PR. J.M.

BOUQUEGNEAU
Faculty of Sciences
Laboratory For Oceanology
UNIVERSITÉ de Liège October 2002
o
O

o TRACE METAL CONTAMINATION

AND DETOXICATION PROCESSES
o IN MARINE MAMMALS FROM
o EUROPEAN COASTS

o
o
o
o
o
o H arbour p o r p o is e - 1. B irks/S W F

o KRISHNA DAS

o
o
o
Dissertation presented in fulfillment for the degree of
Doctor of Sciences (Section Oceanology)

O
 PR. J.M. BOUQUEGNEAU
Faculty of Sciences
Laboratory For Oceanology
UNIVERSITÉ d e L iè g e October 2002

28445

TRACE METAL CONTAMINATION

AND DETOXICATION PROCESSES
IN MARINE MAMMALS FROM
EUROPEAN COASTS

H arbour p o rp o ise - 1 D irks/SW F

KRISHNA DAS

Dissertation presented in fulfillment for the degree of

Doctor of Sciences (Section Oceanology)
 One can meaaune the
Cfneatneeo and the monai pnoo^nece a,
nation âcf ¿oohiny. a t how ¿t tneatâ. hen
animale

'THahatma (phandi
 R e m e r c ie m e n ts

A l ’issu e de cette longue traversée du désert ponctuée d’oasis, de

soleil, de sable fin m ais égalem ent de cham eaux, de scorpions ou
de m irages, je tien s à remercier ceux qui, de près ou de loin,
m ’ont perm is d ’accéder aux rivages de l’océan.

Tout d ’abord, il n ’y a p as de th èse de doctorat sa n s promoteur de

th èse et c ’est avec sincérité et amitié que je remercie le
P rofesseu r Jean-M arie B ouquegneau. Son exigence sa n s faille,
son esprit critique, se s con seils patients ou im patients ont été le
m oteur de cette thèse.
Merci infinim ent de m ’avoir fait confiance et de m ’avoir laissé
toute la liberté voulue pour m ener à terme ce projet tout en
restant disponible pour to u s m es appels à l ’aide tant scientifiques
que financiers.

J e n ’oublie pas m es collègues et amis:

Tous m es rem erciem ents à S y lv ie Gobert pour son aide

de tou s le s in sta n ts et son hum our à toute épreuve malgré
son lourd travail de gestion du laboratoire.

R en zo B ion d o a eu la lourde et pénible responsabilité de

m ’apprendre tant l’art de I’esp re sso italien que le travail de
laboratoire, le s joies de Lotus ou le s scorpions de
Microsoft® ! Ce ne fut p as une sinécure ! Merci pour ta
patience, ton efficacité et ta gentillesse.

Merci égalem ent à R ach el B ou hy pour s a bonne hum eur,

son efficacité, s a disponibilité, et son aide inestim able
d an s le ru sh de fin de thèse. Un grand merci à D en is
V angeluw e pour son aide dan s les dosages finaux de
m étaux traces dan s les poissons.

Tout grand merci à G illes L epoint tant pour la relecture

critique de cette thèse que pour l’initiation au
spectrom ètre de m asse, sa collaboration et son
enthousiasm e pour tou s le s dosages isotopiques, nos
pau se-cafés du m atin et le s petites bières du vendredi
après-midi.

J e remercie T hierry J a u n ia u x et les étu dian ts du C éto-

Club pour avoir réalisé avec entrain et bonne hum eur, à
 m idi ou à m inuit tou tes les autopsies d es anim aux
éch ou és sur les plages belges et avoisinantes et prélevé les
échantillons et données pathologiques, à la base de ce
travail. Merci égalem ent pour tes rem arques constructives
sur les différents asp ects pathologiques de ce travail.

Un grand m erci à Ludo H olsb eek pour n o s d iscu ssion s

souvent anim ées, son esprit critique, les dosages de
mercure et la découverte de Sienne. Autant de souvenirs,
de réflexion scientifiques et de con seils qui ont jalonné et
souten u tout m on parcours depuis le mémoire de licence.

Merci à V igin ie D ebacker pour n o s échanges

bibliographiques et les toutes prem ières analyses de
m étaux lourds et m étallothionéines dans les m amm ifères
marins.

J ’en profite pour saluer le travail réalisé par les autres

m em bres du groupe MARIN : J a n H aelters, J a n
T avem ier, et T hierry J a c q u e s pour leur efficacité et leur
disponibilité dans l’organisation de la logistique des
échouages de m am m ifères m arins.

Je remercie égalem ent m es autres collègues, anciens

étudiants ou m em bres du laboratoire d ’Océanologie Jean -
Marie T h éâ te, Patrick D auby, F ab ien n e N y ssen , Nadège
D em i, F rédéric M ineur, A strid R u tte n , O livier D efaw e,
N athalie A n to in e pour leur bonne hum eur, leur aide et
leurs con seils toujours utiles.

C’e st avec grand plaisir que je remercie toute l’équipe de

HNRS Santé de Montréal pour leur accueil et leur aide
durant m on séjour dan s leur province hivernale en 1999 :
D an iel Cyr, M ichel fo u m ier, J u lie , M arleen, Sam i, A nik
S avoy et S té p h a n e P illet.

De nom breuses personnes ont marqué leur intérêt et leur soutien

à ce travail en me fournissant des échantillons de tou s les
horizons marins:

V éronique L oizeau et A lain A b am ou (IFREMER, Brest)

m ’ont accueilli avec gentillesse à différentes reprises et ont
fourni les échantillons de th on s et de dauphins de
l’Atlantique Nord.
 Thanks a lot to E m er R ogan and S im o n Berrow (Cork
College, Ireland) for providing sam ples from Irish coasts
and for their u sefu l advices on parts of th is m anuscript.

Je remercie égalem ent Gérard M auger (GECC, Cotentin)

pour avoir fourni les échantillons des côtes du Cotentin.
T hanks a lot to A lexei Birkun (Ukrainia) and C laude
J o ir is (VUB, Brussels) for providing marine mamm al
sam p les from the Black Sea.

Je remercie sincèrem ent U rsula Sieb ert (FTZ, Germany)

pour avoir contribué de m anière significative à cette thèse
en me fournissant d es échantillons de m arsouins
d isla n d e, de la Norvège, d es côtes allem andes d es Mers
du Nord et Baltique et pour avoir éclairé de son point de
vue vétérinaire m es résu ltats toxicologiques. Merci
égalem ent pour tou s tes con seils et ta disponibilité dans
l’élaboration de notre projet de collaboration Marie Curie.

Merci à C hris C oorem an et son équipe pour m ’avoir

accueilli sur le R.V. Belgica et m ’avoir aidée pour
l’échantillonnage de poisson s et invertébrés. L aure-Sophie
S c h ie tte k a tte m ’a ramené avec bonne hum eur quelques
m ollusques de se s cam pagnes en Mer du Nord. Je
remercie égalem ent toute l’équipe du T halassa (IFREMER,
Boulogne) qui, à plusieurs reprises, ont accueilli m es
étu dian ts à leur bord.

I w h ish to thank here Dr P.G.H. E van s (Sea Watch

Foundation, Oxford) for h is continuous interest in my
researches, h is m ultiple invitations to the Hebrides Sea,
h is experience and en thusiasm towards marine m am m als
in the field and the wonderful discoveiy of wild seabirds
colonies of Rona, S ula Geir and Shiant Islands, w hich will
remain one of the b est so u ven ir of my career. T hanks also
for providing the nice picture of Jan Birks w hich illustrate
the cover page.

J e dois égalem ent énorm ém ent à m es étudiants qui ont collaboré

à différentes parties de ce travail :

J u lie B ruijninckx, G aëlle M illérioux, V alérie Jacob , Arnaud

D egroof, Yann Leroy. Merci particulièrem ent à M ichaël
F o n ta in e pour n o s d iscu ssion s constructives sur la statistique
multivariée et à C ristin a B eans, notre am éricaine de service,
pour la relecture et le s corrections de l’anglais. En vous
souhaitant à to u s bonne chance pour la suite de votre parcours !

Pour leurs con seils et patience inform atiques ou statistiques, je

remercie
M ichel B ourseau et M ichèle D u pont (UDI) qui ont
répondu autant qu’ils le pouvaient à tou s m es appels à
l ’aide informatique et à M ichel F rankignoulle pour son
précieux logiciel Statistica®, u n pilier de cette thèse ! Merci
a u ssi à P hilipp e M ottet et J o ë lle S a m ed i de 1TJD Spring
pour leur aide dan s m es différentes recherches
bibliographiques et à N icole G érardin du laboratoire de
Biochimie pour se s prêts de matériel, s e s con seils avisés
concernant les m étallothionéines et sa joie de vivre !
Tout grand merci à Frédéric F a m ir du départem ent de
Génétique Quantitative en Faculté de m édecine vétérinaire
qui m ’a conseillé et initié à l’analyse multivariée.

Merci à m a p e tite s œ u r V in ita, à Isab elle, M anon,

N ath an aëlle e t M axim e, F loren ce, N athalie e t to u s le s au tres
pour vos conseils, votre soutien et votre amitié.

C’est du fond du cœ ur que je remercie m on collègue et

com pagnon de chaque in stan t Eric P arm en tier qui par son
soutien quotidien, son se n s critique, s a patience pour toutes m es
im patiences (et m es découragem ents) et son enthousiasm e pour
la recherche m ’a perm is d ’entam er et de finaliser cette traversée
du désert !

D ans to u s les se n s du terme, je ne serais p as là

aujourd’hui sa n s m es p a ren ts qui ont tout supporté depuis m es
m aladies infantiles, m es hurlem ents nocturnes, m es premiers pas
et premières ch u tes, leur dévouem ent pour que je p u isse réaliser
le m étier que j ’ai choisi, to u s leurs dépannages de fin de m ois,
leurs nom breuses bourses de voyage depuis St Malo ju sq u ’à
Vancouver en p assan t par Calvi, Montréal ou Glasgow. Du fond
du cœ ur. Merci de m ’avoir souten ue tout au long d e s m es étu des
en d ’avoir partagé m on enthousiasm e pour l’océanologie!

Ce travail a reçu l ’appui financier du Fond de la

Recherche pour l’Agriculture et l’Industrie (FRIA) et d es Services
Fédéraux pour le s affaires Scientifiques T echniques et Culturelles
(SSTC, contrat M M /D D /50)
 ACKN O W LED G M EN TS
FOREWORD
OBJECTIVES

T CHAPTER 1

G eneral in tro d u c tio n

B 1.1. The N orth S ea

1.2. M arine m a m m a ls in th e
N orth se a
7
18
1.3. Heavy m e ta ls in m a rin e
23
m a m m a ls
1.4. M etallothioneins in 46
m a rin e m a m m a ls
1.5. S tab le iso to p e ecology 60

CHAPTER 2 83

F T una and dolp h in

a sso c ia tio n s in th e N orth­
e a s t A tlan tic: E vidence o f
C d iffe ren t
n ic h e s from
ecological
stable
O iso to p e an d heavy m e ta l
m e a su re m e n ts

TS! CHAPTER 3

M arine m am m als from

103

1 th e N o rth e a st A tlan tic :

E v alu atio n of th e ir
E tro p h ic p o sitio n by S13C
a n d 81SN a n d in flu en ce on
W th e ir tra c e
c o n c e n tra tio n s
m etal

T CHAPTER 4

M arine m am m als from

127

S th e S o u th e rn N orth Sea:
In s ig h ts in th e ir feeding
ecology from 815N a n d
S13C m e a su re m e n ts
CHAPTER 5 157

T race m e ta ls in m arin e
m am m als from th e N orth
S ea a n d a d ja c e n t areas:
R elatio n w ith sta b le carbon
a n d n itro g e n , iso to p es, sex,
m e ta llo th io n e in s a n d body
c o n d itio n
CHAPTER 6 197

W hite-sided d o lp h in m e ta l­
lo th io n e in s: P u rificatio n ,
C h a ra c te risa tio n a n d po­
t e n tia l role
CHAPTER 7 209

G eneral D iscussion and

c o n c lu sio n
APPENDIX
FOREWORD
 Foreword

Marine m am m als are key elem ents in our marine ecosystem s. As

K-selected species, they are likely to have a strong effect in
structuring their environment. As top-predators, they are
potential indicators of ecosystem health (Bouquegneau et al.,
1997; Bowen, 1997; Reijnders and Aguilar, 2002).

Awareness o f the threat of environm ental contam inants to

m arine m am m als is widespread. High concentrations o f certain
com pounds in the tissu e s of th ese a n i m a l s have in the past been
associated with organ anom aly, impaired reproduction and
im m une function, a s show n by large die-offs am ong seal and
cetacean species. Indeed, recent m ass-m ortalities and stock
declining am ong several m arine m am m al populations from highly
polluted areas have am ong others been attributed to the
contam ination by organochlorines su c h a s PCBs (Reijnders and
Aguilar, 2002). Several investigations have been carried out in an
attem pt to evaluate organic contam inant effects at am bient
environm ental levels (Reijnders, 1986; Aguilar and Borrei, 1994;
De Guise et al., 1995; de Swart et ah, 1994). It h a s been
dem onstrated that sea ls fed polluted fish from D utch Wadden Sea
(Southern North Sea) show ed reduced pup production w hen
compared to th ose fed m uch le s s polluted fish from the Northeast
Atlantic (Reijnders et al., 1986). T his study w as the first sign o f a
cau sal relationship betw een naturally occurring levels of
pollutants and a physiological response in marine m am m als.

However, a clear cause and effect relationship betw een residue

levels of organic contam inants and the observed effects h a s been
dem onstrated in a few stu d ies only. A ssessing the im pact of other
contam inants su ch a s trace m etals on marine mammal
populations is even more difficult a s m etals are not strictly man-
m ade chem icals. They are driven to the ocean s by rivers,
dum ping discharges, atm ospheric in p u ts and can be found
naturally in all com partm ents of the biosphere (Clark, 2001).

The m ain reason for the lack of proof o f the im pact of pollution on
marine m am m als is the difficulty or im possibility of
experim enting in laboratory conditions with th ese anim als and
the frequent occurrence of confounding factors that ham per the
establishm ent of a direct cau sal relationship. Exam ples of these
factors are the fact that pollution alw ays occurs in a mixture of a
large num ber o f chem ical com pounds, the lack of data on
biological variables influencing tissu e levels, quality o f sam ples
analysed, the lim ited information on pathology and occurrence of

1
d isea ses in the studied specim en, the absence o f reliable
population data, and the lack o f information on the influence of
other detrim ental factors, su ch a s the im pact of fisheries and
other hum an-related sources of disturbance.

One approach to th is problem is to carry out system atic post­

mortem investigations to establish the disease sta tu s of
contam inated anim als in a relatively large num ber of individuals
from the sam e sp ecies (Hÿvarinen and Sipilä, 1984; Sieberi et ál.,
1999; Jep son et al., 2001). This com plex approach becam e the
warhorse of one group of scien tists strongly backed up by an
essen tial technical and administrative support: the MARIN Group
(Marine Animal R esearch and Intervention Network). This
m ultidisciplinary team , including veterinary pathologists, marine
bird ecologists, ecotoxicologists and oceanographers, aim s to
study the potential ca u ses of death and health sta tu s of the
populations of seabirds and m arine m am m als found stranded
along the Belgian and neighbouring coasts.

During the present work, we have considered the sources of

variation in the trace m etal concentrations of different marine
m am m al sp ecies from the North S ea and adjacent areas, a s well
a s the Black Sea: geographic origin, species, age, sex or body
condition. A special attention h a s been given to the influence of
diet usin g the stable isotope approach (813C and 815N).

The potential role of trace m etal concentrations (Zn, Ni, Cr, Cd,
Fe, Cu, Se, Hg) a s indirectly enhancing marine mammal mortality
w ithin the North S ea and adjacent areas w as also exam ined. Due
to sam pling size, the case of the harbour porpoise (Phocoena
phocoena) h a s been especially discu ssed. When available, trace
m etal levels were exam ined in relation with the general body
condition observed at necropsy. Possible detoxification processes
were investigated through the analyses of m etallothioneins in
livers and kidneys of selected harbour porpoises (Phocoena
p hocoena) and w hite-sided dolphin (Lagenorhynchus acutus).

2
 Foreword

REFERENCES

A guilar, A., Borrell, A. 1994. Je p s o n , P.D., B en n et, P.M., Allchin,

A bnorm ally hig h p olychlorinated C.R., Law, R .J., K uiken, T., B aker,
b iphenyl levels in strip ed d o lp h in s J.R ., R ogan, E., Kirkwood, J.K .
(Stenella coeruleoalba) affected by 1999. In vestigating potential
th e 1990-1992 M ed iterran ean a sso c iatio n s betw een chronic
epizootic. Sei. Total. E nviron. 154: ex p o su re to polychlorinated
23 7-24 7 bip h en y ls a n d infectio u s disease
m ortality in h a rb o u r p o rp o ises from
B o u q u eg n eau , J.M ., D ebacker, V., E n g lan d a n d W hales. Sei. Total.
G obert, S. 1997. Biological E nviron. 2 4 3 -2 4 4 : 33 9 -34 8
oceanography a n d m arin e food
w ebs: role of m arin e m a m m a ls a n d Sieberi, U., Jo iris, C., H olsbeek, L.,
seab ird s. In: (Ja u n ia u x , T., B enke, H., Failing, K., Failing, K.,
B o uquegneau , J.M . , C oignoul, F., Frese, K., Petzinger, E. 1999.
eds). “M arine m am m als, se a b ird s P otential R elation betw een m ercu ry
a n d pollution of m a rin e sy ste m s.” co n c e n tra tio n s and necropsy
P re sse s de la F acu lté de M édecine findings in C eta ce a n s from G erm an
V étérinaire de l ’U niversité de Liège, w ate rs of th e N o rth a n d B altic seas.
15-20 Mar. Pollut. Bull. 38: 28 5 -2 9 5
B owen, W.D. 1997. Role o f m a rin e R eijnders, P .J.H . 1986.
m am m als in a q u a tic eco sy stem s. R eproductive failure in com m on
M ar. Ecol. Prog. Ser. 158: 2 6 7 -2 7 4 sea ls feeding o n fish from polluted
co asta] w aters. N atu re 324: 4 5 6 -4 5 7
C lark, R.B. 2001. M arine Pollution
Oxford U niversity P ress, 2001. 237 R eijnders, P .J.H ., A guilar, A. 2002.
pages. Pollution a n d m a rin e m am m als.
Pps: 9 4 8 -9 5 7 In: P errin, W.E.,
De G uise S., M artin ea u D., B éland W ürsig, B., T hew issen, J.G .M . (Eds)
P., F o u rn ie r M. 1995. Possible Encyclopedia o f m arine m am m als.
m ech a n ism s of a c tio n of A cadem ic P ress. S a n Diego.
en v iro n m en tal c o n ta m in a n ts o n St.
L aw rence b elu g a w hales
(D elphinapterus leucas). Environ.
H ealth Persp. 103 (4): 73 -7 7
De S w art, R.L, R oss, P.S,
T im m erm an, H.H, V edder, L .J.,
T im m erm an, H .H., H eisterk am p , S.,
V an Loveren, H., Vos, J.G .,
R eijnders, P .J.H ., O ste rh a u s.
A.D.M.E. 1994. Im p airm en t of
im m u n e fu n ctio n in h a rb o r sea ls
(Phoca vitulina) feeding o n fish from
polluted w aters. Arnbio 23 (2): 155-
159
H yvärinen, H., Sipilä, T. 1984.
Heavy m eta ls a n d h ig h pup
m ortality in th e S a im a a ringed seal
p o p u latio n in E a s te rn F in lan d . Mar.
Pollut. Bull. 15 (9): 3 3 5 -3 3 7

3
OBJECTIVES
 O bjectives

The aim of the present work is to determ ine the relationships

betw een trace m etal levels and different factors su c h a s species,
geographic origin, diet, age and general body condition of the
a n i m a l s through:

- A system atic determ ination (and comparison) of trace

m etal levels (Zn, Ni, Cr, Fe, Cd, Cu, Se and Hg) in the
livers, kidneys and m u scles of different marine mammal
species, m ainly the harbour porpoise, Phocoena phocoena,
but also the com m on dolphin, D elphinus delphis, the
striped dolphin, Stenella coeruleoalba, the white-beaked
dolphin, L agenorhynchus albirostris, the w hite-sided
dolphin, L agenorhynchus acutus, the harbour seal Phoca
vitulina, and the grey seal Halichoerus grypus. These
anim als have been collected within different parts of the
Northeast Atlantic Ocean, the Irish Sea, the Southern
North S ea and the English Channel, The German Baltic
Sea, Danish, Norwegian and Icelandic coasts and the
Black Sea.

Sampling area of this study

- A system atic analysis of iso topic com position -813C and
815N- in the livers and m u scles of the above m entioned
species in order to estim ate and compare their respective
trophic position.

5
 - The study of the trace m etal (Zn, Cd, Cu and Hg)
spéciation on m étallothioneins in the livers and kidneys of
selected anim als (harbour porpoises and w hite-sided
dolphins) from the North Sea.

- The investigation of the potential relationship existing

between the trace m etal levels in the tissu e s and the
general body condition of the harbour porpoises.

The general introduction and resu lts are presented in different

chapters:

C h apter 1: The general introduction sum m arises som e

information concerning the North Sea, heavy m etals,
m etallothioneins and stable isotopes in relation to marine
m am m als.

C hapter 2: “Tuna a n d dolphin a sso d a tio n s in th e N ortheast

Atlantic: E vidence o f different ecological niches fro m sta b le isotope
an d h e a v y m etal m e a s u r e m e n ts

C h apter 3: “M arine m am m als fro m the N ortheast Atlantic :

E valuation o f their trophic s ta tu s b y S13C a n d S15N a n d influence on
their trace m etal concentrations”

C hapter 4: “M arine m a m m a ls fro m th e Southern North Sea:

insights in their fe e d in g ecology fro m 51SN a n d dl3C m e a su re m e n ts”

C h apter 5: “Trace m etals in m arine m am m als fro m th e North S ea

a n d adjacent areas: Relation w ith sta b le carbon a n d nitrogen
isotopes, sex, m etallothioneins a n d body condition”

C h apter 6 : “W hite-sided dolphin m etallothioneins: Purification,

characterisation a n d potential role”

C hapter 7: General D iscussion a n d Conclusions

6
CHAPTER 1 : GENERAL INTRODUCTION
 Chapter 1

1 .1 . THE NORTH SEA

l . l . l . O c e a n o g r a p h ic features

The Northwest continental sh elf area constitutes major fish-

producing ecosystem s in the world (figure 1.1). The North S ea is a
sem i-enclosed, epi-contiental large ecosystem and, a s defined by
the North S ea Task Force, includes the area south of 62°N (North
S ea Task Force, 1993a;b), the Scandinavian straits (The
Skagerrak and the Kattegat) and the English Channel (Ducrotoy
et al., 2000).

The North Sea (or greater North S ea a s defined by the OSPAR

convention) h a s a m ean depth o f 90 m, op en s broadly to the
Atlantic and receives low-salinity water from the Baltic through
the Kattegat and the Skagerrak, a s well as from rivers. The North
S ea basin is shallow, deepening to the North and is now heavily
sedim ented. Its w estern and northern coasts contain a variety of
rocky and m ountainous shores, sandy beaches, estuaries, and
fjords, while the British east coast is bordered by cliffs, at places
protected by pebbles, with a diversity of geological landscapes. On
the eastern side, sand beaches and d u n es prevail. The Wadden
Sea offers large interdidal areas contained behind a girdle of
barrier islands. The W adden Sea area covers ju st 1% of the total,
about half of which are tidal flats. These are important
production and n u rseiy areas for fish, birds or m arine m am m als
(Sonntag et al., 1999; De Jong et al., 1999).

The topography of the North S ea produces a circulation

influenced by the Coriolis forces. W aters entering from the north
move to the w estern coast while those entering from the Channel
move to the D u tch/B elgian coast (Turrel et al., 1992) creating a
cyclonic (anti-clockwise) circulation. Currents are guided by
topography and by bathym etry in the stratified Northern North
Sea. The southern part o f the North Sea is perm anently mixed,
while the middle and northern parts becom e stratified in
sum mer.

7
 Senera! Introduction

NORWAY

POLAND

Figure 1.1. The North Sea area as defined in the context of the North Sea
Conference (North Sea Secretariat, 1997b)

D epressed oxygen levels are found below 70 m where there is no

wind influence. Flushing tim e for the entire North S ea h a s been
estim ated a s one year to 500 days (Otto et al., 1990 quoted by
Duero toy et al., 2000).

With an annual production in the range of 3 m illion tons, the

North S ea contributes 3-4% of the world’s fish production of
approximately 100 m illion ton s per year and is considered a s the
richest area in the Northeast Atlantic. Indeed, the production of
the North Sea is associated with a relatively shallow depth and
 Chapter 1

the existence o f mixing m echan ism s transporting nutrients from

the nutrient-rich bottom layer to the nutrient-poor upper layers
of the water colum n.

Approximately 230 sp ecies of fish are known to inhabit the North

S ea ranging in size from 5 cm gobies (P om atoschistus sp.) to the
10 m basking shark (Cethorhinidae). Thirteen of th ese fish
sp ecies are the m ain targets of major comm ercial fisheries: cod,
haddock, whiting, saithe, plaice, sole, mackerel, herring, Norway
pout, sprat, sandeel, Norway lobster, and deep-water prawn
(N ephrops sp., P andalus sp.) or brown shrim p (Crangon crangon).
Norway pout, blue whiting, sprat and sandeel are predominantly
the targets of industrial fisheries where the catch is converted
into fish m eal and oil while the other species are u sed for direct
hum an consum ption (Quéro and Vayne; 1997; M uus et al., 1998;
OSPAR, 2000a; ICES, 2001b).
There are also landings from a variety of dem ersal species su ch
a s turbot, anglerfish, gurnards, lem on sole, rays, and sharks
(ICES, 2001b).

Most com m on species are typical of a sem i-enclosed shelf-sea,

although deep-water species are found along the Northern shelf
edge and in the deep-waters of the Norwegian Trench and
Skagerrak. Different assem blages are found in relation to depth
and substrate types.

In the Northern North Sea, on the slope edge, fishes are

dom inated by saithe Pollachius inrens and haddock
M elanogram m us aeglefinus, Norway pout Trisopteris esm arkii,
whiting M erlangius m erlangus, blue whiting M icrom esistius
p o u ta sso u , and cod G adus m orhua (Quéro and Vayne; 1997;
M uus et al., 1998).

In the central North Sea, at depths of 5 0-200 m, the association

is comparable but haddock dom inates, together with whiting and
cod. In shallower parts of the North Sea and the Channel,
com m on dab L im anda lim anda and whiting, together account for
alm ost h alf o f the biom ass. Horse mackerel, Trachurus trachurus
and sandeels A m m o d y te s m arinus and H yperoplus lanceolatus,
also m ake a significant contribution to eastern and southern
populations. Herring and m ackerel Scom ber scom brus show an
interesting migratory spawning, between the North S ea and
adjacent sea s su ch a s the Baltic S ea or Northeast Atlantic, where
they u se the continental slope (ICES, 2001b).

9
 Genera I Introduction

It m u st also be quoted that the North Sea is of global importance

for seabirds feeding and breeding a s 28 species with a total of
4.2 5 m illio n seabirds breed and a further six sp ecies feed here
(D unnei et al., 1990; OSPAR, 2000a).

1 .1 .2 . A n t h r o p o g e n ic im p a c t

Background

The North S ea is often considered a s one of the m ost

polluted se a s in the world (Laane, 1992; North S ea Task Force,
1993a, 1993b, OSPAR, 2000b). Indeed, approximately 185 m illion
people live in the highly industrialised countries bordering the
North Sea. An additional 85 000 000 inhabitants occupy the
Baltic catchm ent w hich flows into the North S ea (Ducrotoy et al.,
2000). D ensities o f populations differ greatly, with over 1000
inhabitants per km2 off the coasts o f Belgium and the
Netherlands to le s s than 50 inhabitants per km2 along the coasts
of Norway and Scotland. Tourism in duces large scale seasonal
m igrations to the coast, am ounting to m illions of people on a
single day.

Several large rivers (Thames, Humber, Elbe, Weser, M euse,

Rhine, Scheldt, Seine) loaded w ith contam inants (both dissolved
and on su spended particulate matter) continuously discharge
into the North S ea where they disperse according to the currents
or accum ulate locally in sedim entation areas su c h a s the
intertidal zones and estuaries (Pedersen, 1996; Radach and
Keyer, 1997).

In addition to heavy anthropogenic in p u ts of pollutants through

its m ain rivers, bu sy shipping routes and off-shore exploitations
(gas and oil), the North S ea also provided w aste incineration
areas until early 1991, w ith su b seq u en t high organochlorine
residue levels in different studied benthic and pelagic organism s
(Dethlesfen et al., 1996).

10
 Chapter 1

Figure 1.2. The North Sea contains some of the busiest shipping route in
the world (picture from UGMM website [Link]

By far the largest industry in the North Sea, off-shore

exploitations land more than 92. IO9 m3 of gas and 183. IO6 ton s
of oil per year, w hich are transported through a 10 000 km long
network of pipe-lines. Other activities, su ch as marine aggregate
extraction, contribute to up to 15 % of som e bordering country
dem ands for sand and gravel. Serious physical im pacts are
related to substratum removal and alteration of the bottom
topography, su ch a s the formation o f temporary plum es in the
water colum n, and re-deposition of material (de Groot, 1996). Re­
su sp en sion of contam inated sedim ents, like those occurring
during dredging activities, especially in harbours and their
vicinity, largely influences pollutant distribution irr the water
colum n, m aking them readily bioavailable to invertebrate
organism s (Fichet et al., 1998). The intensive u se of the North Sea
c a u se s a num ber of problem s in relation to a healthy ecosystem
and sustainable use: a series of old problems continu es to affect
the ecosystem , som etim es showing som e sign s of improvement
w hereas new problem s have also occurred (i.e. detection of new
synthetic compounds) (OSPAR, 2000b).

Fishing activities have 3 m ain effects on North Sea ecosystem s.

The first is due to direct fishing pressure. Capture of fish

and shellfish leads to mortality of both target and non-target
species. The long term effects of high fishing mortality have

11
 General Introduction

resulted in a decrease in abundance in older age groups of target

species. At various time during the la st ten years, the North Sea
stock s of cod, haddock, whiting, saithe, plaice, and herring have
dropped to or below any previously recorded level (5NSC, 1997).
The spawning stock of m ackerel h a s not yet recovered since its
collapse in the m id -1960s. In the case of cod, haddock, and
herring, recovery h a s been in evidence in recent years. However
all the major stock s of roundfish and flatfish and also the herring
stock are considered by ICES to be close to or outside safe
biological limits.

A second effect of fishery on the North S ea ecosystem s is

due to the practise of discarding w hich can significantly im pact
the ecology of scavengers and predators.

Finally, there are direct and indirect effects on benthic

com m unities through direct physical disturbance of sedim ent by
trawling a s well a s increased organic input derived from discard
fish and invertebrates (OSPAR, 2000b).

12
 Chapter 1

Contam inants within the North Sea

Considerable efforts to understand the biogeochem istry of

pollutants in the marine environm ent have been m ade over the
last decades. The fate of contam inants in the North S ea is closely
linked to their distribution betw een the dissolved and the
particulate phase. In general, dissolved su b stan ces will follow the
m ovem ents of the respective water m asses. Due to their strong
association with particulate matter, the fate of m any
contam inants in the Greater North S ea is, however, related to the
transport of (re) su spended solids. In areas of deposition,
sedim ents are loaded with contam inated particles, and it is
estim ated that 70% of the su b stan ces associated with particulate
m atter remain trapped in sedim entation area su ch a s estuaries,
the Wadden Sea, the deepest part of the Kattegat-Skagerrak and
the Norwegian Trench (Laane et al., 1998).

M etals have entered the North S ea ecosystem since historical

tim es, naturally or due to hum an activities. Pollution from metal
m ining in the Pennine ore fields h a s induced contam inant fluxes
to the North S ea for centuries. Industrial pollution and run-off
from urban areas are other important contributors o f m etal
in p u ts a s well a s atm ospheric sources.

Heavy m etals reach the North S ea via both airborne and

waterborne inputs. Inputs are also generated by som e sea-based
activities, su ch a s exploitation of offshore resources and dum ping
of dredged m aterials. Typical atm ospheric deposition levels at
around 500 km from the coast are an order o f m agnitude lower
th an deposition into coastal waters close to industrialised areas.
The riverine fluxes of m etals consist of particulate and dissolved
sp ecies but the particulate phase is predom inant for m ost
elem ents. The spatial distribution of m etals in the coastal zone,
where highest concentrations are found near fresh water outlets
and lowest in the open sea, su ggests that rivers are major
sources of trace m etals. However, the dum ping of dredged
m aterial can disturb th is pattern locally (OSPAR, 2000b).

Many stu dies either u se d sedim ents or fish es a s chronological

indicators (Jorgensen and Pedersen, 1994; Pedersen, 1996;
Callaway et al., 1998; Laane et al., 1998). A decline in the
concentration of various m etals h a s generally been observed in
the surface sedim ent in dynam ic sedim ent zones. This is due to

13
 General Introduction

m ixing with le ss contam inated suspended m atter or the

winnowing of contam inated particles.

The recent history of heavy m etal accum ulation w ithin the North
Sea can be observed in (net) deposition areas su c h a s the
Skagerrak and Norwegian Trench. In the deepest part of the
Skagerrak, Hg concentration in sedim ents have increased
significantly sin ce about 1950, while in creases in Pb and Cu
concentrations began som ew hat earlier, around 1900. In the
North Sea, atm ospheric in p u ts of Pb decreased by 50 - 65%
betw een 1987 and 1995 su ch that the dom inant source is now
considered to be rivers. Decreasing atm ospheric Pb inputs are
reported from several regions of the Northeast Atlantic and
adjacent areas and these have been attributed to the reduction in
u se of alkyl Pb derivatives in petroleum (OSPAR, 2000a,b).

Apart from the changes in the contam inant load to th ese areas
over recent decades, a num ber of other factors have influenced
the observed pattern, including variations in sedim ent transport
and erosion rates, eutrophication and environm ental changes in
the deeper part o f the Skagerrak (OSPAR, 2000a,b). However,
estuaries and their surroundings, receiving large input of m etals
from industrial sources, are still identified a s areas where metal
concentrations in sedim ents are clearly elevated. It h a s for
instance, been estim ated that between the D utch-Belgian border
and the open North S ea roughly h alf of Cd and alm ost 90 % of
the PCB loads en d s up in the sedim ent of the W estern Scheldt.

In general, risks to ecology of the North S ea from m etals appear

to stem from Cu accum ulation (mainly due to effects on the
production at lower trophic levels su ch a s bacteria and
phytoplankton), Cd and Hg (for top-predators), and Pb (on
predators of shellfish). These effects are expected to occur m ost
frequently in estuaries and in the coastal zones. They are due in
large part to the tendency of th ese m etals to bioaccum ulate in
organism s through trophic interactions (OSPAR, 2000a,b).

• C adm ium (Cd)

Atmospheric deposition of Cd is responsible for about one

third of the total Cd input into the greater North Sea while
waterborne in p u ts constitute the remainder. Total Cd input

14
 Chapter 1

reaching the Greater North Sea is estim ated between 79 and

118 t / y depending on the year or the model u sed (OSPAR,
2000a,b). Large decreases of dissolved Cd have been observed
in the North S ea th ese la st 20 years, especially in the
Southern Bight and in the D utch coastal zone, the Tham es
estuary and the Dogger Bank area (Schölten et al., 1988). In
the sedim ent, the Cd content seem s also to have decreased in
areas with previously high concentrations su ch a s the Dutch
coastal zone, the m outh of the Rhine and M euse rivers, the
Schelde estuary and the Wadden Sea. No significant decrease
w as found 2 0-70 km offshore (Laane et al., 1998). Last 20
year-trend in the biota w as exam ined in blue m u sse ls and
significant downward trends were found in blue m u sse ls and
flounder livers from the Netherlands and Norway and in cod
livers from Sw eden (OSPAR, 2000a,b ).

However, Cd concentrations in biota seldom exceed the

Background Reference Concentration (BRC w hich is assum ed
to reflect concentration before h um an contam ination or for
pristine areas) except in punctual locations su c h a s estuaries
(Tay, Forth, Seine) or Norwegian fjords (Sorfjord, due to the
presence of sm elting industries). In the Sorfjord (Norway),
there is still advice against consum ption of blue m u sse ls due
to high Cd (and Pb) levels (OSPAR, 2000b).

• M ercury (Hg)

Riverine and direct in p u ts o f Hg to the Greater North Sea

ranged from approximately 7.6 to 9 t /y in 1996. Calculated
atm ospheric in p u ts w as estim ated to 4 t /y in 1995. Combined
riverine in p u ts from the Rhine, M euse and Elbe rivers
dom inate (OSPAR, 1998). Since the m id-1980s, technical
im provem ents in production technology have lead to a
significant decrease in Hg em issions. This is also due to
important Hg em itters in the former German Democratic
Republic being closed down. Hg in water ranges from 1 ng/1
at the m outh of estuaries to 0.1 ng/1 in open seawater.
Particulate Hg accounts for over 90% o f the total Hg in coastal
w aters and decreases to le ss th an 10% in the le s s turbid open
s e a (OSPAR, 2000b). Dissolved and toxic monomethyl-Hg
concentrations m ay be more than 6 0 tim es higher in estuaries
compared to open se a (600 pg/1 vs 10-60 pg/1 respectively).

15
 Genera I Introduction

In the sedim ent the higher concentrations are found in the

estuaries of the Scheldt, Forth, Elbe, and Tham es a s well as
near-shore stations and dredged spoil disposal sites. In the
Belgian coastal area, an annual 5% decrease w as noted
betw een 1979-95 in som e sewage disposal sites.
In the biota, significant downward trends have been observed
in flounder, plaice and blue m u ssels from the Southern North
Sea.
However, blue m u sse ls from industrial areas of Norway
display high Hg concentrations with respect to BRC.
Enrichm ent factor (mean concentration at site divided by the
BRC) up to 3 can be observed near the German, Belgian and
D utch coasts in som e cod or dab species.

• C opper (Cu)

Cu waterborne in p u ts in the greater North S ea region were

estim ated around 1 1 0 0 t/y in 1996 while m odel calculations
give an estim ate of 5 6 t/y o f Cu reaching the Greater North
Sea via atm ospheric deposits (OSPAR, 2000b). No clear
decrease either for atm ospheric or waterborne Cu in p u ts have
been observed th ese last 10 years party due to the relative
importance o f diffuse sources. However a general decrease
h a s been observed in the sedim ent of the D utch coastal zone,
the m ouths of the Rhine and M euse, in offshore areas with
the highest decrease noticed for the Belgian coast with
concentrations dropping by 65% between 1990 and 1995. In
the biota, decreasing patterns have been show n for m u ssel
and fish tis s u e s from Denmark, Germany, Netherlands as
well a s in the Elbe estuary (which is related to the decline of
chem ical industries in the former GDR). Background
Reference Concentrations are exceeded in som e specific
locations su ch a s the Scheldt, the Tay and the Forth estuaries
(OSPAR, 2000b).

A general lo ss of biodiversity in seaw eeds and seagrasses a s well

a s in benthic com m unities w as observed, with a progressive but
consistent shift towards opportunistic sp ecies (Philippart, 1998 ;
Ducrotoy, 1999; Frid and Haii, 1999; Lindeboom and de Groot,
1999; Wolff, 2000a; 2000b) .

16
 Chapter 1

1 .1 .3 . P r o t e c t io n and m anagem ent

Protection and m anagem ent of the se a s and oceans have greatly

developed during the last 30 years. First concerned by the
m anagem ent of the fisheries resources and the prevention of oil
pollution, international agreem ents soon progressed towards a
global protection of the marine environment.

• In the North Sea, a first international agreem ent regarding

the oil pollution w as signed in the late 1960s (A greem ent
for Co-operation in Dealing w ith Pollution o f th e North Sea
by Oil, Bonn, Ju n e 1969).
• Soon followed the Oslo Convention (Prevention o f Marine
Pollution bu D um ping from Sh ip s a n d Aircraft, 1972)
im plem enting regulations for dum ping w aste at sea.
• The next step w as to prevent m arine pollution from land-
based sources, an agreem ent signed at Paris in 1974
(Paris C onvention for th e Prevention o f M arine Pollution by
L a n d b a se d Sources).
• Several international conferences then took place
(Bremen, 1984; London, 1987; The Hague, 1990;
Copenhagen, 1995; Lisbon, 1998) to co-ordinate, evaluate
and re-adjust th is international joint effort towards the
protection, preservation and sustainable m anagem ent of
the North S ea environm ent (Tromp and Wieriks, 1994).
• All coastal states around the North S ea have ratified the
R a m sa r Convention on W etlands o f International
Im portance. Many of these w etlands (i.e.: the Flem ish
B anks situated w ithin the Belgian area o f interest, 51-52°
N°), together with other sites, qualified a s Special
Protection A reas under the EC Directive on the
Conservation of Wild Birds, 1979 (Carter et al., 1993;
Jacques, 1995).
• In addition to the R a m sa r C onvention. Belgium h a s also
ratified the A greem ent on th e C onservation o f Sm all
C etaceans o f Baltic a n d North S e a (ASCOBANS).
• Furthermore, in accordance with the E uropean
C om m ission H abitats Directive, Belgium h a s also proposed
a large part of the w estern part o f the coast to be included
in the Natura 2 0 0 0 network a s a ‘Special Area for
Conservation’.

17
 General Introduction

1 .2 . MARINE MAMMALS IN THE NORTH SEA

The fertile w aters of the North Sea represent one major life site
for at least four different m arine m am m al species using th is
habitat for breeding and feeding: the harbour porpoise (Phoceona
phocoena), the harbour seal (Phoca vitulina), the grey seal
(Halichoerus grypus) and the bottlenose dolphin (Tursiops
truncatus). The North S ea bottlenose dolphin population is
located in the Moray Firth in Scotland and seem s to be the only
one left within the North Sea since 1937 (Wolff, 2000a; 2000b).
Table 1.1. Estimated number of seals, harbour porpoises, dolphins and
minkc whales in the North Sea (Hammond et al., 1995; OSPAR, 2000b;
Wolff, 2000a; 2000b; )

S p ecies L ocation Number

W est c o a st a n d
N orth S ea co ast, 3400
Oslofjord (Norway)
O rkney, S h etlan d 14100
H arb o u r seal
E a s t S co tlan d a n d
Phoca vitulina 1700
E a s t E ngland (UK)
W adden S ea c o a s t 7040
D a n ish Limfjord 700
K a tte g a t/ S k ag errak 6300
Norway co astlin e '2100
Grey seal UK N orth S ea c o a st 58300
H alichoerus grypus W adden S ea c o a s t 250
K attegat <25
H arb o u r porpoise
N orth S ea 268 300
Phocoena phocoena
M inke w hale
B alaena N orth S ea 7 2 0 0 -2 0 000
acutorostrata
W h ite-b eak ed /
W hite-sided
dolphin
N orth S ea 10 900
L agenorhynchus
albirostris/ L.
acutus
B ottlenose do lp h in Moray F irth
Few
Tursiops truncatus (Scotland)

The harbour porpoise is by far the m ost com m on sp ecies in the

Northeast Atlantic and the North S ea (Hammond et al., 1995).
The harbour porpoise is a sm all (2 m and 50 kg) coastal cetacean

18
 Chapter 1

found over the continental shelves of the tem perate northern

hem isphere (Read, 1999). The sp ecies is relatively short lived (few
anim als live longer than 15 years), attains sexual m aturity at age
3 or 4, and gives birth to a single offspring eveiy year, or eveiy
secon d year, in early sum m er (Read, 1990a; 1990b).

Harbour porpoise represents a major com ponent of the North

S ea food web with an estim ated annual fish consum ption of
approximately 60 000 ton s (by com parison, h u m a n s’ fish
consum ption is around 2.5 x IO6 tons) (Bjôrge and 0ien, 1990
quoted by Hislop et al., 1992). This sp ecies is know n to be
incidentally taken by fishing v e sse ls both in the North Sea and
Baltic Sea (ICES, 2001a; Berggren et al., 2002). It u se d to be a
very com m on species in Dutch coastal waters, but it h a s
disappeared from these waters since the 1960s (Addink and
Sm eenk, 1999; Wolff, 2000a; 2000b). The species seem s now
ab sent from the D utch Delta area and from the D utch and
German Wadden Sea w est of the River Elbe (Vervey and Wolff,
1981a,b). The first c en su s of m arine m am m als in the North Sea
and the Channel (SCANS) show ed con spicu ous absence of
harbour porpoises in the channel and the southern part of the
North Sea (Hammond et al., 1995). Nevertheless, the species
continu es to strand on D utch beaches, although presum ably in
m uch lower num bers than before the second World War (Addink
and Sm eenk, 1999). So, although the harbour porpoise does not
occur in inshore waters any longer, there apparently still exists a
sm all population off the Dutch coast (Wolff, 2000a; 2000b).
Moreover, the increase of porpoise strandings th ese last four
years along the Northern French, Belgian and Dutch coasts
indicates the presence of th is sp ecies in the area (Jauniaux et al.,
2 0 0 2 ).

The harbour porpoise sta tu s in the southern part of the North

and Wadden S eas rem ains unclear: th is species seem s to have
declined severely in the Wadden S ea and its inlets during World
War II and h a s rem ained rare since then (reviewed by Addink and
Sm eenk, 1999). In the beginning of th is centuiy, harbour
porpoises were found throughout the entire North Sea around the
UK and Denmark, off France and Belgium, the Netherlands,
Germany, in the southern, central and northern North Sea, the
Kattegat, the Baltic, in Sw edish and Norwegian waters, up to the
Barents S ea and the White Sea (Reijnders, 1992). Recent studies
have provided evidence that the harbour porpoise of the Southern
North Sea form a separate sub-population (quoted by Addink and
Sm eenk, 1999) and it is concluded that the southern North Sea

19
 General Introduction

porpoises should be considered separately from other

populations.

Figure 1.2. Harbour porpoise, Phocoena phocoena is the main species

stranded along the Southern North Sea coast (picture kindly provided by J.
Haelters, BMM)

The w hite-beaked dolphin (Lagenorhynchus albirostris), the

w hite-sided dolphin (Lagenorhynchus acutus) and the minke
w hale (Balaenoptera acutorostrata) occur regularly over large
parts o f the North Sea in large num ber m ainly to feed (ICES,
2001a).

W hite-beaked dolphins are generally concentrated in a band

across the North Sea between 55° and 60° N, m ostly to the w est
along the eastern British coast (Hammond et al., 1995). However,
extended m ovem ent m ight occur a s well, a s individuals have been
observed in the Southern North S ea and collected stranded on
the Belgian and Dutch coasts (Haase, 1987 ; J. Seys, pers.
comm.).

Harbour seal is one of the m ost widely distributed seal in the

world and the North Sea contains around 10 % of the world
population (North Sea Task Force, 1993a,b). C ounts of comm on
seal num bers (1994-1996) estim ate the current North Sea
population at 36 000 seals. The harbour seal and the grey seal
breed along the coast of the North Sea. An epidem ic o f phocine
distem per virus in 1988 significantly reduced their num bers but

20
 Chapter 1

by the late 1990s they recovered. Since 1989, num bers have
increased to more than 14 000 (Reijnders and Reineking, 1999).

Figure 1.3. The harbour seal, Phoca vitulina (picture from Wandrey, 1999)

Som e species occur in the North S ea on an occasional or

temporary basis. Hooded seal (C ystophora cristata), killer whale
(Orcinus orca), long-finned pilot whale (Globicephala melas),
Sowerby’s beaked whale (M esoplodon bidens), northern bottlenose
w hale (H yperodon ampullatus) and Risso’s dolphin (Grampus
griseus) regularly enter the northw est entrance to the North Sea
(ICES, 2001a).

More occasionally, the larger w hales including the sperm whale

(P hyseter m acrocephalus), se i w hale (Balaenoptera borealis), fin
w hale (Balaenoptera p h y sa lu s) and the blue whale (Balaenoptera
m usculus) also approach the border of the North Sea. The
com m on dolphin (Delphinus delphis) and striped dolphin (Stenella
coeruleoalba) frequently enter the northwest North Sea entrances
and the southern English channel water (Cam phuysen and
Winter, 1995 ; Hammond et al., 1995; ICES, 2001a).

The North S ea is a com plex ecosystem m ade of sand banks,

m udflats, sandy islands and estuaries m aking it unfavourable to
su c h oceanic sp ecies (Hammond et al., 1995). Indeed, winter-
stranded sperm w hales show an absence of food in the gut,
weight lo ss and blubber th ick n ess reductions compatible with an
extended presence in the North S ea where food is inadequate
(Jauniaux et al., 1998).

Marine m am m als have been exploited for centuries for

su b sistence purposes by hum an populations on all continents
except Antarctica. Historical evidence indicates that cetaceans
were abundant in the North S ea and in the English Channel

21
 General Introduction

during the Middle Ages and earlier. Right w hales and perhaps
gray w hales probably were hun ted regularly in th is area from at
least the ninth century onward; harbour porpoises and other
sm all cetaceans were hun ted in th is area before the sixth
century. The im pact on the populations w a s apparently localized
and sm all in scale in com parison to later industrialized
comm ercial hunting (Smet, 1981).

