Environ. Sci. Technol. 2003, 37, 4603-4609
Levels and Temporal Trends of Toxaphene Congeners in Beluga Whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada B R U N O G O U T E U X , † M I C H E L L E B E U F , * ,‡ DEREK C. G. MUIR,§ AND J E A N - P I E R R E G A G N EÄ † Institut des Sciences de la Mer, Universite´ du Que´bec a` Rimouski, 310 Alle´e des Ursulines, Rimouski, Que´bec, Canada, G5L 3A1, Department of Fisheries and Oceans, Maurice Lamontagne Institute, P.O. Box 1000, Mont-Joli, Quebec, Canada, G5H 3Z4, and National Water Research Institute, Environment Canada, Burlington, Ontario, Canada, L7R 4A6
Environmentally relevant toxaphene congeners were determined in blubber samples of stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary (SLE), Canada. The purpose of this study was to evaluate the levels and the temporal trends (1988-1999) of a suite of six chlorobornanes (P26, P40/41, P44, P50, and P62) in the SLE belugas. P26 and P50 mean concentrations were in the same range as those reported for animals living in the Arctic environment suggesting that the atmospheric transport represents the main input of toxaphene to the SLE. A general exponential decline of chlorobornane concentrations in belugas was observed, except for P26 and P50 in males. On average, concentrations decreased by a factor of two in 8.5 years during the 1988-1999 time period. This rate of decline is similar to the reduction of toxaphene emission from agricultural soils in the southern United States reported over the same time period. Some differences in decline rates were observed among the studied CHB congeners. For instance, P62 decreased more rapidly than P26 and P50 in both male and female belugas. Several hypotheses were advanced to explain these differences such as selective metabolism of specific chlorobornanes by SLE belugas or their prey. However, a most likely explanation is the selective degradation of the technical product in soils and atmosphere in the source region.
rodiphenyltrichloroethane (DDT) banned in 1972 (2). Concerns about the toxicity of toxaphene led the U.S. Environmental Protection Agency to progressively ban this product during the 1982-1986 time period (1). In Canada, toxaphene has never been licensed for use, except for special minor applications such as fish eradication from lakes in western Canada (3). Nevertheless, toxaphene is one of the most abundant organochlorine pesticides in biota from Great Lakes, western Canada lakes, the Canadian Arctic, and the St. Lawrence Estuary (SLE) in eastern Canada (4-8). Aerial transport appears to be the most likely pathway for the introduction of toxaphene to Canada (9, 10). This transport is likely to originate mostly from the southern and southeastern United States where residues left in soils after application of toxaphene are a major source of this product (3, 11). After the U.S. ban, a decrease of toxaphene concentrations was expected in biota from Canada. In the Arctic environment, there were no significant temporal trends of toxaphene concentrations in marine mammals such as ringed seals (Phoca hispida), narwhal (Monodon monoceros), or beluga whales (Delphinapterus leucas) between the early 1980s and the mid 1990s (5, 12). In the Great Lakes, no clear temporal trend was observed for lake trout (Salvelinus namaycush) in Lake Superior between 1982 and 1992 (13, 14). However, a significant decrease of toxaphene concentrations was observed in lake trout from the Great Lakes, Michigan, Huron, and Ontario, during the same time period (13). In SLE beluga whales, mean concentrations of toxaphene were significantly higher in the mid 1990s than in the later 1980s, suggesting an increasing temporal trend of the toxaphene contamination in this environment (15). These studies illustrate that temporal trends of toxaphene contamination differ between studied regions in Canada, which likely depend on their distinct contamination history. In addition, temporal trends of toxaphene contamination are complex to interpret mainly suffering from a relatively short time period since the toxaphene ban in the United States. The objective of this research was to evaluate the temporal trends of toxaphene contamination in the SLE after its ban in North America during the 1988-1999 time period. Beluga whale was chosen as an integrator of the past and more recent toxaphene inputs to this ecosystem since this longliving animal resides year-round in the SLE. Technical toxaphene is extensively transformed in the environment leading to simpler residue patterns in environmental samples (16, 17). As a result, this work focuses only on most of the predominant toxaphene congeners found in marine mammals, i.e. congeners P26, P40, P41, P44, P50, and P62 according to the numbering of Parlar (18).
