u t h a n i z e d at the e n d o f the study i n O c t o b e r 1998. S e x w a s determined post-mortem based on m o r p h o l o g y .  A l l treatment a n d h o u s i n g p r o t o c o l s w e r e  a p p r o v e d b y the U n i v e r s i t y o f B r i t i s h C o l u m b i a A n i m a l C a r e C o m m i t t e e ( C e r t i f i c a t e # A 9 7 0043).  112 5.2.4 C h i c k M e a s u r e m e n t s F o r t y - e i g h t L o w e r M a i n l a n d c h i c k s from 25 nests w e r e w e i g h e d a n d m e a s u r e d , a l o n g w i t h 77 O k a n a g a n c h i c k s f r o m 31 different nests. C h i c k s w e r e w e i g h e d a n d b o d y measurements t a k e n a p p r o x i m a t e l y ten d a y s post-hatch, f o l l o w i n g the same p r o t o c o l s as those u s e d for 1997 b i r d s (See C h a p t e r III). W h e n m o r e than one c h i c k w a s c o l l e c t e d f r o m a nest, m e a n s per b r o o d w e r e u s e d for the analyses.  B o d y w e i g h t s a n d m o r p h o m e t r i c measurements w e r e not t a k e n at  later stages. 5.2.5 T h y r o i d H o r m o n e s T h y r o i d h o r m o n e s ( p l a s m a t r i i o d o t h y r o n i n e a n d t h y r o x i n c o n c e n t r a t i o n s ) w e r e assessed b y T r a c y M a r c h a n t at the U n i v e r s i t y o f S a s k a t c h e w a n , S a s k a t o o n , S a s k a t c h e w a n , i n p l a s m a samples c o l l e c t e d from 14 L o w e r M a i n l a n d (8 broods) a n d 58 O k a n a g a n (22 b r o o d s ) b i r d s w h e n they w e r e t e n - d a y s - o l d .  T h e same p r o t o c o l as that u s e d for the 1997 b i r d s w e r e u s e d (See  C h a p t e r III).  5.2.6 I m m u n e R e s p o n s e  5.2.6.1 White Blood Cell Counts B l o o d smears w e r e c o l l e c t e d f r o m the b i r d s w h e n they w e r e t e n - d a y - o l d n e s t l i n g s (47 L o w e r M a i n l a n d c h i c k s f r o m 25 b r o o d s , 73 O k a n a g a n c h i c k s from 30 b r o o d s ) a n d 5 0 - 57 day o l d j u v e n i l e s (43 L o w e r M a i n l a n d b i r d s f r o m 2 2 b r o o d s , 7 0 O k a n a g a n b i r d s f r o m 2 9 b r o o d s ) , u s i n g the same p r o t o c o l as that used for the 1997 b i r d s (See C h a p t e r III).  5.2.6.2 Phytohemagglutinin Skin Test F o r t y - o n e L o w e r M a i n l a n d (21 b r o o d s ) a n d 6 9 O k a n a g a n (31 b r o o d s ) j u v e n i l e s o f b o t h sexes w e r e u s e d for the p h y t o h e m a g g l u t i n i n test, u s i n g the same p r o t o c o l as d e s c r i b e d i n C h a p t e r III.  5.2.6.3 Hematocrits W h e n the b i r d s w e r e 10 (38 L o w e r M a i n l a n d n e s t l i n g s f r o m 21 b r o o d s , 7 6 O k a n a g a n nestlings from 31 b r o o d s ) a n d 50 - 57 days o f age (43 L o w e r M a i n l a n d b i r d s f r o m 2 2 nests, 70 O k a n a g a n b i r d s f r o m 2 9 b r o o d s ) , b l o o d s a m p l e s w e r e c o l l e c t e d i n order to d e t e r m i n e p a c k e d b l o o d c e l l v o l u m e s . T h r e e 4 0 p i h e p a r i n i z e d c a p i l l a r y tubes o f b l o o d w e r e c o l l e c t e d from e a c h b i r d f o l l o w i n g puncture o f the b r a c h i a l v e i n w i t h a 27-gauge needle. T h e ends o f the tubes w e r e p l u g g e d w i t h putty a n d then centrifuged at 11,000 r p m for f i v e m i n u t e s . A v e r a g e s o f the three hematocrits a n d b r o o d means w e r e used for the analyses.  113 5.2.6.4Positive Controls S i x j u v e n i l e r o b i n s a d m i t t e d to M o n i k a ' s W i l d l i f e Shelter f r o m areas a r o u n d the L o w e r M a i n l a n d a n d n o t treated w i t h a n y t y p e o f d r u g w e r e i n c l u d e d as p o s i t i v e c o n t r o l s f o r the i m m u n e tests. T h e s e b i r d s w e r e o r a l l y d o s e d w i t h dexamethasone o n c e a day for t w o days p r i o r to a n d o n the day o f the p h y t o h e m a g g l u t i n i n injections. T w o b i r d s w e r e d o s e d w i t h 1.5 m g / k g b o d y w e i g h t a n d four w i t h 3.7 m g / k g b o d y w e i g h t . 5.2.7 Stress R e s p o n s e Stress response i n 43 L o w e r M a i n l a n d (22 b r o o d s ) a n d 7 0 O k a n a g a n (29 b r o o d s ) b i r d s w a s d e t e r m i n e d u s i n g changes i n c o r t i c o s t e r o n e l e v e l s d u r i n g a restraint stress test ( G a u n t & O r i n g , 1999) w h e n the b i r d s w e r e b e t w e e n 5 0 a n d 57 d a y s o f age ( j u v e n i l e s ) . B l o o d s a m p l e s w e r e c o l l e c t e d i m m e d i a t e l y u p o n capture o f the b i r d s (time 0), after f i v e m i n u t e s ( t i m e 5), ten m i n u t e s ( t i m e 10), t h i r t y m i n u t e s ( t i m e 3 0 ) , a n d s i x t y m i n u t e s ( t i m e 6 0 ) . c o l l e c t i o n s , the b i r d s w e r e h e l d i n a b o x w i t h a c l o t h c o v e r .  Between sample  B l o o d samples were taken and  p r o c e s s e d i n the same w a y as f o r the h e m a t o c r i t s , but o n l y t w o c a p i l l a r y tubes o f b l o o d w e r e collected from five minutes on.  P l a s m a samples were extracted and analyzed using a  r a d i o i m m u n o - a s s a y at the U n i v e r s i t y o f S a s k a t c h e w a n , f o l l o w i n g the p r o t o c o l s o u t l i n e d i n K l o e p p e r - S a m s et a l . (1994) a n d B o r t o l o t t i et a l . (1996). 5.2.8 B l o o d L e a d L e a d l e v e l s w e r e a n a l y z e d i n b l o o d s a m p l e s (14 L o w e r M a i n l a n d a n d 3 0 O k a n a g a n ) c o l l e c t e d w h e n the b i r d s w e r e ten days o l d . A n a l y s e s w e r e c o n d u c t e d at the N a t i o n a l W i l d l i f e R e s e a r c h C e n t e r u s i n g g r a p h i t e furnace s p e c t r o m e t r y .  V a l u e s w e r e e x p r e s s e d as p.g/g, d r y  weight. 5.2.9 T i s s u e W e i g h t s F o l l o w i n g c o m p l e t i o n o f the study ( O c t o b e r 1 9 9 8 ) the b i r d s w e r e e u t h a n i z e d a n d dissected. C o l l e c t e d tissues ( g a l l b l a d d e r , heart, spleen, l i v e r , k i d n e y s , adrenal g l a n d s , gonads, t h y r o i d g l a n d s , t h y m u s , a n d bursa) w e r e w e i g h e d w h e n fresh a n d t h e n f r o z e n o r s t o r e d i n f o r m a l i n for future analyses. S e x w a s also d e t e r m i n e d at the t i m e o f sacrifice.  5.2.10 Statistics D i f f e r e n c e s b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n eggs, n e s t l i n g s , a n d j u v e n i l e s w e r e a n a l y z e d u s i n g o n e - w a y analyses o f v a r i a n c e ( A N O V A s ) a n d nest m e a n s .  T i s s u e w e i g h t s at  t i m e o f s a c r i f i c e w e r e c o r r e c t e d f o r b o d y w e i g h t b y d i v i d i n g the tissue w e i g h t b y the b o d y  114 w e i g h t m i n u s the tissue w e i g h t . D a t a were c o m m o n l o g transformed, w h e r e necessary, i n order to increase n o r m a l i t y . P a i r - w i s e correlations w e r e c o n d u c t e d to determine the effects o f e g g p,p'D D T , o,p'-DDT, p,p'-DDD, o,p'-DDD, p,p'-DDE, and o , p ' - D D E  4  i n the O k a n a g a n b i r d s o n l y .  C o n t a m i n a n t non-detects a n d zeros w e r e r e p l a c e d b y 0 . 0 0 0 0 5 ( h a l f the d e t e c t i o n l i m i t ) f o r the correlations. O u t l i e r s w e r e d e t e r m i n e d u s i n g the M a h a l a n o b i s o u t l i e r distance test. P v a l u e s less than o r e q u a l to 0.05 w e r e c o n s i d e r e d s i g n i f i c a n t . W h e r e p o s s i b l e , data f r o m the 1998 eggs a n d c h i c k s were c o m p a r e d to those from 1997 (see C h a p t e r III) u s i n g the m o d e l : Y  i j k  = p. + L i + T j + ( L T ) y + E  i j k  w h e r e Y i s the parameter b e i n g measured, L refers to whether the b i r d w a s from the O k a n a g a n o r L o w e r M a i n l a n d , T refers to the y e a r the e g g or c h i c k w a s c o l l e c t e d ( 1 9 9 7 o r 1998), a n d L T is the t w o - w a y i n t e r a c t i o n b e t w e e n type a n d year. A l l statistics w e r e c o n d u c t e d u s i n g J M P v e r s i o n 3.2.1 software ( S A S Institute, C a r y , N o r t h C a r o l i n a ) . V a l u e s e x p r e s s e d are ranges a n d m e a n s + standard error. 5.3 R e s u l t s 5.3.1 E g g C o n t a m i n a n t s T a b l e 5-1 illustrates the m e a n s , standard errors, a n d ranges f o r m o i s t u r e content, l i p i d content, p , p ' - D D T , p , p ' - D D D , p , p ' - D D E , o , p ' - D D T , o , p ' - D D D , o , p ' - D D E , a n d the ratios o f D D E to D D T f o r the eggs c o l l e c t e d i n 1998 a n d 1 9 9 7 . T h e c o m p l e t e l i s t i n g o f o r g a n o c h l o r i n e a n d p o l y c h l o r i n a t e d b i p h e n y l s a n a l y z e d i s a v a i l a b l e i n A p p e n d i c e s I a n d II. I n b o t h years, eggs  from  the O k a n a g a n c o n t a i n e d s i g n i f i c a n t l y h i g h e r l e v e l s o f p , p ' - D D E ( F ^ s = 134.7, p < 0 . 0 0 0 1 ) , p , p ' DDT ( F  U  8  = 83.4, p < 0.0001), and p , p ' - D D D ( F i ,  5 8  = 5 7 . 6 , p < 0 . 0 0 0 1 ) t h a n eggs f r o m the  L o w e r M a i n l a n d . O n l y p , p ' - D D E (Fi 8= 8.3, p = 0.006) l e v e l s w e r e h i g h e r i n 1997 t h a n i n 1 9 9 8 j5  eggs.  T h e p , p ' - D D T l e v e l s w e r e also h i g h e r i n 1 9 9 7 t h a n i n 1 9 9 8 , b u t o n l y i f o u t l i e r s w e r e  removed.  T h e L o w e r M a i n l a n d a n d O k a n g a n eggs s h o w e d no significant differences i n  D D E : D D T ratios.  H o w e v e r , w h e n outliers were r e m o v e d , L o w e r M a i n l a n d eggs h a d a  s i g n i f i c a n t l y h i g h e r ratio t h a n the O k a n a g a n eggs.  I n b o t h years, the para,para' i s o m e r s w e r e  found i n a l l L o w e r M a i n l a n d (although only minute amounts i n pooled egg samples) and O k a n a g a n eggs, but the ortho,para' i s o m e r s were o n l y f o u n d i n the O k a n a g a n eggs.  0,p'-DDE  Ortho, para' and para,para' isomers were evaluated separately as they have been shown to have very different effects (e.g., Gaido et al., 1997; Kelce et al., 1995; 1998; Sohoni & Sumpter, 1998). 4  115 Table 5-1: Means (+ standard error) and ranges of DDTs, moisture, and lipids (pig/g) in American robin eggs collected from the Lower Mainland and Okanagan Valley of British Columbia.  % water  % lipid  1997 Lower Mainland n =3 82.0 (0.2)  1998 Lower Mainland n =4  1997 Okanagan n = 31  1998 Okanagan n = 22  Total Lower Mainland n=7  Total Okanagan n = 53  82.0 (0.3)  82.8 (0.3)  81.7(0.4)  82.0 (0.2)  82.4 (0.2)  81.6-82.3  81.1-83.3  80.2-85.3  77.9-85.1  81.1-83.3  77.9-85.3  5.7 (0.2)  5.3 (0.3)  4.3 (0.2)  5.0 (0.2)  5.4 (0.2)  4.6 (0.1)  5.4-6.1  3.8-6.1  1.5-5.8  3.7-7.3  3.8-6.1  1.5-7.3  12.1 (1.6)  6.9(1.8)  0.1 (0.02)  9.9(1.2)  0.9-30.5  0.2-37.3  0.05-0.2  0.2-37.3  0.01 (0.01)  p,p'-DDT  0.1-0.2  p,p'-DDE  p,p'-DDD  0.07 (0.008) 0.05-0.09  1.9(0.7)  0.8 (0.4)  51.7(8.7)  26.2 (2.9)  1.3 (0.4)  41.1 (5.5)  0.9-3.2  0.3-1.9  10.0-245.0  5.4-59.1  0.3-3.2  5.4-245.0  0.009  0.006  1.0(0.3)  0.7 (0.3)  (0.004)  (0.0008)  0.003-0.02  0.005-0.008  o,p'-DDT  ND  ND  2  0.06-8.7  0.02-6.5  0.05 (0.01)  0.05 (0.02)  ND-0.2  ND-0.3  0.007 (0.002) 0.003-0.02  0.9 (0.2) 0.02-8.7 0.05  ND  (0.009) ND-0.3  o,p'-DDE  ND  o,p'-DDD  DDEiDDT  ND  ND  ND  0.004  (0.0006)  (0.001)  ND-0.01  ND-0.02  ND-0.07  0.003  0.004  0.003  (0.0007)  (0.003)  ND-0.02  ND-0.07  0.006 + ND  ND  0.004  (0.001) ND-0.07  12.8 (3.7)  11.3 (4.2)  6.8 (2.0)  11.4 (2.3)  11.9(2.6)  8.7(1.5)  8.0-20.1  6.2-23.7  0.7-63.1  0.7-38.5  6.2-23.7  0.7-63.1  1  ortho,para' a n d para,para' i s o m e r s 2  0.002  N D = not detected  * p < 0.05  116 w a s f o u n d i n 6 2 . 3 % o f the O k a n a g a n eggs, o , p ' - D D D i n 5 6 . 6 % , a n d o , p ' - D D T i n 8 1 . 1 % . T h e r e w e r e n o s i g n i f i c a n t differences b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n eggs i n m o i s t u r e a n d l i p i d content,  a n d there w e r e  no s i g n i f i c a n t c o r r e l a t i o n s o f these parameters w i t h  egg  c o n t a m i n a n t l e v e l s . E g g s c o l l e c t e d i n 1998 h a d s i g n i f i c a n t l y ( F i 6 4 = 8.1, p = 0.006) h i g h e r l i p i d ;  contents than the 1997 eggs.  5.3.2 T h y r o i d H o r m o n e s I n 1 9 9 8 , 10-day o l d O k a n a g a n b r o o d s h a d s i g n i f i c a n t l y h i g h e r t r i i o d o t h y r o n i n e l e v e l s t h a n L o w e r M a i n l a n d b i r d s (F13  = 5.7, p = 0 . 