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Effects of triclosan on reproductive prarmeters and embryonic development of sea urchin, Strongylocentrotus nudus
Ecotoxicology and Environmental Safety 100 (2014) 148–152
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of triclosan on reproductive prarmeters and embryonic development of sea urchin, Strongylocentrotus nudus Jinik Hwang a, Sung-Suk Suh a, Man chang b, So Yun Park c, Tae Kwon Ryu d, Sukchan Lee e, Taek-Kyun Lee a,n a
South Sea Environment Research Department, Korea Institute of Ocean Science & Technology, Geoje 656-830, Republic of Korea Marine Ecosystem Research Division, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of Korea c Risk Assessment Division, National Institute of Environmental Research, Incheon 404-708, Republic of Korea d Medical Research Center of Neural Dysfunction, Gyeonsang National University, Jinju 660-751, Republic of Korea e Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 13 November 2013
Triclosan (TCS, 2,4,4′-trichloro-2′-hydroxydiphenyl ether), a broad-spectrum antibacterial agent, is commonly found in the aquatic environment. In this study, we investigated TCS toxicity with pertaining to gamete viability, fertilization, and embryogenesis up to pluteus stage of the sea urchin, (Strongylocentrotus nudus). When the sperm and eggs were exposed to TCS (0–3.0 μM), the viability of sperm was significantly decreased at molarities higher than 1 μM of TCS. In addition, for exposure of 2.0 μM TCS the viability of eggs was not influenced and none of the sperm was viable. Fertilization rate was significantly decreased when sperm were exposed to 0.5 and 1 μM of TCS (p o0.001) and no fertilization was observed for the exposure of 1.5 μM of TCS. In embryonic development, embryos are treated with higher than 1.0 μM levels of TCS displayed arrested development. For TCS, the EC50 and LOECs values were 1.8, 1.49 and 0.99 μM and 0.53, 0.62 and 0.39 μM for sperm viability, fertilization rate, and larval development to pluteus, respectively. In the recovery test regarding normal development of arrested embryos based upon TCS exposure time, it was observed that embryos exposed to 1 μM TCS for 15 h were normally recovered for normal development, while embryos with more than 30 h exposure were not recovered to normal larvae. Overall, the results of this study strongly suggest that the gametes and embryos of S. nudus can provide the basis for an effective bioassay, with a fast and sensitive means of evaluating TCS contamination in the marine ecosystem. & 2013 Elsevier Inc. All rights reserved.
Keywords: Triclosan Sea urchin Fertilization Early embryonic development Viability
1. Introduction TCS (TCS, 2,2,4′-trichloro-2′-hydroxydiphenyl ether), which is a synthetic broad spectrum antibacterial agent, is utilized in a wide range of products, such as soaps, shampoos, toothpastes, cleaning materials, and cosmetics (Jones et al., 2000). TCS has also been added to polymers and textile fibers as an anti-bacterial consumer products, including toys, undergarments, and cutting boards (Schweizer, 2001). In view of the fact that, TCS is mostly used in the production of consumer products (greater than 95 percent), it is found in domestic sewage and wastewater treatment plants (WWTPs) (Reiss et al., 2002; Lindström et al., 2002; Singer et al., 2002). TCS is extensively utilized and is very difficult to eliminate it completely during the waste water treatment hence about 10.0 ng/L to 98.0 ng/L of TCS has been detected in effluents
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Corresponding authors. Fax: þ 82 55 639 8639. E-mail addresses: [email protected], [email protected] (T.-K. Lee).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.029
(Boyd et al., 2003; Singer et al., 2002). Earlier review reported that TCS was one of the most concentrated chemical substances among various chemical substances that were included in the influents in the waste water treatment plants (Heidler and Halden, 2007). Although TCS is biologically degradable (Singer et al., 2002), it is often detected in rivers and lakes at thenanogram per liter level (Balmer et al., 2004; Bester, 2005; Kolpin et al., 2002; Lindström et al., 2002). Moreover, it has been reported that TCS is a precursor of dioxin congener (2,8-dichlorodibenzo-pdioxin, DCDD) by photolytic degradation (Aranami and Readman, 2007), and may form methyl-TCS via biotransformation (Lindström et al., 2002). TCS is high hydrophobic, with a log Kow value of 4.8, which is the solubility of TCS in octanol/and in water, it likely to be easily accumulated in adipose tissues. methyl-TCS, on the other hand, is relatively more lipophilic compared to TCS, thereby extending its persistence, and resulting in its bio-accumulation in the tissues of many organisms, being detected even in human milk (AdolfssonErici et al., 2002; Balmer et al., 2004). In accordance with the
J. Hwang et al. / Ecotoxicology and Environmental Safety 100 (2014) 148–152
