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Some effects of dehydroabietic acid (DHA) on hydromineral balance and other physiological, parameters… Kruzynski, George M. 1979

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SOME EFFECTS OF DEHYDROABIETIC ACID (DHA) ON HYDROMINERAL BALANCE AND OTHER PHYSIOLOGICAL PARAMETERS IN JUVENILE SOCKEYE SALMON ONCORHYNCHUS NERKA  by  GEORGE M. KRUZYNSKI B.Sc,  M . S c , S i r George W i l l i a m s U n i v e r s i t y ,  1968, 1972  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  THE FACULTY OF GRADUATE STUDIES (Department o f Zoology)  We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1979  (c)  George M. K r u z y n s k i , 1979  In p r e s e n t i n g t h i s  thesis  in partial  an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y the L i b r a r y  s h a l l make  it freely  f u l f i l m e n t o f the requirements f o r of British  available  Columbia,  f o r reference  I agree  that  and s t u d y .  I f u r t h e r 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 t h i s  thesis  f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . of  this  It i s understood that  thesis f o rfinancial  gain shall  written permission.  D e p a r t m e n t o f -Z<5C*tiC>&J The 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  AJQi/ 6 > / 7 9  copying o r p u b l i c a t i o n  n o t be a l l o w e d w i t h o u t my  ABSTRACT  Laboratory abietic acid smolts  (DHA)  experiments were conducted t o study t h e e f f e c t s o f dehydroon the p h y s i o l o g y o f the a d a p t a t i o n o f sockeye salmon  (Oncorhynchus nerka) t o sea water. Dehydroabietic  a c i d occurs  i n the r o s i n o f c o m m e r c i a l l y  important  c o n i f e r o u s t r e e s and i s found i n the u n t r e a t e d e f f l u e n t s o f the p u l p paper i n d u s t r y at concentrations a c u t e l y t o x i c to salmonids. r e s i n a c i d i s known t o be one of kraft mill effluent reduced  (KME)  As  and  this  o f the more p e r s i s t e n t t o x i c components and  by b i o l o g i c a l treatment,  although DHA  i t s concentrations are g r e a t l y  i s n e v e r t h e l e s s d i s c h a r g e d i n the  e f f l u e n t s o f t h e p u l p m i l l s s i t u a t e d on the F r a s e r R i v e r system as w e l l as o f those  l o c a t e d on t h e c o a s t o f B r i t i s h Columbia.  u t i l i z e both  t h e F r a s e r and Thompson R i v e r s d u r i n g t h e i r downstream  m i g r a t i o n , t h i s s p e c i e s may An exposing  As sockeye salmon  attempt was  be  exposed t o DHA  b e f o r e e n t e r i n g the  sea.  made t o s i m u l a t e t h i s s i t u a t i o n i n the l a b o r a t o r y by  sockeye salmon smolts t o a s u b l e t h a l c o n c e n t r a t i o n o f DHA  i n f r e s h water f o r 120 h and c o n t a i n i n g no  then t r a n s f e r r i n g them i n t o sea w a t e r  (28  mg/L) °/oo)  DHA.  H y d r o m i n e r a l b a l a n c e was  , . .  (0.65  ,  o s m o l a l i t y , plasma Na  s t u d i e d by m o n i t o r i n g  changes i n plasma  + + + + + +  , K , Ca  , Mg  and C l , b l o o d h e m a t o c r i t  water c o n t e n t a t the end o f the f r e s h w a t e r  DHA  i n t e r v a l s d u r i n g the a d a p t a t i o n t o sea water water the g i l l p e r m e a b i l i t y t o water and gut were a l s o determined.  Supportive  exposure and (120 h ) .  and  muscle  a t 24 h  A f t e r 24 h i n sea  the water t r a n s p o r t a b i l i t y o f the  experiments measured changes i n the  s i z e o f r e d b l o o d c e l l s , the l e v e l s of plasma b i l i r u b i n as w e l l as the uptake and  t i s s u e d i s t r i b u t i o n o f DHA  i n sockeye salmon s m o l t s .  o f v a r i o u s t i s s u e s were a n a l y z e d  f o r DHA  r e s i d u e s by gas  Lipid extracts chromatography  c o u p l e d w i t h mass spectrometry The  (GC-MS).  exposure o f sockeye salmon to DHA  hydromineral  i n f r e s h water r e s u l t e d i n a  d i s t u r b a n c e c h a r a c t e r i z e d by a drop i n plasma o s m o l a l i t y , sodium,  and c h l o r i d e , i n d i c a t i n g a g e n e r a l h y d r a t i o n which was muscle water c o n t e n t .  A l o w e r i n g o f d i s s o l v e d oxygen t o 75% s a t u r a t i o n  markedly i n c r e a s e d the t o x i c i t y o f DHA a secondary  r e f l e c t e d by i n c r e a s e d  and  the o s m o t i c  imbalance may  r e s u l t o f an a d a p t i v e r e s p i r a t o r y response  brought on by DHA  exposure.  have been  to a hypoxic  stress  I n c r e a s e s i n b l o o d h e m a t o c r i t were caused  s w e l l i n g o f the r e d b l o o d c e l l s r e l a t e d t o lowered  a r i s e i n o s m o l a l i t y was  e l e v a t e d l e v e l s o f a l l the plasma i o n s . in  some m o r t a l i t y and  The  caused  by  added s a l i n i t y  considerably greater excursions  o c c u r r e d i n f i s h which were e x p e r i e n c i n g locomotor  normal l e v e l s .  P r i o r DHA  D u r i n g acute DHA  abnormally  stress  difficulty.  Plasma .  (96 h) t o r e t u r n  exposure i n c r e a s e d the p e r m e a b i l i t y o f the  was  f o l l o w e d by h y p e r s e n s i t i v i t y t o  s t i m u l i and abnormal swimming b e h a v i o r . r e d u c t i o n i n s c h o o l i n g and  gill.  f r i g h t response  mechanical  A f t e r s u b l e t h a l exposure, the g e n e r a l l y became most e v i d e n t  24 h o f sea water a d a p t a t i o n .  These r e s u l t s o f the  a r e d i s c u s s e d i n terms o f the p o s s i b l e r o l e s p l a y e d by the g i l l s , kidney  resulted  exposure i n f r e s h water a g r a d u a l d e t e r i o r a t i o n i n  s c h o o l i n g and f r i g h t response  during the f i r s t  was  i n plasma e l e c t r o l y t e s  magnesium showed the g r e a t e s t e l e v a t i o n and took the l o n g e s t to  a  plasma o s m o l a l i t y .  When these f i s h were t r a n s f e r r e d t o sea water, the h y d r a t i o n r e p l a c e d by d e h y d r a t i o n and  by  i n the DHA-induced p e r t u r b a t i o n s of h y d r o m i n e r a l  balance.  study  gut The  i m p l i c a t i o n s of the accompanying a l t e r a t i o n s i n b e h a v i o r are d i s c u s s e d  and  i n the c o n t e x t o f the e c o l o g i c a l s u r v i v a l o f sockeye  salmon smolts d u r i n g  a d a p t a t i o n t o sea water. Residue  a n a l y s e s showed t h a t sockeye  water t o h i g h l e v e l s i n the b r a i n as w e l l as i n o t h e r t i s s u e s .  salmon accumulated  (954 x ) , l i v e r  The p r e s e n c e  o f DHA  DHA  from  (428 x) and k i d n e y  (404  metabolites i n the  the x)  bile,  which a l s o c o n t a i n e d t h e h i g h e s t DHA  r e s i d u e s (647.3 y g / g ) , i n d i c a t e s t h a t  the h e p a t o b i l i a r y r o u t e i s important  i n the e x c r e t i o n o f DHA  The p o s s i b i l i t y  o f the b i o a c c u m u l a t i o n o f DHA  by  by f i s h i n the w i l d i s  d i s c u s s e d i n r e l a t i o n t o the s e t t i n g o f water q u a l i t y c r i t e r i a mill  effluent.  fish.  f o r pulp  iv  TABLE OF CONTENTS  Page  GENERAL INTRODUCTION  1  GENERAL MATERIALS AND METHODS  18  SYNOPSIS OF STUDIES ON DHA  31  PART I-  PRELIMINARY EXPERIMENTS A.  B.  PART I I  ACUTE TOXICITY OF DHA TO JUVENILE SOCKEYE SALMON INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION •  33 34 37  EFFECTS OF ACUTE DHA EXPOSURE ON OSMOTIC BALANCE INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION  43 43 46  PRINCIPAL EXPERIMENTS A.  B.  EFFECTS OF SUBLETHAL DHA EXPOSURE ON HYDROMINERAL BALANCE IN SOCKEYE SALMON SMOLTS INTRODUCTION MATERIALS AND METHODS RESULTS EFFECTS OF DHA ON GILL AND GUT PERMEABILITY TO WATER INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION  53 54 55  .79 80 87 91  PART I I I ECOLOGICAL IMPLICATIONS  113  APPENDICES  119  LITERATURE CITED  169  V  LIST OF TABLES Pages Table I  Chemical and 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 w e l l water used i n c o n t i n u o u s f l o w b i o a s s a y s w i t h DHA.  20  II  F i s h s i z e and acute b i o a s s a y o p e r a t i n g p a r a m e t e r s .  35  III  C o n d i t i o n f a c t o r s o f u n d e r y e a r l i n g sockeye salmon exposed t o a c u t e l y l e t h a l and s u b l e t h a l c o n c e n t r a t i o n s o f DHA i n f r e s h water (Expts. B and C ) .  47  IV  C o n d i t i o n f a c t o r and t o t a l body water i n u n d e r y e a r l i n g sockeye salmon which d i e d d u r i n g exposure t o 0.95 mg/L DHA i n f r e s h water (Expt. D ) .  48  V  Percentage water o f stomachs d i s s e c t e d from " s w o l l e n " and "normal" salmon exposed t o 1.11 mg/L DHA (Expt. A ) .  49  VI  Muscle water i n u n d e r y e a r l i n g sockeye salmon exposed t o DHA i n f r e s h water and sampled a t t h e d r i f t i n g s t a g e .  50  VII  Muscle water i n u n d e r y e a r l i n g chum salmon exposed t o DHA i n  52  sea water. VIII  S i z e o f t h e sockeye salmon used i n t h e e l e c t r o l y t e b a l a n c e experiments (Expts. 1, 2 and 3 ) .  56  IX  Plasma i o n i c c o m p o s i t i o n , h e m a t o c r i t and muscle water c o n t e n t o f sockeye salmon (Expts. 1, 2 and 3 ) .  59  Plasma e l e c t r o l y t e l e v e l s i n sockeye salmon d u r i n g sea-water a d a p t a t i o n (Expts. 1, 2 and 3 ) .  63  XI  H e m a t o c r i t o f sockeye salmon  65  XII  Percentage water i n g u t o f sockeye salmon  XIII  Percentage water i n muscle o f sockeye (Expts. 2 and 3 ) .  XIV  The plasma i o n i c c o m p o s i t i o n o f an "exposed" f i s h which e q u i l i b r i u m a f t e r 72 h i n s e a water (Expt. 1 ) .  XV  S i z e , p e r c e n t a g e muscle and g u t water and symptoms i n f i s h which appeared t o be s e r i o u s l y a f f e c t e d d u r i n g t h e c o u r s e o f Expt. 2.  XVI  F i s h s i z e and t h e l o s s i n weight d u r i n g sea-water i n c u b a t i o n o f g i l l a r c h e s i s o l a t e d from j u v e n i l e sockeye salmon.  (Expts. 1, 2 and 3 ) .  67  (Expt. 2 ) .  69 lost  71  72  89  VI  Pages  Table F i s h s i z e and t h e l o s s i n weight o f i s o l a t e d i n t e s t i n a l t a k e n from j u v e n i l e sockeye salmon.  XVIII  The e f f e c t o f f i s h l o a d i n g d e n s i t y on c o n c e n t r a t i o n o f DHA i n aquarium water.  122  XIX  Comparison o f c o n c e n t r a t i o n s o f DHA and u n f i l t e r e d water samples.  filtered  125  XX  C o n c e n t r a t i o n s o f DHA  d i r e c t l y s o l u b l e i n f r e s h water a t pH  128  6.76  r e c o v e r e d from  sacs  90  XVII  and 20°C.  XXI  DHA r e s i d u e s i n t h e amphipod Anisogammarus c o n f e r v i c o l u s exposed t o 0.4 mg/L DHA f o r 120 h i n sea water o f s a l i n i t y 10 °/oo.  145  XXII  S i z e , h e m a t o c r i t and plasma i o n i c c o m p o s i t i o n o f f i s h from d a t a o f E x p t . 1.  deleted  152  XXIII  S i z e , h e m a t o c r i t and plasma i o n i c c o m p o s i t i o n o f f i s h from d a t a o f E x p t . 2.  deleted  153  XXIV  B l o o d c h e m i s t r y o f sockeye salmon smolts exposed t o mg/L DHA f o r 5 days i n E x p t . PB-1.  XXV  B l o o d c h e m i s t r y and s i z e o f sockeye salmon smolts exposed t o 0.65 mg/L DHA f o r 5 days i n Expt. PB-2.  161  XXVI  Red b l o o d c e l l dimensions o f j u v e n i l e sockeye salmon exposed t o 0.65 mg/L DHA f o r 120 h i n f r e s h water.  168  0.65  160  vii  LIST OF FIGURES  Pages  Figure 1  Map showing the l o c a t i o n o f p u l p m i l l s i n B r i t i s h Columbia and t h e major sockeye salmon m i g r a t i o n r o u t e s o f t h e F r a s e r R i v e r system.  2  2  S e a s o n a l p r e s e n c e o f sockeye salmon i n t h e F r a s e r R i v e r i n t h e r e g i o n o f t h e k r a f t p u l p m i l l s a t P r i n c e George and Q u e s n e l .  4  3  The s e a s o n a l p r e s e n c e o f sockeye salmon i n t h e Thompson R i v e r downstream from t h e k r a f t p u l p m i l l a t Kamloops.  5  4  S t r u c t u r e s o f some i s o l a t e d components o f k r a f t m i l l waste t o x i c t o salmon.  9  5  Water s u p p l y system as used f o r c o n t i n u i n g f l o w b i o a s s a y s w i t h DHA.  19  6  I l l u s t r a t i o n o f t h e arrangement o f t h e donut t a n k s used f o r c o n t i n u o u s f l o w b i o a s s a y s w i t h DHA.  22  7  A s e c t i o n o f two donut tanks showing d e t a i l s o f t h e water s u p p l y and the DHA m e t e r i n g system.  23  8  The c r o s s - s e c t i o n o f a donut tank g i v i n g p r o p u l s i o n and waste t r a p systems.  24  9  T o x i c i t y c u r v e s i l l u s t r a t i n g 96 h LC50 v a l u e s f o r DHA j u v e n i l e sockeye salmon i n f r e s h water.  d e t a i l s o f t h e water  to  39  10  E f f e c t o f r e d u c e d d i s s o l v e d oxygen i n the water on t h e t o x i c i t y o f DHA t o sockeye salmon s m o l t s .  41  11  I l l u s t r a t i o n o f t h e s w e l l i n g o f sockeye salmon caused by DHA exposure.  44  12  Plasma e l e c t r o l y t e l e v e l s , h e m a t o c r i t and muscle water c o n t e n t i n sockeye salmon exposed t o 0.65 mg/L DHA.  60  13  Change i n plasma e l e c t r o l y t e l e v e l s i n sockeye salmon d u r i n g sea-water a d a p t a t i o n f o l l o w i n g a 120 h exposure t o 0.65 mg/L DHA i n f r e s h water.  62  14  Change o f h e m a t o c r i t and muscle water i n sockeye salmon d u r i n g sea-water a d a p t a t i o n .  66  15  R e l a t i o n s between muscle and g u t water i n moribund f i s h (Expt. 2 ) .  73  16  Comparison o f plasma e l e c t r o l y t e c o n c e n t r a t i o n s measured d r i f t i n g f i s h (Expt. 2)  in 3  75  I l l u s t r a t i o n o f a s e c t i o n o f a donut tank showing t h e p o s i t i o n i n g o f p l a s t i c tubes t o which sockeye salmon were confined. D e t a i l e d i l l u s t r a t i o n o f one o f the p l a s t i c tubes used f o r t h e containment o f sockeye salmon. D i s s s e c t i o n p r o c e d u r e f o l l o w e d t o make the g i l l used i n t h e g i l l p e r m e a b i l i t y experiment.  preparation  Apparatus t o measure weight l o s s o f i s o l a t e d g i l l  filaments.  Diagram o f a p p a r a t u s used f o r i n c u b a t i o n o f i s o l a t e d  intestinal  sacs. E f f e c t s of f i s h loading density i n a s t a t i c bioassay a c u t e t o x i c i t y o f DHA t o j u v e n i l e sockeye.  on t h e  D i s t r i b u t i o n of dehydroabietic a c i d i n pooled t i s s u e s of sockeye salmon s m o l t s . T i s s u e d i s t r i b u t i o n o f d e h y d r o a b i e t i c a c i d i n . a rainbow t r o u t . Salmon m o r t a l i t y d u r i n g the t h r e e e l e c t r o l y t e b a l a n c e experiments (Expts. 1, 2 and 3 ) .  ix  ACKNOWLEDGEMENTS  I thank my s u p e r v i s o r s , Dr. D.J. R a n d a l l and Dr. J . C . D a v i s g r a t e f u l t o both manuscript.  f o r p r o v i d i n g the opportunity,  f o r p r o v i d i n g the f a c i l i t i e s  for this research.  I am  f o r t h e i r guidance and a d v i c e d u r i n g r e v i s i o n s o f the  S p e c i a l thanks a r e o f f e r e d t o Dr. G. G r e e r  f o r many  fruitful  d i s c u s s i o n s d u r i n g t h e r e s e a r c h and e s p e c i a l l y f o r h i s c o n s t r u c t i v e c r i t i c i s m d u r i n g the p r e p a r a t i o n o f t h e t h e s i s . Thanks a r e a l s o extended t o Dr. I.H. Rogers f o r h i s a d v i c e and p r o v i d i n g t h e use o f GC-MS f a c i l i t i e s  and t o Mr. H. Mahood f o r a d v i c e and  t e c h n i c a l a s s i s t a n c e during the r e s i d u e The Wishart  analyses.  t e c h n i c a l a s s i s t a n c e o f Mrs. P. F u t e r , Mr. I . Shand and Miss B. i s a l s o g r a t e f u l l y acknowledged a s i s t h e c o o p e r a t i o n o f t h e  s t a f f a t the P a c i f i c Environment I n s t i t u t e L a b o r a t o r y , West Vancouver, B.C.  where the r e s e a r c h was c o n d u c t e d . T h i s r e s e a r c h was supported  Science Subvention  through F i s h e r i e s and M a r i n e S e r v i c e  Program f u n d i n g t o Dr. D.J. R a n d a l l , a Quebec Government  P o s t Graduate S c h o l a r s h i p t o t h e a u t h o r , of  and b y i n - h o u s e o p e r a t i n g funds  t h e F i s h e r i e s and Marine S e r v i c e . A v e r y s p e c i a l thanks t o lay w i f e Jo-Anne who t y p e d t h e m a n u s c r i p t ,  f o r h e r p a t i e n c e and c o n s t a n t encouragement through t h e d i f f i c u l t  times.  1  GENERAL INTRODUCTION  Wastes from the p u l p and paper i n d u s t r y form the l a r g e s t s i n g l e source of i n d u s t r i a l e f f l u e n t being discharged o f B r i t i s h Columbia. Pacific  i n t o r i v e r s and e s t u a r i n e waters  A t the same time, a l l f i v e s p e c i e s — ^ o f the anadromous  salmon must spend v a r y i n g l e n g t h s o f time i n r i v e r s and  c o n t a i n i n g t h i s p o t e n t i a l l y t o x i c waste.  estuaries  Based on a v a i l a b l e i n f o r m a t i o n i t  appears t h a t i n the f i e l d s a l m o n do not markedly a v o i d d i l u t e k r a f t t  waste and,  i n f a c t , may  mill  sometimes be a t t r a c t e d t o i t (Holland e t a l . , 1960).  In the F r a s e r R i v e r , the f r y o f chum and p i n k salmon as w e l l as y e a r l i n g chinook, coho and  sockeye salmon are exposed t o d i l u t e c o n c e n t r a t i o n s  kraft m i l l effluent sequently,  (KME)  chum f r y and  d u r i n g the p e r i o d of seaward m i g r a t i o n .  of  Sub-  f i n g e r l i n g chinook and coho salmon f e e d i n e s t u a r i n e  areas f o r p e r i o d s of up t o s e v e r a l months  ( W i l l i a m s e t a l . , 1953;  e t a l . , 1978); whereas sockeye salmon smolts may  Dorcey  spend r e l a t i v e l y l i t t l e  i n b r a c k i s h water b e f o r e moving out towards t h e open sea  ( W i l l i a m s , 1969).  In t h e a d u l t s t a g e , a l l f i v e s p e c i e s o f salmon must move t h r o u g h i e s where they spend v a r i a b l e l e n g t h s o f time on t h e i r way  estuar-  t o n a t a l streams.  F o r example, the Adams R i v e r sockeye r u n d e l a y s a p p r o x i m a t e l y the mouth o f the F r a s e r R i v e r p r i o r t o the f r e s h w a t e r  time  t h r e e weeks o f f  transition.  After  e n t r y i n t o f r e s h water, some r a c e s of a d u l t m i g r a n t s spend up t o t h r e e weeks i n the r i v e r b e f o r e r e a c h i n g t h e i r spawning grounds: Bowron s t o c k s  1/  ( F i g u r e 1)  (Killick,  Oncorhynchus nerka sockeye O.kisutch O.keta O.gorbuscha O.tshawytscha  coho chum pink chinook  1955).  e.g.  Stuart  and  2  F i g u r e 1 . Map showing the l o c a t i o n o f p u l p m i l l s i n B r i t i s h Columbia and the major sockeye salmon m i g r a t i o n r o u t e s o f the F r a s e r R i v e r system.  3  During  the p e r i o d o f r i v e r m i g r a t i o n , whether as a d u l t s o r s m o l t s ,  5 f i s h must p a s s through waters r e c e i v i n g a t o t a l o f 4x10  these  3 m  p e r day  o f waste  from the t h r e e p u l p m i l l s a t P r i n c e George and one  a t Quesnel.  concentrations of e f f l u e n t estimated  i n the F r a s e r mainstem i n  t o be p r e s e n t  t h e r e g i o n o f P r i n c e George a r e i l l u s t r a t e d  i n Figure 2 .  During  through t h e Thompson R i v e r , the Adams R i v e r r u n , which p r o v i d e s t o t a l commercial c a t c h o f F r a s e r sockeye the e s t i m a t e d  effluent concentrations  The  maximum  i t s passage 50-75% o f  ( G i l h o u s e n , 1960), would encounter  shown i n F i g u r e  3.  Even though a l l the k r a f t m i l l s on the F r a s e r R i v e r system p r o v i d e logical  (secondary) waste treatment, a study by Gordon and  showed t h a t t r e a t e d e f f l u e n t d i s c h a r g e d t o salmon 90% o f the time.  bio-  (1974)  acutely  a l s o found a t  ( S e r v i z i and Gordon, 1973).  components o f k r a f t m i l l waste are now and  Servizi  a t P r i n c e George was  Substandard treatment was  Kamloops m i l l on the Thompson R i v e r  the  lethal the  Some t o x i c  known to s u r v i v e b i o l o g i c a l  treatment  i n s p i t e of c o n t i n u i n g improvements i n these waste treatment systems, ....  economic f a c t o r s p r e c l u d e effluent.  the complete removal of these  Thus w h i l e the c h r o n i c d i c h a r g e  components i s l i k e l y t o c o n t i n u e , on salmon i n the r i v e r . On  little  the B.C.  t o x i c a n t s from the  of the more p e r s i s t e n t waste  i s known about t h e i r s u b l e t h a l e f f e c t s  c o a s t however, o n l y 2 o f the 11  p r a c t i c e secondary treatment o f e f f l u e n t , c o n s e q u e n t l y much h i g h e r  mills  l e v e l s of  t o x i c a n t s can be expected to r e a c h e s t u a r i n e or marine r e g i o n s u t i l i z e d  by  salmon. Because o f the extreme c o m p l e x i t y whole k r a f t m i l l e f f l u e n t , the p r e s e n t a t i o n o f a s i n g l e component.  and v a r i a b i l i t y i n the c o m p o s i t i o n study was  r e s t r i c t e d t o the  the component chosen  consider-  Although t h i s n e c e s s a r i l y represents a  s i m p l i f i c a t i o n o f the p o s s i b l e b i o l o g i c a l e f f e c t s of whole KME  i n the  ( d e h y d r o a b i e t i c acid-DHA) i s known t o be one  of  of  gross field, the  major t o x i c c o n s t i t u e n t s found i n a wide v a r i e t y o f wastes o r i g i n a t i n g from  F i g u r e 2-.  The s e a s o n a l p r e s e n c e o f sockeye salmon i n t h e F r a s e r River i n the region o f the k r a f t p u l p m i l l s a t P r i n c e George and Q u e s n e l . The maximum t h e o r e t i c a l c o n c e n t r a t i o n s o f e f f l u e n t t o which t h e s e m i g r a t i n g salmon would be exposed , were c a l c u l a t e d on t h e b a s i s o f m i l l d i s c h a r g e r e l a t i v e t o minimum mean monthly r i v e r volume flow'(1950-1970), assuming r a p i d and complete m i x i n g .  F i g u r e 3;  The s e a s o n a l p r e s e n c e o f sockeye salmon i n t h e Thompson R i v e r downstream from t h e k r a f t p u l p m i l l a t Kamloops. The maximum t h e o r e t i c a l c o n c e n t r a t i o n s o f e f f l u e n t t o which these m i g r a t i n g salmon would be exposed were c a l c u l a t e d on t h e b a s i s o f m i l l d i s c h a r g e a s d i l u t e d by t h e minimum mean monthly r i v e r volume f l o w (1911- ' 1958), assuming r a p i d and complete m i x i n g .  6  the f o r e s t p r o d u c t s i n d u s t r y and i s c u r r e n t l y b e i n g d i s c h a r g e d i n s i g n i f i c a n t amounts i n t o waters i n h a b i t e d by m i g r a t i n g salmon. determined  t h e h a l f - l i f e o f DHA  " p e r s i s t e n t " o r g a n i c compound  Brownlee e_t a_l. (1977)  t o be c l o s e t o the 8 week v a l u e d e f i n i n g a ( I n t e r n a t i o n a l J o i n t Commission, 1975)  and  s t r e s s e d the need f o r c h r o n i c e f f e c t s s t u d i e s w i t h a view o f e s t a b l i s h i n g  safe  limits. F o r t h e p r e s e n t study, a p e r s p e c t i v e of the problem r e s e a r c h e d can  be  g a i n e d by c o n s i d e r i n g the c o n f l i c t of i n t e r e s t t h a t e x i s t s i n the use o f the Fraser River.  I n essence, t h i s r i v e r  l a r g e s t producers continuous  o f sockeye  system remains one of the  salmon but a t the same time i t must a s s i m i l a t e a  i n p u t o f i n d u s t r i a l wastes from f i v e p u l p m i l l s .  a p p r e c i a b l e q u a n t i t i e s o f DHA these m i l l s ,  furthermore  world's  I t i s known t h a t  can s u r v i v e the waste treatments p r a c t i c e d  t h e r e c o n t i n u e s t o be a l a c k o f i n f o r m a t i o n on  p o s s i b l e e f f e c t s o f s u b l e t h a l DHA  exposure on salmon.  S i n c e sockeye  by the  salmon  smolts must p a s s through these waters on t h e i r downstream m i g r a t i o n , t h e i r a b i l i t y t o cope w i t h t h e normal s t r e s s e x p e r i e n c e d d u r i n g movement i n t o s a l i n e waters may  be a l t e r e d by exposure t o p u l p m i l l e f f l u e n t .  Although  a r e i g n o r a n t o f the a c t u a l b e h a v i o r o f salmon d u r i n g t h i s f r e s h water t o sea water  (SW)  t r a n s i t i o n i n the w i l d , l a b o r a t o r y experiments  h y d r o m i n e r a l b a l a n c e o f sockeye  (FW)  have shown  t h a t i t i s accompanied by changes i n water and e l e c t r o l y t e b a l a n c e . t h e p r e s e n t s t u d y i n v e s t i g a t e d the e f f e c t s o f s u b l e t h a l DHA  we  Therefore  exposure on  the  smolts i n f r e s h water and d u r i n g a d a p t a t i o n  t o sea water. The  e x p e r i m e n t a l d e s i g n was  based on t h e p r e m i s e  w i t h the mechanisms o f osmotic and  i o n i c homeostasis,  t h a t i f DHA  the n e t r e s u l t w i l l  r e f l e c t e d i n a l t e r e d l e v e l s o f the main plasma e l e c t r o l y t e s . m i n e r a l b a l a n c e was  interferes  Thus  hydro-  s t u d i e d by measurement o f plasma o s m o t i c p r e s s u r e  and  be  7  plasma sodium, potassium,  c a l c i u m , magnesium and c h l o r i d e c o n c e n t r a t i o n s i n  the b l o o d o f salmon d u r i n g DHA  exposure i n f r e s h water and a f t e r t r a n s f e r  sea water; muscle water c o n t e n t was balance.  measured as an i n d i c a t o r o f t i s s u e water  In a d d i t i o n , the gut water t r a n s p o r t a b i l i t y and the g i l l  p e r m e a b i l i t y were determined  into  water  as these organs are i n t i m a t e l y i n v o l v e d i n the  p h y s i o l o g y o f salmonid a d a p t a t i o n t o sea water.  F i n a l l y , o b s e r v a t i o n s were  made on DHA-induced changes i n salmon b e h a v i o r which may s u r v i v a l d u r i n g the t r a n s i t i o n from a  be o f importance  to  f r e s h w a t e r t o a marine e x i s t e n c e .  To keep the e x p e r i m e n t a l d e s i g n r e l e v a n t t o the n a t u r a l s i t u a t i o n , attempts  were made t o s i m u l a t e w i t h i n the c o n s t r a i n t s of a l a b o r a t o r y study,  s e v e r a l o f the c o n d i t i o n s t h a t o c c u r i n the w i l d . DHA  The  f i s h were exposed t o  w h i l e a c t i v e l y swimming f o r a p e r i o d o f time t h a t the Adams R i v e r  m i g r a t i o n i s e s t i m a t e d t o take t o r e a c h the sea t r a n s f e r i n t o sea water was  (5 d a y s ) , and  done a t a r a t e thought  smolt  subsequent  t o be r e p r e s e n t a t i v e o f the  t r a n s i t i o n from r i v e r t o the sea d u r i n g normal m i g r a t i o n . Nature o f the T o x i c a n t The  k r a f t p r o c e s s , i n essence, i n v o l v e s t h e a l k a l i n e d i g e s t i o n o f wood  c h i p s which breaks down the l i g n i n and r e l e a s e s the f i b e r s .  This i s follow-  ed by b l e a c h i n g , u s u a l l y by c h l o r i n a t i o n , t o o b t a i n the d e s i r e d b r i g h t n e s s o f the f i n a l p u l p p r o d u c t s .  During t h i s p r o c e s s a number o f n a t u r a l p r o d u c t s  s y n t h e s i z e d by the l i v i n g t r e e a r e s o l u b i l i z e d , e x t r a c t e d and washed out o f the p u l p .  Common among these are the r e s i n a c i d s and t h e i r  ( a l c o h o l s , aldehydes  and ketones-termed  " u n s a p o n i f i a b l e s " ) which t o g e t h e r  w i t h f a t t y a c i d s o c c u r i n the wood r o s i n o f c o m m e r c i a l l y Depending on t h e i r abundance, these p r o d u c t s may soap.  derivatives  important  conifers.  be r e c o v e r e d as t a l l o i l  In the U S A , r e s i n a c i d from p i n e wood t a l l o i l forms the  b a s i s o f an i n d u s t r y v a l u e d a t $138 m i l l i o n ; i n Canada, where the  lower  8  r o s i n c o n t e n t o f pulpwood and h i g h u n s a p o n i f i a b l e y i e l d p r e c l u d e r e c o v e r y , r e s i n a c i d s a r e u s u a l l y d i s c h a r g e d as wastes  economical  (Swan, 1973).  Wash  water from the b l e a c h i n g p r o c e s s y i e l d s c h l o r i n a t e d l i g n i n d e r i v a t i v e s  such  as t e t r a c h l o r o g u a i a c o l and t e t r a c h l o r o c a t e c h o l which a r e h i g h l y t o x i c t o f i s h and whose s t r u c t u r a l s i m i l a r i t y t o p e n t a c h l o r o p h e n o l  (Fig. 4),a well-  known uncoupler o f o x i d a t i v e p h o s p h o r y l a t i o n , may suggest a s i m i l a r mode o f toxic action. sockeye  S e r v i z i e t a l . (1968) observed i n c r e a s e d oxygen consumption i n  salmon exposed t o 0.1 mg/L o f t e t r a c h l o r o c a t e c h o l .  Webb and B r e t t exposed was  Davis  (1972) observed an i n c r e a s e i n oxygen requirements  t o s u b l e t h a l doses o f k r a f t m i l l wastes.  (1973) and o f salmon  Food c o n v e r s i o n e f f i c i e n c y  lowered and maintenance c o s t s e l e v a t e d by KME i n j u v e n i l e salmon i n  s t u d i e s by E l l i s  (1967) and Webb and B r e t t  (1972), w h i l e S e r v i z i e t a l . (1966) '  observed a r e d u c t i o n i n t h e e f f i c i e n c y o f y o l k u t i l i z a t i o n i n a l e v i n s o f sockeye  and p i n k salmon exposed t o 1% n e u t r a l i z e d b l e a c h waste.  the c o m p o s i t i o n o f t h e mixed waste from a k r a f t p u l p m i l l  However, as  i s extremely  v a r i a b l e and depends t o a l a r g e e x t e n t on t h e wood s p e c i e s b e i n g p u l p e d , t h e p r e c i s e c h e m i c a l s t a t e and i n t e r a c t i o n o f these compounds i n r e c e i v i n g waters  remain  l a r g e l y unknown.  A l t h o u g h r e l i a b l e gas l i q u i d chromatographic  (GLC) methods f o r i d e n t i f i -  c a t i o n o f r e s i n a c i d s were developed 20 y e a r s ago (Hudy, 1959), they were not a p p l i e d t o environmental and s t i l l Thakore,  s t u d i e s u n t i l 1968 by Maenpaa e_t al_. i n F i n l a n d ,  l a t e r i n N o r t h America 1973).  spectrometry  (NCASI 1972, 1975; Rogers,  These t e c h n i q u e s more r e c e n t l y combined w i t h mass  (GC-MS), have c o n f i r m e d t h e p r e s e n c e o f DHA i n e f f l u e n t s  a v a r i e t y of f o r e s t products processes: et a l . ,  1973; Leach and  1968; Rogers,  s u l f i t e and k r a f t m i l l s  1973; Leach and Thakore,  (Maenpaa  1973; Rogers e t a l . ,  m e c h a n i c a l p u l p i n g (Row and Cook, 1971; Leach and Thakore,  1976),  from  1975),  hardboard  9  DEHYDROABIETIC ACID  LEVOPIMARIC ACID  H0 C 2\ o  ^  CH, 3  1-ABIETIC  H0 C  NEOABIETIC ACID  OCH.  CH  o  Cl  OH PENTACHLOROPHENOL  Cl  F i g u r e 4;  0  PALUSTRIC ACID  TETRACHLOROGUAIACDL  TETRACHLOROCATECHOL  ACID  Cl  S t r u c t u r e s o f some i s o l a t e d components o f k r a f t m i l l waste t o x i c t o salmon. P e n t a c h l o r o p h e n o l i s n o t found i n KME and i t s s t r u c t u r e i s shown f o r p u r p o s e s o f comparison o n l y .  10  plants  (Row  storage  and  areas  Cook, 1971;  (Fox,  1977).  Rogers ejt al_., 1977), and  i n water from l o g  Only v e r y r e c e n t l y have t e c h n i q u e s been a v a i l a b l e  t o p r e c i s e l y q u a n t i f y t r a c e s of r e s i n a c i d s i n environmental  samples.  A k r a f t m i l l o f average s i z e and p r a c t i c i n g secondary t r e a t m e n t can 50 kg of mixed r e s i n a c i d s d a i l y because o f the h i g h ( 8 7 x 1 0 discharge  rates  ( H r u t f i o r d e t a l . , 1975), and  standards f o r t o x i c emissions. relatively l i t t l e  the r e c o v e r y  downstream from the  The the  regulatory rates,  However, Maenpaa e t a l . (1968) found 0.17  mg/L  from a k r a f t p u l p m i l l  o f r e s i n a c i d s from the F r a s e r R i v e r 70  t h r e e p u l p m i l l s a t P r i n c e George  communication) p r o v i d e s  3  subsequent b i o l o g i c a l f a t e o f r e s i n  r e s i n a c i d s i n l a k e waters a t a d i s t a n c e o f 4 m i l e s i n F i n l a n d and  m /day) e f f l u e n t  3  In s p i t e o f such h i g h d i s c h a r g e  i s known about the  a c i d s i n the environment.  s t i l l meet the  emit  (I.H. Rogers,  a d d i t i o n a l s i g n i f i c a n c e t o the p r e s e n t  miles personal  investigation.  c h e m i c a l r e a c t i o n s o f r e s i n a c i d s have formed a s i g n i f i c a n t p a r t  l a r g e r study o f t e r p e n e s i n c l a s s i c a l n a t u r a l p r o d u c t s c h e m i s t r y .  of  Some  o f the more important r e s i n a c i d s such as p a l u s t r i c , l e v o p i m a r i c ,  and  neoabietic  isomerize  ( F i g . 4) are c h e m i c a l l y  unstable  and  e i t h e r oxidize or  s p o n t a n e o u s l y t o more s t a b l e forms such as a b i e t i c and (DHA)  d u r i n g wood c h i p s t o r a g e  dehydroabietic  ( Z i n k e l , 1975), o r w i t h heat and  treatment during  the v a r i o u s p u l p i n g p r o c e s s e s  s t a b i l i t y o f DHA  can be  (Lawrence, 1959).  acid  chemical The  a t t r i b u t e d t o the p r e s e n c e o f an a r o m a t i c r i n g which  a l s o can be p r e d i c t e d t o make t h i s compound more water s o l u b l e t h a n the resin  other  acids. Recent s t u d i e s i n d i c a t e t h a t DHA  acids a f t e r discharge sources.  Simoneit  may  be the most p e r s i s t e n t o f the r e s i n  i n t o the environment, b o t h from n a t u r a l and man-made  (1977) r e p o r t e d  DHA  t o be the most common r e s i n a c i d  found i n l i p i d e x t r a c t s o f ocean sediment  samples and  s u g g e s t e d t h a t DHA  may  11  be an e x c e l l e n t n a t u r a l b i o l o g i c a l marker o f r e s i n o u s geochemical s t u d i e s . e s t a b l i s h e d DHA  higher plants  Canadian s t u d i e s i n a Lake S u p e r i o r  as b e i n g  for  ecosystem have  the most important p e r s i s t e n t o r g a n i c  contaminant  2 i n the sediment and water o f a 25 km  zone i n f l u e n c e d by the d i s c h a r g e  mixed-groundwood, k r a f t p u l p i n g p l a n t  (Brownlee and  1977).  Brownlee e t a l . (1977) e s t i m a t e t h a t 340  d a i l y i n t o N i p i g o n Bay  on Lake S u p e r i o r .  removal mechanism  (Fox,  kg of DHA  may  Disappearance due  d e g r a d a t i o n appears slow and d i l u t i o n was short-term  S t r a c h a n , 1977; be  Fox, discharged  to b a c t e r i a l  suggested as the most s i g n i f i c a n t  1977).  As r e s i n a c i d s g e n e r a l l y p o s s e s s a low aqueous s o l u b i l i t y but f r e e l y s o l u b l e i n f a t s o l v e n t s , one r e a d i l y across  the f i s h g i l l  i n t o the b l o o d  Preliminary  f a c t does happen b o t h i n the l a b o r a t o r y and Superior  d i s t a n c e o f 3 km  the  l a b o r a t o r y accumulated DHA  (Fox e t a l . , 1977)  The  studies  and  thus become  indicate that this i n  i n the f i e l d .  In the Lake  study, rainbow t r o u t exposed t o d i l u t i o n s o f whole k r a f t m i l l  e f f l u e n t s i n the water  are  would expect t h e s e compounds t o p a s s  epithelium  d i s t r i b u t e d throughout the body.  of a  and  f i s h are unknown.  The  i s o l a t e d from n a t i v e f i s h c a p t u r e d  e n t r y i n t o f r e s h water and  point  (Brownlee and  b i o l o g i c a l magnification  i n s e c t i c i d e s i s now  s i g n i f i c a n c e to migrating  20 times t h a t i n the  the b i o l o g i c a l s i g n i f i c a n c e o f DHA  delayed u n t i l l i p i d reserves  Conceivably  was  from the m i l l d i s c h a r g e  d i s t r i b u t i o n and  organochlorine  DHA  to a l e v e l  w e l l known and  are u t i l i z e d .  of l i p i d  S t r a c h a n , 1977). residues  their action i s often  a d u l t P a c i f i c salmon which cease f e e d i n g  of upon  as a major energy s o u r c e .  c h l o r i n a t e d l i g n i n d e r i v a t i v e s or r e s i n acids could  s i m i l a r pathways i n f i s h .  within  soluble  Such an a c t i o n c o u l d be  r e l y on l i p i d r e s e r v e s  at a  follow  Storage o f these t o x i c a n t s i n c e l l u l a r l i p i d s  occur w i t h r e s u l t i n g d i s r u p t i o n o f c e l l u l a r f u n c t i o n .  may  Based on widespread  12  h i s t o l o g i c a l damage i n f i s h exposed t o k r a f t m i l l waste i n f i e l d ( F u j i y a , 1961;  1965)  Warner and Tomiyama ( i n F u j i y a , 1965)  experiments  suggested  such a  p o s s i b l e mode o f a c t i o n f o r r e s i n a c i d s . An e a r l y c l u e t o the p o s s i b l e f a t e o f absorbed components o f k r a f t waste was  p r o v i d e d by Hagman (1936) who  a n a l y z e d v a r i o u s organs o f moribund  f i s h c o l l e c t e d from a r i v e r below a k r a f t m i l l . l i v e r , kidney, p a n c r e a s ,  R e s i n a c i d s were found  author  in  and b r a i n t i s s u e , w i t h h i g h e s t l e v e l s i n the  " l i q u i d which surrounds' the b r a i n i n the s k u l l c a v i t y " . c o n c e n t r a t i o n s found  mill  Although  the a c t u a l  a r e not g i v e n , the o b s e r v a t i o n i s s i g n i f i c a n t .  a l s o described a v a r i e t y of biochemical  changes which he  This  attributed  to r e s i n a c i d buildup. The  acute t o x i c i t y o f mixed r e s i n a c i d s t o a q u a t i c organisms has  known f o r many y e a r s .  Hagman (1936) and E b e l i n g  t o f i s h as b e i n g i n the 1-2  mg/L  range.  (1931) r e p o r t e d  More r e c e n t work has  been  toxicity  shown t h a t  mixed r e s i n a c i d s a r e r e s p o n s i b l e f o r much o f the r e s i d u a l t o x i c i t y i n e f f l u e n t s from the b l e a c h e d 1968)  kraft  (Rogers,  and m e c h a n i c a l p u l p i n g p r o c e s s e s  1973), s u l f i t e  (Maenpaa e t a l . ,  (Leach and Thakore, 1976).  Continuous-  f l o w b i o a s s a y s w i t h p u r i f i e d i n d i v i d u a l r e s i n a c i d s have e s t a b l i s h e d 96 h 2/ LC50's— 1975;  G.M.  f o r j u v e n i l e sockeye salmon t o be l e s s t h a n 1 mg/L Kruzynski, unpublished  data).  (Rogers e t a l . ,  Based on acute t o x i c i t y  t h e s e compounds can be c l a s s i f i e d as " h i g h l y t o x i c " contaminants 1967;  alone, (Warner  GESAMP, 1973).  S a l i n i t y Stress A l l anadromous salmonids have the c a p a c i t y o f m a i n t a i n i n g a r e l a t i v e l y c o n s t a n t b l o o d osmotic  p r e s s u r e i n b o t h f r e s h and  s a l i n e w a t e r s , depending  2/ 96 h LC50.  Concentration  l e t h a l t o 50% o f the f i s h i n 96  h.  on  13-  the  stage  i n their l i f e cycles.  D u r i n g m i g r a t i o n and  a complete r e v e r s a l i n i o n i c and  o s m o t i c r e g u l a t o r y f u n c t i o n must take  b o t h i n s m o l t s moving i n t o sea water and I n f r e s h w a t e r , o s m o t i c and with  place  i n a d u l t s e n t e r i n g f r e s h water.  i o n i c g r a d i e n t s a r e s u c h t h a t f i s h are  c o n t i n u a l endosmosis o f w a t e r and  h y p e r o s m o t i c t o the s u r r o u n d i n g  o f t e n w i t h i n a s h o r t time,  water.  l o s s o f s a l t s , as b l o o d As a r e s u l t o f t h e s e  is  faced  maintained  unavoidable  p a s s i v e movements o f e l e c t r o l y t e s , a salmon i n f r e s h w a t e r must a c t i v e l y absorb s a l t s , p r i m a r i l y Na excreted  +  and  Cl  from the w a t e r .  i n l a r g e volumes v i a the k i d n e y s  l o s s of s a l t s .  The  r e n a l and  as d i l u t e u r i n e a l o n g w i t h a  branchial loss of Na  a c t i v e i o n uptake by the g i l l s as w e l l as i n the In sea w a t e r , the freshwater  i o n i c and  Water i s c o n t i n u o u s l y  +  and  osmotic g r a d i e n t s are r e v e r s e d  s i t u a t i o n and water i s l o s t a c r o s s the g i l l s w h i l e N a  water l o s t ,  Na  +  the f i s h  Cl  by t h e g i l l s  (Maetz, 1971). and  e x c r e t e d by a p r o c e s s  The p r i m a r y r o l e s o f the k i d n e y (Mg  by  +  the  and  Cl  compensate f o r the along  i n v o l v i n g the t r a n s p o r t o f  against a concentration gradient  the e x c r e t i o n o f the d i v a l e n t i o n s  by the  To  from  swallows sea water which i s a b s o r b e d by the gut  the s a l t which must be and  i s compensated  diet.  d i f f u s e i n t o t h e b l o o d down a c o n c e n t r a t i o n g r a d i e n t .  