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The risk of non-target species poisoning from brodifacoum used to eradicate rats from Langara Island,… Howald, Gregory Robert 1997

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T H E RISK O F N O N - T A R G E T BRODIFACOUM LANGARA  S P E C I E S POISONING F R O M  USED TO ERADICATE  RATS FROM  I S L A N D , BRITISH C O L U M B I A ,  CANADA  by  GREGORY  ROBERT  HOWALD  B.Sc. (Agr.)(Hons.), University of British Columbia, Vancouver, 1993.  A T H E S I S IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S THE D E G R E E OF M A S T E R OF S C I E N C E in THE FACULTY OF GRADUATE STUDIES Department of A n i m a l S c i e n c e We accept this thesis as conforming to the required standard  The University of British Columbia  August, 1997  © Gregory Robert Howald  FOR  In  presenting this  degree at the  thesis  in  partial  fulfilment  of  the requirements  University  of  British  Columbia,  I agree that the Library shall make it  ;  freely available for reference and study. I further agree that copying  of  department  this thesis for scholarly or  by. his  or  her  for  It  is  understood  head of my  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of  AoiflAL ^  c  ICpOL  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  OCTO&E& 6  /^  advanced  permission for-extensive  purposes may be granted by the  representatives.  an  copying  or  my written  ^  Abstract In 1995, the C a n a d i a n Wildlife S e r v i c e attempted to eradicate introduced  N o r w a y rats (Rattus norvegicus)  from L a n g a r a Island and adjacent C o x a n d L u c y  Islands with the application of the s e c o n d generation anticoagulant rodenticide b r o d i f a c o u m . However, anticoagulant rodenticides are n o n - s p e c i e s specific pesticides a n d p o s e a poisoning risk to non-target s p e c i e s . T h i s t h e s i s a d d r e s s e s the short term poisoning impacts to non-target s p e c i e s from brodifacoum u s e d to e r a d i c a t e the rats from L a n g a r a and L u c y Island. In 1994, during testing of the baiting protocol o n L u c y Island, the native d u s k y s h r e w (Sorex monticolus  elassodon)  population s i z e fell from an estimated 2 5  unique s h r e w s / h a before the baiting to four unique shrews/ha after the baiting. T h i s prompted a monitoring program in three regions o n L a n g a r a Island in 1 9 9 5 . W h i l e s h r e w s w e r e attracted to bait in stations, the decline in their population w a s n o n significant. S h r e w s in breeding condition w e r e at greater risk of poisoning likely d u e to their ability to range widely. S h r e w s entered and c h e w e d o n bait blocks in up to 8 0 % of bait stations. T h e risk of s e c o n d a r y poisoning to avian s c a v e n g e r s from p o i s o n e d toxic rat c a r c a s s e s w a s investigated. In 1994, c o m m o n r a v e n s (Corvus  corax) w e r e  identified a s the most significant s c a v e n g e r of rat c a r c a s s e s . In 1995, two of 15 radio-collared N o r w a y rats p o i s o n e d with brodifacoum died a b o v e g r o u n d a n d o n e w a s s c a v e n g e d . W h o l e body brodifacoum r e s i d u e s from other rats found d e a d a b o v e g r o u n d ranged from 2.40-16.51 mg/kg. B e t w e e n 1995 a n d 1996, 2 0 raven  ii  r e m a i n s w e r e found or reported. In 1995, 13 raven livers tested positive for b r o d i f a c o u m . R a v e n s w e r e secondarily p o i s o n e d from s c a v e n g i n g rat c a r c a s s e s a n d primarily from raiding bait stations. B r o d i f a c o u m w a s detected in Northwestern c r o w s [Corvus  caurinus)  9 months after the c e s s a t i o n of baiting o n L u c y Island in  1994, but before the baiting o n L a n g a r a Island. B r o d i f a c o u m r e s i d u e s w e r e d e t e c t e d in the p l a s m a of 1 5 % of bald e a g l e s [Haliaeetus  leucocephalus)  sampled  (0.037-1.74 ppm). T h e invertebrates a s a s o u r c e of brodifacoum to non-target s p e c i e s w a s investigated. S n a i l s (Vespericola (Ariolimax  sp. and Haplotrema  sp.) a n d b a n a n a s l u g s  sp.) w e r e c o m m o n a n d a b u n d a n t invertebrates found feeding o n bait in  stations. T h e blue coloured bait could be s e e n through the translucent b o d i e s a n d the m o l l u s c s tested positive for brodifacoum. Carrion insects readily c o n s u m e d rat c a r c a s s e s containing brodifacoum. Blowfly larva {Calliphora  sp.) tested positive for  b r o d i f a c o u m residues. T h e invertebrates found feeding o n the bait a n d carrion insects w e r e a s e c o n d a r y and tertiary poisoning risk to non-target s p e c i e s s u c h a s the s o n g s p a r r o w (Melospiza  melodia).  Table of Contents Abstract  ii  T a b l e of C o n t e n t s  iv  List of T a b l e s  vi  List of F i g u r e s  ix  List of A p p e n d i c e s  xii  Abbreviations  xiii  Acknowledgements  xiv  Dedication  xv  C h a p t e r 1. General  Introduction  1  1.1 O b j e c t i v e s of R e s e a r c h  4  1.2 S t u d y A r e a s 1.3 Baiting P r o t o c o l  5 9  C h a p t e r 2. Hemostasis and Mode of Action of the Anticoagulant the Physiological Basis for Non-Target Species Poisoning Concern  Rodenticides: 13  2.1 Introduction 2.2 H e m o s t a s i s 2.3 V i t a m i n K a n d the M o d e of A c t i o n of the A n t i c o a g u l a n t s  13 13 16  1  C h a p t e r 3. The Short Term Impacts of Brodifacoum Mammals 3.1 3.2 3.3 3.4  Small 20  Introduction Materials a n d M e t h o d s Results Discussion  C h a p t e r 4. An Evaluation 4.1 4.2 4.3 4.4  Baiting on the Native  20 22 27 34  of the Secondary  Introduction Materials a n d M e t h o d s Results Discussion  Poisoning  Hazard  to Avian  Wildlife  38 38 41 49 66  iv  C h a p t e r 5. The Uptake of Brodifacoum by Invertebrates Containing the Rodenticide Brodifacoum or Norway Rat Poisoned with Brodifacoum 5.1 5.2 5.3 5.4  Introduction Materials and Methods Results Discussion  C h a p t e r 6. Conclusions  Feeding on Bait carcasses 89 89 90 97 108  and Recommendations  6.1 C o n c l u s i o n s 6.2 R e c o m m e n d a t i o n s  112 112 114  References  120  v  List of Tables Table 3.1. A b u n d a n c e of d e e r mice (Peromyscus maniculatus) /100 T r a p Nights o n G r a h a m Island, before and after the intensive baiting period o n L a n g a r a Island, 1995 27 Table 3.2. D u s k y s h r e w (Sorex monticolus) trap s u c c e s s (# s h r e w s caught/100 T r a p Nights) a n d population estimate before a n d after brodifacoum bait application, L u c y Island, 1994/1995 28 Table 3.3. Proportion of d u s k y s h r e w s (Sorex monticolus) in breeding condition (testes scrotal, nipples large) pre a n d post baiting o n L u c y Island, 1994/1995 (n in brackets) 29 Table 3.4. D u s k y s h r e w (Sorex monticolus) trap s u c c e s s (# c a u g h t / 1 0 0 T r a p Nights) by trap s e s s i o n a n d region on L a n g a r a Island a n d G r a h a m Island, 1995...30 Table 3.5. Proportion of d u s k y s h r e w s (Sorex monticolus) in breeding condition before a n d after intensive baiting, L a n g a r a Island, 1 9 9 5 ( m e a n of 2 grids per region; s a m p l e s i z e in brackets) 32 Table 3.6. N u m b e r of captures, weight, m a x i m u m d i s t a n c e travelled a n d 9 0 % M C P range s i z e for d u s k y s h r e w s (Sorex monticolus) caught five or more times, L a n g a r a Island, 1 9 9 5 33 Table 4.1. B a l d e a g l e (Haliaeetus leucocephalus) trapping s u c c e s s using the floating fish set, L a n g a r a Island, 1995 (n=148 sets) Table 4.2. B a l d e a g l e (Haliaeetus 1995  leucocephalus)  46  trapping results, L a n g a r a Island 46  Table 4.3. Quantitative recovery of brodifacoum from N o r w a y rat (Rattus norvegicus) liver fortified o n L a n g a r a Island in August, 1 9 9 5  48  Table 4.4. C a r c a s s locations of brodifacoum p o i s o n e d radio-tagged N o r w a y rats (Rattus norvegicus), L a n g a r a Island, 1995  50  Table 4.5. Interval between start of poisoning and detected d e a t h of radiot a g g e d N o r w a y rats (Rattus norvegicus), L a n g a r a Island, 1 9 9 5  50  Table 4.6. B r o d i f a c o u m residue concentrations (mg/kg) in N o r w a y rats (Rattus norvegicus) f o u n d d e a d a b o v e ground, L a n g a r a Island, 1995; ( m e a n s , 9 5 % c o n f i d e n c e limits in brackets; m e a n s that do not s h a r e the s a m e l o w e r c a s e letter w e r e significantly different at p<0.05) 53  vi  Table 4.7. B r o d i f a c o u m r e s i d u e s (mg) in N o r w a y rats (Rattus norvegicus) found d e a d a b o v e ground, L a n g a r a Island, 1995; (mean±s.e., range in brackets).  54  Table 4.8. F r e q u e n c y of avian s c a v e n g e r s of u n p o i s o n e d N o r w a y rat (Rattus norvegicus) c a r c a s s e s , L a n g a r a Island, 1994  54  Table 4.9. Identified s c a v e n g e r s of u n p o i s o n e d N o r w a y rat (Rattus c a r c a s s e s , L a n g a r a Island, 1 9 9 4  55  norvegicus)  Table 4.10. B r o d i f a c o u m residues, gut contents, a n d sites of h e m o r r h a g e to c o m m o n r a v e n s (Corvus corax) found d e a d , L a n g a r a Island, 1 9 9 5  57  Table 4.11. C o m m o n raven (Corvus corax) liver brodifacoum residue levels (mg/kg), L a n g a r a Island, 1995 (geometric m e a n ; 9 5 % c o n f i d e n c e interval in brackets)  58  Table 4.12. Primary sites of h e m o r r h a g e in c o m m o n r a v e n s (Corvus d e a d , L a n g a r a Island, 1 9 9 5  58  corax) found  Table 4.13. F o o d remains in the g i z z a r d s a n d intestines of 13 c o m m o n r a v e n s (Corvus corax) found d e a d , L a n g a r a Island, 1 9 9 5  59  Table 4.14. Inactive c o m m o n raven (Corvus 1996  60  corax) nests, L a n g a r a Island,  Table 4.15. D a t e s , location a n d condition of c o m m o n r a v e n s (Corvus or reported d e a d , L a n g a r a Island, 1996  corax) found 62  Table 4.16. B r o d i f a c o u m residue levels in livers of Northwestern C r o w s caurinus), L a n g a r a Island, 1 9 9 5 Table 4.17. B a l d e a g l e (Haliaeetus leucocephalus) a n d prothrombin times, L a n g a r a Island, 1 9 9 5 Table 4.18. B a l d e a g l e (Haliaeetus times  (Corvus 63  p l a s m a brodifacoum r e s i d u e s  leucocephalus)contro\  64 prothrombin 65  Table. 4.19. S p e c i e s a n d L D v a l u e s u s e d for calculating the value that offers 9 5 % bird s p e c i e s protection with 9 5 % a n d 5 0 % confidence limits 77 5 0  Table 5.1. B r o d i f a c o u m residue recovery from fortified s a m p l e s , 1 9 9 4  vii  97  Table 5.2. B r o d i f a c o u m residue recovery rate from fortified s a m p l e s p r e p a r e d o n L a n g a r a Island, 1 9 9 5 98 Table 5.3. B r o d i f a c o u m residues in invertebrates found in bait stations, L a n g a r a Island, 1 9 9 5 102 Table 5.4. B r o d i f a c o u m residues in invertebrates found in bait stations, L u c y Island, 1994 103 Table 5.5. N u m b e r of a n d brodifacoum residues in carrion insects collected from b r o d i f a c o u m p o i s o n e d N o r w a y rats a n d control N o r w a y rats, into 9 5 % ethanol, L a n g a r a Island, 1994 105 Table 5.6. Insects collected and preserved in 9 5 % ethanol with no b r o d i f a c o u m r e s i d u e s d e t e c t e d , L a n g a r a Island, 1995 106 Table 5.7. B r o d i f a c o u m r e s i d u e s in carrion insects collected from b r o d i f a c o u m p o i s o n e d N o r w a y rat c a r c a s s e s , collected fresh, L a n g a r a Island, 1994  107  Table 5.8. B r o d i f a c o u m residues in blowfly larva collected from brodifacoum p o i s o n e d N o r w a y rat c a r c a s s e s , L a n g a r a Island, 1 9 9 5  107  Table A-1. Liver brodifacoum residue in J a p a n e s e Q u a i l after a single oral d o s e of b r o d i f a c o u m ( m e a n ±s.e., n=2 for e a c h time/dose group)  144  Table B-1. B r o d i f a c o u m residues detected in S o n g S p a r r o w s , L a n g a r a Island, 1995  152  Table B-2. M o r p h o l o g i c a l m e a s u r e m e n t s from S o n g S p a r r o w s collected o n L a n g a r a Island, 1 9 9 5  152  viii  List of Figures Figure 1.1. C h e m i c a l structure of brodifacoum. T h e more c o m m o n a n d familiar anticoagulant warfarin is presented for c o m p a r i s o n p u r p o s e s . Note the 4 - h y d r o x y c o u m a r i n ring s y s t e m c o m m o n in both brodifacoum a n d warfarin  3  Figure 1.2. Location of L a n g a r a , L u c y a n d C o x Islands o n the British C o l u m b i a Coast, C a n a d a  7  Figure 1.3. P l a c e n a m e s o n L a n g a r a Island, 1995  8  Figure 1.4. Bait station d e s i g n u s e d on Lucy, L a n g a r a a n d C o x Islands. The stations w e r e m a d e of o r a n g e coloured P V C corrugated d r a i n a g e pipe. T h e lid w a s r e m o v a b l e for p l a c e m e n t of bait and c h e c k i n g of stations. T h e lid s n a p p e d into p l a c e to protect the bait from the w e a t h e r and most non-target s p e c i e s . Each station w a s labelled with a unique identification number. T h e stations w e r e s t a k e d to the g r o u n d with 2-60 c m long wires bent into a h o r s e s h o e s h a p e a n d p l a c e d o n either end.(adapted from T a y l o r 1993) 10 Figure 1.5. Bait station Layout o n L a n g a r a , L u c y and C o x Islands, 1995. E a c h dot r e p r e s e n t s 1 bait station. Total n u m b e r of stations: 3 8 4 8 12 Figure 2.1. A d i a g r a m m a t i c representation of the coagulation c a s c a d e . T h e grey arrows refer to the intrinsic pathway. T h e roman n u m e r a l s represent the clotting factors. T h e lower c a s e a, represents the activated form of that clotting factor. D a s h e d lines represent positive f e e d b a c k loop e n h a n c i n g the coagulation c a s c a d e , (adapted from Sturkie 1986 a n d R a p a p o r t 1987).... 15 Figure 2.2. T h e Vitamin K Metabolic Activities in R a t Liver M i c r o s o m e s . (Black arrows indicate w h e r e the anticoagulants inhibit the cycling of vitamin K) S t e p A is a c a r b o x y l a s e - e p o x i d a s e e n z y m e not inhibited by c o u m a r i n s . S t e p B, is the e p o x i d e r e d u c t a s e e n z y m e w h i c h is inhibited by the anticoagulants. T h i s l e a d s to rapid depletion of V i t a m i n K stores and build up of Vitamin K e p o x i d e . S t e p C is a l s o a r e d u c t a s e e n z y m e inhibited by anticoagulants, however, a n alternative pathway exists allowing this reaction to p r o c e e d . (Adapted from M o u n t et al. 1982; Suttie 1980) 17 Figure 3.1. Location of small m a m m a l trapping grids, L a n g a r a a n d L u c y Island, 1995  23  Figure 3.2. T h e n u m b e r of unique d u s k y s h r e w s (Sorex monticolus) c a p t u r e d o n L u c y Island, 1994 a n d 1995. T h e vertical arrows indicate w h e n the b r o d i f a c o u m baiting for introduced rats b e g a n . T h e bait w a s r e m o v e d in mid A u g u s t , 1994, but w a s reapplied in July 1995 28  ix  Figure 3.3. M e a n unique n u m b e r of s h r e w s captured o n L a n g a r a Island a n d o n the control grids o n G r a h a m Island, 1995. Vertical arrow indicates w h e n b r o d i f a c o u m baiting for introduced rats b e g a n o n L a n g a r a Island 29 Figure 3.4. U n i q u e n u m b e r of d u s k y s h r e w s captured o n e a c h study grid in the 3 regions o n L a n g a r a Island, 1995. Vertical arrows indicate w h e n b r o d i f a c o u m baiting b e g a n . Longworth traps w e r e u s e d o n all grids e x c e p t w h e r e d e n o t e d by a * w h e n s m a l l S h e r m a n n traps w e r e u s e d 31 Figure 3.5. T h e proportion of d u s k y s h r e w s (Sorex monticolus) in breeding condition correlated to the post baiting s h r e w population estimate a s a proportion of the pre- baiting population estimate (r = 0.64) 36 Figure 4.1. L o c a t i o n s of all c a r c a s s e s of c o m m o n raven [Corvus corax) a n d N o r w a y rats (Rattus norvegicus), together with s a m p l i n g locations of bald e a g l e s (Haliaeetus leucocephalus) a n d Northwestern c r o w s (Corvus caurinus) with positive detection of brodifacoum residue, 1995 52 Figure 5.1. C a r r i o n Insect T r a p u s e d to capture a n d hold insects attracted to rat c a r c a s s e s o n L a n g a r a Island, 1994. Note the funnel o v e r the o p e n i n g of the tube o n the left h a n d side, a n d the 2.5 c m diameter P V C tubing exiting the tube into the 5 0 0 ml g l a s s jar for holding insects. T h e g l a s s jar h a s small air holes in the r e m o v a b l e lid. Insects w e r e collected at regular intervals from the g l a s s jar a n d f r o z e n . T h e black triangles w e r e the ventilation holes and w e r e c o v e r e d with n o s e u m netting 93 Figure 5.2. Proportion of bait stations with snails and b a n a n a s l u g s o v e r the c o u r s e of the intensive baiting period, L a n g a r a Island, 1995 (number of bait stations in brackets) 100 Figure 5.3. Proportion of bait stations with the terrestrial snails, Vespericola sp. and Haplotrema sp., o v e r the c o u r s e of the intensive baiting period (number of bait stations in brackets) 101 Figure A-1. Effect of time o n brodifacoum p l a s m a residue concentration (ppm) after a single oral d o s e of brodifacoum at 0.35 and 0.7 mg/kg ( m e a n s that do not s h a r e the s a m e letter w e r e significant at P<0.05)(n=4 at e a c h time point) 142 Figure A-2. Effect of d o s e o n brodifacoum residue concentration (ppm) o v e r 10 d a y s (* significant at P<0.05)(n=2 at e a c h d o s e level) 143 Figure A-3. Effect of time o n the prothrombin time ratio ( P T R ) of J a p a n e s e Q u a i l after a single oral d o s e of brodifacoum (* significant at P<0.05)(n=9 at e a c h time point) 145  x  Figure A-4. Effect of d o s e of the prothrombin time ratio ( P T R ) of J a p a n e s e quail after a single oral d o s e of brodifacoum ( m e a n s that do not s h a r e the s a m e letter w e r e significantly different at P<0.05) 147  xi  List of Appendices Appendix Table 4-1. Dates, locations, weight, a g e a n d s e x of N o r w a y rats (Rattus norvegicus) found d e a d a b o v e ground, L a n g a r a Island, 1 9 9 5 85 Appendix Table 4-2. Dates, locations, a n d s e l e c t e d morphological m e a s u r e m e n t s of bald e a g l e s (Haliaeetus leucocephalus), L a n g a r a Island, 1 9 9 5 86 Appendix Table 4-3. D a t e s , locations a n d s e l e c t e d morphological m e a s u r e m e n t s of Northwestern c r o w s (Corvus caurinus) collected on L a n g a r a a n d L u c y Island, 1995  87  Appendix A. T h e Detection of E x p o s u r e to B r o d i f a c o u m in J a p a n e s e Q u a i l through P l a s m a B r o d i f a c o u m R e s i d u e A n a l y s i s and Prothrombin T i m e Evaluation  134  Appendix B. B r o d i f a c o u m E x p o s u r e in the S o n g S p a r r o w  151  Appendix C. Potential S u b - L e t h a l and L o n g T e r m Effects of B r o d i f a c o u m Exposure  154  Appendix D. E n v i r o n m e n t a l A s p e c t s of B r o d i f a c o u m - Transport, Distribution a n d Transformation 157  xii  Abbreviations  ANOVA  a n a l y s i s of v a r i a n c e  ppm  parts per million  CWS  C a n a d i a n Wildlife S e r v i c e  PT  prothrombin time  dbh  diameter at breast height  PWRC  Pacific Wildlife R e s e a r c h C e n t r e  GIT  gastro-intestinal tract  s  standard deviation  ha  hectare  SAS  trademark, S A S Institute Inc.  HAG  height a b o v e ground  s.e.  standard error  ND  n o n e detected  ug  micro g r a m  NWRC  National Wildlife Research Centre  xiii  Acknowledgements I would like to thank my immediate thesis supervisors, Dr. K i m C h e n g at U B C , Dr. J o h n Elliott at P W R C a n d Dr. Pierre M i n e a u from N W R C , for their support, g u i d a n c e a n d a s s i s t a n c e both in the field and lab. I a m also grateful to G a r y K a i s e r at P W R C for his support throughout. Funding w a s provided by the N e s t u c c a Oil Spill Trust F u n d a n d Environment C a n a d a . P e r m i s s i o n to work in D u u G u u s d Tribal P a r k w a s kindly granted by the Old M a s s e t t V i l l a g e C o u n c i l . C a r l Z e i s s C a n a d a provided o n e pair of 10 x 4 0 binoculars for u s e throughout this project. I would like to a c k n o w l e d g e and thank those that w e r e v a l u a b l e co-workers in the field. B r u c e Fitz-Earle s h a r e d his e n t h u s i a s m a n d humour, outdoor a n d c o o k i n g skills o v e r three long field s e a s o n s . Brent M a t s u d a and T a r a B u r k e are t h a n k e d for their e n t h u s i a s m , s e n s e of humour, and hard work in 1995. M y appreciation a n d s i n c e r e t h a n k s for m a n y nights of shrewing and d a y s of eagling that a p p e a r e d to n e v e r e n d . M a r k D r e v e r a n d R o w l e y Taylor a s s i s t e d with the radio-collaring of rats a n d s h a r e d their expertise in N o r w a y rat ecology. I thank P e t e r B u c k for introducing m e to the rich history of the islands, support in the field, a n d acting a s a travel agent b e t w e e n M a s s e t t a n d L a n g a r a Island. J a m e s H a g e m a n a s s i s t e d in m a n y a s p e c t s of field work. K a r e n T i m m at N W R C w o r k e d through m a n y t i s s u e s a m p l e s in preparation for a n a l y s i s . Dr. M a l c o l m M c a d i e a n d T o m P a c k e r n e c r o p s i e d r a v e n s a n d c o n d u c t e d protein electrophoresis respectively. M.S. Bhatti a n d Dr. J . T h o m p s o n taught m e the fine art of blood s a m p l i n g birds. I w o u l d a l s o like to thank the S c h w e e r s and H o l y o a k families at the L a n g a r a Island Lightstation for o p e n i n g their h o m e s for us to dry off, shower, do laundry a n d eat a h o m e c o o k e d m e a l . L y n n e Holland provided housing a n d a p l a c e for sorting, shipping, a n d c l e a n i n g of equipment in Massett. M o s t importantly, I a m most grateful to my wife Kathryn, for her support a n d patience, a n d our s o n D o u g l a s , for w h o m life is just beginning.  xiv  .to my parents for the many years of love and encouragement.  xv  Chapter 1. General Introduction T h e introduction of rats (Rattus spp.) to o c e a n i c islands c a n h a v e significant c o n s e q u e n c e s for local wildlife populations, particularly for burrow-nesting s e a b i r d s w h i c h c a n d e c l i n e in a b u n d a n c e or eventually be extirpated ( M o o r s a n d A t k i n s o n 1984). Introduced rats h a v e b e e n implicated a s a significant factor in the d e c r e a s e or extirpation of the L a n g a r a Island, British C o l u m b i a , C a n a d a (see section 1.2 a n d Figure 1.2) breeding population of s e a b i r d s that w a s o n c e d e s c r i b e d a s " i m m e n s e " a n d "astronomical" (Drent a n d G u i g u e t 1961). S e a b i r d s that previously utilised L a n g a r a a s a breeding site included: tufted puffins (Lunda cirrhata), petrels (Oceanodroma  leucorha),  auklets (Ptychoramphus  aleutica),  ancient murrelets (Synthliboramphus  L e a c h ' s storm  fork-tailed storm petrels (O. furcata), rhinoceros auklets (Cerorhinca antiquus)  Cassin's  monocerata),  and  ( C a m p b e l l et al. 1990). But, s i n c e  the 1950's, the breeding populations on the Island h a v e d e c l i n e d significantly or h a v e b e e n extirpated. C a s s i n ' s a n d rhinoceros auklets a n d L e a c h ' s a n d fork-tailed storm petrels no longer breed o n L a n g a r a , while only a small breeding population of tufted puffins exists o n nearby C o x Island (Taylor a n d K a i s e r 1993). In 1993, the nesting population of ancient murrelets w e r e estimated to be l e s s than 1 0 % of historical n u m b e r s , a n d h a v e declined by 4 0 % s i n c e the late 1980's (Harfenist 1993; B e r t r a m 1989). T h e ship rat (Rattus rattus), likely introduced during the fur trade in the early 1800's, w a s present o n L a n g a r a Island, but h a s b e e n d i s p l a c e d by the N o r w a y rat (Rattus norvegicus),  likely introduced in the 1940's. T h e introduction of the N o r w a y  1  rat c o i n c i d e d with the decline in the seabird colony (Taylor 1993), a n d there is e v i d e n c e for significant rat predation on murrelet e g g s , chicks, a n d adults (Bertram a n d N a g o r s e n 1995, Harfenist 1993, Bertram 1989). S i m i l a r p r o b l e m s h a v e b e e n reported e l s e w h e r e , but s i n c e 1 9 8 1 , N e w Z e a l a n d biologists h a v e s u c c e s s f u l l y eradicated introduced rats from small o c e a n i c islands with the u s e of anticoagulant rodenticides. R e c e n t rat eradication p r o g r a m s in N e w Z e a l a n d u s e d the anticoagulant brodifacoum, d i s p e n s e d from fixed, e v e n l y s p a c e d bait stations on offshore islands (Taylor and T h o m a s 1993; 1989). In 1995, the C a n a d i a n Wildlife S e r v i c e attempted the c o m p l e t e eradication of N o r w a y rats from L a n g a r a Island a n d adjacent C o x a n d L u c y Islands with the application of the s e c o n d generation anticoagulant, brodifacoum ( I U P A C 3-[3-(4'bromobiphenyl-4-yl)-1,2,3,4-tetrahydro-1-naphthyl]-4-hydroxycoumarin ) (Figure 1.1), using a t e c h n i q u e d e v e l o p e d in N e w Z e a l a n d (Taylor a n d K a i s e r 1993). T h i s w a s the first time this method (Section 1.3) w a s e m p l o y e d in North A m e r i c a a n d it could h a v e potential for other islands in the Q u e e n Charlotte a r c h i p e l a g o (Bertram a n d N a g o r s e n 1995). However, the u s e of brodifacoum to eradicate rats o n offshore i s l a n d s p o s e s a risk of primary and s e c o n d a r y poisoning to non-target s p e c i e s . P r i m a r y poisoning results w h e n the bait and anticoagulant are c o n s u m e d directly by a non-target a n i m a l . S e c o n d a r y poisoning o c c u r s w h e n a primarily p o i s o n e d a n i m a l is c o n s u m e d by a predator or s c a v e n g e r (Colvin et al. 1988).  2  T3 C (D  ro  c ro 13 O  o  z, £  ro o .TO  8i>  ro o .2 E c .ro o E E ro o o E  (D  o o  >« W D) C  d) ro  M. o  E £  GO  8 2 2 4  -Q  (D  O ±= (D (U ZJ O  o ^~ ZJ  •4-1  E o o  •2  ro o o Q. E 5 I O  (D CL  6§ to • 'i_ ^ ro  2  T~  3  CL  E  t.  .P  3  8  1.1  Objectives of Research T h e objective of the C a n a d i a n Wildlife S e r v i c e w a s to e x a m i n e the feasibility  of eradicating introduced rodents from seabird colonies by b a l a n c i n g the long term benefits against the relative costs. T h e benefits are the removal of a significant, exotic predator s p e c i e s from islands w h i c h are important s e a b i r d breeding a r e a s a l o n g the Pacific C o a s t . T h e costs, other than financial, include the effects o n the native a v i a n a n d m a m m a l i a n s p e c i e s that live o n and a r o u n d t h e s e s e a b i r d c o l o n i e s . T h e r e w e r e three main a r e a s of poisoning c o n c e r n for non-target s p e c i e s : L a n g a r a Island h a s a native population of d u s k y s h r e w s (Sorex elassodon  O s g o o d ) . D e e r mice (Peromyscus  maniculatus)  1)  monticolus  w e r e a l s o known to o n c e  inhabit the island. T h e N e w Z e a l a n d baiting model w a s d e s i g n e d to e n s u r e eradication of the introduced rats a n d mice b e c a u s e there w e r e no native s m a l l m a m m a l s to be c o n c e r n e d about. 2) R a t s are known to die a b o v e g r o u n d after ingesting a lethal d o s e of an anticoagulant ( C o x 1990) posing a p o s s i b l e s e c o n d a r y poisoning h a z a r d for native predators. However, few r e s e a r c h e r s h a v e investigated the actual proportion of rats dying a b o v e ground, a n d the importance of rats in the diet of the local predators a n d s c a v e n g e r s o n L a n g a r a Island w a s u n k n o w n . 