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Growth and oxygen consumption in normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolffish (Anarhichas minor Olafsen)
Aquaculture 224 (2003) 105 – 116 www.elsevier.com/locate/aqua-online
Growth and oxygen consumption in normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolffish (Anarhichas minor Olafsen) A. Foss *, T. Vollen, V. Øiestad Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Received 10 May 2002; received in revised form 13 February 2003; accepted 18 February 2003
Abstract The effects of dissolved oxygen (DO) concentration combined with sublethal doses of ammonia on growth and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish. Fish with a mean (S.D.) initial weight of 95.4 (18.0) g were reared in shallow raceways (initial stocking density 10 kg m 2) at normoxic (9.6 mg l 1) and hyperoxic (14.5 mg l 1) conditions, and also under both normoxia and hyperoxia with an additional sublethal concentration of unionised ammonia (0.17 mg NH3 l 1) added, for 8 weeks at 8 jC. There was an interacting effect of oxygen saturation and ammonia level on growth, as growth rate was significantly higher in the hyperoxic/NH3 group compared to the normoxic/NH3 group ( P < 0.001), suggesting that hyperoxic conditions may increase tolerance to unionised ammonia in spotted wolffish. At the end of the experiment, no difference in mean weight was found between fish reared at normoxic and hyperoxic conditions without added ammonia, whereas mean weight was reduced under normoxia with added ammonia as compared to normoxia without added ammonia. Mean daily oxygen consumption was investigated in the nonammonia groups at both normoxic and hyperoxic conditions, and O2 consumption was significantly higher ( P < 0.001) under hyperoxic (100.5 mg O2 kg 1 h 1) compared to normoxic (79.9 mg O2 kg 1 h 1) conditions. A clear diurnal rhythm in the O2 consumption pattern was seen at both O2 saturations, with the most striking observation being a dramatic increase in the morning when the light was turned on. Overall, our results support earlier findings of a high capacity to adapt to variations in environmental water quality parameters, and to restore normal growth rate in the spotted wolffish. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Spotted wolffish; Growth; Oxygen consumption; Hyperoxia; Ammonia * Corresponding author. Present address: Akvaplan-niva, Bergen Office, Nordnesboder 5, 5005 Bergen, Norway. Tel.: +47-55-302250; fax: +47-55-302251. E-mail address: [email protected] (A. Foss). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00209-6
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1. Introduction Spotted wolffish, Anarhichas minor Olafsen, a marine cold-water species, is well suited for farming in intensive land-based rearing systems. To effectively exploit such systems, it is important to determine the environmental water quality standards required by a species. The spotted wolffish has previously exhibited high tolerance to environmental changes in salinity (Foss et al., 2001), dissolved oxygen (DO) content (Foss et al., 2002) and carbon dioxide (Foss et al., 2003), but at high densities in a reuse system, that is, shallow raceways in racks with reuse of water between levels, there will be a gradual decrease in DO and pH, concurrent with a gradual accumulation of natural catabolites such as ammonia and carbon dioxide (Person-Le Ruyet et al., 1997b), which separately or together may affect production characteristics such as growth performance, food conversion efficiency (FCE) and health status. Ammonia is the main end product of nitrogen metabolism in teleosts (Foster and Goldstein, 1969), and it exists in both ionised (NH4+) and unionised (NH3) forms. The toxicity of ammonia to fish and other aquatic organisms is primarily attributed to the unionised form. In intensive reuse systems, ammonia concentrations may increase to levels that can cause reduced growth or even death (Person-Le Ruyet et al., 1997a). Several studies on freshwater fish (Lloyd, 1961; Alabaster et al., 1979; Thurston et al., 1981) and one on gilthead seabream, Sparus aurata (Wajsbrot et al., 1991), have demonstrated an increase in ammonia toxicity at reduced levels of DO. However, in intensive rearing facilities, benefits can be obtained from supplying rearing units with water supersaturated with oxygen as this allows for higher stocking densities, and also maximises utilisation of the water and thus reduces pumping costs. Furthermore, the potential increase in ammonia toxicity caused by low DO will be eliminated. It has been hypothesised that oxygen levels above normal saturation might reduce ammonia toxicity, and Colt et al. (1991) called for an assessment of ammonia toxicity to fish in water with oxygen content ranging from 10 to 30 mg O2 l 1. Although studies on the effects of hyperoxia in fish are limited, high DO has previously been found not to affect growth in spotted wolffish (Foss et al., 2002), turbot, Scophthalmus maximus L., (PersonLe Ruyet et al., 2002) and in some salmonid species (Doulos and Kindschi, 1990; Edsall and Smith, 1990; Caldwell and Hinshaw, 1994). A preliminary study on the effects of ammonia on growth in spotted wolffish at normoxic levels (Foss, unpublished work) indicated a threshold level between 0.07 and 0.2 mg NH3 l 1 at which growth was reduced. The present study was designed to investigate whether O2 concentrations above normal saturation affected tolerance to sublethal doses of unionised ammonia in spotted wolffish. In addition, oxygen consumption was measured to study how this was influenced by water oxygen content.
