Brown algae as heavy metals and nitrogen biomonitors of coastal areas: a review of protocols

TESE DE DOUTORAMENTO
BROWN ALGAE AS HEAVY METALS
AND NITROGEN BIOMONITORS OF
COASTAL AREAS: A REVIEW OF
PROTOCOLS
Ri a Ga cía Seoane
ESCOLA DE DOUTORAMENTO INTERNACIONAL
PROGRAMA DE DOUTORAMENTO EN MEDIO AMBIENTE E RECURSOS NATURAIS
SANTIAGO DE COMPOSTELA
2019
DECLARACIÓN
DO AUTOR/A
DA TESE
B own algae as hea y me als and ni ogen biomoni o s o coas al a eas: a
e iew o p o ocols
Dna. Ri a Ga cía Seoane
P esen o a miña ese, seguindo o p ocedemen o axei ado ao Regulamen o, e
decla o que:
1) A ese aba ca os esul ados da elabo ación do meu aballo.
2) De selo caso, na ese aise e e encia ás colabo acións que i o es e
aballo.
3) A ese é a e sión de ini i a p esen ada pa a a súa de ensa e coincide
coa e sión en iada en o ma o elec ónico.
4) Con i mo que a ese non inco e en ningún ipo de plaxio dou os
au o es nin de aballos p esen ados po min pa a a ob ención dou os
í ulos.
En San iago de Compos ela, No emb o de 2019.
Asdo. Ri a Ga cía Seoane
AUTORIZACIÓN DO DIRECTOR / TITOR DA TESE
B own algae as hea y me als and ni ogen biomoni o s o coas al a eas: a
e iew o p o ocols
D. Jesús Ramón Aboal Viñas
Dna. Ma ía Te esa Boque e Seoane
INFORMA/N:
Que a p esen e ese, co espóndese co aballo ealizado po Dna.
Ri a Ga cía Seoane
, baixo a nosa
di ección, e a
u o izamos
a súa
p esen ación, conside ando
que eúne os
equisi os
esixidos no
R
egulamen o
de Es udos de
Dou o amen o da USC,
e
que
como di ec o es des a
non inco e
nas causas de
abs ención es ablecidas
na Lei
40/2015.
En San iago de Compos ela, No emb o de 2019.
Asdo. Asdo.
D. Jesús Ramón Aboal Viñas Dna. Ma ía Te esa Boque e Seoane

To ca y ou his Doc o al Thesis, Ri a Ga cía Seoane was suppo ed by a
p edoc o al ellowship om he Minis e io de Ciencia, Inno ación y
Uni e sidades (FPU14/02015) (Spanish Go e nmen ).
Du ing he de elopmen o his Doc o al Thesis, wo esea ch s ays we e
done, a he Depa men o In eg a i e Biology (Uni e si y o Sou h
Flo ida, USA) in 2017, and a he Ma ine Biophysics Uni (Okinawa
Ins i u e o Science and Technology G adua e Uni e si y, Japón) in 2018.
Bo h esea ch s ays we e unded by he Minis e io de Ciencia, Inno ación
y Uni e sidades (Spanish Go e nmen ).
The doc o al candida e decla es no con lic s o in e es ela ed o he
Thesis.
The a icles p esen ed in his Thesis ha e he signed consen o all au ho s
who ha e pa icipa ed in hem.
A mis pad es
app oach. Hea y me als as Cd, Cu, Hg, Pb and Zn, which a e pe sis en ,
oxic, and liable o bioaccumula e in o ganisms and ans e o he ophic
chains, a e also included o p o ide a mo e ealis ic es ima e o he impac
o hese bioa ailable elemen s in he aqua ic en i onmen .
Al hough he analysis o ma ine mac oalgae is no equi ed unde he
cu en legisla ion, hese o ganisms ha e been widely used as biomoni o s
o ma ine pollu ion (including hea y me als, o ganic pollu an s o
adioac i e elemen s among o he s) since he middle o he 20 h cen u y.
B own algae ha e demons a ed o s ongly bind a a ie y o me al ions,
making hem one o he mos widely used bioabso ben s (Volesky &
Holan, 1995; Da is e al., 2003). Species such as Fucus esiculosus, F. spi alis
o Ascophyllum nodosum ha e been ou inely used in me al pollu ion
moni o ing p og ammes a ound he wo ld (Ga cía-Seoane e al., 2018a,b).
The use o mac oalgae o e s impo an ad an ages o e ec i e
biomoni o ing, when compa ed o o he ma ine o ganisms included in
some p og ammes, as e.g. ishes o in e eb a es: i) hey a e sessile, easily
iden i ied, collec ed and ansplan ed, ii) hey a e widely dis ibu ed and
a ailable all yea ound, iii) hey ha e a long li e span, i ) hey p esen wide
anges o en i onmen al ole ance, su i ing in highly ad e se and
pollu ed en i onmen al condi ions, ) he concen a ions o pollu an s in
hei issues a e s ongly co ela ed wi h he concen a ion in seawa e and
sedimen s, e c. (e.g. Haug e al., 1974; Phillips, 1980, 1990).
One o he main easons o he non-inclusion o mac oalgae in
en i onmen al moni o ing p og ammes may be he lack o s anda dized
p o ocols desc ibing how his echnique needs o be applied. As i will be
poin ed ou in Chap e s I and VI o his disse a ion, esea che s a e
cu en ly using a wide a ie y o p o ocols di e ing in aspec s such as he
species used, o he way in which he algae a e collec ed, p ocessed and
analyzed. These a ia ions obey mos ly o p ac ical and economic easons
a he han scien i ic c i e ia. The absence o p o ocols based on such
c i e ia limi s he in e p e a ion o he esul s ob ained and hampe s he
compa ison o esul s be ween s udies. Addi ionally, he sca ci y o s udies
add essing me hodological issues also e eals a gene al lack o conce n o

GENERAL INTRODUCTION
17
he s anda diza ion o he p o ocol. Fo his eason, in his disse a ion we
also add essed some basic aspec s o he me hodology o he echnique o
con ibu e o es ablish he use o mac oalgae in ex ensi e moni o ing
s udies egula ed by legisla ion.
The s uc u e o his Thesis esponds o he esea ch gaps iden i ied in
he p e ious pa ag aph. Fi s o all, we pe o med a li e a u e e iew o all
he s udies using mac oalgae as biomoni o s o ma ine pollu ion, including
hose using bo h passi e (collec ion o specimens g owing na u ally in he
a ea o in e es ) and ac i e biomoni o ing ( ansplan ing indi iduals om
hei na u al a eas o he s udy si es). Based on hese e iews, we p oposed
a mo e s anda dized p o ocol ha se ed as he basis o he o he s udies
included in his Resea ch Thesis. Some o he mos impo an
me hodological aspec s o he echnique o which he e was no consensus
ecommenda ion, o which ha e ne e been s udied, we e add essed by
means o expe imen al s udies. Because he use o na i e algae has been
p e e ed o e he use o ansplan ed algae, ou esea ch e o s ocused
on op imizing some me hodological aspec s o he passi e biomoni o ing
echniques: i) numbe o subsamples and subsample collec ion s a egy o
adequa ely ep esen he in a-si e a iabili y in concen a ions (Chap e
III), ii) numbe o samples o signi ican ly di e en ia e he le els o
pollu ion be ween sampling si es (Chap e IV), iii) collec ion pe iod o
samples o be e ep esen empo al a ia ion in concen a ions (Chap e
V), and i ) selec ion o ma e ial o analysis in o de o minimize
di e ences in concen a ions esul ing om issue a ia ion (Chap e V).
In addi ion, he sui abili y o mac oalgae o assessing na u al le els o
pollu ion in he ma ine en i onmen and de ec ing sou ces o pollu ion
was s udied in Chap e II. Finally, a s udy using ansplan a ion echniques
was ca ied ou in Chap e VII o assess he possible a ia ion in he
up ake capaci y o algae unde ch onic me al exposu e.
The species selec ed o ca ying ou he expe imen al s udies ha make
up his Doc o al Thesis is Fucus esiculosus (Linnaeus 1753) because: i) i is
he species o b own algae mos widely s udied in biomoni o ing o wa e
quali y (Ga cía-Seoane e al., 2018b), ii) i mee s he cha ac e is ics o be a
good biomoni o (Ma in e al., 1997), and iii) i is widely sp ead
h oughou he Galician coas . The bladde -w ack, as i is commonly
known, is a pe ennial b own mac oalgae ha belongs o he Phylum
Och ophy a, Class Phaeophyceae, O de Fucales, and Genus Fucus
(S asbu ge e al., 2008). The e ymology ‘ esiculosus’ de i es om he
cha ac e is ic ai bladde s on bo h sides o mid ib ha con e buoyancy o
he halli (Fig. 1B). Al hough his is he main dis inc i e ea u e o he
species, bladde less o ms ha e also been desc ibed on mo e wa e
exposed sho es, e en in he Galician coas (Pazó & Roma ís, 1979;
Bá ba a e al., 1995). The species is widely dis ibu ed in cold wa e s a
bo h sides o he A lan ic Ocean, equen on he A lan ic coas s o
Eu ope (No h Sea, Bal ic Sea, G eenland, Azo es, Cana y Islands and
Madei a), and No h Ame ica ( om Ellesme e Island and Hudson Bay o
No h Ca olina), bu absen in he sou he n hemisphe e (Fig. 1A). Fucus
esiculosus is common on semi-exposed o shel e ed sho es, and dominan
in he mid-in e idal on ocky sho es, o en wi h A. nodosum, and occupying
a posi ion in he in e idal zona ion below he popula ions o F. spi alis,
and in a zone u he up om F. se a us (Ca lson, 1991; Bá ba a e al.,
1995).
As ega ds i s mo phology, his ucoid is cha ac e ized by i s la ened,
and pseudo-dicho omously b anched hallus wi h apical g ow h
c issc ossed by a mid ib. The hallus can each 100 cm in leng h and i is
ixed o he ocky subs a e by means o a conical disc o hold as ( an
den Hoek e al., 1995) (Fig. 1B). As all membe s o he genus Fucus, he
li e cycle o F. esiculosus is oogamous diplon wi h sexual zygo es (Sou h
& Whi ick, 1987; an den Hoek e al., 1995; Se ão e al., 1999). Unlike
o he species, like o example F. spi alis which is monoecious, F. esiculosus
is dioecious, wi h sepa a e male and emale plan s. Du ing he e ile
season he game es a e p oduced by meiosis and mul iplied by mi osis in
he game angia om male plan s (an he idia) and om emale plan s
(oogonia). An he idia and oogonia a e o med in ep oduc i e s uc u es,
called ecep acles, ha de elop apically a he ips o he b anches (Fig.
1C, 1D). Once ma u e, haploid game es (spe ma ozoids and egg cells) a e
GENERAL INTRODUCTION
19
eleased, and diploid zygo es a e p oduced by ex e nal e iliza ion. A e
e iliza ion, zygo es a ach o he subs a e and g ow in o a new diploid
game ophy e ( an den Hoek e al., 1995; Se ão e al., 1996; Ladah e al.,
2003, 2008). Al hough sexual ep oduc ion is he mos common s a egy
in ucoids, asexual p opaga ion by clonal ep oduc ion (i.e. de achmen o
ad en i ious b anches om he pa en al plan ha ac as ege a i e
p opagules) has been desc ibed in species o he genus Fucus, including F.
esiculosus (Be gs öm e al., 2005; Ta a enko e al., 2005).
Fig. 1. A) Dis ibu ion o Fucus esiculosus in he no he n hemisphe e. B) Fucus esiculosus
halli a ached o subs a e. De ailed images o ep oduc i e s uc u es o F. esiculosus,
an he idia (C) and oogonia (D). Images ob ained om www.apho oma ine.com (A),
www.bioweb.uwlax.edu (B), and www.seaweed.ie (C, D).
Gene al Objec i es

GENERAL OBJECTIVES
23
The o e all aim o his PhD esea ch was o e alua e he po en ial o
ma ine mac oalgae o moni o hea y me als, me alloids and ni ogen in
coas al en i onmen s, and o p opose a scien i ically based p o ocol o
he applica ion o his echnique. Fo his pu pose, he in o ma ion
p esen ed in his disse a ion is di ided in o wo pa s, each consis ing o
se e al chap e s. Pa I (Chap e s I o V) encompasses he main body o
his wo k and is dedica ed o he use o na i e mac oalgae as biomoni o s
o pollu ion (passi e moni o ing). Pa II (Chap e s VI and VII) ocuses
on he use o ansplan s o mac oalgae in biomoni o ing s udies (ac i e
moni o ing), as an al e na i e o he use o na i e algae. The speci ic
objec i es o each chap e a e summa ized as ollows:
1. To ca y ou a c i ical e iew o he me hodology used in s udies
conce ning pollu ion biomoni o ing in coas al en i onmen s wo ldwide,
and in ol ing he use o na i e ma ine mac oalgae (passi e
biomoni o ing). The speci ic aims o his e iew we e: o es ablish he s a e
o he a o he me hodology, o iden i y i s deg ee o ha moniza ion, and
o p opose an upda ed p o ocol o moni o ing pollu an s wi h hese
o ganisms (Chap e I).
2. To s udy he po en ial use o he b own mac oalgae Fucus esiculosus
o cha ac e ize he na u al ange o a iabili y in he δ15N signal, and
concen a ions o N and Hg in coas al a eas no a ec ed by local sou ces
o pollu ion, and o de ec small scale sou ces o con amina ion. To
achie e his objec i e, samples o he species we e collec ed in mo e han
150 sampling si es dis ibu ed along he sho eline in 3 s udy zones a ec ed
by di e en deg ees o pollu ion (Chap e II).
3. To p opose a sampling s a egy o biomoni o ing wi h F. esiculosus
ha encompasses he in a-si e a iabili y (local a iabili y), and accoun s
o he spa ial s uc u e in he concen a ions o Al, As, Cd, Co, C , Cu,
Fe, Hg, Ni, Pb, Zn and N, and in he δ15N signal, while op imizing he
numbe o subsamples used o make a composi e sample, he sampling
e o and cos s. Fo his pu pose, 50 subsamples o he algae we e
collec ed a andom using a egula sampling g id design, in each o 3 si es
a ec ed by di e en le els o pollu ion (Chap e III).
4. To de e mine he op imal numbe o samples o F. esiculosus needed
o de ec s a is ically signi ican di e ences in he mean concen a ions o
Al, As, Cd, Co C , Cu, Fe, Hg, Ni, Pb, Zn and N, and in he δ15N signal
be ween 2 sampling si es a ec ed by di e en le els o pollu ion (Chap e
IV). The expe imen al se -up is he same as desc ibed in he p e ious
chap e .
5. To assess he exis ence o empo al a ia ion o bioconcen a ion o
hea y me als, me alloids and ni ogen in F. esiculosus, as well as o s udy
he in a- hallus a iabili y in hese elemen s. Fo his pu pose, he
concen a ions o Al, As, Cd, Co, C , Cu, Fe, Hg, Ni, Pb, Zn, N, and he
δ15N alues we e de e mined in samples o he species collec ed mon hly
o e a pe iod o 3 yea s a 3 sampling si es. The implica ions ha he
empo al (in a-annual/seasonal) and in a- hallus a iabili y in he
concen a ions may ha e on he esul s o biomoni o ing s udies wi h
algae a e discussed. In addi ion, an app op ia e sampling design,
ep esen a i e o he exis ing a iabili y in he issue con en s o elemen s,
is p oposed o applica ion in biomoni o ing s udies. The g ow h o he
species (in e ms o he numbe o dicho omies o med in he hallus
h ough he yea ) is also conside ed, and he implica ions o in e p e a ion
o he esul s o empo al s udies o pollu an s a e also discussed (Chap e
V).
6. To e alua e he essen ial aspec s o he me hodology and he deg ee
o s anda diza ion o he p o ocols used in s udies in ol ing
biomoni o ing o ino ganic pollu an s and nu ien s in coas al
en i onmen s wo ldwide wi h ansplan s o ma ine mac oalgae (ac i e
biomoni o ing). On he basis o he conclusions eached, a scien i ically
based p o ocol is p oposed o applica ion o he biomoni o ing echnique
wi h ansplan ed algae (Chap e VI).
7. To compa e he accumula ion capaci y o me als and me alloids in
specimens o F. esiculosus g owing na u ally unde di e en pollu ion
scena ios. Thalli o he species we e ecip ocally ansplan ed be ween 2
pollu ed and 2 unpollu ed si es. The di e ences in he dynamics o
up ake/ elease a es o Al, As, Cd, Co, C , Cu, Fe, Hg, Ni, Pb and Zn we e
GENERAL OBJECTIVES
25
s udied a e exposu e o he halli in hese en i onmen s o 90 days
(Chap e VII).