0 2 ; F i g u r e 5-1), but there w e r e n o s i g n i f i c a n t  differences i n t h y r o x i n e l e v e l s ( F i g u r e 5-1), n o r w e r e there s i g n i f i c a n t c o r r e l a t i o n s w i t h D D T exposure i n the O k a n a g a n c h i c k s . T h y r o i d h o r m o n e l e v e l s w e r e m e a s u r e d i n m u c h o l d e r b i r d s i n 1997.  A s t h y r o i d hormone levels can change dramatically over time ( W e n t w o r t h & R i n g e r ,  1986), statistical c o m p a r i s o n s b e t w e e n years w o u l d not be m e a n i n g f u l .  5.3.3 I m m u n e R e s p o n s e  5.3.3.1 Hematocrits N o s i g n i f i c a n t differences w e r e f o u n d i n h e m a t o c r i t v a l u e s b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b r o o d s at ten d a y s o r 50 - 57 days.  W h e n the O k a n a g a n b i r d s w e r e j u v e n i l e s ,  h o w e v e r , their h e m a t o c r i t v a l u e s w e r e p o s i t i v e l y c o r r e l a t e d w i t h o , p ' - D D T (r = 0.5, n = 2 2 , p = 0.02) a n d o , p ' - D D E (r = 0.5, n = 2 2 , p = 0.02), but n e g a t i v e l y c o r r e l a t e d w i t h p , p ' - D D E (r = - 0.4, n = 22, p = 0.05).  A c o m p a r i s o n o f the t w o ages r e v e a l e d that h e m a t o c r i t v a l u e s  were  c o n s i d e r a b l y h i g h e r w h e n the b i r d s w e r e j u v e n i l e s than w h e n they w e r e nestlings ( F i j o i - 3 2 3 . 7 , p < 0 . 0 0 0 1 ; F i g u r e 5-2).  H e m a t o c r i t s averaged 4 5 . 0 a n d 49.8 i n the 1.5 m g / k g  dexamethasone  c o n t r o l s , w h i c h w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m the O k a n a g a n a n d L o w e r  Mainland  j u v e n i l e s . H e m a t o c r i t s w e r e not c o l l e c t e d from the 1997 b i r d s .  5.3.3.2 White Blood Cell Ratios A t 10 d a y s o f age, the L o w e r M a i n l a n d n e s t l i n g s h a d h i g h e r e o s i n o p h i l / h e t e r o p h i l to l y m p h o c y t e / m o n o c y t e ratios t h a n their O k a n a g a n counterparts ( F i 6 = 15.5, p = 0 . 0 0 0 1 ) . 1 ;  2  The  difference w a s consistent i n b o t h years. T h e w h i t e b l o o d c e l l ratios w e r e s i g n i f i c a n t l y correlated w i t h p , p ' - D D D e g g l e v e l s (r = - 0.3, n = 5 5 , p = 0 . 0 3 ) f o r the O k a n a g a n b i r d s .  A s juveniles  (1998) a n d post-breeding adults (1997), the b i r d s s h o w e d n o differences i n their w h i t e b l o o d c e l l  117  Figure 5-1: Mean (+ se) triiodothyronine and thyroxine levels (ng/ml) in the blood of 1998 Lower Mainland and Okanagan birds at ten days of age. * p < 0.05  118  60  n  Figure 5-2: Mean (+ se) hematocrit values for 1998 Lower Mainland and Okanagan robins when 10 and 50 -57 days of age.  119 ratios. W h i l e j u v e n i l e s demonstrated n o s i g n i f i c a n t correlations w i t h D D T s , post-breeding adults s h o w e d a p o s i t i v e c o r r e l a t i o n w i t h p , p ' - D D T (r = 0.4, n = 2 5 , p = 0.03). A c o m p a r i s o n o f the t w o ages (1998 b i r d s o n l y ) r e v e a l e d that the b i r d s h a d s i g n i f i c a n t l y h i g h e r ratios as nestlings than as j u v e n i l e s (F1J04 = 9 4 . 0 , p < 0 . 0 0 0 1 ; F i g u r e 5-3). W h i t e b l o o d c e l l ratios w e r e a v a i l a b l e for the t w o 1.5 m g / k g p o s i t i v e c o n t r o l s (0.8 a n d 0.5), a n d they d i d not d i f f e r s i g n i f i c a n t l y f r o m the Okanagan and L o w e r M a i n l a n d birds.  5.3.3.3 Phytohemagglutinin Skin Test W i n g i n d e x m e a s u r e m e n t s i n the 1998 L o w e r M a i n l a n d b i r d s r a n g e d f r o m 0.1 to (0.7 + 0.05), whereas i n the O k a n a g a n b i r d s it r a n g e d f r o m 0.3 to 1.0 (0.6 + 0.03).  1.14  There were  n o s i g n i f i c a n t differences i n response to the p h y t o h e m a g g l u t i n i n test b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n b i r d s . N o s i g n i f i c a n t c o r r e l a t i o n s w i t h in ovo D D T e x p o s u r e w e r e f o u n d either. W i n g i n d e x m e a s u r e m e n t s for the p o s i t i v e c o n t r o l s r a n g e d f r o m 0.2 to 0.9 (0.5 + 0.1) a n d w e r e n o t s i g n i f i c a n t l y different f r o m the O k a n a g a n a n d L o w e r M a i n l a n d b i r d s . demonstrated a greater response to this test than d i d the 1997 birds ( F i j i  =  T h e 1998 b i r d s  8.2, p = 0.006).  As  age, sex a n d y e a r effects w e r e c o n f o u n d e d , it c o u l d n o t be d e t e r m i n e d w h i c h o f these factors w e r e c a u s i n g the difference. H o w e v e r , there w e r e n o s i g n i f i c a n t differences b e t w e e n m a l e s a n d f e m a l e s i n 1998.  T h u s , the d i f f e r e n c e b e t w e e n 1 9 9 7 a n d 1998 m a y be attributable to age  differences. 5.3.4. Stress R e s p o n s e T h e r e w e r e n o s i g n i f i c a n t differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n b r o o d s i n p l a s m a corticosterone l e v e l at any t i m e d u r i n g the restraint test. T h e r e w e r e also n o s i g n i f i c a n t correlations w i t h D D T exposure.  T h e r e m o v a l o f one O k a n a g a n o u t l i e r , h o w e v e r , renders the  differences b e t w e e n L o w e r M a i n l a n d a n d O k a n a g a n b i r d s i n p l a s m a c o r t i c o s t e r o n e at t i m e s 0 (F1.26 = 7 . 0 , p = 0 . 0 1 ) a n d 5 (F1.27 = 4 . 7 , p = 0.04) s t a t i s t i c a l l y s i g n i f i c a n t .  Although  corticosterone l e v e l s were l o w e r i n the O k a n a g a n b i r d s than i n the L o w e r M a i n l a n d b i r d s d u r i n g the first five m i n u t e s o f the test, b o t h groups reached s i m i l a r peak l e v e l s at t i m e 3 0 ( F i g u r e 5-4). R e m o v a l o f the o u t l i e r also resulted i n s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s b e t w e e n e g g p , p ' - D D T a n d corticosterone l e v e l s at t i m e 5 (r = 0.6, n = 2 1 , p = 0.009) a n d t i m e 10 (r = 0.5, n = 2 1 , p = 0.03), o , p ' - D D T and t i m e 5 (r = 0.5, n = 2 1 , p = 0.01) a n d t i m e 10 (r = 0.6, n = 2 1 , p = 0.01), a n d o , p ' - D D E and t i m e 5 (r = 0.6, n = 2 1 , p = 0.008) and t i m e 10 (r = 0.5, n = 2 1 , p = 0.02). T h e r e  120  2.5  A g e (Days)  Figure 5-3: Mean (+ se) eosinophil + heterophil / monocyte + lymphocyte ratios for 1998 Okanagan and Lower Mainland birds when 10 and 50 - 57 days old. * p < 0.05  121 w e r e s i g n i f i c a n t t i m e differences i n corticosterone l e v e l s (¥4^44 = 3 6 . 7 , p < 0 . 0 0 0 1 ; F i g u r e 5-4). Stress response w a s not tested i n the b i r d s c o l l e c t e d i n 1997. 5.3.5 B l o o d L e a d B l o o d l e a d l e v e l s r a n g e d f r o m 0.09 to 1.5 p.g/g (0.4 + 0.1 p.g/g) i n the 1 9 9 8 L o w e r M a i n l a n d c h i c k s a n d 0.2 to 1.3 i i g / g (0.7 + 0.06 p.g/g) i n the O k a n a g a n c h i c k s . T h e s e l e v e l s w e r e s i g n i f i c a n t l y h i g h e r i n the O k a n a g a n than i n the L o w e r M a i n l a n d b i r d s (Fi,4i = 9.0, p = 0.005).  P a i r - w i s e c o r r e l a t i o n s w e r e c o n d u c t e d b e t w e e n b l o o d l e a d l e v e l s i n the ten-day o l d  c h i c k s a n d w h i t e b l o o d c e l l ratios, hematocrits, t h y r o i d h o r m o n e l e v e l s , b o d y w e i g h t s , a n d tarsus lengths at the same age. N o significant correlations w e r e f o u n d . A n o m i n a l l o g i s t i c s c o m p a r i s o n b e t w e e n b l o o d l e a d l e v e l s a n d whether or not the b i r d w a s treated for m y c o p l a s m o s i s w a s also non-significant. B l o o d l e a d l e v e l s were not d e t e r m i n e d for the b i r d s c o l l e c t e d i n 1997.  5.3.6 Infectious Diseases a n d Parasites Mycoplasma  gallisepticum  and/or Mycoplasma  synoviae i n f e c t i o n s w e r e d i a g n o s e d i n  e i g h t L o w e r M a i n l a n d a n d 4 6 O k a n a g a n r o b i n s i n 1998, u s i n g the h e m a g g l u t i n a t i o n i n h i b i t i o n test at the A n i m a l H e a l t h M o n i t o r i n g L a b , B r i t i s h C o l u m b i a M i n i s t r y o f A g r i c u l t u r e , F o o d , a n d Fisheries, Abbotsford, British C o l u m b i a .  T h e s e b i r d s w e r e treated w i t h B a y t r i l ( e n r o f l o x a z i n )  u n t i l they n o l o n g e r s h o w e d s y m p t o m s o f i n f e c t i o n (i.e., s w o l l e n eyes, n a s a l discharge, c o u g h ) , w i t h the first treatments b e g i n n i n g o n J u l y 2 9 , 1998. P l a s m a samples from the b i r d s at ten-days o f age w e r e n e g a t i v e for m y c o p l a s m a , therefore, the b i r d s a c q u i r e d the i n f e c t i o n w h i l e i n captivity.  I n a d d i t i o n , one L o w e r M a i n l a n d b i r d a n d f o u r O k a n a g a n b i r d s w e r e suspected o f  suffering f r o m a s p e r g i l l o s i s (Aspergillus fumigatus)  infections.  T a p e w o r m s w e r e f o u n d i n 16  L o w e r M a i n l a n d a n d 43 O k a n a g a n b i r d s p o s t - m o r t e m , a n d u n i d e n t i f i e d nematodes were f o u n d i n five L o w e r M a i n l a n d a n d ten O k a n a g a n b i r d s .  5.3.7 M o r t a l i t y O f the 38 1998 L o w e r M a i n l a n d b i r d s s t u d i e d , 3 6 ( 9 4 . 7 % ) s u r v i v e d to the e n d o f the study.  O n e L o w e r M a i n l a n d c h i c k d i e d o f u n k n o w n causes w h e n 53 d a y s o f age, a n d one w a s  e u t h a n i z e d w h e n 64 days o f age. e n d o f the study.  F i f t y - e i g h t o f 64 ( 9 0 . 6 % ) O k a n a g a n c h i c k s s u r v i v e d u n t i l the  T w o O k a n a g a n c h i c k s d i e d o f u n k n o w n causes w h e n 6 7 a n d 69 days o f age.  T h r e e O k a n a g a n b i r d s w e r e e u t h a n i z e d ; one at 41 d a y s , o n e at 58 d a y s , a n d o n e at about the same t i m e as the others w e r e s a c r i f i c e d . O n e 55 day o l d O k a n a g a n c h i c k l i k e l y d i e d as a result  Cvl  25  Figure 5-4: Mean (± se) piasma cr.icos.er.ne l e v * (ng/ml) in 1998 Lower MainiandI and Okanagan juvenile robins during a restraint stress test. Outlier removed. * p < 0.05. s  123 o f a mycoplasma infection. 5.3.8 E g g M e a s u r e m e n t s L o w e r M a i n l a n d eggs ( n = 68) w e i g h e d 6.5 + 0.2 g r a m s , a v e r a g e d 29.3 + 0.3 m m l o n g , a n d 21.0 + 0.1 m m w i d e . O k a n a g a n eggs (n = 110) w e i g h e d 6.6 + 0.1 grams, averaged 29.0 + 0.1 mm  l o n g a n d 2 1 . 0 + 0.1 m m w i d e .  There were no significant differences between  Lower  M a i n l a n d a n d O k a n a g a n eggs i n w e i g h t , l e n g t h , o r w i d t h , n o r w e r e there a n y s i g n i f i c a n t c o r r e l a t i o n s w i t h c o n t a m i n a n t l e v e l s w i t h i n the O k a n a g a n eggs.  E g g s c o l l e c t e d i n 1997 a n d  1998 w e r e o f s i m i l a r w e i g h t s a n d w i d t h s , but eggs c o l l e c t e d i n 1997 w e r e l o n g e r than those c o l l e c t e d i n 1998 (Fi m  =  t  3.9, p = 0.05). T h e r e m o v a l o f outliers, h o w e v e r , rendered this result  non-significant. 5.3.9 C h i c k M e a s u r e m e n t s There were no significant differences  between  10-day o l d L o w e r M a i n l a n d  and  O k a n a g a n r o b i n s i n b o d y w e i g h t s , tarsus lengths, or w i n g lengths at this age a n d there w e r e n o differences i n the m e a s u r e m e n t s t a k e n i n 1997 a n d 1998.  L o w e r M a i n l a n d n e s t l i n g s ( n = 58)  w e i g h e d 58.6 ± 1.0 grams (34.0 - 76.0 grams) at ten days, h a d 37.4 + 0.2 m m (32.8 to 41.5 m m ) l o n g tarsus, 21.4 + 0.2 m m (19.0 to 2 6 . 0 m m ) m i d d l e toes, a n d 67.5 ± 1.8 m m (46.0 - 127.0 m m ) w i n g length.  C o r r e s p o n d i n g m e a s u r e m e n t s i n O k a n a g a n n e s t l i n g s ( n = 72) w e r e 60.3 + 0.6  g r a m s (50.1 - 75.1 grams), 37.1 ± 0.2 m m (33.9 to 4 0 . 6 m m ) , 14.9 ± 0.6 m m (6.7 to 2 3 . 0 m m ) a n d 66.2 + 0.7 m m (50.7 - 7 8 . 0 m m ) , r e s p e c t i v e l y . O k a n a g a n c h i c k s h a d shorter m i d d l e toes than L o w e r M a i n l a n d c h i c k s ( F i . m = 3 8 4 . 1 , p < 0.0001), a n d c h i c k s c o l l e c t e d i n 1998 h a d l o n g e r wings ( F i n 1997.  U 2  i = 13.7, p = 0.0003) a n d m i d d l e toes ( F i , i = 2 5 7 . 