Material and Methods Introduction Toxaphene was first produced in the United States in 1947 and was mainly used as an insecticide to protect cotton cultures against pests in the southern United States (1). Historically, the United States was the main producer and user of this product in the world (2). In the mid 1970s, toxaphene became the most widely used pesticide in the United States serving as a substitute insecticide for dichlo* Corresponding author phone: (418)775-0690; fax: (418)775-0542; e-mail:
[email protected]. † Universite ´ du Que´bec a` Rimouski. ‡ Maurice Lamontagne Institute. § National Water Research Institute. 10.1021/es034449e CCC: $25.00 Published on Web 09/19/2003
2003 American Chemical Society
Chemicals. All high-resolution gas chromatograph grade solvents used were provided by EM Science (Gibbstown, NJ). Anhydrous sodium sulfate (BDH, Toronto, ON), alumina oxide (Bio-Rad Laboratories, Hercules, CA), and silica gel (EM Science, Gibbstown, NJ) were extracted three times with dichloromethane (DCM) and n-hexane prior to their use. Bio-Beads SX-3 used in the gel permeation chromatography (GPC) were obtained from Bio Rad Laboratories (Hercules, CA). A mixture of 22 individual toxaphene congeners was purchased from EQ Laboratories (Atlanta, GA). Only the six following chlorobornanes (CHBs) were analyzed: 2-endo,3exo,5-endo,6-exo,8,8,10,10-octachlorobornane (P26), 2-endo,3exo,5-endo,6-exo,8,9,10,10-octachlorobornane (P40), 2-exo,3VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Location of stranded beluga whales collected in the St. Lawrence Estuary (SLE) and the gulf of St. Lawrence. The number of animals collected at each location is indicated in brackets. endo,5-exo,8,9,9,10,10 -octachlorobornane (P41), 2-exo,5,5,8,9,9,10,10-octachlorobornane (P44), 2-endo,3-exo,5-endo,6exo,8,8,9,10,10-nonachlorobornane (P50), and 2,2,5,5,8,9,9,10,10-nonachlorobornane (P62). For simplicity purpose, the Parlar nomenclature is used in the text. D8-4,4′-DDD, used as a quantification standard, and 13C12-PCB #101, used for the instrument performance control, were obtained from Cambridge Isotope Laboratories (Andover, MA). Samples. CHBs were measured in blubber samples of stranded beluga whales (26 males and 26 females) found on the shores of the SLE during the 1988-1999 time period (Figure 1). The age of each animal was determined by counting growth layer groups on sections of teeth (19). Only animals older than 10 years were considered in this study. In general, blubber samples were taken at 60-70% of body length from the nose. Blubber samples, once received at the Maurice-Lamontagne Institute, were stored at -20 °C until analysis. Prior to the analysis, a sample representing the entire depth of the animal blubber layer was taken and homogenized. For five females and six males only the external blubber layer, averaging 3 cm in depth, was sampled. For most of these animals, this layer represents more than half of the entire depth of the blubber layer. Moreover, preliminary results indicated that CHB concentrations were random distributed along the entire depth of the blubber layer for one female (animal #19 in Table 1) and one male (animal #46 in Table 1) belugas. These results are in agreement with the reported lack of stratification of low chlorinated PCBs in the blubber layer of belugas (20). As shown in Figure 1, two animals were collected outside the SLE, on the shores of the Magdalen Islands and Miscou Island (New Brunswick, Canada). Levels and pattern of