SALCOD
The effects of salinity on growth and biology in Atlantic cod
Áhrif seltu á vaxtarhraða og líffræði þorsks
Final activity report: R-065-08
Authors:
Tómas Árnason, Marine Research Institute
Bergljót Magnadóttir, Keldur
Björn Björnsson, Marine Research Institute
Agnar Steinarsson, Marine Research Institute
Björn Þrándur Björnsson, University of Gothenburg and Matís
Lokaskýrsla - Final activity report R-065-08
Abstract
The effects of salinity and temperature on growth, plasma ions, cortisol and immune
parameters were investigated in two experiments. In the first experiment , small, medium
and large juvenile Atlantic cod Gadus morhua (initial weights 2, 8, and 83 g) were reared at
four constant salinities (6 to 32‰) for a short period (19-57 days, depending on size), then
returned to seawater without acclimation and reared for another period (20-391 days). The
highest growth rates were found at 10‰ in all size-classes, and the optimal salinity for
growth was predicted to be 12.5 and 14.8‰ for the medium and large juveniles. After the
fish were returned to seawater the growth rates were inversely related to the change in
salinity in all size-classes. Long-term rearing of the medium sized fish revealed that abrupt
exposure from 6 and 10‰ to seawater may permanently reduce the growth capacity of
juvenile cod. In another long term experiment, there was no significant difference in the
growth of cod (initial weight = 3.4g ) reared at either 13.5 or 32‰ for 187 days, and rearing
at 13.5‰ neither enhanced growth at 6.3 nor 10°C in larger juveniles (>245g) compared to
rearing in seawater. After transfer to seawater on day 187, the lowest growth rates were
found in the groups prevously reared at 13.5‰, but the effects of abrupt increse in salinity
did not differ between groups at 6.3 or 10°C. The study shows that salinity and abrupt
increase in salinity has limited or no effects on stress and immune-related parameters, and
there were no indications of ion regulatory disturbances at salinities as low as 6‰,
suggesting that Atlantic cod is extremely euryhaline.
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Ágrip
Áhrif seltu og hitastigs á vöxt, kortisól, jónir í blóðvökva og vessabundna ónæmisþætti
voru könnuð í tveimur tilraunum. Í fyrri tilrauninni voru lítil, miðlungs og stór þorskseiði
Gadus morhua (upphafsþyngdir 2, 8 og 83 g) alin við fjögur stöðug seltustig (6 til 32‰)
yfir stutt tímabil (19-57 dagar eftir stærð), síðan færð aftur yfir í sjó án aðlögunar og alin í
annað tímabil (20-391 dagar). Í öllum stærðarflokkum var mesti vöxturinn við 10‰ og
áætluð kjörselta til vaxtar var 12.5 og 14.8‰ fyrir miðlungs og stór þorskseiði. Eftir að
seiðin voru færð aftur í sjó var vöxturinn í öfugu hlutfalli við seltubreytinguna í öllum
stærðarflokkum. Langtíma eldi á miðlungs stórum seiðum sýndi að skyndileg seltubreyting
frá 6 og 10‰ í 32‰ getur haft varanleg neikvæð áhrif á vaxtargetu fisksins. Í annari
langtíma tilraun var ekki marktækur munur á vexti þorska (upphafsþyngd = 3.4 g) sem aldir
voru í 187 daga við 13.5 og 32‰. Samanburður á vexti stærri þorskseiða (>245g) milli
sömu seltustiga og tveggja hitastiga (6.3 og 10°C), sýndi enga aukningu á vexti við 13.5‰,
hvorki við 6.3°C né 10°C. Eftir að fiskurinn var fluttur í sjó án aðlögunar var vöxturinn
marktækt lægri í fisknum sem varð fyrir seltubreytingu, en áhrif breytingarinnar voru ekki
ólík milli hitastiga. Rannsóknin sýnir að selta og snöggar seltubreytingar hafa ýmist
takmörkuð eða engin áhrif á stress og vessabundna ónæmisþætti. Það er ekkert sem bendir
til röskunar á jónajafnvægi í seltu niður í 6‰ sem sýnir að Atlantshafs þorskur þrífst við
seltu á mjög breiðu bili.
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Preface
Two chapters are included in this report. The first chapter contains a manuscript submitted
to a peer-reviewed scientific paper and the second chapter contains data which was
excluded from the manuscript.
I.
Effects of salinity and temperature on growth, plasma ions, cortisol and
immune parameters of juvenile Atlantic cod (Gadus morhua)
II.
On growing of Atlantic cod (Gadus morhua) following direct transfer from
different temperatures and salinities to seawater
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Chapter I
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Effects of salinity and temperature on growth, plasma ions,
cortisol and immune parameters of juvenile Atlantic cod (Gadus
morhua)
Tómas Árnasona,*, Bergljót Magnadóttirb, Björn Björnssonc, Agnar Steinarssona,
Björn Thrandur Björnssonde
a
Marine Research Institute, Box 42, IS-240 Grindavík, Iceland
b
Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsvegur,
IS-112 Reykjavík, Iceland
c
Marine Research Institute, Skúlagata 4,Box 1390, IS-121 Reykjavík, Iceland
d
Department of Biology and Environmental Sciences, University of Gothenburg, Box 463,
SE-405 30 Gothenburg, Sweden
e
Matís, Vinlandsleid 12, 113 Reykjavík, Iceland.
Abstract
The effects of salinity and temperature on growth, plasma ions, cortisol and immune
parameters were investigated in two experiments. In the first experiment small, medium
and large juvenile Atlantic cod Gadus morhua (initial weights 1.9 ± 0.4, 8 ± 0.8 and 83 ±
2.9 g, mean ± SEM) were reared at four constant salinities (6 to 32‰) for a short period
(19-57 days, depending on size), then returned to seawater without acclimation and reared
for another period (20-391 days). The highest growth rates were found at 10‰ in all sizeclasses, and the optimal salinity for growth was predicted to be 12.5 and 14.8‰ for the
medium and large juveniles. After the fish were returned to seawater the growth rates were
inversely related to the change in salinity in all size-classes and long-term rearing of the
medium sized fish revealed that abrupt exposure from 6 and 10‰ to seawater may
permanently reduce the growth capacity of juvenile cod. In another long term experiment,
there was no significant difference in the growth of cod (initial weight 3.4 ± 0.55 g) reared
at either 13.5 or 32‰ for 187 days, and rearing at 13.5‰ neither enhanced growth at 6.3°C
nor 10°C in larger juveniles (>245g) compared to rearing in seawater. The study shows that
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salinity and abrupt increase in salinity has limited or no effects on stress and immunerelated parameters, and there were no indications of ion regulatory disturbances at salinities
as low as 6‰, suggesting that Atlantic cod is extremely euryhaline.
Keywords: Atlantic cod, Gadus morhua, growth, temperature, salinity; osmoregulation,
stress, immunology
1. Introduction
Although, the vast majority of Atlantic cod (Gadus morhua) is distributed in oceanic
waters with salinity around 34‰, cod tends to prefer salinities lower than full-strength
seawater if a choice is provided (Claireaux et al. 1995) and tolerates long-term exposure to
low salinities (Odense et al. 1966). Furthermore, Atlantic cod has been found to grow faster
at low (7‰) and intermediate salinities (14 and 15‰) compared to seawater (Lambert et al.
1994; Dutil et al. 1997; Imsland et al. 2011), and it has been postulated that rearing cod at
intermediate salinities could provide an economic advantage compared with rearing cod in
seawater (Lambert et al. 1994).
Marine teleosts such as the Atlantic cod are hypoosmotic regulators, maintaining
plasma osmolality at about one-third of that of full-strength seawater. In order to maintain
hydromineral homeostasis, the osmotic water loss is compensated by ingestion of seawater,
followed by absorption of water and salts over the gastrointestinal tract, and subsequent
secretion of monovalent and divalent ions, over the gills and kidneys, respectively. The
environmental salinity may impact the physiological condition of euryhaline, marine teleost
species in different ways and through various mechanisms, but understanding of this is
currently limited.
