Effects of dietary calcium and phosphorus supp

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 Æ Tosapon Sungsirin Æ 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 1 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:
T. Sungsirin Á S. Piyatiratitivorakul
Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

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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

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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 9 3.0 9 1.0 m) were used as the rearing ponds and the animals were
kept in rearing units (plastic baskets) of 25.0 9 35.0 9 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 2 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 9 2.0 9 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 5 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 1. 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

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Table 1 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

1 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 5 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 9 35.0 9 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

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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 9 (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)] 9 100

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)] 9 100

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 9 P

0.376

0.330

Calcium (Ca)

0.000

2.65 ± 0.06

1.38 ± 0.01

5

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

1.35 ± 0.01

5

2.77 ± 0.17

2.77 ± 0.01

2.82 ± 0.06

2.97 ± 0.02

Final (cm)

3

1.47 ± 0.05

3

1.46 ± 0.01

1.55 ± 0.13

5

1

Two-way ANOVA (P value)

7

4

1.45 ± 0.03

1.45 ± 0.12

1

1

Initial (cm)

P (%)

Ca (%)

3

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 2 Growth performance of juvenile B. areolata fed diets containing different levels of dietary calcium and phosphorus in a recirculating culture system for 180 days

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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)] 9 100;
– 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)] 9 100;
– feed-conversion ratio (FCR) = dry feed fed (g)/wet weight gain (g); and
– survival (%) = 100 9 (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

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Table 2. 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 1 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 1 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 1 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 3 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 1 and 7% supplemental calcium, the spotted babylon had

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Table 3 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

1

30

3.29 ± 0.18

1.43 ± 0.09

2.31 ± 0.04a

3

30

2.84 ± 0.23

1.16 ± 0.12

2.46 ± 0.05a

5

30

2.84 ± 0.11

1.14 ± 0.06

2.50 ± 0.04a

1

30

3.28 ± 0.19

1.31 ± 0.04

2.50 ± 0.04a

3

30

2.62 ± 0.04

0.99 ± 0.07

2.65 ± 0.15a

5

30

2.27 ± 0.22

0.92 ± 0.16

2.53 ± 0.16a

1

30

3.21 ± 0.18

1.31 ± 0.09

2.46 ± 0.04a

3

30

2.81 ± 0.11

1.20 ± 0.08

2.34 ± 0.07a

5

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 9 P

0.108

0.570

0.241

4

7

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 0 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 6 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

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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 2 h, because the spotted

babylon had fast feeding behavior and stopped feeding within 20 min. This means that
little dietary calcium and phosphorus could have leached from the diets within 20 min
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 6 g kg-1 supplementary P. If feed efficiency
and scale mineralization are taken into account, Ca supplementation of 6 g kg-1
(Ca/P = 1) might be the optimum when diet is supplemented with 6 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

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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, for his encouragement and critical reading of
the manuscript.

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