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Mechanism of acidification of expired water in fish Lin, Hong 1989

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MECHANISM OF ACIDIFICATION OF EXPIRED W A T E R IN FISH By HONG LIN B.A.Sc, Zhejiang University, P.R.C., 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) We accept this thesis as coriforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA April 1989 @ HONG LIN, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT i i The effect of varying inhaled water pH on the acidification or alkalization of water as it passes over the gills in rainbow trout (Salmo gairdneri) was investigated by exposing the fish to pH 9.91 and pH 3.88 water. In the control and high pH treatment, water was acidified as it flowed over the gills of the fish because of the dominant effect of CO2 hydration. Water was alkalized as it flowed over the gills of the fish in the low pH treatment due to N H 4 + formation and perhaps H C O 3 ' dehydration. The overall result of CO2 and NH3 excretion is to ameliorate the change of expired water pH in the face of changes in pH of inspired water. Carbon dioxide excretion was not significantly affected by the high or low pH treatments but ammonia was accumulated in fish plasma in both cases. The impact of Na+/H+(NH4+) and Q7HC03~ exchange processes on the acidification or alkalization of expired water has also been examined. Amiloride or SITS was introduced to the water to inhibit the ionic exchange processes and environmental water pH was gradually lowered from neutral to acidic. Under acidic conditions, molecular CO2 and NH3 excretion alone could account for all the proton concentration changes from inspired to expired water, indicating that Na+/H+(NH4+) transport was inhibited by low environmental pH. In neutral environments, the proton concentration changes were the result of molecular CCh and NH3 excretion, and sodium influx in exchange for H+(NH4+). Cr/HCCb'exchange was not an important pathway for carbon dioxide elimination but inhibition of Na+/H+(NH4+) exchange caused a reduction of ammonia excretion. T A B L E OF CONTENTS iii P A G E Abstract ii List of Tables iv List of Figures v Acknowledgements vii 1.0 General Introduction 1 2.0 The effect of varying water pH on the acidifi-cation of water as it passes over the gills of rainbow trout 9 2.1 Introduction 9 2.2 Materials & Methods 10 2.2.1 Animal & Preparation 10 2.2.2 Experimental Protocols & Measurements 10 2.2.3 Calculations & Statistics 13 2.3 Results 14 2.4 Discussion 25 3.0 The effect of Na +/NH4 + and C l T H C O f exchange processes on the acidification of water as it passes over the gills of rainbow trout 29 3.1 Introduction 29 3.2 Materials & Methods 31 3.2.1 Animal & Preparation 31 3.2.2 Experimental Protocols & Measurements 31 3.2.3 Calculations & Statistics 34 3.3 Results 35 3.4 Discussion 43 4.0 General Conclusions & Summary 49 5.0 References 51 LIST OF TABLES TABLE 1 Carbon dioxide excretion during high and low pH treatments in rainbow trout 2 Plasma ammonia levels during high and low pH treatment in rainbow trout 3 Calculated proton concentration increase in expired water V LIST OF FIGURES FIGURE P A G E 1 A simplified cross-section through the gill epithelium, boundary water layer and bulk water flow 2 2 The effect of varying pH on the CO2/HCO3" and NH3/NH4+ ratios in trout plasma 4 3 The apparatus for measuring inspired, expired and stopped-flow water pH 11 4 pH of inspired, expired and stopped-flow expired water of rainbow trout during high pH treatment 15 5 Proton concentration increase in the expired water during high pH treatment 16 6 pH of inspired, expired and stopped-flow expired water of rainbow trout during low pH treatment 17 7 Proton concentration increase in the expired water during low pH treatment 18 8 The relationship of exhalent water to inhalent water pH in rainbow trout 19 9 Dorsal aortic blood pH of rainbow trout 20 10 The recirculating system with a black chamber 32 11 The differences of total CO2 content between inspired and expired water with different environmental pH 36 12 The differences of total ammonia content between inspired and expired water with different environmental pH 37 13 The relationship between measured proton concentration increase in expired water and environmental pH 40 The differences of measured proton concentration increase and calculated proton concentration increase in expired water within low environmental pH range (pH4-6) The differences of measured proton concentration increase and calculated proton concentration increase in expired water within neutral environmental pH range (pH6-7) The relationship between ([H+]meas - [H+]cal) and environmental water pH with experimental data and predicted data ACKNOWLEDGEMENT vii I am very grateful to my supervisor Dr. D.J. Randall for his valuable guidance and support throughout the studies. I thank all the post-doctorals, graduate students and technicians working in our lab, who provided me with helpful suggestions and assistance. Appreciation is also expressed to my committee members for their beneficial corrections and comments to this thesis. 1.0 GENERAL INTRODUCTION The gills of fish are the primary site for oxygen, carbon dioxide and ammonia transfer between water and blood. The surface area of the gills is between 10 and 60 times that of the rest of the body (Randall, 1970). The secondary lamellae represent the major respiratory portion of the gill structure. There is a countercurrent arrangement of blood and water flows in teleost fish, which enables the fish to utilize 60-80% of the oxygen in the water passing over the gills, and blood leaving the gills is usually between 85 and 95% saturated with oxygen (Randall, 1970). Carbon dioxide is the major metabolic endproduct excreted across the gills. The solubility of CO2 in water is 20-30 times greater than that of oxygen (Randall 1970), and the gill epithelium is highly permeable to molecular CO2. Therefore, carbon dioxide can be eliminated from the blood to the water simply via passive diffusion of molecular CO2. Because of the reduced availability of oxygen and increased solubility of carbon dioxide in water, carbon dioxide excretion is usually a less critical problem for water-breathers than oxygen uptake. Water breathers, such as fish, adjust their ventilation to their oxygen requirements (Randall & Cameron, 1973). A unidirectional laminar water flow generated by the breathing movements of the buccal cavity and operculum brings the environmental water into close contact with the gill epithelium. The water next to the mucous coated gill epithelium may have a chemical composition very different from that of the bulk medium (Figure 1; Randall & Wright, 1989). Metabolic end products such as 1 to BULK BOUNDAIY WAT E l d U . BLOOD WATER FLOW IAYE1 EPITHELIUM V 11. HCO. H + NH I N H 3 c o NH Figure 1. A simplified cross-section through the gill epithelium, boundary water layer and bulk water flow showing possible interactions between carbon dioxide and ammonia in the boundary water layer (Randall & Wright, 1989). carbon dioxide and ammonia are excreted into the mucous and boundary water layer. These gases then diffuse into the bulk water and are carried away. Carbon dioxide in water can be hydrated and form bicarbonate and carbonate: CO2 + H 2 O == H C O 3 " + H + == COT + 2fT pKi' = 6.08 , pK2 = 10 (15°C) PKi' is the apparent first dissociation constant of carbonic acid. It varies with temperature, ionic strength and pH because it involves several reactions which are pH dependent. P K 2 is the true second dissociation constant which varies with temperature and ionic strength alone (Boutilier et al, 1985). Except in highly alkaline solutions, the carbonate concentration is negligible. The CO2/HCO3" ratio changes according to pH (Figure 2; Wright & Randall, 1987). In fish plasma, where pH is around 7.85, the CO2/HCO3" ratio is less than 0.1. So most carbon dioxide stores in the body of the fish are in the form of HCO3". An elevation of carbon dioxide levels in the fish body results in an acidosis and vice versa. The term total CO2 refers to the combined concentration of molecular CO2 , bicarbonate (HCCh") and carbonate (CO3 2"). The gill epithelium is highly permeable to molecular CO2 but less permeable to HCCh", and CO2 can dissolve into water easily, therefore, passive diffusion of molecular CO2 is the dominant pathway of carbon dioxide excretion. There is approximately 10% of total carbon dioxide excreted as HCCh" in exchange for Cl" at the gill epithelium in resting fish (Randall & Wright, 1989). Bicarbonate diffusion across the gills may also play a small role 3 Figure 2. The effect of varying pH on the C02/HC03* and NH3/NH4"*" ratios in trout plasma at 15°C. Plasma pH coincides with the point where CO2/HCO3" and NH3/NH4+ ratios are equal (Wright & Randall, 1987). 