9 , p < 0.0001) than those c o l l e c t e d 2 2  T h e r e w a s a s i g n i f i c a n t y e a r b y type i n t e r a c t i o n for toe l e n g t h (^3,122 = 2 9 7 . 3 , p <  0 . 0 0 0 1 ) , w i t h O k a n a g a n b i r d s c o l l e c t e d i n 1997 h a v i n g s i g n i f i c a n t l y shorter m i d d l e toes t h a n L o w e r M a i n l a n d birds.  T h e r e w a s n o s u c h d i f f e r e n c e i n the 1998 b i r d s .  B o d y weight was  p o s i t i v e l y correlated w i t h egg p , p ' - D D E l e v e l s (r = 0.4, n = 5 0 , p = 0.007) a n d w i n g l e n g t h w a s n e g a t i v e l y correlated w i t h p , p ' - D D D levels (r = - 0.3, n = 50, p = 0.02). 5.3.10 T i s s u e W e i g h t s T a b l e 5-2 illustrates the b o d y a n d c o r r e c t e d tissue w e i g h t s for the 1998 L o w e r M a i n l a n d a n d O k a n a g a n b i r d s at sacrifice. O k a n a g a n b i r d s h a d s i g n i f i c a n t l y h e a v i e r l i v e r s (Fi,48 = 0 . 0 0 0 5 ) , k i d n e y s ( F 8 = 7.0, p = 0 . 0 1 ) , a n d spleens ( F i M  ; 4 g  =  13.9, p  = 5.2, p = 0.03) t h a n L o w e r  M a i n l a n d b i r d s . T h e r e m o v a l o f outliers rendered the difference b e t w e e n L o w e r M a i n l a n d a n d  124  Table 5-2: Means (+ se) and ranges of body and tissue weights (grams) of 1998 Okanagan and Lower Mainland broods at time of sacrifice.  body  gall bladder  spleen  bursa  adrenal glands  gonads  thymus  liver  heart  thyroid glands  kidneys  * p < 0.05  Lower Mainland  Okanagan  n = 21  n = 30  81.7 ± 1 . 1  81.8 ± 1.3  66.5-88.9  56.5-90.0  0.001 ± 0 . 0 0 0 1  0.001 ± 0.00007  0.0004 - 0.003  0.0004 - 0.002  0.002 ± 0.0002  0.003 ± 0 . 0 0 0 3 *  0.001 - 0 . 0 0 4  0.001 - 0 . 0 0 8  0.0009 ± 0 . 0 0 0 1  0.0007 ± 0 . 0 0 0 0 7  0 - 0.002  0.0002 - 0.002  0.0002 ± 0.00002  0.0002 ± 0 . 0 0 0 0 1  0.00006 - 0.0004  0.0001 - 0.0003  0.0002 ± 0 . 0 0 0 0 2  0.0002 ± 0 . 0 0 0 0 2  0.0001 - 0 . 0 0 0 4  0.00006 - 0.0006  .001 ± 0 . 0 0 0 0 7  0.001 ± 0 . 0 0 0 0 6  0.005 - 0.002  0.0003 - 0.002  0.03 ± 0.0008  0.04 ± 0 . 0 0 2 *  0.03 - 0.04  0.03-0.08  0.01 ± 0 . 0 0 0 3  0.01 ± 0 . 0 0 0 2  0.009 - 0.02  0.01-0.02  0.0003 ± 0.00002  0.0003 ± 0.00002  0 - 0.0005  0.00006 - 0.0004  0.02 ± 0.0004  0.02 ± 0 . 0 0 0 5 *  0.01-0.02  0.02 - 0.03  125 O k a n a g a n b i r d s ' spleens n o n - s i g n i f i c a n t , but d i d not affect the l i v e r a n d k i d n e y d i f f e r e n c e s . A m o n g the O k a n a g a n b i r d s , p o s i t i v e c o r r e l a t i o n s w e r e f o u n d b e t w e e n b u r s a w e i g h t s a n d p,p'D D E (r = 0.5, n = 2 1 , p = 0.02), l i v e r and p , p ' - D D D (r = 0.6, n = 2 2 , p = 0.002), l i v e r a n d o,p'D D D (r = 0.7, n - 2 2 , p = 0 . 0 0 7 ) , k i d n e y s a n d p , p ' - D D D (r = 0 . 7 , n = 2 2 , p = 0 . 0 0 0 7 ) , a n d k i d n e y s a n d o , p ' - D D D (r = 0.7, n = 2 2 , p = 0.001). S i g n i f i c a n t negative correlations were f o u n d b e t w e e n the g a l l bladder a n d p , p ' - D D E (r = - 0 . 5 , n = 2 2 , p = 0.02) a n d adrenal g l a n d s a n d o,p'D D T (r = -0.4, n = 2 1 , p = 0.05).  O n l y the g a l l b l a d d e r c o r r e l a t i o n w a s s i g n i f i c a n t after the  r e m o v a l o f outliers. D i r e c t c o m p a r i s o n s w i t h the 1997 b i r d s w e r e not p o s s i b l e , as the 1998 b i r d s w e r e s a c r i f i c e d as j u v e n i l e s a n d the 1997 b i r d s w e r e s a c r i f i c e d as t w o to three y e a r o l d adults.  A  total o f 19 L o w e r M a i n l a n d m a l e s , 27 L o w e r M a i n l a n d females, 41 O k a n a g a n m a l e s , a n d 3 7 O k a n a g a n females were sacrificed.  5.4 D i s c u s s i o n  I n b o t h years (1997 and 1998), eggs c o l l e c t e d f r o m o r c h a r d areas o f the O k a n a g a n V a l l e y c o n t a i n e d s i g n i f i c a n t l y h i g h e r l e v e l s o f v a r i o u s D D T i s o m e r s t h a n e g g s f r o m the Mainland.  Lower  R o b i n s f r o m the O k a n a g a n w e r e m o r e s u s c e p t i b l e t o diseases a n d parasites, a n d  suffered h i g h e r m o r t a l i t y t h a n t h e i r L o w e r M a i n l a n d c o n s p e c i f i c s w h e n r e a r e d t o g e t h e r i n c a p t i v i t y . T h e r e w a s n o e v i d e n c e , h o w e v e r , that the O k a n a g a n b i r d s that b e c a m e i n f e c t e d c a m e f r o m nests w i t h h i g h e r l e v e l s o f in ovo D D T exposure.  A t 10 days o f age, r i g h t after c o l l e c t i o n  f r o m their respective nests, O k a n a g a n r o b i n s i n b o t h years h a d s i g n i f i c a n t l y l o w e r h e t e r o p h i l to l y m p h o c y t e ratios t h a n L o w e r M a i n l a n d r o b i n s .  In 1998, O k a n a g a n birds h a d significantly  h i g h e r p l a s m a t r i i o d o t h y r o n i n e l e v e l s than L o w e r M a i n l a n d b i r d s , but h e m a t o c r i t a n d t h y r o x i n e measurements  were  not  different  between  the  two  types  o f birds.  E x c e p t for  the  h e t e r o p h i l / l y m p h o c y t e ratios, n o n e o f the parameters m e a s u r e d at this age w e r e c o r r e l a t e d w i t h in ovo D D T l e v e l s . T h e c o r r e l a t i o n b e t w e e n 10-day h e t e r o p h i l / l y m p h o c y t e ratios a n d p , p ' - D D D a l t h o u g h significant, w a s m a r g i n a l (r = - 0.03). Injecting 2 or 4 m g / k g p , p ' - D D E into Japanese q u a i l (Coturnix  japonica)  c h i c k s h a t c h e d w i t h h i g h e r l e u k o c y t e n u m b e r s ( Q u i n n et a l . , 2 0 0 2 ) . caspia)  eggs resulted i n  C a s p i a n terns  (Sterna  f r o m the G r e a t L a k e s s h o w e d i n c r e a s i n g h e t e r o p h i l / l y m p h o c y t e ratios w i t h i n c r e a s i n g  D D E l e v e l s ( G r a s m a n et a l . , 1996).  C a m p b e l l (1994) c o n c l u d e d that i n g e n e r a l , a n excess o f  either e n d o g e n o u s or e x o g e n o u s g l u c o c o r t i c o i d c o u l d l e a d to s l i g h t to moderate l e u k o c y t o s i s , h e t e r o p h i l i a , a n d l y m p h o p e n i a , w h i c h w o u l d result i n h i g h e r h e t e r o p h i l / l y m p h o c y t e ratios. S m i t s  a n d W i l l i a m s (1999) f o u n d h i g h e r ratios i n 10-day o l d z e b r a f i n c h e s  126 (Taeniopygia guttata)  fed  dexamethasone (a synthetic g l u c o c o r t i c o i d a n d i m m u n o s u p p r e s s a n t ) c o m p a r e d to c o n t r o l s . T h e y f o u n d that stress due to h a n d l i n g i n c r e a s e d t o t a l l e u k o c r i t s , but d e x a m e t h a s o n e i n c r e a s e d the h e t e r o p h i l c o m p o n e n t w h i l e decreasing the l y m p h o c y t e c o m p o n e n t . I n the present study, w e d i d not h a v e 10-day o l d dexamethasone b i r d s to serve as c o n t r o l s , but 10-day o l d O k a n a g a n r o b i n s h a d s i g n i f i c a n t l y l o w e r h e t e r o p h i l / l y m p h o c y t e ratios as c o m p a r e d to L o w e r M a i n l a n d r o b i n s o f the same age. U n f o r t u n a t e l y , o n l y differential w h i t e b l o o d c e l l counts w e r e c o n d u c t e d , not total l e u k o c r i t analyses.  B y h e t e r o p h i l / l y m p h o c y t e ratio measurements, the i m m u n o c o m p e t e n c e o f  y o u n g O k a n a g a n nestlings d i d not seem to have been c o m p r o m i s e d b y in ovo D D T exposure. Jefferies a n d F r e n c h ( 1 9 6 9 ) reported that the t h y r o i d g l a n d s o f feral p i g e o n s  livia)  (Columbia  fed 3 to 36 m g D D T / k g / d a y for s i x w e e k s w e r e t w i c e as h e a v y as those o f c o n t r o l s .  The  t h y r o i d s o f d o s e d birds h a d s m a l l e r f o l l i c l e s , less c o l l o i d , and h y p e r p l a s t i c e p i t h e l i a , regardless o f the dose l e v e l . S i m i l a r results w e r e f o u n d i n b i r d s d o s e d w i t h D D E (Jefferies, 1975). H o w e v e r , c o n t a m i n a n t - i n d u c e d alterations i n t h y r o i d h o r m o n e s i n w i l d l i f e , e x p e r i m e n t a l a n d field studies have p r o d u c e d divergent findings (see r e v i e w b y R o l l a n d , 2 0 0 0 ) . F u r t h e r m o r e , the r e l a t i o n s h i p between thyroid hormones  and i m m u n e response  is mediated b y m a n y endogenous  and  e x o g e n o u s factors a n d m a y not be e a s i l y p r e d i c t a b l e ( F o w l e s et a l . , 1 9 9 7 ; E r f & M a r s h , 1 9 8 9 ; W i l l i a m s o n et a l . , 1990; S m i t s et a l . , 2 0 0 2 ) . I n general, a n i m a l s w i t h l o w t h y r o i d h o r m o n e l e v e l s demonstrated decreased c e l l - m e d i a t e d and h u m o r a l i m m u n e response ( K l e c h a et a l . , 2 0 0 0 ; S m i t s et a l . , 2 0 0 2 ) . I n the present study, 10-day o l d O k a n a g a n r o b i n s h a d s i g n i f i c a n t l y h i g h e r p l a s m a t r i i o d o t h y r o n i n e l e v e l s t h a n s a m e age L o w e r M a i n l a n d r o b i n s i n 1 9 9 8 w h i l e s h o w i n g n o difference i n p l a s m a thyroxine levels.  T h u s , there i s n o e v i d e n c e that the c e l l m e d i a t e d o r  h u m o r a l i m m u n e responses o f y o u n g O k a n a g a n nestlings have been c o m p r o m i s e d by l o w t h y r o i d h o r m o n e l e v e l s . I n fact, the O k a n a g a n r o b i n s m a y b e s h o w i n g m i l d h y p e r t h y r o i d i s m as w a s f o u n d p r e v i o u s l y i n p i g e o n s a n d B e n g a l e s e finches of  D D T dosages  (Jefferies,  1975).  (Lonchura striata)  Unfortunately,  because  e x p o s e d to a v a r i e t y  o f logistic reasons,  p h y t o h e m a g g l u t i n i n s k i n test w a s not p e r f o r m e d o n the b i r d s w h e n they w e r e  the  nestlings.  P h y t o h e m a g g l u t i n i n tests a d m i n i s t e r e d to j u v e n i l e s (1998) a n d adults (1997) r e v e a l e d that there w a s n o difference b e t w e e n the O k a n a g a n a n d L o w e r M a i n l a n d r o b i n s i n t h e i r T - l y m p h o c y t e m e d i a t e d i m m u n e response. N o s i g n i f i c a n t differences were f o u n d i n h e m a t o c r i t v a l u e s b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b r o o d s at ten days or 50 - 57 days, n o r w e r e these v a l u e s s i g n i f i c a n t l y different i n the d e x a m e t h a s o n e  controls.  I n S m i t s a n d W i l l i a m s ' ( 1 9 9 9 ) study, 10-day o l d z e b r a  finch  127 e x p o s e d to d e x a m e t h a s o n e also d i d not h a v e different h e m a t o c r i t v a l u e s t h a n c o n t r o l s .  These  authors c o m m e n t e d that h e m a t o p o i e s i s o f r e d c e l l s i n b i r d s is a v e r y active process d u r i n g the late e m b r y o n i c and e a r l y p o s t - h a t c h p e r i o d s a n d a f u l l c o m p l e m e n t o f r e d b l o o d c e l l s does not o c c u r u n t i l s o m e t i m e b e t w e e n m i d - n e s t l i n g a n d p o s t - f l e d g i n g stage. T h i s i s consistent w i t h o u r f i n d i n g that hematocrit values were c o n s i d e r a b l y h i g h e r w h e n the b i r d s w e r e j u v e n i l e s than w h e n they w e r e n e s t l i n g s .  Hematocrit values taken from j u v e n i l e Okanagan birds were negatively  correlated w i t h o r t h o - D D T isomers ( o , p ' - D D T and o , p ' - D D E ) but p o s i t i v e l y correlated w i t h a p a r a - D D T i s o m e r ( p , p ' - D D E ) . T h e s i g n i f i c a n c e o f these correlations r e m a i n s to be determined. I n i t i a l a n a l y s i s o f the stress response test o f j u v e n i l e r o b i n s r e v e a l e d that there w a s n o s i g n i f i c a n t difference b e t w e e n the O k a n a g a n a n d L o w e r M a i n l a n d b i r d s i n their c o r t i c o s t e r o n e response.  O n e O k a n a g a n female, w h i c h h a d a v e r y h i g h p l a s m a corticosterone l e v e l , w a s f o u n d  d e a d the d a y after the test a n d w a s also i d e n t i f i e d as a n o u t l i e r .  A f t e r the e x c l u s i o n o f this  i n d i v i d u a l f r o m the data set, it w a s f o u n d that L o w e r M a i n l a n d j u v e n i l e r o b i n s h a d a s i g n i f i c a n t l y l o w e r corticosterone response than O k a n a g a n j u v e n i l e s d u r i n g the first 5 m i n u t e s o f exposure to the stress s t i m u l u s . T h i s m a y be a n i n d i c a t i o n that L o w e r M a i n l a n d r o b i n s h a d h i g h e r baseline corticosterone levels than O k a n a g a n birds.  