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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.), University 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 i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology, Resource Ecology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1989 :.=•:;-••/©: Erick Peter Groot, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^Th'yoL&Qy^ The University of British Columbia Vancouver, Canada Date Qrh 6. tqRCj  DE-6 (2/88) ABSTRACT I n t e r t i d a l s pawn ing o f chum sa lmon , Onco rhynchus k e t a . was i n v e s t i g a t e d by m e a s u r i n g egg s u r v i v a l , d e ve l opmen t , and s a l t w a t e r t o l e r a n c e unde r a c t u a l and s i m u l a t e d i n t e r t i d a l c o n d i t i o n s . I n t r a g r a v e l s a l i n i t y and t e m p e r a t u r e were m o n i t o r e d i n t h e i n t e r t i d a l zone o f C a r n a t i o n C r e e k , B r i t i s h C o l u m b i a , u s i n g s a l i n i t y and t e m p e r a t u r e p r o be s i m p l a n t e d i n t h e g r a v e l a t f i v e l o c a t i o n s , r a n g i n g f r om t he 1 .9 - t o 2 . 9 - m e t e r t i d e l e v e l . A r t i f i c i a l l y i m p l a n t e d egg s , p l a c e d a d j a c e n t t o t h e s e p r o b e s , were m o n i t o r e d t o d e t e r m i n e t h e e f f e c t o f o b s e r v e d i n t e r t i d a l c o n d i t i o n s on egg s u r v i v a l and d e v e l o pmen t . I n t r a g r a v e l o xygen and g r a v e l q u a l i t y a l s o were measu red a t t h e s e s t a t i o n s . S a l t w a t e r i n u n d a t i o n o f t h e g r a v e l s t r e ambed r e s u l t e d i n i n t r a g r a v e l s a l i n i t i e s as h i g h as 30°/oo> f o r d u r a t i o n s up t o 8 h , t w i c e d a i l y . Due t o r e g u l a r i n u n d a t i o n o f warmer s a l t w a t e r , egg deve l opmen t a t t h e l o w e r i n t e r t i d a l s i t e s 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 c o n t r o l s i t e s . S u r v i v a l i n t h e i n t e r t i d a l zone ranged, f r om ( 0 - 5 1 . 2 % ) . I n g e n e r a l , no n e g a t i v e e f f e c t s o f s a l t w a t e r e xpo su r e on egg s u r v i v a l were o b s e r v e d . I n s t e a d , egg s u r v i v a l was s t r o n g l y c o r r e l a t e d w i t h i n t r a g r a v e l o x ygen . I t i s s u g g e s t e d t h a t eggs i n t h e i n t e r t i d a l zone may b e n e f i t f r om t he i n t e r c h a n g e o f i n t r a g r a v e l w a t e r and t h e s ub sequen t added a v a i l a b i l i t y o f o x ygen , r e s u l t i n g f r om r e g u l a r i n u n d a t i o n o f s e awa t e r i n t o t h e s t r e a m b e d . L a b o r a t o r y e x p e r i m e n t s were d e s i g n e d t o t e s t s a l t w a t e r t o l e r a n c e o f eggs unde r s i m u l a t e d i n t e r t i d a l c o n d i t i o n s . When eggs were t r a n s f e r r e d t o o r f r om s a l t w a t e r , measurement o f 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 changes showed t h a t i i equilibration with the ambient medium occurred rapidly (15-25 min). This response was modelled and an equation was determined. Eggs were exposed daily to two intermittent exposure regimes (4 and 8 h) and one constant one (24 h) , at six different sali n i t i e s (0, 5, 10, 15, 20, and 30%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 in any of the 3 0 ° /0 0 salinity 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 effects. Eggs exposed to 2 0 ° /0 0 for 4 h also showed no adverse effects, whereas those in the 8 h exposure suffered about 55% mortality. In the constant exposure treatments only eggs in 5°/oo salinity and the control survived to the alevin stage (85-95%). In general, eggs in the higher sa 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 different treatments. Possible mechanisms of saltwater toxicity are discussed and i t is 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 testing sperm motility, sperm v i a b i l i t y and combined sperm and egg v i a b i l i t y in various sa l i n i t i e s (0, 5, 10, .12.5 and 15°/00) . In the sperm motility tests no differences were noted between individual males. However, more vigorous activity and longer periods of motility were observed in sa l i n i t i e s ranging from 5 - 1 0° /0 0 than sa l i n i t i e s of 0 and 1 2 . 5 ° /0 0. No measurable motility was observed in 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 in 12.5 and 15°/oo than was suggested by the motility measurements. High FS (90-95%) occurred in s a l i n i t i e s ranging from 0 - 1 0° /0 0 whereas significantly lower FS i i i occurred in 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 test. However, a lower FS in 15°/oo ^n t n e combined test suggested that saltwater had an added effect on the egg, in addition to i t s inhibitory effect on the sperm. It is concluded that eggs deposited by intertidal 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 v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS . x GENERAL INTRODUCTION 1 CHAPTER I - F i e l d Study 4 INTRODUCTION 4 MATERIALS and METHODS 9 Study Site Description x . 9 Environmental Conditions of the I n t e r t i d a l Zone 13 Intragravel S a l i n i t y and Temperature Measurements . . . . 13 Intragravel Oxygen Measurements 14 Gravel Quality Sampling 14 I n t e r t i d a l Chum Salmon Spawning Survey 16 Egg Capsule Implantation Experiment 17 Data Analysis 19 RESULTS 20 Environmental Conditions of the I n t e r t i d a l Zone . . 20 Intragravel S a l i n i t y 20 Intragravel Temperature 27 Intragravel Oxygen 28 Gravel Quality 30 In t e r t i d a l Salmon Spawning Dis t r i b u t i o n 30 Egg Capsule Implantation Experiment 31 Egg Survival Rates 31 Egg Development Rates 34 DISCUSSION 37 SUMMARY - Chapter I 52 CHAPTER I I - Laboratory Study 55 INTRODUCTION 55 MATERIALS and METHODS 59 S a l i n i t y Tolerance of Salmon Eggs Experiment 59 Gamete Collection and F e r t i l i z a t i o n 59 Incubation Equipment and Conditions 60 Experimental Design and Protocol 62 Sampling Procedures 64 Data Analysis . . . . . 66 P e r i v i t e l l i n e F l u i d Osmolality Tests 68 Measurement of P e r i v i t e l l i n e F l u i d Osmolality . . . . 68 v TABLE OF CONTENTS (cont'd) Modelling Changes i n PVF Osmolality 69 Effects Of S a l i n i t y On F e r t i l i z a t i o n Experiments 69 Gamete Collection 69 Experimental Design 69 Data Analysis 70 Sperm M o t i l i t y Tests 70 Sperm V i a b i l i t y Tests 71 Combined Egg and Sperm V i a b i l i t y Tests 71 RESULTS 74 S a l i n i t y Tolerance of Salmon Eggs 74 P e r i v i t e l l i n e F l u i d Osmolality 74 S a l i n i t y Tolerance and Egg Survival 74 Effects 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 81 Sperm M o t i l i t y 81 Sperm V i a b i l i t y 83 Combined Egg and Sperm V i a b i l i t y 85 DISCUSSION 88 S a l i n i t y Tolerance of Salmon Eggs 88 Effects of Saltwater on the F e r t i l i z a t i o n Process . . . . . . 100 SUMMARY - Chapter I I 108 S a l i n i t y Tolerance of Salmon Eggs 108 Effects 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 I l l GENERAL DISCUSSION 114 REFERENCES . 118 APPENDIX 1 127 Modelling Changes i n P e r i v i t e l l i n e F l u i d Osmolality . . 127 Equation, Parameters, and Residuals 127 Eggs transferred from 0 ° /0 0 to 2 0° /0 0 s a l i n i t y water . . . . 127 Eggs transferred from 2 0° /0 0 to 0 ° /0 0 s a l i n i t y water . . . . 129 v i LIST OF TABLES Table 1. Intragravel dissolved oxygen concentrations and mean p a r t i c l e size at 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 early from the egg capsule implantation experiment 36 Table 3. Summary of data collected 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 di f f e r e n t times of development 80 v i i 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 sections. 10 Figure 3a and 3b. Detailed map and longitudinal 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. Intragravel 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 stations on Dec. 10, 1985 and Oct. 20, 1984 respectively. 21 Figure 5c and 5d. Intragravel s a l i n i t y and temperature p r o f i l e s measured at fi v e i n t e r t i d a l monitoring stations on Jan. 5, 1986 and Jan. 23, 1986 respectively 23 Figure 5e. Intragravel s a l i n i t y and temperature p r o f i l e s measured at fi v e i n t e r t i d a l monitoring stations on Oct. 19, 1984 25 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. . 32 Figure 7. Egg survival rates to the eyed and alevin stages at each of the 11 egg implantation transects 33 Figure 8. Linear regression plot of egg survival on intragravel 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 alevin survival 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 activated 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 osmolality upon transfer 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 osmolality upon transfer 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 i n ambient s a l i n i t i e s ranging from 0 to 15%o 8 2 v i i i 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 test 87 Figure 18. Summary of experimental results 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 t e s t s . . . . . . 102 i x ACKNOWLEDGEMENTS I wish to express my sincere gratitude and appreciation to Dr. Gordon Hartman, my functional supervisor, for his i n i t i a l suggestion of this study and for his relentless guidance and support throughout. His assistance with the planning, implementation, and write-up of this thesis were integral. Also, I am very grateful to Charlie Scrivener for his guidance and assistance in setting up the f i e l d experiments and collecting the results. His willingness to help made a significant difference and was greatly appreciated. I am also thankful for the time and effort 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 for use of their laboratory f a c i l i t i e s at the Pacific Biological Station in Nanaimo, B.C. Further, I am very appreciative of their assistance and useful discussion which was so freely provided throughout the period I spent at the Station. Special thanks go to John Jensen for his unwavering support, both moral and l o g i s t i c a l , during the fin a l stages of the completion of this thesis. I wish to thank my o f f i c i a l university supervisor, Dr. Tom Northcote, for his 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, for their guidance and review of the manuscript. I also would like to express my sincerest thanks to my friends for their support and patience. I am especially grateful to my very special 'friend', x Valerie 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 like to express special thanks to my parents for their 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 Pacific Biological Station (Carnation Creek Project and Incubation and Water Quality section). The Science Council of B.C. provided me with personal financial support for two years through a G.R.E.A.T. scholarship. Both these sources of funding were c r i t i c a l for the completion of this thesis. I am very grateful to them for their 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 of 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 for spawning and rearing of the early l i f e stages are distributed 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 Alaska, USA, including some as far 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). In Asia 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 Si 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 suitable 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, usually return to the trib u t a r y of 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 varies considerably; i n North America i t rarely extends far upstream, except i n Alaska, the Yukon, and the A r c t i c (Bakkala 1970, Scott and Crossman 1973). Some chum, as well as the closely related 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). Tidal water enters the stream channel on a r i s i n g tide and inundates the gravel, exposing these eggs intermittently to seawater (Skud 1954, Hanavan 1954). This portion of the stream, inundated by periodic t i d a l flow, i s referred 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 referred to as i n t e r t i d a l spawners (Hanavan 1954, Hunter 1959, Helle et a l . 1964, Thornsteinson et a l . 1971). Many of southern Alaska's and B.C.'s coastal r i v e r s and streams are short with steep gradients and often have impassable barriers such as waterfalls near t h e i r mouths (Rockwell 1956, Thornsteinson et a l . 1971). These factors combined with 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 for salmon and l i m i t many of the accessible spawning areas to the lower reaches of streams, including the i n t e r t i d a l zones (Hanavan 1954, Helle et a l . 1964). Moreover, chum salmon rarely are intent on overcoming obstacles or barriers 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 exceptional, and knowledge concerning the ecology and physiology of such spawning populations i s l i m i t e d . In Alaska, 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, Helle et a l . 1964). Others have found survival rates of eggs spawned i n these areas to be equal to, or even greater than rates observed i n freshwater areas (Hanavan 1954, Rockwell 1956, Kirkwood 1962). Further, Helle et a l . (1964) and Helle (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 actually are separate populations or races, based upon th 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 literature indicates that this type of spawning is more common in Alaska than in B.C.; however; the literature for B.C. streams is very sparse, largely anecdotal, and therefore not as thorough. Since much of the research on intertidal spawning has been conducted in Alaska on pink salmon, information regarding the ecology of chum salmon and their a b i l i t y to cope with the intertidal environment is limited. This thesis examines the early l i f e stages of intertidal spawning chum salmon. The objectives were to: (1) assess the environmental conditions of the intertidal zone and determine how they differ from those of typical freshwater spawning environments and (2) determine the effect(s) that these differences may have on growth and development of chum salmon eggs and alevins. The study integrated both f i e l d and laboratory experiments, conducted at Carnation Creek, B.C., and at the Pacific Biological Station (PBS) in Nanaimo, B.C., respectively. The f i e l d component examined the egg to alevin survival of eggs a r t i f i c i a l l y implanted in the intertidal zone, and the laboratory experiments determined the salinity tolerance of developing eggs under controlled conditions and the effect 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 (Tait and Kirkwood 1962, Helle et a l . 1964, Noerenberg 1964) and, also occurs i n B.C., although to a lesser extent (Hunter 1959, Fraser et a l . 1974). Eggs i n i n t e r t i d a l spawning beds experience intermittent yet regular exposure to t i d a l saltwater as more dense seawater floods underneath the less dense outflowing fresh stream water (Skud 1954). Observation of th i s type of spawning behaviour led to questions regarding the actual and potential productivity of the i n t e r t i d a l zones (Hanavan 1954, Tait and Kirkwood 1962, Helle et a l . 1964, Fraser et a l . 1974). Fry production for many of the streams i n Prince William Sound, Alaska had been underestimated for 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, Tait and Kirkwood (1962) showed that as much as 74% of the t o t a l Prince William Sound pink and chum salmon f r y production originated i n t e r t i d a l l y . Other researchers examining individual 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, Tait and Kirkwood 1962, Helle et a l . 1964). Further, Noerenberg (1964) reported that 70% of the t o t a l 'even year' pink salmon escapement i n Prince William Sound spawned i n t e r t i d a l l y , ( t o t a l escapement of 80 index streams for 1956 to 1962 ranged from 0.6 to 1.5 m i l l i o n f i s h ) . Stream catalogs for southeastern Alaska, which index 518 streams, indicate that 17 to 34% of these streams have spawning populations of pink and chum that are 4 predominantly i n t e r t i d a l (Martin 1959, O r r e l l and Klinkhart 1963, O r r e l l et a l . 1963, 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 for which de t a i l s are not specified i n the stream catalogs. In an inventory of streams important for chum salmon production on the east coast of Vancouver Island, 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 contributor 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 contributions 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 i t . 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 fluctuations i n sea l e v e l as a result of t i d a l action. 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 exis t to prevent i t s temporary inland movement. The density of the inflowing saltwater i s greater than the density of the outflowing freshwater. Consequently i t moves into the stream channel underneath the stream water. As a result of the different densities, the two bodies of water experience only li m i t e d mixing and largely remain separate due to s t r a t i f i c a t i o n . The extent to which the intruding seawater, referred to as the ' s a l t wedge', moves upstream b a s i c a l l y depends on three factors: tide height, stream discharge, and stream topography. Figure 1 i l l u s t r a t e s 5 schematically the basic 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 interaction between these three major, and many other minor factors 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 intragravel water was approximately equal to that of the overlying s a l t wedge, i . e . , up to 3 0 ° /0 0. This showed that the intragravel environment i s affected by the t i d a l saltwater but did not provide any indication of the duration and frequency of saltwater exposure that eggs i n the gravel would receive. Helle 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 tide levels respectively. However, thei r measurements of intragravel s a l i n i t y , 9.3 and 22.0°/oo a t t n e 3-4- and 2.4-meter tide levels respectively, were much lower than Skud's (1954). The exact manner i n which Helle et a l . (1964) measured tide levels i s not s p e c i f i c a l l y stated. 