AqUaCUltUt%

ELSEVIER Aquaculture 158 (1997) 247-262

Growth of clam seed (Ruditapes decussatus) reared

in the wastewater effluent from a fish farm in
Galicia (N.W. Spain)
Ricardo Jara-Jara, Antonio J. Pazos, Marcelina Abad *,
Leopold0 0. Garcia-Martin, Jo& L. SBnchez
Dpto. Bioquimica y Biologia Molecular, Fucultad de Farmncia, Unicersidud de Santiago de Compostela,
Santiago de Compost& 15706, Spain

Accepted 10 August 1997

Abstract

The use of the effluent from an intensive culture of turbot, Psettu maxima, cultured at Nastos
(Galicia) as a source of nutrients for growing the Ruditupes decussatus seed was evaluated and
compared with the growth under field conditions in a clam-rearing zone (Carril, Galicia). The seed
was placed at a density of 2.5 kg/m2 in trays suspended in four tanks with different flow rates.
Wastewater was changed at the rate of 1 vol./h, 2 vol./h and 4 vol./h. The fourth tank with sea
water was the control. In Carril, the seed was planted at a density approaching 2 kg/m2. After 60
culture days, the slowest growth rate was in the control (K = 0.001) and the fastest in 4 X h tank
(K = 0.03631, with mortality rates less than 18% whereas in Carril, it was approximately 35%.
Significant (P < 0.05) differences of weight and length existed between successive samplings but
not between the three different flow rates. The condition index in the experimental tanks was
similar ranging from I3 to 19. In Carril, the seed reached a similar weight to that registered in the
I X h and 2 X h tanks, although the shell length and the condition index were less. At the end of
the experiment, all biochemical component levels (mg/individual) were higher in the clams fed
with the wastewater, mainly neutral lipids (45.8 pg/mg DW), whereas the clam cultivated in
Carril presented a high concentration of carbohydrates, mostly as polysaccharides (84.3 pg/mg
DW). The clams prefattened with the wastewater also had the greatest energy content (803.1
J/individual) compared to the low values registered in the control tank and Carril (maximum of

* Corresponding author. Tel.: + 34-81563100 ext: 14930; fax: + 34.81596264; e-mail:

bnlina@uscmail.usc.es.

00448486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved.

P/f SOO44-8486(97)00196-S
248 R. Jara-Jara et al./Aquaculture 158 (1997) 247-262

233.0 and 304.8 J/individual, respectively). Only minor differences were observed in the fatty
acid spectrum of the seed reared in the tanks or in Carril. 0 1997 Elsevier Science B.V.

Keywords: Clam; Growth; Prefattening; Ruditapes decussatus; Seed; Turbot; Wastewater

1. Introduction

Galicia (N.W. Spain) trades approximately 16,000 tons of clam per year. However,
this region only yields an average of 4000 tons/yr, hence, Galicia must import over
12,000 tons of clam from other countries each year (CIPEM, 1992). On the other hand,
1300 tons/yr of turbot (Psettu maxima) are produced by Galicia. The fish are grown in
ponds and this type of culture needs to daily pump a considerable quantity of sea water,
which is later poured back into the sea with an important content in particulate organic
matter (Porter et al., 1987).
The depletion of natural and introduced stocks of clams has generated an increase in
the demand of hatchery-produced seed (Laing et al., 1987). The production of larvae
from clam breeders by European hatcheries is well established, but if the seed obtained
(less than 1 mm length) are directly planted into the natural environment, a high
mortality rate occurs due to its small size. De Pauw (1981) and Claus et al. (1983)
reported that a stage of prefattening is necessary until a size equal or higher than lo-15
mm is reached. Nowadays, there is a provision of this seed range on the market, but its
price is still almost inaccessible to clam cultivators. The problem is that the food
supplied at this early stage (mainly microalgae of a high nutritional value) represents a
high capital investment in relationship to the total cost of production (De Pauw, 1981;
Laing et al., 1987; Albentosa, 1994). In this phase, the seed or juvenile filters large
quantities of microalgae, representing about 2% of the live weight of the animal per day
for Ruditapes decussatus (Albentosa, 1994).
To use the wastewater from the intensive culture of turbot to rear clam seeds seems
be a good form of overcoming this costly phase of prefattening, since it is possible to
have large quantities of residual water without additional pumping cost. This water is
rich in dissolved and particulate matter from animal excretion and uneaten food and,
furthermore, contains a microalgae concentration greater than in natural sea water
(Dunstan and Menzel, 1971; Motzkin et al., 1982; Shpigel and Blaylock, 1991). Various
projects related to fish and mollusc polycultures have been carried out by Gordin et al.
(1981), Shpigel and Fridman (1990) Trevor and Iwama (1991) Shpigel and Blaylock
(1991) and Shpigel et al. (1993). These authors and others (Krom et al., 1985a,b; Porter
et al., 1987) analyzed, in detail, the effects of effluents from intensive fish culture in
Eilat (Israel) on the growth and survival rates of several species of bivalve molluscs,
emphasising the great microalgae-richness of these waters.
The clam culture in these special conditions relies upon two important aspects: the
quality of the wastewater (metabolite levels, temperature, oxygen concentration, dis-
solved and particulate organic matter, etc.) and the correct balanced nutrition of the seed.
Although, there is little knowledge about the nutritional requirements of bivalves, it has
been accepted that one of the more important factors is the polyunsaturated fatty acid
 R. Jara-Jara et al./Aquaculture 158 (1997) 247-262 249