4 in CO2 excretion. The rate of the uncatalyzed carbon dioxide hydration/dehydration reaction is of the order of minutes, which is very slow compared to the interlamellar transit time of gill water (100-400 ms) (Wright et al, 1986). Carbonic anhydrase, an enzyme that catalyzes carbon dioxide hydration/dehydration, is located in erythrocytes and gill epithelium including the apical region of the outer layer of gill epithelial cells (Rahim et al, 1988). Wright et al (1986) investigated the downstream pH of expired water and found a significant decrease in water pH as it flowed over the gills which disappeared with the addition of a carbonic anhydrase inhibitor. The carbonic anhydrase catalyzed CO2 hydration not only helps the transfer of CCh from red blood cell to plasma and gill epithelium (Randall & Daxboeck, 1984), but also facilitates the removal of CO2 from the boundary water layer and enhances carbon dioxide ehinination (Randall & Wright, 1989). The conversion of CCh to HCCh" in the boundary water layer will acidify the water. The magnitude of acidification depends on the environmental pH and the buffering capacity of the water (Randall & Wright, 1989). Fish are ammonotelic animals and therefore produce ammonia as the end product of protein catabolism. Ammonia is toxic and has to be excreted or converted to less toxic compounds. Most ammonia is eliminated from the gills but small quantities may also be excreted by the kidneys (Randall & Wright, 1987). Since ammonia is a weak base, it can bind a proton to form ammonium ion in aqueous solution: NH3 + H+ = NH4+ pKamm = 9.58 (15°C) 5 The rate of the reaction in water is extremely rapid and pKamm changes with temperature and ionic strength (Wright & Randall, 1987). The NH3/NH4"1" ratio increases with increasing pH (Figure 2). Within the biological pH range (usually around neutral or slightly alkaline), the predominant form of ammonia is the ionic form. For example, in fish plasma, where pH is almost 2 units lower than pKamm, the NH3/NH4+ ratio is about 0.01. The combined concentrations of non-ionic ammonia (NH3) and ammonium ion (NH4+) in solution will be referred to as total ammonia. Cell membranes are highly permeable to NH3 but relatively impermeable to NH4+ (Wright & Randall, 1987). Therefore, non-ionic ammonia can diffuse passively down its partial pressure gradient from blood to water across the gill epithelium. The excreted NH3 will be converted to NH4+ according to environmental pH (Figure 1), and the boundary water layer will be alkalized. In other words, protons in the boundary water layer will convert NH3 to N H 4 + , and thus maintain a high N H 3 concentration gradient between blood and water and facilitate ammonia excretion (Wright et al, 1989). Although little NH4+ can diffuse across the gills, NH4+ can be excreted branchially by coupling to ion carriers (Figure 1). There is some evidence to suggest that NH4+ is excreted through the gill epithelium in exchange for sodium. This pathway may account for 10-40% of the total branchial ammonia excretion in resting rainbow trout (Randall & Wright, 1989). CCh elimination generates protons in the gill water due to CO2 hydration whereas NH3 excretion consumes protons due to N H 4 + formation. As a result, the excretions of CCh and NH3 are closely 6 linked via chemical reactions in the boundary water layer adjacent to the gills (Wright et al, 1989). The introduction of a carbonic anhydrase inhibitor to the blood reduces not only carbon dioxide excretion but also ammonia excretion (Wright et al, 1989). Increasing the buffering capacity of external water decreases ammonia excretion but does not affect carbon dioxide excretion (Wright et al, 1989). These results indicate that CO2 excretion provides a continual supply of hydrogen ions which are necessary to maintain a sufficient blood-to-water NH3 diffusion gradient for ammonia excretion. The acidification and alkalization of expired water induced by CO2 and N H 3 excretions cancel each other to some extent. Since total carbon dioxide excretion is normally 10 times greater than total ammonia excretion (Wright & Randall, 1987), the overall result will be an acidification of expired water when bulk water pH is around 7. The pH of fish plasma is at the midpoint between p K i c o 2 and pKamm at different temperatures (Randall & Wright, 1989). Both CO2/HCO3" and N H 3 / N H 4 + ratios are very low at this pH (Figure 2), but a maximal and balanced carbon dioxide and ammonia excretion is maintained. Any change in internal or external pH has an effect on carbon dioxide and ammonia elimination. An acidosis will augment HCCh" dehydration in fish blood and enhance CO2 excretion by passive diffusion, but reduce NH3 formation and decrease NH3 excretion. An alkalosis will have the reverse effect. Similarly, high environmental water pH inhibits NH4+ formation and ammonia elimination, but favors CO2 hydration and therefore, carbon 7 dioxide excretion. The gills of fish are also involved in ion transfer and acid-base regulation. The presence of Na +/H +, Na+/NH4+ and Cf/HCCh" exchange processes in gill epithelial cells is well documented (Maetz, 1973; de Ranzis, 1975; Payan, 1978). It is suggested that fish regulate their acid-base status via modulation of Na+/H+,Na+/NH4+ and Cf/HCCh" exchange processes (Girard & Payan, 1980). The Na+/NH4+ exchange process may contribute up to 10-40% of the total ammonia excretion, while Cl/HCCh" exchange process may be responsible for 10% of the total carbon dioxide excretion in resting fish (Randall & Wright, 1989; Figure 1). The phenomenon of expired water acidification was demonstrated by Wright et al (1986). The objectives of the present studies were to examine the mechanisms behind this phenomenon: (1) to investigate the impact of environmental pH on the acidification and alkalization of expired water induced by carbon dioxide and ammonia excretion and (2), to determine the effects of Na+/H+(NH4+) and Cl'/HCCh" exchange across the gills on the proton concentration changes in expired water. 