Environ. Sci. Technol. 2007, 41, 424-430
Levels, Tissue Distribution, and Age-Related Accumulation of Synthetic Musk Fragrances in Chinese Sturgeon (Acipenser sinensis): Comparison to Organochlorines
organic contaminants in aquatic environments (1). Musk xylene was an inducer of mouse CYP2B enzymes, an amine metabolite of musk xylene is a mechanism-based inactivator of mouse CYP2B10 according to in vivo assays (2), and this compound was carcinogenic based on a long-term exposure toxicity study in mice (3). Recent studies reported that some nitro musks such as musk ketone, musk xylene, and their metabolites, 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydeonaphthalene (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[g]-2-benzopyran (HHCB), elicited estrogenic activity in both in vitro and in vivo assays (4-6).
Y I W A N , † Q I W E I W E I , * ,‡ J I A N Y I N G H U , * ,† XIAOHUI JIN,† ZHAOBIN ZHANG,† HUAJUN ZHEN,† AND JIANYI LIU‡ College of Environmental Sciences, Peking University, Beijing, 100871, China, and Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Science, Jingzhou, Hubei 434000, China
Synthetic musk fragrances are used extensively in a variety of personal-care products as surrogates for natural musk, in additives for cigarettes and fish baits, and in technical products such as herbicide formulations and explosives. The worldwide production of nitro musks and polycyclic musks was estimated to be 2500 tons in 1988 (7) and more than 6000 tons in 1996 (1), respectively. In 1981, nitro musks were first identified in the aquatic ecosystem in Japan (8). In the subsequent investigations on the occurrence of musks in the environment, musk compounds were detected in the atmosphere, surface water, sewage, and sediments in Europe and North America, demonstrating that musks are ubiquitous in the environment (1, 9, 10). Because of their properties of lipophilicity (log Kow of polycyclic musks, 4.5-6.3 (1); log Kow of nitro musks, 3.8-4.4 (11)), musk fragrances are bioaccumulative in aquatic biota, which was supported by the residues in fish and invertebrates from both freshwater and marine environment (7, 9, 12, 13). The bioaccumulation factors of HHCB, AHTN, musk xylene, and musk ketone were estimated to be 20-1584, 40-670, 290-40 000, and 60-1380, respectively (14-16), and they were found to be speciesspecific (13, 16). Besides the ubiquitous occurrence of musk in fish and invertebrates, musks have also been detected in high trophic marine organisms such as sharks, seals, and dolphins (17, 18).
Chinese sturgeon (Acipenser sinensis) was listed as a Grade I protected animal in China in 1989, and the observed intersexual phenomenon and sex ratio deviation have suggested that chemicals have posed a risk as environment pollutants. This study analyzed seven musk fragrances in liver, muscle, heart, gonad, stomach, intestines, adipose, gill, pancreas, kidney, gallbladder, and roe from 13 female Chinese sturgeons, and the toxicokinetic behavior of musks were studied and compared with some organochlorines. Of the seven musks, HHCB, AHTN, and musk xylene were detected, and the highest concentrations were observed in adipose tissue: from 33.7 to 62.1 ng/g wet weight (ww), from 1.0 to 5.4 ng/g ww, and from 1.1 to 13 ng/g ww, respectively. Similar to the tissue distribution of DDTs and HCB, musks were detected frequently in high lipid content tissues such as roe, adipose, and liver, suggesting that tissue distribution of musks is controlled by the affinity to lipids. The concentration ratios based on lipid weight between roe and adipose were estimated to be 0.47 for HHCB, 0.58 for AHTN, and 0.51 for musk xylene, and those for the total DDTs and HCB were 0.27 and 0.61, which were relatively low compared with mammals. Relatively high concentrations of p,p′-DDE (68.4-449 ng/g ww) were detected in 10 of total 11 samples, which would cause the feminization of Chinese sturgeons during embryonic development. It was found that lipid-corrected concentrations of HHCB, AHTN, p,p′-DDE, and p,p′-DDD increased with age in female sturgeon, of which the trends were similar to those in fishes and different from those in mammals.
