ACUTE TOXICITY OF WATER-BORN ZINC IN NILE TILAPIA, Oreochromis
niloticus (L.) FINGERLINGS
Mohsen Abdel-Tawwab1*, Gamal O. El-Sayed2, and Sherien H.H.H. Shady1
1
Department of Fish Ecology and Biology, Central Laboratory for Aquaculture Research, Abbassa,
Abo-Hammad, Sharqia, Egypt
2
Chemistry Department, Faculty of Science, Benha University, Benha, Egypt
*Corresponding author email: mohsentawwab@yahoo.com
Abstract
Zinc (Zn) is an essential trace element for most organisms including fish, but above certain
limit Zn will be toxic. The present study was conducted to evaluate the toxic effect of water-born Zn
on Nile tilapia, Oreochromis niloticus (L.) via estimating the acute 96-h median lethal concentration
(LC50) value and behavioral changes. A total 140 of Nile tilapia fingerlings was subjected to 14 20-L
aquaria. Fish were exposed to 0.0, 10, 40, 70, 100,130, or 160 mg Zn/L for 4 days. Each Zn dose was
represented by two aquaria. Fish was daily observed and dead fish were removed immediately. The
data obtained were statistically evaluated using Finney’s Probit Analysis Method and Behrens–Karber’s
Method. The 96 h LC50 value for Nile tilapia was found to be 63.984 mg/L with 95% confidence limits
of 48.029 – 78.372 mg/L. This value was calculated to be 70.0 mg/L with Behrens–Karber’s Method.
The behavioral changes of Nile tilapia were primarily observed as nervous and respiratory
manifestations. It could be concluded that Nile tilapia is a species slightly sensitive to Zn and the two
methods were relatively comparable.
INTRODUCTION
Pollution of the aquatic environment with heavy metals has become a serious health concern
in recent years. These metals are introduced into the aquatic ecosystem through various routes such
as industrial effluents and wastes, agricultural pesticide runoff, domestic garbage dumps and mining
activities (Merian, 1991). Increased discharge of heavy metals into natural aquatic ecosystems can
expose aquatic organisms to unnaturally high levels of these metals (van Dyk et al., 2007). Among
aquatic organisms, fish cannot escape from the detrimental effects of these pollutants, and are
therefore generally considered to be the most relevant organisms for pollution monitoring in aquatic
ecosystems (van der Oost et al., 2003).
It has been reported that heavy metals had a negative impact on all relevant parameters and
caused histo-pathological changes in fish. Some heavy metals are essential elements, while others are
non-essential. Zinc (Zn) is one of the most important trace metals in the body, and participates in the
biological function of several proteins and enzymes (Maity et al., 2008). Despite being an essential
trace element, Zn is toxic to most organisms above certain concentrations (Ho, 2004). Since the
range-finding acute test is conducted to pinpoint exposure concentrations; the definitive acute test is
firstly conducted to estimate LC50 of the chemical to which organisms are exposed (Rand, 2008). Nile
tilapia, Oreochromis niloticus (L.) is an important commercial fish in Egypt and worldwide (El-Sayed,
2006) and it could be used as test organism for evaluation the impact of heavy metals. Consequently,
the objective of this study is to assess the responsiveness of Nile tilapia to Zn through determination
of acute 96-h LC50 value and behavioral responses induced from exposure to different Zn
concentrations.
MATERIALS AND METHODS
Fish management:
Apparently healthy Nile tilapia, O. niloticus (L.) (4.6 ± 0.2 g) were btained from fish hatchery,
Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia, Egypt. Prior to the
experiment, fish were acclimatized for 2 weeks in 14 40-L glass aquaria under laboratory conditions
(natural photoperiod 11.58–12.38 h); 10 fish per each aquarium. The continuous aeration was
maintained in each aquarium using an electric air pumping compressors. Fish were fed daily on
commercial fish diet containing 25% crude protein provided for satiation twice daily at 9:00 and 14:00
h.
