J. exp. Biol. (1976), 64, 159-171
K
I en
ith 3 figures
inted in Great Britain
ACID-BASE BALANCE IN RAINBOW TROUT (SALMO
GAIRDNERI) SUBJECTED TO ACID STRESSES
BY F. B. EDDY*
Research Unit for Comparative Animal Respiration, University of Bristol,
Bristol BS8 1UG, England
(Received 26 June 1975)
SUMMARY
1. The respiratory properties of rainbow-trout blood were investigated in
acid-stressed fish. In the first group acid was introduced into the bloodstream
and in the second the carbon dioxide content of the ambient water was
increased.
2. Initially the introduction of acid to the blood caused a decrease in blood
pH and bicarbonate, and increases in oxygen uptake and ventilation volume.
After 2-3 h these values had returned to the control levels.
3. Trout subjected to high ambient CO2 (about 10 ramrlg) showed a
decrease in blood pH while P c o and bicarbonate increased. After 8 h the
trout began to show signs of compensation to the acidosis.
4. In each experiment the blood P o was little changed but blood Oa
content was decreased and tended not to resume the control value even after
several hours.
5. The results are discussed in terms of the various acid-base mechanisms
thought to be available to the fish. These include branchial ion exchanges
and the possible buffering roles of the extracellular and intracellular fluids.
INTRODUCTION
Detailed mechanisms of acid-base homeostasis in the lower vertebrates are poorly
understood. In the past this subject has received little attention but in recent years
there have been a number of significant studies dealing with the effects of temperature
on the body fluid composition and hydrogen ion balance in various poikilotherms
(Rahn, 1967; Howell, Baumgardner, Bondi & Rahn, 1970; Reeves, 1972).
There have been a considerable number of studies on the in vitro respiratory and
acid-base properties of fish blood (reviewed by Albers, 1970) but the number of in
vivo studies remains, as yet, relatively small. The greater bulk of the work has been
directed towards the elasmobranchs (Piiper & Baumgarten, 1969; Piiper, Meyer &
Drees, 1972; Murdaugh & Robin, 1967; Cross, Packer, Linta, Murdaugh & Robin,
1969; Payan & Maetz, 1973), where some important features in the control of body
fluid composition have been elucidated. In vivo studies of teleost acid-base balance
• Present address: Department of Biological Sciences, University of Dundee, Dundee DDi 4HN,
Scotland.
160
F. B. EDDY
are few though the subject is beginning to attract interest (Cameron & Randall, 1972^
Randall & Jones, 1973; Randall & Cameron, 1973; Cameron & Polhemus, 1974).
In water breathers there are a number of mechanisms whereby the composition of
the body fluids can be regulated. The most important both in fresh water and in
marine fish is thought to be an active and selective exchange of ions between blood
and water at the gill surface, i.e. Na+/H+, Na+/NH4+ and CI-/HCO3- (Maetz, 1971);
obviously the rate of exchange or excretion of these ions could influence the acid-base
status of the blood and body fluids. In most cases the teleost kidney appears to be of
minor importance in this respect.
The bicarbonate concentration and the -Pco, values of fish blood are much lower
than the corresponding values for air breathers. The main reason for this originates
with the high water solubility of COa when compared with that of O2; since the ratio
of CO2 to O2 solubility in water at 15 °C is approximately 30, it follows that the volume
of water passing over the gills sufficient for the uptake of 1 ml O 3 is capable of dissolving about 30 ml CO2. Thus there is a tendency for CO2 to be 'washed out* from
the gills and the resulting blood tensions are low, the P c o gradient between blood
and water being only a few mmHg. This gradient could be increased by a reduction
in ventilation but this would be accompanied by a reduction in O2 uptake. Consequently the bicarbonate buffering system in water breathers is rather weak when
compared to the situation in air breathers.
In the present experiments rainbow trout were exposed to various acid stresses
and the role of blood in buffering was investigated.
