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

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M E C H A N I S M O F ACIDIFICATION OF EXPIRED W A T E R IN FISH By HONG LIN B . A . S c , Zhejiang University, P.R.C.,  1986  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F M A S T E R O F SCIENCE in T H E F A C U L T Y O F 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 C O L U M B I A  April @  1989  H O N G LIN, 1989  In  presenting this  degree  at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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 The or  ii effect  of varying inhaled water pH on the  alkalization of  trout  water  as  it  (Salmo gairdneri) was  passes over  investigated  the  by  acidification  gills  in rainbow  exposing  the  fish  to  pH 9.91 and pH 3.88 water. In the control and high pH treatment, water  was  because  acidified  of  the  as  it  flowed  dominant  effect  over of  the  gills  of  CO2 hydration.  the  fish  Water  was  alkalized as it flowed over the gills of the fish  in the low pH  treatment due to  dehydration. The  NH4  of  +  formation and perhaps  overall  result  change  of expired water  inspired  water.  affected  by  CO2 and  the  high  NH3 excretion  pH in the  Carbon  dioxide or  HCO3'  low  is  face  of  excretion pH  to  ameliorate  changes  was  treatments  in pH of  not but  the  significantly ammonia  was  accumulated in fish plasma in both cases. The on  the  impact of Na /H (NH4 ) and Q7HC03~ exchange processes +  acidification  +  or  +  alkalization  of  expired  water  has  also  been examined. Amiloride or SITS was introduced to the water to inhibit was  the  gradually  conditions, for  ionic  exchange lowered  processes  from  molecular CO2 and  all the  proton  and  neutral  environmental  to  acidic.  NH3 excretion  concentration  changes  from  water, indicating that Na /H (NH4 ) transport +  environmental concentration excretion,  pH. changes  and  +  In were  sodium  the influx  Under could  pH  acidic account  inspired to expired  was inhibited by low  +  neutral  alone  water  environments,  the  proton  of molecular CCh and NH3  result in  exchange  for  H (NH4 ). +  +  Cr/HCCb'exchange was not an important pathway for carbon dioxide elimination  but  inhibition  reduction of ammonia excretion.  of  Na /H (NH4 ) +  +  +  exchange  caused  a  T A B L E O F CONTENTS  iii PAGE  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 acidification 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  2.2.2  Experimental Protocols & Measurements  2.2.3  Calculations & Statistics  2.3  Results  14  2.4  Discussion  25  3.0  The effect of Na /NH4 +  10 10 13  +  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  3.2.1  Animal & Preparation  3.2.2  Experimental Protocols & Measurements  3.2.3  Calculations & Statistics  3.3  Results  35  3.4  Discussion  43  4.0  General Conclusions & Summary  5.0  References  31 31 31 34  49 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 O F FIGURES  FIGURE 1  PAGE 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 4  11  pH of inspired, expired and stopped-flow expired water of rainbow trout during high pH treatment  5  Proton concentration increase  in  the  expired  water during high pH treatment 6  16  pH of inspired, expired and stopped-flow  expired  water of rainbow trout during low pH treatment 7  Proton concentration increase  in  15  the  17  expired  water during low pH treatment 8  The relationship of  18  exhalent water to inhalent  water pH in rainbow trout 9  19  Dorsal aortic blood p H of rainbow trout  20  10  The recirculating system with a black chamber  11  The differences inspired  and  of total expired  CO2 content water  with  32  between different  environmental pH 12  36  The differences of total ammonia content between inspired  and  expired  water  with  different  environmental pH 13  37  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  vii ACKNOWLEDGEMENT  I am very grateful to my supervisor Dr. D.J. Randall for his valuable guidance and support throughout the studies. I thank the  post-doctorals, graduate  our lab, who provided  students  and technicians  all  working in  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  major  (Randall, 1970). The  respiratory  countercurrent  portion  of  arrangement  secondary the  gill  of blood  lamellae represent the structure.  