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Effect of stocking densities on trace metal concentration in three tissues of the brown shrimp Penaeus californiensis
ELSEVIER
Aquaculture
156 (1997) 21-34
Effect of stocking densities on trace metal concentration in three tissues of the brown shrimp Penaeus californiensis Lia Mhdez
*, Baudilio Acosta, Elena Palacios, Francisco Magallh
Centro de Inoestigaciones Biol&icas de1 Noroeste, Apartado Postal 128, L.a Paz, B.C.S. 23000, Mhico Accepted
22 March 1997
Abstract The influence of stocking density in tidal ponds on the concentration of trace metals and macroelements in three shrimp tissues was evaluated. Brown shrimps Penaeus culifonziensis were stocked in three 1 ha earthen-bottom tidal ponds at densities of 4, 8, and 10 shrimps per m*. After 4 months, the animals were collected, weighed, and dissected for metal determination (copper, cadmium, lead, zinc, iron, sodium, potassium, magnesium, and calcium) in the hepatopancreas, muscle, and abdominal cuticle. Although no significant differences were obtained for physicochemical variables in the water, an increase of nitrates and orthophosphates was observed in the pond with the highest density occurring at the end of the experiment. The average body weight of shrimp decreased as the stocking densities increased. The concentrations of Cu and Cd in the hepatopancreas were higher with increasing stocking density. Pb in the hepatopancreas was significantly higher in the animals stocked at the intermediate density. Several correlations between trace metals and macroelements were obtained. Different stocking densities could, over the long term, affect the water chemical speciation and the availability of the elements for the shrimp. 0 1997 Elsevier Science B.V. Keywords: Macroelements; Pollution; Cations; Copper; Cadmium; sis; Shrimp culture
* Corresponding
author. Tel.: +52
Lead; Zinc; Calcium;
112 53633 ext 143; fax: +52
Peruzeus ca[ifonien_
112 54715; e-mail: [email protected]
0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)00078-l
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1. Introduction Although the shrimp industry is widely spread, shrimp nutritional and growout requirements, particularly those concerning minerals and their kinetics, are not completely understood. Most studies on metals in shrimp culture have been made to evaluate pollution problems (Rosas and Ramirez, 1993; Gutierrez-Galindo et al., 1994, PaezOsuna and Ruiz-Femandez, 1995). It has been reported that reducing the levels of some trace elements may increase shrimp production (Castille and Adisson, 1981; Burton and Fisher, 1990; Yuan et al., 1993). Several studies have evaluated the sublethal levels of single and combined heavy metals (Chen and Liu, 1987; Liu and Chen, 1987; Gajbhiye
Table 1 Average, standard deviation and standard error of the physicochemical tidal ponds from October 1993 to February 1994 Parameter
Temperature Average s.d. se
variables
in seawater,
Stocking density 4/m’
S/m2
10/m’
24.28 3.75 1.33
24.19 3.66 1.29
24.21 4.07 1.44
8.38 0.10 0.04
8.34 0.11 0.04
8.41 0.16 0.06
36.63 1.51 0.53
36.50 1.41 0.50
36.75 1.28 0.45
5.20 0.58 0.21
5.83 0.54 0.19
5.93 0.92 0.33
0.10 0.14 0.05
0.16 0.21 0.07
0.20 0.27 0.09
0.504 0.323 0.114
0.560 0.519 0.205
0.770 0.930 0.364
0.031 0.014 0.005
0.03 1 0.016 0.006
0.035 0.014 0.005
0.13 0.15 0.08
0.45 0.36 0.11
0.33 0.41 0.15
(“C)
PH Average s.d. s.e. Salinity (ppt) Average s.d. s.e. Dissolved oxygen (mg/l) Average s.d. s.e. Total ammonia (mg N/l) Average s.d. s.e. Nitrates (mg N/l) Average s.d. s.e. Nitrites (mg N/l) Average s.d. s.e. Orthophosphates (mg N/l) Average s.d. s.e
recorded
in the
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and Hirota, 1990; Rosas and Ramirez, 1993). Trace element concentration in different tissues has been measured in relation to bioaccumulation and the quality of shrimp products (Darmono and Denton, 1990; Davis et al., 1992) and different shrimp populations (Whyte and Boutillier, 1991). Other work considers the nutritional requirements of minerals (Civera and Guillaume, 1989; Davis et al., 1992). Evaluation of the effect of size (Rainbow, 1989; Boyd and Teichert-Coddington, 1995) age (Paez-Osuna and Ruiz-Fernandez, 1995) and reproduction on total body metal concentration (Liu and Chen, 1987) has been done. It is clear that stocking density can affect size, development,
Table 2 Values of the elements Element
in seawater
and sediments
recorded
in the tidal uonds in October
Sediment (mg/kg)
Seawater (mg/l)
Average Range s.d.
14.53 14.26-14.84 0.29
0.0027 0.0021-0.0037 0.0008
Average Range s.d.
2X.55 28.26-28.96 0.36
0.0036 0.0024-0.0056 0.0017
Average Range s.d.
30.78 30.56-3 0.3 1
0.0024 0.0013-0.0033 0.0010
CU
Zn
Fe
Cd Average Range s.d. Pb Average Range s.d. Na Average Range s.d. K Average Range s.d. Mg Average Range s.d. Ca Average Range s.d. N.A. not available.
1.14
6.82 6.68-6.97 0.14 4.67 4.56-4.9 0.2 1
0.0006 0.0000-0.001 0.0005
I
0.001 o.o003-0.00 0.0007
N.A. N.A N.A
10748 10587-10856 142
N.A. N.A. N.A.
415 396-425 16
N.A. N.A. N.A.
1567 1538-1586 25
N.A. N.A. N.A.
400 392-410 9
17
1994
24
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156 (1997) 21-34
and mortality in shrimp (Ray and Chien, 1992; Pinto and Rouse, 1996). However, no work has been done to evaluate the effect of stocking density on metal levels, even though in other organisms, such as mussels (Amiard et al., 1991) and algae (Mallick and Rai, 1993; Voloshko and Gavrilova, 19931, a relationship between population density and body metal concentration has been found. In this study, we report the effect of different shrimp stocking densities on the accumulation of five trace metals and four macroelements in three different tissues.
2. Materials and methods The tidal ponds are located in Ensenada de La Paz, Mexico (between 24”06’ and 24”ll’ north latitude and 1 lO”19’ and 1 lO”25’ west longitude). The experiment was done in three 1 ha, earthen-bottom tidal ponds. All ponds had a maximum depth of 150 cm, similar sediment, aeration using paddles, and each pond was a semi-open system (3 mm
A
P
h~p~tOp~“C~~~*
mY*cla
exOsksls,On
Fig. 1. Mean tissue concentration and standard error of Cu and Cd at three different stocking densities. Solid bars = 4/m*, dashed bars = 8/m*, double-dashed bars = 10/m’. Effect of density = D, effect of tissue = T, interaction of density and tissue effect = DT. Bars not sharing a common superscript are significantly different (P < 0.05).
