Environ. Sci. Technol. 1997, 31, 3103-3109
Butyltin Residues in Deep-Sea Organisms Collected from Suruga Bay, Japan SHIN TAKAHASHI,† S H I N S U K E T A N A B E , * ,† A N D TSUNEMI KUBODERA‡ Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan, and Department of Zoology, National Science Museum, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169, Japan
Aquatic pollution resulting from extensive usage of organotin compounds has been of great concern due to their deleterious effects in organisms. However, organotin contamination in deep-sea ecosystems has not yet been studied. The present study was attempted to determine butyltin compounds (BTs), including mono- (MBT), di- (DBT), and tributyltin (TBT) in deep-sea organisms collected from Suruga Bay, Japan, in order to elucidate the contamination status and accumulation characteristics. The organisms were collected between 135 and 980 m in the aphotic bathyal zone and compared with those collected from shallow waters. Total butyltin (∑BT: MBT + DBT + TBT) concentrations in the tissues of deep-sea fish, crustaceans, cephalopods, echinoderms, and gastropods were up to 980, 460, 460, 130, and 21 ng/g wet wt, respectively. These levels were lower than those in shallowwater organisms from the same bay but comparable to those reported in industrialized areas like Tokyo Bay, suggesting the expansion of BT pollution in deep-sea ecosystems. Deep-sea organisms from Suruga Bay contained much higher levels of BTs than previously reported organochlorine concentrations. BT accumulation appeared to be less lipid dependent. Among BTs, TBT was the predominant compound except in cephalopods, suggesting a fresh input of TBT into the deep-sea environment. To our knowledge, this is the first report on the detection of organotin residues in deep-sea organisms.
Introduction Butyltins (BTs), one of the representative groups of organotin compounds, have been extensively used as polyvinyl chloride (PVC) stabilizers, industrial catalysts, biocides, wood preservatives, and antifouling agents in paints applied for boats and aquaculture nets since 1960s. Aquatic pollution resulting from their usage has been of great concern due to their bioaccumulative potential and deleterious effects in organisms, particularly those by highly toxic trisubstituted organotins such as tributyltin (TBT) typically used as biocides. Earlier studies have demonstrated that TBT exerts chronic toxic effects on susceptible mollusks at water concentrations of a few nanograms per liter (1-3). The toxicities of TBT on embryonic and early life stage organisms also lie in the range * To whom correspondence should be addressed; fax: +81-89946-9904; e-mail address:
[email protected]. † Ehime University. ‡ National Science Museum, Tokyo.
S0013-936X(97)00032-1 CCC: $14.00
1997 American Chemical Society
of a few micrograms per liter or even lower concentrations (4, 5). Concern over the ecotoxicological impacts of TBT led to restriction in most developed countries in the early 1990s. The restriction on TBT usage as an antifouling agent for coastwise boats and aquaculture nets has also been implemented in Japan in 1990. However, it is still being used for oceanliners and far-sea fishery boats and ships. Although a reduction in TBT contamination was recorded after the ban (6), TBT concentrations in Japanese coastal waters still persist at levels considered to be toxic to susceptible organisms. Recently, widespread occurrence of imposex in Japanese gastropods has been shown as a consequence of organotin pollution (7). Furthermore, considerable concentrations of BTs were detected in higher aquatic organisms such as marine mammals and fish-eating birds from the coastal areas in Japan (8-13). Whales and squids collected from open seas also contained detectable concentrations of BTs (8, 14). These findings suggest the possible long-term contamination and toxic threat of BTs in the global marine ecosystem. Despite available monitoring data for coastal and shallow waters, BT contamination in the deep-sea environment has not yet been studied, except for a recent survey that found the presence of BTs in deep-basin sediments collected at a water depth of 377 m (15). It has been emphasized in several studies that deep-sea sediments play a role as a sink for persistent contaminants such as organochlorine compounds (OCs) (16-18). A similar feature is expected for BT contamination; however, the global fate and long-term contamination of BTs is yet unknown due to the lack of data in deep-sea environment. The present study attempted to determine residue levels of BTs including TBT and its degradation products, di- (DBT) and monobutyltin (MBT) in benthic deep-sea organisms collected from Suruga Bay, Pacific coast of Japan, in order to elucidate the status of contamination and to delineate accumulation characteristics. This study integrates our earlier study on residue levels of OCs in deep-sea organisms (19).