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 point i s the 0-meter tide l e v e l . A l l subsequent references to tide levels are referenced to t h i s tide l e v e l . I t i s useful to consider the effects that intermittent saltwater inundation may have on egg survival and development. Limited information i s available regarding the survival 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 survival rates show that 6 LOW TIDE Fig. 1. Schematic diagram of the dynamics of a salt wedge in 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 survival rates were sim i l a r to or higher than those found for eggs deposited i n t o t a l l y freshwater areas within individual 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 effects of saltwater exposure on chum salmon eggs and the factors affecting survival of i n t e r t i d a l l y spawned eggs. Therefore, the f i e l d component of this study dealt 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 alevin incubation period. I related egg to alevin survival 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 into the stream bed, differences i n temperature regimes associated with inundation, intragravel 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, is located on the south side of Barkley Sound on the west coast of Vancouver Island, B.C. (Fig. 2). The creek is a typical second order west coast stream approximately 7 km long which drains a 10 km2 watershed. In 1971, hydrological weirs were constructed on the mainstem and several of the tributaries. During this same year a fish counting fence also was installed near the maximum high tide mark for enumeration of both emigrating and immigrating fish (Fig. 2 and 3). Anadromous fis h populations consist of 400 to 2500 chum salmon (O^ keta), 50 to 200 coho salmon (C\. kisutch), as well as small numbers of sea-run cutthroat trout (Salmo clarki) and steelhead trout.(S^ gairdneri). Prickly sculpin (Cottus asper) , coast range sculpin (C_,_ aleuticus) and threespine stickleback (Gasterosteus aculeatus) also occur in the stream. The marine staghorn sculpin (Leptocottus armatus) resides in 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 intertidal zone, before the fi s h fence was in place. However, since the installation of the fence only about 20% of the chum salmon make the effort to pass through i t to spawn, even though a l l coho salmon and sea-run trout pass through. As a result the majority of chum salmon have not u t i l i z e d spawning habitat above the fence since 1971. Because the fence was constructed near the high tide mark, a l l current chum 9 0- Main Fence F i g . 2. Location of the f i e l d study s i t e , Carnation Creek, and a d e t a i l e d map of the watershed showing the r e l a t i v e location of the I n t e r t i d a l zone below the f i s h counting fence. The freshwater zone was used as a control s i t e f or the egg implantation 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 intertidal zone is 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 level). From this point distances were marked at 10 m intervals by wooden stakes, upstream to the fish fence and downstream for 360 m to the ocean. This series of stakes was designed simply for quick reference to linear 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 partial 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 intertidal zone. Information from this survey was used to prepare an accurate map of the study site (Fig. 3a) and to provide the elevation measurements for construction of a thalweg of the stream (longitudinal profile of maximum cross sectional depth of the stream) (Fig. 3b). Elevations were referenced to the 0-meter tide level. The fi s h counting fence was placed at the maximum high tide mark. Therefore, I defined the intertidal zone as the reach of stream below this fence, and divided i t into upper, middle, and lower zones. The upper intertidal zone extended downstream from the fish 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) Figs. 3a and 3b. Detailed map of the i n t e r t i d a l zone (3a) and an associated longitudinal stream p r o f i l e (thalweg) (3b). In figure 3b the horizontal 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 tide 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 (Fig. 3a). The c r i t e r i a for these divisions were based upon stream topography and elevation of the stream bottom (F i g . 3b). Environmental Conditions of the I n t e r t i d a l Zone Intragravel S a l i n i t y and Temperature Measurements: Five intragravel s a l i n i t y monitoring stations were established 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 (Fig. 3a and 3b). Each station 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 additional probes were implanted at depths of 50 cm and 75 cm. Protective covers, constructed of p l a s t i c piping 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 calibrated p r i o r to being implanted, this was done once they were removed from the gravel. These calibrations indicated that recorded s a l i n i t i e s were at most 2 ° /0 0 lower than the actual values, and that recorded temperatures were within + 0.2°C of the actual values. Further, the presence of the protective 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 salinity and temperature readings were recorded using a Y S I conductivity salinometer (model 33 ) throughout 1984, 1985 and 1986 usually during the egg to alevin development period. Measurements were recorded at the five stations throughout tidal cycles at short time intervals to provide time course data of the hydrodynamics of intragravel seawater during periods of ebb and flood. 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 variety of tide heights over a range of low to high stream flows. Intragravel Oxygen Measurements: Intragravel dissolved oxygen measurements were carried out in mid-February shortly before the f i n a l egg capsules were recovered. Samples were taken adjacent to implanted egg capsules (within 30 cm), along transects across the stream (Fig. 4). Intragravel pore water, extracted using a large stainless steel syringe (80 cm long), was analyzed for dissolved oxygen content with a Y S I oxygen meter (model 57) (calibrated in air saturated water). Temperature and salinity of each sample also was recorded; where necessary, appropriate corrections were made to account for the effect 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 intertidal zone extensive gravel quality information already existed as part of the general Carnation Creek data base. Therefore, gravel samples were taken only at sites where this information was not already available, i.e., in the top of the upper, bottom of the middle, and throughout the lower intertidal zone. A total 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 Fig. 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 in 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 within small areas of stream bed, these samples were taken i n an attempt to define trends i n gravel quality rather than to characterize 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 saline pore water remained i n the gravel. However, the presence of low to intermediate s a l i n i t y water (5 to 20°/oo) did not impede the technique. Analysis 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 into 3 layers; top, middle, and bottom. Each layer was subsampled and passed through a series of sieves to determine proportions of diffe 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 size (Dg, geometric mean) was calculated for each sample using a standardized procedure already i n use for the Carnation Creek data base (Scrivener and Brownlee 1981) . I n t e r t i d a l Chum Salmon Spawning Survey Distributions 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 either 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 trying to discern between test digging s i t e s and actual redd s i t e s ; especially since spawning a c t i v i t y appeared to be most intense at night. As a result of th i s problem, these surveys were used only as general indicators of the location of spawning 16 f i s h , especially i n areas of high spawning density. 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 section was that the f i s h did not necessarily remain on the spawning grounds during the daylight hours but instead t y p i c a l l y schooled i n the deeper pools. 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 control 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 stations ( 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 section 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 either end. Eggs and m i l t were obtained from 3 females and 5 males (mean fork length: 72.3 and 77.5 cm respectively) 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 milt 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 additional test capsules were set up but not implanted to provide an indication of f e r t i l i z a t i o n success following a short period 17 of incubation (36 h ) . After 56 days of incubation (Dec. 18, 1985), 12 representative capsules were recovered to obtain preliminary survival and development rate data for the f i r s t two-thirds of the incubation period. 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 for at least 3 weeks. Even though across-stream transects were established and individual capsules were flagged to f a c i l i t a t e capsule recovery, eight were l o s t . Two of these were found downstream from t h e i r i n i t i a l locations while the others presumably were dug up and not located, or displaced l a t e r a l l y by redd digging a c t i v i t y during the spawning period. In areas of intense spawning, 1 m long metal rods were placed i n front 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 early for the preliminary analysis of development rate and sur v i v a l to the eyed stage, 43 were removed at the end of the experiment for analysis of egg to alevin s u r v i v a l , and 8 were l o s t . Eggs and alevin samples from the recovered egg capsules were processed by evaluating survival to two stages of development; eyed stage (dead or a l i v e with obvious eye pigmentation), and alevin stage (dead or a l i v e well 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 for 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 did not r e a l i z e the potential 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 well as greatly 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 + 0.59% (MEAN + SD) for no broken eggs, down to 39.5 + 21.01% and 2.2 + 0.17% for 1% and 3% broken eggs respectively. Therefore, percent survival calculations for this 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 survival was calculated by dividing the number of l i v e eggs or alevins by 17.7 (58.9% of 30), before multiplying by 100. Data Analysis Analysis of egg s u r v i v a l , intragravel oxygen, and gravel quality data involved pooling individual samples for each transect and testing for between transect differences of each variable using one-way analysis of variance (ANOVA). A l l percent egg survival values were normalized using the arcsine transformation (Snedecor and Cochran 1980). Post-hoc, multiple comparisons of ANOVA results were done using the SYSTAT s t a t i s t i c s package; appropriate levels of significance were determined using the Bonferroni procedure (Wilkinson 1987). Multiple l i n e a r regression analysis was used to test the r e l a t i v e significance of intragravel oxygen, gravel q u a l i t y , and egg capsule location 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 primarily 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 stations nearest to the ocean. The general pattern of saltwater inundation into and out of the Carnation Creek stream bed, during flood and ebb t i d a l cycles, 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 stat i o n at -275 m f i r s t , followed consecutively by the -195, -120, -35, and +40 m stations ( F i g . 5a). Although this pattern was observed at the -120 m s t a t i o n , i t i s not apparent i n figure 5a. Due to the low stream bed elevation i n t h i s area, high intragravel s a l i n i t i e s resulted for extended periods of time, regardless of tide height (Fig . 3b). At a l l of the other stations the intragravel s a l i n i t y probes usually registered measurable s a l i n i t i e s within 5 minutes or less after i n i t i a l coverage by the s a l t wedge. Peak intragravel s a l i n i t i e s were observed usually within 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 intragravel saltwater ranged from 0 to 3 0° /0 0 ( F i g . 5c). 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 basis, even though under certai n conditions the upper stations ( -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 tide height and stream 20 Figs. 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. Solid l i n e and dashed lines represent intragravel s a l i n i t y and temperature respectively. 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 station. discharge during a given t i d a l cycle, some stations, especially +40 and -35 m, would not receive any saltwater exposure at a l l . Unfortunately, the two additional 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 'flushing out' of intragravel saltwater did not occur. Nonetheless, the s a l i n i t i e s recorded by these three probes rarely 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 varied greatly and depended on a number of factors. The station 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 further upstream received b r i e f e r exposures. At the -275 m station coverage by the s a l t wedge commonly occurred for 6 h and sometimes lasted as long as 8 h ( F i g . 5a and 5c). During normal winter stream flows, durations of saltwater exposure often were similar at the -195 and -35 m stations (mean flow = 0.25 cms, during non-storm events ( i . e . , < 0.57 cms)). Saltwater exposure usually lasted for about 6 h or less ( F i g . 5a). When inundation occurred at the +40 m station i t sometimes lasted for 6 h but usually the exposure period was closer to 3 h and i n many cases was nonexistent (e.g., figures 5a and 5b). As with duration of t i d a l exposure, frequency of exposure also depended on the location of a given s i t e with respect to the ocean. Again, those stations closest to the ocean generally received the highest frequency while those further upstream received respectively l e s s . The -120 m s t a t i o n did not f i t t h i s trend, because as mentioned e a r l i e r the stream bed elevation i n t h i s area was lower than i t was further downstream at -195 m. The -275 m 22 Figs. 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. Solid l i n e and dashed lines represent intragravel s a l i n i t y and temperature respectively. 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 station. 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 often, but at times was prevented by above average non-storm event stream flows (0.57-1.41 cms) or lower than average, high tides ( < 2.8 m) ( F i g . 5e). The -120 m station received t i d a l exposure frequencies similar to that of -195 m, but the extended periods of elevated s a l i n i t y i n this low elevation area masked the intermittent i n f l u x otherwise recorded by the intragravel s a l i n i t y probes. The elevation 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 portion of the s a l t wedge tended to sink into the low elevation area around -120 m, further reducing the frequency of inundation at -35 m. Saltwater reached the -35 m station less 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 st a t i o n with saltwater. Higher tides of 3.5 m or more were necessary to produce measurable intragravel 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 this 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 usually reached the -275 m station about halfway into the flood cycle on a 3.0 m t i d e . This was about 3 h after maximum low tide and therefore, 3 h before the next high tide peak. At the -195, -120, and -35 m stations i t was uncommon for inundation to occur more than 3 h before peak high tide and usually i t occurred within 2 h or less of the peak (Fig. 5a and 5b). When saltwater inundation occurred at the +40 m station i t was usually within an hour or less of peak high tide ( F i g . 5a and 5c). 24 0.0 20 H „ 0 o £ 20-< W 20 u i 3 0 tt < 20 0 20 0.0 12.0 e.o "1 -35m i J — , ~ ,i • | i • • 1 -12.0 p a: r8.o 5 "I -I20m(25cm) " 1 r- —\— UJ V-1-12.0 UJ > , ' ' ! • — — -J . 1 . . 1 -8.0 g i i i i i i i r1 r 1 — i 1 1 -1 -195m : L ! rl2.0 < t-2 - - — . - - r / -8.0 -i -275 m ^ * H2.0 — 1 -1 -8.0 0800 HIGH TIDE 1000 1200 1400 LOW TIDE 1600 1800 TIME (h) Fie 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 stations measured on 19 Oct. 1984. Solid l i n e and dashed l i n e s represent intragravel s a l i n i t y and temperature respectively. 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 retreated from the gravel i n much the same manner as i t inundated on a flooding t i d e , but i n reverse. Saltwater retreated f i r s t from the highest station i t had reached followed consecutively by lower downstream stations ( F i g . 5a). However, the flushing process usually occurred over a longer time period especially at the upper i n t e r t i d a l stations. 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 cycle where Inundation did not occur at the two uppermost s i t e s (-35 and +40 m), but shows t y p i c a l patterns of intragravel s a l i n i t i e s for the lower three stations (-120, -195, and -275 m). Most of the intragravel hydrodynamics described so far occurred under r e l a t i v e l y normal or average conditions. However, changes i n tide height and stream discharge greatly 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 tide levels ( > 3.5 m), the s a l t wedge can tra v e l far up the stream, and result i n high and prolonged intragravel s a l i n i t i e s ( F i g . 