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Some aspects of the interrelationship of bacterial kidney disease infection and sodium pentachlorophenate… Iwama, George Katsushi 1977

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SOME ASPECTS OF THE INTERRELATIONSHIP OF BACTERIAL KIDNEY DISEASE INFECTION AND SODIUM PENTACHLOROPHENATE EXPOSURE IN JUVENILE CHINOOK SALMON (ONCORHYNCHUS  TSHAWYTSCHA) by  GEORGE KATSUSHI  IWAMA  B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1975  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f Zoology) We accept t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1977 © George K a t s u s h i Iwama, 1977  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  in p a r t i a l  fulfilment  of  the  requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia, I a g r e e  s h a l l make i t  freely  available  for  that  reference a n d study.  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  this  thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . of  this  thesis for  It  financial  i s understood that copying o r p u b l i c a t i o n gain s h a l l not be allowed without my  written permission.  Zoology Department The  or  '  U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  A p r i l 15, 1977  ABSTRACT  The  i n t e r r e l a t i o n s h i p o f b a c t e r i a l kidney d i s e a s e ,  a c h r o n i c d i s e a s e o f c u l t u r e d salmonids, and an e n v i r o n mental t o x i c a n t , sodium pentachlorophenate, i n j u v e n i l e chinook salmon, over time, was  studied.  T h i s was  by m o n i t o r i n g v a r i o u s h a e m a t o l o g i c a l parameters exposed t o the two  i n the  fish  f a c t o r s , s i n g u l a r l y and i n combination.  An e v a l u a t i o n o f these parameters  as u s e f u l i n d i c e s o f s t r e s s -  f u l s t a t e s imposed by the treatment was thermore,  c a r r i e d out  a l s o attempted.  e x t e r n a l and i n t e r n a l p h y s i c a l examinations, gram  s t a i n e d k i d n e y smears and the occurrence o f m o r t a l i t i e s plemented  sup-  the blood changes i n e v a l u a t i n g the response o f  the f i s h t o these  factors.  Approximately 41 teria,  Fur-  x 10^  v i a b l e kidney d i s e a s e bac-  i s o l a t e d from moribund a d u l t pink salmon, were i n -  j e c t e d i n t r a p e r i t o n e a l l y i n t o the f i s h a f t e r a n a e s t h e t i z a t i o n w i t h n e u t r a l i z e d t r i c a n e methanesulphonate. f i s h were s i m i l a r l y sham i n j e c t e d .  Control  A l l i n j e c t i o n s were c a r -  r i e d out on the same day. Both experimental and c o n t r o l groups o f f i s h were then exposed t o c l e a n water,  i n t e r m e d i a t e and h i g h l e v e l s o f  sodium pentachlorophenate based on the i n c i p i e n t 96 value. LC^Q  The t h r e e l e v e l s were:  and 0.50  x 96  0 x 96  h L C ^ Q , 0.05  h L C ^ Q respectively.  h LC^Q x 96  Four days a f t e r the  b e g i n n i n g o f t o x i c a n t exposure, h a e m a t o c r i t ,  haemoglobin,  red and t o t a l white blood c e l l counts, mean c e l l volume, mean c o r p u s c u l a r haemoglobin  h  c o n c e n t r a t i o n , mean c e l l u l a r  haemoglobin, blood urea n i t r o g e n , t o t a l p r o t e i n and plasma glucose v a l u e s were determined  f o r experimental and c o n t r o l  groups o f f i s h at each t o x i c a n t l e v e l .  Subsequently,  these  d e t e r m i n a t i o n s were made f o r each group o f f i s h every f o u r days.  T h i s was c a r r i e d out f o r 3 ° days a f t e r  beginning  t o x i c a n t exposure u n l e s s m o r t a l i t i e s due t o t h e treatments prevented  sampling.  (two f i s h p e r measurement; 12  Pooled  measurements p e r sample) blood samples from t h e severed caudal peduncle were used f o r these d e t e r m i n a t i o n s . s c r i p t i v e code was developed  A de-  to categorize the progression  o f t h e b a c t e r i a l i n f e c t i o n based on t h e symptoms o f t h i s disease.  T h i s code was a l s o used t o q u a n t i t a t i v e l y compare'  the p h y s i c a l c o n d i t i o n o f each sample f i s h among t h e t h r e e toxicant  levels. G e n e r a l l y , a s y n e r g i s t i c e f f e c t was observed be-  tween t h e b a c t e r i a l i n f e c t i o n and t o x i c a n t exposure i n t h e measured blood parameters,  p h y s i c a l c h a r a c t e r i s t i c s and t h e  occurrence o f m o r t a l i t i e s .  T h i s s y n e r g i s t i c e f f e c t was i n -  d i c a t e d i n t h e measured blood parameters (blood urea  nitro-  gen, mean c e l l volume and white blood c e l l count) by an e a r l i e r d e v i a t i o n o f t h e i n f e c t e d f i s h value from t h e cont r o l f i s h value w i t h t o x i c a n t exposure,  an i n c r e a s e i n t h e  d i f f e r e n c e between i n f e c t e d and c o n t r o l f i s h v a l u e s w i t h t o x i c a n t exposure, sampling p e r i o d .  o r both at any one time o r f o r the e n t i r e The advanced s t a t e o f p h y s i c a l  debilitation  at both l e v e l s o f t o x i c a n t exposed i n f e c t e d f i s h r e l a t i v e t o the r e s p e c t i v e c o n t r o l f i s h a l s o i n d i c a t e d synergism the two f a c t o r s . Furthermore,  between  a catastrophic m o r t a l i t y occurred  0  i n the i n f e c t e d f i s h a t the h i g h t o x i c a n t l e v e l on t h e second sampling day t h a t was i n t e r p r e t e d as a r e s u l t o f a s y n e r g i s t i c e f f e c t o f t h e pathogen and the t o x i c a n t . In  response t o the i n f e c t i o n , depressed haema-  t o c r i t , haemoglobin, r e d blood c e l l count, blood urea n i t r o g e n , t o t a l p r o t e i n and glucose values were observed i n i n f e c t e d f i s h r e l a t i v e t o c o n t r o l f i s h over the sampling period.  Haemodilution due t o pathogenic d e s t r u c t i o n o f osmo-  r e g u l a t o r y t i s s u e s was c o n s i d e r e d t h e primary cause f o r t h e observed r e s u l t s .  I n h i b i t i o n o f e r y t h r o p o i e s i s , leakage o f  p r o t e i n s through open l e s i o n s , d e p l e t i o n o f glycogen s t o r e s by t h e m u l t i p l y i n g b a c t e r i a and c e s s a t i o n o f f e e d i n g were a l s o c o n s i d e r e d as a d d i t i o n a l f a c t o r s t h a t may have c o n t r i buted t o these r e s u l t s . Mean c e l l volume was observed t o i n c r e a s e as a r e s u l t o f the i n f e c t i o n .  E r y t h r o c y t i c s w e l l i n g and i n h i b i -  t i o n o f e r y t h r o p o i e s i s , r e s u l t i n g i n fewer, s m a l l e r immature c e l l s , were c o n s i d e r e d as the c a u s a l f a c t o r s f o r t h i s T o t a l white blood c e l l counts i n i n f e c t e d  result.  fish  were i n i t i a l l y lower than r e s p e c t i v e c o n t r o l f i s h but showed an i n c r e a s i n g t r e n d w i t h t i m e .  T h i s r e s u l t was seen as an  i n i t i a l streee-mediated l e u c o p e n i a f o l l o w e d by a n e u t r o p h i l i a i n response t o the i n c r e a s e i n t i s s u e damage as a r e s u l t o f the d i s e a s e p r o g r e s s i o n . In control fish  response t o t h e t o x i c a n t exposure u n i n f e c t e d showed decreased haematocrits a t the h i g h l e v e l  o f t o x i c a n t exposure.  E l e v a t e d blood urea n i t r o g e n and  glucose v a l u e s were observed i n t h e i n t e r m e d i a t e l e v e l o f  V t o x i c a n t exposed u n i n f e c t e d f i s h r e l a t i v e to the u n i n f e c t e d c o n t r o l s i n c l e a n water.  The  i n t e r p r e t e d as being caused  i n c r e a s e d glucose values were  by an i n c r e a s e i n the s e c r e t i o n  o f " s t r e s s hormones" as a g e n e r a l s t r e s s response. r e s u l t s l e d t o the c o n c l u s i o n t h a t sodium exposure reduced  These  pentachlorophenate  the r e s i s t a n c e o f the f i s h to the  o f the k i d n e y d i s e a s e b a c t e r i a l i n f e c t i o n .  effects  I t was  a l s o con-  cluded t h a t some o f the measured blood parameters are t i v e i n d i c a t o r s o f s t r e s s f u l s t a t e s caused by these Haematocrit, ume  sensi-  factors.  r e d blood c e l l count, mean c e l l  and t o t a l / d i f f e r e n t i a l white blood c e l l count  vol-  measure-  ments are recommended f o r r o u t i n e m o n i t o r i n g o f the  physio-  l o g i c a l c o n d i t i o n s o f f i s h s t o c k s f o r the purposes o f s t r e s s detection.  The  careful evaluation of'the physiological  1  con-  d i t i o n o f f i s h stocks i s recommended as a p a r t o f b i o a s s a y procedures  f o r the purpose o f making meaningful  between t e s t  results.  comparisons  vi ACKNOWLEDGEMENTS  I would l i k e t o express my s i n c e r e g r a t i t u d e t o Dr. G.L. Greer and Dr. D.J. Randall  f o r t h e i r patient  s u p e r v i s i o n and s t i m u l a t i n g guidance through t h i s Their valuable  advice  i n the preparation  i s greatly appreciated.  study.  o f t h i s manuscript  I would a l s o l i k e t o thank Dr. G.  B e l l , Dr. J.C. Davis and Mr. H. Sparrow f o r t h e i r h e l p f u l suggestions and p r o f i t a b l e d i s c u s s i o n s o f v a r i o u s of t h i s investigation.  The advice  aspects  o f Dr. P.A. L a r k i n con-  c e r n i n g t h e s t a t i s t i c a l treatment o f my data i s a l s o appreciated. I am indebted for t h e i r valuable  t o Dr. T. Evelyn, and h i s a s s i s t a n t s  advice  and p a t i e n t guidance  the m i c r o b i o l o g i c a l aspects o f t h i s study.  concerning  I would a l s o  l i k e t o thank Ms. Margaret F i s h e r f o r h e r a s s i s t a n c e i n the p r e p a r a t i o n  and t h e t y p i n g o f t h i s  report.  F i n a l l y , I would l i k e t o thank a l l t h e people a t the P a c i f i c Environment I n s t i t u t e Who d i r e c t l y o r i n d i r e c t l y c o n t r i b u t e d t o t h i s study i n t h e i r v a r i o u s c a p a c i t i e s .  vii TABLE OF CONTENTS Page ABSTRACT ACKNOWLEDGMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF APPENDIX TABLES LIST OF FIGURES LIST OF APPENDIX FIGURES SECTION I - INTRODUCTION SECTION I I - INITIAL EXPERIMENTS I n i t i a l Experiment A - Infection'Experiment Introduction M a t e r i a l and methods Results Discussion I n i t i a l Experiment B - Bioassay Introduction M a t e r i a l and methods R e s u l t s and d i s c u s s i o n  i i vi v i i ix x xi xiii 1  7 # 9 12 14 15 16  SECTION I I I - MATERIALS AND METHODS Experimental design.... Fish Acclimatization Apparatus Infection of fish Toxicant a d m i n i s t r a t i o n Sampling.... Parameter measurement P a t h o l o g i c a l examination Data a n a l y s i s  20 20 20 23 23 24 26 27 28 29  SECTION IV - RESULTS Comparison between u n i n f e c t e d c o n t r o l f i s h and kidney disease uninfected f i s h f o r a l l t o x i cant l e v e l s Comparison among u n i n f e c t e d c o n t r o l f i s h group for a l l toxicant levels Comparison among k i d n e y d i s e a s e i n f e c t e d f i s h for a l l toxicant levels..  32 36 38  SECTION V - DISCUSSION  57  SECTION VI - GENERAL CONCLUSIONS AND RECOMMENDATIONS  67  SECTION V I I - BIBLIOGRAPHY  70  viii Page SECTION V I I I - APPENDICES Appendix I - Sodium pentachlorophenate P r e p a r a t i o n o f stock s o l u t i o n o f NaPCP... Preparation o f bioassay s o l u t i o n s i n m o d i f i e d Mariot b o t t l e s Preparation o f toxicant solutions f o r the main experiment Appendix I I - M i c r o b i o l o g i c a l Procedures P r e p a r a t i o n o f E v e l y n ' s kidney d i s e a s e I s o l a t i o n and growth o f kidney d i s e a s e bacteria , H a r v e s t i n g o f kidney d i s e a s e b a c t e r i a l c e l l s . a n d p r e p a r a t i o n o f inoculum for injection into f i s h V i a b l e counts  77 77 77  79 80 82 S3  Appendix I I I - C a t e g o r i z a t i o n o f p h y s i c a l c h a r a c t e r i s t i c s i n response t o kidney disease i n f e c t i o n  85  Appendix IV - Tables o f mean, standard e r r o r o f t h e mean and grand mean values f o r HCT, Hb, RBC, WBC, MCV, BUN, TP and GLU..  88  Appendix V - R e s u l t s f o r mean c o r p u s c u l a r haemoglobin c o n c e n t r a t i o n and mean c e l l haemoglobin  97  Appendix VI - Photographs o f apparatus used i n b i o a s s a y and t h e main experiment...  105  ix LIST OF TABLES Page TABLE I  I n c u b a t i o n times r e s u l t i n g from d i f f erent c o n c e n t r a t i o n s o f kidney d i s e a s e bacteria injected intraperitoneally into j u v e n i l e coho salmon  10  TABLE I I  Symptoms o f b a c t e r i a l kidney d i s e a s e  10  TABLE I I I  P h y s i c a l c h a r a c t e r i s t i c s code and mort a l i t y f o r the main experiment  39  TABLE IV  Comparison o f v a r i o u s h a e m a t o l o g i c a l v a l u e s measured i n t h i s study w i t h those o f Thomas e t . a l . (1969)  56  X  LIST OF APPENDIX TABLES Page Appendix VI  T a b l e s o f mean,standard e r r o r o f t h e mean and grand mean v a l u e s .  TABLE I  Haematocrit  88  TABLE I I  Haemoglobin  89  TABLE I I I  Red blood c e l l  TABLE IV  T o t a l white blood c e l l  TABLE V  Mean c e l l volume  92  TABLE V I  Blood u r e a n i t r o g e n  93  TABLE V I I  Total protein  94  TABLE V I I I  Glucose  95  Appendix V TABLE I TABLE I I  count  90 count  91  T a b l e s o f mean, standard e r r o r o f t h e mean and grand mean v a l u e s . Mean c o r p u s c u l a r haemoglobin concentration Mean c e l l u l a r haemoglobin  99 100  xi LIST OF FIGURES Page FIGURE l a . FIGURE l b . FIGURE I I  Photograph o f b i o a s s a y a q u a r i a as d e s c r i b e d i n I n i t i a l Experiment B  18  Diagrammatic r e p r e s e n t a t i o n o f one b i o a s s a y aquarium..  18  T o x i c i t y curve showing  19  L T ^ Q ' S  FIGURE I I I F a c t o r i a l d e s i g n used f o r t h e main experiment  21  FIGURE IVa. Photograph o f a p a i r o f t e s t tanks used i n t h e main experiment  22  FIGURE IVb. Diagrammatic r e p r e s e n t a t i o n o f t h e same tanks from t h e back  22  FIGURES V to XII  Graphs o f the responses o f measured h a e m a t o l o g i c a l parameters i n t h e d i f f erent groups o f f i s h t o t h e e x p e r i m e n t a l treatments, b a c t e r i a l k i d n e y d i s e a s e i n f e c t i o n and NaPCP exposure, over t h e sampling period.  FIGURE V  Haematocrit  i  40  FIGURE VI  Haemoglobin  41  FIGURE V I I  Red blood c e l l count  42  FIGURE V I I I T o t a l white blood c e l l count  43  FIGURE IX  Mean c e l l volume  44  FIGURE X  Blood urea n i t r o g e n  45  FIGURE XI  Total protein  46  FIGURE X I I  Glucose  47  FIGURES X I I I t o XX  Histograms showing grand means and means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and experimental f i s h f o r the d i f f e r e n t groups o f f i s h .  FIGURE X I I I Haematocrit  48  FIGURE XIV  49  Haemoglobin  xii LIST OF FIGURES (cont'd) Page FIGURE XV  Red blood c e l l count  FIGURE XVI  White blood c e l l count  .50 51  FIGURE XVII Mean c e l l volume  52  FIGURE XVIII Blood urea n i t r o g e n  53  FIGURE XIX  Total protein  5k  FIGURE XX  Glucose  55  xiii LIST OF APPENDIX FIGURES Page Appendix V  FIGURE I  Graphs o f t h e response o f MCHC and MCH values i n t h e d i f f e r e n t groups o f f i s h t o t h e experimental t r e a t ments. Mean c o r p u s c u l a r haemoglobin concentration  101  FIGURE I I  Mean c e l l u l a r haemoglobin  102  FIGURE I I I  Mean c o r p u s c u l a r haemoglobin concentration..... Mean c e l l u l a r haemoglobin  103 104  FIGURE IV  1a  SECTION  I  INTRODUCTION  1  A l l l i v i n g organisms a r e s u b j e c t t o p e r t u r b a t i o n s i n t h e i r environment which can l e a d t o d e v i a t i o n s from t h e i r normal s t a t e . sical,  The p e r t u r b a t i o n s can be i n t h e form o f phy-  chemical o r b i o l o g i c a l f a c t o r s and be both  and e x t e r n a l t o t h e organism.  internal  Some d e v i a t i o n from a normal  s t a t e a t any g i v e n time i s i n i t s e l f a normal event and r e p r e s e n t s t h e a d a p t i v e responses which t y p i c a l l y  function  to o f f s e t t h e e f f e c t o f p e r t u r b a t i o n s , eg. homeostasis. Consequently, merely t h e presence o f some environmental f a c t o r ( s ) does not mean an abnormal  o r even p a t h o l o g i c a l  s t a t e w i l l ensue u n l e s s t h e f a c t o r s cause t h e normal  range  o f a d a p t i v e responses t o be exceeded. S t r e s s , as d e f i n e d by B r e t t i n t h i s study t o mean:  (1958),  w i l l be used  "...a s t a t e produced by an e n v i r o n -  mental o r o t h e r f a c t o r which  extends t h e a d a p t i v e responses  o f an animal beyond t h e normal range o r which d i s t u r b s t h e normal f u n c t i o n i n g t o such an extent t h a t i n e i t h e r case, the chances o f s u r v i v a l a r e s i g n i f i c a n t l y reduced."  Whether  i t be an environmental s t r e s s o r l o w e r i n g t h e r e s i s t a n c e o f the organism t o a b i o l o g i c a l pathogen o r a pathogenic s t r e s s o r l o w e r i n g t h e r e s i s t a n c e o f the organism t o t h e noxious e f f e c t s o f an environmental t o x i c a n t , the p h y s i o l o g i c a l i n t e r a c t i o n t h a t takes p l a c e between t h e organism and one o r more o f these s t r e s s i n g agents may be important to i t s s u r v i v a l .  2  In d i s c u s s i n g the r o l e o f s t r e s s i n the r e s i s t a n c e i n f i s h , Wedemeyer (1974) s t a t e d :  disease  "Stress r e -  q u i r i n g adjustments t h a t exceed a f i s h ' s a b i l i t y t o accommodate w i l l be l e t h a l . to  Less severe s t r e s s w i l l  predispose  p h y s i o l o g i c a l d i s o r d e r s , or to i n f e c t i o u s diseases i f  f i s h pathogens are p r e s e n t . "  Furthermore, w r i t i n g on  disease mechanisms i n crustaceans  and marine  the  arthropods,  Bang (1970) commented, "Since the time o f M e t c h n i k o f f i t has been r e c o g n i z e d t h a t d i s e a s e mechanisms are a p a r t o f the b i o l o g y o f any organism, and t h a t pathology to  physiology.  ...As  i s related  a r e s u l t o f the s e l e c t i o n f o r c e o f  d i s e a s e s , mechanisms develop t o r e s i s t d i s e a s e .  The  inter-  a c t i o n between such d e s t r u c t i v e f o r c e s and the h o s t ' s sponses to them i s d i s e a s e . "  The  recognition of t h i s  i n t e r a c t i o n i s becoming i n c r e a s i n g l y evident i n the ature.  