Toxicity effects of trace metals on Zebra fish (Danio rerio) embryo

Abstract

Water quality deterioration is a major problem in the most of the countries, due to organic and inorganic contaminants. Among all the toxicant trace metals are threaten to the aquatic ecosystem and organisms. Metals are natural component of aquatic ecosystem. Trace metals such as Copper (Cu), Zinc (Zn) and Chromium (Cr) are important for the metabolic and other biological activities of lives whereas Mercury (Hg), Lead (Pb), Cadmium(Cd) are biologically non-essential metal that can be toxic to biota even at very low concentration. High concentration of some essential trace metals can be toxic when it exceeds the limits(Ebrahimi and Taherianfard, 2011).High accumulation of trace metal in both biotic and abiotic components causes serious health consequences. Thus, assessment of their toxicity has become an important component of water pollution monitoring. Now in most of the toxicity studies Zebra fish (Danio rerio) embryo used as an alternative model for the fish acute toxicity to determine the toxicity of pollutants. Therefore this study was focused on determination of the acute toxicity of Cu, Zn, Cd, As, Pb and Hg that produce lethal effect on zebra fish embryos during four day period.

Materials and Methodology

Zebra fish breeding was carried out and eggs were collected using a small pipette. The stock solution of 1000 ppm of selected Cu, Zn, Pb, Cd, As and Hg were prepared a day before the test by dissolving Copper sulfate (CuSO4.5H2O), Zinc sulfate (ZnSO4. 7H2O), Lead nitrate (Pb(NO3)2, Arsenic pentoxide (As2O5), Cadmium chloride (CdCl2. ½ H2O) and Mercury chloride (HgCl2) in deionized water. The working treatment solution was prepared daily by serial dilution of the stock solution. Initially a range finding test was carried out in the nominal concentrations of (1000, 100, 10, 1 and ) for 96 hours. Ten eggs per concentrations were used. Deionized water was used as a negative control. Each treatment had three replicates. Acute Cu, Zn, Pb, As, Cd and Hg toxicity experiments were performed for a 4-day period using Danio rerio embryo. Ten test concentrations of Cu, Cd (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 10.0 mgL⁻ ), Zn, Pb, As (2.0, 4.0, 8.0, 16.0, 32.0,64.0, 125.0,250.0, 500.0 and 1000.0 mgL⁻ ) and Hg (0.002, 0.004, 0.008, 0.016, 0.032, 0.064, 0.125, 0.250, 0.500 and 1.0 mgL⁻ ) were prepared by diluting the stock solution in deionized water. Deionized water was used as negative control and as internal plate control. As a positive control concentration of 15% ethanol(Hassan et al., 2008) was used with each embryo batch for testing. Fertilized zebra fish embryos were immersed in the test solutions before cleavage, by the 16 cell- stage. At least twice the number of eggs needed per treatment group (40 eggs) was randomly selected and transferred into the respective concentrations and controls within the 90 minutes of post fertilization. 24-well plates were filled with 2 mL per well freshly prepared test solutions. In each plate twenty wells were used for test concentrations and four were used as internal plate control. Another plate was used as positive control. The eggs in standard 24- well plate were covered by shelf adhesive foil and maintain at a temperature to 26 ± 1 °C. The mortality of the fish eggs were recorded at 24, 48, 72 and 96 hours of exposure using a stereo microscope with the indicator of lethality; coagulation of fertilized egg, lack of somite formation, lack of detachment of the tail and lack of heart beat as described in OECD/ OCDE 236, 2013; guideline for the test of chemical, Fish Embryo Acute Toxicity (FET) tests. Dead eggs were removed immediately. Three replicates were performed individually. In this study the acute toxic effect of each metal on the Danio rerio embryo was determined by Probit Analysis LC50 determination method. Data analysis was performed by using statistical software SPSS15.