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

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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).

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.

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.

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 ± 4.17^ab 67 ± 6 31/05/2007

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

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

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

(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

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

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


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