33
CHAPTER II
Applica ion o mac oalgae analysis o assess he
na u al a iabili y in selec ed pollu ion concen a ions (N
and Hg), and o de ec sou ces o i in coas al
en i onmen s
Science o he To al En i onmen , 650 (2019), 1403–1411
R. Ga cía-Seoane1, J.A. Fe nández1, M.T. Boque e2,3, J.R. Aboal1.
1Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Biología, Lope
Gómez de Ma zoa s/n, San iago de Compos ela, 15782, A Co uña, Spain.
2Es ación Biológica de Doñana, CSIC, A enida Amé ico Vespucio 25, Isla de la Ca uja, Se illa 41092,
Spain.
3Depa men o In eg a i e Biology, Uni e si y o Sou h Flo ida, 4202 E Fowle A e, Tampa, FL 33620,
USA.
h ps://doi.o g/10.1016/j.sci o en .2018.09.156
.
Chap e III
37
CHAPTER III
Sampling op imiza ion o biomoni o ing me al
con amina ion wi h ma ine mac oalgae
En i onmen al Pollu ion, 255 (2019), 113349
R. Ga cía-Seoane1, J.A. Fe nández1, Z. Va ela1, C. Real2, M.T. Boque e3,4,
J.R. Aboal1.
1Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Biología, Lope Gómez de
Ma zoa s/n, San iago de Compos ela, 15782 A Co uña, Spain.
2Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Escola Poli écnica Supe io de
Enxeña ía, Rúa Benigno Ledo, 2, Campus Uni e si a io, 27002 Lugo, Spain.
3Es ación Biológica de Doñana, CSIC, A enida Amé ico Vespucio 25, Isla de la Ca uja, Se illa 41092, Spain.
4Depa men o In eg a i e Biology, Uni e si y o Sou h Flo ida, 4202 E Fowle A e, Tampa, FL 33620, USA.
h ps://doi.o g/10.1016/j.en pol.2019.113349

Chap e IV
41
CHAPTER IV
Op imal numbe o
Fucus esiculosus
subsamples o
di e en ia e be ween si es a ec ed by dis inc le els o
hea y me al con amina ion
Submi ed o publica ion.
R. Ga cía-Seoane, J.R. Aboal, J.A. Fe nández.
Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Biología, Lope
Gómez de Ma zoa s/n, San iago de Compos ela, 15782 A Co uña, Spain.
Abs ac
The p esence o ace elemen s in ma ine habi a s is a se ious en i onmen al
p oblem which inc easingly a ec s ecosys em and human heal h. The use o
mac oalgae as con amina ion biomoni o s ep esen s a aluable al e na i e
app oach o adi ional physicochemical me hods. The p esen s udy was ca ied
ou o de e mine he op imal numbe o subsamples o Fucus esiculosus needed o
de ec s a is ically signi ican di e ences in he mean concen a ions o Al, As,
Cd, Co C , Cu, Fe, Hg, Ni, Pb, Zn, N and δ15N be ween wo sampling si es
a ec ed by di e en le els o con amina ion. Fo his pu pose, we plo ed he
densi y dis ibu ions o he concen a ions o he di e en elemen s and
examined he local a iabili y a h ee si es. Fo si es wi h mean concen a ions
di e ing by mo e han 30%, 20 subsamples we e su icien o de ec signi ican
di e ences o all o he elemen s, excep C . The p oposed me hodology could
be used in o he s udies in he absence o speci ic esea ch on each species and
egion.
Keywo ds:
Con amina ion moni o ing; Hea y me als; Coas al en i onmen s;
Algae; Local a iabili y; S anda diza ion.
2
42
2
1
XXY


222
2
1
XXXY


As an explici exp ession is no a ailable o he in e ed squa e oo
ans o ma ion, µY and 𝜎2Y we e calcula ed as he mean and a iance o
he da a ans o med om he aw a iable ( hen yi = xi-0.5, Eqs. 1 and 2),
which al hough no s ic ly co ec , is mo e app op ia e han using he da a
om he o iginal a iable. All s a is ical es s and Box-Cox
ans o ma ions we e pe o med using IBM SPSS S a is ics 24 (SPSS Inc.,
USA).
The densi y unc ions o each elemen we e es ima ed by ke nel
smoo hing, wi h he Ke n-Smoo h package (Wand & Ripley, 2006) in R
(R de elopmen Co e Team, 2008). The echnique was applied wi h a
Gaussian ke nel, he wid h o which was op imised o each da a se by
using di ec in oduc ion wi h wo le els o unc ional es ima ion (Wand
& Jones, 1995).
3. Resul s
The desc ip i e s a is ics o he concen a ions o he elemen s de e mined
in he algae collec ed a he h ee SS s udied a e shown in Table 1. In
gene al, con amina ion le els we e highe a SS3 han a SS1 and SS2. As
ega ds he a iabili y o he da a, he coe icien s o a ia ion and
dispe sion (median absolu e de ia ion/median) we e, in compa ison wi h
he o he elemen s, highe o Al, Fe and Pb in all SS, wi h alues o
a ound 40–50%. Addi ionally, he densi y dis ibu ions o some o he
elemen s and si es s udied a e shown as an example in Fig. 1. These g aphs
con i m ha he concen a ions o mos o he elemen s (o he han N) in
algae we e non-no mally dis ibu ed a all SS (see Fig. 1 and Table 1). In
gene al, polymodal dis ibu ions wi h posi i e asymme y (skewed o he
igh ) we e obse ed (e.g. o δ15N in SS1, and Ni in SS2), and in some

CHAPTER IV
49
cases he skew o he igh was so la ge ha he e appea ed o be some
nega i e asymme y (e.g. As in SS3). These ypes o dis ibu ions a e
indica i e o he exis ence o con amina ion a he SS (Viana e al., 2010).
Fo example, he concen a ions o C , Fe and Zn include an ex eme
alue 3 imes highe han he modal alue o he dis ibu ions in SS2. Some
o hese a ypical alues in he densi y dis ibu ions we e iden i ied in he
same subsamples o some o he elemen s de e mined in he same SS (e.g.
Al and C in SS1, Fig. 1).
The compa isons be ween he densi y dis ibu ions o Cu in SS2–SS3
and Zn in SS1–SS2 a e shown as examples (see Fig. 1) o illus a e he
easoning behind he calcula ion o he minimum numbe o subsamples
equi ed o signi ican ly di e en ia e wo si es in e ms o me al
concen a ions ( alues p esen ed in Table 1). As p e iously explained, he
numbe o subsamples will be de e mined by he d alue (i.e. he di e ence
be ween popula ion means µ1 and µ2) and by he de ia ion o each
dis ibu ion compa ed (σ). Fo Cu, he dis ibu ions compa ed a e clea ly
di e en , wi h means di e ing by ca. 50% (high d alue), whe eas o Zn,
he dis ibu ions almos o e lap and only di e by 5% (low d alue). In
addi ion, o Cu he σ alues o bo h dis ibu ions (~0.8) a e lowe han
hose o Zn (4.9 and 8.8). Wi h his in o ma ion and using he p e iously
de ined equa ions, we ound ha by collec ing a minimum o 3 subsamples
om SS2 and ano he 3 om SS3, we can signi ican ly di e en ia e
(p≤0.05) be ween he mean concen a ions o Cu a bo h SS, as he
minimum de ec able di e ence be ween popula ion means, δ3=2.22, is
lowe han d=2.37. On he con a y, Zn dis ibu ions we e e y a iable
and he mean alues we e e y simila , so ha signi ican di e ences
would no be de ec ed wi h 3 subsamples (as δ3 > d), and a much la ge n
(119 in each SS) would be equi ed o di e en ia e be ween he mean
concen a ions o Zn a SS1 and SS2 (as δ119 < d=1.71). Thus, he mo e
dissimila he means (µ2 - µ1) a e (i.e. he highe he alue o d) and he less
a iable he da a om each dis ibu ion a e (σ1 and σ2) (i.e. he smalle he
alue o σ), he easie i will be o de ec signi ican di e ences be ween
SS (as a small n is equi ed). Likewise, o mos o he emaining elemen s,
when pai s o SS wi h e y di e en means we e compa ed (e.g. Al, As,
Fe, Cu, Hg and Pb, o he SS1–SS3 and SS2–SS3 compa isons), he
numbe o subsamples equi ed was small (be ween 1 and 8), whe eas o
di e en ia e be ween he concen a ions o hose elemen s wi h simila
means in he SS compa ed (e.g. C , Cu and Zn in he SS1–SS2
compa ison), a la ge numbe o subsamples would be equi ed (mo e
han 400 o C and Cu) (Table 1).
Table 1. Desc ip i e s a is ics o concen a ions (µg g-1) o each elemen de e mined in
50 subsamples o Fucus esiculosus collec ed a he sampling si es (SS) unde s udy. Mean
alues and coe icien o a ia ion (%) a e shown in egula ype ace o hose elemen s
ha a e no mally dis ibu ed, and median alues and coe icien s o dispe sion (median
absolu e de ia ion/median, %) a e shown in i alics o hose elemen s and SS ha a e
no no mally dis ibu ed. The minimum numbe o subsamples equi ed o di e en ia e
(p≤0.05) he mean issue concen a ions o he elemen s s udied in F. esiculosus g owing
a he h ee SS compa ed is also shown. Pai s o SS in which he mean concen a ions
di e by mo e han 30% a e shown in bold ype. a: ng g-1; b: %; c: ‰.
Sampling si es
Compa isons
SS1
SS2
SS3
SS1-SS2
SS1-SS3
SS2-SS3
Al
200 (53)
352 (77)
1437 (50)
24
2
2
As
55.6 (10)
51.3 (11)
28.1 (14)
47
2
1
Cd
0.82 (17)
1.02 (20)
0.47 (9)
32
13
9
Co
0.52 (19)
1.18 (16)
0.99 (10)
3
35
48
C
0.31 (24)
0.40 (50)
0.98 (37)
581
29
84
Cu
2.31 (12)
2.53 (17)
4.80 (17)
415
7
3
Fe
191 (35)
299 (58)
1002 (44)
7
1
1
Hga
22.5 (30)
14.5 (20)
59.3 (19)
6
1
1
Ni
1.07 (25)
2.02 (17)
3.75 (6)
9
3
16
Pb
0.40 (41)
0.25 (39)
1.62 (39)
86
8
6
Zn
32.5 (15)
33.1 (10)
46.6 (8)
119
2
4
Nb
0.40 (11)
0.77 (7)
0.59 (8)
2
4
5
δ15Nc
1.94 (4)
2.59 (2)
2.81 (4)
2
12
5
CHAPTER IV
51
Fig. 1. Densi y unc ions o he concen a ions o Al, C and δ15N in sampling si e (SS)
1, Hg and Ni in SS2, and As, Fe and N in SS3, de e mined in he b own algae Fucus
esiculosus. Subsamples showing ex eme concen a ions a e highligh ed in Al and C a
SS1 as examples. The uppe g aphs show he compa isons made be ween wo SS ha
di e in e ms o mean concen a ions (µ1 and µ2) and de ia ions (𝜎1 and 𝜎2) o Cu and
Zn. The size o each a ow indica es he de ec able minimum di e ence in concen a ion
(δ), and he di e ence be ween he mean alues (d) o each pai o SS compa ed, o n=3
and n=119 o Cu and Zn espec i ely. Thin and hick lines ep esen he alues o bo h
popula ions compa ed. Fo each ke nel, e ical lines along he X-axis co espond o he
indi idual alues o he subsamples collec ed a each SS (n=50). The concen a ions a e
exp essed in µg g-1, excep o Hg (ng g-1), N (%) and δ15N (‰).
4. Discussion
The a iabili y in he concen a ions o elemen s in issues o algae la gely
de e mines he numbe o subsamples equi ed o di e en ia e be ween
wo SS sepa a ed in space, o o di e en ia e empo al changes in he same
SS (e.g. in ime end s udies). This a iabili y can be gene a ed by ac o s
ha locally a ec he accumula ion o elemen s in algae (i.e. ac o s causing
in a–SS a iabili y). Some o hese ac o s ha e been in es iga ed in de ail
(e.g. Nickless e al., 1972; Ma in e al. 1997; Ga cía-Seoane e al., 2019)
and a e p ima ily ela ed o he e ec o he e ical posi ion o he algae
on he sho eline and o a ia ions in en i onmen al condi ions (Fuge &
James, 1974; Villa es e al., 2002). In iew o his, and by using he me hod
ecommended by Za (2010), we ha e e i ied ha in o de o de ec
signi ican di e ences be ween SS in which he mean concen a ions o
he elemen unde conside a ion a e e y di e en , e y ew subsamples
a e needed, e.g. 3 o Cu (mean concen a ions di e ing mo e han 50%
be ween SS compa ed, Fig. 1), see Table 1. These esul s a e consis en
wi h hose epo ed by Ba ei o e al. (1993) o F. ce anoides and F.
esiculosus collec ed in he same egion, in which 3–4 subsamples we e
conside ed su icien o de ec signi ican di e ences be ween he mean
concen a ions o Al, Fe, Co, Mn, Ni and Zn, which di e ed by 50% in
he di e en si es. On he o he hand, we obse ed ha he numbe o
subsamples equi ed inc eases subs an ially when he di e ences be ween
he means a e small, leading o imp ac ical sample sizes, e.g. 199
subsamples in he case o Zn (Fig. 1 and Table 1). In ligh o his, we
es ima ed he minimum numbe o subsamples ha would yield he
g ea es numbe o signi ican di e ences be ween SS. We ound ha
collec ion o 20 subsamples a each SS was su icien o de ec signi ican
di e ences (p≤0.05) o all he elemen s whose concen a ions di e ed by
mo e han 30% be ween he SS compa ed (excep o C o SS1–SS3 and
SS2–SS3 compa isons, Table 1). Fo mos o hese elemen s (e.g. Al, As,
Cu, Fe and Hg), a smalle numbe (<10 subsamples) would also ha e been
su icien .
CHAPTER IV
53
These esul s (in e ms o he numbe o subsamples equi ed o
di e en ia e SS) a e only s ic ly applicable o s udies using F. esiculosus
and ca ied ou in he same egion, because as men ioned abo e, he
numbe o subsamples will depend on he exis ing local a iabili y in he
SS and on he simila i y o di e gence in he le el o con amina ion
be ween he SS compa ed. Thus, as he as majo i y o s udies using his
echnique o compa e hea y me al o nu ien le els be ween SS do no
jus i y he sample size chosen, and mos collec a small numbe o
subsamples a each SS (Ga cía-Seoane e al., 2018), he esul s o such
s udies could be ques ioned. In pa icula , in hose cases whe e no
signi ican di e ences we e ound on compa ing con amina ion le els
be ween SS, his may be due o he ac ha he numbe o subsamples
collec ed was no su icien ly ep esen a i e o he high a iabili y exis ing.
Fo example, Rige e al. (1997) examined di e ences in elemen
concen a ions be ween di e en loca ions by collec ing 5 subsamples o
each species (F. esiculosus, F. dis ichus, and A. nodosum) pe si e. Howe e , a
wo-way ANOVA es e ealed ha he concen a ions o he elemen s
displaying he highes le els o na u al a iabili y (i.e. C and Pb), did no
di e signi ican ly be ween any o he 4 loca ions compa ed. Acco ding o
ou es ima ions, he concen a ions o C and Pb in F. esiculosus a e highly
a iable (Table 1), and a leas o his species, 5 subsamples we e no
su icien o di e en ia e he SS wi h ce ain y. We es ima ed ha a leas
30 subsamples would be needed o di e en ia e he si es on he basis o
C concen a ions, and be ween 6 and 86 subsamples would be equi ed
o he same o Pb (Table 1). Likewise, Rainbow e al. (2002) epo ed no
signi ican di e ences (Tukey’s pos -hoc es ) be ween SS wi h simila
mean concen a ions o elemen s such as Pb and Zn, when 10 subsamples
o F. esiculosus we e collec ed in each SS. Acco ding o ou es ima ions
(Table 1), >80 subsamples may ha e been necessa y o de ec di e ences
in Pb and Zn concen a ions be ween SS.