L. M&dez et al./Aquaculture
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25
mesh gate) with seawater flowing freely in and out with the tides. Temperature, oxygen levels (Oximeter YSI 5 IB), and salinity (Aquafauna A-190) were monitored every 4 h. Every two weeks, surface seawater (20 to 30 cm depth) was sampled near the gate of each pond during the incoming tide. The water was collected in polyethylene bottles, previously rinsed with seawater. The samples were filtered through a GF/C membrane and the filtrate analyzed for ammonia (Solorzano, 1969) nitrates (cadmium reduction), nitrites (diazotization), and phosphates (Koroleff, 1983) and the pH recorded (ORION 720). Seawater and sediment samples were analyzed according to Van Loon (1995) for elements, using chloroform as a solvent and sodium diethyldithiocarbamate as a chelant, and analyzed by atomic absorption. The sediment samples were analyzed using aqua regia (3: 1 mixture of hydrochloric and nitric acid) and hydrofluoric acid in teflon bombs.
hDP.tOpmor~as
mulds
aiallelsfm
Fig. 2. Mean tissue concentration and standard error of Zn, Pb, and Fe at three different stocking densities. double-dashed bars = IO/m”. Effect of density = D, effect of Solid bars = 4/m’, dashed bars = 8/m’, tissue = T, interaction of density and tissue ef feet = DT. Bars not sharing a common superscript are significantly different (I’ < 0.05).
26
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Four, eight, and ten animals per m* of P. californiensis (2.5 to 3.0 g> were stocked in each pond in October 1993. All the animals were given food pellets manufactured by Rangen (Idaho, USA) at 10% of estimated biomass per day and were raised in similar conditions until February 1994, when all shrimps were collected with a net and immediately taken to the laboratory. There, 20 animals of each pond were weighed, washed with deionized water, and dissected to remove the hepatopancreas, abdominal muscle, and cuticle from the first abdominal segment. Shrimps in premolt or that had recently moulted were not used. All tissues were dried to constant weight in an oven at 70°C and digested in acid-washed test tubes with a mixture of concentrated nitric and perchloric acid and slowly boiled to dryness on a hotplate. Dried samples were analyzed by atomic absorption (BUCK, Scientific model 200). An air-acetylene flame was used for Cu, Zn, Fe, Ca, Mg, Na, and K, and a graphite furnace (BUCK, Scientific model GFl) for Cd and Pb. Precision was checked against the standard reference material of the National Research Council of Canada (DORM-l for dogfish), and was within the range of certified values. All data were processed and analyzed by STATISTICA Software. Two-way analyses of variance were used to assess significant effects of stocking densities and tissues on
B
rD
p
T
p
bi
DT p
I_ b
D c
Fig. 3. Mean tissue concentration and standard error of K, Na, Ca, and Mg at three different stocking densities. Solid bars = 4/m’, dashed bars = 8/m’, double-dashed bars = 10,’ m*. Effect of density = D, effect of tissue = T, interaction of density and tissue effect = DT. Bars not sharing a common superscript are significantly different (P < 0.05). The values for Ca in the abdominal cuticle are plotted against the right y-axis, which is expressed in thousands
L. M&dez et al./Aquaculture
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metal concentration. Duncan’s multiple-range test was used to test individual differences between tissues and ponds. Regression analyses were made for the tissues’ metal concentration and body size and weight, and for the concentration of metal pairs within the tissues.
3. Results Table 1 depicts the general physicochemical variables in the three ponds. There were no significant changes, although the nitrates and orthophosphates had a tendency to increase with the stocking densities. The initial values for the elements measured in sediment and seawater are shown in Table 2.
Table 3 Correlation matrix of metal pairs in hepatopancreas significance ( * ) was accepted at P < 0.05 4/m’ CU Zn Fe Cd Pb Na K
CU
Fe
Zn
of shrimp cultured at three different densities. The level of
Cd
Pb
Na
K
Ca
Mg
-0.8’
0.7
-0.7
*
Mg Ca
- 0.9 -0.7
8/m’
CU
CU Zn Fe Cd Pb Na K
- 0.9 *
0.7 0.9 Fe
Zn
Pb
0.7 * 0.8” 0.6 *
0.7 * 0.7 * 0.9’
MJ2
Cd
K
Na
0.7
*
0.7 Mg
Ca
Mg
Ca
* 0.7 *
Ca IO/m’
Cu
Zn
Fe
Cd
Pb
Na
K
CU Zn
Fe Cd Pb Na K Mg Ca
0.8 *
0.7
*
0.7
*
L. Mindez et al. / Aquaculture 156 (1997) 21-34
28
As stocking density increased, there was a significant decrease in the length (13.95 f 0.23; 13.36 + 0.09; 12.35 + 0.2; F = 17.56; P < 0.0001) and weight (18.60 f 0.8; 15.53 f 0.3; 12.09 + 0.6; F = 28.98; P < 0.0001) of the shrimp in the 4, 8, and IO/m’ pond. The hepatopancreas contained the higher concentrations of Cu (F = 445.74; P < 0.001; Fig. lA), Zn (F = 113.69; P < 0.001; Fig. 2A), Pb (F = 42.61; P < 0.001; Fig. 2B), Fe (F = 58.05; P < 0.001; Fig. 2C), and Na (F = 89.24; P < 0.001; Fig. 3A). Concentrations of Cd (F = 117.25; P < 0.001; Fig. lB), Mg (F = 469.06; P < 0.001; Fig. 3D), and Ca (F = 465.41; P < 0.001; Fig. 3C) were higher in the abdominal cuticle. Significant differences were found in the metal concentrations in shrimp from each pond. In the hepatopancreas, the concentration of Cu increased significantly (F = 18.71; P < 0.001; Fig. 1A) with the stocking density. A similar tendency was observed for Cd (F = 3.84; P < 0.05; Fig. lB), Zn (F = 3.39; P < 0.05; Fig. 2A), and
Table 4 Correlation significance
matrix of metal pairs in muscle ( * ) was accepted at P < 0.05
4/m’
Cu
CU Zn Fe Cd Pb Na K Mg Ca 8/m’
- 0.9
Zn
Fe
of shrimp Cd
cultured Pb
at three different Na
densities.
The level of
K
Mg
Ca
K
Mg
Ca
Mg
Ca
*
0.8 * 0.7
0.7 0.7 0.8’ 0.8 *
Cu
Zn
-0.7 - 0.6
- 0.7 - 0.7
0.8 * 0.7
0.9 *
0.7 Fe
Cd
Pb
Na
cu Ztl Fe Cd Pb Na K
- 0.6
*
0.7 *
Mg Ca IO/m’
0.7 * Cu
Zn
Cu Zn Fe Cd Pb Na K Mg Ca
Cd
Fe
Pb
Na
-0.6”
0.7 * 0.9 * 0.8
*
0.6
*
0.7 *
K
L. Me’ndez et al./Ayuaculture Table 5 Correlation significance 4/m’ cu Zn Fe Cd Pb Na K
matrix of metal pairs in exoskeleton (* ) was accepted at P < 0.05 cu
Zn
Fe
of shrimp cultured Cd
S/m’
Pb
21,
at three different Na
densities.