Analysis of the water physico-chemical variables:
Water samples were collected from each aquarium prior to Zn exposure. Dissolved oxygen
and temperature were measured on site with an oxygen meter (YSI model 58, Yellow Spring
Instrument Co., Yellow Springs, Ohio, USA). pH value was measured using a pH-meter (Digital MinipH Meter, model 55, Fisher Scientific, Denver, USA). Total alkalinity and total hardness were
measured according to Boyd (1984). The mean values for test water variables were as follows:
dissolved oxygen 5.84±0.72 mg/L, pH 7.5±0.1, water temperature 25.5± 0.1 oC, total alkalinity
153.7±4.8 mg/L as CaCO3, and total hardness 222.5±2.9 mg/L as CaCO3.
Experimental procedures:
The heavy metal Zn in the form of zinc sulfate anhydrous (Analar grade, Merck, Readington
Township, New Jersey, USA) was used in the present study. The acute toxicity test was performed for
4 days in which two replicates of seven different Zn concentrations (0, 10, 40, 70, 100, 130, and 160
mg/L) were used (10 fish for each aquarium). At 24, 48, 72, and 96 h, fish dead were counted in the
different Zn concentrations along with the control group. In this study, the acute toxic effects of Zn on
Nile tilapia were determined by the use of Finney’s Probit Analysis LC50 Determination Method
(Finney, 1971). The computer model (Probit Program Version 1.5 software) was developed by
Environmental Protection Agency (EPA, 1999). It was designed for the analysis of mortality data from
acute toxicity tests with fish and other aquatic life, performed with reference toxicants by regulatory
agencies and permittees under the National Pollutant Discharge Elimination System (NPDES). In
addition, the data were also assessed according to Behrens–Karber’s method using the following
formula (Klassen, 1991):
LC50 = LC100 ∑A x B / N as mg/L;
where LC50 and LC100 indicate the lethal doses for 50% and 100% of the tested fish. Value ‘‘A” gives
the differences between the two consecutive doses, ‘‘B” the arithmetic mean of the mortality caused
by two consecutive doses and ‘‘N” the number of tested fish in each group.
The dead fish were removed immediately. Behavioral changes, clinical toxic signs and postmortem
lesions of tested fish were closely followed up and recorded daily.
RESULTS
The data obtained from the acute toxicity test of water-born Zn for Nile tilapia revealed that
the Zn toxicity increased with increasing concentration and/or exposure time. The number of dead fish
in relation to the Zn concentrations (40, 70, 100, 130 and160 mg/L) were assessed and counted
during the exposure time at 24, 48, 72 and 96 h then they were removed immediately. No mortality
was observed during the 96 h at control (0.0 mg Zn/L) and 100% mortality rate was achieved only at
130 and 160 mg Zn/L (Table 1).
Table 1. The cumulative mortalities and acute 96 h LC50 of water-born Zn in Nile tilapia fingerlings
according to Behrens-Karber's method (Klassen, 1991).
Zn dose
(mg/L)
No. of
exposed
fish
No of dead fish
D1
D2
D3
0
10
40
70
100
130
10
10
10
10
10
10
0
0
0
2
8
8
0
0
1
3
9
10
0
0
2
4
9
10
A
B
AB
D4
Overall
deaths within
96 h
0
0
2
4
9
10
0
0
2
4
9
10
0
10
30
30
30
30
0
0
1.0
3.0
6.5
9.5
0
0
30
90
195
285
∑AB =600
Where A = differences between the two consecutive doses and B = arithmetic mean of the mortality
caused by two consecutive doses.
96 h LC50 = LC100 - ∑ (A x B)/N = 130 – 600/10 = 70.0 ppm.
The relationship between the Zn concentrations and the mortality rate of Nile tilapia
calculated by Finney’s Probit Analysis (Table 2). The mean 96-h LC50 value with 95% confidence
limits for Nile tilapia fingerlings was found to be 63.984 mg/L. This value was estimated to be 70.0
mg/L with the Behrens–Karber’s method (Table 1). The two methods are in good accordance and this
value indicating that the water-born Zn is definitely a slight toxic heavy metal to Nile tilapia.