and water  There  flows  is a  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  availability in  of oxygen  water, carbon  problem such  of molecular  and increased solubility  dioxide  for water-breathers as  fish,  CO2. Because  adjust  excretion than  the reduced  of carbon dioxide  is usually  oxygen  their  of  a  less  critical  uptake. Water breathers,  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 The  water  into  close  contact with  the gill epithelium.  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  BULK  BOUNDAIY WAT E l  WATER FLOW  d U .  IAYE1  BLOOD  EPITHELIUM  V 3  c o  11. to  HCO. H + NH  NH  I NH  Figure  1.  boundary interactions  A water  simplified layer  between  cross-section and  carbon  bulk dioxide  water layer (Randall & Wright, 1989).  through water  and  the  flow  ammonia  gill showing in  the  epithelium, possible boundary  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: C O 2 + H 2 O == H C O 3 " + H  +  ==  COT  pKi' = 6.08 , p K 2 = 10 PKi'  is  the  apparent first  + 2fT  (15°C)  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 ionic  dissociation strength  alkaline  alone  solutions,  CO2/HCO3"  Randall,  constant  (Boutilier  the  et  carbonate  varies al,  In fish  plasma,  with  1985).  where  temperature Except  concentration  ratio changes according to  1987).  CO2/HCO3"  which  is  highly  negligible.  pH (Figure 2; pH is  in  and  The  Wright &  around 7.85,  the  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  C O 2 refers  to  the  combined  concentration of molecular C O 2 , bicarbonate (HCCh") and carbonate (CO3 "). 2  The gill epithelium is highly permeable to molecular C O 2 but less permeable to HCCh", and C O 2 can dissolve into water easily, therefore,  passive  diffusion  of  molecular  C O 2 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  hydration/dehydration is very  the  reaction  slow compared  uncatalyzed  carbon  is of the order  dioxide  of minutes, which  to the interlamellar transit time of gill  water (100-400 ms) (Wright et al, 1986). Carbonic anhydrase, an enzyme located  that  catalyzes  carbon  in erythrocytes  dioxide  and gill  hydration/dehydration, is  epithelium  region of the outer layer of gill  including  epithelial cells  1988). Wright et al (1986) investigated  the apical  (Rahim  the downstream  et al, 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 pKamm  changes  Randall,  1987). The  reaction  with  in water  temperature  and  ratio  NH3/NH4" " 1  is  extremely  ionic  rapid and  strength  increases  with  (Wright  &  increasing pH  (Figure 2). Within the biological pH range (usually around neutral or  slightly  alkaline),  the  predominant  ionic form. For example, in fish  form  of  ammonia  is  the  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  impermeable to NH4 ammonia from  can  are  highly permeable  passively  down  blood to water across the gill  will  be converted to NH4  1),  and  words, protons  boundary  water  in the  boundary  layer  and thus maintain a high  blood  and  water  and  its  partial  pressure  gradient  epithelium. The excreted NH3  according to environmental pH (Figure  +  NH4 , +  NH3 but relatively  (Wright & Randall, 1987). Therefore, non-ionic  +  diffuse  the  to  facilitate  will  water NH3  be  alkalized.  layer will  In  convert  other  NH3 to  concentration gradient between  ammonia  excretion  (Wright et  al,  1989). Although little NH4 be  excreted  branchially  +  can diffuse  by  coupling  across the gills, NH4 to  ion  carriers  There is some evidence to suggest that NH4 the  gill  account  epithelium for  in  10-40%  exchange  of  the  for  total  +  sodium.  branchial  can  +  (Figure  1).  is excreted through This  ammonia  pathway  may  excretion in  resting rainbow trout (Randall & Wright, 1989). CCh CO2  elimination generates protons  hydration whereas  NH3  in the  gill  water  due  excretion consumes protons due to  to  NH4  +  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  Increasing ammonia  also  the  ammonia  buffering  excretion  excretion  capacity  et  (Wright  of  external  but does not affect carbon  al,  water  1989).  decreases  dioxide excretion  indicate that C O 2 excretion  (Wright et al, 1989). These results  provides a continual supply of hydrogen ions which are necessary to maintain  a sufficient blood-to-water N H 3 diffusion  gradient for  ammonia excretion. The  acidification  and alkalization  of expired  water induced  by C O 2 and N H 3 excretions cancel each other to some extent. Since total carbon dioxide excretion is normally total  ammonia  excretion  (Wright  &  10 times greater than  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 pKamm at different temperatures (Randall & CO2/HCO3"  and  NH3/NH4  +  pKico2  and  Wright, 1989). Both  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 passive  in fish  blood  diffusion, but reduce  excretion. An  alkalosis  will  and enhance C O 2 excretion by  N H 3 formation have  the reverse  high environmental water pH inhibits NH4 elimination,  but  favors  C O 2 hydration 7  and decrease N H 3  +  effect. Similarly,  formation and ammonia and  therefore,  carbon  dioxide excretion. The  gills  acid-base  fish  regulation.  