The level of
K
Mg
Ca
K
Mg
Ca
Mg
Ca
0.7 - 0.6
0.9
*
Mg Ca
156 (19971 21-34
0.7 0.9 * cu
0.8* Zn
Fe
0.6 Cd
Pb
Na
cu Zn Fe Cd Pb Na K
0.9 * - 0.7 *
0.7
Mg Ca IO/m’
0.6 cu
Zn
Fe
Cd
Pb
*
* Na
K
cu ZU Fe Cd Pb Na K Mg Ca
0.6 * 0.7 * 0.9 I
0.7
*
0.8
*
Na (F = 24.92; P < 0.001; Fig. 3A). The hepatopancreas of the animals in the medium density pond had the highest Pb concentrations (F = 23.94; P < 0.001; Fig. 2B). In the muscle, concentrations of Zn (F = 3.39; P < 0.001; Fig. 2A) and Na (F = 24.92; P < 0.001;Fig. 3A) were higher in the medium density pond. A similar tendency, although not significant, was observed for Mg and Ca. The concentration of Na in the cuticle increased with density in a significant way (F = 24.92; P < 0.001; Fig. 3A). The concentrations of Cd (F = 3.84; P < 0.05:Fig. 1B) and Fe were higher in the lowest density pond, although the differences in Fe concentration was not significant. For each pond, significant correlation coefficients for several individual concentrations of metal pairs for the hepatopancreas (Table 3), muscle (Table 4), and abdominal cuticle (Table 5) are also shown. Most correlations are between macroelements and trace metals; however, not all the tissues had the same correlations. The pattern was also affected by the stocking densities.
30
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4. Discussion To our knowledge, the present study is the first to address the effect of stocking density on metal accumulation in different tissues of the shrimp. An inverse relationship between stocking density and final size is in agreement with previous reports (Rainbow, 1989; Ray and Chien, 1992; D’Silva and Maughan, 1995; Pinto and Rouse, 1996). This could be an effect of competition for food, either intra- or interspecific, since the ponds were a semi-open system. However, the stocking densities were of the extensive type and food was given several times a day ad libitum. The excretion product of shrimp, ammonia, is known to have toxic effects (Jeney et al., 1992; Tudor et al., 1994) and cause mortality in shrimp larvae (Chen et al., 1991). At a higher stocking density, an increase in the ammonia levels that could slow the shrimp growth might be expected (Cai and Summerdelt, 1992), in particular in P. califomiensis, since it has burrowing habits and ammonia is found in close association with the sediment (Boyd, 1990). Accordingly, Ray and Chien (1992) concluded a higher stocking density in P. monodon can decrease shrimp size and increase mortality caused by deterioration of the pond sediment, which can be inferred by the water physicochemical variables. In our conditions, the effect of density on the size of the shrimp was seen even though the physicochemical variables were not significantly different. The lack of difference between these variables could be an effect of the ponds having a semi-open system and a fairly ‘new’ sediment, hence reducing the eutrophication and deterioration of water and sediment (Ray and Chien, 1992). In this study, differences in the metal concentration of tissues varied with the stocking density. Slight variations between one or several physicochemical variables could affect the activity of free ions because of complexing of the ions with specific ligands (Burton and Stratham, 1991; Rainbow, 1995). Although salinity, reported to affect metal uptake (Wright, 1995; Rainbow, 1995), was similar among the three ponds, the nitrate and orthophosphate levels were different. These differences could be related to the feeding regime. It is not likely that differences in metal concentrations are an effect of competition for ingested mineral contents of the pellets or sediments (Gutierrez-Galindo et al., 1994), because of the reduced competition at low densities and the stability of metal contents of sediments over a long period of time (Cauwet, 1987). In general, the hepatopancreas had the highest concentrations of Cu, Pb, Zn, and Fe, which is in agreement with other results in crustacea (White and Rainbow, 1986; Lindahl and Moksnes, 1993; Marcovecchio, 1994). The increased metal concentration in the hepatopancreas, compared to the other two tissues, probably has to do with its function as a storage and detoxification site (Vogt and Quinitio, 1991; Vogt and Quinitio, 1994) and to its high metallothioneins contents (Lindahl and Moksnes, 1993). Similarly, high concentrations of Ca and Mg were expected in the cuticle because of its composition (Piedad-Pascual, 1989) and its function as an absorption and excretion surface (Castille and Adisson, 1981; Boitel and Truchot, 1989; Whyte and Boutillier, 1991). According to our results, Cu and Cd concentrations are lower in the hepatopancreas when the stocking density is lower. Although Cu is necessary for the respiratory pigment hemocyanin (Ridout et al., 1989; Rainbow, 1991), it is considered lethal for gas
L. M&de? et al./Aquaculture
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exchange in high concentrations (Boitel and Truchot, 1989). In consequence, some chelation (EDTA) and absorption methods have been used to reduce Cu and Cd level in water, even though EDTA in high concentrations can be toxic (Castille and Adisson, 198 1; Burton and Fisher, 1990). For this reason, lower Cu and Cd concentration in hepatopancreas at reduced stocking densities represents an important result of aquaculture interest. However, a decrease in Cu concentration could also be explained as an increase in shrimp size when the stocking density is reduced, since an elevated growth rate could ‘dilute’ the metal content (Rainbow, 1989; White and Rainbow, 1986; Rainbow, 1991). The high Cd levels in the abdominal cuticle of the lowest density pond might be caused by surface adsorption, because the area-volume ratio is the smallest for the animals in this pond (Rainbow, 1991) and Cd body concentration is not regulated (Rainbow, 1991; Blust et al., 1992). Lower Ca levels in the abdominal cuticle of the 4/m’ density pond, could be related to a more advanced molting stage of the shrimp, for it has been reported that previous to a molt, a large amount of Ca is removed from the exoskeleton (Mercado-Allen, 199 1). Differences in molting frequency are expected as an effect of the stocking density, as can be inferred by the growth data in the three ponds. Molting could also affect other element levels in exoskeleton, although Weeks et al. (1992) reported that Cu and Zn levels in amphipods do not present variations in relation to the molt cycle. On the other hand, the concentration of some metals in the water can have an effect on the frequency of the molting cycle (Fingerman et al., 1996) and physicochemical variables, such as pH and carbon dioxide can also affect the Ca and Mg levels in the exoskeleton, as reported by Wickins (1984a,b). Indeed, element concentration in exoskeletons can present variations in relation to shrimp size and in caparace versus abdominal cuticle (Wickins, 1984a). However, since no precise record of the molting cycle was made, we are unable to conclude if differences of Ca and other macroelements observed in the cuticle are related to the molting cycle and growth, or to another cause related to stocking density. Little information is available on the relationship between trace metals and macroelements. Pelgrom et al. (1995) reported a decrease of Na in fish exposed to Cu and a decrease of Ca when fish were exposed to Cd. In our experimental design with shrimp, positive correlations were found for Cu-Na in hepatopancreas of the medium density pond, in muscle for the lowest density pond, and in the cuticle for the highest density pond. Davis et al. ( 1992) reported a deletion of Mg in the diet of P. ~~nnamei produced a significant depression of K in caparace, and a deletion of K lowered levels of Mg in the hepatopancreas. In accordance, we obtained a positive correlation between Mg and K in the abdominal cuticle and the hepatopancreas but only in the medium density pond. Serra et al. (1995) reported a decrease in the levels of Fe in several tissues during Cd exposure in clams. We found a similar negative correlation in muscle for Cd-Fe in the highest density pond. The relationship observed between some of the above metal pairs may be a result of a competition or an additional effect on the metal binders and transporters at a cellular level (Wright, 199.5) or an interaction between the metabolism or excretion of some elements in the hepatopancreas. In conclusion, the present work confirms previous studies on increased growth at lower stocking densities, possibly related to differences in physicochemical variables. In
32
L. M&dez et al./Ayuaculture
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addition, at higher stocking densities, hepatopancreas levels of Cu, Cd, and Zn are increased, a result with possible aquaculture implications that remains to be fully elucidated, but could indicate a toxic accumulation.
