Variation in organochlorine accumulation in relation to the life history of the Japanese eel Anguilla japonica

Marine Pollution Bulletin 80 (2014) 186–193

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Variation in organochlorine accumulation in relation to the life history of the Japanese eel Anguilla japonica Takaomi Arai ⇑ Institute of Oceanography and Environment, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia

a r t i c l e

i n f o

Keywords: Organochlorine compounds Catadromous eel Ecological risk Migration Maturation

a b s t r a c t Members of the catadromous eel live in various fresh, brackish and marine habitats. Therefore, these eels can accumulate organic pollutants and are a suitable bioindicator species for determining the levels of organic contaminants within different water bodies. The ecological risk for organochlorine compounds (OCs) in Anguilla japonica with various migration patterns, such as freshwater, estuarine and marine residences, was examined to understand the specific accumulation patterns. The concentrations of HCB, P P P HCHs, CHLs and DDTs in the silver stage (maturing) eel were significantly higher than those in the yellow stage (immature) eel, in accordance with the higher lipid contents in the former versus the latter. The OC accumulations were clearly different among migratory types in the eel. The ecological risk of OCs increased as the freshwater residence period in the eel lengthened. The migratory histories and the lipid contents directly affected the OC accumulation in the catadromous eel species. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Organochlorine compounds (OCs) are omnipresent in hydrosystems due to daily anthropogenic activities and the persistence of some compounds used in the past. OCs have been used primarily as pesticides since the mid-1940s. They accumulate in living organisms and are toxic to both humans and wildlife. Some of these chemicals are considered to act as environmental hormones that disturb the reproductive cycles of both humans and wildlife (Colborn and Smolen, 1996). Because they might engage in longrange atmospheric transport and cold condensation (Bidleman et al., 1993; Iwata et al., 1993; Wania and Mackay, 1996) and their intensive utilisation in agricultural and industrial activities, OC residues have been widely identified and reported across the world, even in Antarctica and the Arctic Zone (Fu et al., 2001; Chiuchiolo et al., 2004). Another possible transport route for pollutants is biotransport, where migrating animals act as vectors between ecosystems. The migration or movement of contaminated fish can redistribute OCs into uncontaminated areas (Merna, 1986; Lewis and Makarewicz, 1988; Scrudato and McDowell, 1989; Monte, 2002). The decomposition of spawning Pacific salmon affects freshwater streams because various elements, such as carbon and nitrogen, are released from salmon carcasses (Brickell and Goering, 1970; Bilby et al., 1996; Lyle and Elliott, 1998). The recycling of nutrients from carcasses is currently recognised as an essential contributor to the productivity of salmon streams (Cederholm ⇑ Tel.: +60 9 6683195; fax: +60 9 6692166. E-mail address: [email protected] 0025-326X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2014.01.011

et al., 1999; Helfield and Naiman, 2001; Naiman et al. 2002). However sockeye salmon Oncorhynchus nerka carcasses were also recently identified as vectors for the movement of contaminants from the Pacific Ocean into remote freshwater lakes in Alaska; contaminants were remobilized from carcasses after the adults spawn and die (Krümmel et al., 2003). The concentration of the ocean-derived contaminants in the lake sediments was correlated with population density of the spawning fish (O’Toole et al., 2006). Although some studies have been conducted to examine biotransport in several anadromous salmon species, there are few reports regarding the biotransportation of organochlorines by other diadromous fish species, such as the anguillid eel: this species has marine, estuarine and freshwater residence life histories. There have been few studies to date regarding the relationship between OC accumulations and different migratory histories in this species, even though this species may be an excellent biomonitor for the aquatic environment (Maes et al., 2008; de Boer et al., 2010; Ferrante et al., 2010; McHugh et al., 2010). The anguillid eel (genus Anguilla) does not reproduce in fresh and coastal waters in either its yellow (immature) or early silver (mature) stages. Therefore, body burdens are not affected by the reproduction cycle and the associated changes in lipid metabolism (Maes et al., 2008). Furthermore, the yellow eel has a high lipid content that increases with age and reaches a maximum before silvering and emigration. They generally exhibit life-long accumulation and low depuration rates (Larsson et al., 1991; Tulonen and Vuorinen, 1996; Knights, 1997). Before their downstream migration to the offshore spawning area, yellow eels are territorial and maintain local homeranges in rivers and estuaries, residing in