Cf/HCCh"  exchange  documented  (Maetz,  suggested that of  of  +  +  +  The  processes de  regulate +  also  involved  presence  1973;  fish  Na /H ,Na /NH4  are  in  Payan, 1980). The Na /NH4 +  transfer and  Na /H ,  Na /NH4  gill  epithelial  cells  +  1975;  acid-base  and Cf/HCCh"  ion  of  Ranzis, their  in +  +  Payan,  and  +  is  1978).  well It is  status via modulation  exchange processes (Girard &  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 will  in the water, the gill water  due to CO2 hydration  be acidified  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  will  small  acidification with  fraction  be  hydrated.  Thus  of water as it passes over the gills  water pH. The opposite  reaction. Under  acid +  converted to NHt  will  conditions  be true  almost  the extent of will decrease +  for the NH3:NH4  all the N H 3 will  be  but under alkaline conditions only a fraction of  the excreted NH3 will form NH4 " in the gill water. The experiments 4  reported of  in this  section were designed  to investigate the effect  varying inhaled water pH on the acidification of water as  passes over the gills.  9  it  2.2  MATERIALS & METHODS  2.2.1  Animal & Preparation Rainbow trout (Salmo gairdneri), weighing  g, were  324-494  obtained from the West Geek Trout Farm ( Aldergrove, BC ) and housed  in  outdoor  dechlorinated  fiberglass  Vancouver  tanks  tap  water  supplied (pH  with  6.5-6.8;  flowing  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 (1986).  After  the  surgical  of  ventilation  procedure,  as  fish  in Wright  were  left  et al,  to  recover  for 20-40 hours in the van Dam apparatus (Figure 3) supplied with a flowing test solution of 40 mmol.L" NaCl and 0.5 mmol.L CaCh 1  in  dechlorinated tap  water  1  (9.5-11.2°C)  with  a buffering capacity  (p) of 81 u.equiv. L'\pH unit". The test solution had the same 1  ionic  strength  as  the  buffer  electrodes.  By using  this  time  the  electrode,  of  pH  solution  used  test solution,  we  increased  its  to  calibrate  the pH  reduced the  response  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  Peristaltic pump  Dechlorinated tap water Air  Test solution veservoir  Cooling coil 9  Concentrated salt solution Expired water pH meter  Chart Recorder  Inspired water pH meter  Inflow watar  pH i l i c t n i i s valwi  • 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  stopped-flow water pH  water  (pHst) were measured  pH  (pHex)  and  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 G297/G2 capillary  (pHB)  was measured by using a Radiometer  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 UV-visible  of  recording  a  commercial  spectrophotometer  diagnostic  kit  (L-glutamic  with  a  dehydrogenase/  NAD method; Sigma, 1982). 8) Inspired and expired water buffering capacities in  were determined  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 C O 2 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  C O 2 content  total  Henderson-Hasselbalch equation,  using  the  and  pH  by  p K ^ and a  the values  c Q 2  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 through  the gills  , the inspired water was (Figure 4). There was  acidified as it passed  no  significant difference  between pHex and pHst in Cl and E l , but a significant difference was  exhibited in E2  and  C2. The  increase in expired water was  calculated proton concentration  significandy larger when the animal  was exposed to alkaline water (Figure 5). There was significant  difference  in  the  proton  , however, no  concentration  increase  in  expired water between E l and E2, or between Cl and C2. In rather  the low  than  pH  treatment, the inspired water was  acidified, as  There was  it passed  over  the  gills  alkalinized (Figure 6).  no significant difference between pHex and pHst except  in E2. The  decrease in proton concentration in expired water was  larger  the  than  Once again  increase observed  there was  no  in  neutral water  (Figure 7).  