Environ. Sci. Technol. 2002, 36, 4025-4032
Levels of Polybrominated Diphenyl Ether (PBDE) Flame Retardants in Animals Representing Different Trophic Levels of the North Sea Food Web J A N P . B O O N , * ,† W I L M A E . L E W I S , #,† MICHAEL R. TJOEN-A-CHOY,† COLIN R. ALLCHIN,‡ ROBIN J. LAW,‡ JACOB DE BOER,§ CATO C. TEN HALLERS-TJABBES,† AND BART N. ZEGERS† Royal Netherlands Institute for Sea Research (NIOZ), P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands, Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Remembrance Avenue, Burnham-on-Crouch, Essex CMO 8HA, UK, and Netherlands Institute for Fisheries Research (RIVO), P.O. Box 68, IJmuiden, The Netherlands
The levels of individual PBDE congeners were investigated in the invertebrate species whelk (Buccinum undatum), seastar (Asterias rubens), and hermit crab (Pagurus bernhardus), the gadoid fish species whiting (Merlangius merlangus) and cod (Gadus morhua), and the marine mammal species harbor seal (Phoca vitulina) and harbor porpoise (Phocoena phocoena). These species are all important representatives of different trophic levels of the North Sea food web. All six major PBDE congeners detected (BDEs 28, 47, 99, 100, 153, and 154) are most prevalent in the commercial Penta-BDE formulation. There is no evidence for the occurrence of the Octa-BDE formulation in the North Sea food web, since its dominant congener, BDE183, was never detected. BDE209, the main congener (>97%) in the Deca-BDE formulation, was detected only in a minority of the samples and always in concentrations around the limit of detection. Since BDE209 is often the major BDE congener in sediments from the area, the main reason for its low concentrations in biota from the North Sea seems to be a relatively low bioaccumulation potential. This can either be due to a low uptake rate of the very large molecule or a relatively rapid excretion after biotransformation. Since all invertebrates investigated are sentinel species, they are highly representative for the area of capture. The highest lipid-normalized concentrations of PBDEs in the invertebrates occurred near the mouth of the river Tees at the East coast of the UK. The geographical distribution of the PBDEs can be explained by the residual currents in the area. The direction of these currents differs between the summer and the winter season as a result of the presence or absence of vertical summer stratification of the deeper waters north of the Dogger Bank. Summer stratification results in the development of a density-driven bottom water current formed after the onset of vertical stratification of the water column in May leaving the UK coast near Flamborough Head toward the Dogger Bank. In winter, the residual currents run in a more southerly 10.1021/es0158298 CCC: $22.00 Published on Web 08/28/2002
2002 American Chemical Society
direction and follow the UK coastline. The distribution pattern of the PCBs and p,p′-DDE in the invertebrates was entirely different from that of the PBDEs, which could be expected, since the use of these organochlorines in western Europe peaked in the 1960s and 1970s but has been forbidden more than two decades ago, whereas the production and use of the penta-BDE formulation is of a more recent origin. The higher trophic levels of the North Sea food web were represented by the predatory gadoid fish species whiting and cod and the marine mammal species harbor seal and harbor porpoise. The lipid-normalized levels of the six major PBDE congeners in fish were similar to the levels in the invertebrates, but a biomagnification step in concentrations of generally more than an order of magnitude occurred from gadoid fish to marine mammals. Based on the limited number of samples, no differences could be observed between harbor seal and harbor porpoise. In summary, the results in three species of sentinel invertebrates from a network of stations covering a major part of the North Sea basin showed that the estuary of the river Tees at the UK East coast is a major source for tri- to hexa-PBDEs. Throughout the food-chain, the most marked increase in (lipid-normalized) levels of all six PBDE congeners occurred from predatory (gadoid) fish to marine mammals, agreeing with the transition from gillbreathing to lung-breathing animals. This has serious consequences for the route of elimination of POPs, since their elimination from the blood into the ambient seawater via the gill-membrane is no longer possible.