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mud, weed beds or shady pools during the day (Naismith and Knights, 1990). Eels feed on nearby benthic invertebrates and other aquatic fauna (Tesch, 1977). These typical life history characteristics warrant the use of this eel as an indicator for the presence of hazardous chemicals in the environment, particularly substances with a low solubility in water. Therefore, the eel tissue concentration and body burden accurately reflect their environmental exposure and reveal that the tissue concentration is related to the pollution levels of prey species, surface waters and sediments. The Japanese eel Anguilla japonica is widely distributed in East Asia, from Taiwan in the south, through eastern China and Korea up to the Sanriku Coast of northern Honshu Island, Japan (Tesch, 1977). A. japonica generally is a catadromous fish species (McDowall, 1988) that spawns in the North Equatorial Current west of the Mariana Islands. Their transparent leaf–like larvae (leptocephali) are transported from the spawning area toward the coastal waters of East Asia by the North Equatorial and Kuroshio currents, where they metamorphose into glass eels. Generally, the glass eels migrate upstream, growing into the elver and yellow eel stages in freshwater. At maturation, the yellow eels metamorphose into silver eels, migrating downstream toward the ocean to begin their spawning migration (Tesch, 1977). Recently, the migratory history of several species of anguillid eels has been studied using microchemical analytical techniques to determine the ratios of strontium to calcium (Sr:Ca) in the otoliths of fishes. The Sr:Ca ratio in the otoliths of fishes such as anguillid eels differs depending on the amount of time they spend in freshwater vs. seawater (Arai et al., 2004, 2006, 2009; Chino and Arai, 2009; Arai and Chino, 2012). The Sr concentration in seawater is approximately 100-fold greater than that in fresh water (Campana, 1999). Therefore, the Sr:Ca ratios of otoliths may reveal whether individual eels actually move between different habitats with differing salinity regimes. Chino and Arai (2010) used these Sr:Ca ratios to classify the migratory histories of anguillid eels into three migratory types: (1) ‘marine residents’ (spends most of their life in the sea and do not enter freshwater), (2) ‘estuarine residents’ (inhabits estuaries or switches between different habitats), and (3) ‘freshwater residents’ (enters and remains in freshwater river habitats after arrival in the estuary). Therefore, there might be different ecological risks for pollutants, including OCs, for the three migration types of the species. The objective of the present study was to examine differences in the accumulation patterns of OCs such as DDTs, hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), chlordanes (CHLs) and mirex, in the muscles of the three migratory types of Japanese eel A. japonica collected in Japanese waters and to compare the OCs accumulations relative to size and age. The environmental histories of A. japonica were reconstructed using the ontogenic changes in the otolith Sr:Ca ratios along the life history transect to determine the details of eel migration. The results of the present study may provide valuable clues for understanding the ecological risk of OCs according to the migration of diadromous fish.

2. Materials and methods

45°N 40 ° 35 °

Pacific Ocean

30 °

130 ° E 135 ° 140 °

•

B

• A

Katsuura River

145 °

Kii Channel

• C

Fig. 1. Sampling sites in the upper reach (A) and the river mouth (B) of the Katsuura River and the Kii Channel (C) on the eastern part of Shikoku Island in western Japan.

Shikoku Island faces the Inland Sea, and the coast from east to south faces the Kii Channel and the Pacific Ocean. The total length (TL), body weight (BW), gonad weight (GW) and lipid content were measured, and the skin colour was observed to help categorise each specimen as either a yellow (immature) or silver (mature) eel. The sex was determined by examining the gonads. GW (to 0.01 g) and BW (to 0.1 g) were measured to determine the GSI that was calculated as follows:

GSI ¼ GWðgÞ=BWðgÞ  100 All materials used for sample preparation were washed thoroughly with purified water and finally washed with an organic solvent such as acetone. All samples were dissected and muscle tissues were taken. After dissection, they were wrapped in plastic bags and stored frozen at 20 °C until chemical analysis.

