DDT Compounds and PCB Isomers and Congeners in Weddell Seals and Their Fate in the Antarctic Marine Ecosystem

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1 Agricultural and Biological Chemistry ISSN: (Print) (Online) Journal homepage: DDT Compounds and PCB Isomers and Congeners in Weddell Seals and Their Fate in the Antarctic Marine Ecosystem Hideo Hidaka, Shinsuke Tanabe & Ryo Tatsukawa To cite this article: Hideo Hidaka, Shinsuke Tanabe & Ryo Tatsukawa (1983) DDT Compounds and PCB Isomers and Congeners in Weddell Seals and Their Fate in the Antarctic Marine Ecosystem, Agricultural and Biological Chemistry, 47:9, , DOI: / To link to this article: Published online: 09 Sep Submit your article to this journal Article views: 62 View related articles Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at

2 Agric. Bioi. Chem., 47 (9),2009",,2017, DDT Compounds and PCB Isomers and Congeners in. Weddell Seals and Their Fate in the Antarctic Marine Ecosystem Hideo HIDAKA, Shinsuke TANABE and Ryo TATSUKAWA Department ofenvironment Conservation, College ofagriculture, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan Received January 31, 1983 The whole-body burdens and concentrations of IDDT (the sum ofp,p'-dde andp,p'-ddt) and PCBs in the Weddell seal, Leptonychotes weddelli, caught near Syowa Station, Antarctica,were determined by detailed biometric measurements of their organs and tissues and analyses of IDDT and PCBs in them. The concentration levels ofiddt and PCBs in Weddell seals were much lower than those in various species of marine mammals from other oceans. The low levels may be attributable to the low concentration of these chemicals in the food of Weddell seals and in the sea water under antarctic fast ice. However, the concentration ratio of IDDT between the food organisms of seals and sea water under antarctic fast ice was higher than those of other ocean ecosystems. Since Sladen et al. l ) in 1966 reported the low concentrations of DDT compounds in a crabeater seal, there have been only three reports on DDT and PCB residues in the antarctic seal,2-4) in which limited organs and tissue were employed for the chemical analysis, and no analytical data has been reported on the chlorinated hydrocarbons in seal's food and ambient sea water. For evaluating the bioaccumulation of persistent chemicals in antarctic ecosystems, it is desirable to describe their comprehensive behavior and fate in the food chain including sea water, plankton, fish, seal, etc. The comparison of bioaccumulation processes among animal species should be based on both the residual concentration and the burden of chemicals on a whole-body basis. So far, it has been difficult in practice, however, to obtain the whole-body burden or concentration in the case of large-sized animals. In this report we first demonstrate the distribution of LDDT (the sum of p,p'-dde and p,p'-ddt) and PCBs in Weddell seals, Leptonychotes weddelli, caught near Syowa Station (69 00'8, 39 35' E), Antarctica, and estimate their whole-body burdens. Furthermore, the bioaccumulation characteristics of these chemicals in Weddell seals, one of the high trophic level animals in the antarctic marine ecosystem, are examined in relation to the concentration ratio between seals and their food or sea water. The environmental pollution from.lddt and PCBs in the Antarctic is also discussed in comparison with that in other marine ecosystems. MATERIALS AND METHODS Samples. Two adult specimens (male and female) were electrocuted at Tottuki point about 13 km northeast from Syowa Station (Table I). Three newborns were found dead and collected at Tottuki point and Ongulkalven island (about 6 km west of Syowa Station). A newborn male (specimen No.3) seemed to be either stillborn or dead just ~fter birth, since his long umbilical cord was still connected to his mother's amnion. Although this newborn male was in a well-preserved condition at the time of discovery, the other two female (Nos. 4 and 5) were found to be bleeding and dehydrating. The specimens collected were preserved in a cold room at - 18 C or lower until the macroscopic anatomy at Ehime University. All specimens were dissected in detail, and their. organ and tissue weights were measured. The dissected organs and tissues were returned to the cold chamber at -18 C until chemical analysis. The detailed results of the biometry will be published elsewhere. 