Effects of environmental water salinity on blood acid-base status in juvenile turbot (Scophthalmus maximus L.)

Camp. Biochem. Ph.wiol. Vol.

Pergamon

109A,No. 4.pp.985-994,1994

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Effects of environmental water salinity on blood acid-base status in juvenile turbot (Scophthalmus maximus L.) F. Gaumet,*

G. Boeuf,* J.-P. Truchot’f

and G. NonnotteT

*Laboratoire de Recherches Aquacoles, IFREMER Brest, BP 70, 29280 Plouzane, France; and TLaboratoire de Neurobiologie et Physiologie Comparees, URA 1126, CNRS-Universite de Bordeaux I, 33 120 Arcachon, France The time courses of extracellular ionic and acid-base adjustments were studied in juvenile turbot (Scop/ihtRaZmusmaximlrs) following a decrease of water salinity, either abruptly from 32 to 1O%oor after a first step (4 weeks) in 19%0 salinity followed by a direct transfer to 10% brackish water (BW). Net exchanges of acid-base equivalents with the external water were also determined after transfer from 32% SW to 10%0 BW. Direct transfer from seawater (SW) to 10%0 BW induced a transient decrease in plasma osmolarity, plasma sodium and chloride concentrations, associated with a marked and transient metabolic alkalosis in the blood. A significant net outthrx of acidic equivalents was also measured only during the first day in BW. Four weeks preadaptation in 19%0 BW reduced the intensity of the osmotic disturbances elicited by a subsequent abrupt transfer to 10%0 BW. These ionic readjustments were also coupled with minimal acid-base changes, of lesser magnitude than those described after directly from SW to 10%0 BW. Key words: Osmoregulation; Metabolic alkalosis; Plasma

Turbot; Teleost osmolarity.

Comp. Biochem. Physiol. 109A, 985-994,

fish; Scophthalmus maximus; Acid-base;

1994.

Introduction Relationships between ambient salinity and example, Ferraris et al. (1988) on the osmotic regulation of the extracellular com- milkfish (Chanos chanos) or Nordlie et al. partment in fish have been much studied in (1982) on the mullet (Mugil cephalus) have these two last decades (for review see examined the acquisition of osmotic reguEvans, 1984). Although many studies on latory capabilities depending on fish size rapid osmoregulatory responses of fish to and the amplitude of the salinity decrease. salinity changes have been conducted on In a more recent study on Platichthysflesus, salmonids (for review see Boeuf, 1993): juveniles submitted to tidally fluctuating there are only a few papers on other salinity regimes (CL-100% seawater) were teleosts, in particular for juvenile fish. For found to simultaneously modify their water permeability, urine production and drinkCorrespondence to: Dr G. Nonnotte, Laboratoire de ing rate, and to maintain their blood osNeurobiologie et Physiologie Comparkes, URA motic and total water content within 1126, CNRS-UniversitC de Bordeaux I, 33120 narrow limits throughout the range of salArcachon, France. Fax (33) 56 83 03 50. Received 4 September 1993; accepted 10 June 1994. inity (Hutchinson and Hawkins, 1990). CBPA

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Although there is evidence that changes of ambient salinity induce transient acid-base disturbances associated with recovery of the plasma ionic homeostasis in a number of euryhaline animals (for review see Truchot, 1987), the regulatory mechanisms involved remain poorly documented in the case of marine teleosts and very few data are available for the time course of this adjustment. Some basic elements have been previously reported for various marine invertebrates (Truchot, 1987) but only recent work has been dedicated to teleost fish such as Salmo salar (Maxime et al., 1990) or Platichthys j7esu.s (Nonnotte and Truchot, 1990), two euryhaline species. These observations attest to the importance of the extracellular acid-base changes following a salinity transition, that may be linked to extracellular anisosmotic regulation and/or to cellular metabolic adjustments, and compensated partially by ventilatory adjustments. However, the mechanisms involved in these salinity-dependent acid-base disturbances seem rather complex and remain to be elucidated. For a marine teleost, the turbot (Scophthalmus maximus), which at a juvenile stage may be encountered in diluted sea water of estuaries, only few data exist on the physiological adaptive mechanisms induced by changes of ambient salinity. Scherrer (1984) studied the ability of juveniles to tolerate low salinity by evaluating respiratory metabolism and growth potentialities; the optimal range of salinity was estimated for the weight class of 5-20 g to be about 20-25%0 salinity. More recently, ultrastructural studies of the gill epithelium, especially of mitochondria-rich cells, have shown that the turbot is able to adapt to diluted seawater (down to 5%0 salinity) by specific ultrastructural modifications reducing the osmotic permeability of the branchial epithelium (Pisam et al., 1990). However, below 10%0salinity, the survival was limited or uncertain. The present study documents extracellular ionic and acid-base disturbances associated with an abrupt hypoosmotic shock, in order to understand better how juvenile turbot (Scophthalmus maximus) totally adapts to brackish water (BW). The time course of extracellular ionic adjustments and changes in blood pH, PC02 and bicar-

