Environ. Sci. Technol. 1997, 31, 296-301
Elevated Accumulation of Tributyltin and Its Breakdown Products in Bottlenose Dolphins (Tursiops truncatus) Found Stranded along the U.S. Atlantic and Gulf Coasts K . K A N N A N , * ,† K . S E N T H I L K U M A R , ‡ B. G. LOGANATHAN,† S. TAKAHASHI,‡ D. K. ODELL,§ AND S. TANABE‡ Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411, Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan, and Sea World, Inc., 7007 Sea World Drive, Orlando, Florida 32821-8097
Butyltin compounds, including mono- (MBT), di- (DBT), and tributyltin (TBT), were determined in the liver, kidney, and muscle of bottlenose dolphins (Tursiops truncatus) found stranded along the southeast U.S. Atlantic and Gulf coasts during 1989-1994. Total butyltin (BTs: MBT + DBT + TBT) concentrations in dolphin liver ranged between 110 and 11 340 ng/g (wet wt) with a mean value of 1400 ng/ g. Butyltin concentrations in bottlenose dolphins were higher than those reported from other locations. The liver of a adult male dolphin collected in 1989 had the highest BT concentration (11 340 ng/g wet wt) reported. The concentrations of butyltins increased during the early life stages until maturity, for both sexes, and then tended to remain constant. Analysis of fish muscle collected from the Gulf of Mexico indicated the existence of recent inputs of TBT. The biomagnification factor of BTs in dolphins, on average, was 1.0 with the highest value of 6.8. In addition to polychlorinated biphenyls (PCBs), the presence of noticeable concentrations of TBT and DBT, which are potential immunosuppressing agents, might have also contributed to bottlenose dolphin mortality events in the U.S. Atlantic and Gulf coasts.
(Tursiops truncatus) stranded along the mid-Atlantic U.S. coast exhibited PCBs and DDT concentrations in the blubber, which were among the highest reported for cetaceans (8, 9). Similarly, stranded Mediterranean striped dolphins (Stenella coeruleoalba) examined in 1990 carried PCBs and DDT levels that were higher than the levels found in healthy free-ranging animals (3). Studies have shown that the reduced immune response in bottlenose dolphins was correlated with increasing whole blood concentrations of several contaminants (10). Extensive use of tributyltin (TBT) as an antifouling agent since the 1960s in marine paints to prevent the attachment of barnacles and slime on boat hulls and consequent ecotoxicological impacts on oysters and mollusks are well established (11). It has been well documented that TBT is a potential immunotoxicant in mammals (12, 13). Exposure of fish and several marine invertebrates to environmental levels of TBT significantly affected the phagocytic activity and decreased the resistance against pathogenic microorganisms (14, 15). In this context, a high accumulation of butyltin compounds including TBT, DBT, and MBT in marine mammals is of great concern (16-18). Significant accumulation of butyltin compounds in marine mammals has implications for immune suppression and consequent disease outbreak. Although the contamination levels of butyltin compounds in mussels and fish collected from U.S. coastal waters are known (19-21), accumulation features, levels, and possible toxicological implications in marine mammals are not understood. Information on the exposure levels of butyltin compounds in bottlenose dolphins affected by the mass mortality events is needed in order to determine if these contaminants, in addition to PCBs, have contributed to immunosuppression. Therefore, the concentrations of MBT, DBT, and TBT were determined in various tissues and organs of bottlenose dolphins found stranded along the U.S. Atlantic and Gulf coasts during 1989-1994 in order to establish the contaminations and tissue distribution in this affected species. The concentrations of butyltin compounds in pygmy sperm whale (Kogia breviceps) and Atlantic spotted dolphin (Stenella frontalis), which were not affected by the mass mortality event, were also determined. Selected species of fish and shrimp collected in the Gulf of Mexico in 1994 were analyzed to provide information on the trophic transfer/biomagnification of these contaminants.
