Persistent Organic Pollutants in Two Dolphin Species with Focus on

Hollings Marine Laboratory, 331 Fort Johnson Road,. Charleston, South Carolina 29412, and University of. Maryland, Chesapeake Biological Laboratory,...
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Research Persistent Organic Pollutants in Two Dolphin Species with Focus on Toxaphene and Polybrominated Diphenyl Ethers K A R E N J . S . T U E R K , †,‡ J O H N R . K U C K L I C K , * ,† P A U L R . B E C K E R , † H E A T H E R M . S T A P L E T O N , §,| A N D JOEL E. BAKER§ National Institute of Standards and Technology, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, South Carolina 29412, and University of Maryland, Chesapeake Biological Laboratory, 1 Williams Street, Solomons, Maryland, 20688

Assessing trends of persistent organic pollutants (POPs) in marine mammals is difficult due to age, gender, and metabolism influences on accumulation. To help elucidate these effects in dolphins, POP concentrations were determined in the Atlantic white-sided dolphin, Lagenorhynchus acutus, a pelagic delphinid inhabiting North Atlantic waters, and in the rough-toothed dolphin, Steno bredanensis, a pelagic delphinid inhabiting tropical and subtropical waters. The specific objectives of this study were to determine baseline POP concentrations in L. acutus and S. bredanensis blubber samples and to examine the effects of age, gender, and metabolism on POP concentrations in dolphin blubber. Focus was aimed at contaminants of emerging concern, specifically, toxaphene and polybrominated diphenyl ethers (PBDEs). Samples collected from L. acutus (n ) 47) stranding events in Massachusetts (19932000) and S. bredanensis samples (n ) 15) were analyzed for PCBs, toxaphene, and other organic pesticides by gas chromatography/mass spectrometry (GC/MS). Age and gender influences were similar between the two species, with adult females having significantly lower POP concentrations as compared to adult males and juveniles. Mean ∑toxaphene concentrations were highest in juvenile L. acutus, 13.0 (6.7) µg/g wet mass (1 SD), and lowest in adult female S. bredanensis, 1.49 (1.4) µg/g wet mass. ∑PBDE (sum of congeners 47, 99, 100, 153, and 154) concentrations were highest in juvenile L. acutus, 2.41 (1.2) µg/g wet mass, and lowest in adult female S. bredanensis, 0.51 (0.6) µg/g wet mass. POP concentrations did not significantly differ between adult males and juveniles, suggesting metabolism of congeners and/or dilution with growth. PBDE concentrations in juvenile whitesided dolphins were not significantly related to collection * Corresponding author phone: (843)762-8866; fax: (843)762-8742; e-mail: [email protected]. † National Institute of Standards and Technology. ‡ Present address: University of South Carolina, Department of Environmental Health Sciences, 800 Sumter St., Columbia, SC 29208. § University of Maryland. | Present address: National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899. 692

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year, suggesting that there may be a lag period for higher concentrations to be detected in pelagic marine mammals such as L. acutus or that concentrations have already peaked in this species prior to the first collection in 1993.

