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Relationship between lipid composition and organochlorine levels in the tissues of striped dolphin
Volume 19/Number 3/March 1988
0025-326X/88 S3.00+0.00 O 1988 Pergamon Press pie.
Marine Pollution Bulletin, Volume 19, No. 3. pp. 129-133, 1988 Pnnted in Great Britain.
Relationship Between Lipid Composition and Organochlorine Levels in the Tissues of Striped Dolphin S. KAWAI,* M. FUKUSHIMA,* N. MIYAZAKIt and R. TATSUKAWA:~ * Osaka City Institute of Public Health and Environmental Sciences, Tohjo-cho 8-34, Tennofi-ku, Osaka 543," t National Science Museum, Hyakunin-cho 3-23-1, Shinjuku-ku, Tokyo 160; ~Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan
Lipid content and lipid composition were studied in the various tissues of striped dolphins (Stenella coeruleoalba) collected from Japanese waters in order to elucidate the relationship between the levels of organochlorine compounds and the lipid composition. Total lipid content was highest in the adipose tissues such as blubber and melon, followed by mammary gland and milk. Also kidney, pancreas and brain contained about 10% of lipid. Other tissues such as liver, muscle, spleen and lung had low lipid contents ranging from 1-5%. In the tissues with high lipid contents, triglycerides comprised more than 70% of total lipids. Phospholipids and total cholesterol were dominant in brain tissues including cerebrum, cerebellum and medulla oblongata. Organochlorine compounds such as PCBs and DDTs were mainly distributed in tissues with high lipid content and the levels of these compounds were correlated with the content of triglycerides in the tissues. Abnormalities in lipid metabolism such as fatty livers were noticed in individuals with high PCBs and DDTs.
The lipophilic organochlorine compounds such as PCBs, DDTs, and HCH isomers are known to accumulate in adipose tissues of terrestrial and aquatic animals. In addition, abnormal lipid metabolisms such as hyperglyceridemia (Tanaka et al., 1969; Nagai et al., 1971; Uzawa et al., 1969, 1971), fatty liver (Nishizumi et al., 1969; Yoshioka, 1976) and induction of drug metabolizing enzyme activities (Fujita et al., 1971; Kamohara et al., 1974) have been demonstrated to occur in animals including humans exposed to organochlorine compounds. Marine mammals such as seals and cetaceans are known to accumulate higher levels of organochlorine compounds in their subcutaneous fat or lipid fraction of tissues due to their occupation of high trophic levels in marine ecosystems (Sladen et al., 1966; Holden et al., 1967; Wolman et al., 1970; Hall et al., 1971; Gaskin et
al., 1974; Bowes & Jonkel, 1975; Taruski et al., 1975; Reijnders, 1980; O'Shea et al., 1980; Tanabe et al., 1983; Aguilar, 1985). The bioconcentration of these compounds are also not only correlated to the lipid content of the tissues but to the lipid composition. We report in this communication the relationship between the levels of organochlorine compounds and the lipid composition such as triglyceride, non-esterified fatty acids (NEFA), phospholipids and total cholesterol in various tissues of striped dolphins (Stenella coeruleoalba ). Materials and M e t h o d s
Animals The striped dolphins studied here were caught at Taiji (33 ° 36' N, 135 ° 56' E) Wakayama Prefecture, Japan in December of 1978 and 1979, and were stored at -20°C for several months till dissection. After dissection, the samples were repeatedly frozen and thawed for the analyses of various kinds of pollutants. For age determination, the teeth of the striped dolphins were prepared in the longitudinal thin section (10-20 ~tm), decalcified in 5% formic acid, and stained with Mayer's haematoxylin solution based on Kasuya's method (Kasuya, 1976). Age of the striped dolphin was determined by the number of stainable growth layers in dentine for the dolphin less than 10 yr and those in cementum for the dolphin of 10 yr or more. The ages of foetus and newborn calf were estimated by the agebody length regression equation (Miyazaki, 1978). Extraction of lipid 5-10 g of tissues were homogenized with anhydrous sodium sulphate, and lipid was extracted with Folch (Folch et aL, 1955) solution (chloroform :methanol-- 2:1) or n-hexane using a Waring blender. Total lipid Lipid extracted with Folch solution or n-hexane was 129
Marine Pollution Bulletin
determined gravimetrically on an aliquot of the extract.
Lipid composition Triglycerides, non-esterified fatty acids (NEFA), phospholipids and total cholesterol in the extracted lipid were measured by commercial lipid assay kits purchased from Wako Chemical Co. Ltd.
i.d.× 2 m long packed with 2% OV-1 on 80-100 mesh Gas Chrom Q for PCBs analysis, and with a mixture of 1.5% OV-17 and 1.95% QF-1 on 80-100 mesh Chromosorb W, AW DMCS for pesticides analysis.
Results and D i s c u s s i o n
Lipid composition in various tissues of striped dolphins Organochlorine compounds The organochlorine compounds were analysed using the method of Wakimoto & Tatsukawa (1973). The lipids in the extracted n-hexane were removed using a dry florisil column. The organochlorine residues in the extract were separated into two fractions by using a silica-gel column. The first fraction eluted with n-hexane contained PCBs and p,p'-DDE and the second one eluted with 15% diethyl ether in n-hexane contained H C H isomers, p,p'-DDD, p,p'-DDT, and o,p'-DDT. In addition, the second fraction was treated with 7% fuming sulphuric acid. The analyses of PCBs and the pesticides were conducted on a Shimadzu GC4BM Gas Chromatograph equipped with a 63Ni electron captured detector. The glass column was 2 mm
Composition of lipid and fatty acids in blubber (Tsuyuki & Itoh, 1966; Tsuyuki & Itoh, 1967; Itoh & Tsuyuki, 1974; Pilleri, 1971), blood (Demonte & Pilleri, 1977) and milk (Arvy, 1973-4) have been investigated in several seals and cetaceans. However little is known about those in other tissues of the same animals. Fifteen tissues of an immature male (body length, 215 cm; body weight, 106 kg; age, 8.5 yr) and 19 tissues of a pregnant female (body length, 229 cm; body weight, 160 kg; age, 16.5 yr) were examined for the lipid composition. Results were shown in Tables 1 and 2. Total lipid content was highest in either blubber or melon and 8090% of it was triglyceride in both tissues. Brain including cerebrum, cerebellum and medulla oblongata showed rather high lipid content ranging from 9-18%
TABLE 1 Lipid composition in tissues of a mature male striped dolphin.* Tissue Melon Blubber Muscle Kidney Cerebrum Cerebellum Pancreas Spleen Lung Small intestine First stomach Second stomach Heart Diaphragm Blood
Total lipid (rag g-L)
Triglyceride (rag g-i)
NEFA (mg g-i)
980 810 39 82 94 120 94 38 29 31 16 50 44 43 7.2
980 800 4.0 34 0 0 0 6.0 4.0 4.0 2.0 4.0 18 6.0 0.64
0.2 11 2.7 14 8.4 10 48 15 6.1 26 6.5 31 18 14 0.27
Phospholipid (mgg -t) 0 0 16 18 43 49 2O 14 14 13 8.4 23 13 17 2.5
Total cholesterol (mgg -I) 0 0 0 4.8 26 25 2 2.8 2.8 4.8 0 14 0 2.8 1.5
*Body length 215 cm, body weight 106 kg, age 8.5 yr.
