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Comparison of organotin accumulation in the masu salmon Oncorhynchus masou accompanying migratory histories
Comparison of organotin accumulation in the masu salmon Oncorhynchus masou accompanying migratory histories Madoka Ohji a,*, Takaomi Arai a, Nobuyuki Miyazaki b a b
International Coastal Research Center, Ocean Research Institute, The University of Tokyo, 2-106-1 Akahama, Otsuchi, Iwate 028-1102, Japan Center for International Cooperation, Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan Received 8 August 2006; accepted 11 December 2006 Available online 7 February 2007
Abstract In order to examine the accumulation pattern of organotin compounds (OTs) accompanying the migration pattern in diadromous fish, tributyltin (TBT) and triphenyltin (TPT) compounds and their derivatives were determined in the liver, muscle, gill, and ovary tissues of both searun and freshwater-resident masu salmon, which are of the same species, Oncorhynchus masou. Their migratory histories were estimated using strontium (Sr) and calcium (Ca) analysis in the otolith. A significant difference in the mean Sr:Ca ratio from the core to the edge of the otolith was found between sea-run and freshwater-resident masu salmon. The TBT concentration in the liver was significantly higher than that in the other tissues in both sea-run and freshwater-resident fishes. In sea-run masu salmon, the TBT concentrations in all tissues except for the ovary were significantly higher than in those of freshwater-resident individuals. In the sea-run type, the percentage of TBT was higher than that of the freshwater-resident type. The TPT concentration in the liver of the sea-run type was also significantly higher than that in the other tissues, while that in the gill of the freshwater-resident type was significantly higher than that in the other tissues except for the ovary. The TPT concentrations found in the liver and muscle of the sea-run type were significantly higher than those in the freshwater-resident type, whereas the values of the gill in the sea-run type were significantly lower than those in the freshwater-resident fish examined. The percentage of TPT in the sea-run type was higher than that of the freshwater-resident type. These results suggest that the sea-run O. masou has a higher ecological risk of TBT and TPT exposure than the freshwater-residents during their life history. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: tributyltin; triphenyltin; masu salmon; migration; ecological risk
1. Introduction Tributyltin (TBT) and triphenyltin (TPT), which are the representative groups of organotin compounds (OTs), have been used worldwide as the active ingredient in many formulations of marine antifouling paints. These paints are applied to boat hulls and other submerged structures, such as wharves, buoys, and fish pens to prevent fouling, that is, the attachment and growth of marine organisms such as barnacles, mussels, and algae (Snoeij et al., 1987; Blunden and Evans, 1989;
* Corresponding author. E-mail address: [email protected] (M. Ohji). 0272-7714/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2006.12.004
Bosselmann, 1996). These compounds have been of great global concern due to their many deleterious effects on nontarget aquatic life (Fent and Meier, 1994; Ohji et al., 2002, 2003; Grzyb et al., 2003). The use of OTs as an antifouling agent has been regulated in various countries between 1980 and 1990. In October 2001, the International Maritime Organization (IMO) adopted the International Convention on the Control of Harmful Antifouling Systems (AFS Convention), which prohibited the use of OTs as active ingredients in antifouling systems for ships. In spite of efforts to reduce their use in antifouling paints due to these compounds’ extremely high toxicity, considerable levels of TBT and TPT are still present in the aquatic ecosystem (Harino et al., 2000, 2002). Some information is available regarding the effects of TBT and TPT on
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marine and freshwater fishes (Jarvinen et al., 1988; Fent and Meier, 1992, 1994; Fent et al., 1998; Grzyb et al., 2003). However, it is considered that TBT and TPT affect not only marine or freshwater fishes but also diadromous fishes such as the masu salmon Oncorhynchus masou, which have sea-run and freshwater-resident migratory histories. After their emergence from river redds, masu salmon Oncorhynchus masou generally spend 1 or 2 years in freshwater, prior to their seaward migration. After remaining in ocean waters for 1 year, they return to their natal river in spring, at 2.5 to 3.5 years of age (Kubo, 1980). However, some non-migrating O. masou remain in freshwater in a fluvial form. Thus, in spite of the intraspecies relationship between the two types, O. masou have different migratory patterns, i.e. the sea-run (anadromous) type and the freshwater-resident (non-anadromous) type. Therefore, it could be considered that differences of ambient environmental effect exist between the two migration types of the species. Since TBT and TPT have been used primarily in marine environments, it is assumed that the accumulations of these compounds differ between these two migratory types even within the same