2. Materials and methods 2.1. Experimental conditions and origin of fish The eggs from two females inseminated by sperm from six males were incubated from September 2000 at the experimental facilities of the Norwegian College of Fishery
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Science at Ka˚rvika, Tromsø, and hatched in January 2001. After hatching, the larvae were transferred to small raceways with rearing chambers (15 30 cm) (Hansen and Falk-Petersen, 2001) and immediately weaned on a formulated dry food (Nutra Pluss, Skretting) containing 53% protein, 20% fat and 10% carbohydrate. Subsequently, the fish were reared in shallow raceways (0.4 2.2 m) (Øiestad, 1999) with a water level of 8 –10 cm, providing a total volume of 0.08 m3 (80 l) and fed on a commercial formulated floating food (Dan-ex 1547, Dana Feed, Horsens, Danmark), containing 47% protein, 15% fat and 21% carbohydrate (pellet size 4 mm) until and during the experiment. The raceways were supplied with running seawater (salinity 33x, pH 8.0), and water flow was set to 5 l min 1 for each of the experimental units, providing a current speed of 0.25 cm s 1 in the raceways. Two 25-W light bulbs dimmed to 5% of maximum capacity supplied the raceways with low-level light during the experiment. Photon irradiance measured at the raceway bottom was on average 0.2 Amol m 2 s 1, and all groups were maintained under a constant photoperiod of LD 18:6, including 0.5h twilight periods. 2.2. Experimental design On 18 December 2001, 229 fish were tagged intraperitoneally with TrovanR Passive Transponder tags, and on 14 January 2002, the fish (n = 399) were anaesthetised (benzocaine, 0.05 g l 1), weighed individually and total length recorded to the nearest 0.1 g and 0.1 cm, respectively, for all fish. No fish died during tagging. The initial mean weight (S.D.) was 95.4 (18.0) g and did not differ significantly between raceway units. Subsequently, weights and total lengths were recorded after 4 and 8 weeks. Mean initial and final (S.D.) stocking densities in the raceways were 10.2 (0.2) and 13.0 (0.7) kg m 2, respectively, (the fish were restricted to an area of 0.4 1.2 m). The fish were distributed randomly into eight raceways and gradually acclimated to the experimental conditions over 5 days. Treatments consisted of a normoxic group (control), a hyperoxic group, a normoxic group with a sublethal dose of ammonia added and the same sublethal dose added in a hyperoxic group. All treatment groups consisted of fish from two replicate raceways. The requested NH3 levels were obtained by adding a concentrated solution of NH4Cl (100 g NH4Cl l 1 fresh water) by two electromagnetic metering pumps (EH/W, Iwaki) to the header tanks supplying the respective raceways with water. Total ammonia nitrogen (TAN) was measured daily with an ammonia gas sensing combination electrode (Thermo Orion, Model 95-12) connected to an expandable ion analyser (Thermo Orion, EAk920), and the percentage of NH3 was calculated using the equation of Bower and Bidwell (1978), which gives the NH3/TAN ratio as a function of pH, temperature and salinity. Hyperoxic conditions were obtained by passing the inflowing water through an oxygenation cone (Oxy-lab, AGA, Norway) where pure O2 was added. Oxygen level was measured near the water inlet every 10 min using O2 sensors (InPro 6050/120) connected to an O2 transmitter (Mettler Toledo O2 4050). The signals from the transmitter were stored, via a data-logger (Almemo 5590-0), in a computer program (DataControl). Water temperature was set to 8 jC and remained within (S.D.) 0.2 jC of that prescribed. DO levels were set to 9.6 mg l 1 (control) and 14.5 mg l 1 and remained within (S.D.) 0.5 mg l 1, while TAN concentrations averaged 13.5 mg
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l 1, giving a NH3 concentration of 0.17 mg l 1 in the raceways. NH3 concentrations remained within (S.D.) 0.02 mg l 1 of that prescribed throughout the study. Food was provided in excess for 2 h daily (between 0730 and 0830 h and between 1330 and 1430 h), except for Saturdays (fed once) and Sundays (not fed). The food was distributed from automatic feeders, situated close to the water inlet thus enabling the pellets to float downstream. Uneaten pellets were collected 30 min after feeding in the morning and afternoon and counted immediately to measure food intake. All growth estimates in the present study are based on individually tagged fish, whereas results on food conversion efficiency, food consumption, daily feeding rate and protein efficiency ratio (see below) are based on the total biomass. 2.3. Growth, food utilisation and biomass gain Specific growth rate (SGR) was calculated according to the formula of Houde and Schekter (1981): SGR=(e g 1)100, where g=(lnW2 lnW1)(t2 t1) 1, and W2 and W1 are weights (g) at days t2 and t1, respectively. The condition factor (CF) was defined as: CF = 100WLT 3, where W is the weight of the fish and LT the corresponding total length. Food conversion efficiency (FCE) was calculated as: FCE=(W2 W1)C 1 (Kinghorn, 1983), where C is food intake. Total food consumption (CT) was calculated as the difference between food supplied and food waste for the whole experimental period. Daily feeding rate ( F) was calculated from: F = 100CTW 1, where W¯ is the mean daily fish weight over each of the experimental periods. Protein efficiency ratio (PER) was calculated as: PER = weight gain/protein consumed. 2.4. Plasma parameters Blood was sampled from eight fish in each experimental group at start and after 4 and 8 weeks to measure plasma osmolality and plasma chloride levels. Blood (0.4 –0.5 ml) was extracted from the caudal vessels using heparinised syringes, and collected into Eppendorff tubes. The blood was centrifuged for 10 min at 14 000 rpm, and the plasma samples were stored at 80 jC. Plasma osmolality was measured using a Fiske One-Ten osmometer (Fiske Assoc., MA, USA) and plasma chloride levels were determined using a chloride titrator (Corning 925, CIBA Corning Diagnostics). 2.5. Oxygen consumption Oxygen consumption was measured at normoxic and hyperoxic conditions without ammonia present. The specific O2 consumption rate (VO2, mg kg 1 h 1) was calculated according to the formula: V O2 ¼ ðVF dpO2 ÞðBÞ1 (Jobling, 1982; Brix, 1992), where VF is the water flow (l h 1) through the tank, dpO2 the difference in O2 concentration between the inlet and outlet water and B is the total biomass in the tank. The calculations were based on 24-h recordings of O2 levels at the inlet and
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outlet of replicated raceways at 10-min intervals. Measurements of O2 consumption were performed on 22 – 24 January. The fish were not fasted before this period. Corrections for O2 variations between inlet and outlet were performed in raceways without fish after termination of the experiment. 2.6. Statistical methods All statistical analyses were performed with STATISTICAk 5.5 (Statsoft, 1995). To assess normality of distributions, a Kolmogorov – Smirnov test (Zar, 1996) was used, and homogeneity of variances were tested using Levene’s F test (Brown and Forsythe, 1974). Analysis of weight, condition factor, SGR and blood plasma parameters on the various sampling dates were conducted using a three-way nested Model III ANOVA (Zar, 1996), where the replicates were nested within O2 and NH3 levels. Variations in mean daily oxygen consumption between O2 treatments were analysed using a two-way nested Model III ANOVA. For parameters where only group data existed (FCE, PER, F and CT), a two-way ANOVA was applied to test for overall differences between groups. Significant ANOVAs were followed by a Student – Newman – Keuls multiple comparison test (Zar, 1996) to identify differences among treatments. A significance level (a) of 0.05 was used if not stated otherwise.
Fig. 1. Mean F S.E. specific growth rates (SGR) of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia. Different letters denote significant differences (two-way nested ANOVA, P < 0.05) between treatments in each growth period.
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Treatment
Weight
O2 (mg l 1)
NH3 (mg l 1)
n
9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17
56 59 55 59
Length
Date
Condition factor
Date
14 January
11 February
95.5 94.1 95.0 94.5
117.9 101.3 113.2 109.4
(18.7) (15.2) (17.9) (20.1)
(21.2)a (15.6)b (25.4)a (24.5)a
11 March 151.6 131.6 148.3 140.9
(33.7)a (23.1)b (40.6)a (35.8)ab
Date
14 January
11 February
11 March
14 January
11 February
21.5 21.4 21.4 21.4
22.3 21.9 22.1 22.2
23.7 23.1 23.5 23.5
0.95 0.96 0.95 0.95
1.05 0.95 1.03 0.98
(1.1) (1.0) (1.1) (1.3)
(1.1) (1.0) (1.2) (1.4)
(1.3) (1.1) (1.5) (1.6)
(0.07) (0.05) (0.06) (0.06)
(0.08)a (0.06)b (0.10)a (0.08)b
11 March 1.11 (0.11)a 1.05 (0.07)b 1.11 (0.13)a 1.07 (0.10)b
Results are given as mean (S.D.); n denotes number of fish in each experimental group. All results include the data for two replicate raceways. Different letters denote significant differences (Student – Newman – Keuls multiple comparisons, P < 0.05) between treatments.