3) Invertebrates are important in eliminating e x c e s s bait a n d p o i s o n e d c a r c a s s e s from the environment, however, they m a y introduce brodifacoum into the food c h a i n , a n d p o s e a p o s s i b l e s e c o n d a r y or tertiary poisoning risk. T h e overall goal of this thesis w a s to investigate the risk of non-target s p e c i e s poisoning with the u s e of brodifacoum to eradicate rats from L a n g a r a Island. T h e r e  4  w e r e three main objectives of my study:  1) monitor the native small m a m m a l  population o v e r the c o u r s e of the baiting. 2) evaluate the risk of s e c o n d a r y p o i s o n i n g to a v i a n s c a v e n g e r s a n d predators from p o i s o n e d rats, a n d 3) a s s e s s the invertebrates a s a s o u r c e of introduction of brodifacoum into the e c o s y s t e m . In C h a p t e r 2, I s u m m a r i s e the m o d e of action of anticoagulants a n d the physiological b a s i s for the non-target s p e c i e s poisoning c o n c e r n . In C h a p t e r 3,1 e x a m i n e the results of the e n d e m i c small m a m m a l population monitoring before a n d after the intensive baiting period (Section 1.3).  In C h a p t e r 4, I e x a m i n e the role  played by rats in the s e c o n d a r y poisoning h a z a r d to the local avian predators a n d s c a v e n g e r s . In C h a p t e r 5, I determine the transfer of brodifacoum into the e c o s y s t e m , particularly by invertebrates c o n s u m i n g bait a n d p o i s o n e d rat c a r c a s s e s . In C h a p t e r 6,1 s u m m a r i s e the results from the studies and I p r o p o s e r e c o m m e n d a t i o n s for minimising non-target primary and s e c o n d a r y poisoning h a z a r d s w h e n eradicating rats from seabird colonies a l o n g the British C o l u m b i a coast. I a l s o c o n d u c t e d two laboratory experiments to a s s i s t in the evaluation of d a t a collected in C h a p t e r 4 and t h e s e are d e s c r i b e d in A p p e n d i x A.  Environmental  a s p e c t s of brodifacoum are presented in the a p p e n d i c e s .  1.2 Study Areas 1.2.1  Langara Island L a n g a r a Island (54° 14'N, 133° W ) , a l s o known a s Kiis G w a i i or North Island,  is located at the northwestern tip of the Q u e e n Charlotte a r c h i p e l a g o ( H a i d a Gwaii),  5  British C o l u m b i a , C a n a d a (Figure 1.2). T h e Island is 3 3 0 0 ha in s i z e a n d is relatively flat rising to a m a x i m u m elevation of 160 m. T h e shoreline is highly variable ranging from rocky and s a n d y b e a c h e s to steep cliffs a n d bluffs. T h e Island is ringed by three b a n d s of dominant vegetation that include: sitka s p r u c e sitchensis)  predominating along the shoreline, western h e m l o c k  heterophylla),  (Tsuga  a n d western red c e d a r (Thuja plicata) dominating the interior. T h i c k  growths of salal (Gaultheria (Calmagrostis  (Picea  nutkaensis)  shallon) are abundant in a r e a s , while N o o t k a reed g r a s s is found along the shoreline. T h e forest floor is  predominantly o p e n , a n d c o v e r e d with m o s s , and m o s s c o v e r e d logs a n d s t u m p s . R a i s e d b o g s are found inland a m o n g s t the lakes. T h e r e w e r e two a r e a s at which people reside year-round o n the Island. At L a n g a r a Point, o n the north-west tip, is the L a n g a r a Lightstation with two families living permanently (Figure 1.3). At the southern most tip of L a n g a r a Island w e r e the fishing l o d g e s located in and around H e n s l u n g C o v e . T h e r e w e r e two p e r m a n e n t l a n d - b a s e d lodges, W e s t C o a s t Fishing C l u b and L a n g a r a Island L o d g e o n Iphigenia Point. T h r e e other, s e a s o n a l , floating fishing lodges are barged in early spring a n d l e a v e by early fall. Float p l a n e s regularly drop off a n d pick up tourists throughout the fishing s e a s o n . 1.2.2 Lucy Island L u c y Island located in c l o s e proximity to L a n g a r a Island (about 3 0 0 m ) is 4 0 ha in a r e a , 1400 m long a n d 3 0 0 m a c r o s s at its widest. T h e shoreline is highly variable consisting of c o a r s e gravel b e a c h e s and boulders to rocky s h e l v e s a n d outcrops. T h e island is relatively flat with a m a x i m u m elevation of 69 m a b o v e  6  7  8  s e a level. O l d growth forest predominates, but with h e a v y p a t c h e s of windfall containing thick s e c o n d growth running along the central portion of the Island. T h e d o m i n a n t vegetation is similar to that found on L a n g a r a .  1.3 Baiting Protocol T h e following is a s u m m a r y of the baiting protocol of the L a n g a r a Island S e a b i r d Habitat Restoration Project. T h e protocol is d e s c r i b e d in detail in K a i s e r et al. (1997). T h e baiting w a s carried out with up to 70 p e o p l e working out of five field c a m p s d i s p e r s e d around L a n g a r a Island. T h e bait stations w e r e d e p l o y e d approximately e v e r y 1 0 0 m a n d f a s t e n e d d o w n with two 6 0 c m long wires (Figure 1.4). T h e bait blocks m e a s u r e d a p p r o x i m a t e l y 3.5 c m x 3.5 c m x 2 c m , a n d w e i g h e d 2 0 g. T h e bait c o n s i s t e d of b r o d i f a c o u m (Ratak+ ™) at a concentration of 0.005%, or 1 m g b r o d i f a c o u m per bait block. T h e carriers a n d attractants w e r e a mixture of blood, bone, wheat, tallow a n d c a s t o r s u g a r ( K a i s e r et al. 1997). Paraffin w a x bound the bait together a n d "weatherproofed" the block. T h e baiting protocol w a s tested o n L u c y Island beginning J u l y 12, 1994 ( B u c k 1995). E a c h station w a s a r m e d with three bait blocks. T h e stations w e r e c h e c k e d e a c h d a y a n d all activity w a s recorded. Activity included: bait d i s a p p e a r a n c e , c h e w m a r k s by rats, s h r e w s or invertebrates. Baits c h e w e d by s h r e w s or s l u g s w e r e d e s t r o y e d a n d r e p l a c e d . O n d a y 19, baits w e r e placed into plastic b a g s to minimise e x p o s u r e to non-target s p e c i e s . O n A u g u s t 17, 1994, all baits w e r e r e m o v e d from the stations.  9  E o  "o cz  T3  CO  0 Q. h= Q.  •pap ,  Q. CO  o cu sz " -o cu —ZZ CO TO a).5" cu c o i f Z3 .COc o SZ -*—» 1— >+- CO O c "D >o CD O cu ca "co JScu= .2 E a) o o Jcot: CO  _  »ilpiil»l  M  cu  cz  O  M  CU  TO  CO  0  co CO o cu co cu  *:§  CO .ii  >  SZ  CO D ) CO _ CO tz CU CO SZ o  co CO  o co E coLU o O  a  0  _l  > o E  TO  a:  CL  c 0  M M  E o  o  0  5 CU CO  oo O co X  UO  o  .o  CO CD  il _ "O  H P  0  •4—*  ,  CN  0 "O c c TO CO 0 iS o CO -Q c 0 TO CO -t—' c > CO CO co £  V  o  o o E 3R -> ; CD E T3 1  =i  00  c CO CO L_  CO  >o o TD SZ 2 0 T3 ~ "co $ 0 0 0 CO E c o 3£ o  H  0  M—  mm  o "co  g • + < CD O  CD TO «  2 ^  » c T3 CO 0 TO Q0. CO CO Q. CL CO 0 0 0 cz TO 0 SZ CO T3 i— _ cz C3 O 0 ^  -4-*  C D  o o  •F  co •  •  co  'S  c _o .o E® 0 - 0 2 |^ CO 2 § cu TO  i—  =  o  .s>> Li.  10  0)'+;  c  ° n ° £ 8 Q.-i= 0 Q_  id n  C  cz a  0  -D  co  Q_  R a t s w e r e d i s c o v e r e d again on L u c y Island in late A u g u s t 1994, a n d in 1995, the Island w a s re-baited in attempt to eradicate the remaining rats. O n J u l y 11 1995, intensive baiting b e g a n o n L a n g a r a Island. E a c h of the 3 8 4 8 bait stations (Figure 1.5) w e r e a r m e d with 6 to12 bait blocks , monitored e v e r y 2 to 3 d a y s , a n d r e p l e n i s h e d a s n e c e s s a r y until early August. F o r the post-intensive baiting period b e t w e e n A u g u s t 1 9 9 5 a n d A u g u s t 1997, e a c h station o n L a n g a r a L u c y a n d C o x Islands w a s a r m e d with three bait blocks w r a p p e d in plastic p r o d u c e b a g s a n d p l a c e d o n an aluminium or plastic tray to k e e p the bait dry. T h e bait stations w e r e c h e c k e d for e v i d e n c e of rat u s e in S e p t e m b e r 1995, a n d a g a i n in February, M a y , a n d A u g u s t 1996. T h e stations a n d bait w e r e r e m o v e d from the Islands during the s u m m e r of 1997.  11  LANGARA ISLAND  -  J&i L* -  •• i  cox is. "£Jb (i^^u:\::u^44v^.7h :  .Jj£l - *•—*  • •"• t: ?: •::))* sit m  _*  • _• « i «  J  si: *.::::*• ":\  Figure 1.5. Bait station Layout on Langara, Lucy and C o x Islands, 1995. Each dot represents 1 bait station. Total number of bait stations: 3848.  •\  v  .•  '  J  0.5 LUCY IS.  Kilometers  12  Chapter 2. Hemostasis and Mode of Action of the Anticoagulant Rodenticides: the Physiological Basis for Non-Target Species Poisoning Concern. 2.1  Introduction H e m o s t a s i s is o n e of the m a n y physiological characteristics that is s h a r e d by  birds a n d m a m m a l s . H e m o s t a s i s h a s two major functions: (1) to prevent blood loss from sites of v a s c u l a r disruption and (2) prevent pathologic t h r o m b o s i s ( e x c e s s clotting) by limiting clot formation to sites of v a s c u l a r disruption (Brandt 1991). H e m o s t a s i s c a n be v i e w e d a s two forces w h i c h continuously o p p o s e a n d b a l a n c e e a c h other. A n y shift in the b a l a n c e of forces will result in e x c e s s t h r o m b o s i s or bleeding. 2.2  Hemostasis U n d e r b a l a n c e d conditions, hemorrhaging is controlled by clot formation  w h i c h results from a s e r i e s of p r o e n z y m e to e n z y m e transformations culminating in the c o n v e r s i o n of fibrinogen to fibrin ( K a s e et al. 1980). In m a m m a l s a n d birds, this coagulation or e n z y m e c a s c a d e (Macfarlane 1964) c a n be activated by o n e of two w a y s : extrinsically or intrinsically. Intrinsic activation involves contact activation s u c h a s the e x p o s u r e of the blood to the s u b e n d o t h e l i u m in vivo or, contact of blood to a negatively c h a r g e d s u r f a c e s u c h a s g l a s s in vitro (Macfarlane 1964; R a p a p o r t 1987). Recently, the well studied h u m a n coagulation s y s t e m h a s revealed that contact activation is not required for normal h e m o s t a s i s (Jesty and N e m e r s o n 1995). P a s t studies h a v e indicated that birds lack an effective intrinsic clotting m e c h a n i s m (Didisheim et a l . 1959). However, Doerr and Hamilton (1981) have provided e v i d e n c e of a  13  functioning intrinsic m e c h a n i s m in c h i c k e n s , but this plays a minor role in the overall c o a g u l a t i o n function in c o m p a r i s o n to the extrinsic pathway. T h i s is similar to the a c c e p t e d h u m a n m o d e l in that the coagulation c a s c a d e is initiated by t i s s u e factor (thromboplastin) and not n e c e s s a r i l y the intrinsic pathway (Jesty and N e m e r s o n 1995). T h e extrinsic pathway involves a tissue factor (or thromboplastin) that normally r e s i d e s in the endothelium a n d other t i s s u e s s u c h a s the brain (Griminger 1986). T h e thromboplastin is present o n the surface of m a n y cell t y p e s a n d is not normally in contact with the circulating blood (Jesty and N e m e r s o n 1995). U p o n t i s s u e d a m a g e , the thromboplastin, now e x p o s e d to blood, binds with factor VII forming a n enzymatically active c o m p l e x which initiates the clotting c a s c a d e (Rapaport, 1987) (Figure 2.1). T h e m a m m a l i a n a n d avian extrinsic pathway coagulation c a s c a d e s are functionally similar (Griminger 1986; Belleville et al. 1982; K a s e 1978). O n c e the factor Vll-thromboplastin c o m p l e x forms, it initiates the activation of factor X  which  provides a positive f e e d b a c k increasing the e n z y m a t i c activity of the factor V l l thromboplastin c o m p l e x (Rapaport 1987). T h i s c o m p l e x along with activated factor XI then activates factor IX. T h e activated factor Vll-thromboplastin and factor IX c o m p l e x e s are required to activate sufficient factor X to g e n e r a t e e n o u g h thrombin to maintain h e m o s t a s i s (Rapaport 1987). Thrombin (activated factor II) transforms fibrinogen into fibrin followed by the stabilization of the fibrin clot. Interruption of this c a s c a d e at a n y step will prevent the formation of the fibrin clot a n d h e m o r r h a g i n g m a y continue uncontrolled. T h e introduction of an anticoagulant, s u c h a s  14  XI  Intrinsic Pathway  Extrinsic Pathway Thromboplastin Ca++  IX Ca++  Vila  VII  • Xla Ca++  IXa  Ca++  Xa XIII  Ca++  Prothrombin  Thrombin Xllla Ca++  IA Fibrinogen  Fibrin  ' 1B Stabilised Fibrin  Figure 2.1. A d i a g r a m m a t i c representation of the coagulation c a s c a d e . T h e grey arrows refer to the intrinsic pathway. T h e roman numerals represent the clotting factors. T h e lower c a s e a, represents the activated form of that clotting factor. D a s h e d lines represent positive f e e d b a c k loop e n h a n c i n g the coagulation c a s c a d e (adapted from Sturkie 1986 a n d R a p a p o r t 1987).  15  brodifacoum, in sufficient d o s e s , will result in the inhibition of production of s o m e clotting factors required in the extrinsic pathway. 2.3 Vitamin K and the Mode of Action of the Anticoagulants 1  T h e metabolic role of vitamin K., is to activate the vitamin K d e p e n d e n t clotting factors- II (prothrombin), VII, IX, a n d X (Rapaport, 1987; Suttie 1980) by contributing a c a r b o x y side chain by post-translational carboxylation of s e l e c t e d glutamic r e s i d u e s to form g a m m a - c a r b o x y g l u t a m i c acid r e s i d u e s (Figure 2.2). T h e s e a m i n o acid r e s i d u e s are n e c e s s a r y to chelate divalent c a l c i u m to interact with phospholipid containing m e m b r a n e s , their normal site of activation ( G o o d m a n et al., 1985). V i t a m i n K-, now in its oxide form, c a n be reduced to its original form by the e p o x i d e r e d u c t a s e e n z y m e . T h e recycled vitamin K  :  is now available for further  activation of the vitamin K d e p e n d e n t clotting factors. T h e anticoagulants interfere with this p r o c e s s , resulting in the r e l e a s e of inactive clotting factors into the blood stream. All c o u m a r i n s , the group of anticoagulants to w h i c h brodifacoum b e l o n g s , h a v e the s a m e m o d e of action. A n t i c o a g u l a n t s bind to the warfarin binding protein (Thijssen a n d B a a r s 1989) in the e n d o p l a s m i c reticulum of the liver h e p a t o c y t e s ( S e a r c e y et a l . 1977) a n d inhibit the e p o x i d e reductase e n z y m e preventing the reduction of vitamin K -epoxide. T h e 4-hydroxycoumarins d o not n e c e s s a r i l y bind 1  to the r e d u c t a s e e n z y m e , but it h a s b e e n s p e c u l a t e d that they interact with a subunit structure with w h i c h the e n z y m e h a s to interact for normal functioning (Thijssen a n d B a a r s 1989). T h e inihibition of the reductase e n z y m e l e a d s to a build up of hepatic  16  0  T3 0 0  CO CO  .Q  ZJ "O  .ti <o o  sz 0  .E  0  CZ  c c -c -~ •— o TOc oo o co "co 0 o  O) co  E § .52 >»  c  N  CZ  CO  id  0 0  •* o  s °-  Q. -o  « l  II  0  2 CO >, 0  _C0 0 0  e d>  co  0 i  S O !c  CO co CO  CO* O  CO 0  X  o  .Q CD  I*  cz  o "co w X  0  co •2 E _£Z CO _Q x : co •4—• CO O J= CL  o 8 E co 0 E CO  i—  CO  0  >  co tz  T3 N 0 CZ ZJ 0 sn 0 T3  Q.  ^ CO 0 S£ co o ZJ "O  'I -  co  0  o co  -E  Z3  0  o  -I—»  E ~  as  |  ?  CN  00  O CD  CO  X  cz £ "co cz ^ ro 0 cu co 0 cn f=  •~  •i °  0  CZ co co CO 0 CO  Hi •g I 8. E 0 0  CD o _ co CO CD CZ  %o  CO O - cz "cz co DO q Q- 0 £ Q.  > 'szco o  o 5  c §  n  T3 0  0  c CO "O c .E Q. f < CO i csi -I 0 "O CN o) E o c CD 0  ZJ a% CO CO 3 - oo O O T3  t 0 >p O N  ^ CO CO -O 0 w 0  E  CL  17  vitamin K e p o x i d e (Leek a n d P a r k 1981; C a l d w e l l et al. 1974) and the level of 1  activated clotting factor production d e c l i n e s or is inhibited ( C h o o n a r a et a l . 1988; L e e k a n d P a r k 1981). T h e extent to w h i c h the anticoagulant inhibits the production of the clotting factors is d o s e d e p e n d e n t (Thijssen and B a a r s 1989; 1987). In liver, there are specific, high affinity, saturable binding sites for anticoagulants (Thijssen a n d B a a r s 1989; H u c k l e e t a l . 1989a; Huckle et al. 1989b; P a r m a r et a l . 1987). T h e anticoagulants n e e d to saturate the binding sites to initiate the anticoagulant effect ( P a r m a r et al. 1987). Without the carboxylation of the vitamin K d e p e n d e n t clotting factors, they are r e l e a s e d into the blood with little or no e n z y m a t i c viability a n d the level of active factors d e c l i n e s . T h e anticoagulant effect d e v e l o p s gradually o v e r time a s the level of active clotting factors d e c l i n e s according to their different rates of biologic d e c a y ( R a p a p o r t 1987). T h e r e exists a threshold of active clotting factors b e l o w w h i c h significant bleeding will result (Hoffman et al. 1988). Without active clotting factors, a n y t r a u m a induced or s p o n t a n e o u s bleeding is uncontrollable a n d death results from h y p o x i a , a n d h y p o v o l e m i c shock. All birds a n d m a m m a l s s h a r e the coagulation characteristics that m a k e t h e m s u s c e p t i b l e to anticoagulant rodenticides. T h e differences in sensitivity to anticoagulants results primarily from varying ability to metabolise or e x c r e t e t h e s e c o m p o u n d s (Huckle et al. 1989b). In general, birds are l e s s s u s c e p t i b l e to a n t i c o a g u l a n t s a s they are more readily able to metabolise the c o m p o u n d while m a m m a l s are unable to metabolise the anticoagulants before the lethal  18  anticoagulant effect t a k e s place (Huckle et al. 1989b). Further d i s c u s s i o n into potential sub-lethal a n d long term effects of brodifacoum e x p o s u r e is p r e s e n t e d A p p e n d i x C.  19  Chapter 3.0 The Short Term Impacts of Brodifacoum Baiting on the Native Small Mammals. 3.1  Introduction In North A m e r i c a , native small m a m m a l s are a b u n d a n t a n d ubiquitous in a  variety of habitats including offshore islands. T h e u s e of anticoagulants to e r a d i c a t e introduced rats from t h e s e islands m a y alter the a b u n d a n c e a n d c o m p o s i t i o n of the native s m a l l m a m m a l populations. T h e r e is little information on how controlling or eradicating rats affect resident native small m a m m a l populations, particularly o n offshore islands. Historical records indicate that the d u s k y s h r e w a n d the d e e r m o u s e w e r e the only native small m a m m a l s known to inhabit L a n g a r a Island (Foster 1965, I. M c t a g g a r t C o w a n , pers. comm.). B a s e d o n morphological m e a s u r e m e n t s a n d p e l a g e colour, the s h r e w s on L a n g a r a Island h a v e b e e n included with the north M o r e s b y Island r a c e (elassodon)  (Foster 1965). D e e r mice w e r e k n o w n to be  present in the 1940's but had apparently b e e n extirpated by the 1960's w h e n F o s t e r (1965) r e c o r d e d s h r e w s but no d e e r mice. N o e v i d e n c e of d e e r m i c e w a s found during recent trapping c a m p a i g n s (Harfenist 1993; Bertram 1989). T h e d i s a p p e a r a n c e of the d e e r m o u s e c o i n c i d e s with the introduction of the N o r w a y rat (Taylor 1993). However, in S e p t e m b e r 1994, o n e d e e r m o u s e w a s trapped in Lord Bight o n the w e s t c o a s t of L a n g a r a Island (C. F r e n c h , pers. comm.) w h i c h m a y h a v e r e p r e s e n t e d a previously undetected population. T h e native small m a m m a l s are at risk of poisoning b e c a u s e they s h a r e m a n y characteristics with the target rodent s p e c i e s . T h e risk of primary poisoning is  20  related to the palatability a n d availability of the bait in s p a c e a n d time. T h e bait consisting of both animal a n d grain products m a y be attractive to both d u s k y s h r e w s a n d d e e r mice. D u s k y s h r e w s are normally insectivorous, but are k n o w n to c o n s u m e carrion ( C o x 1990; V. C r a i g , U B C , pers. comm.). T h e y are inquisitive a n d readily eat animal c a r c a s s e s , s h o w i n g a preference for organs, particularly the liver, in w h i c h anticoagulants a c c u m u l a t e ( C o x 1990). T h e objective of this study w a s to identify the native small m a m m a l s p e c i e s at risk of poisoning, and monitor the short term population c h a n g e s , a b u n d a n c e a n d c o m p o s i t i o n , o v e r the intensive baiting period.  21  3.2 Materials and Methods Non-target Small Mammal Identification and Population Monitoring In 1994, a 1ha grid of 4 9 trap stations (7x7), 15.2 m apart, with o n e Longworth-style live trap located within 2 m of e a c h station w a s e s t a b l i s h e d o n L u c y Island (Figure 3.1). O n l y o n e grid w a s u s e d d u e to the unavailability of traps, a n d no control grid w a s u s e d .  A three e v e n i n g arming a n d c h e c k period w a s e m p l o y e d .  T h e traps w e r e baited with a mixture of peanut butter a n d oats, a n d c o a r s e brown cotton w a s s u p p l i e d for bedding. T h e traps w e r e a r m e d in the early e v e n i n g a n d c h e c k e d 5-6 hours later, at d u s k or just after. This w a s repeated for the following two e v e n i n g s . Live trapping w a s c o n d u c t e d o n c e before and o n c e after the baiting, in J u l y 1994, 3 d a y s before the start of the baiting a n d in A u g u s t after the bait w a s r e m o v e d from the stations. In M a y 1995 and A u g u s t 1995, a single trap s e s s i o n oo-the e s t a b l i s h e d grid w a s performed. In 1995, L a n g a r a Island w a s divided into three treatment regions: the existing A n c i e n t Murrelet colony at M c p h e r s o n Point (a high population of rats), the e a s t c o a s t in E g e r i a B a y (lower rat population), a n d the w e s t c o a s t region in Lord Bight (possible remnant population of d e e r mice) (Figure 3.1). In e a c h of the regions, two 1-ha grids, e a c h with 4 9 trap stations (7x7), 15.2 m apart, w e r e e s t a b l i s h e d . Longworth style live traps w e r e placed o n all grids e x c e p t for two w h i c h had small S h e r m a n n traps. E a c h grid w a s trapped twice before the baiting b e g a n a n d twice after the intensive baiting period.  22  23  G r a h a m Island, 1 km a c r o s s Parry P a s s a g e from L a n g a r a Island, w a s u s e d a s a control site. T w o control grids w e r e e s t a b l i s h e d , however, o n e w a s r e m o v e d after c o n t i n u o u s disruption by a f e m a l e black b e a r a n d her two c u b s during the s e c o n d trap s e s s i o n in J u n e 1995. A three e v e n i n g arming and c h e c k period w a s e m p l o y e d o n L a n g a r a a n d G r a h a m Islands following the s a m e p r o c e d u r e s u s e d o n L u c y Island. T h i s short arming time w a s utilised b e c a u s e s h r e w s die if left in traps o v e r night (Sullivan 1990). T r a p p i n g w a s performed in the e v e n i n g to i n c r e a s e the c h a n c e s of trapping a n y d e e r m i c e present on L a n g a r a or L u c y Islands. E a c h s h r e w captured w a s w e i g h e d with an A v i n e t spring b a l a n c e , a n d m a r k e d for future identification with a blonde hair d y e in a unique combination of dots o n its fur and/or toe-nail clipped. I c h o s e not to toe-clip b e c a u s e if a n y bait w a s c o n s u m e d , lethal hemorrhaging m a y h a v e b e e n i n d u c e d . D u e to the difficulty in s e x i n g juvenile or non-sexually active s h r e w s , only o b v i o u s reproductive condition w a s noted (Craig 1995; H a w e s 1977). Individuals w e r e r e l e a s e d immediately after p r o c e s s i n g . T h e traps w e r e locked o p e n between trap s e s s i o n s , a n d a l s o b e t w e e n c h e c k s a n d arming times. S h r e w s caught five or more times (Craig 1995) w e r e u s e d for estimation of range s i z e a n d d i s t a n c e traveled calculated with the c o m p u t e r program C A L H O M E (Kie et a l . 1994). R a n g e s i z e w a s calculated using 9 0 % m i n i m u m c o n v e x p o l y g o n s ( M C P ) for c o m p a r i s o n to other studies (Craig 1995; H a w e s 1977).  24  Shrew use of Bait Stations Bait station operators w e r e a s k e d to record s h r e w activity in bait stations. Training in the identification of s h r e w c h e w e d bait blocks w a s provided prior to the baiting. S h r e w c h e w s w e r e identified from the s i z e and pattern of the incisor m a r k s . A n y s h r e w c h e w e d bait block w a s replaced to prevent it being recounted during future visits. However, only d a t a collected by identified, reliable bait station operators w e r e u s e d .  Calculations and Statistical A n a l y s e s Population Size Changes T h e d a t a are reported a s the n u m b e r of individuals/ha a n d unique s h r e w s /100 trap nights (TN). S u c c e s s per 100 T N w a s calculated following N e l s o n a n d C l a r k (1973) a s : C E = A x 100/ (TU - IS/2) W h e r e C E = c a t c h effort; A = n u m b e r o f animals; T U = trap units (TU= P x I x N w h e r e P= n u m b e r of trapping intervals (3 nights); l= length of trapping intervals (1 night); a n d N = n u m b e r of traps (49 traps); S = n u m b e r of traps c l o s e d a n d e m p t y or with a recaptured shrew. W h e r e possible, the S c h n a b e l / S c h u m a c h e r - E s c h m e y e r method w a s u s e d to estimate population s i z e (Krebs 1989). C o n f i d e n c e intervals w e r e calculated using the P o i s s o n distribution. T h e trap s u c c e s s d a t a w e r e s q u a r e root transformed in a n attempt to n o r m a l i z e the data. I u s e d a two w a y A N O V A for repeated m e a s u r e s ( K u e h l 1994).  25  A n a l y s i s w a s carried out with the JMP statistical p a c k a g e ( S A S 1995) with the following statistical m o d e l : Yi  Jk  = H +/?, + Tj + (R7V  E«  k  w h e r e Yj = unique shrews/1 OOTN, R = effect of region, a n d Tj = effect of the jth jk  t  time, a n d (f?7~)jj = the two w a y interaction b e t w e e n time a n d region, a n d E r a n d o m error. A significance level of P<0.05 w a s c h o s e n a priori.  26  i j k  =  3.3 Results O n l y d u s k y s h r e w s (63 unique individuals) w e r e trapped o n L u c y Island in 1994 a n d 1995, a n d o n L a n g a r a Island (182 unique individuals) in 1 9 9 5 . O n G r a h a m Island, d e e r mice (32 unique individuals) w e r e a b u n d a n t o n control grids (Table 3.1), a n d d u s k y s h r e w s (18 unique individuals) w e r e a l s o c a p t u r e d .  Table 3.1. A b u n d a n c e of d e e r mice (Peromyscus maniculatus) /100 T r a p Nights o n G r a h a m Island, before and after the intensive baiting period o n L a n g a r a Island, 1995. Location  Before May  E a s t Grid W e s t Grid  7.9 8.8  Baiting July  Post August  21.0  17.4  Baiting Mid-August  4.3  L u c y Island In 1994, there w a s a decline in the n u m b e r of unique s h r e w s captured after the baiting (Figure 3.2, T a b l e 3.2). B y M a y 1995, trap s u c c e s s r e b o u n d e d to half the pre-baiting 1994 estimate. U n i q u e n u m b e r of s h r e w s captured a g a i n d e c l i n e d o v e r the baiting period in 1995 (Table 3.2). T h e proportion of s h r e w s in breeding condition d e c l i n e d in both y e a r s o v e r the c o u r s e of the baiting period (Table 3.3).  27  F i g u r e 3.2. T h e n u m b e r of unique d u s k y s h r e w s (Sorex monticolus) captured o n L u c y Island, 1994 a n d 1995. T h e vertical arrows indicate w h e n t h e b r o d i f a c o u m baiting for introduced rats b e g a n . T h e bait w a s r e m o v e d in mid A u g u s t , 1994, but w a s reapplied in July 1995.  T a b l e 3.2. D u s k y s h r e w (Sorex monticolus) trap s u c c e s s (# s h r e w s caught/100 T r a p Nights) a n d population estimate before a n d after brodifacoum bait application L u c y Island, 1994/1995.  