sd-sample article B. Edullantes and R. Galapate 25 SCIENCE DILIMAN (JANUARY-JUNE 2014) 26:1 25-40 Embryotoxicity of Copper and Zinc in Tropical Sea Urchin Tripneustes gratilla Brisneve Edullantes* Ritchel ita P. Galapate University of the Philippines Cebu _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online ABSTRACT The study determined the individual toxicity of copper (Cu) and zinc (Zn) i n s e a u r c h i n T r i p n e u s t e s g r a t i l l a . B i o a s s a y u s i n g i n h i b i t i o n s o n fertilization, early cleavage, mid cleavage, late cleavage and blastulation as endpoints involved exposure of viable gametes to Cu and Zn for 0.5, 3 , 6 , 9 a n d 1 2 h , r e s p ec t i ve l y. I n h i b i t i o n s i n c r e a s ed s i g n i f i c a n t l y w i t h concentration of Cu and Zn. Probit analysis estimated EC 50 values for Cu and Zn, respectively, at 32 and 67 µg·L-1 on fer tilization; 31 and 93 µg·L-1 on early cleavage; 43 and 61 µg·L -1 on mid cleavage; 42 and 42 µg·L -1 o n late cleavage; and 20 and 44 µg·L-1 on blastulation. Results showed that t ox i c i t y o f Cu i s s i g n i f i c a n t l y h i g h e r ( p < 0 . 0 5 ) t h a n t h a t o f Zn i n a l l d e v e l o p m e n t a l s t a g e s , e x c e p t i n l a t e c l e a v a g e . A l s o , t h e i n h i b i t i o n s e l i c i t e d b y C u s h o w e d s e n s i t i v i t y t o l i f e s t a g e s . T h i s s t u d y p r o v i d e d evidence on heavy metal species-sensitive, concentration-dependent and stage-specif ic inhibitions on embryonic development in T. gratilla to Cu and Zn. Keywords: Embryotoxicity, sea urchin development , individual toxicity, h e a v y m e t a l s INTRODUCTION Waste disposal from mines and industries discharges complex mixtures of pollutants to coastal areas. These anthropogenic activities expose aquatic wildlife to various heavy metals such as copper and zinc (US EPA 2007), which at elevated levels often subject aquatic organisms to heavy metal poisoning (Eisler 1998). Sea urchins dwell in marine environment, and they respond readily to heavy metal pollution, making them an ideal bioindicator of ecosystem health (Kobayashi and Okamura 2 0 0 4 ) . Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 26 Known to act as teratogen, heavy metals cause developmental delay, malformations and mortalities among exposed aquatic organisms (Eisler 1998). Studies have found that different heavy metals cause developmental anomalies among sea urchins (Kobayashi and Okamura 2004), and elevated concentrations of Cu and Zn inhibit the development of echinoid species (Phillips and others 2003, Kobayashi and Okamura 2004, Kobayashi and Okamura 2005). Although there have been several studies on the effects of toxic heavy metals on marine organisms (see for example King and Riddle 2001, Phillips and others 2003, Kobayashi and Okamura 2005), further research is needed to have a better understanding of the embryotoxic effects of heavy metals. Moreover, endpoints of sea urchin bioassay are limited to spermiotoxicity, inhibitions of fertilization, and malformations. Few studies have been undertaken to investigate the inhibitory effect of Cu and Zn on early stages such as cleavage and blastulation, which are critical stages in sea urchin development. To help address these research gaps, the present study was undertaken. This study may be regarded as an initial attempt to evaluate the inhibitory effects of Cu and Zn on the early life stages of the tropical sea urchin, Tripneustes gratilla, in the Philippines. Using bioassay testing, this study aimed to: (a) determine the percentage of inhibitions on fertilization, early cleavage, mid cleavage, late cleavage, and blastulation; and (b) compare the inhibitions across heavy metal species, concentration, and developmental stages. MATERIALS AND METHODS Preparation of Test Solutions Five nominal concentrations of Cu and Zn (0, 25, 50, 100 and 150 µg·L-1 each) were used to examine the toxicity of heavy metal on sea urchin. The test solutions were prepared by adding copper sulfate and zinc sulfate into f iltered natural seawater. The temperature (28.10 ± 1.84°C), salinity (30.67 ± 0.58 µg·L-1), and pH (6.99 ± 0.6) of the test solutions were maintained. Collection of Sea Urchin Gametes Forty-two adult sea urchins T. gratilla, 6.60 ± 0.47 cm in