bonate concentration were followed under hypoosmotic conditions. These observations were completed by measuring the net exchanges of acid-base equivalents with the external water after direct transfer from full strength SW to 10%0 BW.

Materials and Methods Juvenile turbot, weighing 300-400 g at the beginning of the experiments, were obtained either from a local hatchery (Aqualand, Anglet, France) or from the experimental nursery of IFREMER (Brest, France) and transferred to Arcachon (France) for 12 months of acclimation at 15°C and 32% seawater (SW), in well aerated tanks (2 m’). Fish were fed to satiety with a commercial dry pellet. Half the animals were transferred to 19% brackish water (BW) 1 month before the study started. Fish were starved 2 days before surgery and placed in experimental frames. Fish were anaesthetized by immersion in a phenoxyethanol solution (0.5 ml 1-l) and transferred to an operating table which provided continuous irrigation of the gills with a 0.25 ml 1-l anaesthetic solution. Each fish was placed in a moist tray and chronically implanted with a dorsal aortic cannula which was sutured in place, 10 cm before the end of the tail (Nonnotte and Truchot, 1990). Briefly, the cannula consisted of the cut tip of a 27 gauge needle (internal diameter 0.2 mm) inserted into a short length of an heparinized (175 UI ml-’ of heparine) Ringer-filled polyethylene PE30 catheter (i.d. 0.3 mm) which was itself connected to a PE50 catheter (i.d. 0.4 mm) allowing blood sampling with glass heparinized capillaries. The cannula was implanted in the dorsal aorta between two vertebral arches, rinsed and filled again with 50 UI ml-’ heparinized Ringer’s solution containing (mM): NaCl 160, KC1 3, CaCl, 0.75, MgClz 1. Cannulae were rinsed twice a day during the experiment to prevent coagulation. After surgery, fish were held in experimental boxes with flowing SW for 24 hr before experiments. Apparatus and experimental protocol

In addition to a control experiment with the fish maintained in SW (32%0), two main

987

Acid-base status as a function of water salinity

experimental series were conducted to determine the effect of optimal salinity acclimation on adaptive potentials of turbot. In the first series, the fish was directly transferred from SW (32%0) to 10% BW. In the second series, fish preacclimated for 1 month to 19% SW were exposed to 10%0 BW. To determine the time course of extracellular ionic and acid-base adjustments, blood samples were obtained for both series 1 day before (SW reference) and 2, 4 and 7 days following the transfer. The experimental apparatus allowed simultaneous monitoring of six juveniles. Fish were kept in darkened 25 1 plastic boxes supplied with recirculated filtered water (total volume 270 1; water flow 0.9 1 min-’ per box; 15°C constant; 32 or 19% BW depending on the experiment). A second tank (120 l), where the salinity of water was adjusted to 10% by adding an appropriate volume of freshwater, could be connected to the circulating system through a threeway valve. Abrupt change of ambient water from SW (or 19%o BW) to 10%0 BW was completed within 15 min. Temperature and salinity were checked once a day. The acid-base balance of seawater and brackish water was regulated automatically with a pH-CO2 stat (Dejours et al., 1978). Water carbon dioxide pressure (PCOz) was stabilized at a level of 0.45 Torr by controlling the pH with injection of small amounts of pure CO* into the water through a solenoid valve, and washing out the excess by continuous bubbling of CO*free air. The titratable alkalinity (TAw) was measured twice a day using a modified Gran titration procedure (Culberson et al., 1970) and readjusted to a constant level of 2.15 mEq 1-l in SW and BW by adding appropriate amounts of HCl or NaHCO,. The pH set point, different between SW (or 19% SW) and 10%0 BW, was determined once a day on a water sample after equilibration at PC02 0.45 Torr. The main characteristics of SW and BW in the closed experimental circuit are shown in Table 1. Blood sample collection and measurements The arterial blood samples (about 300 ~1) were drawn from the cannula into five heparinized glass capillaries. The blood volume was maintained constant by injecting an equivalent quantity of heparinized

