Environ. Sci. Technol. 2006, 40, 2937-2943
Remarkable Findings Concerning PBDEs in the Terrestrial Top-Predator Red Fox (Vulpes vulpes) S T E F A N V O O R S P O E L S , * ,† ADRIAN COVACI,† PETER LEPOM,‡ SOPHIE ESCUTENAIRE,§ AND PAUL SCHEPENS† Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium, Umweltbundesamt, P.O. Box 33 00 22, D-14191 Berlin, Germany, and Rabies Department, Pasteur Institute, Engeland Street 642, 1180 Brussels, Belgium
In the present study, we have analyzed muscle, liver, and adipose tissue of 33 red foxes from Belgium for their content of polybrominated diphenyl ethers (PBDEs). Median sums of seven tri- to hepta-BDEs (BDE 28, BDE 47, BDE 99, BDE 100, BDE 153, BDE 154, and BDE 183) were 2.2, 2.4, and 3.4 ng/g lipid weight in adipose tissue, liver, and muscle, respectively. These levels were lower than those found in various species of voles and mice, the main prey species of the red fox. This is probably related to the high capacity of the foxes to metabolize and eliminate lower brominated congeners. BDE 209 generally dominated the PBDE congener profiles in the red fox samples. In samples containing BDE 209, this congener contributed, on the average, approximately 70% to the total PBDE content. BDE 209 was measured in concentrations as high as 760 ng/g lipid weight in the liver, but the detection frequency was not more than 40%. In animals with the highest BDE 209 levels, this congener was detected in muscle, liver, as well as in adipose tissue. Other abundant congeners were BDE 153 and BDE 47, which prevail in other terrestrial species. The particular PBDE congener profile observed in the red fox resembles that seen in grizzly bears from Canada, but differs from those previously reported for terrestrial avian species. Our data confirms unambiguously that BDE 209 does bioaccumulate in terrestrial top predators, such as the red fox.
Introduction Polybrominated diphenyl ethers (PBDEs) were first introduced onto the market in the 1960s and were used since then as flame retardants to improve fire safety in applications, where they are added in concentrations up to 30 wt % (1). A substantial increase in production was seen since the end of the 1970s due to the growing popularity of personal computers and other electronic equipment, and due to stricter fire regulations (2). Since then, environmental levels of PBDEs have been continuously increasing (3, 4). Spillage * Corresponding author phone: +32 3820 2704; fax +32 3820 2722; e-mail:
[email protected]. † University of Antwerp. ‡ Umweltbundesamt. § Pasteur Institute. 10.1021/es060081k CCC: $33.50 Published on Web 03/25/2006
2006 American Chemical Society
and emission during production and use, but also improper product disposal, account for this phenomenon. These chemicals are shown to be persistent and lipophilic, which results in bioaccumulation in fatty tissues of organisms and enrichment throughout the food chain (4). PBDEs have already been identified in tissues of invertebrates and fish (5), aquatic and terrestrial birds (6), marine and terrestrial mammals (4, 7), and humans (8). Several reviews have been dedicated to the toxicological effects of PBDEs in exposed organisms (9-11), while others focused mainly on their endocrine disrupting properties in humans and wildlife (12, 13). The current knowledge on the potential health risks associated with PBDE exposure ascribe these chemicals the potential to disrupt normal thyroid homeostasis, to cause neurological and developmental effects, and to possibly cause cancer in laboratory animals. The observed effects on the thyroid system (12) and the estrogen-mediated gene expression (14) can also be related to the presence of OH-PBDEs, which are biologically formed. It is, therefore, clear that prolonged exposure to these pollutants can interfere with normal physiology and biochemistry. The high toxicological potential of PBDEs can have health consequences for toppredators, such as red foxes, which can theoretically build up substantial amounts of these persistent chemicals in their body following biomagnification through the food chain. Data on PBDE concentrations in terrestrial biota are scarce. Most available