Acknowledgements This research was supported by CONACyT under grant 1895PN, and by CIBNOR proyect IAC-5. Elena Palacios is a fellow of CONACyT. The authors are grateful to Dr. A.M. Ibarra and Dr. I.S. Racotta for comments on the manuscript, to G. Portillo for providing technical data on pond culture, and to Ellis Glazier for English corrections.
References Amiard, J.C., Metayer, C., Baud, J.P., Ribeyre, F., 1991. Influence of some ecological factors on metal bioaccumulation (Cd, Cu, Pb, Zn) in juvenile carpet shells (Ruditapes phikppinarum) during their nursing. Rev. Sci. Eua. 4, 441. Blust, R., Kockelbergh, E., Baillieul, M., 1992. Effect of salinity on the uptake of cadmium by the brine shrimp Artemia franciscana. Mar. Ecol. Prog. Ser. 84, 245. Boitel, F., Truchot, J.P., 1989. Effects of sublethal and lethal copper levels on hemolymph acid-base balance and ion concentrations in the shore crab Cur&us maenas kept in undiluted sea water. Mar. Biol. 103, 495. Boyd, C.E., 1990. Water quality in ponds for aquaculture. Alabama Agricultural Experiment Station, Auburn University, Alabama, p. 25. Boyd, C.E., Teichert-Coddington, D., 1995. Dry matter, ash and elemental composition of pond-cultured Penaeus cwmamei and P. stylirostris. J. World Aquacult. Sot. 26, 88. Burton, D.T.. Fisher, D.J., 1990. Acute toxicity of cadmium, copper, zinc, ammonia, 3,3’-dichlorobenzidine, 2,6-dichloro+nitroaniline, methylene chloride, and 2,4,6-trichlorophenol to juvenile grass shrimp and killfish. Bull. Environ. Contam. Toxicol. 44, 776. Burton, J.D., Stratham, P.J., 1991. Trace metals in seawater. In: R.W. Fumes, P.S. Rainbow (Eds.), Heavy metals in the Marine Environment. CRC Press, Boca Raton, Florida, pp. 5-26. Cai, Y., Summerdelt, C., 1992. Effects of temperature and size on oxygen consumption and ammonia excretion by walleye. Aquaculture 104, 127. Castille, F.L., Adisson, AL., 198 1. The effects of EDTA (ethylendinitrotetraacetic acid) on the survival and development of shrimp nauplii (Penaeus stylirostris Stimpson) and the interactions of EDTA with the toxicities of cadmium, calcium, and phenol. J. World Maricul. Sot. 12, 292. Cauwet, G., 1987. Influence of sedimentalogical features on the distribution of trace metals in marine sediments. Mar. Chem. 22, 221. Chen, J.C., Liu, P.C., 1987. Accumulation of heavy metals in the nauplii of Artemia salina. J. World Aquacult. Sot. 18, 84. Chen, J.C., Liu, P.C., Nan, F.H., 199 I. Acute toxicity of ammonia to larval Merapenaeus ensis. Asian Fish. Sci. 4, 41. Civera, R., Guillaume, J., 1989. Effects of sodium phytate on growth and tissue mineralization of Penaeus japonicus and Penaues mnnamei juveniles. Aquaculture 77, 145. Darmono, D., Denton, G.R.W., 1990. Heavy metal concentrations in the banana prawn, Penaeus merguiensis, and leader prawn, P. monodon, in the townsville region of Australia. Bull. Environ. Contam. Toxicol. 44, 479. Davis, A.D., Addison, A.L., Gatlin, D.M., 1992. Mineral requirements of Penneus mmnamei: A preliminary examination of the dietary essentiality for thirteen minerals. J. World Aquacult. Sot. 23, 8. D’Silva, A.M., Maughan, O.E., 1995. Effects of density and water quality on red tilapia (Oreochromis mossamhicus X 0. urolepis hornorum) in pulsed-flow culture systems. J. Appl. Aquacult. 5, 69
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Fingerman, M., Devi, M., Reddy, P.S., Katyayani, R., 1996. Impact of heavy metal exposure on the nervous system and endocrine-mediated processes in crustaceans. Zoo]. Studies 35, I. Gajbhiye, S.N., Hirota, R., 1990. Toxicity of heavy metals to brine shrimp artemia. J. Indian Fish. Ass. 20, 43. Gutierrez-Galindo, E., Flares-Mutioz, G., Ortega-Lara, V., Villaescusa-Celaya, J.A., 1994. Heavy metals in coastal sediments of the Baja California (Mexico)-California (USA) border zone. Ciencias Marinas 20, 105. Jeney, G., Nemcsok, J., Jeney, Zs., Olah, J., 1992. Acute effect of sublethal ammonia concentrations on common carp (Cyprinus carpio L.). II. Effect of ammonia on blood plasma transaminases (GOT, GPT). GIDH enzyme activity, and ATP value. Aquaculture 104, 149. Koroleff, F., 1983. Determination of phosphorus. In: K. Grasshoff, M. Ehrhardt, K. Kremling (Eds.), Methods of seawater analysis. Verlag Chemie GmbH, Weinheim, pp. 125- 139. Lindahl, U.. Moksnes, P., 1993. Metallothionein as a bioindicator of heavy metal stress in Colombian fish and shrimp: A study of dose-dependent induction. Fish. Dev. Ser. Nat]. Swed. Board Fish. Goeteborg, Sweden Swedmar. 