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Growth of juvenile blacklip abalone (Haliotis rubra) in aquaculture tanks: effects of density and ammonia
Aquaculture 219 (2003) 457 – 470 www.elsevier.com/locate/aqua-online
Growth of juvenile blacklip abalone (Haliotis rubra) in aquaculture tanks: effects of density and ammonia Sylvain M.H. Huchette a,*, C.S. Koh1, Rob W. Day a a
Department of Zoology, The University of Melbourne, Parkville, VIC 3010, Australia
Received 22 April 2002; received in revised form 14 August 2002; accepted 13 November 2002
Abstract Grow-out density is a key factor to consider in abalone mariculture. The growth of 30 – 50-mm Haliotis rubra was reduced at high density. About 1800 juveniles were individually tagged and reared for 5 months in 12 gravity-fed raceway tanks at high and low density. Their growth and distribution in the tanks was monitored monthly. Density was found to affect growth directly through competition for preferred shelter space and indirectly through the degradation of water quality. Initial size was negatively correlated to growth in the first month of the experiment. Ammonia in the water was directly correlated to the number of abalone present upstream. Ammonia was negatively correlated with growth after the effects of density and initial size were removed. Growth appeared to be significantly reduced by this level of chronic ammonia exposure, which is lower than the effective level reported previously (EC5 = 0.004 mg FAN/l). D 2003 Elsevier Science B.V. All rights reserved. Keywords: Abalone; Haliotis rubra; Density; Growth; Ammonia
1. Introduction Abalones (Gastropoda, family Haliotidae) are a highly valued seafood in Asian cultures. To meet the increasing demand of the Asian market, culture of abalone is expanding in land- and sea-based systems in Australia and is expected to produce 425 tonnes of abalone by the year 2000 (Maguire and Hone, 1997). Unfortunately, there are many impediments that hinder economic success and production. The long culture period
* Corresponding author. E-mail address: [email protected] (S.M.H. Huchette). 1 Current address: Block 190-Bukit Batok West Avenue 6 #06-33, 650 190 Singapore. 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00627-0
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and the slow and variable growth (Day and Fleming, 1992) of the juveniles into marketsized abalone during the grow-out phase is important as it potentially could reduce profit to marginal levels in land-based systems. Hence, it is important to understand the factors affecting growth so that optimal conditions can be developed to maximise production. The many factors known to influence the growth of abalone juveniles are water flow (Fleming et al., 1997; Higham et al., 1998), depth (Liu and Chen, 1999), water quality variables like dissolved oxygen (Leitman, 1992; Harris et al., 1999), salinity (Wickins, 1981), temperature (Leighton, 1974; Gilroy and Edwards, 1998), pH (Harris et al., 1998a), nitrogenous waste (Harris et al., 1998b), food quantity and quality (Hooker and Morse, 1985; La Touche et al., 1993; Mercer et al., 1993; Fleming et al., 1998; Tahil and JuinioMenez, 1999), stocking density (Koike et al., 1979; Flassch and Aveline, 1984) and the tank system (Hindrum et al., 1995; Loipersberger, 1997). Many studies have focused on factors such as food quantity and quality, and the components of water quality. However, the effects of stocking density in culture, despite its importance for both growth and economic considerations (per unit production), have not received much attention. Grazing gastropods are known to show density-dependent growth (Creese and Underwood, 1982; Fletcher and Creese, 1985; Fletcher, 1988; Marshall and Keough, 1994). In aquaculture, density may affect growth directly through competition for food or space, which is similar to the natural environment, or indirectly through the accumulation of excretory products. Gastropod excretion is mostly composed of nitrogenous compounds, largely ammonia, while faeces constitute only a marginal source of ammonia (Spotte, 1979; Kinne, 1976). Ammonia is a toxic metabolite and stressor in abalone aquaculture (Basuyaux and Mathieu, 1999; Harris et al., 1998b). Ammonia levels in semienclosed aquaculture are regulated by the water exchange rate, which should be balanced carefully to meet economical and physiological constraints (Ford and Langdon, 2000). The aim of this study was to evaluate the importance of both the direct and indirect effects of density on the long-term growth of juvenile Haliotis rubra in aquaculture tanks.
2. Materials and methods 2.1. Location The study was conducted at Ocean Wave Seafood, located at Lara in Victoria, Australia (longitude E144j27V2U, latitude S38j4V8U). The farm provided the infrastructure, the feed and the abalone for the experiments. 2.2. Experimental animals The juvenile H. rubra originated from a mixture of artificial spawnings. They were 1– 2 years of age and 15– 65 mm in length at the beginning of the experiment. The grow-out system for the abalone involved the use of concrete hides in the tanks, as the juveniles seek shelter from the light during the day. These hides were bridge-shaped with a flat top and two vertical supports that were aligned with the water flow in the tanks. The abalones were transferred into the experimental tanks on these hides to minimize handling stress and they
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were then allowed to settle down for a week before the commencement of the experiment on 16th May 2000. The experiment included five surveys of growth, with the last one being carried out on 31st October 2000. During the experiment, they were fed ad libitum, receiving artificial feed (Adam and Amos abalone food) daily. The feed pellets were uniformly distributed on the top of the hides. To prevent migration out of the tanks, the inlets and outlets were fitted with a guard of oyster mesh. 2.3. Experiment design The experiment was conducted in raceway tanks (2.7 0.5 0.25 m). A series of five tanks were set up at different heights (Fig. 1). Ten concrete hides were placed in a row along each channel, with each hide aligned parallel to the direction of the water flow. The water was pumped from the open sea and was supplied unfiltered. Seawater entered the top tank and then flowed down through the four other tanks. The same flow rate was maintained throughout the study. Four independent raceways were used, making a total of 20 tanks. Two stocking densities were used: 500 abalone were stocked in the high-density tanks and 250 in the low-density ones. Two of the raceways were stocked at high density, while
Fig. 1. Picture and drawing of the 20 tanks set up in four independent raceways used in the experiment and distribution of the treatment in the tanks. Only the shaded tanks were used for data collection.
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the other two were stocked at low density in the first four levels and the bottom tanks were stocked at high density. The bottoms of the four raceways were stocked at the same density in order to compare the effect of abalone excretions independent of density and tank level. High density was preferred because the commercial facilities use high stocking densities and were thus interested in effects at these densities. All tanks were cleaned daily by draining the water and flushing the wastes using a seawater hose. In each tank at the levels 1, 3 and 5 in the raceway, 150 abalone were randomly selected, and tagged by fixing small numbered polyethylene tags (Hallprint, South Australia) to the shell with commercial superglue gel (Shepherd, 1988; McShane and Smith, 1992). The abalones were taken out of the water on the hides and tagged and measured within 10 min to minimize stress. The maximum shell length was recorded to the nearest 0.1 mm using vernier calipers. Subsequent measurements of the tagged abalone were carried out monthly in the same way. During the first survey, 100 abalone were measured a second time after 15-min interval, to estimate the measurement accuracy. The average error in measuring abalone shell length was F 1.0 mm. 2.4. Ammonia In order to find out if the ammonia levels were affected by stocking density, water at the surface above the middle hides in June and August and under the middle hides in August Table 1 Repeated measures analysis of variance for growth increment for June, July, August and the average of September and October Source Between tanks Initial size Treatments Planned comparisons (a) TH vs. TL (b) TH vs. BBH (c) BBH vs. BBL Tank (treatments) Error Within tanks Period Period initial size Partitioning of interaction June vs. other months Between other months Period treatments Period tank (treatments) Error
SS
df
MS
F
P
2.55 81.75
1 5
2.55 16.35
2.89 18.51
0.090 < 0.001
43.72 14.59 6.88 5.29 348.84
1 1 1 6 395
43.72 14.59 6.88 0.88 0.88
50.98 17.02 8.02 1.00
< 0.001 0.006 0.029 0.426
20.52 21.55
3 3
6.84 7.18
4.23 4.44
0.006 0.004
15.29 6.26 30.33 32.44 1917.03
1 2 15 18 1185
17.81 3.13 2.02 1.80 1.62
11.01 1.93 1.12 1.11
0.002 0.145 0.403 0.332
There were 6 treatments and 12 tanks. The initial sizes of the juveniles at the start of the experiment were used as a covariate. Preliminary testing established no significant interaction between initial size and treatments. TH = top tanks high density, TL = top tanks low density, BBH = bottom tanks below high density, BBL = bottom tanks below low density.