2.1. Fish 2.2. Chemical analysis for OCs Forty-seven eels were collected by set nets, eel pots or fishing in the eastern part of Shikoku Island of western Japan (Fig. 1, Table 1). The eels were collected at two sites of the Katsuura River, Tokushima Prefecture; one site was at the upper reach of the river, remained uninfluenced by the tidal effect and was a 0 in salinity (4 specimens); the other site was at the river mouth in the intertidal zone (7 specimens). Eels were also collected off the shore of the Tokushima Prefecture (36 specimens). The northern coast of

In the present study, I determined the concentrations of P hexachlorobenzene (HCB), hexachlorocyclohexanes P (a-HCH + ß-HCH + c-HCH + d-HCH), chlordanes (Heptachlor + Heptachlor epoxide + Oxychlordane + trans-Chlordane + transP Nonachlor + cis-Nonachlor), dichlorodiphenyltrichloroethanes (p,p0 -DDD + o,p0 -DDE + p,p0 -DDE + o,p0 -DDT + p,p0 -DDT) and mirex, as described by Arai and Takeda (2012) and Arai (2013).

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Table 1 Anguilla japonica used for organochlorine compounds and otolith microchemistry analyses. Sampling location

Migration type

Sample size

Katsuura River (upper reach) Katsuura River (river mouth) Kii Channel (off Tokushima)

Freshwater Freshwater Estuarine Freshwater Estuarine Marine

4 4 3 9 18 9

Total length (mm)

Body weight (g)

Age (yr)

Mean ± SD

Range

Mean ± SD

Range

Mean ± SD

Range

Mean ± SD

Range

538 ± 60.4 520 ± 21.2 525 ± 27.1 627 ± 81.5 662 ± 78.7 675 ± 61.2

458–597 501–545 499–553 560–813 484–845 593–766

222 ± 80.5 209 ± 45.7 192 ± 35.3 368 ± 138 452 ± 185 442 ± 128

131–301 160–259 166–232 216–662 224–1080 262–610

9 ± 1.0 8 ± 1.0 7 ± 1.5 9 ± 3.1 8 ± 1.5 9 ± 1.1

8–10 7–9 6–9 7–16 6–10 7–10

1.6 ± 0.3 1.9 ± 0.3 3.3 ± 0.9 1.5 ± 0.2 4.8 ± 0.9 6.2 ± 0.3

1.2–1.7 1.8–2.3 2.7–4.3 1.2–1.8 2.9–5.9 6.0–6.9

Homogenised muscle samples (approximately 5 g) were added to a 45 ml Dionex accelerated solvent extraction (ASE Dionex) cell. 13 C-labeled surrogate standards were added to the ASE cell. The sample was extracted with a mixture of hexane and acetone (1:1) (100 °C, static 5 min, heating 5 min, purge 1 min, 2000 psi). The lipid content for each sample was determined gravimetrically. After the extract was concentrated, the lipids were removed by gel permeation chromatography using a Teflon column (22–25 mm bore diameter, length 50–70 cm) filled with 50 g resin (BioBeads S-X3) suspended in dichloromethane. The column was eluted with dichloromethane and cyclohexane (1:1) at a flow rate of 5 ml/min. The eluent was reduced in volume to approximately 5 ml. The polar compounds were removed and separated by liquid chromatography on FlorisilÒ. The first fraction was eluted using 100 ml of 5% diethyl ether in hexane and contained most of the compounds. The second fraction, eluted with 100 ml of 20% diethylether, contained endrin and dieldrin. After being reduced to approximately 5 ml, the first fraction was transferred to a silica gel column and separated again into two fractions. The first fraction, eluted was using 30 ml hexane and contained HCB. The second fraction was eluted using 25% diethyl ether in hexane and contained the other OCs. The fractions were reduced in volume to 0.5 ml using a stream of dry nitrogen. A syringe spike was added. An Agilent 6890 series gas chromatography/negative chemical ionisation mass spectrometry (Agilent 5973 N) was used for identification and quantification. The separation was carried out with a HT-8 capillary column (SGE, 50 m length  0.22 mm i.d., 0.25 lm film thickness) coated with 8% phenylpolycarborane–siloxane. The column temperature was programmed as follows: 50 °C held for 0.3 min, increased to 200 °C at 20 °C/min and finally increased to 280 °C at 2.5 °C/min. The injector temperature and ion source temperature were 260 °C and 150 °C, respectively. Splitless injection (1 ll) was used for the samples. All the isomers were quantified using the isotope dilution method with the corresponding 13C-labeled congeners. The recovery for each sample was between 50% and 120%.

