Pbdes in serum and blubber of harbor, grey and harp seal pups from Eastern Canada



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PBDEs in serum and blubber of harbor, grey and harp seal pups from Eastern Canada
Héloïse Frouina,b*, Michel Lebeufb, Mike Hammillb, Becky Sjarec, Michel Fourniera

a Institut National de la Recherche Scientifique - Institut Armand-Frappier, Laval, Quebec, H7V 1B7 Canada

b Fisheries and Oceans Canada, Maurice Lamontagne Institute, Mont-Joli, Quebec, G5H 3Z4 Canada

c Fisheries and Oceans Canada, P.O. Box 5667, St.-John’s, Newfoundland, A1C 5X1 Canada
*Corresponding author at: Fisheries and Oceans Canada, Institute of Ocean Sciences, 9860 West Saanich Rd, P.O. Box 6000, Sidney, British Columbia, V8L 4B2, Canada.

Phone: +1-250-363-6551. Fax: +1-250-363-6807.

E-mail address: heloise.frouin@gmail.com

Age determination

Age as a function of total body weight of unknown-age pups was expressed by the formula: age (days) = (weight-birth weight)/weight gain per day.

- Harp seal. We modified the equation of Stewart and Lavigne (Stewart and Lavigne, 1980): age (days) = 0.35*body weight (kg)-3.2. We used the equation age (days) = [body weight (kg)-10]/2.5

- Grey seal. We used the equation age (days) = [body weight (kg)-16]/2.8 determinated from Bowen et al. (Bowen et al., 1992)

- Harbor seal. We used the equation derived from Dubé et al. (2003) : age (days) = [body weight (kg)-11.4]/0.54
Chemical analysis

Seal blubber and serum were analyzed for PBDEs (IUPAC numbers 1, 2, 3, 7, 10, 8/11, 12, 13, 15, 17, 25, 28, 30, 32, 33, 35, 37, 47, 49, 66, 71, 75, 77, 85, 99, 100, 116, 118, 119, 126, 138, 140, 153, 154, 155, 166, 181, 183, 190).



2.1 Blubber samples

Each blubber sample was chemically dried with sodium sulphate before being transferred to a glass column. A single 13C12 PCB (PCB-170) was added to the column before the extraction procedure. Lipids and lipophilic compounds were extracted from the sample with dichloromethane-hexane (50:50). The extraction solution received a mixture of three 13C12 PBDEs (BDE-47, -99 and -153) and was prepared for purification. Lipids were removed from the remaining extract by gel permeation chromatography. The extract was further cleaned by elution through a two-layer column packed with hydrated neutral silica and hydrated alumina. The final extract was reduced in volume and spiked with an instrument performance solution containing two additional 13C12 PCBs (PCB-111 and -189) for a final volume of 100 µL. All solutions were stored in amber glass vials at -20°C.

Quantification of individual PBDEs was performed using either a Varian 3400CX series gas chromatograph equipped with a Varian Saturn TV ion trap, a Varian 1078 split/splitless programmable injector (5 µl injection volume) operated in splitless mode, and a Varian 8200CX autosampler or a ThermoFinnigan Trace 2000 gas chromatograph equipped with a ThermoFinnigan PolarisQ ion trap, a ThermoFinnigan PTV (programmed temperature vaporizer) split/splitless injector (5 µl injection volume) operated in splitless mode, and a ThermoFinnigan 2000 autosampler. Chromatographic separation of PBDEs was achieved using a 30m DB-5MS column (0.25 mm ID, 0.25 µm film thickness) with helium as the carrier gas. The ion source was operated in electron impact ionisation mode and the ion trap in MS/MS mode.

Concentrations of PBDE congeners were calculated using relative response factors (RRFs) determined from a three-point calibration curve. The accuracy of the method was assessed by measuring the recoveries of known quantities of native congeners added to a blank solution, which subsequently underwent all the analytical steps.



2.2 Serum samples

The frozen serum samples were thawed at room temperature. About 2-3 g of precisely weighted serum sample received 50 µl of a surrogate solution containing a mixture of three 13C12-labelled PBDE congeners at a concentration of 200 pg per µL. The serum was denatured with hydrochloric acid (6 M, 1 mL) and isopropanol (3 mL). Hexane:Methyl tert-butyl ether (50:50, 7 mL) was added and the samples were sonicated for 20 min. The phases were separated by centrifugation (189 g, 10 min). The organic phase was transferred and the aqueous phase was extracted two more times. The organic phases were combined and washed with sodium sulftate. The organic phase was transferred to a round-bottom flask and the solvent evaporated. The residues were dissolved in hexane and applied to a pre-washed and conditioned silica solid-phase extraction (SPE) column (Cartouche SPE, J.E. Baker, 500 mg, silica gel SiOH). Elution of the extracts occurred with a total volume of 8 ml of dichloromethane:hexane (15:85). After vaporization of the elution solvent, the extract was spiked with an instrument performance solution containing two additional 13C12 PCBs (PCB-111 and -189) for a final volume of 100 µl. The obtained extract was analyzed by GC/MS/MS as described above.