5. Conclusions
Al hough he echnique o moni o ing con amina ion wi h mac oalgae is
s ill a om being s anda dized, one aspec o he sampling p o ocol has
been de ined o moni o ing hea y me al con amina ion in coas al wa e s.
Despi e he high a iabili y in he concen a ions o he di e en elemen s
(especially Al, Fe and Pb) de e mined in he subsamples o F. esiculosus,
we we e able o es ablish he minimum numbe o subsamples ha should
be collec ed in each SS o allow obus di e en ia ion be ween SS a ec ed
by di e en le els o me al con amina ion (ca. 20 subsamples). F om a
p ac ical poin o iew, his numbe o subsamples can easily be collec ed
du ing sampling su eys.
Al hough he numbe o subsamples was es ima ed using F. esiculosus
and applying ou de ini ion o a subsample (see Ma e ial and me hods),
ou ecommenda ion is alid o s udies ca ied ou in o he egions and
o o he algal species, in he absence o new s udies unde di e en
se ings. Finally, in o de o imp o e he esul s o he s udies and inc ease
he eliabili y and compa abili y o he da a, u he esea ch is needed o
suppo and e ise he cu en me hodology o using algae o moni o
con amina ion le els.
Acknowledgemen s
The au ho s belong o he Galician Compe i i e Resea ch G oup
GRC/GPC2016-002 and o he CRETUS S a egic Pa ne ship
(AGRUP2015/02). Bo h o hese p og ammes a e co- unded by FEDER
(UE). The au ho s a e g a e ul o he RIAIDT-USC o use o analy ical
acili ies. Ri a Ga cía Seoane was in eceip o a g an awa ded by he
Spanish Minis e io de Ciencia, Inno ación y Uni e sidades wi hin he
P og ama de Fo mación de P o eso ado Uni e si a io (FPU 2014).
CHAPTER IV
55
Re e ences
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es ua ies: A ield compa ison o wo b own algae species inhabi ing
uppe es ua ine eaches. En i on. Moni . Assess. 75(2), 121–134.
Ba ei o, R., Real, C., Ca ballei a, A., 1993. Hea y–me al accumula ion by
Fucus ce anoides in a small es ua y in no h–wes Spain. Ma . En i on.
Res. 36(1), 39–61.
Box, G.E., Cox, D.R., 1964. An analysis o ans o ma ions. J. R. S a . Soc.
Se ies B S a . Me hodol. 211–252.
B yan, G.W., Langs on, W.J., Humme s one, L.G., 1980. The use o
biological indica o s o hea y me al con amina ion in es ua ies: wi h
special e e ence o an assessmen o he biological a ailabili y o me als
in es ua ine sedimen s om sou h–wes B i ain. Occas. Publ. Ma . Biol.
Assoc. U.K. 1, 1–73.
Ca o, N., Ga cía I., Ignacio M., Mou ei a A., 2010. Spa ial and empo al
ends o PCBs (polychlo ina ed biphenyls) in mussel om Galician
coas (1998 – 2008). En i on. In . 36, 873–879.
Che no a, E.N., Se gee a, O.S., 2008. Me al concen a ions in Sa gassum
algae om coas al wa e s o Nha T ang Bay (Sou h China Sea). Russ.
J. Ma . Biol. 34(1), 57–63.
Cobelo–Ga cía, A., P ego, R., 2004. In luence o poin sou ces on ace
me al con amina ion and dis ibu ion in a semi–enclosed indus ial
embaymen : he Fe ol Ria (NW Spain). Es ua Coas Shel Sci. 60(4),
695–703.
Cohen, J., 1988. S a is ical Powe Analysis o he Beha io al Sciences. 2nd
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Cohen, J., 1992a. A powe p ime . Psychol. Bull. 112(1), 155–159.
Cohen, J., 1992b. S a is ical Powe Analysis. Cu . Di . Psychol. Sci. 1(3),
98–101.
Con i, M.E., Cecche i, G., 2003. A biomoni o ing s udy: T ace me als in
algae and molluscs om Ty henian coas al a eas. En i on. Res. 93(1),
99–112.
Desu, M., Ragha a ao, D., Bos on, M.A., 1990. Sample size me hodology.
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Deu sch, B., Voss, M., 2006. An h opogenic ni ogen inpu aced by
means o δ15N alues in mac oalgae: esul s om in si u incuba ion
expe imen s. Sci. To al En i on. 366(2 3), 799–808.
Fong, P., Kame , K., Boye , K.E., Boyle, K.A., 2001. Nu ien con en o
mac oalgae wi h di e ing mo phologies may indica e sou ces o
nu ien s o opical ma ine sys ems. Ma . Ecol. P og. Se . 220, 137–
152.
Fuge, R., James, K.H., 1973. T ace me al concen a ions in b own
seaweeds, Ca digan Bay, Wales. Ma . Chem. 1(4), 281–293.
Fuge, R., James, K.H., 1974. T ace me al concen a ions in Fucus om he
B is ol channel. Ma . Pollu . Bull. 5(1), 9–12.
Ga cía-Seoane, R., Fe nández, J.A., Va ela, Z., Real, C., Boque e, M.T.,
Aboal, J.R., 2019. Sampling op imiza ion o biomoni o ing me al
con amina ion wi h ma ine mac oalgae. En i on. Pollu . 255, 113349.
Ga cía–Seoane, R., Fe nández, J.A., Villa es, R., Aboal, J.R., 2018. Use o
mac oalgae o biomoni o pollu an s in coas al wa e s: op imiza ion o
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sedimen s o he Pon e ed a Ria (NW Spain). Soil Sedimen . Con am.
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polychlo ina ed biphenyls and pes icides in di e en species o
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558.
Ma in, M.H., Nickless, G., S enne , R.D., 1997. Concen a ions o
Cadmium, Coppe , Lead, Nickel and Zinc in he alga Fucus se a us in
he Se e n es ua y om 1971 o 1995. Chemosphe e 43(2), 325–334.
Mi amand, P., Ben ley, D., 1992. Hea y me al concen a ions in wo
biological indica o s (Pa ella ulga a and Fucus se a us) collec ed nea he
F ench nuclea uel ep ocessing plan o La Hague. Sci. To al En i on.
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Mohe , D., Dulbe g, C.S., Wells, G.A., 1994. S a is ical powe , sample size,
and hei epo ing in andomized con olled ials. JAMA 272(2), 122–
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Modelling 99Tc concen a ions in Fucus esiculosus om he no h–eas
I ish Sea. J. En i on. Radioac . 77(2), 159–173.
Ne , S., Hen y, F., Rabodoni ina, S., Diop, M., Me haby, D., Mah ouz, C.,
Ama a, R., Ouddane, B., 2015. Accumula ion o PAHs, Me–PAHs,
PCBs and o al me cu y in sedimen s and ma ine species in coas al
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En i on. Res. 9(2), 419–432.
exhibi ed a dec ease in concen a ions om he younges o he oldes
dicho omies. To minimize de e ec o in a-annual a iabili y, a sampling
s a egy consis ing on collec subsamples 2 imes pe yea sepa a ed by 6 mon hs
and combined in a composi e sample was ecommended o biomoni o ing
pu poses wi h his species. To minimize he e ec o in a- hallus a iabili y, i is
p oposed o make composi e samples o he h ee apical dicho omies o hallus.
Keywo ds: Aqua ic con amina ion; Biomoni o ing; Seasonali y; In a- hallus
a iabili y; Algae; Me als.

CHAPTER V
65
1. In oduc ion
F om i s incep ion as use ul en i onmen al ools in biomoni o ing ma ine
pollu ion in he ea ly 1950s (Wo , 1955; Black & Mi chell, 1952),
mac oalgae ha e become one o he mos commonly used bioindica o s
wo ldwide (Malea & Ke ekidis, 2014; Bonanno & O lando-Bonaca,
2018; Ga cía-Seoane e al., 2018). Howe e , he me hods implemen ed in
his ype o s udy ha e no been p ope ly add essed, and many basic
aspec s o he echnique ha e no been aken in o conside a ion despi e
being issues ha a ec he in e p e a ion o he esul s ob ained.
One such aspec is he empo al ep esen a i eness o he
concen a ions o pollu an s in he algae collec ed in a gi en a ea.
Tempo al a iabili y (in a-annual) in elemen concen a ions in algae mus
be cha ac e ized, o he wise samples collec ed wi hin he same si es in
di e en mon hs/seasons will no be compa able, and in e -s udy
compa isons will be also es ic ed. Al hough sample collec ion is usually
limi ed o a pa icula ime o he yea , ypically in summe o p omo e
good sampling condi ions (Ga cía-Seoane e al., 2018), i he
concen a ions o elemen s in algae a y o e ime, he alues ob ained
making a single sampling su ey may no well ep esen he annual mean
alue wi hin he si e, bu an o e o unde es ima ion o i . P e ious s udies
in which samplings we e ca ied ou o e a minimum pe iod o one yea
co e ing all seasons, e ealed in a-annual a iabili y in mo e han 75% o
he elemen s conside ed (Ga cía-Seoane e al., 2018). Fo example,
Fe ei a & Oli ei a (1988) ound coe icien s o a ia ion (CV) o Hg o
45% in samples o he b own algae Fucus esiculosus collec ed h oughou a
yea , and simila ly, Villa es e al. (2013) ound annual CV o 17%, 25%,
32%, 38% and 133% o Mn, N, Fe, Zn and Cu espec i ely o he same
species. These a ia ions in he concen a ions o elemen s in algae ha e
been a ibu ed o changes in en i onmen al ac o s (S oepple e al., 1986;
Ha oon e al., 1995), al hough mos au ho s hink ha biological ac o s,
such as me abolism, ep oduc ion and/o g ow h, cons i u e he mos
de e minan ac o s o he empo al a iabili y (Rao & Indusekha , 1989;
Malea, 1995; W igh & Mason, 1999). Se e al au ho s ha e ecommended
ha o minimize he e ec o his a iabili y, a ious samples should be
collec ed du ing he yea in each si e, commonly wi h mon hly samplings,
and in eg a ed in a single composi e sample, a s a egy also known as
“Time-bulking” o samples (Phillips & Sega , 1986; S oepple e al., 1986).
The main p oblem o his app oach is ha he g ea e he magni ude o
he in a-annual a iabili y (due o no o seasonali y), he la ges he
numbe o samplings would need o be pe o med, in ol ing a huge
sampling and economic e o . Thus, i is necessa y o cha ac e ize he
empo al a iabili y o he concen a ions o elemen s in algae in o de o
es ablish he sampling equency equi ed o yield ep esen a i e alues o
annual concen a ions (Malea e al., 2015; Ga cía-Seoane e al., 2018).
Along wi h he in a-annual a iabili y, o he aspec o he
biomoni o ing echnique wi h mac oalgae ha needs o be closely
in es iga ed in o de o yield ep esen a i e and compa able da a among
s udies is he in a- hallus a iabili y (Rige e al., 1997; Bu ge e al., 2007;
Sáez e al., 2012). Se e al s udies ha e been epo ed o show di e ences
in me al concen a ions be ween di e en pa s o he algal halli,
especially in ucoid species (Ga cía-Seoane e al., 2018), di e ences mainly
ela ed o he age and physiological condi ion o he issues (Fa e o &
F igo, 2002; Sa age & Elmg en, 2004). In consequence, selec ion o he
pa o he algae used as biomoni o should be p e iously s anda dized o
he esul s o be consis en ac oss s udies and no d aw e oneous
conclusions (Sáez e al., 2012). In addi ion, i is s ill no known whe he
he e is any ela ionship be ween in a- hallus and in a-annual a iabili y.
The p esen s udy aims o in es iga e he exis ence o empo al
a ia ion pa e ns o in a-annual bioconcen a ion o ni ogen, δ15N and
ace elemen s in F. esiculosus along wi h he in a- hallus a iabili y in he
concen a ions o hese elemen s o e a h ee-yea pe iod in h ee
sampling si es. The possible implica ions ha he exis ence o empo al
and in a- hallus a iabili y in he issue con en s o hese elemen s would
ha e on he in e p e a ion o esul s in biomoni o ing s udies will be
discussed, and an app op ia e sampling design o ou ine use in
CHAPTER V
67
biomoni o ing p og ammes wi h mac oalgae, ep esen a i e o he in a-
annual and in a- hallus a iabili y, will be p oposed. In addi ion, he
pe iodici y in he occu ence o new dicho omies in he hallus o his
species and hei implica ions in he in e p e a ion o he esul s o
empo ali y s udies o pollu an s will also be discussed.
2. Ma e ial and me hods
2.1 Sampling
Samples o he b own seaweed Fucus esiculosus L. (Class Phaeophyceae)
we e collec ed mon hly om h ee sampling s a ions (SS) loca ed in he
coas o Galicia (NW Spain) om No embe 2015 o No embe 2018.
The SS we e loca ed a om poin sou ces o pollu ion, such as po
in as uc u es o subma ine ou alls. The i s si e (SS1) was loca ed in he
Ría o Fe ol (cen e ed in X = 557811, Y = 4812476; UTM 29N ETRS89),
conside ed one o he mos con amina ed ias by hea y me als in he coas
o Galicia (Cobelo-Ga cía & P ego, 2004). The second si e (SS2) was
selec ed wi hin he Ría de Mu os e Noia (X = 506599, Y = 4737191), an
a ea wi h sca ce indus ial ac i i y. The las si e (SS3) was loca ed in he
Ría de Pon e ed a (X = 523837, Y = 4697147), cha ac e ized by he
p esence o a chlo -alkali indus y and se e al small ci ies in i s ma gins.
In each SS, 30 subsamples consis ed o indi idual halli wi h simila size
a ached o ocks, we e collec ed wi hin h ee 50 m bands pa allel o he
coas line (10 subsamples pe band), and combined in o a single composi e
sample o achie e g ea e ep esen a i eness o he in a-SS a iabili y in
he concen a ions. A mo e de ailed desc ip ion o he sampling p o ocol,
washing and sample p ocessing can be ound in Ga cía-Seoane e al.
(2019).
2.2 Sample p ocessing
Samples we e manually cleaned by emo ing any adhe ing ma e ial and
disca ding ecep acles, damaged o old issues and sec ions hea ily
a ec ed by epiphy es (Ga cía-Seoane e al., 2018). Consecu i e
dicho omous sec ions o he seaweed hallus we e sepa a ed a he base o
e e y pai o ai bladde s unde nea h he angle o med by each o k wi h
a glass spa ula, as illus a ed in Fig. 1, ollowing he me hod used in
p e ious s udies (e.g. Sa age & Elmg en, 2004, S engel e al., 2005;
Ca ballei a e al., 2014). The h ee mos apical dicho omies we e sepa a ed
du ing he i s yea unde s udy. One mo e consecu i e dicho omy (4 h
dicho omy) was sepa a ed he nex yea . The same p ocedu e was ollowed
o e he hi d yea , wi h he 5 h dicho omy being included, making a o al
o 5 dicho omies a he end o he 3 yea s o su ey. P io o analysis, each
dicho omy was indi idually d ied in a o ced ai o en a 40ºC (72 h), hen
homogenized in a angen ial mixe mill wi h zi conium oxide g inding
essels (Re sch MM400), and weighed on a p ecision balance (Me le
ToledoXP26). D ied dicho omies (be ween ca. 2 and 8 g d y weigh , d.w.,
each) we e hen s o ed a oom empe a u e in he me ically sealed ials
un il chemical analysis.
Fig. 1. Diag am showing he di e en sec ions ( om 1s –apical o 5 h–basal) selec ed
o e he h ee-yea su ey in he dicho omously di ided hallus o Fucus esiculosus.
CHAPTER V
69
2.3. G ow h measu emen s
Fucus esiculosus is cha ac e ized by apical g ow h and pseudo-dicho omous
b anching o he hallus. The hallus g ows o ming dicho omous
ami ica ions, leading o a pa allel al hough unequal de elopmen o he
b anches (Hoek e al., 1995). Se e al au ho s ha e sugges ed ha he
b anches in his species a e dicho omously di ided once a yea , which has
led o he in e p e a ion ha each dicho omy co esponds o an annual
g ow h segmen (e.g. Ca lson, 1991; Sa age & Elmg en, 2004; S engel e
al., 2005; Ca ballei a e al., 2014). Howe e , du ing pas ield wo k
campaigns, he au ho s ha e obse ed ha he equency o he hallus
o king is p esumably highe han he p e iously desc ibed in hese
s udies. Because we a e unawa e o any p e ious s udies which add esses
he g ow h pe iodici y o F. esiculosus (in e ms o numbe o dicho omies
o med each yea ), we belie e ha i is con enien o assess whe he each
dicho omy uly co esponds o an age coho o i se e al dicho omous
di isions occu consecu i ely du ing he same yea .
To s udy g ow h o F. esiculosus, a g oup o 30 halli was andomly
selec ed wi hin each SS and indi idually labeled wi h coded plas ic ags in
he basal sec ion o he hallus. The numbe o new dicho omies o med
and he size g ow h we e pe iodically moni o ed by aking pho og aphs o
each hallus agains a backg ound o g aph pape . G ow h moni o ing was
ca ied ou e e y 3 mon hs o a pe iod o 9 mon hs (Janua y 2019 –
Oc obe 2019). All indi iduals we e measu ed o he nea es mm om he
base o he hold as o he ip o he longes ond.
2.4 Chemical analysis
Be o e analysis, samples we e d ied again a 40ºC in a o ced ai o en. The
mine aliza ion o he samples (1 g d.w.) was pe o med in Teflon essels
in a mic owa e o en (CEM MDS2100) in h ee successi e s eps (10 min
a 100ºC, 7 min a 150ºC, 25 min a 190ºC), by adding 10 mL o HNO3
(65%), 2 mL o H2O2 (30%) and 2 mL o MilliQ wa e . The concen a ions

