SC/F16/JR33

Comparison of total Hg levels in O and J type stock ofcommon minke whales based on JARPNII coastal samples
collected in 2012 and 2013
GENTA YASUNAGA AND YOSHIHIRO FUJISEThe Institute of Cetacean Research, 4-5, Toyomi-cho, Chuo-ku, Tokyo, 104-0055, Japan.
Contact e-mail: yasunaga@cetacean.jp

ABSTRACTThe purpose of the present study was to examine differences of total Hg concentrations in muscle and liver ofJ- and O-stocks of common minke whales (Balaenoptera acutorostrata) from the waters off Sanriku.
Concentrations of total Hg in muscle and liver of 35 O- and 24 J-stock immature minke whales taken in the2012 and 2013 JARPNII surveys were measured. Multiple linear regression analyses of total Hg concentrationsof the whales were carried out. These included adjustment for confounders, age index, sex, stock, blubber
thickness and year. Stock had no discernible effect. These findings suggest that there is no stock-dependent
difference of total Hg exposure risk for the minke whales from off Sanriku.

KEYWORDS: COMMON MINKE WHALE; NORTH PACIFIC; MERCURY; STOCK; O-STOCK; JSTOCKINTRODUCTION
The Scientific Committee held two specialized Workshops, one relating to chemical pollutants and cetaceans, and theother on the potential ecological effects on cetaceans of climate change and ozone depletion (IWC, 1994). In the
JARPNII plan, the following objective 2 was addressed, ‘Monitoring environmental pollutants in cetaceans and themarine ecosystem’. This objective contains sub-objective, ‘Pattern of accumulation of pollutants in cetaceans’(Government of Japan, 2002).
Anthropogenic Hg emissions from land to the atmosphere in Asia have increased steadily over the past several decades(Pacyna et al., 2006), whereas those in Europe, Russia and North America peaked in the 1950s to 1970s (UNEP, 2013).
It is important to assess Hg exposure of whales in nearshore waters of Asia for better understanding and the
management of health risks. IWC has raised a concern about the effects of pollutants on cetaceans because of thepossibility that these pollutants negatively affect the whales’ health resulting in a decrease in the abundance of thestocks (IWC, 1994). Furthermore, age, nutritional condition and sex are important to examine accumulation features of
Hg in baleen whales (Fujise et al., 1997; Aguilar et al., 1999; Honda et al., 2006).
In the present study, we measured total Hg concentrations in muscle and liver to evaluate differences of total Hg
exposure in O and J of minke whales from off Sanriku, using multiple regression analysis including adjustment for
confounders, sampling years, age, nutritional condition and sex. Racemization of aspartic acid in lens was used for age
index determination (George et al., 1999; Yasunaga et al., 2014), and blubber thickness was used for nutritional
condition index (Ohsumi et al., 1997; Konishi et al., 2008).

MATERIALS AND METHODS
Samples
Muscle and liver samples were collected for total Hg analyses from 59 immature of common minke whales of both
sexes from the 2012 and 2013 JARPNII coastal survey in coastal waters within 50 n. miles from Ayukawa port in the
Sanriku district (Fig. 1). The muscle tissues were excised from the central dorsal part of the body, liver tissues were
excised from the central part of the organ, and eye lenses were taken from the left eyeball at the research station at
Ayukawa town. Soft tissues and eye lenses were stored in polyethylene bags at -20 and -80°C until analysis,
respectively.
In this study we considered information on the body length and maturity status of male whales. Males and females ofminke whales were defined as sexually mature by testis weight (larger side) of more than 290g and by observation of1

