Aquacult Int (2010) 18:303–313 DOI 10.1007/s10499-009-9244-8 Effects of dietary calcium and phosphorus supplementation on the growth performance of juvenile spotted babylon Babylonia areolata culture in a recirculating culture system Nilnaj Chaitanawisuti Ỉ Tosapon Sungsirin Ỉ Somkiat Piyatiratitivorakul Received: 10 September 2008 / Accepted: 26 January 2009 / Published online: 14 February 2009 Ó Springer Science+Business Media B.V 2009 Abstract A feeding experiment was conducted to determine the effects of dietary calcium and phosphorus, and the interaction between calcium and phosphorus, on the growth of juvenile spotted babylon, Babylonia areolata, cultured in a recirculating culture system Nine isonitrogenous experimental diets supplemented with three levels of calcium (1, 4, and 7%) for each of three levels of phosphorus (1, 3, and 5%) were prepared using fish meal, squid meal, and shrimp meal as the main protein sources Juveniles with an initial average body weight of 0.59 ± 0.09 g were fed to satiation once daily with one of the nine diets for 180 days Absolute and specific growth rates were calculated for both shell length and whole wet body weight Results showed that dietary calcium and phosphorus supplementation significantly affected the growth of juvenile spotted babylon (P \ 0.05), but not survival and feed-conversion ratio The specific growth rate in shell length (SGRL) ranged from 0.32 to 0.39% day-1 No significant difference among phosphorus levels and no significant interaction between calcium and phosphorus in SGRL of the spotted babylon (P [ 0.05) was found, but significant differences were observed among calcium levels, irrespective of phosphorus levels (P \ 0.05) For and 7% supplemental calcium, the spotted babylon had significantly higher SGRL than those fed diets supplemented with 4% calcium However, the specific growth rate in body weight (SGRW) ranged from 0.91 to 1.19% day-1 with no significant difference among calcium and phosphorus levels and no significant interaction between calcium and phosphorus (P [ 0.05) Survival and feedconversion ratio were not significantly affected by dietary calcium and phosphorus levels with ranges from 91.00 to 95.00% and 2.43 to 2.76, respectively At the end of the experiment, shell abnormality of B areolata was found for all feeding trials Keywords Growth Babylonia areolata Á Calcium Á Phosphorus Á Recirculating culture system Á N Chaitanawisuti (&) Aquatic Resources Research Institute, Chulalongkorn University, Bangkok 10330, Thailand e-mail: nilnajc1@hotmail.com T Sungsirin Á S Piyatiratitivorakul Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 123 304 Aquacult Int (2010) 18:303–313 Introduction Spotted babylon Babylonia areolata is a popular marine gastropod cultured in Thailand and a potentially important aquaculture species because of its rapid growth, efficient feed conversion, and high market value Although large-scale rearing of B areolata in Thailand is technically feasible using flow-through systems in concrete/canvass ponds, disadvantages of these systems that must be solved during growout of spotted babylon are: – – – – the systems generally require large quantities of water; location of production systems must be near the sea; stock is vulnerable to external water supply and quality problems; and growth rate is significantly affected by water flow (Chaitanawisuti et al 2002) Waste fish is used as natural food for all commercial growing out of spotted babylon in farms in Thailand The main problems faced are shortage and discontinuous supply, lack of freshness, and inconstant nutritional values of waste fish This situation has led to research on the development of cost-effective artificial feed for farmed spotted babylon (Chaitanawisuti and Kritsanapuntu 1999; Chaitanawisuti et al 2005) In addition, research on a recirculating growout system recently provided major increases in spotted babylon culture intensification, technology, and the understanding of water quality management for maximization of profit by increasing production, lowering costs, and conserving water This system may offer an alternative to pond aquaculture technology for this species The interest in recirculating growout systems is because of their perceived advantages including: – – – – greatly reduced land and water requirements; high environmental control enabling productive-cycle growth at optimum rates; the feasibility of location far from the sea; and high water conservation and reuse However, a major problem in the rearing of