2043

Environmental Toxicology and Chemistry, Vol. 15, No. 11, pp. 2043–2048, 1996Printed in the USA

0730-7268/96 $6.00 1 .00

CHARACTERISTICS OF BUTYLTIN ACCUMULATION AND ITS BIOMAGNIFICATION INSTELLER SEA LION (EUMETOPIAS JUBATUS)

GI BEUM KIM,† SHINSUKE TANABE,*† RYO TATSUKAWA,† THOMAS R. LOUGHLIN‡ and KENJI SHIMAZAKI§†Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan

‡National Marine Mammal Laboratory, National Marine Fisheries Service, 7600 Sand Point Way NE, Seattle, Washington 98115, USA§Research Institute of North Pacific Fisheries, Hokkaido University, Minatocho 3-1-1, Hakodate 041, Japan

(Received 9 September 1995; Accepted 10 May 1996)

Abstract—The present study was conducted to examine sex difference, age, and temporal trends of butyltin accumulation and itsbiomagnification in Steller sea lion (Eumetopias jubatus) collected from Alaska, USA, during 1976–1985 and from Hokkaido, Japan,during 1994–1995. Average concentration of total butyltin compounds (SBTs) in the liver of Steller sea lion from Alaska (19 ng/gwet weight) was much lower than those from western and eastern Hokkaido, Japan (150 and 220 ng/g), respectively. This resultsuggests that Japanese coastal waters are contaminated with BTs in comparison with those of Alaska. In most samples, dibutyltin(DBT) residues were retained at higher levels than tributyltin (TBT), suggesting the degradation of TBT to DBT in the liver. Sexdifference and age-dependent accumulation of BTs residues were not found in Steller sea lion. Similarly, no prominent temporal trendin BT concentrations was observed between 1976 and 1985. Nevertheless, the annual consumption of organotin compound was doubledin the United States during the same period. These results suggest that the butyltin compounds are degraded faster than the intakefrom diet in Steller sea lion. The biomagnification factor of BTs in Steller sea lion was low (0.15–4.6; mean, 0.6), indicating that thisanimal is unlikely to magnify BTs due to rapid degradation and excretion.

Keywords—Butyltin Steller sea lions Age trend Temporal trend Biomagnification

INTRODUCTION

Over the past three decades, tributyltin (TBT) has been usedextensively as an antifouling paint applied on vessels and marinestructures because of its 10 to 20 times higher efficiency overcopper-based antifoulant and also as a weedicide, disinfectant,and biocide for cooling systems. This compound has causeddamage to a wide range of nontarget organisms even at verylow levels, including shell thickening and spat failure in oysters[1,2], imposex in neogastropods and gastropods [3–5], andgrowth retardation in mussels [6]. Due to accelerating ecosystemdegradation caused by TBT, some countries have imposed par-tial regulations on its use. However, TBT inputs from shipslarger than 25 m in length and wastewaters still persist. Inaddition, many countries in the developing world have not im-posed such controls yet. Because of the high toxicity of TBTto some aquatic organisms, accumulation of butyltin compounds(BTs) has been studied extensively in organisms at various tro-phic levels in marine ecosystems, including invertebrates [1–9]and fish [10–15]. Marine mammals, however, have been scarce-ly studied for BT accumulation and effects. Cetaceans and pin-nipeds are known to have reduced capacity to decompose hy-drophobic persistent chemicals such as polychlorinated biphen-yls (PCBs) and dichlorodiphenyltrichloroethane (DDT) andtherefore accumulate high levels of these compounds throughthe food chain [16–18]. The octanol/water partition coefficientof TBT was reported to be in the range of 5,500 to 7,000 [19],suggesting a moderate potential for bioaccumulation [20]. Thesefacts prompted us to examine the occurrence of BT residues inmarine mammals. As expected, Iwata et al. [21] first detectedBTs in the blubber of some cetaceans from Japanese coastalwaters and open seas and also found a very high concentration

* To whom correspondence may be addressed.

of BTs (up to 10 mg/g wet weight) in the liver of a finlessporpoise [22]. Kim et al. [23] further noted that the major portionof the BTs accumulated in the liver of Steller sea lion, reflectingtheir metal-like nature to bind to protein, in contrast to or-ganochlorine compounds, which accumulate in the blubber dueto a high affinity for lipids. However, fundamental studies de-scribing the specific accumulation of BTs according to sex, age,etc., have not been conducted in the higher trophic animals.

