The Effects of Reducing Water pH and Total CO₂ on a Teleost Fish Adapted to an Extremely Alkaline Environment

In rainbow trout, exposure to highly alkaline conditions (pH<9·5) causes severe physiological disturbances, including blood alkalosis, inhibition of Na⁺ influx, impairment of branchial ammonia excretion and a decrease in swimming speed (Wright and Wood, 1985; Ye, 1986). It is unusual, therefore, that a species of teleost fish, Oreochromis alcalicus grahami, is able to thrive in water of pH 10 (Lake Magadi, Kenya). In two previous studies, we have reported that O. a grahami produces urea by the ornithine-urea cycle and excretes all nitrogenous wastes as urea (Randall et al. 1989; Wood et al. 1989), presumably enabling the fish to survive in alkaline water.

Lake Magadi is almost entirely covered by a solid crust of Na₂CO₃/NaHCO₃. The fish live on the perimeter in shallow lagoons fed by underground hot springs (Coe, 1966). The total CO₃²⁻ plus HC0₃⁻ concentration is 184 mmol I⁻¹ and the osmolality is about 50% that of sea water. We propose that, unlike most other aquatic animals, exposure to water of pH 7 will present a number of physiological problems to O. a. grahami. By reducing the pH of Lake Magadi water, the total CO₂ concentration will also be reduced as HCO₃⁻ is titrated to form CO₂, which is then removed from solution by aeration. In neutral water, therefore, the tilapia will be exposed to a reversal of both the H⁺ and HCO₃⁻ gradients. Reite et al. (1974) reported the pH tolerance of O. a. grahami to be between 5 and 11. Their study, however, dealt only with survival and did not address the physiological responses to altered water pH. In the present study, we have determined the acid-base status, blood ion composition, Na⁺ influx rate, O₂ consumption and swimming ability in O. a. grahami at pH 10 and 7 to assess the effects of reduced water pH and total CO₂ levels. As a comparison, we have also studied the effects of alkaline exposure in O. nilotica, a closely related species of tilapia adapted to the neutral waters of the Sagana River, Kenya.

O. a. grahami (1–12g) were collected from Lake Magadi and held outdoors at the Magadi Soda Co. in aerated lake water (water pH=9·98, 30–37°C, total CO₂= 184 mmol l⁻¹, [Na⁺]=342 mmol l⁻¹, [CL] = 108 mmol l⁻¹, [K⁺] = 2·22 mmol I⁻¹, osmolality=525mosmolkg⁻¹). To determine the effects of neutral water pH, lake water was acidified by the addition of concentrated HC1 and vigorously aerated to reduce total CO₂ from the normal value of approximately 180 mmol I⁻¹ to less than 1 mmol I⁻¹, as measured by a Corning 965 CO₂ analyzer. In these experiments it was not possible to separate the effects of reduced water pH from those of reduced total CO₂ levels. Thus, fish were exposed to water of pH 7 and reduced total CO₂ levels of less than 1 mmol I⁻¹ and this water composition will be termed ‘neutral water’.

O. nilotica (6–15 g) were collected from the Sagana River Tilapia Hatchery in Kenya. While this species is nominally O. nilotica, intense cross-breeding with O. zillii and possibly other tilapia species occurs in the hatchery ponds; therefore its exact taxonomic status is uncertain. Fish were held indoors at the University of Nairobi in aerated, dechlorinated Nairobi tapwater (water pH=7·22, 22°C, total CO₂=0·56mmol l⁻¹, [Na⁺]=0·29 mmol I⁻¹, [Cl⁻]=0·09mmol l⁻¹, [K⁺] =0·03 mmol I⁻¹, osmolality <5mosmolkg⁻¹). To determine the effects of alkaline exposure on Sagana tilapia, fish were transferred from Nairobi tapwater at pH 7 (neutral tapwater) to tapwater adjusted to pH 10 with KOH (alkaline tapwater). In this case, there was no change in total water CO₂ levels (approximately 0·5 mmol I⁻¹). A complete description of fish-holding facilities and water composition is given in Randall et al. (1989) and Wood et al. (1989).

