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Intertidal spawning of chum salmon : saltwater tolerance of the early life stages to actual and simulated… Groot, Erick P. 1989

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INTERTIDAL SPAWNING OF  CHUM  SALMON: SALTWATER TOLERANCE OF  THE EARLY LIFE STAGES TO ACTUAL AND SIMULATED INTERTIDAL CONDITIONS by ERICK P. GROOT B.Sc. (hon.), U n i v e r s i t y of V i c t o r i a , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology, Resource Ecology) We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1989 :.=•:;-••/©:  E r i c k Peter Groot, 1989  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  ^Th'yoL&Qy^  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Qrh  6. tqRCj  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  Department of  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT  Intertidal  s p a w n i n g o f chum s a l m o n ,  by measuring egg s u r v i v a l , and s i m u l a t e d i n t e r t i d a l  development,  conditions.  were m o n i t o r e d i n the i n t e r t i d a l using s a l i n i t y locations,  the e f f e c t  these  and  British  conditions  level.  actual  temperature Columbia,  at  five  Artificially  were m o n i t o r e d t o  on egg s u r v i v a l  I n t r a g r a v e l oxygen and g r a v e l q u a l i t y  determine  and  a l s o were measured  at  stations.  Saltwater salinities regular  i n u n d a t i o n of the g r a v e l  a s h i g h a s 30°/oo> f o r  streambed r e s u l t e d i n  durations  i n u n d a t i o n o f warmer s a l t w a t e r ,  intertidal  sites  up t o 8 h ,  i n the  negative  effects  Instead,  e g g s u r v i v a l was s t r o n g l y  intertidal  twice d a i l y .  egg development a t  zone ranged, from (0-51.2%).  of saltwater  s u g g e s t e d t h a t eggs  i n the  from r e g u l a r  Laboratory  exposure on egg s u r v i v a l were  intertidal  z o n e may b e n e f i t  into  e x p e r i m e n t s were d e s i g n e d to t e s t conditions.  measurement o f p e r i v i t e l l i n e  ii  the  Due  to  lower  control  sites.  no  observed.  correlated with intragravel  inundation of seawater  under s i m u l a t e d i n t e r t i d a l saltwater,  the  In general,  oxygen.  from the  i n t r a g r a v e l w a t e r and the subsequent added a v a i l a b i l i t y  resulting  intragravel  was more r a p i d t h a n t h e u p s t r e a m f r e s h w a t e r  Survival  of  tide  to these probes,  of observed i n t e r t i d a l  development.  salinity  implanted i n the g r a v e l  to 2.9-meter  investigated  t o l e r a n c e under  zone o f C a r n a t i o n Creek,  r a n g i n g from the 1.9placed adjacent  and s a l t w a t e r  Intragravel  and temperature probes  implanted eggs,  O n c o r h y n c h u s k e t a . was  of  It  is  interchange  oxygen,  streambed.  saltwater  of  eggs  When e g g s w e r e t r a n s f e r r e d t o o r  from  f l u i d osmolality  tolerance  changes  showed  that  e q u i l i b r a t i o n with the ambient medium occurred rapidly (15-25 min). This response was modelled and an equation was determined. Eggs were exposed d a i l y to two intermittent exposure regimes (4 and 8 h) and one constant one (24 h) , at s i x d i f f e r e n t s a l i n i t i e s  (0, 5, 10, 15, 20, and 3 0 % 0 ) • Survival  was measured from f e r t i l i z a t i o n to 8 d post-hatching. No eggs survived i n any of the 3 0 ° / 0 0 s a l i n i t y treatments. In the intermittent exposure treatments (4 and 8 h) eggs tolerated s a l i n i t i e s of 1 5 ° / 0 0 or less with no adverse e f f e c t s . Eggs exposed to 2 0 ° / 0 0 f o r 4 h also showed no adverse e f f e c t s , whereas those i n the 8 h exposure suffered about 55% mortality. In the  constant exposure treatments only eggs i n 5°/oo s a l i n i t y and the control  survived to the a l e v i n stage (85-95%). In general, eggs i n the higher s a l i n i t i e s hatched f i r s t , although no obvious developmental differences were noted between survivors from the d i f f e r e n t treatments. Possible mechanisms of saltwater t o x i c i t y are discussed and i t i s suggested that eggs provided with a short period of freshwater exposure between saltwater exposures are much more tolerant than eggs exposed to saltwater continuously.  Effects of ambient saltwater on the f e r t i l i z a t i o n process were examined by t e s t i n g sperm m o t i l i t y , sperm v i a b i l i t y and combined sperm and egg v i a b i l i t y i n various s a l i n i t i e s  (0, 5, 10, .12.5 and 15°/00) . In the sperm  m o t i l i t y tests no differences were noted between individual males. However, more vigorous a c t i v i t y and longer periods of m o t i l i t y were observed i n s a l i n i t i e s ranging from 5 - 1 0 ° / 0 0 than s a l i n i t i e s of 0 and 1 2 . 5 ° / 0 0 . No measurable m o t i l i t y was observed i n 15°/oo- Sperm v i a b i l i t y , measured as f e r t i l i z a t i o n success (FS), indicated that sperm were more viable i n 12.5 and 15°/oo than was suggested by the m o t i l i t y measurements. High FS (90-95%) occurred i n s a l i n i t i e s ranging from 0 - 1 0 ° / 0 0 whereas s i g n i f i c a n t l y lower FS  iii  occurred i n 12.5 and 1 5 ° / 0 0 . Combined egg and sperm v i a b i l i t y , also measured as FS, showed a similar response as the sperm v i a b i l i t y t e s t . However, a lower FS i n 15°/oo ^  n t n e  combined test suggested that saltwater had an added  e f f e c t on the egg, i n addition to i t s i n h i b i t o r y e f f e c t on the sperm. I t i s concluded that eggs deposited by i n t e r t i d a l  spawning salmon during times of  saltwater inundation would have low to n i l FS.  iv  TABLE OF CONTENTS ABSTRACT  i i  TABLE OF CONTENTS  v  LIST OF TABLES  ,  v i i  LIST OF FIGURES  viii  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS  .  GENERAL INTRODUCTION  x 1  CHAPTER I - F i e l d Study INTRODUCTION MATERIALS and METHODS Study S i t e D e s c r i p t i o n Environmental Conditions o f the I n t e r t i d a l Zone I n t r a g r a v e l S a l i n i t y and Temperature Measurements I n t r a g r a v e l Oxygen Measurements Gravel Q u a l i t y Sampling I n t e r t i d a l Chum Salmon Spawning Survey Egg Capsule Implantation Experiment Data A n a l y s i s RESULTS Environmental Conditions of the I n t e r t i d a l Zone . . Intragravel S a l i n i t y I n t r a g r a v e l Temperature I n t r a g r a v e l Oxygen Gravel Q u a l i t y I n t e r t i d a l Salmon Spawning D i s t r i b u t i o n  x  .  . . . .  4 4 9 9 13 13 14 14 16 17 19 20 20 20 27 28 30 30  Egg Capsule Implantation Experiment Egg S u r v i v a l Rates Egg Development Rates DISCUSSION SUMMARY - Chapter I  31 31 34 37 52  CHAPTER I I - Laboratory Study INTRODUCTION MATERIALS and METHODS S a l i n i t y Tolerance of Salmon Eggs Experiment Gamete C o l l e c t i o n and F e r t i l i z a t i o n Incubation Equipment and Conditions Experimental Design and Protocol Sampling Procedures Data A n a l y s i s . . . . . P e r i v i t e l l i n e F l u i d Osmolality Tests Measurement o f P e r i v i t e l l i n e F l u i d Osmolality . . . .  55 55 59 59 59 60 62 64 66 68 68  v  TABLE OF CONTENTS (cont'd) Modelling Changes i n PVF Osmolality E f f e c t s Of S a l i n i t y On F e r t i l i z a t i o n Experiments Gamete C o l l e c t i o n Experimental Design Data A n a l y s i s Sperm M o t i l i t y Tests Sperm V i a b i l i t y Tests Combined Egg and Sperm V i a b i l i t y Tests RESULTS S a l i n i t y Tolerance of Salmon Eggs P e r i v i t e l l i n e F l u i d Osmolality S a l i n i t y Tolerance and Egg S u r v i v a l E f f e c t s Of S a l i n i t y On The F e r t i l i z a t i o n Process Sperm M o t i l i t y Sperm V i a b i l i t y Combined Egg and Sperm V i a b i l i t y DISCUSSION S a l i n i t y Tolerance of Salmon Eggs E f f e c t s of Saltwater on the F e r t i l i z a t i o n Process SUMMARY - Chapter I I S a l i n i t y Tolerance of Salmon Eggs E f f e c t s of S a l i n i t y on the F e r t i l i z a t i o n Process  . . . . . .  GENERAL DISCUSSION REFERENCES  69 69 69 69 70 70 71 71 74 74 74 74 81 81 83 85 88 88 100 108 108 I l l 114  .  118  APPENDIX 1 Modelling Changes i n P e r i v i t e l l i n e F l u i d Osmolality Equation, Parameters, and Residuals Eggs t r a n s f e r r e d from 0 ° / 0 0 to 2 0 ° / 0 0 s a l i n i t y water Eggs t r a n s f e r r e d from 2 0 ° / 0 0 to 0 ° / 0 0 s a l i n i t y water  vi  . . . . . . . . . .  127 127 127 127 129  LIST OF TABLES Table 1. I n t r a g r a v e l d i s s o l v e d oxygen concentrations and mean p a r t i c l e s i z e a t 2 freshwater and 9 i n t e r t i d a l zone transects  29  Table 2. Developmental stages of 'well eyed' eggs recovered e a r l y from the egg capsule implantation experiment  36  Table 3. Summary of data c o l l e c t e d at the 2 freshwater and 9 i n t e r t i d a l zone transects  39  Table 4. Percent hatching of F- and B-group eggs measured at three d i f f e r e n t times of development  80  vii  LIST OF FIGURES Figure 1.  Schematic diagram of s a l t wedge hydrodynamics  . . . . . . .  7  Figure 2. Location map of Carnation Creek and the freshwater and i n t e r t i d a l zone study s e c t i o n s .  10  Figure 3a and 3b. D e t a i l e d map and l o n g i t u d i n a l p r o f i l e of the i n t e r t i d a l zone  12  Figure 4. Map showing the transects along which the egg capsules were implanted  15  Figure 5a and 5b. I n t r a g r a v e l s a l i n i t y and temperature p r o f i l e s measured a t f i v e i n t e r t i d a l monitoring s t a t i o n s on Dec. 10, 1985 and Oct. 20, 1984 r e s p e c t i v e l y .  21  Figure 5c and 5d. I n t r a g r a v e l s a l i n i t y and temperature p r o f i l e s measured a t f i v e i n t e r t i d a l monitoring s t a t i o n s on Jan. 5, 1986 and Jan. 23, 1986 r e s p e c t i v e l y  23  Figure 5e. I n t r a g r a v e l s a l i n i t y and temperature p r o f i l e s measured at f i v e i n t e r t i d a l monitoring s t a t i o n s on Oct. 19, 1984 Figure 6. D i s t r i b u t i o n of chum salmon redds i n the i n t e r t i d a l zone.  25 .  32  Figure 7. Egg s u r v i v a l rates to the eyed and a l e v i n stages a t each of the 11 egg implantation transects Figure 8. Linear regression p l o t of egg s u r v i v a l on i n t r a g r a v e l oxygen.  35  Figure 9. Schematic diagram of the exposure regime experienced by eggs i n the s a l i n i t y tolerance experiment.  63  Figure 10. Egg to a l e v i n s u r v i v a l of eggs i n the s a l i n i t y tolerance experiment.  65  Figure 11. Schematic diagram of a f e r t i l i z e d and water a c t i v a t e d salmon egg  67  Figure 12. Changes i n p e r i v i t e l l i n e f l u i d o s m o l a l i t y upon t r a n s f e r of chum salmon eggs from freshwater ( 0 ° / 0 0 s a l i n i t y ) to saltwater (20°/00)  75  Figure 13. Changes i n p e r i v i t e l l i n e f l u i d o s m o l a l i t y upon t r a n s f e r of chum salmon eggs from saltwater (20°/ 0 0 s a l i n i t y ) to freshwater (0%o>  76  Figure 14. Duration of sperm m o t i l i t y from 0 to 1 5 % o  i n ambient s a l i n i t i e s ranging  viii  33  8 2  LIST OF FIGURES (cont'd) Figure 15. Sperm v i a b i l i t y measured as f e r t i l i z a t i o n success i n ambient s a l i n i t i e s ranging from 0 to 1 5 ° / 0 0  84  Figure 16. Combined egg and sperm v i a b i l i t y measured as f e r t i l i z a t i o n success i n ambient s a l i n i t i e s ranging from 0 to 15°/oo  86  Figure 17. Mean weights of eggs measured throughout the combined egg and sperm v i a b i l i t y t e s t  87  Figure 18. Summary of experimental r e s u l t s obtained from the sperm m o t i l i t y , sperm v i a b i l i t y , and combined egg and sperm v i a b i l i t y tests. . . . . .  ix  102  ACKNOWLEDGEMENTS  I wish to express my sincere gratitude and appreciation to Dr. Gordon Hartman, my functional supervisor, f o r h i s i n i t i a l suggestion of this study and f o r h i s r e l e n t l e s s guidance and support throughout. His assistance with the  planning, implementation, and write-up of this thesis were i n t e g r a l .  A l s o , I am very grateful to Charlie Scrivener for h i s guidance and assistance i n s e t t i n g up the f i e l d experiments and c o l l e c t i n g the r e s u l t s . His  willingness to help made a s i g n i f i c a n t difference and was greatly  appreciated. I am also thankful f o r the time and e f f o r t contributed by other Carnation Creek working group members. Thanks to a l l .  I am very grateful to Dr. Don Alderdice, Mr. John Jensen, and Mr. Frank Velsen f o r use of their laboratory f a c i l i t i e s at the P a c i f i c B i o l o g i c a l Station i n Nanaimo, B.C. Further, I am very appreciative of t h e i r assistance and useful discussion which was so f r e e l y provided throughout the period I spent at the Station. Special thanks go to John Jensen f o r h i s unwavering support, both moral and l o g i s t i c a l ,  during the f i n a l stages of the  completion of this t h e s i s .  I wish to thank my o f f i c i a l university supervisor, Dr. Tom Northcote, for h i s patience, support, and c r i t i c a l review of the manuscript. I am also grateful to my other committee members, Dr. Don McPhail and Dr. David Randall, f o r t h e i r guidance and review of the manuscript.  I also would l i k e to express my sincerest thanks to my friends for their support and patience. I am especially grateful to my very special  x  'friend',  V a l e r i e Calderwood. Not only did she provide a c r i t i c a l review of the manuscript, but she also provided me with an endless source of moral support, motivation, and understanding. I also would l i k e to express special thanks to my parents f o r t h e i r continued support, patience, and  acceptance.  Research funds and l o g i s t i c a l support were provided by the Department of Fisheries and Oceans through the P a c i f i c B i o l o g i c a l Station (Carnation Creek Project and Incubation and Water Quality s e c t i o n ) . The Science Council of B.C. provided me with personal f i n a n c i a l support f o r two years through a G.R.E.A.T. scholarship. Both these sources of funding were c r i t i c a l f o r the completion of this t h e s i s . I am very grateful to them f o r t h e i r support.  xi  GENERAL INTRODUCTION  Chum salmon (Oncorhynchus keta) have the widest d i s t r i b u t i o n of the s i x species o f North American and Asian P a c i f i c salmon (Oncorhynchus spp.). In North America, r i v e r s and streams u t i l i z e d f o r spawning and r e a r i n g of the e a r l y l i f e stages are d i s t r i b u t e d from C a l i f o r n i a , USA (37°N l a t . ) to the a r c t i c shores of A l a s k a , USA, i n c l u d i n g some as f a r east as the Mackenzie River on the a r c t i c coast of the Northwest T e r r i t o r i e s , Canada (69°N). I n A s i a t h e i r d i s t r i b u t i o n extends from the a r c t i c coast of S i b e r i a , USSR (73°N l a t . ) south to the Nagasaki Prefecture of Kyushu (33°N l a t . ) i n the Sea of Japan (Sano 1967, Bakkala 1970). This species u t i l i z e s almost a l l s u i t a b l e r i v e r s along the P a c i f i c coasts (1270 streams i n the United States; 880 i n B r i t i s h Columbia (B.C.); 160 i n Hokkaido, Japan) (Bakkala 1970, Scott and Crossman 1973).  Adult P a c i f i c salmon returning to freshwater to breed, u s u a l l y r e t u r n to the t r i b u t a r y o f the r i v e r or stream from which they emerged several years before (Bakkala 1970, Scott and Crossman 1973). For chum salmon the d i s t r i b u t i o n of spawning t e r r i t o r y v a r i e s considerably; i n North America i t r a r e l y extends f a r upstream, except i n Alaska, the Yukon, and the A r c t i c (Bakkala 1970, Scott and Crossman 1973). Some chum, as w e l l as the c l o s e l y r e l a t e d pink salmon (O^ gorbuscha), spawn so close to the ocean that eggs deposited i n the gravel are exposed to t i d a l influence (Neave 1966a, 1966b, Bakkala 1970). T i d a l water enters the stream channel on a r i s i n g t i d e and inundates the g r a v e l , exposing these eggs i n t e r m i t t e n t l y to seawater (Skud 1954, Hanavan 1954). This p o r t i o n of the stream, inundated by p e r i o d i c t i d a l flow, i s r e f e r r e d to as the i n t e r t i d a l zone. Consequently, f i s h spawning i n  1  t h i s zone are r e f e r r e d to as i n t e r t i d a l spawners (Hanavan 1954, Hunter 1959, H e l l e et a l . 1964, Thornsteinson et a l . 1971).  Many of southern Alaska's and B.C.'s c o a s t a l r i v e r s and streams are short w i t h steep gradients and often have impassable b a r r i e r s such as w a t e r f a l l s near t h e i r mouths (Rockwell 1956, Thornsteinson et a l . 1971). These f a c t o r s combined w i t h c h a r a c t e r i s t i c a l l y high annual p r e c i p i t a t i o n can produce d i f f i c u l t migratory routes f o r salmon and l i m i t many of the a c c e s s i b l e spawning areas to the lower reaches of streams, i n c l u d i n g the i n t e r t i d a l zones (Hanavan 1954, H e l l e et a l . 1964). Moreover, chum salmon r a r e l y are i n t e n t on overcoming obstacles or b a r r i e r s of any consequence (Neave 1966a, Scott and Crossman 1973). However, even when upstream freshwater areas are accessible and uncrowded with spawners, some pink and chum salmon s t i l l choose to spawn i n the i n t e r t i d a l zone (Conkle 1961, Thornsteinson et a l . 1971).  I n t e r t i d a l spawning appears to be r e l a t i v e l y common along the coasts' of Alaska and B.C. yet i t i s often regarded as e x c e p t i o n a l , and knowledge concerning the ecology and physiology of such spawning populations i s l i m i t e d . I n A l a s k a , researchers commonly have observed up to 70% of pink salmon populations spawning i n i n t e r t i d a l areas (Tait and Kirkwood 1962, H e l l e et a l . 1964). Others have found s u r v i v a l rates of eggs spawned i n these areas to be equal t o , or even greater than rates observed i n freshwater areas (Hanavan 1954, Rockwell 1956, Kirkwood 1962). Further, H e l l e et a l . (1964) and H e l l e (1970) proposed the p o s s i b i l i t y that i n t e r t i d a l spawners a c t u a l l y are separate populations or races, based upon t h e i r timing and d i s t r i b u t i o n on the spawning grounds. The e x i s t i n g  2  l i t e r a t u r e indicates that this type of spawning i s more common i n Alaska than i n B.C.; however; the l i t e r a t u r e f o r B.C. streams i s very sparse, l a r g e l y anecdotal, and therefore not as thorough. Since much of the research on i n t e r t i d a l spawning has been conducted i n Alaska on pink salmon, information regarding the ecology of chum salmon and t h e i r a b i l i t y to cope with the i n t e r t i d a l environment i s l i m i t e d .  This thesis examines the early l i f e stages of i n t e r t i d a l spawning chum salmon. The objectives were to: (1) assess the environmental conditions of the i n t e r t i d a l zone and determine how they d i f f e r from those of t y p i c a l freshwater spawning environments and (2) determine the e f f e c t ( s ) that these differences may have on growth and development of chum salmon eggs and a l e v i n s . The study integrated both f i e l d and laboratory experiments, conducted at Carnation Creek, B.C., and at the P a c i f i c B i o l o g i c a l Station (PBS) i n Nanaimo, B.C., respectively. The f i e l d component examined the egg to a l e v i n s u r v i v a l of eggs a r t i f i c i a l l y implanted i n the i n t e r t i d a l zone, and the laboratory experiments determined the s a l i n i t y tolerance of developing eggs under controlled conditions and the e f f e c t of ambient saltwater on the f e r t i l i z a t i o n process.  3  CHAPTER I - F i e l d Study  INTRODUCTION  I n t e r t i d a l spawning of pink and chum salmon i s common along the coast of Alaska ( T a i t and Kirkwood 1962, H e l l e e t a l . 1964, Noerenberg 1964) and, a l s o occurs i n B.C., although to a l e s s e r extent (Hunter 1959, Fraser et a l . 1974). Eggs i n i n t e r t i d a l spawning beds experience i n t e r m i t t e n t y e t regular exposure to t i d a l saltwater as more dense seawater floods underneath the l e s s dense outflowing fresh stream water (Skud 1954). Observation of t h i s type of spawning behaviour l e d to questions regarding the a c t u a l and p o t e n t i a l p r o d u c t i v i t y of the i n t e r t i d a l zones (Hanavan 1954, T a i t and Kirkwood 1962, H e l l e e t a l . 1964, Fraser et a l . 1974).  Fry production f o r many of the streams i n Prince W i l l i a m Sound, Alaska had been underestimated f o r many years since t r a d i t i o n a l l y the i n t e r t i d a l zones were not sampled. However, T a i t and Kirkwood (1962) showed that as much as 74% of the t o t a l Prince W i l l i a m Sound pink and chum salmon f r y production o r i g i n a t e d i n t e r t i d a l l y . Other researchers examining i n d i v i d u a l streams commonly found up to 70% or more of a stream's t o t a l spawning population u t i l i z i n g the i n t e r t i d a l spawning grounds (Hanavan 1954, Kirkwood 1962, T a i t and Kirkwood 1962, H e l l e e t a l . 1964). Further, Noerenberg (1964) reported that 70% of the t o t a l 'even year' pink salmon escapement i n Prince W i l l i a m Sound spawned i n t e r t i d a l l y , ( t o t a l escapement of 80 index streams f o r 1956 to 1962 ranged from 0.6 to 1.5 m i l l i o n f i s h ) . Stream catalogs f o r southeastern A l a s k a , which index 518 streams, i n d i c a t e that 17 to 34% of these streams have spawning populations of pink and chum that are  4  predominantly a l . 1963,  i n t e r t i d a l (Martin 1959, O r r e l l and K l i n k h a r t 1963, O r r e l l et  Johnston 1965, Rosier et a l . 1965). Moreover, these s t a t i s t i c s do  not include the many streams that are l i s t e d as supporting minor populations of i n t e r t i d a l spawners but f o r which d e t a i l s are not s p e c i f i e d i n the stream catalogs.  I n an inventory of streams important f o r chum salmon production on the east coast of Vancouver I s l a n d , B.C.,  Fraser et a l . (1974) mention  i n t e r t i d a l spawning as a p o t e n t i a l l y s i g n i f i c a n t c o n t r i b u t o r to spawning populations of t h i s region. However, they stated that the lack of information regarding the biology and d i s t r i b u t i o n of i n t e r t i d a l spawners prevented them from dealing with the respective c o n t r i b u t i o n s of the s p e c i f i c i n t e r t i d a l zones. Province of B.C. stream catalogs provide only cursory evidence of i n t e r t i d a l spawning and i n general make no mention of it.  The i n t e r t i d a l zone of a stream, l i k e the i n t e r t i d a l zone of a beach, i s created by f l u c t u a t i o n s i n sea l e v e l as a r e s u l t of t i d a l a c t i o n . On a high t i d e , seawater i s pushed into the mouths of streams and r i v e r s , where no land masses e x i s t to prevent i t s temporary i n l a n d movement. The density of the i n f l o w i n g saltwater i s greater than the density of the outflowing freshwater. Consequently i t moves i n t o the stream channel underneath the stream water. As a r e s u l t of the d i f f e r e n t d e n s i t i e s , the two bodies of water experience only l i m i t e d mixing and l a r g e l y remain separate due to s t r a t i f i c a t i o n . The extent to which the i n t r u d i n g seawater, r e f e r r e d to as the ' s a l t wedge', moves upstream b a s i c a l l y depends on three f a c t o r s : t i d e h e i g h t , stream discharge, and stream topography. Figure 1 i l l u s t r a t e s  5  schematically the b a s i c process of s a l t wedge movement. I f we assume a high t i d e , then i t becomes apparent how high stream flows and steep gradients would r e s t r i c t upstream movement of the s a l t wedge. In a c t u a l i t y , the i n t e r a c t i o n between these three major, and many other minor f a c t o r s i s very complex.  The degree to which t i d a l water inundates the gravel i s not e n t i r e l y c l e a r . Skud (1954) studied an i n t e r t i d a l pink salmon stream i n Alaska  and  reported that the s a l i n i t y of the i n t r a g r a v e l water was approximately equal to that of the o v e r l y i n g s a l t wedge, i . e . , up to 3 0 ° / 0 0 . This showed that the i n t r a g r a v e l environment i s a f f e c t e d by the t i d a l saltwater but d i d not provide any i n d i c a t i o n of the duration and frequency of saltwater exposure that eggs i n the gravel would r e c e i v e . H e l l e et a l . (1964) observed the percent coverage time of the i n t e r t i d a l zone by the s a l t wedge i n Olsen Creek, Alaska, to be about 15 and 33% at the 3.1- and 2.4-meter t i d e l e v e l s r e s p e c t i v e l y . However, t h e i r measurements of i n t r a g r a v e l s a l i n i t y , 9.3 22.0°/oo  a t  t n e  3-4-  and  and 2.4-meter t i d e l e v e l s r e s p e c t i v e l y , were much lower  than Skud's (1954). The exact manner i n which H e l l e et a l . (1964) measured t i d e l e v e l s i s not s p e c i f i c a l l y s t a t e d . However, i f i t i s the same as Thornsteinson et a l . (1971) used, which seems l i k e l y , then the reference p o i n t i s the 0-meter t i d e l e v e l . A l l subsequent references to t i d e l e v e l s are referenced to t h i s t i d e l e v e l .  I t i s u s e f u l to consider the e f f e c t s that i n t e r m i t t e n t saltwater inundation may have on egg s u r v i v a l and development. Limited information i s a v a i l a b l e regarding the s u r v i v a l of eggs i n t h i s zone and most of i t i s concerned with pink salmon. Measures of egg to f r y s u r v i v a l rates show that  6  LOW TIDE  F i g . 1. Schematic diagram of the dynamics of a s a l t wedge i n a stream channel. The more dense saltwater enters the stream channel along the bottom underneath the less dense outflowing stream water.  pink salmon eggs deposited i n the i n t e r t i d a l zone have good s u r v i v a l , 20 to 54% (Helle et a l . 1964), and 18 to 57% (Thornsteinson  et a l . 1971). These  s u r v i v a l rates were s i m i l a r to or higher than those found f o r eggs deposited i n t o t a l l y freshwater areas w i t h i n i n d i v i d u a l studies (Hanavan 1954, Rockwell 1956, Kirkwood 1962). However, not one of these studies s p e c i f i c a l l y dealt with the e f f e c t s of saltwater exposure on chum salmon eggs and the f a c t o r s a f f e c t i n g s u r v i v a l of i n t e r t i d a l l y spawned eggs.  