gradual elevation of consumption of the products containing TCS, and the accumulated results of studies regarding acute, subacute/ subchronic, and chronic toxicity in detail, the utilization of TCS in food products and personal hygiene products has been restricted recently in many countries, including those of the European Union, as well as Canada and the US (Dayan and Yoshida, 2007). The consequences of TCS on the marine environment have not been sufficiently understood, but recent studies have reported acute and chronic toxicity values for TCS in aquatic organisms (Cortez et al., 2012; Gaume et al., 2012). Based on the results from acute in vitro and in vivo tests, TCS could affect the structures and functions of algal communities (Wilson et al., 2003), crustaceans and fishes (Ishibashi et al., 2004; Orvos et al., 2002). In addition to acute testing, TCS cytotoxicity was evaluated in marine bivalves. TCS reduced lysosomal membrane stability in mussels at low concentrations, and induced the extracellular release of lysosomal hydrolytic enzymes (Canesi et al., 2004). Furthermore, not only the cytotoxic and genotoxic effects of TCS in zebra mussels (Binelli et al., 2009) and zebrafish (Oliveira et al., 2009), but also the biological effects of TCS with regards to fertilization and early embryonic development in sea urchins and marine mussels have been investigated. (Anselmo et al., 2011) Sea urchins are representative marine organisms selected by USEPA, ASTM, and OECD for bioassays such as binding ability of tube feet, cytological and genetic malformations, changes in sperm motility to evaluate marine environmental samples (seawater, effluents, and sediment). The echinoderms are a suitable biological model for environmental toxicity studies because they are convenient to maintain in the laboratory, produce many translucent eggs, and allow in vitro fertilization all year around. Owing to the short pelagic period of sea urchin larvae, as well as the short duration of larval toxicity tests, the embryos and larvae of sea urchins should provide for a suitable biological organism for testing seawater quality standards. The sea urchin, Strongylocentrotus nudus is a typical costal species of Southern Korea. Numbers of sea urchins have gradually decreased due to contamination arising from a human interference. The objective of this study was to investigate the adverse effects of TCS on embryonic development in Korean sea urchins. In particular, sperm and egg viability, fertilization and development rate of S. nudus exposed to TCS were analyzed. Also, recovery tests were employed to obtain information regarding critical TCS exposure time. The results of this study would provide us with a better understanding of the effects of TCS on the development of sea urchin and would clarify the influences of anthropogenic activity in aquatic organisms. 2. Material and method 2.1. Animals and chemicals Adults of S. nudus were collected at the subtidal zone at Geoje in Korea. S. nudus is reproductive from July to September in the waters of Geoje. After the adult sea urchins were transferred to the laboratory, they were reared in aquarium at 18 1C with a 12 h light/dark cycle to prevent natural egg laying. Triclosan (TCS 498 percent purity) was purchased from Tokyo Chemical Industry (Japan), and a stock solution (100 μM) was made in 100 percent ethanol, diluted in Filtered Sea Water (FSW), and used the same day. The ethanol was used for the control in this experiment. All chemicals used in the study were purchased from Sigma Aldrich. 2.2. Gamete collections To obtain eggs and sperm from the sea urchin adults, 1.0 mL of 1 M KCl was injected and was then manually shaken until evenly distributed. KCl-treated males emitted white sperm from the gonopore, which was refrigerated (4.0 1C) after being collected in a small centrifugal tube (1.5 mL). The females emitted yellow eggs. To collect the eggs, clean seawater was added to a 80 mL beaker and the adults were then placed so that the gonopore faced to the seawater in the beaker.
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Released eggs were settled on the bottom of the beaker. Settled eggs were then transferred to a beaker filled with seawater and the floating eggs were then removed in order to select eggs of a good quality.
2.3. Viability test of gametes To study the effects of TCS on egg and sperm viability, 300 mL of egg or sperm dilution (50 mL of sperm in 6.0 mL of FSW) were added to vials containing 5.0 mL of FSW and TCS at different concentrations. The ethanol was used for the control in this experiment. After exposing eggs or sperm to TCS for 20 min, they were transferred to a hemocytometer and were then stained with 0.4 percent trypan blue, followed by counting the number of viable gametes via a microscope. Three replicates of each experiment were performed on different culture batches on different dates.
2.4. Fertilization assay The fertilization rate was evaluated based on USPEA (1995) standard. To test the effects of TCS on fertilization, 500 unfertilized eggs of the same batch and 15.0 mL of sperm dilution was added to vials containing 5.0 mL of FSW and TCS at different concentrations. The ethanol was used for the control in this experiment. Eggs were incubated for 20 min, until control eggs formed a fertilization membrane, after which a few drops of formalin were added to all vials. The fertilization rate (FR, percentage of fertilized eggs) was then calculated. The experiments were repeated for three times on different culture batches at different dates.