Figure S1. Location of capture sites for seal pups in the Estuary and the Gulf of St. Lawrence. 1: Bic Island (near Bic National Park); 2: Magdalen Islands; 3: Amet Island; 4: Hay Island; 5: St. Paul (Newfoundland).

Table S1: Species, sampling location, date and characteristics of pup phocids sampled in the Estuary and Gulf of St Lawrence.


Species Sampling location n Sex Age Blubber lipid Serum lipid Condition Collection date

Sector Region (days) (%) (%) (g/L) index (d/mo/yr)

Harbor seals Bic Estuary 6 5M, 1F 5.8 ± 1.4 a 65 ± 23 1.2 ± 0.4 ab 11.9 ± 4.17ab 67 ± 6 31/05/2007

(1-10) (19-79) (0.9-2) (9-19.9) (62-75)


Harbor seals St. Paul Gulf 6 3M, 3F 6.3 ± 1.2 a 58 ± 9 0.8 ± 0.2a 7.5 ± 2.1a 73 ± 8 24,25/05/2008

(3-9) (46-70) (0.6-1.1) (5.5-10.9) (63-84)


Grey seals Amet Island Gulf 6 3M, 3F 1.5 ± 0.5 b 64 ± 17 1 ± 0.2 ab 10.1 ± 2.2ab 75 ± 8 12/01/2008

& Hay Island (0-4) (43-84) (0.7-1.3) (6.8-12.7) (67-86) 13/01/2008


Harp seals Magdalen Islands Gulf 6 3M, 3F 3 ± 0.8 ab 72 ± 8 1.5 ± 0.3b 14.6 ± 3.2b 83 ± 5 6,7/03/2008

(0-6) (62-85) (1.1-1.9) (10.6-19.3) (75-90)

Values are given as the mean ± standard error.

Species with significant comparisons within a characteristic (blubber lipid, serum lipid or condition index) are labelled with different letters.


Table S2: Mean concentration ± standard deviation (ng/g lipid wt) of five major PBDEs, BDE-47, 99, 100, 153, 154 in blubber and blood of seal species from the Northern Hemisphere




Seal




Sampling










age

lipids

PBDE congeners

∑PBDEs




species

location

period

tissues

sex

n

days

(%)

47

99

100

153

154

Mean

ref




St. Lawrence Estuary

2007

blubber

1F, 5M

6

1-10

65 ± 23

400

41

26

11.1

6.1

540 ± 61

This study




Gulf of St. Lawrence

2008

blubber

3F, 3M

6

3-9

58 ± 9

71

7.1

7.0

1.4

1.3

97 ± 16

This study

Harbor


San Francisco

From eastern Maine to Long Island, New York



1992

1991-2005



blubber

blubber


n.d.

1

13


fetus

pups


67

59


277

2970


86

308


12.4

308


10.5

99


44.6

41.7


430

3645 ± 7388



[1]

[2]


seal

Southern California

1994-2006

blubber

3F, 5M

8

pups

57 ± 25
















2430 ± 2250

[3]




St. Lawrence Estuary

2007

serum

1F, 5M

6

1-10

1.2 ± 0.4

429

76

45.3

26.6

3.7

585 ± 70

This study




Gulf of St. Lawrence

2008

serum

3F, 3M

6

3-9

0.8 ± 0.2

63.9

5.9

3.8

5.7

4.4

84 ± 22

This study




California

2001-2002

blood

F & M

17

pups



















0.07 – 3.02*

[4]




Nova Scotia-Sable Is.

1995

blubber




5

14-24




40

3.9

2.7

1.2

1.3

50 ± 27

[5]




Gulf of St. Lawrence

2008

blubber

3F, 3M

6

0-4

64 ± 17

41

5.2

3.1

2.2

1.4

71± 8.0

This study

grey

Gulf of St. Lawrence

2008

serum

3F, 3M

6

0-4

1 ± 0.2

34

3.2

2.4

1.4

<0.1

41± 7.2

This study

seal

Farne Islands, UK

1998-1999

blubber

F & M

54

pups



















223 ± 150

[6]




North Sea

1998-2000

blubber

F & M

110

pups




210

16

21

11

8

290

[7]

harp

Gulf of St. Lawrence

2008

blubber

3F, 3M

6

1-6

72 ± 8

15

3.5

0.9

0.4

0.4

22 ± 3.4

This study

seal

Gulf of St. Lawrence

2008

serum

3F, 3M

6

1-6

1.5 ± 0.3

23

5.1

1.2

1.4

0.3

31 ± 5.4

This study

hooded

Gulf of St. Lawrence

2001

blubber




6

pups




25

1.2

1.9

1.6

0.1

32

[8]

seal

Gulf of St. Lawrence

2001

plasma




6

pups



















149

[8]

n.d.: not detected; * : wet weight (ng/g whole blood); # : BDE47 + BDE99

References: [1] (She et al., 2002); [2] (Shaw et al., 2008); [3] (Meng et al., 2009); [4] (Neale et al., 2005); [5] (Ikonomou and Addison, 2008); [6] (Hall et al., 2003); [7] (Kalantzi et al., 2005); [8] (Wolkers et al., 2006)

Table S3: Number of detected samples for each congener in seal species and locations (blubber and serum).

