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The effect of water pH on swimming performance, blood pH, red cell pH, ion concentrations and catecholamine… Ye, Xuemin 1986

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The e f f e c t o f water pH on swimming performance, blood pH, red c e l l i o n c o n c e n t r a t i o n s and catecholamine c o n c e n t r a t i o n s i n plasma, and g i l l p o t e n t i a l i n rainbow t r o u t (Salmo g a i r d n e r i ) by Xuemin Ye B.Sc.,Zhongshan U n i v e r s i t y , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i THE FACULTY OF ^  (Department GRADUATE STUDIES 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 standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1986 © Xuemin Ye, 1986. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6(3/81) i i A b s t r a c t The e f f e c t of t r a n s f e r r i n g f i s h from water at pH 7.0 to e i t h e r more a c i d or more a l k a l i n e c o n d i t i o n s was to reduce the maximum c r i t i c a l v e l o c i t y of the f i s h . In water of pH 4.0, 5.0, and 10.0, the maximum c r i t i c a l v e l o c i t y was only 54.5%, 66.5%, and 61% r e s p e c t i v e l y of that recorded for f i s h i n the water of pH 7.0. Thus, both a c i d and a l k a l i n e c o n d i t i o n s i n the water reduce the a e r o b i c swimming c a p a c i t y of t r o u t . Exposure to a c i d c o n d i t i o n s i n c r e a s e d mucus s e c r e t i o n and t h i s was a s s o c i a t e d with an i n c r e a s e i n coughing and b r e a t h i n g frequency i n r e s t i n g f i s h . Coughing r a t e i n c r e a s e d from 41/hr to 592/hr; and r e s p i r a t i o n frequency i n c r e a s e d from 81/min to 104/min when f i s h were t r a n s f e r r e d from water at pH 7.0 to water at pH 4.0. In comparing f i s h exposed to a c i d and a l k a l i n e waters, the r e s u l t s i n d i c a t e s that f i s h have a g r e a t e r c a p a c i t y to r e g u l a t e blood pH i n a c i d than i n a l k a l i n e c o n d i t i o n s . The g i l l p o t e n t i a l was s t r o n g l y dependent on water pH, being negative i n n e u t r a l water, but p o s i t i v e i n a c i d water and more negative i n a l k a l i n e s o l u t i o n . i i i Catecholamine l e v e l s i n c r e a s e d s i g n i f i c a n t l y during a c i d exposure, but were not a l t e r e d d u r i n g a l k a l i n e exposure. The i n c r e a s i n g catecholamine l e v e l s appeared a t d i f f e r e n t time p e r i o d s i n d i f f e r e n t f i s h d u r i n g a c i d exposure and seemed to be a s s o c i a t e d with the death of the f i s h . Na + and C l ~ i o n c o n c e n t r a t i o n s i n plasma decreased s i g n i f i c a n t l y a f t e r 24hrs of a c i d exposure, but d i d not change s i g n i f i c a n t l y i n a l k a l i n e water. This may i n d i c a t e t h a t i o n o r e g u l a t o r y distubance i n plasma i s one of the reasons f o r the decrease i n the maximum c r i t i c a l v e l o c i t y i n a c i d water, but not i n a l k a l i n e water. i v T A B L E OF CONTENTS P a g e A b s t r a c t i i T a b l e o f c o n t e n t s i v L i s t o f t a b l e s v L i s t o f f i g u r e s v i A c k n o w l e d g e m e n t s i x S e c t i o n I I n t r o d u c t i o n 1 S e c t i o n I I M a t e r i a l s a n d M e t h o d s 4 1. E x p e r i m e n t a l a n i m a l s 4 2 . E x p e r i m e n t I : S w i m m i n g v s w a t e r pH 4 3 . E x p e r i m e n t l I : b l o o d s a m p l e v s w a t e r pH 6 S e c t i o n I I I R e s u l t s , 8 1. E x p e r i m e n t I : S w i m m i n g v s w a t e r pH 8 2 . E x p e r i m e n t I I : b l o o d s a m p l e v s w a t e r p H . . 13 2 . 1 E f f e c t o f a c i d w a t e r 13 2 . 2 E f f e c t o f a l k a l i n e w a t e r 16 S e c t i o n I V D i s c u s s i o n 76 1. S w i m m i n g v s w a t e r pH 76 2 . E f f e c t o f a c i d w a t e r 78 3 . E f f e c t o f a l k a l i n e w a t e r 85 S e c t i o n V R e f e r e n c e s 89 V Table 1. E f f e c t of d i f f e r e n t water pH on the swimming performance of rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Table 2. E f f e c t of a c i d water(pH4.0) on the b r e a t h i n g and coughing r a t e of rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Table 3. Blood pH(pHe), red c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium and c h l o r i d e c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) l e v e l s i n plasma and H + , Na + , C l ~ d r i v i n g f o r c e i n c o n t r o l water(pH6.4) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Table 4. E f f e c t of a c i d water(pH4.0) on blood pH(pHe), red c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium and c h l o r i d e c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) l e v e l s i n plasma and H +, Na +, C l ~ d r i v i n g f o r c e i n c o n t r o l water(pH6.4) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Table 5. The whole blood pH, red c e l l pH, catecholamine c o n c e n t r a t i o n s ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) i n plasma, g i l l p o t e n t i a l ( T E P ) , and Na +, C I - c o n c e n t r a t i o n s i n plasma i n rainbow t r o u t , compared the data i n zero c o n t r o l and the data i n maximum catecholamine l e v e l s . * = s i g n i f i c a n t l y d i f f e r e n t from zero control(P<0.05). Table 6. E f f e c t of a l k a l i n e water(pHIO.0) on Blood pH(pHe), red c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium, c h l o r i d e c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) l e v e l s i n plasma and H +, Na +, C l ~ d r i v i n g f o r c e i n c o n t r o l water(pH6.4) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . v i F i g u r e 1. E f f e c t o f d i f f e r e n t water pH on the maximum v e l o c i t y of rainbow t r o u t ( s a l m o g a i r d n e r i ) . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . (P<0.05) F i g u r e 2. E f f e c t o f a c i d water(pH4.0) on blood pH(pHe) i n rainbow t r o u t ( s a l m o g a i r d n e r i ) . * = s i g n i f i c a n t l y d i f f e r e n t from control.(P<0.05) F i g u r e 3. E f f e c t o f a c i d water(pH4.0) on red c e l l pH(pHi) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from control.(P<0.05) F i g u r e 4. E f f e c t o f a c i d water(pH4.0) on g i l l p o t e n t i a l ( T E P ) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from control.(P<0.05) F i g u r e 5. E f f e c t o f a c i d water(pH4.0) on sodium c o n c e n t r a t i o n i n plasma(Na +-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from control.(P<0.05) F i g u r e 6. E f f e c t of a c i d water(pH4.0) on c h l o r i d e c o n c e n t r a t i o n i n plasma(CI~-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from control.(P<0.05) F i g u r e 7. E f f e c t of a c i d water(pH4.0) on n o r a d r e n a l i n e concentrationCNor.] i n plasma ( 1 0 ~ 9 M/L)in rainbow t r o u t , (the mean value of each sampling time vs c o n t r o l group) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 8. E f f e c t o f a c i d water(pH4.0) on n o r a d r e n a l i n e c o n c e n t r a t i o n [ N o r . ] i n plasma ( 1 0 - 9 M/L>in rainbow t r o u t , (the value o f d i f f e r e n t f i s h i n each sampling time vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 9. Noradrenaline c o n c e n t r a t i o n [ N o r . ] i n plasma ( 1 0 - 9 M/L) i n rainbow t r o u t i n c o n t r o l water (pH6.4). (the value o f d i f f e r e n t f i s h i n each sampling time) F i g u r e 10. E f f e c t o f a c i d water(pH4.0) on n o r a d r e n a l i n e t N o r . ] and a d r e n a l i n e [ A d . ] c o n c e n t r a t i o n i n plasma ( 1 0 - 9 M/L)in rainbow t r o u t , (the maximum va l u e s i n d i f f e r e n t f i s h vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). v i i F i g u r e 11. E f f e c t o f a c i d water(pH4.0) on a d r e n a l i n e c o n c e n t r a t i o n Ad.] i n plasma (10~9 M/L)in rainbow t r o u t , (the mean value o f each sampling time vs c o n t r o l group) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 12. E f f e c t of a c i d water(pH4.0) on a d r e n a l i n e c o n c e n t r a t i o n ! A d . ] i n plasma ( 1 0 - ^ M/L)in rainbow t r o u t , (the value o f d i f f e r e n t f i s h i n each sampling time vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 13. Ad r e n a l i n e c o n c e n t r a t i o n [ A d . ] i n plasma (10~^ M/L) i n rainbow t r o u t i n c o n t r o l water (pH6.4). (the value of d i f f e r e n t f i s h i n each sampling time) F i g u r e 14. E f f e c t o f a c i d water(pH4.0) on the whlod blood pH(pHe) and red c e l l pH(pHi) i n rainbow t r o u t , (at the maximum catecholamine val u e s i n d i f f e r e n t f i s h vs a t zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 15. E f f e c t of a c i d water(pH4.0) on g i l l p o t e n t i a l (TEP mV)in rainbow t r o u t . (at the maximum catecholamine values i n d i f f e r e n t f i s h vs a t zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 16. E f f e c t o f a c i d water(pH4.0) on Na + and C l ~ c o n c e n t r a t i o n s i n rainbow t r o u t . (at the maximum catecholamine values i n d i f f e r e n t f i s h vs a t zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). F i g u r e 17. E f f e c t o f a c i d water(pH4.0) on Hydrogen d r i v i n g f o r c e (F-H +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 18. E f f e c t of a c i d water(pH4.0) on sodium d r i v i n g f o r c e (F-Na +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 19. E f f e c t o f a c i d water(pH4.0) on c h l o r i d e d r i v i n g f o r c e (F-C1~) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . v i i i F i g u r e 20. E f f e c t o f a l k a l i n e water(pHIO.0) on blood