Marine Environmental Research 31 (1991) 275 286

Heterogeneous Distribution of Organochiorine Contaminants in the Blubber of Baleen Whales:

Implications for Sampling Procedures

Alex Agui lar & A s s u m p c i 6 Borrell

Department of Animal Biology (Vertebrates), Faculty of Biology, University of Barcelona, 08071 Barcelona, Spain

(Received 13 April 1990; revised version received and accepted 16 May 1991)

A B S T R A C T

The concentration o[" DDTs and PCBs n'as determined #l two strata (imzer and outer) o['the blubber offin and sei whales.,fi'om the eastern North Atlantic. hi the two species residue levels qf all organochlorines in the outer layer were sign(fieantO~ higher than in the inner one. The DDE/'tDDTratio ~fidnot show signOqeant variation between strata, but the tDDT/PCB ratio was higher in the outer layer than in the inner one. Such d(fferences between strata cannot he explained by variation in the quantiO' q[" lipids present in the blubber and probab O' indicate a constant organochlorine distribution pattern in the blubber ¢)[" large whales. The differential role of the blubber strata hz the [~tttening cycle, the heterogeneous lipid composition of blubber throughout its depth and the d_ifferent turnover rates qf pollutants in the layers are the probable factors responsible Jor these observed d(fferences in pollutant distribution between blubber strata. Because ~[" this heterogeneous distribution, blubber samples collected from cetaceans for pollutant anah'ses should include equal representation qf all layers in order to be representative o[the individual's pollutant load. This is especially hnportant when sampling stranded specimens or taking hiopo' samples from largeJree-ranging whales.

I N T R O D U C T I O N

Blubber is a hypodermic tissue which covers the body of cetaceans and serves as an energy reserve deposit and insulating cover. In large whales it reaches a thickness of several centimetres and represents about 20-40% of their total body weight (Lockyer, 1976). The most important biochemical

275 Marine Environ. Res. 0141-1136/91/$03.50 (~ 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain.

276 Alex Aguilar, Assumpci~'~ Borrell

constituents of blubber are lipids, which typically represent about 30-80% of the tissue's wet weight (Ackman et al., 1975; Lockyer et al., 1984; Aguilar & Borrell, 1990). Because of its large contribution to the cetacean's body mass and high lipid content blubber carries most of the load of lipophilic pollutants (such as organochlorines) present in the bodies of large whales. Therefore blubber is the tissue most commonly used for monitoring organochlorine residue levels in cetaceans (Wagemann & Muir, 1984).

However, the blubber of large whales is not a homogeneous tissue throughout its depth but is composed of layers or strata with different roles and biochemical composition (Ackman et al., 1965, 1975: Lockyer et al., 1984). The inner layers are mostly devoted to fat storage, and their lipid content fluctuates greatly following the individual's reproductive and migratory cycle. The outer layers, however, have an insulation function and are little affected by changes in the nutritive condition. As a conse- quence of the differing roles of the strata, the lipid content decreases from the outermost layers to the innermost, the intensity of the gradient depending on the nutritive condition o1" each individual. Thus, in fat, pregnant females, no difference between the strata is usually observed, and the decreasing trend is nonexistent or even reversed. In whales of an intermediate body condition, e.g. most males or immature females, the gradient is almost always apparent, although the difference between strata is not very great. Finally, in lean females, typically those lactating or post- lactating, the gradient is great, and the inner layers contain only about 20% of the lipid present in the outermost external ones (Aguilar & Borrell, 1990l.

Because distribution, metabolism and mobilization of organochlorines are closely linked to those oflipids it has been proposed that organochlorine concentrations may not be uniform in all strata and locations of the blubber in a given animal, even if concentrations are calculated on the basis of the lipid content of the tissue (Aguilar, 1985). A heterogeneous distribution of organochlorines in the blubber strata would have strong implications for the sampling methodology of whales because, given the great thickness that this tissue reaches in some large species, all blubber layers are not usually contained in the piece of tissue excised and analysed for the study. This problem is especially important when samples of the superficial layers of the blubber are collected from free-ranging individuals using a biopsy dart, a technique which has become common in the past few years (Winn et al., 1973: Aguilar & Nadal, 1984; Lambertsen, 1987; Mathews et al., 1988).

In the present study the authors analysed the concentrations of DDTs and PCBs, the two groups of organochlorine compounds most widely found in cetaceans, in different strata of dorsal blubber from fin (Balaenoptera physalus) and sei (Balaenoptera borealis) whales and examined their variation pattern between layers.

