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Ammonia toxicity in rainbow trout (Salmo gairdneri) Hillaby, Betty Ann 1978

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AMMONIA TOXICITY IN RAINBOW TROUT (Salmo gairdneri-) by BETTY ANN HILLABY B.Sc, University of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1978 Q Betty Ann Hi l l a b y , 1978 In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t fr e e l y available for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s under-stood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date : - i i -ABSTRACT Acute ammonia t o x i c i t y i n rainbow t r o u t was s t u d i e d . Th is was c a r r i e d out by i n j e c t i n g f i s h w i t h v a r i o u s c o n c e n t r a t i o n s of ammonia d i s -so lved i n i s o t o n i c s a l i n e . In order to approximate c o n d i t i o n s of n a t u r a l t o x i c i t y , where ammonia would enter the f i s h at the g i l l s , w i thout the a d d i t i o n a l problems of env i ronmenta l f a c t o r s , the ammonium s o l u t i o n s were i n j e c t e d v i a a cannula implanted i n the d o r s a l a o r t a . To determine i f a d i f f e r e n t i a l t o x i c i t y e x i s t e d i n f i s h i n r e l a t i o n to h i g h pH and low pH ammonium s o l u t i o n s , ammonium b i c a r b o n a t e and ammonium c h l o r i d e were chosen f o r i n j e c t i o n . Hydrogen i o n , and t o t a l ammonia, c o n c e n t r a t i o n s were measured i n b lood sampled from the d o r s a l a o r t a , both be fo re and a f t e r i n j e c t i o n . In order to determine i f , d u r i n g the course of the i n j e c t i o n , normal e x c r e t i o n r a t e s would remove a l l of the i n j e c t e d ammonia, t o t a l ammonia and hydrogen i o n c o n c e n t r a t i o n s were measured i n b lood sampled from both the d o r s a l and v e n t r a l a o r t a e , and r a t e s of e x t r a c t i o n of ammonia from blood at the g i l l s were c a l c u l a t e d . Ammonia i s t o x i c to f i s h . 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 dose of NH^Cl and NH^HCO^ which k i l l e d f i s h . T h e r e f o r e , u n l i k e mammals, f i s h e x h i b i t e d no d i f f e r e n t i a l t o x i c i t y to the ammonium compounds t e s t e d . I n j e c t i o n of NH^Cl decreased pHa and i n j e c t i o n of NH^HCO^ inc reased pHa. Both compounds i n c r e a s e d the t o t a l ammonia c o n c e n t r a t i o n i n the b l o o d . A l though i n water the f r a c t i o n of ammonia which e x e r t s the t o x i c e f f e c t s i s u n i o n i z e d ammonia, w i t h i n the f i s h i t i s the i o n i z e d f r a c t i o n which e x e r t s the t o x i c e f f e c t s . The same dose of ammonium k i l l e d f i s h , but NH^HCO^-injected f i s h which s u r v i v e d had a much h igher c o n c e n t r a t i o n of u n i o n i z e d ammonia i n the b lood than N H ^ C l - i n j e c t e d f i s h which d i e d . Ammonia e x t r a c t e d from the b lood i n c o n t r o l f i s h was about one -f i f t h the amount which k i l l e d f i s h . T h i s , together w i t h the measured i n -- i i i -c reases i n b lood ammonia f o l l o w i n g i n j e c t i o n , demonstrate t h a t , a l though ammonia i s a normal e x c r e t o r y product of rainbow t r o u t , the t r o u t cannot i n c r e a s e e x c r e t i o n r a t e s s u f f i c i e n t l y to r i d themselves of an ammonia l o a d . Symptoms observed i n f i s h f o l l o w i n g i n j e c t i o n of ammonium s o l u t i o n s l e d to the c o n c l u s i o n tha t ammonia a c t s at the n e u r a l l e v e l . - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS • i v LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGMENT i x SECTION I INTRODUCTION . . . . 1 SECTION I I MATERIALS AND METHODS 5 1 . Exper imenta l Groups 5 1 . 1 Ammonia E x c r e t i o n Rates 5 1.2 Tox ic L e v e l s of I n j e c t e d Ammonia 5 1 .3 pH E f f e c t 5 2. Operat ion Techniques 6 2 . 1 Cannu la t ions 6 2 .2 D o r s a l A o r t i c Cannu la t ion 6 2 . 3 V e n t r a l A o r t i c C a n n u l a t i o n 6 3 . I n j e c t i o n s and Sampling 11 3 . 1 I n j e c t i o n S o l u t i o n s „ 11 3 . 2 I n j e c t i o n Procedure 11 3 . 3 Sampling Procedure 11 4 . Measurements 12 4 . 1 Ammonia 12 4 . 2 pH 12 5 . Data A n a l y s i s 15 SECTION I I I RESULTS 16 1 . P r e l i m i n a r y Exper iments 16 1 . 1 E f f e c t s of Hand l ing and A n a e s t h e t i c VS. E f f e c t s of NH 4C1 I n j e c t i o n 16 1.2 E f f e c t s of A c i d o s i s vs. E f f e c t s of NH 4C1 I n j e c t i o n 16 - v -Page 2. Ammonia L e v e l s i n Trout Blood and Rates of E x t r a c t i o n 17 3 . Tox ic Symptoms of NH* or HCl I n j e c t i o n 17 3 . 1 R e a c t i o n Due to I n j e c t i o n 17 3 .2 R e a c t i o n to HCl 24 3 . 3 R e a c t i o n to NH 4C1 24 3 .4 R e a c t i o n to NH^HCO 24 3 . 5 D i f f e r i n g R e a c t i o n to HCl VS. Ammonium S o l u t i o n I n j e c t i o n 24 4. E f f e c t s of NH* or HCl I n j e c t i o n on Blood Parameters . 25 4 . 1 HCl I n j e c t i o n 25 4 .2 NH4CI I n j e c t i o n 25 4 . 3 NH4HCO3 I n j e c t i o n 25 4.4 H C l , NHi+Cl, and NHi+HCC^ I n j e c t i o n 35 4 . 5 E f f e c t s of NH^Cl on Blood Parameters 35 SECTION IV DISCUSSION 39 1 . T o x i c i t y of Ammonia to Rainbow Trout 39 2 . Tox ic L e v e l s of Ammonia 40 2 . 1 In Water . 40 2 .2 In Blood . . .* 40 3 . Symptoms of Ammonia T o x i c i t y 42 3 . 1 Symptoms i n Mice and Ch icks 42 3 .2 Symptoms i n F i s h 43 4 . Mechanism of T o x i c i t y 43 4 . 1 B l o o d - B r a i n B a r r i e r 44 4 .2 Ev idence f o r NH4 S u b s t i t u t i o n i n P a s s i v e T ranspor t 45 4 . 3 Ev idence f o r NR"£ S u b s t i t u t i o n i n A c t i v e T ranspor t 45 5 . E f f e c t s of Ammonia on Blood 46 - v i -Page . 6. Other Factors A f f e c t i n g Ammonia T o x i c i t y i n F i s h . • 48 6.1 pH 48 6.2 C0 2 49 6.3 0 2 • • • 52 6.4 N i t r i t e . . 53 7. Ammonia Production and Excretion by F i s h 53 7.1 Enzymes Involved i n Ammonia Production 53 7.2 Major Source of Ammonia 54 7.3 Si t e of Ammonia Excretion 55 7.4 Ammonia Extraction from Blood 55 7.5 Ammonia Excretion Rates 58 8., What Form of Ammonia i s Excreted at the G i l l s ? . . . 59 8.1 NH* Excretion 59 8.2 NH3 Excretion 61 SECTION V LITERATURE CITED 64 SECTION VI APPENDICES 70 I L i s t of Abbreviations and Symbols 70 II Preparation of Blood f o r Ammonia Determination with Orion Model 95-10 Ammonia Electrode 71 I I I C a l i b r a t i o n of Orion Ammonia Electrode, Model 95-10, f or Low Level (10~ 6 to 10" 4 M) Measurements of NH 3 i n f i s h blood 73 - v i i -LIST OF TABLES Table Page 1 - 1 Sources and Types of Ammonia Found i n N a t u r a l Waters 2 I I I - l (a) E f f e c t of NH4CI I n j e c t i o n on F i s h : Less than 5 days recovery t ime vs. 5 or more days recovery t ime . . . 18 (b) E f f e c t of HCl I n j e c t i o n on Plasma pH: Less than 5 days recovery t ime vs. 5 or more days recovery t ime . . . 18 I I I - 2 (a) D o r s a l and V e n t r a l A o r t i c Blood Ammonia and pH i n C o n t r o l F i s h 21 (b) Ammonia E x t r a c t e d from Blood a t the G i l l s 21 I I I - 3 pH, H + , and Blood Ammonia Befo re and A f t e r I n j e c t i o n of NH4CI and NH4HCO3 26 I I I - 4 % Haematocr i t Before and A f t e r I n j e c t i o n of NH^Cl S o l u t i o n . . 38 I V - 1 Threshold LC50 Values of Va r ious S o l u t i o n s of Ammonia f o r Rainbow Trout 41 IV -2 Plasma Ammonia Concent ra t ions Before and A f t e r Exposure to Ammonia at D i f f e r e n t Hydrogen Ion Concent ra t ions 50 IV -3 Comparison of R e s u l t s from A l a b a s t e r and Herber t (1954) w i t h R e s u l t s from L l o y d and Herber t (1960) 51 IV -4 Blood Ammonia Values f o r Va r ious Spec ies of F i s h 56 I V - 5 Ammonia E x c r e t i o n Rates f o r Va r ious Spec ies of F i s h 57 - v i i i -LIST OF FIGURES F i g u r e Page I I - l Diagram of Operat ing Table Showing G i l l s Per fused Backwards . . 8 I I - 2 (a) Diagram of D o r s a l A o r t i c Cannu la t ion 10 (b) Diagram of V e n t r a l A o r t i c Cannu la t ion 10 I I - 3 Per Cent Un ion i zed Ammonia as a F u n c t i o n of pH and Temperature, at 9°C and 13°C 14 I I I - l Days Between C a n n u l a t i o n and I n j e c t i o n vs. F i n a l pH of D o r s a l A o r t i c B lood 20 I I I - 2 Days F o l l o w i n g C a n n u l a t i o n ( - Days of S t a r v a t i o n ) vs. T o t a l Ammonia C o n c e n t r a t i o n of D o r s a l A o r t i c B lood 23 I I I - 3 Change i n Hydrogen Ion C o n c e n t r a t i o n of D o r s a l A o r t i c B lood w i t h Time F o l l o w i n g I n j e c t i o n of HCl S o l u t i o n . . . . . . 28 I I I - 4 Ammonium S o l u t i o n I n j e c t e d vs. T o t a l Ammonia C o n c e n t r a t i o n of D o r s a l A o r t i c Blood F o l l o w i n g I n j e c t i o n 30 I I I - 5 Ammonium S o l u t i o n I n j e c t e d vs. C o n c e n t r a t i o n of Un ion i zed Ammonia of D o r s a l A o r t i c B lood F o l l o w i n g I n j e c t i o n 32 I I I - 6 Change i n Hydrogen Ion C o n c e n t r a t i o n of D o r s a l A o r t i c B lood w i t h Time F o l l o w i n g I n j e c t i o n of NH^Cl S o l u t i o n 34 I I I - 7 T o t a l Ammonia C o n c e n t r a t i o n vs. Hydrogen Ion C o n c e n t r a t i o n of D o r s a l A o r t i c B lood 37 - 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 . R a n d a l l , D r . C. C l a r k e , and Dr . R. B r e t t , f o r t h e i r s u p e r v i s i o n , p a t i e n c e , and g u i d -ance d u r i n g t h i s study1 I would e s p e c i a l l y l i k e to thank Dr . R a n d a l l f o r h i s a d v i c e throughout the course of the exper iments , and i n the p r e p a r a t i o n of t h i s m a n u s c r i p t . I would a l s o l i k e to thank Mr . Bruce H i l l a b y f o r h i s a s s i s t a n c e i n the p r e p a r a t i o n of the f i g u r e s f o r t h i s r e p o r t . - 1 -INTRODUCTION Ammonia found i n n a t u r a l waters i s from a v a r i e t y of s o u r c e s . I t i s the main e x c r e t o r y product of many a q u a t i c organisms. Ammonia may a l s o be d i scharged i n t o water systems as waste from i n d u s t r y and a g r i c u l t u r e , and as unt reated sewerage (Envi ronmental P r o t e c t i o n Agency, 1971; European In land F i s h e r i e s A d v i s o r y Commission, 1973) (Table 1 . 1 ) . Ammonia e x i s t s i n s o l u t i o n as f o l l o w s : N H 3 ( g ) + n H 2 0 ( 1 ) N H 3 - n H 2 0 ( a q ) NH^ + OH" + ( n - l ) H 2 0 ( 1 ) (1) That i s , there i s an e q u i l i b r i u m between d i s s o l v e d u n i o n i z e d ammonia and the hydrated ammonia m o l e c u l e , which i s hydrogen bonded to a t l e a s t th ree water m o l e c u l e s , and between the hydrated ammonia molecu le and i o n i z e d ammonia. The c o n c e n t r a t i o n of u n i o n i z e d ammonia i s dependent not on ly on the t o t a l ammonia c o n c e n t r a t i o n , but a l s o on pH, temperature , and i o n i c s t r e n g t h of the s o l u t i o n . The c o n c e n t r a t i o n of u n i o n i z e d ammonia i n c r e a s e s w i t h i n c r e a s e s i n pH and temperature , and decreases w i t h an i n c r e a s e i n i o n i c s t r e n g t h . The r e d u c t i o n i n per cent u n i o n i z e d ammonia a t t r i b u t e d to up to 300 mg/1 d i s s o l v e d s o l i d s i s n e g l i g i b l e . There i s a s m a l l but s i g n i f i c a n t decrease i n per cent u n i o n i z e d ammonia i n more s a l i n e s o l u t i o n s , or very hard wate r , but t h i s i s g e n e r a l l y ignored i n t a b l e s f o r per cent u n i o n i z e d ammonia (Emerson et dl.t 1975) . The c o n c e n t r a t i o n of u n i o n i z e d ammonia i n s o l u t i o n i s r e l a t e d to pH, as i n d i c a t e d by the f o l l o w i n g : pH = p K ' a + log [NH 3 ] (2) TNHp" and p K ' a i s dependent upon temperature . Tables of per cent u n i o n i z e d ammonia present i n s o l u t i o n as a f u n c t i o n of temperature and pH, such as those p r e -pared by T r u s s e l l (1972) and Emerson et al. (1975) p rov ide a convenient means f o r r e s e a r c h e r s to determine u n i o n i z e d ammonia from measurements of t o t a l ammonia, temperature , and pH. - 2 -TABLE 1-1 SOURCES AND TYPES OF AMMONIA FOUND IN NATURAL WATERS (EPA, 1971) SOURCE TYPE OF AMMONIA Ag r i c u l t u r a l : f e r t i l i z e r s insecticides