MATERIALS AND METHODS
Rainbow trout weighing between 300 and 600 g were obtained from a hatchery at
Nailsworth, Gloucestershire. They were held in 50 gal tanks containing dechlorinated
water at 15 °C and were fed on commercial trout pellets with chopped ox heart.
Trout were anaesthetized with MS 222 (Sandoz) and were then transferred to an
operating table where the dorsal aorta was cannulated by the method of Smith & Bell
(1964); the ventral aorta, the operculum and the buccal cavity were cannulated as
described by Holeton & Randall (1967). The blood cannulae were filled with heparinized fish saline (Wolf, 1963). The fish were allowed to recover for 24 h before the
commencement of an experiment.
The fish was placed in a respirometer through which air-saturated water, temperature 15 °C, flowed at a known rate, usually about 1 1/min. In the first series of experiments involving ten trout, control values were recorded for 2-3 h and then CO2 was
introduced to the respirometer water supply at a controlled rate so that the P c o of
the inflowing water increased to about 10 mmHg after 2 h and then remained
steady. Further measurements were made for the next 4-5 h and in some cases after
24 h. The second series of experiments involved six trout; after control measurements
had been made, about ioo/t-equiv. dilute hydrochloric acid, pH value in the range
pH 3-5, was slowly introduced to the efferent bloodstream. This process normally
took 1-2 min so that a sudden decrease in blood pH was unlikely. Then further
measurements were made for the next 4-5 h.
Acid-base balance in rainbow trout
161
Measurement of blood and water samples
The P O j , P c o , and pH of blood and water samples in the cannulae were determined
using Eschweiler microelectrodes mounted in Eschweiler microelectrode cuvettes
where the temperature was maintained at that of the respirometer, 15 ±1 °C. The
pH electrode was calibrated using Pye Ingold buffers.
After sampling, any blood remaining in the cannula was returned to the fish; this
was achieved by attaching the end of the cannula to a reservoir of fish saline and then
raising the reservoir until fluid flowed down the cannula. In most cases the interface
between blood and saline was diffuse and fluid was allowed to flow until the cannula
was clear of blood. Thus inevitably a small amount of saline was introduced to the
circulation, but this had no noticeable effect on the fish.
The P o > of water entering and leaving the respirometer together with the water
flow rate through it were used to calculate the oxygen uptake of the fish. Ventilation
volume was calculated using the value for oxygen uptake and the P o of water entering
and leaving the gills.
The oxygen and carbon dioxide content of 30 fi\ blood samples were immediately
determined using the Natelson apparatus. In the second series of experiments blood
samples of about 200 fi\ were removed prior to and after the introduction of acid.
These were divided into two portions, the first being equilibrated at 15 °C in a 20 ml
tonometer with air containing 1 or 7 mmHg P C O j ; the blood was then analysed for
O2 and CO2 with the Natelson apparatus. The equilibration time was usually about
10 min, but on a few occasions further determinations were carried out up to 1 h
later. As was previously found (Eddy & Morgan, 1969; Eddy, 1971), equilibration time,
provided it did not exceed about 3 h, had little effect on the oxygen binding characteristics of the blood. During equilibration the blood is suceptible to dehydration and
care was taken to avoid this by making sure that the gas mixtures were completely
saturated with water vapour and that they passed through the tonometer at the slowest
possible rate. The second portion was immediately analysed for Oa and CO2 to determine the in vivo values for the trout. Using these data, a graph of pH against log Poo,
was constructed and the arterial P COi value could be estimated from the arterial
pH value.
Blood oxygen content (C Oi ) is the amount of oxygen contained in a given sample
of blood, and blood oxygen capacity is here defined as the amount of oxygen contained
in the blood when equilibrated with water saturated air, P Of 150 mmHg, P C o, about
0*4 mmHg. These values are expressed as ml/100 ml (or vol. %). Percentage oxygen
saturation can be calculated:
„.
blood Oo content (vol. %) x 100
O/ oof-
/o M
"
x
7
blood O2 capacity (vol. %) '
The oxygen capacity of trout blood is significantly reduced in the presence of low
CO2 tensions, the Root effect (Root, 1931), and possible reasons for this are discussed
by Riggs (1970).