Introduction Historically, polychlorinated biphenyls (PCBs), DDT, chlordanes, hexachlorocyclohexanes (HCHs), and chlorobenzenes and their metabolites have been the main persistent organic pollutants (POPs) quantified in marine mammals (1-4). In recent years, however, greater attention has been given to other organic pollutants, including toxaphene and brominated flame-retardants (5, 6). Toxaphene was introduced in 1949, and following the ban of many persistent chlorinated pesticides a few decades later, it became the most widely used pesticide in the United States and globally. Toxaphene use was curtailed in the United States in 1982 after toxaphene’s persistence and potential for toxicity was recognized. However, toxaphene may still be used in some countries. While toxaphene is generally not routinely determined in biological samples due to analytical difficulties, this compound continues to be of concern. Toxaphene has been identified in numerous aquatic organisms, including marine mammals (6-8). In the northeast North Atlantic, toxaphene was the most prevalent contaminant quantified in white-beaked dolphins (Lagenorhynchus albirostris) and in harbor porpoises (Phocoena phocoena), with concentrations exceeding 50 µg/g wet weight in some blubber samples (9, 10). Toxaphene was also quantified in Hudson Bay beluga whales (Delphinapterus leucas) in concentrations up to 5 µg/g (11). Toxaphene is a known carcinogen in rats and mice and may be mutagenic in fish when present with other contaminants (12). In marine mammals, toxaphene is an inducer of microsomal enzyme activity; for example, toxaphene congeners 32 (2-endo,3-exo,6-exo,8,9,10,10-heptachlorobornane) and 62 (2,2,5,5,8,9,9,10,10-nonachlorobornane) were shown to induce the CYP3B biotransformation enzyme in Phoca vitulina (harbor seal) (13). Polybrominated diphenyl ethers (PBDEs) are used as flame-retardants in electronic circuit boards, textiles, furniture, plastics, and paints and are still widely manufactured with current global production estimated to be 150 000 tons/ year (5, 14-16). PBDEs are additives and are not chemically bonded to products and hence are slowly released into the environment throughout a product’s lifetime. While PBDE production and use, mainly as the deca-PBDE formulation, has largely continued in the United States, restrictions of the product have recently gone into effect in Europe (17). PBDE congeners have recently been identified in numerous biological samples. Several studies demonstrated a temporal increase in PBDE concentrations in biological samples, including human breast milk and beluga whale (Delphinapterus leucas) blubber (18-20). Currently, very few marine mammal samples have been analyzed for these compounds. PBDEs have been identified in several seal species and in long-finned pilot whales (Globicephala melas) with concentrations ranging from 0.8 to 3.2 µg/g (15). In white-beaked dolphins, PBDEs were reported in concentrations exceeding 7 µg/g (sum of three PBDE congeners) (5). The toxic effects of PBDEs, which are structurally similar to the polychlorinated biphenyls (PCBs), are receiving great attention. Structure activity relationships suggest some 10.1021/es0487675 CCC: $30.25

 2005 American Chemical Society Published on Web 12/09/2004

congeners may be carcinogenic or neurotoxic (21). In addition, PBDE metabolites are classified as compounds that may affect steroid hormone regulation (22). Certain PBDE congeners bind to the aryl hydrocarbon-receptor and have weak dioxin-like toxicity and induce the CYP2B enzyme in rats (16, 23). PBDE metabolites formed through this enzymatic pathway may disrupt thyroid hormone regulation (23, 24). For these reasons, PBDE exposure should be determined in marine mammals, especially during early life stages. For this investigation, blubber samples from two dolphin species were examined. Lagenorhynchus acutus, the whitesided dolphin, inhabits cold-temperate waters of the North Atlantic, ranging from Cape Cod, MA, extending up the eastern coast of North America to Greenland, and from Norway to the British Isles. L. acutus is a pelagic, offshore delphinid, reaching 270 cm in length, and may live up to 27 years. Females reach sexual maturity at 201-210 cm (6-12 years of age) with a calving period of approximately 2.5 years (11 months gestation, 18 months lactation) (25). Stomach contents from stranded L. acutus individuals reveal a diet consisting mainly of fish (herring, silver hake, and smelt), squid, and occasionally shrimp (25). Steno bredanensis, the rough-toothed dolphin, has a global distribution in warm subtropical and tropical waters and is predominately associated with waters above 25 °C (26). Like L. acutus, this species is pelagic, feeding mainly on fish and squid (26). While little is know about the life history of this species, both males and females are thought to reach sexual maturity at a length of 180 cm, and maximum length is 280 cm (26). As the life history and trophic status of S. bredanensis are likely similar to those of L. acutus, a comparison of contaminant patterns should help to illustrate differences in marine pollution in two different regions off the coast of the United States. There are no reports of toxaphene or PBDE concentrations in either species.

