Spatial and Temporal Trends of Mercury and Cadmium in Ringed Seals from the Canadian Arctic

Michael and Derek Kwan1 Muir 2

1 Nunavik Research Centre,Kuujjuaq; 2 National Water Research Institute, Burlington, Ontario
Fig. 8. Age-normalization plots of adjusted mean mercury Fig. 5. Temporal trends of mercury in ringed seal liver from Holman,
concentrations in ringed seal liver Sachs Harbour, Arviat and Hudson Strait
Objectives (The y-intercepts of the dash lines from each of the regression lines indicate the age-normalized mean mercury
(S & A, Smith and Armstrong 1976, 1978, arithmetic means; W, Wagemenn, unpubl.data, geometric means;
K&M, Kwan and Muir 2000/05, age-normalized means of a “standard” 7. 4-year-old seal;
concentrations in the liver of a “standard” 7.4-year-old ringed seal. Age-normalized means for each location se, standard error; *, 95% confidence limits for a 7.4-year-old standard seal)
• To investigate the spatial distributions of mercury and cadmium in ringed seals harvested and sampling period were calculated using ANCOVA. Bartlett’s three-group method was used to obtain the
35 Holman 40 Sachs Harbour
Model II regression lines )
from hunting areas of selected major Inuit communities. “standard” 7.4-year-old seal 30 se 35
se *
30 se W
S&A 30
Holman 2001 25
*
• To provide data for facilitating future assessment of temporal trends of mercury and other Arviat 2003
S&A
25 K&M

mercury concentration, ug/g w.w.

20
metals in ringed seals from locations previously studied in the 70’s, the 80’s and the early 25 Pond Inlet 2004 K&M
20

mercury concentration, ug/g ww.

15
90’s.

mercury concentration, ug/g ww.

Grise Fjord 2003 15
10
10
20 5 5
• To investigate the bioaccumulation of mercury and cadmium in various ringed seal tissues. 0
n=83 n=112 n=20 n=45 n=18
0
1
1972/73 2
1977 3
2001 1987
1 2001
2
15
• To provide information on levels and time trends of these contaminants to each community 25
Arviat
18 Hudson Strait
participating in the study on a timely basis. 16
*
10 20
14
*
* 12
15

A Summary of Findings
10
K&M * K&M
5 W 8 *
10
* 6
W
(I) Age-Normalization and Geometric Means: 0
5 K&M 4 K&M

2
Age was found to be an important covariate influencing mercury concentrations in ringed seal liver in any one location. Age-normalized 0 2 4 6 8 10 12 14 16 0
n=15 n=16 n=21
0
n=6 n=17 n=16

mean mercury concentrations in the liver of a “standard”-age seal (fig. 8) was found to be a more satisfactory way than arithmetic or 1992
1 1998
2 2003
3 1989/90
1 1998
2 2002
3
geometric means in expressing the mercury data. The overall mean age of all the seals within the age range of 3 to 14 was 7.4, which was age
designated as the “standard”-age seal in this study. Tissue samples from closed to 500 ringed seals were collected between 1998 and 2004,
only about 68% of these fell within this age range and were used. Older seal were excluded since uncertainty in age determination
increases with older seals. The situation for cadmium was more complicated. Although there was no significant correlation between the
overall cadmium concentrations in the liver within the selected age range (r = 0.029, p> 0.1), three locations showed various degrees of Fig. 9. A comparison of age-normalized mean liver mercury concentrations of a Fig. 6. Temporal trends of mercury in ringed seal liver from Resolute,
positive correlation between liver cadmium concentration and age: Holman 2001 (r = 0.720, p < 0.01), Gjoa Haven 2004 (r = 0.741, 0.01 < p Pond Inlet, Arctic Bay and Grise Fjord
“standard” 7.4-year-old ringed seal with their corresponding geometric (S & A, Smith and Armstrong 1976, arithmetic means; W, Wagemann 1989 and unpubl. data, geometric means;
< 0.05), Hudson Strait 98/99 ( r = 0.675, 0.01 < p < 0.05). To avoid confusion and to allow comparison between locations and within a
means of seals in the age range of between 3 and 14 years K&M, Kwan and Muir 2000/05, age-normalized means of a “standard” 7. 4-year-old seal;
location at different time, it was decided that geometric means (antilog of log10 transformed data) were used for all the cadmium data. se, standard error; *, 95% confidence limits for a 7.4-year-old standard seal)
Indeed it was found that age-normalized means are almost always greater than geometric means (fig. 9). The highly skewed cadmium data 18
Arctic Bay
20
35 Pond Inlet
from some locations also rendered geometric mean a proper choice. 16 18
*
age-normalized mean 16
For both metals, no effect of seal gender on tissue metal concentrations was observed, hence data of both sexes were pooled. 30 geometric mean
14 se *
14
12
25 12

mercury concentration, ug/g ww.