ammonium sulfate ammonium fluoride I n d u s t r i a l : chemical dye and pigment dye and tanning general industry ore processing petroleum photography pottery and porcelain pyrotechnics t e x t i l e plants wood preservation ammonia ammonia n i t r a t e ammonia sul f i d e ammonia thiocyanate aluminum ammonium sulfate ammonium chloride ammonium chloride ammonium sulfate ammonium chloride ammonium molybdate ammonium ammonium carbonate ammonium su l f i d e ammonium thiocyanate ammonium ferrocyanide ammonium dichrornate ammonium molybdate ammonium s u l f i t e ammonium dichrornate ammonium ni t r a t e ammonium thiocyanate ammonium fluoride ammonium thiocyanate ammonium fluoride - 3 -Exposure to either high levels of ammonia, or continuous low l e v e l s , i s believed to be detrimental to f i s h . Sublethal levels of ammonia may cause a reduction i n growth rate (Brockway, 1950; Larmoyeux and Piper, 1973), p r o l i f e r a t i o n of g i l l lamellae, reduced stamina, and increased s u s c e p t i b i l i t y to b a c t e r i a l g i l l disease (Burrows, 1964), thickening of the epithelium of g i l l lamellae, reduction i n lymphoid tissue i n the spleen and haematopoietic tissue i n the kidney (Larmoyeux and Piper, 1973), tissue disintegration, lesions i n blood vessels, and an abundant secretion of mucus ( F l i s , 1963b). However, many external factors, such as temperature, pH, carbon dioxide and oxygen content of the water, and b a c t e r i a l conversion of ammonia to n i t r i t e , contribute to toxic symptoms observed i n f i s h (Wuhrmann and Woker, 1948; Alabaster and Herbert, 1954; Downing and Merkens, 1955; Merkens and Downing, 1957; Lloyd and Herbert, 1960; Lloyd, 1961; Burrows, 1964; B a l l , 1967; Brown, 1968; Wilson, Anderson, and Bloomfield, 1969; Lloyd and Orr, 1969; Larmoyeux and Piper, 1973; EIFAC, 1973; B.C. Research, 1974; Smart, 1975). These ex-ternal factors may i n themselves be toxic to f i s h , or act s y n e r g i s t i c a l l y with ammonia to produce toxic symptoms, and death. Teleost f i s h which are exposed to high levels of ammonia exhibit hyperventilation, h y p e r e x c i t a b i l i t y , loss of equilibrium, convulsions, and death ( F l i s , 1963a; Wilson et al., 1969; Smart, 1975). However, there i s l i t t l e damage to g i l l tissues (Smart, 1976). The toxic effects on f i s h of ammonia i n water have been related to the unionized form (Wuhrman, Zender, and Woker, 1947; Wuhrmann and Woker, 1948; Smart, 1975). Although ammonia i s considered to be toxic to f i s h , i t i s the major form of nitrogen excreted by teleost f i s h . Ammonia i s produced i n the l i v e r (Pequin and Serfaty, 1963; Vellas and Serfaty, 1974), and excreted at the g i l l s (Smith, 1929; Wood, 1958; Goldstein, Forster, and F a n e l l i , J r . , 1964; _ 4 _ Payan and Matty, 1975). The kidney accounts for less than 2% of the t o t a l ammonia excreted by teleost f i s h (Smith, 1929; Wood, 1958; Fromm, 1963; Fromm and G i l l e t t e , 1968; Payan and Matty, 1975). There i s evidence that ammonia crosses the g i l l s i n both the ionized (Maetz and Garcia Romeu, 1964; Maetz, 1972, 1973; Payan and Maetz, 1973; Payan and Matty, 1975) and unionized forms (Goldstein et al., 1964; Fromm and G i l l e t t e , 1968; Maetz, 1973). My experiments were designed to test the t o x i c i t y of ammonia to rainbow trout (Salmo gaivdnevi) by i n j e c t i n g various concentrations of ammonia, dissolved i n isotonic saline, d i r e c t l y into the dorsal aorta, thus avoiding many of the external factors thought to contribute to ammonia tox-i c i t y . As ammonia, i f i t actually enters the f i s h , would enter at the g i l l s , I chose the dorsal aorta, which i s located post-branchially, as the s i t e for in j e c t i o n . In order to test the equipment and methods used, and to compare my results with published data, I also measured ammonia of dorsal and ventral ao r t i c blood and calculated ammonia extracted from the blood at the g i l l s . Ammonia i s also toxic to mammals (jacquez, Poppell, Lawrence, J r . , and Roberts, 1957; Warren, 1958; Warren and Nathan, 1958; Warren and Schenker, 1962; Wilson et al., 1968). Warren (1958) demonstrated a d i f f e r e n t i a l t o x i c i t y of ammonia to mice, depending on the a b i l i t y of the ammonium s a l t to raise blood pH. To test for a d i f f e r e n t i a l t o x i c i t y i n f i s h , my experiments were designed to include ammonia chloride, which lowered blood pH, and ammonium bicarbonate, which increased blood pH, i n the in j e c t i o n solutions. - 5 -MATERIALS AND METHODS 1 . EXPERIMENTAL GROUPS Exper iments were c a r r i e d out from J u l y , 1976 to October , 1977, on rainbow t r o u t (Salmo gaivdnevi) we igh ing between 215 and 555 grams, at water temperatures from 6 to 13.75°C. The f i s h were obta ined from Sun V a l l e y Trout Farms, M i s s i o n , B r i t i s h Co lumbia . The experiments were d i v i d e d i n t o three separate groups. 1 . 1 Ammonia E x c r e t i o n Rates In order to c a l c u l a t e ammonia c l e a r a n c e r a t e s ac ross the g i l l s , experiments were c a r r i e d out to measure t o t a l b lood ammonia and pH from both d o r s a l and v e n t r a l a o r t a e . F i s h used f o r these experiments weighed 364.3± 31.4 g (mean ± 95% conf idence l i m i t s , n = 1 4 ) . Water temperatures were b e -tween 10 and 13.75°C. 1 .2 Tox ic L e v e l s of I n j e c t e d Ammonia Exper iments were performed i n order to determine the t o x i c i n j e c t e d dose of an ammonium s o l u t i o n , the b lood ammonia and pH be fo re and f o l l o w i n g i n j e c t i o n , and to compare the e f f e c t s of i n j e c t i n g a low pH s o l u t i o n , NH^Cl , and a h i g h pH s o l u t i o n , NH^HCO^, i n t o the d o r s a l a o r t a of rainbow t r o u t . NH^Cl i n j e c t i o n experiments were performed on f i s h we igh ing 353.9± 38.7 g (n = 1 5 ) , a t temperatures between 9 and 12.5°C. NH^HCO^ i n j e c t i o n exper iments were performed on f i s h we igh ing 298.6+38.3 g (n = 1 5 ) , a t temper-a tu res between 11 .5 and 12.5°C. 1 .3 pH E f f e c t Data from i n j e c t i o n of ammonium c h l o r i d e s o l u t i o n s showed a severe b lood a c i d o s i s i n i n j e c t e d f i s h , so t h i s se t of exper iments was designed to determine the e f f e c t s of l o w e r i n g b lood pH by i n j e c t i o n of HCl on f i s h s u r -v i v a l . These exper iments were c a r r i e d out on f i s h weigh ing 326.8±41.1 g (n = 1 9 ) , a t temperatures between 6 and 9°C. - 6 -2. OPERATION TECHNIQUES 2 . 1 C a n n u l a t i o n s A f i s h was r a p i d l y a n a e s t h e t i z e d w i t h MS222 ( t r i c a i n e methane s u l p h o n a t e ) , then t r a n s f e r r e d to an o p e r a t i n g t a b l e , where the g i l l s were per fused backwards w i t h r e c i r c u l a t i n g , coo led wate r , which conta ined anaes -t h e t i c ( F igure I I - l ) . D o r s a l and/or v e n t r a l a o r t i c c a n n u l a t i o n s were p e r -formed as f o l l o w s : 2 .2 D o r s a l A o r t i c C a n n u l a t i o n The d o r s a l a o r t a was f i r s t punctured w i t h a Medicut #16 needle (Sherwood M e d i c a l I n d u s t r i e s I n c . ) . The needle was removed and a c a n n u l a , of Int ramedic (C lay Adams, I n c . ) PE 50 t u b i n g , f i l l e d w i t h h e p a r i n i z e d (10 I .U ./ ml) C o r t l a n d s a l i n e (Wolf , 1963) , was i n s e r t e d i n t o the d o r s a l a o r t a through the p l a s t i c s l e e v e of the Medicut n e e d l e . The p l a s t i c s leeve was then removed, a s t i t c h put i n the roof of the mouth to secure the cannula i n p l a c e , and the f r e e end of the cannula passed to the e x t e r i o r through a shor t c o l l a r of PE 190 t u b i n g , which had been i n s e r t e d i n a h o l e punctured between the n a r e s . B lood i n the cannula was r e p l a c e d w i t h h e p a r i n i z e d C o r t l a n d s a l i n e , and the f r e e end of the cannula plugged w i t h a p i n (F igure I I - 2 ( a ) ) . 2 .3 V e n t r a l A o r t i c C a n n u l a t i o n The v e n t r a l a o r t i c c a n n u l a t i o n was performed through the f l o o r of the mouth by p l a c i n g the c a n n u l a , of Int ramedic PE 50 t u b i n g t i p p e d w i t h a #23 Y a l e needle ( B i c t o n , D i c k i n s o n & C o . , Canada, L t d . ) and f i l l e d w i t h h e p a r -i n i z e d s a l i n e , i n t o the v e n t r a l a o r t a , midway between the second and t h i r d g i l l a r c h e s . The f r e e end was plugged w i t h a p i n , and e x i t e d through a s m a l l h o l e cut between the b r a n c h i o s t e g a l r a y s . The cannula was secured w i t h a s t i t c h through the s i d e of the mouth (F igure I I - 2 ( b ) ) . C a n n u l a t i o n s such as these a l l o w b l o o d to be sampled r e p e a t e d l y from an u n a n a e s t h e t i z e d , u n r e s t r a i n e d f i s h , w i t h the l e a s t amount of d i s t u r b -- 7 -FIGURE I I - l DIAGRAM OF OPERATING TABLE SHOWING GILLS PERFUSED BACKWARDS SLING HOOK COOLED ANAESTHETIZED WATER FROM RESERVOIR. WATER RETURNED TO R E S E R V O I R - 9 -FIGURE II - 2(a) DIAGRAM OF DORSAL AORTIC CANNULATION FIGURE II - 2(b) DIAGRAM OF VENTRAL AORTIC CANNULATION - 10 -I. D O R S A L A O R T A - 11 -ance to the f i s h (Smith and B e l l , 1964; Ho le ton and R a n d a l l , 1967; Houston, 1971; S o i v i o , Westman, and Nyholm, 1972) . I f the v e n t r a l a o r t i c c a n n u l a t i o n i s performed be fo re the d o r s a l one, there i s a much l e s s severe l o s s of b l o o d . A f t e r c a n n u l a t i o n , the f i s h were moved to a covered b l a c k Perspex h o l d i n g tank , w i t h i n d i v i d u a l compartments designed f o r minimum d i s t u r b a n c e of the f i s h , from e x t e r n a l s t i m u l i , such as movement or l i g h t . A constant f l o w of d e c h l o r i n a t e d f reshwater was mainta ined through the h o l d i n g tank , w i t h the i n f l o w p r o v i d i n g s u f f i c i e n t a e r a t i o n . The f i s h were a l lowed to recover f o r a t l e a s t 20 hours b e f o r e b lood samples were t a k e n , as the e f f e c t s of the a n a e s t h e t i c p e r s i s t f o r s e v e r a l hours (Houston, Madden, Woods, and M i l e s , 1971) . 3 . INJECTIONS AND SAMPLING 3 . 1 I n j e c t i o n S o l u t i o n s Immediately p r i o r to each i n j e c t i o n , I d i s s o l v e d ammonium c h l o r i d e (BCH Chemica ls , l a b o r a t o r y r e a g e n t ) , ammonium hydrogen carbonate (BDH C h e m i c a l s , a n a l y t i c a l r e a g e n t ) , or h y d r o c h l o r i c a c i d ( M a l l i n c k r o d t , a n a l y t i c a l r e a g e n t ) , i n C o r t l a n d s a l i n e to g i v e the r e q u i r e d c o n c e n t r a t i o n s . These s o l u -t i o n s were mainta ined a t the same temperature as the f i s h . 3 .2 I n j e c t i o n Procedure I n j e c t i o n s v i a the d o r s a l a o r t i c cannula were performed i n the f o l l o w i n g manner. Us ing h e p a r i n i z e d , d i s p o s a b l e s y r i n g e s (Becton , D i c k i n s o n & C o . , Canada, L t d . ) a t tached to three-way v a l v e s , I withdrew b lood u n t i l the cannula was f i l l e d , then exchanged the s a l i n e - f i l l e d s y r i n g e s f o r ones f i l l e d w i t h the i n j e c t i o n s o l u t i o n . I then i n j e c t e d the volume of b lood i n the c a n -n u l a p l u s 1 ml of the i n j e c t i o n s o l u t i o n a t a r a t e of 0 . 1 ml/minute . 3 . 3 Sampling Procedure B lood samples, 0 . 5 ml f o r ammonia and 0 . 1 ml f o r pH a n a l y s i s , were taken w i t h a d i s p o s a b l e , h e p a r i n i z e d s y r i n g e immediate ly b e f o r e , and one - 12 -minute f o l l o w i n g i n j e c t i o n , and , i n some c a s e s , a t i n t e r v a l s a f t e r i n j e c t i o n . B lood was removed a n a e r o b i c a l l y by a p p l y i n g a s l i g h t s u c t i o n to the c a n n u l a . S a l i n e and a s m a l l amount of b lood were d i s c a r d e d be fo re c o l l e c t i n g the sample f o r a n a l y s i s . In between sampl ing , the cannula was f i l l e d w i t h h e p a r i n i z e d s a l i n e and p lugged . For p r e p a r a t i o n of b lood f o r ammonia a n a l y s i s , p l e a s e r e f e r to Appendix I I , page 7 1 . 