5c). This figure shows how even at the +40 m station s a l i n i t y remained r e l a t i v e l y high , > 1 5° /0 0 for nearly two hours. In contrast, figure 5d shows the effect of high stream flow (range for this time period: 1.90-4.56 cms), which apparently overshadowed the 3.5m high tide 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 th i s lowermost station the intragravel s a l i n i t y did not r i s e above 3°/oo> while at the -120 m station the intragravel 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 effect of tide height, figure 5e shows how the lower of the two dai l y high tide peaks (2.7 m) was not high enough to result i n saltwater inundation at the -195 m st a t i o n . A comparison of th 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 tide peak (3.08 m, N=27), indicates that t h i s tide l e v e l was 0.38 m lower than the average. However, during the next high tide cycle i n the l a t t e r portion of this time series, a higher maximum tide l e v e l (3.0 m @ 1946 h) pushed the s a l t wedge further upstream and resulted i n saltwater inundation at the -195 m station about 1.5 h before peak high t i d e . Also note i n figure 5d that as a result of a severe storm event f i v e days p r i o r to the recording of these measurements, the intragravel s a l i n i t y at -120 m was lower than usual (compare to figures 5a and 5c). Intragravel Temperature: Intragravel temperatures, at least during the incubation period of f a l l and winter, increased when the gravel was inundated with saltwater. As intragravel s a l i n i t y increased so did intragravel temperature. The greatest fluctuations occurred at -195 and -275 m, where temperatures increased as much as 4°C above the freshwater intragravel stream temperature (Fig. 5a). At the other stations, -120, -35 and +40 m, intragravel temperature changes also occurred, but usually were only 1 to 2 and sometimes 3°C higher than the stream temperature at low tide (Fig 5a). Intragravel stream temperatures followed the general pattern of intragravel s a l i n i t y increase and decrease but appeared to be affected by additional unmeasured factors. Intragravel temperatures began to increase at the same time as intragravel s a l i n i t y , especially at the -.275 m station ( F i g . 5a). Hence, an increase i n intragravel temperature during a flooding tide was an indicator of saltwater inundation into the stream bed. As the 27 s a l t wedge receded, the reverse of th i s response occurred. Again, t h i s was most obvious at the -275 m station (Fig 5a and 5c). An additional factor affecting intragravel temperature was the presence of warmer groundwater at the +40 m st a t i o n . Intragravel 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 stations; regardless of tide height ( F i g . 5a and 5b). This temperature increase was indicative of an area of warm water seepage or upwelling. However, as shown i n figures 5a and 5c, the upwelling did not prevent saltwater from inundating the gravel when i t did reach this s t a t i o n . Intragravel Oxygen: Differences i n intragravel oxygen levels 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 control 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 intragravel oxygen levels which t y p i c a l l y were within 1 to 4 mg/1 of the surface water levels (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 for the February 20, 1986 samples only (P < 0.001). Differences between the control 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 particle sizes at 11 transects located throughout Carnation Creek, British 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 is the grand mean for the two sample dates. Gravel samples were obtained using the freeze core method (Scrivener and Brownlee 1981) and mean particle size was calculated as the geometric mean (Dg + SD). Transect location (m) Intragravel dissolved oxygen (mg/1) 13/02/86 20/02/86 Mean Mean particle size (mm) +260 +200 9.5 1 + 0.64 12.3 +0.07 10.6 + 1.56 9.4 + 2.12 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 -36 6. .8 + 1, ,06 7, .0 + 3. .66 6 .9 + 2. .41 11 .1 + 1. .51 -110 3, .0 l -- 3. 0 15. 7 l -120 4, ,9 + 0. ,46 4 .9 + 0. .46 11, .2 + 3. ,26 -195 9. .0 + 0. .39 11. ,1 + 0. .16 10 .2 + 1. .15 7, .9 + 0. ,80 -275 9, .5 + 0. .42 11. .3 + .11 10 .6 + 0. .99 7 .6 + 1, .29 1 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 result, the Feb. 13 samples were taken 5 days into a period of relatively low winter flows (Carnation Creek data base, PBS, 1988) and the Feb. 20 29 samples were taken 12 days into the same period. Mean flows for this period were 0.22 + 0.045 (S.D.) cms (Feb.13) and 0.19 + 0.053 cms (Feb.20). Thus intragravel oxygen levels remained high at the two upstream control 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 flow. However, th i s was not the case for the middle and upper s i t e s which experienced reduced mean intragravel oxygen levels 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 intragravel oxygen levels decreased from 7.7 to 3.7 mg/1 within a distance 4 m. A few of the egg capsules retrieved 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 inside the capsules had turned black, presumably from H2S production. Gravel Quality: V a r i a b i l i t y i n mean p a r t i c l e size was high within transects and between transects (range for 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 size 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 Di s t r i b u t i o n Chum salmon spawned throughout the entire 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 portion of this zone, j u s t below the f i s h counting fence. Spawner densities were highest here and i n the top portion 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 (Fig. 6). As mentioned e a r l i e r , the intragravel s a l i n i t y i n the stream section between -100 and -150 m often remained moderately high (15-22%o) -even during ebb tides (see Figs. 5a-5e). Redd digging occurred consistently i n th i s section ( F i g . 6), although i t may have included test digging as well as actual egg deposition. However, below t h i s area of elevated intragravel 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 previously had been implanted. Egg Capsule Implantation Experiment Egg Survival Rates: Mean rates of egg survival to the eyed stage, adjusted for f e r t i l i z a t i o n rate, ranged from 3.5% at -120 m to 67.4% at -195 m. Survival rates to the alevin stage however, were markedly lower and ranged from 0% at -33 and -110 m up to 51.2% at -275 m (Fig. 7). The pattern of egg survival was one of consistently good survival 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 survival i n the upper and middle i n t e r t i d a l areas was lower and more va r i a b l e . Although mean survival 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 transect, as well as between capsules implanted at different transects. 31 Fig. 6. An example of spawner utilization measured as redd distribution of intertidal spawning chum salmon (e.g., Nov. 8, 1985). > or o < > or o or < 90 -i 70 -50 -30 -10 -rfi r i + 260 FRESH WATER" 90 70 50 30 • 10 • - A f t +260 + 200 FRESH W A T E R — * EYED STAGE ft TH r- 100 90 70 50 30 10 0 UPPER-"275 -MIDDLE l-LOWER — •INTERTIDAL-ALEVIN STAGE rti ifL •40 0 "6 "33 "36 "110 "120 "195 "275 — UPPER 1 MIDDLE |-L0WER — | > or z> cn r- 100 90 70 50 30 10 _J > or ZD (f) I N T E R T I D A L-EGG CAPSULE IMPLANTATION SITES Fig. 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. 3 3 Multiple regression analysis of egg survival versus intragravel oxygen, mean gravel p a r t i c l e s i z e , and elevation of the capsule implantation si t e s indicated 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 variables ( F i g . 8). Intragravel 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 . Elevation of capsule location was used as a gross predictor of s a l i n i t y exposure since, as demonstrated e a r l i e r , intragravel s a l i n i t y exposure was a function of proximity to the ocean (except at the -110 and -120 m s i t e s ) . Egg Development Rates: In addition to evaluating differences i n survival rates, I also examined differences i n developmental rates of the eggs obtained from the early recovery capsules (capsules were recovered on Dec. 18, 1985, 56 days after f e r t i l i z a t i o n and implantation). Table 2 shows a downstream trend of increasing development rate, with two exceptions. At +40 m, the s i t e of warm groundwater upwelling, development rates were higher than those at any of the other s i t e s , whereas at -120 m, the s i t e of low stream bed elevation, rates were noticeably lower. The differences 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 control s i t e s (+200 and +260 m) were about two Vernier (1969) stages; stage 28 compared to stage 26 respectively. 34 F i g . 8. Linear regression of egg survival on intragravel oxygen ( r2 - 0.58, N «- 35, P < 0.001). Oxygen measurements were taken adjacent to individual egg capsules, a few days prior to their removal. 35 TABLE 2. Developmental stages of eggs recovered early (56 days after implantation) from the egg capsule implantation experiment. Eggs were located at 11 transects; 2 upstream freshwater control 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 . Egg implantation Egg development No. of transect (m) stage 1 eggs (N) +260 early 26 (22) +200 early 26 (9) +40 29-30 (13) -110 26-27 (19) -120 25 (2) -195 27-28 (36) -275 28 (6) 1 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 intermittently saline environment. Eggs placed i n the i n t e r t i d a l zone of Carnation Creek generally did not show any negative effects from intermittent exposure to saltwater. Even at the lowermost station (-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 for 4-7 h ) , egg survival was good and si m i l a r to that observed at the freshwater control 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 gravel, d i r e c t comparisons of absolute egg survival values with those i n other studies are not possible. Further, these samples were corrected for the observed f e r t i l i z a t i o n rate. Both these factors res u l t i n higher calculated percent survival rates. Nonetheless, my results are s i m i l a r to those of other studies examining egg survival of i n t e r t i d a l spawning pink (Hanavan 1954, Helle et a l . 1964) and chum salmon (Kirkwood 1962) at similar 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 for i n t e r t i d a l eggs of both species i s around the 1.2-meter tide l e v e l (Hanavan 1954, Kirkwood 1962, Helle et a l . 1964). Although some spawning does occur below this l i m i t , especially i n pink salmon, the eggs do not seem to survive. Bailey (1966) demonstrated, using simulated i n t e r t i d a l conditions, that pink salmon eggs subjected to a 37 saltwater exposure representative of the 1.2-meter tide l e v e l (28°/oo f ° r 9.3 h twice daily) suffered 100% mortality. However, at the same s a l i n i t y for shorter durations of 6.7 and 4.0 h twice d a i l y , he observed 50 and 0% mortality respectively. These two exposure times simulated saltwater exposure regimes representative of the 1.8- and 2.4-meter tide levels respectively. The saltwater tolerance response reported by Bailey (1966) coincides with observations reported by others i n f i e l d studies; improved egg survival with increasing elevation within the i n t e r t i d a l zone (Rockwell 1956, Kirkwood 1962, Helle et a l . 1964). Other studies have compared egg survival 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 relationship between i n t e r t i d a l zone elevation or egg implantation location and egg survival i n th i s study, I did observe survival rates at some i n t e r t i d a l zone transects that were si m i l a r to those of the freshwater zone (Fig. 7 and Table 3). The absence of an obvious relationship between location i n the i n t e r t i d a l zone and egg survival i s possibly 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 tide level) examined i n my study, compared to many of the related studies (Hanavan 1954, Helle et a l . 1964, Thornsteinson et a l . 1971). These studies were conducted i n more northerly areas such as Alaska, where tides 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 survival over a broader range of elevations, 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 frequencies). Further, egg survival results i n this study were 38 TABLE 3. Summary of data collected for the f i e l d study at Carnation Creek. Samples were collected at 11 transects, 2 were control sites (+200 and +260 m) located in the freshwater zone and the other 9 were located in the intertidal zone. Samples were pooled for each transect. Mean percent egg survival data were adjusted for 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 particle size was calculated as the geometric mean (Dg + SD). Elevations were determined from figure 3b as the height above the 0-meter tide level. Transect Mean egg Mean egg Mean intragr. Mean part. Eleva-location survival survival oxygen size tion (m) (arcsin %) (%) (mg/1) (mm) (m) +260 33.7 + 5.30 30.8 10.6 9.4 +200 36.9+8.83 36.1 10.7 11.6 +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 -33 6, .8 l 0. 0 6.7 12. 7 2. 4 -36 28. .3 + 10.74 22 .4 6.9 11, .1 2 .8 -110 6, .8 l 0. 0 3.0 15. 7 1. 9 -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 in the upper middle intertidal zone (-33 39 to -80 m). Channelization may have been p a r t i a l l y responsible for the p e r i o d i c a l l y low intragravel oxygen levels observed i n t h i s area (Table 3). S p e c i f i c a l l y , results i n this study show that egg to alevin survival 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 (Fig . 7). Percent survival rates of eggs implanted at 0, -33, -110 and -120 m were low, 3.0, 0, 0, and 2.2 respectively. I t i s u n l i k e l y that these low values are the resul t of high s a l i n i t i e s exclusively, especially 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 mortality since this area experienced prolonged periods of saltwater inundation (Fig. 5a). However, i t i s d i f f i c u l t to separate the effects of l e t h a l s a l i n i t y exposure from other contributing environmental factors such as intragravel oxygen concentration, gravel q u a l i t y , and variations i n stream discharge. I t i s well established that the primary source of oxygen for the intragravel 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 well 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 into and out of the gravel stream bed, i s affected by factors such as gravel permeability, gravel depth, stream bed surface configuration, and stream discharge (Vaux 1962). Interchange ultimately determines the s u i t a b i l i t y of the intragravel environment with 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 well oxygenated water to 40 the egg at 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 et a l . 1963, Sowden and Power 1985). The importance of interchange as the supplier of intragravel oxygen i s refl e c t e d by a commonly observed direct relationship between egg survival and Intragravel oxygen content (Wickett 1954, 1958, Coble 1961, P h i l l i p s and Campbell 1962, Koski 1966, Sowden and Power 1985). Intragravel oxygen concentrations measured i n Carnation Creek were consistent with t h i s relationship 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 results from the i r study on pre-emergent survival 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 less than 5 mg/1 usually were l e t h a l . Intragravel oxygen concentrations and associated egg survival rates measured i n Carnation Creek coincide reasonably well with this value. The fi v e transects with mean oxygen concentrations below 5.1 mg/1 had low egg survivals (Table 3). One exception, at -33 m, had a mean oxygen concentration of 6.7 mg/1 but a survival of 0%. This apparently inconsistent resul t was probably a re s u l t of the across-stream and temporal v a r i a b i l i t y (values ranged from 3.8-8.3 mg/1). Further, i t indicates the need to consider, i n addition to oxygen content, other factors that influence egg survival such as intragravel water v e l o c i t y . Apparent water v e l o c i t y i s an important factor influencing egg survival where oxygen concentrations are not l e t h a l (Coble 1961, S i l v e r et a l . 1963, Shumway et a l . 1964). Sowden and Power (1985) concluded that, provided mean intragravel oxygen exceeded a threshold near 5 mg/1, intragravel 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) at the -33 m s i t e may have been a confounding factor contributing to the lack 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 direct relationship between gravel composition and permeability, and intragravel oxygen and embryo survival (Wickett 1954, 1958, McNeil and Ahnell 1964, Koski 1966, Tappel ,1981). Egg survival and oxygen concentration results from this study however, do not demonstrate t h i s relationship; gravel quality (measured as mean p a r t i c l e size) was not correlated with these parameters (Fig . 8). Sowden and Power (1985) reached a sim i l a r conclusion and indicated that other factors, apparently unrelated to gravel q u a l i t y , could overshadow the effects of gravel quality on intragravel oxygen and egg s u r v i v a l . Intragravel oxygen l e v e l s , and therefore egg survival rates, largely were independent of gravel quality; intragravel concentrations were influenced to a greater extent by low oxygen groundwater. In Carnation Creek, no apparent reason was established for the lack of cor 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 size was high but intragravel oxygen was low (Table 3). As indicated by measurement of warmer intragravel temperatures, t h i s s i t e experienced groundwater upwelling ( F i g . 5a). Although the oxygen content of this groundwater was not determined, other studies indicate that generally i t i s low (2 to 4 mg/1) (Sheridan 1962, Kogl 1965, Sowden and Power 1985). In f a c t , Sheridan (1962) used differences i n oxygen content and temperature ( i . e . , lower oxygen and higher temperature) 42 as a means o f d e t e c t i n g g r oundwa t e r . Thus , l ow oxygen l e v e l s a t +40 m i n t h i s s t u d y , p r o b a b l y were i n f l u e n c e d b y g r oundwa t e r s e epage . However , more d e t a i l e d i n v e s t i g a t i o n i s r e q u i r e d t o p r o v i d e d e f i n i t e c o n c l u s i o n s . L e t h a l o x ygen c o n c e n t r a t i o n s d e t e r m i n e d i n t h e f i e l d r e p r e s e n t an a v e r a g e o f o x ygen measurements 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 o f s i t e s . A c o m p a r i s o n o f l e t h a l oxygen l e v e l s d e t e r m i n e d i n t h e l a b o r a t o r y ( LC50 : 0 . 4 t o 1.6 mg/1 ( A l d e r d i c e e t a l . 1958, S i l v e r e t a l . 1 9 6 3 ) ) , w i t h t h o s e measu red i n t h e f i e l d ( LC100: < 5 mg/1 (Sowden and Power 1 9 8 5 ) ) , shows t h a t f i e l d l e v e l s a r e m a r k e d l y h i g h e r t h a n l a b o r a t o r y l e v e l s . One p o s s i b l e e x p l a n a t i o n i s 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 r e s u l t i n g f r om e xpo su r e t o o xygen c o n c e n t r a t i o n s b e l o w t he c r i t i c a l l e v e l s ( 1 - 7 mg/1 @ 10°C) ( A l d e r d i c e e t a l . 1958 , G a r s i d e 1959 , Shumway e t a l . 1 9 6 4 ) , r e m a i n a l i v e unde r l a b o r a t o r y c o n d i t i o n s b u t n o t i n t h e h a r s h e r n a t u r a l e n v i r o n m e n t ( S i l v e r e t a l . 