EXB 64
162
F. B. EDDY
7
3-5
J? x 30
1 I 2-5
1" E 20
'^
10
~ 7-9
7-8
X
a. 7-7
E
90
C 80
-2-
70
50
40
0
1
2
3
4 5 6
Time (h)
7
8
Fig. i. The effect of acid injection (indicated by vertical bar) on therespirationof rainbow
trout The results are from one experiment and arerepresentativeof theresponsesshown by
the majority of fish. Pg, Ventilation volume (ml/min/kg); I^o,. oxygen uptake (ml/min/kg).
RESULTS
The effect of acid on rainbow trout
Upon the introduction of acid to the blood, the trout responded with increases in
both breathing frequency and amplitude. In some cases the fish struggled vigorously
for a few minutes, undoubtedly adding to the stress already imposed, and probably
leading to increased muscle lactic acid which would later appear in the blood. In
salmonids peak blood lactate concentrations were achieved about 2-5 h after severe
exercise and 8-24 h were required for a return to control values (Black, Manning &
Hayashi, 1966). Thus, in the present experiments, it is likely that blood lactate could
be a significant influence on trout acid-base balance. Usually about 1 h after acid
injection thefishwas again quiet. In two cases thefishreacted violently to acid injection
and died about 1 h later; it was, however, possible to take blood samples before death
occurred.
The results of an experiment which shows most of the characteristics of the response
to acid infusion are shown in Fig. 1. The immediate response was an increase in
ventilation volume and in oxygen uptake, and these were in some cases amplified by
struggling. The P o value of arterial blood remained little changed and in one case it
actually increased, this being best explained by the increased ventilation of the gills.
Blood pH decreased significantly soon after acid infusion but resumed to the control
level after several hours, and blood carbon dioxide content (C c o ) showed a similar
pattern. The results of all the experiments are summarized in Table 1, where values
N
=
S.E.
6 (6)
0'12
I2
I
-
Oxygen uptake (ml/min/kg)
-
Par
-
-
-
h
2.34 (8)'
0'22
oh
3274 (6)'
225
Cco,
2+ h
6.4
1-02
3.9-12.3
we
COS
A
Art. pH
2+ h
7.83 (3)
7.78-7.93
0-1
C%
84.4 (9)
6.25
66-1 18
r
Art. pH
7.55 (7)*
7229.87
Pco,
Ventilation volume (ml/min/kg)
pco, = 7
7.51 (9)
7.307.65
\
Blood pH
pCo2
= 7'
Carbon dioxide
6.5 (10)
0.59
2.8-8.3
Pco, = 7
\
Significantly different from controls at 5 % level, or better.
-
-
Mean
S.E.
Mean
Range
-
A
Blood pH
PC~,
Mean
= 0.3
3+ h
1182 (7)
I 82
PCO,
A
CCO,
7.7 (10)
0.89
3-4-13.2
I
L
OwZen
Oxygen content
Pco, =
oh
944 (9)
121
<
art.
I
7.8 (9)
0.69
3.4-10.6
0 s cap.
pco, = I
7'77 (8)
7.62-7'94
CCO,
8.2 (12)
0.64
89-13
Coa
#
pco, = 0.3
8-30 (7)
8.06-8.47
PCO,
84.7 (10)
3.65
75-103
Par
t
Art. pH
7-83 (6)
7.757'88
Range
S.E.
Mean
r
I
Before acid
cap.
I
Blood DH
Pco, =
L
COO,
6-5 (II)*
0.90
2.9-10.6
I
I
A
Pco,= 7
I
-
4.9 (11).
0.54
227.2
Pco, = 7
Oxygen content
Pco, =
Pco, = 0.3
r
7.9 (3)
0.86
6.7-9'6
0 2
After acid
(All results were obtained from in vivo measurements except for those in columns headed Pco, = 0.3, I or 7 mmHg; these were obtained from blood
equilibrated in tonometers at those CO, tensions. Temperature 15 "C. Values for oxygen uptake and ventilation volume are also shown. Po, and Pco,,
mrnHg. 0, capacity and 0, content (CO,), vol. %. Carbon dioxide content (Cco,), m-mole/l.)