To understand the specific accumulation of BTs in higheranimals, the present study determined their concentration in theliver of Steller sea lion collected from Alaska, USA, and Hok-kaido, Japan, and described their pattern with sex, age, temporal,and geographic factors as well as the status of biomagnification.

MATERIALS AND METHODS

Samples

Male and female Steller sea lions (Eumetopias jubatus) werecollected from Alaska, USA, during 1976–1985 and from Hok-kaido, Japan during 1994–1995 (Fig. 1). The age of the Stellersea lions was determined by counting growth layer groups inthe dentine of their teeth following the procedure described byPitcher and Calkins [24]. Length, age, and sex of the samplesare shown in Table 1. Liver was used for chemical analysis. Todetermine the biomagnification of BTs in Steller sea lion fromHokkaido, fish samples collected from the same area were alsoanalyzed. Fish samples used in this study consisted of the fol-lowing species: greenling (Hexagrammos lagocephalus), Pacificherring (Clupea pallasi), Pacific cod (Gadus macrocephalus),poacher (Podothecus sachi), rockfish (Sebastes schlegeli), saf-fron cod (Eleginus gracilis), salmon (Oncorhynchus masou ma-sou), scorpion fish (Sebastolobus macrochir), sculpin (Myox-ocephalus polyacanthocephalus), smelt (Hypomesus pretiosusjaponicas), and a squid (Loliginidae). The whole body of fish

2044 Environ. Toxicol. Chem. 15, 1996 G.B. Kim et al.

Fig. 1. Sampling area of Steller sea lion used in this study.

Table 1. Biometry of samples used in this study

Species Sampling site Sex nLength(cm)

Age(years)

Marine mammalSteller sea lion Eastern Hokkaido, Japan

Western Hokkaido, Japan

Alaska, USA

MFMFMF

2541

1721

210–223247–280142–300

120170–300197–239

4.5, NINININI0–132–25

FishGreenlingPacific herringPacific codPoacherRockfishSaffron codSalmonScorpionfishSculpinSmelt

Hokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal watersHokkaido coastal waters

121113112

11

39.025–25.541.438.229.0

23.2–25.340.024.5

27.0–28.214.0–16.6

CephalopodSquid Hokkaido coastal waters 1 NI

NI 5 not identified.

and the cephalopod was homogenized and subjected to chemicalanalysis. The list of fish samples is given in Table 1. Amongthese fish, six species fell under the top 10 prey items of Stellersea lions collected in the Gulf of Alaska [25]. After collection,all samples were kept at 2208C in a deep-freezer until analysis.

Chemical analysis

The chemical analysis of BTs followed the method describedby Iwata et al. [21] with some modifications [23]. About 1.5 gof organ or tissue was homogenized with 35 ml of 0.1% tro-polone/acetone and 5 ml of 2 N HCl. The homogenate wascentrifuged at 3,000 rpm, and the supernatant was transferredto 0.1% tropolone/benzene. The moisture was removed with 35g of anhydrous Na2SO4, concentrated to near dryness using arotary evaporator (408C), and made up to 5 ml with benzene.Butyltin compounds in the extract were propylated by adding5 ml of n-propyl magnesium bromide (2 mol/L in tetrahydro-furan (THF) solution, Tokyo Kasei Kogyo Co. Ltd., Tokyo,