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Table 1 Mean weight (g), length (cm) and condition factor of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia
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3. Results 3.1. Growth and plasma parameters The initial mean weight of the fish was not significantly different between treatment groups, and no mortality occurred in any of the experimental groups throughout the study. In the first experimental period, there was a significant interaction between ammonia level and oxygen saturation on growth (three-way nested ANOVA, P < 0.001, Fig. 1), and growth rates were significantly higher in the hyperoxic/NH3 group compared to the normoxic/NH3 group (Student – Newman –Keuls test, P < 0.001). In this period, ammonia levels of 0.17 mg NH3 l 1 significantly reduced growth rates ( P < 0.0001), while no differences in growth rates were observed between treatment groups in the second period. At normoxic conditions, a significant difference in mean weight was found between the normoxic and the normoxic/NH3 treatment from 11 February and onwards (three-way nested ANOVA, P < 0.01, Table 1), while condition factor varied between ammonia/nonammonia groups at both normoxic and hyperoxic conditions in the same period ( P < 0.001). An interactive effect of ammonia level and oxygen saturation was found for both mean weight and condition factor on 11 February ( P < 0.05). Food conversion efficiency, protein efficiency ratio, daily food intake and total food consumption did not vary significantly between treatments (Table 2). Plasma chloride and plasma osmolality levels were affected by ammonia (Table 3). Levels were significantly higher in both the normoxic/NH3 and hyperoxic/NH3 group compared with the non-ammonia groups on 11 February ( P < 0.001). On 11 March, both plasma chloride and plasma osmolality levels had decreased to that of the normoxic group, except for osmolality level in the normoxic/NH3 group which was significantly lower than in all other groups ( P < 0.05). 3.2. Oxygen consumption Oxygen consumption was significantly influenced by O2 saturation level (two-way nested ANOVA, P < 0.001). Mean daily oxygen consumption in the hyperoxic group averaged 100.5 mg O2 kg 1 h 1 compared to 79.9 mg O2 kg 1 h 1 in the normoxic
Table 2 Food conversion efficiency (FCE), protein efficiency ratio (PER), daily feeding rate ( F) and total food consumption (CT) of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia Treatment
FCE
O2 (mg l 1)
NH3 (mg l 1)
9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17
1.16 1.05 1.05 1.10
PER
(0.18) (0.61) (0.36) (0.37)
2.48 2.24 2.23 2.34
F
(0.37) (1.30) (0.77) (0.80)
Results are given as mean (S.D.); n = 4 for each factor combination.
0.55 0.36 0.49 0.45
CT (g) (0.14) (0.17) (0.16) (0.16)
885.0 541.9 762.2 689.8
(315.6) (273.4) (295.7) (289.9)
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Table 3 Plasma osmolality (mOsmol kg 1) and plasma chloride (mmol l 1) concentrations in spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia Measured parameter
Plasma osmolality
Plasma chloride
Treatment group O2 (mg l 1)
NH3 (mg l 1)
9.6 9.6 14.5 14.5 9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17 0.0004 0.17 0.0004 0.17
14 January
11 February
350 (7.8)
348.0 359.3 348.8 367.4 152.2 158.9 152.2 155.9
150.5 (3.3)
(9.6)c (8.2)b (3.9)c (8.5)a (3.0)b (4.4)a (3.6)b (3.6)ab
11 March
348.7 340.6 353.4 350.4 153.0 150.6 150.7 150.9
(4.5)a (6.9)b (10.8)a (8.0)a (2.6) (2.4) (3.7) (1.4)
Values are given as mean (S.D.) (n = 8). Different letters denote significant differences ( P < 0.05) between treatment groups.
group. The fish exhibited a clear diurnal rhythm in their O2 consumption in both groups (Fig. 2), with a dramatic increase in the morning as the light was turned on, followed by a decrease as the fish started feeding. Subsequently, O2 consumption increased in both groups 2 –4 h after feeding, and reached a peak after approximately 6 h. A minimum in consumption was reached during late at night in both groups.
Fig. 2. Diurnal patterns in O2 consumption of spotted wolffish under normoxic (9.6 mg l 1) and hyperoxic (14.5 mg l 1) conditions. Each point represents 18 measurements. Values are given as mean F S.E. Arrows mark feeding time. Shaded areas on the x-axis indicate periods of darkness.
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4. Discussion Tolerance to chronic unionised ammonia exposure varies widely between species, with threshold values for long-term negative effects on growth in fish ranging from 0.01 mg NH3 l 1 in salmonids (Arillo et al., 1981) to 0.30 mg NH3 l 1 in gilthead seabream (Wajsbrot et al., 1993). In the present study, growth was significantly reduced in the first growth period in both groups receiving water with an added concentration of 0.17 mg NH3 l 1, and a moderate but significant increase in plasma osmolality and plasma chloride levels observed on 11 February indicate that the fish need some time to adapt to the increased water ammonia content. However, the high growth rates seen in the second period, combined with a recovery with respect to hydromineral balance, demonstrate that the species has a high capacity to adapt to lasting changes in environmental factors. Previous studies on spotted wolffish have exhibited a high capacity for the species to adapt to both hypoxia and hyperoxia (Foss et al., 2002) as well as changes in environmental hypercapnia (Foss et al., 2003) and salinity (Foss et al., 2001). In the present experiment, growth rates increased in both the normoxic and hyperoxic groups from period 1 to period 2, in contrast to what is usually reported, that is, growth rates decline with increasing fish size. The increase was significant in the hyperoxic group, supporting the foregoing assumption that the fish need some time to adapt to environmental changes. The nonsignificant increase in the normoxic group demonstrates a typical pattern often observed in spotted wolffish. They will adapt to a wide variety of environmental conditions, just given enough time. However, they are sensitive to changes in their daily routines (e.g., temperature and light conditions, feeding regime and tank environment) and may respond with a decrease in food intake (personal observation). Thus, it is possible that moving the fish from the holding facilities to the experimental facilities approximately 4 weeks in advance of start-up, although conditions were almost identical, might have affected food intake even into the first test period. A similar observation was made by Foss et al. (2002) where an increase in spotted wolffish growth rates with increasing fish size was attributed to a change in pellet size 2 weeks before the start of the experiment, a transitional period that may have been too short and which limited food intake in the first growth period. Growth in the hyperoxic/NH3 group was significantly higher compared to the normoxic/NH3 group in the first period, which poses the potentially important question, ‘‘Can tolerance to ammonia be increased by rearing fish at hyperoxic conditions ?’’ The idea was put forward by Colt et al. (1991) who called for an assessment of ammonia toxicity to fish in O2 supersaturated water. It is an established fact that toxicity of ammonia increases with decreasing oxygen level (Lloyd, 1961; Alabaster et al., 1979; Thurston et al., 1981; Wajsbrot et al., 1991), but this is, to the authors’ knowledge, the first study indicating that ammonia tolerance can be increased by rearing fish at DO levels above normal saturation. In general, using water supersaturated with oxygen makes it possible to increase the carrying capacity of a fish culture system, provided that oxygen is the most limiting factor. This may, however, not always be the case, as pointed out by Colt et al. (1991), as there are several other water quality criteria that must be fulfilled in an oxygensupplemented system. The biological effects of increased loading and density may include increased levels of suspended solids, CO2 and ammonia, as well as a potential risk of
