Debromination of Polybrominated Diphenyl Ether Congeners BDE 99

(GLIER), University of Windsor,. Windsor, Ontario, Canada N9B 3P4. Polybrominated diphenyl ether (PBDE) congener patterns in biota are often enriched ...
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Environ. Sci. Technol. 2004, 38, 1054-1061

Debromination of Polybrominated Diphenyl Ether Congeners BDE 99 and BDE 183 in the Intestinal Tract of the Common Carp (Cyprinus carpio) HEATHER M. STAPLETON,† 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, and Great Lakes Institute for Environmental Research (GLIER), University of Windsor, Windsor, Ontario, Canada N9B 3P4

Polybrominated diphenyl ether (PBDE) congener patterns in biota are often enriched in tetra-, penta-, and hexabrominated diphenyl ethers, which is believed to result from the use of the commercial “pentaBDE” formulation. However, our evidence suggests that debromination of PBDEs occurs within fish tissues leading to appreciable accumulation of less brominated congeners. This suggests that PBDE body burdens can reflect both direct uptake from exposure and debromination of more highly brominated congeners. We conducted two independent dietary exposure studies using the common carp (Cyprinus carpio) to trace the fate of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE 99) and 2,2′,3,4,4′,5′,6-heptabromodiphenyl ether (BDE 183) in fish tissues. Carp were fed food spiked with individual BDE congeners for 62 d, and depuration was monitored during the following 37 d. Significant debromination was observed, converting BDE 99 to 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47) and BDE 183 to 2,2′,4,4′,5,6-hexabromodiphenyl ether (BDE 154) and another as yet unidentified hexaBDE congener. The BDE 99 concentration rapidly declined from 400 ( 40 ng/g ww in the food to 53 ( 12 ng/g ww in the gut content material sampled 2.5 ( 1 h following feeding. At least 9.5 ( 0.8% of the BDE 99 mass in the gut was debrominated to BDE 47 and assimilated in carp tissues. In the BDE 183 exposure, approximately 17% of the BDE 183 mass was debrominated and accumulated in carp tissues in the form of two hexa-BDE congeners. In both exposure studies, the concentration of the exposure compound decreased significantly in the gut within 2.5 ( 1 h following ingestion. This rapid decrease in the concentration of the BDE congeners could not be explained entirely by debromination to quantified products or fecal egestion. Reactions occurring within the gut transform BDE congeners to other products that may accumulate or be excreted. Further studies are needed to identify and determine the effects of these BDE metabolites.

* Corresponding author phone: (410)326-7205; fax: (410)326-7341; e-mail: [email protected]. † University of Maryland. ‡ University of Windsor. 1054