Introduction Brominated flame retardants are added to many plastics and printed circuit boards of electronic household equipment and textile and polyurethane foam in furniture and cars for obvious safety reasons. Among the brominated flame retardants (BFRs), there are three commercial formulations that contain the diphenyl ether skeleton. In order of increasing overall bromination, these formulations are named Penta-BDE, Octa-BDE, and Deca-BDE. The global market demands in 1999 were 54 800 metric tons for the Deca-BDE, 8500 tons for the Penta-BDE, and 3825 tons for Octa-BDE (ref: www.bsef.com). PBDEs are very hydrophobic (log Kow values 4-10) and resistant to degradation. The water solubility and vapor pressure of PBDEs decrease with increasing degree of bromination, whereas hydrophobicity increases (1-3). Two major congeners of Penta-BDE, i.e., BDEs 47 and 99 (numbering is according to the IUPAC nomenclature for PCBs), showed even higher bioaccumulation factors than PCBs of similar hydrophobicity, despite a larger molecular size of the brominated compounds (4). The presence of these congeners and BDE100 in tissues of the deep-sea foraging sperm whales indicates that these PBDEs have become * Corresponding author phone: +31 222 369 466; fax: +31 222 319 674; e-mail:
[email protected]. † Royal Netherlands Institute for Sea Research (NIOZ). ‡ Centre for Environment, Fisheries and Aquaculture Science (CEFAS). § Netherlands Institute for Fisheries Research (RIVO). # Present address: TNO-MEP, Dept. for Ecological Risk Studies, P.O. Box 57, 1780 AB Den Helder, The Netherlands. VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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globally dispersed chemicals (5). The temporal trends of the major congeners of the penta-BDE formulation still showed a sharp increase between 1978 and 1998 in lake trout (Salvelinus namaycush) from the Great Lakes (6). The levels in Beluga whale (Delphinapterus leucas) from the St. Lawrence estuary collected between 1997 and 1999 were on average 20 times higher than the levels in animals collected a decade earlier (7), irrespective of sex. Mixed temporal trends were observed in sediments of several UK rivers entering the North Sea or the Irish Sea (8). In the Rhine-Meuse delta, the levels in yellow eel (Anguilla anguilla) from the “Haringvliet” decreased since 1984, and those in eel from the German/ Dutch river Roer, a tributary of the Meuse, since 1993 (8). In Japan, the levels in sea bass and grey mullet from Osaka bay have been rapidly decreasing since 1990 (9). Less is known about the situation for the other two technical PBDE formulations, i.e. the Octa- and the Deca-BDE formulation. Octa-BDE was reported to be absent in freshwater fish from Virginia (10). BDE209 constitutes > 95% of the commercial deca-BDE formulation. The only occasion where this congener and BDE 183 have been reported in relatively high concentrations in wildlife concerns eggs of pererine falcons breeding in Sweden (11). The peregrine falcon (Falco peregrinus) belongs to the top-predators of the terrestrial food-web, since this bird of prey feeds mainly on other birds. The uptake of BDEs 183 and 209 in blood of humans working in a recycling factory of electronic household equipment has also been demonstrated (12). Thus, the assumption that BDE209 cannot bioaccumulate due to its large molecular size is not always correct. This is an important observation, since the concentrations of BDE209 are often much higher than those of the other PBDE congeners in suspended particulate matter and sediments of several continental European, UK, and Irish rivers (8). Although data on PBDE levels in the estuaries of several rivers entering the North Sea have been published (8), a survey of a number of PBDE congeners representative for the different commercial formulations in a number of animal species representing different trophic levels of the North Sea food web has not yet been carried out. For this purpose, the invertebrate species seastar (Asterias rubens), common whelk (Buccinum undatum), and hermit crab (Pagurus bernhardus) and the predatory gadoid fish species whiting (Merlangius merlangus) and cod (Gadus morhua) were sampled between 52 and 58° N and 1°W-10°E, covering a large part of the North Sea basin and the Skagerrak, the connection between the North Sea and the Baltic Sea. The highest trophic levels were represented by harbor seals (Phoca vitulina) from the German Wadden Sea and harbor porpoises (Phocoena phocoena) stranded or by-caught along the Dutch coast. Since the three invertebrate species are sentinel, the geographical distribution of their PBDE levels can give an impression of the location of important input sources into the North Sea basin. For comparison with the PBDEs, the levels of the major PCB congeners CB101, 138/158, and 153 and p,p′-DDE were also analyzed.