Table 2. The acute 96-h LC50 values of Zn and their confidence limits in Nile tilapia fingerlings
according to Finney’s Probit Analysis (EPA, 1999).
Point
Zn concentration
95% Confidence Limits
Slope
Intercept
(mg/L)
Lower
Upper
LC/EC 1.00
24.848
7.968
36.878
5.66±1.47 -5.23±2.74
LC/EC 5.00
32.781
13.835
44.830
LC/EC 10.00
37.999
18.501
49.925
LC/EC 15.00
41.982
22.456
53.816
LC/EC 50.00
63.984
48.029
78.372
LC/EC 85.00
97.516
79.486
147.503
LC/EC 90.00
107.738
86.559
177.222
LC/EC 95.00
124.888
97.285
234.828
LC/EC 99.00
164.756
119.253
404.328
Note: Control group (theoretical spontaneous response rate) = 0.0.
Bold value indicated the acute 96 h LC 50 of Zn and its confidence limits in Nile tilapia fingerlings.
Figure 1 shows that large increases in fish mortality are associated with the increases in
exposure concentrations (r2 = 0.9202). Moreover, the LC50 values and the empirical probit values of
the mortality rate were plotted against the water-born Zn concentrations in Fig. 2, which indicates
that Zn does not have cumulative response to test concentrations.
Fig 2. Plot of adjusted probits and predicted regression line.
It was observed that Nile tilapia individuals exhibited a variety of behavioral changes when
subjected to different Zn concentrations. The behavioral and swimming patterns in the control group
were normal and there were no deaths during the experimental period. The behavioral changes and
clinical toxic symptoms in Nile tilapia subjected to different Zn concentrations are the following:
sluggish movement, loss of equilibrium, and rapid operculum movement as respiratory manifestations.
Variable degrees of fin erosions were seen. Fish died during the experiment were immediately
removed from the aquaria and subjected to a necropsy. The necropsy revealed that there were
general congestion of the kidneys and gills, and spots of congestion on the periphery of the liver at
macroscopic scale.
DISCUSSION
In the present study, Zn toxicity was indicated by fish mortality. Shetty Akhila et al. (2007)
reported that the determination of acute toxicity is usually an initial screening step in the assessment
and evaluation of the toxic characteristics of all compounds. Likewise, De Schamphelaere and Janssen
(2004) reported that fish mortality might be a more sensitive endpoint for assessing effect of Zn
exposure. The acute 96-h LC50 value of water-born Zn for Nile tilapia having an average weight 4.6 g
was calculated as 63.984 mg/L by using Finney’s Probit Analysis and 70.0 mg/L by the use of
Behrens–Karber’s method. The 96-h LC50 values obtained for both methods were found to be
relatively comparable. Similar results were obtained El-Sayed et al. (2009) who used both methods to
evaluate the acute toxicity of ochratoxin-A in sea bass (Dicentrarchus labrax L.).
Bengeri and Patil (1986) found that the 96-h LC50 of Zn for Labeo rohita was 65.0 mg/L.
Hilmy et al. (1987) found that the 96-h LC50 of Zn for Tilapia zillii and Clarias lazera at summer (25.0
o
C) was 13.0 and 26.0 mg/L, respectively. Senthil Murugan et al. (2008) found that the 96-h LC50
concentration of Zn for snakehead, Channa punctatus was 48.68 mg/L. The variation in LC50 values
among the different studies may be due to the variations in kinetic variables that may play a role in
explaining these differences. Moreover, the alkaline and hard water in the present study could be
responsible for being the LC50 herein higher than the other studies. In this regard, Weatherley et al.
(1980) and Wood (2001) stated that Zn bioavailability and toxicity to aquatic organisms are affected
by pH, alkalinity, dissolved oxygen, and temperatures. Alabaster and Lloyd (1982) and Everall et al.