There was however, no difference i n their peak  response l e v e l as w e l l as the t i m e to achieve this l e v e l . T h e i n i t i a l c o r t i c o s t e r o n e response w a s p o s i t i v e l y c o r r e l a t e d w i t h m a n y o f the in ovo D D T isomers. Eastern bluebird  (Sialia sialis)  nestlings that w e r e restrained a n d then c h a l l e n g e d  w i t h adrenocorticotropic hormone exhibited negative correlations between corticosterone and D D E l e v e l s ( M a y n e et a l . , 2 0 0 2 ) . T h i s study, h o w e v e r , w a s d e a l i n g w i t h direct rather than in ovo D D T exposure.  T h e y w e r e also c o n c e r n e d w i t h the l e v e l o f corticosterone response rather than  the rate o f r e s p o n s e .  T h e findings i n this study seem to be n o v e l and deserve  further  investigation. W h i l e D D T s have b e e n s h o w n to i n f l u e n c e s e v e r a l aspects o f the i m m u n e s y s t e m i n a v a r i e t y o f s p e c i e s , i n c l u d i n g b i r d s , a n d in ovo e x p o s u r e s m a y r e s u l t i n m o r t a l i t y , r e d u c e d h a t c h a b i l i t y , w a s t i n g s y n d r o m e , s k e l e t a l a b n o r m a l i t i e s , a n d i m p a i r e d d i f f e r e n t i a t i o n o f the r e p r o d u c t i v e and nervous systems i n o f f s p r i n g ( F r y , 1995; Jefferies, 1 9 7 1 ; Banerjee et a l . , 1996). T h i s s t u d y , h o w e v e r , r e v e a l e d n o e v i d e n c e that i n c a p t i v e O k a n a g a n r o b i n s , the w a s t i n g s y n d r o m e , parasite i n f e c t i o n s , a n d m o r t a l i t y w e r e related to c o m p r o m i s e d i m m u n o c o m p e t e n c e due to in ovo D D T exposure. I n 1998, b l o o d l e a d l e v e l s w e r e a s s a y e d i n 10-day o l d r o b i n n e s t l i n g s a n d O k a n a g a n r o b i n s h a d s i g n i f i c a n t l y h i g h e r b l o o d l e a d than L o w e r M a i n l a n d b i r d s . L e a d has been s h o w n to  128 increase s u s c e p t i b i l i t y to i n f e c t i o u s agents a n d i m p a i r b o t h c e l l - m e d i a t e d a n d h u m o r a l i m m u n i t y ( G r a s m a n & S c a n l o n , 1995; L e e et a l . , 2 0 0 2 ) .  H o w e v e r , G r a s m a n and S c a n l o n (1995) also  c o n c l u d e d that i n Japanese q u a i l , l e a d suppressed a n t i b o d y - m e d i a t e d i m m u n i t y o n l y at dosages that also c a u s e d c l i n i c a l l e a d p o i s o n i n g . F a i r a n d R i c k l e f s ( 2 0 0 2 ) , c o m b i n i n g d o s i n g l e a d w i t h a n i m m u n o l o g i c a l c h a l l e n g e , a l s o f o u n d that l e a d d i d not affect a n t i b o d y p r o d u c t i o n or c e l l m e d i a t e d i m m u n e response i n Japanese q u a i l .  I n the r o b i n study, b l o o d l e a d l e v e l s w e r e not  c o r r e l a t e d w i t h w h i t e b l o o d c e l l ratios, h e m a t o c r i t s , or t h y r o i d h o r m o n e l e v e l s . T h e r e w a s also n o r e l a t i o n s h i p b e t w e e n b l o o d l e a d l e v e l a n d parasite/disease i n f e c t i o n . T h e r e f o r e , there is n o e v i d e n c e i n this study that b l o o d l e a d l e v e l s f o u n d i n the O k a n a g a n b i r d s w a s affecting t h e i r immunocompetence. B y e l i m i n a t i n g alternative h y p o t h e s e s , this study strengthened the n o t i o n put f o r t h i n C h a p t e r III, that L o w e r M a i n l a n d a n d O k a n a g a n r o b i n s are g e n e t i c a l l y different, a n d that the d i f f e r e n c e b e t w e e n the t w o i n d i s e a s e / p a r a s i t e  s u s c e p t i b i l i t y w a s m a i n l y b e c a u s e o f the  adaptability o f L o w e r M a i n l a n d r o b i n s to l o c a l parasite and disease vectors.  129  5.5 References  Banerjee, B . D . (1999). T h e i n f l u e n c e o f v a r i o u s factors o n i m m u n e t o x i c i t y assessment o f pesticide c h e m i c a l s . Banerjee,  Toxicology Letters, 107,  21-31.  B . D . , Koner, B . C , R a y , A . (1996). Immunotoxicity o f pesticides:  Perspectives and trends.  Indian Journal of Experimental Biology, 34,  723-733.  B l u s , L . J . (1996). D D T , D D D , and D D E i n birds. In W . N . Beyer, G . H . H e i n z , and A . W . 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W i l l i a m s o n , R . A . , D a v i s o n , T . F . , & P a y n e , L . N . ( 1 9 9 0 ) . Effects o f t h y r o i d h o r m o n e s o n h u m o r a l a n d c e l l - m e d i a t e d i m m u n e r e s p o n s e i n the d o m e s t i c f o w l  Developmental and Comparative Immunology, 14, W H O . (1989).  (Gallus domesticus).  305-318.  DDT and its derivatives - Environmental Aspects  C r i t e r i a N o . 83). W o r l d H e a l t h O r g a n i z a t i o n .  (Environmental Healt  134 Chapter V I General Discussion  T h e g o a l o f this study  w a s to e x a m i n e  whether  in ovo  and early exposure  to  d i c h l o r o d i p h e n y l t r i c h l o r o e t h a n e ( D D T ) have a n y d e l a y e d o r l o n g - t e r m effects o n the g r o w t h a n d s u r v i v a l , i m m u n e response, b e h a v i o r , r e p r o d u c t i v e success, a n d stress r e s p o n s e o f A m e r i c a n robins  (Turdus migratorius). T h i s w a s done b y c o m p a r i n g c o n t a m i n a t e d O k a n a g a n b i r d s w i t h  uncontaminated L o w e r M a i n l a n d controls, and correlating contamination levels o f Okanagan eggs w i t h a n u m b e r o f parameters i n their c l u t c h mate(s). T h e d e s i g n o f the study w a s based o n three a s s u m p t i o n s :  1) that the O k a n a g a n a n d L o w e r M a i n l a n d b i r d s w e r e o f s i m i l a r genetic  b a c k g r o u n d , 2) the l e v e l o f c o n t a m i n a t i o n i n a l l the eggs i n a c l u t c h w a s s i m i l a r , a n d 3) a m o n g O k a n a g a n b i r d s , parental care and post-hatch D D T exposure ( f r o m h a t c h i n g to about ten-days o f age) w e r e not significant variables. A l t h o u g h A m e r i c a n r o b i n s were once raised as pets ( H o w e l l , 1942) a n d are used for short t e r m c a p t i v e studies (e.g. W e l l e h a n et a l , 2 0 0 1 ; R i c h t e r et a l , 2 0 0 0 ; L e v e y & K a r a s o v , 1992; W o n g & D e s s e r , 1978), this study is the first to c o n d u c t l o n g t e r m o b s e r v a t i o n s a n d l i k e l y the first to report large-scale successful b r e e d i n g o f these b i r d s i n c a p t i v i t y , d e m o n s t r a t i n g that the A m e r i c a n r o b i n c a n be u t i l i z e d as a n o n - d o m e s t i c a t e d passerine m o d e l for l o n g t e r m , m u l t i generation t o x i c o l o g y studies.  6.1 E g g Contaminants  A m e r i c a n r o b i n eggs f r o m o r c h a r d areas i n the O k a n a g a n V a l l e y o f B r i t i s h C o l u m b i a c o n t a i n h i g h l e v e l s o f D D T a n d its m e t a b o l i t e s ( E l l i o t t et a l . , 1 9 9 4 ; G i l l et a l . , 2 0 0 3 ) .  Eggs  c o l l e c t e d f r o m the O k a n a g a n for t h i s study i n 1 9 9 7 a n d 1998 d e m o n s t r a t e d total D D T l e v e l s r a n g i n g f r o m 5.71 to 2 7 7 . 6 2 u g / g , as c o m p a r e d to l e v e l s o f 0.36 to 3.39 p.g/g i n eggs c o l l e c t e d from c o n t r o l areas o f the B r i t i s h C o l u m b i a L o w e r M a i n l a n d .  T h e m a j o r i t y o f the c o n t a m i n a n t  burden  from  found  in  Okanagan  robin  eggs  comes  the  metabolite  p,p'-  d i c h l o r o d i p h e n y l d i c h l o r o e t h y l e n e ( D D E ) , but d i c h l o r o d i p h e n y l d i c h l o r o e t h a n e ( D D D ) , the other m a i n m e t a b o l i t e o f D D T , w a s also f o u n d .  P a r a , p a r a ' - D D E is c o n s i d e r e d the f i n a l b r e a k d o w n  p r o d u c t o f D D T i n l i v i n g b i r d s ( P o l a n d et a l . , 1972), a n d it tends to be lost quite s l o w l y ( S t i c k e l , 1973).  O r t h o , p a r a ' i s o m e r s w e r e not f o u n d i n a n y o f the e g g s c o l l e c t e d f r o m the L o w e r  135 M a i n l a n d , a n d o n l y trace amounts w e r e f o u n d i n the eggs from the O k a n a g a n . Ortho,para' - D D T c o m p o s e s o n l y 10 to 3 0 % o f t e c h n i c a l grade D D T (Jefferies, 1 9 7 5 ; M e l l a n b y , 1 9 9 2 ; W H O , 1989) a n d it tends to be stored less r e a d i l y a n d e l i m i n a t e d m o r e q u i c k l y t h a n p , p ' - D D T ( S t i c k e l , 1973).  A s r o b i n s l i k e l y get the b u l k o f their c o n t a m i n a n t l o a d s f r o m the foods they eat, the  u n e q u a l concentrations o f the v a r i o u s i s o m e r s a n d m e t a b o l i t e s m a y also b e i n f l u e n c e d b y D D T m e t a b o l i s m w i t h i n their invertebrate p r e y species. E a r t h w o r m s , i n particular, have b e e n s h o w n to p l a y a r o l e i n the i n i t i a l d e g r a d a t i o n o f D D T b y c o n v e r t i n g it to D D E a n d D D D ( E d w a r d s & Jeffs, 1974; G i s h & H u g h e s , 1982). T h e s e differences i n the l e v e l s o f the v a r i o u s f o r m s o f D D T are i m p o r t a n t as different i s o m e r s a n d m e t a b o l i t e s h a v e b e e n s h o w n to have different  effects  ( S o h o n i & Sumpter, 1998; S o n n e n s h e i n & Soto, 1998). A s expected, r o b i n eggs f r o m orchards i n the O k a n a g a n h a d s i g n i f i c a n t l y h i g h e r l e v e l s o f D D T s than those c o l l e c t e d f r o m reference sites w i t h i n the L o w e r M a i n l a n d . T h e residue l e v e l s f o u n d i n these eggs w e r e s u r p r i s i n g l y h i g h c o n s i d e r i n g D D T has not b e e n u s e d i n the O k a n a g a n for a p p r o x i m a t e l y t h i r t y years, but w e r e s i m i l a r to l e v e l s f o u n d i n other studies o f O k a n a g a n b i r d s ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 2 ; H a r r i s et a l . , 2 0 0 0 ) . A l s o s u r p r i s i n g , w e r e the l o w D D E to D D T ratios f o u n d i n the eggs from b o t h the O k a n a g a n a n d the L o w e r M a i n l a n d .  These  ratios c o n f i r m the v i e w that D D T degrades v e r y s l o w l y ( C o l b o r n et a l . , 1 9 9 3 ; M u r p h y , 1 9 8 0 ; R e p e t t o & B a l i g a , 1996). T h e O k a n a g a n l i k e l y serves as a D D T s i n k or hot-spot due to h e a v y h i s t o r i c a l use ( E l l i o t t et a l . , 1994; G i l l et a l . , 2 0 0 3 ; H a r r i s , 2 0 0 0 ) . It has b e e n p r e d i c t e d that ratios s h o u l d e x c e e d 20:1 f o l l o w i n g 15 to 2 0 years w i t h o u t D D T a p p l i c a t i o n ( E l l i o t t et a l . , 1994), but this w a s not f o u n d i n the present study. A l t h o u g h the highest l e v e l s o f c o n t a m i n a t i o n i n the r o b i n eggs c a m e f r o m D D T s , these b i r d s w e r e also e x p o s e d to a v a r i e t y o f other current-use a n d h i s t o r i c a l c h e m i c a l s . F o r e x a m p l e , l e a d arsenate w a s u s e d e x t e n s i v e l y as a p e s t i c i d e p r i o r to the i n t r o d u c t i o n o f D D T .  O n e o f the  w a y s l e a d exerts its t o x i c effects is b y a c t i n g as a p o r p h y r i n o g e n i c agent c a u s i n g d e f e c t i v e h e m o g l o b i n i z a t i o n and anemia ( B u n y a n & Stanley, 1982).  T h e organochlorines, chlordane,  heptachlor, m i r e x , a l d r i n , a n d d i e l d r i n were banned m a n y years ago but s t i l l s h o w u p i n soils a n d a n i m a l tissues.  O t h e r o r g a n o c h l o r i n e s s u c h as e n d o s u l f a n , d i c o f o l , l i n d a n e , a n d m e t h o x y c h l o r  are c u r r e n t l y i n use ( C r i s p et a l . , 1998). A p p e n d i c e s I a n d II list the o r g a n o c h l o r i n e c h e m i c a l s e v a l u a t e d for this study.  A s i d e from the D D T s , the o n l y o r g a n o c h l o r i n e s f o u n d at a p p r e c i a b l e  l e v e l s i n the r o b i n e g g s w e r e (TCPM).  Cis-  and  cis-  tomy-nonachlor  and  tr an s-nonachlor  and tris(4-chlorophenyl)methanol  are c o m p o n e n t s o f the c y c l o d i e n e p e s t i c i d e c h l o r d a n e .  T C P M i s a r e l a t i v e l y n e w l y d i s c o v e r e d o r g a n o c h l o r i n e that has b e e n f o u n d i n a n u m b e r o f  136 species.  