with  Cl  slight  (active transport)  a r e the c o n s e r v a t i o n o f water  , S0 , 4  Ca  ) which a r e  absorbed  gut.  The  anadromous l i f e c y c l e o f salmon has  mechanisms t o c o u n t e r In summary  (Wood and  n e c e s s i t a t e d t h e development o f  the p a s s i v e movements o f i o n s and water i n b o t h media. R a n d a l l , 1973a) t h e s e  comprise:  a)  A c t i v e t r a n s p o r t mechanisms i n the g i l l s which can pump i o n s a g a i n s t t h e i r net d i f f u s i o n a l f l u x e s .  b)  E f f i c i e n t kidneys t o e l i m i n a t e osmotic gains i n f r e s h water and t o l i m i t water l o s s i n sea w a t e r .  c)  I n t e s t i n a l mechanisms t o r e p l a c e w a t e r l o s t t o t h e (SW) environment.  hypertonic  14  Thus a s u c c e s s f u l t r a n s i t i o n from one medium t o the o t h e r r e q u i r e s a s e r i e s o f fundamental m o d i f i c a t i o n s i n the p h y s i o l o g i c a l f u n c t i o n o f t h e s e organ systems.  P r i o r t o the c o m p l e t i o n o f t h e s e adjustments, b l o o d and  t i s s u e water and e l e c t r o l y t e l e v e l s d e p a r t s i g n i f i c a n t l y from the s t e a d y s t a t e ; t h e s e a r e t h e n r e t u r n e d t o c l o s e t o p r e - t r a n s i t i o n v a l u e s and a r e s u b s e q u e n t l y m a i n t a i n e d by a v a r i e t y o f r e g u l a t i v e f u n c t i o n s .  The p e r i o d o f  d e p a r t u r e from steady s t a t e v a l u e s t o g e t h e r w i t h the time span r e q u i r e d t o b r i n g e l e c t r o l y t e and water l e v e l s t o a new  s t e a d y s t a t e has been termed  the " a d j u s t i v e phase" w h i l e t h e maintenance o f the new the " r e g u l a t i v e phase"  (Houston, 1959a).  t o l a s t a p p r o x i m a t e l y 36 hours et a l . ,  steady s t a t e i s termed  The a d j u s t i v e phase has been shown  (h) i n coho salmon  (Smith e t a l ^ , 'i971; Conte  1966) and chum f r y (Black, 1951; Houston, 1959b) and 60-100 h i n  rainbow t r o u t Salmo g a i r d n e r i  (Conte et^ a l . ,  1966).  s a l a r ) s m o l t s r e q u i r e d 100 h t o a d j u s t plasma N a  +  A t l a n t i c salmon  back t o normal  (Koch and Evans, 1959) a l t h o u g h plasma o s m o t i c p r e s s u r e was 4 h  (Salmo  levels  adjusted i n only  (Parry, 1960). + D u r i n g t h e s e a d j u s t i v e p e r i o d s , plasma Na  + , K , Cl  -  ++ and Mg  concen-  t r a t i o n s i n c r e a s e r a p i d l y due t o s a l t i n f l u x and l o s s o f plasma water a c r o s s the g i l l s .  C o n t i n u e d d e h y d r a t i o n s t i m u l a t e s the i n g e s t i o n o f sea water,  which l e a d s t o a f u r t h e r r i s e i n plasma i o n i c l e v e l s imposing an added on the newly a c t i v a t e d b r a n c h i a l s a l t e x c r e t o r y mechanisms.  load  F i n a l l y , the  k i d n e y which has had t o s h a r p l y reduce i t s a c t i v i t y as an o r g a n o f water e x c r e t i o n and s a l t r e t e n t i o n b e g i n s t o e x c r e t e the d i v a l e n t i o n s absorbed by t h e g u t (Smith e t a l . ,  1971).  These p r o f o u n d changes i n h y d r o m i n e r a l c o n t r o l as w e l l as the d e p a r t u r e o f b o t h b l o o d and t i s s u e i o n l e v e l s from the s t e a d y s t a t e d u r i n g the a d a p t i v e phase a r e not w i t h o u t consequence.  Houston  (1959b) found t h a t the a c t i v i t y  15  and  c r u i s i n g speed o f chum salmon f r y dropped s h a r p l y upon t r a n s f e r i n t o s e a  water.  T h i s r e d u c t i o n l a s t e d approximately  correspond  36 hours and was found t o  t o i n c r e a s e s i n body c h l o r i d e and d e c r e a s e s i n body water d u r i n g t h e  a d j u s t i v e phase.  When these  l e v e l s r e t u r n e d t o the c h a r a c t e r i s t i c sea water  v a l u e s , swimming performance r e t u r n e d t o normal.  The author i n t e r p r e t e d  these r e s u l t s as an i n h i b i t i o n o f neuromuscular f u n c t i o n caused by e l e c t r o l y t e imbalance d u r i n g the a d a p t i v e  phase.  G i l l p e r m e a b i l i t y has been d e s c r i b e d as a compromise between r e s p i r a t o r y and osmoregulatory needs  (Steen and Kruysse, 1964; R a n d a l l e t a l . , 1967).  As the maintenance o f a l a r g e r e s p i r a t o r y s u r f a c e area f o r gas exchange a l s o p r o v i d e s a l a r g e s u r f a c e f o r p a s s i v e osmotic and i o n i c movements, any  f a c t o r which s e r v e s t o i n c r e a s e t h e p e r f u s i o n o f b l o o d through the  r e s p i r a t o r y l a m e l l a e c a n be expected t o i n c r e a s e these p a s s i v e movements, d i s t u r b i n g hydromineral an i n c r e a s e d N a i n f r e s h water, increased. probably  +  balance.  Wood and R a n d a l l  (1973 a,b,c.) o b s e r v e d  e f f l u x and water i n f l u x i n rainbow t r o u t d u r i n g  exercise  t o compensate f o r the osmotic water g a i n , u r i n e flows then  As t r o u t respond t o h y p o x i a by i n c r e a s i n g v e n t i l a t i o n volume and  t h e p r o p o r t i o n o f b l o o d p a s s i n g through t h e r e s p i r a t o r y l a m e l l a e  (Randall e t a l . , 1967; R a n d a l l , 1970),  concurrent  hydromineral  alterations  c o u l d t h e r e f o r e be expected as a r e s u l t o f t h e response t o hypoxic  conditions.  In a study o f the e f f e c t s o f h a n d l i n g and a n e s t h e s i a on brook t r o u t i n f r e s h water, d e c r e a s e s i n plasma and t i s s u e i o n l e v e l s were the r e s u l t s o f i n completely  compensated endosmosis and i n c r e a s e d b r a n c h i a l and r e n a l  e l e c t r o l y t e e f f l u x d e r i v e d from a p r i m a r y response t o v a s c u l a r h y p o x i a (Houston e t a l . , 1971).  16  K r a f t M i l l Waste and Hydromineral Balance Respiratory manifestations  d i s t r e s s has been shown t o be one o f the most common  o f exposure t o k r a f t p u l p m i l l wastes i n salmonids  ( A l d e r d i c e and B r e t t , 1957; Schaumburg e t a l Davis  M  (1973) has shown t h a t salmon w i l l s h a r p l y  i n response t o s u b - l e t h a l exposure t o KME.  1967; Walden e t a l . , 1970). increase  As reduced a r t e r i a l  observed, t h e f i s h would p r o b a b l y attempt t o i n c r e a s e up  oxygen was  l a m e l l a r flow t o make  f o r the oxygen demand as b l o o d i s u s u a l l y m a i n t a i n e d a t 85-95% s a t u r a t i o n  ( R a n d a l l , 1970). will and  v e n t i l a t i o n volume  Such responses a s s o c i a t e d w i t h enhancement o f gas exchange  lead to increase  of branchial  f l u x e s and r e s u l t i n a l t e r a t i o n s o f water  e l e c t r o l y t e balance. Responses such as i n c r e a s e d  v e n t i l a t i o n volume can have  t o x i c o l o g i c a l as w e l l a s . p h y s i o l o g i c a l i m p l i c a t i o n s . suggested t h a t the m a j o r i t y  o f the i n c r e a s e  the g i l l  epithelium.  (1961) has  i n t o x i c i t y o f p o i s o n s i n water  o f low d i s s o l v e d oxygen i s caused by t h e i n c r e a s e flow, w i t h a consequent i n c r e a s e  Lloyd  serious  i n the r a t e o f r e s p i r a t o r y  i n the r a t e a t which t o x i c substances r e a c h  T h i s a p p l i e s t o any e n v i r o n m e n t a l o r p h y s i o l o g i c a l  f a c t o r which i n c r e a s e s  v e n t i l a t i o n volume; a d i r e c t response o f t h e f i s h t o  oxygen d e f i c i e n c y . Changes i n g i l l p e r m e a b i l i t y  and k i d n e y f u n c t i o n can a l s o o c c u r as a  r e s u l t o f t h e d i r e c t e f f e c t o f t o x i c a n t a c t i o n , e s p e c i a l l y i f the t o x i c a n t combines w i t h membranes o r accumulates t o h i g h c o n c e n t r a t i o n s . can  l e a d t o an i n c r e a s e  across  i n the r a t e o f p a s s i v e  the b r a n c h i a l epithelium,  e l e c t r o l y t e s i n the urine.  Such changes  s a l t and water movements  o r the f i s h may become d i u r e t i c and l o s e  Subsequent d e p a r t u r e s o f plasma and t i s s u e  e l e c t r o l y t e l e v e l s from normal v a l u e s would be governed by t h e d i r e c t i o n o f osmotic and i o n i c g r a d i e n t s  between body f l u i d s and the s u r r o u n d i n g medium  17  once the h o m e o s t a t i c mechanisms were overwhelmed. Smith et_ a l .  (1971) s u g g e s t e d  t h a t any major d e c r e a s e  i n the r a t e  ++ k i d n e y f u n c t i o n c o u l d cause a c c u m u l a t i o n  o f d i v a l e n t i o n s Ca  marine salmon, o r water i n f r e s h w a t e r - a d a p t e d  stages.  of  ++ . , Mg  in  The a c c u m u l a t i o n  of  d i v a l e n t i o n s c a n i n t e r f e r e w i t h swimming performance  (Houston  function of c r i t i c a l  Preliminary observations  of  Smith et_ a l .  exposure  importance  t o m i g r a t i n g salmon.  1959b); a  (1971) showed a drop i n u r i n e p r o d u c t i o n i n salmon upon  t o l o w e r e d d i s s o l v e d oxygen c o n d i t i o n s .  In view o f the  observed  i n t e r f e r e n c e o f some k r a f t m i l l waste components w i t h r e s p i r a t i o n i n s a l m o n i d s , t h e maintenance o f water b a l a n c e may To summarize, sockeye  salmon can e n c o u n t e r  three c r i t i c a l phases of t h e i r l i f e  cycle:  sea water, e n t r y o f sea w a t e r - a d a p t e d  and  entry  subsequent  t r a n s i t i o n between h y p e r t o n i c  i n t e r f e r e n c e w i t h the optimum f u n c t i o n o f  e f f i c i e n c y o f such e c o l o g i c a l l y c r i t i c a l Thompson  and t h e r e b y reduce  these  the  a c t i v i t i e s as swimming, f e e d i n g  (1945) i n d i c a t e d t h a t a d u l t m i g r a n t  d e l a y i n g more t h a n 12 days would f a i l  into  r e g u l a t i o n by a v a r i e t y o f osmo-  systems c o u l d l e a d t o h y d r o m i n e r a l imbalance  and p r e d a t o r e v a s i o n .  The  way.  p u l p m i l l wastes d u r i n g  a d u l t s i n t o f r e s h water, and  £ h y p o t o n i c media r e q u i r e s adjustment The  in a similar  smolt m i g r a t i o n and  upstream m i g r a t i o n t o the spawning grounds.  r e g u l a t o r y systems.  be a f f e c t e d  sockeye,  t o r e a c h t h e spawning g r o u n d s .  Thus f o r  a m i g r a t i n g salmon, the n e t r e s u l t o f a s u f f i c i e n t r e d u c t i o n i n scope f o r activity  ( F r y , 1971)  by an i n d i s c r i m i n a t e s t r e s s such as p u l p m i l l  waste  c o u l d be e q u i v a l e n t t o o u t r i g h t m o r t a l i t y caused by a c u t e t o x i c i t y river  ( B r e t t , 1958). The  some e f f e c t s o f DHA,  experiments  which f o l l o w were d e s i g n e d t o  a major t o x i c component o f p u l p m i l l waste, on  h y d r o m i n e r a l b a l a n c e and r e l a t e d p h y s i o l o g y o f sockeye  i n the investigate the  salmon s m o l t s .  18  GENERAL MATERIALS AND METHODS Water Supply Experiments were conducted i n the laboratories of the P a c i f i c Environment I n s t i t u t e (PEI), West Vancouver, B.C., from 1973-1978.  Fresh water supplied  to the laboratory from a well was processed as shown i n F i g . 5. Water passed through a cartridge f i l t e r  (Cuno-Micro Kleen I I , 125 um) and then through a  bank of aspirators into a series of constant l e v e l foam-insulated header tanks equipped with r e f r i g e r a t i o n units (Frigid Units, Toledo, Ohio) where i t was vigorously aerated and c h i l l e d .  Gas supersaturation was eliminated by  means of a s t r i p p i n g column i n which a counter-current of o i l - f r e e a i r passed through a bed of glass marbles. water supply to saturation.  Air-stones were used to bring the  The chemical composition of the well water  during the study period i s shown i n Table I. The laboratory sea water supply i s drawn from a depth of 18 m i n Burrard Inlet and was also f i l t e r e d , c h i l l e d and aerated before use.  Sea water varied i n s a l i n i t y from 26-29 °/oo (^795o  890 mOsM/kg) had a pH range of 7.6-7.9 and a temperature range of 11.5 ±0.5 C. The system i l l u s t r a t e d i n F i g . 5 supplied f i l t e r e d , a i r equilibrated water to the experimental tanks at constant temperature.  The bottom header  tanks provided each annular tank (donut) with SW and FW through separate l i n e s and flowmeters (Manostat P r e d i c t a b i l i t y Flowmeter, 36-541-31) so that a change from one water supply to the other could be done separately a t each tank by closing one flowmeter and opening the other.  Thus "transfer"  experiments were conducted without disturbance of the f i s h and at the water flow rates used, (500 mL/min) 95% replacement of FW by SW was accomplished within ^ 5.8 h (Sprague, 1969).  19  1 1 2 3 4 5 6 7 8 9 10 11  VALVE CARTRIDGE FILTER ASPIRATORS HEADER TANK 1 HP REFRIGERATION UNIT OVERFLOW GAS STRIPPING COLUMN AIR SUPPLY TO SINTERED GLASS FUNNEL AIR SUPPLY TO AIRrSTONE V3 HP REFRIGERATION UNIT WATER SUPPLY TO FLOWMETERS  WELL WATER  o  t \  S  I™ —  J '  / J&N  1  6  <  11  J  TO FLOWMET ER  <  Figure 5 .  Water s u p p l y system as used f o r c o n t i n u i n g f l o w b i o a s s a y s w i t h DHA.  SEA WATER  Table I.  Chemical and 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 w e l l water used i n continuous f l o w b i o a s s a y s w i t h DHA.  Mean (Range)  mg/L  mEq/L  Na K  +  +  0.79  (0.52-1.26)  0.04  (0.02-0.05)  Ca  + +  0.78  (0.56-1.25)  Mg  + +  0.22  (0.13-0.34)  1.00  (0.71-1.71)  Cl"  Hardness mg/L CaCO^ A l k a l i n i t y mg/L CaCC>  3  C o n d u c t i v i t y umho/cm  18.2 1.43 15.6 2.68  (12.0-29.0) (0.81-2.10) (11.3-25.0) (1.60-4.11)  35.4  (25.0-60.7)  49.2  (41.0-60.1)  27.4  (26.6-28.0)  218.2  (163-302)  6.86-6.96  pH^" range o  11.5 ±0.5  Temperature  C 6.71-6.96  Range o f pH i n e x p e r i m e n t a l tank water  21  The A n n u l a r B i o a s s a y Tank  System  A l l experiments were conducted u s i n g a c o n t i n u o u s - f l o w system.  A series  o f 12 a n n u l a r f i b e r g l a s s t a n k s was s u p p o r t e d on a t w o - t i e r e d wooden frame i n a U-shaped a r r a y  ( F i g . 6) and s u p p l i e d w i t h water as shown i n F i g . 7.  tanks were a m o d i f i c a t i o n o f t h e " a n n u l a r growth Kruzynski  (1972).  Vancouver,  B.C.  chambers" d e s c r i b e d by  The mold was based on a r a d i a l t i r e  90 cm od) and was manufactured Urethane  The  i n n e r tube  (50 cm i d ,  t o s p e c i f i c a t i o n s by E v e r l a s t P l a s t i c s ,  North  foam was s p r a y e d on t h e o u t s i d e f o r i n s u l a t i o n and  the tank stood on wooden b l o c k s as i l l u s t r a t e d  i n F i g . 8, which a l s o shows  c r o s s - s e c t i o n a l view o f t h e d r a i n a r e a . D e t a i l s o f a tank module a r e shown i n composite form i n F i g . 7.  The  t o x i c a n t m e t e r i n g system i s i l l u s t r a t e d a t t h e t o p and c o n s i s t e d o f a 25 L g l a s s M a r i o t t e b o t t l e d e l i v e r i n g a c o n c e n t r a t e d s t o c k s o l u t i o n t o t h e tank through T e f l o n t u b i n g i n a b i u r e t clamp  (1.5 mm i d , 0.4 mm w a l l ) s u p p o r t e d by g l a s s t u b i n g  (Leduc, 1966).  a w e l l water f l o w o f 500 mL/min.  A t o x i c a n t f l o w o f 3 mL/min was mixed The bottom  of F i g . 7 i l l u s t r a t e s  held with  the r e -  c i r c u l a t i n g pump c o n n e c t i o n t o t h e s t a n d p i p e / w a t e r d r i v e u n i t , t h e waste t u b i n g i n t h e waste c o l l e c t i n g p o s i t i o n , ened s c r e e n p o r t i o n  (right).  and f i s h  A water pump  water a t ^ 25L/min and p r o v i d e d a water  s c h o o l i n g under t h e d a r k -  (March MDX-3) r e c i r c u l a t e d t h e  c u r r e n t around t h e tank w i t h a  v e l o c i t y determined by t h e s t a n d p i p e / w a t e r p r o p u l s i o n u n i t used. ments i n which f i s h were f r e e swimming, t h e s t a n d p i p e u n i t p r o v i d e d a c u r r e n t o f ^15 cm/sec w i t h t h e 70 L tank f i l l e d In t h e g i l l p e r m e a b i l i t y experiments  trap  (p.83) i n which  For experi-  shown i n F i g . 8b t o a volume o f 58L.  f i s h were r e s t r i c t e d t o  tubes p l a c e d w i t h i n t h e t a n k , a m o d i f i e d u n i t i l l u s t r a t e d i n F i g . 8c was used t o m a i n t a i n adequate water movement a g a i n s t t h e added r e s i s t a n c e o f t h e t u b e s .  F i g u r e 6.  I l l u s t r a t i o n o f the arrangement of the donut tanks f o r continuous f l o w b i o a s s a y s w i t h DHA.  used  23  .  ^  From water supply  0  Figure  7"".  10cm  A s e c t i o n o f two donut tanks showing d e t a i l s o f the water s u p p l y and the DHA m e t e r i n g system.  25  The r e c i r c u l a t i n g pump was suspended from a n y l o n c o r d a c r o s s the i n t e r n a l diameter o f t h e tank t o e l i m i n a t e v i b r a t i o n .  Each tank was equipped w i t h an  a i r - s t o n e t o m a i n t a i n d i s s o l v e d oxygen l e v e l s above 90% s a t u r a t i o n a f t e r i t appeared t h a t t h e t o x i c a n t i n c r e a s e d  consumption i n exposed  fish.  Covers made o f f i b e r g l a s s mosquito s c r e e n i n g k e p t f i s h from jumping o u t , w h i l e a p i e c e o f b l a c k p l a s t i c p r o v i d e d a shaded a r e a . p o s i t i o n a g a i n s t t h e c u r r e n t under t h i s c o v e r e d t i g h t l y when d i s t u r b e d .  Salmon  a r e a and t h e n  A t f e e d i n g , p e l l e t s o f OMP  maintained schooled  (Oregon M o i s t  Pellet)  were dropped j u s t downstream from t h e stand p i p e o u t l e t and were q u i c k l y consumed by t h e f i s h as t h e p e l l e t s were c a r r i e d around the tank by the current.  The tank i n c o r p o r a t e d a waste t r a p  ( F i g . 8a) which c o l l e c t e d un-  eaten f o o d and f e c e s d r i f t i n g around the i n t e r i o r c i r c u m f e r e n c e wall.  o f the tank  The t r a p was d r a i n e d by a l e n g t h o f Tygon t u b i n g which was kept a t  water l e v e l f o r c o l l e c t i o n accumulated waste.  ( F i g . 7) and lowered t o t h e d r a i n t o siphon t h e  T h i s was done immediately  b e f o r e t h e d a i l y morning f e e d -  i n g t o d r a i n waste accumulated o v e r n i g h t and about f i v e minutes a f t e r i n g t o remove r e m a i n i n g Adjacent  food.  tank modules were s e p a r a t e d by b l a c k p l a s t i c c u r t a i n s t o  prevent v i s u a l disturbance o f the f i s h . 20W f l u o r e s c e n t l i g h t  30-50  Each module was equipped w i t h a  (Duro T e s t V i t a L i t e ) p r o v i d i n g an i l l u m i n a t i o n o f  l x a t t h e water s u r f a c e .  Natural photoperiod  was m a i n t a i n e d  l i g h t s e n s o r mounted on t h e r o o f o f t h e l a b o r a t o r y b u i l d i n g . descent  by a  An i n c a n -  l i g h t b u l b was l e f t on 24 h a day t o p r o v i d e a dim l i g h t s i m u l a t i n g  n i g h t - t i m e outdoor l i g h t i n g c o n d i t i o n s and t o f a c i l i t a t e No m e t a l except supply.  feed-  observations.  f o r s t a i n l e s s s t e e l came i n t o c o n t a c t w i t h t h e water  Header tanks were o f p o l y e t h y l e n e  (Nalgene),  p i p i n g was o f PVC and  a l l t u b i n g d e l i v e r i n g water t o t h e g l a s s f l o w meters was Tygon.  The  r e f r i g e r a t i o n u n i t s had T e f l o n c o a t e d components.  The magnetic d r i v e pump  housings were p o l y p r o p y l e n e and l a t e x rubber t u b i n g s u p p l i e d water t o t h e PVC  stand p i p e u n i t s .  The tanks were epoxy r e i n f o r c e d f i b e r g l a s s w h i l e the  t o x i c a n t was d e l i v e r e d from g l a s s b o t t l e s through T e f l o n t u b i n g . F i s h Holding Sockeye  salmon s t o c k s were k e p t i n t h e outdoor f i s h - h o l d i n g  a t t h e P a c i f i c Environment  Institute.  facility  As f i s h were o b t a i n e d from a v a r i e t y  o f sources and a t v a r i o u s s t a g e s o f development, M a t e r i a l s and Methods s e c t i o n o f each experiment. were o f f i b e r g l a s s c o n s t r u c t i o n , measuring  d e t a i l s are given i n the The outdoor h o l d i n g tanks  3.1 x 1.2 m and were f i l l e d t o a  volume o f 4000 L w i t h w e l l water whose c h e m i c a l c h a r a c t e r i s t i c s have been described i n Table I.  Water temperatures ranged s e a s o n a l l y from 10-12°C and  the f i s h were f e d t w i c e d a i l y on a d i e t o f Oregon M o i s t P e l l e t s . Fish Transfer F i s h were d i p - n e t t e d from t h e o u t s i d e h o l d i n g tanks i n groups  o f ^ 20  and t r a n s f e r r e d i n t o a p o l y e t h y l e n e bucket c o n t a i n i n g 33 mg/L MS-222 o ( T r i c a i n e methanesulfonate, Sandoz) d i s s o l v e d i n water o f s a l i n i t y a t 11°C.  10 /oo  F i s h were then p l a c e d i n t o t h e l a b o r a t o r y donut tanks where t h e y  were h e l d f o r a minimum o f 96 h f o r a c c l i m a t i z a t i o n t o the f l o w i n g  water  c o n d i t i o n s i n the l a b o r a t o r y p r i o r t o t h e s t a r t o f an experiment.  As t h e  a n e s t h e s i a was v e r y l i g h t , f i s h were swimming n o r m a l l y and s c h o o l i n g  under  cover w i t h i n 5 min and would f e e d w i t h i n 1 h o f i n t r o d u c t i o n i n t o t h e tank. F e e d i n g was done once d a i l y w i t h OMP and was d i s c o n t i n u e d 24 h p r i o r t o t h e s t a r t o f experiments. A n e s t h e s i a and B l o o d  Sampling  F i s h were a n e s t h e t i z e d i n two s t a g e s .  A l i g h t a n e s t h e s i a was brought  on by t h e a d d i t i o n o f 2g o f MS-222 t o t h e tank a f t e r s h u t t i n g o f f the water  27  supply and adding an a i r - s t o n e l i g h t l y b u b b l i n g  i n t o t h e water.  The  a n e s t h e t i c was d i s s o l v e d i n I L SW and then s l o w l y added t o t h e tank where thorough m i x i n g was ensured  by t h e r e c i r c u l a t i n g pump.  As soon as t h e f i s h began t o d r i f t w i t h t h e c u r r e n t , two f i s h a t a time were d i p - n e t t e d i n t o a p o l y e t h y l e n e bucket for  rapid terminal anesthesia.  T h i s s o l u t i o n was p r e p a r e d by t h e a d d i t i o n o f  1.2 g MS-222 t o an oxygenated m i x t u r e of  10-12 °/oo a t a pH ^6.3.  hypoxia  c o n t a i n i n g 200 mg/L MS-222  o f 3L SW and 4L FW, y i e l d i n g a  T h i s method ensured  rapid anesthesia,  and was meant t o minimize t h e osmoregulatory  b u f f e r e d MS-222 a n e s t h e s i a  minimized  s t r e s s i n v o l v e d i n un-  (Wedemeyer, 1970) and t h e p h y s i o l o g i c a l  caused by t h e h a n d l i n g o f f i s h i n s o f t water  salinity  stress  (Wedemeyer, 1972).  A f t e r i m m o b i l i z a t i o n , t h e f i s h were r i n s e d w i t h d e i o n i z e d water, measured t o t h e n e a r e s t mm, b l o t t e d and weighed t o t h e n e a r e s t 10 mg on a M e t t l e r P1200 o r t o 100 mg on a PS1200 t o p l o a d i n g b a l a n c e . peduncle  was severed and b l o o d was c o l l e c t e d i n t o N a t e l s o n h e p a r i n i z e d  c a p i l l a r y tubes. chilled  The c a u d a l  The tubes c o n t a i n e d 6 USP ammonium h e p a r i n and were  on i c e p r i o r t o b l o o d c o l l e c t i o n .  Depending on t h e s i z e o f each  f i s h , from 200-500 uL o f b l o o d was c o l l e c t e d and t h e tubes were s e a l e d w i t h C r i t o c a p s (Sherwood M e d i c a l I n d u s t r i e s ) and s t o r e d on i c e u n t i l a l l the f i s h had been p r o c e s s e d . The  tubes were p l a c e d i n c h i l l e d  balsa-wood l i n e r s t o minimize warming  and c e n t r i f u g a t e d a t 1300G f o r 20 minutes i n a DAMON/IEC Model CS centrifuge.  A t o t h e r t i m e s , b l o o d was c o l l e c t e d i n m i c r o h e m a t o c r i t  and c e n t r i f u g a t e d i n an IEC m i c r o h e m a t o c r i t minutes a t 13,000G.  tubes  c e n t r i f u g e Model MB f o r 3  The h e m a t o c r i t was r e c o r d e d , plasma was s e p a r a t e d ,  t r a n s f e r r e d t o 2 mL d i s p o s a b l e c o n i c a l p o l y s t y r e n e sample cups  (Technicon  Auto A n a l y z e r ) and a n a l y z e d immediately o r s t o r e d In  frozen.  cases where t h e water c o n t e n t o f t h e f i s h was determined,  c a r c a s s was b l o t t e d d r y w i t h an absorbent wiper, wet-weighed  each  i n a tared  aluminum d i s h and d r i e d i n a f o r c e d a i r oven a t 110°C t o c o n s t a n t weight. In the  cases where t h e "muscle" water f i s h was used.  c o n t e n t was determined, a c r o s s - s e c t i o n o f  A f t e r t h e c a u d a l peduncle had been t r a n s e c t e d and the  b l o o d sampling completed, p o s t e r i o r t o the v e n t .  a second t r a n s v e r s e c u t was made immediately  T h i s s e c t i o n c o n s i s t e d p r i m a r i l y o f muscle b u t  i n c l u d e d t h e a n a l and a d i p o s e f i n , a s e c t i o n o f t h e v e r t e b r a l column as w e l l as t h e s k i n and s c a l e s . above.  T h i s p r e p a r a t i o n was then d r i e d as d e s c r i b e d  The % water c a l c u l a t e d was termed  "muscle"  water.  Blood E l e c t r o l y t e Determination Plasma c h l o r i d e was measured on a B u c h l e r - C o t l o v e D i r e c t Chloridometer  Reading  ( B u c h l e r Instruments D i v i s i o n , N u c l e a r - C h i c a g o , N.J.)  adapted f o r 10 uL samples w i t h a r h e o s t a t p r o v i d e d by t h e manufacturer. A v a r i a b l e volume B u c h l e r M i c r o p i p e t f i t t e d w i t h a s h o r t l e n g t h o f PE-90 t u b i n g was used t o t r a n s f e r the plasma  sample t o t h e i n s t r u m e n t and de-  i o n i z e d water was used as t h e wash-out s o l v e n t .  The C h l o r i d o m e t e r was  c a l i b r a t e d u s i n g NaCl s o l u t i o n s f o l l o w i n g manufacturer's i n s t r u c t i o n s and r e a d d i r e c t l y i n mEq/L C l . Plasma o s m o l a l i t y was determined on a 1:1 d i l u t i o n w i t h d e i o n i z e d water.  U s i n g an Eppendorf M i c r o p i p e t , a 100 uL a l i q u o t o f plasma was  t r a n s f e r r e d t o an osmometer v i a l f o l l o w e d by 100 uL d e i o n i z e d water u s i n g the  same p i p e t t e t i p .  An Osmette-S-Semi-Automatic Osmometer  (Precision  Systems I n c . , Mass.) c a l i b r a t e d w i t h manufacturer's s t a n d a r d s and o p e r a t e d i n t h e small-sample osmolality  (200 uL) mode, was used t o determine  (milliosmol  (mOsm)/kg w a t e r ) .  plasma  A f t e r a d e t e r m i n a t i o n , t h e sample  was thawed and 100 uL of the mixture was transferred with an Eppendorf Micropipet to a 4 ml conical sample cup containing 2 mL 0.25% strontium chloride (SrCl > dispensed with an Oxford Pipettor Model R, then capped 2  and mixed on a vortex s t i r r e r p r i o r to cation analysis by Atomic Absorption Spectrophotometry  (AAS).  In cases where there was not enough plasma for  osmometry, 50 yL of undiluted plasma was added d i r e c t l y to the SrCl^ solution.  Strontium chloride was used according to the method of Paschen  and Fuchs (1971) f o r suppression of anionic interferences during plasma analysis by AAS.  A single d i l u t i o n sufficed for analysis of the four  + + ++ ++ cations Na , K , Ca , Mg Plasma cations were analyzed on a Perkin Elmer Atomic Absorption Spectrophotometer Ca  , Mg  Model 403, u t i l i z i n g an air/acetylene flame.  +  +  were analyzed at wavelengths (nm) 330 UV, 385 VIS., 211 VIS., and  285 UV respectively.  A standard solution containing (in mEq/L) 140 Na , +  5.0 K , 5.0 C a , and 1.97 M g +  Na , K ,  + +  ++  was prepared according to methods described  i n the instrument manual and then d i l u t e d i n a fashion i d e n t i c a l to the unknown samples.  Absorbance was read and the concentration of the various  ions was calculated and expressed i n mEq/L.  Periodic checks of instrument  performance were made using Hyland I and II and Dade Lab-trol and Patho-trol Chemistry Control Sera. Preparation of DHA f o r F i s h Bioassays Dehydroabietic acid  (DHA) was prepared by the method of Halbrook and  Lawrence (1966) to a p u r i t y of 95.7% as determined by gas l i q u i d chromatography (GLC).  A concentrated stock solution was made by d i s s o l v i n g  the required amount of DHA i n 100 mL ethyl alcohol, adding 2.5 mL 5N NaOH followed by 100 mL d i s t i l l e d water.  Light s t i r r i n g with a magnetic s t i r bar  and slow a d d i t i o n o f t h e water ensured complete  solution.  The m i x t u r e was  then s l o w l y added t o t h e M a r i o t t e b o t t l e c o n t a i n i n g a p p r o x i m a t e l y 20 L d i s t i l l e d water and 2.5 mL 5N NaOH, a g a i n s t i r r i n g concentrated stock s o l u t i o n d i s t i l l e d water. pump  This  100-200 mg/L) was then made up t o 25 L w i t h  The b o t t l e was then connected t o a w a t e r - o p e r a t e d vacuum  ( a s p i r a t o r ) f o r 10-15 min.  vigorous s t i r r i n g  continuously.  T h i s e v a c u a t i o n procedure combined w i t h  e f f e c t i v e l y de-gassed t h e s o l u t i o n .  As t h e M a r i o t t e  b o t t l e system o p e r a t e s under p a r t i a l vacuum, t h i s procedure subsequent problems  eliminated  a r i s i n g from t h e f o r m a t i o n o f m i c r o b u b b l e s which would  c o a l e s c e i n t h e f i n e bore o f t h e t o x i c a n t d e l i v e r y t u b i n g d i s r u p t i n g t h e flow.  U s i n g t h i s d e g a s s i n g p r o c e d u r e , t h e t o x i c a n t f l o w s when once  e s t a b l i s h e d , r e q u i r e d l i t t l e o r no a board a t t h e c e n t e r o f t h e donut  adjustment.  The b o t t l e was p l a c e d on  ( F i g . 7) and a t a f l o w r a t e o f 3 mL/min  p r o v i d e d t o x i c a n t f o r t h e d u r a t i o n o f t h e 120 h exposure p e r i o d w i t h no further disturbance t o the f i s h . (by  I n p r e l i m i n a r y experiments, measurements  GLC) o f t h e c o n c e n t r a t i o n s o f DHA a c t u a l l y p r e s e n t i n t h e water showed  t h a t ^ 90%  o f t h e t h e o r e t i c a l dosage had been a t t a i n e d  (Appendix 1 ) .  31  SYNOPSIS OF STUDIES ON DHA  D u r i n g p r e l i m i n a r y experiments t o e s t a b l i s h t h e a c u t e t o x i c i t y o f DHA t o sockeye salmon i t became n e c e s s a r y t o develop c h e m i c a l methods t o determine the  a c t u a l c o n c e n t r a t i o n s o f DHA p r e s e n t i n t h e water.  These methods (the  e x t r a c t i o n o f DHA from t h e water and i t s q u a n t i f i c a t i o n by g a s - l i q u i d chroma tography  (GLC)) were used t o r e f i n e DHA s o l u b i l i z a t i o n t e c h n i q u e s and  subsequently t o m o n i t o r DHA c o n c e n t r a t i o n s i n f l o w - t h r o u g h b i o a s s a y s .  When  i t became c o n f i r m e d t h a t f i s h were removing DHA from t h e water d u r i n g b i o a s s a y s , e x t r a c t i o n t e c h n i q u e s were a l s o developed t o measure DHA r e s i d u e s i n f i s h t i s s u e and f i n a l l y i n f i s h f o o d organisms.  In a l l , f i v e  experiments  were done and t h e main f i n d i n g s a r e summarized below; t h e d e t a i l s a r e g i v e n i n Appendix I . The s u r v i v a l o f f i s h i n what s h o u l d have been an a c u t e l y t o x i c  con-  c e n t r a t i o n o f DHA d u r i n g a s t a t i c b i o a s s a y was due t o a r a p i d r e d u c t i o n o f the  a c t u a l amount o f DHA p r e s e n t i n t h e water  (Appendix 1-1).  t h i s was l a r g e l y a t t r i b u t a b l e t o t h e p r e s e n c e o f f i s h , the  w a l l s o f t h e t e s t aquarium was a l s o i n d i c a t e d .  Although  some a d s o r p t i o n onto  On t h e b a s i s o f t h e s e  r e s u l t s , a l l subsequent experiments were conducted under c o n t i n u o u s f l o w c o n d i t i o n s e x c e e d i n g t h e t o x i c a n t / w a t e r replacement g u i d e l i n e s g i v e n i n Sprague  (1969).  I n a d d i t i o n , a check o f a c t u a l DHA c o n c e n t r a t i o n s p r e s e n t  i n t h e water d u r i n g t h e s e b i o a s s a y s showed t h a t 90-95% o f t h e t h e o r e t i c a l dose was m a i n t a i n e d d u r i n g t h e d u r a t i o n o f t h e exposure p e r i o d . When DHA was p r e p a r e d and mixed w i t h w e l l water i n t h e form used f o r c o n t i n u o u s - f l o w b i o a s s a y s (as t h e sodium s a l t ) , i t s r e c o v e r y from t h e water  (Appendix I ~ 2 ) .  f i l t r a t i o n had no e f f e c t on  T h i s experiment showed t h a t t h e  r e s i n a c i d was i n s o l u t i o n , and as such s h o u l d be a v a i l a b l e t o t h e f i s h .  32  Another experiment  (Appendix 1-3) determined t h e d i r e c t aqueous s o l u b i l i t y o f  DHA t o be 3.3 mg/L, i n d i c a t i n g t h a t t h e f r e e a c i d can d i s s o l v e d i r e c t l y i n the water t o c o n c e n t r a t i o n s e x c e e d i n g those found t o be a c u t e l y t o x i c t o salmonids. As p r e l i m i n a r y s t u d i e s had shown t h a t DHA was taken up by salmon  during  s u b l e t h a l exposure i n f r e s h water, an experiment was conducted t o determine whether  a c c u m u l a t i o n i n t h e body d i d o c c u r and i f so, t o determine t h e t i s s u e  d i s t r i b u t i o n o f the toxicant  (Appendix 1-4).  The r e s u l t s c o n f i r m e d t h a t DHA  was taken up by f i s h and accumulated t o a l e v e l 30 times h i g h e r than t h a t a v a i l a b l e i n t h e water.  Much h i g h e r b i o c o n c e n t r a t i o n was measured i n  i n d i v i d u a l organs such as the b r a i n  (954 x ) , k i d n e y (428 x ) , l i v e r  the b i l e c o n t a i n e d t h e h i g h e s t o v e r a l l c o n c e n t r a t i o n o f DHA chromatography  (404 x ) ;  (996 x ) .  Gas  c o u p l e d w i t h mass s p e c t r o m e t r y (GC-MS) was used t o d e t e c t  s e v e r a l m e t a b o l i c d e r i v a t i v e s o f t h e p a r e n t DHA m o l e c u l e i n t h e b i l e ,  indicat-  i n g t h a t t h e h e p a t o b i l i a r y r o u t e i s i n v o l v e d i n DHA e x c r e t i o n i n sockeye salmon. The exposure o f a r e p r e s e n t a t i v e f i s h f o o d organism  (the amphipod  Anisogammarus c o n f e r v i c o l u s ) t o DHA r e s u l t e d i n a b i o c o n c e n t r a t i o n o f 21 x t h a t p r e s e n t i n t h e water  (Appendix 1-5).  These r e s u l t s i n d i c a t e t h a t  salmon  may accumulate DHA through t h e f o o d c h a i n as w e l l as d i r e c t l y from t h e water. The  D i s c u s s i o n w i l l go i n t o t h e b i o l o g i c a l s i g n i f i c a n c e o f t h e s e h i g h  DHA r e s i d u e s i n r e l a t i o n t o f e e d i n g b e h a v i o r and p h y s i o l o g i c a l f u n c t i o n o f the  salmon. T h i s c o n c l u d e s t h e summary o f s t u d i e s which were done on t h e t o x i c a n t  and t h e next s e c t i o n w i l l c o v e r experiments which i n v e s t i g a t e d t h e d i r e c t e f f e c t s o f DHA exposure on sockeye  salmon.  PART I . A.  PRELIMINARY EXPERIMENTS  ACUTE TOXICITY OF DHA TO JUVENILE SOCKEYE SALMON INTRODUCTION The acute t o x i c i t y o f DHA t o sockeye  salmon was determined  on t h r e e  s e p a r a t e o c c a s i o n s under c o n t i n u o u s - f l o w c o n d i t i o n s w i t h t h e purpose o f e s t a b l i s h i n g t h e 96 h LC50  i n f r e s h water.  The t o x i c i t y c u r v e s thus  generated were then used t o e s t i m a t e a c o n c e n t r a t i o n o f DHA which would cause n e g l i g i b l e m o r t a l i t y d u r i n g t h e subsequent b a l a n c e experiments.  sublethal  electrolyte  Of t h e t h r e e acute b i o a s s a y s , t h e f i r s t  i n March 1974  (Expt. 74) u t i l i z e d a broad range o f 6 c o n c e n t r a t i o n s (0.47 t o 3.13 mg/L DHA) w h i l e t h e second i n March 1976 (Expt. 76) and t h i r d i n A p r i l 1977 (Expt. 77) employed a more r e s t r i c t e d range o f c o n c e n t r a t i o n s which was expected t o b r a c k e t t h e 96 h LC50. During t h e course o f acute b i o a s s a y s , salmon appeared  t o be under a  r e s p i r a t o r y s t r e s s , as m a n i f e s t e d by f r e q u e n t coughing and v e n t i l a t o r y changes.  Subsequent work showed t h a t an e l e v a t i o n o f h e m a t o c r i t o c c u r r e d  during sublethal hypoxia  exposure  t o DHA, a response t h a t i s known t o o c c u r d u r i n g  (Doudoroff and Shumway, 1970).  I f DHA was i n t e r f e r i n g w i t h normal  gas exchange o r w i t h t h e t r a n s p o r t o f oxygen by t h e b l o o d , then a l o w e r i n g o f d i s s o l v e d oxygen l e v e l s i n t h e water c o u l d i n c r e a s e t h e t o x i c i t y o f t h e resin acid.  To t e s t i f t h i s was t h e case, an experiment  was conducted on  two groups o f salmon smolts exposed t o a n o r m a l l y s u b l e t h a l exposure In one group,  d i s s o l v e d oxygen  (D.O.) l e v e l s were m a i n t a i n e d a t 'v* 75%  s a t u r a t i o n , w h i l e i n t h e o t h e r , d i s s o l v e d oxygen was m a i n t a i n e d ^ 90% saturation.  Both groups o f f i s h were exposed t o a n o r m a l l y  c o n c e n t r a t i o n o f DHA (0.65 mg/L) f o r 120 h i n f r e s h water.  sublethal  t o DHA.  MATERIALS AND  METHODS  The sockeye salmon used i n a l l t h r e e acute b i o a s s a y s were o f the C u l t u s Lake s t o c k .  Expt. 74 was  conducted w i t h f i s h which had been r a i s e d  a t PEI from eggs o b t a i n e d from C u l t u s Lake i n November 1973. were o b t a i n e d as smolts i n January 1976  The 1976  fish  and had been k e p t a t PEI f o r 2  months p r i o r t o the experiment, w h i l e the 1977  f i s h were o b t a i n e d as y e a r -  l i n g s and had been a t PEI f o r 5 months p r i o r t o use.  A t the time o f the  acute b i o a s s a y s a l l f i s h were 17-18 months o l d and had the  silvery  c o l o r a t i o n c h a r a c t e r i s t i c o f sockeye s m o l t s . A f t e r t r a n s f e r from outdoor h o l d i n g tanks a c c o r d i n g t o p r o c e d u r e s d e s c r i b e d i n G e n e r a l Methods, the f i s h were g i v e n 48 h t o a c c l i m a t i z e t o the  l a b o r a t o r y tanks b e f o r e the t o x i c a n t exposure was  started.  In Expt.  the  f i s h had been i n the l a b o r a t o r y tanks f o r 2 months p r i o r t o the b i o a s s a y .  F e e d i n g was d i s c o n t i n u e d 48 h p r i o r t o the s t a r t o f the exposure and were not f e d d u r i n g the experiments. given i n Table I I .  One  74,  fish  T e s t c o n d i t i o n s and f i s h s i z e are  c o n t r o l tank was used i n each experiment  and  r e c e i v e d the s o l v e n t c a r r i e r a t the same r a t e as the t e s t tanks but no  DHA.  Continuous o b s e r v a t i o n s were made d u r i n g the day w h i l e a t n i g h t , m o r t a l i t i e s were r e c o r d e d a t a p p r o x i m a t e l y 4 h i n t e r v a l s . absence o f a l l movement upon h a n d l i n g . f i s h was measured t o the n e a r e s t mm the  Death was  judged by the  The time t o death was  r e c o r d e d , the  ( f o r k l e n g t h ) , b l o t t e d and weighed  to  n e a r e s t 10 mg on a t o p - l o a d i n g b a l a n c e . Log p r o b i t paper was  used t o p l o t c u m u l a t i v e % m o r t a l i t y a g a i n s t time  and the TL50 (time t o 50% m o r t a l i t y ) f o r each c o n c e n t r a t i o n was graphically  (Litchfield,  1949).  