- Email: [email protected]
Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum
Chemosphere 66 (2007) 1353–1357 www.elsevier.com/locate/chemosphere
Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum Suguru Inoue a,b, Yuji Oshima a,*, Hironori Usuki c, Masami Hamaguchi c, Yukio Hanamura c, Norihisa Kai d, Yohei Shimasaki a, Tsuneo Honjo a a
c
Laboratory of Fisheries Environmental Science, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan b Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan National Research Institute of Fisheries and Environment of Inland Sea (FEIS), Fisheries Research Agency (FRA), Maruishi 2-17-5, Hatsukaichi, Hiroshima 739-0452, Japan d Department of Food Science and Technology, National Fisheries University, Nagata-honmachi 2-7-1, Shimonoseki 759-6595, Japan Received 2 February 2006; received in revised form 12 June 2006; accepted 19 June 2006 Available online 4 August 2006
Abstract We investigated the effects of waterborne and maternal exposure to tributyltin (TBT) on veliger larvae of the Manila clam, Ruditapes philippinarum. In a waterborne exposure test, veliger larvae (D-larvae stage: 24 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.055, 0.130, 0.340, and 0.600 lg/l for 13 d. The percentage of normal veliger larvae (the ratio of normal veliger larvae to all larvae) decreased significantly in all TBT treatment groups compared with that in the control group. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, and 0.310 lg/l at 20–22 C for 3 weeks, and the percentage of normal veliger larvae assessed for 13 d. No maternal effects on veliger larvae from TBT were observed in TBT treatment groups as compared with the control group. These results demonstrate that waterborne TBT affects Manila clam veliger larvae, and indicates that TBT may have reduced Manila clam populations by preventing the development and survival of veliger larvae. 2006 Elsevier Ltd. All rights reserved. Keywords: Manila clam (Ruditapes philippinarum); Veliger larvae; Waterborne exposure; Maternal exposure; Tributyltin (TBT)
1. Introduction Tributyltin (TBT) has been widely used as an antifouling agent in marine environments since the early 1960s (Huggett et al., 1992). Some countries have regulated its use as an antifoulant (Champ, 2000) because TBT has been reported to inhibit growth (Triebskorn et al., 1994), behavior (Nakayama et al., 2004), reproduction (Nirmala et al., 1999; Inoue et al., 2004), and sexual differentiation (Shimasaki et al., 2003) in aquatic organisms; in addition, TBT is well known to induce masculinization and inhibit reproduction in gastropods, a condition known as imposex
*
Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima).
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.06.052
(Bryan et al., 1986). However, TBT continues to be detected in water (Inoue et al., 2002), sediment (Inoue et al., 2002), and aquatic organisms (Inoue et al., 2006a). Populations of the Manila clam Ruditapes philippinarum (Adams et Reeve), a very important commercial species in Japan, have decreased markedly over the last two decades (Sekiguchi and Ishii, 2003). This decline is ascribed to factors such as overfishing, habitat reduction, water pollution, and instability of sediments (Kakino, 1992; Toba, 2002; Tsutsumi et al., 2002). Inoue et al. (2006b) reported that TBT may decrease the embryonic development of the Manila clam in the wild and ultimately reduce clam populations in Japan. Similarly, in Europe, Ruiz et al., (1995b,c) suggested that TBT probably reduced populations of the clam Scrobicularia plana in northern Europe in the 1980s by preventing the successful development of embryos.
1354
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
The planktonic veliger larval stage of the Manila clam lasts for 2–4 weeks before settling on sea bottom (Toba et al., 1992). The larvae swim in the seawater as plankton and feed on phytoplankton (Toba et al., 1992). Several studies have reported that development of bivalve veliger larvae is inhibited by TBT (Lapota et al., 1993; Ruiz et al., 1995a; Coelho et al., 2001). Ruiz et al. (1995a) demonstrated that at environmentally relevant concentrations (over 0.05 lg/l), TBT decreased the larval growth rate of S. plana. It has been reported that the decrease in Manila clam populations in Ariake Sea may be caused by a lack of larval recruitment (Ishii et al., 2001; Ishii and Sekiguchi, 2002). TBT may reduce the population by preventing planktonic veliger larvae development, in addition to its impact on embryo development as reported by Inoue et al. (2006b). However, no study has reported on the effect of TBT on Manila clam planktonic veliger larvae. In this study, we investigated the effects of waterborne and maternal TBT exposure on Manila clam planktonic veliger larvae.
and 0.800 lg/l at 23 C with aeration. Each test solution containing TBT was prepared by the addition of TBTO stock solution to seawater. Three days after fertilization, veliger larvae were fed phytoplankton (Pavlova sp., 5 · 103 cells/ml) every morning during exposure periods. The seawater containing TBT was exchanged daily. A sample of the seawater of each TBT treatment group was collected in a 1 l glass bottle every 3 d and stored at 5 C until TBT analysis. About 50 larvae in each treatment group were sampled after 3, 5, 7, 9, 11 and 13 d of exposure. The percentage of normal veliger larvae (normal veliger larvae/total number of larvae · 100) was then assessed under the microscope. Abnormal veliger larvae had an incomplete shell and protruded vellum. All waterborne exposure tests were conducted in triplicate. 2.4. Maternal exposure to TBT
Manila clams (average body weight 12.0 ± 2.9 g) were obtained from the coast of the Seto Inland Sea (Hiroshima Prefecture, Japan) in October and November 2001. Maturity was confirmed by observation of dissected gonads.