1994  1995  Trap Success Population Estimate  Trap Success Population Estimate  Before Baiting  P o s t Baiting  19.9 56.4 (32-155)  2.8 ND  11.8 28.1 (19-185)  a Not Determined. All shrews were trapped on the third evening, b 95% Confidence Interval  28  a  b  b  10.5 13.2 (11-47)  b  Table 3.3. Proportion of d u s k y s h r e w s (Sorex monticolus) in breeding condition (testes scrotal, nipples large) before a n d after baiting o n L u c y Island, 1994/1995 (n in brackets). Pre-Baiting Post-Baiting 1994 1995  0.40 (10) 0.56 (10)  0.25(1) 0.13(2)  Langara Island T h e r e w a s no significant interaction between time a n d regions (Table 3.4). Overall, there w a s no statistical difference in trap s u c c e s s before a n d after the intensive baiting period on L a n g a r a Island (Figure 3.3). T r a p s u c c e s s w a s very low o n the s m a l l S h e r m a n n trap grids (Table 3.4), but removing t h o s e grids from the a n a l y s i s had no effect.  May  July  August  Mid-August  Figure 3.3 M e a n unique n u m b e r of d u s k y s h r e w s (Sorex monticolus) captured o n L a n g a r a Island a n d o n the control grids o n G r a h a m Island, 1 9 9 5 . Vertical arrow indicates w h e n brodifacoum baiting for introduced rats b e g a n o n L a n g a r a Island.  29  Table 3.4. D u s k y s h r e w (Sorex monticolus) trap s u c c e s s (# c a u g h t / 1 0 0 T r a p Nights) by trap s e s s i o n and region o n L a n g a r a Island a n d G r a h a m Island, 1 9 9 5 . Before Region  Grid Location  Baiting  Post  May  July  August  Baiting Mid-August  6.9 6.8  17.1 3.6  L a n g a r a Island Murrelet C o l o n y  No-Name Pt Explorer B a y  14.3 3.4  1.4 9.1  East Coast  E g e r i a North Egeria South  3.0 3.5  11.1 3.5  2.8 1.4  7.0 0.7  West Coast  Lord Bight N o r t h Lord Bight S o u t h  0.7 12.5  1.4 10.3  0.0 3.5  1.4 3.5  G r a h a m Island (Control)  East West  2.4 0.0  _  _  _  5.7  5.7  1.1  a  a  a. Small Shermann traps used on these grids.  W h i l e statistically not significant, the regional n u m b e r of unique s h r e w s captured per hectare declined between the start of the baiting a n d the third trap s e s s i o n in all regions o n L a n g a r a Island. T h e greatest decline o c c u r r e d o n the e a s t coast, dropping by 7 5 % (Figure 3.4). O n the G r a h a m Island control grid, there w a s a n a c c u m u l a t e d trap mortality of three s h r e w s during the s e c o n d and third trap s e s s i o n . During the last trap s e s s i o n , 3 1 , 3 5 a n d 3 6 of the 4 9 traps o v e r the three trap nights, o n the grid w e r e reported c l o s e d a n d empty.  30  Ancient Murrelet Colony  May  July  August  Mid-August  August  Mid-August  August  Mid-August  East Coast  May  July  West Coast  May  July  Figure 3.4. Unique number of dusky shrews {Sorex monticolus) captured on each study grid in the three regions on Langara Island, 1995. Vertical arrows indicate when brodifacoum baiting began. Longworth traps were used on all grids except where denoted by a * when small Shermann traps were used.  31  T h e proportion of s h r e w s captured in breeding condition did not c h a n g e o n the control grid and r e m a i n e d at 5 0 % (Table 3.5).  However, the proportion of  s h r e w s in breeding condition declined at all three baited regions, with the most drastic d e c l i n e (%) occurring on the W e s t C o a s t (Table 3.5).  . Table 3.5. Proportion of d u s k y s h r e w s {Sorex monticolus) in breeding condition before a n d after intensive baiting, L a n g a r a Island, 1995 (mean of two grids per region; s a m p l e s i z e in brackets). Murrelet East Coast West Coast Graham Colony Island (Control) Pre-Baiting Post-Baiting  0.44 (18) 0.39 (17)  0.43 (7) 0.21 (4)  0.49(18) 0.06 (1)  0.50 (5) 0.50 (4)  Shrew Use of Bait Stations S h r e w incisor marks o n bait blocks w e r e small, a n d readily identifiable. T h e s h r e w s c h e w e d the e d g e s of the blocks, working their w a y a r o u n d all e d g e s , n e v e r c o n s u m i n g the w h o l e block. C o n v e r s e l y , rat incisor marks w e r e larger a n d they w o u l d begin a n d continue to c h e w o n o n e side of the bait block. O n L u c y Island s h r e w s c h e w e d 38 bait blocks in 2 8 % of the bait stations in 1994 ( B u c k 1995). S h r e w s w e r e also photographed with automatic c a m e r a s exiting bait stations. B y d a y 20 of the baiting program on L u c y Island in 1995, s h r e w s had visited 8 0 % of 4 2 bait stations a n d c h e w e d o n e or more bait blocks in e a c h station. In H e n s l u n g C o v e , s h r e w s had c h e w e d 157 bait blocks in 5 0 % of the 2 8 stations in that region o v e r the c o u r s e of the intensive baiting period. In the interior of the  32  Island, s h r e w s had c h e w e d 151 bait blocks in 8 0 % of 5 3 stations visited.  On  L a n g a r a Island, bait station operators reported s h r e w s "stumbling" out of stations, indicating p o s s i b l e anticoagulant intoxication ( C o x 1990). A s well, during c h e c k s of the live traps o n the North E g e r i a grid in early August, small, blue colored s h r e w f e c e s w e r e noted in o n e trap indicating that at least o n e s h r e w had b e e n f e e d i n g o n the bait.  Shrew Ranging Distances O n l y four individual s h r e w s w e r e captured five or more times, with the n u m b e r of recaptures ranging from five to eight. T h e s h r e w 9 0 % M C P range s i z e varied b e t w e e n 112 m to 2 7 0 0 m . T h e m a x i m u m d i s t a n c e s traveled b e t w e e n traps 2  2  r a n g e d from 61.8 m to 91.2 m (Table 3.6).  Table 3.6. N u m b e r of captures, weight, m a x i m u m d i s t a n c e traveled a n d 9 0 % M C P range s i z e for d u s k y s h r e w s (Sorex monticolus) c a u g h t five or more times, L a n g a r a Island, 1995. Location Number Weight Shrew Mean Maximum 90% Range of Distance No. Distance Area g Captures m m m 2 5 6 32  Murrelet Colony Murrelet Colony Lord Bight Murrelet Colony  8  8.3  40.3  90.2  2700.0  6  6.8  54.9  91.2  1238.0  5  7.8  75.7  76.5  2475.0  5  5.8  54.6  61.8  112.5  33  3.4  Discussion S h r e w s w e r e a b u n d a n t in the coastal region of L a n g a r a Island prior to the  baiting, but there w a s no e v i d e n c e of d e e r mice. T h e results s u g g e s t that the d u s k y s h r e w w a s able to co-exist with introduced rats, p e r h a p s b e c a u s e of niche differences resulting in low interspecific competition. R a t s m a y h a v e a v o i d e d preying o n s h r e w s b e c a u s e they secrete a distinctive pungent, acrid o d o r m a k i n g t h e m unpalatable (Churchfield 1990; H a w e s 1976). D e e r m i c e w e r e apparently extirpated from the coastal region of L a n g a r a Island either through predation, competition or both. O t h e r islands in the Q u e e n Charlotte a r c h i p e l a g o contain populations of d e e r mice e x c e p t w h e r e there are rats; the exception are the larger islands, G r a h a m , M o r e s b y , Kunghit and Lyell Island that h a v e populations of both d e e r mice a n d rats (Foster 1965). T h e individual d e e r m o u s e trapped in Lord Bight in 1994 m a y h a v e represented a remnant population, or m a y h a v e b e e n accidentally transported from G r a h a m Island by the s u r v e y crew. D e s p i t e u s e of bait stations by shrews, a n d d e c l i n e s o v e r the c o u r s e of the baiting, there w e r e no significant short-term impacts o n s h r e w a b u n d a n c e o n L a n g a r a Island. R a t control p r o g r a m s h a v e b e e n s h o w n to significantly impact other non-target small m a m m a l s p e c i e s . During anticoagulant poisoning for rat control in the spring a n d s u m m e r o n farms in E n g l a n d , non-target w o o d m i c e sylvaticus)  (Apodemus  entered stations a n d fed o n bait ( C o x a n d S m i t h 1990). T h e r e w a s a  7 5 % reduction in the population s i z e of w o o d m i c e after treatment while the control populations i n c r e a s e d ( C o x a n d S m i t h 1990). T h e individual survivorship of m a r k e d  34  individuals w a s between 0-19% on baited farms a n d 5 0 % o n control f a r m s ( C o x a n d S m i t h 1990). N o estimates of survivorship could be m a d e for the s h r e w s o n L a n g a r a Island. A c c o r d i n g to the trapping record, the G r a h a m Island (control grid) d u s k y s h r e w a b u n d a n c e d e c r e a s e d while the L a n g a r a a b u n d a n c e i n c r e a s e d b e t w e e n the third a n d fourth trap s e s s i o n .  It w a s e x p e c t e d that the reverse would be m e a s u r e d ,  i.e., s h r e w s o n L a n g a r a Island would d e c r e a s e in a b u n d a n c e a s c o m p a r e d to the control site. H o w e v e r , o n the control grid, the trapping of non-target d e e r mice, high n u m b e r of c l o s e d but empty traps and trap mortality of s h r e w s could a c c o u n t for the low n u m b e r of s h r e w s trapped o n this grid during the third a n d fourth trap s e s s i o n . T h e L u c y Island population of s h r e w s declined sharply o v e r the c o u r s e of the baiting in 1994 a n d w a s the r e a s o n a monitoring program w a s e s t a b l i s h e d o n L a n g a r a Island in 1 9 9 5 . In 1995, s h r e w s o n L u c y Island did not d e c l i n e in a b u n d a n c e a s in 1994, but the proportion of s h r e w s in breeding condition followed a similar pattern of decline. O n L a n g a r a Island, the greater the regional d e c l i n e in n u m b e r of s h r e w s captured after the intensive baiting, the greater the d e c l i n e in the proportion of s h r e w s in breeding condition (Figure 3.5). T h e r e w a s no c h a n g e in the proportion of b r e e d e r s o n the control grid o v e r the s a m e time period. T h i s d e c l i n e in the proportion of s h r e w s in breeding condition m a y be d u e to s e a s o n a l c h a n g e s s u c h a s recruitment of juveniles or it m a y represent a specific poisoning impact o n s h r e w s in breeding condition. T h i s s e e m s to s u g g e s t that the individual risk of primary poisoning w a s greater for individuals in breeding condition. A s t h e s e  35  Graham Island (Control)  0.5 0.45 0.4  1  Murrelet Colony  #  0.35 0.3 •-  0.25  S  0.2  Lucy Island 1994 East Coast  0.15  • Lucy Island 1995  0.1 I West Coast  0.05  0.2  0.4  0.6  0.8  1  Post Baiting Shrew Population as a Proportion of Pre-Baiting Abundance  Figure 3.5. T h e proportion of d u s k y s h r e w s (Sorex monticolus) in breeding condition correlated to the post baiting s h r e w population estimate a s a proportion of the pre-baiting population estimate (r = 0.64). territories w e r e v a c a t e d , dispersing juveniles and other non-breeding s h r e w s m a y h a v e c o m p e t e d to fill t h o s e v a c a n t territories (Churchfield 1990).  T h i s w o u l d lead to  a n i n c r e a s i n g density of s h r e w s w h i c h m a y explain the i n c r e a s e d population during the fourth trap s e s s i o n in mid-August o n four of the six grids o n L a n g a r a Island. T h e risk of primary poisoning to s h r e w s from gaining a c c e s s to the bait in stations is greater for s h r e w s in breeding condition b e c a u s e they range m o r e than n o n - b r e e d e r s ( H a w e s 1977). T h e greater ranging d i s t a n c e s of breeding s h r e w s i n c r e a s e d t h e c h a n c e of encountering brodifacoum in stations. B r e e d i n g m a l e s m a y  36  be at greater primary poisoning risk than the breeding f e m a l e s , n o n - b r e e d e r s a n d juveniles, a s they range significantly further afield ( H a w e s 1977). A n e x t r e m e m o v e m e n t of o n e scrotal adult male w a s noted in Lord Bight. T h i s individual w a s live-trapped in a field c a m p , m a r k e d a n d r e l e a s e d , and w a s s u b s e q u e n t l y recaptured 7 hours later o n the North Lord Bight grid, 181.5 m a w a y - a l m o s t twice the a v e r a g e d i s t a n c e between bait stations. T h e range s i z e s calculated for d u s k y s h r e w s o n L a n g a r a Island w e r e smaller than the ranges calculated by H a w e s (1977) but similar to t h o s e found by C r a i g (1995). T h e distance b e t w e e n stations w a s a d e q u a t e to spatially e x c l u d e s o m e s h r e w s . T h i s study a d d r e s s e d the short term impacts of brodifacoum baiting o n the s h r e w population. W h a t remains u n k n o w n is the impact of having bait left in bait stations w r a p p e d in plastic b a g s for the 2 y e a r period after this study e n d e d . It c a n be c o n c l u d e d that s h r e w s w e r e attracted to bait in the stations a n d that there w e r e short term impacts o n s h r e w s including breeding individuals. H o w e v e r , they w e r e not extirpated from L a n g a r a or L u c y Island. S h r e w trap s u c c e s s r e b o u n d e d 9 months after bait w a s r e m o v e d from L u c y Island. T h u s , c e s s a t i o n of the baiting could allow for the s h r e w population to i n c r e a s e to pre-baiting levels within 1 year. T h e long term impacts of brodifacoum baiting o n the non-target d u s k y s h r e w w o u l d require further investigation; however, there are unlikely to be a n y significant long term impacts.  37  Chapter 4. A n Evaluation of the Secondary Poisoning Hazard to Avian Wildlife.  4.1 Introduction T h e s e c o n d a r y poisoning risk to birds from feeding o n anticoagulant killed rodents is well known and h a s b e e n demonstrated in the laboratory (Newton et a l . 1990; R a d v a n y i et al. 1988; T o w n s e n d et al. 1981; M e n d e n h a l l a n d P a n k 1980). A s s e s s i n g the actual h a z a r d under field conditions is difficult b e c a u s e p h a r m a c o l o g i c a l susceptibility is not n e c e s s a r i l y an indicator of e c o l o g i c a l susceptibility ( M o o r e 1966) and predators and s c a v e n g e r s are not e x p e c t e d to c o n s u m e only contaminated animals ( T o w n s e n d et al. 1984). T h e risk of s e c o n d a r y poisoning to avian predators and s c a v e n g e r s from brodifacoum p o i s o n e d N o r w a y rats is related to e x p o s u r e factors s u c h a s the behaviour of the target s p e c i e s during the latent period, the location of c a r c a s s e s (above or below ground), the anticoagulant residue loading in the target s p e c i e s , a n d the b e h a v i o u r a n d diet of the non-target s p e c i e s ( R e c o r d and M a r s h 1988; T a y l o r 1993; K a u k e i n e n 1982). A n t i c o a g u l a n t p o i s o n e d N o r w a y rats demonstrate altered b e h a v i o u r w h i c h potentially m a k e s t h e m more susceptible to predation a n d s c a v e n g i n g . N o r w a y rats e x p o s e d to a lethal d o s e of an unidentified anticoagulant s h o w e d r e d u c e d thigmotactic b e h a v i o u r (moving in contact with a vertical surface s u c h a s a wall) a n d s p e n t significantly more time in o p e n a r e a s than under c o v e r a s c o m p a r e d to the controls ( C o x a n d Smith 1992). While in the o p e n , rats w e r e o b s e r v e d sitting m o t i o n l e s s or staggering about shortly before death. O n death, 5 0 % died in o p e n  38  a r e a s , apparently deliberately moving out of nest b o x e s into o p e n a r e a s just before dying. During the field testing of the baiting protocol o n L u c y Island in 1994, o n e of three brodifacoum p o i s o n e d radio-collared N o r w a y rats died a b o v e ground a w a y from a burrow (Howald 1995). C a r c a s s e s of p o i s o n e d rats also w e r e found o n the b e a c h a n d other o p e n a r e a s under the forest c a n o p y . S e c o n d a r y poisoning of non-target s p e c i e s from the u s e of b r o d i f a c o u m to control rats a n d v o l e s h a s b e e n demonstrated in the field. E a s t e r n s c r e e c h - o w l s (Otus asio) died after e x p o s u r e to brodifacoum after b r o a d c a s t application to control v o l e s in o r c h a r d s (Colvin a n d H e g d a l 1988; M e r s o n et al. 1984). In N e w Z e a l a n d , W e s t e r n w e k a (Gallirallus australis  australis australis)  and Stewart Island w e k a  (Gallirallus  scotti) died after c o n s u m i n g rats that had fed on bait containing  b r o d i f a c o u m (Taylor 1984). C o m m o n r a v e n s (Corvus corax), Northwestern c r o w s (Corvus bald e a g l e s (Haliaeetus  leucocephalus)  caurinus)  and  w e r e c o n s i d e r e d to be at risk of s e c o n d a r y  p o i s o n i n g from feeding o n d e a d or dying N o r w a y rats on L a n g a r a Island (Taylor a n d K a i s e r 1993). T h e importance of rats in their diets is u n k n o w n , but, they are opportunistic s c a v e n g e r s and predators that may take a d v a n t a g e of a n e w prey source. T o evaluate the s e c o n d a r y poisoning risk of the three avian s p e c i e s , an integrated study w a s undertaken to determine the extent to w h i c h rats will die a b o v e g r o u n d , a n d to evaluate the poisoning of non-target s p e c i e s . T h e main objective  39  w a s to e v a l u a t e the s e c o n d a r y poisoning risk to avian predators a n d s c a v e n g e r s from the u s e of brodifacoum to control N o r w a y rats o n L a n g a r a Island. T h e specific h y p o t h e s i s tested w a s that dying or d e a d rats would be available to predators a n d s c a v e n g e r s , thus putting t h e m at risk of s e c o n d a r y poisoning.  40  4.2 Materials and Methods 4.2.1 C a r c a s s Locations of Brodifacoum Poisoned Norway Rats Fifty T o m a h a w k live traps w e r e a r m e d for 4 0 0 trap nights & 4 0 0 trap d in early J u l y 1995, between M c p h e r s o n Point and N o - N a m e Point, in a n attempt to capture 5 rats of e a c h a g e (juvenile/adult) and s e x c l a s s . T r a p p e d rats w e r e a n a e s t h e t i s e d with halothane, s e x e d , w e i g h e d with a spring b a l a n c e , e a r t a g g e d with a fingerling tag, and fitted with a P D - 2 C radio collar (Holohil S y s t e m s , Ontario, C a n a d a ) . F o u r adult m a l e s , five adult f e m a l e s , five juvenile m a l e s a n d five juvenile f e m a l e s w e r e radio-collared between three d before the start of the program to the d a y the intensive baiting b e g a n . O n e adult male a n d o n e adult f e m a l e w e r e trapped o n d a y 2 of the program. E a c h rat w a s located at least o n c e per d a y by tracking the signal to its location a n d taking a bearing a n d m e a s u r e m e n t to the nearest bait station. At a n y o n e time, a rat w a s determined to be alive if, w h e n holding the a n t e n n a steady, the signal strength b e c a m e w e a k e r or stronger indicating that the animal w a s moving, or if the rat w a s found to be alive at a future time point. T h e time to death w a s calculated a s the n u m b e r of d e l a p s e d between the start of the baiting p r o g r a m a n d the date of last known activity +24 h.  4.2.2 Brodifacoum Residues in Norway Rats Found Dead A b o v e G r o u n d R a t s found d e a d by bait station operators a n d r e s e a r c h staff w e r e s e x e d , w e i g h e d a n d frozen in pre-labelled whirl-pak bags. Five adult male, four adult  41  female, a n d three juvenile male rats w e r e sent to the National Wildlife R e s e a r c h C e n t r e , Hull, P Q for tissue preparation for brodifacoum residue a n a l y s i s . T h e liver, G I T (gastrointestinal tract, including its contents), a n d the c a r c a s s w e r e individually h o m o g e n i s e d a n d a n a l y s e d for brodifacoum a s d e s c r i b e d below. T h e data w e r e log transformed (X|= log (x+1)). A n a l y s i s of the data w a s carried out using the JMP 10  statistical p a c k a g e ( S A S , 1995) with a two w a y A N O V A . T h e statistical m o d e l w a s : Yj = u. + A Jk  where Y  iJk  +  Tj + (AT)ij + E  ijk  = log brodifacoum residue m e a s u r e d , A, = the effect of the ith a g e / s e x  c l a s s , a n d 7j = effect of the jth tissue, (AT)jj = the two-way interaction b e t w e e n d o s e effect a n d time of blood collection, a n d E  ijk  = random error.  4.2.3 Norway Rat Scavenger Identification In 1994, a y e a r before the baiting operation b e g a n , non-poisoned, s n a p trapped rats w e r e laid out in o p e n , e x p o s e d a r e a s s u c h a s o n b e a c h e s , o p e n a r e a s u n d e r the forest c a n o p y , a n d around bald e a g l e nesting trees. Automatic, infra-red motion s e n s i n g c a m e r a s w e r e u s e d to identify s c a v e n g e r s . A total of 2 9 rats w e r e put out, 18 with c a m e r a s , a n d 11 o n E g e r i a B a y b e a c h w h e r e s c a v e n g i n g s p e c i e s w e r e identified from tracks left behind o n moist s a n d , or by direct o b s e r v a t i o n . T i m e to s c a v e n g i n g w a s only roughly estimated between visits to c a r c a s s sites o r from direct o b s e r v a t i o n .  42  4.2.4 Effects on Predators and Scavengers 4.2.4.1 C o m m o n Ravens All bait station operators, r e s e a r c h and L a n g a r a Lightstation staff w e r e briefed before the baiting program b e g a n and w e r e e n c o u r a g e d to report o b s e r v a t i o n s involving ravens, or to turn in a n y c a r c a s s e s , pellets, or other r e m a i n s they f o u n d . S u c h c a r c a s s e s w e r e labelled a n d frozen for n e c r o p s y at the University of British C o l u m b i a , Department of A n i m a l S c i e n c e by Dr. M a l c o l m M c a d i e , a veterinarian e x p e r i e n c e d in e x a m i n i n g pesticide p o i s o n e d birds. Livers w e r e r e m o v e d a n d frozen for brodifacoum residue analysis.  Source of Brodifacoum  Poisoning  to Ravens  T h e s o u r c e of brodifacoum w a s determined from protein electrophoresis of the g i z z a r d contents a n d by evaluation of the g i z z a r d and intestinal contents for N o r w a y rat hairs a n d bait fragments. E a c h g i z z a r d w a s o p e n e d a l o n g its length a n d the contents, if any, w e r e r e m o v e d and the cutica gastrica rinsed with w a t e r from a s q u e e z e bottle. If there w a s an a d e q u a t e amount, a s a m p l e w a s frozen a n d sent with control s a m p l e s of N o r w a y rat, shrew, raven, snails, a n d bait to the A l b e r t a Natural R e s o u r c e s S e r v i c e , Enforcement-Field S e r v i c e s , F o r e n s i c Laboratory, E d m o n t o n , A l b e r t a for identification by polyacrylamide gel electrophoresis ( M c C l y m o n t et al. 1982). T h e remaining g i z z a r d contents w e r e rinsed through two layers of c h e e s e c l o t h a n d a 60 m e s h s i e v e lined with filter p a p e r before using a d i s s e c t i n g m i c r o s c o p e (7-30x) to identify remains and estimated percent per v o l u m e .  43  T h e intestines w e r e cut into three sections a n d the contents s q u e e z e d into a p r e - w a s h e d g l a s s dish.  T h e contents w e r e e x a m i n e d for hair or bait c r u m b s u n d e r  a d i s s e c t i n g m i c r o s c o p e . All hair w a s identified to s p e c i e s u n d e r a m i c r o s c o p e by c o m p a r i s o n against control s a m p l e s of N o r w a y rat, d u s k y shrew, black-tailed d e e r (Odocoileus  hemionus  columbianus),  h u m a n and published m i c r o g r a p h s of deer,  shrew, a n d N o r w a y rat (Moore et al. 1974; Adorjan 1969).  Raven  Activity-1996 In April 1996, the potential o n g o i n g raven e x p o s u r e to brodifacoum o n  L a n g a r a Island w a s investigated. Formerly active nest sites w e r e visited at M c p h e r s o n Point a n d H a z a r d o u s C o v e . N e w nests w e r e located by w a l k i n g up to 2 0 0 m inland parallel to the shoreline; o n e o b s e r v e r between the b e a c h a n d cliff bottom, the other a l o n g the ridge that rings the island. Regurgitated pellets a n d prey r e m a i n s w e r e collected from under and around the nest sites. A total of 107 bait stations reported active (baits r e m o v e d and/or stations disrupted) w e r e visited. T h e plastic bags, a l u m i n u m trays u s e d to k e e p the bait off the floor of the station, and bait remains w e r e collected a n d e x a m i n e d for b e a k m a r k s . Plastic b a g s w e r e e x a m i n e d under a dissecting m i c r o s c o p e a n d c o m p a r e d against reference e x a m p l e s of raven-torn plastic b a g s . B a g s w e r e e x a m i n e d for s t r e s s bars and/or b e a k marks.  44  4.2.4.2 Northwestern Crows C r o w s (25) w e r e collected by shotgun between M a y a n d A u g u s t 1 9 9 5 . E a c h crow w a s w e i g h e d a n d s e x e d , a n d s e l e c t e d morphological m e a s u r e m e n t s w e r e t a k e n before the livers w e r e r e m o v e d and placed into pre-labelled whirl-pak b a g s a n d f r o z e n . T h e livers w e r e pooled for brodifacoum a n a l y s i s b a s e d o n collection d a t e s and/or location. Control livers from pre-baiting snap-trapped rats w e r e a l s o sent for brodifacoum a n a l y s i s .  4.2.4.3 Bald Eagles U s i n g a fish s n a r e ( J a c k m a n et al. 1993; C a i n a n d H o d g e s 1989), bald e a g l e s w e r e trapped a n d a blood s a m p l e drawn between d a y s 7 a n d 4 7 of the intensive baiting c a m p a i g n (Table 4.1 a n d T a b l e 4.2). T h r e e e a g l e s w e r e trapped prior to the start of the eradication in order to test the trapping m e t h o d s a n d to obtain control bipod. T r a p p i n g w a s concentrated w h e r e e a g l e s c o m m o n l y roosted on the coast, a n d r a n g e d from the L a n g a r a Island Lightstation east to M c p h e r s o n Point, south a l o n g the east c o a s t to Holland Point, and w e s t to C o x Island. T r a p p i n g w a s attempted, but u n s u c c e s s f u l , on the west c o a s t d u e to the i n c e s s a n t northwest/westerly o c e a n swell w h i c h hindered trapping. W e c o m m o n l y o b s e r v e d e a g l e s flying from all regions of the island and most individuals likely w e r e present in our trapping region during the s a m p l i n g period.  45  Table 4 . 1 . B a l d e a g l e (Haliaeetus leucocephalus) trapping s u c c e s s using the floating fish set; L a n g a r a Island, 1995; (n=148 sets). Description  Rate  Induced off R o o s t Attempted to Pick up Bait Trapped  49% 84% 36%  No. Eagles 73 61 22  Table 4.2. B a l d e a g l e (Haliaeetus leucocephalus) trapping results, L a n g a r a Island, 1995. Age Class Adult S u b Adult Old Immature Y o u n g Immature  Number Trapped 13 3 2 4  E a c h e a g l e w a s a s s i g n e d an a g e c l a s s b a s e d o n p l u m a g e patterns (Bortolotti 1984), w e i g h e d with a 10 kg P e s o l a spring s c a l e , a n d the wing chord, tail length, c u l m e n , a n d tarsus diameter m e a s u r e d . S e x w a s determined from the bill depth a n d hallux length m e a s u r e m e n t s (Bortolotti 1984). U p to 10 ml of blood w a s drawn from the brachial vein. T h e blood w a s first collected into heparinized V a c u t a i n e r t u b e s for brodifacoum residue a n a l y s i s , a n d if there w a s e n o u g h , 4.5 ml w a s placed into a V a c u t a i n e r tube containing buffered s o d i u m citrate a n d immediately placed o n wet ice for P T evaluation (Brown 1988). A single drop of blood w a s placed o n to a slide for a s m e a r . T h e bird w a s b a n d e d a n d r e l e a s e d . T h e banding data h a s b e e n submitted to the province of British C o l u m b i a , Ministry of Environment, and the National Wildlife R e s e a r c h C e n t r e , Hull, P Q .  46  T h e blood w a s centrifuged at 2 0 0 0 x g for 15 min a n d the p l a s m a pipetted into pre-labelled 5 ml cryovials, and immediately frozen (-20° C ) . P T s w e r e m e a s u r e d within 6-8 h of collection using the Coulter P/T Fibrinogen following the p r o c e d u r e s for m a n u a l evaluation (Brown 1988). F o r control P T s , blood w a s d r a w n from bald e a g l e s at the O r p h a n e d Wildlife ( O W L ) rehabilitation centre in Delta, B.C. a n d in rehabilitation centres o n V a n c o u v e r Island. T h e P T for e a c h s a m p l e w a s m e a s u r e d three times. N o r m a l h u m a n p l a s m a w a s u s e d before a n d after e a c h s a m p l e to e n s u r e that the test w a s effective. T h e reported P T is an a v e r a g e of the two c l o s e s t times.  