Exposure of brown trout Salmo trutta to a sublethal concentration of copper in soft acidic water: effects upon gas exchange and ammonia accumulation
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* Author for correspondence (e-mail: p.j.butler{at}bham.ac.uk)
Accepted 2 October 2002
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Summary |
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The accumulation of ammonia in the plasma and white muscles during exposure to CLP has already been implicated in reducing the swimming performance of brown trout. Inhibition of cortisol synthesis abolished a large proportion of the increases in both the accumulation and excretion of ammonia that occurred during the second 48 h of the exposure to CLP, but did not inhibit ammonia accumulation completely. It is suggested that CLP not only causes an increase in the rate of production of ammonia, which is enhanced when the level of cortisol starts to increase after 48 h, but that it also inhibits an excretory mechanism (most probably Na+/NH4+ exchange) that is non-obligatory under `normal' conditions (when passive diffusion is sufficient), but is required in order to respond to unusually high ammonia loads.
Key words: copper, low pH, ammonia, gas exchange, swimming, brown trout, Salmo trutta
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Introduction. |
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Exposure to sublethal levels of copper and low pH together can also result
in gill damage, but to a lesser extent. Brown trout Salmo trutta L.,
exposed for 96 h to 0.08 µmol l-1 Cu2+ in acidic
(pH5) softwater, were found to have some minor structural deformations of the
secondary lamellae, but there was no significant difference in the harmonic
mean diffusion distance compared with fish from control conditions
(Taylor et al., 1996). While
swimming performance of fish exposed to copper and low pH was lower than that
of control fish, their rate of oxygen consumption
(
O2) while
routinely active was no different, and there were no differences in the oxygen
content or partial pressure of the arterial blood of exercising fish, which
suggests the absence of an underlying diffusional limitation
(Beaumont et al., 1995a
). A
putative role of increased blood viscosity following haematological changes in
reducing the supply of oxygen to the tissues is unsupported by the
haematological evidence. Plasma sodium, chloride, potassium, haematocrit,
haemoglobin and plasma protein concentrations were not affected by CLP
exposure, there was a lack of further change in variables such as lactate at
the onset of exercise (Beaumont et al.,
2000a
), and the balance of evidence points away from oxygen
delivery being a significant limiting factor during exercise. However, maximum
O2
(
O2max) was not
measured and there were some changes in muscle metabolic status that could be
interpreted as evidence of a degree of hypoxic stress
(Beaumont et al., 2000a
).
A likely alternative is that hyperammonaemia arising from exposure to
copper and low pH is the primary cause of the loss of swimming performance
(Beaumont et al.,
2000a,b
;
Shingles et al., 2001
).
Ammonia is a toxic end product of various metabolic processes, in particular
the hepatic transdeamination of amino acids
(Walton and Cowey, 1982
) and,
during episodes of stress, its production may be stimulated by an elevation in
the circulating levels of catecholamines and cortisol
(Wendelaar Bonga, 1997
;
van Weerd and Komen, 1998
). In
freshwater fish, most (approximately 90%) excretion of ammonia occurs across
the gills, with the remainder being excreted renally or cutaneously (see
Wood, 1993
). The actual
mechanisms of branchial ammonia excretion remain somewhat controversial,
however. Possible routes include the passive diffusion of NH3 or
NH4+, or an active exchange of
NH4+ against a counter-ion such as H+ or
Na+ (Wilson and Taylor,
1992
).
It is generally agreed that, in freshwater fishes, transepithelial
NH4+ diffusion is an unlikely option due to the relative
impermeability of the gill epithelium to the ammonium ion, whereas the passive
diffusion of NH3 is normally accepted as an important pathway (e.g.
Wood, 1993;
Wilkie, 1997
). The question of
an active excretory mechanism has been a matter of argument, however, not
least because of the absence of a method to differentiate between the movement
of NH3 together with a proton and of the NH4+
ion alone. Despite numerous attempts to establish the relative importance of
the various putative mechanisms for the clearance of ammonia across the gills
of fish, the matter is still largely unresolved (e.g.
Cameron and Heisler, 1985
;
Evans and Cameron, 1986
;
Randall and Wright, 1987
).
However, recent opinion seems to be that under most conditions, passive
diffusion of NH3 can account for almost all branchial ammonia
excretion (e.g. Wilkie, 1997
)
with a small role perhaps for a non-obligatory
Na+/NH4+ exchange
(Salama et al., 1999
).
