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Sublethal effects of mercuric chloride on some aspects of behaviour of anadromous fish Prentice, Norman David Joseph 1977

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c . \ THE SUBLETHAL EFFECTS OF MERCURIC CHLORIDE ON SOME ASPECTS OF BEHAVIOUR OF ANADROMOUS FISH by NORMAN DAVID JOSEPH PRENTICE B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August, 1976 Norman David Joseph P r e n t i c e In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t ha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f Z o o l o g y The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Nov 16 1976 Date ABSTRACT The effects of short term exposures to sublethal doses of mercuric chloride were studied using species of anadromous fis h . Aspects of grou/th and behaviour u/ere used as indicators of physiological and psychological mal-functions due to mercurialism. The characteristic migration tendencies ( upstream or downstream ) of Lower Babine and Fulton River alevins were tested in experimental laboratory channels. The were conducted in a chamber in which a day, dawn through dusk, could be simulated. Each stock was divided into three groups and each group was assigned a level of treatment ( control, 0.5 ppm and 1.0 ppm HgCl2 ). The performance of the fish in each group placed in the channels was scored. An analysis of data obtained from the migration experiments indicated that sublethal mercurialism had a disruptive influence on migration patterns of Sockeye salmon alevins. The lower level of intoxication produced a down-stream d r i f t in both stocks while the fish treated at the higher level of intoxication showed an upstream migrational tendency in both stocks. The effects of sublethal mercurialism on growth were studied using Sockeye, Chum, and Coho salmon fry. The fish received standard daily feedings. The fed fish for each of the species were divided into three treatment groups ( control, 0.5 ppm and 1.0 ppm mercuric chloride ) .Weight data were collected and analyzed using regression analysis and the analysis of covariance. The fed Sockeye and Chum salmon fry showed a significant weight loss ((X'= 0.05 ) in the 0.5 ppm and the 1.0 ppm treatments when compared to their respective controls. There was no significant difference in weight between treatments in the fed Coho salmon experiment. A six week starvation experiment was designed to test i f there were any significant differences in weight loss between control fish and fish treat-ed with 0.5 ppm and 1.0 ppm mercuric chloride. Regression analysis and the analysis of covariance did not reveal any s t a t i s t i c a l l y significant differences on the rates of weight loss. A flight responce experiment was conducted using Coho salmon prey and predators. Wild and hatchery Coho salmon prey were used with hatchery Coho predators. Three treatment levels of mercuric chloride were administered ( control, 0.5 ppm and 1.0 ppm ), Prey and predators were placed in an experimental laboratory performance channel and, after a timed exposure to each i i i other, scores indicative of flight responce mere obtained. The data was analyzed using the Kolmogorov-Smirnov Large sample : one tailed test. No significant differences in the responces between treat-ments were noted for the hatchery Coho. The wild Coho prey treated with 0.5 ppm and 1.0 ppm mercuric chloride had a s t a t i s t i c a l l y significant differences when compared to their controls. It was noted that as the level of intoxication increased the the level of flight responce decreased. The results of the above experiments indicate that mercurialism has measurable effects on the physiology and psychology of fish at sublethal level. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF APPENDICES v i i i LIST OF FIGURES * ACKNOWLEDGMENTS x i i PREFACE 1 I SOURCES OF MERCURY IN THE ECOSYSTEM 1 A. N a t u r a l Sources 1 B. The Mining I n d u s t r y 1 C. Mercury i n I n d u s t r y 2 i . The C h l o r - a l k a l i I n d u s t r y . . . . 2 i i . The F u n g i c i d e I n d u s t r y 3 i i i . The Pul p and Paper I n d u s t r y . . . 5 i v . The E l e c t r o n i c s I n d u s t r y 5 v. The P l a s t i c s I n d u s t r y 6 D. F o s s i l F u e l s 9 E. Sewage Sludge 9 F. Mercury P o l l u t i o n i n North America . . . 10 II MERCURY: ITS CHEMISTRY AND MODE OF ACTION WITHIN THE BIOSPHERE 12 A. The Chemistry of Mercury 12 B. The B i o t r a n s f o r m a t i o n of M e r c u r i a l s . . 13 I I I MERCURY IN BIOLOGICAL SYSTEMS 16 A. T r a n s p o r t to Organs 16 B. E x c r e t i o n of M e r c u r i a l s 20 V Page IV SYMPTOMATOLOGY OF MERCURIALISM 24 A. Acute Mercury P o i s o n i n g 24 B. C h r o n i c Mercury P o i s o n i n g 25 C. S e n s i t i v i t y of I n d i v i d u a l s w i t h i n a P o p u l a t i o n 26 D. S u b l e t h a l E f f e c t s 27 I INTRODUCTION 30 II EXPERIMENTAL DESIGN AND RESULTS 32 A. Upstream-Downstream M i g r a t i o n Experiment . . 32 a) E x p e r i m e n t a l F i s h 33 b) E x p e r i m e n t a l Tanks 33 c) The T e s t F i s h 35 d) The Performance Channel f o r the M i g r a t i o n Experiments 36 e) The T e s t Chambers 36 f ) The A l e v i n s i n the Performance Channel 37 g) The S c o r i n g Systems 38 h) The R e s u l t s of the Upstream-Downstream Experiment 39 B. Feeding and S t a r v a t i o n Experiments 40 a) E x p e r i m e n t a l F i s h S p e c i e s 40 b) The E x p e r i m e n t a l Tanks 40 c) E x p e r i m e n t a l Design 41 d) Method o f A n a l y s i s 43 e) R e s u l t s of the Feeding Experiment . . . 45 C. The Pr e d a t o r - P r e y Response Experiments . . . 46 a) The Performance Trough 46 v i Page b) E x p e r i m e n t a l Runs 47 c) Species of F i s h used i n the Experiment 47 d) The Ex p e r i m e n t a l Tanks . 48 e) The E x p e r i m e n t a l Design 48 i . The prey s t o c k s 48 i i . The p r e d a t o r s t o c k 49 f ) The I n t o x i c a t i o n P e r i o d 50 g) The P r e d a t o r - P r e y Response Runs . . . . 50 h) S c o r i n g 51 i ) Method of A n a l y s i s 51 j ) A n a l y s i s of Weight Data 51 k) R e s u l t s of the P r e d a t o r - P r e y F l i g h t Response Experiment .• 52 I I I DISCUSSION 54 A. General D i s c u s s i o n . . . . . . 54 B. D i s c u s s i o n of the Upstream-Downstream M i g r a t i o n Experiment 57 C. D i s c u s s i o n of the Feeding and S t a r v a t i o n Experiments . 62 D. The D i s c u s s i o n of the P r e d a t o r - P r e y F l i g h t Responses 65 IV CONCLUSIONS 67 A. The Upstream and Downstream Experiment . . . 67 B. The Feeding and S t a r v a t i o n Experiments . . . 70 a) The Fee d i n g Experiment 70 b) The S t a r v a t i o n Experiment 71 C. The P r e d a t o r - P r e y F l i g h t Response Experiment 73 a) The Wild Coho Salmon Prey Responses . . 73 v i i Page b) The Hatchery Coho Salmon Prey Responses 73 D. General C o n c l u s i o n s 75 BIBLIOGRAPHY 76 APPENDICES 88 FIGURES 110 v i i i LIST OF APPENDICES App. 1 The mean d a i l y water temperature f o r May, June, J u l y and August f o r the w e l l sources of the Rosewall Creek Hatchery (1971). App. 2 The m o r t a l i t y d a t a f o r the upstream and downstream Sockeye salmon a l e v i n s . App. 3 Worked examples showing maximum, minimum downstream-upstream movement of f i s h i n twenty-two h y p o t h e t i c a l t e s t s u s i n g the formula: ( n x r ) / N T + 12. App. 4 Comparisons among treatment samples of Sockeye salmon (Oncorhynchus nerka) from the downstream m i g r a t o r y F u l t o n R i v e r s t o c k . App. 5 Comparisons among treatment samples of Sockeye salmon (Oncorhynchus nerka) from the upstream m i g r a t o r y Lower Babine R i v e r s t o c k . App. 6 The m o r t a l i t y r a t e f o r both the f e e d i n g and s t a r v a t i o n experiment. App. 7 Comparisons among treatment samples of f e d Chum salmon (Oncorhynchus k e t a ) . App. 8 Comparisons among treatment samples of f e d Sockeye salmon (Oncorhynchus n e r k a ) . App. 9 Comparisons among treatment samples of f e d Coho salmon (Oncorhynchus k i s u t c h ) . App. 10 Comparisons among treatment samples of s t a r v e d Sockeye salmon (Oncorhynchus n e r k a ) . App. 11 Comparisons among treatment samples of s t a r v e d Coho salmon (Oncorhynchus k i s u t c h ) . App. 12 The m o r t a l i t y i n w i l d and h a t c h e r y Coho prey d u r i n g the t hree week exp e r i m e n t a l p e r i o d . App. 13 The m o r t a l i t y d a t a f o r the Coho salmon p r e d a t o r s t o c k . App. 14 Comparisons among samples of w i l d Coho prey (Oncor-hynchus k i s u t c h ) . App. 15 The Kilmogorov-Smirnov Two Sample T e s t ( l a r g e samples: o n e - t a i l e d t e s t ) f o r the h a t c h e r y Coho salmon (Oncor-hynchus k i s u t c h ) f l i g h t response experiment. i x App. 16 The Kilmogorov-Smirnov Two Sample Tes t ( l a r g e samples: o n e - t a i l e d t e s t ) f o r the w i l d Coho salmon (Oncorhyn-chus k i s u t c h ) f l i g h t response experiment. X LIST OF FIGURES F i g . 1 The standard 25-compartment performance c h a n n e l . F i g . 2 The F u l t o n R i v e r Sockeye salmon (Oncorhynchus nerka) c o n t r o l f i s h c o r e s f o r the upstream-downstream m i g r a t i o n tendency experiment. F i g . 3 The F u l t o n R i v e r Sockeye salmon (Oncorhynchus nerka) 0.5 ppm HgCl^ treatment s c o r e s f o r the upstream-downstream m i g r a t i o n tendency experiment. F i g . 4 The F u l t o n R i v e r Sockeye salmon (Oncorhynchus nerka) 1.0 ppm H g C ^ treatment s c o r e s f o r the upstream-downstream m i g r a t i o n tendency experiment. F i g . 5 The Lower Babine R i v e r Sockeye salmon (Oncorhynchus nerka) c o n t r o l f i s h s cores f o r the upstream-down-stream m i g r a t i o n tendency experiment. F i g . 6 The Lower Babine R i v e r Sockeye salmon (Oncorhynchus nerka) 0.5 ppm H g C ^ treatment scores f o r the up-stream-downstream m i g r a t i o n tendency experiment. F i g . 7 The Lower Babine R i v e r Sockeye salmon (Oncorhynchus nerka) 1.0 ppm H g C l 2 treatment scores f o r the up-stream-downstream m i g r a t i o n tendency experiment. F i g . 8 The Chum salmon (Oncorhynchus keta) growth e x p e r i -ment r e g r e s s i o n l i n e s d i p i c t i n g growth r a t e s over a s i x week f e e d i n g p e r i o d . F i g . 9 The Sockeye salmon (Oncorhynchus nerka) growth experi-ment r e g r e s s i o n l i n e s d i p i c t i n g growth r a t e s over a s i x week f e e d i n g p e r i o d . F i g . 10 The Coho salmon (Oncorhynchus k i s u t c h ) growth e x p e r i -ment r e g r e s s i o n l i n e s d i p i c t i n g growth r a t e s over a s i x week f e e d i n g p e r i o d . F i g . 11 The Sockeye salmon (Oncorhynchus nerka) s t a r v a t i o n experiment r e g r e s s i o n l i n e s d i p i c t i n g weight l o s s r a t e s over a s i x week p e r i o d of s t a r v a t i o n . F i g . 12 The Coho salmon (Oncorhynchus k i s u t c h ) s t a r v a t i o n experiment r e g r e s s i o n l i n e s d i p i c t i n g weight l o s s r a t e s over a s i x week p e r i o d of s t a r v a t i o n . F i g . 13 The p r e d a t o r - p r e y f l i g h t response c h a n n e l . F i g . 14 An o u t l i n e map of Babine Lake and ad j a c e n t waters. x i F i g . 15 The Coho salmon (Oncorhynchus k i s u t c h ) h a t c h e r y prey f l i g h t response s c o r e s f o r the c o n t r o l treatment. F i g . 16 The Coho salmon (Oncorhynchus k i s u t c h ) h a t c h e r y prey f l i g h t response s c o r e s f o r the 0.5 ppm m e r c u r i c c h l o r i d e treatment. F i g . 17 The Coho salmon (Oncorhynchus k i s u t c h ) h a t c h e r y prey f l i g h t responses s c o r e s f o r the 1.0 ppm m e r c u r i c c h l o r i d e treatment. F i g . 18 The Coho salmon (Oncorhynchus k i s u t c h ) w i l d prey f l i g h t response s c o r e s f o r the c o n t r o l treatment. F i g . 19 The Coho salmon (Oncorhynchus k i s u t c h ) w i l d prey f l i g h t response s c o r e s f o r the 0.5 ppm m e r c u r i c c h l o r i d e treatment. F i g . 20 The Coho salmon (Oncorhynchus k i s u t c h ) w i l d prey f l i g h t response s c o r e s f o r the 1.0 ppm m e r c u r i c c h l o r i d e treatment. x i i ACKNOWLEDGMENTS The U n i v e r s i t y of B r i t i s h Columbia, the N a t i o n a l Research C o u n c i l of Canada, and the N a t i o n a l F i s h e r i e s Research Board of Canada p r o v i d e d f i n a n c i a l support f o r t h i s s tudy. I wish to express my g r a t i t u d e to my s u p e r v i s o r , Dr. J.R.E. Harger, f o r h i s guidance and c o n s t r u c t i v e c r i t i c i s m s throughout a l l phases of the study and p r e p a r a t i o n of the manuscript. Dr. J.R. C a l a p r i c e of the F i s h e r i e s Research Board at Nanaimo, B.C., granted the use of the f a c i l i t i e s a t the Rose-w a l l Creek Hatchery on Vancouver I s l a n d . I wish to express my g r a t e f u l n e s s to Dr. D. R a n d e l l f o r h i s u n d e r s t a n d i n g , p a t i e n c e and a s s i s t a n c e through the f i n a l s tages of manuscript p r e p a r a t i o n . F i n a l l y , I wish t o thank K a t h i Hughs, a f r i e n d and c o l l e a g u e , f o r her guidance and c o n s t r u c t i v e c r i t i c i s m s of the manuscript. f PREFACE A REVIEW OF LITERATURE RELATED TO MERCURIALISM I. SOURCES.OF MERCURY IN THE ECOSYSTEM A. , N a t u r a l Sources Mercury i s presen t i n s o i l and rock f o r m a t i o n s . The p r i n c i p a l mercury-bearing m i n e r a l s are c i n n a b a r and meta-c i n n a b a r , two polymorphs of mer c u r i c s u l p h i d e (Hgs). Mercury e n t e r s the environment when s o i l and rock undergo weathering, and the r u n - o f f water c a r r i e s m ercuric compounds to streams, l a k e s and oceans where m e r c u r i a l s c o l l e c t i n the.sediments (Jonasson. and Boyle 1971). There are s e v e r a l areas of B r i t i s h Columbia and the Yukon where c o n c e n t r a t i o n s of mercury-bearing m i n e r a l s have been found. Kamloops Lake, Yalakon R i v e r , Bridge R i v e r , A l b e r n i C a n a l , and P i n c h i Lake have l a r g e c i n n a b a r d e p o s i t s (Stevenson 1940). Many areas along the Western C o r d i l l e r a of the Americas have l a r g e m e r c u r i f e r o u s d e p o s i t s . B. The Mining I n d u s t r y In 1968 Cominco brought t h e i r P i n c h i Lake mine back i n t o p r o d u c t i o n i n c o n j u n c t i o n with a r i s i n g demand f o r mer-cury i n Canada. I t has been r e p o r t e d (Anonymous 1968) th a t the mine c o u l d reach an annual p r o d u c t i o n of from 450,000 to 700,000 kg. The p r o x i m i t y o f l o c a l p r o d u c t i o n may encourage the consumption of mercury by B r i t i s h Columbia i n d u s t r y ; how-ever at the date of w r i t i n g t h i s t h e s i s the mine was not i n 2 o p e r a t i o n . P e t e r s o n e t a l (1970) found t h a t s e v e r a l s p e c i e s of f i s h e s c o l l e c t e d from P i n c h i Lake d u r i n g the summers o f 1969 and 1970 had l e v e l s o f mercury f r e q u e n t l y above t h e a c c e p t e d l e v e l s f o r human c o n s u m p t i o n , i . e . 0.5 ppm. L e v e l s of mercury i n f i s h from P i n c h i Lake were g e n e r a l l y h i g h e r than t h o s e from s u r r o u n d i n g l a k e s s u g g e s t i n g the mine as the s o u r c e o f c o n t a m i n a t i o n . M e r c u r y i s o f t e n f o u n d w i t h l e a d and z i n c d e p o s i t s i n M a n i t o b a , O n t a r i o , Quebec, New Br u n s w i c k and Newfoundland. The w e a t h e r i n g o f t a i l i n g s from mines i n t h e s e a r e a s a r e a prime s o u r c e o f heavy m e t a l c o n t a m i n a t i o n o f the a q u a t i c e n v i r o n m e n t . C. Mercury i n I n d u s t r y S e v e r a l i n d u s t r i e s use mercury i n t h e i r p r o c e s s e s , and at t i m e s a p o r t i o n o f t h i s mercury e s c a p e s t o c o n t a m i n a t e the e n v i r o n m e n t . These i n d u s t r i e s w i l l be d i s c u s s e d as t o t h e i r c o n t r i b u t i o n t o e n v i r o n m e n t a l p o l l u t i o n and examples o f how t h i s p o l l u t i o n a f f e c t s a n i m a l and p l a n t l i f e w i l l be g i v e n . i . The C h l o r - a l k a l i I n d u s t r y M e r c u r y c e l l s have come i n t o f a v o u r f o r t h e p r o d u c t i o n of c h l o r i n e and c a u s t i c s o d a . T h i s p r o c e s s which i s used i n Sweden (Hanson 1971) has been shown t o be r e s p o n s i b l e f o r most o f the e m i s s i o n o f mercury i n t o t h e Swedish a q u a t i c e n v i r o n m e n t . T h i s l o s s o f mercury t o t h e env i r o n m e n t d u r i n g 1967 was e s t i m a t e d t o be 100 t o 150 grams per t o n f o r t h e 250,000 t o n s o f c h l o r i n e p r o d u c e d . 3 i i . The F u n g i c i d e I n d u s t r y The development of the o r g a n i c c h e m i c a l i n d u s t r y at the t u r n of the century v a s t l y i n c r e a s e d the number of com-pounds t h a t were a v a i l a b l e to i n d u s t r i a l r e s e a r c h e r s . The f i r s t m e r c u r i a l f u n g i c i d e s were developed i n Germany about 1914, and soon a f t e r t h i s they came i n t o wide use (Novick 1969). The b e g i n n i n g of the Second World War h e r a l d e d p r o d u c t i o n of l i q u i d p r e p a r a t i o n s , such as a l k y l m e r c u r y , which was w i d e l y used i n the treatment of seed g r a i n s . In Sweden the seed d r e s s i n g of c h o i c e was methyl mercury which acted as a wide spectrum f u n g i c i d e , and i t s use c o n t i n u e d u n t i l 1966 when i t was banned by the Swedish govern-ment. Jo h n e l s and Westermark (1969) found t h a t methyl mercury had i n c r e a s e d i n the f e a t h e r s of s e e d - e a t i n g b i r d s s i n c e the i n t r o d u c t i o n of alky1-mercury compounds i n 1940. S i n c e 1966, the mercury c o n t e n t of f e a t h e r s b e l o n g i n g to s e e d - e a t i n g b i r d s has dropped s l i g h t l y but i n c o n t r a s t , the b i r d s of prey examined have shown a c o n t i n u a l i n c r e a s e i n the mercury content of t h e i r f e a t h e r s ( J e r n e l o v 1968a; J o h n e l s e t a l 1966). In A l b e r t a the h u n t i n g season was c l o s e d i n 1969 due to f i n d i n g mercury l e v e l s approaching 0.5 ppm. i n the muscle t i s s u e of pheasants and p a r t r i d g e s (Wishart 1970). The p r e s e n t and p o t e n t i a l t h r e a t of i n g e s t i o n of methyl mercury t r e a t e d seeds by b i r d s , mammals, and t h e i r p r e d a t o r s has been w e l l documen-ted ( F i m r e i t e e t a l 1970; L b f r o t h and D u f f y 1969). Lewis (1971) r e p o r t s t h a t i n 1940, two stenographers i n a C a l g a r y granary d i e d because of a prolonged exposure to 4 an o r g a n i c f u n g i c i d e which was used as a seed p r e s e r v a t i v e ' and s t o r e d i n an area near t h e i r o f f i c e . The two women had been exposed to a l e v e l of one m i l l i g r a m of mercury per c u b i c meter d u r i n g the hot summer months of J u l y and August. T h i s was ten times the recommended i n d u s t r i a l maximum t o l e r a b l e c o n c e n t r a t i o n ( B i d s t r u p 1964). Mercury i s unique among the heavy metals i n t h a t i t has c o n s i d e r a b l e v o l a t i l i t y at o r d i n a r y room temperatures (Goldwater 1957; Copplestone and McArthur 1966). There have been s e v e r a l r e p o r t s of i n t o x i c a t i o n by mercury vapor i n l a b o r a t o r i e s ( M c C a r r o l l 1939; Shepard e t a l 1941; K e n d a l l 1966; Goldwater e t a l 1956; Beauchamp and Tebbens 1951) and i n d u s t r i e s (Hunter 1969). Grant (1971) r e p o r t e d a case of acute mercury p o i s o n -i n g i n a New Mexico f a m i l y which had eaten pork which c o n t a i n e d 27 ppm. of mercury. The hog had been f e d sweepings from a granary f l o o r which i n c l u d e d seed g r a i n which had been t r e a t e d w i t h a m e r c u r i a l f u n g i c i d e . The hog had not developed symp-toms of m e r c u r i a l i s m , but t h r e e c h i l d r e n f e d the meat s u f f e r e d i r r e p a r a b l e n e u r o l o g i c a l damage, and a baby born to the asymptomatic mother had c o n v u l s i o n s at b i r t h which were symptomatic of m e r c u r i a l i s m . The c o n t a m i n a t i o n of streams by r u n - o f f water from f i e l d s which have been p l a n t e d with seed t r e a t e d with m e r c u r i a l f u n g i c i d e s i s o b v i o u s . 