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Use of saline ground water for intensive rearing of Ruditapes philippinarum juveniles in a nursery system
Aquaculture, 88 (1990) 157-178 Elsevier Science Publishers B.V., Amsterdam
157 -
Printed
in The Netherlands
Use of saline ground water for intensive rearing of Ruditapes philippinarum juveniles in a nursery system Jean-Pierre Baud” and Cedric Bacherb “IFREMER Polder des Champs, 85230 Bouin (France) ‘IFREMER La Tremblade, BP 133, 17390 La Tremblade (France) (Accepted 28 September
1989)
ABSTRACT Baud, J.-P. and Bather, C., 1990. Use of saline ground water for intensive rearing of Ruditapesphilippinarum juveniles in a nursery system. Aquaculture, 88: 151-178. Saline ground water was used as a thermal source for intensive rearing of juveniles of Ruditapes philippinarum and as a source of nutrients for phytoplankton production. In experiments concerning growth at different seasons, controlled factors were density ofjuveniles, flow of water, phytoplankton concentration, temperature and frequency of feeding. Their effects on growth and mortality were classified through correspondence analysis, analysis of multiway contingency tables and analysis of variance. An optimal strategy for summer and winter rearing was then defined. The different strategies are discussed in biological terms, with reference to the literature. Observed growth rates in this intensive culture were compared with available data for artificially fed and naturally reared populations.
INTRODUCTION
The ecological and economic importance of filter-feeding molluscs justify the numerous studies undertaken to understand the mechanisms which control the growth of bivalves (Winter, 1978; Bayne and Newell, 1983) and more specifically of species with high sales value such as the Manila clam, Ruditapes philippinarum. To accelerate the complete production cycle from the controlled production of larvae (hatching) up to final growth, a rearing phase for juveniles could be developed during seasons (winter and summer) which are often critical: many authors have pointed out the low growth of filter-feeding bivalves in winter (Walne, 1974; Price et al., 1976; Widdows et al., 1979; Incze et al., 1980) and the major stresses to juveniles under summer conditions (Spencer and Gough, 1978; Claus, 198 1). These disturbances are mainly due to an imbalance between the metabo0044-8486/90/$03.50
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J.-P. BAUD AND C. BACHER
lism of bivalves, sensitive to temperature, and the quantity of available food. During summer, high temperatures enhance the metabolism of bivalves but usually coincide with a decrease in the phytoplankton food in the natural environment. During winter, low temperatures decrease the metabolism of bivalves, and hence their assimilation capacity, while low temperatures combined with poor daylight lead to a near lack of phytoplanktonic food. Consequently, it is necessary to determine the levels of temperature and food for each season in order to rear juveniles satisfactorily. Artificial heating of large volumes of sea water by conventional energy sources (electricity, heat pumps, fuel oil heaters, etc.) seems uneconomical. Large-scale production of phytoplankton often comes up against constraints such as the cost and the quality of the stock. Thus, standard rearing media (Provasoli et al., 1957; Walne, 1974) and media enriched with food (Riva and Lelong, 198 1; Leborgne, 1977) are economically viable only at the hatchery stage or for small production units. Partial solutions have been proposed by some authors, such as the use of thermal power plants (Abbe, 1980; Malouf, 198 1) or the effluents of sewage treatment stations (Grobbelaar et al., 1981; Barnabe, 1986). In the present study we used saline ground water both as a source of thermal energy and as a medium for phytoplankton production. Aquifer resources are estimated at several billion m3 (Bresson, private communication) and are found all around the Bay of Bourgneuf (France). From the winter of 198485 to the summer of 1986, two winter rearing periods of 90 days each and three summer periods of 70 days each were studied for juveniles of the species Ruditapes philippinarum in a nursery with upwelling. The effects of the following controlled factors were assessed: density of juveniles, sea-water flow rate, concentration of phytoplankton, frequency of injection of phytoplankton, and temperature. EXPERIMENTAL
SYSTEM AND METHOD
Rearing technique The physicochemical characteristics of saline ground water were measured in one year. Fifteen samples were obtained by pumping for 30 min at a flow rate of 30 m3/h and the results averaged (Table 1). These measures showed that the saline ground water was unfit for direct use for rearing. Ammonia content was too high, compared to the 10 ppm recommended by Epifanio et al. ( 1975) and pH (7.25) too low to sustain a satisfactory growth rate of filtering bivalves (Kuwatani and Nishii, 1969). Bivalves had therefore to be reared in sea water. However, the ground water which was at a constant temperature ( 13.5 oC all the year round) was used to warm the water of the nurseries during winter and to cool it during summer, through a titanium heat exchanger (Fig. 1). Ground water was, however, a good rearing medium for
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TABLE 1 Physicochemical characteristics and nutrient concentrations of the saline ground water T (“C)
S (%o)
pH
NH: NO, NO, PO:@mole/ 1) @mole/ 1) &mole/ 1) (wok/l)
SiO, (pmole/l)
Total Fe (mg/l)
13.5
30.4
7.25
307.5
178.9
3.3
water
sea
0.2
0.3
24.6
input
water
pond
HEAT
EXCHANGER
\ \ \ \ \ Phytoplankton ponds
II
II
I
upwelling
system
Fig. 1. Design of the intensive experimental nursery system for rearing juvenile clams.
J.-P. BAUD AND C. BACHER
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primary production because of its high content of nutrients and very low contents of mineral particulate matter and bacteria. Low-cost primary production in large volumes was then possible. The diatom Skeletonema costatum was chosen as food in all experiments. It was adapted to the quality of the saline ground water, particularly to the high content of ammonia and silica, and was easy to multiply in large volumes (Goldman and Mann, 1980; De Pauw et al., 1983; Laing, 1985 ). This species was therefore grown in a continuous monospecilic culture in five 50-m3 tanks filled with raw ground water. The microalgal concentrate, when in the exponential growth stage, was injected into the sea water circulating through the nurseries either discontinuously (3 h with food followed by 2 h without food) or continuously ( 14 h with food) so that the same amount was injected in both cases. Three concentrations of food were used: a low value of 20 pg/l (C 1)) a mid value of 40 pg/l (C2) corresponding to the mean phytoplankton bloom observed in spring, and a high value of 80 pg/l (C4). The clam juveniles were spread in vertical, 50-cm-diameter tubes, equipped with a 1-mm-mesh sieve (St-Felix et al., 1984). An upwelling flow brought the necessary oxygen and food, and removed the faeces and pseudo-faeces. At the beginning of the experiments, the size of the populations was homogeneous. The confidence interval, computed on 60 individuals, ranged from 3.9 TABLE 2 Rearing parameters for Ruditapes philippinarum for each tube (50 cm diameter) Flow rate: FRl= 1 m3/h, FR3=3 m3/h Density: D25 = 25 000 individuals, D50 = 50 000 individuals Concentration (Skeletonema costatum): CO=no phytoplankton, Cl ~20 ,ugg/l,C2=40 pg/l, C4=80 fig11 Frequency of food injection: DISC = discontinuous feeding, CONT = continuous feeding Quality of water: NAT = natural water (external temperature ) , EXC = heat-exchanged water (using a titanium heat exchanger) Parameters
Levels
Season
Winter 1
Winter 2
Summer 1
Summer 2
Summer 3
Flow rate of sea water
FRl, FR3
FRI, FR3
FRl, FR3
FR3
FR3
Density
D25, D50
D25, D50
D25
D25, D50
D25
Skeletonema feeding concentration
co, c2
Cl, c2, c4
co, Cl, c2, c4
co, Cl, c2, c4
c2
Feeding frequency
DISC
DISC
DISC
DISC
CONT, DISC
Quality of water
NAT
EXC
NAT, EXC
NAT
NAT
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to 4.3 mm. There were 12 levels of the rearing parameters: density of juveniles per tube, sea-water flow rate, quantity of food injected, frequency of injection, and quality of water (water having undergone thermal exchange or natural water) and five seasons (Table 2). Carrying the experiments over several years enabled us to select the most interesting levels and then to avoid systematic combinations. In total 40 experiments were conducted. The nutritional quality of the sea water, and of the mixture sea water/phytoplankton added, was assessed twice a week during each season by measuring the chlorophyll a and phaeopigments using the Lorenzen ( 1967) method and by counting microalgal cells with a haemocytometer cell (Mallassez cell). Growth and mortality The size frequency histogram at the end of the experiment, based upon all the individuals in each experimental set, showed the average growth performance and the dispersion around this average value, the initial populations being homogeneous in size. Each population of 25 000 or 50 000 clams was sorted by using seven sieves with decreasing mesh size. The number N of clams of each class was derived from the mean weight of 500 individuals:
where IV,= total weight of the size class, and IV, = mean individual weight of the 500 individuals sampled in the same class. The mortality ratio was determined in each tube:
z= (w-No)IxJ, where N,= final number of individuals, and NO= initial number of individuals. METHODS OF ANALYSIS OF GROWTH
Contingency tables Multidimensional contingency table analysis is a convenient way to study relationships between variables (Legendre, 1987 ). It is based on the discretization of the variables, which eliminates the impact of the experimental errors on the computation of the interactions between the variables. Since correlation, regression analysis and analysis of variance are sensitive to the nonlinearity and deviation from a gaussian distribution, contingency table analysis is then a robust alternative to more classical tests (Legendre, 1987). Multidimensional contingency tables were derived from the frequency histograms by repartition of groups of individuals among the combinations of controlled factors (flow, density, quality of water, feed concentration) and of the studied factor (growth). These tables were analysed using linear hierarchized models (Fienberg, 1970; Ku and Kullback, 1974; Sokal and Rohlf, 1981).
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According to these authors, these models test the independence of different parameters and their individual effects. The estimated frequency zijk of the observed frequency yijk is generally written: LOGzij~=C1+Ui+bj+ck+Ub,i+UC,+bCjk, where a, bj and ck are values of the controlled factors A, B, C. The last three terms explain the interactions between factors AB, AC, BC. The goodness of fit of the model is tested with the Wilks ( 1935 ) likelihood ratio:
which approximately follows a chi-square distribution. Studying of interactions requires the building of sub-models (hierarchization) . Two sub-models are compared by calculating Gl - G2 which is a measure of the loss of information from one model to the other. The objective is to obtain a model as simple as possible which takes into account only the significant interactions. Interactions of an order greater than two limit interpretation of the results and lead to the analysis of sub-tables taken from the initial matrix, and this in turn makes the tests less powerful. When the number of combinations is not too high, it is possible from a direct observation of the full matrix to classify an interaction between the explained variable and two controlled factors according to the following categories: (i) increased effect of the second factor versus the levels of the first factor (type I interaction), (ii) inversion of the effect of the second factor versus the levels of the first factor (type II interaction). In our work, the following matrices were then analysed: quality of the water (NAT, EXC) x flow rate (FRl , FR3) x concentration ;co, Cl) in * summer; - concentration (C 1, C2, C4) x flow rate (FRl , FR3 ) in summer with natural water; - concentration (C 1, C2, C4) x density (D25, D50) in summer; - concentration (C 1, C2, C4) x flow rate (FR 1, FR3) x density (D25, D50) in winter with heat-exchanged water. Correspondence analysis The relationships between growth and the controlled factors were observed through simple correspondence analysis. The surviving individuals at the end of growth were classified according to the crossed levels of the variables “number of the experiment” and “size class”. The structure of this population could be visualised by an optimization and projection process. The proximity of certain levels could also, with certain precautions (Volle, 198 1) , be interpreted.