Its o r i g i n s are not clear, h o w e v e r , it is b e l i e v e d to be a n i m p u r i t y i n D D T (de B o e r ,  2 0 0 0 ) . A n u m b e r o f o r g a n o c h l o r i n e pesticides h a v e been l i n k e d to i n c r e a s e d m o r t a l i t y , r e d u c e d r e p r o d u c t i v e s u c c e s s , a l t e r e d i m m u n e f u n c t i o n i n g , a n d o t h e r d e t r i m e n t a l effects  in both  laboratory a n d w i l d l i f e studies (Banerjee et a l . , 1996; B a r n e t t & R o d g e r s , 1994; B l u s & H e n n y , 1997; M u r p h y , 1980; S t i c k e l , 1973; Street, 1 9 8 1 ; T h o m a s , 1975; V o c c i a e t a l . , 1999). A l t h o u g h not i n t e n t i o n a l l y released into the e n v i r o n m e n t , p o l y c h l o r i n a t e d b i p h e n y l s are u b i q u i t o u s contaminants that have been s h o w n to h a v e a n u m b e r o f d e t r i m e n t a l effects o n b i r d s a n d other a n i m a l s .  P o l y c h l o r i n a t e d b i p h e n y l s c a n i n f l u e n c e t h y r o i d g l a n d s a n d h o r m o n e s , the  i m m u n e s y s t e m , the t h y m u s , the l i v e r , t h y r o i d f u n c t i o n i n g , a n d a v a r i e t y o f other  systems  ( B u n y a n & Stanley, 1982; C h e e k et a l . , 1999; F e r n i e et a l . , 2 0 0 1 ; H o f f m a n et a l . , 1996; Jefferies, 1975). T h e most t o x i c p o l y c h l o r i n a t e d b i p h e n y l congeners are p o l y c h l o r i n a t e d b i p h e n y l 126, 7 7 , a n d 169 ( H o f f m a n et a l . , 1996). A n u m b e r o f p o l y c h l o r i n a t e d b i p h e n y l congeners, as w e l l as the p o l y c h l o r i n a t e d b i p h e n y l m i x t u r e A r o c l o r 1260 w e r e m e a s u r e d i n the r o b i n eggs c o l l e c t e d for t h i s study ( A p p e n d i c e s I a n d II).  A l t h o u g h n o n - D D T organochlorines and polychlorinated  b i p h e n y l s were f o u n d i n l o w l e v e l s , it cannot be r u l e d out that some o f these c h e m i c a l s m a y exert a n effect at v e r y l o w c o n c e n t r a t i o n s and/or they m a y interact w i t h the D D T s ( F o s s i , 1 9 9 8 ; S t i c k e l , 1973). T h e m a j o r i t y o f i n s e c t i c i d e s u s e d today are o r g a n o p h o s p h a t e s a n d carbamates. B o t h o f these c h e m i c a l classes act b y i n h i b i t i n g c h o l i n e s t e r a s e a c t i v i t y ( B u n y a n & S t a n l e y , 1 9 8 2 ) . A l t h o u g h these c h e m i c a l s are m u c h less persistent than the o r g a n o c h l o r i n e s , m a n y o f t h e m are c o n s i d e r e d h i g h l y t o x i c , w i t h LD50 l e v e l s less t h a n 2 5 m g / k g i n several species o f b i r d s ( F l u e t s c h & S p a r l i n g , 1994; M u r p h y , 1980).  C h o l i n e s t e r a s e i n h i b i t i o n greater than 2 0 % is i n d i c a t i v e o f  exposure, w h i l e i n h i b i t i o n greater than 5 0 % c a n be lethal ( G i l l et a l . , 2 0 0 0 ) . A s these c h e m i c a l s are often a p p l i e d d u r i n g the b r e e d i n g season they c a n r e d u c e r e p r o d u c t i o n a n d r e c r u i t m e n t b y i n c r e a s i n g m o r t a l i t y , a l t e r i n g adult b e h a v i o r , or d e c r e a s i n g f o o d s u p p l i e s ( F l u e t s c h & S p a r l i n g , 1 9 9 4 ; G i l l et a l . , 2 0 0 0 ; G r a h a m & D e s G r a n g e s , 1 9 9 3 ; P a t n o d e & W h i t e , 1 9 9 1 ; R o n d e a u & D e s G r a n g e s , 1995; T h o m a s , 1975).  T h e y m a y also i n f l u e n c e the i m m u n e s y s t e m (Banerjee et  a l . , 1996; B a r n e t t & R o d g e r s , 1994; Street, 1 9 8 1 ; V o c c i a et a l . , 1999), the t h y r o i d g l a n d a n d its f u n c t i o n i n g ( B i s h o p , V a n D e r K r a a k et a l . , 1998; M a y n e et a l . , 2 0 0 2 ) , a n d other  systems.  A l t h o u g h not m e a s u r e d i n this study, the o r g a n o p h o s p h a t e s a z i n p h o s - m e t h y l ( G u t h i o n ) a n d d i a z i n o n , a n d the c a r b a m a t e c a r b a r y l ( S e v i n ) , a m o n g others, w e r e s p r a y e d i n the O k a n a g a n orchards f r o m w h i c h the r o b i n eggs a n d n e s t l i n g s w e r e c o l l e c t e d .  E g g s a n d c h i c k s w e r e not  137 c o l l e c t e d o n s p r a y i n g days, but m a y have been e x p o s e d to these pesticides j u s t p r i o r to c o l l e c t i o n ( L . W i l s o n , personal communication). B i r d s a n d other a n i m a l s are rarely e x p o s e d to a s i n g l e c h e m i c a l , but rather m i x t u r e s o f t h e m ( B i s h o p , B o e r m a n s et a l . , 1998; M c A r t h u r et a l . , 1 9 8 3 ; T y l e r et a l . , 1998; V o s et a l , 2 0 0 0 ) . A v a r i e t y o f other substances w e r e also s p r a y e d i n the O k a n a g a n orchards, s u c h as d o r m a n t o i l , f e r t i l i z e r s , g r o w t h h o r m o n e s , a n d h e r b i c i d e s . A l l o f these c o u l d h a v e p o t e n t i a l i m p a c t s o n the r o b i n s , either d i r e c t l y ( t o x i c i t y ) or i n d i r e c t l y (reduced f o o d s u p p l y or c o v e r ) .  6.2 Similarities and Differences Between the Lower Mainland and Okanagan Robins D u r i n g the c o u r s e o f the s t u d y , m a n y differences b e t w e e n the O k a n a g a n a n d L o w e r M a i n l a n d r o b i n s were o b s e r v e d (see T a b l e 6-1 for s u m m a r y ) . B e c a u s e not a l l o f the parameters w e r e m e a s u r e d i n the same i n d i v i d u a l s a n d at the same age, a n d because o f s m a l l s a m p l e sizes (e.g., for m a n y parameters, c l u t c h or b r o o d w e r e u s e d to a v o i d p s u e d o - r e p l i c a t i o n ) , it w a s often not p o s s i b l e to integrate the in o v o D D T l e v e l s into a r e g r e s s i o n a n a l y s i s . T h u s , c o r r e l a t i o n a l analyses u s i n g the i n d i v i d u a l D D T i s o m e r s w e r e c o n d u c t e d . T h e s e analyses w e r e b a s e d o n the a s s u m p t i o n that the l e v e l o f c o n t a m i n a t i o n i n a l l the eggs i n a c l u t c h w a s s i m i l a r .  W h i l e most  p r e v i o u s studies also f o l l o w e d this a s s u m p t i o n , there h a v e b e e n suggestions that c o n t a m i n a n t l o a d s m a y v a r y b e t w e e n eggs w i t h i n a c l u t c h ( O h l e n d o r f et a l . , 1 9 8 5 ; O t t i n g e r et a l . , 2 0 0 1 ) . A l t h o u g h o c c a s i o n a l l y m o r e t h a n e g g w a s c o l l e c t e d f r o m a nest f o r this study, c o n t a m i n a n t analyses were o n l y c o n d u c t e d o n one egg per nest. Therefore, c o m p a r i s o n s w i t h i n a c l u t c h were not p o s s i b l e . S i g n i f i c a n t correlations are surnrnarized i n T a b l e 6-2.  6.2.1 E g g and C h i c k M e a s u r e m e n t s A l t h o u g h a l l o f the eggs c o l l e c t e d for this study w e r e w i t h i n the size ranges d e s c r i b e d b y H o w e l l ( 1 9 4 2 ) , f e m a l e s f r o m the L o w e r M a i n l a n d l a i d h e a v i e r a n d w i d e r eggs t h a n t h e i r O k a n a g a n counterparts.  A m o n g the O k a n a g a n females, egg size w a s p o s i t i v e l y correlated w i t h  their in o v o D D T e x p o s u r e .  T h e o c c u r r e n c e o f larger eggs b e i n g l a i d b y m o r e c o n t a m i n a t e d  females m a y be related to the estrogenic effects ( W i l l i a m s , 1999) o f v a r i o u s D D T s ( F r y , 1 9 9 5 ; S o h o n i & S u m p t e r , 1998; T y l e r , 1998), h o w e v e r , this does not a c c o u n t for the fact that L o w e r M a i n l a n d females, as a w h o l e , l a i d e v e n larger eggs. T h e r e are a n u m b e r o f factors that c a n p l a y  138 Table 6-1:  Significant differences found between Lower Mainland and Okanagan birds  and eggs in 1997 and 1998.  Chapter III  IV  V  Parameter  Effect  o,p' isomers  f o u n d o n l y i n O k a n a g a n eggs  egg l i p i d l e v e l s  L o w e r Mainland > Okanagan  10 day m i d d l e toe length  L o w e r Mainland > Okanagan  b o d y w e i g h t at 2 m o n t h s  Okanagan > L o w e r Mainland  b o d y w e i g h t at 7-9 months  L o w e r Mainland > Okanagan  tarsus length at 2 m o n t h s  L o w e r Mainland > Okanagan  tarsus l e n g t h at 5 m o n t h s  L o w e r Mainland > Okanagan  w h i t e b l o o d c e l l ratios at 10 days  L o w e r Mainland > Okanagan  c o c c i d i o s i s infections  Okanagan > L o w e r Mainland  j u v e n i l e deaths  Okanagan > L o w e r Mainland  heart w e i g h t at sacrifice  Okanagan > L o w e r Mainland  males fledging chicks  L o w e r Mainland > Okanagan  egg w e i g h t  L o w e r M a i n l a n d females > O k a n a g a n females  egg w i d t h  L o w e r M a i n l a n d females > O k a n a g a n females  offspring tarsus l e n g t h at 5 days  O k a n a g a n females > L o w e r M a i n l a n d females  o f f s p r i n g w i n g length at 5 days  O k a n a g a n females > L o w e r M a i n l a n d females  o f f s p r i n g deaths at n e s t l i n g stage  L o w e r M a i n l a n d females > O k a n a g a n females  o f f s p r i n g b o d y w e i g h t at sacrifice  same type parents > different type parents  offspring b r a i n w e i g h t at sacrifice  different type parents > same type parents  offspring k i d n e y w e i g h t  O k a n a g a n x O k a n a g a n parents heaviest  offspring l i v e r w e i g h t  O k a n a g a n x O k a n a g a n parents heaviest  drinking behavior  Okanagan > L o w e r Mainland  preening behavior  same type pairs > different type pairs  chukking vocalizations  different type pairs > same type pairs  egg D D T l e v e l s  Okanagan > L o w e r Mainland  10 day t r i i o d o t h y r o n i n e l e v e l s  Okanagan > L o w e r Mainland  10 day w h i t e b l o o d c e l l ratios  L o w e r Mainland > Okanagan  corticosterone l e v e l s t i m e 0  L o w e r Mainland > Okanagan  139 corticosterone levels t i m e 5  L o w e r Mainland > Okanagan  blood lead levels  Okanagan > Lower Mainland  m y c o p l a s m o s i s infections  Okanagan > Lower Mainland  t a p e w o r m s a n d nematodes  Okanagan > Lower Mainland  m i d d l e toe l e n g t h 10 days  L o w e r Mainland > Okanagan  l i v e r w e i g h t at sacrifice  Okanagan > Lower Mainland  k i d n e y w e i g h t at sacrifice  Okanagan > Lower Mainland  140  Table 6-2: Significant correlations between Okanagan egg contaminant levels and other parameters.  DDT  Direction of  Isomer/Metabolite  Parameter  Correlation  Chapter III o,p'-DDD  egg l i p i d levels  negative  p,p'-DDE  10 d a y b o d y w e i g h t  positive  p,p'-DDT  post-breeding w h i t e b l o o d c e l l ratios  positive  p,p'-DDE  g o n a d w e i g h t at sacrifice  negative  o,p'-DDT  o v i d u c t w e i g h t at sacrifice  positive  p,p'-DDT  egg w e i g h t  positive  p,p'-DDD  egg length  positive  p,p'-DDE  egg length  positive  p,p'-DDT  egg w i d t h  positive  o,p'-DDT  egg w i d t h  positive  p,p'-DDT  t r i i o d o t h y r o n i n e l e v e l s , p e r i o d 12  negative  o,p*-DDT  thyroxine levels, period 4  positive  o,p'-DDD  thyroxine levels, period 4  positive  o,p'-DDT  thyroxine levels, period 6  negative  p,p'-DDE  snapping  positive  p,p'-DDD  overall vocalizations  positive  p,p'-DDT  laughing  negative  o,p'-DDT  "other" vocalizations  positive  o,p'-DDD  "other" v o c a l i z a t i o n s  positive  o,p'-DDE  "other" v o c a l i z a t i o n s  positive  p,p'-DDE  eating  positive  p,p'-DDT  flying  (frequency)  positive  p,p'-DDE  flying  (frequency)  positive  p,p'-DDT  f l y i n g (proportion)  positive  p,p'-DDE  f l y i n g (proportion)  positive  Chapter IV  141 Chapter V o,p'-DDT  j u v e n i l e hematocrits  positive  o,p'-DDE  j u v e n i l e hematocrits  positive  p,p*-DDE  j u v e n i l e hematocrits  negative  p,p'-DDD  10 day w h i t e b l o o d c e l l ratios  negative  p,p'-DDT  post-breeding w h i t e b l o o d c e l l ratios  positive  p,p'-DDT  corticosterone l e v e l s , times 5 a n d 10  positive  o,p'-DDT  corticosterone l e v e l s , times 5 a n d 10  positive  o,p»-DDE  corticosterone l e v e l s , times 5 a n d 10  positive  p,p'-DDE  10 day b o d y w e i g h t  positive  p,p'-DDE  10 day w i n g length  negative  p,p'-DDE  g a l l bladder w e i g h t at sacrifice  negative  142 a r o l e i n egg s i z e i n c l u d i n g genetics, f e m a l e c o n d i t i o n ( S t y r s k y et a l . , 2 0 0 2 ) , a n d i n d i v i d u a l differences ( W i l l i a m s , 1999). F e w differences w e r e f o u n d i n the m o r p h o m e t r i c m e a s u r e m e n t s o f the O k a n a g a n a n d L o w e r M a i n l a n d r o b i n s a n d their o f f s p r i n g . O k a n a g a n c h i c k s c o l l e c t e d i n 1997 h a d s i g n i f i c a n t l y shorter m i d d l e toes than the 1997 L o w e r M a i n l a n d b i r d s a n d b o t h groups c o l l e c t e d i n 1998, a n d the y o u n g o f O k a n a g a n females h a d l o n g e r tarsi a n d w i n g s at f i v e - d a y s - o f - a g e .  