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Influx of zinc by channel catfish (Ictalurus punctatus): Uptake from external environmental solutions
0306-4492/92$5.00+ 0.00 0 1992Pergamon Press plc
Camp. Eiochem.Physiol. Vol. lOlC, No. 2, pp. 215-217, 1992 Printed in Great Britain
INFLUX OF ZINC BY CHANNEL CATFISH (ICTALURUS PUNCTATUS): UPTAKE FROM EXTERNAL ENVIRONMENTAL SOLUTIONS PETER J. BENTLEY Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, U.S.A. (Tel.: 919 829-4267) (Received 24 May 1991) Abstract-l. Channel catfish (Icralurus puncratus) take up zinc (measured with 65Zn) from external ambient solutions in a concentration dependent manner. At a concentration of low6 M Zn, this uptake is equivalent to 0.4% of the total body Zn each day. 2. Zinc influx was increased by external acid conditions (decreasing pH from 7.3 to 5). 3. Elevated CaZ+ and Cd*+, but not A13+, concentrations markedly decreased the uptake of Zn. 4. These observations may be relevant to circumstances that occur under natural conditions, and influence the zinc nutrition and toxicity of the fish.
INTRODUCTION
Zinc (Zn) is an essential nutrient (Underwood, 1977), which is a constituent of many enzyme systems and in excess can be toxic. Terrestrial animals mainly accumulate Zn from their food, but fish may also take it up from solution in their external ambient media. The latter process has been demonstrated in plaice (Milner, 1982) and rainbow trout (Spry and Wood, 1989). In the latter, accumulation occurs across the gills (Spry and Wood, 1988). Channel catfish, Ictaluruspunctatus, are indigenous to lakes, rivers, and ponds in the southeastern United States where they are now extensively farmed for food. Zinc has been shown to be an essential nutrient in this species, and its dietary requirements for this mineral have been described (Gatlin and Wilson, 1983). In excess, however, it can be toxic (Lewis and Lewis, 1971). The present observations concern the measurement of the uptake of waterborne Zn by these fish and the effects of the pH and other dissolved ions including Ca*+, Cd*+, and A13+. Such ions commonly contribute to the composition of natural waters.
ZnpuXof bsZn The fish were incubated in 300ml of the artificial pond water, in a polyethylene container, to which was added the appropriate amount of ZnCl, and 65Zn (New England Nuclear, Boston, MA) as a tracer (about O.OS~Ci/ml pond water). This solution was bubbled with air and mixed continually with a magnetic stirrer. Accumulation of Zn was calculated from accumulated whole body counts of the 6JZn and the specific activity of the Zn (counts/mole) in the external medium. At the end of the incubation period, the fish were rinsed 5 times (3 min e&h time) in 50 ml of a solution containing 2 x 10m4M Zn. This procedure was based on that used for comparable measurements of 45Ca influx in fish (Flik et al., 1989) in which a relatively high concentration of “cold” ion is used to displace the isotope from binding to accessible surface sites. (The separated skin of such fish only contained about 5% of the total 65Zn accumulated.) The fish were then killed with 0.1% MS-222 (Sigma Chemical Co., St Louis, MO), weighed, and each was extracted for 7 days in 20 ml of 0.5 N HNO,. This period of time was found to be adequate for equilibration of the isotope in extraction medium. A fragment-free aliquot of this solution was counted in a Beckman Biogamma II counter. RESULTS
MATERIALS AND
METHODS
Acute toxicity of Zn to channel catjish
Fingerling channel catfish (Zctaluruspunctatus) were obtained from a commercial source (Blue Ridge Hatchery, Kernersville, NC). They were maintained in 120-l. tanks, containing aerated well-water, which had a calcium concentration of about 0.2 mM and a pH of 7.3. The Zn concentration in this water (determined by atomic absorption spectrophotometry) was 0.035 pg/ml (0.5 x 10m6M). They were fed a commercial fish chow (Trout Grower, No. 38-480, Ziegler Bros Inc., Gardners, PA). The Zn content of this feed was 233 mg/kg. The fish were kept on a 12 hr light/l2 hr dark cycle at 21°C. The artificial pond water used in the experiments contained (mM): Na, 0.5; Ca, 0.1; Cl, about 3; Mg, 0.01, and K, 0.05. This solution, prepared from glass distilled water, was normally buffered with 2 mM Tris-HCl, and the pH was 7.3. The fish were kept in this solution in the laboratory for 24 hr prior to any experiments.