4 . MEASUREMENTS 4 . 1 Ammonia Ammonia measurements were made w i t h an Or ion ammonia e l e c t r o d e , model 9 5 - 1 0 , connected to F i s h e r Accumet expanded s c a l e pH-mV meter , Model 310 or 320, or to Or ion Research d i g i t a l i o n a l y z e r , model 801A. For c a l i b r a -t i o n p rocedures , p l e a s e r e f e r to Appendix I I I , page 7 2. The ammonia e l e c t r o d e was c a l i b r a t e d d a i l y w i t h a commercial s tandard s o l u t i o n (Or ion) which was mainta ined a t room temperature , the same as the e l e c t r o d e . The standard has a s t a t e d accuracy to w i t h i n ±0.005 m o l e s / l i t r e . Samples were analysed f o r t o t a l ammonia c o n c e n t r a t i o n a t the c a l i b r a t i o n temperature . [NH^] and [NH*] were c a l c u l a t e d from the t o t a l ammonia c o n c e n t r a t i o n , pH, and temperature of the b l o o d , u s i n g per cent u n i o n i z e d ammonia v a l u e s taken from T r u s s e l l (1972) (F igure I I - 3 ) . B lood temperature was assumed to be the same as water tempera-t u r e . 4 . 2 pH pH measurements were made w i t h a Radiometer g l a s s c a p i l l a r y e l e c t r o d e G297/G2, connected to Radiometer PHM 71 a c i d - b a s e a n a l y s e r . The e l e c t r o d e was conta ined i n a thermostated c e l l and mainta ined a t the same temperature as the f i s h . The e l e c t r o d e was c a l i b r a t e d d a i l y w i t h commercial s tandard s o l u t i o n s (Radiometer Copenhagen) which were mainta ined at the same temperature as the e l e c t r o d e . The commercial b u f f e r s o l u t i o n s have a s t a t e d accuracy to w i t h i n ±0.005 pH u n i t s . - 13 -FIGURE II-3 PER CENT UNIONIZED AMMONIA AS A FUNCTION OF pH AND TEMPERATURE, AT 9°C and 13oc (from T r u s s e l l , 1972) - 15 -5 . DATA ANALYSIS I used S t u d e n t ' s t - t e s t to compare the d i f f e r e n c e s between two means, or f o r p a i r e d comparisons (Soka l and R o h l f , 1973) , f o r s t a t i s t i c a l s i g n i f i c a n c e a t P = 0 . 0 5 . Un less o therwise s t a t e d , a l l my v a l u e s are mean ± 95% conf idence l i m i t s (Soka l and R o h l f , 1973) . A l l l i n e s drawn on graphs were f i t t e d by eye. - 16 -RESULTS 1 . PRELIMINARY EXPERIMENTS 1 . 1 E f f e c t s of Hand l ing and A n a e s t h e t i c vs. E f f e c t s of NH^Cl I n j e c t i o n I wanted to look at the e f f e c t s of NH^/NH* on f i s h , r a t h e r than the e f f e c t s of h a n d l i n g and a n a e s t h e t i c , so I c a r r i e d out some p r e l i m i n a r y e x p e r i -ments i n which I i n j e c t e d NH^Cl i n t o the d o r s a l a o r t a of f i s h , a f t e r a l l o w i n g v a r i o u s p e r i o d s of recove ry . 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 the dose which k i l l e d f i s h w i t h l e s s than 5 days recovery t ime and the dose which f i s h w i t h 5 or more days recovery t ime s u r v i v e d (Table I l l - l ( a ) ) . 1.2 E f f e c t s of A c i d o s i s vs. E f f e c t s of NH^Cl I n j e c t i o n I n j e c t i o n of NH^Cl caused a c i d o s i s i n the b l o o d . In order to s e p a r -a te the e f f e c t s of i n c r e a s e d hydrogen i o n c o n c e n t r a t i o n i n the b l o o d from the e f f e c t s of NH^/NH*, I i n j e c t e d f i s h w i t h an HCl s o l u t i o n . R e s u l t s from t h i s set of exper iments , graphed i n F i g u r e I I I - l , i n d i c a t e a d i f f e r e n t e f f e c t on pHa i n f i s h w i t h l e s s than 5 days recovery t ime from h a n d l i n g and a n a e s t h e t i c than i n f i s h w i t h 5 or more days recovery t i m e . 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 the i n j e c t e d dose which a f f e c t e d or k i l l e d the f i s h between these two groups (Table I l l - l ( b ) ) . In the H C l - i n j e c t e d f i s h , a s i m i l a r dose a f f e c t e d or k i l l e d the f i s h r e g a r d l e s s of recovery t i m e , but the e f f e c t was d i f f e r e n t between f i s h w i t h l e s s than 5 days recovery t ime and those w i t h 5 or more days recovery t i m e . In the l a t t e r c a s e , f i s h which s u r v i v e d the i n j e c t i o n showed a much g rea te r r e d u c t i o n i n pHa than f i s h which s u r v i v e d w i t h l e s s than 5 days recovery t i m e . T h e r e f o r e , death was not on ly due to r e d u c t i o n i n pHa i f l e s s than 5 days recovery t ime was a l l o w e d . The pHa of f i s h which s u r v i v e d HCl i n j e c t i o n was much lower than the pHa of f i s h which d i e d from NH^Cl i n j e c t i o n (F igure I I I - 7 ) . - 17 -The results from these two sets of experiments indicate that the effects of handling and anaesthetic could be masking the effects of in j e c t i o n . In order to separate the effects of in j e c t i o n from the effects of handling and anaesthetic, I have analysed my data only for those f i s h which were allowed 5 or more days recovery time. 2. AMMONIA LEVELS IN TROUT BLOOD AND RATES OF EXTRACTION I measured t o t a l blood ammonia concentrations and pH for both the dorsal and ventral aortae. From these values, together with water tempera-ture, and using values for per cent unionized ammonia from Trussell (1972), I calculated the concentration of both ionized and unionized ammonia i n the blood for both ventral and dorsal aortae (Table I I I - 2 ( a ) ) . As the concentration of ammonia i n f i s h blood was quite variable, and increased as the days of starvation increased (Figure I I I - 2 ) , I have only reported values for the dorsal and ventral aortae which were taken from the same f i s h (Part A, Table I I I - 2 ( a ) ) , or for f i s h within the f i r s t 3 days of starvation (Part B, Table I I I - 2 ( a ) ) . From the values l i s t e d i n Table III-2 (a), and using the following formula, C[NH 3 1 V A " [ N I V D A ) x cardiac output* = ammonia extracted from blood (3) I calculated ammonia extracted from the blood at the g i l l s (Table III - 2 ( b ) ) . 3. TOXIC SYMPTOMS OF NH+ OR HCL INJECTION 4 3.1 Reaction Due to Injection Whether the i n j e c t i o n solution was HCl, NH^Cl, or NH^HCO^, some f i s h showed an i n i t i a l reaction to the i n j e c t i o n , which consisted of head shaking, coughing, jumping, and switching ends i n the f i s h box. However, these were very mild reactions which lasted during the f i r s t 1 to 3 minutes of i n j e c t i o n only. Other f i s h remained calm throughout the entire i n j e c t i o n . * Wood (1974), 34.04 ml/kg-min. - 18 -TABLE I I I - l (a) EFFECT OF NH.CL INJECTION ON FISH: LESS THAN 5 DAYS RECOVERY TIME (A) VS. 5 OR MORE DAYS RECOVERY TIME (B)  Weight NH .C l i n j e c t e d I n i t i a l F i n a l E f f e c t (g) ( u mole/100 g) pH [H ] pH_ [H ] n A Died 393.4+ 329.0±211.1 4 112.6 B Surv ived 355.4± 246.1± 6 5 . 1 7 .711 1.993±0.423 7.033 9.997±3.086 7 73 .0 x 1 0 " 8 x 1 0 " 8 B Died 311.9± 522.5±117.7 7.610 2.514±1.052 6.769 1.743±0.643 4 47.7 x 1 0 " 8 x IO" 7 (b) EFFECT OF HCL INJECTION ON PLASMA pH: LESS THAN 5 DAYS RECOVERY TIME (A) VS. 5 OR MORE DAYS RECOVERY TIME (B) Weight HC l i n j e c t e d I n i t i a l F i n a l E f f e c t (g) ( u mole/100 g) pH [H ] pH [H ] n A A f f e c t e d 319.0± 121.0+.27.5 7.768 1.75±0.416 6.710 2.10±1.03 6 and d i e d 8 1 . 8 x 1 0 _ 8 x 1 0 _ 7 33.3% m o r t a l i t y ) B Surv i ved 372.1± 125.0±49.8 7.711 2 .0110.848 6.573 5.91±11.3 4 120.3 x 1 0 ~ 8 x 1 0 " 7 B A f f e c t e d 303.0± 113.9± 7 .3 7.737 1.89±0.449 5.874 3.09±3.95 8 and d i e d 8 2 . 1 x 1 0 _ 8 x 1 0 ~ 6 (12.5% m o r t a l i t y ) - 19 -FIGURE I I I - l DAYS BETWEEN CANNULATION AND HCL INJECTION. VS. FINAL pH OF DORSAL AORTIC BLOOD O f i s h s u r v i v e d C ) f i s h a f f e c t e d # f i s h d i e d r o 7-2J: 6.8 6 .4 o a . 6.0 5.6 4 .8 1 - 20 -© © O © 4 8 TIME (DAYS) - 21 -TABLE III-2 (a) DORSAL AND VENTRAL AORTIC BLOOD AMMONIA AND pH IN CONTROL FISH [NH 3] T [NH3] [NH+] [H +] CM) (,x 10~ 4) (M) (x 10~ 6) (M)(x 10~ 4) pH (M) (x 10" 8) n A Paired VA 5.83 10.20+ 8.51 5.73+0.06 7.910 1.24+ 2.16 2 DA 2.20±5.97 1.98±16.20 2.18±5.84 7.515 3.41±19.00 2 B Paired and unpaired VA 4.69±2.20 7.99± 6.22 4.61±2.14 7.830 1.54± 0.94 4 DA 2.73±2.56 4.611.5.34 2.7H2.54 7.620 2.761 4.63 3 (b) AMMONIA EXTRACTED FROM BLOOD AT THE GILLS [NH 3] T [NH3] [NH+] ymole/lOOg-hr. ymole/lOOg-hr. ymole/lOOg-hr. A Paired 74.1 1.68 72.4 B Paired and unpaired 40.0 0.69 38.8 - 22 -FIGURE I I I - 2 DAYS FOLLOWING CANNULATION (- DAYS OF STARVATION) VS. TOTAL AMMONIA CONCENTRATION OF DORSAL AORTIC BLOOD (mean ± s tandard e r r o r , (n)) - 24 -3 .2 R e a c t i o n to HCl R e a c t i o n to HCl s t a r t e d about 2 minutes from i n i t i a t i o n of i n j e c t -i o n , and c o n s i s t e d of jumping , e r r a t i c v e n t i l a t i o n , and l o s s of e q u i l i b r i u m . The f i s h e i t h e r were v i s i b l y u n a f f e c t e d by the i n j e c t i o n , were a f f e c t e d and recovered c o m p l e t e l y , or w i t h impai red e q u i l i b r i u m , or d i e d d u r i n g the f i r s t hour f o l l o w i n g i n j e c t i o n . Convu ls ions never o c c u r r e d , even f o l l o w i n g a f a t a l i n j e c t i o n of H C l . 3 . 3 R e a c t i o n t o NH.C1 4 R e a c t i o n to NH^Cl began about 5 minutes from i n i t i a t i o n of i n j e c t i o n , and i n c l u d e d g u l p i n g a t the s u r f a c e , coughing , i n c r e a s e d v e n t i l a t i o n f o l l o w e d by sporad ic v e n t i l a t i o n , l o s s of e q u i l i b r i u m , and muscle spasms which ended i n v i o l e n t c o n v u l s i o n s and d e a t h . The f i s h e i t h e r were v i s i b l y u n a f f e c t e d by the i n j e c t i o n , or d i e d . 3 .4 R e a c t i o n to NH.HCCv 4 3 R e a c t i o n to NH^HCO^, which began about 8 minutes from i n i t i a t i o n of i n j e c t i o n , was delayed compared w i t h the r e a c t i o n t imes f o r HCl or NH^Cl i n -j e c t i o n . As w i t h the o ther two i n j e c t i o n s , there was a l o s s of e q u i l i b r i u m , and as w i t h NH^Cl , there were muscle spasms which sometimes l e d to v i o l e n t c o n v u l s i o n s and d e a t h . The f i s h e i t h e r were v i s i b l y u n a f f e c t e d by the i n -j e c t i o n , were a f f e c t e d and r e c o v e r e d , or d i e d . When recovery o c c u r r e d , i t was complete . The c o l o u r of some of the f i s h changed n o t i c e a b l y , from normal , to q u i t e dark , to very l i g h t , and back to normal aga in i f the f i s h r e c o v e r e d . Some f i s h a l s o had n o t i c e a b l y swo l len eyes . 3 .5 D i f f e r i n g R e a c t i o n to HCl vs. Ammonium S o l u t i o n I n j e c t i o n A l l th ree i n j e c t i o n s r e s u l t e d i n the l o s s pf e q u i l i b r i u m i n f i s h , but on ly the i n j e c t i o n of the ammonium s o l u t i o n s r e s u l t e d i n muscle spasms and c o n v u l s i o n s . - 25 -4 . EFFECTS OF NH* OR HCL INJECTION ON BLOOD PARAMETERS 4 In a l l th ree c a s e s , b lood measurements were taken b e f o r e , and one minute f o l l o w i n g i n j e c t i o n . The f i r s t measurements were used as c o n t r o l s . 4 . 1 HCl I n j e c t i o n HCl i n j e c t i o n lowered pHa below c o n t r o l v a l u e s (Par t B, Table I I I - l ( b ) ) . B lood sampled f o l l o w i n g i n j e c t i o n appeared dark red to brown. - 4 Data from one f i s h showed a p r e - i n j e c t i o n [NH^]^ o f 3 .79 x 10 M, - 4 and a p o s t - i n j e c t i o n [NH^]^ of 2 .5 x 10 M, both of which are w i t h i n the range of c o n t r o l v a l u e s from NH^Cl and NH^HCO^ experiments (Table I I I - 3 ) . B lood pH of s u r v i v i n g f i s h re tu rned to normal l e v e l s w i t h i n 40 minutes f o l l o w i n g i n j e c t i o n (F igure I I I - 3 ) . 4 . 2 NH^Cl I n j e c t i o n NH^Cl i n j e c t i o n lowered pHa below c o n t r o l v a l u e s and i n c r e a s e d b lood [ N H 3 J T above c o n t r o l v a l u e s (Table I I I - 3 ) (F igure I I I - 4 ) . B lood sampled f o l -l ow ing i n j e c t i o n appeared ve ry dark r e d . The f i n a l b lood [NH^]^ i n f i s h which d i e d was g r e a t e r than i n those which l i v e d (F igure I I I - 4 ) . However, 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 b lood [NH^]^ and [NH^]. 