Exposure of the clams to TBT was performed using the same methods and under the same conditions as described by Inoue et al. (2006b). In brief, each treatment group of 100 clams was semistatically exposed to TBTO at nominal concentrations of 0, 0.100, or 0.400 lg/l for 6 h per day for 3 weeks. For TBT analysis, a sample of the seawater of each TBT treatment group was collected in 1 l glass bottles once per week and stored at 5 C until TBT analysis. After exposure, spawning induction and fertilized eggs were treated as described above regarding the exposure of veliger larvae to waterborne TBT. They were then resuspended in 30 l of filtered seawater at 23 C, which was replaced 3 times every 1 h. Swimming veliger larvae (Dlarvae stage) were collected 24 h after fertilization, using a 60 lm mesh nylon net. The larvae were divided into groups, suspended in three 1 l beakers containing 1 l seawater at 20 larvae/ml, and then maintained at 23 C with aeration. The veliger larvae were fed phytoplankton (Pavlova sp., 5 · 103 cells/ml) every morning 3 d after fertilization. Seawater was exchanged daily. The veliger larvae were observed every other day for 13 d after fertilization, as described above.
2.3. Exposure of veliger larvae to waterborne TBT
2.5. Analysis of TBT concentration
Fertilized eggs were collected from clams that had not been treated with TBT, using the methods described by Inoue et al. (2006b). In brief, spawning was induced by feeding the plankton at high concentrations. Next, the fertilized eggs were filtered with a 20 lm mesh nylon net and then washed gently 3 times with filtered seawater (1 lm). They were then re-suspended in 30 l of filtered seawater at 23 C, which was replaced 3 times every one hour. Twenty four hours after fertilization, in 1 l glass beakers containing 1 l of seawater, approximately 2.0 · 104 swimming veliger larvae (D-larvae stage) were exposed to TBTO at the following concentrations: 0 (control); 0 + 5 ll DMSO/l (DMSO control); 0.100; 0.200; 0.400;
The extraction and analytical procedures for TBT in seawater was performed as described by Inoue et al. (2006b). In brief, one liter of each water sample was acidified with 10 ml of 1 M hydrochloric acid–methanol solution and then spiked with 1 lg of TBTCl-d27 as an internal standard. The sample was extracted twice with 20 ml of a 0.1% tropolone–hexane solution by stirring for 60 min with a magnetic stirrer. The extract was concentrated to 1 ml at 40 C using a Turbo Vap II (Zymark, Hopkinton, MA, USA) and then made up to 10 ml with hexane. This sample was ethylated by addition of 500 ll of 5% sodium tetraethylborate solution and 10 ml of water and shaken for 30 min. This solution was then hydrolyzed with 10 ml of 1 M potas-
2. Materials and methods 2.1. Reagents Tributyltin oxide (TBTO) was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Tributyltin chloride-d27 (TBTCl-d27) as a surrogate was purchased from Hayashi Pure Chemical Industries, Ltd. (Osaka, Japan). TBTO stock solutions of 10 and 100 lg/ml were prepared by dilution with dimethylsulfoxide (DMSO). The Sep-Pak Florisil cartridge was purchased from Waters (Milford, MA, USA). All organic solvents were of pesticide residue analytical grade. Other reagents used were of analytical grade. 2.2. Manila clams
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
sium hydroxide solution by shaking for 60 min. After centrifugation of the solution at 3000 rpm for 10 min, the hexane layer was collected and the aqueous layer was reextracted with 10 ml of hexane. All hexane layers thus obtained were combined and concentrated to 1 ml at 40 C using a Turbo Vap II. The concentrated hexane was cleaned with a Sep-Pak Florisil cartridge, and the resulting sample was concentrated to 0.5 ml using a Turbo Vap II at 40 C. The TBT concentration was measured with a gas chromatograph equipped with a mass spectrometer (model 6890 gas chromatograph, model 5973 mass spectrometer; Hewlett-Packard, Avondale, PA, USA) equipped with a HP-5MS column (30 m · 0.25 mm I.D. fused silica capillary column; Hewlett-Packard). The detection limit in the seawater sample was 0.005 lg/l. TBT concentrations in the soft body of the clams were analyzed according to Inoue et al. (2006b). In short, about 1–2 g (wet weight) of soft body sample was spiked with 1 lg of TBTCl-d27 as an internal standard and homogenized with 10 ml 1 M hydrochloric acid–methanol solution by polytron homogenizer (PCU11, Kinematica, Lucerne, Switzerland) for 3 min. The homogenate was extracted twice with 10 ml of 0.1% tropolone–hexane solution using an ultrasonicator (B42JH, Branson Ultrasonics, Danbury, CT, USA) for 30 min. Further extraction and measurement of TBT were performed using the methods mentioned above. TBT concentration was expressed on a wet-weight basis in the present study. The detection limit in the soft body of the clam sample was 0.003 lg/g. 2.6. Statistical analysis All percentage data were normalized by arc-sine squareroot transformation. Statistical differences in normal veli-
1355
ger larvae between the control and the TBT-exposed groups were tested with one-way analysis of variance and Dunnett’s multiple comparison test. Differences were considered significant at p < 0.05. All statistical analysis were done using SPSS 10.1 J for Windows software (SPSS, Chicago, IL, USA). 3. Results 3.1. TBT concentrations in experimental seawater In the waterborne exposure test, average TBT concentrations in the experimental seawater of the TBT treatment groups were 0.055 lg/l in the 0.100 lg/l treatment group, 0.130 lg/l in the 0.200 lg/l treatment group, 0.340 lg/l in the 0.400 lg/l treatment group and 0.600 lg/l in the 0.800 lg/l treatment group. TBT was not detected in the seawater of the DMSO control group and the control group (<0.005 lg/l). In the maternal exposure test, average TBT concentrations of the experimental seawater in the 0.100 lg/l and 0.400 lg/l groups were 0.061 lg/l and 0.310 lg/l, respectively. TBT was detected in the seawater of the control groups at 0.007 lg/l. In Sections 3 and 4, we use the measured concentrations of the TBT treatment groups in place of the nominal concentrations. 3.2. Waterborne exposure In both the control and DMSO control groups, over 90% of veliger larvae were observed to be normal throughout the experiment period (Fig. 1). The percentage of normal veliger larvae in the TBT treatment groups 7 d after fertilization had decreased significantly compared with the control group, except for the 0.055 lg/l treatment
Fig. 1. Percentage of observed normal veliger larvae following exposure to TBT for between 3 and 13 d. Asterisks indicate a significant difference from the control. p < 0.05. Data are means ± SD of three tests.
