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Perfluorinated contaminants in fur seal pups and penguin eggs from South Shetland, Antarctica
Science of the Total Environment 407 (2009) 3899–3904
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Perfluorinated contaminants in fur seal pups and penguin eggs from South Shetland, Antarctica A. Schiavone a,⁎, S. Corsolini a, K. Kannan b, L. Tao b, W. Trivelpiece c, D. Torres Jr. d, S. Focardi a a
Department of Environmental Science G. Sarfattiá, University of Siena, via P.A. Mattioli, 4, I-53100 Siena, Italy Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA c U.S. Antarctic Marine Living Resources Division, Southwest Fisheries Science Center, 8604 La Jolla Shores Drive, PO Box 271, La Jolla, CA 92037, USA d Instituto Antartico Chileno (INACH), Plaza Munoz Gamero, 1055, Punta Arenas, Chile b
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 19 December 2008 Accepted 19 December 2008 Available online 24 March 2009 Keyword: Perfluorinated compounds Penguin Egg Seal Antarctica
a b s t r a c t Perfluorinated compounds (PFCs) have emerged as a new class of global environmental pollutants. In this study, the presence of perfluorochemicals (PFCs) in penguin eggs and Antarctic fur seals was reported for the first time. Tissue samples from Antarctic fur seal pups and penguin eggs were collected during the 2003/04 breeding season. Ten PFC contaminants were determined in seal and penguin samples. The PFC concentrations in seal liver were in the decreasing order, PFOS N PFNA N PFHpA N PFUnDA while in Adélie penguin eggs were PFHpA N PFUnDA N PFDA N PFDoDA, and in Gentoo penguin eggs were PFUnDA N PFOS N PFDoDA N PFHpA. The PFC concentrations differed significantly between seals and penguins (p b 0.005) and a species-specific difference was found between the two species of penguins (p b 0.005). In our study we found a mean concentration of PFOS in seal muscle and liver samples of 1.3 ng/g and 9.4 ng/g wet wt, respectively, and a mean concentration in Gentoo and Adélie penguin eggs of 0.3 ng/g and 0.38 ng/g wet wt, respectively. PFCs detected in penguin eggs and seal pups suggested oviparous and viviparous transfer of PFOS to eggs and off-springs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Perfluorinated compounds (PFCs) have emerged as a new class of global environmental pollutants. Several studies have reported their occurrence in wildlife (Giesy and Kannan, 2001; Olivero-Verbel et al., 2006; Van de Vijven et al., 2005), from low latitude to remote areas, suggesting their global distribution including open ocean waters and biota (Prevedouros et al., 2006; Tao et al., 2006; Yamashita et al., 2008). PFCs are persistent and bioaccumulative, although their physicochemical properties are different from other known persistent organohalogens. PFCs such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), unlike organochlorines, do not accumulate in lipids but concentrate in blood and liver tissues (Giesy and Kannan, 2001, 2002; Kannan et al., 2001a). PFOA and PFOS are peroxisome proliferators and elicit potent immunomodulating effects in mice, involving thymic and splenic atrophy, loss of thymocytes and splenocytes, and potent suppression of adaptive immune responses (Yang et al., 2002; Ishibashi et al., 2008a). The exposure of rats to PFOA resulted in the suppression of genes involved in inflammation and immunity (Guruge et al., 2006).
⁎ Corresponding author. Tel.: +39 0577 232 882; fax: +39 0577 232 806. E-mail address: [email protected] (A. Schiavone). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.12.058
Marine mammals are sensitive to accumulation by persistent and bioaccumulative contaminants, because of their high trophic position in the marine food web. Similarly, avian eggs have been used as indicators of environmental contamination. Much of the information on the contamination by PFCs is from biological samples collected in Northern Hemisphere (Kannan et al., 2001a; Van de Vijver et al., 2005; Ishibashi et al., 2008b); little is known about the current status and temporal trend of PFC contamination in Antarctica (Giesy and Kannan, 2001; Kannan et al., 2001b; Tao et al., 2006). In this study, we used tissues of Antarctic fur seals and penguins to determine the concentrations of PFCs in the Antarctic ecosystem. Antarctic fur seals and penguins feed at the top of the polar marine food chain. In addition, they are non-migratory and non-nomadic species breeding in the Antarctic region; the tissue concentrations of chemicals are an indication of local contamination (Hollamby et al., 2006). Tissue samples from Antarctic fur seal pups were collected from the carcasses found dead during a season (2004) of high neonatal seal mortality. Penguin eggs samples were take from unhatched eggs, following permission from the Scientific Committee for Antarctic Research (SCAR) (Ahmed, 2003). The aim of this study was to determine the concentrations of PFCs in Antarctic organisms. The detection of “new contaminants” in remote regions such as Antarctica suggests their widespread distributions and highlighting the need to understand transportation pathways and sources.
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2. Materials and methods Twenty muscle, seventeen liver and five blubber samples were collected from Antarctic fur seal (Arctocephalus gazella) pups between January and February 2004, in the framework of the Italian Program of Research in Antarctica (PNRA) and of a National Science Foundation (NSF) expedition. Samples were collected on Livingston Island, South Shetland, Antarctic Peninsula, (62°39′ S, 60°30′ W). Antarctic fur seal pups were found dead and the tissue samples were taken from the carcasses at the time of necropsy. Samples had no signs of decomposition. Sampling location, gender, body weight, and body length were recorded (Table 1). Pups were aged based on their pelage stage (Kovacs and Lavigne, 1986). Unhatched eggs of Adélie penguins (Pygoscelis adéliae, n = 13) and Gentoo penguins (Pygoscelis papua, n = 13) were collected on King George Island, South Shetland, Antarctic Peninsula (62°10′ S, 58°67′ W), during the 2004/05 field season. All samples were wrapped in polyethylene bags and stored at −20 °C until analysis. 2.1. Analytical methods and instrumental analysis for PFCs PFCs were analyzed following the method described elsewhere (Kannan et al., 2001a; Tao et al., 2006), with some modifications. PFCs were extracted by ion-pairing liquid extraction method. For egg samples, 0.5 g of whole egg homogenate, 3 mL of Milli-Q water, 4 ng of each internal standard (PFBS, 13C4-PFOS, and 13C4-PFOA), 2 mL of 0.25 M sodium carbonate buffer, and 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate solution (adjusted to pH 10) were mixed in a 15 mL polypropylene (PP) tube. The sample was then extracted with 5 mL of methyl-tert-butyl ether (MTBE) by vigorous shaking for 45 min. The MTBE layer was separated by centrifugation at 3500 rpm for 5 min and then transferred into another PP tube (~ 4.5 mL). The aqueous mixture was rinsed with MTBE and separated twice; all rinses were combined in the second polypropylene tube. The MTBE extract was evaporated to near-dryness under a gentle stream of nitrogen and then reconstituted with 1 mL of methanol. For the extraction of liver and muscle samples, a small amount of tissue (about 1 g) was homogenized with 5 mL of Milli-Q water, and then 1 g of the homogenate was transferred into a PP tube and extracted following the procedure described above. Matrix matched calibration standards (seven points ranging from 0.5 to 75 ng/mL) were prepared by spiking different amounts of calibration standards into sample matrix that contained no quantifiable amount of the target analytes; these
Table 1 Details of fur seal pups found dead and used for collecting the samples. Sample
Date of sampling
Tissue
Age
Sex
Length (cm)
Weight (Kg)
08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 February 2004 February 2004 February 2004
M M, M M M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M,
Steel born 2 week old 2 week old 2 week old 1 month old 1 month old 1 month old 1 month old Steel born 45 days old 45 days old 1 week old 45 days old 45 days old 1 month old 45 days old 2 months old 2 months old 2 months old 2 months old
♀ ♂ ♀ ♀ ♂ ♂ ♂ ♀ ♂ ♂ ♂ ♀ ♂ ♀ ♀ ♀ ♂ ♀ ♀ ♀
68 66.5 65 71 77.5 73 74 67 74 77 77 65.5 74 77 71 65 77 77 74 80
4.6 4 4.3 4.2 6 4.6 4.6 3.6 5.7 5.45 6 4 5.3 5.3 4.3 3.65 5.5 4.8 5.6 7.2
(M = muscle, L = liver).