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in the top, middle and bottom tanks of each raceway was sampled. Ammonia levels were determined by spectrophotometry using the phenol – hypochlorite method developed by Solorzano (1969) and modified by Dal Pont et al. (1974). To minimize disturbance in the water flow, all samples were taken with a PVC tube connected to a polypropylene syringe. To exclude large suspended solids such as faeces and food particles, a plankton mesh was fitted at the end of the tube. The water samples were then filtered through Whatman glassfibre GF filters (0.45 Am) at the field site and stored in polyethylene jars washed with 0.1% hydrochloric acid and rinsed thoroughly with distilled water. Samples were kept in a cool and dark place for transport and were processed on the same day. Absorbance was read at ˚ with a DMS 80 spectrophotometer (F 0.1%). The colour was allowed to develop at 640 A room temperature (21 jC) for 1 h. The concentration of ammonia was measured as total ammonia-nitrogen (TAN), and free ammonia-nitrogen (FAN) was calculated from temperature, pH and salinity measured at the time of sampling, following Bower and Bidwell (1978). Because TAN is not affected by pH or temperature, TAN values were preferred to FAN values when comparing the two samplings or evaluating the contribution of excretion. FAN values were however preferred to measure the effect of ammonia on growth. 2.5. Statistical analysis Monthly individual growth rates in all the treatments and the tanks were compared using an ANOVA with repeated measures. Only individuals sampled six times during the experiment were used in the analysis. The initial size was also tested as a covariate. The treatments applied to tanks were compared using planned comparisons to detect effects on
Fig. 2. Average growth increment for each of the six treatments during the 5 months of experiment. Error bars indicate the standard error calculated on the basis of differences between replicate tanks. TL = top tanks low density, ML = middle tanks low density, TH = top tanks high density, MH = middle tanks high density, BBL = bottom tanks below low density and BBH = bottom tanks below high density.
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Fig. 3. Free ammonia nitrogen (FAN) levels in water above and underneath the middle hide for the six treatments on the 29/08/2000. The dotted line shows the EC5 limit determined by Harris et al. (1998a,b) for H. laevigata. Error bars indicate the standard error calculated on the basis of differences between replicate tanks.
growth and FAN of (a) abalone density, (b) tank level in a raceway, (c) the numbers of abalone upstream and (d) the interaction between tank level and abalone density. The experimental design allows a test of (c) that is not confounded with tank level, because the bottom tanks all had the same density of abalone. Ammonia levels were compared between the 2 months for each tank using a paired t-test. Ammonia levels (TAN in mg/l) in August, water velocities (m/s), number of tagged abalone stacking and light intensity were also compared between tanks and treatments using an ANOVA. The effect of chronic ammonia exposure levels was estimated by fitting a linear regression through the average growth increment corrected for density and initial size. The assumptions underlying ANOVA of homoscedasticity of variances and normally distributed residual variation (Sokal and Rohlf, 1981) were checked using boxplots and scatterplots of residuals. All statistical analyses were calculated using the SYSTAT package (Wilkinson et al., 1992).
Table 2 ANOVA of the FAN levels ( 100) on the top and underneath the hides for the 6 treatments in August Source
SS
df
MS
F
P
Treatment Planned comparisons (b) TH vs. BBH (d) TH-MH vs. TL-ML Position Position treatment Tank (treatment) Error
13.098
5
2.620
56.956
< 0.001
2.975 3.085 0.775 0.367 0.274 0.061
1 1 1 5 6 6
2.975 3.085 0.775 0.073 0.046 0.010
23.329 66.054 75.616 7.164 4.450
0.003 < 0.001 0.000 0.016 0.046
TH = top tanks high density, BBH = bottom below high density, MH = middle tanks high density, TL = top tanks low density, ML = middle tanks low density.
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3. Results 3.1. Growth The measurements from September were discarded due to consistent variation between the tanks in size measurements. These variations were probably associated with a change in observer resulting in an underestimation of the size of the animals for half of the tanks. Average monthly growth increments were calculated from measurements in August and October. Individual growth was highly variable within each of the treatments, with a standard deviation averaging 37% of the average growth. The average sizes of the juveniles in the four tanks increased from 33 to 35 mm when the experiment began to 37 –42 mm at the end of October, and the overall size range increased from 15 –65 to 20 – 73 mm.
Fig. 4. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) levels for June and August in the 12 tanks (N = 12). Graphs (A) and (C) show the relationship of TAN with the number of juvenile in upstream tanks. Graphs (B) and (D) show how FAN level correlates with the average monthly growth increment 1 month before and after the sample was taken. In August, both values found in surface water (plain line) and underneath the fifth hide (dotted line) are shown. s = significant ( p < 0.05), hs = highly significant ( p < 0.01).
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The growth rates within treatments varied significantly over time but there were no significant interactions between period and treatments or tanks (Table 1). Partitioning of the significant interaction of period and initial size showed that initial size of the individual abalone affected their growth but only during the first month of the experiment (in June). No interaction between the effect of initial size and treatments was observed ( F5379 = 0.906, p = 0.533), and the overall effect of initial size was not significant. Treatments were found to significantly affect the growth rate of the juvenile abalone (Table 1, Fig. 2). The planned comparisons showed that the low density tanks had significantly greater growth than high density tanks at the top and middle levels, the bottom tanks in the high density raceways had significantly slower growth than the top high density tanks, and the bottom tanks below low densities of abalone had faster growth than those below high densities. 3.2. Ammonia The TAN levels were found to be higher in June (1.51 F 0.09 mgl 1) than in August (0.70 F 0.09 mgl 1) (paired t = 11.07, df = 11, p < 0.001). Note that the tanks had not been cleaned for 2 days before the June measurement whereas the measurement in August was made one night after cleaning. Temperature and pH were found to be uniform between the 12 tanks in June (14 jC, pH = 7.95 F 0.02) and in August (10 jC, pH = 8.17 F 0.01). Free ammonia nitrogen (FAN) levels varied significantly between treatments and positions in the tanks in August. There was also a significant interaction between the factors and between the tanks nested within the treatments (Fig. 3 and Table 2). FAN levels were significantly higher beneath the hides. Planned comparisons showed that FAN levels were increasing faster at high density compared to low density between top and middle tanks and that there was a significant increase of FAN with the tank levels at high density. All the values measured in August were below the EC5 level determined by Harris et al.
