Environ. Sci. Techno/. 1995,29,2959-2962
Hiah Accumulation of Toxic B h i n s in Marine Mammals from Jaoanese Coastal Waters H
HISATO I W A T A , * SHINSUKE T A N A B E , T A K A H I K O M I Z U N O , AND RYO TATSUKAWA Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790,Japan
Organotins are one of the most toxic chemicals in the aquatic environment. While contamination and toxic effects of organotin compounds in lower trophic aquatic organisms have been well-documented, no investigation has focused so far on higher aquatic organisms like marine mammals. W e attempted to analyze butyltin compounds (BTCs), including mono(MBTs), di- (DBTs), and tributyltin compounds (TBTs), in various tissues and organs of finless porpoise (Neophocaenaphocaenoides) collected from Japanese coastal waters and detected these compounds in all the animals. The highest residue levels were found in the liver of a porpoise collected in the inland sea, recording more than 10 ppm BTCs on a wet weight basis. The composition of BTCs was different according to the tissues and organs with higher proportions of DBTs noticed in the liver and blood. Estimation of BTC burdens in tissues and organs indicated that muscle, liver, and blubber retained predominant portion of TBTs, DBTs, and MBTs. The occurrence of higher rates of TBTs on the whole implied the lower metabolic potential of porpoises to BTCs.
Introduction Tributyltin compounds (TBTs),the most toxic substances ever deliberately introduced into natural waters ( I ) , have been used worldwide since the 1960s as antifouling agents in paints used for boats and aquaculture nets. Besides TBTs, MBTs and DBTs have also been consumed as stabilizers and catalysts for chemical products (2). These compounds still contaminate the water-sediment systems (3),and their ecotoxicological impacts have been viewed as significant, as is implicated in morphological and physiological abnormalities in lower trophic aquatic organisms such as mollusks and fish (4-6). Marine mammals such as dolphins and whales, top predators in the aquatic ecosystem, have been noted to accumulate high levels of persistent organochlorines through aquatic food webs. Considering the octanollwater * Address correspondence to this author at the Department of Toxicology, Faculty ofveterinary Medicine, Hokkaido University, N18 W9 North Ward, Sapporo, Japan 060; fax: f81-11-717-7569; e-mail address:
[email protected].
0013-936X/95/0929-2959$09.00/0
C 1995 American Chemical Society
partition coefficient of di- and triorganotins in the range of lo3 and lo5 ( 3 ,which is a factor controlling biomagnification, BTCs may also be accumulated in marine mammals. A large number of laboratory studies using rats and mice have demonstrated the immunotoxicity (8), neurotoxicity (91,teratogenicity (IO), and cutaneous toxicity (11)of organotins. In spite of these physicochemical and toxicological aspects of organotins, data on the contamination of higher trophic wildlife are unavailable, making it difficult to assess the potential risks of exposure to organotins. In a recent study, BTCs were detected in the blubber of marine mammals (12). However, information on BTC levels in tissues other than the blubber is not available. In this study, finless porpoises collected from the Japanese coastal waters in the 1980swere used as a suitable sample to analyze BTCs. This species is primarily a coastal and riverine inhabitant and, therefore, likely to be exposed to the input of anthropogenic chemicals including organotins. We analyzed BTCs including MBTs, DBTs, and TBTs in various tissues and organs of the finless porpoise to understand their distribution in the body. In addition, the body burdens of butyltin species were estimated to elucidate the kinetics of BTCs in marine mammals.