o Al, As, Cd, Co, C , Cu, Fe, Ni, Pb and Zn we e de e mined by ICP–
MS (VARIAN 820-MS ICP quad upole mass spec ome e ) a he
Resea ch Suppo Se ices Uni om Uni e sidade de San iago de
Compos ela. The concen a ions o Hg we e de e mined in an elemen al
analyze (DMA 80 Miles one). Fo de e mina ion o %N and δ15N signal,
3±0.1 mg (d.w.) o sample we e packaged in in capsules (Eu oVec o )
and p ocessed in an elemen al analyse (FlashEA1108 Ca lo E ba
Ins umen s) coupled o a mass spec opho ome e (MAT253
The moFinnigan). De e mina ions we e made a he Uni o Ins umen al
Techniques o Analysis om Uni e sidade da Co uña.
To ensu e he analy ical quali y o he p ocess, one analy ical blank, one
eplica e sample and wo ce i ied e e ence ma e ials, Sea le uce–Ul a
lac uca [BCR–279] and Bladde w ack–Fucus esiculosus [ERM–CD200]
(Ins i u e o Re e ence Ma e ials and Measu emen s, IRMM, Belgium)
we e analyzed once e e y en samples. In he case o %N, only he
e e ence ma e ial BCR–279 was analyzed. The global e o associa ed
wi h he analy ical p ocess was usually lowe han 6%, excep o Hg
(a ound 10%) and Al (a ound 17%). The pe cen age o eco e y om he
e e ence ma e ials anged o BCR–279, be ween 65% (Pb) and 110%
(Hg), usually a ound 85%, and o ERM–CD200, be ween 65% (Pb) and
122% (Hg), usually a ound 90%. De e mina ions we e abo e he
co esponding limi s o quan i ica ion (LOQ), wi h excep ion o Ni (in
5% o he cases), Cu (8%), Co (11%), Cd (15%), Al (33%), Pb (44%) and
C (69%). Da a om hose SS whe e elemen concen a ions we e below
he LOQ in mo e han 30% o he samples, i.e. om SS1 o C , om SS2
o C and Pb, and om SS3 o Al, C and Pb, we e no included in he
da a ea men .
CHAPTER V
71
2.5 Da a analysis
2.5.1 In a- hallus a iabili y
Lillie o s modi ica ions o he Kolmogo o –Smi no es was used o
check he no mali y o he da a. Excep o N and δ15N, which
concen a ions we e no mally dis ibu ed, hose elemen s wi h non-
no mal dis ibu ions we e success ully no malized using Box–Cox
ans o ma ions: log(x) ans o ma ion o As, Cd, Co, Cu, Fe, Ni and Pb,
sq (x) ans o ma ion o Al, and 1/sq (x) ans o ma ion o Hg and
Zn. A Th ee–way ANOVA es was used o es di e ences be ween he
i e dicho omous sec ions o he hallus and s udy he in e ac ion be ween
he ac o s (ca ego ical a iables): “Sampling Mon h * SS * Dicho omy”.
The concen a ions o he elemen s we e conside ed as he dependen
a iables. When he e was no in e ac ion be ween ac o s and when
signi ican di e ences we e de ec ed (p≤0.05), a Tukey’s Pos –Hoc es
was used o iden i y di e ences. Spea man’s ank co ela ion coe icien s
we e also calcula ed o de e mine he ela ionship be ween he
concen a ions o he elemen s and he weigh (d.w.) o he dicho omies.
S a is ical analysis was pe o med using R-3.4.0 (R de elopmen Co e
Team, 2008).
2.5.2 Analysis o he s uc u e o he empo al se ies
The classical analysis o ime se ies is based on he assump ion ha he
alues aken by he a iable a e he consequence o h ee componen s
( end, seasonal and andom componen s) whose combined ac ua ion
esul s in he measu ed alues (B ockwell & Da is, 2002; Cha field, 2003;
Ande son, 2011). To dis inguish be ween end and seasonal componen s,
ends in aw da a we e isola ed by lineal eg ession analysis o he ime
se ies. A de ending p ocedu e was hen applied o emo e he end
componen om hose non-s a iona y ime se ies (wi h end and/o
a iabili y changing sys ema ically o e ime) wi h signi ican ends
(p<0.01) (Cha field, 2003; Box e al., 2014). To assess he deg ee o
dependence in obse a ions o he ime se ies, au oco elog ams we e
calcula ed om he eg ession esiduals in hose de ended se ies, o
di ec ly om aw da a in hose s a iona y se ies (wi h cons an mean and
a iance o e ime), o each dicho omy and o each elemen sepa a ely
a all SS. The “ac ( )” unc ion a ailable in R-3.4.0 (Package gs a ) was used
o plo co elog ams (Pebesma, 2004; G äle e al., 2016). Co ela ions
ou side he 95% con idence in e al (bounds ±1.96/√𝑛, whe e n is he
numbe o lags and 1.96 is he 0.975 quan ile o he s anda d no mal
dis ibu ion) we e deemed signi ican a he 5% signi icance le el
(B ockwell & Da is, 2002). In he absence o da a o he h ee yea s in
he 4 h and 5 h dicho omies, and o ge a mo e consis en empo al analysis,
co elog ams and ime plo s we e only s udied in he h ee mo e apical
dicho omies.
In o de o s udy he seasonal a ia ion and o iden i y he dominan
pe iods (o equencies) o he se ies, pe iodic (cyclic) eg ession models
wi h a sine and a cosine componen we e i ed (Cha field, 2003), using
he “spec um” ( o calcula e spec al densi y and de e mine he pe iods o
he se ies) and “lm” ( o illus a e he model es ima ed) unc ions a ailable
in R-3.4.0 (Package TSA, R de elopmen Co e Team, 2008) (C ye &
Chan, 2008; Shumway & S o e , 2017). A desc ip ion o he pe iodic
eg ession model and i s pa ame e s can be ound in Table S1
(Supplemen a y Ma e ial). Al hough he e ms inside he sine and cosine
unc ions a e known, he eg ession coe icien s a e au oma ically
es ima ed by he eg ession model i ed o each se ies. The i ing o he
models was done by calcula ing de e mina ion coe icien s ( 2), conside ed
signi ican a p<0.01.
3. Resul s
Annual ange o a ia ion in concen a ions o he di e en
elemen s/δ15N signal in he i e dicho omies o he hallus a each SS a e
shown in Table 1. Al hough δ15N signal is no an elemen pe se, bu is an
CHAPTER V
73
iso opic ela ion o elemen s, we will e e om now on o all he elemen s
de e mined (including δ15N signal) as “elemen s”, o simpli y he
eadabili y h oughou he ex . In a-annual a iabili y in he
concen a ions a each dicho omy was s udied by calcula ing he
coe icien s o dispe sion (COD), as he a io be ween he MAD (median
absolu e de ia ion) and he da a median. Concen a ions a ied be ween
3% o δ15N a SS1 ( hi d yea , 1s dicho omy) and 747% o Cd a SS3
( hi d yea , 3 d dicho omy). The COD usually anged be ween 30% and
60% o mos o he elemen s in all he SS, wi h he excep ion o N and
δ15N (usually <20%), and we e especially high (>100%) o Al in SS2, As
and Cd in all SS, and Co, Cu and Fe in SS3. A gene al compa ison be ween
dicho omies showed ha he apical dicho omy was clea ly he leas
a iable in SS1 in he h ee yea s s udied. No di e ences we e appa en ly
obse ed be ween dicho omies in he o he SS.
Table 2. Resul s om signi ican lineal eg ession analysis o ime se ies (p<0.01) in Fucus
esiculosus dicho omies om h ee sampling si es (SS). De e mina ion coe icien s ( 2) and
he slope o each eg ession a e shown. Signi ican eg ession models pa ame e s adjus ed
o he seasonal componen o ime se ies (p<0.01). Sig.: signi ica ion le el; n.s.: non-
signi ican ; β0: mean o he se ies; β1 and β2: eg ession coe icien s; ϵ : esiduals; pe :
pe iod ( u he de ails in Table S1, Suppl. Ma .).
T end componen
Seasonal componen
Dicho omy
2
Slope
2
Sig.
β0
β1
β2
ϵ
pe
1s
0.23
3.44
-
n.s.
-
-
-
-
-
SS1
2nd
-
-
0.22
<0.01
130
8.86
45.8
2.26
10
Al
3 d
-
-
0.42
<0.001
146
29.7
64.5
4.07
10
1s
-
n.s.
-
-
-
-
-
SS2
2nd
0.26
-6.56
-
n.s.
-
-
-
-
-
3 d
0.28
-8.99
-
n.s.
-
-
-
-
-
1s
0.48
2.63
-
n.s.
-
-
-
-
-
SS1
2nd
0.50
1.99
0.24
<0.01
-1.30E-04
12.2
-9.66
-1.99
12
3 d
0.28
0.76
0.33
<0.001
4.56E-05
8.72
-6.37
-1.94
12
1s
0.33
2.26
-
n.s.
-
-
-
-
-
As
SS2
2nd
0.24
1.63
0.59
<0.001
-2.09E-04
-8.95
-32.54
1.33
36
3 d
-
-
0.44
<0.001
44.6
-12.9
-17.7
0.504
36
1s
0.23
1.75
0.36
<0.001
2.99E-04
17.0
-24.7
-1.328
12
SS3
2nd
0.34
1.39
-
n.s.
-
-
-
-
-
3 d
0.19
0.45
0.20
<0.01
-0.424
1.82
-6.61
-1.56
12
1s
0.41
0.05
0.53
<0.001
-1.46E-04
0.664
-0.091
0.040
12
SS1
2nd
0.21
0.01
0.58
<0.001
7.25E-05
0.293
-0.081
-0.021
12
3 d
-
-
0.40
<0.001
0.606
0.223
-0.101
-0.036
12
1s
-
-
0.35
<0.001
1.80
0.209
-0.530
-0.103
12
Cd
SS2
2nd
-
-
0.60
<0.001
0.896
0.193
-0.371
-0.020
12
3 d
-
-
0.44
<0.001
0.766
0.156
-0.330
-0.041
12
1s
-
-
-
n.s.
-
-
-
-
-
SS3
2nd
-
-
0.23
<0.01
0.425
-0.053
-0.126
-0.022
11
3 d
-
-
-
n.s.
-
-
-
-
-
1s
0.28
-0.02
-
n.s.
-
-
-
-
-
SS1
2nd
-
-
0.25
<0.01
1.01
0.406
-0.467
-0.089
12
3 d
-
-
0.31
<0.001
1.35
0.415
-0.705
-0.076
12
1s
0.23
-0.10
-
n.s.
-
-
-
-
-
Co
SS2
2nd
0.24
-0.14
-
n.s.
-
-
-
-
-
3 d
0.21
-0.16
0.16
<0.01
-6.36E-04
0.177
-2.48
-0.518
12
1s
0.26
-0.03
-
n.s.
-
-
-
-
-
SS3
2nd
0.21
-0.03
-
n.s.
-
-
-
-
-
3 d
-
-
0.26
<0.01
0.752
0.100
-0.498
-0.088
12
1s
-
-
0.32
<0.001
3.99
1.37
-0.486
0.087
12
SS1
2nd
-
-
0.20
<0.01
3.72
0.840
-1.01
-0.391
12
3 d
-
-
0.35
<0.001
3.78
0.338
-1.44
-0.340
12
1s
-
-
0.39
<0.001
3.62
0.373
-0.899
-0.227
12
Cu
SS2
2nd
0.31
-0.04
0.58
<0.001
-3.10E-04
0.042
-0.748
0.044
12
3 d
0.40
-0.06
-
n.s.
-
-
-
-
-
1s
-
-
0.35
<0.001
2.38
-0.752
0.310
-0.040
10
SS3
2nd
-
-
0.24
<0.01
1.89
-0.339
-0.353
-0.140
12
3 d
-
-
0.47
<0.001
1.84
-0.596
-0.192
-0.115
12