8 2.0 THE EFFECT OF VARYING WATER PH ON THE ACIDIFICATION OF WATER AS IT PASSES OVER THE GILLS OF RAINBOW TROUT 2.1 INTRODUCTION Fish excrete molecular CCh and NFb into the water as it passes over the gills (Figure 1). CO2 excretion rates are an order of magnitude greater than NH3 excretion rates. Some carbon dioxide is excreted as bicarbonate in exchange for chloride and some ammonia as ammonium ion in exchange for sodium. The former represents about 10% of the total resting carbon dioxide excretion and the latter up to 50% of the total ammonia excretion (See review by Randall and Wright, 1989). Because molecular CO2 is the dominant excretory product appearing in the water, the gill water will be acidified due to CO2 hydration catalysed by carbonic anhydrase in the gill mucus and water boundary layer (Wright et al, 1986) and on the apical surface of the gill epithehum (Rahim et al, 1988). In neutral and alkaline waters most of the excreted CO2 will be converted to bicarbonate, but under acid conditions only a small fraction will be hydrated. Thus the extent of acidification of water as it passes over the gills will decrease with water pH. The opposite will be true for the NH3:NH4+ reaction. Under acid conditions almost all the NH3 will be converted to NHt+ but under alkaline conditions only a fraction of the excreted NH3 will form NH44" in the gill water. The experiments reported in this section were designed to investigate the effect of varying inhaled water pH on the acidification of water as it passes over the gills. 9 2.2 MATERIALS & METHODS 2.2.1 Animal & Preparation Rainbow trout (Salmo gairdneri), weighing 324-494 g, were obtained from the West Geek Trout Farm ( Aldergrove, BC ) and housed in outdoor fiberglass tanks supplied with flowing dechlorinated Vancouver tap water (pH 6.5-6.8; temperature, 8-12°C; hardness, 12 ppm CaCGj). Fish were fed with commercial trout pellets and feeding was suspended at least two days before experimentation. Fish were prepared with a dorsal aortic cannula for sampling blood, an opercular cannula for sampling expired water and a van Dam mask for measurement of ventilation as in Wright et al, (1986). After the surgical procedure, fish were left to recover for 20-40 hours in the van Dam apparatus (Figure 3) supplied with a flowing test solution of 40 mmol.L"1 NaCl and 0.5 mmol.L1 CaCh in dechlorinated tap water (9.5-11.2°C) with a buffering capacity (p) of 81 u.equiv. L'\pH unit"1. The test solution had the same ionic strength as the buffer solution used to calibrate the pH electrodes. By using this test solution, we reduced the response time of the pH electrode, increased its stability and thus obtained more precise water pH measurements. 2.2.2 Experimental Protocols & Measurements Fish were subjected to one of two treatments during the experimental periods: 1) NaOH was added to the reservoir to increase the pH of the test solution to 9.91 ± 0.02. Fish were exposed to the high pH test solution for 90 minutes. 10 Dechlorinated tap water Peristaltic pump Air Test solution veservoir Inflow watar valwi Cooling coil 9 Expired water pH meter Inspired water pH meter Chart Recorder Concentrated salt solution pH i l i c t n i i s • ptrcilar caanala stlrriii bar Stoppeal-f lew apparatas Figure 3. The apparatus for measuring inspired, expired and stopped-flow water pH. Fish were prepared with a van Dam mask, a dorsal aortic cannula and an opercular cannula, and were placed in a two-chambered plexiglass box. 11 2) HCl was added to the reservoir to reduce the pH of the test solution to 3.88 ± 0.02. Fish were exposed to the low pH test solution for 90 minutes. In each series, a half hour control period (Cl), two experimental periods (El, E2), 45 minutes each, and a half hour recovery period (C2), were included. A set of measurements were performed in each of the control, experimental and recovery periods: 1) Inspired water pH (pHin), expired water pH (pHex) and stopped-flow water pH (pHst) were measured as in Wright et al (1986). 2) Ventilation (Vg) was measured by collecting the outflow water over 1 minute periods from the standpipe in the back chamber of the van Dam apparatus. 3) Total carbon dioxide content of the inspired water (CChin) and expired water (CCteex) was measured with a Carle gas chromatograph (Model HI) containing a CCte mscriminating column (porapak Q) (Boutilier et al, 1985; Lenfant & Aucutt, 1966). 4) A blood sample (0.7ml) was withdrawn from the dorsal aortic cannula and replaced with the same amount of heparinized saline. The whole blood pH ( p H B ) was measured by using a Radiometer G297/G2 capillary electrode with a Radiometer PHM-71 acid-base analyzer. 5) The remaining blood sample was centrifuged and the plasma was removed. 6) Total carbon dioxide content of the plasma (C02B) was measured by gas chromatography as described in 4). 7) Total ammonia content (TAmm) of the plasma was measured by 12 micro-modification of a commercial diagnostic kit with a UV-visible recording spectrophotometer (L-glutamic dehydrogenase/ NAD method; Sigma, 1982). 8) Inspired and expired water buffering capacities were determined in acid, alkaline and neutral water by titrating the inspired and expired water using either 0.1N HCl or 0.1N NaOH as titrants. 2.2.3 Calculations & Statistics The rate of carbon dioxide excretion (Mco2) was calculated from the total CO2 contents of the inspired and expired water and ventilation volume per hour by application of the Fick equation, and expressed per gram of fish weight. Free ammonia concentration in plasma (NH3) was calculated from total ammonia concentration in plasma and whole blood pH by the Henderson-Hasselbalch equation, using the appropriate pK value from Cameron & Heisler (1983). Carbon dioxide partial pressure of both plasma and water was calculated from the total CO2 content and pH by the Henderson-Hasselbalch equation, using the p K ^ and a c Q 2 values from Boutilier et at, (1985). The proton concentration increase in expired water was calculated from the appropriate buffer curve and then expressed as the proton concentration increase per hour per gram of fish weight. Data are presented as means ± standard error. To compare the relationships in the data, student's two-tailed t-tests and 1-way and 2-way ANOVAs (analysis of variance) were used. A 5% level of rejection was taken as the statistical limit of significance. 13 2.3 RESULTS 1. Downstream water pH At high pH , the inspired water was acidified as it passed through the gills (Figure 4). There was no significant difference between pHex and pHst in Cl and El, but a significant difference was exhibited in E2 and C2. The calculated proton concentration increase in expired water was significandy larger when the animal was exposed to alkaline water (Figure 5). There was , however, no significant difference in the proton concentration increase in expired water between El and E2, or between Cl and C2. In the low pH treatment, the inspired water was alkalinized rather than acidified, as it passed over the gills (Figure 6). There was no significant difference between pHex and pHst except in E2. The decrease in proton concentration in expired water was larger than the increase observed in neutral water (Figure 7). Once again there was no significant difference in the proton concentration change in expired water between either El and E2 or Cl and C2. Water was acidified at high pH, alkalinized at low pH but at pH 5.3 there was no change in pH of water as it flowed over the gills (Figure 8). 2. Whole blood pH The blood of the fish was alkalinized when the inspired water pH was raised to pH 9.91. There was no significant difference in blood pH between El and E2 (Figure 9). The whole blood pH returned to normal during recovery. No significant change in blood pH was observed when the inspired water pH was dropped to pH 3.88 and then returned to pH 6.37 (Figure 9). 14 0 C1 .30min in E1.45minin E2.90min in C2 .30min in pH 6.80 water pH 9.91 water pH 9.60 water pH 7.25 water • Inspired WM Expired E S Stopped-flow Figure 4. pH of inspired, expired and stopped-flow expired water of rainbow trout during high pH treatment. Measurements were taken 30 minutes after the fish was in the neutral control water, 45 and 90 minutes after the fish was exposed to pH 9.91 experimental water and 30 minutes after the fish was returned to the neutral control water. pHin was significandy different from pHex in all cases while pHst was significandy different from pHex in E 2 and C2. (x ± SE). 