Fig. 5. Effect of average FAN level in surface (dark circles and plain line) and under hide (clear circles and dotted line) flow on the growth adjusted for density and initial size.
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(1998b). TAN was positively correlated with the cumulative number of juveniles present upstream and the resulting FAN was negatively correlated to growth (Fig. 4). The total water flow in the tanks was about 77 F 18 l/min. From the relationship between abalone number upstream and ammonia level in the water (Fig. 4), it can be estimated that a juvenile of 37 F 6 mm produced approximately 0.011 F 0.002 Amol TANg 1h 1. The chronic ammonia exposure level causing a reduction of 5% in growth (EC5) was calculated from the FAN levels measured in the surface water and under the hides from the August sampling (Fig. 5) after adjusting the data for the effects of density and initial size. Our results suggested EC5 values of 0.006 mg FAN/l for surface water and 0.004 mg FAN/ l for the water under the hides.
4. Discussion 4.1. Growth Other studies of the effect of stocking density on growth in abalone and other cultured shellfish have shown that growth rate declined as stocking density increased (Koike et al., 1979; Mgaya and Mercer, 1995; Liu and Chen, 1999; Capinpin et al., 1999). Most papers Table 3 Data from experiments studying the effects of density on the growth of different species of juvenile abalone Reference/species/ number (N) Koike et al. (1979) H. tuberculata Cochard (1980) H. tuberculata Mgaya and Mercer (1995) H. tuberculata Liu and Chen (1999) H. diversicolor supertexta Capinpin et al. (1999) H. discus McCormick et al. (1992) in Aviles and Shepherd (1996) H. rufescens Present study H. rubra
N
Tank design/ duration 6340
0 16,000 0 35
330
– –
4500
‘‘ – ’’ refers to missing information.
0.25 m2 with 4 – 17 hides 4 months 1 m2 with 0.7 m2 of hides 2 months cage
Initial length (mm)
Density/m2
Growth (mm)/ month
%Reduction in growth
9.46
83
2.5
20
9.72 14.7
5000 1000
2.0 3.4
32
14.4 23.7
3000 386
2.3 4.0
52
8 months basket 6 months
23.7 32.4 32.4
1235 177 460
1.9 3.6 2.5
cage 5 months half barrels
19 19 –
113 450 120
4.2 3.6 2.2
240
1.47
85 170
1.3 0.9
raceway 5.5 months
33.91 34.23
31
14 33
24
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reported reductions in growth ranging from 14% to 52% due to 2- to 60-fold increase in density. There was no obvious relationship between the reduction in growth and levels of density used (Table 3). This demonstrates the importance of the rearing technique on growth. The negative relationship between growth and density in gastropods suggests there is density-dependent intraspecific competition for space or food (Stimson, 1970; Branch, 1975; Hughes, 1986; Jarayabhand and Newkirk, 1989; Parsons and Dadswell, 1992; Foster and Stiven, 1996). In the present study, food was given in sufficient quantity to ensure that there was plenty of uneaten food left over in the morning in each tank and so that this would not be considered a limiting factor. Tahil and Juinio-Menez (1999) suggested that food density had a significant effect on food intake when abalone are fed algae. In this study, however, because the number of dry pellets distributed per animal was high, food density was assumed not to affect feeding. Growth significantly decreased with increasing initial sizes, densities and FAN levels. Our results suggest that competition for space may affect growth more than the other two parameters. The reduced growth with initial size suggests that larger individuals were not performing as well as the smaller ones. At high stocking density, abalone may have more difficulty to interfere with each other when they move out from shelter sites and to reach food. This may have affected their feeding and living conditions and growth (Stoner, 1989; Mgaya and Mercer, 1995). Flassch and Aveline (1984) developed a logarithmic relationship between the number of abalone per square meter and their size to describe a density threshold over which growth is reduced significantly. This threshold was established empirically based on the results of a single experiment from Koike et al. (1979) in order to help farmers chose their stocking density. The threshold was theoretically reached when the total surface of shell was equal to half of the surface of the tank bottom. However, the results of Koike et al. (1979) were probably biased because the number of hides per tank was increased with density, reducing the competition for shelter space. Furthermore, the strength of density effects differs widely between studies with different tank designs (Table 3). More work is therefore required in this area to design better management tools. 4.2. Ammonia Water quality is important in providing the optimal conditions for growth. The important water quality parameters include temperature, dissolved oxygen, pH, salinity and nitrogenous wastes (Spotte, 1979). In culture, the level of nitrogenous wastes excreted by aquatic animals is probably the most important parameter once oxygen levels are maintained (Colt and Armstrong, 1981). Within the nitrogenous wastes, ammonia is the major component of the protein catabolism (Kinne, 1976; Colt and Armstrong, 1981; Russo and Thurston, 1991). Chronic exposure can lead to slower growth and ultimately mortality in the cultured animals, thus limiting production in aquaculture (reviewed by Basuyaux and Mathieu, 1999; Hargreaves and Kucuk, 2000). The relative proportions of NH3 (free ammonia) and NH4+ (ammonia) in solution depend on temperature and pH and, to a lesser extent on salinity. Concentrations of NH3 increase with elevated temperatures and pH values and decrease with higher salinities.
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(Downing and Merkens, 1955). Thus, ammonia at low pH is usually toxic only in overwhelming quantities whereas at high pH, much smaller amounts may be lethal (Warren, 1962). In commercial activities, water quality parameters such as temperature, pH and ammonia concentrations may vary greatly over time and between places in the tanks, as suggested in our data, and this variation alone may cause a significant stress and affect the productivity and the health of the animals (Hargreaves and Kucuk, 2000). However, the only studies examining the effects of ammonia on abalone, by Harris et al. (1998b) on juvenile Haliotis laevigata and Basuyaux and Mathieu (1999) on Haliotis tuberculata, have not taken these variations into account. As expected, ammonia showed a strong positive correlation with the biomass in upstream water. The ammonia excretion rate from this relationship is in the range of excretion rates of gastropods given by Kinne (1976) and about two times lower than the one found by Ford and Langdon (2000) for Haliotis rufescens (0.017 to 0.028 Amol TANg 1h 1). This difference may be due to the artificial diet used in the present study or differences in metabolisms due to the temperature. Differences in the ammonia level were found between stocking densities even in the top tanks. The depressed growth rates in the bottom tanks showed that there is an indirect effect of density on the growth of abalone through water quality. However, growth rates in the top and middle tanks suggest that the direct effect of stocking density, probably through competition for space, was more important. There were consistent and significant differences between TAN values in the surface flow and in the water under the hides. Hides were obstacles to the water flow and during the day, abalone aggregated under the hides (Huchette et al., submitted for publication), making further obstruction to the water flows which resulted in the accumulation of faeces. Hides should therefore be used with an appropriate tank system, which would prevent accumulation of faeces and ensure good water circulation. 4.3. Chronic ammonia exposure The level of chronic ammonia exposure found to reduce growth by 5% in this study was 10-fold lower than the one found by Harris et al. (1998b) for H. laevigata. In their study, the juveniles were about 3 years old and 31.8 F 0.1 mm in size. The safe ammonia concentration of 1 mg N l 1 defined by Basuyaux and Mathieu (1999) was reached in all the tanks in June. It is possible but rather unlikely that H. rubra is a more sensitive species to FAN. Another possible explanation of this difference is the duration of the experiments and the number of individuals studied. Indeed, Harris et al. (1998b) and Basuyaux and Mathieu (1999) studied only a few juveniles (N < 200 vs. N = 1800 in this study) for 15 days. Chronic exposure is more likely to show its effect in long-term experiments especially when the main biological parameter studied is growth (Day and Fleming, 1992). Abalone growth is known to be slow and variable, and therefore has to be studied for long periods of time to show significant effects. The chronic ammonia exposure range determined in this study has to be considered carefully, however, as the ammonia level was not monitored regularly and was produced by abalone and not chemically adjusted as in other studies (Harris et al., 1998b; Basuyaux and Mathieu, 1999). The levels given in this
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study were affected by the time since the tanks were last cleaned, resulting in more variable values in the June sampling carried out 2 days after the last cleaning, compared with August when the sampling was carried out only a night after the tanks were cleaned. This suggests that the ammonia level varied within and between tanks from time to time according to the amount of faeces and uneaten food accumulating. In practice; FAN levels will also vary with the temperature, pH and salinity of the water and our low EC5 may be the result of this variation. For an abalone farm, it may be more practical and reliable to evaluate the level of growth depression according to water flow and the number of abalone found in a tank or upstream rather than to look at the chemical components of the excreta. This study provides useful data for this purpose as compared to other studies.