content in the diet (Waldock and Holland, 1984). Certainly, the eicosapentaenoic acid
(20:5n3) and docohexaenoic acid (22:6n3) are considered essential in clams (Laing et
al., 1987). Thus, Laing et al. (1987) carried out different experiments which clearly
showed that the period of hibemal rest of growth in juvenile R. decussatus reared in
natural conditions, could be by-passed in part by supplying microalgae rich in essential
fatty acids. The seasonal variation of the nutritional richness of the wastewater, the
influence of the flow rate through the clam culture systems or the correction of possible
deficiences in essential nutrients are aspects of great importance for the improvement
and the success of this type of culture.
We describe in this report a study with two objectives: (1) to determine the growth
rate and condition of R. decussatus seed fed with different wastewater flows from an
intensive P. maxima culture; and (2) to study the biochemical composition and the fatty
acids profile of the individuals fattened with this wastewater. The results were compared
with those of the same seed grown in a natural environment in the locality of Carril (Ria
de Arousa, Galicia).

2. Material and methods

2. I. Experimental system

The experiments were carried out in Nastos, a fish farm working on the intensive
culture of turbot (P. maxima), situated in Corrubedo (N.W. Galicia). This factory yields
approximately 90 tons of fish annually. The clams were grown in tanks supplied with
wastewater from the fish farm. The seed R. decussatus were grown on wooden trays of
0.6 X 0.4 X 0.1 m with 2 mm nylon netting at the bottom. The trays were placed in four
rectangular tanks constructed of fiberglass with a double bottom (4.0 X 0.4 X 1.4 m).
Each tank was provided with a fixed flow rate of fish pond effluents; the water exchange
rates were one, two and four tank volumes per hour (560 l/h, 1120 l/h and 2240 l/h,
respectively) while the fourth tank was the control inflowing only fresh sea water at the
rate of 2 vol./h. Hereafter, flow rates will be expressed as 1 X h, 2 X h and 4 X h.

2.2. Environmental parameters

Water temperature was daily measured in the experimental tanks with a thermometer
( f 0. 1°C). Oxygen was determined using an ATAGO model S/MILL oxygen-meter and
the oxygen level was always maintained above 7-8 mg/l, injecting oxygen liquid
constantly into the tanks of turbot culture. Salinity was kept constant at 35536%0. The
chlorophyll a concentration was determined every 15 days from June to November
1994. Water samples were taken from the drained channel, just before it entered the
prefattening clam tanks. One to three litre aliquots were concentrated onto GF/C filters
and chlorophyll a was extracted with 90% acetone and assayed according to the
Strickland and Parsons (1968) method.
According to Beveridge et al. (1991) and Iwama (1991) using mass equilibrium
equations, it is possible to estimate the quantity of feces, excretion metabolites and
250 R. Jam-Jara et al./Aquaculture I58 (1997) 247-262

uneaten food flushed from a fish pond. To achieve this estimation, it is necessary to
know the quality and quantity of food delivered to the fish and the conversion food ratio.
In our case the turbot were fed twice a day with a commercial fish pellet (EWOS turbot
4.5 mm and 12% humidity) containing approximately 49% proteins, 14% lipids, 5%
carbohydrates, 12% ash, 1.5% fiber, 6% vitaminic premix, 2% calcium, 1% phosphorus,
0.2% sodium, with a digestible energy of the 17.4 kJ/g.

