Environ. Sci. Technol. 2008, 42, 5562–5567
Examination of Isomer Specific Bioaccumulation Parameters and Potential In vivo Hepatic Metabolites of syn- and anti-Dechlorane Plus Isomers in Juvenile Rainbow Trout (Oncorhynchus mykiss) G R E G G T . T O M Y , * ,†,‡ CADEN R. THOMAS,§ THANE M. ZIDANE,§ KATHRYN E. MURISON,| KERRI PLESKACH,† JONATHON HARE,† GILLES ARSENAULT,⊥ C H R I S H . M A R V I N , # A N D E D S V E R K O #,∇ Department of Fisheries and Oceans, Arctic Aquatic Research Division, Winnipeg, MB, R3T 2N6 Canada, Departments of Chemistry and Environment and Geography, University of Manitoba, Winnipeg, MB R3T 2N2 Canada, Bairdmore School, Winnipeg, MB R3T 5R3 Canada, St. Norbert School, Winnipeg, MB R3V 1H8 Canada, Wellington Laboratories Incorporated, Guelph, ON, N1G 3M5 Canada, Environment Canada, 867 Lakeshore Drive, Burlington, ON L7R 4A6 Canada, and Department of Chemistry, McMaster University, Hamilton, ON, L8S 4L8 Canada
Received January 22, 2008. Revised manuscript received April 24, 2008. Accepted May 12, 2008.
Juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to elevated doses of syn- and anti-isomers of Dechlorane Plus (DP) via their diet for 49 days (uptake phase), followed by 112 days of untreated food (depuration phase) to examine bioaccumulation parameters and possible metabolic products. Three groups of 60 fish were used in the study. Two groups were exposed separately to food fortified with known concentrations of syn- (0.79 ( 0.03 µg/g, lipid weight) and antiDP (1.17 ( 0.12 µg/g, lipid weight) while a third control group was fed unfortified food. Neither isomer reached steadystate after 49 days of exposure. Only the syn-isomer accumulated linearly in the fish (whole-body minus liver) during the dosing phase with a calculated uptake rate constant of 0.045 ( 0.005 (arithmetic mean ( 1 × standard error) nmoles per day. A similar uptake rate was also observed for this isomer in the liver. The elimination of both isomers from the whole fish (minus liver) obeyed first order depuration kinetics (syn-: r2 ) 0.6427, p < 0.001, anti-: r2)0.5350, p < 0.005) with calculated half-lives (t1/2) of 53.3 ( 13.1 (syn-) and 30.4 ( 5.7 (anti-) days. Elimination of the isomers from the liver was * Corresponding author phone: 204-983-5167; fax: 204-984-2403, e-mail:
[email protected]. † Arctic Aquatic Research Division. ‡ University of Manitoba. § Bairdmore School. | St. Norbert School. ⊥ Wellington Laboratories Incorporated. # Environment Canada. ∇ McMaster University. 5562
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difficult to interpret because of suspected enterohepatic circulation and redistribution of the isomers in the liver during clearance from other tissues. The biomagnification factor (BMF, determined in whole fish minus liver) of the syn-isomer (5.2) was greater than the anti-isomer (1.9) suggesting that the former isomer is more bioavailable. A suite of metabolites were screened for in the liver including dechlorinated, hydroxylated, methoxylated and methyl sulfone degradates. Even with the purposely high dose used in the uptake phase, none of these degradates could be detected in the extracts. This suggests that if metabolites of DP are detected in fish from aquatic food webs their presence is likely not from in vivo biotransformation of the parent compound.