pH(pHe), i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 21. E f f e c t o f a l k a l i n e water(pHIO.0) on red c e l l pH(pHi) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 22. E f f e c t o f a l k a l i n e water(pHIO.0) on g i l l p o t e n t i a l ( T E P ) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 23. E f f e c t of a l k a l i n e water(pHIO.0) on sodium c o n c e n t r a t i o n i n plasma (Na-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 24. E f f e c t o f a l k a l i n e water(pHIO.0) on c h l o r i d e c o n c e n t r a t i o n i n plasma (Cl-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 25. E f f e c t o f a l k a l i n e water(pHIO.0) on n o r a d r e n a l i n e c o n c e n t r a t i o n i n plasma [Nor.] i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 26. E f f e c t o f a l k a l i n e water(pHIO.0) on hydrogen d r i v i n g f o r c e (F-H +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 27. E f f e c t o f a l k a l i n e water(pHIO.0) on sodium d r i v i n g f o r c e (F-Na +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . F i g u r e 28. E f f e c t o f a l k a l i n e water(pHIO.0) on c h l o r i d e d r i v i n g f o r c e ( F - C l - ) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . i x ACKNOWLEDGMENT I would l i k e to express my s i n c e r e g r a t i t u d e to Dr. D.J. Randall f o r h i s s u p e r v i s i o n , p a t i e n c e , and guidance d u r i n g t h i s study. I would e s p e c i a l l y l i k e to thank Dr. Randall f o r h i s a d v i c e throughout the course of the experiments, i n the p r e p a r a t i o n o f t h i s manuscript, and p r o v i d i n g the l a b o r a t o r y space and equipment. I would l i k e to express my s i n c e r e g r a t i t u d e to Dr. D.R. Jones, Dr. J . Steeves and Dr. W.K. Milsom f o r t h e i r h e l p i n w r i t i n g t h i s manuscript, and Mr. Harry Chin f o r h i s s c h o l a r s h i p and encouragement. I would l i k e to thank Mr. Dennis Mense, Mr. George Iwama and Miss P a t r i c i a Wright f o r t h e i r a s s i s t a n c e i n my study and t h e i r v a l u a b l e advice i n w r i t i n g t h i s manuscript. F i n a l l y , I would l i k e to thank a l l the people i n Dr. R a n d a l l ' s l a b who d i r e c t l y or i n d i r e c t l y c o n t r i b u t e d to t h i s study i n t h e i r v a r i o u s c a p a c i t i e s . 1 I n t r o d u c t i o n F i s h e s are found i n most n a t u r a l water bodies. The pH of these water bodies v a r i e s , but many have been a c i d i f i e d due to the l a r g e s c a l e p r o d u c t i o n of chemical waste. F i s h are c h r o n i c a l l y exposed to these waters and must o f t e n migrate through waters with marked changes i n pH en route. There i s now an e x t e n s i v e l i t e r a t u r e on the i n t e r a c t i o n of low pH environments with the p h y s i o l o g y of freshwater f i s h (see Wood and McDonald, 1982, f o r review). Many s t u d i e s have t r i e d to i l l u m i n a t e the p h y s i o l o g i c a l mechanisms of a c i d t o x i c i t y to f i s h . Among them, e f f e c t s on normal blood p h y s i o l o g y and d i s t u r b a n c e s to acid-base s t a t e are p a r t i c u l a r l y w e l l known (Packer & Dunson, 1970; N e v i l l e , 1979; Packer, 1979; McDonald, Hobe & Wood, 1980); i n a d d i t i o n , s t u d i e s of the e f f e c t s of a c i d on i o n o r e g u l a t i o n (Wood and McDonald, 1982); and oxygen t r a n s p o r t (Vaala & M i t c h e l l , 1970; Packer 1979) have been r e p o r t e d . L i t t l e i s known, however, about the p h y s i o l o g i c a l mechanisms of a l k a l i n e t o x i c i t y to f i s h ; a l s o l i t t l e i s known about the e f f e c t of a c i d or a l k a l i n e water on the swimming performance i n a d u l t rainbow t r o u t . Swimming performance has been suggested as a s u b l e t h a l c r i t e r i o n f o r measuring e f f e c t s 2 of t o x i c a n t s on f i s h ( B r e t t , 1967; Sprague, 1971). This response would appear to be s e n s i t i v e to a number of t o x i c a c t i o n s , i n c l u d i n g impairment of t r a n s p o r t or exchange of r e s p i r a t o r y gases, a l t e r a t i o n s i n energy t r a n s f o r m a t i o n s or i n h i b i t i o n of nervous or muscular a c t i v i t y (Waiwood and Beamish, 1978) . In a c i d water, impairment of t r a n s p o r t and exchange of r e s p i r a t o r y gases o c c u r s . T h i s i s because: 1) mucus accumulation at the g i l l s has been observed and i s known to i n h i b i t r e s p i r a t o r y gas exchange. 2) blood O2 content does not show a l i n e a r r e l a t i o n s h i p with O2 t e n s i o n , O2 content changes with the slope of the O2 d i s s o c i a t i o n curve(see R a n d a l l , 1970). T h i s slope i s a f f e c t e d by blood pH; C 0 2 t e n s i o n ; temperature and o r g a n i c phosphate l e v e l s . In t e l e o s t blood, i n c r e a s e d CO2 content or decreased pH causes not only a f a l l i n Hb-02 a f f i n i t y ( B o h r e f f e c t ) , but a l s o a decrease i n O2 content(Root e f f e c t : a f a l l i n O2 content even at atmospheric O2 t e n s i o n , see R a n d a l l , 1970; Riggs, 1970). I t has been found that blood pH decrease i n a c i d water, so one might expect that a Root e f f e c t would occur i n a r t e r i a l blood; such an e f f e c t would l i m i t t i s s u e O2 t r a n s p o r t i n the blood and hence a e r o b i c swimming c a p a c i t y . Furthermore, f i s h i n the w i l d must swim a c t i v e l y i n a v a r i e t y of normal behaviours ( f e e d i n g , a v o i d i n g p r e d a t i o n , m i g r a t i o n , spawning e t c . ) ; i f swimming a b i l i t y i s impaired i n a c i d or a l k a l i n e water, the s u r v i v a l of the f i s h p o p u l a t i o n may be i n jeopardy. 3 The purpose of the f i r s t experiment was to f i n d out the e f f e c t of water pH on swimming performance i n rainbow t r o u t (Salmo g a i r d n e r i ) . I f swimming a b i l i t y i s impaired i n a c i d or a l k a l i n e water, what i s the reason f o r t h i s decrease i n the maximum c r i t i c a l v e l o c i t y i n a c i d or a l k a l i n e water? Recently i t has been r e p o r t e d t h a t a d r e n a l i n e ( A d ) can a b o l i s h the Bohr e f f e c t i n t r o u t red c e l l s i n v i t r o d u r i n g an e x t r a c e l l u l a r a c i d o s i s by r e g u l a t i n g e r y t h r o c y t i c pH (Nikinmaa 1983). I t has a l s o been shown t h a t oxygen t r a n s p o r t i s not i n h i b i t e d by e x e r c i s e - i n d u c e d e x t r a c e l l u l a r a c i d o s i s i n t r o u t (Primmett et a l , 1986). The maintenance of blood oxygen content i s a s s o c i a t e d with a maintenance of red c e l l pH and s i g n i f i c a n t i n c r e a s e i n plasma catecholamine l e v e l s . I t i s proposed t h a t a d r e n e r g i c r e g u l a t i o n of red c e l l pH allows normal Hb-02 c a r r i a g e r e g a r d l e s s of the plasma a c i d o s i s . Catecholamines are known to be r e l e a s e d i n t o the c i r c u l a t i o n of f i s h i n response to s t r e s s (Mazeaud & Mazeaud, 1981). A c i d water can be c o n s i d e r e d one kind o f s t r e s s . So i t would be i n t e r e s t i n g to know whether catecholamines are a l s o r e l e a s e d i n t o bloodstream of t r o u t d u r i n g a c i d exposure to r e g u l a t e red c e l l pH and allowed normal Hb-02 c a r r i a g e r e g a r d l e s s of the plasma a c i d o s i s . The second experiment was set up i n order to measure blood pH, red c e l l pH, i o n c o n c e n t r a t i o n s and catecholamine l e v e l s i n plasma, and g i l l p o t e n t i a l i n rainbow t r o u t (Salmo  g a i r d n e r i ) i n a c i d or a l k a l i n e water. 4 M a t e r i a l s and Methods Experimental animals Rainbow t r o u t (Salmo g a i r d n e r i Richardson), weight, 200-300g. le n g t h 20-25cm, of both sexes, were obtained from a commercial hatchery and were kept outdoors i n l a r g e f i b r e g l a s s tanks s u p p l i e d with f l o w i n g d e c h l o r i n a t e d Vancouver tapwater (very s o f t , C a + + 70-150uEq/L). F i s h were fed ad l i b i t u m from automatic feeders c o n t a i n i n g d r i e d t r o u t p e l l e t s . In the l a b o r a t o r y , the f i s h were kept i n blackened a q u a r i a at about 10°C and feeding was suspended 3 days p r i o r to experimentation. Experiment I: Swimming vs water pH In order to measure the maximum c r i t i c a l v e l o c i t y (see Hoar and R a n d a l l , 1978), a B r e t t - t y p e respirometer was used. The water temperature i n the experiment was 16-17°C. The f i s h were e x e r c i s e d i n the respirometer before each experiment i n order to a c c l i m a t e the f i s h to the r e s p i r o m e t e r . A f t e r r e c o v e r y , the f i s h were a c c l i m a t e d i n a h o l d i n g chamber for 14hr before being t r a n s f e r r e d to the r e s p i r o m e t e r , i n aerated water of a known pH. The water from the h o l d i n g chamber was r e c i r c u l a t e d through a b i g bucket 5 and the re s p i r o m e t e r , by using a small water pump. The water pH was reduced or i n c r e a s e d to a giv e n l e v e l (pH4, 5, 6, 7, 9 & 10) f o r experiments by adding concentrated h y d r o c h l o r i c a c i d or sodium hydroxide, and maintained by using a sage s y r i n g e pump and a pH c o n t r o l l e r . At the end of each experiment, the water i n the b i g bucket was r e p l a c e d by f r e s h water. The f i s h were measured f o r fork l e n g t h , h e i g h t , and width, then t r a n s f e r r e d to the re s p i r o m e t e r . A f t e r lOhr recovery, the maximal c r i t i c a l v e l o c i t y of the f i s h exposed to a c e r t a i