Table 1. Water characteristics of the experimental circuits expressed as means f SE Conditions

Before transfer

Salinity (%0) Temp. (“C) PO, PH PCO, (Torr)* TAw (mEq I-‘)

32-l O”Xk 10.2 * 0.1 32.0 + 0.2 14.9 * 0.5 14.9 + 0.5 Air saturation Air saturation 8.10 + 0.03 7.90 +_0.02 0.45 + 0.01 0.45 f 0.01 2.1 f 0.0 2.1 f 0.0

After transfer

Salinity (%o) Temp. (“C) PO2 PH PCO, (Torr)* TAw (mEq I-‘)

19-IO%0 10.2 + 0.1 19.4 f 0.2 14.9 f 0.5 14.9 + 0.5 Air saturation Air saturation 8.20 It_0.03 8.10 + 0.07 0.45 * 0.0 1 0.45 + 0.01 2.1 + 0.0 2.2 +_0.0

(1 Torr = 1 mmHg = 133.322 Pa).

Only four samples were taken from the same fish for acid-base determinations: the amount of the sampled volumes represented less than 10% of the total blood volume of the animal. Measurement of in vivo blood pH was immediately performed at 15°C on the second capillary sample (not air-contaminated) using a Radiometer microelectrode G298A connected to a Radiometer PHM72 pH meter. In vivo arterial partial pressure of carbon dioxide (PCO;?) was determined by the Astrup method (Astrup, 1956). Concentrations of carbonate bicarbonate and plasma ([HCO,-] + COX2-] were calculated by the Henderson-Hasselbach equation using a CO2 solubility coefficient and an appropriate pK’ from Boutilier et al. (1985). Plasma was obtained from blood samples (150 ~1) centrifuged at 7000g for 5 min. Chloride concentrations [Cl-] of water and plasma were determined with a chloride titrator Radiometer CMTlO. Sodium [Na+] and potassium [K+] concentrations were measured by flame photometry (Eppendorf FCM6341). Osmolarity was measured with a vapour pressure osmometer (Wescor 5500). Ringer.

Net H+ juxes determination Exchanges of acid-base equivalents between fish and the ambient water were estimated following transfer from SW to 10%0 BW on six juvenile turbot at 15°C. Before use, specimens were held for 8 days without feeding. Fish were placed in

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individual plastic chambers. Before initiating measurements, the water flow into the fish chambers was stopped and the chamber volume adjusted to a known value (usually 1 1 per 100 g body weight). Chamber aeration was provided at a flow rate of 2-3 1 min-’ by air-lifts which lined the bottom edges of the fish chamber. This served to maintain constant high O2 and low CO2 levels and to provide adequate mixing of the chamber content. Fluxes were measured over 2-hr periods, in SW and at time 1 hr, 4, 7, 23, 26, 30, 46, 50, 70 and 144 hr after transfer to 10%0 BW. Between each flux period, water was recirculated in the fish chambers. Water samples were taken at the beginning and at the end of each flux period and analysed for titratable alkalinity (see above) and for ammonia concentration by a salicylate-hypochlorite method (Bower and Horn-Hansen, 1980). These values were used to calculate fluxes of titratable alkalinity (JTA) and of ammonia (JAmm) in pmol hr-’ 100 g-‘. Ammonia excretion being considered as acid output, the algebraic sum of these two fluxes is equivalent to a net acid flux (see Truchot, 1987). Data analysis All values were expressed as mean + standard error (SE; n = 6). The statistical significance of the changes in variables induced by experimental salinity variations was tested by one-way ANOVA employing Statgraphics, followed by a post hoc multiple comparison test (Tukey’s test). Differences were considered significant at P < 0.05.

