Effects of calcium and magnesium hardness on acute copper toxicity to juvenile channel catfish Ictalurus punctatus

Aquaculture 172 (1999) 275-280

Short communication

Effects of calcium and magnesium hardness on acute copper toxicity to juvenile channel catfish Ictalurus punctatus

Peter W. Perschbacher^a and William A. Wurts^b,*

^aAquaculture and Fisheries Center, Box 4912, University of Arkansas at Pine Bluff, Pine Bluff, AR  71611, USA

^bCooperative Extension Program, Kentucky State University, P.O. Box 469, Princeton, KY 42445-0469, USA

Accepted 19 November 1998

________________________________________________________________________

Abstract

               Two experiments were conducted to evaluate the effects of calcium or magnesium hardness on the acute toxicity of copper sulfate to juvenile channel catfish (Ictalurus punctatus) in low alkalinity environments.  A preliminary bioassay determined the 48-h LC50 of copper sulfate to be 1.25 mg l^-1 for juvenile catfish placed in water with calcium hardness and total alkalinity set at 20 mg l^-1 CaCO₃.  In the first experiment, catfish were exposed to 1.25 mg l^-1 copper sulfate in environments where calcium hardness was varied from 10-400 mg l^-1 CaCO₃.  Total alkalinity was 20 mg l^-1 CaCO₃.  As calcium hardness increased, copper-induced catfish mortalities decreased significantly from 90% at 10 mg l^-1 CaCO₃ to 5% at 400 mg l^-1 CaCO₃.  In the second experiment, catfish were exposed to 1.25 mg l^-1 copper sulfate in environments containing either calcium or magnesium hardness, 20 and 400 mg l^-1 CaCO₃, with total alkalinity set at 20 mg l^-1 CaCO₃.  Survivals in calcium hardness treatments were consistent with those in the first experiment.  However, 100% mortality was observed in both treatments containing magnesium-based hardness.  These data suggest a calcium-specific mechanism with respect to acute copper toxicity in channel catfish. © 1999 Elsevier Science B.V.  All rights reserved.

Keywords:  copper, toxicity, calcium, magnesium, fish

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1. Introduction

        Copper sulfate is routinely used as an algicide in commercial and recreational fish ponds.  It has also been used as an effective treatment for pathogenic protozoan parasites of fish.  It is generally recognized that copper can be highly toxic to teleosts.  However, several studies have reported that either calcium hardness or alkalinity concentrations have significant effects on copper toxicity.  Therefore, recommendations for safe use of copper sulfate have been based on hardness (Inglis and Davis, 1972; Post, 1983; Sawyer et al., 1989) and total alkalinity concentrations of water (MacMillan, 1985; Wellborn, 1985; Reardon and Harrell, 1990).

            Straus and Tucker (1993) reported that total alkalinity and total hardness had significant effects on acute copper toxicity to juvenile channel catfish (Ictalurus punctatus).  Wurts and Perschbacher (1994) observed that alkalinity concentration had the most pronounced effect on acute copper toxicity to juvenile channel catfish when calcium hardness and alkalinity concentrations were treated as independent variables.  Wurts and Perschbacher (1994) also reported a calcium hardness effect, which could affect channel catfish tolerance to copper toxicity in low alkalinity environments.  Miller and Mackay (1980) believed calcium hardness was more important than alkalinity in protecting fish from copper toxicity, based on experiments with juvenile rainbow trout (Oncorhynchus mykiss).  Research about fathead minnows (Pimephales promelas) and rainbow trout, however, found no significant calcium effect on copper uptake sites (Lauren and McDonald, 1987a; Playle et al., 1993a; Zia and McDonald, 1994).  Furthermore, it has been proposed that magnesium hardness also competes with copper for binding sites on the gills (Playle et al., 1993b).

            The present study determined whether acute copper toxicity to juvenile channel catfish was affected by increasing calcium hardness concentrations in low alkalinity waters.  Then by substituting magnesium for calcium at equal hardness concentrations, it was possible to compare the effects of magnesium versus calcium on the acute toxicity of copper to juvenile channel catfish.

2. Methods

            Two bioassays were conducted to facilitate evaluations about calcium and magnesium effects on acute copper toxicity.  The first bioassay determined the amount of copper sulfate needed to effect a 48-h LC₅₀ for 7-10 g juvenile channel catfish in water with calcium hardness and total alkalinity concentrations set at 20 mg l^-1 CaCO₃.  The second bioassay examined whether 48-h survival would be adversely affected if juvenile channel catfish were placed in calcium-free water with high magnesium concentrations and no copper added.  Techniques followed EPA guidelines (U.S. Environmental Protection Agency, 1975).