5 i i i . The P u l p and Paper I n d u s t r y Many pulp and paper f a c t o r i e s i n the past used a mercury compound, phenyl m e r c u r i c a c e t a t e (P.M.A.) to prevent the growth of microorganisms which c l o g machinery and cause d e t e r i o r a t i o n of pulp s t o r e d f o r a time before..being p r o c e s s e d . Phenyl m e r c u r i c a c e t a t e was banned i n Sweden i n 1966 a f t e r Z i e b e l l (1966), and H a s s e l r o t (1964, 1965, 1966) conducted f i e l d t o x i c o l o g y s t u d i e s u s i n g caged f i s h i n e s t u a r i e s and r i v e r s . Z i e b e l l (1966) was a b l e to p i n p o i n t the sources of p o l l u t i o n by p l a c i n g cages of f i s h upstream and downstream wit h r e s p e c t to i n d u s t r i a l o u t f a l l s . H a s s e l r o t (1968) d i s -cussed methods of t r a c i n g mercury p o l l u t i o n and checks on e f f e c t i v e n e s s of measures taken to c o r r e c t such p o l l u t i o n . Wobesser e t a l (1970), sampling f i s h above and below o u t f a l l s , found t h a t i n s e v e r a l cases f i s h caught below i n d u s t r i a l out-f a l l s on the Saskatchewan R i v e r c o n t a i n e d h i g h e r amounts of mercury than f i s h caught above the o u t f a l l s . Canadian pulp and paper manufacturers have reduced t h e i r consumption of P.M.A. i n the pa s t few y e a r s , but s i n c e c a u s t i c soda i s used i n p r o c e s s i n g p u l p , and any c a u s t i c soda from a c h l o r - a l k a l i p l a n t may c o n t a i n m e r c u r i c compounds, the e f f l u e n t from' wood-fiber p l a n t s should be c o n t i n u a l l y moni-t o r e d even i f they have suspended the use of m e r c u r i a l f u n g i c i d e . i v . The E l e c t r o n i c s I n d u s t r y Lowry (1957) r e p o r t s t h a t the p h y s i c a l p r o p e r t i e s of 6 mercury are u n i q u e l y s u i t e d to the demands.of gaseous e l e c -t r o n i c s , and f u r t h e r , t h a t no other element possesses c h a r a c -t e r i s t i c s so p e c u l i a r l y and eminently s u i t e d to the r e q u i r e -ments of a l a r g e number of e l e c t r o n i c d e v i c e s . The mercury-po o l r e c t i f i e r , the i g n i t r o n , hot-cathode r e c t i f i e r s and f t h y r a d t r o n s , the Cooper-Hewitt lamp, f l u o r e s c e n t and g e r m i c i d a l lamps, photo-copying lamps, and the i n c r e a s i n g l y p o p u l a r h i g h - p r e s s u r e mercury lamps are a l l dependent on the unique p h y s i c a l p r o p e r t i e s of mercury. I t i s s e l f - e v i d e n t from the uses of mercury i n e l e c t r o n i c s t h a t t h i s i n d u s t r y p r o v i d e s abundant o p p o r t u n i t i e s f o r c o n t a m i n a t i o n of the environment. v. The P l a s t i c s I n d u s t r y Mercury i s used as a c a t a l y s t i n the p r o d u c t i o n of v i n y l c h l o r i d e . Hanson (1971) r e p o r t e d t h a t spent c a t a l y s t was d i s p o s e d of by dumping s e a l e d b a r r e l s of m a t e r i a l i n t o the North Sea. The metal b a r r e l s i n time r u s t e d away and r e l e a s e d the mercury-laden f l u i d s i n t o the sea. Greve (1971) r e c e n t l y r e p o r t e d f i n d i n g s of r e s e a r c h i n t o the t o x i c i t y of the c ontents of s e v e r a l b a r r e l s of m a t e r i a l which washed up on shore at Den H elder, N e t h e r l a n d s . Although the b a r r e l s d i d not c o n t a i n mercury compounds, t h e i r presence i s i n d i c a -t i v e of the p e r s i s t e n t . u s e of t h i s means of waste d i s p o s a l which can l e a d to g r e a t e r p o l l u t i o n of an a l r e a d y p o l l u t e d s e a . In 1953 an outbreak of human p o i s o n i n g by mercury 7 took p l a c e a t Minamata Bay, J a p a n . The p o i s o n i n g i n v o l v e d 121 i n h a b i t a n t s o f the a r e a , and e x t e n s i v e case h i s t o r i e s e x i s t f o r t h e p e r i o d between 1953 and 1960. The f i s h i n g pop-u l a t i o n o f t h e a r e a was p o i s o n e d by m e t h y l mercury c o n t a i n e d i n the f i s h and s h e l l f i s h w h i c h they c a u g h t i n the bay. The H u n t e r - R u s s e l l syndrome o r s o - c a l l e d Minamata D i s e a s e i s c h a r a c t e r i z e d by c e r e b e l l a r a t a x i a , c o n s t r i c t i o n o f v i s u a l f i e l d s , d y s a r t h r i a , s e n s o r y changes and impairment o f h e a r i n g ( T s u b a k i 1971). T h i s o u t b r e a k o f mercury p o i s o n i n g b r o u g h t about i n t e n s i v e r e s e a r c h by s c i e n t i s t s as t o t h e e t i o l o g y o f t h e d i s e a s e (Irukayama e t a l 1962a, 1962b, 1962c; Irukayama 1966; K u r l a n d e t a l 1960; M a t i d a and Kumada 1969; Rucker 1968; Tokuomi e t a l 1961; U c h i d a e t a l 1961a, 1961b; U i 1969; Y o s h i d a 1967). The s o u r c e o f c o n t a m i n a t i o n was t r a c e d t o a f a c t o r y w h i c h d i s c h a r g e d l a r g e amounts of a m e r c u r i a l com-pound i n e f f l u e n t from a v i n y l c h l o r i d e p r o c e s s . A second o u t b r e a k o f Minamata D i s e a s e was n o t e d i n t h e Agano R i v e r a r e a o f the N i i g a t a P r e f e c t u r e between August 1964 and J u l y 1965. F o r t y - e i g h t p e r s o n s were a f f l i c t e d o f w h i c h s i x have d i e d t o d a t e . The s o u r c e o f e n v i r o n m e n t a l c o n t a m i n a t i o n was an a c e t a l d e h y d e p l a n t w h i c h a l l o w e d mercury c a t a l y s t t o e n t e r the r i v e r a f t e r use. T h i s second o u t b r e a k o f mercury p o i s o n i n g i n t e n s i f i e d t h e r e s e a r c h i n t o t h e e t i o l o g y o f Minamata D i s e a s e . T a k e u c h i e t a l (1962) c o n d u c t e d an e x t e n s i v e s t u d y i n t o the f i r s t Minamata o u t b r e a k . I t was found t h a t a n i m a l s i n the a r e a t h a t f e d on f i s h had symptoms s i m i l a r t o t h o s e found i n humans. Cats were most f r e q u e n t l y a f f e c t e d showing unsteadyness and slow movement, a t a x i c g a i t and c o n v u l s i o n s . Dashing or c i r c l i n g around, tremor and abnormal movements were o c c a s i o n a l l y observed. Takeuchi (1957a) found the h i s t o p a t h o l o g y of the animals to be the same as t h a t of humans which had d i e d from Minamata D i s e a s e . B i r d s found around the bay were observed to develop symptoms,vwhich were c h a r a c t e r i z e d by l o s s of b a l a n c e (Takeuchi 1957b), and mice consuming f i s h from the bay developed symptoms of the d i s e a s e (Takeuchi 1957c). The d i s e a s e a l s o appeared i n f i s h , and upon autopsy, l e s i o n s i n the nervous t i s s u e s with l o s s of granule c e l l s i n the b r a i n were observed. No conspicuous changes i n the r e t i n a o c c u r r e d , but c a t a r a c t s were o f t e n observed (Takeuchi 1960). Matsumoto et a l (1965) d i s c u s s e d Minamata D i s e a s e i n two babies who were found to have l e s i o n s i n the c e n t r a l nervous system c h a r c t e r i s t i c of the d i s e a s e . The c h i l d r e n had never eaten f i s h , but f i s h was an important item i n the d i e t s of the mothers of the c h i l d r e n . I t was concluded t h a t the mercury i n t o x i c a t i o n had taken p l a c e a c r o s s the p l a c e n t a l b a r r i e r . T h i s h'as been proven to be the case f o r methyl mercury which i s the form of mercury most o f t e n encountered i n the food c h a i n s of a n i m a l s . U k i t a e t a l (1967) Suzuki e t a l (1967), and F u j i t a (1969) r e p o r t t h a t a l k y l m e r c u r i a l s c r o s s the p l a c e n t a l b a r r i e r much more e a s i l y than i n o r g a n i c , or a r y l m e r c u r i a l s . 9 D. F o s s i l F u e l s One of the important sources of mercury c o n t a m i n a t i o n of the environment i n v o l v e s combustion of f o s s i l f u e l s . I t has been r e p o r t e d by Wood e t a l (1968) t h a t c o a l i n the U.S. averaged between 0.5 and 3.3 ppm mercury. T h i s , i n terms of p r e s e n t consumption of f u e l , l e a d s to the e s t i m a t e t h a t between 275 and 1,800 tons of mercury i s r e l e a s e d i n t o the environment y e a r l y . E. Sewage Sludge Sludge from sewage p l a n t s has been used as f e r t i l i z e r i n s e v e r a l areas of the w o r l d . R e c e n t l y i n Sweden i t has been found to c o n t a i n amounts of mercury t h a t render i t un-s u i t a b l e f o r a g r i c u l t u r a l use (Grant 1971). Hanson (1971) r e p o r t s t h a t 15 to 20 tons of mercury are used i n making d e n t a l amalgams each year, and t h a t a p a r t of t h i s q u a n t i t y e n t e r s the sewage system d e s p i t e the r e c o v e r y equipment norm a l l y i n s t a l l e d . T h i s i n f o r m a t i o n may be e x t r a p o l a t e d to g i v e e s t i m a t e s of mercury l o s t to the environment i n c o u n t r i e s such as Canada and the U n i t e d S t a t e s . Grant (1971) r e p o r t s t h a t petroleum may c o n t a i n from 1.9 to 21.0 ppm mercury. L i t t l e i s knownabout the r e l e a s e of mercury i n t o the environment d u r i n g the c r a c k i n g p r o c e s s . The end products of c r a c k i n g , such as bitumens, s o l i d hydro-carbons, and a s p h a l t s may c o n t a i n from 2.0 to 900 ppm mercury. The use of a s p h a l t s i n roads and the r e s u l t a n t break-down of t h i s m a t e r i a l by weathering r e l e a s e s mercury i n t o the 10 storm d r a i n s . T h i s mercury, along with the v a r i o u s domestic sources of mercury, become mixed i n combined storm and sewage systems, and the sludge produced i n sewage p r o c e s s i n g c o u l d be expected to be u n s u i t a b l e f o r any a g r i c u l t u r a l use. F. Mercury P o l l u t i o n i n North America In 1970, s e v e r a l l a k e s i n the U.S. (Wallace e t a l 1971; K l e i n e r t and Degurse 1972) and Canada ( B l i g h 1971) were c l o s e d to commercial f i s h i n g because of dangerously high c o n c e n t r a -t i o n s of mercury i n the f i s h t i s s u e . H a r r i s s (1971) r e p o r t s t h a t twenty s t a t e s i n the U.S. have banned the s a l e of f i s h or warned the p u b l i c a g a i n s t consumption of f i s h from waters con-taminated w i t h mercury. The s m a l l e r p l a n k t o n - f e e d i n g f i s h i n p o l l u t e d areas of the Great Lakes show v a l u e s of mercury r a n g i n g from 0.2-1.0 ppm, w h i l e the top p r e d a t o r s such as w a l l e y e , p i k e and white bass show v a l u e s r a n g i n g from 1.0-2.0 ppm of mercury. Water fowl and o t h e r f i s h - e a t i n g animals i n the v i c i n i t y of Lake S t . C l a i r have been d i s c o v e r e d t o c o n t a i n up to 20 ppm of mercury. B l i g h (1971) r e p o r t s l e v e l s of mercury i n c r a b s (1.55-13.4 ppm Hg) and f l o u n d e r (1.00-1.42 ppm) caught at Squamish, B r i t i s h Columbia. These l e v e l s were c o n s i d e r e d h i g h enough to c l o s e Howe Sound to f i s h i n g . Since the concen-t r a t i o n s of mercury were comparable to those on the Agano R i v e r (1.0 to 10.0 ppm as c i t e d by Wallace et a l 1971), i t might be c o n c l u d e d t h a t a d i s a s t e r such as observed i n Japan was avoided o n l y because of the d i f f e r e n c e i n d i e t of North 11 Americans. F i s h were the primary source of p r o t e i n i n the d i e t of the people around Minamata Bay, w h i l e i n the lower mainland of B r i t i s h Columbia f i s h d i d not rank high as a pro-t e i n source. Z i t k o e t a l (1971) r e p o r t e d methyl mercury i n f r e s h -water and marine f i s h i n New Brunswick, the Bay of Fundy, and the Nova S c o t i a Banks. American e e l ( A n g u i l l a r o s t r a t a ) , c h a i n p i c k e r e l (Esox n i g e r ) , white perch (Morone americana), y e l l o w perch (Perca f l a v e s c e n s ), brook t r o u t ( S a l v e l i n u s  f o n t i n a l i s ) , and A t l a n t i c salmon (Salmo s a l a r ) c o n t a i n e d 0.07-2.08, 0.27-1.58, 0.75-1.07, 0.20-1.05, 0.08-0.13 and 0.09 ppm of methyl mercury r e s p e c t i v e l y (expressed as mercury on wet-weight b a s i s ) . In some i n s t a n c e s an i n d u s t r i a l s i t e c o u l d be l i n k e d to f r e s h w a t e r p o l l u t i o n w h i l e o t h e r s i t e s may have been p o l l u t e d by a i r b o r n e mercury. F i m r e i t e (1970) has reviewed the hazards and sources of mercury i n Canada, and A x e l s s o n and F r i b e r g (1960) have d i s c u s s e d the t o l e r a b l e l i m i t s of mercury i n the atmospheric and b i o t i c m i l i e u . 12 I I . MERCURY: ITS CHEMISTRY AND MODE OF ACTION WITHIN THE BIOSPHERE The a c t i v i t y of mercury has been s t u d i e d i n s e v e r a l s p e c i e s , but f i s h s p e c i e s have not been among those which have r e c e i v e d e x t e n s i v e a t t e n t i o n . The e f f e c t of the element upon the b a s i c b i o c h e m i s t r y of organisms appears to be the same i n the animals s t u d i e d thus f a r . A g e n e r a l review of what i s p r e s e n t l y known about mercury and i t s a c t i v i t y w i t h i n b i o l o g i c a l systems can o n l y shed l i g h t upon what one might expect to happen i n anadromous•fishes. A. The Chemistry of Mercury Mercury i s the h e a v i e s t of the t r a n s i t i o n a l elements, r and i t i s p l a c e d with z i n c and cadmium i n p e r i o d i c group 2B. L i k e the o t h e r two members of t h i s group, i t i s d i v a l e n t . Mercury r e a d i l y forms c o v a l e n t l i n k a g e s to s u l p h u r , and i t i s t h i s c h a r a c t e r i s t i c t h at accounts f o r most of the b i o l o g i c a l p r o p e r t i e s of the metal. Hughes (1957) and Friedman (1957) e l u c i d a t e d the r e l a t i o n s h i p between the chemical s t r u c t u r e , and the c h e m i c a l and p h y s i c a l p r o p e r t i e s of mercury compounds as regards t h e i r b i o l o g i c a l a c t i v i t y . When sulphur i s i n the form of s u l p h y d r y l groups, d i v a l e n t mercury r e p l a c e s the hydrogen atoms to form mercaptides (X-Hg-S-R, where X i s an e l e c t r o - n e g a t i v e r a d i c a l and R i s p r o t e i n ) . Organic m e r c u r i a l s form mercaptides of the R-Hg-S-R' type. Mercury i s the o n l y metal t h a t i s i n a l i q u i d s t a t e at normal e a r t h s u r f a c e temperatures. A s l i g h t amount of heat 13 w i l l v a p o r i s e mercury and a l l o w gaseous c o n t a m i n a t i o n of the atmosphere where temperature and p r e s s u r e changes can change mercury vapour back to a s o l i d and the mercury r e t u r n s to the e a r t h ' s s u r f a c e as "dry f a l l o u t . " Rain c o n t i n u a l l y washes mercury vapour out of the atmosphere. Much of the r a i n produced i n our atmosphere f a l l s on the oceans and seas of the world. I t i s est i m a t e d t h a t the oceans of the world now c o n t a i n f i f t y m i l l i o n m e t r i c tons of ele m e n t a l mercury (Montague and Montague 1971). B. The B i o t r a n s f o r m a t i o n of M e r c u r i a l s I n o r g a n i c and phenyl mercury compounds d i s c h a r g e d i n t o the a q u a t i c environment are r a p i d l y absorbed or adsorbed by p a r t i c u l a t e i n o r g a n i c and o r g a n i c m a t e r i a l ( H a r r i s s 1971). When mercury compounds reach the sediments of a q u a t i c e n v i r o n -ments they are conve r t e d by b a c t e r i a to methyl (CH^-Hg-R) or di m e t h y l ^CH-^HgJ mercury ( J e r n e l b v 1968b; Wood e t a l 1968; Jensen and J e r n e l o v 1969). Wood (1972) r e p o r t s t h a t m i c r o-organisms c a p a b l e of v i t a m i n s y n t h e s i s are a l s o c a p a b l e of methyl mercury s y n t h e s i s . I t i s apparent t h a t methanogenic b a c t e r i a c o n v e r t mercury from both n a t u r a l and i n d u s t r i a l s o u r c e s ; and t h a t t h i s c o n v e r t e d mercury, i n the form of methyl mercury, i s t r a n s f e r r e d by b a c t e r i a to those organisms which prey upon them and mercury i s thus moved through the food c h a i n to f i s h (Hannerz 1968; Johnels e t a l 1967, 1968) and to f i s h - e a t i n g animals (Helminen e t a l 1966, 1968; Helminen 1968; ' Takeuchi 1957a). 14 J e r n e l o v (1969) has summarized some of the s t e p s by which i n o r g a n i c mercury and some m e r c u r y - c o n t a i n i n g compounds are c o n v e r t e d i n nature to methyl mercury as f o l l o w s : Dipheny1 mercury ( C 6 H 5 ) 2 Hg H Phenyl mercury C 6H 5 Hg + 1 M e t a l l i c Dimethyl mercury ( C H 3 ) 2 Hg mercury I T o Hg A l k o x i - a l k y l mercury _^ I n o r g a n i c d i v a l e n t mercury Hg Methyl mercury CH 3 Hg + CH 3-0-CH 2-CH 2-Hg + J e r n e l o v s t a t e s the consequences of these t r a n s f o r m i n g r e a c t i o n s to be p a r t i c u l a r l y obvious i n r e l a t i o n to d e p o s i t s of bound mercury i n sediments of l a k e s and r i v e r s , which can be m o b i l i z e d , through c o n v e r s i o n , i n t o the more r e a c t i v e m e r c u r i c forms. Such d e p o s i t s are made up p r i n c i p a l l y from phenyl and methyl mercury i n f i b e r banks and i n o r g a n i c mercury ( d i v a l e n t and m e t a l l i c ) i n bottom sediments. As p r e v i o u s l y noted, the c h i e f sources of mercury are c h l o r i n e f a c t o r i e s and pulp and paper m i l l s where e f f l u e n t s c o n t a i n i n o r g a n i c , phenyl and methyl mercury compounds. The l a t t e r source i s of s p e c i a l i n t e r e s t when c o n s i d e r i n g the waters of Canada, and the i m p l i c a t i o n s of mercury f o r the n a t i o n ' s f i s h e r y . 