CHBs as well as PCBs and organochlorine pesticides (OCs) (M. Lebeuf personal data), observed in these two males (animals #57 and #62 in Table 1), suggest that these individuals were members of the SLE population. Chemical Analysis. The sample cleanup, the GC-MS/MS parameters, and the quantification were done according to the method described in detail by Gouteux et al. (2002), except that the internal quantification standard used was D8-4,4′DDD instead of D8-4,4′-DDT (21). Major steps included the chemical drying of about 1 g wet weight of blubber sample with clean anhydrous sodium sulfate and the extraction of lipids and contaminants with dichloromethane. Then, the extract was fractionated in three parts. The first fraction was 4604
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used to determine the sample extractable lipid content by gravimetry. The second fraction was kept at -20 °C as a backup sample. The third fraction which corresponded to an equivalent wet weight of 0.05 and 0.20 g for males and females, respectively, was further purified. Afterward, lipids were removed from this fraction by GPC followed by a first purification step with a silica/alumina column to collect PCBs and OCs and a second purification step with a silica column to further isolate the CHBs of interest. GC-analyses were performed on a Varian 3400CX (Varian, Walnut Creek, CA) equipped with a Varian 8200 autosampler and a programmable split/splitless injector operated in splitless mode. Chromatographic separations were made on a DB-5MS column (30 m × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific, Folson, CA). Since P40 and P41 were not chromatographically separated, they will be presented as P-40/ 41. The GC was coupled to a Varian Saturn 2000 ion trap mass spectrometer operated in MS/MS mode. The CHB ionization in parent ions was performed by electron impact at 70 eV. Then, parent ions were isolated (isolation window ) 1 m/z) and stabilized in the resonant waveform mode for 5 ms in the ion trap. Collision induced dissociation (CID) of parent ions in daughter ions by helium gas was made during 20 ms at amplitudes varying between 0.20 and 0.35 V depending on the CHB analyzed (21). The concentration of each CHB was calculated using its response factor relative to the one of D8-4,4′-DDD in the same sample. The relative response factor for each CHB was determined from five standard solutions where the D8-4,4′DDD concentration was kept constant at 100 pg/µL, while the CHB concentrations varied from 10 to 500 pg/µL (21). QA/QC. One procedural blank and one Standard Reference Material (SRM) were included with every 10 sample analysis. The SRM, obtained from the National Institute of Standards and Technology (NIST), was a pilot whale (Glopbicephala sp.) blubber sample (SRM 1945). No signals were detected in blanks indicating that procedural contamination was lower than the sample detection limits which varied between 0.3 and 12 pg/g wet weight. Repeated analysis of SRM 1945 (n ) 12) resulted in coefficients of variation for all CHBs which were within 15% of the average values indicating that the precision of the method was satisfactory. The accuracy and the precision of the toxaphene analytical method were confirmed through the Northern Contaminant Program Interlaboratory study NCP II-4 on toxaphene in a
TABLE 1. Collection Date, Main Characteristics, and Chlorobornane Concentrations (ng/g Wet Weight) in Blubber Samples of Stranded Beluga Whales from the St. Lawrence Estuary no.