The present study
The present study was undertaken to investigate a number of aspects of gas
exchange over the gills of brown trout exposed to sublethal copper and low pH.
Specifically, the aim was to address two questions. Firstly, what is the
effect of these pollutants upon
o2 at different levels of
exercise? Secondly, why does ammonia accumulate within these fish when the low
external pH should favour `ammonia trapping' (the conversion of the permeable
NH3 to the impermeable NH4+ ion), thus
maintaining the gradient for the diffusion of this toxic waste product out of
the fish as NH3 (Lin and
Randall, 1991
)? The rate of oxygen consumption during exercise was
measured in a computerised swimming respirometer using fish fitted with
Transonic flow-probes in order that cardiovascular changes could also be
recorded. Ammonia fluxes and the pattern of ammonia accumulation within trout
exposed to copper and low pH were measured, and the role of cortisol
investigated by the use of metyrapone, which inhibits cortisol synthesis
(Milligan, 1997
).
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Materials and methods |
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Rate of oxygen consumption and swimming experiments
Fish were anaesthetised with MS-222 (100 mg l-1, buffered to pH
7.5 with NaHCO3). Once ventilation had ceased, the fish were
transferred to the operating system where the gills were irrigated
continuously with aerated, buffered anaesthetic (50 mg l-1). In a
total of eight fish, the ventral aorta was exposed by making an incision
within the opercular cavity where the vessel could be clearly seen running
under the skin. Connective tissue was eased apart and a factory-calibrated
flow probe (Transonic Systems Inc, Ithaca, NY, USA) placed around the ventral
aorta. The probe was sutured in place and the overlying skin also sutured back
into place. To ensure that the wound was sealed and that the probe would not
move, a polythene patch was secured with polyacrylamide glue over the wound.
The lead from the probe was led from the operculum under the pectoral fin and
up to the dorsal fin. It was secured to the fish by further sutures.
The fish was put into clean water and allowed to begin recovery before
being placed into the respirometer flume. This is a swimming respirometer
developed at the University of Birmingham from an original design provided by
Dr John Steffensen (Steffensen et al.,
1984). Data acquisition and automatic control of the flume were
achieved through a computer with a Lab-PC+ interface card and running software
written in the LabWindows/CVI environment (National Instruments). The fish was
sealed in a plastic tunnel (maximum cross-sectional area 289 cm3)
through which there was a flow of water generated by a propeller and variable
speed, d.c. motor. The water flow was adjusted by shaped honeycomb sections
both before and after the swim chamber, in order to minimise turbulent flow at
all test velocities. The respirometer had a total volume of 80 litres and was
mounted in a larger tank in which the water is cooled to 10±0.2°C
and replaced at 3 l min-1. The respirometer can be converted from
an open to closed system for measurement of oxygen depletion. Between
measurements, water was pumped from the outer tank through the respirometer.
Water was sampled by continual gravity feed from the swim chamber and drawn
past an oxygen electrode (Strathkelvin Instruments 781 meter and 1302
microcathode electrode) also maintained at 10°C.
After 24h, automatic oxygen consumption measurements were begun. For each
measurement, the water pump was switched off, sealing the respirometer. The
pump was restarted before water oxygen saturation in the respirometer fell
below 90%. The software calculated the decline by regression analysis and
stored the value along with goodness of fit. The software then calculated and
stored the rate of oxygen consumption using the appropriate oxygen solubility
coefficient (Boutilier et al.,
1984) and the fish mass.
After a total of 48 h post-operative recovery, exposure to the test water
was begun. For four trout this was 0.08 µmol l-1 copper at pH5
(CLP), and for the remaining four fish, the acclimation water at pH7 was used,
with no added copper (control). At 96h of exposure, critical swimming speed
(Ucrit; Brett,
1964) was determined using intervals of 0.5 m s-1 and
time periods of 45 min to ensure that several respirometry measurements could
be made at each speed. Data from the blood flow probe were time-stamped and
recorded continuously so that they could be analysed in conjunction with those
from the respirometer flume.