15 The i n t e r c o n v e r s i o n o f m e r c u r i a l compounds has been n o t e d by s e v e r a l r e s e a r c h e r s ( M i l l e r e t a l 1960; Gage and Swan 1961; B e r l i n and U l l b e r g 1963; C l a r k s o n 1969). F o r example, i n a n i m a l s , p h e n y l m e r c u r i a l s appear t o be c o n v e r t e d i n t o i n o r g a n i c compounds by m e t a b o l i c p r o c e s s e s . These com-pounds, .thus c o n v e r t e d , show a d i s t r i b u t i o n s i m i l a r , b u t not i d e n t i c a l , t o t h a t o f i n o r g a n i c mercury. The i n t e r c o n v e r s i o n s o f a r y l , a l k y l , and i n o r g a n i c compounds have been summarized by Grant (1969) as f o l l o w s : damages k i d n e y ( a l s o l i v e r , b r a i n , mucosa) damages b r a i n ( a l s o mucosa m u s c l e , a l l t i s s u e ) ARYL INORGANIC ALKYL (ORGANIC) MERCURY (ORGANIC) MERCURY MERCURY t r a n s f o r m e d by body and env i r o n m e n t t r a n s f o r m e d by or g a n i s m m i c r o -h y p e r s e n s i t i v i t y r e a c t i o n s > ( d e l a y e d o r p o s s i b l y immediate i n s k i n , k i d n e y , r e s p i r a t o r y mucosa and p o s s i b l y o t h e r o r g a n s ) 16 I I I . MERCURY IN BIOLOGICAL SYSTEMS A. T r a n s p o r t t o Organs Harvey (1965) s t a t e s t h a t m e r c u r i a l s c a r r i e d by the b l o o d s t r e a m a r e d i s t r i b u t e d t h r o u g h o u t th e body t i s s u e s w i t h i n a few hours of i t s i n t r o d u c t i o n i n t o t h e b l o o d . Harvey f u r t h e r s t a t e s , i n t h e f o l l o w i n g o r d e r of d e s c e n d i n g c o n c e n t r a t i o n , the t i s s u e s i n w h i c h he found m e r c u r i a l s : k i d n e y , l i v e r , b l o o d , s p l e e n , i n t e s t i n a l w a l l , s k i n , h e a r t , s k e l e t a l m u s c l e , and b r a i n . As mercury p a s s e s i n t o the b l o o d o f r a b b i t s , i t becomes f i r m l y bound t o the plasma p r o t e i n s and e r y t h r o c y t e s ( B e r l i n and G ibson 1963). Hughes (1957) s t a t e s t h a t t h e p r i n c i p a l r e a c t i o n o f mercury compounds i s w i t h t h i o l s ( t h a t i s , the f o r m a t i o n o f mercury m e r c a p t i d e ) , and t h a t v a r i a t i o n s i n d i s t r i b u t i o n and e f f e c t a r e dependent upon t h i s r e a c t i o n . Human b l o o d plasma i s 0.5 m i l l i m o l a r t h i o l , and t h e r e d c e l l s c o n t a i n 60 t i m e s the s u l p h y d r y l i n t h e i r haemoglobin (Weiss-man e t a l 1 9 5 0). Hughes (1957) a l s o n o t e d t h a t b o v i n e a l b u m i n , o x y h a e m o g l o b i n , and c a r b o x y l h a e m o g l o b i n bound methyl mercury (w h i c h had been a d m i n i s t e r e d as CHg-Hg-I). M e r c u r y i o n may i n t e r f e r e w i t h t h e p r o d u c t i o n o f haem. Two s t e p s i n t h e b i o s y n t h e s i s o f haem appear v u l n e r a b l e t o the a c t i v i t y o f mercury i o n s , t h e t r a n s f o r m a t i o n o f d e l t a a m i n o l e -v u l i n i c a c i d t o p r o p h o b i l i n o g e n as m e d i a t e d by the enzyme d e l t a a m i n o l e v u l i n i c a c i d d e h y d r a s e ; and t h e t r a n s f o r m a t i o n o f p r o t o p o r p h y r i n IX p l u s i r o n t o haem as mediated by t h e enzyme 17 haem s y n t h e t a s e . The a c t i v e s i g h t i n both enzymes b e i n g f r e e s u l f h y d r y l (S-H) groups. In blood the f u n c t i o n a l e f f e c t would be anemia. Haem i s a l s o an e s s e n t i a l c o n s t i t u e n t of o t h e r r e s p i r a t o r y pigments, the cytochromes, which p l a y key r o l e s i n energy metabolism. Methyl mercury a l s o shows s t r o n g a f f i n i t e s f o r many o t h e r b i o l o g i c a l s u bstances. Hughes (1957) e s t i m a t e d the a f f i n i t y of methyl mercury f o r c a r b o x y l groups as -log^QpC = 4, and the a f f i n i t y f o r amides and amines to be app r o x i m a t e l y the same. Hughes (1957) f u r t h e r s t a t e s t h a t i n i s o l a t e d p r o t e i n systems ( i n the absence of h a l i d e s ) l a r g e amounts of methyl mercury may be found (up to 100 mole of methyl mercury to 1 mole of serum albumin). S o l u b l e i n o r g a n i c m e r c u r i a l s r e a d i l y g a i n a c c e s s to the c i r c u l a t i o n when taken by mouth (Goldwater 1957), although xThe t o x i c o l o g y of methyl mercury h a l i d e s may be d i s c u s s e d i n terms of the a f f i n i t y of the methyl mercury i o n f o r b i o l o g i c a l s u b stances. T h i s a f f i n i t y may be d e s c r i b e d by the f o l l o w i n g e q u a t i o n (CH^-Hg*) + (X~) = CH^-Hg-X. R e l a t i v e a f f i n i t i e s f o r v a r i o u s b i o l o g i c a l substances may then be o b t a i n e d by comparing the e q u i l i b r i u m c o n s t a n t s f o r t h i s r e a c t i o n . [CH3-Hg-X] " [CH 3-Hg +] [X-] 18 a c o n s i d e r a b l e p o r t i o n of the i n g e s t e d mercury may remain f i x e d to the a l i m e n t a r y mucosa and the i n t e s t i n a l c o n t e n t s . The e f f e c t of mercury on d i g e s t i v e enzymes leads to d i g e s t i v e d i s f u n c t i o n which g i v e s r i s e to s t a r v a t i o n . I n s o l u b l e m e r c u r i a l compounds may undergo a degree of o x i d a t i o n which c o n v e r t s them i n t o s o l u b l e , and t h e r e f o r e , absorbable com-pounds. There have been some i n v e s t i g a t i o n s i n t o the a b i l i t y of i n t e s t i n a l b a c t e r i a to methylate mercury. Wood (1972) r e p o r t s t h a t b a c t e r i a which s y n t h e s i z e v i t a m i n c a n a l s o s y n t h e s i z e methyl mercury, and Grant (1971) notes t h a t i f methyl mercury i s present i n the a l i m e n t a r y c a n a l , 95% of i t i s absorbed i n t o the body. I n o r g a n i c m e r c u r i a l s may a l s o be obsorbed through the body s u r f a c e i f they are d i s s o l v e d i n a s u i t a b l e s o l v e n t . Berglund and B e r l i n (1969a, 1969b) and Anonymous (1959) have d i s c u s s e d the r i s k to humans from the many forms of food which c o n t a i n mercury. Rucker and Amend (1969) found t h a t an o r g a n i c m e r c u r i a l p r o p h y l a c t i c used i n f i s h h a t c h e r i e s e n t e r e d rainbow t r o u t , chinook and sockeye salmon v i a t h e i r g i l l t i s s u e . T h i s m e r c u r i c compound ( e t h y l m e r c u r i c phosphate) appeared i n the b l o o d one hour a f t e r the t e r m i n a t i o n of a one-hour exposure to a 2 ppm s o l u t i o n . Tv/elve hours a f t e r the s t a r t of the i n v e s t i -g a t i o n , the mercury co n t e n t of the l i v e r and kidney was 0.9 and 1.0 ppm r e s p e c t i v e l y . The g i l l s r e t u r n e d to the 0.4 ppm l e v e l found as background f o r the f i s h f o u r weeks a f t e r the exposure, and the blood and l i v e r r e t u r n e d to the background 19 l e v e l i n e i g h t weeks and twenty weeks r e s p e c t i v e l y . The kidney s t i l l c o n t a i n e d 1.8 ppm mercury when the i n v e s t i g a t i o n was terminated a f t e r 28 weeks. The authors a l s o exposed a number of t r o u t to the same chemical under a s l i g h t l y d i f f e r e n t e x p e r i m e n t a l regime. Two groups of f i s h were used; one was exposed to one-hour treatments g i v e n once a day f o r e l e v e n days, and the o t h e r group was t r e a t e d f o r one hour per week f o r twelve weeks. These experiments r e v e a l e d t h a t much long e r c l e a r i n g times are r e q u i r e d f o r cumulative m e r c u r i a l c o n c e n t r a t i o n s . Tsuruga (1963) found t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the p a t t e r n s of d i s t r i b u t i o n of mercury i n t i s s u e s of c a r p f e d sea mussel t i s s u e which c o n t a i n e d a m e r c u r i a l , and conger e e l s f e d with clam t i s s u e which a l s o c o n t a i n e d the mercury compound. An accumulation of heavy metal was not found i n the b r a i n , but a h i g h c o n c e n t r a t i o n was noted i n the g i l l s , l i v e r , and kidney as w e l l as the obvious e x i s t e n c e of mercury i n the bone. Rucker and Amend (1969) found mercury i n the b r a i n t i s s u e of t r o u t which they had exposed f o r ten days i n t h e i r e l e v e n day d a i l y dose study. The t i s s u e taken on the t e n t h day c o n t a i n e d 10.1 ppm mercury, and a f t e r f i f t y days the l e v e l had f a l l e n to 5.5 ppm. I t appears t h a t b r a i n t i s s u e may be slow to c l e a r a m e r c u r i a l i f i t gains e n t r y through the b r a i n -b l o o d b a r r i e r . Hughes (1957) found t h a t methyl mercury com-pounds were g e n e r a l l y l i p i d s o l u b l e and l i p i d s o l u b i l i t y might account f o r the r e t e n t i o n of e t h y l mercury phosphate i n the 20 b r a i n t i s s u e of the t r o u t used by Rucker and Amend (1969). B e r l i n and U l l b e r g (1963) a l o n g w i t h Hughes (1957) and Swen-sson e t a l (1959) found t h a t the b r a i n c o n t a i n s a h i g h e r c o n t e n t of methyl mercury r e l a t i v e to the t o t a l body burden when compared to other m e r c u r i a l s t e s t e d . Methyl mercury p e n e t r a t e s the b l o o d - b r a i n b a r r i e r and i s d i s t r i b u t e d i n the b r a i n , p r o d u c i n g symptoms due to the d e s t r u c t i o n of c e l l s i n the c e r e b e l l u m (balance c e n t r e ) , v i s u a l and h e a r i n g c e n t r e s and elsewhere (Grant 1971). B. E x c r e t i o n of M e r c u r i a l s o Mercury, r e g a r d l e s s of the r o u t e of a d m i n i s t r a t i o n or type of compound, i s e x c r e t e d by a l l c h a n n e l s : u r i n e , f e c e s , sweat, s a l i v a , and m i l k . The r a t e of e x c r e t i o n of m e r c u r i a l s v a r i e s w i d e l y a c c o r d i n g to the d i f f e r e n t types of compounds. M e r c u r i a l s a f f e c t s p e c i f i c r e n a l t u b u l a r f u n c t i o n s such as i o n t r a n s p o r t and t u b u l a r s e c r e t i o n and r e - a b s o r p t i o n of o r g a n i c s u b s t a n c e s . M e r c u r i a l s are e x c r e t e d by the body immediately a f t e r a b s o r p t i o n . M e r c u r i a l s used as t h e r a p e u t i c s are e x c r e t e d by the body r e p i d l y . Vogl (1955) r e p o r t e d t h a t 40 to 60% of mercury i s e l i m i n a t e d from the body w i t h i n the f i r s t day, and t h a t by the e i g h t h day a l l a d m i n i s t e r e d mercury was e l i m i n a t e d i n the u r i n e . P i t t s and S a r t o r i u s (1950) c i t e f i v e s t u d i e s which i n d i c a t e t h a t 60 to 100% of m e r a l l u r i d e (a d i u r e t i c ) was e l i m i n a t e d i n 9 hours, and t h a t 99% e x c r e t i o n of t h e r a p e u t i c m e r c u r i a l d i u r e t i c s o c c u r r e d i n 24 hours. Shoemaker (1957) 21 r e p o r t s t h a t 90% had been removed from the body w i t h i n 24 hours. ( M e r c u r i a l d i u r e t i c s are compounds which have the c h a r a c t e r i s t i c s of low t o x i c i t y and low r e t e n t i o n . ) Cafruny and Gussin (1963) l o o k i n g at dogs, and Cafruny (1965) and C a f r u n y and G u s s i n (1967) s t u d y i n g the a g l o m e r u l a r f i s h Lophius americanus suggested t h a t m e r c u r i a l s are p r i m a r i l y found i n the proximal t u b u l e s . Campbell (1960) has i m p l i c a t e d the a c i d s e c r e t o r y system of the proximal t u b u l e s s p e c i f i c a l l y i n mercury e x c r e t i o n . B e r l i n and Gibson (1963) observed i n t h e i r experiments that r e n a l accumulation of mercury exceeded u r i n a r y output and hence they concluded t h a t the accumulation of mercury i n the kidney was due to t u b u l a r r e - a b s o r p t i o n . They concluded mercury e x c r e t e d v i a the proximal t u b u l e s was re-absorbed d i s t a l l y . T h i s c o n c l u s i o n was based on the o b s e r v a t i o n t h a t 50% of the t o t a l dose of i n f u s e d mercury was taken up by the kidney w h i l e o n l y 10% was e x c r e t e d i n the u r i n e of the r a b b i t s i n the e x p e r i m e n t a l s i t u a t i o n . B e r l i n and Gibson found t h a t the u r i n a r y e x c r e t i o n of mercury was propor-t i o n a l to the c o n c e n t r a t i o n of mercury i n the b l o o d , but that i t was not p r o p o r t i o n a l to the c o n c e n t r a t i o n of mercury i n the r e n a l t i s s u e . Passow e t a l (1961) p r e s e n t e d e v i d e n c e t h a t about 90% of the mercury i n the body i s l o c a t e d i n the kidney. I t was concluded that the degree of c l e a r i n g of the body burden of mercury by the kidney was dependent on the a c t i v i t y of the kidney t i s s u e . F r i b e r g (1957) found t h a t phenyl m e r c u r i c a c e t a t e was e x c r e t e d by r a b b i t kidney more r a p i d l y than m e r c u r i c a c e t a t e . Friedman (1957) suggests t h a t 22 f i l t e r i n g by the proximal t u b u l e s appears to be d i f f e r e n t f o r d i f f e r i n g s p a t i a l c o n f i g u r a t i o n s of m e r c u r i a l s . The g a s t r o i n t e s t i n a l t r a c t has been i m p l i c a t e d i n the e x c r e t i o n of mercury. L i e b and Goodwin (1915) found t h a t m e r c u r i a l s were e x c r e t e d by the g a s t r i c mucous membranes. The b i l e must be i n c l u d e d as an agent i n the e x c r e t i o n of mercury. I f any m e r c u r i a l compounds are bound to the s u l p h y d r y l groups of the e r y t h r o c y t e s broken down by the s p l e e n they w i l l appear i n the b i l e s e c r e t e d i n t o the a l i m e n t a r y c a n a l . Aaronson (1971) noted t h i s f u n c t i o n of b i l e but concluded t h a t some of the mercury e x c r e t e d i n b i l e would be re- a b s o r b e d d u r i n g the process of l i p i d d i g e s t i o n and a b s o r p t i o n . The l i p i d s o l u b i l i t y of methyl mercury might l e a d to the r e - e n t r y of any b i l e - b o u n d mercury through the lymphatic system when f a t t y a c i d , g l y c e r o l , and l i k e m a t e r i a l s are absorbed i n t o the body. Lomholt (1920) found t h a t minute q u a n t i t i e s o f mercury were e x c r e t e d by sweat and s a l i v a i n p a t i e n t s t h a t were t r e a t e d w i t h m e r c u r i a l s f o r s y p h i l i s . Increased s a l i v a t i o n has been d e s c r i b e d throughout the h i s t o r y of m e r c u r i a l treatment of s y p h i l i s from i t s f i r s t a p p l i c a t i o n i n the 1500's. L e g a l e s and Duperoy (1900) s t a t e d t h a t mercury c o u l d be found i n the milk of women being t r e a t e d f o r s y p h i l i s . There appears to be a l a g p e r i o d between the exposure to the m e r c u r i a l and i t s e x c r e t i o n i n m i l k . Grant (1971) c i t e d a case of a woman i n the Minamata Bay area who had a c h i l d w i t h m e r c u r i a l i s m . The c h i l d had not eaten any f i s h from the bay and had been f e d e x c l u s i v e l y with i t s mother's m i l k . The author suggested that a reason the mother had been asymptomatic might be based upon the f a c t t h a t the p r o d u c t i o n of milk by the mammary glands bound mercury to the milk p r o t e i n s , and t h e r e -f o r e , c l e a r e d her body of the p o t e n t i a l l y harmful mercury com-pounds . Shoemaker (1957) and B e r l i n (1963b) have reviewed d e t a i l s of a b s o r p t i o n , d i s t r i b u t i o n and e x c r e t i o n of mercury i n d e t a i l . 24 IV. SYMPTOMATOLOGY OF MERCURIALISM Harvey (1965) g i v e s a c o n c i s e d e l i n e a t i o n of the symp-toms of both c h r o n i c and acute m e r c u r i a l i s m . Although the author g i v e s an account of the m a n i f e s t a t i o n s of mercury p o i s o n i n g i n humans i t g i v e s r e a s o n a b l e cause to s p e c u l a t e upon the e f f e c t s of mercury on anadromous f i s h e s and o t h e r animals which l i v e i n p o l l u t e d environments. A. Acute Mercury P o i s o n i n g ( a f t e r Harvey 1965) Syst e m a t i c s i g n s o f acute p o i s o n i n g by el e m e n t a l mercury and e t h y l and methyl m e r c u r i c compounds i n c l u d e g e n e r a l symptoms such as l e t h a r g y , excitement, hyper r e f l e x i a and tremor. The t o x i c a c t i o n of i n o r g a n i c mercury on the kidney i s u s u a l l y the cause of death. The kidney may show ev i d e n c e of d i s r u p t e d f u n c t i o n w i t h i n a few minutes a f t e r the p o i s o n reaches the c i r c u l a t i o n . I f the l o c a l a c t i o n of the compound i s not f o l l o w e d by profound shock and i f c i r c u l a t i o n i s ade-quate, the f i r s t response of the kidney may be d i u r e s i s due to the s u p p r e s s i o n of t u b u l a r r e a b s o r p t i v e a c t i o n . Soon, however, the r e n a l damage becomes so e x c e s s i v e t h a t o l i g u r i a and f i n a l l y a n u r i a r e s u l t s . The r e n a l l e s i o n s produced by mercury are c o n f i n e d l a r g e l y to the t u b u l a r e p i t h e l i u m , but the g l o m e r u l i are a l s o i n j u r e d to some e x t e n t . The events d e s c r i b e d f o r p o i s o n i n g by i n o r g a n i c mercury combine to produce a d i s t o r t i o n i n the e l e c t r o l y t e p a t t e r n and i n the d i s t r i b u t i o n of body f l u i d s . The volume of e x t r a 25 c e l l u l a r f l u i d i s markedly d e p l e t e d as a r e s u l t of v o m i t i n g , d i a r r h e a , and i n i t i a l d i u r e s i s . The d i a r r h e a i s most promi-nent and tends to cause a c i d o s i s , which i s accentuated by the i n a b i l i t y to i n g e s t f o od. P r o t e i n and f l u i d are l o s t through the v e s s e l w a l l s , and the c i r c u l a t o r y volume i s markedly d i m i n i s h e d . The p r o t e i n i n the plasma may be reduced as a r e s u l t of a l b u m i n u r i a and s t a r v a t i o n . The e x c r e t i o n of mercury i n t o the c o l o n r e s u l t s i n c o l i t i s , which i n t e n s i f i e s and prolongs the d i a r r h e a . B. C h r o n i c Mercury P o i s o n i n g ( a f t e r Harvey 1965) C h r o n i c mercury p o i s o n i n g r e s u l t s from exposure to s m a l l amounts of mercury over extended p e r i o d s of time. I t i s encountered i n i n d u s t r i e s t h a t u t i l i z e mercury or i t s s a l t s , and a l s o may r e s u l t from mercury m e d i c a t i o n . The s i g n s and symptoms c h a r a c t e r i z i n g t h i s c o n d i t i o n are g i n g i v i t i s , stoma-t i t i s , l o o s e n i n g of the t e e t h , s a l i v a t i o n , m e t a l l i c t a s t e , c o l i t i s , p r o g r e s s i v e r e n a l damage, l o s s of a p p e t i t e , n u t r i -t i o n a l d i s t u r b a n c e s , anemia, h y p e r t e n s i o n , and p e r i p h e r a l n e u r i t i s . The c e n t r a l nervous system i s e s p e c i a l l y i n v o l v e d , as evidenced by b e h a v i o u r a l changes, mental d e p r e s s i o n , i r r i t a b i l i t y , insomnia, i n t e n t i o n tremors and shaking f a t i g u e and drowsiness, and o c c a s i o n a l l y h a l l u c i n a t i o n s . The e t h y l and methyl mercury compounds mainly cause n e u r o l o g i c a l s i g n s and symptoms, e s p e c i a l l y tremor, a t a x i a , d y s a r t h r i a , c o n s t r i c -t i o n of the v i s u a l f i e l d , and o c c a s i o n a l l y p s y c h o s i s . 26 C. S e n s i t i v i t y of I n d i v i d u a l s w i t h i n a P o p u l a t i o n The s e n s i t i v i t y of i n d i v i d u a l s w i t h i n a p o p u l a t i o n can d i f f e r g r e a t l y . B u c k e l l et a l (1946) and Smith and Moskowitz (1949) observed t h a t i n i n d i v i d u a l cases some high values of mercury i n the u r i n e f r e q u e n t l y occur without evidence of poisoning w h i l e i n others p o i s o n i n g could occur i n a s s o c i a t i o n w i t h r e l a t i v e l y low l e v e l s of u r i n a r y mercury. Many i n d u s t r i a l workers have been exposed to mercury f o r years producing u r i n a r y e x c r e t i o n of mercury up to s e v e r a l m i l l i g r a m s per l i t r e without showing s i g n s of m e r c u r i a l i s m . Grant (1969) reporte d a case of h y p e r s e n s i t i v i t y i n a p h y s i c i a n who complained of severe asthma and s k i n r a s h . The cause of the a l l e r g i c r e a c t i o n was a phenyl mercuric compound present i n an a n t i - b a c t e r i a l f a b r i c s o f t e n e r used i n the h o s p i t a l laundry as part of a s t a p h y l o c o c c a l c o n t r o l program. Ladd e t a l (1963) conducted a study i n a f a c t o r y which produced a v a r i e t y of i n o r g a n i c and organic compounds. The authors found that i n three out of f o u r sets of observations there were i n d i c a t i o n s t h a t those men who had been working the longest w i t h m e r c u r i a l s had the highest u r i n a r y mercury. The f o u r t h set of observations showed a pronounced c o r r e l a t i o n between exposure and u r i n a r y e x c r e t i o n , and t h i s o b s e r v a t i o n was re-enforced by the study of M c G i l l e t a l (1964). B e r l i n and Gibson (1963) observed that the amount of mercury accumulated i n the kidney and u r i n a r y e x c r e t i o n had no d i r e c t r e l a t i o n s h i p . This suggested t h a t u r i n a r y e x c r e t i o n was 27 a poor index of exposure to m e r c u r i a l s , and a c c o r d i n g to Gage (1964), a d i a g n o s i s of mercury p o i s o n i n g c o u l d o n l y be made i f a high c o n c e n t r a t i o n of mercury i n the u r i n e was accompanied by s i g n s and symptoms i n d i c a t i v e of m e r c u r i a l i s m . D. S u b l e t h a l E f f e c t s M e r c u r i a l s , even i n low c o n c e n t r a t i o n s , are c a p a b l e of i n a c t i v a t i n g s u l p h y d r y l enzymes and thus i n t e r f e r i n g w i t h c e l l u l a r metabolism and f u n c t i o n (HeHerman 1937; Barron and K a l n i t s k y 1947; Passow e t a l 1961). Jackim e t a l (1970) found t h a t the a c t i v i t i e s of f i v e l i v e r enzymes ( a c i d and a l k a l i n e phosphatase, c a t a l a s e , x a n t h i n e o x i d a s e , and r i b o n u c l e a s e ) from the f i s h Fundulus h e t e r o c l i t u s were n e g a t i v e l y a f f e c t e d i n t h e i r a c t i v i t y . Enzyme a c t i v i t y was i n v e s t i g a t e d a f t e r 96-hour exposure to s a l t s o f s i x metals ( c o n c e n t r a t i o n s s e t at the mean t o l e r a n c e l i m i t ) . The m e r c u r i c s a l t proved to be a c t i v e a g a i n s t the a c t i v i t i e s of a l l the enzymes except r i b o n u -c l e a s e . Hickman and Trump (1969) i m p l i c a t e a c i d and a l k a l i n e phosphatase a l o n g with a d e n o s i n e t r i p h o s p h a t a s e , cytochrome o x i d a s e , glucose-6-phosphatase and s u c c i n i c dehydrogenase i n the kidney f u n c t i o n s of e u r y h a l i n e t e l e o s t s . The a c t i v i t y of these enzymes may be d i s r u p t e d b y . m e r c u r i a l compounds, and thereby be a component i n kidney d i s f u n c t i o n . Shimizu (1970, 1971) r e p o r t e d f e t a l malformation and death i n the o f f s p r i n g of r a t s when phenyl m e r c u r i c a c e t a t e (P.M.A.) was a p p l i e d to the v a g i n a l t r a c t . I t has been 28 suggested t h a t P.M.A. when used as a c o n t r a c e p t i v e might prove dangerous to women as w e l l as to a f e t u s ( i n case of c o n t r a c e p -t i v e f a i l u r e ) . Mercury compounds have been agents a c t i v e i n chromo-some breakage and n o n - d i s j u n c t i o n . Grant (1971) suggests that the derangement of chromosomes by mercury probably depends on i t s i n t e r a c t i o n with s u l f h y d r y l groups of substances e s s e n t i a l f o r normal s p i n d l e f o r m a t i o n . Genetic mutations and chromo-somal a b e r r a t i o n s have been induced i n p l a n t c e l l s (Ramel 1967, 1969a, 1969b) and i n the gametes of D r o s o p h i l a melanoqaster (Ramel 1967; Ramel and Magnusses 1969). S k e r f u i n g e t a l (1970) have r e c e n t l y presented e v i d e n c e of chromosome breakage i n humans exposed to methyl mercury due to consumption of con-taminated f i s h . Westoo (1969, 1967) r e p o r t e d mercury i n the c o n t e n t s of eggs, and l a t e r Westoo (1969) found t h a t f i s h p rocessed f o r c h i c k e n meal c o n t a i n e d methyl mercury which appeared l a t e r i n c h i c k e n eggs. Stoewsand e t a l (1971) found that e g g s h e l l s of Japanese q u a i l were t h i n n e r a f t e r the b i r d s were f e d m e r c u r i c c h l o r i d e ; t h i s appears as a new aspect of egg c o n t a m i n a t i o n . Jones e t a l (1956) found avoidance r e a c t i o n s i n salmonid f i s h when they were s u b j e c t e d to p u l p m i l l e f f l u e n t which may have c o n t a i n e d m e r c u r i a l compounds. Sprague (1964, 1965) and Sprague et a l (1963) found t h a t m i g r a t i n g a d u l t A t l a n t i c salmon (Salmo s a l a r ) , a p p a r e n t l y voided s u b l e t h a l heavy metal p o l l u t i o n ( z i n c and copper) which was produced by a mine on the M i r a m i c h i R i v e r i n New Brunswick. 