juvenile B areolata in a recirculating system is shell abnormality, mainly characterized by external shell morphology—shell color with dark brown spots changed to pale brown and outer shell layer partially removed Uptake of Ca and other minerals from seawater in a recirculating system may not be sufficient to meet the mineral requirements for shell building of the spotted babylon (Chaitanawisuti et al 2005; Kritsanapuntu et al 2006) However, studies of its mineral requirements are scarce The lack of information on phosphorus (P) and calcium (Ca) dietary requirements and their bioavailability from various sources for aquatic animals can lead to their over-supplementation in formulated feeds Calcium and phosphorus are two of the major inorganic constituents of feeds It seems that almost all aquatic species have a dietary phosphorus requirement, in part because of the very low phosphorus content (typically 0.06 mg l-1) of unpolluted water Phosphorus has an integral role in cellular functions, because it is a key component of nucleic acids, phospholipids, phosphoproteins, ATP, and several key enzymes In addition, phosphate serves as a buffer to maintain optimal pH in body fluids (Coote et al 1996; Tan et al 2001) Phosphorus deficiency can occur in most species and the requirements of each aquatic species seem to differ, most reported values being in the range 0.45–1.5% (Tan et al 2001) In contrast with phosphorus, most aquatic species have no dietary calcium requirement, even though calcium is essential for some important functions, including shell formation, blood clotting, muscle function, osmoregulation, and as a cofactor for enzyme procession and nerve transmission It is extremely difficult to produce a calcium deficiency 123 Aquacult Int (2010) 18:303–313 305 in shellfish, because they can easily absorb dissolved calcium directly from the surrounding water through the gills to fulfill their metabolic requirements (Coote et al 1996; Cheng et al 2006) Some research has reported that dietary calcium affects phosphorus availability in some crustaceans and fish Davis et al (1993) have reported that an excessive level of dietary calcium relative to phosphorus reduces growth and increases mortality of Penaeus vannamei There are few published reports on the effects of dietary calcium and phosphorus on growth of mollusks Coote et al (1996) report that the greenlip abalone (Haliotis laevigata) does not require high levels of calcium (\0.05%) in its diet but that phosphorus supplementation ([0.7% total P) improves growth However, they did not find that dietary calcium levels as high as 1.5% were detrimental Wide variation of the calcium content of feedstuffs, combined with potential over-supplementation, has led to high levels of this mineral in feed In addition potentially excessive levels of calcium may result in increased phosphorus requirements of aquatic species Excessive levels of calcium and phosphorus may increase the cost of feed, increase the input of minerals to the aquatic environment, and possibly affect the bioavailability of other nutrients Therefore, the objective of this study was to determine the effects of calcium and phosphorus supplementation of experimental diets on the growth of juvenile Babylonia areolata cultured in a recirculating culture system Materials and methods Pond preparation and culture management This study was designed to use a recirculating culture system A series of rectangular plastic tanks (1.0 3.0 1.0 m) were used as the rearing ponds and the animals were kept in rearing units (plastic baskets) of 25.0 35.0 25.0 cm which contained numerous pores of 1.5 cm2 (four holes cm-2) on each side The bottom of each rearing unit was covered with coarse sand cm thick as substratum Aeration was provided with an air diffuser This study consisted of nine feeding treatments, and each treatment included three replicates Twenty-seven baskets were assigned to the tanks using a completely randomized design Plastic tanks (3.0 2.0 1.0 m) containing bioballs as biofilter were used as the biological filter tanks Seawater from the rearing pond flowed into the biological filter tank and was pumped back into the rearing pond continuously at a constant rate of 200 l h-1 Rearing units were scrubbed and seawater in the rearing ponds was replaced with new seawater every 30 days, after measurement of length and weight, to minimize accumulation of metabolites and growth of natural food organisms in the culture system During the experimental period, water quality was monitored periodically