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exposing the fish to hyperoxic conditions that are toxic to the fish. Toxicity of oxygen to an organism will depend on species, life stage, environmental conditions and on physiological and nutritional history, but a hyperoxic toxic threshold will exist for all organisms (Colt et al., 1991). The hyperoxic levels used in the present study have previously been shown not to cause any damaging effects in spotted wolffish (Foss et al., 2002), and are also well below the levels put forward as safe by Colt et al. (1991). PersonLe Ruyet et al. (2002) found that O2 saturations of 11.4 and 17.5 mg l 1 did not significantly increase growth in turbot, and similar results have been found for other species (Smart, 1981; Doulos and Kindschi, 1990; Edsall and Smith, 1990; Caldwell and Hinshaw, 1994; Foss et al., 2002). To the authors’ knowledge, there are no reports on increased growth performance of fish reared in water supersaturated with oxygen, although investigations are limited. Mean daily O2 consumption is usually observed to increase with increased food intake, as the metabolic costs associated with digestion will thus increase (Jobling, 1981). Such results have recently been demonstrated for both turbot and sea bass, Dicentrarchus labrax L. (Pichavant et al., 2001). It was, however, unexpected to find that mean daily oxygen consumption was influenced by water oxygen content, that is, consumption was significantly higher in fish experiencing hyperoxic conditions. It is questionable whether this result is a reliable one, as growth was higher in the normoxic group in the period at which the measurements were performed. However, a generally higher swimming activity was observed, although not quantified, in the hyperoxic group, which may account for an increase in O2 consumption rates. In addition, especially in O2 supersaturated water, some of the added oxygen will diffuse from the water to the surrounding air as the water passes through the raceway. This passive diffusion has been corrected for without fish in the raceways, but will probably be intensified by increased fish activity, as the surface where such diffusion can occur will be relatively large compared to the amount of water present. There is also evidence that a higher oxygen consumption may lead to lower growth, as long as the amount of ingested food does not vary between experimental groups (Imsland et al., 1995; Jonassen et al., 2000; Imsland et al., 2001), which was the case in the present study. Thus, it is possible that the reduced growth in the first period was a result of higher routine metabolic rates in the fish experiencing hyperoxic conditions. A few studies have investigated the O2 consumption of fish exposed to O2 supersaturated water (Dejours et al., 1977; Wilkes et al., 1981; Berschick et al., 1987; Person-Le Ruyet et al., 2002). None of these have, however, demonstrated higher O2 consumption rates under hyperoxic, as compared to normoxic conditions. Diurnal variations in fish O2 consumption, on the other hand, have been demonstrated in several marine species, for example, turbot (Imsland et al., 1995), common wolffish, Anarhichas lupus L. (Steinarsson and Moksness, 1996), Atlantic halibut, Hippoglossus hippoglossus L. (Jonassen et al., 2000) and Mediterranean yellowtails, Seriola dumerili Risso (De la Ga´ndara et al., 2002). However, the swift increase observed in the present experiment as the light was turned on in the morning demonstrates the need to resolve the species requirements with respect to light intensity, photoperiod regime and their interaction. The increased ammonia tolerance of fish experiencing hyperoxic conditions demonstrated in the present experiment should also be more thoroughly investigated, as this could be of great importance in intensive fish farming facilities. Further studies should include more detailed observations on
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overall activity as well as map the O2 consumption rate on a wider time and fish size scale.
Acknowledgements The authors thank the technical staff at the Aquaculture Research Station in Ka˚rvika, Tromsø, for valuable assistance before and during the experimental period and AGA Norway for contributing oxygen supplementation equipment. We also thank Dr. A.K. Imsland for valuable comments on an earlier version of this manuscript. Financial support was given by the Norwegian Research Council (NFR 134066/120).
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Houde, E.D., Schekter, R.C., 1981. Growth rates, rations and cohort consumption of marine fish larvae in relation to prey concentrations. Rapp. P.-V. Re´un.-Cons. Int. Explor. Mer 178, 441 – 453. Imsland, A.K., Folkvord, A., Stefansson, S.O., 1995. Growth, oxygen consumption and activity of juvenile turbot (Scophthalmus maximus L.) reared under different temperatures and photoperiods. Neth. J. Sea Res. 34, 149 – 159. Imsland, A.K., Foss, A., Sveinsbø, B., Jonassen, T.M., Stefansson, S.O., 2001. Comparisons of RNA/DNA ratios, growth, and metabolism in different populations of juvenile turbot Scophthalmus maximus reared at four temperatures. J. World Aquac. Soc. 32, 1 – 10. Jobling, M., 1981. The influences of feeding on the metabolic rate of fishes: a short review. J. Fish Biol. 18, 385 – 400. Jobling, M., 1982. A study of some factors affecting rates of oxygen consumption of plaice, Pleuronectes platessa L. J. Fish Biol. 20, 501 – 516. Jonassen, T.M., Imsland, A.K., Kadowaki, S., Stefansson, S.O., 2000. Interaction of temperature and photoperiod on growth of Atlantic halibut, Hippoglossus hippoglossus L. Aquac. Res. 31, 219 – 227. Kinghorn, B.P., 1983. Genetic variation in food conversion efficiency and growth in rainbow trout. Aquaculture 32, 141 – 155. Lloyd, R., 1961. Effect of dissolved oxygen concentration on the toxicity of several poisons to rainbow trout (Salmo gairdneri Richardson). J. Exp. Biol. 38 (2), 447 – 455. Øiestad, V., 1999. Shallow raceways as a compact resource-maximising farming procedure for marine fish species. Aquac. Res. 30, 1 – 10. Person-Le Ruyet, J., Delbard, C., Chartois, H., Le Delliou, H., 1997a. Toxicity of ammonia to turbot juveniles: 1. Effects on survival, growth and food utilisation. Aquat. Living Resour. 