Environ. Sci. Technol. 2005, 39, 3430-3434
Occurrence of Synthetic Musk Fragrances in Marine Mammals and Sharks from Japanese Coastal Waters HARUHIKO NAKATA* Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan
In this study, the occurrence of the polycyclic musk fragrances HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8hexamethylcyclopenta[g]-2-benzopyran) and AHTN (7-acetyl1,1,3,4,4,6-hexamethyltetrahydeonaphthalene) in marine mammals and sharks collected from Japanese coastal waters is reported. HHCB was present in the blubbers of all finless porpoises (Neophocaena phocaenoides) analyzed (n ) 8), at levels ranging from 13 to 149 ng/g on a wet weight basis. A fetus sample of finless porpoise contained a notable concentration of HHCB (26 ng/g wet wt), suggesting transplacental transfer of this compound. Among 12 tissues and organs of a finless porpoise analyzed, the highest HHCB concentration was found in blubber, followed by kidney. This indicates that HHCB accumulates in lipidrich tissues in marine mammals, which is similar to the accumulation profiles of persistent organochlorines, such as PCBs and DDTs. In general, the residue levels of AHTN and nitro musks were low or below the detection limits in finless porpoises, implying either less usage in Japan or high metabolic capacity of these compounds in this animal. HHCB was also found in the livers of five hammerhead sharks (Sphrna lewini) from Japanese coastal waters, at concentrations ranging from 16 to 48 ng/g wet wt. Occurrence of HHCB in higher trophic organisms strongly suggests that it is less degradable in the environment and accumulates in the top predators of marine food chains. This is the first report on the accumulation of synthetic musk fragrances in marine mammals and sharks.
Introduction Synthetic musk fragrances have been used in a wide variety of personal care products, detergents, and household cleaners in the world. In 1981, nitro musks, such as musk xylene and musk ketone, were detected for the first time in the environment, in river water and biota from Tama River and Tokyo Bay, Japan (1). Synthetic fragrances including the polycyclic musks HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8hexamethylcyclopenta[g]-2-benzopyran) and AHTN (7acetyl-1,1,3,4,4,6-hexamethyltetrahydeonaphthalene) have been found in air, sediment, crustacean, mussel, and fish samples in freshwater and marine environment (2-4) as well as human tissues (5-7) from the United States and European countries. Furthermore, high concentrations of musks were detected in the effluents and the sewage sludges of wastewater treatment plants in Germany, Sweden, Netherlands, Swit* Corresponding author phone/fax: +81-96-342-3380; e-mail:
[email protected]. 3430
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zerland, United States and Canada (8-11). These results indicate that discharge from wastewater treatment plants is a major source of synthetic musk fragrances and that these compounds have a potential for bioaccumulation in aquatic system. Regarding the studies of high trophic organisms, musk xylene was identified in the eggs of wild birds from Germany and Norway, at levels in herring gull’s eggs ranging from 2 to 40 ng/g on a lipid weight basis (12). It has been reported that high concentrations of HHCB and AHTN were detected in the liver of Danish otter (Lutra lutra) (12). However, no investigation has focused on the residue levels of synthetic musks in top predators of the marine food web, such as marine mammals and sharks. Because of highly lipophilic character of HHCB and AHTN (log Kow of HHCB and AHTN are 5.9 and 5.7, respectively (13)), they may accumulate in higher trophic animals of the marine environment. Musk xylene, musk ketone, and AHTN have been shown to present estrogenic activity in an assay using human breast cell (14) and transgenic zebrafish (15). It has been demonstrated that musk xylene induces and inhibits the activities of cytochrome P450 enzymes (CYP), especially CYP2B in experimental animals (16). Furthermore, some metabolites and/or transformation products of synthetic musks exhibited a strong potential for environmental persistency (17). While the effective concentrations were generally higher than those levels found in environmental matrixes, only few relevant studies have been carried out on potential persistency and also long-term and species specific adverse effects of synthetic musks. On the basis of these backgrounds, the occurrence of synthetic musk fragrances, such as two polycyclic musks (HHCB and AHTN) and three nitro musks (musk xylene, musk ketone, and musk ambrett) were examined in 11 finless porpoises, including three fetuses, and five hammerhead sharks collected from the Ariake Sea and Yatsushiro Sea, western Japan. To understand the body distribution of musk fragrances in marine mammal, 12 tissues and organs of an adult female finless porpoise were also analyzed.