2.3. Experimental protocol

R. decussatus seed used in this work were supplied by the Ostreira hatchery, with a
initial mean live weight of 28.3 f 11.6 mg and mean shell length of 5.7 + 3.1 mm. For
the acclimation period, the seed were stocked on 30 May 1994 at a density of 2 kg/m2
in two experimental tanks, the wastewater was pumped from the turbot culture pond
2 X h flow.
After 45 days, 16 July 1994, the seed (mean live weight = 162.5 f 58.6 mg and
mean length = 9.6 f 0.9 mm) was placed at a density of 2.5 kg/m2 in wooden trays
which were suspended in the four experimental tanks (three trays per tank). To evaluate
the growth under natural conditions, on the same day, part of seed was planted at an
intertidal zone (40% air exposure) in Carril, the best clam-rearing area in Spain. The
culture density was approximately of 2.5 kg/m2
Fortnightly, 30 individuals were sampled from each tank until 2 November 1994. In
Carril, the samplings were carried out at intervals of approximately 30 days until 20
November 1994. Total length was measured (whole individuals) to the nearest 0.1 mm
using callipers and total live weight using an electronic balance. The meat was separated
from the shell and both were dried until constant weight at 80°C. Afterwards, the meat
samples, in aluminium boats, were cornbusted in a muffle furnace for 8 h at 450°C and
ash samples were then weighed. The total organic matter was calculated as the
difference between the total dry weight and the ash weight. The condition index was
obtained from the formula (Walne and Mann, 1975):

CI = (mean dry meat weight/mean dry shell weight) X 100.

To determine the growth rate in juvenile individuals, Malouf and Bricelj (1989)
recommend the use of the instantaneous growth rate (K), which was calculated using
the equation:

K=lnW,-lnW,/t,-t,

where: W, = the initial mean weight (or other means); W, = the final mean weight;
t, - t, = the elapsed time (generally in days).
The coefficient K, multiplied by 100, yields the percentage of change per day.
The statistical analysis of the results was performed using the one-way ANOVA to
determine the significance at the P < 0.05 level of the differences between samplings
and two-way ANOVA was used to study the effect of the different samplings and flows
on the clam seed growth. The a posteriori test of Duncan (t) was used to check the
differences between successive samplings.
 R. Jan-Jam et aL/Aquaculture 158 (1997) 247-262 251

2.4. Biochemical composition and fatty acids profile

For biochemical analysis, 20 clams were sampled at approximately 2-week intervals