4.2.5 Brodifacoum Residue Analysis F r o z e n t i s s u e s w e r e s h i p p e d to the National Wildlife R e s e a r c h C e n t r e , Hull, P Q for preparation for a n a l y s i s . T i s s u e h o m o g e n i s a t i o n w a s carried out using c h e m i c a l l y c l e a n e d instruments to avoid contamination. Extracts of t i s s u e w e r e a n a l y s e d at N o v a M a n n International, M i s s i s s a u g a , Ontario using high p r e s s u r e liquid c h r o m a t o g r a p h y ( H P L C ) with two detection s y s t e m s : a) post c o l u m n reaction a n d m e a s u r e m e n t of f l u o r e s c e n c e of brodifacoum a n d b) ultraviolet s p e c t r u m s c a n n i n g (for details of procedure s e e Hunter 1983). T h e c o m p o u n d identified a s b r o d i f a c o u m using f l o r e s c e n c e m a t c h e d the U V s p e c t r u m of the b r o d i f a c o u m s t a n d a r d . T h e limit of detection w a s 0.005 mg/kg. A s part of the quality control, N o v a M a n n confirmed quantitative recovery of b r o d i f a c o u m from liver a n d reported m e a n recoveries of 7 6 % for fortifications at the  47  0.5 a n d 1.0 mg/kg levels. Fortified rat liver s a m p l e s w e r e a l s o p r e p a r e d in the field, w r a p p e d in foil packets, placed into pre-labelled polyethylene whirl p a c k b a g s a n d s h i p p e d o n ice. T h e t i s s u e s and foil w e r e rinsed with known v o l u m e s of methanol a n d t i s s u e s h o m o g e n i s e d . T h e h o m o g e n i s e d t i s s u e s w e r e divided into two thirds/one third by weight. T h e larger portion w a s extracted at N o v a M a n n , the s m a l l e r stored at N W R C . T h e m e a n quantitative recovery of brodifacoum w a s 5 4 % for fortifications b e t w e e n 6.4 to 2 3 mg/kg (Table 4.3). All original c h r o m a t o g r a p h y g r a p h i c a l tracings from the instrumental a n a l y s e s w e r e carefully e x a m i n e d at N W R C by Dr. B r y a n W a k e f o r d to e n s u r e both quantitative a n d qualitative a s s e s s m e n t of the b r o d i f a c o u m r e s i d u e s w e r e correct.  Table 4.3. Quantitative recovery of brodifacoum from N o r w a y rat (Rattus norvegicus) liver fortified o n L a n g a r a Island in August, 1995. Tissue Type  Tissue + Methanol Sent  Fortified Level (ug)  Brodifacoum R e p o r t e d (ug)  Percent Recovery  0.0 14.22 16.25 6.97 7.83  0.0 6.94 6.97 6.22 2.79  Control 48.8 42.9 89.2 35.6  (9) Liver Liver Liver Liver Liver  10.3 6.8 5.3 13.7 4.7  B a l d e a g l e p l a s m a w a s a n a l y s e d for brodifacoum residue (Murphy et al. 1989) at the Department of Agriculture, State of Illinois Veterinary D i a g n o s t i c Laboratory, U S A . T h e limit of detection w a s 0.005 p p m . A n aliquot of the s p i k e solution w a s sent to the lab with the s a m p l e s for a n a l y s i s . T h e quantitative recovery w a s 7 8 . 1 % for a concentration of 115 ug/ml s p i k e solution.  48  4.3 Results 4.3.1 Carcass Locations of Brodifacoum Poisoned Norway Rats T h e majority (86.7%) of the radio-collared rats died underground in their burrows a n d therefore w e r e unavailable to avian s c a v e n g e r s (Table 4.4). A t 8 d following initial baiting, an adult f e m a l e w a s found d e a d o n the b e a c h a b o v e the high-tide line in a puddle of water, 24 h after her last known activity. A n o t h e r radio collared adult f e m a l e w a s tracked into the forest c a n o p y 10 d after the start of the baiting. T h e remains, including the h e a d (with the radio collar a r o u n d the neck), forelegs a n d thoracic c a g e (organs r e m o v e d ) w e r e found o n the b r a n c h of a c o a s t a l W e s t e r n H e m l o c k (Tsuga heterophylla)  (dbh = 51 c m ) about 10  m a b o v e the g r o u n d . T h e tree w a s located 36 m south of bait station M C B 1 9 , a n d >225 m from the north shoreline of L a n g a r a Island. It is u n c l e a r w h e t h e r it w a s s c a v e n g e d or preyed o n . T h e r e w a s no e v i d e n c e of ejected pellets or other r e m a i n s u n d e r the tree. T h i s rat w a s known to h a v e b e e n active 4 d before, with no e v i d e n c e of further m o v e m e n t until the signal w a s tracked to the tree. T h e interval between the start of the intensive baiting a n d detected d e a t h for 15 rats r a n g e d from 3 to 9 d (Table 4.5). T h e r e w a s no significant difference (P>0.05) in time to death between s e x and/or a g e c l a s s . T h e s i g n a l s for 2 adult m a l e s could not be detected possibly b e c a u s e of transmitter malfunction, or t h e m having left the region. T h e collar a n d ear-tag of a juvenile f e m a l e w e r e found u n d e r a log, while the radio of another adult f e m a l e w a s found b e c a u s e either it had slipped off, or the rat had b e e n preyed u p o n .  49  Table 4.4. C a r c a s s locations of brodifacoum p o i s o n e d radio-tagged N o r w a y rats (Rattus norvegicus), L a n g a r a Island, 1995. Age Above Below Predated or Unknown Class Ground Ground Scavenged Juvenile Female 0 4 0 1 Juvenile Male 0 5 0 0 Adult F e m a l e 1 2 1 1 Adult Male 0 2 0 2 Total 1 13 1 4 6.7% 86.7% 6.7% a  b  a Found in the forest canopy. It is unclear if it was scavenged or preyed on. b Not included in percentage calculations.  Table 4.5. Interval between start of poisoning a n d detected d e a t h of radio-tagged N o r w a y rats (Rattus norvegicus), Langara Island, 1995. m e a n ± s.e. A g e and S e x M a s s (g) D a y s to D e a t h n Class mean P o s t Start of (range) Baiting" Juvenile Female 90 6 ± 1.15 4 (52-145) 3  3  Juvenile Male  74.4 (50-105)  8 ± 0.32  5  Adult F e m a l e  278.6 (198-353)  6 + 1.47  4  Adult M a l e  237.5 (163-298)  6 + 0.0  2  Mean  7 ±2  a n=5 for all age classes except Adult Male with n=4. b Days to Death= Days to last known activity + 1 day.  50  Other, non-radio t a g g e d rats, of both a g e a n d s e x c l a s s e s w e r e found d e a d a b o v e g r o u n d a n d collected opportunistically by the baiting and r e s e a r c h c r e w s ( A p p e n d i x A ) . T h e locations of all rats found a b o v e ground is in Figure 4 . 1 .  4.3.2  Residues in Norway Rats Found Dead A b o v e Ground T h e brodifacoum residue concentrations in s e l e c t e d rat t i s s u e c a n be found in  T a b l e 4.6. T h e r e w a s a significant interaction between a g e / s e x c l a s s a n d t i s s u e t y p e s (P<0.05) (Table 4.6). C a r c a s s brodifacoum residue concentrations w e r e similar a c r o s s all a g e / s e x c l a s s e s . Liver concentrations w e r e similar a c r o s s all a g e / s e x c l a s s e s . T h e adult f e m a l e liver brodifacoum residue concentration w a s significantly l e s s than the juvenile male but similar to the adult male (Table 4.6). T h e absolute (mg) residue in the t i s s u e s of the rats found d e a d a b o v e ground are p r e s e n t e d in T a b l e 4.7. T h e w h o l e c a r c a s s residue load (mg) of the rats ranged from 0.097-1.809 mg or 0.097-1.809 bait block equivalents.  51  N o - N a m e Pt.  L a n g a r a Pt.  s  M c p h e r s o n Pt.  Explorer B a y  Lord t Bight  ff  m  Fury B a y Egeria Bay #  t  Common Ravens  ^  Bald Eagles  •  Northwestern C r o w s  O  Norway Rats  Hazardous Cove  Figure 4.1. Locations of all c a r c a s s e s of c o m m o n raven (Corvus corax) a n d Norway rats (Rattus norvegicus), together with s a m p l i n g locations of bald e a g l e s (Haliaeetus leucocephalus) a n d Northwestern c r o w s (Corvus caurinus) with positive detection of brodifacoum residue, 1995.  52  t  Henslung Cove  'Holland Pt.  0  oo  l_  CO  CO CD -K  CO  -o ° o 5 -a £ "co ^ sz co 3 CO <.•> c cn co  ICQ _CD  o  D)  oo • o CM CO CO  CM co LO  d> CO  ? l co  0  CO •55  *S O CO CO CO CO  "8  CD  E co j=  °°  oo CO CN CM o 00 CO CM  = LO  CO  £g c  2  0 >  S 0  c E  0  LO  CO CO  w  g LO  CM  CO  1 co  2, CL u  3 co  2 CO  CD 8 g> O£ o  =6 -  2 -d  1  0 CL CO 0  E to g co CO  0  1 ca  CO 0 sz 0  °  o  oo co CM  ^_  <D T3  LO  CM  co T LO LO  2X CO  CO LO  CO  LfT  CM  T —  CD  o LO  CO CM  O co LO co  co •  CM  Sri  _>> -+—'  CO CO  i  CM CM  1  o co c O CD CO o 0 ^ 5^ •3 ~°'cralCO CO CO  45 co  a)  T3  cd co  CO  4.33-4  c -a o o ^ o V 3 LO a. CO ° >  00  co o .o  CD  5  co co  co  o .. C co S  CT>  CO  LO  .O  O CD  OO O0  CO CO LO CM  0 CO ZJ <  ra  CO CM  LO CO CM  0 CO  E 0 u_  •4—'  ZJ <  n  CO CO Cp LO ^ CM CM O 0 CO 0  'E 0 > ZJ  Table 4.7. B r o d i f a c o u m residues (mg) in N o r w a y rats (Rattus norvegicus) found d e a d a b o v e ground, L a n g a r a Island, 1995; (mean+ s.e., range in brackets). Age and S e x Carcass Liver GIT Whole Body Adult M a l e  0.702±0.101 (0.495-1.001)  0.235+0.033 (0.188-0.327)  0.474+0.141 (0.181-0.791)  1.472±0.260 (0.897-1.809)  Adult Female  0.439±0.138 (0.101-0.757)  0.646+0.130 (0.388-0.802)  0.270±0.078 (0.042-0.383)  1.327±0.242 (0.872-1.697)  Juvenile Male  0.058±0.021 (0.037-0.100)  0.039±0.013 (0.015-0.061)  0.108±0.053 (0.045-0.214)  0.205±0.086 (0.097-0.375)  3.3 Scavenger Identification C o m m o n r a v e n s w e r e identified to be the most significant a v i a n s c a v e n g e r (80%) of N o r w a y rats (Table 4.8). Northwestern c r o w s a l s o w e r e p h o t o g r a p h e d at six c a r c a s s sites a n d s u s p e c t e d of s c a v e n g i n g three other rats.  Table 4.8. F r e q u e n c y of avian s c a v e n g e r s of u n p o i s o n e d N o r w a y rat (Rattus norvegicus) c a r c a s s e s , L a n g a r a Island, 1 9 9 4 . Species Raven Crow  Number  Percent  T i m e to S c a v e n g i n g  12 3  80 20  1-24 h 1-14 d  Total  15  100  C r o w s , however, w e r e also attracted to three other N o r w a y rat c a r c a s s e s after they w e r e s c a v e n g e d or buried by the n e c r o p h a g o u s beetle Nicrophorus  sp., or  attacked by other insects (Table 4.9). P h o t o g r a p h s of a n adult bald e a g l e w e r e  54  t a k e n at a c a r c a s s that w a s not s u b s e q u e n t l y s c a v e n g e d , but buried by the beetle Nicrophorus  s p . S o n g s p a r r o w s (Melospiza  melodia) w e r e p h o t o g r a p h e d at three  rat c a r c a s s e s that had b e e n attacked by carrion insects.  Table 4.9. Identified s c a v e n g e r s of u n p o i s o n e d N o r w a y rat (Rattus norvegicus) c a r c a s s e s , L a n g a r a Island, 1994. Scavenger Class Number Percent Avian 15 52 Insect 5 17 Unknown 4 14 Not S c a v e n g e d 3 10 N o Data 2 7 Total 29 100  R a v e n s s c a v e n g e d c a r c a s s e s within 1 h of p l a c e m e n t for six c a r c a s s e s a n d b e t w e e n 4-24 h for the remainder. T h e c a r c a s s e s w e r e s u s p e c t e d to h a v e b e e n s c a v e n g e d by c r o w s a n y w h e r e from 1 h to 14 d after placement. T h e c a m e r a s failed to take pictures of the s c a v e n g i n g s p e c i e s or took pictures for no a p p a r e n t r e a s o n at 11 c a r c a s s e s .  4.3.4 Effects on Predators and Scavengers 4.3.4.1 C o m m o n Ravens A total of 13 r a v e n s w e r e found d e a d during the intensive baiting c a m p a i g n b e t w e e n d a y s 12 and 4 7 after the start of the baiting (Table 4.10 a n d Figure 4.1). T h e coastline b e t w e e n Fury B a y eastward to Iphigenia Point, yielded six p o i s o n e d r a v e n s b e t w e e n d a y s 13 a n d 20, or about 1.14 ravens/km of coastline. T h e e a s t  55  c o a s t from Holland Point north to M c p h e r s o n point yielded four d e a d r a v e n s or 0.38 ravens/km coastline. T h r e e w e r e also found in H e n s l u n g C o v e late in A u g u s t , 1 9 9 5 . All w e r e within 3 0 0 m of the shoreline. Liver residue a n a l y s e s confirmed all 13 ravens w e r e e x p o s e d to b r o d i f a c o u m . T h e level of brodifacoum residue ranged from 0.985 mg/kg to 2.522 mg/kg with a m e a n of 1.353 mg/kg. T h e r e w e r e no significant differences in residue levels b e t w e e n mature a n d immature birds (two tailed t-test P>0.05) or b e t w e e n m a l e a n d f e m a l e s (two tailed t-test; P>0.05) (Table 4.11).  56  CO CO "D C  re  Zf  £  .c £• ro c o E 3  o E  CO E  CO o o ro  CO CD  ro o to  to ro  £ m  ro  ro  o E co  o E  CO  J=  £< ro c  ro c  o E  o E 3  3  CL  CL  CO CO  CL  ro c o 3  CL  o  CO c c CO to E « o o  •ro °to3 E "to XJ  ro CO i  ro "a 3  ro o  73 o o  JD  CO CO  CO CD  CO CD  CO CD  o E  o E  o E  ro  ro  ro 1  CO .c  CO x:  CO .c  ro c  ro c  ro c  £•  o E 3  CL  £•  o E  CL  £•  o E 3  CL  S  0  c  o O  i  +  i  1  1  +  +  1  CM CM m CM  lO CO OJ o  m CO  +  +  CO CM CM  CO CM CM  1  1  1  1  CO CM  o CO CO  CO  CM CM p t—  ra  +  or E  S  I'if  CD  ro  CO p  o CO  o p  2 DC Q)  T3 i_ O CO  CD  XZ  2 !G  o  c o  M—  co CD ' co "O C CO CO  x: £• ro c  CO CO  ro  <2 <S +  c  o E  o E co  CO O)  CO  CO  tz o •E E o o  o E co  0.  o O  > ro i_  ro  3  CO  D)  ro  o E  Q.  S o o  CO  o  T—  to D)  CO  o o O co  -9  .2> co CD  ID 03  CO O  O CM  CO CM  O O  CM CM  O CM  a> CO  CO CM  *s  CM  <u to > £ II .£ T3 T3" "> 1 0  -+—»  c c o o  °  C D  CD ™  M to  <  -+—'  Zl CD  •* 0)  ,_- si  X CD  o  •S  Zl •  o o•  CO  c 3  o o o LL  CL  O  to  Q. O  CD >  o O to 3  O  "2  ro N ro  X  •2  T3 C D•2  CM CM  . ro  P •  >»  d g|  •Q ro (0 CD — I -o  3  —>  c CO  > or ro  CM 3 3  'ra c  Q Z  Q  CO  CO CD Zl •g 'co  Ei  LO  T3  2 to CU a)  CO >  CO > o  o  to 3 o  "E ro N  ro CO £> 3 LL  CO  "D  ro Q  ro m 3  o  3 LL  "E ro N CO  £•  o  CO CM >, 3  —>  00 CM >, 3 ~3  LO  05 CM >, 3 ~3  O) C>% M 3 -3  -  o o o ,_ 0- ro  ro m  I .a Q  X  X  3 ~3  o  ro m  ro  CM >,  u.  LL  ro CO  co  CD  LU  CO  T—  CO  to  3 -3  CD  3 3 <  to 3  CD  3 <  CD  CO  CD  O O  o O  O  >  CD C 3  >  X  CD C 3 (0 c CO X  CO CM  CO C M •4-*  to  c CO  to 3  C 3D  <  to 3  C 3O  <  CM  T3  CO  >  O  CD  C _3  o o O ro cu o <n o  to  c CO  X  to  ^3 **  U  C M •»-> (0 3 | C 3O  ro <o  8§ $ 8  8 0)  < 3 co  .  to c  Q) ' F U s "5 (UC  CO 1  o  C  Table 4.11. C o m m o n raven {Corvus corax) liver brodifacoum residue levels (mg/kg), L a n g a r a Island, 1995 (geometric m e a n ; 9 5 % c o n f i d e n c e interval in brackets). e  Sex  Immature (n=6)  Mature (n=7)  M a l e (n=6)  F e m a l e (n=4)  1.18 (0.98-1.41)  1.45 (1.07-1.91)  1.24 (1.03-1.48)  1.24 (-0.82-28.05)  N e c r o p s y revealed that 6 9 % of the ravens had died from s e v e r e p u l m o n a r y h e m o r r h a g i n g a n d the remainder of intramuscular or intracoelemic h e m o r r h a g i n g (Table 4.12). O n e death could not be determined d u e to autolysis, however, the liver b r o d i f a c o u m concentration w a s similar to the others.  Table 4.12. Primary sites of h e m o r r h a g e in c o m m o n r a v e n s  (Corvus corax) found d e a d , L a n g a r a Island, 1995. Primary Site of Hemorrhage Pulmonary Breast Muscle Intracoelemic Undetermined  Source of Brodifacoum  Percent  No.  69% 15% 8% 8%  9 2 1 1  Poisoning  to Ravens  O n l y s e v e n raven g i z z a r d s contained sufficient material for protein e l e c t r o p h o r e s i s . A protein band matching rat or muskrat h a e m o g l o b i n w a s s e e n in o n e . N o rat s e r u m albumin w a s o b s e r v e d . N o protein b a n d s matching s h r e w controls w e r e o b s e r v e d and no b a n d s from bait or snails. F o u r s a m p l e s s h o w e d protein matching raven albumin.  58  Table 4.13. F o o d remains in the g i z z a r d s a n d intestines of 13 c o m m o n ravens (Corvus corax) found d e a d , L a n g a r a Island, 1995. Contents  Frequency  No.  (%) R a t Hair Bait Invertebrate Marine Other Vegetation Marine Other Vertebrate B o n e Unidentified  38 31  5 4  46 15  6 2  46 46 8 46  6 6 1 5  a  a- Hair characteristic of Norway rat also found in two.  F i v e (38%) of the g i z z a r d a n d intestinal contents contained hair characteristic of N o r w a y rats, including the contents from the raven w h i c h s h o w e d positive for rat or muskrat h a e m o g l o b i n in protein electrophoresis (Table 4.13). T h e g i z z a r d contents of o n e raven contained bait block fragments mixed with 11 unidentified a v i a n b o n e f r a g m e n t s (1.09 ± 0.12 c m long x 0.46 ± 0.05 c m w i d e (mean+s.e.)). N o rat hair w a s found in the g i z z a r d o r intestinal contents. T h r e e regurgitated raven pellets w e r e found in E g e r i a B a y o n d a y 3 5 post start of baiting (16.8 ± 1.9 g, 4.45 ± 0.31 c m long, 2.66 ± 0.08 c m w i d e ( m e a n t s.e.)). All w e r e situated o n logs, two o n the b e a c h a b o v e the high tide line, a n d o n e u n d e r the forest c a n o p y . T h e blue brodifacoum bait predominated in t h e pellets, although hairs characteristic of N o r w a y rat w e r e a l s o found in e a c h .  59  Raven Activity -1996 F i v e nests w e r e visited, however, none w e r e active (Table 4.14). A nest in Dibrell B a y w a s empty and the bark and tops of b r a n c h e s u n d e r the nest w e r e c o a t e d with blue tinted f a e c a l matter. T h e remains of a s c a v e n g e d raven w e r e found nearby. P r e y remains under the nest included chitons a n d 12 regurgitated pellets consisting primarily of bait a n d s e v e n distinct piles (1- >100 p i e c e s ) of bait c r u m b s . S t r e t c h e d p i e c e s of plastic bag that had w r a p p e d t h e bait w e r e found in 5 8 % of the pellets. T h r e e more regurgitated pellets consisting of the blue bait w e r e found b e t w e e n N o - N a m e Point and Dibrell B a y , while s e a r c h i n g for raven n e s t s .  Table 4.14. Inactive c o m m o n raven (Corvus corax) nests, L a n g a r a Island, 1996. Nest # Geographic Y e a r of Last Tree Species Tree Nest Location K n o w n Activity dbh HAG 1 Mcpherson 1995 Sitka S p r u c e 18 m Point a  b  2  Mcpherson Point  -  Western Hemlock  10 m  3  Mcpherson Point  1994  Western Hemlock  7 m  4  Dibrell B a y  -  Sitka S p r u c e  1.16 m  15 m  5  Hazardous Cove  1995  Western Hemlock  0.73 m  7.30 m  a dbh: Diameter at breast height, b HAG: Height above ground.  60  T h e prey r e m a i n s under the nest in H a z a r d o u s C o v e c o n s i s t e d of snails (5 Haplotrema  sp. a n d 1 Vespericola  sp.), two limpet shells, black-tailed d e e r hair,  eight chitons a n d the skull of an adult N o r w a y rat. P l a s t i c b a g s w e r e found outside 54 of 107 bait stations investigated. T e a r patterns characteristic of the raven positive controls w e r e found o n 9 8 % of the b a g s . T h e imprint of a b e a k and/or bill tip of ravens w e r e found o n the a l u m i n u m trays from 12 stations. T h e imprints w e r e within 1.7±0.26 (mean ± s.e.) c m of the tray e d g e w h i c h f a c e d either o p e n i n g of the stations. Bait c r u m b s w e r e found outside stations or o n top of nearby logs at nine bait stations. T h e remains of s e v e n more ravens were found or reported including a fresh, d e a d raven at the top of the b e a c h in H a z a r d o u s C o v e (Table 4.15). N e c r o p s y results confirmed its death w a s d u e to s e v e r e , bilateral pulmonary h e m o r r h a g i n g . F o u r pairs of r a v e n s w e r e o b s e r v e d flying along the north e n d of the island b e t w e e n L a n g a r a Lightstation a n d M c p h e r s o n Point, in E g e r i a B a y , H a z a r d o u s C o v e , a n d at Lord Bight on the w e s t coast. T h i s indicates that s o m e r a v e n s w e r e still alive o n L a n g a r a Island in 1996.  61  E E o o CO  E E o o  CD  £2  1—  CL  co"  u 0 0  ZJ  <  0  CL *f  E E o o o sz -4—  0  1  ZJ  ZJ  <  <  CO  CO  ZJ  <  o CO  i_T 0 CO  'co  o LU -j  SZ  CL  CO 0 £Z  CO  c 'co E 2 CO  -4—  1  0 0  CO  o CD -+—'  CO 0  sz  c 'co E  LL  •3  CO 0  I—  L—  0  sz  l_  CO 0  l_  CO T3 CO  CO  CO  CD  I  -Q  b  o O co' . CO CO CO CO T -  a ?  CL  CO  TO  -4—•  0 CD £Z 0 > CO  0 0  J* CO  o CO c 'o CL  0  c "o CL 0  E co Z  6 z CO  c 'co E 2 CO  o  CD  cz  _ZJ CO  c  0  CO  JXL CO  0 0  0 0  0  CO  CO  TO "i_ 0 CD LU  > o O  CO  CO N CO  0 CD LU  "2  1 CO  CO  XI  C  CO  'l_  co  ZJ CD ZJ  <  • i5  (0 CO — I —I  CO  CO ZJ 0  <  5 2 •8 *  CO  -*—'  0  CO  CO  c 'co E 2  -I—»  0  > o  CO  CM  CO  LO  CO  CM CO  4.3.4.2 Northwestern Crows A s u m m a r y of information o n the c r o w s collected is in A p p e n d i x T a b l e 4-3. B r o d i f a c o u m w a s detected in o n e pooled s a m p l e of crow livers collected from L u c y Island, 12 d after the start of the baiting c a m p a i g n in 1995 (Table 4.16). A c r o w w a s found d e a d in H e n s l u n g C o v e o n A u g u s t 7, 1 9 9 5 but no brodifacoum w a s d e t e c t e d . T r a c e a m o u n t s of brodifacoum (0.048 mg/kg) w e r e detected in o n e crow collected in M a y 1 9 9 5 before the intensive baiting b e g a n o n L a n g a r a Island.  Table 4.16. B r o d i f a c o u m residue levels in livers of Northwestern c r o w s (Corvus caurinus), L a n g a r a Island, 1 9 9 5 . Pool No. C r o w #'s Brodifacoum (mg/kg) 1 1 0.048 a 15, 16 ND 3 ND 2, 3 , 4 , 5 4 6,7,8 0.019 5 9, 10, 11 ND 6 12, 13, 14 ND 7 15, 16 ND 17 8 ND 6 18 ND 9 19,20,21 ND 10 22, 2 3 , 2 5 ND 11 24 LA a  C  2  b  d  a Collected in May 1995. b Found dead in Henslung Cove. c None detected. Detection limit <0.01 mg/kg. d Lost in analysis.  4.3.4.3 Bald Eagles A total of 2 2 bald e a g l e s w e r e trapped; two before the baiting b e g a n , a n d 2 0 o v e r the c o u r s e of the baiting for a 3 6 % trapping s u c c e s s rate. A n o t h e r e a g l e w a s  63  r e s c u e d from a s u r g e c h a n n e l at N o - N a m e Point 3 d before the eradication p r o g r a m . M a i n l y adult birds w e r e targeted although four y o u n g of the y e a r w e r e a l s o t r a p p e d . T h e overall s e x ratio w a s 13 males: 10 f e m a l e s . A detailed s u m m a r y of e a g l e s trapped is in A p p e n d i x T a b l e 4-2. B r o d i f a c o u m w a s detected in the p l a s m a of three individuals (15%).  Each  w a s from a different a g e c l a s s and two w e r e female. T h e greatest p l a s m a r e s i d u e w a s d e t e c t e d in the subadult trapped at C o h o e Point (Table 4.17 a n d F i g u r e 4.1).  Table 4.17. B a l d e a g l e (Haliaeetus leucocephalus) a n d prothrombin times, L a n g a r a Island, 1995. No. 1 2 3 4 5 6 7  Date  Location  Sex  July 5 July 6 July 7 J u l y 18 J u l y 19 July 23 J u l y 31  Margaret Point  M F M M M M F  A A SA A A Ol A  N o - N a m e Point  M  Yl  N o - N a m e Point N o - N a m e Point  F M  SA A  Dibrell B a y N o - N a m e Point Explorer Bay M c p h e r s o n Point Iphigenia P o i n t Iphigenia P o i n t  3  Brodifacoum Residue (mg/kg)  PT (sees)  ND ND ND ND ND ND ND  -  -  261  -  -  N o - N a m e Point  F  A  12 13 14  A u g u s t 10 A u g u s t 10 A u g u s t 10  Egeria Bay C o h o e Point C o h o e Point  F M M  A A A  0.037 ND ND  177 201  15 16 17  A u g u s t 10 A u g u s t 11 A u g u s t 12  Dibrell B a y N o - N a m e Point A n d r e w s Point  F M M  A SA A  ND ND ND  234 224 151  18  A u g u s t 13 A u g u s t 14  F M F  SA Yl Yl  1.74 ND ND  221  A u g u s t 15  C o h o e Point A n d r e w s Point Margaret Point  August 25 August 26 August 26  Iphigenia P o i n t Dadens Henslung Cove  F F M  Ol A Yl  ND ND ND  122 226 122  19 20 21 22 23  1 1 3 4  Age Class  0.041 ND ND ND  8 9 10 11  August August August August  p l a s m a brodifacoum r e s i d u e s  M e a n ± s.e. a A = Adult; S A = S u b - A d u l t ; OI= O l d Immature; Y l = Y o u n g Immature (Bortolotti 1 9 8 4 )  64  -  147 -  284 185  1 9 7 ± 14  P T s w e r e not performed for the adult a n d y o u n g immature bird e x p o s e d to b r o d i f a c o u m either d u e to lack of blood collected, or b e c a u s e lipids in t h e p l a s m a m a d e t h e m unsuitable for testing. T h e subadult e a g l e P T w a s 221 s (Table 4.17). T h e m e a n P T for all e a g l e s w a s 197 s w h i c h w a s significantly longer (one tailed ttest P<0.05) than the m e a n control P T of 125 s (Table 4.18).  Table 4.18. B a l d e a g l e (Haliaeetus control prothrombin times. Number Sex Age a  1 2 3 4 5 6 7 8 9 10 11  M F M M M F F F F M M  YI YI Ol YI YI A Ol YI YI YI YI  leucocephalus) PT (sees) 70 177 151 125 59 149 108 146 145 145 97  m e a n + s.e. a Yl= Young Immature; OI= Old Immature; A= Adult  65  125 ± 11  4.4 Discussion 4.4.1 C a r c a s s Locations of Brodifacoum Poisoned Norway Rats A total of 13.4% of radio collared Norway rats died a b o v e ground and all w e r e adult females. This is likely an underestimate of the actual frequency of death a b o v e ground, b e c a u s e other non-radio collared rats found d e a d a b o v e ground included all a g e and s e x c l a s s e s . T h e location of death d o e s not a p p e a r to be a g e or s e x related. In 1994, o n e of three radio-collared rats died a b o v e ground after the poisoning operation on L u c y Island (Howald 1995). T h e s e results are in apparent contrast to N e w Z e a l a n d operations where all 16 radio-collared Norway rats on Ulva Island died in their burrows (Taylor 1993) and no poisoned rats w e r e found on the surface of H a w e a Island (Taylor and T h o m a s 1989). Further, only four Norway rats w e r e found a b o v e ground o n B r e a k s e a Island in N e w Z e a l a n d after similar brodifacoum baiting for rats (Taylor and T h o m a s 1993). However, there w a s no indication of a quantified s e a r c h method in these studies. During anticoagulant poisoning operations o n farms in E n g l a n d , a small proportion (%) of the Norway rat population w a s found to have died a b o v e ground, the majority died below ground (Harrison et al. 1988; F e n n et al. 1987). In laboratory enclosure trials with wild Norway rats, 6 7 % were reported to have died in o p e n a r e a s after receiving a lethal d o s e of brodifacoum (Cox and Smith 1992). Similarly, G e m m e k e (1990) s h o w e d that captive anticoagulant poisoned rodents died a b o v e ground a s often a s below. More work is required in this a r e a to accurately determine  66  the proportion of rats that die a b o v e ground, thus making t h e m s e l v e s available to scavengers. T h e remains of the adult female found in the tree confirms that s o m e rats w e r e either s c a v e n g e d or preyed o n . T h e s c a v e n g e r or predator could not be identified but the location of the tree 2 2 5 m inland suggests that a raven w a s likely responsible. B a l d e a g l e s w e r e rarely o b s e r v e d under the forest c a n o p y after the ancient murrelets had left the colony in mid-June. R a v e n s , however, were regularly o b s e r v e d in the forest a n d w e r e identified a s the most significant s c a v e n g e r of rats. T h e m e a n time to death for the radio-collared rats w a s 7 d after the start of the baiting program. Although the precise length of the latent period w a s not known, it d o e s indicate that the majority of rats w e r e d e a d and available to s c a v e n g e r s a w e e k after the baiting program b e g a n . For predators, the greatest opportunity to catch a live but toxic rat, w a s within the first w e e k of the baiting c a m p a i g n .  4.4.2 Residues in Norway Rats Found Dead Above Ground T h e p r e s e n c e of brodifacoum in the c a r c a s s e s of poisoned rats found a b o v e ground p o s e s a s e c o n d a r y poisoning hazard to the identified s c a v e n g e r s . T h e liver contained the greatest concentration of brodifacoum a s c o m p a r e d to the c a r c a s s , but w a s not significantly greater than the GIT. This preferential storage in the liver is consistent with other anticoagulants s u c h a s Warfarin, bromadiolone and flocoumafen (Huckle et al. 1989a; Huckle et al. 1989b; Lechevin and Vigie 1992; Newton et al. 1990). T h e liver is the target organ for anticoagulant rodenticides where they act to  67  prevent the reduction of the vitamin K epoxide by inhibiting the epoxide reductase e n z y m e (Mount 1988). However, the liver constitutes only 4 . 