Experimental Section Chemicals. HHCB and AHTN standards were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Musk-xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene), musk ketone (4-tert-butyl-2,6-dimethyl-3,5-dinitroacetophenone), musk ambrett (1-tert-butyl-3,5-dinitro-2-methoxy-4-methylbenzene or 2,6-dinitro-3-methoxy-4-tert-butyltoluene) were purchased from Sigma-Aldrich (St. Louis, MO). The purities of standards were 51, 98, 98, 99, and 98% for HHCB, AHTN, musk xylene, musk ketone, and musk ambrett, respectively. Deuterated polycyclic aromatic hydrocarbon (phenanthrened10) obtained from Cambridge Isotope Laboratory (Andover, MA) was used as the surrogate standard in this study. Samples. Eleven finless porpoises including three pairs of mothers and fetuses were collected from the Ariake Sea and Yatsushiro Sea, western Japan, during 1999 and 2002 (Figure 1). All of the animals were stranded along the coastal area or accidentally caught by fishing nets. Five hammerhead sharks were collected from the Ariake Sea in June 2004. All samples were stored at -20 °C until chemical analysis. Chemical Analysis. The blubbers of finless porpoises were analyzed in this study. However, two fetuses were very immature (body length 25 and 33.5 cm), and the blubber layers were not developed. Therefore, skins were collected and used for chemical analysis in these fetuses. Twelve organs and tissues of a finless porpoisesblubber, liver, kidney, 10.1021/es050199l CCC: $30.25
2005 American Chemical Society Published on Web 04/05/2005
TABLE 1. Recovery Percentage with a Standard Mixture of Known Concentrations compound
tRa (min)
m/z
recovery (%)b
HHCB AHTN musk xylene musk ketone musk ambrett phenanthrene-d10
21.53 21.96 21.88 25.64 20.99 19.04
243 243 282 279 253 188
100 ( 0.4 92 ( 1.6 104 ( 1.3 98 ( 19 108 ( 3.5 107 ( 0.1
a Retention time. b Fifty nanograms of a standard mixture was used for the recovery test.
TABLE 2. Concentrations of HHCB (ng/g wet wt) in Three Finless Porpoises with the Duplicate Analysisa FIGURE 1. Map showing the sampling locations in this study. stomach, muscle, lung, blood, brain, heart, small intestine, ovary, and placentaswere analyzed to understand the body distribution of musk fragrances in the marine mammal. For hammerhead sharks, the liver tissues were analyzed. Approximately 1-4 g of tissues were ground with sodium sulfate and extracted with mixed solvents of dichloromethane and hexane (8:1) for 7 h using a Soxhlet apparatus. A portion of the extract was used for lipid content measurement by evaporating the solvent until a constant weight was obtained. Fifty nanograms of phenantherene-d10 was spiked into an aliquot of the extract as a surrogate standard. Lipid in the sample extract was removed by gel permeation chromatography (GPC) using a Bio-beads S-X3 (Bio-Rad Laboratories, Hercules, CA) packed glass column (380 mm × 22 mm i.d.). A mixture of 50% hexane in dichloromethane (DCM) was used as the mobile phase at a flow rate of 5 mL/min. The first 60 mL of eluate was discarded, and the following 80 mL fraction, which contained HHCB, AHTN, musk xylene, musk ketone, musk ambrett, and surrogate, was collected. The eluted solvent was concentrated and passed through a 1.5 g activated silica gel (Wako-gel S-1, Wako Pure Chemical Co. Ltd, Japan) packed glass column for further fractionation. The first fraction that eluted with 60 mL of hexane was discarded in order to remove some interfering compounds in the next fraction. The second fraction, eluting with 60 mL of 30% dichloromethane in hexane and containing all synthetic fragrances, was collected. This fraction was concentrated to 200 µL and injected into a gas chromatograph interfaced with a mass spectrometer (GC-MS, Agilent 6890 and 5973 Series). Injections were made both in scan and SIM modes. The selected ions were monitored at m/z 243, 258, 213 for HHCB; 243, 258 and 159 for AHTN; 282 and 297 for musk xylene; 279 and 294 for musk ketone; 253 and 268 for musk ambrett; and 188 and 94 for phenenthrene-d10. The GC column used was DB-1 (J&W Scientific Inc.) fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm of film thickness). The oven temperature was programmed from 80 to 160 °C at a rate of 10 °C/min and held for 10 min, the temperature was increased to 300 °C at a rate of 3 °C/min, with a final hold time of 20 min. The temperatures of the injector and detector were set at 270 and 300 °C, respectively. Helium was used as a carrier gas. Quality Control. A standard mixture containing all synthetic musks of interest was used to determine the general recovery rates of the compounds through the analytical procedure. Salad oil (0.3 g), which did not contain detectable quantities of synthetic musks, was spiked with 50 ng of the standard mixture. Three replicate analyses were performed, and the average recoveries of musk fragrances ranged from 92% for AHTN to 108% for musk ambrett (Table 1). In addition
sample
result I
result II
average
FP-1 FP-4 FP-6
20.2 158 11.6
21.5 149 15.1
20.9 153.5 13.4
a Concentrations of musk xylene, musk ketone, and musk ambrett were all less than the detection limits in these samples.