Salinities approaching isoosmotic conditions might improve growth due to lower
energetic costs of osmoregulation, lower standard metabolic rate and a larger proportion of
ingested energy being directed towards growth. Estimations of the energetic costs of
osmoregulation range from 10 to 50% of the total energy budget of the fish (see Boeuf and
Payan, 2001). Higher growth under isoosmotic conditions might also be related to a
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hypothetical conflict between hydromineral and nutritional transport mechanisms in the
intestine, which would be minimized at isoosmotic environmental conditions.
On the other hand, changes in environmental salinity approaching the physiological
tolerance limits of the species are likely to act as a stressor, leading to elevated cortisol
levels which are known to adversely affect immune defence (Harris and Bird, 2000) and
appetite (Gregory and Wood 1999).
It is a well established paradigm that the immune system and disease resistance of
poikilothermic animals like fish is affected by external as well as inherent factors
(Magnadottir, 2010). The link between the environment and the immune defence of fish has
received considerable attention, the main emphasis having been on the effects of
temperature, pollution and seasonal or diurnal changes (Bowden et al., 2007, Bowden,
2008). A close link between the neuroendocrine system and the immune system of fish has
been demonstrated in several studies (Weyts et al., 1999, Harris and Bird, 2000), and most
studied are the suppressive effects of stress, signified by a raised plasma cortisol level, on
the immune response and disease resistance (Fast et al., 2008, Castillo et al., 2009).
While a decreased osmotic gradient may thus promote growth, and may thus be of
applied interest in Atlantic cod aquaculture, such changes in salinity may potentially also
influence other physiological systems. In particular, a question remains at which salinity
the change may induce stress responses in the fish and lead to impaired immune
competence.
The aim of the present study was to elucidate the short and long term effects of salinity
on growth and osmoregulation in Atlantic cod, as well as its effects on stress responses and
immune function. As the impact of salinity on physiology may vary at different life-stages,
three size-classes of cod were reared at four different salinities in order to define optimal
salinities for growth (Sopt.G), and assessing plasma ion and cortisol levels, as well as
immune parameters. As temperature is likely to influence salinity responses, a follow-up
study was carried out, in which cod was reared at optimal salinity and at two temperatures
(6.3 and 10°C).
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2. Materials and methods
2.1. Fish material and rearing conditions
Two experiments were conducted on juvenile Atlantic cod, Gadus morhua, at the
Mariculture Laboratory of the Marine Research Institute at Grindavík, SW Iceland.
Fertilized eggs from the first generation of selectively bred cod from a commercial
breeding selection facility (Icecod Ltd, Iceland) were brought to the laboratory in January
and April 2008, and May 2009, incubated in 150 L silos at 7.5°C, and moved to 3200 L
tanks where the larvae were fed on rotifers and Artemia until weaned on dry feed.
In the experiments, salinity, oxygen and temperature were measured daily, and DST
CTD data storage tags (Star-Oddi Ltd, Iceland) were used to record the temperature and
salinity every hour. Oxygen saturation was close to 100% at all times and the water flow
was adjusted to keep ammonia well below critical levels (Björnsson and Ólafsdóttir, 2006).
Diluted seawater was obtained by mixing ground water (3‰) with seawater (32‰) taken
from a 50-m deep well. All tanks had constant light by incandescent lamps (100-350 lx at
the surface). Excess feeding was maintained in the experiments by stocking automatic
feeders every day with commercial dry feed (Laxá, Iceland). The protein/fat ratio in the
feed was 62/13 for the smallest juveniles (2 – 8 g) and 52/19 for larger fish.
2.2. Weight measurements and sampling
All the experimental fish were individually weighed under anaesthesia (MS222,
tricaine methane sulphonate, 0.1 g L-1, Pharmaq Ltd, UK). Unless otherwise stated, fish
were killed by anaesthesia prior to blood sampling. Blood was collected from the caudal
vessels using heparinized syringes (Sarstedt, Germany), centrifuged at 7000 × g for 10 min,
the plasma collected, placed on dry ice, and stored at -80°C until analysis. During
samplings, an inoculation from the kidney of every other fish was made on blood agar with
1.5% NaCl (BA-S) and incubated at 15°C for 72 h to check for symptomless infections.
2.3. Experiment 1A, B and C: Effects of salinity on juvenile cod at different size/age
The experiment was carried out as three separate trials on (A) 3.5-month old, small (1.87 ±
0.40 g), (B) 4-month old, medium-sized (8.03 ± 0.82 g), and (C) 8.5-month old, large (82.9
± 2.9 g) juveniles. In all three trials (Fig. 1), the fish were separated into four treatment
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groups of which three were gradually acclimatized over a period of two days to lower
salinities, while one group was kept in seawater (32‰). The fish were then kept at these
salinities for a period of time, after which the fish at lower salinities were rapidly (1-2
hours) returned to seawater, and kept there for an additional period of time. All treatment
groups were reared in two replicate tanks. The small and large juveniles were hatched in
14th January 2008 and the medium-sized juveniles in 22nd April 2008. Weight
measurements and blood samples were taken as shown in Fig. 1. No blood samples were
taken in experiment 1A due to the small size of the fish.
In order to assess individual growth rates, 40 individuals from each experimental tank were
PIT-tagged (Trovan Ltd, UK) under anaesthesia one week before the trial was initiated.
However, due to the tag size, only medium-sized and large juveniles were tagged.
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Fig. 1. Design for experiment 1 in which juvenile Atlantic cod were exposed to decreased salinities
for a period of time. (A) Small juveniles exposed to 32, 20, 15 and 10‰ for 19 days and returned to
32‰ for 20 days. (B) Medium-sized juveniles exposed to 32, 20, 10 and 6‰ for 33 days and
returned to 32 %o for 391days. (C) Large juveniles exposed to 32, 20, 10 and 6‰ for 57 days and
returned to 32‰ for 56 days. T, S and B indicate time of individual tagging, size measurement
(body weight) and blood sampling, respectively.
Experiment 1A: From April 25th 2008, 3.5-month old, small juveniles were reared at 10,
15, 20 and 32‰ for 19 days at 11.9°C in rectangular fibreglass tanks (90 × 90× 37 cm, 300
l), initially containing 130 fish per tank. Then, the fish were returned to 32‰ for additional
20 days.
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Experiment 1B: From August 14th 2008, 4-month old, medium-sized juveniles were reared
at 6, 10, 20 and 32‰ for 33 days at 10.9°C, in rectangular fibreglass tanks (90 × 90 × 37
cm, 300 L), initially containing 110 fish per tank. Then, the fish were returned to 32‰ for
additional 36 days. At that point, 27-32 tagged fish from each tank were transferred to one
circular tank (2.9 m diameter and 0.8 m deep, 5300 l) where they were reared in 32‰ for
additional 355 days. The rearing temperature was gradually decreased from 10.9°C to 7°C
as the fish grew larger. Blood samples from twenty fish were taken on day 0 and from 12
fish from each tank on day 33 and 69 (Fig. 1B).
Experiment 1C: From September 24th, 2008, 8.5-month old, large juveniles were reared at
6, 10, 20 and 32‰ for 57 days at 8.7°C in circular fibreglass tanks (5300 l), initially
containing 110 fish per tank. Then, the fish were returned to 32‰ for additional 56 days.
Twenty fish were sampled on day 0 and 12 fish from each tank on day 57 and 113 (Fig.
1C).
2.4. Experiment 2: Effects of salinity and temperature on juvenile cod
Experiment 2 was carried out in two phases (Fig. 2).
Phase 1 (day 0-187): Experimental phase 1 was initiated on August 13th 2009 by
establishing two treatment groups in duplicate tanks by randomly dividing 800 fish with
mean weight of 3.43 ± 0.55 g among four 300 L fibreglass tanks. For two tanks, the salinity
was gradually lowered to 13.5‰ over two days, while keeping it at 32‰ for the other two
tanks. After 69 days, blood samples were collected from 12 fish from each tank. Then, all
the fish were anaesthetized, weighed, PIT-tagged and transferred to larger tanks (2.9 m in
diameter and 0.8 m deep, 5300 l). All fish were weighed again on day 187 and blood
samples collected from 12 fish from each tank after a mild anaesthesia, i.e. without
sacrificing the fish. The fish were reared at 12.4°C for the first 69 days and at 11.6°C from
day 69 to 187.
