Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential

Aquatic Toxicology 51 (2000) 259 – 272 www.elsevier.com/locate/aquatox

Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential M.W. Beaumont *, P.J. Butler, E.W. Taylor School of Biosciences, The Uni6ersity of Birmingham, Edgbaston, Birmingham B15 2TT, UK Received 17 November 1999; received in revised form 27 February 2000; accepted 29 February 2000

Abstract Brown trout acclimated to soft water and exposed for 96 h to a sub-lethal concentration of copper at low pH (0.08 mmol l − 1 Cu, pH 5) have a lower critical swimming speed than fish from copper-free water at neutral pH. This loss of performance is not due to difficulties in oxygen transfer resulting from gill damage since arterial oxygen and carbon dioxide levels remain unaffected. Both red and white muscle showed some metabolic disruptions consistent with local hypoxia, namely a high lactate concentration at rest and, in the white muscle, depletion of glycogen and phosphocreatine. However, a putative role of increased blood viscosity following haematological changes in reducing the supply of oxygen to the tissues is not supported by the current study. Haematocrit, haemoglobin and plasma protein concentrations were not affected by this treatment and a lack of further change in variables such as lactate at the onset of exercise led one to look for an alternative explanation for the effects of copper and low pH upon tissue metabolites. Ammonia concentration, both in the plasma and muscles, is significantly higher in trout exposed to copper and low pH. Ammonia plays a role in the regulation of a number of metabolic pathways and could contribute to the altered metabolic status of these fish. In addition, ammonium ions are known to cause electrophysiological disruptions, particularly the displacement of K+ in ion exchange mechanisms that could lead to the observed loss of swimming performance. Using the measured distribution of ammonia between intracellular and extracellular compartments to estimate membrane potential of resting muscle, a significant depolarisation is predicted in both red and white muscle of fish exposed to copper and low pH. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Copper; Low pH; Ammonia; Swimming; Brown trout; Salmo trutta

1. Introduction

1.1. Background * Corresponding author. Tel.: + 44-121-4143822; fax: +44121-4145925. E-mail address: [email protected] (M.W. Beaumont).

A reduction in the swimming performance of fish following aqueous exposure to a pollutant, is often attributed to disruption of oxygen transfer

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at the gill or to a decrease in the oxygen carrying capacity of the blood. However, although exposure to sub-lethal low pH alone (pH 4.0 – 4.5) and to low pH and copper together (pH 5, 0.08 – 0.47 mmol l − 1 Cu) reduces the critical swimming speed (Ucrit; Brett, 1964) of brown trout, Salmo trutta, there is little evidence that branchial oxygen transfer is the limiting factor (Butler et al., 1992; Butler and Day, 1993; Beaumont et al., 1995a). Three possible alternative hypotheses have been proposed to explain the observed reduction in swimming performance (Beaumont et al., 1995a,b). The first suggested a decrease in the ability of the fish to deliver oxygen to the locomotory muscles due to increased blood viscosity. Sub-lethal changes in blood viscosity might affect exercise by causing a reduced cardiac output, principally due to reduced stroke volume (Randall and Brauner, 1991), or could affect the local circulation of blood through the peripheral capillaries (Wells and Weber, 1991). In brown trout, both low pH and copper exposure caused changes in mean arterial blood pressure (MABP), haemoglobin concentration ([Hb]) and protein concentration ([Pro]) consistent with the possibility of haemoconcentration (Butler et al., 1992; Beaumont et al., 1995a). However, these alterations were not repeatable between treatments, for example sub-lethal pH at 15°C caused elevations in [Hb] and [Pro] but no change in MABP while at 5°C there were smaller changes to [Hb] and no change to [Pro] but MABP was significantly elevated (Butler et al., 1992). The second hypothesis relates to a consistent hyperammonaemia following exposure to these pollutants. Plasma ammonium ion concentration ([NH+ 4 ]) rose by up to 7.5 times in trout exposed to copper in acidic water and a significant, negative correlation was found between Ucrit and 2 [NH+ 4 ] with an r of almost 0.7 (Beaumont et al., 1995b). One probable factor in this hyperammonaemia is an elevation of ammonia production arising from the ‘stress-response’, a mechanism that ensures the provision and reallocation of energy to defend homeostatic equilibrium (Wendelaar Bonga, 1997; van Weerd and Komen, 1998) and this may reasonably be expected to influence the animals capacity for exercise.