            Experiments were conducted to evaluate the mortality response of juvenile channel catfish exposed to a potentially toxic concentration of copper sulfate in waters with differing concentrations of calcium or magnesium hardness and a constant low alkalinity concentration.  Two trials were conducted: one varied calcium hardness and the other varied calcium or magnesium hardness.  Each combination of hardness and alkalinity was replicated in four, aerated, 7.6-l aquaria.  Each aquarium was stocked with seven juvenile channel catfish.  Length and weight averages for catfish were 102 + 5.2 mm and 8.2±0.9g.

            Trial 1 involved exposing fish to 1.25 mg l^-1 copper sulfate in environments with five different concentrations of calcium hardness, ranging from 10 to 400 mg l^-1.  Total alkalinity was held constant at 20 mg l^-1.  Catfish were also observed in a control environment where calcium hardness was 400 mg l^-1 CaCO₃ and total alkalinity was 20 mg l^-1 CaCO₃, and no copper was added.

            Trial 2 examined the relative effects on copper toxicity (1.25 mg l^-1 copper sulfate) of magnesium versus calcium hardness at concentrations of 20 and 400 mg l^-1 CaCO₃.  Total alkalinity was held constant at 20 mg  L^-1 CaCO₃.

Methods used to create and test water treatments, copper toxicity and water quality were the same as those reported by Wurts and Perschbacher (1994).  Magnesium hardness was adjusted to desired concentrations with reagent grade magnesium sulfate.

Fish were not fed 48 h prior to or during each experiment.  Catfish were held for 24 h preceding each experiment in a holding tank with water containing calcium hardness and total alkalinity, set at 20 mg l^-1 CaCO₃.  Water temperature, dissolved oxygen, ammonia-nitrogen (NH₃-N) and pH were measured to monitor water quality.  Mortalities were removed and totaled at regular intervals.

            Survival data were analyzed using PROC GLM and Fischer's LSD (Ott, 1977; SAS, 1989).  Percentile data were transformed using the arc-sine method suggested by Mostellar and Youtz (1961).  Significance was tested at the 0.05 level.

3. Results and Discussion

            A copper sulfate concentration of 1.25 mg l ^-1 was required to effect a 48-h LC₅₀ for juvenile channel catfish placed in water containing total alkalinity and calcium hardness set at 20 mg l^-1 CaCO₃.  Water temperature was 21.5º C.

After 48 h, survival was 100% for juvenile catfish placed in aquaria containing calcium-free water with a magnesium hardness of 400 mg l^-1 CaCO₃.

            It is interesting to note that the copper concentration producing 48-hr LC₅₀ in this study, 1.25 mg l^-1 copper sulfate at low alkalinity (20 mg l^-1 CaCO₃), was substantially lower than that reported by Wurts and Perschbacher (1994) for water of moderate alkalinity (i.e. 28 mg l^-1 CuSO₄, at 75 mg l^-1 CaCO₃).  At a low alkalinity concentration, much less copper was required to produce acute toxicity.

            In general, water quality was poorest in aquaria with the highest survivals (Tables 1 and 2).  Water temperatures ranged from 22.6-23.8ºC in trial 1 and 21.7-22.3ºC in trial 2.  Mean total NH₃-N concentrations ranged from 1.4-1.6 mg l^-1 at 2 h and 2.9-4.0 mg l^-1 at 42 h in the first experiment.  Mean pH ranged from 6.6-7.0 in trial 1 and 6.5-6.9 in trial 2.  Mean dissolved oxygen concentrations ranged from 4.8-6.4 mg l^-1 in trial 1 and 5.0-7.5 mg l^-1 in trial 2.

            In one aquarium each, from trial 1 and trial 2, disruption of aeration occurred for several hours; and two fish jumped from one aquarium in trial 1.  Survival data from these aquaria were treated as missing data in the statistical analyses.

            In trial 1, there were significant differences among experimental groups with respect to survival and calcium hardness concentrations.  As calcium hardness increased, catfish survival improved significantly from 10% at 10 mg l^-1 CaCO₃ to 95% at 400 mg l^-1

Table 1 Mean 48-h survivals and water quality data for juvenile channel catfish exposed to 1.25 mg l-1 copper sulfate at varying calcium hardness concentrations with total alkalinity held constant at 20 mg l-1 CaCO3
Hardness (mg l-1)	42-h pH	42-h NH3-N (mg l-1)	DO		Survivala (%)
			2-h (mg l-1)	18-h (mg l-1)
10b	7.0	2.9	5.5	5.8	10w
20	6.8	3.2	5.3	5.7	32w
50	6.8	3.7	5.6	6.1	71x
200	6.8	3.9	5.1	5.1	93x,y
400b	6.9	3.9	5.7	5.6	95y
400c (control)	6.7	4.0	5.3	4.8	100y
a Values followed by the same superscript were not significantly different at the 0.05 level. b Means for survival, pH and NH3-N within these rows were based on three values rather than four because fish either jumped from or aeration was disrupted in one tank after 18-h. c The control was not exposed to copper sulfate.