29 Avoidance responses are suspected i n l a k e s which have been contaminated by mine t a i l i n g s from mercury mines. 30 I INTRODUCTION Because o f the r e c e n t d i s c o v e r y o f m e r c u r i a l s u b s t a n c e s i n f o o d , f i s h , and game, t h e r e has been an expanding p u b l i c awareness o f t h e problem o f e n v i r o n m e n t a l mercury c o n t a m i n a -t i o n . P h y s i c i a n s , p u b l i c h e a l t h o f f i c i a l s , and o t h e r s argue a g a i n s t u n l i m i t e d and u n t r e a t e d d i s c h a r g e o f i n d u s t r i a l and a g r i c u l t u r a l w a s t e s because o f t h e i r d e l e t a r i o u s e f f e c t s on human w e l f a r e . C o n s e r v a t i o n i s t s p r o t e s t t h e l o s s o f w i l d l i f e and e c o l o g i c a l d i s r u p t i o n c a u s e d by e n v i r o n m e n t a l c o n t a m i n a n t s . Dependent i n d u s t r i e s such as c o m m e r c i a l f i s h e r i e s p r o t e s t the economic l o s s i n c u r r e d when t h e i r c a t c h i s pronounced u n s a f e f o r use as f o o d o r b y p r o d u c t . I t i s an u n f o r t u n a t e f a c t t h a t government a g e n c i e s c h a r g e d w i t h t h e r e s p o n s i b i l i t y f o r e n v i r o n m e n t a l p r o t e c t i o n have u n d e r e s t i m a t e d the p e r v a s i v e n e s s o f mercury and i t s com-pounds, and t h e i r r o l e as e n v i r o n m e n t a l i n t o x i c a n t s . Warnings were sounded by Japanese p u b l i c h e a l t h o f f i c i a l s f o l l o w i n g i n v e s t i g a t i o n s o f the Minamata Bay and N i i g a t a d i s a s t e r s which o c c u r r e d i n 1953 and 1964 r e s p e c t i v e l y . The Japanese were con-c e r n e d w i t h t h e d e l i n e a t i o n o f the human consequences o f organomercury a b s o r p t i o n and t o x i c i t y as w e l l as s t u d y i n g the d i s r u p t i o n o f the b i o t a by m e r c u r i a l s . F i s h and s h e l l f i s h were i m p l i c a t e d as t h e v e c t o r s i n t h e t r a n s f e r o f mercury from the a q u a t i c e n v i r o n m e n t t o humans. Swedish o f f i c i a l s heeded t h e w a r n i n g s and c o n d u c t e d i n v e s t i g a t i o n s o f t h e Swedish e n v i r o n -3 1 ment which, by 1 9 6 6 , had l e a d to the banning of s e v e r a l m e r c u r i a l compounds proven dangerous. S i n c e humans had not been a f f e c t e d by d i s a s t e r s s i m i l a r to those which o c c u r r e d i n Japan, the Swedes concerned themselves with t r a n s p o r t , d i s t r i -b u t i o n and p o t e n t i a l s u b c l i n i c a l e f f e c t s of m e r c u r i a l s . P o l l u t i o n b i o l o g i s t s have developed b i o a s s a y techniques f o r measuring l e t h a l i t y . The parameters u s u a l l y e s t i m a t e d are the LD^Q (mean l e t h a l dose) or EDJ-Q (mean e f f e c t i v e d o s e ) . These are the l e v e l s of which produce a f i f t y per cent m o r t a l i t y i n the t e s t organism i n a f i x e d p e r i o d of time ( 2 4 , 4 8 and 96 hour t e s t s , e t c . ) . B i o a s s a y techniques y i e l d v a l u a b l e i n f o r m a t i o n ; however, e v a l u a t i o n of environmental c o n t a m i n a t i o n and the e v o l u t i o n of p o l i c y designed to c o u n t e r a c t i t can onl y be p a r t i a l l y s u c c e s s -f u l i f acute t o x i c i t y (to a f f e c t e d organisms) i s the o n l y . c r i t e r i o n used to assess p e r m i s s i b l e l e v e l s . I t i s p o s t u l a t e d t h a t l e a r n e d b e h a v i o u r may be e s p e c i a l l y v u l n e r a b l e to the e f f e c t s of m e r c u r i a l i n t o x i c a n t s , and t h a t evidence r e l a t i n g d i s f u n c t i o n of i n a t e and l e a r n e d behaviour to s u b l e t h a l l e v e l s would be i n f o r m a t i v e . F o r the above reasons experiments designed to t e s t the h y p o t h e s i s t h a t s u b l e t h a l doses of a mercury compound (m e r c u r i c c h l o r i d e ) would a f f e c t l e a r n e d and i n a t e b e h a v i o u r were implemented. 32 II EXPERIMENTAL DESIGN AND RESULTS A. Upstream-Downstream M i g r a t i o n Experiment S e v e r a l r i v e r s and streams flow i n t o Babine Lake, B r i t i s h Columbia; t h i s l a k e i s d r a i n e d by the Lower Babine R i v e r . The l a k e p r o v i d e s a r e s i d e n c e f o r l a r g e number of Sockeye Salmon (Oncorhynchus nerka) d u r i n g the f i r s t year of t h e i r development. Sockeye a l e v i n s m i g rate to the l a k e from the v a r i o u s r i v e r s which connect with the Babine Lake system. C a l a p r i c e (1972), working with a l e v i n s from the Babine Lake system, has shown that a l e v i n s from d i f f e r e n t r i v e r s have d i f f e r e n t m i g r a t i o n behaviour with r e s p e c t to the c u r r e n t i n the r i v e r . A l e v i n s which emerge from the g r a v e l of the Lower Babine R i v e r have the g e n e t i c d i s p o s i t i o n to swim upstream a g a i n s t the c u r r e n t to reach the l a k e . A l e v i n s from the F u l t o n R i v e r have a s t r o n g tendency to swim downstream with the c u r r e n t and e n t e r the l a k e . The m i g r a t i o n b e h a v i o u r of the a l e v i n s from the F u l t o n R i v e r and the Lower Babine R i v e r p r o v i d e d a known b e h a v i o u r a l response which c o u l d be used to t e s t the e f f e c t s of s u b l e t h a l doses of m e r c u r i c c h l o r i d e on i n n a t e b e h a v i o u r . An experiment was designed to t e s t the n u l l h y p o t h e s i s t h a t s u b l e t h a l doses of m e r c u r i c c h l o r i d e would not a f f e c t m i g r a t i o n b e h a v i o u r . In f o r m u l a t i n g t h i s h y p o t h e s i s i t was supposed that any d e l e t e r i o u s e f f e c t of m e r c u r i c c h l o r i d e on t e s t f i s h 33 would be r e v e a l e d by d e v i a t e m i g r a t i o n behaviour of t e s t f i s h as compared to c o n t r o l f i s h . a) E x p e r i m e n t a l F i s h Sockeye salmon (Oncorhynchus nerka) a l e v i n s were c o l l e c t e d i n March of 1972 from the Babine Lake a r e a . F i s h from the Lower Babine R i v e r and the F u l t o n R i v e r were s e l e c t e d because of t h e i r c h a r a c t e r i s t i c m i g r a t i o n movements from spawning grounds to Babine Lake. The Lower Babine R i v e r f i s h were known to have s t r o n g upstream t e n d e n c i e s d u r i n g m i g r a t i o n s , w h i l e the F u l t o n R i v e r a l e v i n s were known to migrate downstream to Babine Lake. Redds were dug up i n the two r i v e r s d u r i n g the e a r l y s p r i n g i n o r d e r to o b t a i n a l e v i n s at a time c l o s e to t h e i r normal emergence from the g r a v e l . The a l e v i n s were p l a c e d i n p l a s t i c bags f i l l e d with water and taken to Smithers f o r p r o c e s s i n g . At Smithers the a l e v i n s were s o r t e d and p l a c e d i n t o f r e s h p l a s t i c bags that had been f i l l e d w ith c o l d water. The water had been oxygenated w i t h pure oxygen. The bags were p l a c e d i n i c e c h e s t s and flown to Vancouver from where they were taken by tru c k to Rosewall Creek Hatchery on Vancouver I s l a n d . b) E x p e r i m e n t a l Tanks F i s h tanks at Rosewall Creek Hatchery were used d u r i n g the summer of 1972. The tanks were of uniform d e s i g n and made of f i b e r g l a s s . The f i t t i n g s were P.V.C. ( p o l y v i n a l c h l o r i d e ) . 3 4 I t should be noted that although m e r c u r i a l compounds are used i n the p r o d u c t i o n of P.V.C., i t was c o n s i d e r e d t h a t such a source of m e r c u r i a l c o n t a m i n a t i o n was n e g l i g i b l e . F u r t h e r -more, the source was, i n any case, c o n s i s t e n t throughout a l l the treatments a d m i n i s t e r e d to the t e s t f i s h . The stand pipe of P.V.C. was of f i x e d l e n g t h w i t h the o v e r f l o w h o l e of f i x e d diameter to produce a volume of t h i r t y -t h r e e l i t r e s i n each of the exp e r i m e n t a l tanks. A l l the e x p e r i m e n t a l tanks had the same source o f water which was dispensed through f i x t u r e s of a st a n d a r d a p e r t u r e . The a p e r t u r e s were i n s p e c t e d f o u r times d a i l y f o r any blockage which would a l t e r the water f l o w . ( f r e e f l o w was checked a f t e r the p e r i o d of i n t o x i c a t i o n to ensure the u n i -form and r a p i d f l u s h i n g of the contaminated t a n k s ) . The water used was o b t a i n e d from two w e l l s which were on the hatchery p r o p e r t y . The w e l l water was used i n s t e a d of water from the creek sources because i t e x p e r i e n c e d fewer temperature f l u c t u a t i o n s (Appendix 1: Well water temperature r e c o r d s f o r May through A u g u s t ) . The water flow was i n t e r r u p t e d d a i l y at a s e t time f o r one hour. The doses of m e r c u r i c c h l o r i d e (HgC^) was mixed wit h the s t a n d i n g water i n the tanks. P r e l i m i n a r y t e s t s showed t h a t , f o r t h i s s h o r t p e r i o d of time, the f i s h d i d not s u f f e r due to the i n t e r r u p t i o n of the f l o w of oxygenated water. Two t e s t l e v e l s of m e r c u r i c c h l o r i d e ( HgC^) were used, 0.5 and 1.0 ppm. F i v e tanks f o r each t e s t l e v e l ( c o n t r o l , 0.5 and 1.0 ppm) were used, w i t h random assignments of treatments 35 to the f i f t e e n e x p e r i m e n t a l tanks. The i n t o x i c a t i o n t r e a t -ments were conducted f o r ten days and then the f i s h were put through a performance t e s t . c) The T e s t F i s h The p a r t i c i p a t i n g f i s h were a c c l i m a t i z e d to c o n d i t i o n s i n the hat c h e r y f o r two weeks b e f o r e assignment to t e s t tanks. E i g h t y a l e v i n s were a s s i g n e d to each tank. The a l e v i n s were taken from a pooled s t o c k and a s s i g n e d to the tanks one at a time, moving from tank one to tank f i f t e e n . T h i s form of assignment was repeated u n t i l a l l tanks r e c e i v e d e i g h t y f i s h . Only f o u r tanks of each t e s t l e v e l were used to p r o v i d e f i s h f o r the performance t e s t s . The f i f t h tank of each l e v e l p r o v i d e d replacement f i s h f o r the o t h e r tanks when m o r t a l i t y o c c u r r e d (Appendix 2: M o r t a l i t y Data f o r Upstream and Down-stream Sockeye A l e v i n s ) . The f i f t h tank of each l e v e l had a basket suspended on the stand p i p e i n t o which stock f i s h were p l a c e d to m a i n t a i n e i g h t y f i s h per tank i n the.event t h a t i n t o x i c a t e d animals were removed f o r placement i n e x p e r i m e n t a l tanks. These stock f i s h were segregated to a v o i d mixing w i t h those replacement f i s h a l r e a d y i n t o x i c a t e d . A f u l l complement of f i s h was maintained to absorb the i n t o x i c a n t . Thus, by t h i s s e g r e g a t i o n , the replacement f i s h were i n t o x i c a t e d to a l e v e l s i m i l a r to those they r e p l a c e d i n the e x p e r i m e n t a l tanks. The a l e v i n s were s t a r v e d d u r i n g the p e r i o d of i n t o x i -36 c a t i o n (ten days) because i t was suspected that f e e d i n g would g r e a t l y enhance the c o n d i t i o n of the c o n t r o l f i s h w h i l e p r o v i d i n g n e g l i g i b l e b e n e f i t s f o r the i n t o x i c a t e d f i s h , i . e . the f i s h t r e a t e d with Hg'C^ would be weakened from hunger as w e l l as i n t o x i c a t i o n with the m e r c u r i a l ( t h i s s u s p i c i o n was l a t e r c o n f i r m e d i n growth experiments d e t a i l e d l a t e r ) . d) The Performance Channel f o r the M i g r a t i o n Experiments A performance channel was n e c e s s a r y to d e t e c t the responses of the a l e v i n s t o a d i r e c t e d c u r r e n t . The p e r f o r -mance channel f o r the m i g r a t i o n experiment was d e s i g n e d by C a l a p r i c e (1972). I t c o n s i s t e d of a wooden trough ( F i g u r e 1), 15.4 c e n t i m e t e r s wide by 3.05 meters long and 15.4 c e n t i m e t e r s i n h e i g h t . The l e n g t h was d i v i d e d to p r o v i d e t w e n t y - f i v e compartments of equal volume. A 2.5 c e n t i m e t e r s h o l e l e a d from one compartment to the next i n a staggered f a s h i o n . The h o l e c e n t e r s were of u n i f o r m h e i g h t from the bottom of the trough. The s t a g g e r i n g e f f e c t i v e l y p r e v ented a d i r e c t f low of water from one compartment to the next by i n d u c i n g c i r c u l a r c u r r e n t s i n each compartment b e f o r e water moved i n a down-stream d i r e c t i o n to the next compartment. The t e s t f i s h had to seek out the entrance h o l e to the next compartment and d e c i d e whether the water flow was up-stream or downstream. Twelve performance channels were used i n two banks of s i x . e) The T e s t Chambers Two rooms (approximately 4m. x 4m. x 4m.) were 37 c o n s t r u c t e d u s i n g 2 i n . x 4 i n . beams and % i n . plywood. The i n n e r w a l l s were p a i n t e d w i t h a f l a t non-gloss white p a i n t and i l l u m i n a t e d by a b a t t e r y of l i g h t s . The l i g h t i n g and p a i n t e d w a l l s produced a un i f o r m l i g h t i n t e n s i t y (as checked by a l i g h t m e t e r ) . The l i g h t s were connected to a powerstat o u t s i d e the rooms. The powerstat enabled the author to si m u l a t e a day from dawn to dusk i n seventy minutes. The performance channels i n the rooms were connected to the same head tank and the water f l o w from the head tank to each trough c o u l d be c o n t r o l l e d to produce the d e s i r e d uniform water f l o w . A c o o l i n g u n i t was i n s t a l l e d to c o o l the water to 6° c e n t i g r a d e b e f o r e i t e n t e r e d the head tank. The water flow and the water temperature were a d j u s t e d b e f o r e each e x p e r i m e n t a l run to ensure u n i f o r m i t y i n s e p a r a t e e x p e r i m e n t a l r u n s . f ) The A l e v i n s i n the Performance Channel The chambers of the performance trough were numbered from zero (extreme upstream) to t w e n t y - f i v e (extreme downstream). Twenty a l e v i n s were p l a c e d i n compartment t h i r t e e n . For a c c l i -m a t i z a t i o n , the t e s t f i s h were he l d i n compartment t h i r t e e n , by c o n t r o l gates over the escape h o l e s , f o r ten minutes i n the dark. At the c o n c l u s i o n of the a c c l i m a t i z a t i o n p e r i o d , the gates were removed from the troughs and the room s e a l e d . The l i g h t i n t e n s i t y was i n c r e a s e d from z e r o to 120 watts over f i v e minutes ( i n s t e p s of 24 u n i t s per minute); t h i s was the dawn phase. The l i g h t s were l e f t at 120 watts f o r s i x t y minutes 38 and t h i s c o n s t i t u t e d the day phase. At the end of an hour the l i g h t was t u r n e d down to z e r o watts ( i n s t e p s of 24 u n i t s per minute). T h i s r e p r e s e n t e d the dusk phase. The number of f i s h i n each compartment was noted u s i n g a f l a s h l i g h t . T h i s was an attempt to e l i m i n a t e any movement from one compartment to another due to f r i g h t responses of the f i s h to s e e i n g move-ment above the troughs. Each treatment l e v e l had f o u r runs of twenty f i s h . The t e s t a l e v i n s i n groups of twenty f i s h were as s i g n e d to t e s t troughs randomly. g) The S c o r i n g Systems The numbers of f i s h per chamber were noted i n chambers one (upstream) through t w e n t y - f i v e (downstream). Chamber t h i r t e e n was g i v e n the s c o r e of zero and the chambers moving away from z e r o i n both d i r e c t i o n s (upstream and downstream) were given v a l u e s of one through twelve; upstream having p o s i t i v e v a l u e s and downstream having n e g a t i v e v a l u e s (Appen-d i x 3: Worked examples showing maximum, minimum downstream-upstream movement of f i s h i n twenty-two h y p o t h e t i c a l t e s t s ) . The t o t a l s c o r e was c a l c u l a t e d by m u l t i p l y i n g the s c o r e v a l u e s by the number of i n d i v i d u a l s i n the chambers and d i v i d i n g the sum (a p o s i t i v e or n e g a t i v e v a l u e ) by the number of i n d i v i d u a l s moved from chamber z e r o . A v a l u e of twelve was added to each t o t a l score to a v o i d n e g a t i v e numbers. The maximum downstream sc o r e was z e r o and the maximum upstream s c o r e was twenty-four. 39 The formula f o r t h i s e x p r e s s i o n was: 20 = 1 + 12 NT where n. I number of f i s h per compartment r rank number of c o r r e s p o n d i n g compartments NT the t o t a l number of f i s h t h a t moved from the c e n t r a l chamber d u r i n g the t e s t p e r i o d . F i s h from both the Lov/er Babine R i v e r and the F u l t o n R i v e r were put through the performance c h a n n e l s . There were s u f f i c i e n t f i s h of each type to do s i x t e e n runs of twenty f i s h per t e s t c o n c e n t r a t i o n . A l l the f i s h were run through the channels twice to p r o v i d e a t o t a l of t h i r t y - t w o s c o r e s f o r each c o n c e n t r a t i o n ( c o n t r o l , 0.5 ppm and 1.0 ppm). program designed by Harger (1970) was used to a n a l y s e the d a t a , h) The R e s u l t s of the Upstream-Downstream Experiment The F u l t o n R i v e r a l e v i n s showed s i g n i f i c a n t d i f f e r e n c e s (0< = .05) between treatments (Appendix 4 ) , and treatment s c o r e s have been compiled and are shown as histograms ( F i g u r e s 2, 3, and 4 ) . d i f f e r e n c e s (<*= .01) f o r the c o n t r o l / 0 . 