Materials and Methods Samples. Benthic deep-sea organisms (comprised of fish, crustaceans, cephalopods, echinoderms, and gastropods) were collected from Suruga Bay, Japan (Figure 1), from October to November 1993 and in October 1994 under a research project Study on Deep-Sea Fauna and Preservation of Deep-Sea Ecosystem organized by Department of Zoology, National Science Museum, Tokyo. Suruga Bay is located near the central part of the Pacific coast of Honshu and has a deep and narrow trough along its long axis. The bathyal zone (2001000 m) of the eastern continental slope of the bay has a steep but simple physiography and was selected for sampling (Figure 1). The hydrological circulation within the bay is strongly influenced by the inflow of the oceanic water, the Kuroshio current (20). Fluvial discharge from the Oi, Abe, Fuji, and Kano Rivers acts as a major role of freshwater supply into the bay. The steepness of the bay facilitates the physical and biological material transportation to the deep basin (20). BTs originating from antifouling agents and/or industrial sources could have been released into the bay since maritime and industrial activities are rather high along the coastal area of this bay. Deep-sea organisms were caught using a 1-m dredge or a research bottom trawl. Biometry of the samples are given in Table 1. Except for some squid species such as Japanese common squid, lolignid squid, and cuttlefish that migrate between shallow and deep water, most deep-sea organisms analyzed in this study could be classified as benthic or benthopelagic species feeding at the batyal region (20). The
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FIGURE 1. Map showing the sampling area (shaded portion) in Suruga Bay, Japan. collected samples were placed in butyltin-free polyethylene bags and frozen in a deep-freezer at -20 °C until chemical analysis. The whole body or soft tissues of individuals belonging to the same species were pooled and homogenized to prepare a composite sample. In the case of large specimens (e.g., dory, dogfish shark, lithodid crab, and octopus), representative tissues and organs were taken for chemical analysis. Eleven species of shallow-water fish originating from Suruga Bay were purchased from fisheries in November 1995 (Table 1) and employed for chemical analysis in order to compare the residue levels with deep-sea organisms. All shallow-water fish were identified as benthic species. Using the same methodology as deep-sea fish, shallow-water species were pooled, homogenized, and stored in a deep-freezer until analysis. Chemical Analysis. The analytical procedure for BTs has been described elsewhere (8, 21). Briefly, about 2.0 g (wet wt) of tissue samples was homogenized with 70 mL of 0.1% tropolone-acetone and 5 mL of 2 N HCl. The homogenate was centrifuged at 3000 rpm, and BTs in the supernatant were transferred to 0.1% tropolone-benzene. After elimination of the moisture in the organic layer with anhydrous Na2SO4, the extract was concentrated nearly to dryness using a rotary evaporator (40 °C) and made up to 5 mL with benzene. BTs in the extract were propylated by adding 5 mL of n-propyl magnesium bromide (ca. 2 mol/L in THF solution, Tokyo Kasei Kogyo Co. Ltd., Japan) as a Grignard reagent, and the mixture was shaken at 40 °C for 1 h. After decomposition of the excess Grignard reagent with 20 mL of 1 N H2SO4, the derivatized extract was transferred to 20 mL of 10% benzenehexane and concentrated nearly to dryness; the volume was made up to 5 mL with hexane. The extract was then passed through a 20-g Florisil-packed dry column (eluting with 150 mL of 20% acetonitrile-water) to remove lipids and then purified by eluting through a 6-g Florisil-packed wet column. The final hexane eluate from the cleanup column was concentrated to 5 mL and subjected to GC quantification.