Therefore, the f i e l d component of t h i s study d e a l t with the environmental conditions to which i n t e r t i d a l l y spawned chum salmon eggs were subjected throughout the egg to a l e v i n incubation p e r i o d . I r e l a t e d egg to a l e v i n s u r v i v a l and development of a r t i f i c i a l l y implanted eggs to the unique conditions of the i n t e r t i d a l environment such as, hydrodynamics of saltwater inundation i n t o the stream bed, differences i n temperature regimes associated with inundation, i n t r a g r a v e l oxygen l e v e l s , and gravel q u a l i t y .  8  MATERIALS and METHODS  Study Site Description  The f i e l d study s i t e , Carnation Creek, i s located on the south side of Barkley Sound on the west coast of Vancouver Island, B.C.  ( F i g . 2). The  creek i s a t y p i c a l second order west coast stream approximately 2  which drains a 10 km  7 km long  watershed. In 1971, hydrological weirs were constructed  on the mainstem and several of the t r i b u t a r i e s . During t h i s same year a f i s h counting fence also was  i n s t a l l e d near the maximum high tide mark f o r  enumeration of both emigrating and immigrating f i s h ( F i g . 2 and 3). Anadromous f i s h populations consist of 400 to 2500 chum salmon (O^ keta), 50 to 200 coho salmon (C\. k i s u t c h ) , as well as small numbers of sea-run cutthroat trout (Salmo c l a r k i ) and steelhead trout.(S^ g a i r d n e r i ) . P r i c k l y s c u l p i n (Cottus asper) , coast range sculpin (C_,_ aleuticus) and threespine stickleback (Gasterosteus aculeatus) also occur i n the stream. The marine staghorn sculpin (Leptocottus armatus) resides i n the lower section of the estuary. Occasionally pink salmon (C\_ gorbuscha) and sockeye salmon (0. nerka) enter the stream during autumn.  Chum salmon spawned from about 1 km upstream down to the mouth of Carnation Creek, including the i n t e r t i d a l zone, before the f i s h fence was i n p l a c e . However, since the i n s t a l l a t i o n of the fence only about 20% of the chum salmon make the e f f o r t to pass through i t to spawn, even though a l l coho salmon and sea-run trout pass through. As a r e s u l t the majority of chum salmon have not u t i l i z e d spawning habitat above the fence since Because the fence was  1971.  constructed near the high tide mark, a l l current chum  9  0-  Main  Fence  F i g . 2. L o c a t i o n o f the f i e l d study s i t e , C a r n a t i o n Creek, and a d e t a i l e d map o f the watershed showing the r e l a t i v e l o c a t i o n o f the I n t e r t i d a l zone below the f i s h c o u n t i n g f e n c e . The freshwater zone was used as a c o n t r o l s i t e f o r the egg i m p l a n t a t i o n experiment conducted In the i n t e r t i d a l zone.  salmon spawning areas below i t are subjected to saltwater exposure of varying degrees during the egg to alevin incubation period.  The i n t e r t i d a l zone i s approximately 450 m long and the wetted width varies between 5 and 20 m (Fig 3a). A reference point was  established at the  mean summer high tide mark and defined as '0 meters' (not to be  confused  with the 0-meter tide l e v e l ) . From this point distances were marked at 10 m i n t e r v a l s by wooden stakes, upstream to the f i s h fence and downstream for 360 m to the ocean. This series of stakes was  designed simply f o r quick  reference to l i n e a r distances along the stream channel and was not an accurate  survey.  A more detailed topographical survey of the physical features of the stream was conducted using standard surveying techniques  (theodolite). A  p a r t i a l survey of the area below the fence, from +10 m to -140 m, already existed as part of the Carnation Creek data base; therefore my  survey  continued above and below this section to encompass the entire i n t e r t i d a l zone. Information from this survey was used to prepare an accurate map  of  the study s i t e ( F i g . 3a) and to provide the elevation measurements for construction of a thalweg of the stream (longitudinal p r o f i l e of maximum cross sectional depth of the stream) ( F i g . 3b). Elevations were referenced to the 0-meter tide l e v e l .  The f i s h counting fence was placed at the maximum high tide mark. Therefore, I defined the i n t e r t i d a l zone as the reach of stream below this fence, and divided i t into upper, middle, and lower zones. The upper i n t e r t i d a l zone extended downstream from the f i s h fence at +80 m to -5 m;  11  -20  0  20  40  60  80  100  120  140  160  180  200  220  240  260  280  300  320  DISTANCE (m)  F i g s . 3a and 3b. Detailed map of the i n t e r t i d a l zone (3a) and an associated l o n g i t u d i n a l stream p r o f i l e (thalweg) (3b). In f i g u r e 3b the h o r i z o n t a l to v e r t i c a l scale r a t i o i s 20:1 and the elevations are referenced to the 0-meter t i d e l e v e l . The s a l i n i t y monitoring stations are shown on both maps at +40, -35, -120, -195, and -275 m. Salinity/temperature probes were implanted i n the gravel at depths of about 25 cm.  the middle zone extended from -5 m to -170 m; and the lower zone reached from -170 m to -320 m and beyond ( F i g . 3a). The c r i t e r i a f o r these d i v i s i o n s were based upon stream topography and e l e v a t i o n of the stream bottom ( F i g . 3b).  Environmental Conditions of the I n t e r t i d a l Zone  I n t r a g r a v e l S a l i n i t y and Temperature Measurements:  Five i n t r a g r a v e l  s a l i n i t y monitoring s t a t i o n s were e s t a b l i s h e d throughout the i n t e r t i d a l zone at +40 m, -35 m, -120 m, -195 m and -275 m ( F i g . 3a and 3b). Each s t a t i o n consisted of a s a l i n i t y and temperature probe (Yellow Springs Instrument (YSI)) implanted i n the middle of the stream at an approximate gravel depth of 25 cm. The probe output jacks were passed under the gravel to the stream bank where they were attached to 5x10 cm (2x4 inch) wooden stakes. At the -120 m s t a t i o n , two a d d i t i o n a l probes were implanted at depths of 50 cm and 75 cm. P r o t e c t i v e covers, constructed of p l a s t i c p i p i n g with end caps, were attached to the probes to maintain a water space around the implanted probes. The probes were implanted during the summers of 1983 and 1984. Although the probes were not c a l i b r a t e d p r i o r to being implanted, t h i s was done once they were removed from the g r a v e l . These c a l i b r a t i o n s i n d i c a t e d that recorded s a l i n i t i e s were at most 2 ° / 0 0 lower than the a c t u a l v a l u e s , and that recorded temperatures were w i t h i n + 0.2°C of the a c t u a l v a l u e s . Further, the presence of the p r o t e c t i v e covers around the probes reduced the measured s a l i n i t i e s by no more than l°/oo-  13  Intragravel s a l i n i t y and temperature readings were recorded using a Y S I conductivity salinometer (model 3 3 ) throughout 1984, 1985 and 1986  usually  during the egg to a l e v i n development period. Measurements were recorded at the f i v e stations throughout t i d a l cycles at short time intervals to provide time course data of the hydrodynamics of intragravel seawater during periods of ebb and f l o o d . To observe the interactive effects of tide height and stream discharge on intragravel s a l i n i t i e s , measurements were taken at a v a r i e t y of tide heights over a range of low to high stream flows.  Intragravel Oxygen Measurements:  Intragravel dissolved oxygen  measurements were carried out i n mid-February shortly before the f i n a l egg capsules were recovered. Samples were taken adjacent to implanted egg capsules (within 3 0 cm), along transects across the stream ( F i g . 4 ) . Intragravel pore water, extracted using a large stainless s t e e l syringe (80 cm long), was analyzed for dissolved oxygen content with a Y S I oxygen meter (model 57) (calibrated i n a i r saturated water). Temperature and s a l i n i t y of each sample also was recorded; where necessary, appropriate corrections were made to account f o r the e f f e c t of saline water on the readings of dissolved oxygen content even though any adjustments were minimal.  Gravel Quality Sampling:  In the upper portion of the i n t e r t i d a l zone  extensive gravel quality information already existed as part of the general Carnation Creek data base. Therefore, gravel samples were taken only at s i t e s where this information was not already a v a i l a b l e , i . e . , i n the top of the upper, bottom of the middle, and throughout the lower i n t e r t i d a l zone. A t o t a l of 12 samples was  taken: 2 at +40 m, 1 at -110 m, 2 at -120 m, 3 at  -195 m and 4 at -275 m, using the freeze core method as described by  14  INTERTIDAL  SITES  F i g . 4. Egg capsule implantation transects. Capsules containing 30 f e r t i l i z e d chum salmon eggs each were implanted along these transects. The number i n parentheses indicates the number of capsules implanted at each transect.  Scrivener and Brownlee (1981). Due to the high degree of v a r i a b i l i t y i n gravel q u a l i t y , even w i t h i n small areas of stream bed, these samples were taken i n an attempt to define trends i n gravel q u a l i t y rather than to c h a r a c t e r i z e each s i t e s p e c i f i c a l l y .  Although sampling was conducted s p e c i f i c a l l y during low t i d e , some s a l i n e pore water remained i n the g r a v e l . However, the presence of low to intermediate s a l i n i t y water (5 to 20°/oo) d i d not impede the technique.  A n a l y s i s of the gravel samples was done according to the procedure described by Scrivener and Brownlee (1981). This involved s p l i t t i n g each core sample i n t o 3 l a y e r s ; top, middle, and bottom. Each l a y e r was subsampled and passed through a s e r i e s of sieves to determine proportions of d i f f e r e n t p a r t i c l e s i z e s . Mean p a r t i c l e s i z e (Dg, geometric mean) was c a l c u l a t e d f o r each sample using a standardized procedure already i n use f o r the Carnation Creek data base (Scrivener and Brownlee 1981) .  I n t e r t i d a l Chum Salmon Spawning Survey  D i s t r i b u t i o n s of spawning i n t e r t i d a l chum salmon was recorded during the spawning periods of 1984 and 1985 (October/November). Surveys were conducted by e i t h e r one or two people walking along the stream bank counting f i s h and recording nest s i t e s . D i f f i c u l t i e s arose when t r y i n g to d i s c e r n between t e s t digging s i t e s and a c t u a l redd s i t e s ; e s p e c i a l l y since spawning a c t i v i t y appeared to be most intense a t n i g h t . As a r e s u l t of t h i s problem, these surveys were used only as general i n d i c a t o r s of the l o c a t i o n of spawning  16  f i s h , e s p e c i a l l y i n areas of high spawning d e n s i t y . Due to the behaviour  of  these f i s h , counting the number of redds seemed to be a more informative and accurate measure of the degree of spawner u t i l i z a t i o n than counting the number of apparent spawners. The d i f f i c u l t y with using f i s h numbers per s e c t i o n was that the f i s h d i d not n e c e s s a r i l y remain on the spawning grounds during the d a y l i g h t hours but instead t y p i c a l l y  schooled i n the deeper  p o o l s . Counting numbers of f i s h would not have been representative of the degree of s i t e s p e c i f i c u t i l i z a t i o n .  Egg Capsule Implantation Experiment  Egg capsules, each containing 30 a r t i f i c i a l l y f e r t i l i z e d eggs, were implanted i n the gravel at a t o t a l of 11 s i t e s ; 2 freshwater c o n t r o l s i t e s and 9 i n t e r t i d a l s i t e s . Five of these s i t e s were proximal to s a l i n i t y monitoring s t a t i o n s ( F i g . 4 ) . The egg capsules were the same as those used by Scrivener (1988). Each capsule was made from a 8.0 cm long s e c t i o n of perforated p l a s t i c pipe that was 4.0 cm i n diameter with perforated p l a s t i c caps on e i t h e r end.  Eggs and m i l t were obtained from 3 females and 5 males (mean fork length: 72.3 and 77.5 cm r e s p e c t i v e l y ) during the peak of the Carnation Creek run (Oct. 23, 1985). The gametes were pooled and then f e r t i l i z e d using the dry method ( i . e . , eggs and m i l t were mixed i n the absence of water). A t o t a l of 63 capsules was implanted using the technique described by Scrivener (1988) . Two a d d i t i o n a l t e s t capsules were set up but not  implanted  to provide an i n d i c a t i o n of f e r t i l i z a t i o n success f o l l o w i n g a short period  17  of incubation (36 h ) . A f t e r 56 days of incubation (Dec. 18, 1985), 12 representative capsules were recovered to obtain p r e l i m i n a r y s u r v i v a l and development rate data f o r the f i r s t two-thirds of the incubation p e r i o d . The remaining capsules were removed i n mid-February  (Feb. 19, 1986), once I was  confident that the hatched alevins had been subjected to respective stream conditions f o r at l e a s t 3 weeks. Even though across-stream transects were e s t a b l i s h e d and i n d i v i d u a l capsules were flagged to f a c i l i t a t e capsule recovery, e i g h t were l o s t . Two of these were found downstream from t h e i r i n i t i a l l o c a t i o n s while the others presumably were dug up and not l o c a t e d , or d i s p l a c e d l a t e r a l l y by redd digging a c t i v i t y during the spawning p e r i o d . In areas of intense spawning, 1 m long metal rods were placed i n f r o n t of capsules as suggested by Scrivener (1988). Consequently, none of these capsules was l o s t . Upon f i n a l analysis of the 63 capsules implanted; 12 were recovered e a r l y f o r the preliminary analysis of development rate and s u r v i v a l to the eyed stage, 43 were removed at the end of the experiment f o r a n a l y s i s of egg to a l e v i n s u r v i v a l , and 8 were l o s t .  Eggs and a l e v i n samples from the recovered egg capsules were processed by evaluating s u r v i v a l to two stages of development; eyed stage (dead or a l i v e w i t h obvious eye pigmentation), and a l e v i n stage (dead or a l i v e w e l l developed larvae or a l e v i n s ) . The t o t a l number of eggs i n i t i a l l y  implanted  i n each capsule (30) was adjusted f o r the assessed f e r t i l i z a t i o n rate of 58.9 + 5.6%  (MEAN + SD). This rate of f e r t i l i z a t i o n success (FS) was lower  than expected; rupturing of eggs during the egg taking procedure i s suspected to be the cause. Some broken eggs ( < 1%) were present i n the pooled sample during the f e r t i l i z a t i o n procedure but I d i d not r e a l i z e the p o t e n t i a l consequences at the time. Wilcox et a l . (1984) reported that even  18  a few broken eggs can reduce f e r t i l i t y d r a s t i c a l l y as w e l l as g r e a t l y increase the v a r i a b i l i t y of that f e r t i l i t y . They showed a decrease i n f e r t i l i z a t i o n rate from 93.7 39.5  + 21.01% and 2.2 + 0.17%  + 0.59%  (MEAN + SD) f o r no broken eggs, down to  f o r 1% and 3% broken eggs r e s p e c t i v e l y .  Therefore, percent s u r v i v a l c a l c u l a t i o n s f o r t h i s study were adjusted with respect to the measured f e r t i l i z a t i o n rate of 58.9%, i . e . , percent s u r v i v a l was  c a l c u l a t e d by d i v i d i n g the number of l i v e eggs or a l e v i n s by 17.7  of 30), before m u l t i p l y i n g by  (58.9%  100.  Data Analysis  Analysis of egg s u r v i v a l , i n t r a g r a v e l oxygen, and gravel q u a l i t y data involved pooling i n d i v i d u a l samples f o r each transect and t e s t i n g f o r between transect differences of each v a r i a b l e using one-way a n a l y s i s of variance (ANOVA). A l l percent egg s u r v i v a l values were normalized using the arcsine transformation (Snedecor and Cochran 1980). Post-hoc, m u l t i p l e comparisons of ANOVA r e s u l t s were done using the SYSTAT s t a t i s t i c s package; appropriate l e v e l s of s i g n i f i c a n c e were determined using the Bonferroni procedure (Wilkinson 1987). M u l t i p l e l i n e a r regression a n a l y s i s was used to t e s t the r e l a t i v e s i g n i f i c a n c e of i n t r a g r a v e l oxygen, gravel q u a l i t y , and egg capsule l o c a t i o n i n r e l a t i o n to egg s u r v i v a l .  19  RESULTS  Environmental Conditions of the I n t e r t i d a l Zone  Intragravel S a l i n i t y :  The degree of t i d a l saltwater exposure at a given  monitoring s t a t i o n p r i m a r i l y was  a function of proximity to the ocean. Thus,  the most extreme exposures, i . e . , highest s a l i n i t i e s and most frequent and prolonged exposure, occurred at the s t a t i o n s nearest to the ocean. The general p a t t e r n of saltwater inundation i n t o and out of the Carnation Creek stream bed, during f l o o d and ebb t i d a l c y c l e s , i s i l l u s t r a t e d by  two  examples ( F i g . 5a and 5b). In general, saltwater reached the lowermost s t a t i o n at -275 m f i r s t , followed consecutively by the -195, +40  m s t a t i o n s ( F i g . 5a). Although t h i s pattern was  s t a t i o n , i t i s not apparent i n f i g u r e 5a. Due  -120,  -35,  and  observed at the -120  m  to the low stream bed  e l e v a t i o n i n t h i s area, high i n t r a g r a v e l s a l i n i t i e s r e s u l t e d f o r extended periods of time, regardless of t i d e height ( F i g . 3b). At a l l of the other s t a t i o n s the i n t r a g r a v e l s a l i n i t y probes u s u a l l y r e g i s t e r e d measurable s a l i n i t i e s w i t h i n 5 minutes or l e s s a f t e r i n i t i a l coverage by the  salt  wedge. Peak i n t r a g r a v e l s a l i n i t i e s were observed u s u a l l y w i t h i n one hour of coverage but the times ranged from 15 min to 3.5 h ( F i g . 5a).  The s a l i n i t y of i n t r a g r a v e l saltwater ranged from 0 to 3 0 ° / 0 0 ( F i g . 5 c ) . Stations i n the lower i n t e r t i d a l zone (-120, -195,  and -275 m) were most  l i k e l y to experience the highest s a l i n i t i e s on a regular b a s i s , even though under c e r t a i n conditions the upper s t a t i o n s ( -35 and +40 m)  also  experienced s a l i n i t i e s as high as 2 8 ° / 0 0 . Depending on t i d e height and stream  20  F i g s . 5a and 5b. Intragravel s a l i n i t y and temperature p r o f i l e s from the f i v e s a l i n i t y monitoring stations measured on 10 Dec. 1985 and 20 Oct.. 1984. S o l i d l i n e and dashed l i n e s represent i n t r a g r a v e l s a l i n i t y and temperature r e s p e c t i v e l y . Note r e l a t i v e l y constant s a l i n i t i e s at the -120 m s t a t i o n .  discharge during a given t i d a l c y c l e , some s t a t i o n s , e s p e c i a l l y +40  and  -35 m, would not receive any saltwater exposure at a l l . Unfortunately, the two a d d i t i o n a l s a l i n i t y probes which were implanted 50 and 75 cm below the gravel surface were placed at -120 m, where regular ' f l u s h i n g out' of i n t r a g r a v e l saltwater d i d not occur. Nonetheless, the s a l i n i t i e s recorded by these three probes r a r e l y d i f f e r e d more than a few parts per thousand.  The duration of saltwater inundation at a given s i t e v a r i e d g r e a t l y and depended on a number of f a c t o r s . The s t a t i o n nearest to the ocean (-275  m)  received the most prolonged exposures (excluding -120 m due to the exceptional s i t e - s p e c i f i c topographical conditions i n t h i s area), while those f u r t h e r upstream received b r i e f e r exposures. At the -275 m s t a t i o n coverage by the s a l t wedge commonly occurred f o r 6 h and sometimes l a s t e d as long as 8 h ( F i g . 5a and 5 c ) . During normal winter stream flows, durations of saltwater exposure often were s i m i l a r at the -195 and -35 m s t a t i o n s (mean flow = 0.25  cms, during non-storm events ( i . e . , < 0.57  cms)).  Saltwater exposure u s u a l l y l a s t e d f o r about 6 h or l e s s ( F i g . 5a). When inundation occurred at the +40 m s t a t i o n i t sometimes l a s t e d f o r 6 h but u s u a l l y the exposure period was c l o s e r to 3 h and i n many cases was nonexistent  (e.g., f i g u r e s 5a and  5b).  As with duration of t i d a l exposure, frequency of exposure also depended on the l o c a t i o n of a given s i t e with respect to the ocean. Again, those s t a t i o n s c l o s e s t to the ocean generally received the highest frequency while those f u r t h e r upstream received r e s p e c t i v e l y l e s s . The  -120 m s t a t i o n d i d  not f i t t h i s trend, because as mentioned e a r l i e r the stream bed e l e v a t i o n i n t h i s area was lower than i t was f u r t h e r downstream at -195 m. The  22  -275  m  F i g s . 5c and 5d. Intragravel s a l i n i t y and temperature p r o f i l e s from the f i v e s a l i n i t y monitoring stations measured on 5 Jan. 1986 and 23 Jan. 1986. S o l i d l i n e and dashed l i n e s represent i n t r a g r a v e l s a l i n i t y and temperature r e s p e c t i v e l y . Note r e l a t i v e l y constant s a l i n i t i e s at the -120 m s t a t i o n .  s t a t i o n received saltwater inundation on almost every high t i d e , i . e . , twice d a i l y . At -195 m inundation occurred nearly as o f t e n , but at times prevented by above average non-storm event stream flows (0.57-1.41 cms)  was or  lower than average, high t i d e s ( < 2.8 m) ( F i g . 5e). The -120 m s t a t i o n received t i d a l exposure frequencies s i m i l a r to that of -195 m, but the extended periods of elevated s a l i n i t y i n t h i s low e l e v a t i o n area masked the i n t e r m i t t e n t i n f l u x otherwise recorded by the i n t r a g r a v e l s a l i n i t y probes. The e l e v a t i o n at -35 m was higher than -195 m and correspondingly, the s a l i n i t y exposure frequency was markedly lower than at the -195 m s t a t i o n . The f r o n t a l p o r t i o n of the s a l t wedge tended to sink i n t o the low e l e v a t i o n area around -120 m, f u r t h e r reducing the frequency of inundation at -35  m.  Saltwater reached the -35 m s t a t i o n l e s s than h a l f as often as the -195 m s t a t i o n , i . e . , exposure at -35 m occurred about 2 to 7 times per week. Tides greater than 3.0 m, during times of average flow, were required to inundate the -35 m s t a t i o n w i t h s a l t w a t e r . Higher t i d e s of 3.5 m or more were necessary to produce measurable i n t r a g r a v e l s a l i n i t i e s at the +40 m s t a t i o n . Therefore, saltwater inundation occurred at t h i s s t a t i o n only a l i m i t e d number of times per month (approx. 3 to 8) and i t was prevented e a s i l y by increased stream flow.  The s a l t wedge u s u a l l y reached the -275 m s t a t i o n about halfway i n t o the f l o o d c y c l e on a 3.0 m t i d e . This was about 3 h a f t e r maximum low t i d e and t h e r e f o r e , 3 h before the next high t i d e peak. At the -195, -120, and -35 m s t a t i o n s i t was uncommon f o r inundation to occur more than 3 h before peak high t i d e and u s u a l l y i t occurred w i t h i n 2 h or l e s s of the peak ( F i g . 5a and 5b). When saltwater inundation occurred at the +40 m s t a t i o n i t was u s u a l l y w i t h i n an hour or l e s s of peak high t i d e ( F i g . 5a and 5 c ) .  24  0.0  0.0 12.0  20 H  „  o  £  e.o  0  "1 -35m 20-  -12.0 i  ~ ,i  J—,  i  •|  "  "I -I20m(25cm)  <  •  1  •  p a:  r8.o  1  r- —— \  W 20  1-12.0  5  UJ V-  UJ ui  3 tt <  0-J  .  i 1 -195m  20 0  !  1  i  i  i  i  i  .  .  i  L  :  -i  ' '  ,  1  r  1  • — r  1  —i  -- r -  1  0800 HIGH TIDE  1  1000  1200  TIME (h)  -  -8.0  g  rl2.0 < t2  /  20  —  1  !  --—. -275 m  >  —  ^  1  *  -8.0 H2.0  -8.0 1400 LOW TIDE  1600  1800  F i e 5e Intragravel s a l i n i t y and temperature p r o f i l e s from the f i v e s a l i n i t y monitoring s t a t i o n s measured on 19 Oct. 1984. S o l i d l i n e and dashed l i n e s represent i n t r a g r a v e l s a l i n i t y and temperature r e s p e c t i v e l y . Note r e l a t i v e l y constant s a l i n i t i e s at the -120 m s t a t i o n .  25  On an ebbing t i d e , saltwater r e t r e a t e d from the gravel i n much the same manner as i t inundated on a f l o o d i n g t i d e , but i n reverse. Saltwater r e t r e a t e d f i r s t from the highest s t a t i o n i t had reached followed consecutively by lower downstream s t a t i o n s ( F i g . 5a). However, the f l u s h i n g process u s u a l l y occurred over a longer time period e s p e c i a l l y at the upper i n t e r t i d a l s t a t i o n s . Figure 5b shows the s a l i n i t y p r o f i l e of an ebb t i d a l c y c l e where Inundation d i d not occur a t the two uppermost s i t e s (-35 and +40 m), but shows t y p i c a l patterns of i n t r a g r a v e l s a l i n i t i e s f o r the lower three s t a t i o n s (-120, -195, and -275 m).  Most of the i n t r a g r a v e l hydrodynamics described so f a r occurred under r e l a t i v e l y normal or average c o n d i t i o n s . However, changes i n t i d e height and stream discharge g r e a t l y influenced the extent of saltwater inundation into the stream bed. Under conditions of lower stream flows ( < 0.15 cms) and r e l a t i v e l y high t i d e l e v e l s ( > 3.5 m), the s a l t wedge can t r a v e l f a r up the stream, and r e s u l t i n high and prolonged i n t r a g r a v e l s a l i n i t i e s ( F i g . 5 c ) . This f i g u r e shows how even at the +40 m s t a t i o n s a l i n i t y remained r e l a t i v e l y high , > 1 5 ° / 0 0 f o r nearly two hours. I n c o n t r a s t , f i g u r e 5d shows the e f f e c t of high stream flow (range f o r t h i s time period: 1.90-4.56 cms), which apparently overshadowed the 3.5m high t i d e at the time and prevented the s a l t wedge from entering the stream much beyond the -275 m s t a t i o n . Even at t h i s lowermost s t a t i o n the i n t r a g r a v e l s a l i n i t y d i d not r i s e above 3°/oo> while a t the -120 m s t a t i o n the i n t r a g r a v e l saltwater was flushed out, and the s a l i n i t y decreased to 3 ° / 0 0 . As an example of the simple e f f e c t of t i d e h e i g h t , f i g u r e 5e shows how the lower of the two d a i l y high t i d e peaks (2.7 m) was not high enough to r e s u l t i n saltwater inundation at the -195 m s t a t i o n . A comparison of t h i s s p e c i f i c peak to, the monthly mean of the lower  26  d a i l y high t i d e peak (3.08 m, N=27), indicates that t h i s t i d e l e v e l was 0.38 m lower than the average. However, during the next high t i d e cycle i n the l a t t e r p o r t i o n of t h i s time s e r i e s , a higher maximum t i d e l e v e l (3.0 m @ 1946 h) pushed the s a l t wedge further upstream and r e s u l t e d i n saltwater inundation a t the -195 m s t a t i o n about 1.5 h before peak high t i d e . Also note i n f i g u r e 5d that as a r e s u l t of a severe storm event f i v e days p r i o r to the recording of these measurements, the i n t r a g r a v e l s a l i n i t y at -120 m was lower than usual (compare to f i g u r e s 5a and 5 c ) .  