Acknowledgements Ocean Wave Seafood provided the infrastructure and the animals for this study. The staffs of the farm are especially thanked for their advice, support and daily feeding and cleaning of the experimental tanks. Thanks also to the volunteers who helped with the tagging and collection of the data in often challenging conditions: Patrick Gilmour, Anthony, Sabine Roussel and Huon McDiarmid. We are grateful to the technical staff of the Department of Zoology of the University of Melbourne for their help and support during the project; special thanks to Joe Wedgewood and Viviane Porter. Dr. Mick Keough kindly allowed us to use the Vector velocity meter. Sylvain Huchette acknowledges the financial support provided by the Sadlier –Stokes Scholarship given to encourage young French to tie links with Australia in memory of the Australian veterans who fought in France during WWI. This work was also carried out with the help of International Postgraduate Research Scholarship of the University of Melbourne.
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ammonia in sea water and the preservation of samples for nitrate analysis. CSIRO Division of Fisheries and Oceanography. Report No. 55. Cronulla, Sydney, p. 8. Day, R.W., Fleming, A.E., 1992. The determinants and measurement of abalone growth. In: Shepherd, S.A., Tegner, M.J., Guzman del Proo, S.A. (Eds.), Abalone of the World: Biology, Fisheries, and Culture. Fishing News Books, Blackwell, Oxford, pp. 141 – 168. Downing, K.M., Merkens, J.C., 1955. The influence of dissolved oxygen concentration on the toxicity of unionised ammonia to rainbow trout (Salmo gairdnerii Richardson). Ann. Appl. Biol. 43, 243 – 246. Flassch, J.P., Aveline, C., 1984. Production de jeunes ormeaux a la station experimentale d’Argenton. Rapp. Sci. Tech. CNEXO 50 (67 pp.). Fleming, A., Hone, P., Higham, J., 1997. The effect of water velocity on consumption and growth of greenlip abalone in tanks. In: Hone, P.W., Fleming, A. (Eds.), Proceedings of the 4th Annual Abalone Aquaculture Workshop, Port Fairy, Victoria. FRDC, Canberra, Australia, pp. 16 – 23. Fleming, A.E., Van Barneveld, R.J., Hone, P.W., Vandepeer, M.E., Kruk, J.A., 1998. Complementary additivity of the digestibility coefficients of feed ingredients fed to juvenile greenlip abalone (Haliotis laevigata). J. Shellfish Res. 17, 641 – 647. Fletcher, W.J., 1988. Intraspecific interactions between adults and juveniles of the subtidal limpet, Patella mufria. Oecologia (Berl.) 75, 227 – 272. Fletcher, E.J., Creese, R.G., 1985. Competitive interactions between co-occurring herbivores gastropods. Mar. Biol. 86, 183 – 191. Ford, E., Langdon, C.J., 2000. Co-culture of dulse Palmaria mollis and red abalone Haliotis rufescens under limited flow conditions. Aquaculture 185 (1 – 2), 137 – 158. Foster, B.A., Stiven, A.E., 1996. Experimental effects of density and food on growth and mortality of the Southern Appalachian land gastropod, Mesodon normalis (Pilsbry). Am. Midl. Nat. 136, 300 – 314. Gilroy, A., Edwards, S.J., 1998. Optimum temperature for growth of Australian abalone: preferred temperature and critical thermal maximum for blacklip, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Donovan). Aquac. Res. 29, 481 – 485. Hargreaves, J.A., Kucuk, S., 2000. Effects of diel un-ionized ammonia fluctuation on juvenile hybrid striped bass, channel catfish, and blue tilapia. Aquaculture 195 (1 – 2), 163 – 181. Harris, J.O., Maguire, G.B., Edwards, S., Hindrum, S.M., 1998a. Effect of pH on growth rate and oxygen consumption rate for juvenile greenlip abalone, Haliotis laevigata and blacklip abalone, Haliotis rubra. In: Hone, P.W. (Ed.), Proceedings of the 5th Abalone Aquaculture Workshop, Hobart. FRDC, Canberra, Australia, pp. 103 – 114. Harris, J.O., Maguire, G.B., Edwards, S.J., Hindrum, S.M., 1998b. Effect of ammonia on growth rate and oxygen consumption of juvenile greenlip abalone, Haliotis laevigata Donovan. Aquaculture 160, 259 – 272. Harris, J.O., Maguire, G.B., Edwards, S.J., Johns, D.R., 1999. Low dissolved oxygen reduces growth rate and oxygen consumption rate of juvenile greenlip abalone, Haliotis laevigata Donovan. Aquaculture 174, 265 – 278. Higham, J., Hone, P., Clarke, S., Baudinette, R., Geddes, M., 1998. The effect of flow on growth in juvenile greenlip abalone, Haliotis laevigata (Donovan). In: Hone, P.W. (Ed.), Proceedings of the 5th Annual Abalone Aquaculture Workshop, Hobart. FRDC, Canberra, Australia, pp. 115 – 122. Hindrum, S.M., Cropp, M., Maguire, G.B., 1995. Evolution of abalone growout tank design at Marine Shellfish Hatcheries, Bicheno, Tasmania. Proceedings 2nd Annual FRDC/CRC Workshop on Abalone Culture, CSIRO Marine Laboratories, Hobart. FRDC, Canberra, Australia, pp. 78 – 91. Hooker, N., Morse, D.E., 1985. Abalone: the emerging development of commercial cultivation in the United States. In: Huner, J.V., Brown, E.E. (Eds.), Crustaceans and Mollusk Aquaculture in the United States. AVI Publishing Co., Westport, CT, p. 365. Huchette, S.M.H., Koh, C.S., Day, R.W., submitted for publication. The effects of density on the behaviour and growth of juvenile blacklip abalone (Haliotis rubra). Aquac. Int. Hughes, R.N., 1986. A Functional Biology of Marine Gastropods. Croom Helm, London. Jarayabhand, P., Newkirk, G.F., 1989. Effects of intraspecific competition on growth of the European oyster, Ostrea edulis Linnaeus, 1750. J. Shellfish Res. 8, 359 – 365. Kinne, O. (Ed.), 1976. Marine Ecology, vol. 3. Wiley, London. 1293 pp. Koike, Y., Flassch, J., Mazurier, J., 1979. Biological and ecological studies on the propagation of the ormer,
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Haliotis tuberculata Linnaeus: II. Influence of food and density on the growth of juveniles. La Mer 17, 43 – 52. La Touche, B., Moylan, K., Twomey, W., 1993. Abalone on-growing manual. Aquacult. Expl. 14, 39. Leighton, D.L., 1974. The influence of temperature on larval and juvenile growth in three species of southern California abalones. Fish. Bull. 72, 1137. Leitman, A., 1992. The effects of gas supersaturation on the behaviour, growth and mortality of red abalone, Haliotis rufescens (Swainson). In: Shepherd, S.A., Tegner, M.J., Guzman del Proo, S.A. (Eds.), Abalone of the World: Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 75 – 85. Liu, K.M., Chen, W.K., 1999. Examining the effects of stocking density and depth on growth of intensive cultured abalone, Haliotis diversicolor supertexta Lischke. J. Fish. Soc. Taiwan 26, 23 – 33. Loipersberger, M., 1997. SAABDEV tank report. In: Hone, P.W. (Ed.), Proceedings of the 4th Annual Abalone Aquaculture Workshop, Port Fairy, Victoria. FRDC, Canberra, Australia, pp. 2 – 4. Maguire, G.B., Hone, P., 1997. Port Lincoln abalone culture workshop—R & D aids impressive industry expansion. Austasia Aquac. 