CHAPTER V
81
TABLE 2
(Con inued)
T end componen
Seasonal componen
Dicho omy
2
Slope
2
Sig.
β0
β1
β2
ϵ
pe
1s
-
-
-
n.s.
-
-
-
-
-
SS1
2nd
-
-
0.30
<0.001
141
23.7
38.3
-1.21
10
3 d
-
-
0.36
<0.001
169
38.0
49.9
-3.88
10
1s
-
-
-
n.s.
-
-
-
-
-
Fe
SS2
2nd
0.41
-5.54
-
n.s.
-
-
-
-
-
3 d
0.34
-7.02
-
n.s.
-
-
-
-
-
1s
-
-
0.31
<0.001
68.9
-12.1
-26.1
-6.36
16
SS3
2nd
0.31
-2.35
-
n.s.
-
-
-
-
-
3 d
0.32
-2.46
0.34
<0.001
0.662
0.203
31.2
-1.97
12
1s
0.23
-0.29
-
n.s.
-
-
-
-
-
SS1
2nd
0.48
-0.48
-
n.s.
-
-
-
-
-
3 d
0.28
-0.43
-
n.s.
-
-
-
-
-
1s
-
-
-
n.s.
-
-
-
-
-
Hg
SS2
2nd
0.57
-0.48
-
n.s.
-
-
-
-
-
3 d
0.43
-0.36
-
n.s.
-
-
-
-
-
1s
0.23
-0.43
0.27
<0.01
-1.03E-03
-1.21
-6.38
-0.045
10
SS3
2nd
0.27
-0.47
-
n.s.
-
-
-
-
-
3 d
0.38
-0.46
-
n.s.
-
-
-
-
-
1s
0.36
-0.03
-
n.s.
-
-
-
-
-
SS1
2nd
-
-
0.20
<0.01
1.78
0.858
-0.740
-0.342
12
3 d
-
-
0.25
<0.01
2.67
0.993
-1.27
-0.283
12
1s
0.34
-0.10
-
n.s.
-
-
-
-
-
Ni
SS2
2nd
0.37
-0.18
-
n.s.
-
-
-
-
-
3 d
0.35
-0.20
0.27
<0.01
-8.04E-04
0.683
-2.20
-0.383
12
1s
0.27
-0.05
-
n.s.
-
-
-
-
-
SS3
2nd
0.27
-0.07
-
n.s.
-
-
-
-
-
3 d
-
-
0.21
<0.01
2.35
-0.480
-1.02
-0.300
12
1s
-
-
0.32
<0.001
0.350
0.006
0.090
-0.009
10
Pb
SS1
2nd
-
-
0.32
<0.001
0.457
0.053
0.145
-0.016
10
3 d
-
-
0.23
<0.01
0.604
0.130
0.175
-0.058
10
1s
0.18
-1.26
0.37
<0.001
-0.987
24.3
-8.51
-5.82
12
SS1
2nd
-
-
0.39
<0.001
74.8
44.3
-33.0
-5.09
12
3 d
-
-
0.51
<0.001
95.0
49.3
-48.8
-3.32
12
1s
-
-
0.38
<0.001
43.2
17.7
-19.0
-4.74
12
Zn
SS2
2nd
0.19
-1.77
0.32
<0.001
-2.10E-04
14.7
-28.4
-6.13
12
3 d
-
-
0.48
<0.001
56.2
18.9
-39.8
-4.80
12
1s
0.22
-1.20
0.30
<0.01
3.46E-04
12.3
-15.1
-1.62
12
SS3
2nd
0.24
-1.52
0.32
<0.001
-4.33E-04
7.90
-22.5
-5.57
12
3 d
-
-
0.38
<0.001
51.6
8.17
-26.2
-4.75
12
3.3 G ow h measu emen s
Rega ding g ow h in F. esiculosus, a e y sho ime o expe imen e ealed
he high mo ali y among he indi iduals moni o ed. Se e al indi iduals
had disappea ed a e an in e al o a ew weeks and g adually a some
poin du ing he checkup, so hese could no be ollowed o he en i e
pe iod. Due o a s ong he bi o y in he apical issues o algae and he
signi ican loss o indi iduals in SS3, g ow h measu emen s in his si e
we e only possible a 3 mon hs a e he ini ial ollow-up. In addi ion,
many indi iduals expe ienced signi ican size g ow h ha i was impossible
o ollow he numbe o dicho omies o med om 6 mon hs onwa ds.
Al hough he loss o indi iduals no gua an eed da a o s a is ical
signi icance, i allowed a p elimina y inqui y in o size and dicho omous
b anching a ia ion in his species. Thus, heal hy halli o med 2–3 new
dicho omies e e y h ee mon hs (up o 5 new dicho omies in some cases),
wha is ca. 1 dicho omy/mon h (Fig. S2, Suppl. Ma .). The g ow h in
hallus leng h was mo e a iable be ween measu emen s. Thalli in SS1 and
TABLE 2
(Con inued)
T end componen
Seasonal componen
Dicho omy
2
Slope
2
Sig.
β0
β1
β2
ϵ
pe
1s
-
-
0.65
<0.001
0.719
0.172
-0.083
0.020
12
SS1
2nd
-
-
0.65
<0.001
0.626
0.180
-0.089
0.029
12
3 d
-
-
0.71
<0.001
0.555
0.178
-0.087
0.021
12
1s
-
-
0.67
<0.001
0.932
0.029
-0.133
-0.004
12
N
SS2
2nd
-
-
0.50
<0.001
0.830
0.021
-0.150
0.019
12
3 d
-
-
0.67
<0.001
0.778
0.042
-0.116
0.006
12
1s
-
-
0.71
<0.001
0.757
0.147
-0.149
0.013
12
SS3
2nd
-
-
0.58
<0.001
0.690
0.124
-0.140
-0.003
12
3 d
-
-
0.65
<0.001
0.624
0.097
-0.167
0.012
12
1s
-
-
0.51
<0.001
2.49
0.294
-0.019
0.012
12
SS1
2nd
-
-
0.50
<0.001
2.29
0.269
-0.132
0.011
12
3 d
-
-
0.66
<0.001
2.15
0.244
-0.156
0.016
12
1s
0.36
0.01
-
n.s.
-
-
-
-
-
δ15N
SS2
2nd
0.34
0.01
-
n.s.
-
-
-
-
-
3 d
0.44
0.01
-
n.s.
-
-
-
-
-
1s
-
-
-
n.s.
-
-
-
-
-
SS3
2nd
0.31
0.02
0.21
<0.01
4.72E-05
-0.242
-0.076
-0.028
24
3 d
0.34
0.02
0.23
<0.01
-1.04E-02
-0.230
-0.044
0.009
24
CHAPTER V
83
SS2 had g own 7±3.5 cm (mean±SD) h ee mon hs a e he i s
measu emen and 5±3 cm a e he second measu emen . The g ow h
a e he hi d measu emen was no conside ed gi en he signi ican loss
o indi iduals and he s ong he bibo y in hose who su i ed.
4. Discussion
4.1 In a- hallus a ia ion
The concen a ions o all elemen s a ied signi ican ly depending on he
dicho omy analyzed, bu his a ia ion was no dependen on he
“Sampling mon h” nei he on “SS” (Table S2, Suppl. Ma .). Rega dless he
ype o end in concen a ions obse ed along he hallus, he lack o
signi ican di e ences in concen a ions o mos elemen s be ween he
oldes dicho omies sugges s ha bioconcen a ion a es in F. esiculosus
s abilize wi h he age o he issue.
In a- hallus a ia ion in concen a ions o elemen s was widely
epo ed in algae (Ga cía-Seoane e al., 2018), and p e iously obse ed in
F. esiculosus be ween dicho omies (e.g. Sa age & Elmg en, 2004;
Ca ballei a e al., 2014), and be ween di e en s uc u al pa s o he
hallus (e.g. B yan & Humme s one, 1973; Ca alho e al., 1997).
Howe e , none o hese s udies has applied obus s a is ics o de e mine
whe he concen a ions in he di e en dicho omies o he hallus
signi ican ly in e ac wi h ac o s as he mon h o collec ion o he SS.
Fu he mo e, mos o hem did no e en use s a is ical es s o de e mine
whe he he di e ences obse ed be ween he di e en pa s o he hallus
we e signi ican o no , and nei he c oss-checked he ep esen a i eness
o hei esul s by compa ing among a ious SS (Ga cía-Seoane e al.,
2018). In iew o his, he g adual inc ease in concen a ions o Al, Co, Fe,
Ni, Pb and Zn obse ed om he g owing ips owa ds he olde
dicho omies o he hallus (Fig. S1, Suppl. Ma .) is in acco dance wi h he
esul s epo ed om p e ious s udies o he same elemen s in F.
esiculosus, e.g. o Al, Fe, Pb and Zn (B yan & Humme s one, 1973), Fe,
Ni and Zn (Ba ne & Ashc o , 1985), and Al, Co, Fe, Ni and Zn
(Fö sbe g e al., 1988; Söde lund e al., 1988). Fu he mo e, he dec easing
end in concen a ions o As, Cd, Hg, N and δ15N wi h he age o
dicho omy (Fig. S1, Suppl. Ma .), al hough is opposed o he δ15N alues
ound in he same species by Sa age & Elmg en (2004), is also consis en
wi h he esul s om Ca ballei a e al. (2014) and Viana e al. (2015), who
ound highes δ15N alues in he apical dicho omies compa ed o he es
o he ond. Howe e , al hough hese au ho s no ed he exis ence o
concen a ion g adien s wi hin he hallus o F. esiculosus and, in mos
cases, hei conclusions ag ee wi h hose eached in his s udy, he absence
o s a is ical es s con i ming he exis ence o signi ican di e ences
be ween dicho omies, especially in olde s udies, leads hese esul s o be
conside ed inconclusi e.
The p esence o a concen a ion g adien ela ed wi h he hallus age
could be he esul o se e al ac o s, including: i) he in insic up ake
cha ac e is ics o algae issues (e.g. a ia ions in ca ion exchange capaci y);
ii) he di e en g ow h a es and me abolic ac i i y o issues (e.g. Rice &
Lapoin e, 1981; S engel e al., 2005); iii) bu also he con amina ion by
epiphy es (e.g. Kangas e al., 1982) and ine pa icles (e.g. B yan &
Humme s one, 1973; Fö sbe g e al., 1988; Malino skaya, 1998); and i )
he p esence o p e-exis ing con aminan load in issues. Howe e ,
acco ding o he esul s o he p esen s udy, he p e e en ial concen a ion
o he elemen s in young o old issues canno be explained by a single
ac o . The physicochemical cha ac e is ics o he elemen s may in luence
he capaci y o compe e o binding si es (e.g. S engel e al., 2005; Ryan e
al., 2012), bu he a ia ion in he numbe and/o ype o binding si es
along he hallus may also explain he di e ences obse ed in he
dis ibu ion pa e ns o elemen s. Meanwhile, he di e en me abolic
unc ions o he elemen s ac oss di e en hallus egions can also
con ibu e o he obse ed di e ences (e.g. Rice & Lapoin e, 1981; S engel
e al., 2005).
As some au ho s (e.g. B yan & Humme s one, 1973; Higgins &
Mackey, 1987) sugges ed, he me al up ake capaci y in algae may inc ease
CHAPTER V
85
wi h g ow h a e, as algae g ow h a es de e mine he quan i y o issue pe
uni o ime ha is exposed o he en i onmen al le els o pollu an s.
Al hough he ela ionship be ween bioconcen a ion and g ow h a e is
likely o be me al-speci ic (S engel e al., 2005). Thus, highe g ow h a es
in young issues han in old pa s would lead o a apid inc ease o biomass
and, consequen ly, o a high p opo ion o ee binding si es o cap u e
elemen s (highe ca ion exchange capaci y in issues). Fu he mo e, i is
assumed ha he ac i e up ake and elease p ocesses a e as e in new and
me abolically mo e ac i e issues because he abili y o egula e bo h
p ocesses in he mac oalgae dec ease in he cou se o aging (Malino skaya,
1998). All o he abo e would explain he la ge di e ences obse ed in he
concen a ions o As, Cd, Hg, N and δ15N be ween young and old
dicho omies (Fig. S1, Suppl. Ma .).
Ne e heless, high concen a ions o elemen s such as Al, Co, Fe, Ni,
Pb and Zn in he oldes a eas o he hallus (Fig. S1, Suppl. Ma .) is
p obably mo e ela ed o ac o s as he appea ance o epiphy es
(Malino skaya, 1998) o pa icula e ma e (B yan e al., 1985; Ba ei o e
al., 2002). Bu he possible p e-exis ing con aminan load compa ed o
younges issues o , as sugges ed by Higgins & Mackey (1987), he inc ease
in me al binding si es in olde issues may also cause hose high
concen a ions. Finally, con a y o he men ioned in he p e ious
pa ag aph, some au ho s (e.g. Fuge & James, 1973; Ma kham e al., 1980;
Villa es e al., 2002) ha e poin ed ou ha me al up ake capaci y in algae
dec ease wi h g ow h a e, as highe g ow h a es in apical pa s would
esul in an inc ease in biomass o issue, leading o a dilu ion o
concen a ions by new g ow h, and he e o e o lowe concen a ions in
young han in old issues. Howe e , his heo y can be disca ded based on
ha , al hough young issues g ow p opo ionally mo e han olds, all issues
will up ake/ elease me als un il hey a e balanced wi h he concen a ions
p esen in he en i onmen , e en while he new issues is g owing, so he e
could ne e be a dilu ion by g ow h.
To conclude, he s ong nega i e co ela ions be ween elemen s such
as As and Cd, and he d y weigh o he dicho omies, could indica e ha

he g ea e he amoun o issue, he lowe he bioconcen a ed
concen a ions. This could be ela ed o he amoun o speci ic su ace
a ailable o adso b hese elemen s which, in p opo ion, may be less han
he es o he issue as he algae g ow.
4.2 Tempo al a ia ion
The concen a ions o all elemen s in all he dicho omies o F. esiculosus
a ied conside ably o e yea s. A signi ican dec easing pa e n in
concen a ions o elemen s such as Co, Hg, Ni and Zn was obse ed in
he issues om 2015 o 2018 in all SS (Table 2), wi h concen a ions
dec easing annually be ween 10% and 20%. Al hough Viana e al. (2010)
s udied empo al changes only in apical dicho omies in his species in he
same s udy a ea, hey obse ed a signi ican dec ease in he concen a ions
o Al, Cd, Co, Fe, Hg and Zn o ca. be ween 5% and 10% om 2001 o
2007.
The esul s o he p esen s udy also demons a e a la ge deg ee o
in a-annual a ia ion in he concen a ions o he elemen s de e mined in
he F. esiculosus issue (Table 1), being compa able o he in a-annual
a iabili y ound o he same elemen s in a mul i ude o s udies o
empo al ends using mac oalgae as biomoni o s (i.e. coe icien s o
a ia ion, CV be ween 35% and 58%, excep N, CV <30%) (see Ga cía-
Seoane e al., 2018). Acco ding o he co elog ams calcula ed and o he
models i ed o he ime se ies (Fig. 2, Table 2, Table S3 in Suppl. Ma .),
his in a-annual a ia ion in concen a ions is no andom, and can be
easonably a ibu ed o he exis ence o seasonali y in he
bioconcen a ion p ocess o hese elemen s. Excep o Al, Hg and Pb,
he o he elemen s showed cyclical luc ua ions e e y 12 mon hs in a leas
one o he SS, consis ing on maximum alues in win e and minimum
alues in summe . Concen a ions o Cu, Fe, N and Zn exhibi ed mo e
han wo- old changes be ween bo h seasons. Seasonal a ia ion was
p e iously documen ed in o e 80% o publica ions assessing he e ec
o empo al a iabili y in mac oalgae, and mos o hese concluded, as in
CHAPTER V
87
he p esen s udy, ha he highes concen a ions o me als ypically occu
du ing win e and a e lowe in summe (Ga cía-Seoane e al., 2018).
Seasonali y was obse ed in F. esiculosus o example o As (S oepple e
al., 1986), Cd (Rige e al., 1995), Zn (Rige e al., 1995; Villa es e al., 2013)
and δ15N (Lemesle e al., 2015, 2016), bu also in o he ucoid species, e.g.
o Cd, Zn (Mi amand & Ben ley, 1992; Ma in e al., 1997) and Ni (Ma in
e al., 1997). Howe e , al hough he e has been much discussion abou he
exis ence o seasonal luc ua ions in bioconcen a ion o elemen s in algae,
un il now i has ne e been p o en using au oco ela ion analysis.
Fu he mo e, mos o hose s udies using he e m seasonali y o desc ibe
a ia ions be ween win e and summe , ha e done so e oneously om
ou poin o iew, since in o de o es ablish he exis ence o seasonali y i
is necessa y o collec samples a a high equency (e.g. mon hly) epea edly
o e se e al yea s. Those s udies compa ing concen a ions in di e en
seasons collec ing samples o a one-yea pe iod o less, canno clea ly
s a e ha he a ia ions obse ed co espond o a seasonal pa e n, since
i canno be disca ded ha hese di e ences a e due o a di e en cause,
o example, o an isola ed pollu ion e en a he ime when he highes
concen a ions we e de ec ed. Finally, since he e was no signi ican
in e ac ion be ween “Sampling mon h” and “Dicho omy” o any o he
elemen s, i can be concluded ha he in a- hallus a ia ion occu s
independen ly o he mon h/season o he yea in which he algae a e
collec ed.
Seasonal a ia ion in algae concen a ions may be a ibu ed o changes
in en i onmen al ac o s such as empe a u e, p ecipi a ion, salini y, pH
o ligh condi ions (S oepple e al., 1986; Ha oon e al., 1995; W igh &
Mason, 1999), as he bioa ailabili y o me als in seawa e and sedimen s
changes depending on he physico-chemical condi ions o he
en i onmen (Bu don-Jones e al., 1982; W igh & Mason, 1999).
Al hough seasonal a ia ion could also e lec seasonal changes in he
le els o he elemen s in solu ion due o occasional lu ial and e es ial
inpu s (Lace da e al., 1985; Fink & Manley, 2011). Howe e , al hough
mos au ho s a ibu ed his pa e n o a dilu ing e ec due o g ow h o
algae in he wa mes pe iods (e.g. Rige e al., 1995; Ma in e al., 1997;
Vasconcelos & Leal, 2001; Villa es e al., 2002, 2013), we do no belie e
ha seasonal luc ua ions can be explained by he di e ences in algae
g ow h a es be ween summe and win e , since as i was men ioned
abo e, algal issues up ake/ elease me als un il hey a e balanced wi h he
ambien concen a ions, i espec i e o hei g ow h a e. Thus, he
possibili y ha he seasonali y o concen a ions be due o o he ac o s
in insic o he algae, such as me abolism (pho osyn hesis and espi a ion)
and ep oduc ion canno be disca ded (e.g. Rao & Indusekha , 1989;
W igh & Mason, 1999). In he case o N, as i is he main limi ing nu ien
o p ima y p oduc ion in ma ine coas al wa e s, he occu ence o
minimum alues in summe may sugges s nu ien limi a ion by his
elemen , as i co esponds o he pe iod o maximum p oduc i i y (Villa es
e al., 2013).
Rega dless, any one o hese possibili ies ully explain he seasonal
a ia ion di e ences be ween elemen s. Bioconcen a ion o elemen s in
F. esiculosus is mos likely due o complex in e - ela ions among
en i onmen al and biological ac o s (Vasconcelos & Leal, 2001; Villa es
e al., 2002), and he in e ac ion be ween hese ac o s can esul in
seasonal cycles o di e en pe iod.
Finally, because he halli o F. esiculosus ha e been ound o o m
se e al new dicho omies each yea , dicho omies can no longe be
conside ed equi alen o annual g ow h coho s, as done in biomoni o ing
s udies s udying empo ali y in his species (e.g. Sa age & Elmg en, 2004;
Ca ballei a e al., 2014).
5. Conclusions
The concen a ions o Al, As, Cd, Co, Fe, Hg, Ni, Pb, Zn, N, as well as
δ15N signal depend on he issue age in F. esiculosus. This species
p e e en ially bioconcen a es As, Cd, Hg, N and δ15N in young
dicho omies and Al, Co, Fe, Ni, Pb and Zn in old dicho omies. As he
concen a ions o ni ogen and ace elemen s will la gely depend on he
CHAPTER V
89
dicho omy selec ed o analysis, we assume ha he esul s om
biomoni o ing s udies ha use di e en sec ions o he hallus o F.
esiculosus a e no s ic ly compa able. In addi ion, acco dingly o ou
indings, he concen a ions o ni ogen and ace elemen s in F. esiculosus
a e subjec o high seasonal a ia ion. The e o e, compa ison o samples
collec ed a di e en imes o he yea is no accep able. Thus, he p esen
s udy p oposes he ollowing sampling p o ocol, ha should se e as a
guideline o u u e biomoni o ing s udies using ma ine mac oalgae on he
SS scale:
1. To educe he e ec o a iabili y along F. esiculosus hallus and o
ensu e in e -compa abili y be ween s udies, we p opose o make
composi e samples o he h ee apical dicho omies o hallus (usually
a ailable e en in he younges halli).
2. To minimize he e ec o he seasonal a ia ion and p o ide annual
ep esen a i e da a while op imize he sampling e o , subsamples should
be collec ed 2 imes pe yea sepa a ed by 6 mon hs ( he ime lag be ween
he annual maximum and minimum concen a ions) and combined in a
single composi e sample.
Acknowledgemen s
The au ho s a e membe s o he Galician Compe i i e Resea ch G oup
GRC/GPC2016-002 and o he CRETUS S a egic Pa ne ship
(AGRUP2015/02), which a e co- unded by FEDER (EU). Au ho s
would like o hank he use o RIAIDT-USC analy ical acili ies. Ri a
Ga cía Seoane is g a e ul o he Spanish Minis e io de Ciencia, Inno ación
y Uni e sidades o a g an awa ded wi hin he P og ama de Fo mación de
P o eso ado Uni e si a io (FPU 2014).
Villa es, R., Puen e, X., Ca ballei a, A., 2002. Seasonal a ia ion and
backg ound le els o hea y me als in wo g een seaweeds. En i on.
Pollu . 119(1), 79–90.
Wo , D.J., 1955. The seasonal a ia ion in chemical composi ion o
Mac ocys is in eg i olia and Ne oecys is lue keana in B i ish Colombia coas al
wa e s. Can. J. Bo any 33(4), 323–340.
W igh , P., Mason, C.F., 1999. Spa ial and seasonal a ia ion in hea y
me als in he sedimen s and bio a o wo adjacen es ua ies, he O well
and he S ou , in eas e n England. The Sci. To al En i on. 226, 139–
156.