date
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
88-10-07 88-10-24 88-08-26 89-06-13 89-08-17 90-07-29 92-09-24 92-10-12 93-10-20 94-09-23 95-05-14 95-05-21 97-05-23 97-06-08 97-07-29 97-07-23 97-09-30 97-11-10 98-05-24 98-05-25 98-07-17 99-05-31 99-07-05 99-08-19 99-10-16 99-04-11 88-08-13 89-04-26 89-05-30 90-04-08 90-06-16 90-07-19 93-04-18 93-05-20 93-06-18 94-10-28 94-10-31 94-04-18 95-07-01 97-11-26 97-05-16 97-09-12 98-05-23 98-08-08 98-09-29 98-04-12 98-04-22 98-10-12 99-04-08 99-06-07 99-08-03 99-04-06
agea (y) sex lip (%) 22+ 16 31+ 14+ 25+ 13 21+ 31.5+ 12.5 10 28+ 26+ 28.5 21+ 31+ 22.5+ 30+ 31+ 26+ 20.5+ 11 16 11 10 18+ 15 20+ 27+ 26+ 18+ 23+ 19+ 11+ 25+ 13+ 25+ 27.5 29+ 25+ 26+ 16+ 23.5+ 12+ 17+ 22+ 18+ 22+ 24.5 15+ 17 14+ 19+
F F F F F F F F F F F F F F F F F F F F F F F F F F M M M M M M M M M M M M M M M M M M M M M M M M M M
76 94 80 94 94 94 95 90 94 97 94 95 93 91 90 98 94 90 85 90 96 95 88 88 95 93 87 91 97 88 64 90 92 93 93 96 96 93 93 91 93 87 91 95 76 92 86 75 91 94 76 88
P26 269 230 658 354 1110 154 419 136 167 261 222 482 80 42 133 125 135 247 127 209 103 729 367 410 93 119 700 930 1240 693 617 777 322 905 859 901 1050 790 469 335 478 938 420 576 506 906 967 553 721 805 420 593
P40/41 P44 54 45 106 96 124 49 46 24 28 35 39 56 ndb ndb ndb 30 22 42 23 31 15 66 64 72 17 25 67 117 131 112 48 116 33 90 136 58 113 76 31 ndb 68 107 47 73 38 55 83 ndb 68 58 30 38
39 37 54 67 65 26 28 18 20 19 26 27 8.0 ndb ndb 20 24 18 12 14 8.4 26 36 41 11 13 33 74 78 75 26 79 17 53 44 29 39 36 14 18 43 68 24 58 15 30 32 ndb 27 27 11 13
P50
P62
343 340 1580 622 1690 360 574 249 270 492 197 738 298 142 347 211 419 719 259 387 228 1390 737 563 176 215 1250 1940 2170 1270 1150 1310 563 1710 1550 1690 2170 1510 867 1630 2000 3060 1250 2110 965 1460 1750 1310 1380 1350 747 1120
65 69 65 82 73 56 30 26 23 17 37 17 ndb 13 ndb 15 29 27 3.0 3.0 3.7 13 5.1 30 2.5 4.4 25 52 75 60 23 77 12 33 16 14 38 33 8.1 34 59 69 32 51 7.6 18 23 14 18 11 6.4 11
a + sign indicates that ages may be underestimated due to unreadable dentine layers. b nd ) not detected.
standard solution and in a lipid-free burbot liver extract (21, 22). The CHBs were quantified relative to the internal standard added at extraction, and all reported concentrations were corrected for procedural losses. The mean recovery of the internal standard was 77% (CV ) 29%). Statistical Analysis. All statistical analyses were performed using Systat 10.0 and Microssoft Excel 97 software, with a significance level of P < 0.05. For temporal trend assessment only, CHB concentrations were expressed on a lipid weight basis to avoid the effect of possible loss of water that could occur in long term archived samples comparatively to the more recently collected samples. Results for males and females were treated separately because CHB concentrations
FIGURE 2. Time trends of CHB concentrations in beluga whales from the SLE. Significant regression lines are shown. See Table 3 for regression parameters. were generally greater in males than in females (see Figures 2 and 3). To reduce skewness and kurtosis of the sample distribution, concentration data were natural logarithmtransformed. Data were checked for normality using the D’Agostino-Pearson K2 test on regression residuals and for homoscedasticity using the Kendall rank order test on regression residuals and estimates (23). Temporal trends were assessed using simple linear regression analysis followed by an analysis of variance (ANOVA) to test the significance of VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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CHB levels measured in beluga blubber samples from the SLE are given in Table 1. P26 and P50 were detected in all samples, whereas P40/41, P44, and P62 were below detection limits in a few samples. Male belugas were generally more contaminated than female animals (Table 1). For instance, P50 concentrations ranged from 140 to 1690 ng/g wet weight in female belugas and 560-3060 ng/g wet weight for males. CHB levels were roughly 1.5-3 times higher in males than in females which is comparable to previously reported results for P26, P50, and