determined  Acute t o x i c i t y c u r v e s were then p l o t t e d  u s i n g TL50's v s c o n c e n t r a t i o n on l o g - l o g paper and the 96 h LC50's were  T a b l e I I . F i s h s i z e and acute b i o a s s a y o p e r a t i n g parameters.  Experiment  Fish Size Mean ±SE(n) Fork length Wet weight cm g  Water Characteristics (range)  Number per tank  Loading Temperature density °C L/g fish/day  pH  Dissolved oxygen % 2.  DHA mg/L , Concentrations Estimate of tested 96h LC50  74  11.7 +0.06(141)1 17. 74 ±0.26  20  2.03  11.2 ±0.3  6.32 - 6.40  70-8S  3.13, 1.92, 1.65, 1.16, 0.61, 0.47  0.50  76  12.0 ±0.15( 44)17.97 ±0.52  12  3. 34  10.5 ±0.5  7.04 - 7.13  90-95  1.00, 0.87, 0.65  0.79  77  13.0 ±0.15( 59)127.06 +0.93  15  1. 79  11.2 ±0.5  6.80 " 6.B7  90-95  1.29, 1.00, 0.79, 0.63  0. 88  1 By i n t e r p o l a t i o n from t o x i c i t y  curve.  d i s s o l v e d oxygen dropped t o 70% d u r i n g t h e f i r s t  24 h o f exposure and was r e t u r n e d by s u p p l e m e n t a l a e r a t i o n  to >85% f o r t h e r e m a i n i n g 96 h.  Cn  36  e s t i m a t e d g r a p h i c a l l y by i n t e r p o l a t i o n from t h e t o x i c i t y c u r v e s .  The  absence o f p a r t i a l m o r t a l i t i e s a t 96 h p r e c l u d e d t h e c a l c u l a t i o n o f a more p r e c i s e LC50 e s t i m a t e and c o n f i d e n c e l i m i t s as recommended by Sprague  (1969).  In Expt. 74 t h e t o x i c a n t exposure p e r i o d extended t o 170 h w h i l e i n E x p t s . 76 and 77 t o x i c a n t f l o w was d i s c o n t i n u e d a f t e r 120 h.  At the  c o n c l u s i o n o f t h e exposure i n Expt. 77 t h e water s u p p l y t o the 0.63 tank was s w i t c h e d from FW t o SW t o determine whether. f i s h would t o l e r a t e t h e a d d i t i o n a l osmotic s t r e s s .  mg/L  The s u r v i v i n g DHA-exposed  T h i s r e s u l t would have t o  be taken i n t o c o n s i d e r a t i o n when c h o o s i n g a " s u b l e t h a l " exposure regimen f o r subsequent e l e c t r o l y t e  studies.  F o r t h e h y p o x i a experiment, sockeye salmon the G r e a t C e n t r a l Lake  s m o l t s were o b t a i n e d from  (Vancouver I s l a n d ) r u n a t t h e b e g i n n i n g o f March 1978.  A f t e r 2-1/2 months i n t h e outdoor h o l d i n g t a n k s a t P E I , 21 f i s h were t r a n s f e r r e d t o each o f 2 l a b o r a t o r y tanks by s t a n d a r d i z e d methods p r e v i o u s l y d e s c r i b e d where they were k e p t f o r 1 week p r i o r t o t h e experiment.  Well  water temperature was m a i n t a i n e d a t 11.4 ±0.1 (X ±SE) d u r i n g t h e 5-day exposure p e r i o d t o 0.65 mg/L DHA.  A f t e r a 24 h s t a r v a t i o n p e r i o d , one  tank was s w i t c h e d t o h y p o x i c water  (^ 75% s a t u r a t i o n ) and t h e f i s h were  g i v e n an a d d i t i o n a l 24 h a c c l i m a t i o n t o t h e s e c o n d i t i o n s b e f o r e t h e t o x i c a n t exposure was s t a r t e d .  A f t e r 120 h, t h e t o x i c a n t exposure was  d i s c o n t i n u e d , and t h e normoxic tank  (90-95% s a t u r a t i o n ) was switched t o  hypoxic water t o i n v e s t i g a t e t h e p o s s i b i l i t y o f any l a t e n t s y n e r g i s t i c  toxicity.  F i s h s i z e d u r i n g t h i s experiment was 13.7 ±0.17 cm and 26.3 ±1.09 g (X ± S E ) .  37  RESULTS AND Behavioral  DISCUSSION  Symptoms  Frequent o b s e r v a t i o n s  of f i s h d u r i n g the course o f the acute  made i t p o s s i b l e t o d e t e c t c e r t a i n b e h a v i o r a l changes induced exposure.  The  rate of progression  t o be dose dependent. i n a s c h o o l which was a t most about 1/6th  bioassays  by the  o f these b e h a v i o r a l a l t e r a t i o n s appeared  C o n t r o l f i s h m a i n t a i n e d p o s i t i o n a g a i n s t the centered  toxicant  under the shaded area o f the tank,  o f the c i r c u m f e r e n c e  s c h o o l would immediately t i g h t e n up  o f the donut.  current  covering  If disturbed,  so t h a t a l l the f i s h were under  A t n i g h t , under the c o n t i n u o u s dim  i l l u m i n a t i o n provided,  s c h o o l expanded t o c o v e r about 1/2  o f the c i r c u m f e r e n c e  the  cover.  the s i z e o f  the  with i n d i v i d u a l  f i s h o c c a s s i o n a l l y t u r n i n g t o swim w i t h the c u r r e n t but r a r e l y f o r more than one  circuit. In f i s h exposed t o DHA,  the f i r s t b e h a v i o r a l  symptom t o be observed  a r e d u c t i o n i n the compactness o f the s c h o o l r e s u l t i n g i n a g r a d u a l i n the s e c t o r o c c u p i e d  by the salmon.  ^ 20 h i n f i s h exposed t o 1 mg/L v i s u a l disturbance eliminated.  DHA.  until  i t was  totally  At t h i s time a tap on the tank r e s u l t e d i n a somewhat confused s c h o o l , but the f i s h appeared t o be  problems w i t h muscular c o o r d i n a t i o n . longer maintain  stream.  in  normal c o v e r response upon  became p r o g r e s s i v e l y d i m i n i s h e d  e f f o r t t o a c c e l e r a t e forward and  no  increase  T h i s s c h o o l i n g breakup o c c u r r e d The  was  At t h i s p o i n t , i n d i v i d u a l f i s h  having could  p o s i t i o n a g a i n s t the c u r r e n t and began t o d r i f t down-  At no p o i n t , however, d i d such f i s h abandon t h e i r r h e o t a c t i c  response and  appeared t o be attempting  t o head upstream.  o f t e n r e s u l t e d i n a spasmotic, u n d i r e c t e d movement. manifesting  t h i s behavior  A t a p on the  Eventually a  would l o s e e q u i l i b r i u m and be  tank  fish  swept around the tank  38  with the c u r r e n t .  Attempts a t movement a t t h i s stage r e s u l t e d i n muscular  tremors and death f o l l o w e d a p p r o x i m a t e l y 3 h a f t e r e q u i l i b r i u m l o s s .  If  the t o x i c a n t exposure was d i s c o n t i n u e d when the f i s h c o u l d no l o n g e r m a i n t a i n s t a t i o n , they appeared t o r e c o v e r g r a d u a l l y and i n one case would a c c e p t f o o d a f t e r ^ 24 h i n c l e a n f r e s h water; however, no attempt was made t o a s s e s s t h e r e c o v e r y o f f i s h which were a t a more advanced stage o f d e b i l i t y . The LC50 The r e s u l t s o f the t h r e e acute b i o a s s a y s a r e i l l u s t r a t e d i n F i g . 9 showing 96 h LC50's o f 0.50, 77 r e s p e c t i v e l y .  0.79  and 0.88 mg/L  DHA  f o r E x p t s . 74, 76 and  In Expt. 74, the shape o f t h e t o x i c i t y curve suggests the  p r e s e n c e o f an acute t o x i c i t y t h r e s h o l d i n the 0.4 mg/L lowest c o n c e n t r a t i o n t e s t e d was  0.47 mg/L  DHA.  range; however the  In Expt. 76 and 77 the  t o x i c i t y c u r v e s g e n e r a t e d by the l i m i t e d number o f c o n c e n t r a t i o n s used remained i n the l i n e a r range.  In Expt. 76 the t o x i c a n t exposure  c o n t i n u e d u n t i l complete m o r t a l i t y o c c u r r e d i n the lowest (0.65 c o n c e n t r a t i o n ; t h e l a s t f i s h d i e d a t 262 h.  In Expt. 77,  (^ 27 °/oo ) .  mg/L)  toxicant  exposure was d i s c o n t i n u e d a t 120 h and the water s u p p l y was f r e s h t o sea water  was  s w i t c h e d from  In t h i s case the s l o p e o f the l o g p r o b i t  l i n e used t o determine t h e L T 5 0 ^ f o r t h e 0.79 mg/L  group i n c r e a s e d , s u g g e s t i n g  a l a t e n t and enhanced t o x i c i t y brought on by t h e added s a l i n i t y s t r e s s . the lowest c o n c e n t r a t i o n t e s t e d  (0.63 mg/L), no m o r t a l i t y o c c u r r e d d u r i n g the  120 h exposure p e r i o d ; however, by t h e time the experiment was a f t e r 120 h i n sea water a f u r t h e r 6/14 i n d i c a t e d t h a t the p r i o r  In  f i s h had d i e d .  These  ( s u b l e t h a l ) exposure t o 0.63 mg/L.  discontinued results  DHA  reduced the  subsequent s u r v i v a l o f some sockeye salmon s m o l t s i n c l e a n sea water. were no c o n t r o l m o r t a l i t i e s i n any o f the b i o a s s a y s and i n Expt. 77  There  no  b e h a v i o r a l changes c o u l d be d e t e c t e d i n c o n t r o l f i s h which encountered the 3/  Time t o 50% m o r t a l i t y  (LT 50)  39  1  0.3  I  i  0.4  0.5  |  1  !  !  J  L  V  2  Concentration  F i g u r e 9.  of D H A  mg/L  T o x i c i t y c u r v e s i l l u s t r a t i n g 96 h LC50 v a l u e s f o r DHA t o j u v e n i l e sockeye salmon_in f r e s h water. Confidence l i m i t s (95%) a r e g i v e n by O.  rapid  (.5.8 h f o r 95% replacement) FW->SW t r a n s i t i o n . The 96 h LC50 v a l u e o f 0.50 mg/L  DHA  which was o b t a i n e d i n Expt. 74 i s  somewhat lower than t h a t o b t a i n e d i n Expt. 76 and 77.  Measurements  of  d i s s o l v e d oxygen d u r i n g the c o u r s e o f E x p t . 74 s u g g e s t e d an e l e v a t i o n i n oxygen consumption i n exposed f i s h .  A f t e r 24 h o f exposure, d i s s o l v e d  oxygen l e v e l s i n t h e tanks r e c e i v i n g t h e h i g h e s t dose o f DHA  had dropped  t o 70% s a t u r a t i o n w h i l e t h e c o n t r o l s remained above 90% s a t u r a t i o n .  Supple-  mental a e r a t i o n was added and d i s s o l v e d oxygen l e v e l s remained above 85% f o r the r e s t o f t h e b i o a s s a y .  N e v e r t h e l e s s , as the subsequent experiment showed  t h a t a r e d u c t i o n i n d i s s o l v e d oxygen i n t h e water l e d t o a marked i n c r e a s e i n DHA  t o x i c i t y , t h e LC50 v a l u e o f 0.50 mg/L  DHA  was p r o b a b l y somewhat d e p r e s s e d by reduced 0 . 2  o b t a i n e d d u r i n g Expt. 74 E x p t s . 76 and 77 were  conducted w i t h oxygen l e v e l s i n e x c e s s o f 90% s a t u r a t i o n and i n s p i t e o f d i f f e r e n c e s i n f i s h s t o c k and s i z e , t e s t temperature and pH, the 96 h LC50's  (0.79 and 0.88 mg/L)  were remarkably c l o s e .  E f f e c t s o f Hypoxia The experiment c o n f i r m e d t h e h y p o t h e s i s o f a d i s s o l v e d oxygen and DHA mg/L  DHA  toxicity.  j o i n t a c t i o n o f low  F i s h exposed s i m u l t a n e o u s l y t o  0.65  and reduced oxygen e x p e r i e n c e d 100% m o r t a l i t y w h i l e i n t h e group  exposed a t normal oxygen l e v e l s , o n l y one f i s h d i e d w i t h i n t h e 120 h exposure p e r i o d .  These r e l a t i o n s h i p s are i l l u s t r a t e d  the bottom heavy l i n e d e p i c t s measured D.O.  i n F i g . 10 i n which  l e v e l s , w i t h t h e accompanying  m o r t a l i t y c u r v e (hypoxic) showing 50% m o r t a l i t y a t 76 h. l e v e l s from 78 t o 120 h r e p r e s e n t s the r e d u c t i o n i n 0 the r e d u c t i o n i n the number o f f i s h and once t h e D.O.  2  The r i s e i n  D.O.  consumption due t o l e v e l r e a c h e s ^ 85%  the m o r t a l i t y curve f l a t t e n s o u t somewhat due t o a r e d u c t i o n i n t h e combined s t r e s s of h y p o x i a / t o x i c i t y .  In the case o f the group exposed under normal .  41  Figure  10.  The e f f e c t o f reduced d i s s o l v e d oxygen i n the water on the t o x i c i t y o f DHA (0.65 mg/L) t o sockeye salmon s m o l t s .  D.O. be  conditions, a gradual  increase  i n oxygen consumption by  seen by the s l o p e o f the upper D.O.  when the D.O. represents and  l e v e l reached ^ 85%  curve.  The  f i r s t mortality  saturation.  The  sharp drop a f t e r 120  the measurement 6 h a f t e r the t o x i c a n t f l o w had  the water s u p p l y  s w i t c h e d t o h y p o x i c water.  been  the i n t e r a c t i o n o f DHA  Once the t o x i c a n t was well  discontinued,  a g a i n w i t h i n 24  O2  s a t u r a t i o n and  it  o f DHA  shown p r e v i o u s l y t o be  p r o b a b l y not d e l e t e r i o u s t o the  should be noted t h a t s u b l e t h a l DHA  recover  feeding  Fig.10) i n d i c a t i n g an i n c r e a s e  hypoxia.  salmon  (Davis,  0 -saturated 2  T h i s hypoxia i n 1976).  In a d d i t i o n  exposure i n a normoxic environment 2  from the water  i n the oxygen demand of the  compare f a v o r a b l y w i t h the  of a marked i n c r e a s e  s t r e s s since a  s u b l e t h a l under  caused an i n c r e a s e i n the r a t e o f removal o f 0  observations  began  as a l o a d i n g  c o n d i t i o n s proved l e t h a l when combined w i t h a 30% i t s e l f was  lethality.  h.  These r e s u l t s i n d i c a t e t h a t h y p o x i a a c t e d concentration  occurred  indicates  the r e m a i n i n g f i s h appeared t o  ( b e h a v i o r a l l y normal) even a t 70%  h  discontinued  This  w i t h h y p o x i a which l e d t o  can  occurred  A second m o r t a l i t y  3 h a f t e r the s w i t c h b u t no f u r t h e r m o r t a l i t i e s f o l l o w e d . t h a t i t was  these f i s h  fish.  f i n d i n g s by H i c k s and  i n the acute t o x i c i t y o f whole KME  (dashed  line,  These  DeWitt(1971)  t o j u v e n i l e coho  salmon a t reduced l e v e l s o f d i s s o l v e d oxygen. On  the b a s i s o f t h e s e experiments, 0.65  a concentration  o f DHA  which would be  d u r i n g an exposure p e r i o d o f 120  mg/L  was  chosen t o  represent  s u b l e t h a l t o j u v e n i l e sockeye salmon  h i n w e l l - o x y g e n a t e d f r e s h water.  Sub-  sequent experiments were conducted t o i n v e s t i g a t e the o b s e r v e d i n t e r a c t i o n between p r e v i o u s  DHA  exposure and  salinity  stress.  43  B.  EFFECTS OF ACUTE DHA  EXPOSURE ON OSMOTIC BALANCE  INTRODUCTION During the c o u r s e o f acute t o x i c i t y b i o a s s a y s ,  some sockeye salmon  appeared normal i n s i z e and shape w h i l e o t h e r s d e v e l o p e d a s w o l l e n o r b l o a t e d appearance  (Fig.11a).  D i s s e c t i o n s o f f i s h which were v i s i b l y  s w o l l e n r e v e a l e d an a c c u m u l a t i o n o f f l u i d  i n t h e stomach, i n some c a s e s t o  such a degree t h a t the organ was q u i t e t u r g i d  (Fig.lib).  As t h e s e  symptoms were s u g g e s t i v e o f a water b a l a n c e problem, experiments were conducted t o q u a n t i f y t h i s r e s p o n s e t o DHA (K =  poisoning.  Condition  factors  weight r"~-3 x 100) were c a l c u l a t e d as a measure o f body " f a t n e s s " length• 3  ( L a g l e r , 1969)  and measurements o f t o t a l body water were made t o determine  whether a g e n e r a l h y d r a t i o n was  occurring.  Following these p r e l i m i n a r y  o b s e r v a t i o n s , experiments were c o n d u c t e d t o e s t a b l i s h whether t h i s a p p a r e n t o s m o t i c imbalance extended t o the muscle t i s s u e . muscle o f f i s h exposed t o DHA whereas  T h i s b e i n g the c a s e , the  i n f r e s h water s h o u l d g a i n weight  f i s h exposed i n sea water s h o u l d l o s e water MATERIALS AND  (hydrate),  (dehydrate).  METHODS  Bloating During a p r e l i m i n a r y b i o a s s a y  (Expt. A) i n which a wide range o f  s i z e was used t o i n v e s t i g a t e t h e r e l a t i o n s h i p between DHA  toxicity  eye salmon  (1.1 mg/L),the  t o both sphincters.  f i s h s i z e and acute  b l o a t e d stomachs o f f i v e v i s i b l y  ( f i s h s i z e range, 11.2  s w o l l e n sock-  t o 369 g) were e x c i s e d by a c u t d i s t a l  Each f l u i d - f i l l e d  oven a t 105°C and t h e % water was  sac was  compared  then d r i e d i n a f o r c e d - a i r  t o t h a t o f stomachs taken from  f i s h which had d i e d d u r i n g t h e b i o a s s a y b u t which had m a i n t a i n e d an a p p a r e n t l y normal body  form.  fish  F i g u r e 11.  I l l u s t r a t i o n o f the s w e l l i n g o f sockeye caused by DHA exposure.  salmon  C o n d i t i o n f a c t o r s were c a l c u l a t e d f o r f i s h which had d i e d i n t h r e e p r e l i m i n a r y b i o a s s a y s CExpts. B, C and D): was measured i n Expt. D.  In a d d i t i o n , t o t a l body water  E x p e r i m e n t a l d e t a i l s are o u t l i n e d i n T a b l e s I I I  and IV i n the R e s u l t s s e c t i o n . Muscle Water F o l l o w i n g these p r e l i m i n a r y o b s e r v a t i o n s , the percentage muscle water was  determined i n f i s h exposed  water DHA  (Expt. F ) .  t o DHA  i n f r e s h water  (Expt. E) and i n sea  In Expt. E, u n d e r y e a r l i n g sockeye salmon were exposed  i n f r e s h water and each f i s h was  to  c o l l e c t e d a t the stage when i t c o u l d no  l o n g e r m a i n t a i n r h e o t a x i s i n the a n n u l a r t a n k s . u n d e r y e a r l i n g chum salmon were exposed  t o DHA  In the second  i n sea water.  experiment,  H a l f o f the  f i s h were c o l l e c t e d a t death, w h i l e the o t h e r h a l f were s t i l l  a l i v e when  the b i o a s s a y was d i s c o n t i n u e d . The  sockeye salmon were o f the Great C e n t r a l Lake  s t o c k and were r a i s e d from f e r t i l i z e d eggs a t PEI. experiment,  these f i s h were 8 months o l d .  (Vancouver  A t the time o f t h e  As no sea-water  salmon were a v a i l a b l e , chum salmon from the Inches Creek B.C.)  Island)  adapted  sockeye  s t o c k (Dewdney,  were used and had been hatched a t PEI where they were m a i n t a i n e d  i n sea water  (27-29 °/oo) .  sampling was  done by g e n t l y d i p n e t t i n g i n d i v i d u a l s as they d r i f t e d  the tank.  In the case o f s u r v i v o r s o r d r i f t i n g  fish, around  F i s h were k i l l e d by a sharp f l i c k o f the f i n g e r , b l o t t e d and a  t i s s u e sample was  taken f o r d e t e r m i n a t i o n o f % water.  A transverse cut  was  made through the body immediately p o s t e r i o r t o the v e n t , w i t h a second c u t about 1 cm f u r t h e r p o s t e r i o r l y .  The b u l k o f such a t r u n k s e c t i o n  o f muscle b u t i n c l u d e d s e v e r a l v e r t e b r a e and s k i n .  The  sample was  consisted weighed  i n an aluminum d i s h , d r i e d as d e s c r i b e d p r e v i o u s l y , and the m o i s t u r e c o n t e n t was  e x p r e s s e d as % muscle  water.  RESULTS AND Not a l l  f i s h exposed t o DHA  DISCUSSION  d e v e l o p e d a b l o a t e d appearance nor was the  s w e l l i n g a post-mortem development  as i t was  f r e q u e n t l y observed many  hours b e f o r e e q u i l i b r i u m l o s s i n acute b i o a s s a y s .  In E x p t s . B, C, and D,  c o n d i t i o n f a c t o r s o f sockeye salmon which d i e d d u r i n g DHA s i g n i f i c a n t l y h i g h e r than the c o n t r o l s  (Table I I I and I V ) .  exposure a r e The p r o g r e s s i v e  n a t u r e o f t h i s weight g a i n i s r e f l e c t e d by a g r a d u a l i n c r e a s e i n K f a c t o r b e f o r e and a f t e r e q u i l i b r i u m l o s s l e a d i n g t o death (Table I I I , f o o t n o t e 2 ) . Of the 24 f i s h exposed t o DHA abdomens.  d u r i n g Expt. A, 5 had v i s i b l y  swollen  The a c c u m u l a t i o n o f f l u i d i n the stomachs o f t h e s e f i s h i s shown  i n the r e s u l t s i n T a b l e V.  The stomach o f one f i s h which appeared  p a r t i a l l y s w o l l e n c o n t a i n e d an amount o f water mid-way between t h a t p r e s e n t i n normal and s w o l l e n f i s h . c o n f i r m e d t h a t DHA controls  In Expt. D, t o t a l body water measurements  exposed f i s h c o n t a i n e d s i g n i f i c a n t l y more water than the  (Table I V ) .  These experiments showed t h a t i n some salmon DHA  exposure i n f r e s h  water r e s u l t s i n a d r a m a t i c i n c r e a s e i n water i n g e s t i o n , l e a d i n g t o a v i s i b l e a c c u m u l a t i o n o f water and t h a t i n o t h e r s , a l t h o u g h t h e s w e l l i n g i s n o t apparent, t h e r e o c c u r s a g e n e r a l h y d r a t i o n which i s r e f l e c t e d by e l e v a t ed K - f a c t o r s and i n c r e a s e d t o t a l body water c o n t e n t .  The muscle water  experiments d e s c r i b e d below were conducted t o determine whether edematous c o n d i t i o n extended t o t h e t i s s u e s o f f i s h exposed t o  this DHA.  Muscle Water The r e s u l t s o f t h e sockeye salmon experiment a r e p r e s e n t e d i n T a b l e VI and show t h a t i n f r e s h water.  exposure t o DHA  r e s u l t e d i n a h y d r a t i o n o f muscle  tissue  As t h e s e changes were o b s e r v e d a t a time when t h e f i s h  c o u l d no l o n g e r m a i n t a i n p o s i t i o n a g a i n s t the c u r r e n t perhaps t h i s muscle  47  T a b l e I I I . C o n d i t i o n f a c t o r s o f u n d e r y e a r l i n g sockeye salmon exposed t o a c u t e l y l e t h a l and s u b l e t h a l c o n c e n t r a t i o n s o f DHA i n f r e s h water (Expts. B and C ) .  Mean ±SE  Bioassay  C  2  '  (DHA) mg/L  length cm  weight g  N  1.81 0.83  .8  10.8 ±0.26  17.4 ± 1 . 1 1  1.37*±0.03  7  10.3 ±0.23  15.2 ±1.14  1.36*±0.02  Control  9  10.9 ±0.16  15.6 ±1.09 .  1.32  9  11.9 ±0.65  22.7 ± 1 . 1 3  1.35*±0.02  ' 6  11.2 ±0.18  19.0 ± 0 . 9 1  1.37*±0.02  10  11.8 ±0.33  17.9 ±1.43  1.08 ±0.03  0.57  3  Control  1 X  K  condition^factor K  1.07 ±0.02  weight , = ^-7,3 x 100 length  2 one f i s h one f i s h  sampled b e f o r e l o s s o f e q u i l i b r i u m sampled a f t e r l o s s o f e q u i l i b r i u m  *significantly  different  from c o n t r o l s p<0.05  3  Time to 50% mortality (LT 50)=15.3 days  cm g 12.0/19.6 11.8/20.5  K=1.13 K=1.25  Student's t - t e s t  T a b l e IV.  C o n d i t i o n f a c t o r and t o t a l body water i n u n d e r y e a r l i n g sockeye salmon which d i e d d u r i n g exposure t o 0.95 mg/L DHA i n f r e s h water (Expt. D).  N  length cm  weight g  condition factor K  body water %  Exposed  19  9.6 ±0.24  9.2 ±0.60  1.04* ±0.02  78.55*10.35  Control  10  10.2 ±0.20  9.0 ±0.55  0.85 ±0.01  75.16 ±0.88  1 LT 50 = 62.5 h * s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<0.05.  Student's t - t e s t  T a b l e V.  P e r c e n t a g e water o f stomachs d i s s e c t e d from " s w o l l e n " and "normal" salmon exposed t o 1.11 mg/L DHA (Expt. A ) .  N  % Water (Mean ±SE)  Swollen  ( 5)  91.7*±1.2  Normal  (10)  79.0  Partly Swollen  (1)  84.8  ±1.7  *s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<0.05.  Student's t - t e s t  T a b l e V I . Muscle water i n u n d e r y e a r l i n g sockeye salmon exposed t o DHA i n f r e s h water and sampled a t t h e d r i f t i n g s t a g e . 1  Mean ±SE  N  Length cm  Weight g  M u s c l e Water %  Exposed  20  11.2 ±0.27  13.96 ±0.94  77.05*±0.31  Control  20  11.3 ±0.24  12.89 ±0.85  75.78 ±0.14  ^1.4 mg/L *significantly  different  from c o n t r o l s  p<0.05. S t u d e n t ' s t - t e s t  51  h y d r a t i o n i n t e r f e r e d w i t h normal c o n t r a c t i l e p r o c e s s e s and thus c o n t r i b u t e d t o a r e d u c t i o n i n swimming performance. The r e s u l t s o f t h e exposure o f chum salmon t o DHA  i n sea water a r e  shown i n T a b l e V I I and i l l u s t r a t e a s i g n i f i c a n t d e h y d r a t i o n o f muscle when compared t o the c o n t r o l s .  The g r a d u a l development  of dehydration i s  i l l u s t r a t e d by the r e d u c t i o n i n water c o n t e n t o f salmon which had the 96 h exposure t o DHA.  tissue  survived  T h i s v a l u e suggests t h a t the amount o f d e h y d r a t i o n  i n c r e a s e s w i t h exposure time. These p r e l i m i n a r y experiments i n d i c a t e d t h a t exposure t o DHA  l e d t o an  osmotic imbalance i n a d i r e c t i o n d i c t a t e d by the osmotic g r a d i e n t between body f l u i d s and t h e s u r r o u n d i n g water. be accompanied  As such  an osmotic imbalance c o u l d  by an i o n i c d i s t u r b a n c e , experiments were conducted t o  determine plasma i o n i c c o m p o s i t i o n i n sockeye salmon exposed t o DHA water.  i n fresh  Subsequently, experiments were performed t o measure the h y d r o m i n e r a l  r e g u l a t o r y a b i l i t y o f salmon exposed s u b - l e t h a l l y t o DHA f e r r e d t o sea water.  and then t r a n s -  Table V I I .  Muscle water i n u n d e r y e a r l i n g DHA-*- i n sea water.  chum salmon exposed t o  Mean ±SE Length N  cm  Weight g  M u s c l e Water %  At death  10  10.0 ± 0 . 2 3  7.70 ±0.53  77.09* ±0.36  Survivors  10  10.0 ± 0 . 1 6  7.62 ±0.43  79.53*±0.38  20  10.1 ±0.13  7.92. ±0.33  80.72 ±0.14  Exposed  Controls  1  1.4 mg/L  *significantly  different  from c o n t r o l s p<0.05. S t u d e n t ' s t - t e s t  PART I I . A.  PRINCIPAL EXPERIMENTS  EFFECTS OF SUBLETHAL DHA EXPOSURE ON HYDROMINERAL BALANCE IN SOCKEYE SALMON SMOLTS INTRODUCTION The s u g g e s t i o n made from the p r e v i o u s experiments t h a t osmotic imbalance  was  a l s o accompanied  by an e l e c t r o l y t e d i s t u r b a n c e was  e x p l o r a t o r y experiment  c o n f i r m e d i n an  ( u n p u b l i s h e d o b s e r v a t i o n s ) i n which b l o o d e l e c t r o l y t e  l e v e l s were measured i n sockeye salmon s u b l e t h a l l y exposed t o DHA water.  Exposure t o DHA  r e s u l t e d i n a s i g n i f i c a n t r e d u c t i o n i n the concen-  t r a t i o n s o f the main plasma e l e c t r o l y t e s N a were observed i n plasma K increased.  T h i s complex  i n fresh  +  o r Mg  ++  +  and C l .  However, no  l e v e l s and plasma Ca  ++  changes  • concentrations  response cannot be e x p l a i n e d by a simple  DHA-induced  hydration. To i n v e s t i g a t e t h e s e f i n d i n g s more f u l l y , t h i s experiment was r e p e a t e d and the o b s e r v a t i o n s were a l s o extended i n t o t h e seawater phase. i n the G e n e r a l I n t r o d u c t i o n , p r i o r s u b l e t h a l DHA  As d i s c u s s e d  exposure f o l l o w e d by the  movement o f f i s h i n t o sea water was meant t o s i m u l a t e the exposure o f sockeye salmon t o p u l p m i l l waste i n a r i v e r d u r i n g t h e course o f smolt m i g r a t i o n . Under t h e s e c o n d i t i o n s , the e x t e n t o f t o x i c a n t exposure would be l i m i t e d t o the m i g r a t i o n time i n f r e s h water and subsequent e n t r y i n t o the sea would i n v o l v e a r a p i d t r a n s i t i o n from a h y p o t o n i c t o a h y p e r t o n i c medium.  Although  t h i s seawater a c c l i m a t i o n p e r i o d comprises a " r e c o v e r y phase", as the t o x i c a n t exposure has been d i s c o n t i n u e d , the i n t e r a c t i o n o f the e f f e c t s o f p r i o r exposure w i t h n a t u r a l s a l i n i t y s t r e s s c o u l d have an e f f e c t on hydrom i n e r a l homeostasis. Three experiments were conducted  (Expts. 1,2  and 3) i n which sockeye  salmon smolts were exposed t o a s u b l e t h a l dose o f DHA subsequently " t r a n s f e r r e d " t o sea water.  i n f r e s h water  and  Osmoregulatory performance  was  54  gauged by measuring  plasma e l e c t r o l y t e c o n c e n t r a t i o n s a t the end o f the  t o x i c a n t exposure p e r i o d and, s u b s e q u e n t l y , by f o l l o w i n g the time course o f plasma e l e c t r o l y t e r e g u l a t i o n d u r i n g the t r a n s i t i o n t o sea water. the r e s u l t s o b t a i n e d i n f r e s h water, i t was  h y p o t h e s i z e d t h a t DHA  In view o f exposure  s h o u l d l e a d t o e l e v a t e d plasma i o n i c l e v e l s i n f i s h i n sea water i f the t o x i c a n t a c t e d t o cause a l o s s i n i o n o r e g u l a t o r y p r e c i s i o n . During Expt. 1 however, some o f the f i s h were s u s p e c t e d o f from a c h r o n i c i n f e c t i o n o f b a c t e r i a l kidney d i s e a s e (BKD) the i n f e c t i o n was  suffering  and the presence  of  c o n f i r m e d d u r i n g Expt. 2 which f o l l o w e d immediately.  B a c t e r i a l k i d n e y d i s e a s e i n v o l v e s a g r a d u a l d e s t r u c t i o n o f the k i d n e y which i s i n t i m a t e l y i n v o l v e d w i t h hydromineral balance i n f i s h .  Thus, t o reduce  the  p o s s i b i l i t y o f the d i s e a s e confounding the e f f e c t s caused by the t o x i c a n t , i t seemed i m p e r a t i v e t o d e v i s e a procedure  t o e l i m i n a t e from the d a t a f i s h i n  which the i n f e c t i o n had reached an advanced stage; t h i s was hematocrit values.  done u s i n g  Blood h e m a t o c r i t i s known t o drop g r a d u a l l y as the d i s e a s e  p r o g r e s s e s , t h e r e f o r e d a t a c o l l e c t e d from salmon w i t h abnormally low hematoc r i t s were d e l e t e d .  A thorough d i s c u s s i o n o f b a c t e r i a l k i d n e y d i s e a s e and i t s  i n t e r a c t i o n w i t h DHA  t o x i c i t y , as w e l l as the d e t a i l s o f the s c r e e n i n g method  and t a b l e s c o n t a i n i n g the d e l e t e d d a t a a r e p r e s e n t e d i n Appendix I I . A t a l a t e r date, when a new  s t o c k o f d i s e a s e - f r e e salmon became a v a i l a b l e ,  the e l e c t r o l y t e b a l a n c e experiment  was  r e p e a t e d a t h i r d time  confirmed the r e s u l t s which had been o b t a i n e d i n E x p t s . 1 and MATERIALS AND Three  continuous flow c o n d i t i o n s . t o x i c a n t a d m i n i s t r a t i o n was  mg/L  DHA  and  2.  METHODS  s i m i l a r b u t s e p a r a t e experiments  smolts were exposed t o 0.65  (Expt. 3)  were conducted  f o r 5 days  i n which  sockeye  (120 h) i n f r e s h water under  A t the end o f t h i s s u b l e t h a l exposure p e r i o d , d i s c o n t i n u e d , and the f r e s h water s u p p l y  was  55  s w i t c h e d t o s e a water.  A 95% replacement i n 5.8 h y i e l d e d a s a l i n i t y  26 °/oo a t t h e end o f t h i s time. the  of ^  The f i r s t b l o o d sampling was conducted a t  end o f t h e f r e s h w a t e r DHA exposure p e r i o d  (G h) and s u b s e q u e n t l y f o r 120 h  a t i n t e r v a l s o f 24 h t o f o l l o w the time c o u r s e o f a d a p t a t i o n t o sea water. F i s h h a n d l i n g , b l o o d sampling and a n a l y s i s p r o t o c o l was performed as d e s c r i b e d i n t h e G e n e r a l Methods s e c t i o n .  The s i z e s o f t h e f i s h used i n t h e t h r e e  experiments a r e g i v e n i n T a b l e V I I I . The t h r e e experiments were conducted u s i n g two s e p a r a t e s t o c k s o f sockeye salmon.  E x p t s . 1 and 2 were performed i n May 1977 u t i l i z i n g f i s h which were  o b t a i n e d as y e a r l i n g s from t h e C u l t u s Lake s t o c k and had been k e p t a t PEI f o r 6 months p r i o r t o u s e . b a c t e r i a l kidney disease. Lake  T h i s was t h e s t o c k o f f i s h which d e v e l o p e d In June 1977, sockeye smolts from t h e G r e a t C e n t r a l  (Vancouver I s l a n d ) run were brought t o PEI b u t p r o v e d t o o s m a l l t o conduct  the  c o r r o b o r a t i v e experiment  (Expt. 3 ) .  As a r e s u l t , Expt. 3 was conducted i n  the  f i r s t week o f December 1977 a t which time t h e s e f i s h were 21 months o l d .  Thus w h i l e the s i z e o f f i s h used i n the t h r e e experiments was s i m i l a r , t h e sockeye i n Expt. 3 were o f a d i f f e r e n t s t o c k and were t h r e e months o l d e r . RESULTS L a t e n t Acute For (the  Toxicity  t h e purposes o f t h e s e experiments, " s u b l e t h a l " was d e f i n e d as a dose  p r o d u c t o f c o n c e n t r a t i o n x time) c a u s i n g n e g l i g i b l e m o r t a l i t y i n 120 h.  A c c o r d i n g t o t h i s d e f i n i t i o n 0.65 mg/L DHA was s u b l e t h a l i n E x p t s . 1 and 3. In  Expt. 2  however, m o r t a l i t y reached % 7% d u r i n g t h e exposure p e r i o d .  i n c r e a s e i n t o x i c i t y was caused by t h e BKD i n f e c t i o n and i s d i s c u s s e d i n Appendix I I .  This  •J  T a b l e V I I I . S i z e o f the sockeye salmon used i n the e l e c t r o l y t e b a l a n c e experiments: (Expts. 1,2 and 3) .  Mean* SE(n)  Time in  Control Fork length  hours  0  Experiment 1  0 38(10)  33  cm  s  e  d  Wet weight  97  2 66  14  9  0. 27(10)  34  42  1 83  14  3  0 21(10)  30  71  2 06  14  2  0 50( 7)  29  44  2 75  33  43  1 69  34  02  1 92 '3 23  24  15  0  0 47( 9)  33  93  48  14  8  0 30(10)  32 16  1 96  72  14  6  0 27 ( 9)  31 21  1 98  15  0  0 26( 8)  96  15  2  0 48{ 9)  34  3 54  15  1  0 28( 9)-  61  14  9  0 22(10)  32 48  1 53  15  1  0 40( 7)  35  53  15  7  0 38(10)  40  93  2 33  15  9  0 30(11)  40  65  2 27  24  16  4  0 32( 9)  44  09  2 43  15  4  0 33( 9)  37  06  2 66  48  15  8  0 60 (  6)  40  82  5 25  15  3  0 32(10)  36  75  2 10  72  15 .5  0 30 (  8)  37  06  2 43  15  6  0 24 ( 7)  37  36  1 78  85  2 19  16  0  0 39( 8)  39  69  2 97  0  96  15  8  0 33(10)  39  120  15  6  0 40 ( 8)  38  15  2 46  15  3  0 42 ( 8)  35  90  3 18  15  9  0. 35(11)  36. 68  2 69  15  3  0. 33(12)  31 74  2 18  24  15  0  0. 37(11)  29. 07  2 25  15  3  0 41(12)  32 09  2 58  48  15  8  0 35(12)  33.  71  2 10  15  6  0 47(11)  32 56  2 87  44  1 75  15  5  0 52(10)  32 48  3 79  32 14  2 70  16  0  0 42(10)  36  15  2 56  0 43(12)  34  28  2 30  0  Experiment 3  9  *P°  Fork length  g  3 24  120  Experiment 2  cm  14  E  Wet weight  72  16  0  0 24  96  15  2  0 45(12)  120  16  0  ("ll)  0 32(12)  35  36  04  2 58  15  6  57  The  p r e s e n c e o f a l a t e n t acute  t o x i c i t y o f DHA became apparent when t h e  p r e v i o u s l y exposed f i s h were f a c e d w i t h a s a l i n i t y c h a l l e n g e . i n Fig.25  As i l l u s t r a t e d  (p.155) t h i s combination o f s t r e s s o r s l e d t o m o r t a l i t i e s i n E x p t . l  o n l y a f t e r t h e f i s h had been i n s e a water f o r 24 h. which s t a r t e d d u r i n g t h e f r e s h w a t e r  I n E x p t . 2, m o r t a l i t i e s  exposure p e r i o d appeared t o c o n t i n u e a t  t h e same r a t e f o r t h e f i r s t  24 h i n s e a water, w i t h a subsequent break i n t h e  s l o p e o f t h e t o x i c i t y curve  i n d i c a t i n g an a t t e n u a t i o n o f l a t e n t acute  a f t e r 48 h i n c l e a n sea water. 7/60, 15/75 and 2/72.  A c t u a l m o r t a l i t i e s f o r Expts.  toxicity  1, 2 and 3 were  These f i g u r e s i n c l u d e "moribund" f i s h f o r E x p t . 2 b u t  do n o t i n c l u d e t h e 3 " d r i f t e r s " i n Expt. 3-which were sampled i n t h e e a r l y stages o f i n t o x i c a t i o n .  "Moribund" and " d r i f t e r " f i s h a r e d i s c u s s e d i n t h e  l a s t p a r t o f the Results section. C o n t r o l m o r t a l i t i e s were n e g l i g i b l e , w i t h o n l y 1 o f t h e 204 t o t a l  dying  o f what appeared t o be a fungus i n f e c t i o n o f t h e g i l l s . Sublethal E f f e c t s Behavioral  Observations  Observations  made d u r i n g t h e course  o f t h e t o x i c a n t exposure i n d i c a t e d a  s l i g h t r e d u c t i o n i n t h e compactness o f t h e s c h o o l , a b e h a v i o r to  the f i r s t  stage o f t h e sequence d e s c r i b e d i n t h e B e h a v i o r a l  p o r t i o n o f t h e Acute T o x i c i t y s e c t i o n (p. 37 ) . disturbance  which c o r r e s p o n d s Symptoms  The normal r e s p o n s e t o v i s u a l  (cover response) was o n l y s l i g h t l y reduced i n Expt. 1 and 3  whereas i n E x p t . 2 t h e f i s h g e n e r a l l y r e a c t e d more s l o w l y .  I n E x p t . 2, 15  f i s h were more s e v e r e l y a f f e c t e d a f t e r 103 h exposure; these were sampled p r i o r t o t h e end o f t h e exposure p e r i o d and a r e d i s c u s s e d i n a l a t e r s e c t i o n as "moribund"  fish.  58  T h i s slowness i n response o f DHA-exposed f i s h g e n e r a l l y became more pronounced  d u r i n g the f i r s t 24 h o f seawater a d a p t a t i o n , and many f i s h  d i d not d i s p l a y a normal cover response a f t e r 72 h i n sea water.  still  In c o n t r a s t  the b e h a v i o r o f c o n t r o l f i s h remained unchanged throughout the e n t i r e  exper-  iment . Hydromineral Balance i n F r e s h Water The plasma e l e c t r o l y t e l e v e l s i n sockeye salmon smolts exposed t o 0.65  mg/L  DHA.in f r e s h water are p r e s e n t e d i n T a b l e IX and  i n Fig.12.  In a d d i t i o n t o plasma  i o n s , b l o o d h e m a t o c r i t was  f o r 120  h  illustrated determined i n  a l l t h r e e experiments whereas % muscle water was measured o n l y i n E x p t s . 2 and 3.  T a b l e IX g i v e s  the  means o f the a c t u a l v a l u e s measured i n each o f  the t h r e e experiments, whereas F i g . 1 2 combines these means t o an o v e r a l l  "average  illustrate  response".  S t a t i s t i c a l s i g n i f i c a n c e shown i n F i g . 1 2 was  determined by  combining  the p r o b a b i l i t i e s o b t a i n e d from s e p a r a t e t e s t s o f s i g n i f i c a n c e between " c o n t r o l " and was  "exposed" means i n each experiment.  used and was  The Student's " t " t e s t  c o r r e c t e d when v a r i a n c e s were unequal.  Exact p r o b a b i l i t i e s  were determined f o r each c a l c u l a t e d " t " and then p o o l e d a c c o r d i n g t o the method g i v e n i n S o k a l and R o h l f (1969).  T h i s treatment p r o v i d e s a s i n g l e  t e s t o f s i g n i f i c a n c e o f t h e aggregate based on the p r o d u c t o f the p r o b a b i l i t i e s observed i n d i v i d u a l l y  ( F i s h e r , 1958).  T h i s approach was  used  because, a l t h o u g h t h e t h r e e experiments were s i m i l a r i n d e s i g n , they were not i d e n t i c a l , so t h a t p o o l i n g o f a l l the d a t a f o r j o i n t s t a t i s t i c a l  treatment  was n o t warranted. F i g . 1 2 shows t h a t a 120 h exposure t o a s u b l e t h a l dose o f DHA s i g n i f i c a n t d i s t u r b a n c e i n the l e v e l s o f a l l plasma e l e c t r o l y t e s except f o r sodium.  The d i r e c t i o n o f t h e change however, was  caused a  investigated,  n o t uniform.  59  27X3  275  ~l  2 8 5  260  2 9 0  1 — I ' — | — I — 1 — I — I — I — I — I — I — I — | — I — r  mOsm/kg  -r~l  C O N T R O L  Osmolality  E X P O S E D  11TJ  115  120  " T — I — | — I — I — I — 1 — |  125 I  I — I — I — |  1  C O N T R O L E  X P O S E D  )••  140  1 4 5 I  I  I  I  1  ! 3 0  150 1  1  1~\  1  1  1  1  E Q /  L  Chloride  1 5 5 1  m  1—I—I—I—|  1  160 1  1  I  1  1  mEq/L  C O N T R O L  Sodium  J  E X P O S E D  3.5  3.0  2.5 1  1  •  1  i  «  1  C O N T R O L E X P O S E D  5  0  Potassium  5.5 I  1  1  1  |  6.0 1  1  1  mEq/L  1  mEq/L  1  C O N T R O L  Calcium  E X P O S E D  1.5  1.0 ~~i  r  1  T  1  2 0  mEq/L  r  C O N T R O L ]  Magnesium  E X P O S E D  3 0  4  3 5 4 0 5 5 0 1—I—I—I—|—I—I—<—I—p~1—l—I—I—|—I—I—I—I—|  %  C O N T R O L  Hematocrit  E X P O S E D  75  7 6  T C O N T R O L E  F i g u r e 12.  X P O S E D  |  7 7 I  7 8  |  I  |  7 9 I  |  8 0 '  |  1 Muscle Water  Plasma e l e c t r o l y t e l e v e l s , h e m a t o c r i t and muscle water c o n t e n t i n sockeye salmon exposed t o 0.65 mg/L DHA f o r 120 h i n f r e s h water. V a l u e s a r e means f o r each parameter based on E x p t s . 1, 2 and 3 e x c e p t f o r muscle water which was measured i n E x p t . 2 and 3 o n l y . S i g n i f i c a n c e l e v e l p<0.05^; p<0.'01#4  60  Table IX.  Plasma i o n i c c o m p o s i t i o n , h e m a t o c r i t and muscle water c o n t e n t o f sockeye salmon exposed t o 0.65 mg/L DHA f o r 120 h i n f r e s h water i n E x p t s . 1, 2 and 3.  Experiment 1 Osmolality mOsm/kg  Chloride mEq/L  Sodium mEq/L  Potassium mEq/L  Calcium mEq/L  Magnesium mEq/L  Hematocrit  Muscle Water %  Experiment 2  Control  286.6  1.77(10)  287.6  3.11(10)  285.1  3.23(11)  Exposed  276.4*  i . 