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The effect of water hardness on growth and carapace mineralization of juvenile freshwater prawns, Macrobrachium rosenbergii de Man
329
Aquaculture, 95 ( 1991) 329-345 Elsevier Science Publishers B.V., Amsterdam
The effect of water hardness on growth and carapace mineralization of juvenile freshwater prawns, kfacrobrachium rosenbergii de Man Janet H. Browna, John F. Wickinsb and Marlie H. MacLearP alnstitute ofAquaculture, University ofstirling, Stirling FK9 4LA, UK bMinistry ofAgriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory. Conwy, Gwynedd LL32 8UB, UK (Accepted
19 November
1990)
ABSTRACT Brown, J.H., Wickins, J.F. and MacLean, M.H., 1991. The effect of water hardness on growth and carapace mineralization of juvenile freshwater prawns, Macrobrachium rosenbergii de Man. Aquaculture, 95: 329-345. Growth and carapace mineralization of Macrobrachium rosenbergii juveniles, (0.01-0.5 g) were investigated under conditions of different water hardness from 9 to 326 mg 1-l CaC03. Growth was maximal at < 53 mg 1-l CaCO,, did not change significantly at lower hardness levels but declined at higher levels. Survival was impaired at the highest hardness levels tested. Greater deposition of calcium in the carapace occurred in low-hardness water and the lower levels of calcium found in cast carapaces from these prawns indicated greater withdrawal of calcium from the carapace before moulting.
INTRODUCTION
The giant freshwater prawn, Macrobrachium rosenbergii de Man, is widely cultured throughout the tropics, subtropics and some warm temperate zones of the world, far beyond its natural geographic range. In nature, larval life is completed in brackish water after which the juveniles migrate into fresh water where they grow to maturity. The adult females return to estuarine waters to spawn. Despite widespread commercial interest in the species little information is available on its tolerance to water hardness and on its ability to regulate shell calcium levels. Juveniles and adults moult frequently, between every 5 to 40 days, respectively, and require exogenous sources of cations, particularly calcium for successful shell mineralization. Recorded satisfactory hardness levels for culture range widely; Wickins ( 1982) suggested from 65 to 200 mg 1-l CaC03, New and Singholka ( 1985 ) recommended levels above 40 and below 0044-848619
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J.H. BROWN ET AL.
150 (preferably less than 100 mg l- ’ CaCO, ). Cripps and Nakamura ( 1979) reported greater inhibition of growth at hardness levels above 65 mg 1-l CaC03, while Vasquez et al. ( 1989) showed that while growth was better at 112 than at 20 mg l- ’ CaC03, growth was reduced at 225 and 450 mg l- ’ CaC03. Bartlett and Enkerlin ( 1983) and Howlader and Turjoman ( 1984) reported on growth of M. rosenbergii in high-hardness waters from mainly groundwater sources. While the latter study found that growth was adversely affected by the high hardness levels (688-987 mg l- ’ CaCO,), Bartlett and Enkerlin ( 1983 ) found that growth was not adversely affected by hardness levels between 940 and 1060 mg l- ’ CaC03, but their water had relatively low alkalinity (58-86 mg 1-l when expressed as CaCO,). In Malaysia where natural hardness of culture water can be extremely low ( < 30 mg l- ’ CaCO, ), lime is routinely added to increase hardness up to 50 mg 1-l CaCO, and in French Guyana lime is added to culture ponds to increase hardness levels up to 20 mg l- ’ CaCO,. Johnson ( 1967), however, reported that two species of Macrobrachium, M. geron and M. trompii, were found in Malaysian waters with no detectable calcium content and that M. rosenbergii was also found in very low-hardness waters. In experimental studies Fieber and Lutz ( 1982 ) showed that M. rosenbergii could take up calcium for shell mineralization from an external medium containing 0.45 mmol Ca2+ l- ’ (44.9 mg I- ’ CaC03), and that some shell hardening occurred in solutions when calcium was as low as 0.09 mmol Ca2+ l- ’ (8.98 mg l- ’ CaCO,). Studies on ionic regulation reported by Stem et al. ( 1987), indicated that M. rosenbergii exhibits a high degree of osmoregulatory ability. However, growth and development were found to be reduced in waters of “exotic” salinities and ion composition, in which ionic ratios differ markedly from those found in seawater. There have, in addition, been studies of salinity effects which indirectly involve hardness (in that dilute seawater will contain appreciable amounts of calcium and magnesium) but here interpretation could cause confusion in that Singh (1980), reviewing work by Wickins (1972) and Perdue and Nakamura ( 1976), all of whom found optimal growth in either fresh water or water of low salinity (2%0), suggested that this is related to the ability of the animal to take up more water at ecdysis than animals maintained in more concentrated media. However, Smith et al. ( 1976) recorded “excellent” growth (increases in some male prawns from 0.7 g to 100.6 g in 5 months) in water with a reported average salinity of 7.2%0. In order to evaluate the suitability of waters for M. rosenbergii culture, greater understanding of the species’ tolerance to hardness levels is required. This paper reports on survival, growth, and aspects of shell mineralization in juvenile M. rosenbergii exposed to water hardness levels between 9 and 326 mg l- 1 CaC03.
WATER
H.ARDNESS
AND
GROWTH
AND
CARAPACE
MINERALIZATION
OF FRESHWATER
PRAWNS
331
MATERIALS AND METHODS
Juvenile M. rosenbergii were selected from laboratory stock populations and reared in four experiments at a variety of water-hardness levels. Care was taken to select animals within the narrowest possible size range (Table 1). At the start and end of each trial prawns were blotted dry and weighed to the nearest 0.1 mg on a Mettler AC 100 balance. Carapace lengths (posterior margin of orbit to middorsal margin of carapace) were measured to the nearest 0.0 1 mm using a vernier calliper. The prawns were housed in individual containers, twelve containers per treatment. These were cylindrical plots ( 11-cm diameter) with sides of 1-mm plastic mesh and 2.5mm mesh bottoms, each pot having an air supply and lid. The pots were placed in plastic aquaria containing the test solutions, three pots per 5 litres of test solution. Aquaria were held in 36 x 56 cm tanks in a large recirculation system so that temperature was maintained at 28 ‘C + 1 ‘C. The water in the aquaria was completely changed every 3.5 days. The prawns were fed on a pelleted diet produced in the Institute of Aquaculture (40% protein based on squid, shrimp and fish meal) and were inspected twice daily, so uneaten food could be removed and fresh food added. Moults were recorded and cast carapaces collected, rinsed in distilled water and measured. The carapaces were dried at 60’ C for 24 h, weighed and stored in plastic vials for later analysis. Experiments lasted for a period of time sufficient to allow each individual prawn to moult five times. After live moults the animal was killed halfway through its moult cycle, determined as being half the period of the previous moult cycle, thus ensuring that the final carapaces were sampled at the same stage of the moult cycle. Each prawn was killed by rapid chilling at 4°C for half an hour. It was then blotted dry and weighed. The carapace was dissected off (any attached tissue was carefully removed with fine forceps) and then dried and stored for mineral analysis. Hardnesssolutions The total hardness of water is defined as the sum of the concentrations of calcium and magnesium, both expressed in terms of an equivalent concentraTABLE 1 Size of prawns and average water hardness used in the experiments Experiment
Initial prawn size (g)
I
0.25-0.35 0.1 o-0.20 0.20-0.50 0.10-0.20
2 3 4
“Two tests were run at this hardness.
Average hardness 36
9
13
34 19 30
54 52 58
68 143 316 71” 172 320 74 143 326
J.H. BROWN ET AL.
332
tion of calcium carbonate in mg l- I. When calcium and magnesium concentrations were determined separately the total hardness was calculated from the equation: Total hardness CaC03 mg l- ’ = 2.497 (Ca*+ mg l- ’ ) + 4.118 (Mg*+ mg l- ’ ) While for greater clarity it is probably better to measure the separate levels of the ions, the concept of total hardness is widely used and, more importantly, more easily measured in a field situation, being readily determined by simple calorimetric techniques. For the purpose of these experiments, ion concentrations were measured separately as outlined below. Solutions were made up with deionised water preheated to 28 “C. The recipe used (HMSO, 1969) is shown in Table 2. Analysis of calcium and magnesium in carapaces The stored carapaces were transferred to test tubes held in a heated block where they were digested in 5 ml concentrated Aristar-grade nitric acid. The solutions were made up to 25 ml with distilled water. Analyses were carried out using a Perkin Elmer Atomic Absorption Spectrophotometer (Model 2280) with calcium standard solutions of 4.0 mg 1-l and 2.0 mg l- ’ and magnesium standard solutions of 0.3 and 0.1 mg 1-l. Lanthanum chloride at 5% v/v was added to standards and samples to prevent ionic interference. Presentation of results Results were analysed using an SPSSx statistical program (SPSSx Inc., 1983 ). Analysis of variance (ANOVA) was used to determine whether there was any significant difference between treatments and the differences found were investigated using the multiple-range test, Duncan’s procedure (Duncan, 1955 ) . In the statistical analyses, differences were regarded as signilicant if the level of probability was less than 0.05. In a study of this sort where growth rate will be affected by treatment, some of the effects seen, for example, carapace thickening, may be due to the size TABLE 2 The composition
of stock solutions used to make up the culture media
Stock solution 1
320 g CaC12-6H,O 29 g NaCl 9 g NaNO,
Stock solution 2
15 1g M&O,-7H20 79 g Na,S04
Stock solution 3
27.5 g NaHCO,
Each solution was made up in 1 1 of deionised water. One ml of solutions 1 and 2 with 10 ml of solution 3 in 10 1of deionised water gave a culture medium of 20 mg I-’ CaCOs hardness.