Table I . Blood oaygen, carbon dioxide and hydrogen ion concentrations of rainbm trout before and after acid injection
4
s
o*
a
g
5
-
b
2
164
F.B.EDDY
Table 2. Buffering values of rainbow trout blood expressed as the changes in
carbon dioxide content (Cco , m-mole/1) or Pco with pH
(Exp. refers to a period approximately i h after acid injection when blood samples were taken.
Temperature, i 5 °C. Mean, number of determinations and standard error of the mean are indicated.)
Control
Mean
S.E.
-8-47 (6)
0-74
Exp.
Control
- 2 4 8 (6)
-8-56(5)
093
023
Exp.
-2-39
o - i6
for the control and experimental periods are shown. From these data it can be seen
that arterial blood is almost fully oxygen-saturated during the control period but acid
infusion causes a decrease in oxygen content of about 2 vol. %, or a fall in saturation
to about 78%. In contrast, the value for oxygen capacity is little affected by acid
injection (Table 1); this is because air equilibration removes excess CO2 from the
blood, so raising the pH value and creating conditions most favourable for oxygen
uptake. Thus, on this evidence, the in vitro oxygen carrying capacity of blood is little
altered as a result of acid injection to the fish.
Also indicated in Table 1 are the oxygen, carbon dioxide and pH values for blood
equilibrated at 1 and 7 mmHg P OOi - These were used to give a measure of the buffering
capacity of the blood (rfCCOi/dpH) and these values are shown in Table 2. Since whole
blood was used, CQQ includes buffering due to bicarbonate ions, to non-bicarbonate
buffers such as haemoglobin, and also to other forms of carbon dioxide, e.g. carbamino
CO2. Thus the expression dCC0JdpH is not equivalent to, but is approximately, the
'buffer capacity' or 'buffer index' as defined by Conway (1945) and Burton (1973).
When measured about 1 h after acid injection this value, and also the buffering
expressed d log Pf^JdpH, had changed little (Table 2).
The pHICCOt
diagram
The acid-base balance of trout blood can conveniently be represented by the pH/
C c o diagram and this for two fish is indicated in Fig. 2. In the upper diagram the fish
recovered well from acid injection and the response can be divided into three phases
which are shown on the diagram:
(i) The addition of acid leads to a rapid lowering of arterial pH which tends to
decrease blood bicarbonate.
(ii) The resulting metabolic acidosis is accompanied by hyperventilation which
tends to remove CO2 from the blood circulating in the gills and thus cause an increase
in blood pH. Also, if the gills are the main site for ionic regulation there would be
changes in gill ion fluxes resulting in increased excretion of acid and conservation
of base.
(iii) After about 2 h arterial blood pH and CCOi had returned to approximately the
control values. However, arterial blood oxygen content had not resumed the control
value.
Also in Fig. 2 are the results for a fish which did not survive but died about 1 h
after acid injection. In this case the arterial blood pH dropped to about 7-25 and the
Acid-base balance in rainbow trout
10
165
r
8
1 6
£
i 4
7-4
7-6
7-8
8-0
8-2
8-4
7-8
PH
80
8-2
PH
o
E
4
7-2
7-4
7-6
8-4
Fig. 2. The relationship between carbon dioxide content and pH for blood from a rainbow
trout before (upper line) and about 1 h after acid injection (lower line). Open circles, in vivo
carbon dioxide contents; 1, before, 2, soon after (about i h), and 3, 2 h after acid injection.
Lower diagram, the fish died soon after acid injection.
arterial blood, although retaining the same buffer slope (dCC0 /dpH) as the control
period, had lost some 5 m-mole/1 of bicarbonate. Under these conditions the blood
contained very little oxygen and the arterial P C O j was high, calculated to be
approximately 6 mmHg.
