Global pollution monitoring of butyltin compounds using skipjack tuna as a bioindicator

Environmental Pollution 127 (2004) 1–12 www.elsevier.com/locate/envpol

Global pollution monitoring of butyltin compounds using skipjack tuna as a bioindicator D. Uenoa, S. Inouea, S. Takahashia, K. Ikedab, H. Tanakab, A.N. Subramanianc, G. Fillmannd, P.K.S. Lame, J. Zhenge, M. Muchtarf, M. Prudenteg, K. Chungh, S. Tanabea,* a

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan b National Research Institute of Fisheries and Environment of Inland Sea, Maruishi 2-17-5, Hiroshima, Japan c Center of Advanced Studies in Marine Biology, Annamalai University, Parangipettai 608502, Tamil Nadu, India d Fundac¸a˜o Universidade Federal do Rio Grande, C.P. 474, Rio Grande, RS, 96201-900, Brazil e Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong f Research and Development Center for Oceanology—Indonesian Institute of Sciences, Jl. Pasir Putih 1, Ancol Timur, Jakarta-11048, Indonesia g Science Education Department, De La Salle University, 2401 Taft Avenue 1004 Manila, Philippines h Department of Environmental Toxicology and Public Health, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon, Kyonggi-do 400-746, South Korea Received 28 February 2003; accepted 10 July 2003

‘‘Capsule’’: Global pollution monitoring of butyltin in offshore water and open sea were conducted using skipjack tuna as a bioindicator. Abstract Butyltin compounds (BTs) including mono- (MBT), di- (DBT), tri-butyltin (TBT) and total tin (Sn), were determined in the liver of skipjack tuna (Katsuwonus pelamis) collected from Asian offshore waters (off-Japan, the Japan Sea, off-Taiwan, the East China Sea, the South China Sea, off-Philippines, off-Indonesia, the Bay of Bengal), off-Seychelles, off-Brazil and open seas (the North Pacific). BTs were detected in all the skipjack tuna collected, suggesting widespread contamination of BTs even in offshore waters and open seas on a global scale. Considering specific accumulation, Sex-, body length- differences and migration of skipjack tuna did not seem to affect BT concentrations, indicating rapid reflection of the pollution levels in seawater where and when they were collected. Skipjack tuna is a suitable bioindicator for monitoring the global distribution of BTs in offshore waters and open seas. High concentrations of BTs were observed in skipjack tuna from offshore waters around Japan, a highly developed and industrialized region (up to 400 ng/g wet weight). Moreover skipjack tuna collected from offshore waters around Asian developing countries also revealed the levels comparable to those in Japan (up to 270 ng/g wet weight) which may be due to the recent improvement in economic status in Asian developing countries. High percentages (almost 90%) of BTs in total tin (Sn: sum of inorganic tin+organic tin) were found in the liver of skipjack tuna from offshore waters around Asian developing countries. This finding suggests that the anthropogenic BTs represent the major source of Sn accumulation in skipjack tuna from these regions. # 2003 Elsevier Ltd. All rights reserved. Keywords: Organotin; Butyltin; Fish; Offshore water and open sea; Global pollution monitoring

1. Introduction Butyltin compounds (BTs), which are representative organometallic compounds, have been extensively used for industrial and agricultural purposes, such as poly* Corresponding author. Tel.: +81-89-927-8171; fax: +81-89-9278171. E-mail address: [email protected] (S. Tanabe). 0269-7491/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0269-7491(03)00261-6

vinyl chloride (PVC) stabilizers, industrial catalysts, wood preservatives, and as biocides, since the 1960s. Particularly, toxic TBT (tributyltin) has been used as an effective antifouling agent in paints applied for pleasure boats, large ships and vessels, harbor structures, and aquaculture nets. Aquatic pollution resulting from the usage of TBT has been of great concern in many countries because of its effects on nontarget marine organisms.

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Based on a meeting over the ecotoxicological impacts of TBT, concluded by the International Maritime Organization (IMO) in November 1999, a resolution was prepared and approved by the Marine Environmental Protection Committee proposing a global prohibition on the application of organotin compounds (OTs) as biocides in antifouling systems on ships by January 1, 2003, and a complete prohibition of the presence of OTs by January 1, 2008 (Champ, 2000). It is essential to conduct a comprehensive and continuous monitoring survey of BTs in the ocean environment to evaluate the effectiveness of recently imposed regulatory. It is clear that contamination by BTs has spread all over the world as evidenced by their detection in a wide range of ocean environmental media and biota (e.g. Fent, 1996; Takahashi et al., 1999; Tanabe, 1999). A large number of monitoring surveys have been made using marine organisms such as mussel to assess the BTs pollution in ‘‘coastal ecosystems’’ (e.g. Sudaryanto et al., 2002). On the other hand, despite the fact that ‘‘open seas’’ play a major role as a final sink for persistent toxic contaminants (Iwata et al., 1993), only a few studies reported the contamination status of BTs in ‘‘offshore waters and open sea ecosystems’’ in global terms using sentinel organisms, such as squid (Yamada et al., 1997) and marine mammals (Tanabe et al., 1998). In addition, no data are available in offshore waters around Asian developing countries such as the Indian Ocean. In developing countries along the Asian coast, although no restriction has so far been imposed on the usage of TBT, no monitoring studies of BTs were conducted in offshore waters. Thus, monitoring surveys are required on BTs contamination around Asian developing countries to understand the fate and future trends of these contaminants. In order to elucidate the contamination status of BTs in offshore waters and open sea ecosystem, it is necessary to find out a suitable bioindicator. It has been pointed out that variations in BTs accumulation profile occur even among several species of lower trophic organisms due to the differences in feeding habitat and/ or metabolic capacity (Takahashi et al., 1997, 2001). Therefore, it is required to find a species which can be collected from offshore waters all over the world. Considering these facts, the present study attempted to use skipjack tuna (Katsuwonus pelamis) as a bioindicator for monitoring BT pollution in offshore waters and open seas. Skipjack tuna are principally distributed and migrate only in the offshore waters to open seas in tropical and temperate regions almost all over the world such as the Pacific, Atlantic and Indian Oceans (Collette and Nauen, 1983). It has been reported that skipjack tuna migrates globally from the equatorial spawning areas to the northern and southern temperate regions (Collette

