Aquaculture, 72 (1988) 319-328 319

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Effect of Low Temperature on Sea-water

Tolerance in Rainbow Trout, Salmo guirdneri

BENGT FINSTAD, MAGNE STAURNES and OLA B. REITE’

Department of Zoology, University of Trondheim, AVH, 7055 Dragvoll (Norway)
‘Present address: The Norwegian College of Veterinary Medicine, Box 8146 Dep., 0033 Oslo 1
( Norway 1
(Accepted 30 November 1987)

ABSTRACT

Finstad, B., Staurnes, M. and Reite, O.B., 1988. Effect of low temperature on sea-water tolerance
in rainbow trout, Salmo gairdneri. Aquaculture, 72: 319-328.

Fresh-water-acclimated rainbow trout, Salmo gairdneri (40-180 g), were transferred to fresh
water and sea water (26 %a) at 1 and 8°C. Exposure to fresh water at 1 “C gave a moderate
reduction in plasma osmolality and Na+ and Cl- concentrations. In sea water (26 %o, 8°C) there
was an initial rise in plasma osmolality, plasma concentrations of Na+, Cl- and Mg2+ and tissue
concentrations of Na+ and Cl-. The initial rise was followed by a reduction and stabilization at
levels considerably higher than in fresh water. After transfer to sea water at l”C, there was no
such stabilization. The concentrations continued to increase throughout the experiment, and were
accompanied by marked tissue dehydration. The fish in this group started to die after 2-3 days of
exposure and no fish survived for more than 7 days. The results suggest that low temperatures
affect the mechanisms of active ion transport in gills and kidneys, thus reducing the capacity for
osmotic regulation.

INTRODUCTION

Rainbow trout farming in sea water meets considerable problems at low win-
ter temperatures, involving larger fish in fish cages throughout the year, as
well as juveniles released to fish cages in the autumn. In the farming of Arctic
charr (Salvelinus alpinus) the same problem is present (Finstad and Nilssen,
1987 ) . Low winter temperatures in combination with high salinity probably
create a problem for all salmonids. It is known from Finnmark (Norway), that
during winter, Atlantic salmon (S&no salar) migrate to brackish water and
even swim upstream in the coldest periods (Berg, 1964).
Transfer of rainbow trout from fresh water to sea water involves a lo-day
phase of osmoregulatory adjustment (Gordon, 1959). This period consists of
an initial critical phase (up to 30 h), then a stabilization phase, and finally

0044-8486/88/$03.50 0 1988 Elsevier Science Publishers B.V.

320

after about 10 days a new steady-state period (Bath and Eddy, 1979; Leray et
al., 1981). The transfer can result in a loss of fish during the first days in sea
water (Landless, 1976).
At low temperatures, the ability of fish to regulate water and ions becomes
reduced (Umminger, 1971b; Pucke and Umminger, 1978; Jiirss et al., 1984).
As a consequence, the fish may die in cold sea water due to collapse of their
osmoregulatory mechanisms (Hazel and Prosser, 1974; Vernberg and Silver-
thorn, 1979; Jiirss et al., 1984).
This investigation examines the effect of low temperature on sea-water tol-
erance of rainbow trout after transfer from fresh water. The investigation fo-
cused on possible failure in osmoregulation, and osmotic parameters in blood
plasma and muscle tissue were measured.