from the control and 2 X h tanks. and monthly from Carril. The clams were transported
to the laboratory, placed in filtered sea water at 17°C (24 h) and then dissected. The
meat was freeze-dried and stored at -30°C until analyzed. Distilled and de-ionized
water (500 ~1) was added to the sample (2-3 mg of freeze-dried) and then homogenized
by sonication under refrigeration. The homogenized material was fractionated according
to the procedure of Holland and Gabbott (1971) as modified by Holland and Hannant
(1973). Total carbohydrates and total lipids were determined by the methods described
in Holland and Hannant (1973). Total proteins were determined with an Elemental
analyzer Carlo Erba (model 1108). Energy conversion factors used to calculate the
energy content were: 17.14 kJ/g for carbohydrates (Brody, 1945), 17.97 kJ/g for
proteins (Beukema and De Bruin, 1979) and 35.20 kJ/g for lipids (Beukema and De
Bruin, 1979).
Statistical treatment of data was carried out according to Holland and Gabbott (1971).
Values for the coefficient of variation were less than 0.05. The number of replicate
samples necessary to obtain repeatable biochemical results, determined by using Stu-
dent’s t-test according to Bartlett (1979), were two. When necessary, the data were
treated using the one-way ANOVA analysis and critical significance level was P < 0.05.
Methyl esters of total lipids (FAMES) were prepared by esterification with 2% (w/v)
concentrated sulphuric acid in methanol (Kates, 1972). The FAMES were analyzed,
following a procedure described previously (Abad et al., 1995), by gas chromatography
on a SUPELCOWAX-10 flexible fused capillary column (30 m X 0.25 mm i.d.; 0.25
pm thickness, Supelco) in a Hewlett Packard Model 5890 Series II Chromatograph,
fitted with a split injection system and equipped with a flame ionisation detector (FID).
Helium was the carrier gas and methyl tricosanoate (23:O) was added to each sample as
an internal standard for quantitative determinations. Analyzed solutions (1 ~1) were
separated and identified by comparison with commercial fatty acid standards and FAME
mixtures, which were routinely chromatographed in order to establish retention times
and to determine response factors. To avoid the unnecessary complication of the results,
fatty acids representing less than 0.2% of the total fatty acid content were not
considered.

3. Results

3.1. Environmental and wastewater parameters

Fig. 1 shows the water temperature and chlorophyll a levels in the tanks during the
experimental period. Average temperatures fluctuated between a minimum of 12.7”C
and a maximum of 18.8”C, minor differences were observed throughout the day.
Chlorophyll a levels ranged between 2.25 f 0.58 and 4.89 + 0.56 pg/l, the variations
in the concentration were parallel to bloom-and-crash cycles of the phytoplankton.
252 R. JamJam et al. /Aquaculture 158 (1997) 247-262

18

14

13

122 J J A S 0 N
Fig. 1. Fortnightly mean values for chlorophyll a concentrations (n = 3) in the wastewater and average daily
temperature.

The particulate organic matter content, total nitrogen and phosphorus in the wastewa-
ter from June to November 1994, were 467.5, 24.7 and 3.3 kg/ton of fish, respectively.
The concentration of these components was directly related to the increase of biomass
and growth of the turbot population. There was a decrease in the quantity of waste in
November due to the first sales of the turbot production.
The environmental parameters in Carril were in the same range as in the experimental
tanks: temperature 9-19°C salinity 22-34%0, chlorophyll a 2-5 kg/l and oxygen
5.9-7 pug/l (data from Xunta de Galicia).
 R. Jar-a-Jam et al. /Aquaculture 158 (1997) 247-262 253

3.2. Suruival, growth and condition index (CI)

Mortality was 25-35% in the acclimation stage, and during the study phase was less
than 18% in Nastos and approximately 35% in Carril, where the highest mortality rate
was found in the smaller size seed.
The growth of the seed in the different experimental conditions are summarized in
Table 1 and Fig. 2. The slowest growth rates were in the control (K = 0.001) and the
ANOVA indicated that the only significant (P < 0.05) differences of weight and length

Table 1
Growth and condition index of Ruditcps decussatus reared in a turbot culture effluent water at differing flow
rates and under field conditions (n = 30)
165 IA 9A 16A 1s 9s 16s 20 190 2N 20N

Length (mm) 9.6

Control 10.1 10.6 11.1 11.2 Il.3 11.3 Il.3
lxh 11.5 12.8 14.0 14.3 14.4 14.7 14.7
2Xh 11.6 13.3 14.4 14.7 15.2 15.3 15.3
4xh 11.9 13.6 15.1 15.4 15.6 15.7 xxx
GUti 10.9 11.8 12.9 13.4

Dry meat weight (mg) 2.9

Control 5.6 7.2 11.2 13.0 15.8 15.8 15.8
lxh 16.1 25.1 33.9 38.3 38.8 42.7 43.6
2Xh 17.1 24.6 35.2 41.7 43.0 47.1 48.0
4Xh 19.3 27.7 39.5 47.3 48.7 53.1 xxx
Carril 5.7 9.4 15.8 19.3