diameter, were collected from Marigodon, Lapu-lapu City. Each organism was isolated in a plastic container f illed with seawater to ensure that none of the sea urchins would induce others to spawn. They were transported to the laboratory immediately after sampling. B. Edullantes and R. Galapate 27 Procedures for gamete collection were adapted from the US EPA (1995) protocols, with a few modif ications. Each sea urchin was inverted over a 100 mL beaker fully f illed with f iltered natural seawater. The gonadal openings on the aboral side were immersed in the seawater. About 1 mL of 0.5 M KCl was injected through the tough leathery peristomial membrane into the perivisceral cavity of each sea urchin. This resulted in the contraction of the smooth muscles of the gonad and induced spawning of the specimen. Injections were repeated after 2-5 minutes to induce heavier spawning. The sex of the sea urchin was determined. T. gratilla males ejected cream-colored semen while females released yellow eggs. A drop of the gametes from each sea urchin was examined under the microscope to conf irm its sex. Each spawning sea urchin male was transferred into a petri dish in oral side up position and was allowed to shed into the dish. A drop of the dry sperm (semen) was examined under the microscope to observe the motility of the sperm. The sea urchin males with high sperm motility were used in the test to ensure sperm viability. The viable sperm cells were pooled into a 100 mL beaker, which was covered with paraf ilm to prevent exposure of semen to air that may reduce the viability of the sperm by altering the surrounding pH. Sperm stock was stored at 5°C. Female sea urchins were left to shed eggs into the 100-mL beakers f illed with f iltered natural seawater. A small sample of the eggs from each female was examined under the microscope to determine the presence of mature eggs. Mature eggs were characterized as having a) small nucleus found near the periphery of the cell membrane and b) large amount of cytoplasm. Mature eggs were pooled into a 1 L beaker. The eggs were suspended in 600 mL f iltered seawater, and allowed to settle for 15 minutes. About 500 mL of the overlying water was siphoned off and the volume was brought back to 600 mL with f iltered natural seawater. The eggs were resuspended and allowed to settle for 15 minutes. After siphoning off the overlying 500 mL, the eggs were f inally resuspended in 600 mL f iltered natural seawater. Egg suspension was stored at 12°C. The gametes were used in the toxicity assay after 2 h following the collection. Gametes were exposed to different treatments of Cu and Zn (25, 50, 100 and 150 µg·L^-1 each). Same batch of gametes were exposed to 0 µg·L^-1 Cu and µg·L^-1 Zn, which serve as control. Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 28 Toxicity Assay The exposure experiments were adapted from the protocol used by Kobayashi and Okamura (2004, 2005), with a few modif ications. Inhibitions on fertilization, early cleavage, mid cleavage, late cleavage and blastulation were the endpoints. Exposure experiment for every endpoint was conducted separately in a plastic container with 10 mL of test solution. One drop of dry sperm stock and 1 mL of the egg suspension were added into the container. The gametes that were exposed to different treatments were of the same batch. Incubation temperature (28 ± 2 °C), salinity (30 ± 1 µg·L-1), and pH (7 ± 0.5) were maintained throughout the exposure experiment. Fertilization, early cleavage, mid cleavage, late cleavage, and blastulation were arrested by adding 1 mL of 10% formaldehyde after 0.5, 3, 6, 9 and 12 h exposure to test solutions, respectively. Exposure experiments were triplicated. A drop of the treatment solution was mounted on a slide. About four mounts were prepared for each treatment. Each mount was observed under the compound microscope in a single f ield of vision at 100x magnif ication. One hundred eggs and/or embryos were randomly selected and their development stage, as described in Table 1, was identif ied. Inhibitions on fertilization, early cleavage, mid cleavage, late cleavage, and blastulation were determined. Table 1. Distinguishing features of early developmental stages in sea urchin Stages Description Unfertilized Egg Mature eggs without fertilization cone or envelope Fertilized