In a separate experiment, blood from an uncannulated fish which had been exercised
for about 6 h until exhaustion in a Brett-type respirometer was analysed in the same
way. The results indicated this fish too had lost approximately 5 m-equiv./l of blood
bicarbonate and that arterial pH was estimated to be in the range 7'4-7-6. The acidosis
in this case can be attributed to lactic acid, but the results are similar to those produced
by HC1 injection.
The effect of high ambient CO2 on rainbow trout
In many ways the effect on trout of increasing the environmental P COj to approximately 10 mmHg for long periods was similar to that of acid injection. As was observed
by Van Dam (1938), there were increases in both depth and frequency of respiration;
i66
F. B. EDDY
Fig. 3. The effect of high environmental CO, on the respiration of rainbow trout The results
shown are the mean values for four comparable experiments and indicate the response shown
by the majority of fish. S, Percentage O, saturation of blood; subscript a, arterial, and v,
venous.
in some cases there was vigorous struggling for a few minutes but generally the fish
was quiet again after about -J h. The results of four comparable experiments were
averaged and the results are shown in Fig. 3. Arterial and venous oxygen tensions were
little affected by high COa but the oxygen saturation of the blood showed an initial
decrease, regaining the control value after 24 h. During the control period the venous
P C O i value was higher than the arterial (as was found by Holeton & Randall, 1967),
but in most cases the introduction of 10 mmHg P C O j to the ambient water caused this
situation to be reversed; however, after 24 h a new equilbrium had been reached and
the venous P COt value exceeded the arterial value by 1-2 mmHg. A similar situation
was observed for blood pH where during the experimental period the arterial value
was lower than the venous value (Fig. 3). This suggests that while the blood is in the
gills, little buffering takes place, but once in the tissues some compensation by the
body buffering system occurs, resulting in the removal of hydrogen ions and an
elevated venous pH value. Blood carbon dioxide content increased steadily throughout
Acid-base balance in rainbow trout
167
the experiment, rising from 12-15 rn-mole/1 to more than 20 m-mole/1 after 24 h (a
similar pattern of events was observed by Lloyd & White (1967) and also by Cameron &
Randall (1972)). The averaged results for all fish are shown in Table 3; it is interesting
to note that the P OOi of arterial blood remained slightly higher than that of the environmental water, suggesting that metabolic CO2 is still able to diffuse from the gills and
that the RQ value may be little changed when trout are in water of high COa.
DISCUSSION
In fish the mechanisms of acid-base balance are poorly understood whereas in
mammals these have been studied extensively and much is known about them. In
the mammalian system an acid load to the blood is buffered to some extent by bicarbonate and proteins in the blood plasma, but more important are the blood haemoglobin and the buffers within the body cells. The principle routes for acid excretion
are the lungs where excess CO2 can be expired and the kidneys where acid is removed
in the urine. It is thought that some of these mechanisms are present in fish.
Acid-base balance in fish differs from that in mammals in a number of ways. In
freshwater fish the kidneys are of minor importance in controlling acid-base status,
but serve mainly to remove water which has entered from the hypo-osmotic environment. In the branchial epithelium there is known to be an active and selective exchange
of ions (Na+/H+, Na+/NH4+ and CI-/HCO3-, Maetz (1971, 1973)) which must be an
important factor in controlling acid-base balance. The gills are also the site for
respiratory gas exchange and COa is lost from the blood by diffusion across the
branchial membranes.
Acid injection to the bloodstream caused considerable changes in the acid-base
status of trout. Shortly after injection there was a decrease in blood pH and blood
bicarbonate, but the control values were resumed after 2-4 h; however, the blood
buffering capacity was little altered (Fig. 1; Tables 1, 2). The acid load to the blood
is probably buffered by two main mechanisms. Firstly, branchial ion exchanges (Maetz,
1971) would lead to the elimination of hydrogen ions via the gills, and there would be
a gradual adjustment of the acid-base status back to normal. Payan & Maetz (1973)
working with acid-injected dogfish considered that H + excretion was quantitatively
linked to sodium uptake. Secondly, Piiper & Baumgarten (1969) and Piiper et al.