In order to know the non-toxic external concentration of Zn in the catfish, groups of 3 fish were placed in solutions of the artificial pond water to which Zn was added. A concentration of 10m4M Zn resulted in the deaths of all 3 fish in 24-30 hr. However, all the fish survived a concentration of lo-’ M Zn for at least 7 days. Increasing the Ca concentrations in the pond water from 0.1 mM to 3 mM in the presence of 10e4 M Zn resulted in the survival of all the fish for at least 6 days. Influx of Zn in channel catfish The influx of Zn into catfish, using 65Zn as a tracer (see Materials and Methods), was measured in fish placed in solutions of differing composition (Table 1).
215
PETER J. BENTLEY
216 Table 1. Accumulation
of zinc (using “‘Zn as a tracer) by channel catfish in media of different composition
The influx of Zn was time- and concentrationdependent and was about 6 times greater at a Zn concentration of 10m5M as compared to 10m6M. When the Ca concentration was increased from 0.1 mM to 3 mM the influx of Zn (from 10e6 M) was decreased by 85%. The presence of Cd, 10-j M similarly decreased Zn uptake. Decreasing the pH of the pond water from 7.3 to 5 resulted in a 1.7-times increase in Zn influx. Addition of Al’+ (as Al,[SO&), 10m5M, to this acid pond water did not significantly change the uptake further.
theless suggests that such accumulation of Zn from a 10d6 Zn medium could be nutritionally significant. If the fish were fasting or were growing more slowly, or indeed had a diet with a low Zn content, then such a source of the mineral may be relatively more important. The ionic composition of natural waters can vary considerably often reflecting industrial contamination. Such pollution can have adverse, even toxic effects on the aquatic life. The present results indicate that changes in the pH such as may result from precipitation of acid rain, and the presence of other multivalent ions may influence the accumulation of Zn by such fish. Thus, this process was increased by decreases in the pH of the media such as may result from precipitation of acid rain in poorly buffered lakes and streams. High Ca concentrations as may occur naturally in “hard” waters reduced accumulation of Zn, and also reduced its acute toxicity (see also Jones, 1938). Cadmium, a quite common industrial contaminant (Nriagu and Pacyna, 1988), also reduced Zn uptake in catfish. Industrial acidification of natural waters may result in rises in the concentration of dissolved aluminum by enhancing the dissolution of minerals and it can have toxic effects on fish (Driscoll et al., 1980; Baker and Schofield, 1982). However, this multivalent metal ion had no effect on Zn uptake by catfish.
DISCUSSION
REFERENCES
Channel catfish, like plaice (Milner, 1982) and rainbow trout (Spry and Wood, 1989) can take up waterborne Zn from their ambient media. The external zinc concentration of 10m6M can be considered a “normal” one. It was similar to the town reservoir supply and about double that of local well water. In rainbow trout the gills provide an avenue for this uptake (Spry and Wood, 1988), and it seems likely that the same occurs in the catfish, though a role for the skin cannot be excluded. Influx of Zn was only 6 times greater at lO-5 M Zn as compared to 10e6 M, suggesting the possibility that the saturation of a specific transport process could be occurring. At a Zn concentration of 10m6M, less than 2% of the accumulated Zn can be accounted for by drinking (Bentley, 1990). The total Zn content of these fish is about 0.46 mmol/kg (Bentley, 1991) so that the accumulation of waterborne Zn each day is equivalent to about 0.4% of this total. Insufficient information is available to precisely estimate the contribution of waterborne Zn to the nutritional needs of channel catfish. When kept under optimal conditions, these fish have been shown to grow from 9 g to 570 g in 350 days (Busch, 1985). The increment in total body Zn in this time, based on the Zn content of the young fish (Bentley, 1991), can be estimated to be about 17 mg in each fish. Information is only available about the rate of Zn uptake by small catfish, but if this is applied on a unit weight basis to the fish throughout their period of growth, it would amount to a total of about 12 mg Zn fish (based on an average weight of about 285 g). Such a calculation does not allow for concurrent losses of Zn nor changes in the rate of uptake of waterborne Zn, but it never-
