Copper exposure of freshwater mussels (Anodonta anatina): Some physiological effects
Full text
COPPER EXPOSURE OF FRESHWATER MUSSELS
(Anodon a ana ina):
SOME PHYSIOLOGICAL EFFECTS
Disse a ion submi ed o he
FACULTY OF BIOLOGY, CHEMISTRY, AND GEOSCIENCES
UNIVERSITY OF BAYREUTH, GERMANY
o ob ain he academic deg ee o
D
R
.
RER
.
NAT
.
p esen ed by
A
NDHIKA
P
USPITO
N
UGROHO
M. Sc.
bo n Augus 8, 1976
in Sema ang, Indonesia
Bay eu h, No embe 2011
COPPER EXPOSURE OF FRESHWATER MUSSELS
(Anodon a ana ina):
SOME PHYSIOLOGICAL EFFECTS
by
Andhika Puspi o Nug oho
En i onmen al Chemis y and Eco oxicology
Uni e si y o Bay eu h
Ge many
Supe iso :
P o . D . Ha mu F ank
This doc o al hesis was p epa ed a he Depa men o En i onmen al
Chemis y and Eco oxicology, Uni e si y o Bay eu h, om No embe 2010 un il
No embe 2011, supe ised by P o . D . Ha mu F ank.
This is a ull ep in o he disse a ion submi ed o a ain he academic deg ee
o Doc o o Na u al Sciences (D . e . na .) and app o ed by he Facul y o
Biology, Chemis y and Geosciences o he Uni e si y o Bay eu h.
Ac ing Dean: P o . D . Bea e Lohne
Da e o submission: No embe 9, 2011
Da e o de ense (dispu a ion): June 19, 2012
Doc o al Commi ee
P o . D . Ha mu F ank 1
s
e iewe
P o . D . Klaus H. Ho mann 2
nd
e iewe
P o . D . Ch. E.W. S einbe g 3
d
e iewe
P o . D . Ge ha d Gebaue Chai man
P o . D . S e an Pei e
P o . D . B i a Plane -F ied ich
I
ACKNOWLEDGEMENTS
Many people con ibu ed o his disse a ion in innume able ways, and I am
g a e ul o all o hem. Fi s and o emos I am hea ily hank ul o my supe iso ,
P o . D . Ha mu F ank, o his ime, ad ice, suppo , encou agemen , and
pa ience du ing my esea ch a he Chai o En i onmen al Chemis y and
Eco oxicology, Uni e si y o Bay eu h.
I would like o hank D . Silke Ge s mann o all he guidance, ins uc ion,
and help ul discussion abou my wo k.
The suppo o P o . D . B i a Plane -F ied ich is g ea ly app ecia ed by
allowing me o use he labo a o y acili ies o he mussel exposu e expe imen s
and he p epa a ion o he me al analyses.
I also wan o hank P o . D . Klaus H. Ho mann and D . Ma ina Meye ing-
Vos o all hei help in he labo a o y and gi ing me access o o he acili ies
du ing sample analyses and w i ing my disse a ion.
I am g a e ul o D . Gun e Ilgen and Ba ba a Schei le o assis ing in me al
analyses.
My hanks also go o all o my Indonesian iends o hei nice iendship
gi en o me du ing hese yea s.
I wish o exp ess my app ecia ion o all o my colleagues a he Chai o
En i onmen al Chemis y and Eco oxicology, Uni e si y o Bay eu h. I hank D .
Huong Ngo Thi Thuy o help ul discussions; Fabian Il zsche and S e an Will o
p o iding lo s o echnical assis ance, I mga d Lau e bach o all he help in he
adminis a i e a ai s du ing my s udy; Michael Fische , Ma io Kiesewe e ,
And eas Ban le, and Michael Heye s o all hei suppo wi h labo a o y wo k.
I am pleased o hank o all o my colleagues a he Chai o Animal Ecology
I, Uni e si y o Bay eu h. I hank Ahmad Alame , D . Judi h Lo enz, Hassan El-
Damanhou i, Ma ion P eiß, Do o hea Wiesne , Ca mela He mann, U sula
Wilczek, and In isa Taha o hei kindness and help.
Finally, I would like o hank my pa en s, wi e, pa en s-in-law, b o he s, and
sis e s-in-law o suppo ing and encou aging me always.
II
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
I
TABLE OF CONTENTS
II
LIST OF PUBLICATIONS AND AUTHOR’S CONTRIBUTIONS
V
LIST OF ABBREVIATIONS
VI
SUMMARY
VII
ZUSAMMENFASSUNG
I
X
1.
Gene al in oduc ion
1
1.1. Coppe in aqua ic ecosys ems and i s ans e in ood chains
1
1.2. E ec s o coppe on calcium homeos asis and cellula de ense
mechanisms o eshwa e mussels
2
1.3. F eshwa e mussels and hei s a us .. 4
1.4. Eco oxicological s udies wi h Anodon a ana ina ... 5
1.5. Objec i es o he esea ch 6
1.6. Me hodological equi emen s ... 6
2.
Ma e ials and me hods
7
2.1. Coppe expe imen wi h mic oalgae Pa achlo ella kessle i 7
2.2. Coppe exposu e expe imen o mussels .. 7
2.3. Analy ical me hods ..................................................................... 8
2.4. S a is ical analysis ...................................................................... 8
3.
Resul s
9
3.1. S udies o coppe e ec s on he g een alga Pa achlo ella
kessle i: P oducing Cu-loaded algae o eeding expe imen s ...
9
3.2. S udies o di e en coppe exposu e pa hways on he
eshwa e mussel Anodon a ana ina .........................................
9
3.2.1.
S udies o coppe up ake, dis ibu ion,
bioaccumula ion, and elimina ion ..................................