2.5. Embryogenesis assay For each experiment, eggs and sperm of at least three adults were then transferred to glass Petri dishes containing FSW for cross-fertilization, and were maintained at 15.0 1C. A considerable amount of sperm and debris that were interfered with the embryogenesis assay were filtered out using a 40 mm sieve. Also, floated fertilized eggs on the seawater were removed in order to differentiate healthy eggs. A solution of fertilized eggs with approximately 300 egg mL 1 was used in this study. Fertilized eggs were treated with different concentrations of TCS (0, 0.1, 0.5, 1.0 and 1.5 μM) dissolved in 80 mL of FSW. Fertilized eggs were cultured in a 15 1C incubator with 12 h light/dark cycle and were adjusted to the final density of 100 eggs/mL 1. The beaker was covered with parafilm to minimize evaporation. Samples were collected at the time periods of each developmental stage (15 h, blastula stage; 30 h gastrula stage; 48 h, prism stage; and 72 h, pluteus stage) and were then subjected to analysis under an optical microscope in order to evaluate the development rate in early embryonic development. Development rate was defined as the number of embryos with normal development dividing by the number of embryos exposed. In the abnormal development of embryo exposed, division of development cell did not occur anymore. The experiments were repeated for three times on different culture batches on different dates.
2.6. Recovery test Embryos were exposed to 0.5 or 1.0 μM TCS for four different time periods; 15 h (blastula (hatching)), 30 h (gastrula), 48 h (prism) and 72 h (pluteus). Exposure periods were summarized schematically in Supplementary information 1; TCS exposure from the fertilized egg stage to the blastula; B, TCS exposure from the fertilized egg stage to the gastrula stage; C, TCS exposure from the fertilized egg stage to the prism stage; D, TCS exposure from the fertilized egg stage to the pluteus stage (please see Supplementary information 1). The embryos treated with TCS in each stage were filtered through a sieve (size 100 μm) and were then washed twice with FSW. The experiments were repeated for three times on different culture batches on different dates.
2.7. Statistical analysis All data are presented as means 7 standard deviation (SD). Student's t–test was performed to test difference between the control and each experimental group. Probit analysis (Finney 1971) was carried out to evaluate the concentration that larvae dies as well as the fertilization and development rates. EC50 and LOEC values were also analyzed by probit analysis. One-way ANOVA (Turkey multiple comparison test) was applied for significance tests to verify differences between the control and the tested concentrations. Statistical significance was assigned at p o0.001.
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Fig. 1. Survival rate of sea urchin sperms (a) and eggs (b) exposed to different concentrations of TCS for 20 min. Bars show mean7 standard deviation of three replicate vials. n p o 0.001 tukey test.
Fig. 2. Effects of TCS on fertilization of S. nudus. Bars show mean7 standard deviation of triplicates. np o 0.001 Tukey test.
3. Results 3.1. Viability test Gamete viability of the sea urchin exposed to concentration between 0.1 and 1.5 μM of TCS was investigated. Fig. 1 shows the viability of sperm and eggs. It was found that the viability of sperm was affected by 0.5 μM TCS and was significantly influenced when sperm were exposed to concentrations higher than 1.0 μM TCS. Compared to the control, the sperm viability was decreased by 13 percent and 22 percent at 1.0 μM and 1.5 μM of TSC, respectively. All the sperm treated with higher than 2.0 μM concentrations of TSC died (Fig. 1a). No changes were observed in the viability of eggs at concentrations up to 1.5 μM (Fig. 1b).
3.2. TCS effect on fertilization The effects of TCS on the fertilization rate of the sea urchin were identified by the presence or absence of fertilization membranes. Fertilization rate was influenced at 0.5 μM of TCS and fertilization did not take place at concentrations higher than 1.25 μM of TCS (Fig. 2). It was observed that 97.4 71.21 percent, 92.9 71.80 percent, and 82.7 7 1.57 percent fertilization rates occurred upon exposure to 0.1, 0.5, and 1.0 μM of TCS, respectively. TCS concentration was determined to have a EC50 value of 1.49 μM (1.26–2.26; 95 percent CI) and a LOEC value of 0.618 μM.
Fig. 3. Effects of TCS on early development of S. nudus. Bars show mean 7 standard deviation of three replicate vials. npo 0.001 Tukey test.
Table 1 Toxicololgical estimation using the form of normal development each status in S. nudus exposed to TCS. End-point
EC50 (mM)
95% CI (mM)
LOEC (mM)
Hatched blastula Gastrula Prism Pluteus
1.421 1.037 0.949 0.987
1.232–1.798 0.924–1.211 0.851–1.081 0.895–1.116
0.514 0.340 0.331 0.385
EC50: 50 percent effective concentration, 95 percent CI: 95 percent confidence limit, and LOEC: lowest observed effective concentration.
3.3. Embryotoxicity of TCS Fig. 3 indicates the effects of TCS on the early embryonic development of the sea urchin (15 h, blastula stage; 30 h, gastrula stage, 48 h, prism stage; and 72 h, pluteus stage). Whereas the embryonic development was not effected in the group exposed to low concentrations of TCS up to 0.5 mM, it was significantly inhibited in the embryos treated with 1.0 mM TCS (Fig. 3). Furthermore, the embryonic development from fertilized eggs to pluteus proceeded normally based on the exposure period, while the development in some embryos was delayed or tended to be arrested. In addition, most embryos in each stage exhibited abnormal development at greater than 1.5 mM TCS. EC50 and LOEC values calculated by the Probit analysis are shown in Table 1 (Table 1).