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Following some authors (Lebart et al., 1977; Conan and Comeau, 1986), the factorial axes obtained in that manner can also be used for the projection of the levels of other variables, said to be “illustrative”. These latter levels take no part in the explanation of the variance of the matrix, but it is possible to determine their relationships with the factorial axes thanks to the quality of representation (QR) of each level. Finally, non-linear effects often appear (Guttman effect), information which is difficult to obtain by other methods. The data were analysed with the STATITCF software package ( 1987). In each analysis, the illustrative variables introduced were the parameters of the given set of experiments. Mortality analysis The study of factors influencing mortality brought an additional insight to the response of different populations to positive or negative stress related to different growth conditions. Using analysis of variance (STATITCF, 1987)) the percenta e of mortality tm was studied after transforming the variable by arcsin ( ,B tm) (Lellouch and Lazar, 1974)) in order to stabilize the variance. The influence of the experimental conditions (flow rate, density, concentration and temperature) was then studied. As some combinations of levels were not available, the following designs were analysed: - season x density x concentration, with a flow rate of 3 m 3/h; - flow rate x density x concentration, in winter; - flow rate x density x concentration, in summer. RESULTS
Rapid fluctuations of the temperature of the natural sea water were observed over a time span of a few days in each season. The dampening effect of the heat exchanger was shown by smaller variations of the temperature in the experiment, the efficiency of the heat exchanger being higher in raising the temperature than in lowering it. The heat exchanger kept temperatures lower than 20’ C in summer and higher than 10” C in winter. An ANOVA on the effect of exchanged and natural water in two winter seasons revealed no difference between seasons and a mean difference of 5.7 “C between exchanged and natural water (PC 0.00 1) . There was a difference of nearly 3.2 oC between exchanged and natural water in summer (PC 0.001). A low mean difference was also found between the three summer seasons (less than 1.3 oC, P-c0.01). Since we are interested in the effect of large differences in temperature (3 “C at least), the reproductibility of the thermal gains obtained in winter over 2 years and the similar summer temperature variations over 3 years proved the representativity of each period. The results of the different experiments within each season were then grouped.
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TABLE 3 Mean values of phytoplanktonic cells and pigments (winter and summer) in sea water, enriched with Skeletonema costatum (concentration C2) at a discontinuous frequency. Standard deviations are within parentheses Season
Cell number after injection ( lo6 cell/l)
Pigment after injection (/*g/l )
Pigment of sea water @g/l)
Winter 1 Winter 2 Summer I Summer 2 Summer 3
27.59 (2.47) 22.61 (1.81)
21.6 (2.17) 27.66 (3.67) 31.2 (5.28) 22.88 (2.53) 37.49 (3.27)
4.27 (0.65)
34.99 (1.98) 63.97 (7.5)
8.94 (2.0)
TABLE 4 Mean length and mean weight of Ruditapes philippinarum for each size class corresponding mesh size of the sieves used. The standard deviation is within parentheses Size class
Mean length (mm)
Mean weight (g )
s2 s3 s4 s5 S6 S8 SlO
4.1 6.4 7.2 9.2 12.3 15.3 17.5
0.017 (0.003) 0.109 (0.007) 0.17 (0.01) 0.32 (0.02) 0.39 (0.02) 0.69 (0.03) 1.15 (0.03)
(0.1) (0.2) (0.3) (0.3) (0.3) (0.2) (0.3)
to the
The concentration of total pigments was similar in the two seasons, while the number of microalgal cells was significantly greater in summer than in winter (Table 3). After rearing, populations of juvenile clams were sorted according to size and weight as shown in Table 4. Growth ofRuditapes
philippinarum
in summer
Contingency tables The first two analyses showed that the factors concentration, flow rate, and density were significantly related to growth (Table 5 ) . The quality of water (natural, exchanged) had no independent effect with concentration nor flow rate (Table 6a). In spite of the breakdown of the initial contingency matrix into sub-matrices including flow rate or concentration levels, an order 3 interaction remained significant and pointed to combined effects of external foodstock and temperature (Table 6b). At low flow rates, an increase or a decrease in temperature increased or decreased the growth pattern as food was injected or not (type I interaction). This interaction disappeared at high flow rates, where higher temperatures systematically yielded higher growth.
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TABLE Sa Modelling the effects and interactions of the controlled factors concentration (A), flow rate (B), and the size class (C) of clams in summer. Tables 5a and 5b show the hierarchized models derived from the model containing all the effects and order 2 interactions. G represents the logarithm of the maximum likelihood and allows the goodness of fit of the model to be tested. dG measures the loss of information between the first model and its sub-model. df is the degree of freedom used in testing the significance of G and dG. dG is significant (probability < 1%) -for the sub-models (2a), (2b), (2~) derived from the model ( 1). The interactions BC, AC, AB must then be taken into account. The G test of the model ( 1) is significant (probability> 5%) so that it is considered as valid. In all tables, valid models are underlined. We look for the lowest order valid model AB+AC+BC G=6,8 df
(1)
(la)
(2b)
dG= 137,6 df
dG=325,8
df
dG= 47,2 df
(2c)
AB+AC G= 143,14 df
AB+BC G=331,16
df
AC+BC G=53, 10 df
TABLE 5b Modelling the effects and interactions of the factors A, B, C for the clam in summer. A = concentration, B = density, C = size class. All the order 2 interactions are significant and must be taken into account AB+AC+BC G=10,8df dG=93,4
df
AB+AC G= 103,12 df
dG= 134,8 df
dG=16,2df
AB+BC G= 144,16 df
AC+BC G=26,10
df
Similarly, zero concentration leaded to a temperature x flow rate interaction with inversion of the temperature effect according to flow rate (type II interaction). The addition of food tended to cancel this interaction. The frequency of food injection had no detectable effect on the growth results (Table 7a). However, a comparison of the average growth at the end of the experiments on a sample of 60 individuals tended to show a positive effect of a discontinuous injection of food (Table 7b). Correspondence analysis Three analyses were carried out, showing the effect of the quality of the water, the flow rate, the density and the concentration of the injected phytoplankton. The first axis generally represented the growth gradient, the sizes either increasing or decreasing along that axis. The levels of the illustrative variables were located relative to that gradient, and positive or negative correlation with growth was derived from their position.
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TABLE 6 Modelling the effects and interactions of controlled factors and size class for the growth of clams in summer. No interaction of third order may be deleted (6a), so that sub-tables must be studied according to each level of the temperature (6b), food concentration (6~) or flow rate (6d) 6a. A = quality of water, B = concentration,
C = flow rate, D = size class
ABC+ABD+ACD+BCD G=2.6,4 df dG= 13,4 df
dG=43,4
-BCD G=16,8
-ACD G=47,8
df
6b. A = concentration,
df df
dG=23,4 -ABD G=26,8
df df
dG=lO,
1 df
-ABC G=13,5
df
B = flow rate, C = size class
Heatexchanged water
AB+AC+BC G=9.8,4 df
Natural water
AB+AC+BC G=5.8,4 df dG=35,6
df
dG= 234,4 df
dG= 109,4 df
AB+AC G=41,10
df
AB+BC G= 240,8 df
AC+BC G=115,8df
co
AB+AC+BC
G= 37.2, 4 df
c2
AB+AC+BC G=8,4df
6c. A = quality of water, B = flow rate, C = size class
dG= 59,4 df
dG= 62,4 df
dG=6,
AB+AC G= 67,8 df
AB+BC G= 70,8 df
AC+BC G=14,5df
Flow rate 1
AB+AC+BC
G= 22.2,4 df
Flow rate 3
AB+AC+BC G=4,4df
6d. A = concentration,
1 df
B = quality of water, C = size class
dG=65,4
df
AB+AC G=69,8 df
dG= 199,4 df
dG= 17, i df
AB+BC G=203,8
AC+BC G=21,5 df
df
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TABLE 7a Modelling the effects and interactions of the controlled factors frequency of feeding (A), density (B ) and the size class of clams in summer. After trials, there is no interaction left but density X size class AB+AC+BC G=0.61,2 df dG=23,2df
dG=l,2df
dG=0.2,
AB+AC G=24,4df
AB+BC G= 1.7,4 df
AC+BC G=l, 3df
dG=23,2 AB G=25,6
df df
dG=O.9,2
1 df
df
BC G= 1.9, 5 dG=23,2 A+B+C G=25,7
df df
TABLE lb Comparisons of the mean lengths and weights when the frequency of feeding is continuous or discontinuous. The size of each sample is 60 individuals. The density was 25 000 individuals in each tube (D25 ) . The standard deviation is within parentheses. The differences are significant at a 5% level Frequency
Length (mm)
Weight (mg)
Continuous Discontinuous
13.05 (2.64) 14.55 (1.78)
0.479 (0.214) 0.606 (0.189)
Temperaturexconcentration (CO, C2) xjlow rate. For a fixed density of 25 000, the effects of the injection of phytoplankton and of flow rate were compared for two temperatures (natural water and exchanged water) (Fig. 2). The first axis was explained up to 96% by the S4, S8 and S 10 classes. Size classes were well represented except for the SO class. Projection of the illustrative variables showed firstly that concentration was the factor most correlated with the gradient (quality of the representation was 0.68); and secondly that flow rate and quality of the water had secondary but real effects, although it was not possible to determine which one was the more important (qualities of representation were respectively of 0.15 and 0.18 ) . However, it appeared that the quality of the water had a non-linear effect on growth, since it enhanced the extreme classes. Concentration (Cl, C2, C4) x density in natural sea water. With flow rate fixed at 3 m3/h, the effects of different positive concentrations of injected food were compared (Fig. 3 ) . These effects were clearly visible, the density 25 000
J.-P. BAUD AND C. BACHER
168 0.6 0.5 0.4 -
EXC
0.3 0.2 0.1 -0.1
-
-0.2
-
-0.3
NAT
-0.6
-
-0.7
-
-0.6
-
-0.9
-
-1
-
-1.1
-
-1.2
-
-1.3
1 -1.2
I -0.6
I
I -0.4
I
I
0
I
0.4
,
I
0.6
I
I
1.2
I
-1 1.6
AXIS 1 (70X)
Fig. 2. Correspondence analysis of the growth of clams in summer, with a density D25 (25 000 individuals per tube). The controlled parameters temperature, concentration (0, 2) and flow rate ( 1, 3) are projected as illustrative variables. Notations for Figs. 2-5: Si: size class number i. FRl, FR3: levels of the flow rate ( 1 m3/h, 3 m3/h). D25, D50: levels of the density (25 000, 50 000 individuals per tube). NAT, EXC: natural water, heat-exchanged water. CO, Cl, C2, C4: levels of phytoplanktonic food concentration.
and the concentrations C2 and C4 being favourable factors for growth. Although this projection was not carried out with exchanged water, the conclusions should not be readily changed in that case since the two factors concentration and density seemed to be far more important than the quality of the water studied above. Concentration (Cl, C2, C4) xflow rate. This analysis confirmed the positive effect, even though it was small, of flow rate with a given density of 25 000 and exchanged water (Fig. 4). When a projection was made of the variable “available food”, represented by the ratio concentration/flow rate, no correlation appeared between the growth gradient and the available food. The two factors concentration and flow rate thus appeared to be independent. The water was not only a support of the injected food, the flow itself was an additional, positive factor.