MATERIAL AND METHODS

Fresh-water-acclimated rainbow trout, Salmogairdneri (40-180 g), were ob-

tained from the Institute of Aquaculture Research, Sunndalsora, in autumn
1985 and transported to the Department of Zoology, University of Trondheim,
where the experiments were performed. The fish were kept indoors and accli-
mated to fresh water at 8 a C. After 2 weeks the fish were divided randomly into
groups of 30 fish and transferred to four tanks, each containing 130 1 water,
with the following specifications: I, fresh water, 8°C (control group); II, fresh
water, 1 ‘C; III, 26 ‘.rc sea water, 8’ C; and IV, 26 %Osea water, 1 “C. The water
was recirculated and cleaned by Eheim recirculation pumps (Model 1017 ). A
Grant flow cooler (Model FH 15, FC 15) was used for cooling the water.
After the exposure periods (l-10 days), the fish were killed by a blow to the
head, and body weight and length were measured. Blood samples were collected
from the caudal artery by cutting the caudal fin. Hematocrit was measured
immediately using a Compur M 1100 microcentrifuge. The remainder of the
blood was centrifuged on a Sigma 2 microcentrifuge and plasma was frozen for
later analysis.
Muscle samples were taken from the dorsal muscle close to the dorsal fin.
The pieces were blotted with filter paper and cut into two parts. One part was
used for analysis of Na+ and Cl-, and the other for determination of water
content and concentration of Mg2+ (Lutz, 1972). Samples were taken at the
start and after 1 day (groups II, III and IV), 3 days (groups III and IV ),7 days
(groups I, III and IV) and 10 days (group II).
Plasma osmolality was measured using a Knauer semi-microosmometer (No.
21.20 ) . Plasma and tissue Na’ and Mg2 + were measured by atomic absorption
spectrophotometry (Perkin Elmer 300), and Cl- by Radiometer CMT 10 chlo-
ride titrator. Standard methods were used for all analyses.
Values are given as mean ? standard deviation. For all statistical calcula-
 321

tions Wilcoxon’s two-tailed test was used and a significance level of P~0.05
was chosen.

RESULTS

Mortality occurred only in the group exposed to sea water and low temper-
ature (group IV, Fig. 1) . In this trial the fish lost equilibrium, surfaced, and
swallowed air. In the other trials there were no visible signs of stress.

Blood composition

No significant changes in hematocrit occurred throughout the experimental

period, but there was a trend toward a lower hematocrit value in the two groups
transferred to sea water (Fig. 2).
Exposure to fresh water at 8°C (group I) caused no changes in osmolality

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Fig. 1. Accumulated mortality ( % ) in rainbow trout exposed to 1 ‘C, 26 60 sea water (group IV).
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Fig. 2. Hematocrit (W ) in rainbow trout exposed to fresh water at 8°C (group I, +) and 1 ‘C
(group II, A ) and 26 %o sea water at 8” C (group III, n ) and 1’ C (group IV, . ). Values are mean
i SD (n=4).
322

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Fig. 3. (a) Plasma osmolality (mOsm); (b) plasmasodium (mmol/l); (c) plasmachloride (mmol/
1) and (d) plasma magnesium (mmol/l) in rainbow trout. Symbols are the same as in Fig. 2.
Values are mean k SD (n = 4).

(Fig. 3a); there was a decrease, although not statistically significant, from 310
to 254 mOsm at the end of the experiment in those exposed to fresh water at
1 c C (group II ) . In fish exposed to sea water at 8’ C (group III ) there was an
initial rise in osmolality to 396 mOsm the first day after transfer, but osmolal-
ity then declined and stabilized at a level 17% higher than that of the control
group. Fish transferred to sea water at 1 “C (group IV) displayed no such sta-
bilization, and at the end of the experiment mean osmolality was 512 mOsm
(Fig. 3a).
The development of plasma concentrations of Na+ and Cl- (Fig. 3b and c )
followed much the same pattern as the osmolality, showing a moderate de-
crease in the fishes exposed to cold fresh water, an initial increase followed by
a stabilization in fish transferred to sea water at 8”C, and an uncontrolled
 323

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Fig. 4. Muscle water (76) in rainbow trout exposed to fresh water at 8°C (group I, *) and 1 “C
(group II, A ) and 26 %Osea water at 8°C (group III, n ) and 1 “C (group IV, l ). Values are mean
+ SD (n=4).

increase among those exposed to cold sea water. In this last group (IV), the
plasma concentrations of Na+ and Cl- were 74 and 81%, respectively, above
the values of the control group at the end of the experiment.
There was no change during the experiment in the plasma concentration of
Mg2+ in the two groups of fish retained in fresh water (groups I and II, Fig.
3d). Transfer to sea water without temperature change (group III) caused a
moderate increase in Mg2+ concentration; the fish simultaneously stressed
with low temperature (group IV) had a plasma Mg+ concentration eleven
times higher than that in the control group at the end of the experiment.