Harbor seals Bic Harbor seals NF Harp seals Grey seals

Total n = 6 n = 6 n = 6 n = 6

Blubber

BDE 28 n = 5 n = 4 n = 6 n = 3

BDE 33 n = 1 n = 3 n = 1 n = 2

BDE 35 n = 3 n = 1 n = 0 n = 2

BDE 47 n = 6 n = 6 n = 6 n = 6

BDE 49 n = 5 n = 5 n = 3 n = 2

BDE 99 n = 6 n = 6 n = 6 n = 6

BDE 100 n = 6 n = 6 n = 6 n = 6

BDE 126 n = 0 n = 4 n = 0 n = 3

BDE 138 n = 3 n = 4 n = 4 n = 2

BDE 153 n = 6 n = 6 n = 6 n = 6

BDE 154 n = 6 n = 5 n = 5 n = 5

BDE 183 n = 2 n = 2 n = 0 n = 4

BDE 190 n = 1 n = 0 n = 2 n = 1



Serum

BDE 28 n = 0 n = 0 n = 0 n = 0

BDE 47 n = 6 n = 6 n = 6 n = 6

BDE 99 n = 6 n = 3 n = 6 n = 4

BDE 100 n = 6 n = 2 n = 4 n = 4

BDE 153 n = 6 n = 4 n = 2 n = 2

BDE 154 n = 5 n = 3 n = 2 n = 5


REFERENCES

Bowen, W.D., Stobo, W.T., Smith, S.J., 1992. Mass changes of grey seal Halichoerus grypus pups on Sable Island: differential maternal investment reconsidered. Journal of Zoology 227, 607-622.

Dubé, Y., Hammill, M., Barrette, C., 2003. Pup development and timing of pupping in harbor seals (Phoca vitulina) in the St. Lawrence River estuary, Canada. Canadian Journal of Zoology 81, 188-194.

Hall, A.J., Kalantzi, O.I., Thomas, G.O., 2003. Polybrominated diphenyl ethers (PBDEs) in grey seals during their first year of life--are they thyroid hormone endocrine disrupters? Environmental Pollution 126, 29-37.

Ikonomou, M.G., Addison, R.F., 2008. Polybrominated diphenyl ethers (PBDEs) in seal populations from eastern and western Canada: An assessment of the processes and factors controlling PBDE distribution in seals. Marine Environmental Research 66, 225-230.

Kalantzi, O.I., Hall, A.J., Thomas, G.O., Jones, K.C., 2005. Polybrominated diphenyl ethers and selected organochlorine chemicals in grey seals (Halichoerus grypus) in the North Sea. Chemosphere 58, 345-354.

Meng, X.-Z., Blasius, M.E., Gossett, R.W., Maruya, K.A., 2009. Polybrominated diphenyl ethers in pinnipeds stranded along the southern California coast. Environmental Pollution 157, 2731-2736.

Neale, J.C.C., Schmelzer, K.R., Gulland, F.M.D., Berg, E.A., Tjeerdema, R.S., 2005. Rapid communication: organohalogen levels in harbor seal (Phoca vitulina) pups increase with duration of nursing. Journal of Toxicology and Environmental Health, Part A 68, 687-691.

Shaw, S.D., Brenner, D., Berger, M.L., Fang, F., Hong, C.-S., Addink, R., Hilker, D., 2008. Bioaccumulation of polybrominated diphenyl ethers in harbor seals from the northwest Atlantic. Chemosphere 73, 1773-1780.

She, J., Petreas, M., Winkler, J., Visita, P., McKinney, M., Kopec, D., 2002. PBDEs in the San Francisco Bay Area: measurements in harbor seal blubber and human breast adipose tissue. Chemosphere 46, 697-707.

Stewart, R.E.A., Lavigne, D.M., 1980. Neonatal growth of northwest atlantic harp seals, Pagophilus groenlandicus. Journal of Mammalogy 61, 670-680.

Wolkers, H., Hammill, M.O., van Bavel, B., 2006. Tissue-specific accumulation and lactational transfer of polychlorinated biphenyls, chlorinated pesticides, and brominated flame retardants in hooded seals (Cistophora cristata) from the Gulf of St. Lawrence: Applications for monitoring. Environmental Pollution 142, 476-486.





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