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 b lood [NH^] i n f i s h which l i v e d or those which d ied (F igure I I I - 5 ) . B lood pH of s u r v i v i n g f i s h re tu rned to normal w i t h i n about 1 hour f o l -l ow ing i n j e c t i o n (F igure I I I - 6 ) . [NH^]^ was not f o l l o w e d a f t e r i n j e c t i o n b e -cause removal of such l a r g e b lood volumes may have r e s u l t e d i n death of the f i s h , which may not have been r e l a t e d to i n j e c t i o n of NH^Cl . 4 . 3 NH^HC03 I n j e c t i o n NH^HCOg i n j e c t i o n r a i s e d both b lood pH and [NH^]^, above c o n t r o l v a l u e s (Table I I I - 3 ) . B lood sampled f o l l o w i n g i n j e c t i o n appeared b r i g h t r e d . 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 the f i n a l b lood [NH^]^ between f i s h which l i v e d and those which d ied (F igure I I I - 4 ) . There was no s i g n i f i c a n t d i f f e r e n c e - 26 -TABLE I I I - 3 pH, H , AND BLOOD AMMONIA BEFORE AND AFTER INJECTION OF NH,CL AND NH.HCO N H ^ C l - i n j e c t e d N H 4 H C 0 3 - i n j e c t e d A l i v e Dead A l i v e A f f e c t e d Dead P H 1 7.711 (.7)* 7.610 (4) 7.646 (3) 7.676 (7) 7.739 (5) p H f 7.033 6.769 7.756 7.854 7.766 1.993+0.42 x I O - 8 2.514+1.05 x 1 0 " 8 2.28311.00 x 1 0 " 8 2.14310.39 x 1 0 " 8 1.845+0.36 x 1 0 " 8 [ H + ] f 9.997±3.09 x I O - 8 1.743±0.64 x 1 0 " 7 1.760±0.44 x I O - 8 1.40410.12 x 1 0 " 8 1.75910.53 x IO" 8 [ N H 3 ] T , i 5.934±3.75 x 1 0 " 4 6.003±4.67 x I O - 4 7.53±11.8 x I O - 4 4 . 2 9 1 1 . 9 3 x 1 0 " 4 5.10+4.05 x I O - 4 [ N H 3 ] T , f 2.146±0.40 x I O - 2 3.92±1.07 x I O - 2 1.78±2.20 x 1 0 ~ 2 2 . 7 5 1 1 . 2 1 x IO" 2 2.7410.89 - 2 x 10 [ N H 3 ] ± 5.62±4.30 x IO" 6 5.55±5.44 x I O ' 6 6.5719.67 x I O - 6 4 . 4 4 1 2 . 1 3 x I O - 6 5.7215.15 x I O - 6 [ N H 3 ] f 4.38±1.68 x 1 0 " 5 4.72±2.68 x I O - 5 3.82+9.44 - 4 x 10 4.26+2.02 - 4 x 10 3 .6112.52 x I O - 4 Weight (g) 355.4±73.0 311.9±47.-7 335.51211.5 293.6+69.8 283.5170.9 Dose (ymole/ 100 g) 246.1±65.1 522.5±117.7 253.7+106.6 342.4+55.3 409.41166.6 * n - 27 -FIGURE I I I - 3 CHANGE IN HYDROGEN ION CONCENTRATION OF DORSAL AORTIC BLOOD WITH TIME FOLLOWING INJECTION OF HCL SOLUTION (mean ± standard e r r o r (n)) - 28 -- 29 -FIGURE I I I - 4 AMMONIUM SOLUTION INJECTED VS. TOTAL AMMONIA CONCENTRATION OF DORSAL AORTIC BLOOD FOLLOWING INJECTION (data from Table I I I - 3 ) NH^HC03 i n j e c t i o n : Qfish survived © f i s h affected # f i s h died NH.Cl i n j e c t i o n : Q 0 f i s h survived j ^ f i s h died < to z: 10 -2 200 400 DOSE (/J M O L E / 100 g ) 600 - 31 -FIGURE I I I - 5 AMMONIUM SOLUTION INJECTED vs. CONCENTRATION OF UNIONIZED AMMONIA OF DORSAL AORTIC BLOOD FOLLOWING INJECTION (data from Table I I I - 3 ) NH^HC03 i n j e c t i o n : O f i s h s u r v i v e d © f i s h a f f e c t e d # f i s h d i e d NH^Cl i n j e c t i o n : fP f i s h s u r v i v e d + f i s h d i e d - 32 -I0" 3 8 6 4 I-< to 10 3: 2 8 •4 6 h 10 r5 - 0 -' d -0-200 400 D O S E (/j MOLE / 100 g ) 600 - 33 -FIGURE I I I - 6 CHANGE IN HYDROGEN ION CONCENTRATION OF DORSAL AORTIC BLOOD WITH TIME FOLLOWING INJECTION OF NH..CL SOLUTION (mean ± s tandard e r r o r (n)) T I M E (MIN.) - 35 -i n b lood [NH^]^ and [NH^]. 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 b lood [NH^]. between f i s h which l i v e d and those which d i e d (F igure I I I-5). 4.4 H C l , NH4C1, and NHjHCC^ I n j e c t i o n s The NH^Cl dose which k i l l e d f i s h was not s i g n i f i c a n t l y d i f f e r e n t than the NH 4HC0 3 dose which k i l l e d f i s h (Table I I I-3) . F i n a l b lood [NH^]^ i n f i s h which d i e d from NH^Cl i n j e c t i o n was h igher than from NH^HCO^ i n j e c t i o n (F igure I I I-4) , but the o p p o s i t e was t r u e f o r f i n a l b lood [NH^] (F igure I I I-5). F i n a l b lood pH i n f i s h which d i e d from e i t h e r NH^Cl or NH^HCC^ i n j e c t i o n was h igher than the f i n a l b lood pH i n f i s h which s u r v i v e d HCl i n j e c t i o n (F igure I I I - 7 ) . 4.5 E f f e c t s of NH .C l on Blood Parameters 4 F o l l o w i n g the i n j e c t i o n there was no s i g n i f i c a n t change i n b lood per cent haematocr i t (Table I I I-4) . A b lood sample taken f o l l o w i n g i n j e c t i o n , and looked at under the l i g h t mic roscope , showed no l y s i n g of red b lood c e l l s . - 36 -FIGURE III-7 TOTAL AMMONIA CONCENTRATION VS. HYDROGEN ION CONCENTRATION OF DORSAL AORTIC BLOOD (NH.Cl and NH,HCO„ data from Table I I I - 3 , HCl data from Table I l l - l ( b ) , normal f i s h compiled from control data from the above two Tables) - 37 -I 0 " 8 r < 10 r7 • 10 -6 NORMAL FISH t 8.0 DIED FROM N H 4 H C 0 3 7.0 pH DIED F R O M N H . C L 4 J 6.0 I U I SURVIVED A F T E R H C L 4 8 10 -3 .-2 10 [ N H 3 ] 4 T ( f ) D A - 38 -TABLE I I I - 4 % HAEMATOCRIT BEFORE AND AFTER INJECTION OF NH,CL SOLUTION Befo re I n j e c t i o n A f t e r I n j e c t i o n 22.8±1.65* 23.2±0.534 * mean ± s tandard e r r o r (n = 4) s tandard e r r o r i s from a r c s i n e / x t ransformed data as i t i s improper to perform s t a t i s t i c s on percentage va lues (Sokal and R o h l f , 1973) . - 39 -DISCUSSION Ammonia has been shown to be t o x i c to f i s h . Th is i s a widespread problem i n many f reshwaters because ammonia can accumulate i n a water system due to e x c r e t i o n by f i s h and p r o d u c t i o n by i n d u s t r y (Table 1 - 1 ) . 1 . TOXICITY OF AMMONIA TO RAINBOW TROUT From my i n j e c t i o n exper iments , I conclude that the death of rainbow t r o u t f o l l o w i n g i n j e c t i o n of e i t h e r NH^Cl or NH^HCO^ was due to ammonia t o x i c i t y , and not exper imenta l p rocedure . My i n j e c t i o n experiments were c a r r i e d out on f i s h which had been cannulated f o r not l e s s than 5 days , as my p r e l i m i n a r y exper iments demon-s t r a t e d tha t t h i s was the minimum time r e q u i r e d f o r the f i s h to recover from a n a e s t h e t i c and h a n d l i n g . Some f i s h which I had cannulated s u r v i v e d , w i t h the cannula i n t a c t , f o r up to th ree weeks, t h e r e f o r e death f o l l o w i n g i n j e c t i o n of ammonium s o l u t i o n s was not due to the c a n n u l a t i o n . F i s h which were i n j e c t e d w i t h C o r t l a n d s a l i n e , at a r a t e i n excess of 0 . 1 ml/minute , were not v i s i b l y a f f e c t e d by the i n j e c t i o n . F i s h which, were i n -j e c t e d w i t h s u b - l e t h a l doses of NH^Cl and NH^HCO^ s u r v i v e d the i n j e c t i o n p r o -cedure , t h e r e f o r e the i n j e c t i o n procedure was not the cause of death f o l l o w i n g i n j e c t i o n of ammonium s o l u t i o n s . pHa o f f i s h which s u r v i v e d i n j e c t i o n of HCl was much lower than pHa of f i s h which d i e d f o l l o w i n g NH^Cl or NH^HCO^ i n j e c t i o n (F igure I I I - 7 ) , so death was not due to changes i n pHa caused by i n j e c t i o n of ammonium s o l u t i o n s . I n j e c t i o n of NaHCO^ i n t o rainbow t r o u t (Janssen and R a n d a l l , 1975) , a l though i n c r e a s i n g pHa to l e v e l s s i m i l a r to those I measured f o l l o w i n g NH^-HCO^ i n j e c t i o n , d i d not k i l l t r o u t . F i s h which d ied f o l l o w i n g HCl i n j e c t i o n d i d not e x h i b i t i d e n t i c a l symptoms to those which d i e d f o l l o w i n g NH^Cl i n j e c t -i o n . Thus death f o l l o w i n g i n j e c t i o n of ammonium s o l u t i o n s was not due to the accompanying a n i o n . - 40 -The above r e s u l t s demonstrate that death, f o l l o w i n g i n j e c t i o n of an ammonium s o l u t i o n was not due to c a n n u l a t i o n or i n j e c t i o n t e c h n i q u e s , nor to the e f f e c t of the hydrogen i o n c o n c e n t r a t i o n i n the b l o o d , nor to the an ion accompanying the ammonium i o n . Symptoms which I observed were c o n s i s t e n t w i t h those observed i n o t h e r an imals as a r e s u l t of ammonia t o x i c i t y (Warren, 1958; Warren and Nathan, 1958; F l i s , 1963a; W i l s o n et al., 1968; W i l s o n et al., 1969; Smart, 1975) . 2 . TOXIC LEVELS OF AMMONIA 2 . 1 In Water Wuhrman et aZ . ( 1 9 4 7 ) , Wuhrmann and Woker (1948) , and Smart (1975) have demonstrated t h a t the u n i o n i z e d f r a c t i o n of ammonia i n water i s the t o x i c form, as i t i s a b l e to enter the f i s h . Table I V - 1 c o n t a i n s v a l u e s from the l i t e r a t u r e of t o x i c c o n c e n t r a t i o n s of u n i o n i z e d ammonia which are r e l a t i v e l y c o n s t a n t . I have i n c l u d e d c o n t r o l b lood u n i o n i z e d ammonia c o n c e n t r a t i o n s from my own data and from Smart (1975) , which are much lower than the t o x i c l e v e l s found i n the water . 2 .2 In B lood I have c a l c u l a t e d from my r e s u l t s tha t the t o x i c dose from NH^Cl i n j e c t i o n , 522.5±117.7 ymole/lOO g , i s not s i g n i f i c a n t l y d i f f e r e n t from the t o x i c dose from NH^HCO^ i n j e c t i o n , 409.4±166.6 ymole/100 g . Once the c o n -c e n t r a t i o n of ammonia i n the f i s h i s s u f f i c i e n t to cause d e a t h , i t appears , from my d a t a , that the i o n i z e d form i s t o x i c . NH^HCO^-injected f i s h had a much h i g h e r l e v e l of u n i o n i z e d ammonia i n t h e i r b l o o d , but N H ^ C l - i n j e c t e d f i s h which d i e d from the i n j e c t i o n had a much s h o r t e r r e a c t i o n t i m e . A l s o , the l e v e l s of u n i o n i z e d ammonia i n f i s h which d i e d f o l l o w i n g NH^Cl i n j e c t i o n was much lower than i n f i s h which s u r v i v e d NH/HCO^ i n j e c t i o n (F igure I I I - 5 ) . TABLE I V - 1 THRESHOLD L C c n VALUES OF VARIOUS SOLUTIONS OF AMMONIA FOR RAINBOW TROUT (Salmo gairdneri) Ammonium S o l u t i o n Ammonium c h l o r i d e Ammonium su lphate Ammonium su lphate 7 . 8 6 - 8 . 2 2 8 .10 7 . 0 , 7 . 6 , 8 . 1 Temperature (°C) 1 0 . 5 - 1 1 . 6 10 .5 15.0 Unionized Ammonia (Ac tua l Data) 0 .41 mg N/1 0.39 ppm N Converted f o r Comparison [NH33 (UM) 2 9 . 3 27 .9 0.445 mg NH 3 -N/1 3 1 . 8 C o n t r o l B lood [NHj] (UM) 8 .2 5 . 3 - 5 . 6 Reference B a l l , 1967 L l o y d and O r r , 1969 Smart , 1975 Th is -p-study M - 42 -3 . SYMPTOMS OF AMMONIA TOXICITY I observed h y p e r v e n t i l a t i o n , h y p e r e x c i t a h i l i t y , l o s s of e q u i l i b r i u m , c o n v u l s i o n s , and death i n rainbow t r o u t (S. gairdneri), f o l l o w i n g i n t r a -a r t e r i a l i n j e c t i o n of e i t h e r NH^Cl or NH^HCO^. S i m i l a r responses to ammonia have been observed i n the u r e o t e l i c mouse .(Mus musculus) (Warren, 1958; Warren and Nathan, 1958; W i l s o n et al., 1968) , the u r i c o t e l i c c h i c k (Gallus domesticus) (Wi lson et al., 1968, and ammoneotelic f i s h , carp (Cypvinus earpio) ( F l i s , 1963a) , g o l d f i s h (Carassius auvatus), and channel c a t f i s h (lotaluvus punctatus) (Wi lson et al., 1969) . Because these symptoms are i n d i c a t i v e of n e u r a l d i s -o r d e r , I conclude that ammonia t o x i c i t y i s due to the a c t i o n of ammonia on the c e n t r a l nervous system. As my d a t a i n d i c a t e s tha t there i s no d i f f e r e n t i a l t o x i c i t y i n f i s h between i n j e c t i o n of a low pH or a h i g h pH ammonium s o l u t i o n , a l though a d i f f e r e n t i a l t o x i c i t y has been demonstrated i n mice (Warren, 1958; Warren and Schenker , 1962) , I f u r t h e r conclude tha t the mechanisms of t o x -i c i t y may d i f f e r between s p e c i e s , depending on whether the s p e c i e s i s u r e o -t e l i c , u r i c o t e l i c , or ammoneotel ic . 3 . 1 Symptoms i n Mice and Ch icks Symptoms of t o x i c i t y i n mice appear w i t h i n 2 to 5 seconds f o l l o w i n g a s i n g l e , r a p i d in t ravenous i n j e c t i o n of an ammonium s o l u t i o n (Warren, 1958; Warren and Nathan, 1958; W i l s o n et al„ 1968) . These symptoms i n c l u d e h y p e r -v e n t i l a t i o n and h y p e r e x c i t a h i l i t y f o l l o w e d by a s e r i e s of v i o l e n t , i n v o l u n t a r y muscle spasms which end i n a coma. The coma may l a s t from 30 to 40 minutes , d u r i n g which t ime the mice show no response to touch or l i g h t , but move c o n -v u l s i v e l y to sound. The coma may end w i t h a f a t a l t o n i c extensor c o n v u l s i o n , or the mice may r e g a i n c o n s c i o u s n e s s , i n which case they make a r a p i d and complete recovery (Warren, 1958) . S i m i l a r symptoms occur i n both the c h i c k and the mouse, f o l l o w i n g an i n t r a p e r i t o n e a l i n j e c t i o n , a l though the symptoms are de layed 10 to 15 minutes - 43 -(Wi lson et al., 1968) . W i l s o n et al. (1968) n o t i c e d a d i f f e r e n c e i n symptoms between the two s p e c i e s f o l l o w i n g in t ravenous i n j e c t i o n i n that mice which s u r v i v e d showed no s i g n s of a coma. However, as the coma was observed i n mice by Warren (1958) , the o b s e r v a t i o n of W i l s o n et al. (1968) cou ld be due to dosage d i f f e r e n c e s . 