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Fig. 4. Development rate of S. nudus embryos with different times of recovery from TCS exposure. (a) and (b) Development rates of S. nudus embryos previously exposed to 0.5 or 1.0 mM TCS for 15h, 30 h and 48 h, respectively, followed by removal of TSC by washing them with fresh media without TCS. The development of embryos was monitored under the microscope up to 72 h. The control embryos were continuously exposed to TCS without withdrawal. Bars show mean7 standard deviation of three replicate vials. np o0.001 Tukey test.
3.4. Recovery test To evaluate the sensitivity based on exposure time during the early embryonic development of the sea urchin, the embryos were exposed to TCS 1.0 μM, for 15, 30 or 48 h after fertilization. The embryos of each experimental group were then transferred to TCS-free FSW and it was observed whether embryonic development was normal or not. In the treatment of TSC for 15 h, normally developed embryos tended to be increased by 11 and 40 percent at 48 h and 72 h after TSC removal, respectively (Fig. 4). However, the embryos exposed for over 30 h were not recovered to normal development until 72 h.
4. Discussion The embryonic and larval stages of marine invertebrates are more sensitive to toxicants compared to adults (Huffman Ringwood, 1991). The embryo-larval bioassays of sea urchins have been utilized as a sensitive, simple, and reliable method to monitor and evaluate marine contamination for decades (Böttger et al., 2001; His et al., 1997; Krause, 1994). In the present study, bioassays were carried out by applying gametes viability and the early embryonic development success to observe the response of the TCS treated sea urchin (S. nudus). We found that the swimming speed of sperm was remarkably decreased by TSC in a dose-dependent manner, while the viability of eggs was not affected even when they were exposed to a high concentration of TCS up to 2.0 mM (Fig. 1).These results might be due to the destabilization of sperm plasma membrane in response to TSC, followed by the disturbance in the functional integrity of ́ et al., 2001) and function of egg the cell membrane (Villalaın membrane, which suppresses the absorption of contaminants (Eyster and Morse, 1984; Hollows et al., 2007). This data suggests that sperms are more appropriate to be utilized for studies of the effects of gamete viability of S. nudus, rather than eggs. In addition, we observed that the embryos obtained from the fertilization of gametes became sensitive to TCS, so that were not developed normally (Fig. 3). This indicates that the deleterious effects of TSC on the early embryonic development may take place after fertilization because TSC cannot be accumulated in eggs. In fact, it is certainly possible that TCS was able to get inside the embryo through a change of membrane permeability of embryo treated with TCS (Franchet et al., 1997). On the other hand, hatching time has been widely employed as a marker of toxicity tests on fish early-life stage. Recent studies have reported the sensitivity against
TCS in many aquatic organisms in respect to embryonic hatching, including sea urchins (Psammechinus miliaris), medaka (Oryzias latipes), and zebrafish (Danio rerio) (Anselmo et al., 2011; Ishibashi et al., 2004; Oliveira et al., 2009). For examples, the hatching of fertilized P. miliaris eggs treated with 0.5 mM and 1.0 mM of TCS for 72 h was completely failed, and over 80 percent of abnormalities were shown during exposure to TCS, 0.5 mM (Anselmo et al., 2011). In addition, as freshwater organisms, medaka (Ishibashi et al., 2004) and zebrafish (Oliveira et al., 2009) showed a significant delay in hatching on embryos exposed to 2.16 mM and 2.40 mM of TCS for 72 h, respectively. In this study, we observed that significant delay in the embryonic hatching in response to 1.0 mM of TCS. This data suggests that the embryo of S. nudus is less sensitive to TCS than that of P. miliaris, but more sensitive than that of medaka and zebrafish. In particular, delayed developmental rates as well as abnormalities were observed in P. miliaris exposed to over 0.5 mM of TCS (Anselmo et al., 2011); whereas only delayed development was exhibited in S. nudus in response to 1.0 mM. Interestingly, in the recovery tests to investigate the effects of different exposure periods on the embryonic development, we observed that less drastic effects when exposure period is shorter. For example, S. nudus larvae in the blastula stage, treated with 1.0 mM of TCS for less than 30 h prior to transferal to TCS-free FSW, tended to be recovered to normal larvae over time, while were not recovered at all when exposed to TCS more than 30 h, suggesting that embryos with shorter exposure periods are less affected and can recover to normal embryonic development. Moreover, our study regarding the effects of TCS on the early embryonic development of sea urchins showed that the developmental rate of embryos exposed to 1.0 mM of TCS for 30 h was reduced by approximately 50 percent. However, in the recovery test, we observed an approximate 30 percent of the normal development rate in the same condition. Such discrepancy might be due to the stress-induced decrease in development rate that occurs while transferring from TCS containing culture media to TCS-free FSW and washing. In conclusion, TCS negatively influenced sperm viability, fertilization, and normal embryonic development in sea urchins. Overall, this study implies that gametes and larvae of sea urchins provide a superior organism for the evaluation of TCS for biomonitoring, as a semi-quantitative method.