species. However, organotin accumulation accompanying the migratory pattern is still unknown in diadromous fish. Examination of the ontogenic changes in the levels of otolith strontium (Sr) and calcium (Ca) concentrations is useful in reconstructing the migratory histories of individuals. Wave-length dispersive electron microscope analysis of Sr and Ca concentrations in otoliths has recently been focused on as a method for distinguishing between freshwater and marine migratory phases in diadromous fishes such as masu salmon Oncorhynchus masou (Arai and Tsukamoto, 1998), Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss (Kalish, 1990), sockeye salmon Oncorhynchus nerka (Rieman et al., 1994), chum salmon Oncorhynchus keta (Arai and Miyazaki, 2002), and brown trout Salmo trutta (Arai et al., 2002). This technique should aid in revealing minute OT accumulation patterns accompanying an individual fish’s migratory history as well as in the reconstruction of its migratory history. The aim of the present study is to examine the differences of the accumulation pattern of butyltin compounds (BTs) including TBT and its derivatives, dibutyltin (DBT) and monobutyltin (MBT), and phenyltin compounds (PTs) including TPT and its derivatives, diphenyltin (DPT) and monophenyltin (MPT), between the sea-run and freshwater-resident types of masu salmon Oncorhynchus masou Brevoort. The results of the present study may provide some clues to understanding the ecological risks of OTs in such diadromous fishes. 2. Materials and methods 2.1. Specimens Oncorhynchus masou having the two migratory patterns were collected from marine and freshwater environments. Five large adults (sea-run) (fork length > 400 mm, body
weight > 1400 g), just before upstream migration, were collected by set net at Otsuchi Bay, Iwate Prefecture, Japan (39 220 N, 141 580 E), on 18 April 2003. Three small adult specimens (freshwater-resident) (fork length < 300 mm, body weight < 200 g) were collected by fishing at the Kumano River, Iwate Prefecture (39 110 N, 141 490 E), on 20 May 2003. Liver, muscle, gill, and ovary tissues were dissected from each specimen and all samples were stored in a freezer at 20 C until chemical analysis. 2.2. Chemical analysis of organotins in specimens The method used for determining the concentrations of OTs in the biological samples was based on that described by Ohji et al. (2006a,b). Homogenate soft tissues (5 g) of each fish were placed in a centrifuge tube, with 100 ml of mixed acetone solution including 1 mg ml1 each tributyltin monochloride (TBTCl)-d27, dibutyltin dichloride (DBTCl)-d18, monobutyltin trichloride (MBTCl)-d9, triphenyltin monochloride (TPTCl)-d15, diphenyltin dichloride (DPTCl)-d10, and monophenyltin trichloride (MPTCl)-d5 being added to the tube as a surrogate standard. The mixture was extracted with 25 ml of 1 M HClemethanol/ ethyl acetate (1/1) by shaking for 10 min. After centrifugation for 10 min, the residue was extracted and centrifuged again in the same way. The combined supernatants and 100 ml of saturated NaCl solution were transferred to a separatory funnel. The analytes were extracted twice using 30 ml of ethyl acetate/hexane (3/2) solution. Hexane (100 ml) was mixed with the combined organic layers and let stand for 20 min. After removal of the aqueous layer, the organic layer was dried with anhydrous Na2SO4 and was concentrated up to trace level by a rotary evaporator and further concentrated by means of a nitrogen purge. The analytes were diluted with 5 ml of acetic acidesodium acetate buffer (pH 5.0) and ethylated using 1 ml of 10% NaBEt4. The lipids were saponificated with 40 ml of 1 M KOHeethanol solution by shaking for 1 h. Distilled water (25 ml) and hexane (40 ml) were added to the solution, and ethylated OTs in the mixed sample solution were extracted to an organic layer by shaking for 10 min. The ethylated OT residue in an aqueous layer was extracted again by shaking for 10 min with 40 ml of hexane. The combined organic layers were dried with anhydrous Na2SO4. After being concentrated up to 1 ml by a rotary evaporator and nitrogen gas, the solution was cleaned using a florisil SepPak column (Waters Associates Inc., Milford, Massachusetts, USA) The analytes were eluted with 5% diethyl ether/hexane, and TeBT-d36 and TePT-d20 were then added as an internal standard. The final solution was then concentrated up to 0.2 ml. A Hewlett-Packard 6890 series gas chromatography equipped with a mass spectrometry (5973 N) was used for analysis of the OTs with selected ion monitoring. The separation was carried out in a capillary column coated with 5% phenyl methyl silicone (J&W Scientific Inc., Folsom, California, USA, 30 m length 0.25 mm i.d., 0.25 mm film thickness). The column temperature was held at 60 C for the first