and Nauen, 1983). This species is an important commercial fish, and hence its ecology and biology is well studied (e.g. Collette and Nauen, 1983; Nihira, 1996). Such a wide distribution and the availability of information make it a better species for monitoring global distribution of BTs pollution in the offshore waters and open seas. The objectives of this study are (1) to elucidate the suitability of skipjack tuna as a bioindicator for monitoring BTs by investigating specific accumulation, such as organ and tissue distribution, gender-, growth stagedifference and migration in concentrations, (2) to make clear the global distribution of BTs residue levels in offshore waters and open seas using this species, and (3) to examine the relationship between total tin (Sn: sum of inorganic tin+organic tin) and organotins in skipjack tuna to understand the anthropogenic contribution and natural sources of tin compounds.

2. Materials and methods 2.1. Sample collection Skipjack tuna (Katsuwonus pelamis) were collected from offshore waters of various Asian countries (offJapan, the Japan Sea, off-Taiwan, the East China Sea, the South China Sea, off-Philippines, off-Indonesia, the Bay of Bengal), off-Seychelles, off-Brazil and open sea (the North Pacific) during 1996–2001 (Fig. 1). Some of specimens were obtained from fish markets and fisherman villages after confirming the fishing areas. Skipjack tuna from N-Pacific-1 and-3 were caught during a fishing cruise in research vessels. Detailed information of skipjack tuna used in this study is shown in Table 1. Liver and other tissue samples were taken from these individuals and kept in clean plastic bags and stored at 20  C until analysis. 2.2. Chemical analysis Butyltin compounds (BTs) including mono- (MBT), di- (DBT) and tributyltin (TBT) were analyzed by the method described by Iwata et al. (1995) after slight modification. About 1–2 g of liver sample was homogenized with 70 ml of 0.1% tropolone-acetone and 5 ml of 2 N HCl. The homogenate was centrifuged, and BTs in the supernatant were transferred to 0.1% of tropolone–benzene. After the removal of the moisture in the organic layer with anhydrous Na2SO4, the extract was concentrated to near 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-propylmagnesium bromide ( 2 mol/l in THF solution), and the mixture was shaken at 40  C for 1 h. After decomposition of excess Grignard reagent with 1 N H2SO4, the

D. Ueno et al. / Environmental Pollution 127 (2004) 1–12

derivatived extract was transferred to 20 ml of 10% benzene–hexane and concentrated to near dryness and the volume was made up to 5 ml with hexane. The extract was then passed through 8 g of Florisil-packed wet column for clean-up. For quantification of butyltin compounds, a gas chromatograph equipped with a flame photometric detector (Hewlett-Packard 5890 series II) and a tin filter at 610 nm was used. Chromatographic separation was performed on a DB-1 capillary column (0.25 mm ID and 30 m length) coated with 0.25 mm film thickness. The column oven temperature was programmed from 80  C (1 min hold) to 170  C (1 min hold) at a rate of 15  C/min and then at a rate of 5  C/ min to 210  C (1 min hold) followed by a second raise at 15  C/min to 260  C with a 7 min final hold time. Injector and detector temperatures were held at 200 and 270  C, respectively. Helium was the carrier gas while hydrogen, air and nitrogen were passed at 160, 120 and 10 ml/min to the flame photometric detector. Detection limits of BTs as MBT, DBT and TBT, in liver tissue were 1.8, 2.4 and 2.0 ng/g on a wet-weight basis, respectively. Average recovery rates for monobutyltin trichloride, dibutyltin dichloride and tributyltin monochloride spiked into the liver of an Antarctic minke whale were 85  22, 104 7.6 and 94  11 (n=4), respectively, throughout the analytical procedure.

