Accumulation of Butyltin Compounds in Risso's Dolphin (Grampus

PBDEs, methoxylated PBDEs and HBCDs in Japanese common squid (Todarodes pacificus) from Korean offshore waters. Gi Beum Kim , Heather M. ... Risso's d...
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Environ. Sci. Technol. 1996, 30, 2620-2625

Accumulation of Butyltin Compounds in Risso’s Dolphin (Grampus griseus) from the Pacific Coast of Japan: Comparison with Organochlorine Residue Pattern G . B . K I M , † S . T A N A B E , * ,† R . I W A K I R I , † R. TATSUKAWA,† M. AMANO,‡ N. MIYAZAKI,‡ AND H. TANAKA§ Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan, Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo, Akahama, Otsuchi-cho, Iwate 028-11, Japan, and National Research Institute of Far Seas Fisheries, Orido 5-7-1, Shimizu 424, Japan

Concentrations of butyltins (BTs) in the liver and organochlorine compounds (OCs) in the blubber of Risso’s dolphins collected off Taiji, Japan, in 1991 were determined. Mean and range concentrations (wet weight basis) of these compounds were 3.6 µg/g (0.556.0 µg/g) for BTs, 25 µg/g (1.7-120 µg/g) for PCBs, 17 µg/g (0.45-77 µg/g) for DDTs, 4.0 µg/g (0.19-16 µg/g) for CHLs, and 0.16 µg/g (0.008-0.74 µg/g) for HCHs. OCs concentrations increased with age in males in contrast to that in females, which showed a decreasing trend after maturity. On the other hand, no difference was observed in BT concentrations between male and female, which showed increasing levels until maturity (8-10 years) and then remained constant. It is suggested that, unlike OCs, BTs were less transferable to young ones in reproductive processes. Risso’s dolphins showed higher biomagnification factor (about 6) than Steller sea lion (0.6), implying a slower excretion rate of BTs in cetaceans than in pinnipeds due to the lower degradation capacity of xenobiotics and the lack of physiological processes such as shedding of hair in cetaceans.

Introduction Organotin compounds are one of the representative industrial chemicals with widespread usage as stabilizers of polyvinyl chloride (PVC) and as antifouling biocides in paints. In the early 1980s, about 10% of the total organotin compounds used as biocides and slimicides and in anti* To whom correspondence should be addressed; fax: 81-89946-9904; e-mail address: [email protected]. † Ehime University. ‡ The University of Tokyo. § National Research Institute of Far Seas Fisheries.

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FIGURE 1. Sampling locations of Risso’s dolphin and squids. (A) Risso’s dolphin (Grampus griseus), (B) Japanese flying squid (Todarodes pacificus), (C) purpleback flying squid (Sthenoteuthis oualaniensis) and neon flying squid (Ommastrephes bartrami).

fouling paints were shown to enter the aquatic environment (1). Consequently, the widespread distribution and toxic effects of organotins in aquatic organisms have been well documented (2-6). However, most of these studies have focused on lower trophic organisms in food chain. Our research group first detected butyltin compounds (BTs) in the blubber of marine mammals (7). Further studies showed that BTs accumulated principally in the liver unlike organochlorines (8, 9). In addition, a large amount of BTs was excreted through the shedding of hair and the molting of feathers (9, 10), and in Steller sea lion, BTs concentrations were less variable with age and sex (11). In the course of these studies, it was found that finless porpoise had higher BT concentrations than Steller sea lion (8, 11). This prompted us to consider the presence of a specific mode of BT accumulation in cetaceans in relation to physiological and biological processes. However, to our knowledge, no information is available on the residue pattern of BTs in the process of growth and reproduction in cetaceans. In order to understand a part of these aspects, the present study attempted to analyze BTs in Risso’s dolphin (Grampus griseus) collected from Japanese coastal waters. At the same time, persistent organochlorines such as PCBs, DDTs, HCHs (hexachlorocyclohexanes), and CHLs (chlordane compounds) were also determined in the same animal for comparison.

