J Comp Physiol B (1985) 156:29-34 Journal of Comparative Biochemical, Systemic,

and Environ-

Physiology B m~.,o, Physiology

@ Springer-Verlag 1985

A study of the effects of water carbonate alkalinity on some parameters of blood acid-base status in rainbow trout (SMmo gMrdneri R.)

Serge Thomas and Joseph Poupin Laboratoire de Physiologic Animale*, Facult6 des Sciences et Techniques, F-29283 Brest C+dex, France

Accepted April 2, 1985

Summary. The effects of two levels of water car- bonate alkalinity (CA=0.3-0.5 meq.l-1 and CA = 12-13.5 meq. 1-1) on arterial blood acid-base status (Paco~, pHa, [HCO3-]+[CO3-]), oxygen consumption (Mo2) and plasma ionic composition (Na +, K +, C1-) were investigated in trout living in normoxic-normocapnic water at 15 ~

The results show that a high level of carbonate alkalinity induces a decrease in Paco~ and a situa- tion of mixed respiratory and metabolic acidosis compared to that in low CA water.

These changes are accompanied by significant changes in ionic composition but the levels of oxy- gen consumption are unchanged.

The role of the different capacitance coeffi- cients of water for CO2 and the effects of the differ- ent ionic composition of water on ionic and gas- eous exchanges are discussed.

Introduction

Many factors act directly or indirectly on the acid- base status of the extracellular compartment. Tem-

* Equipe de Recherche associ6e au CNRS N ~ 070622

Symbols and abbreviations: PEco 2 ; Plco~ partial pressure of car- bon dioxide in inspired and expired water; Pino2 ; Pouto2 partial pressure of oxygen in water entering and leaving the respir- ometers; Pwo2 ; Pwco2 ; CWco2 ; pHw partial pressure and con- centration of oxygen and carbon dioxide and pH in water; Pac%; pHa partial pressure of carbon dioxide and pH in arteri- al blood; ~'Wo2 ; 0c0Vco2 oxygen and carbon dioxide solubility coefficients in water; ~Pco2 solubility coefficient of carbon diox- ide ~in blood plasma; flWco2 capacitance coefficient of carbon dioxide in water; TA titration alkalinity in water; CA carbonate alkalinity in water; A)/co2 and Mo2 rate of CO2 output and of 02 uptake; R respiratory exchange ratio; l/w ventilatory flow rate; ~)w water flow rate in the chamber

perature, oxygen and carbon dioxide levels are the most important; for aquatic organisms the water ionic composition is also a major factor.

Among the possible effects of external ionic composition a distinction must be made between salinity (total dissolved salts) and acid-base bal- ance (resulting from the distribution of ions in the medium). The latter determines the pH and the titration alkalinity. In fresh water the major com- ponent of the titration alkalinity is carbonate alka- linity. In different waters of the same carbon diox- ide partial pressure the corresponding pHw value may differ considerably. This variability results in differences in total CO2 content and the capaci- tance of water for CO2.

The present study deals with the possible effect of the water carbonate alkalinity on the blood acid-base status in rainbow trout.

Dejours and Armand (1980, Astacus leptodac- tyIus), Truchot et al. (1980, Scyliorhinus canicula) and Truchot (1981, Carcinus maenas) have shown that a decrease in the titration alkalinity at con- stant water Pco2 causes a decrease in pH and an increase in Pco2 in the extracellular compartment. These results can be explained in two ways:

1) The CO2 and proton excretion are related to the level of chloride and sodium ions in the ambient water. Dejours (1969) showed that in Car- assius auratus, the excretion of CO2 fell in fish living in low [C1-] water. De Renzis and Maetz (1973) have shown that in Carassius auratus the acclimation to C1--free water produces metabolic alkalosis whereas the converse acclimation to sodi- um free water induces metabolic acidosis.

2) In the steady state the respiratory CO2 ex- change can be described by the following relations (Rahn 1966):

Mco = w/ Wco2 (P co - P co) (1)

30 S. Thomas and J. Poupin: Water carbonate alkalinity and blood acid-base status in trout

o r

29/co~ PEco2 = PIco2 +/2w .flWco 2 (2)

It can be assumed (Truchot et al. 1980) that, except in the case of a perfect countercurrent ex- change system, an increase of PEco2 leads to a cor- responding increase in Paco2. Thus the above transfer equations make it possible to predict that when 12 w and ~/co2 are stable the lower flWco~ the greater the d i f f e rence /~co2- PIco~ and the greater also Paco2 �9

These data are illustrated by the surprising re- sults of Johansen et al. (1975) who showed that in Tilapia graham• exposed to water in which free COz can hardly exist because of a very high titra- tion alkalinity, the blood CO2 is correspondingly low resulting in very high pH values.