re-  liter-  W r i t i n g on d i s e a s e r e s i s t a n c e i n f i s h e s , Wedemeyer  (1974) supported cause events  t h i s view t h a t d i s e a s e s are not  single  but are the end r e s u l t o f the i n t e r a c t i o n o f  the d i s e a s e - c a u s i n g agent, the f i s h and the environment. v a r i e t y o f pathogens may f i s h may  be present  not become d i s e a s e d u n t i l  i n the water but  A  the  a s t r e s s i n g agent reduces  the defense system o f the f i s h beyond some c r i t i c a l p o i n t . C u l t u r e d f i s h e s are o f t e n s u b j e c t e d t o a v a r i e t y of  s t r e s s f u l conditions:  e x c e s s i v e h a n d l i n g , crowding,  f l u c t u a t i n g water c o n d i t i o n s and drug treatments.  Under  such c o n d i t i o n s , a number o f the d i s e a s e s encountered be s t r e s s - m e d i a t e d .  may  A number o f s p e c i f i c d i s e a s e s have been  c o r r e l a t e d with the presence o f c e r t a i n  environmental  3 c o n d i t i o n s (Wedemeyer,  1970, 1974).  E p i z o o t i c s a l s o occur i n w i l d stocks o f f i s h t h e i r c o i n c i d e n c e w i t h unusual environmental documented i n f i s h e r i e s l i t e r a t u r e .  (1974) reviewed environmental  and  changes i s w e l l  For example, Snieszko  the c o i n c i d e n c e o f i n f e c t i o u s d i s e a s e s with  s t r e s s caused by temperature, e u t r o p h i c a t i o n ,  sewage, metabolic products o f f i s h , i n d u s t r i a l p o l l u t i o n pesticides.  In t h i s context, he s t a t e d :  " I t i s w e l l known,  from epidemology, t h a t an i n f e c t i o u s agent o f the host i f environmental  and  causes a d i s e a s e  c o n d i t i o n s are r i g h t .  The  i n f l u e n c e o f each subset i s v a r i a b l e - d i s e a s e breaks  out  o n l y i f t h e r e i s a s u f f i c i e n t r e l a t i o n s h i p between them... I f the occurrence o f s t r e s s c o i n c i d e s w i t h the presence pathogenic micro-organisms,  of  i t i s l o g i c a l t o assume t h a t  outbreaks o f d i s e a s e are more l i k e l y t o take p l a c e . " Recognizing the importance  o f the r o l e o f s t r e s s  due t o a b i o t i c f a c t o r s i n d i s e a s e outbreaks  in fish, i t  would be d e s i r a b l e t o be able t o d e t e c t the s t r e s s f u l before the d i s e a s e d s t a t e i s reached and an outbreak The  state occurs.  i n c r e a s i n g volume o f l i t e r a t u r e r e p o r t i n g p h y s i o l o g i c a l  responses o f s t r e s s i n f i s h r e f l e c t s the growing awareness by i n v e s t i g a t o r s o f the importance  o f knowing the p h y s i o -  l o g i c a l c o n d i t i o n o f experimental, c u l t u r e d o r w i l d stocks.  fish  I t has been r e p o r t e d i n t h i s l i t e r a t u r e t h a t a  v a r i e t y o f h a e m a t o l o g i c a l changes occur as a r e s u l t o f the metabolic response  o f the f i s h t o the s t r e s s i n g  Some o f these blood changes may  agent.  be i n d i c a t o r s o f the  4 s t r e s s f u l s t a t e i n f i s h and thus are u s e f u l i n i n i t i a t i n g p r o p h y l a c t i c measures i n f i s h c u l t u r e and p r o v i d e some degree o f assessment o f p h y s i o l o g i c a l s t a t u s o f f i s h s t o c k s . The  g e n e r a l o b j e c t i v e o f the present study was  i n v e s t i g a t e the p o s s i b i l i t y t h a t some h a e m a t o l o g i c a l t e r i s t i c s o f j u v e n i l e chinook might r e f l e c t  to  charac-  salmon (Oncorhynchus tshawytscha)  s t r e s s , as d e f i n e d above.  T h i s was  t o be done  by m o n i t o r i n g v a r i o u s blood parameters d u r i n g exposure t o p o s s i b l e s t r e s s i n g agents, a b a c t e r i a l pathogen and an vironmental t o x i c a n t , presented s i n g l y and The haematocrit  in  en-  combination.  blood parameters t h a t were monitored  (packed  two  were  c e l l volume), haemoglobin, red blood  cell  count, t o t a l white blood c e l l count, mean c e l l u l a r haemoglob i n content, mean c o r p u s c u l a r haemoglobin c o n c e n t r a t i o n , mean c e l l volume, t o t a l plasma p r o t e i n , blood urea n i t r o g e n and plasma g l u c o s e . The b i o t i c agent used kidney d i s e a s e .  Although  i n t h i s study was  acute and subacute  forms o f t h i s  d i s e a s e occur s p o r a d i c a l l y , i t i s mainly c h r o n i c . widespread, Japan.  bacterial  I t i s also  having been r e p o r t e d i n Europe, North America  A l l salmonids  and  are c o n s i d e r e d s u s c e p t i b l e t o kidney  d i s e a s e but i t seldom occurs i n f i s h l e s s than s i x months o l d . The d i s e a s e i s c h a r a c t e r i z e d i n t e r n a l l y by an e n l a r g e d , edematous k i d n e y which u s u a l l y e x h i b i t s o f f - w h i t e l e s i o n s t h a t vary i n s i z e and number and g i v e s r i s e t o the name kidney d i sease..  The  l e s i o n s a l s o can occur i n o t h e r organs such as the  l i v e r , s p l e e n and h e a r t .  A turbid f l u i d  i n the abdominal and  5 p e r i c a r d i a l c a v i t i e s i s o f t e n symptomatic as w e l l .  Externally  f i s h i n advanced.stages o f t h e d i s e a s e u s u a l l y e x h i b i t a d i s tended abdomen, exopthalmia, s k i n p e t e c h i a t i o n , w e l t s , and t i s s u e decay.  No cure f o r t h i s d i s e a s e  lesions  i s known a t p r e -  sent The  t o x i c a n t used i n t h i s study was sodium p e n t a -  chlorophenate (NaPCP).  I t i s a p e s t i c i d a l wood p r e s e r v a t i v e  t h a t i s commonly used i n North America.  In a g r i c u l t u r e , i t  i s used as a d e f o l i a n t and h e r b i c i d e w h i l e i n i n d u s t r y and households i t i s used f o r t r e a t i n g wood a g a i n s t and  termites  as a p r e s e r v a t i v e o f v a r i o u s products prone t o m i c r o -  b i a l attacks  (Hoben e t . a l . , 1976).  A f t e r screening  35  chemicals on t h e b a s i s o f t h e i r chemical p r o p e r t i e s ,  Alderdice  (I963) s e l e c t e d NaPCP as a s u i t a b l e t o x i c a n t f o r f i s h  research  because o f i t s known t o x i c a c t i o n i n f i s h and b e t t e r understanding cants.  o f i t s chemical b e h a v i o r compared t o many other  toxi-  F i n a l l y , NaPCP i s an environmental contaminant which  could f u n c t i o n as an a b i o t i c s t r e s s o r t o f i s h and t h e r e f o r e i s o f p r a c t i c a l i n t e r e s t as w e l l .  Alderdice  (1963)  also  reviewed t h e b i o l o g i c a l a c t i o n o f pentachlorophenol and i t s sodium s a l t , sodium pentachlorophenate (NaPCP). t h a t work on s n a i l t i s s u e and r a b b i t muscle  He  reported  preparations  i n d i c a t e d t h e t o x i c a c t i o n o f both pentachlorophenol and NaPCP was by u n c o u p l i n g o x i d a t i v e p h o s p h o r y l a t i o n .  In a d d i -  t i o n , a d e n o s i n e t r i p h o s p h o t a s e a c t i v i t y and g l y c o l y t i c phosphorylation are i n h i b i t e d . More r e c e n t l y , s e v e r a l s t u d i e s r e p o r t i n g on t h e  6 metabolism o f p e n t a c h l o r o p h e n o l i n f i s h A k i t a k e , 1975 a , b , et.  al.,  c;  (Kobayashi and  Akitake and K o b a y a s h i , 1975; Kobayashi  1975; Kobayashi e t .  a l . , 1975; Kobayashi e t .  al.,1975  Kobayashi e_t. a l . , 1976) i n d i c a t e t h a t decomposition and b i l i a r y excretion, a f t e r  d e t o x i f i c a t i o n by sulphate  t i o n i n the hepatopancreas,  conjuga-  i s the main pathway o f e l i m i n a -  t i o n o f pentachlorophenols i n f i s h . The main f e a t u r e s o f the present seen as a s e r i e s o f more s p e c i f i c accomplished as 1)  study can be  objectives  which were  follows:  to determine i f s e v e r a l o f measured blood parameters  j u v e n i l e Chinook salmon might r e f l e c t  a stressful  in  state im-  posed by e i t h e r the b a c t e r i a l pathogen, the t o x i c a n t o r b o t h , 2)  to c h a r a c t e r i z e both the d i s e a s e process and the t o x i c  effects  o f sodium pentachlorophenate by the measured blood  parameters, 3)  to determine i f s i g n i f i c a n t  change i n these blood p a r a -  meters precede overt symptoms o f the i n f e c t i o n o r response to the t o x i c a n t , thus e v a l u a t i n g the s e n s i t i v i t y o f these parameters 4)  as  i n d i c a t o r s o f the s t r e s s f u l  to i n v e s t i g a t e  s t a t e , and  the p o s s i b i l i t y t h a t the s t r e s s imposed  by the t o x i c a n t might reduce the r e s i s t a n c e o f the f i s h to the d e b i l i t a t i v e e f f e c t s  o f the pathogen.  7a  SECTION I I INITIAL EXPERIMENTS  7 INITIAL EXPERIMENT - A INFECTION EXPERIMENT INTRODUCTION This i n i t i a l  experiment was  c a r r i e d out to d e t e r -  mine the p r o g r e s s i o n o f b a c t e r i a l kidney d i s e a s e f o r d i f f e r ent c o n c e n t r a t i o n s o f b a c t e r i a l suspensions c u l t u r e media and  harvested  injected intraperitoneally.  ment a l s o p r o v i d e d the o p p o r t u n i t y to develop  The a  experi-  coding  system t o d e s c r i b e the symptoms a r i s i n g d u r i n g the of  from  progression  the d i s e a s e f o r the main experiment. Although  j u v e n i l e chinook salmon were used f o r the  subsequent experiments, coho salmon (Oncorhynchus k i s u t c h ) were used f o r t h i s i n i t i a l of  experiment.  Coho was  the  species  c h o i c e f o r the e n t i r e study because they are c u l t u r e d  i n t e n s i v e l y i n the P a c i f i c Northwest r e g i o n and  bacterial  kidney d i s e a s e i n coho i s a c h r o n i c problem i n many f i s h culture operations.  Furthermore, the response o f s e v e r a l  blood parameters to another  environmental  t o x i c a n t s , dehydroi'  a b i e t i c a c i d , has been r e p o r t e d f o r t h i s s p e c i e s (Iwama e t . al.,  1976)  and the present  study would provide a d d i t i o n a l  comparison but w i t h a d i f f e r e n t t o x i c a n t .  The  coho h e l d i n  r e s e r v e f o r the study were, however, l o s t through a c c i d e n t and a replacement stock could not be o b t a i n e d . are c u l t u r e d q u i t e i n t e n s i v e l y as w e l l and  Since chinook  s u s c e p t i b l e to  s i m i l a r c u l t u r i n g problems, t h i s s p e c i e s had p r a c t i c a l est  and was  used to complete the study.  was  j u s t i f i e d on the b a s i s t h a t the i n c u b a t i o n time  inter-  The use o f chinook and  g e n e r a l symptoms o f the d i s e a s e appear t o be s i m i l a r between  8 d i f f e r e n t species and sizes of salmonids f o r a p a r t i c u l a r concentration of experimentally injected kidney disease b a c t e r i a l suspension (T. Evelyn - per. comm.).  I t was  therefore judged that the r e s u l t i n g incubation time determined f o r the coho could be used f o r the subsequent experiment with chinook salmon. MATERIAL AND METHODS FISH Sixty juvenile coho of length 11.0 + 0.75 cm and weight 14.11 + 2.01 g (mean + S.D.) were transferred from a 7000 1 outdoor holding tank receiving Cypress Creek water, 4 to 6 C, to three 64 1 tanks i n the laboratory receiving well water (9-11 C; pH 6.8-6.9; 0  2  8-8.5 mg/1; hardness  54 mg/1 CaC0 ). 3  ACCLIMATIZATION The f i s h were l e f t f o r 48 h to acclimatize to the laboratory environment before d a i l y feeding (5/32 Oregon Moist P e l l e t ) was resumed. PROCEDURE The b a c t e r i a l c e l l suspension f o r i n j e c t i o n into the  f i s h was harvested (App. IIC) from two p e t r i plates of  kidney disease bacteria ( s t r a i n DR151) 15 days a f t e r inocul a t i o n when the c e l l s were i n t h e i r log-phase o f growth (T. Evelyn, per. comm.).  The bacteria were grown on ^  Evelyn's kidney disease media.j--j.-j- and obtained from the Microbiology department at the P a c i f i c B i o l o g i c a l Station  9  (Nanaimo, B.C.).  Three b a c t e r i a l suspensions having o p t i c a l  densities of 0.25, 0.10 and 0.01 (420 nm) were prepared (App. IIC) f o r t h i s  experiment.  After a 24 h starvation period, and before i n j e c t i o n s , three groups of 20 f i s h each were anaesthetized i n 100 mg/1  tricane methanesulphonate (MS 222, Kent Lab. Ltd.  Vancouver,  B.C.)  neutralized with 2N NaOH. Each group of  f i s h received one of the concentrations of b a c t e r i a l suspension by intraperitoneal i n j e c t i o n through the mid-lateral body wall just anterior to the pelvic f i n .  Syringe needles  of 27.5 g were used to eliminate loss of inoculum through the needle puncture a f t e r the needle was withdrawn. RESULTS Table I summarizes the mortality results of the experiment with juvenile coho and shows the respective incubation times observed with the d i f f e r e n t concentrations of injected b a c t e r i a l suspension.  These results indicate  that a b a c t e r i a l c e l l suspension of 0.10 O.D.  (420 nm)  would be the appropriate inoculum concentration f o r the derived 40 day period f o r the main experiment  (App. IIC).  The symptoms of kidney disease observed i n f i s h dying i n t h i s experiment are presented i n Table I I .  10 Table  I  Incubation times trations  r e s u l t i n g from d i f f e r e n t  o f kidney disease bacteria  peritoneally  into  juvenile  coho  concen-  injected  intra-  salmon.  Concentration of kidney disease bacteria injected (optical density)  0.25  0.10  0.01  Incubation time (days; first - last mortality)  34-45  34-49  49-58  Percent successful infection (mortality)  100$  100$  100$  Table  II  Symptoms o f b a c t e r i a l  Observed  kidney  disease  References  (percentage o f t o t a l m o r t a l i t y f o r a l l concentrations )  (9096) B u l l o c k  Exopthalmia  al., 1955;  (40$)  Petechiae  e t . a l . , 1975; E a r p e t .  1955; S n i e s z k o  and G r i f f i n ,  Wood a n d W a l l i s , 1955  B u l l o c k e t . a l . , 1975; E a r p e t . al.,  1955; R u c k e r e t . a l . , 1955;  Snieszko  and G r i f f i n ,  1955;  Wood a n d W a l l i s , 1955 Welts  (4596)  Snout/Upper Jaw Decay Yellow f l u i d  from vent  (75$) (30$)  B u l l o c k e t . a l . , 1975; B e l l , 1961;  Snieszko  1955;  Wood a n d W a l l i s , 1955  This  study  Snieszko  and G r i f f i n ,  and G r i f f i n ,  1955;  Wood a n d W a l l i s , 1955 Distended  abdomen  (100$)  Wood a n d W a l l i s , 1955; S n i e s z k o and G r i f f i n ,  1955; B e l l , 196l  11 O v e r a l l dark c o l o u r i n g  (95%)  Earp e t . a l . , 1955  Haemorrhaging vent  (50$)  Earp e t . a l . , 1955  (85$)  B u l l o c k e t . a l . , 1975  (100$)  B u l l o c k e t . a l . , 1975; Earp  Haemorrhaging i n major organs and body c a v i t y walls Excessive  fluid in  body c a v i t y  et.  a l , 1955; Rucker e t . a l . ,  1955; Enlarged,  pale s p l e e n  (30$)  Wood and W a l l i s , 1955  B u l l o c k e t . a l . , 1975  •Smokey' appearance o f swim  bladder  (50$)  This  study  (95$)  B e l l , 1961; Earp e t . a l . ,  L e s i o n s on h e a r t , l i v e r , s p l e e n and blood  vessels  F r i a b l e , pale  1955;  Snieszko and G r i f f i n ,  1955;  Wood and W a l l i s , 1955  liver  tissue Empty a l i m e n t a r y  canal  Distended h i n d gut  (45$)  T h i s study  (100$)  T h i s study  (65$)  B u l l o c k e t . a l . , 1975  (100$)  Bullock  Swollen pale k i d n e y lumps ( l e s i o n s )  1961;  e t . a l . , 1975; B e l l ,  G r i f f i n , 1954; Earp e t .  a l . , 1955; Rucker e t . a l . , 1954; Bendele and K l o n t z , 1975; Snieszko and G r i f f i n , 1955; Wood and W a l l i s , 1965; Rucker et.  a l . , 1951  Gram p o s i t i v e rods ( D i p l o b a c i l l i ) i n kidneysmears  >  (100$)  A l l references f o r previous symptoms.  12 DISCUSSION The  i n c u b a t i o n times observed i n t h i s  experiment  were found t o be i n g e n e r a l agreement w i t h E v e l y n e t . a l . (1973). and 17  In t h a t study, i n c u b a t i o n times o f 22 t o 36 days t o 31 days were observed i n 86 g (mean) sockeye  (Oncorhynchus  salmon  nerka) s i m i l a r l y i n j e c t e d at o p a c i t i e s o f  and 1 0 . 0 OD at 4 2 0 nm r e s p e c t i v e l y .  1.0  A proportionally longer  i n c u b a t i o n time o f 34 t o 4 9 days r e s u l t e d from a t e n - f o l d lower c o n c e n t r a t i o n , 0 . 1 0 this  OD,  of b a c t e r i a l c e l l s injected i n  study. The m a j o r i t y o f the observed symptoms o f the  d i s e a s e are i n agreement w i t h those r e p o r t e d i n the t u r e , as seen i n Table I I .  The  litera-  symptoms suggest t h a t  this  d i s e a s e i s a systemic i n f e c t i o n t h a t a f f e c t s a l l p a r t s o f the h o s t ' s body.  A l l the r e p o r t e d symptoms r e l a t e d t o t h i s  d i s e a s e were observed i n t h i s experiment  except the f o r m a t i o n  o f w h i t e , f a l s e membranes (Earp e t . a l . , 1 9 5 5 ) • thought, however, t h a t the "smokey" appearance b l a d d e r , p r e v i o u s l y u n r e p o r t e d , may f a l s e membranes.  I t was o f the swim-  i n f a c t have been the  Upon m i c r o s c o p i c examination o f the s u r f a c e  o f the swimbladders,  s m a l l , white, p i n p o i n t l e s i o n s were  observed l i n i n g a l l the blood v e s s e l s , thus g i v i n g a "smokey" appearance.  T h i s suggests t h a t the b a c t e r i a spreads t h r o u g h -  out the body v i a the c i r c u l a t o r y system.  Earp e t . a l . ( 1 9 5 5 )  s t a t e d t h a t the bacterium o f k i d n e y d i s e a s e c o u l d be r e a d i l y i n the c i r c u l a t o r y system o f f i s h i n advanced o f the d i s e a s e .  found stages  T h i s would a l s o e x p l a i n the o b s e r v a t i o n t h a t  the m a j o r i t y o f l e s i o n s were found i n organs t h a t are h i g h l y  13  vascularized  (eg. kidney, l i v e r and s p l e e n ) .  Furthermore,  p e t e c h i a t i o n and haemorrhaging a t the v a r i o u s  sites  indicate  the d e s t r u c t i o n o f v a s c u l a r t i s s u e o f t h e c i r c u l a t o r y system by t h e pathogen.  Destruction  o f the renal t i s s u e i s also a  primary c h a r a c t e r i s t i c o f t h i s disease 1956).  Therefore,  (Wood and Yasutake,  f i s h i n f r e s h water t h a t  osmotically  imbibe water from t h e i r hypotonic environment would more water as t h e i r e x c r e t o r y f e c t e x p l a i n s t h e excessive and  t h e distended  a b i l i t i e s decrease.  gain This e f -  f l u i d i n t h e abdominal c a v i t y  abdomen o f i n f e c t e d f i s h .  The exopthalmia  r e s u l t i n g from t h i s i n f e c t i o n may have been caused by the p r e s s u r e exerted  from the i n c r e a s i n g f l u i d volume 'in t h e  body ..as suggested by Wood and W a l l i s  (1955).  