Introduction The polycyclic chlorohydrocarbon, Dechlorane Plus (DP, C18H12Cl12), has been employed as an additive flame retardant since the 1960s. Its main uses include wire and cable coatings, plastic roofing material, hard connectors in computers, and automotive lubricants. Dechlorane Plus is still marketed today due to its superior performance in certain plastics when compared to current brominated varieties such as, decabromodiphenylether (1). The commercial mixture is comprised of primarily two isomers, syn- and anti-, at a ratio of approximately 1:2 (2), respectively, and plastic formulations can contain as high as 35% (weight basis) of DP. Although not measured in the environment until recently, DP has been shown to be present in air, sediment, biota, and herring gulls within the North American Great Lakes region (2–5). The detection of the isomers in biota suggests that the isomers are bioavailable and bioaccumulative. Furthermore, our earlier work also demonstrated there were clear differences in the bioaccumulation tendencies of the two isomers (2). Our laboratory has been engaged in laboratory-based bioaccumulation experiments on other flame retardants for over four years. The bioaccumulation parameters (depuration and uptake rates, half-life (t1/2), biomagnification factor (BMF)) yielded from studies like these are crucial in furthering our understanding of the environmental fate and behavior of compounds. The current study is a continuation of our work on flame retardants, and the experimental design was similar to our previous studies examining similar kinetics of brominated diphenyl ethers (BDEs), hexabromocyclododecane, and 1,2-bis(2,4,6-tribromophenoxy)ethane (6–8). Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to known amounts of the individual isomers for 49 days to follow uptake kinetics. A depuration period lasting a further 112 days, during which all fish were fed unfortified food, was also included to examine elimination kinetics. A second objective of the study was to examine possible metabolite formation in vivo. Others have reported on metabolites of other cyclic chlorinated compounds like the chlorinated biphenyls (CBs). In addition, Sverko et al. reported on reductive dechlorinated DP metabolites in a sediment core from the Great Lakes (3). Based on these observations, we hypothesize that the isomers of DP may themselves be susceptible to metabolic transformations in fish. As such, liver samples were analyzed for a suite of suspected metabolites.
Materials and Methods Standards and Reagents. Native syn- and anti-DP, BDE congeners 77, 126, 156, 197, and 207 (99.9%) and hydroxylated 10.1021/es800220y CCC: $40.75
2008 American Chemical Society
Published on Web 06/21/2008
chlorinated biphenyl (OH-CB) congeners 120 (2, 3′, 4, 5, 5′) and 187 (2, 3, 3′, 4, 4′, 5) were obtained from Wellington Laboratories (Guelph, ON, Canada). Distilled in glass hexane, dichloromethane (DCM), and acetone were obtained from Caledon (Edmonton, AB, Canada). Corn oil, gelatin, and tricaine methanesulfonate (MS 222) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Ottawa sand (20-30 mesh) was obtained from Fisher Scientific (ON, Canada) Food Preparation. The preparation of the fish food has been described previously (6). Three batches of food were prepared in this study: two of the batches (1.8 kg each) were spiked with a known amount of syn- and anti-DP (3.6 mL of 50 µg/mL), whereas no isomer was added to the third batch (control). Food was stored in the dark at -4 °C to limit the possibility of light-induced degradation of DP. Using the analytical techniques described below, control corrected lipid based concentrations of (arithmetic mean ( 1 × standard error) of syn- and anti-DP in the food were determined to be 0.79 ( 0.03 (n ) 3), 1.17 ( 0.12 µg/g (n ) 3), respectively. Small amounts of the syn-DP were detectable in the unfortified food (1.5 ng/g). Average percent lipid in the food was determined to be 14.3 ( 0.3%. Concentrations of neither DP isomer declined in the food from the start of the exposure experiment (day ) 0) to the end of the clearance phase (day ) 161). Experiment. Juvenile rainbow trout (initial mean weights, 50 ( 5 g) were fed spiked food for 49 days, followed by untreated food for 112 days. The daily feeding rate was equal to 1.0% of the mean weight of fish, adjusted after each sampling period based on the mean weight of the subsample of fish that were sacrificed. Feed was presented by sprinkling at the surface of the water and was generally consumed by each group of fish within 1 min. Sixty fish were used for each treatment, and each treatment was held in separate 200 L fiberglass aquaria receiving 0.3 L UV and carbon dechlorinated Winnipeg city tap water/min (12 °C, pH 7.9-9.1). The dissolved oxygen was always at the level of saturation. A 12 h light:12 h dark photoperiod was maintained throughout the experiment. Four fish were sampled from each tank on days 0, 7, 14, 21, 35, and 49 of the uptake period and on days 7, 22, 35, 49, 70, and 112 days of the depuration period. Fish were always sampled 24 h after the previous feeding. Sampled fish were euthanized with an overdose (0.8 g/L) of pH buffered MS-222. After fin movement ceased (
