Effect of Calcium Pre-Exposure on Acute Copper Toxicity to
Juvenile Nile Tilapia, Oreochromis niloticus (L.)
Mohsen Abdel-Tawwab* and Mamdouh A. A. Mousa
Fish Ecology Department, Central Laboratory for Aquaculture Research,
Abbassa, Abo-Hammad, Sharqia, Egypt.
* Corresponding author email: mohsentawwab@yahoo.com
ABSTRACT
This study was carried out to evaluate the copper toxicity to Nile tilapia; O. niloticus
(L.), and the effect of fish pre-exposure to liming agents and concentrations on copper toxicity
indicated by LC50. Fish weighing 1.8-2.5 g was randomly distributed into the aquaria at a rate
of 50 fish/100 L. The temperature was ranged from 26 to 28 oC. Fish are pre-exposed to
calcium oxide, calcium chloride, calcium sulfate or calcium carbonate at a rate of 100 mg
Ca/L for 4 days. Then, fish are exposed to different concentrations of copper sulfate, and LC 50
are determined. The highest LC50 was obtained with CaO (14.27 mg Cu/L). In the second
experiment, fish are pre-exposed to CaO concentration of 0 (control), 50, 100 or 200 mg Ca/L
for 4 days, and exposed to different concentration of copper sulfate. The obtained results
revealed that the LC50 of fish not exposed to calcium and exposed to copper was 5.03 mg
Cu/L. The pre-exposure of fish to different liming agents for 4 days significantly reduced
copper toxicity. The second experiment showed that pre-exposure to CaO concentration of
50-200 mg Ca/L significantly reduced copper toxicity. The LC50 values are slightly increased
with increasing calcium concentration (P>0.05), and the optimum one was obtained at 50 mg
Ca/L (13.23 mg Cu/L).
Keywords: Calcium, liming agents, Nile tilapia, copper, toxicity.
INTRODUCTION
In aquaculture, copper sulfate is often used as an algaecide in commercial and
recreational fishponds to control growth of phytoplankton and filamentous algae, control
certain fish disease (Boyd, 1990; Tucker and Robinson, 1990). Boyd (1990) stated that the
concentrations of copper sulfate used for phytoplankton control are seldom directly toxic to
fish, but do kill large numbers of invertebrate food organisms such as rotifers, cladocerans
and copepods. However, above a specific concentration, copper is toxic to fish including such
cultured species as salmonids, cyprinids and catfish (Wurts and Perschbacher, 1994). As a
result, treatment recommendation for the use of copper sulfate for finfish are 0.0.5-1.0 mg/L
(Lightner, 1983; Boyd, 1990).
Nile tilapia is a native fish species of Egypt that grows faster in warm months, and
has become more popular allover the world because it is relatively easy in a variety of
aquaculture systems and because tilapia are favorable food fishes (El-Sayed, 2004). However,
Nile tilapia is omniphorous fish and could consume detritus, phytoplankton and zooplankton
(Abdelghany (1993), Abdel-Tawwab, 2000; Abdel-Tawwab and El-Marakby, 2004). Thus,
fish grazed plankton organisms leading to the accumulation of copper inside fish tissues
reaching the toxic concentration.
Copper toxicity is known to be regulated by alkalinity, hardness and pH of water
(Masuda and Boyd, 1993). Therefore, recommendations for safe use of copper sulfate have
been based on hardness (Sawyer et al., 1989; Perschbacher and Wurts, 1998), total alkalinity
of the water (Boyd, 1990; Reardon and Harrell, 1990; Perschbacher and Wurts, 1998), and pH
(Masuda and Boyd, 1993). 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). One means to increase the
uptake of calcium by aquatic organisms is to increase the level of environmental calcium
through application of liming agents. Chakraborti and Mukherjee (1995) found that total
plasma calcium level of common carp (40-50 g) raised in tap water (0.15 mM/L Ca2+)
remains within 3 mmol/L, while fish kept in high calcium freshwater shows larger
hypercalcemic responses. Calcium supplied through liming reduce the uptake of heavy metals
(Raddum et al., 1986; Andersson and Borg, 1988). The objective of the present study was to
determine the acute toxicity of copper to juvenile Nile tilapia, Oreochromis niloticus (L.) preexposed to different calcium sources and concentrations.