during the feeding trials Water temperature and salinity were 29.0–31.0°C and 29.0–30.0 ppt, respectively Salinity was monitored daily, as necessary, to keep the variation within ±2.0 ppt by addition of fresh water to correct for any increased salinity because of water evaporation Dissolved oxygen was not less than mg l-1, and levels of free ammonia and nitrite were negligible Natural light cycle was used in the feeding trials Experimental diet and diet preparation The formulation of the basal diet and proximate analysis are given in Table Dietary treatments were prepared by replacing the filler with graded levels of calcium carbonate (CaCO3) and monobasic potassium phosphate (KH2PO4) KH2PO4 was chosen as the P 123 306 Aquacult Int (2010) 18:303–313 Table Formulation and proximate composition of basal diet for juvenile B areolata Ingredient Dry weight (g kg-1) Fish meal 280 Shrimp meal 230 Squid meal 100 Soybean meal 200 Tuna oil 50 Wheat flour 80 Polymethyl carbamate 20 Mineral mixa 20 Vitamin mixb 20 Proximate composition (%) Crude protein 36.24 Crude lipids 18.64 Ash 12.21 a kg mineral mix consisted of calcium 147 g, iron 2,010 mg, phosphorus 147 g, copper 3,621 mg, zinc 6,424 mg, manganese 10,062 mg, cobalt 105 mg, iodine 1,000 mg, selenium 60 mg b Vitamin A 150,000,000 IU, vitamin D 3,000,000 IU, vitamin E 27.5 g, vitamin K 4.67 g, vitamin B1 25 g, vitamin B2 26 g, vitamin B6 5,000 lg, nicotinamide 20 g, folic acid 0.4 g, vitamin C 143 g, calcium D panthotenate g source because it contains no Ca, is readily soluble, and is likely to be highly bioavailable Nine experimental diets (Table 2) were formulated These contained three levels of supplemental calcium (1, 4, and 7%) for each of three levels of phosphorus (1, 3, and 5%) The diets were prepared by mixing the dry ingredients in a mixer followed by addition of the oil An appropriate amount of deionized water was then added, followed by further mixing The mixed diet was extruded through a 3-mm die in a food grinder to make the particle size suitable for juveniles Pellets were dried overnight at 25°C in an air-conditioned room to a moisture content of 10%; they were then stored at -20°C until use Juvenile rearing Juvenile B areolata used in the feeding trials were purchased from a commercial hatchery in Petchaburi, Thailand, transported to the laboratory, and kept in three 300-l circular plastic tanks for acclimatization During the acclimatization period the snails were fed chopped waste fish mixed with basal diet without Ca and P supplements The amount of waste fish was gradually replaced by the diet until the snails accepted the diet totally The acclimatization period lasted over ten days At the beginning of the experiment, healthy juveniles were sorted into a uniform size to prevent possible growth retardation of small spotted babylon when cultured with larger ones Shell length was measured with calipers to the nearest 0.02 mm and the animals were weighed to the nearest 0.01 g using an electronic balance Initial shell length and whole body weight of juveniles averaged 1.43 ± 0.08 cm and 0.59 ± 0.09 g (mean ± SD, n = 30), respectively, and did not differ significantly (P [ 0.05) among treatments (Table 2) Juveniles were distributed randomly into 27 rearing tanks of 25.0 35.0 25.0 cm (three tanks/diet) at a density of 50 snails per tank At the beginning of the feeding trial, juveniles were hand-fed with the experimental diets in slight excess once daily (10:00 h) The amount of feed was adjusted daily 123 Phosphorus (P) 0.163 0.002 0.851 0.166 0.010 0.21 ± 0.01 0.22 ± 0.02 0.23 ± 0.01 0.18 ± 0.02 0.19 ± 0.01 0.23 ± 0.05 0.22 ± 0.01 0.23 ± 0.01 0.26 ± 0.01 AGRLa (cm month-1) 91.00 ± 1.41 94.99 ± 2.35 1.19 ± 0.08 95.00 ± 7.08 f Feed-conversion ratio (FCR) = dry feed fed (g)/wet weight gain (g) Survival (%) = 100 (final snail number)/(initial snail number) Specific growth rate in weight (SGRW, % day-1) = [(ln final body weight - ln initial body weight)/(feeding trial period, days)] 100 e Absolute growth rate in weight (AGRW, g month-1) = (mean final body weight, g - mean initial body weight, g)/(feeding trial period, months) 0.818 0.891 0.400 95.00 ± 7.08 91.67 ± 2.35 d 0.602 0.490 0.450 1.10 ± 0.01 1.14 ± 0.09 93.33 ± 4.71 0.91 ± 0.11 1.03 ± 0.01 91.67 ± 2.35 95.00 ± 7.08 91.67 ± 2.35 1.10 ± 0.08 1.05 ± 0.06 1.11 ± 0.11 1.19 ± 0.04 SGRWd (% day-1) Specific growth rate in length (SGRL, % day-1) = [(ln final shell length - ln initial shell length)/(feeding trial period, days)] 100 0.025 0.000 0.003 0.57 ± 0.01 0.58 ± 0.02 0.68 ± 0.06 0.46 ± 0.02 0.45 ± 0.04 0.72 ± 0.04 0.59 ± 0.03 0.62 ± 0.01 0.72 ± 0.04 AGRWc (g month-1) c 0.031 0.000 0.009 3.95 ± 0.04 4.03 ± 0.30 4.57 ± 0.37 3.25 ± 0.19 3.34 ± 0.11 5.02 ± 0.42 4.10 ± 0.17 4.29 ± 0.05 4.90 ± 0.16 Final (g) Absolute growth rate in length (AGRL, cm month-1) = (mean final shell length, cm - mean initial shell length, cm)/(feeding trial period, months) 0.650 0.367 0.55 ± 0.02 0.56 ± 0.13 0.53 ± 0.04 0.51 ± 0.04 0.65 ± 0.11 0.70 ± 0.16 0.62 ± 0.04 0.59 ± 0.12 0.59 ± 0.02 Initial (g) Survivale (%) b 0.872 0.641 0.036 0.37 ± 0.01 0.37 ± 0.03 0.37 ± 0.01 0.33 ± 0.01 0.32± 0.04 0.35 ± 0.08 0.36 ± 0.01 0.37 ± 0.04 0.39 ± 0.01 SGRLb (% day-1) Body weight a Values are means from three replicates per treatment Ca P 0.376 0.330 Calcium (Ca) 0.000 2.65 ± 0.06 1.38 ± 0.01 2.74 ± 0.01 2.73 ± 0.04 1.40 ± 0.06 1.41 ± 0.11 2.45 ± 0.01 2.55 ± 0.05 1.35 ± 0.01 2.77 ± 0.17 2.77 ± 0.01 2.82 ± 0.06 2.97 ± 0.02 Final (cm) 1.47 ± 0.05 1.46 ± 0.01 1.55 ± 0.13 Two-way ANOVA (P value) 1.45 ± 0.03 1.45 ± 0.12 1 Initial (cm) P (%) Ca (%) Shell length Dietary supplement 0.559 0.808 0.335 2.57 ± 0.25 2.50 ± 0.26 2.63 ± 0.31 2.76 ± 0.15 2.63 ± 0.21 2.47 ± 0.15 2.43 ± 0.15 2.50 ± 0.27 2.47 ± 0.15 FCRf Table Growth performance of juvenile B areolata fed diets containing different levels of dietary calcium and phosphorus in a recirculating culture system for 180 days Aquacult Int (2010) 18:303–313 307 123 308 Aquacult Int (2010) 18:303–313 on the basis of the amount of food consumed by the snails within 0.5 h on the previous day, to ensure that only a minimal amount of feed was left Uneaten food was removed immediately after the snails stopped eating to prevent water degradation The amount of feed eaten was recorded daily for calculation of feed-conversion ratio The snails were weighed and measured individually (n = 30) at the start of the experiment and every 30 days in feeding trials for growth estimation No chemical and antibiotic agent was used throughout the entire experimental period Grading by size was not carried out in any pond throughout the growing-out period Each feeding trial was terminated after 180 days Sample collection and analysis At the end of the experiment, 20 snails from each feeding trial were removed from the rearing system, weight, measured, and counted Growth was expressed as specific growth rate (SGR), absolute growth rate (AGR), feed efficiency (FE), and survival The calculation formulae were: – absolute growth rate in shell length (AGRL, cm month-1) = (mean final shell length, cm - mean initial shell length, cm)/(feeding trial period, months); – specific growth rate in shell length (SGR, % day-1) = [(ln final shell length - ln initial shell length)/(feeding trial period, days)] 100; – absolute growth rate in body weight (AGRW, g month-1) = (mean final body weight, g - mean initial body weight, g)/(feeding trial period, months); – specific growth rate in weight (SGRW, % day-1) = [(ln final body weight - ln initial body weight)/(feeding trial period, days)] 100; – feed-conversion ratio (FCR) = dry feed fed (g)/wet weight gain (g); and – survival (%) = 100 (final snail number)/(initial snail number) (Tan et al 2001; Ye et al 2006; Liu et al 2006) After the final weighing, ratio of total shell weight to dry tissue weight was calculated to determine whether feeding trial affected shell thickness Soft tissues of snails were removed from each feeding trial (n = 20) at the end of the experiment Soft tissues and empty shells were air dried and weighed individually Shell normality was observed by comparing the external shell morphology of spotted babylon from each feeding trial at the beginning and end of the experiment Shell abnormality were characterized by external shell morphology—shell color with dark brown spots changed to pale brown and outer shell layer partially removed Statistical analysis The feeding trials were arranged in a completely randomized design in a two-factor experiment Three levels of Ca and P were tested Data from each treatment were subjected to a two-way analysis of variance (ANOVA) Tukey’s test was used to detect differences between treatment means because of main effects Differences were considered significant at the 0.05 probability level (P \ 0.05) Results Growth performances of juvenile B areolata fed diets containing different levels of dietary calcium and phosphorus in a recirculating culture system for 180 days are shown in 123 Aquacult Int (2010) 18:303–313 309 Table Dietary calcium and phosphorus supplementation significantly affected