1 9 6 3 ) . F u r t h e r , l a b o r a t o r y measurements a r e made i n d e p e n d e n t o f t h e h i g h v a r i a b i l i t y common t o f a c t o r s i n f l u e n c i n g t h e i n t e r c h a n g e o f i n t r a g r a v e l w a t e r ( M c N e i l 1962 , S h e r i d a n 1962, Sowden and Power 1 9 8 5 ) . E x t e n s i v e s a m p l i n g o f t h e i n t r a g r a v e l e n v i r o n m e n t i s n e c e s s a r y t o a c c u r a t e l y r e p r e s e n t t h e t r u e c o n d i t i o n s ( M c N e i l 1 9 6 2 ) . T h e r e f o r e , i n h e r e n t v a r i a b i l i t y i n t he p h y s i c a l and c h e m i c a l e n v i r o n m e n t s o f t h e egg s , i n c o n j u n c t i o n w i t h r e l a t i v e l y s m a l l samp le s i z e s , may f u r t h e r e x p l a i n i r r e g u l a r r e s u l t s i n t h i s s t u d y ( e . g . , 0% s u r v i v a l a t t h e -33 m s i t e where mean oxygen l e v e l s were 6 .7 m g / 1 ) . Oxygen r e q u i r e m e n t s o f d e v e l o p i n g s a l m o n i d embryos i n c r e a s e o v e r t ime ( A l d e r d i c e e t a l . 1 9 5 8 ) . I n i t i a l l y c r i t i c a l o xygen r e q u i r e m e n t s a r e l ow (1 mg/1 o r l e s s ) , b u t t h r o u g h o u t deve l opmen t t h e y i n c r e a s e t o a maximum a t 43 hatching (7.5-9.6 mg/1), followed by an abrupt drop o f f after 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 th i s study egg survival to the eyed stage was higher than survival 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 especially apparent i n the upper and middle i n t e r t i d a l areas where low and variable oxygen concentrations were measured (Fig. 7 and Table 3). These results suggest that intermediate intragravel oxygen levels i n these areas were s u f f i c i e n t to meet the requirements of early embryos but not l a t e r more advanced ones. Support for t h i s explanation may be found upon closer examination of the r e l a t i v e rates of egg development measured i n the early 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 control s i t e s (+200 and +260 m). They also increased downstream within the i n t e r t i d a l zone, except for three s i t e s (+40, -110 and -120 m). This trend i s explained largely by differences i n intragravel temperatures. However, temperature p r o f i l e s recorded from the probe at -120 m did not indicate any unusually low readings (Figs. 5a to 5e), yet development rates of the early recovery eggs at this 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 result suggests that another factor was influencing the observed development rates. I t i s well established that exposure of salmonid eggs to sublethal oxygen concentrations results 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 result of exposure to sublethal oxygen concentrations. 44 Although intragravel oxygen seems to be the most i n f l u e n t i a l factor a f f e c t i n g egg s u r v i v a l , ultimate egg survival i s determined by the net re s u l t of a complex inter-relationship of environmental factors that influence intragravel water interchange and oxygen concentration. Further investigation, including extensive sampling, i s necessary to elucidate these relationships more precise l y . However, i t i s apparent that i n general, egg sur v i v a l i n the Carnation Creek i n t e r t i d a l zone was not affected negatively by saltwater inundation, and instead was related to intragravel oxygen and other associated factors. V Interchange of intragravel water largely i s the result of water flow over the stream bed (Vaux 1962). However, this process w i l l be altered 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 largely i n h i b i t interchange between the stream water and the intragravel environment. Observations show that as the denser saltwater entered along the stream bottom, over and through the gravel, the downstream flow of freshwater was redirected towards the surface, due to i t s lower density. 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 intragravel water exchange i s dependent, at least 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 eliminated, 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 intragravel oxygen levels during such periods of saltwater inundation (Fraser et a l . 1974). However, eggs survival at the 2.2- and 2.6-45 meter tide levels i n th i s study (-275 and -195 m si t e s respectively) , and si m i l a r i n t e r t i d a l elevations i n other studies (Hanavan 1954, Helle et a l . 1964, Thornstelnson et a l . 1971), indicate that this i s not a major problem. In f a c t , results from the aforementioned studies, as well as t h i s one, repeatedly indicate that egg survival 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 actually benefit eggs incubating i n th i s zone. Perhaps one of these benefits i s the access of eggs to an alternate source of well oxygenated water, namely ocean water. As the seawater moves i n on a flooding t i d e , intragravel water exchanges i n response to the d i f f e r e n t i a l densities. This i s different from the mechanism of exchange during periods of freshwater flow since i t i s not dependent upon the rate of water flow. Intragravel water movement occurs as the less dense freshwater i s displaced by the more dense saltwater. This i s v e r i f i e d by the measurements of intragravel s a l i n i t y shown i n figures 5a to 5e. S i m i l a r l y , as the s a l t wedge retreats on an ebbing t i d e , flowing freshwater begins to d i l u t e and eventually replace the saline intragravel water. In such a way, eggs experience exchanging water over much of the day as the re 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 . , within tolerable l i m i t s of concentration and duration, i n t e r t i d a l eggs can tolerate these conditions on a d a i l y basis. Surface seawater commonly has dissolved oxygen levels near saturation which for inshore waters usually i s around 7-9 mg/1 (Waldichuk 1956, Pickard 46 1961). Regular exposure to intermittent 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 flow, when the oxygen content of stream water often i s low (Table 1) and the intragravel 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 well oxygenated water, especially 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 th e i r sole source of oxygen. Measurements made by McNeil (1962) appear to support t h i s suggestion with regard to intragravel oxygen concentrations at l e a s t . He reported that during times of low flow (summer) intragravel oxygen levels 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 intragravel oxygen levels are less l i k e l y to become depleted as coverage by the s a l t wedge exchanges the intragravel water on a regular basis. 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, this may change during periods of reduced stream flow, especially i n areas of poorer gravel q u a l i t y . I n t e r t i d a l areas usually are associated with low stream gradients and therefore often contain r e l a t i v e l y high percentages of fine 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 size averaged along a transect was s t i l l r e l a t i v e l y large (7.9 mm, Table 3). Nonetheless, this 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 size or increase i n fine sediment reduces gravel permeability and ultimately intragravel water exchange (McNeil and Ahnell 1962, Vaux 1968). These factors combined, can produce s t r e s s f u l l y low oxygen 47 levels for 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 intragravel oxygen levels on an intermittent but regular basis. Thus, the ocean as a source of oxygen, could become an i n f l u e n t i a l factor i n determining the survival of salmon eggs spawned i n the lower reaches of streams, especially 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 dif f e r e n t from the freshwater one upstream. During times of saltwater inundation, intragravel temperatures usually 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 with a s i g n i f i c a n t accumulation of thermal energy over the incubation period. This thermal gain already was apparent i n eggs recovered from the gravel 56 days after 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 stations -195 and -275 m were two Vernier (1969) development stages more advanced than the freshwater eggs located at the upstream stations (+200 and +260 m); stage 28 compared to stage 26 respectively. Mathematically modelled development rate data (Jensen 1988) , indicated that t h i s difference translated into about 56°C-days. (A '°C-day' unit i s the mean da 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 rates, using Jensen's (1988) model), 56°C-days translated into an 8 or 9 day difference for 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 less i n surface ocean water than stream water due to the difference i n r e l a t i v e sizes of the bodies of water. Stream temperatures fluctuate over a wider range than ocean temperatures do. Thus, thermal input from intruding 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 this fact for i n t e r t i d a l pink salmon i n an Alaskan stream. In addition he suggested that warmer seawater temperatures would provide an added buffering effect from low stream temperatures. Especially 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 less l i k e l y to experience deleterious effects 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 subarctic stream (Chena River, Alaska). This suggests that these f i s h were ac t i v e l y selecting spawning areas to minimize deleterious effects from cold stream water. I t may be useful to examine spawning s i t e s e l e ction 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 effects of low temperature on eggs. 49 The net effect of increased development rate, r e s u l t i n g from thermal gains associated with intermittent 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 several southeastern Alaska streams, have reported that sizeable portions of the emerging i n t e r t i d a l f r y were missed. Tait and Kirkwood (1962) based t h e i r pre-season timing of emergence calculations on the temperature regimes of the freshwater areas, and as a result 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 fact that f r y re 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 , raises the question of whether early emergence i s a benefit or a l i a b i l i t y . During the early period of marine l i f e , salmon f r y mortality i s high (Parker 1968, 1971). The mechanisms are not well understood but a siz e - s e l e c t i v e bias towards smaller f r y has been shown (Parker 1971, Healey 1982). One of the major factors affecting early juvenile salmon mortality i s thought to be predation by piscivorous f i s h (Kirkwood 1962, Parker 1968, 1971) and marine birds (Godin 1981). Some studies examining f r y mortality 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 factor i n reducing f r y mortality (Neave 1953, Hunter 1959, Fresh and Schroder 1987). However, i t i s not known i f this 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 for a given r i v e r or stream, early 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 potential mortality by freshwater predators (Parker 1971, Fresh and Schroder 1987) as would other f r y migrating from further upstream. 50 A n o t h e r f a c t o r t o c o n s i d e r , when f r y o r i g i n a t i n g f r om t h e i n t e r t i d a l zone e n t e r t h e ma r i n e e n v i r o n m e n t e a r l i e r , i s f o o d a v a i l a b i l i t y . S i b e r t (1979) r e p o r t e d t h a t t h e s e a s o n a l p a t t e r n o f abundance o f chum sa lmon f r y i n t h e Nanaimo e s t u a r y was t h e same as t h e s e a s o n a l abundance o f h a r p a c t i c o i d c opepod s ; a p r e f e r r e d f o o d i t e m o f t h e f r y . W a l t e r s e t a l . (1978) summar i zed a g e n e r a l r e l a t i o n s h i p be tween abundance o f z o o p l a n k t o n on t h e c o a s t o f B.C. and t h e 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 s a lmon f r y , b u t wa rned t h a t v a r i a t i o n on l o c a l s c a l e s was h i g h due t o p a t c h i n e s s ( L e B r a s s e u r 1 9 6 5 ) . I t i s n o t c l e a r whe t h e r e a r l i e r emergence o f i n t e r t i d a l f r y (up t o a few weeks) w o u l d be a l i a b i l i t y o r a b e n e f i t w i t h r e s p e c t t o f o o d a v a i l a b i l i t y . F u r t h e r s i t e s p e c i f i c s t u d i e s a r e n e c e s s a r y t o answer t h i s q u e s t i o n c o n c l u s i v e l y . 51 SUMMARY - Chapter I 1. The i n t e r t i d a l zone i s a unique environment for developing salmon eggs. Eggs spawned i n t h i s area experienced intermittent exposure to t i d a l seawater at continuously varying frequencies, durations and concentrations. Associated with these t i d a l fluxes, changes i n intragravel temperatures as well as dissolved 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 tide height, stream discharge, stream topography and s p e c i f i c egg location. 3. The range of possible s a l i n i t y exposures varied from short, infrequent, low s a l i n i t i e s (30 min, once weekly to 10°/00) to longer, regular, 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 factor influencing egg survival 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 station where s a l i n i t y probably contributed to low sur v i v a l rates. Due to stream topography, this low elevation area experienced moderate intragravel s a l i n i t i e s (15-22°/00) continuously for extended periods of time (up to 2 weeks). 5. Similar to other studies (Hanavan 1954, Rockwell 1956, Bailey 1966), egg sur v i v a l rates i n the i n t e r t i d a l zone were similar to rates observed i n the upstream freshwater zone. 52 6. Egg survival was d i r e c t l y correlated with intragravel oxygen levels ( r2 = 0.58), whereas no correlation was observed with mean p a r t i c l e size or elevation of egg location. 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 alternate oxygen supply. Especially during times of low stream flow, and i n areas of low gravel permeability, this may become r e l a t i v e l y more important. On a flood t i d e , intragravel freshwater i s displaced by incoming seawater as a res u l t of d i f f e r e n t i a l densities. 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 consistently has moderately high oxygen concentrations. 8. Intragravel temperatures associated with s a l t wedge inundation produced noticeably faster egg development rates. In general, development rates increased with 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 at the -195 and -275 m si t e s compared to the freshwater controls s i t e s (+200 and +260 m), indicated 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 intragravel 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 th i s area. However, i t did not prevent the saltwater from inundating into the gravel when the s a l t wedge reached t h i s area. This observation contradicts the idea 53 that upwelling water necessarily prevents saltwater inundation into intertidal spawning beds. 54 CHAPTER I I - Laboratory Study INTRODUCTION Salmon eggs deposited in intertidal 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 greatly. Bailey (1966) avoided some of this v a r i a b i l i t y by simulating a range of intertidal conditions in 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 °r a s l°ng a s 4 hours twice daily with no adverse effects. Exposures for periods longer than this resulted in reduced survival rates and eventual total mortality. Other studies that have examined salinity tolerance of salmon eggs in 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 is that i t does not accurately represent the conditions experienced in the intertidal zone. Some workers have tried to determine the developmental stage(s) at which salmonids acquire the a b i l i t y to control their 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 ( g i l l s , 55 kidney, and functional gut), workers have investigated alternate mechanisms available to these stages i n attempts to Understand how they regulate t h e i r i n t e r i o r m i l i e u (Leatherland and L i n 1975, Shen and Leatherland 1978b). Even i f embryos and alevins are not exposed to saline conditions, they are s t i l l faced with maintaining ionic and osmotic balances i n a hypo-osmotic freshwater environment. I t i s well 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 ultimately d i e . However, the mechanisms with which they accomplish this balance, especially during the very early stages, are not well understood. In a review on osmotic and ionic regulation i n teleost eggs and larvae, Alderdice (1987) concluded that i n i t i a l regulation i n the teleost 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 fluxes". Membrane permeability may increase s l i g h t l y near the 'eyed' stage, and chloride c e l l s may appear i n the blastoderm ( f i r s t c e l l u l a r layer to overgrow the yolk, i . e . , yolk sac epithelium, during the process of epiboly). Chloride c e l l s , also referred to as 'mitochondria-rich' c e l l s , are one of the osmo-and ionoregulatory mechanisms functioning i n the l a t e r stages of development (Zadunaisky 1984). During overgrowth of the blastoderm, chloride 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 teleost species, Fundulus heteroclitus (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 gairdneri (Shen and Leatherland 1978b) but not i n Oncorhynchus kisutch (Leatherland and L i n 1975), the only P a c i f i c salmon species so far examined. However, chloride c e l l s may not be the only regulatory mechanism available to teleost 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 limi t e d osmo- and ionoregulation p r i o r to formation of the blastoderm and completion of epiboly. Nevertheless, some of the teleost embryos examined, including 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 with respect to ambient conditions, p r i o r to the development of adult phase regulation which occurs on an organistic l e v e l , involving the g i l l s , gut, and kidney. Evidence suggests that the regulatory mechanisms employed by these early l i f e stages are simple ones functioning at the c e l l u l a r and tissue l e v e l s , i . e . , maintenance of 't i g h t ' plasma membranes, possible regulation by blastodermal c e l l s , and i n some species functional chloride c e l l s (Alderdice 1987). When considering s a l i n i t y tolerance of the early l i f e stages of f i s h , the majority of work has focussed on the developing egg and stages beyond. However, researchers have also examined the effects of osmolality and ion content on sperm m o t i l i t y (Baynes et a l . 