10, 307 – 314. Person-Le Ruyet, J., Galland, R., Le Roux, A., Chartois, H., 1997b. Chronic ammonia toxicity in juvenile turbot (Scophthalmus maximus). Aquaculture 154, 155 – 171. Person-Le Ruyet, J., Pichavant, K., Vacher, C., Le Bayon, N., Se´ve`re, A., Boeuf, G., 2002. Effects of O2 supersaturation on metabolism and growth in juvenile turbot (Scophthalmus maximus L.). Aquaculture 205, 373 – 383. Pichavant, K., Person-Le Ruyet, J., Le Bayon, N., Se´ve`re, A., Le Roux, A., Boeuf, G., 2001. Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass. J. Fish Biol. 59, 875 – 883. Smart, G.R., 1981. Aspects of water quality producing stress in intensive fish culture. In: Pickering, A.D. (Ed.), Stress and Fish. Academic Press, New York, pp. 277 – 293. StatSoft, 1995. CSS STATISTICA User’s Guide, Version 5.0. StatSoft, Tulsa, OK. 1064 pp. Steinarsson, A., Moksness, E., 1996. Oxygen consumption and ammonia excretion of common wolffish Anarhichas lupus Linnaeus 1758 in an experimental-scale, seawater, land-based culture system. Aquac. Res. 27, 925 – 929. Thurston, R.V., Russo, R.C., Vinogradov, G.A., 1981. Ammonia toxicity to fishes. Effect of pH on the toxicity of the un-ionized ammonia species. Environ. Sci. Technol. 15 (7), 837 – 840. Wajsbrot, N., Gasith, A., Krom, M.D., Popper, D.M., 1991. Acute toxicity of ammonia to juvenile gilthead seabream Sparus aurata under reduced oxygen levels. Aquaculture 92, 277 – 288. Wajsbrot, N., Gasith, A., Diamant, A., Popper, D.M., 1993. Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects. J. Fish Biol. 42, 321 – 328. Wilkes, P.R.H., Walker, R.L., McDonald, D.G., Wood, C.M., 1981. Respiratory, ventilatory, acid – base and ionoregulatory physiology of the white sucker Catostomus commersoni: the influence of hyperoxia. J. Exp. Biol. 91, 239 – 254. Zar, J.H., 1996. Biostatistical Analysis, 3rd ed. Prentice-Hall, Englewood Cliffs, NJ. 662 pp.
Growth and oxygen consumption in normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolffish (Anarhichas minor Olafsen) A. Foss *, T. Vollen, V. Øiestad Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Received 10 May 2002; received in revised form 13 February 2003; accepted 18 February 2003
Abstract The effects of dissolved oxygen (DO) concentration combined with sublethal doses of ammonia on growth and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish. Fish with a mean (S.D.) initial weight of 95.4 (18.0) g were reared in shallow raceways (initial stocking density 10 kg m 2) at normoxic (9.6 mg l 1) and hyperoxic (14.5 mg l 1) conditions, and also under both normoxia and hyperoxia with an additional sublethal concentration of unionised ammonia (0.17 mg NH3 l 1) added, for 8 weeks at 8 jC. There was an interacting effect of oxygen saturation and ammonia level on growth, as growth rate was significantly higher in the hyperoxic/NH3 group compared to the normoxic/NH3 group ( P < 0.001), suggesting that hyperoxic conditions may increase tolerance to unionised ammonia in spotted wolffish. At the end of the experiment, no difference in mean weight was found between fish reared at normoxic and hyperoxic conditions without added ammonia, whereas mean weight was reduced under normoxia with added ammonia as compared to normoxia without added ammonia. Mean daily oxygen consumption was investigated in the nonammonia groups at both normoxic and hyperoxic conditions, and O2 consumption was significantly higher ( P < 0.001) under hyperoxic (100.5 mg O2 kg 1 h 1) compared to normoxic (79.9 mg O2 kg 1 h 1) conditions. A clear diurnal rhythm in the O2 consumption pattern was seen at both O2 saturations, with the most striking observation being a dramatic increase in the morning when the light was turned on. Overall, our results support earlier findings of a high capacity to adapt to variations in environmental water quality parameters, and to restore normal growth rate in the spotted wolffish. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Spotted wolffish; Growth; Oxygen consumption; Hyperoxia; Ammonia * Corresponding author. Present address: Akvaplan-niva, Bergen Office, Nordnesboder 5, 5005 Bergen, Norway. Tel.: +47-55-302250; fax: +47-55-302251. E-mail address: [email protected] (A. Foss). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00209-6
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1. Introduction Spotted wolffish, Anarhichas minor Olafsen, a marine cold-water species, is well suited for farming in intensive land-based rearing systems. To effectively exploit such systems, it is important to determine the environmental water quality standards required by a species. The spotted wolffish has previously exhibited high tolerance to environmental changes in salinity (Foss et al., 2001), dissolved oxygen (DO) content (Foss et al., 2002) and carbon dioxide (Foss et al., 2003), but at high densities in a reuse system, that is, shallow raceways in racks with reuse of water between levels, there will be a gradual decrease in DO and pH, concurrent with a gradual accumulation of natural catabolites such as ammonia and carbon dioxide (Person-Le Ruyet et al., 1997b), which separately or together may affect production characteristics such as growth performance, food conversion efficiency (FCE) and health status. Ammonia is the main end product of nitrogen metabolism in teleosts (Foster and Goldstein, 1969), and it exists in both ionised (NH4+) and unionised (NH3) forms. The toxicity of ammonia to fish and other aquatic organisms is primarily attributed to the unionised form. In intensive reuse systems, ammonia concentrations may increase to levels that can cause reduced growth or even death (Person-Le Ruyet et al., 1997a). Several studies on freshwater fish (Lloyd, 1961; Alabaster et al., 1979; Thurston et al., 1981) and one on gilthead seabream, Sparus aurata (Wajsbrot et al., 1991), have demonstrated an increase in ammonia toxicity at reduced levels of DO. However, in intensive rearing facilities, benefits can be obtained from supplying rearing units with water supersaturated with oxygen as this allows for higher stocking densities, and also maximises utilisation of the water and thus reduces pumping costs. Furthermore, the potential increase in ammonia toxicity caused by low DO will be eliminated. It has been hypothesised that oxygen levels above normal saturation might reduce ammonia toxicity, and Colt et al. (1991) called for an assessment of ammonia toxicity to fish in water with oxygen content ranging from 10 to 30 mg O2 l 1. Although studies on the effects of hyperoxia in fish are limited, high DO has previously been found not to affect growth in spotted wolffish (Foss et al., 2002), turbot, Scophthalmus maximus L., (PersonLe Ruyet et al., 2002) and in some salmonid species (Doulos and Kindschi, 1990; Edsall and Smith, 1990; Caldwell and Hinshaw, 1994). A preliminary study on the effects of ammonia on growth in spotted wolffish at normoxic levels (Foss, unpublished work) indicated a threshold level between 0.07 and 0.2 mg NH3 l 1 at which growth was reduced. The present study was designed to investigate whether O2 concentrations above normal saturation affected tolerance to sublethal doses of unionised ammonia in spotted wolffish. In addition, oxygen consumption was measured to study how this was influenced by water oxygen content.