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Introduction Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants that are becoming more prevalent in the environment (1-3). Our previous study examining the bioaccumulation of specific BDE congeners has shown that 2,2′,4,4′,5-pentabromodiphenyl ether (BDE 99) does not accumulate in tissues of the common carp (4), similar to several field observations (5-7). This observation is intriguing considering that this congener typically accounts for 1060% of the BDE congener burden in most other biota and is a dominant congener in the commercial products (8). Opperhuizen et al. (9) proposed that organic molecules with a cross-sectional area greater than 9.5 Å are restricted from passing through biological membranes because of their large size. BDE 99 has an effective cross-sectional area of 9.6 Å (10), which suggests that it may be too large to pass the intestinal lining of carp. However, measurable levels of BDE 99 have been observed in other fish species (7, 11, 12) and in humans (13-15), and bioaccumulation has been observed under laboratory exposure conditions (10, 16, 17). In fact, very high bioaccumulation has been observed for both BDE 99 and BDE 47 (13-14 × 105 mL/g dw) in exposed blue mussels relative to some PCB congeners (1.1-2.7 × 105 mL/g dw) (16). In a dietary exposure study, BDE 99 and BDE 47 uptake efficiencies ranged from 60 to 100% in pike (Esox lucius) tissues, while the uptake efficiencies of five other PCB congeners ranged from 45 to 70% (10). These observations imply that BDE 99 is quite capable of passing through biological membranes. Therefore, the most likely reason no appreciable uptake of BDE 99 is observed in carp tissues is due to efficient metabolism. The high assimilation efficiency (93 ( 10%) of 2,2′,4,4′tetrabromodiphenyl ether (BDE 47) observed in our previous carp exposure study (4), combined with the similarities in the structures of BDE 99 and BDE 47, suggests that some mechanism within carp tissues is responsible for removing the meta-substituted bromine atom of BDE 99, resulting in the formation of BDE 47. Debromination of BDE 99 would result in a surplus of BDE 47 that could bioaccumulate in carp tissues. In a previous study, Kierkegaard et al. (18) observed accumulation of several hexa- to nona-BDE congeners following dietary exposure to decabromodiphenyl ether (BDE 209) in tissues of rainbow trout. Their results suggested that debromination of BDE 209 was occurring in rainbow trout tissues. We have recently confirmed BDE 209 debromination in the common carp (22). In a preliminary study, we also observed no accumulation of 2,2′,3,4,4′,5′,6-heptabromodiphenyl ether (BDE 183) following exposure to spiked food. We have therefore conducted two exposure treatments to independently assess the fate of BDE 99 and BDE 183. To gain a better understanding of any potential debromination mechanisms, we amended our sampling protocol to include examination of the food within the intestinal tract of carp following dietary exposure. On each sampling day, carp were sacrificed 2.5 ( 1 h following feeding to examine the changes in exposure concentration of BDE 99 and BDE 183 in the food as it passed through the intestinal tract. Our specific objectives were to quantify the percentage of BDE exposure debrominated and accumulated within carp tissues and gain insight into the most probable pathway by which BDEs may be biotransformed.

Experimental Methods Tank Design. Juvenile carp, approximately 100 mm in length, were purchased from Hunting Creek Fisheries in Thurmont, 10.1021/es0348804 CCC: $27.50

 2004 American Chemical Society Published on Web 01/16/2004

MD, and transferred to the Chesapeake Biological Laboratory in Solomons, MD. Fish were randomly assigned to eight, 132-L, round, polyethylene tanks in a random block design. Filtered well water was heated in a head tank to maintain a constant temperature of 22 °C and supplied to each of the individual tanks at approximately 1.0 L/min. Under these settings, water in the tanks had a 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 1 week to acclimate them to their surroundings prior to beginning the experiment. Food Exposure. Exposure to individual BDE congeners was accomplished by spiking food pellets with the parent compounds dissolved in a fish oil matrix. Frozen blood worms (San Francisco Bay Brand, Newark, CA) were homogenized in a glass blender and mixed with 20% (by mass) fish food pellets (Hunting Creek Fisheries). BDE congeners 99 and 183 (>98% purity, Cambridge Isotopes Laboratories, Andover, MA) were weighed, and each dissolved in separate 20-mL aliquots of cod liver oil. The oil solution was thoroughly blended with the blood worm mixture and stored frozen in plastic bags 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. There were three replicate tanks and one control tank for each exposure, with 8 tanks total and 12 fish in each tank. Fish in the exposure tanks were fed 1.0 g of the spiked food pellets fish-1 d-1 while the two control tanks were fed 1.0 g of the control food fish-1 d-1. In the first treatment, juvenile carp were exposed to BDE 99 at a rate of 400 ( 40 ng d-1 fish-1 with spiked food pellets fed daily for 62 d, while the second treatment was exposed to BDE 183 at a rate of 100 ( 15 ng d-1 fish-1. After 62 d of exposure, the remaining carp were fed clean, control food pellets for 37 d during which depuration over time was monitored. Before exposure began, one carp from each exposure tank was sacrificed and analyzed for background levels of BDEs in their tissues. To ensure that BDE 99 was not lost from the food to the surrounding water in the exposure tanks, the recovery of BDE 99 from food suspended in water for 2 h was measured. Approximately 0.5 g of the BDE 99 spiked food was placed in a test tube filled with 10 mL of a 0.1 M sodium phosphate buffer at a pH of 7.0. After 2 h in the dark, 95 ( 17% (n ) 3) of the original BDE 99 mass remained in the food. We conclude, therefore, that all of the BDE 99 challenge in the food was ingested by the fish during the exposure. The concentration of BDE 99 in the frozen prepared food was also monitored throughout the experiment, and there was no significant change in the concentration of BDE 99 or BDE 183 in the food during storage. Sampling. One fish from each tank was sampled on days 0, 5, 10, 20, 30, 44, 62, 67, 74, 85, and 99 approximately 2.5 ( 1 h following feeding. Fish mass and length were recorded, and fish were killed by cervical dislocation. The viscera of each fish were dissected, and the liver was separated and weighed. Livers from replicate exposures were combined for the analyses of parent exposed compounds. The gastrointestinal mass was removed from the fish, and the intestine was separated from the remaining tissues. The gut content material residing in the intestinal track was removed from the intestinal tissue and frozen for analysis of BDEs. The intestinal tissue was rinsed in DI water and frozen. The remaining carcass was homogenized and stored in frozen precleaned glass jars. During the depuration phase of this study, fish were fed clean, unspiked food pellets and sampled at the same time period post-feeding. Whole body and liver samples were Soxhlet extracted with dichloromethane for BDE analysis. Samples were first ground