(1989) stated that Zn toxicity to fish can be greatly influenced by both water hardness and pH. Hilmy
et al. (1987) found that 96-h LC50 for both fishes increased with the decrease in water temperature.
Eisler (1993) reported that the acute 96-h LC50 values for fish were between 66 and 40,900 µg Zn/L
depending on many factors including pH, alkalinity, dissolved oxygen, and temperatures.
Previous studies have shown that Zn accumulation in fathead minnow, Piinphales promelus,
and common carp, Cyprinus carpio, was reduced in hard water compared with soft waters (Everall et
al., 1989). However, Bradley and Sprague (1985) found that in hard water, Zn accumulation in the
gills of rainbow trout, Salmo gairdneri, was reduced and suggested that water hardness may protect
fish by altering the dynamics of Zn exchange mechanisms. Moreover, the process of metal uptake
may be dependent upon the metal exposure level, its local availability at the sites of uptake and the
duration of exposure. Immediate levels of Zn exposure have been shown to affect the pattern and
rate of metal uptake (Everall, 1987). It is possible that previous metal acclimation may also affect the
pattern and rates of Zn uptake, dependent upon prior tissue loading and depuration (Bradley et al.,
1985).
The loss of positive rheotaxis is a good indication of any toxic response, but in the case of Zn
it takes place when poisoning is already irreversible. Signs of poisoning before loss of positive
rheotaxis are not the same at high and lower concentrations; the air gulping and the increased
opercular movement observed at high concentrations contrast with the general apathy and ataxia, but
without apparent respiration difficulties observed at low concentrations. A comparable behaviour was
reported by Matthiessen (1974) for Sarotherodon mossambicus and Hilmy et al. (1987) for Tilapia zillii
and Clariaz lazera. An interpretation of the toxicity data is that two poisoning mechanism may take
place, one occurring at high concentrations and provoking a rapid suffocation by destruction of the gill
epithelium, the other prevailing at low concentrations and consisting of an inhibition of the main
metabolic pathways.
In conclusion, Nile tilapia is a slightly susceptible species to water-born Zn and the two
methods were relatively comparable and useful. The useful experimental models could be widely used
to assess the aquatic toxicology of heavy metals.
REFERENCES
Alabaster, J.S. and R. Lloyd. 1982. Water Quality Criteria for Freshwater Fish. London: Butterworth, pp
361.
Bengeri, K.V. and H.S. Patil. 1986. Respiration, liver glycogen and bioaccumulation in Labeo rohita
exposed to zinc. Ind. J. Comp. Anim. Physiol. 4: 79-84.
Boyd, C.E. 1984. Water Quality in Warmwater Fishponds. Auburn University Agriculture Experimental
Station, Auburn, Alabama, USA.
Bradley, R.W. and J.B. Sprague. 1985. The influence of pH, water hardness and alkalinity on the acute
lethality of zinc to rainbow trout (Salmogairdneri). Can. J. Fish. Aquat. Sci. 42, 731-736.
Bradley, R.W., C. Duquesnay, and B. Sprague. 1985. Acclimation of rainbow trout, Salmo gairdneri
Richardson, to zinc: kinetics and mechanism of enhanced tolerance induction. J. Fish Biol. 27:
367-379.
De Schamphelaere, K.A., Janssen, C.R., 2004. Bioavailability and chronic toxicity of zinc to juvenile
rainbow trout (Oncorhynchus mykiss): comparison with other fish species and development of
a biotic ligand model. Environ. Sci. Technol. 38: 6201–6209.
Eisler, R. 1993. Zinc Hazards to Fish, Wildlife, and Invertebrates: A Synoptic Review. U.S. Fish and
Wildlife Service, Biological Report 10.
El-Sayed, A.-F.M. 2006. Tilapia Culture. CABI publishing, CABI International Wallingford, Oxfordshire,
UK.