The o v e r a l l  dark c o l o u r i n g , o r m e l a n o s i s , t h a t was observed i n m o r t a l i t i e s and  moribund f i s h was i n t e r p r e t e d as being a n o n - s p e c i f i c  hormonal o r nervous response t o abnormal c o n d i t i o n s i n t e r n a l o r e x t e r n a l environment.  T h i s e f f e c t has been  observed i n f i s h o f the same s p e c i e s toxicant  and age i n response t o  exposure and high l e v e l s o f e x e r c i s e  observations).  in its  (unpublished  14 INITIAL EXPERIMENT - B BIOASSAY INTRODUCTION A b i o a s s a y was LC^Q  conducted t o determine the 96 h  o f NaPCP under a s i m i l a r f i s h l o a d i n g d e n s i t y t h a t  would be used i n the main experiment.  Since at t h i s p o i n t  i n the study, the coho had been l o s t , t h i s experiment had a d d i t i o n a l importance because the t o x i c i t y o f NaPCP f o r j u v e n i l e chinook salmon has not been r e p o r t e d . FISH J u v e n i l e chinook salmon o f l e n g t h 1 0 . 3 and weight 1 1 . 2 5  + 3 . 9 4 g (mean + S.D.)  + 0.8  cm  were r e a r e d i n a  4 0 0 0 1 c i r c u l a r tank i n d o o r s under n a t u r a l p h o t o p e r i o d i n w e l l water  ( 1 0 - 1 1 C; pH 6 . 8 - 6 . 9 ;  54 mg/l CaCO^).  0  2  8-8.6  mg/1;  hardness  Gram s t a i n o f kidney smears a s e p t i c a l l y  taken from a random sample i n d i c a t e d t h a t t h i s stock was free of b a c t e r i a l  pathogens.  APPARATUS S i x 3 2 . 6 1 g l a s s a q u a r i a s e t i n one l a r g e  rectan-  g u l a r f i b e r g l a s s tank were used as t e s t tanks f o r the f l o w through b i o a s s a y s o f NaPCP.  The l a r g e r e c t a n g u l a r tank  p r o v i d e d some degree o f i n s u l a t i o n t o d i u r n a l  temperature  f l u c t u a t i o n s by c o l l e c t i n g the o v e r f l o w from the t e s t a q u a r i a and m a i n t a i n i n g a water depth equal t o approximately 2/3 the a q u a r i a h e i g h t .  The f o u r s i d e s o f each a q u a r i a were  covered w i t h b l a c k p l a s t i c and the t o p covered w i t h smoked  15 p l e x i g l a s t o reduce e x t e r n a l s t i m u l i . connected  An enclosed f u n n e l  t o a g l a s s tube e n t e r i n g the dquarium through  hole i n the p l e x i g l a s top mixed and and d i l u e n t water b e f o r e i t was  a  i n t r o d u c e d the t o x i c a n t  added t o each t e s t tank  (App. VI; F i g . I ) . The  stock s o l u t i o n s o f NaPCP were prepared  d e s c r i b e d by A l d e r d i c e (1963; see a l s o App.  IA).  as  Diluent  water entered the mixing f u n n e l s at a constant flow o f  880  ml/min through  stock  5 mm  diameter  polyethylene tubing.  The  s o l u t i o n o f NaPCP flowed to the mixing f u n n e l s from 25 1 m o d i f i e d M a r i o t b o t t l e s (Leduc, 1966) 3.9 ml/min through tubing ( F i g . I ) .  2 mm,  at a constant r a t e o f  o u t s i d e diameter,  polyethylene  The d i l u e n t water flow p r o v i d e d a  percent replacement  time o f approximately  1.5  90.0  h (Sprague,  1969). PROCEDURE The were 0.06,  c o n c e n t r a t i o n s o f NaPCP used i n the  0.07,  0.08,  0.09,  0.13,  0.19  and 0.27  bioassay mg/1.  Each  b i o a s s a y tank contained 40 f i s h r e s u l t i n g i n a l o a d i n g dens i t y o f approximately  13.8  g / l ; the approximate l o a d i n g den-  s i t y t h a t would be used i n the main experiment. t i o n s i n the t e s t a q u a r i a were maintained 7.0-7.1 and 0  2  8-8.3  mg/1.  Water c o n d i -  at 11.8-12.0 C;  pH  A f t e r the t o x i c a n t flows were  s t a r t e d , the c o n c e n t r a t i o n s i n the t e s t tanks were allowed to e q u i l i b r a t e f o r 4 h, which was  the time, f o r 99.9  percent r e -  placement at the d i l u e n t flows used (Sprague, 1968).  At the  end o f t h i s e q u i l i b r a t i o n p e r i o d , o b s e r v a t i o n s f o r m o r t a l i t y were  16 made at 0 . 2 5 , 0 . 5 , thereafter.  1.0,  2.0,  4 . 0 h and  every 2 + 0 . 5  A f t e r the f i r s t m o r t a l i t y o c c u r r e d ,  t i o n s were made a t more frequent i n t e r v a l s .  Excess water was  was  stopped.  removed from dead f i s h with paper t i s s u e  before o b t a i n i n g the wet DETERMINATION OF THE  weight.  9 6 h LCg  Q  ( L T ^ Q ) was  The median s u r v i v a l time from l o g - p r o b i t p l o t s o f cumulative (Sprague, 1 9 6 9 ) .  observa-  Death  recorded as the time when o p e r c u l a r movements  h  obtained  percent m o r t a l i t y  These data were used t o c o n s t r u c t a t o x i -  c i t y curve by p l o t t i n g log-median s u r v i v a l times a g a i n s t l o g toxicant concentration ( F i g . I I ) . c e n t r a t i o n (LCJJQ) was  determined  The  incipient lethal  con-  from a l o g - p r o b i t p l o t  of  dead a t 96 h ( p r o b i t s c a l e ) a g a i n s t c o n c e n t r a t i o n  percentage  (log scale)(Sprague, 1 9 6 9 ) .  F o l l o w i n g the nomographic  dure o f L i t c h f i e l d and Wilcoxon ( 1 9 4 9 ) a l i n e best  proce-  fitting  t h e p o i n t s was  drawn by m i n i m i z i n g the C h i  v a l u e o f the  line.  T h i s procedure  y i e l d s a r e l a t i v e l y accurate estimate o f the  i n c i p i e n t L C ^ Q value and a l s o a l l o w s the c a l c u l a t i o n o f the 9 5 percent confidence l i m i t s and a slope f u n c t i o n , s. slope f u n c t i o n permits r e p r o d u c t i o n o f the RESULTS AND  line.  DISCUSSION  The chinook  incipient 9 6 h LC^  Q  o f NaPCP f o r j u v e n i l e  salmon a t a l o a d i n g d e n s i t y o f approximately mg/1.  The  95  and 0 . 0 5 7 mg/1.  The  f i s h i n the c o n t r o l tank were  was  0.078  The  13.8  percent confidence l i m i t s were  g/1  0.11  observed  17  to be normal i n b e h a v i o r a l and p h y s i c a l c h a r a c t e r i s t i c s a t the end o f the b i o a s s a y .  The r e l a t i v e l y h i g h l o a d i n g den-  s i t y d i d not seem t o have any obvious adverse the c o n t r o l f i s h . approximates  The 9 6 h L C ^ Q e s t i m a t i o n i n t h i s  those f o r o t h e r salmonids.  NaPCP f o r j u v e n i l e rainbow t r o u t  study  9 6 h L C ^ Q values o f  (Salmo g a i r d n e r i ) , coho  salmon (Oncorhynchus k i s u t c h ) and sockeye nerka) have been r e p o r t e d as 0 . 0 9 8 m g / 1 , mg/1  e f f e c t s on  salmon (Oncorhynchus 0.092  r e s p e c t i v e l y by Davis and Hoos ( 1 9 7 5 ) .  mg/1  and 0 . 1 3 0  As recommended  by t h e g u i d e l i n e s f o r conducting b i o a s s a y s (Sprague, 1 9 6 9 ; Standard Methods, 1 9 7 1 ;  Davis and Mason, 1 9 7 3 ) ,  d e n s i t y o f 0 . 5 mg/1 was used  i n the s t a t i c bioassays involved  i n t h e above study (Davis and Hoos, 1 9 7 5 ) .  The s i m i l a r i t y  between t h e r e s u l t s o f t h e present experiment r e p o r t e d by Davis and Hoos ( 1 9 7 5 ) a b i o a s s a y under f l o w through  a low l o a d i n g  and those  suggests t h a t c a r r y i n g out  conditions with a r e l a t i v e l y  h i g h t o x i c a n t s o l u t i o n replacement  r a t e may a m e l i o r a t e the  e f f e c t s o f using a high loading density.  The p o s s i b l e s t r e s s  t h a t may be imposed on t h e f i s h by a h i g h l o a d i n g d e n s i t y would otherwise be expected t o s i g n i f i c a n t l y lower t h e 9 6 h LCJJQ  value by r e d u c i n g t h e r e s i s t a n c e o f the f i s h t o the  e f f e c t s o f the t o x i c a n t .  18a  n  Photograph cribed  of  in Initial  Diagrammatic assay  test  aquaria  Experiment  showing  tank  and  enclosed modified  containing toxicant.  as  des  B.  representation of  aquarium  funnel, bottle  bioassay  one  bio-  mixing Mariot  18 Figure  I  b.  modified Mariot bottle containing toxicant  19a  Figure  II  T o x i c i t y curve showing L T ^ Q ' S . 96 h L C ^ Q  is  i n d i c a t e d w i t h 95$  determined by nomographic  the i n c i p i e n t  confidence l i m i t s  analysis.  as  19  96 h (5760 min)  E1 E-< M  i n c i p i e n t 96 h L C ^ Q + 95$ confidence l i m i t s = -0.07.8 mg/1 (0.057, 0.11)  < EH Pi O  O  ir\ O EH  I I—1  .  E-i  NaPCP CONCENTRATION - mg/1 Figure I I  20a  SECTION I I I MATERIALS AND METHODS FOR MAIN EXPERIMENT  V  20 EXPERIMENTAL DESIGN A two x t h r e e x nine f a c t o r i a l d e s i g n was used f o r t h i s experiment  ( F i g . I I I ) . The two l e v e l s o f t h e h e a l t h  f a c t o r were u n i n f e c t e d c o n t r o l f i s h and kidney d i s e a s e i n f e c t e d experimental f i s h .  The t h r e e l e v e l s o f t h e e n v i r o n -  mental c o n d i t i o n f a c t o r were 0 , 0 . 0 5 and 0 . 5 0 o f the i n c i p i e n t 9 6 h L C ^ Q value f o r t h e t o x i c a n t NaPCP as determined i n I n i t i a l Experiment  B.  The t h i r d f a c t o r was time, w i t h nine  sampling days a t four-day  intervals.  FISH J u v e n i l e chinook  salmon o f l e n g t h 1 1 . 5 + 0 . 4 cm  and weight 1 5 . 6 2 + 1 . 9 9 g (mean + S.D.) were o b t a i n e d  from  Rosewall Creek on Vancouver I s l a n d where they had been r e a r e d from f r y .  The eggs were from w i l d stock r e t u r n i n g t o the  Qualicum Salmon Hatchery on Vancouver I s l a n d . t r a n s f e r r e d by t r u c k t o the P a c i f i c Environment  The f i s h were Institute  and 2 4 0 f i s h i n t r o d u c e d d i r e c t l y i n t o each o f s i x t e s t r e c e i v i n g w e l l water ( 1 2 - 1 2 . 2 C; 0 hardness  2  8-8.5  mg/1;  pH  tanks  6.5-6.9;  5 4 mg/1 CaCO^).  ACCLIMATIZATION The for  f i s h were a c c l i m a t e d t o the experimental  2 7 days b e f o r e experimental treatment.  commercial f i s h food ( 5 / 3 2 " to  satiation.  tanks  They were f e d a  Oregon Moist P e l l e t ) twice  daily  To c o n d i t i o n the f i s h t o the presence o f t h e  sampling n e t , t h e f i s h were f e d o n l y a f t e r t h e tank  covers  were l i f t e d and t h e sampling net i n t r o d u c e d i n t o the water. I t was thought  t h a t t h i s procedure would reduce t h e time f o r  21a  Figure  III  F a c t o r i a l d e s i g n used f o r the main Factors  are:  H e a l t h (C = u n i n f e c t e d  kidney disease  infected  m e n t a l C o n d i t i o n (1 intermediate  3=  level  high level  days at  = clean water,  sampling day,  exposure;  f o u r day  2 -  intervals).  control fish; E = fish); Environ-  0 x 96  o f NaPCP e x p o s u r e ,  o f NaPCP e x p o s u r e ,  Time ( T I = f i r s t ning toxicant  experimental  experiment.  0.5  0.05 x  h LC^Q; x 96  96 h  f o u r days a f t e r  9 = subsequent  2 =  h LC^Q  LC^Q);  begin-  sampling  21 Figure I I I  EXPERIMENTAL DESIGN HEALTH  IC o  H E—' M Q  o o  IE  \ N  22a  Figure  IV  a.  Photograph used  b.  of a  i n main  Diagrammatic tanks  from  pair  tanks  experiment.  representation  the  back.  voirs,  toxicant  mixing  f u n n e l s and  shown.  of test  head  o f the  Toxicant tank,  diluent  same  reser-  enclosed pipes  are  22  F i g u r e IV  a.  b. d i l u e n t water from constant head tank  enclosed mixing funnel  23 netting the f i s h , thereby reducing the disturbance when regu l a r sampling began.  Oxygen, temperature and pH measure-  ments f o r water i n each tank were taken i n the morning before the f i r s t feeding and at i n t e r v a l s of three to four days during t h i s acclimatization and conditioning period. APPARATUS To provide aerated water at a constant flow to the test tanks, well water (10-11 C; pH 6.8-6.9; 0 mg/1;  hardness 54 mg/1  8-8.5  2  CaCO^) was passed through s i x a s p i r -  ators into a 186 1 f i b e r g l a s head tank.  Additional aeration  was provided with compressed a i r . The flow of diluent water into the mixing funnels located on each of the s i x 186 1 test tanks was regulated independently by valves. The apparatus f o r administering a l l the l e v e l s of NaPCP were the same.  The toxicant was pumped from 100 1  p l a s t i c reservoirs into 10 1 plexiglas head tanks and the overflow returned to the r e s e r v o i r s .  From the toxicant head  tanks, a constant flow of 40 ml/min was led through 5 mm p l a s t i c tubing into the mixing funnels ( F i g . IV). water flow was  Diluent  3.2 l/min and provided a 99.9 percent replace-  ment time of approximately 4.5 h as determined  by the graph-  i c a l method of Sprague (1969). INFECTION OF FISH As a precaution, uninfected control f i s h were a l ways handled before infected f i s h for a l l procedures out the experiment.  through-  Before sham i n j e c t i o n , control f i s h i n  groups of 10 to 15 f i s h were anaesthetized i n 60 1 of 75  ml/1  24 MS 222 n e u t r a l i z e d w i t h 5N NaOH.  The c o n t r o l s o l u t i o n f o r  i n j e c t i n g each f i s h c o n s i s t e d o f 0.1 cc o f s t e r i l e  saline  and peptone (0.$5 and 0.1 percent r e s p e c t i v e l y ) i n j e c t e d i n t r a p e r i t o n e a l l y i n t h e lower m i d - l a t e r a l body w a l l a n t e r i o r to the p e l v i c f i n s .  S t e r i l e , p l a s t i c 1.0 cc s y r i n g e s  were f i l l e d w i t h c o n t r o l s o l u t i o n , f i t t e d w i t h 26.5 gauge needles (both from Becton D i c k i n s o n , R u t h e r f o r d , N.Y.) and kept on i c e u n t i l t h e time o f i n j e c t i o n .  After  injection,  the f i s h were p l a c e d i n a 186 1 r e c o v e r y tank r e c e i v i n g t h e same water as the t e s t t a n k s .  A f t e r a group o f 240 f i s h had  been i n j e c t e d w i t h the c o n t r o l s o l u t i o n , they were t r a n s f e r r e d back i n t o t h e i r t e s t t a n k s .  T h i s procedure was c a r -  r i e d out ttiree times f o r t h e t h r e e c o n t r o l The  groups.  same i n j e c t i o n procedures were f o l l o w e d f o r  the t h r e e experimental groups but w i t h a suspension o f k i d ney d i s e a s e b a c t e r i a (App. I I B and I I C ) .  T h i s inoculum was  prepared w i t h b a c t e r i a h a r v e s t e d (App. I I C ) i n t h e i r l o g phase o f growth from c u l t u r e media (App. I I A ) . mental  The e x p e r i -  f i s h f o r a l l t o x i c a n t l e v e l s o f NaPCP r e c e i v e d the  same volume (0.1 ml) and c o n c e n t r a t i o n (0.1 OD, 420 nm) o f inoculum as determined  i n I n i t i a l Experiment  A.  I n j e c t i o n s f o r both c o n t r o l and experimental groups were c a r r i e d out on t h e same day.  V i a b l e counts  (App. IID)  i n d i c a t e d t h a t approximately 41.28 x 10^ v i a b l e kidney d i s e a s e b a c t e r i a l c e l l s were i n j e c t e d i n t o each experimental  fish.  TOXICANT ADMINISTRATION The time r e q u i r e d f o r blood c o l l e c t i o n , measurement i  ?5 of h a e m a t o l o g i c a l parameters and renewal o f t o x i c a n t s o l u t i o n s p e r m i t t e d sampling be completed  i n one day.  from o n l y two o f the s i x tanks t o Since the time course o f the  experimental treatments was monitored sampling  c o u l d be accomplished  a t four-day  intervals,  f o r a l l tanks over t h i s f o u r  day p e r i o d and t h e i n t e r s a m p l i n g i n t e r v a l f o r each group o f f i s h kept constant f o r t h e d u r a t i o n o f the experiment.  Hence  the i n i t i a l a d m i n i s t r a t i o n o f NaPCP t o the f o u r groups o f fish  ( F i g . I l l ) was staggered t o p r o v i d e equal t o x i c a n t expo-  sure times t o each group.  I t should be noted, however, t h a t  a l l the i n f e c t e d f i s h r e c e i v e d b a c t e r i a l i n j e c t i o n s a t the same t i m e .  Consequently,  a t any g i v e n sampling time the  exposure t o b a c t e r i a l i n f e c t i o n would be unequal  by as much  as t h r e e days between the groups exposed t o the lowest and h i g h e s t c o n c e n t r a t i o n s o f NaPCP w h i l e t o x i c a n t exposure would be the same f o r a l l groups.  Because c o n s i s t e n t v i a b i l i t y o f  s t o r e d inoculum, o r t h e v i a b i l i t y o f separate c u l t u r e s o f b a c t e r i a grown a t i n t e r v a l s t o f i t i n t o the staggered samp l i n g schedule, c o u l d not be assured, i t was judged more p r a c t i c a l t o i n j e c t t h e b a c t e r i a l treatment  groups a t the  same time w i t h one inoculum batch r a t h e r than r i s k  unequal  v i a b i l i t y o f i n o c u l a prepared t o g i v e equal b a c t e r i a l exposure t i m e s .  Sampling  o f t h e u n i n f e c t e d c o n t r o l and kidney  d i s e a s e i n f e c t e d experimental f i s h e i g h t days a f t e r i n j e c t i o n .  i n c l e a n water commenced  Sampling  o f the uninfected  c o n t r o l and i n f e c t e d experimental f i s h a t the i n t e r m e d i a t e (©.05 o f the 96 h L C ^ ) NaPCP l e v e l commenced f o u r days Q  a f t e r t o x i c a n t a d m i n i s t r a t i o n and nine days a f t e r  injection.  26 Sampling  o f the u n i n f e c t e d c o n t r o l f i s h and  infected experi-  mental f i s h at the h i g h (0.5 o f the 96 h L C  5 0  ) NaPCP l e v e l  commenced f o u r days a f t e r t o x i c a n t a d m i n i s t r a t i o n and t e n days a f t e r i n j e c t i o n .  A l l c o n t r o l f i s h were sampled i n the  morning and experimental f i s h i n the a f t e r n o o n . experimental group, sampling was  For each  done at four-day  and a t o t a l o f nine samples were completed  unless  intervals prevented  by m o r t a l i t y . The  i n t e r m e d i a t e and h i g h l e v e l s o f NaPCP admini-  s t r a t i o n commenced on the f i f t h and s i x t h days r e s p e c t i v e l y a f t e r the i n j e c t i o n day.  The  p r e p a r a t i o n and d i l u t i o n  dures f o r the NaPCP are g i v e n i n Appendix IA.'  proce-  The t o x i c a n t  c o n c e n t r a t i o n s i n the t e s t tanks were allowed t o e q u i l i b r a t e to the d e s i r e d l e v e l over one done i n I n i t i a l Experiment  replacement  time  (4.5 h ) , as  B.  SAMPLING Blood f o r the h a e m a t o l o g i c a l measurements  was  c o l l e c t e d from the severed caudal peduncle.  To p r o v i d e  s u f f i c i e n t blood f o r these measurements, two  f i s h were r e -  quired.  Each sample c o n s i s t e d o f 12 pooled  measurements, i e 24 f i s h .  Two  k i l l e d by blows to the head.  haematological  f i s h were n e t t e d t o g e t h e r  and  They were then b l o t t e d w i t h  paper towels and the caudal peduncle wiped w i t h a t i s s u e soaked w i t h 95$  ethanol.  h e p a r i n i z e d N a t e l s o n tubes  Blood was  c o l l e c t e d i n 280 u l  (Sherwood Med.  Ind. S t . L o u i s e  by f i l l i n g t o an a r b i t r a r y mark approximately 1/4 l e n g t h o f the t u b e s .  t o 1/3  A h e p a r i n i z e d microhaematocrit  tube  MO) the  27 ( P r e - c a l , C l a y Adams, N.J.) was a l s o f i l l e d t o l a t e r d e t e r mine t h e haematocrit  (HCT) v a l u e .  