Although hunting h a s stopped and legal protection increased

(IWC, Marine Mammals Protection Act -USA-, A scobans,...) the
populations are currently far from being unaffected by hum an
activities, su ch a s disturbance, change in physical habitats
a n d /o r destruction of habitats, oil spill dam ages, net
entanglem ents, interactions with fisheries and environm ental
pollution. Incidental entanglem ent and mortality in fishing gear is
a global problem affecting m any sp ecies of sm all cetaceans. The
effects of by-catch are probably the m ost important adverse
im pact to sm all cetaceans, especially to harbour porpoises (De
Jong et al., 1999, OSPAR, 2000b; Berggren et al., 2002). Taking
into account abundance estim ates, estim ated by-catch, and
harbour porpoise biology, it is likely that the current by-catch
alone p oses a significant risk to the North and Baltic S eas
populations (OSPAR, 2000b; Berggren et al., 2002). In addition to
entanglem ent, an unknow n num ber of marine m am m als are also
killed by discarded or lost fishing gear worldwide (Berta and
Sum ich, 1999, Ospar, 2000b).

There appears to be an overall increase in cetacean strandings in

the North Sea w hich by hypothesis h a s been related to an overall
increase in population size for som e species, particularly sperm
w h ales (P hyseter m acrocephalus), due to their protected status.
Groups of male sperm w h ales occasionally visit the North Sea,
particularly in the period between November and March during
their southward migration, and strandings have been m ost
frequent during these m onths (Smeenk, 1997).

Occasionally, group strandings typically occur in the southern

region where the coastal topography is characterised by extensive
sandbanks, m udflats and estuaries. Fossil data indicate that,
though relatively rare, th is phenom enon h a s been occurring for
th ou san d s of years (De Sm et, 1997).

22
 Chapter 1

1 .3 . HEAVY METALS IN MARINE MAMMALS

(After: Das, K., Debacker, V., Pillet, S.,

Bouquegneau, J.M. 2002. In: Toxicology of
Marine Mammals (ed. Vos, G., Bossart, G.,
Fournier, M., O’Shea, T.) Taylor and Francis
Publishers Washington D.C. 600 pp. in press

1 .3 .1 . INTRODUCTION

During the past few decades, increasing concern about

environm ental pollution h a s led to m any investigations on heavy
m etals and their distribution in the sea, air or biological
m aterials. The distribution of xenobiotics in the marine
environm ent is not hom ogeneous and a considerable variation of
concentrations m ay occur regionally and temporally. The u se of
bioindicators offers a u sefu l alternative for pollution monitoring
studies. Marine m am m als appear to be potentially valuable
indicators of the level of heavy m etals accum ulated in the marine
environment: due to their top position in the trophic

network, their long life span and their long biological half-tim e for
the elim ination of pollutants, th ese anim als accum ulate high
levels of chem icals, su c h a s organochlorines (Tanabe et al., 1994;
Kamrin and Ringer, 1994), or heavy m etals (André et al., 1991a;
Bouquegneau and Joiris, 1988; 1992; Dietz et al., 1998).

The increased interest in studying contam inants in marine

m am m als is due to large-scale die-offs (Sarokin and Schulkin,
1992; Forcada et al., 1994) or impaired reproduction (De G uise et
al., 1995), w hich could lead to population declines of some
pinniped and sm all cetacean sp ecies in Europe and North
America, and the finding of relatively large contam inant burdens
in these anim als. In m any cases, morbillivirus infections were the
primary cau se of the disease outbreaks (Heide-Jorgensen et al.,
1992; Thom pson and Haii, 1993; de Swart et al., 1995a). These
m ass m ortalities am ong seals and dolphins inhabiting
contam inated marine areas have led to speculation about the

23
 General Introduction

possible involvem ent of im m unosuppression associated with

environmental pollution.

Controlled experim ents are unavailable to estab lish any definite

causal relation betw een th ese pollutant concentrations and any
physiological problem. Moreover, the data are alw ays difficult to
interpret because of the presence of other chem ical contam inants
and other stressors. The physiological sta tu s of th e organism s
(e.g. pregnancy, m oulting, fasting...) also m odulates the toxicity of
heavy m etals. In addition, the available m easurem ents have
alm ost all been performed on anim als found dead, w hich leaves
doubts about the general applicability of collected data at which
an effect at individual or population level m ight be expected.

Several investigations have been carried out in an attem pt to

evaluate contam inant effects at am bient environm ental levels
(Reijnders, 1986; Aguilar and Borrei, 1994; De G uise et al., 1995;
de Swart et al., 1995b; 1996). For exam ple, it h a s been
dem onstrated that sea ls fed polluted fish from the D utch Wadden
S ea show ed reduced pup production w hen compared to those fed
m u ch less polluted fish from the Northeast Atlantic (Reijnders et
al., 1986). This study w as the first sign of a cau sal relationship
betw een naturally occurring levels o f pollutants and a
physiological response in m arine m am m als.
A more recent study over a two year period by de Swart et al.
(1994; 1995b; 1996) h a s dem onstrated an im pairm ent of several
im m une param eters in harbour seals (Phoca vitulina) fed on
herring from the polluted Baltic S ea w hen compared to those fed
with fish from the Atlantic Ocean.
Among impaired param eters, natural killer cell activity plays an
important role in the first line of defence against viral infections
(de Swart et al., 1996). Moreover, those sea ls consum ing
contam inated herring accum ulated higher body burdens of
potentially im m unotoxic organochlorines than seals fed relatively
uncontam inated herring. In the latter study, heavy m etal levels
have unfortunately not been determ ined either in fish or in seals.
Possible im m unosuppressive actions of other groups of
environm ental contam inants, su ch a s heavy m etals, cannot be
ruled out. Indeed, m any laboratories and epidemiological stu dies
have dem onstrated the im m unotoxic effects of heavy m etals in a
variety of sp ecies (Zelikofif and Thomas, 1998). Direct cause and
effect links between a single kind of contam inant and possible
population declines h a s not been establish ed so far, so that many
researchers have proposed the possibility o f a synergistic role of

24
 Chapter 1

different su b sta n ces in increasing the susceptibility of affected

anim als to d isea ses or biotoxins.

In th is chapter we will focu s on heavy m etals and their possible

effects on m arine m am m als. Heavy m etals are usually divided
into essen tial (Zn, Cu, Cr, Se, Ni, Al) and n on -essential m etals
(Hg, Cd, Pb), the latter being potentially toxic even at low
concentrations. Nickel and Cu hazards to wildlife have been
extensively reviewed by Eisler (1997; 1998). W hen considering
m arine m am m als, there are lim ited data about heavy m etals,
except for Hg. Chromium, nickel and lead concentrations are
generally low, rarely exceeding a few Mg.g-1 dry weight (dw) in
m arine m am m al tissu es. No m eaningful assessm en t of their
toxicity in marine m am m als can be m ade as yet (Law, 1996).
However, investigations carried out on a ringed seal population
(Pusa hispida saim ensis) from Finland showed a clear connection
betw een stillbirth of the p u p s and nickel concentrations in the air
(Hÿvarinen and Sipilä, 1984). These authors have underlined the
considerable nickel input in the environm ent from industrial
activity in that particular area. On the other hand, Zn, Cu, Cd
and Hg concentrations often exceed several ten s fig.g 1 dw and so
will be d iscu ssed in particular a s well a s their levels in the
different marine mamm al groups, détoxification m echanism s,
potential hazards and ecological im plications.

25
 General Introduction

1.3 .2 . FACTORS AFFECTING HEAVY METAL CONCENTRATIONS

Som e reviews about heavy m etal contam ination of marine

m am m als have been published (Wagemann and Muir, 1984;
Thom pson, 1990; Kemper et al., 1994; Law, 1994; 1996). Tables
AÍ to A5 (in appendix) present som e selected concentration
valu es and respective references in O dontocetes, M ysticetes,
Pinnipeds, Sirenians and polar bears. It appears that metal
concentrations vary greatly w ithin m arine m am m als, especially
for non-essential m etals su ch a s Cd and Hg (table 1.1).

Table 1.1. Examples of maximum and minimum metal concentrations for

marine mammals. When available, the age o f the animals is given (all the data are expressed
in ug. g"1dry weight, assuming a mean water content of 75% of the tissue).

Metal Minimum Maximum

Hg 0.2 13 156
muscle liver
Pusa hispida Tursiops truncatus
Western Arctic Mediterranean Sea
Wagemann et al., 1996 Leonzio et a t, 1992
Cd 0.007 2324
muscle kidney
Pusa hispida (<ly.) Pusa hispida (8y.)
Northwest Greenland Northwest Greenland
Dietz et a t, 1998 Dietz et a t, 1998
Zn 2 4183
blubber liver
Leptonychotes weddellii (13 y.) Dugong dugong (>30 y.)
Antarctic Australia
Yamamoto et a t, 1987 Denton et a t, 1980
Cu 0.4 600
blood liver
Leptonychotes weddellii (13 y.) Tursiops truncatus
Antarctic Argentina
Yamamoto et a t, 1987 Marcovecchio et at. 1990

The large variation of th ese data (table 1.1 and AÍ to A5)

illustrates the num erous physiological and ecological factors that
m ight affect heavy m etal contamination: geographic location, diet,
age, sex, the tissu e s considered and m etabolic rates. Information
concerning m etabolic rates is largely unavailable for the different
marine m a m m a l s sp ecies so will not be d iscu ssed here. However
it m u st be kept in m ind that ingestion and assim ilation rates
differ between all the marine m am m al sp ecies in relation with
their weight and their m igration or physiological sta tu s (fasting).

26
 Chapter 1

[Link]. Geographic location

Heavy m etal contam ination sources can be both anthropogenic

and natural and distinguishing betw een the two can be very
difficult. The natural background com ponent of the input of
heavy m etals in marine ecosystem s m ay be a s important a s the
anthropogenic one, and in som e areas it appears to be the major
source. This is important because it em p hasises that marine
m am m als have been exposed to heavy m etals long before the
developm ent of hum an activities. It is e.g. the case for the
Mediterranean Sea and the Arctic, w hich are know n for their high
natural m etal levels: Hg in the M editerranean Sea and Cd in the
Arctic.

The Hg levels m easured in dolphins from the Mediterranean S ea

are higher than those encountered in dolphins from the Pacific
coasts of Japan or the Northeast Atlantic (figure 1.5) (André et al.,
1991a; H onda et al., 1983; Leonzio et al., 1992). According to
André et al. (1991b), the origin of high Hg levels observed in
M editerranean dolphins is certainly natural becau se of the large
natural sources present in the Mediterranean basin.

8000-
7000-
6000 -
5 5000-
a1
Ul
4000-
* 3000-
2000 -

1000-
0 -

M editerranean S e a Atlantic

□ m e a n 0 m axim a

Figure 1.5. Mercury levels in livers from striped dolphins (Stenella

coeruleoalba) from the Northeast Atlantic and the Mediterranean Sea
(After André et al., 1991a;1991b). Concentrations are expressed in dry weight assuming a
mean water content o f 75% o f the tissues.

The current state of knowledge of concentrations, spatial and

temporal trends of contam inants including heavy m etals have

27
 General Introduction

been extensively described in the Arctic (Muir et al., 1992; Dietz,

1996; AMAP, 1998). It seem s that Cd concentrations have always
been high in the Greenland Arctic regions a s indicated by the
lack o f obvious temporal trends in sedim ent cores, a s well as
historic hair sam ples from the 15th century from both sea ls and
Inuits (Dietz et al., 1998). Joh an sen et al. (1980) first reported
that Cd levels in tissu e s o f ringed sea ls from Greenland were
higher than previously reported for sea ls from the North Sea. Cd
seem s to accum ulate to higher levels in se a ls living in unpolluted
Arctic waters th an those taking their preys in the North S ea area,
w hich receives large inputs of pollutants. A sim ilar observation
can be m ade for porpoises (figure 1.6).

350-
300 -
250 -
1 200-
2 150-
100-
50
0
Baltic/North S e a GB w a ters S W G reenland

□ m ean m m axim a

Figure 1.6. Cd levels in kidneys of porpoises (Phocoena phocoena) from the

Baltic and North Seas, Great Britain waters and Southwestern Greenland
(Harms et at, 1978; Falconer et at, 1983 ; Paludan-Müller et a t, 1993). Concentrations are
expressed in dry weight assuming a mean water content o f 75% o f the tissues.

W agemann et al. (1996) have investigated the heavy m etal

distribution in belugas (D elphinapterus leucas), narw hals
(M onodon monoceros) and ringed seals w ithin the Arctic region.
Mean Hg concentrations in the livers of belugas and ringed seals
were significantly higher in the W estern Arctic than in the
Eastern Arctic. This com parison w as not possible for narwhals,
as they are not found in the W estern Canadian Arctic. On the
contrary, Cd a s well a s Zn and Cu concentrations in tissu e s (liver
and kidney) of belugas and ringed sea ls were higher in the
Eastern than in the W estern Arctic. These differences in metal
levels between m arine m am m als o f the Eastern and W estern

28
 Chapter 1

Arctic corresponded to the different geological settings and

sedim ent of th ese two regions (Wagemann et al., 1995; 1996).

[Link]. Routes of entry

There exist several different routes of entry of heavy m etals in
marine mammals: uptake from the atm osphere through the
lungs, absorption through the skin, across the placenta before
birth, via milk through lactating, ingestion o f se a water and
ingestion of food. Nevertheless, the major route of heavy m etal
contam ination for m arine m am m als seem s to be via feeding
(André et al., 1990a,b; Augier, 1993b; Law, 1996). Considering
that, the following d iscu ssion will m ainly refer to the diet
(including suckling) and its influence on m etal uptake.

Obviously, m ysticetes are le ss contam inated by heavy m etals

th an odontocetes and pinnipeds a s th ese are located at higher
trophic levels in the marine food web. Cd seem s to be transferred
to the highest trophic levels of the marine food chain mainly by
m olluscs, particularly cephalopods, w hich concentrate Cd in their
viscera (Honda and Tatsukawa, 1983; Bouquegneau and Joiris,
1988; Miles and Hills, 1994). Long-finned pilot w hales
(Globicephala m elas) for exam ple are know n to concentrate Cd in
relation with a preferential cephalopod diet. Elevated levels of Cd
in Pacific w alrus (O dobenus rosm arus divergens) and northern fur
se a ls (Callorhinus ursinus) have been reported in a population
from the Bering Sea, a remote area away from industrial activity
(Miles and Hills, 1994). In an attem pt to find out w hich prey may
transfer Cd to walrus, the m ost com m on preys (mainly bivalves)
found in the stom ach conten ts were analysed for their m etal
concentrations. Am ongst the bivalves analysed, M ya sp. show ed
the highest Cd content suggesting that th is prey could be a Cd
transm itter for w alrus (Miles and Hills, 1994). W agemann and
Stewart (1994) have studied heavy m etal concentrations of w alrus
(O dobenus rosm arus rosmarus) from the Eastern Canadian Arctic
in relation with their food, m ostly bivalves (Mya sp. and Serripes
sp.). These authors show ed that bivalves could also be a source of
lead for walrus, judging the correspondence betw een the high
levels of Pb in both bivalves and w alrus (Wagemann and Stewart,
1994).

However, som e high Cd levels have been reported in certain

m arine m am m als that do not eat Cd-contam inated prey (Denton
et al., 1980). Trace m etals have been determ ined in dugong,

29
 General Introduction

D ugong dugong, w hich m ostly feeds on seagrasses and algae.

High Cd and Zn concentrations have been detected in their liver
and kidney. Denton et al. (1980) have reported renal Cd and
hepatic Zn concentrations reaching respectively 309 ng g"1 and
4183 gg.g 1 dry weight while low levels of th ese m etals are found
in the seagrasses analysed. The authors suggested that the low
levels of Cu in seagrasses m ay influence intestinal absorption of
Zn and Cd in the dugong. Higher than normal Zn and Cd
absorption through the intestinal tract occurs w h en dietary Cu
intake is deficient, due to com petition betw een the m etals for
available binding sites on carrier proteins within the intestine
(Denton et al., 1980). Unlike dugongs o f coastal Australia which
display a Cu deficiency due to seagrass diet, Florida m anatees
[Trichechus m anatus) are considered to be facing the opposite
problem: Cu concentrations in the livers of Florida m anatees were
significantly elevated in areas of high herbicidal Cu usage (O’Shea
et al., 1984). M anatees m ight be inefficient at m aintaining Cu
h om eostasis in the face of dietary excess. The death of a captive
dugong w as attributed to exposure to Cu sulphate added to its
tank a s an algicide (Oke, 1967).

The position of top predators in the food web influences Hg levels

in marine m am m als as th is highly toxic m etal is biomagnified,
w hen available a s methylmercury, through the food web
(Bouquegneau and Joiris, 1988). Diets, and especially those of
marine m a m m a l s relying on fish, are responsible for Hg
contam ination (Svensson et al., 1992; Nakagawa et al., 1997).
Much higher Hg concentrations have been reported in minke
w h ales (Balaenoptera acutorostra) from Greenland (Hansen et al.,
1990) compared to Antarctic m inke w hales (Balaenoptera
bonaerensis), w h ich are several tim es le s s contam inated (Honda
et al., 1986; 1987). H ansen et al. (1990) attributed the lower Hg
concentrations found in tissu e s of Antarctic m inke w hales to
differences in trophic levels and subsequently in the Hg content
of the food items: Northern minke w h ales feed m ainly on fish
while Southern m inke w hales feed on krill. Indeed, the food web
in the Antarctic ecosystem is rather sim ple, as the major food
com ponent is the Antarctic krill. The low trophic position of the
Antarctic minke whale is reflected in the veiy low
bioconcentration factor of Hg in th is animal.

30
 Chapter 1

[Link]. Age

Cd and Hg accum ulated strongly with age in m ost marine

m am m als tis s u e s analysed (e.g.: H am anaka et al., 1982; Honda
and Tatsukawa, 1981; 1983; Honda et al., 1983; Augier et al.,
1993b). This increase is enhanced in the liver in w h ich the
excretion rate m ight be extremely low due to the fossilisation of
Hg under a detoxified form (see below). However, som e exceptions
have been reported in the literature. Honda et al. (1986; 1987)
have studied the heavy m etal distribution in Antarctic minke
w hales, and compared it to their food habit and age. The age of
th ese w hales varies between 1 and 45 years. They found
m axim um concentrations of Cd and Hg in the livers of 20 year old
m inke whales. Both concentrations increase with age u n til about
20 years and thereafter decrease. B ecause there is no evidence
that half-lives o f Cd and Hg change with age, H onda et al. (1986;
1987) suggested a higher food intake for the younger anim als
compared to older. They also suggested that these changes may
be due to the significant decrease in stock s of blue w hales
(Balaenoptera m usculus), and fin w hales, (Balaenoptera p h ysa lu s).
Both species u sed to occupy ecological n ich es similar to those of
the minke w hales in the Antarctic m arine ecosystem s. The young
m inke w hales would be le s s exposed to feeding com petition from
blue and fin w hales, than the older ones. A ccum ulations o f toxic
m etals su ch a s Cd or Hg may also have been influenced by this
ecosystem disruption (Honda et al., 1986; 1987).

High Cu and Zn concentrations have also been observed in very

young anim als and neonates (e.g.: Ju lsh am n et al., 1987;
W agemann et al., 1988; Caurant et al., 1994). These essential
elem ents are known to increase in tissu e s undergoing rapid
developm ent and differentiation (Baer and Thomas, 1991). It h a s
been suggested that these higher levels encountered in young
m ight reflect a specific requirem ent in newborns or a very low
excretion rate o f th ese m etals by the foetus (Wagemann et al.,
1998).

[Link]. Sex
Reproductive activities su ch a s pregnancy, parturition and
lactation can modify m etal levels. Several stu d ies have
dem onstrated that m etal transfer from fem ales to p u p s occurs
through the placenta or lactation: H onda et al. (1987) reported a

31
 General Introduction

hepatic Fe, Co, Pb and Ni transfer from m other to pup. As a

consequence, th ese m etal concentrations decrease in the mature
fem ale with progress of gestation.
Canella and Kitchener (1992) found significantly lower levels of
Hg in pregnant and lactating sperm w hales (Physeter
m acrocephalus) w hen compared with non-breeding fem ales. They
suggested that th is may be due to horm onal changes or stress
causin g the redistribution of Hg in body tissu es.

[Link]. Distribution within organs

The pattern of site distribution of m etals within the organism is
tissu e and m etal specific. For example, Hg is m ostly concentrated
in the liver, with kidney and m uscle having su ccessively lower
levels. On the contrary, the highest Cd concentrations are usually
encountered in kidney due to the presence of m etal binding
proteins. This pattern prevails in m ost m arine mamm al
concentrations (Wagemann and Muir, 1984).

Yamamoto et al. (1987) have studied the distribution of heavy

m etals in the whole organism o f three Weddell seals
(Leptonychotes weddeli) from the Antarctic. T hese authors
estim ated the whole m etal body burden, w hich w as calculated
from the weight o f the different tissu e s and their respective
concentrations. If whole body burdens o f m etal are estim ated for
an adult Weddell seal, Zn is m ostly located in m u scles and in
bones, Cu in m u scles and liver, Hg in liver and m u scles and Cd
in fiver and kidney. The apparent contrast betw een the low
concentration in Hg generally m easured in m arine mammal
m u scles and the high burden encountered is due to the high
m uscle m a ss of th ese anim als. The redistribution of Hg from
highly contam inated organs su ch a s fiver or kidney through
m u scles seem to be a protection m echanism against Hg toxicity
(Cuvin-Aralar and Furness, 1991).

Skin contains generally low Hg concentrations (Yamamoto et al.,

1987). However, the sk in of marine m am m als is not a
hom ogenous tissu e. It co n sists of four distinct layers in w hich Hg
in creases progressively outw ards with a concentration of 6 pg.g-1
(estim ation in dry weight) in the outerm ost layer of Arctic belugas
and narwhals. During the process of m oulting, the last and the
underlying are sh ed and approximately 20% of the total Hg in the
skin is lost annually in belugas and 14 % in narwhals
(Wagemann et al., 1996).

32
 Chapter 1

1 .3 .3 . TOXICITY OF MERCURY AND CADMIUM

As quoted above, the accum ulation through the food chain is a

major risk for top predators. The accum ulation of chem ical
su b sta n ces m ay result in toxic concentrations in organism s
(secondary poisoning) even if the concentration in the
environm ent rem ains below the threshold level for direct toxicity
(Nendza et al., 1997). The finding of high concentrations of m etals
su c h a s Cd or Hg have raised questions about their toxicity.

Reliable toxicity data for predatory m arine m am m als are scarce.

Instead, threshold levels are often extrapolated from terrestrial
species, i.e. interspecies correlations are assum ed to hold for rats
or captive seals. The validity of th ese extrapolations is highly
questionable and can only be justified by the current lack of
better data and by ethic considerations. Indeed, potential effects
of toxic m etals cannot be tested in free-living anim als because
experim ental m anipulations are undesirable. In vitro
experim entations or system atic post-m ortem investigations to
establish the disease statu s of contam inated anim als in a
relatively large num ber of individuals from the sam e species are a
complem entary and valuable alternative way to understand the
num erous processes involved.

[Link]. Mercury toxicity

Hg exists in several interchangeable forms in the biosphere
(Kaiser and Tölg, 1980), but Hg accum ulation through the food
web mainly occurs under an organic form, methylmercury
(MeHg), a s a result of its lipid solubility and preferential
assim ilation during Zooplankton grazing (Mason et al., 1995).
Above Zooplankton, organic Hg is biomagnified along the food
chain up to marine m am m als.

Total Hg concentration is a poor indicator of toxic effects, as

organic Hg com pounds seem to be considerably more toxic to
anim als than inorganic Hg. The biological and toxicological
activity of Hg depends on the form that is taken up, the route of
entry in the body (skin, inhalation or ingestion), and on the
extent to w hich Hg is absorbed (Kaiser and Tölg, 1980). Wolfe et
al. (1998) have recently reviewed the toxicity of Hg on wildlife.

33
 General Introduction

In m a m m al s , m ethylm ercury toxicity is primarily m anifested as

central nervous system dam age including sen so iy and motor
deficits and behavioural impairment. Anim als becom e anorexic
and lethargic. Methylmercury is easily transferred across the
placenta (Wagemann et al., 1988) and th u s concentrates in the
foetal brain (Wolfe et al., 1998). This reproductive effect ranges
from developm ent alterations in the fœ tu s to fœtal death.

Methylmercury is also absorbed by the gastrointestinal tract

while inorganic sa lts of Hg are le ss readily absorbed. It is mainly
a s the m ethylated form that Hg is absorbed from fish, since
alm ost all the Hg present in fish is m ethylated (Svensson et al.,
1992).

Experimental seal intoxication h a s led the anim als to lethargy,

weight lo ss and finally death (Ronald and Tessaro, 1977). Four
harp sea ls (Pagophilus groenlandicus) were intoxicated with
methylmercury by a daily oral intake. Two seals were fed with Hg
d oses of 0.25 [Link]-1 of body weight per day for 6 0 and 90 days.
T hese two seals did not sh ow abnormal blood concentrations but
exhibited a reduction in appetite and consequent weight loss. Two
others sea ls fed 25 [Link]-’ of body weight per day died on day 20
and day 26 of exposure. The m easurem ents of blood param eters
indicated toxic hepatitis, urem ia and renal failure. These
pathologies have been related to high accum ulation o f Hg in these
tissu es. The liver concentrations reached more th an 500 n g .g 1
dry weight after the death. Almost 90% of the Hg analysed in the
liver w as methylmercury. No detoxification m echan ism s were
described in th is case.

However, th is experim ental study did not reproduce the real daily
food intake of marine m am m als in the wild. Nigro and Leonzio
(1993) have calculated a m ean daily food for sm all cetaceans of
approximately 3 kg of fishes and cephalopods with an average Hg
concentration of 0.3 m [Link]-’ fresh weight, the m ean dietary Hg
intake for an adult specim en can be estim ated at 0.9 m g Hg per
day for the whole animal. It is quite far from the 25 [Link]-’ of
body weight and per day adm inistrated in gel caps to those seals.
Another feature that m ight explain the absence of detoxification
is the absence of selenium added to the food. In the wild, if fish
are an source o f exposure to Hg, they are also a source of
selenium (Svensson et al., 1992) and in marine m am m als
dém éthylation m echan ism s occur in the presence of selenium
(see below). In th is experim ental study, the lack of additional

34
 Chapter 1

selenium in the diet w as probably the limiting factor to

detoxification.

V eiy few stu d ies have tried to link m etal concentrations

m easured in free ranging marine m am m als and health statu s
(Hÿvarinen and Sipilä, 1984; Rawson et al., 1993; 1995; Sieberi
et al., 1999; B ennet et al., 2001). Only one case of Hg toxicóse
h a s been reported by Helm inen et al. (1968): the ringed seal
su sp ected of Hg intoxication w as from an area o f heavy industrial
Hg dumping.

Chronic Hg accum ulation w as associated with liver abnorm alities

observed in stranded bottlenose dolphins from the Atlantic. Large
deposits of a brown pigment, identified a s lipofuscin, in the portal
areas of the liver were observed in the livers of nine anim als with
high hepatic Hg levels (> 6 0 Mg/g fresh weight). Analytical
electron m icroscopy carried out on th ese pigm ents dem onstrated
that HgSe w a s the predom inant m aterial (Rawson et al., 1995).
Lipofuscin is believed to be derived from dam aged subcellular
m em branes. T his pigm ent accum ulation strongly correlates with
Hg concentrations. Hg would have inhibited the activity of
lysosom al digestive enzym es and therefore, reduced degradation
of proteins. This h a s led to excessive accum ulation of lipofuscin
w ithin cells and finally cell death. (Rawson et al., 1993).

More recently, Sieberi et al. (1999) exam ined the possible

relationship betw een Hg tissu e concentrations and disease in
harbour porpoises from the German waters o f the North and
Baltic Seas. A higher Hg content h a s been m easured in organs of
the harbour porpoises from the North S ea compared to those of
the Baltic Sea, indicating that Hg is a more important threat for
anim als of the North S ea than for the Baltic Sea. High Hg
concentrations were associated with a prevalence of parasitic
infection and pneum onia.

Bennet et al. (2001) have also u sed th is indirect approach to

investigate the prediction that increased exposure to toxic m etals
resu lts in lowered resistance to infectious disease in harbour
porpoises from the coasts of England and Wales. Mean liver
concentrations of Hg, Se, Hg:Se ratio, and Zn were significantly
higher in the porpoises that died of infectious d isea ses (parasitic,
bacterial, fungal and viral pathogens su ch a s pneum onia),
compared to porpoises that died from physical traum a (most
frequently entrapm ent in fishing gear). Liver concentrations of Pb,
Cd, Cu, and chrom ium did not differ betw een the two groups.

35
 General Introduction

In som e cases, balances between elem en ts seem to be more

important than the absolute concentration w hen the possibility of
toxic effects are considered. High premature birth rates have been
observed and studied between 1968 and 1972 in the Californian
S ea lion (Zalophus californianus) from the southern California
Channel Island rookeries (Martin et al., 1976). T hese premature
p u p s were ataxic, had difficulties in breathing and died shortly
after birth. Heavy m etals were analysed and compared between
norm al and premature pup s and betw een their respective
m others. The resu lts revealed that severe im balance in the
Hg:Se:Br occurred in the livers of the abnormal m others. The
absolute concentrations did not seem to be involved in th is case
a s Hg, Se, and Br were in higher concentrations in the livers of
norm al m others compared to abnormal m others. This su ggests
that the Se:Hg balance is a veiy com plex phenom enon and might
be more important for general health sta tu s than absolute
concentrations.

Som e in intro stu d ies have also been realised to evaluate the
potential hazard o f Hg in marine m am m als. Freem an and
Sangaland (1977) dem onstrated that m ethylm ercuiy alters the in
vitro syn th esis o f steroid horm ones w hich play an important role
in reproduction. G enetic effects of methylmercury on lym phocytes
of one bottlenose dolphin (Tursiops truncatus) have also been
evaluated in vitro by Betti and Nigro (1996). Lymphocytes were
isolated from blood obtained from a 15 year old dolphin (Adriatic
Sea). M ethylm ercuiy in duces DNA single-strand breaks and
cytotoxicity in a dose-dependent m anner. The d o se s of MeHg
u sed in th is study are likely to be in the range of concentrations
(between 1 and 10 ng/ml) naturally occurring in the blood of wild
dolphins found in the Mediterranean S ea (Betti and Nigro, 1996).
It appears that dolphin lym phocytes have a greater resistance
both to the genotoxic and cytotoxic effects of MeHg when
compared to hum an or rat cells. This feature can be interpreted
as an adaptation acquired by dolphins to counter the
m ethylmercury exposure.

[Link]. Cadmium toxicity

Cd is considered a s one of the m ost toxic m etals. High dietary
concentrations of Cd in h u m an s can lead to well know n heavy
skeletal deformities (‘itai-itai’ disease), kidney lesio n s (mainly on

36
 Chapter 1

the proximal tubules) usually preceding lung dam ages,

dysfunction of cardiovascular and hem atopoietic system as well
as carcinogenic, m utagenic and teratogenic effects (Förstner,
1980; Lamphère et al., 1984; Jonnalagadda and Prasada Rao,
1993). Effects of Cd on marine ecosystem s have been recently
reviewed (AMAP, 1998). The renal concentrations can reach levels
a s high a s 2 0 0 0 ng.g-1 dry weight in som e Arctic ringed seals
(Dietz et al., 1998). This is m uch higher th an the critical
concentrations o f approximately 800 ng.g-1 dry weight (200 jig.g 1
fresh weight) associated with kidney damage in m am m als
including h u m an s (WHO, 1992). Moreover, following Elinder and
Järup (1996), th is critical concentration h a s been largely
overestim ated a s Cd-induced renal dysfunctions have been
displayed within order of kidney cortex concentrations of 200
jig.g 1 dry weight (50 ng-g"1 fresh weight). For com parison, in
h um an adults, the renal Cd concentrations am ongst non-
sm okers is le ss than 5 Mg-fT1 fresh weight (Pesch et al., 1989). No
obvious Cd toxic effect h a s been registered till now in marine
m am m als despite the high levels encountered in several species
suggesting highly efficient detoxification m echan ism s (Dietz et al.,
1998).

1 .3 .4 . DETOXIFICATION MECHANISMS

The exposure of marine m am m als to heavy m etals h a s occurred

throughout their evolutionary history, during w hich they have
developed m echan ism s either to control the internal
concentration of certain elem ents or to m itigate their toxic effects.
The m ost obvious case is the one of Hg in dolphins.
Compared to other term inal consum ers like tu n a s or seabirds,
som e marine m am m als accum ulate m uch higher levels of Hg with
biomagnification factors in respect to prey of 500 in dolphins
compared to e.g. 30 in predatory fish (Leonzio, 1996). This can be
explained by physiological differences not only in the involved
uptake and release, but also - and som etim es m ainly - in
detoxification processes.

[Link]. Mercury detoxification

W agemann and Muir (1984) found Hg and selenium
concentrations reaching up to 510 jrg.g-' fresh weight

37
 General Introduction

(approximately 2 0 0 0 ng-g“1 dry weight). Despite su c h extremely

high values, the anim als did not show any overt sign s of Hg or
selenium poisoning because the presence of the two elem ents
together provided protection to the animal. Many stu d ies have
dem onstrated the m utual antagonism between Hg and selenium
(Pelletier, 1985; Cuvin-Aralar and Furness, 1991). This h a s
becom e one of the strongest and m ost general exam ples of
interactions betw een heavy m etals. This phenom enon occur
throughout the anim al kingdom from oysters, shrim ps to marine
m a m m a l s and hum an beings. Koeman et al. (1973; 1975) first
reported the strong correlation between Hg and selenium in fivers
of m arine m a m m al species. A molar ratio Hg:Se o f approximately
1 h a s been observed suggesting Hg detoxification m echan ism s in
presence of selenium .

Different forms o f Hg coexist in the environment. M ethylmercuiy

is know n to be one of the m ost toxic. Hg is transferred up to
m arine m a m m a l s in a m ethylated form. However, very sm all
am ounts of m ethylm ercuiy are generally found in the fiver of
marine m a m m als : le s s than 10% of Hg is present in a m ethylated
form in the fivers of adult m arine m am m als. Hg also occurs in an
inorganic form (Wagemann et al., 1998) w hich im plies that a
dém éthylation process occurs (Joiris et al., 1991). The fate of this
inorganic Hg h a s been mainly elucidated by histological studies
carried out in fivers from specim ens of Cuvier’s beaked whale
(Ziphius cavirostris) and bottlenose dolphins (Martoja and Viale,
1977; Martoja and Berry, 1980). These authors first observed
granules com posed of mercuric selenide (HgSe). Successively,
sim ilar granules were also described in the striped dolphin
(S tenella coeruleoalba). Hg and selenium occurred a s dense
intracellular granules, located m ainly in the fiver m acrophages,
the Kupfer cells, and in the proximal tu b u les of the kidney. These
granules appear a s spherical or polygonal particles ranging from
15 to 80 Á (Augier et al., 1993a; Nigro and Leonzio, 1993; Nigro
and Leonzio, 1996).

More recently, Rawson et al. (1995) found HgSe c iy sta ls in both

the liver and respiratoiy system of the bottlenose dolphin and
short-finned pilot whale and reported HgSe in the lung and hilar
lym ph nodes associated with soot particles. In both the fiver and
hepatic lymph nodes, these crystals were small, averaging 50Á. In
the lung and hilar lym ph nodes, the c iy sta ls were m uch larger,
m easuring 2 5 0 -5 0 0 Â. Abundant carbon w as present in the hilar
nod es while only very sm all am ounts were found in the hepatic
nod es and in the fiver. These findings suggest an alimentary and

38
 Chapter 1

a respiratory entry for Hg in cetaceans: Hg in the liver is likely to

be trophicaUy acquired, passing through the gastrointestinal tract
and carried to the liver by the way of portal veins. In the liver, it
m ay be converted into HgSe accum ulating a s an end product
(Martoja and Berry, 1980). In anim als producing large am ount of
HgSe, some of th is m ay be carried to the hepatic lymph nodes
and even to the spleen (Rawson et al., 1995). HgSe in the lungs
and the hilar nodes appears to be closely associated with carbon
suggesting an atm ospheric association betw een th ese elem ents.
Indeed, Hg and selenium pollution is largely attributed to the
burning of fossil fuel or w aste incinerations and th ese elem ents
tend to aggregate a s particles (Rawson et al., 1995). However, in
vivo precipitation of HgSe into the surfaces of inhaled soot
particles cannot be ruled out and further investigations are
needed to understand this lung accum ulation process.

These mercuric selenide granules seem to be the last step of a

very efficient detoxification m echanism leading to high but non­
toxic concentrations in the organs. The 1 molar ratio h a s been
generally observed in marine m am m als. However, the Hg:Se
molar ratio found in the liver can vary within the range of 0.2
(Hansen et al., 1990) to 2.49 (Caurant et al., 1996).

Palmisano et al. (1995) have explained th is variation: Hg:Se molar

ratio of approximately 1 h a s been observed in striped dolphin
livers only after a certain threshold in the total Hg concentration
(approximately 100 ng Hg.g-1 fresh weight) h a s been exceeded.
Palm isano et al. (1995) have proposed a two-stage m echanism for
the dém éthylation and accum ulation process. At low Hg levels
(first stage) the m etal is retained m ainly in its m ethylated form. At
higher Hg levels (second stage) dém éthylation, w ith a concurrent
accum ulation of Se, seem s to be the prevailing m echanism .
Moreover, above the threshold only a sm all fraction of Hg is
present in a labile form a s Hg ++ and MeHg* bound to the cysteine
residue. These authors have determ ined that 63% of the total Hg
analysed in the liver of one dolphin is involved in the formation of
a very insoluble selenocom pounds, certainly present a s HgSe
(tiemannite) but in addition, Hg can be involved in the formation
of other seleno-com pounds a s H g-selenoproteins.

This hypothesis of a threshold h a s also been suggested in other

stu d ies dealing with Hg spéciation (Sanpera et al., 1993; Caurant
et al., 1996). Sanpera et al. (1993) have found no decrease in the
organic Hg fraction with increasing total Hg concentration in the
livers of fin w hales (Balaenoptera p h y s a lu s ).

39
 &emra\ Introduction

On the contrary, Caurant et al.(1996) found in livers o f longfinned

pilot w hales a decreasing correlation between organic Hg and
total Hg. Organic Hg w as lower th an 2% w hen the total Hg
concentration w a s higher than 100 (ig-g-1 fresh weight (400 Mgg 1
dry weight). Only a sm all fraction (<1%) of total Hg is bound to
heat-stable com pounds (which include the m etallothioneins).
These heat-stable proteins are able to bind bivalent m etals and
are believed to participate in heavy m etal detoxification processes
(see paragraph 1.4.). More than 90% of the Hg w a s in the
insoluble fraction except for two anim als. In th ese two
individuals, insoluble Hg in the liver w a s < 90% and 14% and
35% of Hg were bound to m etallothioneins. These individuals
exhibited total Hg concentrations <50 gg.g 1 fresh weight (200
Hg.g-1 dry weight) in the livers. W hen total Hg w as higher than
th is value, the percentage bound to m etallothioneins w as low
always < 1% whatever the total Hg concentration (Caurant et al.,
1996).

In som e species, Hg concentrations can be low during the entire

life span. Fin w h ales feed at the bottom of the trophic web
(Sanpera et al., 1993) and their Hg levels stay relatively low. In
their livers, the m ean ratio of organic/ total Hg is about 40%. This
result seem s very high compared to other data (see table 1.2). The
authors suggest that dém éthylation is carried out at a constant
rate, probably b ecause concentrations remain low throughout
their life span, betw een 50 and 500 gg.g"1 dry weight (Sanpera et
al., 1993). It seem s in th is case that the specific threshold h as
not been reached and so high percentages of organic Hg are
observed in the livers.

Young marine m a m m a l s can display a high percentage of

m ethylm ercuiy (table 1.2) suggesting that they do not reach a
specific threshold (Palmisano et al., 1995). A second hypothesis is
that young individuals are still unable to dem ethylate Hg
efficiently (Caurant et al., 1996).
 Chapter 1

Table 1.2. Comparison of mean percentage (%) methvlmercury to total

mercury in suckling marine mammals compared to adults.

Species Pups Adults Sources

Ringed seal 70 14 Wagemman et al., 1988

Pagophilus groenlandicus
Striped dolphin 45 2.5 Itano et al., 1984
Stenella coeruleoalba
Long finned pilot whale 55 17 Caurant et al., 1996
Globicephala melas

[Link]. Cadmium detoxification

As quoted above, the concentrations of Cd in kidneys of marine

m am m als can reach levels more than twice the critical
concentrations of approximately 800 g g .g 1 dry weight (200 g g /g
fresh weight) associated with kidney damage in terrestrial
m am m als including h u m an s (WHO, 1992). This raises questions
about the health sta tu s of su ch heavily Cd contam inated marine
m am m als. Dietz et al. (1998) have compared low and high Cd
contam inated kidneys from ringed seals from Northwest
Greenland in an attem pt to do m acroscopic and light microscopic
exam inations. No differences in renal morphology could be
observed betw een experim ental groups. These investigations
indicate that m arine m am m als appear to be able to m aintain
considerable concentrations of Cd w ithout show ing renal damage.
Dietz et al. (1998) have therefore postulated that ringed seals are
adapted to the naturally high Cd levels of the Greenland Arctic
regions.

Metallothioneins

Marine m am m als m ight m itigate the toxic effects of Cd

through binding to sm all specific proteins called m etallothioneins
(MTs) (reviewed by D as et al., 2000). The presence of these low-
m olecular weight proteins h a s been dem onstrated in m any
organism s ranging from blue green algae to sperm w hales (Kägi,
1991; Holsbeek et al., 1998).

41
 (Senera! Introduction

MTs are involved in general essential m etal h om eostasis and non-

essential m etal detoxification. The role of m etallothioneins in
m arine m am m als h a s been recently reviewed (Das et al., 2000)
and constitutes the sixth part of the general introduction of th is
work (see section 1.4. for further inform ation on
m etallothioneins).

Cadmium spherocrystals

Cd-containing granules have been observed in the kidney

of two w hite-sided dolphins (Gallien et al., 2001). These two
individuals with high Cd concentrations exhibited electron dense
m ineral concretions of diam eters up to 300 nm in the basal
m em branes of the proximal tubule. These spherociystals are
m ade up num erous strata of m ineral deposits of calcium ,
phosphorus and Cd. Cd h a s been detected with a molar ratio of
Ca:Cd of 10:1 in the middle of th ese concretions.
The occurrence of m etal-containing granules is well docum ented
in invertebrates (Sim kiss, 1976) but th is is the first report of
granules containing Cd in wild vertebrates. In th ese marine
m am m als exhibiting high levels of Cd, these granules could
constitute a m ean s of im m obilisation and detoxication.

42
 Chapter 1

1 .3 .5 . LIMITS TO DETOXIFICATION AND CONCLUSIONS

Remarkable tolerance of m arine m am m als to heavy m etals h a s

been suggested through several detoxification p rocesses su ch as
tiem m anite storage and binding to m etallothioneins, but is there
a limit to the detoxification process and if so, w hat is the actual
hazard of heavy m etals?

The ratios between different m etals appear more important than

their absolute concentrations (Martin et al., 1976; Becker et al.,
1995). Pups are more affected by th ese m etals a s they exhibit a
higher m ethylmercury ratio compared to total Hg due to their
poorly efficient detoxification m echanism (Wagemann et al.,
1988). Moreover, a depressed molar ratio of Br:Hg:Se in
premature p u p s of California se a lions w a s suggested to be a
m ain death factor (Martin et al., 1976).

Caurant et al. (1996) have proposed that detoxification of Hg

could be lim ited in lactating longfinned pilot w hales . Indeed,
compared to other fem ales, Hg concentrations were m uch higher
in lactating fem ales, while selenium concentrations were lower.
Squid is the major food item o f pilot w hales, but a greater
quantity and variety of fish species have been observed in the diet
o f lactating fem ales. The authors suggested that the energy value
of fish is higher than squid and a higher consum ption of fish
would cover the increasing need of energy to produce milk. The
percentage of seien d es (inorganic form of selenium ) and the
inorganic forms of Hg seem to be higher in sq u id s th an in fish
m u scles where m ethylated Hg is dom inant. Most o f selenium
found in fish occurs in an organic form, w h ich is le s s efficient
th an seien d es in preventing the toxicity of Hg. The different diet
of lactating fem ales could induce both higher levels o f Hg and a
lower efficiency of the detoxification process (Caurant et al.,
1996).

Binding of Cd to m etallothioneins could also be a lim iting factor

to detoxification (Bouquegneau et al., 1997). In debilitated sperm
w h ales stranded on the Belgian coast, only a sm all am ount o f Cd
w as bound to m etallothioneins (Bouquegneau et al., 1997;
Holsbeek et al., 1999). As quoted above, th is could explain the
observed debilitation w hich could have favoured th ed stranding.
Precipitation of Cd under a granule form could also lead to some
toxic effects (Gallien et al., 2001). The authors underlined the fact
that lesion s could be associated to these granules especially in

43
 General Introduction

older anim als exhibiting high Cd concentrations and if so,

detoxification processes could lead to som e toxic effects.

We have to keep in m ind that any detoxification process h a s a

cost for the cell or the organism involved and m ight have a
threshold. This threshold can not be fully defined in term s of
tissu e concentration because of the num ber of param eters that
can interact to lim it physiological pathw ays that lead to
detoxification. For exam ple, gender and horm onal activity can
m odulate the syn th esis o f m etallothioneins (Blazka and Shaikh,
1991). Moreover, detoxification p rocesses can lead to the
formation of com pounds w hich could have toxic effects. The
accum ulation and degradation of Cd-m etallothionein complex
(CdMT) in the renal tubular epithelial cells can induce
nephrotoxicity in m ice counteracted by Zn w hich h a s a protective
effect against th is CdMT-induced nephrotoxicity (Liu et al., 1996;
Tang et al., 1998). As a result of their physiological function in
the h om eostasis o f essen tial m etals, m etallothioneins could be
involved in m any cellular pathways. Thus, they could modulate
physiological p rocesses a s an indirect effect o f heavy m etal
exposure. For exam ple, m etallothioneins have been dem onstrated
a s potential m odulators of som e param eters of the im m une
response (Leibbrandt et al., 1994; Borghesi et al., 1996) .
Detoxification pathw ays could therefore lead to more subtle toxic
effects underlying the complexity of the toxic effects of heavy
m etals.

To conclude, the actual toxic effects of heavy m etals on marine

m am m als remain unclear. Are they responsible - even in part -
for the decline of som e marine m am m al species? As quoted
above, that decline is obviously multifactorial: past overfishing,
present increasing hum an activities, accum ulation of pollutants
am ong w hich heavy m etals can not be neglected. The role of
m arine m am m als on the whole marine ecosystem is still poorly
understood: their contribution in the recycling of nutrients is not
very important, but their part in structuring marine com m unities
and in modifying benthic habitats are more and more evident
(Bowen, 1997). Marine m am m als presently consum e at least
three tim es greater quantity of prey than do hum an fisheries, but
that could be low compared to their ecological role, still poorly
understood. Som e species com pete with fisheries, while others
obviously do not, and yet others partly com pete but could be
u sefu l to fisheries by regulating the developm ent of non­
commercial species, thereby limiting excessive com petition with
com m ercial ones (Bouquegneau et al., 1997a). It is our belief that

44
 Chapter 1

m arine m am m als deserve their place in the ocean s and are worth
being protected.

A cknow ledgem ents. T his w ork w as su p p o rte d by a g ra n t from th e B elgian Office

for Scientific, T echnical a n d C u ltu ra l Affairs (C ontract M N /D D /50). The a u th o rs
w ish to th a n k A. D istèch e , K. F in n so n a n d C. B ean s for re a d in g th e m an u sc rip t.

45
 General Introduction

1 .4 . METALLOTHIONEINS IN MARINE MAMMALS

(After Das K., Debacker VB ouquegneau

J.M. 2000. Metallothioneins in marine
mammals. Cellular and Molecular Biology
46 (2): 283-294

1 .4 .1 . FOREWORD

Many areas have emerged in métallo thio ne in (MTs) research since

their were first discovered (Margoshes and Vallee, 1957), but their
natural function rem ains elusive (Palmiter, 1998). Previous
stu d ies on structure, function, and m olecular regulation have
established a central role for th ese m olecules in the hom eostatic
regulation of essen tial m etals su ch a s zinc (Zn) and copper (Cu).
Thus, it is not surprising that MTs have been detected in both
prokaryotes and eukaryotes including m arine m am m als (reviewed
by Roesijadi, 1992; 1996). Indeed, the u se of m etals a s cofactors
in biochem ical reactions and their toxicity associated with their
affinity for S, N, and O (the predom inant ligands in biomolecular
structures), represent dual asp ects of m etal-biological
interactions. Therefore, m echan ism s that regulate the
intracellular availability of essen tial m etals and protect against
inappropriate and potentially deleterious intracellular
interactions are primordial for efficient biochem ical function. MTs
work a s Zn or Cu donors to other m etalloproteins. They are
induced by and bind excesses of these m etals. Another function
postulated for MTs is the detoxification of non -essen tial m etals
like cadm ium (Cd) and m ercuiy (Hg). As a result of its capacity to
bind cations, MTs are able to bind n on -essential m etals su ch a s
Cd2+ Ag*, Hg2+ and Pb2+ and, in th is way, reduce the
bioavailability of th ese toxic m etals (reviewed by Webb, 1987 and
Roesijadi, 1992).