L
L L L L L L L L L L L L L L L L
standards were passed through the entire analytical procedure along with the samples. Analytes were detected and quantified using an Agilent 1100 series high-performance liquid chromatograph (HPLC) coupled with an Applied Biosystems API 2000 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS). The MS/MS was operated in electrospray negative ion mode. Target compounds were determined by multiple reaction monitoring (MRM). The MRM transitions were 299 N 80 for PFBS, 399 N 80 for PFHS, 499 N 99 for PFOS, 503 N 99 for 13C4-PFOS, 599 N 99 for PFDS, 498 N 78 for PFOSA, 363 N 319 PFHpA, 413 N 369 for PFOA, 417 N372 for 13C4-PFOA, 463 N 419 for PFNA, 513 N 469 for PFDA, 563 N 519 for PFUnDA, and 613 N 569 for PFDoDA. Samples were injected twice to monitor sulfonates and carboxylates separately and PFBS was monitored in both of the injections. A midpoint calibration standard was injected after every 10 samples to check for the instrumental response and drift. Calibration standards were injected daily before and after the analysis. Reported concentrations were not corrected for the recoveries. Blanks were analyzed by passing Milli-Q water and reagent through the entire analytical procedure. Blanks contained trace levels of PFOA in penguin egg analyses and trace of PFOA and PFOS in seals sample analyses. The concentrations reported here were subtracted from the mean value found in blanks. To check the recovery, 13C4-PFOS and 13C4-PFOA were used as internal standards. For penguin eggs and seal tissue samples, mean recoveries of were 61% and 89% for 13C4-PFOS, and 133% and 75% for 13C4-PFOA, respectively. The limit of quantitation (LOQ) was determined based on the linear range of the calibration curve prepared at a concentration range of 0.1 to 20 ng/mL. The tissue samples were compared to this unextracted standard calibration curve. Because of the variety of matrices analyzed and because of evolving analytical methods, the LOQ was variable. The LOQ was determined as the lowest acceptable standard in the calibration curve, deemed acceptable if it was within ±30% of the theoretical value and the peak area of the standard was at least twice as great as the matrix blanks. The LOQ was 0.4 ng/g for all analytes in seal samples, and 0.1 ng/g for all analytes in penguin eggs, except for PFOA and PFHpA, for which the LOQ was 0.2 and 0.5 ng/g respectively. 2.2. Statistical analysis Statistical analyses were performed with STATISTICA 7 for Windows (Ver 7.1; Statsoft, Italia srl) at a significance level of p = 0.05. Statistically significant differences between the mean concentrations of contaminants were investigated by a single factor one-way analysis of variance (ANOVA) and when significant differences were found, they were tested among each other using Tukey's post hoc test. The absolute concentrations were log10-transformed prior to statistical analysis if the data did not follow a normal distribution (Levene test p b 0.05). The transformed data were normally distributed and the Tukey test was used to analyze the differences in concentrations between species, category of species or tissues. The overall significance level of each analysis was set at p b 0.05. The Tukey–Kramer (HSD) test is one of a number of post-hoc methods recommended for testing differences between pairs of means among groups that contain unequal sample sizes (Zar, 1999). The correlations between PFC concentrations and seal age groups were assessed by simple correlation analyses (Zar, 1999). Correlations are expressed using the Pearson correlation coefficient r. If a concentration of PFC was below the LOQ in a sample, the LOQ value itself or a value of half the respective detection limit, was considered prior to statistical analyses. The statistical analysis between seal tissues were limited by the different number of samples (liver samples n = 17, and muscle samples n = 20). 3. Results and discussion Fur seal samples were analyzed for the presence of four perfluorinated sulfonic acids (PFSAs), and six perfluorinated
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Table 2 Concentrations (ng/g ww; mean ± SD) of perfluorinated compounds in Antarctic samples (nd = not detected).
PFHS PFHpA PFOS PFOSA PFOA PFNA PFDA PFDS PFUnDA PFDoDA PFCs
Fur seal pup, muscle
Fur seal pup, liver
% detected
Mean ± SD
% detected
Mean ± SD
% detected
Gentoo penguin egg Mean ± SD
% detected
Mean ± SD
0 80 100 65 50 0 0 0 0 0
nd 0.5 ± 0.3 1.3 ± 0.7 b0.4 0.8 ± 0.8 nd nd nd nd nd 2.7 ± 1
82 100 100 100 6 94 71 6 71 6
b 0.4 1.0 ± 1.9 9.4 ± 3.2 b 0.4 b 0.4 3.3 ± 1.7 0.6 ± 0.5 b 0.4 0.9 ± 0.9 b 0.4 16 ± 6.2
0 15 100 0 8 0 31 Not analyzed 100 54
nd b0.5 0.3 ± 0.1 nd b0.2 nd 0.1 ± 0.2
0 54 100 54 23 8 85 Not analyzed 100 85
nd 2.5 ± 5.5 0.4 ± 0.2 0.2 ± 0.3 b0.2 b0.1 1.3 ± 2.9
carboxylic acids (PFCAs); perfluorooctane sulfonate (PFOS), perfluorhexanesulfonate (PFHS), perfluorooctanesulfonamide (PFOSA), perfluorodecanesulfonate (PFDS), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoA) and perfluoroundecanoic acid (PFUnDA). Penguin samples were analyzed for all of these contaminants except for PFDS (Table 2). 3.1. PFC concentrations in seals: tissue distribution Concentration of PFCs were significantly higher in liver than in muscle (ANOVA p b 0.001), suggesting compound-specific persistence and retention of PFCs in seal liver. Mean concentration of ∑PFSAs (1.4 ng/g ww) was similar to the mean concentration of ∑PFCAs (1.3 ng/g ww) in seal muscle samples. Mean concentration of ∑PFSAs (10.4 ng/g ww) in seal liver samples was 68% higher than that of ∑PFCAs (6.2 ng/g ww), suggesting preferential enrichment of PFOS in seal liver. Hepatic concentrations of PFOS were not correlated (p N 0.05) with age. The PFC concentrations in liver and muscle of seal samples were in the decreasing order, PFOS N PFNA N PFHpA N PFUnDA and PFOS N PFOA N PFHpA N PFOSA, respectively. A similar trend was previously reported in marine mammals (Martin et al., 2004; Van de Vijver et al., 2003), including polar bears (Smithwick et al., 2005; Kannan et al., 2005) from North America, where concentrations decreased with increasing chain length, and the predominant PFC was PFOS. Seal liver samples contained significantly (p b 0.001) higher concentrations of PFOS than the muscle samples, while PFNA was no detected in muscle samples. High PFOS concentrations (range b0.08–3.52 ng/mL ww) were also reported in blood samples of elephant seal pups from the South Shetland Islands (Tao et al., 2006). Previous studies have reported higher PFOS concentrations in dolphins and harbour porpoise pups than in adults (Houde et al., 2005; Van de Vijver et al., 2004; Hart et al., 2008). Some studies have clearly demonstrated a significant placental transfer of PFOS from dam to fetus during gestation (in utero exposure to PFOS) and to the pup during lactation (Luebker et al., 2005a,b; Hart et al., 2008). PFOA was detected at low concentrations (mean 0.8 ng/g ww) in 50% of seal muscle samples, and between 6 and 23% in the other samples analyzed. In general, PFOA has been detected sporadically in marine mammals (Kannan et al., 2002b) and birds (Bossi et al., 2005a; Kannan et al., 2002c; Holmström and Berger, 2008).