Phase 2 (day 187- 278): On day 187, experimental phase 2 was initiated by transferring 8591 randomly selected individuals from each tank (50% of the fish) to tanks with the same
rearing conditions. These fish were then either acclimatized to 6.3 or 10.0°C over a period
of two days, without changing the salinities. Thus, four, duplicate experimental treatments
were created; 10.0°C/13.5‰, 10.0°C/32‰, 6.3°C/13.5‰ and 6.3°C/32‰ (Fig. 2, phase 2).
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The mean weights in the tanks on day 187, after the fish were transferred were from
235±4.2 to 253±4.0 g, but there was no significant difference in body weights between
tanks (p>0.05). The fish were weighed again on day 278, and blood samples collected from
6 randomly selected individuals in each tank without sacrificing the fish as described
above.
Phase 1
2×300 l tanks
32‰
12.4°C
2×5300 l tanks
32‰
10°C
2×5300 l tanks
32‰
11.6°C
2×300 l tanks
13.5‰
12.4°C
Day 0
Aug 13
2009
Phase 2
2×5300 l tanks
32‰
6.3°C
2×5300 l tanks
13.5‰
10°C
2×5300 l tanks
13.5‰
11.6°C
Day 69
Oct 21
2009
2×5300 l tanks
13.5‰
6.3°C
Day 187
Feb 16
2010
Day 278
May 20
2010
Fig. 2. Design for experiment 2 in which juvenile Atlantic cod were kept at two salinities (13.5 and
32‰) during phase 1, day 0-187. During phase 2, day 187-278, the salinity groups were divided
between two rearing temperatures (6.3 and 10 °C). Weight and blood samples were obtained on
days 69, 187 and 278.
2.5. Plasma analysis
Plasma sodium (Na) and calcium (Ca) levels were measured by flame photometry
(Eppendorf ELEX 6361).
Plasma cortisol levels were measured in unextracted plasma using a radioimmunoassay
procedure according to Young (1986) and validated by Sundh et al. (2011). Cortisol
antibody (Code: S020; Lot: 1014-180182) was purchased from Guildhay Ltd (Guildford,
Surrey, U.K.).
Total plasma proteins were analyzed using a protein assay kit from Thermo Scientific (IL,
USA) based on the Bradford method (Bradford, 1976), with bovine serum albumin from
the kit as a standard.
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Natural antibody (IgM) activity was measured using a modified ELISA method previously
described (Magnadottir et al., 1999 a). The modification involved the expression of natural
antibody activity, which was presented as the percentage of the mean optical density value
of plasma from the same six individuals showing a relatively high natural antibody activity
(OD405nm >1.2). The TNP-BSA coating antigen and polyclonal mouse anti-cod IgM
antibody used in the analysis were prepared at the Institute for Experimental Pathology at
Keldur, Iceland.
Anti-trypsin activity was measured in 50% of the samples collected in experiment 1C and
experiment 2. A modification (Magnadottir et al., 1999 a) of the method described by
Bowden et al. was used (Bowden et al., 1997). The method was based on the plasma
inhibition of standard trypsin solution (Sigma, USA), the enzyme substrate being
azocasein (Sigma).
2.6. Statistical methods and data analysis
All data are presented as means ± standard error of the mean (SEM). Specific growth
rate (G) was calculated according to the formula G = 100(lnW2 – lnW1)/(t2-t1), where W1
and W2 are the weights of the fish at times t1 and t2. Tagged fish that were sacrificed for
blood sampling and fish that died during the experiment were excluded from the growth
data analysis. Tank means were calculated for both tagged and un-tagged fish and
measurements of the PIT-tagged fish used to calculate individual growth rates.
In experiment 1, a three parameter model developed by Shepherd (1982) for analyzing
stock-recruitment relationship for fisheries was fitted to the data for the medium and large
juveniles to describe the effect of salinity on growth rate: G = aS/[(1 + (S/b)c], , where G is
the growth rate, S is salinity, and a, b and c are constants estimated by the method of least
square. The relationships between natural antibody activity, plasma protein levels and body
weights were studied with correlation matrix analyses.
The Shapiro-Wilk test was used to analyse whether the data were significantly
different from a standard normal distribution and homogeneity of variances were tested
using the Levene’s test. Data not normally distributed were log-transformed before
statistical analyses. Replicates were pooled prior to analysis since there was no significant
difference between replicates in any treatment. Data which did not conform to normality
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even after log-transformation were analysed using the Kruskal-Wallis ANOVA, and
followed by the Holm post-hoc test if differences between salinities were found. In
experiment 1 and in phase 1 of experiment 2, a two-way ANOVA with time and salinity
was used to test for possible differences in plasma ions, cortisol and immune parameters,
followed by Tukey HSD tests to locate any differences between treatments. Similarly, the
data for growth, plasma ions, cortisol and immune parameters in phase 2 of experiment 2,
were analysed with a two-way ANOVA with temperature and salinity. In all tests, a
probability level of p<0.05 was considered significant. All statistical analyses were done
using R version 2.11.1 (the R foundation for statistical computing).
3. Results
3.1. Experiment 1A, B and C: Effects of salinity on juvenile cod at different size/age
3.1.1. Growth
Experiment 1A: The initial mean weight was significantly higher at 10‰ (1.94 ± 0.38g)
than 32‰ (1.82 ± 0.38 g) (p <0.05). During the 19-day salinity exposure, the fish at 10‰
had the highest growth rate. The growth rate decreased linearly as the salinity increased to
20‰, but similar growth rates were found at 20 and 32‰ (Fig. 3A, Table 1). Optimal
salinity for growth (Sopt.G) could not be calculated as the highest growth rate was found at
the lowest salinity. After being returned to seawater, growth rate was found to be inversely
related to the salinity change (Table 1).
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Fig. 3. Experiment 1: Average specific growth rates of small (A), medium (B) and large (C) cod
juveniles reared at four salinities. The dots represent the overall mean growth rates in each replicate
tank and the lines represent the fit of the model, G = aS/[(1 + (S/b)c]to the data, where G is the
growth rate, S is salinity, and a, b and c are constants determined by the method of least squares.
B: a = 1.0466, b = 4.7906, c = 1.2908
C: a = 0.2781, b = 9.6938, c = 1.5234
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Table 1. Experiment 1A and C: Growth data for the small and large juvenile cod. Values are given
as mean ± standard error of the mean (SEM). Number of fish in each experimental group (n),
overall initial and final mean weights in period 1 (W1 and W2), final mean weight in period 2 (W3),
growth rate (G), and feed conversion ratio (FCR). The growth data for the larger juveniles are from
PIT tagged fish. Different letters denote significant differences between salinity groups (p<0.05).
Different salinities
Seawater
Salinity (‰) n
W1
W2
G(%)
W3
G(%)
a
a
a
10.1
260 1.94 ± 0.38
5.49 ± 0.57
5.48
12.3 ± 0.77
3.85
Small
14.8
260 1.90 ± 0.39ab
5.23 ± 0.56b 5.34
12.0 ± 0.75ab 3.96
(Exp 1A)
20.1
259 1.84 ± 0.43ab
4.95 ± 0.62c 5.20
11.6 ± 0.88b
4.12
b
c
ab
31.8
260 1.82 ± 0.38
4.89 ± 0.59
5.21
12.1 ± 0.78
4.27
c
c
b
5.7
73
83.6 ± 2.35
159 ± 3.49
1.12 ± 0.33
251 ± 5.18
0.82 ± 0.36c
a
a
a
Large
10.0
79
85.7 ± 2.75
193 ± 3.96
1.43 ± 0.18
314 ± 4.63
0.84 ± 0.14bc
(Exp 1C)
20.1
79
85.9 ± 2.58
187 ± 3.88a
1.36 ± 0.18b
299 ± 5.15a
0.85 ± 0.22ab
31.9
79
83.1 ± 2.28
174 ± 3.26b
1.29 ± 0.20b
286 ± 4.32a
0.91 ± 0.20a
Small, day 0 (W1), day 19 (W2) and day 39 (W3); Large, day 0 (W1), day 57 (W2) and day 113 (W3)
Experiment 1B: During the period in different salinities, the relationship between the
overall growth rate and salinity was adequately described with a three parameter model
(Fig. 3B). There is a rapid increase in growth rate as salinity increases from 6‰, passing
through a relatively flat peak at optimal salinity for growth (Sopt.G) and falling gradually as
salinity increases up to 32‰. The model predicts that the growth rates for the medium
juveniles are maximal at 12.5‰.
