The effect of fish stocking density on the growth of California halibut (Paralichthys californicus) juveniles

Aquaculture 265 (2007) 176 – 186 www.elsevier.com/locate/aqua-online

The effect of fish stocking density on the growth of California halibut (Paralichthys californicus) juveniles German E. Merino a,b,⁎, Raul H. Piedrahita c , Douglas E. Conklin d a b

Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Catolica del Norte, Larrondo 1281, Coquimbo, Chile Centro de Estudios Avanzados en Zonas Aridas (CEAZA), Unidad Regional de Desarrollo Científico & Tecnológico (CONICYT), Colina El Pino s/n, La Serena, Chile c Biological and Agricultural Engineering Department, University of California, Davis, California, USA d Department of Animal Science, University of California, Davis, California, USA Received 27 July 2006; received in revised form 12 January 2007; accepted 17 January 2007

Abstract The ability to raise California halibut (Paralichthys californicus) at a relatively high density, thus maximizing culture area usage, is of particular importance for future commercial operations. The effect of stocking densities between 100 and 300% of coverage area (PCA) on growth of juvenile (11.6 g) fish was tested in experiments that lasted 8 to 10 weeks. The experiments were carried out in a recirculation system in small rectangular tanks and raceways with water depths under 6 cm. It was found that maximum fish growth was achieved for fish stocked initially at 100% PCA. The results showed that California halibut can be grown in shallow tanks and raceways at a relatively high stocking density without significantly compromising growth rate and survivability. © 2007 Elsevier B.V. All rights reserved. Keywords: Stocking density; California halibut; Paralichthys californicus; Surface area; Culture density; Flatfish

1. Introduction The ability to raise fish at a relatively high density, thus maximizing usage of the fish production infrastructure, is of importance to commercial aquaculture especially in recirculation systems. Both positive and negative relationships between stocking density and growth have been reported and the pattern of this interaction appears to be species specific. The importance of stocking density on fish growth has been reported for several species, such us ⁎ Corresponding author. Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Catolica del Norte, Larrondo 1281, Coquimbo, Chile. Tel.: +56 51 209 767; fax: +56 51 209 764. E-mail address: [email protected] (G.E. Merino). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.01.028

rainbow trout (Oncorhynchus mykiss) (Trzebioatowski et al., 1981), Nile tilapia (Oreochromis niloticus) (CarroAnzalota and McGuinty, 1986), hamoor (Epinephelus tauvina) (Abdullah et al., 1987), Arctic charr (Salvelinus alpinus) (Christiansen et al., 1992), African catfish (Clarias gariepinus) (Kaiser et al., 1995), gilthead sea bream (Sparus aurata) (Canario et al., 1998), and lake sturgeon (Acipenser fulvescens) (Fajfer et al., 1999). There are a few studies on the effect of stocking densities on the growth of flatfish: turbot (Scophthalmus maximus) (Martinez-Tapia and Fernandez-Pato, 1991; Irwin et al., 1999, 2003), olive flounder (Paralichthys olivaceus) (Jeon et al., 1993), Atlantic halibut (Hyppoglossus hyppoglossus) (Bjørnsson, 1994), and summer flounder (Paralichthys dentatus) (King et al., 1998).

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Flatfish species are bottom dwelling fish and to enhance growth and survival it has been recommended to provide an environment with a high bottom area to water volume ratio (Cripps and Poxton, 1992; Kaiser et al., 1995; Øiestad, 1999; Labatut and Olivares, 2004). Stocking density for flatfish may be expressed either as biomass per unit of bottom area or as a percentage of coverage area (PCA = percent ratio of total fish ventral area to total tank bottom area). Commercial land-based facilities usually stock their fish at densities as high as 40 kg/m2 for olive flounder (Honda, 1998), 70 kg/m2 for sole (Liewes, 1984), and 120 kg/m2 for turbot (Person Le-Ruyet et al., 1991). Bjørnsson (1994) suggested that the optimal stocking density for 2 kg Atlantic halibut is between 25 and 50 kg/m2 and between 50 and 100 kg/m2 for 10 kg Atlantic halibut. However, the normal operational range for flatfish species varies from 15 to 50 kg/m2 depending on rearing conditions (Jones, 1981; Person Le-Ruyet et al., 1991; Liewes, 1984; MartinezTapia and Fernandez-Pato, 1991; Mallekh et al., 1998; Silva and Velez, 1998; Kikuchi, 2000). The suggested safe stocking density, as percentage of coverage area, for flatfish is between 100 and 200% coverage area (Jeon et al., 1993; Bjørnsson, 1994; King et al., 1998). California halibut (Paralichthys californicus) is a gregarious species in the wild and when stocked in tanks, therefore high stocking densities might be possible for a commercial aquaculture system. To date, no studies have been published that examine stocking density for California halibut. The ability to raise California halibut at a relatively high density, thus maximizing culture area usage, is of particular impor-