3 .2 Symptoms i n F i s h Symptoms s i m i l a r to those observed i n the c h i c k and mouse have been observed i n f i s h , r e g a r d l e s s of the method of exposure to ammonia. Carp ( F l i s , 1963a) , and rainbow t r o u t (Smart, 1975) exposed to ammonia i n the water e x -h i b i t e d h y p e r e x c i t a b i l i t y , h y p e r v e n t i l a t i o n , v i o l e n t e r r a t i c movements, coma, and c o n v u l s i o n s . These symptoms were r a p i d l y reversed i f the f i s h were p laced i n ammonia-free w a t e r , o therwise death occur red (Smart, 1975) . The same symptoms appeared i n g o l d f i s h , rainbow t r o u t , and channel c a t f i s h 10 to 15 minutes f o l l o w i n g an i n t r a p e r i t o n e a l i n j e c t i o n (Wi lson et al., 1969. I n t r a -a r t e r i a l i n j e c t i o n of ammonium s o l u t i o n s produced the same symptoms i n r a i n -bow t r o u t ( t h i s s t u d y ; Smart, 1975) . 4. MECHANISM OF TOXICITY A l though s i m i l a r symptoms of ammonia t o x i c i t y are found i n f i s h and m i c e , my data i n d i c a t e s tha t the mechanisms of t o x i c i t y may d i f f e r i n some r e s p e c t s . Warren and Schenker (1962) demonstrated that a decrease i n pHa of mice f o l l o w i n g HCl i n f u s i o n i n c r e a s e d p r o t e c t i o n a g a i n s t ammonium b i c a r b o n a t e t o x i c i t y . Warren (1958) a l s o demonstrated that t o x i c i t y to mice of v a r i o u s ammonium compounds i n c r e a s e d a c c o r d i n g to the a b i l i t y of the compound to i n -c rease pHa. My r e s u l t s i n d i c a t e t h a t , i n f i s h , there i s no d i f f e r e n t i a l t o x i c i t y r e l a t e d to change i n pHa, and no i n c r e a s e d t o x i c i t y w i t h an i n c r e a s e i n u n i o n i z e d b lood ammonia. - 44 -4 . 1 B l o o d - B r a i n B a r r i e r The b l o o d - b r a i n b a r r i e r separates the b r a i n and c e r e b r o s p i n a l f l u i d (CSF) from the b l o o d . I t i s a s e r i e s of r e g u l a t o r y i n t e r f a c e s between b lood and the nervous system. There are r e g i o n s , however, where the b a r r i e r i s not complete (Rapoport , 1976) . Ev idence tha t on ly u n i o n i z e d ammonia can c r o s s the b l o o d - b r a i n b a r r i e r i n mammals was p rov ided by Stabenau et al. (1958) . Measurements of b r a i n and b lood ammonia i n dogs showed tha t when NaOH was i n f u s e d , pHa i n -c r e a s e d , b r a i n and CSF pH showed no change, and the r a t i o s of b r a i n to b lood and CSF to b lood ammonia i n c r e a s e d . When HCl was i n f u s e d there was l i t t l e or no change. However, these experiments were not repeated a f t e r ammonia l o a d i n g . Bromberg et al. (1960) measured t o t a l ammonia c o n c e n t r a t i o n i n b lood and CSF of dogs i n f u s e d w i t h s u b l e t h a l l e v e l s of ammonium a c e t a t e and i n un in fused dogs. They found no s i g n i f i c a n t d i f f e r e n c e i n CSF/blood t o t a l ammonium c o n c e n t r a t i o n r a t i o between the i n f u s e d and un in fused dogs. In f i s h , which have a b r a i n of somewhat d i f f e r e n t s t r u c t u r e than mammals, ammonia- loading cou ld have l o c a l i z -ed e f f e c t s on the b r a i n , e s p e c i a l l y i f the b a r r i e r i s not complete . I t cou ld a l s o cause a break-down i n the systems which he lp to m a i n t a i n homeostasis of the CSF. Ammonia i s s e q u e n t i a l l y metabo l i zed i n the f i s h b r a i n by GDH (Equat ion 4) and g lutamine synthetase (Equat ion 5) (Mehrle and B l o o m f i e l d , 1974) . + + GDH a o x o g l u t a r a t e + NADH + H + NH^ ^ — ^ glutamate + NAD + H 2 0 (4) g lutamate + ATP + NH+ g lutamine synthetase glutamine + ADP + orthophosphate (5) - 45 -H y p e r e x c i t a h i l i t y , c o n v u l s i o n s , and coma ev ident i n f i s h f o l l o w i n g exposure to ammonia are symptomatic of n e u r o l o g i c a l d i s o r d e r . Smart (1975) measured ATP and PC i n the f i s h m e d u l l a , and found both to be s i g n i f i c a n t l y decreased as a consequence of ammonia exposure . He suggested tha t d e p l e t i o n of these b r a i n energy sources c o u l d account f o r the n e u r o l o g i c a l e f f e c t of ammonia. 4 . 2 Evidence f o r NH^ S u b s t i t u t i o n i n P a s s i v e Transport B i n s t o c k and Lecar (1969) have shown, i n the g i a n t squ id axon , that NH^ can s u b s t i t u t e f o r the p a s s i v e movement of both N a + and K + i n a nervous i m p u l s e . Once NH^ i s i n s i d e the axon, i t tends to b l o c k the inward f l o w of N a + . B i n s t o c k and Lecar (1969) suggest tha t the N a - l i k e permeat ion e x p l a i n s NH^ e x c i t a b i l i t y of n e r v e s . 4 . 3 Ev idence f o r NH^ S u b s t i t u t i o n i n A c t i v e T ranspor t Mossberg (1967) , i n s t u d y i n g ammonia a b s o r p t i o n i n the hamster i l e u m , demonstrated tha t net movement of t o t a l ammonia from the mucosal to the s e r o s a l s i d e of the i l eum was independent of the f i n a l pH and f r e e + ammonia g r a d i e n t s , and to account f o r t h i s , suggested NH^ t r a n s p o r t r a t h e r than p a s s i v e NH^ movement ac ross the membrane. He f u r t h e r demonstrated t h a t movement of ammonia ac ross the i l e a l membrane was f a c i l i t a t e d by b i c a r b o n a t e and/or CO2, but cou ld not suggest a mechanism. Ev idence f o r the i n t e r a c t i o n s of NH^~, N a + , and K + i s g i ven by Post and J o l l y (1957) , who demonstrated t h a t , ac ross the human e r y t h r o c y t e membrane, NH^ and K + compete f o r s i t e s both f o r inward movement, and i n the a c t i v a t i o n of N a + t r a n s p o r t out of the c e l l . I n s t u d y i n g the a b s o r p t i o n of sodium by the c r a y f i s h , Shaw (1960) found among the c a t i o n s s t u d i e d (Mg , Ca , K , NH^) tha t NH^ i n the e x t e r n a l medium caused the g r e a t e s t r e d u c t i o n of N a + i n f l u x . He suggested t h a t NH^ competes w i t h N a + f o r t r a n s p o r t s i t e s . Th is type of ev idence suppor ts my f i n d i n g s , tha t NH^ w i t h i n the c i r c u l a t o r y system of f i s h e s i s t o x i c , by showing tha t NH* can be a c t i v e l y moved across membranes. - 46 -5 . EFFECTS OF AMMONIA ON BLOOD Controversy e x i s t s about e f f e c t s of ammonia on both the oxygen c a p a c i t y of f i s h b l o o d , and on b lood pH. My r e s u l t s c o n f i r m the f i n d i n g s of Warren (1958) and Warren and Nathan (1958) , work ing on m i c e , tha t changes i n b lood pH r e s u l t i n g from the i n j e c t i o n of an ammonium s o l u t i o n , i n f i s h as w e l l as i n m i c e , are r e l a t e d to the p a r t i c u l a r ammonium s a l t i n j e c t e d . I n j e c t i o n of NH^Cl r e s u l t e d i n a d e -c rease i n pHa, w h i l e the b lood appeared dark r e d . The dark red c o l o u r can be e x p l a i n e d by the Root and Bohr e f f e c t s due to the decrease i n pH. Converse l y , i n j e c t i o n of NH^HCO^ r e s u l t e d i n an i n c r e a s e i n pHa, w i t h the b lood becoming b r i g h t r e d . The b r i g h t red c o l o u r can be e x p l a i n e d by the r e v e r s e Root and Bohr e f f e c t s , t h a t i s both an i n c r e a s e i n oxygen c a r r y i n g c a p a c i t y of the b l o o d and an i n c r e a s e i n the a f f i n i t y of haemoglobin f o r oxygen, due to the i n c r e a s e i n pH. My r e s u l t s , tha t i n j e c t i o n o f NH^Cl had no e f f e c t on per cent h a e m a t o c r i t , a re i n agreement w i t h exper iments c a r r i e d but by Smart (1975) , that exposure to ammonia i n the water had no e f f e c t on per cent haematocr i t i n t r o u t . Smart (1975) a l s o found tha t ammonia exposure had no e f f e c t on e r y t h r o c y t e count or haemoglobin c o n c e n t r a t i o n . Sousa and Meade (1977) looked a t the a b s o r p t i o n c o n f i g u r a t i o n of haemoglobin from coho (Oncorhynchus kisutch) exposed to ammonia. Over a 14 day p e r i o d , they found a p r o g r e s s i v e t r a n s f o r m a t i o n from an a b s o r p t i o n c o n -f i g u r a t i o n c h a r a c t e r i s t i c of oxygenated haemoglobin to one c h a r a c t e r i s t i c of deoxygenated haemoglobin, w h i l e at the same time pH of the haemoglobin s o l u -t i o n decreased . In t e l e o s t f i s h , a r e d u c t i o n i n b lood pH causes both a r e -d u c t i o n i n the a f f i n i t y of haemoglobin f o r oxygen (Bohr e f f e c t ) and a r e d u c t i o n i n the oxygen c a r r y i n g c a p a c i t y of the b lood (Root e f f e c t ) ( R a n d a l l , 1970) . - 47 -Smart (1975) found t r o u t b lood Po^ decreased f o l l o w i n g exposure of f i s h to ammonia, w i t h no e f f e c t on b lood P^Q' He r e l a t e d the decrease i n Po^ to e i t h e r a h i g h r a t e of oxygen uptake by the t i s s u e s , o r as a consequence of an inba lanced v e n t i l a t i o n / p e r f u s i o n r a t i o due to h i g h g i l l v e n t i l a t i o n and p e r -f u s i o n r a t e s . Smart (1975) de tec ted no change i n pHa of t r o u t exposed to ammonia. He quoted per cent of s u r v i v a l t ime r a t h e r than days of exposure i n t h i s set of exper iments , but o ther exper iments which he c a r r i e d out were on ly of 96 hours d u r a t i o n , so i t i s probable that pH was measured on ly over a r e l -a t i v e l y shor t p e r i o d of t i m e . There i s a p o s s i b i l i t y t h a t , i n cases of c h r o n i c t o x i c i t y , ammonia cou ld have an e f f e c t on f i s h pHa, and t h e r e f o r e , an e f f e c t on the oxygen content of the b l o o d . A l though Brockway (1950) s t a t e d tha t an i n c r e a s e i n e x t e r n a l ammonia reduced b lood 0^ content and i n c r e a s e d b lood content i n f i s h , he i n c l u d e d no a c t u a l d a t a , made no r e f e r e n c e to how b lood gas t e n s i o n s were measured, or how they d i f f e r e d from c o n t r o l v a l u e s . H i s h y p o t h e s i s , that ammonia a f f e c t e d the a b i l i t y of haemoglobin to combine w i t h or to l i b e r a t e cannot be c r i t i c a l l y eva luated due to t h i s l a c k of d a t a . In vitro s t u d i e s by Fromm and G i l l e t t e (1968) , i n which f i s h b lood was spun down and the red b lood c e l l s resuspended i n s a l i n e , i n d i c a t e d tha t a d d i t i o n of ammonia had no s i g n i f i c a n t e f f e c t on the a b i l i t y of haemoglobin to combine w i t h oxygen. In vitro exper iments done by Haswel l (pe rsona l communica-t i o n ) , i n which t r o u t b lood was spun down and red b lood c e l l s resuspended i n s a l i n e , showed tha t the p r o p e r t i e s of the c e l l s , w i t h r e s p e c t to CO^ p r o d u c t i o n , were d i f f e r e n t i n s a l i n e than i n f i s h p lasma. The p o s s i b i l i t y that p r o p e r t i e s of red b lood c e l l s w i t h r e s p e c t to ammonia a re d i f f e r e n t i n s a l i n e than i n f i s h plasma must be checked be fo re Fromm and G i l l e t t e ' s experiments can be accepted . - 48 -Based on the f o r e g o i n g exper iments , I c o n s i d e r that any e f f e c t ammonia may have on the oxygen c a p a c i t y of f i s h b lood i s an i n d i r e c t one r e l a t e d to changes i n pHa which, may r e s u l t from exposure to ammonia, or to the a c t i o n of n i t r i t e , which may be formed by b a c t e r i a l a c t i o n on ammonia. The a c t i o n of n i t r i t e may r e s u l t i n the fo rmat ion of methaemoglobin, and t h e r e f o r e reduce the oxygen c a p a c i t y of the b l o o d . 