Results Transfer from SW to 10%0 BW Abrupt transfer from SW to 10%0 BW resulted in significant decreases in plasma osmolarity and ionic concentrations, as plotted in Fig. 1. Plasma osmolarity decreased markedly (about 6.6%) during the first 48 hr in 10%0 BW (286.4 + 3.2 vs. 301.6 + 3.4 mOsm 1-l) and remained at a significantly lower level than the initial value until day 7. On day 2, plasma sodium and chloride concentrations had also significantly decreased: sodium concentration remained significantly lower than SW value until day 7 (155.1 + 1.3 vs.

l

Na+ b . e ab

b i

320 l

260 L 1 0

Osmolarity

I

2

4

I

J

6

8

Time (days)

Fig. 1. Plasma osmolarity and ion concentrations of turbot in seawater (time 0) and at various times following transfer from seawater (32K salinity) to brackish water (lo%0 salinity). Mean + SE (n = 6). For a given variable, values with the same letter are not significantly different (P < 0.05).

162.5 + 1.3 mmol 1-l). In contrast, chloride concentration returned towards control level by day 4, and was no longer different from the SW reference value by day 7 (147.8 + 1.5 vs. 153.2 + 2.2). Plasma potassium concentration was not significantly affected by transfer, although the last value (day 7) was slightly lower than control. Figure 2 illustrates the time course of changes in acid-base status that occurred when fish were maintained in SW (control) or transferred from SW to 10%0 BW. Whereas acid-base status of control fish did not change during the experiment, the hypoosmotic shock provoked a marked transient metabolic alkalosis characterized by an important rise in pH which peaked on day 2 (whole blood pH: 8.03 + 0.01 vs. 7.84 + 0.01 in SW). It was associated with increased levels of plasma bicarbonate and carbonate [HC03-] + [C03*-]: 9.67 +, 0.45 mmol I-’ on day 2 vs. 6.31 + 0.37 mmol I-’ in SW. The whole blood pH value returned to the control level and was not significantly different from the initial one at the end of the experiment by day 7. Plasma

Acid-base

status as a function

989

of water salinity

82 A

6.0

IL-x - 12.5E g

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Transferfrom32 % SW to 10 %. BW

Time (days)

Time (days)

Fig. 2. Time course of blood acid-base adjustments in turbot for a control experiment in SW (32% salinity) following direct transfer from SW (32%0 salinity) to brackish water (10%0 salinity), and following transfer of fish preacclimated 1 month at 19%0 salinity to brackish water (10%0salinity). Values are expressed as means f SE (n = 6). Water PCO, 0.45 Torr. For a given variable, values with the same letter are not significantly different (P < 0.05).

bicarbonate concentration remained higher than control. In contrast, whole blood PC02 remained unchanged. Figure 3 shows mean measured values for fluxes of titratable alkalinity and ammonia at various times after transfer from SW to 10% BW. In SW, juvenile turbot released alkaline equivalents (JTA) to the medium. However, when this apparent base loss was corrected for ammonia excretion (JAmm),no net output of acidic equivalent (JH+) was found. Following transfer to 10% BW, strongly acid-base fluxes appeared modified. JTA was reversed, i.e. a base influx appeared by l-3 hr, while ammonia ex-

cretion remained unchanged. By 4-9 hr, JTA returned towards SW values, but ammonia excretion increased significantly, reaching more than twice the SW values. As a consequence, calculated net acid efflux was strongly elevated during this period (l-3 hr), essentially at first, by turning JTA efflux to influx and thereafter by increased During J Amm which became predominant. the period 24-48 hr, the efflux of JTA was almost entirely balanced by an efflux of J Amm and the net flux of acidic equivalents was not significantly different from zero. After 48 hr in BW, JTA tended to recover towards SW values, but ammonia excretion

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F. Gaumet et al.

