Environ. Sci. Technol. 2006, 40, 4653-4658
In Vivo and In Vitro Debromination of Decabromodiphenyl Ether (BDE 209) by Juvenile Rainbow Trout and Common Carp H E A T H E R M . S T A P L E T O N , * ,† BRIAN BRAZIL,‡ R. DAVID HOLBROOK,§ CARYS L. MITCHELMORE,| RAE BENEDICT,| ALEX KONSTANTINOV,⊥ AND DAVE POTTER⊥ Duke University, Nicholas School of the Environment and Earth Sciences, Durham, North Carolina, United States Department of Agriculture, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, National Institute of Standards and Technology, Gaithersburg, Maryland, University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Solomons, Maryland, and Wellington Laboratories, Guelph, Ontario, Canada
Decabromodiphenyl ether (BDE 209), the major congener in the high volume industrial flame retardant mixture “DecaBDE”, has recently been shown to be metabolized by carp. To further explore this phenomenon, juvenile rainbow trout were exposed to BDE 209 via the diet for a five month period. Analysis of the whole body homogenate, liver, serum, and intestinal tissues revealed that BDE 209 accumulated in rainbow trout tissues and was most concentrated in the liver. In addition to BDE 209, several hepta-, octa-, and nonaBDE congeners also accumulated in rainbow trout tissues over the same period as a result of BDE 209 debromination. Based on the total body burden of the hepta- through decaBDE congeners, uptake of BDE 209 was estimated at 3.2%. Congener profiles were different among whole body homogenate, liver, and serum, with the whole body homogenates having a greater contribution of the debrominated biotransformation products. Extracts of the rainbow trout whole body homogenates were compared with extracts from a previous experiment with common carp. This comparison revealed that BDE 202 (2,2′,3,3′,5,5′,6,6′-octabromodiphenyl ether) was a dominant debromination product in both studies. To determine whether the observed debromination was metabolically driven, liver microsomal fractions were prepared from both common carp and rainbow trout. Analysis of the microsomal fractions following incubation with BDE 209 revealed that rainbow trout biotransformed as much as 22% of the BDE 209 mass, primarily to octa- and nonaBDE congeners. * Corresponding author phone: (919) 613-8717; fax: (919) 6848741; e-mail:
[email protected]. † Duke University, Nicholas School of the Environment and Earth Sciences. ‡ United States Department of Agriculture, National Center for Cool and Cold Water Aquaculture. § National Institute of Standards and Technology. | University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory. ⊥ Wellington Laboratories. 10.1021/es060573x CCC: $33.50 Published on Web 06/24/2006
2006 American Chemical Society
In contrast, carp liver microsomes biotransformed up to 65% of the BDE 209 mass, primarily down to hexaBDE congeners. These microsomal incubations confirm a metabolic pathway for BDE 209 debromination.
Introduction Decabromodiphenyl ether (BDE 209) is the major component (>97%) of the commercial flame retardant mixture known as decaBDE. Almost half of the annual global decaBDE demand (greater than 56 000 metric tons in 2003 (1)) is used in the flame retarding high impact polystyrene, the plastic resin commonly used in TV and radio cabinets. Other applications of decaBDE include use in polyester fiber additives, coatings for automobile fabrics, polystyrene, and acrylonitrile-butadiene-styrene rubber (ABS) in which decaBDE can be found in concentrations ranging from 6 to 22 wt % (2). DecaBDE is one of three commercial mixtures of a class of brominated flame retardants referred to as polybrominated diphenyl ethers, or PBDEs. The two other commercial mixtures, known as PentaBDE and OctaBDE, have recently been banned from the European Union (3), and production has been voluntarily phased out in the United States (4) over the past few years due to increasing concern over their persistence, bioaccumulation, and potential toxicity. Currently there is no regulation on DecaBDE; however, several agencies are conducting in depth risk assessments to determine its environmental behavior and degradation potential. Concentrations of BDE 209 have been measured and reported in biota, sediments, biosolids, and house dust (59). In the environment, BDE 209 is susceptible to degradation, via debromination, following exposure to UV light (10, 11) and by microorganisms (12). Both of these debromination pathways can potentially lead to the formation of BDE congeners, which are similar to the congeners found in the banned penta- and octaBDE commercial mixtures. In a recent study, the accumulation and debromination of BDE 209 in juvenile carp was assessed (13). Less than 1% of the BDE 209 exposure was assimilated by carp; however, several penta- to octaBDE congeners significantly accumulated during the exposure period. In a separate study, Kierkegaard et al. (14) exposed juvenile rainbow trout to a diet spiked with the commercial decaBDE mixture and also observed apparent debromination of BDE 209 down to hexathrough nonaBDE congeners. Both of these studies suggest that fish have a metabolic capacity to biotransform PBDEs via a debromination pathway. The preferential removal of meta- substituted bromines observed during in vivo studies with carp (13, 15) may implicate a class of selenocysteine proteins known as deiodinase (DI) enzymes which are crucial components for thyroid hormone regulation (16, 17). DI enzymes regulate tissue specific production of triiodothyronine (T3) by removing an iodine atom from the meta position of thyroxine (T4) in a manner very similar to the debromination of PBDE congeners observed in fish tissues. The present study was undertaken to compare the in vivo and in vitro debromination potential of BDE 209 by juvenile rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio). Specifically, we were interested in examining the tissue specific accumulation of BDE 209 and its debrominated products in serum, liver, intestinal tissue, and the whole body homogenate of the rainbow trout. The objectives of this study were (1) to examine the species specific differences in the uptake of BDE 209 from both direct uptake of BDE 209 and through accumulation of its biotransforVOL. 40, NO. 15, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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mation products; (2) to examine tissue specific uptake patterns of BDE congeners in rainbow trout; and (3) to compare in vitro biotransformation of BDE 209 to the in vivo accumulation of BDE 209 in both rainbow trout and common carp. To confirm metabolic debromination of BDE 209, microsomal fractions were prepared from freshly isolated livers of rainbow trout and carp, and incubated with BDE 209 in vitro.