Materials and Methods Three porpoises found stranded along the Japanese coasts were analyzed (Figure 1). All of them were found dead. The animals were brought to the laboratory, dissected, and stored at -20 "C until analysis. The analytical method of BTCs has been described in detail elsewhere (12). About 1-2 g of tissues was homogenized with 1 N HC1 and 0.1% tropolone/acetone. The BTCs in extracts were transferred to 0.1% tropolone/ benzene, and the moisture in the solvent was removedwith anhydrous Na2S04,followed by transferring the solvents to hexane. BTCs in hexane were propylated by adding propylmagnesium bromide as a Grignard reagent. The extract was added on a dry florisil column and passed with nitrogen gas slowly. BTCs adsorbed on the florisil were eluted with 20% water/acetonitrile to remove lipid. The eluate was then subjected to a wet florisil column for further purification. The final extract was injected into a gas chromatograph (GC)with flame photometric detector and a tin mode fiter (610 nm). A fused silica capillary column (DB-1; 0.25 mm i.d. x 30 m length) was used for GC separation. Identification of BTCs was made by assigning peaks in samples to the corresponding peaks of external standard. Peak heights of individual BTCs were used for the quantification. Standard mixtures were prepared with every set of four samples by propylating the known amounts of BTC ion mixtures spiked on a whale liver, which was previously found to contain trace levels of BTCs. Hexyltributyltin was added as an internal standard. In order to examine the recoveries of BTCs through the analytical procedure in tissues except blubber, 0.1 pg of butyltin chloride species dissolved in hexane was spiked into about 2 g of the liver of a whale and analyzed. Concurrently, butyltin chloride mixture without a matrix was also propylated for reference. Recoveries for MBTs, DBTs, and TBTs through the whole analytical procedure
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Japan Sea Seta-,,
Pacific coasl of Chtba
:etaceans (16). Moreover, TBTs were shown to inhibit Gochrome P450 enzymes at elevated concentrations in ish (1 7 ) ; thedeteriorationoftheenzymesystemby elevated xposure to BTCs may accelerate the bioaccumulation of :ontaminants inmarine mammals. Externalmalformations were observed at a few micrograms per gram DBTs in the iver ofrats orallyadministeredwith di-n-butyltin diacetate 18). This suggests that the animals analyzed in this study lave been exposed to the potentially hazardous levels of 3TCs. Immunotoxicity has also been emphasized as one of h e toxic effects ofBTCs. It has been documented that the ‘atsfed di-n-dibutyltin chloride dose dependently reduced he weight of thymus, spleen, and popliteral lymph node, issociated with lymphocyte depletion in the cortex of the hymus and the thymus-dependent areas in the peripheral ymphoid organs (19). In addition to the laboratory sxperiment, mass mortalities of several marine mammal populations in European andNorth American waters since the 1980s haveledtothespeculationthattheenvironmental pollutants impair the immunocompetence ofthese animals andincrease the susceptibilitytoviralinfections (20). While the debate to link pollution with die-offs is still continuing, the impacts of BTCs in marine mammals should also be considered. The accumulation of the highest concentration of BTCs in the liver ofporpoise was different from that of persistent organochlorines,which retained preferentially in the blubber (21). In contrast, the distribution pattern of BTCs was rather similar to that of organic mercurywith larger residues in the liver (22). This indicates that the BTC accumulation is less dependent on the affinity to lipids in tissues and organs. On the contrary, Laughlin et al. (23)reported that the accumulation levels ofTBT in several tissues of marine mussels was correlated with the lipid content. A recent review on the transport mechanism of methylmercury in experimental animals indicated that it forms a complex with glutathione and the complex plays a key role in the body distribution (24). A similar distribution of BTCs to methylmercurymaysuggestthe higher affinityto sulfhydryl groups such as glutathione rather than lipid. Compositions. BTC compositions in samples showed a tissue/organ-specific mode of accumulation. DBTs contributed to a higher proportion in the liver (55-71%) and blood (78%) in comparison with other tissues and organs. The possible distortion ofTBTs to DBTs and MBTs duringthe samplestorageis also conceivable. Nevertheless, the present results exhibited a rather uniform pattern of BTC compositions in three animal tissues, showing no systematic trend with storage periods. This may reflect the tissue- and organ-specificmetaboliccapacity after TBTs exposure or the specific kinetics of each butyltin species depending on their physicochemical parameters. It is noteworthy that, in the intestinal content of the animal collected from Ise Bay in 1994, TBTs were the dominant species (Table 1). suggesting that the uptake of TBTs is at the higher rate through food. If TBT inputs have decreased or stopped in Ise Bay, the proportion of degradation products (DBTs and MBTs) of TBTs would be higher. Several studies have showed the rapid degradation ofTBTs by biotic and abiotic processes in water and sediment (25, 26). The higherproportionofTBTs intheintestinecontent indicates the continuous inputs of TBTs in Ise Bay. Yonezawa et al. (27)estimated the amount ofTBTs loading in port areas of the bay in 1986 to be approximately 9.2-
7
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\
Pacific Ocean
FIGURE 1. Map of Japan showing the sampling sites of finless porpoises.