total toxaphene in SLE belugas (7, 8, 15). The gestational and mainly the lactational transfers of CHBs via lipid rich tissues or milk from females to the fetus and the calf, respectively, were demonstrated in belugas from the West Greenland (25). Similar results were observed in SLE belugas for PCBs and OCs (26, 27). As a result, the reproductively active females were subjected to a depuration of persistent chlorinated organic contaminants relative to the males. P26 and P50 mean concentrations were in the lower range of those previously reported for SLE belugas, especially for females (Table 2). Moreover, P26 and P50 mean concentrations in belugas measured in the present study were in the same order of magnitude as those reported for animals living in the Arctic environment (Table 2). Levels of total toxaphene previously reported in SLE male belugas in the 1980s were also in the same range than those in animals from Arctic (7). In comparison, the ratio SLE/Arctic ranged from about 1630 for ΣPCBs and ΣDDTs, two groups of compounds that were used to a large extent in the populated and industrialized region of the lower St. Lawrence river (7, 28). These observations suggest that atmospheric transport represents the main input of CHBs to these two distinct environments. In the SLE, the local toxaphene usage is considered negligible since it accounted for less than 0.5% of total inputs of toxaphene to the Gulf of St. Lawrence region between 1945 and 2000 estimated by atmospheric transport and deposition modeling (29). In addition, levels of two of the most predominant CHBs in sediments (2-exo,3-endo,6-exo,8,9,10-hexachlorobornane and 2-endo,3-exo,5-endo,6-exo,8,9,10-heptachlorobornane) were below 50 pg/g dry weight in sediments of the SLE, suggesting limited local input of toxaphene to this environment in the past (30). Riverine inputs from the Great Lakes region, the drainage basin of the SLE, represent less than about 10% of the total input of toxaphene to the Gulf of St. Lawrence region (29). This suggests that both internal (local usage) and riverine inputs of toxaphene to the SLE are minor compared to the atmospheric input.
FIGURE 3. Time trends of P26 and P50 (our results + Muir et al. 1996 corrected results) concentrations in beluga whales from the SLE. Corrections on Muir et al. 1996 results are explained in the text. Significant regression lines are shown. See Table 3 for regression parameters. the regression. Slopes of regression lines were compared using an analysis of covariance on regression residuals. As slopes were not all equal, the a posteriori Newman-Keuls test was conducted to determine which of the slopes differ from which others (23).
Results and Discussion Levels and Relative Abundance of CHBs in Belugas from the SLE. In this study, levels of CHBs were determined in stranded beluga whales found on the shores of the SLE. Most of the carcasses examined were in good (code 2) or fair (code 3) conditions according to the classification of Geraci and Lounsbury (24). A study has recently revealed that no significant difference could be made between total toxaphene concentrations measured in biopsied free-ranging animals and in stranded belugas collected during the same time period in the SLE (8).
TABLE 2. Average Concentrations ( SD (ng/g Wet Weight) of Two Major Toxaphene Congeners, P-26 and P-50, in Blubber of Belugas from Arctic and SLE Environmentsa region
sex
period
n
age
P26
P50
reference
nab
468 1300 ( 325c 1250 ( 745 (149-3070) 749 ( 137 (487-932) 710 ( 240 (322-1240) 304 600 ( 310c 493 ( 435 (154-2170) 574 ( 293 (112-832) 280 ( 240 (42-1110)
840 2350 ( 380c 4120 ( 2130 (475-9750) 1510 ( 350 (1060-2090) 1510 ( 530 (563-3060) 630 1050 ( 690c 1770 ( 1460 (322-5840) 1440 ( 783 (355-2320) 520 ( 420 (142-1690)
(47) (48) ( 7)
Arctic (Mackenzie River delta) Arctic (E. Hudson Bay) SLE
M M M
before 1997 before 1992 1987-1990
20 8 15
SLE
M
1993-1994
9
SLE
M
1988-1999
26
Arctic (Mackenzie River delta) Arctic (E. Hudson Bay) SLE
F F F
before 1997 before 1992 1987-1990
5 8 21
SLE
F
1993-1994
7
SLE
F
1988-1999
26
a
Ranges are indicated in brackets when available.
4606
9
b
nab 20.1 ( 7.7 (
