Spatial trends of perfluorochemicals in harbor seals (Phoca vitulina) from Danish waters.

Abstract

Spatial trends of concentrations of perfluorinated chemicals (PFCs) were investigated in harbour seal liver tissue from seven locations in Denmark, ranging from the Wadden Sea in the southern North Sea to the Western Baltic. All samples were collected during the phocine distemper epizootic in 2002 which provided access to a large number of comparable samples over a short time period. PFOS was dominating (mean: 92% of ∑PFC) among the PFCs in the samples, followed by considerably lower concentrations of PFHxS (1.8%), PFDA (1.7%), PFNA (1.6%) PFUnA (1.5%), PFOA (0.9%) and PFOSA (0.5%). The concentrations of all the investigated compounds showed significant differences among the seven locations. PFOS showed the highest concentrations in the Wadden Sea, where high burdens have also been recorded in German seals. Most compounds showed a trend towards higher concentrations at one or both extremes of the geographic range. Two different patterns of relative PFC concentrations were detected; one in the inner Danish waters where PFOSA and PFUnA were more prevalent and another in the Wadden Sea and Limfjord where PFOA, PFHxS and PFNA were found in greater proportions. These patterns probably represent Baltic and North Sea contamination sources.

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Short Communication
Spatial trends of perfluorochemicals in harbor seals (Phoca vitulina) from
Danish waters
Rune Dietza,⁎, Frank F. Rigéta, Anders Galatiusa, Christian Sonnea, Jonas Teilmanna, Rossana Bossib
aDepartment of Bioscience, Aarhus University, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark
bDepartment of Environmental Science, Aarhus University, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark
a b s t r a c ta r t i c l e i n f o
Article history:
Received 6 April 2011
Received in revised form 17 September 2011
Accepted 19 September 2011
Available online 17 November 2011
Keywords:
Denmark
Geographic trend
Harbor seal
Liver
Perfluorinated chemicals
Phoca vitulina
Spatial trends of concentrationsof perfluorinated chemicals (PFCs) were investigatedin harbour seal liver tissue
fromseven locationsinDenmark,rangingfromtheWaddenSea inthesouthern North Sea totheWesternBaltic.
All samples were collected during the phocine distemper epizootic in 2002 which provided access to a large
number of comparable samples over a short time period. PFOS was dominating (mean: 92% of ∑PFC) among
the PFCs in the samples, followed by considerably lower concentrations of PFHxS (1.8%), PFDA (1.7%), PFNA
(1.6%) PFUnA (1.5%), PFOA (0.9%) and PFOSA (0.5%). The concentrations of all the investigated compounds
showed significant differences among the seven locations. PFOS showed the highest concentrations in the Wad-
den Sea, where high burdens have also been recorded in German seals. Most compounds showed a trend to-
wards higher concentrations at one or both extremes of the geographic range. Two different patterns of
relative PFC concentrations were detected; one in the inner Danish waters where PFOSA and PFUnA were
moreprevalentandanother intheWaddenSea andLimfjordwhere PFOA,PFHxS andPFNA werefound ingreat-
er proportions. These patterns probably represent Baltic and North Sea contamination sources.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Perfluorochemicals (PFCs) are found in the environment as end
products of a large range of chemicals produced for applications such
as fire-fighting foams, cleaners, lubricants and coatings (Kissa, 2001).
They fulfill the criteria for persistent organic pollutants as they are
widely spread and persist in the environment where they biomagnify
(Giesy and Kannan, 2001; Kannan et al., 2004; 2005; Yamashita et al.,
2005). Emerging evidence suggests numerous and various toxic effects,
including hepatotoxicity (Miller, 1975; Malinverno et al., 2005), neuro-
toxicity (Johansson et al., 2008; Liu et al., 2010) reproductive toxicity
(Lau et al., 2003; Luebker et al., 2005) and effects on metabolism
(Berthiaume and Wallace, 2002). Unlike most other POPs which accu-
mulate in lipid-rich tissue, PFCs bind to blood proteins and accumulate
mainlyintheliver,kidneysandbilesecretions(Jonesetal.,2003).Given
the persistence and bioaccumulation potential of PFCs, their toxicity to
wildlifeisofconcern.Becausemarinemammalsoccupythehighesttro-
phic levels in the marine food webs, they are exposed to high concen-
trations of anthropogenic bioaccumulating compounds.