177

tance for future commercial operations. Consequently, this research was planned to estimate the effect that stocking density may have on the growth rate of California halibut juveniles. 2. Materials and methods A study was carried out to test the impact of three stocking densities – nominally 100, 200, and 300% PCA – on fish performance. Stocking densities were tested in triplicate in small tanks also referred to as experimental tanks (Fig. 1), attached to the California Halibut Recirculating Hatchery (Conklin et al., 2003; Merino, 2004). Fish morphometrics (total length, TL; total surface area with fins, TSA; body surface area without fins, BSA) were determined at the beginning of the experiment and at Week 8 by image analysis (Merino, 2004). Fish average mass (W, g) was measured at the beginning of the experiment and every other week thereafter. The stocking density experiment lasted 10 weeks. Three of the raceways (Fig. 2) within the California Halibut Recirculating Hatchery (Conklin et al., 2003; Merino, 2004) were stocked at nominally 100, 200, 300% PCA as were the experimental tanks. The purpose was to validate the use of the experimental tanks by comparing W for the fish population from raceways and experimental tanks. Stocking densities were not replicated for the raceways. After Week 6, the raceways stocked at 200 and 300% PCA started to have water quality problems due to limitations in flow capacity and the comparison was terminated.

Fig. 1. Schematic diagram of an experimental tank used for the density experiment. All measurements are in cm. Each slot to introduce nets or weirs is 0.6 cm wide (measurement not shown). Figure is not to scale. Large arrow indicates the direction of the water flow. Water depth was ∼ 6.0 cm (not shown). Tanks were built from dark grey acrylic plate, 0.6 cm thick.

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Fig. 2. Schematic diagram of a raceway. Water flow was from left to right (arrow). Water depth was between 3.5 and 4.5 cm. Figure is not to scale.

maintained at ∼ 6 cm by a weir located at the end of the tank (second weir in Fig. 1). The first weir was placed with the effluent screen (Fig. 1). A quiescent zone was created between the weirs, allowing for observation of the amount of feed uneaten by the fish. Tanks were flushed every day in the morning and before feeding by removing the second weir for a few seconds. The function of the first weir was to keep a constant water level of ∼2 cm during the flushing process. Water velocity in the experimental tanks stocked at 100% PCA was adjusted to 0.08 ± 0.01 body lengths per second (bl/s) to match the corresponding velocity (0.09 bl/s) calculated for the 100% PCA raceway (Table 1). A velocity of 0.09 bl/s corresponded to a flow rate of about 5 L/min for an experimental tank stocked at 100% PCA, resulting in a loading rate of 21.6 L/min kg. This loading rate for the 100% PCA was used to determine the flow rates for the 200% (∼8 L/min) and 300% (∼9 L/min) PCA experimental tanks, resulting in velocities of 0.16 ± 0.01 bl/s and 0.25 ± 0.01 bl/s for the 200 and 300% PCA, respectively. Thereafter and as the fish grew, the experimental tank flow rates were adjusted every other week to keep the initial relative swimming velocities constant over time. Water velocities within experimental tanks induced relative swimming velocities below 1 bl/s as recommended by Merino (2004).

2.1. Fish stock For this study 171 days post hatch (dph) weaned California halibut grown at the UC Davis California Halibut Recirculating Hatchery were used. The juveniles (11.6 g wet mass) were reared in raceways on a recirculating seawater system with a constant temperature of 22 ± 1 °C and salinity of 30 g/L (Gadomski and Caddell, 1991; Madon, 2002). The fish were fed daily at ∼ 1.2 % body mass with commercial dry pelleted feeds (EWOS™ Alpha #2; EWOS™ Pacific #3; http://www. ewos.com). Light was provided by overhead fluorescent tubes on a 16 L: 8 D (L = light; D = dark) photoperiod (Boeuf and Le Bail, 1999; Klokseth and Øiestad, 1999). 2.2. Experimental tanks Rectangular tanks with a total internal length of 60 cm, and an effective experimental length of 41 cm, were used for the stocking density experiment (Fig. 1). Three tank widths, 9.3 cm, 12.3 cm, and 15.3 cm, having a surface area of 381. 3 cm2, 504.3 cm2, and 627.3 cm2, respectively, were used to achieve the desired stocking densities. Screens (5 mm aperture) were placed at both ends of a tank to hold the fish within the experimental area. The water depth in each tank was

Table 1 Culture conditions at time of stocking for raceways stocked at nominal densities of 100%, 200%, and 300% PCA Nominal PCA (%)

Biomass (g)

Fish number

W (g)

TSA (cm2)

TL (cm)

Initial PCA (%)

Flow (L/min)

Loading rate (L/min kg)

Velocity cm/s

bl/s a

100 200 300

2404 4579 6795

214 392 616

11.23 11.68 11.03

30.6 31.4 30.0

9.9 10.1 9.9

107 201 306

9 12 15

3.7 2.6 2.2

1.1 1.4 1.8

0.09 0.13 0.16

Raceway surface area 6105 cm2. a Body length per second.

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2.3. Raceways The raceways used were 2.41 m long, 0.28 m wide with a water depth between 3.5 and 4.5 cm (Fig. 2). A 19 cm quiescent zone was created at the effluent section. The tank surface area available for fish rearing, excluding the quiescent zone was ∼ 6105 cm2. Cylindrical nets (5 mm aperture) were placed at the influent and effluent to prevent fish from escaping and to provide a frame support for a dissolved oxygen sensor. Water flow rates in the raceways were set at stocking time to have ∼5 mg/L of dissolved oxygen at the effluent, resulting in 9, 12, and 15 L/min for the 100%, 200%, and 300% PCA treatment, respectively (Table 1). A maximum flow of 15 L/min per raceway could be achieved in the system. The flow rates were maintained throughout the six week duration of the test. Water velocities within the raceways induced relative swimming velocities below 1 bl/s as recommended by Merino (2004).