Adélie penguin egg
0.6 ± 0.5 0.3 ± 0.8 1.3 ± 1.2
2.3 ± 6.5 0.5 ± 0.4 7.35 ± 9
PFOS. Thus, the mean concentrations of ∑PFCAs in Gentoo penguin (1.08 ng/g ww) and Adélie penguin eggs (6.6 ng/g ww) were an order of magnitude higher than ∑PFSAs (0.29 ng/g ww and 0.56 ng/g ww, respectively) (Fig. 1). PFCA profile was dominated by PFUnDA (mean 0.59 ng/g ww) in Gentoo penguin eggs, and by PFUnDA (2.33 ng/g ww), and PFHpA (mean 2.53 ng/g ww) in Adélie penguin eggs, in which the PFCA concentrations were also higher than that of PFOS (mean 0.29, 0.38 ng/g ww, respectively). High concentrations of long-chain PFCAs in eggs suggest oviparous transfer of these compounds. 3.3. Correlations between PFCs The statistical associations between PFOSA and PFOS have previously been used to demonstrate that PFOSA is a precursor of PFOS (Kannan et al., 2002a; Martin et al., 2004). We found no statistically significant correlation between PFOS and PFOSA in Adélie penguin eggs (p N 0.05). Other reports (Kannan et al., 2001b; Martin et al., 2004; Bossi et al., 2005) have found statistical association between PFOSA and PFOS for different species, but not always consistent. It is not clear whether this association represents metabolism or direct exposure. Statistically significant linear correlations were only found between the concentrations of PFDA and PFUnA (p b 0.05) in the seal liver (Fig. 2), suggesting a common source of these two PFCAs to fur seals. A significant positive relationship between concentrations of PFOS and PFNA was observed in fur seal liver (Fig. 3). These results indicate that the sources of exposure of the seal to PFOS and PFNA are similar, or the coexistence of these compounds in the sources. 3.4. Species-specific differences The PFC concentrations differed significantly between seals and penguins (p b 0.005) and a species-specific difference was found
3.2. PFCs in penguins: long-chain PFCA Concentrations of PFCs were found in the decreasing order PFHpAN PFUnDA N PFDA N PFDoDA in Adélie penguin eggs, and PFUnDA N PFOSN PFDoDA N PFHpA in Gentoo penguin eggs. Several long-chain PFCAs (PFDA, PFUnDA, PFDoDA) were the predominant PFCs detected in penguin egg samples at the concentrations higher or no less than that of
Fig. 1. ∑PFSA and ∑PFCA concentrations (ng/g ww) in seal and penguin samples.
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Fig. 2. Linear regression of PFDA and PFUnDA concentrations in fur seal liver from Antarctica. A log transformation was performed to normalize the data.
between the two species of penguins (p b 0.005) (Fig. 4). Although these organisms share the same environment, differences in diet and metabolism might explain the variations in the concentrations observed. Reid and Arnould (1996) investigated the diet of lactating female Antarctic fur seals (Arctocephalus gazella) in South Georgia, and found that Antarctic krill (Euphausia superba) was the main prey item, followed by fishes and squid. The myctophids Protomyctophum choriodon were the major diet during the lactational period. Outside this period, the nototheniid Lepidonotothen larseni and the channichthyid Champsocephalus gunnari were the dominant prey species (Reid and Arnould, 1996). In contrast, differences in the PFC concentrations between penguin species may be related to diet, reproductive status, ecological niches, and migration. Both penguin species in our study were from the South Shetland Islands, and feed primarily on Euphausia superba, although Gentoo penguins feed more fish (Antarctic silverfish Pleuragramma antarcticum) than Adélie penguins. E. crystallorophias and pelagic and benthic species of amphipods are minor components of the pygoscelid diet (Volkman et al., 1980). Gentoo penguins feed inshore and are deep divers, while Adélie penguins are shallow-diving, offshore foragers (Trivelpiece et al., 1987). Among PFCs, PFOS was detected in all of the samples. PFOS concentrations in the liver and muscle of seal pups were significantly higher (p b 0.001) than those found in penguin eggs. No significant difference was found in PFOS concentrations between eggs from the
Fig. 3. Relationship between concentrations of PFOS and PFNA in fur seal liver from Antarctica.
Fig. 4. Concentrations of perflurooctane sulfonate (ng/g ww) in seal and penguin samples from Antarctica. The straight line is the mean. The dots represent the outlier (N 1.5 interquartile lengths from box edge). Statistically significant difference (p b 0.005) is indicated by ⁎.
two species of penguins (Fig. 5). PFOSA was below the detection limit in seal muscle and liver samples, and in Gentoo penguin eggs, but was detected in Adélie penguin eggs with a frequency of 54%. The PFCA profile was dominated by PFUnDA in penguin eggs, in which the PFCA concentrations were also higher than that of PFOS. Similar results have been reported in other studies on birds (Holmström and Berger, 2008; Verreault et al., 2007). 3.5. Comparison with other studies The presence of PFCs in penguin eggs and Antarctic fur seals is reported here for the first time. In fact, little information has been provided so far on PFC concentrations in these Antarctic organisms (Table 3). One previous study reported PFOS values under the detection limit in Adélie penguin eggs (b0.1 ng/g ww; Tao et al., 2006), while in our study we found a mean concentration of 0.38 ng/g ww. This difference could be due to the different collection period, 1995/1996 and 2004 respectively, suggesting an increase in the environmental levels of these contaminants. Tao et al. (2006) reported a mean PFOS concentration in the blood of elephant seal pups of 0.53 ng/mL — one order of magnitude lower than that found in our fur seal pup liver samples (mean: 9 ng/g ww). This difference may due to the preferential accumulation of PFCs in liver tissues (Van de Vijver et al., 2005).