11, 37 – 39. Marshall, P.A., Keough, M.J., 1994. Asymmetry in intraspecific competition in the limpet Cellana tramoserica (Sowerby). J. Exp. Mar. Biol. Ecol. 177, 121 – 138. McCormick, T.B., Arguirre, A., Mill, T.S., Herbison, K.T., 1992. Growth and survival of red (Haliotis rufescens), green (Haliotis fulgens) and pink (Haliotis corrugata) abalone in land-based and ocean-based cultivation systems. Fish. Res. Pap., Dep. Fish. (S. Aust.) 24, 49 – 60. McShane, P.E., Smith, M.G., 1992. Shell growth checks are unreliable indicators of age of the abalone Haliotis rubra (Mollusca: Gastropoda). Aust. J. Mar. Freshw. Res. 43, 1215 – 1219. Mercer, J.P., Mai, K.-S., Donlon, J., 1993. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata Linnaeus and Haliotis discus hannai Ino: I. Effects of algal diets on growth and biochemical composition. Invertebr. Reprod. Dev. 23, 75 – 88. Mgaya, Y.D., Mercer, J.P., 1995. The effects of size grading and stocking density on growth performance of juvenile abalone, Haliotis tuberculata Linnaeus. Aquaculture 136, 297 – 312. Parsons, G.J., Dadswell, M.J., 1992. Effect of stocking density on growth, production and survival of the giant scallop, Placopecten magellanicus, held in intermediate culture in Passamaquoddy Bay, New Brunswick. Aquaculture 103, 291 – 309. Russo, R.C., Thurston, R.V., 1991. Toxicity of ammonia, nitrite, and nitrate to fishes. In: Brune, D.E., Tomasso, J.R. (Eds.), Aquaculture and Water Quality. The World Aquaculture Society, Baton Rouge, LA, USA, pp. 58 – 89. Shepherd, S.A., 1988. Studies on Southern Australian abalone (Genus Haliotis): VIII. Growth of juvenile H. laevigata. Aust. J. Mar. Freshw. Res. 39, 177 – 183. Sokal, R.R., Rohlf, F.J., 1981. Biometry, 2nd ed. Freeman, New York. 859 pp. Solorzano, L., 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14, 799 – 801. Spotte, S. (Ed.), 1979. Fish and Invertebrate Culture, Water Management in Closed Systems, 2nd ed. Wiley, New York. 179 pp. Stimson, J., 1970. Territorial behaviour of the owl limpet Lottia gigantea. Ecology 51, 113 – 118. Stoner, A.W., 1989. Density-dependent growth and grazing effects of juvenile queen conch Strombus gigas L. in a tropical seagrass meadow. J. Exp. Mar. Biol. Ecol. 130, 119 – 133. Tahil, A.S., Juinio-Menez, M.A., 1999. Natural diet, feeding periodicity and functional response to food density of the abalone, Haliotis asinina L. (Gastropoda). Aquac. Res. 30, 95 – 107. Warren, K.S., 1962. Ammonia toxicity and pH. Nature (Lond.) 195, 47 – 49. Wickins, J.F., 1981. Water quality requirements for intensive aquaculture: a review. In: Tiews, E. (Ed.), Proc. World Symp. on Aquaculture in Heated Effluents and Recirculation Systems, Stavanger, Norway, vol. 1, pp. 17 – 37. Wilkinson, L., Hill, M., Vang, E., 1992. Statistics, Version 5.2. SYSTAT, Evanston, IL. 724 pp.
Growth of juvenile blacklip abalone (Haliotis rubra) in aquaculture tanks: effects of density and ammonia Sylvain M.H. Huchette a,*, C.S. Koh1, Rob W. Day a a
Department of Zoology, The University of Melbourne, Parkville, VIC 3010, Australia
Received 22 April 2002; received in revised form 14 August 2002; accepted 13 November 2002
Abstract Grow-out density is a key factor to consider in abalone mariculture. The growth of 30 – 50-mm Haliotis rubra was reduced at high density. About 1800 juveniles were individually tagged and reared for 5 months in 12 gravity-fed raceway tanks at high and low density. Their growth and distribution in the tanks was monitored monthly. Density was found to affect growth directly through competition for preferred shelter space and indirectly through the degradation of water quality. Initial size was negatively correlated to growth in the first month of the experiment. Ammonia in the water was directly correlated to the number of abalone present upstream. Ammonia was negatively correlated with growth after the effects of density and initial size were removed. Growth appeared to be significantly reduced by this level of chronic ammonia exposure, which is lower than the effective level reported previously (EC5 = 0.004 mg FAN/l). D 2003 Elsevier Science B.V. All rights reserved. Keywords: Abalone; Haliotis rubra; Density; Growth; Ammonia
1. Introduction Abalones (Gastropoda, family Haliotidae) are a highly valued seafood in Asian cultures. To meet the increasing demand of the Asian market, culture of abalone is expanding in land- and sea-based systems in Australia and is expected to produce 425 tonnes of abalone by the year 2000 (Maguire and Hone, 1997). Unfortunately, there are many impediments that hinder economic success and production. The long culture period
* Corresponding author. E-mail address: [email protected] (S.M.H. Huchette). 1 Current address: Block 190-Bukit Batok West Avenue 6 #06-33, 650 190 Singapore. 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00627-0
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and the slow and variable growth (Day and Fleming, 1992) of the juveniles into marketsized abalone during the grow-out phase is important as it potentially could reduce profit to marginal levels in land-based systems. Hence, it is important to understand the factors affecting growth so that optimal conditions can be developed to maximise production. The many factors known to influence the growth of abalone juveniles are water flow (Fleming et al., 1997; Higham et al., 1998), depth (Liu and Chen, 1999), water quality variables like dissolved oxygen (Leitman, 1992; Harris et al., 1999), salinity (Wickins, 1981), temperature (Leighton, 1974; Gilroy and Edwards, 1998), pH (Harris et al., 1998a), nitrogenous waste (Harris et al., 1998b), food quantity and quality (Hooker and Morse, 1985; La Touche et al., 1993; Mercer et al., 1993; Fleming et al., 1998; Tahil and JuinioMenez, 1999), stocking density (Koike et al., 1979; Flassch and Aveline, 1984) and the tank system (Hindrum et al., 1995; Loipersberger, 1997). Many studies have focused on factors such as food quantity and quality, and the components of water quality. However, the effects of stocking density in culture, despite its importance for both growth and economic considerations (per unit production), have not received much attention. Grazing gastropods are known to show density-dependent growth (Creese and Underwood, 1982; Fletcher and Creese, 1985; Fletcher, 1988; Marshall and Keough, 1994). In aquaculture, density may affect growth directly through competition for food or space, which is similar to the natural environment, or indirectly through the accumulation of excretory products. Gastropod excretion is mostly composed of nitrogenous compounds, largely ammonia, while faeces constitute only a marginal source of ammonia (Spotte, 1979; Kinne, 1976). Ammonia is a toxic metabolite and stressor in abalone aquaculture (Basuyaux and Mathieu, 1999; Harris et al., 1998b). Ammonia levels in semienclosed aquaculture are regulated by the water exchange rate, which should be balanced carefully to meet economical and physiological constraints (Ford and Langdon, 2000). The aim of this study was to evaluate the importance of both the direct and indirect effects of density on the long-term growth of juvenile Haliotis rubra in aquaculture tanks.