The Danish waters are situated between two potentially large pol-
lution outlets with discharges from most of northern Europe. To the
east, the inner Danish waters form the transition from the Baltic to
North Sea. Riverine discharges of PFCs to the Baltic are lighter than
in other parts of Europe (McLachlan et al., 2007), but as the Baltic
has a low rate of water exchange with the North Sea, high water con-
centrations of PFCs are found here (0.92–6.24 ng l−1; Ahrens et al.,
2010). To the west, there are considerable discharges of PFCs to the
North Sea from the rivers Elbe and Rhine (McLachlan et al., 2007;
Ahrens et al., 2010). In the only study where PFC-concentrations
have been compared between the Baltic and North Seas within the
same species, Rüdel et al. (2010) found higher PFC loads in North
Sea herring gull (Larus argentatus) eggs than in Baltic Sea eggs.
The harbor seal (Phoca vitulina) is the only pinniped found
throughout the Danish waters. Harbor seals are relatively sedentary
and usually feed close to their haul-out sites. Molecular and satellite
telemetry studies (reviewed by Andersen and Olsen, 2010) indicate
occasional movements of seals between the Wadden Sea and Limf-
jord and between the Kattegat, the Sound and western Baltic respec-
tively (Dietz et al., in review). These data are corroborated by genetic
data implying that seals from the Wadden Sea, Limfjord, Kattegat and
western Baltic constitute separate subpopulations (Olsen et al., 2010).
During the phocine distemper morbillivirus (PDV) epizootic in
2002, 16% of the Baltic and 50–60% of the Skagerrak and North Sea
harbor seals died (Härkönen et al., 2006). This provided a unique op-
portunity to get a string of synchronized samples of harbor seal liver
tissue free of potential temporal trends as documented in polar bears
(Ursus maritimus) (Dietz et al., 2008) for analysis of spatial trends of
PFCs. Samples extended from the Wadden Sea and Limfjord through
Kattegat and the Sound down to the western Baltic.
Science of the Total Environment 414 (2012) 732–737
⁎ Corresponding author. Tel.: +45 87158690; fax: +45 87155015.
E-mail addresses: rdi@dmu.dk (R. Dietz), ffr@dmu.dk (F.F. Rigét), agj@dmu.dk
(A. Galatius), csh@dmu.dk (C. Sonne), jte@dmu.dk (J. Teilmann), rbo@dmu.dk
(R. Bossi).
0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2011.09.048
Contents lists available at SciVerse ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv

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2. Material and methods
2.1. Sampling and age determination
Harbor seals were collected from seven regions (Wadden Sea,
Limfjord, three areas in Kattegat and two areas in Western Baltic)
along the Danish coasts (Fig. 1) during the PDV epizootic in the sum-
mer 2002. Sample sizes ranged from 5 to 13 from each region
(Table 1). Samples were frozen and stored at −20 °C. In the laborato-
ry, liver samples were lightly thawed before chemical analysis. Ages
of the seals were estimated by counting annual layering of cementum
in toluidine blue stained decalcified freeze microtome sections
(14 μm) of canine or premolar teeth, as described by Dietz et al.
(1991). Ages of four juvenile seals were estimated using canine
tooth X-ray (Nørgaard and Larsen, 1991).
2.2. Extraction and analysis
The extraction method was based on ion pairing as described by
Bossi et al. (2005). 13C2-PFDA and 13C4-PFOS (Wellington Laborato-
ries, Guelph, ON, Canada) were used as surrogate standards. Instru-
mental analysis was performed by liquid chromatography-tandem
mass spectrometry (LC-MS-MS) with electrospray ionization (ESI).
