Effects of Extreme pH on the Physiology of the Australian ‘Yabby’ Cherax Destructor: Acute and Chronic Changes in Haemolymph Carbon Dioxide, Acid–Base and Ionic Status

Environmental acidification has become a problem of global proportions, such that large bodies of natural soft water are now acidified to between pH 4.5 and 6.0 (Galloway et al. 1983; Minns and Kelso, 1986). Aquatic organisms at all major trophic levels are affected by decreased pH (e.g. Hargeby, 1990; Rosseland, 1986); however, fish and crustaceans are recognized to be among the most sensitive (Davies, 1989).

Failure of ion regulation at the gill is the predominant cause of acid toxicity (Wood and Rogano, 1986). The toxic effect may, however, disturb a range of physiological functions including fluid-volume distribution, oxygen uptake and transport, and haematological and acid–base homeostasis (Ultsch et al. 1981; Milligan and Wood, 1982). The study reported here considers the consequences of extreme environmental pH for the haemolymph acid–base and ion physiology of the Australian crayfish Cherax destructor (Parastacoidea).

Ion and acid–base regulatory activities are inextricably linked (Henry and Cameron, 1982). Acid–base balance is achieved when the rates of production and elimination of acid–base relevant ions (H⁺, OH⁻ and HCO₃⁻) are equal (Henry and Cameron, 1982; Burnett, 1984). Extra- and intracellular acid–base balance must be maintained in order to ensure optimum enzyme activity, conformational state and the function of basic physiological processes (e.g. Truchot, 1983; Wheatly and Henry, 1992). Therefore, the direct transfer of such ions across the crustacean gill epithelium in acid and alkaline environments is likely to disturb this balance.

The magnitude of disruption to the haemolymph and tissue ionic status, and hence the tolerance to an acid environment, depends on the water hardness and the species (McDonald et al. 1983; Hõbe et al. 1984; Jensen and Malte, 1990). For example, trout, cyprinid fishes and the crayfish Orconectes rusticus exhibit a greater degree of haemolymph ionic status perturbation in acid soft water associated with large equimolar losses of Na⁺ and Cl⁻ (McDonald, 1983; Hõbe et al. 1984). In contrast, animals in hard water show a greater degree of haemolymph acid–base disturbance (Wood and Rogano, 1986) via a net H⁺ uptake resulting from a loss of cations (e.g. Na⁺) in excess of anions (e.g. Cl⁻) (McMahon and Morgan, 1983).

Mobilization of the carapace reservoirs during hypercapnic acidosis has been demonstrated in Orconectes rusticus and Procambarus clarkii during acid exposure (Morgan and McMahon, 1982; Wood and Rogano, 1986). Carapace dissolution may play an essential role in increasing haemolymph pH by providing a source of HCO₃⁻. This mechanism is also available to many other terrestrial (Henry et al. 1981; Wood and Randall, 1981; Morris et al. 1986) and aquatic crustaceans when in air (DeFur et al. 1980). Changes in the strong ion difference (SID) (Stewart, 1978, 1981) and the total concentrations of mineral acids and bases may also be important in reducing acidosis (Heisler, 1986; see Wheatly and Henry, 1992, for a review).

Calcium ions are the major component of water hardness. Their supposed protective effect against severe ion loss in fish, and possibly crayfish, in hard waters may arise from weak ionic interactions with surface ligands on the gill which act to stabilize the apical membrane (McDonald et al. 1983; Booth et al. 1988; McDonald and Milligan, 1988). An objective of this study was to test the hypothesis that water calcium concentration plays a significant role in the regulation of acid–base and ionic equilibria of Cherax in highly acid water.

Some previous studies on Northern Hemisphere crayfish species (Astacoidea) have investigated the acid–base and ion challenges of life in acid water (e.g. DiStefano et al. 1991; Mauro and Moore, 1987; Wood and Rogano, 1986). However, some industrial effluent can increase water pH (e.g. from bleach-craft paper mills), yet there are almost no data on the response to alkaline water. Therefore, further investigation of both environmental acidification and alkalization is imperative.

Animal collection and maintenance

Male and female Cherax destructor (N≈580; 45–60 g) were donated by the commercial crayfish farm Crayhaven (New South Wales). The pH of the ponds at Crayhaven was between 7.23 and 7.60, and the water [Ca²⁺] was approximately 50 μmol l⁻¹. The animals were maintained in a freshwater recirculating aquarium at the University of Sydney and kept under a 16 h:8 h light:dark photoperiod at a room temperature of 20±2°C. Animals were fed meat weekly, except during experimentation. Moulting individuals identified by either brittle (pre-moult) or soft (post-moult) exoskeletons as well as brooding females were excluded from all experimentation.