There were no significant differences in the overall mean weights between groups at
the start of the trial. The PIT-tagged fish at 6‰ and 32‰ were, however, significantly
heavier than fish at 10‰ and 20‰ (Fig. 4, p <0.05), but after 33 days at different salinities,
these weight differences were no longer significant (p >0.05). In the first 36-day period
after the fish were returned to seawater, growth rates were inversely related to the salinity
change (Table 2). From day 69 to end of the 424-day trial, growth rates were over-all fairly
similar among the groups despite marked differences in mean weights. Thus, the negative
effect on growth from abrupt transfer from low salinities to seawater persisted throughout
the trial. At the termination of the trial, the group kept on 32‰ throughout had the highest
mean weight (889 ± 10.4 g), while the group exposed to the lowest salinity 6‰ had the
lowest mean weight (649 ± 9.5 g, Fig. 4).
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Fig. 4. Experiment 1: Mean weights (g) of PIT-tagged cod subjected to four different salinity
treatments. The groups reared at 6 to 20‰ were subjected to abrupt increase in salinity to seawater
on day 33. Means sharing the same letter are not significantly different at the 5% level.
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Table 2. Growth data for PIT tagged medium juveniles in experiment 1B. Body weights (W in g) and specific growth rates between measurements
(G). Values are given as mean ± SEM. All groups were reared in seawater from day 33.
treatment W1
W2
G1-2 (%)
W3
G2-3 (%)
W4
G3-4 (%)
W5
G4-5 (%)
W6
G5-6 (%)
0.44 ± 0.24
6/32‰
8.84 ± 0.88 21.7 ± 1.4
2.72 ± 0.19b 35.6 ± 2.2
1.31 ± 0.45b 211 ± 5.2 1.28 ± 0.18b 291 ± 6.3 0.72 ± 0.41
649 ± 9.5
10/32‰
7.62 ± 0.66 20.1 ± 1.1
2.94 ± 0.16a 34.4 ± 1.7
1.49 ± 0.29b 213 ± 5.3 1.30 ± 0.18ab 301 ± 6.4 0.76 ± 0.30
708 ± 10.5 0.47 ± 0.16
20/32‰
8.03 ± 0.77 20.5 ± 1.4
2.80 ± 0.29b 37.5 ± 1.8
1.68 ± 0.62a 247 ± 5.5 1.35 ± 0.12a 357 ± 6.8 0.81 ± 0.23
793 ± 10.5 0.46 ± 0.19
32/32‰
8.97 ± 0.72 21.7 ± 1.3
2.65 ± 0.23b 42.2 ± 1.8
1.84 ± 0.21a 276 ± 5.2 1.33 ± 0.10ab 390 ± 5.8 0.79 ± 0.27
889 ± 10.4 0.47 ± 0.18
Day 0 (W1), day 33 (W2), day 69 (W3), day 208 (W4), day 251 (W5), day 424 (W6)
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Experiment 1C: The overall initial weight was 82.9 ± 2.9 g and did not differ between
experimental groups (p >0.05). During exposure to different salinities, the highest growth rate
was found in fish reared at 10‰, closely followed by fish reared at 20‰. The relationship
between the overall mean growth rate and salinity could be described using the same model as
used for the medium juveniles, predicting the growth rates to be highest at 14.8‰ (Fig. 3C).
During the recovery period, after all fish were returned to 32‰, the growth rate was lowest in
fish that had been exposed to the lowest salinity and highest in fish kept on 32‰ throughout
(Table 1).
3.1.2. Mortality
Experiment 1A: The overall mortalities of the experimental groups were between 0 and 0.5%.
No samples were collected from this fish for bacterial analysis.
Experiment 1B: The overall mortality rates among the PIT-tagged medium fish exposed to 6,
10, 20 and 32‰ were 11, 13, 7 and 7% respectively. No size-dependent mortality was found
when initial weights were compared to that of the surviving fish (p >0.05). No latent
infections were detected in the fish at any time.
Experiment 1C: Mortality rates in the groups reared at 10, 20 and 32‰ ranged between 0 and
2%, whereas 9% mortality was found in the group reared at 6‰. The highest mortality
incidence in the lowest salinity group was recorded in the first 2 days after initial weighing
(7.3%). No latent infections were detected in the fish at any time.
3.1.3. Experiment 1B: Plasma parameters
Plasma Na and Ca levels did not differ among the salinity treatment groups, neither at the
end of the low-salinity exposure period (Day 33) nor after a period back on seawater (Day
69). There is a time-dependent change in Na plasma levels, with the levels on day 33 (Fig.
5A).
Plasma cortisol levels were generally low (<10 ng ml-1) and did not differ significantly with
salinity treatment or time (Fig. 5B).
Plasma protein levels did not differ significantly between the four salinity groups on day 33
or on day 69 (Fig. 5C). An increase in the plasma protein concentration was seen with
increasing weight from 20.7 mg mL-1 on day 0 to 22 mg mL-1 on day 33 and 25 mg mL-1 on
day 69 (mean values). On day 33, compared to day 0, the increase was statistically significant
only in fish kept at 32‰ while all groups showed significant increase in protein level on day
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69 compared to day 33. For all the data there was a significant positive correlation between
the weight and the plasma protein level (r2 = 0.629, p <0.0001).
Plasma natural antibody activity was significantly higher (55%) on day 33 for the fish kept at
10‰ than fish kept at 20‰ (39%), while the differences between the other groups were
insignificant (Fig. 5D). There was no significant difference between the different groups on
day 69, mean value 52%. An increase in the natural antibody activity was seen with
increasing weight from about 28% on day 0 to 44% on day 33 and 55% on day 69 (mean
values). On day 33, compared to day 0, the increase was statistically significant only in fish
kept at 10‰, while on day 69 the natural antibody activity was significantly higher in all
groups compared to day 0 but the change was not significant when compared to day 33. For
all the data there was a significant positive correlation between weight and natural antibody
activity (r2 = 0.351, p <0.0001).
Fig. 5. Experiment 1B: Temporal changes in plasma ions (A), cortisol (B), total plasma proteins (C)
and natural antibody activity (D) in cod juveniles reared at 6, 10, 20 and 32‰ for 33 days, then reared
onwards at 32‰ for 36 days.
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3.1.4. Experiment 1C: Plasma parameters
Plasma Na and Ca levels were stable throughout the experiment (Fig. 6A).
Plasma cortisol levels at day 0 were significantly higher than at any other point during the
experiment (Fig. 6B). On day 57, plasma cortisol levels were <10 ng ml-1 for all groups
except the group exposed to 6‰ which has significantly higher cortisol levels. On day 113,
after a period in seawater, all groups had similar and low plasma cortisol levels.
Plasma protein levels did not differ significantly between the four salinity groups on day 57
or on day 113 (Fig. 6C). An increase in the plasma protein concentration was seen with
increasing weight from 26 mg mL-1 on day 0 to 38 mg mL-1 on day 57 and 45 mg mL-1 on
day113 (mean values). On day 57, compared to day 0, the increase was statistically significant
in all groups, while only fish kept initially at 6 or 20‰ showed significant increase from day
57 to 113. All the data showed a significant positive correlation between weight and the
protein level (r2 = 0.637, p<0.0001).
Plasma natural antibody activity did not differ significantly between the four salinity groups
on day 57 or on day 113 (Fig. 6D). An increase in natural antibody activity was seen with
increasing weight, from 50% on day 0 to 65% on day 57 and 82% on day 113 (mean values).