Materials and Methods Samples. Risso’s dolphins (G. griseus) were collected off Taiji, Japan, in 1991 (Figure 1). The age of Risso’s dolphin was determined by counting the growth layer groups in the dentine and cement of their teeth by following the procedure of Kasuya (12). The number of samples analyzed was 20 for male and 22 for female. The age of the animals was between 0 and 16.5 years for male and between 0.5 and 34.5 years for female. The body length of males was in the range of 146-287 cm and for females was between 183 and

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TABLE 1

Mean and Range Concentrations of BTs (Cation µg/g wet wt) in the Liver and OCs (µg/g wet wt) in the Blubber of Risso’s Dolphina n

MBT

DBT

TBT

BTsb

n

PCBs

DDTsc

CHLsd

HCHse

male

15 20

1.9 (0.27-3.7) 2.7 (0.37-4.4) 2.4 (0.27-4.4)

0.71 (0.21-1.0) 0.91 (0.30-1.2) 0.82 (0.21-1.2)

2.9 (0.55-5.0) 4.2 (0.70-6.0) 3.7 (0.55-6.0)

13

female

0.29 (0.035-0.53) 0.53 (0.037-1.3) 0.43 (0.035-1.3)

43 (7.4-120) 7.0 (1.7-20) 25 (1.7-120)

29 (3.2-77) 4.2 (0.45-19) 17 (0.45-77)

6.8 (1.3-16) 1.3 (0.19-4.6) 4.0 (0.19-16)

0.27 (0.047-0.74) 0.064 (0.008-0.23) 0.16 (0.008-0.74)

sex

mean

14

a Figures in parentheses indicate the range. b BTs ) MBT + DBT + TBT. c DDTs ) p,p′-DDT + p,p′-DDD + p,p′-DDE. d CHLs ) oxychlordane + trans -nonachlor + cis-nonachlor + trans-chlordane + cis-chlordane. e HCHs ) R-HCH + β-HCH + γ-HCH.

284 cm. Following our previous studies, which revealed higher concentrations of BTs in liver than in other tissues (8, 9), liver was used for BT analysis in the present study. In the case of organochlorine compounds, blubber samples were used for chemical analysis due to their major deposition in this tissue (13). In order to elucidate the bioaccumulation factor and the metabolic capacity of BTs in Risso’s dolphin, three species of squids were collected from Japanese coastal waters and the northwest Pacific in 1993 (in Figure 1) and analyzed. All the samples were stored at -20 °C until chemical analysis. Chemical Analysis. The chemical analysis of BTs followed the method of Iwata et al. (7) with some modifications. About 1.5 g of liver was homogenized with 35 mL of 0.1% tropolone-acetone and 5 mL of 2 N HCl. The homogenate was centrifuged at 3000 rpm, and BTs in the supernatant were transferred to 0.1% tropolone-benzene. The moisture was eliminated with 35 g of anhydrous Na2SO4, then concentrated to near dryness using a rotary evaporator (40 °C), and made up to 5 mL with benzene. BTs in extract were propylated by adding 5 mL of n-propyl magnesium bromide (2 mol/L in THF solution, Tokyo Kasei Kogyo Co. Ltd., Japan) as the Grignard reagent, and the mixture was shaken at 40 °C for 30 min. The excess Grignard reagent was destroyed with 20 mL of 1 M H2SO4, and the propylated mixture was transferred to 20 mL of 10% benzene-hexane/40 mL of hexane-washed water using 10 mL of methanol. The extract was concentrated to near dryness and made up to 5 mL with hexane and then passed through a 6-g activated Florisil-packed wet column for final cleanup using 40 mL of hexane. Final hexane extract was concentrated into 5 mL and subjected to GC quantification. A GC-FPD (Hewlett-Packard 5890 Series II) with a moving needle type injection system and a tin mode filter (610 nm) was used for quantification. Monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) of known amounts (0.2 µg/mL) were spiked into minke whale liver (minke whale from the Antarctic Ocean containing undetectable levels of BTs residues), passed through the whole analytical procedure, and used as an external standard. Concentrations were estimated by comparing peak heights of butyltins in samples with those in external standard. Hexyl-TBT was used as an internal standard to check recovery. The reproducibility of MBT, DBT, and TBT was examined by spiking into the liver of Antarctic minke whale. Recoveries of TBT, DBT, and MBT were 110 ( 9, 98 ( 9, and 83 ( 13%, respectively (n ) 5). Detection limits of TBT, DBT, and MBT were less than 1, 1, and 3 cation ng/g wet wt, respectively. BT concentrations were not corrected for recovery.