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0.7 0.6 -
c4
0.5 0.4 0.3 0.2 0.1 0 -0.1
-
-0.2
-
-0.3
-
-0.4
-
-0.5
-
-0.6
-
-0.7
, -1.2
c2 1
I -0.8
1
, -0.4
I
I 0
I 0.4
I
, 0.8
1 1.2
AXIS 1 (82%)
Fig. 3. Correspondence analysis of the growth of clams in summer, with a flow rate FR3 (3 m3/ h). The controlled parameters concentration (1, 2, 4) and density (25, 50) are projected as illustrative variables (see Fig. 2 for notations).
Growth ofRuditapes
philippinarum
in winter
In winter the temperature factor became of primary importance. The natural sea water temperature was around 5 oC, and was increased to 10°C with the heat exchanger. One can easily see that growth rates were much smaller in winter than in summer and that the use of the exchanger was required in winter. A simple analysis could test the influence of flow rate, density and concentration. Contingency tables The results concerned experiments in warmed-up water. Concentration and density had significant and independent effects (Table 8 ) , whatever the flow rate. Correspondence analysis The antagonistic effects of concentration (positive effect) and density (negative effect ) appeared once again (Fig. 5 ) . There seemed to be no effect of flow rate, judging from the orthogonality of the axes flow rate/growth gradient.
J.-P. BAUD AND C. BACHER
170 2 A3
i .a
1.6 1.4 1.2 1 0.8
A2
0.6 Cl
0.4 0.2 0
iI
-0.2
-
-0.4
-
-0.6
-
-0.8
-
-1
/
FRl
c4
A
-pqS
-1.2
I -1.6
I -1.2
I
I -0.8
I
I -0.4
I
I
0
I
0.4
I
I
0.8
I
I
1.2
AXIS 1 (72%)
Fig. 4. Correspondence analysis of the growth of clams in summer, with a density D25 (25 000 individuals per tube). The controlled parameters concentration ( 1, 2, 4), flow rate ( 1, 3) and the variable A= concentration/flow rate are projected as illustrative variables (see Fig. 2 for notations). TABLE 8 Modelling the effects and interactions of the factors concentration (A), flow rate (B), density (C), and size class (D) of clams in winter. The effect of the flow rate and all third order interactions may be neglected Order 3 G=0.74, 8 df AD+CD G=16,40df -AD G=210,48
df
-CD G= 40,44 df
Mortality results It was possible to compare differences in mortality rates as a function of the five controlled parameters of the media. Not all combinations were tested. Testing the effects of season, density and food concentration showed an effect
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c2
I
FR3
050 0.1 0 -0.1 -0.2
-0.3 -0.4 -0.5 -0.6
c4 , -1
I
I -0.6
I
I -0.2
I
0.2
1
I
0.6
I
I
1
I
1.4
AXIS 1 (90%)
Fig. 5. Correspondence analysis of the growth of clams in winter. The controlled parameters flow rate ( 1, 3), density (25, 50), concentration ( 1,2, 4) are projected as illustrative variables (see Fig. 2 for notations).
of density only, though a small interaction of season~density was noticed (Table 9a). During winter, the systematic use of warmed-up water left only the parameters flow rate, density and concentration, which were combined. None of the factors was significant, which confirmed the difference of the effects of density according to the season. During summer, two designs were analysed. The first design combined concentration and density. The second included the effects of flow rates, quality of water, and concentration (CO, C2 levels) at a density of 25 000. In the former case, clams seemed to be sensitive to the feeding conditions since density and concentration had a significant effect (Table 9b). Multiple range test (Newman-Keuls method) showed the mortality for high concentrations (C2, C4) to be significantly higher than that observed for concentration C 1. In the latter analysis, there appeared to be no significant factor. Though the levels of concentration were not the same, it was seen that concentration did not have the same effect on mortality in the two analyses. There might be an interaction between density and concentration, which could explain the global effect of concentration when the density factor was taken into account. An-
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TABLE 9a ANOVA on the mortality rate tm transformed sity (2), concentration (3)
by arcsin (fi).
Controlled factors: season ( 1 ), den-
Source of variability
Sum of squares (S.S.)
df
F test
Probability
Factor I (season) Factor 2 (density) Factor 3 (cont. ) Interaction 1, 2 Interaction 1, 3 Interaction 2, 3 Residual
618 42 176 138 113 134 2 14
11 1 1 2 1 2 2 2
6.2 25.8 10.1 16.6 9.8 0.12
0.131 0.034 0.091 0.053 0.093 0.890
TABLE 9b Controlled factors: density, concentration density and a low effect of concentration
(Cl, C2, C4), in summer. There is a significant effect of
Source
S.S.
df
F test
Probability
Total Factor 1 (cont.) Factor 2 (density) Residual
0.17 0.07 0.09 0.00
5 2 1 2
22.8 52.9
0.041 0.015
TABLE 10 Percentages of mortality (by ANOVA) clam 3.48 0
winter
4.83
10.10
18.00
24.90
summer 9.1 Cl
c2 concentration
c4
other explanation was the lack of power of the second analysis, since only two levels of concentration were considered. Observed mortalities are summarized in Table 10. Growth dispersion and mortality were not independent, and significant mortality was observed together with high growth rates.
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DISCUSSION
Temperature and quantity of food are the essential parameters governing the growth of bivalves in nurseries. According to Claus ( 198 1)) there is a positive correlation between temperature and growth. Equilibrium between respiration and assimilation is near 13 oC according to Walne ( 1965 ) and 10 ’ C according to Ryther and Tenore ( 1976). The optimal temperature is derived from the response curves of metabolism and of flesh and shell production. It is also affected by the stress found at high temperatures (Mann and Ryther, 1977 ) . Moreover, it depends on whether the growth in flesh or in shell is considered. Mann ( 1979a,b) observed high shell growth rates for Ruditupesphilippinarum and Crassostrea gigas reared at high temperature. On the contrary, lower temperature yields better flesh growth rates. Liang et al. ( 1987 ) conclude that the growth rate for Ruditapes philippinarum increases up to 25 ‘C and decreases beyond that temperature. In our study, the use of the heat exchanger was necessary in winter to obtain a rearing temperature greater than, or equal to 10’ C. In summer, however, average temperatures seldom went beyond 20” C and cooling the water rather tended to slow down the growth when the food was unlimited. Raising the temperature without injection of food was harmful both to the growth and the survival of the clams. However, the positive correlations between growth rate and temperature showed higher growth rates for higher temperatures. In our study, the food consisted of Skeletonemu costatum at high concentration, produced at low cost and discontinuously with ground water. This diatom species multiplies itself easily in media of low salinity with high concentrations of silica (Laing and Jones, 1983; Laing, 1985 ) . It appears to be a dominant species when the ammonia concentration is very high (307.5 patg/ 1) which is a characteristic of the ground water used (Robert, 1987). Skeletonemu cost&urn has a high nutritional value ( Walne, 1970)) particularly for Mercenaria mercenaria and Ruditapes philippinarum (Laing et al., 1987 ) . This corresponds with the satisfactory growth rate observed with Ruditapes philippinarum. This result could be partially due to polyunsaturated acids (Langdon and Waldock, 198 1) . However, growth rates are higher for bivalves fed with a mixture of Skeletonema cost&urn and Tetraselmis (Laing and Millican, 1986 ). This could be due to the low content of tyrosine in SkeZetonema costatum, as detected by Chuecas and Riley ( 1969). The greatest gains in weight and size during the summer rearing were obtained with the injection of a high concentration (C4) of Skeletonema costaturn, together with a sea-water flow rate of 3 m3/h. The concentration resulting from such a dilution corresponded to the optimum level of phytoplankton as given by Matthiessen and Toner ( 1966 ) , but remained below that recommended by Tenore and Dunstan ( 1973 ) (Table 11) .
J.-P. BAUD AND C. BACHER
174 TABLE I1
Computation of the daily available phytoplanktonic food according to different food concentrations and flow rates and according to several authors. Results are expressed in number of cells per ml and per day Matthiessen and Toner ( 1966)
Levels C2, FRl
C4, FRl
1.5X lo5 cell/ml
3 X 1O5cell/ml
2.5 x 10’ cell/day
5 x 10’ cell/day
Tenore and Dunstan ( 1973)
C4, FR3 2X 105cell/ml
1 X 10’ cell/ml 1.7~ lO*cell/day
11 x 10’ cell/day
TABLE 12 Strategies of rearing defined after the analyses of growth and mortality Season
Quality of water
Density
Flow rate
Injected flow rate and concentration
Frequency
Winter
Heat-exchanged water (10°C)
25 OOO/tube 12.7 ind./cm’
1 m3/h per tube 0.67 ml/min per ind.
150 l/h per tube 0.5X lo6 cell/ml
Discontinuous 14 h/24 h
Summer
Natural water (20°C)
25 OOO/tube 12.7 ind./cm’
3 m3/h per tube 2 ml/min per ind.
150 l/h per tube 0.5~ lO”cell/ml
Discontinuous 14 h/24 h
The discontinuous feeding of Ruditapes philippinarum in the rearing stage enhanced growth. Such feeding forces a digestive rhythm upon the juvenile (Langton and Gabbott, 1974; Owen, 1974). According to Epifanio and Mootz ( 1976), the oyster ingests a maximum quantity of algae in a phase of active filtration, then slows down its filtration activity during a phase of digestion. This alternation of activity and rest depends upon the quantity of food in the medium, and the periodicity of this mechanism is dampened when the medium has a low concentration of food, which is the case with the natural sea water. Langton and MacKay ( 1976) suggest that, with higher concentrations in discontinuous feeding, filtration can be stimulated during the feeding period and the efficiency is greater. Feed distribution by an upwelling appears to be a technique very well adapted to the optimization of growth. Contrary to what happens with a laminar flow, all the enriched water irrigates the bivalve population (percolation) . The quantities of juveniles can be increased with this type of flow distribution, and maintenance is easier (Manzi et al., 1984). In our study, the effect of the flow rate was hidden most of the time by the effects of the quantity of food and of the temperature. According to Malouf and Breese ( 1978), the growth of oysters increases with the speed of the water.