73, 36. Liu, P.C., Chen, J.C., 1987. Effects of heavy metals on the hatching rates of brine shrimp Artemia salina cysts. J. World Aquacult. Sot. 18, 78. Mallick, N., Rai, L.C., 1993. Influence of culture density, pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena dolioium and Chlorellu uulgaris. World J. Microbial. Biothechnol. 9, 196. Marcovecchio, J.E., 1994. Trace metals residues in tissues of two crustaceans species from the Bahia Blanca Estuary. Argentina. Environ. Monit. Assess, 29, 65. Mercado-Allen, R., 1991. Changes in the blood chemistry of the American lobster, Homrus americanus, H. Mime Edwards, 1837, over the molt cycle. J. Shellfish Res. 10, 147. Paez-Osuna, F., Ruiz-Fernandez, C., 1995. Trace metals in the Mexican shrimp Penaeus cannamei from estuarine and marine environments. Environm. Pollut. 87, 243. Pelgrom, S.M.G.J., Lock, R.A.C., Balm, P.H.M., Wendelaar-Bonga. S.E., 1995. Effects of combined waterborne Cd and Cu exposures on ionic composition and plasma cortisol in tilapia. Oreochromis mossambicus. Comp. Biochem. Physiol. 11 IC, 227. Piedad-Pascual, F., 1989. Mineral requirements of Penaeids. In: AQUACOP IFREMER (Eds.), Advances in tropical Aquaculture, Tahiti, Feb. 20-March 4, 1989. Actes de Colloque 9, pp. 309-3 18. Pinto, G.F., Rouse, D.B., 1996. Growth and survival of the Australian red claw crayfish Cherax quadricarinatus at three densities in earthen ponds. J. World Aquacult. Sot. 27, 187. Rainbow, P.S., 1989. Copper, cadmium and zinc concentrations in oceanic amphipod and euphausiid crustaceans, as a source of heavy metals to pelagic seabirds. Mar. Biol. 103, 513. Rainbow, P.S., 1995. Physiology, physicochemistry and metal uptake - a crustacean perspective. Mar. Pollut. Bull. 31, 55. Rainbow, P.S., 1991. Heavy metals in the marine environment, R.W. Fumes, P.S. Rainbow (Eds.), CRC Press, Boca Raton, Florida, pp. 67-80. Ray, W.M., Chien, Y.H., 1992. Effects of stocking density and aged sediment on tiger prawn Penaeus monodon, nursery system. Aquaculture 104, 23 1. Ridout, P.S., Rainbow, P.S., Roe, J.H.S., Jones, H.R., 1989. Concentrations of V, Cr, Mn, Fe, Ni, Co, Cu. Zn, As, Cd in mesopelagic crustaceans from the North East Atlantic Ocean. Mar. Biol. 100, 465. Rosas, C., Ramirez, P., 1993. Effect of chromium and cadmium on the thermal tolerance of the prawn Macrobrachium rosenbergii exposed to hard and soft water. Bull. Environ. Contam. Toxicol. 5 I, 568. Serra, R., Carpene, E., Marcantonio, AC., Isani, G., 1995. Cd accumulation and Cd-binding proteins in the bivalve Scapharca inaequit,akis. Comp. Biochem. Physiol. 11 IC, 165. Solorzano, L., 1969. Determination of ammonia in natural waters by the phenolhypoclorite method. Limnol. Oceanogr. 14, 799. Tudor, M., Katavic, I., Marsic-Lucic, J., 1994. Acute toxicity of ammonia to juvenile sea bass (Dicentmrchus labrax L.) at different aeration levels. Aquaculture 128, 89. Van Loon, J.C., 1995. Selected methods of trace metal analysis: biological and environmental samples. Wiley-Interscience, Toronto, 357 pp. Vogt, G., Quinitio, E.T., 1991. Fate and effects of water-borne heavy metals in Penaeus monodon. In: N. DePauw, J. Joyce, (Eds.1, Spec. Pub]. Eur. Aquacult. Sot. 14, pp. 320-321.
34
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Vogt, G., Quinitio, E.T., 1994. Accumulation and excretion of metal granules in the prawn, Penaeus monodon, exposed to water-borne copper, lead, iron and calcium. Aquat. Toxicol. 28, 223. Voloshko, L.N., Gavrilova, O.V., 1993. Response of Anabaena oariabilis to zinc ion. Hydrobiology 30, 39. Weeks, J.M., Rainbow, P.S., Moore, P.G., 1992. The loss, uptake and tissue distribution of copper and zinc during the molt cycle in an ecological series of talitrid amphipods (crustacea: amphipoda), Hydrobiologia 245, 15. White, S. L, Rainbow, P.S., 1986. A preliminary study of Cu., Cd-, and Zn-binding components in the hepatopancreas of Puluemon elegans (crustacea: decapoda). Comp. Biochem. Physiol. 83C, 11 I. Whyte, J.N.C., Boutillier, J.A., 1991, Concentrations of inorganic elements and fatty acids in geographic populations of the spot prawn Pandalus platyceros. Can. J. Fish. Aq. Sci. 48, 382. Wickins, J.F., 1984a. The effect of hypercapnic sea water on growth and mineralization in penaeid prawns. Aquaculture 41, 37. Wickins, J.F., 1984b. The effect of reduced pH on caparace calcium, strontium and magnesium levels in rapidly growing prawns. Aquaculture 41, 49. Wright, D.A., 1995. Trace metal and major ion interactions in aquatic animals. Mar. Pollut. Bull. 31, 8. Yuan, Y., Gao, Ch., Zhang, D., 1993. Effects of heavy metal ions on larval Penaeus chinensis and comparative study on their elimination methods. Acta Oceanol. Sin. Haiyang Xuebao. 12, 285.