TABLE 2 Lipid composition in tissues of a pregnant striped dolphin.* Tissue Melon Blubber Muscle Liver Kidney Cerebrum Cerebellum Medulla oblongata Placenta Pancreas Spleen Lung Small intestine First stomach Second stomach Heart Mammary gland Milk Uterus Blood
Total lipid (rag g-I)
Triglyceride (rag g-L)
NEFA (rag g-I)
940 930 44 34 120 130 130 180 27 156 33 19 36 21 41 42 310 312 20 7.6
940 930 12 2,6 84 2,2 2.2 2.0 1.8 120 6.0 2.0 4.0 0 0 6.0 310 258 2.0 1.0
0.65 1.4 2.4 10 11 21 24 8.2 7.8 46 14 4.6 15 1.7 11 14 4.8 0.1 2.0 0.70
*Body length 229 cm, body weight 160 kg, age 16,5 yr.
130
Phospholipid (mgg -I) 0 0 23 19 11 55 84 73 15 48 26 9.2 14 12 43 32 37 25 11 6.6
Total cholesterol (mg g-t) 0 0 0.8 1.8 4.2 29 29 15 1.4 3.8 4.6 1.6 0.8 0 1.2 1.2 1.6 7.3 0 0.95
Volume 19/Number 3/March 1988
Organochlorine residues in various tissues of striped dolphins
of the tissue. Kidney and pancreas also showed similar lipid contents as brain. The other tissues such as muscle, liver, stomach, spleen, intestine, lung, heart, and diaphragm contained lower levels of lipid ranging from 1-4%. Characteristically mammary glands in matured females showed a high content of lipid (31%) as shown in Table 2. Lipid content in milk of a pregnant female was 31%. It has been reported that milk in cetaceans and seals contained high levels of lipid (30-50%), compared with those of human and cow whose lipid content is about 4% (Jennes et al., 1970). In the tissues of high lipid content such as blubber and melon, triglycerides comprised almost 100% of the tissue lipid. Kidney, pancreas, mammary gland and milk that showed 8-30% of lipid in the tissues also contained much triglycerides ranging 70-80%. On the other hand, triglycerides or NEFA content were low in brain tissues, and most lipids were phosopholipids (5070%) and total cholesterol (15-30%). In other organs containing low levels of lipids mentioned above, triglyceride content was low and phospholipids were generally dominant. About 80% of lipids in blood was comprised of phospholipids and total cholesterol which were less lipophilic than triglycerides.
Distribution of organochlorine compounds in the various tissues of mature male striped dolphins are shown in Table 4. PCB concentrations were high in the blubber. In other tissues, the levels were 1-10% of that in blubber. Organochlorine residues were much lower in a pregnant female than in a mature male as shown in Table 5. This is likely due to the elimination of the residues from the mother dolphin to foetus or calf. Milk and mammary gland in a mature female contained rather high levels of organochlorines. DDT analogues showed similar results like PCBs, but the difference in the distribution of each metabolite in tissues was different. Concentrations of HCH isomers in various tissues were low in comparison to PCBs and DDT compounds even in the adipose tissues. This is presumably due to the fact that H C H isomers show relatively low lipophilicity and more biodegradable properties. Brain showing rather high lipid contents corresponding to those in kidney and pancreas, however, was much lower in the levels of PCBs and DDT compounds. This can be explained by the lipid compositions as described below.
Changes of lipid content and lipid composition in the tissues of striped dolphins with growth
Lipid content, lipid composition and organochlorine compounds in the tissues of striped dolphins
Lipids were assayed in blubber, muscle, and liver of two foetuses (9.6 and 9.9 months after fertilization), one calf female (0.16 yr) and two pregnant females (9.5 and 16.5 yr). Though lipid content was highest in the foetal blubbers, those were only 36-52% in other tissues as shown in Table 3. Possibly, this is due to the fact that differentiation between muscle and blubber is not completed in the stage compared with other striped dolphins. Therefore, muscle of foetuses showed a higher lipid content than immature and mature individuals. Lipid content of liver was lower in foetuses than in other stages. Lipid composition in three kinds of tissues was almost similar in each stage except in the foetal muscle. Triglycerides and NEFA comprised much in foetal stage and this fact might also be explained by the incomplete differentiation of muscle and blubber as mentioned above.