It h a s been suggested that Cd toxicity occurs w hen available MTs

are insufficient to bind all the Cd. Recent experim ents with mice
genetically deprived of MTs due to the lo ss o f functional MT I and
II gen es (coding for the 2 m ain isoform s of MTs involved in the
detoxification process) confirm the protective role of these
proteins against cellular dam ages from m etals su c h as Cd or

46
 Chapter 1

inorganic Hg (Satoh et al., 1997; K lassen and Liu, 1998). This

lead s to consider that these proteins prevent organism s from
toxic hazards that could occur following the high exposure to Cd
and Hg. Recently, K laassen et al. (1999) reviewed the protective
action of MTs against Cd toxicity.
So far, there is little data on MTs in marine m amm als.
Nevertheless, in the framework of heavy m etal ecotoxicology,
m arine m a m m a l s appear a s a choice material, since high levels of
Hg and Cd can be naturally encountered in th ese anim als. While
plankton-eating M ysticetes are generally weakly contam inated by
heavy m etals, fish-eating and squid-eating O dontocetes and
Pinnipeds are heavily contam inated by Hg and Cd respectively
(Bouquegneau and Joiris, 1992). Heavy m etals in marine
m a m m a l s have been recently reviewed by D as et al. (2000).

A maximal Hg concentration of about 13000 p g/g dry weight h a s

been reported in the liver o f one bottlenose dolphin stranded on
the Italian coast (Leonzio et al., 1992). Such high levels of Hg
without overt evidence of deleterious effects m ay only occur if Hg
is detoxified. Koeman et al. (1973; 1975) have first reported a
strong correlation between Hg and selenium in livers of marine
m am m als. A molar ratio Hg:Se o f approximately 1 h a s been
observed suggesting Hg detoxification m echan ism s in presence of
selenium . The fate of Hg h a s been m ainly elucidated by
histological stu d ies carried out in livers from different marine
m am m als sp ecies a s Cuvier’s beaked whale Ziphius cavirostris
and bottlenose dolphins Tursiops truncatus (Martoja and Viale,
1977; Martoja and Berry, 1980; Nigro and Leonzio, 1996). These
authors have observed mercuric selenide granules (HgSe) located
m ainly in the liver m acrophages, the Kupffer cells, and in the
proximal tu b u les of the kidney. The transformation of assim ilated
Hg in tiemm arrite (HgSe) appears to be the last step of
detoxification leading to formation of inert and non-toxic Hg
com pounds.

In the sam e way, Cd renal concentrations can reach levels as

high a s 2 0 0 0 p g .g 1 dry weight in som e Arctic ringed sea ls without
any pathological effects (Dietz et al., 1998). This is m uch higher
th an the critical concentrations of approximately 8 0 0 p g .g 1 dry
weight (200 pg.g 1 wet weight) associated with kidney damage in
m am m als (WHO, 1992). Moreover, following Elinder and Järup
(1996), th is critical concentration h a s been largely overestim ated
a s Cd-induced renal dysfunctions have been observed at kidney
cortex concentrations of 200 p g .g 1 dry weight (50 p g .g 1 wet
weight). For com parison, in h um an adults, the renal Cd

47
 General Introduction

concentrations am ongst non-sm okers is about 1 p g .g 1 wet weight

(Pesch et al., 1989). These investigations indicate that marine
m am m als are able to m aintain considerable concentrations of Cd
w ithout showing renal dam ages. It h a s been postulated that
marine m am m als m itigate the toxic effects of Cd through binding
to MTs. So far, MTs and m etallothionein-like proteins (MTLP)
have been described in 10 marine m am m al sp ecies (table 1.3)
with concentrations ranging from 58 to 7 1 0 pg.g“1 and 140 to
1200 pg.g-1 fw in the liver and kidneys respectively.

1 .4 .2 . CADMIUM BINDING

As quoted above, the renal concentrations of Cd in marine

m am m als can reach levels m uch higher than the critical
concentrations associated with kidney damage in terrestrial
m am m als. The question h a s therefore been raised about anim als
so heavily contam inated with Cd. Dietz et al. (1998) have
compared low and high concentrations of Cd in the kidneys of
ringed seals (Phoca hispida) from Northwest Greenland in an
attem pt to do m acroscopic and light m icroscopic exam inations.
No differences in renal morphology could be observed between
experim ental groups. These investigations indicate that marine
m am m als appeared able to m aintain considerable concentrations
of Cd without show ing renal damage. They therefore postulated
that ringed seals were adapted to the naturally high Cd levels of
the Greenland Arctic regions.

The role of MTs in binding Cd present in the tissu e s m ay vary

widely between different species a s well a s betw een different
individuals from the sam e sp ecies (table 2). This lead s to more or
le ss important spillage of Cd to other m etalloproteins. It is
interesting to note that the low values o f 5, 17 and 18% have
been m easured in the liver of three highly debilitated sperm
w h ales found stranded on the Belgian coast (Bouquegneau et al.,
1997b; Holsbeek et al., 1999). Moreover, a spillage of Cd from
MTs to other soluble com ponents h a s been observed: 34 to 38 %
of the total Cd is bound to soluble proteins other than MTs
(calculated from Holsbeek et al., 1999). This su ggests that Cd w as
not in a detoxified form, either on MT or in lysosom es. These
anim als were seriously debilitated a s indicated by their reduced
blubber th ick n ess and body weight (Jauniaux et al., 1998). Cd
w hich is known to induce debilitation in m am m als can be
considered as one o f the factors responsible for the condition of

48
 Chapter 1

th ese anim als, w hich in addition to stress and starvation, could

have resulted in their stranding (Bouquegneau et al., 1997b).
Table 1.3. Metallothionein and metallothionein-like protein detection and
quantification (metal-free protein) in marine mammals (data about m t
concentrations are either single value or in range, or mean + standard deviation). Metallothionein
concentrations are estimated assuming 7 gram-atoms (Cu, Zn, Cd, Hg) per mole and a molecular
weight o f 7 kDa
Geographic concentration Isoforms
Species Tissue Method References
location (PS-81 ww) detected
California sea Sephadex G-75 or
lion
Oregon 140 MT-1 Sephadex G-200
Zalophus kidney L e e e ta l., 1977
(USA) (n = l) MT-2 W hatman DE 32-
californianus
column
californianus
Ribbon seal Sephadex G-75
MT-1 Mochizuki et
Histriophoca Japan liver nd HPLC-AAS
MT-2 al., 1985
fasciata DEAE-3SW column
Sephadex G-75 Mochizuki et
MT-1
Japan liver nd HPLC-AAS al., 1985
Pinnipeds MT-2
DEAE-3SW column
Harbour seal
Phoca vitulina 240 ± 139
liver
(n=T5) Tohyama et al.,
Japan nd Radioimmunoassay
343 ± 219 1986
kidney
(n=15)
Grey seal Olafeon and
70 Sephadex G-75
Halichoerus Canada liver nd Thompson,
(n = l) followed by G-50
grypus 1974
Pacific fur seal Olafson and
90 G-75 followed by G-
Callorhinum Canada liver nd Thompson,
(n = l) 50
urisnum 1974
167 ± 3 6
(n=7; july
1986) Caurant et al.,
Pilot whale liver Polarography
Faroe 592 ± 200 1996; Amiard
Globicephala nd
Islands (n=7; Triquet and
melas
november Caurant, 1997
1986)
kidney 7 5 1 ± 213
Sephadex G-75
Sephadex G25
DEAE Sephadex-
Striped dolphin A25
Nothwestem 314* MT-1 Kwohn et al.,
Stenella kidney HPLC on GS-320
Pacific (n = l) MT-2 1986; 1988
coeruleoalba column
Amino acid
characterisation and
primary structure
O don tóceles
710' Sephadex G-75,
liver Wagemann et
Narwhal (n = l) Sephadex G-50
al., 1984
M onodon Arctic nd Polyacrylamide gel
1200’ W agemann and
monoceros kidney electrophoresis
(n = l) Hobden, 1986
DEAE Sephadex A25
Harbour
porpoise Antoine et al.,
North Sea liver nd nd AcA-54
Phocoena 1992
phocoena
Sephadex G-75 and Ridlington et
USA fiver nd nd DEAE Sephadex al., 1981
gel
Sperm whale 58'
Physeter fiver Bouquegneau et
(n=3)
macrocephalus al., 1997;
North Sea 468-951 1 nd AcA-54
Holsbeek et al.,
kidney (n -2 ) 1999

49
 General Introduction

However, high levels of Cd with low binding to MTs have been

reported in healthy anim als. Amiard-Triquet and Caurant (1997)
have reported that 51% of total Cd were bound to MTs in the
livers of pilot w hales caught in July 1986 w hereas individuals
caught in November from the sam e year displayed only 6%.
Moreover, individuals caught in winter have low plasm a Cd levels.
According to th ese authors, Cd in the plasm a resulted from a
recent Cd contam in ation. These resu lts are in agreem ent with the
season al availability o f preys: squids w hich are known to
concentrate Cd in their tissu e s, are more abundant in summer.
So it seem s that only recently assim ilated Cd is bound to MTs,
the rest being stored in the insoluble fraction (Amiard-Triquet
and Caurant 1997). In contrast to pilot w hales, in the narwhal,
more th an 70 % of the cytosolic Cd is located in the MT fraction
of the liver, indicating low spillage of Cd to other m etalloproteins
(Wagemann et al., 1984). This could be an adaptation of th is
Arctic species to the high Cd concentrations present in its
environment. However, more data on Cd spéciation in marine
m am m als are needed to get a better com prehension of the precise
role of MTs in detoxification processes.

1.4.3. M e r c u r y b in d in g

Hg also h a s a strong affinity for MTs and high levels o f th is m etal

can be associated with elevated levels of Cd in m arine mammal
tissu e s (Caurant et al., 1996). However, stu dies conducted on
dolphins (Kwohn et al., 1986), California se a lion s (Lee et al.,
1977), pilot w hales (Caurant et al., 1996), narw hals (Wagemann
et al., 1984) and sperm w h ales (Bouquegneau et al., 1997b;
Holsbeek et al., 1999) dem onstrated that only a sm all part of the
total Hg w as bound to MTs (table 1.5). A significant am ount of Hg
m easured in the cytosol of a narwhal liver w as found to be
associated with the high m olecular weight fractions (Wagemann
et al., 1984). According to th ese authors, su ch a spill over of Hg
to the high m olecular weight com ponents would be a normal
occurrence in m arine m am m als and is not related to the
saturation of MTs.

Even though there is a high affinity of Hg for MTs, m ost of the

m etal is bound to com ponents other than MTs. These resu lts are
quite different from those deriving from stu dies carried out on
terrestrial m am m als, in w hich Hg is show n to be particularly
bound to MTs (Whanger and Deagen, 1983). T his striking

50
 Chapter 1

difference betw een terrestrial and marine m am m als is m ainly due

to differences in Hg spéciation in the diet. In the marine
environment, alm ost all the Hg present in fish is m ethylated
(Svensson et al., 1992). M ethylmercuiy, w hose affinity for MTs is
low, cannot be detoxified by th is process. In marine m am m als, it
h a s been show n that the relative MeHg levels decreased from
100% (of the total Hg level) in ju ven iles to only 2 or 3 % in the
liver of adults (Joiris et al., 1991). This reflects the existence of a
slow m ineralisation process without formation of free Hg++ ions
w hich can bind to MTs.

Table 1.4. Cd spéciation in the tissue, the cytosolic fraction and

metallothioneins (Cd concentrations are estimated in ng.g‘‘ dry weight; nd: not determined, dl:
detection limit).

Total Cd % o f Cd in
% cytosolic
Species Tissue n (P g g 1 the cytosolic R eferences
Cd on M T s
dw) fraction
Striped dolphin
Stenella
kidney 4 87 58 98 Kwohn et al., 1986
coeruleoalba

1 37 68 nd
California sea lion kidney
5 65 ± 3 0 63 71 Lee et al., 1977;
Zalophus
1 <dl <dl <dl Ridlington et al., 1981
californianus liver
5 11 ± 7 60 55
7 gestating 548 ±
54 ± 6
kidney females 164
Pilot whale 5 foetuses 1 + 0.8 44+41 Amiard-Triquet and
Globicephala nd
7 gestating 312 ± Caurant, 1997
melas 51 ± 2 0
liver females 124
5 foetuses 0.6 ± 0.7 25 ± 3 4
1 50 92 100 Ridlington et al., 1981
64 53 18
Sperm whale liver
3 71 55 17 Bouquegneau et al.,
Physeter
103 39 5 1997
macrocephalus
225 81 21 Holsbeek et al., 1999
kidney 9
316 85 66
N arw hal kidney 1 332 92 72
Monodon
Wagemann and
monoceros liver 1 176 88 77
Hobden, 1986

The observed high percentage o f Hg bound to the insoluble

fraction of the liver resu lts from the formation of tiem annite
(HgSe) These dense intracellular granules have been observed in
the liver m acrophages and Kuppfer cells, the proximal tu b u les of
the kidney, the lung and hilar lym ph nodes (Martoja and Viale,

51
 General Introduction

1977; Martoja and B en y, 1980; Nigro and Leonzio, 1996; Augier

et al., 1993; Rawson et al., 1995). By th is way, in marine
m am m als, selenium plays a key role in methylmercury
detoxification processes, and hence MTs would play a m inor role,
probably limited to the detoxification of Hg 2+.

Table 1.5. Distribution of mercury on renal and hepatic metallothioneins

Total
% Hg % Hg
Hg
Species Tissue insoluble associated with References
(pg g
fraction MTs
iw)
Pilot whale
Globicephala kidney 6 79 7 Caurant et a t, 1996
(n=7)
melas
California Sea
k' d" e>' 10 54 22
lion
Y1 Lee et at, 1977
Zalophus
!, 61 93 2.6
californianus (n-5)
Narwhal liver 9 88 5
Monodon ,(nr 1} Wagemann et a t, 1984
monoceros kldn,e > 2 73 10
(n=l)
.. 2 85 <1
Spem whale . "Y", 15 95 <0.3 Bouquegneau et at,
Physeter (n > 15 84 <3 1997; Holsbeek et a t,
macrocephalus kidney 2 72 <2 1999
(n=2) 5 70 <6
Striped dolphin
Stenella kidn®y 16 83 6 K w ohnetak. 1986
(n=4)
coeruleoalba

1.4.4. S t r u c t u r e a n d c h a r a c t e r is a t i o n

Comparative sequence stu d ies of MTs from different sp ecies and

organs have revealed remarkable sim ilarities am ong m am m alian
sp ecies (see Binz and Kagi, 1999). About 56% of the 61 am ino
acid residues are conserved in m am m als, am ong them all the 20
cysteine and nearly all lysine and arginine residues (Kojima et al.,
1999).

Characterisation of marine m am m al m etalloproteins were first

attem pted on s e a lion and sperm whale kidney and liver
(Ridlington et al., 1981). Metal binding proteins isolated from
sperm whale liver eluted in a m anner similar to rat MTs, but the
am ino acid analyses yielded only 12% cysteine residues w hereas

52
 Chapter 1

all m ammalian MTs contain approximately 30%. According to the

authors, th is protein w as not likely a m etallothionein but rather a
type of Cu-chelatin. Similar con clu sions were drawn for se a lion
liver and kidney m etal binding proteins a s the amino acid
analysis for the different m etal-binding fractions contained 2 to
15% cysteine residues. It m ust be noted however, that general re­
exam ination of Cu chelatin h a s resulted in its designation a s a
MT (Winge et al., 1981 quoted by Roesijadi, 1992).

However, resu lts reported by Ridlington et al. (1981) are

inconsisten t w ith other marine m amm al MT characterisation.
Indeed, striped dolphin MT I and II isolation and characterisation
have been performed (Kwohn et al., 1986; 1988). According to
th ese authors, 61 am ino acid resid u es are present per mole of
each MT, including 20-21 cysteine residues (table 1.6). The
absence of aromatic am ino acids agrees with the lack of
absorbance at 280 nm. MT am ino acid com position of striped
dolphin compared with rabbit for both isoform s is quite sim ilar.
According to Kwohn et al. (1988), 89% of the MT II amino acid
sequence is conserved between rabbit and dolphin. Neither valine
nor leucine were detected in rabbit both isoform s, w hich su ggests
microheterogeneity betw een the two species.

This discrepancy observed betw een the two characterisations of

marine m am m al MTs m ight be explained, at least in part, by
technical evolution. Indeed, Roesijadi (1992) h a s underlined early
technical difficulties associated w ith purifications of MTs. Studies
that have attem pted to characterise m etal-binding proteins have
often been influenced by the presence of im purities or the
isolation of proteins onto w hich m etals could be redistributed
during sam ple preparation.

Kwohn et al. (1986) have detected two isoform s in the dolphin

kidney identified a s MTs I and II. According to its absorbance at
254 nm, the MT II isoform is m uch more abundant than MT I
with a ratio 1 /1 6 while, for exam ple, it is e.g. only 1 /6 .2 in
equine tissu e s (Kojima et al., 1976 quoted by Kwohn et al., 1986).
Most of the m etals are therefore bound to MT II except Cu which
is preferentially bound to MT I (Kwohn et al., 1986). The sam e
tendency is also observed for horse renal MTs (Kojima et al., 1976
quoted by Kwohn et al., 1986) indicating different cellular
functions for each renal MT of striped dolphins. However, it is
worth noticing that W agemann and Hobden (1986) reported MT I
a s the major form of MTs in the liver and kidney from a narwhal.
The functional significance of m ultiple isoform s of MTs h a s yet to

53
 Genera I Introduction

be dem onstrated for any m am m alian species. The different roles

of MTs in the regulation o f m etals are probably critical to cell
functions but these have yet to be studied in any marine
mammal. It is clear, however, that understanding the MT
function will need to consider the functionality and structure of
each MT isoform.

Table 1.6. Amino acid composition of striped dolphin renal MT I and MT

II (after Kwohn et al., 1986) and rabbit hepatic MT I and MT II (after
Nordberg et al., 1972) *: calculated as metal free molecular weight

Amino acid Dolphin MT I Dolphin MT II Rabbit MT I Rabbit MT II

Asp 3 3 5 5
Thr 3 2 3 4
Ser 7 8 7 9
Glu 2 2 1 3
Gly 6 6 4 4
Ala 6 6 8 8
Cys 21 20 15 19
Val 1 1 / /
Met 1 1 1 1
Ile 1 1 1 1
Leu 1 / / /
Phe / / I /
Lys 6 8 7 9
Arg / / / /
Pro 3 3 5 3
Tyr / / / /
His / / / /
Total 61 61 57 66
Molecular weight * 5986 6013 5608 6616

1.4.5. F a c t o r s m o d u l a t in g m e t a l l o t h io n e in
CONCENTRATIONS

The range of MT concentrations in m arine m am m al kidneys and

liver is widely extended (from 58 to 1200 pg.g fw). Their
concentrations appear to be high compared with those in rats.
Indeed, hepatic levels in norm al rats are about 1 p g .g 1 while it
can reach more than 200 pg.g-1 in Cd-induced anim als (Eaton
and Toal, 1982). Maximum concentrations encountered in marine
m am m als are m uch higher (table 1.1). However, the m odulation
of MT concentrations in marine m am m als is poorly understood
due to a lack of extensive quantifications. In m ost cases, the
factors influencing MT concentrations in the wild are assum ed
after experim ental stu d ies on other m am m als. Till now, only two

54
 Chapter 1

stu d ies have attem pted to correlate MTs with ecological or

ecotoxicological factors (Tohyama et al., 1986; Amiard-Triquet
and Caurant, 1997). According to th ese authors, the MT
concentrations depend more on particular tissu e s, the heavy
m etal concentration, the age and the diet.

[Link]. Tissue
MT concentrations are always higher in the kidneys than in the
livers (table 1.7). Few data are available to allow for com parison,
but MT level in the kidney appears to be 1.2 to 1.6 fold higher
than in the liver (table 1.5) except for sperm w h ales stranded on
the Belgian coast in w hich the ratio may be related with the
debilitated sta tu s of th ese anim als.

Table 1.7. Comparison of the ratio of MT concentrations in the kidney (K)

and liver (L)
Species n Sampling status Ratio K .L 1 Reference
Pilot whale 7 bv-caught 1.2 Amiard-Triquet and Caurant, 1997
Globicephala melas
Harbour seal 15 by-caught 1.4 Tohyama et al., 1986
Phoca vitulina
Narwhal 1 by-caught 1.6 Wagemann et al., 1984
Monodon monoceros
Sperm whale 2 stranded 8.7-12.4 Holsbeek et al., 1999
Physeter macrocephalus

[Link]. Metal level

A study performed on harbour sea ls (Phoca vitulina) caught on
Japanese coasts show that MT concentrations are significantly
correlated with the level of Cd and Zn in the livers and with the
level of Cd, Zn, and inorganic Hg in the kidneys (Tohyama et al.,
1986). These authors suggest that inorganic Hg and Cd could be
sequestered in MT and that Hg toxicity could also be lessen ed by
th is protein.

55
 General Introduction

[Link]. Age
Tohyam a et al. (1986) have pointed out that higher MT levels
have been quantified in the liver and kidney of a seal pup a s
compared to adults. N eonates of various m am m alian species are
know n to have relatively high levels of MTs with associated Zn
and Cu in the liver (Bakka and Webb, 1981, quoted by Tohyama
et al., 1986). Amiard-Triquet and Caurant (1997) have quantified
hepatic MTs in gestating female pilot w h ales and their foetuses.
MT m ean concentrations were always lower in the foetus than in
m other’s livers (mean: 73 pg.g-1 and 167 ng.g-1 respectively).
However, the ratio MT/ total proteins remained unchanged. In
harbour seals, Tohyam a et al. (1986) found a correlation between
MT levels and age. The observed age-dependant ch an ges in renal
and hepatic MTs are associated with Cd accum ulation a s it h as
been show n recently in hum an tissu e (Yoshida et al., 1998).

[Link]. Diet
As quoted above, Caurant et al. (1996) and Amiard-Triquet and
Caurant (1997) have compared m etallothionein-like protein
(MTLP) concentrations between pilot w h ales caught in July and
November 1986 (fig. 1.7).

900

¿ 750 -

600 - n July 1986

□ November 1986
£ 450 -
CL

I^- 300 -
S
150 -

Figure 1.7. Mean concentrations and standard deviations of

metallothionein-like proteins (MTLP) calculated in the livers of pilot
whales (Globicephala melas) caught in the Faroe Islands (data from
Caurant et al., 1996; Amiard-Triquet and Caurant, 1997)

56
 Chapter 1

MT m ean concentrations in the livers of pilot w hales caught in

the su mmer 1986 are more than three fold higher than those
encountered in individuals caught in winter, while Cd
concentrations are sim ilar for both groups. Squid is the major
food item in pilot w hales and is considered a s a significant source
of Cd for several predators (Das et al., 1999b). The lower level of
MTs in w hales caught in November could be related to lower Cd
assim ilation in winter. Indeed, the sum m er diet o f pilot w hales
co n sists m ainly in cephalopods w hereas the winter diet is
characterised by a higher fish input (Caurant et al., 1996 ;
Amiard-Triquet and Caurant, 1997). According to the authors,
the elevated MT concentrations would reflect an induction by a
recent Cd a ssimilation. The m etal could be sequestered later
under another detoxified form {e.g. precipitated within
lysosom es).

1.4.6. M T S AS BIOCHEMICAL INDICATORS OF METAL EXPOSURE

AND TOXICITY

Soon after their discoveiy in aquatic species, the induction of MT

and the analyses of its capacity to bind m etals were proposed as
candidates for biochem ical monitoring of m etal pollution in the
aquatic environm ent (Roesijadi, 1996). Heavy m etal analysis in
the tissu e s reflects the level of contam ination o f a population but
not its response to m etal exposure, a s th ese can be detoxified, at
least in part, through the binding to MTs. The induction of MTs
by non-essential m etals su ch as Cd h a s been considered a s an
a sset in biomonotoring studies. Moreover, with current
m ethodologies, MT induction can be m easured at several levels:
increase in m etal content in the MT pool , increase in MT and
increase in MT mRNA (e.g. Suzuki, 1992; Caurant et al., 1997;
Tom et al., 1998). Each reflects a different level of cellular
regulation and function, and provides complem entary
information. Argum ents against application of MT analysis in
environmental stu d ies resides m ainly in our current lack of
detailed understanding of basal MT function and its relationship
to induction by m etals (Roesijadi, 1992 ; C osson and Amiard,
1998). Through forty years o f MT research, the different
interacting processes appear num erous and complex. C osson and
Amiard (1998) have recently reviewed the utilisation of MTs a s
potential biomarkers o f m etal contam ination in aquatic anim als
including marine m am m als and have underlined the difficulty to

57
 General Introduction

correlate m etal bioaccum ulation and increased MT level. Thus

proposal for the u se of MTs a s biochem ical indicators of m etal
pollution in m arine m am m als m erits continued considerations,
as further investigations are needed to understand the processes
involved in detoxification.

1 .4 ,7 . L im it s t o d e t o x if ic a t io n

Drawing conclu sions concerning the potential role of MTs in

m arine m am m als is not an easy task due to an obvious lack of
recent published stu d ies and extrapolation from other m am m als
are often advanced. Concerning potential role of th ese proteins in
Cd detoxification, opinions are mitigated. The rates of MT
syn th esis can becom e limiting a s exposure concentrations
increase. The related decreasing m etal binding to MTs could
result in the spillage of m etals to other structures that would
include target sites for m etal toxicity (Roesijadi, 1992). Any
detoxification p rocess h a s a cost for the cell or the organism
involved and m ight have a limit. This threshold cannot be fully
defined in term s of tissu e m etal or MT concentrations because of
the num ber of param eters that can interact to limit physiological
pathw ays that lead to detoxification. For exam ple, gender and
horm onal activity can m odulate the sy n th esis of MTs (Blazka and
Shaikh, 1991). Moreover, binding to MTs m ight not be a final
step, and the formation of secon daiy com ponents with toxic
effects have been dem onstrated in other m am m als. The
accum ulation and degradation of Cd-m etallothionein complex
(CdMT) in the renal tubular epithelial cells can induce
nephrotoxicity in m ice counteracted by Zn w hich h a s a protective
effect against th is CdMT-induced nephrotoxicity (Liu et al., 1996;
Tang et al., 1998). As a result of their physiological function in
the h om eostasis o f essen tial m etals, MTs could be involved in
m any cellular pathways. Thus, they could m odulate physiological
processes a s an indirect effect of heavy m etal exposure. For
exam ple, MTs have been dem onstrated a s potential m odulators of
som e param eters o f the im m une response (Leibbrandt et al.,
1994; Borghesi et al., 1996). Detoxification pathw ays could
therefore lead to more subtle toxic effects underlying the
complexity to approach toxic effects of heavy m etals.

58
 Chapter 1

1 .4 .8 . C o n c l u s io n s

Compared w ith other anim als, liver and kidneys of marine

m am m als display high MT concentrations, related with their high
levels of contam ination by heavy m etals (mainly Cd and Hg).
These high heavy m etal concentrations result from both their
hom eotherm y (which requires large food consum ption) and their
position at the top of marine food w ebs. Data about MTs remain
scarce and, until now, d iscu ssion is only possible about their
potential role in the detoxification of Hg and Cd. Obviously, MTs
play a minor role (if any, w hen considering m ethylmercuiy) in the
binding and detoxification of Hg by marine m am m als. On the
contrary, close interactions occur betw een Cd and MT dynam ics.
Cytosolic MTs appear a s a potential short term way of
detoxification of Cd accum ulated from diet. Long-term
detoxification however would imply a sequestration of the m etal
under a precipitated form (e.g. in lysosom es). So many
param eters are likely to m odulate the MT concentration in marine
m am m als tis s u e s that its u se a s a biomarker of heavy m etals
pollution in the marine environm ent rem ains debatable.

A cknow ledgem ents. T his w ork w as su p p o rte d by a g ra n t from th e Belgian Office

for Scientific, T ech n ical a n d C u ltu ra l Affairs (C ontract M N /D D /50). We a re
grateful to P ro fesso r D. Cyr a n d P rofessor R.P. C osson for th e ir co n stru ctiv e a n d
useful co m m en ts o n a previous v ersion o f th is paper.

59
 General Introduction

1 .5 . STABLE ISOTOPE ECOLOGY

The u se of stable isotopes h a s recently evolved to a powerful tool

to study elem ent cycles and various biological, physical, and
chem ical processes in research areas a s diverse a s archaeology,
m edicine, ecology, astronom y and hydrology. This paragraph is
intended to provide an introduction to stable isotopes of carbon
and nitrogen a s th ese isotopes are u se d extensively throughout
further chapters.

1.5 .1 . BACKGROUND

Most elem ents of biological interest have two or more stable

isotopes, w hich differ only in the num ber of neutrons, resulting in
different atomic m asses. Therefore, different stable isotopes of the
sam e elem ent do not react in the sam e m anner in fundam ental
kinetic processes. This phenom enon is called fractionation, w hich
is a function of slight variations in the physical and chem ical
properties of the isotopes and therefore occurs in proportion to
the differences in the isotopic m asses. Each fractionation will
result in a modification of the original ratio betw een the isotopic
sp ecies concerned (Ehleringer and Rundel, 1989). Fractionation
processes of 13C and 15N have been intensively u se d to delineate
trophic relationship in different food w ebs (Gannes et al., 1998;
Hobson, 1999; Kelly, 2000).

Carbon have two stable isotopes with respective different

proportions in nature (table 1.8). Sm all variations in these
abundances occur and th ese can be accurately determ ined with
stable isotope spectrom eters.

Isotopic com position is m easured by determ ining the ratios of the

two stable isotopes present in the sam ple. As the natural
variations in the relative abundance of “light” and “heavy”
isotopes of an elem ent are usually very sm all (e.g. the 13C / 12C
ratio m ay vary between 0 .0 1 0 2 2 5 and 0.011574), it is not very
practical to express these variations a s differences in (ratios) of
the absolute abundance of the isotopes.

60
 Chapter 1

Table 1.8. Average natural abundance of the main stable isotopes of carbon
and nitrogen, according to Ehrlinger and Rundel, 1989

Element /isotope Abundance (%)

Carbon
12C 98.89
13C 1.11
Nitrogen
N 99.63
15n 0.37

The resu lts are generally expressed in a standard “delta-notation”

(8) w hich is based on the m easurem ent of the differences between
the sam ple and a standard defined by the IAEA in Vienna (it is
Pee Dee Belemnite for carbon and atm ospheric nitrogen for
nitrogen)

The stable isotope ratio (8-value) of an elem ent is:

S X = [ ( R s a m p le /R s ta n d a r d ) “ 1 ] X 1 0 0 0

where X is 13 C or ,5N and R is the corresponding ratio 13C/12C or

15N /14N. Results are expressed in %o.

1.5.2. S o u r c e s o f f r a c t io n a t io n in p r im a r y p r o d u c e r s

The u se fullness of stable isotopes in studying elem ent cycles and

p rocesses lies in the fact that sm all but significant variations
observed in nature do not occur at random but are governed by
fractionation processes, i.e. during a reaction (either chemical,
physical or biological) the different isotope m ay react with
different speed and th is may cause the end product to have an
altered isotope com position compared to the source product
(reviewed by Bouillon, 2002).

1 .5 .2 .1 . C a r b o n - 13 (513C )

The first hint o f the utility of carbon isotopes for the study of
trophic ecology came w hen the difference in the isotopic

61
 General Introduction

signatures of C3 and C4 plants w as discovered (reviewed by Kelly,

2000). This utility is derived from two properties: first, som e
sources of dietary carbon have distinct carbon-isotope signatures,
and secon d the isotope signature of a food is incorporated into
the consum er’s tissu e s (DeNiro and Epstein, 1978).

With regards to anim al ecology, the primary process that creates

sou rces with identifiable carbon-isotope signatures is
photosynthesis. Stable carbon isotopes are u sed m ost often to
distinguish carbon fixed by terrestrial C3 plants from that fixed
by C4 plants or marine C3 p lants (table 1.9).

In aquatic plants, 5 13C va lu es show a m uch wider range of

values, w hich are not reliable indicator of the photosynthetic
pathway u sed (reviewed by Bouillon et al., 2002). For instance,
marine phytoplankton u s e s the C3 photosynthetic pathway.
However, its carbon-isotope signature is significantly heavier (-22
%o) than that of terrestrial C3 plants (-24 to -30%o). Benthic
microalgae have been found to be consistently enriched in 13C
relative to their pelagic counterparts by an average of 7%o
(France, 1995c quoted by Bouillon, 2002). This difference
betw een benthic and pelagic microalgae is consistent enough to
be reflected in consom m er ô 13C valu es and h a s been proposed as
a valuable tool to distinguish between benthic and pelagic food
sources.

Table 1.9. Comparison of ö ,3C values in plants using different

photosynthetic pathways (compiled from Descolas-gros, 1985; Lepoint et
al., 2000; Bouillon, 2002). For information, the S13C o f atmospheric CO 2 is -7.8%o.

Photosynthesis carboxylating enzyme Range of 8I3C values

C., plants Phosphoenolpyruvate carboxylase (PEPc) -9 to -199&0

-terrestrial plants:
-24 to —30%o
-phytoplankton:
-18 to -28%o
C 3 plants Ribulose Biphosphate Carboxylase/Oxygenase (RUBISCO) -Antarctic
phytoplankton
-seagrass: -15 to -3%o
-macrophytes: -9 to -
35 %o

62
 Chapter 1

A m ultitude of factors have been found to influence the carbon

isotope com position of aquatic primary producers :

• the type of substrate u se d (CO2 or HCO3 ),

• the concentration and isotopic com position of the
substrate (either CO2 or HCO3 ),
• the growth rate,
• the cell shape and volume,
• the water flow rate and temperature.

B ecause phytoplankton h a s lighter 13C values than m any inshore

p lan ts (e.g., seagrasses, average 813C=-10%o, range=-15 to -3%o),
inshore carbon sources can som etim es be distinguished from
pelagic sources (e.g., Fry, 1983, Hobson et al., 1994; Lepoint et
al., 2000).
All th ese differences in 13C / I2C ratios in primary consum ers are
sufficient enough to influence carbon isotopic com position
(13C/12C) of their respective consum ers and assign them a specific
iso topic signature.

[Link]. Nitrogen-15 (515N)

Unlike carbon isotopes, there is no single process, like
photosynthesis, that creates a large isotopic fractionation of
nitrogen isotop es in plants that can be traced through food webs.
However, 815N signatures o f primary producers in both aquatic
and terrestrial ecosystem s m ay be u sefu l indicators of N sources
and transformations. Fractionation may occur during various
biological reaction su ch as:

• assim ilation of dissolved inorganic nitrogen (DIN) under

NOs', NOU- or NHh form by phytoplankton or
m icroheterotrophs (bacteria, fungi),
• nitrification and denitrification,
• N2 fixation.

Controlled laboratory experim ents have revealed wide variations

w ith growth rate, light intensity, species, N substrate and culture
conditions, resulting in fractionation values for algae between 0.7
and 23%o (for NO.r), 0.7%o (for NO2 ), and between -9 .7 and 14%o
(for NH4+).
However, m ain fractionation processes (and the m ost useful for
our purpose) o c c u l t during trophic interactions (see below).

63
O
 General Introduction

1.5.3. STABLE ISOTOPES AS TRACERS OF ORGANIC MATTER AND

FOODWEB INTERACTIONS

The application of stable isotopes at the natural abundance level

as indicators of the origin of organic m atter and of trophic
interactions is based upon three important h ypotheses (reviewed
by Bouillon et al., 2002):

1. differences (may) exist in the 813C a n d /o r 815N signatures

o f different primary producers,
2. th ese differences are m aintained or altered in a
sufficiently predictable way during degradation processes,
3. consistent and predictable changes in the isotopic
signature occur during transfer to higher trophic levels.
It should also be m entioned that other stable isotopes of other
elem ents su ch a s hydrogen, oxygen or sulphur have also been
proven a s useful ecological tools but are outside the scope of th is
introduction.

S13C:
~ + l% o

S 13C (°/oo)

O ffs h o re /p e la g ic « i n s h o r e / b e n th ic

Figure 1.8. Enrichment of ô ° C and 8 15N from one trophic level to the
other one

64
 Chapter 1

System atic enrichm ent in 13C valu es in marine food ch ain s h a s

been reported (Rau et al., 1983; Boutton, 1991). Most studies
however, indicated that in marine environment, 13C enrichm ent
occurred at low trophic levels but not am ong vertebrates
consum ers (Rau et al., 1983; Wada et al., 1987; Fry, 1988;
Hobson and Welch, 1992; Hobson, 1993; Hobson et al., 1993).
T his minor stepw ise trophic enrichm ent of the carbon isotope
ratio that h a s been docum ented am ong vertebrate consum ers
lim its its u se in a ssessin g trophic level. However, this
characteristic en h an ces the utility o f carbon-isotope ratios for
tracking carbon sources through a food chain (Peterson and Fiy,
1987; Michener and Schell, 1994). Specifically, because there is
little enrichm ent with increase in trophic level the carbon isotope
signature of secondary and tertiary consum ers should reflect the
source of carbon (C3, C4 or marine plants) at the base o f the food
chain.

The variations in nitrogen isotopic com position are largely

determ ined by biologic reactions. Catabolic pathw ays favour the
excretion (through urine, for example) of the lighter isotope,
resulting in an enrichm ent in anim al tissu e s in 15N relative to
plants. This progressive enrichm ent increases along advancing
trophic levels. (Ehleiinger and Rundel, 1989; Minagawa and
Wada, 1984 quoted by Lesage et al., 2001; Peterson and Fry,
1987).

An important advantage of th is approach is that the isotope ratios

from the tissu e s are derived from assim ilated food (and not ju st
ingested food) and therefore reflects dietary input over time (Das
et al., 2000a). This approach can also be a drawback, however, a s
it does not allow u s to identify exactly w hich species h a s been
consum ed. Previous knowledge of possible prey species is
therefore necessary to com plem ent the resu lts obtained through
th ese analyses for trophic level studies.

The u se of stable isotopes to study marine m am m als is recent,

b u t not altogether new. In the past decade we have w itnessed the
increase in the u se o f th is technique for stu d ies concerning
m igrations, trophic ecology, pollution and even paleontology.

Hobson and Welch (1992) characterized the trophic relationships

of the food web in the Canadian arctic region of Lancaster Sound
usin g stepw ise increase in 15N (and to a lesser extent 13C) to

65
 General Introduction

determine the trophic level o f the anim als concerned (zooplankton

to marine m am m als). This past year, (Lesage et al. 2001)
published a stu dy on m arine m am m als and the com m unity
structure of the Estuary and G ulf of St Lawrence in Canada, also
based on stable isotope analysis. They show ed that marine
m am m als occupy the highest trophic levels in the food web (using
15N), although overlapping one another, and that an enrichm ent
in 13C occurs in anim als from the Estuary relative to those from
the Gulf.
Atwell et al. (1998) u sed 515N to characterise the trophic
relationships in an arctic food web (ranging through particulate
organic matter to sea ls and polar bears) in order to determine
that food web’s relationship with total Hg concentrations in the
sam e tissu es, with the objective of studying Hg bioaccum ulation
along the food chain and with age.

Shell et al. (2000) m easured 13C values in keratin in bowhead

w hale baleen from the w estern arctic environm ent (obtained from
hunting records and m u seu m s, records extending to 1947),
w hich can be found in growth layers. These data are
representative of secondary production in the Bering Sea
ecosystem and show that the ecosystem h a s undergone a
significant decrease in average primary productivity.

In recent decades, the u se of stable isotope analysis for ecological

stu d ies h a s been steadily increasing:

- tracer stu d ies to determine migration patterns (reviewed

by Hobson, 1999) a s ‘anim a ls that move betw een isotopically
different food w ebs can retain information of previous feeding
locations for periods that depend on the elem ental turnover rate
for the tissu e of interest’;

- trophic ecology (Hobson and Welch, 1992; Hobson, 1999;

D as et al., 2000a; Kelly, 2000; Lesage et al., 2001; Polischuk et
al., 2001) to understand the relationships between organism s;

- pollution stu d ies (Atwell et al., 1997; Thom pson et al.,

1998a,b; Bearhop et al., 2 0 0 0 , Wayland and Hobson, 2001)
especially pollutant transfer through the ecosystem .

Although th is dramatic increase in the application of stable

isotope analyses to ecological research, th is field is still in its
infancy and continu ous data are needed before getting a better
understanding o f the fundam ental principles governing the

66
 Chapter 1

behaviour o f stable isotopes in ecosystem s and their potential use

in solving ecological questions (Hobson and W assenaar, 1999).

67
 General Introduction

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Vervey, J ., Wolff, W .J. 1981a. The W agem ann, R., S tew art, R.E.A.,
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Wolff, W .J. (Eds). Marine m am m als w h ales (Delphinapterus leucas) from
from the W adden S e a B alkem a, v ario u s lo catio n s in th e C an ad ian
R otterdam , pp. 51-58 Arctic a n d th e S t Law rence River. In:
G eraci, J ., S m ith , T.G. (eds).
W ada, E., T eragaki, M., K obaya, Y., A dvan ces in research on the beluga
Nemoto, T. 1987. >5N a n d « C w hale, D elphinapterus leucas Can.
a b u n d a n c e s in th e Artie O cean w ith Bull. Fish. A quat. Sei. 224: 191-206
e m p h a s is on th e biogeochem ical
s tru c tu re of th e food web. D eep-Sea W agem ann, R ., S tew art, R.E.A.
Res. 34 : 829-841 1994. C o n ce n tra tio n s o f heavy
m eta ls a n d soleni inn in tissu e s an d
W agem ann, R., Snow , N.B., Lutz, A., som e foods o f w a lru s (O dobenus
Scott, D.P. 1983. Heavy m etals in rosm arus rosmarus) from th e
tis s u e s a n d o rg an s o f th e n arw h a l e a ste rn C a n a d ia n Arctic a n d sub-
(Monodon monoceros). C an. J . Fish. Arctic, a n d a sso c ia tio n s betw een
Aquat,. Sei. 40 : 20 6 -2 1 4 m etals, age a n d gender. Can. J .
W agem ann, R. a n d M uir, D.C.G. Fish. A quat. Sei. 51: 4 2 6 -4 3 6
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m etals a n d o rg an o ch lo rin es in W elch, H., In n es, S. 1995. Arctic
m arin e m am m als of N o rth ern m arin e m a m m als a s in teg rato rs
w aters: overview a n d ev aluation. a n d in d ica to rs of m ercury in th e
C an. T ech. Rep. F ish. A quat. Sc. Arctic. W ater, Air, a n d Soil Pollut.
N°1279: v+97p 80: 68 3 -6 9 3
W agem ann, R., H u n t, R., W agem ann, R., In n es, S., R ichard,
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Comp. Biochem . Physiol. 78C: 301-
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G., L ockhart, W.L. 1998.
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m etalloprotein s in tis s u e s of th e Sei. T otal E nviron. 218: 19-31
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Comp. Biochem . Physiol. 84C: 325- W andrey, R. 1999. G uide des
344 m am m ifères m a rin s d u m onde.
D elach au x e t N iestlé SA. 284p

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W ebb, M. 1987. Toxicological

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E x p erien tia 52: 109-134
W hanger, P.D., D eagen, J.T . 1983.
Effect o f dietary m erc u ry levels a n d
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m etallothionein: m ercu ry bin d in g
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WHO 1992. C adm ium .
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World H ealth O rganization, G eneva
Wolfe, M.F., S ch w arzb ach S.,
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com prehensive review. Environ.
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Wolff, W .J. 2000a. T he s o u th ­

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2000 years. Biol. C onserv. 95: 209-
217
Wolff, W .J. 2000b. C a u ses of
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Yam am oto, Y., H onda, K., H idaka,
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164-169
Y oshida, M., O hta, H., Y am auclii,
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Biol. T race Elem. Res. 63: 167-175
Zelikoff, J.T ., T hom as, P.T. 1998.
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82
CHAPTER 2: TUNA AND DOLPHIN A SSO C IA TIO N S
IN THE NORTHEAST ATLANTIC; EVIDENCE OF
DIFFERENT ECOLOGICAL NICHES FROM STABLE
ISOTOPE AND HEAVY METAL MEASUREMENTS

After D as K., Lepoint, G., Loizeau,

V., Debacker, V., Dauby, P.,
B ouquegneau, J.M. 2000. Marine
Pollution Bulletin 40: 102-109
 Chapter 2

ABSTRACT

A ssociations of tu n a s and dolphins in the wild are quite frequent

ev en ts and th e question arises how predators requiring sim ilar
diet in the sam e habitat share their environm ental resources. As
isotopic com position of an anim al is related to that of its preys,
stable isotop es (613C and ô 15N) an alyses were performed in three
predator sp e c ie s from th e Northeast Atlantic: the striped
dolphin, Stenella coeruleoalba, the com m on dolphin D elphinus
delphis, and th e albacore tuna, T hu n n u s alalunga, and compared
to their previously described stom ach content. Heavy m etals (Cd,
Zn, Cu and Fe) are m ainly transferred through the diet and
therefore have also been determ ined in th e tis s u e s of the
anim als. Tuna m u scles display higher 815N th an in com m on and
striped dolphins (mean: 11.4%o us. 10.3%o and 10.4%o,
respectively) w h ich reflects its higher trophic level nutrition.
Higher 813C are found in com m on (~18.4%o) and striped dolphin
(-18.1%o) m u scle s th an in albacore tu n a (-19.3%o) likely in
relation with its m igratoiy pattern. The m ost striking feature is
th e presence of two levels of cadm ium concentrations in the
livers of the tu n a s (32 jig.g 1 vs. 5 p g .g 1 dry weight). T hese two
groups also differ by their iron concentrations and their 81SN and
813C liver valu es. These resu lts su ggest that in th e Biscay Bay,
tu n a s occupy two different ecological n ich es likely based on
different squid input in their diet.

83
 Tunas and dolphins from th e N ortheast Atlantic

2 .1 . INTRODUCTION

Sm all pelagic dolphins are com m only a ssociated w ith tu n a s in

m ixed -sp ecies aggregations. These polyspecific asso cia tio n s have
been extensively stu died in the tropical Pacific (Perrin et al.,
1973; Au and Pitman, 1986; Au, 1991; Scott and Cattanach,
1998) and com prise several sp ecies, like tu n a s, marine
m am m als, seabirds or sharks. In general, th e se polyspecific
aggregations seem to form w h en social sp ecies o f sim ilar foraging
h abits join to form larger groups to increase feeding su c c e s s and
to better avoid predators (Scott and Cattanach, 1998). These
anim als are know n to feed, interact and travel together for
various periods of tim e (Au, 1991). In the feeding aggregations
observed in tropical w aters, prey is driven to the surface by
yellowfin tu n a s and is abundant and diverse en ou gh to allow
dolphins and other sp ecies like seabirds and sh ark s to feed at
the sam e tim e (Au and Pitman, 1986).

In th e Northeast Atlantic three predator sp ecies are often caught

together in th e sam e n e ts during com m ercial sein in g operations:
the albacore tu n a (T h u n n u s alalunga), the striped dolphin
(Stenella coeruleoalba) and the com m on dolphin (Delphinus
delphis). As they are know n to feed together, the q u estion arises
about the p ossib le com petition or com m ensal relationship that
m ight occur betw een them . These kinds of alim entary stu d ies
are often performed by stom ach content an alysis (Perrin et al.,
1973; H assani et al., 1997) or field observations (Au and Pitman,
1986). However, th e se u sefu l data reflect the m ost recent m eal
and m ay not be representative o f the overall diet.

In m arine m am m als, th e u se of naturally occurring stable

isotop es of carbon and nitrogen h a s provided com plem entary
data to their feeding ecology (Hobson and W elch, 1992; Abend
and Sm ith, 1995; Sm ith et al., 1996; H obson et al., 1997; B u m s
et al., 1998). Stable isotope an alysis h a s em erged a s a powerful
tool to trace diet a s isotope ratios of a consum er are related to
th ose of their preys (De Niro and Epstein, 1978; 1981; Peterson
and Fry, 1987). Indeed, the carbon and nitrogen isotope valu es
(813C and 815N) differ betw een organism s and their d iets because
of a slight selective retention of the heavier isotope and excretion

84
 Chapter 2

of the lighter one. As a result, organism s have a higher 8 value

than their diet. N itrogen-15 typically sh ow s a stepw ise increase
w ith trophic level w ithin a food chain (Hobson and Welch, 1992;
Cabana and R asm ussen, 1994; Thom pson et al., 1995) w ith a
trophic enrichm ent value of about 3%o (Hobson and Welch,
1992; M ichener and Schell, 1994). On the contrary, an anim al
h a s 513C v alu es close to that of its diet (De Niro and Epstein,
1978; H obson and Clark, 1992; Thom pson et al., 1995; Sm ith et
al., 1996). T hus, carbon-13, rather th an being a reliable
indicator of trophic level, is generally u se d to indicate relative
contributions to the diet of different potential primary sou rces in
a trophic network (Rau et al., 1992; Havelange et al., 1997;
Dauby et al., 1998), indicating for exam ple the aquatic vs.
terrestrial, inshore vs. offshore, or pelagic vs. benthic
contribution to food intake (Hobson et al., 1995; Sm ith et al.,
1996).