Ammonia accumulation and flux
A catheter was inserted into the dorsal aorta of 16 fish
(Soivio et al., 1972). In six
of these, a catheter was also implanted in the ventral aorta. Cannulae were
made of PE-50 tubing and, in both cases, the cannulae were inserted through
the mouth. The ventral aorta is covered by a layer of cartilage through which
a hole was first made using a needle with its tip removed. Cannulae were
secured with sutures and polyacrylamide glue. The ventral aorta cannulation
had a relatively poor success rate, with some fish losing a significant amount
of blood during the operation. Such trout were rejected from the
experiment.
Trout were placed in polyacrylamide flux boxes of volume approximately 2.5
litres, constructed following a design provided by Dr Gordon McDonald
(McDonald and Rogano, 1986).
Pumps circulated water through each flux box and a stream of air bubbles
ensured adequate mixing and aeration. The fish were allowed to recover for 48h
prior to the commencement of the 96h experimental exposure regime. Five trout
with only a dorsal aortic catheter, and six with both dorsal and ventral
aortic catheters, were exposed to CLP, and five fish, with dorsal aorta
catheter only, were left as controls in the acclimation water only. Other than
during the flux measurements, there was a continuous flow of water through the
flux boxes at a rate of 3 l min-1. Copper was added constantly from
a stock solution of CuCl2 to maintain the desired concentration in
the experimental water. This was regularly monitored by aniodic stripping
voltammetry (Radiometer POL150 polarograph with a hanging-drop mercury
electrode and Tracemaster 5 software), which under our experimental
conditions, had an experimental detection limit of approximately 0.01 µmol
l-1. In the control, artificial softwater, copper concentration was
always below this detection limit. The appropriate pH was maintained by
titration with either 5% NaOH or 5% H2SO4.
Flux measurements were made at 24h intervals. At the beginning of each
measurement period, the pumps were switched off and a 20 ml water sample taken
from each box. Three further samples were taken at intervals of 30-60 min. The
pH of each sample was measured immediately (Radiometer PHM72 meter with
Russell CT757 low-conductivity electrode) and ammonia concentration measured
using a micro-modification of the salicylate method
(van Verdouw et al.,
1978).
Blood samples (0.5-1.0 ml) were taken and haematocrit (Hct) was measured
immediately, using a Hawksley micro-haematocrit centrifuge, in order to
monitor blood loss. Fish were eliminated from the experiment if Hct fell below
20% of the initial, `normal' values (see
Beaumont et al., 2000a). The
remaining sample was centrifuged at 9,000 g in order to
separate plasma and red blood cells, which were subsequently resuspended in
saline and reinjected into the fish. Plasma pH was determined using a Cameron
BGM200 blood gas system thermostatically controlled to 10°C. Plasma carbon
dioxide concentration was determined using a Corning 965 carbon dioxide
analyser calibrated with high precision standards (MultiCal, Ciba-Corning).
Plasma total ammonia concentration [Tamm]
([NH3]+[NH4+]) was measured, within 2 h of
sample collection, using Sigma kit no. 171. The remaining plasma was frozen at
-70°C for later analysis of cortisol concentration by RIA (ICN
immunochem).
Free (NH3) and ionised ammonia (NH4+)
concentrations in water and plasma were calculated from the
HendersonHasselbalch equation:
![]() | (1) |
![]() | (2) |
Cortisol inhibition
Catheters were implanted into the dorsal aorta of six trout, which were
subsequently placed into flux chambers as described above (Ammonia
accumulation and flux). After 48 h recovery, metyrapone (Sigma P856525) was
injected through the catheter (3 mg 100 g body mass-1 in
Courtland's saline) prior to the initiation of CLP exposure. Ammonia flux
measurements, analysis of a plasma sample (for pH, [Tamm]
and cortisol levels), and a further metyrapone injection, were made after each
24 h over a 96 h exposure period.
Ammonia infusion
Three fish had catheters inserted into their dorsal aorta and were allowed
to recover for 48 h in control water in the flux chambers. Ammonia fluxes were
measured and a blood sample taken and analysed for Hct, plasma pH and plasma
[Tamm]. Ammonia was then infused into each animal
via the dorsal aorta. The infusion consisted of 0.5 mol
l-1 NH4HCO3 in Courtland's saline, which was
infused at a rate of 1 ml h-1. After 24 h, ammonia flux was
measured once more and a further blood sample taken for analysis. To avoid
contamination with infusate, the first 1 ml of blood sampled was rejected and
the second sample analysed.