9
3.2.2.
S udies o coppe e ec s ............................................... 9
4.
Gene al discussion
1
0
5.
Conclusions, con ibu ions, and pe spec i es
12
5.1. Conclusions .
12
5.2. Con ibu ions ...
12
5.3. Pe spec i es
13
6
.
Re e ences
13
PUBLICATIONS
Publica ion
I :
P oducing Cu
-
loaded algae o eeding expe imen s:
e ec s o coppe on Pa achlo ella kessle i
20
In oduc i
on
20
Ma e ials and me hods
2
1
Glasswa e and eagen s ... 21
Tes o ganism, cul u e condi ions, and oxici y es ing .
22
De e mina ion o g ow h and biomass 22
III
Coppe de e mina ion 22
De e mina ion o chlo ophyll-a, chlo ophyll-b, and pheophy in-a
con en s ......................................................................................
23
P o ein and polysaccha ide con en s ......................................... 23
Da a analysis ..............................................................................
24
Resul s
24
Discussion
27
Conclusions
28
Acknowledgemen s
29
Re e ences
29
Publica ion II :
Up ake, dis ibu ion, and bioaccumul
a ion o coppe
in he eshwa e mussel Anodon a ana ina
32
In oduc ion
32
Ma e ials and me hods
33
Algal ood p epa a ion ... 33
Iso opic Cu s ock solu ion p epa a ion and labwa e .
33
O ganisms ... 33
Expe imen al design . 34
Me al analyses 34
S a is ical da a analyses
35
Resul s
35
Discussion
39
Conclusions
42
Acknowledgemen s
43
Re e ences
43
Publica ion
III :
E ec s o coppe exposu e on calcium,
ca bohyd a e, and p o ein le els in he eshwa e
mussel Anodon a ana ina
45
In oduc ion
45
Ma e ials and me hods
46
Chemicals 46
Animal and expe imen al design . 46
Calcium de e mina ion .. 47
Coppe de e mina ion 47
P o ein and ca bohyd a e de e mina ions .. 47
S a is ical da a analyses
48
Resul s
48
Discussion
51
Con
clusions
52
Acknowledgemen s
52
Re e ences
52
Publica ion
IV :
E ec s o coppe on
lipid pe oxida ion,
glu a hione,
me allo hionein, and an ioxida i e enzymes in he
eshwa e mussel Anodon a ana ina
55
In oduc ion
55
Ma e ial and me hods
56
Chemicals 56
IV
Animals and expe imen al design ... 56
Analy ical me hods . 57
Sample p epa a ion ... 57
To al coppe ... 57
Lipid pe oxida ion .. 57
Glu a hione . 58
Me allo hioneins .....
58
Enzyme ac i i ies ... 58
P o eins ... 58
S a is ical da a analyses
58
Resul s
60
Discussion
6
2
Conclusions
6
4
Acknowledgemen s
64
Re e ences
64
DECLARATION AND ERKLÄRUNG
67
V
LIST OF PUBLICATIONS AND AUTHOR’S CONTRIBUTIONS
This disse a ion is p esen ed in cumula i e o m. I comp ises ou indi idual
manusc ip s, om which all was published. The au ho ’s con ibu ions o each
manusc ip a e gi en below.
1. Nug oho, A.P., and H. F ank, 2011. P oducing Cu-loaded algae o eeding
expe imen s: e ec s o coppe on Pa achlo ella kessle i. Toxicological and
En i onmen al Chemis y, published (Publica ion I)
Own con ibu ion: idea (60%), me hod de elopmen (90%), da a analysis
and calcula ions (100%), w i ing (100%) and edi ing he pape (60%)
2. Nug oho, A.P., and H. F ank, 2011. Up ake, dis ibu ion, and bioaccumula ion
o coppe in he eshwa e mussel Anodon a ana ina. Toxicological and
En i onmen al Chemis y, published (Publica ion II)
Own con ibu ion: idea (70%), me hod de elopmen (90%), da a analysis
and calcula ions (100%), w i ing (100%) and edi ing he pape (60%)
3. Nug oho, A.P., and H. F ank, 2012. E ec s o coppe exposu e on calcium,
ca bohyd a e, and p o ein le els in he eshwa e mussel Anodon a ana ina.
Toxicological and En i onmen al Chemis y, published (Publica ion III)
Own con ibu ion: idea (70%), me hod de elopmen (100%), da a analysis
and calcula ions (100%), w i ing (100%) and edi ing he pape (60%)
4. Nug oho, A.P., and H. F ank, 2012. E ec s o coppe on me allo hionein,
glu a hione, lipid pe oxida ion, and an ioxida i e enzymes in he eshwa e
mussel Anodon a ana ina. Toxicological and En i onmen al Chemis y,
published (Publica ion IV)
Own con ibu ion: idea (80%), me hod de elopmen (100%), da a analysis
and calcula ions (100%), w i ing (100%) and edi ing he pape (60%)
VI
LIST OF ABBREVIATIONS
APW A i icial pond wa e
ATP Adenosine T iphospha e
BSA Bo ine Se um Albumin
CAT Ca alase
DNA Deoxy ibonucleic Acid
DTNB 5,5'-Di hio-bis-(2-ni obenzoic acid)
DTT Di hio h ei ol
dw D y weigh
EDTA E hylenediamine e aace ic Acid
EF En ichmen Fac o
EPF Ex apallial luid
GHL Gonads, Hea , Labial Palps
GPX Glu a hione Pe oxidase
GR Glu a hione Reduc ase
GSH Glu a hione
HML Hemolymph
IC Inhibi ion Concen a ion
LOEC Lowes -obse ed-e ec concen a ion
MDA Malondialdehyde
MT Me allo hionein
NOEC No-obse ed-e ec concen a ion
OD Op ical densi y
PBS Phospha e-bu e ed Saline
PMSF Phenylme hylsulphonyl Fluo ide
ROS Reac i e Oxygen Species
SOD Supe oxide Dismu ase
TF T ans e Fac o
ww We weigh
2
accumula e coppe om wa e , ans e ing i o g azing species a he nex
ophic le el. This con ibu es o biomagni ica ion along aqua ic ood chains. In
addi ion, g azing species can ake coppe up om he wa e (Connell and
Sande s 1999; Edding and Tala 1996; Pin o e al. 2003). Bioconcen a ion
occu s ia up ake and e en ion o me als om wa e , ac oss gill memb anes o
o he ex e nal body su aces (Kaoud and El-Dahshan 2010). The concen a ion
o he me als in he so issue o pa icula o gans can be conside ed as a
ela i e measu e o ambien concen a ions due o he abili y o mussels o
accumula e coppe (Kuma i and Nai 1992).