1356
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
Normal veliger larvae (%)
100 80 60 40 20 0
3
5
7 9 Days after fertilization Control
0.061 µg/l
11
13
0.310 µg/l
Fig. 2. Percentage of observed normal veliger larvae obtained from Manila clams following exposure to TBT for 3 weeks. Data are means ± SD of three tests.
group at 11 d and 13 d after fertilization. All veliger larvae in the 0.600 lg/l treatment group were dead by 11 d after fertilization. These results indicate that TBT in seawater interferes with development and survival of veliger larvae. 3.3. Maternal exposure The percentage of normal veliger larvae obtained from Manila clams exposed to TBTO was over 90% in all groups throughout the experiment period (Fig. 2). TBT concentrations in the whole body of female clams were 0.470 lg/g in the 0.061 lg/l treatment group and 0.750 lg/g in the 0.310 lg/l treatment group. These results suggest the TBT that accumulated in the females did not affect the development and survival of veliger larvae. 4. Discussion Our results demonstrate that waterborne TBT affects development and survival of veliger larvae. In contrast, accumulation of TBT in females did not inhibit the development and survival of veliger larvae. In the TBT waterborne exposure test, we observed veliger larvae with incomplete shells. Similarly, our previous study (Inoue et al., 2006b) observed D-larvae with incomplete shells in the TBT treatment group and suggested this may have resulted from CaCO3 formation, which was inhibited by TBT. In this study, TBT may also have inhibited shell formation, causing increased numbers of veliger larvae with incomplete shells in TBT treatment groups. During the waterborne exposure test, we observed that the percentage of normal veliger larvae decreased significantly in the 0.055 lg/l treatment group. It has been reported that TBT concentrations in seawater from northern Kyushu, Japan range from 0.005 to 0.091 lg/l (Inoue et al., 2002). Concentrations of TBT in seawater from
Otsuchi Bay ranged from 0.008 to 0.074 lg/l (Harino et al., 1998). Thus, TBT at levels present in seawater in Japanese coastal waters may interfere with development and survival of Manila clam veliger larvae. Ishii and Sekiguchi (2002) reported that the drastic decline in the Manila clam population in Ariake Sea may be caused by a lack of larval recruitment. From our results, we conclude that TBT in seawater may be responsible for decreases in the Japanese Manila clam population by preventing development and survival of planktonic veliger larvae. Although the TBT concentrations declined substantially in the Japanese coastal environment after the regulation of TBT usage was initiated in 1990 (Yamada and Kakuno, 2003), the Manila clam population has not recovered. Similarly, in Europe, Ruiz et al., (1995b,c) showed that the clam (Scrobicularia plana) has also not recovered, despite a decrease in TBT concentrations in seawater after TBT use was regulated. Several studies have demonstrated that invertebrate larvae stay near the surface of seawater (Manuel et al., 1996; Dobretsov and Miron, 2001), and it has been reported that bivalve larvae may come into contact with the surface micro layer (Hardy and Cleary, 1992). TBT concentrations in the surface micro layer have been measured to be 10–1000 times higher than in surface seawater (Watanabe et al., 1989), and may be a threat to the survival, growth and development of planktonic veliger larvae. Consequently, Manila clam populations may have continued to decline due to inhibition by TBT of larval development and survival, even after TBT usage was regulated. Inoue et al. (2006b) reported that TBT that accumulated in female Manila clams was transferred to the eggs and affected D-larvae development. However, in this study, no maternal effects on veliger larvae were observed in TBT treatment groups as compared with the control group. TBT concentrations in the veliger larvae were not analyzed, but we believe that the TBT that accumulated in female clams might not be transferred to veliger larvae. In short, we demonstrated that waterborne TBT, at environmentally relevant concentrations, affects the veliger larvae of the Manila clam. In contrast, no maternal adverse effects of TBT on veliger larvae were observed. We believe that TBT may have reduced the Manila clam population by preventing development and survival of veliger larvae, in addition to its impact on embryo development as reported by Inoue et al. (2006b).
Acknowledgements This study was partly supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 14360112). We thank the staff of the National Research Institute of Fisheries and Environment of Inland Sea and also Mr. Jumpei Arakawa of the Aichi Fisheries Research Institute for their help with our experiment.