MATERIALS AND METHODS
Experimental procedures
Healthy fish of Nile tilapia, Oreochromis niloticus (L.) were collected from fish
hatchery of Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia.
Fish weighing 2-2.5 g/fish were acclimated in indoor tanks for 2 weeks to laboratory
conditions. The fish of mixed sex were distributed randomly in glass aquaria of 150-liter
capacity at a rate of 100 fish/aquarium that containing 140 liter aerated water. Each aquarium
was supplied with compressed air via air-stones from air pumps. Well-aerated water supply
was provided from a storage fiberglass tank. The ambient temperature ranged 26-28 oC.
Two experiments were conducted to establish the effect of fish pre-exposure to
calcium sources or concentrations on the acute copper toxicity in indoor laboratory. The first
experiment was conducted to evaluate the effect of different calcium sources on copper
toxicity indicated by LC50. Fish were kept for 4 days in aquarium, and exposed to 100 mg
Ca2+/L of calcium oxide, calcium sulfate, calcium chloride or calcium carbonate. After fish
exposed to calcium sources, fish were assigned at a rate of 5 fish per glass aquarium
containing 10 liters and exposed to different concentrations of copper sulfate. The dead fish at
each copper concentration was recorded and removed. The 96-hr LC50 for copper was
determined according to Behreus and Karber (1953). From the first experiment, calcium oxide
is the optimum Ca source reduced copper toxicity by increasing LC50 value. In the second
experiment, in the same aquarium at similar condition as previously described, another 100
fish were kept for 4 days, and exposed to different concentrations of calcium oxide equivalent
to either 0 (control), 50, 100 or 200 mg Ca2+/L to obtain the optimum Ca2+ concentration.
After fish exposed to calcium concentrations, fish were assigned in the small glass aquaria
and exposed to different concentrations of copper sulfate to determine the 96-hr LC50 for
copper as described in the first experiment.
During the experiments running, fish were fed frequently a diet contained 35% crude
protein to satiation twice daily. Excreta removing was done by siphoning a portion of
aquarium water and replaced by an equal volume of water containing the same applied
chemicals at the same calcium or copper concentration.
Analysis of water physico-chemical parameters
Water samples for chemical analyses were collected daily at 30 cm depth from each
aquarium. Dissolved oxygen and temperature were measured on site with a YSI model 58
oxygen meter (Yellow Spring Instrument Co., Yellow Springs, Ohio, USA). The pH degree
and ammonia were measured using Hach kits (Hach Co., Loveland, Colorado, USA). Total
alkalinity and total hardness were measured by titration as described by Boyd (1984).
Statistical analysis
The obtained data were subjected to two-way ANOVA and the differences between
means were done at the 5% probability level using Duncan’s new multiple range test.
Correlation analyses were performed by fitting the data into a curve linear selecting the model
giving the best fit. The software SPSS, version 10 (SPSS, Richmond, USA) was used as
described by Dytham (1999).
RESULTS
Dissolved oxygen concentrations ranged from 6.6 to 7.5 mg/L and were above 75% of
saturation in each aquarium for both experiments. The ambient water temperature was
approximately stable for the experimental duration and ranged from 26 to 28oC. In all water
treatments, pH ranged from 8.0 to 8.5, and free ammonia concentration was less than the
critical level. Total alkalinity and total hardness were ranged from 160 to 200 mg/L as CaCO3
and from 120 to 150 mg/L as CaCO3, respectively.
The toxicity of copper of Nile tilapia was determined by determining the LC 50
indicating the toxic concentration at which 50% of fish number died after 96 hours.
Comparing the copper toxicity of Nile tilapia pre-exposed to different calcium sources
equivalent to 100 mg Ca2+/L, Fig 1 shows that the highest LC50 was obtained with calcium
oxide treatment (14.27 mg Cu2+/L), meanwhile calcium sulfate resulted in the les LC50 value
(9.29 mg Cu2+/L; P<0.05). Moreover, data in Fig 2 show the LC50 of Nile tilapia pre-exposed
to different concentrations of calcium oxide. The optimum LC50 was observed at dose of 50
mg Ca2+/L with insignificant differences with calcium treatments (P<0.05). The less LC50 was
obtained at control (5.03 mg Cu2+/L; P<0.05).