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2 min, and then increased to 130 C at 20 C/min, to 210 C at 10 C/min, to 260 C at 5 C/min, and to 300 C at 10 C/min. Finally, column temperature was held at 300 C for 2 min. Interface temperature, ion source temperature, and ion energy were 280 C, 230 C, and 70 eV, respectively. Selected ion monitoring was operated under this program. Splitless injection (1 ml) of the sample was employed. The concentrations of OTs for the biological samples in this study are expressed as Sn4þ on a wet weight basis. In order to examine the quality of the data obtained by the analytical procedure, the soft tissues of the fish were spiked with 1 mg of BTs and PTs. The recoveries of BTs and PTs were in the range of 85.6e100.5% and 71.5e90.1%, respectively, and their relative standard deviations (RSD) were in the range of 2.2e4.6% and 3.8e6.7%. Furthermore, quality control tests for BTs in the biological samples were carried out using the certified reference material (IRMM CRM 477). Our measured BT values agreed with the certification values. The detection limit of each OT for a signalto-noise ratio of 3 was 0.4 ng g1 wet wt for the biological samples. 2.3. Otolith preparation and otolith X-ray microprobe analysis Sagittal otoliths were extracted from each fish, embedded in epoxy resin (Epofix; Struers, Ballerup, Denmark), and mounted on glass slide. The otoliths were then ground to expose the core, using a grinding machine equipped with a diamond cup-wheel (Discoplan-TS; Struers) and polished further with 6 mm and 1 mm diamond paste on an automated polishing wheel (Planopol-V; Struers). Finally, they were cleaned in an ultrasonic bath and rinsed with deionized water prior to examination. For electron microprobe analysis, all otoliths were PtePd coated by a high-vacuum evaporator. Life-history transect analysis of Sr and Ca concentrations in all specimens was measured along a line down the longest axis of each otolith from the core to the edge using a wave-length dispersive X-ray electron microprobe (JXA-8900R; JEOL, Tokyo, Japan) as described in Arai et al. (1997), Arai and Tsukamoto (1998) and Arai et al. (2003). Strontianite (SrCO3) and calcite (CaCO3) were used as standard, and the accelerating voltage and beam current were 15 kV and 1.2 108 A, respectively. The electron beam was focused on points at 10 mm intervals. 2.4. Statistics Differences between data were analyzed using the Manne Whitney U-test. Differences among data were examined by an analysis of variance (ANOVA), and afterwards Scheffe’s multiple range tests for the combination of two data. Significance of the correlation coefficient and regression slope were tested by Fisher’s Z-transformation and an analysis of covariance (ANCOVA) (Sokal and Rohlf, 2003).
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3. Results 3.1. Life history transects All fishes collected from Kumano River maintained a low level of Sr:Ca ratio, averaging 2.5 0.2 103, ranging from 2.3 to 2.7 103 from the core to the edge of otolith (Fig. 1). In contrast, all fishes collected from Otsuchi Bay showed low Sr:Ca ratio from the core to a point 1170e1540 mm, averaging 2.4 0.7 103, ranging from 1.8 to 3.5 103. Thereafter, the ratios increased sharply, averaging 4.5 0.4 103, ranging from 3.9 to 5.0 103. The mean Sr:Ca ratio from the core to the edge of the otolith in all fish from Otsuchi Bay was significantly higher than that from Kumano River (ManneWhitney U-test, p < 0.05). Thus, we could confirm their migratory patterns using otolith Sr:Ca ratio analysis; i.e. all fishes collected from Kumano River were regarded as the freshwater-resident type, and all fishes collected from Otsuchi Bay were regarded as the sea-run type. 3.2. Organotin distribution among tissues of the sea-run type In the sea-run type, TBT concentration in the liver was 12.4 1.3 ng g1 wet wt, and those values were significantly higher than that in muscle (6.8 0.4 ng g1 wet wt), gill (6.2 0.4 ng g1 wet wt), and ovary tissues (3.0 1.2 ng g1 wet wt)(ANOVA, p < 0.0001) (Fig. 2). These values in the ovary were significantly lower than those in the muscle and gill (ANOVA, p < 0.0001), whereas no significant difference was found in TBT concentration between muscle and P gill (ANOVA, p > 0.5). The total butyltin concentrations ( BTs ¼ TBT þ DBT þ MBT) in the liver were also significantly higher than those in the other tissues (Manne Whitney U-test, p < 0.0001e0.05). This value of the ovary was significantly lower than that of the muscle and gill (ANOVA, p
0.5). TBT was the predominant compound in the liverPof the sea-run type, accounting for 50.3 9.8% of the BTs (Fig. 4). In contrast, TBT’s degradation products, DBT and MBT, were the predominant compounds in the muscle, gill, and ovary. The sum of the percentage of DBT and MBT in the muscle, gill, and ovary contributed 65.9 3.3, 64.4 7.5, and 66.9 8.8%, respectively. The P distribution of TPT showed the same trend to TBT and BTs in the sea-run type (Fig. 3). TPT concentration in the liver was also significantly higher than that in the muscle, gill, and ovary (ANOVA, p < 0.0001). This value in the ovary was also significantly lower than that in the muscle and gill, whereas no significant difference was found in TPT concentration between the muscle and gill tissues P (ANOVA, p > 0.5). The total phenyltin concentrations ( PTs ¼ TPT þ DPT þ MPT) in the tissues of the liver and gill were significantly higher than those in the
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Distance from the core (µm) Fig. 1. Oncorhynchus masou. Typical changes in otolith Sr:Ca ratio along line transects from the core (0 mm) to the edge in the sagittal plane of sagittal otoliths of (A) sea-run and (B) freshwater-resident specimens. Specimen identification numbers are given at the upper left.