However, while the changes in hepatic metabolism, mediated initially by catecholamines and chronically by cortisol, are relatively well characterised (van Weerd and Komen, 1998), the stress response in muscle is less well described. Whether or not a stress response leads directly to significant alteration in muscle metabolism, the inability of copper and low pH exposed trout to rid themselves of the ammonia load may itself be a factor in the loss of swimming performance. Ammonium ions themselves play an important regulatory role in a number of biochemical processes. The stimulation of glycolytic flux due to the effect of NH+ 4 ions as an allosteric activator of phosphofructokinase-1 (PFK) (Sugden and Newsholme, 1975) and the possible inhibitory effect of the same ion upon pyruvate carboxylation (Zaleski and Bryla, 1977) may increase the rate of flux through the glycolytic pathway depleting stored glycogen and, furthermore, disrupt its regeneration. Thus the second hypothesis, was that hyperammonaemia has the potential to cause significant impairment of the anaerobic capacity of the locomotory muscle in addition to any effect of stress related metabolic reorganisation. The third hypothesis also arose from the observation of plasma hyperammonaemia. Ammonium ions have the potential to interfere with central or peripheral nervous activity, with transmission at the neuromuscular junction and with excitation/ contraction coupling and muscle electrophysiology (see Beaumont et al., 1995b for discussion). In particular, ammonium ions are able to replace potassium ions in exchange mechanisms which can result in the depolarisation of neurons and muscle fibres (Binstock and Lecar, 1969). In a study of the effect of ammonium ions on frog sartorius muscle, Heald (1975) attributed a decrease in the twitch tension of the whole muscle to a progressive loss of fibres that had become electrically inexcitable which, he concluded, was due to the depolarisation of muscle membrane potential (EM). The third hypothesis was that hyperammonaemia, arising from copper and low pH exposure, may lead to reduced swimming ability due to impairment of the electrophysiological coordination of muscle function.

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1.2. The current study Of the three hypotheses that were put forward (localised hypoxia, metabolic disruption, electrophysiological impairment), the first two may have similar consequences. The main aim of the current study was to determine whether and to what extent the metabolism of locomotory muscle is disrupted by exposure to copper and low pH. Consequently, as a first step in identifying the underlying cause of any observed disruption, measurements of several indicators of haematological status and of red muscle citrate concentration and white muscle PFK activity have been taken. Although there are already similar measurements of the former from trout exposed to copper in acidic water (Beaumont et al., 1995a), as mentioned earlier, these were not repeatable between treatments. With the aim of reducing the complications of temperature and seasonal interaction (see Day and Butler, 1999), the current investigation was conducted at a single temperature (10°C), in the middle of the thermal range of the brown trout, and with a standard photoperiod (12L:12D). Since ammonium ions directly influence isocitrate dehydrogenase and PFK, changes in citrate concentration and PFK activity should be good indicators of a role for hyperammonaemia. While it was not the aim of the current study specifically to investigate the third hypothesis (electrophysiological impairment), the measurement of the distribution of ammonia between the intra- and extracellular compartments has allowed some discussion of this aspect of hyperammonaemia with respect to muscle membrane potential.

2. Materials and methods

2.1. Animal husbandry The animal handling and experimental protocol were similar to those employed in earlier studies (Butler et al., 1992; Beaumont et al. 1995a,b). Adult brown trout, S. trutta (L), (mass, 300 – 600 g) were acclimated to the experimental temperature (1090.2°C; mean9S.E.M.) and soft water

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conditions (composition range, mmol l − 1; Ca2 + , 45–56; Na+, 69–77; K+, 4–6; Mg2 + , 34–48; Cl−, 88–109; SO24 − , 62–68; NO− 3 , 4–7) for a minimum of 4 weeks in continuously flowing water. Following the insertion of a catheter into the dorsal aorta (Soivio et al., 1972), the fish were allowed to recover in the swimming flume for 48 h prior to the commencement of the 96 h experimental exposure regime. Twelve experimental animals were exposed to 0.08 mmol l − 1 copper at pH 5 (CLP) and 12 trout were left in the acclimation water (pH 7, no added copper), as controls. Throughout this period, there was a continuous flow of water through the flumes at a rate of 3 l min − 1. The appropriate pH was maintained by titration with 5% NaOH or 5% H2SO4. Copper was added constantly from a stock solution of CuCl2 to maintain the desired concentration which was regularly monitored by aniodic stripping voltammetry (Radiometer POL150 polarograph with a hanging-drop mercury electrode and Tracemaster 5 software) which, under these conditions, had an experimental detection limit of approximately 0.01 mmol l − 1. In the control, artificial soft water, copper concentration was always below this detection limit.

2.2. Swimming performance After 96 h of exposure, the Ucrit of six fish from each group was determined using the same protocol as in previous experiments (Butler et al., 1992; Beaumont et al., 1995a). An arterial blood sample was taken at Ucrit via the dorsal aortic cannula and analysed as below. Blood samples were also taken from the six remaining trout in each group at rest. Following removal of the arterial blood sample, each fish was rapidly anaesthetised with Saffan (Mallinckrodt Veterinary, Uxbridge) to avoid struggling, and subsequently killed by concussion. Tissue samples (red and white muscle) were excised and ‘freeze-clamped’ using aluminium tongs pre-cooled in liquid nitrogen. The sample was then put into a foil envelope and stored in liquid nitrogen until analysis.

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2.3. Blood analysis Immediate measurements from blood samples included arterial oxygen partial pressure (PO2), using a Cameron BGM200 blood gas system thermostatically controlled to 10°C; oxygen content (CO2), using a Lexington Instruments Lex-O2Con; haematocrit (Hct), using a Hawksley microhaematocrit centrifuge; plasma pH, using the BGM200 and plasma carbon dioxide concentration (CCO2), using a Corning 965 CO2 analyser. Concentrations of haemoglobin ([Hb]), plasma lactate ([Lac]), total ammonia ([Tamm]: [NH3]+ [NH+ 4 ]), glucose and protein ([Pro]) were measured, within two hours of sample collection, using Sigma kits nos. 525, 826-A, 171, 115A and P5656, respectively. The concentrations of plasma catecholamines (adrenaline and noradrenaline) were determined using reverse-phase, ion-pair HPLC with electrochemical detection (Butler et al., 1989). Plasma cortisol concentration was measured using a commercially available RIA (Immuchem, ICN Pharmaceuticals) with standards adjusted to reflect the protein concentration of trout plasma. The concentrations of sodium ([Na+]) and potassium ([K+]) in the plasma were measured using a Pye Unicam SP9 atomic absorption spectrophotometer. Plasma chloride concentration ([Cl−]) was measured with an Aminco chloride titrator.