The caudal peduncle o f  the second f i s h was then severed and t h e columns o f blood i n the Natelson tubes were doubled.  Another  microhaematocrit  tube was f i l l e d f o r HCT d e t e r m i n a t i o n as above. two  One o f t h e  f i s h was put a s i d e f o r f o r k l e n g t h and weight measure-  ments and p a t h o l o g i c a l examination.  The o t h e r f i s h was d i s -  carded.  o f t h e tank cover t o  The t o t a l time from opening  completion  o f blood withdrawal  from t h e two f i s h took  approx-  i m a t e l y f o u r t o s i x minutes. PARAMETER MEASUREMENTS The microhaematocrit  tubes were s e a l e d and imme-  d i a t e l y c e n t r i f u g e d ( I n t . E q u i p t . Co., D i v . o f Damon) f o r 3.5  min a t 13,000 rpm f o r t h e d e t e r m i n a t i o n o f HCT v a l u e s  a c c o r d i n g t o Snieszko  (i960).  The haemoglobin (Hb) d e t e r -  m i n a t i o n was made from a 30 u l a l i q u o t o f whole blood t r a n s f e r r e d from one o f t h e N a t e l s o n tubes by t h e cyanmethaemog l o b i n method (Harry, 1968) u s i n g t h e Accustat Blood system (Becton D i c k i n s o n , M i s s i s s a u g a , O n t a r i o ) . t o t a l r e d and white blood c e l l counts  (RBC and WBC  Analyzer  Blood f o r respec-  t i v e l y ) was o b t a i n e d from an a l i q u o t o f whole blood from t h e first  N a t e l s o n tube.  I t was c o l l e c t e d i n t o a standard r e d  c e l l d i l u t i n g p i p e t t e (1:200 d i l u t i o n ) and d i l u t e d w i t h ReesE c k e r s o l u t i o n ( K l o n t z and Smith, 1968). blood c e l l counts haemocytometer.  Red and t o t a l white  ( i n c l u d i n g thrombocytes) were made w i t h a RBC counts were m u l t i p l i e d by 10,000 and  WBC counts by 500 t o g i v e t h e number o f c e l l s p e r mnr  28  ;  (Hesser, I960). Natelson tubes  Plasma was obtained by c e n t r i f u g i n g t h e (15 min a t 2500 rpm) and t r a n s f e r r e d t o  p l a s t i c v i a l s f o r storage a t -20 C.  Blood u r e a n i t r o g e n  (BUN), glucose (GLU) and t o t a l p r o t e i n (TP) d e t e r m i n a t i o n s were made on t h e s t o r e d plasma samples a t a l a t e r The  date.  d e t e r m i n a t i o n o f BUN, GLU and TP v a l u e s on  p r e v i o u s l y f r o z e n plasma were determined  colorimetrically  u s i n g the A c c u s t a t Blood A n a l y z e r System.  Three e r y t h r o c y t i c  i n d i c e s were c a l c u l a t e d from t h e HCT, Hb and RBC v a l u e s u s i n g the f o l l o w i n g formulae: (MCHC) Mean C o r p u s c u l a r Hb C o n c e n t r a t i o n (pg) = Hb(g/100 ml) x 10 RBC(millions/mm ) 3  (MCH)  Mean C e l l u l a r Hb ($) = Hb(g/100 ml) x 100 HCT  (MCV)  ($)  Mean C e l l Volume ( u ) = 3  HCT($) x 10 -  RBC(millions/mm ) 3  The t o t a l time t o c a r r y out the above procedures  and measure-  ments took approximately 10 t o 12 minutes. PATHOLOGICAL EXAMINATIONS When a sampling and  s e t had been completed,  i n t e r n a l p a t h o l o g i c a l examinations  p a t h o l o g i c a l examinations,  the e x t e r n a l  were c a r r i e d o u t . F o r  t h e s c a l e s o f a l l twelve f i s h  from  29  one sample were removed from one s i d e and t h e s k i n bathed i n 95$ e t h a n o l .  Kidney smears were a s e p t i c a l l y made by  o b t a i n i n g a p i e c e o f k i d n e y t i s s u e on a s t e r i l i z e d  Nichrome  i n o c u l a t i n g l o o p through an i n c i s i o n below and p a r a l l e l t o the l a t e r a l l i n e made w i t h a s t e r i l i z e d s c a l p e l .  The smears  were Gram s t a i n e d and i n s p e c t e d f o r t h e presence o f k i d n e y disease b a c t e r i a .  General notes were made on e x t e r n a l and  i n t e r n a l c h a r a c t e r i s t i c s o f each f i s h w i t h p a r t i c u l a r a t t e n t i o n f o r those p a t h o l o g i c a l symptoms observed i n I n i t i a l Experiment A.  The e x t e r n a l and i n t e r n a l p h y s i c a l  character-  i s t i c s were noted and were grouped a r b i t r a r i l y i n t o s i x main c a t e g o r i e s (number coded one t o s i x ; App. I l l ) a c c o r d i n g t o the s e v e r i t y o f t h e i n f e c t i o n . was  The p h y s i c a l examination  c a r r i e d out f o r each f i s h sampled and a mean was  l a t e d f o r each complete sample o f twelve f i s h .  calcu-  This cate-  g o r i z a t i o n o f p a t h o l o g i c a l c o n d i t i o n s i s t a b u l a t e d i n Table III. DATA ANALYSIS P r e l i m i n a r y computation o f means and a n a l y s i s o f v a r i a n c e was c a r r i e d out u s i n g t h e prepared program, ( U n i v e r s i t y o f B r i t i s h Columbia).  S c h e f f e ' s t e s t was used  t o compare d i f f e r e n c e s between means f o r s t a t i s t i c a l cance a t P = 0.05 (Edwards, 1967).  ANOVAR  signifi-  The r e s u l t s o f s p e c i f i c  comparisons f o r s i g n i f i c a n c e between means a r e shown on t h e graphs o f t h e changes i n t h e h a e m a t o l o g i c a l parameters over time ( F i g s . V - X I I ) . The f o l l o w i n g comparisons were made f o r each parameter a t each sampling time:  between v a l u e s o f  30'  u n i n f e c t e d c o n t r o l f i s h and k i d n e y d i s e a s e i n f e c t e d e x p e r i mental f i s h at each t o x i c a n t l e v e l ;  between v a l u e s o f u n i n -  f e c t e d c o n t r o l f i s h among t h e t h r e e t o x i c a n t l e v e l s and between v a l u e s o f kidney d i s e a s e i n f e c t e d experimental  fish  among t h e t h r e e t o x i c a n t l e v e l s . The  grand mean r e p r e s e n t s t h e mean o f a l l t h e  means f o r a l l sampling  days.  The mean o f a b s o l u t e  differ-  ences between c o n t r o l and experimental f i s h r e p r e s e n t s t h e mean o f a l l t h e a b s o l u t e v a l u e s o f t h e i n d i v i d u a l  differences  between c o n t r o l and experimental means at each sampling f o r a l l sampling days.  time  These two c a l c u l a t e d v a l u e s a r e p r e -  sented i n F i g u r e s X I I I t o XX as a g e n e r a l summary o f t h e r e l a t i v e e f f e c t s t h e v a r i o u s experimental c o n d i t i o n s had on the measured h a e m a t o l o g i c a l parameters f o r t h e experiment as a whole.  I n t h i s treatment  i s f o r t h e experiment sampling t i m e s .  o f data, t h e value d e r i v e d  as a whole r a t h e r than f o r s p e c i f i c  A g r e a t e r departure o f experimental  fish  value from c o n t r o l f i s h value i n t o x i c a n t r e l a t i v e t i c l e a n water i s i n d i c a t e d by a t a l l e r bar f o r t h e mean o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and experimental groups f o r group 2 r e l a t i v e t o group  1.  51a  SECTION IV RESULTS FOR THE MAIN EXPERIMENT  31  Throughout the r e s u l t s and the  symbols IC, 2C,  nate u n i n f e c t e d  3C and  disease  the  intermediate  i n the high t o x i c a n t l e v e l ,  desig-  interand  kid-  i n f e c t e d experimental f i s h i n c l e a n water, i n toxicant l e v e l ,  r e s p e c t i v e l y , as shown i n the The conditioned  3E w i l l be used to  c o n t r o l f i s h i n c l e a n water, i n the  mediate t o x i c a n t l e v e l , ney  I E , 2E,  discussion sections,  i n the high t o x i c a n t  experimental design  level,  (Fig. III).  f i s h i n a l l the tanks appeared to become w e l l  to the presence o f a net  27-day a c c l i m a t i z a t i o n p e r i o d . by the presence o f the o f r u s h i n g to the  The  i n the tanks d u r i n g f i s h were not  the  disturbed  sampling net; t h e i r normal b e h a v i o r  surface  i n a n t i c i p a t i o n o f food each time  the cover was  opened p e r s i s t e d throughout the  o f sampling.  T h e i r c h a r a c t e r i s t i c s c h o o l i n g o f groups 2E  and  early period  IE f i s h seemed to d e t e r i o r a t e , however, b e g i n n i n g  the 12th  1 6 t h (T1+)  (T3) and  creasing reduction  days r e s p e c t i v e l y .  i n f e e d i n g was  i o r a t i o n o f c h a r a c t e r i s t i c s c h o o l i n g was f i s h o f group 3C s t a r t i n g on the 28th day i n groups 2C o r IC.  ness, d i s o r i e n t a t i o n and t u r e became evident followed  An i n -  observed i n groups IE  2E s t a r t i n g about the second sampling day  exposure but not  on  (T2).  The  deter-  a l s o observed i n (T7) o f t o x i c a n t  In a d d i t i o n , s l u g g i s h -  markedly reduced r e s i s t a n c e to  i n a few  and  cap-  i n d i v i d u a l s o f these groups  and  by s e v e r a l days, the appearance o f d e t e r i o r a t i o n o f  schooling behavior.  M o r t a l i t i e s a l s o s t a r t e d to o c c u r at  about these times i n these groups.  I t seemed l i k e l y  that  32 those i n d i v i d u a l s e x h i b i t i n g t h e s l u g g i s h behavior comp r i s e d the m o r t a l i t i e s . During blood c o l l e c t i o n , c l o t t i n g f r e q u e n t l y o c c u r r e d i n a l l groups o f f i s h at the commencement o f the experiment.  However, a f t e r t h e l 6 t h day ( T 4 ) » blood  t i n g was l e s s troublesome  i n both IE and 2E groups.  r e d u c t i o n i n c l o t t i n g tendency  clotThis  a l s o o c c u r r e d i n group 3C  but not u n t i l approximately t h e 25th day o f t o x i c a n t  exposure.  A l l the f i s h i n group 3E d i e d suddenly on t h e s e cond sampling day.(T2).  Although i t was noted on t h e p r e -  v i o u s day t h a t most o f the f i s h were a t the s u r f a c e , f e e d i n g b e h a v i o r , o r t h e i r response t o a r o u t i n e procedure t o which they had been c o n d i t i o n e d , was normal and the sudden mort a l i t y o f t h i s group was unexpected.  Gram s t a i n e d kidney  smears showed t h a t the pathogen had invaded the kidney t i s s u e by t h i s day (14 days a f t e r i n j e c t i o n w i t h kidney disease b a c t e r i a ) .  T h e r e f o r e comparisons  3E were p o s s i b l e f o r o n l y t h e f i r s t  i n v o l v i n g group  sampling day ( T l ) .  Mor-  t a l i t i e s i n groups IE and 2E, due t o the p r o g r e s s i o n o f the d i s e a s e , prevented f u r t h e r sampling o f experimental f i s h i n these groups beyond t h e s i x t h sampling day ( T 6 ) . Since t h e r e were some v a r i a t i o n s i n t h e nature and time courses o f blood changes, they w i l l be d i s c u s s e d separately. 1.  Comparison between u n i n f e c t e d c o n t r o l f i s h and k i d n e y d i s e a s e i n f e c t e d experimental f i s h f o r a l l t o x i c a n t  levels  In g e n e r a l , HCT, Hb, RBC, BUN, TP and GLU a l l showed  33  a g e n e r a l d e p r e s s i o n i n kidney d i s e a s e i n f e c t e d f i s h  rela-  t i v e t o u n i n f e c t e d c o n t r o l f i s h over the experimental period. Haematocrit E x p e r i m e n t a l f i s h i n groups I E , 2E,and 3E a l l showed decreased HCT v a l u e s r e l a t i v e t o sham i n j e c t e d  con-  t r o l groups IC, 2C and 3C over the i n c u b a t i o n time o f the disease.  The d i f f e r e n c e s were s i g n i f i c a n t  from the f i r s t  sampling day ( T l ) f o r a l l groups and i n c r e a s e d with time i n groups IC, IE and 2C, 2E ( F i g . V ) . Haemoglobin E x p e r i m e n t a l f i s h i n groups I E , 2E and 3E a l l showed lowered Hb v a l u e s over t h e i n c u b a t i o n time o f the d i s e a s e r e l a t i v e t o sham i n j e c t e d c o n t r o l groups IC, 2C and 3C ( F i g . V I ) . S i g n i f i c a n t d i f f e r e n c e s were observed between IC and IE groups from the f i r s t t o t h e s i x t h sampling day ( T l to T6).  Although these d i f f e r e n c e s were r e l a t i v e l y  con-  s t a n t throughout the sampling days,, they i n c r e a s e d over the same i n t e r v a l s f o r the 2C and 2E groups.  At t h i s t o x i c a n t  l e v e l , the group 2E Hb value was s i g n i f i c a n t l y g r e a t e r than for  group 2C f i s h on the f i r s t  sampling day ( T l ) .  After  t h i s time, t h e 2E Hb v a l u e s d e c l i n e d s i g n i f i c a n t l y below 2C v a l u e s and continued t o f a l l u n t i l the f i n a l sampling day (T6).  On t h e f i r s t  sampling day ( T l ) the Hb value o f group  3E was s i g n i f i c a n t l y lower than the c o n t r o l group 3C v a l u e . Red blood c e l l  count  E x p e r i m e n t a l f i s h i n groups I E , 2E and 3E a l l showed s i g n i f i c a n t l y lower RBC counts compared t o the sham  34 i n j e c t e d c o n t r o l groups IC, 2C and 3C over the i n c u b a t i o n time o f the d i s e a s e ( F i g . V I I ) .  D i f f e r e n c e s between groups  1G and IE as w e l l as 2C and 2E were s i g n i f i c a n t  on the f i r s t  sampling day ( T l ) and s i m i l a r l y continued t o i n c r e a s e u n t i l the s i x t h  sampling day.  significantly different  RBC v a l u e s o f groups 3C and 3E were on the f i r s t  sampling day ( T l ) .  Blood urea n i t r o g e n The BUN v a l u e s f o r group IE were s i g n i f i c a n t l y lower than IC v a l u e s on the f i r s t two sampling days ( T l and T2; F i g . X ) . T6),  F o r the remainder o f t h e sampling p e r i o d ( T 3 -  the BUN v a l u e s were h i g h l y v a r i a b l e f o r the IE group  showed an i n c r e a s i n g t r e n d towards the IC group v a l u e s about which t h e y f l u c t u a t e d markedly sampling day ( T 3 - T 6 ) . cantly different sixth  from t h e t h i r d t o the s i x t h  Two o f these v a r i a t i o n s were  signifi-  from t h e group IC v a l u e s on t h e f i f t h and  sampling days ( T 5 and T6) when the BUN v a l u e s o f the  f i s h i n group IE f e l l below and then rose above the IC v a l u e s respectively.  The BUN v a l u e s o f f i s h i n group 2E, however,  were s i g n i f i c a n t l y lower than those o f group 3C on the f i r s t sampling day ( T l ) . Total protein E x p e r i m e n t a l f i s h i n group IE showed s i g n i f i c a n t l y lower TP v a l u e s r e l a t i v e t o group IC f o r t h e f i r s t two sampl i n g days ( T l and T2; F i g . X I ) . A f t e r t h i s i n i t i a l d e p r e s s i o n TP v a l u e s i n group IE f i s h were h i g h e r than group IC f i s h on the next two sampling days (T3 and T4) but then d e c l i n e d below the IC group v a l u e s i n t h e l a s t two sampling days (T5 and T6). The d i f f e r e n c e s between the two groups were s i g n i f i c a n t •:: ^  35 on the f o u r t h , f i f t h T6).  and s i x t h sampling days (T4, T5 and  TP v a l u e s f o r group 2 E f i s h were s i g n i f i c a n t l y  than group 2 C f i s h f o r a l l sampling days ( T 1 - T 6 ) .  lower  The TP  value o f group 3E f i s h was s i g n i f i c a n t l y lower than t h e group 3C v a l u e on the f i r s t  sampling day ( T I ) .  Glucose E x p e r i m e n t a l f i s h i n group 2 E showed GLU v a l u e s t h a t were s i g n i f i c a n t l y lower than c o n t r o l f i s h group 2 C f o r all  sampling days ( T 1 - T 6 ) .  The r e s u l t s f o r the IE and IC  groups were s i m i l a r except f o r the f i r s t Fig.  XII).  sampling day ( T I ;  The GLU v a l u e f o r f i s h i n group 3E was a l s o  s i g n i f i c a n t l y lower than the group 3C f i s h on t h e f i r s t sampling day ( T I ) . Mean c e l l volume In  c o n t r a s t t o the t r e n d o f depressed v a l u e s o f  HCT, Hb, RBC, BUN, TP and GLU found f o r experimental f i s h d u r i n g t h e p r o g r e s s i o n o f kidney d i s e a s e , MCV v a l u e s i n i n f e c t e d experimental f i s h i n c r e a s e d above t h e r e s p e c t i v e uninfected control f i s h values.  The MCV v a l u e s i n IE group  f i s h v a r i e d c o n s i d e r a b l y and a s i g n i f i c a n t d i f f e r e n c e compared t o the IC group v a l u e s was not observed u n t i l t h e s i x t h sampling day (T'6) when the group IE f i s h had a h i g h e r MCV ( F i g . I X ) . MCV v a l u e s i n group 2 E f i s h were s i g n i f i c a n t l y h i g h e r than group 2 C f i s h v a l u e s on the t h i r d , and s i x t h sampling p e r i o d s (T3, T 5 and T 6 ) .  fifth  NO s i g n i f i c a n t  d i f f e r e n c e was observed between f i s h i n group 3.E and 3 C on the f i r s t  sampling day ( T l ) . Three blood parameters  (WBC, MCHC and MCH) showed  36 variable  responses  Total white  to kidney disease  blood c e l l  bacterial  counts  E x p e r i m e n t a l f i s h i n group IE had l o w e r WBC o n t h e  first  to  (Fig. VIII).  IC group f i s h  values t i l  s i x t h s a m p l i n g day  s l i g h t l y higher fish,  f o r the  t h e WBC f o r  2C v a l u e .  sampling days  three  2E fish  first  significantly (T1-T3)  relative  A f t e r t h i s t i m e , the  (T6) t h e  than  IE  group  IC group v a l u e s  un-  I E group f i s h had a  .05) WBC.  For the  g r o u p 2E  s i g n i f i c a n t l y l o w e r t h a n g r o u p 2C  (T6) i t was  On t h e  i n group  to the  first  groups  s a m p l i n g day  fish  (P g r e a t e r  t h e WBC was a l s o  values  three  continuously increased toward the  by t h e  infection.  s a m p l i n g days  (T1-T3).  increased u n t i l  Similarly,  by t h e  sixth  s i g n i f i c a n t l y higher than the s a m p l i n g day  (Tl) the  group  experimental  3E showed a s i g n i f i c a n t l y l o w e r WBC r e l a t i v e  g r o u p 3C  value.  Mean c o r p u s c u l a r h a e m o g l o b i n c o n c e n t r a t i o n and mean  cellular  haemoglobin Due t o t h e values fish  pattern  v a r i a t i o n i n t h e MCHC a n d MCH  experimental  c o u l d be s e e n  fish  experiment,  i n Appendix V .  IC,  these  No e a s i l y  re-  discernfor  groups. c o n t r o l f i s h groups  for  all  levels  The c o m p a r i s o n f o r t h e u n i n f e c t e d groups  control  i n t h e MCHC and MCH v a l u e s  C o m p a r i s o n among u n i n f e c t e d toxicant  f i s h and u n i n f e c t e d  sampling p e r i o d o f the  have been p r e s e n t e d  any o f t h e 2.  infected  over the  sults able  of  large  control  fish  2C and 3C i n d i c a t e d t h a t  significant  changes  o c c u r r e d i n H C T , BUN a n d GLU v a l u e s  i n response  to  toxicant  '37 exposure. Haematocrit Although HCT i n f i s h o f group 2C d i d s i g n i f i c a n t l y d i f f e r from group IC f i s h f o r a l l the sampling days (T1-T9),  the group 3C v a l u e s were found t o be s i g n i f i c a n t l y  lower than both IC and 2C v a l u e s from the f i r s t sampling day (T1-T5).  