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
References Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., Burt, G.R., 1986. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J. Mar. Biol. Assoc. UK 66, 611–640. Champ, M.A., 2000. A review of organotin regulatory strategies, pending actions, related costs and benefits. Sci. Total. Environ. 258, 21–71. Coelho, M.R., Guentes, S., Bebianno, M.J., 2001. TBT effects on the larvae of Ruditapes decussates. J. Mar. Biol. Assoc. UK. 81, 259–265. Dobretsov, S.V., Miron, G., 2001. Larval and post-larval vertical distribution of the mussel Mytilus edulis in the White Sea. Mar. Ecol. Prog. Ser. 218, 179–187. Hardy, J.T., Cleary, J., 1992. Surface micro layer contamination and toxicity in the german bight. Mar. Ecol. Prog. Ser. 91, 203–210. Harino, H., Fukushima, M., Yamamoto, Y., Kawai, S., Miyazaki, N., 1998. Contamination of butyltin and phenyltin compounds in the marine environment of Otsuchi Bay, Japan. Environ. Pollut. 101, 209– 214. Huggett, R.J., Unger, M.A., Seligman, P.F., Valkirs, A.O., 1992. The marine biocide tributyltin. Environ. Sci. Technol. 26, 232–237. Inoue, S., Oshima, Y., Imada, N., Honjo, T., 2002. Tributyltin pollution in coastal areas around northern Kyushu, Japan. J. Environ. Toxicol. 5, 43–50, in Japanese. Inoue, S., Oshima, Y., Nagai, K., Yamamoto, T., Go, J., Kai, N., Honjo, T., 2004. Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii). Environ. Toxicol. Chem. 23, 1276–1281. Inoue, S., Abe, S., Oshima, Y., Kai, N., Honjo, T., 2006a. Tributyltin contamination of the bivalves in coastal areas around northern Kyushu, Japan. Environ. Toxicol. 21, 244–249. Inoue, S., Oshima, Y., Usuki, H., Hamaguchi, M., Hanamura, Y., Kai, N., Shimasaki, Y., Honjo, T., 2006b. Effects of tributyltin maternal and/or waterborne exposure on the embryonic development of the Manila clam, Ruditapes philippinarum. Chemosphere 63, 881–888. Ishii, R., Sekiguchi, H., 2002. Larval recruitment of the clam Ruditapes philippinarum in Ariake Sound, southern Jpn. Jpn. J. Benthol. 57, 151– 157 (in Japanese). Ishii, R., Sekiguchi, H., Nakahara, Y., Jinnai, Y., 2001. Larval recruitment of the Manila clam Ruditapes philippinarum in Ariake Sound, southern Japan. Fisheries Sci. 67, 579–591. Kakino, J., 1992. Recent situation on the Japanese littleneck fisheries. Fish. Engineer. 29, 31–39 (in Japanese). Lapota, D., Rosenberg, D.E., Platter-Rieger, M., Seligman, P.F., 1993. Growth and survival of Mytilus edulis larvae exposed to low levels of dibutyltin and tributylin. Mar. Biol. 115, 413–419. Manuel, J.L., Gallager, S.M., Pearce, C.M., Manning, D.A., O’Dor, R.K., 1996. Veligers from different populations of sea scallop Placopecten
1357
magellanicus have different vertical migration patterns. Mar. Ecol. Prog. Ser. 142, 147–163. Nakayama, K., Oshima, Y., Yamaguchi, T., Tsuruda, Y., Kang, I.J., Kobayashi, M., Imada, N., Honjo, T., 2004. Fertilization success and sexual behavior in male medaka, Oryzias latipes, exposed to tributyltin. Chemosphere 55, 1331–1337. Nirmala, K., Oshima, Y., Lee, R., Imada, N., Honjo, T., Kobayashi, K., 1999. Transgenerational toxicity of tributyltin and its combined effects with polychlorinated biphenyls on reproductive processes in Japanese medaka (Oryzias latipes). Environ. Toxicol. Chem. 18, 717–721. Ruiz, J.M., Bryan, G.W., Gibbs, P.E., 1995a. Acute and chronic toxicity of tributyltin (TBT) to the preveliger larvae of the bivalve Scrobicularia plana. Marine Biology 124, 119–126. Ruiz, J.M., Bryan, G.W., Gibbs, P.E., 1995b. Effects of tributyltin (TBT) exposure on the veliger larvae development of the bivalve Scrobicularia plana (da Costa). J. Exp. Mar. Biol. Ecol. 186, 53–63. Ruiz, J.M., Bryan, G.W., Wigham, G.D., Gibbs, P.E., 1995c. Effects of tributyltin (TBT) exposure on the reproduction and embryonic development of the bivalve Scrobicularia plana. Mar. Environ. Res. 40, 363–379. Sekiguchi, H., Ishii, R., 2003. Drastic decreasing of annual catch yields of the Manila clam Ruditapes philippinarum in Ariake Sound, Southern Japan. Oceanogr. Jpn. 12, 21–36 (in Japanese). Shimasaki, Y., Kitano, T., Oshima, Y., Inoue, S., Imada, N., Honjo, T., 2003. Tributyltin causes masculinization in fish. Environ. Toxicol. Chem. 22, 141–144. Toba, M., 2002. Present status of the fishery for the clam Ruditapes philippinarum in Chiba Prefecture, Tokyo Bay. Jpn. J. Benthol. 57, 145–150 (in Japanese). Toba, D.R., Thompson, D.S., Chew, K.K., Anderson, G.J., Miller, M.B., 1992. Guide to Manila Clam culture in Washington. Washington Sea grant Program. University of Washington, pp. 1–80. Triebskorn, R., Ko¨hler, H.-R., Flemming, J., Braunbeck, T., Negele, R.-D., Rahmann, H., 1994. Evaluation of bis(tri-n-butyltin)oxide (TBTO) neurotoxicity in rainbow trout (Oncorhynchus mykiss). I. Behaviour, weight increase, and tin content. Aquat. Toxicol. 30, 189–197. Tsutsumi, H., Ishizawa, K., Tomishige, M., Moriyama, M., Sakamoto, K., Montani, S., 2002. Population dynamics of a clam, Ruditapes philippinarum, on an artificially created sand cover on the tidal flats at the river mouth of Midorikawa River. Jpn. J. Benthol. 57, 177–187 (in Japanese). Watanabe, N., Sakai, S., Takatsuki, H., 1989. Environmental behavior analysis to evaluate the fate of organotin. Environmental science 2, 275–286 (in Japanese). Yamada, H., Kakuno, A., 2003. Present status on the development of alternative tributyltin-free antifouling paints and toxicity of new biocides to aquatic organisms – Review. Bull. Fish. Res. Agen. 6, 56– 72 (in Japanese).
Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum Suguru Inoue a,b, Yuji Oshima a,*, Hironori Usuki c, Masami Hamaguchi c, Yukio Hanamura c, Norihisa Kai d, Yohei Shimasaki a, Tsuneo Honjo a a
c
Laboratory of Fisheries Environmental Science, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan b Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan National Research Institute of Fisheries and Environment of Inland Sea (FEIS), Fisheries Research Agency (FRA), Maruishi 2-17-5, Hatsukaichi, Hiroshima 739-0452, Japan d Department of Food Science and Technology, National Fisheries University, Nagata-honmachi 2-7-1, Shimonoseki 759-6595, Japan Received 2 February 2006; received in revised form 12 June 2006; accepted 19 June 2006 Available online 4 August 2006
Abstract We investigated the effects of waterborne and maternal exposure to tributyltin (TBT) on veliger larvae of the Manila clam, Ruditapes philippinarum. In a waterborne exposure test, veliger larvae (D-larvae stage: 24 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.055, 0.130, 0.340, and 0.600 lg/l for 13 d. The percentage of normal veliger larvae (the ratio of normal veliger larvae to all larvae) decreased significantly in all TBT treatment groups compared with that in the control group. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, and 0.310 lg/l at 20–22 C for 3 weeks, and the percentage of normal veliger larvae assessed for 13 d. No maternal effects on veliger larvae from TBT were observed in TBT treatment groups as compared with the control group. These results demonstrate that waterborne TBT affects Manila clam veliger larvae, and indicates that TBT may have reduced Manila clam populations by preventing the development and survival of veliger larvae. 2006 Elsevier Ltd. All rights reserved. Keywords: Manila clam (Ruditapes philippinarum); Veliger larvae; Waterborne exposure; Maternal exposure; Tributyltin (TBT)
1. Introduction Tributyltin (TBT) has been widely used as an antifouling agent in marine environments since the early 1960s (Huggett et al., 1992). Some countries have regulated its use as an antifoulant (Champ, 2000) because TBT has been reported to inhibit growth (Triebskorn et al., 1994), behavior (Nakayama et al., 2004), reproduction (Nirmala et al., 1999; Inoue et al., 2004), and sexual differentiation (Shimasaki et al., 2003) in aquatic organisms; in addition, TBT is well known to induce masculinization and inhibit reproduction in gastropods, a condition known as imposex
*
Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima).
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.06.052
(Bryan et al., 1986). However, TBT continues to be detected in water (Inoue et al., 2002), sediment (Inoue et al., 2002), and aquatic organisms (Inoue et al., 2006a). Populations of the Manila clam Ruditapes philippinarum (Adams et Reeve), a very important commercial species in Japan, have decreased markedly over the last two decades (Sekiguchi and Ishii, 2003). This decline is ascribed to factors such as overfishing, habitat reduction, water pollution, and instability of sediments (Kakino, 1992; Toba, 2002; Tsutsumi et al., 2002). Inoue et al. (2006b) reported that TBT may decrease the embryonic development of the Manila clam in the wild and ultimately reduce clam populations in Japan. Similarly, in Europe, Ruiz et al., (1995b,c) suggested that TBT probably reduced populations of the clam Scrobicularia plana in northern Europe in the 1980s by preventing the successful development of embryos.
1354
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
The planktonic veliger larval stage of the Manila clam lasts for 2–4 weeks before settling on sea bottom (Toba et al., 1992). The larvae swim in the seawater as plankton and feed on phytoplankton (Toba et al., 1992). Several studies have reported that development of bivalve veliger larvae is inhibited by TBT (Lapota et al., 1993; Ruiz et al., 1995a; Coelho et al., 2001). Ruiz et al. (1995a) demonstrated that at environmentally relevant concentrations (over 0.05 lg/l), TBT decreased the larval growth rate of S. plana. It has been reported that the decrease in Manila clam populations in Ariake Sea may be caused by a lack of larval recruitment (Ishii et al., 2001; Ishii and Sekiguchi, 2002). TBT may reduce the population by preventing planktonic veliger larvae development, in addition to its impact on embryo development as reported by Inoue et al. (2006b). However, no study has reported on the effect of TBT on Manila clam planktonic veliger larvae. In this study, we investigated the effects of waterborne and maternal TBT exposure on Manila clam planktonic veliger larvae.
and 0.800 lg/l at 23 C with aeration. Each test solution containing TBT was prepared by the addition of TBTO stock solution to seawater. Three days after fertilization, veliger larvae were fed phytoplankton (Pavlova sp., 5 · 103 cells/ml) every morning during exposure periods. The seawater containing TBT was exchanged daily. A sample of the seawater of each TBT treatment group was collected in a 1 l glass bottle every 3 d and stored at 5 C until TBT analysis. About 50 larvae in each treatment group were sampled after 3, 5, 7, 9, 11 and 13 d of exposure. The percentage of normal veliger larvae (normal veliger larvae/total number of larvae · 100) was then assessed under the microscope. Abnormal veliger larvae had an incomplete shell and protruded vellum. All waterborne exposure tests were conducted in triplicate. 2.4. Maternal exposure to TBT
Manila clams (average body weight 12.0 ± 2.9 g) were obtained from the coast of the Seto Inland Sea (Hiroshima Prefecture, Japan) in October and November 2001. Maturity was confirmed by observation of dissected gonads.