significant difference in the  proton  concentration change in expired water between either E l 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  water  The  blood of the fish was  pH  was  raised  difference in blood pH blood pH  to  pH  alkalinized when the inspired  9.91.  There  between E l and E2  was  no  significant  (Figure 9). The whole  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  C1.30minin pH 6.80 water  •  Inspired  E1.45minin  E2.90min in  pH 9.91 w a t e r  pH 9.60 water  WM Expired  C2.30min in pH 7.25 water  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 C 2 . (x ± SE).  15  10i  C1.30min in pH6.80 water  E1.45min in pH9.91 water  E2.90min in C2.30min in pH9.60 water 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  10-  x  8--  CL  n in pH 6 . 6 5 w a t e r  •  Inspired  p  H 3 . 8 8 water  • § Expired  pH 3 . 9 9 water  pH 6.37 water  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  1.5 o  cn  2 o  -c  •-  o  1.0 0.5 0.0  c  o  -0.5  O C O O  "O 0) .b  -1.0  c o  CL X  T -1.5 -2.0  C1.30minin pH6.65 water  P  E1.4^r t.l.+5min in H3.88 water  E2^0min in pH3.99 water  +30min in C2. 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).  18  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, r 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. 2  19  8.2 X  CL  TJ  8.1 --  O O  CD  _0)  8.0--  O  7.9-  7.8  t  t  \  T  I  t  1  C1.30min in pH6.80/6,65 water  E1.45min in E2.90min in pH9.91/3.88 pH9.60/3.99 water water  O High pH treatment  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 C O 2 content exposure of  the fish to pH 9.91  during  water (Table 1). Plasma  Pco2  however,  was reduced. Inspired and expired water total C O 2 was  increased  because carbon dioxide  alkaline  water  even though  control values difference within  trapped as  inspired water  (Table 1). There was,  between  the  was  inspired  control  and  and  expired  experimental  was  Pco2  however,  in this  HCO3"  lower than  no change in the  water  between or  Tco2  periods,  that  is  carbon  dioxide excretion was unaffected by the elevation in water pH and Ta>2.  The ventilation  but  there  were  volume  no  (Vg) varied greatly  significant  differences  between animals  during  the  whole  experimental period (Table 1) There CO2  were also no  content,  animal  to  plasma  significant Vg and  Pco2,  acid conditions  (Table  differences  during exposure  Mco2  1).  in plasma total  of  Pco2  the  of the  inspired acid  water was raised to twice that of control water, but total CCte content  decreased.  however,  in  No  Tco2(ex-in)  significant between  difference  fish  in  control  was and  observed, 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 during increased  recovery along  (Table with  2). the  water but returned to normal  Non-ionic  ammonia  [ N H 3 ] / T A m m  ratio,  content [NH3] reflecting  the  elevation in blood pH (Table 2). Total ammonia content in plasma went up continuously during the pH 3.88 21  treatment, increasing by  Table  1.  treatments  Carbon in  dioxide  rainbow  excretion  trout  *  during  indicate  a  high  and  significant  low  pH  difference  from the control (Cl) values, (x ± SE).  High pH Treatment n Total C02  Control 1 Experi mentl Exper i ment2  Control 2  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.ol0. 40 ± 0.02  CmM/LD Expire 7 0. 53 ± 0. 02 0.60 ± 0.Ol 0.64 ± O.ol 0. 54 ± 0. 02 Tco2C ex-in) CmM/LI) Pco2 C Tor r D  7 0. 15 ± O. 01 O. 15 ± O. 02 0. 14 ± 0. 02 0. 14 ± 0. Ol  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 ± 0.060. 00 ± o. 08 0. 01 ± 0.080. 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 ± 0.452. 46 ± 0.28 Low pH Treatment n Total C02  Control 1 Experimentl Experiment2  Control 2  PIasma 6 17. 1 ± 1 . 4 16. 3 ± 1.7 16. 2 ± 1.7 16. 5 ± 1.7 Inspire >e O. 40 ± 0.020. 32 ± 0. ol 0. 32 ± 0.Ol 0. 41 ± 0. 03  CmM/L} Expire 6 0. 54 ± 0. 03 0. 46 ± 0. ot 0. 44 ± 0.ot 0. 52 ± 0.04 Tco2Cex-i nD CmM/L:> Pco2 CTorrD  6 0. 14 ± 0. 02 0. 14 ± 0. 03 0. 12 ± 0. 03 O. 11 ± 0.02  PIasma 6 4. 54 ± 0. 30 4. 51 ± 0. 43 4. 44 ± 0.424.55 ± 0.40 Inspire >& 1.87 ± 0.074. 63 ± O. 2§ 4. 54 ± O.2§ 2. 87 ± O. i S Expire 6 4. 52 ± 0. 28 6. 52 ± 0.5% 6. 25 ± 0.5l4. 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 ± 0.441.85 ± 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 6 48. 9 ± 8. 2 85. O ± 15. % 81.8 ± 17. & 54.5 ± 11.1 i n PI asmaC uM/l„:> [NH3] i n 6 0. 81 ± 0. 15 1. 70 ± 0.3?1. 80 ± 0.360. 88 ± 0. 19 Plasma CuM/LD NH3/Tamm r a t i o  0. 017  O. 020  0. 022  O. 016  Low pH Treatment n  Control 1 Experimentl Exper i ment2 ± 47. 8 213  Total Ammonia 6 57. 3 ± 14. 9 173 i n PlasmaCuM/l  Control 2  ± 49. 3 95. 4 ± 15. 3  CNH3] i n 6 O. 86 ± 0. 24 2. 46 ± O. 6§ 2. 