The recirculating aquarium consisted of four trays (115 cm×60 cm×20 cm), within a stainless-steel frame. Water in the trays was aerated with room air and recirculated through a washed gravel filter system using an all-plastic impeller pump. Clear Perspex lids covered the four trays to prevent evaporation and animal escape, but allowed light penetration. Experiments were carried out using five different combinations of pH and [Ca²⁺]. Conditions were chosen to assess the effects of extreme pH alone on acid–base balance and to examine the influence of different ambient Ca²⁺ concentrations (within the softwater range) on the response to acid exposure. The conditions are given in Table 1.

Animals to be used in the higher-Ca²⁺ experiments were acclimated for 1 week to water at pH 7.1 (treatment 1a) with the following salt composition (in μmol l⁻¹): NaCl, 250; KCl, 500; CaCO₃, 500; MgCO₃, 200; MgSO₄, 500. This served as the ‘control’ condition for the high-Ca²⁺ treatments (500 μmol l⁻¹). Thus, two-way analysis of variance (ANOVA) could be performed, excluding the pre-treatment value, testing between experimental and control (pH 7.1, 500 μmol l⁻¹ Ca²⁺) over the duration of the treatments. Before addition to the aquarium, the water was first ‘bubbled’ with CO₂ gas, to dissolve carbonate salts, and then with air overnight. The water pH was set and monitored at pH 7.1 by daily measurement with an Orion pH meter (model 9204A) connected to an Orion Ross combination pH electrode (model 81-02). The pH of the water in the filter reservoir (400 l) was adjusted by adding NaOH pellets or H₂SO₄ as required.

Two separate ‘experimental’ trays were set up below the recirculating aquarium for both the acid and alkaline water treatments (treatments 1b and 3 respectively). For each tray a ‘pH-stat’ constructed at the University of Sydney automatically regulated the water pH, holding it within ±0.2 units of the required value. Outside this set limit, solenoid valves (Goyen Controls type LP4-6R) regulated the flow of either 0.001 mol l⁻¹ NaOH or 0.001 mol l⁻¹ H₂SO₄ solutions into the water.

Animals to be used in low-Ca²⁺ experiments were acclimated for 1 week to nominally Ca²⁺-free water at pH 7.1 (treatment 2a) with a salt composition as follows (in μmol l⁻¹): NaCl, 250; KCl, 500; Na₂CO₃, 500; MgCO₃, 200; MgSO₄, 500. The Na₂CO₃ replaced the CaCO₃ used in the high-Ca²⁺ treatments. Another tray containing water of the same composition but at pH 4.5 (treatment 2b) was also set up. The water pH was maintained using a pH-stat and [Ca²⁺] was measured daily using atomic absorption spectrophotometry (see below). The water was changed each day in order to maintain a continually low [Ca²⁺] in the water. The [Ca²⁺] varied between 68 and 96 μmol l⁻¹ with a mean of 82.0±13.5 μmol l⁻¹. In the low-Ca²⁺ experiments, ANOVA was routinely carried out with respect to pH 4.5 high-Ca²⁺ as a referrent, i.e analysed for an effect of [Ca²⁺] at low pH.

After initial acclimation to the appropriate control condition, crayfish were transferred to individual mesh cages (15 cm×10 cm×10 cm) and left undisturbed for 12 h prior to experimentation. At time zero (pre-exposure), six animals were carefully removed from their cages and haemolymph samples (approximately 500 μl) were quickly withdrawn (<25 s). Venous samples were withdrawn via the arthrodial membrane at the base of the last walking leg and arterial samples from the pericardial sinus surrounding the heart. Each sample was collected with a 1 ml glass syringe (Suzuki), using 23 gauge needles, and placed on ice to delay haemolymph clotting.

Animals were either transferred within the control condition at pH 7.1 (sham transfer) or to the appropriate treatment tray and left undisturbed until sampling after 0.25, 0.5, 1, 2, 5, 24, 96, 288 or 504 h. Six different animals were used at each time and each was sampled only once to ensure the collection of independent data. The control series served partly as a check on the possible disturbing effects of the brief handling and air-exposure involved in the transfers.