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
333
of the prawn rather than to water hardness directly. Measured factors were therefore each plotted against treatment (water hardness) and against final live weight of the prawns. Slopes differing from 0 at the 95% level of confidence were taken to indicate that a significant relationship existed between the plotted parameters. Where both size and hardness were correlated with a factor, the factor giving the highest correlation was judged to be the more acceptable relationship. Where appropriate, results obtained at similar hardness levels but from separate experiments were combined in order to distinguish the effect of size and hardness on carapace mineral content more clearly. The definitions used in presentation of the results are: growth rate =
final weight (mg) -initial weight (mg) no. of days in experiment for each individual prawn (day 1 of experiment to final sampling)
length-specific dry weight of carapace =
concentration
distribution
dry weight of carapace (mg ) carapace length (mm)
factor =concentration of cations in carapace (mg g- I ) x I ooo concentration of cations in media (mg l- ’ ) coefficient =
concentration factor of magnesium concentration factor of calcium
If the distribution coefficient is less than 1 it indicates selective exclusion of magnesium from the carapace. RESULTS
Survival
Survival was low at the highest hardnesses under test ( > 3 16 mg I- ’ ), average survival times being less than 7 days in the first two experiments, while in the third experiment all but one prawn survived two moults and average survival time was 17.6 days. At the second highest hardness levels ( 143-l 72 mg I-’ CaCO,) survival was more variable (Table 3). In experiment 1, only 8% of the prawns survived to be sampled and the average survival time of the other prawns was just 10 days; however, in experiments 2 and 3 survival at these levels was 8 3% and 9 1.7% respectively. Survival at low hardnesses (experiment 4) ranged from 75% at 9- 13 mg I- ’ CaC03 to 9 1.7% at 3 1 mg I- ’ CaCO,. At intermediate hardness levels, between 30 and 75 mg 1-l CaC03, survival was also good, ranging from 67 to 1000/o_mean 92.5%.
CaCOj)
0.61 0.15
9.63 1.02
Mean final live weight (g) s.d.
Mean final carapacelength s.d.
(mm)
6.2 2.7
83
36
54
7.57 0.95
0.34 0.09
0.1 3.0
91.7
7.43 0.85
0.29 0.07
-0.9 1.4
66.6
68
I
rosenbwgiijuveniles
Mean growth rate (mgday-‘) s.d.
Survival (%)
Hardness (mgg-’
Experiment no.
Survival and growth ofA
TABLE 3
-
-
-
8
-
-
-
0
143 316
7.39 0.63
0.34 0.08
5.8 1.7
100
53
6.83 0.78
0.28 0.10
4.6 2. I
100
71
in waters of different hardness
6.65 0.70
0.26 0.08
3.9 1.3
100
71
2
6.43 0.76
0.23 0.08
2.5 2.2
83
172
-
-
-
0
320 34
9.40 0.60
0.62 0.14
5.4 2.0
100
58
8.95 1.19
0.56 0.24
2.2 1.9
91.7
8.25 1.02
0.41 0.14
2.7 1.3
100
75
3
144
7.90 0.81
0.36 0.11
0.5 0.8
91.7
-
-
-
0
326
9
6.96 0.65
0.30 0.07
4.3 1.7
75
13
6.62 0.74
0.27 0.07
3.8 1.7
75
4
19
6.74 0.55
0.27 0.07
3.1 1.1
83
31
6.74 0.33
0.27 0.03
3.4 0.8
91.7
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
335
Growth rate Where prawns survived high hardness levels (75-172 mg 1-l CaC03), growth was reduced to less than 2.7 mg day-’ (Table 3 ). The growth rates at the lowest hardnesses in experiments 1,2 and 3 were significantly greater than the growth at higher hardness levels whilst in experiment 3, there was a significant difference between each level. No sign&ant differences in growth rate ( 3.4 to 4.5 mg day- ’ ) occurred at low hardnesses over the range 9-3 1 mg 1-l CaCO,. The maximum growth rate achieved (6.2 mg day- ’ at 36 mg l- ’ CaC03 in experiment 1) compares well with the best growth achieved in nutrition trials under similar husbandry conditions but with circulating water (M.R.P. Briggs, pers. comm., 1988). The mean sizes of prawns from experiments 1 to 3 at termination became smaller with increasing hardness while there was an unexpected decrease in the length of the intermoult period also as hardness increased (see below). Moultingfrequency The intermoult period was shorter in prawns exposed to hardness levels above 5 3 mg l- ’ CaCO, even though these animals were growing more slowly than those held at lower levels. In experiment 4 (low hardness) there were no significant differences in intermoult period in the different treatments but all averaged about 7 days between moults compared to 6 days or less for prawns of the same size at hardness levels above 53 mg 1-l CaC03. The regression equation for average values of intermoult period over the full range of hardness (experiments 1,2 and 4) is: intermoult period (days) =7.35-0.011
xhardness
(mg 1-l CaCO,)
{corr = - 0.772, d.f. = 6, P< 0.05} Shell mineralization Intermoult carapaces. Carapaces dissected from intermoult prawns (0.1-0.9 g live weight) at the end of the experiments showed an increase in length-specific dry weight with size. The rates of increase at mean hardness levels of 3 1,55 and 7 1 mg l- ’ CaC03, (experiments 1,2 and 3 ) did not differ significantly and the relationship for the combined data is described by the regression equation: length-specific dry weight = 0.000 15 + 0.00 197 x final weight {corr= +0.891, d.f.= 154, P
all sizes of prawns together the calcium content of intermoult
J.H. BROWN ET AL.
336
carapaces increased from 145 to 223 mg g- ’ as hardness decreased. The regression was: [Ca*+] (mgg-‘)=203.45-0.358xhardness {corr= -0.645,
(mgl-’
CaC03)
d.f.= 13, P-=0.01}
Carapace magnesium content was much lower, 2.5 to 5.2 mg g-r, and seemed independent of hardness down to about 30 mg l- I, below which level it declined. The concentration factors of both calcium and magnesium in intermoult carapaces increased as the water hardness decreased; calcium from 2.7 to nearly 70 ( x 1000) magnesium from 0.3 to 5 ( x 1000) (Fig. la,b). Final prawn size affected the relation between hardness and the mineral content of intermoult carapaces (Table 4). At a mean hardness of 34 mg 1-l CaCO,, calcium content was high (203 mg g- ’ ) but declined as size increased; at 5 5 mg l- ’ CaCO, calcium content was intermediate (mean 182 mg g- ’ ) but no correlation with size was apparent while at 7 1 mg l- ‘, calcium content was lower ( 168 mg g- ’ ) but increased with size. Magnesium content only increased with size at low hardness (Table 4). No correlation with size occurred at intermediate and high hardness levels. The actual concentration of one cation relative to another in the exoskele-
Fig. la. The relationship moult prawn carapaces.
between water hardness and calcium concentration
factor in inter-
WATER
HARDNESS
AND
GROWTH
AND
CARAPACE
MINERALIZATION
OF FRESHWATER
337
PRAWNS
O& i”“1”“i”“1”“,““,““,“~~,~“~,‘~~‘,~’~’,’~~7”’.,.-‘,“..,‘...,.~..,~.~.,
0
10
20
30
40
50
60
70
80
Water hardness
Fig. 1b. The relationship
between water hardness
90
100
110
120
130
140
150
160
170
180
(mg ItaCO,)
and magnesium
concentration
factor in inter-
moult prawn carapaces. TABLE 4 Calcium and magnesium content in relation to final prawn size at three averaged hardness levels (C.L. = carapace length in mm, n.s. = not significant at 95% level) Calcium
Magnesium
34 mg I-’ CaCOS
34 mg 1-l CaCO,
[Ca”] =284.19-9.73 CL. (corr=-0.514,d.f.=28,P<0.005)
[Mg’+] =0.28+0.47 CL. {corr=0.606, d.f.=28,Pt0.001)
55 mg 1-l CaCO,
55 mg 1-l CaCOx
[Ca’+]=214.99-4.19C.L. /corr=-0.152,d.f.=30,P>O.l}n.s.