CaCO₃ (Table 1).  Survival was 100% in the control.  Mean survivals (93 and 95%) at 200 and 400 mg l^-1 calcium hardness were not significantly different from one another or from 100 % survival in the control.  The data indicate a calcium hardness between 50 and 200 mg l^-1 would reduce toxicity and mortality for juvenile channel catfish exposed to a copper sulfate concentration of 1.25 mg l^-1, where total alkalinity is 20 mg l^-1 CaCO₃.

            In trial 2, 100% was mortality in both treatments containing magnesium-based hardness, 20 and 400 mg l^-1 CaCO₃.  Survivals were 48 and 100% in 20 and 400 mg l^-1 calcium hardness treatments, respectively, and were consistent with those in trial 1 (Tables 1 and 2).

            These data suggest a calcium-specific mechanism with respect to acute copper toxicity in juvenile channel catfish.  There is convincing evidence to suggest that copper

Table 2 Mean 48-h survivals and water quality data for juvenile channel catfish exposed to 1.25 mg l-1 copper sulfate at varying calcium or magnesium hardness concentrations with total alkalinity held constant at 20 mg l-1 CaCO3
Hardness (mg l-1)	pH	DO		Survivala (%)
		2-h (mg l-1)	18-h (mg l-1)
Calcium
20b	6.5	6.3	5.0	48x
400	6.7	6.4	5.3	100w
Magnesium
20	6.7	6.4	5.7	0y
400	6.8	6.5	6.6	0y

a Values followed by the same superscript were not significantly different at the 0.05 level. b Mean survival within this row was based on three values rather than four because aeration was disrupted after 18-h in one tank.

disrupts ion homeostasis (Lewis and Lewis,1971; Lauren and McDonald, 1986, 1987b; Reid and McDonald, 1988) and that environmental calcium directly affects osmoregulation, in teleosts (Potts and Flemming, 1971; Bournancin et al., 1972; Flemming et al., 1974; Eddy,1975; Evans, 1975; Isaia and Masoni, 1976; McWilliams and Potts, 1978; Pic and Maetz, 1981).  Indeed, it seems plausible that copper competitively inhibits calcium binding sites, such as those associated with calcium activated channels for monovalent ions (Perez et al., 1994; Vambutas et al., 1994; Levitan and Rogowski, 1996).  Inhibition or suppression of osmoregulatory mechanisms would result in critical losses of serum electrolytes; which in turn, could cause tetany, cardiovascular failure and death.  As observed in this study, a high ratio of the concentrations of calcium to copper ions would minimize the toxic effects of copper (by reducing or preventing competitive inhibition).

            The present research substantiates reports that indicate calcium hardness affects copper toxicity in teleosts.  Calcium hardness significantly affected survival of juvenile channel catfish exposed to a toxic concentration of copper sulfate in low alkalinity water.  But, magnesium hardness provided no protection from copper toxicity.  This study emphasizes the importance of measuring calcium hardness before using copper sulfate in waters with low alkalinity concentrations.

Acknowledgments

            We gratefully acknowledge Dwight Wolfe for his valuable assistance with statistical analyses and UAPB student intern, Lloyd Inman, for assistance with the experimental trials.  We thank Forrest Wynne and Drs. Andrew Goodwin, Bob Durborow, Rebecca Lochmann and Michael Masser for reviewing this manuscript.  This manuscript was assigned publication number 97141 by the Arkansas Agricultural Experiment Station.

References

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Eddy, F.B., 1975.  The effect of calcium on gill potentials and on sodium and chloride fluxes in the goldfish,               Carassius auratus.  J. Comp. Physiol., 96:  131-142.

Evans, D.H., 1975.  Ionic exchange mechanisms in fish gills.  Comp. Biochem. Physiol., 51A:  491-495.

Flemming, W.R., Nichols, J. and Potts, W.T.W., 1974.  The effect of low calcium sea water and         Actinomycin-D on the sodium metabolism of Fundulus kansae.  J. Exp. Biol., 60:  267-273.

Inglis, A. and Davis, E.L., 1972.  Effects of water hardness on the toxicity of several organic and inorganic             herbicides to fish.  U.S. Fish and Wildlife Service Technical Paper 67.

Isaia, J. and Masoni, A., 1976.  The effects of calcium and magnesium on water and ionic permeabilities in               the seawater adapted eel, Anguilla anguilla L.  J. Comp. Physiol., 109:  221-233.