1 % by weight of the rat, while brodifacoum is present throughout the c a r c a s s . Thus, adult rats present more of a h a z a r d than smaller rats b e c a u s e of their weight which allows for greater brodifacoum accumulation. T h e G I T w a s the tissue that resulted in the significant interaction effect b e t w e e n a g e / s e x c l a s s and tissue type. T h i s w a s likely a result of the p r e s e n c e of u n a s s i m i l a t e d bait found in the alimentary tracts of s o m e individuals. O n d i s s e c t i o n , s o m e s t o m a c h s w e r e found to be p a c k e d with bait a n d the intestines w e r e tinted with the colour of the bait. T h e juvenile m a l e s contained the greatest concentration of b r o d i f a c o u m in the GIT suggesting that they preferred and continued to f e e d o n the bait e v e n after hemorrhaging had b e g u n . Adult f e m a l e s a p p e a r e d to contain the least a m o u n t of brodifacoum concentration and this m a y reflect the d e m a n d s p l a c e d o n her, s u c h a s providing for the pups by bringing back bait to the burrows. T h i s could a c c o u n t for the high concentration in the juvenile G I T s . T h e m e a n whole body brodifacoum residue concentrations found in the rats o n L a n g a r a s e e m e d to be the highest yet reported and a p p e a r s to reflect the saturation baiting strategy u s e d . D u b o c k (1984) reported a m e a n c a r c a s s residue level of 3.2 mg/kg in rats after saturation baiting with brodifacoum, although 8% contained a concentration a b o v e 10 mg/kg. M e r s o n et al. (1984) live trapped, e u t h a n i s e d a n d a n a l y s e d the w h o l e body brodifacoum b u r d e n s of m e a d o w v o l e s pennsylvanicus)  (Microtus  and found them to range from 2.07+ 0.17 mg/kg (mean+s.e) to  68  4.07± 0.20 mg/kg after aerial application of bait. In laboratory trials, b r o d i f a c o u m concentrations of v o l e s fed 10 mg/kg brodifacoum ranged from a m e a n of 0.40 to 0.53 mg/kg, while v o l e s fed 50 mg/kg brodifacoum bait ranged from a m e a n of 2.17 to 5.21 mg/kg ( K a u k e i n e n 1982). In general, c a r c a s s residue concentrations are positively related to the rate of anticoagulant application (Merson et al. 1984; D u b o c k 1984). T h e distribution of brodifacoum a c r o s s different tissues m a y provide a d e g r e e of s e c o n d a r y poisoning protection, if there is rejection of any of those tissues. F o r example, golden e a g l e s were observed to reject the s t o m a c h and entrails of strychnine poisoned R i c h a r d s o n ground squirrels (Spermophilus  richardsoni)  resulting  in no detectable strychnine poisoning impact to any e a g l e s ( G r a h a m 1977). If a brodifacoum poisoned rat is eviscerated and the GIT not c o n s u m e d , between approximately 3 0 - 5 0 % of the total brodifacoum residue is thereby a v o i d e d . However, there w a s wide variability between e a c h a g e and s e x c l a s s in tissue retention of brodifacoum. This variation may be d u e to the small s a m p l e size and further work is required.  4.4.3 C o m m o n Ravens C o m m o n ravens were the most significantly impacted s p e c i e s in the study. Although no population estimates were made, the 20 ravens found d e a d between July 1995 and A u g u s t 1996 were likely a minimum number affected. C a r c a s s s e a r c h i n g studies have had low s u c c e s s rates, d u e to the efficiency with which s c a v e n g e r s and  69  predators removed the c a r c a s s e s , as well a s the low efficiency of h u m a n s e a r c h e r s (Linz et al. 1991; Brown et al. 1988; M i n e a u and Collins 1988; B a l c o m b 1986; S t u t z e n b a k e r 1984). Therefore, the impact on the raven population c a n only be estimated. O n Saltspring Island, Brenchley (1985) estimated the minimum raven nesting density to be 26/100 k m . A high estimate relative to E u r o p e where 17 nests/100 k m 2  w a s the highest estimated density (Newton et al. 1982). A s s u m i n g L a n g a r a could sustain similar densities, there may have been five to eight territorial, nesting pairs with between three to s e v e n fledglings e a c h (Ehrlich et al. 1988) before the baiting b e g a n . Therefore, the pre-baiting c o m m o n raven population m a y h a v e b e e n from 20 to 7 2 individuals, and the impact of the poisoning operation on the local raven population a p p e a r s to have been heavy. However, the p r e s e n c e of four pairs in 1996 indicates that s o m e individuals survived, or that empty territories w e r e taken o v e r by immigrants from nearby G r a h a m Island.  Necropsy Results and Liver Residue  Analyses  T h e necropsies confirmed that all ravens died of acute, multifocal hemorrhage, symptomatic of anticoagulant poisoning (M. Mcadie, pers. comm.). T h e s e results are in a c c o r d a n c e with other studies investigating anticoagulant poisoning in avian predators (Mendenhall and P a n k 1980; Newton et al. 1990). T h e body condition s c o r e s indicate that none of the birds were emaciated or in poor condition, a n d there  70  2  w a s no e v i d e n c e of d i s e a s e that may have predisposed the ravens to the effects of brodifacoum. T h e role of brodifacoum poisoning could not be determined w h e n only feathers and/or skeletal remains were found. S o m e mortality may have occurred o v e r the winter, independent of brodifacoum use. However, the locations and e v i d e n c e w h e r e individuals w e r e found suggests that brodifacoum poisoning w a s the ultimate c a u s e of death, e v e n though predation may have been the proximate c a u s e or they w e r e s c a v e n g e d after death. A s brodifacoum binding in the liver is a saturable p r o c e s s resulting in a steep d o s e r e s p o n s e (Thijssen and B a a r s 1989; Godfrey 1985), and death from anticoagulants is d e l a y e d allowing for s o m e metabolism and excretion of the ingested d o s e (Godfrey 1985), the relationship between the concentration m e a s u r e d and the initial e x p o s e d d o s e is complex and difficult to interpret. Godfrey et al. (1985) found that liver levels are unsuitable for quantifying exposure to brodifacoum b e c a u s e of the lack of correlation between liver residue concentration and dosing levels. However, it c a n be concluded that the positive analysis of brodifacoum in the liver together with the n e c r o p s y results are symptomatic of brodifacoum poisoning.  Source of Brodifacoum  Poisoning  to Ravens  S o m e raven mortality from s c a v e n g i n g d e a d rats w a s expected, however, primary poisoning from raiding bait stations w a s not. T h e first indication that primary poisoning contributed to raven mortality c a m e after the bait spill at the L a n g a r a Fishing  71  L o d g e in H e n s l u n g C o v e . T w o to three 20 I buckets containing old bait blocks awaiting incineration, were left o p e n and unattended overnight. T h e following morning, the fishing lodge m a n a g e r reported a "sea of black" ravens picking up bait blocks in their bills and flying off with them. O v e r the next 4 d, three ravens w e r e found d e a d and two were o b s e r v e d falling off their roost and picked up d e a d . T h i s m a y h a v e b e e n an isolated primary poisoning event b e c a u s e it w a s not u n c o m m o n to have piles of g a r b a g e accumulate before incineration, and to o b s e r v e ravens work o p e n holes in b a g s and boxes. T h e p r e s e n c e of the bait buckets with o p e n lids presented an e a s y opportunity for ravens to investigate a new food source. T h e attraction to the bait indicates the palatability of this toxic food s o u r c e to ravens, however, it is not known if ravens were responsible for raiding bait stations during the intensive baiting in 1995. T h e r e were no reports of ravens raiding bait stations during the initial c a m p a i g n and stations were not investigated a s a s o u r c e of poisoning. If ravens w e r e removing bait from stations, it would have b e e n a s s u m e d rats w e r e responsible. T h e beak marks on the aluminum trays, regurgitated pellets of bait containing bits of plastic bag, and torn b a g s found outside bait stations, confirmed that ravens raided bait stations over the winter of 1995/1996. T h e systematic checking of bait stations by operators might have been o b s e r v e d by ravens and led to their investigating the stations, finding the bait blocks, and being primarily p o i s o n e d . R a v e n s preyed o n pinyon jay (Gymnorhinus  cyanocephalus)  e g g s and y o u n g within  24 h after researchers had climbed trees with nests (Marzluff and B a l d a 1992).  72  Similarly, in the O k a n a g a n V a l l e y of British C o l u m b i a , ravens have b e e n o b s e r v e d systematically checking orchards for songbird nests, perhaps imitating researchers studying t h e m (A. Preston pers. comm.). In M a y 1996, eight bait stations, including four all in order and along a line, that w e r e c h e c k e d and re-armed 2 d previously, had the bait removed. T h e design of the stations and how they were staked down, allowed ravens to e m p l o y o n e of three tactics to gain a c c e s s to the bait in them: reaching into the station and pulling the aluminum tray and b a g g e d bait blocks out, lifting o n e end of the station s o that the bait rolled c l o s e r to either opening, or uprooting the stations from the s t a k e s s o the bait rolled out. In N e w Z e a l a n d , western w e k a s and keas (Nestor notabilis) have b e e n o b s e r v e d reaching into the stations and pulling bait out ( E a s o n and S p u r r 1995; Taylor and T h o m a s 1993). However, the action of the ravens o n L a n g a r a is apparently the first reported c a s e of non-target s p e c i e s completely disrupting bait stations to gain a c c e s s to the bait. Clearly, the bait stations w e r e inadequate to exclude c o m m o n ravens and need to be modified for future rat eradication programs. T h e primary and s e c o n d a r y poisoning a p p e a r s to have had a large impact on the local c o m m o n raven population on L a n g a r a Island. In N e w Z e a l a n d , the entire population of western w e k a w a s exterminated from Tawhitinui Island after feeding o n brodifacoum bait intended for ship rats, and by eating d e a d or dying rats (Taylor 1984 in E a s o n and S p u r r 1995). Similarly, 80-90% of Stewart Island w e k a w e r e primarily and secondarily poisoned ( E a s o n and Spurr 1995). Both w e k a s and c o m m o n ravens  73  s h a r e an a g g r e s s i v e and inquisitive behaviour and opportunistic, omnivorous diet (Falla et al. 1983; Ehrlich et al. 1988) which contributed to their decline in population. T h e gizzard analysis confirmed that ravens were not c o n s u m i n g exclusively either rats or bait during the initial c a m p a i g n but the content analysis is not a sensitive e n o u g h indicator to determine the source of poisoning.  N o rat hair, rat protein or bait  w e r e detected in six d e a d ravens in 1995 probably b e c a u s e of the d e l a y e d time to death after ingestion of brodifacoum allowing for evacuation of the GIT contents (Godfrey 1985). Captive barn owls feeding on brodifacoum poisoned rats died between 5-6 d after the last poisoned rat w a s c o n s u m e d (Mendenhall and P a n k 1980). It is possible, however, that brodifacoum exposure may have originated from another s o u r c e s u c h a s invertebrates found feeding o n the bait ( S e e C h a p t e r 5). T h e p r e s e n c e of bait crumbs along with Norway rat hairs m a y indicate that ravens w e r e both primarily and secondarily e x p o s e d to brodifacoum, or that the bait c r u m b s originated from the rats. T h e latter s e e m s more likely b e c a u s e on dissection, rat s t o m a c h s and intestines w e r e laden with bait crumbs. However, the p r e s e n c e of bait c r u m b s without the detection of Norway rat hair, d o e s not preclude the possibility of s e c o n d a r y poisoning. F o r ravens 11 and 12, where only bait c r u m b s w e r e detected in their gut contents, it s e e m s probable that primary poisoning w a s the c a u s e b e c a u s e they w e r e found within 4 d after the bait spill in Henslung C o v e . Secondary Poisoning  and Toxicoiogicai  Significance  R a t s w e r e confirmed a s a source of brodifacoum to ravens, however, it is unclear if o n e or more rats were required to impair hemostasis and c a u s e death. T h e  74  L D , the single acute, oral d o s e required to c a u s e death to 5 0 % of a population, is 5 0  u s e d a s an index of toxicity and demonstrates a pharmacological risk of brodifacoum. The L D  5 0  of brodifacoum for the raven is unknown, however, I calculated a v a l u e that  offers 9 5 % s p e c i e s protection, with 9 5 % and 5 0 % confidence, from 10 published brodifacoum L D  5 0  values representing nine major families of birds (Aldenberg and  S l o b 1993). In other words, the L D  5 0  with 9 5 % and 5 0 % confidence estimate for 9 5 %  s p e c i e s protection b a s e d on published values (Table 4.19). F o r any unknown s p e c i e s the L D  5 0  would be a b o v e 0.56 mg/kg (50% confidence) or 0.105 ( 9 5 % confidence).  T h e closest related s p e c i e s to the raven with published L D  5 0  O r d e r Passeriformes, specifically the house sparrow (Passer  v a l u e s are from the domesticus)  with an L D  >6 mg/kg, blackbird (Turdus merula) and hedge sparrow (Prunella modularis)  5 C  >3  mg/kg (Godfrey 1985). T h e s e values represent the highest d o s e administered. Caution must be u s e d w h e n estimating hazard b e c a u s e the c l o s e t a x o n o m i c relation cannot be consistently u s e d to predict the sensitivity of birds to pesticides (Hill 1994). Therefore, the L D  5 0  range for ravens may be between 0.56 and >6 mg/kg.  With a m e a n weight of 1.19 kg for all ravens found d e a d over the s u m m e r of 1995, a s little a s 0.69 mg to > 7.14 mg brodifacoum could result in a 5 0 % c h a n c e of lethal hemorrhaging. A single adult rat, with a m e a n residue level of 1.4 mg, would result in the equivalent d o s e of 1.17 mg/kg if c o n s u m e d whole. Captive c o m m o n ravens fed diphacinone poisoned adult rats c o n s u m e d everything except the skin and b o n e s (J. Marzluff, pers. comm.). However, small rats were eaten whole and a pellet w a s produced. T h e regurgitation of a pellet would d e c r e a s e the amount of brodifacoum  75  X ingested and possibly, d e c r e a s e the risk of hemorrhaging. B a r n owls feeding o n brodifacoum poisoned mice and producing pellets, reduced the amount of c o n s u m e d rodenticide by an a v e r a g e of 2 5 % , thus lowering their risk of hemorrhage (Gray et al. 1994). A s s u m i n g ravens regurgitated a pellet containing 2 5 % brodifacoum, this would d e c r e a s e the single brodifacoum d o s e to 0.88 mg/kg. This would still be within the estimated L D  5 0  range, thus a single adult rat may pose a s e c o n d a r y poisoning h a z a r d  to ravens.  Primary Poisoning  and Toxicologicai  Significance  T h e toxicologicai significance of bait blocks to ravens is difficult to accurately interpret b e c a u s e ravens regurgitate a pellet (due to the high, indigestible,- paraffin w a x content) that likely contains brodifacoum. E a c h block contains 1 mg brodifacoum, e n o u g h for a raven to have a 5 0 % c h a n c e of haemorrhaging, if all the brodifacoum w a s ingested. Obviously, the c h a n c e of lethal poisoning increases with the n u m b e r of blocks that are c o n s u m e d , and a s e a c h station w a s armed with three bait blocks o v e r the winter months, e a c h raven w a s e x p o s e d to a s m u c h a s 3 mg brodifacoum at e a c h bait station it raided. It s e e m s probable that any o n e raven upon discovery of the e a s y food s o u r c e found in a bait station, continued to raid more than o n e station.  76  Table. 4.19. S p e c i e s and L D values u s e d for calculating the value that offers 9 5 % bird s p e c i e s protection with 9 5 % and 5 0 % confidence limits. Latin Name C o m m o n Name LD (mg/kg) Reference 5 0  5 0  Black-Backed Gull Pukeko California Quail Mallard D u c k  Larus dominicanus P. porphyrio melanotus Callipepla californica Anas platyrhynchos  Harrier Hawk Ring Necked Pheasant Paradise Shelduck Chicken  Circus approximans Phasianus colchicus Tadorna variegata Gallus gallus  >20.0 3.15  Japanese Quail  Coturnix japonica  11.6  a  a  <0.75 0.95 3.3 2.3  Godfrey 1985 Godfrey 1985 Godfrey 1985 Godfrey 1985; Ross et al. 1978 Godfrey 1985 Godfrey 1985  10.0 10.0  Godfrey 1985 Ross et al. 1977; Taylor 1993 Ross e t a l . 1976  a Mean of LD values in literature. 50  C o n f i d e n c e limits for h a z a r d o u s concentrations b a s e d o n logistically distributed brodifacoum L D value calculated following A l d e n b e r g and S l o b (1993). T w o rules w e r e followed a s e m p l o y e d at the National Wildlife R e s e a r c h Centre (P. M i n e a u , pers. comm.): 5 0  1. Include known absolute L D values. D o not u s e < or > L D v a l u e s if they are within the range of the absolute values. 2. If < o r > L D 5 0 values fall outside of absolute L D range, include and new range h a s b e e n established. 5 0  5 0  5 0  T h e 9 5 % a n d 5 0 % confidence limit for brodifacoum L D 0.56 mg/kg.  77  5 0  data is 0.11 mg/kg a n d  4.4.4 Northwestern Crows Brodifacoum w a s detected in low levels in the s a m p l e d crows. T h e crow collected in M a y 1995 w a s e x p o s e d to brodifacoum from L u c y Island b e c a u s e brodifacoum w a s not available on L a n g a r a Island until July 1995. It is unknown if e x p o s u r e occurred during the baiting on L u c y Island in 1994, or if e x p o s u r e occurred after the bait w a s removed from the stations in A u g u s t 1994. T h e biological half-life of brodifacoum h a s b e e n estimated to range between 120-220 d (Godfrey 1985; P a r m a r et al. 1987). With s u c h a range, exposure may have occurred during the baiting on L u c y Island. Brodifacoum w a s still present and available on L u c y Island 9 months after the removal of the bait from the stations. In M a y 1995, four old bait blocks (9.6 g) w e r e found under a log on the west side of Lucy Island and contained 10.99 mg/kg brodifacoum. S n a i l s found near the bait were collected and contained a concentration of 0.910 mg/kg brodifacoum. Therefore, it is possible that crows could h a v e b e e n e x p o s e d to brodifacoum from preying on invertebrates, that had fed o n old bait, during the winter of 1994/1995. Inspection of the adult rat c a r c a s s e s visited by crows, but not s c a v e n g e d , revealed patches of missing hair. T h e rats had also b e e n m o v e d from their original placement. T h e photographs revealed that crows were grabbing the c a r c a s s e s by the tails, legs and flanks. T h e crows were apparently unable to fly off with the larger rats and w e r e unable to break the skin of fresh, d e a d rats. Thus, brodifacoum e x p o s u r e from s c a v e n g i n g rats w a s minimised by the crow's inability to carry whole rats to their  78  p e r c h e s for consumption, and their inability to break the skin to gain a c c e s s to the contaminated flesh.  4.4.5 Bald Eagles B a l d E a g l e s w e r e confirmed to have been e x p o s e d to brodifacoum but no mortality w a s detected during the intensive baiting in 1995. Similarly, no e v i d e n c e of lethal poisoning to N e w Z e a l a n d falcons (Falco novaeseelandiae) novaeseelandiae),  or moreporks (Ninox  the main avian predators at risk, w a s noted o n B r e a k s e a Island in  N e w Z e a l a n d , though they did prey on rats (Taylor and T h o m a s 1993). c o m p a r a b l e numbers of southern great s k u a s (Catharacta  Further,  lonnbergi) and N e w  Z e a l a n d F a l c o n s w e r e s e e n before and after a similar baiting operation o n H a w e a Island in N e w Z e a l a n d (Taylor and T h o m a s 1989), but, t h e s e numbers could h a v e b e e n maintained by immigration. E a g l e s w e r e not attracted to rat c a r c a s s e s put out for them, but m a y h a v e preyed o n rats during the latent period. E a g l e s are known to take rodents and other m a m m a l s a s s o c i a t e d with a body of water, s u c h a s muskrats (Ondatra  zibethicus)  (Knight et al. 1991; W a t s o n et al. 1990). O n the S a n J u a n Islands, rabbits w e r e identified a s the eagle's main prey item (Retfalvi 1970). E a g l e s s p e n d m u c h of their time perching o n trees overlooking the water and shoreline (Gerrard and Bortolotti 1988) and may have opportunistically preyed on a rat if it displayed s y m p t o m s of anticoagulant poisoning near or in the intertidal z o n e . Rats w e r e likely available for diurnal e a g l e s b e c a u s e 3 5 % of rats were live-trapped during daylight h a n d w e r e  79  known to utilise the b e a c h e s , b e c a u s e the contents of a rat burrow on L u c y Island contained a c a r a p a c e and legs of a shorecrab (Howald 1995). O n e of the radiocollared rats (see above) and other rats were found d e a d on or near the b e a c h , a n d two rats w e r e found to have died sometime in the afternoon, including o n e o n the b e a c h e d g e a b o v e the winter high tide line. T h e apparent low brodifacoum exposure of the bald eagle population w a s likely d u e to the highly productive marine environment around L a n g a r a Island w h i c h is the major, if not exclusive, foraging area. Bald eagles were c o m m o n l y o b s e r v e d fishing for Pacific s a n d l a n c e (Ammodytes s a l m o n (Oncorhynchus  hexapterus),  Pacific herring (Clupea harengus)  and  spp.). In 1994, over 100 e a g l e s were o b s e r v e d fishing off the  west coast of L a n g a r a Island. Knight et al. (1990) reported that, for coastal nesting bald eagles, seabirds are the predominant prey item. T h e spatial separation of the foraging and baiting a r e a s reduced the risk of poisoning to bald e a g l e s . Likewise, the potential for barn owl mortality with brodifacoum baiting around farm buildings a p p e a r e d to be low b e c a u s e they preyed on rodents in g r a s s l a n d s a w a y from the buildings (Hegdal and Blaskiewicz 1984). T h e remains of five eagles (4 juveniles and 1 adult) were found in 1996. However, the c a r c a s s e s were too autolytic to determine c a u s e of death. T h e talons exhibited multiple focal areas of reddish discoloration, possibly representing excoriation of s c a l e s or s u b c u t a n e o u s hemorrhage (M. Mcadie, pers. comm.) a n indication of possible brodifacoum exposure. However, the talons w e r e too d e s i c c a t e d to distinguish between lesions and artefacts of post mortem c h a n g e .  80  The  lesions w e r e similar to those s e e n in thermal burns from electrocution (M. M c a d i e , pers. comm.) but there are no electrical power lines on or near L a n g a r a Island. Although eagle mortality w a s detected 10 months after the initiation of the baiting program, the role of brodifacoum poisoning could not be ascertained. B r o d i f a c o u m m a y have been responsible for s o m e mortality b e c a u s e of the w i d e s p r e a d primary poisoning of ravens which, in turn, could have b e e n preyed o n or s c a v e n g e d by e a g l e s . A d e a d bald eagle w a s reported next to the remains of a s c a v e n g e d raven near the lightstation (J. S c h w e e r s , pers. comm.). At least three other remains of ravens were s c a v e n g e d , however, the s c a v e n g e r s could not be identified. F o o d items collected under bald eagle nests in the S a n J u a n Islands included c o m m o n ravens and Northwestern crows (Knight et al. 1990). It is important to note that winter mortality of e a g l e s would likely have occurred independent of brodifacoum use. Overwinter mortality rates for e a g l e s in A l a s k a ranged from 5% to 2 9 % ( B o w m a n et al. 1995). It w a s estimated that 4 6 % of first-year eagles, and 9 % of adults died over the winter in M a i n e ( M c C o l l o u g h 1982; 1986 in Gerrard and Bortolotti 1988). O n L a n g a r a Island, it w a s not u n c o m m o n to find b o n e s and/or c a r c a s s e s of bald eagles before baiting b e g a n . T h e effect of brodifacoum poisoning on the breeding population a p p e a r s to have b e e n negligible. Nesting s u c c e s s declined from 5 7 % in 1995 to 3 5 % in 1996; but there w a s a decline from 3 9 % to 1 0 % nesting s u c c e s s in South M o r e s b y National P a r k in the Q u e e n Charlotte archipelago for the corresponding period (J. Elliott, pers. comm.).  81  Toxicological  Significance  N o brodifacoum L D  5 0  of Residue Levels and Prothrombin  data are available for bald e a g l e s . T h e a b o v e estimate  of 0.56 mg/kg is likely low for bald e a g l e s b e c a u s e the m e a n L D related s p e c i e s , the harrier hawk (Circus approximans) kestrel (Falco sparverius)  Times  5 0  for two more closely  -10.0 mg/kg) and the A m e r i c a n  -8.20 mg/kg (Taylor 1993) is m u c h higher. Thus, using a  m e a n weight of 4.5 kg (Range: 3.6 - 5.6 kg) for all bald e a g l e s s a m p l e d , the d o s e required for a 5 0 % c h a n c e of lethal hemorrhaging may be a s low a s 2.52 mg ( L D : 5 0  0.56 mg/kg) but is more likely around 39 mg brodifacoum. This is equivalent to 1.4 21.6 adult male rats e a c h with a brodifacoum residue loading of 1.809 m g . T h u s , the risk of an eagle hemorrhaging after ingesting a single rat a p p e a r s to be low. T h e 4.3 kg sub-adult eagle trapped on day 3 3 contained the highest concentration of brodifacoum (1.74 mg/l plasma). A s s u m i n g the p l a s m a v o l u m e ( P V ) in bald e a g l e s is similar to that in red-tail h a w k s (mean of 3.5 ml/100g b o d y weight, B o n d and Gilbert 1958), the sub-adult eagle would have a P V of 0.151 litres. T h i s yields a total p l a s m a brodifacoum loading of 0.262 mg at the time w h e n the blood s a m p l e w a s drawn. T h e immature and female bald e a g l e s had total p l a s m a brodifacoum loadings of 0.0052 mg and 0.0073 mg respectively. T h e p l a s m a brodifacoum levels, however, are a poor indicator of the initial d o s e (Appendix 1; Huckle et al. 1989b). T h e p l a s m a brodifacoum loading of J a p a n e s e quail (Coturnix japonica)  24 h post dosing with brodifacoum at 1.4, 0.7 and 0.35  mg/kg, represented only 1.16%, 0.32% a n d 0.19% of the original d o s e respectively (Appendix 1). A s s u m i n g that quail are a representative model and that the e a g l e s  82  w e r e trapped and a blood s a m p l e drawn around 24 h after exposure, a rough estimate of the original d o s e c a n be made. T h e p l a s m a concentration of the sub-adult e a g l e (1.74 ppm) w a s greater than the p l a s m a concentration of the J a p a n e s e quail at 2 4 h post dosing, therefore, it w a s likely e x p o s e d to more than 1.4 mg/kg brodifacoum or more than 6 mg brodifacoum, which is equivalent to more than 3.3 rats (1.809 mg brodifacoum/rat). A g a i n , a single rat w a s not likely the source of brodifacoum, but more likely having c o m e from c o n s u m i n g a raven or several rats. T h e P T is u s e d to screen for deficiencies in the vitamin-K d e p e n d e n t clotting factors. T h e u s e of a m a m m a l i a n thromboplastin for avian P T determinations results in wide variation within and a m o n g s a m p l e s (Appendix A; Griminger 1986). A s well, a sub-lethal e x p o s u r e to brodifacoum would likely not have been detected b e c a u s e a 3 0 % concentration of these factors may provide a normal P T (Brown 1988; Hoffman et al. 1988). A s well, a markedly increased P T would likely have c o r r e s p o n d e d to a positive detection of brodifacoum residue in the p l a s m a . Therefore, it cannot be c o n c l u d e d that the bald e a g l e s w e r e at risk of hemorrhaging b e c a u s e a significantly longer P T w a s detected. S a v a r i e et al. (1979) found golden e a g l e s (Aquila chrysaetos)  experienced increases in the P T from the control time of 2 3 s to 9 0 0 s (39  times longer than the control time) without mortality after feeding o n diphacinone contaminated meat. S o m e of those golden eagles, however, did experience hemorrhaging. T h e effectiveness of the mammalian thromboplastin to detect a significant increase in P T w a s investigated (Appendix A ) . T h e thromboplastin kit w a s able to  83  detect a significant increase in the P T of J a p a n e s e quail d o s e d with brodifacoum. T h e quail had a significant longer P T at 2.5 times the m e a n control time at 7 2 h post d o s e . A s s u m i n g quail are an effective model, no eagles were clinically at risk of hemorrhaging, i.e., all w e r e less than 2.5 times the control time, a s detected by the Coulter P T fibrinogen kit. T h e risk of hemorrhaging to the e x p o s e d individuals a p p e a r e d to be low.  4.5 C o n c l u s i o n s S e c o n d a r y and primary poisoning studies c a n be divided into three levels a s defined by C o l v i n a n d H e g d a l (1987):' hazard to individuals, short term population effects a n d long-term population effects. This study w a s d e s i g n e d to a d d r e s s the h a z a r d to s c a v e n g e r s and predators at a n individual level. A s e c o n d a r y poisoning h a z a r d to avian predators and s c a v e n g e r s d o e s exist from the u s e of brodifacoum to eradicate rats from seabird colonies along the British C o l u m b i a coast. N o r w a y rats w e r e s h o w n to die a b o v e ground and be available for avian s c a v e n g e r s a n d predators. C o m m o n r a v e n s w e r e the s p e c i e s most significantly impacted a s a result of both primary and s e c o n d a r y poisoning. T h e population effect remains u n q u a l i f i e d , however, breeding pairs were o b s e r v e d alive one y e a r after the baiting program w a s initiated. T h e r e w a s a low risk of brodifacoum poisoning to bald e a g l e s from d e a d toxic rats, although p l a s m a brodifacoum levels established that brodifacoum w a s present in the food chain.  