Organohalogen concentrations and a gross and histologic ...

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Age Determination
Individual ages were obtained by counting annual growth layer groups in
the cementum of the I 3 tooth after decalcification, thin sectioning (14 µm)
and staining with toluidine blue using the method described by e.g. Hensel
and Sorensen (1980) and Dietz et al. (1991). To assure the quality of our
readings of GLGs, I 3 teeth from four polar bears of known age (5 to 32 years),
that had lived in Aalborg Zoo, Denmark, were included in our study. In
addition, an intercomparison exercise was conducted using thin-sections (P 1 )
from 23 polar bears that had previously been prepared and their ages estimated
by staff of the Canadian Wildlife Service (CWS, Edmonton). For four
individuals for which teeth were not available, their age was estimated from
the length of the baculum based on a Gompertz “baculum length-on-age”
relationship established for 42 aged male specimens from Scoresby Sound
(data not shown). These four individuals with baculum lengths of 7.5, 11.1,
19.5 and 20.2 cm were estimated to be 0 (cub of the year), 1.2 (yearling), and
more than 10 years old (two animals), respectively.
Contaminant Analysis
All solvents were of analytical grade or better. Chromatographic materials
used for analysis are as follows: Florisil ® (magnesium silicate, F100-500, 60-
100 mesh) and basic aluminum oxide (Brockman activity grade I, 60-325
mesh), purchased from Fisher Scientific Inc. (Ottawa, Ontario, Canada) and
silica gel (Grade 62, 60-200 mesh, 150Å), purchased from Aldrich Chemicals
(Milwaukee, WI, U.S.A.). Deactivation of these materials was achieved with
double-distilled, n-hexane washed H 2 O. Bio-beads S-X3 (200-400 mesh) were
purchased from Bio-Rad Laboratories (Hercules, CA, U.S.A.).
CB and OC standard mixtures were supplied by the Canadian Wildlife
Service (Hull, PQ). The compound 1,3,5-tribromobenzene (Accu-Standard
Inc., New Haven, CT) was used as the CB/OC internal standard. The
method for extraction and clean up of polar bear adipose tissue for OCs and
CB analysis has been described elsewhere (Letcher et al., 1995a, 1995b, 1998;
Norstrom and Won, 1985; Sandala et al., 2004). Briefly, a ca. 0.5 g sample of
polar bear fat was homogenised with sodium sulphate (6:1 ratio by weight),
added to an extraction column containing n-hexane: dichloromethane
(DCM) (1:1), and spiked with the 1,3,5-tribromobenzene internal standard. A
10% portion of the lipid extract volume was used for gravimetric lipid determination.
The remaining extract was concentrated and subjected to gel
permeation chromatography (GPC) for lipid removal. The contaminantcontaining
GPC fraction was reduced in volume and subjected to chromatographic
clean-up with Florisil ® (8.0g, 1.2% deactivated, by weight). Three
fractions were collected, i.e., containing CBs (CB#1), most of the organochlorines
(OC #1), and OC #2 containing heptachlor epoxide and dieldrin.
Each fraction was concentrated to 1 mL for analysis by gas chromatography
with micro electron capture detection (GC-µECD). GC-µECD was performed
on an Agilent 6890 instrument equipped with a 63 Ni ECD detector and Agilent
7673 automated injector using a fused silica DB-5 GC column [(5%
phenyl) methylpolysiloxane (J&W Scientific Inc., Folsom, CA, 30m x 250µm
i.d., 0.25µm film thickness). Further details are described in Sandala et al.
(2004). Throughout the clean-up procedure, no contaminant-containing
fraction was permitted to go to dryness.
An external standard quantification approach was used for quantification of
CBs and OCs based on the peak area of the ECD response. ΣCB is the sum of