Animals in both the control condition (pH 7.1) and acid treatment (pH 4.5) containing 500 μmol l⁻¹ Ca²⁺ were monitored over the full 504 h period, whilst the maximum exposure times for those in the alkaline (pH 8.0, 500 μmol l⁻¹ Ca²⁺) and low Ca²⁺ acid treatment (pH 4.5, 50 μmol l⁻¹ Ca²⁺) were 96 h and 288 h respectively.

Haemolymph samples were immediately measured for carbon dioxide partial pressure and content as well as pH. The and pH of the haemolymph were measured with Radiometer electrodes (types E5047, G229a Radiometer, Copenhagen, Denmark) thermostatted at 20 °C in a BMS3 haemolymph microsystem connected to a PHM73 blood gas analyzer. Total CO₂ content was determined using a Ciba-Corning 965 CO₂ analyzer.

Haemolymph osmolality was immediately determined with a vapour pressure osmometer (Wescor, 5100C) and the remaining haemolymph was frozen (–15 °C). Haemolymph sodium and calcium concentrations were measured using an atomic absorption spectrophotometer (Varian techtron). Frozen haemolymph samples for ion analysis were weighed, dissolved in an equal volume of 0.1 mol l⁻¹ HNO₃ and then mechanically disrupted by centrifugation (10 000 g) for 5 min. The supernatants from denatured haemolymph samples, standard solutions and a blank were diluted with either reverse osmosis water for Na⁺ or LaCl₃.7H₂O (2.66 % w/v) to suppress chemical interference in Ca²⁺ analysis.

The second walking leg was removed from each animal, weighed and digested in 11 mol l⁻¹ HNO₃ for 2 days. Dilutions were carried out for analysis by atomic absorption spectrophotometry and interference was suppressed by the addition of LaCl₃. Both haemolymph and carapace ion concentrations were calculated from regressions of oAb/o[ion] for the standards.

Data have been reported as mean ± standard error unless otherwise stated. Two-way analyses of variance were used to detect differences between arterial and venous haemolymph and amongst treatments. The chosen limit of significance was P=0.05. If necessary, one-way ANOVA with post-hoc comparison of treatment values with the pre-treatment value was also carried out. Post-hoc testing was performed using contrast testing and Tukey’s HSD multiple means comparison test. All analyses were done using the SYSTAT 5.01 statistical package.

The venous carbon dioxide content of crayfish placed in either neutral (treatment 1a) or acid water containing high concentrations of Ca²⁺ (treatment 1b) decreased significantly by 2.55 mmol l⁻¹ and 0.51 mmol l⁻¹, respectively, after 0.25 h (F_9,48=4.27, F_9,50=2.28) (Fig. 1A,C). However, the of acid-exposed animals was significantly lower than that of control animals after 5 and 24 h (F_8,88=2.28, Contrast) (Fig. 1A,B). The arterial-venous (a-v) CO₂ difference of crayfish placed in the acid water (500 μmol l⁻¹ Ca²⁺) increased significantly on average by 0.41 mmol l⁻¹ after 2 h and remained high up to 288 h (F_8,36=7.78, Tukey) (Fig. 1C,D). This a-v difference was not apparent at 504 h.

Fluctuations in the haemolymph were more extreme than those for Acid exposure (500 μmol l⁻¹ Ca²⁺) resulted in a significantly decreased venous partial pressure of carbon dioxide with respect to both the pre-treatment values and control animals; however, this did not occur at all times (F_9,49=6.35, F_9,50=6.60, Tukey; F_8,90=3.68, F_8,90=5.32, Contrast) (Table 2).

The venous haemolymph pH (pHv) of crayfish held in pH 7.1 and pH 4.5 water ([Ca²⁺] maintained at 500 μmol l⁻¹) decreased significantly between the 1 h and 5 h exposure times (F_8,89=6.35) (Fig. 2A). The size of this acidosis was not statistically different between control (pH 7.1) and acid (pH 4.5) treatments and was therefore a general effect. There was no evidence of haemolymph acidosis as a result of exposure to pH 4.5 (500 μmol l⁻¹ Ca²⁺) water at 504 h of exposure (Fig. 2B).