2.3. Otolith preparation, otolith X-ray microprobe analysis and life history analysis Sagittal otoliths were extracted from each eel, embedded in epoxy resin (Struers, Epofix), and mounted on glass slides. The otoliths were then ground to expose the core in the anterior–posterior direction on the frontal plane using a grinding machine equipped with a diamond cup-wheel (Struers, Discoplan-TS) before being polished further using an OP-S suspension on an automated polishing wheel (Struers, RotoPol-35) equipped with a semi-automatic specimen mover (Struers, PdM-Force-20). Finally, the samples were cleaned using distilled water and ethanol and dried at 50 °C in an oven before examination. The ground surfaces of the otoliths were examined at 200 with a light microscope, and photographs were taken to measure the ‘radius’ of the elver mark (the longest distance from the otolith core to the elver mark; Chino and Arai, 2009).

Otolith Sr:Ca ratios

For electron microprobe analyses, all otoliths were coated with Pt–Pd using a high-vacuum evaporator. ‘Life-history transect’ analysis of the Sr and Ca concentrations in all specimens was performed by measuring along a line down the longest axis of each otolith from the core to the edge using a wavelength dispersive X-ray electron microprobe (JEOL JXA-8900R), as described by Chino and Arai (2009). Wollastonite (CaSiO3) and tausonite (SrTiO3) were used as standards, and the accelerating voltage and beam current were 15 kV and 1.2  108 A, respectively. The electron beam was focused on a point 10 lm in diameter, with measurements spaced at 10 lm intervals. Chino and Arai (2009) determined the migratory patterns of Japanese eels using the otolith Sr:Ca ratios outside the ‘high Sr core’ that corresponded to the period of ocean life during the leptocephalus and early glass eel stages (Arai et al., 1997). In accordance with the criteria established by Chino and Arai (2009), we omitted the high Sr core (mean: 150 lm radius from the otolith core), and only values outside the high Sr core were used to obtain a mean otolith Sr:Ca ratio for each specimen. We grouped these specimens into the three general categories using their mean otolith Sr:Ca ratios to enable statistical comparisons to be made between eels with different habitat use histories: ‘marine residents’ (Sr:Ca P 6.0  103), ‘estuarine residents’ (2.5  103 6 Sr:Ca < 6.0  103) and ‘freshwater residents’ (Sr:Ca < 2.5  103). After electron microprobe analysis, the otoliths were polished to remove the coating, etched with 1% HCl and stained with 1% toluidine blue. The age of each specimen was determined by counting the number of blue-stained transparent zones outside the elver mark, as described by Arai et al. (2004) and Chino and Arai (2009). 2.4. Statistics Differences between data were analysed using the Mann–Whitney U-test. Differences among data were also examined using the Kruskal–Wallis test while using the Mann–Whitney U-test for post hoc two-group comparisons. Spearman’s rank test was also used to examine the significance of the correlations (Sokal and Rohlf, 1995). 3. Results 3.1. Migratory patterns In the A. japonica collected in the upper reach of the Katuura River, all eels (four specimens) were identified as freshwater residents (Table 1). There were four freshwater resedences and three estuarine residents among the eels from the river mouth (Table 1). In Kii Cannel, there were nine freshwater residents, eighteen estuarine residents and nine marine residents (Table 1). 3.2. Biological characteristics The total length (TL) and body weight (BW) in A. japonica ranged from 458 to 845 mm and from 131 to 1080 g, respectively