D&tribution q]organochlor&es & whale blubber 277

Fin whale males Fin whale females Sei whale males Sei whale females

T A B L E ! Biological Information from the Whales Analysed

N Body length (m) Age {years) x + SD .v 4__ SD

30 18'7_+0'9 18'5_+ 17"4 59 19"3_+ 1"1 12"1 -+ 8"9 10 13"2_+0'8 14"3_+7'2 13 13"7--+ 1"2 12"0_+5:0

MATERIAL

Large pieces of dorsal blubber were obtained from whales caught off Spain and Iceland by commercial fishing operations. These were collected during flensing procedures, about 15-24h after the death of the animal, and contained the whole blubber depth, including the skin and some of the adjacent basal muscle. All samples were taken from the dorsal region posterior to the dorsal fin. This location was chosen because it is the one typically selected for sampling stranded specimens (Anon., 1985), as well as the one most easily accessible from a vessel during sampling by means of a biopsy dart. In total, samples were obtained from 89 fin whales (30 males and 59 females) caught offSpain in 1984 and from 23 sei whales (10 males and 13 females) caught off Iceland in 1985. Because whaling regulations prevent catching whales of small size the sample was composed almost only of adult individuals. Biological information from the specimens analysed tbr the study is shown in Table 1.

From the original piece of blubber excised, small pieces (about 2 3 g) were drawn from the outer (external) and inner (internal) regions of each sample, and processed independently.

METHODS

Whales were measured and sexed at the flensing platform by experienced people. Age was determined by counting growth layers in the core of the whale's ear plugs following standard procedures (Lockyer, 1984). Samples for organochlorine determination were wrapped in aluminium foil and kept in deep freeze until analysis. For lipid extraction the pieces of blubber were ground with anhydrous sodium sulphate in a mortar, and the mixture extracted with n-hexane for 4 h in a Soxhlet apparatus. The extract was then concentrated to 40ml, from which a 10-ml subsample to determine

278 Alex Aguilar, Assumpci~'~ Borrell

tissue fat content was taken. An aliquot of the remaining extract containing 1 g of lipid was mixed with sulphuric acid for the clean-up following the procedures described by Murphy (1972).

After centrifugation and phase separation, the lipid-free extract was concentrated to 1 ml and injected into a Perkin Elmer Sigma 3B gas chromatograph (injector temperature 250~C), equipped with an electron- capture detector of 63Ni (temperature 350~C), and a Perkin-Elmer Sigma 15 computing integrator. A fused-silica capillary column of 0-25 mm internal diameter, 60 m length, and a stationary phase SPB- 1 with a film thickness of 0.25/~m was used. Pure nitrogen at a flow rate of I ml/min was used as carrier gas.

Temperature was programmed according to the following sequence: injection at 40°C; oven steady for the first 2 rain and then an increase from 40 to 160cC at a rate of 2YC/min; oven maintained at steady temperature for 1 rain and then an increase from 160 to 250°C at a rate of 2°C/min. From this point until the end of the analytical run the column remained isothermal at a temperature of 250~C.

Heptachlor was used as an internal standard to calibrate fluctuations in the operational conditions. The identity of the DDT compounds was confirmed by an alkali conversion to their respective olefins and re-analysis by GLC. PCBs were identified and quantified by their peak characteristics and retention times in relation to a 1:1 standard mixture of Aroclors 1254 and 1260, and confirmed by their resistance to the chemical derivations detailed above. Eight replicates of samples fortified with standards gave the following percentages of recovery for the whole analytical process (mean + coefficient of variation): p,p'-DDE, 72"56% _+ 15'8; p,p'-TDE, 87"41% _+ 8.4; o,p-DDT, 81.91% ± 8"3; p,p'-DDT, 98"13% _+ 7.6; PCB, 90-97% _+ 8.0. Ten replicate chromatographic runs of a mixture of the DDT and PCB standards gave the following coefficients of variation in the quantification: p,p'-DDE, _+7.9%; p,p'-TDE, +6"5%; o,p-DDT, +5-5%; p,p'-DDT, ___8"2%; PCBs (mean of the eight peaks commonly used in the quantification), +8.7%.

All the results are expressed in relation to the total lipid content of the tissue sample. Total DDT (tDDT) levels are calculated as the sum of all the DDT forms (p,p'-DDE, p,p'-DDT, o,p'-DDT and p,p'-TDE).