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The flow of water through the tubes should eliminate organisms in competition for phytoplankton, evacuate the faeces and stimulate nutrition with conditions favouring the filtration mechanisms (Kirby Smith, 1972; Walne, 1972). The different analyses of data clearly showed that the flow rate was not a primary factor but that it did have a positive effect on growth in summer. The opposite effect could be seen in winter, with low temperatures. In summer the upwelling not only served as carried of the food but also contributed to the equilibrium of the food (multi-specific phytoplankton, oligo-elements, dissolved organic materials). The growth rates observed are compatible with published values and lead to the nursing strategies described in Table 12. The study of mortality showed that Ruditapes philippinarum was somewhat sensitive to high concentrations of food (C4) in summer. In that season, the efficiency of rearing was better when using a mean concentration (C2). The analyses showed the value of a high flow rate (3 m3/h) in summer. Finally, the use of the heat exchanger is not required: it brought only a small increase in growth while its operating costs were not negligible. On the contrary, a low flow rate combined with exchanged water can be chosen in winter. In both cases, a low density (D25) and injection of food (phytoplankton) are proposed, so as to obtain satisfactory growth performances. Correspondence analysis gave some information on non-linearity effects of the controlled parameters on growth. Displaying these parameters as illustrative variables showed their effects in a more obvious manner. Moreover, multiway contingency tables analysis gave the statistical justification of these results and measured the interactions between the parameters. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Jean-Paul Dreno for his help in the definition of the experimental design, Dr. Alain Bodoy for his advice on the manuscript and Pierre Bather for the translation into English.
REFERENCES Abbe, G.R., 1980. Non-radiological studies of tray-held oysters, Crassostrea virginica, in the vicinity of the Calvert cliffs nuclear power plan in Chesapeake Bay, 1970- 1979. 72nd Annu. Meet. Natl. Shellfish. Assoc. (abstr.). Barnabe, G., 1986. La collecte de micro-algues. In: G. Barnabe (Editor), Aquaculture, Vol. 1. Lavoisier Technique et Documentation, Paris, pp. 194-l 98. Bayne, B.L. and Newell, R.C., 1983. Physiological energetics of marine molluscs. In: K. Wilbur (Editor), The Mollusca, Vol. 4. Physiology, Part 1. Academic PPess, New York, NY, pp. 407-515. Chuecas, I. and Riley, J.P., 1969. Component fatty acids of the total lipids of some marine phytoplankton. J. Mar. Biol. Assoc. U.K., 49: 97-l 16.
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Claus, C., 198 1. Trends in nursery rearing of bivalve molluscs. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. l-33. Conan, G.Y. and Comeau, M., 1986. Functional maturity and terminal molt of snow crab, Chionoecetes opilio. Can. J. Fish. Aquat. Sci., 43: 17 1O-l 7 19. De Pauw, N., Verboven, J. and Claus, C., 1983. Large-scale microalgae production for nursery rearing of marine bivalves. Aquacult. Eng., 2: 27-47. Epifanio, C.E. and Mootz, C.A., 1976. Growth of oysters in a recirculating maricultural system. Proc. Natl. Shellfish. Assoc., 65: 32-37. Epifanio, C.E., Srna, R. and Pruder, G., 1975. Mariculture of shellfish in controlled environments: a prognosis. Aquaculture, 5: 227-241. Fienberg, SE., 1970. The analysis of multidimensional contingency tables. Ecology, 5 1: 419433. Goldman, J.C. and Mann, R., 1980. Temperature-influenced variation in speciation and chemical composition of marine phytoplankton in outdoor mass cultures. J. Exp. Mar. Biol. Ecol., 46: 29-39. Grobbelaar, J.U., Sveder, C.J. and Toerdien, D.F., 198 1. Waste Water for Aquaculture, Univ. Orange Free State Publ., Ser. C, 3,2 15 pp. Incze, L.S., Lutz, R.A. and Watling, L., 1980. Relationships between effects of environmental temperature and seston on growth and mortality of Mytilus edulis in a temperate northern estuary. Mar. Biol., 57: 147-156. Kirby Smith, W.W., 1972. Growth ofthe bay scallops: the influence of experimental water currents. J. Exp. Mar. Biol. Ecol., 8: 7-18. Ku, H.H. and Kullback, S., 1974. Loglinear models in contingency table analysis. Am. Stat., 28: 115-122. Kuwatani, Y. and Nishii, T., 1969. Effects of pH of culture water on the growth of the Japanese pearl oyster. Bull. Jpn. Sot. Sci. Fish., 35: 342-350. Laing, I., 1985. Factors affecting the large-scale production of four species of commercially important marine algae. Aquaculture, 44: 16 1- 166. Laing, I. and Jones, E., 1983. Large-scale turbidostat culture of marine microalgae. Aquacult. Eng., 2: 203-212. Laing, I. and Millican, P.F., 1986. Relative growth and growth efficiency of Ostrea edulis L. spat fed various algal diets. Aquaculture, 54: 245-262. Laing, I., Utting, S.D. and Kilada, R.W.S., 1987. Interactive effect of diet and temperature on the growth of juvenile clams. J. Exp. Mar. Biol. Ecol., 113: 23-38. Langdon, C.J. and Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigm. J. Mar. Biol. Assoc. U.K., 6 1: 43 l-448. Langton, R.W. and Gabbott, P.A., 1974. The tidal rhythm of extracellular digestion and the response to feeding in Ostrea edulis. Mar. Biol., 24: 18 1- 187. Langton, R.W. and MacKay, G.U., 1976. Growth of Crmsostrea gigas Thunberg spat under different feeding regimes in a hatchery. Aquaculture, 7: 225-233. Lebart, L., Morineau, A. and Tabard, N., 1977. Techniques de la Description Statistique. MCthodes et logiciels pour l’analyse des grands tableaux. Dunod, Paris, 35 1 pp. Leborgne, Y., 1977. L’ecloserie nurserie de la SATMAR et les possibilites actuelles de production de naissain de mollusques bivalves. 3rd Meeting of the ICES working Group of Mariculture. Actes Colloques CNEXO, 4: 353-360. Legendre, L., 1987. Multidimensional contingency table analysis as a tool for biological oceanography. Biol. Oceanogr., 5: 13-28. Lellouch, J. and Lazar, P., 1974. Methodes Statistiques en Experimentation Biologique. Flammarion Medecine-Sciences, Paris, 293 pp.
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Lorenzen, C.J., 1967. Determination of chlorophyll and pheopigments: spectrophotometric equations. Limnol. Oceanogr., 12: 343-346. Malouf, R.E., 198 1. Use of heated effluents for the nursery culture of bivalve molluscs: its problems and potential. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. 171-188. Malouf, R.E. and Breese, W.P., 1978. Intensive culture of the Pacific oyster Crassxtrea gigas (Thunberg) in heated effluents. Oregon State Univ. Sea Grant Program., Publ. no. ORESUT-78-003 Corvallis, OR. Mann, R., 1979a. Some biochemical and physiological aspects of growth and gametogenesis in Crassostrea gigas (Thunberg) and Ostrea edulis L. grown at sustained elevated temperatures. J. Mar. Biol. Assoc. U.K., 59: 95-110. Mann, R., 1979b. The effect of temperature on growth, physiology and gametogenesis in the manila clam Tapes philippinarum. J. Exp. Mar. Biol. Ecol., 38: 12 1- 133. Mann, R. and Ryther, J.H., 1977. Growth of six species of bivalve molluscs in a waste recycling aquaculture system. Aquaculture, 11: 231-245. Manzi, J.J., Hadley, N.H., Battey, C., Haggaty, R., Hamilton, R. and Carter, M., 1984. Culture of the northern hard clam Mercenaria mercenaria (Linne) in a commercial scale, upflow, nursery system. J. Shellfish Res., 4: 119-124. Matthiessen, G.C. and Toner, R.C., 1966. Possible methods of improving the shellfish industry of Martha's Vineyard, Duke’s County, Massachusetts. The Marine Foundation Inc., Edgartown, MA, 138 pp. Owen, G., 1974. Feeding and digestion in the Bivalvia. Adv. Physiol. Biochem., 5: l-35. Price, A.H., Hess, C.T. and Smith, C.W., 1976. Observations of Crassostrea virginica cultured in the heated effluent and discharged radionuclides of a nuclear power reactor. Proc. Natl. Shellfish Assoc., 66: 54-68. Provasoli, L., McLaughlin, J.J.A. and Droop, M-R., 1957. The development of artificial media for marine algae. Arch. Mikrobiol., 25: 392-428. Riva, A. and Lelong, P., 198 1. Growth of juvenile molluscs associated with continuous cultures of natural marine phytoplankton. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. 253-268. Robert, J.M., 1987. Valorisation des sites aquacoles existants ou potentiels de la region des pays de Loire, par utilisation des eaux souterraines pour la production d’algues unicellulaires destin&es a l’alimentation des differentes especes de mollusques au tours de leur cycle d’elevage. Rapport Convention Region des Pays de Loire et Universite de Nantes, 49 pp. Ryther, J.H. and Tenore, K.R., 1976. Integrated systems of mollusc culture. In: 0. Devik (Editor), Harvesting Polluted Waters. Plenum Press, New York, NY, pp. 153-167. St Felix, C., Baud, J.P. and Hommebon, P., 1984. Diversification de la production conchylicole. Elevage de la palourde japonaise en Baie de Bourgneuf. Sci. P&he, 344,345,346: 2-22. Sokal, R.R. and Rohlf, F.J., 1981. Biometry. The Principles and Practice of Statistics in Biological Research, 2nd edn., Freeman, San Francisco, CA, 859 pp. Spencer, B.E. and Gough, C.J., 1978. The growth and survival of experimental batches of hatchery-reared spat of Ostrea edulis L. and Crassostrea gigas Thunberg, using different methods of tray cultivation. Aquaculture, 13: 293-3 12. STATITCF, 1987. Guide de l’utilisateur. ITCF edn., Paris, 150 pp. Tenore, K.R. and Dunstan, W.M., 1973. Comparison of feeding and biodeposition of three bivalves at different food levels. Mar. Biol., 21: 190-195. Volle, M., 198 1. Analyse des Donnees, Economica, Paris, 3 17 pp. Walne, P.R., 1965. Observations on the influence of food supply and temperature on the feeding and growth of Ostrea edulis L. Fish. Invest., Lond., Ser. 2,24 ( 1 ), 45 pp.
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Walne, P.R., 1970. Studies of the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea Mercenaria and Myths. Fish. Invest., Lond., Ser. 2, 26( 5), 62 PP. Walne, P.R., 1972. The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves. J. Mar. Biol. Assoc. U.K., 52: 345-374. Walne, P.R., 1974. Culture of Bivalve Molluscs: 50 years’ Experience at Conway. Fishing News (Books), West Byfleet, 173 pp. Widdows, J., Fieth, P. and Worrall, C.M., 1979. Relationships between seston, available food and feeding activity in the common mussel, Mytilus edulis. Mar. Biol., 50: 195-207. Wilks, S.S., 1935. The likelihood test of independence in contingency tables. Ann. Math. Statist., 6: 190-196. Winter, J.E., 1978. A review of the knowledge of suspension feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture, 13: l-33.