I n t r a g r a v e l Temperature:  Intragravel temperatures, a t l e a s t during the  incubation p e r i o d of f a l l and w i n t e r , increased when the gravel was inundated w i t h saltwater. As i n t r a g r a v e l s a l i n i t y increased so d i d i n t r a g r a v e l temperature. The greatest f l u c t u a t i o n s occurred at -195 and -275 m, where temperatures increased as much as 4°C above the freshwater i n t r a g r a v e l stream temperature ( F i g . 5a). At the other s t a t i o n s , -120, -35 and +40 m, i n t r a g r a v e l temperature changes also occurred, but u s u a l l y were only 1 to 2 and sometimes 3°C higher than the stream temperature at low t i d e (Fig 5a).  I n t r a g r a v e l stream temperatures followed the general pattern of i n t r a g r a v e l s a l i n i t y increase and decrease but appeared to be a f f e c t e d by a d d i t i o n a l unmeasured f a c t o r s . Intragravel temperatures began to increase at the same time as i n t r a g r a v e l s a l i n i t y , e s p e c i a l l y at the -.275 m s t a t i o n ( F i g . 5a). Hence, an increase i n i n t r a g r a v e l temperature during a flooding t i d e was an i n d i c a t o r of saltwater inundation into the stream bed. As the  27  s a l t wedge receded, the reverse of t h i s response occurred. Again, t h i s was most obvious a t the -275 m s t a t i o n ( F i g 5a and 5 c ) .  An a d d i t i o n a l f a c t o r a f f e c t i n g i n t r a g r a v e l temperature was the presence of warmer groundwater at the +40 m s t a t i o n . I n t r a g r a v e l water temperatures at t h i s s t a t i o n commonly were 2 to 3°C, and sometimes as much as 4°C, warmer than the other downstream s t a t i o n s ; regardless of t i d e height ( F i g . 5a and 5b). This temperature increase was i n d i c a t i v e of an area of warm water seepage or upwelling. However, as shown i n figures 5a and 5c, the upwelling d i d not prevent saltwater from inundating the gravel when i t d i d reach t h i s station.  I n t r a g r a v e l Oxygen:  Differences i n i n t r a g r a v e l oxygen l e v e l s between  s i t e s were s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.001). The two upstream c o n t r o l s i t e s , +260 and +200 m, and the two 1 ower i n t e r t i d a l s i t e s , -195 and -275 m, had the highest i n t r a g r a v e l oxygen l e v e l s which t y p i c a l l y were w i t h i n 1 to 4 mg/1 of the surface water l e v e l s (11 to 12 mg/1) (Table 1 ) . Differences between these two s p e c i f i c parts of the stream were not great although they were s t a t i s t i c a l l y s i g n i f i c a n t f o r the February 20, 1986 samples only (P < 0.001). Differences between the c o n t r o l s i t e s and the middle (-6, -33, -36 m) and upper (+40, 0, -6m) i n t e r t i d a l s i t e s were much greater and also were s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.001).  28  TABLE 1. Mean intragravel dissolved oxygen concentrations and mean p a r t i c l e sizes at 11 transects located throughout Carnation Creek, B r i t i s h Columbia. Intragravel oxygen samples were extracted from the gravel at a depth of about 20 cm and averaged for each transect (MEAN + SD). The column of mean oxygen concentrations i s the grand mean for the two sample dates. Gravel samples were obtained using the freeze core method (Scrivener and Brownlee 1981) and mean p a r t i c l e size was calculated as the geometric mean (Dg + SD).  Transect location (m)  Intragravel dissolved oxygen (mg/1)  13/02/86  20/02/86  Mean  +260  9.5 1 + 0.64  +200  8.4 + 0.07  13.1 + 0.14  10.7 + 1.37  11.6 + 1.27  +40  4.,9 + 0.,61  5,.1 + 1..52  5 .0 + 1..24  10,.3 + 2,,79  0  5..0 + 0,.91  5..6 + 1..54  5 .0 + 1..03  6 .7 + 1,,47  -6  5,.1 + 2..06  5 .1 + 2..06  8,.9 + 2,,09  -33  6,.7 + 2..54  6 .7 + 2..54  12,.7 + 2.,98  7,.0 + 3..66  6 .9 + 2..41  11 .1 + 1..51  3. 0  15. 7 l  4 .9 + 0..46  11,.2 + 3.,26  -36  1  Mean p a r t i c l e size (mm)  6..8 + 1,,06  12.3 +0.07  10.6 + 1.56  9.4 + 2.12  -110  3,.0 l  -120  4,,9 + 0.,46  -195  9..0 + 0..39  11.,1 + 0..16  10 .2 + 1..15  7,.9 + 0.,80  -275  9,.5 + 0..42  11..3 +  10 .6 + 0..99  7 .6 + 1,.29  --  .11  Only one sample obtained.  Sampling of intragravel oxygen (Feb. 13 and 20, 1986) occurred during a period of decreasing flow following a storm event (Feb 1, 1986). As a r e s u l t , the Feb. 13 samples were taken 5 days into a period of r e l a t i v e l y low winter flows (Carnation Creek data base, PBS, 1988) and the Feb. 20  29  samples were taken 12 days into the same p e r i o d . Mean flows f o r t h i s period were 0.22 + 0.045 (S.D.) cms (Feb.13) and 0.19 + 0.053 cms (Feb.20). Thus i n t r a g r a v e l oxygen l e v e l s remained high at the two upstream c o n t r o l s i t e s and the two lowermost i n t e r t i d a l s i t e s , even throughout a period of reduced f l o w . However, t h i s was not the case f o r the middle and upper s i t e s which experienced reduced mean i n t r a g r a v e l oxygen l e v e l s ranging from 3.0 to 6.8 mg/1 (Table 1 ) . At several of these s i t e s (-6, 0 and +40 m) there were obvious and sharp across-stream gradients; i n the most extreme case i n t r a g r a v e l oxygen l e v e l s decreased from 7.7 to 3.7 mg/1 w i t h i n a distance 4 m. A few o f the egg capsules r e t r i e v e d from these s i t e s showed signs of anoxia; p l a s t i c 'Peterson' i d e n t i f i c a t i o n tags i n s i d e the capsules had turned b l a c k , presumably from H2S production.  Gravel Q u a l i t y :  V a r i a b i l i t y i n mean p a r t i c l e s i z e was high w i t h i n  transects and between transects (range f o r a l l samples was 1.96 to 21.69 mm). Transect means (Table 1) were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05) and no obvious trend was observed i n mean p a r t i c l e s i z e from upstream (+260 m) to the lower i n t e r t i d a l zone (-275 m) .  I n t e r t i d a l Salmon Spawning D i s t r i b u t i o n  Chum salmon spawned throughout the e n t i r e i n t e r t i d a l zone of Carnation Creek but the highest u t i l i z a t i o n occurred i n the upper p o r t i o n of t h i s zone, j u s t below the f i s h counting fence. Spawner d e n s i t i e s were highest here and i n the top p o r t i o n of the middle i n t e r t i d a l . Other areas of lower  30  u t i l i z a t i o n occurred i n the lower i n t e r t i d a l zone and a few areas of minimal u t i l i z a t i o n occurred i n the middle i n t e r t i d a l zone ( F i g . 6 ) .  As mentioned e a r l i e r , the i n t r a g r a v e l s a l i n i t y i n the stream s e c t i o n between -100 and -150 m often remained moderately high (15-22%o) even during ebb t i d e s (see F i g s . 5a-5e). Redd digging occurred c o n s i s t e n t l y i n t h i s s e c t i o n ( F i g . 6), although i t may have included t e s t digging as w e l l as a c t u a l egg d e p o s i t i o n . However, below t h i s area of elevated i n t r a g r a v e l s a l i n i t y , confirmed spawning a c t i v i t y increased. At the -275 m s t a t i o n , l i v e eggs were found i n the gravel where two capsules p r e v i o u s l y had been implanted.  Egg Capsule Implantation Experiment  Egg S u r v i v a l Rates:  Mean rates of egg s u r v i v a l to the eyed stage,  adjusted f o r f e r t i l i z a t i o n r a t e , ranged from 3.5% at -120 m to 67.4% at -195 m. S u r v i v a l rates to the a l e v i n stage however, were markedly lower and ranged from 0% at -33 and -110 m up to 51.2% at -275 m ( F i g . 7 ) . The pattern of egg s u r v i v a l was one of c o n s i s t e n t l y good s u r v i v a l i n both the upstream freshwater environment (control) and the lower i n t e r t i d a l  environment.  Compared to t h i s , the s u r v i v a l i n the upper and middle i n t e r t i d a l areas was lower and more v a r i a b l e . Although mean s u r v i v a l rates at the lower i n t e r t i d a l s i t e s were s i g n i f i c a n t l y higher (P < 0.05) than those at the upper freshwater s i t e s , the differences were not great. V a r i a b i l i t y was high between adjacent capsules along a given t r a n s e c t , as w e l l as between capsules implanted at d i f f e r e n t t r a n s e c t s .  31  F i g . 6. An example of spawner u t i l i z a t i o n measured as redd d i s t r i b u t i o n of i n t e r t i d a l spawning chum salmon (e.g., Nov. 8, 1985).  90  > or  -i  <  r- 100  STAGE  90  70 50 -  o  EYED  70  ft  rfi r i  50 30  30 -  + 260  10  TH  10 0 UPPER-  -MIDDLE  >  or z> cn  "275  l-LOWER —  FRESH WATER"  •INTERTIDAL-  90  > or  ALEVIN  r- 100  STAGE  90  70  70  50  -Aft  rti  30 •  o or <  50 30 10  10 •  _J > or ZD (f)  ifL +260 2 0 0 +  •40  0  — UPPER FRESH WATER—*  EGG  "6 "33 "36 "110 "120 "195 "275 1  MIDDLE  INTERTIDAL  CAPSULE  |-L0WER — |  -  IMPLANTATION  SITES  F i g . 7. Mean egg survival rates to the eyed and alevin stages (+ 2SE) at each of the egg implantation transects. Upstream freshwater transects were used as controls.  33  M u l t i p l e regression analysis of egg s u r v i v a l versus i n t r a g r a v e l oxygen, mean gravel p a r t i c l e s i z e , and e l e v a t i o n of the capsule implantation s i t e s i n d i c a t e d that oxygen was the only regression c o e f f i c i e n t s i g n i f i c a n t l y d i f f e r e n t from zero (P < 0.05). Therefore, the f i n a l equation shown, only includes oxygen and excludes the other two v a r i a b l e s ( F i g . 8 ) . I n t r a g r a v e l oxygen explained 58.0% of the v a r i a t i o n observed i n egg s u r v i v a l . E l e v a t i o n of capsule l o c a t i o n was used as a gross p r e d i c t o r of s a l i n i t y exposure s i n c e , as demonstrated e a r l i e r , i n t r a g r a v e l s a l i n i t y exposure was a f u n c t i o n of proximity to the ocean (except a t the -110 and -120 m s i t e s ) .  Egg Development Rates:  I n a d d i t i o n to evaluating d i f f e r e n c e s i n s u r v i v a l  r a t e s , I also examined differences i n developmental rates of the eggs obtained from the e a r l y recovery capsules (capsules were recovered on Dec. 18, 1985, 56 days a f t e r f e r t i l i z a t i o n and implantation). Table 2 shows a downstream trend of increasing development r a t e , with two exceptions. At +40 m, the s i t e of warm groundwater upwelling, development rates were higher than those a t any of the other s i t e s , whereas at -120 m, the s i t e of low stream bed e l e v a t i o n , rates were noticeably lower. The d i f f e r e n c e s between the two lower i n t e r t i d a l s i t e s (-195 and -275 m) and the freshwater c o n t r o l s i t e s (+200 and +260 m) were about two Vernier (1969) stages; stage 28 compared to stage 26 r e s p e c t i v e l y .  34  2  F i g . 8. Linear regression of egg s u r v i v a l on intragravel oxygen ( r - 0.58, N «- 35, P < 0.001). Oxygen measurements were taken adjacent to i n d i v i d u a l egg capsules, a few days p r i o r to t h e i r removal.  35  TABLE 2. Developmental stages of eggs recovered e a r l y (56 days a f t e r implantation) from the egg capsule implantation experiment. Eggs were located a t 11 transects; 2 upstream freshwater c o n t r o l s i t e s (+200 and +260 m) and 9 i n t e r t i d a l s i t e s .  1  Egg implantation transect (m)  Egg development 1 stage  No. of eggs (N)  +260  e a r l y 26  (22)  +200  e a r l y 26  (9)  +40  29-30  (13)  -110  26-27  (19)  -120  25  (2)  -195  27-28  (36)  -275  28  (6)  Vernier 1969.  36  DISCUSSION  Perhaps the most unusual aspect of salmon spawning i n the i n t e r t i d a l zone i s the development and growth of a freshwater egg i n an i n t e r m i t t e n t l y s a l i n e environment. Eggs placed i n the i n t e r t i d a l zone of Carnation Creek g e n e r a l l y d i d not show any negative e f f e c t s from i n t e r m i t t e n t exposure to s a l t w a t e r . Even at the lowermost s t a t i o n (-275 m), where the exposure to near f u l l strength seawater was most frequent and prolonged (e.g., 25-28°/oo twice d a i l y f o r 4-7 h ) , egg s u r v i v a l was good and s i m i l a r to that observed at the freshwater c o n t r o l s i t e s (+200 and +260 m) ( F i g . 7 ) . Because the eggs were f i r s t placed i n p l a s t i c egg capsules before being implanted into the g r a v e l , d i r e c t comparisons of absolute egg s u r v i v a l values w i t h those i n other studies are not p o s s i b l e . Further, these samples were corrected f o r the observed f e r t i l i z a t i o n r a t e . Both these f a c t o r s r e s u l t i n higher c a l c u l a t e d percent s u r v i v a l r a t e s . Nonetheless, my r e s u l t s are s i m i l a r to those of other studies examining egg s u r v i v a l of i n t e r t i d a l spawning pink (Hanavan 1954, H e l l e et a l . 1964) and chum salmon (Kirkwood 1962) at s i m i l a r elevations i n the i n t e r t i d a l zone.  The few i n t e r t i d a l spawning studies that have considered chum salmon, report that these f i s h t y p i c a l l y spawn higher i n the i n t e r t i d a l zone than pink salmon (Kirkwood 1962, Thornsteinson et a l . 1971) The lower l i m i t of s u r v i v a l and growth f o r i n t e r t i d a l eggs of both species i s around the 1.2-meter t i d e l e v e l (Hanavan 1954, Kirkwood 1962, H e l l e et a l . 1964). Although some spawning does occur below t h i s l i m i t , e s p e c i a l l y i n pink salmon, the eggs do not seem to s u r v i v e . B a i l e y (1966) demonstrated, using simulated i n t e r t i d a l c o n d i t i o n s , that pink salmon eggs subjected to a  37  saltwater exposure representative of the 1.2-meter t i d e l e v e l (28°/oo f °  r  9.3 h twice d a i l y ) suffered 100% m o r t a l i t y . However, at the same s a l i n i t y f o r shorter durations of 6.7 and 4.0 h twice d a i l y , he observed 50 and 0% m o r t a l i t y r e s p e c t i v e l y . These two exposure times simulated  saltwater  exposure regimes representative of the 1.8- and 2.4-meter t i d e l e v e l s r e s p e c t i v e l y . The saltwater tolerance response reported by B a i l e y (1966) coincides with observations reported by others i n f i e l d studies; improved egg s u r v i v a l with increasing e l e v a t i o n w i t h i n the i n t e r t i d a l zone (Rockwell 1956, Kirkwood 1962, H e l l e e t a l . 1964). Other studies have compared egg s u r v i v a l i n the i n t e r t i d a l zone with freshwater areas and found i t to be as high or higher (Hanavan 1954, Rockwell 1956, Kirkwood 1962). Although I found no apparent r e l a t i o n s h i p between i n t e r t i d a l zone e l e v a t i o n or egg implantation l o c a t i o n and egg s u r v i v a l i n t h i s study, I d i d observe s u r v i v a l rates a t some i n t e r t i d a l zone transects that were s i m i l a r to those of the freshwater zone ( F i g . 7 and Table 3 ) .  The absence o f an obvious r e l a t i o n s h i p between l o c a t i o n i n the i n t e r t i d a l zone and egg s u r v i v a l i s p o s s i b l y due to the generally narrower range of i n t e r t i d a l s i t e elevations (1.9 to 2.9-meter t i d e l e v e l ) examined i n my study, compared to many of the r e l a t e d studies (Hanavan 1954, H e l l e et a l . 1964, Thornsteinson e t a l . 1971). These studies were conducted i n more n o r t h e r l y areas such as Alaska, where t i d e s generally are higher, and they examined i n t e r t i d a l elevations ranging from the 1.8 to 3.7-meter t i d a l l e v e l . Since these studies examined s u r v i v a l over a broader range of e l e v a t i o n s , eggs i n the lower i n t e r t i d a l zone experienced more extreme saltwater exposures ( i . e . , higher concentrations, longer durations and higher f r e q u e n c i e s ) . Further, egg s u r v i v a l r e s u l t s i n t h i s study were  38  TABLE 3. Summary of data c o l l e c t e d f o r the f i e l d study at Carnation Creek. Samples were c o l l e c t e d at 11 transects, 2 were control s i t e s (+200 and +260 m) located i n the freshwater zone and the other 9 were located i n the i n t e r t i d a l zone. Samples were pooled for each transect. Mean percent egg s u r v i v a l data were adjusted f o r an average f e r t i l i z a t i o n success rate of 58.9 + 5.6% and normalized using the arcsine transformation (MEAN + SD) (Snedecor and Cochran 1980). Intragravel oxygen samples were extracted from the gravel and averaged for two separate sample dates (MEAN + SD). Mean p a r t i c l e size was calculated as the geometric mean (Dg + SD). Elevations were determined from figure 3b as the height above the 0-meter tide l e v e l .  Transect location (m)  Mean egg survival (arcsin %)  Mean egg survival (%)  Mean i n t r a g r . oxygen (mg/1)  Mean p a r t . size (mm)  +260  33.7 + 5.30  30.8  10.6  9.4  +200  36.9+8.83  36.1  10.7  11.6  Elevation (m)  +40  14,.4  +  6.33  6 .2  5.0  10 .3  2 .9  0  10..0  +  3.21  3 .0  5.0  6 .7  2 .8  -6  21,.5  +  10.88  13 .5  5.1  8 .9  2 .8  0. 0  6.7  12. 7  2.4  22 .4  6.9  11,.1  2 .8  0. 0  3.0  15. 7  1.9  -33 -36  6,.8 l 28..3  +  10.74  -110  6,.8 l  -120  8,.5  +  1.74  2 .2  4.9  11 .2  2 .1  -195  38 .4  +  5.09  38 .6  10.2  7 .9  2 .6  -275  45,.7  +  11.22  51 .2  10.6  7 .6  2 .2  '0' proportion  - -  l/(4n), when n < 50 (Snedecor and Cochran 1980).  confounded by the low stream bed elevation area around the -120 m station and channeling of the stream flow i n the upper middle i n t e r t i d a l zone (-33  39  to -80 m). Channelization may have been p a r t i a l l y responsible f o r the p e r i o d i c a l l y low i n t r a g r a v e l oxygen l e v e l s observed i n t h i s area (Table 3 ) .  S p e c i f i c a l l y , r e s u l t s i n t h i s study show that egg to a l e v i n s u r v i v a l was lower i n the middle and upper i n t e r t i d a l zones, than i t was i n the lower i n t e r t i d a l and upstream freshwater ones ( F i g . 7 ) . Percent s u r v i v a l rates of eggs implanted at 0, -33, -110 and -120 m were low, 3.0, 0, 0, and 2.2 r e s p e c t i v e l y . I t i s u n l i k e l y that these low values are the r e s u l t of high s a l i n i t i e s e x c l u s i v e l y , e s p e c i a l l y not i n the two upper s i t e s (0 and -33 m). At the lower two (-110 and -120 m), high s a l i n i t i e s probably contributed to the observed m o r t a l i t y since t h i s area experienced prolonged periods of saltwater inundation ( F i g . 5a). However, i t i s d i f f i c u l t to separate the e f f e c t s of l e t h a l s a l i n i t y exposure from other c o n t r i b u t i n g environmental f a c t o r s such as i n t r a g r a v e l oxygen concentration, gravel q u a l i t y , and v a r i a t i o n s i n stream discharge.  I t i s w e l l e s t a b l i s h e d that the primary source of oxygen f o r the i n t r a g r a v e l environment i s the stream (Wickett 1954, Sheridan 1962), except of course i n s p e c i f i c areas where upwelling of w e l l oxygenated groundwater occurs. Generally however, groundwater i s low i n oxygen content (Sheridan 1962, Kogl 1965, Sowden and Power 1985). Interchange, defined as the passage of water i n t o and out of the gravel stream bed, i s a f f e c t e d by f a c t o r s such as gravel p e r m e a b i l i t y , gravel depth, stream bed surface c o n f i g u r a t i o n , and stream discharge (Vaux 1962). Interchange u l t i m a t e l y determines the s u i t a b i l i t y of the i n t r a g r a v e l environment w i t h respect to oxygen a v a i l a b i l i t y . I f eggs deposited i n the stream are to develop normally, interchange must transport the required amount of w e l l oxygenated water to  40  the egg a t s u f f i c i e n t v e l o c i t i e s to ensure normal egg development (Wickett 1954, S i l v e r e t a l . 1963, Sowden and Power 1985).  The importance of interchange as the s u p p l i e r of i n t r a g r a v e l oxygen i s r e f l e c t e d by a commonly observed d i r e c t r e l a t i o n s h i p between egg s u r v i v a l and I n t r a g r a v e l oxygen content (Wickett 1954, 1958, Coble 1961, P h i l l i p s and Campbell 1962, Koski 1966, Sowden and Power 1985). I n t r a g r a v e l oxygen concentrations measured i n Carnation Creek were consistent w i t h t h i s r e l a t i o n s h i p and explained 58.0% of the v a r i a t i o n observed i n egg s u r v i v a l . Sowden and Power (1985) combined r e s u l t s from t h e i r study on pre-emergent s u r v i v a l of salmonid embryos with several others ( P h i l l i p s and Campbell 1962, Turnpenny and Williams 1980), and suggested that mean oxygen concentrations of l e s s than 5 mg/1 u s u a l l y were l e t h a l . I n t r a g r a v e l oxygen concentrations and associated egg s u r v i v a l rates measured i n Carnation Creek coincide reasonably w e l l with t h i s v a l u e . The f i v e transects w i t h mean oxygen concentrations below 5.1 mg/1 had low egg s u r v i v a l s (Table 3 ) . One exception, a t -33 m, had a mean oxygen concentration of 6.7 mg/1 but a s u r v i v a l of 0%. This apparently i n c o n s i s t e n t r e s u l t was probably a r e s u l t of the across-stream and temporal v a r i a b i l i t y (values ranged from 3.88.3 mg/1). Further, i t i n d i c a t e s the need to consider, i n a d d i t i o n to oxygen content, other f a c t o r s that influence egg s u r v i v a l such as i n t r a g r a v e l water velocity.  Apparent water v e l o c i t y i s an important f a c t o r i n f l u e n c i n g egg s u r v i v a l where oxygen concentrations are not l e t h a l (Coble 1961, S i l v e r et a l . 1963, Shumway e t a l . 1964). Sowden and Power (1985) concluded t h a t , provided mean i n t r a g r a v e l oxygen exceeded a threshold near 5 mg/1, i n t r a g r a v e l water  41  v e l o c i t i e s above 5 cm/h improved embryo s u r v i v a l . Therefore, low water v e l o c i t y ( i . e . , < 5 cm/h) a t the -33 m s i t e may have been a confounding f a c t o r c o n t r i b u t i n g to the l a c k of s u r v i v a l , even though the mean oxygen content was intermediate (6.7 mg/1).  Some researchers have observed a d i r e c t r e l a t i o n s h i p between gravel composition and p e r m e a b i l i t y , and i n t r a g r a v e l oxygen and embryo s u r v i v a l (Wickett 1954, 1958, McNeil and A h n e l l 1964, Koski 1966, Tappel ,1981). Egg s u r v i v a l and oxygen concentration r e s u l t s from t h i s study however, do not demonstrate t h i s r e l a t i o n s h i p ; gravel q u a l i t y (measured as mean p a r t i c l e s i z e ) was not c o r r e l a t e d with these parameters ( F i g . 8 ) . Sowden and Power (1985) reached a s i m i l a r conclusion and i n d i c a t e d that other f a c t o r s , apparently unrelated to gravel q u a l i t y , could overshadow the e f f e c t s of gravel q u a l i t y on i n t r a g r a v e l oxygen and egg s u r v i v a l . I n t r a g r a v e l oxygen l e v e l s , and therefore egg s u r v i v a l r a t e s , l a r g e l y were independent of gravel q u a l i t y ; i n t r a g r a v e l concentrations were influenced to a greater extent by low oxygen groundwater. I n Carnation Creek, no apparent reason was e s t a b l i s h e d f o r the l a c k of c o r r e l a t i o n , but some p o s s i b i l i t i e s were considered.  In the area around +40 m, mean p a r t i c l e s i z e was high but i n t r a g r a v e l oxygen was low (Table 3 ) . As i n d i c a t e d by measurement of warmer i n t r a g r a v e l temperatures, t h i s s i t e experienced groundwater upwelling ( F i g . 5 a ) . Although the oxygen content of t h i s groundwater was not determined, other studies i n d i c a t e that generally i t i s low (2 to 4 mg/1) (Sheridan 1962, Kogl 1965, Sowden and Power 1985). I n f a c t , Sheridan (1962) used d i f f e r e n c e s i n oxygen content and temperature ( i . e . , lower oxygen and higher temperature)  42  a s a means o f d e t e c t i n g g r o u n d w a t e r . this  study,  detailed  Thus,  low oxygen l e v e l s  p r o b a b l y were i n f l u e n c e d by groundwater  investigation  is  seepage.  required to provide d e f i n i t e  L e t h a l oxygen c o n c e n t r a t i o n s  a t +40 m i n However,  more  conclusions.  determined i n the f i e l d represent  an  a v e r a g e o f o x y g e n m e a s u r e m e n t s u s u a l l y made o v e r t i m e a t a s e r i e s  of  A comparison of  (LC50:  t o 1.6  lethal  oxygen l e v e l s  mg/1 ( A l d e r d i c e  measured i n the f i e l d field levels  et a l .  1958,  Silver  than laboratory  1958,  laboratory al.  Garside  1963). Further,  variability water  sampling of  1962,  the  study 6.7  i n f l u e n c i n g the  S h e r i d a n 1962,  is necessary  (McNeil 1962). Therefore,  s m a l l sample s i z e s ,  (e.g.,  possible  remain a l i v e  0% s u r v i v a l a t  (Alderdice  (Silver of  the  et high  intragravel  Extensive  to a c c u r a t e l y  inherent v a r i a b i l i t y  of the eggs,  may f u r t h e r  the  of  to  under  environment  interchange  that  from exposure  Sowden a n d P o w e r 1 9 8 5 ) .  environment  shows  ( 1 - 7 mg/1 @ 10°C)  1964),  0.4  those  l a b o r a t o r y m e a s u r e m e n t s a r e made i n d e p e n d e n t  and c h e m i c a l environments  relatively  One  resulting  but not i n the harsher n a t u r a l  intragravel  the true c o n d i t i o n s  levels  Shumway e t a l .  common t o f a c t o r s  (McNeil  physical  1959,  conditions  1963)), with  levels.  t h a t r e t a r d e d o r weak i n d i v i d u a l s  oxygen c o n c e n t r a t i o n s below the c r i t i c a l et a l .  et a l .  ( L C 1 0 0 : < 5 mg/1 ( S o w d e n a n d P o w e r 1 9 8 5 ) ) ,  are markedly higher  explanation is  determined i n the l a b o r a t o r y  sites.  in conjunction  explain irregular  represent in  the  with  results  - 3 3 m s i t e w h e r e mean o x y g e n l e v e l s  in  this  were  mg/1).  