the growth of juvenile B areolata (P \ 0.05) but not survival and feed-conversion ratio (FCR) Absolute growth rate in shell length (AGRL) ranged from 0.18 to 0.26 cm month-1 among feeding trials Two-way analysis of variance indicated no significant difference among phosphorus levels and no significant interaction between calcium and phosphorus in AGRL of the spotted babylon (P [ 0.05), but significant differences were observed among calcium levels regardless of phosphorus levels (P \ 0.05) (Table 2) At and 7% supplemental calcium, the spotted babylon had significantly higher AGRL than those fed diets supplemented with 4% calcium The specific growth rate in shell length (SGRL) ranged from 0.32 to 0.39% day-1 No significant difference among phosphorus levels and no significant interaction between calcium and phosphorus in SGRL of the spotted babylon (P [ 0.05) were found but significant differences were observed among calcium levels regardless of phosphorus levels (P \ 0.05) (Table 2) At and 7% supplemental calcium, the spotted babylon had significantly higher SGRL than those fed diets supplemented with 4% calcium The absolute growth rate in body weight (AGRW) ranged from 0.45 to 0.72 g month-1 among feeding trials Two-way analysis of variance showed that AGRW was significantly affected by dietary calcium and phosphorus and the interaction between calcium and phosphorus (P \ 0.05) (Table 2) Snails fed diets with and 7% calcium supplementation had significantly higher AGRW than those fed diets with 4% calcium AGRW of snails fed diets with 1% phosphorus was significantly higher than those fed diets with and 5% phosphorus at any of the three levels of calcium However, the specific growth rate in body weight (SGRW) ranged from 0.91 to 1.19% day-1 Two-way analysis of variance indicated no significant difference among calcium and phosphorus levels and no significant interaction between calcium and phosphorus in SGRW of the spotted babylon (Table 2) The average ratio of shell weight to dry tissue weight ranged from 2.31 to 2.65 Twoway analysis of variance indicated no significant difference among calcium and phosphorus levels and no significant interaction between calcium and phosphorus in shell weight and dry tissue weight of the spotted babylon (P [ 0.05) (Table 3) However, this study showed that shell abnormality of B areolata was found for all feeding trials This was mainly characterized by external shell morphology—shell color with dark brown spots changed to pale brown and outer shell layer partially removed At the end of the feeding experiment, all feeding trials resulted in high survival (91.00– 95.00%) Two-way analysis of variance indicated no significant difference among calcium and phosphorus levels and no significant interaction between calcium and phosphorus in survival of the spotted babylon (P [ 0.05) (Table 2) Results suggested that survival of the spotted babylon was not affected by dietary treatments Feed-conversion ratio (FCR) ranged from 2.43 to 2.76 among feeding trials with no significant differences among calcium and phosphorus levels (P [ 0.05) (Table 2) Discussion In this study, dietary calcium and phosphorus supplementation significantly affected the growth of juvenile spotted babylon but not survival and feed-conversion ratio Specific growth rate in shell length (SGRL), range from 0.32 to 0.39% day-1, was significantly affected by dietary calcium (P \ 0.05) but not by phosphorus or the interaction between calcium and phosphorus At and 7% supplemental calcium, the spotted babylon had 123 310 Aquacult Int (2010) 18:303–313 Table Average shell weight and dry tissue weight for juvenile B areolata fed diets containing different levels of dietary calcium and phosphorus in a recirculating culture system for 180 days Dietary supplementation n (snails) Shell weight (g) Dry tissue weight (g) Shell weight:dry tissue weight Ca (%) P (%) 1 30 3.29 ± 0.18 1.43 ± 0.09 2.31 ± 0.04a 30 2.84 ± 0.23 1.16 ± 0.12 2.46 ± 0.05a 30 2.84 ± 0.11 1.14 ± 0.06 2.50 ± 0.04a 30 3.28 ± 0.19 1.31 ± 0.04 2.50 ± 0.04a 30 2.62 ± 0.04 0.99 ± 0.07 2.65 ± 0.15a 30 2.27 ± 0.22 0.92 ± 0.16 2.53 ± 0.16a 30 3.21 ± 0.18 1.31 ± 0.09 2.46 ± 0.04a 30 2.81 ± 0.11 1.20 ± 0.08 2.34 ± 0.07a 30 2.89 ± 0.10 1.12 ± 0.14 2.59 ± 0.23a Calcium (Ca) 0.035 0.045 0.148 Phosphorus (P) 0.000 0.002 0.236 Ca P 0.108 0.570 0.241 Two-way ANOVA (P value) Value within the same column followed by different letter superscripts were significantly different (P \ 0.05) Values are means from three replicates per treatment significantly