Data in Fig 3 showed that only calcium oxide and calcium chloride could support the
fish to tolerate the copper toxicity up to 20 mg/L after which fish survival rate decreased with
increasing copper concentration (r2 = - 0.9454 and - 0.8673, respectively). The survival rate of
fish pre-exposed to calcium sulfate or calcium carbonate are the least ones (P<0.05). On the
other hand, all calcium sources supported fish survival rate, with different percentages, up to
40 mg Cu/L after which no fish survived except calcium oxide that supported the fish survival
rate up to 70 mg Cu/L after which no fish survived.
The optimum survival rate in the first experiment was obtained with fish pre-exposed
to calcium oxide. However, in the second experiment, fish are pre-exposed to different doses
of calcium oxide equivalent to 0 (control), 50, 100 or 200 mg Ca2+/L. The obtained results
reveal that all doses of calcium oxide supported the fish survival against copper toxicity better
than control (P<0.05; Fig 4). This support reached up to 20 mg Cu/L except 50 mg Ca2+/L
supported fish up to 30 mg Cu/L after which fish survival rate decreased with increasing
copper toxicity up to 60 mg Cu/L. In control fish, no survival threshold was observed where
copper toxicity inversely affected fish survival up to 40 mg Cu/L after which no fish survival
was observed.
DISCUSSION
Fish are naturally exposed to a variety of metals including both essential and nonessential elements. Copper is one of the essential metals that after absorption from gills and
intestine is transported by metallothionein into blood circulation. Heerden et al. (2004) found
gill damage in rainbow trout (Oncorhynchus mykiss) exposed to copper after 4 h. Cerquiera
and Fernandes (2002) found gill damage in fish exposed to sublethal concentration of copper
for 96 h. disruption of gill function in fish by copper exposure was found on several occasions
(Dang et al., 2000; Daglish and Nowak, 2002). This disruption lead to changes in the
diffusion distance across gill epithelium (Weibel and Knight, 1964), which might impede gas
change, leading to tissue hypoxia (Heerden et al., 2004).
Water chemistry especially pH, alkalinity and hardness could affect heavy metal
toxicity (Miller and Mackay, 1980). Miller and Mackay (1980) observed the incipient LC50 of
copper for juvenile rainbow trout (Salmo gairdneri) increased when hardness was increased
from 12 to 100 mg/L and alkalinity was held at 10-50 mg/L. Also, Wurts and Perschbacher
(1994) observed the LC50 of copper to channel catfish (Ictalurus punctatus), and found that
mortality decreased as calcium hardness levels increased from 20 to 250 mg/L, when
bicarbonate alkalinity was held at 75 mg/L. Copper, as a divalent cation, would have chemical
activity and ionic form similar to the calcium ion (and possibly magnesium). So, in hard
water, copper may compete with calcium for the active sites in the gills, and calcium fail to
significantly reduce copper toxicity. Liming not only supplies calcium for uptake, but
increased levels of environmental calcium that reduce the uptake of heavy metals (Raddum et
al., 1986; Andersson and Borg, 1988). Sorenson et al. (1985) reported that when Cd
concentrations are already present, the protective effect of liming is reduced, as Cd competes
with calcium for uptake. Therefore, pre-exposure of fish to liming agents in heavy metal-free
water would seem to be necessary to prevent effects associated with future exposure to heavy
metals.