Experimental Section Sampling of Invertebrates and Fish. The majority of the samples were taken with a 5 m beam trawl with a mesh size of 3.5 cm during cruise 64PE144 with the RV Pelagia in August-September 1999. The basic data on the position, sampling date, water depth, and temperature, and salinity at the surface and just above the sea-bottom, are given as Supporting Information in Table SI-1. Large differences in temperature and/or salinity between surface and bottom water are indicative for the occurrence of vertical stratification. The presence of a thermocline or a halocline severely inhibits the exchange rates between the sea surface and the sea bottom. The herring samples were caught with a pelagic 4026
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net at 51°34′ N and 2°47′ E in the Southern Bight by the commercial fishing vessel TX 37 in October 2000. Because it is very hard to obtain a good homogenate from entire animals for analysis of POPs, a number of tissues were selected. The following tissues were excised on board immediately after capture and frozen at -20 °C until further analysis: for the sea star the pyloric caeca (part of the intestinal system), for the hermit crab the soft abdomen; for the whelk the whole (soft) body; and for fish the liver and filet from one side of the backbone. No differentiation was made between the sexes except for the whelk. In this case only the males were taken because the females were used for research on levels and effects of organotin compounds. Samples of invertebrates and fish each contained material from five individual animals. From each station, two such samples were analyzed. Samples of Marine Mammals. All samples of marine mammals came from beach-stranded animals or animals drowned in fishing nets. The samples of harbor porpoise originated from the southern North Sea and were obtained from Dr. Chris Smeenk and Drs. Marjan Addink of the Museum of Natural History “Naturalis” in Leiden, The Netherlands. The samples of harbor seals were obtained from Dr. Ursula Siebert of the Centre for Research and Technology in Bu ¨ sum, Germany, and originated from Wadden Sea of Schleswig-Holstein (Germany). For cetaceans and harbor seals, samples of liver and blubber of individual animals were stored for analysis. Extraction Method for the Determination of POPs. Tissue amounts corresponding to approximately 50 mg of lipid were extracted, using an ultra-Turrax method. After extraction with pentane and acetone, the sample was treated with sulfuric acid, and a silica cleanup was performed. The method has been described in more detail elsewhere as the “NIOZ method” (13). Analysis of PBDEs. The levels of 15 individual PBDEs (Cambridge Isotope Laboratories, Inc. Andover, MA) were determined by GC/MS. The GC was a Hewlett-Packard 6890; the mass-selective detector a Hewlett-Packard 5973. GC specifications: split-splitless injection, split valve closed for 1.5 min. Tinjector 270 °C. Column: stationary phase CP Sil-8, 25 m * 0.25 mm * 0.25 µm (Chrompack, NL). Carrier gas He; linear gas velocity 74 cm s-1, constant flow programmed. Oven temperature program: 90 °C (1.5′)/20 °C min-1/190 °C (0′)/4.5 °C min-1/270 °C (5′)/10 °C min-1/320 °C (10′). MSD specifications: negative chemical ionization (NCI) in the SIM mode at the m/z ratios of both bromine isotopes (79 and 81) and m/z ) 487 (for BDE 209 only). Ionization gas CH4. Tion source 210 °C; Ttransferline 320 °C; Tquadrupole 160 °C. The limit of detection (LOD), defined as a signal of three times the noise level, was established at 0.6 ng g-1 lipid for all BDE congeners except BDE209, when an amount of wet tissue containing 50 mg of lipid was extracted. Other amounts of lipid affect this LOD value proportionally. The LOD for BDE209 was 5 ng g-1 lipid. Chromatographic Conditions for the Analysis of PCBs and p,p′-DDE. The levels of individual CBs and p,p′-DDE were determined by gas chromatography on a Carlo Erba 5300 (Italy) with electron capture detection (ECD). The specifications of the GC were as follows: 1 µL split-splitless injection, split valve closed for 4 min, Tinjector ) 300 °C. Column: stationary phase CP Sil-8, 50 m * 0.25 mm * 0.25 µm (Chrompack, NL). Carrier gas H2; constant pressure. Oven temperature program: 90 °C (2′)/10 °C min-1/215 °C (10′)/8 °C min-1/275 °C (17′). The ECD was 300 °C and 90 mL N2 min-1 was used as make up gas. Quality Assurance. Before sample treatment, the internal standards CB112 and decabromobiphenyl (BB209) were added. Although BB209 has been produced in France until recently, no indication of its occurrence has been found in