Results are presented as means ± S.E.M. Unless otherwise stated, significant effects were determined using one- or two-way analysis of variance (ANOVA) and corrected, as appropriate, by post-hoc Bonferroni tests, or by univariate repeated-measures analysis using Systat software (Statsoft Inc). Where data did not have a normal distribution, appropriate logarithmic or arcsine transformations were applied prior to the analyses.
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Results |
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Cardiovascular parameters
In the control trout, cardiac output at Ucrit was
increased by a significant 2.6 times from the routine rate
(Table 1). This was achieved by
a significant 2.4 times increase in heart rate while stroke volume remained
unchanged (Table 1). Routine
cardiac output did not change significantly in trout exposed to CLP
(P=0.13; Table 1) in
comparison to that in control fish. At Ucrit, cardiac
output of CLP exposed trout had increased significantly but was significantly
less than that of control fish at Ucrit (P=0.05).
However, at any given speed, the mean cardiac output of CLP trout was not
significantly different from that of control fish
(Fig. 2). The increase of
cardiac output in CLP exposed fish when exercised was achieved by a
significant, 1.8-fold increase in heart rate, while stroke volume was
unchanged from that at rest (Table
1).
|
Ammonia accumulation and flux
Mean plasma [Tamm] of control fish remained unchanged
during the experiment, while in CLP exposed trout, plasma
[Tamm] increased by almost sixfold from the pre-exposure
level of 130.1±27.1 to 776.9±116.5 µmol l-1
(N=5, P=0.004; Fig.
3). The rate of increase in plasma [Tamm] in
CLP exposed trout from 48 h to 96 h (9.6±1.5 µmol l-1
h-1) was significantly greater (P<0.05) than that from
-0.5 to 48 h (5.8±1.4 µmol l-1 h-1).
|
Net ammonia excretion in trout exposed to control conditions did not change significantly throughout the exposure, ranging from 162.3±15.3 to 105.7±26.7 µmol N kg-1 h-1 (N=5). In those trout exposed to CLP, net excretion rose to a peak at 72 h (429.4±71.6 µmol N kg-1 h-1). After 96 h of exposure to the pollutants, ammonia excretion had declined but was still more than threefold greater than that of control fish (320.0±33.8 µmol N kg-1 h-1, P>0.001; Fig. 3).
The increase in plasma [Tamm] from the dorsal aortae of the six fish with catheters implanted in both the dorsal and ventral aortae followed a similar pattern during CLP exposure to that previously observed in fish with only a single catheter in the dorsal aorta (Fig. 4). In these fish, ammonia accumulated at a rate of 7.44±0.89 µmol N l-1 h-1 (r2=0.96, P=0.004) in the dorsal aorta. [Tamm] in plasma from the ventral aorta was some 50-80 µmol l-1 greater than that from the dorsal aorta and remained so throughout the exposure. This difference was significant (P<0.001). The accumulation rate of ammonia in the plasma of the ventral aorta was 7.58±0.87 µmol N l-1 h-1 (r2=0.96, P=0.003). In the dorsal aorta of control trout, plasma carbon dioxide concentration remained constant throughout the experiment at a mean of 11.1±0.3 mmol l-1. CLP exposure caused no significant difference in plasma carbon dioxide concentration from either aortae (Fig. 4).
|
Plasma cortisol levels were not significantly elevated with respect to pre-exposure values (Fig. 5) until after 48 h of exposure to CLP, when there was a tripling of the mean level (from 4.3±1.5 to 12.6±2.7 µg dl-1, P=0.008, N=5). This peaked at 72 h, with values in some individuals of 20 times the pre-exposure level (mean: 57.8±15.4 µg dl-1, N=5), and remained high at the end of the experiment (49.4±11.1 µg dl-1, N=5).
|
Cortisol inhibition
Trout exposed to CLP but given daily injections of metyrapone displayed no
change in plasma cortisol concentration during the 96 h
(Fig. 6). At the end of the
experiment, plasma cortisol concentration (7.0±3.3 µg
dl-1, N=5) was no different from that of trout kept under
control conditions (see Beaumont et al.,
2000a). However, both plasma [Tamm] and
ammonia efflux were elevated in these animals. Net ammonia efflux had
increased to a similar level after 96 h as that observed in CLP exposed trout
with no metyrapone injections (290.2±50.5 µmol NH3
kg-1 h-1 with metyrapone and 320.0±33.8 µmol
NH3 kg-1 h-1 without metyrapone). However,
the peak in ammonia excretion at 72 h was absent from metyrapone treated fish.