[Mg’+] =4.97-0.03 {corr=-0.034,d.f.=30,
71 mg I-’ CaCO,
71 mg I-’ CaCO,
[Ca’*]=111.81+7.63C.L. jcorr=0.361, d.f.=42, P
[Mg*+]=6.13-0.25 C.L. {corr=-0.241,d.f.=41.P>O.lj
C.L. P>O.l} n.s.
ns.
ton depends not only on the relative cation concentrations in the environment but also on the ability of the prawn to discriminate between them during mineralization; this ability can be expressed as a distribution coefficient (defined in Methods). The low values of the distribution coeffkients (0.02-0.25 ) (Table 5 ) indicate that the prawns were discriminating strongly against mag-
5.19 0.44
0. I 0 0.01
Distribution coefficient s.d.
213.79 17.20
Magnesium (mgkr’) s.d.
Calcium (mgg-‘) s.d.
0.10 0.03
4.65 1.38
190.98 35.46
0.12 0.03
4.19 1.83
170.88 39.16
68
54
Hardness (mg I-’ CaCO,)
36
1
Experiment no.
-
-
-
-
-
-
143 316
0.18 0.03
4.92 I .04
192.35 35.46
53
Mean calcium and magnesium content of carapaces from intermoult
TABLE 5
0.19 0.03
5.23 0.79
167.44 14.01
71
0.14 0.04
4.64 0.87
157.29 25.82
71
2
0.25 0.31
5.03 0.84
128.49 41.89
172
-
-
-
320
0.10 0.02
4.45 0.58
171.67 16.92
34
0.10 0.02
4.43 1.08
162.66 14.49
58
0.07 0.02
3.23 0.69
0.07 0.01
3.53 0.59
181.18 13.40
144
hardness
175.03 12.57
75
3
M. rosenbergir juveniles exposed to waters ofdifferent
-
-
-
326
0.07 0.03
2.64 0.58
182.34 38.28
9
0.07 0.04
2.77 0.40
184.64 55.60
13
4
0.04 0.01
3.06 I .26
222.43 34.89
19
0.03 0.0 1
3.31 1.61
222.43 11.49
31
WATER
HARDNESS
AND
GROWTH
AND
CARAPACE
MINERALIZATION
OF FRESHWATER
PRAWNS
339
nesium with respect to calcium. A positive correlation of the coefficient with hardness (PC 0.02) showed that greater discrimination was occurring at low than at high hardness levels. The distribution coefficient only differed significantly at lower hardnesses -that at 9 mg 1-l CaCO, was significantly lower than at 19 mg l- ’ CaCO, and 3 1 mg l- ’ CaC03, and that at 13 mg l- ’ CaCO, was significantly lower than at 3 1 mg 1-l CaCO,. In other words, there was far greater discrimination in favour of calcium against magnesium at the lowest hardness levels while above 3 1 mg 1-l CaCO, the effect was less pronounced. Cast carapaces. Whilst calcium content of intermoult loosely correlated with size,
carapaces was only
[Caz+] = 109.15+ 10.45 C.L. {corr=0.26,
d.f.= 107, P
the correlation in cast carapaces from first moults was more obvious (experiments 1,2 and 3): [Ca’+] = - 159.0+49.0 {corr=0.88,
C.L.
d.f.=96,P<0.001)
Fig. 2. Differences in calcium relation to water hardness.
concentration
factor between
intermoult
and cast carapaces
in
3.52 1.18
0.07 0.02
Distribution coefficient s.d.
186.77 22.60
Magnesium s’,y,g g- ’ )
Calcium
0.07 0.03
3.18 1.39
185.53 19.40
54
0.08 0.04
31.3 1.42
194.96 35.79
68
36
Hardness (mgl-‘CaC03)
Experiment
I
0.06 0.02
0.68 2.61
175.09 25.34
-
-
-
316
0.21 0.07
0.95 2.75
92.50 24.08
53
0.22 0.08
2.98 1.08
86.29 27.91
71
2
0.17 0.06
3.62 1.59
0.16 0.08
3.22 1.09
100.87 27.78
172
_ -
_
-
320
_ -
_
-
34
_ -
_
-
58
exposed to waters of different
98.92 23.43
71
cast from M. rosenbergii juveniles
143
content of carapaces
no.
Mean calcium and magnesium
TABLE 6
_ -
_
-
75
3
hardness
_ -
_
-
144
_ -
_
-
326
0.17 0.08
2.52 1.21
65.25 13.17
9
0.15 0.05
2.78 1.19
66.94 11.48
13
4
0.10 0.03
2.73 1.08
71.23 11.53
19
0.08 0.02
2.89 1.18
75.85 18.84
31
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
341
This suggested that calcium withdrawal from the carapaces became less pronounced as the animals increased in size. In a comparison of experiments 2 and 3 (of prawns of directly comparable sizes) cast carapaces contained lower calcium concentrations than those dissected from intermoult animals. Difference in calcium content was far greater at hardness levels below 30 mg 1-l CaCO, (average 65%) than at higher levels where average differences were 42%. Mean values (intermoult - cast carapaces) were 133 mg g- ’ and 70.45 mg g-’ Ca2+, respectively. The relationship was most clearly illustrated by plotting the differences between the concentration factors against water hardness levels (Fig. 2 ). From the lowest hardness level up to 35 mg 1-l CaCO,, mean levels of calcium in cast carapaces increased with increasing water hardness levels but there were no significant correlations above this level or with respect to magnesium (Table 6). There were only very small differences in absolute magnesium content between intermoult and cast carapaces ( ~2.5 mg g-’ ) and no significant correlation of differences with hardness levels. Differences between intermoult and cast carapaces when expressed as percentage differences were significant, however, being 35% above 30 mg I- ’ CaC03 hardness and only 6.8% below 30 mg 1-l CaCO,. DISCUSSION
Survival While high hardness has been reported to affect growth in previous studies, it has not previously been shown to affect survival. This could be related to the relative sizes of prawns and the methods used in the different studies. In the tank-based study of Cripps and Nakamura ( 1979 ), the prawns used were very much larger than those used in this study. Other trials have been based on pond studies in which survival is not so easily determined. Growth Growth was maximal at < 53 mg 1-l CaC03 hardness, did not change significantly at lower hardness levels but declined at higher levels. There was no clear indication of how growth rate changes were induced by the different hardness levels. They seemed unlikely to be due to changes in metabolic effort since Greenaway ( 1985) pointed out that the energetic costs of calcium uptake are only a small part of the energy budget for crustaceans from both marine and freshwater habitats. We observed a reduction in moulting rate at low hardness levels but paradoxically this was accompanied by increased growth, presumably mediated through an increased increment at moult. In contrast, Cripps and Nakamura ( 1979) found a reduction in moulting rate and poorer growth at high hardness levels but their prawns were much larger than those in this study. It is
342
J.H. BROWN ET AL.