Fig. 5. Log concentrations of PFOS (ng/g ww) in seal and penguin samples from Antarctica. The straight line is the mean. The dots represent the outlier (N 1.5 interquartile lengths from box edge) Statistically significant difference (p b 0.001) is indicated by ⁎.
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Table 3 Mean (Range) concentration of PFOS (ng/mL in blood or ng/g in other tissues ww) in seals and birds from Antarctica. Matrix
Date of collection
Species
n
Location
PFOS
References
ww Blood
2001 2003/4 and 2004/2005 Not available
Egg
1995/1996 1998/1999 2004/05
Liver
2004/05 Not available
Muscle a
2004/05
Pygoscelis adéliae (Adélie penguin) Mirounga leonine (Elephant seal) Stercorarius maccormicki (Polar skua) Pygoscelis adéliae (Adélie penguin) Stercorarius maccormicki Polar skua Pygoscelis adéliae (Adélie penguin) Pygoscelis papua (Gentoo penguin) Arctocephalus gazella (Antarctic fur seal) Leptonychotes weddellii (Weddell seal) Arctocephalus gazella (Antarctic fur seal)
a
b0.1
2
Admiral Bay South Shetlands Elephant Island South Shetlands Terra Nova Bay
1.2 (b 1 –1.4)
Giesy and Kannan (2001)
6
Edmonson point
b0.1a
Tao et al. (2006)
8 59
3 17
1 20
0.53 (b 0.08–3.52) a
Tao et al. (2006)
2.51 (2.08–3.12) Admiral Bay, King George Is.
20 17
Tao et al. (2006)
0.38 (0.18–0.89)
This study
0.28 (0.13–0.49) Livingston Island
9.01 (1.85–17.25) a
Terra Nova Bay
b35
Livingston Island
1.29 (0.42–3.59)
This study Kannan et al. (2001b) This study
Concentration of PFOS were below LOQ for all samples.
In comparison to seals and birds from the northern hemisphere, the concentrations of PFOS and PFOA in penguins and seals from the South Ocean were 10–100 fold lower. Despite the species differences, concentrations of PFOS measured in the liver of fur seals in our study were two orders of magnitude lower than concentrations in liver of harbor seals (162 ng/g ww) from the northwest Atlantic (Shaw et al., 2006) and one order of magnitude lower than that found in ringed seals (67 ng/g ww) from Greenland (Bossi et al., 2005). The PFOS concentrations found in our penguin eggs were two orders of magnitude lower than the concentrations in herring gull eggs (52 ng/g ww) from northern Norway (Verreault et al., 2007), and three orders of magnitude lower than that found in glaucous gull eggs (104 ng/g ww) from Svalbard (Verreault et al., 2005) and in guillemot eggs (614 ng/g ww) from the Baltic Sea (Holmström et al., 2005). However, comparisons should be interpreted with caution due to interspecies differences in feeding ecology. Although the contamination levels of PFOS and PFOA are low in southern hemisphere fauna, the occurrence of PFCs in these remote locations suggests the widespread distribution of PFCs. Antarctic organisms should continue to be monitored in future due to the global trend of increasing PFC use. 3.6. Ecological implications Acute and chronic dietary exposure studies of PFOS in mallard ducks (Anas platyrhynchos) and northern bobwhite quails (Colinus virginianus) (Newsted et al., 2007, 2006) have recently led to the calculation of PFOS toxicity reference values (TRVs) and predicted noeffect concentrations (PNECs), based on the characteristics of top avian predators (e.g. birds of prey and certain gull species) (Newsted et al., 2005). Conservative egg yolk-based TRVs and PNECs were determined as 1.7 and 1.0 μg PFOS/mL, respectively. In the same study, the lowest observable adverse effect level (LOAEL) in egg yolk was 62.0 μg PFOS/mL. Hence, the mean PFOS concentration evaluated in penguin eggs from Antarctic Peninsula was approximately 3000 times lower than the PFOS TRV, PNEC, and LOAEL values, respectively. From a toxicological standpoint, and assuming that the sensitivity to PFOS exposure of mallard ducks and northern bobwhite quails is similar in penguins, recent concentrations in eggs suggest that PFOS alone would pose a minimal risk to the developing penguin embryo. However, PFOS and other accumulated PFASs in penguins need to be assessed as a part of a broad contaminant cocktail, including chlorine-
and bromine-based chemicals with potential health risks (Newsted et al., 2007, 2006; Molina et al., 2006). Other laboratory studies on rats, monkeys and birds have shown that the toxic effects of PFOA and PFOS occur at tissue concentrations in the range of a few tens to hundreds of μg/g ww (Kennedy et al., 2004; Newsted et al., 2006). Residue concentrations of PFOS and PFOA in our fur seal livers were approximately 3–4 orders of magnitude lower than the effect concentrations found in laboratory animals. The toxic effects of PFCs in seal species are unknown, as only one study has found a significant association of PFOS and PFOA with the presence of disease mortality in sea otters (Kannan et al., 2006): establishment of a cause-effect linkage will require toxicological and controlled animal feeding studies. Further studies are needed on the immunotoxic effects of PFCs and also on the interaction between PFCs and other contaminants found in fur seal tissues. Acknowledgments This research was funded by the Italian National Program of Research in Antarctica (PNRA). The National Science Foundation supported S. Corsolini's stay and travel to and from King George Is. We are very grateful to Daniel Torres and Daniel Torres jr (Instituto Antarctico Chileno, Santiago, Chile) for collecting the fur seal samples during the 2003/04 expedition, and to Wayne Trivelpiece, and Susan Trivelpiece for collecting the penguin eggs samples. We thanks Roger Hewitt, the Agunsa (Punta Arenas, Chile) and Raytheon (USA) for their friendly logistic support. References Ahmed T. “Pygoscelis papua” (On-line), Animal Diversity Web. (2003). Accessed February 18, 2008 at http://animaldiversity.ummz.umich.edu/site/accounts/ information/Pygoscelis_papua.html. Bossi R, Riget FF, Dietz R. Temporal and spatial trends of perfluorinated compounds in Ringed Seal (Phoca hispida) from Greenland. Environ Sci Tecnol 2005;39:7416. Bossi R, Riget FF, Dietz R, Sonne C, Fauser P, Dam M, et al. Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands. Environ Pollut 2005a;136:323–9. Giesy PJ, Kannan K. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 2001;35:1339–42. Giesy JP, Kannan K. Perfluorochemical surfactants in the environment. Environ Sci Technol 2002;36:146A. Guruge KS, Yeung LWY, Yamanaka N, Miyazaki S, Lam PKS, Giesy JP, et al. Gene expression profiles in rat liver treated with perfluorooctanoic acid (PFOA). Toxicol Sci 2006;89:93-107.