2. Materials and methods 2.1. Experimental conditions and origin of fish The eggs from two females inseminated by sperm from six males were incubated from September 2000 at the experimental facilities of the Norwegian College of Fishery
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Science at Ka˚rvika, Tromsø, and hatched in January 2001. After hatching, the larvae were transferred to small raceways with rearing chambers (15 30 cm) (Hansen and Falk-Petersen, 2001) and immediately weaned on a formulated dry food (Nutra Pluss, Skretting) containing 53% protein, 20% fat and 10% carbohydrate. Subsequently, the fish were reared in shallow raceways (0.4 2.2 m) (Øiestad, 1999) with a water level of 8 –10 cm, providing a total volume of 0.08 m3 (80 l) and fed on a commercial formulated floating food (Dan-ex 1547, Dana Feed, Horsens, Danmark), containing 47% protein, 15% fat and 21% carbohydrate (pellet size 4 mm) until and during the experiment. The raceways were supplied with running seawater (salinity 33x, pH 8.0), and water flow was set to 5 l min 1 for each of the experimental units, providing a current speed of 0.25 cm s 1 in the raceways. Two 25-W light bulbs dimmed to 5% of maximum capacity supplied the raceways with low-level light during the experiment. Photon irradiance measured at the raceway bottom was on average 0.2 Amol m 2 s 1, and all groups were maintained under a constant photoperiod of LD 18:6, including 0.5h twilight periods. 2.2. Experimental design On 18 December 2001, 229 fish were tagged intraperitoneally with TrovanR Passive Transponder tags, and on 14 January 2002, the fish (n = 399) were anaesthetised (benzocaine, 0.05 g l 1), weighed individually and total length recorded to the nearest 0.1 g and 0.1 cm, respectively, for all fish. No fish died during tagging. The initial mean weight (S.D.) was 95.4 (18.0) g and did not differ significantly between raceway units. Subsequently, weights and total lengths were recorded after 4 and 8 weeks. Mean initial and final (S.D.) stocking densities in the raceways were 10.2 (0.2) and 13.0 (0.7) kg m 2, respectively, (the fish were restricted to an area of 0.4 1.2 m). The fish were distributed randomly into eight raceways and gradually acclimated to the experimental conditions over 5 days. Treatments consisted of a normoxic group (control), a hyperoxic group, a normoxic group with a sublethal dose of ammonia added and the same sublethal dose added in a hyperoxic group. All treatment groups consisted of fish from two replicate raceways. The requested NH3 levels were obtained by adding a concentrated solution of NH4Cl (100 g NH4Cl l 1 fresh water) by two electromagnetic metering pumps (EH/W, Iwaki) to the header tanks supplying the respective raceways with water. Total ammonia nitrogen (TAN) was measured daily with an ammonia gas sensing combination electrode (Thermo Orion, Model 95-12) connected to an expandable ion analyser (Thermo Orion, EAk920), and the percentage of NH3 was calculated using the equation of Bower and Bidwell (1978), which gives the NH3/TAN ratio as a function of pH, temperature and salinity. Hyperoxic conditions were obtained by passing the inflowing water through an oxygenation cone (Oxy-lab, AGA, Norway) where pure O2 was added. Oxygen level was measured near the water inlet every 10 min using O2 sensors (InPro 6050/120) connected to an O2 transmitter (Mettler Toledo O2 4050). The signals from the transmitter were stored, via a data-logger (Almemo 5590-0), in a computer program (DataControl). Water temperature was set to 8 jC and remained within (S.D.) 0.2 jC of that prescribed. DO levels were set to 9.6 mg l 1 (control) and 14.5 mg l 1 and remained within (S.D.) 0.5 mg l 1, while TAN concentrations averaged 13.5 mg
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l 1, giving a NH3 concentration of 0.17 mg l 1 in the raceways. NH3 concentrations remained within (S.D.) 0.02 mg l 1 of that prescribed throughout the study. Food was provided in excess for 2 h daily (between 0730 and 0830 h and between 1330 and 1430 h), except for Saturdays (fed once) and Sundays (not fed). The food was distributed from automatic feeders, situated close to the water inlet thus enabling the pellets to float downstream. Uneaten pellets were collected 30 min after feeding in the morning and afternoon and counted immediately to measure food intake. All growth estimates in the present study are based on individually tagged fish, whereas results on food conversion efficiency, food consumption, daily feeding rate and protein efficiency ratio (see below) are based on the total biomass. 2.3. Growth, food utilisation and biomass gain Specific growth rate (SGR) was calculated according to the formula of Houde and Schekter (1981): SGR=(e g 1)100, where g=(lnW2 lnW1)(t2 t1) 1, and W2 and W1 are weights (g) at days t2 and t1, respectively. The condition factor (CF) was defined as: CF = 100WLT 3, where W is the weight of the fish and LT the corresponding total length. Food conversion efficiency (FCE) was calculated as: FCE=(W2 W1)C 1 (Kinghorn, 1983), where C is food intake. Total food consumption (CT) was calculated as the difference between food supplied and food waste for the whole experimental period. Daily feeding rate ( F) was calculated from: F = 100CTW 1, where W¯ is the mean daily fish weight over each of the experimental periods. Protein efficiency ratio (PER) was calculated as: PER = weight gain/protein consumed. 2.4. Plasma parameters Blood was sampled from eight fish in each experimental group at start and after 4 and 8 weeks to measure plasma osmolality and plasma chloride levels. Blood (0.4 –0.5 ml) was extracted from the caudal vessels using heparinised syringes, and collected into Eppendorff tubes. The blood was centrifuged for 10 min at 14 000 rpm, and the plasma samples were stored at 80 jC. Plasma osmolality was measured using a Fiske One-Ten osmometer (Fiske Assoc., MA, USA) and plasma chloride levels were determined using a chloride titrator (Corning 925, CIBA Corning Diagnostics). 2.5. Oxygen consumption Oxygen consumption was measured at normoxic and hyperoxic conditions without ammonia present. The specific O2 consumption rate (VO2, mg kg 1 h 1) was calculated according to the formula: V O2 ¼ ðVF dpO2 ÞðBÞ1 (Jobling, 1982; Brix, 1992), where VF is the water flow (l h 1) through the tank, dpO2 the difference in O2 concentration between the inlet and outlet water and B is the total biomass in the tank. The calculations were based on 24-h recordings of O2 levels at the inlet and
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outlet of replicated raceways at 10-min intervals. Measurements of O2 consumption were performed on 22 – 24 January. The fish were not fasted before this period. Corrections for O2 variations between inlet and outlet were performed in raceways without fish after termination of the experiment. 2.6. Statistical methods All statistical analyses were performed with STATISTICAk 5.5 (Statsoft, 1995). To assess normality of distributions, a Kolmogorov – Smirnov test (Zar, 1996) was used, and homogeneity of variances were tested using Levene’s F test (Brown and Forsythe, 1974). Analysis of weight, condition factor, SGR and blood plasma parameters on the various sampling dates were conducted using a three-way nested Model III ANOVA (Zar, 1996), where the replicates were nested within O2 and NH3 levels. Variations in mean daily oxygen consumption between O2 treatments were analysed using a two-way nested Model III ANOVA. For parameters where only group data existed (FCE, PER, F and CT), a two-way ANOVA was applied to test for overall differences between groups. Significant ANOVAs were followed by a Student – Newman – Keuls multiple comparison test (Zar, 1996) to identify differences among treatments. A significance level (a) of 0.05 was used if not stated otherwise.