data are on birds of prey (4, 15, 16), while limited data are available on rabbits (Oryctolagus cuniculus), moose (Alces alces), reindeer (Rangifer tarandus) (17), on hedgehogs (Erinaceus europaeus) (18), and on grizzly bears (Ursus arctos) (7). This lack of data hampers the assessment of PBDE exposure and highlights the need for more research related to terrestrial species. In the past, fox species have already been proposed as a bioindicator for organochlorine pollution (19, 20). They are widely distributed omnivores capable of adapting easily to local food sources, which makes them ideal for monitoring local exposure. However, foxes possess highly developed metabolic systems, as do humans, which can complicate data interpretation through formation of toxicologically potent metabolites that are not always considered in toxicological assessments. Red fox (Vulpes vulpes) are major predators of small mammals including voles, mice, squirrels, hares, and rabbits, which represent 90% of their diet (21). The relevance for the foxes of the present study was confirmed by a detailed investigation on the stomach content of foxes from the same geographical region (22). Additionally, dead animals and domestic garbage, particularly in urban environments, represent additional food resources (23). The red fox used in the present study are, therefore, considered as opportunistic omnivores. In the present study, muscle, liver, and adipose tissue of red fox (Vulpes vulpes) were analyzed for their PBDE, polychlorinated biphenyl (PCB), and organochlorine pesticide (OCP) content. Data on PCBs and OCP were included to support the discussion on PBDEs. Hereby, special emphasis was placed on BDE 209 since the bromine industry and its lobby group Bromine Science and Environmental Forum (BSEF) claims that this fully brominated congener is not bioavailable and, therefore, harmless. This study is the first to report on PBDEs in the red fox and contributes to a better understanding of levels, metabolism, and tissue distribution of PBDEs in terrestrial mammalian wildlife. VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Materials and Methods Sample Collection. Although Belgium was officially declared rabies-free in 2001, the epidemiological surveillance of rabies is still maintained. Therefore, this study was based on foxes processed for a diagnostic screening of rabies infection. Between October 2003 and March 2004, the staff of the Pasteur Institute (Brussels, Belgium) collected thirty-three red foxes (Vulpes vulpes) from the south of Belgium. Data on gender and habitat (urban-rural) were recorded. All 33 foxes included in this study were found dead due to traffic accident traumas or provided by hunters. Liver (right lobe), muscle (left thigh), and abdominal adipose tissue were collected. Due to food deprivation related to trauma or pathology, adipose tissue could be collected only from 27 individuals. In three cases, abdominal damage prevented the collection of the liver (30 livers collected), which was only collected if it was undamaged. All samples were stored at -20 °C until further treatment. Chemicals. All solvents used for the analysis (n-hexane, acetone, dichloromethane, and iso-octane) were of SupraSolv grade (Merck, Darmstadt, Germany). Individual reference standards for each analyte were used for identification and quantification (Wellington, Guelph, ON, Canada; Dr. Ehrenstorfer Laboratories, Augsburg, Germany). Sodium sulfate was heated for at least 6 h at 600 °C, and silica was pre-washed with n-hexane and dried overnight at 60 °C before use. Extraction thimbles were pre-extracted with hexane. Sample Preparation and Analysis. The following PBDE congeners (IUPAC numbering 28, 47, 99, 100, 153, 154, 183, and 209) and brominated biphenyl (BB) 153 were targeted for analysis. BDE 77, BDE 128, BB 155, and 13C-BDE 209 were used as internal standards (IS). Details can be found elsewhere (5, 16). The PCB congeners analyzed were the following: 28/31, 52, 74, 95, 99, 101, 105, 110, 118, 128, 138/ 163, 149, 153, 156, 170, 180, 183, 187, 194, 196/203, and 199. The following OCPs were also determined: R-, β-, and γ-hexachlorocyclohexane (hereafter referred