6. OTHER FACTORS AFFECTING AMMONIA TOXICITY IN FISH The problem of ammonia t o x i c i t y to f i s h has been s t u d i e d e x t e n s i v e -l y . In an attempt to c l a r i f y the r e l a t i o n s h i p between ammonia t o x i c i t y and env i ronmenta l f a c t o r s , r e s e a r c h e r s have looked i n t o the r e l a t i o n s h i p between ammonia and pH (Wuhrmann and Woker, 1948; Smart, 1975) , d i s s o l v e d carbon d i o x i d e ( A l a b a s t e r and H e r b e r t , 1954; L l o y d and H e r b e r t , 1960) , d i s s o l v e d oxygen (Downing and Merkens, 1955; Merkens and Downing, 1957; Larmoyeux and P i p e r , 1973) , and n i t r i t e (B .C . Research , 1974) . 6 . 1 pH Experiments performed by Wuhrmann and Woker (1948) and Smart (1975) , i n which water temperature and t o t a l ammonia c o n c e n t r a t i o n s were kept constant w h i l e pH was v a r i e d , showed tha t s u r v i v a l t ime of rainbow t r o u t decreased w i t h an i n c r e a s e i n water pH. As the c o n c e n t r a t i o n of u n i o n i z e d ammonia i n c r e a s e d w i t h an i n c r e a s e i n pH, they concluded that the u n i o n i z e d f r a c t i o n was t o x i c . Wuhrmann and Woker (1948) suggested t h a t t o x i c i t y was c o r r e l a t e d w i t h g i l l p e r m e a b i l i t y . Th is hypothes i s i s c o n s i s t e n t w i t h the p h y s i c a l p r o p e r t i e s of ammonia. ML^J b e i n g l i p i d s o l u b l e and uncharged, i s more e a s i l y a b l e to d i f f u s e ac ross membranes than the l a r g e r NH*, w h i c h , i n a d d i t i o n to hav ing a charge , has a low l i p i d s o l u b i l i t y . Th is h y p o t h e s i s i s f u r t h e r supported by experiments c a r r i e d out by Smart (.1975). My a n a l y s i s of measurements of plasma ammonia of rainbow t r o u t made by Smart (1975) , both b e f o r e and a f t e r exposure to ammonia, i n d i c a t e two - 49 -e f f e c t s . Plasma ammonia i n c r e a s e d over the c o n t r o l v a l u e f o l l o w i n g 24 hours of exposure to ammonia. Secondly , f o r the same e x t e r n a l ammonia, but a t d i f f e r e n t hydrogen i o n c o n c e n t r a t i o n s , plasma ammonia f o l l o w i n g exposure a t the h igher pH was almost tw ice tha t found at the lower pH (Table I V - 2 ) . 6 .2 C 0 2 A d d i t i o n of CC^ to s o l u t i o n s c o n t a i n i n g ammonia may i n c r e a s e the s u r v i v a l t ime of the f i s h ( A l a b a s t e r and H e r b e r t , 1954) , or i t may decrease the amount of u n i o n i z e d ammonia r e q u i r e d to k i l l the f i s h (L loyd and H e r b e r t , 1960) . Carbon d i o x i d e , a t c o n c e n t r a t i o n s h igher than 30 ppm, i s i n i t s e l f t o x i c ( A l a b a s t e r and H e r b e r t , 1954) . A d d i t i o n of CO^ caused a r e d u c t i o n i n water pH (Equat ion 6) and , t h e r e f o r e , a r e d u c t i o n i n the c o n c e n t r a t i o n of u n i o n i z e d ammonia (Equat ion 2) (Table IV -3 ) (Herbert and A l a b a s t e r , 1954; L l o y d and H e r b e r t , 1960) . C 0 2 + H 2 0 N H + + HC0~ (6) R e s u l t s from Herber t and A l a b a s t e r (1954) show that a d d i t i o n of C 0 2 decreased the c o n c e n t r a t i o n of u n i o n i z e d ammonia i n water w e l l below the lowest l e t h a l c o n c e n t r a t i o n demonstrated by L l o y d and Herber t (1960) (Table I V - 3 ) . From these two exper iments i t i s c l e a r tha t p rov ided the i n c r e a s e i n C 0 2 r e s u l t s i n a decrease of u n i o n i z e d ammonia i n water below t o x i c l e v e l s , s u r v i v a l t ime of f i s h w i l l be i n c r e a s e d . L l o y d and Herber t (1960) found tha t at h i g h C 0 2 c o n c e n t r a t i o n s , l e s s u n i o n i z e d ammonia was r e q u i r e d to k i l l f i s h . They suggested that r e s -p i r a t o r y C 0 2 lowered water pH, and t h e r e f o r e u n i o n i z e d ammonia, a t the g i l l s u r f a c e , w i t h the magnitude of the decrease be ing dependent on the c o n c e n t r a -t i o n of C 0 2 i n the t e s t s o l u t i o n . That i s , the decrease would be g rea te r when the C 0 2 content of the t e s t s o l u t i o n was low. However, t h i s hypothes i s does not take i n t o account the e f f e c t of e x t e r n a l C 0 2 on f i s h b l o o d . An i n c r e a s e i n env i ronmenta l P „ r t r e s u l t s i n an i n c r e a s e i n P a ^ and a decrease i n pHa - 5 0 -TABLE IV-2 PLASMA AMMONIA CONCENTRATIONS BEFORE AND AFTER EXPOSURE TO AMMONIA AT DIFFERENT HYDROGEN ION CONCENTRATIONS (from Smart, 1 9 7 5 ) Water Plasma: [NH 3] T (M x 1 0 ~ 4 ) PH [NH 3] T (M x 1 0 ) pH Before exposure: 4 . 1 4 7 . 8 8 After exposure: 3 . 3 9 5 . 8 1 7 . 1 4 7 . 1 4 8 . 9 3 1 4 . 3 2 8 . 6 2 8 . 6 8 . 2 8 . 1 7 . 6 8 . 0 5 . 6 5 . 6 . 0 7 . 4 8 . 1 4 1 2 . 3 7 . 1 1 1 2 . 8 1 0 . 7 1 3 . 6 7 . 1 1 8 . 1 TABLE IV -3 COMPARISON OF RESULTS FROM ALABASTER AND HERBERT (1954) (a) WITH RESULTS FROM LLOYD AND HERBERT (1960) (b) T o t a l % Un ion ized A c t u a l Un ion i zed Temperature (°C) pH 7.91 Ammonia (ppm N) Ammonia (ppm N) Ammonia (ppm N) Free C 0 2 (ppm) Time (min) E f f e c t (a) 17 .5 30 2.62 0.79 0 720 l e t h a l 17 .5 7.40 30 0 .81 0.24 30 720 none (b) ft* 19.8 8.20 14 .5 5.82 0.84 2 .3 500 l e t h a l 20.7 7.80 23.9 2 .61 0.62 7.7 500 l e t h a l 18 .6 7.37 50 .5 0.84 0.42 21 .5 500 l e t h a l 20 .5 7.00 119.0 0 .41 0.49 4 8 . 0 500 l e t h a l I—1 * approximate va lues c a l c u l a t e d from T r u s s e l l (1972) ** temperatures from L loyd (1961) - 52 -(Janssen and R a n d a l l , 1975; Eddy et dL, 1976) , and , t h e r e f o r e , an i n c r e a s e i n NH~!" i n f i s h b l o o d . 4 Un ion i zed ammonia i n water i s r e f e r r e d to as the t o x i c fo rm, as i t i s t h i s form which i s thought to ente r f i s h (Wuhrmann and Woker, 1948; Smart, 1975) . But my data show that once i n the c i r c u l a t o r y system, the i o n i z e d form i s t o x i c (F igures I I . I -4 and I I I - 5 ) . At h i g h e x t e r n a l CO^, water and b lood pH decrease . Less u n i o n i z e d ammonia would be a v a i l a b l e to enter f i s h , but once i n the b lood a g r e a t e r p r o p o r t i o n would be converted to the t o x i c i o n i z e d f r a c t i o n . C o n v e r s e l y , a t low e x t e r n a l CO^, more u n i o n i z e d ammonia would be a v a i l a b l e to enter the f i s h , and once i n the b lood a g r e a t e r p r o p o r t i o n would remain i n t h i s form. 6.,3 0 2 S u r v i v a l t ime of f i s h exposed to ammonia s o l u t i o n s can be i n c r e a s e d by i n c r e a s i n g the l e v e l s of d i s s o l v e d oxygen (Downing and Merkens, 1955; Merkens and Downing, 1957) . However, t o x i c symptoms a t low l e v e l s of d i s -so lved oxygen may be the r e s u l t of hypox ia r a t h e r than ammonia (Larmoyeux and P i p e r , 1973) , or a combinat ion of both hypox ia and ammonia t o x i c i t y (Merkens and Downing, 1957) . In a water reuse system, f i s h showed a r e d u c t i o n i n growth r a t e , and damage to g i l l , k i d n e y , and l i v e r t i s s u e s , w i t h l e s s than 5 ppm oxygen and more than 0 . 5 ppm ammonia present (Larmoyeux and P i p e r , 1973) . None of these symptoms were apparent i n f i s h w i t h water c o n t a i n i n g more than 7 ppm oxygen and from 0 . 8 to 1 . 0 ppm ammonia. Larmoyeux and P i p e r (1973) suggested the symptoms were due to h y p o x i a , r a t h e r than ammonia t o x i c i t y . Merkens and Downing (1957) a l s o i n v e s t i g a t e d the e f f e c t s of low l e v e l s of u n i o n i z e d ammonia on the r e s i s t a n c e of t r o u t , p e r c h , and roach to h y p o x i a . They found t h a t , i n t r o u t o n l y , u n i o n i z e d ammonia reduced the p e r i o d of s u r v i v a l . Low oxygen causes a decrease i n pHa i n t r o u t (Ho le ton , 1967) . - 53 -Therefore the same argument may apply for low 0^ as was used for high CO^. That i s , a decrease, i n pHa w i l l Increase the amount of ionized ammonia i n the blood, and therefore create toxic ammonium ion levels i n the blood, 6.4 N i t r i t e Ammonia may be oxidized to n i t r i t e by bacteria (B.C. Research, 1974). These bacteria, together with bacteria which convert n i t r i t e to n i t r a t e , are used i n b i o l o g i c a l f i l t e r s to remove ammonia from water (Smart, 1975). In systems, such as f i s h hatcheries, which r e l y on water reuse and b a c t e r i a l f i l t e r s , n i t r i t e concentrations may b u i l d up from excreted ammonia and from incomplete conversion of n i t r i t e to n i t r a t e . N i t r i t e has two main actions on f i s h blood: reduction of t o t a l haemoglobin, and conversion of haemoglobin to methaemoglobin (Brown and McLeay, 1975). The oxygen transporting c a p a b i l i t i e s of f i s h blood are reduced as a result of these actions. Poor growth rate and b a c t e r i a l g i l l disease may be due to n i t r i t e , rather than ammonia, t o x i c i t y (Brown and McLeay, 1975). Exposure to ammonia alone caused no s i g n i f i c a n t change i n methaemoglobin levels i n coho salmon (Sousa and Meade, 1977). 7. AMMONIA PRODUCTION AND EXCRETION BY FISH My measurements of blood ammonia (Table IV-4) show a si g n i f i c a n t decrease i n blood ammonia between the ventral and dorsal aorta, and add sup-port to the hypothesis that most, i f not a l l , of the ammonia excreted at the g i l l s comes from the blood. 7.1 Enzymes Involved i n Ammonia Production Various enzymatic reactions which resu l t i n deamination of amino acids, amides, nucleosides, aminopurines, aminopyrimidines, and hexosamines, can produce ammonia (Cohen and Brown, J r . , 1960). In trout, the major source of ammonia i s from the breakdown of dietary protein. Two of the enzymes thought to be important i n the formation of ammonia i n f i s h are glutamate - 54 -dehydrogenase (GDH) and g lutaminase (Walton and Cowey, 1977) , which c a t a l y s e the f o l l o w i n g r e a c t i o n s : : g lutaminase + g lutamine + H 2 0 ~" glutamate + NH^ (7) g lutamate + NAD + H 2 0 ^ a o x o g l u t a r a t e + NADH + H + NH^ (8) A t h i r d enzyme, g lutamine synthetase (Equat ion 5 ) , i s a c t i v e on ly i n the b r a i n of f i s h and i s cons idered important i n d e t o x i f y i n g , r a t h e r than f o r m i n g , ammonia (Webb and Brown, J r . , 1976) . AMP deaminase, an enzyme i n wh i te muscle of f i s h , i s r e s p o n s i b l e f o r the fo rmat ion of ammonia i n muscle d u r i n g e x e r c i s e ( D r i e d z i c , 1975) . McBean et al. (1966) measured GDH a c t i v i t y i n the l i v e r , k i d n e y , g i l l , and muscle t i s s u e s of the e e l (knguilla rostvata), and found the h i g h e s t a c t i v -i t y i n the l i v e r . Walton and Cowey (1977) measured the a c t i v i t i e s of both GDH and g lutaminase i n the same t i s s u e s i n rainbow t r o u t and found the h i g h e s t a c t i v i t i e s f o r both enzymes i n the l i v e r t i s s u e . AMP deaminase i s not found i n the l i v e r of t r o u t (Walton and Cowey, 1977) . 7 .2 Major Source of Ammonia Pequin and S e r f a t y (1963) measured ammonia i n b lood sampled from the v e n t r i c l e , d o r s a l a o r t a , cauda l v e i n s , c a r d i n a l v e i n s subsequent to l e a v i n g the k i d n e y s , and the suprahepat i c v e i n s , and found the h i g h e s t c o n c e n t r a t i o n i n the v e i n s d r a i n i n g the l i v e r . The h i g h enzyme a c t i v i t i e s i n f i s h l i v e r (McBean et al., 1966; Walton and Cowey, 1977) , together w i t h h i g h c o n c e n t r a t i o n s of ammonia i n v e i n s d r a i n i n g the l i v e r (Pequin and S e r f a t y , 1963; V e l l a s and S e r f a t y , 1974) support the h y p o t h e s i s tha t the l i v e r i s the major source of b lood ammonia i n r e s t i n g f i s h . [NH*] of wh i te muscle i n e x e r c i s i n g f i s h i n c r e a s e s s i g n i f i c a n t l y over that found i n wh i te muscle of r e s t i n g f i s h , due to the a c t i o n of AMP d e -aminase ( D r i e d z i c , 1975) . As no concomitant measurements of b lood ammonia were made, i t i s not known i f t h i s p rocess i s a source of b lood ammonia i n - 55 -e x e r c i s i n g f i s h . 