Fig. 1. Mean F S.E. specific growth rates (SGR) of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia. Different letters denote significant differences (two-way nested ANOVA, P < 0.05) between treatments in each growth period.
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Treatment
Weight
O2 (mg l 1)
NH3 (mg l 1)
n
9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17
56 59 55 59
Length
Date
Condition factor
Date
14 January
11 February
95.5 94.1 95.0 94.5
117.9 101.3 113.2 109.4
(18.7) (15.2) (17.9) (20.1)
(21.2)a (15.6)b (25.4)a (24.5)a
11 March 151.6 131.6 148.3 140.9
(33.7)a (23.1)b (40.6)a (35.8)ab
Date
14 January
11 February
11 March
14 January
11 February
21.5 21.4 21.4 21.4
22.3 21.9 22.1 22.2
23.7 23.1 23.5 23.5
0.95 0.96 0.95 0.95
1.05 0.95 1.03 0.98
(1.1) (1.0) (1.1) (1.3)
(1.1) (1.0) (1.2) (1.4)
(1.3) (1.1) (1.5) (1.6)
(0.07) (0.05) (0.06) (0.06)
(0.08)a (0.06)b (0.10)a (0.08)b
11 March 1.11 (0.11)a 1.05 (0.07)b 1.11 (0.13)a 1.07 (0.10)b
Results are given as mean (S.D.); n denotes number of fish in each experimental group. All results include the data for two replicate raceways. Different letters denote significant differences (Student – Newman – Keuls multiple comparisons, P < 0.05) between treatments.
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Table 1 Mean weight (g), length (cm) and condition factor of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia
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3. Results 3.1. Growth and plasma parameters The initial mean weight of the fish was not significantly different between treatment groups, and no mortality occurred in any of the experimental groups throughout the study. In the first experimental period, there was a significant interaction between ammonia level and oxygen saturation on growth (three-way nested ANOVA, P < 0.001, Fig. 1), and growth rates were significantly higher in the hyperoxic/NH3 group compared to the normoxic/NH3 group (Student – Newman –Keuls test, P < 0.001). In this period, ammonia levels of 0.17 mg NH3 l 1 significantly reduced growth rates ( P < 0.0001), while no differences in growth rates were observed between treatment groups in the second period. At normoxic conditions, a significant difference in mean weight was found between the normoxic and the normoxic/NH3 treatment from 11 February and onwards (three-way nested ANOVA, P < 0.01, Table 1), while condition factor varied between ammonia/nonammonia groups at both normoxic and hyperoxic conditions in the same period ( P < 0.001). An interactive effect of ammonia level and oxygen saturation was found for both mean weight and condition factor on 11 February ( P < 0.05). Food conversion efficiency, protein efficiency ratio, daily food intake and total food consumption did not vary significantly between treatments (Table 2). Plasma chloride and plasma osmolality levels were affected by ammonia (Table 3). Levels were significantly higher in both the normoxic/NH3 and hyperoxic/NH3 group compared with the non-ammonia groups on 11 February ( P < 0.001). On 11 March, both plasma chloride and plasma osmolality levels had decreased to that of the normoxic group, except for osmolality level in the normoxic/NH3 group which was significantly lower than in all other groups ( P < 0.05). 3.2. Oxygen consumption Oxygen consumption was significantly influenced by O2 saturation level (two-way nested ANOVA, P < 0.001). Mean daily oxygen consumption in the hyperoxic group averaged 100.5 mg O2 kg 1 h 1 compared to 79.9 mg O2 kg 1 h 1 in the normoxic
Table 2 Food conversion efficiency (FCE), protein efficiency ratio (PER), daily feeding rate ( F) and total food consumption (CT) of spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia Treatment
FCE
O2 (mg l 1)
NH3 (mg l 1)
9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17
1.16 1.05 1.05 1.10
PER
(0.18) (0.61) (0.36) (0.37)
2.48 2.24 2.23 2.34
F
(0.37) (1.30) (0.77) (0.80)
Results are given as mean (S.D.); n = 4 for each factor combination.
0.55 0.36 0.49 0.45
CT (g) (0.14) (0.17) (0.16) (0.16)
885.0 541.9 762.2 689.8
(315.6) (273.4) (295.7) (289.9)
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Table 3 Plasma osmolality (mOsmol kg 1) and plasma chloride (mmol l 1) concentrations in spotted wolffish reared at normoxic and hyperoxic conditions with and without added ammonia Measured parameter
Plasma osmolality
Plasma chloride
Treatment group O2 (mg l 1)
NH3 (mg l 1)
9.6 9.6 14.5 14.5 9.6 9.6 14.5 14.5
0.0004 0.17 0.0004 0.17 0.0004 0.17 0.0004 0.17
14 January
11 February
350 (7.8)
348.0 359.3 348.8 367.4 152.2 158.9 152.2 155.9
150.5 (3.3)
(9.6)c (8.2)b (3.9)c (8.5)a (3.0)b (4.4)a (3.6)b (3.6)ab
11 March
348.7 340.6 353.4 350.4 153.0 150.6 150.7 150.9
(4.5)a (6.9)b (10.8)a (8.0)a (2.6) (2.4) (3.7) (1.4)
Values are given as mean (S.D.) (n = 8). Different letters denote significant differences ( P < 0.05) between treatment groups.
group. The fish exhibited a clear diurnal rhythm in their O2 consumption in both groups (Fig. 2), with a dramatic increase in the morning as the light was turned on, followed by a decrease as the fish started feeding. Subsequently, O2 consumption increased in both groups 2 –4 h after feeding, and reached a peak after approximately 6 h. A minimum in consumption was reached during late at night in both groups.
Fig. 2. Diurnal patterns in O2 consumption of spotted wolffish under normoxic (9.6 mg l 1) and hyperoxic (14.5 mg l 1) conditions. Each point represents 18 measurements. Values are given as mean F S.E. Arrows mark feeding time. Shaded areas on the x-axis indicate periods of darkness.