muscle and ovary (ANOVA, p < 0.0001e0.005), whereas no significant difference was P found between the liver and gill (ANOVA, p > 0.1). PTs in muscle were higher than those in the ovary (ANOVA, p < 0.0005). The percentages of TPT in all tissues except gill tissue were
extremely higher than that of TBT (Fig. 5). High percentages of TPT were detected in the liver, muscle, and ovary, accounting for 88.5 3.7, 100.0 0.0, and 97.7 5.0% of P the PTs, respectively, while this value in the gill was 50.1 10.4%.
M. Ohji et al. / Estuarine, Coastal and Shelf Science 72 (2007) 721e731 30
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Fig. 2. Oncorhynchus masou. Butyltin concentration in liver, muscle, gill, and ovary of (A) sea-run and (B) freshwater-resident specimens.
3.3. Organotin distribution among tissues of the freshwater-resident type The TBT concentrations were less than the detection limit in all tissues except for liver in freshwater-resident fish (Fig. 2). Significant differences were observed in TBT concentration between the liver and muscle and between the liver and gill (ANOVA, p < 0.05). In the ovary, relatively high concentrations of DBT and MBT were detected, accounting for 23.5 and 25.0 ng g1 wet wt, P respectively. No significant differences were observed in BTs among all tissues except for those of the ovary (ANOVA, p > 0.1e0.5). DBT and MBT were predominant in the liver, with the sum P of the percentage of DBT and MBT being 77.4 0.4% of BTs (Fig. 4). TPT concentration in the gill tissue of freshwater-resident fish was 13.6 1.3 ng g1 wet wt, with these values being
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significantly higher than those in the liver (1.6 1.4 ng g1 wet wt) (ANOVA, p < 0.005), and muscle tissues (3.5 3.1 ng g1 wet wt) (ANOVA, p < 0.01), although no significant differences were found between liver and muscle concentrations (ANOVA, p > 0.5) (Fig. 3). The TPT concenP tration in the ovary was less than the detection limit. PTs in the gill were significantly higher than those in the liver and muscle (ANOVA, p < 0.005), although there was no P difference in PTs between the liver and muscle tissues (ANOVA, p > 0.9). The percentage of TPT in the liver, muscle, gill, and ovary in the freshwater-resident type P was 27.8 25.3, 66.7 57.7, 49.2 9.1, and 0.0 0.0% PTs, respectively (Fig. 5). Statistical analysis of OT concentration in the ovary tissue of freshwater-resident masu salmon could not be conducted because only one ovary sample was analyzed.
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Fig. 3. Oncorhynchus masou. Phenyltin concentration in liver, muscle, gill, and ovary of (A) sea-run and (B) freshwater-resident specimens.
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Fig. 4. Oncorhynchus masou. Butyltin composition in (A) liver, (B) muscle, (C) gill, and (D) ovary of sea-run and freshwater-resident specimens. ND indicates no data due to the lack of samples.
3.4. Difference of organotin accumulation between sea-run and freshwater-resident types TBT concentration in the liver was significantly higher than that in the other tissues in both the sea-run and freshwaterresident types (ANOVA, p < 0.0001e0.05) (Fig. 2). The P BTs in the liver were also significantly higher than those in the other tissues in the sea-run type (ANOVA, p < 0.0001e 0.05), although no significant differences were found in P BTs in freshwater-resident fish among all tissues except for the ovary (ANOVA, p > 0.1e0.5). The TPT concentration in the gill in the freshwater-resident type was significantly higher than that in the other tissues except for the ovary (ANOVA, p < 0.005e0.01), while that in the liver of the sea-run type was significantly higher P than that in the other tissues (ANOVA, p < 0.0001) (Fig. 3). PTs in liver and gill tissues in the sea-run type were significantly higher than those in the muscle and ovary tissues (ANOVA, p < 0.0001e0.005), whereas those in the gill of freshwater-residents were significantly higher than those in the liver and muscle (ANOVA, p < 0.005). These results suggested that the distribution of PTs differed between sea-run and freshwater-resident fish, although both migration types shared the same trend regarding TBT distribution.