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(Lifegard® model AF2 from Aquatic Eco-Systems Inc; http://www.aquaticeco.com/) during the light hours (0900 to 2200 h) 7 days/week for 10 weeks. Total fish mass was measured at stocking time and then every other week to adjust the amount of feed, maintaining a ration of 1.2% body mass per day. Fish mortalities were replaced at sampling time with fish that matched the average population morphological data for the respective replicate. 2.5. Fish growth in raceways This experiment was set up to compare growth tendencies between fish stocked within the experimental Table 2 Summary of data from California halibut reared at three stocking densities in experimental tanks for Week 0 (Initial), Week 8 (Final), and for selected parameters on Week 10 (Final at Week 10) Nominal stocking density as PCA (%)

2.4. Morphometrics and growth in experimental tanks California halibut juveniles to be stocked in the tanks were obtained from a population (n = 1840) with an average W of 11.6 g, determined by counting and weighing the fish as a group. For simplicity and to minimize fish handling, the fish to be stocked in the experimental tanks were selected from the population on the basis of total length (TL). The target TL for Wof 11.6 g was 10.0 cm and was determined as (Merino, 2004): TLðcmÞ ¼ 4:79⁎W ðgÞ0:302 ;

r2 ¼ 0:99:

ð1Þ

The TSA corresponding to an average fish of 11.6 g was 31.2 cm2 and was determined as (Merino, 2004): TSAðcm Þ ¼ 7:08⁎W ðgÞ0:6056 ; 2

r ¼ 0:99: 2

ð2Þ

The number of fish (N) to be stocked in each of the experimental tanks was estimated based on a TSA of 31.2 cm2/fish, the culture area available (A, cm2), and the target stocking density. The three nominal stocking densities, 100, 200 and 300% PCA, were established by stocking 20, 32, and 36 individuals, respectively, into each of three replicate experimental tanks per treatment (Table 2). The percentage of covered area (PCA) was determined as: TSAðcm2 Þ⁎N ⁎100 PCAð%Þ ¼ : Aðcm2 Þ

ð3Þ

The fish were fed commercial dry pelleted feeds (EWOS™ 1 and 2 mm) by automatic carrousel feeders

W (g) Initial Final Final at Week 10 TL (cm) Initial Final TSA (cm2/fish) Initial Final BSA (cm2/fish) Initial Final CV (%) Initial Final Final at Week 10 PCA (%) Initial Final Final at Week 10 Increment (%) BFA (kg/m2) Initial Final Final at Week 10 Increment (%) BFV (kg/m3) Initial Final Final at Week 10 Increment (%)

100

200

Average SD

Average SD Average SD

300

11.8 23.9a 28.5a

0.4 1.0 1.6

11.1 20.9b 24.4b

0.2 11.2 0.4 19.0c 0.6 21.2c

0.4 0.7 0.6

10.4 12.9a

0.03 10.3 0.1 12.5b

0.1 10.3 0.1 12.1c

0.1 0.2

33.3 51.2a

0.3 1.2

33.0 48.1b

0.6 32.9 0.6 44.6c

0.4 1.2

21.7 33.3a

0.5 0.2

21.5 31.9b

0.4 21.4 0.4 29.5c

0.4 0.8

12.7a 26.4a 29.4a

2.3 1.8 2.6

12.9a 23.0b 25.8a

3.3 11.9a 1.1 26.7a 3.6 30.1a

1.8 1.1 2.3

106 163 165 56

1 4 6

209 302 311 49

3 7 5

3.8 7.6 9.1 140

0.1 0.3 0.5

7.1 13.3 15.5 118

0.1 10.6 0.2 17.9 0.4 20.0 89

0.4 0.6 0.6

62.5 127.2 151.6 143

1.9 5.5 8.4

117.5 221.4 274.1 133

1.9 176.0 3.9 298.2 6.2 333.5 90

5.9 10.7 9.8

311 421 425 37

4 12 8

A different letter in a row indicates significant differences ( p b 0.05). SD = Standard deviation; Increment = [((final at week 10) − (initial)) / (initial)] ⁎ 100.

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tanks and fish stocked within the raceways for 6 weeks. California halibut juveniles were taken randomly from a population of 1580 fish with a mean W of 11.6 g and an estimated TSA of 31.2 cm2/fish (Eq. (2)). The number of fish to be stocked in each raceway was determined based on the calculated average TSA of 31.2 cm2/fish, the tank culture area available (6105 cm2), and the nominal stocking density (100, 200, or 300% PCA). Each group of fish was weighed and the average W was used to calculate the corresponding TSA (Eq. (2)) and the actual PCA (Eq. (3)) (Table 1). Raceways were cleaned daily in the morning before feeding. Mortalities were removed and counted daily and were not replaced. Fish biomass was recorded every other week to adjust the amount of feed and maintain a ration of 1.2% body mass per day. The fish were fed commercial dry pelleted feeds (EWOS™ 1 and 2 mm) by automatic belt feeders (model BFS12A from Aquatic Eco-Systems, Inc.) during the light hours (0900 to 2200 h) 7 days/week for 6 weeks.