15 1 0 i C1.30min in pH6.80 water E1.45min in pH9.91 water E2.90min in pH9.60 water C2.30min in pH7.25 water Figure 5. Proton concentration increase in the expired water during high pH treatment of rainbow trout expressed in pMol per hour per gram of fish weight Data were calculated by using the pH values in Figure 2 and buffer curves for water over the appropriate pH range. Significant differences were exhibited between Ei, E2 and Ci, C2. (x ± SE). 16 1 0 -8--x C L n in pH 6 . 65 water p H 3 .88 water pH 3 .99 water pH 6 .37 water • Inspired • § Expired ES2 StoppecLf low Figure 6. pH of inspired, expired and stopped-flow expired water of rainbow trout during low pH treatment Measurements were taken 30 minutes after the fish was in the neutral control water, 45 and 90 minutes after the fish was exposed to pH 3.88 experimental water and 30 minutes after the fish was returned to the neutral control water, pffin was significandy different from pHex in all cases while pHst was significandy different from pHex in E2. (x ± SE). 17 o cn 2 - c o • - o c o O C "O O 0) O . b c o CL X 1.5 1.0 0.5 0.0 - 0 . 5 -1 .0 - 1 . 5 -2 .0 T E1.4^r C1.30minin t.l +5min in pH6.65 water PH3.88 water E2^0min in pH3.99 water + C2. 30min in pH6.37 water Figure 7. Proton concentration increase in the expired water during low pH treatment of rainbow trout expressed in pMol per hour per gram of fish weight Data were calculated by using the pH values in Figure 4 and buffer curves for water over the appropriate pH range. Significant differences were exhibited between Ei, E2 and Ci, C2. (x ± SE). 1 8 Figure 8. The relationship of exhalent water to inhalent water pH of rainbow trout The exhalent water line is the regression line of the mean pH values collected from both high and low pH treatments ( Y = 0.453 X + 2.894, r2 value = 0.96 ). The inhalent and exhalent lines cross at pH 5.3, which may be equivalent to the point where no pH change occurs when water flows over the gills. 19 8.2 X CL TJ O O CD _0) O 8.1 --8.0--7 . 9 -t T t \ I t 7.8 C1.30min in pH6.80/6,65 water E1.45min in pH9.91/3.88 water O High pH treatment 1 E2.90min in pH9.60/3.99 water C2.30min in pH7.25/6.37 water Low pH treatment Figure 9. Dorsal aortic blood pH of rainbow trout Measurements were taken 30 minutes after the fish was in the neutral control water, 45 and 90 minutes after the fish was exposed to pH 3.88 or 9.91 experimental water and 30 minutes after the fish was returned to the neutral control water. * indicate a significant difference from the control (Cl) value, (x ± SE). 20 3. Carbon dioxide excretion There was no change in plasma total CO2 content during exposure of the fish to pH 9.91 water (Table 1). Plasma Pco2 however, was reduced. Inspired and expired water total C O 2 was increased because carbon dioxide was trapped as HCO3" in this alkaline water even though inspired water Pco2 was lower than control values (Table 1). There was, however, no change in the difference between inspired and expired water Tco2 between or within the control and experimental periods, that is carbon dioxide excretion was unaffected by the elevation in water pH and Ta>2. The ventilation volume (Vg) varied greatly between animals but there were no significant differences during the whole experimental period (Table 1) There were also no significant differences in plasma total CO2 content, plasma Pco2, Vg and M co2 during exposure of the animal to acid conditions (Table 1). Pco2 of the inspired acid water was raised to twice that of control water, but total CCte content decreased. No significant difference was observed, however, in Tco2(ex-in) between fish in control and acidified water (Table 1). 4. Plasma ammonia Ammonia was accumulated within the body of the fish during both high and low pH treatments. Total ammonia content of plasma increased 70% in fish in pH 9.91 water but returned to normal during recovery (Table 2). Non-ionic ammonia content [NH3] increased along with the [NH3]/TAmm ratio, reflecting the elevation in blood pH (Table 2). Total ammonia content in plasma went up continuously during the pH 3.88 treatment, increasing by 21 Table 1. Carbon dioxide excretion during high and low pH treatments in rainbow trout * indicate a significant difference from the control (Cl) values, (x ± SE). High pH Treatment n Control 1 Experi mentl Exper i ment2 Control 2 Total C02 CmM/LD PI as ma 6 19. 4 ± 0. 9 20. 1 ±1.0 20.2 ± 1.1 19. 4 ± 0. 9 Inspire <7 0. 38 ± 0. 02 0. 45 ± 0. ol 0. 50 ± 0.ol 0. 40 ± 0.02 Expire 7 0. 53 ± 0. 02 0.60 ± 0.Ol 0.64 ± O.ol 0. 54 ± 0. 02 Tco2C ex-in) CmM/LI) 7 0. 15 ± O. 01 O. 15 ± O. 02 0. 14 ± 0. 02 0. 14 ± 0. Ol Pco2 C Tor r D PI as ma 6 4. 77 ± O. 21 4. 06 ± O. l£ 3. 63 ± 0. 2§ 4. 87 ± 0. 30 Inspi re >7 1.44 ± 0.06 0. 00 ± o. 08 0. 01 ± 0.08 0. 81 ± 0. 0$ Expire 7 4. 43 ± O. 27 1. 61 ± O. 2§ 1.54 ± 0.3$ 3. 51 ± 0. 48 Vg Cml/min) 7 146 ± 38 145 ± 31 117 ± 17 126 ± 15 Mco2CuM/g. hO 7 3. 18 ± 0. 89 2. 91 ± 0. 74 2. 27 ± 0.45 2. 46 ± 0.28 Low pH Treatment n Control 1 Experimentl Experiment2 Control 2 Total C02 CmM/L} PIasma 6 17. 1 ±1.4 16. 3 ± 1.7 16. 2 ± 1.7 16. 5 ± 1.7 Inspire >e O. 40 ± 0.02 0. 32 ± 0. ol 0. 32 ± 0.Ol 0. 41 ± 0. 03 Expire 6 0. 54 ± 0. 03 0. 46 ± 0. ot 0. 44 ± 0.ot 0. 52 ± 0.04 Tco2Cex-i nD CmM/L:> 6 0. 14 ± 0. 02 0. 14 ± 0. 03 0. 12 ± 0. 03 O. 11 ± 0.02 Pco2 CTorrD PIasma 6 4. 54 ± 0. 30 4. 51 ± 0. 43 4. 44 ± 0.42 4.55 ± 0.40 Inspire >& 1.87 ± 0.07 4. 63 ± O. 2§ 4. 54 ± O.2§ 2. 87 ± O. i S Expire 6 4. 52 ± 0. 28 6. 52 ± 0.5% 6. 25 ± 0.5l 4. 95 ± O. 61 Vg Cml/minD 6 110 ± 18 128 ± 22 122 ± 15 126 ± 22 Mco2CuM/g. hD 6 2. 03 ± O. 14 2. 39 ± 0. 46 2. 05 ± 0.44 1.85 ± 0.35 22 Table 2. Plasma ammonia levels during high and low pH treatments in rainbow trout * indicate a significant difference from the control (Cl) values, (x ± SE). High pH Treatment n Control 1 Experimentl Exper i ment2 Control 2 Total Ammoni a i n PI asmaC uM/l 6 „:> 48. 9 ± 8. 2 85. O ± 15. % 81.8 ± 17. & 54.5 ± 11.1 [NH3] in Plasma CuM/LD 6 0. 81 ± 0. 15 1. 70 ± 0.3? 1. 80 ± 0.36 0. 88 ± 0. 19 NH3/Tamm ratio 0. 017 O. 020 0. 022 O. 016 Low pH Treatment n Control 1 Experimentl Exper i ment2 Control 2 Total Ammonia in PlasmaCuM/l 6 57. 3 ± 14. 9 173 ± 47. 8 213 ± 49. 3 95. 4 ± 15. 3 CNH3] in Plasma CuM/LD 6 O. 86 ± 0. 24 2. 46 ± O. 6§ 2. 96 ± O. 6§ 1. 36 ± O.2§ NH3/Tamm ratio 0. 015 0. 014 0. 014 O. 014 23 203.4% and 272.8% above the initial control value in El and E2 respectively. It was still 66.4% higher than the initial control value during recovery (Table 2). [NH3] increased with total ammonia content. There was no change in the [NH3]/TAmm ratio as blood pH remained constant (Table 2). 