The pre-exposure of Nile tilapia to calcium could bind the active sites in the gills, and
the exposure to copper ion after exposure did not find free site to bind. So, copper toxicity
significantly reduced. This hypothesis could explain the result finding in this study where the
pre-exposure of Nile tilapia to calcium irrespective to the source increases the survival rate
more than the control (no pre-exposed to calcium). In this concern, Dutta and Kaviraj (1996)
found that the pre-exposure to liming agents might be effective in reducing the acute toxicity
of Cd to carps. This study has shown that LC50 value of Cd to common carp, Cyprinus carpio)
is 165 mg/L, while toxicity is reduced and the LC value of Cd increases to 235 mg/L when
the fish is pre-exposed to 100 mg/L quick lime. In addition, uptake and distribution of Cd in
fish have shown to be reduced after fish were acclimated to selected calcium concentrations
(Wicklund and Runn, 1988). It has been theorized that calcium-activated proteins control the
passive and energy dependent process regulating ion metabolism at the gills (Evans, 1975;
Wurts and Stickney, 1989). It is likely that copper competes directly with calcium for the
same binding sites on ion regulating proteins. Therefore, high concentrations of calcium
would keep binding sites maximally saturated preventing copper from attaching and
interfering with normal protein functions (i.e. ion metabolism).
Apart from absorption through chloride cells, calcium binds to branchial epithelium
and maintains membrane stability and water permeability (Reid and McDonald, 1991). Fish
adapted to environments of varying calcium concentration modify these processes, resulting
in altered permeability and ion regulation (Gundersen and Curtis, 1995). It has been found
that rainbow trout gills exposed to low Ca2+ at acidic pH exhibits a marked increase in
osmotic permeability (Parker et al., 1985) and net losses Na+ and Cl- (Reid et al., 1991; Freda
et al., 1991). In contrast, a decrease in gill permeability can be expected when it is exposed to
high concentration of Ca2+. Apart from alteration in permeability, competition between the
Ca2+ ion and the other ions entering through the gill was observed (Kaviraj and Dutta, 2000).
There is evidence that acclimation of fish to calcium reduces the uptake of Cd from water and
transfer of Cd from gill to blood (Wicklund and Runn, 1988). This information justify the
reduced tissue concentration of Cd in common carp pre-exposed to calcium oxide (Kaviraj
and Dutta, 2000). They also reported that the reduced tissue concentration of Cd is the
greatest advantage of CaO pre-exposure for common carp culture in polluted water.
In conclusion, the results presented herein indicate that fish pre-exposing to 50-100
mg Ca2+/L could support the fish survival and growth in polluted water. Further work is
needed indicating the changes in biochemical aspects and the growth performance in fish preexposed to calcium and copper toxicity.
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18
a
16
14
b
b
b
LC50
12
10
c
8
6
4
2
0
Control
Ca carbonate
Ca sulfate
Ca chloride
Ca oxide
Calcium sources
Figure 1. Changes in LC50 of Nile tilapia pre-exposed to different calcium sources
followed by exposing to copper for 96 h. Bars assigned with the same letter are
not significantly differed at P<0.05.
18
16
a
a
50
100
a
14
LC 50
12
10
8
b
6
4
2
0
0 (control )
200
Calcium doses (mg/L)
Figure 2. Changes in LC50 of Nile tilapia pre-exposed to different doses of calcium oxide
followed by exposing to copper for 96 h. Points assigned with the same letter are
not significantly differed at P<0.05.
CaCO3
CaSO4
CaCl2
CaO
100
CaO R2 = - 0.9454
2
CaCl2 R = - 0.8673
2
CaSO4 R = - 0.868
60
CaCO3 R2 = - 0.9147
40
(%)
Fish survival
80
20
0
0
10
20
30
40
50
60
70
80
90
Cu dose (mg/L)
Figure 3. Fish survival (%) of Nile tilapia pre-exposed to different Ca sources and
exposed to different doses of copper for 96 h.
0 mg Ca/L
50 mg Ca/L
100 mg Ca/L
100
-1
2
0 mg Ca L R = - 0.9795
-1 2
50 mg Ca L R = - 0.9506
-1 2
100 mg Ca L R = - 0.9345
-1 2
200 mg Ca L R = - 0.918
80
60
40
(%)
Fish survival
200 mg Ca/L
20
0
0
10
20
30
40
50
60
70
80
90
Cu dose (mg/L)
Figure 4. Fish survival (%) of Nile tilapia pre-exposed to different Ca concentrations
and exposed to different doses of copper for 96 h.