Acknowledgment This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by
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the Ministry of Education, Science and Technology (2010-0016829), and by Grants from the Korea Institute of Ocean Science & Technology (No. PE99161).
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of triclosan on reproductive prarmeters and embryonic development of sea urchin, Strongylocentrotus nudus Jinik Hwang a, Sung-Suk Suh a, Man chang b, So Yun Park c, Tae Kwon Ryu d, Sukchan Lee e, Taek-Kyun Lee a,n a
South Sea Environment Research Department, Korea Institute of Ocean Science & Technology, Geoje 656-830, Republic of Korea Marine Ecosystem Research Division, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of Korea c Risk Assessment Division, National Institute of Environmental Research, Incheon 404-708, Republic of Korea d Medical Research Center of Neural Dysfunction, Gyeonsang National University, Jinju 660-751, Republic of Korea e Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2013 Received in revised form 21 October 2013 Accepted 23 October 2013 Available online 13 November 2013
Triclosan (TCS, 2,4,4′-trichloro-2′-hydroxydiphenyl ether), a broad-spectrum antibacterial agent, is commonly found in the aquatic environment. In this study, we investigated TCS toxicity with pertaining to gamete viability, fertilization, and embryogenesis up to pluteus stage of the sea urchin, (Strongylocentrotus nudus). When the sperm and eggs were exposed to TCS (0–3.0 μM), the viability of sperm was significantly decreased at molarities higher than 1 μM of TCS. In addition, for exposure of 2.0 μM TCS the viability of eggs was not influenced and none of the sperm was viable. Fertilization rate was significantly decreased when sperm were exposed to 0.5 and 1 μM of TCS (p o0.001) and no fertilization was observed for the exposure of 1.5 μM of TCS. In embryonic development, embryos are treated with higher than 1.0 μM levels of TCS displayed arrested development. For TCS, the EC50 and LOECs values were 1.8, 1.49 and 0.99 μM and 0.53, 0.62 and 0.39 μM for sperm viability, fertilization rate, and larval development to pluteus, respectively. In the recovery test regarding normal development of arrested embryos based upon TCS exposure time, it was observed that embryos exposed to 1 μM TCS for 15 h were normally recovered for normal development, while embryos with more than 30 h exposure were not recovered to normal larvae. Overall, the results of this study strongly suggest that the gametes and embryos of S. nudus can provide the basis for an effective bioassay, with a fast and sensitive means of evaluating TCS contamination in the marine ecosystem. & 2013 Elsevier Inc. All rights reserved.
Keywords: Triclosan Sea urchin Fertilization Early embryonic development Viability
1. Introduction TCS (TCS, 2,2,4′-trichloro-2′-hydroxydiphenyl ether), which is a synthetic broad spectrum antibacterial agent, is utilized in a wide range of products, such as soaps, shampoos, toothpastes, cleaning materials, and cosmetics (Jones et al., 2000). TCS has also been added to polymers and textile fibers as an anti-bacterial consumer products, including toys, undergarments, and cutting boards (Schweizer, 2001). In view of the fact that, TCS is mostly used in the production of consumer products (greater than 95 percent), it is found in domestic sewage and wastewater treatment plants (WWTPs) (Reiss et al., 2002; Lindström et al., 2002; Singer et al., 2002). TCS is extensively utilized and is very difficult to eliminate it completely during the waste water treatment hence about 10.0 ng/L to 98.0 ng/L of TCS has been detected in effluents
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Corresponding authors. Fax: þ 82 55 639 8639. E-mail addresses: [email protected], [email protected] (T.-K. Lee).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.029
(Boyd et al., 2003; Singer et al., 2002). Earlier review reported that TCS was one of the most concentrated chemical substances among various chemical substances that were included in the influents in the waste water treatment plants (Heidler and Halden, 2007). Although TCS is biologically degradable (Singer et al., 2002), it is often detected in rivers and lakes at thenanogram per liter level (Balmer et al., 2004; Bester, 2005; Kolpin et al., 2002; Lindström et al., 2002). Moreover, it has been reported that TCS is a precursor of dioxin congener (2,8-dichlorodibenzo-pdioxin, DCDD) by photolytic degradation (Aranami and Readman, 2007), and may form methyl-TCS via biotransformation (Lindström et al., 2002). TCS is high hydrophobic, with a log Kow value of 4.8, which is the solubility of TCS in octanol/and in water, it likely to be easily accumulated in adipose tissues. methyl-TCS, on the other hand, is relatively more lipophilic compared to TCS, thereby extending its persistence, and resulting in its bio-accumulation in the tissues of many organisms, being detected even in human milk (AdolfssonErici et al., 2002; Balmer et al., 2004). In accordance with the
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gradual elevation of consumption of the products containing TCS, and the accumulated results of studies regarding acute, subacute/ subchronic, and chronic toxicity in detail, the utilization of TCS in food products and personal hygiene products has been restricted recently in many countries, including those of the European Union, as well as Canada and the US (Dayan and Yoshida, 2007). The consequences of TCS on the marine environment have not been sufficiently understood, but recent studies have reported acute and chronic toxicity values for TCS in aquatic organisms (Cortez et al., 2012; Gaume et al., 2012). Based on the results from acute in vitro and in vivo tests, TCS could affect the structures and functions of algal communities (Wilson et al., 2003), crustaceans and fishes (Ishibashi et al., 2004; Orvos et al., 2002). In addition to acute testing, TCS cytotoxicity was evaluated in marine bivalves. TCS reduced lysosomal membrane