RESULTS AND DISCUSSION

The levels of DDTs and PCBs found in the outer and inner layers of the blubber from fin and sei whales, according to sex, are detailed in Table 2. As was expected, males displayed higher residues of contaminants than females of the same species, a difference attributable to the transfer process that

TA

BL

E 2

E

xtra

ctab

le L

ipid

Co

nte

nt (

EL

P)

and

Org

ano

chlo

rin

e C

on

cen

trat

ion

s (M

ean

and

Ass

ocia

ted

Sta

nd

ard

Dev

iati

ons)

Fo

un

d in

the

Inn

er a

nd O

ute

r L

ayer

s of

the

Blu

bber

of

Bal

een

Wha

les

and

the

Lev

el o

f S

igni

fica

nce

of

the

Dif

fere

nce

Bet

wee

n L

ayer

s A

ccor

ding

to

a t-

Tes

t (c

once

ntra

tion

s ar

e ex

pres

sed

in l

tg/k

g on

an

extr

acta

ble

lipi

d ba

sis)

p,p'

-DD

E

p,p

'-T

DE

o,

p'- D

D T

p,

p'- D

D T

tD

D T

P

CB

s E

LP

Fin

wha

le m

ales

In

ner

42

0 ±

290

15

0 _+

70

300

± 1

80

Out

er

580

+ 3

20

210

± 9

0 42

0_+

230

Si

gnif

ican

ce

0.05

0'

01

0.05

180

± 8

0 1 0

40 ±

600

1 4

30 ±

680

75

.3 ±

14.

1 27

0_+

120

!

490_

+69

0 l

82

0±

730

77

"4±

8-2

0"00

1 0-

02

0"05

N

S ~

100_

+ 50

50

0 _+

270

77

0 ±

410

6

57

± 2

1-6

130

± 7

0 67

0 ±

350

88

0 ±

250

70

'4 ±

5"4

0"

002

0"01

0-

05

NS

120

+ 6

0 67

0 _+

390

60

0 ±

290

15

"7 ±

9'8

17

0 ±

90

970

± 4

90

660

+ 3

00

76.2

± 6

"3

0-05

0"

05

N S

0"

001

Fin

wha

le f

emal

es

Inne

r 17

0 ±

100

80

_+ 5

0 15

0 ±

90

Out

er

240_

+ 1

50

110_

+ 50

2

00

± 1

00

Sig

nifi

canc

e 0"

01

0-02

0"

01

Sei

wha

le m

ales

In

ner

23

0+

150

1

10

± 6

0 2

10

± 1

20

Out

er

360_

+21

0 1

40

± 7

0 3

10

± 1

40

Sign

ific

ance

0.

05

NS

0.

05

Sei

wha

le f

emal

es

Inn

er

60 ±

30

40 +

_ 20

60 ±

30

Out

er

70 ±

50

40 ±

30

60 ±

40

Sig

nifi

canc

e N

S

NS

N

S

"NS

, di

ffer

ence

s no

n-si

gnif

ican

t.

40_+

30

200_

+ 1

10

210_

+90

31

.4_+

19-

6 4

0+

30

2

10

+ 1

50

21

0+

130

8

1.4

+8

.6

NS

N

S

NS

0.

001

,,,,, 2"

2"

b,-)

280 Alex Aguilar, Assumpci6 Borrell

sexually mature females experience during pregnancy and lactation (Aguilar, 1987). In general, the variance of concentrations was very small. In baleen whales of the same sex age is the main source of variation of organochlorine concentrations. However, the effect of age is much greater during the first years of life than in the adult state (Aguilar & Borrell, 1988). Because of size selection by the whaling boats the sample studied here was composed almost entirely of adult specimens, and this undoubtedly reduced its variance.

According to a t-test the residue levels of most pollutants and sample groups analysed were significantly different in the two layers (inner and outer). The only exceptions to this general rule were female sei whales, in which organochlorine concentrations were not dissimilar between the strata. A possible explanation for this apparent exception is that the residue levels in this sample group were so low that they approached detection limits and rendered analytical outputs highly imprecise.

In all the cases in which differences were significant the outer layer carried higher organochlorine concentrations than the inner one. The difference between the inner and outer layers varied largely between compounds and sample groups, ranging from 10% to 73%. Apparently the magnitude of the .variation was not attributable to differences in species, sex, pollutant load or lipid content of the sample. For example, the outer layer of male sei whales contained 44% more DDTs than the inner one but only 10% more PCBs.

