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
Heavy metals have been one of the most threatening problems that greatly affect the diversity of life within the marine ecosystem. They are considered as severe pollutants in the natural environment due to their toxicity, bioaccumulation problems [
Heavy metals could be detected in seawater [
Along these premises, a study on the effects of heavy metals on marine invertebrates could show that they too are as vulnerable as other marine organisms to these contaminants. Sea urchin, a marine invertebrate, demonstrates a model system for analyzing cellular mechanisms during embryonic development due to their rapid differentiation, and to delineate their critical period of developmental vulnerability [
Although some species of sea urchins have already been studied for the toxic effects of heavy metals on their embryonic development like in the short-spine sea urchin (
Thus, this study was conducted to determine and compare the toxic effects of Cd and Zn on the gametes of
A total of 65 adult
All glassware used during induced spawning up to the observation of embryonic development was acid washed. This was done to remove the unwanted heavy metal contaminants that can potentially affect the results and induce bias upon the conduct of the study. Also, prior to the formulation of heavy metal concentrations, seawater was filtered using a system composed of filter flask directly attached to the vacuum pump. A Whatman glass microfiber filter (GF/A) (GE Healthcare Company, UK) with a diameter of 47 mm was added to the filter flask to efficiently filter particles and microorganisms present in the seawater. The filtered seawater was collected into a sterile acid washed container and stored at room temperature.
Gametes were collected through induced spawning by injecting 0.2 mL of 1 M KCl (Anscom Medical Center, Manila) intracoelomically per 2.5 cm of diameter at the perivisceral cavity near the mouth [
For gamete viability assay, pure concentrations of gametes were collected by directly inverting the sea urchin into sterile acid washed containers, covered with aluminum foil, and were immediately plated for the assays. While for fertilization and embryonic development experiments, the method of Rahman et al. [
Cadmium chloride (CdCl2) and zinc sulphate (ZnSO4) were used for the varying heavy metal concentration treatments. One molar CdCl2 (Anscom Medical Center, Manila) and ZnSO4 (Ajax Chemical Inc., Australia) were prepared by dissolving their crystals into filtered seawater. From the prepared 1 M solutions, serial dilutions of 0.5 M, 0.1 M, 5 × 10−2 M, 1 × 10−2 M, 5 × 10−3 M, 1 × 10−3 M, 5 × 10−4 M, and 1 × 10−4 M were made.
RRT was used to test the cytotoxic effects of CdCl2 and ZnSO4 on
Fifty microliters (50
Another set of treatments was done to observe the morphological effects of varying concentrations of heavy metals on sperm cells and egg cells. The procedure was similar to RRT previously done except that no resazurin solution was added. Gametes were smeared into clean glass slides, air-dried, and immediately fixed using absolute methanol. The slides were then stained with Giemsa and washed with slow running distilled water to remove the excess stain. Once dried, the stained smears were mounted with small drop of Canada balsam (Yana Chemodities, Cebu, Philippines), topped with clean cover slips, warmed to evenly spread the mounting medium, and examined under light microscope.
Prior to induced fertilization experiment, gametes were diluted first in filtered seawater following the methods described in Edullantes and Galapate [
The highest CdCl2 and ZnSO4 concentrations tolerable for the gametes particularly to the sperm cells were used in the fertilization experiment following the method of Bielmyer et al. [
To induce the fertilization of the gametes, 1 mL of egg suspension was added to the beakers containing 9 mL of the treatment solutions. Then, 200
To compare the fertilization efficiencies of
The observation of embryonic development was adapted from the method established by Edullantes and Galapate [
For each observation time, three 20
This study followed a completely randomized design (CRD). For the gamete viability assay, analysis of variance (ANOVA) was used to determine the significant difference of the treatments. Post hoc comparison was used to cluster the absorbance of the various treatments following the homogenous subset of Duncan Multiple Range Test. Repeated measures ANOVA with replicated measurements were used to compare the number of deformed embryos on different treatments. All data analyses were carried out using SPSS v.20.
Morphology of gametes,
Results of the resazurin reduction test for the cytotoxic effects of CdCl2 and ZnSO4 in
Results of RRT performed on
Furthermore, percent reduction of resazurin to resorufin was supposed to be computed in all treatment. It was not done for several reasons. First, the microplate reader does not have the wavelength necessary for detecting the absorbance of resorufin (540–570 nm) as needed in the calculation. Secondly, resazurin did not completely mix with the cells on higher concentrations of CdCl2 and ZnSO4 treatments making the lower lethal treatments bluer than expected. However, the microplate reader has available wavelength for resazurin ranging from 600 to 630 nm with the peak at the latter. Since it was necessary to compare the treatments quantitatively, it was decided to do it using treatment absorbance at 630 nm. This quantified the amount of resazurin that is present in the treatment wells. This means that the more viable the cells are, the more they are able to reduce blue resazurin to pink resorufin giving low absorbance at 630 nm. While the treatments are more toxic, the ability of the cells to reduce resazurin to resorufin will be lesser giving higher absorbance at 630 nm. This is with the exception of course of the treatments where cells were suspected to be necrotic resulting for the resazurin to be immiscible because of the viscous media. This would mean that, hypothetically, highest absorbance will be acquired at the treatments where cells are still in the apoptotic phase of cell death and would start to drop at the necrotic phase caused by the higher concentrations of CdCl2 and ZnSO4 treatments. Moreover, the intensity of pink coloration between the untreated (negative control) egg cells (Figures
Moreover, analysis of variance (ANOVA) revealed highly significant difference between the different CdCl2 and ZnSO4 treatments on both sperm cells and egg cells. Absorbances were further homogenized using DMRT for both CdCl2 and ZnSO4 and were clustered according to the closeness of value to each other. Importance was given on determining which concentrations were not significantly different to the negative control since these concentrations were most probably the ones having no toxic effects on the gametes.