On day 57, compared to day 0, the increase was statistically significant in all groups, while
the increase from day 69 to day 113 was not significant. All the data showed a significant
positive correlation between weight and the natural antibody activity (r2 = 0.234, p = 0.0282).
Plasma anti-trypsin activity did not differ significantly between the salinity groups on day 57
or on day 113 and there was no significant change in the anti-trypsin activity between any
dates (Fig. 6E).
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Fig. 6. Experiment 1C: Temporal changes in plasma ions (A), cortisol (B), total plasma proteins (C),
natural antibody activity (D) and anti trypsin activity (E) in cod reared at 6, 10, 20 and 32‰ for 57
days, then returned to seawater and reared onwards for 56 days.
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3.2. Experiment 2: Effects of salinity and temperature on juvenile cod
3.2.1. Growth: The initial mean weights (3.43 g) were not significantly different in the groups
of cod reared at 13.5 and 32‰ (p>0.05, Fig. 7). After 69 days the fish reared at 13.5 and 32‰
weighed 36.0 ± 1.6 g and 35.3 ± 1.5 g on average and 247 ± 4.1 and 243 ± 4.0 g after further
118 days in the same salinities respectively. Thus, there were no significant differences in
mean weights or growth rates between salinity groups during phase 1. At the start of phase 2,
there were no significant differences (p>0.05) in initial mean weights between tanks of
different temperatures and salinities. The final mean weights on day 278 of fish reared at
6.3°C were 437 ± 5.8 and 425 ± 5.9 g at 13.5 and 32‰ respectively, and the mean weights of
fish reared at 10°C were 493 ± 7.0 and 510 ± 6.9 g at the same salinities respectively. There
were no significant differences in the final mean weights between salinity treatments at either
6.3 or 10°C, but fish reared at 10°C weighed significantly more on average than fish reared at
6.3°C (p<0.05, Fig. 7). The mean growth rates of fish reared at 6.3°C were 0.61 ± 0.02 and
0.59 ± 0.02%/day at 13.5 and 32‰ respectively, and were not significantly different. The
growth rates of the fish reared at 10°C, however, were significantly lower (p<0.05) at 13.5
than 32‰, or 0.73 ± 0.02 compared to 0.78 ± 0.02%/day. No significant interaction was found
between the effects temperature and salinity on growth rate (two-way ANOVA, p > 0.05).
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Fig. 7. Experiment 2: Growth of cod reared at two salinities (13.5 and 32‰) for 187 days (August to
February, Phase 1), and for further 93 days (February to May) after the fish were either acclimated to
6.3°C or 10.0°C (Phase 2). Means sharing the same letter are not significantly different at the 5%
level.
3.2.2. Mortality: The overall mortality per treatment from day 1 to 187 (phase 1) was between
0.5 to 1%. No latent infections were detected in the fish at any time. During phase 2, the
mortality rate was between 0 and 0.5% and no latent infections were detected in the fish at
any time.
3.2.3. Plasma Na and Ca levels were neither affected by salinity during phases 1 and 2, nor
by temperature during phase 2 (Fig. 8A).
3.2.4. Plasma cortisol levels were not affected by salinity during the experiment (Fig. 8B).
However, plasma cortisol levels changed significantly with time, being highest at the end of
phase 1. At the end of phase 2, the groups at 6.3°C had significantly higher plasma cortisol
levels than groups at 10°C.
3.2.5. Plasma protein levels did not differ significantly between fish kept at13.5 or 32‰ for
69 days, the mean value being 26 mg mL-1 or on day 187, the mean value being 27 mg mL-1
and the concentration had not changed significantly between the two dates (Fig. 8C).
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At the end of phase 2, on day 278, there was no statistical difference between the four groups,
the mean value being 35 mg mL-1. Compared to the protein level on day 187 the protein level
had increased in all groups on day 278 except in fish kept at 32‰ and 10°C. For all the data
in experiment 2 there was a significant positive correlation between the weight of the fish and
the plasma protein level (r2 = 0.390, p = 0.0007).
3.2.6. Plasma natural antibody activity in phase 1 and 2 were not significantly different
between fish kept at13.5 or 32‰ for 69 days, the mean value being 26 %, or on day 187, the
mean value being 38% (Fig. 8D). The change in natural antibody activity from day 69 to day
187 was not significant in either salinity group. At the end of phase 2, on day 278, there was
no significant difference between the four groups, the mean value being 52%. Compared to
the natural antibody activity at the end of phase 1, the natural antibody activity at the end of
phase 2 had increased significantly in fish kept at 32‰ salinity and 10°C but not in fish kept
at 32‰ and 6°C or in fish kept at 13.5‰ at either temperature. For all the data there was a
significant positive correlation between the weight of the fish and the natural antibody activity
(r2 = 0.507, p< 0.0001).
3.2.7. Plasma anti-trypsin activity was not significantly different between the two salinity
groups after 68 days, the mean activity being 59%, or on day 187, the mean activity being
73% (Fig. 8E). The increase in anti-trypsin activity seen on day 187 compared to day 68 was
statistically significant in both salinity groups. At the end of phase 2 there was no significant
difference between the anti-trypsin activity of the four groups, the mean activity being 46%.
Compared to the anti-trypsin activity at the end of phase 1, the activity had decreased
significantly in all groups. The level was also significantly lower than was observed in both
salinity groups on day 69.
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Fig. 8. Experiment 2: Temporal changes in plasma ions (A), cortisol (B), total plasma proteins (C),
natural antibody activity (D) and anti trypsin activity (E) in cod reared at two salinities (13.5 and
32‰) for 187 days (August to February, Phase 1), and for further 93 days (February to May) after the
fish were either acclimated to 6.3°C or 10.0°C (Phase 2)
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4. Discussion
The present study reveals a size dependent response to salinity on growth of juvenile Atlantic
cod. The highest growth rates were found at 10‰ in all size-classes (experiment 1), but
salinity had the least effect on growth of the smallest juveniles. Moreover, the pattern of
which growth rate of the smallest juveniles changed with salinity indicates that growth may
be maximized at lower salinities in small juveniles (1 – 5 g) than in medium and large
juveniles (10-100 g).
A model originally developed for analysing stock-recruitment relationships for fisheries
(Shepherd, 1982) provided a good fit for the medium and large juveniles. The curves are
relatively flat near the optimal salinity for growth and ± 3‰ deviations from the optimal
salinities (12.5 and 14.8‰) have little effects on the growth rates (0.6-1.4%). Although the
model shows adequately the steep decline in growth rate as the salinity is decreased from 10
to 6‰, it cannot be used to predict growth for lower salinities than 6‰, because the model
assumes that the growth always falls to zero at 0‰ but it is unlikely that cod tolerates much
lower salinities than 6‰ for extended period of time.
Overall, salinity had less effect on the growth rate in the present study compared to
previous studies on cod (Lambert et al. 1994; Imsland et al. 2011) and halibut (Imsland et al.
2008), while studies on turbot (Imsland et al. 2001) and spotted wolffish (Foss et al. 2001)
show comparably low effects. The largest difference in growth rate between 10 and 32‰ in
the current study was 9.8%, whereas, Lambert et al. (1994) found that growth rates in cod at
intermediate salinity (14‰) were between 14 and 63% higher than in seawater (28‰)
depending on feeding regime and season, and Imsland et al. (2011) found that the growth rate
of cod juveniles reared at 15‰ were around 14% higher than in fish reared at 32‰. The
different results between the studies are unlikely to be explained by a single factor, but rather
by complex interactions between the effects of salinity and various factors on growth due to
differences in experimental designs among the studies. Moreover, an interaction between the
effects of salinity and genetic sources (family) on growth has been observed (Imsland et al.
2011), indicating that genetic variation between cod from Canadian (Lambert et al. 1994),
Norwegian (Imsland et al. 2011) and Icelandic origins (current study) may partly explain the
different results.