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Perfluorinated contaminants in fur seal pups and penguin eggs from South Shetland, Antarctica A. Schiavone a,⁎, S. Corsolini a, K. Kannan b, L. Tao b, W. Trivelpiece c, D. Torres Jr. d, S. Focardi a a
Department of Environmental Science G. Sarfattiá, University of Siena, via P.A. Mattioli, 4, I-53100 Siena, Italy Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, USA c U.S. Antarctic Marine Living Resources Division, Southwest Fisheries Science Center, 8604 La Jolla Shores Drive, PO Box 271, La Jolla, CA 92037, USA d Instituto Antartico Chileno (INACH), Plaza Munoz Gamero, 1055, Punta Arenas, Chile b
a r t i c l e
i n f o
Article history: Received 18 November 2008 Received in revised form 19 December 2008 Accepted 19 December 2008 Available online 24 March 2009 Keyword: Perfluorinated compounds Penguin Egg Seal Antarctica
a b s t r a c t Perfluorinated compounds (PFCs) have emerged as a new class of global environmental pollutants. In this study, the presence of perfluorochemicals (PFCs) in penguin eggs and Antarctic fur seals was reported for the first time. Tissue samples from Antarctic fur seal pups and penguin eggs were collected during the 2003/04 breeding season. Ten PFC contaminants were determined in seal and penguin samples. The PFC concentrations in seal liver were in the decreasing order, PFOS N PFNA N PFHpA N PFUnDA while in Adélie penguin eggs were PFHpA N PFUnDA N PFDA N PFDoDA, and in Gentoo penguin eggs were PFUnDA N PFOS N PFDoDA N PFHpA. The PFC concentrations differed significantly between seals and penguins (p b 0.005) and a species-specific difference was found between the two species of penguins (p b 0.005). In our study we found a mean concentration of PFOS in seal muscle and liver samples of 1.3 ng/g and 9.4 ng/g wet wt, respectively, and a mean concentration in Gentoo and Adélie penguin eggs of 0.3 ng/g and 0.38 ng/g wet wt, respectively. PFCs detected in penguin eggs and seal pups suggested oviparous and viviparous transfer of PFOS to eggs and off-springs. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Perfluorinated compounds (PFCs) have emerged as a new class of global environmental pollutants. Several studies have reported their occurrence in wildlife (Giesy and Kannan, 2001; Olivero-Verbel et al., 2006; Van de Vijven et al., 2005), from low latitude to remote areas, suggesting their global distribution including open ocean waters and biota (Prevedouros et al., 2006; Tao et al., 2006; Yamashita et al., 2008). PFCs are persistent and bioaccumulative, although their physicochemical properties are different from other known persistent organohalogens. PFCs such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), unlike organochlorines, do not accumulate in lipids but concentrate in blood and liver tissues (Giesy and Kannan, 2001, 2002; Kannan et al., 2001a). PFOA and PFOS are peroxisome proliferators and elicit potent immunomodulating effects in mice, involving thymic and splenic atrophy, loss of thymocytes and splenocytes, and potent suppression of adaptive immune responses (Yang et al., 2002; Ishibashi et al., 2008a). The exposure of rats to PFOA resulted in the suppression of genes involved in inflammation and immunity (Guruge et al., 2006).
⁎ Corresponding author. Tel.: +39 0577 232 882; fax: +39 0577 232 806. E-mail address: [email protected] (A. Schiavone). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.12.058
Marine mammals are sensitive to accumulation by persistent and bioaccumulative contaminants, because of their high trophic position in the marine food web. Similarly, avian eggs have been used as indicators of environmental contamination. Much of the information on the contamination by PFCs is from biological samples collected in Northern Hemisphere (Kannan et al., 2001a; Van de Vijver et al., 2005; Ishibashi et al., 2008b); little is known about the current status and temporal trend of PFC contamination in Antarctica (Giesy and Kannan, 2001; Kannan et al., 2001b; Tao et al., 2006). In this study, we used tissues of Antarctic fur seals and penguins to determine the concentrations of PFCs in the Antarctic ecosystem. Antarctic fur seals and penguins feed at the top of the polar marine food chain. In addition, they are non-migratory and non-nomadic species breeding in the Antarctic region; the tissue concentrations of chemicals are an indication of local contamination (Hollamby et al., 2006). Tissue samples from Antarctic fur seal pups were collected from the carcasses found dead during a season (2004) of high neonatal seal mortality. Penguin eggs samples were take from unhatched eggs, following permission from the Scientific Committee for Antarctic Research (SCAR) (Ahmed, 2003). The aim of this study was to determine the concentrations of PFCs in Antarctic organisms. The detection of “new contaminants” in remote regions such as Antarctica suggests their widespread distributions and highlighting the need to understand transportation pathways and sources.
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2. Materials and methods Twenty muscle, seventeen liver and five blubber samples were collected from Antarctic fur seal (Arctocephalus gazella) pups between January and February 2004, in the framework of the Italian Program of Research in Antarctica (PNRA) and of a National Science Foundation (NSF) expedition. Samples were collected on Livingston Island, South Shetland, Antarctic Peninsula, (62°39′ S, 60°30′ W). Antarctic fur seal pups were found dead and the tissue samples were taken from the carcasses at the time of necropsy. Samples had no signs of decomposition. Sampling location, gender, body weight, and body length were recorded (Table 1). Pups were aged based on their pelage stage (Kovacs and Lavigne, 1986). Unhatched eggs of Adélie penguins (Pygoscelis adéliae, n = 13) and Gentoo penguins (Pygoscelis papua, n = 13) were collected on King George Island, South Shetland, Antarctic Peninsula (62°10′ S, 58°67′ W), during the 2004/05 field season. All samples were wrapped in polyethylene bags and stored at −20 °C until analysis. 2.1. Analytical methods and instrumental analysis for PFCs PFCs were analyzed following the method described elsewhere (Kannan et al., 2001a; Tao et al., 2006), with some modifications. PFCs were extracted by ion-pairing liquid extraction method. For egg samples, 0.5 g of whole egg homogenate, 3 mL of Milli-Q water, 4 ng of each internal standard (PFBS, 13C4-PFOS, and 13C4-PFOA), 2 mL of 0.25 M sodium carbonate buffer, and 1 mL of 0.5 M tetrabutylammonium hydrogen sulfate solution (adjusted to pH 10) were mixed in a 15 mL polypropylene (PP) tube. The sample was then extracted with 5 mL of methyl-tert-butyl ether (MTBE) by vigorous shaking for 45 min. The MTBE layer was separated by centrifugation at 3500 rpm for 5 min and then transferred into another PP tube (~ 4.5 mL). The aqueous mixture was rinsed with MTBE and separated twice; all rinses were combined in the second polypropylene tube. The MTBE extract was evaporated to near-dryness under a gentle stream of nitrogen and then reconstituted with 1 mL of methanol. For the extraction of liver and muscle samples, a small amount of tissue (about 1 g) was homogenized with 5 mL of Milli-Q water, and then 1 g of the homogenate was transferred into a PP tube and extracted following the procedure described above. Matrix matched calibration standards (seven points ranging from 0.5 to 75 ng/mL) were prepared by spiking different amounts of calibration standards into sample matrix that contained no quantifiable amount of the target analytes; these
Table 1 Details of fur seal pups found dead and used for collecting the samples. Sample
Date of sampling
Tissue
Age
Sex
Length (cm)
Weight (Kg)
08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 January 2004 February 2004 February 2004 February 2004
M M, M M M, M, M, M, M, M, M, M, M, M, M, M, M, M, M, M,
Steel born 2 week old 2 week old 2 week old 1 month old 1 month old 1 month old 1 month old Steel born 45 days old 45 days old 1 week old 45 days old 45 days old 1 month old 45 days old 2 months old 2 months old 2 months old 2 months old
♀ ♂ ♀ ♀ ♂ ♂ ♂ ♀ ♂ ♂ ♂ ♀ ♂ ♀ ♀ ♀ ♂ ♀ ♀ ♀
68 66.5 65 71 77.5 73 74 67 74 77 77 65.5 74 77 71 65 77 77 74 80
4.6 4 4.3 4.2 6 4.6 4.6 3.6 5.7 5.45 6 4 5.3 5.3 4.3 3.65 5.5 4.8 5.6 7.2
(M = muscle, L = liver).