Dry shell weight (mg) 37.3

Control 65.5 78.7 128.5 134.3 135.0 137.5 138.1
lxh 129.0 173.5 216.2 243.6 245.5 251.3 253.3
2xh 112.2 148.5 220.4 242.5 247.8 265.7 266.9
4xh 118.8 178.1 240.4 264.4 272.3 291.3 xxx
Carril 83.6 149.6 245.5 269.0

Condition index 15.0

Control 9.0 9.0 9.0 12.0 10.0 12.0 12.0
Ixh 15.0 13.0 15.0 16.0 16.0 17.0 17.0
2xh 15.0 17.0 16.0 17.0 17.0 18.0 16.0
4xh 17.0 16.0 16.0 18.0 17.0 19.0 xxx
Carril 6.0 6.0 7.0 8.0

165 = 16 July.
IA = 1 August.
9A = 9 August.
16A = 16 August.
1S = 1 September.
9s = 9 September.
16s = 16 September.
20 = 2 October.
190 = 19 October.
2N = 2 November.
20N = 20 November
254 R. Jara-Jam et al./Aquaculture 158 (1997) 247-262

800

700

G 600
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1
300

200

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Fig. 2. Mean live weight (meat + shell) ( f s.d.) for Ruditupes decussatus seed (n = 30). ( 0) 1 x h; (A ) 2 x h;
(0) 4 X h; (0) control; (V ) Carril.

were between the samplings on days 16 July and 1 August, 1 and 16 August, 16 August
and 1 September. In the 1 X h tank, the weight and the length of the seed were
remarkably greater (K = 0.0297) than the control tank. The individuals of the 2 X h tank
underwent a rapid growth in the initial 60 culture days (K = 0.0327). However, the most
rapid growth rate in this period was obtained in the 4 X h tank (K = 0.0363), resembling
the growth curve registered in the 1 X h and 2 X h tanks (Fig. 2). An ANOVA indicated
that the only significant (P < 0.05) differences of weight and length existed between
successive samplings during the initial 60 cultured day in the 1 X h, 2 X h and 4 X h
tanks. Therefore, a two-way ANOVA indicated that there were no significant (P < 0.05)
differences in the growth at the different flow rates during the first 60 days of culture,
unlike the significant (P < 0.05) differences observed on 1 September.
In Carril, the seed reached a similar live weight to that registered in the 1 X h and
2 X h tanks (Fig. 21, although the shell length was greater in the experimental tanks
(Table 1). Under natural conditions, there were significant differences (P < 0.05) in
weight and length between all samplings.
The clam condition index in the control tank (Table 1) fell drastically during the first
two weeks, from 15 to 9, increasing in September with a maximum value of 12. In the
experimental tanks (1 X h, 2 X h and 4 X h) the condition index values were similar
ranging from 13 to 19. The values of CI in the Carril seed were lower, decreasing
sharply after the initial stage from 15 to 6 and then rising slowly to 8.

3.3. Biochemical composition and fatty acid profile

The results of the biochemical analysis of the prefattened seed in the control and
2 X h tanks and in Carril are presented in Table 2, expressed in absolute values:
mg/individual. At the end of the experiment, all biochemical component levels of the
seed from 2 X h tank were higher.
 R. Jara-Jura et al./Aquaculture 158 (1997) 247-262 2%

Table 2
Biochemical composition of Ruditupes decussatus seed (mg/individual) (n = 201
Initial composition Final composition
Control Nastos (2 X h) Carril

Total carbohydrates 0.10 1.52 4.86 2.00

Free reducing sugars 0.05 0.63 1.95 0.38
Polysacchatides 0.06 0.89 2.92 1.63
Total lipids 0.10 1.01 4.67 0.84
Neutral lipids 0.04 0.34 2.35 0.24
Phospholipids 0.06 0.67 2.32 0.60
Proteins 1.63 9.54 30.91 13.40
Ashes 0.48 1.56 5.54 3.45
Organic matter 2.42 14.20 45.79 15.85