Since he middle o he las cen u y, he con inuously inc easing
echnological and indus ial use o coppe has led o globally inc eased mining
and ansloca ion o coppe om he ea h’s c us o he su ace and he
co esponding end o ising concen a ions in all compa men s o he
an h oposphe e o e na u al le els, especially in eshwa e ecosys ems o
indus ialized and indus ializing coun ies. Me allu gic ac i i ies, i s use in
machine y such as elec ically p opelled locomo i es, in he building sec o as
oo ing ma e ial, o wa e pipes and ki chenwa e, o o e land high- ol age
powe lines, and in di ec ly dissipa i e ways o using i as ungicide, algicide,
and molluskicide, he disposal o coppe -con aining was e wa e s, and i s
elease and deposi ion o a mosphe ic pa icula e ma e om coal combus ion
(Mohammed and Ma ke 2006; Momčilo ić 2004), all his has led o a
con inuously inc easing Cu-bu den o he biosphe e. This condi ion en ails
coppe ans e h ough ood chains and i s bioconcen a ion in mussels’ bodies.
Coppe accumula ion in cells o e he physiological equi emen s leads o oxic
e ec s, depending upon i s bioaccumula ion beyond he op imum le el wi hin
he espec i e o ganism (No 1998).
1.2. E ec s o coppe on calcium homeos asis and cellula de ense
mechanisms o eshwa e mussels
Calcium (Ca) is an essen ial mac onu ien o mussels. I has an almos
uni e sal impo ance o ne e conduc ion, mussel con ac ion, as second
messenge o egula ion o ca bohyd a e me abolism such as con olling he
ac i a ion o glycogenesis, egula ion o mi ochond ial elec on anspo , he
me abolism o ca bohyd a e in e media es o he ica boxylic acid cycle, and o
3
almos all aspec s o cellula me abolisms and g ow h (Albe e al. 1994; Sick e
al. 1979). Mo eo e , calcium is equi ed by young and adul mussels o shell
o ma ion (calci ica ion) (Sick e al. 1979). Mussel cells equi e calcium (Ca) in
speci ic limi s o cy osolic concen a ions (≤ 10
-7
M). Calcium homeos asis is
main ained by ex usion and compa men aliza ion sys ems (Via engo e al.
1993). In addi ion, mussels equi e ca bohyd a es as main ene gy sou ce o
hei me abolic p ocesses (Honkoop e al. 1999) and o shell o ma ion (Ma ie
e al. 2007; Ma in and Luque 2004). P o eins a e also equi ed by mussels o
ca alyzing biochemical eac ions, anspo and s o age o molecules in and ou
o wi hin cells, and ha e s uc u al and mechanical unc ions (Albe e al. 1994).
Exposu e o mussels o coppe a 0.35 µmol L
-1
leads o an in e e ence
wi h he sys ems esponsible o main aining Ca homeos asis in gills, diges i e
gland, and kidney (San ini e al. 2011). This is ollowed by uncon olled,
inc eased cy osolic Ca concen a ions ac i a ing a ious Ca-dependen
ca abolic p ocesses such as phospholipid hyd olysis, p o ein deg ada ion, and
DNA agmen a ion, ul ima ely leading o cell dea h (Via engo e al. 2002;
Via engo 1994). In addi ion, high coppe le els can en ail dec eased
ca bohyd a e le els in gills and man le (Sa yapa ameshwa e al. 2006).
Mussels ha e de eloped de oxi ica ion mechanisms o cope wi h coppe
challenge. In he cy osol, glu a hione (GSH), a ipep ide which con ains
sul hyd yl (SH) g oups wi h s ong a ini y o coppe ca ions and ound in high
concen a ions (0.2 – 10 mM) (Monos o i e al. 2009), can p o ide a i s line o
de ense agains inc eased cy osolic le els o ee coppe by binding he me al o
i s SH-g oups. Inc eased coppe also induces syn hesis o me allo hioneins
(MT), speci ic SH- ich p o eins ha ing he capaci y o bind coppe (Conne s and
Ringwood 2000; Via engo e al. 2002).Inc eased cy osolic coppe can induce
oxida i e s ess because coppe may be in ol ed in he o ma ion o eac i e
oxygen species (ROS) by ca alyzing he gene a ion o
•
OH om H
2
O
2
and O
2•-
h ough a Habe -Weiss cycle (Lackne 1998; Pin o e al. 2003). Du ing ae obic
espi a ion, oxygen is educed o wa e h ough ou s eps o elec on ans e
esul ing in oxygen in e media es which a e highly eac i e and oxic ROS, i.e.
he supe oxide anion (O
2•-
), hyd ogen pe oxide (H
2
O
2
), and he hyd oxyl adical
4
(
•
OH) (Lackne 1998). In heal hy ae obic cells, he e is a balance be ween ROS
p oduc ion, molecula oxida ion, and an ioxidan consump ion.