L
L L L L L L L L L L L L L L L L
standards were passed through the entire analytical procedure along with the samples. Analytes were detected and quantified using an Agilent 1100 series high-performance liquid chromatograph (HPLC) coupled with an Applied Biosystems API 2000 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS). The MS/MS was operated in electrospray negative ion mode. Target compounds were determined by multiple reaction monitoring (MRM). The MRM transitions were 299 N 80 for PFBS, 399 N 80 for PFHS, 499 N 99 for PFOS, 503 N 99 for 13C4-PFOS, 599 N 99 for PFDS, 498 N 78 for PFOSA, 363 N 319 PFHpA, 413 N 369 for PFOA, 417 N372 for 13C4-PFOA, 463 N 419 for PFNA, 513 N 469 for PFDA, 563 N 519 for PFUnDA, and 613 N 569 for PFDoDA. Samples were injected twice to monitor sulfonates and carboxylates separately and PFBS was monitored in both of the injections. A midpoint calibration standard was injected after every 10 samples to check for the instrumental response and drift. Calibration standards were injected daily before and after the analysis. Reported concentrations were not corrected for the recoveries. Blanks were analyzed by passing Milli-Q water and reagent through the entire analytical procedure. Blanks contained trace levels of PFOA in penguin egg analyses and trace of PFOA and PFOS in seals sample analyses. The concentrations reported here were subtracted from the mean value found in blanks. To check the recovery, 13C4-PFOS and 13C4-PFOA were used as internal standards. For penguin eggs and seal tissue samples, mean recoveries of were 61% and 89% for 13C4-PFOS, and 133% and 75% for 13C4-PFOA, respectively. The limit of quantitation (LOQ) was determined based on the linear range of the calibration curve prepared at a concentration range of 0.1 to 20 ng/mL. The tissue samples were compared to this unextracted standard calibration curve. Because of the variety of matrices analyzed and because of evolving analytical methods, the LOQ was variable. The LOQ was determined as the lowest acceptable standard in the calibration curve, deemed acceptable if it was within ±30% of the theoretical value and the peak area of the standard was at least twice as great as the matrix blanks. The LOQ was 0.4 ng/g for all analytes in seal samples, and 0.1 ng/g for all analytes in penguin eggs, except for PFOA and PFHpA, for which the LOQ was 0.2 and 0.5 ng/g respectively. 2.2. Statistical analysis Statistical analyses were performed with STATISTICA 7 for Windows (Ver 7.1; Statsoft, Italia srl) at a significance level of p = 0.05. Statistically significant differences between the mean concentrations of contaminants were investigated by a single factor one-way analysis of variance (ANOVA) and when significant differences were found, they were tested among each other using Tukey's post hoc test. The absolute concentrations were log10-transformed prior to statistical analysis if the data did not follow a normal distribution (Levene test p b 0.05). The transformed data were normally distributed and the Tukey test was used to analyze the differences in concentrations between species, category of species or tissues. The overall significance level of each analysis was set at p b 0.05. The Tukey–Kramer (HSD) test is one of a number of post-hoc methods recommended for testing differences between pairs of means among groups that contain unequal sample sizes (Zar, 1999). The correlations between PFC concentrations and seal age groups were assessed by simple correlation analyses (Zar, 1999). Correlations are expressed using the Pearson correlation coefficient r. If a concentration of PFC was below the LOQ in a sample, the LOQ value itself or a value of half the respective detection limit, was considered prior to statistical analyses. The statistical analysis between seal tissues were limited by the different number of samples (liver samples n = 17, and muscle samples n = 20). 3. Results and discussion Fur seal samples were analyzed for the presence of four perfluorinated sulfonic acids (PFSAs), and six perfluorinated
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Table 2 Concentrations (ng/g ww; mean ± SD) of perfluorinated compounds in Antarctic samples (nd = not detected).
PFHS PFHpA PFOS PFOSA PFOA PFNA PFDA PFDS PFUnDA PFDoDA PFCs
Fur seal pup, muscle
Fur seal pup, liver
% detected
Mean ± SD
% detected
Mean ± SD
% detected
Gentoo penguin egg Mean ± SD
% detected
Mean ± SD
0 80 100 65 50 0 0 0 0 0
nd 0.5 ± 0.3 1.3 ± 0.7 b0.4 0.8 ± 0.8 nd nd nd nd nd 2.7 ± 1
82 100 100 100 6 94 71 6 71 6
b 0.4 1.0 ± 1.9 9.4 ± 3.2 b 0.4 b 0.4 3.3 ± 1.7 0.6 ± 0.5 b 0.4 0.9 ± 0.9 b 0.4 16 ± 6.2
0 15 100 0 8 0 31 Not analyzed 100 54
nd b0.5 0.3 ± 0.1 nd b0.2 nd 0.1 ± 0.2
0 54 100 54 23 8 85 Not analyzed 100 85
nd 2.5 ± 5.5 0.4 ± 0.2 0.2 ± 0.3 b0.2 b0.1 1.3 ± 2.9
carboxylic acids (PFCAs); perfluorooctane sulfonate (PFOS), perfluorhexanesulfonate (PFHS), perfluorooctanesulfonamide (PFOSA), perfluorodecanesulfonate (PFDS), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorododecanoic acid (PFDoA) and perfluoroundecanoic acid (PFUnDA). Penguin samples were analyzed for all of these contaminants except for PFDS (Table 2). 3.1. PFC concentrations in seals: tissue distribution Concentration of PFCs were significantly higher in liver than in muscle (ANOVA p b 0.001), suggesting compound-specific persistence and retention of PFCs in seal liver. Mean concentration of ∑PFSAs (1.4 ng/g ww) was similar to the mean concentration of ∑PFCAs (1.3 ng/g ww) in seal muscle samples. Mean concentration of ∑PFSAs (10.4 ng/g ww) in seal liver samples was 68% higher than that of ∑PFCAs (6.2 ng/g ww), suggesting preferential enrichment of PFOS in seal liver. Hepatic concentrations of PFOS were not correlated (p N 0.05) with age. The PFC concentrations in liver and muscle of seal samples were in the decreasing order, PFOS N PFNA N PFHpA N PFUnDA and PFOS N PFOA N PFHpA N PFOSA, respectively. A similar trend was previously reported in marine mammals (Martin et al., 2004; Van de Vijver et al., 2003), including polar bears (Smithwick et al., 2005; Kannan et al., 2005) from North America, where concentrations decreased with increasing chain length, and the predominant PFC was PFOS. Seal liver samples contained significantly (p b 0.001) higher concentrations of PFOS than the muscle samples, while PFNA was no detected in muscle samples. High PFOS concentrations (range b0.08–3.52 ng/mL ww) were also reported in blood samples of elephant seal pups from the South Shetland Islands (Tao et al., 2006). Previous studies have reported higher PFOS concentrations in dolphins and harbour porpoise pups than in adults (Houde et al., 2005; Van de Vijver et al., 2004; Hart et al., 2008). Some studies have clearly demonstrated a significant placental transfer of PFOS from dam to fetus during gestation (in utero exposure to PFOS) and to the pup during lactation (Luebker et al., 2005a,b; Hart et al., 2008). PFOA was detected at low concentrations (mean 0.8 ng/g ww) in 50% of seal muscle samples, and between 6 and 23% in the other samples analyzed. In general, PFOA has been detected sporadically in marine mammals (Kannan et al., 2002b) and birds (Bossi et al., 2005a; Kannan et al., 2002c; Holmström and Berger, 2008).