Exposure of the clams to TBT was performed using the same methods and under the same conditions as described by Inoue et al. (2006b). In brief, each treatment group of 100 clams was semistatically exposed to TBTO at nominal concentrations of 0, 0.100, or 0.400 lg/l for 6 h per day for 3 weeks. For TBT analysis, a sample of the seawater of each TBT treatment group was collected in 1 l glass bottles once per week and stored at 5 C until TBT analysis. After exposure, spawning induction and fertilized eggs were treated as described above regarding the exposure of veliger larvae to waterborne TBT. They were then resuspended in 30 l of filtered seawater at 23 C, which was replaced 3 times every 1 h. Swimming veliger larvae (Dlarvae stage) were collected 24 h after fertilization, using a 60 lm mesh nylon net. The larvae were divided into groups, suspended in three 1 l beakers containing 1 l seawater at 20 larvae/ml, and then maintained at 23 C with aeration. The veliger larvae were fed phytoplankton (Pavlova sp., 5 · 103 cells/ml) every morning 3 d after fertilization. Seawater was exchanged daily. The veliger larvae were observed every other day for 13 d after fertilization, as described above.
2.3. Exposure of veliger larvae to waterborne TBT
2.5. Analysis of TBT concentration
Fertilized eggs were collected from clams that had not been treated with TBT, using the methods described by Inoue et al. (2006b). In brief, spawning was induced by feeding the plankton at high concentrations. Next, the fertilized eggs were filtered with a 20 lm mesh nylon net and then washed gently 3 times with filtered seawater (1 lm). They were then re-suspended in 30 l of filtered seawater at 23 C, which was replaced 3 times every one hour. Twenty four hours after fertilization, in 1 l glass beakers containing 1 l of seawater, approximately 2.0 · 104 swimming veliger larvae (D-larvae stage) were exposed to TBTO at the following concentrations: 0 (control); 0 + 5 ll DMSO/l (DMSO control); 0.100; 0.200; 0.400;
The extraction and analytical procedures for TBT in seawater was performed as described by Inoue et al. (2006b). In brief, one liter of each water sample was acidified with 10 ml of 1 M hydrochloric acid–methanol solution and then spiked with 1 lg of TBTCl-d27 as an internal standard. The sample was extracted twice with 20 ml of a 0.1% tropolone–hexane solution by stirring for 60 min with a magnetic stirrer. The extract was concentrated to 1 ml at 40 C using a Turbo Vap II (Zymark, Hopkinton, MA, USA) and then made up to 10 ml with hexane. This sample was ethylated by addition of 500 ll of 5% sodium tetraethylborate solution and 10 ml of water and shaken for 30 min. This solution was then hydrolyzed with 10 ml of 1 M potas-
2. Materials and methods 2.1. Reagents Tributyltin oxide (TBTO) was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Tributyltin chloride-d27 (TBTCl-d27) as a surrogate was purchased from Hayashi Pure Chemical Industries, Ltd. (Osaka, Japan). TBTO stock solutions of 10 and 100 lg/ml were prepared by dilution with dimethylsulfoxide (DMSO). The Sep-Pak Florisil cartridge was purchased from Waters (Milford, MA, USA). All organic solvents were of pesticide residue analytical grade. Other reagents used were of analytical grade. 2.2. Manila clams
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
sium hydroxide solution by shaking for 60 min. After centrifugation of the solution at 3000 rpm for 10 min, the hexane layer was collected and the aqueous layer was reextracted with 10 ml of hexane. All hexane layers thus obtained were combined and concentrated to 1 ml at 40 C using a Turbo Vap II. The concentrated hexane was cleaned with a Sep-Pak Florisil cartridge, and the resulting sample was concentrated to 0.5 ml using a Turbo Vap II at 40 C. The TBT concentration was measured with a gas chromatograph equipped with a mass spectrometer (model 6890 gas chromatograph, model 5973 mass spectrometer; Hewlett-Packard, Avondale, PA, USA) equipped with a HP-5MS column (30 m · 0.25 mm I.D. fused silica capillary column; Hewlett-Packard). The detection limit in the seawater sample was 0.005 lg/l. TBT concentrations in the soft body of the clams were analyzed according to Inoue et al. (2006b). In short, about 1–2 g (wet weight) of soft body sample was spiked with 1 lg of TBTCl-d27 as an internal standard and homogenized with 10 ml 1 M hydrochloric acid–methanol solution by polytron homogenizer (PCU11, Kinematica, Lucerne, Switzerland) for 3 min. The homogenate was extracted twice with 10 ml of 0.1% tropolone–hexane solution using an ultrasonicator (B42JH, Branson Ultrasonics, Danbury, CT, USA) for 30 min. Further extraction and measurement of TBT were performed using the methods mentioned above. TBT concentration was expressed on a wet-weight basis in the present study. The detection limit in the soft body of the clam sample was 0.003 lg/g. 2.6. Statistical analysis All percentage data were normalized by arc-sine squareroot transformation. Statistical differences in normal veli-
1355
ger larvae between the control and the TBT-exposed groups were tested with one-way analysis of variance and Dunnett’s multiple comparison test. Differences were considered significant at p < 0.05. All statistical analysis were done using SPSS 10.1 J for Windows software (SPSS, Chicago, IL, USA). 3. Results 3.1. TBT concentrations in experimental seawater In the waterborne exposure test, average TBT concentrations in the experimental seawater of the TBT treatment groups were 0.055 lg/l in the 0.100 lg/l treatment group, 0.130 lg/l in the 0.200 lg/l treatment group, 0.340 lg/l in the 0.400 lg/l treatment group and 0.600 lg/l in the 0.800 lg/l treatment group. TBT was not detected in the seawater of the DMSO control group and the control group (<0.005 lg/l). In the maternal exposure test, average TBT concentrations of the experimental seawater in the 0.100 lg/l and 0.400 lg/l groups were 0.061 lg/l and 0.310 lg/l, respectively. TBT was detected in the seawater of the control groups at 0.007 lg/l. In Sections 3 and 4, we use the measured concentrations of the TBT treatment groups in place of the nominal concentrations. 3.2. Waterborne exposure In both the control and DMSO control groups, over 90% of veliger larvae were observed to be normal throughout the experiment period (Fig. 1). The percentage of normal veliger larvae in the TBT treatment groups 7 d after fertilization had decreased significantly compared with the control group, except for the 0.055 lg/l treatment
Fig. 1. Percentage of observed normal veliger larvae following exposure to TBT for between 3 and 13 d. Asterisks indicate a significant difference from the control. p < 0.05. Data are means ± SD of three tests.