Mussel cells ha e a wide ange o an ioxida i e enzymes, neu alizing ROS
and keeping hei concen a ions a e y low le els. Supe oxide dismu ase
(SOD), ca alase (CAT), and glu a hione pe oxidase (GPX) ep esen a g oup o
enzymes ha ing an ioxida i e oles (Isani e al. 2003; Pin o e al. 2003; Via engo
e al. 2002). SOD ca alyzes he disp opo iona ion o O
2•-
o O
2
and H
2
O
2
,
whe eas CAT and GPX ca alyze he p oduc ion o H
2
O om he deg ada ion o
H
2
O
2
and ROOH, espec i ely. SOD is he cell’s i s line o de ense agains
ROS because i con ols O
2•-
which can be a p ecu so o se e al o he highly
eac i e species (Pin o e al. 2003). I hese mechanisms a e challenged beyond
hei p o ec i e capaci ies and ROS p oduc ion a es a e highe han he a es o
i s inac i a ion by an ioxidan de ense sys ems, oxida i e s ess condi ions a ise.
In such case, ee ROS can eac quickly and indisc imina ely wi h biomolecules
such as lipids, p o eins, and nucleic acids, esul ing in lipid pe oxida ion,
o ma ion o p o ein ca bonyl g oups, and DNA s and b eaks. De e mina ion o
lipid pe oxida ion allows o assess oxida i e s ess le els in cells (Company e
al. 2008; Lackne 1998).
1.3. F eshwa e mussels and hei s a us
F eshwa e mussels a e in e eb a e animals which ha e wo shells
(bi al e shell) as mi o images o each o he , connec ed by a hinge-like
ligamen . Adul mussels ha e a a ie y o sizes, colou s, and shapes, depending
on he species. They a e seden a y and inhabi he bo om o eshwa e
ecosys ems such as c eeks, i e s, s eams, ponds, and lakes. They ha e a
muscula oo which helps hem ancho agains s ong cu en s and allows
limi ed mo emen s. Mussels a e ecologically impo an in aqua ic ecosys ems
comp ising a signi ican p opo ion o he o al s anding c op in eshwa e
ben hic communi ies, cycling calcium in lakes, emo ing suspended de i us and
cleaning he wa e , mixing su icial sedimen s h ough bio u ba ion, and se ing
as ood o aqua ic mammals (Box e al. 2006; Naimo 1995; Nedeau and
Vic o ia 2003).
The amily o Unionidae is he mos endange ed o all aqua ic animal
species. Al e a ions o mussel habi a s po en ially in luence he su i al o he
5
mussels because se e al s ages o he mussels’ li e his o ies such as spe m
elease by adul males in o he wa e column, up ake o spe ms by siphoning
emales, e iliza ion o o ae, elease o iable la ae (glochidia) om emales,
and a achmen o glochidia o sui able hos ish by encys a ions o
ans o ma ion o ee-li ing ju enile mussels a e c i ical s ages which ul ima ely
can all con ibu e o a dec eased mussel ep oduc ion and popula ion
de elopmen . Labo a o y expe imen s ha e shown ha he ea ly li e s ages o
eshwa e mussels a e sensi i e o many chemicals including coppe and
ammonia (Cope e al. 2008). Due o he impo ance o mussels in aqua ic
ecosys ems, p o ec i e and conse a i e ac ions a e equi ed o main aining
heal hy mussel popula ions and o eco e ing endange ed ones by
in es iga ion o eshwa e mussel biology, he p ese a ion o wa e and
ipa ian esou ces, and he con ol and/o elimina ion o h ea s o hese animals
(Wa e s e al. 2009).
1.4. Eco oxicological s udies wi h Anodon a ana ina
Eco oxicological in es iga ions conce ning he e ec s o con aminan s a e
de o ed o e eal a which dose o concen a ions hey become oxic.
In es iga ions on con aminan up ake and elimina ion a es, dis ibu ion among
mussel issues and o gans, and he ela ionship be ween con aminan
accumula ion and biological esponses a each le el o o ganiza ion a e
equi ed.
Anodon a ana ina is a eshwa e mussel species o he amily Unionidae
which is widely dis ibu ed in Eu ope in a a ie y o eshwa e ecosys ems and
is used o biological moni o ing su eys (Mäkelä e al. 1995; Mäkelä and Oika i
1990). The species has been used o up ake and body dis ibu ion s udies o
con aminan s such as chlo ina ed phenolics (Mäkela and Oika i 1990), o
accumula ion and moni o ing s udies o 2,4,6- ichlo ophenol (Englund and
Heino 1996), pen acho ophenol (Mäkela and Oika i 1995), o he chlo ina ed
phenolics (Mäkelä e al. 1991),
45
Ca accumula ion (Pynnönen 1991), and
up ake and cadmium accumula ion and depu a ion (Holwe da e al. 1988).
O he eco oxicological s udies on con aminan impac s a biochemical and
physiological le els o A. ana ina ha e been epo ed, such as he e ec s o
coppe on Ca-ATPase and ca bonic anhyd ase (San ini e al. 2011), e ec s o
6
cadmium on calcium me abolism (Ngo e al. 2011), and e ec s o c ude oil on
cy ogene ic damage (Ba šienė e al. 2006).
