Effects of Cadmium and Zinc on the Gamete Viability, Fertilization, and Embryonic Development of Tripneustes gratilla (Linnaeus)

Abstract

Heavy metals are frequently reported for their mutagenic and teratogenic effects on benthic organisms. Thus, this study aimed to determine the toxicity of cadmium (Cd) and zinc (Zn) in the gametes of T. gratilla and to compare its fertilization and embryonic development under the highest nongametotoxic concentrations of these heavy metals. Gamete viability of T. gratilla under CdCl2 and ZnSO4 treatments was assayed through resazurin reduction test (RRT) and was confirmed through gamete morphology assay. ZnSO4 was more toxic to T. gratilla gametes than CdCl2 and egg cells were more sensitive to both than the sperm cells. Higher concentrations of CdCl2 and ZnSO4 induced gamete apoptosis and necrosis while highest nongametotoxic concentrations were determined at 1 × 10(-3) M and 1 × 10(-4) M, respectively, and were used in an in vitro fertilization and embryonic development experiment. ZnSO4 treatment inhibited fertilization more than CdCl2 and yielded more deformed embryos, while both induced abnormalities and hindered further embryonic development. This study gives the first report on the specific concentrations of Cd and Zn that are toxic to T. gratilla gametes and has confirmed the teratogenic effects of these heavy metals.

Figure 1

Results of RRT performed on T. gratilla sperm cells treated with (a) CdCl₂ and (b) ZnSO₄, and egg cells (c and d) treated with the same heavy metal solutions, respectively. M₀ (media control), PC (positive control), T₁ (1 M), T₂ (0.5 M), T₃ (0.1 M), T₄(5 × 10⁻² M), T₅ (1 × 10⁻² M), T₆ (5 × 10⁻³ M), T₇ (1 × 10⁻³ M), T₈ (5 × 10⁻⁴ M), T₉ (1 × 10⁻⁴ M), and NC (negative control). R₁–R₃ are the replicate wells. (e) Mean absorbance at 630 nm of the wells containing T. gratilla gametes with CdCl₂ and ZnSO₄ treatments after 30 mins of incubation with resazurin. (f) A histogram showing the mean absorbance at 630 nm of the wells containing T. gratilla gametes with CdCl₂ and ZnSO₄ treatments after 30 mins of incubation with resazurin.

Figure 2

Representative images showing T. gratilla different gamete morphologies (400x). This network of thread-like blue material (white arrows) strongly indicates that there is a result of leaching DNA materials when a cell undergoes necrosis due to the attachment of Giemsa stain onto sites where adenine and thymine are rich.

Figure 3

The percent successful fertilization and inhibition of fertilization as observed on the gametes after 30 mins exposure to 1 × 10⁻³ M CdCl₂, 1 × 10⁻⁴ M ZnSO₄ and filtered seawater.

Figure 4

Number of deformed embryos observed under the three treatments considered starting from early cleavage up to 2-arm echinopluteus.

Figure 5

The embryonic development of T. gratilla. Normal development observed starting from 2-cell up to 2-arm echinopluteus on the control (filtered seawater; a–e) and abnormalities in the embryos observed under the highest nongametotoxic concentrations of CdCl₂ (f–j) and ZnSO₄ (k–o).