6 3 ( 9)  286.0  3.07(10)  273.8  2.13(12)  Control  124.8  0.39(10)  123.9  Exposed  114.4*  Control  1.51(10)  124.3  1.15(11)  1.35(10)  114.5  a  1.00(11)  110.6*  1.40(12)  153.83  1.31(10)  147.29  1.13(10)  151.94  2.37(11)  Exposed  152.01  1.71(10)  146.08  2.03(11)  154.14  1.61(12)  Control  2.81  0.21(10)  3.63  0.16(10)  3.20  0.14(11) 0.26(12)  Exposed  1.93° 0.24(10)  3.56  0.16(11)  2.68  Control  5.69  5.38  0.07(10)  4.29  0.08(11)  4.78  0.21(12)  0.12(10)  a  Exposed  6.18  0.15(10)  6.18  0.07(11)  Control  1.50  0.03(10)  1.55  0.03(10)  1.37  b  1.52  0.04(10)  1.74  0.04(11)  1.60  Control  33.97  1.63(10)  36.30  1.12(10)  40.56  0.73(11)  Exposed  45.00  2.00(10)  44.76  1.19(11)  47.83  1.04(12)  Control  74.79  0.42(10)  79.50  0.53(11)  Exposed  75.69  0.18(11)  81.17  0.59(12)  p<0.001, p<0.01, ° p<0.02, p<0.05 d i f f e r s s i g n i f i c a n t l y from c o n t r o l t - t e s t b  0.05(11)  Exposed  T o t a l sum o f i o n s a  Experiment 3  d  a  0.03(12)  61  O v e r a l l , DHA  exposure  i n f r e s h water l e d t o most pronounced and c o n s i s t e n t  changes i n plasma l e v e l s o f C l t h r e e experiments, plasma Ca remained remarkably s t a b l e .  ++  and C a and Mg  + +  ++  and i n b l o o d h e m a t o c r i t . i n c r e a s e d whereas plasma Na  The d e c r e a s e i n plasma o s m o l a l i t y was  by an i n c r e a s e i n muscle water  In a l l  +  levels  accompanied  levels.  Hydromineral Balance i n Sea Water  (Plasma E l e c t r o l y t e s )  The e f f e c t s o f the combined s t r e s s o f p r i o r t o x i c a n t exposure and s a l i n i t y on plasma e l e c t r o l y t e s o f sockeye salmon a r e p r e s e n t e d i n T a b l e X and i l l u s t r a t e d i n F i g . 1 3 . T a b l e x  g i v e s the means o f the v a r i o u s plasma  e l e c t r o l y t e d e t e r m i n a t i o n s f o r E x p t s . 1, 2 and 3, w h i l e F i g . 1 3 p r o v i d e s a comparison o f "exposed" r e l a t i v e t o " c o n t r o l " v a l u e s ; c o n t r o l s a r e shown as 100.  Each p o i n t r e p r e s e n t s an average o f t h e combined responses o b t a i n e d i n  E x p t s . 1, 2 and 3, w h i l e the l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e o f the d e p a r t u r e o f each p o i n t from t h e c o n t r o l  (dotted l i n e ) was determined from p o o l e d  p r o b a b i l i t i e s as d e s c r i b e d p r e v i o u s l y . box below each graph.  S i g n i f i c a n c e l e v e l s a r e shown i n t h e  Note t h a t t h e p o i n t s f o r 0 hours SW r e p r e s e n t the  v a r i o u s e l e c t r o l y t e v a l u e s a f t e r 120 h DHA  exposure i n f r e s h water.  Since  they a r e n o t p a r t o f t h e seawater phase, f o r r e a s o n s o f c l a r i t y they a r e n o t connected t o t h e c u r v e s b u t have been shown t o p r o v i d e o f the magnitude o f t h e d i r e c t e f f e c t o f DHA  a relative  indication  on e l e c t r o l y t e b a l a n c e i n f r e s h  water. The r e s u l t s summarized e l e c t r o l y t e s except f o r K exposed t o DHA.  +  i n F i g . 1 3 show t h a t the b a l a n c e o f a l l t h e plasma was a l t e r e d i n f i s h which had p r e v i o u s l y been  The d e p a r t u r e s from t h e c o n t r o l v a l u e s were a l l i n the  p o s i t i v e d i r e c t i o n and s u b s e q u e n t l y r e t u r n e d t o normal l e v e l s a f t e r v a r y i n g intervals.  Plasma C l  c o n c e n t r a t i o n s remained e l e v a t e d  (p<0.001)  f o r 96 h,  62  Figvre 13.  The change in plasma electrolyte levels in sockeye salmon during adaptation to sea water following a 120 h exposure to 0.65 mg/L DHA in fresh water. Points for esch parameter represent the overall mean % change (relative to a control value of ICQ) derived from Experiments 1, 2 and 3. The level of statistical significance of the change is given at the base of each graph.  Table X.  Plasma e l e c t r o l y t e l e v e l s [Mean -SE(N)] i n sockeye salmon d u r i n g sea-water a d a p t a t i o n f o l l o w i n g a 120 h exposure t o 0.65 mg/L DHA i n f r e s h water i n E x p t s . 1, 2 and 3.  EXPT 1  Tine  in  sea water hours  Osmolality  Chloride  mOsjn/kij  mEq/l.  Sodium  Exposed  Exposed  A  calcium  POt&SSluM nEq/L  roEq/L  Exposed  Control  nEq/L  6J( 9)  124 U  0.39(10)  114 4  1 35(10)  153.83  1 11(10)  152 01  1. 71(10)  2. 81  0 21(10)  1 93  2 061 5)  128 3  0.041 9)  146 7° 2 86(10)  162.36  2 49 ( 9)  1.60  0 30 ( 9)  3 40 ( 9)  316 7 10 69 ( 6)  127 2  1.18(10)  137 1* 3 06( 7)  154.26  0 92(10)  2. 42  3 59 ( 7)  JOB 3  4 38 ( 8)  133 2  1.48( 9)  130 8  1 131 8)  159.44  2 23( 9)  179 75* 3. 33(10) d 4. 165 211 7) 16 164 72 2. 89 ( 8)  3 95  295 1  2 06( 9)  293 1  2 091 9)  131 3  1.641 9)  130 9  1 5BI 9)  160.65  2 21 (9)  155 30  1. 51 (9)  3.44  29 5 4  2 48(10)  299 9  1 85( 7)  129 7  0.34(10)  111 4  0 57( 7)  160.82  1 09(10)  161 56  2. 10( 7)  0  266 6  1 77(10)  276 4 " l  24  299 5  1 641 8)  141 2  48  291 1  72  299 1  96 120  Bf-q/L  Expoaod  Exposed  0 24(10)  5 69  0 12110)  6 18"  0 15(10)  1 50  0.03110)  1 52  0.04(10)  1 84  0 17(10)  6 22  0 151 9)  7 15'  0 22(10)  2 19  0.081 9)  1 62*  0.26(10)  0 35(10)  2 83  0 241 7)  5 94  0 06110)  6 26  0 19( 7)  1 89  0.04(10)  3 53  0.491 7)  0 141 9)  3 41  0 201 8)  5 88  0 07( 9)  5 91  0 141 8)  1 99  0.081 9)  2 30  0.191 8)  0 09( 9)  3 62  0 28( 9)  5 86  0 07( 91  5 83  0 07( 9)  1 47  0.091 9)  1 92  0.301 9)  3.67  0 11(10)  3 75  0 111 7)  5 89  0 08(10)  5 99  0 091 7)  1 66  0.04(10)  1 72  0.041 7)  3.63  0 16(10)  3 56  0 16(11)  5 38  0 07(10)  0 07(11)  1 55  0 03110)  1 7?  0.04(11)  0 151 8)  1 94  0 091 9)  3 ie"  0.37( 8)  0 04(10)  1 83  0 04 ( 6)  2 22  0.17(10)  C  d  EXPT 2 0  287 6  3 11(10)  286 0  24  296 2  2 301 9)  325 5  48  306 0  3 72( 6)  305 6  72  289 4  96 120  155.90  1 55( 9)  170 21* 2.891 8)  3.39  0 201 9)  3 85  0 141 8)  5 52  0 071 9)  160.13  2 84 ( 6)  3.31  0 181 6)  3 80  0 20(10)  5 45  0 26( 61  140 3° 1 86( 6)  154.46  1 831 8)  4.01  0 21( 8)  4 17  0 25 ( 6) 5 73  0 331 7)  5 56  0 OBI 6)  1 84  0 061 8)  2 92  0.461 6)  1.11(10)  135 l "  0 84 ( 8)  155.04  1 74(10)  165 09 1.67(10) b 165 3.111 6) 65 157 51 4.2K 8)  a 6 18 d 5 94 5 52  3.61  0 18(10)  3 60  0 151 8)  5 69  0 10(10)  5 48  0 161 8)  1 65  0 05(10)  1 63  0.071 8)  0.861 8)  129 3  1 30 ( 0)  152.03  1 921 8)  152 49  4.02  0 19( 8)  3 94  0 181 8)  5 68  0 141 8)  5 54  0 091 8)  1 64  0 051 B )  1 62  0.051 6)  3.20  0 14(11)  2 68  0 26(12)  4 29  0 08111)  0 21112)  1 37  0 05(11)  a 1 60  0.03(12)  C  0.14112)  a  6 92 ( 8)  130 3  1.781 9)  2 64 ( 9)  132 4  0.911 6)  114 5 1 00111) b 142 2 78( 8) 1 135 0 0 95(10)  2 68 ( 7)  314 o * 3 27( 6)  128 6  1.001 8)  293 8  3 55(10)  301 1  2 09 ( 7)  129 0  295 0  2 24 ( 8)  294 0  1 91 ( 8)  129 8  3 07(10) b  123 9  1.51(10)  147.29  1 13(10)  146 08  2.03111)  1.611 8)  EXPT 3  128 6  1 20(11)  146 3* 3 29(12)  151.76  1 21(11)  168 71* 3.58(12)  3.15  0 22(11)  3 19  0 16(12)  4 70  0 12(11)  1 57(12)  325 9* 7 00(12) d 305 6 5 57(11)  129 3  1 13(12)  153.15  0 95(12)  157 54  3.11(11)  3.28  0 18(12)  3 48  0 14(11)  4 SO  0 06(12)  299 0  3 17(11)  312 6  8 30(10)  130 5  2 22(11)  158.44  1 86(11)  161 65  4.19(10)  3.40  0 14(11)  4 14  0 23(10)  4 99  0 14(11)  4.40  96  288 6  2 37(12)  300 3  3 00(10)  129 2  1 49(12)  137 5° 2 56(11) d 139 6 3 70(10) b 136 3 1 47(10)  d 4.78 c 5.18 4.57  149.33  1 19(12)  153 51  2.14(10)  3.69  0 14(12)  3 88  0 23(10)  4 61  0 08(12)  120  292 3  2 09(12)  291  7  1 67(12)  130  7  1 55(12)  131 5  151.87  0 87(12)  149 22  1.33(12)  3.48  0 15(12)  3 61  0 30(12)  4 38  0 07(12)  0  285 1  3 23(11)  24  290 2  2 07(11)  48  291 7  72  273 8  b  2 13(12)  124 3  1 15(11)  110 6* 1 40(12)  0 78(12)  151.94  2 37111)  154 14  1.61(12)  C  Significance Level P<  0 13(121  2 00  0 14(11)  2 52  0 09111)  1 72  0 09(12)  2 53  b  0 12(101  1 71  0 04111)  2 83  d  4.27°  0 11(10)  1 78  0 08(12)  2 43  0.39110)  4.08*  0 07(12)  1 73  0 06(11)  1.76  0.11(12)  6  0.001  0.0I  0.02  0.05  0.18111) 0.47(10)  64  Na  and Mg  f o r 72 h (p<0.05) w h i l e plasma Ca  c o n t r o l range w i t h i n 48 h.  l e v e l s had r e t u r n e d t o the  Of a l l t h e e l e c t r o l y t e s i n v e s t i g a t e d plasma  Mg  + +  i n c r e a s e d by f a r t h e most, r e a c h i n g a maximum average o f 3.10 mEq/L w i t h i n 24 h as compared Plasma M g  + +  t o 2.04 mEq/L f o r c o n t r o l s w i t h i n 24 h (Table X ) .  l e v e l s s t i l l remained e l e v a t e d a t t h e 96 h sampling p e r i o d  however, t h e g r a d u a l r e t u r n t o c o n t r o l l e v e l s coupled w i t h a h i g h v a r i a t i o n i n t h e exposed groups rendered t h i s d i f f e r e n c e s t a t i s t i c a l l y In g e n e r a l , DHA exposure reduced  t h e p r e c i s i o n o f r e g u l a t i o n a s • i n d i c a t e d by  an i n c r e a s e i n the s t a n d a r d e r r o r s i n T a b l e f o r Expt. 3.  non-significant.  X and i l l u s t r a t e d i n F i g . 16  O v e r a l l , t h e s e changes l e d t o a g e n e r a l i n c r e a s e i n t h e t o t a l  plasma o s m o l a l i t y which p e r s i s t e d f o r 72 h a f t e r t o x i c a n t exposure. Hydromineral  Balance  i n Sea Water  (Hematocrit, Muscle Water,  Gut_Water_Content)  In a d d i t i o n t o the d e t e r m i n a t i o n o f plasma e l e c t r o l y t e c o n c e n t r a t i o n s , measurements were made o f b l o o d h e m a t o c r i t , muscle water and g u t water c o n t e n t . I f t h e f r e s h water DHA exposure l e d t o a subsequent d e h y d r a t i o n o f salmon i n sea water, t h i s c o u l d be r e f l e c t e d i n a l o w e r i n g o f muscle water c o n t e n t c o u p l e d w i t h an i n c r e a s e i n t h e r a t e o f s e a water i n g e s t i o n as a c o u n t e r measure . B l o o d h e m a t o c r i t l e v e l s a t t h e v a r i o u s sampling p e r i o d s a r e p r e s e n t e d i n T a b l e XI  and a comparison between exposed arid c o n t r o l groups i s i l l u s t r a t e d  i n F i g . 14 ( t o p ) .  Hematocrit was measured  muscle water was determined  i n E x p t s . 1, 2 and 3, whereas  i n Expt. 2 and 3.  The % water i n the g u t was  measured o n l y i n Expt. 2; t h e r e s u l t s a r e p r e s e n t e d i n T a b l e X I I and a r e i l l u s t r a t e d i n F i g . 15 which a l s o shows t h e r e l a t i o n o f muscle and g u t water d e t e r m i n a t i o n s i n "moribund" salmon t o those o f c o n t r o l and exposed groups.  Table X I .  Hematocrit of sockeye salmon a f t e r a 5-day exposure t o 0.65 mg/L DHA i n f r e s h water followed by exposure t o sea water i n Expts. 1, 2 and 3.  Hematocrit % Time in sea water  Experiment 1  Experiment 3  33.97  1.63(10)  45.00  2.o o ( i o )  24  45. 19  2.19 ( 9)  49. 36  2.67(10)  48  42. 12  1.06(10)  41.74  1.24( 7)  72  38. 33  0.89( 9)  36. 18  0. 63 ( 8) 1. 22( 9)  t-test  a  96  35. 59  0.96( 9)  120  36..58  1.,06(10)  34..63  0. 85( 7)  36.•  1..12(10)  44..76  1..19(11)  3  0  24  38,.99  0..85( 9)  38..34  2 • 01( 8)  48  37 .28  2 .26( 6)  36..53  1 .47(10)  72  35 .05  1..84( 8)  31 .93  1 .26( 7)  96  35 .35  1 .46(10)  37 .75  1 .38( 8)  120  36 .73  1 -76( 8)  31 .60  1 .44( 7 )  0  40.56  0.73(11)  47.83  1.04(12)" 1.36(12)  24  44.80  1.08(11)  42.30  48  41.28  1.10(12)  41.10  0.65(11) 1.40(10)  72  41.86  0.90(11)  40.55  96  40.48  0.90(12)  39.75  1.34(10)  0.82(12)  36.58  0.92(12)  from control  p<0.001, p<0.05 b  a  35. 57  120  differs significantly  Exposed Mean * SE (n)  0  0  Experiment 2  Control Mean ± SE (n)  n  38.88  b  66  F i g u r e 14.  The change ( r e l a t i v e t o c o n t r o l = 100) o f h e m a t o c r i t and muscle water i n sockeye salmon d u r i n g sea-water adaptation. The f i s h had p r e v i o u s l y been exposed t o 0.65 mg/L DHA f o r 120-h i n f r e s h water. P o i n t s f o r h e m a t o c r i t a r e mean % based on E x p t s . 1 , 2 and 3. P o i n t s f o r muscle water a r e mean % based on E x p t s . 2 and 3. The l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e o f the change i s g i v e n a t t h e base o f each graph.  T a b l e XII.  Percentage water i n gut o f sockeye salmon a f t e r a 5-day exposure t o 0.65 mg/L DHA i n f r e s h water f o l l o w e d by exposure t o s e a water (Expt. 2 ) .  Percentage water i n gut Time i n sea water hours  Control Mean + SE(n)  Exposed Mean + SE(n)  0  76.71  0.97(10)  77.86-  0.74(11)  24  76.11  0.52( 9)  80.24  1.71('9)  48  76.96  1.20( 6)  75.61  0.96(10)  72  77.60  0.70( 8)  79.77  1.33( 7)  96  76.88  0.65(10)  77.35  0.39( 8)  120  77.68  0.52( 8)  80.18  differs significantly t-test  from c o n t r o l p<0.01  a  a  0.57( 8)  68  F i g u r e 14 i n d i c a t e s t h a t t h e b l o o d h e m a t o c r i t was a t the end o f the f r e s h w a t e r exposure phase  consistently elevated  (p<0.001) b u t dropped r a p i d l y t o  c o n t r o l v a l u e s a f t e r t h e f i s h had been i n sea water f o r 24 h.  Subsequently  h e m a t o c r i t remained c l o s e t o o r s l i g h t l y below c o n t r o l v a l u e s , a l t h o u g h  after  120 h i n sea water, the h e m a t o c r i t s o f t h e exposed group a g a i n became lower than i n the c o n t r o l s (p<0.05). P a r a l l e l muscle water d e t e r m i n a t i o n s r e s u l t s t h a t t h e DHA  (Table X I I I ) confirmed p r e v i o u s  exposure l e d t o a h y d r a t i o n i n f r e s h water; t h i s  was  shown t o be f o l l o w e d by a d e h y d r a t i o n a f t e r the f i s h had been i n sea water f o r 24 h  ( F i g . 14).  By 48 h, however, the muscle water l e v e l s were r e s t o r e d  t o the c o n t r o l range a l t h o u g h some f l u c t u a t i o n was e v i d e n t .  In E x p t s . 2 and 3  complete muscle water r e g u l a t i o n was r e g a i n e d by 72 h as i l l u s t r a t e d i n Fig.14. Measurements o f the % water i n the g u t d u r i n g E x p t . 2 ( T a b l e XII)showed t h a t muscle d e h y d r a t i o n i n Expt. 2) was  (which approached b u t d i d n o t meet s t a t i s t i c a l  significance  accompanied by an i n c r e a s e i n g u t water c o n t e n t a t 24 h.  The  amount o f water i n t h e g u t was a g a i n s i g n i f i c a n t l y i n c r e a s e d (p<0.05) a f t e r 120 h i n sea water a l t h o u g h t h e muscle water c o n t e n t was by now back t o control levels.  B l o o d h e m a t o c r i t , however was  exposed f i s h o f Expt. 2 a t t h i s time During Expt. 1 one f i s h was  s i g n i f i c a n t l y reduced i n  (Table X I ) .  sampled when, h a v i n g l o s t e q u i l i b r i u m , i t  appeared t o be h a v i n g s e r i o u s problems w i t h muscular c o o r d i n a t i o n .  These  o b s e r v a t i o n s were made a f t e r t h e f i s h had been i n sea water f o r ^ 70 h. A t the 72 h sampling p e r i o d , t h i s f i s h was was q u i t e r i g i d .  s t i l l b r e a t h i n g r e g u l a r l y but  The r e s u l t s o f plasma e l e c t r o l y t e a n a l y s i s f o r t h i s  Table X I I I . Percentage water i n muscle o f sockeye salmon a f t e r a 5-day exposure t o 0.65 mg/L DHA i n f r e s h water f o l l o w e d by exposure t o sea water (Expts. 2 and 3 ) .  P e r c e n t a g e water i n muscle Time i n sea w a t e r hours  Experiment 2  Experiment 3  Control Mean SE(n)  Exposed Mean SE(n)  0  74.79  0.42(10)  75.69  0.18(11)  24  73.27  0.30( 9)  72.39  0.30( 9)  48  73.39  0.76( 6)  74.52  0.20(10)  72  74.91  0.16( 8)  74.51  0.24( 7)  96  74.99  0.44(10)  74.46  0.19( 8)  120  74.92  0.23( 8)  74.99  0.27( 8)  0  79.50  0.53(11)  81.17  0.59(12)  24  76.22  0.32(11)  75.34  0.43(12)  48  76.86  0.20(12)  76.17  0.78(10)  72  77.21  0.27(11)  77.38  0.49(10)  96  77.18  0.57(12)  77.18  0.31(10)  120  77.35  0.63(12)  77.74  0.22(12)  a D i f f e r s s i g n i f i c a n t l y from c o n t r o l p<0.05.  a  70  moribund f i s h are compared i n T a b l e XIV  t o mean v a l u e s f o r c o n t r o l s and  exposed groups taken a t the 72 h sampling p e r i o d and c l e a r l y i n d i c a t e t h a t t h i s f i s h had  l o s t the a b i l i t y t o r e g u l a t e plasma i o n i c l e v e l s .  o f b e h a v i o r a l symptoms t o h y d r o m i n e r a l d u r i n g E x p t s . 2 and  d i s t u r b a n c e was  further  The  relation  investigated  3.  Moribund and D r i f t i n g  Fish  As the a n a l y s i s o f plasma i o n i c composition was  not conducted  until  after  the completion o f b o t h E x p t s . 1 and 2, the p r e s e n c e o r e x t e n t o f i o n i c d i s t u r b a n c e was  not known d u r i n g the second experiment.  Based on b e h a v i o r a l  o b s e r v a t i o n s , however 15 moribund f i s h were c o l l e c t e d d u r i n g the course o f Expt. 2 a t v a r i o u s s t a g e s o f d e b i l i t y and muscle and gut water d e t e r m i n a t i o n s were conducted.  These r e s u l t s t o g e t h e r w i t h a d e s c r i p t i o n o f symptoms are  p r e s e n t e d i n T a b l e XV  and i l l u s t r a t e d i n F i g . 15 f o r comparison w i t h exposed  and c o n t r o l groups i n Expt.  2.  The r e s u l t s i l l u s t r a t e d i n F i g u r e 15 i n d i c a t e t h a t the muscle water c o n t e n t d u r i n g the f r e s h w a t e r exposure p e r i o d was which was  subsequently  greatly elevated indicating hydration  f o l l o w e d by a t i s s u e d e h y d r a t i o n whose s e v e r i t y  t o be r e l a t e d t o the time o f seawater r e s i d e n c e .  appeared  P a r a l l e l measurements  r e v e a l e d a c o n c u r r e n t r i s e i n t h e amount o f water i n the stomach, a l t h o u g h i t appears  t h a t t h i s water i n g e s t i o n  hours p r i o r t o seawater e n t r y in "inverted" f i s h h y d r a t i o n may The presence T a b l e XV  may  have been e l i c i t e d i n some f i s h s e v e r a l  ( F i g . 15).  ( f i s h which had  Muscle water l e v e l s  (Table  l o s t e q u i l i b r i u m ) suggest t h a t the  a l r e a d y be advanced a t t h i s p o i n t i n the i n t o x i c a t i o n o f much h i g h e r water c o n t e n t i n the gut i n one  which was  sampled immediately  t h a t the water accumulation may  XV)  fish  after equilibrium loss  de-  sequence.  (3.0 h i n  suggests  have been t r i g g e r e d by a combination  of  T a b l e XIV. The plasma i o n i c c o m p o s i t i o n o f an "exposed" f i s h which l o s t e q u i l i b r i u m a f t e r 72 h i n s e a water compared t o mean v a l u e s f o r c o n t r o l s and t h e "normal" exposed f i s h sampled a f t e r 72 h i n s e a water i n Expt. 1.  Control  Exposed  Inverted  Sum o f i o n s mEq/L  304.4  307.1  387.6  Chloride mEq/L  133.2  130.8  177.5  Sodium mEq/L  159.4  164.7  187.3  Potassium mEq/L  3.95  3.41  4.47  Calcium mEq/L  5.88  5.93  7.33  Magnesium mEq/L  1.99  2.30  11.00  Hematocrit %  38.3  36.2  36.4  72  Table XV.  S i z e , percentage muscle and gut water and d e s c r i p t i o n of symptoms i n f i s h which appeared to be s e r i o u s l y affected during the course of Expt. 2.  Time  Fork length  Wet weight  cm  g  hours  Fresh water exposure  85. ,05  YF  81. ,56  Fungus on g i i ;  78. .14  85. .67  MB  78. .34  89. ,67  MB  33. .89 31. .96  74. .01 76. .77  89. .79 87. . 39  MB, SGG  35. .72  74. .13  23. .71  71. .22  MB,, SGG, YP MT, , SGG3  35. .79  72. .89 72 .71  77.  16  36. ,42  77. ,97  15. .4  36. ,80  15. .2  35. .20  15. .1  35. .20  14..5  48. ,47  .0  2..0  115.  .1  115.  .1  120.  15. .5  3..0  15. .4  .  15. .4 Time in sea water  MB , YF  36  13. .5  14. .4  15. .4 15. .4  15. .4 14. .9  19. .6  15, .3  20. .3 24. .4  44. .6 88. .2  13. .2 15, .2 15, .2 14, .7  91. .22 87. .24  33. .15  73, .61  90. .06  36, .10 28, .80  (just)  moribund - s t i l l  breathing  MT  collected after  death  SG  swollen gut  SGG greatly  swollen gut  YF  yellow  YP  yellow plasma  fish  2 Control fish Gut contents 780 mOsmAg (SW 824 mOsm/kg)  3  88. .05  74. .44  HM hemorrhage in fin membranes I (J) inverted  76. .29  75. ,32  40. .68  1  MB  Observations  72. ,78  66  78.  16. .1  .2  Gut Water %  78.  28. ,05  ,2  103.  %  28. ,80  13. ,4  103.  Muscle Water  72, .91  90, .07 80, .23  87, .71 80 .38  MB, YP rigid  IJ  MT, .' SG, HM SG, HM SG  I,  I, MT  SG, YP  SG, HM, YP lhr  73  D)  CD  Ejrposure  Time in s e a w a t e r  (hours) * P <0.05  F i g u r e 15. R e l a t i o n s between muscle (a) and g u t water % (b) d e t e r m i n a t i o n s on moribund f i s h and mean v a l u e s f o r exposed and c o n t r o l groups i n E x p t . 2. V e r t i c a l b a r s i n d i c a t e ±SE o f the mean.  74  t o x i c a n t and seawater exposure, as t h e muscle water c o n t e n t o f t h i s (76.29%) was  s t i l l h i g h e r than t h a t o f t h e c o n t r o l  fish  (74.79%) o r o f the exposed  (75.69%) g r o u p s (Table X I I I ) . Among t h e symptoms l i s t e d i n T a b l e XV i s t h a t o f a y e l l o w i s h t i n g e o f the f i s h which was accompanied by a y e l l o w i s h plasma i n those c a s e s i n which b l o o d samples were t a k e n .  T h i s c o l o r was l a t e r a t t r i b u t e d t o a form o f  t o x i c a n t - i n d u c e d j a u n d i c e and i s d i s c u s s e d i n Appendix I I I . As t h e o b s e r v a t i o n s made on moribund f i s h i n E x p t . 2 s u g g e s t e d a s e r i o u s o s m o t i c i m b a l a n c e ; d u r i n g t h e c o u r s e o f E x p t . 3 t h r e e f i s h were sampled a t 48, 72 and 96 h when i t became a p p a r e n t t h a t they c o u l d no l o n g e r m a i n t a i n p o s i t i o n a g a i n s t the c u r r e n t .  These f i s h were termed " d r i f t e r s " and were  a n a l y z e d s e p a r a t e l y i n the hope o f e s t a b l i s h i n g a l i n k between t h e i r b e h a v i o r and t h e degree o f i o n i c imbalance.  Based on p a s t o b s e r v a t i o n s ,  "drifting"  f i s h g e n e r a l l y e x p i r e d w i t h i n 24-48 h, so t h a t they would have p r o b a b l y added t o t h e o v e r a l l m o r t a l i t i e s by t h e c o n c l u s i o n o f E x p t . 3. The plasma e l e c t r o l y t e l e v e l s o f d r i f t i n g f i s h i n E x p t . 3 a r e compared t o t h o s e o f c o n t r o l and exposed groups i n Figs.16 A t o F and show t h a t a l l the i o n s measured were c o n s i d e r a b l y above t h e i r r e s p e c t i v e "exposed" means. breakdown o f t o t a l i o n s  The  (Fig.16 A) i n d i c a t e s t h a t t h e g r e a t e s t changes appear-  ••i.> ed t o have o c c u r r e d i n plasma M g w i t h t i m e i n sea water.  + +  Plasma C a  (Fig.16 F) which seemed t o be + +  regulation  increasing  (Fig.16 E) i n exposed groups  was r e s t o r e d w i t h i n 48 h and t h e r e was some subsequent overcompensation when compared  to controls.  However, plasma C a  remained r o u g h l y d o u b l e a t t h i s t i m e .  + +  levels i n drifting  The major plasma c a t i o n s N a  +  fish and C l  4 1 0  390  370  h  Total Ions  350  Control  CJ  Exposed  0)  Drif ter  330  310  290  270  »  1  0  1  1  24  48  0.001  0.05  i  72  96  1 . 120  NS  NS  i  Time in sea water (hours) | 0.01 F i g u r e 16, (a-f)  NS  A comparison o f plasma e l e c t r o l y t e c o n c e n t r a t i o n s measured i n 3 d r i f t i n g f i s h , w i t h the means o f c o n t r o l and exposed groups d u r i n g E x p t . 2. V e r t i c a l b a r s i n d i c a t e ±SE o f the mean and the l e v e l o f s i g n i f i c a n c e o f the d i f f e r e n c e between c o n t r o l and exposed means i s g i v e n i n the box below each graph.  Time in sea water (hours)  77  78  persisted at greatly elevated levels i n d r i f t i n g  f i s h a t a time when the  p r e v i o u s l y exposed b u t b e h a v i o r a l l y normal f i s h were r e g u l a t i n g t h e s e i o n s towards c o n t r o l v a l u e s  ( F i g . 16 B,C). While plasma K  +  l e v e l s remained  c l o s e r t o "exposed" means t h a n any o f t h e o t h e r i o n s , t h e y d i d appear t o be i n c r e a s i n g w i t h time i n sea water. These experiments c o n f i r m e d t h a t t h e o s m o t i c imbalance b r o u g h t on by s u b l e t h a l exposure o f salmon t o DHA i n f r e s h water was a l s o accompanied by an e l e c t r o l y t e disturbance.  Furthermore, t h e subsequent t r a n s f e r o f these f i s h  i n t o c l e a n s e a water r e s u l t e d i n a marked l o s s i n t h e p r e c i s i o n o f plasma ion  r e g u l a t i o n and i n a g e n e r a l d e h y d r a t i o n . As t h e maintenance o f h y d r o m i n e r a l b a l a n c e i n salmonids r e l i e s  heavily  on a r e d u c t i o n o f the g i l l p e r m e a b i l i t y and an i n c r e a s e i n t h e g u t p e r m e a b i l i t y t o water, an experiment was conducted t o measure t h e e f f e c t s o f DHA exposure on t h i s a s p e c t o f t h e f u n c t i o n o f t h e s e two o s m o r e g u l a t o r y organs.  A l t e r a t i o n s i n t h e p e r m e a b i l i t y o f e i t h e r organ c o u l d  contribute  t o t h e r a p i d r i s e i n plasma e l e c t r o l y t e l e v e l s which was o b s e r v e d above.  B.  EFFECTS OF DHA  ON GILL AND  GUT PERMEABILITY TO WATER INTRODUCTION  The movement o f e u r y h a l i n e f i s h i n t o sea water from the f r e s h w a t e r environment t r i g g e r s a complex sequence o f a d a p t i v e mechanisms which a c t i n c o n c e r t t o m a i n t a i n m i n e r a l homeostasis.  Although r a p i d dehydration i s  a v o i d e d through a r e d u c t i o n i n the p e r m e a b i l i t y o f t h e b r a n c h i a l t o water  epithelium  (Maetz, 1970a) a n e t water l o s s s t i l l o c c u r s and sea water i s  swallowed, absorbed a c r o s s the gut and s u b s e q u e n t l y p h y s i o l o g i c a l l y " d i s t i l l e d " t o r e p l a c e t h e f r e e water l o s t by t h e g i l l s  (Smith, 1930).  The  r e d u c t i o n i n g i l l p e r m e a b i l i t y s e r v e s t o l i m i t t h i s i n e v i t a b l e water l o s s w h i l e the mechanism  of i n t e s t i n a l  water t r a n s p o r t e n a b l e s the f i s h t o  r e p l a c e b r a n c h i a l and r e n a l water l o s s e s .  Impediment  i n the r e d u c t i o n o f  f r e e water movement a c r o s s t h e g i l l s would p l a c e an a d d i t i o n a l l o a d on t h e gut  t o absorb more water and c o n s e q u e n t l y on the b r a n c h i a l and r e n a l i o n  pumps which d i s p o s e o f t h e e x c e s s s a l t . S i n c e the e l e c t r o l y t e imbalance o b s e r v e d d u r i n g the p r e s e n t study c o u l d have o r i g i n a t e d i n a t o x i c a n t - r e l a t e d i n t e r f e r e n c e w i t h normal p e r m e a b i l i t y changes i n t h e g i l l  and/or g u t , an experiment was conducted t o  t e s t whether s u b l e t h a l exposure o f sockeye salmon t o DHA  i n f r e s h water  i n t e r f e r e d w i t h subsequent p e r m e a b i l i t y changes i n the g i l l s and d u r i n g sea water a d a p t a t i o n .  intestines  The water p e r m e a b i l i t y o f the i s o l a t e d  gill  and the water t r a n s p o r t a b i l i t y o f t h e i s o l a t e d gut were measured i n DHAexposed f i s h a f t e r a 24 h r e c o v e r y p e r i o d i n sea water. Based on the i s o l a t e d g i l l  t e c h n i q u e o f Bellamy (1961) and U t i d a e t a l .  (1967), a p r o c e d u r e was d e v e l o p e d t o measure t h e change i n buoyancy o f an isolated g i l l  a r c h i n c u b a t e d i n sea water f o r 1 h.  T h i s change i n buoyancy  80  was  assumed t o be due t o t h e l o s s o f water a c r o s s t h e g i l l  e p i t h e l i u m making  the  p r e p a r a t i o n more dense and t h e r e f o r e s i n k i n g deeper i n t h e s e a water  r e g i s t e r i n g an apparent weight i n c r e a s e on an e l e c t r o b a l a n c e .  and  The water  t r a n s p o r t a b i l i t y o f t h e g u t was measured by t h e weight changes o f i n t e s t i n a l sacs  (Oide and U t i d a , 1967; U t i d a e t a l . , 1967) f i l l e d w i t h and i n c u b a t e d i n  Cortland saline  (Wolf, 1963).  To enable sampling o f i n d i v i d u a l f i s h w i t h o u t  d i s t u r b a n c e o f t h e o t h e r s , an apparatus was d e s i g n e d i n which e l e c t r o s h o c k was  used t o r a p i d l y s t u n a s e l e c t e d i n d i v i d u a l w i t h no d i s t u r b a n c e o f t h e  o t h e r f i s h i n t h e tank. MATERIALS AND METHODS Two  s i m i l a r experiments were conducted, one i n June and t h e second i n  J u l y 1978.  In both experiments, t e n f i s h were exposed t o 0.4 mg/L DHA i n  f r e s h water f o r 120 h.  An e q u a l number o f f i s h s e r v e d as c o n t r o l s .  t o x i c a n t was then d i s c o n t i n u e d , FW r e p l a c e d by SW, and 24 h l a t e r f i s h were removed and t h e g i l l to  and g u t p r e p a r e d f o r i n c u b a t i o n .  The  individual The exposure  0.65 mg/L DHA which was s u b l e t h a l t o sockeye salmon d u r i n g t h e i o n i c  b a l a n c e experiments p r o v e d t o be l e t h a l t o f i s h c o n f i n e d i n t h e tubes used for  t h e p e r m e a b i l i t y study.  The c h o i c e o f 0.4 mg/L f o r t h e g i l l  experiments was made on t h e b a s i s o f a comparison mortality  permeability  o f t h e time t o 50%  (LT50) o f t h e "tubed" f i s h exposed t o 0.65 mg/L DHA,to t h e  t o x i c i t y curve  ( F i g . 9)  o b t a i n e d p r e v i o u s l y f o r free-swimming  fish.  By  e x t r a p o l a t i o n , 0.4 mg/L DHA was chosen t o ensure 100% f i s h s u r v i v a l d u r i n g a 5-day exposure Sockeye  period.  salmon smolts were o b t a i n e d from t h e G r e a t C e n t r a l Lake r u n i n  March 1978 and k e p t i n outdoor h o l d i n g tanks a t PEI under c o n d i t i o n s d e s c r i b e d i n t h e G e n e r a l Methods s e c t i o n . the  A p p r o x i m a t e l y 25 f i s h were d i p n e t t e d from  outdoor tanks and t r a n s f e r r e d t o t h e l a b o r a t o r y donut tanks i n a bucket  81  c o n t a i n i n g 33 mg/L MS222 d i s s o l v e d i n water o f 10  /oo s a l i n i t y .  F i s h were  g i v e n a minimum o f 96 h t o a c c l i m a t e t o t h e f l o w c o n d i t i o n s i n t h e l a b o r a t o r y , were f e d once d a i l y w i t h OMP, and s t a r v e d f o r 24 h p r i o r t o t r a n s f e r t o t h e experimental tanks.  A t t h e time o f t r a n s f e r , a l i g h t  a n e s t h e s i a was brought  on by t h e a d d i t i o n o f a seawater s o l u t i o n o f 2 g MS222 t o t h e tank  (36 mg/L)  a f t e r s h u t t i n g o f f t h e water s u p p l y and adding an a i r - s t o n e l i g h t l y oxygen i n t o the water.  bubbling  As soon as t h e f i s h began t o d r i f t w i t h t h e c u r r e n t ,  they were n e t t e d and t r a n s f e r r e d t o t h e tubes i n t h e e x p e r i m e n t a l tanks. of  Fish  comparable s i z e were s e l e c t e d and d i s t r i b u t e d a l t e r n a t e l y between t h e  c o n t r o l and t e s t t a n k s .  Each tank c o n t a i n e d 10 f i s h which were p l a c e d i n  i n d i v i d u a l p l a s t i c tubes suspended i n t h e water c u r r e n t (Fig.17 ) .  The up-  stream end o f each tube had a 6 mm mesh n e t t i n g g l u e d t o i t , w h i l e a t t h e downstream end, the n e t t i n g was a t t a c h e d t o a removable s p l i t p l a s t i c  ring.  A f t e r t h e f i s h was i n s e r t e d , t h e r i n g was r e p l a c e d and f o l l o w i n g r e c o v e r y from a n e s t h e s i a , t h e f i s h r e s t e d q u i e t l y on t h e tube bottom o r swam s l o w l y against the current.  A t t a c h e d t o t h e l e n g t h o f each tube were two s t r i p s o f  2.5 cm aluminum tape  (3M S c o t c h No. 425) forming e l e c t r o d e s on e i t h e r s i d e o f  the f i s h  Each tube was w i r e d t o a V a r i a c through a s w i t c h i n g  circuit  (Fig.18 ) .  so t h a t v o l t a g e c o u l d be a p p l i e d t o one tube a t a time.  a l s o covered w i t h b l a c k p l a s t i c t o minimize apparatus,  v i s u a l disturbance.  i n d i v i d u a l f i s h c o u l d be i n s t a n t a n e o u s l y stunned  and removed from t h e water w i t h o u t d i s t u r b i n g t h e upstream The  Each tube was Using  i n their  this  tubes  fish.  f i s h were g i v e n an a c c l i m a t i o n p e r i o d o f 72 h b e f o r e t h e s t a r t o f t h e  t o x i c a n t exposure.  An a i r i - s t o n e ensured t h a t d i s s o l v e d oxygen l e v e l s remained  i n excess o f 90% s a t u r a t i o n throughout maintained  t h e experiments  and t h e temperature  was  a t 11.5 ±0.S^C.After t h e 120 h exposure p e r i o d , f o l l o w e d by 24 h  82  F i g u r e 17.  I l l u s t r a t i o n o f a s e c t i o n o f a donut tank showing the p o s i t i o n i n g o f p l a s t i c tubes t o which sockeye salmon were c o n f i n e d d u r i n g a 120 h exposure t o 0.4 mg/L DHA i n f r e s h water.  Figure  18.  D e t a i l e d i l l u s t r a t i o n o f one o f the p l a s t i c tubes used f o r the containment o f sockeye salmon d u r i n g t h e i r exposure t o 0.4 mg/L DHA p r i o r t o the g i l l p e r m e a b i l i t y experiments.  84  i n sea water, i n d i v i d u a l f i s h were stunned by e l e c t r o s h o c k The tube c o n t a i n i n g t h e i m m o b i l i z e d f i s h was so as n o t t o d i s t u r b the n e i g h b o r i n g f i s h . the  (20V AC f o r 1 0 s . ) .  carefully lifted The f i s h was  from the water  then removed from  tube, stunned by a blow on the head, measured, b l o t t e d and weighed.  e n t i r e b r a n c h i a l b a s k e t was  then r a p i d l y removed as shown i n F i g . 19,  with frequent r i n s i n g with c h i l l e d  (10°C) C o r t l a n d s a l i n e .  b l e e d i n g from the s e v e r e d p s e u d o b r a n c h i a l cautery.  a r t e r y was  A f t e r removal, t h e f i r s t b r a n c h i a l a r c h was  x 1.5  cm) was  checked by means o f t i e d o f f with s i l k A  r i n s e w i t h d e i o n i z e d water, the e n t i r e p r e p a r a t i o n was i n c u b a t i o n apparatus  c o n s t a n t temperature b a t h was beaker was  and  stainless  g l u e d t o the v e n t r a l i n s i d e s u r f a c e o f the  f i r s t g i l l b a r w i t h a drop o f Eastman 910 c y a n o a c r y l a t e a d h e s i v e .  beaker o f the g i l l  1-12  D u r i n g s t e p 3,  s e p a r a t e d from t h e r e s t o f the g i l l b a r s o f the b r a n c h i a l b a s k e t . s t e e l hook (0.46 mm  The  After a  t r a n s f e r r e d t o the  ( F i g . 2 0 ) i n which water from the  c i r c u l a t e d through the beaker water j a c k e t .  f i l l e d w i t h a e r a t e d sea water  (810 mOsM) and temperature  i b r a t e d w h i l e the d i s s e c t i o n s were c a r r i e d o u t . o i n c u b a t e d f o r 1 h a t 11.9 +0.01  The  equil-  The g i l l p r e p a r a t i o n was  then  C  (X ±SE) .  The apparent change i n g i l l  weight  was monitored on a Sanyo 4092U V i d e o M o n i t o r u s i n g a Sanyo V i d e o Camera VC1150 and r e c o r d e d on a Sanyo V i d e o Tape Recorder At  the end o f the g i l l  (VTR 1200)  f o r future processing.  i n c u b a t i o n p e r i o d , the p r e p a r a t i o n was  from the b a l a n c e , the g i l l b a r s d i s s e c t e d away from the c e n t r a l  removed  cartilage  ( F i g . 19, 13-14) p l a c e d i n t a r e d aluminum d i s h e s and oven d r i e d t o c o n s t a n t weight.  The weight change o f t h e g i l l was  e x p r e s s e d i n mg/100 mg  dry  gill  weight/hr. The gut e x t r a c t i o n was way  s t a r t e d as soon as the g i l l  (^ 5 min from f i s h e l e c t r o s h o c k ) .  i n c u b a t i o n was  The abdominal c a v i t y was  under  opened and  D i s s e c t i o n p r o c e d u r e f o l l o w e d t o o b t a i n the f i l a m e n t p r e p a r a t i o n used i n the g i l l p e r m e a b i l i t y experiment. F i g u r e -12 shows the f i n i s h e d f i r s t b r a n c h i a l a r c h u n i t . A f t e r c o m p l e t i o n o f the experiment, the c a r t i l a g e was trimmed o f f ( F i g u r e -13 and -14) p r i o r t o the d e t e r m i n a t i o n o f d r y weight.  F i g u r e 20-  The a p p a r a t u s used t o measure the weight l o s s o f i s o l a t e d sockeye salmon g i l l f i l a m e n t s i n c u b a t e d i n s e a water. The f i s h had been p r e v i o u s l y exposed t o 0.4 mg/L DHA f o r 120 h i n f r e s h - w a t e r .  oo  87  the i n t e s t i n e was t r a n s e c t e d i n t h e r e g i o n o f t h e v e n t ,  f r e e d from t h e  s u p p o r t i n g mesentery a n t e r i o r l y and c u t j u s t p o s t e r i o r t o t h e l a s t  pyloric  o caecae. saline  The g u t was f l u s h e d c l e a n w i t h c h i l l e d  (10 C ) , a e r a t e d  Cortland  (285 mOsM), then f i l l e d w i t h 200 u l s a l i n e by means o f a 1 ml s y r i n g e  f i t t e d w i t h a 21 G needle t i p p e d w i t h 1 cm o f PE 60 (Intramedic)  polyethylene  tubing.  Both ends o f t h e i n t e s t i n a l s a c were t i e d o f f w i t h  adhering  f a t was g e n t l y t e a s e d o f f , and a f t e r b l o t t i n g , t h e p r e p a r a t i o n was  weighed  t o t h e n e a r e s t mg on a M e t t l e r H 43 a n a l y t i c a l  I t was then t r a n s f e r r e d t o t h e i n c u b a t i o n b a t h l i g h t l y a e r a t e d C o r t l a n d s a l i n e a t 11.9 ±0.1°C o f approximately i n the saline.  10 min,  surgical  silk,  balance.  ( F i g . 21) c o n t a i n i n g  (X ±SE)  f o r 1 h.  At intervals  t h e sac was removed, b l o t t e d , weighed, arid r e p l a c e d  A f t e r t h e f i n a l weighing, t h e s i l k was snipped  o f f , and t h e  gut was d r i e d t o c o n s t a n t weight i n a t a r e d aluminum d i s h i n a f o r c e d d r a f t oven a t 105°C.  Weight change was expressed  i n mg/100 mg d r y g u t wt./h.  RESULTS As t h e changes i n weight o f c o n t r o l g i l l s s i g n i f i c a n t l y d i f f e r e n t , t h e d a t a were p o o l e d  i n Expts.  (Table X V I ) .  1 and 2 were n o t The combined  r e s u l t s i n d i c a t e t h a t t h e p r i o r s u b l e t h a l exposure o f sockeye salmon t o DHA i n f r e s h water l e d t o a s i g n i f i c a n t l y g r e a t e r weight l o s s by t h e i r g i l l s i n sea water.  The r e s u l t s o f t h e g u t i n c u b a t i o n experiment i n d i c a t e t h a t  the amount o f water t r a n s p o r t e d by t h e i n t e s t i n e s o f p r e v i o u s l y exposed was  c o n s i d e r a b l y lower t h a n t h e c o r r e s p o n d i n g  c o n t r o l s (Table XVII), the  amount o f v a r i a t i o n i n b o t h groups was l a r g e and t h e d i f f e r e n c e was n o t significant.  while fish  Figure  21.  Diagram showing t h e a p p a r a t u s used f o r the i n c u b a t i o n o f i s o l a t e d i n t e s t i n a l s a c s taken from sockeye salmon which had p r e v i o u s l y been exposed t o 0.4 mg/L DHA f o r 120 h.  T a b l e XVI.  F i s h s i z e and the l o s s i n weight (during sea water i n c u b a t i o n ) o f g i l l arches i s o l a t e d from j u v e n i l e sockeye salmon which had been p r e v i o u s l y exposed t o 0.4 mg/L DHA i n f r e s h water f o r 5 days* . 3  (Mean ±SE)  Fork  length cm  Wet  Weight g  G i l l weight l o s s mg/100 mg d r y weight/h  Control (19)  14.3  ±0.16  30.48 ±0.87  5.685 ±0.27  Exposed (17)  14.7  ±0.18  31.99 ±1.00  6.471  a  Weight o f water  ±0.25  lost.  G i l l s were e x t r a c t e d  from f i s h which had been i n SW f o r 24  t o 36 h. °Differs s i g n i f i c a n t l y from c o n t r o l p<0.05  (t-test).  T a b l e XVII. F i s h s i z e and the l o s s i n weight o f i s o l a t e d i n t e s t i n a l s a c s t a k e n from j u v e n i l e sockeye salmon which had p r e v i o u s l y been exposed t o 0.4 mg/L DHA i n f r e s h water f o r 5 d a y s . ' a  b  (Mean ±SE) Fork length cm  Wet  Weight g  Gut weight  loss  mg/100 mg d r y weight/h  Control  (14)  14.1 ±0.17  29.44 ±0.87  45.18 ±4.37  14.7 ±0.18  31.99 ±1.00  37.73 ±4.31  Exposed  (17)  I n t e s t i n e s were e x t r a c t e d from f i s h which had been i n SW f o r 24 t o 36 h. I n t e s t i n a l s a c s f i l l e d w i t h and i n c u b a t e d i n C o r t l a n d  saline.  91  DISCUSSION  Water and  e l e c t r o l y t e homeostasis i n f i s h i s a c h i e v e d  n a t e d f u n c t i o n s o f the g i l l ,  gut and k i d n e y .  through the  coordi-  S i n c e plasma e l e c t r o l y t e l e v e l s  normally  are m a i n t a i n e d w i t h i n a r e l a t i v e l y narrow r a n g e , t h e i r measurement  provides  a broad assessment o f the performance o f t h i s i n t e g r a t e d homeostatic  function.  