B ecause stable isotop es ratios of con su m ers’ tis s u e s are derived

from assim ilated food, the tissu e reflects dietary input integrated
over tim e, not ju s t th e last food intake. The m etabolic rates and
th e su b seq u en t turn-over of elem en ts differ betw een liver and
m uscle, allowing the evaluation o f diet integrated betw een
different periods of tim e (e.g., days, w eek or m onths) (Tieszen et
al., 1983; H obson et al., 1996; 1997).

We had the opportunity to u s e sam p les from three sp ecies from

the Northeast Atlantic: the striped dolphin, the com m on dolphin
and the albacore tuna, w h ose stom ach co n ten ts have been
previously determ ined by H assani et al. (1997). In order to
contribute to the know ledge of ecological a sp ects of polyspecific
associations, we have stu died their isotopic com position (515N
and S13C). Com bined to the isotopic ratio determ inations, heavy
m etals analysis have b een performed in the tis s u e s of the three
predators. Heavy m etals are m erely transferred through the diet.
Indeed, heavy m etal levels found in m arine organism s depend
not only on th e environm ental c o n t a m i n a t i o n but also on several
other ecological or physiological factors (Bouquegneau and
Joiris, 1988; André et al., 1990a; 1990b, Caurant et al., 1994),
am ong w hich th e diet and the position in the trophic web are
determ ining elem en ts (Bouquegneau and Joiris, 1992; Cabana
and R asm ussen, 1994; Kidd et al., 1995; Stewart et al., 1997).

By u sin g a com bination of stable isotop es and heavy m etals

analysis, our aim w as to compare th e diet and the position in the

85
 Tunas and dolphins from th e N ortheast Atlantic

trophic web of three predators from the Northeast Atlantic, the

striped dolphin, th e com m on dolphin and the albacore tu n a and
to contrast our resu lts w ith stom ach conten t an alysis previously
described by H assani et al. (1997).

2 .2 . MATERIALS AND METHODS

2 .2 .1 . C o l l e c t io n and storage

The 23 striped dolphins (Stenella coeruleoalba) (10 fem ales and

13 males) and 10 com m on dolphins (D elphinus delphis) (5
fem ales and 5 m ales) u se d in th is study originated from the Bay
of Biscay in the Northeast Atlantic. The locations of th e captures
covered an area betw een 46° to 50° North and 9° to 20° West.
They were accidentally captured during com m ercial tu n a seining
operations from May to Septem ber 1993, from w h ich 20 albacore
tu n a s (T hunnus alalunga) (9 fem ales and 11 m ales) were
collected. This collection w as conducted by the Institut Français
d e R echerche p o u r l’Exploitation d e la M er (IFREMER) from Brest
(France) to a s s e s s ecological risk linked to the Northeast Atlantic
French drif net activity (Goujon et a l, 1993; Goujon, 1996).

T issue sam plings were performed at the fishing site and frozen
im m ediately in liquid nitrogen and th en kept in a freezer (-20°C)
until storage in the O ceanology Laboratory (Liège, Belgium).

Dolphin se x and age determ inations were realised by Collet

(1993a; 1993b) who show ed an exponential length-age
relationship (see also Di-Méglio et al., 1996). The ages of
dolphins were estim ated by counting tooth growth layer groups
(GLGs). The total len gth from the sn o u t to fluke notch were
determ ined for each individual by G oujon (1996). All tu n a
sp ecim en s are ju v e n ile s (4-5 years old) with len gth ranging from
75 to 81 cm w hile the ages of the dolphins range from 0.1 to 20
years old (Collet, 1993a; b).

86
 Chapter 2

2 .2 .2 . A n a l y t i c a l m e t h o d s

Zn, Cd, Fe and Cu analyses

After being w eighed and dried for 48 h at 110°C, sam p les were
digested w ith a m ixed solu tion of hydrochloric (Merck 317) and
nitric (Merck 456) acids (1:3, v:v) and slow ly heated to 100°C
un til com plete digestion. Atomic absorption spectrophotom etry
(ARL 3510) w a s u se d to determ ine heavy m etal concentrations
(Cu, Zn, Cd, Fe). Pb, Ni, Cr con ten ts were also determ ined but
the resu lts m ost often were below th e detection lim its
(unpublished data). C oncentrations are exp ressed a s fig-IT1 dry
w eight (dw).

Parallel to the sam ples, a se t of certified m aterial sam p les (CRM

278 Com m unity Bureau of Reference, C om m ission of the
European Com m unities) w a s also analysed to en su re the
m ethod’s sensitivity. Recoveries ranged from 92 to 102 % for Cu,
Zn, and Fe and 88% for Cd. Limits of detection were 0.01 fig g-1
dw for Cu, 0.33 for Zn, and 0.22 for Cd.

Stable isotope m easurem ents

O rganism s m ay vary in their concentrations of lipids. As lipids

have been show n to be depleted in 13C relatively to the diet
(Tieszen et ál., 1983), they were extracted from sam p les u sin g
repeated rin ses with 2:1 chloroform : m ethanol prior to analysis.
After drying at 50°C (48h), sam p les were ground into a
h om ogen eou s powder. Carbon dioxide and nitrogen g a s were
analysed on an V.G. Optima (Micromass) IR-MS coupled to a N-
C-S elem ental analyser (Carlo Erba). Routine m easu rem en ts are
precise to w ithin 0.5%o for 13-carbon and 0.6%o for 15-nitrogen.
Stable iso to p es ratios were expressed in 5 notation according to
the following:

S X = [ ( R s a m p l e / R s ta n d a r d ) - 1] X 1000

w here X is 13 C or 15N and R is th e corresponding ratio 13C / 12C or

15 N / 1 4 N>

Carbon and nitrogen ratios are expressed relative to the VPDB

(Vienna Peedee Belem nite) standard and to atm ospheric
nitrogen, respectively.

87
 Tunas and dolphins from th e Northeast Atlantic

2 .2 .3 . D a t a treatm ent

Parametric and n on parametric te sts were u se d to compare

different groups: Kolmogorov-Smirnov te st w as u se d to assu m e
the norm ality of the data. ANOVA followed by p ost-hoc m ultiple
com parison te s ts have b een u se d to compare the data betw een
the different sp ecies. W hen the n ecessary a ssu m p tion s to realise
ANOVA were not gathered (normality o f the variables and
hom ogeneity of variances), Kruskall-W allis w as u se d followed by
m ultiple com parisons based on the Kruskall-Wallis rank su m s to
test for pairwise differences am ong sp ecies. M ann-W hitney U-
test w as performed to compare differences am ong s e x e s or
groups w h en variances were not hom ogenous. Spearm an
coefficient h a s been u se d to te st correlations betw een the
values. R esults were judged significant w h en P<0.01 (Scherrer,
1984).

2 .3 . RESULTS

2 .3 .1 . M e t a l level in t h e t iss u e s

In dolphin and tu n a tis s u e s , Zn, Cd and Cu and Fe m ean

concentrations are higher in liver than in m u scle (Table 2.1). In
dolphins, Cd displays the h ig h est m ean concentration in kidney.
Fe is always higher in th e blood due to the haem oglobin
respiratory pigm ent. In both m am m al glands and te ste s, iron
and zinc were m ore concentrated th an copper and cadm ium . Zn
concentrations in the liver are sim ilar for the three sp ecies
(ANOVA, F=1.6, p>0.5). Hepatic copper levels are not
significantly different for th e two dolphin sp ecies but tu n a s
display lower copper concentrations th an com m on dolphins
(ANOVA: F=3.07, p<0.01). Hepatic iron concentrations are
con sisten tly greater th an th ose in com m on dolphins, w h ich in
turn are greater than th ose in albacore tu n a s (ANOVA: F=0.94,
p=0.04). Cadmium concentrations in the livers are not
significantly different for th e three sp ecies (ANOVA: F=0.3,
p>0.05). The standard deviations of cadm ium m ean

88
 Chapter 2

concentrations both in kidney and livers of dolphins (Table 2.1)

are very large.

Table 2.1. Trace element concentrations Qig.g1 dw), 5 1JC and 51SN (%o) of
three main predators (Stenella coeruleoalba, Delphinus delphis, Thunus
alalunga) of the Northeast Atlantic expressed as a mean ± standard
deviation, range of concentrations (minimum-maximum); n: number of
samples; nd: non determined

Species n Zn Cd Cu Fe 0 13C ô15N

167 ± 7 8 1 7 1 15 43154 974 1 363 -17.81 05 1 0.810.6

Liver 22
(33-385) (0.2-51) (7-272) (393-1792) (-18.9/-17.08) (9.4-11.9)
41 ± 16 0 .3 7 1 1 6 1 1 .4 5 771221 -18.110.5 10.410.75
Muscle 23
(27-101) (<0.05-5) (1-9) (96-1131) (-19.27-17.2) (8.8-12.1)
113124 91157 1916 7161226
Striped dolphin Kidney 23
(76-168) (0.1-199) (9-30) (413-1413)
Stenella coeruleoalba
71130 1.3 1 0 .9 4 .5 1 1 273169
Mammal gland 4
(47-115) (0.4-2) (3-6) (187-344) nd
Testis 2 45-157 0.6-3.2 3-3.2 120-215
2 3 1 12 0.6510.7 2 .8 1 1 .3 25101264
Blood 12
(12-52) (<0.05-2.5) (1.5-6) (2034-2972)
153127 15117 32112 7061227 -17.81 0.5 11.11 0.4
Liver 10
(117-197) (0.4-58) (19-52) (326-1096) (-18.9/-17.3) (10.4-11.8)
50146 0 .1 1 0.2 511 470 1 95 -18.41 0.5 1 0.310.3
Muscle 10
(27-181) (<0.05-0.5) (3-7) (278-560) (-19.1/-17.7) (9.8-10.8)
109121 7 1 1 61 201 8 7071224
Common dolphin Kidney 10
(86-146) (3-169) (27-181) (396-1032)
Delphinus delphis
74117 0 .6 1 0.9 3 .5 1 1 .4 1881 9 0
Mammal gland 4
(56-97) (<0.09-2) (2-5) (110-305) nd
Testis 1 99 3.1 4.2 278
2119 11 1 412 22891 504
Blood 8
(13-40) (<0.07-4) (2-9) (1153-2729)
1521 46 17 1 14 1216 4 2 9 1 343 -18.41 0.8 10.81 0.8
Liver 20
Albacore tuna (107-265) (3-41) (5-29) (101-1391) (-19.8/-16.9) (9.4-12.7)
Thunnus alalunga 1613 1 1 0 .6 49110 -19.31 0.6 11.41 0.4
Muscle 12 <0.1
(12-21) (0.5-3) (28-65) (-20.4/-18.5) (10.9-12.2)

Metal levels are sim ilar for both genders in the three sp ecies
(data not shown), but a significant increase of th is m etal level
w ith length of individuals appears in the livers of both striped
and com m on dolphins (figure 2.1).

89
 Tunas and dolphins from th e N ortheast Atlantic

60
striped dolphin-Spearm an R=0.
“( X com m on dolphin-Spearm an R=
50

40
?
T3
V
D)
30
CD
3
T3 2 0
Ü
•O
10

oo
0
80 100 120 140 160 180 200 220 240
length (cm)

Figure 2.1. Relationship between the length of the dolphins and cadmium
concentrations in livers

Su ch a relationship w as not found betw een the size of th e tu n a s

and their hepatic cadm ium concentration s (figure 2.2).

45
40
35
„ 30
S
^ 25
àCJ) 20
CL
^ 15
O
10
5
0
73 74 75 76 77 78 79 80 81 82
length (cm)

Figure 2.2. Relationship between the length of the tunas and the cadmium
concentrations in the liver and. The black plots are the cadmium
contaminated group.

The m ost striking feature of figure 2.2 is that the tu n a group is

heterogeneous. One group o f tu n a s is heavily contam inated by

90
 Chapter 2

cadm ium (mean 32 ng.g-1 dw) w hile the other one displays lower
cadm ium levels (mean: 5 Mg.g-1 dw; M ann-W hitney U test:
p<0.001). Zinc and copper are sim ilar for both groups w hile iron,
515N and 513C are significantly different (Table 2.2). In the
m u scles, there are no significant differences betw een the two
groups of tu n a s w here cadm ium concentrations were always
under the detection limit.

Table 2.2. Zn, Cu, Cd, Fe (g g .g 1 dw), 5 1SN and S13C values in the livers of
the two groups of tunas

Cd contaminated group Low Cd contaminated group

n=l 1 n=9
Zn 159 ± 4 146 ±41
Cu 13 + 5 10 ± 7
Cd* 32 ± 4 5± 1
Fe* 722 + 312 189 ± 7 2
5 ,SN* 11.5 ± 0.6 10.2 + 0.5
S°C* -18.1 ±0.9 -18.7 ±0.5
‘significantly different with p<0.001 (Mann-Whitney U test)
mmp=0.05

W hen the three sp ecies are com pared together (Table 2.1), tu n as
display lower zinc and copper concentrations in m u scles than
the two dolphins sp e c ie s (Kruskall-Wallis: p<0.001and ANOVA:
F=1.4, p<0.001, respectively).

2 .3 .2 . S t a b l e is o t o p e a n a l y s e s

Isotopic com positions are sim ilar for both s e x e s in the three
sp ecies and no effect of age h a s b een observed (p>0.05, data not
show n).

Tunas display significantly higher 815N in m u scle than striped

and com m on dolphins: 11.4%o vs. 10.4 and 10.3%o,
respectively (ANOVA: F=3.3, p<0.0001 and p< 0.001 respectively,
Table 2.1). The m ean 6 15N v alu es in m u scle s do not vary
significantly betw een the dolphin species: 5,5N of +10.3%o in the
m u scle s of com m on dolphins and +10.4%o in the m u scles of
striped dolphins (ANOVA: p>0.05).

91
 Tunas and dolphins from th e N ortheast Atlantic

T unas display a m u scle 513C significantly lower th an dolphins

(-19.3%o versu s -18.1 and 18.4%o for striped and com m on
dolphins respectively (ANOVA: F=0.6, p<0.001 and p=0.001
respectively).

Individual resu lts of 515N and S13C are plotted in figures 2.3 and
2.4. In the m u scles, specific regrouping appears: tu n a s are
com pletely isolated from dolphins w ith a higher 815N and a lower
S13C (figure 2.3).

13

12

10

0 strip ed dolphins
9 O com m on dolphin:
+ tu n a s

■21 -20 -19 -18 -17

8i3C ( % o)

Figure 2.3. 8 13C and 8 lsN in the muscles of tunas, striped and common
dolphins from the Northeast Atlantic

The com m on dolphin data are also concentrated in a sm all area

of the figure (m edium S15N and 813C values) w hile the striped
dolphin displays a wide range of 815N v a lu es overlapping the two
other species.
In livers, no regrouping betw een individuals from the sam e
sp ecies occurs (figure 2.4).

92
 Chapter 2

13

12

Z 11
o
+ +
QB
10
+ H*
0 strip ed dolphins
O com m on dolphins • V
+ tu n a s

-21 -20 -19 -18 -17

S13C (%o)

Figure 2.4. S13C and 81SN in the livers of tunas, striped and common
dolphins from the Northeast Atlantic

However, tu n a s with low cadm ium contam ination display lower

515N valu es th an contam inated tu n a s (ANOVA: p<0.0001),
striped and com m on dolphins (ANOVA: p=0.02 and p<0.001
respectively; Table 2.2, figure 2.5 and 2.6). Com m on dolphins
display m edium 515N m ean va lu es betw een striped dolphins and
tu n a s with high cadm ium levels, th e difference betw een th ese
latest being significant (ANOVA, F= 1.17, p=0.002).

o C (%o)

Figure 2.5. Comparison of the stable isotope values in livers between highly
(•) and low-contaminated groups of tunas (o)

93
 Tunas and dolphins from the N ortheast Atlantic

813C m ean valu es in the livers are significantly lower for tu n a s

w ith low cadm ium levels compared to other tu n a s (ANOVA:
p=0.02), striped dolphins (p<0.001) and com m on dolphins
(p<0.002). The 813C m ean v alu es are sim ilar for tu n a s
contam inated by Cd, striped and com m on dolphins (p>0.05).

2 .3 . DISCUSSION

W hen sp ecies require sim ilar resou rces in the sam e habitat a s
striped and com m on dolphins and albacore do in the Biscay Bay,
they ten d to reduce th e com petition by occupying different
physical locations or feeding on different preys, a s it h a s already
been observed for other sm all od on tocetes (Gowans and
W hitehead, 1995). A comparative dietaiy stu dy of albacore and
both dolphin sp ecies from Northeast Atlantic h a s b e e n realised
and published by H assani et al. (1997). These auth ors have
show n that com m on and striped dolphins have a more
preferential cephalopod diet. Fish ranked secon d in th e food of
the com m on dolphin and only third in the striped dolphin, in
w hich cru stacean s were more important in relative abundance.
Fish present in dolphin stom ach were m ostly sm all m esopelagic
fish a s Myctophidae or Sternoptychidae. The food o f albacore
w as m ainly fish (m ostly the sm all Sternoptychidae M aurolicus
muelleri) and to a lesser extent, cru stacean s (H assani et al.,
1997). However, the com m on dolphin sam p les includ ed all the
fish sp ecies found in the diet of the albacore w h ereas only one
sp e c ie s of prey w as found in both striped dolphin and tu n a s
stom achs. Each sp e c ie s se em s to have preferential preys but it
h a s b een dem onstrated that striped and com m on dolphins are
quite opportunistic feeders and can take advantage of season ally
or locally abundant prey (Würtz and Marrale, 1993; Blanco et al.,
1995; Young and Cockroft, 1995). For exam ple, in th e Northwest
Atlantic, the diet of com m on dolphins c o n sists m ainly of bigger
fish a s cap elins (Mallotus villosus) and m ackerels (Scom ber
scom brus) (Overholtz and Waring, 1991; Ostrom et al., 1993).
Isotopic data on delphinid s are scattered (Hobson and W elch,
1992; Ostrom et al., 1993). Ostrom et al. (1993) have found in
the m u scles of one com m on dolphin from the W estern North
Atlantic a 815N and a 813C of 14.8 and -17.8% o, respectively. Our

94
 Chapter 2

isotope v alu es in m u scle s of com m on dolphin are low compared

to th ese data. T hese high S15N v alu es m easured by th e se authors
are con sisten t w ith a higher trophic levels and th e piscivorous
diet of com m on dolphin in the stu died geographical area (Ostrom
et al., 1993).

Our isotopic data in m u scles w h ich integrate a long-term

nutrition, also su ggest a specific diet: tu n a s are com pletely
isolated from the other two sp ecies with a higher 815N w hich
reflects their higher trophic level nutrition com pared with
dolphins (figure 2.3). The com m on dolphin data are also
concentrated in a sm all area o f the figure w hile the striped
dolphin displays a wide range of S15N. This large dispersion of
the individual data may reflect more opportunistic trophic habits
of striped dolphins compared to com m on dolphin.

The trophic position of the tu n a is rather u ncom m on becau se

generally, in stable isotope stu d ies, m arine m am m als display the
h igh est 515N (Hobson and Welch, 1992; Ostrom et al., 1993;
H obson et al., 1995). Moreover, tu n a s exhibit lower 513C valu es
th an both dolphin sp ecies. Both 515N and 813C v a lu es in m u scles
indicate that different sou rces o f food take part in the diet of
tu n a s compared to striped and especially com m on dolphins
despite a su b stan tial overlap in diet. T unas presen t a strong
m igratoiy cycle: during the spring, they are found offshore from
the Azores and th en migrate w ithin the Bay of Biscay during May
to A ugust (quoted by Goujon, 1996). M easured lower S13C data
presum ably reflect a greater reliance on offshore food in relation
w ith th is migration. Stable carbon isotop es have proven m ost
u sefu l in identifying where particular organism s feed, and 813C
v alu es are typically higher in coastal or benthic food w eb s than
in pelagic food w eb s (Hobson et al., 1995; 1997).
The considerable overlap in valu es am ong th e three sp ecies and
the absence of specific regrouping in the livers (figure 2.4)
indicate isotopic s imilarity am ong their respective recent diets.

95
 Tunas and dolphins from the N ortheast Atlantic

Figure 2.6. Comparison o f 51 5 n in livers o f striped dolphins (s), common dolphins (c), tunas
contaminated by cadmium (t*) and other tunas (t). Arrows indicate significant decreasing
difference between groups (ANOVA).

From an ecotoxicological point of view, anim als feeding on

cephalopods ten d to concentrate higher cadm ium content
(B ouquegneau and Joiris, 1992; Caurant et al., 1994;
B ouquegneau et al., 1997). Indeed, cephalopods have been
show n to concentrate high level of th is m etal (Law et al., 1997).
Therefore, it is quite striking to find sim ilar cadm ium levels in
the livers of th e three sp ecies a s veiy few squids are found in the
tu n a diet (H assani et al., 1997). However w h en we go through
individual tu n a resu lts (figure 2.2 and table 2.2), it appears that
there are two levels of iron and cadm ium in the livers of tunas:
one group is highly contam inated by cadm ium a s the other one
displays low levels. There is no relationship betw een the size of
the tu n a s and the hepatic cadm ium concentrations. On the
other hand, there is an increase of hepatic cadm ium
concentrations of both dolphin sp ecies w ith len gth (figure 2.1),
w h ich reflects a con stan t intake o f cadm ium contam inated preys
associated w ith a long half-tim e o f elim ination of th e pollutant
(10 to 30 yr. a s quoted by W agem ann et al., 1990). The absence
of su c h a correlation in the tu n a s su g g e sts that th e high hepatic
cadm ium conten t m ight n ot reflect a chronic contam ination but
be th e resu lt o f a p un ctual in gestion o f cadm ium contam inated
preys. These r esu lts are en h anced by S15N and S13C differences
in th e livers of tu n a s (figure 2.5 and table 2.2). It clearly appears
that isotopic com position differs strongly betw een th e cadm ium
contam inated group of tu n a s and the secon d one, indicating that
two different food habits were coexisting sym patrically and
temporally. Differences in iron concentrations betw een the two
groups of tu n a s are still to be elucidated. Cadmium levels and

96
 Chapter 2

isotope ratios in m u scles are sim ilar in the two groups of tu n as

suggestin g that th ese feeding h ab its m ight be relatively recent.
D espite an apparent large overlap in stable iso to p es v alu es in
livers of the three sp e c ie s (figure 2.4), different groups can be
pointed out (figure 2.6). T unas w ith low cadm ium contam ination
display lower SI5N and 813C m ean v a lu es th an others tu n a s and
both dolphins, su ggesting a greater contribution of lower trophic
level preys in their diet. On the contrary, com m on dolphins and
tu n a s contam inated by Cd display sim ilar 813C and §15N valu es
in their liver confirm ing that tu n a s share more prey sim ilarities
w ith com m on dolphins th an striped dolphins (H assani et al.,
1997). In fact, com m on dolphins seem to occupy a m edium
trophic position betw een striped dolphins and tu n a s with high
Cd levels (figure 2.6). These observed differences com bined with
cadm ium concentrations in livers likely resu lt from differential
reliance on squids. Our observations are en h an ced by stom ach
an alyses carried out by H assani et al. (1997) who have suggested
that the diet of the tu n a may differ: w h en tu n a w as captured
alone or in the sam e n e ts a s com m on dolphins, fish w as
predom inant in the stom ach as described earlier. However, th ese
authors have noticed that, w h en tu n a s were captured in the
sam e n e ts th an striped dolphins, their stom ach c o n ten ts becam e
sim ilar to dolphins with a high squid content suggestin g that
feeding behaviour of albacore m ay differ w h en it co-occurs with
striped dolphin. On the contrary, the p resen ce of com m on
dolphins w ould have no influence on the feeding habits of tu n as
(H assani et al., 1997).

515N, S13C, Fe and Cd also su ggest different feeding habits

betw een th ese three predators. While striped and com m on
dolphins seem to differ little in their diet, tu n a s seem to occupy
two potential different ecological n ic h e s in th e Bay of Biscay.
T hese different n ic h e s are based on at least two typ es of preys,
influencing both their isotope v a lu es and their cadm ium and
iron content in livers. These two striking behaviours were
relatively recent from the collection of the sam p les, in agreem ent
w ith the rapid turn-over of livers compared to m u scles. An
experim ental stu dy carried on gerbils (M eriones unguiculatus)
h a s determ ined a faster turn-over rate of carbon in livers (half-
life: 6 .4 days) than in m u scles (half-life: 27.6 days) (Tieszen et
al., 1983). This behaviour is likely to be in relation with the
migration pattern of the tunas. W hen tu n a s get through the
Biscay Gulf, som e individuals becom e associated with dolphins

97
 Tunas and dolphins from the N ortheast Atlantic

sharing sim ilar prey preferences a s sq u id s suggestin g a potential

com petition. For unclear reason s, som e tu n a s display different
feeding particularities m ainly based on sm all Sternoptychidae.
As tu n a feeding behaviour may differ in relation w ith the dolphin
sp e c ie s it is associated to, two h yp o th eses can be form ulated
considering th e se tu n a s w h ich recently fed on Sternoptychidae :

1. T unas and com m on dolphins were hun ting together prey

that were abundant and varied enough to allow specific
preferences w ithin a com m on d o lp h in /tu n a sch ool or, as
su ggested by H assani et al. (1997)

2. These tu n a s were not associated w ith dolphins.

To conclude, com bined stable iso to p es and heavy m etals

a n alyses appear a s powerful tool for delineating trophic
relationship w ithin th e se m ixed -sp ecies aggregations. Further
research on other organism s from th is area will help to get a
better understanding on the transfer of heavy m etals and
different alim entary relations w ithin high trophic levels.

Acknowledgem ents. T h a n k s to M r Alain A b am o u (IFREMER, B rest) a n d h is

com m ents: w ith o u t him tliis w ork would n o t have b een possible. We a ie g rateful to
Mr Biondo (Oceanology Laboratory, Liège, Belgium) for h is v alu ab le technical
a ss ista n c e a n d to Mr. M ichel G oujon, Loïc A ntoine (IFREMER, B rest, France),
V incent Ridoux a n d S am i H a ssan i (Oceanopolis, B rest, France) w ho collaborated to
perform all th e sam p lin g a n d collect th e biological inform ation. T he p erso n al
co m m en ts of S am i H a ssa n i o n dolphin a n d tu n a d iets helped u s to im prove th is
m a n u sc rip t. We also w ish to th a n k Mr H olsbeek a n d Pi' Jo iris from VUB [Belgium)
for th e ir valuable advice. T h a n k s also to M iss N. Poisseroux for h e r logistical
assistan ce.
T his study w as su p p o rted by g ra n ts from Modèle - Environnement , Fondation
Lefranc a n d from th e Belgian Office for Scientific, T echnical a n d C u ltu ral Affairs
(contract M N /D D /50).

98
 Chapter 2

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M esoplodon bidens, based on c o n c e n tratio n s in S h etlan d seabirds.
isotopic com p ariso n s am ong n o rth ­ Mai-, Ecol. Prog. Ser. 151: 237-244
w e ste rn A tlantic c eta cean s. Can. J .
Zool. 71: 858-861 T hom pson, D.R., F u rn e ss, R.W.,
Lewis, S.A. 1995. D iets a n d long­
Overholtz, W .J., W aring, G.T. 1991. term ch a n g e s in 815N a n d 813C
D iet com position o f pilot w hales v alu es in n o rth e rn fu lm a rs Fulm arus
G lobicephala sp. and com m on glacialis from tw o n o rth e a s t A tlantic
d o lp h in s D elphinus delphis in th e colonies. Mar. Ecol. Prog. Ser. 123:
m id-A tlantic B ight d u rin g sp rin g 3-11
1989. Fish. Bull. U.S. 89: 723-728
Tieszen, L.L., B outton, T.W.,
P errin, W.F., W arner, R.R., F iscus, T esdahl, K.G., Slade, N.A. 1983.
C .H., Holts, D.B. 1973. S to m ach F ractio n atio n and tu rn -o v e r of
c o n te n ts of porpoise, Stenella sp ., sta b le ca rb o n isotopes in an im al
and yellowfin tu n a , T hunnus
tissu es: im plication for 513C an aly sis
albacares in m ixed species of diet. Oecologia 57: 32-37

101
 Tunas and dolphins from th e N ortheast Atlantic

W agem ann, R., S te w a rt R.E.,

B éland, P., D esjard in s, C. 1990.
Heavy m etals a n d selen iu m in
tissu e s of b elu g a w hales,
Delphinapterus leucas, from th e
C an ad ian Arctic a n d th e St.
Law rence e stu a iy . C an. J . Fish.
Aquat. Sei. 224: 191-206

W iirtz, M., M arrale, D. 1993. Food of

strip ed dolphin, Stenella
coeruleoalba, in th e L igurian Sea. J .
Mar. Biol. Assoc. UK 73: 571-578

Young, D.D., C ockioft, V.G. 1995.

S to m ach c o n te n ts of stra n d e d
com m on d olphins Delphinus delphis
from th e so u th -e a st o f S o u th e rn
Africa. Z. Säugetierkd. 60: 343-351

102
CHAPTER 3: MARINE MAMMALS FROM NORTHEAST
ATLANTIC : EVALUATION OF THEIR TROPHIC
STATUS BV S13C AND 015N AND INFLUENCE ON
THEIR TRACE METAL CONCENTRATIONS

After K. Das, Beans, C., Holsbeek,

L., Mauger, G., Berrow, S.D., Rogan,
E. and Bouquegneau, J.M. Marine
Environmental Research. Accepted.
 Chapter 3

ABSTRACT

The relationship betw een trophic position through 513C and 815N
and trace m etal concentrations (Zn, Cd, Cu and Hg) h a s been
investigated in the tis s u e s of 6 m arine m am m al sp e c ie s from the
Northeast Atlantic: striped dolphin Stenella coeruleoalba,
com m on dolphin, D elphinus delphis, Atlantic w hite-sided dolphin
L agenorhynchus acutus, harbour porpoise Phocoena phocoena,
w hite beaked-dolphin Lagenorhynchus albirostris, grey seal
H alichoerus g ry p u s stranded on French C hannel and Irish
coasts.
W hite-beaked dolphins, harbour porpoises, white sided
dolphins, com m on and striped dolphins display the sam e
relative and decreasing trophic position, a s m easured by 815N
valu es, along both the Irish and French ch an n el coasts,
reflecting conservative trophic h abits betw een th e se two places.
Hepatic and renal Cd concentrations were significantly
correlated to m u scle 813C and 815N v alu es w hile Hg, Zn and Cu
did not. These resu lts su ggest that Cd accum ulation is partly
linked to the diet w hile other factors su c h a s age or body
condition m ight explain Hg, Zn or Cu variability in marine
mam m als. Com bined stable isotope and trace m etal analyses
appear to be u sefu l tools for the study of m arine m ammal
ecology.

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 Marine mammals from th e N ortheast Atlantic

3 .1 . INTRODUCTION

Over 40 sp ecies of cetacean s and pinnipeds occur throughout

th e Northeast Atlantic a s defined by the OSPAR Convention
(OSPAR, 2000). Among th ese sp ecies, the com m on dolphin,
D elphinus delphis, the striped dolphin, Stenella coeruleoalba, the
harbour porpoise, Phocoena phocoena, the w h ite-beaked dolphin,
L agenorhynchus albirostris, the w h ite-sided dolphin,
Lagenorhynchus acutus and the grey seal, H alichoerus g ryp u s are
regularly sighted w ithin the C hannel and Celtic S ea (Hammond
et al., 1995; Rogan and Berrow, 1996; OSPAR, 2000).

T hese anim als are considered to be top predators and their

primordial role in the structure of the m arine ecosystem h a s
often been su ggested (Bowen, 1997; B ouquegneau et al., 1997;
Pauly et al., 1998). Published inform ation on diet com position
and trophic sta tu s of th e se sp ecies in Irish w aters and the
French C hannel is sparse (Rogan and Berrow, 1996) and is
obviously n eeded to understand the role of m arine m am m als in
ecosystem dynam ics. Moreover, in m arine m am m als, diet is the
m ain pollutant contam ination pathway and m ight influence their
contam inant load (reviewed by Aguilar et al., 1999, D as et al.,
2 0 0 2 ).

Dietary stu d ies are often performed by stom ach conten t an alysis
or field observations. In m arine m am m als, the u se o f naturally
occurring stable isotop es of carbon (13C) and nitrogen (15N) h a s
recently provided n ew in sig h ts in the study of their feeding
ecology (e.g. H obson and W elch, 1992; Abend and Sm ith, 1995;
Sm ith et al., 1996; H obson et al., 1997; B u m s et al., 1998; Kelly,
2000; Lesage et al., 2001). The m ethod is b ased on the
dem onstration that stable isotope ratios of a consum er are
related to th ose of their prey (De Niro and Epstein, 1978; 1981;
Peterson and Fry, 1987). N itrogen-15 typically sh ow s a stepw ise
increase with trophic level w ithin a food chain (Hobson and
W elch, 1992; Cabana and R asm ussen, 1994; Thom pson et al.,
1995).

104
 Chapter 3

The carbon-13 value is close to that o f th e diet and is

preferentially u se d to indicate relative contributions to the diet
of different potential primary so u rces in a trophic network,
indicating for exam ple th e aquatic vs. terrestrial, inshore vs.
offshore, or pelagic vs. benthic contribution to the food intake
(Hobson et al., 1995; Sm ith et al., 1996; Dauby et al., 1998).

W hen u sin g stable isotop es to a s s e s s diets of anim als feeding at

or near the top of the trophic w eb on several and on different
prey item s, m any of w hich may have sim ilar isotopic signatures,
clear d istinctions about the diet are more difficult to determ ine.
However, it m ay be possible to infer th e general trophic level at
w h ich anim als are feeding, by applying and com paring 1SN (and
to a lim ited extent 13C) step-w ise enrichm ent v a lu es (Kurle and
Worthy, 2001). Furthermore, stable isotope an alysis is often
u se d to provide a con tin u ou s variable with w h ich to a s s e s s both
trophic level (Michener and Schell, 1994; H obson et al., 1995)
and trophic transfer of contam inants (Kidd et al., 1995; D as et
al., 2000).

In previous stu d ies, stable isotope ratios and trace m etal

concentrations were determ ined in com m on and striped dolphin
tis s u e s from the Northeast Atlantic (chapter 2) and high renal Cd
concentrations encountered were assu m ed to be related to the
diet.

In th is study, w e exam ine closer and compare the relationship

am ong 6 m arine m am m al sp ecies from the Irish and French
channel coasts, the grey seal, the harbour porpoise, the striped
dolphin, th e com m on dolphin, th e w h ite-sided dolphin and the
w hite-beaked dolphin, u sin g a m ultidisciplinaiy approach based
on stable isotope (5I3C and 515N) and trace m etal analyses. We
also exam ined mercury (Hg), cadm ium (Cd), zinc (Zn) and copper
(Cu) concentrations for evidence of diet transfer, specific
bioaccum ulation or biom agnification p ro cesses and differences
betw een th e two regions.

105
 Marine mammals from th e N ortheast Atlantic

3 .2 . MATERIALS AND METHODS

3 .2 .1 . C O L L E C T IO N A N D S T O R A G E

Liver, kidney and m u scle sam p les were collected from 7 striped
dolphins, 24 com m on dolphins, 13 harbour porpoises, 4 white-
beaked dolphins, 5 w hite-sided dolphins and two grey sea ls
found stranded (while som e of them are be caught suspected) in
fishing n e ts along the Irish coast betw een 1989 and 1993
(cou nties of Cork, Galway, Kerry, M eath, Clare and Waterford)
and the northern French Atlantic coast (region of Cotentin)
betw een 1998 and 2001.

3 .2 .2 . A N A L Y T IC A L M E T H O D S

Zn, Cd and Cu analyses

After being w eighed and dried for 48 h at 1 10°C, sam p les were
digested with a solu tion of nitric acid (Merck 456) and slowly
heated to 100C° u n til com plete digestion. Atomic absorption
spectrophotom etry (ARL 3510) w as u se d to determ ine Cu, Zn
and Cd concentrations. C oncentrations are exp ressed a s ng g 1
dry w eight (dw).
Parallel to the sam ples, a set o f certified m aterial sam p les (CRM
2 78 Com m unity B ureau o f Reference, C om m ission of the
European Com m unities) w as also analysed to ensure the
m ethod’s sensitivity. Recoveries ranged from 92 to 102 % for Cu
and Zn, and 88% for Cd. Limits of detection were 0.01 ng g-> dw
for Cu, 0.33 for Zn, and 0.22 for Cd.

Hg analyses

Hg w as analysed by flam eless atom ic absorption

spectrophotom etry (Perkin-Elmer MAS-50A) after sulfuric acid

106
 Chapter 3

digestion, a s described by Joiris et al., 1991). Quality control

m easurem en ts for total m ercuiy included replicate analysis
resulting in coefficients of variation <10% and analysis of
certified m aterial (DORM-1, NRC, Canada).

Stable isotope m easurem ents

O rganism s m ay vary in their concentrations of lipids. As lipids

have been show n to be depleted in 13C relatively to the diet
(Tieszen et al., 1983), they were extracted from sam p les usin g
repeated rin ses w ith 2:1 chloroform: m ethanol prior to analysis.
After drying at 50°C (48h), sam p les were ground w ith a mortar
and pestle into powder. Stable isotope m easu rem en ts were
performed on a V.G. O ptim a (Micromass) IR-MS coupled to a N-
C-S elem ental analyser (Carlo Erba) for autom ated analyses.
Routine m easu rem en ts are precise to w ithin 0.3 ° / o o for 13-
carbon and 15-nitrogen. Stable isotope ratios were expressed in
8 notation according to the following equation:

5X —[(R s a m p le / R sta n d a r d )- 1] X X000

w here X is 13 C or 15N and R is the corresponding ratio 13C / 12C or

15N / 14N.
Carbon and nitrogen ratios are expressed relative to the V-PDB
(Vienna Peedee Belem nite) standard and to atm ospheric
nitrogen, respectively. Reference m aterials were IAEA-N1 (S1SN =
+0.4 ± 0.2%o) and IAEA CH-6 (sucrose) (5i3C = -1 0 .4 ± 0.2%o).

3 .2 .3 . D A T A T R E A T M E N T

Kolmogorov-Smirnov te st w a s u se d to te st for data departure to

normality. W hen not distributed norm ally the variables were log-
transformed to norm alize their distribution. Effect of sp ecies and
sam pling location on S13C and 5I5N v alu es or trace m etal
concentrations were tested sim u ltan eou sly u sin g m ultivariate
an alysis of variance (2-way MANOVA) followed by post-hoc
m ultiple com parison te s ts (LSD test). Parametric Spearm an-
coefficient h a s been u se d to test correlations betw een the
values. R esults were ju d ged significant w h en p<0.01 u n le ss
otherw ise stated.

107
 Marine mammals from th e N ortheast Atlantic

3 .3 . RESULTS

3 .3 .1 . S T A B L E IS O T O P E A N A L Y S IS

515N and 813C a n alyses were performed in the m u scle s of six

m arine mamm al sp ecies (table 3.1). The grey seal and the w h ite­
sided dolphin were exclud ed from statistical treatm ent due to
the sm all sam ple size and unavaibility for the two regions.

Table 3.1. 8 1SN and 8 13C values in muscles of marine mammals from the
French Channel and Irish coasts. Data is given as average (median) + standard deviation,
(minimum - maximum); n: number of samples, na: not available.

C hannel coast Irish coast

8 13C 8 1SN S '3C 8 i5N

-16.7 ± 0 .4 11 ± 1.8 -17.5 ± 0 .1 10.8 + 0.6

Striped dolphin
(-17.1 / -16.4) (9 .8 -1 3 .1 ) (-1 7 .7 /-1 7 .4 ) (1 0 .2 -1 1 .3 )
Stenella coeruleoalba
n=3 n=3 n=3 n=3

-16.5 ± 0 .5 12.1 ± 0 .4 -17.1 ± 0 .4 12.2 ± 1

Common dolphin
(-17.1 /-1 5 .6 ) (1 1 .4 -1 2 .6 ) (- 1 8 / -16.5) (1 0 -1 4 .3 )
Delphinus delphis
n=8 n=8 n=14 n=14

-17.0 ± 0 .5 12.7 ± 0 .5
Atlantic w hite-sided dolphin
na (-1 7 .4 /-1 6 .4 ) (1 2 .1 -1 3 .4 )
Lagenorhynchus acutus
n=4 n=4

-16.1 ± 0 .6 16.5 ± 2 .4 -16.5 ± 0 .7 14.1 ± 1.6

Harbour porpoise
(-17.1 / -15.8) (1 3 .2 -1 8 .8 ) (-17.2 / -15.1) (1 2 .0 -1 7 .2 )
Phocoena phocoena
n=4 n=4 n=7 n=7

-16.3 ± 0 .3 15.8 ± 2 .3
W hite-beaked dolphin -15.4 16.5
(-1 6 .6 /-1 6 .0 ) (1 3 .3 -1 7 .8 )
Lagenorhynchus albirostris n=l n=l
n=3 n=3

Grey seal -15.4 18.3

na
Halichoerus grypus n=l n=l

108
 Chapter 3

813C m easurem en ts varied significantly w ith both sp ecies and

sam pling location w hile m ean 515N value rem ained similar
b etw een French C hannel and Irish c o a sts (2-way MANOVA,
univariate resu lts see table 3.4). In both ecosystem s, the lower
S 15N valu es were displayed by th e striped dolphin, significantly
depleted compared to harbour porpoise (post-hoc LDS test,
p<0.0001) and w hite-beaked dolphin (post-hoc LSD test,
p<0001). Mean S15N data did n ot differ significantly betw een
striped and com m on dolphins (post-hoc LSD test, p=0.06). For
both ecosystem s, striped dolphins were significantly depleted in
carbon-13 com pared to w hite-beaked dolphin (post-hoc LSD
test, p<0.0005) and harbour porpoises (post-hoc LSD test,
p<0.005) but were s imilar to com m on dolphins (post-hoc LSD
test, p = 0 .1). M ean §13C v alu es did not differ significantly betw een
harbour porpoise and w hite-beaked dolphin (p>0.1).
The m ean m u scle and liver 513C v alu es were significantly more
negative for the anim als from the Irish c o a sts compared to
anim als from th e Northern French coast (post-hoc LSD test,
p<0.0002)

20

# Irish coasts
18
O F rench Channel coasts
HO wbd
16
wbd

14
wsd
12

10

18 -17 -16 -15

513C (%>)

Figure 3.1. Muscle 8 13C and 5 lsN in marine mammals from Irish, French
Channel coasts (sd: striped dolphin; cd: common dolphin; wsd: white-sided dolphin; hp: harbour
porpoise; wbd: white-beaked dolphin, gs: grey seal

109
 Marine mammals from th e N ortheast Atlantic

3 .3 .2 . M E T A L L E V E L IN T H E T IS S U E S

Geographic location did not affect trace m etal concentrations

w hile Zn, Cu and Cd displayed strong-interspecific differences
(table 3.2 and 3.3). Hg rem ained sim ilar betw een sp ecies and
locations.

Table 3.2. Zn, Cd, Cu and Hg concentrations (p g.g1 dry weight) in the
liver, muscle and kidney of marine mammals from the French Channel
CO aStS, data is given as a mean (median) ± standard deviation, range of concentrations (minimum-
maximum); n: number of samples; na: not available

Zn Cd Cu Hg
140 + 41 35 + 55 26 ± 9.2 37 ± 2 5
Liver (9 4 -1 7 1 ) (3 - 9 9 ) (1 8 - 3 6 ) (11 -6 0 )
n=3 n=3 n=3 n=3
33 ± 0.9 <0.1 ± 0 .2 7.8 ± 1 .2 3.7 ± 1.5
Striped dolphin
Muscle (33 - 34) (<0.1 / 0.3) (6 .9 -9 .1 ) ( 2 - 4 .8 )
Stenella coeruleoalba
n=3 n=3 n=3 n^S
112 + 3 5 71 ± 1 0 4 16 ± 2 .6 8 + 4.3
Kidney (73 -1 4 0 ) (<0.1 /1 9 0 ) (1 4 - 1 9 ) ( 3 .9 - 1 3 )
n=3 n=3 n=3 n=3
141 ± 31 1 .5 + 1.3 17 ± 3 1 2 4 ± 156
Liver (1 0 6 -1 7 7 ) (0.3 -3 .7 ) (14 - 22) (2.2 - 320)
n=5 n=5 n=5 n=5
32 + 6 <0.1 5.1 + 1.4 2.7 ± 2 .9
C ommon dolphin
Muscle (20 - 39) (<0.1) ( 3 .8 - 7 ) ( 0 .3 - 9 .1 )
Delphinus delphis
n=8 n -8 n=8 n=8
84+23 8 .7 + 8 .1 13 (14) ± 5 .6 13 ± 5.1
Kidney (55 - 108) (1-22) ( 7 .5 - 2 1 ) (6.8 - 20)
n=5 n=5 n=5 n=5
258 + 246 0.3 ± 0.2 111 ± 6 9 8.9 ± 1 1
Liver (1 2 6 -6 2 8 ) (0.1 - 0 .5 ) (36 - 203) (3 -2 6 )
n=4 n=4 n=4 n=4
126 ± 4 9 <0.1 12 + 4.6 3.5 ± 2.4
Harbour porpoise
Muscle (101 -1 9 9 ) (<0.1) ( 5 .2 - 1 5 ) (1 .8 -5 .2 )
Phocoena phocoena
n=4 n=4 n~4 n=2
99 ± 8 1.5 ± 2 .7 32 + 16 4 + 2 .5
Kidney (9 3 -1 1 1 ) (<0.1 / 5.6) (1 8 -5 5 ) ( 2 .3 -7 .6 )
n -4 n=4 n=4 n=4
96 0.4 27 229
Liver
n=l n=l n=l n=l
W hite-beaked dolphin
67 0.9 5.5
Lagenorhynchus Muscle 4.2
n=l n=l n=l
albirostris n=l
37 0.9 8.2 5.6
Kidney
n=l n=l n=l n=l
204 ± 5 9 1.5 ± 1.0 51 ± 7 368 ± 59
Liver (163 - 246) (0.8 - 2.2) (46 - 56) (326 - 409)
n=2 n=2 n=2 n=2
Grey seal
92 0.3 3.7 7.6
Halichoerus grypus Muscle
n=l n=l n=l n=l
150 5.7 17 49
Kidney
n =l n=l n=l n=l

110
 Chapter 3

Table 3.3. Zn, Cd, Cu and Hg concentrations (fig-g1 dry weight) in the
liver, muscle and kidney of marine mammals from the Irish coasts Data is
given as a mean (median) ± standard deviation, range of concentrations (minimum-maximum); n:
number of samples; na: not available

Zn Cd Cu Hg
185 ± 6 0 38 ± 1 3 39 ± 9 41 ± 3 1
Liver (1 3 3 -2 5 0 ) (2 5 -5 1 ) (30 - 49) (1 9 -6 3 )
n=3 n=3 n=3 n=2
47 + 9 0.2 ± 0 .2 5.9 ± 1 .7 4 ± 0 .9
Striped dolphin
Muscle (42 - 58) (<0.1 -0 .4 ) (4.7 - 7.8) (3.4 - 4.6)
Stenella coeruleoalba
n=3 n=3 n=3 n=2
150 ± 15 150 ± 3 5 18 ± 4 .6 15 ± 5
Kidney (1 3 6 -1 6 3 ) (1 1 8 -1 9 9 ) (1 3 -2 4 ) (1 1 -1 8 )
n=4 n=4 n=4 n=2
150 ± 3 5 6.8 ± 6 .9 20 ± 4 .6 46 ± 5 5
Liver (81 -2 2 0 ) (1 .2 - 2 7 ) (1 3 -2 8 ) (4 -1 63)
n=14 n=14 n=14 n=8
48 ± 13 0.3 ± 0.3 6 ± 1.7 2.7 ± 1 .2
C ommon dolphin
Muscle (35 - 80) (<0.1-1) (3.6 - 9.8) (1.5 4.9)
Delphinus delphis
n=14 n=14 n=14 n=3
97 ± 2 3 33 ± 2 5 13 ± 3.2 8± 5
Kidney (53 - 130) (6 -7 2 ) ( 8 .2 - 1 9 ) (2 -1 4 )
n=12 n~12 n=12 n=7
136± 140 2.8 ± 4 18 ± 2 2
150
Liver (37 - 235) (<0.1 / 5.7) (2.7 - 33)
n—1
n=2 n=2 n=2
41 ± 2 4 3.6 ± 6 .7 4.8 ± 1.7 2.7 ± 1 .2
W hite-sided dolphin
Muscle (22 - 75) (0.1 -1 4 ) ( 3 .2 - 6 .4 ) ( 1 .5 - 3 .9 )
Lagenorhynchus acutus
n=4 n=4 n=4 n=3
97 ± 10 9.6 ± 14 7.7 ± 6 .2
7.8
Kidney (9 0 -1 0 4 ) (<0.1 / 19) ( 3 .4 - 1 2 )
n=l
n~2 n=2 n=2
173 ± 9 8 0.6 ± 0.4 22 ± 1 1 24 ± 4 2
Liver (91 -3 8 0 ) (<0.1 /1 .1 ) (3 - 39) (4.1 - 9 9 )
n -8 n=8 n=8 n=5
45 ± 8 <0.1 ± 0 .1 4.6 ± 1 .6 3.6 ± 1 .4
Harbour porpoise
Muscle (32 - 56) (<0.1 / 0.2) ( 1 .2 - 6 ) (2.5 - 5.6)
Phocoena phocoena
n=7 n=7 n=7 n=4
90 ± 1 6 3.9 ± 3 .4 13 ± 1.6 2.2 ± 0 .6
Kidney (7 0 -1 1 2 ) ( 0 .4 -1 0 ) (1 1 -1 5 ) ( 1 .6 - 2 .9 )
n=7 n=7 n=7 n=4

iii
 Marine mammals from th e Northeast Atlantic

Table 3.3. (continued). Zn, Cd, Cu and Hg concentrations (g g .g 1 dry

weight) in the liver, muscle and kidney of marine mammals from the Irish
COäStS Data is given as a mean (median) ± standard deviation, range of concentrations (minimum-
maximum); n: number of samples; na: not available
Zn______________Cd______________Cu________ H g
101 ± 2 8 0.3 ±0.1 24 ± 14
Liver (8 1 -1 2 1 ) (0.2 - 0.3) (1 4 -3 4 )
n=2 n=2 n=2
97 ± 3 9 <0.1 5 ±2
W hite-beaked dolphin
Muscle (5 3 - 127) (<0.1) (3.5 - 7.2)
Lagenorhynchus albirostris
n=3 n=3 n=3
73 ± 41 0.3 ± 0 .0 3 7.6 ± 6 .1
Kidney (4 4 - 102) (0.2 - 0.3) ( 3 .3 - 1 2 )
n=2 n=2 n=2

Striped dolphin displayed higher renal Zn concentrations than

harbour porpoise (post-hoc LSD test, p<0.01) and com m on
dolphins (post-hoc LSD test, p<0.005), w h ich in turn were higher
th an th ose of w hite-beaked dolphin (post-hoc LSD test, p<0.05).