Oxygen r e q u i r e m e n t s (Alderdice  et a l .  1958).  o f d e v e l o p i n g s a l m o n i d embryos Initially  ( 1 mg/1 o r l e s s ) , b u t t h r o u g h o u t  critical  development  43  increase  oxygen requirements they  increase  over are  time low  t o a maximum a t  hatching (7.5-9.6 mg/1), followed by an abrupt drop o f f a f t e r hatching which eventually reaches a s t a b i l i z e d l e v e l (2.3-4.8 mg/1)  (Rombough 1988). In  t h i s study egg s u r v i v a l to the eyed stage was higher than s u r v i v a l to the a l e v i n stage i n the i n t e r t i d a l zone. This was e s p e c i a l l y apparent i n the upper and middle i n t e r t i d a l areas where low and v a r i a b l e oxygen concentrations were measured ( F i g . 7 and Table 3 ) . These r e s u l t s suggest that intermediate i n t r a g r a v e l oxygen l e v e l s i n these areas were s u f f i c i e n t to meet the requirements of e a r l y embryos but not l a t e r more advanced ones.  Support f o r t h i s explanation may be found upon c l o s e r examination of the r e l a t i v e rates of egg development measured i n the e a r l y recovery egg capsules. Generally development rates were higher i n the i n t e r t i d a l zone than they were i n the freshwater c o n t r o l s i t e s (+200 and +260 m). They also increased downstream w i t h i n the i n t e r t i d a l zone, except f o r three s i t e s (+40, -110 and -120 m). This trend i s explained l a r g e l y by d i f f e r e n c e s i n i n t r a g r a v e l temperatures. However, temperature p r o f i l e s recorded from the probe at -120 m d i d not i n d i c a t e any unusually low readings ( F i g s . 5a to 5e), yet development rates of the e a r l y recovery eggs at t h i s s i t e and the nearby -110 m s i t e were among the slowest of any s i t e (Table 2 ) . This r e s u l t suggests that another f a c t o r was i n f l u e n c i n g the observed development r a t e s . I t i s w e l l e s t a b l i s h e d that exposure of salmonid eggs to s u b l e t h a l oxygen concentrations r e s u l t s i n reduced growth and development rates (Alderdice et al.1958, S i l v e r et a l . 1963, Shumway et a l . 1964). Since retarded development rates observed at these s i t e s apparently were not caused by temperature e f f e c t s , i t i s probable that instead they were the r e s u l t of exposure to s u b l e t h a l oxygen concentrations.  44  Although i n t r a g r a v e l oxygen seems to be the most i n f l u e n t i a l f a c t o r a f f e c t i n g egg s u r v i v a l , ultimate egg s u r v i v a l i s determined by the net r e s u l t of a complex i n t e r - r e l a t i o n s h i p of environmental f a c t o r s that influence i n t r a g r a v e l water interchange and oxygen concentration. Further i n v e s t i g a t i o n , i n c l u d i n g extensive sampling, i s necessary to e l u c i d a t e these r e l a t i o n s h i p s more p r e c i s e l y . However, i t i s apparent that i n general, egg s u r v i v a l i n the Carnation Creek i n t e r t i d a l zone was not a f f e c t e d negatively by saltwater inundation, and instead was r e l a t e d to i n t r a g r a v e l oxygen and other associated f a c t o r s . V Interchange of i n t r a g r a v e l water l a r g e l y i s the r e s u l t of water flow over the stream bed (Vaux 1962). However, t h i s process w i l l be a l t e r e d during periods of saltwater inundation. I t seems l o g i c a l that the presence of the s a l t wedge i n the stream channel would l a r g e l y i n h i b i t interchange between the stream water and the i n t r a g r a v e l environment.  Observations show  that as the denser saltwater entered along the stream bottom, over and through the g r a v e l , the downstream flow of freshwater was r e d i r e c t e d towards the surface, due to i t s lower d e n s i t y . The rate at which the s a l t wedge entered the stream channel was very slow, much slower than the usual rate of freshwater flowing out. Since the rate of i n t r a g r a v e l water exchange i s dependent, at l e a s t p a r t i a l l y , on water v e l o c i t y (Vaux 1962), one would expect interchange to be reduced considerably as the s a l t wedge enters the stream. Consequently, the a v a i l a b i l i t y of outflowing freshwater would be e l i m i n a t e d , as long as the s a l t wedge remained i n the stream. Some researchers have expressed concern over t h i s , and suggested the p o s s i b i l i t y of reduced i n t r a g r a v e l oxygen l e v e l s during such periods of saltwater inundation (Fraser et a l . 1974). However, eggs s u r v i v a l at the 2.2- and 2.6-  45  meter t i d e l e v e l s i n t h i s study (-275 and -195 m s i t e s r e s p e c t i v e l y ) , and s i m i l a r i n t e r t i d a l elevations i n other studies (Hanavan 1954, H e l l e e t a l . 1964, Thornstelnson e t a l . 1971), i n d i c a t e that t h i s i s not a major problem. In f a c t , r e s u l t s from the aforementioned s t u d i e s , as w e l l as t h i s one, repeatedly i n d i c a t e that egg s u r v i v a l i n areas of regular t i d a l influence can be as high or higher than that observed i n freshwater areas. This suggests that one or more aspects of the environmental conditions i n the i n t e r t i d a l zone a c t u a l l y b e n e f i t eggs incubating i n t h i s zone. Perhaps one of these b e n e f i t s i s the access of eggs to an a l t e r n a t e source of w e l l oxygenated water, namely ocean water.  As the seawater moves i n on a f l o o d i n g t i d e , i n t r a g r a v e l water exchanges i n response to the d i f f e r e n t i a l d e n s i t i e s . This i s d i f f e r e n t from the mechanism of exchange during periods of freshwater flow since i t i s not dependent upon the rate of water flow. I n t r a g r a v e l water movement occurs as the l e s s dense freshwater i s displaced by the more dense s a l t w a t e r . This i s v e r i f i e d by the measurements of i n t r a g r a v e l s a l i n i t y shown i n f i g u r e s 5a to 5e. S i m i l a r l y , as the s a l t wedge r e t r e a t s on an ebbing t i d e , flowing freshwater begins to d i l u t e and eventually replace the s a l i n e i n t r a g r a v e l water. I n such a way, eggs experience exchanging water over much of the day as the r e s u l t of a ' d i f f e r e n t i a l density exchange mechanism'. As long as the saltwater exposures are not too severe, i . e . , w i t h i n t o l e r a b l e l i m i t s of concentration and d u r a t i o n , i n t e r t i d a l eggs can t o l e r a t e these conditions on a daily basis.  Surface seawater commonly has d i s s o l v e d oxygen l e v e l s near s a t u r a t i o n which f o r inshore waters u s u a l l y i s around 7-9 mg/1 (Waldichuk 1956,  46  Pickard  1961). Regular exposure to i n t e r m i t t e n t seawater could become c r i t i c a l i n a b e n e f i c i a l manner during times of low stream f l o w , when the oxygen content of stream water often i s low (Table 1) and the i n t r a g r a v e l exchange rates are reduced (Vaux 1962). At c r i t i c a l times such as these, i n t e r t i d a l eggs would be assured of a regular source of w e l l oxygenated water, e s p e c i a l l y i n areas of regular s a l t wedge coverage; those i n freshwater areas on the other hand, would not. Instead, they s t i l l would be dependent upon stream water as t h e i r sole source of oxygen. Measurements made by McNeil (1962) appear to support t h i s suggestion w i t h regard to i n t r a g r a v e l oxygen concentrations at l e a s t . He reported that during times of low flow (summer) i n t r a g r a v e l oxygen l e v e l s were s i g n i f i c a n t l y higher i n the i n t e r t i d a l sampling area compared to the upstream freshwater area. I n t e r t i d a l i n t r a g r a v e l oxygen l e v e l s are less l i k e l y to become depleted as coverage by the s a l t wedge exchanges the i n t r a g r a v e l water on a regular b a s i s .  D i f f e r e n t i a l density exchange as described above may be r e l a t i v e l y unimportant during times of normal stream flow i n areas of reasonably good gravel q u a l i t y . However, t h i s may change during periods of reduced stream flow, e s p e c i a l l y i n areas of poorer gravel q u a l i t y . I n t e r t i d a l areas u s u a l l y are  associated w i t h low stream gradients and therefore often contain  r e l a t i v e l y high percentages of f i n e sediment (Helle 1970). In the Carnation Creek i n t e r t i d a l zone t h i s c h a r a c t e r i s t i c was not apparent since the smallest observed mean p a r t i c l e s i z e averaged along a transect was  still  r e l a t i v e l y large (7.9 mm, Table 3 ) . Nonetheless, t h i s c h a r a c t e r i s t i c decrease i n p a r t i c l e s i z e or increase i n f i n e sediment reduces gravel p e r m e a b i l i t y and u l t i m a t e l y i n t r a g r a v e l water exchange (McNeil and A h n e l l 1962, Vaux 1968). These factors combined, can produce s t r e s s f u l l y low oxygen  47  l e v e l s f o r incubating salmon eggs (Wickett 1954, Coble 1961, McNeil 1962). However, i f these eggs were i n areas of regular saltwater inundation, ' d i f f e r e n t i a l density exchange' p o t e n t i a l l y could improve the ambient i n t r a g r a v e l oxygen l e v e l s on an i n t e r m i t t e n t but regular b a s i s . Thus, the ocean as a source of oxygen, could become an i n f l u e n t i a l f a c t o r i n determining the s u r v i v a l of salmon eggs spawned i n the lower reaches of streams, e s p e c i a l l y during times of low stream discharge or i n areas of low gravel q u a l i t y .  The temperature regime of the i n t e r t i d a l environment i s very d i f f e r e n t from the freshwater one upstream. During times of saltwater inundation, i n t r a g r a v e l temperatures u s u a l l y increased i n response to the i n f l u x of warmer seawater. In Carnation Creek, temperatures increased by as much as 2 to 4°C, whereas i n an Alaskan stream they increased up to 5.6°C above freshwater stream temperatures (Helle et a l . 1964). Regular changes such as these may occur as often as twice d a i l y , providing eggs w i t h a s i g n i f i c a n t accumulation of thermal energy over the incubation p e r i o d .  This thermal gain already was apparent i n eggs recovered from the gravel 56 days a f t e r f e r t i l i z a t i o n ; development i n the i n t e r t i d a l eggs obviously was more advanced than those from the freshwater s i t e s . I n t e r t i d a l eggs located at s t a t i o n s -195 and -275 m were two Vernier (1969) development stages more advanced than the freshwater eggs located at the upstream s t a t i o n s (+200 and +260 m); stage 28 compared to stage 26 r e s p e c t i v e l y . Mathematically modelled development rate data (Jensen 1988) , i n d i c a t e d that t h i s d i f f e r e n c e t r a n s l a t e d into about 56°C-days. (A '°C-day' u n i t i s the mean d a i l y incubation temperature above 0°C (Foerster 1968). For example, a mean  48  r  d a i l y temperature of 6°C over a period of 10 days would equal 60°C-days). At mean stream temperatures of 6-7°C (as determined from the observed egg development r a t e s , using Jensen's (1988) model), 56°C-days t r a n s l a t e d into an 8 or 9 day d i f f e r e n c e f o r eggs at these stages. This estimation does not include the added thermal gain these eggs would have experienced had they remained i n the gravel u n t i l emergence (approximately two more months).  Seasonal temperature v a r i a t i o n i s l e s s i n surface ocean water than stream water due to the d i f f e r e n c e i n r e l a t i v e s i z e s of the bodies of water. Stream temperatures f l u c t u a t e over a wider range than ocean temperatures do. Thus, thermal input from i n t r u d i n g seawater would become r e l a t i v e l y more important as the a i r and stream temperatures decreased during the colder winter months. Hanavan (1954) mentioned t h i s f a c t f o r i n t e r t i d a l pink salmon i n an Alaskan stream. In a d d i t i o n he suggested that warmer seawater temperatures would provide an added b u f f e r i n g e f f e c t from low stream temperatures. E s p e c i a l l y i n areas where winter stream temperatures drop to near zero or lower, and ocean temperatures remain above zero, i n t e r t i d a l eggs are l e s s l i k e l y to experience deleterious e f f e c t s r e s u l t i n g from extremely low stream temperatures. Kogl (1965) found that chum salmon l i m i t e d t h e i r spawning d i s t r i b u t i o n to areas of warm water upwelling or seepage i n a s u b a r c t i c stream (Chena R i v e r , A l a s k a ) . This suggests that these f i s h were a c t i v e l y s e l e c t i n g spawning areas to minimize deleterious e f f e c t s from c o l d stream water. I t may be u s e f u l to examine spawning s i t e s e l e c t i o n by i n t e r t i d a l spawners with respect to the same c r i t e r i o n ; minimizing negative e f f e c t s of low temperature on eggs.  49  The net e f f e c t of increased development r a t e , r e s u l t i n g from thermal gains associated with i n t e r m i t t e n t t i d a l water exposure, presumably would be e a r l i e r emergence of i n t e r t i d a l f r y . Studies, assessing the f r y production of s e v e r a l southeastern Alaska streams, have reported that s i z e a b l e portions of the emerging i n t e r t i d a l f r y were missed. T a i t and Kirkwood (1962) based t h e i r pre-season timing of emergence c a l c u l a t i o n s on the temperature regimes of the freshwater areas, and as a r e s u l t large numbers of i n t e r t i d a l f r y emerged and emigrated e a r l i e r than expected. Hanavan (1954) also reported e a r l i e r emergence of i n t e r t i d a l f r y .  The f a c t that f r y r e s u l t i n g from eggs incubated i n the i n t e r t i d a l zone emerge e a r l i e r , r a i s e s the question of whether e a r l y emergence i s a b e n e f i t or a l i a b i l i t y . During the e a r l y period of marine l i f e , salmon f r y m o r t a l i t y i s high (Parker 1968, 1971). The mechanisms are not w e l l understood but a s i z e - s e l e c t i v e b i a s towards smaller f r y has been shown (Parker 1971, Healey 1982). One of the major f a c t o r s a f f e c t i n g e a r l y j u v e n i l e salmon m o r t a l i t y i s thought to be predation by piscivorous f i s h (Kirkwood 1962, Parker  1968,  1971) and marine b i r d s (Godin 1981). Some studies examining f r y m o r t a l i t y i n freshwater report that swamping predators with high numbers of f r y i s a c r i t i c a l f a c t o r i n reducing f r y m o r t a l i t y (Neave 1953, Hunter 1959,  Fresh  and Schroder 1987). However, i t i s not known i f t h i s response also applies to the estuary and ocean environments. I f so, i t suggests that as long as i n t e r t i d a l f r y production i s high f o r a given r i v e r or stream, e a r l y emergence i s p o t e n t i a l l y b e n e f i c i a l . Further, i n t e r t i d a l f r y would not be exposed to the same p o t e n t i a l m o r t a l i t y by freshwater predators (Parker 1971, Fresh and Schroder 1987) as would other f r y migrating from f u r t h e r upstream.  50  Another zone e n t e r (1979)  factor  to consider,  when f r y o r i g i n a t i n g  the marine environment e a r l i e r ,  reported that  is  from the  food a v a i l a b i l i t y .  a p r e f e r r e d food item of  a general relationship  the f r y .  w o u l d be a l i a b i l i t y specific  et a l .  salmon f r y ,  s c a l e s was h i g h due t o p a t c h i n e s s  i s not c l e a r whether e a r l i e r  site  Walters  studies  emergence o f  intertidal  (1978)  or a b e n e f i t w i t h respect  summarized  (LeBrasseur  B.C.  that 1965).  It  (up t o a f e w w e e k s )  to food a v a i l a b i l i t y .  a r e n e c e s s a r y t o answer t h i s  51  but warned  fry  in  harpacticoid  between abundance o f z o o p l a n k t o n on the c o a s t o f  and the e m i g r a t i o n t i m i n g o f F r a s e r R i v e r v a r i a t i o n on l o c a l  Sibert  t h e s e a s o n a l p a t t e r n o f a b u n d a n c e o f chum s a l m o n f r y  t h e N a n a i m o e s t u a r y was t h e same a s t h e s e a s o n a l a b u n d a n c e o f copepods;  intertidal  question  Further  conclusively.  SUMMARY - Chapter I  1.  The i n t e r t i d a l zone i s a unique environment f o r developing salmon eggs.  Eggs spawned i n t h i s area experienced i n t e r m i t t e n t exposure to t i d a l seawater at continuously varying frequencies, durations and concentrations. Associated with these t i d a l f l u x e s , changes i n i n t r a g r a v e l temperatures as w e l l as d i s s o l v e d oxygen concentrations occurred.  2.  The degree of s a l i n i t y exposure experienced by eggs i n the i n t e r t i d a l  zone of Carnation Creek depended upon t i d e h e i g h t , stream discharge, stream topography and s p e c i f i c egg l o c a t i o n .  3.  The range of p o s s i b l e s a l i n i t y exposures v a r i e d from s h o r t , infrequent,  low s a l i n i t i e s (30 min, once weekly to 10°/00) to longer, r e g u l a r , high s a l i n i t i e s (6-7 h, twice d a i l y to 2 8 % 0 ) •  4.  S a l i n i t y was not a major environmental f a c t o r i n f l u e n c i n g  egg s u r v i v a l  i n the implanted egg capsules, even at the lowermost s i t e (-275 m) which received the most extreme s a l i n i t y exposures. One exception to t h i s was noted at the -120 m s t a t i o n where s a l i n i t y probably contributed to low s u r v i v a l r a t e s . Due to stream topography, t h i s low e l e v a t i o n area experienced moderate i n t r a g r a v e l s a l i n i t i e s (15-22°/00) continuously f o r extended periods of time (up to 2 weeks).  5.  S i m i l a r to other studies (Hanavan 1954, Rockwell 1956, B a i l e y 1966), egg  s u r v i v a l rates i n the i n t e r t i d a l zone were s i m i l a r to rates observed i n the upstream freshwater zone.  52  6. (r  Egg s u r v i v a l was d i r e c t l y c o r r e l a t e d with i n t r a g r a v e l oxygen l e v e l s 2  = 0.58), whereas no c o r r e l a t i o n was observed w i t h mean p a r t i c l e s i z e or  e l e v a t i o n o f egg l o c a t i o n .  7.  Eggs i n the i n t e r t i d a l zone may u t i l i z e t i d a l seawater as an a l t e r n a t e  oxygen supply. E s p e c i a l l y during times of low stream flow, and i n areas of low gravel p e r m e a b i l i t y , t h i s may become r e l a t i v e l y more important. On a f l o o d t i d e , i n t r a g r a v e l freshwater i s displaced by incoming seawater as a r e s u l t o f d i f f e r e n t i a l d e n s i t i e s . The opposite process occurs on an ebb t i d e . A 'density dependent exchange mechanism' would not r e l y on the volume or rate of stream flow, as otherwise i s the case. Moreover, ocean surface water c o n s i s t e n t l y has moderately high oxygen concentrations.  8.  I n t r a g r a v e l temperatures associated with s a l t wedge inundation produced  n o t i c e a b l y f a s t e r egg development r a t e s . I n general, development rates increased w i t h a progression from the upstream freshwater s i t e s down towards the lower i n t e r t i d a l ones. Converting into days the thermal gain (56°C-days) r e a l i z e d by eggs a t the -195 and -275 m s i t e s compared to the freshwater c o n t r o l s s i t e s (+200 and +260 m), i n d i c a t e d that a few weeks p r i o r to hatching the i n t e r t i d a l eggs already were 8 to 9 days more advanced.  9.  Upwelling i n t r a g r a v e l water was observed i n the upper i n t e r t i d a l zone,  below the f i s h counting fence, Warmer temperatures of the upwelling water markedly increased the development rates of eggs implanted i n t h i s area. However, i t d i d not prevent the saltwater from inundating i n t o the gravel when the s a l t wedge reached t h i s area. This observation c o n t r a d i c t s the idea  53  that upwelling water necessarily prevents saltwater inundation into i n t e r t i d a l spawning beds.  54  CHAPTER I I - Laboratory Study  INTRODUCTION  Salmon eggs deposited i n i n t e r t i d a l spawning beds experience saltwater inundation and exhibit very reasonable survival rates. From a physiological perspective, this raises important questions about the egg's a b i l i t y to withstand saltwater exposure. However, due to the inherent v a r i a b i l i t y of the intragravel environment (McNeil 1962), ' f i e l d ' measurements of survival can vary g r e a t l y . Bailey (1966) avoided some of this v a r i a b i l i t y by simulating a range of i n t e r t i d a l conditions i n a controlled f i e l d study. He showed that pink salmon eggs tolerated exposure to saltwater concentrations as high as 28°/oo f °  ra s  n  l° g  a s  4 hours twice d a i l y with no adverse e f f e c t s .  Exposures f o r periods longer than this resulted i n reduced s u r v i v a l rates and eventual t o t a l mortality.  Other studies that have examined s a l i n i t y tolerance of salmon eggs i n the laboratory have used only constant exposure regimes (Rockwell 1956, Weisbart 1968, Shen and Leatherland 1978a). The d i f f i c u l t y with trying to extrapolate from a constant treatment to an intermittent one i s that i t does not accurately represent the conditions experienced i n the i n t e r t i d a l zone. Some workers have t r i e d to determine the developmental  stage(s) at which  salmonids acquire the a b i l i t y to control t h e i r internal ionic and osmotic environments, independently of ambient conditions (Rockwell 1956, Parry 1960, Kashiwagi and Sato 1969, Shen and Leatherland 1978a, Weisbart 1968, among others). Since at these early stages the embryo has not yet developed the adult organs necessary to f u l f i l l their regulatory requirements  55  (gills,  kidney, and f u n c t i o n a l g u t ) , workers have i n v e s t i g a t e d a l t e r n a t e mechanisms a v a i l a b l e to these stages i n attempts to Understand how i n t e r i o r m i l i e u (Leatherland and L i n 1975,  they regulate t h e i r  Shen and Leatherland 1978b). Even  i f embryos and a l e v i n s are not exposed to s a l i n e c o n d i t i o n s , they are  still  faced w i t h maintaining i o n i c and osmotic balances i n a hypo-osmotic freshwater environment. I t i s w e l l established that i f these animals do  not  maintain a c e r t a i n l e v e l of mineral balance they u l t i m a t e l y d i e . However, the mechanisms with which they accomplish t h i s balance, e s p e c i a l l y during the very e a r l y stages, are not w e l l understood.  In a review on osmotic and i o n i c r e g u l a t i o n i n t e l e o s t eggs and Alderdice  larvae,  (1987) concluded that i n i t i a l r e g u l a t i o n i n the t e l e o s t embryo was  due to " r e s i s t i v e maintenance of the i n t e g r i t y of the egg proper, achieved through the presence of a t i g h t plasma membrane and l i m i t e d transmembrane water and ion f l u x e s " . Membrane permeability may 'eyed' stage, and c h l o r i d e c e l l s may  increase s l i g h t l y near the  appear i n the blastoderm ( f i r s t  c e l l u l a r l a y e r to overgrow the y o l k , i . e . , yolk sac epithelium, during the process of e p i b o l y ) . Chloride c e l l s , also r e f e r r e d to as c e l l s , are one of the osmo-and ionoregulatory  'mitochondria-rich'  mechanisms f u n c t i o n i n g i n the  l a t e r stages of development (Zadunaisky 1984). During overgrowth of the blastoderm, c h l o r i d e c e l l s have been i d e n t i f i e d i n a number of anadromous or estuarine t e l e o s t species, Fundulus h e t e r o c l i t u s (Guggino 1980), P o e c i l i a r e t i c u l a t a (Depeche 1973,  c i t e d from Alderdice 1987)  and Salmo g a i r d n e r i  (Shen and Leatherland 1978b) but not i n Oncorhynchus k i s u t c h (Leatherland and L i n 1975), the only P a c i f i c salmon species so f a r examined. However, c h l o r i d e c e l l s may  not be the only regulatory mechanism a v a i l a b l e to t e l e o s t  embryos since as Alderdice  (1987) reported, Jones et a l . (1966) were not  56  able to f i n d them i n Clupea harengus. a marine species capable of l i m i t e d osmo- and i o n o r e g u l a t i o n p r i o r to formation of the blastoderm and completion of e p i b o l y .  Nevertheless, some of the t e l e o s t embryos examined, i n c l u d i n g  salmonids  (Weisbart 1968, Shen and Leatherland 1978a), are able to regulate t h e i r i n t e r n a l environment w i t h respect to ambient c o n d i t i o n s , p r i o r to the development of adult phase r e g u l a t i o n which occurs on an o r g a n i s t i c l e v e l , i n v o l v i n g the g i l l s , gut, and kidney. Evidence suggests that the regulatory mechanisms employed by these e a r l y l i f e stages are simple ones f u n c t i o n i n g at the c e l l u l a r and t i s s u e l e v e l s , i . e . , maintenance of ' t i g h t ' plasma membranes, p o s s i b l e r e g u l a t i o n by blastodermal c e l l s , and i n some species f u n c t i o n a l c h l o r i d e c e l l s (Alderdice 1987).  When considering s a l i n i t y tolerance of the e a r l y l i f e stages of f i s h , the m a j o r i t y of work has focussed on the developing egg and stages beyond. However, researchers have a l s o examined the e f f e c t s of o s m o l a l i t y and i o n content on sperm m o t i l i t y (Baynes et a l . 1981, Morisawa et a l . 1983) and o c c a s i o n a l l y v i a b i l i t y (Werner 1934, R i e n i e t s and M i l l a r d 1987). However, most of these t e s t s were conducted with low i o n i c concentrations s o l u t i o n s , u s u a l l y l e s s than or equal to the o s m o l a l i t y of blood plasma or seminal f l u i d (270-300 mosmol/kg, Morisawa et a l . 1983). Limited information i s a v a i l a b l e on the e f f e c t of higher concentrations of i o n i c s o l u t i o n s , e s p e c i a l l y w i t h respect to the complete f e r t i l i z a t i o n process i n v o l v i n g both gametes.  57  Available information suggests that i n t e r t i d a l spawners do not deposit t h e i r eggs during times of saltwater inundation. However, spawning behaviour s p e c i f i c to i n t e r t i d a l spawning salmon apparently has not been examined i n d e t a i l . Therefore, I wished to determine whether or not f e r t i l i z a t i o n  was  even f e a s i b l e given the environmental conditions of the i n t e r t i d a l spawning area during saltwater inundation.  