The extracts (20 μL injection volume) were chromatographed on a
C18 Betasil column (2.150 mm, Thermo Hypesil-Keystone, Bellefonte,
PA) using an Agilent 1100 Series HPLC (Agilent Technologies, Palo
Alto, CA). The HPLC was interfaced to a triple quadrupole API 2000
(Sciex, Concord, ON, Canada) equipped with a TurboIon Spray source
operating in negative ion mode. Instrument set-up, quality assurance
and calibration procedures as well as the standards and reagents used
are described in detail by Bossi et al. (2005). Recoveries of the
analytes (as given in Bossi et al., 2005) were between 80 and 111%,
determined by spiking bovine liver.
2.3. Statistical methods
Nonparametric statistical tests (Kruskal–Wallis rank sum and
Spearman rank correlation) were preferred to test geographic differ-
ences and correlations among compounds since normality and vari-
ance homogeneity could not be verified in all cases, even after log-
transformations. These tests also reduce the influence of extreme
values and values below detection limits (DL) of some compounds
(see results). Results below DL were included in the analyses with a
value given as half the DL of the relevant compound. When a com-
pound was not detected in a sample, the value ‘0’ was used in the
analyses.
To investigate the relative contributions of the PFCs and their co-
variance pattern in relation to geography, a principal components
analysis (PCA) was performed on the covariance matrix of the con-
centration data. To standardize the impact of each compound on the
PCA, variance was normalized to unit. As the concentrations approx-
imately had a log-normal distribution, all values were log trans-
formed (LOG10(x+0.1)).
The statistical analyses were performed using the statistical pack-
ages R (R Development Core Team, 2008).
3. Results and discussion
3.1. Presence of the various PFCs
Perfluorooctanesulfonic acid (PFOS) was the major contributor
(on average 92.0% of ∑PFC (95% CI: 91.0–93.1); detected in all
15°E14°E13°E12°E11°E10°E9°E8°E7°E 6°E
59°N
58°N
57°N
56°N
55°N
54°N
Wadden
Sea
Limfjord
Northern
Kattegat
Central
Kattegat
Southern
Kattegat
The
Sound
Western
Baltic
Germany
Sweden
Denmark
Norway
Fig. 1. Map showing the seven regions in Danish waters from where seals were sampled in 2002 and consecutively analyzed for PFCs.
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samples) to the PFC profiles which is in agreement with previous har-
bor seal studies (Van de Vijver et al., 2003, 2005; Shaw et al., 2009;
Ahrens et al., 2009). In decreasing order, PFOS was followed by per-
fluorohexane sulfonate (PFHxS; on average 1.8% of ∑PFC (95% CI:
1.5–2.1); detected in 98.3% of samples), perfluorodecanoic acid
(PFDA; on average 1.7% of ∑PFC (95% CI: 1.5–1.9); detected in
98.3% of samples), perfluorononanoic acid (PFNA; on average 1.6%
of ∑PFC (95% CI: 1.3–2.0); detected in 86.7% of samples), perfluor-
oundecanoic acid (PFUnA; on average 1.5% of ∑PFC (95% CI: 1.2–
1.8); detected in 94.9% of samples). Perfluorooctanoic acid (PFOA;
on average 0.9% of ∑PFC (95% CI: 0.4–1.4; detected in 42.4% of sam-
ples) and perfluorooctanesulfonamide (PFOSA; on average 0.5% of
∑PFC (95% CI: 0.4–0.6, detected in 83.3% of samples) were also fre-
quently detected. Basic statistics regarding concentrations of the ana-
lyzed compounds are listed in Table 1.
Odd-number chain-length PFCA concentrations were generally
similar to or greater than the corresponding even-number chain-
length PFCA (i.e., PFNANPFOA, PFUnANPFDA), while the concentra-
tions of PFUnA and PFDA were closely correlated (Spearman
ρ=0.58; pb0.01). This indicates that common sources of fluorotelo-
mer alcohols as the origin of a large part of these compounds in the
samples (Martin et al., 2004; Smithwick et al., 2005). A similar pat-
tern was also detected in harbor porpoises (Phocoena phocoena)
from the Danish North Sea (Galatius et al., 2011).