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Hexyltin was added as an internal standard to all the samples analyzed, and its recovery through all analytical batches was 104 4.3%. Accuracy of the analytical method was checked using a certified biological reference material (National Institute for Environmental Studies, Ibaraki, Japan [NIES] No. 11), and our result (1.3  0.02 mg TBTCl/g, n=3) showed a good agreement with the certified value (1.3  0.1 mg TBTCl/g). Concentrations in samples were not corrected for the recoveries of internal standards. Concentrations of BTs are reported as nanograms of cation per gram on a wet weight basis, unless specified otherwise. In addition, concentrations were also calculated on a dry-weight basis based on the moisture contents of the samples (the percentages of moisture contents were estimated from the weight of oven dried samples). The analytical procedure for total tin (Sn) was based on the method described elsewhere (Le et al., 1999) with slight modification. Briefly, tissue samples were dried at 80  C for 12 h and then homogenized into powder. About 0.1 g of sample was weighed into 8 ml polytetrafluoroethylene (PTFE) tubes. Digestion was carried out with purified HNO3 ( 63%) in a microwave oven at 200 W. After digestion, the sample was transferred into a measuring flask and diluted with Milli-Q1 water (Millipore, Bedford, MA, USA). Tin

Fig. 1. Map showing sampling locations of skipjack tuna.

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Probability values less than 0.05 were considered as significant using the Mann–Whitney U test and Spearman rank correlation.

concentrations in samples were determined using inductively coupled plasma mass spectrometry (ICPMS) (Hewlett-Packard, HP 4500). Detection limit was 10 ng Sn/g on a dry-weight basis. The recovery of Sn in this method was 107  1.2% (n=3). Accuracy of the analytical method was checked by analyzing a certified biological reference material (NIES No. 11), and our result (2.5  0.1 mg Sn/g, n=4) agreed well with the certified value (2.4  0.1 mg Sn/g). TBT, DBT and MBT concentration (ng/g wet wt) were converted to Sn concentration (ng Sn/g dry wt) for data comparison using respective dividing factors to subtract the molecular weight of butyl group(s).

3. Results and discussion 3.1. Contamination status Concentrations of BTs detected in skipjack tuna collected from offshore waters of Asia, off-Seychelles, offBrazil and open seas are shown in Tables 1 and 2. BTs were detected in the organs and tissues of skipjack tuna

Table 1 Mean and range of butyltins and total tin concentrations in the liver of skipjack tunas collected from Asian offshore waters, off-Seychelles, off-Brazil and open seasa Location

Month/ n Year

BL (cm)

BW (kg)

Moisture MBT (%)

DBT

TBT

BTs

(ng/g wet wt) N-Pacific-1

08/1998 4

44 (43–45) N-Pacific-2 05/1997 10 43 (40–44) 07/1999 5 63 (61–64) N-Pacific-3 03/1998 5 79 (75–80) Off-Japan-1 10/1997 5 49 (49–50) Off-Japan-2 10/1997 5 52 (49–55) Off-Japan-3 10/1997 5 60 (60–61) 10/1998 10 53 (51–59) Japan Sea 10/1997 5 63 (62–65) E-China Sea-1 10/1997 5 58 (55–60) E-China Sea-2 10/1997 5 61 (60–63) Off-Taiwan-1 10/1998 5 61 (59–63) Off-Taiwan-2 06/1999 5* 42 (40–45) S-China Sea 06/2001 1 31

1.8 (1.7–2.0) 1.6 (1.3–1.7) 5.7 (5.7–5.8) 10.5 (9.0–12.0) 2.6 (2.6–2.7) 3.3 (3.0–3.7) 4.5 (4.5–5.0) 3.0 (2.6–4.0) 5.6 (5.5–5.8) 3.9 (3.4–4.5) 5.2 (4.9–5.4) 5.0 (4.9–5.0) 1.3 (1.2–1.3) 0.5

Off-Philippines 12/1997 5

1.5 (1.3–1.6) 2.0 (2.0–2.0) 1.9 (1.7–2.0) 3.6 (3.4–3.8) 3.7 (3.1–4.4)

Bay of Bengal

10/1998 2

Off-Indonesia

01/1999 5

Off-Seychelles

01/1999 3

Off-Brazil**

08/2001 5

a

42 (41–45) 47 (46–47) 47 (45–48) 55 (54–55) 55 (53–57)

60 (40–76) 40 (39–41) 67 (63–71) 63 (61–65) 44 (39–50) 40 (40–41) 39 (34–46) n.a.

TBT/ BTs (%)

BTs

Sn

(ng Sn/g dry wt)

BTs/ Sn (%)

2.7 ( < 2.4–4.4) 9.1 (5.4–16) 20 (9.9–33) 7.4 ( < 2.4–18) 6.7 (3.8–12) 47 (14–76) 82 (25–120) 47 (14–120) 89 (48–120) 110 (75–170) 74 (26–160) 15 (12–19) 60

13 (6.9–23) 4.9 (22–63) 33 (17–43) 8.9 (3.8–13) 45 (29–71) 100 (89–120) 85 (27–120) 86 (45–120) 100 (74–130) 200 (170–220) 100 (56–190) 50 (36–59) 80

24 (6.9–26) 60 (35–73) 56 (30–77) 18 (3.8–30) 54 (33–86) 160 (130–200) 170 (73–250) 140 (61–240) 200 (150–260) 330 (280–400) 180 (110–350) 67 (52–75) 150

72 (22–100) 81 (65–91) 60 (52–66) 59 (39–100) 84 (80–88) 67 (59–77) 50 (46–66) 46 (46–80) 51 (41–62) 60 (53–69) 59 (45–77) 74 (69–80) 55

20 (7.8–30) 49 (41–54) 77 (55–110) 23 (4.0–39) 39 (37–44) 130 (98–150) 150 (53–210) n.a.