1.5. Objec i es o he esea ch
This p esen esea ch p ojec aims o s udy he impo ance o wo di e en
coppe exposu e pa hways, i.e. ia wa e o ood, on up ake, dis ibu ion,
accumula ion, and elimina ion in he eshwa e mussel Anodon a ana ina, and
hei po en ial physiological impac s. In o de o e eal he e ec s o coppe ia
ood, he mussel mus be ed by coppe -con aining algae which ha e no mal
nu i ional alue o a oid seconda y e ec s. The e o e, be o e he ac ual mussel
expe imen s, mic oalgae Pa achlo ella kessle i a e aised while being exposed
o a ious coppe concen a ions, o ind he limi a which he physiological s a e
and nu i ional alue is compa able o non-exposed algae and o be used as
63
Cu-loaded ood o he mussels (Publica ion I). In he ollowing expe imen s, A.
ana ina a e exposed o Cu ia wa e o ood. Dis ibu ion, bioaccumula ion, and
elimina ion o he ace me al among he o gans o he exposed mussels a e
in es iga ed (Publica ion II). The e ec s o ele a ed coppe le els on he le els
o calcium, soluble ca bohyd a es and p o eins (Publica ion III), on
me allo hionein induc ion and glu a hione le els, on he ac i i ies o an ioxida i e
enzymes, and on lipid pe oxida ion (Publica ion IV) in a ious o gans and
issues a e examined.
1.6. Me hodological equi emen s
Since mussels con ain endogenous coppe , he s able iso ope
63
Cu is used
as ace o di e en ia e be ween endogenous and exogenous coppe .
Acco ding o C o eau e al. (2004), s able iso opes o me als can be used as
ma ke s o help in es iga ing di ec ional up ake pa hways and hei
bioaccumula ion and elimina ion om aqueous and die a y sou ces. Use o
induc i ely-coupled plasma mass spec ome y (ICP-MS) as analy ical ool
allows o de e mine he iso opes a low concen a ions as indi idual masses.
7
2. Ma e ials and me hods
2.1. Coppe expe imen wi h mic oalgae Pa achlo ella kessle i
P. kessle i (SAG Cul u e Collec ion, Uni e si y o Goe ingen, Ge many) is
exposed o Cu a a ious concen a ions o 96 hou s o ind he mos sui able
Cu concen a ion which does no a ec i s nu i ional alues, being used o
p oduce
63
Cu-loaded algae as ood o mussel expe imen . De ails o coppe
expe imen s wi h he algae a e desc ibed in Publica ion I.
2.2. Coppe exposu e expe imen o mussels
The duck mussel A. ana ina can ake coppe up om wa e o ood, so
h ee g oups o 21 mussels each a e used, one as con ol and he wo o he
g oups o be exposed o he s able iso ope
63
Cu ia wa e o ood o 24 days
ollowed by 12 days o depu a ion. Se en samplings o h ee mussels each
e e y six h days a e aken o s udy he ime-dependen Cu accumula ion and
elimina ion. Coppe exposu e o 24 days ep esen s a long- e m coppe
exposu e (Company e al. 2008), while he depu a ion pe iod o 12 days allows
o in es iga e how as he le els e u n o con ol alues, e lec ing also he hal -
li e o coppe . De ails o coppe exposu e expe imen s wi h he mussel a e
desc ibed in Publica ion II.
Du ing he expe imen s, o al coppe and iso opic coppe (
63
Cu and
65
Cu)
a e de e mined in he hemolymph (HML), he ex apallial luid (EPF), gills,
man le, kidney, diges i e gland, oo , adduc o s, in es ines, and he collec i e
emaining o gans, i.e. gonads, hea , and labial palps (GHL) (Publica ion II).
E ec s o coppe on calcium homeos asis, p o eins, and ca bohyd a es a e
s udied in all hese body compa men s. The ela ionships be ween coppe and
Ca, ca bohyd a e, and p o ein le els espec i ely, and be ween calcium and
p o eins a e examined in he compa men s (Publica ion III). E ec s o coppe
on me allo hionein, glu a hione, lipid pe oxida ion, and an ioxida i e enzymes
a e also examined (Publica ion IV).
8
2.3. Analy ical me hods
2.3.1. De e mina ions o o al Cu, iso opic Cu, and o al calcium
To al Cu and iso opic Cu in lyophilized issue ac ions a e de e mined by
induc i ely-coupled plasma mass spec ome y (ICP-MS), o al Ca by
induc i ely-coupled plasma a omic emission spec oscopy (ICP-AES).
De e mina ions and calcula ions o he concen a ions o he elemen s a e
desc ibed in de ail in Publica ions II and III.
2.3.2. De e mina ions o ca bohyd a es and p o eins
Ca bohyd a es a e de e mined by he phenol-sul u ic acid assay (Masuko
e al. 2005), p o eins by he dye-binding assay (K uge 1994). De ails o he
de e mina ions a e desc ibed in Publica ion III.
2.3.3. De e mina ion o me allo hionein
Me allo hionein (MT) concen a ions in he gills, man le, diges i e gland,
and kidney a e de e mined using he spec opho ome ic me hod desc ibed by
Via engo e al. (1997) and modi ied by Ve leca e al. (2008). De ails o
me allo hionein de e mina ion a e desc ibed in Publica ion IV.
2.3.4. De e mina ions o glu a hione, an ioxida i e enzyme ac i i ies, and lipid
pe oxida ion
Glu a hione le els a e de e mined acco ding o Ande son (1985). The
ac i i ies o he an ioxida i e enzymes ca alase, glu a hione pe oxidase, and
supe oxide dismu ase a e assayed acco ding o he me hods o Rao e al.
(1996), Paglia and Valen ine (1967), and Beauchamp and F ido ich (1971),
glu a hione educ ase ac i i ies a e assayed ollowing he me hod o Massey
and William (1965). Fo lipid pe oxida ion assay, he me hod o Buege and Aus
(1978) is employed. De ails o he de e mina ions a e desc ibed in Publica ion
IV.