10 K&M

mercury concentration, ug/g ww.

(II) Spatial Trends: 8
K&M *
10
20 W
8
Wide spatial variations in both mercury and cadmium concentrations in seal liver were observed (figures 1 to 4). Relatively high liver 6 K&M 6
*
mercury levels were found in seals from the Central / Western Arctic (Holman and Sachs Harbour) as well as those from the Eastern 15 4
se
K&M
4
Hudson Bay (Inukjuak), the Ungava Bay and Cumberland Sound (Pangnirtung). Samples from the Central High Arctic such as Arctic 10 2 2
S&A

Bay, Grise Fjord, Gjoa Haven, Resolute and to some extent, Pond Inlet were comparatively low in their liver mercury levels. Intermediate 0
n=15 n=18 n=22
0
n=33 n=17 n=21

liver mercury levels were found in seals from Labrador, Hudson Strait and Arviat. The highest liver cadmium concentrations were found 5 1
1983 2
2000 3
2004
1976
1 2000
2 2004
3

in seals from the Cumberland Sound (Pangnirtung), which is 1.6 times higher than those from the second highest location, Arviat 1998.
0 Grise Fjord
Samples from Gjoa Haven had distinctly lower cadmium concentrations (geometric mean = 0.07 ug/g) than all other locations. In contrast 25
Resolute
18

with mercury, samples from Sachs Harbour and Holman were relatively low in cadmium. Geologic makeup of a sampling location is likely 16 *

Po lut 04

ut 00
do 003

8
ay 99
rc en 2
Ba 004
Ha an 3

9
4

00
2
o u 01

ak 1

A Inle 99
La rvia 04

Ba 000
99

99
0
0

Pa Ar 98/9
I n 00
S t 200
ud kju 200

0
ch olm 20

20

20
ud a B 98/
rb 20

9
se se

S t 98/
to play an important role in explaining the wide spatial variations observed. 20

es y 2

es y 2
t2

t1
2
14

nd g 1
t2

tic t 2
nd e 2
d

e
a ait
r

ng via
r

it
le
or

Po tun
ra
Each bar in figs. 1 - 4 represents 15 to 32 seals, depending on the number available fell within the age range 3 to 14 years. *
r

v 12

ol
Fj

U br a
A
tic

r
H
u

ni
15
e

S&A K&M
H

rc
In
r is

10
so

R
a
so
jo

ng
W
s
G

* * 8
(III) Temporal Trends:
K&M
H
Sa

H
10
6
Samples from the Central High Arctic (Resolute, Arctic Bay, Grise Fjord and Pond Inlet) consist of data from two sampling periods. The K&M
K&M
4
5
significantly lower (0.001 < p < 0.01) cadmium levels from the 2004 samples (fig. 4) in comparison with the earlier samples from these 2 n=18
locations are difficult to explain considering the two sampling periods were only 4 to 5 years apart. In contrast, no significant difference (p n=27 n=27 n=18 n=20 n=21
0 0
> 0.05) in liver cadmium levels was found between the two sampling periods for both Arviat and Hudson Strait (fig. 3). 1976
1 1993
2 2000
3 2004
4 1998
11 2004
22

A similar picture was observed for mercury in the liver of seals from Arctic Bay, Pond Inlet and Grise Fjord for which significantly lower Fig. 10. Correlations between mercury and cadmium concentrations in the liver
(0.01 < p < 0.05) mercury levels from the 2004 samples compared with the earlier samples (fig. 2). However, no significant difference
between the two sampling periods was found for Resolute. In contrast with cadmium, significantly lower mercury levels from the more
and that in other tissues of ringed seals of 3 to 14 years old Fig. 7. Temporal trends of cadmium in ringed seal liver from Holman, Fig. 11. Percentage organic mercury in tissues of Size exclusion chromatograms of the cytosolic extract of
recent samples than those collected in 1998/99 were found for Arviat ( p < 0.001) and Hudson Strait (0.001 < p < 0.01). (Bartlett’s three-group method was used to obtain the Model II regression lines and equations) Arctic Bay, Resolute and Hudson Strait the ringed seal as a function of age the liver of a 11-year-old ringed seal
7.0
3.5 Cadmium in kidney (M, Macdonald 1986, arithmetic mean; F, Fallis unpubl. data, arithmetic mean; W, Wagemann, 1989, unpubl.data, geometric means;
An attempt was made to compare our recent data with findings available from past studies to throw some light on possible temporal Mercury in kidney
loge cadmium concentration in kidney, ug/g

y = 0.864x + 1.743 K&M, Kwan and Muir 2000/05, geometric means; se, standard errors; *, 95% confidence limits for the geometric means)
loge mercury concentration in