Figure 1 summarizes the relationship between lipid content, lipid composition, and PCBs levels in various tissues of a pregnant female (age, 16.5 yr). In such tissues as blubber, melon, pancreas, kidney, and mammary gland with higher concentrations of PCBs, most lipid was of triglycerides, whereas in the brain where 90% o f the lipid composition was either phospholipids or total cholesterol, the PCBs concentration was low. It is likely that PCBs concentrate less in polar lipids such as phospholipids and cholesterol. On the other hand, H C H isomers in brain showed an interesting feature as shown in Table 6. Except in brain, the more lipophilic [3-HCH was detected at higher concentrations in all other tissues, whereas, brain with more phospholipids and total cholesterol accumulated the more polar ~t-HCH isomer. Similar results were reported by Tanabe et al. (1981). The findings of Le
TABLE 3 Changes in lipid composition with growth in tissues of striped dolphins. Relative content (%) Age (yr)
Status
Sex
Foetus
Female
9.6M*
Foetus
Male
9.9M*
Calf
Female
0.16
Pregnant
Female
9.5
Pregnant
Female
16.5
Tissue
Total lipid ( m g g -1)
Triglyceride
NEFA
Phospholipid
Total cholesterol
Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver
520 70 25 360 83 28 860 39 59 940 30 43 780 45 34
95 49 "6 92 51 5 95 11 10 98 19 4 100 32 4
2 23 35 3 16 44 3 37 43 1 19 42 0 7 39
3 28 60 5 30 52 1 57 47 0 62 50 0 59 51
1 () 0 0 2 0 1 0 0 1 0 4 0 2 6
*Agesof foetusesexpressedas months(M) after fertilization. 131
Marine PollutionBulletin TABLE 4
TABLE 5
Concentrations of PCBs, DDTs, and HCHs in tissues of a matured male striped dolphin.*
Concentrations of PCBs, DDTs, and HCHs in tissues of a pregnant striped dolphin.*
Tissue Muscle Blubber Liver Kidney Lung Heart .Small intestine First stomach Cerebrum Cerebellum Medulla oblongata Blood
Lipid content (%)
Concentration (l~gg-~ wet wt) PCBs DDTs HCHs
1.7 73.6 4.6 8.1 1.2 1.9 1.7 0.74 8.7 9.1 17.0 0.13
0.335 22.600 0.304 0.940 0.137 0.223 0.165 0.067 0.122 0.139 0.205 0.016
0.464 32.900 0.959 2.680 0.166 0.326 0.262 0.097 0.187 0.242 0.320 0.036
Tissue Muscle Blubber Melon Liver Kidney Pancreas Lung Heart Spleen Small intestine First stomach Second stomach Diaphragm Cerebrum Cerebellum Mammary gland Placenta Uterus Milk
0.010 0.470 0.057 0.061 0.013 0.017 0.012 0.026 0.021 0.022 0.040 0.001
*Body length 238 cm, body weight 161 kg, age 16.5 yr. B o e u f & B o n n e l (1971) in C a l i f o r n i a sea lions a n d H o l d e n (1978) in grey seals f u r t h e r s u p p o r t this conclusion. O t h e r tissues i n c l u d i n g muscle, liver, heart, lung, intestine, s t o m a c h a n d b l o o d s h o w e d low lipid contents ( 0 . 8 - 4 . 4 % ) a n d low levels of PCBs. In these tissues, the percentages of triglycerides or N E F A in the total lipids were low a n d r a n g e d 1 - 2 0 % . D D T s showed similar results. A s s h o w n in Fig. 1, milk also showed high lipid c o n t e n t a n d high c o n c e n t r a t i o n of PCBs. In addition, a b o u t 9 0 % of lipids of milk was c o m p o s e d of triglycerides. T h e lipid c o m p o s i t i o n of milk and m a m m a r y gland, where the milk was synthesized, resembled each other very m u c h . It is very i m p o r t a n t that p o l l u t a n t s such as P C B s in m a m m a l s , partition to triglycerides of milk a n d s u b s e q u e n t l y t r a n s p o r t e d to the calf through lactation. F r o m these results, we c o n c l u d e that the levels of o r g a n o c h l o r i n e c o m p o u n d s such as PCBs, D D T s , a n d H C H isomers d e p e n d entirely o n the lipid c o m p o s i t i o n , especially o n the triglyceride c o n t e n t in tissues. A b n o r m a l i t y in lipid m e t a b o l i s m d u e to the a c c u m u lation of o r g a n o c h l o r i n e c o m p o u n d s were not so clear in this study. But s o m e aspects of a b n o r m a l i t y such as Total Lipid (mg g-l)
Tissue
Lipid content (%) 1.6 94 98 3.5 20 18 1.6 3.4 2.0 2.0 0.2 2.3 2.3 20 11 13 1.4 0.5 33
*The same individual as shown in Table 2. N D ='Not Detected' ( < 0.001 ~g g-f wet wt). TABLE 6
HCH concentrations in the muscle, kidney, and brain of a matured male striped dolphin.* Lipid content HCHsconcentration (ng g-~ wet wt) Tissue (%) ct-HCH [3-HCH y-HCH Total Muscle 1.7 1.14 8.80 0.54 10.5 (ll) (84) (5) Kidney 8.1 2.75 57.1 1.06 6(}.9 (5) (94) (2) Cerebrum 8.7 16.0 4.7 0.6 21.3 (75) (22) (3) Cerebellum 9.1 16.3 5.3 0.6 22.2 (73) (24) (3) Medullaoblongata 17.1 33.3 6.2 0.7 40.2 (83) (16) (2) *The same individualas shown in Table 5. Values in parentheses indicate the percentages of HCH isomer 6HCH not detected in these tissues.
PCBs pg g-i wet wt.
Lipid Composition
(%)
Melon
940
2.70
Blubber
930
2.53
Milk
280
0.507
W//AI
Kidney
120
0.494
I~//////I
Mammary gland
310
0.333
Pancreas
160
0.282
V/z~2~ ~///////////J ~/////I//////I//I////AI
Muscle
44
0.057
Heart
42
0.065
Lung Small
intestine
Liver
19
0
36
o. o 21
34
l I
~/////////////////////I .
I o. o 9 o I
0
3
1
.......
~
I//////////////////////71
~/////////////////////////////////~
Cerebrum
130
0
.
0
4
9
~
Cerebellum
130
0
.
0
4
3
~
Second stomach Blood
41 8
o. o17 ~ / / / / / / / / / / / / / / / / / / Z ~ 0.010
. .