Japan) as the Grignard reagent, and the mixture was shaken at408C for 30 min. The excess Grignard reagent was destroyedwith 20 ml of 1 M H2SO4, and the propylated mixture wastransferred to 20 ml of 10% benzene/hexane and 40 ml of hex-ane-washed water using 10 ml of methanol. The extract wasconcentrated to near dryness and made up to 5 ml with hexane.The solution was added on a glass column packed with 20 gof dry Florisil, and then nitrogen was passed gently throughthis column for 3 h. Butyltin compounds adsorbed on Florisilwere eluted with 150 ml of 20% acetonitrile/water to removefat. The eluate was collected in a separatory funnel containing100 ml of 10% benzene/hexane and 600 ml of hexane-washedwater, and the BTs were transferred to benzene/hexane, con-centrated to 5 ml, and then passed through a 6-g activated Flor-isil-packed wet column for final cleanup using 40 ml of hexane.The final hexane extract was concentrated to 5 ml and subjectedto quantification with gas chromatography.

A gas chromatograph with a flame photometric detector

Butyltin accumulation and biomagnification in Steller sea lion Environ. Toxicol. Chem. 15, 1996 2045

Table 2. Mean and range of concentrations (cation ng/g wet weight) of butyltin compounds in liverof Steller sea lions from Hokkaido and Alaska

Sampling site Sex n MBT DBT TBT BT

Eastern Hokkaido, Japan M

F

M 1 F

2

5

7

82(63–100)a

93(52–130)

90(52–130)

110(89–130)

100(51–140)

110(51–140)

18(16–20)

23(16–31)

21(16–31)

210(170–250)

220(180–300)

220(170–300)

Western Hokkaido, Japan M

FM 1 F

4

15

58(28–110)

6760

(28–110)

68(39–130)

10075

(39–130)

8(6–10)3413(6–34)

130(75–250)

200150

(75–250)

Alaska, USA M

F

M 1 F

17

21

38

5.3(ld–5.5)

7.1(ld–7.1)

5.9(ld–7.1)

7.2(ld–14)

7.6(ld–20)

7.5(ld–20)

3.3(1.9–5.1)

3.5(2.1–5.6)

3.4(1.9–5.6)

16(1.9–22)

18(2.2–24)

17(1.9–24)

a Figures in parentheses indicate the range of BT concentrations.ld 5 Less than the detection limit (see Materials and Methods).

(Hewlett-Packard 5890 Series II), a moving needle-type injec-tion system, and a tin mode filter (610 nm) was used for quan-tification. Monobutyltin (MBT), dibutyltin (DBT), and TBT ofknown amounts (0.2 mg/ml) were spiked into whale liver (minkewhale from the Antarctic Ocean containing undetectable levelsof BT residues), passed through the whole analytical procedure,and used as an external standard. Concentrations were estimatedby comparing peak heights of butyltins in samples with thosein external standard. Hexyltributyltin was used as an internalstandard to check recovery. The reproducibility of MBT, DBT,and TBT was examined by spiking the liver of Antarctic minkewhale. Recoveries of TBT, DBT, and MBT were 110 6 9, 986 9, and 83 6 13%, respectively (n 5 5). Detection limits ofTBT, DBT, and MBT were less than 1, 3.3 to 5.5, and 4.9 to16 ng/g, respectively.