The overall variation between layers appeared greater for all the compounds of the DDT family than for the PCBs. Thus, in the sample groups in which differences proved significant, tDDT were 13-44% greater in the outer layer than in the inner one, while corresponding values for PCBs ranged only from 10% to 27%. As shown in Fig. 1, this produced differences in the tDDT/PCB ratio between strata (significant at p < 0"01 for male and female fin whales and male sei whales). In all these cases the tDDT/PCB ratio was higher in the outer layer than in the inner one.

Contrary to this, the various DDT forms distributed between strata in a similar manner, and the isomeric composition of DDTs remained very much alike in the two strata. As a consequence the DDE ratio ( p , p ' - D D E / t D D T ) , a sensitive index of DDT metabolic activity (Addison et al., 1984; Aguilar, 1984; Borrell & Aguilar, 1987), was not significantly different between the inner and outer layers in most of the sample groups (Fig. 2). Again the only exception to this general pattern were female sei whales which, as mentioned earlier, may have yielded spurious analytical results because of the extremely low levels of pollutants present in their tissues.

These observed differences in the distribution of pollutants between blubber layers are difficult to explain, but three physiological processes may be responsible for them: (i) the differential role of the blubber strata in the

Distribution of organochlorines in whale blubber 281

Fig. 1.

frequency 2 0 ] ~ Fin whele femele,s (n-59)

1 0

5

o ~

T Fin whale male8 (n-a0)

4

40 50 60 70 SO 00 100 110 120 1SO 140 2- ~"~4 _1 ]~ ~ Sel whsle female8 (n-131 I

" , , , , rl ! . . . . 8el whale, male8 (n=lO)

0 E ~ L

,o -o oo ,oo .o , . , ; o . o , ; o io , ,o ~o ~o

% tDDT/PCB

EZ~ Inner I outer

Frequency distribution of the tDDT/PCB ratio in the outer and inner strata of blubber from fin and sei whales.

fattening cycle; (ii) the heterogeneous lipid composition of blubber throughout its depth; and (iii) the different turnover rate of pollutants in the layers.

The first factor--the different role of blubber strata in the fattening cycle--may explain part of the variation in the total load of organochlorines between layers but not the differences in the proportions between organochlorines (i.e. in the ratios between compounds). The inner zones adjacent to the muscle are the sites of reception and mobilization of lipids, while the outermost external ones are responsible for thermoregulation

282 Alex Aguilar, Assumpci6 Borrell

Fig. 2.

frequency 2 5 ] 1 FEn whale ferna[es (n=,59)

20-

15'

1@

10

8

25 80 $5 40 48 50 58 80 4

3- Se[ whale female8 (n=13)

1

0

Sel whale males (n=lO)

4 7

8 -

2 -

' n D D E / t D D T

[Z~ Inner I outer

Frequency distribution of the DDE/tDDT ratio in the outer and inner strata of blubber from fin and sei whales.

(Ackman et al., 1975; Lockyer et al., 1984; Aguilar & Borrell, 1990). The two whale species studied here were sampled during the summer season, which is the time of their intensive feeding. Hence it is reasonable to expect that the inner region of the blubber had been considerably expanded because of massive fat deposition, thus producing a dilution of lipophilic pollutants and, therefore, an apparent reduction in the tissue residue levels.

A similar physiological process has been observed in other mammal species during the stages of body growth or expansion of the fat tissue, especially during infancy (Hayes, 1975; Hansen & Welborn, 1977). In seals several researchers have also observed that organochlorine residue

Distribution (ff organochlorines in whale blubber 283

concentrations correlated negatively with blubber thickness, and thus with the nutritive condition of the specimens sampled (Addison & Smith, 1974; Drescher et al., 1977; Donkin et al., 1981).

The second factor that may account for the differential distribution of organochlorines observed between layers is the heterogeneous lipid composit ion of blubber throughout its depth. Expressing pollutant concentrations in relation to the total lipid content of the tissue avoids the problem of comparing samples with different quantitative contents of lipids but does not take into account variations in lipid polarity or tissue capacity for lipophilic pollutant retention. Affinity of the different organochlorine compounds is not identical for all lipid forms and this appears to be one of the main factors responsible for the distribution of lipophilic chemicals among the various body compartments (Tanabe et al., 1981; Aguilar, 1985).