84  Appendix Table 4-1. Dates, locations, weight, age and sex of Norway rats (Rattus norvegicus) found dead above ground, Langara Island, 1995. Rat # Date Location Found Weight Age Class Sex Reported/ (g) Turned In 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35  -  -  July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July  13 14 14 15 15 15 15 16 16 18 18 18 18 19 19 20 20 20 20 20 20 20 20 20 20 21 22 24 25 25  Mcpherson Pt. Mcpherson Pt. Mcpherson Pt. Mcpherson Pt. Lord Bight Hazardous Cove No-Name Pt. Hazardous Cove Mcpherson Pt. Mcpherson Pt Mcpherson Pt. No-Name Pt. Mcpherson Pt No-Name Pt. No-Name Pt Hazardous Cove Mcpherson Pt. Mcpherson Pt. Mcpherson Pt. Mcpherson Pt. No-Name Pt. No-Name Pt. 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The Uptake of Brodifacoum by Invertebrates Feeding on Bait Containing the Rodenticide Brodifacoum or Norway Rat c a r c a s s e s P o i s o n e d with Brodifacoum. 5.1  Introduction Invertebrates play a n important role in the nutrient cycling p r o c e s s by  c o n s u m i n g animal c a r c a s s e s and organic matter, releasing nutrients for primary c o n s u m e r s . T h e bait u s e d a s the rodenticide carrier and attractant provides a rich a n d readily available food s o u r c e for invertebrates (Stejskal et a l . 1994). Similarly, a n i m a l c a r c a s s e s resulting from brodifacoum poisoning are likely attractive to carrion insects. Invertebrates that feed o n the bait and/or c a r c a s s e s , therefore, m a y be a s e c o n d a r y or tertiary poisoning risk to non-target s p e c i e s that m a y otherwise be at low risk of p o i s o n i n g . To-date, the uptake of brodifacoum r e s i d u e s by invertebrates from anticoagulant p o i s o n e d c a r c a s s e s h a s not b e e n studied. T h e objective of this study w a s to identify the invertebrates that are attracted to a n d c o n s u m e brodifacoum p o i s o n e d rats and the bait blocks within bait stations. T h e specific hypothesis tested w a s that invertebrates c o n s u m i n g b r o d i f a c o u m p o i s o n e d rat c a r c a s s e s and/or bait would carry detectable levels of b r o d i f a c o u m . In 1994, different m e t h o d s of collecting and preserving invertebrates for b r o d i f a c o u m a s s a y w e r e tested a s there w a s no e s t a b l i s h e d method available in the literature. T h e results allowed me to establish a protocol for t i s s u e collection. In 1995, invertebrates w e r e again collected using the protocol e s t a b l i s h e d a n d a n a l y s e d for brodifacoum residue.  89  5.2. Materials and Methods 5.2.1  Invertebrate Collections 5.2.1.1 Collection of Invertebrates at Bait Stations In 1994, invertebrates found in bait stations o n L u c y Island w e r e collected a n d frozen (- 5 C e l s i u s ) individually in plastic b a g s . In 1995, o n L a n g a r a Island, bait station operators w e r e requested to d o c u m e n t the p r e s e n c e of a n y invertebrates present in stations. S a m p l e s of invertebrates found in the bait stations w e r e collected during the baiting c a m p a i g n . T h e s a m p l e s w e r e pooled and frozen for a n a l y s i s ( S e e below).  5.2.1.2 Collection of Carrion Insects In 1994, different m e t h o d s of collection and preservation of invertebrates e x p o s e d to brodifacoum w e r e tried:  1. Preservation in 95% Ethanol 1994 pilot study:  Ethanol is an effective preservative for t i s s u e s a m p l e s  w h e n they cannot be quick frozen in the field. However, it is a l s o a solvent a n d m a y l e a c h brodifacoum from tissue s a m p l e s . Rat c a r c a s s e s w e r e laid out in three habitats: along the shoreline in N o o t k a reed g r a s s , in the ancient murrelet colony, a n d in the interior.  Within e a c h location, two c a r c a s s e s , o n e  s n a p trapped rat (unpoisoned) and o n e brodifacoum p o i s o n e d rat w e r e s p a c e d approximately 50 m apart, and c o v e r e d with a c a g e m a d e with  90  h a r d w a r e cloth (1 c m x 1 c m m e s h ) to e x c l u d e a v i a n s c a v e n g e r s .  Each  c a r c a s s w a s c h e c k e d daily or every other d a y until sufficient n u m b e r s of insects w e r e present feeding on the c a r c a s s e s . T h e c a r c a s s a n d a s h o v e l full of soil around the c a r c a s s w e r e carefully placed into a (30 c m X 2 0 c m ) w o o d e n box a n d w r a p p e d with a cotton shirt to allow for g a s a n d moisture e x c h a n g e . Insects w e r e allowed to exit the box from a 5 c m d i a m e t e r hole through a down-pipe e l b o w into a g l a s s jar filled with 9 5 % ethanol. Insects w e r e allowed to c o n s u m e the c a r c a s s for approximately 12 wk before the lid w a s r e m o v e d , and remaining insects collected a n d p l a c e d into the ethanol filled jars. 1995:  carrion insects w e r e again collected using the previous b o x e s .  The  d e s i g n of the box w a s modified to minimise handling of c a r c a s s a n d insects to prevent contamination. T h r e e brodifacoum p o i s o n e d rats w e r e p l a c e d individually into b o x e s with a s h o v e l full of soil a n d c o v e r e d with n o s e u m netting. A funnel w a s placed o n o n e e n d for an entrance. T h e exit o p e n i n g w a s a 5 c m diameter d o w n p i p e e l b o w into a jar filled with 9 5 % ethanol. O n l y insects that voluntarily left the c a r c a s s into the ethanol w e r e a n a l y s e d . D u e to the low n u m b e r of insects collected, insects from all three s a m p l e s w e r e pooled for a n a l y s i s .  91  2. Preservation by Freezing 1994 pilot study: T h e L a n g a r a Lightstation staff offered the u s e of a -5 C e l s i u s f r e e z e r and I d e c i d e d to run the insect collection trials a g a i n a n d freezing the s a m p l e s . T h e fresh collected carrion insect experiment w a s c o n d u c t e d between July and A u g u s t 1994. T h r e e brodifacoum p o i s o n e d a n d six u n p o i s o n e d rat c a r c a s s e s w e r e e a c h placed in a 10 c m diameter, 30 c m long P V C drainage pipe t u b e s (Figure 5.1). T h e tube had a ventilation hole cut in the top a n d c o v e r e d with n o s e u m netting, while small holes w e r e cut in the bottom to prevent accumulation of rainwater. A funnel with the s a m e d i a m e t e r a s the tube w a s p l a c e d o v e r o n e end for an entrance.  O n the  opposite e n d , a 2.5 c m diameter clear P V C tubing (10 c m long) w a s c o n n e c t e d to the tube a n d led into a g l a s s jar. Insects that w e r e in the g l a s s jar w e r e collected every 7 d for 4 wk, sorted by s p e c i e s , a n d frozen for future a n a l y s i s . D u e to the low n u m b e r s of individuals collected e a c h day, individuals of e a c h s p e c i e s w e r e pooled a c r o s s time for brodifacoum r e s i d u e analysis.  3. Preservation by Hexane Cleaned Aluminum Foil and Freezing T h e results of the carrion insect collection trials in 1994 r e v e a l e d that there w a s contamination of control s a m p l e s from both ethanol a n d f r o z e n preservation. In 1995, carrion insects w e r e opportunistically collected from  92  CO -3  E co oC0T3 CO C CO  E co  O  gV- ,_  CO  CO  co c\i .VL™, rn CO CO c S CO CO  S(5  "  E o o  1  O±3_C0J0 cn C O - n CD CD CZ $ £ <D C D co -C-iz CD -I-*-  ro S-g ra  .EE C  P =J  CO — CO  •c6"° a co g  CO CD  o co 2  CO _  £ C0>  -b-fz-c=-5 CD  cog-2S>8 g cu.ratS a> W-Cl  CD CD •D CDi? O - O  c  2  co  M- P  E o o  5  CO  0  CO  CD 'JLZ CD CO c O . r z . b C CD  o  - > CD-S CO > 5 l CD £ ZJ o —  CO  CD  CO C D £ _ Q CD  • cz co "g cz o lug It  C CD> , CO JZ CD *O- *-".^ ZJ 1  / > u _  — c  • •-—•- •—  CD CD .55 CD t z _ r z _ t  co — 1  ^ CD~ CD - t t ^ S l T 3 C D _ CZ 0  ro  S i SI §  E o o  93  b r o d i f a c o u m p o i s o n e d rat c a r c a s s e s . S u r f a c e feeding carrion insects w e r e collected with chemically c l e a n e d (hexane) instruments a n d w r a p p e d in c h e m i c a l l y c l e a n e d a l u m i n u m foil to prevent contamination. T h e p a c k e t s w e r e slipped into pre-labelled whirl-pack b a g s and f r o z e n .  5.2.2 Sample Preparation S a m p l e s w e r e s h i p p e d frozen to N W R C , Hull, P Q . After the s a m p l e s w e r e prepared, one-third (by weight) of all t i s s u e s w e r e archived at N W R C .  5.2.2.1 Bait Station Invertebrates T h e snails a n d slugs had their shells r e m o v e d a n d the soft t i s s u e w e i g h e d . T h e shells w e r e rinsed with methanol in a 1 m m d i a m e t e r kitchen strainer into a c u p holding the soft tissue.  5.2.2.2 Carrion Insects 1.  Samples Preserved in 95% Ethanol T h e insects w e r e s e p a r a t e d and the ethanol w a s allowed to e v a p o r a t e from the insects for approximately 1 h a n d the contents w e i g h e d . T h e insects w e r e h o m o g e n i s e d a n d an aliquot of both insects a n d the ethanol w a s a n a l y s e d for brodifacoum residue ( S e e below).  94  2. Frozen Samples T h e insects w e r e r e m o v e d from containers, counted a n d w e i g h e d .  Methanol  w a s u s e d to rinse a n y brodifacoum residue from original container.  The  insects w e r e c h o p p e d into fine p i e c e s with c h e m i c a l l y c l e a n e d s c i s s o r s to facilitate h o m o g e n i s a t i o n .  3.  Samples Frozen in Hexane Cleaned Aluminum Foil  T h e insects w e r e s e p a r a t e d from the foil p a c k a g e s a n d w e i g h e d . T h e foil w a s rinsed with methanol into a c u p holding the insects. Insects w e r e c h o p p e d with chemically c l e a n e d s c i s s o r s into fine p i e c e s to facilitate h o m o g e n i s a t i o n . H o m o g e n i s a t i o n w a s d o n e in c h e m i c a l l y c l e a n e d g l a s s jars. A c h e m i c a l l y c l e a n e d shaft h o m o g e n i s e r w a s u s e d for all s a m p l e s .  5.2.3 Brodifacoum A s s a y Extraction of brodifacoum residues from prepared s a m p l e s w a s carried out at N o v a M a n n International, M i s s i s s a u g a , Ontario, following the p r o c e d u r e e s t a b l i s h e d by Hunter (1983). T h e limit of detection w a s 0.01 p p m . A s part of the quality control, c l e a n invertebrate and C a n a d a g o o s e canadensis)  [Branta  liver s a m p l e s w e r e fortified with brodifacoum a n d s h i p p e d a l o n g with  treatment invertebrates in 1994. In 1995, c l e a n snails a n d s l u g s w e r e collected from G r a h a m Island, 1 k m a c r o s s from L a n g a r a Island, a n d fortified with b r o d i f a c o u m in the field. S a m p l e s w e r e individually w r a p p e d in c h e m i c a l l y c l e a n e d foil p a c k e t s a n d  95  slipped into pre-labelled polyethylene whirl p a c k b a g s . T h e s e control s a m p l e s w e r e h a n d l e d the s a m e w a y a s t h o s e invertebrates collected from rat c a r c a s s e s a n d bait stations. All original c h r o m a t o g r a p h y graphical tracings from the instrumental a n a l y s e s w e r e verified by B r y a n W a k e f o r d at the N W R C laboratory.  96  5.3. 5.3.1  Results Brodifacoum A s s a y  Quality Control:  In 1994, the brodifacoum residue recovery from fortified  s a m p l e s w a s 1 4 % from insect s a m p l e s a n d 0.0% from the liver s a m p l e (Table 5.1). In 1 9 9 5 , recovery of brodifacoum ranged between 0.0% a n d 1 2 7 % (Table 5.2). All control s a m p l e s did not test positive for brodifacoum.  Table 5.1. B r o d i f a c o u m residue recovery from fortified s a m p l e s , L a n g a r a Island, 1994. Tissue Type Fortified Level Tissue Brodifacoum % Recovery ug B r o d i f a c o u m Weight g R e p o r t e d ug Liver  5.4  2.2  N o n e Detected  0.0  Insect  9.9  4  1.4  14%  97  Table 5.2. B r o d i f a c o u m residue recovery rate from fortified s a m p l e s p r e p a r e d o n L a n g a r a Island, 1995. Tissue Type  Spike Solution A d d e d (ul) 115 ug/ml  ug Brodifacoum Added  Fortified Level (ug/g)  Brodifacoum Reported (ug)  Percent Recovery  Vespericola Snail  sp.  56  6.4  0.30  7.16  111.2%  Vespericola Snail  sp.  54  4.14  0.34  5.26  127.1%  Vespericola Snail  sp.  100  11.5  0.40  11.91  103.5%  Vespericola Snail  sp.  100  11.5  1.46  5.42  47.11%  Ariolimax s p . Slug  100  11.5  0.85  0.00  0.00%  Ariolimax sp. Slug  74  8.51  Ariolimax sp. Slug  90  10.35  0.34  3.02  29.2%  Ariolimax sp. Slug  52  5.98  0.26  4.00  66.9%  93.14  81.0%  S p i k e Solution  -  a  115 ug/ml  Haplotrema s p . Control S n a i l  0.00  0.00  0.00  0.00  Ariolimax s p . Control S l u g  0.00  0.00  0.00  0.00  Ariolimax s p . Control S l u g  0.00  0.00  0.00  0.00  a Not Processed because of leakage of contents in transport, b Control Sample.  98  b  b  b  5.3.2 Bait Station Invertebrates T h e terrestrial molluscs, b a n a n a slugs (Ariolimax sp. a n d Haplotrema  sp.) a n d snails  (Vespericola  sp.) w e r e the most c o m m o n and a b u n d a n t invertebrates found  in bait stations and feeding o n the bait. T h e blue coloured bait could be readily s e e n through the translucent body o n t h o s e individuals found feeding o n bait. B l u e c a s t s from both s n a i l s a n d slugs w e r e evident around stations o n l e a v e s a n d m o s s , a s well a s inside stations. T h e m e a n daily reported proportion of bait stations with s l u g s a n d snails w e r e similar at 8.9% and 8.2% respectively (two-tailed t-test; P>0.05) (Figure 5.2). T h e proportion of bait stations with o n e or more snails ranged from 1.9% to a high of 17.4%. T h e brown shelled snail, Vespericola  sp. w a s reported in a m e a n of 5.8%  bait stations daily, significantly more than the yellow/green coloured snail, Haplotrema  sp. (3.0%) (one-tailed t-test; P<0.05) (Figure 5.3). T h e b r o d i f a c o u m  residue levels for the terrestrial m o l l u s c s ranged from a low of 0.54 ug/g in b a n a n a s l u g s to a high of 4.13 ug/g in Haplotrema  sp. snails (Table 5.3).  O t h e r invertebrates found in bait stations included crickets (Order Orthoptera) two millipedes ( C l a s s Diplopoda, Harpaphe  sp. a n d o n e unidentified)  a n d h a r v e s t m e n (Order P h a l a g i d a ) although they w e r e u n c o m m o n . T h e blue c o l o u r e d bait could be s e e n through the translucent body of the crickets found in bait stations. T h e cylindrical millipedes burrowed into the bait blocks. G e n e r a l l y the millipedes w e r e found burrowed into the bait blocks alone, h o w e v e r they w e r e a l s o  99  0,18-,-  15 16 17 18 19 20 21 22 23 24 25 26 27 28 31 (412) (353) (367) (352) (328) (267) (444) (333) (552) (312) (397) (306) (369) (264) (139) Date, July 1995  Figure 5.2. Proportion of bait stations with snails and b a n a n a s l u g s o v e r the c o u r s e of the intensive baiting period, L a n g a r a Island, 1995 (number of bait stations in brackets).  100  0.12  T  17 18 19 20 21 22 23 24 25 26 27 28 31 (367) (352) (328) (267) (444) (333) (552) (312) (397) (306) (369) (264) (139) Date, July 1995  Figure 5.3. Proportion of bait stations with the terrestrial snails, Vespericola s p . a n d Haplotrema sp., o v e r the c o u r s e of the intensive baiting period (number of bait stations brackets).  101  Table 5.3. B r o d i f a c o u m residues in invertebrates found in bait stations, L a n g a r a Island, 1995. Common Name  No. Individuals  Taxonomy  Mean Weight  Brodifacoum ug/g  ug Brodifacoum per individual  g Banana Slug  Ariolimax  Snail  Vespericola  Snail  Haplotrema  sp.  13 13  10.2 17.1  2.90 0.54  29.55 9.21  sp.  13 40  0.85 0.62  1.53 1.42  1.29 0.88  sp.  9 30 15  1.16 0.87 0.91  3.57 4.13 4.04  4.12 3.58 3.66  Cricket  Order Orthoptera  10  0.04  8.17  0.33  Cylindrical Millipede  C l a s s Diplopoda  30  0.08  2.74  0.22  4  0.2  3.70  0.74  Millipede  Harpaphe  sp.  Predatory Centipede  Class Chilopoda  1  0.2  0.0  0.0  Ground Beetle  Family Carabidae  3  0.1  0.0  0.0  Daddy Long Legs  Order Phalangida  7  0.014  266.4  3.81  102  reported in g r o u p s of three or more. H a r v e s t m e n or d a d d y long legs w e r e found to contain the highest concentration of brodifacoum (Table 5.3.) O n l y terrestrial m o l l u s c s and o n e millipede w e r e found to f e e d o n bait in stations o n L u c y Island in 1994. B r o d i f a c o u m residues could not be d e t e c t e d in Haplotrema  sp. snails but w e r e detected in Vespericola  sp. (Table 5.4).  B r o d i f a c o u m r e s i d u e s in b a n a n a slugs w e r e at detection limits.  Table 5.4. B r o d i f a c o u m residues in invertebrates found in bait stations, L u c y Island, 1994. Genus Common No. Mean Brodifacoum ug Name Individuals W e i g h t Brodifacoum (ug/g) per individual (g) Snail  Vespericola  sp.  46  0.40  2.462  0.984  Snail  Haplotrema  sp.  6  0.27  0.000  0.000  Banana Slug  Ariolimax  sp.  8  3.94  0.002  0.002  Millipede  Harpaphe  sp.  1  0.56  1.607  0.900  103  5.3.2. Carrion Insects 5.3.2.1 Preservation in 95% Ethanol T h e r e w e r e four g e n u s that w e r e the most a b u n d a n t at rat c a r c a s s e s ; t h e s e include three g e n u s from the O r d e r C o l e o p t e r a (Beetles): Nicrophorus Necrophilus  sp.,  sp., and Catops sp. T h e fourth group w e r e the blowflies or blue-bottle  flies from the O r d e r Diptera, Family Calliphoridae, G e n u s  Calliphora.  In 1994, brodifacoum residues w e r e detected in the pooled insects collected from b r o d i f a c o u m p o i s o n e d and control rat c a r c a s s e s (Table 5.5). B r o d i f a c o u m r e s i d u e s w e r e a l s o detected in the ethanol u s e d for the preservation of insects. B r o d i f a c o u m r e s i d u e s w e r e not detected in insects collected into ethanol in 1 9 9 5 (Table 5.6). 5.3.2.2 Preservation by Freezing In 1994, brodifacoum r e s i d u e s w e r e detected only in Nicrophorus  s p . larvae  (Table 5.7). B r o d i f a c o u m residues w e r e not detected in the more c o m m o n a n d a b u n d a n t blowfly larva or adult forms. B r o d i f a c o u m r e s i d u e s w e r e detected in adult blowflies collected from a control c a r c a s s (Table 5.7). 5.3.2.3 Preservation in Chemically Cleaned Aluminum Foil and Freezing B r o d i f a c o u m r e s i d u e s w e r e detected in the blowfly larvae collected from the radio-collared N o r w a y rat c a r c a s s e s in 1995 (Table 5.8). T h e concentration w a s variable a n d ranged from 0.27-11.39 ug/g larva. S m a l l ants (Family F o r m i c i d a e ) w e r e collected from two rat c a r c a s s e s , but no brodifacoum r e s i d u e s w e r e d e t e c t e d .  104  oo  co  CO  ll  CN +-» CO  co o o  LO CN  "t  LO  CD  or  O O  or  O O CO  o ^ W  T -  C O C N ^ C N O O C N $ 2 o O  CO  CD  " t LO  CD  CD CD  E  co  CO CO CD T -  T -  T -  o  O) CM O " t CN t -  CN  o  CM  CD  si  CN  CO  O  O O O  CM CO •si- C D CO  i i  b TO CO CO O —  c  CM CN CO  2  8 | CO  CD  2 o E £  •  3 CO  O O CO  CD CO  ± i CO ±t ±^ CO ± i CO ± i Z J ^ Z l Z l ^ Z i ^ Z l  5 c 6 5 5 c o 5 c o 5  ZJ - C  8 o .CO "D O  v O  LO CD  CO  -D .F C . CO CO  Zl  c  CD  "5 2  o  CL  CL CO  CO  ! CD  CO  CO  cL5 CO  O  co - §  CL  co  2 o •c .Q. "co O  O  if) o> 8 •S ~° (0 c h- CO  E o c o  X CO  •4-*  o  CD  CD CO  c  CD  -g 'c  i— CO  CD T3  £  Q.  o  _CD O  O  CO 1_  CD Q.  b  CO  o  3 D) 'CD  o c CO  c  LU  E  E  Zl  o o  o o  2  O  Z)  co ^ •  CO  "O  CO  Zl  1_  CD  Table 5.6. Insects collected and preserved in 9 5 % ethanol with no b r o d i f a c o u m r e s i d u e s d e t e c t e d , L a n g a r a Island, 1995. Order Family/Genus Common Name Association Number Coleoptera (Beetles)  Catops sp. Necrophilus sp. Nicrophorus sp. Family Rhizophagidae Family Dermistidae  Carrion beetle Carrion beetle Carrion beetle Root-Eating beetles Skin beetles  Unidentified Diptera (Flies)  Calliphora sp. Family Ptychopteridae Family Trichoceridae Family Phoridae Family Platypezidae Family Asilidae Family Anthomyiidae Unidentified  Carrion Carrion Carrion Decaying wood Carrion & Vegetation  -  Blowflies Phantom Crane flies Winter Crane flies Humpbacked flies Fiat-Footed flies Robber flies  -  Carrion Decaying Vegetation Decaying Vegetation Decaying Vegetation Vegetation Insect Larva  Anthomyiid flies -  -  184 2 1 3 18 54 4 5 1 7 3 1 2 2  Collembola  Springtails  Soil  >63  Diplura  Diplurans  Soil  4  Lepidoptera  Moths  Vegetation  1  Araneae  Spiders  Insects  5  Acari  Spiders  Insects  2  >362  Total Brodifacoum (mg/kg)  None Detected  106  Table 5.7. B r o d i f a c o u m residues in carrion insects collected from b r o d i f a c o u m p o i s o n e d N o r w a y rat c a r c a s s e s , collected fresh, L a n g a r a Island, 1994. Genus Life Mean Brodifacoum ug Stage No. Weight (ug/g) Brodifacoum (g) p e r Individual Nicrophorus  sp.  Larva  13  1.02  0.860  0.877  Necrophilus  sp.  Adult  18  0.18  ND  < Detection Limits  Adult  193  0.002  ND  < Detection Limits  Catops s p .  a  Calliphora  sp.  Larva  204  0.02  ND  < Detection Limits  Calliphora  sp.  Adult  25  0.14  ND  < Detection Limits  a None Detected  Table 5.8. B r o d i f a c o u m residues in blowfly larva collected from b r o d i f a c o u m p o i s o n e d N o r w a y rat c a r c a s s e s , L a n g a r a Island, 1995. No. Larva in P o o l  Mean Larva W e i g h t (g) /  ug B r o d i f a c o u m p e r Individual Larva  Brodifacoum Concentration ug/g L a r v a  100 100 15 56 40 25 33 60  0.05 0.06 0.05 0.06 0.02 0.09 0.08 0.02  0.24 0.15 0.12 0.02 0.26 0.18 0.06 0.05  4.81 2.65 2.54 0.27 11.39 2.09 0.75 2.28  107  5.4.  Discussion In 1 9 9 5 , collection a n d preservation protocols w e r e e s t a b l i s h e d to rectify  the contamination problem e n c o u n t e r e d in 1994. However, the level of. b r o d i f a c o u m residue detected from pooled s a m p l e s w a s highly variable mostly b e c a u s e of the variable recovery rates from s a m p l e to s a m p l e by the a s s a y p r o c e d u r e u s e d . T h e procedure for a s s a y i n g the 4-hydroxycoumarin anticoagulants h a s b e e n published with m e a n recovery rates b e t w e e n 8 9 - 9 1 % (Hunter 1983). However, the results provided by N o v a M a n n Laboratories s h o w e d recovery rates ranging from 0 to 1 2 7 % from the s p i k e d s a m p l e s .  No  explanation w a s offered by N o v a M a n n . A n o t h e r factor w h i c h m a y h a v e contributed to the variability is that the b r o d i f a c o u m r e s i d u e s detected m a y represent unassimilated b r o d i f a c o u m in the gut of s o m e invertebrate s a m p l e s and assimilated brodifacoum into the t i s s u e s of other s a m p l e s . T h e control s a m p l e s did not test positive for brodifacoum, thus, I a m confident that the qualitative brodifacoum residue a n a l y s i s w a s s o u n d a n d b r o d i f a c o u m w a s detected from s a m p l e s in 1995. T h e d a t a p r e s e n t e d indicate that invertebrates feeding o n bait a n d brodifacoum p o i s o n e d rats did contain d e t e c t a b l e levels of brodifacoum residue. C a r r i o n insects w e r e a potential tertiary poisoning risk and the invertebrates feeding on the bait w e r e a  '  s e c o n d a r y poisoning risk to non-target s p e c i e s . In N e w Z e a l a n d , invertebrates w e r e o b s e r v e d to f e e d on baits, and brodifacoum r e s i d u e s w e r e found in beetles  108  collected from bait stations containing bait intended for rats o n Stewart Island, however, no d a t a w e r e presented ( E a s o n a n d S p u r r 1995). T h e only reported s e c o n d a r y poisoning of insectivorous birds w a s in a z o o w h e r e birds in a n aviary died after f e e d i n g o n ants and c o c k r o a c h e s that had eaten bait containing b r o d i f a c o u m (Godfrey 1985). T h e blue coloured bait could be readily s e e n through the translucent b o d i e s of the snails a n d slugs found feeding o n the bait. T h e b r o d i f a c o u m r e s i d u e s in the gut of N o r w a y rats represented between 3 0 - 5 0 % of the w h o l e b o d y residue level ( S e e C h a p t e r 4). T h e blowfly larva collected likely had a gut full of carrion containing brodifacoum residues. T h e implication is that the secondary/tertiary poisoning h a z a r d to non-target s p e c i e s is greater from a n invertebrate that h a s b e e n recently feeding o n bait/carrion containing b r o d i f a c o u m v e r s u s o n e that h a s had time to excrete the contents containing b r o d i f a c o u m . In other words, the poisoning risk c a n be defined a s short a n d long term. T h e short term risk m a y be greater than the long term risk b e c a u s e of the p r e s e n c e of brodifacoum in the gut of the invertebrate.  In order to elucidate  the a b o v e possibility, known a m o u n t s of brodifacoum could be fed to invertebrates a n d the residue levels from pooled or individual s a m p l e s c a n be a n a l y s e d at v a r i o u s time points after feeding including w h e n the bait h a s p a s s e d through the gut. T h i s would allow for m e a s u r e m e n t of the brodifacoum r e s i d u e s in the gut v e r s u s levels of brodifacoum retained in the t i s s u e s .  109  T h e attractiveness of the bait to snails and slugs indicate that a n y e x c e s s bait d i s p e r s e d by rats o v e r the c o u r s e of the intensive baiting period w o u l d be c o n s u m e d . T h e a m o u n t of bait d i s p e r s e d by rats a n d not c o n s u m e d is u n k n o w n . However, bait c r u m b s w e r e found in and around burrows, u n d e r logs, a n d a l o n g runs u s e d by rats. O n L u c y Island in 1994, a Vespericola  sp. snail  w a s found to be feeding on the bait c r u m b s outside a burrow a n d had e x c r e t e d a blue cast.  