5 ) The age of the adult specimens was determined by Dr. Y. Naito, National Institute of Polar Research, using the

3 2010 H. HIDAKA, S. TANABE andre TATSUKAWA TABLE I. SAMPLING DATA AND ORGAN AND TISSUE WEIGHTS OF WEDDELL SEALS Specimen No Sampling date Oct. 9. '81 Oct. 9. '81 Oct. 27. '81 Nov. 7. '81 Nov. 12. '81 Location Tottuki Tottuki Ongulkalven Ongulkalven Tottuki Sex female male male female female Age 3y. & 11m. 13 or 14y. newborn newborn newborn Body length (cm) Body weight (kg) Organ and tissue weight (kg) Blubber 94.0 (29.7) (30.2) 3.37 (12.9) 2.19 (12.1) 1.24 (6.42) Muscle (33.4) (36.1) 9.01 (34.5) 5.98 (33.0) 8.59 (44.5) Liver 6.87 (2.17) 8.35 (2.18) 0.77 (2.94) 0.51 (2.81) 0.48 (2.49) Kidneys 1.19 (0.37) 1.53 (0.39) 0.30 (1.13) 0.14 (0.77) 0.17 (0.88) Heart (0.55) 1.49 (0.39) 0.17 (0.65) 0.17 (0.92) 0.18 (0.94) Lungs 3.49 (1.10) 5.30 (1.38) 0.57 (2.18) 0.45 (2.46) 0.60 (3.11) Spleen 5.16 (1.63) 4.82 (1.26) 0.25 (0.95) 0.15 (0.81) 0.09 (0.45) Stomach 1.62 (0.51) 2.02 (0.53) 0.15 (0.58) 0.11 (0.59) 0.10 (0.53) Intestine 4.45 (1.40) 5.87 (1.53) 0.51 (3.67) 0.97 (5.34) 0.79 (4.07) Diaphragm 1.86 (0.59) 2.13 (0.56) 0.14 (0.54) 0.09 (0.50) 0.11 (0.57) Brain 0.50 (0.16) 0.50 (0.13) 0.38 (1.44) 0.34 (1.88) 0.28 (1.47) Blood 39.2 (12.4) 36.6 (9.56) 0.96 (3.68) 0.28 (1.55) 0.18 (0.93) Bone 11.8 (3.72) 12.9 (3.37) 2.67 (10.2) 1.93 (10.7) 3.09 (16.0) Others 27.0 (8.53) 35.0 (9.14) 4.55 (17.4) 3.09 (17.1) 3.30 (17.1) Total (96.2) (96.7) 23.8 (9L2) 16.4 (90.6) 19.2 (99.5) Figures in parentheses indicate the percentage of organ and tissue weight to body weight. growth layer of the canine tooth. The adult female (No.1, 3 years and 11 months old) has no parity. Chemical analysis. The analyses of l7ddt and PCBs were performed following the alkaline digestion method. 6 ) An organ and tissue sample ( g) was refluxed in 1N KOH-ethanol solution for 1hr. The ethanol extract was transferred to hexane, and then a cleaning process was carried out using silica gel column chromatography. The final hexane eluate was concentrated to 6 ml and, if necessary, cleaned by shaking with a solution containing 5% fuming sulfuric acid. For the analysis of low concentration samples, the final hexane extract was further concentrated to 100 JlI in a microconcentrator under a stream of purified nitrogen gas..the determinations of l7ddt and PCBs were conducted on a Shiniadzu GC-5A and GC-8A gas chromatograph equipped with a 63Ni electron capture detector. The column consisted of a 1.8mx2mm Ld. glass column packed with 2% QF % OV-17 for DDT analysis and a 5.0mx2mm i.d. glass column packed with purified 2% Apiezon L grease for PCB analysis. The support was 100 to 120 mesh Chromosorb W AW HMDS for both columns. Some representative samples were injected into a gas chromatograph-mass spectrometer equipped with a selected-ion monitor (Shimadzu GC-MS 9020 DF) for PCB isomer and congener analysis. Details of the GC and GC-MS analysis conditions are the same as those previously reported. 7 ) l7ddt which was detected as a single peak (p,p'-dde) in the chromatograms includes bothp,p' DDE and p,p'-ddt, since p,p'-ddt is converted into p,p'-ddein the alkaline digestion method. RESULTS AND DISCUSSION Accumulation of EDDT and PCBs in Weddell seals The representative organs and tissues to be analyzed were selected from 60 dissected parts of one seal's body. The residual concentrations and amounts of EDDT and PCBs in the organs and tissues of an adult male (specimen No.2) and a newborn male (No.3) are given in Tables II and III respectively. Blubber and muscle, which constitutes a large portion of the body weight, were taken from several areas in the seal's body for checking the variance of organochlorine distribution. Those organs which were assumed to be with low lipid content were not employed for the chemical analysis. The total weight of analyzed organs