0

5

10

20

25

30

45

50

70

145

150

Time (hours)

Fig. 3. Mean values of the flux of titratable alkalinity (JrJ, total ammonia excretion (JAmm) and the net flux of acidic equivalents (.I”+) in juvenile turbot adapted to seawater of 32% salinity (ref) and at various times after transfer from 32K to 10% salinity. Positive and negative values denote net influx and efflux, respectively. Mean f SE (n = 6); a same letter per column denotes values not significantly different (P < 0.05). For a given variable, values with the same letter are not significantly different (P < 0.05).

remained high. After 6 days in BW, JTA and JH+ had recovered but JAmm was always significantly higher than SW control values. Transfer from 19 to 10%0 B W

In fish acclimated for 1 month to 19% BW, plasma osmolarity and concentration of Na+, Cl- and K+ were not significantly different from SW (32% salinity) control values (compare Figs 1 and 4). Transfer of these fish to 10% BW elicited extracellular ionic disturbances. After 2 days, plasma

osmolarity decreased by about 5.0% (282.0 f 8.4 vs. 297.4 f 3.8 mOsm 1-l) and remained unchanged up to 6 days following transfer (Fig. 4). Chloride and sodium concentrations reached a minimum value on days 2 and 4, respectively ([Na+] 146.8 f 2.5 vs. 160.2 + 1.7 mmol 1-l; [Cl-] 132.8 f: 2.4 vs. 145.2 + 1.1 mmol l-l), then increased slowly until day 6 without recovering initial levels (Fig. 4). Blood pH, [HCOj-] + [CO,*-] and PC02 did not differ significantly from control SW

Acid-base status as a function of water salinity

I

.

v j

Na*

l K+

:i-. 3

I

2LI 320

260

;

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i-1 0

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1 6

Time(days) Fig. 4. Plasma osmolarity and ion concentrations of turbot preacclimated 1 month to 19%0 salinity (time 0) and at various times following transfer from this medium to brackish water (10%0 salinity). Mean + SE (n = 6). For a given variable, values with the same letter are not significantly different (P < 0.05).

(32%0salinity) values after 1 month acclimation to 19%0 (Fig. 2). Upon transfer to 10%0 BW, blood pH and PCO, did not change significantly. Plasma bicarbonate and carbonate concentrations showed a slight increase on day 2 (9.15 f 0.59 vs. 6.63 f 0.54 mmol 1-l) (Fig. 2).

Discussion In a previous investigation of the effects of hypo- or hyper-osmotic environment on the acid-base status of the flounder Platichthys jesus, Nonnotte and Truchot (1990) observed that plasma ion concentrations and haematocrit were not seriously modified by the cannulation procedure. Moreover, Waring et al. (1992) demonstrated in Atlantic salmon S. salar and flounder that the blood characteristics of cannulated animals were similar to those of “normal” confined ones, thus excluding a possible post-operative trauma. Therefore, we assume that the cannulation technique used in this work also provides anaerobic blood samples for in uiuo pH measurements.

991

The extracellular acid-base balance of aquatic animals is well known to be strongly affected by the water acid-base status and especially by the PCO, and the titration alkalinity TAw (Truchot, 1987). In the present experiments, a pH-CO,-stat system and frequent control of TAw allowed water PC02 to be kept constant. Thus the acid-base disturbances we observed can be confidently attributed to the effects of changes of ambient salinity alone. The present observations clearly confirm the ability of turbot to withstand large and rapid variations in ambient salinity, at least in the juvenile stage. After transfer from 32%0 SW to 10%0 BW, it took a few days (less than 8 days) after the osmotic shock for the extracellular ionic balance to return to a stable level, on average less than 10%0 lower than the SW reference, as it was previously reported for a few euryhaline species by several authors: Ferraris et al. (1988) on milkfish C. chums, Talbot and Potts (1989) and Maxime et al. (1990) on salmon S. salar and Nonnotte and Truchot (1990) on flounder P.JEesus. Moreover, long term acclimation to an intermediate salinity (19%0SW) led to unchanged plasma osmolarity and ion concentrations and reduced extracellular electrolyte and acid-base disturbances upon further transfer to a lower salinity (10%0 SW). In addition, other experiments (unpublished results) performed on juveniles of the same size, showed that turbot was also able to develop in the long term similar growth potentialities in diluted SW (10%0salinity) than in full strength SW. The observed extracellular alkalosis induced by transfer to reduced ambient salinity was similar to that reported in the literature for marine organisms such as invertebrates (Truchot, 1981, 1987, 1992) and also euryhaline fish. For example, Nonnotte and Truchot (1990) observed that an abrupt transfer of the flounder P. Jesus from SW to freshwater (FW) induced a transient decrease in plasma osmolarity and a concomitant metabolic alkalosis associated with a marked persistent hypercapnia. In adult Atlantic salmon S. salar, the increase in blood pH observed during the first 15 days of transfer to FW corresponded to a mixed metabolic/respiratory alkalosis, with a transient hypocapnia (Maxime et al., 1990). Conversely, FW species tend to