Ackman et al. (1965) and Lockyer e t al. (1984) have shown that, in fin whales, free fatty acids of shorter chain length predominate in the inner layer, while those of longer chain length are more prevalent in the outer one. This may lead to differences in the capacity for dilution of organochlorines by strata, an effect which can be noticed in the differential distribution of DDTs and PCBs between layers, as mentioned above. Because the type of PCB congeners commonly detected in fin whales have a high chlorine content and are less polar than DDTs (Chiou et al., 1977: Ernst, 1980) the difference in pollutant concentrations observed between strata would agree with a higher lipophilicity of the inner layer constituents.

The third factor which may be responsible for the differences observed between layers is the different turnover rate of pollutants in the blubber regions. The pollutants initially deposited in the blubber are transferred back and forth to blood following changes in the ingestion or excretion rates of organochlorines, The intensity of this exchange between compartments is certainly dependent on the efficiency of the circulation network in a particular region of the tissue and, since the inner layers of blubber are much better supplied with blood than the outer, the pollutants present in the first strata will be more easily transferred than those deposited in the latter. In addition, the deposition/mobilization mechanisms may be enhanced or diminished by the polarities of the organochlorine compounds, these also being potentially responsible for the variation observed in the relative proportions of chemicals between strata. Therefore the differences observed between layers may be explained by recent ingestion of low-polluted food and subsequent mobilization and reversion to blood of part of the organochlorines initially deposited in the inner layers of the blubber.

It is difficult to decide which of these three factors is most responsible for the differences observed between strata. Probably the three interact and have synergistic effects which are difficult or impossible to apportion. The dilution

284 Alex Aguilar, Assumpci6 Borrell

of organochlorines in the inner layer caused by the expansion of the basal blubber deposits during the period of hyperphagia is undoubtedly the factor which may produce changes of a greater amplitude in the absolute concentrations of pollutants. However, the variation in the relative proportion of the different chemicals suggests that polarity or the differential exchange rate with the mainstream blood also plays a significant role in the compartmentation of organochlorines within the blubber.

Implications for sampling procedures

These differences between strata, both in absolute organochlorine concentrations and their ratios, have consequences for the sampling of blubber for pollutant analyses. In general, the outer layers had levels between 30% and 50% higher than the inner ones, but larger variations may be expected under particular physiological or environmental conditions. For example, the variation between layers may probably be enhanced in stranded specimens, which are often found after having suffered a period of stringent fat mobilization and in an almost exhausted energetic condition.

Sampling of free-ranging specimens using biopsy darts shot from a vessel may also be critical. The darts commonly in use (Winn et al., 1973; Aguilar & Nadal, 1984; Lambertsen, 1987; Mathews et al., 1988) do not penetrate the whole blubber depth but rather collect a small piece of superficial blubber. This type of sampling will produce results which are not representative of the specimen's blubber pollutant load, especially in cetaceans of large body size with blubber attaining a thickness of several centimetres. None of the blubber layers is necessarily more representative of the total body pollutant burden than the others, for which reason the most suitable procedure for obtaining representative results is to analyse a sample containing an equal contribution from all the strata. When procedures used in the sample collection render this objective impossible, reservations should be expressed about the overall representativeness of the pollutant concentrations determined.

ACKNOWLEDGEMENTS

The blubber samples used in this study were collected at the factories of the whaling companies Industria Ballenera SA in Spain and Hvalur HF in Iceland. Thanks are expressed to the staffofthese companies for the facilities and co-operation given during the field work. Thanks are also extended to Esteve Grau for his assistance in the field work, especially in Iceland; to Johann Sigurj6nsson for his co-operation and support for our sampling

Distribution of organochlorines in whale blubber 285

programme; and to Christina Lockyer for assistance in the age determi- nation. Partial funding for this research was made available by the Fisheries Secretariat, Spain's Ministry of Agriculture and Fisheries.

R E F E R E N C E S

Ackman, R. G., Eaton, C. A. & Jangaard, P. M. (1965). Lipids of the fin whale (Balaenoptera physalus) from North Atlantic waters. Fatty acid composition of whale blubber and blubber sections. Can. J. Biochem., 43, 1513 20.

Ackman, R. G., Hingley, J. H., Eaton, C. A., Sipos, J. C. & Mitchell, E. D. (1975). Blubber fat deposition in mysticeti whales. Can. J. Zool., 53, 1332--9.

Addison~ R. E & Smith, T. G. (1974). Organochlorine residue levels in Arctic ringed seals: variations with age and sex. Oikos, 25, 335 7.

Addison, R. F., Brodie, E F., Zinck, M. E. & Sergeant, D. E. (1984). DDT has declined more than PCBs in eastern Canadian seals during the 1970s. Era'iron. Sci. Technol., 18, 935-7.