4 DDT and PCB in Weddell Seals 2011 TABLE II. LIPID CONTENTS (%), l'ddt AND PCBs CONCENTRATIONS (ng/g ON FRESH WEIGHT BASIS) AND RESIDUAL AMOUNTS (J-tg) IN ORGANS AND TISSUES OF AN ADULT MALE (SPECIMEN No.2) Organs and tissues Lipid content (%) l'ddt PCBs l'ddt PCBs (ng/g) (ng/g) (J-tg) (%) (J-tg) (%) Blubber front center rear mean ,700 (98.3) 4370 (97.5) Muscle dorsal front ventral front dorsal center ventral center dorsal rear ventral rear mean (1.24) 93.6 (2.09) Liver (0.11) 7.7 (0.16) Kidneys «0.01) 0.3 «0.01) Heart (0.03) 1.4 (0.03) Lungs ( <0.01) 0.3 ( <0.01) Spleen (0.03) 1.5 (0.03) Stomach ( <0.01) 0.1 «0.01) Intestine (0.07) 1.8 (0.04) Diaphragm ( <0.01) 0.3 ( <0.01) Cerebrum ( <0.01) 0.1 «0.01) Blood (0.02) 1.1 (0.02) Penis «0.01) 0.1 «0.01) Bone ribs tibia total 29.6 (0.15) 6.2 (0.14) Total burdens 20,000 (100) 4480 (100) Figures in parentheses indicate the percentage ofresidual amounts ofl'ddt and PCBs in organs and tissues to total burdens. and tissues in the adult and newborn males were 84.4 and 56.7% of the body weight respectively. The highest concentration of.eddt and PCBs was found in blubber where the lipid content was also the highest. The variation of lipid content and, concentrations of.eddt and PCBs in three areas of blubber were small. However, the concentration of these chemicals in muscle varied and was much lower than that in blubber. Measurable concentrations of.eddt and PCBs were also found in other organs and tissues. The heart and liver in the adult male ranked next to the blubber in regard to the concentration on a fresh weight basis. The skeletal bones in the adult male were separated into 22 parts, in which the tibia and ribs as representatives of the respective. bones with high and low lipid contents were subjected to chemical analysis. The concentration level of.eddt and PCBs in organs and tissues depends roughly on their lipid contents as known in many other cases. 8,9) The whole-body burdens of.eddt and PCBs were obtained by multiplying the weight of organs and tissues by the concentration of chemicals in them. In the case of the adult male, more than 97% of the total residual amounts of either.eddt and PCBs was retained in his blubber. On the other hand, the

5 2012 H. HIDAKA, S. TANABE and R. TATSUKAWA TABLE III. l:ddt AND PCBs CONCENTRATIONS (ng/g ON FRESH WEIGHT BAStS) AND RESIDUAL AMOUNTS (J.lg) IN THE ORGANS AND TISSUES OF A NEWBORN MALE (SPECIMEN No.3) Organs and tissues l:ddt PCBs l:ddt PCBs (ng/g) (ng/g) (J.lg) (%) (J.lg) (%) Bl~bber front center rear mean (84.7) 14.7 (87.8) Muscle dorsal front ventral front dorsal center ventral center dorsal rear ventral rear mean (14.7) 2.04 (12.2) Liver 0.27 < (0.22) 0.01 (0.03) Kidneys 0.10 < (0.03) Heart (0.13) < 0.01 (0.02) Lungs 0.20 < (0.12) Spleen 0.13 < (0.03) Cerebrum 0.21 < (0.07) Total burdens 94.3 (100) 16.7 (100) Figures in parentheses indicate the percentage ofresidual amounts ofl:ddt and PCBs inorgans and tissues to total burdens. Lipid contents in blubber and muscle were 29.6 and 1.9%, respectively. newborn male contained somewhat smaller amounts of 17DDT (85%) and PCBs (88%) in its blubber, which seems to be due to both the comparatively low percentage of blubber weight to body weight (Table I) and the smaller amount of lipid contents (29.6%) in the blubber. The residual amounts of17ddt and PCBs in the other organs and tissues analyzed were extremely small in both specimens, and those in the non-analyzed organs and tissues can be expected to be negligible in terms of wholebody burden, because the weights and lipid contents of these organs and tissues are very small. For the adult and newborn males, the residual amounts ofeither 17DDT and PCBs contained in both the blubber and muscle were more than 99% of the total burdens. Therefore, the sum of the residues in both blubber and muscle can be considered as a whole;.body burden in these animals. Figure 1 shows. the composition of individual chlorobiphenyls in the blubber of the adult and newborn males. The PCBs found in both seals consisted ofmany compounds mostly with five and six chlorine atoms. The PCBs in the newborn male, however, were composed ofslightly higher amounts oflower chlorinated biphenyls than those in the adult male. A similar observation has been reported in the PCB compositions of mother and her fetus of the striped dolphin,!!) in which the lower chlorinated biphenyls in the mother transferred more easily to her fetus. The significant amount of lower chlorinated biphenyls in the newborn Weddell seal might have been transferred from its mother through transplacental action. Figure 2 shows the PCB compositions in sea water, fish and Weddell seal (adult male) in the Antarctic with respect to their chlorine numbers. The major components in sea water were lower chlorinated biphenyls with two and three chlorine atoms. The fish which ingest PCBs from both foods and sea water, contain-

6 DDT and PCB in Weddell Seals 2013 ed not only penta- and hexa-chlorobiphenyls but/also a considerable amount of lower chlorinated biphenyls. The PCBs in Weddell seal consisted mainly of higher chlorinated biphenyls with more than five chlorine atoms and a much lower amount of lower chlorinated biphenyls, indicating that lower chlori- ~ o -.-1.j.J C'j ~.j.j ~ Q) ~ 1.0 o u Q) :> '.-1.j.J C'j..--1 ~ 0.5 Adult Newborn '}! 3! i C1 Number C1 content:58.3 % C1 content:57.2 % FIG. 1. PCB Isomer and Congener Compositions in the Blubber of Adult (Specimen No.2) and Newborn (No.3) Males of Weddell Seal. Each bar shows the relative abundance of individual PCB compounds. Details of each peak and its chemical structure are given in a previous report. 10 ) nated biph~nyls were actively metabolized in Weddell seal. The whole-body concetrations and burdens of l7ddt and PCBs were estimated for all specimens and are given in Table IV. The concentrations of EDDT and PCBs in the newborn samples were found to be approximately one order of magnitude lower than those in the adult seals. The observation at the time of sampling indicated that the newborn (No.3) seemed not to have been lactated and other two newborns (Nos. 4 and 5) were little lactated, ifat all. So the transplacental transfer rate of chlorinated hydrocarbons in the Weddell seal was estimated to be less than 2% of the mother's burden, assuming that the newborns (Nos. 3, 4 and 5) were born of the adult female (No.1). It has been reported that the transplacental transfer rates of chlorinated hydrocarbons are 4.7% for DDT compounds and 4.0% for PCBs in mother striped dolphin,!!) and less than 1% for PCBs in TABLE IV. CONCENTRATIONS (ng/g ON WHOLE BODY BASIS) AND BURDENS (Jig) OF EDDT AND PCBs IN WEDDELL SEALS Specimen LDDT PCBs No. Sex Conc. Burden Conc. Burden 1 F M , M F F Sea Water Fish (T.bernacchii) Weddell Seal a, , % FIG. 2. Chlorobiphenyl Compositions (%) of PCBs in the Antarctic Marine Ecosystem. All samples were collected at Tottuki point near Syowa Station from July to December Sea water and fish data were documented by Tanabe et al. 12 )and Subramanian et al.,13) respectively. The PCB composition in Weddell seal is in the blubber of adult male.