1356
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
Normal veliger larvae (%)
100 80 60 40 20 0
3
5
7 9 Days after fertilization Control
0.061 µg/l
11
13
0.310 µg/l
Fig. 2. Percentage of observed normal veliger larvae obtained from Manila clams following exposure to TBT for 3 weeks. Data are means ± SD of three tests.
group at 11 d and 13 d after fertilization. All veliger larvae in the 0.600 lg/l treatment group were dead by 11 d after fertilization. These results indicate that TBT in seawater interferes with development and survival of veliger larvae. 3.3. Maternal exposure The percentage of normal veliger larvae obtained from Manila clams exposed to TBTO was over 90% in all groups throughout the experiment period (Fig. 2). TBT concentrations in the whole body of female clams were 0.470 lg/g in the 0.061 lg/l treatment group and 0.750 lg/g in the 0.310 lg/l treatment group. These results suggest the TBT that accumulated in the females did not affect the development and survival of veliger larvae. 4. Discussion Our results demonstrate that waterborne TBT affects development and survival of veliger larvae. In contrast, accumulation of TBT in females did not inhibit the development and survival of veliger larvae. In the TBT waterborne exposure test, we observed veliger larvae with incomplete shells. Similarly, our previous study (Inoue et al., 2006b) observed D-larvae with incomplete shells in the TBT treatment group and suggested this may have resulted from CaCO3 formation, which was inhibited by TBT. In this study, TBT may also have inhibited shell formation, causing increased numbers of veliger larvae with incomplete shells in TBT treatment groups. During the waterborne exposure test, we observed that the percentage of normal veliger larvae decreased significantly in the 0.055 lg/l treatment group. It has been reported that TBT concentrations in seawater from northern Kyushu, Japan range from 0.005 to 0.091 lg/l (Inoue et al., 2002). Concentrations of TBT in seawater from
Otsuchi Bay ranged from 0.008 to 0.074 lg/l (Harino et al., 1998). Thus, TBT at levels present in seawater in Japanese coastal waters may interfere with development and survival of Manila clam veliger larvae. Ishii and Sekiguchi (2002) reported that the drastic decline in the Manila clam population in Ariake Sea may be caused by a lack of larval recruitment. From our results, we conclude that TBT in seawater may be responsible for decreases in the Japanese Manila clam population by preventing development and survival of planktonic veliger larvae. Although the TBT concentrations declined substantially in the Japanese coastal environment after the regulation of TBT usage was initiated in 1990 (Yamada and Kakuno, 2003), the Manila clam population has not recovered. Similarly, in Europe, Ruiz et al., (1995b,c) showed that the clam (Scrobicularia plana) has also not recovered, despite a decrease in TBT concentrations in seawater after TBT use was regulated. Several studies have demonstrated that invertebrate larvae stay near the surface of seawater (Manuel et al., 1996; Dobretsov and Miron, 2001), and it has been reported that bivalve larvae may come into contact with the surface micro layer (Hardy and Cleary, 1992). TBT concentrations in the surface micro layer have been measured to be 10–1000 times higher than in surface seawater (Watanabe et al., 1989), and may be a threat to the survival, growth and development of planktonic veliger larvae. Consequently, Manila clam populations may have continued to decline due to inhibition by TBT of larval development and survival, even after TBT usage was regulated. Inoue et al. (2006b) reported that TBT that accumulated in female Manila clams was transferred to the eggs and affected D-larvae development. However, in this study, no maternal effects on veliger larvae were observed in TBT treatment groups as compared with the control group. TBT concentrations in the veliger larvae were not analyzed, but we believe that the TBT that accumulated in female clams might not be transferred to veliger larvae. In short, we demonstrated that waterborne TBT, at environmentally relevant concentrations, affects the veliger larvae of the Manila clam. In contrast, no maternal adverse effects of TBT on veliger larvae were observed. We believe that TBT may have reduced the Manila clam population by preventing development and survival of veliger larvae, in addition to its impact on embryo development as reported by Inoue et al. (2006b).
Acknowledgements This study was partly supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 14360112). We thank the staff of the National Research Institute of Fisheries and Environment of Inland Sea and also Mr. Jumpei Arakawa of the Aichi Fisheries Research Institute for their help with our experiment.