higher SGRL than those fed diets supplemented with 4% calcium However, specific growth rate in body weight (SGRW) ranged from 0.91 to 1.19% day-1 with no significant difference among calcium and phosphorus levels Coote et al (1996) reported that although dietary Ca supplementation did not affect the growth of abalone Haliotis laevigata the specific growth rate of abalone fed diets supplemented with P was 7.9% higher than that of abalone fed diets without P supplement They also suggested that abalone did not require high levels of Ca in their diet Supplementation with CaCO3 was unnecessary, but P supplementation (C0.7% total P) can improve growth rates The Ca:P ratio of feed was not important within the range assessed (0.72:1 to 2.68:1) Tan et al (2001) also reported that the weight gain rate and daily increment in shell length of juvenile abalone Haliotis discus hannai were significantly affected by dietary phosphorus levels (0–2.0%) but survival ranged from 94.7 to 100% with no significant difference among dietary treatments The calcium/phosphorus ratio of the diets was not important within the range assessed (0.1:1–9.0:1) For other aquatic animals, Penaflorida (1999) reported that comparison between and 1.5% Ca levels showed similar percentage weight gain of shrimp Peneaus monodon fed diets supplemented with 0, 1.5, or 2% P The reduction in percentage weight gain at high P levels was presumably caused by a shift in the pH of the diet, increased potassium (K) level, and an interaction with other nutrients With the shift from pH to 5.5 (low to high P diets), Ca probably acted as a buffer reducing the pH shift the diet An increase in P level also increased K, the source being KH2PO4 This increase can affect magnesium (Mg) level, which is essential in cell respiration and phosphate transfer reaction, because Mg complexes with adenosine tri, di, and monophosphates Davis et al (1993) reported an excessive level of dietary calcium relative to phosphorus increased mortality and reduced growth of Penaeus vannamei Vielma and Lall (1998) indicated high calcium/phosphorus ratio is unlikely to interfere with dietary 123 Aquacult Int (2010) 18:303–313 311 phosphorus utilization in Atlantic salmon Salmo solar Cheng et al (2006) suggested that dietary Ca/P ratio should be considered as well as individual dietary levels of the animals Excessive dietary Ca may result in increased P requirements of shrimp, which would increase the cost of feeds and output of minerals to the rearing media, and inhibit the bioavailability of other nutrients Differences in dietary requirements for Ca and P between species may be because of physiological differences, particularly the presence or absence of an acidic stomach, because absorption of both Ca and P is facilitated by low pH In particular, species lacking a stomach may be less able to absorb P in fishmeal or soybean (Coote et al 1996) In this study, survival and feed-conversion ratio were not significantly affected by dietary calcium and phosphorus levels in the ranges 91.00 to 95.00% and 2.43 to 2.76, respectively Leaching of the experimental diets was not done in this study, because all experimental diets had maximum water solubility of h, because the spotted babylon had fast feeding behavior and stopped feeding within 20 This means that little dietary calcium and phosphorus could have leached from the diets within 20 immersion in seawater All of the animals showed no sign of stress or boredom with diets; they consumed all diets and grew well The experimental diets in this study contained 36% crude protein, which was reported by Zhou et al (2007) to be the optimal dietary protein requirement for growth and feed efficiency of juvenile ivory shell B areolata Ye et al (2006) indicated that dietary Ca supplementation increased feed efficiency of juvenile grouper Epinephelus coioides at the level of g kg-1 supplementary P If feed efficiency and scale mineralization are taken into account, Ca supplementation of g kg-1 (Ca/P = 1) might be the optimum when diet is supplemented with g kg-1 P They also reported that when P was not supplemented, juvenile grouper E coioides fed diets with Ca supplementation had comparable growth with fish fed without Ca supplementation, but had lower feed intake, and consequently resulted in elevated feed efficiency In this study, shell abnormality of B areolata was found in all feeding trials It was mainly characterized by external shell morphology—shell color with dark brown spots changed to pale brown and the outer shell layer