7 2014 H. HIDAKA, S. TANABE and R. TATSUKAWA humans. 14 ) Characteristic bioaccumulation of I:DDT and PCBs in antarctic marine ecosystems The DDT and PCB concentrations in the blubber of pinnipeds and small cetaceans so far published are summarized in Table V. All the specimens presented here are adult male in order to exclude variations due to parturitional and lactational losses of DDTs and PCBs. The concentrations of DDTs and PCBs in marine mammals living in the oceans of TABLE v. MEAN CONCENTRATIONS OF DDTs AND PCBs IN THE BLUBBER OF PINNIPEDS AND SMALL CETACEANS ON A FRESH-WEIGHT BASIS Area Species n DDTs (Jlgjg) PCBs (Jlgjg) Ref. Arctic Canadian Ringed seal ) Northern Atlantic Newfoundland Harp seal ) Gulf of St. Lawrence Harp seal ) Nova Scotia Atlantic white-sided dolphin ) Rhode Island Striped dolphin ) Caribbean Long-snouted dolphin ) Northern Pacific California Common dolphin ) Hawaii Rough-toothed dolphin ) Northwestern Pacific Striped dolphin ) Striped dolphin ) Finless porpoise ) Common dolphin ) Eastern Tropical Pacific Fraser's dolphin ) Striped dolphin ) Southern Atlantic Uruguay Franciscana dolphin ) Antarctic Weddell seal ) Weddell seal Present study All specimens are adult male. TABLE VI. CONCENTRATIONS (Jlgjliter OR ngjg) AND CONCENTRATION RATIOS OF l'ddt AND PCBs IN THE MARINE FOOD CHAINS OF THE ANTARCTIC AND NORTHWESTERN PACIFIC OCEANS Antarctic Northwestern Pacific l'ddt PCBs l'ddt PCBs Sea water Cone. 1.3 x X 10-5 Sea water Cone. 7.5 x X 10-4 (under fast ice) Krill Cone. 2.6 x X 10-2 Zooplankton Cone x 10-1 CR (OjW) 2.0 x X 10 2 CR (OjW) 2.5 x X 10 3 Squid Cone. 2.2 x x 10 CR (OjW) 2.9 x lw 2.3 X 105 Benthic fish Cone. 6.5 x X 10-1 Myctophid Cone. 4.4 x x 10 (T. bernacchii) CR (OjW) 5.0 x X 10 3 CR (OjW) 5.8 x X 105 Weddell seal Cone. 5.2 x x 10 Striped Cone. 6.0 x X 10 3 CR (OjW) 4.0 x X 105 dolphin CR (OjW) 7.9 x X 10 7 CR (MjF) 8.0 x x 10 CR (MjF) 1.4 x x 10 CR (OjW) = Cone. in organismjconc. in water, CR (MjF) = Cone. in mammaljconc. in fish. The data excluding those of Weddell seals are cited from our previous reports.l )

8 DDT and PCB in Weddell Seals 2015 middle latitudes of the northern hemisphere are the highest, which reflects the extensive production and consumption of these chemicals in the developed countries of this area to date. In contrast, much lower concentrations were found in arctic seals and even lower levels were obtained from Weddell seals living around the antarctic fast ice. Concentrations of l'ddt and PCBs in antarctic Weddell seals were found to be two or three orders of magnitude lower than those in marine mammals from the middle latitudes ofthe northern hemisphere. To make clear the reason why the concentrations of L'DDT and PCBs in antarctic Weddell seals are so low, the ecosystem dynamics of the organochlorines will be discussed. Table VI shows the concentrations of L'DDT and PCBs in' several marine organisms and ambient water in the Antarctic and Northwestern Pacific oceans, and also presents the concentration ratios (CRs) of L'DDT and PCBs in organisms to those in ambient water [CR (O/W)] and of these chemicals in mammals to those in fish [CR (M/F)]. Much lower concentrations of L'DDT and PCBs were found not only in Weddell seal but also in other antarctic organisms and ambient water compared with those in the Northwestern Pacific. 0ritsland 22 ) has reported that the estimated daily food of the Weddell seal consists of 53% fish, 11 % cephalopods and 36% of other invertebrates. When assuming that benthic fish (Trematomus bernacchii) is representative of both fish and cephalopods, and krill of the invertebrates, the concentrations of L'DDT and PCBs in the foods of Weddell seal were estimated to be 0.43 and 0.15ng/g on a fresh weight basis respectively. On the other hand, striped dolphins feed mainly on squid. Consequently, the CRs between the Weddell seal and their foods (120 for L'DDT and 78 for PCBs) were nearly the same as those for the striped dolphin (270 for L'DDT and 64 for PCBs). This evidence strongly suggests that the low concentrations of L'DDTand PCBs in antarctic Weddell seals are attributable to the much lower concentration in their foods. The CRs (O/W) of L'DDT and PCBs between fish or plankton and ambient water vary with the two oceans, whereas this CRs (M/F) are almost the same in both oceans. The CRs (O/W) of L'DDT in the antarctic organisms were one order ofmagnitude higher than those in the Northwestern Pacific. This