96 ± O. 6§ 1. 36 ± O.2§ Plasma CuM/LD NH3/Tamm r a t i o  0. 015  0. 014  23  0. 014  O. 014  203.4% and 272.8% above the initial control value in E l and E2 respectively. value  It  during  was  still  recovery  66.4% (Table  higher 2).  than  [NH3]  ammonia content. There was no change in the blood pH remained constant (Table 2).  24  the  initial control  increased  with  [NH3]/TAmm  total  ratio as  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  essentially  all  of the  the  excreted  molecular  NH3 will  form  carbon dioxide  NH4 , +  excreted  whereas  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  +  however,  will  occur because  almost all the excreted  formation,  ammonia is  converted to NH4 . As a result, expired water pH showed a small +  but significant increase. In observed  all experiments, pHst and pHex values were similar, as by  Wright,  hydration/dehydration  et  reaction  al, is  (1986),  indicating  catalysed  by  that  the CCh  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 converted to HCCh",  of the excreted CCh will be  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  C O 2 hydration.  of  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  explanations of  NH4  latter  of  1.5  this  times  observed  CCh increase  excretion. are  Other  a decreased formation  at high pH, and/or a reduced HCO3"  +  occurs,  excretion  of  C O 2 excretion molecular  will  CO2  be  excretion. If the  maintained by  augmented  by  possible  the  an elevated  increased  PC02  difference between water and blood (Table 1). The gills,  alkaline equivalents added to the water passing over the  when fish  somewhat  were  exposed  larger than the  to  acid waters  expected  (Figure 7),  ammonia excretion,  were  which is  known to be reduced in acidic environments (Wright and Wood, 1985).  Bicarbonate  process  and  may  dehydration account  for  will the  also  contribute  unexpectedly  high  to  this  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);  will be  [NH3]  times greater than that in the control inspired water  (pH  100 6.80).  This will greatly inhibit the passive diffusion of NH3 out of the fish.  In  Na /NH4 +  pH.  +  The  therefore, blood  addition, Wright  and Wood  concluded  (1985)  that  the  exchange mechanism was inhibited by high environmental observed  accumulation  was expected  N H 3 / T A m m  in fish  of  total  exposed  ammonia  to  in  plasma,  high water pH. The  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,  27  was  very  small.  This  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  concentrations in  blood  in  total  increased  the  ammonia excretion. longer  the  acid  Ammonia exposure  lasted.  [NH3]  in  plasma also increased because of total ammonia accumulation. The N H 3 / T A m m  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  THE EFFECT OF Na /NH4 AND Cl'/HCOa" EXCHANGE PROCESSES  3.0  +  +  ON THE ACIDIFICATION OF WATER AS IT PASSES OVER THE GILLS OF RAINBOW TROUT  3.1  INTRODUCTION Considering  forms,  the  CO2 excretion  excretion of  only  induces  the  passive  induces  diffusion  an  of  acidification  the  non-ionic  while  ammonia  an alkalization to expired water.  acidification/alkalization  depends  on  The magnitude  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  however,  2).  probably  Na /NH4 +  also  and  +  contribute  C17HC03" to  total  exchange carbon  pathways,  dioxide  and  ammonia excretion and the impact of the elimination of these ionic forms  on  the  very different  acidification/alkalization from that of  of  expired  the non-ionic  forms.  water  will  be  Bicarbonate will  bind a proton under acid condition and ammonium ion will donate a proton  under  alkaline  inhibitor,  inhibits  transport  sites.  condition.  epithelial It  is  Na  known  Amiloride, transport  +  that  the  by  a  cation  competing  addition  of  transport for Na  amiloride  +  to  water will reduce sodium uptake in the gill epithelium of fish by 84%  (Perry  &  Randall,  1981).  4-acetamido-4'-iso-thiocyanatostilbene-2,2'  The  anion  transport  disulphonic  inhibitor, 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 fish  gills  effects by  on chloride  altering  1981). The blockage amiloride excretion  gill of  epithelial  Na /NH4 +  also leads to (Payan,  and sodium cell  In  respectively  pH (Perry  in  & Randall,  exchange pathways induced by  +  a significant  1978).  