The f i r s t part of the laboratory component of this study was designed to examine the s u r v i v a l of eggs exposed to controlled intermittent s a l i n i t y treatments. Treatment conditions were established to cover a wide range of s a l i n i t i e s at a number of exposure durations that bracketed actual conditions observed i n the i n t e r t i d a l zone of Carnation Creek. Further, eggs from two supposedly separate spawning areas of a stream, i n t e r t i d a l and freshwater areas, were tested for d i f f e r e n t i a l s a l i n i t y tolerance. The question examined was whether eggs from the i n t e r t i d a l area were more s a l i n i t y tolerant than those from freshwater  areas.  The second part of the laboratory component of this study was devised to examine the e f f e c t s of saltwater exposure on the f e r t i l i z a t i o n process. Experiments were designed to test the effects over a range of s a l i n i t i e s . Three aspects of f e r t i l i z a t i o n and the associated effects of saltwater exposure were examined: (1) sperm m o t i l i t y , (2) sperm v i a b i l i t y , and combined egg and sperm v i a b i l i t y .  58  (3)  MATERIALS and METHODS  S a l i n i t y Tolerance of Salmon Eggs Experiment  Gamete C o l l e c t i o n and F e r t i l i z a t i o n :  Gametes were taken from chum salmon  spawning i n i n t e r t i d a l and freshwater areas of Goldstream R i v e r , which i s located 10 km north of V i c t o r i a , B.C. In November 1985, eggs and m i l t were c o l l e c t e d from 4 females and 4 males i n each area and t r a n s f e r r e d to the laboratory at PBS i n a cooler maintained between 2 and 5°C. Gametes c o l l e c t e d from the i n t e r t i d a l s i t e were designated as B-group eggs, w i t h respect to the b r a c k i s h water of t h i s area, and gametes c o l l e c t e d from the freshwater s i t e were designated as F-group eggs, with respect to the freshwater of t h i s area. Unfortunately however, l o g i s t i c a l problems were encountered i n catching true i n t e r t i d a l spawners, therefore I am not confident about the source of these eggs. Once these f i s h were disturbed from the redds, they mixed w i t h other f i s h that may have been schooling i n t h i s area before moving f u r t h e r upstream.  P r i o r to f e r t i l i z a t i o n the eggs were inspected f o r i n d i v i d u a l s that were water hardened or broken. Any such eggs were removed and discarded. S i m i l a r l y the m i l t was inspected f o r unusual c o l o u r , and tested f o r m o t i l i t y using a l i g h t microscope (100X m a g n i f i c a t i o n ) . Vigour and duration of sperm m o t i l i t y was assessed f o l l o w i n g the a d d i t i o n of water. Normal m o t i l i t y was c h a r a c t e r i z e d by very vigorous movement maintained f o r 15 to 25 s by the m a j o r i t y of the sperm.  59  The eggs and m i l t were pooled together w i t h i n both groups (B and F ) . Batches o f about 1500 eggs, taken from each of these two p o o l s , were separated and f u r t h e r d i v i d e d f o r s i x s a l i n i t y treatments (0, 5, 10, 15, 20, and 30°/oo) ^  n e e  Sg  s  were f e r t i l i z e d by the dry method w i t h an egg to m i l t  r a t i o o f about 500:1. For each s a l i n i t y the B-group and F-group eggs were f e r t i l i z e d simultaneously. For d i f f e r e n t s a l i n i t i e s , the sequence of f e r t i l i z a t i o n was randomized ( i . e . , c h r o n o l o g i c a l order of f e r t i l i z a t i o n was 10, 20, 0, 15, 30, and 5 % o ) •  A f t e r f e r t i l i z a t i o n , but before the s t a r t of the saltwater exposure r  treatments, a l l of the eggs were placed i n freshwater (0°/oo) f ° 12 h to allow water a c t i v a t i o n and hardening to occur without any confounding e f f e c t s from saltwater exposure. Once water hardened, the eggs were placed i n t h e i r respective treatments.  Incubation Equipment and Conditions:  The eggs were contained i n small  incubators constructed from 3.2 mm t h i c k a c r y l i c and p l a s t i c n e t t i n g with 1 mm square h o l e s . Each rectangular incubator was 8.6 cm long and 7.9 cm wide and consisted of a bottom and a l i d s e c t i o n . These two sections were 1.9 and 0.3 cm high r e s p e c t i v e l y . Each s e c t i o n was covered with p l a s t i c n e t t i n g so that when placed together they formed a completely enclosed box w i t h mesh on the top and bottom. Handles were glued to the bottom s e c t i o n . The l i d s were h e l d i n place by four 2.2 cm long pieces of a c r y l i c dowel l o c a t e d i n each corner. A few days p r i o r to hatching the l i d s were secured to the bottom sections by s t r e t c h i n g small e l a s t i c s around i n d i v i d u a l u n i t s . The incubators were supported on racks also made from 3.2 mm t h i c k a c r y l i c  60  that were designed to hold 12 incubators and to f i t i n t o temperature c o n t r o l l e d tanks a t the PBS l a b o r a t o r y .  Each of the 10 tanks used was a completely closed system with a volume of 40 1 and dimensions of 50 x 29 x 28 cm (1 x w x h ) . Water c i r c u l a t i o n was maintained by 18 cm long airstones placed across the back of the tanks. The racks were designed so that water flow would be forced down through the mesh covered incubators. C i r c u l a t i o n was tested using dye p r i o r to the beginning of the experiment and standardized f o r a l l tanks. Temperatures were maintained a t 9.0 + 0.1°C by c o l d water c o o l i n g c o i l s and t h e r m o s t a t i c a l l y c o n t r o l l e d e l e c t r i c heating elements. Temperature gradients w i t h i n tanks were tested and found to be n e g l i g i b l e (+ 0.02°C).  The freshwater used was dechlorinated and f i l t e r e d Nanaimo c i t y water. T h e . c h a r a c t e r i s t i c s of t h i s water were as f o l l o w s : t o t a l hardness as CaC03, +2  +  19.2 mg/1; s p e c i f i c conductance, 59 umhos/cm; pH, 6.9; Ca , 6.6 mg/1; Na , +2  +  3.2 mg/1; Cl"- 2.2 mg/1; Mg , 0.62 mg/1; K , 0.3 mg/1; and t o t a l N as n i t r i t e and n i t r a t e , 0.04 mg/1. The saltwater used was f i l t e r e d ocean water pumped from a depth of 18 m. Since t h i s water u s u a l l y was 2 8 - 2 9 ° / 0 0 s a l i n i t y a small a d d i t i o n of Instant Ocean Synthetic Sea S a l t ™ was used to produce 30°/oo s a l i n i t y s a l t w a t e r . Except f o r the f i l l i n g of freshwater tanks, a l l water mixtures were premixed v o l u m e t r i c a l l y i n 50 1 carboys to allow f o r coarse adjustment of temperature and s a l i n i t y . Further f i n e adjustments were made i n the tanks. S a l i n i t y was measured using the low p r e c i s i o n t i t r a t i o n method described by S t r i c k l a n d and Parsons (1968). The water i n each tank was changed about every eight days depending on the degree of usage. S a l i n i t y was checked every four days on average.  61  Experimental Design and Protocol: the  This experiment was designed to test  saltwater tolerance of two sources or groups (B and F) of chum salmon  eggs at 6 s a l i n i t i e s and 3 exposure times. For each group, tolerance was tested at two intermittent and one constant exposure regime, using s a l i n i t i e s of 0, 5, 10, 15, 20, and 30°/oo- ^  e  intermittent treatments  consisted of either 4 or 8 h exposures per 24 h period and the constant treatments were continuous exposures (Fig. 9 ) . Two r e p l i c a t e incubators were used f o r every treatment, each one containing about 200 eggs. Due to space r e s t r i c t i o n s , r e p l i c a t e incubators were kept i n the same tanks and by s t r i c t d e f i n i t i o n should be referred to as subsamples.  Ten tanks were set up; 6 treatment tanks were used f o r the test s a l i n i t i e s of 0 to 30°/oo and 4 freshwater tanks were used f o r holding purposes. The freshwater tanks were required f o r holding the 4 and 8 h treatment incubators during the respective 20 and 16 h per day that they were not exposed to the saline test conditions (Fig. 9 ) .  The condition of intermittent saltwater i n f l u x i n the i n t e r t i d a l zone was simulated by manually transferring the incubators from freshwater to t h e i r respective test s a l i n i t i e s . Once the exposure time period of 4 or 8 h was complete the incubators were returned to freshwater. Care was taken to minimize mechanical shock during the transfers by avoiding movements that j a r r e d the eggs or caused them to r o l l around. Any accidental shocks were recorded f o r future reference. Before returning the incubators to freshwater, they were rinsed i n a separate freshwater bath to minimize saltwater contamination of the freshwater tanks. To equalize any p o t e n t i a l  62  F i g . 9. Exposure regime experienced by eggs i n the s a l i n i t y tolerance experiment conducted i n the laboratory study. Treatments involved 6 a t s a l i n i t i e s (0, 5, 10, 15, 20, and 30°/oo) 3 exposure times; 2 intermittent (4 and 8 h) and 1 constant exposure (24 h ) . The 15°/oo s a l i n i t y treatment regime i s provided as an example.  63  b i a s introduced by movement e f f e c t s , c o n t r o l incubators i n the 0 / 0 0 treatment tank were mock t r a n s f e r r e d at the appropriate time i n t e r v a l s , i . e . , they were l i f t e d but not t r a n s f e r r e d . For the same reason, constant s a l i n i t y treatment incubators also were l i f t e d and replaced i n t o t h e i r respective tanks. Thus a l l incubators were t r a n s f e r r e d , or mock t r a n s f e r r e d , twice during every 24 h p e r i o d .  P h y s i c a l movement of the eggs apparently was not a problem since i n the c o n t r o l treatments and many of the lower s a l i n i t y treatments, egg s u r v i v a l s were high ranging from 90-98% ( F i g . 10). Even i f minor movement e f f e c t s d i d occur they would have been d i s t r i b u t e d evenly across a l l treatments since a l l incubators b a s i c a l l y were subjected to the same degree of movement.  Biases between the l o c a t i o n s of the tanks were minimized by keeping the order random and changing the l o c a t i o n s every 6 to 10 days. W i t h i n tanks, p o t e n t i a l small scale differences i n water c i r c u l a t i o n were accounted f o r by r o t a t i n g the 10 to 12 incubators i n each tank; each incubator occupied every one of the 12 l o c a t i o n s on the supporting rack once every 12 days.  Sampling Procedures:  Samples were taken to assess f e r t i l i z a t i o n success  (FS) 18 h a f t e r f e r t i l i z a t i o n . Five eggs were sampled from each incubator and preserved i n Stockard's s o l u t i o n (40 ml g l a c i a l a c e t i c a c i d , 50 ml formaldehyde, 60 ml g l y c e r o l , and 850 ml d i s t i l l e d water). I n t o t a l , 30 eggs were sampled from each batch of simultaneously f e r t i l i z e d eggs. Eggs were considered to be ' f e r t i l i z e d ' when a 4 or 8 c e l l b l a s t u l a was apparent. These t e s t s revealed that 98.3-100.0% of the eggs were f e r t i l i z e d .  64  F i g . 10. Egg to a l e v i n stage survival rates (8 d post-hatching) of the freshwater source (F-group) and i n t e r t i d a l source (B-group) eggs at 6 d i f f e r e n t s a l i n i t i e s and 3 exposure times. Each point represents the mean (+ 2SE) of 2 r e p l i c a t e incubators containing about 200 eggs each. Open c i r c l e s represent recalculated mean survival rates (see t e x t ) .  Therefore, no c o r r e c t i o n s were made i n c a l c u l a t i n g the f i n a l s u r v i v a l values.  M o r t a l i t y samples were picked and preserved i n Stockard's s o l u t i o n every second or t h i r d day. An egg was c l a s s i f i e d  as 'dead' when the plasma  membrane surrounding the egg proper ( F i g . 11), or the y o l k sac epithelium i n l a t e r developmental stages, was obviously damaged ( i . e . , egg appeared whitened and opaque to the naked eye due to coagulation o f y o l k m a t e r i a l i n the p e r i v i t e l l i n e  f l u i d when i n freshwater; i n saltwater however, the yolk  m a t e r i a l d i d not turn white i n c o l o u r ) . Dead eggs were examined f o r signs of development, obvious abnormalities, and p o s s i b l e causes of death.  Hatching rates o f the eggs were assessed by noting the time of the f i r s t hatched a l e v i n (larvae) i n each incubator and subsequently estimating the percentage o f hatched eggs u n t i l the process was complete.  The experiment was terminated a t about one week post-hatching (1600 h elapsed time o f development (ET) @ 9.0°C) and f i n a l egg to a l e v i n s u r v i v a l rates were c a l c u l a t e d from f e r t i l i z a t i o n to t h i s time.  Data A n a l y s i s :  Percent values were normalized using the arcsine or  angular transformation before conducting s t a t i s t i c a l analyses. Percent egg s u r v i v a l r e s u l t s o f the two groups o f i n t e r t i d a l  source and freshwater  source eggs were analyzed by one-way ANOVA's f o r each exposure time (4, 8, and 24 h) w i t h i n each group (B and F ) . M u l t i p l e post-hoc comparisons were done using the SYSTAT s t a t i s t i c s package and the Bonferroni procedure (Wilkinson 1987).  66  F i g . 11. Schematic diagram of a f e r t i l i z e d and water activated salmon (not to s c a l e ) .  P e r i v i t e l l i n e F l u i d Osmolality Tests  Measurement of P e r i v i t e l l i n e F l u i d Osmolality:  Measurements of  p e r i v i t e l l i n e f l u i d (PVF) were made on eggs t r a n s f e r r e d from 0 ° / 0 0 to 2 0 ° / 0 0 and back again. P e r i v i t e l l i n e f l u i d i s the often viscous l i q u i d that f i l l s the p e r i v i t e l l i n e space and surrounds the egg proper. I t i s contained by the e x t e r n a l egg membrane ( F i g . 11) (Groot and Alderdice 1985). Spare F-group eggs, f e r t i l i z e d a t the same time as those used i n the main experiment, were used i n these t e s t s . These eggs were w e l l eyed and a t about 700 h ET a t 9°C. Osmolality of PVF was measured using a Wescor vapour pressure osmometer (model 5100C) (Wescor Inc.,  Logan, Utah, USA). M i c r o c a p i l l a r y tubes (10 nl)  were used f o r c o l l e c t i n g and dispensing a l l standard and t e s t samples into the  osmometer.  P e r i v i t e l l i n e f l u i d was sampled by c a r e f u l l y making a very small hole i n the e x t e r n a l egg membrane and withdrawing the f l u i d i n t o the c a p i l l a r y tube. A b a s e l i n e o s m o l a l i t y f o r eggs i n freshwater was e s t a b l i s h e d i n i t i a l l y by sampling eggs i n 0 ° / 0 0 water f i r s t . Eggs then were t r a n s f e r r e d to 2 0 ° / 0 0 and measurements s t a r t e d immediately. These were continued, as r a p i d l y as the technique allowed, u n t i l osmolality changes i n the PVF had l e v e l e d o f f . E q u i l i b r a t e d eggs also were tested at 1 and 4 h i n t e r v a l s to observe i f any a d d i t i o n a l changes occurred over time. A s i m i l a r sampling procedure was done when the eggs were returned t o freshwater (0°/00) from 2 0 ° / 0 0 . Five sampling runs were conducted f o r the t r a n s f e r of eggs i n t o 2 0 ° / 0 0 and three f o r the t r a n s f e r back to freshwater.  68  Modelling Changes i n PVF Osmolality :  Changes i n PVF o s m o l a l i t y as a  f u n c t i o n of time were modelled using a general growth model (Schnute 1981). The computer program of the model was accessed through the 'user l i b r a r y ' of the PBS computer department. The model, w i t h constants a and b not equal to zero, i s described by equation (1):  b  b  Y ( t ) = [ Y, + ( Y,  b  - Y,  )  j . I  .  a fc T1  e e  -< - >  ]  -a(T2-Tl)  ( 1 )  where T and T2 are the i n i t i a l and f i n a l times (min.), and Y x and Y2 are the 1  p r e d i c t e d PVF o s m o l a l i t i e s (mmol/kg) at those times. The p r e d i c t e d values of Y ( t ) were determined by a minimization process using a nonlinear parameter estimation technique (simplex) and the associated software package ( M i t t e r t r e i n e r and Schnute 1985).  E f f e c t s Of S a l i n i t y On F e r t i l i z a t i o n Experiments  Gamete C o l l e c t i o n :  Chum salmon gametes were c o l l e c t e d from B i g Qualicum  F i s h hatchery on Vancouver I s l a n d , B.C. i n November 1986. Eggs were s t r i p p e d from 3 females and m i l t was c o l l e c t e d from 5 males. The gametes were t r a n s f e r r e d w i t h i n a cooler (2-5°C) to PBS where they were checked f o r a b n o r m a l i t i e s . Except f o r the sperm m o t i l i t y t e s t s , eggs and m i l t were pooled f o r a l l experiments.  Experimental Design:  Three separate experiments were conducted to t e s t  the e f f e c t s of various concentrations of ambient saltwater on (1) sperm  69  m o t i l i t y , (2) sperm v i a b i l i t y , and (3) combined egg and sperm v i a b i l i t y . A l l samples were maintained at low temperatures i n the range of 3-5°C.  Data Analysis:  As was done with the s a l i n i t y tolerance experiment data,  a l l percent data were normalized using the arcsine transformation before conducting s t a t i s t i c a l manipulations. One-way ANOVA's were done f o r a l l three experiments. Post-hoc m u l t i p l e comparisons were done according to the procedure o u t l i n e d i n the s a l i n i t y tolerance experiment.  Sperm M o t i l i t y Tests  The m o t i l i t y of sperm was examined i n 6 s a l i n i t i e s ; 0, 5.0, 7.5,  10.0,  12.5, and 15.0°/oo- Three d i f f e r e n t males were tested separately and each t e s t was r e p l i c a t e d 5 times.  A very small droplet of sperm, as much as would adhere to the end of a sharp d i s s e c t i n g probe, was placed i n the center of a hemacytometer s l i d e and a c o v e r s l i p l a i d over top. Once the s l i d e was placed under a l i g h t microscope (100X), a drop of t e s t s a l i n i t y water was added onto the s l i d e beside the c o v e r s l i p . Surface tension q u i c k l y d i s t r i b u t e d the water evenly over the s l i d e . The sperm droplet needed to be small enough to allow f o r complete and near simultaneous d i l u t i o n by the water drop. Upon a c t i v a t i o n of the sperm ( i n i t i a t i o n of vigorous movement), the timer was s t a r t e d . The duration of sperm m o t i l i t y was recorded as the i n t e r v a l from i n i t i a l sperm a c t i v a t i o n to about 95% i m m o t i l i t y . Immotility was defined as the cessation of vigorous movement and u s u a l l y included a period of slow sedentary v i b r a t o r y movements.  70  Sperm V i a b i l i t y Tests The v i a b i l i t y of sperm i n s a l i n e water was tested by premixing sperm, pooled from 3 males, w i t h various concentrations of s a l i n e water before using i t to f e r t i l i z e a small beaker of eggs (25 ml).  The t e s t s a l i n i t i e s  0  used were 0, 5.0, 10.0, 12.5, and 15.0 /oo- A f t e r 5 seconds the water and sperm mixture was added to a 150 ml beaker containing about 30 eggs (15 ml) The f i n a l egg to sperm d i l u t i o n r a t i o was 650:1. A f t e r l e t t i n g i t stand f o r 1 minute the contents were poured c a r e f u l l y i n t o a d i v i d e d compartments i n heath t r a y (Heath Tecna Corp., Tacoma, Washington, USA) f u l l of 9°C freshwater. Three r e p l i c a t e s were tested f o r each s a l i n i t y . The order f o r t e s t i n g the d i f f e r e n t s a l i n i t i e s was randomized.  An a d d i t i o n a l experiment, s i m i l a r to the previous one, examined the v i a b i l i t y of sperm i n various concentrations of saltwater f o l l o w i n g a longe p e r i o d of sperm a c t i v a t i o n . The sperm and saltwater mixture was l e f t f o r 15 s instead of only 5 s before adding i t to the eggs.  Sperm v i a b i l i t y was evaluated by examining the f e r t i l i z a t i o n success of the  eggs 18 h a f t e r f e r t i l i z a t i o n when development had reached the 4 to 8  c e l l stage. Samples of 15 eggs were picked from each r e p l i c a t e f o r examination of FS.  Combined Egg and Sperm V i a b i l i t y Tests The v i a b i l i t y of both gametes was examined by simultaneously adding egg and sperm, pooled from 3 parents each, to various concentrations of s a l t w a t e r . Within each t e s t s a l i n i t y , f e r t i l i z e d eggs were maintained i n t h e i r respective s a l i n i t i e s f o r e i t h e r 1, 15, 60, or 240 minutes. This  71  experiment simulated the p o t e n t i a l s i t u a t i o n of i n t e r t i d a l chum salmon spawning during a f l o o d t i d e when any gametes released into the stream would encounter s a l i n e c o n d i t i o n s .  About 50 eggs (20ml) and 0.2ml sperm were added simultaneously to 50 ml of t e s t s a l i n i t y ; 0, 5.0, 10.0, 12.5, or 15.0%o- A f t e r mixing gently they were allowed to remain f o r 1 minute. Depending on the treatment, the eggs were e i t h e r t r a n s f e r r e d d i r e c t l y to freshwater (1 minute  post-fertilization  exposure), or t r a n s f e r r e d to a tank containing the respective t e s t s a l i n i t y and maintained f o r 15, 60, or 240 minutes (15, 60, and 240 minute exposures r e s p e c t i v e l y ) . Three r e p l i c a t e s were e s t a b l i s h e d f o r each combination of s a l i n i t y and exposure time t e s t e d .  A d d i t i o n a l measurements were conducted on two representative treatments (15 and 240 min exposures) to assess the e f f e c t of saltwater on the water a c t i v a t i o n process of the egg. Egg weight was used as an i n d i c a t o r of the amount of water imbibed by the egg. Five eggs from each s a l i n i t y (0, 5, 10, 12.5, and 15°/oo) were weighed at 3 or 4 time i n t e r v a l s depending on the duration of the saltwater exposure. For the 15 min exposure treatment, eggs were weighed before a c t i v a t i o n , 15 min a f t e r being placed i n t o the t e s t s a l i n i t i e s , and more than 12 h a f t e r being t r a n s f e r r e d from the t e s t s a l i n i t i e s to freshwater. For the 240 min exposure treatment, eggs were weighed before a c t i v a t i o n , 150 and 240 minutes a f t e r being placed into the t e s t s a l i n i t i e s , and more than 12 h a f t e r being t r a n s f e r r e d back to freshwater.  72  Egg and sperm v i a b i l i t y was evaluated i n the same manner as f o r the sperm v i a b i l i t y experiment. Samples of 15 eggs were picked from each r e p l i c a t e at 18 h p o s t - f e r t i l i z a t i o n and examined f o r percent FS.  73  RESULTS  Salinity Tolerance of Salmon Eggs  Perivitelline Fluid Osmolality P e r i v i t e l l i n e f l u i d osmolality increased r a p i d l y i n response to increased ambient o s m o l a l i t y . Within 20 to 25 min of being t r a n s f e r r e d to 20°/oo s a l i n i t y water, the PVF osmolality had reached 95% of the maximum and was completely e q u i l i b r a t e d by 50 min ( F i g . 12). Eggs that were returned to 0°/oo water revealed that the opposite response, e f f l u x of s a l t w a t e r , occurred i n about 13 min, h a l f the time of the i n f l u x ( F i g . 13). The l i n e s drawn through the data points represent the p r e d i c t e d values of the model used to describe the processes of saltwater i n f l u x and e f f l u x (equation 1, M a t e r i a l s and Methods). Model parameters, predicted values and r e s i d u a l s are included i n Appendix 1.  Salinity Tolerance and Egg Survival The s a l i n i t y tolerance of eggs i n the F-group was r e l a t i v e l y i n v a r i a b l e . Eggs exposed to the i n t e r m i t t e n t treatments (4 and 8 h) a t s a l i n i t i e s of 15°/oo or l e s s , had s u r v i v a l rates that were s t a t i s t i c a l l y the same as the freshwater c o n t r o l s (P > 0.05). At 20°/oo> the 4 h exposure also had high s u r v i v a l s s i m i l a r to the c o n t r o l s , whereas the 8 h exposures d i d not. Eggs subjected to 20°/oo f °  r  8 n  P  er  ^ay showed a s i g n i f i c a n t decrease i n s u r v i v a l  (P < 0.001) ( F i g . 10). No eggs survived i n the 30°/oo treatments even a t the l e a s t severe 4 h exposure.  74  F i g . 12. O b s e r v e d and p r e d i c t e d changes i n p e r i v i t e l l i n e f l u i d (PVF) o s m o l a l i t y u p o n t r a n s f e r o f chum s a l m o n e g g s f r o m f r e s h w a t e r (0°/ 0 0 s a l i n i t y ) t o s a l t w a t e r (20°/oo)• ^he p o i n t s r e p r e s e n t t h e o b s e r v e d d a t a a n d the s o l i d curved l i n e represents the p r e d i c t e d values obtained u s i n g a g e n e r a l g r o w t h m o d e l ( s e e t e x t a n d A p p e n d i x 1). ' A m b i e n t o s m o l a l i t y ' r e f e r s to the o s m o l a l i t y of the s a l t w a t e r .  PVF OSMOLALITY ( m m o l / k g )  600 - > 500 400 -  TIME (min.)  F i g . 13. Observed and p r e d i c t e d changes i n p e r i v i t e l l i n e f l u i d (PVF) o s m o l a l i t y upon t r a n s f e r o f chum salmon eggs from s a l t w a t e r (20°/oo s a l i n i t y ) t o f r e s h w a t e r ( 0 / ) • The p o i n t s r e p r e s e n t the observed d a t a and the s o l i d c u r v e d l i n e r e p r e s e n t s the p r e d i c t e d v a l u e s o b t a i n e d u s i n g a g e n e r a l growth model (see t e x t and Appendix 1 ) . 'Ambient o s m o l a l i t y ' r e f e r s t o the o s m o l a l i t y o f the f r e s h w a t e r . 0  0 0  Eggs i n the B-group responded much more v a r i a b l y to the s a l i n i t y treatments, even though the trends were the same as the F-group eggs. S u r p r i s i n g l y , the s u r v i v a l rates of the controls i n the B-group were lower than the rates observed at 5 and 1 0 ° / 0 0 f o r both of the  intermittent  exposures (4 and 8 h) ( F i g . 10). An i n i t i a l wave of m o r t a l i t i e s , which was e s p e c i a l l y prominent i n the freshwater c o n t r o l s , occurred during the f i r s t 3 days of i n c u b a t i o n . Recalculating  the s u r v i v a l rates f o r both groups of eggs  w i t h these i n i t i a l m o r t a l i t i e s removed ( i . e . , subtracting  these m o r t a l i t i e s  from both the f i n a l t o t a l number of m o r t a l i t i e s and the i n i t i a l  t o t a l number  of eggs), r e s u l t e d i n markedly higher s u r v i v a l s but d i d not account e n t i r e l y for  the differences  observed between the c o n t r o l s u r v i v a l rates and those of  the 5 and 10°/oo treatments. Figure 10 demonstrates how t h i s s e r i e s of i n i t i a l m o r t a l i t i e s was r e s t r i c t e d l a r g e l y to the controls and how i t was much l e s s apparent i n the other B-group treatments and b a s i c a l l y nonexistent i n the F-group treatments.  