ovary, respectively (Bando et al., unpublished data). Common minke whales from the Okhotsk Sea/Western Pacific(‘O’ stock) and the Yellow Sea/East China Sea/Sea of Japan (‘J’ stock) were identified by microsatellite analysis(Kanda et al., 2009).
Laboratory analysis
For total Hg analysis, the tissue homogenate samples (approximately100 mg) were set on a ceramic boat and subjected
to cold-vapor atomic absorption spectrometry with heat-vaporization, gold amalgamation method (Nippon Instruments
Co., MA-2000). Accuracy and precision of the methods were confirmed using standard materials, DORM-2 (NRCC:
muscle of dogfish).
For age index determination by aspartic acid racemization in lens, the lens samples were stripped externally to avoid
contamination and homogenized with Tris-buffer (200mM Tris, 150mM NaCl, pH 8.0) using ultrasonic disruptors. The
homogenate was centrifuged at 15,000 × g for 15 min. at 4°C, then desalted with acetone and air-dried. The sample redissolved in water was then set up within a glass tube for hydrolysis and freeze-dried. The purified samples werelyophilized in tubes and were hydrolyzed in a gas-phase 6N-HCl for 7 hr at 108°C (Pico Tag Work Stations, Waters,
Tokyo). The hydrolysates were evaporated under reduced pressure. The hydrolysates were dissolved and incubated in
0.1N-HCl and Borate buffer (0.1M, pH10.4), and were further incubated with o-phthalaldehyde (OPA) and n-tertbutyloxycarbonyl-L-cysteine (Boc-L-Cys) to form diastereoisomers. The D/L ratio of aspartic acid was determinedusing RP-HPCL (Alliance ® HPLC systems e2696, Waters) with a Nova-Pak ODS column (3.9mm ×300mm, Waters)
using fluorescence detection (344 nm excitation wavelength and 433 nm emission wave length). Elution was carried out
with a simple isocratic adsorption of 3% acetonitril+3% tetrahydrofuran /0.1M acetate pH6.0 buffer solution in 45 min
at a flow rate of 0.8 ml/min, at 23°C. Sensitivity of intensity for both aspartic acid enantiomers was corrected by
L50ppm/D2.5ppm-Aspartic acid (minimum 99% TLC, SIGMA-ALDRICH) solution.
Statistical analysis
Factors affecting total Hg concentrations in whales were assessed using multiple linear regression analyses (Zar, 1999).
The following independent variables: “sampling year (2012=0; 2013=1)”, “age index”, “sex (male=0; female=1)”,“stock (O stock=0, J stock=1)” and “blubber thickness”, were allowed, and all parameters were normalized. Thesestatistical analyses were executed with SPSS ver.11 for Windows (SPSS Co. Ltd.).
RESULTS AND DISUCSSION
Total Hg levels in muscle and liver of common mike whales were 0.06 ± 0.05 and 0.15 ± 0.11 ppm wet wt.,
respectively. Plots of total Hg concentrations against their age index are shown for muscle (Fig. 2) and liver (Fig. 3). To
examine the factors affecting Hg concentrations in minke whales, multiple regression linear analyses were conducted.
The results for total Hg in muscle and liver of minke whales are shown in Tables 2 and 3, respectively. Regression
models were significant for total Hg concentrations in both organs. For muscle of minke whales, age index (+) and sex(male < female) were significantly selected (Table 1), and for liver of whales, only sex (male < female) weresignificantly selected (Table 2).
Significant association between total Hg concentrations in muscle of minke whales and their age index were observed.
This result is consistent with previous studies (Honda et al., 1983; Aguilar et al., 1999). However, concentrations inliver of minke whales were not significantly associated, whereas there seems to be association between total Hg levels
in liver and age index. The reasons are likely sample size and use of immature individuals. Female was a positive factor
for total Hg concentrations in both muscle and liver of minke whales. The results are also consistent with the previous
study (Aguilar et al., 1999). On the other hand, stock of minke whales was not significantly associated with total Hgconcentrations. These results suggest that there is no difference of total Hg exposure for both stocks of the minke
whales from off Sanriku. In addition, total Hg levels in both stocks from off Sanriku were extremely lower than thatwhich would have adverse effects on whales.
REFERENCES
Aguilar, A., Borrell, A. and Pastor, T. 1999. Biological factors affecting variability of persistent pollutant levels incetaceans. J. Cetacean Res. Manage., (Special Issue 1):83-116.
Fujise, Y., Honda, K., Yamamoto, Y., Kato, H., Zenitani, R. and Tatsukawa, R. 1997. Changes of hepatic mercury
accumulations of southern minke whales in past fifteen years. Paper SC/M97/20 presented to the IWC
Intercessional Working Group to Review Data and Results from Special Permit Research on minke whales in the
Antarctic, May 1997 (unpublished). 16pp.
George, J.C., Bada, J., Zeh, H., Scott, L., Brown, S.E., O'Hara, T. and Suydam, R. 1999. Age and growth estimates ofbowhead whales (Balaena mysticetus) via aspartic acid racemization. Can. J. Zool. 77:571-80.
Government of Japan. 2002. Research Plan for Cetacean Studies in the Western North Pacific under Special Permit(JARPNII). Paper SC/54/O2 presented to the IWC Scientific Committee, May 2002 (unpublished). 46pp.
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Honda, K., Tatsukawa, R., Itano, k., Miyazaki, N. and Fujiyama, T. 1983. Heavy metal concentrations in muscle, liverand kidney tissue of striped dolphin, Stenella coeruleoalba, and their variations with body length, weight, age andsex. Agric. Biol. Chem. 47:1219-1228.
Honda, K., Aoki, M. and Fujise, Y. 2006. Ecochemical approach using mercury accumulation of Antarctic minke whale,
Balaenoptera bonaerensis, as tracer of historical change of Antarctic marine ecosystem during 1980-1999. Bull.
Environ. Contam. Toxicol. 76:140-7.
International Whaling Commission. 1994. Chairman’s Report of the Forty-Fifth Annual Meeting, Appendix 12.
Resolution on research on the environment and whale stock. Rep. int. Whal. Commn. 44:35.
Kanda et al. 2009. Individual identification and mixing of the J and O stocks around Japanese waters examined by
microsatellite analysis. Paper SC/J09/JR26 presented to the JARPNII Review Workshop, Tokyo, January 2009
(unpublished). 9pp.
Konishi, K., Tamura, T. Zenitani, R., Bando, T., Kato, H. and Walløe, L. 2008. Decline in energy storage in the
Antarctic minke whale (Balaenoptera bonaerensis) in the Southern Ocean. Polar Biol., 31: 1509–1520.
Ohsumi, S., Fujise, Y., Ishikawa, H., Hakamada, T, Zenitani, R. and Matsuoka, K. 1997. The fattyness of the Antarctic
minke whale and its yearly change. Paper SC/M97/18 presented to the IWC Intersessional Working Group to
Review Data and Results from Special Permit Research on Minke whales in the Antarctic, May 1997
(unpublished). 21pp.
Pacyna, E., Pacyna, J., Steenhuisen, E. and Wilson, S. 2006. Global anthropogenic mercury emission inventory for 2000.
Atmos. Environ. 40:4048-4063.
UNEP. 2013. Global Mercury Assessment 2013: Sources, emissions, releases, and environmental transport. Pp42.
http://www.unep.org/publications/contents/pub_details_search.asp?ID=6282
Yasunaga, G., Bando, T. and Fujise, Y. 2014. Preliminary estimation of the age of Antarctic minke whales based on
aspartic acid racemization. Paper SC/F14/J12 presented to the JARPA II Review Workshop, Tokyo, February
2014 (unpublished). 10pp.
Zar, J.H. 1999 Biostatistical analysis, 4th ed., Prentice Hall, New Jersey, USA, 663pp.