Striped dolphin displayed th e h igh est hepatic and renal Cd

concentrations significantly higher th an th ose o f com m on
dolphins (post-hoc LSD test, p<0.0006), w hich in turn were
higher th an th o se of harbour porpoise (post-hoc LSD test,
p<0.0001) and w hite beaked dolphin (post-hoc LSD test, p<0.01).
The h igh est hepatic Cu w as m easured in the liver of the harbour
porpoise and the low est in the com m on dolphin (post-hoc LSD
test, p<0.005). Other sp ecies displayed sim ilar Cu
concentrations.

112
 Chapter 3

Table 3.4. Influence of species and sampling locations (2-way MANOVA

results, univariate specific effects) on metal concentrations and stable
isotope ratios in the tissues, ns not significant, p> 0 . 1 ; log: indicate the data were log-
transformed before statistical treatment to ensure a normal distribution

Factors species geographic location

Liver
Zn (log) F3 -23 =1.4, p>0.2 Fi^3=0.001, p>0.9
Cu (log) FWj=4.1, p<0.02 Fi,23 =0.9, p>0.3
Cd (log) F.,,26=20.2, [Link] F i.26=0.6, p>0.4
Hg (log) 1' 3 .14=1 2. p>0.3 Fi.14 =0.7, p>0.4
Kidney
Zn (log) F3J3=5.9, p<0.005 F i.23=0.16, p>0.6
Cu (log) F3^3=4.7, p<0.015 F i,23=3.9, p>0.06
Cd (log) F3j6=20.3, p<0.0001 F i 26=1.15, p>0.2
Hg .............. F3 ,i4=0.8, p>0.5 FU4=0.05, p>0.8
Muscle
Zn (log) F3j 3=12.2, p<0.0001 F i,23=2.98, p>0.09
Cu (log) F 3.23 =0.6, p>0.6 F i .23=3.3, p>0.08
Hg (log) F3,14=0.06, p>0.9 F,,„=0.001, p>0.9
Sl3C Fm5=6.4, ¡K 0 . 0 0 2 Fi,35=11.7, p<0.002
515N F3,35=22.01, p<0.0001 F,,35 =2,p>0.1

3 .3 .3 . R E L A T I O N S H I P B E T W E E N S T A B L E IS O T O P E S
A N D H E A V Y M E T A L S

No clear relationship w as observed betw een hepatic or m uscular

813C and 515N and hepatic, renal or m uscular Hg concentrations,
neither w ithin a sp e c ie s nor for all sp ecies considered (p>0.01).

113
 Marine mammals from th e N ortheast Atlantic

• com m on dolphin
+ w h ite -b e a k e d dolphi
+ % h arb o u r p o rp o ise
O strip ed dolphin

O O
• e
o (,t

S15N ( % o)
Figure 3.2. Relationship between muscle 81SN and hepatic Hg
concentration using a log-scale in marine mammals from Irish and French
Channel coasts

A decreasing relationship w as observed betw een m uscle 8 15N

and cadm ium concentrations (log-transformed) in the liver
(Pearson Product-M om ent Correlation, r=-0.68, p< 0.0001, n=35)
and in the kidney (Pearson Product-M om ent Correlation, r=-
0.76, p< 0.0001, n=31, figure 3.3).

# com m on dolphin
+ w h ite -b ea k ed dolphii
100.0 # h a rb o u r p o rp o ise
O strip ed dolphin

T53
10.0
D>
a.
-o
U
CO
c
0
O'

9 11 13 15 17 19 21

S,5N ( % o)

Figure 3.3. Relationship between muscle 81SN and renal Cd concentration

using a log-scale in marine mammals from Irish and French Channel
coasts

114
 Chapter 3

A sim ilar correlation w as found betw een 513C v alu es and

cadm ium concentrations (log-transformed) in th e liver (Pearson
Product-M om ent Correlation, r=-0.47, p<0.005, n=35) and in the
kidney (Pearson Product-M oment Correlation, r=-0.56, p<0.001,
n=31, figure 3.4).

• common dolphin
+ white-beaked dolphin
100.0 m harbour porpoise
O striped dolphin

10.0
D>
rai>
■
O a
c
a<u 1.0

0.1
-19 -18 -17 -16 -14

513C (%o)

Figure 3.4. Relationship between muscle S13C and renal Cd concentration

using a log-scale in marine mammals from Irish and French Channel
coasts

Zn and Cu in liver, kidney or m u scle were never correlated with

either m uscle S13C and 815N values.

115
 Marine mammals from th e N ortheast Atlantic

3 .4 . DISCUSSION

The u se fu ln e ss of 513C and 815N sign atu res a s a m easure of

trophic sta tu s in stu d ie s of mercury accum u lation h a s been
reported recently for a variety of sp ecies, includ ing marine
m am m als (Kidd et al., 1995; Atwell, 1998; T hom pson et al.,
1998a,b; Bearhop et al., 2000; Braune et al., 2002) w hile the
relationship w ith other m etals received le s s attention (Camuso et
al., 1998; D as et al., 2000). Marine m am m als generally have high
m ercuiy and cadm ium concentrations in their tis s u e s compared
to other marine groups, w h ich is thought to be a con seq u en ce of
their high position in the food w eb (Thompson, 1990, Atwell et
al., 1998; Jarm an et al., 1998). Although differences in m ean
mercury levels am ong sp e c ie s were not statistically significant,
high mercury concentrations have b een m easured in th e livers of
the w hite-beaked dolphin and the grey seal from the channel
coast (table 3.2.). Elevated Hg v alu es have b een described
previously in one w hite-beaked dolphin from the Irish Sea
(around 108 Mg.g' dw a s s um ing a m ean water con ten t of 75%
for the tissu es) and in 12 grey se a ls from the Liverpool Bay
(mean: 590 jig.g-' dw) (Law et al., 1992). Positive correlations
betw een §15N v a lu es and Hg concentrations have in the past
been described for freshwater fish sp e c ie s (Kidd et al., 1995), in
the blood of th e great sk u a ch ick s (Bearhop, et al., 2000) or more
generally w ithin a trophic web (Atwell et al., 1998), su ggesting
that part of the Hg variation can be linked to a bioam plification
process.

In the present study we did not find any positive relationship

betw een 815N (or 813C) v alu es and m ercu iy concentrations either
for all sp ecies together (figure 3.2), or w ithin a sin gle sp ecies
while th e wide range of 813C and 815N v a lu es observed am ong the
six marine m a m m al sp e c ie s supports the h yp oth esis of a
different trophic sta tu s (figure 3.1). Indeed, w h en considering all
th e individuals together, ö 15N va lu es range from 9%o to more
th an 19%o.

The 813C and 815N value in m arine predator tis s u e s is determ ined
initially by the isotopic com position of the baseline phyto- and
Zooplankton sou rces, technically m easured in the particulate

116
 Chapter 3

organic m atter (POM). No POM data are available for the Celtic
S ea or the French Channel. A m ean 515N value of 5%o is
generally u se d for offshore POM (Tucker et al., 1999) but data
may vary w ith their sam pling origin, from 4 .5 %o in th e G ulf of
St-Lawrence (Northwest Atlantic) to 5.0±1.2 %o in som e salt
m arshes w ithin the Northeast French Atlantic (Lesage et al.,
2001; Riera et al., 1999), reaching even higher v a lu es (up to
9%o) in the coastal part of the North S ea (Mariotti et al., 1994;
Middelburg and N ieuw enhuize, 1998). As a resu lt, part of the
815N interspecific variation in m arine m am m als m ight be related
to coastal v e rsu s offshore 815N signature of th e primary
producers. Indeed, som e sp ecies su c h a s the striped dolphin are
typically oceanic w hile others, su c h a s th e harbour porpoise, are
a more coastal (Evans et al., 1987). However, th e se geographic
differences observed for particulate organic m atter (POM) do not
explain the large 515N range displayed by the 6 m arine m ammal
sp ecies of th is study and th e se 515N v alu es reflect specific
trophic statu s. In both areas, high m u scle and hepatic §15N
v a lu es encountered su ggest a higher trophic position of white
beaked dolphins and harbour porpoises com pared to com m on or
striped dolphins. A high 8I5N value h a s also b een m easured in
the grey se a ls but m u st be confirm ed on a larger sam pling
(figure 3.1). Moreover, w hite-beaked dolphins, harbour
porpoises, w hite sided dolphins, com m on and striped dolphins
display th e sam e relative and decreasing trophic position, as
m easured by 815N values, along both the Irish and French
C hannel coasts, reflecting conservative trophic h abits betw een
th e se two places.

While previous stu d ies su ggest that m ercuiy levels tend to be

greater in tis s u e s of higher trophic level organism s, it is unclear
to w hat extent th is is the resu lt of biom agnification through the
food web or bioaccum ulation w ithin organism s over tim e (Atwell
et al., 1998). This is difficult to discern a s top-predators su ch as
m arine m am m als are long-living sp e c ie s and resu lts are
w eakened by the absen ce o f age data. R elationships betw een Hg
and age have b een reported for various porpoise (Sieberi et al.,
1999), dolphin (Honda and Tatsukawa, 1981; H onda et al., 1983)
or seal sp e c ie s (Anan et al., 2002). In th is study, total
concentrations of m ercuiy were m easured, but it is w ell know n
that organic and inorganic sp e c ie s o f m ercuiy have v e iy different
dynam ics in marine m am m als. Methy 1-mercury is the m ain form
p resen t in the prey (fish and invertebrates) and th en is stored

117
 Marine mammals from the Northeast Atlantic

indefinitely a s tiem m anite (HgSe) in the liver of m arine m am m als

(Nigro and Leonzio, 1996).

In contrast to Hg, a significant decreasing relationship betw een

m uscular 513C and S15N va lu es and renal Cd su ggest that som e
of th e variation can be linked to dietary sp ecialisation (figures
3.3 and 3.4). The ten dency observed is, a s m u scle 515N and S13C
increase, cadm ium concentrations decrease, with v a lu es ranging
betw een those o f striped dolphins (maximum renal cadm ium ,
m inim um m u scle 815N and 813C) and grey se a ls and white-
beaked dolphins (m inim um renal cadm ium , m axim um m uscle
S15N and 8i3C).

S13C is more u sefu l to indicate the origin of carbon sou rces than
a s an indicator of the trophic level. The general pattern of
inshore, ben th os linked food w eb s being more enriched in 13C
compared with offshore, pelagic food w eb s p resen ts a potentially
u sefu l tool. For exam ple, S13C valu es are typically higher in
coastal or benthic food w ebs th an in offshore food w eb s (Hobson,
1999). A clear relation is observed betw een increasing m uscle
813C of the six m arine m am m al sp e c ie s and oceanic versus
coastal habitat preference (figure 3.1). The 813C depletion
observed for striped, com m on and w h ite-sided dolphins would
therefore presum ably reflect a greater reliance on offshore food
w hile the higher m ean va lu es observed for harbour porpoise,
w hite-beaked dolphin and grey seal correspond to their
preference for a m ore coastal habitat (figure 3.1). The white-
beaked dolphin h a s le s s oceanic preferences than th e Atlantic
w hite-sided dolphin (Cipriano, 2002; Kinze, 2002) w h ich is in
agreem ent with its higher 813C values. High Cd concentrations
have b een reported previously in the livers and kidneys of by­
caught striped and com m on dolphins from the Northeast
Atlantic (Das et al., 2000) and oceanic cephalopods con stitu ted a
significant part o f their diet (H assani et al., 1997). Previous
stu d ies have highlighted the ability of cephalopods to
concentrate cadm ium in th e digestive gland, even in unpolluted
areas like the K erguelen Island s (Bustam ante, et al., 1998b).
O ceanic cephalopods are, indeed, considered an essen tia l link
for cadm ium transfer in m arine trophic food ch ain s (Law et al.,
1997; B ustam ante e t al., 1998a). Our resu lts su g g est that the
high Cd valu es encountered in striped and com m on dolphins
from the Irish and the French C hannel c o a sts are partly diet
related a s a resu lt of in gestion of prey displaying low 81SN and

118
 Chapter 3

S13C valu es and high Cd levels. S u ch high Cd levels can be found

in oceanic cephalopods (Bustam ante et al., 1998) w hich
furthermore display typical low 515N valu es (Ostrom et al., 1993,
Hooker et al., 2001) compared to individuals collected w ithin the
Southern North S ea bight (see chapter 4). In contrast harbour
porpoises, w hite-beaked dolphins and grey se a ls are likely to
rely more on fish species. A previous stom ach content study
reported that fish sp e c ie s su c h a s Gadidae and Clupeidae form
th e bulk of harbour porpoise diet (Rogan and Berrow, 1996).

No relation w a s found betw een isotopic com position and either

Zn or Cu in th e tis s u e s su ggestin g that the large value range
observed for th e se sp ecies is not diet related. Other factors su ch
a s body condition influenced by nutritional str e ss are likely to be
involved. Zn, Cu and Cd v alu es m easured in th e se stranded
com m on and striped dolphins are sim ilar to that reported for the
sam e sp ecies individuals by-caught in the Bay of Biscay in 1993
(Das et al., 2000). Harbour porpoises that died from infectious
d isea se s displayed significantly higher Zn and Hg concentrations
than healthy porpoises that died from physical traum a while Cu
and Cd did n ot differ betw een the two groups (Bennet et al.,
2001). Previous stu d ies have also docum ented an increase of
815N valu es in starving anim als a s they m ight u se their proteins
for survival (G annes et al., 1998). In birds, nutritional stress
cau sed su b stan tial in creases in diet-fractionation valu es due
either to m obilization and redeposition of proteins elsew here in
the body or am ino acid com position ch an ges in the tissu e s
(Hobson and Clark, 1992; G annes et al., 1998). In contrast,
Arctic ground squirrels (Sperm ophilus parryii plesius) in poor and
excellent body condition had sim ilar 515N v a lu es (Ben-David et
al., 1999). Similarly, m uscle 815N and 813C v alu es do not differ
betw een porpoises from the North S ea displaying a poor,
moderate of good body condition allowing the u se of their
m u scle tissu e for stable isotope stu d ie s (see chapter 5).

To conclude, moreover, w h ite-beaked dolphins, harbour

porpoises, w hite sided dolphins, com m on and striped dolphins
display the sam e relative and decreasing trophic position, as
m easured by 515N valu es, both th e Irish and French channel
coasts, reflecting conservative trophic habits betw een th ese two
places.
Hepatic and renal Cd concentrations were significantly
correlated to m u scle 513C and S15N v alu es w hile Hg, Zn and Cu
did not. These resu lts su ggest that Cd accum ulation is partly

119
 Marine mammals from th e N ortheast Atlantic

linked to the diet w hile other factors su c h a s age or body

condition m ight explain Hg, Zn or Cu variability in marine
m am m als. Com bined stable isotope and trace m etal analyses
appear a s prom ising and powerful tools for the stu d y of marine
m am m al ecology. Further work sh ou ld concentrate on u sin g the
stable isotope m ethod to further explore the behaviour and
transfer of trace m etals in th e m arine environm ent. However,
further data on other trophic com p onents from th e Northeast
Atlantic should allow a better understanding of trophic
contam inant transfer.

Aknowledgm ents. We a re g ratefu l to D r Thierry J a u n ia u x (M arine A nim al R esearch

an d In tervention Network) w ho realised th e necropsy of two strip e d dolp h in s
stra n d e d on th e N o rth ern F re n c h co asts. T h a n k s to M rs R achel Bouhy a n d Mr
Renzo Biondo (Oceanology L aboratory, Liège, Belgium) for th e ir v aluable tech n ical
a ssista n c e . T h an k s also to Fabrice K erleau an d Philippe de M eersm an (GECC) for
th e ir logistical coordination. T he field w ork h a s been m ade possible by th e
collaboration of th e s e differen t d e p artm e n ts: T he Préfecture maritime d e la Manche
et d e la Mer du Nord, th e Directions D épartem entales d e s Services d ’Incendie et de
Secours de Normandie, th e Gendarmeries D épartem entales d e Normandie, a n d ali
th e GECC v eterin aria n a n d v olunteers.
T his stu d y w as su p p o rte d by th e B elgian Office for Scientific, T echnical a n d
C u ltu ral Affairs (co n tract M N /D D /50). K rish n a D as h a s received a g ra n t from
FRIA (Fond p o u r la R echerche d a n s l’In d u strie e t d a n s l’A griculture). C ristin a
B eans h a s received a n exchange fellow ship from th e MAE (Ministerio d e A suntos
Exteriores, Spain) a n d th e CGRI (C om m issariat G énéral a u x R elations
In tern atio n ales de la C o m m u n au té F ran ç aise de Belgique). E m er Rogan a n d Sim on
Berrow w ere funded by th e H eritage C ouncil of Ireland.

120
 Chapter 3

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T echn. 30: 654-660
relatio n sh ip s w ith in a h ig h Arctic
food web u sin g 813 C a n d S15 N Jo iris, C.R., H olsbeek, L.,
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M ar. Ecol. Prog. Ser. 128: 1-10 avian and m am m alian tro p h ic
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H obson; K.A., S ease; J.L ., M errick;
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H onda, K., T atsu k aw a, R., Itano, K.,
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Heavy m etal c o n c e n tra tio n s in K ennedy S., M ilnes R., M orris R .J.
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Law, R .J., M orris, R .J., Allchin, m arin e m am m als. ICES J . Mar. Sei.
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chlorobiphenyls in tissu e s of sperm
w hales (Physeter macrocephalus) P eterso n, B .J., Fry, B. 1987. Stable
and o th e r ce ta c e a n s species isotopes in ecosystem stu d ies. A nnu.
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T.G., L am bertsen, R.H. (eds). Sperm
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Lesage, V., H am m il, M.O., Kovacs, in v e rteb ra tes in a sa lt m a rsh
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E stu a ry a n d G ulf of S t Law rence, im p o rtan ce o f locally produced
C an ad a : evidence from stab le sources. Mar. Ecol. Prog. Ser. 187:
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Rogan, E., Berrow , S.D. 1996. A
M ariotti, A., Lancelot, C., Billen, G. review of h a rb o u r porpoises,
1994. N atu ral isotopic com position Phocoena Phocoena in Irish w aters.
o f n itro g en a s a tra c e r o f origin for Rep. Int. W hal. C om m n. 46: 595-605
su sp en d ed organic m a tte r in th e
S ch eld t estu ary . Geochim. S ieb eri, U., Jo iris, C., H olsbeek, L.,
Cosm ochim . Ac. 48: 549-555 B enke, H., Failing, K., Failing, K.,
Frese, K., Petzinger, E. 1999.
M ichener, R.H., Schell, D.M. 1994. P o ten tial R elation betw een m ercu ry
S table isotopes ra tio s a s tra c e rs in c o n c e n tra tio n s and necropsy
m arin e aq u atic foodwebs. In: findings in C e tacean s from G erm an
L attjh a, K., M ichener, R.H. (eds.) w a te rs of th e N orth a n d B altic seas.
Stable isotopes in ecology and M ar. PoUut. Bull. 38: 285-295
environmental science. Blackwell
Scientific P ublications, Oxford, U.K. S m ith , R .J., H obson, K.A.,
pp. 138-186 K oopm an, H.N., Lavigne, D.M. 1996.
D istin g u ish in g b etw een pop u latio n s
M iddelburg, J .J ., N ieuw enhuize, J . of fresh- a n d sa lt-w a te r h a rb o u r
1998. C arbon a n d n itro g en stab le sea ls (Phoca vitulina) u sin g stab le -
isotopes in su sp en d ed m a tte r a n d isotope ratio s a n d fatty acid profiles.
sed im en ts from th e S chelde estu ary . C an. J . Fish. A quat. Sei. 53: 272-
Mar. Chem . 60: 217-225 279
Nigro, M., Leonzio, C. 1996. T hom pson, D.R. 1990. M etal levels
In tracellu lar storage of m ercu ry a n d in m arin e v erteb ra tes. In: F u rn e ss
selen ium in different m arin e R.W ., R ainbow , P.S. (eds). H eavy
v erteb rates. Mar. Ecol. Prog. Ser. m etals in the marine environment.
135 : 137-143 CRC P ress, pp 143-182
OSPAR, 2000. Q uality S ta tu s report. T hom pson, D.R., F u rn e ss, R.W.,
OSPAR C om m ission, London 108 + Lewis, S.A. 1995. D iets a n d long­
Vii pp. term ch an g es in 81SN a n d 813C
v alu es in n o rth e rn fu lm ars Fulmarus

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glacialis from tw o n o rth e a s t A tlantic

colonies. Mar. Ecol. Prog. Ser. 123:
3-11

T hom pson, D.R., B earhop, S.,

S p eakm an, J.R ., F u rn e ss, R.W.
1998. F eath e rs a s a m e a n s of
m onitoring m ercu ry in seabirds:
in sig h ts from stab le isotope
analysis. E nviron. Pollut. 101: 193-
200

T ieszen, L.L., B outton, T.W.,

T esdahl, K.G., Slade, N.A. 1983.
F ractionation and tu m -o v e r of
stab le carb o n isotopes in an im al
tissu es: im plication for §13C a n aly sis
of diet. Oecologia, 57, 32-37.

Titllem ier, S.A., Fisk, A.T., H obson,

K.A., N orstrom , R..J. 2002.
E xam ination of th e b io accu m u latio n
of halogenated dim ethyl bipyrroles
in a n arctic m a rin e food w eb u sin g
stab le nitro g en an aly sis. Environ.
Pollut. 116 : 85-93

T ucker, J ., S h e a ts, N., Giblin, A.E.,

H opkinson, C .S., M ontoya, J.P .
1999. U sing sta b le isotopes to tra ce
sew age-derived m aterial th ro u g h
B oston H arb o u r a n d M a ssa c h u se tts
Bay. Mar. E nviron. Res. 48: 353-375

125
CHAPTER 4: MARINE MAMMALS FROM THE
SOUTHERN NORTH SEA: IN SIG H T S IN THEIR
FEEDING ECOLOGY FROM ô15N AND 013C
MEASUREMENTS

After D as K., Lepoint G., Leroy Y.,

Bouquegneau J.M. Marine Ecology
Progress Series. Submitted.
 Chapter 4

ABSTRACT

The harbour porpoise Phocoena phocoena, the harbour seal

Phoca vitulina, th e grey seal Halichoerus g ry p u s and th e white-
beaked dolphin L agenorhynchus albirostris are regularly found
stranded along the Southern North S ea coast. More occasionally,
offshore sp e c ie s su c h as the w hite-sided dolphin
L agenorhynchus acutus, the sperm w hale P h yseter m acrocephalus
or th e fin w hale B alaenoptera p h y s a lu s are also found stranded.
In order to trace their diet, 815N and 813C m easu rem en ts have
b een performed in their organs a s well a s in 49 invertebrate and
fish sp ecies collected in the Southern North Sea. 815N data
su ggest that harbour seal, grey seal and w hite-beaked dolphin
occupy the h igh est trophic position along w ith ichtyophageous
fish su ch a s gadids (m ean m u scle values: 18.7%o, 17.9%o,
18.8%o and 17.9%o respectively). Harbour porpoise occu p ies a
slightly lower trophic position (m ean 815N value: 16.2%o)
reflecting a higher am ount of zooplanktivorous fish in their diet
(m ean S15N: 14.7 %o). Suckling harbour porpoises display a
significant S15N enrichm ent of 2.8%o com pared to adult fem ales.
Adult fem ales are 15N -enriched com pared to adult m ale harbour
porpoises. Sperm w hales, fin w h ales and w hite-sided dolphins
are 13C-depleted compared to Southern North S ea POM and
sp e c ie s su ggesting that despite regular sightings, they basically
(if at all) do not feed w ithin the Southern North S ea area. Fish
relying on plankton su c h as herring, sprat or sa n d eels represent
a major link betw een the base of the food w eb and top-predators
su c h a s harbour porpoise, grey seal, harbour seal, w hite-beaked
dolphin and carnivorous fish, raising the n eed of an appropriate
m anagem ent of th ese sm all com m ercial fish species.

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 Marine mammals from th e Southern North Sea

4 .1 . INTRODUCTION

The fertile w aters of the North S ea represent a major life site for
at lea st four different m arine m amm al species: th e harbour
porpoise, Phocoena phocoena, the harbour seal, Phoca vitulina,
the grey seal, Halichoerus g ry p u s and th e w hite-beaked dolphin,
Lagenorhynchus albirostris (Hammond et al., 1995). Harbour
porpoise and harbour seal are the m ost com m on sp e c ie s in the
Northeast Atlantic and the North S ea (Hammond et al., 1995;
Boran et al., 1998). Their southern distribution se em s to be
lim ited to the D u tch W adden S ea w hile w hite-beaked dolphins
are generally concentrated in a band across the North Sea
betw een 55° and 60°N, m ostly to the W est along th e eastern
British coast (Hammond et al., 1995; De Jong et al., 1999). Grey
seal hauling and breeding s ite s are w ell know n and described
along the Northern UK coast (Nigel Bonner, 1989; Reijnders et
al., 1995; OSPAR, 2000). However, som e individuals have
already b een observed or are regularly found stranded in the
Southern part o f the North S ea suggestin g m ore extended
m ovem ents for th e se sp e c ie s (Haase, 1987; Leopold and
Couperus, 1995; Abt et al., 2002; Jau n iau x et al., 2002).

Other sp ecies like w hite-sided dolphins L agenorhynchus acutus,

sperm w h ales P h yseter m acrocephalus and fin w h ales
Balaenoptera p h y s a lu s can be occasionally sighted or found
stranded, but are still considered veiy rare in the Southern
North S ea (C am phuysen and Winter, 1995; Hammond e t a l,
1995). This area is characterized by intricate sy stem s of sand
banks, m udflats, sandy isla n d s and estuaries, obviously an
unfavourable environm ent for su ch oceanic sp ecies (Hammond
et al., 1995).

The distribution of m arine m am m als is strongly influenced by

the distribution o f their prey (Gowans and W hitehead, 1995;
G annon et al., 1997). However, despite regular and new
observations in the Southern North Sea, veiy few data dealing
with m arine m am m al diet w ithin th is area are available (Hislop,
1992; Santos et al., 1999). Strandings offer a good opportunity
for sc ie n tists to collect biological data but in m ost c a ses, either
stranded anim al stom ach s are empty, or digested m aterial is not

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 Chapter 4

suitable for diet research (Santos et al., 1994; Jau n iau x et al.,
2002). Moreover, strandings m ight represent potentially biased
sam p les of anim als a s sick or injured anim als m ay not be
feeding norm ally prior to death (Sekiguchi et al., 1992; Santos et
al., 1994).

The u se of naturally occurring stable isotop es of carbon and

nitrogen h a s provided com plem entary data to m arine mammal
feeding ecology (Hobson and Welch, 1992; Abend and Sm ith,
1995; Sm ith et a l.,1996; H obson et al., 1997; B u m s et al., 1998;
H obson and Schell, 1998; D as et al., 2000). Indeed, the carbon
and nitrogen isotope ratios (13C / 12C and 15N / 14N) in a consum er
reflect th ose o f its diet w ith a slight selective retention of the
heavier isotope and excretion of the lighter one. A s a result,
th e se ratios (in delta notation: S15N and 513C) typically sh ow a
trophic enrichm ent value of about 2-3°/oo and l°/oo respectively
(De Niro and Epstein, 1978; 1981; H obson and Welch, 1992;
M ichener and Schell, 1994). Stable isotop es o f nitrogen can be
u se d to quantitatively a s s e s s th e trophic level w hile the 13C,
rather than being a reliable indicator of trophic level, is generally
u se d to indicate relative contributions to th e diet of different
potential prim aiy so u rces in a trophic network, indicating for
exam ple the inshore vs. offshore, or pelagic vs. benthic
contribution to food intake (Rau et al., 1992; H obson et al.,
1995; Sm ith et al., 1996; Lepoint et al., 2000).

B ecause stable isotope ratios in th e tissu e of a consum er are

derived from assim ilated food, the tissu e reflects dietary input
integrated over tim e, not ju s t th e la st m eal before stranding
w h ich m ight be considered as biased. The m etabolic rates and
th e su b seq u en t turn-over of elem en ts differ betw een liver and
m u scle, allowing the evaluation of diet integrated betw een
different periods of tim e (e.g., days, w eek or m onths; T ieszen et
al., 1983; H obson et al., 1996; 1997).

In order to contribute to the know ledge of their feeding ecology,

815N and 813C have been determ ined and com pared in the livers
and m u scles of sev en m arine m am m al sp e c ie s beach ed along the
French Channel, Belgian and D utch c o a sts betw een 1994 and
2000: the harbour porpoise, the harbour seal, th e grey seal, the
w hite-beaked-dolphin, th e w hite-sided dolphin, th e sperm whale
and the fin w hale. Stable isotope m easu rem en ts have also been
performed in 15 invertebrate and 34 fish sp ecies collected in the

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 Marine mammals from th e Southern North Sea

Southern North S ea to delineate trophic relation sh ips betw een

marine m am m als and other sp e c ie s from th is area.

We have also addressed the q u estion of w hether m ore occasional

sp ecies su c h a s sperm w hale, fin whale or w h ite-sided dolphin
actually feed w ithin th e North S ea Southern Bay su c h a s
com m only sighted sp ecies, nam ely the harbour porpoise, the
harbour seal, the grey seal and the w hite-beaked dolphin.

4 .2 . MATERIAL AND METHODS

4.2.1. Marine mammal sampling

Liver and m uscle of 46 harbour porpoises Phocoena phocoena, 23

harbour se a ls Phoca vitulina, 6 grey se a ls H alichoerus grypus, 7
w hite-beaked dolphins L agenorhynchus albirostris, 2 w hite-sided
dolphins L agenorhynchus acutus, 7 sperm w h ales P hyseter
m acrocephalus and 3 fin w h ales Balaenoptera p h y s a lu s stranded
on the French C hannel, Belgian and D utch co a sts betw een 1994
and 2 0 0 0 have b een sam pled and stored at -20°C u n til analysis
(for necropsy d etails see J au n iau x et al., 1998; 2001; 2002).

4.2.2. Invertebrate and fish sampling

15 invertebrate and 34 fish sp ecies (sp ecies d etails in table 4.1)

were collected w ith th e Southern Part of the North S ea (betw een
51° and 56°N) during three cru ises of the R.V. Belgica (Belgium)
in Septem ber 2 0 00, February and May 2001 and one cruise of
the Thalassa (IFREMER, France) in March 2001 respectively. All
sam p les were frozen and stored at -20°C u n til analysis.

4.2.3. Stable isotope measurements

O rganism s may vary in their concentrations of lipids. As lipids

have been show n to be depleted in I3C relatively to the diet

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 Chapter 4

(Tieszen et a l, 1983), they were extracted from sam p les usin g

repeated rin ses with 2:1 chloroform: m ethanol prior to analysis.
After drying at 50°C (48h), sam p les were ground into a
h om ogen eou s powder. After grinding, sam p les containing
inorganic carbonates were acidified with HC1 (IN). As
recom m ended by Pinnegar and Polunin (1999), w h en sam p les
were acidified, 15N / 14N ratios were m easured before acidification
due to significant m odifications of th e se ratios after HC1
treatm ent (Bunn et al., 1995).
Stable isotope m easu rem en ts were performed on a V.G. Optima
(Micromass) IR-MS coupled to a N-C-S elem en tal analyser (Carlo
Erba) for autom ated analyses. Routine m easu rem en ts are
precise to w ithin 0.3 °/oo for both 13-carbon and 15-nitrogen.
Stable isotope ratios were exp ressed in 5 notation according to
th e following equation:

5X = [(R sample/R standard)- 1] X 1000

where X is 13C or 15N and R is th e corresponding ratio 13C / 12C or

15N/14N.
Carbon and nitrogen ratios are exp ressed relative to the V-PDB
(Vienna Peedee Belem nite) standard and to atm ospheric
nitrogen, respectively. Reference m aterials were IAEA-N1 (515N =
+0.4 ± 0.2%o) and IAEA CH-6 (sucrose) (813C = -1 0 .4 ± 0.2%o).
Isotopic m odel

M uscle 815N sign atu res of harbour porpoise, grey seal, harbour
seal and w hite-beaked dolphin were converted to trophic
position (TP) u sin g the above equation (after H obson and Welch,
1992; Lesage et al., 2001):

T P = 2 + (Dm - PO M - TEFmmt)/ T E F

Where Dm = 515N value in m arine m amm al m u scle, POM = 815N

value of m arine particulate organic m atter of th e Southern North
S ea (fixed to 9%o after Middelburg and N ieuw enhuize, 1998),
and TEF = trophic enrichm ent factor in 815N for a specific tissu e
(Hobson and W elch, 1992). This latter value w a s se t to a m ean
3.4%o for all com m unity com p onents (Lesage et al., 2001) except
for marine m am m als, for w hich a TEF value (TEFmmt) of 2.4%o
w as obtained in the m u scle s of 2 harbour se a ls fed on a constan t
herring diet (Hobson et al., 1996).

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 Marine mammals from th e Southern North Sea

4.2.4. Data treatment

Parametric and n o n parametric te s ts were u se d to compare

different groups: Kolmogorov-Smirnov te st w as u se d to assu m e
the normality of th e data. ANOVA followed by post-h oc m ultiple
com parison te s ts (LSD test) have been u se d to com pare the data
betw een the different sp ecies. W hen the n ecessary assu m p tion s
to realise ANOVA were n ot gathered (normality of th e variables
and hom ogeneity of variances), Kruskall-W allis w as u se d
followed by m ultiple com parisons b ased on the Kruskall-Wallis
rank su m s to te st for pairwise differences am ong sp ecies. Mann-
W hitney U -test w a s performed to compare differences am ong
groups w hen variances were not hom ogenous. Spearm an
coefficient h a s b een u se d to test correlations betw een the
values. R esults were judged significant w h en p<0.05.

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 Chapter 4

4 .3 . RESULTS

R esults on isotopic com position o f invertebrates, fish and marine

m am m al sp e c ie s are sum m arized in table 4.1 and 4.2.

Table 4.1. Length (cm), S1JC and 51SN values (%o) and feeding type (FT) of
selected invertebrates and fish of the Southern North Sea.

Values are given as mean ± St) in 0/<h>. (Zof: Fish feeding on Zooplankton, Ct: ctenophore (*: pool of 7
individuals), Omi: omnivorous invertebrates, Sf: suspension feeders, Gi: grazer invertebrates, Bifc:
crustaceans feeding on benthic invertebrates, Mf: mollusc feeder, Bif: fish feeding on benthic
invertebrates, Cf: carnivorous fish), nd: not determined. When length was not available weight is given
between brackets.

Species
n Length 5 13C 5 I5N FT

Ctenophores
Pleurobrachia pileus Sea Gooseberry 1* nd -12.9 16.6 Ct
Molluscs
Buccinum undatum Whelk 2 nd (37-45 g) -15.1 ±0.2 13.7 ±0.1 Omi
Solen marginatus Grooved razor shell 2 10.6-11.2 -16.6 + 0.3 11.2 ± 0 .1 Sf
Spisula solida Thick trough shell 2 nd (9-11 g) -17.4 ± 0.8 11.0 1 1.2 Sf
Sepia officinalis Common cuttlefish 5 nd (2.3-3.2 g) -15.9 ± 0 .6 16.1 ± 0 .6 B if
Loligo vulgaris Common squid 9 nd (2.2-45 g) -15.9 ± 0 .6 17.2 ± 1.3 B if

Crustaceans
Crangon crangon Common shrimp 1 n d ( l.6-2.6 g) -17.2 17.5 Bifc
Palaemon Cerratus Common prawn 1 6-8 -15.8 14.6 Omi
Carcinus maenas Common shore crab 3 nd (5-11 g) -17.4 ± 0 .3 15.5 ± 0 .5 Omi
Liocarcinus holasatus Swimming crab 2 nd (15-19 g) -15.0 ± 0.1 16.1 ± 0 .5 Omi
Pagurus berhardus Hennit crab 2 nd (4.6-7.6 g) -15.3 ± 0 .6 15.0 ± 0 .3 Omi
Echinoderms
Asteria ruben Common starfish 3 nd (46-182 g) -13.8 ± 1.5 13.3 ± 0 .5 Mf
Ophiura ophiura Sand-star 1 nd (0.8 g) -15.8 11.7 Sf
Echinocardium cordatum Sea potato 1 nd (22.6 g) -17.4 10.6 Sf
Psammechinus miliaris Sea urchin 1 nd (14.7 g) -14.1 12.1 Gi

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 Marine mammals from th e Southern North Sea

Table 4.1. (continued) : Length (cm), 513C and S1SN values (%o) and
feeding type (FT) of selected invertebrates and fish of the Southern North
Sea.

Values are given as mean + SD in °/oo. (Zof: Fish feeding on Zooplankton, Ct: ctenophora, Omi:
omnivorous invertebrates, Sf: suspension feeders, Gi: grazer invertebrates. Bifc: crustaceans feeding on
benthic invertebrates, Mf: mollusc feeder, Bif: fish feeding on benthic invertebrates, Cf: carnivorous
fish), nd: not determined. When length was not available weight is given between brackets.

Species n Length §13C s ,5n FT

Elasmobranchs
Raja clavata Thomback ray 5 37-94 -15.0 ± 0.6 14.9 ± 0.4 B if
Raja montagui Spotted ray 3 56-65 -16.6 ± 1 .1 15.3 ± 0 .7 B if

Raja radiata Starry ray 3 41-49 -16.5 ± 0 .6 13.5 ± 0 .2 B if

Scyliorhinus canicula Small spotted catshark 1 24-70 -15.6 18.2 B if
Mustelus asterias Stellate smooth-hund 2 70-81 -1 5 .2 ± 0.13 16.2 ± 0 .0 4 B if
Clupeiformes
Clupea harengus Herring 3 10-30 -16.7 ± 0.1 14.4 ± 0 .2 Zof

Engraulis encrasicolus Anchovy 2 10-12 -16.8 ± 2 15.2 ± 0 .2 Zof

Sprattus sprattus Sprat 5 7-9 -17.3 ± 0 .2 16.7 ± 0 .5 Z of
Pleuronectiformes
Buglossidium luteum Solenette 1 4.5 -16.8 14.8 B if
Limanda limanda Dab 7 7-20 -15.9 ± 1.6 16.9 ± 0 .5 B if
Microstomus kitt Lemon sole 1 16-42 -16.1 14.0 B if
Platichthysflesus Flounder 4 27-42 -16.9 ± 2 .7 17.5 ± 1.9 B if
Pleuronectes platessa Plaice 2 19-31 -15.6 ± 0 .4 16.9 ± 0 .5 B if
Solea (Pegusa)lascaris Sand sole 5 10-12 -15.5 ± 0 .5 17.4 ± 0 .6 B if
Solea solea (vulgaris) Common sole 8 9-19 -15.7 ± 1.5 17.5 ± 0 .9 B if

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 Chapter 4

Table 4.1. (continued) : Length (cm), 5 13C and 8lsN values (%o) and
feeding type (FT) of selected invertebrates and fish of the Southern North
Sea.

Values are given as mean ± SD in °/oo. (Zof: Fish feeding on Zooplankton, Ct: ctenophore, Omi:
omnivorous invertebrates, Sf: suspension feeders, Gi: grazer invertebrates, Bifc: crustaceans feeding on
benthic invertebrates, Mf: mollusc feeder, Bif: fish feeding on benthic invertebrates, Cf: carnivorous
fish), nd: not determined. When length was not available weight is given between brackets.

Species n Length 5 13C s 15n FT

Scorpaeniformes
Agonus cataphractus Pogge 5 7-7.5 -16.4 ± 0 .9 16.5 ± 0 .4 B if
Aspitriglia cuculus Red gurnard 6 18-23 -15.8 ± 0 .3 16.2 ± 0 .5 B if
Eutriglia gurnardus Grey gurnard 5 7-21 -15.3 ± 0 .7 16.7 ± 0 .8 Cf
Liparis liparis Common seasnail 5 7-9 -15.1 ± 0 .4 17.6 ± 0 .4 Cf
Trigla lucerna Tub gurnard 1 26 -15.4 18 Cf
Perciformes
Scomber scombrus Mackerel 2 27-42 -17.0 ± 0 .1 16.1 ±0.1 B if
Ammodytes tobianus Lesser sandeel 5 15-19 -16.9 ± 1 .5 15.9 ± 0 .8 Zof
Hyperoplus lanceolatus Greater sandeel 4 22-24 -16.0 ± 1.1 17.2 ± 0 .4 Cf
Callionymus lyra Common dragonet 5 13-18 -17.3 ± 0 .5 17.0 ± 0 .3 B if
Mullus surmuletus Striped red mullet 3 16-20 -16.3 ± 0 .4 17.5 ± 0 .3 B if
Pomatoschistus sp Goby 9 5-8 -17.1 ± 0 .5 17.7 ± 0 .8 B if
Trachurus trachurus Atlantic Horse mackerel 5 26-29 -16.3 ± 0 .5 18.2 ± 0 .8 Cf
Echiichtys vipera Lesser weever 5 12-13 -16.3 ± 0 .3 18.7 ± 0 .2 Cf
Beloniformes
Belone belone Garfish 1 49 -15.9 18.0 Cf
Gadiformes
Melanogrammus aeglefinus Haddock 1 40 -16.9 14.8 Zof
Merlangius merlangus Whiting 8 13-27 -16.3 ± 0 .6 19.1 ± 0 .7 Cf
Trisopterus luscus Bib 5 15-17 -15.6 ± 0 .4 19.1 ± 0 .2 Cf
Gadus morhua Cod 6 37-95 -16.3 ± 1.4 19.2 ± 1.4 Cf
Anguilliformes
Anguilla anguilla eel 1 38 -17.3 19.6 Cf

4.3.1. Stable isotopic composition of marine mammals

In th e m u scles, harbour seal, grey seal and w hite-beaked

dolphin displayed sim ilar m ean 815N values, all significantly
higher than that of harbour porpoise, sperm w hale, w hite-sided-
dolphin and fin whale (ANOVA, p<0.002, F=2.0, table 4.2, figure

135
 Marine mammals from th e Southern North Sea

4.1). 815N valu es were sim ilar for harbour seal, grey seal and
w hite beaked-dolphin (ANOVA, F=2.0, p>0.1). In turn, m ean 815N
v a lu es displayed by harbour porpoise were significantly higher
than sperm w hale, w hite-sided dolphin and fin w hale (ANOVA,
F=2.0, p<0.05). 815N did not differ significantly betw een w hite­
sid ed dolphin and fin w hale (ANOVA, F=2.0, p>0.1).

Table 4.2. 8 UC and 8 1SN values in the muscles and livers of marine
mammals collected along the Southern North Sea coast

Species n Muscle S,3C Muscle 8 15N Liver SI3C Liver 5 1SN

Fin whale
3 -18.5 ± 0.9 9 .6 1 1 .3 -18.710.4 11.312.7
Balaenoptera physalus
White-sided dolphin
2 -19.2 ±0.1 1 0.810.4 -17 .8 1 0 .4 12.310.4
Lagenorhynchus acutus
Sperm whale
7 -19.010.9 14.610.6 -18.410.3 13.210.6
Physeter macrocephalus
Harbour porpoise
46 -16.4 ± 1.6 16.2 ± 1.6 -15.8 ± 1.6 16.7 ± 1.8
Phocoena phocoena
Grey seal
6 -15.6 db 1.6 17.9 ±2.1 -15.3 ± 2.6 18.2 ± 0.9
Halichoerus grypus
Harbour seal
23 -16.2 ± 1.3 18.7 ±2.5 -15.9 ± 1.1 17.9 ± 1.9
Phoca vitulina
White-beaked dolphin
7 -15.8 ± 0.7 18.8 ± 1.1 -15.6 ± 0 .7 17.2 ±2.1
Lagenorhynchus albirostris

M uscle m ean 813C did not differ significantly betw een harbour
porpoise, harbour seal, grey seal and w hite-beaked dolphin
(ANOVA, F=1.0, p>0.1). Mean SI3C v a lu es displayed by w hite­
sid ed dolphin, sperm w hale and fin w hale did not differ
significantly (ANOVA, F=1.0, p>0.5) but were significantly lower
th an th ose of other sp e c ie s (ANOVA, F = 0.1, p<0.02).

B ased on their m u scle 815N valu es, trophic levels were estim ated
for harbour porpoise, grey seal, harbour seal and w hite-beaked
dolphin (table 4.3). Trophic levels were n ot estim ated for sperm
w hale, fin whale and w hite-sided dolphin a s their depletion in
813C strongly su ggested that they did not feed w ithin th is area
(see d iscu ssio n below ).

136
 Chapter 4

In the livers, isotopic patterns were quite sim ilar to m u scles with
harbour seal, grey seal and w hite-beaked dolphin displaying
higher m ean 815N valu es compared to other sp ecies (table 4.2,
figure 4.1 and 4.3). 515N v a lu es were sim ilar betw een harbour
seal, grey seal and w hite-beaked dolphin (ANOVA, F=1.5, p>0.5).
Harbour porpoise displayed significantly lower va lu es than
harbour seal (ANOVA, F=1.5, p<0.02) but its 515N v alu es did not
differ significantly from that of grey seal (p=0.06) or w hite-beaked
dolphin (p=0.5). Hepatic 515N va lu es were sim ilar for w hite-sided
dolphin, fin whale and sperm w hale (ANOVA, F= 1.5, p>0.5) and
were significantly lower than th ose of other sp e c ie s (ANOVA,
F=1.5, [Link]).

Hepatic 813C v alu es were sim ilar for harbour seal, grey seal,
w hite-beaked dolphin and harbour porpoise (ANOVA, F=1.4,
p>0.5). These 513C v alu es were significantly higher th an those
displayed by w hite-sided dolphin, sperm w hale and fin w hale
(ANOVA, F=1.4, p<0.05).

Hepatic com pared to m u scle S13C and 815N v a lu es were similar

for harbour seal and grey seal (T-student te st for dependant
sam p les, p>0.1, figure 4.2 and 4.3). No te s ts w a s performed for
w hite-sided dolphin and fin w hale a s only two liver sam p les were
available. M uscle 8I5N valu es of w hite-beaked dolphin were
significantly higher than hepatic o n e s (T-student for dependant
sam ples, p<0.005) while S13C v alu es were sim ilar (p>0.1). M uscle
815N valu es displayed by sperm w hale were significantly higher
th an hepatic o n e s (T-student for dependant sam p les, p<0.02). By
contrast, hepatic m ean 813C and 815N v alu es of harbour porpoise
were significantly higher than m u scle v alu es (T-Student, p<0.01
and p=0.05 respectively, figures 2 and 3). The two sm allest
porpoises (80 and 87 cm) displayed high 815N v alu es (19.3 and
18.1%o respectively) com pared to adult fem ales and m ales
(figure 4.4). However, due to the sm all sam ple size, no statistical
te st w as performed. For harbour porpoises, adult fem ales
displayed higher m u scle 8I5N than adult m ales (figure 4.4, T-
stu d en t for independent sam ples, p<0.05) w hile ju venile isotopic
v alu es were sim ilar betw een s e x e s (p>0.5).