6.0
trends. In these past studies, only geometric or arithmetic means were reported and more complete data sets were often unavailable for 3.0
y = 0.200x + 1.118
2
R = 0.528 2.0 1.8
more detailed statistical analysis or age-adjustment. Great cautions have to be taken when making comparison between age-normalized 2.5 R2 = 0.149 5.0 O.D. 280nm
Cadmium 1.6
100
kidney, ug/g d.w.

Absorbance, 254nm, 280nm

mean and geometric and arithmetic means. With the limited amount of data available, some preliminary observations were made: (1) O.D. 254nm
1.4
2.0 Holman Arctic Bay 1.5

organic mercury as a percentage of

4.0
there seems to be an overall decrease in liver mercury concentration in seals from a number of locations studied: More noticeably,

cadmium, nmol/ml
Cd nmol/ml
90
d.w.

6 18
* 1.2
Holman between 1972/73 and 2001 (fig. 5), Resolute between 1976 and 2000 (fig. 6). For cadmium, downward trends were evident for 1.5 3.0 se 16 se
5
Resolute between 1993 and 2004, and Arctic Bay between 1983 and 2004 (fig. 7). (2) A possible increase in liver cadmium levels was 14 1.0
observed between 1981 and 2001 in Holman (fig.7). (3) Mercury levels in seal livers from Pond Inlet were 3-fold higher in 2000 than in
1.0 2.0
4
K&M
12 80 1.0
0.8
W
10
1976 and then dropped by more than half between 2000 and 2004 (fig.6). A similar trend was also observed in Hudson Strait in which 0.5 1.0 3
W 70 kidney 0.6

total mercury, %
8 F
between 1989/90 and 1998, liver mercury concentrations were almost double between 1989/90 and 1998, then these dropped by half

cadmium concentration, ug/g w.w.

* 0.5

cadmium concentration, ug/g ww.

0.0 2 M 6 0.4
0.0
between 1998 and 2002 (fig. 5). K&M
0.0 2.0 4.0
loge mercury concentration in liver, ug/g d.w.
6.0 0.0 1.0 2.0 3.0 4.0
loge cadmium concentration in liver,
5.0
1
4 K&M *
60 muscle 0.2
ug/g d.w. 2
n=38 n=7 n=18 n=17 n=15 n=20 n=22
At this stage, it must be stressed that any interpretation of temporal trends has to be preliminary and not conclusive. Not until more data 0.0 0.0
are available over a more extended period (several more decades) would a better elucidation of temporal trends be possible. loge cadmium concentration in liver, ug/g d.w. loge cadmium concentration in liver,
0
1981
1 1993
2 2001
3
0
1974
1 1983
2 2000
3 2004
4 50 liver 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64
ug/g d.w.
2.0 35

Absorbance, 254nm, 280nm

0.0
0.0 1.0 2.0 3.0 4.0 5.0
0.0
0.0 1.0 2.0 3.0 4.0 5.0
40 cerebrum Mercury 30

mercury, picomol/ml
-0.5 Cadmium in muscle Cadmium in cerebrum
30 1.5
loge cadmium concentration in

-0.5
Resolute
loge cadmium concentration in

25
(IV) Mercury and Cadmium Accumulation in Various Tissues: -1.0
y = 1.152x - 5.551
R2 = 0.671
-1.0
y = 0.733x - 4.595
2
R = 0.526
18
16 se
16 Hudson Strait
20
cerebrum, ug/g d.w.
muscle, ug/g d.w.