Tissue composition

The water content in white muscle tissue of fresh water fishes was stable at
about 77% (Fig. 4). In fish transferred to sea water at 8°C there was a reduc-
tion to about 75% the first day, followed by a stabilization to a level slightly
lower than that in the fresh water groups. However, among those exposed to
cold sea water (group IV ) the water content continued to drop and at the end
of the exposure period it was about 70%.
The trends of Na+ and Cl- concentration in muscle tissue (Fig. 5a and b)
followed largely the same pattern as in the plasma. In cold fresh water (group
II) there were significant declines in both Na+ and Cl- concentration (9 and
16%, respectively) from the beginning to the end of the exposure period. In
group III (transfer to sea water without temperature change) there was an
increase in the concentration of Na+ (25% ) and Cl- (22% ) after the third day
of the exposure period. Thereafter there was a decrease and stabilization at
values significantly higher than those of the control group. In the sea-water
group at 1°C there was an increase in the concentrations of Na+ and Cl- in
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Fig. 5. (a) Muscle sodium (mmol/kg wet weight); (b) muscle chloride (mmol/kg wet weight)
and (c) muscle magnesium (mmol/kg wet weight) in rainbow trout. Symbols are as in Fig. 4.
Values are mean & SD (n ~4).

muscle tissue throughout the experiment, and at the end both values were about
125% higher than those of the control group.
There was no significant change in the muscle concentration of Mg’+, in
either group II ( 1 ‘C, fresh water) or group III (8” C, 26 %Osea water, Fig. 5c ).
However, in group IV (sea water, low temperature) there were significantly
higher values than in the other groups.

DISCUSSION

A sudden temperature drop from 8 to 1 ‘C in fresh water caused no mortality.