influxes  order  reduction to  of  total ammonia  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  acidification/alkalization manner, focusing passing  over  environmental  of  this the  section  expired  examine  water  in  a  quantitative  on the changes in proton concentration the  pH,  gills as  of  well  fish, as  under  following  neutral amiloride  the  in water  and and  acidic 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 confined, recover  but  not  physically  restrained,  for 20-36 hours. This  outflowing  aerated  dechlorinated  in  a  black  black chamber was tap  water  were then chamber to  supplied with  during  the  recovery  period. Three hours prior to the experiment, the water supply was switched to the aerated test solution (40 mmol.L  1  mmol.L"  CaCb  1  adjusted  to  in  dechlorinated  ambient  temperature  tap  water).  (6.5°-8.5°C)  NaCl  and 0.5  Temperature  was  a cooling  coil  with  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  In  each  test set  solution of  to  give  experiments  final the  concentrations fish  was  of  exposed  lO^mol.L" . 1  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  were  prepared  with  an  water  samples  were  withdrawn  electrode chambers.  opercular  cannula.  from  the  a black chamber. Fish Inspired oudets  and of  the  expired glass  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  exposure.  the  beginning  The amount of  of  each  different  environmental pH  acid added was measured precisely and  pH was recorded to construct buffer curves. The water pH of the recirculating minutes)  all  system was the  time  changing slighdy because  of  ammonia  accumulation and/or  air.  Inspired  and  dioxide  samples  (approximately 5ml each) were withdrawn from the outlets  the electrode  with  pH unit per 30  carbon  of  exchange  (<0.3  glass chambers at the end of  expired  water  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  salicylate-hypochlorite  measured assay  by  with  a  frozen  micro-modification water  samples  of  the  (Verdouw,  van Echted & Dekker, 1978). To ensure there was no ammonia loss from  water  in  the  recirculating 33  system,  two  experiments  were  carried out in which known amounts of  were added to the  NH4CI  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  calculated  from the  (assuming  difference  no  in  total  HCCh"  excretion),  CCh content  was  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  calculated  by  from  the  the  fish  (assuming  difference  in  no total  NH4  +  excretion),  ammonia  content  was of  inspired and expired water and pHex by the Henderson-Hasselbalch equation, using the The water,  pKamm  calculated  [H ]cal, +  was  value from Cameron & Heisler (1983). proton  obtained  concentration by  increase  subtracting the  in  expired  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 analyses  are  were  presented used  to  as  means  describe  +  standard error.  relationships  between  Regression parameters.  Student's 2-tailed t-test and 1 & 2-way ANOVA were used to test for significant  differences  between  means  and  regression  Tests of significance were conducted at the 5% level of rejection. 34  equations.  3.3  RESULTS  1. Carbon dioxide excretion The differences and  expired  significant  water  in total CCh content between inspired water are  difference  presented  in  in  Figure  [CCh]ex-[CCh]in  11.  as  There was  environmental  no 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  amiloride treatment relative to the SITS treatment. significant  difference  in  [CCh]ex-[CCh]in  with  the  There was no  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.  significantly  In  pH  lower in the  6-7  water,  [Amm]ex-[Amm]in  amiloride treatment  was  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  increase in expired water ([H ]meas) +  pH  (pHin) are presented  curves.  The  regression  in figure curve  for 35  measured  proton concentration  and the environmental water 13 with two-degree regression control  values  was  not  80  Figure 11. The differences of total and expired water with different amiloride and SITS treatments. difference between amiloride and indicates a significant difference only, (x ± SE).  36  CO2 content between inspired environmental pH in control, * indicates a significant both control and SITS; ** between amiloride and SITS  Figure 12. The differences inspired and expired water control, amiloride and SITS difference between amiloride and  of total Ammonia content between with different environmental pH in treatments. * indicates a significant both control and SITS, (x ± SE).  