5 ppm' stock and the 0.5 ppm/-1.0 ppm s t o c k . There were no s i g n i f i c a n t d i f f e r e n c e s d e t e c t e d between the c o n t r o l group and the 1.0 ppm group (Appendix 5 ) . The scores have been compiled and are shown i n histograms ( F i g u r e s 5, 6, and 7 ) . The s c o r e s were c a l c u l a t e d and an a n a l y s i s of v a r i a n c e The Lower Babine R i v e r a l e v i n s showed s i g n i f i c a n t 40 B. Feeding and S t a r v a t i o n Experiments Harvey (1965) r e p o r t s t h a t c h r o n i c exposure to m e r c u r i a l compounds causes the l o s s of a p p e t i t e and weight as w e l l as d i s r u p t e d m e t a b o l i c processes i n humans. A f e e d i n g experiment was designed to t e s t the n u l l h y p o t h e s i s t h a t there would be no s i g n i f i c a n t d i f f e r e n c e i n the growth of f i s h s u b j e c t e d to three treatment l e v e l s of me r c u r i c c h l o r i d e ( c o n t r o l , 0.5 ppm and 1.0 ppm HgC^ K t h e s u b l e t h a l doses given over a prolonged p e r i o d of t i m e ) . A s t a r v a t i o n experiment was desig n e d to t e s t the n u l l h y p o t h e s i s t h a t there would be no s i g n i f i c a n t d i f f e r e n c e i n weight l o s s of f i s h s u b j e c t e d to three treatment l e v e l s of mer c u r i c c h l o r i d e ( c o n t r o l , 0.5 ppm and 1.0 ppm HgC ^ K t h e s u b l e t h a l doses g i v e n over a prolonged p e r i o d of t i m e ) . In f o r m u l a t i n g these hypotheses i t was supposed t h a t e f f e c t s of m e r c u r i c c h l o r i d e would be r e v e a l e d by lower body weights as compared to t h a t of c o n t r o l s . a) E x p e r i m e n t a l F i s h S p e c i e s Experiments were conducted u s i n g Sockeye salmon (Oncorhynchus n e r k a ) , Cotho salmon (Oncorhynchus k i s u t c h ) , and Chum salmon (Oncorhynchus k e t a ) . The Sockeye salmon were pooled stock taken from the Lower Babine R i v e r and both the Coho and Chum f r y were o b t a i n e d from stock at Rosewall Creek Hatchery, Vancouver I s l a n d . b) The Ex p e r i m e n t a l Tanks The e x p e r i m e n t a l tanks, hardware and water source 41 were the same as those used i n the m i g r a t i o n experiments. c) Experimental Design The f i s h used i n the f e e d i n g and s t a r v a t i o n e x p e r i -ments were s e l e c t e d from a c c l i m a t i z e d s t o c k s at the Rosewall Creek Hatchery. F i s h i n poor c o n d i t i o n , or which were o u t s i d e an e s t a b l i s h e d range i n s i z e , were d i s c a r d e d . There were s u f f i c i e n t numbers of Sockeye salmon and Coho salmon to conduct both f e e d i n g and s t a r v a t i o n experiments. T h i r t y - o n e tanks were a s s i g n e d to each of these s p e c i e s . The f r y were a s s i g n e d one at a time to the t h i r t y - o n e tanks and the c y c l e was re p e a t e d u n t i l e i g h t y f i s h had been a s s i g n e d to a l l the t a n k s . For each s p e c i e s f i f t e e n tanks were a l l o t t e d to each of the fed and s t a r v e d groups. Each bank of f i f t e e n tanks was s u b - d i v i d e d i n t o three groups to g i v e f i v e tanks f o r each of the l e v e l s o f i n t o x i c a t i o n ( c o n t r o l , 0.5 ppm and 1.0 ppm me r c u r i c c h l o r i d e ) . Only f o u r tanks of each treatment l e v e l were used i n the weighings. The f i f t h tank of each treatment l e v e l was used to p r o v i d e replacement f i s h f o r the o t h e r f o u r . The t h i r t y f i r s t tank h e l d e i g h t y f i s h which were weighed to o b t a i n base weight d a t a . There were i n s u f f i c i e n t Chum salmon to conduct both f e e d i n g and s t a r v a t i o n experiments. S i x t e e n tanks were as s i g n e d f o r a f e e d i n g study ( f i v e c o n t r o l s , f i v e 0.5 ppm, and f i v e 1.0 ppm l e v e l s of m e r c u r i c c h l o r i d e ) . The f i f t h tank of each l e v e l p r o v i d e d replacement f i s h f o r the o t h e r f o u r 42 tanks undergoing analogous treatment. The s i x t e e n t h tank of the Chum group h e l d e i g h t y f i s h which were weighed to o b t a i n base weight d a t a . The f i s h were a s s i g n e d to the tanks u s i n g the same procedure as used i n a s s i g n i n g the Sockeye and Coho salmon. The f r y were allowed an a c c l i m a t i z a t i o n p e r i o d of one week, d u r i n g which time m o r t a l i t y was r e c o r d e d (Appendix 6 ) . At the end of a c c l i m a t i z a t i o n e i g h t y f r y of each s p e c i e s were weighed to o b t a i n base weight d a t a . The f i s h were not f e d d u r i n g the a c c l i m a t i z a t i o n p e r i o d . In a l l cases the treatments r e c i e v e d by the f i s h i n each tank were as s i g n e d randomly. The water flow to a l l tanks was stopped f o r one hour per day. During t h i s time the p r e s c r i b e d treatments and f e e d i n g s were a d m i n i s t e r e d . The r a t i o n of food a l l o t t e d to the f i s h a s s i g n e d to the f e e d i n g experiment was e s t a b l i s h e d by u s i n g a st a n d a r d hatchery f e e d i n g formula (the minimum food to o b t a i n maximum growth). The c a l c u l a t i o n took i n t o account the t o t a l number of f i s h i n the treatment tank, the t o t a l weight of e i g h t y f i s h ( o b t a i n e d from the base d a t a ) , and the temperature of the water. These f a c t o r s ivere used i n c o n j u n c t i o n with a c h a r t which gave a percentage of the t o t a l body weight to be given i n f o o d . The d e l i v e r y of food occured on a d a i l y b a s i s d u r i n g the p e r i o d of i n t o x i c a t i o n and the amount of food g i v e n d a i l y remained c o n s t a n t throughout the term of the experiment. The f i s h were f e d d u r i n g a p e r i o d of s t i l l water to a v o i d food l o s s by the o v e r f l o w of r u n n i n g water i n t o the stand p i p e s . i 43 Twenty f i s h from each tank were weighed every f o u r t e e n days f o r s i x weeks f o l l o w i n g the e s t a b l i s h m e n t of base d a t a . The f o u r tanks per treatment (twenty o b s e r v a t i o n s per tank) p r o v i d e d e i g h t y o b s e r v a t i o n s per treatment every two weeks. In weighing f i s h , the a n e s t h e t i c , 2-phenoxyethanol (CgH 5OCH 2CH 2OH) was used (0.5 ml per 1,000 ml of w a t e r ) . T h i s water was a d j u s t e d so t h a t i t s temperature would be s i m i l a r to t h a t of the t e s t tanks. T h i s p r e c a u t i o n was taken to a v o i d temperature shock. Cages were suspended from the stand pipes of each tank and the f i s h used i n the weighings were p l a c e d i n these cages f o r s e g r e g a t i o n from the unweighed f i s h . T h i s s e g r e g a t i o n separated the f i s h which may have s u f f e r e d i l l e f f e c t s from e i t h e r the a n e s t h e t i c or h a n d l i n g d u r i n g the weighing p r o c e s s . Replacement f i s h were p l a c e d i n the a p p r o p r i a t e tanks when m o r t a l i t y o c c u r r e d . Stock f i s h were p l a c e d i n cages of the replacement tanks to r e p l a c e those f i s h which had been t r a n s -f e r r e d to treatment tanks. d) Method of A n a l y s i s A program designed by Harger (1970) was used to a n a l y s e data o b t a i n e d over a s i x week p e r i o d i n which the weights of the f i s h were r e c o r d e d . An a n a l y s i s of c o v a r i a n c e was executed on the s l o p e s of the r e g r e s s i o n l i n e s r e l a t i n g weight (dependent v a r i a b l e ) to treatment (independent v a r i a b l e ) at a s p e c i f i c p o i n t i n time ( t h r e e weeks a f t e r the i n i t i a t i o n 44 s i x week p e r i o d were s i g n i f i c a n t l y d i f f e r e n t (oC= 0.01). The i n d i v i d u a l d e g r e e s of freedom t e s t on t h e a d j u s t e d means showed s i g n i f i c a n t d i f f e r e n c e s (0( = 0.05) between th e c o n t r o l and the 0.5 ppm H g C l 2 t r e a t m e n t s , the c o n t r o l and t h e 1.0 ppm HgCl2 t r e a t m e n t s and the c o n t r o l and the combined 0.5 ppm H g C l 2 and 1.0 ppm H g C l 2 t r e a t m e n t s (Appendix 8). There was no s i g n i f i c a n t d i f f e r e n c e between t h e 0.5 ppm H g C l 2 and 1.0 ppm H g C l 2 t r e a t m e n t s . The mean w e i g h t of the f i s h a t the o n s e t o f the t r e a t m e n t s was 0.142 gms. F i g u r e 9- i n d i c a t e s a s t e a d y w e i g h t g a i n i n t h e c o n t r o l f i s h and a d e c l i n e i n w e i g h t o f f i s h t r e a t e d w i t h 0.5 ppm H g C l 2 and 1.0 ppm H g C l 2 ( t h e l a t t e r h a v i n g t h e more s e v e r e w e i g h t l o s s ) . A l t h o u g h t h e c o n t r o l Coho salmon showed a s t e a d y p o s i t i v e growth t h e d i f f e r e n t t r e a t m e n t s o f f e d Coho salmon (Oncorhynchus k i s u t c h ) showed no s i g n i f i c a n t d i f f e r e n c e (0< = 0.05) (Appendix 9 ) . The mean w e i g h t o f the f i s h a t the o n s e t o f the t r e a t m e n t s was 0.725 gms. The s t a r v e d Sockeye salmon (Oncorhynchus n e r k a ) and t h e s t a r v e d Coho salmon (Oncorhynchus k i s u t c h ) showed no s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t s ( A p p e n d i c e s 10 and 11 r e s p e c t i v e l y ) . The r e g r e s s i o n l i n e s f o r the t r e a t m e n t s f o r each o f the above s p e c i e s used i n the s t a r v a t i o n e x p e r i m e n t s a r e g i v e n i n F i g u r e s 11 and 12 r e s p e c t i v e l y . I n t h e s t a r v a t i o n e x p e r i m e n t s b o t h s p e c i e s (Oncorhyn-chus n e r k a and Oncorhynchus k i s u t c h ) showed a s t e a d y w e i g h t l o s s a t a l l l e v e l s o f t r e a t m e n t ( F i g u r e s 11 and 1 2 ) . 45 o f t r e a t m e n t s ) . The s t r a i g h t r e g r e s s i o n l i n e s g e n e r a t e d were l i n e s b e s t f i t f o r d a t a w h i c h was s l i g h t l y c u r v i l i n e a r ; how-e v e r , t h i s l a t t e r component was s m a l l r e l a t i v e t o the o v e r a l l t r e n d s ( F i g u r e s 8, 9, 10, 11 and 1 2 ) . e) R e s u l t s of t h e F e e d i n g E x p e r i m e n t The f e d Chum salmon (Oncorhynchus k e t a ) showed a s i g n i f i c a n t d i f f e r e n c e i n growth r a t e s between t r e a t m e n t s ( c o n t r o l , 0.5 ppm H g C ^ and 1.0 ppm H g C ^ ) . The s l o p e s o f the r e g r e s s i o n l i n e s g e n e r a t e d from the d a t a c o l l e c t e d o v e r the s i x week p e r i o d were s i g n i f i c a n t l y d i f f e r e n t ( # = 0.01). The i n d i v i d u a l d e g r e e s o f freedom t e s t on t h e a d j u s t e d means showed s i g n i f i c a n t d i f f e r e n c e s (& = 0.05) between the c o n t r o l and the 0.5 ppm H g C ^ t r e a t m e n t s , the c o n t r o l and the 1.0 ppm H g C ^ t r e a t m e n t s , and the c o n t r o l and t h e combined 0.5 ppm H g C l j and 1.0 ppm H g C ^ t r e a t m e n t s (Appendix 7 ) . There was no s i g n i f i c a n t d i f f e r e n c e between the 0.5 ppm HgCl2 and 1.0 ppm H g C ^ t r e a t m e n t s . The mean w e i g h t o f the f i s h a t the o n s e t o f the t r e a t m e n t s was 0.308 gms. F i g u r e 8 i n d i c a t e s a s t e a d y w e i g h t g a i n i n t h e c o n t r o l f i s h and a d e c l i n e i n w e i g h t o f f i s h t r e a t e d w i t h 0.5 ppm H g C ^ and 1.0 ppm H g C ^ ( t h e l a t t e r h a v i n g t h e more s e v e r e w e i g h t l o s s ) . The f e d Sockeye salmon (Oncorhynchus nerka) showed a s i g n i f i c a n t d i f f e r e n c e i n growth r a t e s between t r e a t m e n t s ( c o n t r o l , 0.5 ppm H g C l 2 and 1.0 ppm H g C l 2 ) . The s l o p e s o f the r e g r e s s i o n l i n e s g e n e r a t e d from the d a t a c o l l e c t e d o v e r the 46 C. The Pr e d a t o r - P r e y Response Experiments The p r e d a t o r - p r e y experiment was designed to measure the escape responses of prey exposed to p r e d a t o r s . The n u l l h y p o t h e s i s t e s t e d was t h a t s u b l e t h a l doses of mer c u r i c c h l o r i d e would not e f f e c t the escape responses to i n t o x i c a t e d f i s h . In f o r m u l a t i n g t h i s h y p o t h e s i s i t was supposed t h a t any d e l e t e r i o u s e f f e c t of m e r c u r i c c h l o r i d e would be r e v e a l e d by a reduced response i n i n t o x i c a t e d f i s h as compared to c o n t r o l f i s h . a) The Performance Trough ( F i g u r e 13) A performance trough 385 cm. i n l e n g t h was made of P.V.C. p i p e . The t o t a l l e n g t h was s u b d i v i d e d by a b a r r e d p a r t i t i o n i n t o two s e c t i o n s (360 and 25 cm. i n l e n g t h ) . The bars of the b a r r e d p a r t i t i o n were one cm. a p a r t . T h i s allowed the f r y to escape from the p r e d a t o r s down the trough w h i l e c o n t a i n i n g the p r e d a t o r s w i t h i n the i n t e r a c t i o n chamber. The 25 cm. long s e c t i o n of the trough was d i v i d e d i n t o two areas: one of 10 cm. i n l e n g t h (prey chamber) and another of 15 cm. i n l e n g t h ( p r e d a t o r chamber). Two w a l l s of P.V.C. s h e e t i n g were arranged so as to swing i n and out of the trough. These w a l l s allowed the s e g r e g a t i o n and containment of the p r e d a t o r s and t h e i r prey. 47 b) E x p e r i m e n t a l Runs An e x p e r i m e n t a l run c o n s i s t e d of p l a c i n g f i v e prey and two p r e d a t o r s i n t h e i r r e s p e c t i v e chambers. The w a l l s e p a r a t i n g the p r e d a t o r s and prey was removed and the d i s t a n c e each prey f i s h escaped down the trough runway was noted. There was a s c a l e marked on the s i d e of the trough and the d i s t a n c e s t r a v e l l e d were noted f i v e seconds a f t e r the w a l l was removed. c) Species of F i s h used i n the Experiment Sockeye salmon were s e l e c t e d f o r use as prey. These f i s h were a s s i g n e d to e x p e r i m e n t a l tanks and treatment begun. An outbreak of f u n g a l d i s e a s e produced an i n c r e a s i n g l y high m o r t a l i t y i n the c o n t r o l t a n k s . The m e r c u r i c c h l o r i d e i n the 0.5 ppm and 1.0 ppm treatment tanks a p p a r e n t l y acted as a p r o p h y l a c t i c s i n c e the m o r t a l i t y was much lower i n these tanks. The e x c e s s i v e l y high m o r t a l i t y of the c o n t r o l f i s h f o r c e d the abandonment of Sockeye salmon as a prey s p e c i e s . A stock of Coho salmon f r y was on hand at the h a t c h e r y and a few hundred w i l d Coho f r y were o b t a i n e d from a cree k i n the mountains of Vancouver I s l a n d . These f i s h were used as prey. The d a t a o b t a i n e d from experiments u s i n g h a t c h e r y and w i l d stock allowed f o r a comparison of the r e a c t i o n s of stock r e a r e d i n w i l d and a r t i f i c i a l environments. An attempt to c a p t u r e w i l d p r e d a t o r s ( t r o u t ) was made i n the mountain creek but a s u f f i c i e n t number of t r o u t c o u l d 48 not be o b t a i n e d . Many of the t r o u t d i d not s u r v i v e the shock treatment used to capture w i l d f i s h (the e l e c t r i c shocker equipment was p r o v i d e d by the P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B.C.). Two year o l d Coho salmon were o b t a i n e d from the Rose-w a l l Creek Hatchery f o r use as p r e d a t o r s . These f i s h were s t a r v e d f o r a p e r i o d of f o u r weeks and then exposed to l i v i n g prey ( t a d p o l e s , small sockeye f r y and i n s e c t s ) i n an attempt to enhance t h e i r p r e d a t o r y b e h a v i o u r . The t r a n s i t i o n from hatchery food to l i v e prey was slow but once these f i s h developed t h e i r p r e d a t o r y s k i l l s they would s t r i k e at almost any food s o u r c e . As f a r as c o u l d be a s c e r t a i n e d by i n c i d e n t a l o b s e r v a t i o n , the p r e d a t o r s seemed to be r e l a t i v e l y non s p e c i f i c i n t h e i r food p r e f e r e n c e s . d ) The E x p e r i m e n t a l Tanks The t e s t tanks, hardware and water source were the same as i n the p r e v i o u s experiments. I t should be noted that at the c o n c l u s i o n of each experiment a l l the equipment a s s o c i a -ted with the t e s t tanks and the t e s t tanks themselves were washed down wit h E t h y l e n e d i a m i n e t e t r a A c e t i c A c i d (E.D.T.A.) s o l u t i o n . The E.D.T.A. wash was done twice and t h e n • f o l l o w e d by s e v e r a l washings with c o p i o u s amounts of water. e) The Ex p e r i m e n t a l Design ~ ~ ~ W~ i ) The prey s t o c k s (hatchery and w i l d Coho salmon) Three i n t o x i c a t i o n l e v e l s of m e r c u r i c c h l o r i d e ( H g C l 2 ) were used ( c o n t r o l , 0.5 ppm and 1.0 ppm). The treatments were 49 g i v e n f o r an hour i n s t a n d i n g water once a day f o r twenty-one days. Si x tanks were a s s i g n e d to each treatment l e v e l . F i v e of the tanks were used to hol d f i s h which were to be used as prey ( t w e n t y - f i v e hatchery or w i l d Coho f r y ) . The s i x t h tank c o n t a i n i n g t w e n t y - f i v e f r y was used to p r o v i d e r e p l a c e -ment f i s h when m o r t a l i t y o c c u r r e d i n the t e s t tanks (Appendix 12 ). The s i x t h tank of each treatment l e v e l ( f o r both the hatchery and w i l d Coho s t o c k s ) had a cage suspended from the stand p i p e . The o r i g i n a l consignment of t w e n t y - f i v e f i s h was p l a c e d o u t s i d e the cage. When f i s h were removed from these tanks to r e p l a c e f i s h which had d i e d i n t h e i r r e s p e c t i v e t r e a t -ment l e v e l s , stock f i s h were p l a c e d i n the cages to m a i n t a i n t w e n t y - f i v e f i s h i n each tank. The t w e n t y - f i v e prey a s s i g n e d to each tank were p l a c e d i n the tanks one at a time s t a r t i n g w i t h the f i r s t tank and p r o g r e s s i n g to the l a s t tank. The c y c l e was rep e a t e d u n t i l a f u l l complement had been a s s i g n e d . There was a random a s s i g n -ment of treatments to the tanks. i i ) The predator stock Six tanks were a s s i g n e d to h o l d the pr e d a t o r s t o c k . Ten p r e d a t o r s were p l a c e d i n each of s i x tanks. F i v e tanks p r o v i d e d f i s h to be used as p r e d a t o r s i n the experiment. The s i x t h tank p r o v i d e d replacement f i s h i n cases of m o r t a l i t y i n the other f i v e tanks. 50 The p r e d a t o r f i s h were as s i g n e d u s i n g the same t e c h -niques as was used to a s s i g n f i s h to the prey tanks. The tanks used to h o l d t e s t groups and the replacement groups were s e l e c t e d randomly. The m o r t a i l i t y data was noted f o r the pr e d a t o r f i s h over the t h r e e week ex p e r i m e n t a l p e r i o d (Appendix 13) . Both the predator and prey f i s h were s t a r v e d d u r i n g the course of the experiment. f ) The I n t o x i c a t i o n P e r i o d A t h r e e week p e r i o d of i n t o x i c a t i o n was e s t a b l i s h e d f o r the prey s t o c k s ( c o n t r o l , 0.5 ppm and 1.0 ppm m e r c u r i c c h l o r i d e ) . The three week p e r i o d of i n t o x i c a t i o n was chosen to a l l o w f o r a s u f f i c i e n t exposure to m e r c u r i c c h l o r i d e w h i l e not being of such d u r a t i o n as to o v e r l y weaken the s t a r v e d f i s h . T h i s d e c i s i o n was i n f l u e n c e d by the data o b t a i n e d i n the f e e d i n g and s t a r v a t i o n experiments. I t was c o n s i d e r e d b e t t e r to s t a r v e the f i s h r a t h e r than have s i z e d i s c r e p a n c i e s which would f o l l o w t h r e e weeks of f e e d i n g . g) The P r e d a t o r - P r e y Response Runs A s e r i e s of runs was conducted on the w i l d Coho prey and were f o l l o w e d by a s e r i e s of runs u s i n g the ha t c h e r y s t o c k . An ex p e r i m e n t a l run used f i v e prey f i s h and two p r e d a t o r f i s h . Each tank c o n s e c u t i v e l y c o n t r i b u t e d a r u n . The treatments were p r e v i o u s l y randomized. When one c y c l e of runs was com-p l e t e d a new c y c l e was s t a r t e d from tank one. These c y c l e s c o n t i n u e d u n t i l a l l the f i s h had been t e s t e d . A f t e r the f i s h 51 were used i n runs they were p l a c e d back i n t h e i r r e s p e c t i v e t a n k s . A w i r e cage suspended from the stand pipe was used to segregate t e s t e d f i s h from u n t e s t e d f i s h . A l l the f i s h p a r t i c i p a t e d i n the f l i g h t response experiment t w i c e . T h i s y i e l d e d a t o t a l of 250 s c o r e s f o r each treatment. The second time the f i s h were put through the experiment they were taken from the cages, t e s t e d and r e t u r n e d to the tank. The p r e d a t o r f i s h were s e l e c t e d , used and segregated i n a l i k e manner. h) S c o r i n g A measuring tape marked o f f i n cm. u n i t s was a t t a c h e d to the s i d e w a l l of the f l i g h t response trough. The tape s t a r t e d at the b a r r e d gate and ended a t the f a r end of the trough (360 cm.). F i v e seconds a f t e r the prey were exposed to the p r e d a t o r s the d i s t a n c e down the trough each f i s h had t r a v e l e d was noted. A f i s h which d i d not pass through the gate was g i v e n a score of z e r o . i ) Method of A n a l y s i s The non-parametric Kolmogorov-Smirnov Two-Sample T e s t (Large samples: o n e - t a i l e d t e s t , 0< = 0.01) was used to analyze the data o b t a i n e d from the p r e d a t o r prey f l i g h t response e x p e r i -ment. j ) A n a l y s i s of Weight Data The p r e d a t o r s and prey were weighed upon the c o n c l u s i o n of the p r e d a t o r - p r e y f l i g h t response s t u d y . An a n a l y s i s 5 2 of v a r i a n c e was done on the data to determine i f any s i g n i f i -c a n t d i f f e r e n c e s e x i s t e d between the treatment groups (Appendix 1 4 ) . Ic) R e s u l t s of the P r e d a t o r - P r e y F l i g h t Response Experiment The r e s u l t s f o r the hatchery Coho stock are g i v e n i n F i g u r e s 1 5 , 1 6 and 1 7 . The s t a t i s t i c a l a n a l y s i s of the data showed no s i g n i f i -c a n t d i f f e r e n c e s between treatments ( tX = 0 . 