Lauren, D.J. and McDonald, D.G., 1986.  Influence of water hardness, pH, and alkalinity on the mechanisms        of copper toxicity in juvenile rainbow trout, Salmo gairdneri.  Can J. Fish. Aq. Sci., 43: 1488-1496.

Lauren, D.J. and McDonald, D.G., 1987a.  Acclimation to copper by rainbow trout, Salmo gairdneri         Richardson.  J. Comp. Physiol., 155:  635-644.

Lauren, D.J. and McDonald, D.G., 1987b.  Acclimation to copper by rainbow trout, Salmo gairdneri:          physiology.  Can J. Fish. Aq. Sci., 44:  99-105.

Levitan, I.B. and Rogowski, M.A., 1996.  Potassium channels (editorial).  Neuropharmacology, 35(7):  759.

Lewis, S.D. and Lewis, W.M., 1971.  The effect of zinc and copper on the osmolality of blood serum of the     channel catfish, Ictalurus punctatus Rafinesque, and golden shiner, Notemigonus chrysoleucas Mitchell.     Trans. Am. Fish. Soc., 100:  639-643.

McWilliams, P.G. and Potts, W.T.W., 1978.  The effects of pH and calcium concentrations on gill potentials             in the brown trout.  J. Comp. Physiol., 126:  277-286.

MacMillan, J.R., 1985.  Infectious diseases.  In: C.S. Tucker (Editor), Channel Catfish Culture.  Elsevier,               Amsterdam, pp. 405-496.

Miller, T.G. and Mackay, W.C., 1980.  The effects of hardness, alkalinity and pH of test water on the toxicity of copper to rainbow trout (Salmo gairdneri).  Water Res. 14:  129-133.

Mostellar, F. and Youtz, C., 1961.  Tables of the Freeman-Tukey transformations for the binomial and         Poisson distributions.  Biometrika, 48:  433-440.

Ott, L. 1977.  An introduction to statistical methods and data analysis.  Wadsworth Publishing Company,            Inc., Belmont, CA, 730 pp.

Perez, G., Lagrutta, A., Adelman, J.P. and Toro, L., 1994.  Reconstitution of expressed KCa channels from       Xenopus oocytes to lipid bilayers.  Biophys. J., 66(4):  1022-1027

Pic, P. and Maetz, J., 1981.  Role of external calcium in sodium and chloride transport in the gills of         seawater-adapted Mugil capito.  J. Comp. Physiol., 141:  511-521.

Playle, R.C., Dixon, D.G. and Burnison, K., 1993a.  Copper and cadmium binding to fish gills: estimates of         metal-gill stability constants and modeling of metal accumulation.  Can. J. Fish. Aquat. Sci., 50:  2678-      2687.

Playle, R.C., Dixon, D.G. and Burnison, K., 1993b.  Copper and cadmium binding to fish gills: modification       by dissolved organic carbon and synthetic ligands.  Can. J. Fish. Aquat. Sci., 50:  2667-2677.

Post, G., 1983.  Textbook of Fish Health.  T.F.H. Publications, Neptune City, NJ.

Potts, W.T.W. and Fleming, W.R.,  1971.  The effect of environmental calcium and ovine prolactin on         sodium balance in Fundulas kansae.  J. Exp. Biol., 55:  63-75.

Reardon, I.S. and Harrell, R.M., 1990.  Acute toxicity of formalin and copper sulfate to striped bass         fingerlings held in varying salinities.  Aquaculture 87:  255-270.

Reid, S.D. and McDonald, D.G., 1988.  Effects of cadmium, copper, and low pH on ion fluxes in the rainbow                trout, Salmo gairdneri.  Can. J. Fish. Aq. Sci., 45:  244.

SAS, 1989.  SAS Institute, Inc.:  Version 6.08.  Cary, NC.

Sawyer, M.D.J., Reader, J.P. and Morris, R., 1989.  The effect of calcium concentration on the toxicity of    copper, lead and zinc to yolk-sac fry of brown trout, Salmo trutta L., in soft, acid water.  J. Fish Biol. 35:               323-332.

Straus, D.L. and Tucker, C.S., 1993.  Acute toxicity of copper sulfate and chelated copper to channel catfish   Ictalurus punctatus.  J. World Aquaculture Soc., 24(3):  390-395.

U.S. EPA, 1975.  Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians.  EPA-      660/3-75-009, Nat. Tech. Info. Ser., Washington, DC.

Vambutas, V., Tamir, H. and Beattie, D.S., 1994.  Isolation and partial characterization of calcium-activated              chloride ion channels from thylakoids.  Arch. Biochem. Biophys., 312(2):  401-6.