Baker J. P. and Schofield C. L. (1982) Aluminum toxicity to fish in acidic waters. Water, Air, Soil, Pollut. 18, 289-309. Bentley P. J. (1990) Unidirectional fluxes of Na+, Cl-, and water in fingerling channel catfish, Ictalurus punctatus. Comp. Biochem. Physiol. 9lA, 195-199. Bentley P. J. (1991) A high-affinity zinc binding protein in channel catfish (Ictalurus punctatus). Biochem. Physiol. (In press). Busch R. L. (1985) Channel catfish culture in ponds. In Channel Catfish Culture (Edited by Tucker C. S.), pp. 13-84. (Developments in Aquaculture and Fisheries Science 15). Elsevier, Amsterdam. Driscoll C. T., Baker J. P., Bisogni J. J. and Schofield C. L. (1980) Effect of aluminum on fish in diluted acidified waters. Nature, Lond. 2&i, 161-164. Flik G. L., Fenwick J. C. and Wendelaar Bonga S. E. (1989) Calcitropic actions of prolactin in freshwater North American eel (Anguilia rostrata LeSuer). Am. J. Physiol. 257, Rl44R79. Gatlin D. M. and Wilson R. P. (1983) Dietary zinc requirement of fingerling channel catfish. J. Nutr. 113,630-635. Jones J. R. E. (1938) The relative toxicity of salts of lead, zinc and copper to the stickleback (Gusterosteus aculeatus L.) and the effect of calcium on the toxicity of lead and zinc salts. J. exp. Biol. 15, 394-407. Lewis S. D. and Lewis Wm. (1971) The effect of zinc and copper on the osmolality of blood serum of the channel catfish, Ictalurus punctatus Rajinesque, and golden shiner, Notemigonus crysoleucas Mitchell. Trans. Am. Sot. Fish 100, 639-643. Milner N. J. (1982) The accumulation of zinc by O-group plaice, Pleuronekes platessa (L), from high concentrations in sea water and food. J. Fish Biol. 21, 325-347. Nriagu J. 0. and Pacyna J. M. (1988) Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature, Lond. 333, 134-139. Spry D. J. and Wood C. M. (1988) Zinc influx across the
Conditions’
Zinc uptake nmol’kg-“hr-’
IO-‘M Zn (8) 10-6M Zn (8) + Ca, 3 mM (8) +Cd, lO-‘M (8) + pH 52 (8) +Al, lo-‘M (7)
440 f 19* I2 f 15 f 135 f 155 f
44 11 1*** 2*** 19’ 13
*P ~0.05, ***P
Zn inffux in catfish isolated, perfused head preparation of the rainbow trout (Salmo gairdneri) in hard and soft water. Can. J. Fish Aguat. Sci. 45, 22062215. Spry D. J. and Wood C. M. (1989) A kinetic method for measurement of zinc influx in u&o in the rainbow trout.
217
and the effects of waterborne calcium on flux rates. J. exp. Biol. 142, 425-446. Underwood E. J. (1977) In Trace Elements in Animal and Human Nutrition, 4th Edn, pp. 196-242. Academic Press, London.
Camp. Eiochem.Physiol. Vol. lOlC, No. 2, pp. 215-217, 1992 Printed in Great Britain
INFLUX OF ZINC BY CHANNEL CATFISH (ICTALURUS PUNCTATUS): UPTAKE FROM EXTERNAL ENVIRONMENTAL SOLUTIONS PETER J. BENTLEY Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, U.S.A. (Tel.: 919 829-4267) (Received 24 May 1991) Abstract-l. Channel catfish (Icralurus puncratus) take up zinc (measured with 65Zn) from external ambient solutions in a concentration dependent manner. At a concentration of low6 M Zn, this uptake is equivalent to 0.4% of the total body Zn each day. 2. Zinc influx was increased by external acid conditions (decreasing pH from 7.3 to 5). 3. Elevated CaZ+ and Cd*+, but not A13+, concentrations markedly decreased the uptake of Zn. 4. These observations may be relevant to circumstances that occur under natural conditions, and influence the zinc nutrition and toxicity of the fish.
INTRODUCTION
Zinc (Zn) is an essential nutrient (Underwood, 1977), which is a constituent of many enzyme systems and in excess can be toxic. Terrestrial animals mainly accumulate Zn from their food, but fish may also take it up from solution in their external ambient media. The latter process has been demonstrated in plaice (Milner, 1982) and rainbow trout (Spry and Wood, 1989). In the latter, accumulation occurs across the gills (Spry and Wood, 1988). Channel catfish, Ictaluruspunctatus, are indigenous to lakes, rivers, and ponds in the southeastern United States where they are now extensively farmed for food. Zinc has been shown to be an essential nutrient in this species, and its dietary requirements for this mineral have been described (Gatlin and Wilson, 1983). In excess, however, it can be toxic (Lewis and Lewis, 1971). The present observations concern the measurement of the uptake of waterborne Zn by these fish and the effects of the pH and other dissolved ions including Ca*+, Cd*+, and A13+. Such ions commonly contribute to the composition of natural waters.