The main energetic reserves (polysaccharides and neutral lipids) are presented in Fig.
3 expressed in relative values: pg/mg of dry meat weight. The individuals of the 2 X h
tank accumulated the greatest quantity of neutral lipids (45.8 pg/mg DW at the end of
experiment), whereas the higher concentration of carbohydrates was stored in the Carril
seed, mainly as polysaccharides (84.3 pg/mg DW). The clams grown with the
wastewater also had the greatest energy content, reaching 803 J/individual, in compari-
son with the low values registered in the control tank and Carril (maximum of 233 and
30.5 J/individual, respectively).
The variations in the fatty acid composition of the clams grown in Carril and at

60
1
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2 70-
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$ 50-
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J
, A
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Fig. 3. Variations of neutral lipid and polysaccharide levels expressed as pg/mg of dry meat weight. (0)
2 X h; (0) control; (v) Carril.
256 R. Jura-Jara et al./Aquaculture 158 (19971247-262

Table 3
Variations in the fatty acid comuosition in the total liaids of Ruditaaes decussatus seed (n = 20)

Control Nastos (2 X h) Carril

16.J 16A 16s 190 2N 16A 16s 190 2N 9A 9s 190 20N

‘3Saturated 23.1 32.9 22.8 24.0 20.8 28.3 31.0 27.0 27.9 21.8 18.5 20.8 21.1

PIt7 9.6 11.1 12.0 18.8 12.7 12.9 11.5 12.9 13.9 6.5 2.2 5.3 4.2
s n9 7.8 6.9 8.3 4.2 7.9 9.2 9.2 8.8 8.9 6.7 6.9 5.4 5.9
‘2MU FA 18.4 19.3 21.3 24.7 23.3 22.5 21.5 22.3 24.0 15.3 11.5 12.8 12.5

16:4n3 6.9 4.4 6.7 5.8 8.1 3.7 6.1 4.9 5.4 11.0 10.0 9.8 10.7
18:3n3 2.0 1.2 1.4 - 1.8 1.1 2.5 3.3 2.0 - - - -
18:4n3 3.1 0.9 0.8 - 3.4 1.2 1.2 1.6 0.9 - - 1.3 1.6
205113 9.8 5.2 13.9 5.3 5.4 12.4 10.8 12.6 11.1 7.7 6.9 8.4 2.5
22:5n3 2.3 3.0 4.1 3.7 3.4 3.3 - 3.3 3.6 4.5 6.3 3.2 2.8
22:6n3 22.8 11.9 18.0 11.9 14.0 15.9 15.8 14.7 15.5 15.1 17.1 22.8 26.4
B n) 46.9 26.1 45.0 26.7 36.1 37.6 36.4 40.5 38.4 38.3 40.4 45.5 44.0

18:2n6 1.4 0.6 0.6 2.6 - 0.8 0.8 1.0 0.8 0.5 - 0.8 -
20:2n6 0.7 0.8 0.5 - 0.8 0.6 - 0.6 0.4 1.4 - 0.9 -
20:4n6 2.2 4.6 2.6 4.5 3.6 2.7 2.5 2.5 2.5 4.8 4.0 3.2 2.0
22:4n6 _ _ _ _ _ _ _ _ _ 3.5 3.4 2.7 1.6
225n6 1.2 _ _ _ _ _ _ _ - - 5.5 1.8 4.1
B n6 5.4 6.0 3.6 7.2 4.3 4.1 3.3 4.2 3.7 10.3 12.9 9.4 7.7

22:2 i 2.2 1.7 2.9 2.1 2.8 2.5 3.2 2.3 2.2 1.6 5.0 1.9 2.3
22:2j 2.3 4.2 2.4 12.1 9.2 2.2 2.3 1.9 2.7 9.1 7.4 6.2 6.8

B P”FA 56.8 44.7 53.9 54.5 53.5 47.9 45.3 48.8 47.0 60.3 65.7 63.1 60.7

MUFA = monounsaturated fatty acids.