2.4. S a is ical analysis
Da a o mussel expe imen s a e ans o med o log (X+1) uni s be o e
s a is ical analysis o homogenei y o a iance and no mali y. The a iabili y o
all pa ame e s wi h exposu e ime and coppe exposu e pa hways a e es ed in
9
each o gan by wo-way analysis o a iance (ANOVA). De ails o s a is ical
analyses we e desc ibed in Publica ions II and III, and Manusc ip IV.
3. Resul s
3.1. S udies o coppe e ec s on he g een alga Pa achlo ella kessle i:
P oducing Cu-loaded algae o eeding expe imen s
Exposu e o he algae o coppe abo e 6 µmol L
-1
leads o inc eased Cu
le els in he algae, inhibi ion o algal g ow h, and signi ican al e a ions o
biochemical-physiological pa ame e s, s onges e ec s being obse ed a
highes concen a ion (Publica ion I, Figu e 1, 2, and 3, Table 1). Chlo ophyll
con en s and g ow h a e a e he mos sensi i e indica o s. A 5.9 µmol L
-1
Cu,
he obse ed pa ame e s do no di e signi ican ly om con ol alues.
3.2. S udies o di e en coppe exposu e pa hways on he eshwa e
mussel Anodon a ana ina
3.2.1. S udies o coppe up ake, dis ibu ion, bioaccumula ion, and elimina ion
Mobiliza ion o he s able iso ope
63
Cu among mussel o gans e eals ha
Cu up ake om wa e occu s ia he gills and man le and om he ood ia he
diges i e gland (Publica ion II, Figu e 4). Exogenous Cu (
63
Cu) and o al
(exogenous and endogenous) Cu inc ease in all body compa men s, highes
le els being obse ed a day 24. Upon exposu e ia he wa e , high o al Cu
le els a e ound in he gills, man le, diges i e gland, kidney, and GHL while
upon exposu e ia he ood highes le els a e ound in he diges i e gland and
kidney (Publica ion II, Figu e 1, 2, 3, and 4). Du ing depu a ion, o al and
exogenous Cu dec ease in all body compa men s, excep o o al Cu in he
man le and in es ines o which e en u he inc eases we e obse ed wi hin he
i s six days o depu a ion.
3.2.2. S udies o coppe e ec s
3.2.2.1. Change in Ca le els
Ca le els in all body compa men s inc ease in pa allel o inc eased Cu
concen a ions, eaching highes le els a day 24 (Publica ion III, Figu e 1 and
2). Upon depu a ion, Ca concen a ions in he body luids decline as , e u ning
10
o con ol alues wi hin he i s six days al hough Cu le els a e s ill ele a ed. In
he o gans, Ca le els end o no malize, al hough no ully back o con ols.
3.2.2.2. Changes in ca bohyd a e and p o ein le els
Soluble ca bohyd a es and soluble p o eins decline in all o gans upon Cu
exposu e and in pa allel o i s concen a ions, eaching lowes le els a day 24
(Publica ion III, Figu e 3). When Cu adminis a ion is e mina ed, he le els in
he s udied o gans s a o inc ease, al hough no ully back o con ol wi hin he
12 days.
3.2.2.3. E ec s on me allo hionein, glu a hione, lipid pe oxida ion, and
an ioxida i e enzymes
Exposu e o A. ana ina o coppe induces inc eases in me allo hionein
(MT) in all o gans, eaching highes le els a day 24 (Publica ion IV, Figu e 1).
Fo glu a hione (GSH), he le els dec ease a simila a es in all o gans,
eaching lowes le els a day 24. Simul aneously wi h he Cu elimina ion, MT
le els dec ease in all o gans, o GSH being inc eased slowly. Thioba bi u ic
acid- eac i e subs ances (TBARS) inc ease s ongly upon Cu exposu e ia he
wa e , eaching highes le els a day 24. Du ing depu a ion, TBARS le els
dec ease slowly.
Ac i i ies o supe oxide dismu ase (SOD), ca alase (CAT), glu a hione
pe oxidase (GPX), and glu a hione educ ase (GR) a e exp essed in wo ways,
i.e. ela i e o p o ein con en s and o issue we weigh . In ela ion o p o ein
con en s, all enzyme ac i i ies inc ease eaching highes le els a day 24.
S onges e ec s we e ound in he diges i e gland (Publica ion IV, Figu e 2). In
e ms o issue we weigh , ac i i ies o SOD, GPX, and GR decline in all o gans,
eaching lowes le els a day 24. Fo CAT, he ac i i ies emain unchanged o
bo h pa hways excep o he kidney in which he ac i i y is inc eased, eaching
highes le el a day 18. Du ing depu a ion, mos pa ame e s end o no malize
bu do no e u n o con ol alues.
4. Gene al discussion
Declines in chlo ophyll con en s o P. kessle i upon Cu exposu e sugges
h ee possibili ies, i.e. inhibi ion o chlo ophyll syn hesis, inc eased lipid
11
pe oxida ion on chlo oplas memb anes, and deg ada ion o chlo ophyll-a
con i med by inc eased pheophy in-a (Sandmann and Böge 1980; T ipa hi and
Gau 2006). This condi ion may a ec pho osyn hesis a es, lowe ing glucose
syn hesis and ul ima ely esul ing in inhibi ion o algal g ow h. Dec eases in
ca bohyd a es and p o eins may be a esul o inc eased hyd oxyl adical
o ma ions induced by coppe ia he Habe -Weiss eac ion. The adicals a e
highly eac i e, oxidizing, and b eaking apa biological mac omolecules
(Nikooka e al. 2005).