n experimental pH was measured by using increments of 0.5 body lengths per second at 0.5 hr i n t e r v a l s u n t i l the f i s h was exhausted. For p r e c i s e measurement of swimming speed, i t i s necessary to c o r r e c t a l s o f o r the e f f e c t of the f i s h body on water v e l o c i t y , the f i s h causes a narrowing of the a v a i l a b l e water channel, r e s u l t i n g i n an a c c e l e r a t i o n of flow over the body, This e r r o r can be c o r r e c t e d using the equation g i v e n i n Smit et a l . (1971) : Uc=Us(l+Ai/Aii) where, Uc, c o r r e c t e d v e l o c i t y ; Us, v e l o c i t y i n the absence of a f i s h ; A i i , c r o s s - s e c t i o n a l area of the swimming chamber; A i , c r o s s - s e c t i o n a l area of the f i s h , which i s assumed to appromimate an e l l i p s e and thus equal 3.14/0.5d/0.5w, where d 6 and w r e p r e s e n t the maximum body he i g h t and width, r e s p e c t i v e l y (see Hoar and R a n d a l l , 1978). At pH4.0 and pH7.0, the b r e a t h i n g and cough frequency of f i s h were recorded before e x e r c i s e . Experiment I I : blood samples vs water pH The second experiment was s e t up to measure blood and e r y t h r o c y t i c pH, sodium and c h l o r i d e c o n c e n t r a t i o n s i n plasma and i n water, catecholamine c o n c e n t r a t i o n s i n plasma, and g i l l t r a n s e p i t h e l i a l p o t e n t i a l i n a c i d (pH 4.0) or a l k a l i n e (pHlO.O) c o n d i t i o n s . The trout(250-400g) were a n a e s t h e t i s e d with MS222 (1:10000, pH7.5) and implanted with c h r o n i c d o r s a l a o r t i c cannulae (PE50 p o l y e t h y l e n e tubing) i n a manner s i m i l a r to Smith and B e l l (1964). Subsequent to the c a n n u l a t i o n procedure, the f i s h were allowed to recover f o r 2 days i n an i n d i v i d u a l chamber. The water from the i n d i v i d u a l chamber was r e c i r c u l a t e d through a b i g tank (-120L) us i n g a small water pump. The water i n the b i g tank was a e r a t i n g and was c o n t i n u o u s l y r e p l a c e d by tapwater. The water temperature was 10+.1°C. The water pH was a d j u s t e d u s i n g c o n c e n t r a t e d h y d r o c h l o r i c a c i d or sodium hydroxide and maintained at a c o n s t a n t l e v e l by u s i n g a p e r i s t a l t i c pump and a pH c o n t r o l l e r . At each sampling time, 600ul of blood was removed from the f i s h . T h i s blood was r e p l a c e d with c o r t l a n d s a l i n e . The whole blood pH (pHe) was measured u s i n g a Radiometer 7 PHM-71 acid-base a n a l y z e r and a s s o c i a t e d micro-pH e l e c t r o d e s . The remaining whole blood was then c e n t r i f u g e d . l O u l of plasma was mixed with 1ml d e i o n i z e d water for l a t e r Na+ measurement us i n g a Perkin-Elmer atomic a b s o r p t i o n spectrophotometer. 20ul of plasma was used to measure C l ~ ion c o n c e n t r a t i o n using a CMT10 c h l o r i d e t i t r a t o r . The remaining plasma (350-500ul) was taken up and t r a n s f e r r e d to an Eppendorf v i a l f o r sto'rage at -70°C, subsequently analyzed f o r catecholamines content ( n o r a d r e n a l i n e [Nor.], and a d r e n a l i n e [Ad.]), using high pressure l i q u i d chromatoqraphy (see Woodward 1982). E r y t h r o c y t e pH was measured u s i n g the quick f r e e z i n g method of Z e i d l e r & Kim (1977). At the same time, a water sample was taken, Na+ and Ca++ c o n c e n t r a t i o n s i n water were measured using a Perkin-Elmer atomic a b s o r p t i o n spectrophotometer. Cl~ c o n c e n t r a t i o n i n water was measured using a Buchler Cotlove c h l o r idometer. The g i l l p o t e n t i a l s (TEP) were recorded on a 602 s o l i d s t a t e e l e c t r o m e t e r , two e l e c t r o d e s were connected to the e x t e r n a l bath and the cannula v i a 3M KCl-agar b r i d g e s . A t h i r d KCl-agar b r i d g e c o n n e c t i n g the two e l e c t r o d e s provided a r e f e r e n c e zero p o t e n t i a l . H +' Na + and CI~ d r i v i n g f o r c e were c a l c u l a t e d by F=TEP-E, E=(RT/nf)*LN(Ci/Co), where F, d r i v i n g f o r c e ; C i i s blood and Co i s water c o n c e n t r a t i o n . A l l data from the above procedures are g i v e n as a r i t h m e t i c means+ S.E.. The s i g n i f i c a n c e of value changes from the c o n t r o l c o n d i t i o n ( * ) (P<0.05) was determined using the p a i r e d (or unpaired as a p p r o p r i a t e ) student's t - T e s t . 8 R e s u l t s Experiment I: Swimming vs water pH The e f f e c t s o f d i f f e r e n t water pH on the maximum c r i t i c a l v e l o c i t y of rainbow t r o u t are shown i n Table' 1 and Fi g u r e 1. At water pH 7.0, the maximum c r i t i c a l v e l o c i t y was 4.11+0.26 BL/second. At water pH 6.0 and pH 9.0, the maximum c r i t i c a l v e l o c i t i e s were 3.63+0.26 BL/second and 3.92+0.26 BL/second, r e s p e c t i v e l y , the r e d u c t i o n of the maximum swimming speeds were not s i g n i f i c a n t compared with the normal water group. The maximum c r i t i c a l v e l o c i t i e s were, however, 2.24+0.24 BL/second, 2.73+0.30 BL/second and 2.50+0.18 BL/second at water pH4.0, 5.0, and 10.0, r e s p e c t i v e l y , that was only 54.5%, 66.5% and 61% r e s p e c t i v e l y of that recorded f o r f i s h i n water of pH 7.0. The r e d u c t i o n i n swimming speed was, i n t h i s case, s i g n i f i c a n t l y d i f f e r e n t (P<0.05) from that of pH 7.0. Exposure to a c i d c o n d i t i o n s (pH4.0) i n c r e a s e d coughing and b r e a t h i n g frequency i n r e s t i n g f i s h . Coughing r a t e i n c r e a s e d from 41/hr to 592/hr, and b r e a t h i n g r a t e i n c r e a s e d from 81/min to 104/min when f i s h was t r a n s f e r r e d from water at pH7.0 to water at pH4.0 (Table 2). 9 Table 1. E f f e c t of d i f f e r e n t water pH on the maximum c r i t i c a l v e l o c i t y (MCV) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t (P<0.05) from c o n t r o l (pH 7.0) water pH 4.0 5.0 6.0 7.0 9.0 10.0 MCV (BL/S) 2.24* 2.73* 3.63 4.11 3.92 '2.50* S.E. +0.24 +0.30 +0.26 +0.26 +0.26 +0.18 n 9 7 8 8 8 6 10 Table 2. E f f e c t of a c i d water(pH4.0) on the b r e a t h i n g . and coughing r a t e o f rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . (P<0.05) water pH 7.0 4.0 cough r a t e 40.56 589.56* (times/hour) S.E. +14.16 +140.4 n 8 9 brea t h F. 80.5 104.4* (times/min) S.E. +6.1 +8.8 n 8 9 11 F i g u r e 1. E f f e c t of d i f f e r e n t water pH on the maximum c r i t i c a l v e l o c i t y of rainbow trout(Salmo g a i r d n e r i ) . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P<0.05) I 8.0 I 9.0 i 10.0 I 7.0 Water PH 13 Experiment I I : blood samples vs water pH Table 3 shows the blood pH, red c e l l pH, g i l l p o t e n t i a l , sodium, c h l o r i d e c o n c e n t r a t i o n s and catecholamines ( n o r a d r e n a l i n e [Nor.] & a d r e n a l i n e [Ad.]) c o n c e n t r a t i o n s i n plasma, and H +, Na +, C l ~ d r i v i n g f o r c e i n c o n t r o l water (pH6.4) i n rainbow t r o u t . E f f e c t of a c i d water (pH 4.0) The e f f e c t s of a c i d water on blood pH, g i l l p o t e n t i a l , Na + ' C l ~ i o n c o n c e n t r a t i o n s , and catecholamines ( n o r a d r e n a l i n e [Nor.] & a d r e n a l i n e [Ad.]) c o n c e n t r a t i o n s i n plasma and H +' Na +, and C l ~ d r i v i n g f o r c e s are shown i n Table 4. There was a s m a l l , but s i g n i f i c a n t d e c l i n e i n blood pH from 7.937+0.02 i n c o n t r o l water to 7.909+0.02 a f t e r 8hr a c i d exposure, but l a r g e s i g n i f i c a n t d e c l i n e i n blood pH to 7.759+0.03 a f t e r 24hr a c i d exposure ( f i g u r e 2). In most cases e r y t h r o c y t i c pH was u n a f f e c t e d except t h a t there was a s i g n i f i c a n t decrease i n e r y t h r o c y t i c pH from 7.367+0.01 i n c o n t r o l water to 7.325+0.02 a f t e r 24hrs exposure to a c i d c o n d i t i o n ( f i g u r e 3). The e f f e c t of a c i d water on g i l l p o t e n t i a l i s shown i n f i g u r e 4. Whenever the water pH was decreased to pH 4.0, the g i l l p o t e n t i a l s h i f t e d from negative (-6.6+1.1 mV) to 14 p o s i t i v e (15.5+1.0 mV a f t e r 2hr a c i d exposure) and remained steady d u r i n g a c i d exposure. The e f f e c t o f a c i d water on Na+ c o n c e n t r a t i o n i n plasma i s shown i n f i g u r e 5. Na + c o n c e n t r a t i o n i n plasma d i d not change w i t h i n 8 hr, but decreased s i g n i f i c a n t l y from 135.7+7.0 mEq/L i n c o n t r o l water to 120.1+2.6 mEq/L a f t e r 24hr a c i d exposure. The e f f e c t o f a c i d water on C l ~ c o n c e n t r a t i o n i n plasma i s shown i n f i g u r e 6. As observed f o r Na + i o n , C l ~ c o n c e n t r a t i o n i n plasma d i d not change w i t h i n 8 hr, but decreased s i g n i f i c a n t l y from 117+6.1 mEq/L i n c o n t r o l water to 98+2.8 mEq/L a f t e r 24 hr a c i d exposure. The a c i d water d i d have e f f e c t on catecholamine c o n c e n t r a t i o n s i n plasma (Table 4). Noradrenaline (Nor.) l e v e l s i n c r e a s e d i r r e g u l a r l y d u r i n g a c i d exposure. The maximum n o r a d r e n a l i n e l e v e l appeared a t d i f f e r e n t exposure times i n each f i s h ( f i g u r e 8). F i g u r e 9 shows the n o r a d r e n a l i n e c o n c e n t r a t i o n i n plasma i n f i s h i n c o n t r o l water. Although the mean value o f n o r a d r e n a l i n e a t each sampling time was i n s i g n i f i c a n t l y higher than the zero c o n t r o l value ( f i g u r e 7), the maximum n o r a d r e n a l i n e l e v e l s i n each f i s h were s i g n i f i c a n t l y higher than the zero c o n t r o l value (from 3.5+0.5 10-9 