Aquaculture
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Effect of stocking densities on trace metal concentration in three tissues of the brown shrimp Penaeus californiensis Lia Mhdez
*, Baudilio Acosta, Elena Palacios, Francisco Magallh
Centro de Inoestigaciones Biol&icas de1 Noroeste, Apartado Postal 128, L.a Paz, B.C.S. 23000, Mhico Accepted
22 March 1997
Abstract The influence of stocking density in tidal ponds on the concentration of trace metals and macroelements in three shrimp tissues was evaluated. Brown shrimps Penaeus culifonziensis were stocked in three 1 ha earthen-bottom tidal ponds at densities of 4, 8, and 10 shrimps per m*. After 4 months, the animals were collected, weighed, and dissected for metal determination (copper, cadmium, lead, zinc, iron, sodium, potassium, magnesium, and calcium) in the hepatopancreas, muscle, and abdominal cuticle. Although no significant differences were obtained for physicochemical variables in the water, an increase of nitrates and orthophosphates was observed in the pond with the highest density occurring at the end of the experiment. The average body weight of shrimp decreased as the stocking densities increased. The concentrations of Cu and Cd in the hepatopancreas were higher with increasing stocking density. Pb in the hepatopancreas was significantly higher in the animals stocked at the intermediate density. Several correlations between trace metals and macroelements were obtained. Different stocking densities could, over the long term, affect the water chemical speciation and the availability of the elements for the shrimp. 0 1997 Elsevier Science B.V. Keywords: Macroelements; Pollution; Cations; Copper; Cadmium; sis; Shrimp culture
* Corresponding
author. Tel.: +52
Lead; Zinc; Calcium;
112 53633 ext 143; fax: +52
Peruzeus ca[ifonien_
112 54715; e-mail: [email protected]
0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)00078-l
22
L. Mthdez
et al./Aquaculture
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1. Introduction Although the shrimp industry is widely spread, shrimp nutritional and growout requirements, particularly those concerning minerals and their kinetics, are not completely understood. Most studies on metals in shrimp culture have been made to evaluate pollution problems (Rosas and Ramirez, 1993; Gutierrez-Galindo et al., 1994, PaezOsuna and Ruiz-Femandez, 1995). It has been reported that reducing the levels of some trace elements may increase shrimp production (Castille and Adisson, 1981; Burton and Fisher, 1990; Yuan et al., 1993). Several studies have evaluated the sublethal levels of single and combined heavy metals (Chen and Liu, 1987; Liu and Chen, 1987; Gajbhiye
Table 1 Average, standard deviation and standard error of the physicochemical tidal ponds from October 1993 to February 1994 Parameter
Temperature Average s.d. se
variables
in seawater,
Stocking density 4/m’
S/m2
10/m’
24.28 3.75 1.33
24.19 3.66 1.29
24.21 4.07 1.44
8.38 0.10 0.04
8.34 0.11 0.04
8.41 0.16 0.06
36.63 1.51 0.53
36.50 1.41 0.50
36.75 1.28 0.45
5.20 0.58 0.21
5.83 0.54 0.19
5.93 0.92 0.33
0.10 0.14 0.05
0.16 0.21 0.07
0.20 0.27 0.09
0.504 0.323 0.114
0.560 0.519 0.205
0.770 0.930 0.364
0.031 0.014 0.005
0.03 1 0.016 0.006
0.035 0.014 0.005
0.13 0.15 0.08
0.45 0.36 0.11
0.33 0.41 0.15
(“C)
PH Average s.d. s.e. Salinity (ppt) Average s.d. s.e. Dissolved oxygen (mg/l) Average s.d. s.e. Total ammonia (mg N/l) Average s.d. s.e. Nitrates (mg N/l) Average s.d. s.e. Nitrites (mg N/l) Average s.d. s.e. Orthophosphates (mg N/l) Average s.d. s.e
recorded
in the
L. M&dez et al./Aquaculture
156 (1997) 21-34
23
and Hirota, 1990; Rosas and Ramirez, 1993). Trace element concentration in different tissues has been measured in relation to bioaccumulation and the quality of shrimp products (Darmono and Denton, 1990; Davis et al., 1992) and different shrimp populations (Whyte and Boutillier, 1991). Other work considers the nutritional requirements of minerals (Civera and Guillaume, 1989; Davis et al., 1992). Evaluation of the effect of size (Rainbow, 1989; Boyd and Teichert-Coddington, 1995) age (Paez-Osuna and Ruiz-Fernandez, 1995) and reproduction on total body metal concentration (Liu and Chen, 1987) has been done. It is clear that stocking density can affect size, development,
Table 2 Values of the elements Element
in seawater
and sediments
recorded
in the tidal uonds in October
Sediment (mg/kg)
Seawater (mg/l)
Average Range s.d.
14.53 14.26-14.84 0.29
0.0027 0.0021-0.0037 0.0008
Average Range s.d.
2X.55 28.26-28.96 0.36
0.0036 0.0024-0.0056 0.0017
Average Range s.d.
30.78 30.56-3 0.3 1
0.0024 0.0013-0.0033 0.0010
CU
Zn
Fe
Cd Average Range s.d. Pb Average Range s.d. Na Average Range s.d. K Average Range s.d. Mg Average Range s.d. Ca Average Range s.d. N.A. not available.
1.14
6.82 6.68-6.97 0.14 4.67 4.56-4.9 0.2 1
0.0006 0.0000-0.001 0.0005
I
0.001 o.o003-0.00 0.0007
N.A. N.A N.A
10748 10587-10856 142
N.A. N.A. N.A.
415 396-425 16
N.A. N.A. N.A.
1567 1538-1586 25
N.A. N.A. N.A.
400 392-410 9
17
1994
24
L. Mindez et al. / Aquaculture
156 (1997) 21-34
and mortality in shrimp (Ray and Chien, 1992; Pinto and Rouse, 1996). However, no work has been done to evaluate the effect of stocking density on metal levels, even though in other organisms, such as mussels (Amiard et al., 1991) and algae (Mallick and Rai, 1993; Voloshko and Gavrilova, 19931, a relationship between population density and body metal concentration has been found. In this study, we report the effect of different shrimp stocking densities on the accumulation of five trace metals and four macroelements in three different tissues.
2. Materials and methods The tidal ponds are located in Ensenada de La Paz, Mexico (between 24”06’ and 24”ll’ north latitude and 1 lO”19’ and 1 lO”25’ west longitude). The experiment was done in three 1 ha, earthen-bottom tidal ponds. All ponds had a maximum depth of 150 cm, similar sediment, aeration using paddles, and each pond was a semi-open system (3 mm
A
P
h~p~tOp~“C~~~*
mY*cla
exOsksls,On
Fig. 1. Mean tissue concentration and standard error of Cu and Cd at three different stocking densities. Solid bars = 4/m*, dashed bars = 8/m*, double-dashed bars = 10/m’. Effect of density = D, effect of tissue = T, interaction of density and tissue effect = DT. Bars not sharing a common superscript are significantly different (P < 0.05).
L. M&dez et al./Aquaculture
156 (1997) 21-34
25
mesh gate) with seawater flowing freely in and out with the tides. Temperature, oxygen levels (Oximeter YSI 5 IB), and salinity (Aquafauna A-190) were monitored every 4 h. Every two weeks, surface seawater (20 to 30 cm depth) was sampled near the gate of each pond during the incoming tide. The water was collected in polyethylene bottles, previously rinsed with seawater. The samples were filtered through a GF/C membrane and the filtrate analyzed for ammonia (Solorzano, 1969) nitrates (cadmium reduction), nitrites (diazotization), and phosphates (Koroleff, 1983) and the pH recorded (ORION 720). Seawater and sediment samples were analyzed according to Van Loon (1995) for elements, using chloroform as a solvent and sodium diethyldithiocarbamate as a chelant, and analyzed by atomic absorption. The sediment samples were analyzed using aqua regia (3: 1 mixture of hydrochloric and nitric acid) and hydrofluoric acid in teflon bombs.
hDP.tOpmor~as
mulds
aiallelsfm
Fig. 2. Mean tissue concentration and standard error of Zn, Pb, and Fe at three different stocking densities. double-dashed bars = IO/m”. Effect of density = D, effect of Solid bars = 4/m’, dashed bars = 8/m’, tissue = T, interaction of density and tissue ef feet = DT. Bars not sharing a common superscript are significantly different (I’ < 0.05).