N o n e o f these  differences persisted into a d u l t h o o d . W h i l e s o m e o f this d i s c r e p a n c y m a y be due to changes i n m e a s u r i n g p r a c t i c e s or i n the p e o p l e c o n d u c t i n g the m e a s u r e m e n t s ,  it is a l s o p o s s i b l e that  e n v i r o n m e n t a l c o n d i t i o n s or parental care p l a y e d a r o l e . B o d y w e i g h t s a n d w i n g measurements w e r e s i m i l a r to those r e p o r t e d b y H o w e l l ( 1 9 4 2 ) , a l t h o u g h tarsus lengths i n this study w e r e s l i g h t l y larger. T h e m a j o r i t y o f studies do not report toe lengths, so the s i g n i f i c a n c e o f this size difference is not clear. It is p o s s i b l e that in ovo D D T exposure i n f l u e n c e d the g r o w t h o f the O k a n a g a n c h i c k s . F o r e x a m p l e , b o d y w e i g h t at ten days o f age w a s p o s i t i v e l y c o r r e l a t e d w i t h p , p ' - D D E e g g l e v e l s a n d w i n g l e n g t h w a s n e g a t i v e l y c o r r e l a t e d w i t h p , p ' - D D D i n the 1997 O k a n a g a n b i r d s .  These  b i r d s also s h o w e d a d e l a y i n their tarsus g r o w t h r e l a t i v e to the L o w e r M a i n l a n d c o n t r o l s . It i s not p o s s i b l e , unfortunately, to separate the effects o f c o n t a m i n a n t exposure f r o m that o f parental care, genetics, a n d other factors, so one c a n not be certain w h a t r o l e D D T p l a y e d i n the g r o w t h o f these b i r d s . O v e r a l l , the m a l e to f e m a l e ratio o f the c h i c k s c o l l e c t e d i n 1997 a n d 1998 w a s 0.76 for the L o w e r M a i n l a n d b i r d s a n d 1.15 for the O k a n a g a n b i r d s , w h i c h is not a s i g n i f i c a n t difference. T h e r e w e r e also n o s i g n i f i c a n t differences i n the sex ratios o f the o f f s p r i n g o f the 1997 b i r d s . L o w e r M a i n l a n d females h a d a m a l e to female ratio o f 0.81 whereas O k a n a g a n females s h o w e d a 1.56 ratio. S e x ratios i n the w i l d are not w i d e l y reported ( S a l l a b a n k s & James, 1999), h o w e v e r , Y o u n g ( 1 9 5 5 ) s u g g e s t e d that the average is 1:1.  A l t h o u g h D D T may feminize and D D E  d e m a s c u l i n i z e b i r d s , this w a s l i k e l y not a n issue i n this study as m o s t o f the c h i c k s w e r e s e x e d g e n e t i c a l l y a n d the f e m i n i z i n g / d e m a s c u l i n i z i n g effects o f D D T a n d D D E m o s t l i k e l y act o n s e x u a l differentiation o f the b r a i n , gonads, a n d secondary s e x u a l characteristics, not o n the genes t h e m s e l v e s ( M c L a c h l a n & A r n o l d , 1996). C o m p l e t e sex r e v e r s a l has also not b e e n w i t n e s s e d i n any b i r d species studied ( F r y & T o o n e , 1981).  143 6.2.2 B e h a v i o r a n d R e p r o d u c t i o n I n C h a p t e r III it w a s r e p o r t e d that the 1997 O k a n a g a n a n d L o w e r M a i n l a n d b i r d s d i s p l a y e d n o significant differences i n their a b i l i t i e s to b u i l d nests, l a y eggs, h a t c h eggs, or fledge chicks.  T h e t i m i n g o f r e p r o d u c t i v e events w a s a l s o s i m i l a r b e t w e e n the t w o g r o u p s a n d  p r e v i o u s l y s t u d i e d b i r d s ( G i l l et a l . , 2 0 0 3 ; K e m p e r , 1971).  T h e r e p r o d u c t i v e success o f these  r o b i n s i m p r o v e d d r a m a t i c a l l y d u r i n g t h e i r s e c o n d b r e e d i n g season.  T h i s m a y be due to  experience, changes i n the pens, different mates, and/or e n v i r o n m e n t a l c o n d i t i o n s . F e w differences were f o u n d i n the b e h a v i o r s o f the 1997 L o w e r M a i n l a n d a n d O k a n a g a n birds.  O k a n a g a n birds drank more frequently than L o w e r M a i n l a n d birds, same type pairs  ( L o w e r M a i n l a n d x L o w e r M a i n l a n d , O k a n a g a n x O k a n a g a n ) p r e e n e d m o r e than different type p a i r s ( L o w e r M a i n l a n d x O k a n a g a n , O k a n a g a n x L o w e r M a i n l a n d ) , a n d different type p a i r s exhibited more c h u k k i n g v o c a l i z a t i o n s than same type pairs. differences, h o w e v e r , is unclear.  T h e i m p o r t a n c e o f these  W h i l e d r i n k i n g a n d p r e e n i n g h a v e o b v i o u s s u r v i v a l benefits,  the purpose o f c h u k k i n g has not b e e n w e l l established. It is also not k n o w n i f e x c e s s i v e d i s p l a y s o f these b e h a v i o r s i n d i c a t e b o r e d o m or frustration, or a c t u a l l y represent d e h y d r a t i o n , feather parasites, or other c o n d i t i o n s . D e s p i t e the fact that there w e r e f e w d i f f e r e n c e s b e t w e e n the t w o g r o u p s , w i t h i n the O k a n a g a n b i r d s several b e h a v i o r s w e r e s i g n i f i c a n t l y correlated w i t h in ovo D D T exposure.  The  fact that s o m e o f the m o s t h i g h l y c o n t a m i n a t e d b i r d s demonstrated h i g h frequencies o f c e r t a i n b e h a v i o r s (e.g., nest b u i l d i n g , m o u n t i n g ) suggests that there m a y be t h r e s h o l d l e v e l s b e l o w w h i c h r o b i n b e h a v i o r is not affected.  T h e s e thresholds l i k e l y v a r y d e p e n d i n g o n sex, age, the  b e h a v i o r tested, a n d e v e n i n d i v i d u a l differences.  I n order to test this, one w o u l d n e e d sufficient  s a m p l e sizes o f birds w i t h s i m i l a r contaminant l e v e l s or treat b i r d s or eggs w i t h D D T s i n order to e x a m i n e dose-response curves.  6.2.3 H o r m o n e s A l t h o u g h O k a n a g a n b i r d s h a d h i g h e r t r i i o d o t h y r o n i n e l e v e l s t h a n their L o w e r M a i n l a n d counterparts at ten days o f age, no s i g n i f i c a n t differences i n p l a s m a t r i i o d o t h y r o n i n e or t h y r o x i n e w e r e f o u n d at any other age tested. T h e r e w e r e also n o s i g n i f i c a n t correlations w i t h in ovo D D T e x p o s u r e for the O k a n a g a n c h i c k s . T h e O k a n a g a n c h i c k s m a y h a v e b e e n e x p e r i e n c i n g a m i l d h y p e r t h y r o i d i s m , as seen i n other species e x p o s e d to a v a r i e t y o f D D T dosages (Jefferies, 1975). A l t h o u g h t h y r o i d h o r m o n e s p l a y a r o l e i n g r o w t h ( B o l a n d e r , 1994; N e l s o n , 2 0 0 0 ; S i n g h et a l . , 1968) a n d i m m u n i t y (Jefferies, 1975), a m o n g other processes, these w e r e n o t s y s t e m a t i c a l l y tested.  T h e r e w e r e , h o w e v e r , s i g n i f i c a n t differences across the testing p e r i o d s , w h i c h is not  144 s u r p r i s i n g g i v e n that t h y r o i d h o r m o n e l e v e l s c a n v a r y d r a m a t i c a l l y o v e r t i m e ( W e n t w o r t h & R i n g e r , 1986). C o r t i c o s t e r o n e l e v e l s w e r e evaluated i n the 1998 b i r d s d u r i n g a restraint stress c h a l l e n g e . P l a s m a l e v e l s o f corticosterone were s i g n i f i c a n t l y l o w e r i n the O k a n a g a n b i r d s than i n the L o w e r M a i n l a n d b i r d s d u r i n g the f i r s t f i v e m i n u t e s  o f the test.  T h i s s u g g e s t s that  c o r t i c o s t e r o n e c o n c e n t r a t i o n s w e r e l o w e r i n the O k a n a g a n b i r d s .  B o t h groups,  baseline however,  d i s p l a y e d s i m i l a r peaks i n corticosterone at 30 minutes. It is not clear w h y the O k a n a g a n b i r d s h a d l o w e r c o r t i c o s t e r o n e l e v e l s at the b e g i n n i n g o f the test. d i f f e r e n c e s or p e r h a p s D D T e x p o s u r e .  It m a y be r e l a t e d to g e n e t i c  C o r t i c o s t e r o n e l e v e l s at five a n d ten m i n u t e s w e r e  p o s i t i v e l y correlated w i t h b o t h p , p ' - D D T a n d o , p ' - D D T exposure.  H o w m u c h o f the m e a s u r e d  corticosterone is b o u n d , rather than free, is also not k n o w n a n d c o u l d be i n f l u e n c i n g these g r o u p differences.  6.2.4 I m m u n i t y Immune  response  w a s tested u s i n g d i f f e r e n t i a l w h i t e b l o o d c e l l c o u n t s  p h y t o h e m a g g l u t t i n i n s k i n test.  and  the  A t t e n d a y s o f age, L o w e r M a i n l a n d c h i c k s h a d h i g h e r  e o s i n o p h i l / h e t e r o p h i l to l y m p h o c y t e / m o n o c y t e ratios than O k a n a g a n b i r d s . A s these ratios w e r e n e g a t i v e l y c o r r e l a t e d w i t h p , p ' - D D D l e v e l s , it is p o s s i b l e that in ovo e x p o s u r e p l a y e d a r o l e i n this effect.  W h e t h e r o r not the O k a n a g a n b i r d s w e r e at a d i s a d v a n t a g e i n terms o f i m m u n e  response c a n not be determined f r o m this test, as l y m p h o c y t e and h e t e r o p h i l n u m b e r s increase i n response to different i n f e c t i o u s agents ( S i e g e l , 1980). T h e h i g h e r l e v e l s o f l y m p h o c y t e s i n the O k a n a g a n b i r d s m a y h a v e m a d e t h e m m o r e resistant to v i r a l i n f e c t i o n s but m o r e susceptible to bacterial invasion.  T h e h e t e r o p h i l to l y m p h o c y t e ratio q u a n t i f i e s the b a l a n c e b e t w e e n  the  n o n s p e c i f i c , fast-acting defenses o f heterophils a n d the a n t i g e n - s p e c i f i c , s l o w e r a c t i n g defenses o f l y m p h o c y t e s ( G r a s m a n et a l . , 1996). N o significant differences i n w h i t e b l o o d c e l l ratios w e r e f o u n d b e t w e e n the b i r d s w h e n they w e r e tested as j u v e n i l e s or adults. T h e r e w e r e , nonetheless, d i f f e r e n c e s b e t w e e n the different t e s t i n g p e r i o d s .  B o t h j u v e n i l e s and adults  demonstrated  d r a m a t i c a l l y l o w e r w h i t e b l o o d c e l l ratios than they d i d as n e s t l i n g s . T h e s e age differences are l i k e l y the result o f n o r m a l changes i n the i m m u n e s y s t e m ( S m i t s & W i l l i a m s , 1999; S t u r k i e & G r i m i n g e r , 1986). T h e p h y t o h e m a g g l u t i n i n s k i n test reflects a c o m p l e x series o f p h y s i o l o g i c a l events ( G r a s m a n et a l . , 1996; L o c h m i l l e r et a l . , 1993). T h e d e l a y e d (24 h o u r ) response tested here is due to a l o c a l i n f l u x o f T c e l l s r e c r u i t i n g i n f l a m m a t o r y c e l l s to the c h a l l e n g e site ( P a r m e n t i e r et  145 a l , 1998; S m i t s et a l . , 1999). A l t h o u g h G r a s m a n et a l . ( 1 9 9 6 ) f o u n d a decrease i n w i n g i n d e x measurements w i t h i n c r e a s i n g D D E l e v e l s i n g u l l s a n d terns, the r o b i n s i n this study s h o w e d n o differences i n response, despite their h i g h l e v e l s o f D D E e x p o s u r e .  T h i s w a s true b o t h o f the  1998 j u v e n i l e s a n d the 1997 adult m a l e s . It i s p o s s i b l e that in ovo c o n t a m i n a t i o n h a d n o effect o n T l y m p h o c y t e m e d i a t e d i m m u n i t y i n these b i r d s . H o w e v e r , as dexamethasone treatment d i d not i n f l u e n c e the p h y t o h e m a g g l u t i n i n response o f the 1998 p o s i t i v e c o n t r o l s , it is p o s s i b l e that this particular i m m u n e test is not appropriate for r o b i n s . H e m a t o c r i t v a l u e s w e r e not s i g n i f i c a n t l y different b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s w h e n they w e r e n e s t l i n g s o r j u v e n i l e s .  V a l u e s were, however, significantly  h i g h e r i n the b i r d s w h e n they w e r e j u v e n i l e s t h a n w h e n they w e r e t e n - d a y - o l d n e s t l i n g s . S i g n i f i c a n t c o r r e l a t i o n s w i t h e g g c o n t a m i n a n t s w e r e f o u n d i n the b i r d s as j u v e n i l e s , w h i c h suggest that D D T exposure has the potential to i m p a c t p a c k e d r e d b l o o d c e l l v o l u m e s . O k a n a g a n r o b i n s appeared to be far m o r e susceptible to infectious disease than the L o w e r M a i n l a n d birds.  