High Blood CO₂ Levels in Rainbow Trout Exposed to Hypercapnia In Bicarbonate-Rich Hard Fresh Water - A Methodological Verification

Rainbow trout, weighing 250 –350 g, were acclimated to flowing Uppsala tap water (8−10°C, pH7·88, ⁠, [O₂] = 11mgr⁻¹, [Na⁺] = 0·6mmoll⁻¹, [CL] = 0·55mmoll⁻¹, [Ca²⁺] = 2·60mmoll⁻¹, [HCO₃⁻] =4mmoll^−l) for at least 3 months.

Three weeks before the experiments, fish were anaesthetized with MS-222, and the dorsal aorta was cannulated as described by Soivio, Nyholm & Westman (1975). Six fish were then transferred to 100−1 test aquaria with water of the same quality as that used during acclimation, except that the pH was 7·65 and the was 0·57 kPa. 48 h before the experiment began the fish were enclosed in individual restrainers. At the start of the experiment the fish were exposed to 1·73 kPa by using hydrochloric acid to acidify continuously the flowing aerated water until it reached pH 7·0. Samples were taken at 0, 4 and 48 h. Approximately 0’3 ml of arterial blood was removed and replaced by an equivalent amount of Ringer’s solution (Wolf, 1963). Blood acid—base variables were analysed tonometrically according to the method of Astrup (1956).

Uncannulated and unrestrained fish were pre-acclimated for 3 weeks and then exposed to 1·73 kPa as in the previous experiment. Venous blood samples were taken from six trout with a syringe via the ductus Cuvieri, and then immediately killed. The venous sampling procedure, including capture, lasted about 45 s. Blood acid—base variables were analysed tonometrically.

Five fish were exposed to 3·47 kPa to investigate the possible maximum blood CO₂ level attained during hypercapnia. Only uncannulated trout were used and venous blood samples were taken as before. Trout were pre-acclimated for 3 weeks in test aquaria and then subjected to steadily increasing hypercapnia for 3 days, reaching a final level of 3·47kPa (water pH = 6·70). This level of hypercapnia was then maintained for an additional 3 days. Total blood CO₂ content was analysed both tonometrically and gasometrically.

Blood pH values were measured in samples using a micro-pH electrode unit (Radiometer, G297/G2). Blood [HCO₃⁻] and total [CO₂] were calculated according to the Hendersson—Hasselbalch equation, using values of pKa and CO₂ solubility from Boutilier, Heming & Iwama (1984). When gasometry was used for the total blood CO₂ determination, samples of 50–100 μ1 of whole blood were acidified with 0−1 mol l⁻¹ HC1 in a Warburg flask. The amount of CO₂ evolved from the blood was compared with a standard curve using Na₂CO₃ as a reference. In addition, samples were also treated with 20 % KOH in the Warburg flask to correct for the O₂ content in the blood.

Results were statistically analysed using Wilcoxon rank-sum or signed-rank tests (Colquhoun, 1971); 5 % was taken as the fiducial limit of significance.

The effect of 1·73 kPa external on acid—base variables followed the ‘classical’ response: the induced acidosis returned to normal values as blood HCO₃⁻ levels increased (Table 1). Both blood sampling procedures revealed that the hypercapnic treatment elevated blood HCO₃⁻ levels to about 55 mmol l⁻¹. In the third experimental series, with 3·47 kPa hypercapnia, venous blood pH was 7·634 ±0·052, blood rose to 4·56 ±0·47 kPa ⁠, and blood CO₂, which approximately equals the HCO₃⁻ concentration, increased to as high as 66 mmol l⁻¹ (means ± S.D., N = 5). There was no statistically significant difference between tonometrically and gasometrically obtained blood CO₂ values.

From the present results it is evident that blood CO₂ concentration is not affected by the ‘grab and stab’ technique as applied under the conditions used in this study. Moreover, the consistency between the tonometric and the gasometric estimates of blood CO₂ concentration excludes any sort of analytical error attributable to the Astrup method (1956), used by Börjeson (1977) and Dimberg & Höglund (1987).

Finally, for comparison, the concentration of CO₂ in blood from rainbow trout adapted to Uppsala tap water diluted 1:10 with deionized water for several months at pH 7·60 and was measured and found to be about 10 mmol l⁻¹. This is in agreement with the data given by Perry (1982) for control fish adapted to water with a low and low bicarbonate concentration. It therefore seems reasonable to conclude that the high blood CO₂ concentration demonstrated in this study was real, although it is confusing that the level is at least twice as high as that found in most other investigations. However, as discussed by Heisler (1986), the bicarbonate in the hypercapnic fish has to be increased by the same factor as blood to achieve complete pH compensation. Accordingly, if the blood is increased from 0·6 to 1·79kPa, i.e. about three times, a fish with an initial blood CO₂ concentration of 19 mmol l⁻¹ has to increase the CO₂ content to 57 mmol l⁻¹. This is indeed the case in the present study with 1·73 kPa hypercapnia. An explanation for the discrepancy between the present high level of blood CO₂ with that value in the literature may be the use of the Uppsala water, characterized by high bicarbonate concentration and relatively high ⁠. The importance of the ion composition and buffering capacity of the external medium during hypercapnia has been discussed previously by Heisler & Neuman (1977) and Perry (1982). However, the possible maximal blood CO₂ level attained in hypercapnic rainbow trout and to what extent the external bicarbonate concentration interact with the adaptory process must be explored further.

This work was supported by a grant from the Hierta Retzius Foundation. Thanks are given to Docent L. B. Höglund for his comments on the manuscript.