PCBs and organochlorine insecticides (OCs) were analyzed following the method described by Tanabe et al. (14). Samples were homogenized with anhydrous Na2SO4 and transferred to a Soxhlet apparatus with 300 mL of diethyl ether and 100 mL of hexane for extraction. The concentrated extracts were passed through a Florisil packed dry column to remove fat and eluted with 150 mL of 20% hexane-washed water in acetonitrile. The eluant was collected in a separatory funnel containing 100 mL of hexane and 600 mL of hexane-washed water. After shaking, the hexane layer was concentrated to 6 mL, cleaned with sulfuric acid, and separated with 12 g of Florisil-packed glass column into two fractions. The first fraction, eluted with hexane, consisted of PCBs, trans-nonachlor, p,p′-DDE, and HCB (hexachlorobenzene). The second fraction, eluted with 150 mL of 20% dichloromethane in hexane, was comprised of HCH isomers, chlordane compounds, and the remaining DDTs. The quantification of OCs was made on a gas chromatograph (Hewlett-Packard 5890 Series II) equipped with a 63Ni electron capture detector and a moving needle-type injection port. For the quantification of PCBs, an equivalent mixture of Kanechlor 300, 400, 500, and 600 was used as a standard. The sum of individually resolved peaks was obtained to estimate total PCB concentration. The concentrations of OC insecticides were quantified from individually resolved peak heights with the corresponding peak heights of authentic standards. Recoveries of OCs ranged from 90 to 100% (n ) 3). OC concentrations were not corrected for recovery.

Results and Discussion Residue Pattern and Composition of Butyltins and Organochlorines. The mean and range concentrations of BTs and OCs are given in Table 1. BT concentrations in the liver of Risso’s dolphin were in the range of 0.55-6.0 µg/g with a mean value of 3.6 µg/g. These values are comparable or higher than those of bivalves collected from TBT-polluted bays and estuaries (15-19), indicating that TBT contamination is also serious in higher trophic marine organisms and is extended from regional to global problem. To our knowledge, the information on the concentration of BTs in marine mammals is few except studies conducted so far in our laboratory (8, 9, 11). BT concentrations in Risso’s dolphins were comparable to those of finless porpoises inhabiting coastal waters, but apparently higher than those of Steller sea lion from Hokkaido, Japan, and Alaska (Table 2). Although the available data to compare the residue levels of BTs in marine mammals are small, it is likely that cetaceans have higher concentrations of BTs than pinnipeds. This is probably due to the lower capacity to degrade

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TABLE 2

Butyltin Concentrations (Cation µg/g wet wt) in the Liver of Marine Mammals species Risso’s dolphin (Grampus griseus) finless porpoise (Neophocaena phocaenoides) Steller sea lion (Eumetopias jubatus)

location Taiji, Japan Chiba, Japan Seto-inland sea, Japan Ise Bay, Japan Hokkaido, Japan Alaska

sex

n

concn (range)

ref

M F M M F M F M F

15 20 1 1 1 6 6 17 21

2.9 (0.55-5.0) 4.2 (0.70-6.0) 1.1 10 3.3 0.16 (0.075-0.25) 0.22 (0.18-0.30) 0.016 (0.002-0.022) 0.018 (0.002-0.024)

this study this study 8 8 8 11 11 11 11

TABLE 3

PCBs and DDTs Concentations (µg/g wet wt) in the Blubber of Risso’s Dolphin Collected from Various Locationsa sampling year

sex

n

PCBs

DDTs

ref

Pacific Coast of Japan

1991

French Coast (Atlantic and Mediterranean) Italian Coast South Africa South Atlantic South Atlantic

1979 1992 1971 1984 1986

M F na M, Fc na M F

13 14 3 2 na 1d 1e

43 (7.4-120) 7 (1.7-20) 68 ((23b) 610, 20 nd 2.7 0.38

29 (3.2-77) 4.2 (0.45-19) 70 ((34b) 400, 5.2 0.26 7.5 1.3

this study this study 24 25 26 27 27

location

d

a Figures in parentheses indicate a range. na, not available; nd, not detected. Body length was 3.14 m (mature). e Body length was 1.29 m (immature).

FIGURE 2. Composition pattern of BTs in marine mammals and their prey (whole body). Footnote 1 indicates that the data are cited from Kim et al. (11).

BTs in cetaceans than in pinnipeds, as reported in a case of PCBs (20-23). This could also be noticed from the composition of BTs in the present study, which showed higher TBT and lower MBT proportions in Risso’s dolphin than in Steller sea lion; nevertheless, their prey had a similar composition of BT compounds (Figure 2). Kim et al. (9) reported that Steller sea lion excreted about 25% of total BT body burden through the shedding of hair once a year. This suggests that cetaceans with no body hair may have a less excretory route of BTs than pinnipeds and, thus, contribute to the relatively higher accumulation of BTs in porpoises and dolphins. Among organochlorines, the highest concentration was found for PCBs ranging from 1.7 to 120 µg/g (mean, 25 µg/g wet wt) followed by DDTs ranging from 0.45 to 77 µg/g (mean, 17 µg/g), CHLs from 0.19 to 16 µg/g (mean, 4 µg/g), and HCHs from 0.008 to 0.74 µg/g (mean, 0.16 µg/g) (Table 1). Although the number of samples analyzed was small, comparison of the concentrations of PCBs and DDTs in Risso’s dolphin with those from other areas (Table 3) suggested that the status of contamination in the Pacific Coast of Japan was comparable to those in French and Italian Coasts, but much higher than those off South Africa and in the South Atlantic. Aguilar (28) suggested the DDE/

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b

Standard deviation. c Male is mature and female is immature.