Blood samples (15–200μl fish⁻¹) for acid-base, ammonia, urea and ionic analyses were drawn anaerobically from stunned Magadi and Sagana tilapia. Blood samples were collected from both species of tilapia in their normal environmental water within 24 h of captivity. In transfer experiments, blood samples were taken after 12 h in neutral water (O. a. grahami) and after 10 h in alkaline tapwater (O. nilotica). Fish were exposed for several minutes to the air during sampling. Samples were taken by blind caudal puncture of the haemal arch. For details and discussion on the validity of the blood-sampling protocol and measurements, the reader is referred to Wood et al. (1989).

Magadi tilapia were swum in a miniature swim tunnel to determine the effect of neutral water on swimming performance. The apparatus was constructed from the barrel of a disposable 20 ml syringe connected to a 3 A submersible pump (Little Giant), with the plunger-end of the syringe connected to the outlet of the pump. A wire mesh was placed between the pump and syringe, and the needle-end of the syringe was perforated in several places to create a more even cross-sectional flow. The pump and syringe were placed in a 201 reservoir (O₂ tension=16±2kPa, 30·9±0·4°C). Flow was varied by attaching the pump to a rheostat. Velocity of flow was determined by collecting the volume of water per unit time leaving the syringe. Fish of similar size (length 4·3±0·2cm, mass 1·4±0·1g, N=5) were divided into two groups. One group was kept in Lake Magadi water, the other group was transferred to neutral water for 2–3 h before the start of the experiment. Fish were placed individually in the syringe and swum for 30 min at low velocity, followed by 15 min at each increased water velocity until the fish fatigued and was no longer able to maintain its position in the current. Critical swimming velocity was calculated as described by Brett (1964).

Oxygen (O₂) consumption rate and Na⁺ influx were assessed in O. a. grahami transferred from Lake Magadi water to neutral water. To measure O₂ consumption, fish were placed in individual sealed chambers. After an initial 10 min period of adjustment, water samples (1ml) were removed at 0 and 20 min for measurement of water O₂ consumption was calculated from the change in over the 20 min experimental period using appropriate O₂ solubility coefficients (Boutilier et al. 1984).

To determine Na⁺ influx ⁠, 10 μCi of ²²Na (as NaCl, Amersham) was added to 250 ml of water in aerated beakers. After a 10 min mixing period, water samples (10ml) were removed at Omin and 3h. The fish were killed with an excess of anaesthetic (MS-222). was determined by the appearance of ²²Na in the fish, taking into account water Na⁺ specific activity.

Water and plasma ammonia and urea levels and blood acid-base parameters were measured as described by Wood et al. (1989). and HCO₃⁻ levels were calculated by the Henderson-Hasselbalch equation using values of pK’ and CO₂ appropriate to the temperature and ionic strength of the samples (Boutilier et al. 1984). Values of were similarly calculated using appropriate constants from Cameron and Heisler (1983). Plasma protein levels were determined using a Goldberg refractometer (American Optical). Plasma samples and whole fish were frozen on dry ice and transported to the University of Ottawa where ion concentrations, osmolality and ²²Na activities were measured. Plasma [Na⁺] and [K⁺] were determined by atomic absorption spectroscopy (Varian AA-10), plasma [Cl⁻] by coulometric titration using a chloridometer (Buchler-Cotlove), and plasma [Ca²⁺] by spectrophotometry using a commercial assay kit (Sigma). Plasma osmolality was measured on 7 μl samples using a vapour pressure osmometer (Wescor 5100B). Water and whole-body ²²Na activities were determined using a gamma counter.

Values are reported as means±l S.E.M. (N), where N represents the number of fish (flux experiments and blood acid-base data) or the number of sample pools (plasma ammonia, urea and ion data). Student’s paired or unpaired t-tests were employed to evaluate the significance of differences between mean values (P<0·05).

The transfer of Magadi tilapia from alkaline to neutral water resulted in a severe metabolic acidosis (–0·43 pH units), an eightfold decrease in total CO₂, and a threefold decrease in calculated after 12 h (Table 1). In contrast, when Sagana tilapia were transferred to alkaline tapwater, there was no significant change in acid-base status after 10h. Acute transfer (1 h) to Lake Magadi water, however, resulted in a severe metabolic alkalosis measured shortly after death. This was reflected in a +0·16 unit rise in pH and a more than threefold increase in total CO₂ (Table 1). Chronic measurements could not be made as the fish died within 1 h of exposure to Lake Magadi water.