26
L. M&dez
et al./Aquaculture
156 (19971 21-34
Four, eight, and ten animals per m* of P. californiensis (2.5 to 3.0 g> were stocked in each pond in October 1993. All the animals were given food pellets manufactured by Rangen (Idaho, USA) at 10% of estimated biomass per day and were raised in similar conditions until February 1994, when all shrimps were collected with a net and immediately taken to the laboratory. There, 20 animals of each pond were weighed, washed with deionized water, and dissected to remove the hepatopancreas, abdominal muscle, and cuticle from the first abdominal segment. Shrimps in premolt or that had recently moulted were not used. All tissues were dried to constant weight in an oven at 70°C and digested in acid-washed test tubes with a mixture of concentrated nitric and perchloric acid and slowly boiled to dryness on a hotplate. Dried samples were analyzed by atomic absorption (BUCK, Scientific model 200). An air-acetylene flame was used for Cu, Zn, Fe, Ca, Mg, Na, and K, and a graphite furnace (BUCK, Scientific model GFl) for Cd and Pb. Precision was checked against the standard reference material of the National Research Council of Canada (DORM-l for dogfish), and was within the range of certified values. All data were processed and analyzed by STATISTICA Software. Two-way analyses of variance were used to assess significant effects of stocking densities and tissues on
B
rD
p
T
p
bi
DT p
I_ b
D c
Fig. 3. Mean tissue concentration and standard error of K, Na, Ca, and Mg at three different stocking densities. Solid bars = 4/m’, dashed bars = 8/m’, double-dashed bars = 10,’ m*. Effect of density = D, effect of tissue = T, interaction of density and tissue effect = DT. Bars not sharing a common superscript are significantly different (P < 0.05). The values for Ca in the abdominal cuticle are plotted against the right y-axis, which is expressed in thousands
L. M&dez et al./Aquaculture
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27
metal concentration. Duncan’s multiple-range test was used to test individual differences between tissues and ponds. Regression analyses were made for the tissues’ metal concentration and body size and weight, and for the concentration of metal pairs within the tissues.
3. Results Table 1 depicts the general physicochemical variables in the three ponds. There were no significant changes, although the nitrates and orthophosphates had a tendency to increase with the stocking densities. The initial values for the elements measured in sediment and seawater are shown in Table 2.
Table 3 Correlation matrix of metal pairs in hepatopancreas significance ( * ) was accepted at P < 0.05 4/m’ CU Zn Fe Cd Pb Na K
CU
Fe
Zn
of shrimp cultured at three different densities. The level of
Cd
Pb
Na
K
Ca
Mg
-0.8’
0.7
-0.7
*
Mg Ca
- 0.9 -0.7
8/m’
CU
CU Zn Fe Cd Pb Na K
- 0.9 *
0.7 0.9 Fe
Zn
Pb
0.7 * 0.8” 0.6 *
0.7 * 0.7 * 0.9’
MJ2
Cd
K
Na
0.7
*
0.7 Mg
Ca
Mg
Ca
* 0.7 *
Ca IO/m’
Cu
Zn
Fe
Cd
Pb
Na
K
CU Zn
Fe Cd Pb Na K Mg Ca
0.8 *
0.7
*
0.7
*
L. Mindez et al. / Aquaculture 156 (1997) 21-34
28
As stocking density increased, there was a significant decrease in the length (13.95 f 0.23; 13.36 + 0.09; 12.35 + 0.2; F = 17.56; P < 0.0001) and weight (18.60 f 0.8; 15.53 f 0.3; 12.09 + 0.6; F = 28.98; P < 0.0001) of the shrimp in the 4, 8, and IO/m’ pond. The hepatopancreas contained the higher concentrations of Cu (F = 445.74; P < 0.001; Fig. lA), Zn (F = 113.69; P < 0.001; Fig. 2A), Pb (F = 42.61; P < 0.001; Fig. 2B), Fe (F = 58.05; P < 0.001; Fig. 2C), and Na (F = 89.24; P < 0.001; Fig. 3A). Concentrations of Cd (F = 117.25; P < 0.001; Fig. lB), Mg (F = 469.06; P < 0.001; Fig. 3D), and Ca (F = 465.41; P < 0.001; Fig. 3C) were higher in the abdominal cuticle. Significant differences were found in the metal concentrations in shrimp from each pond. In the hepatopancreas, the concentration of Cu increased significantly (F = 18.71; P < 0.001; Fig. 1A) with the stocking density. A similar tendency was observed for Cd (F = 3.84; P < 0.05; Fig. lB), Zn (F = 3.39; P < 0.05; Fig. 2A), and
Table 4 Correlation significance
matrix of metal pairs in muscle ( * ) was accepted at P < 0.05
4/m’
Cu
CU Zn Fe Cd Pb Na K Mg Ca 8/m’
- 0.9
Zn
Fe
of shrimp Cd
cultured Pb
at three different Na
densities.
The level of
K
Mg
Ca
K
Mg
Ca
Mg
Ca
*
0.8 * 0.7
0.7 0.7 0.8’ 0.8 *
Cu
Zn
-0.7 - 0.6
- 0.7 - 0.7
0.8 * 0.7
0.9 *
0.7 Fe
Cd
Pb
Na
cu Ztl Fe Cd Pb Na K
- 0.6
*
0.7 *
Mg Ca IO/m’
0.7 * Cu
Zn
Cu Zn Fe Cd Pb Na K Mg Ca
Cd
Fe
Pb
Na
-0.6”
0.7 * 0.9 * 0.8
*
0.6
*
0.7 *
K
L. Me’ndez et al./Ayuaculture Table 5 Correlation significance 4/m’ cu Zn Fe Cd Pb Na K
matrix of metal pairs in exoskeleton (* ) was accepted at P < 0.05 cu
Zn
Fe
of shrimp cultured Cd
S/m’
Pb
21,
at three different Na
densities.
The level of
K
Mg
Ca
K
Mg
Ca
Mg
Ca
0.7 - 0.6
0.9
*
Mg Ca
156 (19971 21-34
0.7 0.9 * cu
0.8* Zn
Fe
0.6 Cd
Pb
Na
cu Zn Fe Cd Pb Na K
0.9 * - 0.7 *
0.7
Mg Ca IO/m’
0.6 cu
Zn
Fe
Cd
Pb
*
* Na
K
cu ZU Fe Cd Pb Na K Mg Ca
0.6 * 0.7 * 0.9 I
0.7
*
0.8
*
Na (F = 24.92; P < 0.001; Fig. 3A). The hepatopancreas of the animals in the medium density pond had the highest Pb concentrations (F = 23.94; P < 0.001; Fig. 2B). In the muscle, concentrations of Zn (F = 3.39; P < 0.001; Fig. 2A) and Na (F = 24.92; P < 0.001;Fig. 3A) were higher in the medium density pond. A similar tendency, although not significant, was observed for Mg and Ca. The concentration of Na in the cuticle increased with density in a significant way (F = 24.92; P < 0.001; Fig. 3A). The concentrations of Cd (F = 3.84; P < 0.05:Fig. 1B) and Fe were higher in the lowest density pond, although the differences in Fe concentration was not significant. For each pond, significant correlation coefficients for several individual concentrations of metal pairs for the hepatopancreas (Table 3), muscle (Table 4), and abdominal cuticle (Table 5) are also shown. Most correlations are between macroelements and trace metals; however, not all the tissues had the same correlations. The pattern was also affected by the stocking densities.