24 2.4 DISCUSSION 1. Downstream water pH In inspired water of approximately neutral pH, expired water was acidified by excreted CCh, which formed HCO3" and protons, the reaction catalyzed by carbonic anhydrase (Wright et al., 1986). At the same time, protons were consumed by excreted NH3 which formed NH4+. As CCh excretion is about 10 times greater than ammonia, the overall result is acidification of the expired water. When fish were exposed to pH 9.91 water, the alkalization of expired water due to NH4+ formation was reduced because at this pH less than half of the excreted NH3 will form NH4+, whereas essentially all the molecular carbon dioxide excreted will form bicarbonate, and a portion will form carbonate. In pH 3.88 water, on the other hand, acidification caused by HCQf formation was negligable, for no more than 1% of the total excreted CCh will be converted to HCCh" at this pH. Alkalization by NH4+ formation, however, will occur because almost all the excreted ammonia is converted to NH4+. As a result, expired water pH showed a small but significant increase. In all experiments, pHst and pHex values were similar, as observed by Wright, et al, (1986), indicating that the CCh hydration/dehydration reaction is catalysed by carbonic anhydrase at the gill surface over a wide range of water pH. When we plot inhalent water pH and exhalent water pH against inspired water pH, the two lines cross at pH 5.3 (Figure 8). At this pH value, about 1/10 of the excreted CCh will be converted to HCCh", acidifying the expired water, but almost all the excreted ammonia, about 1/10 of the amount of excreted CCh, 25 will be converted to NH4+ and raise expired water pH. The overall result, therefore, is no change in water pH as it flows over the gills. The total of acid equivalents added to the neutral water as it passed over the gills was in the same range as carbon dioxide excretion, indicating they, were the result of CO2 hydration. Exposure to alkaline water resulted in a marked increase in the acid equivalents added to the water (Figure 5) without any change in carbon dioxide excretion (Table 1). This is to be expected because of a marked increase in proton production due to carbonate formation (pK2=10). As a result, proton production at this pH will be approximately 1.5 times CCh excretion. Other possible explanations of this observed increase are a decreased formation of N H 4 + at high pH, and/or a reduced HCO3" excretion. If the latter occurs, CO2 excretion will be maintained by an elevated excretion of molecular CO2 augmented by the increased PC02 difference between water and blood (Table 1). The alkaline equivalents added to the water passing over the gills, when fish were exposed to acid waters (Figure 7), were somewhat larger than the expected ammonia excretion, which is known to be reduced in acidic environments (Wright and Wood, 1985). Bicarbonate dehydration will also contribute to this process and may account for the unexpectedly high levels of alkaline equivalents added to the water under acid conditions. 2. C02 excretion In high pH water, a decrease in water and, therefore, blood Pco2 was expected. When the water pH was increased from 6.80 to 9.91, the CCh/HCCh" ratio went down from about 0.2 to 0.002. As a 26 result, there was a decrease of water P002 when pH was increased. This significant decrease in inspired water Pco2 was associated with a lowered plasma Pco2. In low pH water, however, Pco2 was expected to increase for only 1% of the total CO2 will be as HCCh". This in fact, occurred but had no impact on blood Pa>2. Changes in water pH, whether acid or alkaline, had no effect on carbon dioxide excretion by the fish. It appears that Cl/HCCh" exchange across the red blood cell membrane is the rate limiting step in carbon dioxide excretion (Perry et al, 1982) and this will be unaffected by changes in water pH. This may also explain the absence of any effect of a rise in water Pco2 on plasma Pco2 in acid water. 3. Ammonia At an environmental water pH of 9.91, more than half of the ammonia will be in a non-ionic state (NH3); [NH3] will be 100 times greater than that in the control inspired water (pH 6.80). This will greatly inhibit the passive diffusion of NH3 out of the fish. In addition, Wright and Wood (1985) concluded that the Na+/NH4+ exchange mechanism was inhibited by high environmental pH. The observed accumulation of total ammonia in plasma, therefore, was expected in fish exposed to high water pH. The blood NH3/TAmm ratio was increased during the experimental period because of the increase in blood pH. This will facilitate ammonia excretion and ameliorate the rise in ammonia in plasma. In water of pH 6.65 (control) the ratio of NH3/NH4+ is 0.02 but when the water pH was reduced to 3.88 the ratio was only 0.00005. The actual [NH3] difference between water of pH 3.88 and pH 6.65 at constant total ammonia content, however, was very small. This 27 small decrease in water [Nrfa] might be expected to reduce blood [NH3] but in fact blood [NH3] increased despite the reduction in water [NH3] when the fish was exposed to acid conditions. Wright and Wood (1985), however, showed that Na+/NH4+ exchange was abolished in fish exposed to water of pH 3.88 which led to a reduction in total ammonia excretion. Ammonia concentrations in blood increased the longer the acid exposure lasted. [NH3] in plasma also increased because of total ammonia accumulation. The NH3/TAmm ratio, however, was unchanged since there was no change in blood pH. The overall effect of CCh and NH3 excretion is to ameliorate the magnitude of the change in water pH next to the gills in the face of "changes in pH of inspired water. Inspired water pH was varied from pH 3.88 to pH 9.91 but expired pH varied from only pH 4.33 to pH 7.10. 28 3.0 THE EFFECT OF Na+/NH4+ AND Cl'/HCOa" EXCHANGE PROCESSES ON THE ACIDIFICATION OF WATER AS IT PASSES OVER THE GILLS OF RAINBOW TROUT 3.1 INTRODUCTION Considering only the passive diffusion of the non-ionic forms, CO2 excretion induces an acidification while ammonia excretion induces an alkalization to expired water. The magnitude of the acidification/alkalization depends on the environmental water pH, which governs the C02/HC03"and NH3/NH4+ ratios in the aquatic medium (Section 2). The change from acidification to alkalization of expired water occurs at a bulk water pH of about 5.3 (Section 2). Na+/NH4+ and C17HC03" exchange pathways, however, probably also contribute to total carbon dioxide and ammonia excretion and the impact of the elimination of these ionic forms on the acidification/alkalization of expired water will be very different from that of the non-ionic forms. Bicarbonate will bind a proton under acid condition and ammonium ion will donate a proton under alkaline condition. Amiloride, a cation transport inhibitor, inhibits epithelial Na + transport by competing for Na + transport sites. It is known that the addition of amiloride to water will reduce sodium uptake in the gill epithelium of fish by 84% (Perry & Randall, 1981). The anion transport inhibitor, 4-acetamido-4'-iso-thiocyanatostilbene-2,2' disulphonic acid (SITS), inhibits anion transport by binding to a specific membrane protein. Introduction of SITS to water results in a 71% reduction of chloride uptake by gills (Perry & Randall, 1981). Although Na+ and Cl" transport systems are considered to be independent of each 29 other, both amiloride and SITS have been shown to have secondary inhibitory effects on chloride and sodium influxes respectively in fish gills by altering gill epithelial cell pH (Perry & Randall, 1981). The blockage of Na+/NH4+ exchange pathways induced by amiloride also leads to a significant reduction of total ammonia excretion (Payan, 1978). In order to separate the different pathways of CCh and ammonia excretion from each other, amiloride and SITS were introduced to the bathing water of the fish. The studies reported in this section examine the acidification/alkalization of the expired water in a quantitative manner, focusing on the changes in proton concentration in water passing over the gills of fish, under neutral and acidic environmental pH, as well as following amiloride and SITS treatments. The effects of amiloride and SITS on branchial carbon dioxide and ammonia excretions have also been investigated. 