The pre-acclimation of Cherax to low-Ca²⁺ water (50 μmol l⁻¹) at a constant pH 7.1 decreased the mean by 2.59 mmol l⁻¹ and by 1.9 mmol l⁻¹ (Fig. 1C, time 0). Reducing the pH of the low-Ca²⁺ water to pH 4.5 was associated with a further significant decrease in both and (2 h cf. time 0). In fact, both the and of animals placed in lower-Ca²⁺ acid water were significantly lower than in those animals placed in higher-Ca²⁺ acid water for the entire 288 h treatment period (F_8,88=5.62, Contrast) (Fig. 1C,D). Furthermore, the arterial-venous difference of animals in low-Ca²⁺ acid water decreased significantly after 1 h compared with the initial value and remained small, even after 288 h (F_8,36=11.26, Tukey).

Both the and of Cherax held in acid lower-Ca²⁺ water were significantly higher than those of crayfish in high-Ca²⁺ water at almost all times over the 288 h exposure period, except initially and after 2 h (F_8,88=6.33, Contrast) (Table 2). Consequently, after 288 h, the of animals in low-Ca²⁺ water was unusually high and on average 4.38 mmHg greater than that of animals in higher-Ca²⁺ water and 1.73 mmHg higher than the pre-treatment condition (F_8,43=7.62, Tukey).

The decreased values of crayfish in low-Ca²⁺ water were associated with pHv values 0.1 unit higher than in those animals in high-Ca²⁺ water from the start of the experiment. This difference in pH correlated with [Ca²⁺] (Fig. 2C,D) varied with time, but the pronounced trend continued, even after 288 h (12 days) (F_1,90=60.23).

Cherax held in pH 8.0 water exhibited a significant decrease in after only 0.25 h compared with the initial value (F_7,40=24.40), which remained low throughout the 96 h treatment period (mean 3.9 mmol l⁻¹) compared with the control animals (mean 7.6 mmol l⁻¹) (F_6,68=4.05, Contrast) (Fig. 1A,B). Only the 0.5 h value showed an arterial-venous difference different from the initial value (F_7,32=60.04, Tukey) and at this time the a-v difference was not significant.

In contrast to acid-exposure, exposure to alkaline water produced and values significantly higher than in control animals in the first 5 h of exposure (F_6,70=4.95, F_6,70=4.88, Contrast) (Table 2; mean 2.79 mmHg, pH 7.1 and 3.82 mmHg, pH 8.0). The subsequently decreased in a similar manner to and could not be statistically separated from pH 7.1 exposure values for the remainder of the exposure.

Animals placed in pH 8.0 water, unlike those in neutral and acid water, exhibited an apparent minor venous haemolymph alkalosis in the first 5 h of exposure (Fig. 2A), which increased the pHv 0.09 units above the initial value. This change was similar to the variance of the mean and therefore was not statistically significant. However, alkaline-exposed animals had significantly higher haemolymph pH values than control animals after 1, 2 and 5 h (F_6,70=6.28, Contrast), but after this time the measured venous pH of alkaline-exposed animals was within the variance of control values (Fig. 2B).

Crayfish held in pH 7.1, pH 4.5 and pH 8.0 high-Ca²⁺ water (500 μmol l⁻¹) exhibited only small, yet similar, decreases in haemolymph osmolality in the first 5 h of exposure. Despite these decreases, neither 504 h (21 days) of exposure of Cherax destructor to either neutral or acid water nor 96 h (4 days) of exposure to alkaline water caused significant changes in haemolymph osmolality (Table 3).

In contrast, exposure of Cherax destructor to 50 μmol l⁻¹ Ca²⁺ acid water for 12 days significantly decreased haemolymph osmolality below the initial value after 24 h (F_8,45=3.44, Tukey).

The haemolymph [Na⁺] of Cherax in neutral water (treatment 1a) was constant over a 504 h exposure period, but decreased in animals exposed to acid water (pH 4.5, 500 μmol l⁻¹) after 96 h (mean decrease 98.88 mmol l⁻¹) (F_9,50=3.41, Tukey) (Fig. 3A,B). The [Na⁺] subsequently returned to near the pre-experimental values (0 h) by 288 h. Animals in acid water only had significantly increased Na⁺ levels compared with the haemolymph from control animals after 1 h (F_12,102=2.52, Contrast).