30
L. Me’ndez et al./Aquaculture
156 (1997121-34
4. Discussion To our knowledge, the present study is the first to address the effect of stocking density on metal accumulation in different tissues of the shrimp. An inverse relationship between stocking density and final size is in agreement with previous reports (Rainbow, 1989; Ray and Chien, 1992; D’Silva and Maughan, 1995; Pinto and Rouse, 1996). This could be an effect of competition for food, either intra- or interspecific, since the ponds were a semi-open system. However, the stocking densities were of the extensive type and food was given several times a day ad libitum. The excretion product of shrimp, ammonia, is known to have toxic effects (Jeney et al., 1992; Tudor et al., 1994) and cause mortality in shrimp larvae (Chen et al., 1991). At a higher stocking density, an increase in the ammonia levels that could slow the shrimp growth might be expected (Cai and Summerdelt, 1992), in particular in P. califomiensis, since it has burrowing habits and ammonia is found in close association with the sediment (Boyd, 1990). Accordingly, Ray and Chien (1992) concluded a higher stocking density in P. monodon can decrease shrimp size and increase mortality caused by deterioration of the pond sediment, which can be inferred by the water physicochemical variables. In our conditions, the effect of density on the size of the shrimp was seen even though the physicochemical variables were not significantly different. The lack of difference between these variables could be an effect of the ponds having a semi-open system and a fairly ‘new’ sediment, hence reducing the eutrophication and deterioration of water and sediment (Ray and Chien, 1992). In this study, differences in the metal concentration of tissues varied with the stocking density. Slight variations between one or several physicochemical variables could affect the activity of free ions because of complexing of the ions with specific ligands (Burton and Stratham, 1991; Rainbow, 1995). Although salinity, reported to affect metal uptake (Wright, 1995; Rainbow, 1995), was similar among the three ponds, the nitrate and orthophosphate levels were different. These differences could be related to the feeding regime. It is not likely that differences in metal concentrations are an effect of competition for ingested mineral contents of the pellets or sediments (Gutierrez-Galindo et al., 1994), because of the reduced competition at low densities and the stability of metal contents of sediments over a long period of time (Cauwet, 1987). In general, the hepatopancreas had the highest concentrations of Cu, Pb, Zn, and Fe, which is in agreement with other results in crustacea (White and Rainbow, 1986; Lindahl and Moksnes, 1993; Marcovecchio, 1994). The increased metal concentration in the hepatopancreas, compared to the other two tissues, probably has to do with its function as a storage and detoxification site (Vogt and Quinitio, 1991; Vogt and Quinitio, 1994) and to its high metallothioneins contents (Lindahl and Moksnes, 1993). Similarly, high concentrations of Ca and Mg were expected in the cuticle because of its composition (Piedad-Pascual, 1989) and its function as an absorption and excretion surface (Castille and Adisson, 1981; Boitel and Truchot, 1989; Whyte and Boutillier, 1991). According to our results, Cu and Cd concentrations are lower in the hepatopancreas when the stocking density is lower. Although Cu is necessary for the respiratory pigment hemocyanin (Ridout et al., 1989; Rainbow, 1991), it is considered lethal for gas
L. M&de? et al./Aquaculture
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31
exchange in high concentrations (Boitel and Truchot, 1989). In consequence, some chelation (EDTA) and absorption methods have been used to reduce Cu and Cd level in water, even though EDTA in high concentrations can be toxic (Castille and Adisson, 198 1; Burton and Fisher, 1990). For this reason, lower Cu and Cd concentration in hepatopancreas at reduced stocking densities represents an important result of aquaculture interest. However, a decrease in Cu concentration could also be explained as an increase in shrimp size when the stocking density is reduced, since an elevated growth rate could ‘dilute’ the metal content (Rainbow, 1989; White and Rainbow, 1986; Rainbow, 1991). The high Cd levels in the abdominal cuticle of the lowest density pond might be caused by surface adsorption, because the area-volume ratio is the smallest for the animals in this pond (Rainbow, 1991) and Cd body concentration is not regulated (Rainbow, 1991; Blust et al., 1992). Lower Ca levels in the abdominal cuticle of the 4/m’ density pond, could be related to a more advanced molting stage of the shrimp, for it has been reported that previous to a molt, a large amount of Ca is removed from the exoskeleton (Mercado-Allen, 199 1). Differences in molting frequency are expected as an effect of the stocking density, as can be inferred by the growth data in the three ponds. Molting could also affect other element levels in exoskeleton, although Weeks et al. (1992) reported that Cu and Zn levels in amphipods do not present variations in relation to the molt cycle. On the other hand, the concentration of some metals in the water can have an effect on the frequency of the molting cycle (Fingerman et al., 1996) and physicochemical variables, such as pH and carbon dioxide can also affect the Ca and Mg levels in the exoskeleton, as reported by Wickins (1984a,b). Indeed, element concentration in exoskeletons can present variations in relation to shrimp size and in caparace versus abdominal cuticle (Wickins, 1984a). However, since no precise record of the molting cycle was made, we are unable to conclude if differences of Ca and other macroelements observed in the cuticle are related to the molting cycle and growth, or to another cause related to stocking density. Little information is available on the relationship between trace metals and macroelements. Pelgrom et al. (1995) reported a decrease of Na in fish exposed to Cu and a decrease of Ca when fish were exposed to Cd. In our experimental design with shrimp, positive correlations were found for Cu-Na in hepatopancreas of the medium density pond, in muscle for the lowest density pond, and in the cuticle for the highest density pond. Davis et al. ( 1992) reported a deletion of Mg in the diet of P. ~~nnamei produced a significant depression of K in caparace, and a deletion of K lowered levels of Mg in the hepatopancreas. In accordance, we obtained a positive correlation between Mg and K in the abdominal cuticle and the hepatopancreas but only in the medium density pond. Serra et al. (1995) reported a decrease in the levels of Fe in several tissues during Cd exposure in clams. We found a similar negative correlation in muscle for Cd-Fe in the highest density pond. The relationship observed between some of the above metal pairs may be a result of a competition or an additional effect on the metal binders and transporters at a cellular level (Wright, 199.5) or an interaction between the metabolism or excretion of some elements in the hepatopancreas. In conclusion, the present work confirms previous studies on increased growth at lower stocking densities, possibly related to differences in physicochemical variables. In
32
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addition, at higher stocking densities, hepatopancreas levels of Cu, Cd, and Zn are increased, a result with possible aquaculture implications that remains to be fully elucidated, but could indicate a toxic accumulation.
Acknowledgements This research was supported by CONACyT under grant 1895PN, and by CIBNOR proyect IAC-5. Elena Palacios is a fellow of CONACyT. The authors are grateful to Dr. A.M. Ibarra and Dr. I.S. Racotta for comments on the manuscript, to G. Portillo for providing technical data on pond culture, and to Ellis Glazier for English corrections.
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