137
 Marine mammals from th e Southern North Sea

4.3.2. Fish and invertebrate muscle isotopic composition.

The h igh est m ean 815N valu es w as m easured in the eel while the
low est m ean value h a s b een recorded in the echinoderm
Echinocardium cordatum (table 4.1). The range of v a lu es w as wide
a s individual 8!5N data ranged from 8.6%o in the m u scle of one
fin w hale to 21.9% o in th e m u scle of one goby.

B ased on their gu t content com position and their lifestyle

(personal observations, G reenstreet et al., 1997; Miller and
Loates, 1997; Quéro and Vayne, 1997), the sp ecies were
classified into feeding typ es (table 4.1). The majority o f the
macro- and m egafaunal taxa investigated proved to be either
Zooplankton feeder invertebrates (Ct) su c h a s the Ctenophore
Pleurobrachia pileus, zooplanktivrous fish (Zof), su sp e n sio n
feeders (Sí), om nivorous invertebrates (crustacean s su c h a s the
com m on shore crab), cru stacean s feeding on benthic
invertebrates (Bifc), m ollusc feeders (Mí), grazer invertebrates
(Gi, m ainly the s e a urchin P sam m echinus miliaris), fish feeding
on benthic invertebrates (Bif) or carnivorous fish (Cf, m ainly fish
or sq u id s feeders). Mean isotopic com position va lu es had been
calculated for each feeding type and com pared to marine
m am m al m uscle data (figure 4.1).

The su sp en sio n feeders displayed the low est m ean S15N value
(mean: 11.1%<>) followed by invertebrate grazers (mean: 12.1%o)
and m ollusc feeders (mean: 13.2 %<>). Carnivorous fish displayed
higher m ean 8I5N valu es th an fish (and squids) feeding on
benthic invertebrates, fish feeding on Zooplankton and
om nivorous invertebrates (figure 4.1, ANOVA, F=2.0, p<0.001).
However, data did n ot differ significantly betw een carnivorous
fish (mean: 17.8%o), cru stacean s feeding on benthic
invertebrates (mean: 17.3 %o) or the Ctenophore Pleurobrachia
p ile u s (mean: 16.6%o, ANOVA, F= 2.0, p>0.1). O m nivorous
invertebrates were significantly enriched in 13C com pared to fish
feeding on Zooplankton (ANOVA, F=1.0, p<0.0001) w hile 815N
data did not differ significantly betw een th ese two feeding
groups.
The Ctenophore Pleurobrachia pileus, th e grazer invertebrate,
P sam m echinus miliaris and the m ollusc feeder A steria rubens
were considerably enriched in 13C (m ean 813C value: -12.9% o, -

138
 Chapter 4

14.1%o and -13.8% o respectively) compared to other feeding

ty p es (table 4.1, figure 4.1).

Herring sam pling w as performed during two cru ises in

Septem ber 2 0 0 0 and in May 2001. Stable isotope data and
len gth differed significantly betw een th e se two sam plings:
herrings caught in May were significantly longer and depleted in
13C and 15N compared to herrings caught in Septem ber (figure
4 .5, T -student for independent sam ples, p<0.001).

4.3.3. Comparison with marine mammals

515N valu es displayed by carnivorous fish did n ot differ

significantly from that of grey se a ls or w h ite-beaked dolphins
(Mann-W hitney U -test, p>0.1, figure 4.1). However, carnivorous
fish 515N data were significantly higher th an that of harbour
porpoises (Mann W hitney U -test, p<0.001) and lower than that of
harbour se a ls (Mann-W hitney U test, p<0.01). W ithin the fish
sp ecies, only the Zooplankton feeders su c h a s Clupeiform s or the
lesse r sandeel A m m o d y te s tobianus displayed lower m ean §15N
and S13C v alu es than harbour porpoises (Mann- W hitney U test,
p<0.005 and p<0.0001 respectively). Sperm w h ales, fin w h ales
and w hite-sided dolphins were significantly depleted in 13C
compared to other feeding groups or m arine m am m als from the
Southern North S ea (figure 4.1 and figure 4.2, Kruskall Wallis,
pcO.001).

4 .4 . DISCUSSION

4.4.1. PATTERN OF CARBON ISOTOPE SIGNATURES

813C sign atures vary widely (range up to 9 .4 %o) am ong

organism s collected w ithin the Southern North S ea (table 4.1,
figure 4.1).

139
 Marine mammals from th e Southern North Sea

Sperm w hales, w hite-sided dolphins and fin w h a les are highly

13C -depleted relative to th e POM, invertebrate, fish and other
m arine mammal sp e c ie s (table 4.1, table 4.2, figure 4.1 and 4.2).
This low 813C m easured in the m u scles and livers of sperm
w hales, w hite-sided dolphins and fin w h ales can be related to a
more oceanic feeding.
Stable carbon isotope ratios have proven m ost u sefu l in
identifying w here particular organism s feed a s 813C v alu es are
typically higher in sp e c ie s from coastal or benthic food w ebs
than in offshore food w eb s (Hobson, 1999; Lesage et al., 2001).
No flesh h a s b een found in the digestive tract o f th e sperm
w h ales and th is se em s to indicate that the w h ales had not been
feeding w ithin the Southern North S ea prior to stranding
(Jauniaux et al., 1998).

Male sperm w h ales are recorded a s including a significant

proportion of squids and fish in their diet in the deep waters of
North Atlantic and Arctic w aters (Santos et al., 1999). In the
Northern hem isphere, they leave warm w aters at the beginning
of th e sum m er to reach feeding grounds on the perim eter of the
polar zone, returning again in w inter (Santos et al., 1999). From
our isotopic data, it appears that despite regular sigh tings of
sperm w h ales w ithin the Southern part of the North Sea, they do
not feed m ainly w ithin th is area, even on local cephalopods.
Indeed, the squid sp e c ie s sam pled in the Southern North se a as
Loligo vulgaris or Sepia officinalis display higher 813C (and S15N)
than sperm w h ales (table 4.1 and 4.2). O ceanic or abyssal
cephalopods have quite a different isotopic signature more
sim ilar to that of sperm w h ales (Ostrom et al., 1993; Abend and
Sm ith, 1995; Iken et al., 2001). Sim ilar co n clu sio n s can be
drawn for fin w h ales and w hite-sided dolphins stranded along
the Belgian and D utch coasts. The depletion in 13C observed for
the two w h ite-sided dolphins m ight also be linked to a more
offshore feeding. 813C of th e se anim als are sim ilar to that
recorded in other parts of the Northeast Atlantic, along the Irish
co a sts (see chapter 3).

140
 Chapter 4

22

20 • M a r in e m am m als

18 H F ish an d in v e rte b r a te s (F T )
B ife
m ft -
S R
'w' 16 hp
Z
Vi
sw O bi i

MD
14 /o f
I -----1 M f -
12 wsd

10
fw TOM
8
-20 -18 -16 -14 -12
513C (%o)
Figure 4.1. Muscle Sl3C and 81SN mean values (± SD) of selected
invertebrate, fish and marine mammals from the Southern North Sea

fw 0 M u scles
a Livers

SW

w sd

w
•2 w bd
o
a.
V)
gs

hs

hp

-23 -21 -19 -17 -15 -13 -11

S13C (%«)
Figure 4.2. Mean and range of 513C values in the livers and muscles of
marine mammals from the Southern North Sea coasts. *: p<0.05, T-test for
dependant samples

fw. fin whale, sw: sperm whale, wsd: white-side dolphin, wbd: white-beaked dolphin, gs: grey seal, hs:
harbour seal, hp: harbour porpoise, Zof: fish feeding on Zooplankton, Omi: omnivorous invertebrates,
Sf: suspension feeders, Ct: ctenophores, Gi: grazer invertebrates, liifc: crustaceans feeding on benthic
invertebrates, Mf: mollusc feeders, Bif: Fish and cephalopods feeding on benthic invertebrates, Cf:
carnivorous fish, POM: particulate organic matter from the Southern North Sea (data from Middelburg
and Nieuwenhuize, 1998).

141
 Marine mammals from th e Southern North Sea

W hite-sided and w hite-beaked dolphins have already b een

sighted in m ixed sp ecies aggregations in the Southern North S ea
(Haase, 1987). T hese temporary association s are n o t likely to be
diet related a s S13C strongly differs betw een th e se two sp ecies
suggestin g two different feeding habits. W hite-beaked dolphin
h a s a more coastal feeding a s su ggested by its enrichm ent in 13C
both in livers and m u scle s (figure 4.2). Similar isotopic
observations have b een done on w hite-beaked and w hite-sided
dolphins collected along the Irish c o a sts (see chapter 3).

Com parison of 813C sign atu res in m u scle s and livers allow s a
long and short term diet com parison a s m am m al m uscle is
know n to have a longer turn over than liver (Tieszen et al., 1983,
H obson and Clark, 1992; H obson et al., 1996). The range of S13C
v alu es in m arine m am m als d o es not differ strongly betw een
livers and m u scles (figure 4.2). Harbour porpoises display higher
513C valu es in the fivers su ggestin g that the anim als were
feeding closer inshore before stranding. No statistical test could
be performed for the two w h ite-sided dolphins due to the lim ited
sam ple size but 513C are also higher in the fivers com pared to
m u scle s reflecting a shift in the diet prior to stranding. A large
range o f 513C v alu es is observed in the fivers of grey se a ls with
data ranging from -1 8 .4 to -1 0 .5 %o w h ich su g g e sts that som e
individuals m ight feed quite offshore w hile others have fed very
near the coast prior to death. The large S13C range observed for
grey se a ls is likely to reflect a m ixture betw een resident se a ls
from the Southern North S ea (likely the W adden S ea colonies)
and temporary or sea so n a l im m igrants from UK coast (Abt et al.,
2 0 0 2 ).
Indeed, long-distance travel outside th e breeding sea so n is not
uncom m on in grey se a ls (McConnel et al., 1999). Som e grey
se a ls occurring w ithin the sou th -eastern North S ea during the
spring (after their moult) are assu m ed to com e from more
northern hau l-ou t site s su c h a s Scotland, Faroe Island s or from
the Humber estuary, along the UK c o a sts (Abt et al., 2002).
Resident grey se a ls are also increasingly observed during th is
last decade along th e W adden S ea c o a sts (Reijnders et al., 1995;
Abt et al., 2002). The six grey se a ls from th is study were
collected along th e Belgian and French C hannel c o a sts betw een
2 0 0 0 and 2001. No stranding h a s b een recorded previously, at
lea st for the Belgian coast. This apparent rate of increase could
be related to the dispersing of the eastern UK stock into the
sou th -eastern North S ea a s observed season ally in other areas
(Abt et al., 2002).

142
 Chapter 4

Fish and invertebrates from the Southern North S ea are more

enriched in 13C com pared to m arine POM data from the
Southern North S ea (Middelburg and N ieuw enhuize, 1998).
Considerable overlap betw een sp e c ie s is observed (figure 4.1).
B ecau se 813C o f an anim al is largely determ ined by th e S13C of its
diet, th ese inter-taxa overlaps in isotope abundance indicate
isotopic sim ilarity am ong the respective diets of m any of th ese
sp ecies. As expected, su sp e n sio n feeders are 13C-enriched
com pared to POM. Among the different feeding types, th e grazer
invertebrates, th e m ollusc feeders and strikingly th e Ctenophore
are strongly 13C -enriched. D eposit feeders have been show n to
be more enriched in 513C compared to su sp e n sio n feeders
suggestin g two different isotopic carbon sign atures for
su sp en d ed particulate m atter and a m ixture of su sp en d ed and
sedim entary organic m atter respectively (Dauby et al., 1998).
Coastal or continental in p u ts are im portant w ithin th is area
leading to 13C enriched valu es for th e particulate m atter of the
C hannel and th e North S ea com pared to the Bay o f Biscay
(Dauby et al., 1994). However, the reason of the h igh enrichm ent
o f th e ctenophore Pleurobrachia p ile u s is unclear. It strongly
differs from other zooplanktivorous anim als su c h a s th e herring
or the lesser san d eel (table 4.1, figure 4.1).

813C differs also betw een herring sam pled in Septem ber 2000
and May 2001 (figure 4.5). Moreover, the m ean len gth of th ese
fish is higher in May th an in Septem ber. The structure of herring
stock in the Northeast Atlantic is com plex w ith different su b ­
population, age and feeding regrouping (Jennings et al., 2001).
Anim als captured in May are ad ults displaying an oceanic
carbon signature compared to anim als collected in Septem ber
w hich are ju v en iles with a typical coastal 813C enrichm ent
compared to POM.

4.4.2. PATTERNS OF NITROGEN ISOTOPE SIGNATURES

Trophic levels of marine mammals

Trophic p osition s (TP) were estim ated according to the m odel
described by Lesage et al. (2001) for harbour porpoise, harbour
seal, grey seal and w hite-beaked dolphin. TP were not evaluated
for w hite-sided dolphins, fin w h ales and sperm w hales. Indeed,

143
 Marine mammals from th e Southern North Sea

their 13C-depletion strongly su g g e sts that they feed out of the

Southern North S ea (figure 4.1 and 4.2). A consum er isotopic
signature is determ ined initially by th e isotopic com position of
the b aseline phyto- and Zooplankton so u rces w h ich m ight vary
widely w ith the sam pling origin (Lesage et al., 2001; Riera et al.,
1999; Middelburg and N ieuw enhuize, 1998). Southern North Sea
POM v alu es is not extrapolable to su c h oceanic sp ecies.

W ithin the North Sea, harbour seal, grey seal and w hite-beaked
dolphin seem to occupy a sim ilar trophic position at the top of
the food web a s su ggested by their high 815N v a lu es in both
m u scle s and livers (table 4.2 and 4.3 and figure 4.1).

Table 4.3. Trophic level comparison of harbour porpoise, grey seal,

harbour seal and white-beaked dolphins (Pauly et al., 1998; this study)

Species Pauly et al. (1998) This study

Harbour porpoise
4.1 3.4
Phocoena phocoena
Grey seal
4.0 3.9
Halichoerus grypus
Harbour seal
4.0 4.1
Phoca vitulina
White-beaked dolphin
Lagenorhynchus 4.2 4.2
albirostris

Estim ated trophic levels calculated by Pauly et al. (1998), based

on stom ach conten t data indicate that trophic levels of harbour
porpoise, harbour seal, grey seal and w hite-beaked dolphin are
sim ilar ranging betw een 4 .0 and 4.2 (table 4.3). T his extensive
stu dy h a s calculated a m ean trophic level for each of the 97
m arine mammal sp ecies and Pauly et al. (1998) have em phasized
the tentative character o f the m odelling. TP estim ated from 815N
v alu es are in good agreem ent w ith data described by Pauly et al.
(1998) except for harbour porpoises w h ich display a lower TP
th an the three other sp ecies (table 4.3). This discrepancy reflects
a high proportion o f prey displaying a low trophic level su c h as
zooplanktivorous fish in the diet of harbour porpoise from the
Southern North Sea. Furthermore, porpoises and dolphins are
opportunist feeders taking advantage of local abundance of prey

144
 Chapter 4

(Lick, 1991; Rogan and Berrow, 1996; C ouperus, 1997;H assani

et al., 1998).

0 Muscles
& Livers

flllllll
6 8 10 12 14 16 18 20 22 24

ölsN (%o)

Figure 4.3. Mean and range of 5,SN values in the livers and muscles of
marine mammals stranded along the French, Belgian and southern Dutch
coasts. *: p<0.05, T-test for dependant samples
fw: fin whale, sw: sperm whale, wsd: white-side dolphin, wbd: white-beaked dolphin, gs: grey seal, hs:
harbour seal, hp: harbour porpoise

145
 Marine mammals from th e Southern North Sea

Adult fem ales porpoises feed at a higher trophic level than adult
m ales while ju venile porpoises display no differences betw een
se x e s (figure 4.4). Previous stu d ie s have described that pregnant
or lactating fem ales m ight have a higher consum ption, feed on
larger prey or forage on different prey sp e c ie s (quoted by
Aarefjord et éd., 1995). The 515N high value encountered for the
two harbour porpoise p u p s m ight be due to their reliance on
their m other for su b sisten ce, either m ilk or nutrition via the
placen ta (Hobson et al., 1997). Indeed, the length o f th e se two
p u p s corresponds to their suckling period ju s t after their birth
(Aarefjord et al., 1995). A m ean enrichm ent o f 2 .5 %o is observed
b etw een th ese two p u p s and adult fem ales w h ich is in
agreem ent with previous stu d ie s carried on northern fur se a ls
(Hobson et al., 1997) or black bears (Hobson et al., 2000) who
reported a m ean 51SN enrichm ent of 1.9 and 2.5 %o respectively
b etw een suckling ju v e n ile s and adult fem ales. However th is
trophic enrichm ent betw een pup and m other is strongly specific
and n e e d s further investigations before u sin g stable isotop es to
quantify w eaning or other lactation p ro cesses (Jen kin s et al.,
2 0 0 1 ).

20

19

18

O 17
p<0.02

16
'ë b

15

13
pups(n=2) adult females (n=9) adult males (n=8)

Figure 4.4: Muscle 8 1SN (mean ± SD) in pups, adult male and adult female
harbour porpoises. Number of individuals are given between brackets

146
 Chapter 4

Trophic relationships
W ithin the North Atlantic, herring, cod, sandeel, w hiting goby
and sole represent major prey for m arine m am m als with large
intraspecific variations. Indeed, m arine mamm al diet h a s been
show n to vary according to age of the individuals, abundance of
prey sp ecies, se a so n s or geographic location (Evans, 1987; Lick,
1991; Pierce et al., 1991b; Aareijord et al., 1995; Tollit et al.,
1997; 1998).

In the North Sea, the harbour porpoise is know n to feed on a

wide range of pelagic and dem ersal fish sp e c ie s su c h a s cod,
herring, sole, goby or dab (Lick, 1991). Expressed a s biom ass of
fish, sole and cod represented 41% and 25% respectively of the
stom ach conten t of harbour porpoises from German waters. In
contrast, in the Baltic sea, cod can represent 70% o f the harbour
porpoise diet biom ass. In you n g porpoises, goby is the m ain prey
by num ber and w eight (Lick, 1991). Harbour se a ls usu ally feed
on clupeids, gadoids, cephalopods or san d eels depending on the
prey avaibility. In the W adden Sea, th e harbour seal se em s to
prefer flatfish sp ecies (North S ea Task Force, 1993; Tollit et
al., 1998). A large proportion of th e grey seal diet in clu d es
san d eels (Ammodytidae), w h ich can constitute over 70% of their
diet depending on location and se a so n (Prime and Hammond,
1990; Pierce et al., 1991a; Hammond et al., 1994).

Carnivorous fish su c h a s G adids display sim ilar 815N to grey

seals, harbour se a ls and w hite-beaked dolphins su ggesting that
they occupy a sim ilar trophic level at the top of the food web
(figure 4.1). Moreover, th e m ean v a lu es of fish sp ecies usu ally
described a s potential prey for North S ea m arine m am m a ls are
h igh compared to harbour porpoise, harbour seal, grey seal and
w hite beaked dolphin data (figure 4.5).

147
 Marine mammals from th e Southern North Sea

22

20 # M arin e m am m als

wbd
18 O Fish
So
G al
o
Ls
& Ach'
z 14 cr
H e (1 1 cm )

12 -

_ I le (27 cm )

10
TOM O
8
-20 -18 -16 -14 -12
813C (% o )
Figure 4.5: Muscle 813C and 81SN mean values (± SD) in marine mammals
and selected fish from the Southern North Sea.

fw: fin whale, sw: sperm whale, wsd: white-side dolphin, wbd: white-beaked dolphin, gs: grey seal, hs:
harbour seal, hp: harbour porpoise Co: cod. So: sole, Go: goby, Ls: lesser sandeel, Ach: anchovy, He:
herring, POM: particulate organic matter from the Southern North Sea (POM data from Middelburg
and Nieuwenhuize. 1998).

As a 815N trophic enrichm ent o f 2.4 %o is expected betw een

potential prey and m arine m a m m a l s (Hobson et al., 1996), u su a l
described prey s u c h as cod, other gadids, goby or sole are not
likely to form the bulk of their diet. Indeed, m ean 815N valu es of
goby, sole or cod are higher th an that of harbour porpoises.
Gadids, goby and sole con stituted a significant part o f the diet of
the North S ea harbour porpoise analysed by Lick (1991) but from
isotopic data, they are not likely to con stitu te the m ain part of its
diet w ithin the Southern North Sea. Cod valu es are even higher
th an that of the two seal and w hite-beaked dolphin sp ecies
(figure 4.4). However, a relationship betw een body size and 815N
value h a s been show n for several m arine sp e c ie s (Jennings et
al., 2002) com plicating the data interpretation. Even if the range
of fish len gth s are sim ilar to that described for m arine m am m al
preys (Aarefjord et al., 1995; G annon et al., 1997), it is not
excluded that sm aller fish individuals w ith lower S15N valu es
could be preyed by m arine m am m als.

148
 Chapter 4

On contrast, zooplanktivorous fish su c h a s herring but also

lesser san d eel or sprat, have lower 815N (and 813C) v alu es of
about 2 to 4 %o and are likely to represent a major link betw een
th e base of th e food w eb, w hich in clu d es various bacterio, phyto-
and Zooplankton, and m arine m am m als or carnivorous fish
(figure 4.1 and figure 4.5). Harbour and grey se a ls display higher
trophic position s th an harbour porpoises su ggestin g that higher
trophic level fish su c h a s lesser (or greater) sa n d eels may be
preyed upon. Sandeel is one of the m ost com m on fish sp ecies on
the continental sh e lf of th e Northwest Europe, com prises 10 to
15% o f the total fish b iom ass in the North S ea and is currently
the target o f the largest single sp e c ie s fishery in the North Sea
(quoted by Rindorf et al., 2000). Sandeel avaibility h a s been
show n to have major effects on breeding s u c c e s s of other marine
predators su c h a s seabirds (Rindorf et al., 2000). Mean 813C and
S15N valu es are sim ilar betw een grey and harbour seal
su ggesting that the range of prey sp e c ie s taken by th ese two seal
foraging areas m ight overlap in th is part o f th e North Sea.
Similar observations based on faecal sam p lings have been
performed in th e sou th-w estern North S ea along the UK W ash
co a sts (Haii et al., 1998). Harbour se a ls are know n to travel ten
kilom etres to feed (Thompson and Miller, 1990) w h ereas grey
se a ls may travel far greater d istan ces (Me C onnel et al., 1999).
Additional partition of resou rces m ay resu lt from differential
foraging in offshore and coastal areas.

Our isotopic data clearly su ggest that pleuronectiform es, cod

and other gadids represent minor contributions to the diet of
Southern North S ea m arine m am m als. The reason w hy th ese
high trophic level fish do not represent a major part of marine
mamm al diet is unclear. The range of sp ecies preyed by marine
m am m als can be wide a s more th an 30 fish prey sp e c ie s can be
identified in the diet of m arine m am m als (Lick, 1991). However,
diet preferences seem to be oriented towards lower trophic level
prey su ch a s clu p eid s or san d eels. Previous stu d ie s indicate that
during the p ast decades, intensive and size selective fishing h a s
changed th e size-structure of the North S ea fish com m unity
resulting in a general body size decrease. Sm aller and early-
m aturing sp e c ie s have increased in relative abundance as
su ggest by th e absolute and relative catch es of sm aller pelagic
industrial sp e c ie s com pared to large dem ersal sp ecies su c h as
cod. (Fifth North S ea Conference Secretariat, 2002).

149
 Marine mammals from the Southern North Sea

4 .5 . CONCLUSIONS

D espite occasional sigh tin gs of sperm w hale, fin whale and

w hite-sided dolphin in th e Southern Bight of the North Sea, they
m ainly feed offshore e.g. w ithin the North Atlantic. On the other
hand, harbour porpoise, harbour seal, grey seal and white-
beaked dolphin belong to th e Southern North S ea food web.
W hite-beaked dolphin, grey seal and harbour seal feed on higher
trophic level prey than harbour porpoise but som e diet overlap
m ight occur betw een th e se species. Intraspecific variations have
b een observed in harbour porpoises: adult fem ales display
higher m uscle 515N than adult m ales while no differences appear
betw een ju ven iles. The lower trophic position occupied by
harbour porpoise reflects a higher input in its diet of lower
trophic level prey su c h a s herring or sa n d eels w h ich represent
an important com ponent in th is ecosystem according to stable
isotope values.

Acknowledgem ents We a re g ratefu l to D r T hierry J a u n ia u x (M ariae A nim al

R esearch an d Interv en tio n Network) w ho necropsied th e m a rin e m am m als from
th is study. T h a n k s to J a n T av e m ie r a n d J a n H aelters for logistical coordination.
T h a n k s to R achel B ouhy, D enis V angeluw e a n d Renzo B iondo (Oceanology
L aboratory, Liège, Belgium) for th e ir valu ab le tec h n ica l a ssista n c e . The a u th o rs
a re grateful to K. C oorem an a n d h is scientific a n d fish erm en te a m (C entrum voor
L andbouw kundig O nderzoek, O ostende, Belgium) for th e ir c o n tin u o u s su p p o rt in
sam ple collection a n d species d eterm in atio n . We ar e also g ratefu l to th e crew s of
th e R.V. Belgica a n d R.V. T h a la ssa (IFREMER, Boulognes, France) for satisfying
cru ises. T his study w as funded by th e B elgian Office for Scientific, T echnical a n d
C u ltu ral Affairs (co n tract M N /D D /50). K. D as a n d G. Lepoint received g ra n ts fiora
th e "Fonds pour la Recherche d a n s l'Agriculture et l'Industrie" (F.R.l.A).

150
 Chapter 4

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Mar. Chem . 60: 217-225 th e d iet o f com m on seals (Phoca
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Miller, P .J., Loates, M .J. 1997. F ish S cotland. J . Zool. 223:641-652
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Pocket Guide. H arp e r Collins P in n eg ar, J.K ., P olunin, N.V.C.
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513C a n d S1SN am o n g fish tissu e s:
Nigel B onner, W. 1989. T he N atu ral im plicatio n s for th e stu d y of tro p h ic
h isto ry of seals. C h risto p h e r Helm in te ra ctio n s. F u n c t. Ecol. 13: 225-
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Prim e, J.H ., H am m ond, P.S. 1990.
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Progress report. F ifth In te rn atio n al W estern N orth S ea asse sse d from
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N orth Sea, 20-21 M arch 2002. faeces. J . App. Ecol. 27: 435-447.
B ergen, Norway. 2 10p
Q uéro, J.C .; V ayne, J . J . 1997. Les
N orth S ea T ask Force 1993. N orth poisons de m er d es pêches
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a n d P aris C om m ission, London. L au san n e-P aris. 304p
O lsen & O lsen, Fredensborg,
D enm ark. 132 + VIp R au, H., Ainley, D .G., B engtson,
J.L ., T orres, J . J . , H opkins,T.L.
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Weddell seab ird s, seals, a n d fish: al. (eds.). Benguela trophic

im plications for d ie t a n d tro p h ic functioning. S. Afr. J . m ar. Sei. 12:
stru c tu re . M ar. Ecol. Prog. Ser. 84: 843:861
1-8
S m ith , R .J., H obson, K.A.,
R eijnders, P J.H ., V an Dijk, J ., K oopm an, H.N., Lavigne, D.M. 1996.
K uiper, D. 1995. R ecolonization of D istin g u ish in g betw een p o p u latio n s
tile D u tch W adden S e a by th e grey o f fresh - a n d salt-w a te r h a rb o u r
seal Halichoerus grypus. Biol. sea ls (Phoca vitulina) u sin g stable-
C onserv. 71: 231-235 isotope ra tio s a n d fatty acid profiles.
Can. J . F ish. A qua. Sei. 53: 272-279
R iera, P., Stal, L .J., N ieuw enhuize,
J ., R ichard, P., B lan ch ard , G., Tieszen, L.L., B outton, T.W.,
G entil, F. 1999. D eterm in atio n o f T esdahl, K.G., Slade, N.A. 1983.
food sources for b en th ic F ractio n atio n and tu m -o v e r of
in v erteb rates in a sa lt m a rsh stab le ca rb o n isotopes in an im al
(Aiguillon Bay, France) by c arb o n tissu e s: im plication for 513C a n aly sis
and nitrogen sta b le isotopes: of diet. Oecologia 57: 32-37
im portance o f locally produced
Tollit, D .J., Black, A.D., T hom pson,
sources. Mar. Ecol. Prog. Ser. 187:
P.M., M ackay, A., Corpe, H.M.,
301-307
W ilson, B., V an Parijs, S .M., G rellier
R indorf, A., W anless, S., H arris, K., P arian e, S. 1998. V ariation in
M.P. 2000. Effects o f ch an g es in h a rb o u r seal Phoca vitulina diet a n d
san d eel avaibility on th e d iv e-d ep th s in relatio n to foraging
reproductive o u tp u t of seab ird s. h a b ita t. J . Zool. 244: 209-222
Mar. Ecol. Prog. Ser. 202: 241-252
Tollit, D .J., G reen street, S.P.R.,
Rogan, E., Berrow , S.D. 1996. A T hom pson, P.M. 1997. Prey selection
review of h a rb o u r porpoises, by h a rb o u r seals, Phoca vitulina in
P hocoena P hocoena in Irish w aters. relatio n to v ariatio n s in prey
Rep. Int. W hal. Com m n. 46: 595-605 a b u n d a n c e . C an. J . Zool. 75: 1508-
1518
S an to s, M.B., Pierce, G .J., Boyle,
P.R., Reid, R .J., R oss, H.M., T hom pson, P.M., Miller, D. 1990.
P atterso n , I.A.P., Kinze, C.C., S u m m er foraging activity and
T ougaard, S., Lick, R., Piatkow ski, m ovem ents of radio-tagged com m on
U., H em and ez-G arcia, V. 1999. seals (Phoca vitulina, L.) in th e
S to m ach c o n te n t o f sp erm w hales Moray firth , N.E. S cotland. J . Appl.
P h yseter macrocephalus stra n d e d in Ecol. 27: 492-501
th e N orth Sea 1990-1996. Mar. Ecol.
Prog. Ser. 183: 281-294

S an to s, M.B., Pierce, G .J., R oss,

H.M., Reid, R .J., W ilson, B. 1994.
D iets of sm all c e ta c ea n s from th e
S co ttish coast. ICES C.M. 1994/
N: 11 16p

Sekiguchi, K., Klages, N.T.W., B est,

P.B. 1992. C om parative an a ly sis of
th e d iets of sm aller O dontocete
ce ta c e a n s along th e c o ast of
S o u th e rn Africa. In: Payne, A.I.L. e t

155
CHAPTER 5: TRACE METALS I N MARINE MAMMALS
FROM THE NORTH SEA AND ADJACENT AREAS:
Relation with s ta b l e carbon and nitrogen isotopes, sex,
metallothioneins and nutritional s t a t u s

In preparation
 Chapter 5

ABSTRACT

In a first a p p ro a c h , tra c e m e ta l c o n c e n tra tio n s w ere exam in ed in 5

sp ecies of m a rin e m a m m a ls fo u n d s tr a n d e d along th e B elgian a n d
F re n c h C h a n n e l co ast: th e h a rb o u r p o rp o ise P hocoena phocoena, th e
h a rb o u r se a l P hoca vitulina, th e g rey se a l H a lic h o e ris g rypus, the
w h ite -b e a k e d d o lp h in L a g en o rh yn ch u s albirostris a n d th e w h ite­
sid e d d o lp h in L a g en o rh yn ch u s acutus. T hen, in trasp ecific so u rc e s of
tra c e m e ta l v a ria tio n s w ere in v estig ated u sin g th e h a rb o u r porpoise
a s a m odel. P o rp o ises from th e B elgian c o a sts w ere c o m p ared to
in d iv id u als from th e G e rm a n N orth S e a a n d B altic c o a sts, D en m ark ,
N orw ay a n d Icelan d . Toxicological re s u lts w ere co n fro n ted to m o st
com m on p ath o lo g ical findings, n am ely a s em aciatio n , lesions of th e
re sp ira to ry s y ste m or p a ra sitism . In flu en ce of d ie t th ro u g h stab le
c a rb o n a n d n itro g e n a n a ly sis (813C a n d S15N), age a n d sex have also
b e e n co n sid ered .
T race m etal c o n c e n tra tio n s v ary w idely a m o n g sp ecies, especially Cd
co n c e n tra tio n s w h ich a re v ery h ig h in th e k id n ey o f th e two w h ite­
sid ed d o lp h in s (88 pg.g-1 dw for b o th individuals). For all m arin e
m a m m a l sp e c ie s, re n a l C d c o n c e n tra tio n s w ere significantly
co rre la ted w ith m u sc le 815N m e a n in g th a t Cd a c c u m u la tio n is partly
d ie t related a s a re s u lt of ocean ic ce p h alo p o d ingestion. The
n u tritio n a l s ta tu s of s tra n d e d h a r b o u r po rp o ise from th e S o u th e rn
N orth S e a is p oor c o m p a re d to b y -c a u g h t ind iv id u als from Norway
a n d Iceland, a s reflected by th e ir b lu b b e r th ic k n e ss, w eight to length
ratio a n d h e p a tic to to tal body m a s s ratio. The p o rp o ises collected
along th e S o u th e rn N orth S e a c o a s t d isp lay h ig h e r Zn a n d Hg
c o n c e n tra tio n s c o m p a re d to in d iv id u a ls collected in m ore p reserv ed
a re a s from th e N orth A tlantic. M oreover, sig n ificant Zn, Hg a n d Se
levels w ere o b serv ed w ith in c re a sin g e m aciatio n severity. P orpoises
d isp lay in g sev ere b ro n c h o p n e u m o n ia also h av e h ig h er Zn
c o n c e n tra tio n s p ro b a b ly d u e to th e asso ciatio n prev io u sly d escrib ed
of em aciatio n and b ro n c h o p n e u m o n ia . T h ese in cre asin g
c o n c e n tra tio n s a re n o t re la te d to a s h rin k in g of liver m a ss a s it
re m a in s strik in g ly u n c h a n g e d d u rin g th e e m aciatio n p ro c ess. As a
re s u lt, h e p a tic tra c e m etal load in c re a se s also. T h ese observ atio n s
te n d to in d ic a te a g e n e ra l re d is trib u tio n of heavy m e ta ls w ithin th e
o rg a n s (m u scles to livers), w h ich r e s u lt from p ro te in catabolism .
S u c h a re -d istrib u tio n co u ld w ell b e a n a d d itio n a l s tre s s for
po rp o ises a lre a d y ex p erien cin g s tre s s fu l con d itio n s (organochlorines,
etc...). In c o n tra s t, C u a n d C d w ere n e v er a sso c ia te d to em aciation.
O th e r p a ra m e te rs s u c h a s age c la ss o r d iet a re m ore likely to be
involved.

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 Marine mammals, trace metals and nutritional status

5 .1 . INTRODUCTION

Since 1998, th e sou th ern part of th e North S ea including the

Northern French, Belgian and D utch c o a sts h a s been
characterised by an increased num ber of stranded m arine
m am m als su c h a s the harbour porpoise, Phocoena phocoena
(Jauniaux et al., 2002). These anim als are final lin k s in the
Southern North S ea (see chapter 4) and therefore raise som e
concern about potential im pact of p ollu tants on their health
statu s. The q u estion o f whether environm ental pollution may
affect the dynam ics and the h ealth of m arine mamm al
popu lation s is unresolved (Harveii et al., 1999; Anan et al.,
2002; Geraci and L ounsbuiy, 2002). Several investigations
have b een carried out in an attem pt to evaluate organic
contam inant effects at am bient environm ental levels (e.g.
Reijnders, 1986; Aguilar and Borrei, 1994; De G uise et al.,
1995; de Swart et al., 1994; J ep so n et al., 1999) w hile fewer
stu d ie s have tried to link m arine m am m al health sta tu s and
m etal levels w ithin th e North S ea and adjacent areas
(Hÿvarinen and Sipilä, 1984; Siebert et al., 1999; B enn et et
al., 2001). W ithin the North Sea, heavy m etals su ch as
cadm ium (Cd), mercury (Hg), or copper (Cu), rem ain
su b sta n c es for priority action under th e OSPAR strategy with
regards to hazardous su b sta n ces (OSPAR, 2000). Chronic
exposure to m etals has b een show n to cause
im m unosup pression or health deterioration in laboratory
anim als during controlled experim ental condition (Krämer et
al., 1993; Debacker et al., 2001;W right and W elboum , 2002).
However, a clear cau se and effects relationship betw een the
residue levels of contam inants in m arine m am m als and the
observed effects h a s only been dem onstrated in only a few
stu d ie s (Reijnders and Aguilar, 2002). The m ain reason s for
the lack o f proof of the im pact of th o se p ollu tan ts in m arine
m am m als are the difficulty of experim enting in laboratory
conditions w ith th ese anim a ls and the frequent occurrence of
confounding factors that ham per th e estab lish m en t of cau se-
effect relationships (Reijnders and Aguilar, 2002). An
alternative to undesirable experim ental m anipulation on
m arine m am m a ls is to carry out system atic post-m ortem
investigations to estab lish the health sta tu s o f stranded and

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 Chapter 5

by-caught anim als coupled to system atic p ollu tants analysis

(Jepson et al., 1999; Siebert et al., 1999; B enn et et al., 2001).
We proposed here a further in sigh t in the relationship
b etw een trace m etal concentrations, sp ecies, diet, nutritional
sta tu s and m etallothioneins (MTs) u sin g m ultiple stable
isotope (S13C and 515N), m ultiple tissu e (liver, m uscle and
kidney) and m ultiple m etal (Zn, Cu, Cd, Fe, Hg, Se) approach.

- In the in te r sp e c ific part of th is study, trace m etals are

compared betw een the tis s u e s o f harbour porpoise Phocoena
phocoena, harbour seal Phoca vitulina, grey seal Halichoerus
grypus, w hite-beaked dolphin L agenorhynchus albirostris and
w hite-sided dolphin Lagenorhynchus acutus found stranded
along the Northern French and Belgian coasts, using
previously reported 513C and §I5N stable isotope
m easurem en ts to infer specific diet influence on m etal levels
(see chapter 4). The u s e of stable isotop es in dietary stu d ies is
w ell estab lish ed (G annes et al., 1998; Kelly, 2000) and is
based on the dem onstration that stable isotope ratios (13C / 12C
and 15N / HN) in a consum er are related to th o se of their prey
(De Niro and Epstein, 1978; 1981).

- in tr a sp e c ific variations of m etal concentrations and

potential relationships w ith the health sta tu s are investigated
in th e harbour porpoise tis s u e s by com bining toxicological
resu lts, biom etric data, stable isotope m easu rem en ts and
m ost com m on pathological findings, nam ely em aciation,
parasitism , and le sio n s of the respiratory system , previously
described (Siebert et al., 1999; 2001; Jau n iau x et al., 2002).
Harbour porpoises from the Southern North S ea are
com pared to th ose from the German North and Baltic Seas,
Denmark, Iceland and Norway. Furthermore, the spéciation of
Zn, Cu, Cd and Hg on m etallothioneins h a s b een studied in
the liver and kidney o f selected porpoises from the Southern
North S ea to get a furher in sigh t on th ese protein role in
m etal hom eostasis.

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 Marine mammals, trace metals and nutritional status

5 .2 . MATERIAL AND METHODS

5.2.1. SAMPLE COLLECTION AND STORAGE

49 harbour porpoises, 26 harbour sea ls, 6 grey sea ls, 2 w hite­

sid ed dolphins and 9 w hite-beaked dolphins found stranded
along the Belgian coastline and northern France have been
sam pled betw een Decem ber 1993 and May 2001 (tables 5.1
and 5.2). Moreover 75 harbour porpoises respectively
collected along c o a sts of th e German North S ea (11 stranded),
German Baltic S ea (8 by-caught), Denm ark (15 by-caught),
Iceland (11 by-caught) and Norway (23 by-caught) have been
added to th is study (see table 5.2 for details). Post-m ortem
investigations were performed according to the protocol
described by Kuiken and Garcia-Hartmann (1993). Sam ples of
liver, kidney and m u scle were taken and stored at -20°C until
analyses. The nutritional statu s, the lesio n s o f th e respiratory
tract and the parasitism were previously described by Siebert
et al. (1999; 2001).

- The nutritional sta tu s (body condition) w as ju d ged on the

b a sis o f blubber th ick n ess, m easured at four different
locations (sternal, caudodorsal, caudolateral and
caudoventral to th e dorsal fin) and on the state of m u scles
and divided into three categories: good, m oderate, em aciated.

- The lesio n s o f the respiratory system were grouped into mild

(pulmonary oedem a, pulm onary congestion, no nem atode, no
pneum onia), m oderate (pulmonary oedem a, pulm onary
congestion, m ild nem atode infestation and m ild pneum onia)
and severe lesio n s (pulmonary oedem a, pulm onary
congestion, severe nem atode infestation and severe
pneum onia).

- Parasitism w as divided into 4 broad categories: absen ce of

parasitism , mild, m oderate and severe based on observations
during necropsy (Siebert, personal com m unication).

The ages of the porpoises were estim ated by counting tooth

growth layer groups (GLGs) according to Lockyer (1995).

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 Chapter 5

5 .2 .2 . A N A L Y T IC A L M E T H O D S

Zn, Cd, Fe and Cu analyses

After being w eighed and dried for 48 h at 1 10°C, sam p les were
digested w ith a solution of nitric acid (Merck 456) (1:3, v:v)
and slow ly heated to 100C° until com plete digestion. Atomic
absorption spectrophotom etry (ARL 3510) w a s u se d to
determ ine heavy m etal concentrations (Cu, Zn, Cd, Fe).
C oncentrations are expressed a s pg.g 1 dry w eight (dw).
Parallel to the sam ples, a set o f certified m aterial sam p les
(CRM 278 Com m unity Bureau of Reference, Com m ission of
th e European Com m unities) w as also analysed to ensure the
m ethod’s sensitivity. Recoveries ranged from 92 to 102 % for
Cu, Zn, and Fe and 88% for Cd. Limits of detection were 0.01
Hg g-1 dw for Cu, 0.33 for Zn, 0.16 for Fe and 0 .2 2 for Cd.

Total mercury analysis

The total m ercury concentration w as determ ined by specific
atom ic absorption spectrom etry on a Perkin-Elmer Coleman
Ma s -50 Mercury analyser after the m ethod described in detail
in Joiris et al. (1991). Quality control includ ed procedural
blanks, replicate an alyses and an alyses o f standard
references tissu e , u sin g an external standard curve. The
absolute detection lim it is 10 ng Hg corresponding to 0.3 Hgg"
1 for an average 0.3 g dw sam ple.

Selenium analysis
Selen ium w as analysed by fluorimetry following com plete
digestion of th e tissu e by nitric, perchloric and hydrochloric
acids, coupling to EDTA and 2,3-diam inonaphtalène and
extraction by cyclohexane (modified from Mejuto Marti et al.,
1987). The fluorimetre w as set at excitation w avelength of
364n m and em ission w avelength o f 523nm w ith an em ission
slit of 2m m and an integration tim e for read m ode of 2s.

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 Marine mammals, trace metals and nutritional status

Metallothionein analysis
3 to 4 grains of kidney were hom ogenized u sin g an Ultra-
Turrax in a 0.01 Mol am m onium formiate (pH = 7.4)
containing 10 mM sodium azide and 0.01% dithithreitol and
centrifuged at 26 000 g (60 m in, 4°C). The supernatant w as
filtered on an AcA 54 at 4°C. Fractions were collected and
absorbance profile read at 215, 2 5 4 and 280 nm u sin g a
spectrophotom eter (Beckm an DU 530).
After adding nitric acid (65%) to each fraction and to the
pellet, hom ogenate and supernatant, all were slow ly heated to
100°C until com plete digestion. Sam ples were diluted u sin g
deionised water and filtered prior to heavy m etal an alysis (Zn,
Cu and Cd) by atom ic absorption spectrophotom etiy (ICPS:
ARL 3510). Hg w as analysed by ftam eless atom ic absorption
(Perkin-Elmer MAS-50A) a s described above.

Stable isotope m easurem ents

O rganism s m ay vary in their concentrations of lipids. As lipids
are in 13C relatively to the diet (Tieszen et al., 1983), they were
extracted from sam p les u sin g repeated rin se s with 2:1
chloroform : m ethanol prior to analysis. After drying at 50°C
(48h), sam p les were ground into a h om ogen eou s powder.
Carbon dioxide and nitrogen g a s were analysed on an V.G.
O ptim a (Micromass) IR-MS coupled to a N-C-S elem ental
analyser (Carlo Erba). Routine m easu rem en ts are precise to
0.3 °/oo for both 13-carbon and 15-nitrogen. Stable isotope
ratios were exp ressed in 8 notation according to th e following:

5X = [(R sample/R standard)“ 1] X 1000

where X is 13 C or 15N and R is th e corresponding ratio 13C / 12C

or is N / 14 N.
Carbon and nitrogen ratios are exp ressed relative to the VPDB
(Vienna Peedee Belem nite) standard and to atm ospheric
nitrogen, respectively.

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 Chapter 5

Isotopic model
M uscle 515N sign atures of harbour porpoises were converted
to trophic position, TP, u sin g the above equation (after
H obson and Welch, 1992; Lesage et al., 2001):

T P = 2 + (Dm - PO M - TEFmmt)/ T E F

Where Dm = 515N value in porpoise m u scle, POM = 515N value

of particulate organic m atter (fixed to 9%o after Middelburg
and N ieuw enhuize (1998) for Southern North S ea marine
particulate organic matter), and TEF = trophic enrichm ent
factor in 8I5N for a particulate tissu e (Hobson and Welch,
1992). This latter value w as set to a m ean 3.4%o for all
com m unity com p onents (Lesage et al., 2001) except for
m arine m am m als, for w hich a TEF value (TEFmmt) of 2.4%o
w as obtained in the m u scles of 2 harbour se a ls fed on a
constan t herring diet (Hobson et al., 1996).

5.2.3. DATA TREATMENT

Kolmogorov-Smirnov te st w as u se d to assu m e th e normality

of th e data. W hen not distributed norm ally th e variables were
log-transform ed to normalize their distribution. Multivariate
an alysis of variance (n-way MANOVA) followed by post-hoc
m ultiple com parison te s ts (LSD test) have b een u se d to
compare the data betw een the different groups. Parametric
Spearm an- and n on parametric Pearson- coefficients have
b een u sed to test correlations betw een th e values. R esults
were judged significant w h en p<0.05 u n le s s otherw ise stated.
Com parisons betw een sp ecies a s w ell a s com parison betw een
locations were m ade u sin g the different tis s u e s available:
fiver, kidney and m uscle for heavy m etals and fiver and
m u scle for stable isotope ratios. Effect of sp e c ie s on heavy
m etals and stable isotope ratios were tested u sin g a
m ultivariate an alysis of variance (one way-MANOVA). For Hg
and Cd, m ultifactorial an alysis of covariance
(ANCOVA/MANCOVA) w as u se d to adjust the effects for age
(covariate).
Effects of em aciation (good, m oderate, em aciated), lesio n s of
th e respiratory system (absence, mild, m oderate, severe) and
parasitism (absence, mild, m oderate, severe) on trace m etal

163
 ^ Marine mammals, trace metals and nutritional status

concentrations have b een tested sim u ltan eou sly (three-way

MANOVA) on th e liver, kidney and m u scle o f th e porpoises
from the German Baltic Sea, th e German North Sea, the
D anish, Norway and Icelandic coast, excluding porpoises from
th e Belgian and Northern France coasts. For th e latter, only
the effect of em aciation (one-way MANOVA) w a s judged, as
data for the respiratoiy system and parasitism were not
comparable to other locations.

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 Chapter 5

5 .3 . RESULTS

5.3.1. INTERSPECIES COMPARISON

Hepatic, renal and m u scle m etal levels were compared

betw een harbour porpoises, harbour and grey se a ls and
w hite-beaked dolphins found stranded along th e Northern
French and Belgian c o a sts (tables 5.1 and 5.3). Main
statistical r esu lts are sum m arised in the table 5.5. Only two
w h ite-sided dolphins were available and so, were excluded
from the statistical analysis.