TBT concentrations in all tissues except for the ovary in sea-run masu salmon were significantly higher than those of freshwater-resident fish (ManneWhitney U-test, p < 0.05) P (Fig. 2). BTs in the muscle tissue of the sea-run type were significantly higher than those in freshwater-resident fish (ManneWhitney U-test, p < 0.05), although no significant difference was found in the other tissues except for the ovary (ManneWhitney U-test, p > 0.05e0.1). The percentage of TBT in the sea-run type was higher than that of the freshwater-resident type (Fig. 4). The TPT concentrations in the liver and muscle tissues of the sea-run type were significantly higher than those in the freshwater-resident type (ManneWhitney U-test, p < 0.05), whereas those values in the gill of the sea-run type were significantly lower than those of freshwater-resident fish (ManneWhitP ney U-test, p < 0.05) (Fig. 3). PTs in all tissues except the ovary in the sea-run type were significantly higher than those of the freshwater-resident type (ManneWhitney U-test, p < 0.05). The percentage of TPT in the sea-run type was higher than that of the freshwater-resident type (Fig. 5). These results suggest that sea-run masu salmon has a higher risk for TBT and TPT than freshwater-resident individuals.
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Fig. 5. Oncorhynchus masou. Phenyltin composition in (A) liver, (B) muscle, (C) gill, and (D) ovary of sea-run and freshwater-resident specimens. ND indicates no data due to the lack of samples.
3.5. Relationship between Sr:Ca ratio and OT concentrations Positive linear relationships were found between otolith Sr:Ca ratios and TBT concentrations in the liver, muscle, and gill tissues (ANCOVA, p < 0.05), although no significant difference was found in the ovary (ANCOVA, p > 0.1) (Fig. 6). No correlations were found between Sr:Ca ratios and TPT concentrations in the tissues of the liver, muscle, gill, and ovary (ANCOVA, p > 0.05e0.5). AsPwell, no relationship was found between Sr:Ca ratios and BTs, and P between Sr:Ca ratios and PTs (ANCOVA, p > 0.05e0.5). These results suggest that the accumulation of TBT increases with increasing Sr:Ca ratio, and that this phenomenon differed between TBT and TPT. 4. Discussion 4.1. Organotin accumulation accompanying migration The present study demonstrated that the TBT and TPT concentrations in sea-run masu salmon were significantly higher than those in freshwater-resident fish, in spite of their intraspecies relationship. The metabolic capacity of TBT and TPT is
considered to be the same between sea-run and freshwaterresident masu salmon as they belong to the same species. Therefore, the proportion of OTs in each fish might be affected by the present status of OTs in the fish’s water environment. This suggests that the difference in the migratory history between sea-run and freshwater-resident fish was reflected in the accumulation profile of TBT and TPT of each migration type. Since TBT and TPT have been used mainly in marine environments, it is considered that the sea-run individuals experience greater exposure to TBT and TPT during the period of migration in coastal and ocean waters. In contrast, since freshwater-resident individuals remain in a freshwater environment throughout their life history, they were exposed to TBT and TPT to a lesser extent. Furthermore, the relationship between otolith Sr:Ca ratio and TBT concentration clearly showed that TBT concentrations increased significantly with increasing Sr:Ca ratio. Although no correlations were observed in Sr:Ca ratio and TPT concentration, the TPT concentrations in sea-run individuals were significantly higher than those in freshwater-resident fish. Therefore, the risk for TBT and TPT in sea-run fish is higher than that in freshwaterresident fish. The present study found that the general composition of P TBT in sea-run fish was 26.4e63.5% of the BTs. The
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Fig. 6. Oncorhynchus masou. Relationship between otolith Sr:Ca ratio and TBT concentrations in (A) liver, (B) muscle, (C) gill, and (D) ovary of sea-run and freshwater-resident specimens.