Lowering the [Ca²⁺] of the water had a significant effect on the haemolymph [Na⁺] of Cherax destructor at all times (F_8,86=10.23) (Fig. 4). At the start of the experiment, the [Na⁺] of animals in the control condition (treatment 2a) was 50 % of that of crayfish acclimated to 500 μmol l⁻¹ Ca²⁺ water (mean 161.03 and 324.66 mmol l⁻¹ respectively) (Fig. 4). Thus, water [Ca²⁺] significantly affected haemolymph [Na⁺] irrespective of water pH. In addition, the haemolymph [Na⁺] of animals held in low-Ca²⁺ water, unlike that of crayfish in higher-Ca²⁺ water, was significantly greater after the 288 h exposure period (Fig. 4), especially after 5 h and 288 h (F_8,43=21.34).

Animals placed in pH 8.0 water (treatment 3) had a significantly increased haemolymph [Na⁺] between 2 h and 5 h compared with the initial value (F_7,39=7.27). Alkaline-exposed crayfish also had significantly higher [Na⁺] concentrations than those in the control condition after just 1 h and at all times up to 24 h (F_12,102=2.52, Contrast) (Fig. 3). This was most apparent after 2 h, when animals held in alkaline water had 36 % higher Na⁺ concentrations than control animals.

The haemolymph Ca²⁺ concentrations of Cherax kept in both neutral and acid water containing 500 μmol l⁻¹ Ca²⁺ were similar over the 504 h treatment period (F_2,102=13.99, Tukey) (Fig. 5A,B).

The initial haemolymph [Ca²⁺] of crayfish in 50 μmol l⁻¹ Ca²⁺ water was not significantly below that of animals held in higher-Ca²⁺ water (Fig. 5C; 18.77 and 19.42 mmol l⁻¹ respectively). Interestingly, haemolymph [Ca²⁺] increased significantly after 24 h, 96 h and 288 h of acid exposure (50 μmol l⁻¹ Ca²⁺) (F_8,90=9.37, Tukey), despite the very low concentrations in the surrounding water (Fig. 5D). Subsequently, these animals had significantly higher haemolymph Ca²⁺ levels than animals placed in higher-Ca²⁺ acid water after 24 h and up until 288 h (F_7,78=6.18, Contrast), when the haemolymph calcium concentration of acid-exposed animals in low-Ca²⁺ water was 6.15 mmol l⁻¹ greater than that of animals in 500 μmol l⁻¹ Ca²⁺ water (Fig. 5D).

Within 0.25 h, animals held in pH 8.0 water had significantly increased haemolymph Ca²⁺ concentrations compared with both control and acid-exposed animals during the 4 day exposure period (mean 18.36 mmol l⁻¹ and 20.15 mmol l⁻¹ respectively; Fig. 5A,B) (F_2,102=13.99, Tukey). The elevated haemolymph [Ca²⁺] of animals exposed to pH 8.0 water peaked at 1 h, at which time this was also a significant increase with respect to the time zero condition (F_7,38=5.19, Tukey).

Animals held in higher-Ca²⁺ neutral water exhibited little change in carapace [Ca²⁺], but the carapace Ca²⁺ content of acid-exposed animals decreased significantly after 96 h with respect to the initial value (F_9,100=13.79, Tukey) (mean decrease 0.64 mmol g⁻¹) (Fig. 6). After 96 h and 288 h of exposure to acid water, Cherax had significantly lower carapace [Ca²⁺] than control animals (F_8,86=13.32, Contrast). Interestingly, there was substantial recovery of carapace [Ca²⁺] after 3 weeks (504 h) of exposure.

Acid–base and ion balance in Cherax destructor is clearly influenced by changes in the environmental pH. Whilst few previous studies have considered responses beyond 96 h (4 days) of exposure, this study shows that the longer-term effects can be different and important. For example, the recovery of Na⁺ homeostasis in animals exposed to pH 4.5 water with 500 μmol l⁻¹ Ca²⁺ was only evident after 288 h.

Measurements indicated Cherax to be quite ‘acid-tolerant’. The common response of European and Northern American species to acid water and relatively low [Ca²⁺] (pH≈4.0, [Ca²⁺]≈100 μmol l⁻¹) is a combined metabolic and respiratory acidosis (Table 4) associated with an initially elevated Increased in Orconectes propinquus accounted for 30–40 % of the total pH depression in the first 48 h of exposure to pH 4.0, 100 μmol l⁻¹ Ca²⁺ water (Wood and Rogano, 1986). A similar elevation was observed in O. rusticus throughout 96 h of acid exposure, but not in Procambarus clarkii (Morgan and McMahon, 1982; McMahon and Morgan, 1983). Subsequent chronic decreases in ⁠, via hyperventilation, may reverse the metabolic depression by 25 % (Wood and Rogano, 1986; Jensen and Malte, 1990). The large ranges and occasionally high values (2.4–7.3 mmHg) suggest that the various haemolymph CO₂ species (i.e. CO₂, HCO₃⁻, CO₃²⁻) may not be in complete equilibrium (see Cameron, 1986, for a discussion on CO₂ disequilibrium and acid–base balance; see also DeFur et al. 1980; Patterson and DeFur, 1988).