possible that the reduction in moult frequency we observed could have arisen if an increased time was required for completing the exoskeletal mineralization processes under low hardness conditions. Even so, calcium uptake is rapid in Mucrobruchium ( 7.5 pmoles g- ’ h- ‘, Fieber and Lutz, 1982 ) , about four times more rapid than in the crayfish Austropotamobius pallipes (2 pmoles g-l hh’, Greenaway, 1974). Unfortunately the rates of mineral deposition were not measured in the experiments reported here. Mineralization The absence of pronounced differences in mineral content in cast carapaces collected from consecutive moults suggested that prawns responded quickly to external hardness levels and supported the contention that by the time the intermoult carapaces were collected (after live moults) the prawns had achieved a “steady state” with respect to internal mineral reserves. Observed differences between minerals were affected therefore by levels in the water rather than internal reserves, diets in all treatments being the same. Prawns grown in low-hardness water deposited more calcium in the carapace than those at higher hardnesses. The lower levels of calcium in cast carapaces, particularly those from prawns reared in low-hardness water may indicate that the prawns were attempting to conserve calcium by withdrawing the mineral from the old carapace prior to moulting. (It is also possible that some calcium was lost passively from the cast carapaces after moulting at rates related to the different calcium concentrations in the water, although the frequent collection of cast shells should have minimized this loss). These results suggest that calcium is actively conserved when external levels are low and they are in line with those of Chaisemartin, ( 1962a,b, 1967, cited in Greenaway, 1985 ) who found that total body calcium was higher in crayfish from soft waters [0.135 mmol Ca 1-l (5.42 mg 1-l CaC03) ] than from hard waters [2.35 mmol Ca 1-l (234.53 mg 1-l CaCO,) 1. The need for increased deposition and subsequent withdrawal of calcium by prawns kept in low-hardness waters may have been responsible for the observed prolongation of the intermoult period. Dal1 ( 195 8 ) found that carapace calcium content in Metupenaeus sp. varied from 10% wet weight of cuticle when the time from the last moult was short, and up to 30% wet weight of cuticle in animals where moulting had been delayed. He suggested that this indicated that the amount of calcification depended to a large extent on the length of time between moults. The positive correlation of calcium content with size in the cast carapaces indicates either that calcium withdrawal becomes less critical with increasing age or that it becomes more difficult for the animal to withdraw and store calcium from the cast exoskeleton with increasing size. Compared to small prawns, large prawns may rely to a greater extent on uptake of calcium ions from the external media than on storage. In this respect Fieber and Lutz
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
343
( 1982 ) demonstrated that adult Mucrobrachium ( 16-96 g live weight ) needed to obtain at least 43% of carapace calcium from exogenous sources. These results may be compared and contrasted with those obtained from studies of mineralization in Penaeus mono&n (Wickins, 1984a,b). Carapace calcium levels in intermoult prawns were similar, around 150 to 220 mg g- ‘, despite large differences in the calcium concentrations of seawater (400 mg l- ’ ) and fresh water, (0.7 to 100 mg l- ’ in the present study). A major difference was that the cast penaeid carapaces contained a higher concentration of calcium than did those dissected from intermoult prawns while in Macrobruchium the opposite was true, reflecting perhaps the greater need and ability of juvenile A4acrubruchium to mobilize and conserve calcium. The high levels of magnesium normally occurring in seawater (ca 1100 mg l- I ) also contrasted with those in fresh water (0.4-25 mg 1-l ). Magnesium content was lower in intermoult Macrobruchium carapaces (2.5-5.0 mg 1-l ) than in the penaeids (8. l-l 1.6 mg l- ’ ) and differed little in cast carapaces. In contrast, the penaeid cast carapaces contained significantly higher concentrations of magnesium ( 10.4- 19.9 mg l- ’ ) than intermoult carapaces. Such a loss of magnesium in cast shells at moulting would be beneficial in marine forms since magnesium continuously enters the body and must be excreted (Hagerman, 1980). Stern et al. ( 1987) found that M. rosenbergii always kept blood calcium concentration hyperionic to the external medium and magnesium hypoionic to the external medium. In high-hardness waters it could be that the excretion of magnesium is an energy drain. However, high calcium levels alone inhibited growth in studies where only calcium salts were added to the water (Cripps and Nakamura, 1979; Vasquez et al., 1989). It seems very likely that if calcium is always kept hyperionic to the external medium in M. rosenbergii, then higher external calcium levels in the presence of elevated levels of bicarbonate may result in excessive deposition of excess calcium in the carapace, either passively or as a control method (Nash and Brown, in prep. ). The results of Wickins ( 1984a) and Bartlett and Enkerlin ( 1983 ) suggest that high external calcium levels are only likely to cause excessive mineralization if alkalinity is also high. The results of the present study demonstrate the sensitivity of juvenile Mucrobruchium to elevated water hardness levels which are shown to affect both growth and survival. The data support the experiences of New and Singholka ( 1985 ) who recommended levels between 40 and 100 mg 1-i CaCO, for successful farming operation. Low calcium content has often been considered a constraint to fresh-water crustacean distribution, but in the case of M. rosenbergii, the effects of high-hardness water appear to be more of a handicap, particularly when carbonate alkalinity is also elevated. Our results highlight the need to determine the extent to which crustacean mineralization and growth are affected by the balance between levels of hardness (calcium and
344
J.H. BROWN ET AL.
magnesium) and alkalinity (carbonate and bicarbonate) in the water. Further clarification of the extent to which size influences the degree to which prawns are affected by the water hardness and alkalinity levels is also needed if reliable recommendations are to be made concerning the suitability of a particular water source for prawn-farm development. ACKNOWLEDGEMENTS
The prawn research at the Institute of Aquaculture is funded by the Overseas Development Administration of the Foreign and Commonwealth Office, UK (Natural Resources Grants R3874 and R422 1) and additional financial help has been received from the European Community under the Science and Technology for Development Programme (Grants TSD A 200UK (H) and TS2 0099 M (H) ) . This support for the work is gratefully acknowledged. It is a pleasure also to acknowledge the contribution to this study made by a former student of the Institute of Aquaculture, Mr. Weidong Zhou, MSc.
REFERENCES Bartlett, P. and Enkerlin, E., 1983. Some results of rearing giant prawn, Macrobrachium rosenbergii in asbestos asphalt ponds in hard water and on a low protein diet. Aquaculture, 30: 353-356. Cripps, M.C. and Nakamura, R.M., 1979. Inhibition of growth of Macrobrachium rosenbergii by calcium carbonate water hardness. Proc. World Maricult. Sot., 10: 575-580. Dall, W., 1958. Observations on the biology of the greentail prawn, Metapenaeus mastersii (Haswell) (Crustacea: Decapoda: Penaeidae). Aust. J. Mar. Freshwater Res., 9: 11 l-l 34. Duncan, D.B., 1955. Multiple range and multiple Ftests. Biometrics, 11: l-42. Fieber, L.A. and Lutz, P.L., 1982. Calcium requirements for moulting in Macrobrachium rosenbergii. J. World Maricult. Sot., 13: 21-27. Greenaway, P., 1974. Calcium balance at the postmoult stage of the freshwater crayfish Austropotamobiuspallipes (Lereboullet). J. Exp. Biol., 61: 35-45.. Greenaway, P., 1985. Calcium balance and moulting in the Crustacea. Biol. Rev., 60: 425-454. Hagerman, L., 1980. The regulation of calcium and magnesium in the urine during the moulting cycle of Crangon vulgaris (Fabr.). J. Exp. Mar. Biol. Ecol., 46: 5 l-58. HMSO, 1969. Fish toxicity tests. Report of the Technical Committee. Ministry of Housing and Local Government, London, 15 pp. Howlader, M.S. and Turjoman, A.M.A., 1984. Some results of rearing giant prawn, Macrobrachium rosenbergii, in oasis water of Al-Hassa, Saudi Arabia. J. World Maricult. Sot., 15: 92-94. Johnson, D.S., 1967. Some factors influencing the distribution of freshwater prawns in Malaya. Proc. Symp. Crustacea Mar. Biol. Assoc. India, 1: 418-433. Nash, G. and Brown, J.H., in prep. Pathology associated with culture in hard water in Macrobrachium rosenbergii de Man. New, M.B. and Singholka, S., 1985. Freshwater Prawn Farming. A Manual for the Culture of Macrobrachium rosenbergii. FAO Fish. Tech. Pap., (225) Rev. 1, 118 pp.
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Perdue, J.A. and Nakamura, R., 1976. The effect of salinity on the growth of Macrobrachium rosenbergii. Proc. World Maricult. Sot., 7: 647-654. Singh, T., 1980. The isosmotic concept in relation to the aquaculture of the giant prawn, Macrobrachium rosenbergii. Aquaculture, 20: 25 l-256. Smith, T.J., Sandifer, P.A. and Trimble, W.C., 1976. Pond culture of the Malaysian prawn Macrobrachium rosenbergii (de Man) in South Carolina. Proc. World Maricult. Sot.. 7: 625645. SPSSx Inc., 1983. SPSSx User’s Guide. McGraw-Hill, New York, NY, 806 pp. Stern, S.. Borut, A. and Cohen. D., 1987. Osmotic and ionic regulation of the prawn, Macrobrachium rosenbergii (de Man) adapted to varying salinities and ion concentrations. Comp. Biochem. Physiol., 86A: 373-379. Vasquez, O.E., Rouse, D.B. and Rogers, W.A., 1989. Growth response ofMucrobrachium rosenbergii to different levels of hardness. J. World Aquacult. Sot., 20: 90-92. Wickins, J.F., 1972. Experiments on the culture of the spot prawn, Pandalus plafyceros Brandt, and the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Fish Invest., G.B., Ser. II, 27: l-23. Wickins, J.F., 1982. Opportunities for farming crustaceans in Western temperate regions. In: J.F. Muir and R.J. Roberts (Editors), Recent Advances in Aquaculture. Croom Helm, London, pp. 87- 177. Wickins, J.F., 1984a. The effect of hypercapnic sea water on growth and mineralization in penaeid prawns. Aquaculture, 41: 37-48. Wickins, J.F., 1984b. The effect of reduced pH on carapace calcium, strontium and magnesium levels in rapidly growing prawns (Penaeus monodon Fabricius). Aquaculture, 41: 49-60.