highest percentages were found in liver tissues. The dominance of the parent compound in sea-run masu salmon contrasts with the relative percentages found in P other fish species. TBT accounted for about 25% of the BTs in the livers of Lateolabrax japonicus, Pennehia argentatus, and Seriola quinqueradiata (Harino et al., 2000), and MBT burdens were higher than those of TBT in the livers in various Australian fish (Nemadactylus douglasii, Aptchotrema rostrata, Achoerodus viridis, Mugil cephalus, Lutjanus vitta, Salmo salar, Platycephalus fuscus, Caranx sexfasciatus, and Dicentrarcus labrax) (Kannan et al., 1995). The DBT levels in pike liver tissues were 15- to 30-fold higher than those of the parent compound (Sta¨b et al., 1996). Since TBT is degraded in the livers of all fish, the ability of masu salmon to metabolize TBT might be relatively low by comparison, as can be seen from the higher percentage of TBT. In contrast, the proportion of TBT in freshwater-resident fish was lower than that in sea-run individuals. Since the metabolic capacity of TBT between these two migratory types is considered to be the same due to their intraspecies relationship, the accumulation profile of OTs in each fish might be reflected by OTs in
the water environment. These results suggest that the sea-run masu salmon has a higher ecological risk for TBT than the freshwater-resident type. 4.2. Organotin distribution among tissues In the present study, differences in OT burdens were found among the tissues of masu salmon. TBT concentration in the liver was the highest among the tissues of the sea-run and freshwater-resident types. A similar condition was found in a previous study (Sta¨b et al., 1996). OTs in the livers of pike, eel, and ruffe collected from a shallow freshwater lake in the Netherlands were in general 2- to 5-folds higher than those in the muscle tissue. It has also been reported that TBT concentrations in liver tissue were 1.5e4.5 times higher than those in the muscle tissue of the eel Anguilla anguilla (Harino et al., 2002). This suggests that OTs accumulate at high levels in the liver. Furthermore, di-OT/tri-OT ratios in the liver are generally higher than those in muscle tissue. The liver is clearly one of the organs in which the biodegradation of TBT and TPT takes place. Similar results have shown
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high concentrations of TBT degradation products in the liver and gall bladder (Martin et al., 1989), suggesting that TBT is dealkylated in the liver and extracted via the bile. Furthermore, among the tissues examined in the previous study, TBT and TPT in ovary were the lowest. These results indicate that TBT and TPT are not transferred from mother to egg in Oncorhynchus masou. It has been reported that TBT was transferred from mother to offspring in viviparous surfperch (Ohji et al., 2006c). Therefore, it might be considered that transfer of TBT as well as TPT differs between oviparous and viviparous fish. 4.3. Comparison of accumulation pattern among organotin compounds The present study found differences between BT and PT compositions. In BTs, TBT and its metabolites, DBT and MBT, were found in approximately equal percentages in sea-run fish, while TBT was below the detection limit in freshwater-resident individuals. In contrast, TPT in both fish types was, in general, predominant. In the muscle, TPT alone was found in all individuals in both sea-run and freshwater-resident types, except for one individual. A similar condition was found in a previous study (Sta¨b et al., 1996). High DBT and MBT levels among BTs were detected in the liver tissue of the pike, while high TPT concentration among PTs was also found. A different metabolic capacity to degrade TBT and TPT in the European eel Anguilla anguilla and the rainbow trout Oncorhynchus mykiss was found in a previous in vitro experiment (Fent and Bucheli, 1994). The liver microsomes in eel and rainbow trout were affected by TBT and TPT, and TPT inhibited the metabolic system more strongly than TBT. At TBT concentrations of 9.9 107 ng l1 for the eel and 2.1 107 ng l1 for rainbow trout, the cytochrome P-450 enzymes of each eel and rainbow trout were 50% inactivated, while both fish showed higher sensitivity to TPT, with 50% P-450 inhibition at a concentration of only 9.5 106 ng l1. In the present study, the high concentrations of the degradation products of TBT, and high concentrations of TPT were found in not only liver but also in the other tissues. These might result from the fact that TBT is more easily dealkylated in the liver and excreted via the bile than TPT because the liver’s metabolic capacity to degrade TBT is higher than its capacity to degrade TPT in O. masou. Experiments regarding the expose of the European minnow Phoxinus phoxinus in its early life stages to TBT and TPT have been reported (Fent, 1992; Fent and Meier, 1992, 1994). Essentially, the toxicities of both compounds were found to be similar in terms of survival data, morphology, and histopathology. Similar cellular alterations in the same target tissues were noted after exposure to both tributyltin and triphenyltin chlorides, but potencies towards some of these organs differed. The acute toxicity of TPT was slightly low compared to that of TBT (Fent, 1992; Fent and Meier, 1992). Furthermore, the effects of TPT on the skin, muscle, kidneys, and eye tissues (cornea, retina, pigment layer) were less marked, but the lens was seriously affected. Triphenyltin chloride also showed