Materials and Methods Blubber samples for this study were collected from 47 stranded L. acutus and 14 stranded S. bredanensis using protocols established by the National Institute of Standards and Technology (27). Morphometric information was collected for each animal, including total length, blubber thickness, gender, and reproductive status. Teeth from 19 L. acutus and 14 S. bredanensis were available for aging. The age estimation protocol has been described previously for L. acutus and S. bredanensis (27, 28). Animals generally stranded in mass, and all L. acutus stranding events occurred on the Massachusetts coast, near Cape Cod. L. acutus individuals generally stranded in good condition. S. bredanensis samples were collected from a single mass stranding event (December 15, 1997) on the Gulf Coast of Florida, near Apalachicola (28, 29). Blubber samples were cryohomogenized and stored according to established procedures (30, 31). Sample preparation has been described in detail elsewhere (27, 28, 32). Briefly, samples (approximately 1 g, exact weight known) were mixed with Na2SO4 and added to a pressurized fluid extraction (PFE) cell along with an internal standard solution and were extracted with CH2Cl2 using PFE. High molecular weight compounds were removed by size-exclusion chromatography using CH2Cl2 as the mobile phase. The extract was then fractionated using a semipreparative aminopropylsilane column into relatively lower and higher polarity fractions (F1 and F2, respectively). After the extracts were analyzed for PCB congeners and pesticides, the extracts were recombined for the analysis of toxaphene and PBDEs, as both were found in F1 and F2. PCBs, PBDEs, and chlorinated pesticides (excluding toxaphene) were determined using an Agilent 6890/5973 (Palo Alto, CA) GC/MS instrument operating in the electron impact

mode. Details of PCB and pesticide analysis have been described in detail elsewhere (27, 32). Toxaphene congeners were determined using an Agilent 6890/5973 GC/MS instrument operating in the negative chemical ionization (NCI) mode. Four calibration solutions for total toxaphene were prepared by weighing (to the nearest 0.0001 g) portions of Standard Reference Material (SRM) 3067 (technical toxaphene in methanol) into weighed portions of isooctane. The calibration curve ranged from approximately 100 to 11000 ng dissolved in approximately 0.5 mL of isooctane. Four individual toxaphene congeners were targeted for quantification in addition to total toxaphene. Three calibration solutions were prepared by weighing (to the nearest 0.0001 g) portions of a commercial standard solution: 2-endo,3-exo,5-endo,6-exo,8,8,10,10-octachlorobornane (congener 26), 2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-nonachlorobornane (congener 50), congener 62, and congener 32 (Promochem, Germany). GC/MS conditions are given elsewhere (33, 34). Total toxaphene (∑toxaphene) is the sum of hexa- through decachlorobornanes. PBDEs. PBDE congeners were determined using Agilent 6890/5973 and 5890/5972 GC/MS instruments using electron impact ionization in the selected ion-monitoring mode. Detailed methods, as well as ions used to determine the analytes, are given elsewhere (33). A calibrant solution containing congeners PBDE 47 (2,2′,4,4′-tetraPBDE), PBDE 99 (2,2′,4,4′,5-pentaPBDE), PBDE 100 (2,2′,4,4′,6-pentaPBDE), PBDE 153 (2,2′,4,4′,5,5′-hexaPBDE), PBDE 154 (2,2′,4,4′,5,6′hexaPBDE), and PBDE 183 (2,2′,3,4,4′,5′,6-heptaPBDE) (Cambridge Isotope) was used to quantify congeners in blubber samples. Briefly, samples were injected (2 µL, splitless injection) onto a 60 or 30 m DB-5ms capillary column (0.25 mm i.d. × 0.25 µm film thickness). The injector temperature was 260 °C/min. Helium was used as the carrier gas at a constant flow rate of 30 cm/s. The initial column temperature was 60 °C with a 1 min hold. The temperature was then ramped to 200 °C at 25 °C/min and then to 300 °C at 3 °C/ min with a 20 min hold. The ion source temperature was 250 °C. Statistical Analyses. To compare concentrations among life history classes, male and female juveniles were pooled, and multivariate analysis of variance (MANOVA) was used to compare mean contaminant concentrations between five life history/species groups; adult females of both species, juveniles of both species, and adult male L. acutus (R ) 0.05). Only one adult male S. bredanensis was sampled, so this individual was omitted from the comparisons. As toxaphene and PBDE values have not been investigated in S. bredanensis, or for many tropical or subtropical marine mammals, individual toxaphene and PBDE congeners were investigated in greater detail between the two species. Contaminant concentrations were log transformed to fit the assumption of normality. Individual analyses of variance (Welch’s ANOVA for unequal variances) were then used to determine which compounds were significantly different, using contrasts (Tukey-Kramer) to determine concentration differences among groups. MANOVA was also used to compare concentrations of three toxaphene congeners and five PBDE congeners (congeners 47, 99, 100, 153, and 154) among the life history classes described previously. Relative proportions of individual toxaphene and PBDE congeners between the two species were examined as described previously. No life history classes were considered in these analyses, so results are given for pooled S. bredanensis individuals and L. acutus individuals. To evaluate the apparent biotransformation of toxaphene and PBDE congeners, ratios of individual congeners to metabolically resistant PCB 153 (adapted from 35) were calculated, and linear regressions were performed on ratios against body length as described previously (27). Only L. VOL. 39, NO. 3, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Summary of Mean Organohalogen Concentration among Life History Classes in L. acutus and S. bredanensis Blubbera compound(s)