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(Table 1). The age of the specimen was based on the number of annual rings in the otolith in A. japonica and ranged from 6 to 16 yr (Table 1). The GSI values in A. japonica ranged from 0.00 to 3.98. Based on the index and morphological observation, all specimens from the Katsuura River (11 specimens) were in the yellow (immature) stage and all specimens from the Kii Channel (36 specimens) were in the silver (mature) stage, in accordance with the data from Matsui (1972) and Utoh et al. (2004). In the present study, all specimens from the Katuura River and the Kii Channel were identified as females. The lipid contents in the yellow and silver stage eels ranged from 3% to 4.3% and 8.6% to 26.7%, respectively, with means ± SD of 3.7 ± 0.9% and 16.3 ± 5.5%, respectively (Table 1). The lipid contents in the silver stage eels were significantly higher than in the yellow stage eels (Mann–Whitney U-test, p < 0.05).

There was no relationship between each organochlorine compound level and TL (Spearman’s rank test, p > 0.05) (Fig. 2). There were also no relationships between each organochlorine compound level and BW (Spearman’s rank test, p > 0.05) (Fig. 2). No P P relationship was found between the HCB, CHLs and DDTs levels and age in A. japonica (Spearman’s rank test, p > 0.05) (Fig. 2).

3.3. OC accumulation in eels

3.5. OCs accumulation in maturation stage

P P Among five OCs compound groups (HCB, HCHs, CHLs, DDTs and mirex), heptachlor was not detected in any specimen and d-HCH was not dected in the Katsuura River eels (Table 2). P The concentrations of DDT were the highest of any chemical anaP P lysed (Table 2). The concentrations of HCB, HCHs, CHLs and P DDTs in the Kii Channel eels were significantly higher than the eels in the Katsuura River (Mann–Whitney U-test, p < 0.0001). The residue levels of DDT and its metabolites occurred in the P following order for both sited: DDE > DDD > DDT. The DDT/ DDT ratio in A. japonica from all sites ranged from 0.00 to 0.49, suggesting that no new DDT was added to their habitats. In the HCH residue, ß-HCH was the dominant isomer constituting more than 65% in both sites, followed by a-HCH and c-HCH, while d-HCH was not detected in most eels. Therefore, HCH was previously used in the Tokushima area. For the CHL isomers, trans-nonachlor was the major constituent of the compounds at more than 60%. The second most abundant chlordane was trans-chlordane (Table 2) followed by oxychlordane, cis-nonachlor and heptachlor epoxide. Trans-nonachlor was the most persistent and trans-chlordane was the dominant compound in a chlordane technical mixture. The higher trans-nonachlor/trans-chlordane (N/C) ratios suggest the recent input of

P In the yellow stage eels (11 specimens), the HCB, HCHs, P P CHLs and DDTs concentrations ranged from 0.30 to 1.56, 0.13 to 1.00, 1.15 to 23.24 and 1.08 to 59.00 ng g1 wet wt, respectively, with means ± SD of 0.69 ± 0.46, 0.51 ± 0.29, 7.07 ± 6.93 and P P 14.73 ± 17.47 ng g1 wet wt, respectively. The HCB, HCHs, CHLs, P and DDTs concentrations in the silver stage eels (36 specimens) ranged from 0.30 to 4.46, 0.34 to 15.53, 3.08 to 105.62 and 8.90 to 450.95 ng g1 wet wt, respectively, with means ± SD of 1.29 ± 0.81, 1.88 ± 2.52, 32.37 ± 27.57 and 59.56 ± 81.45 ng g1 wet wt, respectively (Table 2). All OC concentrations in the silver stage eels were significantly higher than were observed in the yellow stage eels (Mann–Whitney U-test, p < 0.01) (Fig. 3). The accumulation of OCs in the muscle depends on the organ lipid contents.