Table 5.1. Trace element concentrations (p g.g1 dry weight) in the liver,
kidney and muscle of marine mammal species stranded along the
Belgian and Northern French coasts expressed as a mean ± standard
deviation, range of concentrations (minimum-maximum); n: number of
samples; nd: non determined
Harbour seal Grey seal W hite-beaked dolphin W hite-sided dolphin

liver 122 ± 5 8 125 ± 21 1 5 5 ± 103 167 ± 2 5

(48-296) (91-153) (78-382) (149-185)
n=26 n=6 n=9 n=2
kidney 108 ± 4 6 86 ± 15 90 ± 3 2 118 ± 19
Zn (67-268) (67-111) (26-119) (105-131)
n=24 n=6 n=9 n=2
muscle 136 ± 6 2 109 ± 2 3 56 ± 1 6 28 ± 6
(53-271) (79-143) (28-95) (24-32)
n=27 n=6 n=9 n=2
liver 0.1 ± 0 .2 <0.05 0.1 ±0.1 25 ± 4
(< 0.05-0.9) (<0.05-0.1) (< 0.05-0.3) (22-28)
n=26 n=6 n=9 n=2
kidney 1.0 ± 3 0.3 0.5 ± 0.5 88 ± 0.3
Cd (<0.05-12) (0.05-0.6) (<0.05-1.3) (88.0-88.4)
n=6
1!

n=9 n=2
S
3

muscle <0.05 <0.05 0.4 ± 1.2 0.3 ± 0.03

(<0.05-0.7) (<0.05-0.3) (<0.05-4) (0.25-0.3)
n=27 n=6 n=9 n=2
liver 2726 ± 2088 1439 ± 761 1411± 638 1215 ± 54
(413-8162) (739-2886) (683-2377) (1176-1253)
n=26 n=6 n=9 n=2
kidney 726 ± 220 534 ± 145 458 ± 101 7 5 7 ± 183
Fe (306-1237) (299-721) (336-608) (627-886)
n=23 n=6 n=9 n=2
muscle 785 ± 266 559 ± 2 0 3 612± 164 734 ± 153
(400-1387) (214-803) (302-880) (625-842)
n=27 n=6 n=9 n=2

165
 Marine mammals, trace metals and nutritional status

Table 5.1 (continued). Trace element concentrations (p g.g1 dry weight)

in the liver, kidney and muscle of marine mammal species stranded
along the Belgian and Northern French coasts expressed as a mean ±
standard deviation, range of concentrations (minimum-maximum); n:
number of samples; nd: non determined

H arbour seal Grey seal W hite-beaked dolphin W hite-sided dolphin

liver 21 ± 2 2 37 ± 18 26 ± 7 27 ± 6
(5-118) (7-57) (10-36) (22-31)
n=26 n=6 n= 9 n=2
kidney 20 ± 13 12.5 ± 3 .5 11 ± 5 13 ± 1
Cu (9-74) (7-15) (4-19) (13-14)
n=24 n=6 n=9 n=2
muscle 8± 3 6 ± 1.2 8± 3 5± 2
(4-15) (4-7) (3-14) (4-7)
n= 27 n=6 n=8 n=2
liver 8± 7 8±6 51 ± 6 5 23 ± 12
(1-30) (4-14) (1-157) (14-31)
n=22 n=3 n=7 n-2
kidney 5±4 10 ± 3 3± 3 4± 4
Se (0.7-13) (7-13) (0.2-9) (1-7)
O
IO

n=3 n=8 n -2
It
3

muscle 1.5 ± 0 .5 2 ± 0 .7 1.7 ± 0.1 3

(1-2.2) (1.5- 2.8) (1.5-2.0)
11=8 n=3 n~3 n=l
liver 7± 5 8 ± 11 74 ± 9 4 13 8 ± 114
(1-19) (0.8-24) (7-140) (56-219)
n=4 n=2 n=2
3
II

kidney 5± 5 8±4 4.5 ± 4 10

Hg (1-16) (5-11) (2-7)
n=l 1 n=2 n=2 n=l
muscle 3±4 3 ± 1.9 4± 4 4 ± 0 .5
(0.9-14) (1.5-4.2) (0.9-7) (3 .4 4 .1 )
n = ll n=2 n=2 n=2

- Z n differed significantly betw een sp ecies (one way-MANOVA,

F9,i8o=6.7 p<0.0001). T hese differences were driven by hepatic
and m uscle Zn concentrations w hile renal Zn concentrations
did not differ significantly (table 5.5). Specifically, harbour
porpoise hepatic Zn concentrations were significantly higher
than those of harbour seal (post-hoc LSD test, p<0.0005). The
h igh est m u scle Zn level w as found in harbour seal,
significantly higher th an that of harbour porpoise (post-hoc
LSD test, p<0.00001) and w hite-beaked dolphin (post-hoc
LSD test, p<0.00001).

- C d levels differed betw een sp e c ie s (one-w ay MANOVA,

Fó,ioo=5.5, p<0.0001). Differences appeared both in liver and

166
 Chapter 5

kidney (table 5.5). M uscle concentration were below or close

to the detection lim it and were exclud ed from statistical tests.

V eiy high Cd levels were found in the liver and kidney of the
two w hite-sided dolphins (table 5.1). Harbour porpoises,
harbour and grey se a ls and w hite-beaked dolphins displayed
far lower concentrations. Among th e se four sp ecies, harbour
porpoise displayed the h igh est m ean hepatic and renal
concentrations while grey seal displayed th e low est, close to
th e detection limit.

- Fe concentrations differed betw een sp e c ie s (one-way-

MANOVA, Fg,180=3.1, p<0.005). Specifically, differences were
driven by hepatic levels (table 5.5). The h igh est m ean hepatic
concentrations w as found in th e harbour seal, significantly
higher th an that of harbour porpoise (post-hoc LSD test,
p<0.0005) and w hite-beaked dolphin (post-hoc LSD test,
p<0.05).

- Cu concentrations differed significantly betw een sp ecies

(one-way-MANOVA, Fç>,i77=4.8, p<0.00001). D ifferences were
driven by hepatic and renal concentrations w hile m uscle Cu
concentrations rem ained sim ilar betw een sp e c ie s (table 5.5).
Specifically, harbour seal displayed higher hepatic Cu
concentrations than grey seal (post-hoc LSD test, p<0.05) or
harbour porpoise (post-hoc LSD test, p<0.0005). The h ighest
renal Cu concentration w as observed for the harbour seal and
harbour porpoise and, both were significantly higher than
that of w hite-beaked dolphin (post-hoc LSD test, respectively
p<0.005 and p<0.05). Grey seal displayed also significantly
lower renal Cu concentrations th an harbour seal (post-hoc
LSD test, p<0.05).

- S e and Hg concentrations rem ained sim ilar betw een sp ecies

in the liver, kidney and m uscle. However, the interpretation of
the resu lts w as w eakened by the absen ce of age data for the
two seal and dolphin species.

- R ela tio n sh ip w ith sta b le iso to p e s

Considering the w hole data together (harbour porpoises,

harbour seals, grey seals, w hite-beaked and w hite-sided
dolphins), a significant relationship w as observed betw een
m u scle 515N m easu rem en ts previously reported (see chapter
4) and hepatic (Bravais-Pearson correlation, r=-0.65,

167
 Marine mammals, trace metals and nutritional status

p<0.0001, n=64) and renal (Bravais-Pearson correlation, r=-

0 .6 5 , [Link], n=62) Cd (log-normalised) concentrations
(figure 5.1). No relationship w as observed with other m etals.

1 0 0 .0 0
m harbour porpoise
A harbour seal
▲ grey seal
□ white-beaked dolphin
■ white-sided dolphin
10.00
• mgs •

'ui
Ó
n) 1.00
■O
O
<c<v0
a:
0.10

0.01
8 10 12 14 16 18 20 22 24

51SN (%o)
Figure 5.1. Relationship between muscle 51SN and renal Cd
concentrations of marine mammals stranded along the Belgian and
neighbouring coasts using a log-scale

100.00

10.00

1.00

C
O
n
<
Lu
Q German North Sea
0.10 German Baltic Sea
Denmark
Iceland
Norway
Belgian coasts
0.01
10 12 14 16 18 20 22

51SN (%«)
Figure 5.2. Relationship between muscle 5,SN and renal Cd
concentrations of harbour porpoises from the Northeast Atlantic using
a log-scale

168
 Chapter 5

5.3.2. INTRASPECIFIC VARIATIONS: THE CASE OF

THE HARBOUR PORPOISE

T hese resu lts includ ed porpoises from the Belgian and

Northern French coasts, w hich were com pared to porpoises
from the c o a sts of the German North and Baltic Seas,
Denmark, Norway and Iceland (tables 5.2 and 5. 3).

Table 5.2. Sampling description of harbour porpoises from the North

Sea and adjacent areas ( 2 juvenile porpoises were not evaluated for nutritional status).

sex nutritional status

stranded by-caught
male fem ale non-em aciated m oderate em aciated

3 foetuses 1 2 1 2 3(100% ) 0 0

11 neonates 10 1 6 5 5 (46%) 3 (27%) 3 (27%)

82 juveniles 43 39 36 46 46 (56%) 7 (8.5%) 27 (33%)

38 adults 20 18 19 19 18(47% ) 8 (21%) 12(32% )

TOTAL: 135 74 61 63 72 73 (5 4 % ) 18 (1 3 % ) 4 2 (4 1 % )

Considering all the porpoises together, hepatic Zn and Cu

concentrations differed betw een the foetu ses, n eo n a tes (<90
cm), ju ven iles (<130 cm) and ad ults (> 130cm) (1-way
MANOVA, all effects p< 0.0001, Fe,234=12.6 and 22.6).
Specifically, fo etu ses displayed significantly higher hepatic Cu
concentrations than n eon ates (post-hoc LSD te s ts p<0.04)
w hich in turn had higher Cu concentrations th an ju ven iles
and adults (post-hoc LSD test, p<0.0001). N eonates displayed
significantly lower Zn concentrations th an fo etu ses (post-hoc
LSD test , p=0.05), ju v en iles (post-hoc LSD te st p<0.007) and
ad u lts (post-hoc LSD test p<0.01). Juvenile and adult
porpoises displayed sim ilar Cu and Zn concentrations (post-
h oc LSD test p>0.6). For other statistical tests, the 3 fo etu ses
and 11 n eon ates will be excluded.

169
 Marine mammals, trace metals and nutritional status

Table 5.3. Trace element concentrations (mean ± SD, range and

number of samples) in harbour porpoises from different North Sea and
Atlantic areas

B elgian and N orth Sea Baltic Sea North Sea

Iceland Norwegian
Northern France German German Danish coasts coasts
coasts coasts coasts coasts

liver 234 ± 179 217± 175 135 ± 5 6 117 ± 44 135 ± 26 111 ± 47

(40-684) (71-727) (78-242) (74-261) (114-187) (69-248)
n=49 n=15 n=9 n=17 n = ll n=23
kidney 107 ± 2 7 106 ± 2 6 90 ± 2 0 92 ± 2 3 101 ± 16 96 ± 2 5
Zn (68-201) (69-157) (68-136) (74-166) (78-120) (73-164)
n=48 n=13 n=9 n=15 n=l 1 n=22
muscle 74 ± 3 3 83 ± 2 7 58 ± 2 7 71 ± 3 4 4 4 ± 13 53±40
(24-193) (51-134) (27-124) (39-148) (20-61)) (19-163)
n=51 n=14 n=9 n=15 n=10 n=23
liver 0.5 ± 0.6 0.6 ± 1.3 0.2 ± 0.2 0.2 ± 0 .1 6 ± 11 0.4 ± 0 .5
(<0.05-2.5) (< 0.05-5) (< 0.05-0.5) (<0.05-0.4) (0.4-39) (<0.05-2)
n = 49 n=15 n=9 n=17 n = ll n=23
kidney 3.1 ± 3 .1 4± 9 1.1 ± 1.5 1.1 ± 1.0 19 ± 17 6 ± 4 .5
Cd (<0.05-12) (<0.05-33) (<0.05-5) (0.1-3.5) (2-58) (<0.05-16)
n=48 n=13 n=9 n=15 n = ll n=22
muscle <0.05 <0.05 <0.05 0.1 ± 0 .3 0.1 ± 0 .1 5 <0.05
(<0.05-0.2) (<0.05-0.2) (<0.05) (<0.05-1.2) (<0.05-0.4) (<0.05-0.8)
n=51 n=14 n=9 n=15 n=10 n=23
liver 1435 ± 995 1182 ± 7 3 5 962 ± 461 1287 ± 5 7 3 1567 ± 5 4 4 1249 ± 3 0 0
(324-4490) (235-2556) (349-1826) (527-2588) (697-2378) (792-1774)
n = 49 n=15 n=9 n=17 n = ll n=23
kidney 6 2 1 ± 375 518 ± 2 0 9 914± 1091 7 5 0 ± 183 819 ± 2 5 5 815 ± 2 5 0
Fe (267-2539) (219-844) (425-3808) (541-1203) (511-1368) (489-1681)
n=48 n=13 n=9 n=15 n=l 1 n=22
muscle 752 ± 246 638 ± 250 4 6 7 ± 193 5 4 4 ± 112 651 ± 143 563 ± 163
(262-1204) (260-1168) (225-802) (327-671) (373-842) (256-887)
n=51 n=14 n=9 n=15 n=10 n=23

170
 Chapter 5

Table 5.3 (continued). Trace element concentrations in harbour

porpoises from different North Sea and Atlantic areas

B elgian and N orth Sea B altic Sea North Sea

Iceland Norwegian
Northern France German German Danish coasts coasts
coasts coasts coasts coasts

39 ±3 8 64 ±53 62 ± 7 7 28 ± 16 30 ± 11 48 ±61
liver (9-257) (20-169) (18-260) (15-88) (13-53) (12-217)
n=49 n=15 n= 9 n=17 n-11 n=22
17± 11 15 ± 2 16 ± 3 15 ± 6 16 ± 4 16 ±4.5
Cu kidney (7-73) (11-19) (13-21) (11-36) (11-23) (10-32)
n= 48 n=13 n=9 n=15 n=U n=22
7±4 7±2 7± 2 5 ±1.3 5 ± 1.5 6±2
muscle (2-22) (3-11) (4-10) (3-8) (1.8-7) (2-9)
n = 51 n= 14 n=9 n=15 n=10 n=23
14± 21 11 ±11 6± 3 9±7 10 ±8.5 13 ± 7
liver (0.6-99) (1-39) (2-10) (3-32) (2.5-26) (4-34)
n=37 n=13 n=8 n=14 n=6 n=23
6 ±4 17 ± 9
Se kidney (1-21) nd nd nd nd (3-33)
CO
"ff

n=21
II
C

3.8 ±8.5 5 ± 15
muscle (0.4-39) nd nd nd nd (0.2-71)
n=20 n=22
80 ± 279 13 ± 17 4.5 ± 3.6 22 ± 3 6 16 ± 14 14 ± 10
liver (0.6-1681) (1-56) (0.9-12) (1-147) (1.4-44) (1-32)
n-37 n=15 n -9 n-17 n= ll n-22
8 ±13 6±9
Hg kidney (0.9-*2) nd nd nd nd (1-43)
n-18 n=21
5±7 9 ± 31
muscle (0.7-28) nd nd nd nd (0-146)
n=17 n=22

Zn. Geographic location of collections

significantly affected Zn concentrations (1-way MANOVA all
effects Fis,281=4.68, p<0.00001; figure 5.3), specifically in liver
and in m uscle while renal Zn rem ained sim ilar (table 5.5).
Hepatic Zn concentrations did not differ significantly betw een
porpoises from Belgian and German parts of the North Sea,
w h ich in turn were significantly higher th an th ose from
German Baltic S ea (post-hoc LSD test, p=0.05 and p<0.05 for
Belgian and German porpoises respectively), Denm ark (post-
hoc LSD test, p<0.005) and Norway (post-hoc LSD test,
p<0.0005).

171
 Marine mammals, trace metals and nutritional status

□ non -emaciated
i moderate
memaciated

Figure 5.3. Hepatic Zn concentration in relation to the nutritional

status and sampling location considered (B: Belgian and French coasts; GNS:
Gennan North Sea coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N: Norway. The
3 foetuses and 11 neonates are excluded)

□ non -emaciated
0 moderate
@emaciated

Figure 5.4. Hepatic Zn load in relation to the nutritional status and

Sampling location considered (B: Belgian and French coasts; GNS: German North Sea
coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N: Norway. The 3 foetuses and 11
neonates are excluded)

172
 Chapter 5

□ absence
omild
æ moderate
□severe
3 500

b 400

X
UL*
i 1 2 9 il 2 i3
GNS GBS

Figure 5.5. Hepatic Zn concentration in relation to the lesions of the

respiratory system and sampling location considered (GNS: German North
Sea coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N: Norway. The 3 foetuses and
1 1 neonates are excluded)

200
a n o n -emaciated
— 180
□ moderate
□ emaciated

o> 120

GNS GBS DK

Figure 5.6. Hepatic Hg concentration in relation to the nutritional

status and sampling location considered (B: Belgian and French coasts GNS:
German North Sea coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N: Norway. The
3 foetuses and 11 neonates are excluded)

173
 Marine mammals, trace metals and nutritional status

Sim ilarly, higher Zn m u scle concentrations were found in

porpoises from the Belgian and German areas of the North
S ea com pared to porpoises from Iceland (post-hoc LSD
test, p<0.002) and Norway (post-hoc LSD test, p<0.001).
Moreover, German porpoises from th e North S ea displayed
higher m uscle Zn concentrations th an German porpoises
from th e Baltic S ea (post-hoc LSD test, p<0.03).

Nutritional sta tu s significantly affected Zn concentrations

(table 5.5, figure 5.3). T hese differences were driven by
hepatic concentrations w h ich were significantly higher in
porpoises displaying a good (post-hoc LSD test, p<0.0006) and
m oderate body condition (p<0.003) but no effect on renal or
m u scle Zn concentrations were observed (p>0.5). Parallel to
the concentrations, hepatic Zn load increased w ith degrading
body condition (figure 5.4).

Lesions of the respiratory system and parasitism significantly

affected Zn concentrations (table 5.5, F9,io7= 3 .4 , p<0.0015
and F3,44=4.9, p<0.005 respectively). Porpoises displaying
severe lesio n s of th e respiratory system had significantly
higher hepatic Zn concentrations th an porpoises w ithout
(post-hoc LSD test, p<0.00005), or displaying m ild (post-hoc
LSD test, p<0.00001) or m oderate (post-hoc LSD test,
p<0.00003) respiratory le sio n s (figure 5.5). Porpoises
displaying severe parasitism had higher hepatic Zn
concentrations than porpoises w ithout (post-hoc LSD test,
p<0.01) or with m ild (post-hoc LSD test, p<0.0001) or
m oderate (post-hoc LSD test, p<0.001) parasitism .

Cu. Hepatic, renal and m u scle Cu

concentrations rem ained sim ilar betw een the six locations
(table 5.5). Hepatic, renal and m u scle Cu concentrations were
n ot affected by the nutritional sta tu s or le sio n s of the
respiratory system . The degree of parasitism (absence, mild,
m oderate or severe) affected significantly the Cu
concentration. Porpoises displaying mild or moderate
parasitism display lower hepatic and m uscle concentration
th an porpoises w ithout parasitism .

Cd. Geographic location of collections

significantly affected hepatic and renal cadm ium

174
 Chapter 5

concentrations adjusted for age (table 5.3 and 5.5, 1-way

MANCOVA adjusted effects, Rio,i22=7.5, p<0.0001).
Specifically, hepatic and renal concentrations in porpoises
from Iceland were significantly higher th an th ose from other
locations (post-hoc LSD test, p<0.02). The low est Cd
concentrations were found in the fiver and kidney of
porpoises from the German Baltic S ea and D an ish coasts.
T hese two locations did not differ significantly betw een them
(post-hoc LSD test, p>0.5). Porpoises from Norway, German
North S ea and Belgian coasts display sim ilar hepatic and
renal Cd concentrations (post-hoc LSD test, p>0.5), in an
interm ediate position betw een German Baltic and D anish
porpoises (post-hoc LSD test, p<0.05) and Icelandic porpoises
(post-hoc LSD test, p<0.02). No statistical test w a s performed
in m uscle a s th e concentrations were below or close to the
detection limit.
The nutritional statu s, lesio n s o f th e respiratory system and
parasitism did not affect hepatic or renal Cd concentrations in
porpoises (p>0.5).

F e. M uscle Fe concentration s differed

significantly betw een sam pling location s w hile hepatic and
renal concentrations did not (table 5.5). Renal Fe
concentrations were related to the nutritional status.
Specifically, porpoises in good body condition display
significantly higher renal Fe th an porpoises displaying a
m oderate (post-hoc LSD test, p<0.02) or em aciated body
condition (post-hoc LSD test, p<0.002).

Renal Fe concentrations are also related to lesio n s of the

respiratory system a s porpoises w ithout bronchopneum onia
displayed higher renal Fe concentrations th an porpoises
characterized by m ild (post-hoc LSD test, p<0.05), moderate
(post-hoc LSD test, p<0.05) or severe (post-hoc LSD test,
p<0.0005) lesion s. No relationship betw een parasitism and
hepatic, renal or m u scle Fe concentration w a s observed.

Hg, Geographic location affected hepatic Hg

concentrations standardised for age (1-way ANCO VA, all
effects, Fs, 76=2.48, p<0.04, figure 5.6).

Porpoises from Belgian and French c o a sts carried the h ighest

Hg concentrations, significantly higher than porpoises from

175
 Marine mammals, trace metals and nutritional status

the German Baltic Sea (post-hoc LSD test, p=0.005). One six-
year-old em aciated fem ale porpoise displayed an hepatic Hg
concentration of 1681 H g g ’1 dw (table 5.3) but th is
exceptionally high concentration w a s exclud ed from statistical
com parison to avoid potential b iased resu lts. Considering all
the sam pling locations sim ultan eou sly, neither nutritional
sta tu s or lesio n s of the respiratoiy system were related to the
hepatic m ercuiy concentrations (table 5.5). However,
considering only Belgian and German North S ea porpoises
together, Hg ten ded to increase w ith degrading body condition
(one-way ANCOVA, adjusted effects for age, p<0.07).

Harbour porpoises displaying m oderate or severe parasitism

have higher m ercuiy concentration th an porpoise w ithout or
w ith mild parasitism (3-way ANCOVA, F3,45=2.8, p<0.05,
adjusted effects for age).

Se. Se concentrations did not differ

significantly betw een sam pling location s (one-way ANCOVA,
F5,63=0.7, p>0.6, adjusted effects for age). Neither body
condition (F2,38=l, p>0.3), lesio n s of the respiratory system
(F3,38=1.2, p>0.2) or parasitism (F3,38=2.31, p>0.09) affected Se
concentrations (three-way ANCOVA, adjusted effect for age).
However, considering only Belgian and German North S ea
porpoises together, hepatic Se ten d ed to increase with
degrading body condition (one-way ANCOVA, adjusted effects
for age, p<0.002). Se w as significantly correlated with Hg
concentrations in each country excep t for Iceland porpoises
(Pearson product-m om ent correlation, r=0.64, n=6, p<0.2) for
w hich the sm all sam ple size m ight have w eakened the test.

M e ta llo th io n e in s. Hepatic MTs bound 51%

o f the total zinc and 48% of the total Cd w hile Hg is detected
m ainly in the in solub le fraction of the tissu e (table 5.4).
Increase of Zn concentration in the liver w as followed by an
increase of the % of Zn bound to MTs (r=0.8, p=0.0002) while
the percentage of Zn bound to hepatic high m olecular weight
proteins d ecreases significantly (r=-0.73, p=0.01, figure 5.7).

176
 Chapter 5

70
HMWPs
60 MTs

50

40

30

20

10

0
0 100 200 300 400 500 600 700 800
Total hepatic Zn (pg.g-1 dw)

Figure 5.7. Relationship between the total hepatic Zn concentration and

the fraction bound to metallothioneins (MTS) and high molecular
weight proteins (HMWPs)

800 non-emaciated
700
# moderate
600
® emaciated
500
400

£ 300

‘co

'■*= 100
a57
90
80
I 70
60
50
40

30
1 2 3 4 5 6

Liver to body m ass ratio (%)

Figure 5.8. Relationship between the ratio liver to total body mass and
zinc concentrations using log-scale

177
 Marine mammals, trace metals and nutritional status

Table 5.4. Distribution of Zn, Cu, Cd and Hg in the centrifugation

pellet, soluble fraction, high molecular weight proteins (HMWP),
metallothioneins (MTs), and low molecular weight proteins (LMWP) of
harbour porpoises stranded along the Belgian coasts

soluble fraction
peeilet
HMWP MTs LMWP
Liver 86 ± 58 53 ± 1 9 144 ± 165 1.8 ± 117
Zn
(30-228) (29-104) (16-595) (< 0 . 1 -6 ,2 )
n=14
30% 19% 51% -0%
14 ± 12 5± 3 10 ± 11 <0 . 1
Cu
(6-43) (1.3-12) (2-39) (<0 . 1 -6 . 1 )
n=14
48% 18% 34% -0%

0 .2 ± 0 .2 0.5 ± 0 .6 1 .1 ± 0 .8 0.5 ± 1
Cd
(0 .0 1 -0 .6 ) (<0 . 1 - 1 .8 ) (0.1-2.7) (<0.1-3.4)
n=14
9% 21.5% 48% 21.5%
43 ±51 0 .1 ± 0 .1 <0 . 1 <0 . 1
Hg
(0.5-167) (<0.1-0.3) (< 0 . 1 -0 .2 ) (< 0 . 1 )
n=14
99.5% -0.25% 0.25% -0%
kidney 55 ± 12 40 ±21 16 ± 5 4.4 ± 7
Zn
(36-69) (23-105) (6-28) (<0.1-27)
ii=14
48% 35% 14% <4%
8 .8 ± 2 .2 2 .8 ± 1 .8 2.9 ± 1 .6 <0 . 1
Cu
(6-13) (0.4-6.1) (0 .6 -6 ) (< 0 . 1 )
n=14
61% 19% 20% -0%

1 .1 ± 1 ,8 0 .2 ± 0 ,6 1.9 ± 2 0 .2 ± 0 ,8
Cd
(<0,1 - 5,6) (<0,1 - 0 ,9 ) (< 0 ,1 -5 ) (<0,1 - 1,7)
n=14
33% 6% 56% 5%
3.2 ±3.3 0.5±0.3 0.3±0.2 0 .2 ± 0 .1
Hg
(0.7-12) (0 .2 - 1 . 1 ) (0.1-0.4) (<0.1-0.5)
n=10
76% 12% 7% 5%

178
 Chapter 5

S13C and 5 1SN m ea su r e m en ts. Geographic

location significantly affected S13C and 815N m easu rem en ts
(figure 5.9). Porpoises from the German Baltic North Sea
displayed significantly higher 815N v a lu es th an porpoises from
B elgian co a sts (post-hoc LSD test p<0.02), Denm ark (post-hoc
LSD test p<0.01), German Baltic S ea (post-hoc LSD test
p<0.005), Norway (post-hoc LSD te s t p<0.0001) and Iceland
(post-hoc LSD test p<0.0001). Porpoises from Belgian coasts
were significantly enriched in 13C com pared to individuals
from Denmark (post-hoc LSD te s t p<0.005), Germ an Baltic
S ea (post-hoc LSD test p<0.0005), Norway (post-hoc LSD test
p<0.0001) and Iceland (post-hoc LSD te st p<0.005).

M uscle S13C and 815N (figure 5.13) were com pared betw een
harbour porpoises from Belgian and German c o a sts o f the
North S ea displaying a good, m oderate and em aciated body
condition. Stable isotope ratios rem ained sim ilar betw een
th e se three different categories (table 5.5).

20
GNS
19
18
17
16
GBS DK
15
14
13
12
11
-19 -18 -17 -16 -15
8 1JC (% o)

Figure 5.9. Muscle 8 13C and ö I5N values of harbour porpoises from the
North Sea and adjacent arcas

B: Belgian and French coasts (see chapter 4); GNS: German North Sea
coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N: Norway

179
 ~^ Marine mammals, trace metals and nutritional sta tu s
Table 5.5. Influence of species, age, body condition, lesions of the respiratory system and parasitism on metal concentrations in
the tissues of the harbour porpoise. The symbols IT or U indicate an increasing or decreasing trend; ns not significant; nd: not
determined. <LD: below the detection limit so not included in the statistical treatment; log: indicate the data were log-transformed
before statistical treatment to ensure a normal distribution

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 Chapter 5

5 .4 . DISCUSSION

5 .4 .1 . IN T E R S P E C IE S V A R IA T IO N S

Strong interspecific differences were observed for the different

marine m am m al sp ecies collected along the Belgian coast
(tables 5.1 and 5.5). Zn, Cu, Fe and Cd differed significantly
betw een sp e c ie s w hile Hg and Se did not. However, the
interpretation of the resu lts w a s w eakened by the sm all
sam ple size and absence o f age data for th e two seal and
dolphin sp ecies, especially for m etals displaying an age
accum ulation su c h a s Cd, Se or Hg (reviewed by D as et al.,
2002). Strikingly, the h igh est Cd concentrations were
observed for the two adult w hite-sided dolphins (table 5.1).
This m ean concentration of 88 jrg.g1 dw is m uch higher than
that described for one adult w h ite-sided dolphin collected
along the Irish coast (19 ng.g-1 dry weight; see chapter 3) and
for 2 adult individuals from the U.S. Atlantic c o a sts (about 2
u g .g 1 dry w eight, Kuehl et al., 1994). In the Southern North
Sea, higher Cd concentration were only described in livers
and kidney of 5 sperm w h ales found stranded along the
Belgian and D u tch coast in 1995 (H olsbeek et al., 1999). Both
sperm whale and w hite-sided dolphin were depleted in 813C
compared to other trophic com p onents o f the Southern North
S ea indicating that they fed m ainly on pelagic prey (see
chapter 4). Previous stu d ies have recorded cephalopods as
th e m ost im portant prey category for sperm w h ales (Santos et
al., 1999). Moreover, the high Cd level encountered in th ese
two sp ecies can be related to oceanic cephalopods a s th ese
invertebrates have been show n to concentrate Cd in their
digestive glands (Bustam ante et al., 1998). This observation is
en h anced by the overall decreasing relationship found
betw een 815N and Cd (log-) concentrations in livers and
kidneys of harbour porpoises and other m arine m am m al
sp e c ie s from th is study (figures 5.1 and 5.2). S u ch a
relationship h a s been previously described (see chapter 3).
Our resu lts confirm that high Cd v alu es encountered in som e
m arine m amm al sp ecies are partly diet related a s a resu lt of
an ingestion of prey displaying typical low 815N and 813C
va lu es and high Cd levels su c h a s oceanic cephalopods

181
 Marine mammals, trace metals and nutritional status

(Ostrom et al., 1993; B ustam ante et al., 1998;, Hooker et al.,

2 0 0 1 ).

Hg concentrations were higher in the 2 w hite-beaked

dolphins com pared to the two seal sp ecies w h ereas they
display sim ilar 515N v alu es (see chapter 4). This is likely an
age-effect a s th e harbour se a ls of th is study are ju ven iles. A
Hg concentration of 229 gg.g-1 dw h a s b een reported in the
liver of one w hite-beaked dolphin found stranded along the
French C hannel coast (see chapter 3). Moreover, no
relationship h a s b een observed betw een hepatic and m uscle
815N and m ercury concentrations w hich have already been
reported in a previous study (see chapter 3). M ercuiy levels in
m arine m am m als are strongly influenced by age and the high
Hg concentrations found in the stu died m arine m am m als
probably reflect a contam ination over the anim al’s whole life
rather than a biom agnification process.

BODY CONDITION OF HARBOUR PORPOISES

Season al ch an ges in body fat condition in relation to food

consum ption have already been observed in captive and free-
living harbour porpoises (Kastelein et al., 1997; Lockyer et al.,
2001). These season al ch an ges of total body m a ss and
blubber th ick n ess are correlated w ith water tem perature in
relation with the functional role of blubber a s in su lation and
energy reserves (Iverson, 2002). However, the blubber
th ick n ess decrease can also be linked to em aciation
characterized by a m oderate to severe am yotrophy (Siebert et
al., 2001; J au n iau x et al., 2002). Indeed, the m ean blubber
th ick n ess in creases from the Southern North S ea to the
Northern Norway or Iceland and as expected, d ecreases
drastically w ith increasing em aciation severity (figure 5.10).

182
 Chapter 5

g non -emaciated
E s moderate
E
memaciated
«i
« 25
o 20
! 15
<L>
S io
D
m

B GNS GBS DK N

Figure 5.10. Blubber thickness (mm) of harbour porpoises in relation to

the nutritional status and sampling location considered. B: Belgian and
French coasts; GNS: German North Sea coasts; GBS: German Baltic coasts;
DK: Denmark, I: Iceland; N: Norway

3000
non-emaciated
m moderate
2500 @ emaciated

2000
S
ui
ñ 1500
E
B
ä 1000

500

80 100 120 140 160 180

Length (cm)

Figure 5.11. Relationship between the length and liver mass of the
harbour porpoise in relation to the nutritional status

183
 ^ Marine mammals, trace metals and nutritional status

0 non -emaciated
5
1 rroderate
® emaciated
4

o
B GNS GBS DK I N

Figure 5.12. Liver to total body mass (%) of harbour porpoises in

relation the nutritional status and sampling location considered. B:
Belgian and French coasts; GNS: German North Sea coasts; GBS: German Baltic coasts; DK:
Denmark, 1: Iceland; N: Norway

Figure 5.13. Muscle 81SN of harbour porpoises in relation to the

nutritional status and sampling location considered B: Belgian and French
coasts; GNS: German North Sea coasts; GBS: German Baltic coasts; DK: Denmark, I: Iceland; N:
Norway

184
 Chapter 5

D ue to th e se geographical variations, blubber th ic k n ess alone

is a poor indicator of the nutritional sta tu s o f the harbour
porpoise. B enn et et al. (2001) u se d the body m a ss to body
len gth ratio to estim ate nutritional statu s. However, th is
factor varies w ith the age of the individual especially during
their growth. In m am m als and birds, fasting resu lts in a
significant reduction of body and liver m a sse s (Krämer et al.,
1993; Debacker et al., 2000; 2002). Liver m a ss w as reduced of
2 / 3 in experim entally em aciated rats com pared to controls
(Hashimoto et al., 1998), resu ltin g from glycogenolysis and
protein catabolism (Krämer et al., 1993). The liver m a ss of the
harbour porpoises in creases with the len gth of th e individuals
but rem ains strikingly u n chan ged betw een porpoises in good
or m oderate body condition, or em aciated (figure 5.11).

This m ean s that during the starvation process, lipids of the

blubber are preferentially m etabolized (M arkussen, 1995). The
ratio of the liver to the total body m a ss in creases significantly
w ith degrading body condition (figure 5.12) a s a resu lt o f the
lo ss of total body m ass. In contrast to total body m ass, th is
ratio is independent of length or age and so appears a s a
com plem entary con tin u ou s datum to characterize body
condition. The poor nutritional sta tu s of the porpoises h a s
b een previously related to a reduced food intake linked to
parasitism (Sieberi et al., 1999), ulceration of the upper
gastro-in testinal tract or bronchopneum onia (Jauniaux et al.,
2 0 0 2 ).

TOXICOLOGY

Short-term fasting or prolonged starvation, through

m obilization of body reserves, are know n to influence the
m etabolism of trace elem en ts su c h a s Zn or Cu (Filteau and
Woodward, 1982; Spencer et al., 1985; Eisler et al., 1993;
Krämer et al., 1993; Debacker et al., 2000; 2001). Previous
stu d ie s m entioned higher m etal levels associated with
stranded or debilitated birds and m arine m am m als (Sieberi et
al., 1999; Debacker et al., 2000; B enn et et al., 2001). The
re su lts of the p resen t study further confirm th e se resu lts and,
more important, clearly p oints out a general rise o f Zn and Hg

185
 Marine mammals, trace metals and nutritional status

in the livers of harbour porpoises a s the em aciation worsens:

th e more th e harbour porpoises are em aciated, the higher Zn
and Hg levels in their tis s u e s (figures 5.3, 5.6, 5.8) while renal
Fe decreases in relation to em aciation. Cu and Cd levels in
tis s u e s do not seem to be influenced by the body condition
(table 5.5). In contrast to previous stu d ies (Debacker et al.,
2000; 2001; Krämer et al., 1993), th e increasing
concentrations are not linked to the lo s s of the individual liver
m a ss w hich rem ains con stan t throughout the whole
em aciation p rocess (figures 5.11 and 5.12).

Zn absorption from the in testin e can increase in sta tes of

m alnutrition (Spencer et al., 1985). Furthermore, a large
proportion of the m uscle m a ss is lost during em aciation and
Zn bound to m u scle m etalloproteins can be redistributed,
after proteolysis, in the plasm a and other tis s u e s su c h a s the
liver (Spencer et al., 1985; Krämer et al., 1993).

However, heavy m etals su c h a s Zn and Hg m ay lead to a

decrease of th e total body m a ss in various w ildstock or
laboratory anim als (Eisler, 1993; Debacker et al., 2001). Zn
exerts also a critical influence on m am m alian im m une system
(Salgueiro et al., 2000; Rink and Gabriel, 2001). Adverse
biochem ical effects of a high Zn intake on Cu m etabolism
have been reported (Spencer et al., 1985, Fosm ire, 1990). A
high Zn intake m ay cau se su bstantial change of the Z n /C u
ratio in various tissu e s. In m arine m am m als, Zn is generally
correlated w ith Cu a s a resu lt of both an antagonistic
behavior and binding to m etallothioneins. This relationship
h a s been observed for the by-caught porpoises from Norway
but not for th e porpoises found stranded along the Southern
North S ea coast reflecting a severe h om eostasis disturbance
(figure 5.14).

186
 Chapter 5

a
so
y= 5+ 0.2x
C orrelation: r =

40

*
-a
> 30
oai;>

« 20
15
Q.
»
X
10

60 80 1 00 120 140
Hepatic Zn (pg.g1 dw)

b
100

80

qi 60
d)
=t

ro
a.
a
X
20

0 100 200 300 400 500 600 700 800

Hepatic Zn (pg.g1 dw)

Figure 5.14. Relationship between hepatic Zn and Cu concentrations in

porpoises caught accidentally in fishing nets (a, Norway) and found
stranded along the Belgian coast (b)

Trace m etals su c h a s Zn or Hg could be particularly toxic for

harbour porpoises and could w ell represent an additional
source of stress to porpoises from th e Southern North S ea
already facing stressfu l condition su c h as elevated levels of
organochlorines (Covaci et al., 2002).

187
 Marine mammals, trace metals and nutritional status

W hen considering m etallothioneins and their p ossib le role a s

a detoxifying m echanism , our resu lts indicate that th ese
proteins are involved in Cd detoxication (table 5.4). Hg is
m ainly presen t in th e in solub le fraction of th e liver and
kidney confirm ing that m etallothioneins are not involved in
Hg detoxication. The relationship betw een Hg and Se is in
agreem ent w ith the form ation of tiem m anite w ell described in
m arine m am m als (Nigro and Leonzio, 1996). The percentage of
total Zn bound to m etallothioneins in crea ses from 20 to
around 70% concom itant to total hepatic Zn concentrations
(figure 5.7).

Additional param eters could contribute to explain the m etal

levels encountered su c h a s age or diet variation (table 5.5).
For in stan ce fo etu ses display high levels of Cu and Zn in their
liver. These m etals are know n to play a key-role in growing
tis s u e s (Eisler, 1993).
Renal Cd concentrations of harbour porpoises increase with
decreasing 815N and 513C valu es (figure 5.2). Similar
observations have b een m ade in other m arine mamm al
sp ecies (figure 5.1) confirm ing the u se fu ln e ss o f com bined
stable isotope and trace m etal m easu rem en ts for tracing the
diet.

TROPHIC POSITION OF HARBOUR PORPOISES

Stable carbon and nitrogen ratios do not vary w ith degrading

body condition (figure 5.13) while an increase o f 815N value
h a s been reported for birds during experim ental fasting
(Hobson et al., 1993). Marine and hibernating m am m als may
p o s s e ss biochem ical pathw ays different from th o se o f birds, in
w h ich no additional fractionation of nitrogen occurs (Ben-
David et al., 1999). These resu lts allow stable isotope
com parisons betw een stranded and by-caught marine
m am m als. Porpoises from the German North S ea seem to feed
at a higher trophic level th an individuals from other locations
(figure 5.9). Porpoises from the German Baltic Sea, D anish,
and Belgian c o a sts display sim ilar 815N v a lu es while 813C
varies widely betw een locations. The ô 15N and 8 13C of marine
predator tis s u e s are determ ined initially by the isotopic

188
 Chapter 5

com position of th e baseline phyto- and Zooplankton sources.

515N and 813C v alu es for Atlantic phytoplankton data m ay vary
w ith their sam pling origin. Trophic position s were estim ated
according to th e m odel described by Lesage et al. (2001) for
harbour porpoises from th e Southern North Sea, German
Baltic S ea and Norway co a sts for w hich 515N v alu es in the
particulate organic m atter (POM) were available (Middelburg
and N ieuw enhuize, 1998; V elinsky and Fogel, 1999; Vos et
al., 2000). A m ean trophic position of 3.4 h a s b een calculated
for harbour porpoises from the Belgian part o f th e Southern
North Sea. A ssum ing a sim ilar S15N value around 9%o for the
German North S ea POM, porpoises from th is area occupy a
trophic position of 3.7 w h ich is higher than anim als from the
Belgian coast. In contrast, porpoises from Norwegian coasts
display a lower trophic position of 3.2.
The depletion in S13C observed for individuals from Norway
and Iceland can be related to a more offshore feeding a s the
continental sh e lf is considerably reduced along the c o a sts of
Norway and Iceland compared to th e Southern North S ea
bight. These resu lts are en h an ced by the h igh hepatic and
renal Cd concentrations observed in porpoises from Norway
and Iceland, su ggesting a significant contribution of oceanic
cephalopods to their diet.

CONCLUSIONS

Heavy m etal absorption dep en d s on a variety of factors often

directly related to the m etabolism and physiology of the
animal. Marine m am m als from th e southern North S ea
display strong intra- and inter-specific variations linked to the
geographic location, the nutritional statu s, th e age and the
diet. As expected, by-caught anim als from Norway and
Iceland display a better nutritional sta tu s th an stranded
porpoises from the Southern North S ea a s show n by their
higher blubber th ick n ess, total body m a ss to len gth ratio and
hepatic to total body m a ss ratio. This may reflect the ability of
healthy porpoises to m obilize fat from their blubber a s a
primary energy source and spare their protein reserves while
porpoises in poor body condition depend primarily on m uscle

189
 Marine mammals, trace metals and nutritional status

protein catabolism . In th is view, Zn, Hg, Se and Fe

concentrations are clearly linked to em aciation, w h ich in turn
is linked to le sio n s of the respiratory system and parasitism
as previously described (Sieberi et al., 1999; Jau n iau x et al.,
2002). In addition, the constan cy of liver m a ss during
em aciation and the increase of hepatic Zn load s indicate a
specific m etabolism facing starvation, different from other
m am m als. Porpoises from th e Southern North S ea (Belgian
and German coasts) are carrying th e h igh est burdens of Hg
and Zn. In contrast, porpoises from Norwegian c o a sts display
lower levels of th e se m etals. These differences m ight be linked
to both a difference in the anthropogenic im pact of studied
locations and nutritional sta tu s of the individuals. E xcess of
Zn and Hg could represent an additional source o f stress to
porpoises. Cd levels vary also greatly w ith locations with
how ever no relation w ith the nutritional statu s. The
relationship observed betw een Cd levels and stable isotop es
for harbour porpoises and other sp ecies su g g e sts that th ese
variations are diet-related and that porpoises have a locally
specific diet throughout their distribution area.

A cknow ledgem ents: T he a u th o rs a re gratefu l to F rédéric F a m ir (G énétique

Q u an titativ e, Liège U niversity) for h is efficient h elp co n cern in g th e sta tistic a l
ap p ro ach , a n d to R achel Bouhy a n d Renzo B iondo for th e ir valu ab le tech n ical
assista n c e . T h a n k s to J a n tav en ier a n d J a n H aelters for collecting th e sam p les
along th e Belgian c o a st a n d to E iner Rogan for th e porpoise age d eterm in atio n .
T his stu d y w as fu n d ed by th e Belgian Office for Scientific, T echnical an d
C u ltu ral Affairs (co n tract M N /D D /50).

190
 Chapter 5

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Bull. 38:304-313 S taal, C., N ieu w straten , S.H.
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O strom , P., S m ith , S.C. 2001. Diet phocoena). In: Read, A .J.,
of n o rth e rn b o ttlenose w hales W iepkem a, P.R., N achtigall, P.E.
inferred from fatty acid a n d stab le (eds). The biology o f the harbour
isotope an aly ses of biopsy sam ples. porpoise. T he Spil P u b lish ers,
C an. J . Zool. 79: 1442-1454
W oerden, The N eth erlan d s pp.
H yvärinen, H., Sipilä, T. 1984. 179-199
Heavy m etals a n d hig h p u p Kelly, J.F . 2000. S table isotopes of
m ortality in th e S aim aa ringed
c a rb o n a n d nitro g en in th e study
seal populatio n in E a ste rn
of av ia n a n d m am m a lia n trophic
F inland. Mar. Pollut. Bull. 15 :
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K räm er, K., M arkw itan, A., M enne,
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Coignoul, F. 2002. Post-m ortem
1987/8 8 m ass m ortality.
findings an d c a u se s of d e a th of
C h em osphere 28: 1245-1253
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phocoena) stra n d e d from 1990 to K uiken, T., G arcia-H artm an n ,
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Belgium an d N o rth ern F rance. J . d issection tec h n iq u e s a n d tissu e
Comp. Pathol. 126: 243-253 sam pling. P roceedings of th e
E u ro p ean C etacean Society
Je p s o n , P.D ., B ennet, P.M.,
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Allchin, C.R., Law, R .J., K uiken,
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Kirkwood, J.K . 1999. In vestigating K.M. 2001. M arine m am m als an d
p o ten tial asso ciatio n s betw een th e com m unity s tru c tu re of th e
chronic exp o su re to E stu a ry a n d G ulf o f S t Lawrence,
polychlorinated b iphenyls and C anada: evidence from stable
infectious d isea se m ortality in isotope a n aly sis Mar. Ecol. Prog.
h a rb o u r porpoises from E ngland Ser. 210: 203-221
an d W ales. Sei. T otal Environ.
2 4 3 /2 4 4 : 339-348 Lockyer, C. 1995. In vestigation of
a sp e c ts of th e life h istory of th e
Jo iris C. R., H olsbeek L., h a rb o u r porpoise, Phocoena
B ouquegneau J.M ., B o ssicart M. phocoena, in B ritish w aters. Rep.
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th e h a rb o u r porpoise Phocoena 16): 189-197
phocoena a n d o th e r ceta ce a n s
from th e N orth S ea a n d th e Lockyer, C., D esportes, G.,
K attegat. W ater Air Soil Pollut. 56: A nderson, K., A ndreasen, H.,
283-293 L abberté, S., S ieb eri, U. 2001.
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h a rb o u r porpoise (Phocoena P ollution a n d m a rin e m am m als.
Phocoena) from free-living an d Pps: 948-957 In: P errin , W .E.,
captive stu d ies. 14th B iennial W ürsig, B., T hew issen, J.G .M .
C onference on th e Biology of Encyclopedia o f marine mammals.
m arin e m am m als. N ovember 28- A cademic Press. 1414 pps.
D ecem ber 3, 2001, V ancouver
R ink, L., G abriel, P. 2001.
B .C., C anada. Book of a b stra c ts.
E x trac ellu lar a n d im m unological
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a c tio n s o f zinc. B iom etals 14: 367-
M arkussen, 1995. C han g es in 383.
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S algueiro, M .J., Zubillaga, M.,
com position d u rin g sta rv a tio n an d
Lysionek, A., C rem aschi, G.,
sem istarv atio n in h a rb o u r seals,
G oldm an, G., Caro, R., De Paoli,
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T., H ager, A., Weill, R., Boecio, J .
O. (Eds), D evelopm ents in m arin e
2000. Zinc s ta tu s a n d im m u n e
biology, w hales, S eals, F ish a n d
sy stem relatio n sh ip . A review. Biol.
M an, Vol. 4, E lsevier, A m sterdam ,
Trace. Ele. Res. 76: 193-205.
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S a n to s, M.B., Pierce, G .J., Boyle,
M ejuto M arti, C.M., Bollain,
P.R., Reid, R .J., R oss, H.M.,
R odriguez, M.H., M artinez, F.B.
P a tte rso n , I.A.P., Kinze, C.C.,
1987. F luorim etric d eterm in atio n
T o u g aard , S., Lick, R., Piatkow ski,
o f selenium in h u m a n tissu e s.
U., H em and ez-G arcia, V. 1999.
A nalysis 15: 420-423
S to m ach c o n te n t o f sp erm w hales
M iddelburg, J .J ., N ieuw enhuize, J . P h yseter macrocephalus stra n d e d
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S ieberi, U., Jo iris, C., Holsbeek,
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L., B enke, H., Failing, K., Failing,
Nigro, M. a n d Leonzio, C. 1996. K., F rese, K., Petzinger, E. 1999.
In tracellu lar sto rag e o f m ercury P o ten tial R elation betw een
a n d selenium in differen t m arin e m ercu ry c o n c e n tra tio n s an d
v erteb rates. M ar. Ecol. Prog. Ser. necropsy findings in C etaceans
135: 137-143 from G erm an w a te rs of th e N orth
a n d B altic seas. M ar. Pollut. Bull.
OSPAR, 2000. Q uality S ta tu s
38: 285-295
re p o rt 2000, R egion II - G reater
N orth Sea. OSPAR C om m ission, S ieb eri, U., W u n sc h m a n n , R.,
London. 136 + xiii pp. W eiss, R., F ran k , H., B enke, H.,
F rese, K. 2001. Post-m ortem
O strom P.H., Lien J ., Macko S.A.
findings in h a rb o u r porpoises
1993. E valuatio n of th e d iet of
(Phocoena phocoena) from th e
Sow erby’s beak ed w hale,
G erm an N orth a n d B altic S eas. J .
M esoplodon biden s, b ased on
Comp. P ath . 124: 102-114
isotopic co m p ariso n s am ong
n o rth -w e ste rn A tlantic cetacean s. S p en cer, H., K ram er, L., O sis, D.
C a n ..J. Zool. 71: 858-861 1985. Z n m etabolism in m an . J .
E nviron. Pathol. Toxicol. Oncol.
R eijnders, P .J.H . 1986. 86: 864-867
R eproductive failu re in com m on
seals feeding on fish from polluted T ieszen, L.L., B outton, T.W.,
co astal w aters. N atu re 324: 456- T esd ah l, K.G., Slade, N.A. 1983.
457 F ractio n a tio n a n d tu rn -o v e r of

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stab le carb o n isotopes in an im al

tissu es: im plication for 513C
a n aly sis of diet. Oecologia 57: 32-
37

Velinsky, D .J., Fogel, M.L. 1999.