Use o he s able iso ope
63
Cu
can ep esen exogenous Cudis ibu ion and
he edis ibu ion o endogenous Cu among body compa men s. Redis ibu ion
o endogenous Cu causes p onounced al e a ions in o al Cu in he o gans,
such as inc eases in he man le and in es ines upon exposu e ia he ood
(Publica ion II, Figu e 2, 3, and 4).
Coppe elimina ion can occu due o he di e ence in g adien Cu
concen a ion be ween he mussel and APW (Publica ion II, Figu e 2 and 3).
High Cu le el in he kidney indica es ha his o gan plays an impo an ole in
elimina ion. Declines o exogenous
63
Cu le els du ing depu a ion indica e ha
he iso ope is in a ela i ely easily exchangeable o m while he endogenous Cu
is mo e enaciously e ained (Publica ion II, Figu e 4).
Dis u bance o Ca homeos asis upon Cu exposu e (Publica ion III, Figu e 1
and 2) can occu due o mobiliza ion o CaCO
3
om he shell, mos likely due o
Cu-induced me abolic acidosis (An unes e al. 2002; Faubel e al. 2008; Lopes-
Lima e al. 2008). Inhibi ion o Ca ex usion and o in acellula
compa men aliza ion sys ems may be ano he complica ion (Pa naik e al.
2007; Via engo e al. 2002; Via engo e al. 1994). Dec eased p o ein le els
(Publica ion III, Figu e 3) may be due o inc eased Ca le els, ac i a ing Ca-
dependen ca abolic p ocesses such as p o ein deg ada ion (Via engo e al.
1994). A s ong dec ease o ca bohyd a e le els is a ibu ed o cell hypoxia
caused by coppe , leading o inc eased ac i i ies o glycoly ic enzymes in ol ed
in anae obic ATP p oduc ion (Sa yapa ameshwa e al. 2006; Ma ίnez e al.
2006).
Inc eases o MT le els in all obse ed o gans upon Cu exposu e
(Publica ion IV, Figu e 1) con i m he ole o MT in coppe me abolisms. S ong
dec eases in GSH le els wi hin he i s 6 days o exposu e indica e ha GSH is
18
Pin o, E., T.C.S. Sigaud-Ku ne , M.A.S. Lei ão, O.K. Okamo o, D. Mo se, and P.
Colepicolo. 2003. Hea y me al-induced oxida i e s ess in algae. Jou nal
o Phycology 39:1008-1018.
Pynnönen, K. 1991. Accumula ion o
45
Ca in he eshwa e unionids Anodon a
ana ina and Unio umidus, as in luenced by wa e ha dness, p o ons, and
aluminum. Jou nal o Expe imen al Zoology 260:18-27.
Rao, M.V., G. Paliya h, and D.P. O m od. 1996. Ul a iole -B and ozone-
induced biochemical changes in an ioxidan enzymes o A abidopsis
haliana. Plan Physiology 110:125-136.
Sandmann, G., and P. Böge . 1980. Coppe de iciency and oxici y in
Scenedesmus. Zei sch i ü P lanzenphysiologie 98:53-59.
San ini, O., N. Chahbane, P. Vasseu , and H. F ank. 2011. E ec s o low-le el
coppe exposu e on Ca
2+
-ATPase and ca bonic anhyd ase in he
eshwa e bi al e Anodon a ana ina. Toxicological and En i onmen al
Chemis y 93:1826-1837.
Sa yapa ameshwa , K., T.R. Reddy, and N.V. Kuma . 2006. S udy o
ca bohyd a e me abolism in selec ed issues o eshwa e mussel,
Lamellidens ma ginalis unde coppe sulpha e oxici y. Jou nal o
En i onmen al Biology 27:39-41.
Sick, L.V., C.C. Johnson, and C.A. Sieg ied. 1979. Fluxes o dissol ed and
pa icula e calcium in selec ed issues o C assos ea i ginica. Ma ine
Biology 54:293-299.
T ipa hi, B.N., and J.P. Gau . 2006. Physiological beha iou o Scenedesmus
sp. du ing exposu e o ele a ed le els o Cu and Zn and a e wi hd awal o
me al s ess. P o oplasma 229:1–9.
Ve leca , X.N., K.B. Jena, and G.B.N. Chainy. 2008. Modula ion o an ioxidan
de ences in diges i e gland o Pe na i idis (L.), on me cu y exposu es.
Chemosphe e 71:1977-1985.
Via engo, A., B. Bu lando, and C. Bolognesi. 2002. Cellula esponses o coppe
in aqua ic o ganisms. In Handbook o Coppe Pha macology and
Toxicology, ed. E.J. Massa o, 417-427. New Je sey: Humana P ess, Inc.
Via engo, A., E. Ponzano, F. Donde o, and R. Fabb i. 1997. A simple
spec opho ome ic me hod o me allo hionein e alua ion in ma ine
o ganisms: an applica ion o Medi e anean and An a c ic mollusks. Ma ine
En i onmen al Resea ch 44:69 – 84.
19
Via engo, A. 1994. Hea y me al cy o oxici y in ma ine o ganisms: E ec s on
Ca
2+
homeos asis and possible al e a ion o signal ansduc ion pa hways.
Ad ances in Compa a i e and En i onmen al Physiology 20:85-110.
Via engo, A., G. Mancinelli, M. Pe ica, R. Fabb i, and M. O unesu. 1993.
E ec s o hea y me als on he Ca
2+
-ATPase ac i i y p esen in gill cell
plasma-memb ane o mussels (My ilus gallop o incialis Lam.).
Compa a i e Biochemis y and Physiology 106C:655-660.
Wa e s, G.T., M.A. Hogga h, and D.H. S ansbe y. 2009. The eshwa e
mussels o Ohio. Ohio: The Ohio S a e Uni e si y P ess.