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Table 1. Biological data and total mercury concentrations in muscle and liver of common minke whales (males) fromwestern North Pacific during 1994 and 2014
Year StockSexnBody length
Blubber thickness
Hg in muscle
Hg in liver
Age Index*(m)(cm)(ppm wet wt.)
(ppm wet wt.)
3.1 ± 0.6
2012
O
Male 84.59 ± 0.39
0.0393 ± 0.0020
0.03 ± 0.05
0.093 ± 0.112( 4.02 - 5.31 ) ( 0.0361 - 0.0416 ) ( 2.1 - 4.1 ) (<0.001 - 0.14 ) ( 0.013 - 0.35 )0.17 ± 0.12
Female 94.99 ± 0.53
0.0407 ± 0.0032
3.2 ± 0.7
0.05 ± 0.05
( 4.37 - 5.97 ) ( 0.0361 - 0.0454 ) ( 2.3 - 4.3 ) (<0.001 - 0.12 ) ( 0.003 - 0.32 )J
Male 74.73 ± 0.52
0.0402 ± 0.0032
3.3 ± 0.6
0.06 ± 0.07
0.20 ± 0.18
( 3.92 - 5.44 ) ( 0.0344 - 0.0448 ) ( 2.8 - 4.4 ) (<0.001 - 0.17 ) ( 0.007 - 0.51 )Female 55.11 ± 0.60
0.0411 ± 0.0035
3.4 ± 0.4
0.07 ± 0.03
0.16 ± 0.05
( 4.61 - 6.1 ) ( 0.0374 - 0.0456 ) ( 2.9 - 3.9 ) ( 0.041 - 0.1 ) ( 0.10 - 0.22 )2013
OMale 10 4.63 ± 0.68
0.0426 ± 0.0027
2.8 ± 0.5
0.03 ± 0.03
0.11 ± 0.07
( 3.73 - 5.73 ) ( 0.0390 - 0.0477 ) ( 1.9 - 3.4 ) ( 0.004 - 0.1 ) ( 0.029 - 0.23 )Female 85.32 ± 0.55
0.0449 ± 0.0026
3.1 ± 0.4
0.07 ± 0.06
0.16 ± 0.13
( 4.14 - 5.88 ) ( 0.0411 - 0.0491 ) ( 2.4 - 3.8 ) ( 0.009 - 0.19 ) ( 0.059 - 0.45 )J
Male 44.80 ± 0.97
0.0433 ± 0.0023
2.7 ± 0.6
0.06 ± 0.03
0.18 ± 0.08
( 3.70 - 5.66 ) ( 0.0414 - 0.0453 ) ( 2.0 - 3.3 ) ( 0.03 - 0.093 ) ( 0.11 - 0.28 )Female 8
5.27 ± 0.55
0.0464 ± 0.0045
2.7 ± 0.7
0.09 ± 0.03
0.20 ± 0.06
( 4.72 - 6.14 ) ( 0.0416 - 0.0556 ) ( 1.8 - 3.7 ) ( 0.038 - 0.13 ) ( 0.081 - 0.28 )Total
59 4.92 ± 0.62
0.0423 ± 0.0038
3.0 ± 0.6
0.06 ± 0.05
0.15 ± 0.11
( 3.70 - 6.14 ) ( 0.0344 - 0.0556 ) ( 1.8 - 4.4 ) ( 0.00 - 0.19 ) ( 0.003 - 0.51 )*: Age index=Ln (1+AspD/L)/(1-AspD/L)