Plasma [Tamm] in the metyrapone treated fish rose during
exposure to almost 3.5 times its pre-exposure levels (from 169.3±33.7
to 459.0±40.6; P<0.001, N=6), but this was still
less than two-thirds that of the untreated, CLP exposed animals (see
Ammonia accumulation and flux).
|
Ammonia infusion
After 24 h of infusion, net ammonia excretion had risen by fivefold to
832.1±40.5 µmol NH3 kg-1 h-1 (from
163.2±20.0 µmol NH3 kg-1 h-1;
N=3, P=0.003) and plasma ammonia concentration more than
doubled from 112.1±9.1 to 298.7±12.1 µmol l-1.
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Discussion |
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Ammonia accumulation and excretion
Exposure to CLP caused ammonia to accumulate in trout and, since ammonia
excretion in these fish is greater than that of trout in control conditions,
it is also apparent that this is in some part a consequence of an increase in
ammonia production. From the current study, it is clear that there are two
phases in the accumulation of ammonia in CLP exposed trout. There is a steady
accumulation, which started within the first 24 h of exposure, continued
throughout the experiment and did so even in the absence of a change in plasma
cortisol concentration (Fig.
6). After 48 h, there is an acceleration in ammonia accumulation
associated with an increase in plasma cortisol levels. This latter phase was
absent when cortisol production was inhibited. If the bulk of ammonia is
excreted across the gills by passive diffusion of NH3, then its
rate of excretion
NH3 can be
described by Fick's equation in terms of diffusive conductance and the
difference in partial pressure:
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It is worth noting that the delayed increase in plasma cortisol in the CLP
fish is probably related to the fact that they were kept in soft water. Brown
et al. (1989) reported that
exposure of brown trout to acid (pH 4.0-4.6) conditions in water with a high
concentration (2.8 mmol l-1) of calcium caused a significant
increase in plasma cortisol concentration within 2 days, which returned to the
baseline level within 7 days. However, the opposite occurred in fish kept in
acid water with a low concentration (0.05 mmol l-1) of calcium;
there was no change in plasma cortisol concentration within 2 days, but there
was a large, 20-fold, increase within 7 days. Brown et al.
(1989
) discuss the possible
reasons for the difference.
Control conditions
Over the 96 h exposure period, GNH3 of trout
in control conditions declined from 37.9±4.9 to 16.4±5.4 µmol
N kg-1 h-1 mPa-1. This occurred due to a fall
in NH3 without a
corresponding change in
PNH3 (from
4.6±0.78 to 6.2±0.86 mPa). Fish were unfed during these
experiments and there was probably an associated decline in ammonia
production, which would account for the fall in excretion
(Brett and Zala, 1975
).
Conductance of NH3 depends upon available surface area and
epithelial thickness as well as the permeability of the gill to ammonia. At
rest, some 40% of the gill lamellae may be redundant
(Booth, 1979
). In addition to a
lower effective surface area, blood flow may be distributed to the basal
channels of the lamellae (Farrell et al.,
1980
), which tend to be buried in filamental tissue. During
activity, there is not only an increase in effective surface area of the
gills, but their epithelium may also be stretched to accommodate the increased
thickness of the vascular sheet, thus causing a narrowing of the diffusion
distance between blood and water (Farrell
et al., 1980
). As activity declines, therefore, a decrease in
effective surface area and an increase in epithelial thickness might lead to a
decrease in GNH3, accounting for a lower rate
of excretion in the absence of a change in
PNH3.
CLP exposure with cortisol inhibition
After 96 h, GNH3 of trout treated with
metyrapone and exposed to CLP was 15.4±2.8 µmol N kg-1
h-1 mPa-1, half that of the pre-exposure value of
31.4±5.2 µmol N kg-1 h-1 mPa-1.