RESULTS AND DISCUSSION

Status of BT contamination

Tributyltin was detected in all the liver samples, ranging from1.9 to 5.6 ng/g wet weight in Steller sea lion collected fromAlaska. Dibutyltin concentrations ranged from less than the de-tection limit (,3.3;,5.5) to 20 ng/g wet weight, and MBTfrom less than the detection limit (,4.9;,16) to 7.1 ng/g wetwt (Table 2). Among 38 samples analyzed, DBT and MBT weredetected in 34 and 4 samples, respectively. Excluding the sam-ples with undetectable DBT and MBT, mean concentrations ofTBT, DBT, and MBT were 3.4, 7.5, and 5.9 ng/g, respectively,with a SBT concentration of 17 ng/g. Although the mean MBTand DBT concentrations might be an overestimate of the realvalue (due to the exclusion of nondetects for estimating meanvalues) in Steller sea lion from Alaska, the mean SBT concen-tration was significantly lower (p , 0.001, Mann-Whitney Utest) than those from Hokkaido, where animals from the easternand western parts had SBT concentrations of 220 and 150 ng/g,respectively (Table 2). The reason for the differences in theresidue levels of SBT between Hokkaido and Alaska Steller sealions is unclear. However, the possible explanation might be laidon the greater usage of TBT and the greater number of pollutionsources (e.g., harbors) in Hokkaido coastal waters than in theAlaskan waters. In fact, the BT level in bivalve mollusks in

Alaska was the lowest among the East and West coasts of theUnited States [8], suggesting that Alaska is relatively cleanercompared to other regions of the United States. On the contrary,Horiguchi et al. [9] reported that the rate of occurrence of im-posex in Thais clavigera and T. bronni was 100% from mostlocations surveyed (32 sites) in Japan. This report indicated thesignificant BT contamination throughout Japan, although no BTresidue data are available for Hokkaido coastal waters. There-fore, the variation in the contamination status of BTs in Stellersea lion between two sampling sites seems to be due to thegeographic gap of their environmental contamination.

In Hokkaido samples, the spatial variation in SBT concen-tration between western and eastern parts was not so significant,although Steller sea lions collected in each area were consideredto belong to different breeding groups (one group comes fromSakhalin and the other from the Kuril islands) [26]. This ob-servation suggests that the coastal region of Hokkaido has arelatively uniform contamination status of BTs.

To our knowledge, only one report has been published on theoccurrence of BT residues in the liver of marine mammals otherthan Steller sea lion. This report noted that finless porpoise(Neophocaena phocaenoides) from Japanese coastal waters hadSBT concentrations ranging from 1.1 to 10 mg/g in the liver[22]. Compared to this value, Steller sea lion showed one tothree orders of magnitude lower SBT concentration. Finlessporpoise was collected from highly industrialized coastal wa-ters, whereas Steller sea lion was from less contaminated coastalwaters. Such a situation might be a plausible explanation forlower SBT levels in Steller sea lion. In addition, the excretionof BTs through shedding of hair might also be considered. Inan earlier study, Kim et al. [23] found that about 25% of thewhole-body burden of BTs was excreted through the sheddingof hair in Steller sea lion. Therefore, this should also be con-sidered for the low accumulation of BTs in Steller sea lion whencompared with finless porpoise, which have no body hair.

Total butyltin compound concentrations in the whole bodyof fish and a cephalopod from Hokkaido coastal waters areshown in Table 3 and were compared with those in fish fromother regions in Japan. In Hokkaido, sculpin retained the lowestvalue of SBTs (6.3 ng/g), and Pacific herring had the highest

2046 Environ. Toxicol. Chem. 15, 1996 G.B. Kim et al.

Table 3. Butyltin compound concentrations (cation ng/g wet weight) in fish andsquid collected from Japanese coastal waters

Speciesn

Totaln MBT DBT TBT BTs

Bay or inshorea

Offshorea

Tokyo Bayb

Hokkaido(this study)

28

14

18

11

70

36

164

25

NA

NA

11(,1–32)

5.3(2.9–9.9)

NA

NA

20(5–35)

8.1(1.1–37)

82(ND–380)c

52(ND–190)

90(6–420)

36(1.3–160)

120(11–470)

49(6.3–200)

a [11].b [12].c Figures in parentheses indicate the range of BT concentrations.

NA 5 not analyzed; ND 5 not detected.

Fig. 2. Concentration of DBT plus TBT according to age in the liverof Steller sea lion. Only the samples with detectable levels of bothDBT and TBT are given in this figure (V 5 female; v 5 male).

value (200 ng/g). Average SBT concentration was 49 ng/g.Among the data reported so far, fish samples from Hokkaidocoast were ranked as the group with lower SBT residue levels,indicating that Hokkaido coastal waters are relatively pristinein terms of BT pollution in Japan.