with sodium sulfate, spiked with a surrogate standards (2,4,4′, 6-tetrabromodiphenyl ether, BDE 75), and then extracted for approximately 24 h. Gut content material and intestinal tissues were also ground with sodium sulfate to remove excess water but were then sonicated for 20 min in a test tube using 25 mL of dichloromethane. After 20 min of sonication, each test tube was centrifuged for 5 min at 3000 rpm, and the solvent was collected into a round-bottom flask. This process was repeated two more times for each gut content and intestinal tissue sample. All extracts were concentrated using rotoevaporation and then cleaned up using gel permeation chromatography, followed by Florisil chromatography. An internal standard of 2,2′,3,4,4′,5,6,6′-octachlorobiphenyl (PCB 204) was added to each sample for quantification of BDEs. Recovery of the surrogate standards averaged 78 ( 5% for BDE 75. Sodium sulfate blanks were extracted and quantified for BDEs alongside the fish extracts. There was some minor laboratory contamination for BDEs 47 and 99, but levels were sufficiently low to exclude blank correction procedures in these samples. Concentrations of BDEs in control fish were very low, ranging from below detection limits to 1.62 ng/g ww throughout the entire experiment, indicating that all BDE levels observed in the exposed fish were a result of the spiked food treatments. Method detection limits (MDLs) for BDEs were 1.0 ( 1.2, 2.0 ( 2.2, 0.07 ( 0.02, and 0.10 ( 0.03 ng for BDE 47, 99, 154, and 183, respectively. Quantification. All samples were quantified for BDEs using Hewlett-Packard 5890N gas chromatograph equipped with a Hewlett-Packard 5973 mass spectrometer operated using negative chemical ionization. A 30-m DB-5 capillary column was used in the GC, and the injection port temperature was maintained at 260 °C while the detector temperature was maintained at 320 °C. The oven temperature was initially held at 80 °C for 2 min followed by a ramp of 30 °C/min to a temperature of 200 °C, followed by another ramp of 5 °C/min to a final temperature of 300 °C, which was then held for an additional 7 min. The mass spectrometer was operated in selective ion monitoring (SIM) mode, and ions -79 and -81 were monitored for all BDEs. Confirmation of all BDE congeners was accomplished by running a subset of samples on a GC/HRMS at the National Water Research Institute in Burlington, ON, using the method developed by Alaee et al. (19).