Cycling of dissolved an d
p a rticu late nitro g en a n d ca rb o n in
th e F ram v aren Fjord, Norway:
stab le isotopic v ariatio n s. Mar.
Cheni. 67: 161-180

Vos, M., L arsen, B., Leivuori, M.,

V allius, H. 2000. S tab le isotope
sig n als of eu tro p h icatio n in B altic
S ea sedim ents. J . Mar. Sys. 25:
287-298

W right, D.A., W elboum , P. 2002.

C h a p te r 6: M etals a n d o th e r
inorganic chem icals. In: W right,
D.A., W elboum , P. (eds) 2002.
Environmental Toxicology . 629 pp.
C am bridge E n viro n m en tal
C hem istry Series; 11. C am bridge
U niversity P ress pp. 249-348.

195
CHAPTER 6: W H ITE-SID ED DOLPHIN
METALLOTHIONEINS: PURIFICATION, CHA-
RACTERISATION AND POTENTIAL ROLE

After Das K., Jacob, V.,

Bouquegneau J.M. Comparative
Biochem istry and Physiology Part C
 Chapter 6

ABSTRACT

M etallothioneins (MTs) have been characterised in the kidneys of

a w hite-sided dolphin L agenorhynchus a cutus stranded along the
Belgian coast, displaying high levels of cadm ium (Cd) and
m ercury (Hg) in liver and kidney. The protein h a s two isoforms:
MT-1 and MT-2. MT-1 binds Cu, Zn, Hg and Cd while MT-2 only
binds Zn, Hg and Cd. This suggest different m etabolic functions
for the two isoforms: MT-1 is m ainly involved in Cu hom eostasis;
MT-2, which w as four tim es more abundant than MT-1, detoxifies
m ost of the accum ulated cadm ium .

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 Metallothioneins in Atlantic white-sided-dolphin

6 .1 . INTRODUCTION

Since their first discoveiy by M argoshes and Vallee (1957),

m etallothioneins (MTs) have been detected studied in both
prokaryotes and eukaryotes (Roesijadi, 1992; 1996) through more
than 5000 research papers. MTs received their designation from
their high m etal and sulfur content. The m am m alian forms are
characterised by a low m olecular m a ss of 6 0 0 0 -7 0 0 0 Da,
containing 60 to 68 am ino acid residues, am ong w hich 20
cysteines. MTs bind a total of 7 equivalents of bivalent m etal ions.
However, the natural function of m etallothioneins rem ains
elusive. Previous stu d ies on its structure, function, and
m olecular regulation have established a central role for these
sm all proteins in the hom eostatic regulation of essen tial m etals
including zinc (Zn) and copper (Cu) and detoxication of non-
essen tial m etals su c h a s mercury (Hg) or cadm ium (Cd) (Klaassen
et al., 1999). In order to understand the functions of MTs, the
functionality and structure of each MT isoform need to be
considered. Indeed, in various m am m al organs, su c h a s liver or
kidney, two major isoform s distinguishable by their charge, are
present at low basal levels, MT-1 and MT-2 (Kägi, 1993). To date,
however, no functional difference between the two isoform s h a s
been firmly established (Moffat and Denizeau, 1997).
In the framework of heavy m etal ecotoxicology, m arine m am m als
appear a s a choice material, since high levels of Hg and Cd can
be naturally encountered in these anim als (Das et al., 2001). The
potential role of m etallothioneins in marine m am m als h a s been
recently reviewed by D as et al. (2000). It appears that very few
characterisations of marine m am m al m etallothioneins have been
attem pted. The first attem pt w as realised on se a lion, Zalophus
californianus, and sperm whale kidney and liver, P hyseter
m acrocephalus (Ridlington et al., 1981). Thereafter, MT I and II
isolation and characterisation have been performed in the kidney
of striped dolphin, Stenella coeruleoalba (Kwohn et al., 1986;
1988).

More recently, Pillet et al. (2000) have described the presence of

both isoform s in the blood leucocytes of the grey seal,
H aliochoerus grypus.

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 Chapter 6

In order to obtain a better understanding on the role of

m etallothioneins in the detoxication and the dynam ics of trace
m etals in marine m am m als, detection, purification and trace
m etal content of w hite-sided-dolphin renal m etallothioneins have
been performed and compared with published data.

6 .2 MATERIAL AND METHODS

The kidney and liver of an adult male w hite-sided dolphin

L agenorhynchus a cu tu s found dead along the Belgian coasts on
the 22nd Septem ber 1999 were sam pled and stored at -20°C .
These dolphin tissu es were remarkably fresh (Jauniaux, personal
comm unication) allowing protein analysis. The renal tissue
contained 78% of water.

6.2.1. ACA 54 CHROMATOGRAPHY

3 to 4 grams of kidney were homogenized usin g an Ultra-Turrax

in a 0.01 Mol am m onium formiate (pH = 7.4) containing 10 mM
sodium azide and 0.01% dithithreitol and centrifuged at 26 000 g
(60 min, 4°C). The supernatant w as filtered on an AcA 54 gel
colum n (1.6 X 64 cm) at 4°C. Fractions were collected and
absorbance profile read at 215, 254 and 280 nm.

6 .2 .2 . H e a v y m etal a n a l y s is and MT c o n c e n t r a t io n

ASSESSMENT

After adding nitric acid (65%) to each fraction and to the pellet,
hom ogenate and supernatant, all were slowly heated to 100°C
until complete digestion. Sam ples were diluted using deionised
water and filtered prior to heavy m etal analysis (Zn, Cu and Cd)
by atomic absorption spectrophotom etry (ICPS: ARL 3510). Hg
w as analysed by flam eless atomic absorption (Perkin-Elmer MAS-
50A) as described by Joiris et al. (1991). Quality control
m easurem ents for total mercury included replicate analysis
resulting in coefficients of variation <10% and analysis of certified

199
 Metallothioneins in Atlantic white-sided-dolphin

material (DORM-1, NRC, Canada) as described by Debacker et al.

(1997).
The Hg absolute detection limit is 10 ng corresponding to 0.13
pg.g 1 fw for an average of 1.5 g fw. Detection lim its for Cu, Cd
and Zn were, respectively 0.18, 0.1 8 and 0.17 g g .g 1. Quality of
the analyses w as controlled through participation in an
intercalibration program (22). Recovery rates ranged from 94, 97,
100 and 102 % for Hg, Cu, Zn and Cd, respectively. Metal
concentrations are expressed a s gg.g 1 fresh weight (fw.). From
the heavy metal analyses, the MT concentration w as estim ated
considering that Cu occurs in the divalent form and that 1 mole
MT with a 6 8 0 0 Da m olecular m ass typically binds 7 bivalent
m etal ions (7 gram -atom s per MT mole) (Kägi, 1993; Kojima et al.,
1999).

6.2.3. DEAE sep h ad ex c h r o m a to g ra p h y

Anion exchange chromatography w as performed according to

Kwohn et al. (1986). Briefly, after ACA 54 gel chromatography,
the fractions with a low absorbance at 280 nm and a high
absorbance at 254 nm were pooled and lyophilised (VIRTIS,
12XL). To elim inate the NaCl resulting from the lyophilisation, the
proteins were chromatographed on a Sephadex G25 Medium
colum n (1.5 x 28 cm). M etallothionein separation w as performed
on an anion-exchange Sephadex A25 (1.6 x 70 cm) colum n
equilibrated with ImM Tris-HCL buffer (pH 8.6). The colum n w as
eluted in a gradient mode with 1 to 250 mM Tris-HCl buffer, pH
8.6.
The ultraviolet spectra of renal MT-1 and MT-2 were recorded
using the highest 0.2 nm resolution. DEAE Sephadex, Cd, Zn and
Cu analyses, and UV spectra have been carried out in triplicate.

6 .3 . RESULTS

AcA-54 gel chromatography (figure 6.1. a) of the supernatant

reveals a large band of metal (Zn, Cd, Cu) in the 10 kDa region of

200
 Chapter 6

the profile, consistent with the presence of m etallothioneins. This

m etal peak is associated with a high absorbance at 254 nm and
low absorbance at 280 nm related with the lack of aromatic
am ino acids of these proteins. This metallic peak show s a
remarkably high temperature tolerance after a 3 m inute heat
treatm ent at 89°C followed by centrifugation (figure 6.1.b).

a b

3 3-

1
O'
e
g
-2 i 2
i
.a B
c
i
C o
3
I

P_ o
1 2 3 4 5 1 2 3 4 5
Ve/ Vo Ve/ Vo

Figure 6.1. Elution profile on AcA 54 column of the water soluble fraction extracted
from the kidney of the white-sided dolphin Lagenorhynchus acutus before (a) and
after a 89°C heat treatment of the cytosolic fraction (b). The three types of bold lines
represent the metal concentrations (-: Zn, Cd, Cu) of the fractions. The normal lines
represent the protein absorption (-: 215, 250, 280 nm) Ve: elution volume; Vo: void
volume of the column, mean volume per fraction: 2 ml

Table 6.1. sh ow s the m etal (Cd, Zn, Hg, Cu) distribution in the
insoluble and soluble fractions including the m etals bound on
high m olecular weight proteins, métallo thionein-like proteins,
and smaller m olecules. High cadm ium concentrations are found
in the kidney (more than 12 fig.g-1 fw) and liver (5 pg.g 1 fw; data
not shown). Alm ost 60% of the total Cd, 30% of the total Zn and
12% of the total copper are bound to cytosolic m etallothioneins.
The am ount o f m etallothioneins in the kidney w as calculated

201
 Metallothioneins in Atlantic white-sided-dolphin

from the m etal content, assum ing that the protein w as fully
saturated (7 equivalent of bivalent ions or 7 g-atom s per MT mole)
with a m olecular m a ss of 6 8 0 0 kDa. A 172 p g .g 1 fw concentration
of m etallothionein w as calculated. After DEAE Sephadex A25
chromatography, the level of both isoform s estim ated from trace
m etal data differ (figure 6.2.): MT-2 concentration w a s four tim es
higher than that of MT-1.

Table 6.1. Zn, Cd, Cu, and Hg renal content in the insoluble fraction (pellet), cytosol
(soluble proteins) containing the high molecular weight proteins (HMWP), the
metallothioneins (MTs) and lower molecular weight proteins (LMWP). nd: not
determined; the concentrations are expressed in pg.g" fresh weight

Metals Total Insoluble fraction cytosol Protein type

(pellet) (supernatant)
HMWP 4
Zn 19 g 11 MTs 6 .6 (35%)
( 1 0 0 %) (42%) (58%) LMWP 0.7
HMWP <0 . 2
Cd 12 3 9 MTs 8.30 (71%)
( 1 0 0 %) (25%) (75%) LMWP 0.3
HMWP 0.14
Cu 2 .2 1 .6 0 .6 MTs 0.4(18% )
( 1 0 0 %) (72%) (18%) LMWP <0 . 2
HMWP nd
Hg 2 1.5 0 .6 MTs nd
( 1 0 0 %) (75%) (25%) LMWP nd

Figure 6.2 show s the elution profile of the DEAE Sephadex A-25
ion exchange chromatography. The profile reveals three m etal
containing fractions indicating that the MT w as resolved into two
isoproteins along the NaCl gradient. The first m etal containing
fraction (named I on the figure) did not bind to the colum n matrix
and w as eluted with ImM Tris-HCl (pH 8.6) before the gradient
started. This peak contains Zn, Cd, and Hg. The last two fractions
were, in accordance with earlier experim ents on m am m alian liver,
kidney and intestine, designated a s MT-1 and MT-2 (Kwohn et al.,
1986; Suzuki, 1991; Moffet and Denizeau, 1997). Hg
concentrations were determ ined in pooled fractions (0.016 and
0.03 jig-atoms for MT-1 and MT-2 respectively).

202
 Chapter 6

0.075

215 nm Zn Cu
254 nm ““ Cd ““ Hg

1.0
0.050

Hg-atoms
o
t/5 MT-1
Xi
<
0 .5 -
0.025

0.0 0.000
0 10 20 30 40 50 60 70 80 90 100 110 120
Fractions

Figure 6.2. DEAE Sephadex A-25 chromatography profile of white-sided-dolphin

metallothioneins.
Pooled and concentrated metallothionein fractions from AcA 54 column were applied to
a DEAE Sephadex A-25 column. The three types of bold lines represent the Zn, Cd, and
Cu concentrations of the fractions. Metal concentrations are expressed in pg-atoms
(concentration expressed in pg.g'*divided by respective metal atomic mass). The
horizontal dot lines represent the Hg concentration in the three pooled fractions. The
normal lines represent the 215 and 250 nm protein absorption.

Furthermore, we observed that the m etal com position of white­

sided dolphin isom etallothioneins varies in the different isoforms:
MT-1 contains Zn, Cd, Hg and Cu while MT-2 contains Zn, Cd
and Hg. Copper could not be detected in the second isoform (table
6 . 2 ).

203
 Metallothioneins in Atlantic white-sided-dolphin

Table 6.2. The molar ratio of metals to thionein (g-atoms/6000 g thionein) in striped
(after Kwohn et aL, 1986) and white-sided dolphins (mean ± standard deviation of
the measure ; this work) . n: number of replicates.

Striped dolphin White-sided dolphin n

MT-1 Cd 2.41 2.57 ±0.3 3

Hg 0.06 0.39 1

Zn 2.09 2.47 ±0.2 3

Cu 0.98 1.82 ± 0.7 3
MT-2 Cd 2.93 2.99 ± 0.4 3
Hg 0.04 0 .2 2 1

Zn 2 .8 6 3.88 ±0.5 3
Cu 0.16 <0.04 3

The ultraviolet spectrum reveals a very low absorbance at 280 nm

and a shoulder at 2 5 4 nm m ainly for MT-X (figure 3). The 230,
250, 270, and 3 1 0 nm absorbances reflects Zn-S, Cd-S, Cu-S and
Hg-S binding.

220 250 2 80 310

Wavelength (nm )

Figure 6.3: Ultraviolet spectrum of white-sided dolphin MT-1 and MT-2.

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 Chapter 6

6 .4 . DISCUSSION

Cadmium is know n to accum ulate preferentially in the kidneys,

bound in large am ounts to MTs w hich also contain Zn and Cu.
Renal and hepatic Cd concentrations m easured in the male
w hite-sided dolphin kidney are high (table 1). Indeed, the hepatic
Cd concentration is ten tim es higher than the 0.5 H g.g1 (fw)
m easured previously in the liver o f an adult w hite-sided dolphin
from the U.S. Atlantic coast (Kuehl et al., 1994).
81% of the renal cadm ium were found in the soluble fraction of
the tissu e bound m ostly to low m olecular weight soluble and
heat-stable proteins (figure 6.1 and table 6.1) with high
absorbance at 254 nm and lack of absorbance at 280 nm in
agreem ent with MT specific features. The heat tolerance of these
proteins is remarkable and im plies that a single dosage of Cd, Zn,
Cu and Hg in the heat-treated supernatant followed by
centrifugation could allow the determ ination of the MT-bound
m etals and hence the renal MT concentrations. Complementary
stu d ies on other species are carried out in our laboratory to test
the accuracy of su ch a m ethod to quantify MTs in marine
m am m als.
Mercury is preferentially distributed in the insoluble fraction of
the kidney (pellet, table 6.1), probably resulting from the well
know n formation of tiem annite (HgSe) in marine m am m al livers
and kidneys (Das et al., 2000; 2001).

Mammal kidney m etallothioneins con sist of two isoforms, MT-1

and MT-2 nam ed in the order of their elution from anion-
exchange colum n (Suzuki, 1991). The resolution o f white-sided
dolphin renal MT into two isoproteins (MT-1 and MT-2, Figure
6.2) on ion exchange chromatography corroborates the
observations m ade on kidney and liver MT o f m onkey (Paliwal et
al., 1982; 1986), hum an (Hellemans et al., 1999) or striped
dolphin (Kwohn et al., 1986).
Cd, Zn and Hg bind to both isoform s while copper is not detected
on MT-2. These resu lts are consistent with a previous study on
striped dolphin, stenella coeruleoalba, w hich h a s show n that MT-
2 bind Cu in lower am ount than MT-1 (Kwohn et al., 1986; table
6.2). These resu lts seem to indicate a specific role of renal MT-1

205
 Metallothioneins in Atlantic white-sided-dolphin

in copper hom eostasis, copper being an essen tial m etal involved

in m any enzymatic processes (Eisler, 1997).
MT-2 is the major isoform present in the w hite-sided dolphin
kidneys with a ratio MT-2/MT-1 concentration equal to 4.2. A
ratio of 16 h a s been m easured in striped dolphin kidney and 6.2
in equine kidneys (Kwohn et al., 1986). However, it is worth
noticing that W agemann et al. (1986) reported MT I a s the major
form of MTs in the liver and kidney from a narwhal. In fact, the
relative abundance of each isoform can differ according to
species, age, tissu e and the nature of the inducers (Moffat and
Denizeau, 1997). The higher MT-2 concentration probably resu lts
from a difference in the rate of syn th esis of hepatic MT-1 and MT-
2 w hich lead to a greater and more prolonged induction of MT-2
following adm inistration of trace m etals (Lehman-McKeeman et
al., 1991) and a faster degradation rate o f MT-1 compared to MT-
2 (Mehra and Brenmer, 1985). As a whole, th is lea d s to a more
important role o f MT-2 in the sequestration of Cd.

6 .5 . CONCLUSION

To conclude, w hite-sided dolphin m etallothioneins display two

renal isoform s MT-1 and MT-2, characterised by different
m etabolic functions in relation with copper cellular hom eostasis
(MT-1) and cadm ium detoxification (MT-2). MTs appear to play a
minor role in the binding and detoxification of Hg by marine
m am m als. Moreover, after appropriate heat treatm ent, trace
m etals m easured in the supernatant are those bound to MTs,
leading to a potential rapid and sim ple m ethod to quantify these
proteins in marine m amm al tissu es. Further experim ents are
needed to test the suitability of these m ethod to others tissu e s
and species.

206
 Chapter 6

REFERENCES

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D as, K, D ebacker, V, B o u q u eg n eau , K.T. S u zu k i, N. Im u ra a n d K im u ra
JM . 2000. M etallothioneins in (eds). B irk h ä u s e r Verlag, B asel
m arin e m am m als: a review. Cell. S w itzerland, pp. 29-55.
Mol. Biol. 46: 28 3 -2 9 4
K laassen, CD, Liu, J , C h o u d h u ri, S.
D as, K, D ebacker, V, Pillet, S, 1999. M etallothionein: An
B o uquegneau , JM . 2002. Heavy in tra ce llu lar pro tein to p ro te c t
m etals in m a tin e m am m als. In: a g a in s t c a d m iu m toxicity. A nnu.
Toxicology o f Marine Mammals, Rev. P harm acol. Toxicol. 39: 267-
Taylor an d F ra n c is p u b lish e rs 294
W ashington D.C. Invited p aper.
Kojirna, Y.,. Binz, P-A, Kägi, J.H .R .
D ebacker, V., H olsbeek, L., T apia, N om enclature of m etallothionein:
G., G obert, S., Jo iris, C.R., p ro p o sal for a revision. In: K laassen ,
J a u n ia u x , T., Coignoul, F., C.D., ed. M etallothionein IV (pp 3-6)
B ouquegneau , J.M . 1997. 1999, B irk h ä u s e r Verlag, Basel.
Ecotoxicological a n d pathological
stu d ie s of com m on guillem ots Uria K uehl, D.W., H aebler, R., P o tter, C.
aalge beached o n th e Belgian c o a st 1994. C o p la n a r a n d m etal re sid u e s
d u rin g six su ccessiv e w intering o n d o lp h in s from th e U.S. A tlantic
periods (1989-90 to 1994-95) Dis. c o a st in clu d in g A tlantic B ottlenose
A quat. Org. 29: 159-168 o b tain ed d u rin g 1987-88 m a ss
m ortality. C h em o sp h ere 28: 1245-
E isler, R. 1997. C opper h a z a rd s to 1253
fish, wildlife a n d in v erteb rates: A
synoptic review. U.S. Geological Kwohn, YT, O kubo, A, H irano, H,
survey, Biological R eso u rces Kagawa, H, Y am azaki, S, Toda, S.
Division, Biological Science R eport 1988. P rim ary s tru c tu re o f strip ed
U S G S /B R D /B S R -1997-0002. 98 d olp h in re n a l m etallo th io n ein II.
pp. Agrie. Biol. C hem . 52: 837-841
H ellem ans, G, Soum illion, A, Proost, Kwohn, Y.T., Y am azaki, S., O kubo,
P, V an D am m e, J , V an Poppel, H., A., Y oshim ura, E., T atsu k aw a, R,
B aert, L., De Ley, M. 1999. Toda, S. 1986. Isolation an d
M etallothioneins in h u m a n kidney c h a ra c te riza tio n o f m etallo th io n ein
a n d asso ciate d tu m o rs. N ephron 83: from kidney of striped dolphin,
33 1-34 0 Stenella coeruleoalba. Agrie. Biol.
Chem . 50: 2 8 8 1 -2 8 8 5
Jo iris, J.C ., H olsbeek, L.,
B ouquegneau , J.M ., B o ssicart M. L ehm an-M cK eem an, LD, K ershaw ,
1991. M ercury c o n ta m in a tio n o f th e KC, K laassen , CD. 1991. S pecies
h a rb o u r porpoise Phocoena differences in m etallo th io n ein s
phocoena a n d o th e rs c e ta c e a n s from regulation: A co m p ariso n of th e
th e N orth S ea a n d th e K attegat. in d u c tio n o f iso m etallo th io n ein s in
W ater Air Soil Pollut. 56: 2 8 3 -2 9 3 r a ts a n d m ice. In: M etallothionein in
Biology and M edicine (C.D.
Kägi, JH R . 1993. Evolution, K laassen, a n d K.T. S uzuki, eds.),
s tru c tu re a n d ch em ical activity of CRC P ress, B oca R aton, F.L. pp.
cla ss I m etallo th io n ein s. An 121-131

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L ehm an-M cK eem an, LD, K laassen, a q u a tic an im als. A quat. Toxicol. 22:
CD. 1987. In d u ctio n of 8 1 -1 1 4
m etallo th io n ein -1 an d
m etallothionein-2 in ra ts by R oesijadi, G. 1996. M etallothionein
cad m iu m an d zinc. Toxicol. Appl. and its role in toxic m etal
P harm acol. 88: 195-202 regulation. Comp. B iochem . Physiol.
113C: 117-123
M argoshes, M, Vallee, BL. 1957. A
cad m iu m pro tein from eq u in e S uzuki, K.T. 1991. P urificatio n o f
kidney cortex. J . Am. C hem . Soc. v e rte b rate m etallo th io n ein s. M eth.
79: 4 8 1 3 -4 8 1 4 Enzim ology 205: 2 5 2 -2 6 3

M ehra, R.K, B renm er, I. 1985. W agem ann, R, H obden, B. 1986.

S tu d ie s on th e m etab o lism of r a t Low m o lecu lar w eight
liver copper-m etabolism . Biochem . m étallo p ro te in s in tis s u e s of th e
J . 227: 903-908 n a rw h a l (Monodon monoceros).,
Comp. B iochem . Physiol. 84C : 325-
Moffat, P, D enizeau, F. 1997. 344
M etallothionein in physiological a n d
physiopathological p ro cesses. D rug
M etabolism Reviews 29: 26 1 -3 0 7
Paliwal, VK, Kohli, KK, S h a rm a , M,
N ath, R. 1986. P urificatio n a n d
c h aracterizatio n of m etallo th io n ein
from liver of cad m iu m exposed
R h e su s m onkeys (M acaca mulatto).
Mol. Cel. Biochem . 71: 139-147
Paliwal, V.K., Lyall, V., P rasad , R.,
G ulati S., S h a rm a M., N ath R. 1982.
P urification a n d c h a ra c te riz a tio n of
m onkey kidney cad m iu m
m etallothionein a n d its selenium
binding properties. Biochem . Int. 4:
399-408
Pillet, S., F o u rn ier, M.,
B o uquegneau, J.M ., Cyr D. 2000.
M odulation by zinc a n d e strad io l of
m etallothionein levels in grey seal
p e rip h eral blood leukocytes. Com p.
Biochem . Physiol. P a rt A (ab stracts):
p. 121
Q uevauviller, P. 1997. The
QUASIMEME P roject from a
E u ro p ean U nion View point. Mar.
Poll. Bull. 35: 140-145.
R idlington, JW , C h a p m a n , DC,
Goeger, DE, W hanger, PD. 1981.
M etallothionein a n d C u -ch elatin :
c h aracterizatio n of m etal-b in d in g
p ro tein s from tis s u e s o f fo u r m arin e
an im als. Comp. Biochem . Physiol.
70B: 9 3 -1 0 4
R oesijadi, G. 1992. M etallothioneins
in m etal regulation a n d toxicity in

208
CHAPTER 7: GENERAL D ISC U SSIO N AND
CONCLUSIONS
 Chapter 7

The objective o f the present work w as to determ ine the

relationships betw een trace m etal levels and different factors
su c h a s species, geographic origin, diet, age and the nutritional
sta tu s of m arine m am m als living in the north-w estern European
continental shelves. This final chapter is intended to provide:

(1) an integrated overview of the resu lts presented in

previous chapters, in order to present an overall image of
trace m etal concentrations and potential sources of
variations. Heavy m etal data in Black S ea harbour
porpoises and selected fish species are integrated in th is
discu ssion.

(2) som e consideration on the relationship between heavy

m etals and the health statu s

(3) and finally, som e conclusions.

209
 General discussion and conclusions

Map 7.1. Example of variations (mean ± sd) for hepatic Zn (blue bars) and renal Cd (red bars) in harbour porpoises

210
 Chapter 7

7 .1 . SOURCES OF VARIATIONS

During the present work, we have considered the sources of

variation of trace m etal concentrations in different marine
m am m al sp ecies from the North S ea and adjacent areas, as well
a s from the Black S ea (map 7.1): geographic origin, species, age,
se x or body condition. Special attention h a s been given to the
influence of diet using the stable isotope approach (813C and
515N).

7.1.1. THE DIET

Stable carbon isotop es (13C / 12C) are clearly powerful tracers of

the source of organic carbon sustaining com m unities, provided
that the primary carbon sources are adequately characterized
and differ in their 813C signatures. Stable isotopes of nitrogen
(15N / MN) u su ally have little value a s an indicator of the primary
nitrogen source of a consum m er’s diet, but have been proven to
be an indicator of the trophic level of an organism due to the
more pronounced fractionation that occurs betw een trophic
levels.

However drawbacks in its application m ust be m entioned: (1) the

degree o f fractionation show s rather large sp ecies variability, (2)
the m echan ism s underlying the fractionation of 15N are still
poorly understood. Therefore, w hen detailed inform ation on the
trophic position of a specific organism is required, it may be
needed to first determ ine the actual degree of fractionation in
laboratories or controlled field conditions (Hobson et al., 1996).
Furthermore (3), baseline information on phytoplankton 815N is
obviously needed to calculate a realistic predator’s trophic
position.

In m any c a ses however, S15N data of consum ers can still provide
usefu l information. In our data set on Southern North S ea fish
and invertebrates (chapter 4), for example, consisten t differences
in S15N valu es were clearly seen between higher and lower trophic
level species or feeding groups.

211
 General discussion and conclusions

Even more u sefu l than the trophic statu s, 515N and S13C have
provided a continu ous data set in order to obtain a further
understanding in the trace m etal bioaccum ulation and
bioamplification p rocesses (chapters 3 and 5).

For example, marine m am m als display strong inter- and intra­

specific Cd concentrations. O ceanic species su c h a s sperm
w h ales stranded on the Belgian coast (Holsbeek et al., 1999),
striped and com m on dolphins from th e Bay of Biscay, Irish and
the French Channel coasts (chapters 2 and 3) displayed elevated
renal Cd levels compared to harbour porpoises, grey and harbour
se a ls and white-beaked dolphins (chapters 3 and 5).
Unexpectedly, two adult w hite-sided dolphins stranded on the
Belgian coast displayed very high Cd level (both SSug.g-1 dw).

Furthermore, harbour porpoises from the Icelandic coasts

experienced more hepatic and renal Cd concentrations than
porpoises collected along the Belgian coast (map 7.1, chapter 5).
A significant relationship h a s been observed betw een both 513C
and 515N values and Cd concentrations (liver and kidney)
suggesting that part of th is accum ulation is diet related a s a
resu lt of ingestion of prey experiencing high Cd level in their
tissu e s and a depletion in both 513C and 815N com pared to som e
marine m am m als species. S uch an isotopic signature and high
Cd levels have been reported for various cephalopod species
(Ostrom et al., 1993; B ustam ante et al., 1998; Hooker et al.,
2 0 0 1 ).

No su c h obvious relationship h a s been observed betw een stable

isotopes and other trace m etals m eaning that other factors are
involved in the large variations observed for Zn, Hg, Se, Cu or Fe.

7.1.2. THE A G E

As expected, Cd and Hg accum ulate strongly with age (or length

where ages were not available) in m ainly all analysed marine
m am m al species. Cu and Zn concentrations are also high in the
liver of foetus in relation to their involvem ent in tissu e
development.

212
 Chapter 7

7.1.3. ENVIRONMENTAL CONTAMINATION

Zn and Hg concentrations m easured in harbour porpoises

stranded on the French Channel, Belgian and German North Sea
c o a sts coasts were higher than those from Norway, Iceland and
the Baltic sea. Part of the Zn and Hg variations are likely to be
linked to the general body conditon of the harbour porpoises (see
chapter 5 and paragraph 7.2. below) but th ese m etal levels
rem ain high even in anim als displaying a good body condition.
However m etal data on by-caught porpoises from the Southern
North Sea should help to confirm th ese results.

The Black S ea contam ination deserves special attention, since

environm ental pollution of the surrounding m ainland by heavy
m etals and organochlorines are expected to be high due to
historical, agricultural and industrial habits, a s well a s limited
water exchanges with the Mediterranean S ea (Mee, 1992).
Annually the Danube alone discharges 60 ton s of m ercuiy and
6 0 0 0 tons of zinc, w hich exceeds the entire river load to the North
S ea (Mee, 1992). Surprisingly, hepatic Zn, Cu and Se
encountered in by-caught harbour porpoises from the Ukraine
were the low est values of th is study (map 7.1). Similar
observations have been drawn for mercury, at about one order of
m agnitude lower than that in the North S ea (Joiris et al., 2001).
Factors other th an environm ental contam ination are likely to be
involved: an abundant particulate matter within the North Sea on
w hich m etals get adsorbed (Martin et al., 1973 quoted by Dauby
et al., 1994, Mart et al., 1982) and on the other han d the special
hydrological conditions in the Black Sea with an important
deeper anoxic zone. This m ight constitute an im portant sin k of
organic particulate matter (and m etals adsorbed) and explain the
low level of trace m etals encountered in porpoises .

7.1.4. METALLOTHIONEINS INVOLVEMENT

Harbour porpoise m etallothioneins represent 1.3% of the total

protein concentrations. D espite this weak percentage,
m etallothioneins appear to have a key role in m etal hom eostasis
a s in the liver they bind 50% of the total Zn while only 19% is

213
 General discussion and conclusions

present on the high m olecular weight proteins (chapter 5).

M etallothioneins are also involved in Cd detoxication a s they bind
56% of the total renal Cd. Both in liver and kidney,
m etallothioneins appear to have a w eak role in Hg detoxication as
th is m etal is m ainly distributed in the pellet, m ost probably
associated with selenium a s tiem m anite (HgSe). A significant
relationship h a s been observed between total hepatic Zn and
m etallothioneins concentrations related to the Zn induction
function in the cell.

Moreover, w hen Zn increases in the liver, the percentage bound

to metallothioneins in creases (from 20 to nearly 70%), suggesting
that th ese proteins m ight take in charge the Zn overload resulting
from the em aciation process.

M etallothioneins have also been characterised in the kidneys of a

w hite-sided dolphin displaying high levels of cadm ium and
m ercury in liver and kidney (see chapter 6). These preliminary
resu lts suggest that the protein h a s two isoforms: MT-1 and MT-
2. MT-1 binds Cu, Zn, Hg and Cd while MT-2 only binds Zn, Hg
and Cd. These two isoform s are likely to have different functions:
MT-1 is mainly involved in Cu hom eostasis; MT-2, w hich w as
four tim es more abundant th an MT-1, detoxifies m ost of the
accum ulated cadm ium .

7 .2 . TRACE METALS AND HEALTH STATUS

Trace m etal levels were exam ined in relation to the general

nutritional sta tu s of harbour porpoises described previously
(figure 7.1.) (Sieberi et al., 1999; 2001; Jau n iau x et al., 2002).
Other necropsy data su ch a s lesion s o f the respiratory system s
and total parasite burden were confronted to our toxicological
results.

214
 Chapter 7

Figure 7.1. Harbour porpoises displaying a good (a) and emaciated (b)
body condition (picture kindly provided by T. Jauniaux, ULG)

Previous stu d ies m entioned higher m etal levels associated with

stranded or debilitated birds and m arine m am m als (Sieberi et al.,
1999; Debacker et al., 2001a,b, Bennet et al., 2001). The resu lts
of the present study further confirm these resu lts and, more
important, clearly point out a general rise of Zn and Hg in the
liver of harbour porpoise a s the em aciation worsens: the more the
harbour porpoises get em aciated, the higher Zn and Hg levels in
their tissu e, while renal Fe decreases in relation to emaciation.
Cu and Cd levels in tissu e s do not seem to be influenced by the
body condition (chapter 5). In contrast to previous stu dies
(Debacker et al., 2 0 0 1 a ,b; Krämer et al., 1993), the increasing
concentrations are not linked to the lo ss o f the individual liver
m ass, w hich rem ains constant throughout the whole em aciation
process. High Zn concentrations encountered in em aciated
porpoises could be well related to a redistribution of zinc from
other tissu e s su ch a s m uscle and so be a consequence of the
em aciation p rocess instead of a direct cause.

However, heavy m etals (such a s Zn and Hg) contam ination may

lead to a decrease of total body m a ss in various wildstock or
laboratory anim als (Ronald et al., 1977; Eisler et al., 1993;
Debacker et al., 2 0 0 la,b). Zn also exerts a critical influence on
the m am m alian im m une system (Salgueiro et al., 2000; Rink and
Gabriel, 2001). Siebert et al. (1999) observed that harbour

215
 General discussion and conclusions

porpoises from German North S ea are carrying a significant

burden of mercury. The higher mercury content in organs from
harbour porpoises from the North S ea indicated that mercury is a
more important threat for anim als of th is region th an for anim als
from the Baltic Sea. Hg concentrations could represent an non­
specific morbidity factors of harbour porpoises. Similar
con clu sions were drawn for another species wintering in the
Southern North Sea, the com m on guillem ot Uria aalge. High
heavy m etal levels (Zn, Hg) in tissu e associated to fasting could
favor a general debilitation process (Debacker et al., 2 0 0 la,b).
The Southern North S ea is know n to be contam inated by m etals
and higher Zn concentrations can also be found in fish species
(some of them are potential prey for harbour porpoises),
compared to more open areas su ch a s the Northeast Atlantic
(table 7.1.)

Table 7.1. Muscle Zn and Hg (mean expressed in p g .g 1 dw) in selected fish

species from the North Sea and adjacent areas

fish species Location Zn Hg Reference

Southern North Sea 70 0 1 this work
herring
Northeast Scotland 6 0.05 Brown and Balls, 1997
Clupea harengus
Northeast Atlantic 19 0 . 2 Cossa et al., 1990
cod Central North Sea 3.6 0.18 Brown and Balls, 1997
Gadus Morhua Southern North Sea 63 0.5 this work
sandeel Southern Nort Sea 207 0 . 1 this work
Ammodites sp
whiting Southern North Sea 55 0 .1 this work
Merlangius merlangus Northeast Atlantic 17 0.4 Cossa et al., 1990
sole Southern North Sea 127 0 . 0 2 this work
Solea solea Northeast .Atlantic 22 0.4 Cossa et al., 1990

Harbour porpoises are more likely to be susceptible to the

contam inant potential detrim ental effects compared to laboratory
anim als a s they have to face a range of other natural and
anthropogenic stressors su ch a s capture in fishing n e ts and high
levels of organochlorines (Covaci et al., 2002; Van de Vijver et al.,
submitted).

216
 Chapter 7

7 .3 . CONCLUSIONS

Within their Northeast Atlantic and Black Sea distribution area,

marine m am m als display strong intra- and interspecies
variations in trace m etal levels due to geographic origin, age, diet
and trophic position but also by nutritional sta tu s of the
individuals. For exam ple, Zn and Hg concentrations are higher in
harbour porpoises from the Southern North S ea compared to
other areas. Som e individuals are severely em aciated a s show n by
their m uscle atrophy and reduced blubber thickn ess. Hepatic Zn,
Se and Hg concentrations are significantly higher in em aciated
porpoises than in normal anim als.

It is generally assum ed that anim als exposed to high levels of

m etals for a long time have evolved m echan ism s to minimize the
potential detrim ental effects of th ese contam inants. However, this
does not exclude the possibility that the m etals m ight affect the
anim als. Obviously, m etallothioneins appear to have a key role in
the h om eostasis of Zn and Cu and in the detoxication of Cd. In
contrast, Hg is mainly present in the insoluble fraction of the
tissu e in relation with the tiem m anite formation. The
m etallothioneins appear to be involved in the physiological
response to the general heavy m etal hom eostasis disruption
induced in the em aciated porpoises stranded along the Belgian
coasts. The high Zn concentrations encountered in th ese anim als
are clearly linked to a general m étallothionein synthesis. However
the question rises and rem ains debatable to know at w hat cost
debilitated individuals can still afford m etallothioneins syn thesis
while consum ing their protein reserves.

Although not at risk on a toxicological b a sis under normal

conditions (robustness) the porpoises could well be adversely
affected by Zn, Se and Hg with degrading body condition. Further
investigations are needed before we can reach any definitive
conclu sions but we cannot reject the hypothesis that trace m etal
exposure m ay influence marine m am m al health and contribute to
the high mortality observed these last few years.

Apart from capture in fishing nets, w hich rem ains an important

direct cause of mortality, marine m am m als living near the coasts
have to face various other stressful conditions su c h a s trace
m etal and organochlorine contam ination, habitat destruction,
prey depletion and noise pollution. The Southern North Sea

217
 G eneral discussion and conclusions

obviously appears a s an unfavourable environm ent for marine

m am m als as it com bines all th ese detrim ental stress factors and
the question arises about the future and m anagem ent of these
species and their habitat.
 Chapter 7

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D eterm in atio n o f o rg an o h alo g en ated V., Ali, I., Roose, P., N ellissen, J.P .,
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w h ales stra n d e d in th e S o u tirera
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G obert, S., B o u q u eg n eau , J.M . w inter. Mar. Pollut. Bull. 38:304-
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a n d p a rtic u la te Al, Cd, Cr, Cu, Pb,
Ti, Z n an d 513C in th e E nglish H ooker, S .K., Iverson, S .J ., O strom ,
C h an n el and a d ja c e n t a re a s. P., S m ith , S.C. 2001. D iet of
O ceanol. A cta 17: 64 3 -6 5 7 n o rth e rn b o ttle n o se w h ales inferred
from fatty acid a n d stab le isotope
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2001a. C om bined effects of
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a n d starv atio n o n q u a il’s body H aelters, J ., T avernier, J ., Coignoul,

219
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P. 2002. P ost-m ortem findings an d S ieberi, U., Jo iris, C., H olsbeek, L.,
causes of d e a th of h a rb o u r B enke, H., Failing, K., Failing, K.,
porpoises (Phocoena phocoena) Frese, K., Petzinger, E. 1999.
stra n d e d from 1990 to 2 0 0 0 along P o ten tial R elation betw een mercury'
th e co astlin es o f B elgium a n d c o n c e n tra tio n s a n d necro p sy fin­
N o rth ern F rance. J . Com p. P athol. din g s in C e ta ce a n s from G erm an
126: 243-2 5 3 w a te rs o f th e N o rth a n d B altic seas.
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Jo iris, C., H olsbeek, L., bolba, D.,
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2 20
APPENDIX
Table A l: Heavy metal mean and range concentrations in livers ( and kidney for Cd) from odontocetes: selected references. When the range was not available, standard deviation was
used (all the data are expressed in dry weight assuming a mean water content o f 75% of the tissues.

n CHj-
Species Location Hg-total Cd liver Cd kidney Se Cu Zn Source
(age estimation) ...
2 330±16
Pontoporia blainvillei Argentina 15±6 nd 13±6 40±16 nd 64±9 Marcovecchio etal., 1990
(2and3y.) 0

0.1 2 207 126

Platanista gangetica India 4 nd nd nd Kannanetfl/., 1993
<0.04-0.15 <0,04-6 9-400 64-210
1 310: 785±13
Argentina 344±29 nd 3±1 114±17 nd Marcovecchio e ta l, 1990
(lOy.) 1 6
South Carolina, 71 0.2 38 43 227
Tursiops truncatus 34 nd nd Beck etal., 1997
USA <2-586 0 .0 2 - 1 0.7-189 5-316 46-1084
10 180 2 74
Gulf of Mexico nd nd nd nd Kuehl and Haebler, 1995
Adult males 20-351 0.4-5 41-133
Alaska 180 52
11 nd nd nd nd nd
4-448 6-114
852±77
Hg: n = 8 308 344 nd nd nd Caurant etal., 1996
Globicephala melas Faroe Islands 6 17±15%
Cd: n= 28 6 -6 6 8 6-976 0-480 8-60 100-900 Caurant and Amiard-Triquet., 1995
Newfoundland, 63 78 43 31 17 264
26 nd Muir et al., 1988
Canada 0.3-298 0-190 0 -1 0 2 3-113 9-35 68-716
Hg: n = 8 206 17 91 43 167 Das et al., 2000
Northeast Atlantic nd nd
Others: n=22 5-348 0.2-51 0.1-199 7-272 33-385 André et al., 199 l b
Stenella coeruleoalba
820±40 194±11
Pacific Ocean, Japan mature dolphins n=15 3.4% nd nd nd nd Itano etal., 1984
8 5
28 128 6 13 12 143
Delphinus delphis Northeast Atlantic 7% nd Holsbeek et al., 1998
Stranding 3-631 0-96 0.4-81 3-32 65-293
Lagenorhynchus 3 2 14 8 20 100
Newfoundland 26 nd Muir et al., 1988
albirostris 0.5-6 .
0 2 -8 2-44 4-12 3-32 43-136
Table A l (continued).

Species Location n Hg-total c h 3- Cd liver Cd kidney Se Cu Zn Source

Hg
Monodon monoceros Baffin Island 38 24±12 / 133±130 298±192 16±7 21±13 151±40 Wagemann etal., 1983
West Greenland n>48 211 / 431 156' 131 / 144* Hansen et al., 1990
<0.02-171 <0.06- <0.06-500 <0.8-144 53-271
295
Delphinapterus leucas West Greenland 40 7 / 9 41 15 / 114 Hansen ei aí., 1990
0.3-123 <0.06-34 2 -1 1 1 87-181
St. Lawrence, Canada 30 134 / 0.6 6 / / Wagemann et al, 1990
1.5-808 0.004-2 0.004-15
Canadian western Arctic 77 108 / 9 39 75 45 112 Wagemann etal., 1996
1-464 0.5-27 15-88 3-235 3-140 43-185
Canadian eastern Arctic 73 41 / 26 90 21 77 115 Wagemann etal., 1996
5-154 0.03-103 0.3-314 1.5-91 3-1324 41-361
Kogia breviceps Argentina 1 47 / 30 1650 / 40 652 Marcovecchio e ta l, 1990
Physeter North Sea 6 41 5% 82 258 18 8 104 Bouquegneau et al., 1997b
macrocephalus 9-61 2 -8 % 52-175 133-426 6-43 5-12 90-125 Holsbeek ei al., 1999
Phocoena phocoena North Sea 5 170 37% / / / / / Joiris et al., 1991
1-504
Baltic sea 4 / / 0.3 1.5 / 24 120 Szefer etal., 1994
0.3-0.4 0.2-3 18-6 96-144
West Greenland 44 16 / 13 53 11 48 200 Paludan-Müller er al., 1993
2-80 0.2-45 0.4-290 2-36 2 0 -2 0 0 145-
370
Phocoenoides dalli Nothwestem Pacific 3 6 / / / / / / Fujise er al., 1988
(n— 1 ; fœtus) 0-84 0-136 20-329 HO-
186

1 Median
Table A2: Heavy metal mean and range concentrations in livers from mysticetes: selected references. Cadmium concentrations are also given for the kidney. All the
data are expressed in dry weight, assuming a mean water content of 75% o f the tissues, nd: not determined

Species n Location Hg-total CH j-H e Cd liver Cd kidney Se Cu Zn Source

0.06 4 4 2 9 99
Eschrichtius robustus 10 Western North America nd Varanasi etal., 1994
0 .0 1 -0 . 1 0.06-6 0 .1 -6 0.3-3 0.6-25 2-160
0.2 38 17 146
135 Antarctic nd nd nd Honda etal., 1986; 1987
0.1-0.5 9-133 9-34 99-232
Balaenoptera acutorostrata
21 41 151 6‘ 1381
17 West Greenland nd nd Hansen et al„ 1990
1 -1 1 2 -6 7-22 4-10 106-192
2
Balaenoptera physalus 11 Northeast Atlantic 40% nd nd nd nd nd Sanpera etal., 1993
0.6-5
0.2 31 4 20 137
Balaena mysticetus 20 Alaska nd nd Krone etal., 1999
0.08-0.4 2 -8 8 1-9 12-40 88-261
: median
Table A3. Heavy m etal mean and range concentrations in livers from pinnipeds: selected references. Cadmium concentrations are also given for the
kidney. W hen the range was not available, standard deviation was used. All the data are expressed in dry weight, assuming a mean water content o f
75% o f the tissues.

Hg- CH3- Cd Cd
Species Location n Se Cu Zn Source
total Hg liver kidney
232 1596
Northwest Greenland 5 / / 108- 1036- / / / Dietz et al., 1998
436 2324
Pusa hispida
76 128
176 2.6 8 18
Swedish coasts 4 / 11- 72- Frank et al., 1992
19-348 0.6-3.4 5-22 15-19
112 180
44 216
104 0.4 2 34
Swedish coasts 8 / 16- 76- Frank et al., 1992
5-264 0.2-0.7 1-3 6-52
Phoca vitulina 104 248
24 171
Arctic 12 77 14 26
13 0.9- / 121- Wagemann, 1989
1-39 2 -2 2 2 2 -6 6 12-84
173 287
56 184
104 0.7 5 56
Swedish coasts 9 / 39- 140- Franketal., 1992
92-368 0.3-0.8 2 -1 1 28-72
Halichoerus 208 304
grypus 218
Northwest England 590 2 53
12 / / / 88- Law et al., 1992
(Liverpool Bay) 6-1720 <0.06-4 9-112
356
Leptonychotes 80 191
16 3 17
Antarctica 3 / / 76- 166- Yamamoto et al., 1987
weddelli 0.2-34 <0.02-5 <0.02-40
103 220
Table A3. Continued

Hg CHj- Cd Cd
Species Location n Se Cu Zn Source
total »'• Uver kidney
263 384
Arctocephalus 215 350
gazella
Georgia 11 52-334
/
55-684
/ / 132- 259- Malcom et al., 1994
438 643
0.5 ±
Pups : 9± 6 / / / /
Arctocephalus
Chile n=27 1
0.2 Sepulveda et al., 1997
philippii
Adult :n=l 75 / / / /
2
4 151
Odobenus 114 38 244 10 32 Wagemann and
Arctic 0.03- ! 50-
rosmarus
19
0.1-137 0.1-564 2-20 6-137
300
Stewart, 1994
O Q O O Ü O O O O O O O O O Q O O O