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4. Discussion Tolerance to chronic unionised ammonia exposure varies widely between species, with threshold values for long-term negative effects on growth in fish ranging from 0.01 mg NH3 l 1 in salmonids (Arillo et al., 1981) to 0.30 mg NH3 l 1 in gilthead seabream (Wajsbrot et al., 1993). In the present study, growth was significantly reduced in the first growth period in both groups receiving water with an added concentration of 0.17 mg NH3 l 1, and a moderate but significant increase in plasma osmolality and plasma chloride levels observed on 11 February indicate that the fish need some time to adapt to the increased water ammonia content. However, the high growth rates seen in the second period, combined with a recovery with respect to hydromineral balance, demonstrate that the species has a high capacity to adapt to lasting changes in environmental factors. Previous studies on spotted wolffish have exhibited a high capacity for the species to adapt to both hypoxia and hyperoxia (Foss et al., 2002) as well as changes in environmental hypercapnia (Foss et al., 2003) and salinity (Foss et al., 2001). In the present experiment, growth rates increased in both the normoxic and hyperoxic groups from period 1 to period 2, in contrast to what is usually reported, that is, growth rates decline with increasing fish size. The increase was significant in the hyperoxic group, supporting the foregoing assumption that the fish need some time to adapt to environmental changes. The nonsignificant increase in the normoxic group demonstrates a typical pattern often observed in spotted wolffish. They will adapt to a wide variety of environmental conditions, just given enough time. However, they are sensitive to changes in their daily routines (e.g., temperature and light conditions, feeding regime and tank environment) and may respond with a decrease in food intake (personal observation). Thus, it is possible that moving the fish from the holding facilities to the experimental facilities approximately 4 weeks in advance of start-up, although conditions were almost identical, might have affected food intake even into the first test period. A similar observation was made by Foss et al. (2002) where an increase in spotted wolffish growth rates with increasing fish size was attributed to a change in pellet size 2 weeks before the start of the experiment, a transitional period that may have been too short and which limited food intake in the first growth period. Growth in the hyperoxic/NH3 group was significantly higher compared to the normoxic/NH3 group in the first period, which poses the potentially important question, ‘‘Can tolerance to ammonia be increased by rearing fish at hyperoxic conditions ?’’ The idea was put forward by Colt et al. (1991) who called for an assessment of ammonia toxicity to fish in O2 supersaturated water. It is an established fact that toxicity of ammonia increases with decreasing oxygen level (Lloyd, 1961; Alabaster et al., 1979; Thurston et al., 1981; Wajsbrot et al., 1991), but this is, to the authors’ knowledge, the first study indicating that ammonia tolerance can be increased by rearing fish at DO levels above normal saturation. In general, using water supersaturated with oxygen makes it possible to increase the carrying capacity of a fish culture system, provided that oxygen is the most limiting factor. This may, however, not always be the case, as pointed out by Colt et al. (1991), as there are several other water quality criteria that must be fulfilled in an oxygensupplemented system. The biological effects of increased loading and density may include increased levels of suspended solids, CO2 and ammonia, as well as a potential risk of
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exposing the fish to hyperoxic conditions that are toxic to the fish. Toxicity of oxygen to an organism will depend on species, life stage, environmental conditions and on physiological and nutritional history, but a hyperoxic toxic threshold will exist for all organisms (Colt et al., 1991). The hyperoxic levels used in the present study have previously been shown not to cause any damaging effects in spotted wolffish (Foss et al., 2002), and are also well below the levels put forward as safe by Colt et al. (1991). PersonLe Ruyet et al. (2002) found that O2 saturations of 11.4 and 17.5 mg l 1 did not significantly increase growth in turbot, and similar results have been found for other species (Smart, 1981; Doulos and Kindschi, 1990; Edsall and Smith, 1990; Caldwell and Hinshaw, 1994; Foss et al., 2002). To the authors’ knowledge, there are no reports on increased growth performance of fish reared in water supersaturated with oxygen, although investigations are limited. Mean daily O2 consumption is usually observed to increase with increased food intake, as the metabolic costs associated with digestion will thus increase (Jobling, 1981). Such results have recently been demonstrated for both turbot and sea bass, Dicentrarchus labrax L. (Pichavant et al., 2001). It was, however, unexpected to find that mean daily oxygen consumption was influenced by water oxygen content, that is, consumption was significantly higher in fish experiencing hyperoxic conditions. It is questionable whether this result is a reliable one, as growth was higher in the normoxic group in the period at which the measurements were performed. However, a generally higher swimming activity was observed, although not quantified, in the hyperoxic group, which may account for an increase in O2 consumption rates. In addition, especially in O2 supersaturated water, some of the added oxygen will diffuse from the water to the surrounding air as the water passes through the raceway. This passive diffusion has been corrected for without fish in the raceways, but will probably be intensified by increased fish activity, as the surface where such diffusion can occur will be relatively large compared to the amount of water present. There is also evidence that a higher oxygen consumption may lead to lower growth, as long as the amount of ingested food does not vary between experimental groups (Imsland et al., 1995; Jonassen et al., 2000; Imsland et al., 2001), which was the case in the present study. Thus, it is possible that the reduced growth in the first period was a result of higher routine metabolic rates in the fish experiencing hyperoxic conditions. A few studies have investigated the O2 consumption of fish exposed to O2 supersaturated water (Dejours et al., 1977; Wilkes et al., 1981; Berschick et al., 1987; Person-Le Ruyet et al., 2002). None of these have, however, demonstrated higher O2 consumption rates under hyperoxic, as compared to normoxic conditions. Diurnal variations in fish O2 consumption, on the other hand, have been demonstrated in several marine species, for example, turbot (Imsland et al., 1995), common wolffish, Anarhichas lupus L. (Steinarsson and Moksness, 1996), Atlantic halibut, Hippoglossus hippoglossus L. (Jonassen et al., 2000) and Mediterranean yellowtails, Seriola dumerili Risso (De la Ga´ndara et al., 2002). However, the swift increase observed in the present experiment as the light was turned on in the morning demonstrates the need to resolve the species requirements with respect to light intensity, photoperiod regime and their interaction. The increased ammonia tolerance of fish experiencing hyperoxic conditions demonstrated in the present experiment should also be more thoroughly investigated, as this could be of great importance in intensive fish farming facilities. Further studies should include more detailed observations on
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overall activity as well as map the O2 consumption rate on a wider time and fish size scale.
Acknowledgements The authors thank the technical staff at the Aquaculture Research Station in Ka˚rvika, Tromsø, for valuable assistance before and during the experimental period and AGA Norway for contributing oxygen supplementation equipment. We also thank Dr. A.K. Imsland for valuable comments on an earlier version of this manuscript. Financial support was given by the Norwegian Research Council (NFR 134066/120).
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