DDTs ratio as an index to understand the input of DDT in the environment. In the present study, the DDE/DDTs ratio of 0.77 implied no recent input of DDT in the coastal areas of Japan. However, high concentrations of PCBs and DDTs impose serious threats and possible toxic effects in Risso’s dolphin. Regardless of sex, β-HCH, trans-nonachlor, and p,p′-DDE showed the highest proportion in HCHs, CHLs, and DDTs compositions, respectively (Table 4). The composition pattern of CHLs was similar to that of other cetaceans as reported by Kawano et al. (29), and the percentage of oxychlordane, a metabolite of chlordane, was apparently lower in Risso’s dolphin than in pinnipeds (23, 30, 31), further suggesting a lower metabolic capacity of cetaceans. Age Trend and Male-Female Difference of Butyltin and Organochlorine Accumulation. The concentrations of PCBs and DDTs increased with age in males, whereas in females the decreasing pattern was observed after maturity (Figure 3). The age trend of OCs observed in the present study was consistent with previous observations, where male cetaceans and pinnipeds accumulated OCs continuously throughout life and females exhibited a decreasing trend due to their transfer from mother to fetus with gestation and infant with lactation (14, 23, 31-33). In contrast to organochlorines, the concentration of BTs increased with age until maturity (9-10 years) (34) and then tended to be stable (Figure 3). No significant difference was observed between both sexes in the residue pattern of BT concentrations with age. Considering these results, it can be suggested that, unlike OCs, BTs are less transferable from mother to fetus and infant through gestation and lactation. Less transfer of BTs during reproductive process was also found in a laboratory experiment with rats (35, 36). The differences in the accumulation of BTs and OCs might have arisen from the chemical properties of these compounds. BTs were suggested to combine with proteins having -SH and dNH groups (37-39), whereas OCs are

TABLE 4

Composition (%) of Organochlorine Compounds in the Blubber of Risso’s Dolphin chlordanesa

HCHs

DDTs

sex

r

β

γ

oxy

t-chl

c-chl

t-nona

c-nona

p,p′-DDE

p,p′-DDD

p,p′-DDT

male female mean

4 12 8

93 83 88

2 5 4

5 4 4

2 4 3

10 12 11

65 65 65

18 16 17

82 73 77

8 11 9

10 16 13

a

Terms from left to right represent oxychlordane, trans-chlordane, cis-chlordane, trans-nonachlor, and cis-nonachlor, respectively.

FIGURE 3. Accumulation pattern of BTs and OCs concentrations with age (O, male; b, female).

FIGURE 4. Variations in the proportions of TBT in BTs, r-HCH in HCHs (r + β + γ isomers) and p,p′-DDT in DDTs (p,p′-DDT + p,p′-DDD + p,p′-DDE) with age (O, male; b, female).

retained in lipids (13, 40). Interestingly, the age trend of BTs in Risso’s dolphin was different from that of Steller sea lion, which contained an apparently lower concentration (mean, 17 ng/g wet wt) of BTs and a comparable level regardless of age and sex (11). Risso’s dolphin showed no difference of BT accumulations between both sexes similar to Steller sea lion but exhibited a specific age trend with increasing concentration until maturity. Such a trend is probably attributable to the balance of BT intake and excretion. As mentioned earlier, in Risso’s dolphin, low BT residue levels in infants seem to be a result of their less transfer in the reproductive process. In addition, in immature animals, the intake of BTs through feeding is suggested to be larger than excretion by metabolic degradation, hence the increasing pattern of BT accumulations was found. Unlike pinnipeds, the increasing trend of BTs in the immature stage of Risso’s dolphin was probably accelerated by the lack of excretory processes such as the shedding of hair. Considering the constant feeding rate at mature stage, the intake amount of BTs is likely to be balanced by the rate of metabolic degradation, and thus uniform levels of BTs were attained after maturity. The composition of BTs was the same in both sexes but varied with age (Figure 4). After birth, the TBT proportion