El-Sayed, Y.S., R.H. Khalil, and T.T. Saad. 2009. Acute toxicity of ochratoxin-A in marine water-reared
sea bass (Dicentrarchus labrax L.). Chemosphere 75: 878–882.
EPA 1999. LC50 Software Program, Version 1.50. Ecological Monitoring Research Division,
Environmental Monitoring Systems Laboratory, EPA, Cincinnati, Ohio 45268, USA.
<http://www.epa.gov/nerleerd/stat2.htm>.
Everall, N.C., 1987. The effects of water hardness and pH upon the toxicity of zinc to the brown trout
Salmo trutta. Ph.D. thesis, Trent Polytechnic, Nottingham, UK, pp 242.
Everall, N.C., N.A.A. Macfarlane, and R.W. Sedgwi. 1989. The effects of water hardness upon the
uptake, accumulation and excretion of zinc in the brown trout, Salmo trutta L. J. Fish Biol. 5:
881-892.
Finney, D.J. 1971. Probit Analysis. Cambridge University Press, New York, USA, pp 337.
Hilmy, A.M., N.A. El-Domiaty, A.Y. Daabees, and H.A. Abdel Latife. 1987. Toxicity in Tilapia zillii and
Clarias lazera (Pisces) induced zinc, seasonally. Camp. Biochem. Physiol. 86C: 263-265.
Ho, E. 2004. Zinc deficiency, DNA damage and cancer risk. J. Nutr. Biochem. 15: 572–578.
Klassen, C.D. 1991. Principles of toxicology. In: Gilman, A.G., Tall, T.W., Nies, A.S., Taylor, P. (Eds.),
Pharmacological Basis of Therapeutics, eighth ed. McGraw-Hill, Berlin, pp. 49–61.
Maity, S., S. Roy, S. Chaudhury, and S. Bhattacharya. 2008. Antioxidant responses of the earthworm
Lampito mauritii exposed to Pb and Zn contaminated soil. Environ. Pollut. 151: 1–7.
Matthiessen, P. 1974. Some effects of slow release bis(tri-n-butyltin) oxide on the tropical food fish,
Tilapia mossambica (Peters). In: Controlled Release Pesticide Symposium, Report No. 25, The
University of Akron, Ohio, USA.
Merian, E. 1991. Metals and their Compounds in the Environment. Occurrence, Analysis and Biological
Relevance. VCH: Weinheim.
Rand, G.M. 2008. Fish toxicity studies. In: Di Giulio, R.T., Hinton, D.E. (Eds.), The Toxicology of
Fishes. CRC Press, New York, USA, pp. 659–682.
Senthil Murugan, S., R. Karuppasamy, K. Poongodi, and S. Puvaneswari. 2008. Bioaccumulation
pattern of zinc in freshwater fish Channa punctatus (Bloch.) after chronic exposure. Turk. J.
Fish. Aquat. Sci. 8: 55-59.
Shetty Akhila, J., Deepa, S., Alwar, M.C., 2007. Acute toxicity studies and determination of median
lethal dose. Current Science 93(7): 917 – 920.
van der Oost, R., J. Beyer, and N.P.E. Vermeulen. 2003. Fish bioaccumulation and biomarkers in
environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13: 57–149.
van Dyk, J.C., G.M. Pieterse, and J.H.J. van Vuren. 2007. Histological changes in the liver of
Oreochromis mossambicus (Cichlidae) after exposure to cadmium and zinc. Ecotoxicol.
Environ. Safety 66: 432–440.
Weatherley, A.H., P.S. Lake, and S.C. Rogers. 1980. Zinc pollution and the ecology of the freshwater
environment. In: J. O. Nriagu (ed.), Zinc in the environment. Part I: Ecological Cycling. John
Wiley, New York, USA, pp 337-417.
Wood, C.M. 2001. Toxic responses of the gill. In: Schlenck, D., Benson, W.H. (Eds.), Target Organ
Toxicity in Marine and Freshwater Teleosts Vol. 1, Organs. Taylor and Francis, London, New
York, pp. 1–89.