992

F. Gaumet er al.

become acidotic when acclimated to increased salinities (Truchot, 1981; Wilkes and MacMahon, 1986; Nonnotte and Truchat, 1990). Nevertheless, these salinity-induced acid-base disturbances are not general for teieost fish, since they appear to be absent in rainbow trout for example, when transferred from FW to SW (Bath and Eddy, 1979). On the basis of data for euryhaline teleost as P.JEesus (Nonnotte and Truchot, 1990) it has been suggested that the CO2 exchange system can compensate, at least transiently, for metabolic disturbances when the mechanisms of ionic exchange involved in the control of extracellular acid-base balance are already engaged in the ionoregulatory responses to a reduction of external salinity. However, the present results on juvenile turbot differ from that found in the flounder. The salinity-induced changes of extracellular pH occurred without significant alterations of blood PC02, whereas the bicarbonate concentration of the blood increased at lower salinity. Thus, no ventilatory mechanisms seem to compensate for acid-base disturbances in this species. A recent review (Goss et al., 1992) presented evidence that transfer of acid-base equivalents coupled to ion exchanges with ambient water are variably utilized by freshwater fish to regulate acid-base disturbances induced by numerous experimental procedures such as hyperoxia (Wood et al., 1984) hypercapnia (Petty et al., 1987) and HCl or NaHCO, infusion. However, the mechanisms accounting for the metabolic alkalosis induced in fish by a decrease in ambient osmolarity remain presently largely unsolved. In a pioneering work on the euryhaline crab Carcinus mamas submitted to l/3 SW, Truchot (1981) described a typical metabolic alkalosis, but associated this with a large apparent base output, suggesting that the metabolic acid-base disturbances observed were of tissular origin and probably linked to metabolic adjustments ensuring isosmotic intracellular regulation. However, in another euryhaline crustacean species, the Chinese crab (Eriocheir sinensis) transferred from SW to FW (Truchot, 1992), measurements of external acid-base exchanges supported the view that both extracellular adjustments and cellular metabolism could explain the

acid-base disturbances following a salinity transition. To elucidate the origin of acid-base readjustments found in juvenile turbot after transfer to BW, we determined the net flux of acid-base equivalents as the sum of a titratable component and ammonia excretion which must be considered as an acid output (see Truchot, 1987). Two phases were apparent (Fig. 3). At first, during the early hours after transfer of seawater-adapted turbot into BW (10% salinity) an important net outflux of acidic equivalents was observed, which probably contributed to the build up of the blood alkalosis. This acid outflux was first associated with a reversal of the titratable flux component which transiently turned to an influx of alkaline equivalents, perhaps linked to gill diffusional efflux of chloride (see Evans, 1984) balanced by an equal HC03- input, and to the reversal of the branchial transepithelial potential which occurs when SW fish are rapidly transferred by hypoosmotic water (Potts, 1984). Then, the titratable flux component recovered toward control values, the net H+ efflux being mainly caused by an increase of ammonia excretion. During a second phase starting on the second day after transfer in BW, a net efflux of acidic equivalents remained weak or even absent, and any extracellular acid-base change occurring at this time can hardly be explained by acid-base exchanges with the ambient water. As a consequence of the decrease of water salinity, there was an important rise of the ammonia excretion. This increase started in the early hours after transfer of the fish to BW and was sustained during all the experiment. It was counterbalanced by an excretion of basic equivalents only after 24 hr in BW. An increase of ammonia excretion after transfer to diluted SW or to FW was already reported in many euryhaline invertebrates (Emerson, 1969; Truchat, 198 1, 1992) but not in fish up to now. It may be indicative of enhanced catabolism of the pool of free amino acids acting as intracellular osmolytes (Gilles, 1979) which may result in a net production of metabolic acid or base (Brosnan et al., 1988). In addition, transmembrane ion exchanges involved in the restoration of cell volume may also contribute to the development of salinity-dependent acid-base disturbances