3.2. Age relationship
Spearman correlations revealed no significant relationships be-
tween age and any of the seven analyzed compounds in the pooled
samples (all pN0.10). We had three seals determined to be b1 year
old and two seals without age determinations that were in the length
range of b1 year olds. These five seals tended to have higher PFOS-
levels than the average for the other groups (mean=769 ng g–1ww;
range=218–1209;remaining
range=27–1324), while levels of other compounds were within the
general range. Therefore, these five specimens were excluded from
the analysis of geographical trends. Ahrens et al. (2009) and Shaw
et al. (2009) likewise detected significantly higher PFOS levels in
b1 year old harbor seals than in older seals.
samplemean=387 ng g–1ww;
3.3. Geographic differences
Statistically significant differences were found among the seven
subareas for all seven analyzed compounds (Kruskal–Wallis tests;
all pb0.01; Fig. 2). Post hoc tests (Sokal and Rohlf, 1995) for signifi-
cance (α=0.05) revealed differences between the Wadden Sea and
the other locations for PFOS; for PFOSA, C. and S. Kattegat samples
had higher concentrations than all other locations. For PFHxS, the
Wadden Sea and Limfjord showed higher concentrations than the
Table 1
Basic data and PFC data (ng/g ww) from the harbor seals from the seven regions in Danish waters including age range, mean, SD, median and range.
Wadden SeaLimfjordN KattegatC KattegatS KattegatThe sound W BalticAll
N 131158105759
Ages2–73–61–91–111–71–41–10 1–11
PFOS Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
Mean
SD
Med
Min
Max
689.1
236.2
634.0
430.0
1284.2
295.0
234.1
199.0
77.3
908.2
1.3
1.3
0.8
bDL
4.4
5.9
3.5
5.0
0.0
12.2
3.3
1.9
3.5
bDL
5.9
3.9
1.9
4.9
ND
7.0
3.7
1.8
3.2
1.3
7.7
2.2
1.6
1.6
ND
4.9
315.2
240.6
218.9
92.8
942.9
144.9
131.5
105.0
26.9
371.2
0.8
0.5
0.9
bDL
1.5
2.7
1.8
3.2
0.9
5.4
1.2
1.8
0.6
ND
4.7
1.5
1.0
1.7
ND
2.6
2.7
1.1
2.8
1.3
4.6
3.4
1.8
3.7
1.0
5.6
157.5
134.3
116.4
37.6
388.9
243.6
152.6
224.5
104.2
604.2
1.9
0.9
2.0
bDL
3.6
4.7
2.8
4.2
1.1
9.9
1.6
0.9
1.5
bDL
3.2
3.2
2.8
2.3
ND
8.1
2.9
1.9
3.0
bDL
5.8
2.5
1.8
2.5
bDL
6.0
260.4
151.0
239.7
125.7
618.0
481.6
448.4
285.5
82.3
1324.2
280.4
184.0
362.2
71.6
499.2
1.6
1.3
1.7
bDL
3.1
3.0
1.4
2.6
1.9
5.4
2.6
2.4
3.5
ND
5.3
4.8
3.1
4.1
ND
8.5
5.8
4.6
4.7
1.3
11.4
9.8
7.6
6.1
2.9
18.9
308.0
198.5
387.5
83.2
545.4
336.8
68.2
316.0
281.2
475.2
1.1
0.4
1.1
0.8
1.7
5.5
4.2
4.5
2.2
14.7
2.6
2.4
3.5
ND
5.3
4.8
3.1
4.1
1.7
8.5
5.8
4.6
4.7
1.3
11.4
9.8
7.6
6.1
2.9
18.9
322.8
147.2
334.3
0.0
524.6
397.5
302.8
330.0
26.9
1324.2
PFOSA0.9
0.7
0.7
2.9
1.8
2.7
1.5
1.3
1.1
ND
bDLND
2.9
16.3
7.9
14.6
6.5
32.4
1.8
1.5
1.8
5.7
4.7
3.1
3.5
2.0
11.5
0.5
0.6
5.7
7.1
6.5
4.8
0.0
32.4
1.6
1.7
1.0
PFHxS
PFOA
bDL
ND ND ND
6.1
8.7
3.6
9.0
1.4
5.3
9.1
2.6
6.1
5.6
5.0
4.7
0.7