Adélie penguin egg
0.6 ± 0.5 0.3 ± 0.8 1.3 ± 1.2
2.3 ± 6.5 0.5 ± 0.4 7.35 ± 9
PFOS. Thus, the mean concentrations of ∑PFCAs in Gentoo penguin (1.08 ng/g ww) and Adélie penguin eggs (6.6 ng/g ww) were an order of magnitude higher than ∑PFSAs (0.29 ng/g ww and 0.56 ng/g ww, respectively) (Fig. 1). PFCA profile was dominated by PFUnDA (mean 0.59 ng/g ww) in Gentoo penguin eggs, and by PFUnDA (2.33 ng/g ww), and PFHpA (mean 2.53 ng/g ww) in Adélie penguin eggs, in which the PFCA concentrations were also higher than that of PFOS (mean 0.29, 0.38 ng/g ww, respectively). High concentrations of long-chain PFCAs in eggs suggest oviparous transfer of these compounds. 3.3. Correlations between PFCs The statistical associations between PFOSA and PFOS have previously been used to demonstrate that PFOSA is a precursor of PFOS (Kannan et al., 2002a; Martin et al., 2004). We found no statistically significant correlation between PFOS and PFOSA in Adélie penguin eggs (p N 0.05). Other reports (Kannan et al., 2001b; Martin et al., 2004; Bossi et al., 2005) have found statistical association between PFOSA and PFOS for different species, but not always consistent. It is not clear whether this association represents metabolism or direct exposure. Statistically significant linear correlations were only found between the concentrations of PFDA and PFUnA (p b 0.05) in the seal liver (Fig. 2), suggesting a common source of these two PFCAs to fur seals. A significant positive relationship between concentrations of PFOS and PFNA was observed in fur seal liver (Fig. 3). These results indicate that the sources of exposure of the seal to PFOS and PFNA are similar, or the coexistence of these compounds in the sources. 3.4. Species-specific differences The PFC concentrations differed significantly between seals and penguins (p b 0.005) and a species-specific difference was found
3.2. PFCs in penguins: long-chain PFCA Concentrations of PFCs were found in the decreasing order PFHpAN PFUnDA N PFDA N PFDoDA in Adélie penguin eggs, and PFUnDA N PFOSN PFDoDA N PFHpA in Gentoo penguin eggs. Several long-chain PFCAs (PFDA, PFUnDA, PFDoDA) were the predominant PFCs detected in penguin egg samples at the concentrations higher or no less than that of
Fig. 1. ∑PFSA and ∑PFCA concentrations (ng/g ww) in seal and penguin samples.
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Fig. 2. Linear regression of PFDA and PFUnDA concentrations in fur seal liver from Antarctica. A log transformation was performed to normalize the data.
between the two species of penguins (p b 0.005) (Fig. 4). Although these organisms share the same environment, differences in diet and metabolism might explain the variations in the concentrations observed. Reid and Arnould (1996) investigated the diet of lactating female Antarctic fur seals (Arctocephalus gazella) in South Georgia, and found that Antarctic krill (Euphausia superba) was the main prey item, followed by fishes and squid. The myctophids Protomyctophum choriodon were the major diet during the lactational period. Outside this period, the nototheniid Lepidonotothen larseni and the channichthyid Champsocephalus gunnari were the dominant prey species (Reid and Arnould, 1996). In contrast, differences in the PFC concentrations between penguin species may be related to diet, reproductive status, ecological niches, and migration. Both penguin species in our study were from the South Shetland Islands, and feed primarily on Euphausia superba, although Gentoo penguins feed more fish (Antarctic silverfish Pleuragramma antarcticum) than Adélie penguins. E. crystallorophias and pelagic and benthic species of amphipods are minor components of the pygoscelid diet (Volkman et al., 1980). Gentoo penguins feed inshore and are deep divers, while Adélie penguins are shallow-diving, offshore foragers (Trivelpiece et al., 1987). Among PFCs, PFOS was detected in all of the samples. PFOS concentrations in the liver and muscle of seal pups were significantly higher (p b 0.001) than those found in penguin eggs. No significant difference was found in PFOS concentrations between eggs from the
Fig. 3. Relationship between concentrations of PFOS and PFNA in fur seal liver from Antarctica.
Fig. 4. Concentrations of perflurooctane sulfonate (ng/g ww) in seal and penguin samples from Antarctica. The straight line is the mean. The dots represent the outlier (N 1.5 interquartile lengths from box edge). Statistically significant difference (p b 0.005) is indicated by ⁎.
two species of penguins (Fig. 5). PFOSA was below the detection limit in seal muscle and liver samples, and in Gentoo penguin eggs, but was detected in Adélie penguin eggs with a frequency of 54%. The PFCA profile was dominated by PFUnDA in penguin eggs, in which the PFCA concentrations were also higher than that of PFOS. Similar results have been reported in other studies on birds (Holmström and Berger, 2008; Verreault et al., 2007). 3.5. Comparison with other studies The presence of PFCs in penguin eggs and Antarctic fur seals is reported here for the first time. In fact, little information has been provided so far on PFC concentrations in these Antarctic organisms (Table 3). One previous study reported PFOS values under the detection limit in Adélie penguin eggs (b0.1 ng/g ww; Tao et al., 2006), while in our study we found a mean concentration of 0.38 ng/g ww. This difference could be due to the different collection period, 1995/1996 and 2004 respectively, suggesting an increase in the environmental levels of these contaminants. Tao et al. (2006) reported a mean PFOS concentration in the blood of elephant seal pups of 0.53 ng/mL — one order of magnitude lower than that found in our fur seal pup liver samples (mean: 9 ng/g ww). This difference may due to the preferential accumulation of PFCs in liver tissues (Van de Vijver et al., 2005).