stability in mussels at low concentrations, and induced the extracellular release of lysosomal hydrolytic enzymes (Canesi et al., 2004). Furthermore, not only the cytotoxic and genotoxic effects of TCS in zebra mussels (Binelli et al., 2009) and zebrafish (Oliveira et al., 2009), but also the biological effects of TCS with regards to fertilization and early embryonic development in sea urchins and marine mussels have been investigated. (Anselmo et al., 2011) Sea urchins are representative marine organisms selected by USEPA, ASTM, and OECD for bioassays such as binding ability of tube feet, cytological and genetic malformations, changes in sperm motility to evaluate marine environmental samples (seawater, effluents, and sediment). The echinoderms are a suitable biological model for environmental toxicity studies because they are convenient to maintain in the laboratory, produce many translucent eggs, and allow in vitro fertilization all year around. Owing to the short pelagic period of sea urchin larvae, as well as the short duration of larval toxicity tests, the embryos and larvae of sea urchins should provide for a suitable biological organism for testing seawater quality standards. The sea urchin, Strongylocentrotus nudus is a typical costal species of Southern Korea. Numbers of sea urchins have gradually decreased due to contamination arising from a human interference. The objective of this study was to investigate the adverse effects of TCS on embryonic development in Korean sea urchins. In particular, sperm and egg viability, fertilization and development rate of S. nudus exposed to TCS were analyzed. Also, recovery tests were employed to obtain information regarding critical TCS exposure time. The results of this study would provide us with a better understanding of the effects of TCS on the development of sea urchin and would clarify the influences of anthropogenic activity in aquatic organisms. 2. Material and method 2.1. Animals and chemicals Adults of S. nudus were collected at the subtidal zone at Geoje in Korea. S. nudus is reproductive from July to September in the waters of Geoje. After the adult sea urchins were transferred to the laboratory, they were reared in aquarium at 18 1C with a 12 h light/dark cycle to prevent natural egg laying. Triclosan (TCS 498 percent purity) was purchased from Tokyo Chemical Industry (Japan), and a stock solution (100 μM) was made in 100 percent ethanol, diluted in Filtered Sea Water (FSW), and used the same day. The ethanol was used for the control in this experiment. All chemicals used in the study were purchased from Sigma Aldrich. 2.2. Gamete collections To obtain eggs and sperm from the sea urchin adults, 1.0 mL of 1 M KCl was injected and was then manually shaken until evenly distributed. KCl-treated males emitted white sperm from the gonopore, which was refrigerated (4.0 1C) after being collected in a small centrifugal tube (1.5 mL). The females emitted yellow eggs. To collect the eggs, clean seawater was added to a 80 mL beaker and the adults were then placed so that the gonopore faced to the seawater in the beaker.
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Released eggs were settled on the bottom of the beaker. Settled eggs were then transferred to a beaker filled with seawater and the floating eggs were then removed in order to select eggs of a good quality.
2.3. Viability test of gametes To study the effects of TCS on egg and sperm viability, 300 mL of egg or sperm dilution (50 mL of sperm in 6.0 mL of FSW) were added to vials containing 5.0 mL of FSW and TCS at different concentrations. The ethanol was used for the control in this experiment. After exposing eggs or sperm to TCS for 20 min, they were transferred to a hemocytometer and were then stained with 0.4 percent trypan blue, followed by counting the number of viable gametes via a microscope. Three replicates of each experiment were performed on different culture batches on different dates.
2.4. Fertilization assay The fertilization rate was evaluated based on USPEA (1995) standard. To test the effects of TCS on fertilization, 500 unfertilized eggs of the same batch and 15.0 mL of sperm dilution was added to vials containing 5.0 mL of FSW and TCS at different concentrations. The ethanol was used for the control in this experiment. Eggs were incubated for 20 min, until control eggs formed a fertilization membrane, after which a few drops of formalin were added to all vials. The fertilization rate (FR, percentage of fertilized eggs) was then calculated. The experiments were repeated for three times on different culture batches at different dates.
2.5. Embryogenesis assay For each experiment, eggs and sperm of at least three adults were then transferred to glass Petri dishes containing FSW for cross-fertilization, and were maintained at 15.0 1C. A considerable amount of sperm and debris that were interfered with the embryogenesis assay were filtered out using a 40 mm sieve. Also, floated fertilized eggs on the seawater were removed in order to differentiate healthy eggs. A solution of fertilized eggs with approximately 300 egg mL 1 was used in this study. Fertilized eggs were treated with different concentrations of TCS (0, 0.1, 0.5, 1.0 and 1.5 μM) dissolved in 80 mL of FSW. Fertilized eggs were cultured in a 15 1C incubator with 12 h light/dark cycle and were adjusted to the final density of 100 eggs/mL 1. The beaker was covered with parafilm to minimize evaporation. Samples were collected at the time periods of each developmental stage (15 h, blastula stage; 30 h gastrula stage; 48 h, prism stage; and 72 h, pluteus stage) and were then subjected to analysis under an optical microscope in order to evaluate the development rate in early embryonic development. Development rate was defined as the number of embryos with normal development dividing by the number of embryos exposed. In the abnormal development of embryo exposed, division of development cell did not occur anymore. The experiments were repeated for three times on different culture batches on different dates.