157 -
Printed
in The Netherlands
Use of saline ground water for intensive rearing of Ruditapes philippinarum juveniles in a nursery system Jean-Pierre Baud” and Cedric Bacherb “IFREMER Polder des Champs, 85230 Bouin (France) ‘IFREMER La Tremblade, BP 133, 17390 La Tremblade (France) (Accepted 28 September
1989)
ABSTRACT Baud, J.-P. and Bather, C., 1990. Use of saline ground water for intensive rearing of Ruditapesphilippinarum juveniles in a nursery system. Aquaculture, 88: 151-178. Saline ground water was used as a thermal source for intensive rearing of juveniles of Ruditapes philippinarum and as a source of nutrients for phytoplankton production. In experiments concerning growth at different seasons, controlled factors were density ofjuveniles, flow of water, phytoplankton concentration, temperature and frequency of feeding. Their effects on growth and mortality were classified through correspondence analysis, analysis of multiway contingency tables and analysis of variance. An optimal strategy for summer and winter rearing was then defined. The different strategies are discussed in biological terms, with reference to the literature. Observed growth rates in this intensive culture were compared with available data for artificially fed and naturally reared populations.
INTRODUCTION
The ecological and economic importance of filter-feeding molluscs justify the numerous studies undertaken to understand the mechanisms which control the growth of bivalves (Winter, 1978; Bayne and Newell, 1983) and more specifically of species with high sales value such as the Manila clam, Ruditapes philippinarum. To accelerate the complete production cycle from the controlled production of larvae (hatching) up to final growth, a rearing phase for juveniles could be developed during seasons (winter and summer) which are often critical: many authors have pointed out the low growth of filter-feeding bivalves in winter (Walne, 1974; Price et al., 1976; Widdows et al., 1979; Incze et al., 1980) and the major stresses to juveniles under summer conditions (Spencer and Gough, 1978; Claus, 198 1). These disturbances are mainly due to an imbalance between the metabo0044-8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
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J.-P. BAUD AND C. BACHER
lism of bivalves, sensitive to temperature, and the quantity of available food. During summer, high temperatures enhance the metabolism of bivalves but usually coincide with a decrease in the phytoplankton food in the natural environment. During winter, low temperatures decrease the metabolism of bivalves, and hence their assimilation capacity, while low temperatures combined with poor daylight lead to a near lack of phytoplanktonic food. Consequently, it is necessary to determine the levels of temperature and food for each season in order to rear juveniles satisfactorily. Artificial heating of large volumes of sea water by conventional energy sources (electricity, heat pumps, fuel oil heaters, etc.) seems uneconomical. Large-scale production of phytoplankton often comes up against constraints such as the cost and the quality of the stock. Thus, standard rearing media (Provasoli et al., 1957; Walne, 1974) and media enriched with food (Riva and Lelong, 198 1; Leborgne, 1977) are economically viable only at the hatchery stage or for small production units. Partial solutions have been proposed by some authors, such as the use of thermal power plants (Abbe, 1980; Malouf, 198 1) or the effluents of sewage treatment stations (Grobbelaar et al., 1981; Barnabe, 1986). In the present study we used saline ground water both as a source of thermal energy and as a medium for phytoplankton production. Aquifer resources are estimated at several billion m3 (Bresson, private communication) and are found all around the Bay of Bourgneuf (France). From the winter of 198485 to the summer of 1986, two winter rearing periods of 90 days each and three summer periods of 70 days each were studied for juveniles of the species Ruditapes philippinarum in a nursery with upwelling. The effects of the following controlled factors were assessed: density of juveniles, sea-water flow rate, concentration of phytoplankton, frequency of injection of phytoplankton, and temperature. EXPERIMENTAL
SYSTEM AND METHOD
Rearing technique The physicochemical characteristics of saline ground water were measured in one year. Fifteen samples were obtained by pumping for 30 min at a flow rate of 30 m3/h and the results averaged (Table 1). These measures showed that the saline ground water was unfit for direct use for rearing. Ammonia content was too high, compared to the 10 ppm recommended by Epifanio et al. ( 1975) and pH (7.25) too low to sustain a satisfactory growth rate of filtering bivalves (Kuwatani and Nishii, 1969). Bivalves had therefore to be reared in sea water. However, the ground water which was at a constant temperature ( 13.5 oC all the year round) was used to warm the water of the nurseries during winter and to cool it during summer, through a titanium heat exchanger (Fig. 1). Ground water was, however, a good rearing medium for
INTENSIVE
REARING
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TABLE 1 Physicochemical characteristics and nutrient concentrations of the saline ground water T (“C)
S (%o)
pH
NH: NO, NO, PO:@mole/ 1) @mole/ 1) &mole/ 1) (wok/l)
SiO, (pmole/l)
Total Fe (mg/l)
13.5
30.4
7.25
307.5
178.9
3.3
water
sea
0.2
0.3
24.6
input
water
pond
HEAT
EXCHANGER
\ \ \ \ \ Phytoplankton ponds
II
II
I
upwelling
system
Fig. 1. Design of the intensive experimental nursery system for rearing juvenile clams.
J.-P. BAUD AND C. BACHER
160
primary production because of its high content of nutrients and very low contents of mineral particulate matter and bacteria. Low-cost primary production in large volumes was then possible. The diatom Skeletonema costatum was chosen as food in all experiments. It was adapted to the quality of the saline ground water, particularly to the high content of ammonia and silica, and was easy to multiply in large volumes (Goldman and Mann, 1980; De Pauw et al., 1983; Laing, 1985 ). This species was therefore grown in a continuous monospecilic culture in five 50-m3 tanks filled with raw ground water. The microalgal concentrate, when in the exponential growth stage, was injected into the sea water circulating through the nurseries either discontinuously (3 h with food followed by 2 h without food) or continuously ( 14 h with food) so that the same amount was injected in both cases. Three concentrations of food were used: a low value of 20 pg/l (C 1)) a mid value of 40 pg/l (C2) corresponding to the mean phytoplankton bloom observed in spring, and a high value of 80 pg/l (C4). The clam juveniles were spread in vertical, 50-cm-diameter tubes, equipped with a 1-mm-mesh sieve (St-Felix et al., 1984). An upwelling flow brought the necessary oxygen and food, and removed the faeces and pseudo-faeces. At the beginning of the experiments, the size of the populations was homogeneous. The confidence interval, computed on 60 individuals, ranged from 3.9 TABLE 2 Rearing parameters for Ruditapes philippinarum for each tube (50 cm diameter) Flow rate: FRl= 1 m3/h, FR3=3 m3/h Density: D25 = 25 000 individuals, D50 = 50 000 individuals Concentration (Skeletonema costatum): CO=no phytoplankton, Cl ~20 ,ugg/l,C2=40 pg/l, C4=80 fig11 Frequency of food injection: DISC = discontinuous feeding, CONT = continuous feeding Quality of water: NAT = natural water (external temperature ) , EXC = heat-exchanged water (using a titanium heat exchanger) Parameters
Levels
Season
Winter 1
Winter 2
Summer 1
Summer 2
Summer 3
Flow rate of sea water
FRl, FR3
FRI, FR3
FRl, FR3
FR3
FR3
Density
D25, D50
D25, D50
D25
D25, D50
D25
Skeletonema feeding concentration
co, c2
Cl, c2, c4
co, Cl, c2, c4
co, Cl, c2, c4
c2
Feeding frequency
DISC
DISC
DISC
DISC
CONT, DISC
Quality of water
NAT
EXC
NAT, EXC
NAT
NAT
INTENSIVE REARING OF RVDZTAPES
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to 4.3 mm. There were 12 levels of the rearing parameters: density of juveniles per tube, sea-water flow rate, quantity of food injected, frequency of injection, and quality of water (water having undergone thermal exchange or natural water) and five seasons (Table 2). Carrying the experiments over several years enabled us to select the most interesting levels and then to avoid systematic combinations. In total 40 experiments were conducted. The nutritional quality of the sea water, and of the mixture sea water/phytoplankton added, was assessed twice a week during each season by measuring the chlorophyll a and phaeopigments using the Lorenzen ( 1967) method and by counting microalgal cells with a haemocytometer cell (Mallassez cell). Growth and mortality The size frequency histogram at the end of the experiment, based upon all the individuals in each experimental set, showed the average growth performance and the dispersion around this average value, the initial populations being homogeneous in size. Each population of 25 000 or 50 000 clams was sorted by using seven sieves with decreasing mesh size. The number N of clams of each class was derived from the mean weight of 500 individuals:
where IV,= total weight of the size class, and IV, = mean individual weight of the 500 individuals sampled in the same class. The mortality ratio was determined in each tube:
z= (w-No)IxJ, where N,= final number of individuals, and NO= initial number of individuals. METHODS OF ANALYSIS OF GROWTH
Contingency tables Multidimensional contingency table analysis is a convenient way to study relationships between variables (Legendre, 1987 ). It is based on the discretization of the variables, which eliminates the impact of the experimental errors on the computation of the interactions between the variables. Since correlation, regression analysis and analysis of variance are sensitive to the nonlinearity and deviation from a gaussian distribution, contingency table analysis is then a robust alternative to more classical tests (Legendre, 1987). Multidimensional contingency tables were derived from the frequency histograms by repartition of groups of individuals among the combinations of controlled factors (flow, density, quality of water, feed concentration) and of the studied factor (growth). These tables were analysed using linear hierarchized models (Fienberg, 1970; Ku and Kullback, 1974; Sokal and Rohlf, 1981).
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According to these authors, these models test the independence of different parameters and their individual effects. The estimated frequency zijk of the observed frequency yijk is generally written: LOGzij~=C1+Ui+bj+ck+Ub,i+UC,+bCjk, where a, bj and ck are values of the controlled factors A, B, C. The last three terms explain the interactions between factors AB, AC, BC. The goodness of fit of the model is tested with the Wilks ( 1935 ) likelihood ratio:
which approximately follows a chi-square distribution. Studying of interactions requires the building of sub-models (hierarchization) . Two sub-models are compared by calculating Gl - G2 which is a measure of the loss of information from one model to the other. The objective is to obtain a model as simple as possible which takes into account only the significant interactions. Interactions of an order greater than two limit interpretation of the results and lead to the analysis of sub-tables taken from the initial matrix, and this in turn makes the tests less powerful. When the number of combinations is not too high, it is possible from a direct observation of the full matrix to classify an interaction between the explained variable and two controlled factors according to the following categories: (i) increased effect of the second factor versus the levels of the first factor (type I interaction), (ii) inversion of the effect of the second factor versus the levels of the first factor (type II interaction). In our work, the following matrices were then analysed: quality of the water (NAT, EXC) x flow rate (FRl , FR3) x concentration ;co, Cl) in * summer; - concentration (C 1, C2, C4) x flow rate (FRl , FR3 ) in summer with natural water; - concentration (C 1, C2, C4) x density (D25, D50) in summer; - concentration (C 1, C2, C4) x flow rate (FR 1, FR3) x density (D25, D50) in winter with heat-exchanged water. Correspondence analysis The relationships between growth and the controlled factors were observed through simple correspondence analysis. The surviving individuals at the end of growth were classified according to the crossed levels of the variables “number of the experiment” and “size class”. The structure of this population could be visualised by an optimization and projection process. The proximity of certain levels could also, with certain precautions (Volle, 198 1) , be interpreted.