the present set of samples, which were run simultaneously without the addition of this internal standard. Moreover, PBBs were also not detected in any sample from a large set from Dutch coastal waters either (14). Our participation in the first interlaboratory study, which has been organized for PBDEs, showed a good performance for the samples of biota and sediment. Until now, no certified reference materials are available for the determination of PBDEs. Extraction Method for the Determination of Tissue Lipid Contents. The levels of all POPs have been expressed on the basis of extractable lipids extracted from the same tissue as used for the POP analyses, since the POPs of interests are all very hydrophobic and thus they prefer to accumulate in fatty tissues (15, 16). However, the extraction method used for the determination of the different POPs, extracts especially the polar phospholipids incompletely. Therefore a separate extraction of the tissues was performed for the determination its total lipid content, using a dichloromethane/methanol/ water solvent system (17). Especially in lean tissues, this method produced substantially higher values than the acetone/pentane/water solvent system used for the extraction of the POPs. For comparison, a table with data of the lipid contents as obtained with both methods is given as Supporting Information in Table SI-2. Multivariate Statistical Analysis. The data of the six major PBDEs, three major PCBs, and p,p′-DDE were subjected to Principal Component Analysis (PCA) to find the underlying pattern and correlation structure of the data set. PCA was performed on the correlation matrix of the lipid-based concentrations. The data were 10log-transformed prior to the calculations to obtain a greater homogeneity of variance. The results have been graphically displayed in the form of a principal components bi-plot. From such figures, several things can be read. A: The Position of Each Sample in the Plane. Each sample can be conceived as a point in a k-dimensional space, where k is the number of compounds taken into account. The first two principal components (PCs 1 and 2) span the plane on which the projected points show the highest variance. The position of each projected point () individual sample) on this plane can be visualized in the form of a so-called covariance biplot (18). The x and y coordinates of each data point represent the scores for the first and second PC, respectively. The coordinates of each data point can be read in the bi-plot from the lower x-axis for the value of the first PC and the left y-axis for the value of the second PC. B: The Vectors. The biplot also shows the values of the correlation (r) between each originalsin this case the logarithm concentration of each compoundsand the first and second principal components by means of a vector. The x and y coordinates of the endpoint of each vector represent the loading. The squared length of each vector shows the goodness of fit and is equivalent to the fraction of the total variance that is explained by the sum of the first and second PC (In formula: (lvector)2 ) R2). Thus, if a vector of a compound reaches the drawn circle of unit variance (circle R2 ) 1), then all variance in the concentration is explained by this sum. The values for the individual correlations with the PCs plotted can be read by orthogonal projection of the endpoint of each vector on the upper X-axis in the plot for the first PC and on the right y-axis for the second second PC. The correlation r between the concentrations of two different compounds is equal to the product of the length of their vectors and the cosine of the angle R between them. Thus, the concentrations of two compounds of which the vectors touch the unit-circle (i.e. all variance can be explained by the sum of the first two PCs), and point in the almost the same direction (cosine R approaches 1), show a very high positive correlation. In contrast, orthogonal vectors indicate a zero correlation (cosine 90° ) 0); a negative correlation is indicated by angles
between 90 and 180°. C: The Relation between the Location of the Datapoints and the Vectors. The orthogonal projection of each datapoint () individual sample) on each vectorsor its extension on the other side of the center of the plotsshows the relative concentration of the accompanying compound in the sample with respect to the mean value, which is represented by the center of the plot.