Chap e VI
99
CHAPTER VI
Biomoni o ing coas al en i onmen s wi h ansplan ed
mac oalgae: a me hodological e iew
Ma ine Pollu ion Bulle in, 135 (2018), 988–999
R. Ga cía-Seoane1, J.R. Aboal1, M.T. Boque e2,3, J.A. Fe nández1.
1Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Biología, Lope Gómez
de Ma zoa s/n, San iago de Compos ela, 15782, A Co uña, Spain.
2Es ación Biológica de Doñana, CSIC, A enida Amé ico Vespucio 25, Isla de la Ca uja, Se illa 41092,
Spain.
3Depa men o In eg a i e Biology, Uni e si y o Sou h Flo ida, 4202 E Fowle A e, Tampa, FL 33620,
USA.
h ps://doi.o g/10.1016/j.ma polbul.2018.08.027
Chap e VII

103
CHAPTER VII
Pheno ypic di e ences in hea y me al accumula ion in
popula ions o he b own mac oalgae
Fucus esiculosus
:
a ansplan a ion expe imen
Accep ed in Ecological Indica o s.
R. Ga cía-Seoane1, J.R. Aboal1, M.T. Boque e2,3, J.A. Fe nández1.
1Ecology Uni , Dep . Func ional Biology, Uni e sidade de San iago de Compos ela, Fac. Biología, Lope Gómez
de Ma zoa s/n, San iago de Compos ela, 15782, A Co uña, Spain.
2Es ación Biológica de Doñana, CSIC, A enida Amé ico Vespucio 25, Isla de la Ca uja, Se illa 41092,
Spain.
3Depa men o In eg a i e Biology, Uni e si y o Sou h Flo ida, 4202 E Fowle A e, Tampa, FL 33620,
USA.
Abs ac
The concen a ions o Al, As, Cd, Co, C , Cu, Fe, Hg, Ni, Pb and Zn in he halli
o Fucus esiculosus ansplan ed ecip ocally among ou si es a ec ed by di e en
deg ees o me al pollu ion ( wo unpollu ed and wo indus ial en i onmen s)
we e measu ed wi h he aim o compa ing he capaci y o he algae o accumula e
hese elemen s unde such condi ions. A he beginning o he expe imen , he
concen a ions o all elemen s di e ed signi ican ly be ween he indi iduals om
a leas one o he unpollu ed and one o he pollu ed si es. A e exposu e o he
algae o 90 days, he concen a ions o all o he elemen s excep As, Cd and Cu
in indi iduals ansplan ed om he unpollu ed si es o he mos pollu ed si e
inc eased o highe le els han in he algae ansplan ed wi hin he si e o o igin.
The same was obse ed o all elemen s excep As and Cd in he halli
ansplan ed om one o he unpollu ed si es o he second mos pollu ed si e.
By con as , he concen a ions o he elemen s in ansplan s om he pollu ed
si es exposed in he unpollu ed si es usually dec eased o app oxima ely he same
le els as in he au o ansplan s. These esul s sugges ha he hea y me al up ake
capaci y may be limi ed in F. esiculosus popula ions exposed o long- e m
pollu ion as an adap i e esponse o oxici y by me als. We he e o e ecommend
he use o algal ansplan s o s udy wa e quali y in highly pollu ed si es, a he
han o na i e algae al eady g owing in he si es, o a oid he possible e ec s o
such adap a ion.
Keywo ds:
Biomoni o ing; Ma ine pollu ion; Hea y me als; Fucus; T ansplan s;
Adap a ion.
CHAPTER VII
105
1. In oduc ion
Biomoni o a e o ganisms ha p o ide quan i a i e in o ma ion
(measu able esponses) abou en i onmen al changes, whe he na u al o
human-induced, h ough chemical analysis o hei issues (He z, 1991;
Ca ballei a e al., 2000). In he con ex o hea y me al pollu ion in ma ine
en i onmen s, an ideal biomoni o should be a ne accumula o o me als,
i.e. an o ganism in which a simple co ela ion be ween he concen a ions
o me als in i s issues and he bioa ailable concen a ions in i s
su ounding en i onmen is obse ed (Phillips, 1980; Rainbow, 1995;
Ma ke e al., 1999). The linea ela ionship be ween exposu e
concen a ions and body bu den should be main ained o p o ide
unequi ocal in o ma ion abou he le els o me als a ailable in he
en i onmen (S engel & D ing, 2000).
Ma ine mac oalgae a e o en used in biomoni o ing p og ammes o
es ima e me al pollu ion in a wide ange o en i onmen s (Phillips, 1980).
Some species o b own algae (Class Phaeophyceae) a e able o su i e in
highly pollu ed en i onmen s, such as a eas ecei ing was e om ish
a ms (Rönnbe g e al., 1992), di e en ypes o indus ies (Ma sden e al.,
2003), and sewage ea men plan s (Oczkowski e al., 2008), and can
accumula e high le els o hea y me als in hei issues. Al hough some
me als play impo an oles in me abolic p ocesses ( iz. Cu, Fe and Zn),
hey may ha e oxic e ec s when p esen a high concen a ions and cause
physiological s ess in algae (Gau & Rai, 2001; Pea son e al., 2010),
a ec ing g ow h a es (e.g. Collén e al., 2003), su i al a es (e.g. Ma sden
e al., 2003; Sales e al., 2011) and co e (e.g. Ma sden e al., 2003).
Acco ding o se e al au ho s, in algae excess me al can lead o he
subs i u ion o essen ial co- ac o s and hus o he inac i a ion o enzymes
and p o eins and he subsequen inhibi ion o pho osyn hesis (Plö z, 1991;
Küppe e al., 2002).
Se e al s udies ha e demons a ed he exis ence o esis ance
mechanisms in algal popula ions con inuously exposed o high le els o
concen a ions o me als in he samples ansplan ed om P2 ( o all
elemen s excep As, Co and Pb), om U1 ( o Co, Fe, Hg and Ni), and
om U2 ( o Co, Hg and Ni) o P1 we e, signi ican ly highe han he
le els eached in he au o ansplan s. A simila pa e n o inc ease in he
concen a ions was obse ed in halli ans e ed o P2, o ewe elemen s
(i.e. Al, C , Cu, Fe, Hg, Pb, and Zn), and wi h he c oss ansplan s om
U1 ( o Al, Cu and Hg) and om P1 ( o Pb) eaching signi ican ly highe
concen a ions han in he au o ansplan s.
On he o he hand, he concen a ions o all elemen s in
c oss ansplan s om P1 exposed in U1 and U2 ended o dec ease o
app oxima ely he same le els as in he au o ansplan s. The dec ease was
o mo e han hal he ini ial alues o C , Ni, Pb and Zn, and up o ca.
six old imes o Co and Cu. The concen a ions o As, Cu, Hg and Zn
( o Zn only in U2) dec eased in he algae ansplan ed om P2 o U1 and
U2. Fo he o he elemen s (i.e. Al, Cd, Co, C , Fe, Ni, Pb), he
concen a ions in he c oss ansplan s om P2 ended o inc ease, a he
han dec ease. By con as , he inal concen a ions o Cd we e
signi ican ly lowe in c oss ansplan s om P1 and U2 exposed in U1, and
om P1 exposed in U2 han in he co esponding au o ansplan s. As an
excep ion, he concen a ions o Cd in he c oss ansplan s om U1 and
U2 exposed in P1 and P2 we e lowe han in he au o ansplan s, whe eas
he concen a ions in he algae ansplan ed om P2 o U1 and U2 ended
o inc ease abo e he concen a ions in he au o ansplan s.
Finally, he wo–way ANOVA es applied o he inal concen a ions
(see Table 1) also e ealed a signi ican exposu e si e*o igin si e in e ac ion
o Al, Cd, Cu, Fe and Hg. The exposu e si e had a di e en e ec on he
inal concen a ion o me als depending on he si e o o igin o he algae.
Al hough no clea pa e n was obse ed ega ding he signi ican
in e ac ions, he Simple E ec s Tes showed ha he e ec in he
concen a ions was associa ed wi h exposu e si es P1 and P2
(excep ionally also U1 and U2 in he case o Cd).
The up ake/ elease a es o each elemen and ansplan , along wi h
he a iabili y exp essed as he coe icien o a ia ion (CV), we e also

CHAPTER VII
113
calcula ed (Table 2). T ansplan s exposed in unpollu ed si es (U1 and U2)
showed nega i e a es ( elease) o all elemen s, which we e especially high
in ansplan s om P1. Release a es we e also obse ed o As and Cd in
ansplan s ans e ed o P2. On he con a y, he highes posi i e a es
(up ake) we e obse ed in ansplan s exposed in he pollu ed si es (mainly
o Al, Cu, Fe and Zn). The CV e ealed g ea e a iabili y in he
concen a ions o some elemen s in he au o ansplan s om P1 ha in
he c oss ansplan s ( o Al, Co, C , Cu, Fe, Ni, Pb and Zn). The same was
ound in P2 o o he elemen s (i.e. As, Cd and Hg). In U1 and U2, au o
and c oss ansplan s we e no clea ly dis inguished in e ms o a iabili y.
Fig. 1. Changes in he mean concen a ions (µg g-1 d.w. ± SE, n=5) o Al, As, Cd, Co,
C and Cu in ansplan s o Fucus esiculosus exposed in wo pollu ed si es (P1 and P2) and
in wo unpollu ed si es (U1 and U2). Squa es and con inuous lines: specimens
ansplan ed om he pollu ed si es (black lines: P1; g ey lines: P2). T iangles and
discon inuous lines: specimens ansplan ed om he unpollu ed si es (black lines: U1;
g ey lines: U2). As e isks indica e he compa isons in which he mean concen a ions a
ini ial ime we e signi ican ly di e en (p≤0.05) among si es ( esul s o K uskal–Wallis
es ). No e ha he scale on he o dina e axis is di e en o pollu ed and unpollu ed
si es. 0: ini ial ime; : inal ime (90 days).
CHAPTER VII
115
Fig. 2. Changes in he mean concen a ions (µg g-1 d.w. ± SE, n=5) o Fe, Hg, Ni, Pb
and Zn in ansplan s o Fucus esiculosus exposed in wo pollu ed si es (P1 and P2) and in
wo unpollu ed si es (U1 and U2). Squa es and con inuous lines: specimens ansplan ed
om he pollu ed si es (black lines: P1; g ey lines: P2). T iangles and discon inuous lines:
specimens ansplan ed om he unpollu ed si es (black lines: U1; g ey lines: U2).
As e isks indica e he compa isons in which he mean concen a ions a ini ial ime we e
signi ican ly di e en (p≤0.05) among si es ( esul s o K uskal–Wallis es ). No e ha he
scale on he o dina e axis is di e en o pollu ed and unpollu ed si es. 0: ini ial ime; :
inal ime (90 days).
Table 1. Resul s o he wo–way ANOVA compa ing inal concen a ions ( ) in
au o ansplan s and c oss ansplan s o Fucus esiculosus, acco ding o he exposu e and
o igen si e. F = F s a is ic; d = deg ees o eedom; and p = p alue. Signi ican p alues
a e shown in bold i alics (p<0.001), in bold ype (p<0.01) and in i alics (p<0.05).
Final concen a ions
Sou ce o a ia ion
Dependen
a iable
d
Mean squa es
F
Sig. (p)
Exposu e si e
Al
3
1.09E+07
53.43
<0.001
As
3
7613
30.44
<0.001
Cd
3
1.708
64.20
<0.001
Co
3
96.81
87.46
<0.001
C
3
30.31
32.03
<0.001
Cu
3
9412
216.8
<0.001
Fe
3
1.42E+07
89.35
<0.001
Hg
3
1992
192.5
<0.001
Ni
3
66.02
33.76
<0.001
Pb
3
99.34
5.38
0.003
Zn
3
3.31E+05
67.66
<0.001
O igin si e
Al
3
7.26E+05
3.569
0.020
As
3
2218
8.869
<0.001
Cd
3
0.378
14.22
<0.001
Co
3
0.809
0.731
0.538
C
3
1.513
1.599
0.200
Cu
3
218.3
5.028
0.004
Fe
3
8.72E+05
5.474
0.002
Hg
3
29.09
2.810
0.048
Ni
3
4.646
2.376
0.080
Pb
3
4.612
0.250
0.861
Zn
3
2.10E+04
4.291
0.009
Exposu e si e * O igin si e
Al
9
5.50E+05
2.703
0.011
As
9
327.0
1.308
0.254
Cd
9
0.094
3.546
0.002
Co
9
1.025
0.926
0.510
C
9
1.170
1.237
0.293
Cu
9
244.6
5.635
<0.001
Fe
9
3.45E+05
2.163
0.039
Hg
9
45.69
4.414
<0.001
Ni
9
1.853
0.948
0.493
Pb
9
13.07
0.708
0.699
Zn
9
4287
0.876
0.552
E o
Al
55
2.04E+05
As
55
250.1
Cd
55
0.027
Co
55
1.107
C
55
0.947
Cu
55
43.42
Fe
55
1.59E+05
Hg
55
10.35
Ni
55
1.955
Pb
55
18.47
Zn
55
4894
CHAPTER VII
117
Table 2. Mean up ake/ elease a es (ng g-1 day-1) o he elemen s bioconcen a ed in ansplan s o Fucus esiculosus exposed o
90 days a each s udy si e (n=5 eplica es). Fo Al, Fe and Zn, he a es a e exp essed in µg g-1 day-1. Value o he coe icien o
a ia ion: CV (%); pollu ed si es: P1, P2; unpollu ed si es: U1, U2.