In trout in less extreme conditions the pHa, Paco~ and [ H C O ~ ] + [ C O ~ - ] values obtained in reference conditions (normoxia and normocapnia) vary from one author to another (Table 1). Pre- sumably the various ionic compositions of the waters are related to this diversity but unfortunate- ly the exact ionic compositions of the waters have rarely been mentioned.

The aim of this paper is to show the effect of the water carbonate alkalinity on the acid-base sta- tus of trout blood. For this purpose the best dem- onstration would be direct measurements of every factor appearing in Eqs. 1 and 2 in addition to the blood characteristics Pao2, Paco~, pHa, [C1-], [Na+], and [K+]. In fact, it is impossible to mea-

sure 2~/co2 and 12 w because of the great difficulty in obtaining good samples of expired water in te- leost fish. So, in view of the fact that the fish were in steady state because of a long acclimation peri-

od, the ratio ~;/co~ �9 was assumed to be constant M o 2

and possible variation of the metabolic CO2 pro- duction was estimated by measurement of 3)/o2.

Materials and methods

Animals. Trout in the range 250-300 g obtained from a hatchery (Sizun, Brittany) were divided into two groups and acclimated for three weeks in water of low or high carbonate alkalinity. The experiments were performed in the period September to November. Water was maintained at 15_+0.5 ~ and the fish were fed daily. At the end of the acclimation period the fish in each group were prepared for two types of experiments :

1) Fifteen trout were chronically implanted with a catheter (Clay-Adams PE 50) in the subclavian artery (Thomas and Le Ruz 1982) to monitor the blood acid-base characteristics. Be- fore surgery, trout were starved for 48 h, then anaesthetized with neutralized MS 222 (0.1 g.1 -~ in water). Throughout the operation they were kept in oxygenated water containing a maintenance dose of MS 222 (0.05 g.l-1). The fish were then allowed to recover in running acclimation water for 48 h before sampling.

2) Ten fish were starved for 48 h, then placed for one week in respirometers supplied with the acclimation water to measure the oxygen consumption of each group.

Water. At a given temperature, water contains strong electro- lytes completely dissociated into cations (Na § K +, Ca ++, Mg ++ . . . . ) and anions (C1-, NO3, S O 2 - , ...) and partially dissociated weak electrolytes such as bicarbonate and carbon- ate, borate, phosphate and silicate.

Table 1. pHa and Paco2 values obtained by different authors for trout living in different waters. For the purpose of comparison the pHa values marked (*) are the calculated values corresponding to a temperature of 15 ~

Experimental Origin Period of pHa Paco 2 pHa* Reference conditions of fish year (Torr)

Tw = 13-/- 1 ~ Sun Valley B.C., 8.02_+0.02 1.78_+0.11 7.98 Cameron and Pwco2 =0.3 Torr Canada Randall (1972)

Tw = 8.9 • 0.1 ~ Sun Valley B.C., 7.93 2.5 7.83 Janssen and Pwco =0.3 Tort Canada Randall (1975)

Tw= 15 ~ Gloucestershire, 7.83-7.84 1.01-3.2 7.83-7.84 Eddy (1976) England

Tw = 10 ___ 0.5 ~ Laukaa station, August 7.74-7.86 3.60-4.3 7.65-7.77 Soivio et al. p H w = 6.3 6.8 Central Finland November (1977)

T~,= 15• 1.5 ~ Ontario, Canada 7.82+_0.01 2.51 • 7.77 Wood and Jackson (1980)

Tw= 10• ~ Sun Valley B.C., 7.89-7.90 2.56-2.59 7.81--7.82 Perry et al. (1981) Canada

Tw=9• ~ Laukaa station, November 7.85 1.4 -1.6 7.75 Soivio et al. (1981) pHw = 6.9 Central Finland

S. Thomas and J. Poupin: Water carbonate alkalinity and blood acid-base status in trout 3t

rnL-~ ~ : 1 5 C ~a~-er2 : : .A .2 13.3'mEq.L-' I

l total m r . o l . U ' . torV I

I c-0

"" - - -__ pH water 1 8

i . . . . . . . i ....... 0

0.5

CO:~ - 0

i i

HCO~ / # [ ~ w c o 2 : 0 . 0 6 4

total CO 2 mrnol. L4.torr -'

water 1 : C.A.= 0.4 rnf:q.L -r

diss. CO 2

0.1 012 0.3 PCO 2 tort

Fig. 1. pH values, total CO2, HCO~-, CO~--, and dissolved CO2 concentrations as functions of the COz partial pressure of the two kinds of water used in the experiments. Water 1, with a normal carbonate alkalinity had a capacitance of 0.064 mmol. 1-1. Torr-1 at the Pco~ prevailing in the aquarium. Water 2, with an artificially increased carbonate alkalinity pre- sented a much greater flWc% of 1.936 mmol. 1-1. Torr - ~ corre- sponding to the actual Pco~ of this water. For a purpose of presentation the lower scale is twenty times expanded which shows the extremely great difference of CO2 content existing in both waters