were 96.9 f 26.1, 102 f 8.7, and 91.1 i 13.3% (n = 3). respectively. In addition, hexyltributyltin was also added into the liver, and the recovery was 104 5 12.8%. All the concentration data are given as nanogram of butyltin ion/ gram on a wet weight basis, if not specified. Detection limits of BTCs were 5 ng/g for MBTs, 1 ng/g for DBTs. and 0.5 ng/gforTBTs. For blubbersamples, therecoveriesand detection limits have been reported in our previous study (12). The concentrations of BTCs in the samples were not corrected based on the result of spiking experiment.
Results and Discussion Residue Levels. BTCs were detected in all the tissues and organs of porpoise (Table 1). The highest BTC concentration was found in the liver at a ppm level on a wet weight basis, followed bythe kidney. Particularly, a M e s s porpoise collected in the Seto-inland Sea was highly contaminated with 10.2 and 3.2 pg/g BTCs in the liver and kidney, respectively. Residue levels of BTCs in the blubber were lower than those in these organs. It was also noted that the blood contained significant levels of BTCs. The hepatic BTC concentrationsin porpoiseswerehigher than the residue levels [astotal Sn) reported for the Pacific oyster (Crassostrea gigas1 collected from Arcachon Bay, France, in the early 1980s. where poor shell growth and shell malformations were observed (4). According to a monitoring surveyconducted by the EnvironmentalAgency of Japan in the coastal marine environment (131,the soft tissues of mussels collected from the Ise Bay in 1989-1992 contained 50-160 ng of TBTOlg on a wet weight basis. Most of the organs of porpoise from the same bay showed higher TBT concentrations than in the mussels. Furthermore, the porpoise liver was found to contain higher concentrations than the liver of several fish species [sum of MBTs, DBTs, and TBTs: 158-508 ng of hutyltin chloride/g on a wet weight basis) collected in 1993 from the Tokyo Bay with a large number of maritime facilities (141.
It has been assumed that the BTC concentrations in rats and mice are low due to rapid metabolism. Hence, the capacity for bioaccumulation of BTCs in lower trophic organisms has been considered to be much greater than in the mammals (15). The present results suggest that the assumption may not be true for marine mammals. A high accumulation of BTCs in porpoise might partly be due to the low cytochrome P450 enzyme activities in small 2960 m ENVIRONMENTAL SCIENCE 8 TECHNOLOGY I VOL. 29, NO. 12, 1995
TABLE 1
Concentrations (ng of Butyltin lonlg Wet Weight Basis) of BTCs in Tissues and Organs of Finless Porpoises sex
tissues and organs
MBTs
DBTs
TBTs
CBTs
151
d
Seto-inland Sea
162
d
Ise Bay
139
Q
muscle blubbera liver kidney heart lung stomach muscle blubbera liver kidney heart lung stomach brain testis muscle blubber liver kidney heart lung stomach brain adrenal gland spleen boneb esophagus pancreas intestine urinary bladder eye blood intestine content
18 91 130 47 19 12 11 67 210 3000 340 130 44 150 350 120 22 73 680 55 12 90 18 9 130 33 14 19 72 9 26 15 82 6
12