30.8
6.6
5.7
4.6
PFNA
NDND
15.1
14.8
6.1
14.7
7.1
28.3
5.1
1.8
5.5
2.0
8.7
757.8
246.8
720.0
457.7
1364.7
30.8
4.5
3.5
3.5
1.4
12.3
7.4
6.0
5.3
1.5
20.5
506.8
462.2
302.3
90.3
1364.9
PFDA
bDL
28.3
5.2
4.3
4.5
PFUnA
ND
20.5
417.3
320.7
343.9
0.0
1364.9
∑PFC
bDL: below detection limit; ND: not detected
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other locations. For PFOA, the Limfjord and Sound samples had higher
concentrations than most other locations. For PFNA and PFDA, the
Wadden Sea and western Baltic showed higher concentrations than
the other locations, while Western Baltic showed higher concentra-
tions than all other locations for PFUnA. The high PFOS levels in the
Wadden Sea relative to the other locations are consistent with data
from herring gull eggs between 1991 and 2008 (Rüdel et al., 2010).
Several of the compounds (PFOS, PFHxS, PFNA, PFDA, PFUnA)
showed higher concentrations at one or both ends of the geographic
range; the Wadden Sea to the west and/or the Western Baltic Sea to
the east (Fig. 2). The westernmost of our sampling sites, the Danish
Wadden Sea is situated approximately 150 km north of the mouth
of the River Elbe, a known source PFC contamination in the southern
part of the North Sea (Ahrens et al., 2010). To the east, in the Baltic,
there seems to be more diffuse sources of PFC contamination, but
the limited amount of water exchange means that concentrations
are relatively high here (Ahrens et al., 2010). Studies of water ex-
change patterns have shown that water at our intermediate sampling
sites in the Kattegat is a combination of water from the North Atlantic,
the southern North Sea, the Baltic, and to a lesser extent, fresh water
from rivers around Kattegat (Rydberg et al., 1996). Water from the
North Atlantic and from the less densely populated catchment areas
in Norway and Sweden may be expected to be less heavily polluted
than water from the North Sea and Baltic. Thus, a dilution of contam-
inants from the Baltic and/or southern North Sea in Kattegat is not
unexpected.
To investigate the relative contributions of the PFCs and their co-
variance pattern in relation to geography, a PCA on the standardized,
log-transformed individual concentrations was performed (Fig. 3,
Table 2). PC1 was closely correlated with the sum of log-
0
5
10 15
20 25 30
PFNA (ng/g ww)
0
5
10
15
20
25
PFDA (ng/g ww)
0
5
10
15
20
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
PFUnA (ng/g ww)
0
1
2
3
4
5
6
PFOA (ng/g ww)
0
400
800
1200
PFOS (ng/g ww)
0
5
10
20
30
PFHxS (ng/g ww)
0
1
2
3
4
5
PFOSA (ng/g ww)
Fig. 2. Regional differences of PFC medians (bold horizontal line), 95% confidence intervals (box), range (whiskers) and outliers (open circles) from the seven regions in Danish
waters from which seals were sampled in 2002 and consecutively analyzed for PFCs.