I n 1997, 13 O k a n a g a n b i r d s d i e d as a result o f s t a r v a t i o n , l i k e l y due to the  i n t e s t i n a l c o c c i d i o s i s parasites  Eimeria  and  Isospora.  N o n e o f the L o w e r M a i n l a n d b i r d s ,  h o u s e d under the same c o n d i t i o n s , w e r e affected. T w o o f the infected birds were s h o w n to carry p r o t o z o a n b l o o d parasites  (Leucocytozoon).  b i r d s w e r e treated for i n f e c t i o n s c a u s e d b y  synoviae.  I n 1998, eight L o w e r M a i n l a n d a n d 4 6 O k a n a g a n  Mycoplasma gallisepticum  and/or  Mycoplasma  O n e L o w e r M a i n l a n d and four O k a n a g a n birds w e r e suspected o f h a v i n g a s p e r g i l l o s i s  (Aspergillus fumigatus),  a n d t a p e w o r m s a n d u n i d e n t i f i e d n e m a t o d e s w e r e d i s c o v e r e d i n several  birds, e s p e c i a l l y those f r o m the O k a n a g a n . C a p t i v e b i r d s are prone to a n u m b e r o f diseases a n d parasites, a n d one w o u l d expect that a l l the birds w o u l d be e q u a l l y susceptible. It is p o s s i b l e that O k a n a g a n b i r d s s u c c u m b e d to these infections m o r e r e a d i l y because their early D D T exposure c o m p r o m i s e d their i m m u n e systems i n s o m e w a y that w a s not r e a d i l y apparent g i v e n the tests u s e d i n this study.  G e n e t i c differences  m a y also have p l a y e d a r o l e . A s the birds were a l l r a i s e d i n the L o w e r M a i n l a n d , it m a y be that the O k a n a g a n b i r d s d i d not have the a b i l i t y to cope w i t h the p a r t i c u l a r infectious agents f o u n d i n this area. T h e L o w e r M a i n l a n d b i r d s , i n contrast, m a y h a v e d e v e l o p e d i m m u n i t i e s to t h e m o v e r successive generations.  P e r h a p s i f the b i r d s h a d b e e n r a i s e d i n the O k a n a g a n , the L o w e r  M a i n l a n d birds w o u l d h a v e been m o r e susceptible. A s a l l o f the b i r d s w e r e j u v e n i l e s at the t i m e o f i n f e c t i o n , they m a y have been less able to m o u n t adequate i m m u n e responses than i f they h a d b e c o m e infected as adults. It is not k n o w n i f these infections or the B a y t r i l treatments i n f l u e n c e d other aspects o f the study.  146  6.2.5 M o r t a l i t y a n d T i s s u e W e i g h t s C a u s e o f death for m a n y o f the b i r d s i n this study w a s not d e t e r m i n e d . N e a r l y 3 7 % o f the b i r d s that w e r e c o l l e c t e d i n 1997 s u r v i v e d u n t i l the t i m e o f s a c r i f i c e i n 2 0 0 0 , whereas the rest d i e d as a result o f accidents, depredation, disease ( c o c c i d i o s i s ) , or u n k n o w n causes. R a t s  norvegicus)  (Rattus  w e r e the m a i n predators i n the r o b i n pens, a n d a c c i d e n t a l deaths i n c l u d e d d r o w n i n g s  i n water dishes, c o m p l i c a t i o n s d u r i n g b l o o d s a m p l i n g , a n d a v a r i e t y o f other misfortunes. T h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s i n cause a n d age o f d e a t h b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s , l i k e l y due to the s t a r v a t i o n deaths o f j u v e n i l e O k a n a g a n b i r d s . In ovo D D T l e v e l s d i d not s e e m to i n f l u e n c e either cause o r age o f death. T h e b i r d s c o l l e c t e d i n 1998 w e r e s a c r i f i c e d less t h a n s i x m o n t h s after b e i n g t a k e n into c a p t i v i t y , so it is not s u r p r i s i n g that m o r e t h a n 9 0 % o f t h e m s u r v i v e d to the t i m e o f s a c r i f i c e . T h r e e o f the b i r d s i n this group d i e d o f u n k n o w n causes, four w e r e e u t h a n i z e d due to d e b i l i t a t i n g injuries, a n d one O k a n a g a n b i r d is b e l i e v e d to have d i e d as a result o f a m y c o p l a s m a i n f e c t i o n . A m o n g the o f f s p r i n g h a t c h e d b y the 1997 b i r d s , m o r e L o w e r M a i n l a n d f e m a l e s t h a n O k a n a g a n females h a d c h i c k s die w i t h i n the first t w o w e e k s post-hatch. A g a i n , cause o f death w a s often not k n o w n .  C h i c k s that d i s a p p e a r e d w e r e l i k e l y depredated b y rats.  demonstrated bruises and wounds.  Several chicks  A l t h o u g h p r e d a t o r s m a y h a v e i n f l i c t e d these i n j u r i e s ,  predators w o u l d m o r e l i k e l y c a r r y the c h i c k a w a y a n d eat it. O n e m a l e w a s w i t n e s s e d s h a k i n g a n d b i t i n g h i s o f f s p r i n g , a n d some females w e r e seen p e c k i n g at y o u n g i n the nest. T h u s , at least s o m e o f these injuries m a y have b e e n due to parental a g g r e s s i o n . It i s not k n o w n for sure h o w m a n y o f the injuries w e r e i n c u r r e d p r i o r to as o p p o s e d to after death n o r w h y the parents b e h a v e d so a g g r e s s i v e l y . H e a r t w e i g h t s , a n d to s o m e extent l i v e r w e i g h t s , w e r e h i g h e r i n 1997 O k a n a g a n b i r d s t h a n i n the L o w e r M a i n l a n d b i r d s .  T h e gonads o f O k a n a g a n birds from heavily p , p ' - D D E  c o n t a m i n a t e d nests w e r e l i g h t e r t h a n those from less c o n t a m i n a t e d nests, whereas o v i d u c t s w e r e h e a v i e r i n b i r d s f r o m nests w i t h h i g h l e v e l s o f o , p ' - D D T .  A s p , p ' - D D E is k n o w n to act as a n  a n t i - a n d r o g e n ( G a i d o et a l . , 1997; K e l c e et a l . , 1 9 9 5 ; 1998), it is not s u r p r i s i n g that h i g h l e v e l s w o u l d suppress g o n a d size, at least i n m a l e s . A l t e r e d g o n a d a l m o r p h o l o g y has b e e n s h o w n i n g u l l s a n d c h i c k e n s e x p o s e d in ovo o r d u r i n g d e v e l o p m e n t to D D T s ( B a l a s u b r a m a n i a m & Sundararaj,  1993; Burlington & L i n d e m a n ,  1950; F r y & Toone, 1981; Stickel,  1973).  O r t h o , p a r a ' - D D T a n d other f o r m s o f D D T , o n the other h a n d , are estrogen agonists a n d h a v e  147 b e e n s h o w n to i n f l u e n c e the female r e p r o d u c t i v e s y s t e m i n a n u m b e r o f species ( F r y & T o o n e , 1 9 8 1 ; G a i d o et a l . , 1997; M c L a c h l a n & A r n o l d , 1996; S t i c k e l , 1973). T h e l i v e r s a n d k i d n e y s o f the 1998 O k a n a g a n b i r d s w e r e heavier than those o f the L o w e r M a i n l a n d birds.  W h i l e e n l a r g e d hearts a n d l i v e r s h a v e b e e n l i n k e d to the effects o f D D T o n  t h y r o i d g l a n d f u n c t i o n i n g (Jefferies, 1975), n o s i g n i f i c a n t differences w e r e f o u n d i n t h y r o i d g l a n d w e i g h t s . T h e r e c o u l d , h o w e v e r , have b e e n m o r p h o l o g i c a l differences i n the t h y r o i d glands that w e r e not e x a m i n e d . A l t h o u g h heavier hearts, l i v e r s , a n d k i d n e y s i n the O k a n a g a n b i r d s m a y represent genetic differences b e t w e e n the t w o groups o f b i r d s , the b i r d s c o l l e c t e d i n 1997 d i d not e x h i b i t the same a s y m m e t r i e s as the 1998 b i r d s . A g e m a y p l a y a role i n this y e a r effect, as the 1997 birds were s a c r i f i c e d as adults but the 1998 b i r d s w e r e s a c r i f i c e d as j u v e n i l e s . A m o n g the o f f s p r i n g o f the 1997 b i r d s , the y o u n g o f O k a n a g a n parents tended to h a v e h e a v i e r k i d n e y s than those o f L o w e r M a i n l a n d or m i x e d t y p e parents.  S a m e t y p e parents h a d  y o u n g w i t h h e a v i e r b o d y w e i g h t s at the t i m e o f s a c r i f i c e , w h e r e a s different t y p e parents h a d y o u n g w i t h heavier brains.  A l t h o u g h these results suggest p a r e n t a l i n f l u e n c e s o n o f f s p r i n g  p h y s i o l o g y l o n g after the b i r d s have b e c o m e independent a n d s e c o n d generation effects o f early c o n t a m i n a n t e x p o s u r e , the s a m p l e s i z e w a s quite s m a l l a n d i n c l u d e d s i b l i n g s . T h u s , n o c l e a r c o n c l u s i o n s c a n be d r a w n .  6 . 3 Problems  L i k e a l l research, this study h a d its share o f d i f f i c u l t i e s .  A l t h o u g h not a l l factors c a n be  c o n t r o l l e d i n a n experiment, there are some things that c o u l d perhaps have b e e n done differently a n d s o m e things that c o u l d not be h e l p e d . F o r e x a m p l e , the b i r d s i n this study w e r e e x p o s e d to a v a r i e t y o f c h e m i c a l s i n a d d i t i o n to D D T . It w o u l d be v i r t u a l l y i m p o s s i b l e to f i n d a w i l d species that w a s not p r e v i o u s l y e x p o s e d to pesticide c o n t a m i n a t i o n to s o m e extent. C o l l e c t i n g eggs a n d c h i c k s f r o m nests i n the w i l d i s a t i m e - c o n s u m i n g a n d c o s t l y enterprise.  T h e s e p r o b l e m s w e r e exacerbated b y the fact that p e a k e g g l a y i n g p e r i o d s do not  o c c u r at the same t i m e i n the O k a n a g a n a n d L o w e r M a i n l a n d ( C a m p b e l l et a l . , 1997).  Sample  sizes m a y not be e q u a l a n d it m a y not be p o s s i b l e to a l w a y s get b o t h an egg a n d c h i c k s from the same nests as a result o f t i m i n g p r o b l e m s , d i f f i c u l t y  finding  the nests, a n d d e p r e d a t i o n o f nests.  A l t h o u g h it w o u l d have b e e n n i c e to a n a l y z e c o n t a m i n a n t l e v e l s i n a l l o f the eggs i n d i v i d u a l l y , this w a s not p o s s i b l e due to the h i g h costs i n v o l v e d a n d the fact that v e r y l o w l e v e l s o f D D T s w e r e expected to be f o u n d i n the L o w e r M a i n l a n d eggs.  148 Inconsistencies b e t w e e n years m a y h a v e p l a y e d a r o l e i n s o m e o f the r e p r o d u c t i o n a n d b e h a v i o r results f o u n d i n this study. N o t o n l y w e r e the 1 9 9 7 b i r d s i n e x p e r i e n c e d d u r i n g their first b r e e d i n g season, so w a s the experimenter. T h u s , s o m e o f the b e h a v i o r a l s c o r i n g m a y have c h a n g e d s l i g h t l y b e t w e e n years.  T h e pens u n d e r w e n t m a j o r r e n o v a t i o n s p r i o r to the s e c o n d  b r e e d i n g season i n c l u d i n g the e l i m i n a t i o n o f the i n d o o r pens, i m p r o v e m e n t s to the o u t d o o r pens to ensure m o r e c o n s i s t e n c y b e t w e e n pens, a n d h i g h e r v i g i l a n c e against rats.  Environmental  effects s u c h as temperature a n d w e a t h e r c o n d i t i o n s m a y h a v e i n f l u e n c e d the o u t c o m e o f this study, but they w e r e not taken into a c c o u n t as b o t h L o w e r M a i n l a n d a n d O k a n a g a n b i r d s w o u l d h a v e b e e n e x p o s e d to the same c o n d i t i o n s . T h e o r i g i n a l intent o f this study w a s to e x a m i n e the effects o f D D T as a n e n d o c r i n e disruptor.  U n f o r t u n a t e l y , there w a s a m i s c o m m u n i c a t i o n w i t h the l a b o r a t o r y c o n d u c t i n g the  h o r m o n e assays a n d the p l a s m a s a m p l e s w e r e a n a l y z e d f o r t h y r o i d h o r m o n e s rather estradiol a n d testosterone.  