37  Table 3. Calculated proton concentration in expired water CuMol/L.kg) pH 5-6  pH 6-7 Control Ami l o r i d e SITS  increase  pH 4-5  0.205 ± 10.399  -21.382 ± 6.421  -20.279 ± 7.424  2.908 ± 6.301  -10.756 ± 2.601  -10.271 ± 1.416  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 increase  differences and  calculated  between  measured  proton  concentration  proton  concentration  increase  in  expired  water, [H ]meas-[H ]cal, was around zero when environmental p H was +  under  6  positive were  (Figure  14).  differences greatest  treatment large  +  and  variation  in  In  (Figure the  smallest in  water  CO2  of  15).  The  control in and  neutral pH, however, magnitude  experiment,  the  amiloride  ammonia  were not significant  39  there  were  of  the  differences  smaller  in  the  treatment  contents,  these  Because  SITS of  differences  »- Control  200-  b. Amiloride  5 6 Environmental water pH  5 6 Environmental water pH  SITS  200 150.100 -  in  cn  £  \  o _*  + x  5  50-  o 3  -100  6  5 6 Environmental water pH  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  150100-  -100 4.0  4.5  5.0  5.5  6.0  E n v i r o n m e n t a l water pH •  Control  Figure increase water (pH4-6).  14. and  • Amiloride  The  differences  calculated  ([H ]meas-[H ]cal) +  The  +  regression  of  proton  zero.  41  measured  concentration  within line  •  is  low not  SITS  proton  concentration  increases  environmental significantly  in pH  different  expired range from  Control  Amiloride  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  chloride  effects of amiloride and SITS on branchial sodium and 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  similar  consequences.  excreted  through  If  of these  a certain  two drugs  percentage  carbon  the Cl7HC03~exchange pathway,  leads to dioxide  is  the addition of  these drugs to the bathing water should lead to a reduction of total C O 2 excretion. The same will be true for ammonia excretion. The  fact  [C02]ex-[C02]in  that  indicates  SITS  did not have  that  CO2  any impact  excretion  via the  on the C1/HCO3"  exchange pathway is unimportant, if SITS did not alter ventilation of  the fish. Alternatively, if 10% of the total C O 2 excretion is  in the form of  HCO3"  exchange pathway  in exchange for Cl", then blockage of this  may result  in an elevation  plasma, which in turn will enhance Since  the bicarbonate  elimination  of  Pco2  in fish  the molecular C O 2 diffusion.  pathway  is  responsible  for no  more than 10 percent of the total C O 2 excretion, 30 minutes should be enough for the development gradient  and  the  of the higher blood-to-water  re-establishment  of  the  normal  Pco2  total C O 2  excretion rate. The  amiloride treatment however, did result in a decrease of  [C02]ex-[C02]in. CI/HCO3"  This  is  very  difficult  to  explain.  Since  exchange appears not to play an important role in carbon 43  dioxide  excretion,  process  by  Also,  then  blockade  amiloride could not  the results  Wright,  the  et  preparation,  (1989).  they  because  [C02]ex-[C02]in  that  the  Cl'/HCCh" for  are in conflict  Using  found  the  be responsible  of our study  al,  of  a  the  blockade  of  trout  treatment  Na /H +  decrease.  with those from  blood-perfused  amiloride  exchange  +  head  increased  and Na /NH4 +  +  exchange processes induced an acidosis in the gills which in turn reduced C O 2 study  stores. We don't know why amiloride in this in vivo  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  amiloride  fact  did  representative of  not  that  in time, reduced  alter  ventilation  ammonia excretion)  and will  ammonia excretion  (if  [Amm]ex-[Amm]in  was  lead to an elevation of  ammonia content in fish plasma, which in turn will increase the blood-to-water excretion. and  Thus,  eventually  Nfb  diffusion  gradient,  and  enhance  total ammonia store  in the fish  ammonia  was  production. As the Na /NH4 +  excretion +  matched  ammonia  body increased, to  ammonia  exchange process may contribute up to  40% of the total ammonia excretion and the ammonia excretion rate is low compared with total ammonia stores, re-establishment be  longer  of  the  normal  total  than that required to 44  the time needed for  ammonia excretion  re-establish  rates will  CCh excretion rates  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  Thus  the  inhibition  reduction would  in  be  [Amm]ex-[Amm]in  less  severe  with  due  SITS  the gill.  to Na /H (NH4 ) +  than  +  +  with amiloride  treatment. A  reduction  in  ammonia  excretion  environmental pH decreased, for Na /NH4 +  was  also  expected  as  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  treatment  At  process  pH  environmental  was  blocked  in  was  similar  pH under all  in  5,  treatments,  control  the and  and  SITS  Na /NH4 exchange +  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  salmon. In our study, feeding  hours was  after  suspended  feeding  in  sockeye  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  carbon dioxide and ammonia by molecular C O 2 45  that  fish  excreted  and N H 3  diffusion  only,  and that acid influx  was  negligible.  