42 (32–54) 100 (86–130) 210 (110–290) 36 (10–61) 85 (52–130) 310 (280–370) 320 (160–440) n.a.

44 (25–58) 48 (41–60) 38 (32–45) 59 (11–100) 53 (34–79) 42 (34–54) 43 (33–58) n.a.

39 (37–41) 39 (37–43) 40 (39–41) 66 (65–67) 68

0.5 ( < 1.8–2.3) 2.1 ( < 1.8–5.1) 3.2 ( < 1.8–6.9) 1.6 ( < 1.8–7.9) 2.4 ( < 1.8–3.5) 6.2 ( < 1.8–15) 6.1 ( < 1.8–9.7) 4.9 (2.0–8.0) 8.5 (5.9–11) 17 (6.7–38) 5.7 ( < 1.8–13) 2.2 ( < 1.8–3.3) 5.4

140 (110–150) 260 (220–330) 150 (81–280) 87 (75–98) n.a.

340 (270–390) 560 (520–600) 310 (190–480) 200 (120–250) n.a.

40 (38–40) 46 (39–55) 46 (38–58) 47 (28–62) n.a.

n.a.

< 1.8

24

150

170

88

n.a.

n.a.

n.a.

64 (62–67) 66 (64–68) 62 (60–64) n.a.

1.9 ( < 1.8–5.7) 14 ( < 1.8–29) < 1.8

180 (78–270) 120 (60–180) 76 (13–150) 24 (18–33) 78 (24–140)

80 (75–86) 67 (43–90) 67 (54–88) 55 (53–58) 64 (45–100)

230 (140–320) 89 (n.a.–89) 110 (79–210) n.a.

88 (68–100) 88 (n.a.–88) 77 (35–110) n.a.

< 1.8

150 (62–200) 66 (54–77) 48 (7.9–88) 13 (11–17) 46 (21–83)

220 (95–310) 78 (n.a.–78) 89 (15–180) n.a.

n.a.

35 (16–66) 40 (5.7–74) 27 (4.7–61) 11 (7.8–16) 40 ( < 2.4–58)

n.a.

n.a.

n.a.

< 1.8

Figures in parentheses indicate the range. n: Number of samples. BL : Fork length. BW : Body weight. BTs: MBT+DBT+TBT. N-Pacific : the North Pacific. E-China Sea: the East China Sea. S-China Sea : the South China Sea. * : Pooled samples. **: The muscle (back) were employed for chemical analysis, and recalculated to liver concentration using conversion factors of 2.5 for TBT and 18 for DBT (see the text and Table 3). n.a. : No data available. Detectiom limit: MBT < 1.8, DBT < 2.4, MBT < 2.0.

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from all the sampling locations investigated. Considering the results of previous studies in mussel (Sudaryanto et al., 2002), coastal fish (Kannan et al., 1995a), squid (Yamada et al., 1997), marine mammal (Tanabe et al., 1998) and also this study, it is evident that BTs are widespread along coastal to offshore waters on global scale. Concentrations of BTs (TBT+DBT+MBT) in the tissues of skipjack tuna range 3.8–400 ng/g wet weight (Tables 1 and 2). Among BTs, TBT was detected at relatively higher concentrations in all locations, whereas the concentrations of DBT and MBT were lower. This may suggest fresh inputs of TBT into the aquatic environment and the presence of recent sources in the Asian offshore waters, off-Seychelles, off-Brazil and open seas. In addition, skipjack tuna, similar to other mollusks and fishes (Lee, 1996), may have a limited ability to metabolize TBT to DBT and MBT. 3.2. Specific accumulation of BTs 3.2.1. Organ and tissue distribution The organ and tissue distribution of BTs was examined in skipjack tuna collected off Japan-3 in 1997 (Table 2 and Fig. 2). BT s were detected in all the tissues analyzed. BTs residue levels (on wet weight basis)

were in the order of liver 5kidney=pyloric coecum > gonad=spleen=gill=muscle (red) > muscle (back) =muscle (stomach). No gender difference in concentrations was observed in the order of body distribution. Higher concentrations were observed in liver and kidney. This result agrees with the previous studies (Yamada et al., 1994; Harino et al., 2000). Interestingly, concentrations of BTs in the pyloric coecum were comparable to those in the liver (Table 2 and Fig. 2). Pyloric coecum is an organ involved in the digestion, absorption and storage. Therefore, levels in pyloric coecum reflect BTs concentration in stomach content. Almost half of the levels of liver concentrations were detected in gonad, spleen, gill and red muscle (Table 2 and Fig. 2). Among muscles, concentrations in red muscles were higher than those in back and stomach muscles, and similar to those in the spleen. It may be due to the fact that red muscle contains a higher amount of blood pigment than the other muscles, the same as the spleen. Even though, BTs are lipophilic, concentrations in the abdominal muscle (high lipid) were similar to those in the dosal muscle (less lipid). This is different to that of persistent organochlorine residues such as PCB and DDT whose levels always depend on lipid contents in the tissues. No correlation between BTs concentrations

Table 2 Tissue distribution of butyltins concentrations in skipjack tuna collected off-Japan-3, 1997 Tissue

Weight (g)

MBT

DBT

TBT

BTs

TBT/BTs (%)