2.4. Tissue analysis The tissue was ground to powder under liquid nitrogen using a mortar and pestle. Tissue ammonia was measured using the glutamate dehydrogenase method of Kun and Kearney (1974). Glycogen and free glucose were measured in PCA extracted tissue homogenates using the protocol described by Keppler and Dekker (1974) and tissue lactate using that of Gutmann and Wahlefeld (1974). Phosphocreatine (PCr), ATP and glucose 6 phosphate (G6P) were measured in the coupled assay system described by Lamprecht et al. (1974). Pyruvate analysis was performed following the methods of Czok and Lamprecht (1974). The concentration of citrate in red muscle samples was determined using citrate lyase (Da-

gley, 1974). All spectrophotometric analysis was performed at 25°C using a Shimadzu UV-160A spectrophotometer. PFK activity was determined in muscle following homogenisation (20 mM potassium phosphate, 15 mM 2-mercaptoethanol, 5 mM EDTA, 5 mM EGTA, 1 mM phenyl-methyl-sulfonyl fluoride) using standard assay conditions for trout PFK (final volume 1 ml containing 20 mM imadzole buffer at pH 7.2, 0.15 mM b-NADH, 5 mM MgCl2, 3.4 mM ATP, 0.16 mM fructose-6-phosphate, 2 U aldolase, 2 U triosephosphate isomerase, 1 U a-glycerophosphate dehydrogenase; Su and Storey, 1995). Ammonia was removed from the auxiliary enzymes by centrifugation through Sephadex G25. Enzyme activity at 10°C was determined from the rate of change in absorbance at a wavelength of 340 nm recorded using a Shimadzu UV-160A spectrophotometer with a CPS-240A thermal control unit. Protein concentration in the homogenate was measured with Coomassie blue (Sigma 610-A). To investigate the effect of additional ammonia in the assay cuvette, muscle homogenates from one of the resting control trout were also assayed for PFK activity in the same conditions as above but with the addition of 2 and 5 mmol l − 1 NH4Cl. Tissue intracellular pH (pHi) was determined using the metabolic inhibition method of Po¨rtner et al. (1990) and using the Cameron BGM200 blood gas system at 10°C. For determination of white muscle ion concentrations, samples were digested for 24 h in a 1:10 mass:volume dilution with 1 mol l − 1 nitric acid. The concentrations of sodium ([Na+]) and potassium ([K+]) in the neutralised supernatants were measured using a Pye Unicam SP9 atomic absorption spectrophotometer. Chloride concentration ([Cl−]) was measured in the same samples with an Aminco chloride titrator. Intracellular and extracellular fluid volume (ICFV and ECFV, respectively) were measured using tritiated polyethylene glycol ([3H]PEG, Dupont) which was dissolved in Young’s teleost saline (Hale, 1965) and injected via the cannula into the dorsal aorta at a rate of 0.925 MBq kg − 1 body mass. On the basis of trials previously conducted in the laboratory of equilibration times for [3H]PEG in rainbow and brown

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trout tissues (Taylor, S.E., Mair, J.L., Beaumont, M.W., unpublished data), these injections were made 18 h prior to the termination of the experiment. Plasma and tissue samples were solubilised in ‘Optisolv’ (LKB Scintillation Products), neutralised with glacial acetic acid and added to ‘HiSafe 3’ scintillation cocktail (LKB Scintillation Products). Radioactivity was counted using a Beckman LS 1701 counter against a pre-prepared quench curve for trout tissue. Samples of tissue were also dried to a constant weight at 70°C to determine water content.

2.5. Calculations Free (NH3) and ionised ammonia (NH+ 4 ) concentrations in water and plasma were calculated from the Henderson – Hasselbalch equation: [NH+ 4 ]=

Tamm ´ 1+ 10pH − pK

[NH3]= Tamm − [NH+ 4 ] Values of pK% were estimated from the nomogram of Cameron and Heisler (1983). The NH3 and CO2 concentrations and appropriate solubility coefficients (aNH3, aCO2 determined from Cameron and Heisler (1983) and Boutilier et al. (1984)) were used to calculate the partial pressures of ammonia (PNH3) and carbon dioxide (PCO2). Extracellular fluid volume was calculated from the ratio of disintegrations per minute (DPM) measured in tissue and plasma samples and intracellular fluid volume by subtraction from total tissue water. ECFV(ml g − 1)=

DPMtissue g − 1 wet weight DPMplasma ml − 1

ICFV (ml g − 1)= total tissue water −ECFV

2.6. Statistics Results are presented as mean9 S.E. Significant effects were determined using one- or twoway analysis of variance (ANOVA) and corrected Bonferroni post hoc tests as appropriate performed using Systat software (Statsoft). Where

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the data deviated from normal, appropriate logarithmic or arcsine transformations were applied prior to analysis.