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transformed concentrations of individual compounds (Pearson's
r=0.91) and can be regarded as a general contamination index. PC1
and PC2 combined explained 69% of the overall variance. The remain-
ing PCs did not show geographical trends. PC2 showed a positive
trend against PC1 in the Wadden Sea and the Limfjord seals (upper
right and left quadrant, respectively) and a negative one in the Katte-
gat (North, Central and South), Sound and western Baltic seals
(Fig. 3). These differences in PC2 indicate two geographically different
patterns of PFC-contamination. One in the Wadden Sea and Limfjord,
where PFOA, PFHxS and PFNA (positive loadings on PC2; see Table 2)
are more prevalent, and another in the eastern areas, where PFOSA
and PFUnA (negative loadings on PC2) are more prevalent.
3.4. Considerations
A previous study of sea otters (Enhydra lutris) has recorded elevat-
ed PFC levels in animals that have died from infectious diseases
(Kannan et al., 2006) as is the case with the current study. However,
the PFC concentrations recorded in this study are in ranges similar to
what has been recorded previously in harbor seals from the German
Bight neighboring the Danish Wadden Sea (Ahrens et al., 2009). Fur-
ther south, in the Dutch Wadden Sea, concentrations are lower. The
median PFOS value from Dutch seals, likewise from the 2002 epizoot-
ic, was 161 ng g−1ww (Van de Vijver et al., 2005) compared to
634 ng g−1ww in our Danish Wadden Sea sample. Van de Vijver
et al. (2005) also reported PFNA as the dominant PFCA in the Dutch
sample, whereas PFDA was found in higher concentrations in our
Wadden Sea sample. Thus, there may be different contamination pat-
terns even within the Wadden Sea area. In the western Atlantic, Shaw
et al. (2009) recorded lower PFOS concentrations in harbor seals
along the northern Atlantic coast of USA (~100 ng g−1ww in adults)
compared to our samples, but similar concentrations of long-chained
PFCAs (CN8).
Acknowledgements
We thank the Danish Ministry of Environment for providing fund-
ing for the sampling program. Center for Game Health and Dep. of En-
vironmental Science, Aarhus University provided funding for the
analyses. Colleagues Susi Edrén, Oluf Henriksen and Thomas Dau Ras-
mussen (Dep. of Bioscience) and Svend Tougaard and Thyge Jensen
(formerly the Fisheries and Maritime Museum, Esbjerg) and game
wardens Anton Linnet, (the Limfjord), Peter Have, Læsø, (Northern
Kattegat), Morten Abildstrøm (Anholt, Central Kattegat), Niels
Worm and Hans Henrik Erhardi (Sjællands Odde and Tisvilde, South-
ern Kattegat), Sven Norup (Copenhagen, The Sound) and Svend Niel-
sen (Falster, western Baltic) did a tremendous job collecting the seals.
Jeppe Møhl (formerly Natural History Museum, University of Copen-
hagen) helped with dissections and disposal of the seal carcasses.
Thomas Dau Rasmussen and Kristin Johansson performed the age de-
terminations. For four juvenile seals the X-ray equipment at the Nat-
ural History Museum, Copenhagen was used with support from
Tammes Menné. Inga Jensen skillfully conducted the PFC analyses at
the Department of Environmental Science laboratory.
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−604
−4
1
PC 1 (46.65%)
PC 2 (22.6%)
Limfjord
North Kattegat
Central Kattegat
South Kattegat
The Sound
West Baltic
Wadden Sea
2
3
−3
−2
−1
−4
−2 2
0
Fig. 3. Scores along PC1 and PC2 from the principal component analysis of harbor seals from the seven regions in Danish waters plotted against each other. Dots indicate individual
scores, squares indicate centroid positions of the regions and ellipses indicate the 95% confidence intervals of the centroid positions.
Table 2
Component loadings of the seven analyzed PFCs on principal components 1 and 2 from
the principal component analysis (see text).
CompoundPC1PC2
PFOS
PFOSA
PFHxS
PFOA
PFNA
PFDA
PFUnA
0.76
0.16
0.75
0.09
−0.57
0.42
0.76
0.30
0.02
−0.60
−0.34
0.82
0.87
0.49
736
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