Depending on the species, the decrease in haemolymph pH varies between 0.4 and 0.8 units (Table 4). In contrast, the acid–base response of Cherax in pH 4.5, 50 μmol l⁻¹ Ca²⁺ water was quite different. There was no evidence of the initial hypercapnia or severe acidosis reported for other species. While it is more difficult to generalise as to the response in haemolymph [HCO₃⁻], a respiratory acidosis would normally increase blood ⁠, whereas Cherax exhibited the opposite trend.

The decrease in appears to be a result of a potential metabolic acidosis compensated by a mixed alkalosis, consistent with a small acid influx, significantly reduced haemolymph CO₂ content and the hypometabolic state of crayfish (Ellis and Morris, 1995). While this circumstance is reminiscent of O. propinquus (Wood and Rogano, 1986), the net result for Cherax is the maintenance of a stable haemolymph pH.

Interestingly, acclimation to low-Ca²⁺ water at pH 7.1 had a marked effect on acid–base state, inducing a marked alkalosis, decreased ⁠, loss of a-v difference and possibly lower and thus increased pH. It would seem that the response to reduced [Ca²⁺] is in many ways a pre-adaptation countering the ‘normal’ effects of acid exposure on crayfish in low-Ca²⁺ water. This control of acid–base state appears to be at the expense of ion regulation.

Maintenance of stable haemolymph pH during environmental acidification could be achieved by a reduction in H⁺ permeability or stimulation of H⁺ excretion. Transbranchial ion exchange also seems to be important in acid-stressed Pacifastacus leniusculus (Wheatly, 1989). Alternative suggestions include a shut-down of Cl⁻/HCO₃⁻ exchange, conserving HCO₃⁻ in the haemolymph and buffering influxed H⁺ (McDonald, 1983; Wood and Rogano, 1986; Audet et al. 1988). Mobilisation of carapace CaCO₃ could contribute to pH maintenance via relative elevation of haemolymph [HCO₃⁻], explaining the 3.2 mmol l⁻¹ increase in haemolymph [Ca²⁺] for Cherax in acid water (see Wheatly and Henry, 1992, for a review). However, that there was no real increase in circulating argues against this mechanism and implies that Ca²⁺ comes from some other source. Clearly the importance of HCO₃⁻/Cl⁻ exchange in the recovery response to acid stress requires further investigation. If, as it appears, ionoregulatory mechanisms are employed by Cherax in low-Ca²⁺ acid water to maintain acid–base status, one result is likely to be a permanent ion disequilibrium.

While Cherax can maintain haemolymph pH at low water Ca²⁺ concentration (50 μmol l⁻¹), environmental calcium may play an important role in the maintenance of haemolymph ion status. Indeed, acclimation of Cherax to low-Ca²⁺ water at pH 7.1 resulted in the depletion of haemolymph [Na⁺] of both control and acid-exposed animals relative to those in higher-Ca²⁺ water.

Previous studies have claimed acid–base imbalance to be more severe with increasing environmental [Ca²⁺] (Wood and Rogano, 1986; McDonald, 1983). Nevertheless, acid–base perturbations of Cherax in water containing Ca²⁺ at 500 μmol l⁻¹ were comparatively minor (Table 4) and of short duration. The only significant decreases in pHv were at 2 and 5 h; however, compensation was achieved within 24 h and a steady pH of approximately 7.55 was maintained. The only change in was a significant decrease at 0.25 h, suggesting a mixed metabolic acidosis and respiratory alkalosis similar to that in Cherax in low-Ca²⁺ acid water. Similar changes in the control series suggest, however, that the response may be in part a disturbance effect (see Wilkes and McMahon, 1982). The eventual elevation of from the 0.25 h minimum to the 504 h maximum was significant, but not with respect to the initial condition. The transient acidosis apparent at 2 and 5 h appeared to be almost entirely respiratory in origin, but was not accompanied by significant changes in CO₂ content.