7 .3 S i t e of Ammonia E x c r e t i o n Exper iments performed by Smith (1929) on f i s h i n which water c i r c u l -a t i n g through the g i l l s was separated from water sur rounding the r e s t of the body, and ammonia measured i n both compartments, c l e a r l y demonstrated tha t the g i l l s are the major s i t e of ammonia e x c r e t i o n i n t e l e o s t f i s h . Subsequent e x -per iments by Wood (1958) , Fromm (1963) , Fromm and G i l l e t t e (1968) , and Payan and Matty (1975) support these f i n d i n g s , and i n d i c a t e tha t the k idney accounts f o r l e s s than 3% of the t o t a l ammonia e x c r e t e d . 7.4 Ammonia E x t r a c t i o n from Blood G o l d s t e i n " and F o r s t e r (1961) suggested tha t the amount of ammonia exc re ted by the marine f i s h Myoxocephalus saovpi-us cou ld be accounted f o r by the combined a c t i o n of g lutaminase and GDH i n g i l l t i s s u e e x t r a c t s , but l a t e r s t u d i e s , on the same s p e c i e s ( G o l d s t e i n et al., 1964) and on rainbow t r o u t (Walton and Cowey, 1977) , demonstrated no s i g n i f i c a n t d i f f e r e n c e i n b lood g lutamine a f f e r e n t and e f f e r e n t to the g i l l s . B lood ammonia was a l s o measured i n the l a t t e r two s t u d i e s , and i n carp (Pequin and S e r f a t y , 1963) (Table I V - 4 ) . These measurements, on th ree d i f f e r e n t s p e c i e s of f i s h , showed that b lood ammonia was s i g n i f i c a n t l y h igher i n the v e n t r a l a o r t a than i n the d o r s a l a o r t a . Exper iments c a r r i e d out on a per fused t r o u t head p r e p a r a t i o n (Payan and M a t t y , 1975) demonstrated t h a t up to 80% of the ammonia exc re ted a t the g i l l s cou ld be e x t r a c t e d from the p e r -f u s i o n f l u i d , w i t h the remainder be ing formed i n the g i l l t i s s u e s ( b a s a l ammonia e x c r e t i o n ) ( T a b l e I V - 5 ) . A comparison of ammonia e x c r e t i o n r a t e s w i t h ammonia e x t r a c t e d from the b lood a t the g i l l s (Table IV -5) shows tha t most , i f not a l l , ammonia excre ted a t the g i l l s comes from the b l o o d . V a r i a t i o n s i n b lood ammonia l e v e l s s e t out i n Table IV -4 are due to d i f f e r e n c e s i n temperature (Pequin and S e r f a t y , 1963) , measurements of plasma TABLE IV -4 BLOOD AMMONIA VALUES FOR VARIOUS SPECIES OF FISH Spec ies Temper-a tu re (°C) Myoxocephalus. 15-18 soorpius Cyprinus oarp-io Salmo gaivdneri 7, 20 15 15 11-13 11-13 6 -13 S ta rva -t i o n (days) fed fed 1-3 1 Weight Ig) 250-700 500 250-280 200 404 407.6 396.9 n S i t e of Measure-ment A c t u a l Data 2 bulbus a r t e r i o -sus DA 0 . 2 4 - 0 . 3 1 0 . 1 2 - 0 . 0 8 ymole/100 ml v e n t r i c l e 2 . 8 9 , 4 .03 DA 0 . 6 8 , 0 .70 yg/ml Converted f o r Comparison (M x 1Q-1*) 2 . 4 - 3 . 1 1 . 2 - 0 . 8 2 . 0 6 , 2.88 0 . 4 9 , 0 .50 10 DA bulbus a r t e r i o -sus DA VA DA 5.80 mg NH 3 -N/1 23 10 ymole/100 ml plasma 4 VA 3 DA 4.14 2 .3 1 .0 5 .83 2.20±0.47 4 .6910.69 2 .7310.60 Ammonia E x t r a c t e d from Blood (ymole/100 g - h r ) Reference 2 2 . 8 - 2 6 . 4 1 6 . 0 , 4 8 . 6 ' 26.5 7 4 . 1 J 40.0 1,2 G o l d s t e i n et dl., 1964 Pequ in and S e r f a t y , 1963 Smart , 1975 Walton and Cowey, 1977 Th is study O N 1 p a i r e d measurements 2 unpai red measurements TABLE IV -5 AMMONIA EXCRETION RATES FOR VARIOUS SPECIES OF FISH Spec ies Carassius auratus Cyprinus carpio Salmo gairdneri Temper-a tu re (°C) Myotocephalus 15-18 scorpius 21-16 7 20 12-14 17 S ta rva -t i o n (days) fed d a i l y unfed fed Weight Ifi) 250-700 102-222 500 126 150 Ammonia E x c r e t i o n : n A c t u a l Data 1 0 - 15 - -31 32 5 5 350-356 ymole/kg-hr 1 0 - 1 8 . 2 mg/100 g-24 hr 19.7±2.13 97.3±7.29 ymole/100 g -h r 25 -30 mg/24 hr 70-80 mg/24 hr 190 mg/kg-day" 12 26.3±3.55 14 21.4±3.04 12 5.9±0.89 ymole/100 g -h r C a l c u l a t e d (ymole/100 g -hr ) 3 5 . 0 - 3 5 . 6 1 2 4 . 5 - 4 4 . 6 " 19.7 97 .3 2 ,3 1 4 . 9 - 1 7 . 9 : 4 1 . 7 - 4 7 . 6 ' 2 A 136 mg/kg-day 23.6 370 mg/kg-dayJ 1 0 6 . 8 ' 5 4 . 9 ' 2,7 26 .3 21.4 5 .9 7 ,8 7 ,9 Ammonia E x t r a c t e d from Blood (ymole/100 g -h r ) 2 2 . 8 - 2 6 . 4 1 6 . 0 48 .6 7 4 . 1 4 0 . 0 21 .4 1 t o t a l ammonia excreted i n t o wate r , i n c l u d e s g i l l and u r i n a r y ammonia 2 g i l l ammonia e x c r e t i o n 3 f i s h ammonia-loaded by i n t r a p e r i t o n e a l i n j e c t i o n of ammonium su lphate (1 meq/lOOg) 4 t o t a l N excre ted i n t o water - 3% u r i n a r y N x 60% = g i l l ammonia N e x c r e t i o n 5 es t imated from graph 6 my d a t a , f o r comparison 7 per fused head 8 e x t r a c t e d from p e r f u s a t e 9 b a s a l ammonia Reference G o l d s t e i n et al., 1964 Thornburn and M a t t y , 1963 Maetz , 1972 I I Pequin and S e r f a t y , 1963 Fromm, 1963 Payan and M a t t y , 1975 - 58 -ammonia ve rsus whole b lood ammonia ( S e l i g s o n and H i r a h a r a , 1957; Bromberg et al, 1960; Maetz , 1973) , and n u t r i t i o n a l s t a t u s (Fromm, 1963) . G o l d f i s h (Maetz , 1973) have a r a t i o of whole b lood to plasma ammonia of approx imate ly 2 . Assum-i n g a s i m i l a r r a t i o f o r t r o u t , my b lood ammonia measurements are comparable w i t h those of Walton and Cowey (1977) , but are low compared to those of Smart (1975) (Table I V - 4 ) , f o r the same s p e c i e s , even t a k i n g i n t o account that my techniques r e s u l t e d i n an u n d e r e s t i m a t i o n of ammonia l e v e l s by approx imate ly 13% (Appendix I I ) . My measurements of d o r s a l a o r t i c b lood ammonia show an i n i t i a l i n -c rease i n b lood ammonia c o n c e n t r a t i o n w i t h s t a r v a t i o n . Th is i n c r e a s e appears to l e v e l o f f , a f t e r about 6 days of s t a r v a t i o n , a t a c o n c e n t r a t i o n which i s h igher than tha t measured the f i r s t day of s t a r v a t i o n (F igure I I I - 2 ) . When t r o u t are not f e d , they u t i l i z e t h e i r own muscle p r o t e i n f o r energy, and t h i s cou ld account f o r the i n c r e a s e i n b lood ammonia w i t h s t a r v a t i o n . Th is i n -c rease i n b lood ammonia cou ld account f o r the h igher d o r s a l a o r t i c b lood ammonia v a l u e s of Smart (1975) . 7 .5 Ammonia E x c r e t i o n Rates Ammonia e x c r e t i o n r a t e s f o r a number of s p e c i e s are l i s t e d i n Table I V - 5 . These r a t e s do not n e c e s s a r i l y r e f l e c t what the an imal i s capable of e x c r e t i n g , but are o n l y a r e f l e c t i o n of the n u t r i t i o n a l and p h y s i o l o g i c a l c o n d i t i o n of the an imal a t the t ime of the exper iments . Ammonia e x c r e t i o n r a t e s v a r y w i t h n u t r i t i o n a l s t a t u s . F i s h show an i n i t i a l decrease i n ammonia e x c r e t i o n d u r i n g the f i r s t 6 days of s t a r v a t i o n , which l e v e l s o f f to a f a i r l y constant r a t e f o l l o w i n g 6 to 14 days of s t a r v a t i o n (Fromm, 1963; De Vooys, 1968) . My r e s u l t s f o r ammonia e x t r a c t e d from the b l o o d , 74 to 40 umole/100 g of f i s h per hour , a re w i t h i n the range , 100 to 49 umole/100 g of f i s h per hour , I have c a l c u l a t e d u s i n g d a t a from Fromm (1963) (106.8 to 5 4 . 9 umole/100 - 59 -g - h r ) , c o r r e c t e d f o r b a s a l e x c r e t i o n ( 5 . 9 umole/100 g -h r ) (Payan and M a t t y , 1975) (Table I V - 5 ) , f o r the same s p e c i e s under s i m i l a r c o n d i t i o n s . 8 . WHAT FORM OF AMMONIA IS EXCRETED AT THE GILLS? Ammonia e x i s t s i n s o l u t i o n as d i s s o l v e d u n i o n i z e d ammonia, as the hydrated ammonia m o l e c u l e , and i n the i o n i z e d form (Equat ion 1 ) . The concen -t r a t i o n of u n i o n i z e d ammonia depends on the t o t a l ammonia c o n c e n t r a t i o n , pH, and temperature of a s o l u t i o n (Equat ion 2) (Emerson et al., 1975; T r u s s e l l , 1972) . P i s r e l a t e d to the c o n c e n t r a t i o n of u n i o n i z e d ammonia as f o l l o w s : NH 3 '™3> - £ 3 . . V , »> (Maetz, 1973) . An i n c r e a s e i n pH w i l l r e s u l t i n an i n c r e a s e i n the c o n c e n t r a -t i o n of u n i o n i z e d ammonia, and t h e r e f o r e , i n P._„ . NH 3 Ammonia i s measured by i n c r e a s i n g the pH of a s o l u t i o n to conver t i o n -i z e d ammonia i n t o u n i o n i z e d ammonia. The u n i o n i z e d ammonia i s then'measured, by ammonia probe , c o l o u r o m e t r i c , or o ther methods. Wi th the c u r r e n t methods of a n a l y s i s , i t i s p o s s i b l e to measure on ly t o t a l ammonia and, from the pH and temperature of the o r i g i n a l s o l u t i o n , to c a l c u l a t e the i o n i z e d and u n i o n i z e d f r a c t i o n s . Maetz and G a r c i a Romeu (1964) , Maetz (1972; 1973) , and Payan and Maetz (1973) p o s t u l a t e d tha t NH^ i s the main form e x c r e t e d , w i t h NH 3 e x c r e t i o n o c c u r r i n g under c e r t a i n c o n d i t i o n s . G o l d s t e i n et al. (1964) and Fromm and G i l l e t t e (1968) p o s t u l a t e d that u n i o n i z e d ammonia d i f f u s e s down a c o n c e n t r a -t i o n g r a d i e n t from b lood to w a t e r . + 8 . 1 NH. E x c r e t i o n 4 Maetz and G a r c i a Romeu (1964) p o s t u l a t e d a c h l o r i d e - b i c a r b o n a t e e x -change and a sodium-ammonium exchange to e x p l a i n the independence of a b s o r p t i o n of sodium and c h l o r i d e by the g o l d f i s h g i l l (Ga rc ia Romeu and Maetz , 1964) . T h e i r r e s u l t s showed that w h i l e N a + uptake at the g i l l was i n h i b i t e d by an i n c r e a s e i n NH^ i n e x t e r n a l w a t e r , i t i n c r e a s e d f o l l o w i n g i n t r a p e r i t o n e a l NH^ - 60 -i n j e c t i o n . They concluded from these r e s u l t s tha t NH* was excreted at the g i l l s i n exchange f o r Na* from the wate r . No s t o i c h i o m e t r i c r e l a t i o n s h i p b e -tween ammonia e x c r e t i o n and sodium uptake e x i s t s (Maetz, 1972) u n l e s s H* e x -c r e t i o n ac ross the g i l l i s a l s o taken i n t o account (Maetz, 1973) . In s tudy ing the e f f e c t of e x t e r n a l c a t i o n s on the a b s o r p t i o n of Na* by the c r a y f i s h , Shaw (1960) a l s o demonstrated NH* i n h i b i t i o n of Na* i n f l u x , but he a t t r i b u t e d t h i s to c o m p e t i t i o n between Na* and NH* f o r t r a n s p o r t s i t e s . These exper iments o f f e r an a l t e r n a t e e x p l a n a t i o n f o r the r e s u l t s of Maetz and G a r c i a Romeu (1964) . I f u n i o n i z e d ammonia, r a t h e r than i o n i z e d ammonia, i s e x c r e t e d , an a l t e r n a t e e x p l a n a t i o n f o r enhancement of Na* uptake by NH* i n j e c t i o n i s as f o l l o w s . I n j e c t i o n of NH* r a i s e s the t o t a l b lood ammonia c o n c e n t r a t i o n ( t h i s s t u d y ; Maetz , 1973) . E x c r e t i o n of NH^ a t the g i l l would r e s u l t i n NH^ be ing formed i n the blood to r e p l a c e that l o s t . NH* ^ NH + H* (10) 4 3 + + H formed would then be a v a i l a b l e f o r exchange w i t h Na . Maetz (1972; 1973) concluded that because g o l d f i s h exc re ted an am-monia s p e c i e s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t of f r e e ammonia, e x c r e t i o n of the u n i o n i z e d form was u n l i k e l y . T h i s c o n c l u s i o n was based on measurements, both e x t e r n a l and i n t e r n a l , of ammonia, pH, and temperature , from which the r e l a t i v e amounts of NH. and NH* were c a l c u l a t e d . Maetz then c a l c u l a t e d P „ „ 3 4 NH^ of both water and b l o o d . H i s c a l c u l a t i o n s i n d i c a t e d tha t the c o n c e n t r a t i o n g r a d i e n t f o r p a s s i v e ammonia d i f f u s i o n was from water to b l o o d , r a t h e r than from b lood to w a t e r . There are two p o s s i b l e sources of e r r o r i n Maetz ' measurements which c o u l d have l e d him to an i n c o r r e c t c o n c l u s i o n . F i r s t l y , Maetz (1973) s t a t e d tha t the r a t i o of whole b lood to plasma ammonia c o n c e n t r a t i o n was 2 . He measured plasma ammonia. Th is would r e s u l t i n a l a r g e u n d e r e s t i m a t i o n of u n -- 61 -i o n i z e d ammonia, and t h e r e f o r e , u n d e r e s t i m a t i o n of b lood P,„ T . Secondly , NH3 blood was sampled from the cauda l a r t e r y , an e x t e n s i o n of the d o r s a l a o r t a . My r e s u l t s (Table I I I - 2 ( a ) ) i n d i c a t e t h a t , i n t r o u t b l o o d , pH and ammonia c o n c e n t r a t i o n s are h igher i n the v e n t r a l a o r t a than i n the d o r s a l a o r t a . Measurements of b lood from the cauda l a r t e r y cou ld r e s u l t i n an u n d e r e s t i m a -t i o n of both pH and ammonia c o n c e n t r a t i o n of b lood i n the g i l l s . Thus, i t appears tha t the P c a l c u l a t e d by Maetz (1972; 1973) f o r g o l d f i s h b lood was low, and the a c t u a l c o n c e n t r a t i o n g r a d i e n t f o r u n i o n i z e d ammonia d i f f u s i o n cou ld have been from b lood to w a t e r . 