Aquaculture, 95 ( 1991) 329-345 Elsevier Science Publishers B.V., Amsterdam
The effect of water hardness on growth and carapace mineralization of juvenile freshwater prawns, kfacrobrachium rosenbergii de Man Janet H. Browna, John F. Wickinsb and Marlie H. MacLearP alnstitute ofAquaculture, University ofstirling, Stirling FK9 4LA, UK bMinistry ofAgriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory. Conwy, Gwynedd LL32 8UB, UK (Accepted
19 November
1990)
ABSTRACT Brown, J.H., Wickins, J.F. and MacLean, M.H., 1991. The effect of water hardness on growth and carapace mineralization of juvenile freshwater prawns, Macrobrachium rosenbergii de Man. Aquaculture, 95: 329-345. Growth and carapace mineralization of Macrobrachium rosenbergii juveniles, (0.01-0.5 g) were investigated under conditions of different water hardness from 9 to 326 mg 1-l CaC03. Growth was maximal at < 53 mg 1-l CaCO,, did not change significantly at lower hardness levels but declined at higher levels. Survival was impaired at the highest hardness levels tested. Greater deposition of calcium in the carapace occurred in low-hardness water and the lower levels of calcium found in cast carapaces from these prawns indicated greater withdrawal of calcium from the carapace before moulting.
INTRODUCTION
The giant freshwater prawn, Macrobrachium rosenbergii de Man, is widely cultured throughout the tropics, subtropics and some warm temperate zones of the world, far beyond its natural geographic range. In nature, larval life is completed in brackish water after which the juveniles migrate into fresh water where they grow to maturity. The adult females return to estuarine waters to spawn. Despite widespread commercial interest in the species little information is available on its tolerance to water hardness and on its ability to regulate shell calcium levels. Juveniles and adults moult frequently, between every 5 to 40 days, respectively, and require exogenous sources of cations, particularly calcium for successful shell mineralization. Recorded satisfactory hardness levels for culture range widely; Wickins ( 1982) suggested from 65 to 200 mg 1-l CaC03, New and Singholka ( 1985 ) recommended levels above 40 and below 0044-848619
l/$03.50
0 199 1 -
Elsevier Science Publishers
B.V.
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J.H. BROWN ET AL.
150 (preferably less than 100 mg l- ’ CaCO, ). Cripps and Nakamura ( 1979) reported greater inhibition of growth at hardness levels above 65 mg 1-l CaC03, while Vasquez et al. ( 1989) showed that while growth was better at 112 than at 20 mg l- ’ CaC03, growth was reduced at 225 and 450 mg l- ’ CaC03. Bartlett and Enkerlin ( 1983) and Howlader and Turjoman ( 1984) reported on growth of M. rosenbergii in high-hardness waters from mainly groundwater sources. While the latter study found that growth was adversely affected by the high hardness levels (688-987 mg l- ’ CaCO,), Bartlett and Enkerlin ( 1983 ) found that growth was not adversely affected by hardness levels between 940 and 1060 mg l- ’ CaC03, but their water had relatively low alkalinity (58-86 mg 1-l when expressed as CaCO,). In Malaysia where natural hardness of culture water can be extremely low ( < 30 mg l- ’ CaCO, ), lime is routinely added to increase hardness up to 50 mg 1-l CaCO, and in French Guyana lime is added to culture ponds to increase hardness levels up to 20 mg l- ’ CaCO,. Johnson ( 1967), however, reported that two species of Macrobrachium, M. geron and M. trompii, were found in Malaysian waters with no detectable calcium content and that M. rosenbergii was also found in very low-hardness waters. In experimental studies Fieber and Lutz ( 1982 ) showed that M. rosenbergii could take up calcium for shell mineralization from an external medium containing 0.45 mmol Ca2+ l- ’ (44.9 mg I- ’ CaC03), and that some shell hardening occurred in solutions when calcium was as low as 0.09 mmol Ca2+ l- ’ (8.98 mg l- ’ CaCO,). Studies on ionic regulation reported by Stem et al. ( 1987), indicated that M. rosenbergii exhibits a high degree of osmoregulatory ability. However, growth and development were found to be reduced in waters of “exotic” salinities and ion composition, in which ionic ratios differ markedly from those found in seawater. There have, in addition, been studies of salinity effects which indirectly involve hardness (in that dilute seawater will contain appreciable amounts of calcium and magnesium) but here interpretation could cause confusion in that Singh (1980), reviewing work by Wickins (1972) and Perdue and Nakamura ( 1976), all of whom found optimal growth in either fresh water or water of low salinity (2%0), suggested that this is related to the ability of the animal to take up more water at ecdysis than animals maintained in more concentrated media. However, Smith et al. ( 1976) recorded “excellent” growth (increases in some male prawns from 0.7 g to 100.6 g in 5 months) in water with a reported average salinity of 7.2%0. In order to evaluate the suitability of waters for M. rosenbergii culture, greater understanding of the species’ tolerance to hardness levels is required. This paper reports on survival, growth, and aspects of shell mineralization in juvenile M. rosenbergii exposed to water hardness levels between 9 and 326 mg l- 1 CaC03.
WATER
H.ARDNESS
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MATERIALS AND METHODS
Juvenile M. rosenbergii were selected from laboratory stock populations and reared in four experiments at a variety of water-hardness levels. Care was taken to select animals within the narrowest possible size range (Table 1). At the start and end of each trial prawns were blotted dry and weighed to the nearest 0.1 mg on a Mettler AC 100 balance. Carapace lengths (posterior margin of orbit to middorsal margin of carapace) were measured to the nearest 0.0 1 mm using a vernier calliper. The prawns were housed in individual containers, twelve containers per treatment. These were cylindrical plots ( 11-cm diameter) with sides of 1-mm plastic mesh and 2.5mm mesh bottoms, each pot having an air supply and lid. The pots were placed in plastic aquaria containing the test solutions, three pots per 5 litres of test solution. Aquaria were held in 36 x 56 cm tanks in a large recirculation system so that temperature was maintained at 28 ‘C + 1 ‘C. The water in the aquaria was completely changed every 3.5 days. The prawns were fed on a pelleted diet produced in the Institute of Aquaculture (40% protein based on squid, shrimp and fish meal) and were inspected twice daily, so uneaten food could be removed and fresh food added. Moults were recorded and cast carapaces collected, rinsed in distilled water and measured. The carapaces were dried at 60’ C for 24 h, weighed and stored in plastic vials for later analysis. Experiments lasted for a period of time sufficient to allow each individual prawn to moult five times. After live moults the animal was killed halfway through its moult cycle, determined as being half the period of the previous moult cycle, thus ensuring that the final carapaces were sampled at the same stage of the moult cycle. Each prawn was killed by rapid chilling at 4°C for half an hour. It was then blotted dry and weighed. The carapace was dissected off (any attached tissue was carefully removed with fine forceps) and then dried and stored for mineral analysis. Hardnesssolutions The total hardness of water is defined as the sum of the concentrations of calcium and magnesium, both expressed in terms of an equivalent concentraTABLE 1 Size of prawns and average water hardness used in the experiments Experiment
Initial prawn size (g)
I
0.25-0.35 0.1 o-0.20 0.20-0.50 0.10-0.20
2 3 4
“Two tests were run at this hardness.
Average hardness 36
9
13
34 19 30
54 52 58
68 143 316 71” 172 320 74 143 326
J.H. BROWN ET AL.
332
tion of calcium carbonate in mg l- I. When calcium and magnesium concentrations were determined separately the total hardness was calculated from the equation: Total hardness CaC03 mg l- ’ = 2.497 (Ca*+ mg l- ’ ) + 4.118 (Mg*+ mg l- ’ ) While for greater clarity it is probably better to measure the separate levels of the ions, the concept of total hardness is widely used and, more importantly, more easily measured in a field situation, being readily determined by simple calorimetric techniques. For the purpose of these experiments, ion concentrations were measured separately as outlined below. Solutions were made up with deionised water preheated to 28 “C. The recipe used (HMSO, 1969) is shown in Table 2. Analysis of calcium and magnesium in carapaces The stored carapaces were transferred to test tubes held in a heated block where they were digested in 5 ml concentrated Aristar-grade nitric acid. The solutions were made up to 25 ml with distilled water. Analyses were carried out using a Perkin Elmer Atomic Absorption Spectrophotometer (Model 2280) with calcium standard solutions of 4.0 mg 1-l and 2.0 mg l- ’ and magnesium standard solutions of 0.3 and 0.1 mg 1-l. Lanthanum chloride at 5% v/v was added to standards and samples to prevent ionic interference. Presentation of results Results were analysed using an SPSSx statistical program (SPSSx Inc., 1983 ). Analysis of variance (ANOVA) was used to determine whether there was any significant difference between treatments and the differences found were investigated using the multiple-range test, Duncan’s procedure (Duncan, 1955 ) . In the statistical analyses, differences were regarded as signilicant if the level of probability was less than 0.05. In a study of this sort where growth rate will be affected by treatment, some of the effects seen, for example, carapace thickening, may be due to the size TABLE 2 The composition
of stock solutions used to make up the culture media
Stock solution 1
320 g CaC12-6H,O 29 g NaCl 9 g NaNO,
Stock solution 2
15 1g M&O,-7H20 79 g Na,S04
Stock solution 3
27.5 g NaHCO,
Each solution was made up in 1 1 of deionised water. One ml of solutions 1 and 2 with 10 ml of solution 3 in 10 1of deionised water gave a culture medium of 20 mg I-’ CaCOs hardness.