The water acid-base balance results from three physical laws : Conservation of mass; weak electrolyte dissociation equi- libria; electroneutrality.

The electroneutrality can be written as:

[strong cations[ - [strong anions] + [H +] - [OH-]

- [HCO ;] - [CO3- -] - [H2BO~ "1 - [e] = 0

where the brackets [ ] express the concentrations in meq.1- and e is the sum of the remaining trace elements.

In this equation [H+], [OH-] and [e] are negligible. The difference [strong cations] minus [strong anions] is then bal- anced by the sum of weak anions. This term has been defined in several ways: complement of weak anions (CWA; Dejours 1978) or titration alkalinity (TA; Truchot and Duhamel-Jouve 1980).

Table 2. Titration alkalinity (TA), total CO2 or carbonate alka- linity (CA) in the two experimental waters obtained by different methods

Method Parameter Water 1 Water 2 determined

Strickland and Parson (1972)

Cameron (1971)

Cameron (1971); Riley and Skirrow (1975)

pHw =f Pc% + Henderson- Hasselbalch

TA 0.3-0.5 13.32 _+ 0.02 * (meq. 1-1) n = 8 n = 5

total CO2 - 12.54_+0.22 (mmol . l - 1) (out of range) n = 7

CA - 13.24 • 0.25 (meq-1-1) (out of range) n = 7

CA 0.46_+0.05 12.30_+0.07 (meq.1 1) n = 5 n = 5

In fresh water [H2CO~-] is about nil and the titration alka- linity can be set equal to CA, the carbonate alkalinity.

The acid-base characteristics of water can be experimen- tally determined by three methods :

a) Strickland and Parson (1972): this assay determines the total alkalinity in meq. 1-i in the range 0.5-2.8 meq.1-1.

b) Cameron (1971): the carbonate alkalinity can be calcu- lated from the direct measurement of the total COz content with the aid of the tables of Riley and Skirrow (1975).

c) Measuring pH value as a function of Pco2. For a given temperature and strong ion composition the curve p H w = f(Pwco~) is representative of the acid-base status of a water. The use of the Henderson-Hasselbalch equation makes it possi- ble to calculate [HCO3] and [CO~--] for different Pwco2 values.

Water 1 . This water is city tap water and comes from a ground of granitic origin. Its composition is (in meq- l - l ) : Na § 1.0; K + 0; Ca ++ 0.8; Mg ++ 0.5;C1- 1.0; SO 2 - 0.6; NO~- 0.3. Fig- ure 1 shows the relationship at 15 ~ between pHw, [HCO3], [CO 3 -] and dissolved CO2 on the one hand and the imposed Pwco~ on the other. Thus the pHw value is 7.9 when this tap water is air-equilibrated at a Pco2 value of about 0.25 Torr.

As shown in Table 2, the Cameron (1971) method for de- termination of CO2 content cannot be used in such a low CO~ water. Both the other methods give similar values for the car- bonate alkalinity (between 0.3 and 0.5 meq. 1-1).

In our aquarium the mean pHw value was actually 7.77 corresponding to a Pc% value of 0.37 Torr. This light hypercap- nia was due to the animals' CO2 production which was not completely removed despite efficient air bubbling. The value for the capacitance of water for CO2 i.e., the value of the de- rived function for Pco~=0.37 Torr (fl=ACco2/APco~) at this point is equal to the s~ope of the curve Cco2=f(Pc%). Thus flWco~ = 0.064 mmol. 1- l . Tor r - 1 and AC = 0.45 meq. 1-1

Water 2: This water was produced from the first type of water by adding sodium carbonate (1.2 g, 1-1) and bicarbonate (0.2 g- 1-1) which theoretically corresponds to an increase in CA of 16mmol '1-1. The measured values were actually lower and varied between 12.00 and 13.5 mmol-1-1. This discrepancy can be accounted for by crystallization on the walls of the tanks which reduced the total amount of dissolved carbonate and bicarbonate.

Figure I shows that changes in Pwc% in this water produce completely different effects on pHw, [HCO~-] and [CO ~- -].