Results and Discussion Growth. On each sampling day throughout the experiment, the mass and length of each fish were recorded (see Supporting Information). All statistical analyses were conducted using the mixed procedure model in the software package SAS (version 8.0) to test for significant differences among exposure treatments, replicate tanks, and time. No significant differences (p < 0.05) were observed in the growth rates between the control and the exposed treatments nor among replicate tanks during the exposure period. There were no mortalities in the control or BDE 99-exposed tanks; however, three deaths in two of the BDE 183-exposed tanks appeared to be caused by an intestinal parasitic infection rather than BDE exposure. Lipid contents of whole body tissues did not change significantly during the experiment, nor were they significantly different among treatments (p < 0.05). Lipid averaged 2.3 ( 1.4, 2.2 ( 1.2, and 2.8 ( 1.5% among the control, BDE 99-exposed, and BDE 183-exposed groups, respectively. Intestinal lipid contents averaged 2.7 ( 1.4, 2.5 ( 1.0, and 2.7 ( 1.2% among control, BDE 99-exposed, and BDE 183exposed groups, respectively, and were not different among the treatments. Exposure to BDE 99. On the first sampling point (day 5), one fish from each tank was sacrificed 2.5 ( 1 h following VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Recovered mass of BDE 99 and its debrominated metabolite, BDE 47, in the undigested food material and intestinal tissue 2.5 h post-feeding.

FIGURE 1. Relative concentrations of the exposure compound, BDE 99, among tissues demonstrating the change in concentration of BDE 99 and the appearance of the debrominated product, BDE 47, in the undigested food material and intestinal tissues approximately 2 h post-feeding. Levels of BDE 47 in whole body tissues reflect the entire body burden accumulated through the first 5 d of exposure. (Error bars represent standard deviation of three replicates.) feeding. Before feeding, the concentration of BDE 99 in the exposure food was 400 ( 40 ng/g ww. The concentration of BDE 99 in the recovered undigested food material on day 5 measured 51 ( 8 ng/g ww (Figure 1), 2.5 ( 1 h after passing through the intestinal tract of the carp. Additionally, BDE 47 was detected in the undigested food, suggesting that debromination was occurring possibly due to mediation by microbrial fauna and/or endogenous enzyme systems. The change in the concentration of BDE 99 in the food following feeding was observed on each subsequent sampling day during the exposure. This drop in concentration was consistent throughout the exposure and, if following a firstorder loss rate, is equivalent to 0.81 h-1 (based on the change in concentration at time 0 and 2.5 h), indicating rapid transformation. As seen in Figure 2, the BDE 99 challenge was rapidly degraded in the intestine to less than 40 ng or 10% of the 400 ng fed to each fish. One gram of spiked food fish-1 d-1 was added to each tank and upon sampling the carp 2.5 ( 1 h post-feeding, approximately 0.56 ( 0.24 g of wet undigested food (gut contents) was recovered from the intestinal cavity of each fish. Therefore, assuming that 1 g was ingested by each fish, approximately 50% of the food mass had been evacuated or metabolized and absorbed from the intestine in this time. Gut evacuation studies on carp measured in situ have determined that food in the gut has a residence time following a first-order rate loss at 0.31 h -1 at 19.8 °C (19). This agrees well with our observations taken at 22 °C. Specziar (20) also demonstrated that 90% of the mass ingested by carp is evacuated within 7 h of feeding. These data indicate that the resulting decrease in concentration of BDE 99 does not result from dilution of previously 1056