Results and Discussion PBDE Congeners Detected. Of the 16 congeners analyzed, six were present as major compounds. The basic statistical data (mean, median, standard error, minimum-maximum values) of the levels of these congeners in the different species and tissues are given in Table 1. The general order of decreasing concentrations is BDE47 > BDE99, BDE100 > BDE153, BDE154 > BDE28. An exception to this rule is the sea star, where BDE100 > BDE47, BDE99. In contrast to the situation in the sea star, BDE100 is remarkably low in the harbor seal tissues, which might indicate the occurrence of a certain biotransformation capacity in this species. In the invertebrates, the amount of BDE47 is generally < 50% of ΣPBDE except for the shrimps, whereas in fish it is always > 50%. In harbor seal blubber the contribution of BDE47 to ΣPBDEs was somewhat higher than in harbor porpoise with values for respectively the mean ( SEM/median/min-max range of 63 ( 3.6%/66%/42-80% for harbor seal blubber and 51 ( 3.9%/49%/32-68% for harbor porpoise blubber (n ) 9 for both species). However, far fetched conclusions should not be drawn from these differences in relatively small data sets, since for anotherslargersdata set of harbor porpoise blubber, the values 60 ( 1.4%/59%/39-88% have been reported (19), which is very similar to our results for the harbor seal. The congeners BDE66, BDE75, BDE77, BDE119, and BDE138 were present at concentrations around the limit of detection (LOD; 0.6 ng g-1 lipid in a tissue containing 50 mg of extractable lipid). The congeners BDE71, BDE85, BDE183, and BDE190 were never detected. BDE209 was occasionally present in concentrations just above its LOD of 5 ng g-1 lipid. However, when the samples contained parts of the digestive system, a presence of BDE209 just above the detection limit cannot be interpreted as unambiguous proof for a real uptake by the organism. Instead, the levels may represent remainders of food present in the digestive system. Since BDE209 is a major compound in suspended particles and sediments from the North Sea and related environments (14), it does apparently not bioaccumulate to a high degree (20). Thus, either its large molecular size decreases the uptake rates, or a relatively rapid biotransformation increases its degradation rate. The literature presents evidence for both sides. A number of field studies showed that the bioaccumulative properties of BDE209 were much lower than those of the tetra- and pentabrominated congeners that dominate in the Penta-BDE formulation (21, 22). The only occasion where this congener has been reported (together with BDE183) in relatively high concentrations in wildlife concerns eggs of pererine falcons breeding in Sweden (11). The peregrine falcon (Falco peregrinus) belongs to the top-predators in the terrestrial food-web, since this bird of prey feeds mainly on other birds. The uptake of BDEs 183 and 209 in blood of humans working in a recycling factory of electronic household equipment has also been demonstrated (12). However, as a result of biotransformation processes, BDE209 had a half-life of less than 10 days in these humans. A relatively rapid metabolism of this compound has also been shown in rats (23). A slow but measurable uptake of BDE209 has also been demonstrated in laboratory studies with rainbow trout (24) and juvenile salmon (25). Laboratory studies with 14C-labeled BDE47 in marine inVOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Lipid-Normalized Concentrations in ng g-1 of the Six Major PBDE Congeners Encountered in Animals of Different Trophic Levels of the North Sea Food Weba BDE28 Br subst.: (2,2′,4-) species
mean/ median (min-max)
BDE47 (2,2′,4,4′-) mean/ median
(min- max)
BDE100 (2,2′,4,4′,6-)
BDE99 (2,2′,4,4′,5-)
mean/ mean/ median (min- max) median
BDE154 (2,2′,4,4′,5,6′-)
(min-max)
mean/ median
BDE153 (2,2′,4,4′,5,5′-) mean/ median (min-max)
(min-max)
Invertebrates sea star (pc) hermit crab (abd) whelk (sp) shrimp (wb)
0.6/0.5 1.4/1.6 1.2/1.0 2.6/2.6
(0.4-1.1) (0.8-1.7) (1.0-1.6) (2.4-2.7)
22/19 38/29 10/5.5 37/37
(3.4-56) (8.6-118) (2.6-30) (35-39)
23/9.1 15/11 4.2/2.8 6.9/6.9
(2.2-82) (2.8-40) (1.5-9.7) (5.1-8.7)
9.3/2.9 16/9.2 6.1/4.0 5.1/5.1
(1.0-49) (1.7-71) (2.9-17) (4.6-5.5)
9.0/1.1 10/5.0 4.1/4.1 3.9/3.9
(0.4-41) (1.5-56) (2.0-6.3) (3.9-3.9)
2.6/2.6 3.7/1.9 7.1/5.8