4. Discussion
In he p esen s udy we used an expe imen al design based on a
ansplan a ion echnique wi h he objec i e o s udying he di e ences in
hea y me al accumula ion capaci ies among ansplan s o F. esiculosus
exposed in di e en en i onmen s. To accomplish his, we selec ed a
numbe o si es wi h high and low le els o con amina ion and exposed a
each o hose si es, algal samples om o he si es (c oss ansplan s) and
na i e algae om he exposu e si e (au o ansplan s). We ound ha ou
app oach (in ela ion o he choice o si es) was adequa e o he
es ablished objec i es, because he di e ences in he concen a ions
among si es a he beginning o he expe imen con i med ha P1 was he
mos pollu ed si e, ollowed by P2, and ha U1 and U2 we e no pollu ed,
o we e a ec ed by much lowe le els o pollu ion han P1 and P2 (Figs.
2 and 3).
Ou me hodology is an imp o emen on ha used in o he s udies
assessing he esponse o algae o en i onmen al hea y me al exposu e
using ansplan a ion echniques (e.g. Eide e al., 1980; Ho, 1984; Amado
Filho e al., 1999; Hédouin e al., 2008; Sáez e al., 2015), because hese
esea che s did no use au o ansplan s, bu compa ed he concen a ions
in c oss ansplan s wi h hose in he na i e algae g owing in he si e.
Howe e , as p e iously men ioned, his compa ison is no s ic ly alid
because he algae collec ed di ec ly om na u al popula ions and analysed
a e no subjec ed o he e ec o he ansplan a ion, ha may add an ex a
laye o a ia ion in he inal concen a ions ob ained (Sáez e al., 2015).
A e compa ing he inal concen a ions o hea y me als in
au o ansplan s and c oss ansplan s eached a e 3 mon hs o exposu e
in he di e en si es, we obse ed he ollowing: i) he concen a ions in
he ansplan ed algae om he unpollu ed si es (U1 and U2) and om he
pollu ed si e P2 (gene ally less con amina ed han P1) inc eased and
exceeded hose in he au o ansplan s om P1 o all elemen s s udied, in
a leas one o he au o ansplan s/c oss ansplan s compa isons, and ii)
algae om P1 and P2 ansplan ed o he unpollu ed si es esponded
CHAPTER VII
119
apidly o he low me al concen a ions, wi h a la ge educ ion in he
me allic con en o hei issues o alues close o o e en lowe han he
au o ansplan s o all elemen s (excep Cu and Hg in ansplan s om P2)
(Figs. 2 and 3). These obse a ions con i m, i s o all, ha me al le els
in algae a y dynamically, i.e. algae a e able o modi y hei me al bu den
in esponse o he le els o hese elemen s in hei su ounding
en i onmen . Secondly, he ac ha ansplan s o iginally g owing in less
pollu ed en i onmen s a e capable o aking up highe le els o me als
han na i e algae when g owing unde he same condi ions, sugges s ha
popula ions o F. esiculosus di e in hei capaci y o ake up hea y me als.
Al hough i was ini ially hough ha he accumula ion p ocess en ailed
such a s ong bond ha he elease o me als o seawa e was no possible
(Munda, 1978), educ ion ( elease) o me al concen a ions in algal
specimens ans e ed om pollu ed o unpollu ed si es was p e iously
obse ed in b own algae (e.g. Eide e al., 1980; And ade e al., 2006) and
desc ibed as a me al de oxi ica ion mechanism, p obably associa ed wi h
exuda ion o chela ed me al compounds (e.g. Ka ez & Pe ei a, 1995;
Gledhill e al., 1999; Vasconcelos & Leal, 2001; Pin o e al., 2003). O he
au ho s such as Eide e al. (1980) and Amado Filho e al. (1999) a ibu ed
he educ ion in me al le els in wo species o b own algae o a dilu ion
e ec associa ed wi h issue g ow h du ing exposu e in a me al– ee
en i onmen . Howe e , ou indings a e no consis en wi h he dilu ion
e ec hypo hesis as we did no obse e a educ ion in he concen a ions
o me als in he c oss ansplan s o au o ansplan s exposed in he
unpollu ed si es. On he con a y, concen a ions in au o ansplan s om
U1 and U2 emained basically unchanged o inc eased sligh ly be ween 0
and , he inal concen a ions we e no signi ican a ec ed by g ow h o
he algae.
The o al me al con en in he algae is hus he esul o he inpu s and
ou pu s om di e en cellula compa men s (i.e. ex acellula ,
in e cellula and in acellula ) plus he pa icula e ma e adhe ed o he
su ace o he hallus and in acellula pa icles. Me als can also be
adso bed ex acellula ly o he cell wall o aken up and seques e ed in cells
(Mo is e al., 1999; Cos as & López, 2001; Fink & Manley, 2011).
Polyanionic polysaccha ides (nega i ely cha ged and composed mainly o
alginic acids and sulpha ed ucans) a e he main cons i uen s o he cell
wall and in e cellula ma ix and ac as ion-exchange and ionic ba ie
(Pe ci al, 1979; Ve oy e al., 1980; And ade e al., 2004). These
compounds a e able o e ain me al ca ions om he su ounding
en i onmen by exchanging hem wi h coun e ions (e.g. Na+, Ca+2 and
Mg+2) bounded o polyanionic si es, such as amino, ca boxyl, phospha e
and sulpha e unc ional g oups (Pe ci al & McDowell 1967; Mykles ad e
al., 1978; Żbikowski e al., 2007). The con en o a pa icula me al in he
cellula wall will be he e o e de e mined by he ion selec i i y o he
polyanions and he compe i ion among me allic ions (Haug & Smids ød,
1967; Sinno , 2007; Ryan e al., 2012). When abso bed in he cy oplasm,
me al ions a e chela ed by in acellula mac omolecules such as
polyphenols, me allo hioneins and phy ochela ins (Mykles ad e al., 1978).
I has been sugges ed ha polyphenols, which ha e a high capaci y o
chela e hea y me als and a e p esen in high p opo ions in b own algae
(Ragan, 1976; Ta ge e al., 1992), may accumula e some me als in b own
algal issues (e.g. Ragan e al., 1979; Pede sén, 1984; Ka ez & Pe ei a, 1995;
Hédouin e al., 2008).
Based on ou knowledge o he mechanisms in ol ed in me al up ake
in algae, and on ou p e ious expe ience, we belie e ha high p opo ions
o me als in algae a e bound o he cell wall polysaccha ides and a e no
localized in he in acellula compa men o he cells, as concluded by
Ryan e al. (2012). Ou indings in a ecen ield s udy wi h F. esiculosus
ansplan s (unpublished da a), showed ha he species is able o ake up
and elease Hg in only 5 days; his would p obably no be possible i mos
o he Hg we e localized in acellula ly, as i would possibly en ail a high
cos in e ms o ene gy. We he e o e hypo hesize ha he di e ences we
obse ed in he capaci y o he di e en popula ions o ake up/ elease
hea y me als (in aspeci ic a iabili y) may be due o he modi ica ion o
one o mo e s uc u al componen s o he cellula compa men s in ol ed
in me al up ake, and p obably o di e ences in he cell wall polysaccha ide
CHAPTER VII
121
composi ion. Na i e algae om he mos pollu ed si es (P1 in he p esen
s udy), subjec ed o ch onic ele a ed me al exposu e, would ha e su e ed
such changes in esponse o pollu ed-induced s ess, and would ha e a
lowe me al up ake capaci y han hose li ing in sligh ly pollu ed o
unpollu ed en i onmen s (e.g. Mo is e al., 1999; Ma e al., 2000; Hédouin
e al., 2008). Some au ho s ha e sugges ed ha adap a ion o his ype o
en i onmen s may imply he gene ic di e en ia ion o o ganisms
subjec ed o he selec i e p essu e gene a ed by con amina ion (Hédouin
e al., 2008; Ri e e al., 2010; Sáez e al., 2015), gi ing ise o eco ypes o
he same species wi h di e en ial ole ance o hea y me al excess (Moenne
e al., 2016). Fo his eason, algal ansplan s may he e o e be mo e
sensi i e biomoni o s han na i e algae, e lec ing mo e accu a ely he
le el o me al pollu ion in he en i onmen , as hey will no be a ec ed by
he esis ance mechanisms ha may occu in na i e popula ions (e.g.
Hédouin e al., 2008; Pe ei a e al., 2014; Ga cía-Seoane e al., 2018), in
addi ion o o he ad an ages o e he use o na i e mac oalgae, as e.g. he
abili y o be exposed in places whe e esiden species do no occu (e.g.
Sønde gaa d e al., 2014; Jona-Lasinio e al., 2015).
5. Conclusions
The linea ela ionship be ween me al concen a ions in algae and in he
su ounding en i onmen is used o jus i y he use o hese o ganisms as
pollu ion biomoni o s. Howe e , ou indings sugges ha his
ela ionship may no occu in algae con inuously exposed o high
concen a ions o me als. Hence, we ecommend he use o ansplan s
(ac i e biomoni o ing) a he han na i e algae (passi e biomoni o ing) o
assess he le el o me al con amina ion, in o de o p e en he e ec o
changes in he me al up ake capaci y ( ole ance/ esis ence mechanisms)
as a esul o ch onic exposu e o con amina ion.
p o eomics highligh s local adap a ion o wo s ains o he model
b own alga Ec oca pus siliculosus. P o eomics 10, 2074–2088.
Rönnbe g, O., Ådje s, K., Roukola hi, C., Bondes am, M., 1992. E ec s
o ish a ming on g ow h epiphy es and nu ien con en o Fucus
esiculosus L. in he Åland a chipelago, no he n Bal ic Sea. Aqua . Bo .
42(2), 109–120.
Ryan, S., McLoughlin, P., O'Dono an, O., 2012. A comp ehensi e s udy
o me al dis ibu ion in h ee main classes o seaweed. En i on. Pollu .
167, 171–177.
Sáez, C.A., González, A., Con e as, R., Moody, J., Moenne, A., B own,
M.T., 2015. A no el ield ansplan a ion echnique e eals in a-
speci ic me al-induced oxida i e esponses in s ains o Ec oca pus
siliculosus wi h di e en pollu ion his o ies. En i on. Pollu . 199, 130–
138.
Sales, M., Ceb ian, E., Tomas, F., Balles e os, E., 2011. Pollu ion impac s
and eco e y po en ial in h ee species o he genus Cys osei a (Fucales,
He e okon ophy a). Es ua . Coas . Shel S 92(3), 347–357.
Sinno , M., 2007. Ca bohyd a e Chemis y and Biochemis y: S uc u e
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o abso bed coppe in Fucus. Physiol. Plan . 66(4), 692–698.
Sønde gaa d, J., Bach, L., Gus a son, K., 2014. Measu ing bioa ailable
me als using di usi e g adien s in hin ilms (DGT) and ansplan ed
seaweed (Fucus esiculosus), blue mussels (My ilus edulis) and sea snails
(Li o ina saxa ilis) suspended om moni o ing buoys nea a o me
lead–zinc mine in Wes G eenland. Ma . Pollu . Bull. 78(1–2), 102–109.
S engel, B.D., D ing, M.J., 2000. Coppe and i on concen a ions in
Ascophyllum nodosum (Fucales, Phaeophy a) om di e en si es in

CHAPTER VII
129
I eland and a e cul u e expe imen s in ela ion o hallus age and
epiphy ism. J. Exp. Ma . Biol. Ecol. 246(2), 145–161.
Ta ge , N.M., Coen, L.D., Boe che , A.A., Tanne , C.E., 1992.
Biogeog aphic compa isons o ma ine algal polyphenolics: e idence
agains a la i udinal end. Oecologia 89(4), 464–470.
Vasconcelos, M.T.S., Leal, M.F.C., 2001. An agonis ic in e ac ions o Pb
and Cd on Cu up ake, g ow h inhibi ion and chela o elease in he
ma ine algae Emiliania huxleyi. Ma . Chem. 75(1–2), 123–139.
Ve oy, R.L., Mon a, N., de Guzman, M.L.B., Lase na, E.C., Cajipe, G.J.,
1980. S udies on he binding o hea y me als o algal polysaccha ides
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A., 2010. Use o mac oalgae s o ed in an En i onmen al Specimen
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320–332.
131
APPENDIX.
SUPPLEMENTARY MATERIAL
APPRENDIX. SUPPLEMENTARY MATERIAL (CHAPTER V)
Fig. S1. Boxplo s o he elemen concen a ions in he i e dicho omies o he hallus o
Fucus esiculosus (1s –apical, 5 h–basal) om all si es unde s udy. The whiske s ex end om
1.5 IQR (in e qua ile ange) o he lowe qua ile o he 1.5 IQR o he uppe qua ile.
Those da a poin s ha exceed hese limi s a e d awn ou side he boxplo . Di e en
le e (s) abo e he e o ba s deno e signi ican di e ences o mean concen a ions
among dicho omies based on esul s om Tukey’s Pos –Hoc es (p≤0.05). The
concen a ions a e exp essed in µg g-1 (excep *: % and **: ‰). In each boxplo : n=108
(1s , 2nd and 3 d dicho omies), n=72 (4 h dicho omy), and n=36 (5 h dicho omy). Fo Al
and Pb, n alue is espec i ely one- hi d and wo- hi ds lowe in each dicho omy, as some
o he sampling si es we e elimina ed om da a analysis.
133
Fig. S2. G ow h p og ession in one Fucus esiculosus indi idual om si e 2 (SS2). A)
Janua y; B) Ap il; C) July. G ow h moni o ing was no possible in Oc obe .
Table S1. Desc ip ion o he gene al pe iodic eg ession model and i s pa ame e s i ed
o he ime se ies.