32 S. Thomas and J. Poupin: Water carbonate alkalinity and blood acid-base status in trout

The pH prevailing in the aquarium was 9.10 corresponding to Pwco~ and flWco~ values of 0.39 Torr and 1.936 mmol.1 ~. Torr-~, respectively. It is to be noted that this flWco~ value is twenty-three times higher than the previous one.

Analytical methods

Series 1: Blood acid-base characteristics: After three weeks of acclimation fish were sampled within 48 h following surgery for measurements of pHa, Paco2 (Radiometer BMS2 MK2 + Metrohm instruments), C1- (Radiometer chloridometer CMT 10), Na + and K + (Flame spectrophotometry, Beckman Klina Flame).

The values of Paco~ and pHa were plotted on diagrams expressing [HCO~-]+[CO ~ ~] as functions of blood pH and drawn with a CO2 solubility coefficient taken from Severing- haus et al. (1956a, b) and Wood and Johansen (1973) (ePco~ = 0.054mmol.l-a.Torr -1 at 15 ~ and pK[ and pK~ values (6.24 and 9.80) experimentally determined using the method of Siggaard-Andersen as modified by Truchot (1974).

Series 2: Oxygen consumption: The trout were fed during the three weeks of acclimation. At the end of this time two groups of 10 fish were placed in a respirometer and starved. Their weight was determined before and after the 9 days experimental period. Oxygen consumption was obtained from the decrease in P% between the water entering and leaving the respirometer (Yellow Spring instruments meter):

Qw x (Pino2 - Pouto~) x ~Vo~

k:/o2 - weight

5 /

o i . L t L

Z7 Z 8 7 . 9 8 . 0 p H

Fig. 2. In vivo arterial blood pH plasma [HCO3-]+[CO~- ] diagram with isopleths of Paco2 at 15 ~ The points represent values of Paco2, pHa and [HCOf]+[CO~-] obtained by As- trup's method. Filled circles correspond to fish adapted to low carbonate alkalinity, open circles to fish adapted to high car- bonate alkalinity water. Two buffer lines are presented. They were obtained by pooling the blood samples of each series

Po2 values were monitored every twelve minutes for the dura- tion of the experiment.

Results and Discussion

Series 1

The representative points of blood acid-base status of the fish living in both waters are given in Fig. 2 where [HCO3]+[CO~-] is expressed as a func- tion of pH. The fish living in low CA have mean values of 7.87__+0.03 for pHa, 1.76_+0.09 for Pco2 and 4.19_+0.30 for [HCO3]+[CO3-] (n=15). The fish living in high CA have significantly differ-

ent values (P<0.01) for Paco2:0.82___0.08 and [ H C O 3 ] + [ C O 3 q : 2.29_+0.19 and (P<0.05) for pHa: 7.95_+0.03.

The mean buffer lines in Fig. 2 show that the second group of fish is in a situation of mixed respiratory alkalosis and metabolic acidosis as compared to the first group.

Table 3 shows that these changes in acid-base status are accompanied by significant changes in Na + and C1- concentrations. Fish living in high CA exhibit a rise in plasma [Na +] and conversely a decrease in [C1-].

Table3. Mean values (+SEM) of arterial blood plasma characteristics (pH, Pco~, [HCO~-]+[CO3-], [CI-], [Na+], [K+]) in both series of fish. The significance (P < 0.05 or P < 0.01) of the changes was tested using Student's t-test. NS = not significant

Water Water CA Plasma values (Strickland and Parson) n pH Pc% meq- 1- t (Torr)

[HCO 3] [Cl-] [Na + ] [K + ]

(meq-1-1)

0.40

13.32 (0.02)

i5 7.87 1.76 (0.03) (0:09)

P<0.05 P<0.01

15 7.95 0.82 (0.03) (0.08)

4.19 138.00 134.87 3.50 (0.30) (1.38) (1,,.88) {0.33)

P<0.01 P<0.05 P<0.01 NS

Z29 134.05 141.60 3.52 (0.19) (1.60) (0.87) (0.45)

S. Thomas and J. Poupin: Water carbonate alkalinity and blood acid-base status in trout 33

/~02 pmoP. hr- ' . g-'

0 �9

�9 g ~ o o �9 $

1 2 3 4 5 6 Time , day

T= 15C

�9 g o

Fig. 3. Time course of oxygen consumption in the two series of fish. Filled circles correspond to mean values obtained each day (one value obtained every 12 min) in a group of ten fish adapted to low carbonate alkalinity water. Open circles, fish adapted to water of high carbonate alkalinity

Series 2

Figure 3 shows the results of the oxygen consump- tion measurements. Even after three weeks accli- mation in two different waters no significant chan- ges can be observed. ~;/o~ fluctuates between 2 and 3 gmol' h -1 .g -1 . Each point in this figure corre- sponds to the mean value of 120 measurements each day (every 12 rain). These results show that there is no difference in the metabolic rates in the two groups of fish.