Wellborn, T., 1985.  Control and therapy.  In: Principal Diseases of Farm Raised Catfish.  Southern Coop.     Series Bull. 225:  50-67.

Wurts, W.A. and Perschbacher, P.W., 1994.  Effects of bicarbonate alkalinity and calcium on the acute         toxicity of copper to juvenile channel catfish (Ictalurus punctatus).  Aquaculture, 125:  73-79.

Zia, S. and McDonald, D.G., 1994.  Role of the gills and gill chloride cells in metal uptake in the freshwater-          adapted rainbow trout, Oncorhynchus mykiss.  Can. J. Fish. Aquat. Sci. 51:  2482-2492.

Effects of bicarbonate alkalinity and calcium on the acute toxicity of copper to juvenile channel catfish (Ictalurus punctatus)

Effects of bicarbonate alkalinity and calcium on the acute toxicity of copper to juvenile channel catfish (Ictalurus punctatus)

William A. Wurts^a,*, Peter W. Perschbacher^b

^a Cooperative Extension Program, Kentucky State University, P.O. Box 469, Princeton, KY 42445-0469, USA

^b University of Arkansas at Pine Bluff, Agriculture Experiment 

Station Center, Box 40005, Pine Bluff, AR 71601, USA

Accepted 1 March 1994

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Abstract

Three experiments were conducted to evaluate the relative importance of calcium hardness and bicarbonate alkalinity to the acute response of juvenile channel catfish (Ictalurus punctatus) exposed to a toxic concentration of copper sulfate. A preliminary bioassay revealed 28 mg∙l^-1 copper sulfate caused 50% mortality within 48 h (48-h LC₅₀) in juvenile channel catfish placed in water with calcium hardness and bicarbonate alkalinity, set at 75 mg∙l^-1 CaC0₃. Catfish were then exposed to 28 mg∙l^-1 copper sulfate concentrations in environments where hardness or alkalinity concentrations were varied. Bicarbonate alkalinities above 75 mg∙l^-1 CaC0₃, with calcium hardness held at 20 mg∙l^-1 CaC0₃, significantly reduced catfish mortalities from 97-100% to 63-70%. Copper-induced mortalities were 100% for all fish placed in calcium hardness treatments (20-250 mg∙l^-1 CaC0₃) in which bicarbonate alkalinity was held at 20 mg∙l^-1 CaC0₃. When bicarbonate alkalinity was held constant at 75 mg∙l^-1 CaC0₃ and calcium hardness was varied from 20 to 250 mg∙l^-1 CaC0₃, copper related catfish mortalities displayed high variability and means ranged from 6.7 to 60%. Mortalities decreased as calcium concentrations increased. Although differences in mortalities were not statistically significant, the latter hardness findings appear to suggest a biologically significant calcium effect on copper toxicity in the presence of sufficient alkalinity concentrations. _____________________________________________________________________________________

1. Introduction

Copper sulfate has been used to control protozoan diseases in fish and is used extensively in ponds as an algicide. However, above a specific concentration, copper is toxic to fish including  such  cultured  species  as  salmonids, cyprinids and catfish. Recommendations for

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*Corresponding author.

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W.A. Wurts, P.W. Perschbacher / Aquaculture 125 (1994) 73-79
74        

safe use have been based on water hardness (Inglis and Davis, 1972; Sawyer et al., 1989) and, more recently, on alkalinity (Post, 1983; MacMillan, 1985; Reardon and Harrell 1990; Wurts,1990).

Alkalinity is generally recognized as influencing copper toxicity to fish through the formation of less toxic copper-base complexes (Stiff, 1971; Pagenkopf et al., 1974). High concentrations of calcium, a major component of hardness, are also thought to limit copper toxicity by protecting the ion-regulating mechanisms at the gills from the disruptive effects of copper (Pagenkopf, 1983). Experiments evaluating the effects of hardness and alkalinity on copper toxicity have been conducted with salmonids in soft water. Studies with rainbow trout (Salmo gairdneri) demonstrated that a threshold level of calcium hardness must be present before alkalinity will influence copper toxicity (Miller and Mackay, 1980).

Information about the relative effects of bicarbonate alkalinity and calcium hardness on copper toxicity to channel catfish (Ictalurus punctatus) is inconclusive. The purpose of our research was to determine whether alkalinity, calcium concentration or an interrelationship of the two had the most pronounced effects on the acute toxicity of copper sulfate to channel catfish fingerlings.