3. Results Swimming performance was significantly lower (Ucrit 1.119 0.2 bl s − 1, n= 6) in the CLP fish compared to that of the untreated controls (Ucrit 2.029 0.1 bl s − 1, n= 6). This sub-lethal level of copper and acid was without apparent effect upon the ability of fish to extract oxygen from the water as indicated by the maintenance of both arterial oxygen partial pressure and content (Table 1). While there was a small but significant effect of exercise upon arterial PCO2 (ANOVA P= 0.037), there was no significant difference in this variable between control and CLP exposed fish. There was no effect of exposure to copper in acidic water upon resting plasma lactate concentration and there were also no effects of this treatment upon plasma protein concentration, haematocrit, haemoglobin concentration or MCHC (Table 1). There was no significant difference between levels of adrenaline and noradrenaline measured in fish from control conditions (respectively 4.29 0.7, 3.690.9 nmol l − 1, n= 3) in comparison to those of CLP exposed fish (respectively 5.59 0.6, 3.890.9 nmol l − 1, n= 4). Plasma cortisol concentration was significantly elevated in CLP exposed fish in comparison to their appropriate controls (CLP 53.19 16.9 ng ml − 1, control 8.69 1.9 ng ml − 1), while exercise had no significant effect under either experimental condition (CLP 52.49 19.3 ng ml − 1, control 14.09 4.4 ng ml − 1). Plasma [Tamm] was higher in fish exposed to copper and acid for 4 days. Levels in CLP trout at rest were greater by almost 5 times in comparison to control fish at rest and the exercised group had approximately 8 times more circulating ammonia than counterparts in neutral water with no copper (Fig. 1). The mean plasma [Tamm] of CLP exposed trout that had been exercised to Ucrit was also significantly greater than that of the CLP trout at rest. There was no significant effect of exercise upon plasma [Tamm] between the control groups. In the muscle, there were similar changes in

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Table 1 Blood respiratory, haematological and metabolic data from groups of brown trout at rest or after exercise to critical swimming speed and following 96 h of exposure to either circum-neutral, copper-free water or to water containing 0.08 mmol l−1 Cu at pH 5a

Oxygen partial pressure, PO2 (kPa) Oxygen content, CO2 (mmol l−1) Carbon dioxide content, CCO2 (mmol l−1) Carbon dioxide partial pressure, PCO2 (kPa) Haemoglobin concentration, [Hb] (g dl−1) Haematocrit, Hct (%) Mean corpuscular haemoglobin, MCHC Plasma protein (mg ml−1) Plasma pH Plasma lactate (mmol l−1) Plasma glucose (mmol l−1)

Control (pH 7, no copper)

CLP (pH 5, 0.08 mmol l−1 Cu)

Rest

Exercise

Rest

Exercise

14.79 0.5 4.5 90.3 8.6 9 0.9 0.279 0.02 7.8991.04 24.192.3 0.339 0.03 37.393.5 7.979 0.04 0.679 0.07 4.39 0.4

15.3 91.0 4.2 9 0.8 6.9 9 1.3 0.36 9 0.07 8.35 9 1.11 25.3 92.6 0.57 90.09 35.2 94.2 7.67 9 0.11 2.4090.60*** 5.99 2.5

15.3 9 0.7 3.8 90.5 7.0 9 0.5 0.23 90.02 7.56 90.55 23.1 92.4 0.33 9 0.02 30.5 9 2.9 7.95 90.04 0.75 90.11 25.7 9 5.7‡

16.2 90.5 5.8 9 1.0 7.3 9 0.9 0.45 9 0.13 9.5 9 0.65 28.3 9 1.3 0.34 9 0.02 37.8 92.2 7.75 90.09 1.49 9 0.50‡ 18.4 9 6.3‡

Values are means 9 S.E.M. * Significant effect of exercise in a given water quality. ‡ Significant effect of copper and low pH upon resting or exercised trout. One, two or three symbols signify PB0.05, 0.01 or 0.001 respectively. a

[Tamm] (Fig. 1). After correction for extracellular contamination, mean red muscle [Tamm] was 7.149 0.93 and 7.6091.19 mmol l − 1 ICF in resting and exercised CLP trout respectively, i.e. between 2 and 3 times that of control trout. CLP trout at rest had a mean white muscle [Tamm] of 5.009 0.62 mmol l − 1 ICF which is also over double that of control fish. However, exercised control trout had a significantly higher white muscle ammonia concentration than trout from the same conditions at rest, while there was no difference in [Tamm] of this tissue between resting or exercised CLP trout. Red muscle pHi did not differ significantly from the resting control value of 7.18 9 0.05 except in the exercised CLP animals, in which the red muscle became more alkaline (Table 2). Under control conditions, an acidosis occurred in the white muscle at Ucrit, but no change was observed in the CLP trout that were exercised (Table 2). Lactate levels in red muscle were 2.4 times greater in resting CLP fish than in those from neutral, copper free water (Table 2). However, while red muscle lactate in exercised trout from the latter conditions was almost 3-fold greater than those at rest, CLP fish swum to Ucrit had a mean red muscle lactate concentration no higher

than the resting level of CLP trout. Pyruvate concentration in red muscle from CLP trout was significantly greater by some 40–75% than that of control fish but was unaffected by exercise in

Fig. 1. Total ammonia concentration, [Tamm], measured in the plasma, white and red muscle of brown trout sampled at rest or after exercise to Ucrit and following 96 h of exposure to either circa-neutral, copper-free water (control) or to water containing 0.08 mmol l − 1 Cu at pH 5 (CLP). Values are means9 S.E.M (n =6) and units are mmol l − 1 for plasma and mmol l − 1 ICF for tissues. For explanation of symbols, see Table 1.