ZnpuXof bsZn The fish were incubated in 300ml of the artificial pond water, in a polyethylene container, to which was added the appropriate amount of ZnCl, and 65Zn (New England Nuclear, Boston, MA) as a tracer (about O.OS~Ci/ml pond water). This solution was bubbled with air and mixed continually with a magnetic stirrer. Accumulation of Zn was calculated from accumulated whole body counts of the 6JZn and the specific activity of the Zn (counts/mole) in the external medium. At the end of the incubation period, the fish were rinsed 5 times (3 min e&h time) in 50 ml of a solution containing 2 x 10m4M Zn. This procedure was based on that used for comparable measurements of 45Ca influx in fish (Flik et al., 1989) in which a relatively high concentration of “cold” ion is used to displace the isotope from binding to accessible surface sites. (The separated skin of such fish only contained about 5% of the total 65Zn accumulated.) The fish were then killed with 0.1% MS-222 (Sigma Chemical Co., St Louis, MO), weighed, and each was extracted for 7 days in 20 ml of 0.5 N HNO,. This period of time was found to be adequate for equilibration of the isotope in extraction medium. A fragment-free aliquot of this solution was counted in a Beckman Biogamma II counter. RESULTS
MATERIALS AND
METHODS
Acute toxicity of Zn to channel catjish
Fingerling channel catfish (Zctaluruspunctatus) were obtained from a commercial source (Blue Ridge Hatchery, Kernersville, NC). They were maintained in 120-l. tanks, containing aerated well-water, which had a calcium concentration of about 0.2 mM and a pH of 7.3. The Zn concentration in this water (determined by atomic absorption spectrophotometry) was 0.035 pg/ml (0.5 x 10m6M). They were fed a commercial fish chow (Trout Grower, No. 38-480, Ziegler Bros Inc., Gardners, PA). The Zn content of this feed was 233 mg/kg. The fish were kept on a 12 hr light/l2 hr dark cycle at 21°C. The artificial pond water used in the experiments contained (mM): Na, 0.5; Ca, 0.1; Cl, about 3; Mg, 0.01, and K, 0.05. This solution, prepared from glass distilled water, was normally buffered with 2 mM Tris-HCl, and the pH was 7.3. The fish were kept in this solution in the laboratory for 24 hr prior to any experiments.
In order to know the non-toxic external concentration of Zn in the catfish, groups of 3 fish were placed in solutions of the artificial pond water to which Zn was added. A concentration of 10m4M Zn resulted in the deaths of all 3 fish in 24-30 hr. However, all the fish survived a concentration of lo-’ M Zn for at least 7 days. Increasing the Ca concentrations in the pond water from 0.1 mM to 3 mM in the presence of 10e4 M Zn resulted in the survival of all the fish for at least 6 days. Influx of Zn in channel catfish The influx of Zn into catfish, using 65Zn as a tracer (see Materials and Methods), was measured in fish placed in solutions of differing composition (Table 1).
215
PETER J. BENTLEY
216 Table 1. Accumulation
of zinc (using “‘Zn as a tracer) by channel catfish in media of different composition
The influx of Zn was time- and concentrationdependent and was about 6 times greater at a Zn concentration of 10m5M as compared to 10m6M. When the Ca concentration was increased from 0.1 mM to 3 mM the influx of Zn (from 10e6 M) was decreased by 85%. The presence of Cd, 10-j M similarly decreased Zn uptake. Decreasing the pH of the pond water from 7.3 to 5 resulted in a 1.7-times increase in Zn influx. Addition of Al’+ (as Al,[SO&), 10m5M, to this acid pond water did not significantly change the uptake further.
theless suggests that such accumulation of Zn from a 10d6 Zn medium could be nutritionally significant. If the fish were fasting or were growing more slowly, or indeed had a diet with a low Zn content, then such a source of the mineral may be relatively more important. The ionic composition of natural waters can vary considerably often reflecting industrial contamination. Such pollution can have adverse, even toxic effects on the aquatic life. The present results indicate that changes in the pH such as may result from precipitation of acid rain, and the presence of other multivalent ions may influence the accumulation of Zn by such fish. Thus, this process was increased by decreases in the pH of the media such as may result from precipitation of acid rain in poorly buffered lakes and streams. High Ca concentrations as may occur naturally in “hard” waters reduced accumulation of Zn, and also reduced its acute toxicity (see also Jones, 1938). Cadmium, a quite common industrial contaminant (Nriagu and Pacyna, 1988), also reduced Zn uptake in catfish. Industrial acidification of natural waters may result in rises in the concentration of dissolved aluminum by enhancing the dissolution of minerals and it can have toxic effects on fish (Driscoll et al., 1980; Baker and Schofield, 1982). However, this multivalent metal ion had no effect on Zn uptake by catfish.