Plasma urea and total ammonia levels did not significantly change in Magadi tilapia after 12 h of exposure to neutral water (Table 2). In Sagana tilapia, plasma urea and ammonia concentrations also did not change after 10 h of exposure to alkaline tapwater. Mean plasma NH₃ tensions were calculated from mean total ammonia (Table 2) and pH (Table 1) values. levels decreased threefold in Magadi fish exposed to neutral water, primarily as a result of the plasma acidosis.

Plasma ionic composition, osmolality and protein concentration for Magadi tilapia in Lake Magadi water and neutral water are given in Table 3. Exposure to neutral water resulted in a significant decrease in Na⁺ and an increase in Cl⁻, as well as a very large decrease in HCO₃⁻ levels. There were no significant changes in K⁺, Ca²⁺ and NH4⁺ levels and osmolality. Plasma K⁺ values, however, were higher than expected for extracellular fluid (Guyton, 1981) in all fish, which may be related to the stress associated with the blood sampling method (for discussion see Wood et al. 1989). The plasma protein level decreased by 30% in neutral water, suggesting that a haemodilution occurred. This is supported by the observation that fish exposed to neutral water for 48 h had an extremely bloated appearance. Plasma ionic composition for Sagana tilapia is also given in Table 3 for comparison. The values are typical of other freshwater teleosts, but [Na⁺], [Cl⁻], [Ca²⁺] and osmolality are lower than in Magadi tilapia in either alkaline or neutral water.

In neutral water, was 55 % higher compared to control rates [Lake Magadi water, 2·1±0·2μmolg⁻¹h⁻¹versus neutral water, 3·3±0·1μmolg⁻¹h⁻¹ (N=6)]. There was no increase in metabolic rate, as transfer from alkaline to neutral water did not change O₂ consumption [Lake Magadi water, 9·06±1·42mmolkg⁻¹h⁻¹versus neutral water, 11·39±0·82mmolkg⁻¹ h⁻¹ (N=6)].

We measured critical swimming velocity in Magadi tilapia to determine the effect of neutral water on swimming ability. In Lake Magadi water, Magadi tilapia swam efficiently in the swimming apparatus [3·2±0·2 body lengths⁻¹], but when transferred to neutral water, critical swimming velocity decreased fourfold [0·7±0·2 body lengths⁻¹ (N=5)].

O. a. grahami are remarkable in their ability to survive in an extremely alkaline environment (pH=10, total CO₂=184mmol l⁻¹). The results of this study show that, unlike the majority of aquatic animals, O. a. grahami do not function well in neutral water (pH 7, total CO₂ <1 mmol I⁻¹). A good indicator of overall performance in fish is swimming ability. Critical swimming velocity was severely reduced when O. a. grahami were transferred from Lake Magadi to neutral water. In addition, there was a large metabolic acidosis and an ionic and fluid volume disturbance. Clearly, Magadi tilapia can survive but not function adequately in neutral water.

The question as to why Magadi tilapia suffer a number of adverse affects in neutral water must be related to water composition. Acidification and subsequent aeration of Lake Magadi water resulted in the following changes: (1) a reduction in total CO₂ levels (184 to <1 mmol I⁻¹), (2) a reduction in calculated [HCO₃⁻] (64 to <1 mmol I⁻¹, see Materials and methods), (3) a 1000-fold increase in water H⁺ levels (pH 10 to pH 7), and (4) a calculated fourfold increase in Cl⁻ levels (108 to about 400 mmol I⁻¹). When Magadi tilapia were exposed to neutral water, the HCO₃⁻ gradient (now blood-to-water), H⁺ gradient (now water-to-blood) and Cl⁻ gradient (now water-to-blood) were reversed compared to the gradients in Lake Magadi water. Thus, one would predict the efflux of HCO₃⁻ from the plasma to the environment and the entry of Cl⁻ and H⁺. The data support this prediction: plasma [HCO₃⁻] and pH decreased significantly, and [Cl⁻] increased significantly in neutral water (Tables 1 and 3).