juvenile L. acutus n ) 23

female L. acutus n)9

male L. acutus n ) 15

juvenile S. bredanensis n)7

female S. bredanensis n)6

29a ( 9.14 14.5a ( 4.09 7.75a ( 2.9 0.20a ( 0.05 0.20a ( 0.06 1.39a ( 0.37 0.07a,c ( 0.02 10.7a ( 3.98 1.82a ( 0.76

24.1a ( 10.5 12.0a ( 7.23 3.58a,b ( 2.82 0.038c ( 0.06 0.04b ( 0.01 0.35b ( 0.39 0.33d ( 0.16 3.33b ( 1.49 1.36a ( 0.57

10.9b ( 9.38 5.41b ( 5.3 1.82b ( 1.4 0.02c ( 0.02 0.02c ( 0.07 0.15c ( 0.18 0.17c ( 0.15 1.49c ( 1.42 0.51b ( 0.57

99b,c ( 65 250b ( 140 48b ( 19

55c ( 58 130c ( 130 23c ( 19

Polybrominated Diphenyl Ether Congeners (ng/g wet mass) 260b ( 210 990a ( 370 690a ( 310 85b,c ( 57 220a ( 97 140b ( 59 110b ( 99 350a ( 160 260a ( 120 74b ( 94 120a,b ( 81 67a,b ( 25 83b,c ( 55 150a,b ( 54 210a ( 63

260b ( 330 56c ( 66 100b ( 120 25c ( 23 77c ( 30

ΣPCBs ΣDDT Σchlordanes ΣHCH HCB dieldrin mirex Σtoxaphene ΣPBDEs

29.4a ( 15.1 15.9a ( 9.65 8.8a ( 5.4 0.30a ( 0.17 0.24a ( 0.15 1.81a ( 0.84 0.06a ( 0.03 13.0a ( 6.67 2.41a ( 1.16

9.41b ( 8.22 4.09b ( 4.22 2.2b ( 2.15 0.09b ( 0.05 0.05b ( 0.05 0.29b,c ( 0.32 0.04b ( 0.02 3.56b ( 3.97 0.61b ( 0.52

26 50 62

750a ( 440 1040a ( 560 200a ( 110

Toxaphene Congeners (ng/g wet mass) 190b ( 220 620a ( 220 270b,c ( 300 790a ( 230 55b ( 43 120a ( 24

47 99 100 153 154

1460a ( 680 290a ( 150 390a ( 160 120a ( 68 150a,b,c ( 110

a Values are the mean ( 1 standard deviation. Concentrations were log transformed prior to statistical analysis. Welch’s ANOVA was used due to heteroscedasticity. MANOVA results: 1 ) 0.014, F ) 8.84, p < 0.0001. Groups sharing the same superscript letter are not significantly different. Concentrations are µg/g wet mass unless otherwise noted.

acutus males were included in this analysis, as metabolism is the dominant mechanism for POP excretion in adult males and the sample size for male S. bredanensis was not large enough. To determine whether there was a temporal trend in PBDE concentrations in L. acutus, mean concentrations were compared across collection years for juveniles and the means were compared using ANOVA and linear regression.