G e n e r a l d e v i a t i o n s from the normal range do n o t e x p l a i n which  c o n t r o l mechanisms have been d i s t u r b e d but  the p a t t e r n s o f osmotic and  change i n r e l a t i o n t o the e x t e r n a l environment can may  be  involved.  osmotic and  suggest which mechanisms  However,the s p e c i f i c mechanisms by which the t o x i c a n t  a f f e c t s h y d r o m i n e r a l b a l a n c e were n o t The p r e s e n t  ionic  (DHA)  s t u d i e d i n the p r e s e n t i n v e s t i g a t i o n .  study e s t a b l i s h e d t h a t s u b l e t h a l exposure t o DHA  altered  i o n i c b a l a n c e i n sockeye salmon smolts i n f r e s h water.  In  a d d i t i o n , when p r e v i o u s l y exposed f i s h were t r a n s f e r r e d i n t o sea water, hydrom i n e r a l b a l a n c e was normal.  a g a i n d i s t u r b e d f o r s e v e r a l days b e f o r e  Exposure t o DHA  a l s o l e d t o a change i n g i l l p e r m e a b i l i t y .  o b s e r v e d accumulation o f f l u i d i n the gut c o u l d not be d i f f e r e n c e i n the water a b s o r p t i v e represent  returning to An  a t t r i b u t e d to a  f u n c t i o n o f the i n t e s t i n e but  could  an i n c r e a s e i n the i n g e s t i o n o f sea water i n response t o dehy-  d a t i o n i n the h y p e r t o n i c medium.  S t u d i e s on DHA  a c i d accumulates s e v e r a l - h u n d r e d - f o l d the k i d n e y ;  uptake showed t h a t the  i n the t i s s u e s , n o t a b l y  the g i l l  resin and  which are organs d i r e c t l y i n v o l v e d i n e l e c t r o l y t e b a l a n c e i n  salmon. A c o n s i d e r a t i o n o f the o v e r a l l changes o b s e r v e d i n h y d r o m i n e r a l b a l a n c e and  i n several hematological  toxic action:  1)  DHA  parameters suggests t h r e e p o s s i b l e p a t t e r n s  directly affects g i l l  i n c r e a s e s the p a s s i v e movements o f water and  and gut p e r m e a b i l i t y electrolytes.  2)  DHA  of  and exposure  92  l e a d s t o h y p o x i a and t h e observed changes i n h e m a t o c r i t r e p r e s e n t a secondary response; 3) it  DHA a f f e c t s kidney  i s o f course p o s s i b l e t h a t a l l t h r e e o f these  and e l e c t r o l y t e s function.  a c t i o n s occur  Although  simultaneously,  they n e v e r t h e l e s s have been p a r t i t i o n e d f o r t h e purposes o f t h e d i s c u s s i o n and  as a r e s u l t some r e p e t i t i o n has been  unavoidable.  Changes i n P e r m e a b i l i t y - G i l l Among t h e mechanisms u t i l i z e d by e u r y h a l i n e f i s h t o m a i n t a i n i o n i c balance  i n media d i f f e r i n g w i d e l y  i n osmotic p r e s s u r e  s i t i o n i s t h a t o f a change i n g i l l p e r m e a b i l i t y . osmotic p e r m e a b i l i t y o f the g i l l  osmotic and  and i o n i c compo-  The maintenance o f low  i s e s s e n t i a l t o m i n i m i z e water g a i n i n f r e s h  water and thus reduces t h e amount o f work r e q u i r e d by t h e k i d n e y . t o water b a l a n c e .  maintain  When e u r y h a l i n e f i s h move from f r e s h water i n t o t h e marine  environment, a r e d u c t i o n i n g i l l p e r m e a b i l i t y f u n c t i o n s t o d i m i n i s h t h e osmotic l o s s o f water due t o t h e g r a d i e n t between t h e b l o o d plasma mOsM/kg) and sea water  <> 1000 mOsM/kg).  Evans  (^ 300  (1969) r e p o r t e d a lower  gill  water p e r m e a b i l i t y i n SW-adapted as compared t o FW-adapted f l o u n d e r and e e l s . However, t h e r e e x i s t s a c e r t a i n time l a g b e f o r e these a d a p t i v e changes a r e complete.  permeability  The r a t e o f b r a n c h i a l water l o s s i n e e l s j u s t  after  t r a n s f e r from FW t o SW was almost t r i p l e t h a t o b s e r v e d a f t e r 3 days i n SW (Oide and U t i d a , 1968) and Kamiya  (1967) found t h a t g i l l s e x c i s e d from e e l s  a f t e r 6-12 h a d a p t a t i o n t o SW l o s t s i g n i f i c a n t l y l e s s water than FW g i l l s and if  t h e f i s h were g i v e n 24 h i n SW, t h e i r g i l l s behaved as SW g i l l s .  o f t h i s time l a g i n t h e e s t a b l i s h m e n t i n the present  study  of g i l l permeability  Because  characteristics,  sockeye salmon were g i v e n a t l e a s t 24 h o f sea water  adaptation p r i o r t o g i l l  and g u t e x t r a c t i o n .  The p e r i o d i n sea water v a r i e d  from 24 ^ 36 h as i t took about 75 min f o r a complete experiment t o be performed on one f i s h .  93  A f t e r e n t r y o f t h e f i s h i n t o t h e s e a , osmotic l o s s o f water i s r e c o v e r e d by i n g e s t i o n o f SW which i s then absorbed a c r o s s the i n t e s t i n e and s u b s e q u e n t l y physiologically  " d i s t i l l e d " with the bulk of Na  across the g i l l s . ational  and C l  +  excretion occurring  As t h e s e mechanisms take some time t o become f u l l y o p e r -  ( K i r s c h and Mayer-Gostsn, 1973), t h e r e d u c t i o n i n g i l l p e r m e a b i l i t y  be an e f f e c t i v e and  e n e r g e t i c a l l y i n e x p e n s i v e method o f l i m i t i n g  upon e n t r y i n t o sea water.  may  dehydration  The s t r a t e g y used t o a c c o m p l i s h a d a p t a t i o n t o sea  water appears t o v a r y w i t h s p e c i e s .  Lahlou e t a i .  (1975) showed t h a t the  t r o u t Salmo i r r i d e u s depends p r i m a r i l y on r e d u c t i o n o f p e r i p h e r a l water  loss  and t h e r e f o r e l e s s on t h e a c t i v e uptake o f i o n s and water i n the i n t e s t i n e . T h i s e n e r g e t i c a l l y e c o n o m i c a l s t r a t e g y was  l i n k e d t o the o b s e r v a t i o n  r e l a t i v e l y slow s t a r t - u p time f o r t h e g i l l  Na  +  e x c r e t o r y mechanism.  of a In t h i s  case, l i m i t i n g the l o s s o f water appears t o be "cheaper" than r e p l a c i n g i t after loss.  The maintenance o f an e s s e n t i a l l y homoiosmotic i n t e r n a l concen-  t r a t i o n d u r i n g a d a p t a t i o n t o sea water i n t h e rainbow t r o u t r e l i e s on a r a p i d r e d u c t i o n o f osmotic p e r m e a b i l i t y  (Gordon, 1963), much f a s t e r  than some o t h e r e u r y h a l i n e s p e c i e s such as t h e e e l , f l o u n d e r 1966) and t h e k i l l i f i s h In  (Maetz e t a l . ,  heavily  (Motais e t  1967).  t h e p r e s e n t study, normal sockeye salmon were o b s e r v e d t o m a i n t a i n  e s s e n t i a l l y homoiosmotic r e g u l a t i o n d u r i n g a d a p t a t i o n t o sea water. o t h e r hand the salmon which had been pre-exposed t o DHA  sea water.  On the  i n f r e s h water showed  a c o n s i d e r a b l e l o s s o f plasma e l e c t r o l y t e r e g u l a t o r y p r e c i s i o n a f t e r to  al.,  transfer  I f sockeye salmon r e l y on a r e d u c t i o n i n p e r m e a b i l i t y t o a  s i m i l a r e x t e n t as do t r o u t , the o b s e r v e d i o n i c and o s m o t i c d i s t u r b a n c e be l i n k e d i n p a r t t o a t o x i c a n t - i n d u c e d i n c r e a s e i n g i l l p e r m e a b i l i t y . more pronounced water l o s s a c r o s s i s o l a t e d g i l l s p r e v i o u s l y exposed t o DHA  may The  o f salmon which had been  supports t h i s hypothesis.  94  In many s p e c i e s , d u r i n g t h e time t h a t osmotic p e r m e a h i l i t y i s b e i n g reduced, t h e r e i s a p r o g r e s s i v e augmentation o f b r a n c h i a l p e r m e a b i l i t y t o Na  +  and C l  (Maetz, 1974)  pumping e f f i c i e n c y and 1969).  as w e l l as an i n c r e a s e i n the i n t e s t i n a l  i t s attendant  In t h r e e e u r y h a l i n e  species  v e r y low r a t e o f sodium t u r n o v e r 29.5  t o 46.9%  i n sea water  s o l u t e - l i n k e d water f l o w  to maintain  i n FW  (0.01  t o 0.55%  turnover  These s p e c i e s r e l y on the r a p i d the  t o sea water, the  c o n s i d e r a b l y from FW  c o n t r a s t , the rainbow t r o u t m a i n t a i n s  a low N a  as w e l l as an o n l y s l i g h t l y i n c r e a s e d C l  +  levels.  turnover  turnover  rate  electrolyte In marked  i n sea water  (Lahlou e_t al^. ,  +  i n f l u x i n c r e a s e d by 4-5  times  o u t f l u x o n l y doubled, l e a d i n g t o a net g a i n o f sodium u n t i l death. t r o u t , the s t r a t e g y appears t o be p r i m a r i l y one water l o s s d u r i n g a d a p t a t i o n  t o sea water.  advantageous, such a s t r a t e g y w i l l render  of l i m i t i n g  1975;  In  while the  s a l t loading  and  Thus a l t h o u g h e n e r g e t i c a l l y s p e c i e s u s i n g i t more v u l n e r a b l e  to  d i s t u r b a n c e by f a c t o r s which d i r e c t l y or i n d i r e c t l y l e a d t o  i n c r e a s e d g i l l p e r m e a b i l i t y , as the h o m e o s t a t i c mechanisms which s a l t s and  (4-10%/h)  L a h l o u e t a l . (1975) a l s o found t h a t when t r o u t were m i l d l y  s t r e s s e d -during seawater a d a p t a t i o n , N a  hydromineral  intestine  a c c o r d i n g l y , t h e i r p e r m e a b i l i t y to both  during adaptation  l e v e l s i n the b l o o d plasma d e p a r t  to  +  s a l t pumping mechanism i n the g i l l s and  s a l t and water i s h i g h and  the  i n t e r n a l Na /h)rose  (Maetz, 1970b), w i t h t h e r a t e o f  s a l t and water b a l a n c e ;  Gordon, 1963).  (Skadhauge,  (the f l o u n d e r , e e l and k i l l i f i s h )  generally d i r e c t l y proportional to s a l i n i t y . a c t i v a t i o n of a powerful  salt  absorb water may  be e a s i l y  excrete  overloaded.  I f the s t r a t e g y f o l l o w e d by the rainbow t r o u t i s r e p r e s e n t a t i v e o f salmonids i n g e n e r a l , then a r a p i d s a l t l o a d i n g o f DHA-exposed sockeye salmon may  o c c u r , p l a c i n g an e x c e s s i v e burden on osmoregulatory mechanisms which are  95  designed  t o a v o i d s a l t l o a d i n g r a t h e r than t o d e a l w i t h i t .  A l t h o u g h the p e r m e a b i l i t y o f the g i l l s t o s a l t was study,  not determined i n t h i s  an augmentation i n b r a n c h i a l i o n i c p e r m e a b i l i t y c o u l d e x p l a i n the r a p i d  r i s e i n plasma e l e c t r o l y t e l e v e l s i n DHA-exposed salmon undergoing  adaptation  to  permeability  sea water.  induced by DHA  The mechanisms behind exposure a r e unknown.  accumulated by the g i l l s  the apparent i n c r e a s e i n g i l l  Residue a n a l y s i s showed t h a t DHA  (Appendix 1-4)  so a d i r e c t a c t i o n on the  c h a r a c t e r i s t i c s o f t h e b r a n c h i a l e p i t h e l i u m may  have o c c u r r e d .  was  permeability  Another  p o s s i b i l i t y c o u l d be t h a t i n response t o DHA-induced h y p o x i a a g r e a t e r e f f e c t i v e exchange a r e a was  p e r f u s e d w i t h b l o o d a t the time t h a t the  were e x t r a c t e d and i n c u b a t i o n o f g i l l  arches  gills  i n sea water l e d t o a more  r a p i d water l o s s . Increases  i n t h e f u n c t i o n a l s u r f a c e a r e a o f the g i l l s have been shown t o  o c c u r i n response t o h y p o x i c R a n d a l l , 1967a) and ( L o r e t z , 1979).  s t r e s s (Steen and K r u y s s e , 1964;  Both o f these phenomena would be expected t o render  seawater a d a p t a t i o n .  and  can r e s u l t i n an i n c r e a s e i n p a s s i v e water movements  e f f e c t i v e the r e d u c t i o n i n b r a n c h i a l p e r m e a b i l i t y n e c e s s a r y  water l o s s and  Holeton  The  for successful  e f f e c t o f such a d y s f u n c t i o n would be t o  s a l t g a i n and may  less  increase  have c o n t r i b u t e d t o the e l e v a t i o n i n plasma  e l e c t r o l y t e l e v e l s o f sockeye salmon observed i n the sea water stage o f present  study.  The  time course  of recovery of hydromineral  balance  the  as  i n d i c a t e d by the g r a d u a l r e t u r n o f plasma e l e c t r o l y t e s t o c o n t r o l v a l u e s s u g g e s t s t h a t g i l l p e r m e a b i l i t y may DHA to  exposure.  A l l plasma e l e c t r o l y t e and muscle water l e v e l s had  normal by 120  occurred.  be d i s t u r b e d f o r ^ 3 days a f t e r s u b l e t h a l returned  h i n sea water i n d i c a t i n g t h a t i r r e v e r s i b l e changes had  not  96  The p r e s e n c e o f c a l c i u m i o n s appears t o be e s s e n t i a l i n f i s h e s t o ensure s u c c e s s f u l a d a p t i v e p e r m e a b i l i t y changes t o b o t h water and CPotts and F l e m i n g , 1970,  1971).  T h i s e f f e c t may  be  electrolytes  largely a passive  process  r e l a t e d t o changes i n the s t r u c t u r a l p r o p e r t i e s o f the d i f f u s i o n b a r r i e r the g i l l s ,  of  such as s t a b i l i z a t i o n o f the mucus c o a t o r the c e l l membrane  (Mashiko and Jozuka, 1962).  The  a d d i t i o n o f c a l c i u m t o f r e s h water i n amounts  e q u i v a l e n t t o seawater c o n c e n t r a t i o n reduced the u r i n e f l o w i n the brown t r o u t (Salmo t r u t t a ) by 32%  (Oduleye, 1975).  osmotic p e r m e a b i l i t y i n f r e s h w a t e r  As u r i n e f l o w i s a measure o f  f i s h , t h i s r e d u c t i o n i n f l o w may  a s i g n i f i c a n t d e c r e a s e i n the work r e q u i r e d o f the The  observed e l e v a t i o n o f plasma C a  + +  the  represent  kidney.  l e v e l s may  be an a d a p t i v e  response  i n an e f f o r t t o reduce the p e r m e a b i l i t y o f the g i l l s t o water i n f l u x . the c a l c i u m c o n t e n t  o f the f r e s h water used i n the p r e s e n t  (0.8 mEq/L) i t i s d o u b t f u l t h a t t h e r i s e i n plasma C a uptake.  + +  study was  was  due  (Mashiko and  Jozuka, 1962)  which can serve as a  source when r e q u i r e d d u r i n g times o f s t r e s s (Ichikawa, Weiss and Watabe, 1978).  1953;  t o an  low  increased  l i t e r a t u r e on whether an e l e v a t i o n o f plasma C a  a similar effect. maintained and Mg  calcium  Urist,  Plasma C a  + +  l e v e l s i n freshwater  + +  not a b l e t o  l e v e l s from w i t h i n  f i s h are known t o  by p r o l a c t i n s e c r e t i o n , which i n t u r n i s c o n t r o l l e d by concentrations.  o f i s o l a t e d g i l l s incubated  the find has  be  environmental  Ogawa (1974) showed t h a t p r o l a c t i n i n j e c t i o n s  i n t o e e l s and rainbow t r o u t p r i o r t o s a c r i f i c e  l o w e r i n g o f plasma Ca  1966;  A l t h o u g h the r e d u c t i o n i n g i l l p e r m e a b i l i t y by  p r e s e n c e o f e x t e r n a l c a l c i u m has been w e l l e s t a b l i s h e d , I was  Ca  very  More l i k e l y i s the m o b i l i z a t i o n o f c a l c i u m from r e s e r v o i r s such as  s c a l e s and bone  any  As  i n d e i o n i z e d water.  l e v e l s may  r e d u c e d the water p e r m e a b i l i t y I t appears p r o b a b l e  that a  t r i g g e r p r o l a c t i n s e c r e t i o n l e a d i n g t o an  97  i n c r e a s e i n plasma Ca  c o n c e n t r a t i o n and p o s s i b l y i t s b i n d i n g a t the  which then i n f l u e n c e s plasma i o n i c composition  and o s m o l a l i t y  gills  (Wendelaar  Bonga, 1978). Changes i n P e r m e a b i l i t y - Gut The a b i l i t y o f t h e i s o l a t e d i n t e s t i n e t o t r a n s p o r t water appeared t o be little  a f f e c t e d i n f i s h exposed t o DHA  sea water f o r 24 h.  i n f r e s h water and then t r a n s f e r r e d t o  However, t h e i n c r e a s e d water c o n t e n t  salmon which were dehydrated may  i n the stomachs o f  i n d i c a t e t h a t the r a t e o f water  absorption  a c r o s s the g u t c o u l d n o t keep up w i t h the r a t e o f water l o s s a c r o s s t h e i n these  fish.  Under normal c i r c u m s t a n c e s ,  the p r o c e s s  o f seawater  t i o n i n v o l v e s an i n i t i a l movement o f plasma water i n t o the gut 1967;  Skadhauge, 1969); e x c e s s i v e  and s a l t  absorp-  (Utida e t a l . ,  lead to a further  dehydration  ( K i r s c h and Mayer-Gostan, 1973).  Dehydrated f i s h  cannot g a i n more water by d r i n k i n g more as t h e r e i s a l i m i t  t o the amount o f  salt  l o a d i n g o f plasma  i n g e s t i o n may  gills  and i t s a t t e n d a n t water f l o w t h a t t h e i n t e s t i n e i s capable  e s p e c i a l l y d u r i n g t h e e a r l y stages o f a d a p t a t i o n t o sea water 19691. gills  of  absorbing,  (Skadhauge,  T h i s emphasizes t h e importance o f r e d u c i n g o s m o t i c p e r m e a b i l i t y o f t h e d u r i n g sea water a d a p t a t i o n  ( K r i s t e n s e n and Skadhauge, 1974).  A l t h o u g h the i n g e s t i o n o f some f r e s h water by t e l e o s t s has been w e l l documented,  (Maetz and Skadhauge, 1968; G a i t s k e l l and C h e s t e r - J o n e s ,  P o t t s and F l e m i n g , 1970) and i s n o t c o n s i d e r e d advantageous  (Shuttleworth  1971;  t o be e n e r g e t i c a l l y d i s -  and Freeman, 1974), the i n g e s t i o n o f abnormal  amounts o f f r e s h water d i d l e a d t o a r a p i d g e n e r a l h y d r a t i o n i n e e l s ( K i r s c h and Mayer-Goston, 1973).. o f water was  In t h e p r e s e n t  experiment, the i n c r e a s e d i n g e s t i o n  f r e q u e n t l y observed d u r i n g f r e s h w a t e r  The d i s s e c t i o n o f f i s h h a v i n g f i l l e d w i t h water.  exposure o f salmon t o  DHA.  a b l o a t e d appearance r e v e a l e d a t u r g i d stomach  As t h e maintenance o f water p e r m e a b i l i t y , i n i t i a t i o n o f  98  branchial salt  e x c r e t i o n , and  the d r i n k i n g r e f l e x have been shown t o be  the c o n t r o l o f the nervous system  (Mayer-Gostan and  Hirano,  i n d i c a t e d s t i m u l a t i o n o f d r i n k i n g i n freshwater-adapted be r e l a t e d t o t h e a c c u m u l a t i o n  1976), the  sockeye salmon  o f h i g h c o n c e n t r a t i o n s o f DHA On a wet  c o n t a i n e d t h e h i g h e s t l e v e l s o f DHA  o f a l l the organs a n a l y z e d  DHA  organs.  Such a m e c h a n i c a l  flow of b i l e  weight b a s i s , the b r a i n t i s s u e  In a d d i t i o n , the stomach t u r g i d i t y  exposure c o u l d be expected  in this  study  f r e q u e n t l y observed  t o e x e r t c o n s i d e r a b l e p r e s s u r e on  blockage  may  i n the b r a i n o r  o t h e r p a r t s o f the nervous system.  (Appendix 1-4).  under  during  internal  c o u l d have i n t e r f e r e d w i t h the normal  and c o n t r i b u t e d t o a form o f o b s t r u c t i v e j a u n d i c e observed  in  DHA-exposed f i s h and which i s d e s c r i b e d i n Appendix I I I . Blood Hematocrit  and Plasma E l e c t r o l y t e Changes  The b l o o d sampled from salmon exposed s u b l e t h a l l y t o DHA  i n f r e s h water  f r e q u e n t l y appeared t o be more v i s c o u s and d a r k e r i n c o l o r than t h a t taken from c o n t r o l f i s h , i n d i c a t i n g t h a t some form o f hemoconcentration occurred.  Hemoconcentration was  f u r t h e r i n d i c a t e d by the h i g h l y  i n c r e a s e i n b l o o d h e m a t o c r i t observed  i n f i s h a f t e r 120  h o f DHA  On the o t h e r hand, the c o n c u r r e n t r e d u c t i o n i n plasma o s m o l a l i t y t h a t h e m o d i l u t i o n had o c c u r r e d , as might be expected an osmotic  imbalance i n f r e s h water.  had significant exposure. suggested  in fish suffering  A possible explanation of this  from  apparent  d i s c r e p a n c y i s o f f e r e d a f t e r a d i s c u s s i o n o f t h r e e mechanisms which can a c t to  increase hematocrit  in fish.  An i n c r e a s e o f h e m a t o c r i t (Doudoroff  and Shumway, 1970)  i s a well-known response  hypoxia  which can be an a d a p t i v e measure t o i n c r e a s e  the oxygen c a r r y i n g c a p a c i t y o f the b l o o d . contribute to a r i s e  of f i s h to  i n hematocrit:  1)  A t l e a s t t h r e e mechanisms can  a decrease  i n plasma volume,  99  2) an a c t u a l i n c r e a s e i n r e d b l o o d c e l l numbers i n r e d c e l l s i z e due t o s w e l l i n g .  (polycythemia)  3) an i n c r e a s e  Any one o r c o m b i n a t i o n o f t h e s e changes  w i l l l e a d t o an e l e v a t i o n i n h e m a t o c r i t . A d e c r e a s e i n plasma volume  i n response t o h y p o x i a can r e s u l t from  movement o f water from t h e b l o o d t o t i s s u e s  ( H a l l , 1928; B l a c k e t a l . ,  S o i v i o e t a l . , 1974c) o r an i n c r e a s e d d i u r e s i s  1959;  ( W e s t f a l l , 1943; Hunn, 1969;  S w i f t and L l o y d , 1974; L l o y d and S w i f t , 1976).  These f l u i d s h i f t s can r e s u l t  i n hemoconcentration and a r i s e i n h e m a t o c r i t .  In the p r e s e n t study, nemo-  c o n c e n t r a t i o n due t o f l u i d  s h i f t s from t h e plasma i n t o the t i s s u e s can be  r u l e d out s i n c e the plasma o s m o l a l i t y i n DHA dropped  exposed f i s h i n f r e s h water  ( F i g . 12). r a t h e r than i n c r e a s e d as might be expected i f plasma water  were b e i n g removed  from t h e c i r c u l a t i o n .  plasma o s m o l a l i t y was  A f t e r t r a n s f e r t o sea water,  s i g n i f i c a n t l y e l e v a t e d a t 24 h b u t h e m a t o c r i t was n o t .  However, when plasma o s m o l a l i t y o f b o t h groups became e s s e n t i a l l y  identical  a t 120 h, the h e m a t o c r i t s o f exposed f i s h were s i g n i f i c a n t l y below those o f controls. An a c t u a l i n c r e a s e i n t h e numbers o f r e d b l o o d c e l l s can r e s u l t from a s t i m u l a t i o n o f h e m a t o p o i e t i c a c t i v i t y and/or r e l e a s e o f e r y t h r o c y t e s from s t o r a g e depots such as t h e head k i d n e y (Ostroumova, 1957; Z a n j a n i e t a l . , Fromm, 19-77)..  Some a u t h o r s have suggested t h a t t h e s p l e e n may  1969;  a l s o s e r v e as  a s t o r a g e organ f o r e r y t h r o c y t e s which can be r e l e a s e d i n t o the c i r c u l a t i o n i n response t o h y p o x i c s t r e s s  (Dawson, 1935; L l o y d and S w i f t , 1976).  It is  d o u b t f u l however, t h a t s p l e n i c c o n t r a c t i o n a l o n e c o u l d c o n t r i b u t e enough r e d b l o o d c e l l s t o a l t e r h e m a t o c r i t s i g n i f i c a n t l y as Stevens (1968) determined t h e b l o o d volume o f the s p l e e n i n rainbow t r o u t t o be 70 y l / 1 0 0 g body weight. C o n s i d e r i n g a b l o o d volume o f 5% o f t h e body weight  (Smith, 1966)  and an  100  i n i t i a l h e m a t o c r i t o f 30%, (assuming  even a complete emptying o f t h e s p l e n i c  i t s e n t i r e b l o o d volume comprised  the h e m a t o c r i t t o 31.3%  i n a 100 g t r o u t .  contents  e r y t h r o c y t e s ) would o n l y r a i s e I t i s more l i k e l y then t h a t the  s p l e e n i s i n v o l v e d i n the uptake and d e s t r u c t i o n o f r e d b l o o d c e l l s f u n c t i o n s as a s t o r a g e s i t e f o r i r o n i n the form o f h e m o s i d e r i n (Grover, 1968;  Yu e t a l . ,  1971)  and responds  and  bodies  t o h y p o x i a by s u p p l y i n g the  r e q u i r e d f o r hemoglobin s y n t h e s i s i n the h e m a t o p o i e t i c t i s s u e o f the (Ostroumova, 1957).  The  evidence  kidney  s u g g e s t s t h a t the c o n t r i b u t i o n o f the s p l e e n  t o r a i s i n g h e m a t o c r i t i s p r o b a b l y i n d i r e c t and not I f as suggested, DHA  immediate.  exposure i n f r e s h water l e d t o h y p o x i a , a s t i m u l a t i o n  o f e r y t h r o p o i e s i s (red b l o o d c e l l formation) may  have o c c u r r e d w i t h r e d b l o o d  c e l l s b e i n g added t o the c i r c u l a t i o n i n s u f f i c i e n t numbers t o account higher hematocrit.  f o r the  A l t h o u g h r e d b l o o d c e l l s counts were not performed,  i s u n l i k e l y t h a t the observed c o u l d have been due  iron  r i s e i n hematocrit  (from 34% t o 45%,  it  typical)  t o p o l y c y t h e m i a because w i t h i n 24 h o f r e c o v e r y i n sea  water, the h e m a t o c r i t o f exposed and c o n t r o l f i s h was  similar.  T h i s would  have r e q u i r e d a r a p i d removal o f a l a r g e number o f r e d b l o o d c e l l s from circulation.  Although  such a f u n c t i o n was  i m p l i e d f o r the l i v e r and  the  the  s p l e e n by S w i f t and L l o y d (1974), based on b l o o d volume d e t e r m i n a t i o n s f o r these organs  (Stevens, 1968)  b l o o d c e l l s would o c c u r .  i t seems u n l i k e l y t h a t such a s t o r a g e o f r e d  S o i v i o e t a l . (1974a) c o u l d not f i n d any  particular  a r e a i n the t r o u t k i d n e y s p e c i a l i z e d f o r b l o o d s t o r a g e nor any evidence  for a  r e l e a s e o f e r y t h r o c y t e s from t h a t organ d u r i n g h y p o x i a . The r i s e i n h e m a t o c r i t d u r i n g t o x i c a n t exposure i n f r e s h water f o l l o w e d by a r a p i d drop a f t e r the f i s h were t r a n s f e r r e d t o sea water, when c o n s i d e r e d t o g e t h e r w i t h changes i n plasma o s m o l a l i t y , r a t h e r suggests a s w e l l i n g and  101  shrinking o f erythrocytes.  I f i n d e e d DHA exposure c o n t r i b u t e d  t o a hypoxic  c o n d i t i o n i n sockeye salmon t h r o u g h some i n t e r f e r e n c e w i t h gas exchange mechanisms, then a b u i l d u p the s w e l l i n g o f r e d b l o o d by  small increases  1941).  o f CO2 c o u l d o c c u r i n t h e b l o o d .  c e l l s i n r e s p o n s e t o h y p o x i c s t r e s s can be  i n plasma CO2 l e v e l s  C e l l u l a r swelling contributed  rainbow t r o u t  (Holeton  In s a l m o n i d s ,  and R a n d a l l ,  caused  ( B e n d i t t e t a l . , 1941 ; I r v i n g e t a l . ,  to increased  hematocrits i n hypoxic  1967a) and S o i v i o e t a l . (1974b,c)  c o n f i r m e d t h a t t h e s w e l l i n g o b s e r v e d i n v i v o i n t r o u t c o u l d a l s o be demons t r a t e d i n v i t r o a t reduced C>2 l e v e l s .  The i n c u b a t i o n o f s w o l l e n  erythrocytes  i n O2 d i d n o t r e s u l t i n complete volume r e g u l a t i o n and these a u t h o r s c o n c l u d e d t h a t the volume change was a complex response i n v o l v i n g c h a n g e s . i n pH, PCO2 and l a c t i c a c i d as w e l l as some unknown m e t a b o l i t e . reported  commersoni)led t o a 30% i n c r e a s e  buildup Eddy  and l e d t o a sharp r i s e i n h e m a t o c r i t .  in_ v i t r o a d d i t i o n o f l a c t i c a c i d t o t h e b l o o d  swelling  (Catastomus  i n h e m a t o c r i t a l s o as a r e s u l t o f e r y t h r o c y t e  (Black and I r v i n g , 1938). o f these m e t a b o l i t e s  o f the sucker  Hypoxic c o n d i t i o n s may c o n t r i b u t e t o a  i n the t i s s u e s or i n the r e d blood  (1977). determined t h a t t h e O2 uptake by t h e b l o o d  portion  (1977)  t h a t a l o c a l i z e d t i s s u e hypoxia a f t e r muscular e x e r t i o n i n t r o u t  resulted i n a swelling o f erythrocytes The  C a s i l l a s and Smith  c e l l s per se.  formed a s i g n i f i c a n t  (4.5% a t 20°C) o f t h e t o t a l energy consumption o f t r o u t , and may be  associated with the metabolic processes o f the r e d blood  cell.  Recent work h a s shown t h a t volume changes i n r e d b l o o d  c e l l s o f f i s h can  a l s o be b r o u g h t about by osmotic and i o n i c movements as p a r t o f t h e mechanism of "isosmotic  intracellular regulation'.  resulted i n swelling o f flounder  A d e c r e a s e i n plasma  erythrocytes  (Fugelli,  osmolality  1967) and C a l a  (1977)  showed t h a t subsequent c e l l volume r e g u l a t i o n was accomplished by means o f a d i r e c t change i n t h e p e r m e a b i l i t y  o f the membrane t o Na  or K  .  Net water  102  f l o w was secondary t o n e t i n o r g a n i c c a t i o n f l u x .  Changes i n t h e i o n i c  c o m p o s i t i o n o f plasma must be accompanied by i p n i c s h i f t s w i t h i n t o m a i n t a i n i o n i c and osmotic e q u i l i b r i u m O i k a r i , 1978).  There i s a l s o c o n s i d e r a b l e  erythrocytes  (Munroe and Poluhowich, 1974; e v i d e n c e t o i n d i c a t e t h a t even i n  r e l a t i v e l y s t a b l e o s m o r e g u l a t o r s , f l u c t u a t i o n s i n plasma o s m o l a l i t y r e s u l t i n s i g n i f i c a n t shrinking o r swelling of r e d blood 1975).  cells  (Schmidt-Nielsen,  Thus i t appears t h a t s u c c e s s f u l o s m o r e g u l a t i o n i n t e l e o s t s undergoing  a change i n s a l i n i t y i n v o l v e s b o t h e x t r a c e l l u l a r and i n t r a c e l l u l a r In t h e p r e s e n t  study, a drop i n plasma o s m o l a l i t y a t the end o f the DHA  exposure p e r i o d was accompanied by an i n c r e a s e o f t h e dimensions o f r e d b l o o d The  components.  i n hematocrit.  Measurements  c e l l s showed t h a t s w e l l i n g had indeed  occurred.  response o f sockeye salmon t o s u b l e t h a l DHA exposure i n f r e s h water  probably represents  a complex p h y s i o l o g i c a l r e a c t i o n t o the t o x i c a n t s t r e s s .  A l t h o u g h t h e a c t u a l mechanisms o f t o x i c i t y were n o t i n v e s t i g a t e d , t h e d a t a suggest t h a t a form o f h y p o x i c s t r e s s i s i n v o l v e d which r e s u l t s i n , o r i s accompanied by an osmotic i n f l u x o f water. were accompanied by an i n c r e a s e  R e d u c t i o n s i n plasma o s m o l a l i t y  i n s i z e o f the r e d blood  i n c r e a s e which f o r some r e a s o n was n o t r e g u l a t e d .  cells;  a volume  T h i s c o u l d r e s u l t from the  p a r t i t i o n i n g o f DHA i n t o t h e l i p i d components o f t h e r e d c e l l membrane a l t e r i n g i t s permeability  and/or i n t e r f e r i n g w i t h t h e i o n pumping mechanism  i n t h e p r o c e s s o f volume r e g u l a t i o n . continued  involved  A f t e r t h e t o x i c a n t exposure was d i s -  and t h e f i s h t r a n s f e r r e d t o sea water, t h e r i s e i n plasma o s m o l a l i t y  would f a c i l i t a t e water o u t f l u x from t h e e r y t h r o c y t e s medium and thus account f o r a l o w e r i n g  i n t o the hypertonic  of the hematocrit.  The d e p r e s s i o n o f  h e m a t o c r i t measured i n exposed f i s h a f t e r 120 h i n sea water may r e p r e s e n t shrinking o f red blood  cells.  As plasma o s m o l a l i t y  a  was i n the normal range  a t t h i s t i m e , t h i s may be due t o inadequate volume r e g u l a t i o n a f t e r hyperosmotic  103  s t r e s s , perhaps due t o an a l t e r a t i o n i n c e l l membrane p e r m e a b i l i t y .  Some  e v i d e n c e f o r DHA-induced h y p o x i a was p r o v i d e d d u r i n g the course o f acute DHA  bioassays.  Symptoms such as an i n c r e a s e d b r e a t h i n g amplitude and f r e q u e n t  coughing i n d i c a t e d t h a t the salmon were undergoing r e s p i r a t o r y d i s t r e s s . was  c o n f i r m e d d u r i n g the h y p o x i a b i o a s s a y which demonstrated  a dramatic  i n c r e a s e i n t o x i c i t y o f a n o r m a l l y s u b l e t h a l dose o f DHA  brought on by a  r e d u c t i o n o f d i s s o l v e d oxygen l e v e l s t o 75% s a t u r a t i o n .  These  suggested t h a t DHA  may  This  results  i n t e r f e r e w i t h the mechanism o f oxygen uptake by the  b l o o d , l e a d i n g t o hypoxemia.  A d d i t i o n a l support f o r t h i s view was p r o v i d e d  d u r i n g the g i l l p e r m e a b i l i t y experiments when i t became apparent t h a t the confinement o f f i s h t o the tubes i n c r e a s e d t h e i r s u s c e p t i b i l i t y t o DHA.  As  d i s s o l v e d oxygen l e v e l s were m a i n t a i n e d i n excess o f 90% s a t u r a t i o n i n t h e s e experiments, i t i s u n l i k e l y t h a t the apparent i n c r e a s e i n t o x i c i t y was to  reduced oxygen a v a i l a b i l i t y .  However, i f DHA  t r a n s f e r o f oxygen over the g i l l s ,  due  were t o i n t e r f e r e w i t h  normal  the r e d u c t i o n i n swimming a c t i v i t y o f  fish  c o n f i n e d t o the tubes would have reduced ram v e n t i l a t i o n and c o u l d have l e d to the  hypoxemia.  Because o f the added r e s i s t a n c e o f the f i s h r e t a i n i n g s c r e e n s ,  v e l o c i t y o f the water c u r r e n t p a s s i n g through the tubes was  of  t h a t a g a i n s t which  in  the donut t a n k s .  DHA  about  sockeye salmon m a i n t a i n e d p o s i t i o n when swimming  half free  Iwama e t a l . (1976) observed an i n c r e a s e i n t o x i c i t y o f  t o j u v e n i l e coho salmon a t reduced a c t i v i t y l e v e l s .  In a l l the  experiments  conducted, sockeye salmon attempted t o m a i n t a i n p o s i t i v e r h e o t a x i s d u r i n g acute stages o f DHA the  toxicity.  A l t h o u g h one would assume t h a t swimming w i t h  c u r r e n t would have been e n e r g e t i c a l l y much e a s i e r , t h i s d i d n o t o c c u r .  A c o n c e r t e d e f f o r t t o head i n t o the c u r r e n t may i n c r e a s e p a s s i v e l y the volume o f water  be an a d a p t i v e response t o  i r r i g a t i n g the  gills.  104  A l t h o u g h no i n f o r m a t i o n resin acid toxicity,Davis  i s a v a i l a b l e on a h y p o x i a - i n d u c e d i n c r e a s e o f  (1973) found t h a t sockeye salmon exposed t o s u b l e t h a l  l e v e l s of bleached k r a f t m i l l e f f l u e n t  (BKME) responded w i t h an i n c r e a s e i n  v e n t i l a t i o n volume and coughing r a t e .  In s p i t e o f an i n c r e a s e  consumption, t h e r e was an average 20% r e d u c t i o n a r t e r i a l blood. and  i n oxygen  i n t h e oxygen s a t u r a t i o n o f  The e f f l u e n t used i n t h a t study had been a e r a t e d ,  neutralized,  f i l t e r e d so t h a t t h e r e s i d u a l t o x i c i t y was p r o b a b l y a t t r i b u t a b l e t o t h e  more p e r s i s t e n t t o x i c components such as r e s i n a c i d s and c h l o r i n a t e d I.H.  Rogers  (personal  organics.  communication) l a t e r c o n f i r m e d t h e presence o f DHA  (1-2 mg/L) i n e f f l u e n t from the same p u l p m i l l . Davis  (1973) suggested t h a t p a r t o f t h e observed r e d u c t i o n  f i c i e n c y o f gas exchange may have been due t o an e l a b o r a t i o n gill  e p i t h e l i u m , a s Walden and Howard  (1968) r e p o r t e d  production a t the g i l l s o f underyearling The  e f f e c t s o f an i n c r e a s e  r e c e n t l y been e s t i m a t e d .  i n the e f -  o f mucus by t h e  an i n c r e a s e  i n mucus  salmon exposed t o n e u t r a l i z e d BKME.  i n d i f f u s i o n distance  on f i s h r e s p i r a t i o n have  Based on t h e assumption t h a t t h e t h i c k n e s s  l a m e l l a r d i f f u s i o n b a r r i e r was ^ 5 p  (Steen, 1971) t h e e l a b o r a t i o n  of the of a  5 um t h i c k l a y e r o f mucus on a secondary l a m e l l a was c a l c u l a t e d t o l e a d t o an 81% i n c r e a s e  o f the d i f f u s i o n resistance  t o oxygen  ( U l t s c h and Gros, 1979).  They a l s o suggested t h a t t h e p r e s e n c e o f t h i s mucus would r e s u l t i n the reduction  o f t h e e f f e c t i v e r e s p i r a t o r y water f l o w by h a l f .  hyperventilation  The compensatory  w i t h i t s a t t e n d a n t oxygen demand a t a time when 0^ f l u x i n t o  the b l o o d was b e i n g r e s t r i c t e d by a d d i t i o n a l mucus c o r r e s p o n d s q u i t e to the observations o f increased a r t e r i a l P0  2  closely  v e n t i l a t i o n , 0^ consumption and d e p r e s s e d  i n salmon exposed s u b l e t h a l l y t o BKME (Davis,  1973).  In the  p r e s e n t study, no e x c e s s mucus p r o d u c t i o n was noted, a l t h o u g h an i n c r e a s e  105  in thickness of the order described by TJltsch and Gros (1979) could have occurred i n response to DHA exposure. Additional evidence f o r DHA-induced hypoxia may be suggested from the pattern of plasma e l e c t r o l y t e changes i n salmon exposed to DHA i n fresh water which followed closely that reported i n the l i t e r a t u r e f o r hypoxic f i s h . the end of the freshwater exposure period plasma Na elevated and K  +  was s i g n i f i c a n t l y lowered.  by Soivio ejt a l .  +  was  An i d e n t i c a l pattern was observed  (1975) during iri v i t r o incubation of rainbow trout blood under  f a l l i n g oxygen tension. an i n f l u x of K  was unaltered, Mg  At  A progressive r i s e i n hematocrit was accompanied by  into the erythrocytes from the plasma.  Platner (1950) found  that a combination of hypoxic stress and lowered temperature resulted i n a marked increase i n plasma M g a leakage of M g  ++  ++  l e v e l s i n the g o l d f i s h which he attributed to  from the erythrocytes into a plasma d i l u t e d by water uptake.  In the present experiments the lowering of plasma osmolality and elevation of muscle water concentrations indicate a water retention and/or an increase i n water uptake from the environment by DHA-exposed f i s h . Kidney function As no attempts were made to measure kidney function i n the present investigation, the following discussion i s based on an interpretation of the changes observed i n plasma e l e c t r o l y t e s i n the l i g h t of the known role of the kidney i n hydromineral balance. The kidney plays an indispensable r o l e i n osmoregulatory adaptations which determine euryhalinity i n t e l e o s t fishes such as the salmon. In fresh water, i n spite of a r e l a t i v e l y low o v e r a l l permeability, there i s a continuous osmotic i n f l u x of water into the f i s h due to the osmotic gradient between the plasma  300 mOsM/kg) and the medium (< 5 mOsM/kg).  The kidney  106  counteracts  t h i s i n f l u x o f water by p r o d u c i n g  an abundant, s t r o n g l y  (to plasma) u r i n e and t o g e t h e r w i t h t h e u r i n a r y b l a d d e r of f i l t e r e d  ions.  achieves  conservation  In sea water t h e osmotic g r a d i e n t between t h e plasma  mOsM/kg) and the medium  (^ 300  1000 mOsM/kg) i s c o n s i d e r a b l y g r e a t e r and i n t h e  r e v e r s e d i r e c t i o n so t h a t t h e f i s h tends t o become dehydrated. o f t h e kidney  hypotonic  and b l a d d e r  The f u n c t i o n s  i n s e a water a r e t o minimize water l o s s by r e -  a b s o r p t i o n o f water and monovalent i o n s , t o produce a u r i n e i s o t o n i c t o t h e plasma, and t o e x c r e t e d i v a l e n t i o n s which e n t e r w i t h swallowed s e a water (Smith,  1930).  The  i m p l i c a t i o n t h a t DHA exposure o f salmon t o f r e s h water r e s u l t s i n  h y p o x i a has a l r e a d y been d i s c u s s e d . f i s h respond t o hypoxic area o f the g i l l s  One o f t h e a d a p t i v e mechanisms by which  s t r e s s i s that o f i n c r e a s i n g the f u n c t i o n a l surface  (Steen and Kruysse, 1964; Holeton  While an i n c r e a s e i n t h e p e r f u s i o n o f g i l l it  and R a n d a l l ,  lamellae f a c i l i t a t e s  1967a). oxygen uptake,  c o n c u r r e n t l y augments t h e s u r f a c e a r e a a v a i l a b l e f o r p a s s i v e o s m o t i c and  i o n i c movements. i n f r e s h water  T h i s l e a d s t o an i n c r e a s e d uptake o f water over t h e g i l l s  ( L o r e t z , 1979).  I n hypoxic  t r o u t L l o y d and S w i f t  (1976) found  an i n c r e a s e d p e r m e a b i l i t y t o water which p e r s i s t e d beyond t h e time when v e n t i l a t i o n had r e t u r n e d t o normal.  