1981, Morisawa et a l . 1983) and occasionally v i a b i l i t y (Werner 1934, Rieniets and M i l l a r d 1987). However, most of these tests were conducted with low ionic concentrations solutions, usually less than or equal to the osmolality of blood plasma or seminal f l u i d (270-300 mosmol/kg, Morisawa et a l . 1983). Limited information i s available on the effect of higher concentrations of ionic solutions, especially with respect to the complete f e r t i l i z a t i o n process involving both gametes. 57 Available information suggests that intertidal spawners do not deposit their eggs during times of saltwater inundation. However, spawning behaviour specific to intertidal spawning salmon apparently has not been examined in de 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 feasible given the environmental conditions of the intertidal spawning area during saltwater inundation. The f i r s t part of the laboratory component of this study was designed to examine the survival of eggs exposed to controlled intermittent salinity 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 in the intertidal zone of Carnation Creek. Further, eggs from two supposedly separate spawning areas of a stream, intertidal and freshwater areas, were tested for differential salinity tolerance. The question examined was whether eggs from the intertidal area were more salinity tolerant than those from freshwater areas. The second part of the laboratory component of this study was devised to examine the effects 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 motility, (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 . 58 MATERIALS and METHODS S a l i n i t y Tolerance of Salmon Eggs Experiment Gamete Collection 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 River, 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 collected from 4 females and 4 males i n each area and transferred to the laboratory at PBS i n a cooler maintained between 2 and 5°C. Gametes collected from the i n t e r t i d a l s i t e were designated as B-group eggs, with respect to the brackish water of this area, and gametes collected from the freshwater s i t e were designated as F-group eggs, with respect to the freshwater of this 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 with other f i s h that may have been schooling i n t h i s area before moving further upstream. Pr i o r to f e r t i l i z a t i o n the eggs were inspected for individuals 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 for unusual colour, and tested for m o t i l i t y using a l i g h t microscope (100X magnification). Vigour and duration of sperm m o t i l i t y was assessed following the addition of water. Normal m o t i l i t y was characterized by very vigorous movement maintained for 15 to 25 s by the majority of the sperm. 59 The eggs and mi l t were pooled together within both groups (B and F). Batches of about 1500 eggs, taken from each of these two pools, were separated and further divided for s i x s a l i n i t y treatments (0, 5, 10, 15, 20, and 30°/oo) ^n e eSgs were f e r t i l i z e d by the dry method with an egg to mi l t r a t i o of 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 different 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 . , chronological order of f e r t i l i z a t i o n was 10, 20, 0, 15, 30, and 5%o) • After f e r t i l i z a t i o n , but before the st a r t of the saltwater exposure treatments, a l l of the eggs were placed i n freshwater (0°/oo) f °r 12 h to allow water ac t i v a t i o n and hardening to occur without any confounding effects 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 thick a c r y l i c and p l a s t i c netting with 1 mm square holes. Each rectangular incubator was 8.6 cm long and 7.9 cm wide and consisted of a bottom and a l i d section. These two sections were 1.9 and 0.3 cm high respectively. Each section was covered with p l a s t i c netting so that when placed together they formed a completely enclosed box with mesh on the top and bottom. Handles were glued to the bottom section. The l i d s were held i n place by four 2.2 cm long pieces of a c r y l i c dowel located 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 stretching small e l a s t i c s around individual u n i t s . The incubators were supported on racks also made from 3.2 mm thick a c r y l i c 60 that were designed to hold 12 incubators and to f i t into temperature controlled tanks at the PBS laboratory. 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. Ci 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 for a l l tanks. Temperatures were maintained at 9.0 + 0.1°C by cold water cooling c o i l s and thermostatically controlled e l e c t r i c heating elements. Temperature gradients within tanks were tested and found to be negligible (+ 0.02°C). The freshwater used was dechlorinated and f i l t e r e d Nanaimo c i t y water. The.characteristics of th i s water were as follows: t o t a l hardness as CaC03, 19.2 mg/1; s p e c i f i c conductance, 59 umhos/cm; pH, 6.9; Ca+2, 6.6 mg/1; Na+, 3.2 mg/1; Cl"- 2.2 mg/1; Mg+2, 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 usually was 28-29°/0 0 s a l i n i t y a small addition of Instant Ocean Synthetic Sea Salt ™ was used to produce 30°/oo s a l i n i t y saltwater. Except for the f i l l i n g of freshwater tanks, a l l water mixtures were premixed volumetrically i n 50 1 carboys to allow for coarse adjustment of temperature and s a l i n i t y . Further fine adjustments were made i n the tanks. S a l i n i t y was measured using the low precision t i t r a t i o n method described by Strickland 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: This experiment was designed to test the 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 replicate incubators were used for every treatment, each one containing about 200 eggs. Due to space restrictions, replicate incubators were kept in the same tanks and by s t r i c t definition should be referred to as subsamples. Ten tanks were set up; 6 treatment tanks were used for the test s a l i n i t i e s of 0 to 30°/oo and 4 freshwater tanks were used for holding purposes. The freshwater tanks were required for 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 influx in the intertidal zone was simulated by manually transferring the incubators from freshwater to their 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 jarred the eggs or caused them to r o l l around. Any accidental shocks were recorded for future reference. Before returning the incubators to freshwater, they were rinsed in a separate freshwater bath to minimize saltwater contamination of the freshwater tanks. To equalize any potential 62 Fig. 9. Exposure regime experienced by eggs in the salinity tolerance experiment conducted in the laboratory study. Treatments involved 6 sa l i n i t i e s (0, 5, 10, 15, 20, and 30°/oo) a t 3 exposure times; 2 intermittent (4 and 8 h) and 1 constant exposure (24 h). The 15°/oo salinity treatment regime is provided as an example. 63 bias introduced by movement e f f e c t s , control incubators i n the 0 /0 0 treatment tank were mock transferred 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 transferred. 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 into t h e i r respective tanks. Thus a l l incubators were transferred, or mock transferred, twice during every 24 h period. Physical movement of the eggs apparently was not a problem since i n the control treatments and many of the lower s a l i n i t y treatments, egg survivals were high ranging from 90-98% (Fig. 10). Even i f minor movement effects did occur they would have been distributed 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 locations of the tanks were minimized by keeping the order random and changing the locations every 6 to 10 days. Within tanks, potential small scale differences i n water c i r c u l a t i o n were accounted for by rotating the 10 to 12 incubators i n each tank; each incubator occupied every one of the 12 locations 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 after 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 solution (40 ml g l a c i a l acetic acid, 50 ml formaldehyde, 60 ml gl y c e r o l , and 850 ml d i s t i l l e d water). In 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 tests revealed that 98.3-100.0% of the eggs were f e r t i l i z e d . 64 Fig. 10. Egg to alevin stage survival rates (8 d post-hatching) of the freshwater source (F-group) and intertidal source (B-group) eggs at 6 different s a l i n i t i e s and 3 exposure times. Each point represents the mean (+ 2SE) of 2 replicate incubators containing about 200 eggs each. Open circles represent recalculated mean survival rates (see text). Therefore, no corrections were made i n calculating the f i n a l survival values. Mortality samples were picked and preserved i n Stockard's solution 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 (Fig. 11), or the yolk sac epithelium i n la t e r developmental stages, was obviously damaged ( i . e . , egg appeared whitened and opaque to the naked eye due to coagulation of yolk material 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 material did not turn white i n colour). Dead eggs were examined for signs of development, obvious abnormalities, and possible causes of death. Hatching rates of the eggs were assessed by noting the time of the f i r s t hatched al e v i n (larvae) i n each incubator and subsequently estimating the percentage of hatched eggs u n t i l the process was complete. The experiment was terminated at about one week post-hatching (1600 h elapsed time of development (ET) @ 9.0°C) and f i n a l egg to alevin survival rates were calculated from f e r t i l i z a t i o n to th i s time. Data Analysis: 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 survival results of the two groups of i n t e r t i d a l source and freshwater source eggs were analyzed by one-way ANOVA's for each exposure time (4, 8, and 24 h) within each group (B and F). Multiple 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 Fig. 11. Schematic diagram of a f e r t i l i z e d and water activated salmon (not to scale). 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 transferred 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 external egg membrane (Fig. 11) (Groot and Alderdice 1985). Spare F-group eggs, f e r t i l i z e d at the same time as those used i n the main experiment, were used i n these t e s t s . These eggs were well eyed and at about 700 h ET at 9°C. Osmolality of PVF was measured using a Wescor vapour pressure osmometer (model 5100C) (Wescor Inc., Logan, Utah, USA). Microcapillary tubes (10 nl) were used for c o l l e c t i n g and dispensing a l l standard and test 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 ca r e f u l l y making a very small hole i n the external egg membrane and withdrawing the f l u i d into the c a p i l l a r y tube. A baseline osmolality for eggs i n freshwater was established 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 transferred to 2 0° /0 0 and measurements started immediately. These were continued, as rapidly as the technique allowed, u n t i l osmolality changes i n the PVF had leveled o f f . Equilibrated eggs also were tested at 1 and 4 h intervals to observe i f any additional changes occurred over time. A similar sampling procedure was done when the eggs were returned to freshwater (0°/00) from 2 0 ° /0 0. Five sampling runs were conducted for the transfer of eggs into 2 0° /0 0 and three for the transfer back to freshwater. 68 Modelling Changes i n PVF Osmolality : Changes i n PVF osmolality as a function 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, with constants a and b not equal to zero, i s described by equation (1): Y(t) = [ Y,b + ( Y,b - Y,b ) j . e -a<fc-T1> ] ( 1 ) I . e - a ( T 2 - T l ) where T1 and T2 are the i n i t i a l and f i n a l times (min.), and Yx and Y2 are the predicted PVF osmolalities (mmol/kg) at those times. The predicted values of Y(t) were determined by a minimization process using a nonlinear parameter estimation technique (simplex) and the associated software package (Mittertreiner and Schnute 1985). Effects 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 Collection: Chum salmon gametes were collected from Big Qualicum Fish hatchery on Vancouver Island, B.C. i n November 1986. Eggs were stripped from 3 females and m i l t was collected from 5 males. The gametes were transferred within a cooler (2-5°C) to PBS where they were checked for abnormalities. Except for the sperm m o t i l i t y t e s t s , eggs and m i l t were pooled for a l l experiments. Experimental Design: Three separate experiments were conducted to test the effects 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 for a l l three experiments. Post-hoc multiple comparisons were done according to the procedure outlined i n the s a l i n i t y tolerance experiment. Sperm Motility 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 different males were tested separately and each test was replicated 5 times. A very small droplet of sperm, as much as would adhere to the end of a sharp dissecting probe, was placed i n the center of a hemacytometer s l i d e and a coverslip 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 test s a l i n i t y water was added onto the s l i d e beside the coverslip. Surface tension quickly dis 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 for complete and near simultaneous d i l u t i o n by the water drop. Upon activation of the sperm ( i n i t i a t i o n of vigorous movement), the timer was started. 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 ac t i v a t i o n to about 95% immotility. Immotility was defined as the cessation of vigorous movement and usually included a period of slow sedentary vibratory 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 saline water was tested by premixing sperm, pooled from 3 males, with various concentrations of saline water before using i t to f e r t i l i z e a small beaker of eggs (25 ml). The test s a l i n i t i e s used were 0, 5.0, 10.0, 12.5, and 15.00/oo- After 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. After l e t t i n g i t stand for 1 minute the contents were poured c a r e f u l l y into a divided compartments i n heath tray (Heath Tecna Corp., Tacoma, Washington, USA) f u l l of 9°C freshwater. Three replicates were tested for each s a l i n i t y . The order for testing the dif f e r e n t s a l i n i t i e s was randomized. An additional experiment, si 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 following a longe period of sperm a c t i v a t i o n . The sperm and saltwater mixture was l e f t for 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 after 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 replicate for 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 saltwater. Within each test s a l i n i t y , f e r t i l i z e d eggs were maintained i n th e i r respective s a l i n i t i e s for either 1, 15, 60, or 240 minutes. This 71 experiment simulated the potential 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 flood tide when any gametes released into the stream would encounter saline conditions. About 50 eggs (20ml) and 0.2ml sperm were added simultaneously to 50 ml of test s a l i n i t y ; 0, 5.0, 10.0, 12.5, or 15.0%o- After mixing gently they were allowed to remain for 1 minute. Depending on the treatment, the eggs were either transferred d i r e c t l y to freshwater (1 minute p o s t - f e r t i l i z a t i o n exposure), or transferred to a tank containing the respective test s a l i n i t y and maintained for 15, 60, or 240 minutes (15, 60, and 240 minute exposures respe c t i v e l y ) . Three replicates were established for each combination of s a l i n i t y and exposure time tested. Additional measurements were conducted on two representative treatments (15 and 240 min exposures) to assess the effect of saltwater on the water ac t i v a t i o n process of the egg. Egg weight was used as an indicator 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 intervals 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 after being placed into the test s a l i n i t i e s , and more than 12 h after being transferred from the test 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 after being placed into the test s a l i n i t i e s , and more than 12 h after being transferred back to freshwater. 72 Egg and sperm v i a b i l i t y was evaluated i n the same manner as for the sperm v i a b i l i t y experiment. Samples of 15 eggs were picked from each repl 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 for 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 rapidly i n response to increased ambient osmolality. Within 20 to 25 min of being transferred to 20°/oo s a l i n i t y water, the PVF osmolality had reached 95% of the maximum and was completely equilibrated 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 saltwater, occurred i n about 13 min, ha l f the time of the i n f l u x ( F i g . 13). The lines drawn through the data points represent the predicted 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, Materials and Methods). Model parameters, predicted values and residuals 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 invariable. Eggs exposed to the intermittent treatments (4 and 8 h) at s a l i n i t i e s of 15°/oo or l e s s , had survival rates that were s t a t i s t i c a l l y the same as the freshwater controls (P > 0.05). At 20°/oo> the 4 h exposure also had high survivals s i m i l a r to the controls, whereas the 8 h exposures did not. Eggs subjected to 20°/oo f °r 8 n Pe r ^ay showed a s i g n i f i c a n t decrease i n survival (P < 0.001) (F i g . 10). No eggs survived i n the 30°/oo treatments even at the least 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 upon t r a n s f e r o f chum sa lmon eggs f r om 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 and t h e 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 t h e 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 g r ow th mode l ( see t e x t and Append i x 1). ' Amb i e n t o s m o l a l i t y ' r e f e r s t o t h e o s m o l a l i t y o f t h e s a l t w a t e r . PVF OSMOLALITY (mmol/kg) 600 - > 500 -400 -TIME (min.) F i g . 13. Observed and predicted changes i n p e r i v i t e l l i n e f l u i d (PVF) osmolality upon transfer of chum salmon eggs from saltwater (20°/oo s a l i n i t y ) to freshwater ( 0 0 / 0 0 ) • The points represent the observed data and the s o l i d curved l i n e represents the predicted values obtained using a general growth model (see text and Appendix 1). 