2. Methods

A preliminary bioassay revealed that 28 mg·l^-1 copper sulfate was required to effect 50% mortality in 3-6 g juvenile channel catfish within 48 h (48-h LC₅₀) in water with predetermined concentrations of bicarbonate alkalinity and calcium hardness. Increments of copper concentrations used to determine this 48-h LC₅₀ followed EPA recommendations (US Environmental Protection Agency, 1975). Test water contained 75 mg·l^-1 calcium hardness and 75 mg·l^-1 bicarbonate alkalinity. Water temperature was 20°C.

The preliminary bioassay was conducted to facilitate comparisons of the relative importance of calcium hardness versus bicarbonate alkalinity on acute copper toxicity to juvenile channel catfish. These experiments were not intended as an examination of species resistance to copper or to establish specific recommendations for copper use.

Controlled, laboratory experiments were conducted to evaluate the mortality response of juvenile channel catfish exposed to a 28 mg·l^-1 concentration of copper sulfate in waters with differing concentrations of bicarbonate alkalinity and calcium hardness. Three independent trials were conducted: one which varied alkalinity and two which varied hardness. Each combination of hardness and alkalinity was replicated in three, aerated, 7.6-liter aquaria.

Each aquarium was stocked with 10 juvenile channel catfish ranging in size from 3.0-5.6 g and 55-70 mm TL. Averages were 4.4 ± 0.8 g and 64 ±4.4 mm. Fish were not fed for 48 h before experiments were begun. Catfish were maintained for 24 h preceding each experiment in a holding tank with water at the same temperature and containing the median concentrations of alkalinity and hardness for the subsequent trial. After stocking, juvenile catfish were held in test aquaria environments for 2 h before adding copper sulfate. Water temperature, dissolved oxygen, and pH were measured in all aquaria before the addition of copper and at 24-h intervals. Mortalities were removed regularly and totaled at 18, 24 and 48 h.

W.A. Wurts, P.W. Perschbacher / Aquaculture 125 (1994) 73-79                                 75

In all experiments, calcium hardness and bicarbonate alkalinity were adjusted to desired levels with reagent-grade calcium sulfate dihydrate and food-grade sodium bicarbonate. Copper concentrations for each experiment were added using a 3000 mg·l^-1, stock solution of reagent-grade cupric sulfate pentahydrate. Reagent grade potassium chloride was added to all experimental treatments to create a concentration of 5 mg·l^-1 as recommended by EPA guidelines (US Environmental Protection Agency, 1975). Water treatments and stock solutions were made with distilled water.

Copper sulfate concentrations in test environments were verified by measuring free or non-complexed, dissolved cuprous and cupric ions using a bicinchoninate photometric technique (Hach Co., Loveland, CO). Bicarbonate alkalinity and calcium concentrations were measured as mg·l^-1 calcium carbonate (mg·l^-1  CaCO₃) with bromcresol green-methyl red and EDTA titration techniques, respectively.

Trial 1 involved exposing fish to copper in environments with 5 concentrations of bicarbonate alkalinity ranging from 20 to 250 mg·l^-1. Calcium hardness was held at 20 mg·l^-1. This experiment was conducted to determine the relative importance of bicarbonate alkalinity to copper toxicity.

Trial 2 examined the effects of copper on fish in environments with 5 levels of calcium hardness ranging from 20 to 250 mg·l^-1. Bicarbonate alkalinity was held constant at 20 mg·l^-1. The objective of this experiment was to ascertain the relative importance of calcium to copper toxicity.

Trial 3 subjected fish to copper in environments with 5 calcium hardness concentrations ranging from 20 to 250 mg·l^-1. Bicarbonate alkalinity was held constant at 75 mg·l^-1. The purpose of the experiment was to expand the findings of trial 2.

Analysis of variance and Fischer's LSD (Ott, 1977) were performed on mortality data. Percentile data were transformed using the arc-sine method suggested by Mostellar and Youtz (1961). Significance was tested at the 0.05 level. Regression analyses examined mortality as a function of alkalinity and hardness.

3. Results

Dissolved oxygen concentrations ranged from 6.6 to 8.2 mg·l^-1 and were above 75% of saturation in each aquarium for all experiments. In all water treatments, pH ranged from 6.95 to 7.8. The water temperature reported for each trial was stable for the duration of that experiment.

In trial 1, there were significant differences among experimental groups with respect to mortality and bicarbonate alkalinity concentrations (Table 1). At 48 h, mortality was significantly higher for fish exposed to 28 mg·l^-1 copper sulfate in environments with alkalinity concentrations of 75 mg·l^-1 or less. Water temperature was 24°C.

In trial 2, all experimental groups exhibited 100% mortality at 48 h (Table 2). Calcium hardness concentrations from 20 to 250 mg·l^-1 did not protect catfish fingerlings from the toxic effects of copper with bicarbonate alkalinity held constant at 20 mg·l^-1. Water temperature was 21°C.