than  W h i l e this o p e n e d a n e x c i t i n g n e w avenue for research, it r e q u i r e d a  dramatic shift i n the focus o f the study. G i v e n s o m e o f the differences seen i n r e p r o d u c t i o n a n d b e h a v i o r , it w o u l d have been interesting to relate t h e m to differences i n g o n a d a l h o r m o n e l e v e l s . T h e m a i n p r o b l e m o f c o n c e r n i n this study is the p o s s i b i l i t y that g e n e t i c d i f f e r e n c e s b e t w e e n the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s m a y o v e r s h a d o w a n y effects e a r l y D D T e x p o s u r e m a y h a v e h a d . It is l i k e l y that the L o w e r M a i n l a n d a n d O k a n a g a n b i r d s represent t w o different subspecies o f A m e r i c a n r o b i n ( A l d r i c h & James, 1 9 9 1 ; S a l l a b a n k s & James, 1999). W h i l e t h i s m a y b e a n arbitrary d i s t i n c t i o n , b a s e d o n g e o g r a p h i c a l l o c a t i o n a n d differences i n morphometric measurements  a n d p l u m a g e ( S a l l a b a n k s & J a m e s , 1 9 9 9 ) , it c o u l d p o t e n t i a l l y  account for m a n y o f the differences seen b e t w e e n the t w o groups. It w o u l d be interesting to see h o w c l o s e l y related the t w o groups are g e n e t i c a l l y . I n order to a v o i d this p r o b l e m , one w o u l d h a v e to dose r e l a t i v e l y c l e a n L o w e r M a i n l a n d b i r d s w i t h D D T or use o n l y b i r d s f r o m the Okanagan.  E l l i o t t et a l . ( 1 9 9 4 ) c o m p a r e d b i r d s f r o m c o n v e n t i o n a l l y m a n a g e d  Okanagan  o r c h a r d s that h a d b e e n treated w i t h D D T to ones f r o m o r g a n i c o r c h a r d s that h a d not b e e n e x p o s e d to pesticides for at least f i v e years. T h e present study, h o w e v e r , does demonstrate that the t w o sub-species c a n inter-breed.  6.4 Implications  D e s p i t e its s h o r t c o m i n g s , this study p r o v i d e d v a l u a b l e i n s i g h t s into the effects o f D D T s o n a m o d e l passerine.  T h e effects o f direct e x p o s u r e to D D T , i n its v a r i o u s f o r m s , h a v e b e e n  149 b r o a d l y studied, b u t studies o n the m o r e l o n g - t e r m , latent effects as seen i n second-generations have been sparse.  T h i s study i n d i c a t e d that l o n g - t e r m , latent effects d o exist but m a y b e subtle,  a n d m a y p r i m a r i l y b e seen i n h i g h l y c o n t a m i n a t e d b i r d s .  A l t h o u g h it is not clear h o w m u c h  i n f l u e n c e g e n e t i c s h a d o n t h e f i n d i n g s p r e s e n t e d h e r e , b a s e d o n the n u m b e r o f s i g n i f i c a n t correlations w i t h in ovo D D T exposure, i t i s o b v i o u s that D D T s have the p o t e n t i a l to p l a y a role i n several aspects o f these b i r d s ' l i v e s . T h e r o b i n s i n this study are interesting i n that they w e r e e x p o s e d n o t o n l y to in ovo c o n t a m i n a t i o n v i a the e g g y o l k , b u t a l s o d i r e c t l y t h r o u g h t h e i r i n g e s t i o n o f c o n t a m i n a t e d e a r t h w o r m s d u r i n g their first t e n d a y s post-hatch. demonstrate b o t h l o n g - t e r m effects, a n d as c h i c k s , r e l a t i v e l y i m m e d i a t e effects.  T h u s , they m a y The long-term  effects o f D D T e x p o s u r e m a y r e l y o n a n u m b e r o f different m e c h a n i s m s o r p a t h w a y s . may  exert  their  influences  through  their  alterations  o f enzymes,  genes,  DDTs  hormones,  neurotransmitters, and other c e l l s , r e s u l t i n g i n changes i n the g r o w t h a n d d e v e l o p m e n t o f organs, tissues, a n d g l a n d s . other f u n c t i o n s .  T h e s e changes, i n t u r n , m a y affect s u r v i v a l , r e p r o d u c t i o n , b e h a v i o r , a n d  A l t e r a t i o n s i n o n e s y s t e m c a n h a v e p r o f o u n d effects o n other  systems.  Therefore, i t i s u n l i k e l y that the effects early D D T exposure h a d o n these b i r d s c a n b e attributed to particular m e c h a n i s m s . O n a p o p u l a t i o n l e v e l , A m e r i c a n robins f r o m the O k a n a g a n do not appear to be d e t r i m e n t a l l y affected b y e a r l y D D T e x p o s u r e . their h i g h levels o f contamination.  T h e y c o n t i n u e to t h r i v e a n d r e p r o d u c e despite  A f t e r several generations o f D D T e x p o s u r e , r o b i n s i n the  O k a n a g a n m a y h a v e d e v e l o p e d a tolerance o r resistance to the effects o f D D T , s i m i l a r to that seen i n other species ( W H O , 1989).  T h i s a b i l i t y to a c c u m u l a t e D D T s , h o w e v e r , m a k e s these  b i r d s p o t e n t i a l sources o f secondary p o i s o n i n g for their predators, i n m u c h the same w a y as the e a r t h w o r m s they t h e m s e l v e s c o n s u m e .  T h i s i s e s p e c i a l l y p r o b l e m a t i c f o r susceptible r a p t o r i a l  species s u c h as the p e r e g r i n e f a l c o n (Falco peregrinus; D e W e e s e et a l . , 1986; M o r a , 1 9 9 7 ) . D D T l e v e l s as l o w as 1 u g / g c a n be detrimental to peregrines ( M o r a , 1997), a n d l e v e l s f o u n d i n r o b i n s a n d other p r e y species are often c o n s i d e r a b l y h i g h e r than that ( B l u s , 1996; H a r r i s et a l . , 2000) A n u m b e r o f questions have arisen f r o m this study that warrant further i n v e s t i g a t i o n . F o r e x a m p l e , i t w o u l d b e w o r t h w h i l e to see i f the c o l l e c t e d tissues f r o m these b i r d s s t i l l c o n t a i n detectable l e v e l s o f D D T s . It has been suggested that i n s o m e species it c a n take u p to 988 days for 9 5 % o f a bird's b o d y b u r d e n o f D D T to b e e l i m i n a t e d ( S t i c k e l et a l . , 1984). It w o u l d also b e interesting to d e t e r m i n e i f D D T e x p o s u r e d u r i n g d e v e l o p m e n t i n f l u e n c e d the h i s t o l o g y o f the g o n a d s , b r a i n , t h y r o i d , a n d a d r e n a l g l a n d s , as these are a l l p o t e n t i a l targets o f D D T a n d  150 a l t e r a t i o n s i n t h e i r d e v e l o p m e n t c a n h a v e p r o f o u n d effects o n the a n i m a l t h r o u g h o u t  life  ( B i e s m a n n & v o n F a b e r , 1 9 8 1 ; F r y & T o o n e , 1 9 8 1 ; Jefferies, 1975; K e l c e et a l . , 1998; L a t i m e r & S i e g e l , 1974; L o r e n z e n et a l . , 1999; P a r m i g i a n i et a l . , 1998; S p y k e r , 1975). D o s i n g studies c o u l d be e m p l o y e d i n order to determine i f there are, i n fact, t h r e s h o l d l e v e l s a b o v e w h i c h r o b i n b e h a v i o r , r e p r o d u c t i o n , a n d other functions are d e t r i m e n t a l l y affected.  D o s i n g the c o n t r o l b i r d s  w i t h D D T / D D E or f e e d i n g t h e m e a r t h w o r m s f r o m the O k a n a g a n w o u l d demonstrate w h e t h e r a l l r o b i n s are s o m e w h a t i m m u n e to the effects o f D D T s or i f this is a trait f o u n d o n l y i n b i r d s that have been e x p o s e d o v e r several decades. R a i s i n g the b i r d s i n c a p t i v i t y a l l o w e d for the measurement o f a n u m b e r o f parameters that w o u l d h a v e b e e n v e r y d i f f i c u l t to study i n the f i e l d .  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T . , D e s s e r , S. S. ( 1 9 7 8 ) . U l t r a s t r u c t u r a l o b s e r v a t i o n s o n renal s c h i z o g o n y o f L e u c o c y t o z o o n d u b r e u i l i i n the A m e r i c a n r o b i n . J o u r n a l o f P r o t o z o o l o g y , 25(3 P t 2), 3 0 2 - 1 4 . W H O . (1989).  DDT and its derivatives - Environmental Aspects  (Environmental Healt  C r i t e r i a N o . 83). W o r l d H e a l t h O r g a n i z a t i o n . Y o u n g , H . ( 1 9 5 5 ) . B r e e d i n g b e h a v i o r a n d n e s t i n g o f the E a s t e r n R o b i n .  Midland Naturalist, 53(2),  329-352.  The American  158  Appendix I  M e a n o r g a n o c h l o r i n e a n d p o l y c h l o r i n a t e d b i p h e n y l ( P C B ) l e v e l s (p.g/g, w e t w e i g h t ) f o u n d i n A m e r i c a n r o b i n eggs c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r M a i n l a n d i n 1997.  Okanagan  Lower Mainland  n = 31  n =3  1,2,4,5-tetrachIorobenzene  < 0.00010(0)  < 0.00010(0)  1,2,3,4-tetrachlorobenzene  < 0.00010(0)  < 0.00010(0)  pentachlorobenzene  < 0.00010(0)  < 0.00010(0)  a-hexachlorocyclohexane  < 0.00010(0)  < 0.00010(0)  8- hexachlorocyclohexane  < 0.00010(0)  < 0.00010(0)  y- hexachlorocyclohexane  < 0.00010(0)  < 0.00010(0)  0.00010(1)  0.00053 (2)  < 0.00010(0)  < 0.00010(0)  heptachlor epoxide  0.00033 (3)  0.0024 (3)  oxychlordane  0.00086 (2)  0.0030 (1)  trans-chlordane  < 0.00010(0)  < 0.00010(0)  cis-chlordane  < 0.00010(0)  < 0.00010(0)  0.0026(11)  0.0022 (3)  0.0026 (8)  0.0090 (2)  dieldrin  < 0.00010(0)  < 0.00010(0)  photomirex  < 0.00010(0)  < 0.00010(0)  mirex  < 0.00010(0)  < 0.00010(0)  0.055 (15)  0 . 0 0 0 7 0 (1)  p,p'-DDT  12.08 (31)  0.14(3)  p,p'-DDE  51.66 (31)  1.89 (3)  p,p'-DDD  1.01 (31)  0.0090 (3)  o,p'-DDT*  0.049 (23)  < 0.00010(0)  o,p'-DDE*  0.0024 (14)  < 0.00010(0)  +  hexachlorobenzene octachlorostyrene  trans-nonachlor cis-nonachlor  tris (4-chlorophenyl) methanol  1  159 o,p'-DDD*  0.0025 (15)  < 0.00010(0)  64.87  2.05  0.00029 (3)  < 0.00010(0)  0.0018(2)  0.0016(1)  pentachlorobiphenyls  0.021 (31)  0.020 (3)  hexachlorobiphenyls  0.043 (31)  0.058 (3)  heptachlorobiphenyls  0.032 (31)  0.048 (3)  octachlorobiphenyls  0.011 (15)  0.011 (3)  0.0040 (4)  < 0.00010(0)  0.11  0.14  0.10(30)  0.092 (3)  total organochlorines trichlorobiphenyls  1  tetrachlorobiphenyls  2  3  4  5  6  nonachlorobiphenyls  7  total PCBs Aroclor 1260 T  N u m b e r s in brackets represent the number of samples containing each chemical at levels greater  than 0.00010 pg/g *non-detects replaced with zeros in order to determine means 'trichlorobiphenyls = P C B 16/32, 17, 18, 2 2 , 2 8 , 3 1 , 3 3 / 2 0 tetrachlorobiphenyls = P C B 4 2 , 4 4 , 4 7 / 4 8 , 4 9 , 5 2 , 5 6 / 6 0 , 6 4 , 6 6 , 7 0 / 7 6 , 7 4  2  pentachlorobiphenyls = P C B 8 5 , 8 7 , 9 2 , 9 5 , 9 7 , 9 9 , 101/90, 105, 110, 118  3  Vxachlorobiphenyls = P C B 128, 130, 137, 138, 141, 146, 149, 151, 153, 156, 157, 158 heptachlorobiphenyls = P C B 170/190, 1 7 1 , 172, 174, 176, 177, 178, 179, 180, 183, 187  5  Octachlorobiphenyls = P C B 194, 195, 196/203, 2 0 0 , 2 0 1 , 2 0 2 'nonachlorobiphenyls = P C B 206, 207, 208  160  A p p e n d i x II  M e a n organochlorine and polychlorinated biphenyl ( P C B ) levels (jig/g, wet weight) found i n A m e r i c a n r o b i n eggs c o l l e c t e d f r o m the O k a n a g a n a n d L o w e r M a i n l a n d i n 1998.  Okanagan n =  t  31  Lower Mainland n =  3  1,2,4,5-tetrachlorobenzene  < 0.00010(0)  < 0.00010(0)  1,2,3,4-tetrachlorobenzene  < 0.00010(0)  < 0.00010(0)  pentachlorobenzene  < 0.00010(0)  < 0.00010(0)  a-hexachlorocyclohexane  < 0.00010(0)  < 0.00010(0)  (3- h e x a c h l o r o c y c l o h e x a n e  < 0.00010(0)  < 0.00010(0)  y - hexachlorocyclohexane  < 0.00010(0)  < 0.00010(0)  0.00045 (8)  0.00088 (2)  < 0.00010(0)  0.00033 (1)  0.00060 (8)  0.073 (1)  0.0010 (5)  0.0019(1)  trans-chlordane  < 0.00010(0)  < 0.00010(0)  cis-chlordane  < 0.00010(0)  < 0.00010(0)  trans-nonachlor  0.0023 (17)  0.0015 (4)  cis-nonachlor  0.00014 (3)  0.0011 (1)  dieldrin  < 0.00010(0)  < 0.00010(0)  photomirex  < 0.00010(0)  0.0043 (1)  mirex  < 0.00010(0)  0.011 (1)  0.042 (19)  0.00085 (2)  p,p'-DDT  6.91 (22)  0.071 (4)  p,p»-DDE  2 6 . 2 2 (22)  0.85 (4)  p,p'-DDD  0.66 (22)  0.0063 (4)  o,p'-DDT*  0.046 (20)  < 0.00010(0)  o,p'-DDE*  0.0040(19)  < 0.00010(0)  hexachlorobenzene octachlorostyrene heptachlor epoxide oxychlordane  tris (4-chlorophenyl) methanol  1  161  o,p'-DDD*  0.0043 (15)  < 0,00010(0)  33.89  0.95  < 0.00010(0)  < 0.00010(0)  < 0.00010(0)  < 0.00010(0)  pentachlorobiphenyls  0.0046 (22)  0.0070 (4)  hexachlorobiphenyls  0.0097 (22)  0.028 (4)  0.0064 (22)  0.016 (4)  0.0013 (8)  0.0047 (4)  < 0.00010(0)  < 0.00010(0)  0.022  0.055  0.020 (21)  0.039 (4)  total organochlorines trichlorobiphenyls  1  tetrachlorobiphenyls  2  3  4  heptachlorobiphenyls  5  octachlorobiphenyls  6  nonachlorobiphenyls  7  total PCBs Aroclor 1260  lumbers in brackets represent the number of samples containing each chemical at levels greater than 0.00010 pg/g *non-detects replaced with zeros in order to determine means 'trichlorobiphenyls = P C B 16/32, 17, 18, 2 2 , 2 8 , 3 1 , 3 3 / 2 0 tetrachlorobiphenyls = P C B 4 2 , 4 4 , 4 7 / 4 8 , 4 9 , 5 2 , 5 6 / 6 0 , 64, 6 6 , 70/76, 7 4  pentachlorobiphenyls = P C B 8 5 , 87, 9 2 , 9 5 , 9 7 , 9 9 , 101/90, 105, 110, 118  3  hexachlorobiphenyls = P C B 128, 130, 137, 138, 141, 146, 149, 151, 153, 156, 157, 158 'heptachlorobiphenyls = P C B 1 7 0 / 1 9 0 , 1 7 1 , 1 7 2 , 1 7 4 , 1 7 6 , 1 7 7 , 1 7 8 , 1 7 9 , 1 8 0 , 1 8 3 , 1 8 7 Octachlorobiphenyls = P C B 194, 195, 196/203, 2 0 0 , 2 0 1 , 2 0 2 'nonachlorobiphenyls = P C B 206, 207, 208  

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