DISCUSSION
REFERENCES
Channel catfish, like plaice (Milner, 1982) and rainbow trout (Spry and Wood, 1989) can take up waterborne Zn from their ambient media. The external zinc concentration of 10m6M can be considered a “normal” one. It was similar to the town reservoir supply and about double that of local well water. In rainbow trout the gills provide an avenue for this uptake (Spry and Wood, 1988), and it seems likely that the same occurs in the catfish, though a role for the skin cannot be excluded. Influx of Zn was only 6 times greater at lO-5 M Zn as compared to 10e6 M, suggesting the possibility that the saturation of a specific transport process could be occurring. At a Zn concentration of 10m6M, less than 2% of the accumulated Zn can be accounted for by drinking (Bentley, 1990). The total Zn content of these fish is about 0.46 mmol/kg (Bentley, 1991) so that the accumulation of waterborne Zn each day is equivalent to about 0.4% of this total. Insufficient information is available to precisely estimate the contribution of waterborne Zn to the nutritional needs of channel catfish. When kept under optimal conditions, these fish have been shown to grow from 9 g to 570 g in 350 days (Busch, 1985). The increment in total body Zn in this time, based on the Zn content of the young fish (Bentley, 1991), can be estimated to be about 17 mg in each fish. Information is only available about the rate of Zn uptake by small catfish, but if this is applied on a unit weight basis to the fish throughout their period of growth, it would amount to a total of about 12 mg Zn fish (based on an average weight of about 285 g). Such a calculation does not allow for concurrent losses of Zn nor changes in the rate of uptake of waterborne Zn, but it never-
Baker J. P. and Schofield C. L. (1982) Aluminum toxicity to fish in acidic waters. Water, Air, Soil, Pollut. 18, 289-309. Bentley P. J. (1990) Unidirectional fluxes of Na+, Cl-, and water in fingerling channel catfish, Ictalurus punctatus. Comp. Biochem. Physiol. 9lA, 195-199. Bentley P. J. (1991) A high-affinity zinc binding protein in channel catfish (Ictalurus punctatus). Biochem. Physiol. (In press). Busch R. L. (1985) Channel catfish culture in ponds. In Channel Catfish Culture (Edited by Tucker C. S.), pp. 13-84. (Developments in Aquaculture and Fisheries Science 15). Elsevier, Amsterdam. Driscoll C. T., Baker J. P., Bisogni J. J. and Schofield C. L. (1980) Effect of aluminum on fish in diluted acidified waters. Nature, Lond. 2&i, 161-164. Flik G. L., Fenwick J. C. and Wendelaar Bonga S. E. (1989) Calcitropic actions of prolactin in freshwater North American eel (Anguilia rostrata LeSuer). Am. J. Physiol. 257, Rl44R79. Gatlin D. M. and Wilson R. P. (1983) Dietary zinc requirement of fingerling channel catfish. J. Nutr. 113,630-635. Jones J. R. E. (1938) The relative toxicity of salts of lead, zinc and copper to the stickleback (Gusterosteus aculeatus L.) and the effect of calcium on the toxicity of lead and zinc salts. J. exp. Biol. 15, 394-407. Lewis S. D. and Lewis Wm. (1971) The effect of zinc and copper on the osmolality of blood serum of the channel catfish, Ictalurus punctatus Rajinesque, and golden shiner, Notemigonus crysoleucas Mitchell. Trans. Am. Sot. Fish 100, 639-643. Milner N. J. (1982) The accumulation of zinc by O-group plaice, Pleuronekes platessa (L), from high concentrations in sea water and food. J. Fish Biol. 21, 325-347. Nriagu J. 0. and Pacyna J. M. (1988) Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature, Lond. 333, 134-139. Spry D. J. and Wood C. M. (1988) Zinc influx across the
Conditions’
Zinc uptake nmol’kg-“hr-’
IO-‘M Zn (8) 10-6M Zn (8) + Ca, 3 mM (8) +Cd, lO-‘M (8) + pH 52 (8) +Al, lo-‘M (7)
440 f 19* I2 f 15 f 135 f 155 f
44 11 1*** 2*** 19’ 13
*P ~0.05, ***P
Zn inffux in catfish isolated, perfused head preparation of the rainbow trout (Salmo gairdneri) in hard and soft water. Can. J. Fish Aguat. Sci. 45, 22062215. Spry D. J. and Wood C. M. (1989) A kinetic method for measurement of zinc influx in u&o in the rainbow trout.
217
and the effects of waterborne calcium on flux rates. J. exp. Biol. 142, 425-446. Underwood E. J. (1977) In Trace Elements in Animal and Human Nutrition, 4th Edn, pp. 196-242. Academic Press, London.