PUFA = polyunsaturated fatty acids.
165 = 16 July.
9A = 9 August.
16A = 16 August.
9S = 9 September.
16s = 16 September.
190 = 19 October.
2N = 2 November.
20N = 20 November.

experimental conditions are summarized in the Table 3. To simplify the interpretation of

the data, only the most relevant results are presented. The fatty acid composition of the
lipids in the clams grown in effluent water agreed with a normal marine distribution.
There were minor differences between the fatty acid composition, for example 22:4n6
and 225n6 were present in Carril but not in 2 X h tank, whereas 18:3n3 was present in
2 X h tank but not in Carril. The clams from Nastos were richer in series n - 7 than
series n - 9 (Table 31, whereas in Carril the percentages were similar. In the analysis,
two C,, dienoic acids, 22121’ and 22:2j, were found, which were considered to be
22:2(7,13) and 22:2(7,15) respectively. The percentage of PUFA was higher in the seed
 R. Jam-Jam et al./Aquaculture I58 (1997) 247-262 257

growth in Carril (56.865.7%) than in 2 X h tank (45.3-56.8%) and the control

(44.7-56.8%). The high levels of PUFA in Carril was mainly due to the series II - 6
and 22:2j fatty acid.

4. Discussion

Two important parameters having a direct influence on the growth of bivalve

molluscs are: the temperature and the available food (Sastry, 1979; Maitre-Allain, 1982;
Beninger and Lucas, 1984; Body et al., 1986; Laing et al., 1987). In our study, the
temperature used is considered an optimum for the culture of R. decussatus, according
to Laing et al. (1987) who found that the greatest growth-rate coefficients for R.
decussatus juveniles occurred at 12-20°C. Other factors, such as salinity and oxygen,
can affect the growth but do not appear to have a notable effect on our results, since they
were very stable and adequate for the clam growth.
The quality of an intense fish culture effluent, as a source of nutrients, depends on a
great number of variables (Beveridge et al., 1991). The turbot farm produced a
supplementation to the available food in the water by the introduction of pelleted feed
and fecal wastes as well as detritus, phytoplankton and bacteria. The content of nitrogen
(3.3 kg/ton of fish), phosphorous (24.7 kg/ton of fish) and particulate organic matter
(467.5 kg/ton of fish) in the wastewater employed in this study were similar to those
reported by other authors (Sweden, 1983; Bergheim and Forsberg, 1993).
Culture density is an important parameter to be taken into account when evaluating
the growth rate of a population. The final density (kg of biomass/m’) reached in the
present work was 5.5 kg/m* (flow 4 X h) greater than that normally used in extensive
culture in a natural environment, which is not usually higher than 2.5 kg/m2 according
to Perez-Camacho and Cuba (1987) Cerviiio et al. (1993) Robert et al. (1993) and Pech
et al. (1993). Thus, the decrease observed in the growth rate from 1 September in the
different clam tanks, might be due to the increase of the culture density (kg of
biomass/m’), which involved higher competition for food. This suggestion agrees with
the significant differences in the clam growth (P < 0.05) found from 1 September
onwards between the three different flow rates of wastewater (Fig. 2).
The condition index of the seed fattened with the wastewater was higher than that of
the seed reared under natural conditions. Ansell (1968) studying the growth of Merce-
naria mercenaria clam cultured in the thermal effluent of an electric power station,
observed low condition indices due to a great increase in the size and dry shell weight
without a similar increase in dry meat weight. Similar results were reported by Salo and
Leet (1969) and Brown and Hartwick (1988) for Crussostrea gigas, these authors found
that when the available food is insufficient, the dry meat weight is low in relationship to
the dry shell weight and the internal shell volume decreases whereas the thickness
increases. This might explain the low condition index found in the seed from Carril,
because in Nastos, the seed stayed constantly submerged and with a continuous food
supply, whereas the Carril seed was subject to the tidal cycles as well as food
fluctuations.
258 R. Jam-Jam et al./Aquaculture 158 (1997) 247-262