2. Materials and methods 2.1. Location The study was conducted at Ocean Wave Seafood, located at Lara in Victoria, Australia (longitude E144j27V2U, latitude S38j4V8U). The farm provided the infrastructure, the feed and the abalone for the experiments. 2.2. Experimental animals The juvenile H. rubra originated from a mixture of artificial spawnings. They were 1– 2 years of age and 15– 65 mm in length at the beginning of the experiment. The grow-out system for the abalone involved the use of concrete hides in the tanks, as the juveniles seek shelter from the light during the day. These hides were bridge-shaped with a flat top and two vertical supports that were aligned with the water flow in the tanks. The abalones were transferred into the experimental tanks on these hides to minimize handling stress and they
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were then allowed to settle down for a week before the commencement of the experiment on 16th May 2000. The experiment included five surveys of growth, with the last one being carried out on 31st October 2000. During the experiment, they were fed ad libitum, receiving artificial feed (Adam and Amos abalone food) daily. The feed pellets were uniformly distributed on the top of the hides. To prevent migration out of the tanks, the inlets and outlets were fitted with a guard of oyster mesh. 2.3. Experiment design The experiment was conducted in raceway tanks (2.7 0.5 0.25 m). A series of five tanks were set up at different heights (Fig. 1). Ten concrete hides were placed in a row along each channel, with each hide aligned parallel to the direction of the water flow. The water was pumped from the open sea and was supplied unfiltered. Seawater entered the top tank and then flowed down through the four other tanks. The same flow rate was maintained throughout the study. Four independent raceways were used, making a total of 20 tanks. Two stocking densities were used: 500 abalone were stocked in the high-density tanks and 250 in the low-density ones. Two of the raceways were stocked at high density, while
Fig. 1. Picture and drawing of the 20 tanks set up in four independent raceways used in the experiment and distribution of the treatment in the tanks. Only the shaded tanks were used for data collection.
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the other two were stocked at low density in the first four levels and the bottom tanks were stocked at high density. The bottoms of the four raceways were stocked at the same density in order to compare the effect of abalone excretions independent of density and tank level. High density was preferred because the commercial facilities use high stocking densities and were thus interested in effects at these densities. All tanks were cleaned daily by draining the water and flushing the wastes using a seawater hose. In each tank at the levels 1, 3 and 5 in the raceway, 150 abalone were randomly selected, and tagged by fixing small numbered polyethylene tags (Hallprint, South Australia) to the shell with commercial superglue gel (Shepherd, 1988; McShane and Smith, 1992). The abalones were taken out of the water on the hides and tagged and measured within 10 min to minimize stress. The maximum shell length was recorded to the nearest 0.1 mm using vernier calipers. Subsequent measurements of the tagged abalone were carried out monthly in the same way. During the first survey, 100 abalone were measured a second time after 15-min interval, to estimate the measurement accuracy. The average error in measuring abalone shell length was F 1.0 mm. 2.4. Ammonia In order to find out if the ammonia levels were affected by stocking density, water at the surface above the middle hides in June and August and under the middle hides in August Table 1 Repeated measures analysis of variance for growth increment for June, July, August and the average of September and October Source Between tanks Initial size Treatments Planned comparisons (a) TH vs. TL (b) TH vs. BBH (c) BBH vs. BBL Tank (treatments) Error Within tanks Period Period initial size Partitioning of interaction June vs. other months Between other months Period treatments Period tank (treatments) Error
SS
df
MS
F
P
2.55 81.75
1 5
2.55 16.35
2.89 18.51
0.090 < 0.001
43.72 14.59 6.88 5.29 348.84
1 1 1 6 395
43.72 14.59 6.88 0.88 0.88
50.98 17.02 8.02 1.00
< 0.001 0.006 0.029 0.426
20.52 21.55
3 3
6.84 7.18
4.23 4.44
0.006 0.004
15.29 6.26 30.33 32.44 1917.03
1 2 15 18 1185
17.81 3.13 2.02 1.80 1.62
11.01 1.93 1.12 1.11
0.002 0.145 0.403 0.332
There were 6 treatments and 12 tanks. The initial sizes of the juveniles at the start of the experiment were used as a covariate. Preliminary testing established no significant interaction between initial size and treatments. TH = top tanks high density, TL = top tanks low density, BBH = bottom tanks below high density, BBL = bottom tanks below low density.
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in the top, middle and bottom tanks of each raceway was sampled. Ammonia levels were determined by spectrophotometry using the phenol – hypochlorite method developed by Solorzano (1969) and modified by Dal Pont et al. (1974). To minimize disturbance in the water flow, all samples were taken with a PVC tube connected to a polypropylene syringe. To exclude large suspended solids such as faeces and food particles, a plankton mesh was fitted at the end of the tube. The water samples were then filtered through Whatman glassfibre GF filters (0.45 Am) at the field site and stored in polyethylene jars washed with 0.1% hydrochloric acid and rinsed thoroughly with distilled water. Samples were kept in a cool and dark place for transport and were processed on the same day. Absorbance was read at ˚ with a DMS 80 spectrophotometer (F 0.1%). The colour was allowed to develop at 640 A room temperature (21 jC) for 1 h. The concentration of ammonia was measured as total ammonia-nitrogen (TAN), and free ammonia-nitrogen (FAN) was calculated from temperature, pH and salinity measured at the time of sampling, following Bower and Bidwell (1978). Because TAN is not affected by pH or temperature, TAN values were preferred to FAN values when comparing the two samplings or evaluating the contribution of excretion. FAN values were however preferred to measure the effect of ammonia on growth. 2.5. Statistical analysis Monthly individual growth rates in all the treatments and the tanks were compared using an ANOVA with repeated measures. Only individuals sampled six times during the experiment were used in the analysis. The initial size was also tested as a covariate. The treatments applied to tanks were compared using planned comparisons to detect effects on
Fig. 2. Average growth increment for each of the six treatments during the 5 months of experiment. Error bars indicate the standard error calculated on the basis of differences between replicate tanks. TL = top tanks low density, ML = middle tanks low density, TH = top tanks high density, MH = middle tanks high density, BBL = bottom tanks below low density and BBH = bottom tanks below high density.
