Environ. Sci. Technol. 2004, 38, 112-119
Debromination of the Flame Retardant Decabromodiphenyl Ether by Juvenile Carp (Cyprinus carpio) following Dietary Exposure HEATHER M. STAPLETON,† MEHRAN ALAEE,‡ ROBERT J. LETCHER,§ AND J O E L E . B A K E R * ,† Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, Solomons, Maryland 20688, National Water Research Institute, Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6, and Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario, Canada N9B 3P4
The congener 2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether (BDE 209) is the primary component in a commonly used flame retardant known as decaBDE. This flame retardant constitutes approximately 80% of the world market demand for polybrominated diphenyl ethers (PBDEs). Because this compound is very hydrophobic (log Kow ∼ 10), it has been suggested that BDE 209 has very low bioavailability, although debromination to more bioavailable metabolites has also been suggested to occur in fish tissues. In the present study, juvenile carp were exposed to BDE 209 amended food on a daily basis for 60 days, followed by a 40-day depuration period in which the fate of BDE 209 was monitored in whole fish and liver tissues separately. No net accumulation of BDE 209 was observed throughout the experiment despite an exposure concentration of 940 ng/day/fish. However, seven apparent debrominated products of BDE 209 accumulated in whole fish and liver tissues over the exposure period. These debrominated metabolites of BDE 209 were identified as penta- to octaBDEs using both GC/ECNI-MS and GC/HRMS. Using estimation methods for relative retention times of phenyl substitution patterns, we have identified possible structures for the hexa- and heptabromodiphenyl ethers identified in the carp tissues. Although exposure of carp to BDE 209 did not result in the accumulation of BDE 209 in carp tissues, our results indicate evidence of limited BDE 209 bioavailability from food in the form of lower brominated metabolites.
Introduction Decabromodiphenyl ether (BDE 209) is the principal component in commercial flame retardant formulations referred to as “decaBDE” (1). Additionally, it is a member of the class of compounds collectively known as polybrominated diphenyl ethers (PBDEs), which are becoming increasingly prevalent * Corresponding author phone: (410)326-7205; fax: (410)326-7341; e-mail:
[email protected]. † University of Maryland Center for Environmental Science. ‡ National Water Research Institute. § University of Windsor. 112
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 1, 2004
in the environment (2-5). DecaBDE comprises approximately 80% of the world market demand for PBDEs, which in 2001 was reported at 56 100 metric tons (6). Much concern has been focused on the fate and behavior of lower brominated BDEs in the environment as levels of tetra-, penta-, and hexabromodiphenyl ethers appear to be exponentially increasing in human milk and biota in the environment (4, 7, 8). These lower brominated compounds have log Kow values ranging from 6.8 to 7.8 (9) and biomagnify in aquatic systems (10). In contrast, BDE 209 has a log Kow value of approximately 10 (1) and is not considered to be readily bioavailable (11). Despite the large molecular size and hydrophobicity of BDE 209, low levels have been identified in the tissues of fish from the river Elbe, in Germany (12), and in the blood serum of workers in an electronics dismantling plant (13), suggesting that this congener can be accumulated in tissues. The heavy application of this compound in electronic equipment results in slow leaching of BDE 209 from these products into the environment where it can be transported in the atmosphere (14, 15) and lead to its ubiquitous presence throughout the world. High levels of BDE 209 have also been measured in river sediments (16) and land-applied sewage sludge (17). The stability of BDE 209 in the environment is poorly understood. Very few studies have examined the metabolism of PBDEs in the environment (18) or the abiotic degradation of individual congeners (19). A few studies have demonstrated that BDEs can be debrominated to less brominated BDE congeners photolytically by both UV light, and under certain conditions, by natural sunlight with half-lives ranging from 15 min up to 81 h (19, 20). Several studies have shown an inverse relationship between potential toxicity and number of bromine atoms among the BDE congeners (21,22). Therefore, debromination of BDE 209 in the environment could lead to potentially more toxic degradation products. In a previous laboratory study, Kierkegaard et al. (23) exposed rainbow trout to the commercial formulation decaBDE. They observed very minimal accumulation of BDE 209 and greater accumulation of several hexa- to nonabromodiphenyl ethers in trout tissue. The authors were unable to determine if the lower brominated PBDEs appeared due to debromination of the BDE 209 compound or accumulation of minor components present in the commercial product. In the current study we have exposed juvenile common carp to food spiked exclusively with BDE 209 (>98% purity as reported by Cambridge Isotope Laboratories), not a commercial formulation that may contain debrominated impurities. Hale et al. (2) measured common carp (Cyprinus carpio) PBDE concentrations in Virginia and observed the highest reported concentration detected in fish to date. Additionally, carp appeared to have an unusual BDE congener accumulation pattern suggestive of BDE metabolism. On the basis of this evidence, the present study examines the fate of BDE 209 in exposed carp with respect to bioavailability and possible debromination to lower brominated, and more accumulative, congeners within fish tissues.