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
333
of the prawn rather than to water hardness directly. Measured factors were therefore each plotted against treatment (water hardness) and against final live weight of the prawns. Slopes differing from 0 at the 95% level of confidence were taken to indicate that a significant relationship existed between the plotted parameters. Where both size and hardness were correlated with a factor, the factor giving the highest correlation was judged to be the more acceptable relationship. Where appropriate, results obtained at similar hardness levels but from separate experiments were combined in order to distinguish the effect of size and hardness on carapace mineral content more clearly. The definitions used in presentation of the results are: growth rate =
final weight (mg) -initial weight (mg) no. of days in experiment for each individual prawn (day 1 of experiment to final sampling)
length-specific dry weight of carapace =
concentration
distribution
dry weight of carapace (mg ) carapace length (mm)
factor =concentration of cations in carapace (mg g- I ) x I ooo concentration of cations in media (mg l- ’ ) coefficient =
concentration factor of magnesium concentration factor of calcium
If the distribution coefficient is less than 1 it indicates selective exclusion of magnesium from the carapace. RESULTS
Survival
Survival was low at the highest hardnesses under test ( > 3 16 mg I- ’ ), average survival times being less than 7 days in the first two experiments, while in the third experiment all but one prawn survived two moults and average survival time was 17.6 days. At the second highest hardness levels ( 143-l 72 mg I-’ CaCO,) survival was more variable (Table 3). In experiment 1, only 8% of the prawns survived to be sampled and the average survival time of the other prawns was just 10 days; however, in experiments 2 and 3 survival at these levels was 8 3% and 9 1.7% respectively. Survival at low hardnesses (experiment 4) ranged from 75% at 9- 13 mg I- ’ CaC03 to 9 1.7% at 3 1 mg I- ’ CaCO,. At intermediate hardness levels, between 30 and 75 mg 1-l CaC03, survival was also good, ranging from 67 to 1000/o_mean 92.5%.
CaCOj)
0.61 0.15
9.63 1.02
Mean final live weight (g) s.d.
Mean final carapacelength s.d.
(mm)
6.2 2.7
83
36
54
7.57 0.95
0.34 0.09
0.1 3.0
91.7
7.43 0.85
0.29 0.07
-0.9 1.4
66.6
68
I
rosenbwgiijuveniles
Mean growth rate (mgday-‘) s.d.
Survival (%)
Hardness (mgg-’
Experiment no.
Survival and growth ofA
TABLE 3
-
-
-
8
-
-
-
0
143 316
7.39 0.63
0.34 0.08
5.8 1.7
100
53
6.83 0.78
0.28 0.10
4.6 2. I
100
71
in waters of different hardness
6.65 0.70
0.26 0.08
3.9 1.3
100
71
2
6.43 0.76
0.23 0.08
2.5 2.2
83
172
-
-
-
0
320 34
9.40 0.60
0.62 0.14
5.4 2.0
100
58
8.95 1.19
0.56 0.24
2.2 1.9
91.7
8.25 1.02
0.41 0.14
2.7 1.3
100
75
3
144
7.90 0.81
0.36 0.11
0.5 0.8
91.7
-
-
-
0
326
9
6.96 0.65
0.30 0.07
4.3 1.7
75
13
6.62 0.74
0.27 0.07
3.8 1.7
75
4
19
6.74 0.55
0.27 0.07
3.1 1.1
83
31
6.74 0.33
0.27 0.03
3.4 0.8
91.7
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
335
Growth rate Where prawns survived high hardness levels (75-172 mg 1-l CaC03), growth was reduced to less than 2.7 mg day-’ (Table 3 ). The growth rates at the lowest hardnesses in experiments 1,2 and 3 were significantly greater than the growth at higher hardness levels whilst in experiment 3, there was a significant difference between each level. No sign&ant differences in growth rate ( 3.4 to 4.5 mg day- ’ ) occurred at low hardnesses over the range 9-3 1 mg 1-l CaCO,. The maximum growth rate achieved (6.2 mg day- ’ at 36 mg l- ’ CaC03 in experiment 1) compares well with the best growth achieved in nutrition trials under similar husbandry conditions but with circulating water (M.R.P. Briggs, pers. comm., 1988). The mean sizes of prawns from experiments 1 to 3 at termination became smaller with increasing hardness while there was an unexpected decrease in the length of the intermoult period also as hardness increased (see below). Moultingfrequency The intermoult period was shorter in prawns exposed to hardness levels above 5 3 mg l- ’ CaCO, even though these animals were growing more slowly than those held at lower levels. In experiment 4 (low hardness) there were no significant differences in intermoult period in the different treatments but all averaged about 7 days between moults compared to 6 days or less for prawns of the same size at hardness levels above 53 mg 1-l CaC03. The regression equation for average values of intermoult period over the full range of hardness (experiments 1,2 and 4) is: intermoult period (days) =7.35-0.011
xhardness
(mg 1-l CaCO,)
{corr = - 0.772, d.f. = 6, P< 0.05} Shell mineralization Intermoult carapaces. Carapaces dissected from intermoult prawns (0.1-0.9 g live weight) at the end of the experiments showed an increase in length-specific dry weight with size. The rates of increase at mean hardness levels of 3 1,55 and 7 1 mg l- ’ CaC03, (experiments 1,2 and 3 ) did not differ significantly and the relationship for the combined data is described by the regression equation: length-specific dry weight = 0.000 15 + 0.00 197 x final weight {corr= +0.891, d.f.= 154, P
all sizes of prawns together the calcium content of intermoult
J.H. BROWN ET AL.
336
carapaces increased from 145 to 223 mg g- ’ as hardness decreased. The regression was: [Ca*+] (mgg-‘)=203.45-0.358xhardness {corr= -0.645,
(mgl-’
CaC03)
d.f.= 13, P-=0.01}
Carapace magnesium content was much lower, 2.5 to 5.2 mg g-r, and seemed independent of hardness down to about 30 mg l- I, below which level it declined. The concentration factors of both calcium and magnesium in intermoult carapaces increased as the water hardness decreased; calcium from 2.7 to nearly 70 ( x 1000) magnesium from 0.3 to 5 ( x 1000) (Fig. la,b). Final prawn size affected the relation between hardness and the mineral content of intermoult carapaces (Table 4). At a mean hardness of 34 mg 1-l CaCO,, calcium content was high (203 mg g- ’ ) but declined as size increased; at 5 5 mg l- ’ CaCO, calcium content was intermediate (mean 182 mg g- ’ ) but no correlation with size was apparent while at 7 1 mg l- ‘, calcium content was lower ( 168 mg g- ’ ) but increased with size. Magnesium content only increased with size at low hardness (Table 4). No correlation with size occurred at intermediate and high hardness levels. The actual concentration of one cation relative to another in the exoskele-
Fig. la. The relationship moult prawn carapaces.
between water hardness and calcium concentration
factor in inter-
WATER
HARDNESS
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MINERALIZATION
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337
PRAWNS
O& i”“1”“i”“1”“,““,““,“~~,~“~,‘~~‘,~’~’,’~~7”’.,.-‘,“..,‘...,.~..,~.~.,
0
10
20
30
40
50
60
70
80
Water hardness
Fig. 1b. The relationship
between water hardness
90
100
110
120
130
140
150
160
170
180
(mg ItaCO,)
and magnesium
concentration
factor in inter-
moult prawn carapaces. TABLE 4 Calcium and magnesium content in relation to final prawn size at three averaged hardness levels (C.L. = carapace length in mm, n.s. = not significant at 95% level) Calcium
Magnesium
34 mg I-’ CaCOS
34 mg 1-l CaCO,
[Ca”] =284.19-9.73 CL. (corr=-0.514,d.f.=28,P<0.005)
[Mg’+] =0.28+0.47 CL. {corr=0.606, d.f.=28,Pt0.001)
55 mg 1-l CaCO,
55 mg 1-l CaCOx
[Ca’+]=214.99-4.19C.L. /corr=-0.152,d.f.=30,P>O.l}n.s.