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Table 2 Metabolite concentrations in red and white muscle of brown trout at rest or after exercise to critical swimming speed and following 96 h of exposure to either circum-neutral, copper-free water or to water containing 0.08 mmol l−1 Cu at pH 5a Control (pH 7, no copper)

CLP (pH 5, 0.08 mmol l−1 Cu)

Rest

Exercise

Rest

Exercise

White muscle Glycogen Glucose Glucose 6 phosphate intracellular pH Lactate [Lac] Pyruvate [Pyr] [Lac]/[Pyr] Adeonsine triphosphate Phosphocreatine

31.49 3.5 1.14 9 0.12 1.61 9 0.15 7.25 9 0.03 7.6390.32 0.079 0.02 128.79 11.4 12.6190.55 16.63 9 2.48

27.4 9 8.3 2.33 9 0.66 3.43 9 0.14* 6.83 9 0.11* 18.77 9 1.68* 0.11 90.02 177.2 920.6 9.84 91.51 4.85 91.22**

15.2 9 2.5‡ 1.26 9 0.16 1.46 90.51 7.17 9 0.04 12.8 92.36 0.11 90.02 122.1 99.9 11.48 9 0.30 8.99 9 2.11‡

21.0 9 1.8 2.4790.62 2.08 9 0.49 7.0290.09 12.43 9 1.10‡ 0.099 0.02 155.0 918.7 10.21 91.11 6.60 9 3.05

Red muscle Glycogen Glucose Glucose 6 phosphate intracellular pH Lactate [Lac] Pyruvate [Pyr] [Lac]/[Pyr] Adenosine triphosphate Phosphocreatine Citrate

26.3 9 1.8 1.3 9 0.2 1.18 9 0.30 7.18 9 0.05 2.1590.25 0.1290.01 18.79 1.7 7.33 90.70 2.8190.59 0.8090.03

19.5 9 1.5 1.69 0.3 1.20 9 0.14 7.18 9 0.05 6.29 90.80*** 0.149 0.02 45.6 95.8*** 5.88 9 0.95 1.56 9 0.57 0.60 90.04*

26.2 91.8 1.6 90.3 1.03 9 0.35 7.15 90.06 5.15 90.97‡ 0.21 90.01‡ 23.2 9 3.3 6.36 90.79 2.71 90.68 1.12 90.06‡

23.9 91.9 1.4 9 0.4 0.9690.30 7.42 9 0.07*,‡ 5.02 9 0.80 0.20 90.01‡ 24.7 9 3.5‡ 6.089 0.68 2.07 9 0.49 1.00 90.05‡

Values are means 9 S.E.M and units are mmol g−1 tissue wet weight. * Significant effect of exercise in a given water quality. ‡ Significant effect of copper and low pH upon resting or exercised trout. One, two or three symbols signify PB0.05, 0.01 or 0.001 respectively. a

either water quality (Table 2). Consequently, the ratio of lactate and pyruvate concentration increased only in the red muscle of exercised trout from control conditions. White muscle lactate concentration was affected in a similar manner to that of red muscle by exposure to copper and low pH, although levels in CLP trout at rest were quite variable and therefore not significantly different from those of control fish (Table 2). The lactate concentration of white muscle from exercised trout in neutral water was again almost 3-fold higher than that of resting fish and, as for red muscle, this difference between resting and exercised fish was absent in CLP trout. White muscle pyruvate was unaffected by either exercise or CLP exposure (Table 2). While analysis of variance indicates that the ratio of [Lac]:[Pyr] is significantly larger in trout that

have been exercised (P = 0.016), the pattern of variation is such that differences between individual group means are not revealed by post hoc tests. Mean glucose concentration in both muscles was unaffected by exposure to copper at low pH or by exercise, although CLP trout had a significantly higher plasma glucose concentration than trout from control conditions (Table 2). The glycogen concentration in the white muscle of resting CLP trout was half that of trout from neutral copper free water while mean red muscle glycogen was no different between any of the groups (Table 2). Mean ATP concentration also did not differ in either red or white muscle and PCr in red muscle was similarly unaffected by any treatment (Table 2). However, there were some significant differences between treatment groups

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Fig. 2. Concentration of sodium, chloride and potassium ions in (A) plasma and (B) white muscle of brown trout sampled at rest following 96 h of exposure to either circa-neutral, copperfree water (control) or to water containing 0.08 mmoll − 1 Cu2 + at pH 5 (CLP). Values are means 9S.E.M (plasma n=6; white muscle n = 4). For explanation of symbols, see Table 1.