P

CHLs were recently released into the environment (Iwata et al., 1993; de Brito et al., 2002). In the present study, the N/C ratio in all eels had a mean ± SD of 30.6 ± 132. The high N/C value in A. japonica indicates that they were exposed to relatively new sources of CHLs in this region. 3.4. Relationships between OCs accumulation and each biological characteristic

3.6. OCs accumulation in migratory types P P P Comparisons between each HCB, HCHs, CHLs, and DDTs concentration and each migratory type (freshwater, estuarine rand marine residents) could be conducted in the silver stage but not the yellow stage due to the limited number of samples. The HCB, P P P HCHs, CHLs and DDTs concentrations in the freshwater, estuarine and marine residents ranged from 0.93 to 2.95, 1.37 to 15.54,

Table 2 Concentrations of organochlorines (ng g1 wet wt.) in muscle of Anguilla japonica. Compounds

a-HCH b-HCH c-HCH d-HCH Total HCH HCB p,p0 -DDD o,p0 -DDE p,p0 -DDE o,p0 -DDT p,p0 -DDT Total DDT Heptachlor Heptachlor Epoxide trans-Chlordane Oxychlordane cis-Nonachlor trans-Nonachlor Total CHL Mirex ND: not detected.

Katsuura River (upper reach)

Katsuura River (river mouth)

Kii Channel (off Tokushima)

Mean ± SD

Range

Mean ± SD

Range

Mean ± SD

Range

0.06 ± 0.06 0.18 ± 0.08 0.03 ± 0.02 ND 0.26 ± 0.05 0.47 ± 0.41 ND ND 2.08 ± 0.86 ND ND 2.08 ± 0.86 ND 0.02 ± 0.04 0.30 ± 0.28 0.23 ± 0.17 0.17 ± 0.09 2.07 ± 1.28 2.76 ± 1.81 0.08 ± 0.02

0.01–0.15 0.09–0.25 0.01–0.05

0.10 ± 0.07 0.52 ± 0.21 0.04 ± 0.02 ND 0.66 ± 0.27 0.81 ± 0.47 1.58 ± 0.67 0.12 ± 0.12 14.49 ± 12.14 1.27 ± 1.88 3.23 ± 2.59 21.96 ± 18.45 ND 0.17 ± 0.07 1.92 ± 1.09 0.95 ± 0.71 0.83 ± 0.72 5.83 ± 4.81 9.53 ± 7.23 0.20 ± 0.12

0.04–0.19 0.09–0.77 ND – 0.07

0.38 ± 0.42 1.29 ± 2.19 0.11 ± 0.09 0.09 ± 0.13 1.86 ± 2.53 1.29 ± 2.19 17.04 ± 26.75 0.07 ± 0.17 46.93 ± 86.09 3.89 ± 4.71 9.13 ± 26.17 77.34 ± 114.1 ND 1.16 ± 1.63 5.05 ± 9.40 3.48 ± 3.11 3.82 ± 3.73 27.56 ± 57.01 40.78 ± 62.08 0.00 ± 0.01

0.09–2.13 0.18–2.48 0.05–0.51 ND – 0.52 0.34–4.06 0.30–4.46 ND – 132.2 ND – 0.67 0.03–157.0 ND – 15.08 ND – 148.1 0.12–451.2 ND 0.07–7.40 0.08–46.66 0.11–15.16 0.09–13.52 0.09–347.7 0.44–365.5 ND – 0.07

0.18–0.30 0.04–1.02

1.08–3.02

1.08–3.02 ND – 0.07 0.05–0.69 0.07–0.47 0.09–0.29 0.93–3.90 1.15–5.36 0.06–0.11

0.13–1.00 0.30–1.24 0.85–2.86 ND – 0.27 2.78–37.9 ND – 4.55 1.36–8.84 5.23–59.00 ND 0.08–0.29 0.74–3.60 0.39–2.27 0.19–2.27 1.44–15.10 2.77–23.24 0.10–0.39

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P P P Fig. 2. Relationship between the OCs such as HCB, HCHs, CHLs and DDTs concentrations in the muscles and biological characteristics such as TL, BW and age in Anguilla japonica.