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SC/F16/JR33
Table 2. Results of multiple linear regression analyses with "total Hg levels in muscle of common minke
whales from off Sanriku" as the dependent variable.
a) Model of regression
Model
R
1
0.856

R2
0.747

R2'
0.724

b) Analysis of Variance TableModel
Sum of Squares
Regression
0.103
Residual
0.035
Total
0.138

DF
5
53
58

Mean Square
0.021
0.001

F value
31.377

P value<0.001c) Variables
Model
Constant
Age index
sex
stock
blubber thickness
year

SE
0.052
0.003
0.005
1.079
0.007
0.007

B'

T
3.004
5.175
3.609
-0.0770.502
0.200

P value
0.004<0.001
0.001
0.939
0.618
0.842

B
0.155
0.016
0.018
-0.083
0.004
0.001

0.555
0.362
-0.0060.038
0.015

Table 3. Results of multiple linear regression analyses with "total Hg levels in liver of common minke
whales from off Sanriku" as the dependent variable.
a) Model of regression
Model
R
1
0.854

R2
0.729

R2'
0.703

b) Analysis of Variance TableModel
Sum of Squares
Regression
0.503
Residual
0.187
Total
0.691

DF
5
53
58

Mean Square
0.101
0.004

F value
28.477

P value<0.001c) Variables
Model
Constant
Age index
sex
stock
blubber thickness
year

SE
0.120
0.007
0.011
2.502
0.017
0.017

B'

T
3.579
1.470
7.297
-0.469-0.202-0.171

P value
0.001
0.148<0.001
0.641
0.841
0.865

B
0.428
0.011
0.082
-1.173-0.003-0.0030.163
0.758-0.040-0.016-0.0135

50˚N

Area 345˚Area 1

Area 240˚

35˚
Figure 1. Research area of the JARPNII coastal survey off Sanriku

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Hg concentrations in muscle (ppm wet wt.)

SC/F16/JR33

0.20

O stock-maleO stock-female

0.15

J stock-male
J stock-female

0.10
0.05
0.00
0.030

0.035

0.040

0.045

0.050

0.055

0.060

Age index: Ln((1+AspD/L)/(1-AspD/L))

Hg concentrations in liver (ppm wet wt.)

Figure 2. Relationship between Hg concentrations (ppm wet wt.) in muscle and age index (Ln ((1+AspD/L)/(1AspD/L))) in common minke whales from off Sanriku

0.60

O stock-maleO stock-female
J stock-male

0.40

J stock-female
0.20

0.00
0.030

0.035

0.040

0.045

0.050

0.055

0.060

Age index: Ln((1+AspD/L)/(1-AspD/L))
Figure 3. Relationship between Hg concentrations (ppm wet wt.) in liver and age index (Ln ((1+AspD/L)/(1-AspD/L)))in common minke whales from off Sanriku

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