-1.5 14
Fig. 10. A significant positive correlation between mercury concentrations in the liver and the kidney (r = 0.386, p < 0.005) was found. In -2.0
-1.5
14
12 *
20 1.0
contrast, no significant correlation was observed between liver mercury concentrations and those in the muscles and the cerebrum. For -2.0 12 W
se 15
-2.5 10
cadmium, strong positive correlations (p < 0.005) were found between liver cadmium concentrations and those in the kidney ( r = 0.727 ), -3.0
-2.5 10 * 10 0.5 10
* 8 W
the muscles ( r = 0.819) and the cerebrum ( r = 0.725 ). -3.5 -3.0 8 K&M
5
-4.0 -3.5
6
K&M
6
K&M
0
Fig. 11. The percentage of mercury accumulated as organic mercury (% org-Hg) in seal liver, kidneys and, to a lesser extent the cerebrum 4 * 4 0.0 0
varies with age and total mercury concentration in the tissues. In the liver and the kidneys, % org-Hg decreases sharply with age and total
-4.5 -4.0
2 n=15 n=13 K&M
2 0 2 4 6 8 10 12 14 16 1 7 13 19 25 31 37 43 49 55 61
-5.0 -4.5 n=16 n=10 n=15 n=14
0 0
mercury concentration. A very gradual increase in % org-Hg with age in the cerebrum and the % org-Hg in muscles remains by and large
unchanged at between 80 and 100%.
1993
1 2000
2 2004
3 1989
1 98/99
2 2002
3 age 2.0 0.40
Selenium 0.35

Absorbance, 254nm, 280nm

Fig. 12. A majority of cadmium in the liver and the kidneys was bound to methallothionein (MT). A strong positive correlation between

selenium, picomol/ml
1.5 0.30
MT concentration in the liver cadmium concentration (r = 0.930). In the kidney, MT level increased sharply with tissue cadmium
concentration and then plateau-off as the kidney cadmium concentration increased further. The MT levels in the cerebrum were much
lower than those in the liver and the kidneys and by and large remain unchanged with cadmium concentration in the cerebrum. Very little
Metallothionein Determination Fig. 12. Metallothionein concentrations in the tissues of the ringed seal
1.0
0.25

0.20
MT was found in the muscles. as a function of tissue cadmium concentrations 0.15

A silver saturation method (Scheuhammer and Cherian 1991) was used. The 0.5 0.10
Fig. 13. Mercury and selenium in the seal liver was found to associate with high molecular weight cytosolic proteins ( > 100kD). A
substantial amount of mercury in the liver cytosolic extract is associated with a protein peak of about 6.5kD (containing the MT peak). method based on the complete displacement of all metals bound to MT by silver. At 700 1400

Future Sampling
0.05

This peak also responsible for all the cadmium presents in the cytosolic extract. saturation, 17mol of Ag bound to each mol of MT. After removing all unbound Ag by Liver Kidney 0.0 0.00
metallothionein concentration,

600
metallothionein concentration,

thermal denaturation in the presence of haemoglobulin. The soluble phase contains Ag-MT 1200
1 4100
7 10 15016
13 19 200
22 25 28250
31 34 300
37 40 43350 400 55 58 450
46 49 52 61 64
complexes which are thermally stable. Silver concentration in the soluble phase is 500 1000
determined by AAS and MT concentration can be calculated. Future sampling was planned for the following locations. Past data or archived V0 elution volume, ml
ug/g dry wt.

MT
ug/g dry wt.

400 800 samples are exist for one or more points in time for all the location selected.
Ref. Scheuhammer, A.M. and M.G. Cherian (1991) Quantification of metallothionein by silver 300
Sampling year Location Last sampling year
Methodology
600
saturation. In: Methods in Enzymology Vol. 205, Metallobiochemistry Part B, Methallothionein and
Related Molecules. Edited by J.F. Riordan and B.L. Vallee. Academic Press, London, pp 78-83. 200
400
100
2005 Arviat* 2003
200
Sachs Harbour 2001
(I) Total Mercury and Cadmium (II) Total Organic Mercury 0
0 10 20 30 40 50 60
0 Broughton Island* 1987
Tissues were homogenized and digested in protease (2mg/ml) in Tris-HCl buffer (50mM, pH cadmium concentration, ug/g dry wt.
0 100 200 300 400 Nain* 1998
Tissues were initially digested in 70% w/v nitric acid in Teflon digestion bombs at
120oC cadmium concentration, ug/g dry wt.
8.5). An alkaline cysteine reagent (1% w/v cysteine in 40% w/v sodium hydroxide) was added to chelate all
overnight. The primary digests were diluted with Type I water prior to cadmium determination. Aliquots of mercury from the tissue digest. Cupric sulfate and acidic sodium bromide reagent (28mM sodium bromide
primary digests were further subjected to a more vigorous acid oxidation prior to total mercury in 8% v/v sulfuric acid) were added to liberate alkylmercury from their cysteine complexes. The 250 20 2006 Pangnirtung 2002
determination. The aliquots were digested for 4 hours in a mixture of nitric, sulfuric and hydrochloric acids alkylmercury bromides formed were then quantitatively extracted into toluene and subsequently back- Cerebrum Holman 2001
at 75oC in an open system of boiling tubes on aluminum heating blocks. The secondary digests were diluted 18 Muscle
metallothionein concentration,