0 5 ) . The n u l l h y p o t h e s i s was accepted. I t appeared t h a t s u b l e t h a l doses of merc u r i c c h l o r i d e d i d not a f f e c t the f l i g h t responses of the hatchery Coho prey (Appendix 1 5 ) . The r e s u l t s f o r the w i l d Coho stock are g i v e n i n F i g u r e s 1 8 , 1 9 and 2 0 . A s t a t i s t i c a l a n a l y s i s of the w i l d Coho d a t a r e v e a l e d a s i g n i f i c a n t d i f f e r e n c e (<X = 0 . 0 1 ) i n the responses of f i s h t r e a t e d with 0 . 5 ppm and 1 . 0 ppm HgCl (Appendix 1 6 ) . An i n s p e c t i o n of the F i g u r e s 1 5 , 1 6 and 1 7 r e v e a l e d an i n v e r s e r e l a t i o n s h i p between the amount of i n t o x i c a n t a d m i n i s t e r e d and the l e v e l of response (the f i s h t r e a t e d with 1 . 0 ppm H g C l 2 r e s -ponded l e a s t i n the f l i g h t t e s t s ) . Some m o r t a l i t y o c c u r r e d i n the prey s t o c k s i n the t h i r d week of treatment (Appendix 1 2 ) but i t was n e g l i g i b l e con-s i d e r i n g the number of f i s h used i n the experiment. There was no m o r t a l i t y i n the p r e d a t o r f i s h d u r i n g the term of the e x p e r i -ment (Appendix 1 3 ) . The r e s u l t s of the a n a l y s i s of weight data r e v e a l e d no 53 s i g n i f i c a n t d i f f e r e n c e s i n the weights of f i s h used i n the t h r e e treatments f o r both the w i l d and hatchery Coho prey (Appendix 14). The a n a l y s i s of the weight data from the f i v e tanks p r e d a t o r s r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s i n the weight among the f i v e groups (Appendix 14). 54 I I I DISCUSSION A. General D i s c u s s i o n Methyl mercury i s the most t o x i c m e r c u r i a l encountered i n the environment. When mercury of any form i s dumped i n t o a storm sewer, stream or l a k e , b i o l o g i c a l systems s l o w l y c o n v e r t a p o r t i o n of i t i n t o the deadly p o i s o n , methyl mercury. Microorganisms present i n mud m e t a b o l i z e m e t a l l i c and i n o r g a n i c mercury to produce methyl mercury. The methyl mercury i s i n e v i t a b l y e x c r e t e d i n t o the water where i t reaches h i g h e r animals. Animals which prey on the b a c t e r i a , and which are then preyed upon, move the methyl mercury through the food c h a i n toward the top p r e d a t o r s . J e r n e l o v (1972) found t h a t microorganisms i n the slime c o a t i n g on f i s h methylated mercury. The microorganisms present i n the slim e on f i s h i n the e a r l y s p r i n g were found to co n v e r t 90 per cent of the i n o r g a n i c mercury, to which they had been exposed, to methyl mercury. Subsequent experiments d u r i n g the s p r i n g and summer r e v e a l e d l e v e l s of c o n v e r s i o n of 30 and 10 per cent r e s p e c t i v e l y . The s l i m e f l o r a changed with the season and had a d e c r e a s i n g a b i l i t y to methylate i n o r g a n i c mercury. Those microorganisms which methylated m e r c u r i a l com-pounds (some a t a very high r a t e ) d i d so o n l y when grown on a f i s h e x t r a c t s u b s t r a t e . No growth was noted when a meat e x t r a c t s u b s t r a t e was used to c u l t u r e the b a c t e r i a . I t appears 55 t h a t f i s h t i s s u e c o n t a i n s f a c t o r s which augment th e growth o f m e t h y l a t i n g b a c t e r i a ( J e r n e l o v , 1972). M e r c u r i c c h l o r i d e was used as a s o u r c e o f mercury because i t was l e s s t o x i c t h a n methyl mercury and t h e r e f o r e ' p r e s e n t e d l e s s o f a h e a l t h h a z a r d t o t h e h a t c h e r y s t a f f and h a t c h e r y a n i m a l s . The a u t h o r depended on n a t u r a l b i o t r a n s f o r m a t i o n s t a k i n g p l a c e w i t h i n o r on the f i s h and w i t h i n the h o l d i n g tank e n v i r o n m e n t t o p r o v i d e t h e m e t h y l mercury f o r i n t o x i c a t i o n . The methyl mercury thus produced c o u l d pass t h r o u g h the e p i t h e l i a l t i s s u e ( e s p e c i a l l y the g i l l t i s s u e ) t o t h e c i r c u l a -t o r y system w h i c h c o u l d p e r f o r m t h e d i s t r i b u t i o n o f t h e m e r c u r i a l t h r o u g h o u t the a n i m a l s ' body. The b i o t r a n s f o r m a t i o n o f m e r c u r i c c h l o r i d e t o m e t h y l mercury i s a p r o c e s s w h i c h r e q u i r e s t i m e . T h e r e f o r e a p o r t i o n o f the h i g h l y s o l u b l e m e r c u r i c c h l o r i d e c o u l d be e x p e c t e d t o e n t e r the f i s h e s ' body d i r e c t l y t h r o u g h membranes. T h i s m e r c u r i c c h l o r i d e c o u l d undergo t r a n s f o r m a t i o n s w i t h i n the body t o produce m e t h y l mercury. The o r i g i n a l e x p e r i m e n t a l d e s i g n c o n c e p t c a l l e d f o r a c o n t i n u a l m i x i n g o f a d i l u t e m e r c u r i c c h l o r i d e s o l u t i o n w i t h w ater from t h e head tank t o produce c o n s t a n t t e s t c o n c e n t r a t i o n s o f 0.02 and 0.04 ppm. The c o s t o f p r o d u c i n g a d o s i n g a p p a r a t u s w h i c h c o u l d dose a l a r g e number of t a n k s , and the subsequent problem of d e t o x i f i c a t i o n o f the r u n o f f w a t e r , prompted the use of a s h o r t e r p e r i o d o f i n t o x i c a t i o n (one hour per day) i n s t a n d i n g w a t e r a t l e v e l s o f 0.5 and 1.0 ppm. These l e v e l s o f 56 i n t o x i c a t i o n c o u l d be o b t a i n e d by p l a c i n g an a l e q u o t of c o n c e n t r a t e d m e r c u r i c c h l o r i d e s o l u t i o n i n the s t a n d i n g water of the f i s h t a n k s . The water i n the f i s h tanks c o n t a i n e d s u f f i c i e n t oxygen to m a i n tain the f i s h f o r an hour. When the head tank water was turned on, a f t e r the hour treatment, the m e r c u r i c c h l o r i d e was f l u s h e d out of the tank and allowed to run i n t o a f i l t r a t i o n t r e n c h (100 meters l o n g ) . The trench c o n t a i n e d sand and g r a v e l . The m i n e r a l c o n t e n t of the f i l t r a t i o n t r e n c h was thought to be s u f f i c i e n t to b i n d m e r c u r i c c h l o r i d e and o t h e r m e r c u r i a l by-products of the experiment, thus a l l o w i n g f o r t h e i r slower r e l e a s e i n t o the environment. The Sockeye salmon a l e v i n s used i n the m i g r a t i o n e x p e r i -ment were j u s t at the " b u t t o n i n g up" stage and t h e r e f o r e had egg yolk m a t e r i a l s u f f i c i e n t to m a i n t a i n them d u r i n g the course of the experiment. 57 B. D i s c u s s i o n of the Upstream-Downstream M i g r a t i o n Experiment The c o n t r o l a l e v i n s from the Lower Babine R i v e r and the F u l t o n R i v e r e s t a b l i s h e d scores (32 r e p l i c a t e s of each t e s t l e v e l ) from which a grand mean score was a s c e r t a i n e d . The grand mean s c o r e was i n d i c a t i v e of the upstream-downstream ten d e n c i e s of the f i s h . A s c o r e of 12.00 u n i t s i n d i c a t e d no upstream or downstream m i g r a t i o n (Appendix 3 ) . The upstream-downstream behaviour was the dependent v a r i a b l e and the t r e a t -ment c o n c e n t r a t i o n s ( c o n t r o l , 0.5 ppm HgCl^ and 1.0 ppm HgC^) were the independent v a r i a b l e s . The Lower Babine R i v e r c o n t r o l f i s h had a grand mean score of 16.71 u n i t s , i n d i c a t i v e of an upstream m i g r a t o r y tendency, w h i l e the F u l t o n R i v e r c o n t r o l f i s h had a grand mean score of 9.46 u n i t s i n d i c a t i n g downstream m i g r a t i o n (Appendices 4 and 5 ) . Both the Lower Babine and F u l t o n R i v e r a l i v e n s t r e a t e d w i t h 0.5 ppm m e r c u r i c c h l o r i d e had grand mean s c o r e s lower (10.16 and 4.55 r e s p e c t i v e l y ) than the c o n t r o l f i s h from the two s t o c k s . These s c o r e s were s i g n i f i c a n t l y d i f f e r e n t from t h e i r r e s p e c t i v e c o n t r o l s c o r e s ((X = 0.01) (Appendices 4 and 5 ) . The a l e v i n s from the Lower Babine R i v e r which had been t r e a t e d with 1.0 ppm m e r c u r i c c h l o r i d e had a grand mean score of 16.84 u n i t s . T h i s s c o r e was i n d i c a t i v e of an upstream movement which was onl y s l i g h t l y g r e a t e r than the grand mean score (16.71 u n i t s ) of the Lower Babine R i v e r c o n t r o l f i s h . S t a t i s t i c a l l y t h e r e was no s i g n i f i c a n t d i f f e r e n c e between the c o n t r o l and the t e s t f i s h t r e a t e d with 1.0 ppm m e r c u r i c c h l o r i d e . 58 The a l e v i n s from the F u l t o n R i v e r which were t r e a t e d w i t h 1.0 ppm m e r c u r i c c h l o r i d e had a grand mean scor e of 13.13 u n i t s f o r the t h i r t y - t w o r e p l i c a t e s which i n d i c a t e d an upstream m i g r a t i o n tendency. T h i s grand mean score was s i g n i -f i c a n t l y d i f f e r e n t from the F u l t o n R i v e r c o n t r o l s ((X = 0.05). The downstream tendency of both the Lower Babine R i v e r and F u l t o n R i v e r f i s h t r e a t e d with 0.5 ppm mer c u r i c c h l o r i d e may have been caused by f a t i g u e and l e t h a r g y , which are sympto of c h r o n i c m e r c u r i a l p o i s o n i n g . Methyl mercury causes n e u r o l o g i c a l damage which i s manifested as mental d i s f u n c t i o n . In a l l p r o b a b i l i t y , methyl mercury was the a c t i v e agent i n the i n t o x i c a t i o n of the t e s t f i s h . The t e s t f i s h of both s t o c k s which were t r e a t e d with 0.5 ppm m e r c u r i c c h l o r i d e tended to be found downstream from the s t a r t i n g p o i n t and most probably d r i f t e d with the c u r r e n t to t h e i r p o s i t i o n s at the t e r m i n a t i o n of the e x p e r i m e n t a l runs The author observed the f i s h d u r i n g the p e r i o d of i n t o x i c a t i o n (10 d a y s ) . A l l the tanks were covered w i t h l i d s except d u r i n g the p e r i o d s of i n t o x i c a t i o n . At that time a l l the covers of the c o n t r o l , 0.5 ppm and 1.0 ppm t e s t tanks were removed. The f i s h i n the c o n t r o l tanks,became very a c t i v e and were seen swimming around the tank. The f i s h i n the tanks which were r e c e i v i n g the 0.5 ppm treatments tended to be found at the bottom of the tank and were r e l a t i v e l y i n a c t i v e w h i l e the f i s h undergoing treatment with 1.0 ppm mercuric c h l o r i d e e x h i b i t e d a g r e a t e r l e v e l of a c t i v i t y than the 0.5 ppm t r e a t e d 59 f i s h . These o b s e r v a t i o n s , although q u a l i t a t i v e , tend to augment the argument of l e t h a r g y and f a t i g u e i n the 0.5 ppm t r e a t e d f i s h . The f i s h from both s t o c k s which were t r e a t e d with 1.0 ppm mercuric c h l o r i d e showed r e l a t i v e upstream t e n d e n c i e s . The Lower Babine R i v e r f i s h j u s t surpassed the c o n t r o l s of t h e i r stock w h i l e the f i s h from the F u l t o n Rive s i g n i f i c a n t l y surpassed the c o n t r o l f i s h of t h e i r stock (Appendix 4 ) . The author observed the g i l l t i s s u e of the c o n t r o l and t e s t f i s h from both the Lower Babine R i v e r and the F u l t o n R i v e r s t o c k s w h i l e they underwent t h e i r r e s p e c t i v e treatments. Because of the methanogenic a c t i v i t y of b a c t e r i a i n the s l i m e c o a t of the g i l l s and the p r o x i m i t y of c i r c u l a t i n g b l o o d to the e x t e r i o r , i t was suspected t h a t the most l i k e l y e n t r y f o r m e r c u r i a l s would be the g i l l t i s s u e . Other o b s e r v a t i o n s were made of the f i s h i n t h e i r treatment t a n k s . I t was observed that the g i l l s of the c o n t r o l f i s h remained a b r i g h t pink w h i l e the g i l l s of the t e s t f i s h , f o r both l e v e l s of m e r c u r i a l treatment, and both s t o c k s , tended to become a p a l e r pink with time. A l s o , g r e a t e r mouth and g i l l movement was observed f o r the i n t o x i c a t e d f i s h . Furthermore, a tendency to swim i n t o the c u r r e n t c r e a t e d by the pipe d e l i v e r i n g water to the h o l d i n g tanks from the head tank above was observed. Methyl mercury i o n (CH^Hg*) may i n t e r f e r e with the p r o d u c t i o n of haem. Two s t e p s i n the b i o s y n t h e s i s of haem appear v u l n e r a b l e to the a c t i v i t y of methyl mercury i o n s : 60 the t r a n s f o r m a t i o n of d e l t a a m i n o l e v u l i n i c a c i d to p o r p h o b i -l i n o g e n mediated by d e l t a a m i n o l e v u l i n i c a c i d dehydrase, and the t r a n s f o r m a t i o n of p r o t o p o r p h y r i n IX p l u s i r o n to haem mediated by haem symthetase (the a c t i v e s i g h t of both enzymes being f r e e s u l f h y d r y l (-SH) g r o u p s ) . In the b l o o d the f u n c t i o n a l e f f e c t would be anemia and m e t a b o l i c d i s t u r b a n c e s (haem being an e s s e n t i a l c o n s t i t u e n t i n o t h e r r e s p i r a t o r y pigments which p l a y key r o l e s i n energy metabolism, i . e . the cytochromes). A p o s s i b l e e x p l a n a t i o n f o r the upstream tendency of both the Lower Babine R i v e r and the F u l t o n R i v e r f i s h ( t r e a t e d with 1.0 ppm m e r c u r i c c h l o r i d e ) would be t h a t they were attempting to o b t a i n more oxygen by swimming i n t o the c u r r e n t and were thereby i n c r e a s i n g the flow of oxygenated water over the g i l l t i s s u e . The p h y s i o l o g i c a l requirement f o r an i n c r e a s e d oxygen supply must have superseded mental d i s f u n c t i o n and l e t h a r g y i n the animals which were i n t o x i c a t e d and s t r e s s e d by the higher l e v e l of m e r c u r i c c h l o r i d e . I n i t i a l d i u r e s i s , d i s t o r t i o n of e l e c t r o l y t e p a t t e r n , a c i d o s i s , a l b u m i n u r i a , c o l i t i s and p r o g r e s s i v e r e n a l damage may a l s o have been c o n t r i b u t i n g f a c t o r s i n the d e t e r i o r a t i o n of the c o n d i t i o n of the i n t o x i c a t e d f i s h . These changes are c h a r a c t e r i s t i c a l l y found i n cases of c h r o n i c m e r c u r i a l p o i s o n i n g . However, the f i s h i n t h i s experiment were not examined f o r these p a r t i c u l a r p h y s i o l o g i c a l changes. The n u l l h y p o t h e s i s was r e j e c t e d . S u b l e t h a l doses of m e r c u r i c c h l o r i d e appeared to have a d i s r u p t i v e e f f e c t on the 61 upstream-downstream m i g r a t i o n t e n d e n c i e s of the a l e v i n s t e s t e d . 62 C. D i s c u s s i o n of the F e e d i n g and S t a r v a t i o n Experiments In a l l s p e c i e s of f i s h (Oncorhynchus k e t a , Oncorhynchus  nerka and Oncorhynchus k i sutch) used i n f e e d i n g experiments, the c o n t r o l animals showed pronounced growth under the f e e d i n g program. T h i s growth was c o n s i s t e n t w i t h the type of growth seen i n f i s h throughout the hatchery undergoing a s i m i l a r f e e d i n g program. An o v e r a l l d e c l i n e i n the weight of the f i s h t r e a t e d w i t h m e r c u r i c c h l o r i d e (0.5 ppmHgCl 2 and 1.0 ppm H g C l 2 was noted. The weight l o s s i n t e s t f i s h ( a l l three s p e c i e s ) appeared to be a f u n c t i o n of the c o n c e n t r a t i o n of the m e r c u r i a l ( H g G l 2 ) i n t h a t those f i s h undergoing treatment with 1.0 ppm m e r c u r i c c h l o r i d e showed a g r e a t e r weight l o s s than those t r e a t e d with 0.5 ppm m e r c u r i c c h l o r i d e . The Chum and Sockeye salmon, t r e a t e d with 0.5 ppm and 1.0 ppm H g C l 2 showed a s i g n i f i c a n t weight l o s s ((X = 0.01). Although the Coho-salmon t r e a t e d with 0.5 ppm and 1.0 ppm showed a d e c l i n e i n weight i t was not a s i g n i f i c a n t d e c l i n e ( 0< = 0.05). D i f f e r e n c e s i n body weight may have c o n t r i b u t e d to the r e s u l t . The Coho salmon were the l a r g e s t and h e a v i e s t of the f i s h used i n the f e e d i n g study. The body burden of m e r c u r i a l (ppm m e r c u r i a l per u n i t weight) may have been d i f f e r e n t between s p e c i e s g i v e n the same l e v e l of i n t o x i c a t i o n f o r l i k e p e r i o d s of time ( i . e . the g r e a t e r the biomass the g r e a t e r the t o l e r a n c e to m e r c u r i a l i n t o x i c a t i o n ) . 63 The t h r e s h o l d l e v e l f o r the appearance of symptoms of m e r c u r i a l i s m may be d i f f e r e n t between s p e c i e s , but the biomass of i n d i v i d u a l s i s more l i k e l y to determine when symptoms appear. The r e s u l t s of the f e e d i n g experiments u s i n g Sockeye and Chum salmon l e a d to the r e j e c t i o n of the n u l l h y p o t h e s i s ( t h a t a prolonged exposure to s u b l e t h a l doses of m e r c u r i c c h l o r i d e ( c o n t r o l , 0.5 ppm and 1.0 ppm HgC^)would not modify growth). S u b l e t h a l doses of m e r c u r i c c h l o r i d e (0.5 ppm and 1.0 ppm) appear to be r e s p o n s i b l e f o r a n e g a t i v e growth p a t t e r n i n i n t o x i c a t e d f i s h . T h i s i s i n marked c o n t r a s t to the pronounced p o s i t i v e growth seen i n c o n t r o l s . Although the Coho salmon d i d not show s i g n i f i c a n t d i f f e r e n c e s i n growth between treatments ( a f t e r t h r e e weeks of t r e a t m e n t ) , F i g u r e 10 i n d i c a t e s a c o n t i n u a l weight l o s s i n the fish... t r e a t e d w i t h 0.5 and 1.0 ppm H g C ^ and a c o n t i n u a l weight g a i n i n the c o n t r o l group. A l o s s of a p p e t i t e along with a d i s r u p t e d d i g e s t i v e metabolism (symptoms of m e r c u r i a l i s m ) are the most probable causes of the weight l o s s i n the i n t o x i c a t e d f i s h . One must conclude the m e r c u r i c c h l o r i d e treatments given to the f i s h were r e s p o n s i b l e f o r the d i f f e r e n t growth r a t e s . Both the Coho and Sockeye salmon used i n the s t a r v a -t i o n study showed a d e c l i n e i n weight f o r a l l t r e a t m e n t s . There was no s i g n i f i c a n t d i f f e r e n c e i n weight l o s s between treatments a f t e r three weeks of i n t o x i c a t i o n . 