Results and Discussion In any given region, the suite of contaminants in the water column depends on physical and chemical properties of the compounds, transport rates, source and contaminant input, and degradative processes acting on the chemical (36, 37). Marine mammals in different areas should be expected to have different patterns of POP contamination (26, 38). Therefore, it may be expected that L. acutus, inhabiting cool waters in the northern North Atlantic, and S. bredanensis, inhabiting warm tropical and subtropical waters, would exhibit different POP patterns and concentrations despite having similar life histories and trophic status. POP concentrations revealed significant differences among age classes and between species (p < 0.0001) (Table 1). In L. acutus, juveniles and adult males were not significantly different for any compound or compound class with the exception of PBDE 99, which was significantly lower in adult males. POP concentrations in juvenile bottlenose dolphins (Tursiops truncatus) were also not significantly different between juveniles and adult males for samples collected from animals inhabiting waters adjacent to the East Coast of the United States (39). Female blubber concentrations of POPs were significantly lower than those of juveniles or adult males within a species, except for the ∑chlordanes and ∑HCHs in S. bredanensis and PBDE congeners 153 and 154 in L. acutus. The trend of lower levels in females than adult males or juveniles results mainly from lactational offloading, as suggested by other studies (e.g., ref 40). Significant differences (p < 0.05) in POP levels in blubber were seen between the two species. For juveniles and males, which are the best represented and not significantly different within a species, ∑PCBs, ∑DDT, ∑chlordanes, ∑PBDEs (sum of congeners 47, 99, 100, 153, and 154; congener 183 was 694

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below the limit of detection in all samples), and PBDE congeners 47, 100, 153, and 154 were not significantly different between S. bradanensis and L. acutus. ∑HCH, HCB, dieldrin, total toxaphene (∑toxaphene), toxaphene congeners 50 and 62, and BDE congener 99 concentrations were significantly lower in S. bradenensis than in L. acutus blubber. Mirex was the only compound significantly higher in S. bredanensis than in L. acutus, likely due to the past use of mirex in the Southeast United States for fire ant control. The between species concentration differences are likely due to usage and mobility of the compounds in the region these animals inhabit and probably not trophic differences, since the two species likely feed at similar trophic levels. Proportions of individual toxaphene (congeners 26, 50, and 62; congener 32 was below the limit of detection in all samples) and PBDE congeners relative to the sums of toxaphene and PBDE congeners, respectively, in the two species (individuals pooled) were examined, as there have been few investigations of these compounds in toothed whales. Individuals within each species were pooled to simplify the between species comparison (Table 1). Significant differences were observed between the proportion of toxaphene and PBDE congeners in the two species (p < 0.0001) (Figure 1). For PBDEs, the relative proportion of PBDE 47 was significantly higher in L. acutus than in S. bredanensis; however, the proportion of PBDE 154 was significantly higher in S. bredanensis. No other significant species differences were observed for congeners 99, 100, or 153. For toxaphene, the proportion of congener 26 was significantly higher in L. acutus, and the proportion of congener 50 was significantly higher in S. bredanensis. While the proportion of congener 62 appeared much higher in S. bredanensis than in L. acutus (16 and 11%, respectively), this difference was not significant due to the large standard deviation for S. bredanensis, likely due to the pooling of adult females with juveniles. While ∑PBDE concentrations in the animals did not generally differ between L. acutus and S. bredanensis, individual PBDE congeners did. PBDE 47, a tetra-BDE, was present in significantly higher proportions in L. acutus. Congener 154, a hexa-BDE, was present in significantly higher proportions in S. bredanensis. Similarly, blubber samples