In M a y 1995, 9 months after the removal of the bait from the stations,  four old bait blocks (9.6 g) were found under a log on the west side of L u c y Island and contained 10.986 ug/g brodifacoum. S n a i l s found near the bait w e r e collected a n d contained a concentration of 0.910 mg/kg brodifacoum. T h u s , the s n a i l s a n d s l u g s could be an o n g o i n g s e c o n d a r y poisoning risk until the bait h a s d e g r a d e d or is c o n s u m e d . N o brodifacoum residues w e r e detected in the ethanol p r e s e r v e d insects in 1 9 9 5 . T h i s m a y be d u e to the low c o n s u m p t i o n of the c a r c a s s e s by carrion insects. T h e c a r c a s s e s w e r e not being c o n s u m e d by carrion insects a s found the previous y e a r using a similar d e s i g n trap. T h i s m a y be d u e to the high n u m b e r of other wild rat c a r c a s s e s that w e r e available to insects or the altered d e s i g n of the trap attracted fewer carrion insects. M o s t of the other insects w e r e a s s o c i a t e d with vegetation and soil, and w e r e a result of placing a s h o v e l full of soil in with the c a r c a s s . T h e brodifacoum residue data from blowfly larva collected in 1 9 9 5 w e r e m o r e reliable d u e to the different collection technique a n d i n c r e a s e d attention to  110  minimising or eliminating contamination d u e to collection. It c a n be c o n c l u d e d that blowfly larva c o n s u m i n g rat c a r c a s s e s containing brodifacoum p o s e a tertiary poisoning risk to non-target s p e c i e s . In 1994, s o n g s p a r r o w s a n d Northwestern c r o w s w e r e photographed at rat c a r c a s s e s that had b e e n attacked by carrion beetles ( C h a p t e r 4). T h e y w e r e at risk of tertiary poisoning in 1995, if the carrion insects c o n s u m i n g c a r c a s s e s did carry a load of brodifacoum residue. P o o l e d s a m p l e s of s o n g sparrow livers tested positive for b r o d i f a c o u m ( A p p e n d i x 2). Unfortunately, the highly variable quantitative brodifacoum residue d a t a preclude estimating actual risk of poisoning from the invertebrates. T h e confirmation of brodifacoum residues in invertebrates indicates that insectivorous non-target s p e c i e s may be e x p o s e d to brodifacoum a n d n e e d to be c o n s i d e r e d in future eradications. T h e saturation baiting strategy u s e d o n L a n g a r a Island provided a constant food supply for snails, s l u g s a n d other invertebrates. T h e carrion insects rapidly a n d readily c o n s u m e d brodifacoum p o i s o n e d rat c a r c a s s e s and s u b s e q u e n t l y attracted s o n g s p a r r o w s a n d Northwestern c r o w s ( C h a p t e r 4). T h e a b o v e data indicate that to minimise potential non-target s p e c i e s poisoning, less brodifacoum in the form of bait and/or p o i s o n e d c a r c a s s e s should be available to invertebrates. In c o n c l u s i o n , brodifacoum residues w e r e detected in all invertebrates attracted to bait in the stations. T h e positive detection of brodifacoum r e s i d u e s in carrion insects a n d bait station invertebrates indicates that they w e r e a tertiary a n d s e c o n d a r y poisoning risk to insectivorous s p e c i e s .  111  Chapter 6. C o n c l u s i o n s and Recommendations T h e overall objective of this thesis w a s to investigate the short term poisoning h a z a r d to non-target s p e c i e s from brodifacoum bait u s e d to eradicate rats from L a n g a r a a n d surrounding L u c y a n d C o x Islands. B r o d i f a c o u m r e s i d u e s w e r e d e t e c t e d in e v e r y level investigated: carrion insects, terrestrial m o l l u s c s , s o n g b i r d s , r a v e n s , Northwestern crows, a n d bald e a g l e s - the top of the food c h a i n . T h e results p r e s e n t e d in this thesis provide the b a s i s for future work by identifying p a t h w a y s a n d s p e c i e s at risk of poisoning during similar operations a l o n g the British C o l u m b i a coast.  6.1  Conclusions  6.1.1  Native Small Mammal Study  1. W h i l e D u s k y s h r e w s w e r e attracted to the bait in the stations, the d e c l i n e in their population w a s non-significant in all regions o n L a n g a r a Island, indicating they w e r e at low risk of extirpation. 2. Adult breeding D u s k y s h r e w s a p p e a r e d to be at greater risk of p o i s o n i n g than n o n - b r e e d e r s a n d juveniles.  6.1.2 Secondary Poisoning Risk to Avian Scavengers and Predators 1. R a t s dying a b o v e ground are a c o n s e q u e n c e of anticoagulant poisoning a n d is not a g e or s e x related.  112  2. T h e m e a n w h o l e body brodifacoum residue concentrations in N o r w a y rats found d e a d a b o v e ground w e r e the highest yet reported in the literature. 3. C o m m o n r a v e n s w e r e at a n extreme risk of primary a n d s e c o n d a r y poisoning: i. R a v e n s w e r e able to gain a c c e s s to the bait in the stations. ii. R a v e n s w e r e identified a s the most significant s c a v e n g e r of N o r w a y rat carcasses. 4. B a l d e a g l e s w e r e e x p o s e d to brodifacoum, however, no mortality w a s d e t e c t e d . 5. B r o d i f a c o u m w a s detected in Northwestern c r o w s 9 months after the L u c y Island baiting in 1994.  6.1.3 Study of Brodifacoum Transport into the E c o s y s t e m 1. T h e bait blocks w e r e highly attractive to terrestrial snails a n d s l u g s . 2. B r o d i f a c o u m r e s i d u e s w e r e detected in snails, slugs, blowfly larva, a n d other s p e c i e s , however, it is unclear if it w a s unassimilated brodifacoum in the gut and/or brodifacoum retained in the t i s s u e s .  6.1.4 Detection of Brodifacoum Exposure through Plasma Residue A n a l y s i s and Prothrombin Time Evaluation. 1. P l a s m a brodifacoum residue a n a l y s i s is an effective indicator of e x p o s u r e in birds. 2. T h e u s e of a m a m m a l i a n derived thromboplastin is only a n effective tool to detect a s e v e r e l y affected bird.  113  6.2 Recommendations T h e following eight r e c o m m e n d a t i o n s are presented to minimise the n o n target s p e c i e s poisoning during similar rat eradication operations in the future. T h e y are p r e s e n t e d from a non-target s p e c i e s perspective and s o m e r e c o m m e n d a t i o n s m a y not be e c o n o m i c a l l y or operationally feasible under all conditions.  1. S w i t c h from saturation baiting to pulsed baiting. Utilising a p u l s e d baiting strategy, w h e r e the bait is available in limited quantities a n d is only r e p l e n i s h e d at pre-specified intervals, h a s b e e n s h o w n to reduce the residue load of target rodents ( M e r s o n et al. 1984; K a u k e i n e n 1982). Alternatively, reduction of the concentration of the active ingredient h a s a l s o b e e n s h o w n to r e d u c e the target s p e c i e s residue level ( K a u k e i n e n 1982). T h e pulsed baiting strategy w o u l d a l s o r e d u c e the total a m o u n t of brodifacoum r e l e a s e d into the environment, a n d available for potential transport into the e c o s y s t e m by invertebrates. T h e cost is a n i n c r e a s e d time to eradication which could translates into an i n c r e a s e d e c o n o m i c cost.  2. Shift the intensive baiting period to the time of the y e a r w h e n the rat population is at its lowest. T h i s would r e d u c e the absolute n u m b e r of rats dying a b o v e g r o u n d a n d therefore, d e c r e a s i n g availability to s c a v e n g e r s a n d predators. T h e late winter months are likely the time rat populations are lowest. H o w e v e r , it m a y not  114  be f e a s i b l e to h a v e people working o n and around offshore islands during winter m o n t h s off the c o a s t of British C o l u m b i a w h e n s e v e r e storms are c o m m o n .  3. T h e u s e of anticoagulants that are less toxic to non-target s p e c i e s should be investigated and considered. B r o d i f a c o u m is very effective for controlling rodents d u e to its high toxicity to the target s p e c i e s . However, it is a l s o highly toxic to non-target bird s p e c i e s at d o s e levels that w e r e readily available during the L a n g a r a Island S e a b i r d Habitat Restoration Project. T h e u s e of a less toxic anticoagulant would reduce the primary and s e c o n d a r y poisoning risk to birds. But it is generally avoided b e c a u s e of the risk of rats developing bait s h y n e s s or resistance. O n the other hand, there are other anticoagulants that are a s toxic a s brodifacoum to rats, but less toxic to birds. F o r example, flocoumafen h a s a L D  5 0  of 0.25-0.56 mg/kg for Norway rats (similar to brodifacoum) (Huckle et al. 1989b) but is approximately 26 times less toxic to J a p a n e s e quail (flocoumafen L D = 5 0  >300 mg/kg (Huckle et al. 1989b); brodifacoum L D = 11.6 mg/kg ( R o s s et al. 5 0  1976)).  4. R e - d e s i g n bait stations to exclude C o m m o n R a v e n s . Studies with different bait station d e s i g n s s u c h a s an S-shape or simply, a longer station should be undertaken to determine the s h a p e that d o e s not c o m p r o m i s e a c c e p t a n c e by rats but e x c l u d e s ravens.  115  5. U s e bait formulations that are less attractive to birds. T h e u s e of a bait formulation that would m a k e the baits unpalatable to birds but maintains high attractiveness to the target s p e c i e s would be ideal. In the United States, methyl anthranilate is u s e d a s a n effective non-lethal bird repellent. It has been u s e d effectively a s a nonlethal bird repellent in the field ( A s k h a m 1994; A v e r y 1992; C u m m i n g s et al. 1991; G l a h n e t a l . 1989). It is not yet registered for u s e in C a n a d a (P. M i n e a u , pers. comm.), however, further study into the effectiveness of this product, or others, to prevent primary poisoning to birds otherwise attracted to the bait is warranted.  6. U s e non-removable bait blocks in stations. E v e n if recommendation 4 is followed, rats may disperse large quantities of bait and potentially m a k e t h e m a c c e s s i b l e to non-target s p e c i e s . T h e bait blocks should be fastened d o w n s o that they cannot be r e m o v e d but must be c o n s u m e d within the stations thus minimising dispersal. Alternatively, the bait could be reformulated into a brick (15 c m x 15 c m x 2.5 cm) that cannot be physically removed from the station but requires rats to feed directly o n the bait in station. Either option would reduce the number of visits to a station by an operator, thus, reducing the number of crew required to c h e c k stations. O n the d o w n side, fixing the bait may lead to dominant rats defending stations a n d preventing conspecifics from gaining a c c e s s to bait. A s a c o n s e q u e n c e , the time to eradication may be lengthened.  116  7. Monitor non-target s p e c i e s before, during, and after eradication c a m p a i g n s . i. Avian scavengers.  C o m m o n ravens are the non-target s p e c i e s at greatest  risk of poisoning. R a v e n s are c o m m o n s c a v e n g e r s in seabird colonies in the Q u e e n Charlotte archipelago ( R o d w a y et al. 1990;1988). Their a g g r e s s i v e and inquisitive behaviour put them in the highest risk of poisoning of all s p e c i e s studied a n d thus, could be u s e d a s an indicator of non-target s p e c i e s poisoning. T h e y could indicate the availability of brodifacoum to non-target s p e c i e s . Population estimates before, during and post eradication should be m a d e a n d c o m p a r e d against o n e or more control site(s). If an effective trapping method c a n be developed, blood sampling of adult and juvenile ravens during and post eradication c a n be u s e d to monitor the e x p o s u r e of individuals to brodifacoum. If future eradications are pursued in late winter/early spring w h e n ravens are nesting, pre-fledging raven chicks m a y be a readily a c c e s s i b l e s o u r c e of p l a s m a . T h e p l a s m a brodifacoum residue analysis is a n effective, inexpensive indicator of exposure. A laboratory that is already set up for the analysis should be u s e d to ensure quality control. ii. Native small mammals.  M a n y seabird colonies in the Q u e e n Charlotte  archipelago harbor populations of native d e e r mice and/or d u s k y shrews. S h r e w s are unlikely to be extirpated from islands following the regime of the L a n g a r a Island S e a b i r d Habitat Restoration Project. However, the operation h a s the potential to alter population dynamics. S m a l l m a m m a l populations should be monitored for c h a n g e s in the short term and long term. V a r i a b l e s s u c h a s population size,  117  d e m o g r a p h i c s including a g e and s e x structure c h a n g e s should be m e a s u r e d and quantified. A control site, with similar small m a m m a l composition and island size, should be monitored concurrently.  8. Training in the safe handling and disposal of bait blocks is essential. At least three of the 13 ravens found d e a d were primarily poisoned a s a direct result of a bait spill. T h i s incident indicates that a strict protocol in the handling and d i s p o s a l of bait blocks is required. A training program specifically d e s i g n e d to provide training for island restoration crews dispensing and disposing of bait should be d e v e l o p e d .  A d h e r e n c e to all or s o m e of t h e s e r e c o m m e n d a t i o n s would r e d u c e the n o n target s p e c i e s poisoning risk substantially. In particular, if only the first four r e c o m m e n d a t i o n s are followed, the non-target s p e c i e s poisoning risk s h o u l d a l r e a d y be l e s s e n e d significantly.  6.3 Future Directions T h i s t h e s i s only investigated the short term impacts of the p o i s o n i n g operation a n d d o e s not a d d r e s s the long term impacts. O v e r 14, 0 0 0 bait b l o c k s w e r e d i s p e n s e d from bait stations a c r o s s the island o v e r the intensive baiting period. H o w e v e r , the 1,100 active bait stations w e r e concentrated primarily within a few h u n d r e d meters of the shoreline, leaving the possibility of o n g o i n g primary a n d s e c o n d a r y poisoning risks to non-target s p e c i e s . T h e long-term effects of the  118  baiting o n both the s h r e w a n d raven population should be investigated. E n d p o i n t s s u c h a s mortality s h o u l d be continued to be monitored but a l s o sub-lethal effects s u c h a s reproduction. T h e u s e of radio-telemetry would be useful to monitor the h a z a r d to individual c o m m o n r a v e n s . T h i s technology allows for the determination of the location, time, a n d potentially, c a u s e of d e a t h . If brodifacoum p o i s o n e d r a v e n s w e r e preyed o n or s c a v e n g e d by e a g l e s , radio telemetry could possibly a n s w e r this q u e s t i o n . Laboratory investigation into the L D  5 0  of brodifacoum to local s p e c i e s , s u c h  a s the c o m m o n raven, would be useful for determining risk of poisoning from b r o d i f a c o u m r e s i d u e s found within s p e c i e s investigated in this thesis. Similarly, laboratory investigation into the biological significance of brodifacoum p l a s m a r e s i d u e s o n prothrombin time (a m e a s u r e of risk of hemorrhaging) in birds w o u l d be useful to interpret residue data. Further investigations into modelling accumulation and transfer of r e s i d u e s , s u c h a s that d e v e l o p e d by S m i t h et al. 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T h e acute oral toxicity ( L D ) of P P 5 8 1 to the C h i c k e n . Imperial C h e m i c a l Industries. U n p u b l i s h e d . 5 0  R o s s , D.B., Roberts, N.L. a n d C a m e r o n , D.M. 1976. T h e acute oral toxicity ( L D ) of P P 5 8 1 to the J a p a n e s e Q u a i l . Imperial C h e m i c a l Industries. U n p u b l i s h e d . 5 0  S A S . 1 9 9 5 . J M P Statistical P a c k a g e . S A S Institute Inc. C a r e y , North Carolina.  130  S a v a r i e , P.J., H a y e s , D.J., M c B r i d e , R.T., a n d Roberts, J.D. 1 9 7 9 . Efficacy a n d safety of d i p h a c i n o n e a s a predacide. In: A v i a n a n d m a m m a l i a n Wildlife T o x i c o l o g y . E.E. K e n a g a , Editor. A m . S o c . Test. Mat. S p e c . T e c h . P u b l . No. 6 9 3 : 69-79. S c h a r d e i n , J.L. 1985. Anticoagulants. In: C h e m i c a l l y induced birth defects. N e w York, B a s e l , M a r c e l D e k k e r Inc. pp. 89-100. (Cited in I P C S 1995). S e a r c e y , M.T., G r a v e s , C . B . a n d O l s o n , R.E. 1977. Isolation of a Warfarin binding protein from liver e n d o p l a s m i c reticulum of S p r a g u e - D a w l e y a n d Warfarin-resistant rats. J o u r n a l of Biological C h e m i s t r y . 252(18):62606267. Shirer, M. 1 9 9 2 . In poison's d e f e n c e . Terra N o v a 17:3. Smith, R.H., C o x , P.R. a n d R a m p a u d , M. 1990. R o d e n t i c i d e ecotoxicology: s y s t e m s a n a l y s i s a n d simulation. P r o c . Vert. P e s t Conf. 14:47-54. Stejskal, V., K u c e r o v a , Z. a n d Z u s k a , J . 1994. R o d e n t bait containing b r o d i f a c o u m a s a d e v e l o p m e n t a l m e d i u m for a population of Ptinus tectus ( C o l e o p t e r a , Ptinidae) yielding teratological individuals. O c h r . R o s t l . 30(1 ):11-13. Stopforth, A. 1970. A study of coagulation m e c h a n i s m s in d o m e s t i c c h i c k e n s . J . C o m p . P a t h . 80:525-533. S t u t z e n b a k e r , C D . , B r o w n , K. a n d Lobpries, D. 1984. S p e c i a l R e p o r t : A n a s s e s s m e n t of the a c c u r a c y of d o c u m e n t i n g waterfowl die-offs in a T e x a s c o a s t a l m a r s h . In: W o r k s h o p o n L e a d P o i s o n i n g in Wild Waterfowl, S y m p o s i u m s P r o c e e d i n g s . 88-95. Sullivan, T . P . 1990. D e m o g r a p h i c r e s p o n s e s of small m a m m a l populations to a herbicide application in coastal coniferous forest: population density a n d resiliency. C a n . J . Z o o l . 68:874-883. Suttie, J . W . 1 9 8 0 . T h e metabolic role of vitamin K. Federation P r o c e e d i n g s . 39(10):2730-2735. T a h i r a , N., D u b e , B. a n d A g r a w a l , G . P . 1977. B l o o d coagulation studies in s o m e wild Indian birds: effect of different tissue thromboplastins. J . C o m p . P a t h . 87:451-457. Taylor, D.P. 1984. T h e identification a n d detection of the rats in N e w Z e a l a n d a n d the eradication of ship rats on Tawhitinui Island. U n p u b l i s h e d dissertation for D i p l o m a in P a r k s and R e c r e a t i o n , Lincoln C o l l e g e , Canterbury, N e w Z e a l a n d . 73 pp. (Cited in E a s o n a n d S p u r r 1995). 131  Taylor, R.H. 1993. T h e Feasability of Rat Eradication on. L a n g a r a Island, British C o l u m b i a , C a n a d a . U n p u b l i s h e d report. 2 3 pp. Taylor, R.H. a n d Kaiser, G.W. 1993. Trip Report: L u c y a n d C o x Island at the North E n d of H a i d a G w a i i ( Q u e e n Charlotte lslands)-31 M a y to 6 J u n e 1 9 9 3 . U n p u b l i s h e d report. 11 pp. Taylor, R.H. a n d T h o m a s , B.W. 1993. R a t s eradicated from rugged b r e a k s e a island (170 ha), Fiordland, N e w Z e a l a n d . Biological C o n s e r v a t i o n 65:191-198. Taylor, R.H. a n d T h o m a s , B.W. 1989. Eradication of N o r w a y R a t s (Rattus Norvegicus) from H a w e a Island, Fiordland, using B r o d i f a c o u m . N e w Z e a l a n d J o u r n a l of E c o l o g y . 12:23-32. T h i j s s e n , H.H.W. a n d B a a r s , L . G . M . 1989. M i c r o s o m a l Warfarin binding a n d vitamin K 2,3-epoxide reductase. B i o c h e m i c a l P h a r m a c o l o g y . 38(7):1115-1120. T h i j s s e n , H.H.W. a n d B a a r s , L . G . M . 1987. Hepatic uptake a n d storage of Warfarin. T h e relation with the target e n z y m e vitamin K 2,3-epoxide r e d u c t a s e . J o u r n a l of P h a r m a c o l o g y and E x p e r i m e n t a l T h e r a p e u t i c s . 243(3): 1 0 8 2 - 1 0 8 8 T o w n s e n d M.G., B u n y a n P.J., O d a m , E.M., S t a n l e y P.I., a n d W a r d a l l H.P. A s s e s s m e n t of s e c o n d a r y poisoning h a z a r d of Warfarin to least w e a s e l s . J . Wildl. M a n a g e . 48(2):628-632. T o w n s e n d , M.G., Fletcher, M.R., O d a m E.M., a n d Stanley, P.I. 1 9 8 1 . A n a s s e s s m e n t of the s e c o n d a r y poisoning h a z a r d of Warfarin to tawny owls. J . Wildl. M a n a g e . 45(1):242-247. V a n H a a r l e m , L.J.M., K n a p e n , M.H.J., Hamulyak, K. a n d V e r m e e r , C. 1988. Circulating o s t e o c a l c i n during oral anticoagulant therapy. T h r o m b . H a e m o s t . 60: 7 9 - 8 2 . W a l z , D.A., Kipfer, R.K. a n d O l s o n , R.E. 1975. Effects of vitamin K deficiency Warfarin, a n d inhibitors of protein s y n t h e s i s u p o n the p l a s m a levels of vitamin K-dependent clotting factors in the chick. J . Nutr. 105:972-981. W a t s o n , J.W., Garrett, M.G. a n d Anthony, R.G. F o r a g i n g e c o l o g y of bald e a g l e s in the C o l u m b i a R i v e r Estuary. J . Wildl. M a n a g e . 55(3):492-499.  132  Worthing, C R . a n d Walker, S . B . 1987. T h e P e s t i c i d e M a n u a l . T h e British C r o p Protection C o u n c i l , Thornton Heath, E n g l a n d . L a v e n h a m P r e s s . (Cited in T a y l o r 1993).  133  A p p e n d i x A . The Detection of Exposure to Brodifacoum in J a p a n e s e Quail through Plasma Brodifacoum Residue Analysis and Prothrombin Time Evaluation  Introduction In 1995, the C a n a d i a n Wildlife S e r v i c e attempted to eradicate introduced N o r w a y rats from 3,300 hectare L a n g a r a Island at the north-western tip of British C o l u m b i a ' s Q u e e n Charlotte archipelago by u s e of the anticoagulant b r o d i f a c o u m . T h e island, however, is also h o m e to breeding B a l d E a g l e s , C o m m o n R a v e n s  and  other wildlife w h i c h prompted c o n c e r n s of non-target s p e c i e s p o i s o n i n g . B a l d E a g l e s , the top predator on L a n g a r a Island, w e r e identified to be at risk from s e c o n d a r y poisoning from potentially scavenging/preying o n rats a n d non-target s p e c i e s e x p o s e d to brodifacoum. B a l d E a g l e s w e r e trapped a n d blood s a m p l e s d r a w n for prothrombin time (PT) determinations a n d p l a s m a brodifacoum residue a n a l y s i s to determine if e x p o s u r e o c c u r r e d . T h e P T s w e r e e v a l u a t e d using the c o m m e r c i a l l y available Coulter PT/Fibrinogen kit, a m a m m a l i a n (rabbit brain) thromboplastin. T h e validity of using a m a m m a l i a n s o u r c e thromboplastin for a v i a n P T determinations for brodifacoum e x p o s e d birds n e e d s to be e v a l u a t e d . O n e of the m a n y tests to a s s e s s the hemostatic s y s t e m , the P T is u s e d to indicate the relative risk of hemorrhaging of individuals s u s p e c t e d of anticoagulant e x p o s u r e . T h e P T m e a s u r e s the extrinsic portion of the coagulation c a s c a d e ( B r o w n 1988), a n d is an indicator of the vitamin K d e p e n d e n t clotting factor activity ( R a p a p o r t 1987). After w h o l e blood is collected and the c a l c i u m is b o u n d by s o d i u m  134  citrate to prevent coagulation, tissue thromboplastin is mixed with p l a s m a , a n d time to clotting is noted (Brown 1988). T h e P T is c o m p a r e d against a known normal a n d indicates the d e g r e e of clinical anticoagulation. A P T c a n be determined for avian s p e c i e s using h o m o l o g o u s or h e t e r o l o g o u s thromboplastins. T h e P T w h i c h most accurately reflects the in-vivo s y s t e m , however, is best estimated with a h o m o l o g o u s thromboplastin. D i d i s h e i m et. a l . (1959) found that w h e n h o m o l o g o u s thromboplastin is u s e d for P T determinations in a v i a n p l a s m a , the prothrombin time is no longer than m a n y m a m m a l i a n s p e c i e s . H o w e v e r , the P T is prolonged w h e n a m a m m a l i a n s o u r c e thromboplastin is u s e d for a v i a n P T determinations ( K a s e 1978; T a h i r a et al. 1977; D i d i s h e i m et a l . 1959). T h e literature is unclear, but there is a s u g g e s t i o n that factor VII is in low concentration or a b s e n t in avian blood (Walz et a l . 1975; Stopforth 1970), w h i c h m a y a c c o u n t for the i n c r e a s e d P T if avian thromboplastin h a s no physiological n e e d to bind with factor VII. K a s e (1978) found e v i d e n c e of factor VII in a v i a n p l a s m a while Belleville et al. (1982) d e m o n s t r a t e d the m a m m a l i a n equivalent of factors V , VII, IX, a n d X in J a p a n e s e Q u a i l p l a s m a . T h e resulting prolonged prothrombin times, w h e n using a m a m m a l i a n s o u r c e thromboplastin in a v i a n p l a s m a (and v i c e versa), m a y reflect a difference between avian a n d m a m m a l i a n coagulation s y s t e m s , or that they are l e s s specific manifestations of the c l a s s specificity of t h e s e protein interactions ( K a s e et al. 1980). However, further studies by Belleville et a l . (1982) a n d Doerr and Hamilton (1981), h a v e d e m o n s t r a t e d a f u n d a m e n t a l l y similar c o a g u l a t i o n pathway a s in the well studied h u m a n s y s t e m , thus the differences are  135  likely a result of the c l a s s specificity of the protein reactions. Therefore, the u s e of a m a m m a l i a n thromboplastin for evaluating avian P T m a y be a d e q u a t e for c o m p a r i s o n p u r p o s e s if the P T are reproducible between individuals. T h e objective of this e x p e r i m e n t w a s to evaluate if brodifacoum residue could be detected in the p l a s m a of birds, a n d if the prothrombin test kit detect an i n c r e a s e in prothrombin time. T h e s e c o n d a r y objective w a s to a s s e s s w h e t h e r brodifacoum p l a s m a residue levels or prothrombin time c h a n g e is a more sensitive indicator of e x p o s u r e to low levels of brodifacoum.  136  Materials and Methods 1.0  Plasma Brodifacoum Residues E i g h t e e n male J a p a n e s e quail (Coturnixjaponica)  w e r e obtained from the  U B C Q u a i l G e n e t i c R e s o u r c e C e n t r e . T h e birds w e r e put o n a 12L/12D light regime with w a t e r a n d food provided ad libitum.  After a 6 d a y acclimatisation period the  birds w e r e randomly divided into six treatment g r o u p s of two with six a s r e s e r v e s . T h e e x p e r i m e n t a l d e s i g n w a s a 2 x 3 factorial, with two d o s e levels of b r o d i f a c o u m a n d three time points for blood collection. T h e two d o s e levels w e r e 0.7 mg/kg a n d 1.4 mg/kg ( 6 % a n d 1 2 % of the L D  5 0  respectively ( R o s s et al. 1976). T h e three time  points for blood collection w e r e 24 h, 5 d a n d 10 d post d o s i n g . After p r o c e s s i n g the 24 hour groups, 2 of the 1.4 mg/kg group had died between 24-36 h post d o s i n g , 1 by 3 d, a n d 1 by 4 d post d o s i n g . N e c r o p s y results revealed m a s s i v e localised h e m o r r h a g i n g o n the top of the h e a d a n d side of the neck. J a p a n e s e Q u a i l tend to j u m p in reaction to noise and possibly c a u s i n g t h e m to hit their h e a d s o n the ceiling of the battery. T h u s , the remaining 6 reserve quail w e r e d o s e d at a lower level of 0.35 mg/kg a n d blood collected from 2 birds e a c h at 24 h, 6 d a n d 10 d post d o s i n g . Trunk blood w a s collected into heparin c o a t e d test t u b e s a n d transferred into 1 ml cryovials for centrifuging. T h e p l a s m a w a s pipetted into 1 ml prelabelled cryovials a n d frozen at -20 C until shipment with the livers to the D e p a r t m e n t of Agriculture, State of Illinois, Veterinary Laboratory S e r v i c e for H P L C a n a l y s i s (Murphy et al. 1989; Hunter 1983). T h e detection limit w a s 0.002 p p m . All birds w e r e e x a m i n e d for signs of internal bleeding a n d livers extracted a n d  137  f r o z e n for residue a n a l y s i s . T h e 0.7 a n d 0.35 mg/kg (instead of 1.4 mg/kg) d o s e g r o u p s d a t a w e r e treated a s a 2 ( d o s e levels) x 3 (time points) experiment for a n a l y s i s . A n a l y s i s of t h e d a t a w a s carried out using the JMP statistical p a c k a g e ( S A S , 1995) with t h e following statistical m o d e l : Y  ijk  = u + D, + 7j + (DT)jj + E  where Y  ijk  = R e s i d u e level, D, = the effect of the ith d o s e level, a n d TJ = effect of t h e  ijk  jth time point of blood collection, (DT)jj = t h e two-way interaction b e t w e e n d o s e effect a n d time of blood collection, a n d E  ijk  = r a n d o m error. T h e data w a s c o m m o n log  t r a n s f o r m e d in attempt to normalise the data a n d the a n a l y s i s rerun using t h e a b o v e model. A n A N O V A w a s u s e d to a n a l y s e the 1.4, 0.7 a n d 0.35 mg/kg d o s e g r o u p s at 2 4 h post d o s i n g .  