BSA) were gathered through image analysis (Merino, 2004) only for fish within the experimental tanks at initial stocking and at Week 8. Fish were not fed 24 h before sampling. Feeding was resumed the day after sampling. Fish growth as percent gain with respect to the values at experiment initiation was calculated based on mass (PWG, %), total surface area (TSAG, cm2), and body surface area (BSAG, cm2). Other values determined were specific growth rate (SGR, %/day, Fonds et al., 1995), feed efficiency (relative to feed offered, FE, g/g, Forster and Ogata, 1996), coefficient of variation for the fish within the culture tanks (mass based, CV, %), culture density as biomass of fish per area (BFA, kg/m2) or per water volume (BFV, kg/m3 ), and Fulton's condition factor (K) (Innis, 1990) (Eq. (4)). K¼

W ðgÞ ⁎ 100 TLðcmÞ3

ð4Þ

2.8. Statistical analysis 2.6. Water quality measurements Dissolved oxygen (DO) concentrations in the effluents from the tanks were measured three times per day. In the case of the raceways, effluent oxygen concentrations were measured every 10 min with dissolved oxygen sensors (Sensorex model DO6000; http://www.sensorex.com). Temperature was monitored with a copper-constantan thermocouple (Omega®; http://www.omega.com) placed in the system reservoir. Data from oxygen sensors and thermocouples were recorded with a micrologger (Model 21X, Campbell Scientific, Inc.; http://www.campbellsci.com). TAN, nitrite and nitrate were measured using a Hach™ Odyssey spectrophotometer (model DR/2500; http:// www.hach.com). Readings of pH were taken with a Fisher Scientific Accumet pHmeter (model 50; https:// www1.fishersci.com). Alkalinity was measured by titration (La Motte Chemical test kit, model WAT-DR; http://www.lamotte.com). Salinity was measured with a YSI SCT meter (model 33; http://www.ysi.com). TAN, nitrite, nitrate, pH, salinity, and alkalinity were monitored daily at 9:00 h before fish feeding. Sodium bicarbonate was added daily at 9:00 h and 17:00 h to maintain alkalinity. 2.7. Fish sampling All fish were weighed as groups every other week to determine biomass so that feeding rations could be adjusted. Individual morphological data (TL; TSA; and

The response variables analyzed were W, TL, TSA, BSA, SGR, PWG, TSAG, BSAG, FE and CV. Response variables were analyzed for significant differences among treatments using a one-way complete model analysis of variance (Dean and Voss, 1999) with SAS statistical software, release 8.02 Level 02M0 (SAS Institute Inc., Cary, NC; http://www.sas.com). The stated null hypothesis (Ho) was that there is no difference in fish response between stocking density levels, and the corresponding alternative hypothesis (Hi) was that at least one level is different. Significance was analyzed using Duncan's multiple range test and Tukey's studentized range test at a significance of 0.05 (Dean and Voss, 1999). The three raceways stocked at densities similar to those of the experimental tanks were used to validate the use of the small tanks by comparing the tendencies for growth data from raceways and experimental tanks stocked at similar densities. 3. Results Salinity during the experiments was 30.1 ± 1.3 g/L, and temperature was 22.4 ± 0.7 °C. Temperature increased during Week 6 and remained at 24.1 ± 1.0 °C until Week 9 due to a chiller failure. During Weeks 9 and 10 the temperature was lowered to 19.6 ± 1.2 °C. During the higher temperature period fish behaved normally but TAN concentration increased from less than 1.0 mg/L to 5.5 mg/L. Nitrite nitrogen concentration varied between

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Fig. 3. Mean mass (W ) of California halibut juveniles reared at three densities during 10 weeks. Vertical lines represent standard deviations.

0.2 and 0.9 mg/L. Water pH remained unchanged over time, having an average of 7.3 ± 0.2. Alkalinity varied between 2 and 3 meq/L and had an average value of 2.46 ± 0.3 meq/L. The lowest dissolved oxygen in the influent water to both the experimental tanks and raceways at stocking time was 6.4 ± 0.1 mg/L. Effluent dissolved oxygen concentrations from the raceways reached 4.2 ± 0.3, 4.4 ± 0.4, and 4.9 ± 0.4 mg O2/L for 300% PCA, 200% PCA, and 100% PCA, respectively, during Week 6, when the raceway experiment was terminated. The lowest dissolved oxygen concentration in the effluents from the experimental tanks was 5.8 ± 0.2 mg/L and was observed at the end of the experiment (Week 10) in the tanks stocked at 300% PCA. 3.1. Morphometrics and growth in experimental tanks A total of five fish were replaced in replicates of the 300% PCA treatment, two of which died in the tanks and the other three jumped out of the tanks. Five fish were replaced in replicates of the 200% PCA treatment, one of which died in the tank and the others jumped out of the culture tanks. No fish had to be replaced in replicates of the 100% PCA treatment. Some fish in the 300% PCA treatment tended to attach themselves to the side walls of the tanks. A total of three fish with damaged tails were found in the 300% PCA treatment tanks from Weeks 2 to 10. No injured fish were seen in any of the 100% and 200% PCA treatment tanks.