may be explained as follows: (1) Antarctic zooplankton and fish contain a larger amount of lipids in their bodies, and/or (2) Antarctic fish con- sume a larger amount of oxgen compared with fish elsewhere,23) and therefore, the uptake of l'ddt from water through the gill membrane rnay be larger. On the contrary, the CRs (O/W) of PCBs in the antarctic organisms were lower than those in the Northwestern Pacific. It has already been reported that the chlorine content of PCBs in antarctic water under fast ice (37.9%)12) is' markedly smaller than that in northwestern Pacific water (46.3%).21) The lower chlorinated biphenyls are more biodegradable and less bioaccumulative in higher animals. 10,21,24) Considering all these facts, the low chlorine content in antarctic water under fast ice may be responsible for the low CR values of PCBs in antarctic fish and zooplankton. Global transport of DDTs and PCBs to the Antarctic Based upon the global surveillance conducted by the author's laboratory, a conceptual scheme for the global transport of DDTsand PCBs, with special reference to the antarctic ecosystem, is shown in Fig. 3. DDTs andpcbs discharged into theterrestrial and surrounding environment are transported mainly through the atmosphere to the open ocean. Atmospheric DDTs and PCBs over the open ocean are present mostly in the gas phase, and enter the sea water by gas exchange at the sea/water interface. Since the Antarctic is covered with ice and snow almost all the year around, the input of DDTs and PCBs into sea water is blocked by the thick fast ice. During the short austral summer, the melting snow and ice act as a medium for supplying DDTs

9 2016 H. HIDAKA, S. TANABE and R. TATSUKAWA ',' Fast Ice ". -, -.",' :...:. Open Ocean... : : ::.....:~i(.:.~~ ad tjdo tjo q Antarctica FIG ~ ~ Conceptual Drawing of the Global Transport of DDTs and PCBs to the Antarctic. and PCBs into the antarctic sea water under fast ice. We have noted in a previous paperthat the concentrations of DDTs and higher chlorinated biphenyls in sea water under fast ice were evidently lower than those in surface water from the outer margin of pack ice. 12 ) DDTs and higher chlorinated biphenyls with very low water solubility are easily adsorbed onto suspended materials and readily removed from the sea surface by the sinking of these particles. The removal rates of these chemicals depend on the primary productivity in the oceans. 25 ) Since the primary productivity of water under fast ice in the antarctic ocean is several times higher than that of the outer margin of pack ice,26) an active downward transport of DDTs and higher chlorinated biphenyls can be strongly expected in the antarctic water around the fast ice area. Such a specific process seems to have led to the low concentrations of DDTs and PCBs in sea water under fast ice, and subsequently caused the low accumulation.level of these chemicals in the antarctic marine ecosystem under fast ice. The atmospheric concentrations of DDTs and PCBsin the Antarctic are very low,27) and this too is one of the factors affecting the low levels of DDTs and PCBs in the antarctic hydrosphere and biosphere. It may be most likely, however, that the active removal of these chemicals accompanied by the precipitation of suspended materials is a critical factor. Acknowledgments. Weddell seals and other antarctic materials were collected under a project of the 22nd Japanese Antarctic Research Expedition (JARE-22). The authors are grateful to Professor Y. Yoshida, leader of JARE-22, and Dr. K. Kaminuma, subleader of JARE-22, both from National Institute of Polar Research, for their kind arrrangements and encouragement in this survey. We wish to thank Mr. N. Kurihara, Radio Research Laboratories, and Mr. R. Sakai, National Institute of Polar Research, for their devoted support in sampling, and also other members of JARE-22 for their cooperation. Many thanks are due to the officers and crew of the Icebreaker Fuji for their assistance on board. The authors are indebted to Dr. Y. Naito, National Institute of Polar Research, for the age determination of adult Weddell seals. We are pleased to acknowledge the kind support of Professor T. Hoshiai, National Institute of Polar Research, and Professor T. Torii, Chiba Institute of Technology, for our participation in this project. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (project No ).

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