calculated  Thus  proton  represented  the  or efflux  if  our  concentration net  proton  increase  assumptions  increase  gain  dioxide and ammonia excretions, concentration  in  through the  in  are  +  from the  the  +  introduction  of  water  interaction  the which  of carbon  should equal the measured proton  expired  and Na /NH4  +  correct,  expired  water.  This  occurred  environmental water pH was under 6 (Figure 14), both Na /H  gill epithelium  when  indicating that  exchange were inhibited. The fact that  +  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 supports the contention that Cl/HCOf minor role,  because in  this  a positive  value  and this  exchange was playing only a  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 control  than  [H ]meas different  in  in expired water  the  SITS  and [H ]cal  +  was  increase  +  treatments  playing  in  would be  treatment.  low  The  zero  environmental  smaller in the difference  pH with  all three  also suggested that HCCh" and NH4  very  +  little  role  in  carbon  dioxide  of  diffusion  and ammonia  excretion. In [H ]cal +  be  neutral water pH, the differences were positive  a proton  efflux  between [H ]meas and +  in all treatments  indicating that there must  through  epithelium.  the  gill  The value  of  [H ]meas-[H ]cal was higher in control than in amiloride and SITS r  treatments,  r  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 different  influx  across  environmental  (1986).  The protons  pH  gills was  in  trout  reported  excreted by  fish  batfiing by  in  Randall  through  water of & Wright  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  influx to proton efflux.  coupling of Na  +  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  measured  and  water  were  together (Figure calculated  similar  to  proton that  which indicates that Na /H +  called  background  proton  +  16).  The difference  concentration  predicted  +  from  between the  increase the  in  Na influx +  expired data,  exchange may be responsible for the so concentration  in fish exposed to water of neutral pH.  47  increase  in  expired  water  200 150-  5  6  Environmental water pH o Experimental data • Predicted data Figure  16.  environmental control  The  relationship  water  experiments)  pH and  between  with predicted  +  experimental data  data reported in Randall & Wright, 1986).  48  ([H ]meas data  (obtained  -  [H ]cal) +  (obtained from  Na  +  and from influx  GENERAL CONCLUSIONS & SUMMARY  4.0  It has been demonstrated diffusion carbon  of  molecular  dioxide  equivalents  CO2, the  excretion,  added to  in these studies that the  the  was  predominant  the  water  major  passing  pH  and the  Cf/HCOi  protons in the anhydrase  inhibitor SITS.  boundary water  and  the  extent  layer,  of  of  total  of  the  acid  gills  of  fish.  source  over  Carbon dioxide excretion was not affected  pathway  the  passive  by environmental water  CO2 hydration, generating was  catalyzed  hydration  was  by carbonic  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 diffusion  elimination  of  un-ionized  generated  alkaline  ammonia  equivalents  (NH3)  in  by  passive  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  the  blood-to-water  availability  (Wright  et  al,  (Wright  et  al,  of  NHa partial pressure  hydrogen  1989). 1989),  ions  in  Therefore,  a  inhibition  of  the  gradient  depended on  boundary  reduction carbonic  of  water  layer  CO2 excretion  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 inhibited  by  amiloride,  ammonia excretion pathway can be  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  state of surface active toxicants acting on the gill fish.  50  evaluating the epithelium of  5.0  REFERENCES  Brett, J.R. and Zala, C A . (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). arterial pH as temperature changes in gairdneri). Am. J. Physiol. 225:997-1001.  Respiratory control of rainbow trout (Salmo  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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