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Table 2 lists the growth performance of the fish reared at the three stocking densities. At stocking time the fish were chosen to provide a uniform TSA. There were no significant differences ( p N 0.05) between treatment means for W, TL, TSA, BSA, and CV at the start of the experiment. Mean W was significantly different for the three treatments for Weeks 8 ( p b 0.05) and 10 ( p b 0.05), with the mean W for the 100% PCA treatment being greater than that for the 200% PCA treatment, which was in turn greater than that for the 300% PCA (Table 2). Significant differences ( p b 0.05) between treatments were seen on Week 8 for TL, TSA, BSA, and CV (Table 2). The coefficient of variation (CV) increased significantly ( p b 0.05) from the start of the experiment to Week 10 for the three stocking densities. However, there was no clear trend in terms of the evolution over time of CV differences between the three stocking densities. In fact, although CVs were significantly different ( p b 0.05) among stocking densities at Week 8, no differences were found at Week 10 (Table 2). At the end of the experiment the largest fish were those stocked originally at 100% PCA, and the smallest those stocked at 300% PCA. The increase in fish biomass per area (BFA) at Week 10 was 140%, 118%, and 89% for the 100% PCA, 200% PCA, and 300% PCA, respectively (Table 2). The increase in fish biomass per volume (BFV) at Week 10 was 143%, 133%, and 90% for the 100% PCA, 200% PCA, and 300% PCA, respectively (Table 2). Growth in W for the duration of the experiment was represented well by exponential curves (Fig. 3). Average PWG, SGR, TSAG, and BSAG were affected significantly by stocking density (Table 3). Significant decreases ( p b 0.05) in PWG, SGR, TSAG, and BSAG were observed as density was increased.

Table 3 Fish growth and feed efficiency for California halibut reared at three stocking densities for 8 weeks in experimental tanks (mean value±standard deviation) Nominal stocking density (%) 100

200

300

Average SD Average SD Average SD PWG (%) SGR (%/d) TSAG (%) BSAG (%) FE (g biomass/g feed)

103.5a 1.18a 53.9a 53.4a 1.22a

3.8 0.03 3.9 2.3 0.03

88.4b 1.06b 45.9b 48.2a 1.10b

3.4 0.03 1.4 1.6 0.02

69.5c 0.88c 35.5c 37.7b 0.89c

6.4 0.06 2.7 2.8 0.06

Values followed by a different letter on the same row are significantly different ( p b 0.05). SD = Standard deviation.

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The FE was affected significantly by stocking density (Table 3). A significant decrease ( p b 0.05) in FE occurred as stocking density was increased, from a maximum of 1.22 g/g in fish initially stocked at 100% PCA to 0.89 g/g for fish stocked at 300% PCA (Table 3). The condition factor, K, at stocking time for all PCAs was 1.0. At Week 8, K was 1.11, 1.06 and 1.08 for 100, 200, and 300% PCA, respectively. 3.2. Fish growth comparison between raceways and experimental tanks This experiment was terminated on Week 6 due to low oxygen concentrations in the raceways stocked at 200 and 300% PCA. Growth data for fish reared at three stocking densities in the raceways and experimental tanks are shown in Table 4. Fish were stocked at about the same BFA in both vessel types but water depths were lower in the raceways resulting in higher volumetric densities in comparison to the experimental tanks (Table 4). The overall tendency for W at Week 6 was to decrease with stocking density with the exception of fish stocked in raceways at 100 and 200% PCA for which the W were Table 4 Growth summary and observed volumetric density for California halibut juveniles reared during six weeks at three stocking densities within two types of culture vessels Nominal stocking density (%) 100 Average Raceways W (g) Initial Final BFA (kg/m2) Initial Final BFV (kg/m3) Initial Final

200 SD

Average

300 SD

Average

SD

11.2 20.6

⁎ ⁎

11.7 20.5

⁎ ⁎

11.0 18.2

⁎ ⁎

3.9 7.1

⁎ ⁎

7.5 13.1

⁎ ⁎

11.1 17.5

⁎ ⁎

101 181

Experimental tanks W (g) Initial 11.8 Final 20.7 BFA (kg/m2) Initial 3.7 Final 5.6 BFV (kg/m3) Initial 62.5 Final 109.8

⁎ ⁎

165 286

⁎ ⁎

228 359

⁎ ⁎

0.4 0.9

11.1 18.3

0.2 0.4

11.2 17.0

0.4 0.7

0.1 0.2

7.1 9.8

0.1 0.4

10.6 14.2

0.4 0.4

1.9 4.7

117.5 193.9

1.9 4.5

176.0 267.6

5.9 10.3

Tank depth was 6 cm and raceway depth was 3.6–4.5 cm. ⁎ There was no SD for raceways as they were not replicated.