Egg Mature eggs with fertilization cone or envelope Early Cleavage 2- and 4-cell stage embryos Mid Cleavage 8- and 16-cell stage embryos Late Cleavage 32- and 64-cell stage embryos Blastulation Embryos with a sphere of cells surrounding a cavity Data Analyses The toxicity responses were reported as percent inhibitions on fertilization (IF), early cleavage (IEC), mid cleavage (IMC), late cleavage (ILC), and blastulation (IB) using the following formulas: = 100 Eq. 1 B. Edullantes and R. Galapate 29 = + + + 100 = + + + + 100 Eq. 4 Eq. 5 where U is the number of unfertilized eggs, F is the number of fertilized eggs, EC is the number 2- and 4-cell stage embryos, MC is the number 8- and 16-cell stage embryos, LC is the number of 32- and 64-cell stage embryos, and N is the total number of eggs and/or embryos evaluated. The toxicity responses were f itted against the concentration through a probit model to estimate the concentration at which 50% inhibition is observed (EC 50 ). Kruskal - Wallis ANOVA was used to compare the toxicity responses among treatments. All statistical analyses were done using the IBM SPPSS Statistics Version 20 software, at 95% conf idence interval. RESULTS The effects of varying concentrations of Cu and Zn on the different developmental stages of T. gratilla are shown in Figures 1 to 5. Both Cu and Zn elicited logarithmic concentration-dependent inhibitions on T. gratilla fertilization, early cleavage, mid cleavage, late cleavage and blastulation (Figures 1 to 5, respectively). Comparison between EC 50 of Cu and Zn across different embryonic stages is shown in Figure 6. Inhibitions on Fer til ization (IF) IF increased with increasing Cu and Zn concentration at 0.5 h exposure (Figure 1). All treatments of Cu and Zn elicited a signif icantly higher IF than in control (17 ± 7%). IF at 25 µg·L-1 Cu increased threefold from the control (62 ± 3%). Increasing the Cu concentration to 50 and 100 µg·L-1 elicited 70 ± 6% and 75 ± 3% IF, respectively, which were four times higher than in control. At 150 µg·L-1, a f ivefold increase in IF was observed (87 ± 3%). In zinc treatment, IF doubled at 25 µg·L-1 Zn (39 ± 3%). It increased threefold at 50 and 100 µg·L-1 Zn, eliciting 56 ± 3% and 61 ± 2% IF, respectively. It was signif icantly high at 150 µg·L-1 Zn (73 ± 5%), which was four = + 100 = + + 100 Eq. 2 Eq. 3 Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 30 times higher than in control. The EC 50 of Cu (32 ± 11 µg·L-1) was signif icantly lower than Zn (67 ± 3 µg·L-1) (Figure 6), suggesting that Cu is twice as toxic as Zn in eliciting inhibitions on fertilization. Inhibitions on Early Cleavage (IEC) Concentration-dependent inhibitions on early cleavage of T. gratilla were also observed at 3 h exposure period to increasing Cu and Zn concentration (Figure 2). IEC in all Cu treatments were signif icantly higher than in control (15 ± 5%). At 25 µg·L-1 Cu, IEC increased threefold to 50 ± 6%. Increasing the concentration to 50 µg·L-1 Cu elicited 68 ± 7% IEC, which was four times higher than in control. IEC increased six times from the control at more elevated concentration (>90%). IEC at 25 and 50 µg·L-1 Zn showed no signif icant difference from the control. At higher Zn concentrations, IEC increased more than threefold, with values signif icantly higher than those in control. EC 5 0 of Cu and Zn on IEC were 31 ± 3 µg·L - 1 and 93 ± 43 µg·L -1, respectively (Figure 6). There was a signif icant difference between the EC 50 of Cu and Zn in eliciting inhibitions on early cleavage, indicating that Cu is three times more toxic than Zn. Figure 1. Inhibitions on fertilization to varying concentrations of Cu (black circles) and Zn (white circles). B. Edullantes and R. Galapate 31 Figure 2. Inhibitions on early cleavage to varying concentrations of Cu (black circles) and Zn (white circles). Figure 3. Inhibitions on mid cleavage to varying concentrations of Cu (black circles) and Zn (white circles). Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 32 Inhibitions on Mid Cleavage (IMC) The inhibitions on mid cleavage were evaluated at 6 h exposure to varying concentrations of Cu and Zn (Figure 3). Similar to the previous observations, IMC increased with increasing concentration of Cu and Zn. IMC in both treatments did not vary signif icantly from the control (23 ± 13%) at 25 µg·L-1, but showed a signif icant difference from the control at elevated concentrations. In Cu treatment, IMC doubled at 50 µg·L-1 (59 ± 7%). Increasing the Cu concentration to 100 µg·L-1 elicited 75± 5%, which was threefold higher than in control. IMC increased four times at 150 µg·L-1 (93 ± 9%). There was a twofold increase in IMC at 50 and 100 µg·L-1 Zn (51 ± 3% and 63 ± 5%, respectively). At 150 µg·L-1 Zn, IMC had increased to 81 ± 6%, which was three times higher than in control. The EC 50 of Cu (43 ± 11 µg·L-1) was signif icantly lower than Zn (61 ± 4 µg·L-1) (Figure 6), which suggests that Cu is more toxic than Zn in inhibiting mid cleavage. Inhibitions on Late Cleavage (ILC) Inhibitions on T. gratilla late cleavage were evaluated at 9 h exposure to Cu and Zn (Figure 4). Trends similar to those for the previous developmental stages were observed between ILC and concentration of Cu and Zn. All treatments showed a Figure 4. Inhibitions on late cleavage to varying concentrations of Cu (black circles) and Zn (white circles). B. Edullantes and R. Galapate 33 signif icant difference from the control (17 ± 7 %). ILC at 25 µg·L-1 Cu increased twofold from the control (39 ± 5%). Increasing the Cu concentration to 50 µg·L-1 caused 52 ± 10% ILC, which was three times higher than in control. At higher concentration, Cu elicited a f ivefold increase in ILC (>90%). In zinc treatment, ILC doubled at 25 µg·L-1 Zn (40 ± 7%). It increased threefold at 50 µg·L-1 Zn, producing 57 ± 6% inhibitions. ILC at 100 µg·L-1 Cu quadrupled from the control (81 ± 2%). It was signif icantly high at 150 µg·L-1 Zn (98 ± 2%), which was f ive times higher than in control. Cu and Zn elicited EC 50 at 42 ± 4% and 42 ± 9%, respectively (Figure 6). No signif icant difference was observed between EC 50 of Cu and Zn, which indicate that Cu is as toxic as Zn. This is a different trend from that obtained in the previous observations. Inhibitions on Blastulation (IB) The percent inhibitions on blastulation were determined at 12 h exposure to varying concentrations of Cu and Zn (Figure 5). Similar trends of inhibitions were observed in the blastulation, which was found to increase with increases in the concentration of Cu and Zn. IB in all Cu treatments varied signif icantly from the control (17 ± 6%). At 25 µg·L-1 Cu, IB was quadrupled (70 ± 6%). At elevated Cu concentration, IB was f ive times higher compared to control (>82%). IB at low concentration of Zn showed Figure 5. Inhibitions on blastulation to varying concentrations of Cu (black circles) and Zn (white circles). Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 34 no signif icant difference from control. Concentration at 100 µg·L-1 Zn elicited an IB of 73 ± 4%, which was four times higher than in control. Increasing the concentration to 150µg·L-1 Zn increased the IB to f ive times (86 ± 17%). The EC 50 of Cu and Zn were 20 ± 7 µg·L -1 and 44 ± 6 µg·L-1, respectively (Figure 6). Based on this EC 50 values, Cu appeared to be twice as toxic as Zn in inhibiting blastulation. DISCUSSION Industrial and agricultural wastes discharge heavy metals such as Cu and Zn that pollute coastal areas and endanger aquatic organisms, including the sea urchins. Elevated concentrations of these heavy metals may cause adverse effects on the growth, survival and reproduction of the echinoid species (Thongra-ar 1997, Kobayashi and Okamura 2004). Findings of this study showed that elevated concentrations of Cu and Zn lead to signif icantly higher inhibitions on fertilization, early cleavage, mid cleavage, late cleavage and blastulation in T. gratilla. The signif icant variation of percent inhibitions between control and treatments manifests toxicity of Cu and Zn in early developmental stages. Previous studies reported similar f indings on embryotoxicity of heavy metal when elevated from their natural Figure 6. Comparison of EC 50 values of Cu and Zn in eliciting inhibitions of different stages of embryonic development in sea urchin T. gratilla. Lower-case letters indicate significant difference between inhibitory effects of Cu and Zn for a particular embryonic stage. Black and white circles indicate signif icant difference between the inhibitions of two stages for a certain heavy metal. B. Edullantes and R. Galapate 35 concentrations in seawater (US EPA 