Analyzing the r e c a l c u l a t e d B-group values i n d i c a t e d that the c o n t r o l s u r v i v a l rates f o r the 4 and 8 h exposures were s t i l l lower (P < 0.001) than the rates at 5 to 2 0 ° / 0 0 . However, the s u r v i v a l rate i n the 2 0 ° / 0 0 s a l i n i t y treatment at 8 h was s i g n i f i c a n t l y lower than the rates at 5, 10, and 15°/oo-  Compared to the F-group v a l u e s , percent s u r v i v a l rates i n the B-group were lower and generally more v a r i a b l e i n a l l s a l i n i t i e s , e s p e c i a l l y f o r r  eggs exposed to 10 and 15°/oo f ° & h. S u r v i v a l of eggs exposed to 2 0 ° / 0 0 f o r 8 h i n the F-group was s i m i l a r to the s u r v i v a l of eggs i n the same condition i n the B-group ( F i g . 10). As i n the F-group, a l l eggs died i n each of the 3 0 ° / 0 0 treatments i n the B-group a l s o .  77  The majority of dead eggs had developed abnormally. Therefore, observed d i f f e r e n c e s i n s u r v i v a l rates i n the various s a l i n i t y treatments l a r g e l y were due to d i f f e r e n c e s i n the numbers of abnormally developed eggs. Only small numbers of dead eggs appeared to be normally developed. Undeveloped eggs were observed most commonly i n the 3 0 ° / 0 0 and the constant exposure 20°/oo treatments. Occurrence of undeveloped eggs i n any of the other s a l i n i t i e s was rare and u s u a l l y amounted to no more than 1 or 2%. However, the c e r t a i n t y w i t h which I could accurately c l a s s i f y the m o r t a l i t i e s was low due to (1) inherent d i f f i c u l t i e s i n detecting signs of development during the e a r l i e r stages of development (50-110 h ET), (2) v a r y i n g degrees of decomposition of the eggs while they were s t i l l i n the incubators, and (3) t i s s u e d i s t o r t i o n p o t e n t i a l l y r e s u l t i n g from osmotic s t r e s s i n eggs exposed to higher s a l i n i t i e s (20-30°/oo)- As a r e s u l t , c a t e g o r i z i n g m o r t a l i t i e s provided only general i n d i c a t i o n s of the cause of death.  In the constant exposure treatments of the B- and F-group eggs, only those i n 0 and 5°/oo survived; a l l of the other s a l i n i t y treatments r e s u l t e d i n 100% m o r t a l i t y ( F i g . 10). With the B-group eggs, a r e s u l t s i m i l a r to the i n t e r m i t t e n t treatments was seen; c o n t r o l s u r v i v a l s were markedly lower than those i n the 5°/oo treatment. Again, a large proportion of t h i s reduced s u r v i v a l was due to i n i t i a l m o r t a l i t i e s i n the f i r s t few days (see broken l i n e i n F i g . 10). I t i s obvious from the responses i n both groups that a constant exposure of 5 ° / 0 0 d i d not have any negative e f f e c t on egg s u r v i v a l . In both groups of eggs they were not s i g n i f i c a n t l y d i f f e r e n t than the c o n t r o l s (P > 0.05). For the B-group eggs t h i s treatment may even have had a m i t i g a t i n g e f f e c t considering the problems observed with these c o n t r o l s .  78  Eggs i n 10, 15, and 20°/oo constant exposures developed f o r varying e  periods of time before dying. I n 10 and 15°/oo g g  s  began to d i e i n large  numbers between 400-800 h ET. Many of the f i n a l m o r t a l i t i e s i n these two s a l i n i t i e s were 'eyed' (stage 21, Vernier 1969) although o f t e n epiboly was incomplete. I n many cases a seemingly permanent hole e x i s t e d a t the p o s t e r i o r region of the y o l k sac where normally, i n the f i n a l stage of e p i b o l y , the y o l k sac e p i t h e l i a l c e l l s converge and form a complete l a y e r around the y o l k . I n some cases the yolk a c t u a l l y was protruding from t h i s hole as i f the e p i t h e l i a l c e l l s had formed around the p r o t r u s i o n . I n other cases, even i f the c e l l s had formed a complete l a y e r , the area appeared malformed and abnormal i n s t r u c t u r e . I t often resembled scar t i s s u e , c o n s i s t i n g of t r a c t s of b u i l t - u p t i s s u e r a d i a t i n g out from a c e n t r a l p o i n t . In constant 2 0 ° / 0 0 exposure, eggs d i d not develop past 50 h ET (stage 7, Vernier 1969), even though I could not i d e n t i f y them p o s i t i v e l y as m o r t a l i t i e s u n t i l about 200-300 h ET.  Hatching rates of the eggs d i f f e r e d f o r the 6 s a l i n i t i e s . Hatching occurred e a r l i e r and was complete sooner i n the s a l i n i t y treatments than i n the freshwater c o n t r o l treatments (Table 4 ) . Except f o r the 5°/oo s a l i n i t y treatments, data are presented only f o r i n t e r m i t t e n t s a l i n i t y exposure treatments since a l l of the eggs i n the constant treatment s a l i n i t i e s higher than 5°/oo died p r i o r to hatching (Table 4 ) . Eggs from the F-group hatched  79  TABLE 4. Percent hatching measured for the F- and B-group eggs at three different times of development. Data for treatment s a l i n i t i e s of 3 0 ° / were not included since none of these eggs survived to hatching. 0 0  Treatment Exposure Time  F-GROUP  B-GROUP  Percent Hatch (%) Treatment S a l i n i t y (°/oo) 0 5 10 15 20  Percent Hatch (%) Treatment S a l i n i t y ( ° / ) 0 5 10 15 20 0 0  ETM314h 24h  0  7  NS  NS  NS  0  2  NS  NS  NS  8h  0  0  9  4  7  0  0  4  3  4  4h  0  0  0  1  1  1  0  1  4  1  24h  30  65  NS  NS  NS  23  48  NS  NS  NS  8h  10  99  . 98  88  75  23  45  65  65  70  4h  45  96  97  80  92  15  25  90  34  80  24h  95  100  NS  NS  NS  82  100  NS  NS  NS  8h  80  100  100  100  100  83  96  99  96  99  4h  86  100  100  100  100  82  94  98  92  100  2  ET=1392h  ET=1410h  1 2  ET is the Elapsed Development Time i n hours since f e r t i l i z a t i o n . NS denotes no s u r v i v a l to the hatching stage.  more quickly than those from the B-group. Within the F-group, eggs i n the 5 and 10°/oo intermittent s a l i n i t y treatments hatched more quickly than those i n the 15 and 20°/oo treatments. eggs (Table 4).  This trend was not observed i n the B-group  These results may have been confounded by the fact that a l l  of the eggs were relocated to an adjacent laboratory with the same  80  experimental conditions during the time of hatching due  to unavoidable space  r e s t r i c t i o n s . It i s well established that physical movement may  induce eggs  to hatch more quickly i f they already are near that stage of development (J.O.T. Jensen, 1987,  pers. comm., P a c i f i c B i o l o g i c a l Station, Nanaimo,  B.C.).  E f f e c t s Of S a l i n i t y On The F e r t i l i z a t i o n Process  Sperm M o t i l i t y Duration of sperm m o t i l i t y did not vary s i g n i f i c a n t l y between the 3 d i f f e r e n t males tested within a given s a l i n i t y (P > 0.05). However, sperm m o t i l i t y varied greatly between the d i f f e r e n t test s a l i n i t i e s . Chum salmon sperm were motile for a s i g n i f i c a n t l y shorter time i n freshwater than i n the 5,  7.5  and 10%o  s a l i n i t i e s (P < 0.001) ( F i g . 14).  period u n t i l 95% immotility was In 1 5 ° /  0 0  s a l i n i t y m o t i l i t y was  a c t i v i t y was  In 12.5%o the time  about h a l f that observed i n 5 to 1 0 ° / . 0 0  assessed as n i l , since no true swimming  observed ( F i g . 14).  However, i n addition to quantitative differences of a c t i v i t y between the test s a l i n i t i e s , q u a l i t a t i v e differences also existed. The general response, i n s a l i n i t i e s of 0 to 12.5°/oo ^  n  which a c t i v i t y occurred, was  an  initial  period of vigorous a c t i v i t y followed by a period of decreasing a c t i v i t y t r a i l i n g o f f to t o t a l immotility. Even once the sperm had stopped swimming forward, they usually continued to vibrate i n one spot for 1-5 becoming t o t a l l y i n a c t i v e . The most vigorous a c t i v i t y was  min. before  observed i n  s a l i n i t i e s ranging from 5 to 1 0 ° / . In s a l i n i t i e s above t h i s , 1 2 . 5 ° / , 0 0  0 0  81  and  F i g . 14. Duration of sperm motility i n response to various ambient s a l i n i t i e s (see text for d e f i n i t i o n of m o t i l i t y ) . The duration of m o t i l i t y was assessed from the i n i t i a t i o n of m o t i l i t y following the addition of the test water, to about 95% immotility. No m o t i l i t y per se was observed at 15°/oo s a l i n i t y but some movement was recorded. Each point i s the mean (+ 2SE) of 5 replicates from 3 different males. No difference i n m o t i l i t y . was observed between the individual males.  82-  below i t , 0°/oo> there was a n o t i c e a b l y slower a c t i v a t i o n time and a shorter m  duration o f a c t i v i t y . I n 15°/oo> °re than 99% of the sperm f a i l e d to i n i t i a t e m o t i l i t y , even though they d i d v i b r a t e s l i g h t l y . Occasionally I observed a s i n g l e sperm swimming v i g o r o u s l y f o r a few seconds. I n 2 0 ° / 0 0 , there were very s l i g h t v i b r a t o r y movements i n a few of the r e p l i c a t e t e s t s but u s u a l l y no movement occurred.  Sperm V i a b i l i t y  V i a b i l i t y , assessed as FS a f t e r 18 h of development, of sperm mixed f o r 5 s i n saltwater of 5 or 1 0 ° / 0 0 s a l i n i t y was not s i g n i f i c a n t l y lower than p  that of the c o n t r o l (0°/oo) ( > 0.05). V i a b i l i t y d i d not decrease u n t i l s a l i n i t i e s of 12.5°/oo  o r  higher were used to premix the sperm before  f e r t i l i z i n g the eggs ( F i g . 15). However, only at 15°/oo  w a s  FS s i g n i f i c a n t l y  lower than the value a t 1 0 ° / 0 0 (P < 0.05).  This same r e l a t i o n s h i p d i d not hold when the sperm were premixed f o r 15 s i n the various t e s t s a l i n i t i e s . These sperm showed nearly equal v i a b i l i t i e s f o r the whole range of s a l i n i t i e s t e s t e d , 0 - 1 5 ° / 0 0 . Although some v a r i a b i l i t y occurred, the b a s i c response was a FS ranging from 60-70% (Fig.15). The t e s t at 5 ° / 0 0 was excluded from c a l c u l a t i o n s and s t a t i s t i c a l analyses because the premixing time period inadvertently was extended to 25 s instead of 15 s. As a r e s u l t , the f e r t i l i z a t i o n rates were much lower (24.6 + 4.91%, mean + SD). This value revealed that even a s l i g h t added delay beyond 15 s r e s u l t e d i n further reduction of f e r t i l i z a t i o n r a t e s .  83  F i g . 15. Sperm v i a b i l i t y measured as f e r t i l i z a t i o n success (FS) i n various ambient s a l i n i t i e s . V i a b i l i t y was tested by premixing sperm i n various s a l i n i t i e s f o r e i t h e r 5 s ( s o l i d l i n e ) or 15 s (broken l i n e ) , before adding the mixture to eggs. Each point i s the mean (+ 2SE) of 3 r e p l i c a t e s comprised of 15 eggs each.  8H  Combined Egg and Sperm V i a b i l i t y V i a b i l i t y of eggs and sperm exposed simultaneously to various s a l i n i t i e s was not a f f e c t e d i n lower s a l i n i t i e s ranging from 5-10°/oo (P < 0.001), whereas higher s a l i n i t i e s of 12.5 and 15°/oo r e s u l t e d i n reduced v i a b i l i t y (P < 0.05) ( F i g . 16). These r e s u l t s were s i m i l a r to the sperm v i a b i l i t y t e s t s ; percent FS rates e s s e n t i a l l y were the same at a l l of the s a l i n i t i e s except 15°/oo- At t h i s concentration the percent FS was much lower, 20.9% + 0.20% (mean + SD) compared to 53.5% + 0.46% ( F i g . 16).  Eggs maintained i n the respective t e s t s a l i n i t i e s f o r durations longer than 1 min, i . e . , 15, 60, and 240 min., d i d not s u f f e r any  additional  e f f e c t s due to prolonged exposures ( F i g . 16). Eggs developed normally to 18 h ET w i t h no apparent problems i n those s a l i n i t i e s that d i d not i n h i b i t f e r t i l i z a t i o n . Two-way ANOVA indicated that only s a l i n i t y had a s i g n i f i c a n t e f f e c t (P < 0.001), whereas duration of exposure and the i n t e r a c t i o n term were not s i g n i f i c a n t (P > 0.05).  However, eggs l e f t i n 12.5 and 1 5 ° / 0 0 f o r 240 min d i d show signs of i n h i b i t i o n of water hardening, even a f t e r exposure to freshwater f o r more than 12 h. In the higher s a l i n i t i e s these eggs had not increased i n weight as much as the eggs i n e i t h e r (1) lower s a l i n i t i e s (0-10°/00) f o r a s i m i l a r duration (240 min) or (2) s i m i l a r s a l i n i t i e s (12.5 and 15°/00) f o r a shorter duration (15 min.) ( F i g . 17). However, due to the v a r i a b i l i t y i n egg weights w i t h i n the i n d i v i d u a l groups weighed, t h i s response was not s t a t i s t i c a l l y s i g n i f i c a n t (P > 0.05).  85  Fig. 1 6 . Combined egg and sperm viability measured as fertilization success ( F S ) in various salinities. Viability was tested by adding eggs and sperm simultaneously to various salinities and maintaining eggs in the test salinities for four different time intervals. Each point is the mean ( + 2 S E ) of 3 replicates comprised of 1 5 eggs each. Solid circles represent 1 min time interval; open circles represent 1 5 min interval; open triangles represent 6 0 min interval; and open squares represent 2 4 0 min ( 4 h) interval.  PRE-FERT'N  O  (min)  5%o TREATMENT  0 150  0%o  3-<  I5H  (min)  (min)  5%oTREATMENT  015  PRE-FERT'N  ' 3  °  o  15  150 240-  (min)  FRESHWATER 12-16(h) PRE-FERT'N (min)  5%o TREATMENT  n  0  3  r _  !2.5%o  l  I5-|  5%o TREATMENT  0  10%,  3^  3  1  3-<  3 3^  i 12.5%,  1504 240  FRESHWATER 12-16(h) (min)  3—•  • 12-16-  (min)  PRE-FERT'N  5%,  1 12-16I0%o  3^  n  0 150240  5%a  FRESHWATER 12-16 (h) 5 %o TREATMENT  3 ^  12-16-  (min)  (min)  0°/cos_  240-  FRESHWATER 12-16 (h) PRE-FERT'N  3 -  nU  -< 12-16 l5%o  3-<  15  (min)  FRESHWATER 12-16(h) 240  OH R-* 150 333 • 240  15%,  H 12-16280 320 360 EGG WEIGHT (mg) 15 MIN EXPOSURE  240  280 320 360 EGG WEIGHT (mg)  400  240 MIN EXPOSURE  F i g . 17. Mean weights of eggs during combined egg and sperm v i a b i l i t y experiment. Weights were measured for the 15 and 240 min time exposures at various times: before f e r t i l i z a t i o n , at the end of the saltwater exposure ( i . e . , 15 or 240 min), and after more than 12 h i n freshwater. Each point i s the mean (+ 2SE) of 5 eggs.  81  DISCUSSION  S a l i n i t y Tolerance of Salmon Eggs  Chum and pink salmon embryos, a l e v i n s , and f r y are able to t o l e r a t e constant s a l i n e conditions (31.8°/oo) b e t t e r than coho (CL. k i s u t c h ) . chinook (0. tshawytscha) and sockeye salmon (Oj. nerka) (Weisbart 1968) . Although chum and pink salmon embryos and a l e v i n s are not t r u l y euryhaline, Weisbart (1968) showed that t h e i r increased r e s i s t a n c e to near f u l l strength seawater was a r e s u l t of b e t t e r osmoregulatory  c a p a b i l i t i e s compared to the three  other Oncorhynchus species. The f r y of these two species on the other hand were t r u l y euryhaline. He measured LC50 values f o r embryos and a l e v i n s and reported that chum and pink had the longest s u r v i v a l times. Unfortunately, t h i s information does not provide any i n s i g h t into the c r i t i c a l exposure time a t which i r r e p a r a b l e damage occurs and death becomes imminent, even i f the embryos were returned to freshwater. When o s m o t i c a l l y stressed eggs, a l e v i n s , or f r y are returned to a n o n - s t r e s s f u l environment before some threshold or c r i t i c a l p o i n t , u s u a l l y they w i l l recover completely  (Rockwell  1956, H o l l i d a y and B l a x t e r 1960). I n l i g h t of the reported d i f f e r e n c e s i n saltwater t o l e r a n c e , the question a r i s e s whether t h i s point would be d i f f e r e n t f o r the f i v e species of Oncorhynchus?  B a i l e y (1966) examined the s a l i n i t y tolerance of pink salmon eggs a l e v i n s and f r y i n a simulated i n t e r t i d a l environment. He observed no adverse e f f e c t s a t moderate exposure l e v e l s ; 4 h or l e s s twice d a i l y i n s a l i n i t i e s of 1 0 - 1 5 ° / 0 0 . I n high s a l i n i t i e s (28°/00) however, he observed 0% s u r v i v a l during the f i r s t 12 days f o r eggs exposed f o r 9.3 h twice d a i l y .  88  Exposures of 6.7 and 4.0 h to the same s a l i n i t y r e s u l t e d i n egg s u r v i v a l s of 50 and 100% r e s p e c t i v e l y . These r e s u l t s suggest that c r i t i c a l exposures to saltwater f o r pink salmon eggs begin i n s a l i n i t i e s greater than 2 8 ° / 0 0 at durations between 4.0 and 6.7 h twice d a i l y .  Results from my study are consistent with B a i l e y ' s work. My r e s u l t s i n d i c a t e that the c r i t i c a l s a l i n i t y f o r chum salmon eggs i s between 20 and 3 0 ° / 0 0 . No reduced s u r v i v a l was observed i n 2 0 ° / 0 0 at 4 h per day (94.7%), whereas exposure to the same s a l i n i t y f o r 8 h per day r e s u l t e d i n 48.0  and  41.2% s u r v i v a l (F and B groups r e s p e c t i v e l y ) . I observed no s u r v i v a l i n 3 0 ° / 0 0 f o r e i t h e r exposure time, 4 or 8 h.  A comparison of the i n t e r m i t t e n t s a l i n i t y tolerance of chum and pink salmon eggs i n d i c a t e s that l i t t l e d i f f e r e n c e e x i s t s at moderate s a l i n i t i e s . Whereas at higher s a l i n i t i e s , pink salmon eggs appear to be more t o l e r a n t to both concentration and duration of saltwater exposure. However, before accepting the observed differences between my r e s u l t s and B a i l e y ' s (1966) as the r e s u l t of i n t e r s p e c i f i c differences i n osmoregulatory a b i l i t y , i t may be necessary to consider a few other p o s s i b l e explanations.  P o t e n t i a l l y , the most important of these i s the manner i n which the eggs were exposed to the treatment s a l i n i t i e s . Eggs i n my experiment were placed p h y s i c a l l y i n t o tanks containing the t e s t s a l i n i t i e s and as a r e s u l t experienced very abrupt changes i n ambient s a l i n i t y . The abruptness of these changes may have had adverse e f f e c t s on the embryos. Although d e t a i l s are not g i v e n , i t seems l i k e l y that eggs i n B a i l e y ' s (1966) study experienced l e s s abrupt changes i n ambient s a l i n i t y . Weisbart (1968) examined t h i s  89  p o t e n t i a l problem when he tested the e f f e c t of an abrupt change from 0 ° / 0 0 i n t o 3 1 . 8 ° / 0 0 s a l i n i t y , compared to a stepwise one over a four day p e r i o d i n t o the same f i n a l s a l i n i t y . He showed no d i f f e r e n c e i n LC50 values of a l e v i n s exposed to the two exposure regimes. Since he found the a l e v i n stage of development to be the most s e n s i t i v e to saltwater exposure, the absence of any d i f f e r e n t i a l  s u r v i v a l suggests that e f f e c t s from an abrupt change  probably are n e g l i g i b l e . However, i t may not be reasonable to extrapolate from Weisbart's one time abrupt exposure to the repeated abrupt exposures conducted i n my study.  Another p o s s i b l e explanation f o r the observed d i f f e r e n c e s i n s u r v i v a l rates between B a i l e y ' s study (1966) and mine, i s a temperature r e l a t e d one. Rockwell (1956) reported that eggs and a l e v i n s incubated a t lower temperatures had a higher tolerance to saltwater exposure than those incubated at higher temperatures. Therefore, i f B a i l e y (1966) maintained lower temperatures i n h i s t e s t s a l i n i t i e s than I d i d i n mine (9.0 + 0.1 C ) , the  observed d i f f e r e n c e s i n s u r v i v a l i n the two experiments could be, a t  l e a s t i n p a r t , due to the d i f f e r e n t i a l e f f e c t s of temperature on s a l i n i t y t o l e r a n c e . I t i s quite p o s s i b l e that B a i l e y ' s (1966) water was cooler since he was pumping saltwater i n from a bay and using stream water f o r h i s freshwater source. These p o s s i b l e causes remain speculative since i n s u f f i c i e n t d e t a i l of B a i l e y ' s experimental procedure were provided to make any conclusive statements regarding procedural d i f f e r e n c e s . Further, the p r e c i s e e f f e c t s of temperature on saltwater tolerance are not w e l l understood.  90  Rockwell (1956) a l s o presented data that i n d i c a t e d s i m i l a r i n t e r s p e c i f i c d i f f e r e n c e s over a range of s a l i n i t i e s and temperatures, even though he d i d not provide any d i s c u s s i o n of i t . Weisbart (1968) a l s o reported a d i f f e r e n c e between chum and pink salmon saltwater tolerance; LC50 values f o r pink salmon a l e v i n s were s l i g h t l y higher than those f o r chum salmon. Furthermore, h i s time s e r i e s data provide p h y s i o l o g i c a l evidence of a p o s s i b l e d i f f e r e n c e i n saltwater tolerance between chum and pink salmon embryos and a l e v i n s . A f t e r 8 and 12 h exposure to 31.8°/oo> pfnk salmon g e n e r a l l y had lower blood +  osmotic, Na , and C l " concentrations than chum salmon, even though pink demonstrated among the highest l e v e l s i n i t i a l l y . Although the d i f f e r e n c e s were not e s p e c i a l l y l a r g e , they suggest a f u n c t i o n a l b a s i s f o r p o s s i b l e d i f f e r e n t i a l saltwater tolerances between pink and chum salmon.  The d i s t r i b u t i o n of these two species on the i n t e r t i d a l spawning grounds d i f f e r s . Generally chum salmon choose the upper and middle portions of the i n t e r t i d a l zone whereas pink salmon seem l e s s p a r t i c u l a r and w i l l spawn throughout the whole zone, i n c l u d i n g the lower portions (Thornsteinson et a l . 1971). T y p i c a l l y , the lower l i m i t of spawning f o r i n t e r t i d a l pink salmon i s f u r t h e r downstream than chum. Therefore i n t e r t i d a l pink salmon eggs are more l i k e l y to experience longer durations, greater frequencies and p o s s i b l y higher concentrations of saltwater exposure than i n t e r t i d a l chum salmon. Thus the reported d i f f e r e n c e s i n saltwater tolerance between chum and pink salmon eggs may be r e a l and i n f a c t r e f l e c t d i f f e r e n c e s i n the l i f e h i s t o r i e s of these two species of salmon.  Except f o r B a i l e y (1966), other studies examining s a l i n i t y tolerance of salmon eggs have a l l used constant exposure treatments even though the  91  r a t i o n a l e o f t e n was introduced from the perspective of i n t e r t i d a l spawning. Rockwell (1956) provided a thorough and i n t e r e s t i n g h i s t o r i c a l review of experiments d e a l i n g with the e f f e c t s of saltwater on salmonid eggs. From t h i s i t i s apparent that many pre-1956 researchers, i n c l u d i n g Rockwell, chose not to make a d i s t i n c t i o n between t e s t i n g the e f f e c t s of constant versus i n t e r m i t t e n t saltwater exposure. The o v e r a l l conclusion from these and more recent studies (Weisbart 1968, Kashiwagi and Sato 1969, Shen and Leatherland 1978a) was that salmonid eggs and a l e v i n s cannot survive prolonged periods of time i n concentrations of saltwater greater than i s o s m o t i c i t y (approx. 10-12°/oo> Shen and Leatherland 1978a). Salmonid eggs and a l e v i n s are not able to f u l l y iono- and osmoregulate i n hyperosmotic c o n d i t i o n s . Yet, depending on exposure conditions and species i n v o l v e d , these embryos and a l e v i n s are able to survive i n intermediate to f u l l strength seawater (up to 30°/00) f o r l i m i t e d periods of time.  S u r v i v a l data, corroborated by measurements of i o n i c and osmotic t i s s u e concentrations, suggest that the a b i l i t y of salmonid eggs to withstand l i m i t e d saltwater exposure i s the r e s u l t of t i s s u e tolerance i n some species and p a r t i a l r e g u l a t i o n i n others. Information from my study and B a i l e y ' s (1966) i n d i c a t e s that eggs can survive exposure to higher s a l i n i t i e s f o r weeks at a time when provided with a regular freshwater i n t e r l u d e between exposures. A p e r i o d of recuperation i n freshwater during which embryonic t i s s u e s can balance t h e i r osmotic and i o n i c concentrations appears to be e s s e n t i a l . In l i g h t of the regulatory mechanisms a v a i l a b l e to salmonid embryos and a l e v i n s t h i s conclusion seems reasonable.  92  As mentioned e a r l i e r , the young t e l e o s t embryo probably regulates i t s i n t e r n a l i o n i c and osmotic environments l a r g e l y through passive maintenance of t i g h t plasma membranes. At the eyed stage t h i s tightness decreases s l i g h t l y and ions are absorbed u n t i l hatching  (Alderdice  1987). In some species, i n c l u d i n g rainbow t r o u t (Shen and Leatherland 1978b) but not coho salmon (Leatherland and L i n 1975), c h l o r i d e c e l l s have been located i n the y o l k sac epithelium surrounding the y o l k , which i n the e a r l i e r stages of development was contained only by a plasma membrane. I f c h l o r i d e c e l l s do f u n c t i o n as osmo- and iono-regulatory c e l l s , then the absence of these c e l l s i n coho salmon i s at l e a s t consistent with the a v a i l a b l e information. Weisbart (1968) found that t h i s species could not c o n t r o l i t s i n t e r n a l i o n i c and osmotic concentrations. Therefore, i n view of h i s f i n d i n g s that pink and chum salmon embryos could at l e a s t p a r t i a l l y c o n t r o l t h e i r i n t e r n a l i o n i c and osmotic concentrations, i t would be u s e f u l to conduct a s i m i l a r search f o r c h l o r i d e c e l l s i n these two  species.  Although the p o t e n t i a l f o r r e g u l a t i o n by s p e c i f i c c e l l s e x i s t s i n t e l e o s t post-eyed embryos and a l e v i n s , i t i s not known to what extent t h i s p o t e n t i a l i s r e a l i z e d s p e c i f i c a l l y i n salmonids.  