M/L i n c o n t r o l water to 38.07+7.07 10-9 M/L i n maximum va l u e s , f i g u r e 10). As observed f o r n o r a d r e n a l i n e . A d r e n a l i n e (Ad.) l e v e l s i n c r e a s e d i r r e g u l a r l y and were a s s o c i a t e d with changes i n n o r a d r e n a l i n e l e v e l s d u r i n g a c i d exposure. The maximum 15 a d r e n a l i n e l e v e l appeared at d i f f e r e n t exposure times i n d i f f e r e n t f i s h ( f i g u r e 12). F i g u r e 13 shows the a d r e n a l i n e c o n c e n t r a t i o n i n plasma i n d i f f e r e n t f i s h i n c o n t r o l water. Although the mean value o f a d r e n a l i n e at each sample time was i n s i g n i f i c a n t l y higher than the zero c o n t r o l value ( f i g u r e 11), the maximum a d r e n a l i n e l e v e l s i n d i f f e r e n t f i s h at d i f f e r e n t sampling times were s i g n i f i c a n t l y higher than the zero c o n t r o l values (from 0.5+0.07 1 0 - 9 M ' L i n c o n t r o l water to 18.41+3.88 1 0 - 9 M/L i n maximum va l u e s , f i g u r e 10). The e f f e c t o f a c i d water on the e l e c t r o c h e m i c a l g r a d i e n t f o r H + i s shown i n f i g u r e 17. The H + i n f l u x d r i v i n g f o r c e i n c r e a s e d s i g n i f i c a n t l y i n a c i d water from 73.8+3.34 mV i n c o n t r o l water to 203.5+1.14 mV a f t e r 2hr a c i d exposure. The e f f e c t of a c i d water on the Na + d r i v i n g f o r c e i s shown i n f i g u r e 18. The Na + e f f l u x d r i v i n g f o r c e showed only a s m a l l , but s i g n i f i c a n t i n c r e a s e from 204.0+1.5 mV i n c o n t r o l water to 210.0+2.8 mV a f t e r 8hr a c i d exposure, and decreased ag a i n to almost normal (206.8+1.5 mV) a f t e r 24hr a c i d exposure. The e f f e c t o f a c i d water on the C l ~ d r i v i n g f o r c e i s shown i n f i g u r e 19. The C l ~ e f f l u x d r i v i n g f o r c e decreased s i g n i f i c a n t l y a f t e r a c i d exposure, from 223.1+.2.6 mV i n c o n t r o l water to 137.3+.2.2 mV a f t e r 2hr a c i d exposure. 16 E f f e c t s of a l k a l i n e water (pHlO.O) o E f f e c t s of a l k a l i n e water on blood pH, red c e l l pH, g i l l p o t e n t i a l , Na + ' d ~ c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e [Nor.] & a d r e n a l i n e [Ad.]) c o n c e n t r a t i o n s i n plasma, and H +' Na + and C l ~ d r i v i n g f o r c e s were shown i n Table 6. The e f f e c t of a l k a l i n e water on blood pH i s shown i n f i g u r e 20. Blood pH i n c r e a s e d s i g n i f i c a n t l y w i t h i n 2hr of a l k a l i n e exposure, being 7.863+0.01 i n c o n t r o l water and 8.15+0.05 a f t e r 2hr of a l k a l i n e exposure. The e f f e c t of a l k a l i n e water on red c e l l pH i s shown i n f i g u r e 21. Red c e l l pH a l s o i n c r e a s e d s i g n i f i c a n t l y a f t e r 2hr of a l k a l i n e exposure, from 7.304+0.02 i n c o n t r o l water to 7.48+0.03 a f t e r 2hr of a l k a l i n e exposure. The e f f e c t of a l k a l i n e water on g i l l p o t e n t i a l i s shown i n f i g u r e 22. G i l l p o t e n t i a l decreased s i g n i f i c a n t l y from -9.5 + 1.4 mV i n c o n t r o l . water to -22.7 + 2.3' mV a f t e r 2 hr of a l k a l i n e exposure, and remained steady dur i n g the exposure p e r i o d . The e f f e c t of a l k a l i n e water on Na + c o n c e n t r a t i o n i n plasma i s shown i n f i g u r e 23. Na + c o n c e n t r a t i o n i n plasma showed a s m a l l , slow and gradual i n c r e a s e d u r i n g a l k a l i n e exposure, from 142.0+3.58 mEq/L i n c o n t r o l water, Na + l e v e l s i n c r e a s e d s i g n i f i c a n t l y to 155.1+3.7 mEq/L a f t e r 72h a l k a l i n e exposure. 17 The e f f e c t o f a l k a l i n e water on C l ~ c o n c e n t r a t i o n i n plasma was shown i n f i g u r e 24. C h l o r i d e c o n c e n t r a t i o n i n plasma, l i k e Na +, a l s o showed a s m a l l , slow and gradual o i n c r e a s e d u r i n g a l k a l i n e exposure, from 110.6+2.40 mEq/L i n c o n t r o l water, C l ~ l e v e l s i n c r e a s e d s i g n i f i c a n t l y to 128.0+.5.5 mEq/L a f t e r 72hr a l k a l i n e exposure. The e f f e c t of a l k a l i n e water on catecholamine c o n c e n t r a t i o n s i n plasma i s shown i n f i g u r e 25. Noradrenaline l e v e l s d i d not change d u r i n g a l k a l i n e exposure. A d r e n a l i n e l e v e l s were too low to measure duri n g the exposure p e r i o d . The e f f e c t of a l k a l i n e water on the H + d r i v i n g f o r c e i s shown i n f i g u r e 26. The H + d r i v i n g f o r c e s h i f t e d from negative i n s i d e the f i s h , to p o s i t i v e d u r i n g a l k a l i n e exposure, from -98.58 + 1.50 mV i n c o n t r o l water to 81.17+_3.35 mV a f t e r 2hr of a l k a l i n e exposure. The e f f e c t of a l k a l i n e water on the Na + d r i v i n g f o r c e i s shown i n f i g u r e 27. The Na + d r i v i n g f o r c e decreased s i g n i f i c a n t l y d u r i n g a l k a l i n e exposure, from 202.4+.1.10 mV i n c o n t r o l water to 121.9+2.1 mV a f t e r 2hr of a l k a l i n e exposure. The e f f e c t o f a l k a l i n e water on the C I - d r i v i n g f o r c e i s shown i n f i g u r e 28. The C l ~ e f f l u x d r i v i n g f o r c e i n c r e a s e d s i g n i f i c a n t l y a f t e r 2hr a l k a l i n e exposure, from 220.6 + 1.9mV i n c o n t r o l water to 233.8+_3.97 mV a f t e r 2hr a l k a l i n e exposure. 18 Table 3. Blood pH(pHe), red c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium, c h l o r i d e c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e [ N o r . ] & a d r e n a l i n e [ A d . ] ) c o n c e n t r a t i o n s i n plasma and H +, Na +, C l ~ d r i v i n g f o r c e i n c o n t r o l water(pH6.4) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . (P<0.05) Time(hrs) 0 2 4 8 24 48 72 pHe 7.913 7. 941 8. 004 7. 935 7. 894 7.925 7.910 S.E. +0.02 +.0.0 2 + 0 .03 + 0.02 + 0.02 +0 .03 + 0 .02 n 16 15 5 15 13 15 15 pHi 7. 389 7. 416 7. 413 7.406 7. 376 7. 388 7. 385 S.E. +0.01 +0.01 +0 .01 + 0.01 +0.01 +0.02 +.0.0 2 n 16 15 5 15 13 15 15 TEP (mV) -6.2 -5. 3 -0.9 -3.6 10.0* -4.7 S.E. + 1. 8 +.2.0 + 2.1 + 2.7 + 3.6 + 1.9 n Na + -p mEq/L n 146. 6 + 4.7 7 144 . 9 +4 . 3 7 149. + 3, 147 + 4 146. 8 +5.0 146. 6 +6.7 6 C l ~ - p mEq/L n 117. 4 ±3.5 7 115. 1 +4 . 1 7 117. 6 +4 . 0 7 121. 4 + 4.2 7 120 +4 117.0 +7.8 6 [Nor.] 2. 987 5. 318 10 _ 9M/L + 0 . 58 + 1. 22 n 14 12 [Ad.] 0 . 489 0 . 373 10"9M/L +0 . 09 + 0 . 07 n 14 12 F-H+mV -69.83 -89.76 S.E. +10.12 + 3.02 n 15 15 F-Na+mV 209. 0 202. 1 S.E. + 0 . 3 +0 . 5 n 7 7 F-Cl -mV -218.8 -219.0 S.E. + 8.5 +5.1 n 7 7 2. 578 + 1. 24 5 0 . 306 +0. 07 5 5.069 3. 368 6.902 5.097 + 1. 32 ±0 .74 +.1.63 + 1 . 46 13 12 12 12 0. 353 0. 335 0. 318 0. 361 + 0.53 ±0 . 06 + 0 . 05 + 0 . 10 12 11 12 12 87. 32 -84 . 92 -81.50 -88.94 + 3. 76 15 204. 3 + 2.5 7 -219.8 + 2.9 7 + 3. 58 13 208. 8 +7.9 7 +4.52 15 219. 1 + 3.6 7 -233.2 -215.7 +0.6 +4.4 7 7 + 3. 29 15 201. 1 + 3.0 6 -229. 0 + 3.2 ' 6 19 Table 4. E f f e c t o f a c i d water(pH4.0) on blood pH(pHe), r e d c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium, c h l o r i d e c o n c e n t r a t i o n s and catecholamine ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) c o n c e n t r a t i o n s i n plasma and H +, Na +, C l ~ d r i v i n g f o r c e i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero control(P<0.05). Time(hrs) 0 2 4 8 24 48 72 pHe 7. 937 7.901* 7. 958 7.909* 7.759* 7.669*- 7.601* S.E. + 0.02 +0.02 +0 .03 +0.02 +0.03 +0.06 + 0. 10 n 29 26 9 16 20 12 4 pHi 7. 367 7. 381 7. 433* 7.331 7.325* 7. 341 7. 327 S.E. + 0 .01 +0 .01 +0 .02 + 0 .02 +0 .02 +0.0 2 +0.01 n 29 26 9 16 20 9 4 TEP (mV) -6.6 15. 5* 14. 6* 13.6* 12.9* 13.0* 8.9* S.E. + 1. 1 + 1.0 + 1.9 + 1.5 + 1.3 + 2. 3 + 2.5 n 29 26 9 16 20 9 4 Na + -p 135.7 143. 5 139. 5 120.1* 105.5* 109. 1 mEq/L + 7.0 + 2.0 + 2.8 + 2.6 + 3.8 + 3.4 n 21 17 8 14 6 2 C l ~ - p 117. 0 117. 0 117. 0 98. 0* 83.0* 76. 0 mEq/L + 3.5 +4. 1 + 4 . 0 +4. 2 +4 . 6 +7.8 n 21 17 8 14 6 2 [Nor. ] 3. 509 10 . 38 2. 830 13.09 9. 108 12. 10 26. 90 10" 9M/L + 0 . 49 + 4.42 + 1 .03 + 6.58 + 1.90 + 3.73 + 8.82 n 25 21 6 13 16 9 4 [Ad. ] 0.497 2. 026 0 . 963 3. 473 2. 148 6. 681 23. 30 10-9M/L + 0 . 07 + 1. 15 ±o . 74 + 2.01 + 0 . 75 + 2.56 + 11. 6 n 23 21 6 13 17 9 4 F-H+mV -73.87 -203.5* -205.9* -198.2* -196.2* -193.3* S.E. + 3. 34 + 1. 14 + 1.74 + 2. 10 + 3.33 + 3.7 n 15 15 15 13 15 15 F-Na+mV 204. 0 216.1* 210.0* 206. 8 204. 2 205. 0 S.E. + 1.5 + 1.5 + 2 . 8 + 1.5 + 2.6 + 3.9 n 21 17 8 14 ~~ 6 2 F-Cl~mV -223.1 -137.3* _ 137.9* -138.0* -146.5* -143.6 S.E. + 2.6 + 2. 2 + 2.5 + 2.4 + 1.7 + 3.4 n 21 17 8 14 6 2 20 Table 5." The whole blood pH, red c e l l pH, catecholamine c o n c e n t r a t i o n s ( n o r a d r e n a l i n e t N o r . ] & a d r e n a l i n e [ A d . ] ) i n plasma, g i l l p o t e n t i a l ( T E P ) , and Na +, C l ~ c o n c e n t r a t i o n s i n plasma i n rainbow t r o u t , compared the data i n zero c o n t r o l and the data i n maximum catecholamine l e v e l s . * = s i g n i f i c a n t l y d i f f e r e n t from zero control(P<0.05). pHe pHi [Nor.] [Ad.] TEP [Na +] -.{CI "3 10~ 9M/L 10 - 9M/L mV mEq/L mEq/L C o n t r o l 7.921 7.384 4.132 0.455 -5.7 126.3 145.3 S.E. +0.0 2 +0.0 2 +0.79 +0.0 8 +2.1 +3.5 +4.0 n 13 13 13 12 13 10 10 Max.Cate 7.644* 7.368 S.E. +0.06 +0.02 n 13 13 38.07* 18.41* 9.79* +7.07 +3.88 +1.96 13 13 12 90.8* +5. 