BT composition

Dibutyltin was the dominant component in the liver of Stellersea lion, followed by MBT and TBT (Table 2). The relativelyhigher proportion of TBT in Alaska than in Hokkaido might beexplained by the fact that the Alaskan samples were collectedprior to the restriction on the use of TBT imposed in 1987 [27],whereas Hokkaido samples were collected in 1994 and 1995,when the import and usage of TBT had already been regulated[28]. In general, many reports have shown that TBT residuelevels in fish were higher than those of DBT and MBT, irre-spective of the species and sampling sites of fish in Japan[10,12,29–31]. Even in an in vivo study, Tsuda et al. [32] re-ported that in carp (Cyprinus carpio), the concentration of TBTin muscle was 10 times higher than concentrations of its me-tabolites. In addition, the present study showed that most of thefish samples analyzed also had a high proportion of TBT (Table3). On the other hand, the highest proportion of BTs in the liverof Steller sea lion was DBT, followed by MBT and TBT (Table2). This pattern was the same in Alaskan and Hokkaido samplesregardless of a large difference in SBT concentration. Pitcher[25] examined the stomach contents of 250 Steller sea lionscollected from the Gulf of Alaska over the year and elucidatedthat fish made up 95.7% of their diet. These observations implythat a part of TBT residues absorbed into the body of Stellersea lion through feeding on fish is transformed metabolicallyinto DBT and MBT in the liver. Higher proportions of DBTand MBT than TBT have been found not only in Steller sealion but also in finless porpoise [22]. These results indicate thatmarine mammals possess the metabolic capacity to degrade TBTin the liver.

Variation of BT concentration according to age, sex, andtemporal factors

In the case of organochlorines such as PCBs and DDTs, age-dependent accumulation has been well demonstrated in malemarine mammals, while decreasing concentrations are rathercommon in adult females due to the lactational transfer of thesepersistent lipophilic contaminants in large quantities[16,17,33,34]. In fact, Steller sea lion from Alaska analyzed inthis study also revealed the age-dependent accumulation of

PCBs and DDTs in male, with an apparent male/female differ-ence in the residue levels [35]. On the contrary, no age-depen-dent accumulation of BT residues was noticed in both the sexesof Steller sea lion, and no significant difference between BTlevels was observed between sexes (Table 2 and Fig. 2). Al-though female Steller sea lions reach sexual maturity at about4.5 years [24,36], BT concentrations were constant regardlessof age. These observations suggest that BTs are transferred lessthrough gestation/lactation from mother to fetus/pup in Stellersea lion, and probably also in other marine mammals.

The mean concentration of DBT plus TBT in Steller sea lioncollected from Alaska in 1985 was 11 6 6.1 ng/g, which wascomparable to the mean value of 11 6 3.7 ng/g in 1976–1977(Fig. 3a). Since TBT compounds were first registered in theUnited States in the early 1960s, the number of these formu-lations has increased to 364, and their consumption has alsoincreased gradually from 1965 to 1986 (Fig. 3b) [37]. However,the increasing trend of DBT plus TBT concentrations from 1976to 1985 was not found in Steller sea lion from Alaska (Fig. 3a).Careful consideration of the present results is needed becauseof much lower levels of BT concentration in Steller sea lion;however, it may remain a possibility that such a temporal trendof BT accumulation might be attributed to the high metaboliccapacity of Steller sea lion to degrade BTs in their liver, asexplained based on BT composition. In addition, there is another

Butyltin accumulation and biomagnification in Steller sea lion Environ. Toxicol. Chem. 15, 1996 2047

Fig. 3. (a) Temporal trend of BT concentrations (DBT plus TBT) in the liver of Steller sea lions from 1976 to 1985. Only the samples withdetectable levels of both DBT and TBT are given in this figure V 5 individual data; v 5 mean; — 5 standard deviation). (b) Annual consumptionof alkyltin compounds in the United States. [37].