30 3.2 MATERIAL & METHODS 3.2.1 Animal & Preparation Rainbow trout (Salmo gairdneri), 282-548 g, were maintained in outdoor fiberglass tanks supplied with flowing dechlorinated Vancouver tap water. Fish were fed daily with commercial trout pellets and feeding was suspended 48 hours prior to experimentation. Surgery was performed on each fish under general anaesthesia (1:10,000 MS222 solution, pH adjusted to 7.5 with NaHCQj) to fix an opercular cannula for sampling expired water. Fish were then confined, but not physically restrained, in a black chamber to recover for 20-36 hours. This black chamber was supplied with outflowing aerated dechlorinated tap water during the recovery period. Three hours prior to the experiment, the water supply was switched to the aerated test solution (40 mmol.L1 NaCl and 0.5 mmol.L"1 CaCb in dechlorinated tap water). Temperature was adjusted to ambient temperature (6.5°-8.5°C) with a cooling coil during recovery, acclimation and experimental periods. 3.2.2 Experimental Protocols & Measurements Three sets of experiment were carried out using a recirculating system with the black chamber (Figure 10). One was a control experiment with the test solution only. The other two were amiloride and SITS treatments, with either amiloride or SITS added to the test solution to give final concentrations of lO^mol.L"1. In each set of experiments the fish was exposed to four environmental pH levels:pH 7, pH 6, pH 5 and pH 4, for a 30 minute period. The volume of the recirculating system was six liters and 31 pH electrodes Figure 10. The recirculating system with a black chamber. Fish were prepared with an opercular cannula. Inspired and expired water samples were withdrawn from the oudets of the glass electrode chambers. was aerated and temperature controlled. A magnetic stirring bar was used in the mixing reservoir to ensure complete mixing. The pH of the test solution was adjusted to 7 by adding 0.1N NaOH or 0.1N HQ and then gradually but quickly lowered to 6, 5 and 4 by adding 0.1N HCl at the beginning of each different environmental pH exposure. The amount of acid added was measured precisely and pH was recorded to construct buffer curves. The water pH of the recirculating system was changing slighdy (<0.3 pH unit per 30 minutes) all the time because of ammonia accumulation and/or carbon dioxide exchange with air. Inspired and expired water samples (approximately 5ml each) were withdrawn from the outlets of the electrode glass chambers at the end of each 30 minute exposure. Between each exposure there was a 15 minutes exchange time during which the water was mixed. Inspired and expired water pH were followed during the whole experimental period with combination glass pH electrodes housed in two water-jacketed glass chambers. Inspired pH (pHin) and expired pH (pHex) values were recorded at the beginning and end of each 30 minutes exposure. Total carbon dioxide content of inspired water (C02in) and expired water (C02ex) was measured immediately with a Carle gas chromatograph (Model HI) containing a CCh discriminating column (porapak Q). Total ammonia content of inspired water (Amrnin) and expired water (Ammex) was measured by a micro-modification of the salicylate-hypochlorite assay with frozen water samples (Verdouw, van Echted & Dekker, 1978). To ensure there was no ammonia loss from water in the recirculating system, two experiments were 33 carried out in which known amounts of NH4CI were added to the system without fish. No loss from the system occurred. 3.2.3 Calculations & Statistics Bicarbonate concentration [HCCh"] increase in expired water, which was equivalent to the calculated proton addition due to CCh excretion by the fish (assuming no HCCh" excretion), was calculated from the difference in total CCh content of inspired and expired water and pHex by the Henderson-Hasselbalch equation, using the pKco2' and 0x02 values from Boutilier et al, (1985). Carbonate formation is negligible over this pH range. Ammonium ion concentration [NH4+] increase in expired water, which was equivalent to the calculated proton consumption due to NH3 excretion by the fish (assuming no NH4+ excretion), was calculated from the difference in total ammonia content of inspired and expired water and pHex by the Henderson-Hasselbalch equation, using the pKamm value from Cameron & Heisler (1983). The calculated proton concentration increase in expired water, [H+]cal, was obtained by subtracting the calculated proton consumption, [NH4+] from the calculated proton addition [HCCh"]. The measured proton concentration increase in expired water, [H+]meas, was calculated from the appropriate buffer curve and inspired and expired water pH. Data are presented as means + standard error. Regression analyses were used to describe relationships between parameters. Student's 2-tailed t-test and 1 & 2-way ANOVA were used to test for significant differences between means and regression equations. Tests of significance were conducted at the 5% level of rejection. 34 3.3 RESULTS 1. Carbon dioxide excretion The differences in total CCh content between inspired water and expired water are presented in Figure 11. There was no significant difference in [CCh]ex-[CCh]in as environmental pH changed from 7 to 4 within control, amiloride or SITS treatments. In pH 6-7 water, [CCh]ex-[CCh]in was significantly lower with the amiloride treatment than during control and SITS treatments. In pH 5-6 water, [CCh]ex-[CCh]in was significantly lower with the amiloride treatment relative to the SITS treatment. There was no significant difference in [CCh]ex-[CCh]in between control, amiloride or SITS treatments in pH range of 4-5. 2. Ammonia excretion The differences of total ammonia content between inspired water and expired water are presented in Figure 12. There was no significant difference in [Amm]ex-[Amm]in as inspired water pH was decreased from 7 to 4 within control, amiloride or SITS treatments. There was a great variation in ammonia content between individual fish. In pH 6-7 water, [Amm]ex-[Amm]in was significantly lower in the amiloride treatment than in the control and the SITS treatment. 3. Proton increase in expired water The values of the calculated proton concentration increase in expired water are presented in table 3. The relationships between the measured proton concentration increase in expired water ([H+]meas) and the environmental water pH (pHin) are presented in figure 13 with two-degree regression curves. The regression curve for control values was not 35 80 Figure 11. The differences of total CO2 content between inspired and expired water with different environmental pH in control, amiloride and SITS treatments. * indicates a significant difference between amiloride and both control and SITS; ** indicates a significant difference between amiloride and SITS only, (x ± SE). 36 Figure 12. The differences of total Ammonia content between inspired and expired water with different environmental pH in control, amiloride and SITS treatments. * indicates a significant difference between amiloride and both control and SITS, (x ± SE). 37 Table 3. Calculated proton concentration increase in expired water CuMol/L.kg) pH 6-7 pH 5-6 pH 4-5 Control 0.205 ± 10.399 -21.382 ± 6.421 -20.279 ± 7.424 Ami loride 2.908 ± 6.301 -10.756 ± 2.601 -10.271 ± 1.416 SITS 12.035 ± 8.031 -8.611 ± 2.340 -6.304 ± 3.793 38 statistically different from that for values obtained with amiloride and SITS treatments. The differences between measured proton concentration increase and calculated proton concentration increase in expired water, [H+]meas-[H+]cal, was around zero when environmental pH was under 6 (Figure 14). In water of neutral pH, however, there were positive differences (Figure 15). The magnitude of the differences were greatest in the control experiment, smaller in the SITS treatment and smallest in the amiloride treatment Because of large variation in CO2 and ammonia contents, these differences were not significant 39 »- Control 200-b. Amiloride 5 6 Environmental water pH SITS 200 150-.100 -in cn o _* £ \ 5 0 -+ x o 3 - 1 0 0 5 6 Environmental water pH 5 6 Environmental water pH 5 6 Environmental water pH Figure 13. The relationships between measured proton concentration increase in expired water ([H+]meas) and environmental pH (pHin) in (a) control experiment, (b) amiloride treatment and (c) SITS treatment. The two-degree regression curves for all three treatments are shown on the same axis in (d). 