3 11 120.0* + 5.9 11 21 Table 6. E f f e c t of a l k a l i n e water(pHIO.0) on Blood pH(pHe), red c e l l pH(pHi), g i l l p o t e n t i a l ( T E P ) , sodium, c h l o r i d e c o n c e n t r a t i o n s and catecholamine l e v e l s i n 0 plasma and H +, Na +, C l ~ d r i v i n g f o r c e i n c o n t r o l water(pH6.4) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Time(hrs) 0 2 pHe 7.863 8.150* S.E. ±0.01 ±0.05 n 10 7 pHi 7.304 7.480* S.E. ±0.02 ±0.03 n 10 7 TEP (mV) -9.5 -22.7* S.E. ±1.4 ±2.3 n 10 7 Na + -p 142.0 139.4 S.E. ±3.58 ±4.7 n 10 7 C l _ - p 110.6 108.0 S.E. ±2.40 ±4.6 n 10 7 [Nor.3 2.21 5.05 10 _ 9M/L ±0.42 ±0.98 n 9 6 [Ad.] N.S N.S. 10 - 9M/L F-H+mV -98.58 81.17* S.E. ±1.50 ±3.35 n 10 7 F-Na+mV 202.4 121.9* S.E. ±1.10 ±2.5 n 10 7 F-Cl-mV -220.6 -233.8* S.E. ±1.90 ±3.97 n 10 7 24 48 72 8.350* 8.240* 8.230* ±0.04 ±0.05 ±0.06 7 7 6 7.470* 7.410* 7.400 ±0.04 ±0.0 2 ±0.01 7 7 6 -20.4* -18.6* -15.8* ±1.9 ±1.6 ±2.2 7 7 6 144.6 146.1 155.1* ±3.5 ±3.1 ±3.7 7 7 6 111.6 116.6 128.0* ±4.8 ±5.5 ±5.5 7 7 6 2.64 3.84 3.50 ±0.58 ±0.67 ±0.58 7 6 6 N.S. N.S. N.S. 72.22* 80.36* 83.91* ±2.05 ±2.48 ±3.00 7 7 6 122.9* 125.6* 127.8* ±1.9 ±1.7 ±2.0 7 7 6 -242.6* -248.5* -248.0* ±2.52 ±2.92 ±4.4 7 7 6 22 F i g u r e 2. E f f e c t of a c i d water (pH4.0) on blood pH (pHe) i n rainbow t r o u t (salmo g a i r d n e r i ) . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). ) © H d 24 F i g u r e 3. E f f e c t of a c i d water (pH4.0) on red c e l l pH (pHi) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). ) 26 F i g u r e 4. E f f e c t of a c i d water (pH4.0) on g i l l p o t e n t i a l (TEP) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). ALU (|DftUO}Od | D U » l f t l d 9 8 U D J ) ) d 3 1 28 F i g u r e 5. E f f e c t o f a c i d water (pH4.0) on sodium c o n c e n t r a t i o n i n plasma (Na +-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). Time of exposure (hour) 30 F i g u r e 6. E f f e c t o f a c i d water (pH4.0) on c h l o r i d e c o n c e n t r a t i o n i n plasma (Cl~-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05) Time of exposure (hour) 32 F i g u r e 7. E f f e c t of a c i d water (pH4.0) on n o r a d r e n a l i n e c o n c e n t r a t i o n [ N o r . ] i n plasma ( 1 0 ~ 9 M/L)in rainbow t r o u t , (the mean value of each sampling time vs c o n t r o l group) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). 28 0 2 4 8 24 48 72 Time of exposure (hour) 34 F i g u r e 8. E f f e c t of a c i d water (pH4.0) on n o r a d r e n a l i n e c o n c e n t r a t i o n [Nor.] i n plasma (10~ 9 M/L)in rainbow t r o u t , (the value of d i f f e r e n t f i s h i n each sampling time vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). Time of exposure (Hour) 36 F i g u r e 9. Noradrenaline c o n c e n t r a t i o n [Nor.] i n plasma ( 1 0 - y M/L) i n rainbow t r o u t i n c o n t r o l water (pH6.4). (the value of d i f f e r e n t f i s h i n each sampling time) ) 38 F i g u r e 10. E f f e c t of a c i d water (pH4.0) on n o r a d r e n a l i n e [Nor.] and a d r e n a l i n e [ A d . ] c o n c e n t r a t i o n i n plasma (1 0 ~ 9 M/L)in rainbow t r o u t , (the maximum values i n d i f f e r e n t f i s h vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). ) J \ 3 0) I o r-0 E 0 o a 3 4 L. 0 z 0.0: Control, 1.0: Cate. Max. 40 F i g u r e 11. E f f e c t of a c i d water (pH4.0) on a d r e n a l i n e c o n c e n t r a t i o n [Ad.] i n plasma ( 1 0 - 9 M/L)in rainbow t r o u t , (the mean value of each sampling time vs c o n t r o l group) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). 24 0 2 4 8 24 48 72 Time of exposure (hour) F i g u r e 12. E f f e c t of a c i d water (pH4.0) on a d r e n a l i n e c o n c e n t r a t i o n [Ad.3 i n plasma (1 0 ~ 9 M/L)in rainbow t r o u t , (the value of d i f f e r e n t f i s h i n each sampling time vs zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). 43 V W 6-01 D O i « D | d u | [-pv] 44 F i g u r e 13. A d r e n a l i n e c o n c e n t r a t i o n f A d . ] i n plasma (10~ y M/L) i n rainbow t r o u t i n c o n t r o l water (pH6.4). (the value of d i f f e r e n t f i s h i n each sampling time) Time of exposure (hour) 46 F i g u r e 14. E f f e c t of a c i d water (pH4.0) on the whole blood pH (pHe) and red c e l l pH (pHi) i n rainbow t r o u t , (at the maximum catecholamine v a l u e s i n d i f f e r e n t f i s h vs at zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05) . 0.0: Control, 1.0: Cote. Max. 48 F i g u r e 15. E f f e c t of a c i d water (pH4.0) on g i l l p o t e n t i a l (TEP mV)in rainbow t r o u t . (at the maximum catecholamine values i n d i f f e r e n t f i s h vs a t zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). Gtll potential (TEP) mV. 50 F i g u r e 16. E f f e c t of a c i d water (pH4.0) on Na + and C l ~ c o n c e n t r a t i o n s i n rainbow t r o u t . (at the maximum catecholamine values i n d i f f e r e n t f i s h vs a t zero c o n t r o l ) * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l (P<0.05). 0.0: Control, 1.0: Cote. Max. 52 F i g u r e 17. E f f e c t of a c i d water (pH4.0) on hydrogen d r i v i n g f o r c e (F-H +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Time of exposure(hour) 54 F i g u r e 18. E f f e c t of a c i d water (pH4.0) on sodium d r i v i n g f o r c e (F-Na +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . ) 250 240 Time of exposure (hour) 56 F i g u r e 19. E f f e c t of a c i d water (pH4.0) on the c h l o r i d e d r i v i n g f o r c e ( F - C l - ) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) Time of exposure (hour) 58 F i g u r e 20. E f f e c t of a l k a l i n e water (pHlO.O) on the whole blood pH (pHe) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) ) Time of exposure(hour) 60 F i g u r e 21. E f f e c t of a l k a l i n e water (pHlO.O) on red c e l l pH (pHi) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) ) 62 F i g u r e 22. E f f e c t of a l k a l i n e water (pHlO.O) on g i l l p o t e n t i a l (TEP) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) ) 63 64 F i g u r e 23. E f f e c t of a l k a l i n e water (pHlO.O) on sodium c o n c e n t r a t i o n i n plasma (Na-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) ) Time of exposure(hour) 66 F i g u r e 24. E f f e c t of a l k a l i n e water (pHlO.O) on c h l o r i d e c o n c e n t r a t i o n i n plasma (Cl-p) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) ) Time of exposure(hour) 68 F i g u r e 25. E f f e c t o f a l k a l i n e water (pHlO.O) on n o r a d r e n a l i n e c o n c e n t r a t i o n i n plasma [Nor.] i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . ) Time of exposure (hour) 70 F i g u r e 26. E f f e c t o f a l k a l i n e water (pHlO.O) on the hydrogen d r i v i n g f o r c e (F-H +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) Time of exposure(hour) 72 F i g u r e 27. E f f e c t of a l k a l i n e water (pHlO.O) on the sodium d r i v i n g f o r c e (F-Na +) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) AUJ + D N — d 74 F i g u r e 28. E f f e c t of a l k a l i n e water (pHlO.O) on the c h l o r i d e d r i v i n g f o r c e ( F - C l - ) i n rainbow t r o u t . * = s i g n i f i c a n t l y d i f f e r e n t from zero c o n t r o l . (P<0.05) Time of exposure(hour) 76 Discuss ion Swimming vs water pH The r e s u l t s i n the f i r s t experiment have demonstrated that both a c i d and a l k a l i n e waters reduce the a e r o b i c swimming c a p a c i t y of t r o u t . Waiwood and Beamish (1978) found: In the absence of copper, c r i t i c a l speed was not a p p r e c i a b l y i n f l u e n c e d by hardness, pH, or time of exposure. But the water pH they used was from pH 6.0 to 8.0. In my experiment , maximum c r i t i c a l speed was not a p p r e c i a b l y decreased i n water pH from 6.0 to 9.0, but out of t h i s range, the maximum c r i t i c a l v e l o c i t y decreased s i g n i f i c a n t l y . We conclude that i f the water pH i s l e s s than 6.0 or g r e a t e r than 9.0, s u r v i v a l of the f i s h may be i n jeopardy. I f we co n s i d e r that pH 7.0 water i s normal, then both pH 9.0 water and pH 5.0 water are two pH u n i t s o f f the pH 7.0 water. The e f f e c t s of these two waters, however, are q u i t e d i f f e r e n t . In the pH 9.0 group, the maximum c r i t i c a l v e l o c i t y decreased i n s i g n i f i c a n t l y , from 4.11+0.26 BL/S to 3.92+0.26 BL/S, whereas i n the pH 5.0 group, the maximum c r i t i c a l v e l o c i t y decreased s i g n i f i c a n t l y , from 4.11±0.26 BL/S to 2.73+0.3 BL/S. This means that the pH 5 water i s more t o x i c than the pH 9 water. The reason f o r t h i s maybe: 1) When f i s h e x e r c i s e , they produce a l a r g e amount of a c i d metabolic 77 products such as carbon d i o x i d e and l a c t i c a c i d . F i s h e x e r c i s e d i n a c i d water are more l i k e l y to generate an a c i d o t i c s t a t e , which would i n f l u e n c e O2 t r a n s p o r t v i a Root e f f e c t . 2) The t o x i c i t y o f H + and 0H~ maybe d i f f e r e n t . P h y s i o l o g i c a l mechanisms of OH - t o x i c i t y to f i s h i s not c l e a r yet. Graham and Wood (1980) measured the e f f e c t * o f low pH on f i n g e r l i n g rainbow t r o u t , weight: 3.50+0.09g, l e n g t h : 7.59+0.30cm, using s u l f u r i c a c i d a c i d i f i c a t i o n . They found t h a t , below pH 4.6 ( s o f t water) or 4.4 (hard water), c r i t i c a l v e l o c i t y d e c l i n e d l i n e a r l y by about 4% per 0.1 pH u n i t . In my experiment, the maximum c r i t i c a l v e l o c i t y decreased s i g n i f i c a n t l y below pH 5.0 water. The r e s u l t s were a l i t t l e d i f f e r e n t . T h i s i s most probably because of the f i s h s i z e , the a c i d type, and the water we used were d i f f e r e n t . In a d d i t i o n , the a c c l i m a t i o n time should be a l s o c o n s i d e r e d . In Graham and Wood's (1980) study, the f i s h were e x e r c i s e d as soon as they were exposed to the low pH water. In the study of X i q i n He e_t al_. ( 1984, personal communication), the f i s h were e x e r c i s e d as soon as they were exposed to low pH (pH4.0), and the r e d u c t i o n of maximal c r i t i c a l v e l o c i t y was l e s s than 20%. In my experiment, the r e d u c t i o n was 45.5%. So i t i s concluded that the degree of high pH or low pH, as w e l l as the a c c l i m a t i o n time i n such pH, a l s o i n f l u e n c e s the maximum c r i t i c a l v e l o c i t y of f i s h . I t i s l i k e l y t h a t the e x e r c i s e c a p a b i l i t i e s of f i s h 1 s u r v i v i n g i n low pH or high pH environments f o r longer p e r i o d s are reduced more than seen i n these experiments. 