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Following some authors (Lebart et al., 1977; Conan and Comeau, 1986), the factorial axes obtained in that manner can also be used for the projection of the levels of other variables, said to be “illustrative”. These latter levels take no part in the explanation of the variance of the matrix, but it is possible to determine their relationships with the factorial axes thanks to the quality of representation (QR) of each level. Finally, non-linear effects often appear (Guttman effect), information which is difficult to obtain by other methods. The data were analysed with the STATITCF software package ( 1987). In each analysis, the illustrative variables introduced were the parameters of the given set of experiments. Mortality analysis The study of factors influencing mortality brought an additional insight to the response of different populations to positive or negative stress related to different growth conditions. Using analysis of variance (STATITCF, 1987)) the percenta e of mortality tm was studied after transforming the variable by arcsin ( ,B tm) (Lellouch and Lazar, 1974)) in order to stabilize the variance. The influence of the experimental conditions (flow rate, density, concentration and temperature) was then studied. As some combinations of levels were not available, the following designs were analysed: - season x density x concentration, with a flow rate of 3 m 3/h; - flow rate x density x concentration, in winter; - flow rate x density x concentration, in summer. RESULTS
Rapid fluctuations of the temperature of the natural sea water were observed over a time span of a few days in each season. The dampening effect of the heat exchanger was shown by smaller variations of the temperature in the experiment, the efficiency of the heat exchanger being higher in raising the temperature than in lowering it. The heat exchanger kept temperatures lower than 20’ C in summer and higher than 10” C in winter. An ANOVA on the effect of exchanged and natural water in two winter seasons revealed no difference between seasons and a mean difference of 5.7 “C between exchanged and natural water (PC 0.00 1) . There was a difference of nearly 3.2 oC between exchanged and natural water in summer (PC 0.001). A low mean difference was also found between the three summer seasons (less than 1.3 oC, P-c0.01). Since we are interested in the effect of large differences in temperature (3 “C at least), the reproductibility of the thermal gains obtained in winter over 2 years and the similar summer temperature variations over 3 years proved the representativity of each period. The results of the different experiments within each season were then grouped.
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TABLE 3 Mean values of phytoplanktonic cells and pigments (winter and summer) in sea water, enriched with Skeletonema costatum (concentration C2) at a discontinuous frequency. Standard deviations are within parentheses Season
Cell number after injection ( lo6 cell/l)
Pigment after injection (/*g/l )
Pigment of sea water @g/l)
Winter 1 Winter 2 Summer I Summer 2 Summer 3
27.59 (2.47) 22.61 (1.81)
21.6 (2.17) 27.66 (3.67) 31.2 (5.28) 22.88 (2.53) 37.49 (3.27)
4.27 (0.65)
34.99 (1.98) 63.97 (7.5)
8.94 (2.0)
TABLE 4 Mean length and mean weight of Ruditapes philippinarum for each size class corresponding mesh size of the sieves used. The standard deviation is within parentheses Size class
Mean length (mm)
Mean weight (g )
s2 s3 s4 s5 S6 S8 SlO
4.1 6.4 7.2 9.2 12.3 15.3 17.5
0.017 (0.003) 0.109 (0.007) 0.17 (0.01) 0.32 (0.02) 0.39 (0.02) 0.69 (0.03) 1.15 (0.03)
(0.1) (0.2) (0.3) (0.3) (0.3) (0.2) (0.3)
to the
The concentration of total pigments was similar in the two seasons, while the number of microalgal cells was significantly greater in summer than in winter (Table 3). After rearing, populations of juvenile clams were sorted according to size and weight as shown in Table 4. Growth ofRuditapes
philippinarum
in summer
Contingency tables The first two analyses showed that the factors concentration, flow rate, and density were significantly related to growth (Table 5 ) . The quality of water (natural, exchanged) had no independent effect with concentration nor flow rate (Table 6a). In spite of the breakdown of the initial contingency matrix into sub-matrices including flow rate or concentration levels, an order 3 interaction remained significant and pointed to combined effects of external foodstock and temperature (Table 6b). At low flow rates, an increase or a decrease in temperature increased or decreased the growth pattern as food was injected or not (type I interaction). This interaction disappeared at high flow rates, where higher temperatures systematically yielded higher growth.
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TABLE Sa Modelling the effects and interactions of the controlled factors concentration (A), flow rate (B), and the size class (C) of clams in summer. Tables 5a and 5b show the hierarchized models derived from the model containing all the effects and order 2 interactions. G represents the logarithm of the maximum likelihood and allows the goodness of fit of the model to be tested. dG measures the loss of information between the first model and its sub-model. df is the degree of freedom used in testing the significance of G and dG. dG is significant (probability < 1%) -for the sub-models (2a), (2b), (2~) derived from the model ( 1). The interactions BC, AC, AB must then be taken into account. The G test of the model ( 1) is significant (probability> 5%) so that it is considered as valid. In all tables, valid models are underlined. We look for the lowest order valid model AB+AC+BC G=6,8 df
(1)
(la)
(2b)
dG= 137,6 df
dG=325,8
df
dG= 47,2 df
(2c)
AB+AC G= 143,14 df
AB+BC G=331,16
df
AC+BC G=53, 10 df
TABLE 5b Modelling the effects and interactions of the factors A, B, C for the clam in summer. A = concentration, B = density, C = size class. All the order 2 interactions are significant and must be taken into account AB+AC+BC G=10,8df dG=93,4
df
AB+AC G= 103,12 df
dG= 134,8 df
dG=16,2df
AB+BC G= 144,16 df
AC+BC G=26,10
df
Similarly, zero concentration leaded to a temperature x flow rate interaction with inversion of the temperature effect according to flow rate (type II interaction). The addition of food tended to cancel this interaction. The frequency of food injection had no detectable effect on the growth results (Table 7a). However, a comparison of the average growth at the end of the experiments on a sample of 60 individuals tended to show a positive effect of a discontinuous injection of food (Table 7b). Correspondence analysis Three analyses were carried out, showing the effect of the quality of the water, the flow rate, the density and the concentration of the injected phytoplankton. The first axis generally represented the growth gradient, the sizes either increasing or decreasing along that axis. The levels of the illustrative variables were located relative to that gradient, and positive or negative correlation with growth was derived from their position.
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J.-P. BAUD AND C. BACHER
TABLE 6 Modelling the effects and interactions of controlled factors and size class for the growth of clams in summer. No interaction of third order may be deleted (6a), so that sub-tables must be studied according to each level of the temperature (6b), food concentration (6~) or flow rate (6d) 6a. A = quality of water, B = concentration,
C = flow rate, D = size class
ABC+ABD+ACD+BCD G=2.6,4 df dG= 13,4 df
dG=43,4
-BCD G=16,8
-ACD G=47,8
df
6b. A = concentration,
df df
dG=23,4 -ABD G=26,8
df df
dG=lO,
1 df
-ABC G=13,5
df
B = flow rate, C = size class
Heatexchanged water
AB+AC+BC G=9.8,4 df
Natural water
AB+AC+BC G=5.8,4 df dG=35,6
df
dG= 234,4 df
dG= 109,4 df
AB+AC G=41,10
df
AB+BC G= 240,8 df
AC+BC G=115,8df
co
AB+AC+BC
G= 37.2, 4 df
c2
AB+AC+BC G=8,4df
6c. A = quality of water, B = flow rate, C = size class
dG= 59,4 df
dG= 62,4 df
dG=6,
AB+AC G= 67,8 df
AB+BC G= 70,8 df
AC+BC G=14,5df
Flow rate 1
AB+AC+BC
G= 22.2,4 df
Flow rate 3
AB+AC+BC G=4,4df
6d. A = concentration,
1 df
B = quality of water, C = size class
dG=65,4
df
AB+AC G=69,8 df
dG= 199,4 df
dG= 17, i df
AB+BC G=203,8
AC+BC G=21,5 df
df
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TABLE 7a Modelling the effects and interactions of the controlled factors frequency of feeding (A), density (B ) and the size class of clams in summer. After trials, there is no interaction left but density X size class AB+AC+BC G=0.61,2 df dG=23,2df
dG=l,2df
dG=0.2,
AB+AC G=24,4df
AB+BC G= 1.7,4 df
AC+BC G=l, 3df
dG=23,2 AB G=25,6
df df
dG=O.9,2
1 df
df
BC G= 1.9, 5 dG=23,2 A+B+C G=25,7
df df
TABLE lb Comparisons of the mean lengths and weights when the frequency of feeding is continuous or discontinuous. The size of each sample is 60 individuals. The density was 25 000 individuals in each tube (D25 ) . The standard deviation is within parentheses. The differences are significant at a 5% level Frequency
Length (mm)
Weight (mg)
Continuous Discontinuous
13.05 (2.64) 14.55 (1.78)
0.479 (0.214) 0.606 (0.189)
Temperaturexconcentration (CO, C2) xjlow rate. For a fixed density of 25 000, the effects of the injection of phytoplankton and of flow rate were compared for two temperatures (natural water and exchanged water) (Fig. 2). The first axis was explained up to 96% by the S4, S8 and S 10 classes. Size classes were well represented except for the SO class. Projection of the illustrative variables showed firstly that concentration was the factor most correlated with the gradient (quality of the representation was 0.68); and secondly that flow rate and quality of the water had secondary but real effects, although it was not possible to determine which one was the more important (qualities of representation were respectively of 0.15 and 0.18 ) . However, it appeared that the quality of the water had a non-linear effect on growth, since it enhanced the extreme classes. Concentration (Cl, C2, C4) x density in natural sea water. With flow rate fixed at 3 m3/h, the effects of different positive concentrations of injected food were compared (Fig. 3 ) . These effects were clearly visible, the density 25 000
J.-P. BAUD AND C. BACHER
168 0.6 0.5 0.4 -
EXC
0.3 0.2 0.1 -0.1
-
-0.2
-
-0.3
NAT
-0.6
-
-0.7
-
-0.6
-
-0.9
-
-1
-
-1.1
-
-1.2
-
-1.3
1 -1.2
I -0.6
I
I -0.4
I
I
0
I
0.4
,
I
0.6
I
I
1.2
I
-1 1.6
AXIS 1 (70X)
Fig. 2. Correspondence analysis of the growth of clams in summer, with a density D25 (25 000 individuals per tube). The controlled parameters temperature, concentration (0, 2) and flow rate ( 1, 3) are projected as illustrative variables. Notations for Figs. 2-5: Si: size class number i. FRl, FR3: levels of the flow rate ( 1 m3/h, 3 m3/h). D25, D50: levels of the density (25 000, 50 000 individuals per tube). NAT, EXC: natural water, heat-exchanged water. CO, Cl, C2, C4: levels of phytoplanktonic food concentration.
and the concentrations C2 and C4 being favourable factors for growth. Although this projection was not carried out with exchanged water, the conclusions should not be readily changed in that case since the two factors concentration and density seemed to be far more important than the quality of the water studied above. Concentration (Cl, C2, C4) xflow rate. This analysis confirmed the positive effect, even though it was small, of flow rate with a given density of 25 000 and exchanged water (Fig. 4). When a projection was made of the variable “available food”, represented by the ratio concentration/flow rate, no correlation appeared between the growth gradient and the available food. The two factors concentration and flow rate thus appeared to be independent. The water was not only a support of the injected food, the flow itself was an additional, positive factor.