'Ambient osmolality' r e f e r s to the osmolality of the freshwater. Eggs i n the B-group responded much more variably to the s a l i n i t y treatments, even though the trends were the same as the F-group eggs. Surprisingly, the survival rates of the controls i n the B-group were lower than the rates observed at 5 and 1 0° /0 0 for 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 especially prominent i n the freshwater controls, occurred during the f i r s t 3 days of incubation. Recalculating the survival rates for both groups of eggs with these i n i t i a l m o rtalities removed ( i . e . , subtracting these mortalities from both the f i n a l t o t a l number of mortalities and the i n i t i a l t o t a l number of eggs), resulted i n markedly higher survivals but did not account e n t i r e l y for the differences observed between the control survival rates and those of the 5 and 10°/oo treatments. Figure 10 demonstrates how t h i s series 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 largely to the controls and how i t was much less 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 recalculated B-group values indicated that the control s u r v i v a l rates for 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 survival 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 values, percent survival rates i n the B-group were lower and generally more variable i n a l l s a l i n i t i e s , especially for eggs exposed to 10 and 15°/oo f °r & h. Survival of eggs exposed to 2 0° /0 0 for 8 h i n the F-group was si m i l a r to the survival of eggs i n the same condition i n the B-group (Fig. 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 also. 77 The majority of dead eggs had developed abnormally. Therefore, observed differences i n survival rates i n the various s a l i n i t y treatments largely were due to differences 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 usually amounted to no more than 1 or 2%. However, the certainty with which I could accurately c l a s s i f y the mor 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) varying degrees of decomposition of the eggs while they were s t i l l i n the incubators, and (3) tissue d i s t o r t i o n p o t e n t i a l l y resulting from osmotic stress 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 , categorizing m o r t a l i t i e s provided only general indications 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 resulted i n 100% mortality (Fi g . 10). With the B-group eggs, a result s i m i l a r to the intermittent treatments was seen; control survivals were markedly lower than those i n the 5°/oo treatment. Again, a large proportion of t h i s reduced survival was due to i n i t i a l m ortalities 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 did not have any negative effect 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 controls (P > 0.05). For the B-group eggs this treatment may even have had a mitigating effect considering the problems observed with these controls. 78 Eggs i n 10, 15, and 20°/oo constant exposures developed for varying periods of time before dying. In 10 and 15°/oo eggs began to die i n large numbers between 400-800 h ET. Many of the f i n a l m ortalities i n these two s a l i n i t i e s were 'eyed' (stage 21, Vernier 1969) although often epiboly was incomplete. In many cases a seemingly permanent hole existed at the posterior region of the yolk sac where normally, i n the f i n a l stage of epiboly, the yolk sac e p i t h e l i a l c e l l s converge and form a complete layer around the yolk. In some cases the yolk actually 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 protrusion. In other cases, even i f the c e l l s had formed a complete layer, the area appeared malformed and abnormal i n structure. I t often resembled scar t i s s u e , consisting of tracts of built-up tissue radiating out from a central point. In constant 2 0° /0 0 exposure, eggs did not develop past 50 h ET (stage 7, Vernier 1969), even though I could not id e n t i f y them p o s i t i v e l y as mort a l i t i e s u n t i l about 200-300 h ET. Hatching rates of the eggs differ e d for 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 control treatments (Table 4). Except for the 5°/oo s a l i n i t y treatments, data are presented only for intermittent 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 sa l in i t i e s of 3 0 ° / 0 0 were not included since none of these eggs survived to hatching. F-GROUP B-GROUP Percent Hatch (%) Percent Hatch (%) Treatment Treatment Sal inity (°/oo) Treatment Sal ini ty ( ° / 0 0 ) Exposure Time 0 5 10 15 20 0 5 10 15 20 ETM314h 24h 0 7 NS2 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 ET=1392h 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 ET=1410h 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 1 ET is the Elapsed Development Time in hours since f e r t i l i z a t i o n . 2 NS denotes no survival to the hatching stage. more quickly than those from the B-group. Within the F-group, eggs in the 5 and 10°/oo intermittent sa l in i ty treatments hatched more quickly than those in the 15 and 20°/oo treatments. This trend was not observed in the B-group eggs (Table 4). 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 restrictions. It is 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., Pacific Biological Station, Nanaimo, B.C.). Effects Of Salinity On The Fer t i l i z a t i o n Process Sperm Motility Duration of sperm motility did not vary significantly between the 3 different males tested within a given salinity (P > 0.05). However, sperm motility varied greatly between the different test s a l i n i t i e s . Chum salmon sperm were motile for a significantly shorter time in freshwater than in the 5, 7.5 and 10%o s a l i n i t i e s (P < 0.001) (Fig. 14). In 12.5%o the time period u n t i l 95% immotility was about half that observed in 5 to 1 0 ° / 0 0 . In 1 5 ° / 0 0 s a l i n i t y motility was assessed as n i l , since no true swimming activity was observed (Fig. 14). However, in addition to quantitative differences of activity between the test s a l i n i t i e s , qualitative differences also existed. The general response, in s a l i n i t i e s of 0 to 12.5°/oo ^n which activity occurred, was an i n i t i a l period of vigorous activity followed by a period of decreasing activity t r a i l i n g off to total immotility. Even once the sperm had stopped swimming forward, they usually continued to vibrate in one spot for 1-5 min. before becoming totally inactive. The most vigorous activity was observed in s a l i n i t i e s ranging from 5 to 1 0 ° / 0 0 . In s a l i n i t i e s above this, 1 2 . 5 ° / 0 0 , and 81 Fig. 14. Duration of sperm motility in response to various ambient sa l i n i t i e s (see text for definition of motility). The duration of motility was assessed from the initiation of motility following the addition of the test water, to about 95% immotility. No motility per se was observed at 15°/oo salinity but some movement was recorded. Each point is the mean (+ 2SE) of 5 replicates from 3 different males. No difference in motility . was observed between the individual males. 82-below i t , 0°/oo> there was a noticeably slower activation time and a shorter duration of a c t i v i t y . In 15°/oo> m°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 did vibrate s l i g h t l y . Occasionally I observed a single sperm swimming vigorously for a few seconds. In 2 0 ° /0 0, there were very s l i g h t vibratory movements i n a few of the replicate tests but usually 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 after 18 h of development, of sperm mixed for 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 that of the control (0°/oo) (p > 0.05). V i a b i l i t y did 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 (Fig. 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 at 1 0° /0 0 (P < 0.05). This same relationship did not hold when the sperm were premixed for 15 s i n the various test 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 for the whole range of s a l i n i t i e s tested, 0 - 1 5°/0 0. Although some v a r i a b i l i t y occurred, the basic response was a FS ranging from 60-70% (Fig.15). The test at 5 ° /0 0 was excluded from calculations 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 resulted i n further reduction of f e r t i l i z a t i o n rates. 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 for either 5 s ( s o l i d line) or 15 s (broken l i n e ) , before adding the mixture to eggs. Each point i s the mean (+ 2SE) of 3 replicates 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 affected 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 resulted i n reduced v i a b i l i t y (P < 0.05) ( F i g . 16). These results were sim i l a r to the sperm v i a b i l i t y tests; 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% (Fig. 16). Eggs maintained i n the respective test s a l i n i t i e s for durations longer than 1 min, i . e . , 15, 60, and 240 min., did not suffer any additional effects due to prolonged exposures ( F i g . 16). Eggs developed normally to 18 h ET with no apparent problems i n those s a l i n i t i e s that did 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 effect (P < 0.001), whereas duration of exposure and the interaction 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 for 240 min did show signs of i n h i b i t i o n of water hardening, even after exposure to freshwater for 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 either (1) lower s a l i n i t i e s (0-10°/00) for a sim i l a r duration (240 min) or (2) sim i l a r s a l i n i t i e s (12.5 and 15°/00) for a shorter duration (15 min.) (Fig. 17). However, due to the v a r i a b i l i t y i n egg weights within the individual 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 (min) 5%o TREATMENT (min) O I5H FRESHWATER 12-16 (h) PRE-FERT'N 0-(min) 5%oTREATMENT 15 (min) FRESHWATER 12-16 (h) PRE-FERT'N n (min) ° 5 %o TREATMENT 15 (min) FRESHWATER 12-16-(h) PRE-FERT'N n r ( m i n ) 0 _ l 5%o TREATMENT I5-| (min) FRESHWATER 12-16-(h) PRE-FERT'N n (min) U 5%o TREATMENT 15 (min) FRESHWATER 12-16-(h) 3-< 0%o 0 150 240-12-16-' 5%a 0 150-240 3 1 12-16-3 ^ I0%o o 150 240-3 • 12-16-!2.5%o 0 1504 240 -< 12-16 3-< l5%o OH 150 240 H 12-16-3 - 0°/c os_ 3 ^ 5%, 3—• 3 ^ 10%, 3 1 3-< 3 i 3 ^ 12.5%, R-* 333 • 15%, 240 280 320 360 EGG WEIGHT (mg) 15 MIN EXPOSURE 240 280 320 360 400 EGG WEIGHT (mg) 240 MIN EXPOSURE Fig. 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 in freshwater. Each point is 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, alevins, and f r y are able to tolerate constant saline conditions (31.8°/oo) better than coho (CL. ki s u t c h) . chinook (0. tshawytscha) and sockeye salmon (Oj. nerka) (Weisbart 1968) . Although chum and pink salmon embryos and alevins are not t r u l y euryhaline, Weisbart (1968) showed that t h e i r increased resistance to near f u l l strength seawater was a re s u l t of better 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 for embryos and alevins and reported that chum and pink had the longest survival times. Unfortunately, t h i s information does not provide any insight into the c r i t i c a l exposure time at which irreparable damage occurs and death becomes imminent, even i f the embryos were returned to freshwater. When osmotically stressed eggs, alevins, or f r y are returned to a non-stressful environment before some threshold or c r i t i c a l point, usually they w i l l recover completely (Rockwell 1956, Holliday and Blaxter 1960). In l i g h t of the reported differences i n saltwater tolerance, the question arises whether t h i s point would be diffe r e n t for the f i v e species of Oncorhynchus? Bailey (1966) examined the s a l i n i t y tolerance of pink salmon eggs alevins and f r y i n a simulated i n t e r t i d a l environment. He observed no adverse effects at moderate exposure levels; 4 h or less twice d a i l y i n s a l i n i t i e s of 10 - 1 5°/0 0. In high s a l i n i t i e s (28°/00) however, he observed 0% surv i v a l during the f i r s t 12 days for eggs exposed for 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 resulted i n egg survivals of 50 and 100% respectively. These results suggest that c r i t i c a l exposures to saltwater for 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 Bailey's work. My results indicate that the c r i t i c a l s a l i n i t y for chum salmon eggs i s between 20 and 3 0 ° /0 0. No reduced survival 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 for 8 h per day resulted i n 48.0 and 41.2% survival (F and B groups respectively). I observed no survival i n 3 0° /0 0 for either exposure time, 4 or 8 h. A comparison of the intermittent s a l i n i t y tolerance of chum and pink salmon eggs indicates that l i t t l e difference exists 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 tolerant to both concentration and duration of saltwater exposure. However, before accepting the observed differences between my results and Bailey's (1966) as the result 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 possible 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 physically into tanks containing the test s a l i n i t i e s and as a result experienced very abrupt changes i n ambient s a l i n i t y . The abruptness of these changes may have had adverse effects on the embryos. Although d e t a i l s are not given, i t seems l i k e l y that eggs i n Bailey's (1966) study experienced less abrupt changes i n ambient s a l i n i t y . Weisbart (1968) examined this 89 potential problem when he tested the effect of an abrupt change from 0 ° /0 0 into 3 1 . 8°/0 0 s a l i n i t y , compared to a stepwise one over a four day period into the same f i n a l s a l i n i t y . He showed no difference i n LC50 values of alevins exposed to the two exposure regimes. Since he found the alevin stage of development to be the most sensitive to saltwater exposure, the absence of any d i f f e r e n t i a l survival suggests that effects 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 possible explanation for the observed differences i n survival rates between Bailey's study (1966) and mine, i s a temperature related one. Rockwell (1956) reported that eggs and alevins incubated at lower temperatures had a higher tolerance to saltwater exposure than those incubated at higher temperatures. Therefore, i f Bailey (1966) maintained lower temperatures i n his test s a l i n i t i e s than I did i n mine (9.0 + 0.1 C), the observed differences i n survival i n the two experiments could be, at least i n part, due to the d i f f e r e n t i a l effects of temperature on s a l i n i t y tolerance. I t i s quite possible that Bailey's (1966) water was cooler since he was pumping saltwater i n from a bay and using stream water for his freshwater source. These possible causes remain speculative since i n s u f f i c i e n t d e t a i l of Bailey's experimental procedure were provided to make any conclusive statements regarding procedural differences. Further, the precise effects of temperature on saltwater tolerance are not well understood. 90 Rockwell (1956) also presented data that indicated s i m i l a r i n t e r s p e c i f i c differences over a range of s a l i n i t i e s and temperatures, even though he did not provide any discussion of i t . Weisbart (1968) also reported a difference between chum and pink salmon saltwater tolerance; LC50 values for pink salmon alevins were s l i g h t l y higher than those for chum salmon. Furthermore, hi s time series data provide physiological evidence of a possible difference i n saltwater tolerance between chum and pink salmon embryos and alevins. After 8 and 12 h exposure to 31.8°/oo> pfnk salmon generally had lower blood osmotic, Na+, and Cl" concentrations than chum salmon, even though pink demonstrated among the highest levels i n i t i a l l y . Although the differences were not especially large, they suggest a functional basis for possible 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 less p a r t i c u l a r and w i l l spawn throughout the whole zone, including 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 for i n t e r t i d a l pink salmon i s further 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 possibly higher concentrations of saltwater exposure than i n t e r t i d a l chum salmon. Thus the reported differences i n saltwater tolerance between chum and pink salmon eggs may be real and i n fact r e f l e c t differences i n the l i f e h i s t o r i e s of these two species of salmon. Except for Bailey (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 rationale often was introduced from the perspective of i n t e r t i d a l spawning. Rockwell (1956) provided a thorough and interesting h i s t o r i c a l review of experiments dealing with the effects of saltwater on salmonid eggs. From thi s i t i s apparent that many pre-1956 researchers, including Rockwell, chose not to make a d i s t i n c t i o n between testing the effects of constant versus intermittent saltwater exposure. The ov 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 alevins cannot survive prolonged periods of time i n concentrations of saltwater greater than isosmoticity (approx. 10-12°/oo> Shen and Leatherland 1978a). Salmonid eggs and alevins are not able to f u l l y iono- and osmoregulate i n hyperosmotic conditions. Yet, depending on exposure conditions and species involved, these embryos and alevins are able to survive i n intermediate to f u l l strength seawater (up to 30°/00) for l i m i t e d periods of time. Survival data, corroborated by measurements of ionic and osmotic tissue 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 resul t of tissue tolerance i n some species and p a r t i a l regulation i n others. Information from my study and Bailey's (1966) indicates that eggs can survive exposure to higher s a l i n i t i e s for weeks at a time when provided with a regular freshwater interlude between exposures. A period of recuperation i n freshwater during which embryonic tissues can balance t h e i r osmotic and ionic concentrations appears to be e s s e n t i a l . In l i g h t of the regulatory mechanisms available to salmonid embryos and alevins this conclusion seems reasonable. 