S. Inoue et al. / Chemosphere 66 (2007) 1353–1357
References Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., Burt, G.R., 1986. The decline of the gastropod Nucella lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. J. Mar. Biol. Assoc. UK 66, 611–640. Champ, M.A., 2000. A review of organotin regulatory strategies, pending actions, related costs and benefits. Sci. Total. Environ. 258, 21–71. Coelho, M.R., Guentes, S., Bebianno, M.J., 2001. TBT effects on the larvae of Ruditapes decussates. J. Mar. Biol. Assoc. UK. 81, 259–265. Dobretsov, S.V., Miron, G., 2001. Larval and post-larval vertical distribution of the mussel Mytilus edulis in the White Sea. Mar. Ecol. Prog. Ser. 218, 179–187. Hardy, J.T., Cleary, J., 1992. Surface micro layer contamination and toxicity in the german bight. Mar. Ecol. Prog. Ser. 91, 203–210. Harino, H., Fukushima, M., Yamamoto, Y., Kawai, S., Miyazaki, N., 1998. Contamination of butyltin and phenyltin compounds in the marine environment of Otsuchi Bay, Japan. Environ. Pollut. 101, 209– 214. Huggett, R.J., Unger, M.A., Seligman, P.F., Valkirs, A.O., 1992. The marine biocide tributyltin. Environ. Sci. Technol. 26, 232–237. Inoue, S., Oshima, Y., Imada, N., Honjo, T., 2002. Tributyltin pollution in coastal areas around northern Kyushu, Japan. J. Environ. Toxicol. 5, 43–50, in Japanese. Inoue, S., Oshima, Y., Nagai, K., Yamamoto, T., Go, J., Kai, N., Honjo, T., 2004. Effect of maternal exposure to tributyltin on reproduction of the pearl oyster (Pinctada fucata martensii). Environ. Toxicol. Chem. 23, 1276–1281. Inoue, S., Abe, S., Oshima, Y., Kai, N., Honjo, T., 2006a. Tributyltin contamination of the bivalves in coastal areas around northern Kyushu, Japan. Environ. Toxicol. 21, 244–249. Inoue, S., Oshima, Y., Usuki, H., Hamaguchi, M., Hanamura, Y., Kai, N., Shimasaki, Y., Honjo, T., 2006b. Effects of tributyltin maternal and/or waterborne exposure on the embryonic development of the Manila clam, Ruditapes philippinarum. Chemosphere 63, 881–888. Ishii, R., Sekiguchi, H., 2002. Larval recruitment of the clam Ruditapes philippinarum in Ariake Sound, southern Jpn. Jpn. J. Benthol. 57, 151– 157 (in Japanese). Ishii, R., Sekiguchi, H., Nakahara, Y., Jinnai, Y., 2001. Larval recruitment of the Manila clam Ruditapes philippinarum in Ariake Sound, southern Japan. Fisheries Sci. 67, 579–591. Kakino, J., 1992. Recent situation on the Japanese littleneck fisheries. Fish. Engineer. 29, 31–39 (in Japanese). Lapota, D., Rosenberg, D.E., Platter-Rieger, M., Seligman, P.F., 1993. Growth and survival of Mytilus edulis larvae exposed to low levels of dibutyltin and tributylin. Mar. Biol. 115, 413–419. Manuel, J.L., Gallager, S.M., Pearce, C.M., Manning, D.A., O’Dor, R.K., 1996. Veligers from different populations of sea scallop Placopecten
1357
magellanicus have different vertical migration patterns. Mar. Ecol. Prog. Ser. 142, 147–163. Nakayama, K., Oshima, Y., Yamaguchi, T., Tsuruda, Y., Kang, I.J., Kobayashi, M., Imada, N., Honjo, T., 2004. Fertilization success and sexual behavior in male medaka, Oryzias latipes, exposed to tributyltin. Chemosphere 55, 1331–1337. Nirmala, K., Oshima, Y., Lee, R., Imada, N., Honjo, T., Kobayashi, K., 1999. Transgenerational toxicity of tributyltin and its combined effects with polychlorinated biphenyls on reproductive processes in Japanese medaka (Oryzias latipes). Environ. Toxicol. Chem. 18, 717–721. Ruiz, J.M., Bryan, G.W., Gibbs, P.E., 1995a. Acute and chronic toxicity of tributyltin (TBT) to the preveliger larvae of the bivalve Scrobicularia plana. Marine Biology 124, 119–126. Ruiz, J.M., Bryan, G.W., Gibbs, P.E., 1995b. Effects of tributyltin (TBT) exposure on the veliger larvae development of the bivalve Scrobicularia plana (da Costa). J. Exp. Mar. Biol. Ecol. 186, 53–63. Ruiz, J.M., Bryan, G.W., Wigham, G.D., Gibbs, P.E., 1995c. Effects of tributyltin (TBT) exposure on the reproduction and embryonic development of the bivalve Scrobicularia plana. Mar. Environ. Res. 40, 363–379. Sekiguchi, H., Ishii, R., 2003. Drastic decreasing of annual catch yields of the Manila clam Ruditapes philippinarum in Ariake Sound, Southern Japan. Oceanogr. Jpn. 12, 21–36 (in Japanese). Shimasaki, Y., Kitano, T., Oshima, Y., Inoue, S., Imada, N., Honjo, T., 2003. Tributyltin causes masculinization in fish. Environ. Toxicol. Chem. 22, 141–144. Toba, M., 2002. Present status of the fishery for the clam Ruditapes philippinarum in Chiba Prefecture, Tokyo Bay. Jpn. J. Benthol. 57, 145–150 (in Japanese). Toba, D.R., Thompson, D.S., Chew, K.K., Anderson, G.J., Miller, M.B., 1992. Guide to Manila Clam culture in Washington. Washington Sea grant Program. University of Washington, pp. 1–80. Triebskorn, R., Ko¨hler, H.-R., Flemming, J., Braunbeck, T., Negele, R.-D., Rahmann, H., 1994. Evaluation of bis(tri-n-butyltin)oxide (TBTO) neurotoxicity in rainbow trout (Oncorhynchus mykiss). I. Behaviour, weight increase, and tin content. Aquat. Toxicol. 30, 189–197. Tsutsumi, H., Ishizawa, K., Tomishige, M., Moriyama, M., Sakamoto, K., Montani, S., 2002. Population dynamics of a clam, Ruditapes philippinarum, on an artificially created sand cover on the tidal flats at the river mouth of Midorikawa River. Jpn. J. Benthol. 57, 177–187 (in Japanese). Watanabe, N., Sakai, S., Takatsuki, H., 1989. Environmental behavior analysis to evaluate the fate of organotin. Environmental science 2, 275–286 (in Japanese). Yamada, H., Kakuno, A., 2003. Present status on the development of alternative tributyltin-free antifouling paints and toxicity of new biocides to aquatic organisms – Review. Bull. Fish. Res. Agen. 6, 56– 72 (in Japanese).