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Sample extracts were analyzed for BTs using capillary gas chromatography with flame photometoric detection (GCFPD). Chromatographic separation was performed on a Hewlett-Packard 5890 Series II gas chromatograph with a 30 m × 0.25 mm (i.d.) DB-1 capillary column coated at 0.25 µm film thickness (J&W Scientific Co., Folsom CA, 100% dimethyl polysiloxane). The flame photometer was operated using a hydrogen-air-nitrogen flame and equipped with a 610-nm bandpass filter selective for tin-containing compounds. Monobutyltin trichloride, dibutyltin dichloride, and tributyltin chloride of known amounts (0.1 µg each) spiked into the liver of Antarctic minke whale containing undetectable levels of BT residues was concurrently run with samples through the whole analytical procedure for use as an external standard. Only freshly derivatized external standards prepared along with samples were used to estimate concentrations. Concentrations were quantified by comparing peak heights of each BT species in samples with those in the external standards. Procedural blanks were included with every batch of six samples to check for interfering compounds and to correct sample values, if necessary. Monobutyltin, probably originating from commercial solvents or reagents that came into contact with PVC containing this compound as a stabilizer, was found at trace levels in reagent blanks. The values obtained for MBT in samples were therefore corrected for blank concentration. Detection limits of BTs in samples were assigned twice the values of procedural blanks. Detection limits of MBT, DBT, and TBT in tissues were 15.0, 4.0, and 2.0 ng/g wet wt, respectively. The average recovery rates for monobutyltin trichloride, dibutyltin dichloride, and tributyltin chloride dissolved and spiked into the liver of Antarctic minke whale were 104 ( 19.6%, 117 ( 14.3%, and 108 ( 5.2% (n ) 4), respectively, through the whole analytical procedure. In addition, hexyl tributyltin was added as an internal standard in the samples analyzed, and its recovery through the analytical procedure was 92 ( 20% (n ) 96). The concentrations of BTs are reported as nanograms of corresponding ion per gram on a wet weight basis in this study.
TABLE 1. Sample Details of Deep-Sea and Shallow-Water Organisms from Suruga Bay, Japan species
sampling depth (m)
mean length (cm)
mean weight (g wet wt.)
2 2 2 (2)a 3 2 2 (15) (7)
220-540 220-540 400-740 400-740 310-680 220-540 200-540 200-250
26.4 59.5 29.4 (24.3) 66.3 53.5 40.3 (12.6) (22.8)
334.7 513.7 440.7 (244.4) 610.6 571.4 364.4 (22.9) (56.3)
3 5 (14) 13 (19) 8 (11) 5 (1) 10 (7)
740-800 400-740 380-980 350-600 350-600 350-600
8.9b 9.2 (10.9) 13.0 (13.0) 15.3 (16.4) 17.2 (14.0) 10.5 (10.2)
397.2 25.5 (25.8) 18.5 (12.3) 30.0 (33.5) 76.2 (35.1) 14.9 (10.7)
1 2 (2)
285-480 200-540
46.0 49.9 (45.2)
825.7 322.3 (245.2)
4 (5) 2 (9)
135-350 135-350
25.5 (22.5) 13.5 (9.7)
104.4 (72.4) 99.3 (16.5)
(12)
310-680
(7.6)
(19.4)
(2) (2)
310-680 310-680
(29.5) (14.5)
(172.5) (105.7)
(3)
180-450
(11.5)
n Deep-Sea Organisms
fish dory, Zenopsis nebulosa dogfish shark, Deania calcea scorpionfish, Helicolenus hilgendorfi bonefish, Pterothrissus gissu grenadier, Coelorinchus sp. cusk eel, Hoplobbrotula armata greeneye, Chlorophthalmus albatrossis argentine, Glossanodon semifasciatus crustaceans lithodid crab, Paralomis multispina isopoda, Bathynomus doederleini stout red shrimp, Aristeus virilis gaint red shrimp, Aristeomorpha foliacea Japanese lobster, Nephrops japonicus glyphocrangon shrimp, Glyphocrangon hastacauda cephalopods octopus, Octopus tenuicirrus Japanese common squid, Todarodes pacificus lolignid