Materials and Methods Introduction Since 1987, large-scale mortalities of dolphins have been reported along the Atlantic coast of North America, in the Gulf of Mexico, and in the Mediterranean Sea (1-3). Dolphins collected from these large-scale mortality events along the U.S. Atlantic and Gulf coasts and in the Mediterranean Sea exhibited bacterial and viral infections indicative of immune dysfunction and were therefore less capable of surviving infectious diseases (4-6). Since these incidences have occurred mainly in industrialized coastal areas, chronic exposure to immunosuppressive pollutants such as polychlorinated biphenyls (PCBs) and DDT has been suggested as a possible cause of this epidemic (7). Bottlenose dolphins * Author to whom correspondence should be addressed at his present address: 201B Pesticide Research Center, Michigan State University, East Lansing, MI 48824; telephone: 517-353-9195; fax: 517-353-5598; e-mail:
[email protected]. † Skidaway Institute of Oceanography. ‡ Ehime University. § Sea World, Inc.
296
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 1, 1997
Samples. Marine mammal tissues were acquired from the Southeastern United States Marine Mammal Stranding Network and had been originally collected under letters of authorization issued by the National Marine Fisheries Service, Southeast Region. Bottlenose dolphins were found stranded along the Atlantic and Gulf coasts of Florida (Figure 1) from November 1989 to August 1994. Atlantic spotted dolphin and pygmy sperm whale were found beached along the Atlantic coast of Florida during 1991-1994. All the samples were acquired from fresh strandings, or those which had undergone only minimal decomposition (minor bloating and skin peeling). Animals were dissected, and tissues were wrapped in aluminium foil, placed in air-tight plastic bags, and frozen immediately at -20 °C until analysis. The length of individuals was used to determine approximate age groups (22, 23). Bottlenose dolphins included 9 male (2 neonates, 2 calves, a juvenile, and 4 adults) and 10 female animals (a neonate, 5 calves, 2 juveniles, and 2 adults). Animals of e120 cm are considered neonates, 120-190 cm are calves (about 2 years old), 190-220 cm are juveniles, and >220 cm are adults. Atlantic spotted dolphin (2 calves) and pygmy sperm
S0013-936X(96)00657-8 CCC: $14.00
1996 American Chemical Society
FIGURE 1. Dolphin and whale sampling locations (dotted area) in the Atlantic and Gulf coasts of Florida. whale (2 adults and a calf) were males. Tissue samples were cut from the internal portion of the original thawed sample with a clean stainless steel scalpel to avoid surface contamination. Liver, kidney, and muscle were analyzed for most samples whereas blubber, melon, heart, and brain were also examined in a few samples. Blue catfish (Ictalurus furcatus), gafftopsail catfish (Bagre marinus), hardhead catfish (Arius felis), brown shrimp (Penaeus aztecus), white shrimp (Penaeus setiferus), and croaker (Micropogonias undulatus) were collected from various locations in the Gulf of Mexico (Table 4) in 1994. Muscle tissue from several individuals of the same species was pooled and stored at -20 °C until analysis. For shrimp, the whole body was used for analysis. Analysis. Butyltin compounds including MBT, DBT, and TBT were analyzed by following the method described by Kannan et al. (24). Approximately 4-5 g of tissues was homogenized and extracted twice with 35 mL of 0.1% tropolone-acetone and 5 mL of 2 N HCl. The combined extract was transferred to 100 mL of 0.1% tropolone-benzene and 500 mL of hexane-washed water. After shaking and partitioning, 35 g of anhydrous Na2SO4 was added to the