92 As mentioned e a r l i e r , the young teleost embryo probably regulates i t s internal ionic and osmotic environments largely through passive maintenance of tight plasma membranes. At the eyed stage this 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, including rainbow trout (Shen and Leatherland 1978b) but not coho salmon (Leatherland and L i n 1975), chloride c e l l s have been located i n the yolk sac epithelium surrounding the yolk, which i n the e a r l i e r stages of development was contained only by a plasma membrane. I f chloride c e l l s do function 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 least consistent with the available information. Weisbart (1968) found that t h i s species could not control i t s int e r n a l ionic and osmotic concentrations. Therefore, i n view of his findings that pink and chum salmon embryos could at least p a r t i a l l y control t h e i r internal ionic and osmotic concentrations, i t would be useful to conduct a s i m i l a r search for chloride c e l l s i n these two species. Although the potential for regulation by s p e c i f i c c e l l s exists i n teleost post-eyed embryos and alevins, i t i s not known to what extent t h i s potential 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 fluxes across the plasma membrane i n the early embryo, i s dependent upon low plasma membrane permeability. Drawing a p a r a l l e l between the amphibian egg and the f i s h egg, Alderdice (1987) summarized that the membrane permeability c o e f f i c i e n t (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 affected 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 internal hydrostatic pressure of the egg. Egg hydrostatic 93 pressure i s related 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 cav i t y , the permeability c o e f f i c i e n t (Pd) i s high. However, once passed out of this osmotically controlled environment, Pd remains high only for a short period following f e r t i l i z a t i o n before i t drops rapidly to a very low value ( i . e . , an i o n i c a l l y and osmotically tight 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 result of water imbibation across the external egg membrane from the ambient environment. In response to the osmotically active 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 hydrostatic pressure develops i n the egg and rises to an equilibrated l e v e l , ranging from 30-90 mm Hg i n the fi v e species of north american P a c i f i c salmon (Alderdice et a l . 1984). This internal pressure exerts a tension on the external egg membrane and varies considerably depending on species, stage of development, and external conditions, 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 affects plasma membrane permeability. For example, hyperosmotic media that produce a decrease i n internal egg pressure, result i n an increase i n membrane permeability. Another important factor affecting plasma membrane permeability i s temperature. Although the interactions are very complex the basic effect i s increased permeability i n response to increased temperature (Alderdice 1987). 94 How do the physiological effects associated with saltwater exposure, intermittent or constant, relate to observed survival responses of eggs exposed to such conditions? Assuming plasma membrane permeability i s increased by a lowering of the internal 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 effects on the young developing embryo. In general, survival curves of teleost embryos exposed to various s a l i n i t i e s indicate 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 tolerant (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 sublethal or chronic effects (these may result i n death for a portion of the test 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 effect i s acute or l e t h a l ( a l l of the f i s h die, usually within a short period after f e r t i l i z a t i o n ) . Evidence for sharp mortality response curves i n f i s h eggs exposed to saltwater can be found i n a number of studies including this one. Chum salmon eggs exposed intermittently for 8 h per day i n my experiments, showed no adverse effects up to s a l i n i t i e s of 15°/oo> a chronic or sublethal effect at 20°/oo> and an acute or l e t h a l effect 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. Bailey (1966) recorded survivals of 100% i n exposures of less 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 resulted i n decreasing survivals that reached 0% at 9.3 h twice d a i l y and produced a marked increase i n developmental abnormalities. Shen and Leatherland (1978a) reported survivals of 79.6 and 28.1% at 10 days post-hatching i n rainbow trout alevins exposed to 11 and 95 1 3° /0 0 continuously throughout development. Many of the eggs that did hatch i n the 1 3° /0 0 treatment were deformed and usually did not survive beyond 12h post-hatching. Other studies concerning freshwater species (Parry 1960, Kashiwagi and Sato 1969, Watanabe et a l . 1985) and marine species (Holliday 1965, Alderdice et a l . 1979) have reported results demonstrating si 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 related to plasma membrane permeability. I t seems reasonable to speculate that eggs exposed to 2 0° /0 0 for 8 h d a i l y i n this study, experienced temporarily lowered inte r n a l egg pressures as a result of the increased t o n i c i t y of the external medium. I f we accept that the previously discussed relationship of lowered internal 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 permeabilities of the embryonic c e l l s . Reduced membrane tightness would allow for increased ion and water fluxes along the ex 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 nl y 4 b dai 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 or, the exposure to higher internal ionic concentrations for 4 instead of 8 h, was not long enough to produce irreparable c e l l u l a r damage. Due to an .incomplete understanding of the toxic effects of saltwater exposure and the chronological development of osmo- and ionoregulatory mechanisms i n the early l i f e stages of salmonids, these suggestions must remain speculative. 96 In view of the effects that intermittent compared to continuous saltwater exposure has on egg development, one might ask whether i t i s the duration of exposure at any one given time or, 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 for 4 h twice d a i l y exhibit the same survival rate as eggs exposed to 20°/oo for 4 h only once d a i l y (94.7%) or, would the rate be si m i l a r to eggs exposed to 20°/oo f °r ^  h once d a i l y (45%)? I t i s also possible that the survival rate would be intermediate between the two. Thus i t i s useful to determine the importance of the return of an egg to freshwater between saltwater exposures, i n r e l a t i o n to the actual saltwater exposure i t s e l f . I t i s not obvious whether i t i s this period of freshwater reprieve that i s c r i t i c a l or, 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 reprieve. Why do some members of a test group f a i l to survive test conditions while others seemingly remain unaffected? Knowledge of the s p e c i f i c mechanisms that produce the toxic or negative effects of saltwater exposure also may provide some insights into variable survival rates w i t h i n , as well as between test treatments. Realization of t h i s , could possibly help explain the variable egg survival rates observed i n this study for the B-group eggs compared to the F-group. Variable and low rates were especially prominent i n the control treatment eggs of B-group (0°/oo) (Fig- 10)- An i n i t i a l wave of mort a l i t i e s within the f i r s t 3 days accounted for much of the observed difference but the cause remains unexplained. Some potential causal factors 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% for a l l incubators i n both groups), (2) incubation conditions 97 (these were the same as the F-group control eggs, which showed consistently high s u r v i v a l , 92-97%), and (3) movement effects ( a l l incubators were given the same movement treatments, moreover, v a r i a b i l i t y between replicate incubators and exposure time treatments generally was low). The eggs i n question (control treatment) were from the same pooled source as those used for a l l of the other B-group s a l i n i t y treatments, yet the effect of lowered survival was seen only i n these control 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 incubation, the data show that the B-group eggs survived better i n low levels of s a l i n i t y than i n freshwater. One possible interpretation of this observation suggests that these eggs actually required low to intermediate levels of s a l i n i t y to develop normally. Or, perhaps i t was a gamete source problem that affected 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 conjectural. The experiment designed to test whether i n t e r t i d a l eggs were more s a l i n i t y tolerant than freshwater eggs was not successful 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 obtaining eggs from true i n t e r t i d a l spawning parents, and second were the previously discussed problems with the controls and o v e r a l l survival of the B-group eggs. In spite of these problems, the results show that under the experimental conditions tested, there was no indication of lower s a l i n i t y tolerance i n the freshwater source eggs. Perhaps potential differences i n s a l i n i t y tolerance, 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 isolated 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 further 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. Alt e r n a t e l y , one might consider testing f i s h i n a r i v e r system where discrete i n t e r t i d a l and freshwater spawning populations already are thought to ex 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 inte r m i t t e n t l y , tolerated higher concentrations than those exposed continuously. Eggs exposed to 20°/oo o r less for 4 h d a i l y showed no noticeable difference i n su r v i v a l rates compared to the controls. However, eggs exposed to 20°/oo f °r twice as long, 8 h, showed a marked decrease i n survival (45%). None of the eggs survived when exposed to 30°/oo f °r 4 or 8 h per day. Except for some inexplicable v a r i a t i o n observed i n the B-group eggs, survival rates were high for a l l intermittent treatment s a l i n i t i e s of 15°/oo o r l e s s , irrespective of the duration. 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 li 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, usually with deformed yolk sacs resulting from abnormal epiboly), and high su r v i v a l and normal development i n 0 and 5 ° /0 0. A comparison of these results to the only other intermittent exposure study i n the l i t e r a t u r e , suggests possible i n t e r s p e c i f i c differences i n saltwater tolerance between pink and chum salmon embryos. D i f f e r e n t i a l s urvival rates suggest that pink salmon embryos and alevins are more tolerant than chum salmon to both duration and concentration of intermittent saltwater exposure. Further, i n t e r t i d a l spawning di s t r i b u t i o n s of these two 99 species indicate that the observed interspecific differences may be a reflection of different l i f e histories; pink salmon typically spawn further down in the intertidal zone than chum salmon. Regulatory mechanisms available to salmonid embryos and alevins are relatively simplistic in that they function at a cellular level and do not involve organs per se. In the early embryo especially, 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 survival. Membrane permeability is affected directly and indirectly by many factors including osmotic pressure of the external medium (e.g., salinity) and temperature. Based on results from this study and others, dysfunction of the plasma membrane in hyperosmotic media is speculated to be an important contributing factor in the deleterious nature of prolonged saltwater exposure. As speculated earlier, eggs may need a time period of freshwater exposure that is sufficiently long to re-establish their 'interior milieu' to preferred levels. Not u n t i l this requirement is met w i l l they be able to tolerate repeated exposures to saltwater without the occurrence of irreparable embryonic damage. Effects of Saltwater on the Fer t i l i z a t i o n Process Apparently, salmon that spawn in the intertidal 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 in 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 interested i n testing whether or not the presence of saline 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? Quantitative and qu 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. Similar results were summarized by Scott and Baynes (1980) i n a review on sperm biology. The cause was speculated to be the resul t of reduced osmotic stress i n low s a l i n i t y water compared to freshwater. In contrast, negligible m o t i l i t y ( « 1%) was observed at 15°/oo- The requirements for i n i t i a t i o n of sperm m o t i l i t y are well established; the most important i s a decrease i n K+ ion concentration below 2-5 millimoles/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+ concentration of about 9-13 mM (Bidwell and Spotte 1985, Morisawa 1987) which i n 1 5 %0 s a l i n i t y water would be reduced by about h a l f to 4-6 mM. Further, i n the presence of Na+ ions, s l i g h t l y higher levels of K+ 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 possible 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+ ion levels above the ac t i v a t i o n threshold. The fact that the K+ ion concentration of 1 5° /0 0 was close to the i n h i b i t i n g concentration may explain the variable 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 immotility with occasional bursts of a c t i v i t y by individual 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 results obtained from testing the effects 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 (Figs. 15 and 16). The s o l i d triangles 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 (Fi g . 14). that sperm m o t i l i t y was a reasonable indicator of f e r t i l i z a t i o n success. Generally t h i s relationship i s well established (Scott and Baynes 1980, Moccia and Munkittrick 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 relationship (Scott and Baynes 1980). In th i s study v i a b i l i t y of sperm was less sensitive to s a l i n i t y than would have been predicted 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 less vigor observed i n the 0 ° /0 0 was of no consequence to sperm v i a b i l i t y ( F i g . 18) (at least not under the test conditions used). Further, m o t i l i t y measurements indicated 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 test eggs i n the sperm v i a b i l i t y tests were f e r t i l i z e d ( F i g . 18). The procedure used to test sperm m o t i l i t y i n this study may have produced ambiguous r e s u l t s . I t i s not clear 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 li m i t e d when tested under a microscope s l i d e coverslip. 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 coverslip, whereas the same sperm activated i n an open vessel remained motile for at least 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 coverslip, and measured maximum durations of m o t i l i t y that were no greater than 25 s. This value i s similar to measurements observed i n th i s and other studies that have used coverslips (Baynes et a l . 1981, Rieniets and M i l l a r d 1987). Thus, testing of sperm m o t i l i t y on a microscope s l i d e with a coverslip appears to be a routinely used technique and at l e a s t , produces results that are comparable to other studies u t i l i z i n g this same technique. 103 In a review on salmonid spermatozoa, Scott and Baynes (1980) indicated that the duration of sperm motility varied considerably between different studies in the literature. They concluded that although some of these disparities were due to different c r i t e r i a for the assessment of motility, the majority were due to differences in the techniques used to assess motility. This suggests that the measurements of duration and vigor of motility conducted in this study are comparable only to measurements within this study and others using similar experimental procedures. But, in treatments where reduced or negligible motilities were observed in apparent response to test conditions, the responses probably were not confounded by procedural effects. These observations should remain valid and be comparable with other studies. In general however, comparison of results between studies requires a standardized method to minimize procedural biases. Considering the general lack of measurable motility in 1 5 ° /0 0, the FS rate of 53.5% was surprisingly high (Fig. 18). This suggests that either occasionally active sperm ( « 1%) observed in the motility tests at this s a l i n i t y were sufficient to f e r t i l i z e the 50 or so eggs tested in each replicate or, 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 after the i n i t i a l period of forward swimming act i v i t y , were no longer viable. These wavering movements may be similar to the vibratory movements seen in this study at the 15°/oo exposure. If so, one would expect a similar response of non-viability. However, Stoss et a l . (1978) found that sperm with low levels of motility (1-10% of f u l l y motile samples) retained high v i a b i l i t y . It is not totally 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 successful 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 results are si m i l a r to those observed here. The difference between these results and Rockwell's (1956) suggest that either considerable i n t e r s p e c i f i c differences e x i s t within the salmonids as a group or that assessment techniques were so different 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 inh i b i t o r y effect 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 addition to the effect 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 test 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 test s a l i n i t y . The l a t t e r experiment simulated most closely the natural spawning process and indicated that the presence of saltwater, 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 natural 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 negligible at t h i s concentration. The fact that v a r i a b i l i t y between replicates was not high, 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 did not provide any further insight into what that mechanism(s) might be. I f eggs i n the natural environment were f e r t i l i z e d under saline conditions, p o t e n t i a l l y they could be subjected to these conditions for 4 h or more after 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 indicated that the duration of saltwater exposure after f e r t i l i z a t i o n had no additional negative effect on FS, above and beyond that of the i n i t i a l exposure ( i . e . , the effect of the test s a l i n i t y after a 1 min exposure). One exception was noted however, eggs maintained i n 1 5° /0 0 for 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 general, these results indicated that eggs f e r t i l i z e d i n the w i l d under saline conditions probably would not suffer further deleterious effects from saltwater exposure during the remainder of the high tide cycle. However, th 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 after 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 did not test 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 additional effect 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 affected by prolonged exposure (4 h) to intermediate s a l i n i t i e s . Eggs maintained i n 12.5 and 15%o f o r 2 4 0 min after f e r t i l i z a t i o n had f i n a l weights that were noticeably 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 for a si m i l a r time period i n lower s a l i n i t i e s (0-10°/00) and (2) 106 eggs kept i n si m i l a r s a l i n i t i e s for only 15 min (Fig. 17). I f impairment of the water imbibation mechanism was r e a l , i t may have resulted 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 or, (2) a possible chemical f i x i n g of the external egg membrane (Zotin 1958, Kobayashi 1982). Actual hardening of the egg membrane while the egg remained f l a c c i d i n saltwater would physically prevent the egg from imbibing water and swelling upon return to freshwater. In conclusion, results obtained here indicate 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 affect the a b i l i t y of eggs to water harden normally, even after they return to freshwater. This information indicates that eggs spawned during high or flooding tides and i n the presence of the s a l t wedge, would experience low to n i l survival as a result of adverse effects 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 flood tides 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 alevin 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 o r less for 4 h/day or, s a l i n i t i e s of 15°/oo o r less for 8 h/day. Eggs exposed to 2 0° /0 0 for 8 h/day resulted i n s i g n i f i c a n t l y lower survival (41.0-48.0%) (P < 0.001). Most of the eggs that did not survive i n t h i s treatment were developed abnormally. In eggs exposed to 30°/oo f °r either 4 or 8 h/day, no survival and only very early embryonic development was observed. 