Acid-base status as a fuunction of water salinity

(Livne and Hoffmann, 1990). Further investigations are still necessary to clarify these problems. In summary, a decrease of ambient salinity induces in the juvenile turbot a transient metabolic alkalosis in the extracellular fluid, as previously described in some marine vertebrates and invertebrates. This disturbance can be attenuated by prior acclimation of fish to intermediate strength seawater. Salinity-dependent acid-base reponses seem to be complex and probably involve different physiological mechanisms resulting from interactions between the extracellular compartment and both external and cell compartments. Acknowledgements-This investigation was supported by a grant from IFREMER and Communaute Urbaine de Brest to F. Gaumet. This study was developed in the Laboratoire de Neurobiologie et Physiologie Comparees, Universite de Bordeaux I, URA CNRS 1126. The authors would like to thank D. A. Colin for helpful discussion and C. Veillard for his expert technical assistance for the illustrations.

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Importance de la regulation de l’tquilibre acide-base de l’eau ambiante pour l’etude des Cchanges respiratoires et ioniques des animaux aquatiques. C. R. Acad. Sci., Paris 287, 1397-l 399. Emerson D. N. (1969) Influence of salinity on ammonia excretion rates and tissue constituents of euryhaline invertebrates. Comp. Biochem. Physiol. 29, 339-358.

Evans D. H. (1984) The roles of gill permeability and transport mechanisms in euryhalinity. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. Xb, pp. 239-283. Academic Press, New York. Ferraris R. P., Almendras J. M. and Jazul A. P. (1988) Changes in plasma osmolarity and chloride concentration during abrupt transfer of milkfish (Chanos chanos) from seawater to different test salinities. Aquaculture 70, 145-157. Gilles R. (1979) Intracellular organic osmotic effectors. In Mechanisms of Osmoregulation in Animals (Edited by Gilles R.), pp. 11I-154. John Wiley & Sons, Chichester. Goss G. G., Perry S. F., Wood C. M. and Laurent P. (1992) Mechanisms of ion and acid-base regulation at the gills of freshwater fish. J. exp. Zoo/. 263, 143-159. Hutchinson S. and Hawkins L. E. (1990) The influence of salinity on water balance on O- group flounders, Platichthysjesus (L). J. Fish Biol. 36, 751-764.

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Bath R. N. and Eddy F. B. (1979) Ionic and respiratory regulation in rainbow trout during rapid transfer to seawater. J. camp. Physiol. 134, 351-357. Boeuf G. (1993) Salmonid smolting: a pre-adaptation to the oceanic environment. In Fish Ecophysiology (Edited by Rankin J. C. and Jensen F. B.), Vol. 9, lard lir.)i136. Fish and Fisheries Series, Chapman

Livne A. and Hoffmann E. K. (1990) Cytoplasmic acidification and activation of Na+/H+ exchange during regulatory volume decrease in Ehrlich ascites tumor cells. J. Membr. Biol. 114, 153-157. Maxime V., Peyraud-Waitzenegger M., Claireaux G. and Peyraud C. (1990) Effects of rapid transfer from seawater to freshwater on respiratory variables, blood acid-base status and 0, affinity of haemoglobin in Atlantic salmon (Salmo safar L.). J. camp. Physiol. 16OB, 31-39. Nonnotte G. and Truchot J. P. (1990) Time course of extracellular acid-base adjustments under hypo- or hyperosmotic conditions in the euryhaline fish Platichthysjesus. J. Fish. Biol. 36, 181-190. Nordlie F. G., Szelistowski W. A. and Nordlie W. C. (1982) Ontogenesis of osmotic regulation in the stripped mullet, Mugil cephalus L. J. Fish. Biol. 20, 79-86.

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