T h i s i n c r e a s e d p e r m e a b i l i t y was r e f l e c t e d  i n compensatory i n c r e a s e s i n u r i n e f l o w r a t e as w e l l as a r e d u c t i o n o f t h e a c t i v e uptake o f sodium and c h l o r i d e by t h e g i l l s Increased kidney Mg  + +  ( S w i f t and L l o y d ,  1974).  d i u r e s i s combined w i t h a r e d u c t i o n o f s a l t r e a b s o r p t i o n by t h e  l e d t o t h e e x c r e t i o n o f abnormally h i g h c o n c e n t r a t i o n s  and C l  i n t h e u r i n e o f rainbow t r o u t a f t e r h y p o x i c  In t h e p r e s e n t  study,  of Na , K , +  +  s t r e s s (Hunn, 1969).  arguments have a l r e a d y been p r e s e n t e d  t o support  the view t h a t DHA exposure i n f r e s h water l e d t o an i n c r e a s e i n water l o a d i n g p o s s i b l y v i a the mechanism d i s c u s s e d above.  The r a p i d i n f l u x o f water  across  107  the g i l l s and p o s s i b l y a l s o a c r o s s the gut may o f the k i d n e y t o m a i n t a i n  water b a l a n c e .  have exceeded the  capacity  At h i g h r a t e s o f d i u r e s i s , the  e f f i c i e n c y o f the r e n a l s a l t r e a b s o r p t i v e mechanism i s reduced due  t o shortened  urine residence  bladder.  As n o r m a l l y  time b o t h i n the k i d n e y t u b u l e s and  between 80-90% o f NaCl i s r e a b s o r b e d  i n the u r i n a r y  (Lahlou, 1970)  this  r e s u l t i n the e x c r e t i o n o f u r i n e c o n t a i n i n g abnormal c o n c e n t r a t i o n l y t e s , e s p e c i a l l y c h l o r i d e , such as has been shown t o o c c u r s t r e s s and Grafflin,  " l a b o r a t o r y d i u r e s i s " ( F o r s t e r , 1953; 1931;  Holmes, 1961).  i n DHA-exposed f i s h d u r i n g  The  F o r s t e r and  r e s i d e n c e may  be due  of e l e c t r o -  a f t e r handling  c h a r a c t e r i s t i c lowering  freshwater  can  Berglund,  1956;  o f plasma C l t o an  unavoidable  i n c r e a s e i n the r e n a l e x c r e t i o n o f t h i s i o n accompanying a compensatory in urine  production.  Another p o s s i b i l i t y , o t h e r than a simple  overloading  which c o u l d c o n t r i b u t e t o a r e n a l i n s u f f i c i e n c y may h i g h l e v e l s o f DHA  on the k i d n e y f u n c t i o n p e r  exposure employed i n the p r e s e n t 278.1  yg/g.  The  study, DHA  se.  o f the  kidney,  be a d i r e c t e f f e c t D u r i n g the  are unknown but  reached could  Based on the u n i t s u r f a c e  a v a i l a b l e f o r s a l t t r a n s p o r t , the c e l l s o f the r e n a l t u b u l e s are the o f i n t e n s e exchanges  i n v o l v e d i n a c t i v e t r a n s p o r t may Jones  area  sites  (Lahlou, 1970), which have been shown t o i n v o l v e N a - K +  (Jampol and E p s t e i n , 1970,  Trump and  of  sublethal  l e v e l s i n the k i d n e y s  e f f e c t s o f such a c o n c e n t r a t i o n  p o s s i b l y d i s r u p t normal c e l l u l a r f u n c t i o n s .  ATP-ase  rise  E p s t e i n e t al.,1969).  be  Enzyme systems  s e n s i t i v e to high concentrations  of  DHA.  (1977) d e s c r i b e d a fundamental c o r r e l a t i o n between the  i n h i b i t i o n o f a c t i v e t r a n s p o r t and u l t r a s t r u c t u r a l changes i n c e l l s o f  the  t e l e o s t nephron.  of  These were r e l a t e d t o the a l t e r a t i o n i n p e r m e a b i l i t y  the plasma membrane and m i t o c h o n d r i a  +  by a v a r i e t y o f t o x i c agents.  Studies  108  on organ h i s t o p a t h o l o g y were n o t conducted  i n t h e p r e s e n t study b u t F u j i y a  (1965) r e p o r t e d kidney t u b u l e n e c r o s i s i n marine f i s h taken from waters adjacent to a k r a f t pulp  mill.  DHA-induced k i d n e y d y s f u n c t i o n c o u l d have e q u a l l y important for  consequences  f i s h moving i n t o s e a water as the s u c c e s s f u l t r a n s i t i o n o f a m i g r a t i n g  salmon from a r i v e r t o t h e marine environment depends t o a l a r g e e x t e n t on the a b i l i t y o f i t s k i d n e y t o assume a v e r y d i f f e r e n t osmoregulatory o f t e n w i t h i n a m a t t e r o f hours.  The c r i t i c a l adjustment r e q u i r e d o f the  kidney i n a f i s h f a c e d w i t h i n c r e a s e d s a l i n i t y u r i n e f l o w t o l i m i t water l o s s .  role,  i s a prompt r e d u c t i o n i n  T h i s i s accomplished  by a r e d u c t i o n i n  glomerular  f i l t r a t i o n r a t e (GFR) (Hickman and Trump, 1969).  accomplish  this quickly w i l l  lead to a rapid  Failure to  dehydration.  A f t e r t h e t r a n s f e r o f DHA-exposed sockeye salmon t o sea water, h y d r a t i o n was  r e p l a c e d by a r a p i d d e h y d r a t i o n as demonstrated by a r i s e i n plasma  o s m o l a l i t y and a l l measured i o n s .  T h i s was f o l l o w e d by a g r a d u a l r e g u l a t i o n  back down t o normal l e v e l s a f t e r 120 h i n s e a water. i n f r e s h water, u r i n e f l o w s c o u l d be expected the t o x i c a n t - i n d u c e d h y d r a t i o n .  In DHA-exposed  fish  t o be e l e v a t e d t o contend  with  I f such a f i s h i s r a p i d l y t r a n s f e r r e d t o  sea water, i t i s c o n c e i v a b l e t h a t t h e r e might be a d e l a y i n t h e n e c e s s a r y a d a p t i v e r e d u c t i o n i n GFR.  I n i t i a l l y t h i s would a i d i n the d i s p o s a l o f  excess water b u t i f c o n t i n u e d c o u l d l e a d t o a r a p i d I t i s probable  t h a t t h e changes observed  dehydration.  a t t h e 24 h sampling  period  do n o t r e p r e s e n t t h e maximum e x c u r s i o n s i n plasma e l e c t r o l y t e s which occurred.  Utida  ( i n F o n t a i n e , 1975) observed  t h a t d u r i n g the t r a n s f e r o f  smolts o f t h e salmon Oncorhynchus masou from FW t o SW, maximum plasma  109  o s m o l a l i t y was reached w i t h i n 8 h and was s u b s e q u e n t l y r e g u l a t e d back down t o normal seawater v a l u e s .  I n DHA-exposed sockeye salmon the measurements o f  plasma e l e c t r o l y t e s i n b e h a v i o r a l l y abnormal f i s h r e v e a l e d t h a t they were n o t able t o regain c o n t r o l o f hydromineral balance a f t e r t h i s r a p i d departure normal e l e c t r o l y t e l e v e l s . be r e l a t e d t o t h e e x t e n t  The s e v e r i t y o f locomotory d i f f i c u l t y appeared t o  o f t h i s departure  measured i n a f f e c t e d ( b u t s t i l l : (i.e.  could not maintain  equilibrium plasma |/  + +  from t h e plasma i o n i c  regulating)fish.  levels  I n f i s h which were d r i f t i n g  p o s i t i o n against the current)  o r had j u s t  lost  e l e c t r o l y t e l e v e l s were g r o s s l y i n c r e a s e d above b o t h t h e  c o n t r o l s and exposed plasma M g  from  (but r e s i s t i n g ) groups.  Of a l l t h e i o n s measured,  showed t h e l a r g e s t r e l a t i v e i n c r e a s e and t h e slowest  T h i s key f i n d i n g p r o v i d e s more d i r e c t support  f o r t h e involvement o f t h e k i d n e y  i n DHA-induced h y d r o m i n e r a l b a l a n c e o b s e r v e d i n salmon d u r i n g adaptation.  Beyenbach  equilibrium)  to Mg  + +  regulation.  (1974) found t h a t t h e t o l e r a n c e  seawater  (before l o s s o f  i n rainbow t r o u t was d i r e c t l y r e l a t e d t o t h e c a p a c i t y  o f t h e r e n a l s e c r e t o r y system t o m a i n t a i n During the adaptation  magnesium homeostasis.  o f f i s h t o s e a water, t h e t e m p o r a l l y  second most  important f u n c t i o n o f t h e k i d n e y becomes t h a t o f d i v a l e n t i o n s e c r e t i o n ; e s p e c i a l l y o f magnesium and s u l f a t e which a r e absorbed a c r o s s t h e i n t e s t i n e from i n g e s t e d s e a water. ratios 1968;  Tubular  secretion of Mg  maintained urine/plasma  (U/P) i n t h e 50:1 t o 100:1 range i n seawater-adapted f l o u n d e r Lahlou,  rainbow t r o u t  1970) coho salmon  (Oncorhynchus k i s u t c h  (Beyenbach and K i r s c h n e r ,  Some DHA-exposed salmon a p p a r e n t l y still  + +  i n freshwater  (Hickman,  ( M i l e s , 1971) and  1975). i n c r e a s e d water i n g e s t i o n w h i l e  and thus c o u l d have had mucosal p e r m e a b i l i t i e s  by t h e d i r e c t c o n t a c t w i t h t h e l i p i d - s o l u b l e r e s i n a c i d . i n g u t p e r m e a b i l i t y c o u l d i n c r e a s e t h e uptake o f M g  + +  increased  Such an i n c r e a s e  from s u b s e q u e n t l y  110  swallowed s e a water. Although the i n t e s t i n a l absorption route i s g e n e r a l l y considered the most important,  K i r s c h n e r e t a l . (1974) found t h a t t h e g i l l  e x h i b i t e d a low b u t d e f i n i t e p e r m e a b i l i t y t o M g . ++  t o be  epithelium  As e q u i l i b r a t i o n between  plasma and s e a water a c c o r d i n g t o t h e e l e c t r o c h e m i c a l g r a d i e n t would in  a 10-20 f o l d i n c r e a s e i n plasma M g  is  imperative  that f i s h maintain  + +  concentration  result  (Beyenbach, 1974), i t  a low g i l l p e r m e a b i l i t y t o t h i s i o n . In  view o f t h e changes i n g i l l p e r m e a b i l i t y t o water i t may be t h a t p r i o r DHA exposure i n c r e a s e d g i l l p e r m e a b i l i t y t o M g  Even though i t i s n o t known whether M g in  i n s e a water.  + +  + +  uptake v i a t h e g i l l s  increased  f i s h p r e v i o u s l y exposed t o DHA, t h e i n c r e a s e d e n t r y t h r o u g h t h e g u t alone  would p r o b a b l y dehydration  s u f f i c e t o r a p i d l y e l e v a t e plasma M g  + +  concentrations.  The  o c c u r r i n g i n sockeye salmon which were t r a n s f e r r e d t o s e a water  a f t e r s u b l e t h a l DHA exposure was shown t o be accompanied by a compensatory i n c r e a s e i n sea-water i n g e s t i o n (Table X I I ). of the ingested normal t r o u t  (SW) M g  + +  was shown t o o c c u r  Since  a n e t a b s o r p t i o n o f 44%  across the i n t e s t i n e o f  (Shehadeh and Gordon, 1969) an i n c r e a s e i n s e a water i n g e s t i o n  beyond normal l e v e l s c o u l d be e x p e c t e d t o r a p i d l y r a i s e plasma M g  + +  concen-  trations. As an i n c r e a s e d uptake o f M g  + +  can o n l y be e l i m i n a t e d by t h e k i d n e y , and  because t h i s i o n must be t i g h t l y r e g u l a t e d , t h e t e l e o s t k i d n e y a powerful  tubular Mg  + +  secretory function.  Magnesium i n f u s i o n i n a  v a r i e t y o f e u r y h a l i n e f i s h s p e c i e s b r i n g s about a p o w e r f u l which p e r s i s t s u n t i l plasma M g  + +  l e v e l s r e t u r n t o normal  B r u l l and Cuypers, 1955; F o r s t e r , 1953; Hickman, 1968). found s i m i l a r r e s u l t s i n m i g r a t i n g  has d e v e l o p e d  d i u r e t i c response ( B i e t e r , 1933;  Natochin  e t a l . (1970)  sockeye salmon smolts which they  loaded  ++ e x p e r i m e n t a l l y w i t h Mg  .  As t h e r a t e o f u r i n e f l o w i n s e a water i s determined  Ill  by t h e i n t e n s i t y o f M g to  + +  excretion, Mg  aggravate d e h y d r a t i o n i n sea water Plasma M g  + +  + +  l o a d i n g o f salmon can be expected  1974).  (Beyenbach,  l e v e l s a r e n o r m a l l y r e g u l a t e d w i t h i n narrow l i m i t s and s m a l l  v a r i a t i o n s a r e known t o b l o c k neuromuscular t r a n s m i s s i o n  ( d e l C a s t i l l o and  Engbaek, 1954) s u g g e s t i n g t h a t t h e o b s e r v e d changes i n locomotor performance of  DHA exposed f i s h may be r e l a t e d t o myoneural b l o c k a g e by M g . ++  Juvenile  p i n k salmon s u b j e c t e d t o t h e s t r e s s o f s c a l e l o s s i n sea water became unr e s p o n s i v e t o v i s u a l and m e c h a n i c a l s t i m u l a t i o n a f t e r plasma Mg  levels  i n c r e a s e d by 63% ( p e r s o n a l communication, Dr. L.S. Smith, U n i v e r s i t y o f Washington). the  These symptoms were f o l l o w e d by a p r o g r e s s i v e p a r a l y s i s .  In  p r e s e n t study i d e n t i c a l symptoms were o b s e r v e d i n DHA-exposed., sockeye  salmon i n t h e f i r s t  48 h a f t e r sea water t r a n s f e r .  by^50%.  these f i s h increased plasma M g  + +  Block e t a l .  Plasma M g  increase of a l l  levels i n  F i s h which were d r i f t i n g o r moribund had  l e v e l s 3 t o 4 t i m e s h i g h e r than t h e exposed  (1978)  + +  found t h a t plasma M g  e l e c t r o l y t e s measured  + +  (but r e s i s t i n g )  l e v e l s underwent  fish.  the greatest  i n w h i t e p e r c h (Morone americana)  exposed t o c h l o r i n e i n e s t u a r i n e w a t e r s .  These changes were a t t r i b u t e d t o  a l t e r a t i o n s i n b r a n c h i a l p e r m e a b i l i t y due t o damage t o t h e g i l l  epithelium  by t h e t o x i c a n t . In Mg  + +  t h e p r e s e n t study, a comparison o f t h e o b s e r v e d changes i n plasma  l e v e l s w i t h those d e s c r i b e d i n t h e l i t e r a t u r e on r e n a l f u n c t i o n suggests  t h a t DHA exposure o f salmon reduces t h e e f f i c i e n c y o f k i d n e y f u n c t i o n i n sea water and/or i n c r e a s e s g i l l p e r m e a b i l i t y t o M g  + +  r e n a l e x c r e t o r y mechanisms may become o v e r l o a d e d .  t o such an e x t e n t  that  Based on t h e l i t e r a t u r e ,  ++ e l e v a t e d plasma Mg  l e v e l s may be r e s p o n s i b l e f o r many o f t h e b e h a v i o r a l  changes o b s e r v e d i n DHA exposed sockeye salmon d u r i n g seawater  adaptation.  112  Presumably the r e c o v e r y  o f f i s h thus a f f e c t e d i n v o l v e s a s u c c e s s f u l r e d u c t i o n  i n GFR and g i l l p e r m e a b i l i t y , f o r sea water i n g e s t i o n . plasma M g  + +  t h e r e b y r e d u c i n g water l o s s and  T h i s would be f o l l o w e d by a g r a d u a l  l e v e l s t o normal.  the n e c e s s i t y return of  113  PART I I I .  ECOLOGICAL IMPLICATIONS  E n v i r o n m e n t a l s t r e s s has  been d e f i n e d  e n v i r o n m e n t a l f a c t o r which extends the o r which d i s t u r b s o f s u r v i v a l are ophy f u r t h e r ,  s i g n i f i c a n t l y reduced"  the o f t e n  subtle  ( B r e t t , 1958).  Sprague  to s u c c e s s i v e l y  expected t o c o n t r i b u t e  any  chances  Pursuing t h i s p h i l o s -  study and  interpretation  changes measured i n  of  the  higher e c o l o g i c a l l e v e l s of i n t e g r a t i o n .  to a reduction  animal,  (1971) emphasized the need t o r e l a t e  toxicant-induced physiological  words, i s the measured change w i t h i n  nation  t o such an e x t e n t t h a t the  i n a r e v i e w o f methods used i n the  effects of toxicants,  organism?  "a s t a t e produced by  normal a d a p t i v e r e s p o n s e s o f an  the normal f u n c t i o n i n g  sublethal  laboratory  as  In o t h e r  the normal a d a p t i v e range or can i n the  i t be  s u r v i v a l p o t e n t i a l o f the  entire  T h i s d e c i s i o n p o s e s a s i n g u l a r l y d i f f i c u l t problem as a d e t e r m i -  o f the e c o l o g i c a l s i g n i f i c a n c e o f l a b o r a t o r y  be made i n the therefore  f i n d i n g s u s u a l l y has  f a c e o f l i m i t e d knowledge o f what happens i n the  requires  to  field  and  a h i g h e r than u s u a l l e v e l o f p e r s o n a l judgement by  the  investigator. In the p r e s e n t study, the a period  Subsequent t r a n s f e r o f t h e s e f i s h i n t o sea water a g a i n  i n marked changes i n h y d r o m i n e r a l b a l a n c e i n the  osmotic and  ionic gradients.  The  m a j o r i t y o f the  d i r e c t i o n of  f i s h so-affected  t o r e t u r n plasma e l e c t r o l y t e l e v e l s t o c o n t r o l v a l u e s w i t h i n toxicant  exposure had  reduction  been d i s c o n t i n u e d , i n d i c a t i n g t h a t no  i n osmoregulatory c a p a c i t y  temporary l o s s i n r e g u l a t o r y f i s h one  for  o f 5 days l e d t o s i g n i f i c a n t d e p a r t u r e s o f plasma e l e c t r o l y t e s from  "normal" v a l u e s . resulted  exposure of j u v e n i l e sockeye salmon t o DHA  had  occurred.  To  120  implications.  managed  h after  the  permanent  suggest whether such a  p r e c i s i o n i s m e a n i n g f u l t o the  s u r v i v a l of  must c o n s i d e r not o n l y the p h y s i o l o g i c a l e f f e c t s but  ecological  the  also  the  the  114  S p e c i a l e f f o r t s were made d u r i n g t h i s study t o attempt t o changes i n plasma e l e c t r o l y t e l e v e l s , gut water c o n t e n t and l e v e l s w i t h observed changes i n g e n e r a l b e h a v i o r o f the o f t h e s e parameters a t g r a d u a l l y i.e.  d r i f t i n g , equilibrium  s e r i o u s l y the  measured v a r i a b l e s the  from the  the  mortality,  based on the  Although a cause/effect  dysfunction,  T h i s was + +  the  e s p e c i a l l y true  showing the  r e l a t i o n s h i p was  not  after  greatest established,  l i t e r a t u r e i t i s suggested t h a t the observed locomotory be  due  concentrations.  to high M g  + +  r e l a t e d t o an  interference In a d d i t i o n  w i t h neuromuscular the h i g h DHA  i n b r a i n t i s s u e c o u l d have l e d t o d y s f u n c t i o n  One  Measurement  g r e a t e r were the d e p a r t u r e s of  control values.  d i f f i c u l t i e s may  contributed  salmon.  c o n f i r m e d t h a t the more  t r a n s f e r o f f i s h t o sea water, w i t h plasma M g  p e r c e n t change.  muscle water  more severe stages o f locomotory  l o s s , and  f i s h were a f f e c t e d ,  correlate  a t the  residues  first  noticeable  g r a d u a l break up o f the  schooling  increased  and  schooling  i n f i s h i s known t o be  also involved  e f f e c t s of sublethal response.  a progressive reduction  DHA  Initially,  t o x i c i t y was  initial  a  f i s h - t o - f i s h distances  i n c o v e r response developed.  In the  and  co-ordination.  under v i s u a l c o n t r o l , the  (Pitcher et al.,1976).  occurring  integrative level  t o locomotor d i f f i c u l t i e s t h r o u g h poor muscle  o f the  function  Although  lateral line  stages o f  is  intoxication,  salmon which were more w i d e l y spaced s t i l l m a n i f e s t e d a c o v e r response  to  v i s u a l stimulation,  At  i n d i c a t i n g t h a t v i s i o n was  l a t e r s t a g e , when c o v e r response was  p r o b a b l y not  impaired.  c o m p l e t e l y reduced, a tap on  the  would t r i g g e r an u n d i r e c t e d spasmotic swimming b e h a v i o r s u g g e s t i v e o f h y p e r s e n s i t i v i t y and may Gardner  indicate a dysfunction  (1975) l i n k e d b e h a v i o r a l  o f the  a l t e r a t i o n s to h i s t o p a t h o l o g i c a l  p a t t e r n s was  tank a  l a t e r a l l i n e system..  marine t e l e o s t s exposed t o a number o f water-borne t o x i c a n t s . a b i l i t y to maintain schooling  a  changes i n  A loss in  r e l a t e d t o l e s i o n s i n the  the  olfactory  115  epithelium of A t l a n t i c s i l v e r s i d e the  laboratory.  ( M e n i d i a menidia) exposed t o crude o i l i n  In t h e f i e l d , t h e u n n a t u r a l b e h a v i o r and h y p e r s e n s i t i v i t y t o  m e c h a n i c a l s t i m u l a t i o n i n menhaden  (Brevoortia tyrannus) c o l l e c t e d i n the  e f f l u e n t d i s c h a r g e a r e a o f a n u c l e a r g e n e r a t i n g s t a t i o n were l i n k e d t o l e s i o n s i n t h e l a t e r a l l i n e organs. of  j u v e n i l e A t l a n t i c salmon  l a b o r a t o r y and i n f i e l d  Gardner  (1975) a l s o r e p o r t e d t h a t  exposure  (Salmo s a l a r ) t o p u l p m i l l waste b o t h i n  s t u d i e s r e s u l t e d i n l e s i o n s i n o l f a c t o r y organs.  The changes i n b e h a v i o r o f DHA-exposed sockeye salmon may from t h e t o x i c a n t - i n d u c e d changes d i s c u s s e d above.  have a r i s e n ,  i n part,  On the o t h e r hand, t h e  r e s p o n s e s c o u l d have o r i g i n a t e d i n d y s f u n c t i o n o f the nervous system as was  shown t o accumulate t o h i g h l e v e l s i n b r a i n  tissue.  U n t i l r e c e n t l y the e s t a b l i s h m e n t o f water q u a l i t y c r i t e r i a has  depended  almost e x c l u s i v e l y on t h e r e s u l t s o f a c u t e o r l o n g - t e r m c h r o n i c l i f e bioassays to For  (Sprague, 1976).  Only a few workers have d i r e c t e d t h e i r  cycle  attention  the e c o l o g i c a l s i g n i f i c a n c e o f s u b t l e , t o x i c a n t - i n d u c e d b e h a v i o r a l example,  Basch and Truchan  DHA  (1976) o b s e r v e d g u l l s f e e d i n g on s m a l l  changes. fish  f l o u n d e r i n g near the s u r f a c e d u r i n g a p e r i o d o f c o o l i n g water c h l o r i n a t i o n a t a Lake M i c h i g a n power p l a n t .  While t h e temporary exposure o f a p r e y s p e c i e s t o  a p h y s i o l o g i c a l l y s u b l e t h a l c o n c e n t r a t i o n o f a c h e m i c a l t o x i c a n t may cause l i t t l e  directly  more than abnormal b e h a v i o r , i f g r e a t e r p r e d a t i o n i s the c o n s e -  quence t h e n a s u b l e t h a l e f f e c t has i n d i r e c t l y become l e t h a l t o the p r e y (Goodyear, 1972). " e c o l o g i c a l death". by F a r r to  Mirex  (1977) who  These o b s e r v a t i o n s form a good example o f the c o n c e p t o f Another example was p r o v i d e d by an experiment r e p o r t e d exposed g r a s s shrimp  (Palaemonetes v u l g a r i s )  sublethally  (an o r g a n o c h l o r i n e i n s e c t i c i d e ) and found t h a t a f t e r 13 days  was no d i f f e r e n c e i n s u r v i v a l between c o n t r o l s and exposed g r o u p s .  there  However,  116  w i t h i n 24 h o f the i n t r o d u c t i o n o f p r e d a t o r y p i n f i s h  (Lagodon rhomboides)  s u r v i v a l o f the p r e v i o u s l y exposed shrimp dropped p r e c i p i t o u s l y .  Similar  r e s u l t s have been demonstrated u s i n g o t h e r simple f i s h p r e d a t o r / p r e y (Goodyear, 1972;  K a n i a and O'Hara, 1974;  S c h o o l i n g b e h a v i o r i s w e l l developed  the s e a r c h f o r food  (Eggers, 1976) -  systems  S u l l i v a n and A t c h i s o n , 1978). i n sockeye  salmon smolts and  have a d a p t i v e v a l u e a t t h e e s t u a r i n e s t a g e o f the l i f e in  the  Very  little  cycle  i t may  (Hoar, 1958)  or  i s known about the  m i g r a t o r y b e h a v i o r , r o u t e s f o l l o w e d , o r p h y s i o l o g i c a l and b e h a v i o r a l r e s p o n s e s d u r i n g e n t r y i n t o the sea  (Northcote, 1974;  suggests t h a t F r a s e r R i v e r sockeye environment r a t h e r q u i c k l y " based  ..."  Hanamura, 1966).  Ricker  (1966)  move out i n t o t h e o f f s h o r e p e l a g i c  on t h e i r absence from s e i n e c a t c h e s .  In  i n t e r p r e t i n g the s e l e c t i o n b e h a v i o r o f salmon i n a l a b o r a t o r y s a l i n i t y ent, Williams  (1969) suggested  t h a t sockeye  gradi-  salmon smolts move r a p i d l y out  into  the S t r a i t o f G e o r g i a w i t h i n s e v e r a l h o u r s o f e n t e r i n g the F r a s e r R i v e r Estuary.  Williams  (1969) a l s o found t h a t smolts from the C u l t u s Lake run were  capable o f s u r v i v i n g a d i r e c t t r a n s f e r i n t o sea water. t h e s e l a t t e r f i n d i n g s s i n c e i n t h e p r e s e n t experiments  My  results  support  c o n t r o l f i s h a t the  smolt stage m a i n t a i n e d t h e i r b l o o d e l e c t r o l y t e c o n c e n t r a t i o n s w i t h i n v e r y o narrow l i m i t s a f t e r t r a n s f e r t o 28 exposed f i s h c o u l d n o t . observed  /oo  sea water.  On the o t h e r nand,  I f the changes i n s c h o o l i n g and swimming b e h a v i o r  i n the l a b o r a t o r y were t o o c c u r i n the f i e l d upon e n t r y i n t o  water, sockeye  smolts c o u l d s u f f e r a heavy p r e d a t i o n .  salmonids  is  of natural predation  salmon smolts on t h e i r seaward m i g r a t i o n .  O f t e n n e g l e c t e d i n the i n t e r p r e t a t i o n o f s t u d i e s on e l e c t r o l y t e of  sea  However,very l i t t l e  known a t t h e p r e s e n t time about t h e e x t e n t o r importance on normal sockeye  DHA-  i s the c o n s i d e r a t i o n o f s a l i n i t y .  balance  Many l a b o r a t o r i e s , the p r e s e n t  117  one  included,  u t i l i z e c o a s t a l sea water  investigations. strength  ( s a l i n i t y 25-30 ^/oo ) i n t h e i r  In the w i l d , m i g r a t i n g sockeye salmon may  sea water s h o r t l y a f t e r l e a v i n g the r i v e r and  considerably  higher s a l i n i t i e s  move i n t o  would be  full  exposed  (y 35 °/oo). Boeuf e t a l . (1978) found  that  coho salmon smolts which remained i n an e s s e n t i a l l y homoiosmotic s t a t e t r a n s f e r i n t o sea water o f 30 °/oo  s a l i n i t y suffered  h y d r o m i n e r a l imbalance when p l a c e d  i n waters o f 35 °/oo  exposed sockeye salmon moving i n t o waters o f the considerably The  accumulate DHA  function reported  in this thesis.  pulp m i l l s at Prince  KME  George and  i n t h e s e f i s h nor  these r i v e r s must be t o x i c i t y was  of saturation,  long these residues on  can  exert  the l e v e l s o f DHA  that  the p o s s i b l e  not  their toxic  or o t h e r r e s i n River past  Quesnel o r down the Thompson R i v e r i n t e r a c t i o n s o f DHA  i n the r i v e r s .  The  the past  with other  addressed.  Results  reported  remarkably i n c r e a s e d  a drop w e l l w i t h i n  natural  by  known t o x i c i t y o f DHA,  safe  of  d i s s o l v e d oxygen i n  i n the p r e s e n t study showed  a reduction  o f oxygen t o  75%  fluctuations. bioaccumulation p o t e n t i a l ,  l i m i t s o f d i s c h a r g e o f t h i s , and  o t h e r r e s i n a c i d s , would b e t t e r be  been  question of a p o t e n t i a t i o n  f l u c t u a t i o n s i n temperature and  In view o f the demonstrated p e r s i s t e n c e , and  k i d n e y and  known t o be p r e s e n t i n k r a f t m i l l waste have not  t o x i c i t y by n a t u r a l  t h a t DHA  rapidly  However,DHA d e p u r a t i o n r a t e s were  information  p r e s e n c e and  persistent toxicants studied  be  l e v e l s are p r o b a b l y r e l a t e d t o the p h y s i o l o g i c a l dys-  i s t h e r e any  The  DHA-  shown.  t h a t salmon can  a c i d s i n sockeye salmon which have moved down the F r a s e r  Kamloops.  Thus  S t r a i t of Georgia could  exposure experiments e s t a b l i s h e d  measured, so i t i s not known how a c t i o n , nor  salinity.  i n major organs such as the b r a i n , l i v e r and  these h i g h r e s i d u e  after  from a s i g n i f i c a n t  more s e r i o u s l y a f f e c t e d than the p r e s e n t study has  laboratory  to  determined by e s t a b l i s h i n g an  possibly "Ecological  118  L i m i t " 4/ —  f o r these t o x i c a n t s .  b i o a c c u m u l a t i o n , and discharge date on  levels.  T h i s concept t a k e s i n t o account p e r s i s t e n c e ,  sublethal thresholds  to c a l c u l a t e environmentally  A l t h o u g h t h e r e appears t o be  sublethal thresholds  v a r i e t y o f p h y s i o l o g i c a l and  o f any  no p u b l i s h e d  information  r e s i n acid, sublethal thresholds  behavioral  safe  for a  f u n c t i o n s have been determined f o r  salmonids exposed t o whole k r a f t m i l l e f f l u e n t (Davis, 1976).  Perhaps t h e s e  e s t i m a t e s c o u l d be used f o r e s t a b l i s h i n g E c o l o g i c a l L i m i t s w h i l e s t u d i e s r e s i n acid sublethal thresholds T h i s study has concentrations  are being  demonstrated t h a t c h r o n i c exposure t o s u b l e t h a l  can r e s u l t i n s u b s t a n t i a l t o x i c a n t a c c u m u l a t i o n .  should  a f t e r t h e i r discharge  consider the b i o c o n c e n t r a t i o n  p o t e n t i a l of r e s i n acids  exposure was  (1958) d e f i n i t i o n o f an  The  present  study, w h i l e l i m i t e d  i n i t s scope,underlines  the  general  sockeye salmon  i n t e r a c t i o n s w i t h p u l p m i l l wastes  Furthermore, the study has  during  elucidated sublethal e f f e c t s of  on salmonids and p o s s i b l e mechanisms n o t p r e v i o u s l y d e s c r i b e d .  the magnitude and  behavior  then i t i s h i g h l y  on the p h y s i o l o g i c a l e c o l o g y o f the  e s p e c i a l l y i n r e l a t i o n t o the  DHA  field,  i f 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 r e d u c e d " .  paucity of information  migration.  environmental  shown t o " d i s t u r b the normal f u n c t i o n i n g "  l a b o r a t o r y a l s o o c c u r s i n the  p r o b a b l y t h a t the  Regulatory  "safe"  o f the sockeye salmon h y d r o m i n e r a l h o m e o s t a t i c mechanism and observed i n the  DHA  i n t o r e c e i v i n g waters.  When viewed i n t h e l i g h t o f B r e t t ' s s t r e s s , s u b l e t h a l DHA  on  conducted.  a g e n c i e s a t t e m p t i n g t o s e t water q u a l i t y c r i t e r i a u t i l i z i n g concentrations  to  While  scope f o r d e l e t e r i o u s e f f e c t s o f such t o x i c a n t s upon F r a s e r  R i v e r s t o c k s has n o t been d i r e c t l y i n v e s t i g a t e d , the p o t e n t i a l f o r such e f f e c t s upon a s e n s i t i v e l i f e stage o f m i g r a t o r y salmonids has  been demonstrated.  4/ — E c o l o g i c a l L i m i t (EL) concept was proposed i n the N e t h e r l a n d s by and S l o o f and i s d i s c u s s e d i n van E s c h (1978).  Canton  119  APPENDIX 1-1.  REMOVAL OF DHA FROM FRESH WATER BY SOCKEYE SALMON  In t h i s experiment, t h e acute t o x i c i t y o f DHA was i n v e s t i g a t e d i n r e l a t i o n t o f i s h loading density during a s t a t i c bioassay. a n a l y t i c a l methods were developed  In a d d i t i o n ,  which p e r m i t t e d t h e m o n i t o r i n g  o f the  a c t u a l c o n c e n t r a t i o n o f DHA i n t h e aquarium water. MATERIALS AND METHODS The  experiment was conducted u s i n g u n d e r y e a r l i n g sockeye salmon which  had been hatched stock.  a t PEI from eggs o b t a i n e d from t h e C u l t u s Lake 1973 brood  A t t h e time o f t h e experiment t h e f i s h were 4 months o l d and had  been t r a n s f e r r e d t o l a b o r a t o r y tanks  from outdoor f a c i l i t i e s a week e a r l i e r .  In t h e l a b o r a t o r y , they were kept i n tanks p r o v i d e d w i t h a continuous o f w e l l water a t t h e same temperature as o u t s i d e  (10.5 ±0.5°C).  flow  Daily feed-  i n g w i t h OMP was d i s c o n t i n u e d 24 h p r i o r t o t r a n s f e r o f t h e f i s h t o g l a s s aquaria f i l l e d  t o a volume o f 30 L w i t h w e l l water k e p t a t 10.5 ±0.5°C by  means o f a water b a t h .  An a i r - s t o n e m a i n t a i n e d  d i s s o l v e d oxygen l e v e l s  above 90% s a t u r a t i o n d u r i n g t h e study. A f t e r a 24 h a c c l i m a t i o n p e r i o d , t h e f i s h were g e n t l y t r a n s f e r r e d t o i d e n t i c a l t e s t a q u a r i a c o n t a i n i n g a t h e o r e t i c a l DHA c o n c e n t r a t i o n o f 1.88 mg/L.  One aquarium c o n t a i n e d t h e r e s i n a c i d b u t no f i s h , t o determine t h e  e x t e n t o f normal a d s o r p t i o n and/or d e g r a d a t i o n tank was s t o c k e d w i t h 5 f i s h (high l o a d i n g d e n s i t y ) .  o f t h e t o x i c a n t , a second  (low l o a d i n g d e n s i t y ) and a t h i r d w i t h 10 f i s h  A f o u r t h aquarium was used as a c o n t r o l and  c o n t a i n e d s i m i l a r d i l u t i o n s o f o n l y t h e DHA c a r r i e r s o l v e n t s and 10 f i s h . Times t o d e a t h  (as judged by c e s s a t i o n o f o p e r c u l a r movement) were  recorded,  and dead f i s h were weighed and measured. DHA was p r e p a r e d was  t o a p u r i t y o f 94% and a c o n c e n t r a t e d  stock  solution  made by d i s s o l v i n g 640 mg o f DHA i n 5 mL e t h a n o l f o l l o w e d by 5 mL 5N NaOH  120  and made up to 1000 mL with d i s t i l l e d water.  The addition of 94 mL of t h i s  solution to the aquarium water yielded a t h e o r e t i c a l concentration of 1.88 mg/L DHA. At 24 and 96 h, water samples (500 mL) were taken from both aquaria for determination of DHA concentrations.  Water samples were a c i d i f i e d with 1  drop of cone. H2SO4 and extracted with chloroform separatory funnel.  (4 x 50 mL)  i n a l L  The chloroform extracts were evaporated to dryness under  vacuum, re-dissolved i n d i e t h y l ether, methylated with diazomethane and dried under a stream of nitrogen.  Methanol d i l u t i o n s of these extracts were  analyzed by GLC and quantified by comparison of integrated peak areas to a c a l i b r a t i o n curve based on an external DHA standard.  Analyses were done on a  Hewlett Packard 7620A gas chromatograph equipped with an integrator and f i t t e d with a 1.2 m x 3 mm stainless s t e e l column packed with 10% S i l a r 5CP on Gas Chrom Q (80-100 M) .  Oven temperature was 240°C while i n j e c t i o n port  and detector (flame ionization) were operated at 250°C.  Nitrogen was the  c a r r i e r gas. RESULTS AND DISCUSSION At the low loading density, a l l f i s h were dead by 54.3 h (3255 min) whereas at the high loading density only 3 out of 10 f i s h died i n the f i r s t 39.3 h (2355 min)  and no further m o r t a l i t i e s occurred during the remainder  of the 96 h exposure period (Fig.22).  This high rate of s u r v i v a l of  f i s h exposed to 1.88 mg/L DHA can be a t t r i b u t e d to t h e i r rapid reduction of DHA concentration to a sublethal range (0.86 mg/L) a f t e r 24 h followed by a further reduction to 0.29 mg/L a f t e r 96 h (Table XVIII).On the other hand, a f t e r 24 h at the lower loading density, DHA l e v e l s were s t i l l at 1.16 mg/L; a concentration r a p i d l y l e t h a l to the salmon.  In addition, a 32% reduction  i n DHA concentration i n the tank containing no f i s h indicates that some  o  High Loading Density  A Low Loading Density  2 . 2 g/|_ 1.1 g/L  The e f f e c t s o f f i s h l o a d i n g d e n s i t y in. a s t a t i c b i o a s s a y on the acute o f DHA  (1.88 mg/L)  t o j u v e n i l e sockeye salmon.  toxicity  Table  XVIII.  The e f f e c t o f f i s h l o a d i n g d e n s i t y on c o n c e n t r a t i o n o f DHA i n aquarium water  Loading d e n s i t y  DHA  concentration  g/L  "initial  mg/L 24 h  96 h  0  1.44  0.98  1.1  1.16  0.77  2.2  0.86  0.29  theoretical  concentration  1.88  mg/L.  123  adsorption or degradation  took p l a c e .  These r e s u l t s showed t h a t a r e d u c t i o n i n DHA o c c u r r e d and t h a t t h i s r e d u c t i o n was fish.  However because DHA  been removed by simple  may  l e v e l s i n the water  g r e a t l y enhanced by the p r e s e n c e o f  n o t have been a c t u a l l y i n s o l u t i o n o r may  a d s o r p t i o n t o the tank w a l l s o r t o the s u r f a c e o f  f i s h , experiments were conducted t o answer these q u e s t i o n s . are d e s c r i b e d i n the r e s t o f Appendix I .  have the  These experiments  124  APPENDIX 1-2.  Although  EFFECTS OF FILTRATION ON DHA RECOVERY FROM FRESH WATER  DHA r e c o v e r y experiments c o n f i r m e d  t h a t t h e r e s i n a c i d was  p r e s e n t i n t h e water a t c l o s e t o t h e t h e o r e t i c a l c o n c e n t r a t i o n s , t h e y d i d n o t i n d i c a t e whether t h e t o x i c a n t was i n s o l u t i o n . been adsorbed  The r e s i n a c i d c o u l d have  t o p a r t i c u l a t e s p r e s e n t i n t h e water o r c o u l d simply have been  p r e s e n t i n suspension able t o the f i s h .  and as such might have been bound o r n o t f r e e l y  avail-  As these p a r t i c u l a t e s s h o u l d be r e a d i l y f i l t e r a b l e , a  comparison o f DHA r e c o v e r y from f i l t e r e d and u n f i l t e r e d s o l u t i o n s was made. METHODS R e p l i c a t e s o l u t i o n s o f DHA were made up u s i n g l a b o r a t o r y w e l l water as used i n b i o a s s a y experiments. which had been p r e p a r e d  A concentrated stock s o l u t i o n  (238.5 mg/L DHA)  f o r a b i o a s s a y was t h e source o f t h e r e s i n a c i d and  the same d i l u t i o n r a t i o as used i n c o n t i n u o u s - f l o w (3 mL stock/500 mL w a t e r ) .  b i o a s s a y s was u t i l i z e d  Four i d e n t i c a l s o l u t i o n s were p r e p a r e d  g l a s s b o t t l e s which were s t o p p e r e d ,  shaken t o ensure m i x i n g  i n 1000 mL  and then l e f t f o r  o 36 h i n a water b a t h a t 10.5 ±0.5 C.  Two o f t h e samples were then  acidified  w i t h 1 drop cone. H2SO4 and e x t r a c t e d w i t h c h l o r o f o r m a s p r e v i o u s l y d e s c r i b e d w h i l e t h e o t h e r two were s u c t i o n f i l t e r e d through before e x t r a c t i o n .  a 0.22 ym Nucleopore  filter  The e x t r a c t s were then a n a l y z e d by GLC as b e f o r e . RESULTS AND DISCUSSION  F i l t r a t i o n had no e f f e c t on t h e r e c o v e r y o f DHA from t h e water  (Table X I X ) .  As t h e o r i g i n a l t h e o r e t i c a l c o n c e n t r a t i o n was 1.35 mg/L DHA, and as t h e mean percentage  r e c o v e r y was 86.9% some l o s s d i d o c c u r d u r i n g t h e experiment,  p r o b a b l y by a d s o r p t i o n t o t h e g l a s s i n t h e b o t t l e s as w e l l as some l o s s d u r i n g the e x t r a c t i o n p r o c e s s .  As "apparent  s o l u b i l i t y " has been o p e r a t i o n a l l y  d e f i n e d by f i l t r a t i o n through a 0.45 ym membrane f i l t e r  ( S u f f e t and F a u s t ,  T a b l e XIX.  Comparison o f c o n c e n t r a t i o n s o f DHA r e c o v e r e d from f i l t e r e d and u n f i l t e r e d w a t e r samples.  Replicate samples  Filtered  Unfiltered  DHA  concentration m  9/  A  A  ' O r i g i n a l c o n c e n t r a t i o n 1.3 5  L  Recovery %  1.17  86.7  1.18  87.4-  1.18  87.4  1.16  85.9  mg/L.  1972)  and i n the p r e s e n t experiment  concluded t h a t the DHA a v a i l a b l e t o the  fish.  was  a 0.22  um  f i l t e r was  used,  i t was  indeed d i s s o l v e d and as such s h o u l d be  freely  127  APPENDIX 1-3.  ESTIMATE OF THE DIRECT SOLUBILITY OF DHA IN FRESH WATER  A l t h o u g h DHA may be c o n s i d e r e d the c h e m i c a l  definition  as b e i n g water i n s o l u b l e a c c o r d i n g t o  (Grant, 1944), t h e e x t e n t o f t h i s s o l u b i l i t y may  n e v e r t h e l e s s be s u f f i c i e n t t o be o f t o x i c o l o g i c a l and t h e r e f o r e b i o l o g i c a l significance. was  To t e s t t h i s h y p o t h e s i s ,  t h e d i r e c t aqueous s o l u b i l i t y o f DHA  determined by gas chromatographic methods. METHODS Dehydroabietic  acid  (95% p u r i t y ) c r y s t a l s were ground t o a f i n e powder  u s i n g a mortar and p e s t l e and t h r e e samples were weighed on an e l e c t r o b a l a n c e ( P e r k i n Elmer Autobalance Model AD^2) t o 0.01 mg and t h e n ' t r a n s f e r r e d t o 500 mL w e l l water  (pH 6.76) i n 1000 mL g l a s s b o t t l e s .  used t o keep t h e m i x t u r e i n suspension. b o t t l e , now a t room temperature  Magnetic s t i r b a r s were  A f t e r 24 h, t h e c o n t e n t s  o f each  (20°C) were f i l t e r e d through a 0.45 um f i l t e r .  