0
Triglyceride I'
I,NEFA ~
,
50
Phospholipid 5m22727~,Total cholesterol l
Fig. 1 Relationship between PCBs concentration and lipid composition in the tissues of a pregnant striped dolphin. 132
Concentration (gg g-' wet wt) PCBs DDTs HCHs 0.057 0.438 0.018 2.530 2.180 0.147 2.700 3.060 (I.262 0.090 0.034 0.006 0.494 0.493 0.020 0.282 0.318 0.019 0.031 0.030 ND 0.065 0.044 0.003 0.035 0.031 ND 0.020 0.020 0.002 0.004 0.001 ND 0.007 0.004 ND 0.007 0.004 ND 0.049 0.023 0.002 0.043 0.018 0.001 0.333 0.358 0.018 0.007 0.004 ND 0.075 0.041 ND 0.507 0.438 0.018
i00
Volume 19/Number 3/March 1988
fatty liver, which has been considered as a characteristic symptom of animals exposed to high levels of PCBs (Yoshioka, 1976), were noticed in several striped dolphins. For example, the liver of a pregnant female (232 cm body length, 163 kg body weight and 25.5 yr) contained much organochlorine compounds and total lipid (12%), compared with other individuals. Moreover, lipid of the liver was mostly NEFA. The individual analysed here was sampled in 1978, and was repeatedly frozen and thawed for the analysis of various kinds of pollutants. Therefore, triglycerides might be changed to NEFA by some enzymes. Similar results were obtained in other striped dolphins, and livers that showed rather high levels or organochlorine compounds generally contained higher concentration of lipids. From these results, abnormal lipid metabolism might be induced in the striped dolphins by the accumulation of much PCBs and DDTs. This study was supported in part by a Grant-in-Aid for Scientific Research (Project No. 343056) from the Ministry of Education, Science and Culture of Japan. Aguilar, A. (1985). Compavtmentation and reliability of sampling procedure in organochlorine pollution surveys of cetaceans. Residue Rev. 95, 91-114. Arvy, L. (1973/4). Mammary glands in cetaceans. Vol. V. In Investigation on Cetacea (G. Pilleri, ed.), p. 165. University Berne. Bowes, G. W. & Jonkel, C. J. (1975). Presence and distribution of polychlorinated biphenyls (PCB) in Arctic and Subarctic marine food chains. J. Fish. Res. Board Can. 32, 2111-2123. De Monte. T. & Pillcri, G. (1977). Some blood chemistry values obtained from Stenella coeruleoalba (Meyen, 1833) and Delphi delphis (Linnaeus, 1758) from the Western Mediterranean. In Investigation on Cetacea, Vol. III (G. Pilleri, ed.), pp. 223-230,383. Folch, J., Lees, M. & Slone, G. H. (1957). A simple method for the isolation and purification of total lipid from animal tissues. J. BioL Chem. 226,497-506. Fujita, S., Tsuji, H., Kato, K., Saeki, S. & Tsukamoto, H. (1971). Effect of biphenyt chlorides on rat liver microsomes. Fukuoka Acta Medica 62, 30-34. Gaskin, D. E., Smith, G. J. D., Arnold, P. W., Louisy, M. V., Frank, R., Holdrinet, M. & McWade, J. W. (1974). Mercury, DDT, dieldrin, and PCB in two species of odontoceti (Cetacea) from St. Lucia, Lesser Antilles. J. Fish. Res. Board Can. 31, 1235-1239. Hall, J. D., Gilmartin, W. G. & Mattson, J. L. (1971). Investigation of a Pacific pilot whale stranding on San Clemente Island. Z Wildlife Diseases 7,324-327. Holden, A. V. & Marsden, K. (1967). Organochlorine pesticides in seals and porpoises. Nature 216, t274-1276. Holden, A. V. (1978). Pollutants and seals--A review. Mammal Rev. 8, 53-66. Itoh, S. & Tsuyuki, H. (1974). Fatty acid composition of different blubber oil of finless porpoise. Sci. Rep. Whales Res. Inst. 26, 303306.
Jenncs, R. & Sloan, R. E. (1970). The composition of milks of various species. A review. DairySci. Abstr 32,599-612. Kamohara, K.. Fujiwara, K. & Itokawa, Y. (1974). Studies on experimental PCB poisoning--Dose response effect of PCB and sinergistic effect with PCB and ABS. Jap. J. Hyg. 29,321-327. Kasuya, T. (1976). Reconsideration of life history parameters of the spotted and striped dolphins based on cemental layers. Sci. Rep. Whales Res. Inst. 28, 73-106. Miyazaki, N. (1978). Growth and reproduction of Stenella coeruleoalba off the Pacific coast of Japan. Sci. Rep. Whales Res. Inst. 29, 21-48. ke Boeuf, B. J. & Bonnel, M. L. (197l). DDT in California sea lions. Nature 234, 108-109. Nagai, J., Furukawa, M., Yae, Y. & Higuchi, K. (1971). The influence of chlorobiphenyls (Kanechlor) administration on the organ lipids of rats. Fukuoka Acta Medica 62, 42-47. Nishizumi, M., Kohchi, S. & Kuratsune, M. (1969). An experimental study on "Yusho'" or chlorobiphenyls poisoning (Preliminary Report). Fukuoka Acta Medica 60,539-543. O'Shea, T. J., Brownell, R. L. Jr., Clark, D. R. Jr., Walker, W. A., Gay, M. L. & Lamont, T. G. (1980). Organochlorine pollutants in small cetaceans from the Pacific and South Atlantic Oceans, November 1968-June 1976. Pest. Monit. J. 14, 35-46. Pilleri, G. (1971). Preliminary analysis of the lipids present in the blubber of Platanista indi and gangetica. In btvestigations on Cetacea, (G. Pilleri, ed.), p. 179. University Berne. Reijnders, P. J. H. (1980). Organochlorine and heavy metal residues in harbour seals from the Wadden Sea and their possible effects on reproduction. Netherland J. Sea Res. 14, 30-65. 81aden, W. J. L., Menzie, C. M. & Reichel, W. L. (1966). DDT residues in Adelie penguins and a crabeater seal from Antarctica. Nature 2 !0, 67{)-673. Tanabe, S., Tatsukawa, R., Tanaka, H., Maruyama, K., Miyazaki, N. & Fujiyama, T. (i981). Distribution and total burdens of chlorinated hydrocarbons in bodies of striped dolphins (Stenella coeruleoalba). Agric. Biol. Chem. 45, 2569-2578. Tanabe, S., Mori, T. & Tatsukawa, R. (1983). Global pollution of marine mammals by PCBs, DDTs and HCHs (BHCs). Chemosphere 12, 1269-1275. Tanaka, K.. Fujita, S., Komatsu, K. & Tamura, N. (1969). Experimental subacute poisoning of chlorobiphenyls, particularly the influence on the serum lipids in rats. Fukuoka Acta Medica 60, 544-547. Taruski, A. G., Olney, C. E. & Winn, H. E. (1975). Chlorinated hydrocarbons in cetaceans, J. Fish. Res. Board Can. 32, 22{)5-2209. Tsuyuki, H. & Itoh, S. (1966). Studies on the oils contained in the blubber of a southern elephant seal. Sci. Rep. Whales Res. Inst. 20, 213-221. Tsuyuki, H. & hob, S. (1967). Fatty acid composition of finless porpoise oil. Sci. Rep. Whales Res. h2st. 21,131-136. Uzawa, H., Ito, Y., Notomi, A. & Katsuki, S. (1969). Hyperglyceridemia resulting from intake of rice oil contaminated with chlorinated biphenyls. Fukuoka Acta Medica 60,449-454. Uzawa, H., Ito, Y., Notomi, A., Hori, S., Ikeura, Y. & Katsuki, S. (1971). Clinical and experimental studies on the hyperglyceridemia induced by oral ingestion of chlorinated biphenyls. Fukuokak Acta Medica 62, 66-73. Wakimoto, T. & Tatsukawa, R. (1973). Analytical methods of PCB (in Japanese). PPM 4, 46-5 I. Wolman, A. A. & Wilson, A. J. Jr (1970). Occurrence of pesticides in whales. Pest. Monit. J, 4, 8-10. Yoshioka, M. (1976). Effects of polychlorinated biphenyls (PCB) and alkylbenzene sulfonic sodium salt (ABS) on the lipid metabolism in rats. Jap. J./Jyg. 3 l, 575-582.