The high growth rate of the seed fattened with the wastewater could fundamentally be
due to the great content of organic matter in the water, together with the considerable
microalgal concentrations which are an important source of PUFAs (Laing et al., 1987).
Various authors suggest that the accumulation of the different biochemical components
occurs in the periods of greater abundance of food in the environment (Gabbott, 1975;
Beninger, 1982, 1984; Ruiz et al., 1992). These energy reserves will later be used in
moments of nutritional stress (winter) and/or during the gametogenesis (Gabbott, 1985;
Pazos, 1993). It is generally accepted that glycogen is the principal energetic reserve in
bivalves while in the oocytes it is lipids (Giese, 1966; Holland, 1978; Gabbott, 1985;
Pazos, 1993). In optimum culture conditions, proteins are not an important energy
source but are used for the growth and gametogenic development. Thus, proteins do not
have variations so marked as carbohydrates and lipids and tend to increase progressively
during the animal growth.
The seed from Carril mainly accumulated glycogen since the availability of food was
less and, furthermore, this seed underwent the tidal exposure. In aerobic conditions, the
neutral lipids are an important source of energy but in anaerobiosis the carbohydrates are
the principal fuel, especially the glycogen (but also some amino acids). On the other
hand, the growth ceases during the phases of anoxia and the metabolism is maintained at
the expense of the glycogen and proteins.
The fatty acid composition of the bivalves varies according to species (Watanabe and
Ackman, 1974), but in the same species there are variations due to internal factors, such
as the gametogenic cycle and, externally, the temperature, salinity and food, which have
a great influence on the lipid metabolism and the composition of the biological
membranes. In our study the temperatures registered in Carril and Nastos were similar,
only a little lower in Carril, thus the greater influence on the clam fatty acid composition
in the seed would be the food, which supplies the energy necessary for the maintenance
and growth of the individual but also provides the essential metabolites. For the bivalve
molluscs, the 20:5n3 and 22:6n3 fatty acids are considered essential (Langdon and
Waldock, 1981; Joseph, 1982; Ackman, 1983; Waldock and Holland, 1984; Bell et al.,
1986; Sargent et al., 1990; Delaunay, 1992) due to the void or the almost inability of
elongating and desaturating the 18:3n3 acid from the diet. The results obtained by
Albentosa (1994) for R. decussatus seed confirm this hypothesis. On the other hand, this
author also indicated that the absence of 20:5n3 or 22:6n3 fatty acids in the diet is not a
limiting factor to growth of the R. decussatus seed but can cover the PUFA requirement
in these clams. However, Laing et al. (1987) reported that algae that contain relatively
high amounts of 20:5n3 or 22:6n3 support much better growth than species without
either.
Although the food produced differences in the fatty acid spectra, a similar pattern was
observed in the three culture conditions. When we compare the fatty acid spectrum of
the seed prefattened in Carril and Nastos some differences can be noted, for example the
absence of 18:3n3 in Carril, which might be due to the low percentage of this fatty acid
in the diet, or its immediate assimilation. This deficiency of 18:3n3 seems to be
compensated by an increase in the percentage of 16:4n3 and 22:6n3. The 22:4n6 and
22:5n6 present in the seed from Carril, could come from red algae, which is abundant in
the upwelling at the end of summer and autumn. These algae contain high percentages
 R. Jara-Jara et al./Aquaculture 158 (1997) 247-262 259

of 20 and 22 C fatty acids with 4, 5 and 6 double bonds (Wood, 1974). Furthermore, the
predominance of C,, PUFA in Carril could be the result of a selective retention of these
fatty acids from the diet. On the other hand, the individuals grown in the wastewater
maintained a very good relationship of n3/n6 (8.6-11.7), while in Carril were much
lower (3.7-5.7). We did not find branching fatty acids, typical of the bacterial lipids
(Kharlamenko et al., 1995), which might indicate that the contribution of the bacteria on
the clam seed nutrition in the study conditions was very low.
The growth of the seed in a suspended culture system presents some advantages, such
as an easy and fast cleaning of the trays and a rapid removal or collection. Furthermore,
the seed will not suffer the typical and natural movements produced in suspended
culture on a raft in the sea. The high densities reached and the positive increases of dry
weight, using trays without substrate, reinforces the advantages of this type of system
for the prefattening of clam at industrial scale. Moreover, the optimum size demanded
by clam-cultivators (15 mm of length) was reached in the first 45 days, just before the
growth becomes slower.

Acknowledgements

This research was supported, in part, by a grant from Ministerio de Industria,

Comercio y Turismo, Spain (CDTI-709/91) and by fellowship to R. Jara-Jara from
Instituto de Cooperaci6n Iberoamericana.

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