3.08 to 105.34 and 11.14 to 105.34 ng g1 wet wt, from 0.38 to 1.96, 0.34 to 3.72, 0.44 to 105.62 and 0.12 to 451.24 ng g1 wet wt and from 0.30 to 4.46, 0.74 to 1.34, 11.44 to 365.48 and 17.51 to 546.83 ng g1 wet wt, respectively, with means ± SD of 1.67 ± 0.70, 3.58 ± 4.61, 37.00 ± 23.33 and 49.25 ± 36.31 ng g1 wet wt, 1.04 ± 0.41, 1.40 ± 1.03, 24.97 ± 27.65 and 71.84 ± 106.13 ng g1 wet wt and 1.43 ± 1.31, 1.07 ± 0.16, 76.19 ± 112.72 and 116.44 ± 170.36 ng g1 wet wt, respectively (Table 2). For the HCB, the freshwater residents had significantly higher concentrations than the estuarine residents (Kruskal–Wallis test, p < 0.01), while no significant differences were found in any other combinations P (Kruskal–Wallis test, p > 0.05) (Fig. 4). In HCHs, the freshwater residents had significantly higher concentrations than the estuarine or the marine residents did (Kruskal–Wallis test, p < 0.05) (Fig. 4). There were no significant differences between migratory types

P P P Fig. 3. Concentrations of OCs such as HCB, HCHs, CHLs and DDTs concentrations in the yellow (immature) and the silver (mature) stage eels (Anguilla japonica).

regarding the (Fig. 4).

P P CHLs and DDTs (Kruskal–Wallis test, p > 0.05)

4. Discussion 4.1. OC accumulation relative to fish size and age The present study suggested that the accumulation of organochlorine compounds such as HCB, HCHs, CHLs, and DDTs, in the catadromous eel A. japonica did not depend on TL, BW or age. DDT and its metabolites increased with age, TL and BW in the European eel A. anguilla, although the concentrations of lindane (c-HCH) displayed a negative relationship with age (Larsson

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tion between the concentration of OCs and fish size was reported for the red mullet Mullus barbatus and sea bass Dicentrarchus labrax (Pastor et al., 1996), as well as for lindane in A. anguilla. Albaigfs et al. (1987) suggested that this concentration decrease in the Mediterranean red mullet might be caused by increased metabolic capacity with age. Another reasonable explanation might be a simple dilution effect produced by the increased body weight (Niimi and Oliver, 1983; Rossel et al., 1987). Furthermore, the OC concentrations did not vary with size in the Greenland shark Somnious microcephalus (Fisk et al., 2002). Therefore, the relative importance of age and fish size on OCs accumulation varies with the OC characteristics of fish species and sites examined. In the present study, the mean age of the examined A. japonica was 7–9 yr, and most of the eels were less than 10 yrs old (Table 1): therefore, the OC accumulation might display a correlation with the TL, BW or age of the eels because the eels were much younger than 12 yr. The levels of HCB (0.47–1.29 ng g1 ww), HCHs (0.26–1.86 ng g1 ww), CHLs (2.76–40.78 ng g1 ww), and DDTs (2.08–77.34 ng g1 ww) have lower levels than those reported in Belgium (HCB; 0.003– 192.0 ng g1 ww, DDTs; 1.5–3995.4 ng g1 ww) (Maes et al., 2008), Italy (HCB; 0.3–16.5 ng g1 ww, DDTs; 36.4–248.5 ng g1 ww) (Ferrante et al., 2010), and Poland (HCB; 4.0–533.9 ng g1 ww, HCHs; 9.8–273.9 ng g1 ww, DDTs; 0.4–23.8 ng g1 ww) (SzlinderRichert et al., 2010). In addition, we primarily detected metabolites for each organochlorine compounds. Therefore, the OCs might be eliminated or attain less accumulate if the eels are not continuously exposed lowering the correlation between the OCs accumulation and the size and age. 4.2. OCs accumulation in maturation stage

P P P Fig. 4. Concentrations of OCs such as HCB, HCHs, CHLs and DDTs in the three migratory types of silver stage eels (Anguilla japonica): freshwater, estuarine and marine residence eels.