[Mg’+] =4.97-0.03 {corr=-0.034,d.f.=30,
71 mg I-’ CaCO,
71 mg I-’ CaCO,
[Ca’*]=111.81+7.63C.L. jcorr=0.361, d.f.=42, P
[Mg*+]=6.13-0.25 C.L. {corr=-0.241,d.f.=41.P>O.lj
C.L. P>O.l} n.s.
ns.
ton depends not only on the relative cation concentrations in the environment but also on the ability of the prawn to discriminate between them during mineralization; this ability can be expressed as a distribution coefficient (defined in Methods). The low values of the distribution coeffkients (0.02-0.25 ) (Table 5 ) indicate that the prawns were discriminating strongly against mag-
5.19 0.44
0. I 0 0.01
Distribution coefficient s.d.
213.79 17.20
Magnesium (mgkr’) s.d.
Calcium (mgg-‘) s.d.
0.10 0.03
4.65 1.38
190.98 35.46
0.12 0.03
4.19 1.83
170.88 39.16
68
54
Hardness (mg I-’ CaCO,)
36
1
Experiment no.
-
-
-
-
-
-
143 316
0.18 0.03
4.92 I .04
192.35 35.46
53
Mean calcium and magnesium content of carapaces from intermoult
TABLE 5
0.19 0.03
5.23 0.79
167.44 14.01
71
0.14 0.04
4.64 0.87
157.29 25.82
71
2
0.25 0.31
5.03 0.84
128.49 41.89
172
-
-
-
320
0.10 0.02
4.45 0.58
171.67 16.92
34
0.10 0.02
4.43 1.08
162.66 14.49
58
0.07 0.02
3.23 0.69
0.07 0.01
3.53 0.59
181.18 13.40
144
hardness
175.03 12.57
75
3
M. rosenbergir juveniles exposed to waters ofdifferent
-
-
-
326
0.07 0.03
2.64 0.58
182.34 38.28
9
0.07 0.04
2.77 0.40
184.64 55.60
13
4
0.04 0.01
3.06 I .26
222.43 34.89
19
0.03 0.0 1
3.31 1.61
222.43 11.49
31
WATER
HARDNESS
AND
GROWTH
AND
CARAPACE
MINERALIZATION
OF FRESHWATER
PRAWNS
339
nesium with respect to calcium. A positive correlation of the coefficient with hardness (PC 0.02) showed that greater discrimination was occurring at low than at high hardness levels. The distribution coefficient only differed significantly at lower hardnesses -that at 9 mg 1-l CaCO, was significantly lower than at 19 mg l- ’ CaCO, and 3 1 mg l- ’ CaC03, and that at 13 mg l- ’ CaCO, was significantly lower than at 3 1 mg 1-l CaCO,. In other words, there was far greater discrimination in favour of calcium against magnesium at the lowest hardness levels while above 3 1 mg 1-l CaCO, the effect was less pronounced. Cast carapaces. Whilst calcium content of intermoult loosely correlated with size,
carapaces was only
[Caz+] = 109.15+ 10.45 C.L. {corr=0.26,
d.f.= 107, P
the correlation in cast carapaces from first moults was more obvious (experiments 1,2 and 3): [Ca’+] = - 159.0+49.0 {corr=0.88,
C.L.
d.f.=96,P<0.001)
Fig. 2. Differences in calcium relation to water hardness.
concentration
factor between
intermoult
and cast carapaces
in
3.52 1.18
0.07 0.02
Distribution coefficient s.d.
186.77 22.60
Magnesium s’,y,g g- ’ )
Calcium
0.07 0.03
3.18 1.39
185.53 19.40
54
0.08 0.04
31.3 1.42
194.96 35.79
68
36
Hardness (mgl-‘CaC03)
Experiment
I
0.06 0.02
0.68 2.61
175.09 25.34
-
-
-
316
0.21 0.07
0.95 2.75
92.50 24.08
53
0.22 0.08
2.98 1.08
86.29 27.91
71
2
0.17 0.06
3.62 1.59
0.16 0.08
3.22 1.09
100.87 27.78
172
_ -
_
-
320
_ -
_
-
34
_ -
_
-
58
exposed to waters of different
98.92 23.43
71
cast from M. rosenbergii juveniles
143
content of carapaces
no.
Mean calcium and magnesium
TABLE 6
_ -
_
-
75
3
hardness
_ -
_
-
144
_ -
_
-
326
0.17 0.08
2.52 1.21
65.25 13.17
9
0.15 0.05
2.78 1.19
66.94 11.48
13
4
0.10 0.03
2.73 1.08
71.23 11.53
19
0.08 0.02
2.89 1.18
75.85 18.84
31
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
341
This suggested that calcium withdrawal from the carapaces became less pronounced as the animals increased in size. In a comparison of experiments 2 and 3 (of prawns of directly comparable sizes) cast carapaces contained lower calcium concentrations than those dissected from intermoult animals. Difference in calcium content was far greater at hardness levels below 30 mg 1-l CaCO, (average 65%) than at higher levels where average differences were 42%. Mean values (intermoult - cast carapaces) were 133 mg g- ’ and 70.45 mg g-’ Ca2+, respectively. The relationship was most clearly illustrated by plotting the differences between the concentration factors against water hardness levels (Fig. 2 ). From the lowest hardness level up to 35 mg 1-l CaCO,, mean levels of calcium in cast carapaces increased with increasing water hardness levels but there were no significant correlations above this level or with respect to magnesium (Table 6). There were only very small differences in absolute magnesium content between intermoult and cast carapaces ( ~2.5 mg g-’ ) and no significant correlation of differences with hardness levels. Differences between intermoult and cast carapaces when expressed as percentage differences were significant, however, being 35% above 30 mg I- ’ CaC03 hardness and only 6.8% below 30 mg 1-l CaCO,. DISCUSSION
Survival While high hardness has been reported to affect growth in previous studies, it has not previously been shown to affect survival. This could be related to the relative sizes of prawns and the methods used in the different studies. In the tank-based study of Cripps and Nakamura ( 1979 ), the prawns used were very much larger than those used in this study. Other trials have been based on pond studies in which survival is not so easily determined. Growth Growth was maximal at < 53 mg 1-l CaC03 hardness, did not change significantly at lower hardness levels but declined at higher levels. There was no clear indication of how growth rate changes were induced by the different hardness levels. They seemed unlikely to be due to changes in metabolic effort since Greenaway ( 1985) pointed out that the energetic costs of calcium uptake are only a small part of the energy budget for crustaceans from both marine and freshwater habitats. We observed a reduction in moulting rate at low hardness levels but paradoxically this was accompanied by increased growth, presumably mediated through an increased increment at moult. In contrast, Cripps and Nakamura ( 1979) found a reduction in moulting rate and poorer growth at high hardness levels but their prawns were much larger than those in this study. It is
342
J.H. BROWN ET AL.