organic layer to remove moisture. The benzene extract was rotary evaporated at 40 °C almost to dryness, and the volume was made up to 5 mL with hexane. The extract was derivatized by the addition of 5 mL of n-propyl magnesium bromide (ca. 2 mol/L in THF solution) as the Grignard reagent. The excess Grignard reagent was destroyed with 20 mL of 1 N H2SO4, and the derivatized extract was passed through a 6-g Florisil packed wet column for cleanup. The eluate from the cleanup column was rotary evaporated to produce a final volume of 5 mL. Sample extracts were injected into a gas chromatograph equipped with a flame photometric detector (GC-FPD). Chromatographic separation was performed on a HewlettPackard 5890 Series II gas chromatograph with a 30 m × 0.25 mm (i.d.) DB-1 capillary column coated at 0.25 µm film thickness. The column oven temperature was programmed from 80 °C (1 min hold) to 170 °C (1 min hold) at a rate of 15 °C/min and then at a rate of 5 °C/min to 210 °C (1 min hold) followed by a second raise at a rate 15 °C/min to a final temperature of 260˚C with a 7-min final hold time. Helium was used as the carrier gas at 1.3 mL/min. Injector and detector temperatures were held at 200 and 270 °C, respectively. The flame photometer was operated using a hydrogen-air-nitrogen flame and was equipped with a 610-nm bandpass filter that is selective for tin-containing compounds. Butyltin trichloride, dibutyltin dichloride, and tributyltin chloride (0.1 µg each) were spiked into the liver of Antarctic minke whale (Balaenoptera acutorostrata), containing butyltins below the limit of detection. These samples were passed through the whole analytical procedure and were used as external standards. Only freshly derivatized external standards prepared along with every set of eight samples were used to estimate concentrations. Concentrations were quantified by comparing peak heights of butyltins in samples with those in the external standards. Tributylhexyltin (synthesized in laboratory as an internal standard by reaction of n-hexyl magnesium bromide with tributyltin chloride) was added to each sample as an internal standard before extraction. Procedural blanks were included with every batch of four samples to check for interfering compounds and to correct sample values. Monobutyltin, probably originating from commercial solvents or reagents that came into contact with PVC containing this compound as a stabilizer, was found at trace levels (ca. 1 ng) in reagent blanks. The values obtained for MBT in samples were, therefore, corrected for blank concentrations. However, blanks never contained TBT. The detection limits (three times the standard deviation of the matrix spikes) of MBT, DBT, and TBT in tissues were 5, 1, and 1 ng/g wet wt, respectively. The average recovery rates for monobutyltin trichloride, dibutyltin dichloride, and tributyltin chloride dissolved in hexane, spiked into the muscle of cod (Gadus morhua), and passed through the whole analytical procedure were 85 ( 10, 106 ( 11, and 93 ( 5% (n ) 8), respectively. The recoveries of matrix spikes were calculated based on freshly propylated mixture of external standards. All concentrations refer to butyltin species as the corresponding ion, and they were corrected for the recovery of the internal standard (which was, on average, 82 ( 12%).
Results and Discussion Concentrations and Accumulation Characteristics in Dolphins. Liver contained the highest concentrations of total butyltins (BTs) in dolphins and whales (Table 1). The concentrations of BTs in livers were positively correlated with those in kidneys (p < 0.01) and muscles (p < 0.01). Kidneys recorded the second highest concentration among the various tissues analyzed. Butyltin concentrations in kidneys were 7-10 times lower than those in livers (Figure 2). In an adult female bottlenose dolphin, the concentrations and composition of butyltin compounds in right (500 ng/g wet wt) and left kidneys (520 ng/g wet wt) were comparable. Butyltin
VOL. 31, NO. 1, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
297
TABLE 1. Concentrations of Butyltin Compounds (ng/g wet wt) in Cetaceans Collected from the U.S. Atlantic and Gulf Coasts n
MBT
liver
17
kidney
16
muscle
11
340 (32-2260)b 93 (12-390) 18 (5.8-44) 78 140 20 38
tissue
a
blubberc melon heart brain
1 1 1 1
liver
2
kidney
2
muscle
2
blubber
1
liver
3
kidney
2
muscle
2
DBT Bottlenose Dolphin 960 (54-8300) 67 (5.6-310) 6.4 (2.2-20) 310 4.6 8.4 24
100 (5.8-770) 41 (3.2-220) 16 (4-46) 240 47 22 44
1400 (110-11340) 200 (25-670) 41 (13-110) 630 190 50 110
Atlantic Spotted Dolphin 220 (56-380) 7 (6.6-7.4) 3.2 (2.6-3.6) 12
44 (11-76) 12 (11-13) 9.2 (6.6-12) 80
360 (83-630) 32 (31-33) 21 (14-28) 200
120 (86-160) 20 (14-26) 11 (11-12)
Pygmy Sperm Whale 260 (240-290) 36 (35-38) 5.2 (3.2-7.2)
9 (5-12) 5.8 (4.4-7.2) 5.0 (2.2-7.6)
390 (350-410) 62 (59-65) 21 (16-26)
BTs ) MBT + DBT + TBT (BT values were rounded).