t o the f i f t h  On t h e s i x t h sampling day (T6) the  HCT value i n group 3C was s i g n i f i c a n t l y lower than the 2C ^  group value o n l y ( F i g . V ) . Blood urea n i t r o g e n BUN v a l u e s f o r group 2C f i s h were s i g n i f i c a n t l y h i g h e r than group IC and 3C f i s h on t h e f i r s t  f o u r sampling  days (T1-T4;  d i f f e r e n c e was  F i g . X).  However no s i g n i f i c a n t  observed between t h e BUN v a l u e s o f IC and 3C over the same interval. Glucose D e s p i t e the l a r g e v a r i a t i o n s i n GLU v a l u e s i n a l l c o n t r o l groups, t h e r e appeared t o be a s i g n i f i c a n t  depression  i n group 3C r e l a t i v e t o groups IC and 2C on the f i r s t sampling days (T1-T4; (TI)  Fig. XII).  On the f i r s t  four  sampling day  GLU v a l u e s i n f i s h o f group 3C were s i g n i f i c a n t l y  than those o f group 2C.  lower  However, on t h e second, t h i r d and  f o u r t h sampling days (T2, T3 and T4) the group 3C v a l u e s were s i g n i f i c a n t l y lower than those o f both' 2C and IC groups. I n s p e c t i o n o f F i g u r e XI  shows t h a t although the glucose  v a l u e s o f u n i n f e c t e d c o n t r o l f i s h i n group IC v a r i e d t h i s parameter  little,  i n groups 2C and 3C was more v a r i a b l e .  group 2C f i s h however, t h e r e appeared t o be a t r e n d o f  In  3* i n c r e a s i n g GLU  values,  e s p e c i a l l y a f t e r the f i f t h  day  (T5).  3•  Comparison among kidney disease toxicant  infected fish for a l l  levels,  E x p e r i m e n t a l f i s h i n groups I E , 2E and t h a t no c o n s i s t e n t p a t t e r n occurred blood  sampling  3E showed  i n any o f the measured  parameters over a l l the sampling days (T1-T6; F i g s .  V - XII). The Fig.  histograms ( F i g s . XVII, XVIII and  IV) show t h a t t o x i c a n t a d m i n i s t r a t i o n  App.  appears t o have  caused a g r e a t e r d i f f e r e n c e between the c o n t r o l and mental f i s h values level  o f MCV,  BUN  i n the  o f t o x i c a n t t r e a t e d groups, 2C and  the c l e a n water groups IC and period  and MCH  (T1-T6).  V,  2E,  experi  intermediate r e l a t i v e to  IE over the whole sampling  TABLE I I I P h y s i c a l c h a r a c t e r i s t i c s code and m o r t a l i t y f o r the main experiment. 4 days  12 days  16 days  20 days  24 days  2 8 days  32 days  36 days  0 0  0 0  0 0  0 0  0 0  0 0  0 0  0 0  *  *  0 0  0 0  8 days  c  0  TP Hi  1.4 0  1.7 0  2.2 0  2.8 4  3.3 5  c  0 0  0 0  0 0  0 0  0 0  z>  1.8 0  2.3 0  2.9 1  3.1 2  4.8  c  0 0  0 0  0 3  0  IT  2.2 0  cw  INT  HIGH  b  216  *  1  14  0 0  CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate L e v e l NaPCP (0.05 x 96 h L C ^ Q ) 5 Q  )  C = U n i n f e c t e d C o n t r o l Group E = Kidney Disease I n f e c t e d Experimental Group  52 0 '3.  0 0 5.9 48 0 0  *  #  HIGH = High L e v e l NaPCP (0.5 x 96 h L C  5.3 33  *  31  11  0  *  1  0  0 8  X  n = 12 (two f i s h pooled f o r each measurement. )  # -  Key f o r Code i n Appendix I I I  a = Physical  Characteristics  b' = M o r t a l i t y f o r t h a t day ^ = No data due t o m o r t a l i t y  40a  Figures V to XII Graphs o f t h e responses o f measured h a e m a t o l o g i c a l parameters  i n the d i f f e r e n t  groups o f f i s h t o the e x p e r i -  mental treatments, b a c t e r i a l kidney d i s e a s e i n f e c t i o n and NaPCP exposure,  over the sampling p e r i o d .  Each p o i n t  r e p r e s e n t s a mean + 1 . 9 6 standard e r r o r o f the mean (n=12).  Responses i n u n i n f e c t e d c o n t r o l f i s h are d e s i g -  nated by s o l i d l i n e s and kidney d i s e a s e i n f e c t e d by broken l i n e s , 96 h L C ^ Q ) , 96 h L C J Q )  fish  a, b and c r e p r e s e n t c l e a n water ( 0 x ( 0 . 0 5x  i n t e r m e d i a t e l e v e l o f NaPCP exposure  ( 0 . 5 x 96 h  and h i g h l e v e l o f NaPCP exposure  LC^Q) respectively.  T I r e p r e s e n t s the f i r s t  sampling  day, f o u r days'tafter b e g i n n i n g t o x i c a n t exposure. 9 r e p r e s e n t subsequent  sampling days.  Symbols on t o p  o f each sampling day designate s t a t i s t i c a l (P=0.05),  2 to  significance  by S c h e f f e ' s t e s t , between means o f d i f f e r e n t  groups o f f i s h :  * = between c o n t r o l and experimental  f i s h , 1 = i n a, 2 = i n b, 3 = i n c; H = i n c o n t r o l  fish  groups, A = between a and b, B = between b and c, C = between a and c; D = i n kidney d i s e a s e i n f e c t e d Figure V  Haematocrit  Figure VI  Haemoglobin  Figure VII  Red Blood C e l l Count  Figure VIII  T o t a l White Blood C e l l Count  F i g u r e IX  Mean C e l l Volume  Figure X  Blood Urea N i t r o g e n  F i g u r e XI  Total Protein  Figure XII  Glucose  fish.  40  41 12  F i g u r e VI  12  12  12 H  B  12 . BC H  12 H  C  BC  l  12 10 8  * -4 ^  6 4 2  O O  bfl  PQ  o  §  c 1CL  + Tl  4  SAMPLING PERIOD - 4 day i n t e r v a l s  8  9  42 123  gure V I I  •  12  12  -  12 H  12 12  C  a 18CL  140  100 O  o o o  <  60 b 180_  W H H  o  O  o  140  100  .-3 .-1 O  o o  60  PQ  Q  c 180_  P3  140  100L I  1  1  Tl  2  3  I  I  L  J  7  L  8  $  SAMPLING PERIOD - 4 day i n t e r v a l  43/ *123 *12 *12  Figure VIII  'AC a  6.  c  7  o o \  O  E-i to O  o  1-3 1-1  w  o  o o o  1-1  m  w  E-" H Hi  o  SAMPLING PERIOD - k- day i n t e r v a l s  44  *2  gure IX  H  AC  *12 H  B  a 360^  OA  I  t-1 O >  FI  I-1  w  o  c 300_ 240  180L  L  Tl  I  2  1  3 4  »  1  5  I  6  I _ l I  SAMPLING PERIOD - 4 day i n t e r v a l s  7 8 9  45  *  123 AB  *  12 AB  *  2 AB  *  *  *  2 12-12 "ABC "AC  n  BC  SAMPLING PERIOD - 4 day i n t e r v a l s  46 F i g u r e XI  *123 *12 ABC BC BC  H  D  Tl  H  H  *2 BC A D  2  H  *12 *12 AB BC A  *12  H  D  3  4  I  5  I  6  SAMPLING PERIOD - 4 day i n t e r v a l s  1  7  L_  8  47 *23 *12 *12 *12 *12 *12 AB BC BC BC AB  Figure XII  H  a  H  H  H  'ABC  75 65 55 45 35 25l  b 110 105 S  o o  S  95 85 75 65  W  CO  O O !=> O  55 45 35 25  SAMPLING PERIOD - 4 day i n t e r v a l s 1  48a  F i g u r e s X I I I t o XX  Histograms showing grand means and means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and e x p e r i mental f i s h f o r the d i f f e r e n t groups o f f i s h . Sample s i z e s used t o d e r i v e these values a r e i n d i c a t e d on t o p o f b a r s . Figure  XIII  Haematocrit  F i g u r e XIV  Haemoglobin  F i g u r e XV  Red  F i g u r e XVI  T o t a l White Blood C e l l Count  F i g u r e XVII  Mean C e l l Volume  F i g u r e XVIII  Blood Urea  F i g u r e XIX  Total Protein  F i g u r e XX  Glucose  Blood  C e l l Count  Nitrogen  HAEMATOCRIT g r a n d means ( $ )  •  o  i  "I  I  ^  '  i 0 0  i  i  '  £  ;  * means o f a b s o l u t e  differences  between c o n t r o l and experimental  fish.  HAEMOGLOBIN g r a n d means (g/100 m l )  * means o f a b s o l u t e  differences  between c o n t r o l a n d experimental  fish.  *  means o f between  absolute control  experimental  differences  and  fish.  WHITE BLOOD. CELL COUNT grand means  o 1  (cells/mnr/500)  ho  •1  1  1  CD  Co  >0  co CD Co  o * means o f a b s o l u t e d i f f e r e n c e s between c o n t r o l and experimental  fish.  *  means o f between  absolute control  experimental  differences  and.  fish.  BLOOD UREA NITROGEN grand means (mg/100 ml)  * means o f absolute d i f f e r e n c e s between c o n t r o l and experimental  fish.  TOTAL PROTEIN grand means (gm/lOO ml)  rO CD h-f  *  o CO  col *  N5 * means o f a b s p l u t e d i f f e r e n c e s between c o n t r o l and experimental  fish.  *  means o f ences  absolute  between  experimental  differ-  control fish.  and  56  Table IV  Comparison o f v a r i o u s h a e m a t o l o g i c a l v a l u e s measured i n t h i s study w i t h those o f Thomas e t .  This  study  (grand means o f uninfected  control  a l . (1969). Thomas e t . a l .  (1969)(means; male - female)  f i s h i n c l e a n water) HCT  ($)  Hb  (g/100 ml)  RBC MCV MCHC MCH TP GLU  34.64  (cells/mm /lO,000) 3  (u ) 3  (pg) ($) (g/100 ml) (mg/100 ml)  3 , 2 6  150.93  237.08  55.92 23.96 3.78  56.69  41.0 - U.6 7.9 8.2 157.40 - 156.10 261 - 268 "50.1 - 52.8 19.2  -  19.7  4.02 - 4.12 100.8 - 99.8  57a  SECTION V DISCUSSION  57  The  r e s u l t s o f t h e main experiment i n d i c a t e t h a t  the experimental f a c t o r s o f NaPCP exposure and k i d n e y d i sease i n f e c t i o n imposed s t r e s s f u l c o n d i t i o n s on the f i s h and t h a t these f a c t o r s acted  synergistically.  The o c c u r -  rence o f m o r t a l i t i e s , as shown i n Table I I I , supports t h i s contention. and  The f a c t t h a t the m o r t a l i t i e s f o r groups IE  2E s t a r t e d t o occur a t approximately the same time  suggests t h a t the intermediate not  enhance the p r o g r e s s i o n  t o x i c a n t l e v e l exposure d i d  o f the kidney d i s e a s e t o a  p o i n t where a d i f f e r e n c e i n commencement o r r a t e o f mort a l i t y occurred. occurred  However, the c a t a s t r o p h i c m o r t a l i t y t h a t  i n group 3E f i s h i n d i c a t e s t h a t the high l e v e l o f  NaPCP imposed a s t r e s s on t h e f i s h i n t h i s group, i n a d d i t i o n t o the i n f e c t i o n , t h a t was beyond t h e i r a b i l i t i e s , thus c u l m i n a t i n g  adaptive  i n death.  Earp et_. a l . (1955) r e p o r t e d  several  catastrophic  m o r t a l i t i e s i n a d u l t chinook salmon due t o t h i s In one o f these i n s t a n c e s , the u n c o n t r o l l e d break occurred  sudden o u t -  a f t e r these f i s h were t r a n s p o r t e d  pered i n t o sea water.  disease.  and tem-  The t r a n s p o r t a t i o n and h a n d l i n g  have caused a s t r e s s t h a t played  may  a role s i m i l a r to that of  the high l e v e l o f t o x i c a n t exposure i n t h i s experiment i n c o n t r i b u t i n g t o the c a t a s t r o p h i c m o r t a l i t y . m o r t a l i t i e s occurred  and i n c r e a s e d  The f a c t  that  i n f i s h i n groups 2C and  3C indica'tes t h a t the two c o n c e n t r a t i o n s  o f NaPCP i n t h i s  study became s t r e s s f u l t o the f i s h w i t h continuous exposure over t i m e .  The e a r l i e r commencement and g r e a t e r magnitude  5* o f t h e m o r t a l i t i e s i n g r o u p 3C f i s h fish  i n d i c a t e that the s t r e s s  t i o n a l to the  compared t o g r o u p 2C  i m p o s e d b y NaPCP was p r o p o r -  concentration.  The p h y s i c a l c h a r a c t e r i s t i c s o f t h e shown i n T a b l e I I I ,  indicate that  disease  infection,  p r o g r e s s i o n was  s l i g h t l y more a d v a n c e d i n t o x i c a n t  exposed  fish  i n clean water.  compared t o e x p e r i m e n t a l f i s h  experimental This  suggests that the progression of kidney disease  was  h a n c e d w i t h NaPCP e x p o s u r e a n d t h a t t h i s e f f e c t  appears  have been p r o p o r t i o n a l t o t h e stress  c o n c e n t r a t i o n o f NaPCP.  of the  infected  experimental f i s h  and 3E t o t h e d e b i l i t a t i v e e f f e c t s enhancing the progression o f the Although the have IE,  to The  the  i n groups  of the pathogen,  2E  thus  disease.  experimental treatments  imposed v a r y i n g degrees o f s t r e s s  on f i s h  appear in  to  groups  2C, 2E, 3C and 3E, t h e a m b i e n t c o n d i t i o n s o f t h e  ment w e r e fish  en-  o f NaPCP e x p o s u r e was t h o u g h t t o h a v e r e d u c e d  resistance  experi-  c o n s i d e r e d not t o have s i g n i f i c a n t l y s t r e s s e d  i n any o f t h e g r o u p s .  that there  T h i s was d e t e r m i n e d o n t h e  i s a g e n e r a l agreement  parameter values  between t h e  (1969;  Table l y )  of the  same s p e c i e s  Slight  d i s c r e p a n c i e s between t h e v a l u e s  ambient c o n d i t i o n s as w e l l as d i f f e r e n c e s techniques.  of  and  o f t h e two  were a t t r i b u t e d t o t h e p o s s i b l e d i f f e r e n c e  Thomas age. studies  i n measurement  t e s t t a n k s and t h e a c c l i m a t i z a t i o n p r o c e d u r e s were that  basis  in uncontrolled  The r e l a t i v e l y h i g h f l o w - t h r o u g h r a t e s  t o have a m e l i o r a t e d f a c t o r s  the  haematological  o b t a i n e d i n t h i s s t u d y and t h a t for fish  as  o t h e r w i s e may h a v e  i n the considered imposed  59' a d d i t i o n a l s t r e s s f u l c o n d i t i o n s f o r the f i s h . As i n d i c a t e d i n Table I I I , the GLU v a l u e s o b t a i n e d i n t h i s study were lower than t h e value o b t a i n e d by Thomas (1969) f o r t h i s s p e c i e s .  T h i s d i f f e r e n c e was a l s o  attri-  buted t o some p e r s i s t e n t d i f f e r e n c e i n ambient c o n d i t i o n s i n t h e two s t u d i e s .  As w i l l be d i s c u s s e d i n t h i s  GLU l e v e l s have been found t o be v e r y s e n s i t i v e of  section,  indicators  s t r e s s f u l c o n d i t i o n s and t h a t hyperglycemia i s u s u a l l y  a metabolic response t o s t r e s s i n f i s h .  BUN and WBC count  v a l u e s have not been p r e v i o u s l y r e p o r t e d i n t h e l i t e r a t u r e f o r t h i s s p e c i e s a t t h i s age. E i g h t o f the t e n h a e m a t o l o g i c a l parameters were measured i n t h e main experiment  that  showed c o n s i s t e n t p a t -  t e r n s over the sampling p e r i o d i n response t o the e x p e r i mental t r e a t m e n t s .  These s u b l e t h a l blood changes w i l l be  d i s c u s s e d a c c o r d i n g t o the nature o f t h e i r responses. Haemodilution was thought t o be t h e primary  cause  f o r t h e g e n e r a l l y lower HCT, Hb, RBC, BUN, TP and GLU v a l u e s f o r k i d n e y d i s e a s e i n f e c t e d experimental f i s h compared t o sham i n j e c t e d c o n t r o l f i s h over t h e sampling p e r i o d VI, V I I , X, XI and X I I ) .  Wood and Yasutake  ( F i g s . V,  (1955) s t a t e d  t h a t they thought the t i s s u e o f the a n t e r i o r head kidney, t h e major haematopoietic t i s s u e i n f i s h , was q u i t e l i k e l y t h e f i r s t t i s s u e a f f e c t e d i n kidney d i s e a s e i n f e c t i o n . t o p o i e t i c t i s s u e was always  i n an advanced  The haema-  stage o f d e s t r u c -  t i o n even i n t h e e a r l i e s t  stages o f t h e d i s e a s e .  served e x t e n s i v e f i b r o t i c  l e s i o n s enveloping r e n a l t i s s u e  i n t h i s part o f the kidney.  They ob-  60 Furthermore,  as t h e d i s e a s e progressed, they observed  d e s t r u c t i o n o f t h e p o s t e r i o r p o r t i o n s o f the kidney which took the form o f a more g e n e r a l t i s s u e r e a c t i o n .  The  p o s t e r i o r kidney i s p r i m a r i l y i n v o l v e d i n r e g u l a t i n g body f l u i d s and e l e c t r o l y t e s as w e l l as b e i n g an e x c r e t o r y organ  (Hickman and Trump, 1 9 6 9 ) .  Wood and Yasutake  a l s o observed severe d e s t r u c t i o n o f s p l e n i c t i s s u e ,  (1955) another  haematopoietic t i s s u e i n f i s h , as a consequence o f kidney disease  infection. The  symptoms o f kidney d i s e a s e observed  t i a l Experiment  A and t h e main experiment  in Ini-  i n t h i s study are  i n agreement w i t h t h e o b s e r v a t i o n s o f Wood and Yasutake (1955).  The p h y s i c a l c h a r a c t e r i s t i c s , as presented i n Table  I I I , o f these experimental f i s h on the f i r s t  sampling day  ( T l ) i n d i c a t e s t h a t some degree o f breakdown i n body f l u i d r e g u l a t i o n was present at t h i s time.  With the degeneration  o f e x c r e t o r y t i s s u e , f i s h i n freshwater would tend t o imbibe more water from t h e i r hypotonic environment  than they c o u l d  e x c r e t e , thus r e s u l t i n g i n a h a e m o d i l u t i o n . The  i n h i b i t i o n o f e r y t h r o p o i e s i s by t h e d e s t r u c t i o n  o f haematopoietic t i s s u e would c o n t r i b u t e t o t h i s  dilution  e f f e c t w i t h regards t o HCT, Hb and RBC count v a l u e s i n i n f e c t e d experimental f i s h r e l a t i v e t o u n i n f e c t e d c o n t r o l fish.  A d d i t i o n a l f a c t o r s such as leakage through open l e s i o n s  and p o s s i b l e reduced l i v e r f u n c t i o n were c o n s i d e r e d t o have c o n t r i b u t e d t o the s i g n i f i c a n t l y lower TP v a l u e s i n kidney d i s e a s e i n f e c t e d experimental f i s h compared t o u n i n f e c t e d c o n t r o l f i s h over time.  Hunn (1964) observed depressed TP  61  values  i n kidney d i s e a s e d  trol fish.  He  f i s h r e l a t i v e to uninfected  a l s o a t t r i b u t e d t h i s e f f e c t to the  mentioned h e r e .  Reduced TP v a l u e s  o t h e r pathogens have been r e p o r t e d and Maclean, 1 9 7 6 ; Mulcahy, 1 9 6 7 ,  con-  factors  i n f i s h stressed  by  by s e v e r a l workers 1971;  (Shieh  Yamashita, 1 9 6 7 ;  Einszporen-Orecka, 1 9 7 0 ) . There were two  a d d i t i o n a l f a c t o r s t h a t were con-  s i d e r e d to have c o n t r i b u t e d to the GLU  values  s i g n i f i c a n t l y depressed  i n i n f e c t e d experimental f i s h compared t o  fected control f i s h .  These were the p o s s i b l e d e p l e t i o n  glycogen s t o r e s as a r e s u l t o f the demand the  fact that cessation of feeding occurred  values  i n kidney d i s e a s e  i n c r e a s e was cells  sampling p e r i o d considered  imbibing  infected experi-  ( F i g . IX).  This  significant  increase  c a l c u l a t e d MCV  values,  f o r the  f i s h r e l a t i v e t o c o n t r o l f i s h , would r e s u l t . s w e l l i n g i s one occur.  way  was  to the haemo-  I f the r a t e o f decrease i n HCT  l e s s than the r a t e o f decrease i n RBC  i n the  blood  water from the surrounding plasma t h a t  becoming i n c r e a s i n g l y hypotonic t o the c e l l s due  values was  control  t o be the r e s u l t o f the red  d i l u t i o n already discussed.  the  day.  mental f i s h were g e n e r a l l y h i g h e r than u n i n f e c t e d f i s h over the  and  i n f i s h i n groups  2E from about the second sampling MCV  of  multiplying  pathogens imposed on t h e i r h o s t s ' energy resources  IE and  unin-  i n which t h i s d i f f e r e n t i a l  counts, an  experimental Erythrocytic decrease  can  Furthermore, i n h i b i t i o n o f e r y t h r o p o i e s i s by haema-  t o p o i e t i c t i s s u e damage would tend to cause l a r g e r , more mature e r y t h r o c y t e s  to predominate i n the  peripheral  '  62  blood p i c t u r e . to  T h i s l a t t e r f a c t o r may  the i n c r e a s e d MCV  values i n kidney  a l s o have c o n t r i b u t e d disease i n f e c t e d f i s h  compared t o u n i n f e c t e d c o n t r o l f i s h . The  v a r i a b l e response seen i n WBC  counts to  kidney  d i s e a s e i n f e c t i o n over the sampling p e r i o d ( F i g . V I I I ) a t t r i b u t e d t o a p o s s i b l e stress-mediated w i t h haemodilution,  i n the i n i t i a l  leucopenia  was  coupled  sampling days, f o l l o w e d  by  a p o s s i b l e i n c r e a s e i n c i r c u l a t i n g n e u t r o p h i l e s i n response to  the  was  i n c r e a s i n g t i s s u e damage caused by the pathogen.  