200 1 5 0 -1 0 0 --100 4.0 4.5 5.0 5.5 6.0 Env i ronmenta l water pH • Cont ro l • Ami lor ide • SITS Figure 14. The differences of measured proton concentration increase and calculated proton concentration increases in expired water ([H+]meas-[H+]cal) within low environmental pH range (pH4-6). The regression line is not significantly different from zero. 41 C o n t r o l A m i l o r i d e SITS Figure 15. The differences of measured proton concentration increases and calculated proton concentration increases in expired water ([H+]meas-[H+]cal) within neutral environmental pH range (pH6-7). 42 3.4 DISCUSSION The effects of amiloride and SITS on branchial sodium and chloride influx have been investigated by Perry and Randall (1981). Amiloride had an immediate inhibitory effect not only on Na + influx but also on Cf influx, by 84% and 54% respectively. SITS also induced an instantaneous 71% reduction in Cl" influx and 81% reduction in Na + influx (Perry & Randall, 1981). Although the Na+/H+(NH4+) and Cl/HC03~ exchange pathways are independent, the linkage of the inhibitory effects of these two drugs leads to similar consequences. If a certain percentage carbon dioxide is excreted through the Cl7HC03~exchange pathway, the addition of these drugs to the bathing water should lead to a reduction of total CO2 excretion. The same will be true for ammonia excretion. The fact that SITS did not have any impact on the [C02]ex-[C02]in indicates that CO2 excretion via the C1 /HCO3" exchange pathway is unimportant, if SITS did not alter ventilation of the fish. Alternatively, if 10% of the total CO2 excretion is in the form of H C O 3 " in exchange for Cl", then blockage of this exchange pathway may result in an elevation of Pco2 in fish plasma, which in turn will enhance the molecular CO2 diffusion. Since the bicarbonate elimination pathway is responsible for no more than 10 percent of the total CO2 excretion, 30 minutes should be enough for the development of the higher blood-to-water Pco2 gradient and the re-establishment of the normal total CO2 excretion rate. The amiloride treatment however, did result in a decrease of [C02]ex-[C02]in. This is very difficult to explain. Since CI/HCO3" exchange appears not to play an important role in carbon 43 dioxide excretion, then the blockade of the Cl'/HCCh" exchange process by amiloride could not be responsible for the decrease. Also, the results of our study are in conflict with those from Wright, et al, (1989). Using a blood-perfused trout head preparation, they found that amiloride treatment increased [C02]ex-[C02]in because the blockade of Na + /H + and Na+/NH4+ exchange processes induced an acidosis in the gills which in turn reduced C O 2 stores. We don't know why amiloride in this in vivo study had the opposite effect-reducing [C02]ex-[C02]in and why the effect vanished when water pH was 5 or below. It is possible that amiloride triggered an increase of ventilation in the animal which lowered the CCh content in the expired water while CCh excretion did not change. A reduction of [Amm]ex-[Amm]in occurred with the amiloride treatment, presumably due to Na+/NH4+ inhibition. When water pH was reduced, the [Amm]ex-[Amm]in increased slightiy. This might be due to the fact that in time, reduced ammonia excretion (if amiloride did not alter ventilation and [Amm]ex-[Amm]in was representative of ammonia excretion) will lead to an elevation of ammonia content in fish plasma, which in turn will increase the blood-to-water Nfb diffusion gradient, and enhance ammonia excretion. Thus, total ammonia store in the fish body increased, and eventually ammonia excretion was matched to ammonia production. As the Na+/NH4+ exchange process may contribute up to 40% of the total ammonia excretion and the ammonia excretion rate is low compared with total ammonia stores, the time needed for re-establishment of the normal total ammonia excretion rates will be longer than that required to re-establish CCh excretion rates 44 following the SITS treatment SITS, whose inhibitory effect on Cl/HC03~ exchange was greater than that of amiloride (Perry & Randall, 1981), might have induced an alkalosis and raised the NH3/NH4+ ratio in plasma which in turn would result in a higher NH3 gradient across the gill. Thus the reduction in [Amm]ex-[Amm]in due to Na+/H+(NH4+) inhibition would be less severe with SITS than with amiloride treatment. A reduction in ammonia excretion was also expected as environmental pH decreased, for Na+/NH4+ exchange was reported to be inhibited by low pH (Wright & Wood, 1985; section 2). We believed this was the case in our control study. The differences between the mean values of [Amm]ex-[Amm]in were not significant was due to the large variation of ammonia excretion between individual fish. The tendency for [Amm]ex-[Amm]in to decrease with decreased environmental pH was similar in control and SITS treatment At environmental pH under 5, the Na+/NH4+exchange process was blocked in all treatments, and the differences in [Amm]ex-[Amm]in between treatments decreased. One of the reasons there was such a large variation in ammonia levels was that the fish we used were subjected to different periods of starvation. According to Brett & Zala, (1975), the ammonia excretion rate will return to a baseline level 24 hours after feeding in sockeye salmon. In our study, feeding was suspended at least two days before surgery. Even so, the fish starved for 48-72 hours had a ammonia excretion rate 2-3 times higher than those starved longer. When calculating [H+]cal, we assumed that fish excreted carbon dioxide and ammonia by molecular C O 2 and N H 3 diffusion 45 only, and that acid influx or efflux through the gill epithelium was negligible. Thus if our assumptions are correct, the calculated proton concentration increase in expired water which represented the net proton gain from the interaction of carbon dioxide and ammonia excretions, should equal the measured proton concentration increase in expired water. This occurred when environmental water pH was under 6 (Figure 14), indicating that both Na + /H + and Na+/NH4+ exchange were inhibited. The fact that the introduction of amiloride made no difference was not surprising because it appears to have the same effect as low pH on Na+/H+(NH4+) exchange (Payan, 1978; Wright & Wood, 1985). The introduction of SITS did not lead to a positive value and this supports the contention that Cl/HCOf exchange was playing only a minor role, because in this pH range, if there was any CCh excreted in the form of HCCh", it would be converted to CCh, protons would be consumed, and the value of the measured proton concentration increase in expired water would be smaller in the control than in the SITS treatment. The zero difference of [H+]meas and [H+]cal in low environmental pH with all three different treatments also suggested that HCCh" and NH4+ diffusion was playing very little role in carbon dioxide and ammonia excretion. In neutral water pH, the differences between [H+]meas and [H+]cal were positive in all treatments indicating that there must be a proton efflux through the gill epithelium. The value of [Hr]meas-[Hr]cal was higher in control than in amiloride and SITS treatments, indicating the presence of a Na+/H+(NH4+) exchange. The importance of Cl'/HCCh" exchange cannot be assessed from this 46 data, but may be a minor contributing factor. Sodium influx across gills in trout batfiing in water of different environmental pH was reported by Randall & Wright (1986). The protons excreted by fish through the Na+/H+(NH4+) exchange pathway in the gill epithehum can be predicted from the Na + influx, if we assume that there is a 1:1 coupling of Na+ influx to proton efflux. In addition, if we assume the ventilation rate of the fish was 150ml/min (see Section 2), we can convert the Na + influx data into the same units as the [H+]meas-[H+]cal data and plot them together (Figure 16). The difference between the measured and calculated proton concentration increase in expired water were similar to that predicted from the Na+influx data, which indicates that Na + /H + exchange may be responsible for the so called background proton concentration increase in expired water in fish exposed to water of neutral pH. 47 2 0 0 1 5 0 -5 6 Environmental water pH o Experimental data • Predicted data Figure 16. The relationship between ([H+]meas - [H+]cal) and environmental water pH with experimental data (obtained from control experiments) and predicted data (obtained from N a + influx data reported in Randall & Wright, 1986). 