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ingested food or excreta, as gut contents are likely to have been purged between the 24-h feeding period. While measurable amounts of BDE 99 were present in the intestinal tissue, BDE 99 was not detected in liver tissue or whole body tissues throughout the experiment. The low levels of BDE 99 measured in intestinal tissue during the exposure were consistent over time (Figure 2). In fact, the ratio of BDE 99 in the intestine to that in the gut content material (0.15 ( 0.03; ww basis) was consistent over time and may indicate simple partitioning behavior, suggesting that the mass of BDE 99 in the intestinal tissue may be residue from the material removed upon sampling. Although every attempt was made to rinse the intestinal tissue with deionized water following extraction from the body cavity and to remove the material within the intestinal villi, this hydrophobic compound could readily adsorb to the lining of the intestine and would not be removed by a water rinse. Because no increase in intestinal BDE 99 concentration was observed, we suggest that either microbrial fauna are extremely efficient at metabolizing this congener prior to uptake across the gut wall or that the endogenous intestinal enzyme systems are involved in the debromination. However, we note that the liver in carp is closely intertwined with the intestine. Although we never detected BDE 99 in the liver tissue, it is possible the observed debromination of BDE is due to enzyme systems in the liver. In contrast, we observed increasing levels of BDE 47 in the intestine with an uptake rate of 1.5 ( 0.4 ng (g of wet body tissue)-1 d-1. Carp whole body tissues measured on day 5 showed appreciable body burdens (116 ( 40 ng) of BDE 47 after only 4 full days of exposure to BDE 99 (Figure 1). BDE 47 continued to accumulate in whole body tissues throughout the exposure at a rate of 1.6 ( 0.4 ng g-1 d-1 or 36 ( 7 ng fish-1 d-1 (Figure 3). Additionally, BDE 28 was measured in carp tissues ranging from 0.23 ( 0.20 on day 20 to 1.19 ( 0.30 ng/g ww on day 62. Although these levels are very low, they may suggest that BDE 47 is further debrominated to BDE 28, which in turn is rapidly converted to other products. No other BDE congeners were detected in any of the tissue types. Because rapid uptake of BDE 47 was observed in intestinal and whole body tissue and there was no increase in concentration of BDE 99 within the intestinal tissues, the results implicate endogenous intestinal/liver enzyme systems in mediating the debromination and/or breakdown of BDE 99 in the gut. If a microbial community was responsible for the observed debromination in the gut, we would expect to see some minor accumulation of BDE 99 in the carp tissues. The most probable mechanisms seems to be an enzyme system located in the intestinal and/or liver tissues. High

FIGURE 3. Increase in BDE 47 concentrations observed in whole body, liver, and intestinal tissues. (Error bars on whole body and intestinal symbols represent one standard deviation for the three replicate samples; liver samples represent a composite of three replicates.) glucuronidation activity observed in carp intestinal microsomes (21) indicates that carp may have higher metabolic activities relative to other fish for removing and breaking down endogenous and exogenous substances in the gut. Carp are stomachless fish, as are other members of the Cyprinidae family. The evolution of the stomachless digestive system may have resulted in higher intestinal metabolic activities in these fish as they compensated for the loss of a stomach, which typically serves as a venue for digestion. The highest concentrations of BDE 47 were observed on the last exposure day. The percentage of the BDE 99 exposure that was debrominated and accumulated in tissues was estimated using the following equation: % assimilated ) average BDE 47 body burden on day 62 (nmol) cumulative BDE 99 exposure mass from day 1 to day 62 (nmol)

(1) At least 9.5 ( 0.8% of the BDE 99 exposure mass was debrominated and accumulated in the form of BDE 47 within carp tissues. The debromination rate is a conservative estimate as some of the BDE 47 formed was present in the undigested food material in the gut and could have been further metabolized and/or excreted. In an earlier study (4), we monitored the uptake of a suite of BDE congeners in the common carp under similar exposure conditions. The suite of BDE congeners used in that study included both BDE 99 and BDE 47, and we observed a 93 ( 10% net assimilation efficiency of BDE 47 in carp tissues. Our present results indicate that some of the accumulated BDE 47 burden resulted from debromination of BDE 99. Using the observed debromination rate in this study, the assimilation efficiency of BDE 47 can be recalculated from direct dietary exposure. The assimilation efficiency of BDE 47 in our previous experiment increased from 15 ( 6% on day 5 to 93 ( 14% on day 60 (Figure 4), which was a result of both direct assimilation of BDE 47 from the food and debromination of BDE 99 to BDE 47. Assuming that 10% of the BDE 99 exposure in that experiment was accumulated in the form of BDE 47, we now conclude that BDE 47 had a net assimilation efficiency of approximately 68 ( 14% on day 60, which is still significantly higher than the assimilation efficiencies of all three PCB congeners in that study. However, as evident in the bottom graph of Figure 4, the net assimilation efficiency of BDE 47 continued to increase during the exposure period and never reached a steady state as was