(ng/g wet wt) Male (BL 60cm, BW 5.0kg) Liver Kidney Pyrloric coecum Gonad (testis) Spleen Gill Muscle (red) Muscle (stomach) Muscle (back) Whole body* Stomach content**

45 51 70 5 6 190 160 580 620 1727 350

9.7 5.2 5.6 13 5.9 4.7 <1.8 <1.8 <1.8 1.2 8.3

120 23 46 27 24 9.0 17 6.2 5.8 13 18

120 130 200 100 99 88 75 41 40 60 160

250 150 250 140 130 100 93 47 45 74 190

48 82 80 72 77 87 81 87 87 83 86

Female (BL 61cm, BW 4.5kg) Liver Kidney Pyrloriccoecum Gonad (egg) Spleen Gill Muscle (red) Muscle (stomach) Muscle (back) Whole body* Stomach content**

29 36 59 23 9 240 140 500 570 1606 260

<1.8 3.2 <1.8 3.3 2.4 <1.8 2.7 3.0 <1.8 1.3 3.8

25 14 20 7.7 9.2 <2.4 7.0 <2.4 2.7 3.5 7.4

48 72 54 20 27 35 24 13 15 21 130

73 89 74 31 39 35 34 16 18 26 140

66 80 73 64 70 100 72 81 85 81 92

BL : Fork length. BW : Bodyweight. BTs: MBT+DBT+TBT. *: Estimated concentrations using BTs concentrationa and weight in each tissue. **: Small fishes

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and lipid contents were reported in fish (Kannan et al., 1995b; Harino et al., 2000) and marine mammal (Iwata et al., 1995). BTs concentrations in the whole body were estimated using BT concentration and weight of each tissue in skipjack tuna (Table 2). A rough estimate of food chain magnification of BTs was obtained by calculating biomagnification factor (BMF). BMF was calculated using BT residues in stomach contents (small fishes) and estimated concentration in the whole body. Average BMF values of TBT, DBT and MBT were 0.24, 0.58 and 0.27, respectively (Table 3), indicating that no considerable biomagnification occurred for these compounds through the food chain. These values were comparable or lower than those in fish from the laboratory experiment (Yamada et al., 1994) and wild fish (Takahashi et al., 1999). It is quite different from persistent organochlorines such as PCB and DDT (e.g. Tanabe et al., 1984).

Moreover, the concentration ratios between whole body and different tissues were calculated. Concentration ratios for TBT, DBT and MBT were 1.1–2.9, 6.1–21 and 5.4–8.0, respectively (Table 3). Using these ratios, it is assumed that concentrations in some tissues of skipjack tuna can be used to estimate the concentrations in other tissues. For example, BT concentrations in the muscle of skipjack tuna from off-Brazil were used to estimate the levels in the liver by multiplying a factor of 2.5 for TBT and 18 for DBT which is the ratio of liver to muscle concentrations (back and stomach) (Tables 1 and 3). These results indicate that samples of skipjack tuna in which tissues are limited such as muscles for food or those kept in a museum can be used for data comparison by converting the BT concentrations to those in tissues needed. Among BT compositions, TBT residues were predominant (more than 70%) over its metabolites (DBT

Fig. 2. Relative concentrations of butyltin compounds in several tissues of skipjack tuna collected off-Japan-3, 1997. Vertical bars represent concentrations in individual tissues relative to the liver.

Table 3 Concentration ratios (conversion factor*) of butyltins between whole body and several tissues of skipjack tuna collected off-Japan-3, 1997a Tissue

MBT

DBT

TBT

BTs

6.5 (6.1–7.0) 18 (16–21) 11 (10–12) 0.58 (0.48–0.68)

1.3 (1.1–1.6) 2.5 (1.8–2.9) 1.6 (1.3–2.0) 0.27 (0.16–0.38)

2.4 (2.0–2.7) 4.8 (3.9–5.5) 3.0 (2.7–3.4) 0.29 (0.19–0.39)

(Concentration ratio) Liver/Muscle (red)

5.4

Liver/Muscle (back and stomach)

5.4

Liver/Whole body**

8.0

Whole body**/ Stomach content (BMF***)

0.24 (0.15–0.34)

a Figures in parentheses indicate the range. BTs: MBT+DBT+TBT. *:Conversion factor to estimate BTs concentrations in other tissues (see text and Table 1, off-Brazil). ** : Estimated concentrations in whole body (see Table 2) were employed for calculation. *: Biomagnification factor. n.a.: no data available

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D. Ueno et al. / Environmental Pollution 127 (2004) 1–12 Table 4 Sex differences of butyltins concentrations in the liver of skipjack tuna collected off-Japan-3, 1998a Sex

n

BT (kg)

BL (cm)

MBT

DBT

TBT

BTs

(ng/g wet wt) Male Female a

5 5

3.1 (2.6–4.0) 2.9 (2.7–3.1)

54 (51–59) 52 (52–53)

4.7 (2.0–6.0) 5.1 (2.3–8.0)

41 (27–69) 53 (14–110)

85 (52–120) 86 (45–110)

130 (88–1 70) 140 (61–240)

Figures in parentheses indicate the range. n: Number of samples. BL : Fork length. BW:Bodyweight. BTs: MBT+DBT+TBT