In experiment 1, the growth rates in the three size classes were consistently higher in fish
kept at 10‰ compared to seawater. Therefore, it was surprising that rearing cod at 13.5‰ had
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little or no long term effects on the growth in experiment 2 (phase 1), and the same salinity
neither enhanced growth at 6.3°C nor 10°C in larger juveniles (>245g, phase 2) compared to
cod reared in seawater. The reason for this discrepancy is not clear, but it may be related to
the different time spans of the experiments. The period between the first and the second
weighting in experiment 2 was 69 days and by that time the fish were on average ten times
heavier than in the beginning of the experiment, whereas the fish in experiment 1 roughly
doubled their weight during the growth periods in different salinities. Thus, the relatively
small growth enhancing effects of rearing cod at 13.5‰ may have lasted for a short period
and fish reared in seawater may have caught up with those reared at 13.5‰ in the long run.
Atlantic cod has been found to be very tolerant of low salinities and is able to withstand
direct transfer from seawater to salinities as low as 7‰ without any mortalities or significant
indications of stress (Dutil et al. 1992). Moreover, a direct transfer of 500 g cod to 5‰ has
been found to result in only slightly increased mortalities (Provencher et al. 1993), whereas
1700-1800 g cod exposed to gradual salinity decrease started to show agitated behaviour at
4.6‰ (Odense et al. 1966). In the present study, the fish were acclimated for two days to the
different salinity regimes. No abnormal behaviour was seen in the medium and large juveniles
during the two day acclimation to the lowest salinity (6‰). However, some of the large
juveniles showed abnormal behaviour (e.g. swimming upside down) immediately after the
initial weight measurements and most of these fish were dead within few days. Although the
large juveniles showed no osmoregulatory disturbances after 57 days at 6‰, they showed
plasma cortisol levels that were around 10 ng/mL higher compared to other salinities, and also
the lowest growth rates. The same increase in cortisol levels has been found to indicate
chronic stress situation in salmonids (Pickering and Pottinger, 1989). Thus, the data suggests
that 6‰ is near the limit of tolerance for large cod juveniles of Icelandic origin and that cod is
sensitive to handling under such conditions. Similarly, survival rates after handling have been
found to be influenced by salinity in American shad (Alosa sapidissima) and striped bass
(Morone saxatilis) (Chittenden 1973; Wallin and Van Den Avyle, 1995).
There was a marked decrease in growth rate in all size-classes after the fish were exposed
to abrupt salinity increase, and the persistence of the effect was correlated to the salinity
change. For the medium sized-juveniles, an increase in salinity from 20 to 32‰ appeared to
have relatively transient effects, whereas an increase in salinity from 6 and 10‰ to 32‰
appeared to have a prolonged effect on growth rate. Thus, the specific growth rates of fish
transferred from 6 and 10‰ to seawater never exceeded the growth rate in the group at 32‰
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during the 424 day trial despite having substantially lower mean body mean weight in all
measurements. This indicates that an abrupt exposure from low salinity to seawater may
permanently reduce the growth capacity of juvenile cod. Although not examined in the
present study, it is possible that similar mechanisms exist as when anadromous salmonids are
transferred from freshwater to seawater prematurely, prior to completion of smoltification.
These salmonids cease to grow due to growth hormone (GH) resistance, indicated by
extremely high plasma GH levels (Björnsson et al., 1988, Bolton et al., 1987) caused by
down-regulation of hepatic GH receptors (Gray et al., 1992).
Although prematurely transferred salmonids may also experience osmoregulatory
problems in seawater (Björnsson et al., 1989), there is no indication that the transferred cod of
the present study are experiencing any significant disturbances of their hydromineral balance,
nor is there any indication, based on plasma cortisol levels, that abrupt transfers between
salinities induced chronic stress.
The presents study shows, that different salinities ranging from 6 to 32‰ and abrupt
increase in salinity had limited or no effects on plasma protein concentration, natural antibody
activity or anti-trypsin activity in cod. Plasma protein levels and natural antibody activity
were positively correlated to fish size in both experiment 1 and 2, and this is in accordance to
a previous study on wild Atlantic cod (Magnadottir et al., 1999 b). Despite higher body
weights, the fish in experiment 2 generally showed lower plasma protein levels and natural
antibody activity compared to fish in experiment 1B and C. Moreover, the positive correlation
between the weight of the fish and the plasma protein level was weaker in experiment 2
compared to experiment 1, whereas the correlation was stronger between weight and antibody
activity in experiment 2. This discrepancy is unlikely caused by stress or infection which is
known to influence the plasma protein and natural antibody activity in cod (Magnadottir et al.,
2001; Magnadottir et al., 2010; Magnadottir et al. 2011), and inherent factors which have
been found to influence the immune system in many species (Magnadottir 2010) are also
unlikely, since fertilized eggs and sperm were collected from many males and females in each
experiment. However, plasma protein levels in cod have been found to be influenced by
season (Magnadottir et al., 2001) and thus it is possible that the difference in protein levels in
the present study may be attributed to the larger time span of experiment 2, primarily during
the winter and spring.
Seasonal influence is also suspected in the changes observed in the anti-trypsin activity.
The anti-trypsin activity in experiment 2 was relatively high, especially in fish sampled in
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February and comparable to the samples taken in the winter in experiment 1. However,
relatively low values were seen in samples collected in May. Thus, it is likely that the reason
for this variation may be related to the large time span and the concomitant seasonal changes
in light intensity due to exposure from sunlight coming through windows on the roof (6
meters above the experimental tanks). Similar seasonal variation in anti-trypsin activity was
observed in a previous long standing (18 months) experiment on cod kept under similar
conditions (Magnadottir et al., 2001).
Various studies have shown that environmental temperature modulates the immune
system of fish (Bowden, 2008, Bowden et al. 2007). Maintaining cod at 1°C, 7°C and 14°C
for 12 months has been shown to affect the protein level, the natural antibody activity and the
anti-trypsin activity of cod serum (Magnadottir et al., 1999 a). However, the changes were
only significant between cod kept at 1°C and 14°C and not between cod kept at 7°C and
14°C. Hence, maintaining cod at 6.3°C or 10°C for 3 months had no marked effects on these
parameters in the present study.
In conclusion, the present study shows that Atlantic cod is extremely euryhaline and
showed no ion regulatory disturbances at salinities as low as 6‰, and there are no indications
that low salinities have detrimental effects on immunity-related parameters. Growth rates are
fairly similar over the salinity range studied, but the highest growth rates were estimated at
about 13.5‰. The growth was, however, only enhanced by salinity in short term experiments
but not when reared at 13.5‰ over extended time. The same salinity neither enhanced growth
at 6.3 or 10°C in larger cod (>245g). Abrupt transfer from low salinities to full-strength
seawater has clearly long-term negative effects on growth. Therefore, cod juveniles reared in
salinities lower than seawater should be given gradual acclimation to seawater if the
husbandry practice calls for a later transfer to sea-cages.
Acknowledgements
The authors thank Njáll Jónsson and Kristján Sigurdsson for taking care of the experimental
fish, Sigridur Steinunn Audunsdottir for assisting with the blood sampling and serological
analysis, Sigurdur Helgason for bacterial analysis, Linda Hasselberg for the ion analysis,
Henrik Sundh for the cortisol analysis, and Hreiðar Þór Valtýsson for help with the with
regression model analyzes. Financial support was given by the Icelandic AVS fund (R06508).
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Humoral immune parameters in Atlantic cod (Gadus morhua L.): II. The effects of
size and gender under different environmental conditions. Comparative Biochemistry
and Physiology Part B: Biochemistry and Molecular Biology 122, 181-188.
Magnadóttir, B., Jónsdóttir, H., Helgason, S., Björnsson, B., Solem, S. T., Pilström. L., 2001.
Immune parameters of immunized cod (Gadus morhua L.). Fish & Shellfish Immunology
11, 75-89.
Magnadottir, B., Audunsdottir, S.S., Bragason, B.Th., Gisladottir, B., Jonsson, Z.O.,
Gudmundsdottir, S., 2011. The acute phase response of Atlantic cod (Gadus morhua):
Humoral and cellular response. Fish & Shellfish Immunology 30, 1124-1130.