thought t h a t i n the i n i t i a l  pathogen o r one  sampling days (T1-T3),  It the  o r more o f the p h y s i o l o g i c a l e f f e c t s o f the  i n f e c t i o n e l i c i t e d a n o n - s p e c i f i c s t r e s s , response i n e x p e r i mental  fish. The  to  phenomenon o f WBC  count d e p r e s s i o n  i n response  s t r e s s f u l s i t u a t i o n s and t o a d r e n o c o r t i c o t r o p h i c hormone  (ACTH) o r c o r t i c o s t e r o i d a d m i n i s t r a t i o n i n o t h e r t e l e o s t s have been r e p o r t e d by s e v e r a l workers (Weinreb, 1958; 1973 (1973  a, b, 1975  McLeay,  a, b; Benet and N e v i l l e , 1975). McLeay  a ) , u s i n g the technique  of d i f f e r e n t i a l c e l l  determined the primary cause o f l e u c o p e n i a  counting,  i n coho salmon  s t r e s s e d with a 12 h exposure t o one h a l f the 96 h L C ^  of  Q  k r a f t pulp m i l l e f f l u e n t to a r e d u c t i o n i n the number o f c i r c u l a t i n g s m a l l lymphocytes. penic response was  due  He  suggested t h i s lympho-  to a s t r e s s - m e d i a t e d  c o r t i c o s t e r i o d s e c r e t i o n by the i n t e r r e n a l Both s t r e s s and  increase i n tissue.  c o r t i c o s t e r i o d a d m i n i s t r a t i o n have  been shown t o cause lymphopenia i n salmonids (Weinreb, McLeay, 1970,  1973  a, b) and  i n other vertebrates  1958.  (Dougherty,  63 I960; Benett and H a r b o t t l e , 1968; where the lymphocytes to  Benett e t . a l . , 1972)  have been shown t o be  l y s i s by c o r t i c o s t e r o i d s .  a f t e r the t h i r d sampling day  susceptible  The ascending WBC  counts  (T3) i n i n f e c t e d experimental  f i s h were thought t o be due t o an i n c r e a s e i n c i r c u l a t i n g n e u t r o p h i l e s i n response t o the i n c r e a s i n g t i s s u e damage caused by the p r o l i f e r a t i o n o f the k i d n e y d i s e a s e b a c t e r i a throughout the f i s h ' s body w i t h t i m e . McLeay (1973 a) s t a t e d t h a t  "In both mammals and  t e l e o s t f i s h , the number o f c i r c u l a t i n g n e u t r o p h i l e s are a p p a r e n t l y u n a f f e c t e d by c o r t i c o s t e r o i d a d m i n i s t r a t i o n a l though  e l e v a t e d by both s t r e s s and ACTH i n j e c t i o n s  and White, 1943,  (Dougherty  1944; Weinreb, 195#); the n e u t r o p h i l e , t h e r e -  f o r e i s not thought t o be under d i r e c t a d r e n o c o r t i c a l trol."  con-  I t i s known i n mammalian p h y s i o l o g y t h a t a marked  i n c r e a s e i n c i r c u l a t i n g n e u t r o p h i l e s occurs i n response t o t i s s u e damage.  In mammals, a g l o b u l i n substance known as  l e u c o c y t o s i s promoting  f a c t o r i s produced and r e l e a s e d by  most damaged o r inflamed t i s s u e s and causes a r e l e a s e o f " r e s e r v e d " n e u t r o p h i l e s from the bone marrow which may  har-  bour up t o 30 o r 40 times the number o f c i r c u l a t i n g n e u t r o p h i l e s as w e l l as an i n c r e a s e i n the p r o d u c t i o n o f t h i s type of  repair  cell.  Although the responses o f the blood  parameters  observed i n t h i s study are i n agreement with those r e p o r t e d by o t h e r workers, t h i s study.  s e v e r a l c o n t r a r y o b s e r v a t i o n s were made i n  For example, depressed BUN  v a l u e s f o r kidney  64'  disease are  infected fish  compared t o u n i n f e c t e d c o n t r o l f i s h  contrary to the observations  elevated  of several reports  BUN v a l u e s h a v e b e e n o b s e r v e d i n f i s h  pathological  characteristics  Yamashita,  1976;  -  Field  1967;  known h i s t o p a t h o l o g i c a l e f f e c t s as d i s c u s s e d a b o v e ,  (Shieh,  However t h e  1 9 4 4 ) .  of this specific  pathogen,  support t h e explanation presented  as a p o s s i b l e mechanism o f t h e o b s e r v e d r e s u l t . v a t e d BUN l e v e l s  where  exhibiting  due t o o t h e r d i s e a s e s et. a l . ,  i n g r o u p 2C f i s h  here  The e l e -  r e l a t i v e t o g r o u p 1G a n d  3C f i s h may h a v e b e e n a s p e c i f i c  p h y s i o l o g i c a l response t o  t h e NaPCP o r t o a g e n e r a l  response.  found t h a t  stress  Wedemeyer  a uremia developed i n rainbow t r o u t  excretion.  (1970)  exposed t o  u n n e u t r a l i z e d MS 222. I t was u n c l e a r i n ' h i s s t u d y t h i s t r e n d was d u e t o a c c e l e r a t e d  (Fig.X)  whether  production o r retarded  He c o n s i d e r e d d e c r e a s e d  gill  t r a n s p o r t due t o  t h e MS 222 ( s u l f o n i c a c i d ) u n l i k e l y s i n c e p l a s m a c h l o r i d e l e v e l s were  unaffected.  W i t h t h e v e r y l i m i t e d knowledge o f t h e e f f e c t s o f t h e t o x i c a n t , NaPCP, o n t h e p h y s i o l o g y o f f i s h , icult  to speculate  for the elevated  o n t h e p o s s i b l e mechanisms  BUN l e v e l s .  The f a c t  that  i t is diff-  responsible  an e l e v a t i o n o f  t h i s parameter occurred i n i n f e c t e d experimental f i s h i n g r o u p I E s u g g e s t s t h a t t h i s e l e v a t i o n may n o t h a v e b e e n d u e to the singular effect that the levels deviate the  of the toxicant.  for fish  from t h e f i s h  fourth, fifth  The p e c u l i a r  fact  i n g r o u p 3C d i d n o t s i g n i f i c a n t l y  i n group I C , w i t h t h e e x c e p t i o n o f  and seventh  s a m p l i n g days  w h i c h p r o b a b l y were a consequence  (T4, T5 a n d T7)  o f t h e severe  fluctuations,  65 i s not e x p l a i n e d . The elicited  intermediate l e v e l o f toxicant  a s l i g h t hyperglycemic response i n group 2C f i s h  r e l a t i v e t o group IC f i s h a f t e r t h e f i f t h (Fig.  exposure  XII).  sampling day (T5)  T h i s i s i n agreement w i t h s e v e r a l workers who  have r e p o r t e d hyperglycemia i n s e v e r a l s p e c i e s o f f i s h i n response t o v a r i o u s s t r e s s f u l f a c t o r s such as t r a n s p o r t , muscular  e x e r c i s e , h a n d l i n g , capture and a n a e s t h e s i a ( S c o t t ,  1921; Simpson, 1926; Menten, ,1927; Chavin and Young, 1970; Black, 1957 a, b, c; Black e t . a l . , I960; Wedemeyer, 1972; Wardle, 1972 and Houston e t . a l . , 1971 a, b; S o i v i o and O i k a r i , 1976). Hyperglycemia  has a l s o been r e p o r t e d as response i n  f i s h t o t o x i c a n t exposure 1975).  (Hunn, 1972; McLeay, 1973, 1974,  McLeay (1975) i n t e r p r e t e d t h i s hyperglycemia as being  the r e s u l t o f g l u c o c o r t i c o i d and catecholamine s e c r e t i o n the i n t e r r e n a l t i s s u e i n response t o s t r e s s f u l s t i m u l i . s l i g h t hyperglycemic response i n group 2C f i s h i s a l s o  from The  inter-  p r e t e d as being due t o a s t r e s s f u l c o n d i t i o n d e v e l o p i n g with time and t h e p o s s i b l e i n c r e a s e i n t h e s e c r e t i o n o f these " s t r e s s hormones" from t h e i n t e r r e n a l  tissue.  C o n t r a r y t o these o b s e r v a t i o n s , t h e GLU value i n group 3C f i s h was s i g n i f i c a n t l y lower than t h e GLU v a l u e i n group IC f i s h on t h e f i r s t  sampling day ( T l ) .  This observa-  t i o n was i n t e r p r e t e d as b e i n g due t o t h e o x i d a t i v e u n c o u p l i n g a c t i o n o f t h e NaPCP ( A l d e r d i c e , 1963) i n c r e a s i n g t h e metabolic r a t e o f t h e f i s h and t h e r e b y i n c r e a s i n g t h e uptake o f GLU from the p e r i p h e r a l c i r c u l a t o r y  system.  66 As seen i n t h e m o r t a l i t i e s and p h y s i c a l c h a r a c teristics served  (Table  I I I ) , a s y n e r g i s t i c e f f e c t was a l s o ob-  between t h e experimental f a c t o r s o f k i d n e y  disease  i n f e c t i o n and NaPCP exposure i n t h e s u b l e t h a l responses o f s e v e r a l o f t h e h a e m a t o l o g i c a l parameters over t i m e . The  accentuated d i f f e r e n c e i n MCV and BUN  values  between groups 2C and 2E f i s h compared to groups IC and IE ( F i g s . XVII and XVIII) f i s h i n d i c a t e t h a t the t o x i c a n t s t r a t i o n enhanced the p r o g r e s s i o n kidney disease being  infection.  admini-  and subsequent e f f e c t s o f  T h i s was i n t e r p r e t e d as p o s s i b l y  due t o the s t r e s s o f NaPCP exposure r e d u c i n g  the r e s i s -  tance o f the i n f e c t e d experimental f i s h t o the d e b i l i t a t i v e e f f e c t s o f t h e pathogen. teristics  On the b a s i s o f t h e p h y s i c a l c h a r a c -  presented i n Table I I I , i t was thought t h a t  renal  damage due t o the i n f e c t i o n was s l i g h t l y more advanced i n group 2E f i s h than group IE f i s h .  I f t h i s were the case,  the r e s u l t i n g h a e m o d i l u t i o n would be g r e a t e r i n group 2E f i s h compared t o group IE f i s h r e s u l t i n g i n a g r e a t e r i n the blood  o f t h e f i s h i n t h e former group.  h y p o t o n i c i t y could The  cause t h e g r e a t e r  hypotonicity  This  increased  MCV.  f a c t t h a t the ascending t r e n d  i n WBC counts o f  group 2E f i s h s t a r t e d e a r l i e r i n time and continued  at a  g r e a t e r r a t e compared t o group IE f i s h a l s o suggests t h a t toxicant administration process. has  been  enhanced t h e e f f e c t s o f t h e d i s e a s e  The p o s s i b l e mechanism by which t h i s could discussed.  occur  67a  SECTION  GENERAL  CONCLUSIONS  AND  VI  RECOMMENDATIONS  -67  The  f o l l o w i n g g e n e r a l c o n c l u s i o n s were made based  on the r e s u l t s o f t h i s The two determined to  study.  noxious agents employed i n t h i s study were  t o be s t r e s s f u l based on m o r t a l i t i e s i n response  both t r e a t m e n t s .  In response  t o the s t r e s s o f i n f e c t i o n ,  c o n s i s t e n t and s i g n i f i c a n t t r e n d s o c c u r r e d i n e i g h t (HCT, RBC,  BUN,  TP,  GLU,  MCV  and WBC)  Hb,  out o f the t e n blood p a r a -  meters measured i n experimental f i s h r e l a t i v e to.sham i n jected control f i s h . t h r e e (HCT,  BUN  In response t o the t o x i c a n t  and GLU)  out o f the t e n blood parameters o f  u n i n f e c t e d c o n t r o l f i s h showed s i g n i f i c a n t t r e n d s . f o r e , i t was  exposure,  There-  concluded t h a t c e r t a i n blood parameters o f  j u v e n i l e chinook  salmon y e a r l i n g s drh e x h i b i t a response  to  the s t r e s s imposed by the b a c t e r i a l pathogen and t h e e n v i r o n mental t o x i c a n t used The  in this  study.  f a c t t h a t most o f the d i f f e r e n c e s between the  blood parameter values.~of experimental and s i g n i f i c a n t on the f i r s t  c o n t r o l f i s h were  sampling day when no o v e r t symptoms  o f the d i s e a s e o r the t o x i c a n t were evident suggests t h a t these parameters have a p o t e n t i a l i n being used as  early  indicators of stressful states in this species. S e v e r a l o f the blood parameters showed accentuated d i f f e r e n c e s between experimental and t o x i c a n t a d m i n i s t r a t i o n (BUN  and MCV)  control values with and s i g n i f i c a n t  erences among u n i n f e c t e d c o n t r o l v a l u e s (HCT,  BUN  w i t h the t h r e e l e v e l s o f t o x i c a n t a d m i n i s t r a t i o n .  and The  s i c a l c h a r a c t e r i s t i c s a l s o i n d i c a t e t h a t the s t a t e o f  diffGLU) phy-  68 p h y s i c a l d e b i l i t a t i o n caused by the pathogen was more a d vanced  i n the t o x i c a n t a d m i n i s t e r e d experimental f i s h com-  pared t o t h e experimental f i s h i n c l e a n water.  Further-  more, although t h e m o r t a l i t i e s i n the experimental f i s h i n c l e a n water and t h e i n t e r m e d i a t e t o x i c a n t l e v e l s t a r t e d t o occur a t approximately t h e same time, the c a t a s t r o p h i c mort a l i t y i n t h e experimental f i s h at the h i g h t o x i c a n t  level  was a t t r i b u t e d t o the s y n e r g i s t i c e f f e c t s o f t h e kidney d i s e a s e b a c t e r i a l i n f e c t i o n and NaPCP exposure.  These  con-  s i d e r a t i o n s l e d t o the main c o n c l u s i o n t h a t the e n v i r o n mental  s t r e s s reduced the r e s i s t a n c e o f the f i s h t o the  e f f e c t s o f t h e pathogen. S e v e r a l recommendations can be made w i t h regard t o e s t i m a t i n g the p h y s i o l o g i c a l c o n d i t i o n o f f i s h s t o c k s (experimental, cultured o r w i l d ) . were used  The f o l l o w i n g  i n s e l e c t i n g the best parameters  criteria  f o r r o u t i n e moni'  t o r i n g o f the health status i n t h i s species: - s e n s i t i v i t y t o s t r e s s f u l environmental c o n d i t i o n s - low v a r i a b i l i t y - measurement techniques which r e q u i r e r e l a t i v e l y i n expensive m a t e r i a l s t h a t a r e r e a d i l y a v a i l a b l e commercially - t e c h n i c a l l y simple t o measure Haematocrit, r e d blood c e l l count, mean c e l l volume and t o t a l / d i f f e r e n t i a l white blood c e l l count were c o n s i d e r e d t o be the best parameters t i n e measurements o f these parameters  determinations  t o monitor.  Rou-  once a week d u r i n g  the summer and w i n t e r months and t w i c e t o t h r e e times a  69  week d u r i n g the f a l l  and s p r i n g months, when environmental  c o n d i t i o n s are more v a r i a b l e , would enable the e a r l y tion of stressful  s t a t e s , t h a t may predispose  detec-  the f i s h  to  d i s e a s e , and the e a r l y development o f remedial programmes t o combat -or remove the The r e s u l t s  stress..  o f the present  study i n d i c a t e  that  the p h y s i o l o g i c a l c o n d i t i o n o f f i s h i s an important t o c o n s i d e r i n t o x i c i t y s t u d i e s such as For the purposes between d i f f e r e n t  i n c o n s i d e r i n g the v a r i o u s f a c t o r s v a r i a b i l i t y i n the r e s u l t s . fish loading density, handling,  bioassays.  o f making meaningful  bioassay r e s u l t s ,  comparisons-  care must be that  pH and a e r a t i o n  Evidence i s presented  i n t h i s study t h a t  are:  photoperiod,  exposure t o v i s u a l s t r e s s , temperature,  t i o n , water hardness,  exercised  c o n t r i b u t e t o the  Some o f these f a c t o r s  f i s h s i z e and age,  factor  fish  acclima-  (Davis and Hoos,  1975).  indicates that  the  h e a l t h s t a t u s o f the t e s t f i s h may a l s o be an important factor  i n t h i s regard.  smears o f t e s t  I n s p e c t i n g gram s t a i n s o f k i d n e y  f i s h as a r o u t i n e procedure i n b i o a s s a y s may  a i d i n the d e t e c t i o n o f abnormal s t o c k s o f f i s h and may a i d the meaningful-comparisons o f d i f f e r e n t future.  test results  i n the  70a  SECTION v n BIBLIOGRAPHY  70" A k i t a k e , H. and K. Kobayashi.  1975.  S t u d i e s on t h e meta-  b o l i s m o f chlorophenols i n f i s h - I I I .  Isola-  t i o n and i d e n t i f i c a t i o n o f a conjugated PGP exc r e t e d by g o l d f i s h .  B u l l . Jap. Soc. S c i . F i s h . ,  41(3): 321-327. A l d e r d i c e , D.F. 1963.  Some e f f e c t s o f simultaneous  a t i o n i n s a l i n i t y , temperature  vari-  and d i s s o l v e d oxy-  gen on t h e r e s i s t a n c e o f j u v e n i l e coho salmon (Oncorhynchus k i s u t c h ) t o a t o x i c Ph.d.  substance.  t h e s i s , U n i v e r s i t y o f Toronto, 177 p.  American P u b l i c H e a l t h Assoc. 1971. the Examination  "Standard Methods f o r  o f Water and Wastewater", 13th E d .  J o i n t P u b l i c a t i o n o f American P u b l i c Health  Assoc.,  American Waterworks Assoc. and the Water P o l l u t i o n C o n t r o l F e d e r a t i o n , Washington, D.C., 874 p. Bang, F.B. 1970.  Disease mechanisms i n crustaceans and Pages 3#3-404 in_ S t a n i s l a s F.  marine a r t h r o p o d s .  Snieszko, ed. A symposium on d i s e a s e s o f f i s h e s and s h e l l f i s h e s . B e l l , G.R. 196l.  Am. F i s h . Soc. Spec. P u b l . No. 5.  Two epidemics o f apparent  kidney d i s e a s e  i n c u l t u r e d Pink salmon (Oncorhynchus J . F i s h . Res. Board Bendele,  gorbuscha).  Can. 18(4): 559-562.  R.A. and G.W. K l o n t z . 1975.  Histopathology o f  t e l e o s t kidney d i s e a s e s .  Pages 365-383 i n W.E.  R u b e l i n and G. M i g a k i eds. The Pathology o f F i s h e s . The U n i v e r s i t y o f Wisconsin P r e s s , Madison. Benett, M.F. and H a r b o t t l e . 1968. The e f f e c t o f h y d r o c o r t i s o n e on t h e blood o f t a d p o l e s and f r o g s , Rana c a s t e s b e i a n a . B i o l . B u l l . 135: 92-95. Benett, M.F., C.A. Gaudio, A.O. Johnson, and J.H. S p i s s o . 1972. Changes i n t h e blood o f newts, Notophthalmus descens,  viri-  f o l l o w i n g t h e a d m i n i s t r a t i o n o f hydro-  cortisone.  J . Comp. P h y s i o l . #0: 233-237.  Benett, M.F. and C.G. N e v i l l e . 1975.  E f f e c t s o f c o l d shock  on t h e d i s t r i b u t i o n o f l e u c o c y t e s i n g o l d f i s h , Carassius auratus.  J . Comp. P h y s i o l . 98: 213-216.  71 B l a c k , E.C. 1957 a.  Alterations  i n t h e blood l e v e l o f l a c t i c  a c i d i n c e r t a i n salmonoid  fishes following  mus-  c u l a r a c t i v i t y . I . Kamloops t r o u t , Salmo g a i r d n e r i . J.  F i s h . Res. Board Can. 14: 117-134. 1957  b.  Alterations  i n t h e blood l e v e l o f l a c t i c  a c i d i n c e r t a i n salmonoid  fishes following  c u l a r a c t i v i t y . I I . Lake t r o u t , S a l v e l i n u s cush. 1957  J . F i s h . Res. Board c.  Alterations  musnamay-  Can. 14: 645-649.  i n t h e blood l e v e l o f l a c t i c  - a c i d i n c e r t a i n salmonoid  f i s h e s f o l l o w i n g mus-  c u l a r a c t i v i t y . I I I . Sockeye salmon, Oncorhynchus nerka.  J . F i s h . Res. Board  Black, E.C., A.C. Robertson, Alterations  Can. 14: 807-314.  A.R. H a n s l i p and W.G. C h i u . I960.  i n glycogen, glucose and l a c t a t e i n  rainbow and Kamloops t r o u t , Salmo g a i r d n e r i , lowing muscular a c t i v i t y .  