A f t e r a c i d i f i c a t i o n , t h e f i l t r a t e s were e x t r a c t e d and a n a l y z e d  as p r e v i o u s l y  described. RESULTS AND DISCUSSION The  r e c o v e r y o f DHA from t h e water showed t h a t t h e r e s i n a c i d was  d i r e c t l y s o l u b l e i n w e l l water t o an average o f 3.3 mg/L (Table XX ). Nyren and Back  (1958) determined t h e t o t a l s o l u b i l i t y  u n i o n i z e d DHA) t o be 6.6 mg/L w h i l e  (sum o f i o n i z e d and  f o r t h e u n i o n i z e d a c i d i t was 4.9 mg/L  and demonstrated t h a t t h e s o l u b i l i t y was a f u n c t i o n o f pH. suggested t h a t t h e a r o m a t i c  r i n g o f DHA rendered  o t h e r r e s i n a c i d s such as a b i e t i c . suggest t h a t DHA may be capable  authors  i t more h y d r o p h i l i c than  The r e s u l t s o f t h e p r e s e n t  experiment  under c e r t a i n c o n d i t i o n s , o f d i s s o l v i n g  d i r e c t l y i n water t o a l e v e l a c u t e l y t o x i c t o salmonids. "insoluble", dehydroabietic  These  a c i d can be c o n s i d e r e d  While  chemically  toxicologically "soluble"  128  Table XX .  Replicate samples  B  C o n c e n t r a t i o n s o f DHA d i r e c t l y s o l u b l e i n f r e s h water a t pH 6.76 and 20°C.  C o n c e n t r a t i o n o f DHA i n o r i g i n a l mixture mg/L  C o n c e n t r a t i o n o f DHA i n solution mg/L  29.78  3.15  31.68  3.20  33.26  3.61  as the f r e e a c i d . s t a t u s o f DHA  As d i s c u s s e d i n t h e I n t r o d u c t i o n , the p r e c i s e  i n r e c e i v i n g waters has n o t been e s t a b l i s h e d a l t h o u g h i t i s  u s u a l l y d i s c h a r g e d as a sodium s a l t . be determined  chemical  The e x a c t c h e m i c a l s p e c i e s w i l l  by r e c e i v i n g water c h e m i s t r y .  conducted d u r i n g the p r e s e n t study, DHA b e f o r e a d d i t i o n t o the aquarium water.  was  In a l l b i o a s s a y  then  experiments  p r e p a r e d as the sodium  salt  130  APPENDIX 1-4.  UPTAKE OF DHA FROM FRESH WATER AND ITS DISTRIBUTION IN THE TISSUES OF JUVENILE SOCKEYE SALMON AND A MATURE RAINBOW TROUT  INTRODUCTION The  f i s h - l o a d i n g d e n s i t y experiment d e s c r i b e d i n Appendix 1-1 showed  t h a t DHA was removed from t h e water by t h e p r e s e n c e o f t h e salmon, however t h i s removal c o u l d be a c c o m p l i s h e d b y simple  adsorption t o the surface o f the  f i s h o r b y an a c t u a l a b s o r p t i o n i n t o t h e f i s h . It  i s known t h a t i n f i s h e s t h e e n t r y o f a t o x i c a n t a c r o s s t h e g i l l  membranes i s r e l a t e d t o i t s p a r t i t i o n c o e f f i c i e n t and i t s pKa (Hunn and A l l e n , 1974).  Dehydroabietic  a c i d i s a weak o r g a n i c a c i d o f v e r y low  aqueous s o l u b i l i t y b u t which i s f r e e l y s o l u b l e i n l i p i d chloroform.  s o l v e n t s such a s  Although no p u b l i s h e d i n f o r m a t i o n on t h e p a r t i t i o n  coefficient  o f DHA c o u l d be found i n t h e l i t e r a t u r e , t h e p a r t i t i o n c o e f f i c i e n t s o f a wide spectrum o f compounds a r e i n v e r s e l y r e l a t e d t o t h e i r aqueous and  i n t u r n determine b i o m a g n i f i c a t i o n and l i p o p h i l i c  1977;  N e e l y e t a l . , 1974; Yang and Sun, 1977).  storage  solubility  (Chiou e_t a l . ,  Based on i t s s o l u b i l i t y  p r o p e r t i e s and a t t h e pH o f w e l l water DHA c o u l d be e x p e c t e d t o p a s s r e a d i l y through t h e g i l l  epithelium  (McLeay e_t a l . , 1979) .  A f t e r e n t e r i n g t h e v a s c u l a r system, l i p i d  s o l u b l e compounds a r e  thought t o d i s s o l v e i n plasma l i p o p r o t e i n s (Fromm, 1970) and a r e then b u t e d throughout t h e body by t h e b l o o d deposited  i n tissues o f high l i p i d  distri-  (Holden, 1962) where they may be  content  (Branson e t a l . , 1975; F r e e d  e t a l . , 1976). As a p r e l i m i n a r y experiment taken up and c o n c e n t r a t e d bioassay,  (unpublished)  showed t h a t DHA was i n d e e d  from t h e water by sockeye salmon d u r i n g an acute  two d e t a i l e d experiments were performed t o measure t h e e x t e n t o f  DHA accumulation  d u r i n g s u b l e t h a l exposure and t o determine t h e t i s s u e  131  d i s t r i b u t i o n o f these residues. MATERIALS AND METHODS F i s h and T e s t C o n d i t i o n s Two s e p a r a t e experiments were conducted, one w i t h sockeye salmon and one w i t h a s i n g l e rainbow t r o u t  (Salmo g a i r d n e r i ) .  o b t a i n e d from t h e G r e a t C e n t r a l Lake the  PEI outdoor h o l d i n g f a c i l i t y  female rainbow t r o u t  Sockeye  salmon  smolts were  (Vancouver I s l a n d ) m i g r a t i o n and kept i n  f o r 2 months p r i o r t o use.  A s i n g l e mature  ( f o r k l e n g t h 47 cm, 1520 g wet weight) was used f o r t h e  t r o u t experiment and was p a r t o f a s t o c k o b t a i n e d from a commercial i n M i s s i o n , B.C.  These f i s h were 2+ y e a r s o l d and had been h e l d a t PEI f o r  an a d d i t i o n a l 18 months b e f o r e t h e experiment. 11.5  ±0.5°C i n w e l l water In  supplier  The f i s h were h e l d a t  (see T a b l e I ) .  t h e salmon experiment, 10 f i s h  (15-20 g) were t r a n s f e r r e d t o t h e  l a b o r a t o r y donut t a n k s r e c e i v i n g w e l l water a t t h e same temperature and were h e l d t h e r e f o r a f u r t h e r 72 h b e f o r e t h e t o x i c a n t exposure began.  The f i s h  were n o t f e d d u r i n g t h e a c c l i m a t i o n p e r i o d o r d u r i n g t h e 120 h exposure period.  Ten salmon were exposed t o 0.65 mg/L DHA i n one tank and another 10  s e r v e d as c o n t r o l s i n a second tank; a t h i r d tank c o n t a i n e d the s i n g l e bow t r o u t .  rain-  A c o n t i n u o u s c u r r e n t o f 15 cm/sec was m a i n t a i n e d and the f l o w o f  w e l l water a t a r a t e o f 500 mL/min p r o v i d e d a 90% replacement time o f ^ 4 h (Sprague, 1969).  The f i s h l o a d i n g d e n s i t y f o r t h e salmon was 3.6 L water/g  f i s h / d a y and 0.47 L/g/day f o r t h e t r o u t . Toxicant The DHA t o x i c a n t was p r e p a r e d and metered system by p r o c e d u r e s d e s c r i b e d p r e v i o u s l y .  i n t o t h e incoming water  A GLC check o f t h e DHA c o n c e n t r a -  t i o n a c t u a l l y p r e s e n t i n the water showed t h a t ^ 90% o f t h e t h e o r e t i c a l dosage had been a t t a i n e d .  The pH o f t h e b i o a s s a y water a f t e r a d d i t i o n o f the  132  s t o c k s o l u t i o n was temperature was  6.96  f o r the DHA  maintained  and 6.97  f o r the c o n t r o l tank.  The  water  a t 11 ±0.5°C and d i s s o l v e d oxygen > 90% s a t u r a t i o n .  Fish tissue preparation A t the c o n c l u s i o n o f the exposure p e r i o d , the salmon were a n e s t h e t i z e d w i t h 200 mg/L B l o o d was  MS-222, w h i l e the t r o u t was  c o l l e c t e d from severed c a u d a l p e d u n c l e s o f both  each salmon was smears and pooled  stunned by a blow on the head. Blood  from  s p o t t e d on a m i c r o s c o p e s l i d e f o r the p r e p a r a t i o n o f b l o o d  a f t e r c e n t r i f u g a t i o n the h e m a t o c r i t was  f o r the d e t e r m i n a t i o n  b l o o d c e l l s were p o o l e d rainbow t r o u t , DHA  of osmolality.  The  measured and  the plasma  was  In the salmon the packed r e d  f o r the d e t e r m i n a t i o n  o f DHA  r e s i d u e s w h i l e f o r the  r e s i d u e s were determined i n a sample o f whole b l o o d .  The g a l l b l a d d e r b i l e was Vacutainers.  species.  c o l l e c t e d from both  l i v e r , kidney,  spleen, g i l l ,  s p e c i e s u s i n g 3 mL  B-D  gut and b r a i n were r a p i d l y  removed from b o t h s p e c i e s and i n the salmon, t h e remainder was  termed the  "carcass". I n d i v i d u a l t i s s u e s were p o o l e d  and  f r e e z e - d r i e d t o c o n s t a n t weight.  the t r o u t , a d d i t i o n a l samples o f ova and c a r c a s s was  not a n a l y z e d .  l a t e r a l muscle were taken but  In the salmon, the e n t i r e b r a n c h i a l b a s k e t  u t i l i z e d w h i l e i n the t r o u t o n l y t h e g i l l muscle sample was  f i l a m e n t s were used.  removed from t h e d o r s o - l a t e r a l muscle mass  p o s t e r i o r t o the d o r s a l f i n and t h e s k i n was  The  trout  immediately  removed b e f o r e weighing.  t o be determined b e f o r e f r e e z e - d r y i n g so t h a t the % water c o u l d be weights o f p o o l e d  The weights  determined.  t i s s u e s were not measured,  wet  weights were approximated by u s i n g t h e salmon dry t i s s u e weights which been measured and  a p p l y i n g the % water o b t a i n e d f o r the t r o u t d a t a .  case o f salmon b i l e , e r y t h r o c y t e s , g i l l s and  c a r c a s s , the  the was  l a r g e mass o f the v a r i o u s t i s s u e s i n the t r o u t p e r m i t t e d a c c u r a t e wet  In salmon, where the wet  For  had  In the  relationship  133  between d r y and wet weight was e s t a b l i s h e d from c o n t r o l Chemical  samples.  Extraction  Dry t i s s u e samples were ground by mortar and p e s t l e w i t h sodium a c i d i f i e d w i t h a drop o f 10% H^SO^, packed on a chromatographic  sulfate,  column  (1 cm x 20 cm o r 2 cm x 30 cm, depending on sample volume) and e x t r a c t e d w i t h 100-200 mL p e s t i c i d e grade methylene  chloride  (Burdick & Jackson L a b o r a t o r i e s  Inc., M i c h i g a n , USA). The e x t r a c t s were evaporated t o d r y n e s s under vacuum, r e d i s s o l v e d i n d i e t h y l e t h e r , m e t h y l a t e d w i t h diazomethane, hexane d i l u t i o n s were then a n a l y z e d by GLC. samples  and s u i t a b l e  A n a l y s i s o f two c o n t r o l  s p i k e d w i t h DHA y i e l d e d 92.2 and 99.3% r e c o v e r y f o r t h i s  procedure.  liver  extraction  B i l e i n t h e V a c u t a i n e r v i a l s was decanted 5 times w i t h  methylene  c h l o r i d e , a c i d i f i e d , e v a p o r a t e d t o d r y n e s s , m e t h y l a t e d and then a n a l y z e d by GLC. Analytical  Procedure  The p r e s e n c e o f DHA was c o n f i r m e d i n hexane d i l u t i o n s o f t h e f i s h e x t r a c t s by gas chromatography-mass s p e c t r o m e t r y (GC-MS), u s i n g a Hewlett Packard HP 5992A f i t t e d w i t h a 1.8 m x 6 mm g l a s s column packed w i t h 3% OV-101 on S u p e l c o p o r t 80-100 mesh.  The oven temperature was programmed  from 150 t o 250°C a t 8 C/min a f t e r an i n i t i a l  i s o t h e r m a l h o l d o f 2 min.  o D e t e c t o r and i n j e c t i o n p o r t temperature was 250 C and h e l i u m was t h e c a r r i e r gas.  An e x t e r n a l DHA s t a n d a r d was used f o r comparison o f t h e mass s p e c t r a .  Ions were scanned from 40 t o 400 mass u n i t s w i t h a s i n g l e i o n b e i n g monitored a t m/e 239 CEnzell and Wahlberg,  1969).  Q u a n t i t a t i v e d e t e r m i n a t i o n o f DHA was made by GLC on a Hewlett Packard HP 5700 f i t t e d w i t h a flame i o n i z a t i o n d e t e c t o r u t i l i z i n g a s p l i t r a t i o o f 100:1  and a 30 m x 0.2 mm column w a l l - c o a t e d w i t h OV-101.  The oven  temperature was programmed from 124 t o 205 C U  i s o t h e r m a l h o l d o f 2 min.  a t 8 C/min a f t e r an  initial  D e t e c t o r and i n j e c t i o n p o r t temperature  was  o 250 C and h e l i u m was DHA  t h e c a r r i e r gas.  DHA  was  used as an e x t e r n a l s t a n d a r d .  Residue C a l c u l a t i o n R e s i n a c i d r e s i d u e s a r e e x p r e s s e d b o t h as ug DHA/g d r y t i s s u e and  t i s s u e i n o r d e r t o f a c i l i t a t e the comparison o f c o n c e n t r a t i o n (tissue  : water).  wet  factors  The salmon wet t i s s u e weights a r e e s t i m a t e s d e r i v e d from  measured d r y w e i g h t s as p r e v i o u s l y d e s c r i b e d .  The whole body r e s i d u e s f o r  the sockeye salmon were determined by summing the dry weight o f a l l the t i s s u e s t o r e c o n s t i t u t e the " o r i g i n a l " f i s h .  As the % water f o r the c a r c a s s  was known from c o n t r o l samples, t h e o r i g i n a l t o t a l wet weight o f t h e exposed f i s h c o u l d be b a c k - c a l c u l a t e d from t h e d r y w e i g h t s . r e c o v e r e d from the v a r i o u s t i s s u e s was  The t o t a l DHA  residue  summed and d i v i d e d by the wet  weight  o f the f i s h . F o r the t r o u t , the t o t a l body r e s i d u e s were c a l c u l a t e d by summing t h e i n d i v i d u a l t i s s u e DHA weight.  r e s i d u e s and d i v i d i n g by the o r i g i n a l t o t a l f i s h  E s t i m a t e s were made f o r b l o o d and muscle a c c o r d i n g t o p r o p o r t i o n s  i n the l i t e r a t u r e .  T o t a l b l o o d r e s i d u e s were e s t i m a t e d on the b a s i s o f a  b l o o d volume o f 3% o f body weight residues weight  wet  (Smith, 1966)  while t o t a l  "muscle"  were c a l c u l a t e d on t h e b a s i s o f a muscle mass o f 67% o f body  (Stevens, 1968).  As the t r o u t was  g r a v i d and h e a v i l y l a d e n w i t h  eggs, "body weight", f o r t h e purposes o f t h e s e c a l c u l a t i o n s , was  taken as  the t o t a l wet weight minus eggs. RESULTS Both f i s h s p e c i e s accumulated DHA weight b a s i s , t h e sockeye salmon DHA rainbow t r o u t c o n t a i n e d 22 mg/kg.  from t h e water.  On a whole body, wet  r e s i d u e s were 19.2 mg/kg w h i l e the  The DHA  d i s t r i b u t i o n i n various  tissues  135  i s shown f o r salmon wet  weight and  (Fig.23)  and  dry weight b a s i s  f o r rainbow t r o u t ( l e f t and  r i g h t ordinate  A l l c a l c u l a t i o n s which f o l l o w are based on wet As i n the the  shown i n F i g . -23, salmon b i l e  the h i g h e s t  the k i d n e y  r a t i o s o f 954,  428,  and  404  and  o f DHA  ratio  was  found  o f 996.  l a r g e s t amount (619.8 yg/g)  l i v e r (262.5 yg/g)  respectively.  The  yielding  carcass,  On  a wet  the head, s k e l e t o n ,  weight b a s i s , the  bioconcentration  which c o n s i s t s  muscle, s k i n e t c . , c o n t a i n e d 7.7  i n t e r n a l organs and  the  t o t a l f i s h weight y e t c o n t a i n e d 29%  and  k i d n e y accounted f o r 40%  of  The  rainbow t r o u t r e s i d u e  (290.6 yg/g) residues,  kidney  and  y i e l d i n g bioconcentration  b i l e c o n t a i n e d 17.9  the  t r o u t , the  l i v e r and  yg/g  t i s s u e s represent only  DHA.  17%  The  brain  of  liver  burden.  t i s s u e d i s t r i b u t i o n (Fig.24 ) shows the  (.182.5 yg/g)  The  there-  yg/g  o f the t o t a l body r e s i d u e .  o f the organ DHA  Of follow-  the o r i g i n a l body minus the t i s s u e s l i s t e d t o the r i g h t i n F i g . 2 3 and fore includes  a  t i s s u e weights.  (647.3 yg/g); t h i s y i e l d s a b i l e / w a t e r  (278.1 yg/g)  e x p r e s s e d on  scale, respectively).  o v e r a l l concentration  salmon organs, the b r a i n c o n t a i n e d the  ed by  (Fig.24)  (154.3 yg/g)  f a c t o r s o f 447,  281  r e s u l t i n g i n a bile/water  k i d n e y accounted f o r 56%  c o n t a i n e d the and  238  liver highest  respectively.  r a t i o o f 27.5.  o f the  For  t o t a l organ  DHA  burden. While the whole body b i o c o n c e n t r a t i o n rainbow t r o u t amounted t o 30 and  34  r e s p e c t i v e l y , i t can  i n d i v i d u a l organs, f o r example i n the the  0.65  f a c t o r s f o r sockeye smolts seen t h a t  in  l i v e r , DHA  was  p r e s e n t a t 400  times  yg/mL l e v e l p r e s e n t i n the water d u r i n g  the  120  period.  B i l e samples o f b o t h the  salmon and  d e r i v a t i v e s was o f the  molecule.  i d e n t i f i e d as m e t h y l a b i e t a t e t r a e n o a t e .  t i s s u e s taken from c o n t r o l  h exposure  t r o u t were found t o c o n t a i n  l e a s t t h r e e m e t a b o l i c d e r i v a t i v e s o f the p a r e n t DHA  i n any  be  and  fish.  DHA  One was  of  not  at the detected  136  r4.2 4.0 3.8  DRY  |  | WET  3.6 o  3.4  x  co O  x  -5!  N:  z7g  z o H.2S  cr >  t—  rZ  z LU  1.0  UJ UJ  8 m A.  a  CO  r0.6^ UJ  UJ CO  cr, 1< < O  Figure  23.  LL)  IOB"  co  s -  1  3.2 =<• 1.4  :  I r—  cr  3  CJ  < cr CO  LU  m  0.4'  < X  -0.2 Q  LU  D i s t r i b u t i o n o f d e h y d r o a b i e t i c a c i d i n p o o l e d t i s s u e s of sockeye salmon smolts a f t e r a 5 day l a b o r a t o r y exposure t o 0.65 mg/L DHA. . .  137  -1.2  DRY WET  1.0  o  p  X  X  CO  2  O  <  rx  < rx  LU  o o LU CO CO :  "  5 %  2^ Z LU LU  < D  LU  LU  Q.  co D  2 o  24;  O O  o o  -0.4 £ >•  3  < LU I  n  I LU Z3 CO  LU  LU  Figure  -0.6  CO  rx  Q < I Q  >-  LiJ >  0.2  3  _ i  CD  T i s s u e d i s t r i b u t i o n of d e h y d r o a b i e t i c a c i d i n a rainbow t r o u t a f t e r a 5 day l a b o r a t o r y exposure t o 0.65 mg/L DHA.  138  DISCUSSION D e h y d r o a b i e t i c a c i d i s a weak o r g a n i c a c i d which,based on i t s aqueous solubility  (Appendix 1-3), can be c l a s s i f i e d  (Grant, 1944).  On p h y s i c o c h e m i c a l  would be expected  c h e m i c a l l y as  grounds DHA  water  "insoluble"  a t the pH o f w e l l water  t o p a r t i t i o n r a p i d l y from the water, a c r o s s g i l l  epithelia,  i n t o t h e b l o o d and from t h e r e be d i s t r i b u t e d t o the v a r i o u s l i p i d p o o l s w i t h i n the body  (Hunn and A l l e n , 1974;  t i a t e d by the presence  o f h i g h DHA  i n salmon and t r o u t i n the p r e s e n t  McLeay e t a l . , 1979).  T h i s i s substan-  residues i n l i p i d extracts of tissues study.  No p u b l i s h e d i n f o r m a t i o n i s a v a i l a b l e on l e v e l s o f DHA organs o f f i s h .  in individual  Hagman (1936) i n a n a l y s e s o f moribund f i s h c o l l e c t e d down-  r i v e r from a s u l f a t e p u l p m i l l ,  found a p o s i t i v e  (colorimetrie) reaction for  r e s i n a c i d s i n e x t r a c t s of l i v e r , pancreas,  k i d n e y and mucus, w i t h  e s p e c i a l l y s t r o n g r e a c t i o n f o r the " l i q u i d  s u r r o u n d i n g the b r a i n " .  p r e s e n t study has h i g h e s t DHA  an The  shown t h a t the b r a i n o f the sockeye salmon accumulated the  r e s i d u e s o f any organ.  Tomiyama ( i n F u j i y a 1965)  p e n e t r a t i o n o f r e s i n a c i d s i n t o f i s h t i s s u e s may  suggested  that  have l e d t o c h r o n i c t o x i c  e f f e c t s such as the h i s t o p a t h o l o g i c a l changes r e p o r t e d by F u j i y a . noted a v a r i e t y o f n e c r o t i c changes i n the k i d n e y ,  Fujiya  i n t e s t i n e , pancreas  and  g i l l s o f f i s h taken from waters r e c e i v i n g k r a f t p u l p m i l l waste, w i t h p a r t i c u l a r l y s e r i o u s e f f e c t s on the l i v e r  ( F u j i y a 1961,  p r e s e n t work, the l i v e r c o n t a i n e d among the h i g h e s t DHA two  salmonid  1965).  In the  r e s i d u e s i n the  species tested.  The h i g h l e v e l o f DHA more p o l a r m e t a b o l i t e s  i n the b i l e  o f salmon as w e l l as the presence  i n b o t h f i s h s p e c i e s suggests  excretion route f o r t h i s r e s i n a c i d .  a hepatobiliary  In f i s h e s , t h i s r o u t e o f x e n o b i o t i c  of  139  e x c r e t i o n i s now  w e l l e s t a b l i s h e d , w i t h the l i v e r p l a y i n g a fundamental r o l e  i n biotransformation  o f l i p i d s o l u b l e compounds i n t o more p o l a r , water  s o l u b l e forms which are then e x c r e t e d K l a a s s e n 19751. and  v i a the b i l e  (Adamson and  Sieber  A number o f p e s t i c i d e s , p o l y c h l o r i n a t e d b i p h e n y l s ,  phenols,  d e t e r g e n t s have been i s o l a t e d from the b i l e o f a v a r i e t y o f f i s h  (Lech e t a l . , 1973;  Gakstatter,  e t a l . , 1973;Lech, 1973;  1968;  Melancon and  T o v e l l e t a l . , 1975).  among the requirements f o r e x t e n s i v e p o l a r i t y and  Lech, 1976;  Smith  1974;  species  Statham  (1971) r e p o r t e d  that  b i l i a r y e x c r e t i o n , a b a l a n c e between  n o n - p o l a r i t y , m o l e c u l a r weight o f 300-400, and  the p r e s e n c e o f  an e a s i l y i o n i z a b l e group are important p h y s i c o c h e m i c a l c h a r a c t e r i s t i c s . DHA,  w i t h a m o l e c u l a r weight o f 300  t h e s e requirements.  Hydroxylation  and  a carboxyl  group would appear t o f i t  by the l i v e r would i n c r e a s e  weight, make the r e s i n a c i d more p o l a r and  thus f a c i l i t a t e  molecular  its biliary  excretion. B i l e f l o w i s a l s o an important d e t e r m i n a n t o f the r a t e a t which many compounds are c l e a r e d from the plasma and S c h o t t e l i u s , 1969).  excreted  D u r i n g the exposure t o DHA,  a marked y e l l o w i s h t i n g e which was  i n the b i l e  the  sockeye salmon developed  p a r t i c u l a r l y noticeable  o f the p e c t o r a l and p e l v i c f i n s as w e l l as i n the plasma. were found t o be due plasma b i l i r u b i n .  t o a form o f j a u n d i c e  These r e s u l t s are d i s c u s s e d  b i l i r u b i n i s reduced by acute l i v e r d i s e a s e (Nosslin,1960).  B i l e i s known t o be  when r e l e a s e d i n t o the t i s s u e s d u r i n g  These symptoms  i n depth i n Appendix I I I . clearance  o r o b s t r u c t i o n o f the  acute o b s t r u c t i v e j a u n d i c e  highly caustic to trout l i v e r t i s s u e .  of bile  t o x i c t o human l i v e r parenchyma  19681; s i m i l a r l y H e n d r i c k s e t a l . , (1976) r e p o r t e d was  i n the membranes  caused by a DHA-induced r i s e i n  J a u n d i c e i n mammals i s known t o r e s u l t when h e p a t i c  duct  ( T u t t l e and  (Sherlock,  t h a t rainbow t r o u t b i l e  In the p r e s e n t  experiment,  the  140  f a i l u r e o r o v e r l o a d i n g o f the h e p a t o b i l i a r y system may  have c o n t r i b u t e d t o the a c c u m u l a t i o n o f DHA  v a r i o u s organs.  Presumably when uptake  accumulation o f DHA tissue  and/or b i l e  i n salmon exposed t o  DHA  residues to toxic l e v e l s i n  exceeds metabolism  i n the l i v e r may  and  excretion,  o c c u r and r e s u l t i n l o c a l i z e d  necrosis.  The rainbow t r o u t d i d n o t e x h i b i t j a u n d i c e .  I t s much l a r g e r s i z e  have enabled i t t o s t o r e o r m e t a b o l i z e more o f the DHA e f f e c t s become apparent;  e f f e c t s of The  before i t s t o x i c  the r e s u l t s o f p r e l i m i n a r y acute b i o a s s a y s  l i s h e d ) i n d i c a t e d t h a t l a r g e r f i s h may  may  (unpub-  be more r e s i s t a n t t o the acute  toxic  DHA.  s u b l e t h a l exposure  o f f i s h t o a t o x i c a n t can l e a d t o i t s a c c u m u l a t i o n  a t h i g h c o n c e n t r a t i o n s i n v a r i o u s i n t e r n a l organs which subsequently undergo h i s t o p a t h o l o g i c a l changes.  T h i s mode o f t o x i c a c t i o n has been w e l l documented  f o r p e s t i c i d e s and r e l a t e d c h e m i c a l s accumulated (Buhler e t a l . , 1969; or  i n the w i l d  Eller,  1971;  (Johnson, 1968;  by f i s h i n the l a b o r a t o r y  K r u z y n s k i , 1972;  Mathur, 1962;  Kennedy e t a l ^ . , 1970) .  and pronounced changes o c c u r i n the l i v e r most o f the organ systems a r e a f f e c t e d  (Couch,  Mount,  In many cases  1975)  1962)  rapid  although f r e q u e n t l y  (see Walsh and R i b e l i n , 1975  for  review). The accumulation o f heavy m e t a l s  such as mercury and cadmium has been  shown t o r e s u l t i n damage t o f i s h k i d n e y t u b u l e s  (Trump e t a l . , 1975)  as i n r e n a l l e s i o n s and l i v e r and gonad d e g e n e r a t i o n Summerfelt, 1975).  (Tafanelli  as w e l l  and  These a u t h o r s c o n c l u d e d t h a t the h i s t o p a t h o l o g i c a l  changes and subsequent  p h y s i o l o g i c a l d y s f u n c t i o n were caused d i r e c t l y  the accumulation o f l a r g e organ r e s i d u e s a f t e r s u b - l e t h a l  exposure.  by  141  Although h i s t o p a t h o l o g i c a l observations present  study,  were n o t attempted i n the  i t would be i l l u m i n a t i n g t o determine whether t h e h i g h DHA  r e s i d u e s can be l i n k e d t o i n t e r n a l organ h i s t o p a t h o l o g y ; Fujiya  such as r e p o r t e d by  (1961) i n f i s h exposed t o k r a f t m i l l waste i n t h e v i c i n i t y o f a  coastal pulp m i l l .  Renal damage may be one o f t h e main c o n t r i b u t i n g f a c t o r s  t o t h e hydromineral  imbalance observed i n the DHA-exposed  The  fish.  c a l c u l a t e d whole body wet weight DHA r e s i d u e s o b t a i n e d  compare f a v o r a b l y w i t h those  I found i n a p r e l i m i n a r y  Using a s e m i - q u a n t i t a t i v e t e c h n i q u e  i n this  (unpublished)  study  study.  (Mahood and Rogers, 1975) t o achieve  the s e p a r a t i o n o f i n t e r f e r i n g f a t t y a c i d s from DHA p r i o r t o GLC a n a l y s i s I measured whole body r e s i d u e s o f 27.3 mg/g DHA i n sockeye salmon which had d i e d d u r i n g freshwater  exposure t o 1.1 mg/L DHA.  This represents a b i o -  c o n c e n t r a t i o n o f ^ 25 x t h a t t h e o r e t i c a l l y p r e s e n t i n the water. Fox e t a l . (1977) exposed 200 g rainbow t r o u t t o d i l u t i o n s o f k r a f t m i l l waste  (KME) and found whole f i s h r e s i d u e s o f DHA approximately  the DHA c o n c e n t r a t i o n s p r e s e n t f i g u r e s , a 48 h continuous  i n the d i l u t e d e f f l u e n t .  20 times  Based on t h e i r  exposure o f t r o u t t o KME c o n t a i n i n g a mean  c o n c e n t r a t i o n o f 0.67 ug/mL DHA r e s u l t e d i n r e s i d u e s o f 10 yg/g. In t h e p r e s e n t  study,  an exposure o f 120 h to.0.65 yg/mL DHA r e s u l t e d  i n r e s i d u e s o f 22 yg/g f o r t h e rainbow t r o u t and 19.2 yg/g f o r t h e sockeye salmon.  I f t h e uptake r a t e o f DHA f o r rainbow t r o u t as r e p o r t e d by Fox  - e t a l . (1977). was l i n e a r , then a f t e r 120 h approximately  25 yg/g DHA.  r e s u l t s obtained  This estimate  i n the present  t h e i r f i s h should have  contained  compares f a v o r a b l y w i t h the  experiment.  I f such a comparison i s v a l i d ,  then t h e time f o r e q u i l i b r a t i o n o f t h e uptake r a t e would exceed 5 days. T e c h n i c a l l y , the s y n t h e s i s o f r a d i o - l a b e l l e d DHA would g r e a t l y s i m p l i f y t h e e s t i m a t i o n o f uptake and d e p u r a t i o n  rates of this resin acid.  142  APPENDIX 1-5.  THE ACCUMULATION OF DHA IN SALMON VIA THE DIETARY ROUTEEVIDENCE OF ACCUMULATION IN THE SALMON FOOD ORGANISM Anisogammarus c o n f e r v i c o l u s .  INTRODUCTION In a d d i t i o n t o a b s o r b i n g DHA  d i r e c t l y from the water, salmon may  on organisms which have accumulated the r e s i n a c i d above ambient  feed  levels.  The uptake and b i o c o n c e n t r a t i o n o f some p e s t i c i d e s by members o f the v a r i o u s t r o p h i c l e v e l s o f the a q u a t i c food c h a i n has been w e l l documented 1969;  Johnson et_ a i . ,  i s thought  1971;  Schoenthal,  1963)  and  (Hatfield,  i n some cases, t h i s  t o be the major source o f c o n t a m i n a t i o n  route  f o r f i s h i n n a t u r a l waters  (Macek and Korn, 1970). F i e l d s t u d i e s on f e e d i n g h a b i t s have e s t a b l i s h e d the g e n e r a l o f e s t u a r i n e amphipods i n the d i e t o f j u v e n i l e salmon 1972;  L e v i n g s , 1973).  Healey  o f sockeye salmon smolts  (1978) found  (Goodman and Vroom,  t h a t amphipods dominated the  sampled i n G e o r g i a S t r a i t , B.C.  Anisogammarus c o n f e r v i c o l u s was the d i e t o f j u v e n i l e chinook  i n f l u e n c e of a c o a s t a l pulp m i l l  tshawytscha  diet  and the amphipod  found t o be the most important  salmon O.  importance  food i t e m i n  w i t h i n the a r e a o f  (Kask and P a r k e r ,  1972).  As an i n c r e a s e i n the abundance o f t h e s e f o o d organisms has been observed  i n the v i c i n i t y o f p u l p m i l l o u t f a l l s and l o g s t o r a g e  ( B i r t w e l l , 1978;  Harger and Nassichuk,  1974;  i t i s c o n c e i v a b l e t h a t j u v e n i l e salmonids may  areas  Waldichuk and B o u s f i e l d , be  1962)  " a t t r a c t e d " i n t o waters  c o n t a i n i n g r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f t o x i c waste components. a d d i t i o n , i f f o o d organisms such as amphipods were t o accumulate  In  these  t o x i c a n t s above ambient l e v e l s , t h e r e would a r i s e the p o t e n t i a l f o r b i o c o n c e n t r a t i o n o f the more p e r s i s t e n t t o x i c components by f i s h u t i l i z i n g contaminated  food  source.  this  143  As DHA  i s known t o be one o f the more p e r s i s t e n t t o x i c a n t s p r e s e n t i n  k r a f t p u l p m i l l waste, a l a b o r a t o r y study was t h i s r e s i n a c i d was organism;  conducted t o determine whether  taken up from the water by a r e p r e s e n t a t i v e salmon food  the amphipod A.  confervicolus. MATERIALS AND  METHODS  Amphipods were c o l l e c t e d from t r o u g h s d r a i n i n g the outdoor f i s h h o l d i n g tanks a t PEI.. Water i n the t r o u g h s growth o f mussels  ( s a l i n i t y ^ 10 °/oo ) supported a t h i c k  ( M y t i l u s e d u l i s ) among which l i v e d the amphipods and  i s o p o d Gnorimosphaeroma o r e g o n e n s i s .  the  No e f f o r t was made t o s e p a r a t e the  amphipods from the mussel mass and a p p r o x i m a t e l y e q u a l amounts o f the mixture were p l a c e d i n t o two g l a s s c y l i n d e r s 9.2  x 40.7  covered by f i b e r g l a s s mosquito  Each c y l i n d e r was  netting.  cm  (2.76 L) w i t h the ends then l a i d f l a t  on  the bottom o f s e p a r a t e donut tanks i n which the r e c i r c u l a t i n g pump m a i n t a i n e d a c o n t i n u o u s water c u r r e n t o f ^ 15 cm/sec.  Approximately  Fucus v e s i c u l o s u s c o l l e c t e d from the beach was provide a d d i t i o n a l  30 g o f the a l g a  added t o each c y l i n d e r t o  shelter.  A f t e r a 48 h a c c l i m a t i o n p e r i o d , t h e amphipods were exposed mg/L  DHA  i n b r a c k i s h water  (10 ±1 °/oo ; 10.5  ±0.5°C; pH 6.97;  oxygen>90% s a t u r a t i o n ) under c o n t i n u o u s - f l o w c o n d i t i o n s . 740 mL/min p r o v i d e d a 95% replacement  time o f  4.5  h  to  0.4  dissolved  A flow rate of  (Sprague, 1969).  c o n t r o l tank r e c e i v e d the d i l u e n t s o l v e n t s w i t h o u t the r e s i n  The  acid.  A t the c o n c l u s i o n o f the 120 h exposure p e r i o d , the c y l i n d e r s were l i f t e d o u t o f the t a n k s , the c o n t e n t s emptied were p i c k e d o u t w i t h f o r c e p s .  i n t o a t r a y and the amphipods  D u r i n g the s o r t i n g p r o c e d u r e , the amphipods  were p l a c e d i n t o a t r a y c o n t a i n i n g uncontaminated s o r t i n g was  complete,  b r a c k i s h water.  After  they were p l a c e d on a s c r e e n , r i n s e d w i t h d e i o n i z e d  water, b l o t t e d and then f r o z e n r a p i d l y w i t h l i q u i d CC^.  Each amphipod mass  144  was  weighed and  and  dry weight.  then f r e e z e d r i e d t o determine the r e l a t i o n s h i p between  A f t e r g r i n d i n g w i t h Na2S0^, the sample was c h l o r i d e and p r o c e s s e d i n the f i s h samples. weight and  A f t e r GLC  wet  e x t r a c t e d w i t h methylene  same manner as p r e v i o u s l y d e s c r i b e d  f o r the  a n a l y s i s , the r e s u l t s were e x p r e s s e d on b o t h a  wet  dry weight b a s i s . RESULTS  S h o r t l y a f t e r t h e i r t r a n s f e r i n t o the l a b o r a t o r y donut t a n k s , amphipods appeared t o have s e t t l e d i n t o the v a r i o u s were a c t i v e l y f e e d i n g .  c r e v i c e s and  the  the  mussels  D u r i n g the course o f the experiment, no dead or  b e h a v i o r a l l y abnormal amphipods were observed and  the accumulation o f  f e c a l p e l l e t s downstream o f the g l a s s c y l i n d e r s i n d i c a t e d t h a t a c t i v e continued The  throughout the exposure p e r i o d . r e s u l t s show t h a t the amphipods c o n t a i n e d  t h e o r e t i c a l l y present  i n the water  (Table, XXI).  On  DHA  a t a l e v e l 21 x t h a t  a whole body wet-weight  b a s i s , t h i s f i g u r e compares f a v o r a b l y w i t h the 2 9 . 5 - f o l d DHA  by  sockeye salmon exposed t o 0.65  l e n g t h o f time.  residues  control  mg/L  concentration  information  on the  shape o f the uptake curve so  a t e q u i l i b r i u m are not known.  of  o f the r e s i n a c i d f o r the same  As b o t h exposures were o f r e l a t i v e l y s h o r t d u r a t i o n  d a t a do not p r o v i d e DHA  feeding  No DHA  was  detected  the that  i n the  samples. DISCUSSION  T h i s experiment i n d i c a t e s t h a t t h e r e e x i s t s a p o t e n t i a l f o r bio-accumul a t i o n o f DHA  v i a the d i e t a r y r o u t e , however the e s t i m a t e o f i t s r e l a t i v e  importance must await uptake and organisms such as amphipods may  assimilation studies. be exposed t o DHA  As  fish-food  from t h e i r own  food  supply  145  T a b l e XXI.  DHA r e s i d u e s i n the amphipod Anisogammarus c o n f e r v i c o l u s exposed t o 0.4 mg/L DHA f o r 120 h i n sea water o f s a l i n i t y 10 °/oo .  T o t a l Weight o f sample  N  DHA  Concentration ' Factor 2  Wet  Dry  g  g  Wet  Dry  yg/g  yg/g  29.03  Exposed  6.8400  1.9760  133  8.39  Control  10.7136  3.0793  208  ND  21  1 E s t i m a t e based on i n d i v i d u a l wet weight p o o l e d wet weight o f 100 amphipods.  (51.6 mg)  c a l c u l a t e d from the  2 Wet weight b a s i s r e l a t i v e t o maximum t h e o r e t i c a l c o n c e n t r a t i o n i n the water.  146  as w e l l as d i r e c t l y from the water, such s t u d i e s c o u l d b e s t be done i n a model ecosystem u t i l i z i n g Nevertheless, m i l l s may  s e v e r a l members o f the a q u a t i c f o o d c h a i n .  r e c e n t s t u d i e s have shown t h a t the r e g i o n s around p u l p  be a t t r a c t i v e t o j u v e n i l e salmonids because o f the p r e s e n c e o f a  more r e a d i l y a v a i l a b l e f o o d supply.  Under l a b o r a t o r y c o n d i t i o n s , the  a d d i t i o n o f d i l u t e KME  freshwater  to a r t i f i c i a l  stream communities l e d t o  an enhancement o f the biomass o f the amphipod Crangonyx  (Ellis,  i t appears t h a t s i m i l a r e f f e c t s do o c c u r i n the f i e l d .  Kelso  suggested  t h a t the i n t e n s e a g g r e g a t i o n  1967)  and  (1977)  o f the f i s h community near a k r a f t  p u l p m i l l o u t f a l l i n Lake S u p e r i o r was. due  t o the extreme abundance o f  b e n t h i c i n v e r t e b r a t e s a d j a c e n t t o the p o i n t o f e n t r y o f the e f f l u e n t plume. He  i n d i c a t e d t h a t the f e e d i n g response t o a h i g h b e n t h i c biomass may  r i d e any  avoidance r e a c t i o n t o the e f f l u e n t .  over-  I n c r e a s e d numbers o f j u v e n i l e  chinook salmon i n the a r e a a d j a c e n t t o a c o a s t a l p u l p m i l l a t P o r t A l b e r n i B.C.  may  1978).  have been due F i s h thus  t o such an enhancement o f the f o o d supply  (Birtwell,  " a t t r a c t e d " i n t o r e g i o n s o f i n c r e a s e d food supply would  exposed t o e l e v a t e d l e v e l s o f t o x i c a n t s i n the water as w e l l as i n the Although  (1968) conducted a l a b o r a t o r y growth  on j u v e n i l e chinook salmon f e d on t u b i f i c i d worms ( o l i g o c h a e t e s ) . on worms which had p r e v i o u s l y been exposed t o f u l l s t r e n g t h KME  consumed e q u a l amounts o f f o o d but had much lower growth r a t e s than controls.  diet.  the consequences o f consuming contaminated f i s h f o o d organisms  i n the w i l d a r e unknown, Tokar  fed  be  T h i s was  food u t i l i z a t i o n . s t r o n g l y suggest  study Salmon f o r 24  h  the  a t t r i b u t e d t o a s t r i k i n g r e d u c t i o n i n the e f f i c i e n c y o f As the f i s h were kept i n e f f l u e n t - f r e e water these  effects  a s u b l e t h a l t o x i c a c t i o n by some component accumulated from  the e f f l u e n t and absorbed by the  fish.  147  S i n c e s p e c i e s such as chinook, coho and chum salmon can spend  extended  p e r i o d s o f time f e e d i n g i n e s t u a r i n e h a b i t a t s which are a l s o b e i n g used f o r the d i s c h a r g e o f p u l p m i l l wastes  ( B i r t w e l l , 1978;  Davis e t a l . ,  S i b e r t and Kask, 1978), the i n g e s t i o n o f DHA-contaminated food  1978; organisms  c o u l d be o f c o n s i d e r a b l e s i g n i f i c a n c e t o these as w e l l as t o o t h e r species.  I f the r a t e o f t r a n s i t o f sockeye  e s t u a r i e s i s as r a p i d as t h a t thought  salmonid  salmon smolts through o t h e r  t o o c c u r i n the F r a s e r R i v e r E s t u a r y ,  ( W i l l i a m s , 1969), i t i s u n l i k e l y t h a t the consumption o f DHA-contaminated amphipods would p l a y a s i g n i f i c a n t p a r t i n the b u i l d u p o f DHA the o t h e r hand, w h i l e s t i l l aquatic insects  i n the r i v e r , sockeye  (Goodman, 1964)  p o t e n t i a l source o f DHA  residues.  smolts f e e d p r i m a r i l y  on  so t h a t t h i s f o o d source c o u l d be a  contamination.  I t i s n o t known whether the p o t e n t i a l f o r b i o - a c c u m u l a t i o n o f suggested by t h i s p r e l i m i n a r y study i s r e a l i z e d i n the f i e l d However c i r c u m s t a n t i a l e v i d e n c e i n d i c a t e s t h a t t h i s may Brownlee e t a l . (1977) found DHA 15 km  On  i n bottom sediments  DHA  situation.  indeed be the case.  a t a d i s t a n c e o f up t o  from the o u t f a l l o f a k r a f t p u l p and paper m i l l i n Lake S u p e r i o r .  Such sediment-bound DHA  may  be taken up by b e n t h i c organisms  t r a n s l o c a t e d t o bottom-feeding i n the sucker  (Catastomus);  fish.  Brownlee and Strachan  and  subsequently  (1977) found  a bottom f e e d e r , and i n y e l l o w p e r c h  DHA  (Perca  f l a v e s c e n s ) which f e e d on a q u a t i c i n v e r t e b r a t e s as w e l l as s m a l l f i s h e s . I t i s p o s s i b l e t h a t these f i s h accumulated  DHA  d i r e c t l y from the water  i n the r e g i o n o f the p u l p m i l l , but based on the amphipod d a t a o b t a i n e d i n the p r e s e n t study, t h e r e appears  t o be l i t t l e  reason t o e x c l u d e the f o o d  c h a i n r o u t e from a c o n t r i b u t i n g r o l e i n t h e b u i l d u p o f DHA  i n wild  fish.  14!  APPENDIX I I .  BACTERIAL KIDNEY DISEASE(BKD)  Salmonid b a c t e r i a l k i d n e y disease(BKD) i s caused by a genus o f Corynbacterium and  a f f e c t s a l l f i v e s p e c i e s o f P a c i f i c salmon, the A t l a n t i c  salmon, as w e l l as most o f the commonly c u l t u r e d t r o u t s p e c i e s . present  i n the c h r o n i c  stages,  f r e q u e n t l y reaches e p i z o o t i c p r o p o r t i o n s  As BKD  no t r e a t m e n t has  As a r e s u l t , BKD  a number o f U.S.  (Suzomoto  disease  the e n t i r e i n f e c t e d s t o c k i s r o u t i n e l y  i n f e c t i o n s have s e r i o u s l y a f f e c t e d p r o d u c t i o n  trout hatcheries  and marine n e t  infected  dying.  y e t been found, so t h a t when the  (Snieszko  e t a l . , 1955)  o f the most s e r i o u s problems f a c i n g P a c i f i c  hatcheries  and  are  in  currently  salmon c u l t u r e b o t h i n  pens.  A l t h o u g h most p r e v a l e n t BKD  u n t i l some o f the more s e v e r e l y  e x t e r n a l symptoms o r s t a r t  a r i s e s i n aquaculture operations  one  chronic  i n f e c t i o n s a r e h i g h l y r e s i s t a n t t o a n t i m i c r o b i a l drugs  e t a l . , 1977)  destroyed.  