64 The r e s u l t s of the s t a r v a t i o n experiments l e a d to the acceptance of the n u l l 1 h y p o t h e s i s t h a t t h e r e would be no s i g n i f i c a n t d i f f e r e n c e i n weight l o s s between treatments ( c o n t r o l , 0.5 ppm H g C ^ and 1.0 ppm HgClg) d u r i n g a p e r i o d of exposure to s u b l e t h a l doses of the m e r c u r i a l . I t s h o u l d be noted t h a t the f e e d i n g and s t a r v a t i o n s t u d i e s were conducted over a s i x week p e r i o d to e s t a b l i s h r e g r e s s i o n l i n e s i n d i c a t i v e of t r e n d s . The r e g r e s s i o n and c o v a r i a n c e a n a l y s i s was done on data a f t e r three weeks of treatments. The P r e d a t o r - P r e y experiment was designed to have a t h r e e week p e r i o d of i n t o x i c a t i o n . I t was necessary to e s t a b l i s h whether or not to feed the f i s h d u r i n g the p e r i o d of i n t o x i c a t i o n . The r e s u l t s of the f e e d i n g and s t a r v a t i o n experiments i n d i c a t e d t h a t f e e d i n g c o u l d i n f l u e n c e the c o n d i t i o n of f i s h undergoing i n t o x i c a t i o n , i . e . c o n t r o l f i s h appeared to be l a r g e r and i n b e t t e r c o n d i t i o n than those undergoing i n t o x i c a t i o n w i t h mercuric c h l o r i d e . 65 D. The D i s c u s s i o n of the P r e d a t o r - P r e y F l i g h t Responses The w i l d Coho f r y used as prey c o n t r o l s d i s p l a y e d a pronounced f l i g h t response when they were c o n f r o n t e d with the p r e d a t o r Coho. In comparison to the c o n t r o l prey, s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s (& = 0.01) were seen i n the f l i g h t responses of the w i l d Coho prey t r e a t e d with 0.5 ppm and 1.0 ppm H g C ^ . An i n s p e c t i o n of F i g u r e s 18, 19 and 20 i n d i c a t e d t h a t the g r e a t e r the amount of m e r c u r i a l p r e s e n t i n the t r e a t -ment the g r e a t e r the r e t a r d a t i o n of the f l i g h t r e s p o n s e . The w i l d Coho salmon were o b t a i n e d from a mountain stream on Vancouver I s l a n d . These f i s h had been exposed to p r e d a t o r s r e s i d i n g i n the stream up u n t i l the time of t h e i r c a p t u r e , and t h e r e f o r e had w e l l developed f l i g h t r e s p o n s e s . The symptoms of m e r c u r i a l i s m d e s c r i b e d by Harvey (1965) i n c l u d e : tremor, a t a z i a , d y s a r t h r i a , c o n c e n t r i c c o n s t r i c t i o n of the v i s u a l f i e l d , numbness, i n a b i l i t y to c o n c e n t r a t e , f a t i g u e and impairment of memory. Some or a l l of these symp-toms c o u l d have been p r e s e n t i n the i n t o x i c a t e d f i s h and c o u l d account f o r the imp a i r e d responses of the f i s h t r e a t e d w i t h mercuric c h l o r i d e . The s t a t i s t i c a l a n a l y s i s of the data o b t a i n e d i n the p r e d a t o r - p r e y f l i g h t response experiment u s i n g h a t c h e r y Coho f r y showed no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s (0( = 0.05) between tr e a t m e n t s . The h a t c h e r y Coho f r y had been r a i s e d from eggs i n the a r t i f i c i a l and c o n t r o l l e d environment of the Rosewall Creek Hatchery. These f r y had never been exposed to a 66 p r e d a t o r b e f o r e the study. I t appears t h a t much of the f l i g h t response i s l e a r n e d b e h a v i o u r . The lack of knowledge of response a p p r o p r i a t e behaviour may have been l a r g e l y r e s p o n s i b l e f o r the poor f l i g h t responses seen i n the c o n t r o l and i n t o x i c a t e d hatchery s t o c k . There may have been o t h e r c o n t r i b u t i o n f a c t o r s f o r the l e s s r e s p o n s i v e behaviour of the hatc h e r y Coho salmon. The hatc h e r y stock were somewhat l a r g e r than the w i l d Coho stock and t h e r e f o r e they may have been l e s s a t t r a c t i v e to the p r e d a t o r s due to some s i z e p r e f e r e n c e . The p r e d a t o r stock had been used f o r the f u l l e x p e r i -mental run with the w i l d Coho stock b e f o r e t h e i r use with the hatchery Coho. The p r e d a t o r s may have l e a r n e d that the b a r r e d gate prevented s u c c e s s f u l c a p t u r e of the prey and t h e r e f o r e they may not have been as a g g r e s s i v e i n t h e i r a t t a c k s . 67 IV CONCLUSIONS A. The Upstream and Downstream Experiment The r e s u l t s of the upstream and downstream m i g r a t i o n experiments i n d i c a t e d t h a t s u b l e t h a l l e v e l s of m e r c u r i a l i n t o x i c a t i o n have d i s r u p t i v e e f f e c t s on the behaviour of m i g r a t i n g a l i v e n s . The Sockeye salmon a l e v i n s (Oncorhynchus nerka) from the Lower Babine R i v e r spawning area ( F i g u r e 14) migrate to Babine Lake and r e s i d e t h e r e f o r a year b e f o r e commencing t h e i r seaward m i g r a t i o n . In the case of the a l e v i n s from the Lower Babine R i v e r , the downstream tendency of those animals t r e a t e d with 0.5 ppm m e r c u r i c c h l o r i d e c o u l d l e a d to t h e i r d r i f t i n g toward the sea p r e m a t u r e l y . These f i s h would most probably f i n d themselves i n a much more h o s t i l e environment than Babine Lake. Those animals t r e a t e d with the h i g h e r l e v e l of m e r c u r i c c h l o r i d e (1.0 ppm) showed an upstream tendency. These f i s h m igrated a g a i n s t the c u r r e n t and, i n the w i l d , t h i s would take them to Babine Lake. Although the upstream migra-t i o n tendency was s i m i l a r to the n a t u r a l m i g r a t i o n tendency of the stock these f i s h may have s u f f e r e d i r r e p a r a b l e n e u r a l and p h y s i c a l damage which c o u l d render them more prone to environmental hazards, i . e . i n c r e a s e d v u l n e r a b i l i t y to p r e d a t o r s . 68 The Sockeye salmon a l e v i n s from the F u l t o n R i v e r spawning area ( F i g u r e 14) migrate downstream to Babine Lake and r e s i d e t h e r e f o r a year b e f o r e t h e i r seaward m i g r a t i o n . The a l e v i n s t r e a t e d w i t h 0.5 ppm m e r c u r i c c h l o r i d e showed a downstream tendency which, i n the n a t u r a l s e t t i n g , would take them to Babine Lake. I t i s suggested t h a t l e t h a r g y i s the primary symptom of m e r c u r i a l i s m at lower l e v e l s of i n t o x i c a t i o n . The f i s h t r e a t e d with the lower dose of m e r c u r i c c h l o r i d e would f i n d t h e i r way to Babine Lake but because of impaired mental f u n c t i o n s they would be more prone to p r e d a t i o n . The a l e v i n s t r e a t e d with 1.0 ppm m e r c u r i c c h l o r i d e showed an upstream tendency which was c o u n t e r to the n a t u r a l m i g r a t i o n tendency of the s t o c k . These a l e v i n s would have proceeded upstream toward a l a r g e f a l l s on the F u l t o n R i v e r and a hazardous environment. For both sto c k s i t appeared t h a t the lower l e v e l of i n t o x i c a t i o n (0.5 ppm H g C ^ ) d u l l e d the mental f u n c t i o n s of the a l e v i n s . The h i g h e r l e v e l o f i n t o x i c a t i o n (1.0 ppm H g C ^ ) may a l s o have had a d u l l i n g mental e f f e c t . However, the tendency of the f i s h from both s t o c k s to swim a g a i n s t the c u r r e n t suggests some added mental and/or p h y s i c a l s t r e s s . I t i s probable t h a t oxygen d e p r i v a t i o n f o r c e d the f i s h to swim a g a i n s t the c u r r e n t i n order to i n c r e a s e the f l o w of oxygen b e a r i n g water over the g i l l s . The p r e v i o u s d i s c u s s i o n of the a c t i o n of m e r c u r i a l s on blood c h e m i s t r y and the anaemic appearance of the g i l l s support t h i s s u g g e s t i o n . The r e s u l t s of the upstream and downstream m i g r a t i o n experiment p o i n t out the d i s r u p t i v e p o t e n t i a l of s u b l e t h a l l e v e l s of m e r c u r i a l p o l l u t i o n on animals i n the a q u a t i c environment. 70 B. The Feeding and S t a r v a t i o n Experiments a) The Feeding Experiment The c o n t r o l animals f o r a l l s p e c i e s used i n the f e e d i n g experiment showed a.steady p o s i t i v e growth which was c o n s i s t e n t with growth seen i n other h a t c h e r y animals fed the same food w i t h a l i k e r a t i o n i n g f o r m u l a . The t e s t animals ( t r e a t e d with 0.5 ppm and 1.0 ppm H g C ^ ) l o s t weight throughout the e x p e r i m e n t a l time p e r i o d . F i s h t r e a t e d w i t h the h i g h e r dosage of m e r c u r i a l showed a s l i g h t l y g r e a t e r r a t e of weight d e c l i n e . The Sockeye and Chum f r y t r e a t e d with the m e r c u r i a l showed a s i g n i f i c a n t d e c l i n e i n weight when compared to t h e i r c o n t r o l s . The Coho salmon f r y d i d not show a s i g n i f i c a n t weight d e c l i n e when compared to t h e i r c o n t r o l s . The Coho salmon f r y were l a r g e r than the Sockeye and Chum f r y and appeared to w i t h s t a n d the s t r e s s of m e r c u r i a l i s m b e t t e r . I t was concluded t h a t s i z e and biomass were f a c t o r s i n r e d u c i n g the impact of the mercuric c h l o r i d e . I t i s suggested t h a t a mental and/or p h y s i c a l s t r e s s was r e s p o n s i b l e f o r the weight l o s s . T h i s s t r e s s may have r e t a r d e d a p p e t i t e and/or i n h i b i t e d the proper a s s i m i l a t i o n of any food consumed. Lethargy and l o s s of a p p e t i t e are n e u r a l symptoms of m e r c u r i a l i s m . The f i s h may not have been i n t e r e s t e d i n food due to t h e i r mental c o n d i t i o n . A more probable e x p l a n a t i o n i s t h a t , along with a 71 lowered i n t e r e s t i n food, a d i s r u p t i o n i n normal d i g e s t i v e c h e m i s t r y o c c u r r e d . S e v e r a l d i g e s t i v e enzymes have a c t i v e s u l f h y d r y l groups. These enzymes would be rendered i n a c t i v e by the non-competitive i n h i b i t o r R-Hg +. I t i s a l s o p o s s i b l e they enzymes a c t i v e i n the t r a n s f e r of molecules through the i n t e s t i n a l w a l l c o u l d be i n h i b i t e d i n a l i k e manner. Prolonged s u b l e t h a l exposures to a m e r c u r i a l such as me r c u r i c c h l o r i d e would u l t i m a t e l y l e a d to the death of the animals i n t o x i c a t e d . The weight l o s s of the animals along with reduced mental and p h y s i c a l c a p a b i l i t i e s , would render these f i s h more prone t o p r e d a t i o n or some oth e r form of e a r l y d e ath. b) The S t a r v a t i o n Experiment In the Sockeye salmon a l l treatments ( c o n t r o l , 0.5 ppm and 1.0 ppm H g C ^ ) showed a d e c l i n e i n weight i n the s t a r v a t i o n experiment. The 0.5 ppm H g C ^ treatment showed a g r e a t e r d e c l i n e i n weight than the c o n t r o l . The 1.0 ppm H g C ^ t r e a t -ment showed a g r e a t e r d e c l i n e than the 0.5 ppm H g C ^ treatment. There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the r a t e s of weight d e c l i n e between treatments. The Coho salmon showed a s i m i l a r weight l o s s f o r a l l treatments, and no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n weight l o s s r a t e s were noted. A p p a r e n t l y the presence of m e r c u r i c c h l o r i d e d i d not enhance the s t a r v a t i o n p r o c e s s d u r i n g the time span of the t e s t . The r e s u l t s of the s t a r v a t i o n study suggest t h a t i n t o x i -72 c a t i o n experiments should be conducted on s t a r v e d f i s h . T h i s should be done to a v o i d s i z e , weight, and c o n d i t i o n d i f f e r e n c e s due to f e e d i n g . 73 C. The P r e d a t o r - P r e y F l i g h t Response Experiment a) The Wild Coho Salmon Prey Responses The w i l d Coho prey t r e a t e d w i t h m e r c u r i c c h l o r i d e e x h i b i t e d s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s (cX = 0.01) i n f l i g h t response behaviour when compared to t h e i r c o n t r o l s . The e f f e c t s of m e r c u r i a l p o i s o n i n g were v e r y apparent i n the f i s h exposed to 0.5 ppm and 1.0 ppm m e r c u r i c c h l o r i d e . An examination of the F i g u r e s 18, 19, and 20 suggested t h a t the g r e a t e r the i n t o x i c a t i o n l e v e l the more r e t a r d e d the a b i l i t y of f r y to a v o i d and/or escape p r e d a t o r a t t a c k s . The m e r c u r i a l may have e f f e c t e d the n e u r a l t i s s u e of the b r a i n and the nervous system i n such a way as to cause a r e d u c t i o n i n the c a p a c i t y to r e a c t to the a g g r e s s i v e behaviour of a p r e d a t o r . I t i s a l s o p o s s i b l e t h a t the neuro-muscular response mechanisms needed f o r r a p i d r e t r e a t were d i s r u p t e d . I t appears that p s y c h o l o g i c a l and p h y s i o l o g i c a l damage common to m e r c u r i a l i s m was r e s p o n s i b l e f o r the r e t a r d e d f l i g h t r e s p o n s e s . b) The Hatchery Coho Salmon Prey Responses There were no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ((X = 0.05) between the treatments i n the hatchery Coho f l i g h t response experiment. These f i s h had no p r e v i o u s knowledge of the behaviour of p r e d a t o r s , although t h e r e i s l i t t l e doubt t h a t they c o u l d develop response a p p r o p r i a t e behaviour with exposure to a p r e d a t o r p o p u l a t i o n . 74 Other c o n t r i b u t i n g f a c t o r s may have i n f l u e n c e d the response of the hatchery c o n t r o l f i s h . The hatchery Coho prey were l a r g e r than the w i l d Coho prey. The p r e d a t o r Coho may have had a s i z e p r e f e r e n c e as to prey, or the hatc h e r y Coho may have been too l a r g e f o r the p r e d a t o r s to a t t a c k . The p r e d a t o r Coho salmon had been used i n the w i l d Coho prey experiment b e f o r e they were used with the hatc h e r y Coho prey. The p r e d a t o r s may have l e a r n e d t h a t the b a r r e d gate p r o h i b i t e d t h e i r s u c c e s s f u l a t t a c k and p u r s u i t of prey. I t appears t h a t the hatchery stock prey were a poor c h o i c e f o r the f l i g h t response study. 75 D. General C o n c l u s i o n s F i s h exposed to s u b l e t h a l amounts of the m e r c u r i a l m e r c u r i c c h l o r i d e f o r s h o r t p e r i o d s of time develop symptoms of m e r c u r i a l i s m . 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The G e n e t i c and E n v i r o n m e n t a l Mechanisms C o n t r o l l i n g t h e Lakeiuard M i g r a t i o n o f Young Rainbow T r o u t ( Salmo g a i r d n e r ) from O u t l e t and I n l e t R e a r i n g S t r e a m s . 88 APPENDICES 89 APPENDIX 1 The mean d a i l y water temperature f o r May, June, J u l y and August f o r the w e l l sources of the Rosewall Creek Hatchery (1971) Day May June J u l y August 1 6.7 6.0 5.8 5.4 2 6.0 6.1 5.7 5.5 3 5.9 6.1 5.7 5.6 4 5.9 6.0 5.8 5.7 5 5.9 6.0 5.8 5.7 6 5.8 6.0 5.7 5.7 7 5.8 6.0 5.7 5.7 8 5.8 6.0 5.6 5.8 9 5.8 6.0 5.6 5.8 10 5.8 5.9 5.7 5.8 11 5.9 5.9 5.9 5.9 12 5.9 5.9 6.4 5.9 13 5.8 6.2 6.4 5.7 14 5.8 6.1 6.4 6.0 15 5.8 6.0 6.5 6.1 16 5.9 6.0 6.5 6.0 17 5.9 6.0 6.5 6.1 18 5.9 6.0 6.5 *7.0 19 5.9 6.0 6.5 7.2 20 5.9 6.0 6.6 7.3 21 5.9 6.1 6.6 7.3 22 5.9 6.1 6.6 7.4 23 5.9 6.2 6.6 7.4 24 5.9 6.2 6.6 7.4 25 5.8 6.2 6.7 6.8 26 5.8 6.2 6.7 6.7 27 5.8 *5.5 6.8 6.7 28 5.8 "5.5 6.8 6.7 29 5.8 5.6 6.8 6.6 30 6.2 5.7 6.8 6.7 31 6.1 6.9 6.8 •Change i n source w e l l (the hatchery had two w e l l s i n d i f f e r -ent l o c a t i o n s on hatchery p r o p e r t y ) . APPENDIX 2 The ..mortality data f o r the Upstream and Downstream Sockeye salmon alevins ( s c o r e s over a two week p e r i o d ) F u l t o n R i v e r Stock Lower Babine R i v e r Stock C o n t r o l 0.5 ppm HgC,l 2 1.0 ppm H g C l 2 C o n t r o l 0.5 ppm H g C l 2 1.0 ppm H g C l 2 T o t a l number of deaths per tank over the two week p e r i o d 1,0,1,2 2,0,2,3 3,3,3,4 0,0,1,2 2,2,0,2 3,3,3,3 The per cent m o r t a l i t y of the t o t a l treatment p o p u l a t i o n 1.25% 2.19% 4.06% 0.94% 1.88% 3.75% APPENDIX 3 Worked examples showing maximum, minimum downstream-upstream movement of f i s h i n twenty-two h y p o t h e t i c a l t e s t s u s i n g the formula: (nxr)/Ni> + 12 where n = number of f i s h per compartment, r = rank number, N T = t o t a l number of f i s h used i n the t e s t . ( after Kelso 1972 ) Rank No. 12 11 10 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 Upstream Score Downstream Score Net Score Compart- . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ment No. T e s t l ' 0 20 12.000 24.000 0.000 2 1 19 12.600 23.400 1.200 3 2 18 13.200 22.800 . 2.400 4 3 17 13.800 22.200 3.600= 5 4 16 14.400 21.600 4.800 6 5 15 15.000 21.000 6.000 7 8 6 7 14 13 15.600 16.200 20.400 19.800 7.200 8.400 9 8 12 16 .800 19.200 9.600 10 9 11 17.400 18.600 10.800 11 10 10 18.000 18.000 12.000 12 ' 11 9 18.600 17.400 13.200 13 12 8 19.200 16.800 14.400 14 13 7 19.800 16.200 15.600 15 14 6 20.400 15.600 16.800 16 15 5 21.000 15.000 18.000 17 16 4 21.600 14.400 19.200 18 17 3 22.200 13.800 20.400 19 18 2 22.800 13.200 21.600 20 19 1 23.400 12.600 22.800 21 20 0 24.000 12.000 24.000 22 0 2 1 1 4 3 2 1 4 12.833 17.944 6.889 92 APPENDIX 4 Comparisons among treatment samples of Sockeye salmon (Oncor-hynchus nerka) from the downstream m i g r a t o r y F u l t o n R i v e r s t o c k . The treatments ( c o n t r o l , 0.5 ppm H g C l 2 , and 1.0 ppm HgCl„) and independent v a r i a b l e s and the behaviour s c o r e s are the dependent v a r i a b l e s . Three a s t e r i s k s (***) r e p r e s e n t a s i g n i f i c a n t d i f -f e r e n c e of 0.001 l e v e l of p r o b a b i l i t y , two a s t e r i s k (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Group Mean S t d . Dev. Sample S i z e C o n t r o l 9 .46 2. 60 32 0.5 ppm HgCl 2 4 .55 1. 96 32 1.0 ppm HgCl 2 13 .13 2. 11 32 A n a l y s i s of V a r i a n c e Source S. S. D.F. M. S o F Lev. S i g . Groups 1186 .45 2 593. 23 118 .10 * * E r r o r 467 .15 93 5. 02 T o t a l 1653 .60 93 I n d i v i d u a l D . F . T e s t s Source S.S. D.F. M.S e F Lev. S i g . Co n t r o l / 0 . 5 ppm H g C l 2 386 ,17 1 386. 17 76. 88 * * 0.5 ppm/1.0 ppm H g C l 2 1178 .11 1 1178. 11 234. 54 * * C o n t r o l / 1 . 0 ppm H g C l 2 215 ,28 1 215. 28 42. 86 * 93 APPENDIX 5 Comparisons among treatment samples of Sockeye salmon (Oncor-hynchus nerka) from the upstream m i g r a t o r y Lower Babine R i v e r s t o c k . The treatments ( c o n t r o l , 0.5 ppm H g C l 2 , and 1.0 ppm HgClp) are independent v a r i a b l e s and the be h a v i o u r s c o r e s are the dependent v a r i a b l e s . Three a s t e r i s k s (***) r e p r e s e n t a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l of p r o b a b i l i t y , two a s t e r -i s k s (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Group Mean S t d . Dev. Sample S i z e C o n t r o l 16.71 2.04 32 0.5 ppm H g C l 2 10.76 2.75 32 1.0 ppm H g C l 2 16.84 2.22 32 A n a l y s i s of V a r i a n c e Source S.S. D.F • M.S. F Lev. S i g . Groups 932.25 2 466.12 84. 13 E r r o r 515.27 93 5.54 T o t a l 1447.52 93 I n d i v i d u a l D.F. T e s t s Source S.S . D.F. M.S. F Lev. S i g . C o n t r o l / 0 . 5 ppm H g C l 2 685. 25 1 685.25 123.68 ** 0.5 ppm/1.0 ppm H g C l 2 712. 68 1 712.68 128.63 ** C o n t r o l / 1 . 0 ppm H g C l 2 0. 27 1 o.27 0 . 0 5 N . S . APPENDIX 6 The m o r t a l i t y r a t e f o r both the f e e d i n g and s t a r v a t i o n experiment Fed Coho Salmon Starved Coho Salmon The A c c l i m a t i z a t i o n Week C o n t r o l 0.5 ppm H g C l 2 1.0 ppm H g C l 2 C o n t r o l 0.5 pprn HgCl2 1.0 ppm H g C l 2 0% 0% 0% 0% 0% 0% The S i x Week T e s t P e r i o d C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 3.6% 5.2% 7.2% 8.8% 10.0% 14.4% Fed Sockeye Salmon Starved Sockeye Salmon The A c c l i m a t i z a t i o n Week C o n t r o l 0.5 ppm HgCl2 1.0 ppm HgCl2 C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 0% 0.3% 0% 0% 0.3% 0% The S i x Week T e s t P e r i o d C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 C o n t r o l 0.5 ppm H g C l 2 1.0 ppm H g C l 2 7.5% 7.8% 7.2% 7.5% 10.3% 10.8% Fed Chum Salmon The A c c l i m a t i z a t i o n Week C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 0% 0% . 