This is similar to the change that occurred in control trout. Unlike the
situation in control trout, PNH3 rose in
the CLP/metyrapone fish (from 6.2±1.24 to 19.0±0.49 mPa), but
was not matched by a proportional increase in
NH3. Since
oxygen consumption was unchanged by 96 h exposure to CLP/metyrapone, one might
speculate that perfusion of the gill follows the same pattern as that of
control trout. The observed excretion rates are indeed similar to those
predicted by the Fick equation using the appropriate daily mean
GNH3 from control trout
(Fig. 7).
|
CLP exposure with cortisol production
In trout exposed to CLP alone, the difference between the magnitude of the
rise in PNH3 (from 5.3±0.62 to
32.7±5.29 mPa) and of the increase in ammonia excretion was larger
still. GNH3 fell by a third from
34.1±5.6 to 10.9±1.9 µmol N kg-1 h-1
mPa-1. Moreover, since data for cardiac output and the difference
in arterio mixed venous [Tamm] are available for
trout exposed to this treatment, it is possible to use the Fick principle of
convection to calculate the rate of branchial ammonia excretion
(Table 2):
![]() | (4) |
|
On the basis of this change in the route of ammonia excretion,
GNH3 of CLP exposed trout at 96 h was
7.6±1.3 µmol N kg-1 h-1 mPa-1, less
than a quarter of the pre-exposure value and considerably lower than that of
control fish. In trout exposed to CLP alone, observed excretion rates were
considerably lower than those predicted from the Fick equation using the
appropriate daily mean GNH3 from control trout
(Fig. 7). Both copper and low
pH exposures can induce gill damage (e.g. hyperplasia, increased mucus
secretion, epithelial thickening and vacuolation), which could result in
greater diffusion distances and hence a decreased
GNH3. However, the levels of copper and low pH
used in the present study have produced no evidence of such damage in previous
investigations (Taylor et al.,
1996). It also seems unlikely that NH3 diffusion would
be affected in this manner in the absence of similar effects upon the
exchanges of oxygen and carbon dioxide (Figs
2 and
4).
The infusion experiments demonstrate that trout, unexposed to pollutants,
have the capacity to elevate the net rate of ammonia excretion by at least
fivefold in response to an infusion of ammonia at a rate that was equivalent
to a sixfold increase in ammonia production. It is clear that CLP exposed fish
do increase ammonia excretion, but not sufficiently to prevent accumulation.
It is interesting to examine the magnitude of the discrepancy. In the present
study, ammonia was found to accumulate in the plasma of these fish at a rate
of 7.17 µmoll-1 h-1. This is just 1-3% of the
excretion rate. Given that ammonia accumulates in other tissues, data from
previous studies (Beaumont et al.,
2000a) can be used to estimate the rates of accumulation in red
and white muscle as 31.56 and 20.52 µmol N kg-1 h-1.
Using measurements of various tissues as percentages of body mass from the
same studies and overestimating accumulation in the remainder of the tissues
by using the value for red muscle, whole body ammonia accumulation rate is at
most 24.2 µmol N kg-1 h-1. Even this overestimate
represents only 5-10% of the amount of ammonia being excreted at any given
time and demonstrates that, whatever the mechanism of the effect of exposure
to CLP, it has a relatively minor effect upon instantaneous excretion rates,
but becomes important over time.
In conclusion, previous studies (Beaumont et al.,
1995a,
2000a
) have inferred an absence
of an effect of CLP exposure upon the branchial uptake of oxygen from an
absence of effect upon arterial oxygen partial pressure and content. The
present study provides evidence of a corresponding lack of effect upon the
oxygen transport system. Thus, in answer to the first questioned posed, heart
rate, cardiac output and oxygen consumption of CLP exposed trout are no
different from those of control fish, at any given level of activity. It is
argued that maximum rates are not achieved in CLP exposed fish due to an
absence of demand. The answer to the second question is that hyperammonaemia,
a possible factor in the loss of exercise performance, arises from two phases
of ammonia production. There is a steady increase that occurs in the absence
of cortisol production. Once the changes in branchial diffusive conductance
observed in control fish during the experiment are taken into account, this
ammonia accumulation may simply represent the readjustment of equilibrium, an
increase in plasma ammonia to increase the difference in
PNH3 and, therefore, ammonia excretion by passive
diffusion. The second phase of accumulation, associated with a rise in
cortisol, does not lead to the level of excretion that would be predicted from
simple passive diffusion. Ammonia infusion experiments show that control fish
can respond to such an ammonia load. This observation may indicate the
presence of an excretory mechanism (most probably
Na+/NH4+ exchange) that is non-obligatory in
`normal' conditions but that is required (and inhibited by CLP exposure) in
order to respond to unusual ammonia loads.
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Acknowledgments |
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References |
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