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Fig. 3. Free ammonia nitrogen (FAN) levels in water above and underneath the middle hide for the six treatments on the 29/08/2000. The dotted line shows the EC5 limit determined by Harris et al. (1998a,b) for H. laevigata. Error bars indicate the standard error calculated on the basis of differences between replicate tanks.
growth and FAN of (a) abalone density, (b) tank level in a raceway, (c) the numbers of abalone upstream and (d) the interaction between tank level and abalone density. The experimental design allows a test of (c) that is not confounded with tank level, because the bottom tanks all had the same density of abalone. Ammonia levels were compared between the 2 months for each tank using a paired t-test. Ammonia levels (TAN in mg/l) in August, water velocities (m/s), number of tagged abalone stacking and light intensity were also compared between tanks and treatments using an ANOVA. The effect of chronic ammonia exposure levels was estimated by fitting a linear regression through the average growth increment corrected for density and initial size. The assumptions underlying ANOVA of homoscedasticity of variances and normally distributed residual variation (Sokal and Rohlf, 1981) were checked using boxplots and scatterplots of residuals. All statistical analyses were calculated using the SYSTAT package (Wilkinson et al., 1992).
Table 2 ANOVA of the FAN levels ( 100) on the top and underneath the hides for the 6 treatments in August Source
SS
df
MS
F
P
Treatment Planned comparisons (b) TH vs. BBH (d) TH-MH vs. TL-ML Position Position treatment Tank (treatment) Error
13.098
5
2.620
56.956
< 0.001
2.975 3.085 0.775 0.367 0.274 0.061
1 1 1 5 6 6
2.975 3.085 0.775 0.073 0.046 0.010
23.329 66.054 75.616 7.164 4.450
0.003 < 0.001 0.000 0.016 0.046
TH = top tanks high density, BBH = bottom below high density, MH = middle tanks high density, TL = top tanks low density, ML = middle tanks low density.
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3. Results 3.1. Growth The measurements from September were discarded due to consistent variation between the tanks in size measurements. These variations were probably associated with a change in observer resulting in an underestimation of the size of the animals for half of the tanks. Average monthly growth increments were calculated from measurements in August and October. Individual growth was highly variable within each of the treatments, with a standard deviation averaging 37% of the average growth. The average sizes of the juveniles in the four tanks increased from 33 to 35 mm when the experiment began to 37 –42 mm at the end of October, and the overall size range increased from 15 –65 to 20 – 73 mm.
Fig. 4. Total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) levels for June and August in the 12 tanks (N = 12). Graphs (A) and (C) show the relationship of TAN with the number of juvenile in upstream tanks. Graphs (B) and (D) show how FAN level correlates with the average monthly growth increment 1 month before and after the sample was taken. In August, both values found in surface water (plain line) and underneath the fifth hide (dotted line) are shown. s = significant ( p < 0.05), hs = highly significant ( p < 0.01).
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The growth rates within treatments varied significantly over time but there were no significant interactions between period and treatments or tanks (Table 1). Partitioning of the significant interaction of period and initial size showed that initial size of the individual abalone affected their growth but only during the first month of the experiment (in June). No interaction between the effect of initial size and treatments was observed ( F5379 = 0.906, p = 0.533), and the overall effect of initial size was not significant. Treatments were found to significantly affect the growth rate of the juvenile abalone (Table 1, Fig. 2). The planned comparisons showed that the low density tanks had significantly greater growth than high density tanks at the top and middle levels, the bottom tanks in the high density raceways had significantly slower growth than the top high density tanks, and the bottom tanks below low densities of abalone had faster growth than those below high densities. 3.2. Ammonia The TAN levels were found to be higher in June (1.51 F 0.09 mgl 1) than in August (0.70 F 0.09 mgl 1) (paired t = 11.07, df = 11, p < 0.001). Note that the tanks had not been cleaned for 2 days before the June measurement whereas the measurement in August was made one night after cleaning. Temperature and pH were found to be uniform between the 12 tanks in June (14 jC, pH = 7.95 F 0.02) and in August (10 jC, pH = 8.17 F 0.01). Free ammonia nitrogen (FAN) levels varied significantly between treatments and positions in the tanks in August. There was also a significant interaction between the factors and between the tanks nested within the treatments (Fig. 3 and Table 2). FAN levels were significantly higher beneath the hides. Planned comparisons showed that FAN levels were increasing faster at high density compared to low density between top and middle tanks and that there was a significant increase of FAN with the tank levels at high density. All the values measured in August were below the EC5 level determined by Harris et al.
Fig. 5. Effect of average FAN level in surface (dark circles and plain line) and under hide (clear circles and dotted line) flow on the growth adjusted for density and initial size.
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(1998b). TAN was positively correlated with the cumulative number of juveniles present upstream and the resulting FAN was negatively correlated to growth (Fig. 4). The total water flow in the tanks was about 77 F 18 l/min. From the relationship between abalone number upstream and ammonia level in the water (Fig. 4), it can be estimated that a juvenile of 37 F 6 mm produced approximately 0.011 F 0.002 Amol TANg 1h 1. The chronic ammonia exposure level causing a reduction of 5% in growth (EC5) was calculated from the FAN levels measured in the surface water and under the hides from the August sampling (Fig. 5) after adjusting the data for the effects of density and initial size. Our results suggested EC5 values of 0.006 mg FAN/l for surface water and 0.004 mg FAN/ l for the water under the hides.
4. Discussion 4.1. Growth Other studies of the effect of stocking density on growth in abalone and other cultured shellfish have shown that growth rate declined as stocking density increased (Koike et al., 1979; Mgaya and Mercer, 1995; Liu and Chen, 1999; Capinpin et al., 1999). Most papers Table 3 Data from experiments studying the effects of density on the growth of different species of juvenile abalone Reference/species/ number (N) Koike et al. (1979) H. tuberculata Cochard (1980) H. tuberculata Mgaya and Mercer (1995) H. tuberculata Liu and Chen (1999) H. diversicolor supertexta Capinpin et al. (1999) H. discus McCormick et al. (1992) in Aviles and Shepherd (1996) H. rufescens Present study H. rubra
N
Tank design/ duration 6340
0 16,000 0 35
330
– –
4500
‘‘ – ’’ refers to missing information.