Experimental Methods Exposure Tanks. Juvenile carp, approximately 100 mm in length, were purchased from Hunting Creek Fisheries in Thurmont, MD, and transferred to the Chesapeake Biological Laboratory for the experiment. Fish were randomly separated into five, 132-L, round polyethylene tanks. Filtered well water heated to a constant temperature of 22 °C flowed through each tank at approximately 1 L/min, resulting in an hydraulic 10.1021/es034746j CCC: $27.50
2004 American Chemical Society Published on Web 11/04/2003
residence time of approximately 2 h. Air stones were placed in each tank to maintain oxygen saturation in the water and temperature and flow rates were monitored throughout the duration of the experiment. Fish were fed clean food for a week to acclimate them to their new surroundings prior to beginning the experiment. Food Exposure. Fish exposure to BDE 209 was accomplished via consumption of spiked food pellets. Frozen blood worms purchased from San Francisco Bay Brand in Newark, CA, were homogenized in a glass blender and mixed with 20% (by mass) fish food pellets purchased from Hunting Creek Fisheries. BDE 209 was purchased from Cambridge Isotopes Laboratories (>98% pure, Andover, MA), weighed out, and dissolved in 20 mL of cod liver oil to create a concentration of 940 ng/g wet weight. This concentration was chosen to simulate the level of exposure and thus rate of potential uptake that is typically observed in sediments in the environment (20). The oil solution was thoroughly blended with the blood worm mixture and then stored in plastic bags at -20 °C until use. Control food was prepared by homogenizing blood worms with 20% (by mass) fish food pellets and spiking with 20 mL of pure cod liver oil. Fish were fed 1 g/day/fish of either the spiked food pellets or the control food, depending on the tank, for 60 days. After 60 days, all the remaining fish in the tanks were fed control food for 40 days to monitor depuration. Two tanks were used as control exposures and three tanks were used as replicate exposure tanks. Chemicals. All solvents used for extraction and clean up of the extracts were analytical grade solvents purchased from J. T. Baker. BDE congener standards were purchased from Cambridge Isotope Laboratories in Andover, MA. These standards included 2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether (BDE 209, >98% purity), 2,4,4′,6-tetrabromodiphenyl ether (BDE 75), 2,2′,3,4,4′,5,5′,6-octabromodiphenyl ether (BDE 203), and a standard solution containing 39 BDE congeners ranging from mono- to heptaBDEs (EO-5113). Sampling. One fish from each tank was sampled on Days 0, 5, 10, 20, 30, 45, 60, 69, 85, and 100. Fish mass and length were recorded and then euthanized by cervical dislocation. In each fish, the stomach cavity contents were removed and discarded. The liver was isolated, its mass was recorded, and livers from replicate exposures were combined for the analyses of parent exposure compounds. Remaining whole body samples were then homogenized individually in minichoppers and stored in precleaned glass jars at -20 °C until analysis. Whole body and liver samples were extracted for BDE analysis using Soxhlet extraction. A complete report of methods used can be found in (24) and will only be discussed here briefly. Samples were first ground with sodium sulfate, spiked with the surrogate standard (BDE 75), and extracted with dichloromethane in Soxhlets for approximately 24 h. Lipid content was determined on the extracts using gravimetric analysis. Afterward, samples were concentrated in volume using rotoevaporation and then cleaned using alumina chromatography, followed by Florisil chromatography. An internal standard of PCB 204 (2,2′,3,4,4′,5,6,6′octachlorobiphenyl) was added to quantify BDEs. Recovery of the surrogate standard averaged 93 ( 12%. Quantification. BDE 209 is a fully brominated diphenyl ether, and because of its high degree of substitution, it is prone to thermal decomposition under high temperatures typically found in GC injection ports. Recent research attempts to measure BDE 209 using GC/MS techniques have encountered analytical difficulties such as peak broadening, decomposition, and reduced precision (25). To minimize the potential for decomposition of BDE 209, we developed a method using a cool on-column injection gas chromatograph coupled to an electron capture detector (ECD). Using
this method, we could ensure that all the BDE 209 reached the head of the capillary column and reduce decomposition of the standard that typically occurs in high-temperature injection ports. The temperature of the oven was then ramped to elute the compound through the column. Hydrogen and nitrogen were employed as the carrier and makeup gases respectively, and the flow rate of the carrier gas was set to 3 mL/min. Separation was made using a 15 m × 0.25 mm DB-5-MS (0.25-µm film thickness) capillary column. The inlet temperature was set to track the oven temperature program: 80 °C held for 2 min followed by a temperature ramp of 12 °C/min to 140 °C, followed by a temperature ramp of 5 °C/ min to a final temperature of 280 °C, which was held for 20 additional minutes. The detector temperature was set at 300 °C. Using a calibration standard, we observed some minimal decomposition of BDE 209 to several nonaBDEs at low concentrations of BDE 209. The nonlinear response of the nonaBDEs in our calibration curve suggested that the nonaBDEs were therefore a result of decomposition within the GC and not necessarily present in the standard. We thereby conclude that carp were only challenged with BDE 209 in their food. To quantify BDE 209, we applied a power function to our standard calibration curve to estimate concentrations of BDE 209 at low concentrations (