Aguilar, A. (1984). Relationship of DDE/tDDT in marine mammals to the chronology of DDT input into the ecosystem. Can. J. Fish. Aquat. Sci., 41, 840--4.

Aguilar, A. (1985). Compartmentation and reliability of sampling procedures in organochlorine pollution surveys of cetaceans. Res. Rev., 95, 91- 114.

Aguilar, A. (1987). Using organochlorine pollutants to discriminate marine mammal populations: a review and critique of the methods. Mar. Mamnz. Sci.. 3, 242 62.

Aguilar, A. & Borrell, A. (1988). Age- and sex-related changes in organochlorine compound levels in fin whales, Balaenoptera physalus, from the eastern North Atlantic. Mar. Environ. Res., 25, 195-211.

Aguilar, A. & Borrell, A. (1990). Patterns of lipid content and stratification in the blubber of fin whales (Balaenoptera physalus). J. Mamm., 71,544 54.

Aguilar, A. & Nadal, J. (1984). Obtenci6n de biopsias hipod6rmicas de cetficeos en libertad. Inv. Pesq., 48, 23-9.

Anon. (1985). Recommendations for procedures in surveys of organochlorine pollution in cetaceans. Rep. Int. Whal. Commn., 35, 150.

Borrell, A. & Aguilar, A. (1987). Variations in DDE percentage correlated to total DDT burden in the blubber of fin and sei whales. Mar. Pollut. Bull., 18, 70 4.

Chiou, C. T., Freed, V. H., Schmedding, D. W. & Kohnert, R. M. (1977). Partition coefficient and bioaccumulation of selected organic chemicals. Environ. Sci. Technol., 11,475 8.

Donkin, P., Mann, S. V. & Hamilton, E. I. (1981). Polychlorinated biphenyl, DDT and Dieldrin residues in grey seals (Halichoerus grypus) males, females and mother-foetus pairs sampled at the Farne Islands, England, during the breeding season. Sci. Total Environ., 19, 121-42.

Drescher, H. E., Harms, U. & Huschenbeth, E. (1977). Organochlorines and heavy metals in the harbour seal Phoca vitulina from the German North Sea coast. Mar. Biol., 41, 99-106.

Ernst, W. (1980). Effects of pesticides and related organic compounds in the sea. Helgol. Wiss. Meeresunters., 33, 301-12.

286 Alex Aguilar, AssumpciD Borrell

Hansen, L. G. & Welborn, M. E. (1977). Distribution, dilution and elimination of polychlorinated biphenyl analogs in growing swine. J. Pharm. Sci., 66, 497-501.

Hayes, W. J. (1975). Toxicology c?/'Pesticides. Williams & Wilkins, Baltimore, M D, 580 pp.

Lambertsen, R. H. (1987). A biopsy system for large whales and its use for cytogenetics. J. Mamm., 68, 443-5.

Lockyer, C. (1976). Body weights of some species of large whales. J. Cons. Int, Explor. Met, 36, 259-73.

Lockyer, C. H. (1984). Age determination by means of the ear plug in baleen whales. Rep. Int, Whal. Commn., 34, 692-6.

Lockyer, C., McConnell, L. C. & Waters, T. D. (1984). The biochemical composition of fin whale blubber. Can. J. Zool., 62, 2553-62.

Mathews, E. A., Keller, S. & Weiner, D. B. (1988). A method to collect and process skin biopsies for cell culture from free-ranging gray whales (Eschrichtius robustus). Mar. Mamm. Sci., 4, 1 12.

Murphy, R G. (1972). Sulphuric acid for the clean-up of animal tissues for analysis of acid stable hydrocarbon residues. J. Assoc. Q~." Anal. Chem., 55, 1360-2.

Tanabe, S., Tatsukawa, R., Tanaka, H., Maruyama, K., Miyazaki, N. & Fujiyama, T. (1981). Distribution and total burdens of chlorinated hydrocarbons in bodies of striped dolphins (Stenella coeruleoalba). Agric. Biol. Chem., 45, 2569 78.

Wagemann, R. & Muir, D. G. C. (1984). Concentrations of heavy metals and organochlorines in marine mammals of northern waters: overview and evaluation. Can. Tech. Rep. Fish. Aquat. Sci., 1279, 1 97.

Winn, H. E., Bischoff, W. L. & Taruski, A. G. (1973). Cytological sexing of cetacea. Mar. Biol., 23, 343-6.