to as “HCHs”), trans-chlordane (TC), cis-chlordane (CC), trans-nonachlor (TN), oxychlordane (OxC) (hereafter referred to as “CHLs”), 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (p,p′-DDT), 2,2bis(4-chlorophenyl)-1,1-dichloroethylene (p,p′-DDE), 2,2bis(4-chlorophenyl)-1,1-dichloroethane (p,p′-DDD), (hereafter referred to as “DDTs”), and hexachlorobenzene (HCB). The method used for analysis of fox tissues has been previously described (5) and is briefly summarized below. Depending on the type of tissue, 0.2 g (adipose tissue), 2 g (liver), or 6 g (muscle) of homogenized sample were mixed with anhydrous Na2SO4, spiked with IS, and Soxhlet extracted with hexane/acetone (3:1, v/v). An aliquot of the extract was used for gravimetrical lipid determination. The extracts were cleaned-up on silica impregnated with concentrated sulfuric acid (48%, w/w) and analytes were eluted with hexane and dichloromethane. The cleaned extract was evaporated to dryness and reconstituted in iso-octane. PBDEs were analyzed by gas chromatography-electron capture negative ionization mass spectrometry (GC/ECNIMS) operated in selected ion monitoring (SIM) mode. Details of the instrumental conditions were published elsewhere (5). For tri- to hepta-BDE congeners and BB 153, a 25 m 0.22 mm × 0.25 µm HT-8 capillary column (SGE, Zulte, Belgium) was used and ions m/z ) 79 and 81 were monitored. For the analysis of BDE 209, a 12 m × 0.18 mm × 0.10 µm AT-5 capillary column (Alltech, Lokeren, Belgium) was used and ions m/z ) 484.7/486.7 and 494.7/496.7 were monitored for BDE 209 and 13C-BDE 209, respectively. PCBs were analyzed by GC-electron impact MS (GC/EIMS) operated in SIM-mode using a 30 m × 0.25 mm × 0.25 µm DB-1 capillary column (J&W, Folsom, CA). OCPs were analyzed by GC/ECNI-MS operated in SIM-mode on a 25m 2938
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× 0.22 mm × 0.25µm HT-8 capillary column (SGE, Zulte, Belgium). Instrumental conditions for PCB and OCP analysis were published elsewhere (24). A subset of 10 samples were sent to the Laboratory for Water Analysis of the German Environment Agency (Umweltbundesamt, Berlin, Germany) for confirmatory analysis of BDE 209. These samples were extracted by accelerated solvent extraction with toluene followed by a multilayer silica gel column chromatography and gel permeation chromatography cleanup (25). Analyses were conducted using a short 2 m × 0.25 mm × 0.25 µm RTXCLPesticides column (Restek, Germany), which provides sufficient selectivity and high sensitivity (Lepom and Sawal, unpublished results). The carrier gas was helium at constant flow (4.7 mL/min), and the oven was programmed from 150 °C to 300 °C at 25 °C/min. Ions m/z ) 484.7/486.7 and 494.7/ 496.7 were monitored for BDE 209 and 13C-BDE 209, respectively. Quality Assurance and Quality Control. Instrumental QC was done by regular injection of solvent blanks and standard solutions, while the analyst and method QC was ensured through replicate sample analyses (RSD < 5%) and procedural blanks (RSD < 12%). Therefore, the mean procedural blank value was used for subtraction. After blank subtraction, the limit of quantification (LOQ) was set at 3 times the standard deviation of the blank, which ensures >99% certainty that the reported value is originating from the sample. This implies varying LOQs (between 2 and 500 pg/g ww) depending on the sample intake and on the PBDE congener. Absolute recoveries for individual PBDE congeners were between 87 and 104% (RSD < 12%). The efficiency of the method was demonstrated by successful participation in several international interlaboratory studies on the determination of PBDEs in biota (QUASIMEME and NIST). Due to analytical difficulties and low concentrations which have to be measured, reports on the presence of BDE 209 in biological tissues often provoke some skepticism. Therefore, confirmatory analysis of a subset of 10 samples covering a wide range of BDE 209 concentrations ranging from not detected to several hundred ng/g lw was done in the Laboratory for Water Analysis of the German Environment Agency. The agreement of results in this small interlaboratory exercise was excellent (differences were