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0.7 0.6 -
c4
0.5 0.4 0.3 0.2 0.1 0 -0.1
-
-0.2
-
-0.3
-
-0.4
-
-0.5
-
-0.6
-
-0.7
, -1.2
c2 1
I -0.8
1
, -0.4
I
I 0
I 0.4
I
, 0.8
1 1.2
AXIS 1 (82%)
Fig. 3. Correspondence analysis of the growth of clams in summer, with a flow rate FR3 (3 m3/ h). The controlled parameters concentration (1, 2, 4) and density (25, 50) are projected as illustrative variables (see Fig. 2 for notations).
Growth ofRuditapes
philippinarum
in winter
In winter the temperature factor became of primary importance. The natural sea water temperature was around 5 oC, and was increased to 10°C with the heat exchanger. One can easily see that growth rates were much smaller in winter than in summer and that the use of the exchanger was required in winter. A simple analysis could test the influence of flow rate, density and concentration. Contingency tables The results concerned experiments in warmed-up water. Concentration and density had significant and independent effects (Table 8 ) , whatever the flow rate. Correspondence analysis The antagonistic effects of concentration (positive effect) and density (negative effect ) appeared once again (Fig. 5 ) . There seemed to be no effect of flow rate, judging from the orthogonality of the axes flow rate/growth gradient.
J.-P. BAUD AND C. BACHER
170 2 A3
i .a
1.6 1.4 1.2 1 0.8
A2
0.6 Cl
0.4 0.2 0
iI
-0.2
-
-0.4
-
-0.6
-
-0.8
-
-1
/
FRl
c4
A
-pqS
-1.2
I -1.6
I -1.2
I
I -0.8
I
I -0.4
I
I
0
I
0.4
I
I
0.8
I
I
1.2
AXIS 1 (72%)
Fig. 4. Correspondence analysis of the growth of clams in summer, with a density D25 (25 000 individuals per tube). The controlled parameters concentration ( 1, 2, 4), flow rate ( 1, 3) and the variable A= concentration/flow rate are projected as illustrative variables (see Fig. 2 for notations). TABLE 8 Modelling the effects and interactions of the factors concentration (A), flow rate (B), density (C), and size class (D) of clams in winter. The effect of the flow rate and all third order interactions may be neglected Order 3 G=0.74, 8 df AD+CD G=16,40df -AD G=210,48
df
-CD G= 40,44 df
Mortality results It was possible to compare differences in mortality rates as a function of the five controlled parameters of the media. Not all combinations were tested. Testing the effects of season, density and food concentration showed an effect
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0.8 0.7 0.6 0.5
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JUVENILES
c2
I
FR3
050 0.1 0 -0.1 -0.2
-0.3 -0.4 -0.5 -0.6
c4 , -1
I
I -0.6
I
I -0.2
I
0.2
1
I
0.6
I
I
1
I
1.4
AXIS 1 (90%)
Fig. 5. Correspondence analysis of the growth of clams in winter. The controlled parameters flow rate ( 1, 3), density (25, 50), concentration ( 1,2, 4) are projected as illustrative variables (see Fig. 2 for notations).
of density only, though a small interaction of season~density was noticed (Table 9a). During winter, the systematic use of warmed-up water left only the parameters flow rate, density and concentration, which were combined. None of the factors was significant, which confirmed the difference of the effects of density according to the season. During summer, two designs were analysed. The first design combined concentration and density. The second included the effects of flow rates, quality of water, and concentration (CO, C2 levels) at a density of 25 000. In the former case, clams seemed to be sensitive to the feeding conditions since density and concentration had a significant effect (Table 9b). Multiple range test (Newman-Keuls method) showed the mortality for high concentrations (C2, C4) to be significantly higher than that observed for concentration C 1. In the latter analysis, there appeared to be no significant factor. Though the levels of concentration were not the same, it was seen that concentration did not have the same effect on mortality in the two analyses. There might be an interaction between density and concentration, which could explain the global effect of concentration when the density factor was taken into account. An-
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J.-P. BAUD AND C. BACHER
TABLE 9a ANOVA on the mortality rate tm transformed sity (2), concentration (3)
by arcsin (fi).
Controlled factors: season ( 1 ), den-
Source of variability
Sum of squares (S.S.)
df
F test
Probability
Factor I (season) Factor 2 (density) Factor 3 (cont. ) Interaction 1, 2 Interaction 1, 3 Interaction 2, 3 Residual
618 42 176 138 113 134 2 14
11 1 1 2 1 2 2 2
6.2 25.8 10.1 16.6 9.8 0.12
0.131 0.034 0.091 0.053 0.093 0.890
TABLE 9b Controlled factors: density, concentration density and a low effect of concentration
(Cl, C2, C4), in summer. There is a significant effect of
Source
S.S.
df
F test
Probability
Total Factor 1 (cont.) Factor 2 (density) Residual
0.17 0.07 0.09 0.00
5 2 1 2
22.8 52.9
0.041 0.015
TABLE 10 Percentages of mortality (by ANOVA) clam 3.48 0
winter
4.83
10.10
18.00
24.90
summer 9.1 Cl
c2 concentration
c4
other explanation was the lack of power of the second analysis, since only two levels of concentration were considered. Observed mortalities are summarized in Table 10. Growth dispersion and mortality were not independent, and significant mortality was observed together with high growth rates.
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DISCUSSION
Temperature and quantity of food are the essential parameters governing the growth of bivalves in nurseries. According to Claus ( 198 1)) there is a positive correlation between temperature and growth. Equilibrium between respiration and assimilation is near 13 oC according to Walne ( 1965 ) and 10 ’ C according to Ryther and Tenore ( 1976). The optimal temperature is derived from the response curves of metabolism and of flesh and shell production. It is also affected by the stress found at high temperatures (Mann and Ryther, 1977 ) . Moreover, it depends on whether the growth in flesh or in shell is considered. Mann ( 1979a,b) observed high shell growth rates for Ruditupesphilippinarum and Crassostrea gigas reared at high temperature. On the contrary, lower temperature yields better flesh growth rates. Liang et al. ( 1987 ) conclude that the growth rate for Ruditapes philippinarum increases up to 25 ‘C and decreases beyond that temperature. In our study, the use of the heat exchanger was necessary in winter to obtain a rearing temperature greater than, or equal to 10’ C. In summer, however, average temperatures seldom went beyond 20” C and cooling the water rather tended to slow down the growth when the food was unlimited. Raising the temperature without injection of food was harmful both to the growth and the survival of the clams. However, the positive correlations between growth rate and temperature showed higher growth rates for higher temperatures. In our study, the food consisted of Skeletonemu costatum at high concentration, produced at low cost and discontinuously with ground water. This diatom species multiplies itself easily in media of low salinity with high concentrations of silica (Laing and Jones, 1983; Laing, 1985 ) . It appears to be a dominant species when the ammonia concentration is very high (307.5 patg/ 1) which is a characteristic of the ground water used (Robert, 1987). Skeletonemu cost&urn has a high nutritional value ( Walne, 1970)) particularly for Mercenaria mercenaria and Ruditapes philippinarum (Laing et al., 1987 ) . This corresponds with the satisfactory growth rate observed with Ruditapes philippinarum. This result could be partially due to polyunsaturated acids (Langdon and Waldock, 198 1) . However, growth rates are higher for bivalves fed with a mixture of Skeletonema cost&urn and Tetraselmis (Laing and Millican, 1986 ). This could be due to the low content of tyrosine in SkeZetonema costatum, as detected by Chuecas and Riley ( 1969). The greatest gains in weight and size during the summer rearing were obtained with the injection of a high concentration (C4) of Skeletonema costaturn, together with a sea-water flow rate of 3 m3/h. The concentration resulting from such a dilution corresponded to the optimum level of phytoplankton as given by Matthiessen and Toner ( 1966 ) , but remained below that recommended by Tenore and Dunstan ( 1973 ) (Table 11) .
J.-P. BAUD AND C. BACHER
174 TABLE I1
Computation of the daily available phytoplanktonic food according to different food concentrations and flow rates and according to several authors. Results are expressed in number of cells per ml and per day Matthiessen and Toner ( 1966)
Levels C2, FRl
C4, FRl
1.5X lo5 cell/ml
3 X 1O5cell/ml
2.5 x 10’ cell/day
5 x 10’ cell/day
Tenore and Dunstan ( 1973)
C4, FR3 2X 105cell/ml
1 X 10’ cell/ml 1.7~ lO*cell/day
11 x 10’ cell/day
TABLE 12 Strategies of rearing defined after the analyses of growth and mortality Season
Quality of water
Density
Flow rate
Injected flow rate and concentration
Frequency
Winter
Heat-exchanged water (10°C)
25 OOO/tube 12.7 ind./cm’
1 m3/h per tube 0.67 ml/min per ind.
150 l/h per tube 0.5X lo6 cell/ml
Discontinuous 14 h/24 h
Summer
Natural water (20°C)
25 OOO/tube 12.7 ind./cm’
3 m3/h per tube 2 ml/min per ind.
150 l/h per tube 0.5~ lO”cell/ml
Discontinuous 14 h/24 h
The discontinuous feeding of Ruditapes philippinarum in the rearing stage enhanced growth. Such feeding forces a digestive rhythm upon the juvenile (Langton and Gabbott, 1974; Owen, 1974). According to Epifanio and Mootz ( 1976), the oyster ingests a maximum quantity of algae in a phase of active filtration, then slows down its filtration activity during a phase of digestion. This alternation of activity and rest depends upon the quantity of food in the medium, and the periodicity of this mechanism is dampened when the medium has a low concentration of food, which is the case with the natural sea water. Langton and MacKay ( 1976) suggest that, with higher concentrations in discontinuous feeding, filtration can be stimulated during the feeding period and the efficiency is greater. Feed distribution by an upwelling appears to be a technique very well adapted to the optimization of growth. Contrary to what happens with a laminar flow, all the enriched water irrigates the bivalve population (percolation) . The quantities of juveniles can be increased with this type of flow distribution, and maintenance is easier (Manzi et al., 1984). In our study, the effect of the flow rate was hidden most of the time by the effects of the quantity of food and of the temperature. According to Malouf and Breese ( 1978), the growth of oysters increases with the speed of the water.