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a higher affinity to neural tissues, but caused lesser irritation to surface epithelia. The reasons for these differences are not known, but they might be related to these compounds’ different molecular structures, octanolewater partition coefficients (log Kow), and modes of action. The log Kow values for tributyltin chloride and triphenyltin chloride at pH 7.8 have been shown to be 3.83 and 3.46, respectively (Tsuda et al., 1990). It should be emphasized, however, that TBT and TPT share similar toxicities, target organs, and pharmacodynamics (Fent et al., 1991; Fent, 1992; Fent and Meier, 1992), rather than marked differences. Organotins are used as biocides, heat and light stabilizers for polyvinyl chloride (PVC), and industrial catalysts. In organotin compounds, triorganotins, such as TBT, are used as biocides in such materials as marine antifouling paints, agricultural pesticides, and wood preservatives. Organotins used as catalysts and stabilizers are mainly mono- and di-organotins, such as MBT and DBT. The use of TBT in antifouling paints is still important for its application on large sea-going vessels, resulting in environmentally significant TBT water concentrations in the open sea (Rivaro et al., 1999). In the present study, higher levels of MBT and DBT were detected despite the presence of TBT not being detected in all samples except in the livers of freshwater-resident fish. Therefore, it is considered that (1) MBT and DBT may accumulate in freshwater-resident fish as derivatives of TBT used as the usage of agricultural pesticides and/or wood preservatives, or (2) MBT and DBT may find their way into fish due to the substances’ use as catalysts or stabilizers. Generally, the study of organotins focuses primarily on TBT because it is far more toxic to aquatic organisms than are DBT and MBT (Vighi and Calamari, 1985; Maguire, 1996). However, some information is available regarding the toxic effects of MBT and DBT on fishes, e.g. MBT’s cytotoxic effects on bluegill sunfish Leopomis macrochirus (Babich and Borenfreund, 1988); DBT’s histopathological effects on the guppy Poecilia reticulata (Wester and Canton, 1987), its cytotoxic effects on the bluegill sunfish (Babich and Borenfreund, 1988), and its effects on the liver and gas gland as a result of impaired glycogen breakdown in Japanese medaka Oryzias latipes (Wester et al., 1990). The present study suggests that these derivative compounds of TBT might have potential toxic effects on Oncorhynchus masou. Further study is needed to examine the biological effects of these derivatives on the masu salmon. In the present study, TPT was detected in not only sea-run fish but also freshwater-resident individuals. Differences of utility between TBT and TPT have been known. TBT is used mainly in antifouling paint, although it is also used in wood preservation. Therefore, it is considered to contaminate marine environments. In contrast, apart from its occasional use in antifouling preparations (less common than TBT), TPT is also used in agricultural applications such as fungicides for the protection of crops against fungal disease, as rodent and insect repellants, and as molluscicides. Both direct release into the water from antifouling paint and agricultural applications in rice fields, as well as runoff from agricultural fields, are prone to contaminate aquatic environments. In the present
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study, TPT was detected in freshwater-resident fish, and its concentrations in the gill were significantly higher than the concentrations found in sea-run individuals. These results indicate that not only sea-run type of Oncorhynchus masou, but also the freshwater-resident type might be affected by being exposed to TPT in their life history. 4.4. The risk of organotin compounds in Oncorhynchus masou The present study indicates that diadromous fish, such as Oncorhynchus masou, have a different risk of TBT and TPT accompanying intraspecies variation of the migratory pattern. It has been reported that TBT has adverse effects on many fish species even at ambient water levels; i.e. TBT has been found to cause thymus atrophy, an increase in granulocytes, accumulation of glycogen and fat in the liver, and changes in the cornea, retina, and skin of the guppy (Wester and Canton, 1987). Furthermore, TBT causes a number of histopathological effects in rainbow trout and in the European minnow in their early life stages (de Vries et al., 1991; Fent and Meier, 1992; Schwaiger et al., 1992). Survival reduction and morphological and histopathological alterations have been found in response to exposure to TPT in the European minnow in its larval stages (Fent and Meier, 1994). Exposure to TPT induces chronic effects on growth and survival in the larvae of the fathead