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0025-326X/88 S3.00+0.00 O 1988 Pergamon Press pie.
Marine Pollution Bulletin, Volume 19, No. 3. pp. 129-133, 1988 Pnnted in Great Britain.
Relationship Between Lipid Composition and Organochlorine Levels in the Tissues of Striped Dolphin S. KAWAI,* M. FUKUSHIMA,* N. MIYAZAKIt and R. TATSUKAWA:~ * Osaka City Institute of Public Health and Environmental Sciences, Tohjo-cho 8-34, Tennofi-ku, Osaka 543," t National Science Museum, Hyakunin-cho 3-23-1, Shinjuku-ku, Tokyo 160; ~Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan
Lipid content and lipid composition were studied in the various tissues of striped dolphins (Stenella coeruleoalba) collected from Japanese waters in order to elucidate the relationship between the levels of organochlorine compounds and the lipid composition. Total lipid content was highest in the adipose tissues such as blubber and melon, followed by mammary gland and milk. Also kidney, pancreas and brain contained about 10% of lipid. Other tissues such as liver, muscle, spleen and lung had low lipid contents ranging from 1-5%. In the tissues with high lipid contents, triglycerides comprised more than 70% of total lipids. Phospholipids and total cholesterol were dominant in brain tissues including cerebrum, cerebellum and medulla oblongata. Organochlorine compounds such as PCBs and DDTs were mainly distributed in tissues with high lipid content and the levels of these compounds were correlated with the content of triglycerides in the tissues. Abnormalities in lipid metabolism such as fatty livers were noticed in individuals with high PCBs and DDTs.
The lipophilic organochlorine compounds such as PCBs, DDTs, and HCH isomers are known to accumulate in adipose tissues of terrestrial and aquatic animals. In addition, abnormal lipid metabolisms such as hyperglyceridemia (Tanaka et al., 1969; Nagai et al., 1971; Uzawa et al., 1969, 1971), fatty liver (Nishizumi et al., 1969; Yoshioka, 1976) and induction of drug metabolizing enzyme activities (Fujita et al., 1971; Kamohara et al., 1974) have been demonstrated to occur in animals including humans exposed to organochlorine compounds. Marine mammals such as seals and cetaceans are known to accumulate higher levels of organochlorine compounds in their subcutaneous fat or lipid fraction of tissues due to their occupation of high trophic levels in marine ecosystems (Sladen et al., 1966; Holden et al., 1967; Wolman et al., 1970; Hall et al., 1971; Gaskin et
al., 1974; Bowes & Jonkel, 1975; Taruski et al., 1975; Reijnders, 1980; O'Shea et al., 1980; Tanabe et al., 1983; Aguilar, 1985). The bioconcentration of these compounds are also not only correlated to the lipid content of the tissues but to the lipid composition. We report in this communication the relationship between the levels of organochlorine compounds and the lipid composition such as triglyceride, non-esterified fatty acids (NEFA), phospholipids and total cholesterol in various tissues of striped dolphins (Stenella coeruleoalba ). Materials and M e t h o d s
Animals The striped dolphins studied here were caught at Taiji (33 ° 36' N, 135 ° 56' E) Wakayama Prefecture, Japan in December of 1978 and 1979, and were stored at -20°C for several months till dissection. After dissection, the samples were repeatedly frozen and thawed for the analyses of various kinds of pollutants. For age determination, the teeth of the striped dolphins were prepared in the longitudinal thin section (10-20 ~tm), decalcified in 5% formic acid, and stained with Mayer's haematoxylin solution based on Kasuya's method (Kasuya, 1976). Age of the striped dolphin was determined by the number of stainable growth layers in dentine for the dolphin less than 10 yr and those in cementum for the dolphin of 10 yr or more. The ages of foetus and newborn calf were estimated by the agebody length regression equation (Miyazaki, 1978). Extraction of lipid 5-10 g of tissues were homogenized with anhydrous sodium sulphate, and lipid was extracted with Folch (Folch et aL, 1955) solution (chloroform :methanol-- 2:1) or n-hexane using a Waring blender. Total lipid Lipid extracted with Folch solution or n-hexane was 129
Marine Pollution Bulletin
determined gravimetrically on an aliquot of the extract.
Lipid composition Triglycerides, non-esterified fatty acids (NEFA), phospholipids and total cholesterol in the extracted lipid were measured by commercial lipid assay kits purchased from Wako Chemical Co. Ltd.
i.d.× 2 m long packed with 2% OV-1 on 80-100 mesh Gas Chrom Q for PCBs analysis, and with a mixture of 1.5% OV-17 and 1.95% QF-1 on 80-100 mesh Chromosorb W, AW DMCS for pesticides analysis.
Results and D i s c u s s i o n
Lipid composition in various tissues of striped dolphins Organochlorine compounds The organochlorine compounds were analysed using the method of Wakimoto & Tatsukawa (1973). The lipids in the extracted n-hexane were removed using a dry florisil column. The organochlorine residues in the extract were separated into two fractions by using a silica-gel column. The first fraction eluted with n-hexane contained PCBs and p,p'-DDE and the second one eluted with 15% diethyl ether in n-hexane contained H C H isomers, p,p'-DDD, p,p'-DDT, and o,p'-DDT. In addition, the second fraction was treated with 7% fuming sulphuric acid. The analyses of PCBs and the pesticides were conducted on a Shimadzu GC4BM Gas Chromatograph equipped with a 63Ni electron captured detector. The glass column was 2 mm
Composition of lipid and fatty acids in blubber (Tsuyuki & Itoh, 1966; Tsuyuki & Itoh, 1967; Itoh & Tsuyuki, 1974; Pilleri, 1971), blood (Demonte & Pilleri, 1977) and milk (Arvy, 1973-4) have been investigated in several seals and cetaceans. However little is known about those in other tissues of the same animals. Fifteen tissues of an immature male (body length, 215 cm; body weight, 106 kg; age, 8.5 yr) and 19 tissues of a pregnant female (body length, 229 cm; body weight, 160 kg; age, 16.5 yr) were examined for the lipid composition. Results were shown in Tables 1 and 2. Total lipid content was highest in either blubber or melon and 8090% of it was triglyceride in both tissues. Brain including cerebrum, cerebellum and medulla oblongata showed rather high lipid content ranging from 9-18%
TABLE 1 Lipid composition in tissues of a mature male striped dolphin.* Tissue Melon Blubber Muscle Kidney Cerebrum Cerebellum Pancreas Spleen Lung Small intestine First stomach Second stomach Heart Diaphragm Blood
Total lipid (rag g-L)
Triglyceride (rag g-i)
NEFA (mg g-i)
980 810 39 82 94 120 94 38 29 31 16 50 44 43 7.2
980 800 4.0 34 0 0 0 6.0 4.0 4.0 2.0 4.0 18 6.0 0.64
0.2 11 2.7 14 8.4 10 48 15 6.1 26 6.5 31 18 14 0.27
Phospholipid (mgg -t) 0 0 16 18 43 49 2O 14 14 13 8.4 23 13 17 2.5
Total cholesterol (mgg -I) 0 0 0 4.8 26 25 2 2.8 2.8 4.8 0 14 0 2.8 1.5
*Body length 215 cm, body weight 106 kg, age 8.5 yr.