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The flow of water through the tubes should eliminate organisms in competition for phytoplankton, evacuate the faeces and stimulate nutrition with conditions favouring the filtration mechanisms (Kirby Smith, 1972; Walne, 1972). The different analyses of data clearly showed that the flow rate was not a primary factor but that it did have a positive effect on growth in summer. The opposite effect could be seen in winter, with low temperatures. In summer the upwelling not only served as carried of the food but also contributed to the equilibrium of the food (multi-specific phytoplankton, oligo-elements, dissolved organic materials). The growth rates observed are compatible with published values and lead to the nursing strategies described in Table 12. The study of mortality showed that Ruditapes philippinarum was somewhat sensitive to high concentrations of food (C4) in summer. In that season, the efficiency of rearing was better when using a mean concentration (C2). The analyses showed the value of a high flow rate (3 m3/h) in summer. Finally, the use of the heat exchanger is not required: it brought only a small increase in growth while its operating costs were not negligible. On the contrary, a low flow rate combined with exchanged water can be chosen in winter. In both cases, a low density (D25) and injection of food (phytoplankton) are proposed, so as to obtain satisfactory growth performances. Correspondence analysis gave some information on non-linearity effects of the controlled parameters on growth. Displaying these parameters as illustrative variables showed their effects in a more obvious manner. Moreover, multiway contingency tables analysis gave the statistical justification of these results and measured the interactions between the parameters. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Jean-Paul Dreno for his help in the definition of the experimental design, Dr. Alain Bodoy for his advice on the manuscript and Pierre Bather for the translation into English.
REFERENCES Abbe, G.R., 1980. Non-radiological studies of tray-held oysters, Crassostrea virginica, in the vicinity of the Calvert cliffs nuclear power plan in Chesapeake Bay, 1970- 1979. 72nd Annu. Meet. Natl. Shellfish. Assoc. (abstr.). Barnabe, G., 1986. La collecte de micro-algues. In: G. Barnabe (Editor), Aquaculture, Vol. 1. Lavoisier Technique et Documentation, Paris, pp. 194-l 98. Bayne, B.L. and Newell, R.C., 1983. Physiological energetics of marine molluscs. In: K. Wilbur (Editor), The Mollusca, Vol. 4. Physiology, Part 1. Academic PPess, New York, NY, pp. 407-515. Chuecas, I. and Riley, J.P., 1969. Component fatty acids of the total lipids of some marine phytoplankton. J. Mar. Biol. Assoc. U.K., 49: 97-l 16.
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Claus, C., 198 1. Trends in nursery rearing of bivalve molluscs. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. l-33. Conan, G.Y. and Comeau, M., 1986. Functional maturity and terminal molt of snow crab, Chionoecetes opilio. Can. J. Fish. Aquat. Sci., 43: 17 1O-l 7 19. De Pauw, N., Verboven, J. and Claus, C., 1983. Large-scale microalgae production for nursery rearing of marine bivalves. Aquacult. Eng., 2: 27-47. Epifanio, C.E. and Mootz, C.A., 1976. Growth of oysters in a recirculating maricultural system. Proc. Natl. Shellfish. Assoc., 65: 32-37. Epifanio, C.E., Srna, R. and Pruder, G., 1975. Mariculture of shellfish in controlled environments: a prognosis. Aquaculture, 5: 227-241. Fienberg, SE., 1970. The analysis of multidimensional contingency tables. Ecology, 5 1: 419433. Goldman, J.C. and Mann, R., 1980. Temperature-influenced variation in speciation and chemical composition of marine phytoplankton in outdoor mass cultures. J. Exp. Mar. Biol. Ecol., 46: 29-39. Grobbelaar, J.U., Sveder, C.J. and Toerdien, D.F., 198 1. Waste Water for Aquaculture, Univ. Orange Free State Publ., Ser. C, 3,2 15 pp. Incze, L.S., Lutz, R.A. and Watling, L., 1980. Relationships between effects of environmental temperature and seston on growth and mortality of Mytilus edulis in a temperate northern estuary. Mar. Biol., 57: 147-156. Kirby Smith, W.W., 1972. Growth ofthe bay scallops: the influence of experimental water currents. J. Exp. Mar. Biol. Ecol., 8: 7-18. Ku, H.H. and Kullback, S., 1974. Loglinear models in contingency table analysis. Am. Stat., 28: 115-122. Kuwatani, Y. and Nishii, T., 1969. Effects of pH of culture water on the growth of the Japanese pearl oyster. Bull. Jpn. Sot. Sci. Fish., 35: 342-350. Laing, I., 1985. Factors affecting the large-scale production of four species of commercially important marine algae. Aquaculture, 44: 16 1- 166. Laing, I. and Jones, E., 1983. Large-scale turbidostat culture of marine microalgae. Aquacult. Eng., 2: 203-212. Laing, I. and Millican, P.F., 1986. Relative growth and growth efficiency of Ostrea edulis L. spat fed various algal diets. Aquaculture, 54: 245-262. Laing, I., Utting, S.D. and Kilada, R.W.S., 1987. Interactive effect of diet and temperature on the growth of juvenile clams. J. Exp. Mar. Biol. Ecol., 113: 23-38. Langdon, C.J. and Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigm. J. Mar. Biol. Assoc. U.K., 6 1: 43 l-448. Langton, R.W. and Gabbott, P.A., 1974. The tidal rhythm of extracellular digestion and the response to feeding in Ostrea edulis. Mar. Biol., 24: 18 1- 187. Langton, R.W. and MacKay, G.U., 1976. Growth of Crmsostrea gigas Thunberg spat under different feeding regimes in a hatchery. Aquaculture, 7: 225-233. Lebart, L., Morineau, A. and Tabard, N., 1977. Techniques de la Description Statistique. MCthodes et logiciels pour l’analyse des grands tableaux. Dunod, Paris, 35 1 pp. Leborgne, Y., 1977. L’ecloserie nurserie de la SATMAR et les possibilites actuelles de production de naissain de mollusques bivalves. 3rd Meeting of the ICES working Group of Mariculture. Actes Colloques CNEXO, 4: 353-360. Legendre, L., 1987. Multidimensional contingency table analysis as a tool for biological oceanography. Biol. Oceanogr., 5: 13-28. Lellouch, J. and Lazar, P., 1974. Methodes Statistiques en Experimentation Biologique. Flammarion Medecine-Sciences, Paris, 293 pp.
INTENSIVE REARING OF RUDITAPES
PHILIPPINARUM
JUVENILES
177
Lorenzen, C.J., 1967. Determination of chlorophyll and pheopigments: spectrophotometric equations. Limnol. Oceanogr., 12: 343-346. Malouf, R.E., 198 1. Use of heated effluents for the nursery culture of bivalve molluscs: its problems and potential. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. 171-188. Malouf, R.E. and Breese, W.P., 1978. Intensive culture of the Pacific oyster Crassxtrea gigas (Thunberg) in heated effluents. Oregon State Univ. Sea Grant Program., Publ. no. ORESUT-78-003 Corvallis, OR. Mann, R., 1979a. Some biochemical and physiological aspects of growth and gametogenesis in Crassostrea gigas (Thunberg) and Ostrea edulis L. grown at sustained elevated temperatures. J. Mar. Biol. Assoc. U.K., 59: 95-110. Mann, R., 1979b. The effect of temperature on growth, physiology and gametogenesis in the manila clam Tapes philippinarum. J. Exp. Mar. Biol. Ecol., 38: 12 1- 133. Mann, R. and Ryther, J.H., 1977. Growth of six species of bivalve molluscs in a waste recycling aquaculture system. Aquaculture, 11: 231-245. Manzi, J.J., Hadley, N.H., Battey, C., Haggaty, R., Hamilton, R. and Carter, M., 1984. Culture of the northern hard clam Mercenaria mercenaria (Linne) in a commercial scale, upflow, nursery system. J. Shellfish Res., 4: 119-124. Matthiessen, G.C. and Toner, R.C., 1966. Possible methods of improving the shellfish industry of Martha's Vineyard, Duke’s County, Massachusetts. The Marine Foundation Inc., Edgartown, MA, 138 pp. Owen, G., 1974. Feeding and digestion in the Bivalvia. Adv. Physiol. Biochem., 5: l-35. Price, A.H., Hess, C.T. and Smith, C.W., 1976. Observations of Crassostrea virginica cultured in the heated effluent and discharged radionuclides of a nuclear power reactor. Proc. Natl. Shellfish Assoc., 66: 54-68. Provasoli, L., McLaughlin, J.J.A. and Droop, M-R., 1957. The development of artificial media for marine algae. Arch. Mikrobiol., 25: 392-428. Riva, A. and Lelong, P., 198 1. Growth of juvenile molluscs associated with continuous cultures of natural marine phytoplankton. In: C. Claus, N. de Pauw and E. Jaspers (Editors), Nursery Culturing of Bivalve Molluscs. EMS Spec. Publ. no. 7. European Mariculture Society, Bredene, Belgium, pp. 253-268. Robert, J.M., 1987. Valorisation des sites aquacoles existants ou potentiels de la region des pays de Loire, par utilisation des eaux souterraines pour la production d’algues unicellulaires destin&es a l’alimentation des differentes especes de mollusques au tours de leur cycle d’elevage. Rapport Convention Region des Pays de Loire et Universite de Nantes, 49 pp. Ryther, J.H. and Tenore, K.R., 1976. Integrated systems of mollusc culture. In: 0. Devik (Editor), Harvesting Polluted Waters. Plenum Press, New York, NY, pp. 153-167. St Felix, C., Baud, J.P. and Hommebon, P., 1984. Diversification de la production conchylicole. Elevage de la palourde japonaise en Baie de Bourgneuf. Sci. P&he, 344,345,346: 2-22. Sokal, R.R. and Rohlf, F.J., 1981. Biometry. The Principles and Practice of Statistics in Biological Research, 2nd edn., Freeman, San Francisco, CA, 859 pp. Spencer, B.E. and Gough, C.J., 1978. The growth and survival of experimental batches of hatchery-reared spat of Ostrea edulis L. and Crassostrea gigas Thunberg, using different methods of tray cultivation. Aquaculture, 13: 293-3 12. STATITCF, 1987. Guide de l’utilisateur. ITCF edn., Paris, 150 pp. Tenore, K.R. and Dunstan, W.M., 1973. Comparison of feeding and biodeposition of three bivalves at different food levels. Mar. Biol., 21: 190-195. Volle, M., 198 1. Analyse des Donnees, Economica, Paris, 3 17 pp. Walne, P.R., 1965. Observations on the influence of food supply and temperature on the feeding and growth of Ostrea edulis L. Fish. Invest., Lond., Ser. 2,24 ( 1 ), 45 pp.
178
J.-P. BAUD AND C. BACHER
Walne, P.R., 1970. Studies of the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea Mercenaria and Myths. Fish. Invest., Lond., Ser. 2, 26( 5), 62 PP. Walne, P.R., 1972. The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves. J. Mar. Biol. Assoc. U.K., 52: 345-374. Walne, P.R., 1974. Culture of Bivalve Molluscs: 50 years’ Experience at Conway. Fishing News (Books), West Byfleet, 173 pp. Widdows, J., Fieth, P. and Worrall, C.M., 1979. Relationships between seston, available food and feeding activity in the common mussel, Mytilus edulis. Mar. Biol., 50: 195-207. Wilks, S.S., 1935. The likelihood test of independence in contingency tables. Ann. Math. Statist., 6: 190-196. Winter, J.E., 1978. A review of the knowledge of suspension feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture, 13: l-33.