8 .2 NH 3 E x c r e t i o n My data p rov ide i n d i r e c t ev idence f o r NH^ e x c r e t i o n . I n j e c t i o n of NH^HCO^ i n c r e a s e d pHa, w h i l e i n j e c t i o n of NH^Cl lowered pHa. NH^HCO^-injected f i s h were sub jec ted to h igher ambient temperatures than N H ^ C l - i n j e c t e d f i s h . T h e r e f o r e , NH^HCO^-injected f i s h had a g r e a t e r p r o p o r t i o n of t o t a l b lood am-monia i n the u n i o n i z e d form (F igure I I I - 5 ) , and should have been capable of e x c r e t i n g more ammonia than N H ^ C l - i n j e c t e d f i s h , p r o v i d i n g the i n j e c t i o n dose was the same. In c o n s i d e r i n g a l l of my data from ammonium i n j e c t i o n , 39.9% of the i n j e c t e d ammonia i s r e t a i n e d i n the b lood one minute f o l l o w i n g t e r m i n a t i o n of i n j e c t i o n of NH^Cl , w h i l e 37.5% i s r e t a i n e d f o l l o w i n g i n j e c t i o n of NH^HCO^. T h i s d i f f e r e n c e i s not s i g n i f i c a n t . However, i n c o n s i d e r i n g the h igher i n -j e c t i o n doses o n l y , the 37.2% r e t e n t i o n of i n j e c t e d ammonia f o l l o w i n g NH^Cl i n j e c t i o n i s s i g n i f i c a n t l y h igher than 33.4% r e t e n t i o n f o l l o w i n g NH^HCO^ i n -j e c t i o n . T h i s d i f f e r e n c e , together w i t h the h igher p r o p o r t i o n of u n i o n i z e d ammonia i n the b lood f o l l o w i n g NH^HCO^ i n j e c t i o n , supports the hypothes is tha t u n i o n i z e d ammonia i s exc re ted a t the g i l l s . A l though Maetz (1972; 1973) suggested that NH* was the main form e x -c r e t e d , he s t a t e d tha t NH^ e x c r e t i o n occur red when the e x t e r n a l medium was a c i d i f i e d , or i n the absence of e x t e r n a l sodium r e g a r d l e s s of pH. Maetz (1973) - 62 -measured an i n c r e a s e i n the r a t e of ammonia e x c r e t i o n upon a c i d i f i c a t i o n of the e x t e r n a l medium, and suggested tha t i n some f a s h i o n the g i l l became p r e f e r e n t i a l -l y permeable to u n i o n i z e d ammonia upon exposure to a c i d . He a t t r i b u t e d the t r a n s i t o r y nature of t h i s i n c r e a s e to a decrease i n water P „ T due to the a d d i t -NH 3 i o n of a c i d . Ammonia e x c r e t i o n r a t e s re tu rned to normal once b lood and water P—, reached a new e q u i l i b r i u m . I f there was a b lood to water P„_ T g rad ien t NH3 NH3 befo re a c i d i f i c a t i o n of the wate r , then a f t e r a c i d i f i c a t i o n t h i s g r a d i e n t would be even g r e a t e r , a l l o w i n g augmented ammonia e x c r e t i o n . The e x p l a n a t i o n of p r e f e r e n t i a l g i l l p e r m e a b i l i t y to u n i o n i z e d ammonia i s t h e r e f o r e unnecessary . Maetz (1973) observed tha t t o t a l ammonia e x c r e t i o n i n c r e a s e d i n the absence of e x t e r n a l sodium, and o f f e r e d t h i s o b s e r v a t i o n as f u r t h e r ev idence f o r the e x c r e t i o n of u n i o n i z e d ammonia under s p e c i a l c o n d i t i o n s . He suggested that when there i s no e x t e r n a l i o n to exchange w i t h the ammonium i o n , movements of ammonia occur e x c l u s i v e l y i n the u n i o n i z e d fo rm. G o l d s t e i n et dl. (1964) showed a d i f f e r e n c e of b lood ammonia a f f e r e n t and e f f e r e n t to the g i l l s , but d i d not c a l c u l a t e c o n c e n t r a t i o n g r a d i e n t s b e -tween b lood and wate r . They suggested tha t d i f f u s i o n of u n i o n i z e d ammonia down a c o n c e n t r a t i o n g r a d i e n t from b lood to water cou ld account f o r t h e i r measured d i f f e r e n c e s . Fromm and G i l l e t t e (1968) demonstrated tha t ammonia e x c r e t i o n i n rainbow t r o u t was i n h i b i t e d by i n c r e a s e d e x t e r n a l ammonia, and suggested t h i s i n h i b i t i o n supported the hypothes i s of p a s s i v e d i f f u s i o n of u n i o n i z e d ammonia down a c o n c e n t r a t i o n g r a d i e n t . Payan and Matty (1975) observed an a c t i v a t i o n by CC^ of ammonia e x -t r a c t i o n by the t r o u t g i l l from the p e r f u s a t e i n an i s o l a t e d head p r e p a r a t i o n . They a l s o observed a decrease i n the r a t e of e x t r a c t i o n , which was independent of ammonia c o n c e n t r a t i o n i n the p e r f u s a t e , when CC^ was removed from the p e r -f u s a t e . pH of the a f f e r e n t p e r f u s i o n f l u i d was main ta ined constant r e g a r d l e s s of whether C0 0 was present or n o t . - 63 -Wi th 5% CC>2 i n the p e r f u s a t e , Payan and Matty (1975) measured an i n c r e a s e i n hydrogen i o n c o n c e n t r a t i o n i n the e x t e r n a l medium, and a decrease i n hydrogen i o n c o n c e n t r a t i o n i n the e f f e r e n t p e r f u s a t e . With 0% CCv,, the o p p o s i t e o c c u r r e d . In the f i r s t i n s t a n c e , w i t h 5% CC^, the i n c r e a s e i n pH as the p e r f u s a t e passed through the g i l l s would r e s u l t i n a g r e a t e r p r o p o r t i o n of ammonia b e i n g i n the u n i o n i z e d form and a v a i l a b l e f o r e x c r e t i o n . With 0% ClX,, the decrease i n pH as the p e r f u s a t e passed through the g i l l s would r e s u l t i n a g rea te r p r o p o r t i o n of ammonia be ing i n the i o n i z e d fo rm, and t h e r e f o r e u n a v a i l a b l e f o r e x c r e t i o n . The r e s u l t s of Payan and Matty (1975) , tha t ammonia e x c r e t i o n was a c t i v a t e d by the presence of CC^ and depressed i n the absence of CC^, are c o n s i s t e n t w i t h the hypothes is tha t ammonia i s removed a t the g i l l s i n the u n i o n i z e d fo rm. - 64 -A l a b a s t e r , J . S . , and D.W.M. H e r b e r t . (1954) . I n f l u e n c e of carbon d i o x i d e on the t o x i c i t y of ammonia. N a t u r e . 174: 404. B a l l , I .R . (1967) . The r e l a t i v e s u s c e p t i b i l i t i e s of some s p e c i e s of f r e s h -water f i s h to p o i s o n s . I. ammonia. Water Res . 1 : 7 6 7 - 7 7 5 . B i n s t o c k , L . , and H. L e c a r . (1969) . 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V i e r t e l j a h r s s c h r i f t der N a t u r f . G e s e l l s c h a f t i n Z u r i c h . 92 : 198-204 Wuhrmann, K . , and H. Woker. (1948) . B e i t r a g e zur T o x i k o l o g i e der F i s c h e . I I . E x p e r i m e n t e l l e Untersuchungen i iber d i e Ammoniak- und B l a u s a u r e v e r g i f t u n g . Schweiz . Z . H y d r o l . 1 1 : 210 -244 . - 7.0 " APPENDIX I LIST OF SYMBOLS AND ABBREVIATIONS NH.C l = ammonium c h l o r i d e 4 NH^HCO^ = ammonium bicarbonate/ammonium hydrogen carbonate [NH^]^ = t o t a l ammonia c o n c e n t r a t i o n , i n moles per l i t r e [NH^] = u n i o n i z e d ammonia c o n c e n t r a t i o n , i n moles per l i t r e [NH^] = i o n i z e d ammonia c o n c e n t r a t i o n , i n moles per l i t r e K ' a = apparent d i s s o c i a t i o n constant of a weak a c i d p K ' a = l o g a r i t h m i c t r a n s f o r m a t i o n of K' (Lehn inger , 1972) pHa = pH of a r t e r i a l b lood VA = v e n t r a l a o r t a DA = d o r s a l a o r t a i = i n i t i a l measurement be fo re i n j e c t i o n f = f i n a l measurement f o l l o w i n g i n j e c t i o n Po^ = p a r t i a l p ressure of oxygen (mm Hg) P = p a r t i a l p ressure of ammonia (mm Hg) 3 [ N H 3 ] = • P N H 3 (Equat ion 9) the c o n c e n t r a t i o n of ammonia ( m o l e s / l i t r e ) i s equal to the s o l u b i l i t y of ammonia (a = Bunsen s o l u b i l i t y c o e f f i c i e n t , l i t r e / l i t r e - m m Hg) d i v i d e d by 2 2 . 1 ( l i t r e (STPD) per mole NH^ gas) t imes the p a r t i a l p ressure of NH^ gas (mm Hg) . - 71 -APPENDIX I I PREPARATION OF BLOOD FOR AMMONIA DETERMINATION WITH ORION MODEL 95-10 AMMONIA ELECTRODE The f o l l o w i n g procedure , m o d i f i e d from W i l l i a m s o n and Corky (1969) , removes b lood p r o t e i n s which i n t e r f e r e w i t h the e l e c t r o d e membrane. 1 . Add b lood sample to equa l volume of 16% HCIO^. 2. C e n t r i f u g e 10 minutes a t 1700 G, a t room temperature . 3 . Decant supernatant , r e c o r d volume. 4 . N e u t r a l i z e supernatant w i t h 3 M K^CO^ i n 0 . 5 M t r i m e t h a n o l -amine h y d r o c h l o r i d e to pH 5 . 5 to 6 . 0 (use pH paper) by slow a d d i t i o n , and reco rd t i t r e . 5 . C e n t r i f u g e 10 minutes a t 1700 G a t room temperature . 6. Decant s u p e r n a t a n t , reco rd volume, add to d i s t i l l e d water and NaOH s o l u t i o n r e f e r r e d i n p a r t 2d of Appendix I I I . O r ion Research recommends t h a t b lood samples be kept on i c e and water u n t i l a n a l y s i s , and W i l l i a m s o n and Corky (1969) recommend that samples be c e n t r i f u g e d at -4°C . Or ion (1975) found a 50% l o s s of ammonia from a s t i r r e d , b a s i c 100 ml sample i n 6 hours . A v a i l a b i l i t y of equipment, and t i m i n g of experiments made i t d i f f i c u l t to f o l l o w t h i s p rocedure , so I ran samples at room temperature (20 to 22°C). To t e s t f o r l o s s of ammonia from my samples a t room temperature , I added a known c o n c e n t r a t i o n of ammonium c h l o r i d e to samples of f i s h b l o o d , c a r r i e d out the s teps l i s t e d above, and determined the f i n a l c o n c e n t r a t i o n of ammonia. The c o n c e n t r a t i o n of ammonia i n the sample a f t e r a d d i t i o n of ammonium c h l o r i d e was i n excess of normal b lood ammonia l e v e l s , so that the c o n t r i b u -t i o n from b lood ammonia would be n e g l i g i b l e . F o l l o w i n g a d d i t i o n of 0 . 1 M NH^Cl to the b l o o d , my f i n a l measurement-was 0.087+0.003 M, showing a l o s s of 13%. T h e r e f o r e , my r e s u l t s from c a r r y i n g out the procedure a t room temperature are an u n d e r e s t i m a t i o n of ammonia l e v e l s by approx imate ly 13%. De Vooys (1969) u s i n g m i c r o d i f f u s i o n techniques to measure ammonia i n b lood to which a q u a n t i t y of ammonium su lphate had been added, had l o s s e s from the b lood from 14 to 30%. - 7 3 -APPENDIX I I I CALIBRATION OF ORION AMMONIA ELECTRODE, MODEL 9 5 - 1 0 , FOR LOW LEVEL (10"6 to 10"^ M NH„) MEASUREMENTS OF NH 3 IN FISH BLOOD 1 . Have a l l s o l u t i o n s at room temperature (20 -22°C) . R e p r o d u c i b i l i t y of low l e v e l measurements cannot be obta ined at f i s h temperatures ( 9 - 1 4 ° C ) . 2 . Prepare s o l u t i o n s and c a r r y out c a l i b r a t i o n procedure as o u t l i n e d by O r i o n , 1975, but note the f o l l o w i n g : a . E l e c t r o d e must be l e f t i n ammonia - f ree , pH4 b u f f e r at l e a s t 5 minutes be fo re c a l i b r a t i o n . b. E l e c t r o d e must be a l lowed to s t a b i l i z e i n the d i s t i l l e d water and NaOH s o l u t i o n (5 -10 m i n u t e s , r e c o r d mV reading) be fo re a d d i t i o n of the standard s o l u t i o n . c . A f t e r c a l i b r a t i o n , e l e c t r o d e should be kept i n pH 4 b u f f e r f o r a t l e a s t 5 minutes between measurements. d . Be fo re adding prepared b lood (Append ix I I ) , aga in a l l o w e l e c t r o d e to s t a b i l i z e i n d i s t i l l e d water and NaOH s o l u t i o n u n t i l w i t h i n +2 mV of . . r e a d i n g obta ined i n s tep b above. At ve ry low l e v e l measurements, the i n i t i a l p o r t i o n of the c a l i b r a t i o n curve i s dependent upon t h i s v a l u e . 

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