b
Range. c Blubber was from the animal that contained the highest BTs.
compounds were also found in blubber, melon, heart, and brain (Table 1). This feature of high accumulation of butyltin compounds in liver and kidney was similar to those observed for trace metals (such as cadmium, lead, and nickel), but different from those of organochlorine contaminants that concentrate in the lipid-rich blubber of marine mammals. Preferential accumulation of butyltin compounds in the liver as compared to other tissues has been reported in our earlier studies (16, 17). The tendency of butyltin accumulation in the liver and kidney of higher trophic vertebrates may be associated with the presence of and affinity toward sulfydryl groups of glutathione present in these organs (17). Glutathione probably conjugates with electrophilic compounds such as butyltins and may reduce their reactivity and aid in their excretion. The concentrations of BTs in the liver of bottlenose dolphins ranged from 110 to 11 340 ng/g wet wt (mean, 1400 ng/g) (Table 1). In general, the mean concentration of BTs
9
BTsa
94 (16-170) 13 (11-15) 8.8 (4.8-13) 110
FIGURE 2. Relationship of butyltin concentrations between livers and kidneys/muscles of bottlenose dolphins from southeast U.S. coastal waters (sample containing the highest BT concentration was excluded).
298
TBT
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 1, 1997
in bottlenose dolphins from the U.S. Atlantic coast was higher than those of the diseased Mediterranean Sea animals (17) and finless porpoise (Neophocaena phocaenoides) and Risso’s dolphins (Grampus griseus) from Japanese coastal waters (18). A concentration of 11 340 ng/g wet wt found in the liver of a adult male bottlenose dolphin collected in 1989 on Hutchinson Island, Atlantic Ocean, is the highest value reported for a cetacean. This was greater than the previously reported highest concentration of 10.2 µg/g (wet wt) in the liver of a finless porpoise found stranded along the Seto Inland Sea of Japan (16). Butyltin concentrations in the livers of spotted dolphin and pygmy sperm whale were 3-4 times lower than in bottlenose dolphins. Both spotted dolphin and pygmy sperm whale are off-shore species; therefore, the exposure to BTs is expected to be minimal. In contrast, the bottlenose dolphin is primarily a coastal species occupying bay and estuaries and inshore channels between islands. Therefore, the exposure to BTs from antifouling paints on boats and ships might be higher. Total butyltin concentrations in the liver, kidney, and muscle of a captive adult (242 cm; 21 yr) female bottlenose dolphin were 78, 19, and 13 ng/g wet wt, respectively, which were 50-100-fold lower than the concetrations found in freeranging dolphins. This dolphin was collected from the Mississippi Sound in 1978 for the U.S. Navy and was in captivity in New England aquarium and in Sea World of Florida until 1991. The BTs concentration recorded in captive bottlenose dolphin can be considered as a background value in cetaceans collected from Atlantic coastal areas. The composition of TBT and its breakdown products varied depending on the tissue/organ (Figure 3). The percentage of DBT in total butyltin concentration was higher in the liver (61-69%) than in other tissues and organs. This can be explained by the metabolic degradation of TBT to DBT and MBT by cytochrome P-450 enzymes present in the liver (25). Although a distortion in butyltin composition during the decomposition of carcass is expected (24), the composition in various tissues of stranded dolphins was similar to that observed in the captive bottlenose dolphin, which was fresh
TABLE 2. Concentrations of Butyltin Compounds (ng/g wet wt) in the Liver of Bottlenose Dolphins from Different Age Groups age group
sex
n
MBT
DBT
adult
M
4
F
2
juvenile
M
1 2
calf
M
2
F
4
M F
1 1
750 (120-2260)b 430 (100-760) 68 340 (310-360) 170 (44-290) 150 (32-350) 56 148
2390 (450-8300) 930 (290-1570) 230 700 (670-720) 220 (54-370) 560 (70-880) 130 560
newborn a
BTs ) MBT + DBT + TBT (BT values were rounded).