similar and larger than for the fish stocked at 300% PCA. 4. Discussion Water velocities within the tanks and raceways induced swimming velocities which were less than 1.0 bl/s. Merino (2004) found that California halibut juveniles grew faster and made more efficient use of the feed provided at relative swimming velocities of 0.5 and 1.0 bl/s than at 1.5 bl/s. Bengston and Alves (2002) found that summer flounder (124 g, 257 g, and 387 g), grew best when reared at ∼ 0.5 bl/s in comparison to those grown at relative swimming velocities less than 0.3 bl/s or greater than ∼ 1.3 bl/s. Ogata and Oku (2000) found that water flow velocities corresponding to a relative swimming velocity beyond 2.1 bl/s (19.5 to 23.5 °C) led to growth retardation and lower feed efficiency for all olive flounder sizes tested and reported that the optimal water flow velocity for growth occurred at about 1.0 bl/s for a 12.5 cm/body length (5.7 g/fish). Therefore, there is enough evidence to support that the swimming velocities used in these experiments do not affect the growth performance of California halibut juveniles. All water quality parameters were within values recommended in the literature for other flatfish species. In the case of TAN, the worst condition measured was 5.5 mg TAN/L (salinity 30.1 g/L; 24.1 °C; pH 7.3) which is equivalent to about 0.05 mg/L un-ionized ammonia as nitrogen (NH3–N), Juvenile turbot (∼ 20 g) reduced their food intake when un-ionized ammonia was over 0.117 mg NH3–N/L, and a reduction in body mass gain per day occurred when the level was over 0.108 mg NH3–N/L (pH 8, 16 °C, 28 g/L salinity) (Rasmussen and Korsgaard, 1996). Person-Le Ruyet et al. (1997) reported that for 13, 23, and 104 g turbot reared in water having in the effluent over 80% oxygen saturation (∼6 mg/L), the growth was not affected at concentrations of 0.21, 0.18, and 0.09 mg NH3–N/L, while growth stopped immediately for all groups above 0.8 mg NH3–N/L (∼ pH 8, ∼ 17 °C, 34.5 g/L salinity). Turbot survival was not affected at levels below 0.4 mg NH3–N/L (13 mg TAN/L) (Person-Le Ruyet et al., 1997). Therefore the worst concentration of un-ionized ammonia as nitrogen recorded in this experiment is within a safe range. In the case of dissolved oxygen, the stocking density trial carried out in the raceways was terminated at the end of Week 6 when dissolved oxygen in the raceway effluent dropped below 60% of oxygen saturation, the value recommended as an allowable limit for turbot (Brown et al., 1984). Effluent dissolved

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oxygen in the experimental tanks remained above 75% of saturation throughout the experiment. Fish stocked in the experimental tanks at 200% and 300% PCA did not eat all the feed offered between Weeks 8 and 10. The increase in pellets left in the quiescent zones of the tanks loaded at 200% and 300% PCA started when the stocking density reached 283% PCA and 397% PCA, respectively. California halibut reared in the experimental tanks had a drop in SGR during the last 2 weeks of experimentation, regardless of the tank stocking density. A reduction of feeding activity and a drop in SGR can indicate a possible stressful condition. The culture density of flatfish is frequently reported as the PCA (Bjørnsson, 1994; Klokseth and Øiestad, 1999). A common practice to estimate surface area of flatfish in aquaculture is by placing the fish over a paper grid, drawing the fish edges, and counting the area enclosed by the drawing (Bjørnsson, 1994; King et al., 1998; Fairchild and Howell, 2001). The limits of this current methodology are numerous. Foremost is that since the process is labor intensive and time consuming only a few animals can be sampled. Sampled fish are subjected to excessive manipulation, with a high potential for injury. Merino (2004) used photographic and computer image analysis techniques to perform an accurate and fast method to estimate surface area of flatfish which he called total surface area (TSA) (area of fish edges) as well as body surface area (BSA) (excluding the fins) of California halibut. Fulton's condition factor (K ) is an indicator of general fish well-being. California halibut in the present study had a K value between 1.06 and 1.11, which according to Fulton's condition definition will be considered as a healthy fish. 4.1. Morphometrics and growth in experimental tanks Morphometric analysis was planned originally for a period of 10 weeks, but a mechanical problem with the chiller resulted in an increase in the water temperature from 22 to 24 °C during Week 6. Therefore, it was decided to do a morphometric analysis at the next scheduled sampling (beginning of Week 8). Madon (2002) who experimented with California halibut juveniles of similar sizes to the ones used in this research did not detect differences in growth for temperatures ranging from 20 to 28 °C. In the experiments described here, fish stocked at 100% PCA (final 163% PCA; 127.2 kg/m3) grew larger than those stocked at 200% PCA (final 302% PCA; 221.4 kg/m3 ) and 300% PCA (final 421% PCA;