0.25 m2 with 4 – 17 hides 4 months 1 m2 with 0.7 m2 of hides 2 months cage
Initial length (mm)
Density/m2
Growth (mm)/ month
%Reduction in growth
9.46
83
2.5
20
9.72 14.7
5000 1000
2.0 3.4
32
14.4 23.7
3000 386
2.3 4.0
52
8 months basket 6 months
23.7 32.4 32.4
1235 177 460
1.9 3.6 2.5
cage 5 months half barrels
19 19 –
113 450 120
4.2 3.6 2.2
240
1.47
85 170
1.3 0.9
raceway 5.5 months
33.91 34.23
31
14 33
24
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reported reductions in growth ranging from 14% to 52% due to 2- to 60-fold increase in density. There was no obvious relationship between the reduction in growth and levels of density used (Table 3). This demonstrates the importance of the rearing technique on growth. The negative relationship between growth and density in gastropods suggests there is density-dependent intraspecific competition for space or food (Stimson, 1970; Branch, 1975; Hughes, 1986; Jarayabhand and Newkirk, 1989; Parsons and Dadswell, 1992; Foster and Stiven, 1996). In the present study, food was given in sufficient quantity to ensure that there was plenty of uneaten food left over in the morning in each tank and so that this would not be considered a limiting factor. Tahil and Juinio-Menez (1999) suggested that food density had a significant effect on food intake when abalone are fed algae. In this study, however, because the number of dry pellets distributed per animal was high, food density was assumed not to affect feeding. Growth significantly decreased with increasing initial sizes, densities and FAN levels. Our results suggest that competition for space may affect growth more than the other two parameters. The reduced growth with initial size suggests that larger individuals were not performing as well as the smaller ones. At high stocking density, abalone may have more difficulty to interfere with each other when they move out from shelter sites and to reach food. This may have affected their feeding and living conditions and growth (Stoner, 1989; Mgaya and Mercer, 1995). Flassch and Aveline (1984) developed a logarithmic relationship between the number of abalone per square meter and their size to describe a density threshold over which growth is reduced significantly. This threshold was established empirically based on the results of a single experiment from Koike et al. (1979) in order to help farmers chose their stocking density. The threshold was theoretically reached when the total surface of shell was equal to half of the surface of the tank bottom. However, the results of Koike et al. (1979) were probably biased because the number of hides per tank was increased with density, reducing the competition for shelter space. Furthermore, the strength of density effects differs widely between studies with different tank designs (Table 3). More work is therefore required in this area to design better management tools. 4.2. Ammonia Water quality is important in providing the optimal conditions for growth. The important water quality parameters include temperature, dissolved oxygen, pH, salinity and nitrogenous wastes (Spotte, 1979). In culture, the level of nitrogenous wastes excreted by aquatic animals is probably the most important parameter once oxygen levels are maintained (Colt and Armstrong, 1981). Within the nitrogenous wastes, ammonia is the major component of the protein catabolism (Kinne, 1976; Colt and Armstrong, 1981; Russo and Thurston, 1991). Chronic exposure can lead to slower growth and ultimately mortality in the cultured animals, thus limiting production in aquaculture (reviewed by Basuyaux and Mathieu, 1999; Hargreaves and Kucuk, 2000). The relative proportions of NH3 (free ammonia) and NH4+ (ammonia) in solution depend on temperature and pH and, to a lesser extent on salinity. Concentrations of NH3 increase with elevated temperatures and pH values and decrease with higher salinities.
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(Downing and Merkens, 1955). Thus, ammonia at low pH is usually toxic only in overwhelming quantities whereas at high pH, much smaller amounts may be lethal (Warren, 1962). In commercial activities, water quality parameters such as temperature, pH and ammonia concentrations may vary greatly over time and between places in the tanks, as suggested in our data, and this variation alone may cause a significant stress and affect the productivity and the health of the animals (Hargreaves and Kucuk, 2000). However, the only studies examining the effects of ammonia on abalone, by Harris et al. (1998b) on juvenile Haliotis laevigata and Basuyaux and Mathieu (1999) on Haliotis tuberculata, have not taken these variations into account. As expected, ammonia showed a strong positive correlation with the biomass in upstream water. The ammonia excretion rate from this relationship is in the range of excretion rates of gastropods given by Kinne (1976) and about two times lower than the one found by Ford and Langdon (2000) for Haliotis rufescens (0.017 to 0.028 Amol TANg 1h 1). This difference may be due to the artificial diet used in the present study or differences in metabolisms due to the temperature. Differences in the ammonia level were found between stocking densities even in the top tanks. The depressed growth rates in the bottom tanks showed that there is an indirect effect of density on the growth of abalone through water quality. However, growth rates in the top and middle tanks suggest that the direct effect of stocking density, probably through competition for space, was more important. There were consistent and significant differences between TAN values in the surface flow and in the water under the hides. Hides were obstacles to the water flow and during the day, abalone aggregated under the hides (Huchette et al., submitted for publication), making further obstruction to the water flows which resulted in the accumulation of faeces. Hides should therefore be used with an appropriate tank system, which would prevent accumulation of faeces and ensure good water circulation. 4.3. Chronic ammonia exposure The level of chronic ammonia exposure found to reduce growth by 5% in this study was 10-fold lower than the one found by Harris et al. (1998b) for H. laevigata. In their study, the juveniles were about 3 years old and 31.8 F 0.1 mm in size. The safe ammonia concentration of 1 mg N l 1 defined by Basuyaux and Mathieu (1999) was reached in all the tanks in June. It is possible but rather unlikely that H. rubra is a more sensitive species to FAN. Another possible explanation of this difference is the duration of the experiments and the number of individuals studied. Indeed, Harris et al. (1998b) and Basuyaux and Mathieu (1999) studied only a few juveniles (N < 200 vs. N = 1800 in this study) for 15 days. Chronic exposure is more likely to show its effect in long-term experiments especially when the main biological parameter studied is growth (Day and Fleming, 1992). Abalone growth is known to be slow and variable, and therefore has to be studied for long periods of time to show significant effects. The chronic ammonia exposure range determined in this study has to be considered carefully, however, as the ammonia level was not monitored regularly and was produced by abalone and not chemically adjusted as in other studies (Harris et al., 1998b; Basuyaux and Mathieu, 1999). The levels given in this
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study were affected by the time since the tanks were last cleaned, resulting in more variable values in the June sampling carried out 2 days after the last cleaning, compared with August when the sampling was carried out only a night after the tanks were cleaned. This suggests that the ammonia level varied within and between tanks from time to time according to the amount of faeces and uneaten food accumulating. In practice; FAN levels will also vary with the temperature, pH and salinity of the water and our low EC5 may be the result of this variation. For an abalone farm, it may be more practical and reliable to evaluate the level of growth depression according to water flow and the number of abalone found in a tank or upstream rather than to look at the chemical components of the excreta. This study provides useful data for this purpose as compared to other studies.
Acknowledgements Ocean Wave Seafood provided the infrastructure and the animals for this study. The staffs of the farm are especially thanked for their advice, support and daily feeding and cleaning of the experimental tanks. Thanks also to the volunteers who helped with the tagging and collection of the data in often challenging conditions: Patrick Gilmour, Anthony, Sabine Roussel and Huon McDiarmid. We are grateful to the technical staff of the Department of Zoology of the University of Melbourne for their help and support during the project; special thanks to Joe Wedgewood and Viviane Porter. Dr. Mick Keough kindly allowed us to use the Vector velocity meter. Sylvain Huchette acknowledges the financial support provided by the Sadlier –Stokes Scholarship given to encourage young French to tie links with Australia in memory of the Australian veterans who fought in France during WWI. This work was also carried out with the help of International Postgraduate Research Scholarship of the University of Melbourne.
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