FIGURE 4. Net assimilation efficiency of BDE 47 calculated on sampling points throughout the cocktail exposure in our previous experiment (4) (A), the percent of BDE 99 exposure accumulated in the form of BDE 47 from the present study (B), and the recalculation of the net assimilation efficiency of BDE 47 from the cocktail study assuming some of the accumulation was due to debromination of BDE 99 (C). (Error bars represent the standard deviation of three replicates.) observed with the five other congeners. This suggests the presence of yet another source of BDE 47 to carp in that experiment. Very little (20%) in all species except the deepwater sculpin (Myoxocephalus thompsoni), which had no detectable traces of BDE 99 (34). In our previous investigations into PCB dynamics in the Great Lakes, we determined that deepwater sculpin metabolized PCBs to methylsulfone PCBs, presumably by specific CYP2B-like isoenzymes (35). These results suggested that deepwater sculpin have unique biotransformation capabilities for both PCBs and BDEs. As mentioned previously, carp are stomachless fish, and perhaps the intestine of carp has evolved into a more efficient digestive and absorptive organ with enhanced metabolic activities relative to other fish species. Other species may have similar biotransformation and/or biodegradation capacities as the common carp. All three species identified as lacking accumulation of BDE 99, which may indicate high BDE biotransformation capacities, are epibenthc. Close associations with sediments may have resulted in enhanced abilities to enzymatically degrade exogenous substances often found in sediment or sediment associated bacteria may be incorporated into their gut flora in a mutualistic relationship. Alternatively, it is possible that all organisms may be able to debrominate BDEs but only differ in the rates and efficiency of the transformations. BDE 99 is the dominant congener in 1060

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commercial penta-BDE formulations and is also typically identified as the dominant congener in sediment samples (7, 36). Yet, in almost all biota samples BDE 47 is the dominant congener (8, 11, 12). This relative trend among sources, abiotic compartments, and biotic compartments can be explained by differences in assimilation efficiencies between BDE 47 and 99, by debromination of BDE 99 to BDE 47 in organisms, or by combinations of both. A similar relationship may exist between BDE 183 and its debromination products, BDE 154 and Hexa-3, but more environmental data and speciesspecific studies are needed to address these issues. Implications. The results from this experiment demonstrate that BDE congeners experience a previously unidentified biotransformation pathway in some fish tissues. More work is needed to determine the pathway by which BDE congeners are debrominated in fish tissues and determine the potential for debromination in other species. Clearly the accumulation of BDE congeners in organisms will be affected by this transformation pathway, and attempts to correlate congener patterns between commercial sources and tissue patterns will be compromised. In addition, more work is needed to assess the potential toxicity and/or stress induced in fish by this debromination mechanism.

Acknowledgments The authors thank Dr. Mehran Alaee and Ivy D’Sa of the National Water Research Institute in Burlington, ON, for the use of their GC/HRMS. We also thank the following for their aid in the experiment: Richard Kraus, Megan Toaspern, Susan Klosterhaus, and Eileen Beard. This is the University of Maryland Center for Environmental Science Contribution No. 3702.

Supporting Information Available Two files detailing information on fish mass, length, and lipid and BDE concentrations over time. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review August 8, 2003. Revised manuscript received November 30, 2003. Accepted December 2, 2003. ES0348804

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