This work was based upon the hypothesis that a change in water carbonate alkalinity would cause acid-base changes in the extracellular compartment in two ways: (i) an effect of water composition on the efficiency of CO2 excretion; (ii) an effect of water composition on the ionic exchanges in the gills.

Effect o f water composition on the efficiency o f C02 excretion

The transfer equation PEco~-- PIco2 = ~ rco j l)'w' flWco2 predicts that a change in flWco2 resulting from a modification in ionic composition of the water might change Paco~, if we assume that both 29/co~ and l~w are constant.

The results above clearly show that the differ- ence between Paco~ and Pwco~ in the series (1) fish is much greater than the difference in series (2) fish, the values being 1.42 Torr and 0.43 Torr, respectively a 3.5-fold decrease. At the same time flWco~ increases thirty-fold; the results of-g/co~ in

Fig. 3 show that the assumption of a constant value for ~/co2 is acceptable as in such a prolonged experiment the respiratory quotient ~/co2/A;/o~ must reach a steady state. The change in the capa- citance value explains the simultaneous change in Paco~. Nevertheless this change is much smaller than predicted by theory if f'w were assumed con- stant, which was, however, not demonstrated.

This discrepancy shows that the effect of water CO2 capacitance is limited. Other physical factors necessarily reduce the extent of the capacitance ef- fect. First, Vw might decrease, because of the de- crease in PEco~ and then of Paco~. Compared to the initial situation the expired water is now hypo- capnic and even if not detectable, a small change in 12 w is likely. Secondly, CO2 is lost mainly by diffusion (Cameron 1979). This means that after passing through the gill membranes the molecules of CO2 combine with water: C O 2 + H 2 0 ~,~_ H2CO3. When not catalysed this reaction is slow; its half-reaction time is 53.3 s at 15 ~ which is very long compared to the speed of water passage across the gill (Roughton et al. 1967; Drenth and Kwart 1980).

This raises the question of the location of car- bonic anhydrase in the external wall of the gill epithelium. If this enzyme is actually present, the carbon dioxide would be instantaneously hydrated and the capacitance effect would be maximal. If absent the effect would be negligible. The present results do not answer this question because the observed changes in Paco2 are significantly corre- lated with the changes of flWco2 but are not large enough to be definitive.

Effect o f water composition on the ionic exchanges in the gill

It has been shown by De Renzis and Maetz (1973) and by Dejours (1969) that the acid-base status of the extracellular compartment varies as a func- tion of the ionic composition of the ambient medi- um. Thus a Na-free medium provokes a metabolic acidosis. Moreover, freshwater compensate for a loss of Na + and C1- ions by reabsorbing these ions independently. This occurs by a specific ex- change mechanism located in the gills, such as Na + /H + or C1-/HCO 3- (Maetz and Garcia-Romeu 1964; Garcia-Romeu and Motais 1966; Maetz 1971; Evans 1980).

In our experiments the sodium concentration in the water was considerably increased and the proton concentration lowered. Both these changes could have enhanced sodium-proton exchange re- sulting in a rise in extracellular [Na+]. Further-

34 S. Thomas and J. Poupin: Water carbonate alkalinity and blood acid-base status in trout

more, the exchange of endogenous HCO 3 against CI- would have been reduced because of the tre- mendous increase in external [HCO3]. The signifi- cant rise in the plasma level of Na + and the con- comitant reduction of chloride observed in these experiments demonstrate that the hypothesis stated above is probably correct.

In conclusion, we have described the plasma ion balance of trout during prolonged exposure to water of high carbonate alkalinity. This situa- tion results in a significant decrease in Pac% and [HCO3], a rise in [Na +] and a fall in [C1-]. These results support the possible effect of the increased water capacitance for CO2, however, the situation is complicated by concomitant changes in sodium and chloride balance occurring in the gills.

Further experiments must be devised which would distinguish between the two effects either by replacing sodium by another cation, or by mak- ing the same observations at an increased Pwc02 where flWc% becomes independent from carbonate alkalinity.

Acknowledgements. We are indebted to Professors Barthelemy and Peyraud for fruitful discussions and to Professor Truchot for helpful criticism. This work was supported by a grant from the Centre National pour l'Exploitation des Ocbans N ~ 81/8493.

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