b
BTsa
TBT 250 (5.8-770) 98 (26-170) 22 110 (94-130) 45 (40-50) 52 (10-110) 22 8
3390 (570-11340) 1460 (420-2500) 310 1150 (1120-1160) 440 (140-710) 760 (110-1260) 210 720
Range.
FIGURE 4. Relationship of butyltin concentrations in the liver of bottlenose dolphins with length. FIGURE 3. Composition (%) of TBT and its breakdown products in tissues and organs of cetaceans. when sampled (respective compostion of MBT, DBT, and TBT in captive dolphin was 23, 64, and 13% in liver; 49, 41, and 10% in kidney; and 48, 38, and 14% in muscle). The presence of higher proportions of TBT in muscle, blubber, heart, and brain (Figure 3) suggested the distribution of butyltin compounds throughout the body tissues, possibly due to their binding with erythrocytes. Adult male dolphins contained higher concentrations of BTs than in females. However, in immature stages, females had relatively higher concentrations than males (Table 2). However, the variation in butyltin concentrations between sexes was not significant (p > 0.05), implying that the transfer of butyltin compounds through lactation and gestation may be minimal. Examination of the milk of Ganges river dolphin (Platanista gangetica) showed the presence of very low concentrations of butyltin compounds (26). Butyltin accumulation with the age of bottlenose dolphins was nonlinear (Figure 4). The concentrations increased with length, for both sexes, during early life stages until maturity and then tended to remain constant (Figure 4). This pattern was different from those observed for organochlorines such as PCBs and DDTs, which tend to increase with age during the juvenile stage, both in males and females, because the rate of ingestion of pollutants surpasses the rates of their excretion and metabolization. In adult males, this imbalance is maintained throughout life, resulting in a progressive accumulation of organochlorines with age whereas in females concentrations gradually decrease as a consequence of transfer during
TABLE 3. Annual Variations in Butyltin Concentrations (ng/g wet wt) in Liver of Bottlenose Dolphins BTsa
year
n
1989 1990 1993 1994
Adult 1 2260 8300 770 11340 1 370 1210 120 1700 1 760 1570 170 2500 3 160 450 46 650 (100-250)b (290-610) (5.8-110) (420-960)
1990 1 1994 2 a
MBT
360 190 (68-310)
DBT
TBT
Juvenile 670 130 480 58 (230-720) (22-94)
1160 725 (310-1120)
BTs ) MBT + DBT + TBT (BT values were rounded).
b
Range.
gestation and lactation (27). Similar to our results, butyltin concentrations in the liver of Risso’s dolphins collected from Japanese coastal waters increased until maturity and then reached steady state (18). These results suggest that the factors controlling the accumulation and thereby the toxicity of organotins are different from those of organochlorines. Butyltin compounds are relatively biodegradable, and therefore, the recorded concentrations are a measure of an equilibrium between uptake and elimination rates. Upon exposure, butyltin compounds bind to cell receptors and exert acute toxic effects, including the destruction of plasma membranes followed by lymphocyte depletion and decreased phagocytic activity (13, 14, 28). Peripheral blood neutrophils and lymphocytes were suppressed in channel catfish after a
VOL. 31, NO. 1, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
299
TABLE 4. Concentrations of Butyltin Compounds (ng/g wet wt) in Muscle of Fish Collected from the Gulf of Mexico in 1994 species blue catfish [6]b blue catfish [10] Gafftopsail catfish [10] hardhead catfish [10] brown shrimp [28] brown shrimp [18] white shrimp [15] croaker [5] croaker [6] croaker [12] a
BTs ) MBT + DBT + TBT.