To disregard the effect of chloride and sulphate constituents of Cd and Zn, another set of RRT was conducted on the gametes. The Cl2 and SO4 compounds with nonheavy metal constituent were used. These were NaCl and Na2SO4. Results (pictures not shown) revealed that there was a significant difference between the effects of the same molar concentrations of CdCl2 and ZnSO4 and NaCl and Na2SO4 on
The variation of the absorbance among the different treatments of CdCl2 and ZnSO4 is influenced by the capacity of the gametes to maintain metabolic activity [
Results revealed that both egg cells and sperm cells treated with CdCl2 and ZnSO4 underwent apoptosis and necrosis proving the earlier suspicion (Figure
Representative images showing
Table
Summary of viability assay on
CdCl2 | ZnSO4 | |||
---|---|---|---|---|
Sperm | Egg | Sperm | Egg | |
Normal | 1 × 10−4 M–1 × 10−3 M |
1 × 10−4 M | 1 × 10−4 M |
1 × 10−4 M–5 × 10−4 M |
Apoptosis | 5 × 10−3 M–0.1 M | 5 × 10−4 M–0.1 M | 5 × 10−4 M–0.1 M | 1 × 10−3 M–0.1 M |
Necrosis | 0.5 M–1 M | 0.5 M–1 M | 0.5 M–1 M | 0.5 M–1 M |
Figure
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.
A study conducted by Gopalakrishnan et al. [
As the time of observation increased, ZnSO4 yielded more deformed embryos than CdCl2 as shown in Figure
Number of deformed embryos observed under the three treatments considered starting from early cleavage up to 2-arm echinopluteus.
Table
The morphological observations on the embryos of
Time of observation | Treatments | ||
---|---|---|---|
FSW (filtered seawater) | Cadmium chloride (CdCl2) | Zinc sulphate (ZnSO4) | |
3 hrs | |||
2-cell | SCD | SCD | SCD |
4-cell | SCD | SCD | SCD |
6 hrs | |||
8-cell to 16-cell | SCD | SCD, DE, EPC | SCD, DE, EPC |
9 hrs | |||
32-cell to 64-cell | SCD | ESD, DE | ESD, DE |
12 hrs | |||
Blastula | SCD | NFD, DE | NFD, DE |
24 hrs | |||
Gastrula | SCD | NFD | NFD |
2-arm echinopluteus | SCD | NFD | NFD |
General observation | No abnormalities | Formation of apoptotic bodies, cell fragmentation | Blebbing and deformed embryos |
EPC = embryos with pigmented cells, SCD = successful cell division, ESD = embryo stop dividing, NFD = no further development, and DE = deformed embryos.
The embryonic development of
Moreover, the embryos treated with the highest nongametotoxic concentration of CdCl2 were observed to be deformed throughout the observation time as shown in Figures
A study conducted by Migliarini et al. [
Zn affects embryos through the disruption of ribosomal RNA synthesis and inhibits the development of endoderm and mesenchyme derivatives resulting in abnormalities (Pirrone et al. 1970, Timourian 1968, as cited by Edullantes and Galapate [
Voronina and Wessel [
The highest nongametotoxic concentration of CdCl2 and ZnSO4 used for the
Furthermore, the amount of Cd detected in seawater near the industrial sites ranges from 0.002
Conversely, no available information was gathered on the amount of Zn effluents allowed by DENR; however, several studies suggest that the tolerable amount of Zn in the marine environment should not exceed 4.9
The performance of embryos treated with 2.8
Following a unique experimental approach aimed at providing a more realistic context of an environmental milieu, this study demonstrated that Cd and Zn have toxic effects on the gametes of
These results have huge impact in the formulation of environmental policies for the regulation of the amount of the heavy metals Cd and Zn in industrial effluents. This is especially because the current policy on the allowable amount of Cd imposed by the DENR regulated industrial effluents at disturbingly higher levels than what was being shown as toxic in this study. Conversely, no policy concerning the allowable amount of Zn in industrial effluents is currently available in the Philippines.
The authors declare that they have no competing interests.
The authors would like to extend their gratitude to all individuals who made this study a reality: to Dr. Takeshi Tomiyama and Dr. Lawrence Liao of Graduate School of Biosphere Science, Hiroshima University, Hiroshima, Japan, for providing the necessary reagent for the resazurin reduction test experiment; to Dr. Edgardo E. Tulin for allowing them to use the microplate reader of the Biotechnology Laboratory; to Statistical Consulting Unit of Department of Mathematics, Physics and Statistics (DMPS), Visayas State University, for running statistical analyses; to Professor Jacob Glenn Jansalin for ensuring that the conversion of heavy metal concentrations is correct; and to Dr. Analyn Mazo for her help extended in Invertebrate Biology.