FIGURE 1. Relative proportions of (A) PBDE congeners and (B) toxaphene congeners to the sum of the five individual PBDE congeners (47, 99, 100, 153, 154) or three individual toxaphene congeners (26, 50, and 62) measured in L. acutus and S. bredanensis. Error bars are 1 SD. Significant differences (p < 0.05) were observed between the two species for PBDE 47, PBDE 154, and toxaphene congener 26. collected from beluga whales in the Canadian Arctic had lower concentrations of PBDEs with more than four bromine atoms (19). These differences are likely due to physicochemical properties of individual PBDE congeners (49). Tittlemeir et al. (41) measured the liquid-phase vapor pressures of several PBDE congeners and showed that vapor pressure values decrease with an increase in the number of bromine atoms. Vapor pressures also increase as the temperature increases, favoring volatilization in warm environments and condensation in cooler environments, such as the northwest North Atlantic Ocean. The same was true for toxaphene congener 26, which has higher vapor pressures than congeners 50 and 62 and was found in higher proportions in L. acutus than in S. bredanensis (Figure 1) (42). Concentrations of total toxaphene and PBDEs in L. acutus show some similarity to values determined on other species of toothed whale in the northern North Atlantic Ocean. The highest concentrations of toxaphene reported in a delphinid were for male L. albirostris from the Newfoundland coast where the mean (1 SD) concentration was 46.0 mg/kg (22.1 mg/kg) (wet basis) (9). Concentrations of toxaphene observed in the present study in L. acutus (Table 1) were more comparable to those observed in harbor porpoise (P. phocoena (10)) or pilot whale (Globicephala malaena (9)), where concentrations in adult males (wet basis) were 15.2 mg/kg (7.1 mg/kg) and 11.7 mg/kg (7.1 mg/kg), respectively. The concentrations of toxaphene in juvenile S. bredanensis blubber (3.3 mg/kg (1.49 mg/kg)), which are likely similar to those in adult males, were statistically lower than those in

any L. acutus age class. Voldner and Schroeder (43) suggested that air currents function in transporting contaminants northwards where they preferentially deposit in cooler regions such as the northwestern North Atlantic Ocean. This is also the rationale for why cold lakes, such as Lake Superior, sometimes have toxaphene concentrations in the food web that are unexpectedly high (44). Two of the most comprehensive published studies to date on concentrations of PBDEs are for harbor porpoise (Phocoena phocoena) collected as bycatch from the coast of England and Wales (45) and stranded beluga whales from the St. Lawrence Estuary. In the study by Law et al. (45), the geometric mean (1 SD) of total PBDEs (sum of 47, 99, 100, 153) in male harbor porpoise was 1.9 µg/g wet mass (1.6 µg/g wet mass, n ) 21) as compared to 2.16 µg/g wet mass (0.92 µg/g wet mass) found by the present study in male L. acutus. The geometric mean of total PBDEs (sum of congeners 28, 47, 49, 66, 99, 100, 153, 154, 155, and 183) in male beluga whales from the St. Lawrence Estuary was 0.19 µg/g wet mass (0.18 µg/g wet mass, n ) 28). It is interesting that ∑PBDE levels in St. Lawrence beluga whales were so much lower than in P. phocoena or L. acutus given previously reported levels of organohalogens in St. Lawrence beluga whales (e.g., average ∑PCB of 80 µg/g wet mass in males (7)). Metabolism. Little is known about the metabolism of PBDEs and toxaphene in cetaceans. However, the metabolism of PBDEs is assumed to be similar to that of PCBs, since they are structurally similar. To investigate if specific PBDE and toxaphene congeners are metabolized by male L. acutus, the ratios of individual congener concentrations to PCB 153 concentrations were calculated. Ratios were then regressed against length for male L. acutus (Figure 2). Because of a small sample size (n ) 1), this analysis could not be performed for male S. bredanensis. Length has been shown to serve as a proxy for age estimation for these species (27). Among toxaphene congeners, both toxaphene congener 50/PCB 153 and toxaphene congener 62/PCB 153 decreased significantly with length. Among PBDE congeners, PBDE 47/PCB 153, PBDE 99/PCB 153, and PBDE 100/ PCB 153 decreased significantly with increasing length. Additionally, the ratio of ∑toxaphene and ∑PBDEs to PCB 153 decreased significantly with length (Figure 2). Metabolism of toxaphene and PBDE congeners has not been extensively investigated until recently. The regression of toxaphene congener 62/PCB 153 shows a significant decrease with length in male L. acutus. In a study of gray seals (Halichoerus grypus), van Hezik et al. (46) determined that CYP3A enzymes may aid in metabolism of toxaphene congeners 32 and 62. Similarly, Boon et al. (47) found that harbor seals (Phoca vitulina) are able to metabolize congeners 32 and 62, while white-beaked dolphins (Lagenorynchus albirostris) could only metabolize congener 32. No metabolism of congener 50 was noted (47). Congener 32 was not detected in all L. acutus samples, suggesting metabolism of this congener. However, this study did not determine contaminants in prey of L. acutus; therefore, it is not known whether congener 32 was present in its prey. Very little data are available regarding PBDE toxicity and metabolism, and the bulk of studies examining the toxicological effects of PBDEs have been conducted on rodents. Whole-body autoradiography revealed that 14C-labeled PBDE 47 was metabolized by rats, but the parent compound concentration was higher than metabolite concentrations in urine and feces (78 and 21%, respectively) (15). In a similar study, however, it was found that mice were able to efficiently metabolize congener 47, with higher metabolite concentrations relative to those of the parent compound (85 and 15%, respectively) (15). In a study of PBDE 99 metabolism in rats, 90% of the compound excreted in feces was the parent compound, with only 10% consisting of metabolites (15). VOL. 39, NO. 3, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Ratios of (A) ∑toxaphene, (B) toxaphene congener 50, (C) toxaphene congener 62, (D) ∑PBDEs, (E) PBDE congener 47, (F) PBDE congener 99, and (G) PBDE congener 100 to PCB 153 plotted against body length for male L. acutus. Concentrations were log transformed prior to calculating ratios. This suggests that this congener is not readily metabolized. In the same study, PBDE congener 153 was not found to induce mixed function oxidase response. Results from this study suggest that PBDE 47, PBDE 99, and PBDE 100 may all be metabolized by L. acutus, while PBDE 153 and PBDE 154 do not appear to be metabolized. Temporal Increase in PBDEs. To determine whether PBDE concentrations have increased in L. acutus with time, mean concentrations were compared across the year of collection using juveniles, as concentrations in juveniles 696