78 The maximum c r i t i c a l v e l o c i t y i s an index f o r f i s h swimming a b i l i t y . F i s h swimming a b i l i t y i s important f o r s u r v i v a l . F i s h i n the water must swim a c t i v e l y f o r f e e d i n g , a v o i d i n g p r e d a t i o n , m i g r a t i o n , spawning and other normal behaviours. A l l these behaviours would not be s u c c e s s f u l i f the maximal c r i t i c a l v e l o c i t y was decreased by the t o x i c i t y of a c i d or a l k a l i n e water. So i n a c i d water or a l k a l i n e water, f i s h p o p u l a t i o n s w i l l be decreased f o r t h i s , as well as other reasons. Why does the maximum c r i t i c a l swimming v e l o c i t y decrease i n a c i d water or a l k a l i n e water? An examination of changes i n the blood may i l l u m i d a t e t h i s problem. E f f e c t o f a c i d water The f i s h g i l l e p i t h e l i u m i s very permeable to hydrogen ions (McWilliams & P o t t s , 1978). In a c i d water, hydrogen i o n c o n c e n t r a t i o n i s much higher i n the water than i n f i s h blood. Hydrogen ions may d i f f u s e a c r o s s the g i l l membrane i n t o the f i s h body producing a p o s i t i v e s h i f t i n TEP. Any f a c t o r c a u s i n g a change i n the p o t e n t i a l between blood and water should a f f e c t the i o n movement acr o s s the g i l l . A n a l y s i s of the d r i v i n g f o r c e i n d i c a t e s that the H + i n f l u x d r i v i n g f o r c e i n c r e a s e d s i g n i f i c a n t l y i n a c i d water. The incoming H + ions would combine with b i c a r b o n a t e ions i n the blood to produce C02 and water: H + + HCO3- H20 + CO2. 79 A c c o r d i n g to Eddy (1976), l e s s than 20% of the incoming H + ions are b u f f e r e d by blood HCO3- i o n s , the r e s t being n e u t r a l i z e d by the i n t r a c e l l u l a r compartments. In t h i s case, we can expect that the blood pH c o u l d be kept n e a r l y constant f o r a p e r i o d o f time. I t i s i n t e r e s t i n g at t h i s p o i n t to note t h a t : In my experiment, 1) The decrease i n blood pH was small from 7.937+0.02 i n c o n t r o l water to 7.909+0.02 a f t e r " 8 h r a c i d exposure and was decreased to 7.759+0.03 onl y a f t e r 24hr of a c i d exposure ( f i g u r e 3). 2) Exposure of rainbow t r o u t to a c i d water r e s u l t s i n an i n c r e a s e i n v e n t i l a t i o n r a t e (from 80.5+6.1 breath/min i n c o n t r o l water to 104.4+8.8 breath/min a f t e r 24hr a c i d exposure), which w i l l a s s i s t i n the removal of excess CO2 from the blood r e s u l t i n g from a combination of H + and HC03_ ions (Table 2). Sodium and c h l o r i d e i o n c o n c e n t r a t i o n s i n plasma decreased s i g n i f i c a n t l y a f t e r 24hr a c i d exposure. These r e s u l t s c o n f i r m those r e p o r t e d by McDonald and Wood (1980). The reasons f o r the decrease i n Na + and C l ~ i o n c o n c e n t r a t i o n s i n plasma would be: 1) Water entered i n t o the bloodsteam from the i n t r a c e l l u l a r f l u i d or from the water environment. 2) Na + and C l ~ s h i f t e d from the plasma i n t o the i n t r a c e l l u l a r compartments or i n t o the water environment. 3) Na + and C I - uptake v i a the g i l l were i n h i b i t e d . I t has been suggusted t h a t osmotic water e n t r y a t the g i l l s decreased as plasma osmotic pressure f e l l with p r o g r e s s i v e i o n l o s s (Wood and McDonald, 1982). They a l s o found: 1) measurements of the v a r i o u s f l u i d compartments r e v e a l e d no change i n t o t a l 80 body water content, but a l a r g e s h i f t o f f l u i d from the e x t r a c e l l u l a r f l u i d volume i n t o the i n t r a c e l l u l a r f l u i d volume. I n t r a c e l l u l a r water content i n white muscle i n c r e a s e d s i g n i f i c a n t l y while i o n l e v e l s f e l l . Plasma volume d e c l i n e d i n p r o p o r t i o n to the o v e r a l l e x t r a c e l l u l a r f l u i d volume change. 2) Na + and C l ~ l o s s e s from the i n t r a c e l l u l a r compartment were almost as l a r g e as from the e x t r a c e l l u l a r f l u i d . -A n a l y s i n g the a c i d e f f e c t s on b r a n c h i a l and r e n a l i o n f l u x e s . Wood and McDonald (1982) found: The major route o f i o n l o s s i s c l e a r l y b r a n c h i a l and not r e n a l . The dominance of b r a n c h i a l route f o r i o n l o s s was even more pronounced i n s o f t water. There are two main p o s s i b l e reasons f o r i o n l o s s from the g i l l : 1) An i n h i b i t i o n of i o n i n f l u x ; 2) an inc r e a s e of i o n e f f l u x , or both changes may occur. As f o r Na + i o n , i t has been found t h a t severe a c i d exposure a b o l i s h e d Na + i n f l u x , while p a s s i v e Na + e f f l u x was i n c r e a s e d s u b s t a n t i a l l y (Packer & Dunson, 1970; McWilliams & P o t t s , 1978; Wright & Wood, 1985). A n a l y s i s of the d r i v i n g f o r c e i n d i c a t e d t h a t the Na + e f f l u x d r i v i n g f o r c e showed a small s i g n i f i c a n t i n c r e a s e o n l y w i t h i n 8hr, and was unchanged l a t e r ( f i g u r e 18). This means that i n c r e a s i n g p a s s i v e Na + e f f l u x must be due to i n c r e a s e the b r a n c h i a l p e r m e a b i l i t y i n a c i d water. I t seems that H + ions might d i s p l a c e c a l c i u m ions i n the g i l l and produce an o v e r a l l i n c r e a s e i n p e r m e a b i l i t y to a l l ions (Fromm 1980). There i s no d i r e c t evidences concerning the mechanism of i n h i b i t i o n o f Na + 81 i n f l u x by high [ H + ] . Fromm (1980) c o n s i d e r e d : i t i s p o s s i b l e that sodium uptake i s reduced i n a c i d water due to an inc r e a s e d load on the sodium pump mechanism i n which i f Na + i s exchanged f o r H +, the hydrogen ions must be e x p e l l e d a g a i n s t steep c o n c e n t r a t i o n g r a d i e n t s . The reduced uptake of Na + may be due to the i n c r e a s e d load on the c a r r i e r system as a consequence of the s h i f t i n TEP. One might a l s o speculae that a) H + ions i n t e r f e r e with Na + uptake d i r e c t l y by competing f o r c a r r i e r s i t e with Na + ; b) H + ions may a l t e r the p r o p e r t i e s o f the t r a n s p o r t mechanism; c) H + ions depress the metabolic r a t e of t r a n s p o r t i n g c e l l s . Less i s known of the e f f e c t s of low pH on g i l l C l _ f l u x e s . C I - e f f l u x d r i v i n g f o r c e decreased s i g n i f i c a n t l y from 223.1 + 2.6 mV i n c o n t r o l water to 137.3+_2.2 mV a f t e r 2hr a c i d exposure ( f i g u r e 19). This means that C I - d i f f u s i o n a l e f f l u x would be probably decreased d u r i n g a c i d exposure. However, under the c o n d i t i o n o f low water pH, the e x c r e t i o n of HCO3- i n exchange f o r C l ~ would exacerbate the acid-base d i s t u r b a n c e s . The a c t i v e C I " uptake would be reduced, shut down or even r e v e r s e d at a l r e a d y lowered plasma [HCO3-] i n f i s h exposed to a c i d water. In a d d i t i o n , the i n c r e a s i n g b r a n c h i a l p e r m e a b i l i t y would o f f s e t the d e c r e a s i n g C l ~ e f f l u x d r i v i n g f o r c e . So the drop i n plasma CC1~] c o u l d be e x p l a i n e d by C l ~ e f f l u x exceeding a c t i v e uptake, even there was a decrease i n C l ~ e f f l u x d r i v i n g f o r c e i n a c i d water. Packer and Dunson (1972) observed a decreased oxygen consumption i n brook t r o u t exposed to low environmental pH and 82 suggested t h a t the reduced r a t e of O2 uptake was r e l a t e d to mucus c o a g u l a t i o n on the g i l l s . Janssen and Randall (1975) observed a s i m i l a r mucus b u i l d up on the rainbow t r o u t g i l l s which c o u l d have i n t e r f e r e d with O2 t r a n s f e r . The i n c r e a s e i n the cough frequency i n t r o u t i n pH 4.0 water (Table 2) i s most probably because the mucus c o a g u l a t i o n on the g i l l s . B r a n c h i a l mucus s e c r e t i o n i s a common response i n f i s h exposed to a c i d water. Mucus may serve to p r o t e c t the f i s h by l i m i t i n g p a s s i v e b r a n c h i a l i o n l o s s . T h i s would be enhance f i s h s u r v i v a l . On the other hand, mucus may l i m i t O2 uptake, which w i l l r e s u l t i n an in c r e a s e of b r e a t h i n g and coughing frequency. The r e s u l t s of t h i s study show that catecholamine l e v e l s i n c r e a s e d i r r e g u l a r l y d u r i n g a c i d exposure. The maximum catecholamine l e v e l s seemed to appear only once i n each f i s h at a d i f f e r e n t exposure p e r i o d and were a s s o c i a t e d with the death of f i s h . The reasons f o r the m o b i l i z a t i o n of catecholamines i n t o the bloodstream are s t i l l not c l e a r . I t has been found t h a t acute e x t r a c e l l u l a r a c i d o s i s , whether of endogenous or exogenous o r i g i n , promotes the r e l e a s e of catecholamines i n rainbow t r o u t (Nakano and Tomlinson 1967; Primmett et a l 1985; B o u t i l i e r , Iwama & R a n d a l l , 1986). In t h i s study, the maximum catecholamine l e v e l s were a s s o c i a t e d with a decreased blood pH ( f i g u r e 14), a s h i f t i n the TEP from negative to p o s i t i v e ( f i g u r e 15), and a decrease i n Na + and C l ~ i o n c o n c e n t r a t i o n s i n plasma ( f i g u r e 16). I t i s unclear whether they are reasons f o r the m o b i l i z a t i o n of catecholamines i n t o the bloodstream. 