Fig. 5. Log concentrations of PFOS (ng/g ww) in seal and penguin samples from Antarctica. The straight line is the mean. The dots represent the outlier (N 1.5 interquartile lengths from box edge) Statistically significant difference (p b 0.001) is indicated by ⁎.
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Table 3 Mean (Range) concentration of PFOS (ng/mL in blood or ng/g in other tissues ww) in seals and birds from Antarctica. Matrix
Date of collection
Species
n
Location
PFOS
References
ww Blood
2001 2003/4 and 2004/2005 Not available
Egg
1995/1996 1998/1999 2004/05
Liver
2004/05 Not available
Muscle a
2004/05
Pygoscelis adéliae (Adélie penguin) Mirounga leonine (Elephant seal) Stercorarius maccormicki (Polar skua) Pygoscelis adéliae (Adélie penguin) Stercorarius maccormicki Polar skua Pygoscelis adéliae (Adélie penguin) Pygoscelis papua (Gentoo penguin) Arctocephalus gazella (Antarctic fur seal) Leptonychotes weddellii (Weddell seal) Arctocephalus gazella (Antarctic fur seal)
a
b0.1
2
Admiral Bay South Shetlands Elephant Island South Shetlands Terra Nova Bay
1.2 (b 1 –1.4)
Giesy and Kannan (2001)
6
Edmonson point
b0.1a
Tao et al. (2006)
8 59
3 17
1 20
0.53 (b 0.08–3.52) a
Tao et al. (2006)
2.51 (2.08–3.12) Admiral Bay, King George Is.
20 17
Tao et al. (2006)
0.38 (0.18–0.89)
This study
0.28 (0.13–0.49) Livingston Island
9.01 (1.85–17.25) a
Terra Nova Bay
b35
Livingston Island
1.29 (0.42–3.59)
This study Kannan et al. (2001b) This study
Concentration of PFOS were below LOQ for all samples.
In comparison to seals and birds from the northern hemisphere, the concentrations of PFOS and PFOA in penguins and seals from the South Ocean were 10–100 fold lower. Despite the species differences, concentrations of PFOS measured in the liver of fur seals in our study were two orders of magnitude lower than concentrations in liver of harbor seals (162 ng/g ww) from the northwest Atlantic (Shaw et al., 2006) and one order of magnitude lower than that found in ringed seals (67 ng/g ww) from Greenland (Bossi et al., 2005). The PFOS concentrations found in our penguin eggs were two orders of magnitude lower than the concentrations in herring gull eggs (52 ng/g ww) from northern Norway (Verreault et al., 2007), and three orders of magnitude lower than that found in glaucous gull eggs (104 ng/g ww) from Svalbard (Verreault et al., 2005) and in guillemot eggs (614 ng/g ww) from the Baltic Sea (Holmström et al., 2005). However, comparisons should be interpreted with caution due to interspecies differences in feeding ecology. Although the contamination levels of PFOS and PFOA are low in southern hemisphere fauna, the occurrence of PFCs in these remote locations suggests the widespread distribution of PFCs. Antarctic organisms should continue to be monitored in future due to the global trend of increasing PFC use. 3.6. Ecological implications Acute and chronic dietary exposure studies of PFOS in mallard ducks (Anas platyrhynchos) and northern bobwhite quails (Colinus virginianus) (Newsted et al., 2007, 2006) have recently led to the calculation of PFOS toxicity reference values (TRVs) and predicted noeffect concentrations (PNECs), based on the characteristics of top avian predators (e.g. birds of prey and certain gull species) (Newsted et al., 2005). Conservative egg yolk-based TRVs and PNECs were determined as 1.7 and 1.0 μg PFOS/mL, respectively. In the same study, the lowest observable adverse effect level (LOAEL) in egg yolk was 62.0 μg PFOS/mL. Hence, the mean PFOS concentration evaluated in penguin eggs from Antarctic Peninsula was approximately 3000 times lower than the PFOS TRV, PNEC, and LOAEL values, respectively. From a toxicological standpoint, and assuming that the sensitivity to PFOS exposure of mallard ducks and northern bobwhite quails is similar in penguins, recent concentrations in eggs suggest that PFOS alone would pose a minimal risk to the developing penguin embryo. However, PFOS and other accumulated PFASs in penguins need to be assessed as a part of a broad contaminant cocktail, including chlorine-
and bromine-based chemicals with potential health risks (Newsted et al., 2007, 2006; Molina et al., 2006). Other laboratory studies on rats, monkeys and birds have shown that the toxic effects of PFOA and PFOS occur at tissue concentrations in the range of a few tens to hundreds of μg/g ww (Kennedy et al., 2004; Newsted et al., 2006). Residue concentrations of PFOS and PFOA in our fur seal livers were approximately 3–4 orders of magnitude lower than the effect concentrations found in laboratory animals. The toxic effects of PFCs in seal species are unknown, as only one study has found a significant association of PFOS and PFOA with the presence of disease mortality in sea otters (Kannan et al., 2006): establishment of a cause-effect linkage will require toxicological and controlled animal feeding studies. Further studies are needed on the immunotoxic effects of PFCs and also on the interaction between PFCs and other contaminants found in fur seal tissues. Acknowledgments This research was funded by the Italian National Program of Research in Antarctica (PNRA). The National Science Foundation supported S. Corsolini's stay and travel to and from King George Is. We are very grateful to Daniel Torres and Daniel Torres jr (Instituto Antarctico Chileno, Santiago, Chile) for collecting the fur seal samples during the 2003/04 expedition, and to Wayne Trivelpiece, and Susan Trivelpiece for collecting the penguin eggs samples. We thanks Roger Hewitt, the Agunsa (Punta Arenas, Chile) and Raytheon (USA) for their friendly logistic support. References Ahmed T. “Pygoscelis papua” (On-line), Animal Diversity Web. (2003). Accessed February 18, 2008 at http://animaldiversity.ummz.umich.edu/site/accounts/ information/Pygoscelis_papua.html. Bossi R, Riget FF, Dietz R. Temporal and spatial trends of perfluorinated compounds in Ringed Seal (Phoca hispida) from Greenland. Environ Sci Tecnol 2005;39:7416. Bossi R, Riget FF, Dietz R, Sonne C, Fauser P, Dam M, et al. Preliminary screening of perfluorooctane sulfonate (PFOS) and other fluorochemicals in fish, birds and marine mammals from Greenland and the Faroe Islands. Environ Pollut 2005a;136:323–9. Giesy PJ, Kannan K. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 2001;35:1339–42. Giesy JP, Kannan K. Perfluorochemical surfactants in the environment. Environ Sci Technol 2002;36:146A. Guruge KS, Yeung LWY, Yamanaka N, Miyazaki S, Lam PKS, Giesy JP, et al. Gene expression profiles in rat liver treated with perfluorooctanoic acid (PFOA). Toxicol Sci 2006;89:93-107.
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