Table 4. Biomagnification factor of BTs based on a whole-bodybasis in Steller sea lions and fish from Hokkaido, Japan

Species Concn. (cation ng/g) BMF

Steller sea lionsFish (11 species)b

2949 (6.3–200)

0.59 (0.15–4.6)a

a Figures in parentheses indicate the range of BMFs and BT concen-trations.

b Including squid.

possibility that the temporal pattern on the usage of TBT inAlaska might be different from general U.S. statistics.

Biomagnification of BT in Steller sea lion

In order to estimate the biomagnification factor (BMF) of BTsin Steller sea lion from Hokkaido, 11 species of fish collectedfrom the same area were analyzed. Based on the results of aprevious study estimating SBT concentrations in several tissues/organs of Steller sea lion [23], SBT concentration on a whole-body weight basis of Steller sea lion was calculated to be 29ng/g. Fish showed a range of SBT concentrations on a whole-body basis of 6.3 to 200 ng/g and a mean of 49 ng/g (Table 4).Comparing the above SBT concentration in Steller sea lion withthose in fish, the BMF was estimated to be between 0.15 and4.6 (mean, 0.59). This value was much lower than those oforganochlorine compounds in marine mammals [35,38–40], in-dicating a great deal less biomagnification potential of BTs inthe body of higher trophic animals.

In addition, in order to understand the accumulation rate ofBTs in the body of Steller sea lion, the amount of BT intakethrough feeding was estimated by the following equation:

4.5

I 5 (A 3 B 3 C 3 365 3 t) dtt E0

where

It 5 the total amount of BT intake (mg),A 5 the growth rate of Steller sea lion (62 kg/year) (calculated

from the slope of the relationship between body weightand age of Steller sea lion analyzed in this study),

B 5 the daily consumption of prey per body weight (extrap-olated from a value of 9.4% that was reported for a 2-year-old Steller sea lion [41]),

C 5 the mean concentration (49 mg/kg) of SBTs in prey col-lected from Hokkaido (Table 4), and

t 5 age (years).

Using these values, the cumulative amount (It) of BT intake fora 4.5-year-old Steller sea lion was estimated to be 11 3 105 mg.In fact, the whole-body burden of BTs in the same species (4.5years) from Hokkaido was 6 3 103 mg [23]. Considering thesevalues, it is likely that Steller sea lions accumulate less than1% of the total amount of BT intake. This result suggests thatmost BT intake through feeding was rapidly metabolized andthen excreted from their bodies, which is also supported by thecomposition pattern and the temporal trend of BTs in the liverof Steller sea lion. The shedding of hair may also be part ofthe excretory route of BTs in Steller sea lion. Such excretionof BTs was also noted in birds during moulting [42]. Low ac-cumulation of BTs in Steller sea lion was also supported by thestudy of Evans et al. [43], which indicated that more than 90%of BT intake was excreted through feces and urine. On thecontrary, excreted organochlorine compounds were only 9 to32% of the total intake in humans [44]. These observations,including the results of the present study, suggest that unlikeorganochlorines, BTs are largely excreted from the body ofSteller sea lion and thus accumulated less in this animal. Al-though certain aspects, such as absorption efficiency of BTs,exact intake rate and diet, etc., are not followed in the presentestimation, it can be safely concluded that BTs are less bioac-cumulative in higher trophic organisms.

Acknowledgement—The authors wish to acknowledge K. Kannan forcritical reading of this manuscript, D. Calkins for providing most ofthe Alaskan samples, and B. Robson for logistical support. This researchwas supported by a Grant-in-Aid from the Scientific Research Program(Projects 06405006, 06454097, and 07660245) of the Ministry of Ed-ucation, Science, and Culture of Japan. This study was financed in partby a scholarship from the Ministry of Education, Korea, awarded toG.B. Kim.

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