83 G e n e r a l l y speaking, the i n c r e a s e d catecholamines i n the plasma w i l l enhance oxygen exchange a c r o s s the g i l l s and oxygen t r a n s p o r t to the t i s s u e s . Because: 1) Catecholamines have been shown to i n c r e a s e g i l l blood flow, and l a m e l l a r r e c r u i t m e n t (see R a n d a l l , 1982 f o r revi e w ) , and t h e r e f o r e l i k e l y p l a y s a r o l e i n the e l e v a t i o n o f oxygen uptake. 2) Catecholamines may i n c r e a s e a r t e r i a l blood pressure and heart r a t e . 3) Catecholamine causes the m o b i l i z a t i o n o f red c e l l s from the s p l e e n ( N i l s s o n and Grove, 1974). 4) Catecholamines r e a c t with b e t a - r e c e p t o r s on the red c e l l membranes and a c t i v a t e H + e f f l u x a l l o w i n g e r y t h r o c y t e pH r e g u l a t i o n d u r i n g e x t r a c e l l u l a r a c i d o s i s (Nikinmaa, 1982; Table 5, F i g u r e 14), these s t u d i e s have been s u b s t a n t i a t e d by l a t e r o b s e r v a t i o n s (Heming, 1984; Nikinmaa & H u e s t i s , 1984) that a d r e n a l i n e s t i m u l a t e s net H + and HCO3- e f f l u x and net Na + and C l ~ i n f l u x across t r o u t r e d c e l l membranes i n v i t r o . NaCl i n f l u x promotes c e l l u l a r H 2 0 uptake, and i n c r e a s e i n e r y t h r o c y t e volume. The r i s e i n red c e l l pH and volume i s expected to i n c r e a s e Hb-0 2 a f f i n i t y and oxygen contend v i a re v e r s e d Bohr and Root e f f e c t s , and by d i l u t i o n of c e l l u l a r o r g a n i c phosphates, r e s p e c t i v e l y . U n f o r t u n a t e l y , the catecholamines r e l e a s e d i n t o the plasma are r a t h e r l a b i l e d u r i n g a c i d exposure. The maximum catecholamine l e v e l s appeared only once and f o r j u s t a s h o r t p e r i o d ( f i g u r e 8, 12). There was s t i l l a s i g n i f i c a n t decrease i n red c e l l pH a f t e r 24hr exposure. This means that Bohr and Root e f f e c t s were not completely erased by the e f f e c t s of catecholamine d u r i n g a c i d exposure, one of the 84 reasons f o r decreased maximum c r i t i c a l v e l o c i t y i n a c i d water c o u l d s t i l l be a decreased oxygen d e l i v e r y c a p a c i t y due to the Root e f f e c t . Besides, catecholamines may i n c r e a s e the g i l l p e r m e a b i l i t y and i n h i b i t the g i l l C l ~ uptake (Vermette and Per r y , 1986) which would exacerbate the acid-base and i o n o r e g u l a t o r y d i s t u r b a n c e s . Catecholamines may a l s o i n c r e a s e the metabolic r a t e and oxygen consumption. So i t seems th a t , d u r i n g a c i d exposure, the blood pH and Na +, C l -c o n c e n t r a t i o n s i n plasma g r a d u a l l y decreased, the red c e l l pH i s a l s o decreased. The l a t t e r e f f e c t might decrease oxygen d e l i v e r y c a p a c i t y v i a Root e f f e c t . As the d i s t u r b a n c e became more severe, the catecholamines might be r e l e a s e d i n t o the bloodstream. Although catecholamines might improve the oxygen uptake from the g i l l s and oxygen t r a n s p o r t to t i s s u e s , the e f f e c t of t h i s c o u l d not be maintained, s i n c e the i n c r e a s e catecholamine l e v e l s was f o r j u s t a s h o r t p e r i o d . On the other hand, catecholamines would i n c r e a s e the g i l l p e r m e a b i l i t y which would exacerbate the acid-base and i o n o r e g u l a t o r y d i s t u r b a n c e s , i n c r e a s e d a r t e r i a l blood pressure and heart r a t e , i n c r e a s e d metabolic r a t e and oxygen consumption. Those e f f e c t s might become s e l f - r e i n f o r c i n g , r e s u l t i n g i n sudden c a r d i o v a s c u l a r c o l l a s p s e , and the death of the f i s h . 85 E f f e c t o f a l k a l i n e water The t o x i c i t y of 0H~ on f i s h i s l e s s c l e a r . In a l k a l i n e water, hydrogen i o n d r i v i n g f o r c e s h i f t e d from negative to p o s i t i v e . T h i s means: Hydrogen i o n may be d i f f u s i n g from f i s h a c r o s s the g i l l membrane i n t o water. On the other hand, OH - i o n c o n c e n t r a t i o n i n water i s much l a r g e r than i n f i s h blood, 0H~ i o n would move i n t o the f i s h body, r e s u l t e d i n a more negative g i l l p o t e n t i a l ( f i g u r e 22). Compared to blood and red c e l l pH i n a c i d water, blood and red c e l l pH changed f a s t e r and l a r g e r i n f i s h exposed to a l k a l i n e water than a c i d water. T h i s i n d i c a t e s t h a t f i s h have a g r e a t e r c a p a c i t y to r e g u l a t e pH i n a c i d compared with a l k a l i n e water. Na + and C l _ c o n c e n t r a t i o n s i n plasma show a small but s i g n i f i c a n t i n c r e a s e a f t e r 72hrs o f a l k a l i n e exposure. The p o s s i b l e reasons f o r i n c r e a s i n g Na + and C l ~ c o n c e n t r a t i o n s i n plasma would be: 1) water l o s t from plasma i n t o the water environment or i n t o the i n t r a c e l l u l a r compartments. 2) Na + and C l ~ s h i f t e d i n t o plasma from the water environment or from the i n t r a c e l l u l a r compartments. 3) Na + and C l ~ e f f l u x were decreased. There i s not enough i n f o r m a t i o n to e x p l a i n why Na + and C l ~ c o n c e n t r a t i o n s i n c r e a s e d u r i n g a l k a l i n e exposure. Wright and Wood (1985) found t h a t high pH water' (pH9.54) i n h i b i t e d Na + uptake about 55% and i n c r e a s e d 86 Na + e f f l u x i n rainbow t r o u t . T h e i r t e s t s were i n r e l a t i v e l y hard water, and the f i s h were exposed i n a l k a l i n e water for only a short p e r i o d ( w i t h i n 3hrs). Maetz & De Renzis (1978) o found a 1 0 - f o l d s t i m u l a t i o n of Na + i n f l u x i n T i l a p i a mossambica r a i s e d from water pH=7.8 to 9.7, t h e i r t e s t s , however, were conducted i n 10% sea water. In my experiment, however, there was an i n i t i a l small decrease i n Na + c o n c e n t r a t i o n i n plasma a f t e r 2hrs a l k a l i n e exposure and a small s i g n i f i c a n t i n c r e a s e i n Na + c o n c e n t r a t i o n a f t e r 72hr a l k a l i n e exposure. I t i s p o s s i b l e that the e f f e c t s of a l k a l i n e water i n d e c r e a s i n g Na + uptake and i n c r e a s i n g Na + e f f l u x i s maintained f o r only a short p e r i o d and t h i s would e x p l a i n the i n i t i a l f a l l i n plasma Na + c o n c e n t r a t i o n observed i n my experiment i n a l k a l i n e water. The p o s s i b l e reasons for the subsequent i n c r e a s e i n Na + c o n c e n t r a t i o n i n plasma d u r i n g a l k a l i n e exposure are: 1) the u r i n a r y e x c r e t i o n r a t e of Na + d e c l i n e d . 2) Na + m°ved i n t o plasma from i n t r a c e l l u l a r compartments. 3) water was l o s t from plasma. 4) net Na + uptake inceased due to the s t i m u l a t i o n of Na + i n f l u x or the decrease of Na + e f f l u x d r i v i n g f o r c e , t h e r e f o r e , d e c r e a s i n g Na + e f f l u x i f the g i l l p e r m e a b i l i t y d i d not change. C I - e f f l u x d r i v i n g f o r c e showed a small s i g n i f i e s nt i n c r e a s e from 220.6+.1.90 mV i n c o n t r o l water to 233.8+.2.1 mV a f t e r 2hr a l k a l i n e esposure. t h e r e f o r e , there might be a small o i n c r e a s e i n C l ~ e f f l u x d u r i n g a l k a l i n e esposure i f the g i l l p e r m e a b i l i t y d i d not change. The C l ~ i o n c o n c e n t r a t i o n , however, d i d not change much w i t h i n 48hr i n a l k a l i n e water, and showed a small s i g n i f i c a n t i n c r e a s e 87 from 110.6+2.40 mEq/L i n c o n t r o l water to 128.0+J5.5 mEq/L a f t e r 72hr a l k a l i n e exposure. The reasons f o r i n c r e a s i n g C l ~ c o n c e n t r a t i o n d u r i n g a l k a l i n e exposure are not c l e a r . I t could o be dut to: 1) the u r i n a r y e x c r e t i o n r a t e of C I - d e c l i n e d . 2) C l ~ i o n moved i n t o the plasma from i n t r a c e l l u l a r compartments. 3) water was l o s t from plasma. 4) i t i s a l s o p o s s i b l e that e l e v a t e d plasma bicar b o n a t e w i l l s t i m u l a t e the C1~/HC03~ exchange process and t h e r e f o r e C I ~ i n f l u x . Catecholamines d i d not change i n f i s h exposed to pH 10.0 water. The red c e l l pH i n c r e a s e d s i g n i f i c a n t l y with the blood a l k a l o s i s due to a l k a l i n e exposure. I t seems that f i s h have no s i m i l a r e r y t h r o c y t e pH r e g u l a t i o n mechanisimes i n blood a l k a l o s i s as i n blood a c i d o s i s . The d e t a i l s of the causes of the r e d u c t i o n of maximal c r i t i c a l v e l o c i t y i n f i s h exposed to low or high pH i s s t i l l unknown, e s p e c i a l l y i n high pH water. Further work should be done. In t h i s experiment, the blood pH and red c e l l pH in c r e a s e d s i g n i f i c a n t l y i n a l k a l i n e water. So the acid-base distubance would be one of the reasons to decrease maximum c r i t i c a l v e l o c i t y i n a l k a l i n e water. At low pH water, many p h y s i o l o g i c a l changes were observed: decreased oxygen consumption (Packer & Dunson, 1972); i n c r e a s e d b r a n c h i a l mucus s e c r e t i o n which i n f l u e n c e s r e s p i r a t o r y gases exchange (Janssen & R a n d a l l , 1975). decreased 0 2 d e l i v e r y to the t i s s u e s ; a 88 f a l l i n blood pH; decreased plasma Na + and C l ~ c o n c e n t r a t i o n s ; A l l o f those would d i s t u r b the microenviroment f o r many enzyne r e a c t i o n s i n the t i s s u e s and muscles, and when f i s h e x e r c i s e , the s i t u a t i o n may be worse. I t i s easy to assume t h a t , under such circumstances, f i s h c o u l d not reach the maximal c r i t i c a l v e l o c i t y observed i n normal pH water. 