Materials and Methods In Vivo Fish Exposure. Juvenile rainbow trout (av weight 91.2 ( 5.8 g) were reared at the U.S. Department of Agriculture’s National Center for Cool and Coldwater Aquaculture in Kearneysville, WV. Sixty fish were randomly separated into four square polyethylene flow-through tanks. Fish in all tanks were acclimated to the control diet (nonspiked food) for a period of approximately three weeks prior to the start of the exposure in which experimental fish were fed BDE 209 amended food. One tank was fed a control diet throughout the experiment and three tanks were fed a diet spiked with decabromodiphenyl ether (BDE 209). The BDE 209 concentration in the spiked food was 940 ( 14 ng/g wet weight. See Supporting Information for details on the preparation of the control and spiked food mixtures. During the five month study period, fish were fed spiked or control food at a rate of 1% of their body weight/day, Monday through Friday, of each week. The weights of all fish in the tanks were recorded once a week during the first two months, and once a month during the last three months of the study. The average fish mass/tank was used to calculate the feeding rate of 1% body weight/day. One fish from each tank was sampled on nine different time points throughout the five month exposure. Fish were sacrificed using MS-222 as a euthanizing agent. Fish mass and length were recorded and then individual fish were dissected to isolate the liver and intestinal tissues. Blood was drawn from individual fish prior to the initiation of the exposure and on three different time points during the last three months of the exposure. Heparinized syringes were used to draw the blood from the dorsal aorta and transferred to evacuated containers, centrifuged for twenty minutes to isolate the serum, and then stored at -20 °C until analysis. Blood samples drawn from the fish prior to the exposure were pooled and analyzed to determine the background levels in the rainbow trout serum. The remaining fish carcass was homogenized and stored at -20 °C prior to analysis for PBDEs. Sample extraction and analysis was similar to our previous studies (13, 15), and further information on the methods and quality assurance can be found in the Supporting Information. In Vitro Incubations. Rainbow trout livers were obtained from three individuals at the USDA Cold Water Aquaculture Facility, and common carp livers were obtained from three individual carp reared at the Chesapeake Biological Laboratory (see Supporting Information). Livers were removed and snap frozen in liquid nitrogen with storage at -80 °C until microsomal preparation. Microsome preparation was adapted from the procedures of Nilsen et al., 1998 (18); further information on the microsomal preparation and incubation can be found in the Supporting Information.
Results and Discussion In Vivo Experiment. Fish mass and lipid content were continually monitored during the experiment as an indication of fish growth health. Information on the growth rates and lipid content of the fish can be found in the Supporting Information. The concentration of BDE 209 in the spiked food was measured both prior to the initiation of the experiment (939 ( 14 ng/g wet weight; n ) 3) and after the completion of the experiment (936 ( 13 ng/g wet weight; n 4654
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FIGURE 1. BDE 209 concentrations (ng/g wet weight) measured in liver, serum, and carcass of the exposed fish (A). BDE 209 concentrations plotted in (B) are the same values including a comparison of BDE 209 levels measured in control fish carcasses. Values represent the mean and standard deviation of three replicates. ) 3) to ensure no degradation of BDE 209 occurred during the course of the experiment. Figure 1 displays the concentration of BDE 209 measured in the whole body homogenate of both the exposed and control fish, in addition to levels measured in the serum and liver tissue of the exposed group. Liver displayed the greatest accumulation of BDE 209 during the exposure study increasing from