in mean PCr concentration of the white muscle. Both exercised and CLP exposed trout had a lower PCr concentration in this tissue in comparison to that of resting control fish. CLP exposed fish had a small but significantly higher level of citrate in their red muscle (Table 2). Ion concentrations were measured in the white muscle of four resting control fish and the same number of CLP trout at rest. In these fish, there were no significant differences between exposure groups in plasma Na+, Cl− or K+ concentration or in white muscle Na+ (Fig. 2). White muscle [K+] was some 15% lower in CLP fish whilst [Cl−] was 2-fold higher. PFK activity is shown in Fig. 3. White muscle PFK activity was significantly greater than that of the red muscle (ANOVA PB 0.1%). There was no significant difference in PFK activity due to any treatment. However, the addition of ammonia directly to the assay did result in elevated activity levels. This was particularly marked in the white muscle sample which had a PFK activity of 0.40 U mg − 1 protein with no added ammonia and 0.90 and 1.70 U mg − 1 protein with 2 and 5 mM NH4Cl respectively. The effect of added ammonia on the red muscle sample was smaller (PFK activity 0.33, 0.39 and 0.50 U mg − 1 protein with 0, 2 and 5 mM NH4Cl, respectively).

4. Discussion

Fig. 3. Phosphofructokinase (PFK) activity in red and white muscle of brown trout sampled at rest or after exercise following 96 h of exposure to either circa-neutral, copper-free water (control) or to water containing 0.08 mmol l − 1 Cu at pH 5 (CLP). Values are means 9 S.E.M. Symbols (§) indicate a significant difference between red and white muscle. One or two symbols signify P B 0.05, or 0.01 respectively.

Just as at 5°C in winter and 15°C in summer (Beaumont et al., 1995a), swimming performance of brown trout acclimated to 10°C and a constant 12L:12D photoperiod was significantly lower in conditions of low pH and copper. The current study also confirmed the apparent absence of an effect of this level of copper and low pH upon the ability of the fish to extract oxygen from the water as indicated by the maintenance of both arterial oxygen partial pressure and content (Table 1). As CCO2 and, particularly, PCO2 were no higher in CLP exposed fish than in control trout there is no evidence for a branchial limitation to the diffusion of either respiratory gas.

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4.1. Tissue hypoxia In highly aerobic tissues such as red muscle, there is normally no change in the carbohydrate stores or energy charge during mild environmental hypoxia (Dunn and Hochachka, 1986). However, when challenged with a level of hypoxia that is severe or nearly lethal, these tissues are unable to maintain their energy status, and glycogen, ATP and PCr concentrations all fall (Johnston, 1975; van Raaij et al., 1994). Since all of these factors were similar in the red muscle of resting CLP trout compared to those of control fish, it was concluded that this tissue did not face severe hypoxia under these conditions. However, red muscle lactate was higher in CLP fish in comparison to that in control trout. Since there was no corresponding increase in circulating lactate concentration, this is most likely endogenously derived from increased anaerobic metabolism and could indicate the presence of some moderate constraint upon the delivery of oxygen to the mitochondria in the red muscle. Resting lactate concentration was also higher in white muscle of CLP fish and concentrations of glycogen and PCr were lower compared to those in control fish. White muscle has a high capacity for anaerobiosis and, even during near fatal hypoxia, both ATP and energy charge are maintained through elevated glycogenolysis and the use of PCr energy stores (Dunn and Hochachka, 1986; van Raaij et al., 1994). A moderate level of tissue hypoxia could therefore be the cause of the differences (elevated lactate, lowered glycogen and PCr) between the white muscle of CLP and control trout at rest. Thus it seems that the muscle metabolite data from resting trout support the hypothesis that CLP exposed trout could be subject to a moderate level of localised tissue hypoxia. However, in neither tissue is the lactate:pyruvate ratio increased thus implying the maintenance of oxidative balance. Moreover, one might expect a limitation in oxygen delivery due to CLP exposure to be reflected in further increases in lactate following exercise i.e. when a ‘functional hypoxia’ is superimposed on any existing problem. Lactate concentration was no higher in either muscle of CLP

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trout despite, in the case of the white muscle, being below that normally seen after exhaustion at Ucrit. The red muscle lactate concentration of the resting CLP fish was already at a similar level to that of control fish exercised to exhaustion, although extreme exhaustion induced by chasing may cause considerably higher increases (Day and Butler, 1996). PCr concentration was similarly no lower in either muscle of exercised CLP trout in comparison to CLP trout at rest. More significantly perhaps, the osmoregulatory and haematological data from this experiment do not support the hypothesis that increased blood viscosity is the basis of CLP induced tissue hypoxia. In the current study, there were no changes in plasma ion concentrations and nor were there changes in Hct, [Hb] or [Pro] associated with exposure to copper at low pH and it seems unlikely that blood viscosity increased significantly. Moreover, Gallaugher et al. (1995) have now shown that, despite a positive relationship between Hct and viscosity, elevation of Hct up to 55% actually increases the Ucrit of rainbow trout.