fol-  J . F i s h . Res. Board Can.  17: 487-500. B u l l o c k , G.L., H.M. Stuckey and K. Wolf. 1975. kidney d i s e a s e o f salmonid F i s h . Dept. L e a f l e t  : 41  Chavin, W. and J.E. Young. 1970.  fishes.  Bacterial  U.S. Dept. I n t .  7 p.  F a c t o r s i n t h e determina-  t i o n o f normal serum glucose l e v e l s o f g o l d f i s h , C a r a s s i u s auratus L.  Comp. Biochem. P h y s i o l .  33: 629-653. Dougherty, T.F. and A. White. 1943. c o r t i c a l secretions  Influence o f adrenal  on blood elements.  Science,98:  367-369. . 1944. on lymphoid  I n f l u e n c e o f hormones  tissue structure  and f u n c t i o n .  The  r o l e o f t h e p i t u i t a r y a d r e n o t r o p h i c hormone i n the r e g u l a t i o n  o f t h e lymphocytes and o t h e r  u l a r elements o f t h e b l o o d .  cell-  E n d o c r i n o l o g y , 35:  1-14. Dougherty, T.F. I960.  Lymphocytokaryorrhetic  adrenocortical  steroids.  effects of  Pages 112-124 i n J.W.  Rebuck, ed. The lymphocyte and lymphocytic Harper (Hoeber), New York, N.Y.  tissue.  72 Davis. J.C. and R.A.W. Hoos. 1975. Use of sodium pentachlorophenate and dehydroabietic acid as r e f e r ence toxicants f o r salmonid bioassays. J . F i s h . Res. Board Can. 32: 411-416. Earp, B.J., C.H. E l l i s and E.J. Ordal. 1955. Kidney disease i n young salmon. Wash. Dept. Fish. Spec. Rep. Ser. No. 1, 74 p. Einszdorn-Orecka, T. 1970. Quantitative changes i n the c i r culating blood of tenches (Tinea tinea L . ) - i n fested by Ergasilus s i e b o l d i Nordm. P o l . Arch. Hydrobiol. 17(30): 463-481. Evelyn, T.P.T., G.E. Hoskins, and G.R. B e l l . 1973. First record of b a c t e r i a l kidney disease i n an apparently wild salmonid i n B r i t i s h Columbia. J . F i s h . Res. Board Can. 30(10): 1578-80. F i e l d , J.B., LL. Gee, C.A. Evehjem and C. Juday. 1944. The blood picture i n frunculosis induced by Bacterium salmonicida i n f i s h . Arch. Biochem. 3: 277-284. Henry, T. „ 1968. C l i n i c a l Chemistry - P r i n c i p l e s and Technics. Harper and Row, N.Y., 742 p. Hesser, E.F. I960. Methods f o r routine f i s h haematology. Prog. Fish Cult. 22: 164-171. Hickman, C P . and B.F. Trump. 1969. The Kidney. Pages 9-239 i n W.S. Hoar and D.J. Randall, ed. Fish Physiology. Vol. 1. Academic Press, Inc., New York. H i l l , H.W. 1908. The mathematics of b a c t e r i a l count. Anu-: Publ. Health Ass. Rep. Bost. 33(2): 110-120. Hoben, H.J., S.A. Ching, L.J. Casarett, R.A. Young. 1976. Study of pentachlorophenol by r a t s . I. Method f o r determination of pentachlorophenol i n rat plasma urine and tissue and i n aerosol samples. Bull. Environ. Contamin. T o x i c o l . 15(1): 78-36. Houston, A.H., M.A. DeWilde and J.A. Madden. 1971 a. Some physiological effects of handling and tricane methanesulphonate anaesthetization upon the brook t r o u t . J . F i s h . Res. Board Can. 28: 625-633.  V  73 Houston, A.H., J.A. Madden, R.J. Woods and H.M. M i l e s . 1971 b. Variations i n the blood and tissue chemistry of brook trout, Salvelinus f o n t i n a l i s , subsequent to handling, anaesthesia and surgery. J . F i s h . Res. Board Can. 28: 635-642. Hunn, J.B. 1972. Blood chemistry values f o r some fishes of the upper M i s s i s s i p p i River. J . Minn. Acad. S c i . 38: 19-21. 1964. Some patho-physiologic effects of b a c t e r i a l kidney disease i n brook trout. Proc. Soc. Exp. B i o l . Med. 117: 383-3^5. Iwama, G.K., G.L. Greer and P.A. Larkin. 1976. Changes i n some haematological c h a r a c t e r i s t i c s of coho salmon (Oncorhynchus kisutch) i n response to active exposure to dehydroabietic acid (DHAA) at different exercise l e v e l s . J . F i s h . Res. Board Can. 33(2): 285-289. Klontz, G.W. and L.S. Smith. 1968. Methods of using f i s h as b i o l o g i c a l research subjects. Pages 325-385 i n W.I. Gay, ed. Methods o f animal experimentation. Vol I I I . Academic Press. Inc., New York, N.Y. Kobayashi, K. and H. Akitake. 1975 a. Studies on the metabolism of chlorophenols i n f i s h - I . Absorption and excretion of PCP by g o l d f i s h . B u l l . Jap. Soc. S c i . F i s h . 4KD: 87-92. . 1975 b. Studies on the metabolism of chlorophenols i n f i s h - I I . Turnover of absorbed PCP i n g o l d f i s h . Ibid. 41(1): 93-99. . 1975 c. Studies on the metabolism of chlorophenols i n f i s h - IV. Absorption and excretion of phenol by'goldfish. Ibid. 41(12): 1271-1276. Kobayashi, K., H. Akitake, C. Matsuda and S. Kimura. 1975. Studies on the metabolism o f chlorophenols i n f i s h V. Isolation and i d e n t i f i c a t i o n of a conjugated phenol excreted by g o l d f i s h . Ibid. 41(12): 1277-1282.  V  74  Kobayashi, K., H. Akitake and S. Kimura. 1975. Studies on the metabolism of chlorophenols i n f i s h - VI. Turnover of absorbed phenol i n g o l d f i s h . I b i d . 42(1): 45-50. Kobayashi, K., S. Kimura and H. Akitake. 1976. Studies on the metabolism of chlorophenols i n f i s h - VII. Sulfate conjugation of phenol and PCP i n f i s h l i v e r s . Ibid. 42(2): 171-177. McLeay, D.J. 1970. A histometric investigation of the act i v i t y of the p i t u i t a r y - interrenal axis i n juvenile coho salmon, Oncorhynchus kisutch Walbaum. Ph.d. Thesis. University of B r i t i s h Columbia, Vancouver, B.C. 189 p. . 1973 a. E f f e c t s of a 12-hour and 25-day exposure to k r a f t pulp m i l l effluent on the blood and tissues of juvenile coho salmon (Oncorhynchus kisutch). J . F i s h . Res. Board Can. 30: 395-400. . 1973 b. E f f e c t s o f ACTH on the p i t u i t a r y interrenal axis and abundance o f white blood c e l l types i n juvenile coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 21: 431-440. . 1975 a. S e n s i t i v i t y o f blood c e l l counts i n juvenile coho salmon (Oncorhynchus kisutch) to stressors including sublethal concentrations of pulp m i l l effluent and zinc. J . F i s h . Res. Board Can. 32: 2357-2364. . 1975 b. Variations i n the p i t u i t a r y - i n t e r renal axis and the abundance of c i r c u l a t i n g bloodN  c e l l types i n juvenile coho salmon, Oncorhynchus kisutch, during stream migration.  Can. J . Zool.  53: 1832-1891. Menten, M.L. 1927. Changes in:,the blood sugar o f the cod, sculpin and pollack during asphyxia. J . B i o l . Chem. 72: 249-253. Mulcahy, M.F. 1967. Serum protein changes i n diseased A t l a n t i c salmon. Nature 215: 143-144. \  75 Mulcahy, M.F.  1971.  Serum protein changes associated with  u l c e r a t i v e dermal necrosis (UDN) Salmo t r o t t a L. Rucker, R.R.,  i n the trout  J . Fish B i o l . 3: 199-201.  A.F. Bernier, W.J. Whipple, and R.E. Burrows.  1951.  Sulfadiazine  f o r kidney disease.  Progressive F i s h - c u l t u r i s t Rucker, R.R.,  13(3):  135-137.  B.J. Earp and E.J. Ordal. 1954.  diseases of P a c i f i c Salmon.  The  Infectious  Trans. Am.  F i s h . Soc.  83: 297-312. Scott, E.L. 1921.  Sugar i n the blood of the dog-fish and of  the sand shark. Shieh-, H.S.  Am.  J . Physiol. 55:  and J.R. Maclean. 1976.  349-354.  Blood changes i n brook  trout induced by i n f e c t i o n with Aeromonas s a l o n i c i d a. J . W i l d l i f e Diseases. 12: 77-82. Simpson, W.W.  1926.  The e f f e c t s of asphyxia and isletectomy  on the blood sugar of Myoxocephalus and Am.  Amerius.  J . Physiol. 77: 409-418.  Snieszko, S.F. and P.J. G r i f f i n . 1955. brook trout and i t s treatment. 17(1):  Kidney disease i n Prog. F i s h - c u l t u r i s t .  3-13.  Snieszko, S.F. I960.  Microhaematocrit as a t o o l i n fishery  research and management.  U.S.  Fish W i l d l . Serv.,  Spec. S c i . Rep. - Fish. 341: 15 p. . 1974. The e f f e c t s of environmental stress on outbreaks of infectious diseases of f i s h e s .  J.  F i s h . B i o l . 6: 197-208. Soivio, A. and A. O i k a r i . 1976. Haematological effects of stress i n a t e l e o s t , Esox lucius L.  J. Fish. Biol.  8: 397-411.  Sprague, J.B. 1969.  Measurement of pollutant t o x i c i t y to  f i s h - I.  Bioassay methods f o r acute t o x i c i t y .  Water Res. 3:  793-821.  Thomas, A.E., J.E. E l l i o t t , and J.L. Banks. 1969.  Haemato-  l o g i c a l and chemical c h a r a c t e r i s t i c s associated with precocious male chinook salmon f i n g e r l i n g s . Trans. Amer. Fish. Soc. 98: 23-26.  76  Wardle, C.S. 1 9 7 2 . The changes i n blood glucose i n Pleuronectes platessa following capture from the wild: a stress reaction. J . Mar. B i o l . Ass. U.K. 52:  635-651.  Wedemeyer, G.A. and J.W. Wood. 1 9 7 4 . Stress as a p r e d i s posing factor i n f i s h diseases. U.S. Dept. Int., F i s h . Dept. Leaflet - 3 8 . 8 p. Wedemeyer, G.A. 1 9 7 0 . The role o f stress i n the disease resistance of f i s h e s . Pages 3 0 - 3 5 i n Stanislas F. Snieszko, ed. A Symposium on diseases o f fishes and s h e l l f i s h e s . Am. F i s h . Soc. Spec. Publ. No. 5 . . 1 9 7 0 . Stress of anaesthesia with MS 222 and benzocaine i n rainbow trout (Salmo gairdneri) J . F i s h . Res. Board Can. 2 7 : 9 0 9 - 9 1 4 . . 1 9 7 2 . Some physiological consequences of handling stress i n the juvenile steelhead trout (Salmo gairdneri) and coho salmon (Oncorhynchus k i s u t c h ) . J . F i s h . Res. Board Can. 2 9 : 1 7 8 0 - 1 7 8 3 . Wood, J.W. and J . W a l l i s . 1 9 5 5 . Kidney disease i n adult chinook salmon and i t s transmission by feeding to young chinook salmon. Oreg. Fish Comm. Res. Briefs 6:  32-40.  Wood, E.M. and W.T. Yasutake. 1 9 5 5 . Histopathology of kidney disease i n f i s h .  Amer. J . Pathol. 3 2 ( 4 ) :  845-857.  Yamashita, H. 1 9 6 7 . Haematological study of a species of Rockfish - I I . Changes of the moisture content of blood, s p e c i f i c gravity, serum protein, haematocrit value and urea nitrogen l e v e l of serum i n the specimens affected by u l c e r s . B u l l . Jap. Soc. S c i . Fish. 3 3 : 995-1001.  77a  SECTION VIII APPENDICES  77 APPENDIX I SODIUM PENTACHLOROPHENATE (NaPCP) A.  Preparation of stock solution of NaPCP This was  i d e n t i c a l to the procedure of Alderdice  (1963) except f o r the fact that the amount of NaOH was doubled. B.  Preparation of bioassay toxicant solutions i n modified Mariot bottles 2 ml of 5N NaOH was added to 1 1 of d i s t i l l e d  water i n 25 1 glass carboys.  To these solutions, the appro-  priate volumes of the 14.413 g/1 NaPCP stock solution described above were added and the r e s u l t i n g mixtures made up to 25 1 and mixed with a magnetic s t i r r e r f o r 5 minutes. ing solutions had a pH of at least C.  The  result-  3.5.  Preparation of toxicant solutions f o r the main experiment Two  experiment.  concentrations of NaPCP were used i n the main The test tank concentration f o r the intermediate  l e v e l of toxicant was 0.05 96 h L C  5 0  and 0.50  (0.0039 mg/1)  (0.039 mg/1)  the high l e v e l of toxicant. of 3.2 1/min  of the incipient  of t h i s value was used f o r  With a constant diluent flow  and a constant toxicant flow of 0.04  l/min at  the mixing funnel ( F i g . IV), toxicant concentrations of 0.312  mg/1  and 3.12  toxicant reservoirs. of 5N NaOH was reservoir.  mg/1  were required i n the 100 1 p l a s t i c  To achieve these concentrations, 4.0  ml  f i r s t added to 10 1 of d i s t i l l e d water i n each  Then, f o r the intermediate toxicant l e v e l  78 reservoir, 1.08 ml of the 14.413 g/1 NaPCP solution was added to t h i s solution.  10.8 ml of t h i s same stock solu-  t i o n was added to t h i s solution i n the high toxicant l e v e l reservoir.  The r e s u l t i n g solutions i n both reservoirs were  then brought up to 100 1 with well water and mixed f o r 15 minutes.  When the reservoir solutions were approximately  h a l f depleted, the l e v e l s were brought up by momentarily stopping the toxicant  flow and pouring 0.54 ml of the 14.418  g/1 NaPCP stock solution with 2.0 ml of 5N NaOH into the intermediate toxicant l e v e l reservoir and 5.40 ml of the same NaECP stock solution with 2.0 ml of 5N NaOH into the high toxicant l e v e l reservoir, bringing the l e v e l s i n both reservoirs back up to 100 1 while mixing and resuming the toxicant flows.  The t o t a l time i n which the toxicant  was stopped was about 10 minutes. reservoirs was at least 8 . 5 .  \  flow  The r e s u l t i n g pH i n the  79 APPENDIX II MICROBIOLOGICAL PROCEDURES A»  Preparation of Evelyn's kidney disease media-j^ The media  KDM-J--J--J-  ^KDM-J-JJ)  consists of Media-j- plus f o e t a l  c a l f serum and has the following composition: Peptone Yeast extract Cysteine - HC1 Agar Foetal c a l f serum in d i s t i l l e d water 1.  Preparation  10.00$ 0.05$ 0.10$ - 1.50$ 20.00$  of Media-j-  0.313 g of cysteine-hydrochloride (Fisher Chem. Co.) was dissolved i n 250 ml of d i s t i l l e d water without heat and the pH adjusted to 6.5 + 0 . 2 with 2N NaOH.  3.125 g of peptone  (Difco Lab.), 0.156 g of yeast extract (Difco Lab.) and 4.688 g of agar (Difco Lab.) were then added to t h i s solution with heat and constant s t i r r i n g u n t i l the agar was completely d i s solved.  This Media-j- was poured into a 500 ml screw cap  bottle and s t e r i l i z e d i n an autoclave at 15 p s i and 250° F for 15 min. 2.  Preparation of KDM-j.-j.-j. 62.5 ml aliquots of s t e r i l e , virus screened f o e t a l  c a l f serum (Microcan Research Ltd., Calgary, Alta.) were stored at -20 C.  At the end of the s t e r i l i z a t i o n process,  both Mediaj and one 62.5 ml aliquot of f o e t a l c a l f serum, thawed at room temperature, were brought to 45 C i n a  80 temperature controlled water bath.  At the end of t h i s  e q u i l i b r a t i o n procedure, a l l of the f o e t a l c a l f serum was - poured into the 500 ml bottle containing 250 ml of Mediaj. The r e s u l t i n g KDM^j-j- was mixed thoroughly i n the bottle f o r one minute.  Approximately 15 ml of  KDM^JJ  was poured into  each of 21 p l a s t i c disposable p e t r i plates on a l e v e l surface and l e f t f o r 16 h at room temperature f o r s o l i d i f i c a t i o n and evaporation of excess moisture. B.  Isolation and growth of kidney disease bacteria Where the v i a b i l i t y of the bacteria was desirable,  a l l procedures were carried out using s t e r i l e techniques and at low temperatures (0-3 C). 1.  Source of bacteria Viable kidney disease bacteria were isolated from  a moribund, yearling pink salmon (0. gorbuscha) of length 19.0 cm and weight 118.2 g, reared at the P a c i f i c Environment I n s t i t u t e (W. Vancouver, B.C.) under natural l i g h t i n a 4000 1 s a l t water tank i n which flow and temperature were maintained at 30 1/min  and 12 C.  Gram stained kidney smears  taken a s e p t i c a l l y from a sample of moribund, f i s h from the same tank showed an abundance of gram-positive rods (mostly in p a i r s ) .  P r i o r to transfer to the P a c i f i c Environment  I n s t i t u t e , these f i s h had been incubated and reared at an elevated water temperature (12 C) at the P a c i f i c B i o l o g i c a l Station (Nanaimo, B.C.) from eggs taken at Jones Creek, near Hope, B.C., and acclimatized to s a l t water.  81 2.  Bacterial cultures After k i l l i n g the f i s h by concussion and removing  the scales from one side, the whole f i s h was bathed i n 95 percent ethanol, wiped and bathed again and f i n a l l y covered with tissue soaked with 95 percent ethanol.  Using s t e r i l e  techniques, a 5 -cm beveled cut was made along the mid-lateral body wall about 1 cm above the l a t e r a l l i n e exposing a port i o n of the kidney.  Approximately 0.75 g of kidney tissue  was a s e p t i c a l l y excised and dropped into a tissue homogenizer containing 15 ml of cold, s t e r i l e .saline and peptone (0.$5 and 0.1 percent respectively) solution.  Eight 10-fold d i l u -  tions of t h i s o r i g i n a l suspension were made with t h i s same solution i n s t e r i l e , screw-capped test tubes. —8 the 10  0.1 ml of  suspension was spread over an area approximately  6.0 cm diameter on each of 12 s t e r i l e disposable p e t r i plates containing  KDM-J-J-J-.  The 12 inoculated plates were placed on a l e v e l surface i n an incubator at 15 C f o r 24 h to allow evaporat i o n of excess moisture from the inoculum.  They were then  wrapped i n a p l a s t i c sheet i n pairs and incubated upside down f o r 14 days at the same temperature.  The inoculated  plates were examined every f i v e days f o r the presence of contaminating organisms.  Contaminants were 'excised using  s t e r i l e techniques. After 10 days of incubation, the kidney disease bacteria started to appear i n colonies on the surface of the media.  However, instead of being i n discrete colonies the  growth was confluent and presented a ''ground glass"  6*2 appearance indicating a very heavy growth on a l l plates. The  c e l l s were harvested on the 14th day o f incubation when  the c e l l s were i n t h e i r log-phase o f growth (T. Evelyn, per. comm.)  C.  Harvesting o f kidney disease bacteria t i o n o f inoculum f o r i n j e c t i o n into Kidney disease bacteria  growth (14 days on  KDMJ-J-J  at 15  C;  c e l l s and prepara-  fish  i n t h e i r log-phase o f T. Evelyn per. comm.) ?  were washed , into 25 ml o f c h i l l e d s t e r i l e saline and peptone 1  solution  (0.85 and 0.1 percent respectively).  The following  procedures were carried out at room temperature to derive the o p t i c a l density of t h i s milky suspension o f c e l l s . A l l suspensions were thoroughly agitated before aliquots were extracted f o r d i l u t i o n s . Eight 1:1 d i l u t i o n s , i n the saline and peptone solution above, were made from a 3 ml aliquot o f t h i s o r i g i n a l suspension.  The o p t i c a l density was determined f o r  each d i l u t i o n using the offset method on a spectrophotometer (Guilford Instr. Inc., Ohio; model 240) at 420 nm. Of these d i l u t i o n s , one that gave an o p t i c a l density reading between 0.20 and 0.70 was used as a reference (T. Evelyn, per. comm.) and  a l i n e a r extrapolation was made to determine the o p t i c a l  density o f the o r i g i n a l suspension.  This was done by m u l t i -  plying the d i l u t i o n factor by the o p t i c a l density o f that reference d i l u t i o n .  I t was found that a d i l u t i o n o f 1/64  gave an o p t i c a l density o f 0.22. Therefore, i t was  33  determined that the o p t i c a l density of the o r i g i n a l suspension was 1 4 . 0 . It was found i n I n i t i a l Experiment A that an o p t i c a l density of 0 . 1 f o r the i n j e c t i o n inoculum f o r the main experiment would be s a t i s f a c t o r y because i t resulted i n an incubation time of approximately 4 0 days.  This duration  for the main experiment was decided on the basis of the least number of desired sampling days, the size of tanks and the number and size of available f i s h f o r the experiment. Therefore,  the o r i g i n a l suspension, c h i l l e d on i c e , was  d i l u t e d 1 4 0 times with cold, s t e r i l e saline and peptone solution to y i e l d 1 4 0 ml of inoculum with an opacity of 0.1  OD at 4 2 0 nm.  