In trial 3, no significant differences were found among experimental groups with respect to mortality   and   calcium   hardness   (20-250 mg·l^-1)   with   bicarbonate   alkalinity   held   at  75

 76                   W.A. Wurts, P.W. Perschbacher / Aquaculture 125 (1994) 73-79 

Table 1 Mean mortalities of juvenile channel catfish exposed to 28 mg·l-1 copper sulfate at varying bicarbonate alkalinity concentrations and a fixed, low concentration of calcium hardness
Alkalinity	Hardness	pH	DO	Mortality1
(mg·l-1)	(mg·l-1)		(mg·l-1)	18-h	24-h	48-h
					(%)
20	20	7.15	6.6	100.0w	100.0w	100.0w
50	20	7.15	7.1	100.0w	100.0w	100.0w
75	20	7.22	7.2	90.0w	96.7w	96.7w
125	20	7.48	7.5	36.7x	53.3x	70.0x
250	20	7.81	7.3	16.7x	20.0x	63.3x
1Values within a specific time period (18-, 24- or 48-h) and followed by the same superscript were not significantly different at the 0.05 level.

Table 2 Mean mortalities of juvenile channel catfish exposed to 28 mg·l-1 copper sulfate at varying calcium hardness concentrations and a fixed, low concentration of bicarbonate alkalinity
Alkalinity	Hardness	pH	DO	Mortality1
(mg·l-1)	(mg·l-1)		(mg·l-1)	18-h	24-h	48-h
					(%)
20	20	7.14	7.2	100.0w	100.0w	100.0w
20	50	7.12	7.1	100.0w	100.0w	100.0w
20	75	7.00	7.4	100.0w	100.0w	100.0w
20	125	7.00	7.2	100.0w	100.0w	100.0w
20	250	6.95	6.9	93.3w	100.0w	100.0w
1Values within a specific time period (18-, 24- or 48-h) and followed by the same superscript were not significantly different at the 0.05 level.

Table 3 Mean mortalities of juvenile channel catfish exposed to 28 mg·l-1 copper sulfate at varying calcium hardness  concentrations and a fixed, moderate concentration of bicarbonate alkalinity
Alkalinity	Hardness	pH	DO	Mortality1
(mg·l-1)	(mg·l-1)		(mg·l-1)	18-h	24-h	48-h
					(%)
75	20	7.24	8.0	30.0w	53.3w	60.0w
75	50	7.27	8.1	6.7w	16.7w	20.0w
75	75	7.45	8.1	10.0w	10.0w	13.3w
75	125	7.48	8.2	10.0w	17.7w	20.0w
75	250	7.53	7.9	6.7w	6.7w	6.7w
1Values within a specific time period (18-, 24- or 48-h) and followed by the same superscript were not significantly different at the 0.05 level.

W.A. Wurts, P.W. Perschbacher / Aquaculture 125 (1994) 73-79                      77

Table 4 Correlation coefficients comparing 18-, 24-, and 48-h mortality and concentrations of alkalinity (Trial 1) or hardness (Trial 3) for juvenile channel catfish exposed to 28 mg·l-1 copper sulfate
Trial	n	r
		18-h	24-h	48-h
1. Alkalinity	15	-0.88	-0.92	-0.74
3. Hardness	15	-0.30	-0.44	-0.47

mg·l^-1   (Table 3). Although percent mortality was highly variable, 48-h mortality was the lowest (mean, 6.7%; range, 0-20%) in environments with the highest hardness (250 mg·l^-1). Mortality was the highest (mean, 60%; range, 20-90%) in replicates with the lowest hardness concentrations (20 mg·l^-1). Temperature was 16°C.

Correlations between mortality and alkalinity or hardness are presented in Table 4. A

significant (P < 0.01) linear relationship was demonstrated between increasing alkalinity and decreasing mortality in trial 1 at all time intervals. However, no statistically significant relationship could be established between mortality and calcium concentration.

4. Discussion

The close inverse relationship between alkalinity and mortality observed in trial 1 appeared to have resulted from the increased bases, associated with elevated alkalinity concentrations, which combined with copper to form less toxic compounds (Pagenkopf et al., 1974; Boyd, 1979). While an increase in alkalinity diminishes the toxicity of copper, an increase in water temperature generally exacerbates the toxic effects (Sorensen, 1991). It is likely that the lower temperature (16°C) in Trial 3 contributed to the reduction in mortalities as well as the high variability observed.