possible that the reduction in moult frequency we observed could have arisen if an increased time was required for completing the exoskeletal mineralization processes under low hardness conditions. Even so, calcium uptake is rapid in Mucrobruchium ( 7.5 pmoles g- ’ h- ‘, Fieber and Lutz, 1982 ) , about four times more rapid than in the crayfish Austropotamobius pallipes (2 pmoles g-l hh’, Greenaway, 1974). Unfortunately the rates of mineral deposition were not measured in the experiments reported here. Mineralization The absence of pronounced differences in mineral content in cast carapaces collected from consecutive moults suggested that prawns responded quickly to external hardness levels and supported the contention that by the time the intermoult carapaces were collected (after live moults) the prawns had achieved a “steady state” with respect to internal mineral reserves. Observed differences between minerals were affected therefore by levels in the water rather than internal reserves, diets in all treatments being the same. Prawns grown in low-hardness water deposited more calcium in the carapace than those at higher hardnesses. The lower levels of calcium in cast carapaces, particularly those from prawns reared in low-hardness water may indicate that the prawns were attempting to conserve calcium by withdrawing the mineral from the old carapace prior to moulting. (It is also possible that some calcium was lost passively from the cast carapaces after moulting at rates related to the different calcium concentrations in the water, although the frequent collection of cast shells should have minimized this loss). These results suggest that calcium is actively conserved when external levels are low and they are in line with those of Chaisemartin, ( 1962a,b, 1967, cited in Greenaway, 1985 ) who found that total body calcium was higher in crayfish from soft waters [0.135 mmol Ca 1-l (5.42 mg 1-l CaC03) ] than from hard waters [2.35 mmol Ca 1-l (234.53 mg 1-l CaCO,) 1. The need for increased deposition and subsequent withdrawal of calcium by prawns kept in low-hardness waters may have been responsible for the observed prolongation of the intermoult period. Dal1 ( 195 8 ) found that carapace calcium content in Metupenaeus sp. varied from 10% wet weight of cuticle when the time from the last moult was short, and up to 30% wet weight of cuticle in animals where moulting had been delayed. He suggested that this indicated that the amount of calcification depended to a large extent on the length of time between moults. The positive correlation of calcium content with size in the cast carapaces indicates either that calcium withdrawal becomes less critical with increasing age or that it becomes more difficult for the animal to withdraw and store calcium from the cast exoskeleton with increasing size. Compared to small prawns, large prawns may rely to a greater extent on uptake of calcium ions from the external media than on storage. In this respect Fieber and Lutz
WATER HARDNESS AND GROWTH AND CARAPACE MINERALIZATION OF FRESHWATER PRAWNS
343
( 1982 ) demonstrated that adult Mucrobrachium ( 16-96 g live weight ) needed to obtain at least 43% of carapace calcium from exogenous sources. These results may be compared and contrasted with those obtained from studies of mineralization in Penaeus mono&n (Wickins, 1984a,b). Carapace calcium levels in intermoult prawns were similar, around 150 to 220 mg g- ‘, despite large differences in the calcium concentrations of seawater (400 mg l- ’ ) and fresh water, (0.7 to 100 mg l- ’ in the present study). A major difference was that the cast penaeid carapaces contained a higher concentration of calcium than did those dissected from intermoult prawns while in Macrobruchium the opposite was true, reflecting perhaps the greater need and ability of juvenile A4acrubruchium to mobilize and conserve calcium. The high levels of magnesium normally occurring in seawater (ca 1100 mg l- I ) also contrasted with those in fresh water (0.4-25 mg 1-l ). Magnesium content was lower in intermoult Macrobruchium carapaces (2.5-5.0 mg 1-l ) than in the penaeids (8. l-l 1.6 mg l- ’ ) and differed little in cast carapaces. In contrast, the penaeid cast carapaces contained significantly higher concentrations of magnesium ( 10.4- 19.9 mg l- ’ ) than intermoult carapaces. Such a loss of magnesium in cast shells at moulting would be beneficial in marine forms since magnesium continuously enters the body and must be excreted (Hagerman, 1980). Stern et al. ( 1987) found that M. rosenbergii always kept blood calcium concentration hyperionic to the external medium and magnesium hypoionic to the external medium. In high-hardness waters it could be that the excretion of magnesium is an energy drain. However, high calcium levels alone inhibited growth in studies where only calcium salts were added to the water (Cripps and Nakamura, 1979; Vasquez et al., 1989). It seems very likely that if calcium is always kept hyperionic to the external medium in M. rosenbergii, then higher external calcium levels in the presence of elevated levels of bicarbonate may result in excessive deposition of excess calcium in the carapace, either passively or as a control method (Nash and Brown, in prep. ). The results of Wickins ( 1984a) and Bartlett and Enkerlin ( 1983 ) suggest that high external calcium levels are only likely to cause excessive mineralization if alkalinity is also high. The results of the present study demonstrate the sensitivity of juvenile Mucrobruchium to elevated water hardness levels which are shown to affect both growth and survival. The data support the experiences of New and Singholka ( 1985 ) who recommended levels between 40 and 100 mg 1-i CaCO, for successful farming operation. Low calcium content has often been considered a constraint to fresh-water crustacean distribution, but in the case of M. rosenbergii, the effects of high-hardness water appear to be more of a handicap, particularly when carbonate alkalinity is also elevated. Our results highlight the need to determine the extent to which crustacean mineralization and growth are affected by the balance between levels of hardness (calcium and
344
J.H. BROWN ET AL.
magnesium) and alkalinity (carbonate and bicarbonate) in the water. Further clarification of the extent to which size influences the degree to which prawns are affected by the water hardness and alkalinity levels is also needed if reliable recommendations are to be made concerning the suitability of a particular water source for prawn-farm development. ACKNOWLEDGEMENTS
The prawn research at the Institute of Aquaculture is funded by the Overseas Development Administration of the Foreign and Commonwealth Office, UK (Natural Resources Grants R3874 and R422 1) and additional financial help has been received from the European Community under the Science and Technology for Development Programme (Grants TSD A 200UK (H) and TS2 0099 M (H) ) . This support for the work is gratefully acknowledged. It is a pleasure also to acknowledge the contribution to this study made by a former student of the Institute of Aquaculture, Mr. Weidong Zhou, MSc.
REFERENCES Bartlett, P. and Enkerlin, E., 1983. Some results of rearing giant prawn, Macrobrachium rosenbergii in asbestos asphalt ponds in hard water and on a low protein diet. Aquaculture, 30: 353-356. Cripps, M.C. and Nakamura, R.M., 1979. Inhibition of growth of Macrobrachium rosenbergii by calcium carbonate water hardness. Proc. World Maricult. Sot., 10: 575-580. Dall, W., 1958. Observations on the biology of the greentail prawn, Metapenaeus mastersii (Haswell) (Crustacea: Decapoda: Penaeidae). Aust. J. Mar. Freshwater Res., 9: 11 l-l 34. Duncan, D.B., 1955. Multiple range and multiple Ftests. Biometrics, 11: l-42. Fieber, L.A. and Lutz, P.L., 1982. Calcium requirements for moulting in Macrobrachium rosenbergii. J. World Maricult. Sot., 13: 21-27. Greenaway, P., 1974. Calcium balance at the postmoult stage of the freshwater crayfish Austropotamobiuspallipes (Lereboullet). J. Exp. Biol., 61: 35-45.. Greenaway, P., 1985. Calcium balance and moulting in the Crustacea. Biol. Rev., 60: 425-454. Hagerman, L., 1980. The regulation of calcium and magnesium in the urine during the moulting cycle of Crangon vulgaris (Fabr.). J. Exp. Mar. Biol. Ecol., 46: 5 l-58. HMSO, 1969. Fish toxicity tests. Report of the Technical Committee. Ministry of Housing and Local Government, London, 15 pp. Howlader, M.S. and Turjoman, A.M.A., 1984. Some results of rearing giant prawn, Macrobrachium rosenbergii, in oasis water of Al-Hassa, Saudi Arabia. J. World Maricult. Sot., 15: 92-94. Johnson, D.S., 1967. Some factors influencing the distribution of freshwater prawns in Malaya. Proc. Symp. Crustacea Mar. Biol. Assoc. India, 1: 418-433. Nash, G. and Brown, J.H., in prep. Pathology associated with culture in hard water in Macrobrachium rosenbergii de Man. New, M.B. and Singholka, S., 1985. Freshwater Prawn Farming. A Manual for the Culture of Macrobrachium rosenbergii. FAO Fish. Tech. Pap., (225) Rev. 1, 118 pp.
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HARDNESS
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MINERALIZATION
OF FRESHWATER
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Perdue, J.A. and Nakamura, R., 1976. The effect of salinity on the growth of Macrobrachium rosenbergii. Proc. World Maricult. Sot., 7: 647-654. Singh, T., 1980. The isosmotic concept in relation to the aquaculture of the giant prawn, Macrobrachium rosenbergii. Aquaculture, 20: 25 l-256. Smith, T.J., Sandifer, P.A. and Trimble, W.C., 1976. Pond culture of the Malaysian prawn Macrobrachium rosenbergii (de Man) in South Carolina. Proc. World Maricult. Sot.. 7: 625645. SPSSx Inc., 1983. SPSSx User’s Guide. McGraw-Hill, New York, NY, 806 pp. Stern, S.. Borut, A. and Cohen. D., 1987. Osmotic and ionic regulation of the prawn, Macrobrachium rosenbergii (de Man) adapted to varying salinities and ion concentrations. Comp. Biochem. Physiol., 86A: 373-379. Vasquez, O.E., Rouse, D.B. and Rogers, W.A., 1989. Growth response ofMucrobrachium rosenbergii to different levels of hardness. J. World Aquacult. Sot., 20: 90-92. Wickins, J.F., 1972. Experiments on the culture of the spot prawn, Pandalus plafyceros Brandt, and the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Fish Invest., G.B., Ser. II, 27: l-23. Wickins, J.F., 1982. Opportunities for farming crustaceans in Western temperate regions. In: J.F. Muir and R.J. Roberts (Editors), Recent Advances in Aquaculture. Croom Helm, London, pp. 87- 177. Wickins, J.F., 1984a. The effect of hypercapnic sea water on growth and mineralization in penaeid prawns. Aquaculture, 41: 37-48. Wickins, J.F., 1984b. The effect of reduced pH on carapace calcium, strontium and magnesium levels in rapidly growing prawns (Penaeus monodon Fabricius). Aquaculture, 41: 49-60.