+MBT), except in the liver (Table 2 and Fig. 2). These proportions were similar to those in stomach contents (small fish). This indicates that skipjack tuna has a limited ability to metabolize TBT to DBT and MBT (Lee, 1996), therefore it reflects the BT composition in stomach contents. While most tissues revealed the concentrations in the order of TBT > DBT > MBT, the metabolically activity tissue as liver was noted to have higher levels of metabolites (DBT and MBT) than those of TBT (50%) (Table 2 and Fig. 2). These profiles agree with previous reports on fish (Harino et al., 2000) and mammal (Iwata et al., 1995). 3.2.2. Gender difference, growth trend and migration Due to the high accumulative nature of BTs in the liver, accumulation of BTs in this tissue was examined to evaluate gender differences and growth trend (bodylength differences) in skipjack tuna. Gender differences of BT concentrations were studied using the liver of samples collected off-Japan-3, 1998 (Table 4). As shown in Table 4, no significant differences of BTs and individual compound (TBT, DBT and MBT) concentrations were found between male and female (P > 0.5, n=10, Mann–Whitney U test), and mean and range of these concentrations were rather similar. Moreover, growth trend (body-length differences) of BT residue levels were examined in the liver of skipjack tuna collected from N-Pacific-2, 1997 and 1999 (Fig. 3). Sex differences were

Fig. 3. Relationship between butyltin concentrations and body-length of skipjack tuna collected N-Pacific-2, 1997 and 1999. The p value indicates Spearman’s rank correlation..

not taken into consideration to calculate growth trend because gender differences are no significant. Results of statistical analysis showed no significant relationship between body-length and BT concentrations, and the concentrations were rather uniform (P > 0.5, n=15, Spearman’s rank correlation). Similar results were observed for the accumulation of organochlorines such as PCB and DDT in skipjack tuna (Ueno et al., 2003). It is also supported by the fact that BMF of BTs in this species was 0.2–0.4 (Table 3), and no considerable biomagnification was found to occur through the food chain. Harino et al. (2000) also obtained similar results that gender and body-length did not affect BT residue levels in fish. This feature of BTs in skipjack tuna is different from that of organic Hg (methyl mercury) in fish, which showed significant increasing trend with body-length (e.g. Al-Majed and Preston, 2000). Overall, gender and body-length differences did not seem to affect BT concentrations in the liver of skipjack tuna, and the concentrations were rather uniform in the samples collected from the same location. Gender- and growth stage- differences in BT concentrations in skipjack tuna will not be considered for further discussions. Moreover, rapid uptake and elimination of TBT (biological half life: 7.4–28.8 days) were reported in several fish (Yamada et al., 1992). It has been reported that gillbreathing organisms have the ability to respond rapidly to changes in the ambient levels of these organic contaminants based on the concept of equilibrium partitioning between ambient water and body (Tanabe et al., 1984). As an evidence, positive correlations were found between organochlorine concentrations (such as PCBs and DDTs) in skipjack tuna and those in seawater collected from Asia-Pacific region (Ueno et al., 2003). These results suggest skipjack tuna rapidly reflect BT levels in seawater where and when they were collected even though they migrate globally. Considering these facts, changes in the residue levels of BT in tuna during their migration may not affect their suitability for pollution monitoring of BTs on a global scale. Skipjack tuna is distributed only in the offshore water and open sea of tropical to temperate regions almost all over the world. These results suggest that skipjack tuna is a suitable bioindicator for monitoring global distribution of BT pollution in offshore waters and open seas.

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3.3. Geographical distribution of BT concentrations BT concentrations in relation to body distribution, sex, growth and rapid reflection of ambient levels in skipjack tuna suggest better use of liver samples, regardless of sex, body length and migration while considering the global monitoring of BT contamination. Using skipjack tuna collected from Asian offshore waters, off-Seychelles, off-Brazil and open seas, global distribution of BT residues in this species was examined. Geographical distribution of BT concentrations in the liver of skipjack tuna is shown in Fig. 4. BTs were detected in livers of all skipjack tuna examined, indicating widespread distribution of BTs even in offshore waters and open seas on a global scale. In particular, skipjack tuna collected from offshore waters around Japan contained the highest BTs concentrations about 400 ng/g on a wet weight basis (E-China Sea-1). Yamada et al. (1997) studied the global monitoring of organotin compounds using squid liver, showing the highly polluted nature of developed and industrialized areas, such as offshore waters around Japan and France. Harino et al. (1999) reported decreasing trends of BTs in water and mussels from Osaka Bay after TBT usage was regulated in 1990 in Japan. Despite the decreasing trend reported in this area, skipjack tuna from offshore waters around Japan still containing elevated levels of BTs. It might be attributed to the recent TBT input from antifouling paints from foreign vessels, and also illegal usage for some maritime activity in Japan. Existing sources of BTs in Japan and wide spread nature of BTs in offshore waters are also suggested from our study. In addition to skipjack tuna from waters around Japan, samples from E-China Sea-1 and Japan Sea showed relatively higher levels of BTs (Table 1 and Fig. 4). One of the highest levels of BTs in