to the recovery test, the reproducibility of synthetic musks concentrations in samples was examined. The blubbers of three finless porpoises (FP-1, -4, -6) were analyzed in duplicate. The average concentrations of HHCB were 20.9 ng/g wet wt for FP-1, 153.5 ng/g for FP-4, and 13.4 ng/g for FP-6, and variations of the concentrations between results 1 and 2 were relatively small (Table 2). This suggests that the analytical method is suitable for routine analysis of synthetic musks in biological samples. A procedural blank was analyzed with every set of six samples to confirm interfering peaks in chromatograms and to correct sample values, if necessary. Blank peaks were observed in the chromatograms, as discussed below. The concentrations were reported as below the detection limit if the peak height was not greater than the specified threshold (signal-to-noise ratio of 3 in the blank chromatogram). The detection limits for HHCB, AHTN, musk xyene, musk ketone, and musk ambrett in samples were 8.8, 9.1, 1.9, 2.3, and 2.5 ng/g, respectively.
Results and Discussion Residue Levels. GC-MS chromatograms and fragmentation patterns of HHCB in finless porpoise and standard mixture are shown in Figure 2. The retention time and fragmentation patterns of HHCB in finless porpoise blubber agreed well with those in the standard mixture. While the blank contained HHCB, the abundance of this under SIM using m/z 243, 258, and 213 was approximately 2 orders of magnitude less than that in samples and standard. HHCB was detected in blubber samples of all finless porpoises (n ) 8), ranging in concentrations from 13 to 149 ng/g on a wet weight basis (Table 3). The highest concentration was found in an adult female. Despite the occurrence of HHCB in finless porpoise, AHTN was detected only in an adult female, and the concentration was low, 9.6 ng/g wet wt, close to the limit of detection. Concentrations of musk xylene, musk ketone, and musk ambrett were all below the detection limits in finless porpoises. In the United States and European countries, musk xylene and musk ketone are ubiquitous contaminants in the environment as well as in water and sludge samples from the sewage treatment plants. For example, concentrations of musk ketone in fish, mussels, and lobsters from the eastern coast of Canada and Lake Ontario are 1-2 orders of magnitude higher than the levels of musk xylene and polycyclic musks (4). Significant conVOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. GC-MS chromatograms (SCAN mode) and fragment patterns of HHCB in the blubber of finless porpoise and a standard mixture of musk fragrances.
TABLE 3. Concentrations of Synthetic Musk Fragrances (ng/g wet wt) in Finless Porpoises and Their Fetuses Collected from the Ariake Sea and Yatsushiro Sea, Japan FP-1 sampling year sex length (cm) weight (kg) tissue analyzed lipid content (%) HHCB AHTN musk xylene musk ketone musk ambrett recovery (%)b a
FP-2
FP-3
FP-4
FP-5
FP-8
FPF-1a
FPF-2a
FPF-3a
2000
1999
1999
2001
1999
2001
2002
2000
1999
1999
female 110 23.4 blubber 84 22