decreased until maturity and then remained constant. This pattern is probably caused by the balance of BT residues in Risso’s dolphin and their diet. As shown in Table 5 and Figure 2, BTs in squids consisted largely of TBT. In immature Risso’s dolphin with lower BT burden, the composition of BTs in diet influences greatly the dolphins. On the other hand, unlike immature animals, BT composition in adult animals is not likely to be affected by those in their diet (squids) because of the relatively higher concentration of BTs in Risso’s dolphin than in squids. A similar feature was also observed for OCs composition. As shown in Figure 4, the proportions of R-HCH and p,p′DDT tended to increase with age in females after maturity. This is due to a much lower concentration of these contaminants in females than in males as a result of lactational and gestational transfer, and thus the composition of contaminants in females was influenced greatly by those in diet, which had a higher proportion of R-HCH and p,p′-DDT (29). Another possible explanation for this variable composition with age might be due to the changes in metabolic potential according to age. However, liver microsomal monooxygenase activity in marine mammals was generally constant regardless of age (41), and therefore this possibility was excluded.

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TABLE 5

Mean and Range Concentrations (Cation µg/g wet wt) of Butyltin Compounds in the Liver of Squids, Prey of Risso’s Dolphin species

sampling site

sex

n

purpleback flying squid (Sthenoteuthis oualaniensis) neon flying squid (Ommastrephes bartrami) Japanese flying squid (Todarodes pacificus)

Northwest Pacific

M

3

Northwest Pacific

M

6

Japanese coastal water

nib ni

2 2c

a

BTs ) MBT + DBT + TBT.

b

MBT

DBT

TBT

BTsa

mature

9 (7-14)

24 (12-32)

28 (19-41)

61 (37-87)

immature

8 (5-10)

21 (18-26)

19 (8-42)

47 (38-67)

63 (55-71) 6

140 (120-150) 12

maturity

ni ni

Acknowledgments The authors would like to thank Dr. K. Kannan (Skidaway Institute of Oceanography) for a critical reading of this manuscript. This research was supported by a grant-in-

9

380 (300-460) 69

ni, not identified. c Whole body was analyzed.

Bioaccumulation of Butyltins. Cephalopods appear to form the entire diet of Risso’s dolphin. Stomach contents of only the remains of cephalopods were recorded in stranded or captured animals from the Mediterranean (42) and the Eastern Pacific (43, 44) and off Japan (45, 46), and no fish remains have been encountered. Tsutsumi et al. (45) noted that Risso’s dolphin in captivity would feed only on squid. Taking into account the above information, the biomagnification factor (BMF) of BTs in Risso’s dolphin was estimated based on the analysis of three species of squid collected from Japanese coastal waters and the northwest Pacific Ocean. BT concentrations in the liver of a squid collected from Japanese coastal waters were five times higher than those from the open seas (Table 5). Based on the analysis of liver and whole body of T. pacificus, it is evident that squid accumulates higher concentration of BTs in the liver than in other parts, as in marine mammals (8, 9). The liver weight of Risso’s dolphin was recorded to be about 2.2% of the whole body (12). In the case of finless porpoise, the liver accounted for 2.3% of the body weight and retained 19% of the whole body burden of BTs (8). Based on this information, the whole body concentration of BTs in Risso’s dolphin was estimated to be 400 ng/g. Assuming that Risso’s dolphin in the Western Pacific feeds principally on T. pacificus, which migrates in Japanese coastal waters between the East China Sea and about 50 °N in the Pacific Ocean (47), the BMF was estimated to be about 6. The BMF would be greater if the dolphins migrate to an open sea such as the Eastern Pacific and feed on squid with lower concentrations of BTs. The BMF value of 6 in Risso’s dolphin was apparently higher than the value of 0.6 in Steller sea lion (10). This may again suggest a slower excretion rate (or little excretion pathway) of BTs in Risso’s dolphin, which resulted from lower degradation capacity and the lack of a shedding process of hair when compared with pinnipeds. BT residue levels were higher in Risso’s dolphin than in Steller sea lion, which was probably caused by lower metabolic capacity and lack of excretory pathway of BTs through hair shedding. These observations strongly suggest that cetaceans accumulate BTs more than pinnipeds due to some specific biological and physiological differences. Therefore, adequate attention should be paid with respect to BT contamination in these animals, similar to organochlorines.

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aid from the Scientific Research Programme (Project Nos. 06405006, 06454097, and 07660245) of the Ministry of Education, Science and Culture of Japan. This study was also conducted partly by the financial support in the form of scholarship from the Ministry of Education, Korea, awarded to G.B.K.

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Received for review January 18, 1996. Revised manuscript received April 15, 1996. Accepted April 16, 1996.X ES9600486 X

Abstract published in Advance ACS Abstracts, June 15, 1996.

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