1987, Nakamura and others 1989, King and Riddle 2001). The observed inhibitions on fertilization can be attributed to the spermiotoxic effects of Cu and Zn. Motility and fertilizing capacity of T. gratilla spermatozoa are reduced by these toxicants (Thongra-ar 1997), hence lowering the fertilization success. Results also revealed inhibitions on cleavage and blastula stages, which are clear mitotoxic responses of Cu and Zn. Exposure to high levels retards the division of cells, thus delaying the formation of blastula (Kobayashi and Okamura 2004). Copper may elicit inhibitions on the early life stages in sea urchin by (1) respiratory acidosis (Bielmyera and others 2005) or (2) disruption of ionic balances by alteration of ATPases (Li and others 1996) and carbonic anhydrase (Zimmer and others 2012). Zinc, on the other hand, possibly causes embryotoxic effects by: (1) inhibition of glucose-6-phosphate dehydrogenase that transforms carbohydrate via the pentose phosphate pathway (Durkina and Evtushenko 1991), (2) inhibition of the synthesis of ribosomal RNA (Pirrone and others 1970), (3) restriction of the development of endoderm as well as mesenchyme derivatives causing abnormalities to developing embryos (Timourian 1968), and (4) intervention with the action of cortical granule- derived protease that inhibit the formation of the fertilization membrane in sea urchin eggs (Nakamura and others 1989). The results of this study provide evidence of concentration-dependent inhibitions caused by Cu and Zn on the embryonic development of sea urchin (Figures 1 to 5). Generally, inhibitions on developmental stages in sea urchin followed a logarithmic pattern when plotted against the concentration of Cu and Zn. Inhibitions increased exponentially at low concentration but slowed at elevated concentration. Although the effects of other stressors (e.g. particulate materials) on the inhibitions could not be completely excluded, these were minimized in the experiment. In fact, samples of natural seawater were collected in a pristine area and were f iltered to remove particulate materials. Hence, any variation between the nominal and actual would have been kept at minimum; the same would be true for the observed concentration-response relationships. One limitation of this study is that inhibitions in the control group (>15%) were higher than the value (<10%) ideal for toxicity testing. As such, it could be argued that sources of stress other than Cu and Zn could have contributed to the Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 36 concentration-response relationship observed. Also, levels of Cu and Zn were not determined in the test solutions, consequently casting some questions regarding the accuracy of the EC 50 values. Corollary to this, some inhibitions in Cu treatments showed higher than 50% for all concentrations (e.g. , Figures 1, 2 and 5). A possible explanation for this is that the EC 50 might be overestimated due to lack of information on the inhibitions at concentrations lower than the tested levels. It must be pointed out, however, that although range f inding tests were not conducted before the def initive tests, the exposure experiments were designed to determine inhibitions at concentrations within the range of the EC 50 values specif ied in the literature (see for example Kobayashi 1990, King and Riddle 2001, Phillips and others 2003). Hence, any differences in the threshold values between experiments with or without range f inding test would be insignificant. There would be a negligible difference in EC 50 values between def initive tests conducted with range f inding test and def initive tests conducted without range f inding test since the concentration used in the def initive tests were at concentrations within the range in the literature. As observed, Cu was toxic within the range of 20 to 43 µg·L -1. EC 50 of Cu. Comparatively, this is below (King and Riddle 2001, Phillips and others 2003), within (Heslinga 1976, Pagano and others 1986) and above (Kobayashi 1985, Ramachandran and others 1997) the observed threshold range reported in other studies. On the other hand, EC 50 of Zn from the past studies is below (Kobayashi 1990), within (Phillips and others 2003) and above (Bay and others 1993, Thongra-ar 1997, King and Riddle 2001) the observed range of EC 50 of