1.2 Constant exposure to 5°/oo produced no adverse effects on survival (86.5-97.3%) and was not s i g n i f i c a n t l y different than the controls (P > 0.05). In contrast, eggs incubated i n 10 or 1 5° /0 0 showed advanced development (well eyed stage) but did not survive to hatching. Many of the embryos appeared normal except for abnormally developed yolk sacs. These abnormalities appeared to be the result of incomplete epiboly (blastoderm overgrowth of the yolk sac). Embryonic development stopped at an early stage (morula stage) i n eggs exposed to 2 0° /0 0 constantly and was neg 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 survivals i n 0 ° /0 0 (control) than 5°/oo *-n t n e 4 and 8 h exposures. On the whole, survival of thi 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 results of th i s abnormality were most noticeable i n the 108 freshwater treatment; although the nature of this abnormality was not established. 1.4 The intermittent s a l i n i t y tolerance of chum salmon eggs i n this study was di f f e r e n t than the tolerance of pink salmon eggs tested by Bailey (1966). Pink salmon embryos and alevins appeared to be more tolerant 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 difference was an i n t e r s p e c i f i c one. However, since some po t e n t i a l l y s i g n i f i c a n t procedural variations existed between the studies, i t was d i f f i c u l t to make direct 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 differences 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 et 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 differences i n s a l i n i t y tolerance noted i n th i s study were refl e c t i o n s of differences 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 Bailey (1966) indicated that a regular freshwater interlude between saltwater exposures was a key factor i n s a l i n i t y tolerance of chum and pink salmon eggs. Embryos and alevins were able to tolerate exposure to higher s a l i n i t i e s when provided with regular 109 freshwater reprievals. 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 for re-establishment of the organism's i n t e r i o r m i l i e u . Once th i s requirement i s met, embryos and alevins 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 regulation mechanisms develop and become functional. 1.7 Possible osmo- and iono-regulatory mechanisms i n salmon embryos and alevins are; ' t i g h t ' embryonic c e l l plasma membranes and chloride c e l l secretion. H i s t o l o g i c a l and physiological evidence from rainbow trout embryos and alevins indicates the presence of chloride c e l l s . In l i g h t of th i s information and physiological evidence from chum and pink salmon, the presence of chloride c e l l s also i s probable i n chum and pink, especially i n advanced embryos and alevins. 1.8 The hypothesis that eggs obtained from i n t e r t i d a l spawning parents (B-group) would have better s a l i n i t y tolerance that those obtained from freshwater upstream parents (F-group) was not tested as rigorously 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 actual i n t e r t i d a l spawners. Further, inexplicable differences i n 'control' egg survivals between the two groups of eggs made comparisons inappropriate. However, aside from these problems, no indications 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 tested. 110 Effects 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 s t a t i s t i c a l l y (P > 0.05), a s l i g h t reduction i n duration and vigor 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 immotility, as 95% immotility, misrepresented observations i n t h i s highest s a l i n i t y since occasional spermatozoa exhibited 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 levels of K+ ion i n the saltwater. In 1 5° /0 0 s a l i n i t y , the concentration of this ion was near the established l i m i t for prevention of m o t i l i t y ( > 2-5 mM) (Morisawa 1987). 2.2 Actual sperm v i a b i l i t y was underestimated using sperm m o t i l i t y as a predictor 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 differences 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 nonsignificant 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 this l e v e l was surprisingly 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. Either the occasionally active sperm observed at 15°/oo ^n t n e m o t i l i t y tests 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 test eggs or, immotile sperm exhibiting only vibratory movements were capable of gaining access to the eggs v i a the raicropyle to carry out f e r t i l i z a t i o n . I l l 2.3 Measurements of FS of sperm and eggs placed in various s a l i n i t i e s simultaneously, suggested that dilute saltwater had an added effect on the egg, in addition to i t s effect on the sperm. The responses essentially were the same as in the previous sperm v i a b i l i t y experiment (Summary 2.2), in 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 diluted in the test salinity before being added to the eggs. 2.4 Eggs maintained in their respective test s a l i n i t i e s for various time intervals (1, 15, 60, and 240 min) following addition of sperm, did not result in differential rates of FS. Eggs kept in 1 5 ° /0 0 for 240 min showed some signs of reduced FS compared to those kept for only 1, 15, or 60 min in the same s a l i n i t y , but the results were not conclusive. 2.5 Eggs kept in 12.5 and 15°/oo salinity 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. It is possible that either (1) perivitelline f l u i d colloids were affected by prolonged exposure (240 min) to intermediate sa l i n i t i e s (12.5 and 15°/oo) or> (2) short-term chemical changes in the egg membrane as a result of water hardening, prevented the egg from imbibing water normally upon return to freshwater. 2.6 In conclusion, experiments assessing the effect 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 in s a l i n i t i e s greater than 10°/oo- Not surprisingly, sperm appeared to be more susceptible than the much larger eggs; yet the data suggested adverse effects of 112 s a l i n i t y on the egg as well. It was concluded that intertidal chum salmon spawning in 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 intertidal zone, i t is not common to observe salt wedge sal i n i t i e s as low as 10°/oo during flood tides, at least not in 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 this 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 alternate source of oxygen during times when intragravel levels were low. On a flood 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 di f f e r e n t densities. Measurements of PVF osmolality of individual eggs indicated 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 inside the egg. Thus, during saltwater exposure on a flood t i d e , an i n t e r t i d a l egg experiences an exchange of the surrounding water as well as an exchange i n the ion 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 with the intragravel water as i t i s replaced by fresh stream water. Thus, density dependent water exchange functions on two lev e l s ; i n the intragravel 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. Intragravel flow i n streams largely i s due to gravity fed water flow produced by the volume of water passing through the stream (McNeil 1962, Vaux 1962). The degree of intragravel interchange depends on characteristics of the gravel and topography of the stream bed (Vaux 1962, 1968). In contrast, density dependent water exchange i s not dependent on the volume of stream flow and i s influenced less by physical characteristics 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 fines i n the gravel (McNeil and Ahnell 1964, Helle et a l . 1970), low permeability gravel could produce intragravel exchange problems. However, water interchange resulting from density differences probably i s less susceptible•to low gravel permeabilities, than flow dependent intragravel interchange of non-i 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 less prone to low intragravel dissolved 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 this study provide preliminary support for t h i s speculation but further investigation i s required to provide conclusive evidence. Direct 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 this study i s not completely appropriate. I f one temporarily ignores a l l of the additional environmental factors af 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 for the entire experiment (67 d). These incubation conditions were more severe than those at the -195 or -275 m egg implantation s i t e s , especially for 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, duration, and s a l i n i t y of exposure varied continuously and depended upon the inter-relationships between tide height, stream discharge, and egg location. However, i f one acknowledges a l l of the other environmental factors that ' i n stream' eggs were exposed to, such as fluctuations i n dissolved oxygen concentration, variations i n stream flow, predators, etc., then the f i e l d conditions probably were more adverse. 115 The laboratory setting provided an opportunity to control the confounding factors and concentrate on a s p e c i f i c test condition, i n this case s a l i n i t y . The s a l i n i t y tolerance tests i n the laboratory indicate that chum salmon eggs do not experience any adverse effects from intermittent saltwater exposures to 1 5° /0 0 s a l i n i t y or less for 4 or 8 h per day, and to 2 0° /0 0 for 4 h per day. Evidence from these results 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 tolerate periodic exposures up to 30°/oo D u t these s a l i n i t i e s did not p e r s i s t for 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 unl i k e l y that s a l i n i t i e s would exceed 3 3° /0 0 for open coastal areas and 3 0° /0 0 for inner coastal areas (Waldichuk 1956, Pickard 1961). Evidence from Rockwell (1956), Bailey (1966), Weisbart (1968), as well as th i s study indicates 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 affect the survival of chum or pink salmon eggs. I t seems that a key factor i n the s a l i n i t y tolerance of eggs, under an alternating 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, Bailey 1966) of freshwater reprieval between saltwater exposures. I f th 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 internal osmotic and ionic environments well enough to provide short-term tolerance to fluctuations i n ambient s a l i n i t y throughout development. This does not 116 hold true for the other three North American species of P a c i f i c salmon (Weisbart 1968). Chum and pink salmon appear to have an added physiological potential which allows them to spawn successfully i n the lower reaches of ri v e r s and streams that experience t i d a l influence. Geographically, chum and pink salmon are the most widely distributed species of 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 into r i v e r s on the northern coasts of the USSR, USA, and Canada. Chum salmon share a si 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 of 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 et a l . (1967) stated that pink and chum salmon could survive i n streams subjected to extreme floods and other physical disturbances. Perhaps the abundance of these f i s h i s related to th e i r a b i l i t y to adapt to or tolerate a wide variety of environmental conditions; one of which i s the incubation conditions of the i n t e r t i d a l zone. 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The mechanism of hardening of the salmonid egg membrane after 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 in Perivitelline Fluid Osmolality: General Growth Model - Equation. Parameters, and Residuals (see text in Materials and Methods, and Results sections of Chapter 2 for details) Test Condition: Eggs transferred from 0°/oo t o 20°/oo sa l in i ty water Equation: Y(t) = [35.79 2- 0 9 + (600.692 0 9 - 35.792 0 9 ) 1 - e -°-"<t-°> ]i/2-09 1 . e -0.10(180.0-0) Parameters: Low Xx= 0.00 High X2= 180.00 Yx= 35.79 Y2= 600.69 a = 0.10 b = 2.09 No. of records = 59 Minimized SS = 15603.55 X Y observed Y predicted residuals (min.) (mmol/kg) (mmol/kg) (mmol/kg) 0. .00 43, .00 35. .79 7. .215 0, .00 39, .00 35. .79 3. .215 0, .00 40. .00 35. .79 4. .215 0. .00 35, .00 35. .79 -0. .785 0. ,00 35, .00 35. .79 -0. .785 0. .00 37, .00 35. .79 1. .215 0, .00 37. .00 35. .79 1. .215 0. .00 40, .00 35. .79 4. .215 0. .00 43, .00 35. .79 7. .215 0, .00 41. .00 35. .79 5. .215 0. .00 40. .00 35. .79 4. .215 0. .00 25, .00 35; .79 -10. .785 0. .00 31, .00 35. .79 -4. .785 0. .00 33, .00 35. .79 -2. .785 0, .00 27, .00 35, .79 -8. .785 127 X Y observed Y predicted residuals (min.) (mmol/kg) (mmol/kg) (mmol/kg) 0 .00 31 .00 35, .79 -4 .785 0 .00 30 .00 35 .79 -5 .785 0 .00 30 .00 35 .79 -5, .785 0 .30 149 .00 116, .54 32 .459 0 .30 123 .00 116, .54 6 .459 0, .40 154, .00 132, .08 21, .922 0 .40 106 .00 132, .08 -26 .078 0, .90 176, .00 189, .07 -13, .073 1 .90 306 .00 262, .07 43, .931 2 .00 283, .00 267, .86 15, .137 2 .20 243, .00 278. .90 -35 .896 2 .70 284, .00 303. .74 -19 .740 4, .70 385 .00 377. .70 7, .300 4, .90 344, .00 383. .56 -39, .558 5, .00 390, .00 386. .41 3, .593 5, .00 365, .00 386. .41 -21, .407 5. 00 383, .00 386. .41 -3, .407 6. 20 399, .00 417, .00 -17, .998 6, .90 462, .00 432. .26 29. .745 7. 00 445, .00 434. .30 10, .697 7. 60 438. .00 445. .96 -7. .962 7. 80 436, .00 449. .62 -13. .625 9. 20 461. .00 472. .55 -11. .553 9, .50 470, .00 476. .92 -6. .918 11. .00 525. .00 496. .34 28. .663 11. .80 538. .00 505. .27 32. .731 12. .90 504. .00 516. .20 -12. .196 13. .80 545. .00 524. .12 20. .882 15. .00 550. .00 533. .45 16. .548 15. .20 531. .00 534. .89 -3. .885 15. .90 549. .00 539. .65 9. 351 16. .90 573. .00 545. .83 27. .172 18. .60 531. .00 554. .86 -23. ,864 22. .00 562. .00 568. .58 -6. .582 22. .30 569. ,00 569. ,57 -0. ,567 24. .80 558. .00 576. ,66 -18. ,662 26. .70 571. .00 580. .93 -9. .930 31. .80 594. .00 588. ,96 5. ,037 39. .90 588. .00 595. ,54 -7. .542 50. .50 590. .00 598. ,93 -8. ,930 60. .10 598. .00 600. ,02 -2. .025 80. .20 602. .00 600. ,61 1. 394 125. .00 601. .00 600. ,69 0. 307 180. ,00 613. .00 600. 69 12. .306 128 Test Condition: Eggs transferred from 20°/0o t o °°/oo salinity water Equation: Y(t) = [601.70"1-*9 + (22.14'1*9 - 601.70-1-*9) 1 - e"(-°-0 1 ) ( t"0 ) I1'1-*9 I _ e-(-0.01)(55.0-0) 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 Y observed Y predicted residuals (min.) (mmol/kg) (mmol/kg) (mmol/kg) 0. .00 612. .00 601, .70 10. .303 0, .00 609. .00 601, .70 7, .303 0. .00 598. .00 601, .70 -3. .697 0. .00 611. .00 601, .70 9, .303 0, .00 602. .00 601, .70 0. .303 0, .00 601. .00 601 .70 -0, .697 0. .00 613. .00 601, .70 11. .303 0. .00 602. .00 601, .70 0, .303 0. .20 415. .00 487, .89 -72. .887 0. .30 456. .00 448, .29 7, .707 2. .30 252. .00 197, .26 54, .741 3. .00 235. .00 169. .76 65. .236 5. ,30 100. .00 120. .37 -20, .373 6. .70 114. .00 103. .70 10. .303 8. .60 78. .00 88. .09 -10, ,091 8. .80 50. ,00 86. .76 -36. .759 11. .20 59. .00 73. .78 -14. ,783 13. .00 53. ,00 66. .62 -13. ,619 14. .00 40. .00 63. .28 -23. .282 15. .50 39. ,00 58. .93 -19. .929 16. .50 42. .00 56, .38 -14. .383 17. .60 36. ,00 53. .85 -17. .852 19. .30 43. .00 50, .40 -7. .401 24. .70 33. ,00 42. .05 -9. .053 25. .00 45. .00 41. .68 3. .324 29. .80 36. ,00 36, .48 -0. .484 129 X Y observed Y predicted residuals (min.) (mmol/kg) (mmol/kg) (mmol/kg) 32. .00 43. .00 34. .53 8. 469 36. .00 39. .00 31. .48 7. 520 39. .00 40. .00 29, .53 10. .473 43. .00 35. .00 27. .27 7. 730 44, .00 35. .00 26. .76 8. 242 49. .00 31. .00 24. .45 6. 548 51. .00 33. .00 23. .63 9. 368 52. .00 27. .00 23. .24 3. 758 54. .00 31. .00 22. .50 8. 504 55. .00 30. .00 22. .14 7. 861 130 

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