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tended to be less variable as a group than those in mature females or males (Table 1). Juveniles are also a good group to examine for POP temporal trends, as concentrations are not statistically different between males and females; hence, pooling of more individual samples is possible. In addition, metabolic capabilities may not be fully developed in young animals, thereby limiting modification of POP patterns. Contrary to most other studies examining temporal trends of PBDEs in marine mammals, there was no significant temporal trend in ∑PBDE or individual congener concentra-

FIGURE 3. Mean values (1 SD) for ∑PBDE concentrations in juvenile L. acutus that were collected between 1993 and 2000. tions from 1993 until 2000 for L. acutus (Figure 3). Temporal trends in PBDE concentrations seem to be site specific. Ikonomou et al. (48) found a positive temporal trend in PBDE concentrations in porpoises within a period of 4 years, and several studies on beluga whales also show positive correlations of ∑PBDEs with collection year (see ref 20 for a review). Studies in humans generally reveal a temporal increase in PBDE concentration especially in the U.S. population (18). Several studies reviewed by Watanabe and Sakai (49) have measured PBDEs in biological samples, including guillemot eggs in the Baltic sea, human blood in Sweden, and fish tissue in Osaka Bay, Japan, showing a significant increase from the 1970s to the early 1990s, followed by a decrease in the late 1990s. Decreases in PBDE congeners have been observed in sediment cores collected from Western Europe (e.g., ref 50). There may be a lag period for higher concentrations to be detected in pelagic marine mammals such as L. acutus, or concentrations may have already peaked in this species. Examining specimens whose collection years span a greater range would help to resolve this.

Acknowledgments This work was made possible through the support of the Marine Mammal Health and Stranding Response Program (Teresa K. Rowles), National Marine Fisheries Service (NMFS) Office of Protected Resources. The authors thank Greg Early, the Cape Cod Stranding Network, and the New England Aquarium for aid in the collection of the white-sided dolphin samples; Blaire Mase, NMFS Southeast Regional Stranding Coordinator; the Florida Stranding Network; Ruth Ewing, NMFS Southeast Fisheries Science Center; Bill Fable, NMFS Panama City Laboratory; Tim Nelson, DVM; Catherine Bray Nelson, Teri and Scott Calleson, and Misty Nabers; Wayne McFee, the National Ocean Service; and the Florida State Park Rangers for aid in collecting the rough-toothed dolphin samples. Certain commercial equipment or instruments are identified in the paper to specify adequately the experimental procedures. Such identification does not imply recommendations or endorsement by NIST, nor does it imply that the equipment or instruments are the best available for the purpose.

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Received for review August 6, 2004. Revised manuscript received October 20, 2004. Accepted October 22, 2004. ES0487675