Zn (42 - 93 µg·L -1). Findings showed the sensitivity of sea urchin bioassay to the heavy metal species (Figure 6). Generally, EC 50 of Cu was signif icantly lower than that of Zn in all developmental stages, except in late cleavage. This comparison of Cu and Zn toxicity tests on developmental stages in T. gratilla suggests that Cu is more toxic than Zn. The potential toxicity of Cu to T. gratilla was found to be 2–4 times greater than Zn. Previous studies also observed the same trend (Thongra-ar 1997, Kobayashi and Okamura 2004 and 2005). Responses of embryonic development to toxicants are stage specif ic (Pagano and others 1986, Dinnel and others 1987, Bay and others 1993). This can be seen in the f indings regarding the inhibitions caused by Cu in the different developmental B. Edullantes and R. Galapate 37 stages of T. gratilla (Figure 6). EC 50 of Cu in blastulation was signif icantly higher compared to the threshold value in mid and late cleavage, suggesting that Cu is more toxic during blastulation than during the earlier developmental stages. Kobayashi (1980) reported that Cu is more disruptive in the later embryonic stages than in the earlier stages. In contrast to the f indings for Cu, inhibitions elicited by Zn did not vary signif icantly across developmental stages, which indicate that the toxicity of Zn is not stage-dependent. Accumulative trend in the inhibitions from early to late embryonic development was not observed in the present study. That is, it appears that the toxicity endpoints are independent of each other. The inhibitions in early development do not seem to influence the inhibitions in the latter stages. One possible explanation is that heavy metal exposure experiments were not carried out continuously. In continuous bioassay testing, it is expected that inhibitions from fertilization to blastula will show remarkable differences. CONCLUSION The study examined the inhibitory effect of Cu and Zn on fertilization, early cleavage, mid cleavage, late cleavage and blastulation of T. gratilla. The inhibitions exhibited logarithmic concentration dependence where it increases exponentially at low concentration, but more slowly at elevated concentrations of heavy metals. The f indings conf irmed the sensitivity of sea urchin bioassay to heavy metal pollution, with Cu eliciting greater toxicity than Zn in the early developmental stages of T. gratilla. Also, the study revealed the sensitivity of the assay to the developmental stages, although only Cu showed stage-specif ic inhibitions. Generally, the study provided a clear evidence of the dependence of heavy metal toxicity on heavy metal species, their concentration and the developmental stage they inhibit. The f indings may contribute to the improvement of the bioassay, particularly the use of the sea urchin T. gratilla in the assessment of toxicity of harmful anthropogenic substances. ACKNOWLEDGMENTS The authors are grateful to the reviewers and the editor whose critical review helped improve this manuscript. 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Aquatic Toxicology 122-123: 172-180. Embryotoxicity of Copper and Zinc in Tropical Sea Urchin 40 _______________ Brisneve Edullantes is currently an Assistant Professor of Biology of the University of the Philippines Cebu. His research interests are aquatic ecology, water quality assessment and environmental toxicology. His research outputs have been presented in international and local scientif ic conferences. He received his Bachelor’s degree in Biology from University of the Philippines Cebu. He earned his Master’s degree in Environmental Engineering from Mokpo National Maritime University, South Korea. Ritchel ita P. Galapate is currently an Associate Professor of Environmental Science of the University of the Philippines Cebu. Her research interests are water quality assessment, water pollution, water treatment, and environmental toxicology. She has published in international and local refereed journals of which the international publications have received at least 84 citations. Her research outputs have likewise been presented in international and local conferences of professional and scientif ic organizations. She received her Master’s and Doctoral degrees in Engineering (major in Environmental Science) from Hiroshima University, Japan.