minnow Pimephales promelas (Jarvinen et al., 1988). Thus, the higher ecological risk posed by TBT and TPT to sea-run type individuals than that to freshwater-residents may result in the former being more conspicuously affected by TBT and TPT. However, the effects of TBT and TPT might not be only on the sea-run type, but also on the freshwaterresident type. After sea-run masu salmon complete their oceanic migration, they return to their natal river, and participate in reproductive activity with freshwater-resident masu salmon. It has been reported that TBT exposure detrimentally affects the reproductive activity of spermatozoa, i.e. decreases the duration and intensity of motility, as well as reduces the ATP levels in the African catfish (Rurangwa et al., 2002), and decreases the viability of spermatozoa in the herring Clupea harengus (Grzyb et al., 2003). It has also been reported that embryonic exposure to TBT as well as to TPT results in delays in hatching, decreases in hatching success, and increases in the mortality in the European minnow (Fent, 1992; Fent and Meier, 1992, 1994). In the sea urchin, the high sensitivity of its embryos towards triorganotins is to be expressed because different targets (inhibition of DNA and protein synthesis, alteration of cellular Ca2þ homeostasis) are involved during embryonic development (Girard et al., 1997), while sperm toxicity presumably involves both the inhibition of the acrosomal reaction (Giudice, 1986) and a direct inhibition of the energy functions of cells (Argese et al., 1998). Furthermore, it has been reported that the reproductive toxicity of triorganotin induces population disturbance. Masculinization (imposex) induced in female gastropods by TBT and TPT exposure has been found to lead to reproductive failure and subsequent population decline (Bryan et al., 1986; Gibbs and Bryan, 1986,
1987; Bettin et al., 1996; Horiguchi et al., 1997; Matthiessen and Gibbs, 1998). Therefore, TBT and TPT exposure might also affect the reproductive systems of sea-run masu salmon, and the risks involved in the exposure of the sea-run masu salmon to TBT and TPT may in turn influence freshwater-resident individuals, resulting in a disturbance of the maintenance of the O. masou population. Acknowledgements The present study was partially supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Nos. 12NP0201, 13760138, 15780130, and 15380125). Partial support was also given in the form of a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists to M. Ohji. References Arai, T., Miyazaki, N., 2002. Analysis of otolith microchemistry of chum salmon, Oncorhynchus keta, collected in Otsuchi Bay, northeastern Japan. Otsuchi Marine Science 27, 13e16. Arai, T., Tsukamoto, K., 1998. Application of otolith Sr:Ca ratios to estimate the migratory history of masu salmon, Oncorhynchus masou. Ichthyological Research 45, 309e313. Arai, T., Otake, T., Tsukamoto, K., 1997. Drastic changes in otolith microstructure and microchemistry accompanying the onset of metamorphosis in the Japanese eel, Anguilla japonica. Marine Ecology Progress Series 161, 17e22. Arai, T., Kotake, A., Aoyama, T., Hayano, H., Miyazaki, N., 2002. Identifying sea-run brown trout, Salmo trutta, using Sr:Ca ratios of otolith. Ichthyological Research 49, 380e383. Arai, T., Kotake, A., Ohji, M., Miller, M.J., Tsukamoto, K., Miyazaki, N., 2003. Occurrence of sea eels of Anguilla japonica along the Sanriku Coast of Japan. Ichthyological Research 50, 78e81. Argese, E., Bettiol, C., Ghirardini, A.V., Fasolo, M., Giurin, G., Ghetti, P.F., 1998. Comparison of in vitro submitochondrial particle and microtoxÒ assays for determining the toxicity of organotin compounds. Environmental Toxicology and Chemistry 17, 1005e1012. Babich, H., Borenfreund, E., 1988. Structureeactivity relationships for diorganotins, chlorinated benzenes and chlorinated anilines established with bluegill sunfish BF-2 cells. Fundamental and Applied Toxicology 10, 295e301. Bettin, C., Oehlmann, J., Stroben, E., 1996. TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgola¨nder Meeresuntersuchungen 50, 299e317. Blunden, S.J., Evans, C.J., 1989. Organotin compounds. In: Hutzinger, O. (Ed.), The Handbook of Environmental Chemistry, Part E. Anthropogenic Compounds, vol. 3. Springer-Verlag, Berlin, pp. 1e44. Bosselmann, K., 1996. Environmental law and tributyltin in the environment. In: de Mora, S.J. (Ed.), Tributyltin: Case Study of an Environmental Contaminant. Cambridge University Press, Cambridge, pp. 237e263. 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. Journal of the Marine Biological Association of the United Kingdom 66, 611e640. Fent, K., Lovas, R., Hunn, J., 1991. Bioaccumulation, elimination and metabolism of phenyltin chloride by early life stages of minnows in Phoxinus phoxinus. Naturwissenschaften 78, 125e127. Fent, K., 1992. Embryotoxic effects of tributyltin on the minnow Phoxinus phoxinus. Environmental Pollution 76, 187e194. Fent, K., Meier, W., 1992. Tributyltin-induced effects on early life stages of minnows, Phoxinus phoxinus. Archives of Environmental Contamination and Toxicology 22, 428e438.
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