b
wt (g)
location
MBT
DBT
TBT
BTsa
101 (30-207)c 60 (29-272) 104 (22-272) 28 (21-35) 5.7 (1.8-10) 6.2 (2.5-9.8) 10 (7.4-17) 21 (9.2-33) 11 (8.4-15) 43 (20-69)
Mississippi River Estuary, Louisiana
28
86
114
228
Vermilion Bay, Louisiana
22
31
120
173
Lake Barre, Louisiana
29
36
129
194
Lavaca Bay, Texas
33
60
196
289
Calcasieu Lake, Louisiana
43
67
253
363
Galveston Bay, Texas
13
22
59
94
Mississippi River Estuary, Louisiana
22
36
88
146
Mobile Bay, Alabama
44
76
110
230
Lake Borgne, Louisiana
23
35
100
158
Pensacola Bay, Florida
31
35
130
196
Number of samples pooled. c Range.
single injection of 1 mg of TBT/kg body weight (29). TBT concentration of 290 ng/g (1 µM) inhibited phagocytosis and exocytosis of leukocytes in rabbits, and at 1.45 µg/g (5 µM) cytolysis occurred (12). On the contrary, persistent and lipophilic contaminants such as organochlorines tend to bioaccumulate in fatty tissues over a period of time and exert chronic toxic effects in marine mammals. Although the number of samples is small to discern temporal variations, it was noticed that the concentrations of BTs in adult dolphins were lower in 1994 than in earlier years (Table 3). In juveniles, the recorded concentration in 1994 was 27-97% (mean, 62%) of that observed in 1990 whereas in adults it was reduced by 25-57% (mean, 39%). In the United States, several states banned TBT in 1988, and in all states in 1989 TBT was banned in paints applied to boats less than 25 m, but it is still being used on larger vessels and on aluminium-hulled boats. TBT concentrations in Gulf of Mexico oysters in 1990 and 1991 decreased after the regulation of TBT in the United States in 1989 (30). Butyltin compounds persist in sediments over the years, and therefore the exposure to biota persists (31). Despite the reduction in butyltin concentration over the years, the observed values in 1994 are still higher than in off-shore dolphins and suggest the continuing exposure of near-shore dolphins to butyltin compounds. The BTs concentrations observed in the liver of dolphins and whales from the southeast U.S. coasts were compared with those reported from Japan (Figure 5). Cetaceans from Japanese coastal waters included finless porpoise, Risso’s dolphin, dall’s porpoise (Phocoenoides dalli), dwarf sperm whale (Kogia simus), and Ginkgo-toothed whale (Mesoplodon ginkgodens) (16, 18). No data were found on the contamination levels of butyltins in dolphins from other parts of the world. Butyltin concentrations in U.S. dolphins were comparable or higher than those reported for Japan, suggesting a higher exposure in U.S. samples. Relatively higher concentrations in the U.S. dolphins might be related to a higher usage of TBT in the United States. The United States is the major producer and consumer of organotin compounds, accounting for about 35% of the global consumption. The use rate of marine paints containing TBT in the United States was estimated to be 140 t/year by the late 1980s (32). Concentrations in Fish and Trophic Transfer. The concentrations of butyltin compounds in fish muscle and in shrimp collected from the Gulf of Mexico were between 94 and 363 ng/g wet wt (Table 4). Butyltin concentrations in
300
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 31, NO. 1, 1997
FIGURE 5. Comparison of total butyltin concentrations in cetacean livers and fish muscles from the United States and Japan. Values for cetaceans and fish from Japan were cited from refs 18 and 33, respectively. fish muscle were comparable to those reported for Japanese market fish collected in 1991 (Figure 5), but higher than those of fish from Asian countries (24). The concentration range of butyltins in the liver of Gulf coast fish was