TABLE 2 Lipid composition in tissues of a pregnant striped dolphin.* Tissue Melon Blubber Muscle Liver Kidney Cerebrum Cerebellum Medulla oblongata Placenta Pancreas Spleen Lung Small intestine First stomach Second stomach Heart Mammary gland Milk Uterus Blood
Total lipid (rag g-I)
Triglyceride (rag g-L)
NEFA (rag g-I)
940 930 44 34 120 130 130 180 27 156 33 19 36 21 41 42 310 312 20 7.6
940 930 12 2,6 84 2,2 2.2 2.0 1.8 120 6.0 2.0 4.0 0 0 6.0 310 258 2.0 1.0
0.65 1.4 2.4 10 11 21 24 8.2 7.8 46 14 4.6 15 1.7 11 14 4.8 0.1 2.0 0.70
*Body length 229 cm, body weight 160 kg, age 16,5 yr.
130
Phospholipid (mgg -I) 0 0 23 19 11 55 84 73 15 48 26 9.2 14 12 43 32 37 25 11 6.6
Total cholesterol (mg g-t) 0 0 0.8 1.8 4.2 29 29 15 1.4 3.8 4.6 1.6 0.8 0 1.2 1.2 1.6 7.3 0 0.95
Volume 19/Number 3/March 1988
Organochlorine residues in various tissues of striped dolphins
of the tissue. Kidney and pancreas also showed similar lipid contents as brain. The other tissues such as muscle, liver, stomach, spleen, intestine, lung, heart, and diaphragm contained lower levels of lipid ranging from 1-4%. Characteristically mammary glands in matured females showed a high content of lipid (31%) as shown in Table 2. Lipid content in milk of a pregnant female was 31%. It has been reported that milk in cetaceans and seals contained high levels of lipid (30-50%), compared with those of human and cow whose lipid content is about 4% (Jennes et al., 1970). In the tissues of high lipid content such as blubber and melon, triglycerides comprised almost 100% of the tissue lipid. Kidney, pancreas, mammary gland and milk that showed 8-30% of lipid in the tissues also contained much triglycerides ranging 70-80%. On the other hand, triglycerides or NEFA content were low in brain tissues, and most lipids were phosopholipids (5070%) and total cholesterol (15-30%). In other organs containing low levels of lipids mentioned above, triglyceride content was low and phospholipids were generally dominant. About 80% of lipids in blood was comprised of phospholipids and total cholesterol which were less lipophilic than triglycerides.
Distribution of organochlorine compounds in the various tissues of mature male striped dolphins are shown in Table 4. PCB concentrations were high in the blubber. In other tissues, the levels were 1-10% of that in blubber. Organochlorine residues were much lower in a pregnant female than in a mature male as shown in Table 5. This is likely due to the elimination of the residues from the mother dolphin to foetus or calf. Milk and mammary gland in a mature female contained rather high levels of organochlorines. DDT analogues showed similar results like PCBs, but the difference in the distribution of each metabolite in tissues was different. Concentrations of HCH isomers in various tissues were low in comparison to PCBs and DDT compounds even in the adipose tissues. This is presumably due to the fact that H C H isomers show relatively low lipophilicity and more biodegradable properties. Brain showing rather high lipid contents corresponding to those in kidney and pancreas, however, was much lower in the levels of PCBs and DDT compounds. This can be explained by the lipid compositions as described below.
Changes of lipid content and lipid composition in the tissues of striped dolphins with growth
Lipid content, lipid composition and organochlorine compounds in the tissues of striped dolphins
Lipids were assayed in blubber, muscle, and liver of two foetuses (9.6 and 9.9 months after fertilization), one calf female (0.16 yr) and two pregnant females (9.5 and 16.5 yr). Though lipid content was highest in the foetal blubbers, those were only 36-52% in other tissues as shown in Table 3. Possibly, this is due to the fact that differentiation between muscle and blubber is not completed in the stage compared with other striped dolphins. Therefore, muscle of foetuses showed a higher lipid content than immature and mature individuals. Lipid content of liver was lower in foetuses than in other stages. Lipid composition in three kinds of tissues was almost similar in each stage except in the foetal muscle. Triglycerides and NEFA comprised much in foetal stage and this fact might also be explained by the incomplete differentiation of muscle and blubber as mentioned above.