When Sagana tilapia were exposed to Lake Magadi water, they quickly died, exhibiting a marked metabolic alkalosis (Table 1). When Sagana tilapia were exposed to alkaline tapwater, however, the effects on blood acid-base balance were negligible (Table 1), indicating that toxicity was related in some way to the high water carbonate alkalinity rather than to pH per se.

Exposure to neutral water resulted in a fluid volume disturbance in Magadi tilapia. There are two pieces of evidence that point to an increase in blood volume and possibly extracellular fluid volume. First, plasma protein levels decreased and, second, fish in neutral water had a bloated appearance. This expansion was due to an uptake of both ions and water as there was no change in osmolality in Magadi tilapia exposed to neutral water.

Changes in plasma acid-base status and ionic composition may have affected metabolic function. O. a. grahami excrete all nitrogenous wastes as urea (Randall et al. 1989) and, when exposed to neutral water, urea excretion was completely inhibited (Wood et al. 1989). The inhibition of urea excretion may have been due to an inhibition of urea production via the ornithine-urea cycle (Randall et al. 1989; Wood et al. 1989). The large decrease in plasma [HCO₃⁻] (Table 1) may have resulted in an inhibition of liver urea metabolism. In toadfish hepatocytes, a decrease in extracellular [HCO₃⁻] reduces the rate of urea synthesis (Walsh et al. 1989). Moreover, these data indicate an inhibition of protein metabolism because plasma ammonia levels (Table 2) and excretion rates (Wood et al. 1989) did not rise, despite an inhibition of urea production. Nitrogenous wastes in Sagana tilapia were excreted predominantly as ammonia, but during alkaline exposure, urea excretion increased threefold (Randall et al. 1989; Wood et al. 1989), with no change in plasma [HCO₃⁻] (Table 1) but an increase in plasma [ammonia] (Table 2). This response is found in some ammoniatelic teleost fish which are able to increase urea production slightly via uricolysis in response to elevated body ammonia levels (Olson and Fromm, 1971).

Exposure to neutral water significantly increased Na⁺ influx in O. a. grahami. To our knowledge, Na⁺ influx rates in O. a. grahami have not been previously reported. In freshwater rainbow trout, Na⁺ influx is pH sensitive: the optimum pH is between 7 and 8, and Na⁺ influx decreases in acid or alkaline water (Wright and Wood, 1985; Randall and Wright, 1986). The pH sensitivities of the Na⁺ transporters in trout and Magadi tilapia appear to be similar, but Na⁺ flux rates in trout are an order of magnitude lower.

In summary, the results of this study show that exposure of O. a. grahami to neutral water results in a metabolic acidosis, an ionic and fluid volume disturbance, an increase in Na⁺ influx, and a large reduction in critical swimming velocity. Reite et al. (1974) report the pH tolerance of O. a. grahami to be between 5 and 11: however, they did not measure any physiological parameters and were only interested in survival for 24h. The adverse response of O. a. grahami to neutral conditions is unique since the majority of aquatic animals have an environmental pH optimum between 6 and 8. Finally, our results also indicate that Lake Magadi water is far more ‘toxic’ to O. nilotica adapted to neutral conditions than neutral water is to O. a. grahami adapted to Lake Magadi water. The Lake Magadi toxicity appears to be related to the high bicarbonate alkalinity rather than to pH per se. Thus, urea production via the ornithine cycle in O. a. grahami can be viewed as a way to maintain nitrogenous excretion by consuming NH4⁺ and blood pH by consuming HCO₃⁻.

We thank George Mathuria and Steven Wantee from the University of Nairobi for their valuable assistance, the management and staff of Magadi Soda Co. for their hospitality, and Professor G. M. O. Maloiy of the University of Nairobi for his help throughout the project. We also thank R. S. Munger, J. Curtis, and R. Rhem at McMaster University for technical assistance. Supported by NSERC and International Collaborative Research Grants and Operating Grants to CMW, SFP and DJR, and grants from McMaster University, University of Ottawa, University of Wyoming and Living Lakes Inc.