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298.2 kg/m3) (Table 2). Fish held at the lowest densities had the largest SGR (Table 3). Most studies on flatfish stocking densities have found that higher densities result in lower growth. Irwin et al. (1999) reported that turbot, with initial biomass of 8.62 g/fish, were stocked at four different densities (10%, 14%, 17%, and 21% PCA), and after 45 days fish reared at the higher stocking densities (final density 149.3% PCA) showed significantly slower growth rates than those held at lower densities (final density 67.4% PCA). Jeon et al. (1993) reported that olive flounder with initial biomass of 42 g/ fish were stocked at five different densities (33%, 50%, 100%, 200%, and 300% PCA), and after 35 days of experimentation it was found that fish at the highest stocking density (final density 460% PCA) showed a significantly slower growth rate than those held at the lowest density (final density 45% PCA). King et al. (1998) summer flounder with initial biomass of 7.8 g/ fish, were stocked at three different densities (100%, 150%, and 200% PCA), and after 58 days the fish reared at the highest stocking density (final density 493% PCA) showed significantly slower growth rates than those held at the lowest density (final density 355% PCA). Feed efficiency was affected when California halibut were stocked at higher densities as has been described for olive flounder (Jeon et al., 1993). In the current study, a considerable amount of feed was found in the quiescent zone of tanks stocked at 200% and 300% PCA starting on Week 8. There are reports that with rising stocking density, an increase in aggressive behavior, growth, and feed efficiency was observed for the dominant fish while stress, fin damage, and metabolic rates increased in subordinate fish (Refstie and Kittelsen, 1976; Purser and Hart, 1991; Canario et al., 1998). A study done with turbot did not find differences on growth nor aggressive behavior between equally sized or mixed animals (Sunde et al., 1998). Fish stocked in the present study were pre-selected by size and only one case of damaged tail was observed in each of the 300% PCA replicates. Bjørnsson (1994) found that for Atlantic halibut, more of the feed consumed was used for metabolism than for growth with increasing stocking densities. Thus it is possible that an increase in metabolic rate, which was not measured, at higher stocking densities might have been the cause for the feed efficiency and growth reduction found here with California halibut. A direct effect of stocking density on growth variability has been reported by a number of authors (Andrews et al., 1971; Irwin et al., 1999; Lambert and Dutil, 2001). The relative size difference between

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members of a population usually increases with increasing stocking density due to social interactions, development of hierarchies, and establishment of territorial borders (Lambert and Dutil, 2001). Furthermore, an increase in the CV for fish within a population is considered to be indicative of the establishment of hierarchies (Jobling, 1994; Irwin et al., 1999; Lambert and Dutil, 2001). The CV for turbot was found to increase more rapidly in groups held at higher stocking densities (Irwin et al., 1999). It was also found that the CV for turbot increased with increasing size of fish both for graded and ungraded groups (Sunde et al., 1998). In this study, the CV increased significantly for all groups of California halibut during the 10 weeks of experimentation, indicating that growth rates were affected at all stocking densities analyzed. However, there was no clear trend in terms of the evolution over time of CV differences among stocking densities (Table 2). 4.2. Fish growth comparison between raceways and experimental tanks Experimental tanks were used in triplicate to set up a density experiment and three raceways were stocked at the same densities (100%, 200%, and 300% PCA) and used as controls against the experimental tanks. Results for the experimental tanks agreed well with those from the raceways (Table 4). Raceways, which were 5.6 times larger, 2–3 times wider, and stocked with 9 to 16 times more biomass than experimental tanks had growth results similar to those in the tanks. Because of the shallowness of the water in the tanks and raceways used, BFV in this research (Table 4) are larger than the 75 kg/m3 maximum reported for turbot (Irwin et al., 1999; Martinez-Tapia and Fernandez-Pato, 1991). In addition BFV and BFA in the current research were also larger than the 200.8 kg/m3 and 8.03 kg/m2, respectively, reported recently by Labatut and Olivares (2004) for 7.63 g turbot reared at 262 PCA% in shallow raceways with a rearing area of ∼36,000 cm2 and a velocity of 0.64 bl/s. Areal densities reported for conventional aquaculture vessels are up to 29.5 kg/m2 for juvenile summer flounder (King et al., 1998), 36.3 kg/m2 for olive flounder (Chang et al., 1995), 68 kg/m2 for turbot (Martinez-Tapia and Fernandez-Pato, 1991), and 95 kg/ m2 for halibut (Bjørnsson, 1994) which suggests that flatfish can be raised at high densities.

angular tanks and raceways at a relatively high stocking density without significantly compromising survivability. In this study, the number of fish was held constant for the duration of the experiment, and biomass density (BFA and BFV) was allowed to increase with time due to the growth of the fish. Initial stocking densities of 100% PCA produced a higher individual fish growth over time than the other stocking densities tested, which indicates that stocking density does affect the growth of California halibut juveniles. The results indicate that, of the densities tested, the optimal one is somewhere between 100% and 200% PCA. Therefore, tanks with California halibut juveniles should be split when the stocking density reaches a maximum target PCA to reduce the BFA and prevent a significant reduction in SGR. Volumetric biomass densities for California halibut juveniles can be relatively high. In this study, BFV reached values between 151.6 and 333.5 kg/m3, which are larger than those reported for other flatfish (Irwin et al., 1999; Iglesias et al., 1987; Jones et al., 1981; Labatut and Olivares, 2004). The high BFV reached in this study were the result of rearing the fish in shallow tanks. In this study relatively low BFA of 9.1 and 20.0 kg/m2 were tested due to the small size of the fish used, but work with olive flounder (Honda et al., 1993; Honda, 1998), turbot (Person Le-Ruyet et al., 1991), and Atlantic halibut (Bjørnsson, 1994) suggest that values of 48 (for a 120 g fish), 25 to 50 kg/m2 (for fish N 300 g), and 25 to 50 (for a 2 kg fish), respectively, are possible. The BFA can be estimated by knowing W, the corresponding TSA, and the PCA. The TSA can be measured for flatfish by using image analysis (Merino, 2004). For California halibut, TSA can be estimated using Eq. (2) (Merino, 2004). Having W and TSA, an

4.3. Practical implications The practical implications of the present results are that California halibut can be grown in shallow rect-

Fig. 4. Projected stocking density of California halibut corresponding to 100%, 200%, and 300% PCA related to biomass of fish.