89 References B r e t t , J.R. 1964. The r e s p i r a t o r y metabolism and swimming performance of young sockeye salmon. J . F i s h . Res. Bd. Canada 21: 1183-1226. B r e t t , J.R. 1967. Swimming performance of sockeye salmon (Oncorhynchus nerka) i n r e l a t i o n to f a t i g u e time and temperature. J . F i s h . Res. Bd. Can. 24:1731-1741. B o u t i l i e r , R.G., Iwama, G.K. & R a n d a l l , D.J., 1986. Acute e x t r e c e l l u l a r a c i d o s e s promote catecholamine r e l e a s e i n rainbow t r o u t (Salmo g a i r d n e r i ) : I n t e r a c t i o n s between red c e l l pH and 02_Hb c a r r y i n g c a p a c i t y , i n p ress. Cameron, J.N. 1978. R e g u l a t i o n of blood pH i n t e l e o s t f i s h . R e s p i r . P h y s i o l . 33:129-144. Cuthbert, A.W. and Maetz, J.1972. The e f f e c t s of c a l c i u m and magnesium on sodium f l u x e s through the g i l l s of C a r a s s i u s auratus. J . P h y s i o l . (London) 221:633-643. D i v e l y , J.L., Mudge, J.E., Neff, W.H. and Anthony, A. 1977. Blood PO2 PCO2 and pH changes i n 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 ) exposed to s u b l e t h a l l e v e l s of a c i d i t y . Comp. Biochem. P h y s i o l . 57A:347-351. Eddy, F.B. 1976. Acid-base balance i n rainbow trout(Salmo ga i r d n e r i i ) s u b j e c t e d to a c i d s t r e s s . J . exp. B i o l . 64:159-171. 90 Fromm, P.O. 1980. A review of some p h y s i o l o g i c a l and t o x i c o l o g i c a l responses of freshwater f i s h to a c i d s t r e s s . Env. B i o l . F i s h 5:79-93. Guido Van Den T h i l l a r t , David Randall and L i n hoa-Ren. 1983. CO2 and H + e x c r e t i o n by swimming Coho salmon(Oncorhynchus K i s u t c h ) . J . exp. B i o l . 107:169-180. Graham, M.S. and Wood, CM. 1981. T o x i c i t y of environmental a c i d to the rainbow t r o u t : i n t e r a c t i o n s of water hardness, a c i d type, and e x e r c i s e . Can J . Zool. 59:1518-1526. Heming, T.A. 1984. The r o l e of f i s h e r y t h r o c y t e s i n t r a n s p o r t and e x c r e t i o n of carbon d i o x i d e . Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia. Hoar, W.S. and R a n d a l l , D.J. 1978.Editors, F i s h P h y s i o l o g y : Locomotion, V o l . 7. New york: Academic Press. Janssen, R.G. and R a n d a l l , D.J. 1975. The e f f e c t s of changes i n pH and PCO2 i n blood and water on b r e a t h i n g i n rainbow t r o u t , Salmo g a i r d n e r i . R e s p i r . P h y s i o l . 25:235-245. Maetz, J . and De Renzis, G. 1978. Aspects of the a d a p t a t i o n of f i s h to high e x t e r n a l a l k a l i n i t y : comparison of T i l a p i a grahami and T. mossambica. In Comparative P h y s i o l o g y : Water, Ions, and F l u i d Mechanics, (eds K. Schmidt-Nielsen, L. B o l i s & S.H.P.Maddrel1), pp. 1 213-228. Cambridge: Cambridge U n i v e r s i t y Press. 91 McDonald, D.G., Hobe, H., and Wood, CM. 1980. The i n f l u e n c e of c a l c i u m on the p h y s i o l o g i c a l responses of the rainbow t r o u t . Salmo g a i r d n e r i , to low environmental pH. J . exp. B i o l . 88:109-131. McDonald, D.G. and Wood, CM. 1981. B r a n c h i a l and r e n a l a c i d and i o n f l u x e s i n the rainbow t r o u t . Salmo g a i r d n e r i . at low environmental pH. J . exp. B i o l . 93:101-118. McWilliams, P.G. and P o t t s , W.T.W. 1978. The e f f e c t s o f pH and ca l c i u m on g i l l p o t e n t i a l s i n brown t r o u t Salmo  t r u t t a . J . comp. P h y s i o l . 126:277-286. Nakano, T. and Tomlinson, N. 1967. Catecholamine and carbohydrate c o n c e n t r a t i o n s i n rainbow t r o u t (Salmo  g a i r d n e r i ) i n r e l a t i o n to p h y s i c a l d i s t u r b a n c e . J . F i s h . Res. Bd. Can. 24, 1701-1715. N e v i l l e , CM. 1979a. S u b l e t h a l e f f e c t s of environmental a c i d i f i c a t i o n on rainbow t r o u t Salmo g a i r d n e r i . J . F i s h . Res. Board Can. 36:84-87. N e v i l l e , CM. 1979b. I n f l u e n c e of mild hypercapnia on the e f f e c t s of environmental a c i d i f i c a t i o n on rainbow t r o u t Salmo g a i r d n e r i . J . exp. B i o l . 83:345-349. N e v i l l e , CM. 1979c. V e n t i l a t o r y response of rainbow t r o u t Salmo g a i r d n e r i to i n c r e a s e d H + ion c o n c e n t r a t i o n i n blood and water. Comp. Biochem. P h y s i o l . 63A:373-376. Nikinmaa, M. 1982. Adrenergin r e g u l a t i o n of haemoglobin oxgen a f f i n i t y i n rainbow t r o u t red c e l l s . J . comp. P h y s i o l . 152: 67. 92 Nikinmaa, M. 1983. E f f e c t s o f a d r e n a l i n e on red c e l l volume and c o n c e n t r a t i o n g r a d i e n t of protons a c r o s s the red c e l l membrane i n the rainbow t r o u t Salmo g a i r d n e r i . Molec. P h y s i o l . 2: 287-297. Nikinmaa, M. & H u e s t i s , W.H. 1984. Adre n e r g i c s w e l l i n g of nucleate d e r y t h r o c y t e s : c e l l u l a r mechanism i n a b i r d , domestic goose, and two t e l e o s t s , s t r i p e d bass and rainbow t r o u t . J . comp. P h y s i o l . N i l s s o n , S. and Grove, D.J. 1974. Adrenergic and c h o l i n e r g i c i n n e r v a t i o n of the spleen of the cod: Gadus morhua. European J . Pharmacol. 28: 135-143. Packer, R.K. 1979. Acid-base balance and gas exchange i n 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 ) exposed to a c i d i c environments. J . exp. B i o l . 79:127-134. Packer, R.K. and Dunson, W.A. 1970. E f f e c t s of low environmental pH on blood pH and sodium balance of brook t r o u t . J . exp. Zoo l . 174:65-72. Packer, R.K. and Dunson, W.A. 1972. Anoxia and sodium l o s s a s s o c i a t e d with the death of brook t r o u t at low pH. Comp. Biochem. P h y s i o l . 41A:17-26. Primmett, D.R.N. 1982. the r o l e of n o r a d r e n a l i n e i n i n t r a c e l l u l a r pH r e g u l a t i o n i n rainbow.trout (Salmo g a i r d n e r i ) . B.Sc. T h e s i s . U n i v e r s i t y of B r i t i s h Columbia. R a n d a l l , D.J. 1970. Gas exchange i n f i s h . In: F i s h P h y s i o l o g y . v o l . IV (eds. W.S. Hoar & D.J. Randall) pp. 253 - 2 9 2 , Academic Press, N.Y. 93 Riggs, A. 1970. P r o p e r t i e s of f i s h hemoglobins. In: F i s h Physiology, v o l . IV (eds. W.S. Hoar & D.J. Randall) pp. 209-252, Academic pr e s s , N.Y. Smith. L.S. and B e l l , G.R. 1964. A technique f o r prolonged blood sampling i n free-swimming salmon. J . F i s h Res. Bd. Can. 21:1775-1790. Sprague, J.B. 1971. Measurement fo p o l l u t a n t t o x i c i t y to f i s h . I l l . S u b l e t h a l e f f e c t s and " s a f e " c o n c e n t r a t i o n s . Water Research 5:245-266. U l t s c h , G.R., Ott, M.E. and H e i s l e r , N. 1980. Standard metabolic r a t e , c r i t i c a l O2 t e n s i o n , and a e r o b i c scope fo r spontaneous a c t i v i t y fo trout(Salmo  q a i r d n e r i ) and c a r p ( C y p r i n u s c a r p i o ) i n a c i d i f i e d water. Comp. Biochem. P h y s i o l . 67A:329-335. U l t s c h , G.R., Ott, M.E., and H e i s l e r , N. 1981 Acid-base and e l e c t r o l y t e s t a t u s i n c a r p ( C y p r i n u s carpio)exposed to low environmental pH. J . exp. B i o l . 93:65-80. Vaala, S.S. and M i t c h e l l , R.B. 1970, Blood oxygen t e n s i o n changes i n a c i d exposed brook t r o u t . Proc. Pa. A c i d . S c i . 44:41-44. Vermtte, M.G. and S.F. Perry, 1986. The e f f e c t s of continuous e p i n e p h r i n e i n f u s i o n on the p h y s i o l o g y of the rainbow t r o u t , (Salmo g a i r d n e r i ) I I . B r a n c h i a l sodium f l u x e s . J . exp. B i o l . Ms. submitted. 94 Waiwood, K.G. and Beamish, F.W.H. 1978. E f f e c t s of copper, pH, and hardness on the c r i t i c a l swimming perfomance of rainbow trout(Salmo g a i r d n e r i Richardson). Water Research. 12:285-287. Wood. CM. and C a l d w e l l , F.H. 1978. Renal r e g u l a t i o n of acid-base balance i n a freshwater f i s h . J . exp. Zool . 205:301-307. Wood, CM. and McDonald, D.G. 1982. P h y s i o l o g i c a l mechanisms of a c i d t o x i c i t y to f i s h . In Acid R a i n / F i s h e r i e s , Proceedings of an I n t e r n a t i o n a l Symposium on A c i d i c P r e c i p i t a t i o n and F i s h e r y Impacts i n North-Eastern North America, (ed R.E.Johnson), pp. 197-226. Bethesda: American F i s h e r i e s S o c i e t y . Wood, CM. and R a n d a l l , D.J. 1973. The i n f l u e n c e of swimming a c t i v i t y on sodium balance i n the rainbow trout(Sa1 mo  g a i r d n e r i ) J . comp. P h y s i o l . 82:207-233. Woodward, J . J . 1982. Plasma catecholamines i n r e s t i n g t r o u t , Salmo g a i r d n e r i Richardson, by high pressure l i q u i d chromatography. J . F i s h B i o l . 21: 429-432. Wright, P.A. and Wood, CM. 1985 An a n a l y s i s of b r a n c h i a l ammonia e x c r e t i o n i n the freshwater rainbow t r o u t : E f f e c t s of enviromental pH change and sodium uptake blockade. J . exp. B i o l . 114:329-353. Z e i d l e r , R. and Kim, H.D. 1977. P r e f e r e n t i a l hemolysis of post n a t a l c a l f red c e l l s induced by i n t e r n a l a l k a l i n i z a t i o n . J . gen. P h y s i o l . 70: 385-401. 

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