Table S2. Resul s o he Th ee-way ANOVA es compa ing he concen a ions o
elemen s in he i e dicho omous sec ions o he hallus o Fucus esiculosus. d =deg ees
o eedom; SS=sum o squa es; MS=mean squa e; F=F s a is ical alue; Sig.=s a is ical
signi icance le el (p– alue). Signi ican p– alues a e shown in bold ype.
Sou ce
Va iable
d
SS
MS
F
Sig.
Mon h
Al
11
267
24.3
1.41
.166
As
11
1.16
0.105
1.93
.036
Cd
11
4.44
0.404
10.0
.000
Co
11
18.8
1.72
14.2
.000
Cu
11
2.62
0.238
13.3
.000
Fe
11
0.992
0.090
2.21
.014
Hg
11
0.308
0.028
7.35
.000
Ni
11
9.61
0.874
9.77
.000
Pb
11
0.676
0.061
1.09
.373
Zn
11
0.380
0.034
54.2
.000
N
11
7.05
0.641
79.1
.000
δ15N
11
3.57
0.324
3.75
.000
Si e
Al
2
3007
1504
87.7
.000
As
2
2.56
1.28
23.5
.000
Cd
2
7.32
3.66
90.8
.000
Co
2
19.4
9.69
80.2
.000
Cu
2
5.92
2.96
165
.000
Fe
2
7.41
3.71
90.9
.000
Hg
2
0.149
0.075
19.6
.000
Ni
2
0.794
0.397
4.44
.012
Pb
2
19.9
9.96
176
.000
Zn
2
0.092
0.046
72.2
.000
N
2
3.53
1.76
218
.000
δ15N
2
24.1
12.0
139
.000
Dicho omy
Al
4
187
46.9
2.73
.029
As
4
10.9
2.73
50.2
.000
Cd
4
13.1
3.28
81.3
.000
Co
4
10.7
2.68
22.2
.000
Cu
4
0.542
0.135
7.56
.000
Fe
4
3.11
0.777
19.1
.000
Hg
4
0.072
0.018
4.70
.001
Ni
4
20.6
5.16
57.7
.000
Pb
4
5.55
1.39
24.5
.000
Zn
4
0.087
0.022
34.3
.000
N
4
2.17
0.542
66.9
.000
δ15N
4
9.26
2.32
26.8
.000
135
TABLE S2
(Con inued)
Sou ce
Va iable
d
SS
MS
F
Sig.
Mon h * Si e
Al
22
711
32.3
1.88
.011
As
22
0.282
0.013
0.235
1.000
Cd
22
1.96
0.089
2.21
.002
Co
22
1.69
0.077
0.635
.897
Cu
22
1.49
0.068
3.78
.000
Fe
22
1.76
0.080
1.97
.007
Hg
22
0.099
0.005
1.19
.255
Ni
22
2.87
0.130
1.46
.089
Pb
22
1.66
0.075
1.33
.151
Zn
22
0.034
0.001
2.41
.000
N
22
1.23
0.056
6.93
.000
δ15N
22
5.81
0.264
3.05
.012
Mon h * Dicho omy
Al
44
200
4.54
0.265
1.000
As
44
0.335
0.008
0.140
1.000
Cd
44
0.663
0.015
0.373
1.000
Co
44
1.63
0.037
0.307
1.000
Cu
44
0.693
0.016
0.879
.689
Fe
44
0.349
0.008
0.195
1.000
Hg
44
0.075
0.002
0.451
1.000
Ni
44
1.61
0.037
0.410
1.000
Pb
44
0.618
0.014
0.248
1.000
Zn
44
0.030
0.001
1.08
.341
N
44
0.130
0.003
0.364
1.000
δ15N
44
1.51
0.034
0.398
1.000
Si e * Dicho omy
Al
8
191
23.9
1.39
.200
As
8
0.122
0.015
0.280
.972
Cd
8
0.248
0.031
0.769
.630
Co
8
0.143
0.018
0.148
.997
Cu
8
0.186
0.023
1.30
.244
Fe
8
0.095
0.012
0.293
.968
Hg
8
0.018
0.002
0.608
.771
Ni
8
0.305
0.038
0.426
.905
Pb
8
0.208
0.026
0.460
.884
Zn
8
0.008
0.001
1.65
.110
N
8
0.021
0.003
0.328
.954
δ15N
8
0.113
0.014
0.163
.995
TABLE S2
(Con inued)
Sou ce
Va iable
d
SS
MS
F
Sig.
Mon h * Si e * Dicho omy
Al
88
714
8.11
0.473
1.000
As
88
0.475
0.005
0.099
1.000
Cd
88
1.13
0.013
0.318
1.000
Co
88
1.29
0.015
0.121
1.000
Cu
88
0.703
0.008
0.446
1.000
Fe
88
1.12
0.013
0.313
1.000
Hg
88
0.112
0.001
0.334
1.000
Ni
88
1.18
0.013
0.150
1.000
Pb
88
1.77
0.020
0.355
1.000
Zn
88
0.010
0.000
0.177
1.000
N
88
0.236
0.003
0.331
1.000
δ15N
88
1.39
0.016
0.184
1.000
Residuals
Al
233
3993
17.1
As
252
13.7
0.054
Cd
251
10.1
0.040
Co
250
30.2
0.121
Cu
250
4.48
0.018
Fe
252
10.3
0.041
Hg
250
0.953
0.004
Ni
251
22.4
0.089
Pb
252
14.3
0.057
Zn
251
0.162
0.001
N
252
2.04
0.008
δ15N
252
21.8
0.086
137
Table S3. Desc ip ion o he co ela ion pa e ns in ime se ies o he di e en elemen s, o each dicho omy o he Fucus
esiculosus hallus a he sampling si es (SS). Co ela ions a e showed om lag 1 o lag 35. Upwa d and downwa d a ows ep esen
posi i e and nega i e co ela ion coe icien s, espec i ely. Signi ican co ela ions (whe he posi i e o nega i e) a e showed in
bold ype. Backg ound shading has been included o acili a e in e p e a ion o co ela ion pa e ns.
hose in he su ounding en i onmen (Philips, 1990). The esul s epo ed
in Chap e VII indica e ha he linea i y o he bioconcen a ion p ocess
in algae g owing nea by pollu ion sou ces has been al e ed, b eaking one
o he basic p inciples o biomoni o ing. Fo his eason, we p oposed he
use o algae ansplan s o e he use o na i e algae o he quan i a i e
e alua ion o wa e quali y in coas al sys ems a leas when sou ces o
pollu ion a e known in he a ea.
Finally, despi e he ad ances epo ed in his disse a ion, and as s a ed
in he wo li e a u e e iews (Chap e s I and VI), some o he key
me hodological aspec s o he biomoni o ing echnique using mac oalgae
s ill emain o be s anda dized. Some aspec s ela ed o he use o na i e
algae comp ise e.g. issues ela ed wi h he sample collec ion (as he size o
he SS), and o he aspec s associa ed wi h he sample p ocessing (as he
sample cleaning me hod be o e chemical analysis o emo e pa icula e
ma e ial adhe ed o he hallus su ace). Al hough he use o
ansplan a ion echniques has been less equen ly implemen ed in
biomoni o ing s udies (see Chap e VI), i o e s ce ain ad an ages o e
he use o na i e plan s as e.g.: i) enables s udies o be ex ended o a eas
whe e na i e popula ions o he algae species o in e es a e absen
(Fe nandes e al., 2012; Jona-Lasinio e al., 2015) and, ii) a oids he
in luence o some ac o s ha may a ec he in e p e a ion o he esul s,
since educes he e ec o he possible spa ial a iabili y o concen a ions
in na i e algae a he SS (Alqueza e al., 2013), imp o es he empo al
in e p e a ion o he esul s because he ime o exposu e is known (Sáez
e al., 2015) and, as discussed in Chap e VII, elimina es he possible
pheno ypic a ia ion o na i e algae in highly pollu ed si es (Hédouin e
al., 2008). The e o e, u u e e o s o s anda dize p ocedu es should be
di ec ed owa ds he op imiza ion o he p o ocols implemen ed in
moni o ing echnique using algae ansplan s a he han na i e algae.

Gene al Conclusions
GENERAL CONCLUSIONS
147
I has been demons a ed ha Fucus esiculosus can be used o es ablish he
na u al ange o a iabili y in concen a ions o δ15N, N and Hg in coas al
a eas. The esul s also showed ha a la ge pa o his na u al a iabili y is
explained by he p esence o a spa ial pa e n (de e minis ic componen o
he a iabili y) ha is cha ac e is ic o each zone and each con aminan
unde s udy. The e alua ion o he na u al ange o a iabili y in
concen a ions o δ15N using his species was use ul o de ec he p esence
o sou ces o small scale con amina ion by δ15N, bu also enabled o
de e mine he spa ial ep esen a i eness o he sampling si e (SS) ( he SS
can me mo ed a dis ance o up o 900 m wi hou yielding la ge a ia ions
in he δ15N signal, a leas o he SS s udied).
High in e -indi idual a iabili y was obse ed be ween subsamples o
F. esiculosus a 3 di e en SS, wi h concen a ions a ying longi udinally
and ans e sally along he SS. This e ealed ha he posi ion o he
subsamples wi hin he SS in luences he bioconcen a ed concen a ions
in he issues. The indings e idenced ha , o minimize he spa ial in a-
SS a iabili y in he concen a ions and adequa ely ep esen he
concen a ions wi hin he SS, a minimum o 30 subsamples e enly
dis ibu ed should be collec ed wi hin h ee bands pa allel o he coas line
along a 30–50 m wide s e ch (10 subsamples in each band), and also a
di e en heigh s on he ocks i possible, and bulked in a single composi e
sample.
The abili y o di e en ia e signi ican ly he mean concen a ions o any
elemen be ween wo SS using F. esiculosus will depend on he numbe o
samples o his species collec ed and indi idually analyzed a each SS. The
e idences p o ided in his Thesis allowed o conclude ha he numbe o
samples needed o de ec such di e ences will be la gely de e mined by
he mean and a iance in he concen a ions o elemen s in algae issues
a each SS. Taking in o accoun he esul s, a minimum o 20 samples
should be collec ed a each SS o de ec signi ican di e ences o elemen s
whose concen a ions di e ed by mo e han 30% be ween he SS
compa ed.
Fac o s as he ime o collec ion and he pa o he algal hallus selec ed
o chemical analysis should be aken in o accoun when s anda dize he
me hodology o moni o ing pollu ion le els using algae. Ou indings
demons a ed he exis ence o high in a-annual/seasonal, as well as high
in a- hallus a ia ion in he concen a ions o elemen s in F. esiculosus
issues. In o de o minimize he in luence o hese sou ces o a iabili y
on he da a ob ained in biomoni o ing s udies and o ensu e a good
ep esen a i eness o he esul s, we sugges ed ha subsamples should be
collec ed 2 imes pe yea sepa a ed by 6 mon hs and combined in a
composi e sample. To minimize he e ec o a iabili y along F. esiculosus
hallus, i is p oposed o make composi e samples o he h ee apical
dicho omies o hallus.
The abili y o F. esiculosus o ake up and/o elease elemen s can be
al e ed when na i e popula ions o his algae g ow subjec ed o long- e m
and high hea y me al pollu ion le els. We hypo hesized ha he s ess
induced by me allic oxici y has al e ed some o he mechanisms in ol ed
in he p ocess o bioconcen a ion o hese elemen s, es ic ing hei
abili y o concen a e hese compounds. This pheno ypic al e a ion is
in e p e ed as a esponse o he algae o o e come such ex eme
condi ions. The e o e, we ecommend o use algae ansplan s a he han
na i e algae o biomoni o ing s udies o wa e quali y in o de o p e en
he possible e ec o such adap a ions.
An exhaus i e s udy o he me hods used in passi e biomoni o ing s udies
wi h mac oalgae e ealed ha , up o da e, he e is no s anda d p o ocol ha
enables he widesp ead implemen a ion o his echnique. This lack o s anda d
me hodology hampe s ou ine use o hese o ganisms in biomoni o ing
p og ammes. Thus, wi h he objec i e o no malizing he use o na i e algae as
en i onmen al ools o wa e quali y assessmen , we p oposed a s anda d
p o ocol. Al hough mos o he me hodological aspec s included in his p o ocol
ha e no ye been ho oughly in es iga ed, ou ecommenda ions a e based on
he conclusions o p e ious s udies in ol ing his echnique, especially hose who
conside ed me hodological issues and, o cou se, in he esul s om he
expe imen al s udies ca ied ou in he cou se o his Doc o al Thesis ( he la e
GENERAL CONCLUSIONS
149
a e shown in bold ype in he Recommenda ions sec ion). The p o ocol is
desc ibed below:
Me hodological issues
Recommenda ions
1. P e-sampling issues
1.1 Sampling design
Regula sampling (g ids) o ansec s
1.2 Numbe o sampling si es (SS)
As many as possible (based on he scope, deg ee o
spa ial esolu ion, economical and ope a ional
cons ain s o he su ey)
1.3 Sampling densi y
1 SS/16 km o 1 SS/32 km2 (1 SS pe 16/2n km o 1
SS pe 32/2n km2, when mo e esolu ion is needed)
1.4 Su ey equency
As equen as possible (economic limi a ions), bu
i s ly check o seasonali y and/o in a-annual
a ia ion
1.5 Sampling pe iod
2 samplings pe yea sepa a ed by 6 mon hs
(annual composi e sample)
1.6 Algae species
1 species (Ul a sp. o Fucus sp.). I mo e han 1 species
is used, quali a i e in e p e a ion
2. Sample collec ion
2.1 Nº o samples/subsamples
To signi ican ly di e en ia e he le els o
pollu ion be ween SS: minimum 20 samples
(indi idually analyzed) pe SS
To ep esen he in a-si e a iabili y in
concen a ions: composi e sample (minimum 30
subsamples)
2.2 Amoun o sample collec ed
Minimum o 5 g d y weigh (d.w.)
2.3 Subsamples collec ion s a egy
E enly dis ibu ed in he SS wi hin 3 bands
pa allel o he coas line (10 subsamples/band),
a di e en heigh s on he ocks i possible
2.4 A ea occupied by he SS
Be ween 200 and 500 m2
2.5 Size o he SS
30–50 m wide s e ch
2.6 Momen o collec ion
Low ide (in e idal zone)
2.7 Collec ion dep h
Less han 1 m deep in in e idal zone a low ide
2.8 Spa ial ep esen a i eness o he SS
SS a 900 m away om he o iginal SS
2.9 Dis ance be ween SS and pollu ion ocus
Fu he esea ch
2.10 Sampling p ecau ions
Use o plas ic glo es (p e-cleaned and ace me al
ee)/sample only ma e ial a ached o subs a e/no
smoking
2.11 In si u cleaning
Washed ho oughly in seawa e in he ield
2.12 T anspo a ion
In sealed plas ic bags in e ige a ed condi ions
2.13 S o age p e-p ocessing
A 4°C no mo e han 6 days. Ne e eeze samples
be o e analysis
3. Sample p ocessing
3.1 Cleaning
Manual cleaning and sc aping o epiphy es, wi hou
washing
3.2 Selec ion o ma e ial o analysis
Composi e sample o 3 apical dicho omies in
Fucus
sp.
3.3 D ying
Lyophilizing o d ying a 40°C + an aliquo a 105ºC
(d.w.)
3.4 Homogeniza ion
G ound in me al ee mills (no sie e)
3.5 Ce i ied e e ence ma e ial o analysis
Ce i ied e e ence ma e ial o mac oalgae
4. Addi ional issues
4.1. Co ec ion o pa icula e con amina ion
Apply a leas once pe si e o ule ou he possible
con ibu ion o sedimen

Since he de elopmen o me hodology o applica ion o he ac i e
biomoni o ing echnique wi h algae is p ac ically inexis en , he p o ocol
p oposed below is suppo ed by he indings epo ed in he a ailable
li e a u e. In his espec , me hodological issues which ha e ne e been
s udied in de ail, like he p epa a ion o he algae o ansplan (including
acclima ion, cloning and cul u e in he labo a o y), he i al s a us o he
ansplan s (whe he using li ing o de i alized ma e ial), and exposu e o
he ansplan s (e.g. du a ion o exposu e and exposu e condi ions) should
be add essed in he nea u u e.
Me hodological issues
Recommenda ions o
species wi h lamina g ow h
Recommenda ions o
species wi h apical g ow h
1. Selec ion and p epa a ion o he mac oalgae
1.1 Moni o ing species
1 single species (Ul a sp.)
1 single species (Fucus sp.)
1.2 Selec ion o ma e ial o ansplan
Discs
Whole halli
2. P epa a ion o he ansplan s
2.1 P e-exposu e ea men s
Acclima ion o low nu ien
condi ions in s udies on
nu ien s
Acclima ion o low nu ien
condi ions in s udies on
nu ien s
Fu he esea ch
Fu he esea ch
2.2 Sample cleaning
Washed in seawa e in he field
Washed in seawa e in he field
2.3 Cloning and cul u e in he labo a o y
Fu he esea ch
Fu he esea ch
2.4 Type o ansplan
Cages
Bags
2.5 Amoun o algae/ ansplan
1 g esh weigh ( .w.)/30 cm3
1 g .w./100 cm2
2.6 Vi al s a us
Fu he esea ch
Fu he esea ch
3. Exposu e o he ansplan s
3.1 Suppo
Buoys
Buoys
3.2 Dep h o exposu e
ca. 1 m
ca. 1 m
3.3 Du a ion o exposu e
Fu he esea ch
Fu he esea ch
3.4 Numbe o eplica es/ ansplan
Minimum o 3
Minimum o 3
3.5 Ini ial concen a ions and con ols
3–5 ini ial imes and 3 con ols
3–5 ini ial imes and 3 con ols
4. Pos -exposu e ea men s
4.1 T anspo a ion
Unde e ige a ion
Unde e ige a ion
4.2 S o age p e-p ocessing
A 4°C no mo e han 6 days.
Samples ne e ozen be o e
analysis
A 4°C no mo e han 6 days.
Samples ne e ozen be o e
analysis
4.3 Cleaning
Washed in seawa e in he field
and manual cleaning
Washed in seawa e in he field
and manual cleaning
4.4 Selec ion o ma e ial o analysis
Algal discs
G een pa s o he shoo o
apical segmen s (2–3 cm)
4.5 D ying
Lyophilizing o d ying a 40°C
o 24 h + an aliquo a 105ºC
(d.w.)
Lyophilizing o d ying a 40°C
o 24 h + an aliquo a 105ºC
(d.w.)
4.6 Homogeniza ion
Milling in me al ee mills
Milling in me al ee mills
4.7 Ce i ied e e ence ma e ial o analysis
Ce ified e e ence ma e ial o
mac oalgae
Ce ified e e ence ma e ial o
mac oalgae
Gene al Re e ences
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