4.2. Metabolic consequences of CLP exposure With the first hypothesis, local hypoxia, rejected, the next question is whether the metabolic changes observed in CLP exposed fish might have arisen from a generalised stress response. Hyperglycaemia, as exhibited by the CLP trout, is often considered to be indicative of such a response and plasma cortisol was also elevated in these animals. While mean liver glycogen concentration was not different from that of the control group, these levels are notoriously variable in fish (e.g. Wright et al., 1989) and the current result may simply reflect the lack of knowledge of the status of liver glycogen in individual fish prior to the start of the experiment. With respect to the response of muscle metabolism to stress, it seems that elevated cortisol alone does not cause significant changes in muscle glycogen concentration (Mu¨ller and Hanke, 1974) and its glucocorticoid effects are aimed toward the modulation of hepatic gluconeogenesis and chronic maintenance of energy stores (Wendelaar Bonga, 1997). In the present study, although muscle lactate concentration in

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CLP exposed fish tended to be higher and white muscle glycogen concentration significantly lower compared to those of control fish, red muscle glycogen concentration was not significantly different and no difference in catecholamine concentration was detected to account for these changes. It is possible that, since the adrenergic response of fish is usually very rapid and transitory (Randall and Perry, 1992), catecholamine concentrations may have returned to pre-exposure levels by the time the plasma was sampled after 96 h of exposure. Glycogen depletion in the white muscle could have occurred in this initial response and reserves remained low due to the suppression of glycogen synthase activity by cortisol (Milligan, 1997) which was elevated in CLP exposed fish. Additionally, the inability of CLP exposed fish to rid themselves of their ammonia load may deepen the consequences of any hormonally regulated stress response perhaps shifting the balance from adaptive to maladaptive. While there was no significant difference between PFK activity of muscle from control or CLP exposed trout, despite the significantly elevated ammonia concentration measured in the latter tissues, this observation may be an artefact arising from the dilution of the tissue ammonia in the assay cuvette. With ammonia added to the assay medium at approximately the concentrations observed in control (2 mM) and CLP exposed (5 mM) fish, PFK activity was elevated (Fig. 3). This activation was greatest in the white muscle where PFK activity was almost doubled. Ammonia also inhibits pyruvate carboxylase, thus, in the liver at least, impairing the synthesis of glycogen through gluconeogenesis (Zaleski and Bryla, 1977). PCr concentration in the white muscle of CLP trout was lower than that of control trout at rest which may represent the cost of maintaining ATP concentration in the face of some futile cycling in the glycolytic/gluconeogenic pathway. Unlike that in white muscle, glycogen concentration in red muscle was not significantly lowered by CLP exposure although lactate concentration was elevated. What, therefore, is the source of the lactate in red muscle and why is glycogen not depleted by an ammonia-stimulated increase in glycolytic flux? Perhaps glycolytic flux may not be

increased in red muscle despite an increase in [Tamm]. Storey (1991) found that PFK extracted from the liver of rainbow trout was not significantly activated by NH+ and, in the current 4 study, addition of ammonium ions to muscle in vitro had less effect on the PFK activity of red muscle than of white. Red muscle PFK may have more similarity to the liver isozyme than to that of white muscle in this respect and so glycolysis in red muscle is relatively insensitive to hyperammonaemia. A second, not necessarily exclusive, possibility is that red muscle is using an alternative to stored glycogen as a fuel source. Red muscle has a greater hexokinase activity than that of white muscle (Knox et al., 1980) and a considerable scope for an increase in uptake and utilisation of circulating glucose above that normally found in unstressed resting fish (West et al. 1993). CLP trout did show a significant plasma hyperglycaemia and it is possible that, at rest, the red muscle of these fish is using this exogenous source of glucose to maintain glycogen stores and fuel an increased flux through the glycolytic pathway. Under aerobic conditions, the products of glycolysis in the red muscle would normally be expected to be completely oxidised to CO2 by the TCA cycle and not to accumulate as lactate. However, the first step in this process is the oxidative decarboxylation of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex (PDHC) which can be inhibited in the presence of high ammonia (Katunuma et al., 1966). In the current study, CLP exposed fish had significantly higher red muscle pyruvate and lactate concentrations than control trout, which may indeed reflect an impaired transfer into the TCA cycle. Exhaustive exercise is powered by the breakdown of glycogen to lactate and incremental speed tests such as Ucrit induce a significant anaerobic component. Burgetz et al. (1998) showed that rainbow trout are required to use anaerobic metabolism to support swimming speeds greater than 70% of their Ucrit and, in the brown trout at least, white muscle fibre recruitment begins at even lower speeds (Day and Butler, 1996). The loss of white muscle glycogen stores occurred also in trout exposed to low pH alone (Day and Butler, 1996) and an inability to recruit these

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fibres could account for the loss of swimming performance. Carbohydrates are a relatively minor fuel for the aerobic red fibres (Moyes and West, 1995) and the oxidation of substrates such as amino and fatty acids does not involve the glycolytic pathway but insert directly into the TCA cycle. There is, however, some scope for impaired metabolic function in red muscle since ammonium ions have the potential to directly regulate TCA cycle activity as an inhibitor of isocitrate and a-ketoglutarate dehydrogenases (Katunuma et al., 1966; Lai and Cooper, 1991) and also indirectly through inhibition of enzymes involved in the malate-aspartate shuttle responsible for the provision of reducing equivalents for the TCA cycle and the regeneration of cytosolic NAD+ (Ratna Kumari et al., 1986; Lai and Cooper, 1991). In the present study, citrate in red muscle of CLP trout was slightly, but significantly, higher than that of control fish at rest. The current study, therefore, provides evidence to support the second hypothesis of a disruption of the energetic status of the locomotory muscle and a possible role of hyperammonaemia in that disruption. From the present experiment, one cannot identify which of the factors (‘stress’ or hyperammonaemia) is most significant nor demonstrate a simple link between the energetic status of the fish and its swimming performance. Future experiments involving the infusion of ammonia into fish not previously exposed to copper may be productive in this respect.