A 31 % decrease in haemolymph [Na⁺] at 96 h of acid-exposure (500 μmol l⁻¹ Ca²⁺) occurred without any affect on ⁠, or pH. Similar decreases have been reported for the crayfish Cambarus bartonii bartonii (35 %) and Astacus astacus (30 %) (Appelberg, 1985) in much softer water ([Ca²⁺]≈100 μmol l⁻¹). Decreased haemolymph [Na⁺] has been explained by increases in external [H⁺] reversing the normal Na⁺/H⁺ exchange, promoting Na⁺ loss (Holeton et al. 1983) and, in Orconectes, by reducing Na⁺ influx but maintaining Na⁺ efflux (Wood and Rogano, 1986). Recovery of both haemolymph pH and Na⁺ concentration to pre-exposure values after 96 h may, therefore, have been due either to activation of the ‘normal’ Na⁺/H⁺ exchange mechanism or to changes in the affinities and/or numbers of Na⁺ carriers (Wood and Rogano, 1986; Audet et al. 1988). A similar compensatory trend has been observed in the crayfish species Procambarus clarkii after longer periods of sublethal acid exposure (Bennett and Walker, 1992; DiStefano et al. 1991). Haemolymph [Na⁺] of Cherax in only 50 μmol l⁻¹ Ca²⁺ did not recover and remained significantly lower for the duration of the treatment. Thus, Ca²⁺ seems to have some role in mediating Na⁺ and H⁺ movements across the apical exchanger of the gill epithelia. Relatively minor disruptions in Ca²⁺ metabolism have been implicated as a critical factor for maintenance of crayfish populations in soft water acid environments (Mills et al. 1976; France, 1987).

The carapace may be an important contributor to extracellular acid–base status (Wheatly, 1989), providing HCO₃⁻ for ion exchange during decalcification via transfer of CO₃²⁻ and Ca²⁺ into the haemolymph. The decreased carapace calcium content of Cherax destructor after 96 h of exposure to pH 4.5 ([Ca²⁺]=500 μmol l⁻¹) supports the participation of carapace carbonates in buffering haemolymph acidosis (for a review, see Wheatly and Henry, 1992). Subsequent recovery of carapace calcium content may therefore result from restoration of normal pH after the transient acidosis, halting any further efflux of Ca²⁺ from the relatively basic carapace fluid compartment (Cameron, 1985; Cameron and Wood, 1985; Wheatly et al. 1991).

The relative acid-tolerance of Cherax compared with European and American species may be associated with the relative acidity of its haemolymph. The pH of 7.6 of venous haemolymph in resting Cherax destructor from 500 μmol l⁻¹ Ca²⁺ acid water was unusually acidic compared with that of Northern Hemisphere crayfish species (pH 7.9; Table 4). Cherax belong to the Parastacidae, rather than the Astacidae family in which the other species are found, so ‘acidic’ haemolymph may prove to be a feature of the Southern Hemisphere Parastacoidea.

Differences between the results of this and other studies may be due, in part, to the pH of the acclimation water (Table 4). Acclimation pH in some studies (e.g. Mauro and Moore, 1987) was equivalent to that used as an alkaline exposure in the present study (pH 8.0), the clear effects of which suggest that this pH does not represent a true ‘control’ condition. Acclimating the animals to pH values above 7.4 is probably atypical of many freshwater bodies and is certainly not normal on the basis of data obtained from water bodies near Sydney (Ball, 1991; Mackay, 1991). Similarly, exposure to pH values below 4.0 inaccurately represents realistic levels of environmental acidification, since such extreme pH values are rare in natural waters (Wood and Rogano, 1986).

The specific effect of acid-exposure on the ionoregulatory system of Cherax destructor in soft water varies with the environmental Ca²⁺ concentration. The ionoregulatory response of Cherax in acid water containing 500 μmol l⁻¹ Ca²⁺ was similar to those reported for other crayfish species in softer water containing approximately 100 μmol l⁻¹ Ca²⁺, yet at a Ca²⁺ concentration of 50 μmol l⁻¹ (this study), the response was very different. 50 μmol l⁻¹ Ca²⁺ is near or below the minimum equilibrium concentration for efficient calcium metabolism (Greenaway, 1974), which may explain the similarity between the responses of Cherax destructor in high-Ca²⁺ water and those studies that used 100 μmol l⁻¹ Ca²⁺. Studies of rainbow trout suggest that calcium ions function in stabilizing biological membranes, increasing the tightness of intercellular tight junctions and thereby controlling ion and water permeability across the gill epithelium (McDonald et al. 1983; McDonald and Milligan, 1988). The calcium ions probably interact directly with the transport sites or affect the transport affinity of ion-exchange systems (McDonald et al. 1983).