2.6. Recovery test Embryos were exposed to 0.5 or 1.0 μM TCS for four different time periods; 15 h (blastula (hatching)), 30 h (gastrula), 48 h (prism) and 72 h (pluteus). Exposure periods were summarized schematically in Supplementary information 1; TCS exposure from the fertilized egg stage to the blastula; B, TCS exposure from the fertilized egg stage to the gastrula stage; C, TCS exposure from the fertilized egg stage to the prism stage; D, TCS exposure from the fertilized egg stage to the pluteus stage (please see Supplementary information 1). The embryos treated with TCS in each stage were filtered through a sieve (size 100 μm) and were then washed twice with FSW. The experiments were repeated for three times on different culture batches on different dates.
2.7. Statistical analysis All data are presented as means 7 standard deviation (SD). Student's t–test was performed to test difference between the control and each experimental group. Probit analysis (Finney 1971) was carried out to evaluate the concentration that larvae dies as well as the fertilization and development rates. EC50 and LOEC values were also analyzed by probit analysis. One-way ANOVA (Turkey multiple comparison test) was applied for significance tests to verify differences between the control and the tested concentrations. Statistical significance was assigned at p o0.001.
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Fig. 1. Survival rate of sea urchin sperms (a) and eggs (b) exposed to different concentrations of TCS for 20 min. Bars show mean7 standard deviation of three replicate vials. n p o 0.001 tukey test.
Fig. 2. Effects of TCS on fertilization of S. nudus. Bars show mean7 standard deviation of triplicates. np o 0.001 Tukey test.
3. Results 3.1. Viability test Gamete viability of the sea urchin exposed to concentration between 0.1 and 1.5 μM of TCS was investigated. Fig. 1 shows the viability of sperm and eggs. It was found that the viability of sperm was affected by 0.5 μM TCS and was significantly influenced when sperm were exposed to concentrations higher than 1.0 μM TCS. Compared to the control, the sperm viability was decreased by 13 percent and 22 percent at 1.0 μM and 1.5 μM of TSC, respectively. All the sperm treated with higher than 2.0 μM concentrations of TSC died (Fig. 1a). No changes were observed in the viability of eggs at concentrations up to 1.5 μM (Fig. 1b).
3.2. TCS effect on fertilization The effects of TCS on the fertilization rate of the sea urchin were identified by the presence or absence of fertilization membranes. Fertilization rate was influenced at 0.5 μM of TCS and fertilization did not take place at concentrations higher than 1.25 μM of TCS (Fig. 2). It was observed that 97.4 71.21 percent, 92.9 71.80 percent, and 82.7 7 1.57 percent fertilization rates occurred upon exposure to 0.1, 0.5, and 1.0 μM of TCS, respectively. TCS concentration was determined to have a EC50 value of 1.49 μM (1.26–2.26; 95 percent CI) and a LOEC value of 0.618 μM.
Fig. 3. Effects of TCS on early development of S. nudus. Bars show mean 7 standard deviation of three replicate vials. npo 0.001 Tukey test.
Table 1 Toxicololgical estimation using the form of normal development each status in S. nudus exposed to TCS. End-point
EC50 (mM)
95% CI (mM)
LOEC (mM)
Hatched blastula Gastrula Prism Pluteus
1.421 1.037 0.949 0.987
1.232–1.798 0.924–1.211 0.851–1.081 0.895–1.116
0.514 0.340 0.331 0.385
EC50: 50 percent effective concentration, 95 percent CI: 95 percent confidence limit, and LOEC: lowest observed effective concentration.
3.3. Embryotoxicity of TCS Fig. 3 indicates the effects of TCS on the early embryonic development of the sea urchin (15 h, blastula stage; 30 h, gastrula stage, 48 h, prism stage; and 72 h, pluteus stage). Whereas the embryonic development was not effected in the group exposed to low concentrations of TCS up to 0.5 mM, it was significantly inhibited in the embryos treated with 1.0 mM TCS (Fig. 3). Furthermore, the embryonic development from fertilized eggs to pluteus proceeded normally based on the exposure period, while the development in some embryos was delayed or tended to be arrested. In addition, most embryos in each stage exhibited abnormal development at greater than 1.5 mM TCS. EC50 and LOEC values calculated by the Probit analysis are shown in Table 1 (Table 1).