(1972) analysed the blood of dogfish which had been subjected to severe exercise;
their results suggested that some of the hydrogen ions generated by lactate could be
balanced by changes in plasma sodium and chloride concentration, and that while a
portion of the H + was excreted via the gills to the sea water a significant proportion
was retained and buffered in the tissues. Thus intracellular buffering appears to be a
significant mechanism in fish.
In the present experiments, approximately 100 /j-equiv. HC1 was introduced to the
bloodstream of trout weighing about 500 g. Assuming blood volume to be 5% of the
body weight then the increase in acidity is 100 /£-equiv./25 ml or 4/i-equiv./ml blood.
This situation may exist for a few hours immediately after the introduction of acid,
before it has penetrated the other body fluid compartments and become diluted. In
most cases arterial blood was analysed about \ h after acid injection, when the acid
would tend to be distributed throughout the body fluid compartments. Also, by this
168
F . B. EDDY
time some of the acid would probably have been eliminated via the gills. The arterial blood
bicarbonate had dropped from 6-09 m-mole/1 to 5-44 m-mole/1 (Table 1), equivalent to
a loss of 0-65 /i-equiv./ml bicarbonate and suggesting that less than 20 % of the acid
load is buffered in this way. The remainder is probably buffered in the intracellular
compartment. A similar situation was observed in acid-injected dogs (Pitts, 1965),
where it was found that 15-20 % of the administered acid load was neutralized by the
plasma buffers, the remainder being neutralized by interstitial buffers and buffers in
cells and bones, where hydrogen ions were exchanged for cellular sodium and
potassium.
Similar buffering mechanisms must be available to trout subjected to high ambient
CO2. Here, COa diffuses across the gills and is hydrated in the blood plasma; some of
the hydrogen ions generated are buffered in the plasma. In the tissues, plasma hydrogen
ions are likely to be exchanged for cellular Na + and K+ and venous blood emerges
relatively well buffered. This is supported by data in Fig. 3 which shows that venous
blood has lower H+ and P C O t values than arterial blood.
During hypercapnia, the fish accumulate plasma bicarbonate at the expense of
chloride (Lloyd & White, 1967), but the resulting increase in buffering is insufficient
for complete compensation; the blood pH never resumes the control value as the fish
attempts to adjust to a permanent respiratory acidosis which is later complicated by
a metabolic acidosis as well.
In both groups of experimental fish, respiration rate and ventilation volume initially
increased. In acid-injected fish this response may have aided the removal of excess
CO2 from the blood. However, in fish exposed to high CO2 the response appeared to
serve no useful purpose, because it is not possible to reduce blood P OOj by diffusion
to a value lower than that of the surrounding water. This point is discussed by Randall
& Shelton (1963), who suggest that high CO2 in the tissues is usually associated with
the hypoxic response; also, Randall & Jones (1973) suggest that blood O2 rather than
blood pH and P COl , is the dominant factor determining changes in ventilation in fish.
Oxygen transport by the blood
Both experimental procedures increased the acidity and COa tension in the blood;
conditions unfavourable for maximum oxygen uptake by the blood haemoglobin. In
each case the arterial oxygen saturation was reduced some 20-30 % below the control
value by the Root effect. It is of interest that the blood oxygen capacity, which in
practice is almost identical to the control period oxygen content, was more or less
unaltered by the experimental procedures (Tables 1, 3). This indicates that no irreversible changes have occurred in the ability of the haemoglobin to take up oxygen.
The depleted in vivo oxygen content of blood may indicate that the intracellular
compartment, particularly the red cells, still contain significant quantities of H+ and
that the restoration of cations to the cells remains uncompleted. This idea receives
support from the data of Schaefer, Messier & Morgan (1970), who exposed rats and
guinea-pigs to 15% CO2 for several days. Their results showed that the pH values of
both plasma and red-cell haemolysates were lowered, and also the red-cell cation
concentration decreased significantly, this being mainly a loss of cellular potassium.