Some researchers have noted that a minimum concentration of free calcium was necessary for alkalinity to have an effect on copper or zinc toxicity. For example, Jones (1938) found that a calcium hardness of 25 mg·l^-1 was necessary to increase survival of sticklebacks (Gasterosteus aculeatus) exposed to toxic levels of zinc. Miller and Mackay (1980) observed the incipient LC₅₀ (ILC₅₀) of copper for juvenile rainbow trout (Salmo gairdneri) did not change when alkalinity concentrations were increased from 10 to 50 mg·l^-1 and hardness was held at 12 mg·l^-1. However, the ILC₅₀ increased 3-fold when hardness was increased from 12 to 100 mg·l^-1 and alkalinity was held at 10 mg·l^-1.

In trial 3, mortality decreased as calcium hardness levels increased from 20 to 250 mg·l^-1, when bicarbonate alkalinity was held at 75 mg·l^-1 (Table 1). The statistical analysis for trial 2 and the analysis for trial 3 did not detect significant differences among mean mortalities for either individual experiment. However, a relative comparison of mean, 48-h mortalities between the two studies (100 vs. 6.7-60.0%, respectively) suggests calcium was biologically important when sufficient alkalinity (20 vs. 75 mg·l^-1, respectively) was present. Miller and Mackay (1980) reported the ILC₅₀ copper concentration for juvenile rainbow trout was significantly greater at 100 mg/l hardness when alkalinity was increased
78                        W.A. Wurts, P. W. Perschbacher /Aquaculture 125 (1994) 73-79

from 10 to 50 mg·l^-1. The present findings suggest that a calcium hardness between 20 and 250 mg·l^-1 may minimize mortality in juvenile channel catfish exposed to toxic concentrations of copper at specific alkalinity concentrations. These observations with channel catfish are consistent with those for trout.

  Juvenile channel catfish were remarkably resistant to a 48-h exposure to copper, requiring a concentration of 28 mg·l^-1 copper sulfate to effect a LC₅₀ at 20°C (hardness and alkalinity, 75 mg·l^-1). Miller and Mackay (1980) calculated the ILC₅₀ at 13°C for juvenile rainbow trout to be 80 µg·l^-1 copper at 50 mg·l^-1 alkalinity and 75 mg·l^-1 hardness—a considerably lower copper concentration. However, it would be inappropriate to make direct comparisons between these findings with channel catfish and those for juvenile trout. Experimental designs were dissimilar (acute vs. chronic exposure) and there appear to be species-related differences in sensitivity to copper.

   While alkalinity reduces toxicity by combining chemically with copper, high calcium concentrations apparently block or minimize the effects of copper at the sites of toxic action. Copper, a divalent cation, would have chemical activity and ionic form similar to the calcium ion (and possibly magnesium). It has been theorized that calcium-activated proteins control the passive and energy-dependent processes regulating ion metabolism at the gills (Evans, 1975; Wurts and Stickney, 1989). Pic and Maetz (1981) observed that an environmental calcium concentration of 40 mg·l^-1 was necessary to sustain maximum function of the mechanisms associated with the energy-dependent exchange of sodium and potassium ions in mullet (Mugil capito). It is likely that copper competes directly with calcium for the same binding sites on ion regulating proteins. Therefore, high concentrations of calcium (i.e., a high ratio of calcium to copper ions) would keep binding sites maximally saturated, preventing copper from attaching and interfering with normal protein functions (i.e., ion metabolism). Sorensen (1991) indicated that both calcium and magnesium may confer similar protection. Further research in this area is warranted.

   The present study supports recommendations and research which indicate that alkalinity is the primary factor affecting acute copper toxicity in aquatic environments. A minimum calcium hardness concentration between 20 and 250 mg/1 may be important to the maintenance of normal ion metabolism in juvenile channel catfish exposed to toxic concentrations of copper. These experiments emphasize the importance of controlling alkalinity concentration, as an independent variable, before attempting to evaluate a calcium effect on acute copper toxicity to fish. The acceptance of alkalinity-based recommendations (Wellborn, 1985; MacMillan, 1985) for the use of copper in fish ponds seems prudent.

References

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Evans, D.H., 1975. Ionic exchange mechanisms in fish gills. Comp. Biochem. Physiol, 51A: 491-495.

Inglis, A. and Davis, E.L., 1972. Effects of water hardness on the toxicity of several organic and inorganic herbicides to fish. U.S. Fish and Wildlife Service Technical Paper 67.

Jones, J.R.E., 1938. The relative toxicity of salts of lead, zinc and copper to the stickleback (Gasterosteus aculeatus L.) and the effect of calcium on the toxicity of lead and zinc salts. J. Exp. Biol, 4: 394-407.

MacMillan, J.R., 1985. Infectious diseases. In: C.S. Tucker (Editor), Channel Catfish Culture. Elsevier, Amsterdam, pp. 405-496.

     

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