Maintenance of l i m i t e d trans-membrane ion and water f l u x e s across  the  plasma membrane i n the e a r l y embryo, i s dependent upon low plasma membrane p e r m e a b i l i t y . Drawing a p a r a l l e l between the amphibian egg and the f i s h A l d e r d i c e (1987) summarized that the membrane permeability c o e f f i c i e n t  egg, (Pd)  i n the f i s h egg, s i m i l a r to that i n the amphibian egg, i s a f f e c t e d i n d i r e c t l y by tension on the external egg membrane. This tension i n turn i s dependent upon the i n t e r n a l h y d r o s t a t i c pressure of the egg. Egg h y d r o s t a t i c  93  pressure i s r e l a t e d to egg s i z e , properties of the p e r i v i t e l l i n e c o l l o i d s , and t o n i c i t y of the external medium.  In mature f l a c c i d oocytes contained i n the female body c a v i t y , the permeability c o e f f i c i e n t (Pd) i s h i g h . However, once passed out of t h i s o s m o t i c a l l y c o n t r o l l e d environment, Pd remains high only f o r a short period f o l l o w i n g f e r t i l i z a t i o n before i t drops r a p i d l y to a very low value ( i . e . , an i o n i c a l l y and o s m o t i c a l l y t i g h t s t a t e ) . These changes coincide with establishment of the p e r i v i t e l l i n e space as a r e s u l t of water imbibation across the e x t e r n a l egg membrane from the ambient environment. I n response to the o s m o t i c a l l y a c t i v e c o l l o i d s present i n the p e r i v i t e l l i n e f l u i d , a h y d r o s t a t i c pressure develops i n the egg and r i s e s to an e q u i l i b r a t e d l e v e l , ranging from 30-90 mm Hg i n the f i v e species of north american P a c i f i c salmon (Alderdice e t a l . 1984). This i n t e r n a l pressure exerts a tension on the e x t e r n a l egg membrane and v a r i e s considerably depending on species, stage o f development, and external c o n d i t i o n s , such as s a l i n i t y (Alderdice 1987). Thus the s a l i n i t y of the external medium i n d i r e c t l y a f f e c t s plasma membrane p e r m e a b i l i t y . For example, hyperosmotic media that produce a decrease i n i n t e r n a l egg pressure, r e s u l t i n an increase i n membrane permeability.  Another important f a c t o r a f f e c t i n g plasma membrane permeability i s temperature. Although the i n t e r a c t i o n s are very complex the b a s i c e f f e c t i s increased permeability i n response to increased temperature (Alderdice 1987).  94  How do the p h y s i o l o g i c a l e f f e c t s associated with saltwater exposure, i n t e r m i t t e n t or constant, r e l a t e to observed s u r v i v a l responses of eggs exposed to such conditions? Assuming plasma membrane permeability i s increased by a lowering of the i n t e r n a l egg pressure i n response to increased t o n i c i t y of the external medium (e.g., s a l i n i t y ) , one can speculate about the e f f e c t s on the young developing embryo. In general, s u r v i v a l curves of t e l e o s t embryos exposed to various s a l i n i t i e s i n d i c a t e a threshold defined by a r e l a t i v e l y narrow s a l i n i t y range. Often there i s a range of s a l i n i t i e s at which the embryos are completely t o l e r a n t (no adverse e f f e c t s ) , followed by a r e l a t i v e l y narrow range of s a l i n i t i e s at which they experience s u b l e t h a l or chronic e f f e c t s (these may r e s u l t i n death f o r a p o r t i o n of the t e s t group due to advanced developmental stage abnormalities) and f i n a l l y a range of s a l i n i t i e s at which the e f f e c t i s acute or l e t h a l ( a l l of the f i s h d i e , u s u a l l y w i t h i n a short period a f t e r f e r t i l i z a t i o n ) .  Evidence f o r sharp m o r t a l i t y response curves i n f i s h eggs exposed to saltwater can be found i n a number of studies i n c l u d i n g t h i s one. Chum salmon eggs exposed i n t e r m i t t e n t l y f o r 8 h per day i n my experiments, showed no adverse e f f e c t s up to s a l i n i t i e s of 15°/oo> a chronic or s u b l e t h a l e f f e c t at 20°/oo> and an acute or l e t h a l e f f e c t at 30°/oo-  A t  2 0 ° / 0 0 exposure 8 h  d a i l y , about 45% of the eggs survived and hatched while the majority of the 55% m o r t a l i t i e s developed abnormally. B a i l e y (1966) recorded s u r v i v a l s of 100% i n exposures of l e s s than 4 h at 2 8 ° / 0 0 twice d a i l y . Durations longer than 4 h twice d a i l y r e s u l t e d i n decreasing s u r v i v a l s that reached 0% at 9.3 h twice d a i l y and produced a marked increase i n developmental a b n o r m a l i t i e s . Shen and Leatherland (1978a) reported s u r v i v a l s of 79.6 and 28.1% at 10 days post-hatching i n rainbow trout a l e v i n s exposed to 11 and  95  1 3 ° / 0 0 continuously throughout development. Many of the eggs that d i d hatch i n the 1 3 ° / 0 0 treatment were deformed and u s u a l l y d i d not survive beyond 12h post-hatching. Other studies concerning freshwater species (Parry 1960, Kashiwagi and Sato 1969, Watanabe e t a l . 1985) and marine species ( H o l l i d a y 1965, Alderdice et a l . 1979) have reported r e s u l t s demonstrating s i m i l a r responses i n s a l i n i t y tolerance.  These data suggest there i s a threshold beyond which development cannot proceed normally. Perhaps that threshold i s r e l a t e d to plasma membrane p e r m e a b i l i t y . I t seems reasonable to speculate that eggs exposed to 2 0 ° / 0 0 for  8 h d a i l y i n t h i s study, experienced temporarily lowered i n t e r n a l egg  pressures as a r e s u l t of the increased t o n i c i t y of the e x t e r n a l medium. I f we accept that the p r e v i o u s l y discussed r e l a t i o n s h i p of lowered i n t e r n a l pressure r e s u l t i n g i n increased membrane permeability i s v a l i d , then these eggs would experience a concomitant increase i n plasma membrane p e r m e a b i l i t i e s o f the embryonic c e l l s . Reduced membrane tightness would allow f o r increased ion and water f l u x e s along the e x i s t i n g concentration gradients, i . e . , most ions would pass inward and water outward. On the other hand, eggs exposed to 20°/oo f °  r o n  l y 4 b d a i l y showed no adverse e f f e c t s .  Perhaps the exposure time was not s u f f i c i e n t to cause a marked increase i n membrane permeability o r , the exposure to higher i n t e r n a l i o n i c concentrations f o r 4 instead of 8 h, was not long enough to produce i r r e p a r a b l e c e l l u l a r damage. Due to an .incomplete understanding of the t o x i c e f f e c t s of saltwater exposure and the c h r o n o l o g i c a l development of osmo- and ionoregulatory mechanisms i n the e a r l y l i f e stages of salmonids, these suggestions must remain s p e c u l a t i v e .  96  In view of the e f f e c t s that i n t e r m i t t e n t compared to continuous saltwater exposure has on egg development, one might ask whether i t i s the duration of exposure a t any one given time o r , the t o t a l duration of exposure over a given length of time that i s most s i g n i f i c a n t . For example, would eggs exposed to 2 0 ° / 0 0 f o r 4 h twice d a i l y e x h i b i t the same s u r v i v a l rate as eggs exposed to 20°/oo f o r 4 h only once d a i l y (94.7%) o r , would the r  rate be s i m i l a r to eggs exposed to 20°/oo f ° ^ h once d a i l y (45%)? I t i s a l s o p o s s i b l e that the s u r v i v a l rate would be intermediate between the two. Thus i t i s u s e f u l to determine the importance of the r e t u r n of an egg to freshwater between saltwater exposures, i n r e l a t i o n to the a c t u a l saltwater exposure i t s e l f . I t i s not obvious whether i t i s t h i s p e r i o d of freshwater reprieve that i s c r i t i c a l o r , i f i t i s the concentration and duration of the saltwater exposure that combine to determine egg s u r v i v a l , independent of freshwater r e p r i e v e .  Why do some members of a t e s t group f a i l to survive t e s t conditions while others seemingly remain unaffected? Knowledge of the s p e c i f i c mechanisms that produce the t o x i c or negative e f f e c t s of saltwater exposure a l s o may provide some i n s i g h t s i n t o v a r i a b l e s u r v i v a l rates w i t h i n , as w e l l as between t e s t treatments. R e a l i z a t i o n of t h i s , could p o s s i b l y help e x p l a i n the v a r i a b l e egg s u r v i v a l rates observed i n t h i s study f o r the B-group eggs compared to the F-group. V a r i a b l e and low rates were e s p e c i a l l y prominent i n the c o n t r o l treatment eggs of B-group (0°/oo) (Fig- 10)- An i n i t i a l wave of m o r t a l i t i e s w i t h i n the f i r s t 3 days accounted f o r much of the observed d i f f e r e n c e but the cause remains unexplained. Some p o t e n t i a l causal f a c t o r s i n i t i a l l y considered but subsequently discounted, were: (1) f e r t i l i z a t i o n rate (98-100% f o r a l l incubators i n both groups), (2) incubation conditions  97  (these were the same as the F-group c o n t r o l eggs, which showed c o n s i s t e n t l y high s u r v i v a l , 92-97%), and (3) movement e f f e c t s ( a l l incubators were given the same movement treatments, moreover, v a r i a b i l i t y between r e p l i c a t e incubators and exposure time treatments generally was low). The eggs i n question ( c o n t r o l treatment) were from the same pooled source as those used f o r a l l of the other B-group s a l i n i t y treatments, yet the e f f e c t of lowered s u r v i v a l was seen only i n these c o n t r o l treatment eggs. This i n i t i a l l y suggests that t h i s was not a simple gamete source problem. I f I assume that no procedural biases were introduced during i n c u b a t i o n , the data show that the B-group eggs survived b e t t e r i n low l e v e l s of s a l i n i t y than i n freshwater. One p o s s i b l e i n t e r p r e t a t i o n of t h i s observation suggests that these eggs a c t u a l l y required low to intermediate l e v e l s of s a l i n i t y to develop normally. Or, perhaps i t was a gamete source problem that a f f e c t e d the whole group of eggs but the associated consequences simply were most apparent i n freshwater. Due to i n s u f f i c i e n t information these questions remain unanswered and the explanations remain c o n j e c t u r a l .  The experiment designed to t e s t whether i n t e r t i d a l eggs were more s a l i n i t y t o l e r a n t than freshwater eggs was not s u c c e s s f u l due to l o g i s t i c a l problems. F i r s t and foremost among these were the d i f f i c u l t i e s encountered i n o b t a i n i n g eggs from true i n t e r t i d a l spawning parents, and second were the p r e v i o u s l y discussed problems with the c o n t r o l s and o v e r a l l s u r v i v a l of the B-group eggs. In s p i t e of these problems, the r e s u l t s show that under the experimental conditions t e s t e d , there was no i n d i c a t i o n of lower s a l i n i t y tolerance i n the freshwater source eggs. Perhaps p o t e n t i a l d i f f e r e n c e s i n s a l i n i t y t o l e r a n c e , i f they e x i s t , would not be measurable unless one compared more s p a t i a l l y i s o l a t e d spawning populations i n a given r i v e r  98  system. For example, using freshwater spawners that t r a v e l f u r t h e r upstream (50-100 km) i n a r i v e r system where i n t e r t i d a l spawners also occur. A l t e r n a t e l y , one might consider t e s t i n g f i s h i n a r i v e r system where d i s c r e t e i n t e r t i d a l and freshwater spawning populations already are thought to e x i s t (e.g., Olsen Creek, Alaska) (Helle et a l . 1964, 1970, Thornsteinson et a l . 1971).  In summary, eggs exposed to saltwater i n t e r m i t t e n t l y , t o l e r a t e d higher concentrations than those exposed continuously. Eggs exposed to 20°/oo  o r  l e s s f o r 4 h d a i l y showed no noticeable d i f f e r e n c e i n s u r v i v a l rates compared to the c o n t r o l s . However, eggs exposed to 20°/oo f °  r  twice as long,  8 h, showed a marked decrease i n s u r v i v a l (45%). None of the eggs survived when exposed to 30°/oo f °  r  4 or 8 h per day. Except f o r some i n e x p l i c a b l e  v a r i a t i o n observed i n the B-group eggs, s u r v i v a l rates were high f o r a l l i n t e r m i t t e n t treatment s a l i n i t i e s of 15°/oo  o r  l e s s , i r r e s p e c t i v e of the  d u r a t i o n . Eggs exposed to constant s a l i n i t i e s showed no s i g n i f i c a n t development i n 30°/oo> very l i m i t e d development i n 20°/oo (morula stage), advanced but incomplete and abnormal development i n 10 and 1 5 ° / 0 0 (eyed eggs, u s u a l l y w i t h deformed yolk sacs r e s u l t i n g from abnormal e p i b o l y ) , and high s u r v i v a l and normal development i n 0 and 5 ° / 0 0 .  A comparison of these r e s u l t s to the only other i n t e r m i t t e n t exposure study i n the l i t e r a t u r e , suggests p o s s i b l e i n t e r s p e c i f i c d i f f e r e n c e s i n saltwater tolerance between pink and chum salmon embryos. D i f f e r e n t i a l s u r v i v a l rates suggest that pink salmon embryos and a l e v i n s are more t o l e r a n t than chum salmon to both duration and concentration of i n t e r m i t t e n t saltwater exposure. Further, i n t e r t i d a l spawning d i s t r i b u t i o n s of these two  99  species indicate that the observed i n t e r s p e c i f i c differences may be a r e f l e c t i o n of d i f f e r e n t l i f e h i s t o r i e s ; pink salmon t y p i c a l l y spawn further down i n the i n t e r t i d a l zone than chum salmon.  Regulatory mechanisms available to salmonid embryos and alevins are r e l a t i v e l y s i m p l i s t i c i n that they function at a c e l l u l a r l e v e l and do not involve organs per se. In the early embryo e s p e c i a l l y , maintenance of tight plasma membranes with respect to ion and water fluxes seems to be c r i t i c a l for normal development and s u r v i v a l . Membrane permeability i s affected d i r e c t l y and i n d i r e c t l y by many factors including osmotic pressure of the external medium (e.g., s a l i n i t y ) and temperature. Based on r e s u l t s from this study and others, dysfunction of the plasma membrane i n hyperosmotic media i s speculated to be an important contributing factor i n the deleterious nature of prolonged saltwater exposure. As speculated e a r l i e r , eggs may need a time period of freshwater exposure that i s s u f f i c i e n t l y long to ree s t a b l i s h t h e i r ' i n t e r i o r m i l i e u ' to preferred l e v e l s . Not u n t i l this requirement i s met w i l l they be able to tolerate repeated exposures to saltwater without the occurrence of irreparable embryonic damage.  E f f e c t s of Saltwater on the F e r t i l i z a t i o n Process  Apparently, salmon that spawn i n the i n t e r t i d a l zone do so during ebb tides only. Therefore, actual gamete deposition does not occur during times of saltwater inundation. Although I have no evidence to contend this statement, personal observation of chum salmon i n Carnation Creek indicates deposition of gametes during a flood tide to be a p o s s i b i l i t y . Therefore, I  100  was i n t e r e s t e d i n t e s t i n g whether or not the presence of s a l i n e water would i n h i b i t the f e r t i l i z a t i o n process. I f so, what s a l i n i t i e s were preventive and what stages of the process were affected?  Q u a n t i t a t i v e and q u a l i t a t i v e measures of sperm a c t i v i t y showed that a c t i v i t y was most prolonged and vigorous i n s a l i n i t i e s ranging from 5 - 1 0 ° / 0 0 . S i m i l a r r e s u l t s were summarized by Scott and Baynes (1980) i n a review on sperm b i o l o g y . The cause was speculated to be the r e s u l t of reduced osmotic s t r e s s i n low s a l i n i t y water compared to freshwater. In c o n t r a s t , n e g l i g i b l e motility ( «  1%) was observed at 15°/oo- The requirements f o r i n i t i a t i o n of  sperm m o t i l i t y are w e l l e s t a b l i s h e d ; the most important i s a decrease i n K i o n concentration below 2-5 m i l l i m o l e s / 1 (mM)  (Morisawa 1987). F u l l strength  seawater (30-33°/ 0 0 s a l i n i t y ) t y p i c a l l y has a K 9-13 mM  +  Na  concentration of about  ( B i d w e l l and Spotte 1985, Morisawa 1987) which i n 1 5 % 0  water would be reduced by about h a l f to 4-6 mM. +  i o n s , s l i g h t l y higher l e v e l s of K  +  +  salinity  Further, i n the presence of  ion are required to i n h i b i t sperm  m o t i l i t y (Baynes et a l . 1981). Therefore, i t i s p o s s i b l e that i n h i b i t i o n of m o t i l i t y at 15°/oo  w a s  the r e s u l t of elevated K  a c t i v a t i o n t h r e s h o l d . The f a c t that the K  +  +  ion l e v e l s above the  i o n concentration of 1 5 ° / 0 0  was  close to the i n h i b i t i n g concentration may e x p l a i n the v a r i a b l e observations of sperm m o t i l i t y at t h i s s a l i n i t y (e.g., general i m m o t i l i t y w i t h occasional bursts of a c t i v i t y by i n d i v i d u a l sperm c e l l s ) .  A comparison of the three responses of sperm m o t i l i t y , sperm v i a b i l i t y , and combined egg and sperm v i a b i l i t y to various s a l i n i t i e s ( F i g . 18), showed  101  F i g . 18. Summary of the experimental r e s u l t s obtained from t e s t i n g the e f f e c t s of various ambient concentrations of saltwater on the f e r t i l i z a t i o n process. The s o l i d c i r c l e s and squares represent the f e r t i l i z a t i o n success (%FS) measured i n the combined egg and sperm, and sperm v i a b i l i t y experiments respectively ( F i g s . 15 and 16). The s o l i d t r i a n g l e s represent the duration of sperm m o t i l i t y measured i n the sperm m o t i l i t y experiment ( F i g . 14).  that sperm m o t i l i t y was a reasonable i n d i c a t o r of f e r t i l i z a t i o n success. Generally t h i s r e l a t i o n s h i p i s w e l l established Moccia and M u n k i t t r i c k  (Scott and Baynes 1980,  1987), but since m o t i l i t y and f e r t i l i t y reside i n  separate parts of the spermatozoan i t i s not a simple r e l a t i o n s h i p  (Scott  and Baynes 1980). I n t h i s study v i a b i l i t y of sperm was l e s s s e n s i t i v e to s a l i n i t y than would have been p r e d i c t e d from the m o t i l i t y t e s t s . For example, the s l i g h t l y shorter duration and l e s s v i g o r observed i n the 0 ° / 0 0 was o f no consequence to sperm v i a b i l i t y ( F i g . 18) (at l e a s t not under the t e s t conditions used). Further, m o t i l i t y measurements i n d i c a t e d no m o t i l i t y at 15°/oo whereas i n a c t u a l i t y up to 53.5% of the 50 t e s t eggs i n the sperm v i a b i l i t y t e s t s were f e r t i l i z e d ( F i g . 18).  The procedure used to t e s t sperm m o t i l i t y i n t h i s study may have produced ambiguous r e s u l t s . I t i s not c l e a r i n the l i t e r a t u r e whether measurement of sperm m o t i l i t y i s l i m i t e d when tested under a microscope s l i d e c o v e r s l i p . Terner and Korsch (1963) reported that trout sperm m o t i l i t y was l i m i t e d to 30 s under a c o v e r s l i p , whereas the same sperm a c t i v a t e d i n an open v e s s e l remained motile f o r at l e a s t h a l f an hour. However, Morisawa et a l . (1983) tested sperm m o t i l i t y of rainbow trout sperm on a microscope s l i d e without a c o v e r s l i p , and measured maximum durations of m o t i l i t y that were no greater than 25 s. This value i s s i m i l a r to measurements observed i n t h i s and other studies that have used c o v e r s l i p s (Baynes e t a l . 1981, R i e n i e t s and M i l l a r d 1987). Thus, t e s t i n g of sperm m o t i l i t y on a microscope s l i d e w i t h a c o v e r s l i p appears to be a r o u t i n e l y used technique and at l e a s t , produces r e s u l t s that are comparable to other studies u t i l i z i n g t h i s same technique.  103  In a review on salmonid spermatozoa, Scott and Baynes (1980) indicated that the duration of sperm m o t i l i t y varied considerably between d i f f e r e n t studies i n the l i t e r a t u r e . They concluded that although some of these d i s p a r i t i e s were due to d i f f e r e n t c r i t e r i a f o r the assessment of m o t i l i t y , the majority were due to differences i n the techniques used to assess m o t i l i t y . This suggests that the measurements of duration and vigor of m o t i l i t y conducted i n this study are comparable only to measurements within t h i s study and others using similar experimental procedures. But, i n treatments where reduced or n e g l i g i b l e m o t i l i t i e s were observed i n apparent response to test conditions, the responses probably were not confounded by procedural e f f e c t s . These observations should remain v a l i d and be comparable with other studies. In general however, comparison of r e s u l t s between studies requires a standardized method to minimize procedural b i a s e s .  Considering the general lack of measurable m o t i l i t y i n 1 5 ° / 0 0 , the FS rate of 53.5% was s u r p r i s i n g l y high ( F i g . 18). This suggests that either occasionally active sperm ( «  1%) observed i n the m o t i l i t y tests at this  s a l i n i t y were s u f f i c i e n t to f e r t i l i z e the 50 or so eggs tested i n each r e p l i c a t e o r , those sperm exhibiting vibratory movements were s t i l l capable of f e r t i l i z i n g eggs. Ginzburg (1968) stated that sperm exhibiting wavering movements a f t e r the i n i t i a l period of forward swimming a c t i v i t y , were no longer v i a b l e . These wavering movements may be s i m i l a r to the vibratory movements seen i n this study at the 15°/oo exposure. I f so, one would expect a s i m i l a r response of n o n - v i a b i l i t y . However, Stoss et a l . (1978) found that sperm with low l e v e l s of m o t i l i t y (1-10% of f u l l y motile samples) retained high v i a b i l i t y . I t i s not t o t a l l y clear whether or not immotile sperm are  104  v i a b l e . I f they are, the question remains how they reach the egg c e l l to complete the f e r t i l i z a t i o n process.  Rockwell (1956) reported good f e r t i l i z a t i o n success ( > 70%) i n s a l i n i t i e s up to 18°/oo with pink salmon and noted some f e r t i l i z a t i o n success i n s a l i n i t i e s as high as 30°/oo (about 20%). Ginzburg (1968) however, reported s u c c e s s f u l f e r t i l i z a t i o n of salmonid eggs i n s a l i n i t i e s no higher than 13°/oo- The l a t t e r r e s u l t s are s i m i l a r to those observed here. The d i f f e r e n c e between these r e s u l t s and Rockwell's (1956) suggest that e i t h e r considerable i n t e r s p e c i f i c differences e x i s t w i t h i n the salmonids as a group or that assessment techniques were so d i f f e r e n t as to render the studies incomparable.  Results from the combined egg and sperm v i a b i l i t y experiment suggest that there i s an added i n h i b i t o r y e f f e c t of saltwater on the egg or f e r t i l i z a t i o n process as a whole, i n a d d i t i o n to the e f f e c t on the sperm ( F i g . 18). F e r t i l i z a t i o n  success at 15°/oo  w a s  l°wer i n the combined v i a b i l i t y  experiment (12.5-35.0%) i n comparison to the sperm v i a b i l i t y  experiment  (53.5%). In the sperm v i a b i l i t y experiment, sperm was premixed i n the t e s t s a l i n i t y p r i o r to adding i t to the eggs, whereas i n the combined egg and sperm experiment the gametes were added simultaneously to the t e s t s a l i n i t y . The l a t t e r experiment simulated most c l o s e l y the n a t u r a l spawning process and i n d i c a t e d that the presence of s a l t w a t e r , at s a l i n i t i e s greater than 10°/oo> probably would begin to i n h i b i t successful f e r t i l i z a t i o n i n a n a t u r a l s i t u a t i o n . Although a s a l i n i t y of 2 0 ° / 0 0 was not tested the observed trend suggests that f e r t i l i z a t i o n success would be n e g l i g i b l e at t h i s concentration. The f a c t that v a r i a b i l i t y between r e p l i c a t e s was not h i g h ,  105  suggests that the i n h i b i t o r y mechanisra(s) i s a consistent one. However, the design of these experiments d i d not provide any f u r t h e r i n s i g h t i n t o what that mechanism(s) might be.  I f eggs i n the n a t u r a l environment were f e r t i l i z e d under s a l i n e c o n d i t i o n s , p o t e n t i a l l y they could be subjected to these conditions f o r 4 h or more a f t e r f e r t i l i z a t i o n . Experiments simulating t h i s s i t u a t i o n i n d i c a t e d that the duration of saltwater exposure a f t e r f e r t i l i z a t i o n had no a d d i t i o n a l negative e f f e c t on FS, above and beyond that of the i n i t i a l exposure ( i . e . , the e f f e c t of the t e s t s a l i n i t y a f t e r a 1 min exposure). One exception was noted however, eggs maintained i n 1 5 ° / 0 0 f o r 240 min (4 h) experienced s l i g h t l y lower FS than the other exposure times at the same s a l i n i t y . In g e n e r a l , these r e s u l t s i n d i c a t e d that eggs f e r t i l i z e d i n the w i l d under s a l i n e conditions probably would not s u f f e r f u r t h e r d e l e t e r i o u s e f f e c t s from saltwater exposure during the remainder of the high t i d e c y c l e . However, t h i s conclusion does not take into account the p o s s i b i l i t y of the ambient s a l i n i t y increasing a f t e r the i n i t i a l time of f e r t i l i z a t i o n . Although the experiments i n t h i s study d i d not t e s t t h i s scenario, the response of eggs and sperm to saltwater during the f e r t i l i z a t i o n process suggests that no a d d i t i o n a l e f f e c t would occur with respect to FS.  