Whilst Cherax in higher-Ca²⁺ acid water showed only transient changes in haemolymph [Na⁺] and carapace [Ca²⁺], exposure of Cherax to acid water containing only low calcium concentrations (50 μmol l⁻¹) resulted in both net increases in haemolymph [Ca²⁺] and reductions in [Na⁺]. It is thus at the expense of ion balance that Cherax maintains acid–base homeostasis over a very large range of [Ca²⁺], such that even in high-Ca²⁺ acid water the disturbance to blood pH is minimal.

With respect to acid–base state Cherax continued to behave to some extent as if it were in soft water even when exposed to 0.5 mmol l⁻¹ Ca²⁺. There is evidence that CaCO₃ is mobilised in some crayfish species during acid-exposure at water Ca²⁺ concentrations of 100 μmol l⁻¹ (DiStefano et al. 1991; Wood and Rogano, 1986), but this study cannot conclude whether CaCO₃ mobilisation is important for buffering in water containing either 50 or 500 μmol l⁻¹ Ca²⁺, since there was no evidence for metabolic alkalosis.

Cherax appears able to maintain acid–base homeostasis in acidic environments which have proved challenging to both European and North American crayfish species. It seems likely that water [Ca²⁺] must decrease significantly below 50 μmol l⁻¹ before Cherax will exhibit the perturbations observed in other species.

The decrease in haemolymph was substantial, especially within the first 30 min of exposure (mean decreases, 4.4 mmol l⁻¹ venous; 4.1 mmol l⁻¹ arterial), but with only a transient hypercapnia at 5 h and no significant change in pH. The obvious hypocapnic alkalosis in the haemolymph of Cherax exposed to pH 8.0 was primarily respiratory in origin, while the reduction in total CO₂ content is clearly the result of an addition of acid equivalents to the haemolymph, although no lactacidosis was evident in Cherax (Ellis and Morris, 1995). Any initial respiratory alkalosis of the haemolymph must decrease the haemolymph [HCO ⁻] capacity. If HCO ⁻ cannot leave the haemolymph, then must rise. In either case, CO₂ excretion either by HCO₃⁻/Cl⁻ exchange or by diffusion of gaseous CO₂ is encouraged. Subsequent fluctuations in the acid–base status of Cherax were only small, even after 96 h. Consequently, Cherax made rapid respiratory and metabolic adjustments in the face of alkaline stress to maintain a relatively constant haemolymph pH at the cost of decreased [HCO₃⁻] and appeared to maintain this for at least 4 days.

The 36 % increase in haemolymph [Na⁺] after only 2 h in alkaline water may reflect a loss of H⁺ to the relatively alkaline water. The haemolymph pH of Cherax destructor in alkaline water increased, however, by only 0.1 unit above that in control animals. The longer-term maintenance of haemolymph Ph could be by subsequent active pumping of H⁺ into the haemolymph (metabolic acidosis). Increased HCO₃⁻/Cl⁻ exchange would explain the decreased bicarbonate levels of the animal and the net increase in haemolymph Na⁺ levels. Rainbow trout also demonstrate a significant decrease in haemolymph [HCO₃^-] when exposed to pH 10.1 (Yesaki and Iwama, 1992). The haemolymph Na⁺ and Ca²⁺ concentrations show similar trends and follow a time course much like that of the pH, suggesting major changes in strong ion difference during the alkaline exposure. All these variables tend to return towards initial values after 24 h in pH 8.0 water, such that ion balance is re-established without loss of acid–base regulation.

The maintenance of a steady-state pH in alkaline water despite respiratory disturbances to the acid–base status suggests a strong buffering capacity by both intra-and extracellular compartments. Investigation of intracellular pH during environmental pH challenge must be a high future priority. Further work is required to determine the source of the sustained metabolic acidosis, since there was no obvious contribution from lactic acid, while the elevation of both [Na⁺] and [Ca²⁺] appears to implicate major changes in strong ion difference.

We would like to thank Robert and Cheryl McCormack of Crayhaven Aquacultural Industries for the supply of animals and their support during this study. We would also like to thank Agnieszka Adamczewska for her assistance in processing the haemolymph samples. This project was supported by a University of Sydney Research Grant and the Australian Research Council.