extracted into sodium thiosulphate (5mM). Phase separation was effected by centrifugation (6200 x g, 20
metallothionein concentration,

with acidic potassium dichromate solution and measured for mercury. Each sample was digested in min.). The thiosulphate extraxts were digested with a mixture of nitric, sulfuric and hydrochloric acids at 200 16
duplicate. 75oC in test tubes on aluminum heating blocks. Mercury in the acid digests was determined by CVAAS. 14 2007 Kangiqsualujjuaq 2002
Cold-vapour atomic absorption spectrometry (CVAAS) was used to determine total mercury Certified Reference Materials (DORM2, DOLT2 and Mussel 2976 from the National Research Council of Inukjuak 2002
ug/g dry wt.

ug/g dry wt.

150 12
concentrations in the secondary digests. A model 4110ZL Zeeman atomic absorption spectrometer (Perkin Canada) and methylmercury chloride standards were routinely used in quality control.
Elmer) equipped with an electrodeless discharge (EDL) mercury lamp and a FIAS-100 flow injection system 10
(Perkin Elmer) were used. A 10% w/v tin (II) chloride in 30% v/v hydrochloric acid was used as reductant, (III) Age Determination 100 8 2008 Gjoa Haven 2004
and a 10% v/v hydrochloric acid as the carrier. Normal calibration method with a standard curves was used 6 detection lim it of MT, 8.4
in quantitation of absorbance signals. Measurements were made in duplicate for each digest. The typical Thin longitudinal sections of the canine (about 1/50 mm thick) were cut using a low-speed
detection limit achieved was about 0.05 ng/mL.
jewelry saw. Age was estimated by counting annual lays in the dentine under a stereo-microscope. 50 4 ug/g dry w t. for m uscle
2009 Pond Inlet 2004
Graphite furnace atomic absorption spectrometry (GFAAS) was used to determine cadmium
concentrations in the primary digests. A 4110ZL Zeeman atomic absorption spectrometer, equipped with a (IV) Size Exclusion Chromatography 0
2
0
(* sample collecting has been completed and samples are being analyzed)
Acknowledgements
transversely-heated graphite atomizer (THGA) was used. Chemical interference was suppressed by using an 0 0.1 0.2 0.3 0.4 0 1 2 3 4
ammonium phosphate-magnesium nitrate matrix modifier. Background absorption was corrected using the The following conditions were used :
cadmium concentration, ug/g dry wt. cadmium concentration, ug/g dry wt.
longitudinal inverse a.c. Zeeman effect background corrector of the spectrometer. Atomization and ashing
1. Cytosol extraction: Homogenize sample in 2 volumes of eluent (plus 0.5mM PMSP) at
4oC under nitrogen for 3 min. using a polytron at 30,000 rpm.
We are indebt to the Inuit hunters and local Hunters, Trappers Organization in Nunavik,
temperatures used were 1800oC and 560oC respectively. Nunavut and Labrador for their efforts in sampling and bio-data recording. We extend our
Centrifuge at 13,000G for 20 min. at 4oC. Supernatant stored at
Internal QA/QC: For each batch of samples, two certified reference materials (dogfish muscles
DORM-2 and dogfish livers DOLT-2 from the National Research Council of Canada) were digested and
minus 80oC. Thawed and filtered through 0.22µm PVDF membrane
prior to applying to the column.
thanks to researchers who provided us with archived samples: Drs. Ian Stirling and Nick
analyzed in duplicate. A recovery of the analyte to within 10% of its certified value was used as a criterion 2. Column: Sephacryl S-100HR, 1kD to 100kD, 26/100 bed dimension Lund (Canadian Wildlife Services), Dr. Kunito (Ehime University, Japan) and Dr. Arron Fisk
for validation of the batch. Digestion blanks in duplicate were included in each batch of samples digested. (Pharmacia-Biotech Ltd.)
External QA/QC: NCP QA/QC exercise and the QUASIMEME Laboratory Performance 3. Eluent: 50mM Tris-HCl, 140mM NaCl, 2.5mM dithiothreitol, pH 7.8 at a (National Water Research Institute, Canada). Last but not least, the Northern Contaminants
Studies. flow rate of 0.85 ml/min. Program for funding the study.