2.0 Prothrombin Time Validation T w e n t y s e v e n , 4 month old m a l e J a p a n e s e quail (Coturnix japonica)  were  o b t a i n e d from the U B C Q u a i l G e n e t i c R e s o u r c e C e n t r e . T h e birds w e r e put o n a 12L/12D light regime with water a n d food provided ad libitum.  After a 6 d a y  acclimatisation period t h e birds w e r e randomly divided into nine treatment g r o u p s of three. T h e e x p e r i m e n t a l d e s i g n w a s a 3 x 3 factorial, with three d o s e levels of b r o d i f a c o u m a n d three time points post-exposure for prothrombin time e v a l u a t i o n . T h e three d o s e levels w e r e 0.0 mg/kg (control), 0.7 mg/kg (a low a n d sub-lethal  138  d o s e ) , a n d 1.4 mg/kg (a high and potentially lethal d o s e a s d e t e r m i n e d from a pilot study c o n d u c t e d earlier). T h e three time points for prothrombin time evaluation w e r e 2 4 h, 7 2 h a n d 120 h post d o s i n g (Stopforth, 1970). After p r o c e s s i n g the 2 4 h and 72 h treatment groups, it w a s o b v i o u s that the highest d o s e group w e r e not showing signs of internal bleeding or morbidity after e x p o s u r e . T h e 120h g r o u p s w e r e therefore not p r o c e s s e d , a n d the birds w e r e given a 7 w e e k rest period (to minimise any effects of prior e x p o s u r e ) a n d r e d o s e d at 13.5 mg/kg (the u p p e r 9 5 % c o n f i d e n c e interval of the L D  5 0  ( R o s s et a l . 1976)). B l o o d  w a s collected from the jugular vein but trunk blood w a s collected from t h o s e with w h i c h difficulties w e r e e n c o u n t e r e d in bleeding. B l o o d collected w a s immediately transferred into a test tube containing buffered s o d i u m citrate. Prothrombin t i m e s w e r e m e a s u r e d within 6 h of collection using the C o u l t e r P/T Fibrinogen kit (Lot # N 1 2 2 2 2 9 5 ) for m a n u a l evaluation (tilt-tube method) (Brown, 1988). All birds w e r e sacrificed after blood collection a n d e x a m i n e d for s i g n s of internal bleeding. S i n c e blood collected from decapitation s h o w e d a significantly shorter P T time than blood collected via the jugular vein, the P T m e a s u r e m e n t s w e r e c o n v e r t e d to prothrombin time ratio ( P T R ) to eliminate the bias d u e to collection m e t h o d (Miletitch 1995). T h e P T R is the ratio of the d o s e group P T to the m e a n control P T ( P T R = S a m p l e PT/ M e a n Control P T ) . T h e d a t a w a s s q u a r e root ( S Q R T ) transformed for a n a l y s i s . Initial a n a l y s i s s h o w e d that there w a s no difference in the P T R in the control g r o u p s of the first a n d  139  the a d d e d treatment, the data w e r e therefore treated a s a 4 ( d o s e levels) x 2 (time points) e x p e r i m e n t for analysis. A n a l y s i s of the data w a s carried out u s i n g the  JMP  statistical p a c k a g e ( S A S 1995) with the following statistical m o d e l : Y where Y  ijk  ijk  = n + D| + 7j + (DT)ij + E  i j k  = ( S Q R T ) P T R m e a s u r e d , D, = the effect of the ith d o s e level, a n d 7] =  effect of the jth time point of blood collection, (DT)jj = the two-way interaction b e t w e e n d o s e effect a n d time of blood collection, a n d E  140  i i k  = r a n d o m error.  Results 1.0 Plasma Brodifacoum Residues L o g transformation had no effect on a n a l y s i s . T h e d a t a is p r e s e n t e d using the arithmetic v a l u e s . 1.1 Plasma Residue  Levels  T h e r e w a s no significant interaction between time a n d d o s e . 1.1.1  Time post  Exposure  T h e r e w a s no significant difference in brodifacoum p l a s m a r e s i d u e s at 2 4 h b e t w e e n the 1.4, 0.7, and 0.35 mg/kg d o s e groups. T h e p l a s m a r e s i d u e s d e c l i n e d significantly ( P O . 0 5 ) between d 1 (0.028 ± 0.005 ppm) a n d 5 (0.005 ± 0.005 ppm) d post d o s i n g , but not between d 5 and 10 (0.002 ± 0.005 ppm) (Figure A-1).  1.1.2 Effect of dose level T h e r e w a s a significant d o s e effect. T h e brodifacoum p l a s m a residue w a s significantly l e s s (P<0.05) in the 0.35 mg/kg d o s e group (0.005 ± 0.004) than the 0.7 mg/kg group (0.018 ± 0.004) (Figure A-2).  1.2 Liver Residue Levels T h e r e w a s no significant interaction effect, or significant effect of time or d o s e o n the level of residue between g r o u p s (Table A-1).  141  0.04 0.035 1 0.03  a -|—  0.025 0.02 0.015 0.01 0.005  24  132  240  Time Post Exposure (Hours)  Figure A - 1 . Effect of time o n brodifacoum p l a s m a residue concentration (ppm) after a single oral d o s e of brodifacoum at 0.35 a n d 0.7 mg/kg ( m e a n s that do not s h a r e the s a m e letter w e r e significant at P<0.05) (n=4 at e a c h time point).  142  g-  0.021  0.016  0.011  0.006  0.001  +  0.7  0.35  Dose Brodifacoum mg/kg  Figure A - 2 . Effect of d o s e o n brodifacoum residue concentration (ppm) o v e r 10 d (* significant at P<0.05) (n=2 at e a c h d o s e level).  143  Table A-1. Liver brodifacoum residue in J a p a n e s e Q u a i l after a single oral d o s e of b r o d i f a c o u m (mean ±s.e., n=2 for e a c h time/dose group). Days Post  D o s e (mg/kg)  Dose  1.4  1  0.700 ±0.100  0.52 ± 0.110  0.487 ± 0.12  5  -  0.443 ±0.122  0.402 ± 0.003  10  -  0.354 ± 0.043  0.373 ± 0.037  0.7  0.35  1.3 Necropsy Results Q u a i l in the 1.4 mg/kg group s h o w e d hemorrhaging a r o u n d the c r a n i u m a n d neck, a n d into the a b d o m i n a l cavity. T h e 0.7 a n d 0.35 mg/kg g r o u p s s h o w e d no s i g n s of h e m o r r h a g i n g .  2.0 Prothrombin Time Validation T h e r e w a s no significant interaction between time post e x p o s u r e a n d d o s e level. 2.1 Time post  exposure  O n e of the 4 birds d o s e d with 13.5 mg/kg brodifacoum s h o w e d s i g n s of morbidity by 4 8 h a n d the remaining w e r e bled for P T evaluation before 7 2 h post e x p o s u r e . T h e s e w e r e included in the 7 2 h group for a n a l y s i s . T h e P T R of birds bled at 2 4 h (1.11 ± 0.068 s e c ) w a s significantly (P<0.02) l e s s than t h o s e bled at 4 8 or 7 2 h post e x p o s u r e (1.37±0.062 s e c ) (Figure A-3). 2.2 Effect of dose level T h e r e w a s a significant (P<0.02) d o s e effect. T h e P T R for the control  144  1.45 1.4 g  1.35  03 CD  2  1.3  co _i  c? l— CL  r |  ro  cl  1.25  1.2  1-15 1  -  1  1.05 1 48  24 Time Post Dose (hours)  Figure A-3. Effect of time o n the prothrombin time ratio ( P T R ) of J a p a n e s e Q u a i l after a single oral d o s e of brodifacoum (* significant at P<0.05) (n=9 at e a c h time point).  145  (1.00+0.082 s e c ) w a s not significantly different from t h o s e of the 0.7 mg/kg group (1.17±0.088 s e c ) . T h e P T R of the 13.5 mg/kg d o s e group (1.47+0.099 s e c ) w a s significantly longer than the control a n d 0.7 mg/kg groups but not the 1.4 mg/kg group (1.31 +0.099 s e c ) . T h e 1.4 mg/kg a n d 0.7 mg/kg w e r e not significantly different from e a c h other (Figure A-4).  2.3 Necropsy  Results  T h e control, 0.7 mg/kg a n d the 1.4 mg/kg groups s h o w e d no s i g n s of h e m o r r h a g i n g . T h e 13.5 mg/kg group s h o w e d s i g n s of h e m o r r h a g i n g . O n e individual w a s found d e a d at 4 8 h post d o s i n g with e x t e n s i v e h e m o r r h a g i n g in the breast m u s c l e a s well a s frank blood in the a b d o m i n a l a n d thoracic cavity. A n o t h e r individual w h i c h h a d not died at 4 8 h, s h o w e d signs of h e m o r r h a g i n g o n the left body wall of the a b d o m e n .  146  1.8 1.7 co 1-6  n=5  1.5  ab  1.4  n=5  be  o 1.3 o  n=6  a: £  co CO  1.2  n=7  1.11 1 0.9  1  13.5  1.4  0.7  Dose Brodifacoum mg/kg  Figure A - 4 . Effect of d o s e of the prothrombin time ratio ( P T R ) of J a p a n e s quail after a single oral d o s e of brodifacoum ( m e a n s that d o not s h a r e t h e s a m e letter w e r e significantly different at P<0.05).  147  Discussion T h e results of the experiment indicate that the C o u l t e r PT/Fibrinogen kit is a b l e to detect a n i n c r e a s e in the P T of avian p l a s m a after e x p o s u r e to b r o d i f a c o u m . H o w e v e r , the kit w a s unable to detect a low, sublethal e x p o s u r e to b r o d i f a c o u m . B r o d i f a c o u m residue w a s detected in the p l a s m a at all d o s e levels for up to 10 d. In the P T experiment both time and d o s e w e r e significant, likely from the 1.4 a n d 13.5 mg/kg d o s e s . T h e lack of significant interaction b e t w e e n time a n d d o s e m a y be a result of the low s a m p l e s i z e in e a c h group and/or variation b e t w e e n s a m p l e s . T h e variability is likely d u e to the u s e of m a m m a l i a n thromboplastin for a v i a n P T determinations a n d not the technique (Griminger 1986). Dorn a n d Muller (1965), u s i n g a rabbit brain thromboplastin, did not detect a significant i n c r e a s e in the P T s of c h i c k s until 6 d of feeding vitamin K deficient a n d 0 . 1 % s u l f o n a m i d e f e e d . C o n v e r s e l y , the u s e of h o m o l o g o u s thromboplastin detected a significant rise in the P T at 3 d. T h u s , a m a m m a l i a n thromboplastin would not be a s sensitive a s a n a v i a n thromboplastin for avian P T determinations. It would h a v e t a k e n a bigger d e c r e a s e in prothrombin levels before it c a n be detected by m a m m a l i a n thromboplastin. In other words, the PT/Fibrinogen kit m a y be useful to detect a high d o s e of b r o d i f a c o u m but would not be sensitive e n o u g h to detect a low d o s e exposure. T h e PT/Fibrinogen kit w a s unable to detect a low d o s e of b r o d i f a c o u m that did not result in a n i n c r e a s e d P T or a high d o s e until the P T rose, 48-72 h after e x p o s u r e . T h e s e results coincide with the a c c e p t e d model that after an  148  anticoagulant binds in the liver a n d inhibits the recycling of vitamin K, e n o u g h time is required for the p l a s m a concentration of the vitamin K d e p e n d e n t clotting factors to d e c l i n e to a point below w h i c h the P T i n c r e a s e s (Rapaport 1987; Hoffman et a l . 1988). T h e i n c r e a s e d P T R indicates a risk of bleeding but not that h e m o r r h a g i n g will occur. T h e 1.4 mg/kg a n d 13.5 mg/kg d o s e groups had similar P T R s at 48/72 h although only the latter group s h o w e d a n y signs of bleeding. In the b r o d i f a c o u m residue experiment, individuals in the 1.4 mg/kg group, lethally h a e m o r r h a g e d apparently brought on by trauma. T h i s indicates that trauma m a y be required to i n d u c e h e m o r r h a g i n g in individuals that h a v e s u p p r e s s e d prothrombin levels. T h e prothrombin levels m a y h a v e b e e n e n o u g h to stop minor bleeding s u c h a s from s p o n t a n e o u s h a e m o r r h a g e s , but not a "major" w o u n d (Doerr a n d Hamilton 1981). T h e 13.5 mg/kg group prothrombin levels m a y h a v e b e e n s u p p r e s s e d to the point b e y o n d w h i c h minor, s p o n t a n e o u s hemorrhaging could not be controlled. T h e P T R of the 1.4 mg/kg group at 7 2 h s u g g e s t s that t h e s e individuals w e r e at risk of h e m o r r h a g i n g although the conditions in the laboratory setting did not i n d u c e a n y detectable bleeding. B r o d i f a c o u m w a s rapidly r e m o v e d from the blood s t r e a m b e t w e e n d a y s 1 a n d 5 post d o s i n g followed by a slower insignificant elimination to the e n d of the study at d a y 10. In m a m m a l s e r u m , brodifacoum follows an exponential d e c a y , persisting for 2-3 w ( M u p h y et a l . 1985). E a s o n et al. (1996) reported that sub-lethal levels of b r o d i f a c o u m (0.1 mg/kg) w e r e detectable in p l a s m a of p o s s u m s  149  (Trichosurus  vulpecula)  for 3 5 d after oral administration of a sub-lethal d o s e .  T h e r e s i d u e s in quail liver indicate that brodifacoum is slowly eliminated. F o r a c l o s e l y related c o m p o u n d , flocoumafen, the elimination half-life for quail liver is >100 d (Huckle et al. 1989b). In m a m m a l s , brodifacoum is extremely slowly eliminated (Mosterd a n d T h i j s s e n 1991). G o d f r e y (1985) estimated the half-life of b r o d i f a c o u m to be in e x c e s s of 150-200 d. Multiple long-term e x p o s u r e to sub-lethal d o s e s of brodifacoum h a v e the potential to a c c u m u l a t e a n d s u p p r e s s factor levels from a partially d e p r e s s e d state a n d m a y result in significant bleeding (Hoffman et al. 1988). A l t h o u g h the test results are from a small s a m p l e size, they indicate that the u s e of a m a m m a l i a n derived thromboplastin u s e d for the P T determination of bald e a g l e s o n L a n g a r a Island in 1995 w a s valid and would h a v e detected a s e v e r e l y anticoagulated bird. However, a high P T would likely c o r r e s p o n d to a positive detection of p l a s m a brodifacoum residue. W h e n u s e d in combination, the p l a s m a b r o d i f a c o u m residue a n d P T d a t a c a n confirm e x p o s u r e a n d a s s o c i a t e d risk of h e m o r r h a g i n g . However, p l a s m a brodifacoum residue a n a l y s i s is a more sensitive indicator of e x p o s u r e in birds.  150  Appendix B. Brodifacoum Exposure in the S o n g Sparrow. Introduction S o n g s p a r r o w s w e r e photographed at three rat c a r c a s s e s p l a c e d to identify s c a v e n g e r s of rats ( C h a p t e r 4). T h e y w e r e apparently attracted to insects that b e g a n to c o n s u m e the rat c a r c a s s e s . In chapter 5, carrion insects w e r e found to test positive for brodifacoum after c o n s u m i n g b r o d i f a c o u m p o i s o n e d rats and p o s e a tertiary poisoning risk to non-target s p e c i e s .  The  objective of this section w a s to determine if s o n g s p a r r o w s w e r e e x p o s e d to brodifacoum.  Methods S o n g s p a r r o w s w e r e collected by shotgun after the intensive baiting period beginning mid A u g u s t 1995. After s e l e c t e d morphological m e a s u r e m e n t s w e r e t a k e n , the livers w e r e r e m o v e d a n d frozen for a n a l y s i s . T h e livers w e r e p o o l e d a c r o s s time or location for a n a l y s i s by H P L C a s d e s c r i b e d in C h a p t e r 4.  Results B r o d i f a c o u m r e s i d u e s w e r e detected in s p a r r o w s collected (Table A 2 . 1 ) . S e l e c t e d morphological m e a s u r e m e n t s a n d collection locations c a n be found in table A 2 . 2 .  151  Table B-1. B r o d i f a c o u m residues detected in S o n g S p a r r o w s , L a n g a r a Island, 1995. Pool  Sparrow  Location  Date  1 2 3 4 5  1,4 2,9 6,7 10, 11, 12 3, 5, 8, 13  North E g e r i a B a y North E g e r i a B a y South Egeria Bay Lord Bight E g e r i a Bay/Lord Bight  A u g u s t 14 A u g u s t 16 A u g u s t 14/16 A u g u s t 21 August 14/16/21  Tissue Analysed  Brodifacoum (ppm)  Liver Liver Liver Liver Body  ND 0.643 ND 0.567 0.058  Table B-2. M o r p h o l o g i c a l m e a s u r e m e n t s from S o n g S p a r r o w s collected o n L a n g a r a Island, 1 9 9 5 . No  Date Collected  1 2 3 4 5 6 7 8 9 10 11 12 13  August August August August August August August August August August August August August  14 16 16 14 14 14 16 16 16 21 21 21 21  Sex  Wing Chord (cm)  M M  6.8 6.3  7.1 5.9  0.8  -  -  -  -  M  6.6 6.2 7.0 6.4 6.3 6.9 7.2 7.0 6.6 7.0  7.6 7.0 6.9 5.8 7.2 7.6 5.7 6.5 5.6 6.0  0.78  -  F M -  M M M M -  Tail Length (cm)  Hallux (cm)  -  -  0.86 -  0.81 0.86 0.82 0.80 0.79 0.74  152  Bill Depth (cm) 0.61 0.55 0.59 0.60 0.70 0.65 0.62 0.54 0.72 0.62 0.70 0.61 -  Tarsus Length (cm)  Tarsus Diameter (cm)  -  -  2.20  0.12  -  -  2.34  0.18  -  -  2.72 2.48 2.36 2.31 2.64 2.50 2.44 2.47  0.20 0.17 0.16 0.18 0.16 0.16 0.16 0.16  Discussion S o n g s p a r r o w s w e r e e x p o s e d to brodifacoum during the baiting c a m p a i g n a n d w e r e at risk of poisoning. It is unclear if the risk w a s from c o n s u m i n g carrion insects c o n s u m i n g brodifacoum p o i s o n e d rat c a r c a s s e s or primarily from eating c r u m b s of bait that w a s found scattered around bait stations a n d a l o n g rat runs. In N e w Z e a l a n d , both granivorous a n d insectivorous birds w e r e primarily p o i s o n e d after feeding on aerial distributed bait pellets intended for introduced rodents ( E a s o n a n d S p u r r 1995). S o n g s p a r r o w s are primarily insectivores, however, they are known to take s e e d s (Ehrlich et al. 1988). A s well, they m a y h a v e preyed o n snails or other invertebrates feeding o n the bait in the stations. Therefore, s o n g s p a r r o w s w e r e at risk of primary, s e c o n d a r y a n d tertiary p o i s o n i n g . Primary poisoning from the bait crumbs, s e c o n d a r y from the invertebrates feeding on the bait, and tertiary from carrion insects. T h e pooling of s a m p l e s precludes quantifying the brodifacoum residue in individual c a r c a s s e s a s the brodifacoum m a y h a v e originated from a single s p a r r o w or all, likely diluting the concentration. However, it c a n be c o n c l u d e d that s p a r r o w s w e r e at risk of brodifacoum poisoning and the primary, s e c o n d a r y a n d tertiary poisoning risk should be further investigated in future eradication operations.  153  Appendix C. Potential Sub-Lethal and Long Term Effects of Brodifacoum Exposure T h e r e is no d a t a from the literature o n the long term effects of b r o d i f a c o u m or the other anticoagulants o n target or non-target s p e c i e s . T h i s a p p e n d i x identifies p o s s i b l e sub-lethal effects and potential long term effects of brodifacoum e x p o s u r e to non-target s p e c i e s . T h e anticoagulant rodenticides are k n o w n to s u p p r e s s clotting factor levels, affect b o n e m a s s , reproductively toxic, a n d are teratogenic with p o s s i b l e d e v e l o p m e n t effects. T h e s u p p r e s s i o n of the clotting factor levels w a s the m e c h a n i s m f o c u s e d o n in this thesis, a n d the m o d e of action is d i s c u s s e d in C h a p t e r 2. However, there are long term implications of sub-lethal or repeated e x p o s u r e to b r o d i f a c o u m in that an increasingly s m a l l e r d o s e would potentiate the anticoagulant effect b e c a u s e of the a l r e a d y s u p p r e s s e d level of clotting factors. A normal P T m a y be obtained from h u m a n p l a s m a with between 3 0 - 1 0 0 % of normal vitamin K d e p e n d e n t clotting factor concentrations (Hoffman et al. 1988). S u p p r e s s i o n of the clotting factor concentration below the threshold would result in an i n c r e a s e d P T a n d possibly uncontrollable h e m o r r h a g i n g . A significant d e c r e a s e in the levels of the vitamin K d e p e n d e n t clotting factors lasted 4 3 d a y s after ingestion of brodifacoum (Hoffman et a l . 1988). B o n e m a s s m a y be c o m p r o m i s e d by sub-lethal or long term e x p o s u r e to oral anticoagulants. B o n e contains the vitamin K d e p e n d e n t protein o s t e o c a l c i n w h i c h is part of the b o n e matrix, a n d levels in the blood m a y be u s e d a s an indicator of active b o n e d e p o s i t i o n . T h e oral anticoagulants ( p h e n p r o c o u m o n a n d a c e n o c o u m a r o l ) r e d u c e the activity of o s t e o c a l c i n , w h i c h is activated by a vitamin K d e p e n d e n t reaction  154  ( V a n H a a r l e m et a l . 1988). Patients o n long term oral anticoagulant therapy s h o w e d significantly lower b o n e m a s s than controls (Fiore et a l . 1990; R e s c h et a l . 1991). Furthermore, it w a s found that a poor vitamin K status w a s a s s o c i a t e d with a high urinary c a l c i u m loss ( K n a p e n et al. 1993). Therefore, y o u n g individuals with growing b o n e s a n d f e m a l e birds a n d m a m m a l s with high c a l c i u m d e m a n d s in the breeding s e a s o n are at greatest risk of sub-lethal effects of brodifacoum e x p o s u r e . T h e c o u m a r i n anticoagulants h a v e b e e n s h o w n to be e m b r y o t o x i c a n d teratogenic to rats. In rats, warfarin induced i n c r e a s e d rates of embryolethality, h e m o r r h a g e a n d g r o s s structural malformations (internal h y d r o c e p h a l u s a n d a n o m a l i e s of skeletal ossification) (Mirkova and A n t o v 1983). Similarly, indiscriminate u s e of b r o d i f a c o u m in fields w a s related to i n c r e a s e d i n c i d e n c e s of abortions a n d h e m o r r h a g e in s h e e p a n d goats in Egypt ( F e i n s o d et a l . 1986). In contrast, other laboratory studies h a v e not demonstrated reproductive toxicity or teratogenicity of b r o d i f a c o u m to rats or rabbits at various d o s e levels ( H o d g e et a l . 1 9 8 0 a , 1980b, 1980c). A n t i c o a g u l a n t s h a v e d e v e l o p m e n t a l impacts o n the h u m a n fetus. In h u m a n s , there are two t y p e s of anticoagulant induced defects, d e p e n d i n g o n the time of administration of the anticoagulant: fetal warfarin s y n d r o m e a n d fetal w a s t a g e ( A n o n y m o u s 1976). T h e most consistent feature of fetal warfarin s y n d r o m e is n a s a l h y p o p l a s i a leading to respiratory difficulty. Fetal w a s t a g e results a l s o in central n e r v o u s s y s t e m a n o m a l i e s . Other c o m m o n features includes b o n e abnormalities of the axial a n d a p p e n d i c u l a r skeleton ( I P C S 1995), opthalmological malformations  155  leading to blindness, d e v e l o p m e n t a l delay, low birth weight, premature birth, mental retardation, a n d e a r a n o m a l i e s ( S c h a r d e i n 1985). A l t h o u g h the a b o v e s u m m a r y indicates that brodifacoum m a y h a v e potential long term effects from sub-lethal e x p o s u r e to non-target s p e c i e s , more work in t h e s e a r e a s are required to confirm potential impacts. However, this s u m m a r y identifies potential a r e a s of investigation that m a y be c o n d u c t e d both in laboratory a n d field e x p e r i m e n t s . Clearly, the mortality endpoint a s a result of the e x p o s u r e to b r o d i f a c o u m , the main f o c u s of this thesis, is not the only non-target s p e c i e s impact that could be m e a s u r e d during future island restoration projects.  156  Appendix D. Environmental A s p e c t s of Brodifacoum - Transport, Distribution and Transformation.  Air, Water and Soil Air A n t i c o a g u l a n t rodenticides h a v e low volatility a n d i n c r e a s e d levels in the air are unlikely ( I P C S 1995). Water B r o d i f a c o u m is slightly soluble in water (<10 mg/l at 2 0 °C, p H 7). T h e v a p o r p r e s s u r e is <0.13 m P a at 25°C ( I P C S 1995). It is a w e a k acid a n d d o e s not readily form water-soluble salts (Worthing and W a l k e r 1987). H o w e v e r , it is k n o w n to be toxic to fish. T h e brodifacoum 96-h L C  5 0  to rainbow trout is 0.051  mg/l (Hill et al. 1976). T h i s is equivalent to 1 bait block (20 g bait with b r o d i f a c o u m at 0.005%) per 2 0 I of water that could be c o n s i d e r e d a toxic h a z a r d to fish. Soil T h e adsorption and desorption of C - b r o d i f a c o u m in laboratory conditions 14  h a s b e e n investigated. B r o d i f a c o u m binds very strongly to soil particles a n d equilibria is e s t a b l i s h e d fairly rapidly with larger w a t e n s o i l ratios despite v e r y low b r o d i f a c o u m w a t e r solubility ( N e w b y and White 1978). Binding i n c r e a s e s with greater o r g a n i c matter in the soil (ICI 1984). O n c e b o u n d to the soil particles, 14  C - B r o d i f a c o u m is effectively immobile and desorption is very s l o w ( J a c k s o n  a n d Hall 1992; N e w b y and White 1978). L e s s than 2% of brodifacoum, a d d e d to  157  soil (0.6 a n d 6.0 kg/ha) with a p H from 4.3-7.1, organic matter from 6.8-72.1% a n d c l a y content from 5-19%, l e a c h e d more than 2 c m (ICI 1984). T h i s s u g g e s t s that mobility of brodifacoum would be restricted to erosion p r o c e s s e s , traveling with soil particles. O n c e b o u n d to soil particles, slow microbial degradation r e d u c e s b r o d i f a c o u m to C 0  2  and water (Taylor 1993; Shirer 1992). T h e half life of  b r o d i f a c o u m in soil r a n g e s between 12-25 w e e k s , d e p e n d i n g o n soil conditions (ICI 1984). Hall a n d Priestley (1992) monitored the m e t a b o l i s m of  1 4  C-  B r o d i f a c o u m in soil under a e r o b i c conditions for 52 w e e k s . A m e a n of 3 5 . 8 % of radioactivity r e c o v e r e d w a s  1 4  C 0 . R a d i o l a b e l e d brodifacoum w a s the major 2  r a d i o l a b e l e d c o m p o n e n t in the soil extracts. A half-life of brodifacoum in soil w a s c a l c u l a t e d to be 157 d a y s . N o information o n the abiotic degradation of brodifacoum is available. H o w e v e r , abiotic degradation of related second-generation anticoagulants h a v e b e e n d e s c r i b e d . B r o m a d i o l o n e d e g r a d e s rapidly o n e x p o s u r e to sunlight, with a half-life of 2.1 h ( I P C S 1995). T h e photolytic half life of d i f e n a c o u m at p H 5, 7, a n d 9 o v e r 2 4 hours w a s calculated to be 3.3, 8.1 and 7.3 h (Hall et a l . 1992). T h e hydrolytic half-life of brodifacoum w a s found to be in e x c e s s of 30 d a y s , but no p r e c i s e estimation w a s m a d e ( J a c k s o n et al. 1991). T h e s e d a t a indicate that brodifacoum is a lipophilic c o m p o u n d relatively resistant to b r e a k d o w n in soil. A n y brodifacoum that is not c o n s u m e d by target or non-target o r g a n i s m s and that is left in the environment will be present a n d  158  potentially available for transport into the e c o s y s t e m long after the bait h a s b e e n r e m o v e d from the stations.  Biological Retention of Brodifacoum Biologically, brodifacoum is a lipophilic anticoagulant rodenticide relatively resistant to m e t a b o l i s m .  M o s t (75%) of a 2 5 mg/kg d o s e of b r o d i f a c o u m g i v e n  to rats w a s retained principally in the liver, p a n c r e a s and the salivary g l a n d s , at ten d a y s post d o s i n g w h e n the study e n d e d (Godfrey 1985). T h e biological halflife w a s found to be 150-200 d a y s (Godfrey 1985). P a r m a r et al. (1987) estimated the liver brodifacoum half-life to be 130 d a y s in rats. B r o d i f a c o u m w a s detected in s h e e p liver 128 d a y s post d o s i n g at 0.2 a n d 2 mg/kg ( L a a s et a l . 1985). Similarly, s h e e p c o n s u m i n g a single sub-lethal d o s e of 2 mg brodifacoum/kg s h o w e d 2 mg/kg in the liver 4 months after d o s i n g ( R a m m e l l et al. 1984). T h e s e d a t a indicate that sub-lethally e x p o s e d m a m m a l s a n d likely birds, p o s e a long term poisoning risk to s p e c i e s preying o n t h e m . L o n g term retention of b r o d i f a c o u m m a y a l s o lead to accumulation of brodifacoum initiating the anticoagulant effect leading to hemorrhaging and d e a t h .  159  

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