squid, Loligo bleekeri cuttlefish, Sepiidae sp. echinoderms echinothuirid sea urchin, Phormosona bursarium sun star, Solaster uchidai goniasterid sea star, Ceramaster japonicus gastropods Hirase’s volute, Musashia hirasei knifejaw, Oplegnathus fasciatus morweng, Goniistius quadricornis halfmoon, Microcanthus strigatus gurnard, Lepidotrigla sp.1 sweetlip, Plectorhynchus cinctus gurnard, Lepidotrigla sp.2 pinecornfish, Monocentris japonicus blackall, Plectorhynchus pictus scorpionfish, Sebastiscus marmoratus croaker, Nibea sp. cornetfish, Fistularia sp. a
Shallow-Water Fish 1 1 2 2 1 2 1 1 1 1 1
17.0 27.5 17.0 17.3 34.3 16.1 12.4 21.2 20.2 50.9 72.6
(28.9c)
154.1 238.8 132.4 62.6 393.7 54.5 73.6 720.8 146.2 309.8 92.2
Figures in parentheses indicate the data of whole body samples pooled. b Width of shell. c Weight of soft tissue.
Results and Discussion Contamination Status. BTs were detected in all deep-sea organisms in the bathyal region and shallow-water fish from Suruga Bay (Table 2). To our knowledge, this is the first report on the detection of organotin residues in deep-sea organisms. Total butyltin (∑BT: MBT + DBT + TBT) concentrations in the tissues of deep-sea fish, crustaceans, cephalopods, echinoderms, and gastropods were up to 980, 460, 460, 130, and 21 ng/g wet wt, respectively. These levels were comparable to those found in coastal shallow-water organisms from Tokyo Bay that were strongly influenced by industrial and human activities (6, 11, 22, 23). These results suggest the expansion of BT contamination in deep-sea environment, despite the restriction of TBT usage as an antifouling agent. Shallow-water fish collected from Suruga Bay also contained much higher concentrations (with up to 4300 ng/g wet wt) than those from Tokyo Bay. In order to elucidate the magnitude of contamination in Suruga Bay, the BT concentrations in deep-sea and shallowwater fish were compared with those in shallow-water fish from Asian and Oceanian countries (21), the United States (24-26), Canada (27), The Netherlands (28), Italy (29), and Japan (22, 23). BT concentrations in deep-sea fish from
Suruga Bay were comparable to those in shallow-water fish from developed nations where the usage of TBT was extensive (Figure 2). Furthermore, shallow-water fish from Suruga Bay had higher ∑BT concentrations among the various locations studied so far. As Suruga Bay seems to be one of the polluted site similar to other coastal waters in developed nations, the deep-sea ecosystem in this bay may be affected by BT contamination. Residue levels of TBT found in deep-sea organisms were close to reported values of estimated effective concentrations (20-100 ng/g wet wt) based on chronic toxicity end points in susceptible organisms such as oysters and gastropods (4, 7). Recent studies on in vitro cytotoxicity of organotin compounds emphasized that trisubstituted organotins such as TBT and TPT exert high cytotoxic effects in fish hepatoma cells even at nanogram per gram levels (30). Based on this information, it is plausible that deep-sea organisms are affected by BT contamination. Further studies are needed to understand the impacts of BT contamination in the bathyal and abyssal fauna. In contrast to BTs, OC contamination in Suruga Bay organisms was lower than those of fish from Tokyo Bay and deep-sea organisms from the Atlantic Ocean (19). In the case
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TABLE 2. Butyltin Concentrations (ng/g wet wt) in Deep-Sea and Shallow-Water Organisms from Suruga Bay species
tissue/ organb fat (%) moisture (%)
DBT
TBT
∑BTc
TBT/∑BT (%)d
7.5 (6.3-8.7)a 51 (29-72) 12 (11-12) 11 (7.4-14) 17 (16-18) 13 (10-16) 29 (26-32)