mussels was found in Korea (Hong et al., 2002), and higher concentrations of BTs were found in cod from the Japan Sea than in those from the Pacific Ocean (deBrito et al., 2002). Higher levels of BTs in samples from the Japan Sea and the East China Sea seem to reflect the elevated levels of BTs in Korea and/or limited exchange of water mass between the Japan Sea and the East China Sea compared with those in the Pacific Ocean. Skipjack tuna collected from offshore waters around Asian developing countries, such as S-China Sea, offPhilippines, off-Indonesia and Bay of Bengal, showed comparable levels of BTs residues to those from Japan (Fig. 4). Elevated levels of BTs were also detected in mussels collected from the coast of India and Malaysia in 1997–1998 (Sudaryanto et al., 2002) and Philippines in 1994 (Prudente et al., 1999). Sudaryanto et al. (2002) reported that almost all the locations with high concentrations of BTs in mussels had intensive maritime activities, such as the vicinity of large harbors, major shipping traffic and aquaculture areas. Considering these results, it can be suggested that maritime activities contribute to BT sources in these countries, like significant usage of TBT as a biocide in antifouling paints on ship hulls and /or other marine structures in harbors (Sudaryanto et al., 2002). From the late 1970s to early 1990s, apparently lower concentrations of BTs were found in fish (Kannan et al., 1995a) and marine mammal (Tanabe, 1999) collected from coastal waters around Asian developing countries than those from industrialized countries such as Japan and the USA, indicating significant and continuous input of BTs in the coastal waters of industrialized nations while lesser usage in developing countries is implied at least at present. However, Tanabe (1999) suggested that contribution by developed nations on the control of usage of BTs is necessary to prevent further contamination in

Fig. 4. Geographical distribution of total butyltin concentrations in the liver of skipjack tuna collected from Asian offshore waters, off-Seychelles, off-Brazil and open seas.

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Asian developing countries, because future loading of BTs is expected to increase due to the increasing demand for antifouling paints in the Asia–Pacific region (Reisch, 1996). As mentioned above, Sudaryanto et al. (2002) found high BTs pollution in Asian developing countries using mussel as indicators, and suggested that contamination by BTs in these countries may increase with high economic growth ratio (GNP). It might indicate that those countries having better economic status tend to increase the usage of TBT with the increase in boating, shipping and aquaculture activities. Similar trends in BTs pollution in coastal waters were also observed in skipjack tuna from offshore waters. Collectively, these results suggest that BTs used for maritime activities, such as antifouling paint for international vessels around Asian developing countries, are widely distributed in offshore areas, and these contaminants are highly accumulated in skipjack tuna. It is clearly indicating of ongoing contamination by BTs and widespread distribution to offshore waters and open seas on a global scale. The residue levels of BTs were apparently lower in the open seas (N-Pacific-1,-2 and-3) compared to those in water around Japan (Table 1 and Fig. 4). The clear differences in BT residues between coastal and open sea were also recorded in the global monitoring study using squid (Yamada et al., 1997) and marine mammal (Tanabe, 1999). Moreover, similar patterns of distribution were found for PCDDs in cetaceans (Kannan et al., 1989) and DDTs in skipjack tuna (Ueno et al., 2003). Much lower levels of PCDDs and DDTs in these specimens from open seas were explained by the lower transportability of PCDDs and DDTs through air and water due to their low vapor pressure, low water solubility and high particle affinity (Ueno et al., 2003; Kannan et al., 1989), thereby resulting in elevated residue levels of PCDDs and DDTs in the coastal ecosystems. Similar to PCDDs and DDTs, BTs have a less transportable nature and highly localized characteristics, which explains the lower BT concentrations that were found in skipjack tuna from open seas. BTs concentrations in skipjack tuna from the southern hemisphere, e.g., off-Indonesia, off-Seychelles and off-Brazil, were lower than those from the northern hemisphere (Table 1 and Fig. 4). Lower residue levels of BTs in the southern hemisphere were also reported in the global monitoring study using squid (Yamada et al., 1997). These observations plausibly explain larger usage of these compounds in the northern hemisphere. 3.4. Geographical distribution of BT compositions Geographical distribution of BT compositions in the liver of skipjack tuna was shown in Fig. 5. Among BT derivatives, TBT residues were predominant than its metabolites, MBT and DBT. High proportions of TBT

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Fig. 5. Composition of butyltin compounds in the liver of skipjack tuna collected from Asian offshore waters, off-Seychelles and open seas.

(up to 90%) in skipjack tuna from offshore waters around Japan suggest fresh TBT input still exists in Japan. Specimens collected in offshore waters around Asian developing countries, such as S-China Sea, offPhilippines, off-Indonesia and Bay of Bengal, also showed a higher ratio of TBT (up to 90%), supporting recent usage of TBT for maritime activity in coastal regions of these countries. Despite lower concentrations of BTs being detected in open sea (Table 1 and Fig. 4), a higher percentage of TBT was observed in specimens from N-Pacific-1,-2 and-3 (Fig. 5) which may reflect the continuous input of TBT from antifouling paints from large international vessels throughout over the North Pacific Ocean. Relatively high ratios of TBT (50%) were found in samples from off-Indonesia and off-Seychelles (Fig. 5), suggesting recent usage of TBT in southern hemisphere.