Odense, P., Bordeleau, A., Guilbault, R., 1966. Tolerance levels of cod (Gadus morhua) to
low salinity. Journal of Fisheries Research Board of Canada 23, 1465-1467.
Pickering, A.D., Pottinger, T.G., 1989. Stress responses and disease resistance in salmonid
fish: effects of chronic elevation of plasma cortisol. Fish Physiol. Biochem. 7, 253-258.
Pilstrom, L., Warr, G.W., Stromberg, S., 2005. Why is the antibody response of Atlantic cod
so poor? The search for a genetic explanation. Fisheries Science 71, 961-971.
Provencher, P., Munro, J., Dutil J.-D., 1993. Osmotic performance and survival of Atlantic cod
(Gadus morhua) at low salinities. Aquaculture 116, 219-231.
Shepherd, J.G., 1982. A versatile new stock-recruitment relationship for fisheries, and the
construction of sustainable yield curves. J. Cons.Int. Explor. Mer. 40(1), 67-75
Sundh, H., Calabrese, S., Jutfelt, F., Niklasson, L., Olsen, R.E., Sundell, K., 2011.
Translocation of infectious pancreatic necrosis virus across the intestinal epithelium of
Atlantic salmon (Salmo salar L.). Aquaculture 321, 85–92.
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Wallin, J.E., Van Den Avyle, M.J., 1995. Interactive effects of stocking site salinity and
handling stress on survival of striped bass fingerlings. Transactions of the American
Fisheries Society 124, 736-745.
Weyts, F.A.A., Cohen, N., Flik, G.,Verburg-Van Kemenade, B.M.L., 1999. Interactions
between the immune system and the hypothalamo-pituitary-interrenal axis in fish. Fish
& Shellfish Immunology 9, 1-20.
Young, G., 1986. Cortisol secretion in vitro by the interregnal of coho salmon (Oncorhynchus
kisutch) during smoltification: relationship with plasma thyroxine and plasma cortisol.
Gen. Comp. Endocrinol. 63, 191– 200.
Young, G., Björnsson, B.Th., Prunet, P., Lin, R.J., Bern, H.A., 1989. Smoltification and
seawater adaptation in coho salmon (Oncorhynchus kisutch): Plasma prolactin, growth
hormone, thyroid hormones, and cortisol. General and Comparative Endocrinology 74,
335-345.
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Chapter II
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On growing of Atlantic cod (Gadus morhua) following direct
transfer from different temperatures and salinities to seawater
Abstract
The growth of Atlantic cod was monitored following transfer from different
salinity/temperature combinations (13.5‰/6°C, 13.5‰/10°C, 32‰/6°C and 32‰/10°C) to
seawater. The fish from the salinity/temperature groups were either transferred to a sea-cage
(A) or to seawater in indoor tanks (B).
A: Approximately 70% of the fish in each salinity/temperature group were transferred to the
sea-cage on 20th May when the salinity and temperature in the fjord was 35‰ and 6.2°C. All
groups showed almost no growth in the sea-cage. However, the highest mean growth was
found in the group exposed to the least changes in environmental conditions (32‰/6°C) and
the lowest mean growth rate was found in the group of fish subjected to changes in both
temperature and salinity (13.5‰/10°C).
B: The remaining fish from each salinity/temperature group were directly transferred to two
tanks with seawater, one at 7.2°C and the other at 10°C. The fish previously kept at 6°C were
transferred to the tank at 7.2°C. Cod subjected to abrupt increase in salinity from 13.5‰ to
seawater showed significantly lower final mean weights and specific growth rates compared
to the groups exposed to no change in salinity in seawater. The effects of the abrupt increase
in salinity on growth were not different between temperatures.
Introduction
Several authors have studied the influence of salinity on growth and various physiological
factors in Atlantic cod (Gadus morhua) (Lambert et al. 1994; Imsland et al. 2011; Árnason et
al. in press). In most cases, the highest growth rates and feed conversion efficiencies were
found at intermediate salinities around 15‰, but the benefits of reduced salinities on growth
were highly variable between authors. Using brackish water as a rearing medium for cod
juveniles may have practical implications for the farming of juvenile cod in land-based farms
but under normal circumstances cod juveniles are eventually transferred to sea-cages in order
to limit the production costs. Most studies on the ability of fish to tolerate transfer from fresh
to seawater have been done on salmonids (see reviews by Folmar and Dickhoff 1980;
McCormick and Saunders 1987) whereas, little is known about the effects of direct transfer
from low salinities to seawater on euryhaline marine fish. In the preceding study (chapter I)
we found that direct transfer of cod juveniles (20 g) from 6 and 10‰ to seawater resulted in
substantial long-term reduction in growth rates (Árnason et al. in press). The aim of this study
is to investigate effects of abrupt transfer from different salinity-temperature combinations on
the growth, plasma ions and immune parameters in larger cod (423-532 g).
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Materials and methods
Experimental design
In chapter I, two experiments were described. In experiment 1 the optimal salinities for
growth were found for two size-classes of cod and in experiment 2 the long term benefits of
rearing cod at optimal salinity vs. seawater were evaluated. In this chapter, we present results
from two follow up trials (A and B) which were carried out after the termination of
experiment 2.
A. In trial A approximately 70% of the fish in experiment 2 were transferred to a sea-cage
in Álftafjörður, NW Iceland (Fig. 1, A).
B. In trial B the remaining fish were transferred to tanks with seawater at either 7.2°C or
10°C (Fig. 1, B) with no acclimatisation for the fish kept at 13.5‰.
Since both trials were carried out in continuation of experiment 2, we refer to day 278 as the
first day in the trials. The main focus will be on the period after the fish were transferred to
sea-water from day 278 to 420, but the growth in the period from day 1 to 278 will also be
presented. Overview of the experimental design is given in Figure 1.
Figure 1. Overview of the experimental design. The letters A and B denote the trials presented here.
Trial A: On growing in a sea-cage
At the start of trial A on day 278, 108-115 of PIT-tagged fish from each of the four salinitytemperature combination groups (13.5‰/6.3°C, 13.5‰/10.0°C, 32‰/6.3°C and 32‰/10.0°C)
in experiment 2 were transported from Grindavík to Súðavík by a fish transport vehicle in
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tanks containing 7°C sea-water (32‰). The groups were not acclimatized to the different
temperature and/or salinity in the fish transport tanks and were therefore subjected to abrupt
changes in temperature and/or salinity. At the arrival to Súðavík the fish were transferred by a
boat in tanks with seawater (35‰ and 6°C) to a sea-cage (20 m circumference and 6 m deep)
in the fjord, Álftafjörður and kept there from 20th May to 5th October 2010 (139 days). The
temperature which was measured two meters below the surface at the sea-cage, 6 days a week
ranged between 6.2 and 13.4°C (mean = 9.9°C) (Fig. 2). The fish were hand fed on dry feed
(Laxá Ltd.) according to appetite two times a day and 6 days a week. All fish were
slaughtered at the termination of the trial and transported to Reykjavík where whole body
weight (g), total length (cm) and PIT-number of each individual were recorded. All fish had
been measured previously on day 69, 187 and 278 (Experiment 2, Fig. 1). The data from these
measurements were used to trace the growth performances of each group back to the tagging
day (day 69).
16
14
Temperature (°C)
12
10
8
6
4
2
0
20-May
09-Jun
29-Jun
19-Jul
08-Aug
28-Aug
17-Sep
07-Oct
Day
Figure 2. The temperature profile, two meters below the surface at the sea-cage in Álftafjörður from 20th
May to 5th October 2010.
Trial B: On growing in sea-water in tanks at 7.2 and 10.0°C
At the start of trial B on day 278 (Fig. 1), subsamples of 24-29 tagged fish from each tank (49
– 56 per salinity-temperature group) were randomly selected and transferred to two 5300 L
tanks with seawater, one at 7.2°C and the other at 10°C. The fish previously kept at 6°C were
transferred to the tank at 7.2°C. Thus, half of the fish were exposed to abrupt increase in
salinity from 13.5‰ to 32‰ at either 7.2°C or 10°C whereas the other half experienced little
or no change in salinity or temperatures. After 139 days (on day 420), all the experimental
fish were killed by anaesthesia and their weight and length recorded. Blood samples were
collected from 12 fish from each group (i.e. 24 samples per tank). Plasma ion levels (Na and
Ca), the natural antibody activity and plasma protein levels were measured in all blood
samples collected using the same methods as described in chapter I.