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equation can be written that relates fish biomass (W ) with stocking density: BFA ¼ a⁎



 W ðgÞ ⁎k TSAðcm2 Þ

ð5Þ

where α is the stocking factor and is equal 1 for 100% PCA, 2 for 200% PCA, and so on; and k is a conversion factor (10) to express g/cm2 as kg/m2. Increases in W will result in the corresponding rises in BFA for a given PCA as shown in Fig. 4. The graph does not represent growth, merely the calculated biomass of fish stocked per surface area (BFA) for a given fish size (W ) and desired PCA. For example it would take 10 kg of 200 g California halibut (50 fish) for a 100% PCA or 20 kg of 1000 g California halibut (20 fish) for a 100% PCA. Thus one layer of small fish will weigh less than one layer of large fish. 5. Conclusions Growth of California halibut decreased as stocking density was increased from 100% to 300% PCA. Maximum SGRs were obtained when California halibut juveniles were initially stocked at 100% PCA. The results of this study could be used to plan yieldmaximizing farming strategies for juvenile California halibut. Results from this study are appropriate for the size of fish tested. Requirements and growth of California halibut may change for fish of different sizes. Further observations and research will refine the understanding of optimum stocking density for other California halibut sizes. Ultimately, the optimal stocking density from the point of view of a fish farmer will depend on capital cost per unit of rearing area, availability and cost of juveniles, fish growth, market price and size of fish, among other factors, to maximize the biomass and size of fish produced for a target market. Acknowledgements The fish used in this work were supplied as eggs or larvae by the California Halibut Hatchery Program in Redondo Beach, California. The assistance of Jean Benoit Muguet, Doug Bush, Dr. Margarita Cervantes-Trujano, and Dr. Enric Gisbert is gratefully acknowledged. This work was primarily funded by the National Sea Grant College Program of the U.S. Department of Commerce's National Oceanic and Atmospheric Administration under NOAA Grant no. NA06RG0142, project number R/A-116, through the California Sea Grant College Program, and in part by the California

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State Resources Agency. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies. The U.S. Government is authorized to reproduce and distribute for governmental purposes. Other sponsors were the Chilean Government through the Beca Presidente de la Republica, the Organization of American States (OAS) through a fellowship under the Regular Training Program (PRA), and the University of California at Davis through a Jastro-Shields Research Scholarship. References Abdullah, M., Wuan, T., Kawahara, S., 1987. Preliminary studies on stocking density and production of hamoor Epinephelus tauvina in PVC-lined raceways. Journal of the World Aquaculture Society 18, 237–241. Andrews, J., Knight, L., Page, J., Matsuda, Y., Brown, E., 1971. Interactions of stocking density and water turnover on growth and food conversion of channel catfish reared in intensively stocked tanks. The Progressive Fish Culturist 33, 197–203. Bengston, D., Alves, D. Effects of water velocity on conditioning of summer flounder for net pens. OAA Progress Report for the period 1/01/01 through 12/31/01. Retrieved December 16, 2002 from the www.oaa.unh.edu/finfish/2001_bengston/2001_bengston_report. html. Bjørnsson, B., 1994. Effects of stocking density on growth rate of halibut (Hippoglossus hippoglossus L.) reared in large circular tanks for three years. Aquaculture 123, 259–270. Boeuf, G., Le Bail, P., 1999. Does light have an influence on fish growth? Aquaculture 177, 129–152. Brown, J.A.G., Jones, A., Matty, A.J., 1984. Oxygen metabolism of farmed turbot (Scophthalmus maximus). I. The influence of fish size and water temperature on metabolic rate. Aquaculture 36, 273–281. Canario, A., Condeca, J., Power, D., 1998. The effect of stocking density on growth in the gilthead sea-bream, Sparus aurata (L.). Aquaculture Research 29, 177–181. Carro-Anzalota, A., McGuinty, A., 1986. Effects of stocking density on growth of Tilapia nilotica culture in cages in ponds. Journal of the World Aquaculture Society 17, 1–4. Chang, Y., Kim, S., Yang, H., 1995. Culture of the olive flounder (Paralichthys olivaceous) in a semi-closed recirculating seawater system. Journal of Korean Fisheries Society 28, 457–468. Christiansen, J., Svendsen, Y., Jobling, M., 1992. The combined effects of stocking density and sustained exercise on the behaviour, food intake, and growth of juvenile Arctic charr (Salvelinus alpinus L.). Canadian Journal of Zoology 70, 115–122. Conklin, D., Piedrahita, R., Merino, G., Muguet, J., Bush, D., Gisbert, E., Rounds, J., Cervantes-Trujano, M., 2003. Development of California halibut, Paralichthys californicus, culture. Journal of Applied Aquaculture 14, 143–154. Cripps, S., Poxton, M., 1992. A review of the design and performance of tanks relevant to flatfish aquaculture. Aquaculture Engineering 11, 71–91. Dean, A., Voss, D., 1999. Design and Analysis of Experiments. Springer-Verlag New York, Inc. 740 pp. Fajfer, S., Meeyers, L., Willman, G., Carpenter, T., Hansen, M., 1999. Growth of juvenile lake sturgeon reared in tanks at three densities. North American Journal of Aquaculture 61, 331–335.

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