4.3. Tissue ammonia and muscle membrane potential The ratios of [Tamm] between intracellular and extracellular fluid ([Tamm]i/[Tamm]e) of trout at rest and in control conditions were 28.49 6.1 and 33.6 97.8 for white and red muscle, respectively. Similarly high values have been found by several authors (e.g. Wright et al., 1988; Wright and Wood, 1988; Tang et al., 1992) and this suggests that ammonia is distributed in these tissues according to their membrane potential, EM (see Wood, 1993 for review). In this case, EM can be determined by using the observed distribution of NH+ 4 and the Nernst equation (Eq. (1)). Using

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this approach results in estimates of − 83.09 5.9 and − 79.39 5.5 mV for red and white muscle EM, respectively. These values fall within the range of vertebrate and more specifically fish muscle measured under resting, steady state conditions (e.g. Altringham and Johnston, 1988). EM = −

RT [NH+ 4 ]i ln zF [NH+ 4 ]e

(1)

where R is the gas constant, T the temperature, F the Faraday constant, z the valency and the subscripts i and e denote the intracellular and extracellular concentrations respectively. The mean [Tamm]i/[Tamm]e ratios of the red and white muscle of CLP exposed trout at rest (15.39 2.1, 10.59 1.2, respectively) are significantly lower than the observed ratios of control fish. Since there are no differences in plasma, red or white muscle pH of control or CLP trout at rest, there can be only two explanations for this decrease in [Tamm] ratio; either there has been a change in the relative permeability of the membrane for the ammonium ion or the voltage gradient across the membrane decreased. In the latter case, it is possible to estimate the size of the depolarisation from the distribution of ammonia and the Nernst equation. The calculated mean membrane potential of white muscle from CLP exposed trout at rest is − 57.49 3.2 and − 65.69 3.0 mV for red muscle. Depolarisation may account for the poor swimming performance of these fish due to a loss of fibres recruited for swimming activity. In frog muscle fibres, Jenerick (1959) reported the onset of loss of electrical excitability at values above − 60 mV with complete loss from − 55 to − 45 mV (Jenerick, 1956), an effect probably arising from the inactivation of the voltage-gated sodium channel. Using frog sartorius muscle, Heald (1975) demonstrated the functional significance of such a depolarisation, showing that there was a reduction in twitch tension as fibres became electrically inexcitable. That this was a membrane phenomenon was demonstrated by the presence of contractures in fibres treated with caffeine or in which outward current was injected. The higher estimates of voltage gradient in red muscle from CLP exposed trout (i.e. less depolarised) in comparison to those of the white muscle may provide

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the answer to the question of why, during exposure to low pH alone, red muscle apparently continues to function despite an increase in ammonia, while white muscle does not (Day and Butler, 1996). However, there has been some theoretical criticism of the hypothesis of distribution according to EM due to the additional stress placed upon cellular pH regulation by proton cycling (Heisler, 1990). Moreover, [Tamm]i/[Tamm]e has not been found to be high in all studies (e.g. Mommsen and Hochachka, 1988) and recently it has been suggested that the situation may be more plastic, in that the pH gradient has the dominant effect upon ammonia distribution at rest and the effect of muscle membrane potential is dominant following exercise, i.e. that the relative membrane permeability to NH3 and NH+ 4 can change under some circumstances (Wang et al., 1994, 1996). The effect of CLP exposure upon muscle membrane potential requires further investigation. To conclude, the supply of oxygen to the exercising tissues has often been thought of as the limiting factor in fish exposed to metals or to low pH, either due to the effects of these pollutants upon the ability of fish to extract oxygen from their environment or to transport oxygen in the circulatory system. However, at the sub-lethal combination of low pH and copper toxicity used in these studies, swimming performance is impaired and yet such a limitation to oxygen supply does not seem to exist. It has been previously proposed that loss of swimming performance may instead may arise either from metabolic changes as a consequence of a general stress response exacerbated by hyperammonaemia and/or due to electrophysiological disruption, also a result of elevated ammonia (Beaumont et al., 1995b). In the current study, evidence is presented that adds further support to both these hypotheses. It is unclear, however, why this increased ammonia load is not simply excreted. The low pH of the environment should enhance the excretion of ammonia by simple diffusion of NH3 across the gill (due to ‘’ammonia-trapping’) and the absence of effects upon the respiratory gases make a branchial diffusional limitation seem unlikely. This aspect of the effect of exposure to copper

and low pH is currently under investigation in a separate study in the laboratory. Preliminary data show that while ammonia accumulates in the plasma by 7–9-fold, its excretion rate only doubles at most.

Acknowledgements This project was supported by the Natural Environment Research Council.

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