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Results and discussion
Trial A: On-growing in a sea-cage
The fish exhibited high growth-rates in the period at the Marine Research Institute (day 1-278,
Fig. 3). The mean weight on day 278 in the lower temperature groups were 449 and 436 g in
fish acclimated to 13.5‰ and 32‰ respectively, and 508 and 526 g in the higher temperature
groups at the same salinities respectively. Soon after the fish were transferred to the sea-cage
it became evident that they were not behaving normally. Under normal circumstances cod will
swim towards the surface when feed is being dispersed into sea-cages. In the present study,
however, the fish seemed stressed and remained close to the bottom of the cage at all times.
Thus, it is likely that most of the feed sunk through the cage floor before it was eaten. As a
result, the growth in most individuals stagnated and many fish lost weight (Fig. 3). We can
only speculate as to what caused the abnormal behaviour in the fish, but it is very likely
related to the small size of the cage. For example, wind and sunlight has greater effect on the
fish in shallow cages such as the one used here (6 m deep) with a diameter of only 6 m
compared to conventional sea-cages which are much deeper and larger in diameter. Moreover,
the accessibility to the feed was sometimes limited because of strong currents which carried
the feed through the cage walls before it reached the bottom.
800
700
13.5‰/10°C
32‰/10°C
13.5‰/6°C
32‰/6°C
Untagged
Weight (g)
600
500
400
Fish transferred
to sea-cage
a
ab
bc
c
Temperature
changed
300
200
100
0
0
50
100
150
200
250
300
350
400
450
Days
Figure 3. Growth of cod reared at two salinities (13.5 and 32‰) for 187 days, then for 93 days after the
fish were either acclimated to 6.3 or 10.0°C, and for 139 days after the fish were transferred to a sea-cage.
All fish were tagged on day 69. There was no difference in growth rates between salinities before the fish
were tagged on day 69. Means sharing the same letter in a row are not significantly different (P>0.05).
Despite very low growth rates in the sea-cages there were marked differences in growth
between groups. Fish acclimated to 32‰ and 6°C gained more weight in the sea-cage than the
fish acclimated to 32‰ and 10°C (Fig. 3.), suggesting that the transfer from 10.0°C to the
relatively low ambient temperature (6.2°C) had negative effect on the growth. The group
exposed to the least changes in environmental conditions (32‰/6°C) exhibited the highest
mean growth rate in the sea-cage (G = 0.09%), followed by the group acclimated to 10°C and
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Lokaskýrsla - Final activity report R-065-08
32‰ (G = 0.04%). The groups subjected to abrupt increase in salinity from 13.5‰ to
seawater showed the lowest mean growth rates, or 0.01% and 0.00% for the groups
transferred from 6°C and 10°C to seawater, respectively (Fig. 4). Thus, the negative effect of
abrupt increase in salinity on growth was greater than the abrupt decrease in temperature, and
the lowest growth was found for the group subjected to changes in both temperature and
salinity. The mortality in the sea-cage was from 1 to 7 fish per treatment (1 to 6 %).
0.09
c
Specific growth rate (%/day)
0.08
0.07
0.06
0.05
0.04
a
0.03
0.02
0.01
a
a
0.00
13.5‰/10.0°C
32‰/10.0°C
13.5‰/6.3°C
32‰/6.3°C
Figure 4. Specific growth rates in groups transferred from different salinity/temperature combinations to
the sea-cage. Means sharing the same letter in a row are not significantly different (P>0.05).
Trial B: On growing in sea-water in tanks at 7.2 and 10.0°C
As in trial A the fish exhibited high growth-rates in the period at the Marine Research
Institute (day 1-278, Fig. 5). The mean weights on day 278 in the lower temperature groups
were 423 and 430 g in fish acclimated to 13.5 and 32‰ respectively, and the mean weights in
the higher temperature groups were 532 and 510 g in fish acclimated to the same salinities
respectively. There were no significant differences in mean weights on day 278 between
salinity groups within the temperature treatments (ANOVA, P>0.05). The final mean weights
(day 420) of the fish exposed to abrupt increase in salinity from 13.5 to 32‰ at 7°C was 872
g while the mean weight of fish reared at a relatively constant salinity of 32‰ was 942 g. At
10°C the final mean weight of the group exposed to the same increase in salinity was 1040 g
and the final mean weights of the group reared at a constant salinity in seawater was 1080 g.
At both temperature treatments, there were no significant differences in the final mean
weights between fish subjected to increase in salinity from 13.5 to 32‰ compared to the
groups exposed to no change in salinity in seawater (P<0.05, Fig. 5). However, the mean
specific growth rates in fish subjected to increase in salinity were significantly lower than in
fish kept at constant salinity in seawater (P>0.05). The mortality ranged between 1 and 3 fish
per treatment from day 278 to 420 (2 – 6%). At the end of the trial there were no significant
differences in plasma Na (mean = 164 mM), Ca (2.9 mM), protein levels (33 mg mL-1) or
natural antibody activity (64%) between salinities or temperatures.
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1200
800
Weight (g)
a
ab
bc
c
13.5‰/10°C
32‰/10°C
13.5‰/6°C
32‰/6°C
Untagged
1000
Salinity
increased
600
Temperature
changed
400
200
0
0
50
100
150
200
250
300
350
400
450
Days
Figure 5. of cod reared at two salinities (13.5 and 32‰) for 187 days, then for 93 days after the fish were
either acclimated to 6.3°C or 10.0°C, and for 139 days after the fish were trasferred to sea-water at either
7.2°C or 10.0°C. Means sharing the same letter in a row are not significantly different (P>0.05).
0.58
c
Specific growth rate (%/day)
0.56
0.54
abc
ab
0.52
0.5
0.48
a
0.46
0.44
0.42
13.5‰/10.0°C
32‰/10.0°C
13.5‰/6.3°C
32‰/6.3°C
Figure 6. Specific growth rates in groups transferred from different salinity/temperature combinations to
tanks with seawater at either 10°C or 7.2°C. Means sharing the same letter in a row are not significantly
different (P>0.05).
Concluding remarks



Transfer from 13.5‰ to seawater had negative effect on growth rate but the effects
were not significantly different between temperatures.
Acclimatization is important when cod are transferred from intermediate salinity to
seawater.
There were no significant differences in plasma ion levels, plasma protein levels or
natural antibody activity between salinity or temperature groups.
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References
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Folmar, L.C., Dickhoff, W.W., 1980. The parr-smolt transformation (smoltification) and
seawater adaptation in salmonids. A review of selected literature. Aquaculture 21: 1-37.
Imsland, A.K., Koedijk, R., Stefansson, S.O., Foss, A, Hjörleifsdóttir, S., Hreggviðsson, G.Ó.,
Otterlei E., Folkvord, A. 2011. A retrospective approach to fractionize variation in body
mass of Atlantic cod Gadus morhua. Journal of Fish Biology 78, 251-264.
Lambert, Y., Dutil, J.-D., Munro, J., 1994. Effects of intermediate and low salinity conditions
on growth rate and food conversion of Atlantic cod (Gadus morhua). Can. J. Fish .
Aquat. Sci. 51, 1569-1576.
McCormick, S.D., Saunders, R.L. 1987. Preparatory physiological adaptations for marine life
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Acknowledgements
The authors would like to express their thanks to the staff of the company Hraðfrystihúsið
Gunnvör for taking care of the fish in the sea-cage, and we thank Mr Njáll Jónsson, Mr
Kristján Sigurdsson and Mr Matthías Oddgeirsson, Marine Research Institute, for their
assistance with the experiment in Grindavík. We also thank Sigridur Steinunn Audunsdottir,
Keldur, for assisting with the blood sampling and serological analysis and Linda Hasselberg
for the ion analysis. This study was financed by the Icelandic AVS fund (R065-08).
42