F i n a l weights of the eggs from the combined v i a b i l i t y  experiment  suggested that the water hardening process may have been a f f e c t e d by prolonged exposure (4 h) to intermediate s a l i n i t i e s . Eggs maintained i n 12.5 and 1 5 % o  f o r  2 4 0  min a f t e r f e r t i l i z a t i o n had f i n a l weights that were n o t i c e a b l y lower but not s t a t i s t i c a l l y d i f f e r e n t , compared to (1) eggs maintained f o r a s i m i l a r time period i n lower s a l i n i t i e s 106  (0-10°/00) and (2)  eggs kept i n s i m i l a r s a l i n i t i e s f o r only 15 min (Fig. the  17). I f impairment of  water imbibation mechanism was r e a l , i t may have r e s u l t e d from (1)  permanent reduced osmotic a c t i v i t y of the p e r i v i t e l l i n e c o l l o i d s o r , (2) a p o s s i b l e chemical f i x i n g of the external egg membrane ( Z o t i n 1958, Kobayashi 1982). A c t u a l hardening of the egg membrane while the egg remained f l a c c i d i n saltwater would p h y s i c a l l y prevent the egg from imbibing water and s w e l l i n g upon r e t u r n to freshwater.  In c o n c l u s i o n , r e s u l t s obtained here i n d i c a t e that ambient s a l i n i t i e s greater than about 1 0 ° / 0 0 would s i g n i f i c a n t l y reduce f e r t i l i z a t i o n success and could a f f e c t the a b i l i t y of eggs to water harden normally, even a f t e r they r e t u r n to freshwater. This information i n d i c a t e s that eggs spawned during high or f l o o d i n g t i d e s and i n the presence of the s a l t wedge, would experience low to n i l s u r v i v a l as a r e s u l t of adverse e f f e c t s from the ambient seawater. As mentioned e a r l i e r (Chap. 1 ) , s a l i n i t i e s on the i n t e r t i d a l spawning grounds during f l o o d t i d e s often are as high as 20-30°/oo and only under s p e c i f i c conditions do they remain below 1 0 ° / 0 0 .  107  SUMMARY - Chapter I I  S a l i n i t y Tolerance of Salmon Eggs  1.1  Chum salmon eggs survived to the a l e v i n stage (8 days post-hatching,  92.6-96.0%) and developed normally when exposed to s a l i n i t i e s of 20°/oo l e s s f o r 4 h/day o r , s a l i n i t i e s of 15°/oo  o r  o r  l e s s f o r 8 h/day. Eggs exposed  to 2 0 ° / 0 0 f o r 8 h/day r e s u l t e d i n s i g n i f i c a n t l y lower s u r v i v a l (41.0-48.0%) (P < 0.001). Most of the eggs that d i d not survive i n t h i s treatment were r  developed abnormally. I n eggs exposed to 30°/oo f ° e i t h e r 4 or 8 h/day, no s u r v i v a l and only very e a r l y embryonic development was observed.  1.2  Constant exposure to 5°/oo produced no adverse e f f e c t s on s u r v i v a l  (86.5-97.3%) and was not s i g n i f i c a n t l y d i f f e r e n t than the c o n t r o l s (P > 0.05). I n c o n t r a s t , eggs incubated i n 10 or 1 5 ° / 0 0 showed advanced development ( w e l l eyed stage) but d i d not survive to h a t c h i n g . Many of the embryos appeared normal except f o r abnormally developed y o l k sacs. These abnormalities appeared to be the r e s u l t of incomplete epiboly (blastoderm overgrowth of the yolk s a c ) . Embryonic development stopped a t an e a r l y stage (morula stage) i n eggs exposed to 2 0 ° / 0 0 constantly and was n e g l i g i b l e i n eggs exposed to 3 0 ° / 0 0 .  1.3  The i n t e r t i d a l , B-group eggs showed markedly lower s u r v i v a l s i n 0 ° / 0 0 nt n e  (control) than 5°/oo *-  4 and 8 h exposures. On the whole, s u r v i v a l of  t h i s group was lower than the F-group. Since a l l of the B-group eggs came from the same pooled source, i t was speculated that they were not t o t a l l y normal. The r e s u l t s of t h i s abnormality were most noticeable i n the  108  freshwater treatment; although the nature of t h i s abnormality was not established.  1.4  The i n t e r m i t t e n t s a l i n i t y tolerance of chum salmon eggs i n t h i s study  was d i f f e r e n t than the tolerance of pink salmon eggs t e s t e d by B a i l e y (1966). Pink salmon embryos and a l e v i n s appeared to be more t o l e r a n t to high s a l i n i t i e s than chum salmon. Evidence from these studies and others (Rockwell 1956, Weisbart 1968) suggested that the d i f f e r e n c e was an i n t e r s p e c i f i c one. However, since some p o t e n t i a l l y s i g n i f i c a n t procedural v a r i a t i o n s e x i s t e d between the s t u d i e s , i t was d i f f i c u l t to make d i r e c t comparisons.  1.5  Information from the l i t e r a t u r e on the d i s t r i b u t i o n of chum and pink  salmon on the i n t e r t i d a l spawning grounds supported the p o s s i b i l i t y that the i n t e r s p e c i f i c d i f f e r e n c e s seen i n the laboratory were r e a l . Pink salmon t y p i c a l l y have a lower l i m i t of spawning i n the i n t e r t i d a l zone (Thornsteinson e t a l . 1971). As a r e s u l t , pink salmon eggs deposited i n i n t e r t i d a l areas are l i k e l y to receive more frequent, prolonged, and concentrated saltwater exposures than chum salmon eggs deposited i n i n t e r t i d a l areas. Perhaps the i n t e r s p e c i f i c d i f f e r e n c e s i n s a l i n i t y tolerance noted i n t h i s study were r e f l e c t i o n s of d i f f e r e n c e s i n the l i f e h i s t o r i e s of these two species.  1.6  Data from t h i s study and B a i l e y (1966) i n d i c a t e d that a regular  freshwater i n t e r l u d e between saltwater exposures was a key f a c t o r i n s a l i n i t y tolerance of chum and pink salmon eggs. Embryos and a l e v i n s were able to t o l e r a t e exposure to higher s a l i n i t i e s when provided w i t h regular  109  freshwater r e p r i e v a l s . I t was speculated that the duration of freshwater exposure needed to be s u f f i c i e n t l y long to allow f o r re-establishment of the organism's i n t e r i o r m i l i e u . Once t h i s requirement i s met, embryos and a l e v i n s should be able to withstand repeated exposures to high s a l i n i t i e s u n t i l adult r e g u l a t i o n mechanisms develop and become f u n c t i o n a l .  1.7  P o s s i b l e osmo- and iono-regulatory mechanisms i n salmon embryos and  a l e v i n s are; ' t i g h t ' embryonic c e l l plasma membranes and c h l o r i d e c e l l s e c r e t i o n . H i s t o l o g i c a l and p h y s i o l o g i c a l evidence from rainbow t r o u t embryos and a l e v i n s i n d i c a t e s the presence of c h l o r i d e c e l l s . I n l i g h t of t h i s information and p h y s i o l o g i c a l evidence from chum and pink salmon, the presence of c h l o r i d e c e l l s also i s probable i n chum and pink, e s p e c i a l l y i n advanced embryos and a l e v i n s .  1.8  The hypothesis that eggs obtained from i n t e r t i d a l spawning parents (B-  group) would have b e t t e r s a l i n i t y tolerance that those obtained from freshwater upstream parents (F-group) was not tested as r i g o r o u s l y as i n i t i a l l y planned. Due to l o g i s t i c a l problems i n c o l l e c t i n g the s p e c i f i c f i s h types, I was not confident that I had obtained eggs from a c t u a l i n t e r t i d a l spawners. Further, i n e x p l i c a b l e differences i n ' c o n t r o l ' egg s u r v i v a l s between the two groups of eggs made comparisons inappropriate. However, aside from these problems, no i n d i c a t i o n s were found that suggested d i f f e r e n t i a l s a l i n i t y tolerance between the two groups of eggs t e s t e d .  110  E f f e c t s of S a l i n i t y on the F e r t i l i z a t i o n Process  2.1  Sperm a c t i v i t y and m o t i l i t y was highest and most vigorous i n s a l i n i t i e s  ranging from 5-10°/oo- Although i t was not s i g n i f i c a n t  statistically  (P > 0.05), a s l i g h t reduction i n duration and v i g o r of m o t i l i t y was noted i n 0°/oo- *  n  12.5°/oo a s t a t i s t i c a l l y s i g n i f i c a n t reduction i n m o t i l i t y was  noted whereas no measurable m o t i l i t y was observed i n 15°/oo- Applying  a  subjective measure of i m m o t i l i t y , as 95% i m m o t i l i t y , misrepresented observations i n t h i s highest s a l i n i t y since occasional spermatozoa e x h i b i t e d bursts of forward movement. I t was suggested that i n h i b i t i o n of m o t i l i t y was due to the elevated l e v e l s of K  +  i o n i n the s a l t w a t e r . I n 1 5 ° / 0 0 s a l i n i t y , the  concentration of t h i s i o n was near the e s t a b l i s h e d l i m i t f o r prevention of m o t i l i t y ( > 2-5 mM) (Morisawa 1987).  2.2  A c t u a l sperm v i a b i l i t y was underestimated using sperm m o t i l i t y as a  p r e d i c t o r of v i a b i l i t y . No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n v i a b i l i t y , measured as f e r t i l i z a t i o n success (FS), were observed i n the lower s a l i n i t i e s 0-10%o (80.0-100.0%) (P > 0.05). A s t a t i s t i c a l l y n o n s i g n i f i c a n t decrease i n FS occurred i n 12.5°/oo (75.5%). In 15°/oo s a l i n i t y , FS was s i g n i f i c a n t l y lower than at 12.5°/oo (P < 0.05), but t h i s l e v e l was s u r p r i s i n g l y high (53.5%) i n view of the assessed lack of m o t i l i t y i n the f i r s t experiment. E i t h e r the o c c a s i o n a l l y a c t i v e sperm observed at 15°/oo ^  n t n e  m o t i l i t y t e s t s were s u f f i c i e n t to f e r t i l i z e the low number of  t e s t eggs o r , immotile sperm e x h i b i t i n g only v i b r a t o r y movements were capable of gaining access to the eggs v i a the raicropyle to carry out fertilization.  Ill  2.3  Measurements of FS of sperm and eggs placed i n various  salinities  simultaneously, suggested that d i l u t e saltwater had an added e f f e c t on the egg, i n addition to i t s e f f e c t on the sperm. The responses e s s e n t i a l l y were the same as i n the previous sperm v i a b i l i t y experiment (Summary 2.2), i n a l l s a l i n i t i e s except 15°/oo-  I n  this concentration of saltwater, FS was  lower  (12.0-35.0%) compared to the previous experiment (53.5%), where the sperm f i r s t was  2.4  d i l u t e d i n the test s a l i n i t y before being added to the eggs.  Eggs maintained i n t h e i r respective test s a l i n i t i e s for various time  i n t e r v a l s (1, 15, 60, and 240 min) result i n differential  following addition of sperm, did not  rates of FS. Eggs kept i n 1 5 ° / 0 0 for 240 min showed  some signs of reduced FS compared to those kept for only 1, 15, or 60 min i n the same s a l i n i t y , but the results were not conclusive.  2.5  Eggs kept i n 12.5  and 15°/oo s a l i n i t y water for 240 min showed signs of  lower f i n a l weights once they were returned to freshwater. Due  to the  v a r i a b i l i t y between eggs, no s t a t i s t i c a l differences were observed. I t i s possible that either (1) p e r i v i t e l l i n e  f l u i d c o l l o i d s were affected by  prolonged exposure (240 min) to intermediate s a l i n i t i e s  (12.5 and 15°/oo)  or  >  (2) short-term chemical changes i n the egg membrane as a r e s u l t of water hardening, prevented the egg from imbibing water normally upon return to freshwater.  2.6  In conclusion, experiments assessing the e f f e c t of saltwater on the  f e r t i l i z a t i o n process indicated that problems w i l l occur i n s a l i n i t i e s greater than 10°/oo- Not s u r p r i s i n g l y , sperm appeared to be more susceptible than the much larger eggs; yet the data suggested adverse e f f e c t s of  112  s a l i n i t y on the egg as w e l l . I t was concluded that i n t e r t i d a l chum salmon spawning i n a stream during times of saltwater inundation, would experience low to n i l f e r t i l i z a t i o n success. Except for the upper i n t e r t i d a l zone, i t i s not common to observe s a l t wedge s a l i n i t i e s as low as 10°/oo during flood t i d e s , at least not i n Carnation Creek.  113  GENERAL DISCUSSION F i e l d and Laboratory Studies Integrated  I speculated i n the f i e l d study of t h i s thesis that eggs located i n the lower i n t e r t i d a l zone of Carnation Creek could u t i l i z e t i d a l saltwater as an a l t e r n a t e source of oxygen during times when i n t r a g r a v e l l e v e l s were low. On a f l o o d t i d e , gravel pore water i s displaced as the denser seawater enters the stream. As a r e s u l t , water surrounding the eggs i s exchanged due to the d i f f e r e n t d e n s i t i e s . Measurements of PVF osmolality of i n d i v i d u a l eggs i n d i c a t e d that a change i n ambient s a l i n i t y produced an equal change i n osmotic pressure of the PVF i n s i d e the egg. Thus, during saltwater exposure on a f l o o d t i d e , an i n t e r t i d a l egg experiences an exchange of the surrounding water as w e l l as an exchange i n the i o n and water f r a c t i o n of the PVF bathing the embryo. The opposite process occurs when the t i d a l water i s flushed out of the gravel on an ebb t i d e . S a l i n i t y of the PVF w i l l change w i t h the i n t r a g r a v e l water as i t i s replaced by f r e s h stream water. Thus, density dependent water exchange functions on two l e v e l s ; i n the i n t r a g r a v e l environment surrounding the egg and i n the p e r i v i t e l l i n e space enveloping the embryo.  I n t r a g r a v e l flow i n streams l a r g e l y i s due to g r a v i t y fed water flow produced by the volume of water passing through the stream (McNeil 1962, Vaux 1962). The degree of i n t r a g r a v e l interchange depends on c h a r a c t e r i s t i c s of the gravel and topography of the stream bed (Vaux 1962, 1968). I n c o n t r a s t , density dependent water exchange i s not dependent on the volume of stream flow and i s influenced l e s s by p h y s i c a l c h a r a c t e r i s t i c s of the  114  stream. Since i n t e r t i d a l areas t y p i c a l l y have increased f i n e s i n the gravel (McNeil and A h n e l l 1964, H e l l e et a l . 1970), low permeability gravel could produce i n t r a g r a v e l exchange problems. However, water interchange  resulting  from density d i f f e r e n c e s probably i s l e s s s u s c e p t i b l e • t o low gravel p e r m e a b i l i t i e s , than flow dependent i n t r a g r a v e l interchange of noni n t e r t i d a l areas. Thus, embryos developing i n productive portions of the i n t e r t i d a l zone may be l e s s prone to low i n t r a g r a v e l d i s s o l v e d oxygen problems, regardless of whether these problems are due to low stream flow or reduced gravel q u a l i t y . Results from t h i s study provide p r e l i m i n a r y support f o r t h i s speculation but f u r t h e r i n v e s t i g a t i o n i s required to provide conclusive evidence.  D i r e c t comparison of the s a l i n i t y tolerance of chum salmon eggs i n the f i e l d and laboratory components of t h i s study i s not completely  appropriate.  I f one temporarily ignores a l l of the a d d i t i o n a l environmental f a c t o r s a f f e c t i n g eggs i n the stream, eggs i n the laboratory received more extreme s a l i n i t y exposures than those i n the f i e l d . Eggs i n the laboratory were exposed abruptly to the exact same s a l i n i t y and the same duration d a i l y f o r the e n t i r e experiment (67 d ) . These incubation conditions were more severe than those at the -195 or -275 m egg implantation s i t e s , e s p e c i a l l y f o r laboratory eggs i n the 8 h/day exposures at the higher s a l i n i t i e s (20-30°/ 00 ). In the stream, frequency, d u r a t i o n , and s a l i n i t y of exposure v a r i e d continuously and depended upon the i n t e r - r e l a t i o n s h i p s between t i d e h e i g h t , stream discharge, and egg l o c a t i o n . However, i f one acknowledges a l l of the other environmental f a c t o r s that ' i n stream' eggs were exposed t o , such as f l u c t u a t i o n s i n d i s s o l v e d oxygen concentration, v a r i a t i o n s i n stream flow, predators, e t c . , then the f i e l d conditions probably were more adverse.  115  The laboratory s e t t i n g provided an opportunity to c o n t r o l the confounding f a c t o r s and concentrate on a s p e c i f i c t e s t c o n d i t i o n , i n t h i s case s a l i n i t y . The s a l i n i t y tolerance t e s t s i n the laboratory i n d i c a t e that chum salmon eggs do not experience any adverse e f f e c t s from i n t e r m i t t e n t saltwater exposures to 1 5 ° / 0 0 s a l i n i t y or l e s s f o r 4 or 8 h per day, and to 2 0 ° / 0 0 f o r 4 h per day. Evidence from these r e s u l t s and the f i e l d study suggest that eggs can withstand higher s a l i n i t i e s , than those observed i n the laboratory study, i f the conditions are not permanent. Eggs i n the f i e l d were able to t o l e r a t e p e r i o d i c exposures up to 30°/oo did  D u t  these s a l i n i t i e s  not p e r s i s t f o r extended periods of time.  S a l i n i t y tolerance information, summarized from t h i s study and others, suggests that the duration of exposure to saltwater i s more c r i t i c a l than the absolute concentration. Maximum s a l i n i t i e s of the s a l t wedge i n Carnation Creek were measured at 30°/oo- I t i s u n l i k e l y that s a l i n i t i e s would exceed 3 3 ° / 0 0 f o r open c o a s t a l areas and 3 0 ° / 0 0 f o r inner c o a s t a l areas (Waldichuk 1956, P i c k a r d 1961). Evidence from Rockwell (1956), B a i l e y (1966), Weisbart (1968), as w e l l as t h i s study i n d i c a t e s that short duration exposure to s a l i n i t i e s as high as 3 0 ° / 0 0 do not s i g n i f i c a n t l y a f f e c t the s u r v i v a l of chum or pink salmon eggs. I t seems that a key f a c t o r i n the s a l i n i t y tolerance of eggs, under an a l t e r n a t i n g s a l t - and freshwater exposure, i s a s u f f i c i e n t l y long period (more than 8 h, B a i l e y 1966) of freshwater r e p r i e v a l between saltwater exposures. I f t h i s requirement i s f u l f i l l e d , these two species have the a b i l i t y to regulate t h e i r i n t e r n a l osmotic and i o n i c environments w e l l enough to provide short-term tolerance to f l u c t u a t i o n s i n ambient s a l i n i t y throughout development. This does not  116  h o l d true f o r the other three North American species o f P a c i f i c salmon (Weisbart 1968). Chum and pink salmon appear to have an added p h y s i o l o g i c a l p o t e n t i a l which allows them to spawn s u c c e s s f u l l y i n the lower reaches o f r i v e r s and streams that experience t i d a l i n f l u e n c e .  Geographically, chum and pink salmon are the most widely d i s t r i b u t e d species o f Oncorhynchus (Bakkala 1970, Scott and Crossman 1973). Pink salmon spawn i n r i v e r s and streams ranging from North Korea and North Japan, (Hokkaido, about 40°N l a t . ) , around the P a c i f i c Rim to C a l i f o r n i a , USA, (about 38°N l a t . ) (Takagi et a l . 1981). They also extend i n t o r i v e r s on the northern coasts o f the USSR, USA, and Canada. Chum salmon share a s i m i l a r d i s t r i b u t i o n except they extend further south on the asian side o f the P a c i f i c Ocean to south Japan (Kyushu, approximately 33°N l a t i t u d e , Sano 1967). Atkinson e t a l . (1967) stated that pink and chum salmon could survive i n streams subjected t o extreme floods and other p h y s i c a l disturbances. Perhaps the abundance o f these f i s h i s r e l a t e d to t h e i r a b i l i t y to adapt to or t o l e r a t e a wide v a r i e t y of environmental conditions; one of which i s the incubation conditions o f the i n t e r t i d a l zone. Other species of P a c i f i c salmon appear to f i n d the i n t e r t i d a l zone unacceptable as spawning h a b i t a t . I speculate that the broad d i s t r i b u t i o n o f chum and pink salmon i s r e l a t e d to t h i s a b i l i t y to u t i l i z e the i n t e r t i d a l zone as a productive breeding ground.  117  REFERENCES  A l d e r d i c e , D.F. 1987. Osmotic and i o n i c r e g u l a t i o n i n t e l e o s t l a r v a e . I n : F i s h P h y s i o l o g y . (W.S. Hoar and D . J . R a n d a l l , p a r t A , p p . 1 6 3 - 2 5 1 . A c a d e m i c P r e s s . 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B i o l o g i c a l c h a r a c t e r i s t i c s of i n t e r t i d a l and freshwater spawning pink salmon a t Olsen Creek, Prince W i l l i a m Sound, A l a s k a , 1962-63. U.S. F i s h . W i l d l . Serv. Spec. S c i . Rep. F i s h . No. 602. H e l l e , J.H., R.S. Williamson, and J.E. B a i l e y . 1964. I n t e r t i d a l ecology and l i f e h i s t o r y of pink salmon a t Olsen Creek, Prince W i l l i a m Sound Alaska. U.S. F i s h W i l d l . Serv. Spec. S c i . Rep. F i s h . No. 483. H o l l i d a y , F.G.T. and J.H.S. B l a x t e r . 1960. The e f f e c t s o f s a l i n i t y on the developing eggs and larvae of the h e r r i n g . J . Mar. B i o l . Assn. U.K. 39:591-603. H o l l i d a y , F.G.T. 1965. Osmoregulation i n marine t e l e o s t eggs and larvae. C a l i f . Coop. Oceanic F i s h . Invest. 10:89-95. Hunter, J.G. 1959. S u r v i v a l and production of pink and chum salmon i n a c o a s t a l stream. J . F i s h . Res. Bd. Can. 16(6):835-886. 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Inshore-marine and freshwater l i f e h i s t o r y of the pink salmon, Oneorhvnchus gorbuscha (Walbaum), and the chum salmon, C\_ keta (Walbaum), i n Prince W i l l i a m Sound, A l a s k a . Ph.D. t h e s i s , U n i v e r s i t y of Kentucky, L o u i s v i l l e , KY, USA. 300p. Kobayashi, W. 1982. The f i n e structure and amino a c i d composition of the envelope o f the chum salmon egg. J . Fac. S c i . Hokkaido Univ. Ser. 6,23:1-12. K o g l , D.R. 1965. Springs and groundwater as f a c t o r s a f f e c t i n g s u r v i v a l of chum salmon spawn i n a subarctic stream. M.Sc. t h e s i s , U n i v e r s i t y of A l a s k a , AK, USA. 59p. K o s k i , K.V. 1966. The s u r v i v a l of coho salmon (Oncorhynchus k i s u t c h ) from egg d e p o s i t i o n to emergence i n three oregon c o a s t a l streams. M.Sc. t h e s i s , Oregon State U n i v e r s i t y , OR, USA. Leatherland, J.F. and L. L i n . 1975. 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F i s h . Res. Bd. Can. 15(5):1103-1126. Wilcox, K.W., J . Stoss, and E.M. Donaldson. 1984. Broken eggs as a cause of i n f e r t i l i t y of coho salmon gametes. Aquacult. 40:77-87. W i l k i n s o n , L. 1987. SYSTAT: The system f o r s t a t i s t i c s . Systat I n c . , Evanston, I L , USA. Zadunaisky, J.A. 1984. The c h l o r i d e c e l l : The a c t i v e transport of chloride and the p a r a c e l l u l a r pathways. In: F i s h Physiology, (W.S. Hoar and D.J. R a n d a l l , eds.). V o l . 10, part B, pp. 129-176. Academic Press, New York, NY, USA. Z o t i n , A . I . 1958. The mechanism of hardening of the salmonid egg membrane a f t e r f e r t i l i z a t i o n or spontaneous a c t i v a t i o n . J . Embryol. Exp. Morphol. 6:546-568.  126  APPENDIX 1 Modelling Changes i n P e r i v i t e l l i n e F l u i d Osmolality:  General Growth Model - Equation. Parameters, and Residuals (see text i n Materials and Methods, and Results sections of Chapter 2 for details) Test Condition: Eggs transferred from 0°/oo  t  o  20°/oo s a l i n i t y water  Equation: Y(t)  = [35.79 2  09  + (600.69  2  0 9  - 35.79  2 09  )  1 - e -°-"<t-°> 1  .  e  Parameters: Low X = 0.00 High X = 180.00 Y= 35.79 Y = 600.69 a = 0.10 b = 2.09 x  2  x 2  No. of records = 59 Minimized SS = 15603.55  X Y observed Y predicted (min.) (mmol/kg) (mmol/kg) 0..00 0,.00 0,.00 0..00 0.,00 0..00 0,.00 0..00 0..00 0,.00 0..00 0..00 0..00 0..00 0,.00  43, .00 39,.00 40. .00 35,.00 35,.00 37,.00 37..00 40, .00 43, .00 41..00 40. .00 25,.00 31,.00 33,.00 27,.00  35..79 35..79 35..79 35..79 35..79 35..79 35..79 35..79 35..79 35..79 35..79 35;.79 35..79 35..79 35,.79  residuals (mmol/kg) 7..215 3..215 4..215 -0. .785 -0. .785 1..215 1..215 4..215 7..215 5..215 4..215 -10. .785 -4. .785 -2. .785 -8. .785  127  -0.10(180.0-0)  ]i/2-09  X Y observed Y predicted (min.) (mmol/kg) (mmol/kg)  0.00 0.00 0.00 0.30 0.30 0, .40 0.40 0,.90 1.90 2.00 2.20 2.70 4,.70 4,.90 5, .00 5, .00 5. .00 6. .20 6, .90 7. .00 7. .60 7. .80 9. .20 9, .50 11..00 11..80 12..90 13..80 15..00 15..20 15..90 16..90 18..60 22..00 22..30 24..80 26..70 31..80 39..90 50..50 60..10 80..20 125..00 180.,00  31.00 30.00 30.00 149.00 123.00 154,.00 106.00 176,.00 306.00 283,.00 243,.00 284,.00 385.00 344,.00 390,.00 365,.00 383,.00 399,.00 462,.00 445,.00 438..00 436,.00 461..00 470,.00 525..00 538..00 504..00 545..00 550..00 531..00 549..00 573..00 531..00 562..00 569.,00 558..00 571..00 594..00 588..00 590..00 598..00 602..00 601..00 613..00  35,.79 35.79 35.79 116,.54 116,.54 132,.08 132,.08 189,.07 262,.07 267,.86 278..90 303..74 377..70 383..56 386..41 386..41 386..41 417,.00 432..26 434..30 445..96 449..62 472..55 476..92 496..34 505..27 516..20 524..12 533..45 534..89 539..65 545..83 554..86 568..58 569.,57 576.,66 580..93 588.,96 595.,54 598.,93 600.,02 600.,61 600.,69 600. 69  residuals (mmol/kg)  -4.785 -5.785 -5,.785 32.459 6.459 21,.922 -26.078 -13,.073 43,.931 15,.137 -35.896 -19.740 7, .300 -39,.558 3, .593 -21,.407 -3,.407 -17,.998 29..745 10,.697 -7..962 -13..625 -11..553 -6..918 28..663 32..731 -12..196 20..882 16..548 -3..885 9. .351 27..172 -23.,864 -6..582 -0.,567 -18.,662 -9..930 5.,037 -7..542 -8.,930 -2..025 1..394 0..307 12..306  128  Test Condition:  Eggs transferred from 20°/0o  t o  °°/oo s a l i n i t y water  Equation: 1  9  1  Y(t) = [601.70" -* + (22.14' *  9  -1  9  (  - 601.70 -* )  1 - e" -°I  Parameters: Low X:= 0.00 High X2= 55.00 Yx= 601.70 Y2= 22.14 a -0.01 b = -1.49 No. of records = 36 Minimized SS = 17647.97  X (min.) 0..00 0,.00 0..00 0..00 0,.00 0,.00 0..00 0..00 0..20 0..30 2..30 3..00 5.,30 6..70 8..60 8..80 11..20 13..00 14..00 15..50 16..50 17..60 19..30 24..70 25..00 29..80  Y observed (mmol/kg) 612..00 609..00 598..00 611..00 602..00 601..00 613..00 602..00 415..00 456..00 252..00 235..00 100..00 114..00 78..00 50.,00 59..00 53.,00 40..00 39.,00 42..00 36.,00 43..00 33.,00 45..00 36.,00  Y predicted (mmol/kg) 601,.70 601,.70 601,.70 601,.70 601,.70 601 .70 601,.70 601,.70 487,.89 448,.29 197,.26 169..76 120..37 103..70 88..09 86..76 73..78 66..62 63..28 58..93 56,.38 53..85 50,.40 42..05 41..68 36,.48  residuals (mmol/kg) 10..303 7,.303 -3..697 9,.303 0..303 -0,.697 11..303 0,.303 -72..887 7,.707 54,.741 65..236 -20,.373 10..303 -10,,091 -36..759 -14.,783 -13.,619 -23..282 -19..929 -14..383 -17..852 -7..401 -9..053 3..324 -0..484  129  01)(t  "  0)  _ e -(-0.01)(55.0-0)  1  1  I ' -*  9  X Y observed Y predicted (min.) (mmol/kg) (mmol/kg)  32..00 36..00 39..00 43..00 44,.00 49..00 51..00 52..00 54..00 55..00  43..00 39..00 40..00 35..00 35..00 31..00 33..00 27..00 31..00 30..00  34..53 31..48 29,.53 27..27 26..76 24..45 23..63 23..24 22..50 22..14  residuals (mmol/kg)  8..469 7..520 10..473 7..730 8..242 6..548 9..368 3..758 8..504 7..861  130  

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