Introduction Synthetic musk compounds, including nitro musks and polycyclic musks, have recently been recognized as important * Corresponding authors phone and fax: 86-10-62765520; e-mail:
[email protected]. Phone: 86-716-8128881; fax: 86-7168228212; e-mail:
[email protected]. † Peking University. ‡ Yangtze River Fisheries Research Institute. 424
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Chinese sturgeon (Acipenser sinensis) is predatory fish and feed at the top of the aquatic food web (19). As one of the 25 extant sturgeon species protected under the convention of the International Trade of Endangered Species, Chinese sturgeon is listed as a Grade I protected animal in China Red Data Book of Endangered Animals (20) due to the rapid decline in its population. While the declining population was attributed to overfishing and dam construction by the public and the Chinese government (21, 22), the risk of environmental pollutants such as endocrine-disrupting chemicals has been neglected (23). Recent studies have clarified the increase of female-male sex ratios (from 0.79 in 1981-1993 (21) to 5.9 in 2003-2004 (24)), the declining activity of sperm, and intersexual phenomenon (21, 25), implying that environmental pollution poses a risk to Chinese sturgeon. In this study, we analyzed seven musk fragrances (HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butylindan (ADBI), musk ketone, musk xylene, musk ambrette, musk tibeten) in liver, muscle, heart, gonad, stomach, intestines, adipose, gill, pancreas, kidney, gallbladder, and roe from 13 Chinese sturgeons, and then the tissue distribution, maternal transfer, and age-related accumulation of musk fragrances were estimated and compared with organochlorines, including hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its related chemicals. 10.1021/es061771r CCC: $37.00
2007 American Chemical Society Published on Web 12/09/2006
TABLE 1. Details of Chinese Sturgeon Samples Used in This Study length (cm) sample date of code collection A0466
2003
A0406 A0410
2004 2004
A0412
2004
A0414 A0408 A0447 A0445 A0403 A0444 A0452 A0449 A0500
2004 2004 2005 2005 2005 2005 2005 2005 2005
tissuea L, M, H, Go, St, I, A, Gi, K R, L, M, H, Go, St, P R, L, M, H, Go, St, I, A, Gb R, L, M, H, Go, St, I, Gi, P R, L, M, I, A, Gi R R, L, M, H, Go, I, Gi, L, M, H, Go, I, A, Gi R R R R R
age wt total (year) (kg) length
body length
24
254
339
285
18 17
174 140
297 288
245 246
24
230
334
287
25 22 19 18 24 23 23 22 22
263 230 192 187 260 224 207 252 227
337 312 303 285 338 320 322 327 317
285 258 247 237 280 270 282 275 261
a L: liver; M: muscle; H: heart; Go: gonad; St: stomach; I: intestines; A: adipose; Gi: gill; P: pancreas; K: kidney; Gb: gallbladder; R: roe. All sturgeons were female.
Materials and Methods Sample Collection. Chinese sturgeon are typical anadromous fish. They live in the sea until the initial reproduction of fish at an average age of ∼14.3 years (21). At the end of spring (June-July) every year, maturing adults stop feeding, leave the ocean, and ascend the main channel of the Yangtze River to spawning grounds. Prespawners (sturgeons with immature gonads) mature for ∼1 year in the Yangtze River to reproduce during the spawning season (early October-early November) of the next year and then return to the sea (21, 26). Offspring arrive in the estuary of the Yangtze River along the main channel of the river the next May. Since the migration route of Chinese sturgeon was blocked by the Gezhou Dam in 1981, the population of fish has declined sharply (22), and artificial propagation was begun to conserve this endangered species (26). Because this sturgeon is listed as a Grade I protected animal (the highest level of protection) in China, the capture of Chinese sturgeon only for scientific purposes must be authorized by the Ministry of Agriculture of the People’s Republic of China, and the number is limited: eight to ten every year in recent years. After artificial propagation, the live sturgeon must be released into the river under the supervision of the local government, but some sturgeon would die. In this study, the roes before artificial fecundation and tissues from the dead fish were collected during 20032005 and frozen immediately at -20 °C until analysis. Ages of fish were determined by counting growth layers in clreithrum, as described previously (19, 21, 24). Details of the samples analyzed in this study are shown in Table 1. Sample Preparation. Approximately 0.5-10 g of tissues (about 0.5-1 g for adipose, 1-2 g for roe and liver, and 5-10 g for other tissues) mixed with ∼20 g of Na2SO4 were spiked with surrogate standard (phenanthrene-d10, PCB 121, and PCB 198) and Soxhlet extracted for 24 h using 250 mL of dichloromethane/methanol (7:3 v/v) mixture solution. Twenty percent of the extract was used to determine the lipid percentage. The extracts were rotated to dryness and heated at 65 °C for ∼30 min, and lipid amounts were determined gravimetrically. The remaining 80% of the extract was subjected to acetonitrile partitioning to remove lipid as described previously (27). The extracts were rotoevaporated and reconstituted by 1 mL of hexane and then added to a separatory funnel containing 50 mL of hexane-saturated acetonitrile and 20 mL of hexane. After the solution was shaken for 5 min and partitioned, the acetonitrile layer was