Changes of this sort in trout red cells would certainly lead to conditions of depressed
oxygen uptake in both the acid injection and high carbon dioxide experiments.
All CO, data
Time after COB
0-3 h
Control
I 0.4
20
0.85
3
0.86
I 1.7
0.63
9'44
9
4
2.48
10'0
0
--
-
(Mean, number of determinations and standard error of the mean are indicated. Values obtained during the control period are shown; these are
followed by values obtained during exposure to high CO, for increasing lengths of time; only results from comparable experiments being included.
Finally, the averaged results for all experimental period data are shown. Temperature 15 "C. Po, and Pco,,rnmHg; Coo,(carbon dioxide content of
blood), rn-rnole/l; H+, n-mole/l.)
Blood pH and hydrogen ions
Carbon dioxide
A
L
-,
I
-,
f
I
Arterial
Venous
Arterial
Arterial
Venous
Venous
&
Pco, of
PH
H+
PH
H+
water
Po,
% sat.
Po,
% sat.
Pco,
Cco,
Table 3. The effect of high environmental carbon dioxide on in vivo values for blood oxygen, carbon dioxide
and pH in rainbow trout blood
170
F . B. E D D Y
Thus under the experimental conditions, which are in fact essentially hypoxic1
conditions, the response by the fish was hyperventilation. If the fish had been in
oxygen-deficient waters, this response would improve the oxygen supply to the gills,
but in the present experiments the water was not oxygen-deficient, so the response
was of little value because in this way very little additional oxygen could be added to
the blood. Thus in the efferent gill blood vessels, partially arterialized blood with
increased P C O j and H+ values passes via the dorsal aorta to the peripheral circulation.
Because the blood is poorly oxygen-saturated, smaller than normal quantities of
oxygen will be unloaded to the tissues by means of the Bohr effect. Greater quantities
could be unloaded if the blood-to-tissue POf gradient were to be increased, and this
situation could arise if the tissues were very oxygen depleted, their P o value having
fallen to an extremely low level; this condition, if prolonged, could be detrimental to
the tissues, and would certainly impose limits on the scope for activity by the fish.
Mixed venous blood, with oxygen saturation reduced from about 50 to 25 % by
the experimental procedures, enters the heart and ventral aorta, indicating that the
tissues are likely to be hypoxic. It is noteworthy that neither venous nor arterial Po
values were altered very much by the experimental procedures; this stresses the point
that hypoxia is not caused by unavailability of oxygen, but by the inability of the blood
to combine with and transport oxygen sufficient for the needs of the fish.
In Fig. 3 it can be seen that after 24 h the oxygen saturation of the blood, having
been depressed by high CO2, once again approaches the control value. Earlier experiments (Eddy & Morgan, 1969) have shown that after 24 h in water of high CO2,
rainbow-trout blood no longer showed a marked Root effect and the blood oxygen
affinity had apparently increased compared with the control value, these changes
possibly being mediated by changes in the haemoglobin binding of organic phosphorus
compounds (Riggs, 1970). Similar results were obtained by Schaefer et al. (1970),
who exposed rats and guinea-pigs to 15 % COa for several days. They also observed
an increase in blood oxygen affinity, possibly mediated by changes in haemoglobin
binding of 2,3-diphosphoglycerate.
This work was supported by a N.E.R.C. grant to Professor G. M. Hughes and this
is gratefully acknowledged. Advice from Dr R. F. Burton and also from Professor
G. M. Hughes is acknowledged with thanks.
REFERENCES
ALBERS, C. (1970). Acid-base balance. In Fish Physiology, vol. 4 (ed. W. S. Hoar and D. J. Randall),
pp. 173-208. New York and London: Academic Press.
BLACK, E. C , MANNING, G. T. & HAYASHI, K. (1966). Changes in levels of haemoglobin, oxygen,
carbon dioxide, pyruvate and lactate in venous blood of rainbow trout {Salmo gairdneri) during and
following severe muscular activity. J. Fish. Res. BdCan. 23, 783—95.
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