i n the  Because i t i s b a s i c a l l y asymptomatic i n the  i t u s u a l l y passes unnoticed  f i s h begin developing  be  stage and p r o g r e s s s l o w l y w i t h no apparent symptoms  f o r much o f the y e a r b u t e a r l y s p r i n g months.  I t may  i n f i s h culture operations,  i t is likely  that  a l s o a f f e c t s n a t u r a l s t o c k s o f salmon a l t h o u g h the consequences on marine  s u r v i v a l are unknown. B.C.  The  (Evelyn e t a l . , 1973)  Sound, Washington  d i s e a s e has and  r e c e n t l y i n a " w i l d " c h i n o o k salmon i n Puget  ( E l l i s e t a l . , 1978).  sampled r e t u r n i n g t o the C a p i l a n o a d u l t s were found t o be Station, personal t o BKD  been found i n " w i l d " rainbow t r o u t i n  River  i n f e c t e d w i t h BKD  communication).  i n coho salmon may  In 1977,  11 o f 41 coho salmon  (West Vancouver, B.C.) (G.E.  as  spawning  Hoskins, P a c i f i c B i o l o g i c a l  Recent e v i d e n c e suggests t h a t  be g e n e t i c a l l y determined  resistance  (Suzomoto e_t a l . , 1977).  149  Once the d i s e a s e i s e s t a b l i s h e d , i t s course and s e v e r i t y appear t o be a f f e c t e d by a v a r i e t y o f f a c t o r s such as water temperature season, degree o f crowding and d i e t . in  37  U.S.  and  hardness,  In a c o n s i d e r a t i o n o f water c h e m i s t r y  salmonid h a t c h e r i e s , Warren  (1963)  found t h a t as the c o n s t i t u e n t  l o a d o f the water d e c r e a s e d , the s e v e r i t y o f c o r y n b a c t e r i a l k i d n e y d i s e a s e increased.  However an e t i o l o g i c r e l a t i o n s h i p was  (1976)  et a l .  not e s t a b l i s h e d .  Wedemeyer  suggested t h a t i n c r e a s e d m o r t a l i t i e s i n s o f t waters may  t o i n c r e a s e d energy requirements A l t h o u g h BKD  be  due  f o r osmoregulation.  i s termed k i d n e y d i s e a s e , i t i s a c t u a l l y a s y s t e m i c  i n f e c t i o n and can a f f e c t most o f the v i t a l organs, a l t h o u g h the h e m a t o p o i e t i c t i s s u e o f the k i d n e y and s p l e e n are among the f i r s t t i s s u e s i n f e c t e d and Yasutake,  1956;  Snieszko e t a l . ,  1955).  (Wood  In more acute s t a g e s , the i n -  f e c t i o n l e a d s t o n e c r o s i s o f the e n t i r e k i d n e y and can extend t o the  gills,  liver,  (Bell,  s p l e e n , eyes, m u s c u l a t u r e and a n t e r i o r g a s t r o i n t e s t i n a l t r a c t  1961) .  Young and Chapman  (.1978)  d e s c r i b e d u l t r a - s t r u c t u r a l changes i n the  glomerulus and r e n a l t u b u l e s o f BKD  i n f e c t e d brook t r o u t  (Salvelinus  f o n t i n a l i s ) which were i n t e r p r e t e d as s i g n s o f i r r e v e r s i b l e c e l l  injury.  The p r o g r e s s i v e d e s t r u c t i o n o f k i d n e y t i s s u e can be expected t o l e a d t o osmoregulatory d y s f u n c t i o n and i t has been p o s t u l a t e d t h a t e v e n t u a l d e a t h be due t o r e n a l i n s u f f i c i e n c y  (1956)  (Bendele and K l o n t z ,  1975).  Wood and  may  Yasutake  suggested t h a t the complex and e x t e n s i v e m o r p h o l o g i c a l changes  observed i n o t h e r organs were p r o b a b l y n o t a d i r e c t r e s u l t o f i m p a i r e d r e n a l f u n c t i o n as the e x c r e t o r y system was  one o f the l a t t e r t i s s u e s t o be  by the d i s e a s e , but t h a t each organ was  affected  a f f e c t e d d i r e c t l y by the b a c t e r i a .  These a u t h o r s a t t r i b u t e d the o f t e n - r e p o r t e d edema t o damage t o the system, e s p e c i a l l y the m e s e n t e r i c b l o o d v e s s e l s .  circulatory  A reduction i n hematocrit,  15C  hemoglobin, of  and plasma p r o t e i n i n i n f e c t e d f i s h was  osmoregulatory d y s f u n c t i o n  i s d i f f i c u l t t o determine how  (Hunn, 1964;  i n t e r p r e t e d as a symptom  Suzumoto e t a l . , 1977).  While i t  f a r a l o n g the d i s e a s e has p r o g r e s s e d b e f o r e the  e x c r e t o r y f u n c t i o n o f t h e k i d n e y i s s e r i o u s l y a f f e c t e d i t i s known t h a t i t s h e m a t o p o i e t i c f u n c t i o n i s reduced e a r l y i n the i n f e c t i o n .  The p r o g r e s s i o n from  c h r o n i c s u b c l i n i c a l t o the m a n i f e s t e d _stages i s t h e r e f o r e accompanied ing  o f h e m a t o c r i t (Suzomoto e t a l . , 1977;  The Occurrence o f BKD In  by a lower-  Iwama, 1977).  D u r i n g E l e c t r o l y t e Balance  Experiments  t h e p r e s e n t s t u d y , i n d i c a t i o n s o f the p r e s e n c e o f a c h r o n i c  BKD  i n f e c t i o n i n the sockeye salmon smolts became apparent d u r i n g b l o o d sampling i n E x p t . 1.  S e v e r a l f i s h had lowered h e m a t o c r i t s and one showed a s w o l l e n  abdomen which  i s one o f the v i s i b l e  symptoms o f i n f e c t i o n .  However no  m o r t a l i t i e s were o b s e r v e d i n t h e s t o c k h o l d i n g t a n k s c o n t a i n i n g ^ 2000 smolts i n the week p r i o r t o o r d u r i n g E x p t . 1.  H e m a t o c r i t s o b t a i n e d i n Expt.  which f o l l o w e d immediately, i n d i c a t e d t h a t the d i s e a s e was r a p i d l y and a c h r o n i c m o r t a l i t y tanks a t t h i s time. the  salmon was  (.1-2  2,  p r o g r e s s i n g more  f i s h p e r day) began i n the s t o c k h o l d i n g  S h o r t l y a f t e r , a c o n f i r m a t i o n o f the p r e s e n c e o f BKD  in  o b t a i n e d through the D i a g n o s t i c S e r v i c e o f t h e F i s h H e a l t h  Program, P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B.C.  and the e n t i r e s t o c k  destroyed.  low  A t t h i s time tank m o r t a l i t i e s remained  s t o c k o f ^ 2 0 0 0 ) , t y p i c a l o f the c h r o n i c stage o f BKD i m p o s s i b l e t o judge t h e s e v e r i t y o f the c h r o n i c BKD E x p t s . 1 and 2 u n t i l  a f t e r t h e b l o o d sampling was  (2-3 f i s h p e r day i n a  infection.  As i t was  i n f e c t i o n i n f i s h used i n  completed,  lowered h e m a t o c r i t were judged t o be i n an advanced  was  fish  showing  stage of chronic  i n f e c t i o n and were d e l e t e d from the s t a t i s t i c a l c o n s i d e r a t i o n o f the d a t a . T h i s p r o c e d u r e was  based on two assumptions:  A) t h a t as the d i s e a s e p r o -  g r e s s e d i n s e v e r i t y , h e m a t o c r i t dropped and B) t h a t the i n c i d e n c e o f i n f e c t i o n  151  was  n o r m a l l y d i s t r i b u t e d i n t h e f i s h used i n t h e experiments.  the f i r s t  assumption  assumption  Support f o r  was p r o v i d e d above, w h i l e t h e b a s i s f o r t h e second  was ensured by a random sampling o f f i s h from t h e s t o c k tanks f o r  use i n E x p t s . 1 and 2. Hoffman  (1963), u s i n g t h e p r o p e r t i e s o f t h e normal d i s t r i b u t i o n , de-  s c r i b e d a g r a p h i c a l method which s e p a r a t e s c l i n i c a l l y h e a l t h y from d i s e a s e d persons.  By u s i n g a r i t h m e t i c p r o b a b i l i t y paper,  a normal d i s t r i b u t i o n c a n be  t r a n s f o r m e d i n t o a s t r a i g h t l i n e by p l o t t i n g c u m u l a t i v e f r e q u e n c i e s , e x p r e s s e d as p e r c e n t a g e s  o f t h e t o t a l f r e q u e n c y a g a i n s t t h e end p o i n t s o f t h e c l a s s  intervals. When t r e a t e d i n t h i s manner, a composite cally  d i s t r i b u t i o n comprising  clini-  "normal" and " s i c k " components can be g r a p h i c a l l y s p l i t i n t o two b y  eye f i t t i n g  a s t r a i g h t l i n e t o t h e component which r e p r e s e n t s t h e c l i n i c a l l y  normal d a t a w i t h maximum weight b e i n g g i v e n t o t h e p o i n t s around 50%.  Normal  l i m i t s a r e then a r b i t r a r i l y d e f i n e d t o be t h e v a l u e s which g r a p h i c a l l y e n c l o s e 95% o f those o b t a i n e d by t e s t i n g c l i n i c a l l y the p r e s e n t experiment  for to  In  t h e r e f o r e , f i s h were d e f i n e d as " d i s e a s e d " i f t h e i r  h e m a t o c r i t s f e l l i n t h e lower 2.5% o f t h e c l i n i c a l l y Separate  "normal" s u b j e c t s .  "normal" v a l u e s .  c u r v e s were p l o t t e d u s i n g t h e h e m a t o c r i t s o f a l l t h e " c o n t r o l "  fish  Expt. 1 and Expt. 2, t h e lower l i m i t s were e s t a b l i s h e d and then a p p l i e d e l i m i n a t e f i s h from b o t h c o n t r o l and e x p e r i m e n t a l groups h a v i n g hemato-  c r i t s below t h e lower l i m i t s . w h i l e f o r Experiment  I n Experiment  2 i t was 25.2%.  1 t h e lower  The d a t a f o r t h e f i s h which were t h u s  e l i m i n a t e d a r e p r e s e n t e d i n T a b l e XXII and XXIII Due  l i m i t was 26.8%  respectively.  t o t h e p r o g r e s s i v e n a t u r e o f BKD, t h i s s c r e e n i n g method was c o n s i d e r -  ed t o e l i m i n a t e f i s h which were i n t h e advanced c h r o n i c o r s e v e r e stages o f a d i s e a s e w h i c h may compromise osmoregulatory  performance.  This screening  T a b l e X X I I . S i z e , h e m a t o c r i t and plasma i o n i c c o m p o s i t i o n o f f i s h d e l e t e d s u s p e c t e d advanced i n f e c t i o n o f b a c t e r i a l k i d n e y d i s e a s e .  Sampling Time (hours)  c Group  Fish size Fork l e n g t h Wet w e i g h t cm g  Hematocrit  Osmolality  %  mOsm/kg  Chloride mEg/1  from d a t a of Expt.  Sodium mEq/1  1  because o f a  Potassium  Calcium  Magnesii  mEq/1  mEq/1  mEq/1  24  C  14.5  33.2  26.4  298  126.0  159.7  2.78  5.57  1.82  48  E  15.8  36.4  26.3  298  140.0  152.0  3.23  4.90  3.79  72  E  14.8  31.7  25.9  264  113.0  150.5  3.21  6.31  1.52  96  C  14.2  28.1  20.8  284  129.5  148.8  3.33  5.27  1.59  120  E  14.6  29.2  25.5  298  135.5  161.0  3.41  5.25  1.68  Based on h e m a t o c r i t <  26.8%  ^Sockeye salmon smolts were exposed t o sea water a f t e r  a 120  h exposure t o 0.65  mg/L  DHA  i n f r e s h water.  "C=control, E=exposed. to  T a b l e X X I I I . S i z e , h e m a t o c r i t and plasma i o n i c c o m p o s i t i o n o f f i s h d e l e t e d ' s u s p e c t e d advanced i n f e c t i o n o f b a c t e r i a l k i d n e y d i s e a s e .  Sampling Time (hours)  Group  Fish size Fork length Wet weight cm g  Hematocrit %  Muscle Water %  Gut Water %  Osmolality mOsm/kg  Chloride mEq/1  from data o f Expt.2  Sodium mEq/1  Potassium mEq/1  because o f a  Calcium mEq/1  Magnesium mEq/1  0  C C  16.4 17.2  47. 7 52. 9  21. 7 17. 7  75. 69 75. 38  77. 93 79. 72  290 274  113 .0 114 ..0  148. 2 144. 2  4. 47 3. 66  5. 48 6. 13  1.56 1.56  24  C C  16.5 16.4 13.6  45. 5 40. 5 28. 1  21. 3 23. 39.o  71. 81 73. 31 71. 29  73. 61 76. 77 72. 76  300 334 350  130. 5 131,.0 162. 5  148. 1 170.9 172. 6  3. 29 4. 36 4. 17  5. 53 7. 30 3. 61  2.65 4.11 2.09  c c  14.0 15.7 13.8  28.5 41.,2 28. 6 .  20.,2 22..7 19.,9  73. 55 75.,25 74. 47  77. 27 79. 07 81. 17  294 304 306  129.,5 131.,5 127.,5  156. 7 • 158.,3 160.,0  4.,74 3.,45 4.,45  5.,49 5.,10 5..51  1.59 1.91 1.73  C C C  16.4 14.9 14.7 17.7 16.6  48.,7 33..4 29.,1 58.,0 43..1  20..6 19..8 22..8 24..0 23..2  74.,95 74..50 75..92 74..90 75..03  77.,98 79..44 79.,46 84..17 80..03  306 304 292 302 232  129 .0 122,.5 129 .0 138,.5 100 .0  150..1 161..0 154.,3 157..6 122,.4  4..26 3,.99 3,.97 4..03 2 .59  5..13 5..91 5,.58 5,.03 4,.18  1.58 1.89 1.95 1.76 2.45  17.6 15.8 16.0 14.9  53,.0 41..1 36,.0 37,.3  9..3 18,.6 21 .4 20 .6  74,.67 74,.93 75 .12 74 .75  77,.15 80,.24 79 .71 81,.39  304 296 304 282  132 .0 133 .5 134 .0 131 .5  153,.7 158,.8 144 .3 150 .2  3 .25 5 .53 3 .62  5 .13 5 .26 4 .85 5 .13  1.76 1.51 2.20 2.43  14.1 16.0 15.9 15.0  27 .7 37 .8 40 .8 37 .3  24 .5 18 .4 22 .5 21 .9  75 .15 75 .88 74 .50 81 .20  77 .15 79 .43 80 .31 75 .41  316 300 298 282  130 .0 124 .5 129 .5 127 .5  156 .9 146 .2 153 .4 150 .7  4 .47 4 .25 4 .55 4 .06  6 .74 5 .95 5 .29 4 .43  1.75 1.72 1.61 1.62  c 48  C  72  E E 96  C C  E E 120  c C C  E  d  4 .02  Based on h e m a t o c r i t < 25.2%. Sockeye  salmon s m o l t s were exposed t o s e a water a f t e r a 120 h exposure t o 0.65 mg/L  C = c o n t r o l , E=exposed. ^ E l i m i n a t e d on b a s i s o f BKD  symptoms; f l u i d  filled  abdomen.  DHA  i n f r e s h water.  154  method c o u l d n o t e l i m i n a t e c h r o n i c a l l y i n f e c t e d f i s h so t h a t the r e s u l t s o f Expt. 2 may The  be viewed as an i n t e r a c t i o n o f d i s e a s e  I n t e r a c t i o n o f BKD  w i t h the T o x i c i t y o f  The p r e s e n c e o f a c h r o n i c BKD tibility  o f sockeye salmon t o DHA  with healthy  toxicant.  DHA  i n f e c t i o n appeared t o i n c r e a s e the toxicity  (Fig.25).  t o x i c a n t exposure p e r i o d and  seawater  recovery  detected,  the d i s e a s e  t o x i c i t y o f DHA,  phase.  In E x p t . 1, i n which the BKD  near the end  a t o t a l o f 6/60  d i f f e r e n t manner than f o r E x p t s .  h i n the  i n f e c t i o n was  first  i n c r e a s e i n the l a t e n t  exposed f i s h d y i n g  1 and  i n this period.  generated i n a  slightly  3 i n which a c t u a l t i m e s t o death were  In Expt. 2 f o u r f i s h were c o l l e c t e d a f t e r d e a t h b u t e i g h t  were sampled i n a "moribund" c o n d i t i o n t o p e r m i t the d e t e r m i n a t i o n gut w h i l e  s t i l l alive.  would have d i e d w i t h i n 3-4 the t o x i c i t y curve,  hours but  sampling time was  Based on p a s t o b s e r v a t i o n s ,  fish  o f % water these  t a k e n t o be  time t o death.  The  l i n e ; s h i f t i n g i t s l i g h t l y t o the r i g h t  ( F i g . 25) .  at  l o s t e q u i l i b r i u m ) b u t were not  " i n v e r t e d " stage  ( f i s h which had  t o p l o t the t o x i c i t y c u r v e . was  ing  In Expt. 2,  r e a c h i n g an advanced c h r o n i c stage i n  l o a d i n g due  t o the d i s e a s e and  b o t h e a r l i e r and  addition  the seawater r e c o v e r y  the  Three f i s h were sampled  i n which the BKD  used  infection  some f i s h , the combined s t r e s s  t o x i c a n t was  shown by m o r t a l i t i e s o c c u r r -  i n g r e a t e r numbers, as i l l u s t r a t e d  That the s l o p e o f the m o r t a l i t y curve  fish  f o r the p u r p o s e s o f the c o n s t r u c t i o n o f  o f s e v e r a l hours would have made a s m a l l d i f f e r e n c e i n the l o c a t i o n o f  the  of  t h a t i s , m o r t a l i t i e s began o n l y a f t e r the f i s h had been i n  " m o r t a l i t y " c u r v e shown f o r Expt. 2 was  * i n muscle and  suscep-  In E x p t . 3 conducted  the second a f t e r 65.5  appeared t o c o n t r i b u t e t o an  sea water f o r 24 h w i t h  recorded.  the  f i s h , o n l y 2 out o f the 72 exposed f i s h d i e d ; one  the freshwater  The  and  in Fig.  25.  does not b r e a k u n t i l 24 h i n t o  p e r i o d i n d i c a t e s a p e r s i s t e n c e i n the enhancement  155  Figure  25.  The e x t e n t o f salmon m o r t a l i t y d u r i n g the t h r e e e l e c t r o l y t e b a l a n c e experiments (Expts. 1, 2 and 3) i n which sockeye salmon smolts were exposed t o sea water a f t e r a 120 h exposure t o 0.65 mg/L DHA i n f r e s h water.  156  o f t h e s u b l e t h a l t o x i c i t y o f DHA. t o t a l o f 15/75.  A f t e r t h e break, two more f i s h d i e d f o r a  E v i d e n c e t h a t t h e BKD i n f e c t i o n p e r se o r i n combination w i t h  the s a l i n i t y s t r e s s was n o t l e t h a l i s p r o v i d e d  by t h e t o t a l absence o f  c o n t r o l m o r t a l i t y i n Expt. 1 and o n l y 1 m o r t a l i t y o u t o f 72 c o n t r o l f i s h i n Expt. 2 i n which t h e d i s e a s e was more advanced. In t h e p r e s e n t  context  "acute t o x i c i t y " i s d e f i n e d as t h a t  d u r i n g t h e 96-h t o x i c a n t exposure p e r i o d .  occurring  The " s u b l e t h a l " exposure used f o r  the e l e c t r o l y t e b a l a n c e experiments was p u r p o s e l y  chosen t o cause n e g l i g i b l e  m o r t a l i t y i n an a d d i t i o n a l 24-h exposure t o DHA.  The m o r t a l i t y d a t a  t h a t t h i s d e s i g n was a c h i e v e d  confirm  and t h a t depending on t h e s e v e r i t y o f the BKD  i n f e c t i o n , t h i s " s u b l e t h a l " exposure s h i f t e d c l o s e r t o t h e "acute" l e v e l o f exposure and t h e i n t e r a c t i o n o f s t r e s s e s o f d i s e a s e / t o x i c a n t / s e a contributed  t o an i n c r e a s e i n t h e l a t e n t t o x i c i t y o f DHA.  t o a s h i f t t o t h e l e f t o f t h e acute t o x i c i t y curve b a s i s f o r the choice o f a concentration  water  T h i s would amount  (Fig.9) which was t h e  o f DHA which would be " s a f e " f o r 120 h.  157  APPENDIX I I I .  PLASMA BILIRUBIN  INTRODUCTION D u r i n g t h e c o u r s e o f exposure t o DHA, j u v e n i l e sockeye salmon were seen t o d e v e l o p a j a u n d i c e d appearance c h a r a c t e r i z e d by a y e l l o w i s h t i n g e  which  became p a r t i c u l a r l y  As  n o t i c e a b l e i n t h e membranes o f t h e p a i r e d f i n s .  j a u n d i c e i n mammals i s brought about by a b u i l d u p o f b i l i r u b i n i n the b l o o d , plasma b i l i r u b i n  levels  o f exposed f i s h were compared t o those o f c o n t r o l s .  In v e r t e b r a t e s , b i l i r u b i n i s a n a t u r a l end-product o f t h e c a t a b o l i s m o f hemoglobin,  formed i n t h e r e t i c u l o e n d o t h e l i a l system, d e l i v e r e d t o the b l o o d  stream and c l e a r e d by uptake i n t h e l i v e r c e l l s . recognized:  unconjugated  normally c i r c u l a t i n g s o l u b l e i n water.  (UCB) and c o n j u g a t e d (CB) b i l i r u b i n .  i n t h e b l o o d stream  In t h e l i v e r , i t undergoes  forms t h e b a s i s o f t h e " d i r e c t "  - soluble  f o r b i l i r u b i n and t h e r e l a t i v e  Bilirubin  (UCB) i s p r o t e i n bound and i n a c o n j u g a t i o n and i s thus  rendered s o l u b l e before e x c r e t i o n i n t o the b i l e .  test  Two t y p e s o f b i l i r u b i n a r e  This d i f f e r e n t i a l  (CB) v s . " i n d i r e c t "  solubility  - insoluble(UCB)  p r o p o r t i o n s o f CB/UCB a r e used t o  c h a r a c t e r i z e t h e type o f j a u n d i c e i n v o l v e d .  F o r example, a r i s e  i n serum  UCB w i t h o u t a concomitant r i s e i n CB can i n d i c a t e an i n c r e a s e i n b l o o d d e s t r u c t i o n o r h e m o l y s i s b u t t h a t t h e h e p a t o b i l i a r y system i s s t i l l bilirubin  i n a normal  excreting  fashion.  As normal serum c o n t a i n s n e g l i g i b l e t o v e r y low amounts o f CB, i t s r i s e i s i n t e r p r e t e d as a s i g n o f a h e p a t o b i l i a r y d i s o r d e r such as acute disease or b i l i a r y obstruction  parenchymal  (Gray, 1961; N o s s l i n , 1960; Wintrobe,  1961).  To determine whether t h e apparent j a u n d i c e was r e l a t e d t o e l e v a t e d bilirubin  levels,  b o t h CB and UCB c o n c e n t r a t i o n s were measured i n sockeye  salmon smolts i n two experiments  (Expt. PB-1 and PB-2).  158  MATERIALS AND METHODS In b o t h experiments a 5-day exposure o f smolts t o 0.65 mg/L water was  DHA  i n fresh  f o l l o w e d by t e r m i n a l b l o o d sampling and a d e t e r m i n a t i o n o f plasma  bilirubin levels.  In Expt. PB-1  b i l i r u b i n was  determined i n b l o o d p o o l e d  from 10 f i s h which had been exposed d u r i n g the DHA (Appendix 1-4). exposure was  t i s s u e r e s i d u e experiment  When e l e v a t e d plasma b i l i r u b i n l e v e l s were c o n f i r m e d , the  r e p e a t e d u s i n g 20 f i s h .  experiment, b i l i r u b i n was  In a d d i t i o n t o the c o n t r o l s f o r each  a l s o measured i n a sample o f plasma p o o l e d from  5 salmon taken from the l a b o r a t o r y s t o c k tank d u r i n g p r e l i m i n a r y work. The sockeye salmon smolts used i n t h e s e experiments were from the same s t o c k as used i n the DHA  t i s s u e r e s i d u e experiment and h o l d i n g ,  t o x i c a n t exposure and b l o o d sampling p r o t o c o l was  acclimation,  the same as d e s c r i b e d i n  the G e n e r a l Methods. A f t e r the measurement o f h e m a t o c r i t (Hct) o f each f i s h , the b l o o d plasma was p o o l e d f o r measurement o f o s m o l a l i t y (CB) b i l i r u b i n as w e l l as plasma i r o n  (mOsM), t o t a l  (Fe).  (CB+UCB) and  direct  Plasma Fe was measured t o  determine whether the b i l i r u b i n e m i a c o u l d be a t t r i b u t e d t o i n c r e a s e d hemog l o b i n breakdown as o c c u r s i n h e m o l y t i c j a u n d i c e (Smith, 1973). Plasma b i l i r u b i n was measured by the m o d i f i e d d i a z o procedure developed by M i c h a e l s s o n (1961) f o r the d e t e r m i n a t i o n o f serum b i l i r u b i n i n newborn i n f a n t s .  However, r a t h e r than u s i n g 1 mL o f 1/10  _ 50 uL o f u n d i l u t e d plasma was  diluted  serum,  added t o the r e a g e n t s h a l v e d i n volume.  Absorbance was measured a t 600 nM on a Pye Unicam o r a G i l f o r d 2400 Spectrophotometer. was  To check the a c c u r a c y o f the method, a c a l i b r a t i o n curve  c o n s t r u c t e d u s i n g t o t a l b i l i r u b i n l e v e l s g i v e n i n human serum c a l i b r a -  tion references  (General D i a g n o s t i c s , C a l i b r a t e I , I I , I I I ) . As t h i s showed  159  t h a t the a b s o r p t i o n law was b e i n g f o l l o w e d , t h e c a l i b r a t i o n c o n s t a n t o f 43 was used as d e s c r i b e d by M i c h a e l s s o n  (1961).  Plasma i r o n was measured i n p o o l e d samples o f b l o o d taken from i n E x p t s . PB-1 and 2, as w e l l as from a s i n g l e rainbow t r o u t the development o f t h e a n a l y t i c a l p r o c e d u r e s . those d e s c r i b e d f o r human serum  (1400 g) d u r i n g  P r e p a r a t i v e methods f o l l o w e d  ( P e r k i n Elmer, 1971) and plasma i r o n  measured by Atomic A b s o r p t i o n Spectrophotometry a t 248.3 nm  salmon  was  u s i n g an a i r - a c e t y l e n e flame  UV. RESULTS  No m o r t a l i t i e s were observed exposure p e r i o d .  i n e i t h e r experiment  d u r i n g the 5-day  While b l o o d plasma o f c o n t r o l f i s h was e s s e n t i a l l y  t h a t o f t h e DHA-exposed f i s h developed  clear,  the c h a r a c t e r i s t i c yellow tinge.  The  r e s u l t s o f t h e b l o o d c h e m i s t r y a n a l y s e s a r e shown i n T a b l e XXIV f o r Expt. PB-1 and i n T a b l e XXV results. the d i r e c t  f o r Expt.  PB-2.  Both experiments  show s i m i l a r  Plasma b i l i r u b i n l e v e l s a r e e l e v a t e d i n f i s h exposed t o DHA, (CB) a c c o u n t i n g f o r 81.6% and 69.7% o f t h e t o t a l  (Expts. PB-1 and 2 r e s p e c t i v e l y ) . There appears plasma i r o n , w h i l e h e m a t o c r i t was e l e v a t e d r e s i n a c i d exposure.  with  bilirubin  t o be v e r y l i t t l e  change i n  and o s m o l a l i t y lowered by the  In t h e b l o o d plasma p o o l e d from the 5 salmon taken  t h e s t o c k h o l d i n g tank, c o n j u g a t e d b i l i r u b i n was n o t d e t e c t a b l e w h i l e b i l i r u b i n was 0.30 mg%.  from  total  The plasma o f the s i n g l e rainbow t r o u t c o n t a i n e d  30 ug% i r o n . DISCUSSION An i n c r e a s e i n plasma b i l i r u b i n can be caused by i n c r e a s e d b l o o d d e s t r u c t i o n o r by the r e d u c t i o n i n the c a p a c i t y o f t h e l i v e r t o remove the pigment from t h e b l o o d and e x c r e t e i t i n t h e b i l e .  However, t h e l i v e r i s g e n e r a l l y  T a b l e XXIV.  B l o o d c h e m i s t r y o f sockeye salmon s m o l t s exposed 0.65 mg/L DHA f o r 5 days i n E x p t . PB-1.  P o o l e d Plasma Bilirubin Total  yg%  Blood Osmolality  Hematocrit  mOsm/kg  %  N  Exposed =10  2.06  1.68  46  282  49.0  ±1.55  Controls N=10  0.19  0.11  42  290  41.7  ±0.69  S i z e range 15-20 b  Direct mg%  Iron  to  Mean  g  ±SE  S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s p<  0.05  ( t-test )  T a b l e XXV.  B l o o d c h e m i s t r y and s i z e o f sockeye salmon smolts exposed t o 0.65 mg/L DHA f o r 5 days i n Expt.PB 2.  Pooled Bilirubin Total  Iron  Fish  Blood  Plasma Osmolality  Direct mg%  yg%  mOsm/kg  Hematocrit  Fork length cm  Wet weight x ±SE  Exposed N=20  2.28  1.59  38  288  52.4 ±0.8  13.7  Controls N=18  0.47  ND  38  297  43.0 ±0.6  14.0 ±0.2  Not  detectable  ^ D i f f e r s s i g n i f i c a n t l y from c o n t r o l p<0.05  (t-test)  ±0.3  g  22.41 ±1.57  22.0 ±1.01  162  c o n s i d e r e d t o have a l a r g e r e s e r v e c a p a c i t y and an i n c r e a s e i n plasma b i l i r u b i n can u s u a l l y be l i n k e d t o a d y s f u n c t i o n i n t h e h e p a t i c e x c r e t i o n o f the  pigment  (Wintrobe, 1961).  N e v e r t h e l e s s , t h e l e v e l s o f plasma b i l i r u b i n  have been s e l e c t e d a s a measure o f i n c r e a s e d Hb d e g r a d a t i o n i n rainbow t r o u t and coho salmon exposed t o f o r m a l i n  (Wedemeyer,1971).  Hemoglobin i s broken down i n t o g l o b i n , i r o n and b i l i r u b i n .  In t h e  rainbow t r o u t , most o f t h e i r o n i s bound t o t h e plasma p r o t e i n  transferrin  (Fromm, 1977), however d u r i n g i n c r e a s e d r e d b l o o d c e l l d e s t r u c t i o n ,  this  b i n d i n g c a p a c i t y may be exceeded and l e a d t o i n c r e a s e d plasma Fe l e v e l s . The method used i n t h e p r e s e n t study measured t h e l e v e l s o f unbound plasma Fe and t h e r e s u l t s i n d i c a t e no measurable e f f e c t o f DHA on t h i s parameter i n sockeye salmon. the  The range o f plasma i r o n v a l u e s  mean o f 55 y g % o b t a i n e d f o r t h e rainbow t r o u t In  (38-46 yg%) i s lower than (Fromm, 1977).  t h e p r e s e n t study i t i s u n l i k e l y t h a t t h e o b s e r v e d i n c r e a s e i n plasma  b i l i r u b i n was due t o i n c r e a s e d r e d b l o o d c e l l d e s t r u c t i o n o r h e m o l y s i s . The exposure o f salmon t o DHA i n f r e s h water i n v a r i a b l y l e d t o an e l e v a t i o n , n o t a l o w e r i n g o f h e m a t o c r i t , no change o r a s l i g h t drop i n plasma K  +  and v i r t u a l l y  no change i n plasma Fe l e v e l s ; b o t h o f which c o u l d be e x p e c t e d t o r i s e as a r e s u l t of increased erythrocyte destruction.  The o b s e r v e d r i s e i n plasma  b i l i r u b i n can t h e r e f o r e be a t t r i b u t e d t o a r e d u c t i o n i n t h e e f f i c i e n c y o f i t s excretion. The r e l i a b l e measurement o f t r a c e amounts o f b i l i r u b i n i n plasma i s d i f f i c u l t and c o n s i d e r a b l e work has been done towards t h e development o f methodology, m o s t l y because o f t h e importance o f a c c u r a t e d e t e r m i n a t i o n s i n i n f a n t s s u f f e r i n g from n e o n a t a l j a u n d i c e . i s c o n s i d e r e d t o be t h e most r e l i a b l e  The method o f M i c h a e l s s o n (1961)  (With, 1968) and was a p p l i e d t o t h e  163  measurement o f salmon b i l i r u b i n i n t h i s study.  Due t o t h e use o f d i f f e r e n t  methods, w i d e l y v a r y i n g l e v e l s o f plasma b i l i r u b i n have been r e p o r t e d f o r fish.  S a k a i and Kawazu (1978) r e p o r t e d 0.025 mg% f o r t h e c a r p  . c a r p i o ) and 0.032 mg% f o r t h e rainbow t r o u t . Buckley  (Cyprinus  S a t i a e t a l (1974) and  (1976) r e p o r t e d 0.05 mg% f o r rainbow t r o u t and coho salmon r e s p e c t i v e l y  w h i l e Wedemeyer  (1971) found plasma b i l i r u b i n  t r o u t and 1.6 mg% f o r coho salmon.  l e v e l s o f 0.6 mg% f o r rainbow  In t h e p r e s e n t study, t o t a l plasma  r u b i n l e v e l s i n c o n t r o l sockeye salmon averaged  0.33 mg%.  bili-  The sample o f  plasma p o o l e d from 5 salmon taken from t h e s t o c k tank y i e l d e d 0.30 mg%. accompanying v e r y low o r n o n - d e t e c t a b l e  The  l e v e l s o f conjugated b i l i r u b i n i n  c o n t r o l samples a r e c o n s i s t e n t w i t h t h e mammalian l i t e r a t u r e  (Gray, 1961;  Wintrobe, 1961). When t h e r e i s i n c r e a s e d b l o o d d e s t r u c t i o n , t h e conjugated  form o f  b i l i r u b i n c o n s t i t u t e s l e s s than 15% o f t h e t o t a l ; h i g h e r p r o p o r t i o n s a r e u s u a l l y a t t r i b u t e d t o the r e g u r g i t a t i o n o f the conjugated g l u c u r o n i d e by t h e l i v e r  ( M i c h a e l s s o n , 1961).  bilirubin  In salmon exposed t o DHA,  t h e r e was a d r a m a t i c r i s e i n t h e t o t a l b i l i r u b i n , o f which the b u l k was a t t r i b u t e d t o t h e c o n j u g a t e d  form.  (70-82%)  T h i s c o n s t i t u t e s f u r t h e r evidence  a g a i n s t i n c r e a s e d b l o o d d e s t r u c t i o n and t h e s e symptoms t h e r e f o r e can be i n t e r p r e t e d as a r e s u l t o f o b s t r u c t i v e j a u n d i c e . Two main t y p e s o f o b s t r u c t i v e j a u n d i c e a r e r e c o g n i z e d : due  A)  Mechanical  -  t o e x t r a - h e p a t i c o b s t r u c t i o n o f b i l e f l o w and B) Parenchymatous - due t o  i n t r a - h e p a t i c o b s t r u c t i o n stemming from t h e d i s t o r t i o n o f l i v e r a r c h i t e c t u r e by l o c a l n e c r o s i s o f l i v e r c e l l s  (Gray, 1961).  cell  Extra-hepatic  o b s t r u c t i o n l e a d s t o the f o r c e d r e t e n t i o n o f b i l e i n the l i v e r , d i s t e n d i n g the b i l i a r y passages and e v e n t u a l l y r u p t u r i n g t h e b i l e c a p i l l a r i e s .  This r e s u l t s  i n t h e passage o f b i l e i n t o t h e s i n u s o i d s and back i n t o t h e b l o o d .  As t h e  164  bile  i s t o x i c t o human l i v e r parenchymal c e l l s , i t s leakage o u t o f t h e b i l e  canaliculi  can l e a d t o l o c a l  (1976) r e p o r t e d  necrosis  (Sherlock,  1968).  Hendricks e t a l .  t h a t rainbow t r o u t b i l e was h i g h l y c a u s t i c t o t r o u t  liver  tissue. The  jaundice  both types.  o b s e r v e d i n salmon s u b l e t h a l l y exposed t o DHA c o u l d be o f  I n f i s h which swallowed l a r g e amounts o f f r e s h water t h e i n c r e a s e d  h y d r o s t a t i c p r e s s u r e o f a t u r g i d stomach may have reduced b i l e  flow.  In f i s h  i n which no excess water i n t a k e was observed, i n t r a - h e p a t i c o b s t r u c t i o n i s suggested and may be r e l a t e d t o l i v e r  histopathology.  Residue a n a l y s e s showed t h a t t h e l i v e r had among the. h i g h e s t o f DHA o f a l l t h e t i s s u e s i n v e s t i g a t e d concentrations  (Fig.23  and F i g . 2 4 ) .  o f DHA i n t h e l i v e r , t h e j a u n d i c e  The h i g h  and t h e suggested importance  of the h e p a t o - b i l i a r y route f o r e x c r e t i o n o f the t o x i c a n t tempting t o s p e c u l a t e  residues  t h a t an i n t e r r e l a t i o n s h i p e x i s t s .  (p.138) make i t  Fujiya  (1961, 1965)  observed n e c r o t i c changes i n many o f t h e major organs, e s p e c i a l l y t h e l i v e r , p a r t i c u l a r l y around t h e b i l i a r y k r a f t p u l p m i l l waste.  ducts,  o f f i s h taken from waters r e c e i v i n g  Tomiyama (1965) p r o p o s e d t h a t these h i s t o p a t h o l o g i c a l  changes may have been caused by r e s i n a c i d s . observations  Although h i s t o p a t h o l o g i c a l  were n o t conducted i n t h e p r e s e n t study, DHA a c c u m u l a t i o n may  r e s u l t i n n e c r o t i c changes i n t h e l i v e r which c o u l d account f o r t h e observed obstructive On  jaundice.  t h e o t h e r hand, t h e a c c u m u l a t i o n o f DHA may be secondary t o t h e  obstructive  jaundice.  which many x e n o b i o t i c s excreted  i n the b i l e  Bile  f l o w i s an important d e t e r m i n a n t o f t h e r a t e a t  ( f o r e i g n compounds) a r e c l e a r e d from t h e plasma and ( T u t t l e and S c h o t t e l i u s , 1969).  The f a i l u r e  o r over-  l o a d i n g o f t h e h e p a t o b i l i a r y system i n f i s h exposed t o DHA may have  contributed  165  t o the accumulation  of t h i s t o x i c a n t i n v a r i o u s organs.  e s p e c i a l l y marked i f t h e b i l e t o n e c r o t i c changes and i n e x c r e t i o n o f DHA  This could  be  l e a k e d out i n t o t h e l i v e r parenchyma l e a d i n g  a reduction in physiological function.  A.reduction  v i a t h e b i l e c o u l d compromise the f u n c t i o n o f  other  organs. The DHA-induced j a u n d i c e may  have o t h e r c h r o n i c e f f e c t s ,  t h e d i r e c t i n f l u e n c e o f f r e e b i l i r u b i n on t i s s u e metabolism. plasma b i l i r u b i n  levels  such as o c c u r  in_ v i t r o  oxidative phosphorylation  i n i s o l a t e d mitochondria indirectly,  (Gray, t o one  1961).-,These o f the p o s s i b l e  as p r o p o s e d  by  t h e s e mechanisms r e m a i n s p e c u l a t i v e , h y p e r b i l i r u b i n e m i a i s  f r e q u e n t l y r e l a t e d t o a reduced  c l e a r a n c e due  to hypoxia  and  w i t h the c e l l volume r e g u l a t o r y mechanism o f r e d b l o o d c e l l s In the p r e s e n t  study,  shown t o be c h a r a c t e r i s t i c  hypoxic  of  (1965) .  Although  salmon.  jaundice,  accumulates i n b r a i n t i s s u e .  modes o f t o x i c a c t i o n o f k r a f t m i l l e f f l u e n t on f i s h ,  1961).  to  elevated  s t u d i e s have demonstrated the u n c o u p l i n g  f i n d i n g s are remarkably c l o s e , a l b e i t  Warner  At  i n i n f a n t s during neonatal  b i l i r u b i n c r o s s e s t h e b l o o d / b r a i n b a r r i e r and A t these l e v e l s ,  related  As one  and  symptoms o f s u b l e t h a l DHA  o f the suggested  s t r e s s , t h e r e may  the accumulation  increased hematocrit  be  of b i l i r u b i n  mechanisms i s one  can  interfere  (Wintrobe,  j a u n d i c e have been  t o x i c i t y t o sockeye  of toxicant-induced  a r e l a t i o n s h i p between h y p o x i a ,  hematocrit,  i n t h e b l o o d o f salmon exposed t o  DHA.  and  166  APPENDIX IV.  THE RED  EFFECTS OF SUB-LETHAL DHA EXPOSURE IN FRESH WATER ON BLOOD CELL DIMENSIONS OF SOCKEYE SALMON SMOLTS  In the p r e s e n t t o DHA  study,  THE  the s u b l e t h a l exposure o f j u v e n i l e sockeye salmon  i n f r e s h water i n v a r i a b l y r e s u l t e d i n e l e v a t e d b l o o d h e m a t o c r i t  As a r i s e i n h e m a t o c r i t may  values.  r e s u l t from an i n c r e a s e i n r e d b l o o d c e l l  size,  measurements were made o f t h e l e n g t h and w i d t h o f e r y t h r o c y t e s taken from exposed and  control fish. MATERIALS AND  Blood  METHODS  smears were made d u r i n g b l o o d sampling a t t h e c o n c l u s i o n o f  exposure p e r i o d i n t h e DHA  r e s i d u e experiment  (Appendix 1-4).  smears were a i r d r i e d , f i x e d i n m e t h y l a l c o h o l and (Hesser,  1960).  The  the  Duplicate  s t a i n e d w i t h Giesma  l e n g t h and w i d t h o f 25 r e d b l o o d c e l l s was  determined  on  each s l i d e u s i n g an o c u l a r micrometer i n a m i c r o s c o p e under 400  x magnifi-  cation . C e l l areas were c a l c u l a t e d u s i n g the f o r m u l a ( l e n g t h the r a t i o o f width/length Burton, 1979).  was  used as a measure o f c e l l roundness  and  (Murray  and  the means were c a l c u l a t e d  on the b a s i s o f t h e measurements o f 50 c e l l s p e r f i s h . roundness f o r exposed and  Student's t - t e s t .  c o n t r o l f i s h were then compared u s i n g  had been determined f o r i n d i v i d u a l  RESULTS AND results  Grand means f o r c e l l  In a d d i t i o n , the o s m o l a l i t y o f p o o l e d plasma samples  measured a f t e r the h e m a t o c r i t s  The  7r)and  As p r e l i m i n a r y c a l c u l a t i o n s showed no d i f f e r e n c e between  r e p l i c a t e s f o r each f i s h , the v a l u e s were p o o l e d  a r e a and  x w i d t h x 0.25  was  fish.  DISCUSSION  (Table XXVI) show t h a t DHA-exposed salmon d i s p l a y e d two  of  the  symptoms c h a r a c t e r i s t i c o f s u b l e t h a l exposure t o t h i s t o x i c a n t i n f r e s h water; a l o w e r i n g o f plasma o s m o l a l i t y and a r i s e i n h e m a t o c r i t .  Elevated  hematocrit  167  was  accompanied by a h i g h l y s i g n i f i c a n t i n c r e a s e i n the a r e a o f the r e d  cell  (Table XXVI) but t h i s o c c u r r e d w i t h o u t  roundness.  any  As the o b s e r v e d r i s e i n h e m a t o c r i t  g r e a t e r than the measured change i n c e l l a r e a probably  change i n the degree o f  due  (17.4%) was (10.9%),  blood cell  considerably  the d i f f e r e n c e i s  t o a concomitant i n c r e a s e i n c e l l t h i c k n e s s  (height).  An a p p r o x i m a t i o n o f the change i n c e l l volume can be c a l c u l a t e d by t a k i n g the c e l l h e i g h t t o be ^ 4 p i n c r e a s e i n h e i g h t was  (Eddy, 1977)  and  assuming t h a t  the  p r o p o r t i o n a t e l y s i m i l a r t o t h a t observed f o r the  l e n g t h and w i d t h dimensions.  By a p p l y i n g the formula  f o r a p r o l a t e sphere  (V=4/3 TT lwh), where 1, w and h r e f e r t o the r a d i i o f the r e d b l o o d c e l l ,  the  3 mean volume f o r the c o n t r o l c e l l s i s 211 ^10%,  ym  .  I f the c e l l h e i g h t  the mean volume o f the "exposed" c e l l s would be  an 18%  i n c r e a s e i n volume and  i n blood  367  ym^.  increased  This  represents  c o r r e s p o n d s w e l l t o the observed r i s e o f  17.4%  hematocrit.  Based on the r e s u l t s o f t h i s experiment as w e l l as on a more thorough d i s c u s s i o n o f o t h e r mechanisms which c o u l d l e a d t o hemoconcentration i t can be concluded  (p.98  t h a t the s u b l e t h a l exposure o f sockeye salmon t o DHA  ),  in  f r e s h water l e a d s t o a d i l u t i o n o f the b l o o d plasma and t o a s w e l l i n g o f r e d b l o o d c e l l s which c o n t r i b u t e s t o an i n c r e a s e i n b l o o d  hematocrit.  3" o f j u v e n i l e sockeye salmon exposed t o 0.65 mg/L DHA f o r 120 h T a b l e XXVI. Red b l o o d c e l l d i m e n s i o n s i n f r e s h water.  Blood  Erythrocyte Roundness  Hematocrit %  125.03 ±0.94  0.552 ±0.005  48.98 ±1.55  112.74 ±1.82  0.556 ±0.005  41.72 ±0.69  Length ym  Width ym  Area ym  Exposed  17.02 ±0.08  9.36 ±0.06  Control  16.06 ±0.52  8.94 ±0.10  a  2  b  Based on t h e means o f measurements  a  o f 50 r e d b l o o d c e l l s f o r each o f 10 f i s h .  'Pooled plasma Differs  s i g n i f i c a n t l y from c o n t r o l  3  p<0.001  p<0.01  Student's t - t e s t  Plasma Osmolality mOsM/kg  282  290  169 LITERATURE CITED Adamson, R.H., and S.M. S i e b e r . 1974. The d i s p o s i t i o n o f x e n o b i o t i c s by fishes, p.203-211. I n : M.A.Q. Khan and J.B. Bederka, J r . , eds. S u r v i v a l i n t o x i c environments. Academic P r e s s , New York. A l d e r d i c e , D.F. and J.R. B r e t t . 1957. Some e f f e c t s o f k r a f t m i l l e f f l u e n t on young P a c i f i c salmon. J . F i s h . Res. Board Can. 14: 783-795. Basch, R.E. and J.G. Truchan. 1976. T o x i c i t y o f c h l o r i n a t e d power p l a n t c o o l i n g waters t o f i s h . U.S. E n v i r o n . P r o t . Agency E c o l . Res. S e r . No. R-800700. 105pp. B e l l , G.R. salmon  1961. Two e p i d e m i c s o f apparent k i d n e y d i s e a s e i n c u l t u r e d p i n k (Oncorhynchus g o r b u s c h a ) . J . F i s h . Res. 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