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Fig. 4. Development rate of S. nudus embryos with different times of recovery from TCS exposure. (a) and (b) Development rates of S. nudus embryos previously exposed to 0.5 or 1.0 mM TCS for 15h, 30 h and 48 h, respectively, followed by removal of TSC by washing them with fresh media without TCS. The development of embryos was monitored under the microscope up to 72 h. The control embryos were continuously exposed to TCS without withdrawal. Bars show mean7 standard deviation of three replicate vials. np o0.001 Tukey test.
3.4. Recovery test To evaluate the sensitivity based on exposure time during the early embryonic development of the sea urchin, the embryos were exposed to TCS 1.0 μM, for 15, 30 or 48 h after fertilization. The embryos of each experimental group were then transferred to TCS-free FSW and it was observed whether embryonic development was normal or not. In the treatment of TSC for 15 h, normally developed embryos tended to be increased by 11 and 40 percent at 48 h and 72 h after TSC removal, respectively (Fig. 4). However, the embryos exposed for over 30 h were not recovered to normal development until 72 h.
4. Discussion The embryonic and larval stages of marine invertebrates are more sensitive to toxicants compared to adults (Huffman Ringwood, 1991). The embryo-larval bioassays of sea urchins have been utilized as a sensitive, simple, and reliable method to monitor and evaluate marine contamination for decades (Böttger et al., 2001; His et al., 1997; Krause, 1994). In the present study, bioassays were carried out by applying gametes viability and the early embryonic development success to observe the response of the TCS treated sea urchin (S. nudus). We found that the swimming speed of sperm was remarkably decreased by TSC in a dose-dependent manner, while the viability of eggs was not affected even when they were exposed to a high concentration of TCS up to 2.0 mM (Fig. 1).These results might be due to the destabilization of sperm plasma membrane in response to TSC, followed by the disturbance in the functional integrity of ́ et al., 2001) and function of egg the cell membrane (Villalaın membrane, which suppresses the absorption of contaminants (Eyster and Morse, 1984; Hollows et al., 2007). This data suggests that sperms are more appropriate to be utilized for studies of the effects of gamete viability of S. nudus, rather than eggs. In addition, we observed that the embryos obtained from the fertilization of gametes became sensitive to TCS, so that were not developed normally (Fig. 3). This indicates that the deleterious effects of TSC on the early embryonic development may take place after fertilization because TSC cannot be accumulated in eggs. In fact, it is certainly possible that TCS was able to get inside the embryo through a change of membrane permeability of embryo treated with TCS (Franchet et al., 1997). On the other hand, hatching time has been widely employed as a marker of toxicity tests on fish early-life stage. Recent studies have reported the sensitivity against
TCS in many aquatic organisms in respect to embryonic hatching, including sea urchins (Psammechinus miliaris), medaka (Oryzias latipes), and zebrafish (Danio rerio) (Anselmo et al., 2011; Ishibashi et al., 2004; Oliveira et al., 2009). For examples, the hatching of fertilized P. miliaris eggs treated with 0.5 mM and 1.0 mM of TCS for 72 h was completely failed, and over 80 percent of abnormalities were shown during exposure to TCS, 0.5 mM (Anselmo et al., 2011). In addition, as freshwater organisms, medaka (Ishibashi et al., 2004) and zebrafish (Oliveira et al., 2009) showed a significant delay in hatching on embryos exposed to 2.16 mM and 2.40 mM of TCS for 72 h, respectively. In this study, we observed that significant delay in the embryonic hatching in response to 1.0 mM of TCS. This data suggests that the embryo of S. nudus is less sensitive to TCS than that of P. miliaris, but more sensitive than that of medaka and zebrafish. In particular, delayed developmental rates as well as abnormalities were observed in P. miliaris exposed to over 0.5 mM of TCS (Anselmo et al., 2011); whereas only delayed development was exhibited in S. nudus in response to 1.0 mM. Interestingly, in the recovery tests to investigate the effects of different exposure periods on the embryonic development, we observed that less drastic effects when exposure period is shorter. For example, S. nudus larvae in the blastula stage, treated with 1.0 mM of TCS for less than 30 h prior to transferal to TCS-free FSW, tended to be recovered to normal larvae over time, while were not recovered at all when exposed to TCS more than 30 h, suggesting that embryos with shorter exposure periods are less affected and can recover to normal embryonic development. Moreover, our study regarding the effects of TCS on the early embryonic development of sea urchins showed that the developmental rate of embryos exposed to 1.0 mM of TCS for 30 h was reduced by approximately 50 percent. However, in the recovery test, we observed an approximate 30 percent of the normal development rate in the same condition. Such discrepancy might be due to the stress-induced decrease in development rate that occurs while transferring from TCS containing culture media to TCS-free FSW and washing. In conclusion, TCS negatively influenced sperm viability, fertilization, and normal embryonic development in sea urchins. Overall, this study implies that gametes and larvae of sea urchins provide a superior organism for the evaluation of TCS for biomonitoring, as a semi-quantitative method.
Acknowledgment This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by
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the Ministry of Education, Science and Technology (2010-0016829), and by Grants from the Korea Institute of Ocean Science & Technology (No. PE99161).
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