4. Anthropogenic sources In order to understand the portion of anthropogenic input of tin (Sn), concentrations of total Sn (Sn: sum of inorganic Sn+organic Sn) were determined in the liver of skipjack tuna, and compared with those of BTs (Table 1 and Fig. 6). The highest Sn concentration (600 ng/g dry weight) was found in skipjack tuna which showed the highest concentration of BTs (Table 1). The concentrations of Sn were generally related to those of BTs; higher concentrations of Sn being found in locations where higher BT contaminations were found. These observations indicate that anthropogenic sources play a major role in Sn accumulation in skipjack tuna. In fact, as seen in Fig. 6, BTs occupied considerably

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Fig. 6. Percentage of butyltin compounds in Sn in the liver of skipjack tuna collected from Asian offshore waters, off-Seychelles and open seas.

higher percentages in Sn concentrations in skipjack tuna collected from offshore waters around Asian developing countries having high concentrations of BTs. It is clearly suggesting that hepatic Sn exists primarily in organic form as BTs that originate from anthropogenic sources, even in those samples from offshore waters. A similar conclusion was obtained in our previous studies with mussels (Sudaryanto et al., 2002) and higher trophic marine mammals (Le et al., 1999) from coastal waters around Asian countries. This feature of Sn in skipjack tuna is different from that for Hg and Cd, which had mostly originated from a natural background (Muir et al., 1992; Dietz et al., 1996). Although BTs were the major components of Sn compounds accumulated in skipjack tuna from polluted locations such as waters around Asian developing countries, relatively low proportions of BTs in Sn were noted in samples from offshore waters around Japan, showing higher concentrations of BTs (Table 1 and Fig. 6). This result suggests that these specimens were exposed to other organic tins (OTs) and inorganic tin compounds in waters around highly industrialized countries like Japan. The pollution of other OTs (such as phenyl-, octyl- and methyltin compounds) has originated from many anthropogenic sources because these compounds are used as stabilizers for polyvinyl chloride (dimethyl- and dioctyltin), fungicides (triphenyltin), and antifouling agents (triphenyltin) (Fent, 1996). It has been documented that these OTs were found in some aquatic ecosystems (Fent, 1996; Shawky and Emons, 1998). On the coast of Japan, remarkably high concentrations of phenyltins (12,000 ng/g wet wt) were detected in horseshoe crabs (Kannan et al., 1995c). Moreover, it is known that OTs, including BTs, will finally be degraded to inorganic Sn by the environmental microbial process (Fent, 1996). Therefore, there

is a possibility that a higher percentage of other Sn compounds in skipjack tuna from offshore waters around Japan were also comprised of inorganic Sn originated from microbially degraded OTs, because inorganic Sn seems to be accumulated in the waters around Japan due to the extensive and historical usage of OTs in this region since the 1960s. On the contrary, lower ratios of BTs to Sn were found in skipjack tuna from the open sea (N-Pacific-1,-2 and-3) even though BT residue levels were apparently lower than those from offshore waters around Japan (Fig. 6). It seems that inorganic Sn originating from natural background contributes greatly to Sn due to low concentrations of BT residues in open seas. In addition, there is a possibility that skipjack tuna accumulated naturally generated organic Sn, such as methyltins which originate mainly from natural microbial processes (Yemennicioglu et al., 1997).

5. Conclusion BT concentrations in relation to body distribution, sex, growth and rapid reflection of ambient levels in skipjack tuna suggest better use of liver samples, regardless of sex, body-length and migration while considering the global monitoring of BTs contamination. It seems that skipjack tuna is a suitable bioindicator for monitoring BTs pollution in offshore water ecosystem on a global basis. Results of global monitoring using this species clearly indicated widespread BT contaminations and the presence of recent BT pollution sources even in offshore waters and open seas. In particular, elevated levels of BT contamination are evident in offshore waters around Asian developing countries. High percentages of BT in Sn (almost 90%) were

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found in the liver of skipjack tuna, suggesting that anthropogenic BTs represent the major source of Sn accumulation. Continuous monitoring of BTs contamination in offshore waters and open seas can be conducted using skipjack tuna as a bioindicator to make decisions on the future issues of BT contamination in oceans.

Acknowledgements We thank Dr T.B. Minh, Ehime University, Japan for critical reading of the manuscript. The authors thank Dr A. Nihira (Ibaraki Prefectural Fisheries Experimental Station, Japan), Dr J. Takeuchi (Wakayama Research Center of Agriculture, Forestry and Fisheries, Japan), Dr H. Tameishi (Japan Fisheries Information Service Center, Japan) and Dr I. Nakamura (Kyoto University, Japan) for providing the ecological information of skipjack tuna and Mr M. Nakamura and Ms M. Takahashi (Hiraki-no-Takahashi, Co. Ltd., Japan) for collecting skipjack tuna from various regions of Japan. The authors thank Y. Taki (Ehime University, Japan) for BTs analysis of skipjack tuna. This study was supported partly by a Fund ‘‘Development of Advanced Technology for Monitoring the Marine Pollution by Toxic Contaminants Using Organisms as Bioindicators’’ and ‘‘Japan–Korea Cooperative Joint Research Program on Endocrine Disrupting Chemicals’’ from the Ministry of the Environment, Japan. This study was supported by Grant-in-Aid for Scientific Research (A) (Grant No. 12308030) and Grant-in-Aid for JSPS Fellows from the Japan Society for the Promotion of Science. This study was also supported partly by Grant-in-Aid for Scientific Research on Priority Areas (A) (Grant No. 13027101) and ‘‘21st Century COE Program’’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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