0% The S i x Week T e s t P e r i o d C o n t r o l 0.5 ppm HgCl2 1.0 ppm H g C l 2 5.0% 7.2% 10.0% 95 APPENDIX 7 Comparisons among treatment samples of f e d Chum salmon (Oncorhynchus k e t a ) . In t h i s • t a b l e the independent v a r i a b l e i s the i n t o x i c a n t l e v e l s ( c o n t r o l , 0.5 ppm HgCl2 and 1.0 ppm HgCl2) and the dependent v a r i a b l e i s wet body weight i n grams. Three a s t e r i s k s (***) r e p r e -sent a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l of p r o b a b i l i t y , two a s t e r i s k s (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Treatment R e g r e s s i o n E q u a t i o n F2,317 s l o p e s A d j u s t e d means X = 1.5 F2,317 1) C o n t r o l Y=0.3138+0.0375X 191.88 * 0.370 274.92 ** 2) 0.5 ppm H g C l 2 Y=0.3228-0.0085X 0.310 3) 1.0 ppm H g C l 2 Y=0.3223-0.0198X 0.293 I n d i v i d u a l D.F. T e s t on A d j . means Fl,317 1 / 2 299.08 * 1 / 3 500.01 * 2 / 3 25.67 N.S. 1 / 2 , 3 524.17 * 96 APPENDIX 8 Comparisons among treatment samples of f e d Sockeye salmon (Oncorhyn-chus n e r k a ) . In t h i s t a b l e the independent v a r i a b l e i s the i n t o x i c a n t l e v e l ( c o n t r o l , 0.5 ppm HgCl2 and 1.0 ppm HgCl2) and the dependent v a r i a b l e i s wet body weight i n grams. Three a s t e r i s k s (***) r e p r e -sent a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l of p r o b a b i l i t y , two a s t e r i s k s (•*), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Treatment R e g r e s s i o n E q u a t i o n F2,317 s l o p e s A d j u s t e d means X = 1.5 F2,317 1) C o n t r o l Y=0.1325+0.0149X 229.32 * * 0.155 227.06 ** 2) 0.5 ppm H g C l 2 Y=0,1418-0.0100X 0.127 3) 1.0 ppm H g C l 2 Y=0.1442-0.0145X 0.122 I n d i v i d u a l D.F. T e s t on A d j . means Fl,317 1 / 2 283.66 * 1 / 3 395.08 * 2 / 3 9.21 N.S. 1 / 2 , 3 449.42 * 97 APPENDIX 9 Comparisons among treatment samples of f e d Coho salmon (Oncorhynchus  k i s u t c h ) . In t h i s t a b l e the independent v a r i a b l e i s the i n t o x i c a n t l e v e l ( c o n t r o l , 0.5 ppm HgCl2 and 1.0 ppm HgCl2) and the dependent v a r i a b l e i s wet body weight i n grams. Three a s t e r i s k s ( * * * ) r e p r e -sent a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l o f p r o b a b i l i t y , two a s t e r i s k s (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Treatment Re g r e s s i o n E q u a t i o n F2,317 s l o p e s A d j u s t e d means F2,317 X = 1.5 1) C o n t r o l Y=0.4058+0.0716 66.18 N.S 0.513 82.31 N.S. 2) 0.5 ppm H g C l 2 Y=0.4300-0.0042 0.424 3) 1.0 ppm H g C l 2 Y=0.4266-0.0202 0.396 I n d i v i d u a l D.F. T e s t on A d j . means Fl,317 1/2 79.17 N.S. 1 / 3 115.90 N.S. 2 / 3 1 3.49 N.S. 1 / 2 , 3 128.88 N.S. 9 8 APPENDIX 10 Comparisons among treatment samples of s t a r v e d Sockeye salmon (Oncorhynchus n e r k a ) . In t h i s t a b l e the independent v a r i a b l e i s the i n t o x i c a n t l e v e l ( c o n t r o l , 0.5 HgCl2 and 1.0 ppm HgCl2) and the dependent v a r i a b l e i s wet body weight i n grams. Three a s t e r i s k s (***) r e p r e s e n t a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l of p r o b a b i l i t y , two a s t e r i s k s (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Treatment R e g r e s s i o n E q u a t i o n F2,317 s l o p e s A d j u s t e d means F2,317 X = 1.5 1) C o n t r o l Y=0.1402-0.0104X 11.30 N. S. 0.125 10.19 N.S. 2) 0.5 ppm H g C l 2 Y=0.1413-0.0109X 0.125 3) 1.0 ppm H g C l 2 Y=0.1426-0.0149X 0.120 I n d i v i d u a l D.F. 1 / 2 1 / 3 2 / 3 1 / 2 , 3 T e s t on A d j . means Fl,317 0.310 N.S. 19.05 ..N.S. 14.53 N.S. 8.06 N.S. 99 APPENDIX 11 Comparisons among treatment samples of s t a r v e d Coho salmon (Oncorhyn-chus k i s u t c h ) . In t h i s t a b l e the independent v a r i a b l e i s the i n t o x i -c a n t l e v e l ( c o n t r o l , 0.5 ppm HgCl2 and 1.0 ppm'HgCl2) and the dependent v a r i a b l e i s wet body weight i n grams. Three a s t e r i s k s (***) r e p r e s e n t a s i g n i f i c a n t d i f f e r e n c e of 0.001 l e v e l of p r o b a b i -l i t y , two a s t e r i s k s (**), the 0.01 l e v e l and "one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . Treatment R e g r e s s i o n E q u a t i o n F2,317 s l o p e s A d j u s t e d means F2,317 X = 1.5 1) C o n t r o l Y=0.4259-0.0136X 1.03 N.S 0.406 2.13 N.S. 2) 0.5 ppm H g C l 2 Y=0.4281-0.0113X 0.411 3) 1.0 ppm HgCl2 Y=0.4264-0.0221X. 0.393 I n d i v i d u a l D.F. T e s t on A d j . means Fl,317 1 /2 0.08 N.S. 1 / 3 1.15 N.S. 2 / 3 1.86 N.S. 1 / 2 , 3 0.21 N.S. APPENDIX 12 The m o r t a l i t y i n w i l d and hatchery Coho prey d u r i n g the three week experimental p e r i o d W i l d Coho Prey Hatchery Coho Prey The per cent m o r t a l i t y f o r the t e s t p e r i o d C o n t r o l 0.5 ppm HgCl,-, 1.0 ppm H g C l 2 C o n t r o l 0.5 ppm H g C l 2 1.0 ppm H g C l 2 1.6% 3.2% 5.6% 2.4% 2.4% 5.6% 101 APPENDIX 13 The m o r t a l i t y data f o r the Coho salmon p r e d a t o r stock C o n t r o l P r e d a t o r s The m o r t a l i t y f o r the p r e d a t o r stock 0% 102 APPENDIX 14 Comparisons among samples of w i l d Coho prey (Oncorhynchus k i s u t c h ) Source S.S. D.F. M.S. F Lev. S i g . Groups 0.14 2 0.07 0.96 N.S. E r r o r • 24.47 3 72 0.07 T o t a l 27.61 374 Comparisons among samples of h a t c h e r y Coho prey (Oncorhynchus k i s u t c h ) Source S.S. D.F. M.S. F Lev. S i g . Groups 0.04 2 0.04 0.49 N.S. E r r o r 15.51 372 0.04 T o t a l 15.55 374 Comparisons among samples of h a t c h e r y Coho p r e d a t o r s (Oncorhynchus k i s u t c h ) Source S.S D.F. M.S. F Lev. S i g . Groups 0.33 4 0.08 0.99 N.S. E r r o r 3.74 45 0.08 T o t a l 4.07 49 Three a s t e r i s k s (***) r e p r e s e n t a s i g n i f i c a n t d i f f e r e n c e of , 0.001 l e v e l of p r o b a b i l i t y , two a s t e r i s k s (**), the 0.01 l e v e l and one a s t e r i s k (*), the 0.05 l e v e l . N.S. r e p r e s e n t s not s i g n i f i c a n t . 103 APPENDIX 15 The Kilmogorov-Smirnov Two Sample Test (large samples: one-tailed test) for the hatchery Coho salmon (Oncorhynchus kisutch) f l i g h t response experiment. The treatment l e v e l s were the independent variables and the f l i g h t response the dependent v a r i a b l e . A. The f l i g h t re sponse of the f i s h treated with 0.5 ppm HgCl 2 versus the con t r o l f i s h Control 0.5 ppm HgCl2 Control 0.5 ppm HgCl 2 treatment treatment treatment treatment S250(X)-S a250(X) S 1250(X) S250(X) S 1250(X) S250(X) 79 250 90 250 0.316 0.360 0.044 101 250 118 250 0.404 0.472 0.068 121 250 141 250 0.484 0.564 0.080 140 250 160 250 0.560 0.640 0.080 160 250 178 250 0.640 0.712 0.072 179 250 195 250 0.716 0.780 0.064 194 2-50 211 250 0.776 0.844 0.068 205 250 222 250 0.820 0.888 0.068 215 250 231 250 0.860 0.924 0.064 225 250 237 250 0.900 0.978 0.048 232 241 0.928 0.964 0.036 250 250 239 25 0 244 250 0.956 0.976 0.020 243 250 247 250 0.972 0.988 0.016 245 250 249 250 0.980 0.996 0.016 248 250 250 250 0.992 1.000 0.008 249 250 250 250 0.996 1.000 0.004 250 250 250 250 1.000 1.000 1.000 104 APPENDIX 15 c o n t i n u e d A. The f l i g h t response of the f i s h t r e a t e d w i t h 0.5 ppm H g C ^ v e r s u s the c o n t r o l f i s h D = maximum ( s 1250 (X )-S250 (X )) = 0.080 The maximum v a l u e of D i n the p r e d i c t e d d i r e c t i o n was 0.080. The v a l u e of X^ was computed u s i n g the f o r m u l a : X 2 = 4 D 2 n l n 2 n l + n 2 ... = 4 C 0 . 0 8 0 ) 2 ( 2 5 Q ^ 2 5 0 ) 250 + 250 = 3.20 . . 2 The X v a l u e of 3.20 (2 d . f . , o n e - t a i l e d t e s t ) was not s i g n i f i c a n t a t the 0.05 l e v e l of s i g n i f i c a n c e . B. The f l i g h t response of 1.0 ppm HgCl2 v e r s u s the f i s h t r e a t e d w i t h the c o n t r o l f i s h C o n t r o l treatment s l 2 5 0 ( X ) 1.0 ppm H g C l 2 treatment S 2 5 0 ( X ) C o n t r o l treatment S a 2 5 0 ( X ) 1.0 ppm H g C l 2 treatment S 2 5 0 ( X ) S 2 5 0 ( X ) -- S a 2 5 0 ( X ) 79 250 90 250 0. .316 0. 360 0. .044 101 250 119 250 0. ,404 0. 476 0. .072 121 250 143 250 0. .484 0. 5 72 0. .088 140 250 16 5 "250" 0. .560 0. .660 0. .100 160 250 185 250 0. .640 0. .740 0. .100 179 250 202 250 0 .716 0. .808 0 .092 194 250 217 250 0 .7 76 0. .868 0 .092 205 250 230 "250" 0 .820 0. .920 0 .100 215 250 238 250 0 .860 0. .952 0 .092 225 250 243 250 0 .900 0 .972 0 .072 232 250 245 250 0 .928 0. .980 0 .052 105 APPENDIX 15 c o n t i n u e d B. The f l i g h t response of the f i s h t r e a t e d w i t h 1.0 ppm H g C l 2 v e r s u s the c o n t r o l f i s h C o n t r o l 1.0 ppm H g C l 2 C o n t r o l 1.0 ppm H g C l 2 treatment treatment treatment treatment -S a250(X) S 1250(X) S250(X) Sl250(X) S250(X) S250(X) 239 250 248 250 0.956 0.992 0 .036 243 250 249 250 0.972 0.996 0 .024 245 250 250 250 0.98.0 1.000 0 .020 248 250 250 250 0.992 1.000 0 .008 249 250 250 250 0.996 1.000 0 .004 250 250 250 250 1.000 1.000 0 .000 D = maximum [s a250 (X )-S250 (X )] = 0.100 The maximum v a l u e of D i n the p r e d i c t e d d i r e c t i o n was 0.100. The value of X 2 was computed u s i n g the for m u l a : X 2 = 4D 2 n l n2 n^+n 2 = 4(0.100) = 5.00 2 (250)(250) 250 + 250 The X v a l u e of 5.00 (2 d . f . , o n e - t a i l e d t e s t ) was not s i g n i f i c a n t a t the 0.05 l e v e l of s i g n i f i c a n c e . 106 APPENDIX 16 The K i l m o g o r o v - S m i r n o v Two Sample T e s t ( l a r g e s a m p l e s : o n e - t a i l e d t e s t ) f o r t h e w i l d Coho salmon (Oncorhynchus k i s u t c h ) f l i g h t r e s p o n s e e x p e r i m e n t . The t r e a t m e n t l e v e l s w e r e • t h e i n d e p e n d e n t v a r i a b l e s and the f l i g h t r e s p o n s e t h e dependent v a r i a b l e . A. The f l i g h t r e s p o n s e o f the f i s h t r e a t e d w i t h 0.5 ppm H C C I 2 v e r s u s the c o n t r o l f i s h C o n t r o l t r e a t m e n t s^socx) ,5 ppm HgCl2 t r e a t m e n t S250(X) C o n t r o l t r e a t m e n t S 1 2 5 0 ( X ) 0.5 ppm HgCl2 t r e a t m e n t S250(X) S250(X)-S" L250(X) 16 250 31 250 45 250 57 250 70 250 •SI 250 93 250 104 250 114 250 125 250 135 250 145 250 154 250 163 250 172 250 182 250 190 250 197 250 205 250 211 250 218 250 29 250" 51 250 69 250 85 250 102 250 118 250 129 250 140 250 150 250 161 "250" 171 250 181 250 189 250 196 "250" 204 250 210 250 216 250 224 250 229 250 234 250 2 3 7' 250 0.064 0.124 0.180 0.228 0.280 0.324 0.372 0.416 0.456 0.500 0.540 0.580 0.616 0.652 0.688 0.728 0.760 0.788 0.820 0.844 0.872 0.116 0.204 0.276 0.340 0.408 0.472 0.516 0.560 0.600 0.544 0.684 0.724 0.756 0.784 0.816 0.840 0.864 0.896 0.916 0.936 0.948 0.052 0.080 0.096 0.112 0.128 0.148 0.144 0.144 0.144 0.144 0.144 0.144 0.140 0.132 0.128 0.112 0.104 0.108 0.096 0.092 0.076 107 APPENDIX 16 continued A. The f l i g h t response of the f i s h t r e a t e d w i t h .0.5 ppm HgCl2 v e r s u s the c o n t r o l f i s h C o n t r o l treatment S 1250(X) 0.5 ppm HgCl2 treatment S250(X) C o n t r o l treatment Sl250(x) 0.5 ppm H g C l 2 treatment S250(X) S250(X)-S 1250(X) 227 250 225 250 228 250 232 250 234 250 236 250 237 250" 238 250 239 250 241 750 242 250 244 250 245 250 247 250 250 250 241 250 245 250 247 250 248 250 249 250 249 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 0.888 0.900 0.912 0.928 0.936 0.944 0.948 0.952 0.956 0.964 0.968 0.976 0.980 0.988 1.000 0.964 0.980 0.988 0.992 0.996 0.996 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0. 0, 0, 0, 0, 0, 0. 0. 0, 0, 0, 0, 0, 0, 0. 076 080 076 064 060 052 052 048 044 036 032 024 020 012 000 D = maximum [s 1250 (X)-S250 (X )] = 0.148 The maximum v a l u e of D i n the p r e d i c t e d d i r e c t i o n was 0.148. The value of X^ was computed u s i n g the f o r m u l a : X 2 = 4D 2 n l n2 n^+n 2 . 4 ( 0 . 1 4 8 ) ( " ° ) ( " ° ) . 250 .+. 250 . = 10.952 The X 2 value o f 10.952 (2 d . f . , o n e - t a i l e d t e s t ) was s i g n i f i c a n t at the 0.01 l e v e l of s i g n i f i c a n c e . 108 APPENDIX 16 co n t i n u e d B. The f l i g h t response of the f i s h t r e a t e d w i t h 1.0 ppm H g C l 2 v e r s u s the c o n t r o l f i s h C o n t r o l 1.0 ppm H g C l 2 C o n t r o l 1.0 ppm H g C l 2 treatment treatment treatment treatment S250(X)-S 1250(X) sl250(X) S250(X) S 1250(X) S250CX) 16 250 52 250 0.064 0.208 0.144 31 250 75 250 0.124 0.300 0.176 45 250 91 250 0.180 0.364 0.184 57 250 105 250 0.228 0.420 0.192 70 250 118 250 0.280 0.472 0.192 81 250 131 250 0.324 0.524 0.200 93 250 142 250 0.372 0.568 0.196 104 250 154 250 0.416 0.616 0.200 114 250 166 250 0.456 0.664 0.208 125 250 177 250 0.500 0. 708 0.208 135 250 187 250 0.540 0.748 0.208 145 250 194 250 0.580 0.776 0.196 . 154 250 202 250 0.616 0.808 0.192 163 250 210 250 0.652 0.840 0.188 172 250 218 250 0.688 0.872 0.184 182 250 224 250 0.728 0.896 0.168 190 250 229 250 0.760 0.916 0.156 197 250 233 250 0.788 0.932 0.144 205 .250 237 250 0.820 0.948 0.128 211 250 242 250 0.844 0.968 0.124 218 "250" 246 250 0.872 0.984 0.112 222 250 247 250 0.888 0.988 0.100 109 APPENDIX 16 con t i n u e d B. The f l i g h t response of the f i s h t r e a t e d w i t h 1.0 ppm H g C l 2 v e r s u s the c o n t r o l f i s h C o n t r o l treatment Sl250(X) 1.0 ppm H g C l 2 treatment S250(X) C o n t r o l treatment S a250(X) 1.0 ppm H g C l 2 treatment S250(X) S250(X)-S X250(X) 225 250 228 250 232 250 234 250 236 250 232 250 238 250 239 250 241 250 247 250 244 250 245 250 247 250 250 250 248 250 249 250 249 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 250 0.900 0.912 0.928 0.936 0.944 0.948 0.952 0.956 0.964 0.968 0.976 0.980 0.988 1.000 0.992 0.996 0.996 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0, 0, 0, 0, 0. 0, 0. 0. 0. 0. 0. 0. 0. 0. 092 084 071 064 056 052 048 044 036 032 024 020 012 000 D = maximum [s 1250 (X )-S250 (X )) = 0.208 The maximum v a l u e of D i n the p r e d i c t e d d i r e c t i o n was 0.208. The v a l u e of X 2 was computed u s i n g the f o r m u l a : 4D 2 n l n2 n 1 + n 2 4(0.208) = 21.632 2 (250)(250) 250 + 250 The X v a l u e of 21.632 (2 d . f . , o n e - t a i l e d t e s t ) was s i g n i f i c a n t at the 0.01 l e v e l of s i g n i f i c a n c e . 110 FIGURES I l l Fig.1 The standard 25-compartment performance channel (entrance and exit of each compartment staggered). The c i r c l e i n the upper plan view shows the compart-ment used tc release f r y . ( af t e r kelso 1972 ) Outlet trap Inlet Outlet EH release point Inlet trop' Figure 2. The Fulton River Sockeye salmon ( Oncorhynchus nerka ) control f i s h scores f o r the upstream-douinstr8am migration tendency experiment. 10 ro 7 8 9 113 11 12 13 14 15 16 17 , 17 Upstream-downstream migration tendency scores. 18 19 20 21 22 23 24 Figure 3. The Fulton River Sockeys salmon ( Oncorhynchus nerka ) 0.5 p.p.m. HgCl 2 treatment scores f o r the upstrean-douinstream migration tendency experiment. H i H i LO 8 9 10 11 12 13 14 15 TS 17 18 Upstream-domnstream migration tendency scores. 19 20 21 22 23 24 Figure 4. The F u l t o n R i v e r Sockeye salmon ( Oncorhynchus nerka ) 1.0 p.p.m. HgCl2 treatment scores f o r the upstream-dotunstream migration tendency experiment. r - i H i 2 0 21 2 2 2 3 2 4 3 9 Tu—Ti — T 3 — — — T T T 7 — T T T S " Upstream-downstrean migration tendency scores. Figure 5. Th8 Louier Babine River Socksye salmon ( Oncorhynchus nerka ) c o n t r o l f i s h scores f or the upstrsam-dou;nstream migration tendency experiment. H i H i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Upstream-doiunstream migration tendency scores. F i g u r e 6. The Loiuer Babine R i v e r Sockeye salmon ( Oncorhynchus nerka ) 0.5 p.p.m. HgC^ treatment scores f o r the upstream-douinstream migration tendency experiment. H i 8 9 10 11 12 13 14 15 16 17 18 Upstream-domnstream migration tendency scores. 19 20 21 22 23 24 Figure 7. The Loioer Babine River Sockeye salmon ( Oncorhynchus nerka ) 1.0 p.p.m. HgCl2 treatment scores for the upstream-doiunstream migration tendency experiment. H i H i -4 8 9 10 11 12 13 14 15 16 17 18 19 20 Upstream-domnstream migration tendency experiment. 21 22 2 3 24 0.600 Figure 8. The Chum salmon ( Oncorhynchus keta ) growth experiment regression l i n e s d i p i c t i n g growth rates over a s i x meek feeding period. Treatments Control 0.5 p.p.m. HgCl^ 1.0 p.p.m. HgCl Y = 0.3138 + 0.0375 X N = 320 Y = 0.3228 - 0.0085 X N = 320 Y = 0.3223 - 0.0198 X N = 320 0.500 0.400 co E (0 u • H 0.300 - p JZ CT •H CD 3 ( Control ) ( 0.5;-p.p.m. HgCl ) ( 1.0 p.p.m. HgCl 2 ) I - i H i 00 0.200 0.100 0.000 Time i n masks. Figure 9. The Sockeye salmon ( Oncorhynchus narka ) grouith experiment regression l i n e s d i p i c t i n g grouith rates over a s i x week feeding period. 0.400 Treatments Control 0.5 p.p.m. HgCl, 1.0 p.p.m. HgCl, Y =0.1325 + 0.0149 X N = 320 Y = 0.1418 - 0.0100 X N = 320 Y = 0.1442 - 0.0145 X N = 320 0.350 0.300 o e to u cn x: cn - H CO 3 0.250 0.200 0.150 0.100 0.050 ( Control ) ( 0.5 p.p.m. HgCl ( 1.0 p.p.m. HgCl r - i Hi CD 0.000 Time i n weeks. Figure 10. The Coho salmon ( Oncorhynchus kisutch ) grouith experiment regression l i n e s d i p i c t i n g growth rates over a s i x meek feeding period. 1.200 r Treatments Control 0.5 p.p.m. 1.0 p.p.m. Y = 0.4058 + 0.0716 X N = 320 HgCl Y = 0.4300 - 0.0042 X N = 320 HgCl^ Y = 0.4266 - 0.0202 X N = 320 1.000 0.800 0) E a <-> at • P cn 0.600 0.400 (Control ) H i ro o ( 0.5 p.p.m. HgCl 2 ) ( 1.0 p.p.m. HgCl 2 ) 0.200 0.000 6 Time i n weeks. 0.400 I-0.350 Figure 11. The sockeye salmon ( Oncorhynchus nerka ) starvation experiment regression lines dipicting u/eight loss rates over a six week period of starvation. Treatments Control Y 0.5 p.p.m. HgCl Y 1.0 p.p.m. HgCl, Y 0.1402 - 0.0104 X N = 320 0.1413 - 0.0109 X N = 320 0.1426 - 0.0140 X N = 320 0.300 . 0.250 (0 u 0.200 - P .c a> • H CD 3 H i ro H i 0.150 0.100 ( Control ) ( 0.5 p.p.m. ( 1.0 p.p.m. HgCl ) HgCl, ) 0.050 0.000 Time in weeks. F i g u r e 1 2 . The Coho s a l m o n ( O n c o r h y n c h u s k i s u t c h ) s t a r v a t i o n e x p e r i m e n t r e g r e s s i o n l i n e s d i p i c t i n g w e i g h t l o s s r a t e s o v e r a s i x week p e r i o d o f s t a r v a t i o n . 1 .200 • T r e a t m e n t s C o n t r o l 0 . 5 p . p . m . H g C l 2 1.0 p . p . m . H g C l „ Y = 0 . 4 2 5 9 - 0 . 0 1 3 6 X N = 320 Y = 0 .4281 - 0 . 0 1 1 3 X N = 320 Y = 0 . 4 2 6 4 - 0 .0221 X N = 320 1 .000 0 . 8 0 0 0 . 6 0 0 0 . 4 0 0 ( 0 . 5 p . p . m . ( C o n t r o l ) ( 1.0 p . p . m . H g C l , H g C l , 0 . 2 0 0 0 . 0 0 0 T ims i n w e e k s . 360 cm:- -25 cm.-20 cm. ro F igure 13 . The p r e d a t o r - p r e y f l i g h t response c h a n n e l . The channel was emptied between each of the exper imenta l r u n s . Fresh water at 6 C. was p laced i n the channel to ensure t h a t both the oxygen and temperature c o n d i t i o n s were uni form throughout the exper iment . hinged p a r t i t i o n -11) cm j - * •15 cm5 prey chamber predator chamber 124 79 Figure 15. The Coho salmon ( Oncorhynchus k i s u t c h ) hatchery prey f l i g h t response scores f o r the c o n t r o l treatment. 30- U 25-20-u c CD CT CO u ca u o u in r - i ro 15-10-5-0- i i i i i 50 100 190 200 1 250 i i i 300 i i. 350 Distance i n centimeters 90 30-Figure 16. The Coho salmon ( Oncorhynchus kieutch ) hatchery prey f l i g h t response scores f or the 0.5 p.p.m. mercuric chloride treatment. 25-20->» o c m D CT CO £ 15-CO u o u cn 10-5- r ro CD 0- i i, i i i i i i i i i • • • • 3&T I I I 1 1 5TT 100 150 200 250 Distance i n centimeters. 350 90 Figure 17. The Coho salmon ( Oncorhynchus k i s u t c h ) hatchery prey f l i g h t responses scores f o r the 1.0 p.p.m. mercuric c h l o r i d s treatment. • r i • . • • t i t I i 1 l ' l l • 0 50 100 150 200. 250 500 350 Distance i n centimeters. Figure 18. The Coho salmon ( Oncorhynchus kisutch ) mild prey f l i g h t response scores for the c o n t r o l treatment. n • i i % > 50 > » ' »—'—i- -*—i—>-100 150 200 250 Distance i n centimeters 300 350 Figure 19. The Coho salmon ( Oncorhynchus kisutch, ) uiild prey f l i g h t response scores f or the 0.5 p.p.m. mercuric chloride treatment. I n . 0 300 350 Distance i n centimeters. 30-52 Figure 20. The Coho salmon ( Oncorhynchus kisutch ) wild prey flight response scores for the 1*0 p.p.m. mercuric chloride treatment. 25- r 20-o c CD 3 CT m <>-o u o o cn 15-10-5-i_r LT IV CO o • • • l I I ' • • 50 100 150 2 0D 250 Distance in centimeters. 300 350 

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