et al., 1991). The largest increase in DDT uptake was found for eels aged 12 years and older, while from 6 to 12 years, the uptake of OCs was lower and increased gradually (Larsson et al., 1991). Therefore, the accumulation pattern of OCs varied with the growth stage. Fish accumulate persistent pollutants from water and food (Spigarelli et al., 1983). Uptake from water is a fast process that occurs over hours or days, depending on the chemical properties of the pollutants (Pizza and O’Connor, 1983). However, uptake from food is a slower process, and hydrophobic pollutants accumulate over years (Spigarelli et al., 1983). The exposure time governs the uptake process (Larsson et al., 1991). However a negative correla-

Clearly, OC accumulation in the silver stage (maturing) eels was higher than in the yellow stage (immature) eels. The mean lipid content in the silver stage eels was five times higher than in the yellow stage eels. The OCs examined in this study are highly lipid soluble, and thus are significantly influenced by lipid dynamics. The biochemical features of lipid storage during the growth of the shortfinned eel (Anguilla australis) have been reported previously (De Silva et al., 2002). The lipid content increased as the eel growth progressed, and higher amounts of fatty acids in the total lipid, saturates and monoenes were found during the final stage of eel growth. Eels might not feed just before their spawning migration and oocytes are not fully developed during the migration (Lokman et al., 1998). A certain proportion of the reserves will need to be channeled to sustain the eel during this period. Therefore, biochemical changes, including the build-up of large energy reserves, occur before the long oceanic spawning migration. A situation similar to that regarding lipid content in A. australis might be considered for A. japonica because they are in the same genus. To obtain the energy necessary for migration, gamete production and spawning, the total stored lipids in eels must exceed 20% of their body weight (Boetius and Boetius, 1980). In the present study, several eels stored lipids at nearly 20% of their body weight, and these eels might be ready for spawning migration. Because OCs are hydrophobic, they will be retained in lipids in the muscle. Therefore, a higher lipid content in eels might lead to elevated OCs levels in A. japonica. 4.3. OCs accumulation versus migratory patterns In all migratory types of A. japonica, OCs were detected in the present study. The OC residues indicate that there is a persistent presence of OCs in sea, estuary and freshwater environments despite the ban on their usage. Furthermore, freshwater resident eels accumulated higher OCs levels indicating that the OC concentrations increased significantly over longer freshwater residence

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periods. The same tendency has been reported in freshwater fish because they contained higher concentrations of OCs than marine fish, implying that OCs originate from inland watersheds (Monirith et al., 1999). Consuquently, the present results also suggest that the previous intensive agricultural activity in the inland area might explain the elevated OC concentration in the freshwater residents. The risk of OCs in freshwater residents is higher than in the estuarine and a marine residents. Therefore, the risk of OCs varies depending on the migratory types, even within the same species. The yellow stage eel has been used as a biomonitor for the aquatic environment for numerous reasons. After a long journey from the open ocean, juvenile glass eels settle at a certain locations in seas, estuaries or rivers without further significant migration for a time. Subsequently, they then metamorphose into silver stage eels and become fatter because they build an energy reserve for the long journey back to the offshore spawning area. The absence of spawning during their growth before the journey is a major advantage for monitoring trends because there is almost no seasonal disturbance in the contamination pattern during their entire life span (De Boer et al., 2010). Furthermore, the eels live in the upper sediment layer; therefore they have a perfect exchange with the aquatic environment close to the contaminated sediment making them better representiatives of the local contaminant situation than other fish species. In the present study, three migratory types appeared in Kii Channel, while two types appeared in the mouth of the Katsuura River. Therefore, the life histories of the eels varied even though they were collected from a relatively small area. Therefore, a comparison between the OC contamination levels using anguillid eels as a biomonitoring indicator should be conducted relative to the migratory history of the specimens to understand the details and mechanism for the bioaccumulation of OCs because the accumulation patterns of these eels were different relative to their habitat.

Acknowledgements The author thanks Yoko Ikeda for assistance with the organochlorine analyses. This work was supported in part by Grant-inAid No. 20688008 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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