D.  Viable counts The procedures involved i n determining the actual  number of viable bacteria c e l l s that were injected into each f i s h consisted o f making d i l u t i o n s of the i n j e c t i o n inoculum (App. IIC), p l a t i n g accurately measured volumes at each o f these d i l u t i o n s on KDMj-j--j- and counting the numbers of colonies plated (between 3 0 and 3 0 0 ; H i l l , 1 9 0 3 ) and assuming each colony arose from one c e l l .  Once the number o f  viable c e l l s per volume was known f o r a s p e c i f i c d i l u t i o n , the number of viable c e l l s per volume f o r the i n j e c t i o n inoculum was determined by multiplying the d i l u t i o n factor by the number of viable c e l l s at that d i l u t i o n . Eight 1 0 - f o l d d i l u t i o n s of the i n j e c t i o n inoculum were made using a cold, s t e r i l e saline and peptone  ( 0 . 3 5 and  84 0.1 percent respectively) solution on i c e . From each d i l u t i o n , seven 25 u l aliquots of c e l l suspension were spotted onto each of f i v e p e t r i plates containing  KDM-QT  (App. I I A ) .  This yielded 35 r e p l i c a t e counts f o r each d i l u t i o n . plates were incubated as described above (App. IIB).  The Viable  counts were determined on the 14th and 21st days on incubation from plates showing discrete colonies.  85 APPENDIX I I I CATEGORIZATION OF PHYSICAL CHARACTERISTICS IN RESPONSE TO KIDNEY DISEASE INFECTION Physical Characteristics no external symptoms some f l u i d i n abdominal cavity alimentary canal p a r t i a l l y empty above plus: s l i g h t l y bloated abdomen s l i g h t l y enlarged kidney and hind gut haemorrhaging i n i n t e r n a l body walls (esp. at injection site bloated abdomen moderate amount of f l u i d i n abdominal cavity haemorrhaging i n l i v e r t i s s u e , gonads and i n t e r nal body walls alimentary canal empty enlargement of kidney and hind gut pale head kidney bloated abdomen as i n #3 some yellow f l u i d exuding from vent moderate amount of f l u i d i n abdominal cavity as haemorrhaging as alimentary canal pale head kidney kidney and l i v e r small lesions on  i n #3 as i n #3 as i n #3 enlargement as i n #3 kidney  bloated abdomen as i n #3 excessive f l u i d i n abdominal cavity few unbroken welts some yellow f l u i d exuding from vent as i n #4 haemorrhaging as i n #3  86  enlarged and pale spleen smokey appearance of swimbladder swollen, pale kidney - lumpy with lesions i n severe cases lesions appearing on other organs ( l i v e r , spleen) #6  as i n #5 plus: broken welts petechiae haemorrhaging vent i n t e r n a l haemorrhaging spread to other tissues non-specific moderate autolysis  X  37  APPENDIX IV Tables of mean, standard error of the mean and grand mean values f o r HCT, Hb, RBC, WBC, MCV, BUN, TP and GLU  APPENDIX IV, TABLE I Haematocrit (HCT; %) 4 days  8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days 35.74 .756  35.44 .938  36.21 1.92  35.68 1.20  31.59 1.02  32.94 1.23  28". 13 .794  25.66 .921  20.54 .904  23.22 .814  21.92 1.70  36.28 .603  34.77 .918  31.80 .533  34.05 1.09  34.29 .432  35.63 .696  30.38 1.02  26.19 .811  25.63 .745  23.60 1.09  13.73 1.19  20.13 .961  34.47 .366  32.42 1.06  33.45 31.43 .751 ' .545  32.53 .641  31.03 .631  37.08 .6l3  a  r C  CW F E  INT E  HIGH E  h b  33.75 .557 #  33.80 .681  32.47 .996  *  #  36.13 .523  34.71 .774  32.90 .676  Grand Mean 34.64 25.40  34.51 24.12  33.62 .742  35.30 .445  34.33 1.12  33.19 23.39  23.39 .366  CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h L C ) 5 0  C = Uninfected Control Group E = Kidney Disease Infected Experimental Group  n = 12 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard E r r o r  * =  No data due to mortality  Haemoglobin (Hb; mg/100 ml) 4 days 10.23 , .101  8 days 12 days 16 days 20 days 24 days 23 days 32 days 36 days  a  rt  U  CW  b  9.29 .193  9.07 .237  7.95 .165  7.93 .147  7.35 .116  T? Ill  7.93 .237  6.43 .193  6.63 .196  5.23 .305  5.39 .211  4.13 .274  n  3.30 .223  3.13 .266  3.36 .217  3.13 .332  3; 63 .133  3.54 .147  T?  Hi  3.33 .165  6.63 .176  6.13 .274  5.02 .320  4.13 .277  4.93 .199  r< L>  3.33 .349  3.34 .303  3.65 .176  7.23 .167  7.42 .292  7.35 .165  F. Ill  7.02 .346  INT  HIGH  *  *  *  *  CW = Clean Water (0 x 96 h L C ) 50  INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h L C ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group 5 Q  *  7.19 .113  * 3.23 .274  7.50 .237 x  3.20 .217 x  7.07 .139  7.27 .130 x  7.74 .233 x  7.67 .237 x  6.30 .199 x  Grand Mean 3.26 5.99  3.32 5.97  7.61 7.02  n = 12 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard E r r o r * = No data due to mortality  APPENDIX IV, TABLE I I I Red blood c e l l count (RBC; no. of cells/mmVlO,000) 4 days  8 days 12 days 16 days 20 days 24 days 2 8 days 32 days 36 days  147.83? 6.82B  147.83 6.47  147.42 7.82  E  122.92 5.22  117.33 6.77  97.17 4.75  C  149.42 5.45  137.67 6.76  146.67  E  129.50 6.90  94.34 8.06  94.67  139.67  143.75 6.91  150.33  CW  INT  C HIGH E  8.08  113.17 6.93  *  8.26 5.18  6.18  159.17 5.48  153.07  6.46  6.35  92.00  99.42 4.37  73.42  147.17 8.02  163.58 3.25  151.92 7.37  94.92 6.44  71.75 5.78  71.50 5.58  156.58 5.12  148.83 3.75  4.84  136.OO 6.04  *  *  151.00  5.46  *  #  CW = Clean Water ( 0 x 96 h L C ) INT = Intermediate Level NaPCP ( 0 . 0 5 x 96 h L C ) HIGH = High Level NaPCP ( 0 . 5 x 96 h L C ^ Q ) C = Uninfected Control Group 50  50  E = Kidney Disease Infected Experimental Group  149.17 6.14  * 153.17 5.19  * 148.08  153.71 4.95  * 142.86 7.37  * 1:52.43  149.14 6.18  * 152.72 7.78  * 162.86  8.43  4.38  4.92  *  *  *  Grand Mean 150.93  100.38  149.46 92.78  148.73  113.17  n = 1 2 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard Error * _ No data due to mortality  APPENDIX IV, TABLE IV Total white blood c e l l count (WBC; no. of c e l l s / m m / 5 0 0 ) 3  4 days C CW  .352°  3 days 1 2 days 16 days 20 days 2 4 days 2 3 days ;32 days 36 days 3.17  4.50  3.42  4.17 .378  4.33 .791  .600  .621  .621  3.67  .667  E  1.58 .378  2.00 .407  1.83. .528  2.33 .450  3.00  4.42 1.04  x  C  4.25 .494  4.67  4.42  4.00 .739  4.03 .557  4.53 .357  3.25 .739  E  2.33  1.58  1.92  3.58  5.67 1.06  7.42 .653  C  3.17 .491  4.42  4.03  4.42  E  1.58 .358  x  INT  HIGH  .396  .782  .260  .621  .782  .525  3.92 .543  x  .626  .667 3.33 .655  .794  *  .932  x  CW = Clean Water ( 0 x 9 6 h L C ^ ) INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h L C ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group 50  4.57 .223  x  X  2.50 .436 X  2.00 .407  Grand Mean  4.43 ^ .404  x  2.43 .497  3.39  2.53  3.74  x  *  3.75  2.00  2.50  3.37  .647  .471  x  X  1.53  n = 12 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard Error x _ No data due to mortality  APPENDIX IV, TABLE V Mean c e l l volume (MCV; 4 days  u ) 3  8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days  C  256.03^ 11.42°  248.79 15.49  246.71 12.89  230.23 14.44  240.85 13.19  E  271.60 12.80  245.87 11.93  273.25 18.74  228.28 12.81  236.27 - 310.02 24.86 9.13  C  247.13 11.70  256.64 9.45  223.66 11.93  236.05 10.51  210.70 5.51  239.57 10.23  E  239.56 11.11  298.55 26.58  277.34 12.15  257.19 15.17  271.13 16.08  292.32 15.81  C  254.16 13.12  231.28 13.63  227.35 7.02  235.81 10.27  203.09 11.21  209.89 5.91  E  266.00 18.28  *  CW  INT  HIGH  *  *  x  CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h L C ) C = Uninfected Control Group 5 0  E = Kidney Disease Infected Experimental Group  205.59 12.50  229.93 8.39  * 239.15 9.53 x  234.15 12.16 x  x  n a b x  254.64 23.04 x  221.41 5.94 x  233.23 6.29 x  220.94 10.12 x  220.84 11.07 x  212.79 9.38 x  Grand Mean 237.03 260.33  232.79 272.63  226.36 266.00  12 (two f i s h pooled f o r each measurement. ) Mean Standard E r r o r No data due to mortality  APPENDIX IV, TABLE VI Blood urea nitrogen (BUN; mg/100 ml) 4 days <  n \J  cw TT*  Cl  rt U  INT T?  Ci  rt  HIGH V. ill  8 days 12 days 16 days 20 days 24 days 28 days 32 days 36 days  8.38 .595  a  4.73 .517  4.92 .814  5.38 .433  6.23 .499  5.41 .424  4.18 .167  3.02 .199  3.85 .370  6.57 .782  4.54 .318  6.75 .629  8.08 .849  11.16 .759  5.25 ^.344  2.83 .199  2.18 .335  3.52 .176  3.59 .277  4.34 .196  8.73 .612  4.24 .124  5.13 .606  3.98 .367  9.79 1.37  6.38 .710  10.91 1.11  6.57 .453  x  x  14.36 2.05  10.04 1.06  x  x  CW = Clean Water (0 x 96 h L C ^ Q ) INT = Intermediate Level NaPCP (0.05 x 96 h L C ^ Q ) HIGH = High Level NaPCP (0.5 x 96 h L C ) C = Uninfected Control Group E = Kidney Disease Infected Experimental Group  5.59 .664  x  4.78 .753 x  3.90 .159 x  7.52 .473 X  3.09 .329 X  2.89 .338 X  3.46 .476 X  Grand Mean  3.87 .375 X  3.43 .257 X  3.40 .306 X  5.20 4.80  7.81 3.62  5.45 6.57  n  12 (two f i s h pooled f o r each measurement. )  a  Mean Standard E r r o r No data due to mortality  5 Q  b x  APPENDIX I V . TABLE V I I T o t a l p r o t e i n ( T P ; g/100 4 days  8 d a y s 12 d a y s 16 d a y s 20 d a y s 24 d a y s 28 d a y s 32 d a y s 36 d a y s 3.79  3.86 .121  3.13 .110  4.03  4.31 .156  2.86 .147  2.61  .208  3.47 .225  4.34 .147  2.30  .217  2.71 .156  4.73 .245  4.17 .245  4.18 .294  4.73 .240  4.59 .364  3.68 .225  3.18 .153  2.60 .211  2.77 .205  3.01 .326  3.16  .193  2.64 .297  U  2.93 .251  3.15 .191  3.23  3.33 .251  3.18 .124  3.77 .196  T?  2.09 .271  L.  3.49 .185  £»  a  CW  ri  0  INT TT*  £j  rt  HIGH Hi  ml)  h b  .136  .245  CW = C l e a n W a t e r (0 x 96 h INT = I n t e r m e d i a t e  *  #  LC  c n  *  )  L e v e l NaPCP ( 0 . 0 5  HIGH = H i g h L e v e l NaPCP ( 0 . 5  .179  x 96 h L C ^ Q )  C = U n i n f e c t e d C o n t r o l Group E = Kidney Disease  I n f e c t e d E x p e r i m e n t a l Group  x  3.77 .260  3.96 .147  = 12  3.76  3.76  .404  .113  x  x  3.83 .600 x  x  3.17 .508 x  x  X  n x 96 h L C ^ Q )  3.93 .179  G r a n d Mean  3.40 .136 x  4.04 .233 x  (two f i s h p o o l e d f o r measurement.  a = Mean b = Standard  Error  x = No d a t a due t o  mortality  3.73 3.05  4.12 2.39  3.42 2.09  each  )  Glucose (GLU; mg/100 ml) 4 days  8 days 1 2 days 1 6 days 2 0 days 24 days 2 8 days 3 2 days 3 6 days  C  60.81*  62.38  54.40  2.79  2.46  2.49  E  56.46  35.58  33.92  1.49  2.45  1.39  C  76.79 3.55  58.17 2.35  52.08  E  60.03 2.50  C  53.00 5.38  E  41.04 3.00  GW  INT  HIGH  59.25 3.77  64.29 1.49  62.07 1.43  59.69  *  *  *  38.66  79.29 2.32  52.92 3.45  68.57 4.55  38.54 2.52  38.79 3.37  #  *  *  62.25 2.16  55.67 3.40  57.92  79.93 3.48  49.93 3.27  *  *  60.43 3.33  56.54 2.66  57.04  38.63 2.22  30.21  37.17 2.28  62.08 2.58  61.50  2.96  1.87  43.50 2.18  35.50 2.10  35.42 2.12  46.00  45.92  3.24  2.17  37.46 .950  *  Grand Mean  1.65  #  CW = Clean Water ( 0 x 9 6 h L C ) CA  INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h L C ) 5 Q  C = Uninfected Control Group E = Kidney Disease Infected Experimental Group  1.94  2.13  101.07 6.39  *  68.05  41.97  54.23  41.04  n = 1 2 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard Error * _ No data due to mortality  96  )  APPENDIX V Results f o r Mean Corpuscular Haemoglobin Concentration and Mean C e l l Haemoglobin  97  The r e s u l t s f o r MCHC and MCH were highly variable in a l l groups over the entire sampling period.  In some cases,  these r e s u l t s seemed to even contradict those patterns that would be expected from the trends i n the values used to der i v e the MCHC and MCH values.  For example, experimental  f i s h i n group IE showed i n i t i a l l y lower, but not s i g n i f i c a n t , MCHC values r e l a t i v e to sham injected controls i n group IC on the f i r s t two sampling days ( T l and T2; App. V F i g . I ) . Thereafter, the MCHC values i n group IE f i s h increased and remained above control f i s h values o f group IC u n t i l the s i x t h sampling day (T6).  The MCHC values o f group 2E declined,  r e l a t i v e to group 2C values, from being s i g n i f i c a n t l y higher on the f i r s t two sampling days ( T l and T2) to being only s l i g h t l y lower (P greater than .05) than group 2C values on the fourth sampling day ( T 4 ) .  After the decline by time T 4 ,  the MCHC f o r experimental f i s h increased u n t i l they were s i g n i f i c a n t l y higher than the 2C group values on the sixth sampling day.  No s i g n i f i c a n t difference was observed be-  tween group 3E and 3C MCHC values on the f i r s t sampling day (Tl).  The MCH values i n group IE f i s h were found to be  s i g n i f i c a n t l y lower on the f i r s t two sampling days ( T l and T2; App. V F i g . II) r e l a t i v e to group IC values on the same days. The IE group values then increased u n t i l they were s i g n i f i cantly higher than the values of f i s h i n group IC on the fourth sampling day ( T 4 ) .  After the MCH values f o r group IE  dropped, once again, to being s i g n i f i c a n t l y lower than the. control group value by the sixth sampling day (T6). The  98 The reverse trend seemed to occur i n f i s h of groups 2E and 2C.  The MCH values of group 2E started out significantly-  higher than group 2C values on the f i r s t two sampling days (TI and T2) after which they dropped to be s i g n i f i c a n t l y lower than group 2C values by the fourth sampling day (T4). Then, the group 2E values rose to a s l i g h t l y higher, but not s i g n i f i c a n t , l e v e l r e l a t i v e to 2C values on the sixth sampling day (T6).  N O s i g n i f i c a n t difference occurred  between MCH values of f i s h i n groups 3E and 3C on the f i r s t sampling day ( T I ) . Due to the marked fluctuations and inconsistent trends i n the mean corpuscular haemoglobin concentration and i n the mean c e l l u l a r haemoglobin values i n a l l groups of f i s h over the experimental period, no attempt was made to draw any conclusions from these  results.  APPENDIX V, TABLE I Mean corpuscular haemoglobin concentration (MCHC; pg) 4 days  8 days 1 2 days 1 6 days 2 0 days 2 4 days 2 8 days 3 2 days 3 6 days  3.43  50.43 1.56  53.77 2.13  47.64 2.29  43.94 1.73  53.40 1.33  52.64 2.59  62.30 3.00  70.06 4.04  57.93 2.31  54.66 1.35  53.05 2.73  x  x  x  56.10 3.46  60.22 3.34  62.22 3.45  56.55 2.49  53.34 1.39  57.50 2.51  54.26 2.33  52.74 1.15  51.13 2.01  TP  Cl  70.46 3.64  75.15 5.16  65.95 1.73  53.33 1.30  59.34 3.39  71.74 3.39  x  x  x  r> O  60.90 1.94  53.37 2.43  53.53 2.42  51.32 4.21  47.69 1.97  49.52 1.20  49.13 2.54  43.03 1.37  42.14 1.76  TP  64.71 5.20  x  *  *  *  x  x  70.99? 2.99°  64.25 3.15  61.23  TP Hi  65.87 2.92  r> O  n  CW  INT  HIGH Hi  *  CW = Clean Water ( 0 x 9 6 h L C ^ Q ) INT = Intermediate Level NaPCP ( 0 . 0 5 x 9 6 h L C ^ Q ) HIGH = High Level NaPCP ( 0 . 5 x 9 6 h LC, ) C = Uninfected Control Group n  E = Kidney Disease Infected Experimental Group  Grand Mean 55.92  61.43  '  56.00  66.03  51.35  64.71  n = 1 2 (two f i s h pooled f o r each measurement. ) a = Mean b = Standard E r r o r x — No data due to mortality  APPENDIX V, TABLE I I Mean c e l l u l a r haemoglobin (MCH; $) 4 days n O  CW E  rt 0  INT TT* Cl  r>  HIGH F. Ill  3 days 1 2 days .16 days 2 0 days 2 4 days 28 days 3 2 days 3 6 days 23.56 .924  21.34 .315  23.44 1.04  19.66 1.44  x  24.12 .889  25.35 .341  24.07 .551  24.07 .707  21.23 .979  22.03 .482  24.80 1.09  25.95 .583  23.03  22.89 1.10  *  *  27.81 , .401°  26.07 .502  25.64 .970  22.49 1.02  22.70  24.33 .499  23.10  26.11 .987  25.90 1.43  22.91  23.63  .722  27.93 .722  29.47 .875  25.56 .756  24.30 .771  25.98 1.09  a  .632  24.42  1.19  .546  .494  .993  X  CW = C l e a n Water (0 x 9 6 h L C ^ Q ) INT = I n t e r m e d i a t e L e v e l NaPCP (0.05 x 9 6 h L C ^ Q ) HIGH = H i g h L e v e l NaPCP (0.5 x 9 6 h L C ^ Q ) C = U n i n f e c t e d C o n t r o l Group E = K i d n e y D i s e a s e I n f e c t e d E x p e r i m e n t a l Group  Grand Mean  23.85  23.96  x  x  23.76  24.24 .352  23.30 .456  24.25  x  x  x  24.69  23.72 .505  21.08 .473  20.64  x  x  x  22.74 .580  21.76 .941  .468  .396  19.91 .580 x  23.05 24.42  n = 12 (two f i s h p o o l e d f o r each measurement. ) a = Mean b = Standard E r r o r x - No d a t a due t o m o r t a l i t y  101a  Appendix V, Figures I and I I Graphs of the response of MCHC and MCH values i n the d i f f e r e n t groups of f i s h to the experimental t r e a t ments, b a c t e r i a l kidney disease i n f e c t i o n and NaPCP exposure, over the sampling period. Each point represents a mean + 1.96 standard error of the mean (n=12). Responses i n uninfected control f i s h are designated by s o l i d l i n e s and kidney disease infected f i s h by broken l i n e s , a, b and c represent clean water (0 x 96 h L C ^ ) , intermediate l e v e l of NaPCP exposure (0.05 x 96 h L C ^ Q ) , and high l e v e l of NaPCP exposure (0.5 x 96 h LC^ ) r e s p e c t i v e l y . T l represents the f i r s t sampling day, four days a f t e r beginning toxicant exposure. 2 to 9 represent subsequent sampling days. Symbols on top o f each sampling day designate s t a t i s t i c a l significance (P=0.05), by Scheffe's t e s t , between means of d i f f e r e n t groups of f i s h : * = between control and experimental f i s h , 1 = in a, 2 = i n b, 3 = i n c; H = i n control f i s h groups, A = between a and b, B = between b and c, C = between a and c; D = i n kidney disease infected f i s h . Q  Q  Appendix V, Figure I Appendix V, Figure II  Mean Corpuscular Haemoglobin Concentration Mean C e l l u l a r Haemoglobin  101 12  Appendix V, BC  H  Figure I  H  H  B  B  H  BC  80_  60  o E-i  a w o a o o  m  o  40L b 85_  65  a  w  ac  •CE! < J=> O  45  CO !=> OH  Ct! O O  c 75_  55  35 TI  2  3  4  5  6  7  SAMPLING PERIOD - 4 day i n t e r v a l s  8  9  102 Appendix V, H  Figure II  D  a  29>  b  31  12 12 AC A B °A  2  12. H  2 1 BC B H  D.  E-H  •S E-H  O  o  27L  PQ O hi  a  23 pc; <  19  hi  w o  c  28  24  20 I  Tl  1  2  I  3  L  4  J  5  L  6  J  7  L  8  SAMPLING PERIOD - k day intervals  103a  Appendix V, Figures I I I and IV Histograms showing grand means and means of absolute differences between control and experimental f i s h f o r the different groups of f i s h . Sample sizes used to derive these values are indicated on top of bars. Appendix V, Figure I I I  Mean Corpuscular Haemoglobin Concentration  Appendix V, Figure IV  Mean C e l l u l a r Haemoglobin  MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION•g r a n d means ( p g )  i— o 1  I  L_  O  ^  L  * means o f a b s o l u t e  j  o  differences  between c o n t r o l and e x p e r i m e n a l fish.  MEAN CELLULAR HAEMOGLOBIN CONTENT -  grand means (%)  •  o  O  11  !  o  o  o 1  O i  00  O  I  NO  J oJ la  fD  mm  * i  O fD  CO  n  co fD  3-  CO  *  N3  CO * means of absolute differences between control and experimental f i s h . *  105  APPENDIX VI PHOTOGRAPHS OF APPARATUS USED IN BIOASSAYS AND THE MAIN EXPERIMENT  106  

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