48 4.0 GENERAL CONCLUSIONS & SUMMARY It has been demonstrated in these studies that the passive diffusion of molecular CO2, the predominant pathway of total carbon dioxide excretion, was the major source of the acid equivalents added to the water passing over the gills of fish. Carbon dioxide excretion was not affected by environmental water pH and the Cf/HCOi inhibitor SITS. CO2 hydration, generating protons in the boundary water layer, was catalyzed by carbonic anhydrase and the extent of hydration was dependent on environmental water pH. Thus the magnitude of the acidification in expired water decreased with decreasing inspired water pH. The Cl7HC03" exchange process appears to be unimportant for total CO2 excretion. The elimination of un-ionized ammonia (NH3) by passive diffusion generated alkaline equivalents in expired water. Ammonium ion (NH4+) formation, similar to CO2 hydration, was pH dependent and the magnitude of alkalization in expired water increased with decreasing environmental water pH. The maintenance of the blood-to-water NHa partial pressure gradient depended on the availability of hydrogen ions in the boundary water layer (Wright et al, 1989). Therefore, a reduction of CO2 excretion (Wright et al, 1989), inhibition of carbonic anhydrase activity (Wright et al, 1989), increase of water buffering capacity (Wright et al, 1989), increase of environmental water pH and blockade of H efflux in exchange for Na , all had an inhibitory effect on NHb excretion. The elimination of NH4+ in exchange for Na+ played a role in 49 total ammonia excretion. This ammonia excretion pathway can be inhibited by amiloride, SITS and acid and alkaline water conditions. If water pH was below 6, then pH changes in water passing over the gills could be accounted for by reactions resulting from CCh and NH3 alone, indicating inhibition of Na + /H + and Na+/NH4+ exchange, and that the gill epithelium is not permeable to NH4+ or HCCh" ions. If water pH was above 6, then acid equivalents were added to the water passing over the gills, in addition to those generated by CCh hydration. The source of these additional protons could be Na + /H + exchange. Cl'/HCCh" exchange appears to play only a minor role in the removal of acid equivalents from the water. These studies have described the role of carbon dioxide and ammonia excretion in the pH changes occurring in the water as it passes over the gills of trout. The overall effect is to ameliorate the effect of changes in water pH on the pH of water in contact with the gills. An understanding of the chemistry of the water in contact with the gills is important in evaluating the state of surface active toxicants acting on the gill epithelium of fish. 50 5.0 REFERENCES Brett, J.R. and Zala, CA. (1975). Daily pattern of nitrogen excretion and oxygen consumption of sockeye salmon (Oncorhynchus nerka) under controlled conditions. J.Fish. Res. Biol. Can. 32: 2479-2486. Boutilier, R.G., Iwama, G.K., Heming, T.A. and Randall, D.J. (1985). The apparent pK of carbonic acid in rainbow trout blood plasma between 5 and 15°C. Respir. Physiol. 61:237-254. Cameron, J.N. and Heisler, N. (1983) Studies of ammonia in the rainbow trout: physiochemical parameters, acid-base behaviour, and respiratory clearance. J. exp. Biol. 105: 107-125. De Renzis, G. (1975). The branchial chloride pump _ in the goldfish Carassius auratus: relationship between C1/HCO3" and Cf/Cl" exchanges and the effect of thiocyanate. J. exp. Biol. 63: 587-602. Girard, J.P. and Payan, P. (1980). Ion - exchanges through respiratory and chloride cells in freshwater- and seawater-adapted teleosteans. Am. J. Physiol. 238: 260-268. Lenfant, C. and Aucutt, C. (1966). Measurement of blood gases by gas chromatography. Respir. Physiol. 1:398-407. Maetz, J. (1973). Na+/NH4+, Na + /H + exchanges and NH3 movement across the gill of Carassius auratus. J. exp. Biol. 58: 225-275. Payan, P. (1978) A study of the Na+/NH4+ exchange across the gill of the perfused head of the trout (Salmo gairdneri). J. Comp. Physiol. 124: 181-188. Perry, S.F., Davie, P.S., Daxboeck, C. and Randall, D.J. (1982). A comparison of CCte excretion in a spontaneously ventilating blood-perfused trout preparation and saline-perfused gill preparation: contribution of the branchial epithelium and red blood cell. J. exp. Biol. 101: 47-60. Perry, S.F and Randall, D.J. (1981). Effects of arniloride and SITS on branchial ion fluxes in rainbow trout, (Salmo gairdneri). J. exp. Zool. 215:225-228. Rahim, S., Delaunoy, J.P. and Laurent, P. (1988). Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostean fish erythrocyte and gill epithelia. Histochem. 89: 451-459. Randall, DJ. (1970). Gas exchange in fish. In Fish Physiology (ed. W.S. Hoar and D.J. Randall), Vol. 4, pp. 253-292. New York: Academic Press. 51 Randall, DJ. and Cameron, J.N. (1973). Respiratory control of arterial pH as temperature changes in rainbow trout (Salmo gairdneri). Am. J. Physiol. 225:997-1001. Randall, DJ. and Daxboeck, C. (1984). Oxygen and carbon dioxide transfer across fish gills. In Fish Physiology (ed. W.S. Hoar and DJ. Randall), Vol. 10A, pp. 263-314. New Youk: Academic Press. Randall, DJ. and Wright, P.A. (1986). Ammonia production and excretion in fish. Proceedings of the US-USSR Symposium on Aquatic Toxicity. Borok, Yaraslavl, USSR. EPA/600/9-86/024. Randall, DJ. and Wright, P.A. (1987). Ammonia distribution and excretion in fish. Fish Physiol.Biochem. 3:107-120. Randall, DJ. and Wright, P.A. (1989). The interaction betreen carbon dioxide and ammonia excretion and water pH in fish. Can. J. Zool. In press. Sigma (1982). The quantitative ultraviolet determination of ammonia in plasma at 340 nm. Sigma Technical bulletin 170-UV. Sigma Chemical Co., St. Louis. Verdouw, H , van Echted, CJ.A. and Dekkers, E.MJ. (1978). Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12: 399-402. Wright, P.A., Heming, T.A. and Randall, DJ. (1986). Downstream pH changes in water flowing over the gills of raibow trout. J. Exp. Biol. 126: 499-512. Wright, P.A. and Randall, DJ. (1987). The interaction between ammonia and carbon, dioxide stores and excretion rates in fish. Annie Sor. r. zool. Belg. 117:321-330. Wright, P. A., Randall, DJ. and Perry, S.F. (1989). Fish gill water boundary layer: site of linkage between carbon dioxide and ammonia excretion. J. Comp. Physiol. In press. Wright, P.A. and Wood, C.M. (1985) An analysis of branchial ammonia excretion in the freshwater rainbow trout: effects of environmental pH change and sodium uptake blockage. J. Exp. Biol. 126: 329-353. 52 

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