Also, the fish tolerated direct transfer from fresh water to 26 %O sea water
without visible signs of stress. However, mortality was observed initially after
2 days in the fish exposed to both sea water and low temperature. This agrees
with the results of Spannhof (1982) who reported that the tolerance limit of
rainbow trout at 1 o C was 13 %o. He observed no mortality in rainbow trout at
0°C in fresh water or in brackish water of less than 10 %o. Saunders et al.
 (1975 ) examined brook trout (Salvelinus fontinalis), Atlantic salmon (Salmo
salar) and rainbow trout at temperatures close to 0°C in 30 %G sea water. All
three species showed high mortality rates, brook trout being the least cold tol-
erant of these species.
Fish transferred from fresh water at 8°C to fresh water at 1 ‘C (group II)
showed a tendency to a decrease in osmolality and ionic concentrations in both
blood plasma and muscle tissue. This response of fresh-water fish previously
has been thought to be due to a reduced ability of these fishes to osmoregulate
in cold water (Umminger, 1969). Furthermore, this lower osmotic gradient
between the fish and the environment has been attributed to an adaptive en-
ergy-saving response as a result of reduced energy production in cold water
(Prosser et al., 1970; Umminger, 1971a; Prosser and Nelson, 1981).
Previous experiments have shown that when salmonids are transferred from
fresh water to sea water, there is an initial critical phase characterized by sig-
nificant increases in Na+, Cl- (Bath and Eddy, 1979; Leray et al., 1981; Vir-
tanen and Oikari, 1984) and Mg2+ (Miles and Smith, 1968) concentrations in
blood plasma, and also increases of Na+ and Cll (Houston, 1973) concentra-
tions in muscle tissue in the first days. An initial reduction in muscle water
has also been observed (Virtanen and Oikari, 1984 ). After about 10 days it was
found that all of these parameters were regulated back to a level normal for
salmonids in sea water. Our results from group III (transfer without temper-
ature change), with high plasma and tissue concentrations after 24 h, followed
by a reduction and stabilization from the third to the seventh day, are therefore
in accordance with what has been found in previous experiments. The plasma
concentrations of Na+ and Cl- after 7 days in sea water were about 175 and
150 mmol/l, respectively (group III, Fig. 3b and c ). This is very similar to those
concentrations found in long-term sea-water-acclimated rainbow trout and
Atlantic salmon (Finstad and Staurnes, unpubl., 1987). However, while the
concentration of plasma Mg2+ in these long-term acclimated fishes was not
greater than 1 mmol/l, the concentration after a week in the blood plasma of
fish at 26 % was three to four times higher. This may indicate that the regu-
lation of Mg2+ in rainbow trout takes longer than the regulation of Na+ and
Cl-, or alternatively, that the fish in our experiment were unable to regulate
the Mg2+ concentration to normal levels in 26 % sea water. In some prelimi-
nary experiments on the same species, all fish died during the first 2 days when
transferred to 34 %, and mortality was also observed at 28 %a. Therefore, it
seems that in our experiment transfer to 26 % is near the tolerance limit, and
also that regulation of Mg2+ may be more critical than regulation of Na+ and
Cl-.
When fish were transferred from fresh water at 8°C to sea water at 1 “C
(group IV), the initial increase in osmolality, shown in group III, was not reg-
ulated back after 2-3 days. There was a steep increase in osmolality, and fish
started to die after 2 days when blood osmolality exceeded 420 mOsm. Conte
and Wagner (1965) mentioned 450 mOsm as being the lethal osmolality for
rainbow trout. Osmolality thus seems to be a good indicator of stress in sal-
monids. A steep increase in osmolality has also been found in the killifish,
Fund&s diaphanus exposed to 30 ?k sea water at 0.5”C (Ahokas and Sorg,
1977 ) and in Baltic salmon exposed to 29 %0 sea water at 1.5’ C (Virtanen and
Oikari, 1984).
The concentrations of Na+ and Cl in plasma and tissue followed the same
pattern as osmolality. There are several examples of increased levels of Na+
and Cl- in plasma of the killifish (Pucke and Umminger, 1978), Baltic salmon
(Virtanen and Oikari, 1984) and in muscle tissue of rainbow trout (Houston,
1973) when exposed to cold sea water. Fish in group IV (1 “C, 26 %Osea water)
totally lost their capacity for magnesium regulation. Virtanen and Oikari (1984)
measured an increase to a level of 1.5 mmol/l in the plasma concentration of
Mg2+ in Baltic salmon after 9 days exposure to sea water (29 %O) at 1.5”C,
but this increase was less than that observed in rainbow trout (group IV) in
the present experiment.
The occurrence of muscle dehydration indicates that there is an insufficient
drinking rate, or an insufficient uptake of water in the gut. This water uptake
is mediated by a Na-K-ATPase-dependent transport of Na+ and Cl- across the
gut wall (Kirsch et al., 1985). The gut activity of this enzyme is probably very
temperature sensitive, as is the gill activity of Na-K-ATPase in both rainbow
trout (Jiirss et al., 1984) and kill&h (Pucke and Umminger, 1978). In the
gills this enzyme plays a major role in the extrusion of excess Na+ and Cl- in
sea water (Maetz, 1974; Silva et al., 1977; Degnan, 1985). The temperature
sensitivity of this enzyme may therefore result in reduced activity levels where
the fish is unable to cope with the salt load at low sea water temperatures.
As mentioned above, the results indicate that in sea water the regulation of
Mg2+ may be more critical than the regulation of Na+ and Cl-. Therefore, the
mechanism in the kidney responsible for Mg2+ extrusion (see Evans, 1979)
may be even more vulnerable than the gill extrusion mechanisms of Na+ and
Cl- when the fish are exposed to cold sea water.
The salt extrusion processes in both kidneys and gills, as well as water up-
take in the gut, are mediated by membrane-bound enzymes. Therefore, the
effects of low temperature on these processes are probably due both to reduc-
tion in the activity of the enzymes themselves and changes in the properties of
the membranes. The lipid composition of membranes is strongly temperature
dependent, and in general, changes in lipid composition of membranes seem
to be important in adaptation to low temperature (Hazel and Prosser, 1974;
Sinensky, 1974; Hazel, 1984). Studies on the effects of low temperature on
membranes in salt-transporting tissue, and in this respect the importance of
the lipid composition of the feed, should be examined in future work on winter
mortality problems in Norwegian rainbow trout farming in sea water.
 327

ACKNOWLEDGEMENTS

The authors thank Arne Kittelsen and Terje Refstie at the Institute of Aqua-
culture Research, Sunndalwra, for kindly supplying fish for this experiment,
and Dr. Karl E. Zachariassen for comments and criticism of the manuscript.

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