partially removed Shell abnormality may because of insufficient of calcium and other elements in a recirculating system because of depletion of these elements for shell building or loss of calcium from the shell to outside medium, because of the equilibrium concentration of calcium between the blood and outside medium These elements in diets cannot compensate for no bioavailability for use in shell building Calta (2000) reported that a number of aquatic molluscs are able to absorb most of their calcium directly from the surrounding water Calcium is very important element for fish and shellfish because it is necessary for a variety of functions such as bone and scale growth, shell building, muscle contraction, transmission of nerves impulses, intercellular signaling, hormone secretion, and against osmotic and ionic gains and losses Calcium enters the fish through the gills, intestines, and skin The gills are a particularly important calcium uptake site In comparison with other mineral elements calcium is required at rather high levels by aquatic animals These requirements are met by dietary resources; however, dissolved calcium is readily taken up by gills of fish/shellfish and some species can acquire 65–80% of their metabolic needs from the water Greenaway (1971) summarized that influx and net uptake of calcium by the freshwater snail Limnaea stagnalis are related to external calcium concentration in a non-linear manner Calcium depletion does not significantly alter the normal influx or net uptake rate of calcium and the calcium concentration in the blood remains constant during net uptake from, and net loss to, the medium Hincks and Mackie (1997) reported that maximum growth of zebra mussel (Dreissena polymorpha) occurred at calcium levels of 32 mg Ca l-1, alkalinity of 65 mg CaCO3 l-1, and total hardness of 100 mg CaCO3 l-1 There was negative growth at 123 312 Aquacult Int (2010) 18:303–313 calcium levels less than 31 mg CaCO3 l-1, and positive growth of juvenile zebra mussel only occurred at pH greater than 8.3 They also stated that mollusc shells are composed primarily of crystals of CaCO3 (96.3% CaCO3 and 0.34% MgCO3 in zebra mussel) bound together in an organic matrix Most of the calcium (80%) deposited in the shell is actively taken up from the seawater Crystallization removes calcium and carbonate ions from the fluid and the reaction proceeds to add new shell layers However, these reactions are reversible, and under certain conditions calcium may be removed from the shell, which may explain degrowth in some of the mussels In addition, they suggested that normal calcium metabolism occurs at 10–12 mg l-1 Below these levels the mussels lose calcium to the external medium Presumably, low calcium had an effect on juvenile growth rates because there was not enough calcium for shell building Compared with this study, growth rate in shell length was significantly affected by dietary calcium In this study, seawater temperature, salinity, pH, dissolved oxygen, nitrite-nitrogen, and ammonia-nitrogen throughout the experiment were in the ranges 27.33–27.92°C, 36.33–40.65 ppt, 7.51–7.97, 6.04–6.55 mg l-1, 0.2653–0.4811 mg l-1, and 0.2026–0.3334 mg l-1, respectively The greatest change of water quality was only found in the alkalinity (58.76–84.06 mg l-1) which was approximately 50% less than that of natural ambient seawater (110 mg l-1) Abnormal shell building of the spotted babylon may, then, be because of mineral deficiency owing to depletion in the recirculating system, because this study had a complete seawater exchange every 30 days Perry et al (2001) reported that exoskeletal calcification in blue crab Callinectes sapidus is achieved predominantly with calcium absorbed from seawater and seawater with calcium levels reduced to 60–80% of normal decreased the calcification rate Further work is needed in order to discern the effect of dietary calcium and phosphorus on the bioavailability of other minerals such as magnesium, zinc, and manganese, and tissue mineralization and shell/muscle calcium and phosphorus deposition, as indicators of dietary calcium and phosphorus supplementation for the juvenile B areolata cultured in both flow-through and recirculating systems Acknowledgments This study was supported by the National Research Council of Thailand (NRCT), who provided funding for this research in the fiscal years 1996–2007 We are especially grateful to Professor Dr Yutaka Natsukari, Faculty of Fisheries, Nagasaki University, 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