collected. This procedure was repeated, and a total of 100 mL of hexane-saturated acetonitrile was collected and transferred to a 1-L separation funnel containing 500 mL of 5% NaCl solution and 50 mL of hexane. After the solution was shaken, the water layer was removed and the hexane was collected. These procedures were repeated, and a total of 100 mL of hexane was obtained. About 10 g of Na2SO4 was used to remove moisture, and then the hexane was concentrated to exactly 50 mL. Half of the hexane was stored, and the rest was concentrated to ∼1 mL and passed through a glass column (10-mm i.d.) containing 10 g of 5% H2Odeactivated neutral Al2O3 (200 mesh size, Shanghai Ludu Chemicals, Shanghai, China) for cleanup. This column was eluted with 30 mL of high-purity hexane and 30 mL of hexane/ dichloromethane (3:1 v/v). The eluant was dried and dissolved in 0.2 mL of hexane for gas chromatography/mass spectrometry (GC/MS) analysis. The information about chemicals and standards and chemical analysis is provided in Supporting Information. Quantitation and Quality Assurance Quality Control. All equipment rinses were done with methanol to avoid sample contamination, and a procedural blank was analyzed with every set of seven samples. Quantification of the musks, HCB, HCHs, and DDTs in biota samples was carried out by using relative response factors determined from calibration standard runs. To automatically correct the losses of analytes during extraction or sample preparation, as well as for variations in instrument response from injection to injection, surrogate standards were used in this study. Phenanthrened10, PCB 121, and PCB 198 do not exist in the environment and were used as surrogate standard for musks and organochlorines, respectively, in previous studies (18, 28-30). In the present study, HCBs, HCB, p,p′-DDMU, o,p′-DDD, p,p′DDD, o,p′-DDE, and p,p′-DDE were quantified in sample extracts relative to PCB 121, and o,p′-DDT, p,p′-DDT were quantified relative to PCB 198. Phenanthrene-d10 was used as a surrogate standard for musks as exemplified in previous papers (17, 18), because the surrogate shows similar physicochemical properties such as logarithm octanol-water partition coefficient (4.57 for phenantharene (31) and 4-6 for musks (1, 11)) and retention time on GC column with those of musks. For calibration purposes, four different concentrations of compounds (10, 25, 50, and 100 µg/L for musks, o,p′-DDT, and p,p′-DDT; 5, 20, 50, and 100 µg/L for HCB, HCHs, and other DDTs) and their respective surrogate standards were analyzed by GC/MS to obtain relative response factors for all standards. For sample analysis, the procedure described above was validated for recovery experiment by choosing a roe sample (A0410) with low pollution level as spiked sample. Analyte addition was made with the criterion of at least three times the original concentration. The six replicate roe samples, spiked with 80 ng of musks, 200 ng of DDTs and HCHs, and 600 ng of HCB, and one roe sample (matrix blank sample) were analyzed to determine the general recovery rate. The recoveries for spiked samples were 89-120% for HCHs and HCB, 99-113% for DDTs, 74-111% for musks, 70-102% for PCB 121, 66-89% for PCB 198, and 70-94% for phenanthrene-d10. Since we found that lipid amount was the main factor affecting the recovery for analysis because of the emulsion during the extraction, the amount of a sample for each tissue was controlled to make the lipid amount lower than that in the spiked roe samples. For all the biota samples for analysis, the recoveries of PCB 121, PCB 198, and phenanthrene-d10 were 84 ( 14, 70 ( 15, and 85 ( 15%, respectively. For HHCB and AHTN, the blank samples were used to correct the sample value due to the ubiquity of these chemicals, and the limits of quantitation (LOQs) were set to be the three concentrations in the blank samples. The VOL. 41, NO. 2, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. GC/MS chromatograms of HHCB, AHTN, and musk xylene in adipose samples of Chinese sturgeon. (a) Musk xylene standards; (b) musk xylene in samples; (c) HHCB and AHTN standard mixture; (d) HHCB and AHTN in samples; identification ions were m/z 243 and 258 for HHCB and AHTN and m/z 282 and 297 for musk xylene. detection limits for HHCB, AHTN, ADBI, musk ambrette, musk xylene, musk tibeten, and musk ketone were 1.3, 0.5, 0.2, 0.5, 0.5, 0.2, and 0.5 ng/g, respectively. And the detection limits were 0.05 ng/g for p,p′-DDMU, o,p′-DDD, p,p′-DDD, o,p′-DDE, and p,p′-DDE, 0.2 ng/g for o,p′-DDT and p,p′DDT, 0.07 ng/g for HCB, and 0.1 ng/g for HCHs.
Results and Discussion Residue Levels. Figure 1 shows the GC/MS chromatograms of HHCB, AHTN, and musk xylene in an adipose sample (A0410 in Table 1). Of the seven musk fragrances, only HHCB, AHTN, and musk xylene were detected mainly in liver, adipose, and roe (Table 2), and the concentrations of other musk fragrances were all below the detection limit. While HHCB was detected in three liver samples (n ) 7;