Figure 1 summarizes the relationship between lipid content, lipid composition, and PCBs levels in various tissues of a pregnant female (age, 16.5 yr). In such tissues as blubber, melon, pancreas, kidney, and mammary gland with higher concentrations of PCBs, most lipid was of triglycerides, whereas in the brain where 90% o f the lipid composition was either phospholipids or total cholesterol, the PCBs concentration was low. It is likely that PCBs concentrate less in polar lipids such as phospholipids and cholesterol. On the other hand, H C H isomers in brain showed an interesting feature as shown in Table 6. Except in brain, the more lipophilic [3-HCH was detected at higher concentrations in all other tissues, whereas, brain with more phospholipids and total cholesterol accumulated the more polar ~t-HCH isomer. Similar results were reported by Tanabe et al. (1981). The findings of Le
TABLE 3 Changes in lipid composition with growth in tissues of striped dolphins. Relative content (%) Age (yr)
Status
Sex
Foetus
Female
9.6M*
Foetus
Male
9.9M*
Calf
Female
0.16
Pregnant
Female
9.5
Pregnant
Female
16.5
Tissue
Total lipid ( m g g -1)
Triglyceride
NEFA
Phospholipid
Total cholesterol
Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver Blubber Muscle Liver
520 70 25 360 83 28 860 39 59 940 30 43 780 45 34
95 49 "6 92 51 5 95 11 10 98 19 4 100 32 4
2 23 35 3 16 44 3 37 43 1 19 42 0 7 39
3 28 60 5 30 52 1 57 47 0 62 50 0 59 51
1 () 0 0 2 0 1 0 0 1 0 4 0 2 6
*Agesof foetusesexpressedas months(M) after fertilization. 131
Marine PollutionBulletin TABLE 4
TABLE 5
Concentrations of PCBs, DDTs, and HCHs in tissues of a matured male striped dolphin.*
Concentrations of PCBs, DDTs, and HCHs in tissues of a pregnant striped dolphin.*
Tissue Muscle Blubber Liver Kidney Lung Heart .Small intestine First stomach Cerebrum Cerebellum Medulla oblongata Blood
Lipid content (%)
Concentration (l~gg-~ wet wt) PCBs DDTs HCHs
1.7 73.6 4.6 8.1 1.2 1.9 1.7 0.74 8.7 9.1 17.0 0.13
0.335 22.600 0.304 0.940 0.137 0.223 0.165 0.067 0.122 0.139 0.205 0.016
0.464 32.900 0.959 2.680 0.166 0.326 0.262 0.097 0.187 0.242 0.320 0.036
Tissue Muscle Blubber Melon Liver Kidney Pancreas Lung Heart Spleen Small intestine First stomach Second stomach Diaphragm Cerebrum Cerebellum Mammary gland Placenta Uterus Milk
0.010 0.470 0.057 0.061 0.013 0.017 0.012 0.026 0.021 0.022 0.040 0.001
*Body length 238 cm, body weight 161 kg, age 16.5 yr. B o e u f & B o n n e l (1971) in C a l i f o r n i a sea lions a n d H o l d e n (1978) in grey seals f u r t h e r s u p p o r t this conclusion. O t h e r tissues i n c l u d i n g muscle, liver, heart, lung, intestine, s t o m a c h a n d b l o o d s h o w e d low lipid contents ( 0 . 8 - 4 . 4 % ) a n d low levels of PCBs. In these tissues, the percentages of triglycerides or N E F A in the total lipids were low a n d r a n g e d 1 - 2 0 % . D D T s showed similar results. A s s h o w n in Fig. 1, milk also showed high lipid c o n t e n t a n d high c o n c e n t r a t i o n of PCBs. In addition, a b o u t 9 0 % of lipids of milk was c o m p o s e d of triglycerides. T h e lipid c o m p o s i t i o n of milk and m a m m a r y gland, where the milk was synthesized, resembled each other very m u c h . It is very i m p o r t a n t that p o l l u t a n t s such as P C B s in m a m m a l s , partition to triglycerides of milk a n d s u b s e q u e n t l y t r a n s p o r t e d to the calf through lactation. F r o m these results, we c o n c l u d e that the levels of o r g a n o c h l o r i n e c o m p o u n d s such as PCBs, D D T s , a n d H C H isomers d e p e n d entirely o n the lipid c o m p o s i t i o n , especially o n the triglyceride c o n t e n t in tissues. A b n o r m a l i t y in lipid m e t a b o l i s m d u e to the a c c u m u lation of o r g a n o c h l o r i n e c o m p o u n d s were not so clear in this study. But s o m e aspects of a b n o r m a l i t y such as Total Lipid (mg g-l)
Tissue
Lipid content (%) 1.6 94 98 3.5 20 18 1.6 3.4 2.0 2.0 0.2 2.3 2.3 20 11 13 1.4 0.5 33
*The same individual as shown in Table 2. N D ='Not Detected' ( < 0.001 ~g g-f wet wt). TABLE 6
HCH concentrations in the muscle, kidney, and brain of a matured male striped dolphin.* Lipid content HCHsconcentration (ng g-~ wet wt) Tissue (%) ct-HCH [3-HCH y-HCH Total Muscle 1.7 1.14 8.80 0.54 10.5 (ll) (84) (5) Kidney 8.1 2.75 57.1 1.06 6(}.9 (5) (94) (2) Cerebrum 8.7 16.0 4.7 0.6 21.3 (75) (22) (3) Cerebellum 9.1 16.3 5.3 0.6 22.2 (73) (24) (3) Medullaoblongata 17.1 33.3 6.2 0.7 40.2 (83) (16) (2) *The same individualas shown in Table 5. Values in parentheses indicate the percentages of HCH isomer 6HCH not detected in these tissues.
PCBs pg g-i wet wt.
Lipid Composition
(%)
Melon
940
2.70
Blubber
930
2.53
Milk
280
0.507
W//AI
Kidney
120
0.494
I~//////I
Mammary gland
310
0.333
Pancreas
160
0.282
V/z~2~ ~///////////J ~/////I//////I//I////AI
Muscle
44
0.057
Heart
42
0.065
Lung Small
intestine
Liver
19
0
36
o. o 21
34
l I
~/////////////////////I .
I o. o 9 o I
0
3
1
.......
~
I//////////////////////71
~/////////////////////////////////~
Cerebrum
130
0
.
0
4
9
~
Cerebellum
130
0
.
0
4
3
~
Second stomach Blood
41 8
o. o17 ~ / / / / / / / / / / / / / / / / / / Z ~ 0.010
. .
0
Triglyceride I'
I,NEFA ~
,
50
Phospholipid 5m22727~,Total cholesterol l
Fig. 1 Relationship between PCBs concentration and lipid composition in the tissues of a pregnant striped dolphin. 132
Concentration (gg g-' wet wt) PCBs DDTs HCHs 0.057 0.438 0.018 2.530 2.180 0.147 2.700 3.060 (I.262 0.090 0.034 0.006 0.494 0.493 0.020 0.282 0.318 0.019 0.031 0.030 ND 0.065 0.044 0.003 0.035 0.031 ND 0.020 0.020 0.002 0.004 0.001 ND 0.007 0.004 ND 0.007 0.004 ND 0.049 0.023 0.002 0.043 0.018 0.001 0.333 0.358 0.018 0.007 0.004 ND 0.075 0.041 ND 0.507 0.438 0.018
i00
Volume 19/Number 3/March 1988
fatty liver, which has been considered as a characteristic symptom of animals exposed to high levels of PCBs (Yoshioka, 1976), were noticed in several striped dolphins. For example, the liver of a pregnant female (232 cm body length, 163 kg body weight and 25.5 yr) contained much organochlorine compounds and total lipid (12%), compared with other individuals. Moreover, lipid of the liver was mostly NEFA. The individual analysed here was sampled in 1978, and was repeatedly frozen and thawed for the analysis of various kinds of pollutants. Therefore, triglycerides might be changed to NEFA by some enzymes. Similar results were obtained in other striped dolphins, and livers that showed rather high levels or organochlorine compounds generally contained higher concentration of lipids. From these results, abnormal lipid metabolism might be induced in the striped dolphins by the accumulation of much PCBs and DDTs. This study was supported in part by a Grant-in-Aid for Scientific Research (Project No. 343056) from the Ministry of Education, Science and Culture of Japan. Aguilar, A. (1985). Compavtmentation and reliability of sampling procedure in organochlorine pollution surveys of cetaceans. Residue Rev. 95, 91-114. Arvy, L. (1973/4). Mammary glands in cetaceans. Vol. V. In Investigation on Cetacea (G. Pilleri, ed.), p. 165. University Berne. Bowes, G. W. & Jonkel, C. J. (1975). Presence and distribution of polychlorinated biphenyls (PCB) in Arctic and Subarctic marine food chains. J. Fish. Res. Board Can. 32, 2111-2123. De Monte. T. & Pillcri, G. (1977). Some blood chemistry values obtained from Stenella coeruleoalba (Meyen, 1833) and Delphi delphis (Linnaeus, 1758) from the Western Mediterranean. In Investigation on Cetacea, Vol. III (G. Pilleri, ed.), pp. 223-230,383. Folch, J., Lees, M. & Slone, G. H. (1957). A simple method for the isolation and purification of total lipid from animal tissues. J. BioL Chem. 226,497-506. Fujita, S., Tsuji, H., Kato, K., Saeki, S. & Tsukamoto, H. (1971). Effect of biphenyt chlorides on rat liver microsomes. Fukuoka Acta Medica 62, 30-34. Gaskin, D. E., Smith, G. J. D., Arnold, P. W., Louisy, M. V., Frank, R., Holdrinet, M. & McWade, J. W. (1974). Mercury, DDT, dieldrin, and PCB in two species of odontoceti (Cetacea) from St. Lucia, Lesser Antilles. J. Fish. Res. Board Can. 31, 1235-1239. Hall, J. D., Gilmartin, W. G. & Mattson, J. L. (1971). Investigation of a Pacific pilot whale stranding on San Clemente Island. Z Wildlife Diseases 7,324-327. Holden, A. V. & Marsden, K. (1967). Organochlorine pesticides in seals and porpoises. Nature 216, t274-1276. Holden, A. V. (1978). Pollutants and seals--A review. Mammal Rev. 8, 53-66. Itoh, S. & Tsuyuki, H. (1974). Fatty acid composition of different blubber oil of finless porpoise. Sci. Rep. Whales Res. Inst. 26, 303306.
Jenncs, R. & Sloan, R. E. (1970). The composition of milks of various species. A review. DairySci. Abstr 32,599-612. Kamohara, K.. Fujiwara, K. & Itokawa, Y. (1974). Studies on experimental PCB poisoning--Dose response effect of PCB and sinergistic effect with PCB and ABS. Jap. J. Hyg. 29,321-327. Kasuya, T. (1976). Reconsideration of life history parameters of the spotted and striped dolphins based on cemental layers. Sci. Rep. Whales Res. Inst. 28, 73-106. Miyazaki, N. (1978). Growth and reproduction of Stenella coeruleoalba off the Pacific coast of Japan. Sci. Rep. Whales Res. Inst. 29, 21-48. ke Boeuf, B. J. & Bonnel, M. L. (197l). DDT in California sea lions. Nature 234, 108-109. Nagai, J., Furukawa, M., Yae, Y. & Higuchi, K. (1971). The influence of chlorobiphenyls (Kanechlor) administration on the organ lipids of rats. Fukuoka Acta Medica 62, 42-47. Nishizumi, M., Kohchi, S. & Kuratsune, M. (1969). An experimental study on "Yusho'" or chlorobiphenyls poisoning (Preliminary Report). Fukuoka Acta Medica 60,539-543. O'Shea, T. J., Brownell, R. L. Jr., Clark, D. R. Jr., Walker, W. A., Gay, M. L. & Lamont, T. G. (1980). Organochlorine pollutants in small cetaceans from the Pacific and South Atlantic Oceans, November 1968-June 1976. Pest. Monit. J. 14, 35-46. Pilleri, G. (1971). Preliminary analysis of the lipids present in the blubber of Platanista indi and gangetica. In btvestigations on Cetacea, (G. Pilleri, ed.), p. 179. University Berne. Reijnders, P. J. H. (1980). Organochlorine and heavy metal residues in harbour seals from the Wadden Sea and their possible effects on reproduction. Netherland J. Sea Res. 14, 30-65. 81aden, W. J. L., Menzie, C. M. & Reichel, W. L. (1966). DDT residues in Adelie penguins and a crabeater seal from Antarctica. Nature 2 !0, 67{)-673. Tanabe, S., Tatsukawa, R., Tanaka, H., Maruyama, K., Miyazaki, N. & Fujiyama, T. (i981). Distribution and total burdens of chlorinated hydrocarbons in bodies of striped dolphins (Stenella coeruleoalba). Agric. Biol. Chem. 45, 2569-2578. Tanabe, S., Mori, T. & Tatsukawa, R. (1983). Global pollution of marine mammals by PCBs, DDTs and HCHs (BHCs). Chemosphere 12, 1269-1275. Tanaka, K.. Fujita, S., Komatsu, K. & Tamura, N. (1969). Experimental subacute poisoning of chlorobiphenyls, particularly the influence on the serum lipids in rats. Fukuoka Acta Medica 60, 544-547. Taruski, A. G., Olney, C. E. & Winn, H. E. (1975). Chlorinated hydrocarbons in cetaceans, J. Fish. Res. Board Can. 32, 22{)5-2209. Tsuyuki, H. & Itoh, S. (1966). Studies on the oils contained in the blubber of a southern elephant seal. Sci. Rep. Whales Res. Inst. 20, 213-221. Tsuyuki, H. & hob, S. (1967). Fatty acid composition of finless porpoise oil. Sci. Rep. Whales Res. h2st. 21,131-136. Uzawa, H., Ito, Y., Notomi, A. & Katsuki, S. (1969). Hyperglyceridemia resulting from intake of rice oil contaminated with chlorinated biphenyls. Fukuoka Acta Medica 60,449-454. Uzawa, H., Ito, Y., Notomi, A., Hori, S., Ikeura, Y. & Katsuki, S. (1971). Clinical and experimental studies on the hyperglyceridemia induced by oral ingestion of chlorinated biphenyls. Fukuokak Acta Medica 62, 66-73. Wakimoto, T. & Tatsukawa, R. (1973). Analytical methods of PCB (in Japanese). PPM 4, 46-5 I. Wolman, A. A. & Wilson, A. J. Jr (1970). Occurrence of pesticides in whales. Pest. Monit. J, 4, 8-10. Yoshioka, M. (1976). Effects of polychlorinated biphenyls (PCB) and alkylbenzene sulfonic sodium salt (ABS) on the lipid metabolism in rats. Jap. J./Jyg. 3 l, 575-582.
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