PUBLICATIONS
Toxicological & En i onmen al Chemis y
Vol. 93, No. 3, Ma ch 2011, 537–548
P oducing Cu-loaded algae o eeding expe imen s: e ec s o coppe on
Pa achlo ella kessle i
Andhika Puspi o Nug oho
ab
and Ha mu F ank
a
*
a
Depa men o En i onmen al Chemis y and Eco oxicology, Uni e si y o Bay eu h,
95440 Bay eu h, Ge many;
b
Facul y o Biology, Labo a o y o Ecology, Gadjah Mada Uni e si y,
Yogyaka a 55281, Indonesia
(Recei ed 18 Sep embe 2010; inal e sion ecei ed 2 No embe 2010)
Mic oalgae equi e se e al essen ial me als o op imum g ow h, which a
ele a ed concen a ions may in e e e wi h biochemical and physiological
p ocesses, one o hem being coppe (Cu). The aim o his s udy is o aise
Cu-loaded Pa achlo ella kessle i as eed o mussels. In o de o spike he algae
wi h Cu wi hou lowe ing hei nu i ional quali y, i is impo an o know he
highes Cu-concen a ion a which he main pa ame e s emain una ec ed,
especially in espec o p o eins and polysaccha ides. The dependence o g ow h
a e, biomass, chlo ophyll-a and -b, pheophy in-a, p o ein, and polysaccha ide
con en s on Cu concen a ions a e de e mined. The es s show ha P. kessle i is
la gely unchanged in i s nu i ional alue when exposed o Cu a le els o up o
6mmol L
1
. Abo e 10 mmol L
1
, oxic e ec s become ob ious, wi h chlo ophyll
con en s and g ow h a e being he mos sensi i e indica o s.
Keywo ds: coppe ; Pa achlo ella kessle i; g ow h a e; biomass; chlo ophyll-a;
chlo ophyll-b; pheophy in-a; p o eins; polysaccha ides
In oduc ion
Mic oalgae a e undamen al cons i uen s o ood chains in almos all aqua ic ecosys ems,
se ing as ood o o ganisms o he nex ophic le els and as sou ce o oxygen o
espi a ion. Fo op imum g ow h, algae equi e a numbe o essen ial me als, some o
which may be oxic abo e ce ain le els. A ypical example is Cu; usually, i is p esen in
na u al esh wa e s a concen a ions anging om 0.02 o 2 nmol L
1
(0.001–0.1 mgL
1
)
and in ocean wa e s om 0.5 o 10 nmol L
1
(0.03–0.6 mgL
1
). To cope wi h i s low
a ailabili y, algae ha e mechanisms o ac i e up ake and accumula ion (Debelius e al.
2009; Lim e al. 2006; W igh and Welbou n 2002), based upon he s ong complexa ion
wi h unc ional hiol g oups o he p o eins in ol ed in he ac i e up ake o Cu (Le y e al.
2008; Nalimo a e al. 2005; S aube and Flo ence 1987; Yan and Pan 2002).
Coppe is equi ed as a co ac o o enzymes pa icipa ing in oxygen me abolism and in
edox eac ions, e.g., plas ocyanin, polyphenol oxidase, supe oxide dismu ase, asco ba e
oxidase, cy och ome oxidase, lysyl oxidase, and diamine oxidase (Nalimo a e al. 2005;
Yilmaz, Is ik, and Sayin 2005). Mollusks and o he in e eb a es also equi e Cu as a
componen o hemocyanine; he animals ecei e i om he wa e as well as om
Cu-con aining ood (Amia d-T ique e al. 2006; Company e al. 2008).
*Co esponding au ho . Email: [email p o ec ed]
ISSN 0277–2248 p in /ISSN 1029–0486 online
2011 Taylo & F ancis
DOI: 10.1080/02772248.2010.537859
h p://www.in o mawo ld.com
20
Due o i s use as ungicide, in he building sec o as oo ing ma e ial, o o e land high-
ol age powe lines, and i s p esence in municipal and indus ial was e wa e s, Cu
concen a ions in i e s, lakes, and es ua ies ha e inc eased se e al old o e na u al le els
(Mohammed and Ma ke 2006; Yilmaz, Is ik, and Sayin 2005). High concen a ions ha e
been epo ed in i e s o Japan (1.1–3.5 mmol L
1
(0.07–0.22 mg L
1
), Pawlik-Skow on
´ska
and Skow on
´ski 2001), China (Yang se i e 0.2–0.8 mmol L
1
(15–50 mg L
1
), Xu e al.
2000), o B azil (Ju ujuba Sound 0.08–3.3 mmol L
1
(5–210 mg L
1
), Ne o, Smi h, and
Mc Allis e 2000). A such le els, Cu can ha e oxic e ec s o p oduce s and consume s on
he a ious s ages o he aqua ic ood chain. Fo algae, oxici y h esholds (NOEC, no
obse ed e ec concen a ion) ha e been epo ed (Le y, S aube , and Jolley 2007) o be in
he ange o 0.003–0.14 mmol L
1
(0.2–9 mgL
1
), lowes obse ed e ec concen a ions
(LOECs) in he ange o 0.004–0.63 mmol L
1
(0.3–40 mgL
1
), and he 72 h IC
50
be ween
0.009 and 8.3 mmol L
1
(0.6–530 mgL
1
), depending on he s ain.
The p ima y oxic e ec s o Cu on algal cells a e al e ing he a e o deoxy ibonucleic
acid (DNA) syn hesis, in e e ing wi h p o ein and ca bohyd a e me abolisms, mi ochon-
d ial elec on anspo , and adenosine iphospha e (ATP) p oduc ion and espi a ion,
dis up ing cell di ision, and in e e ing wi h he up ake o Ca and Mg (A unakuma a and
Xuecheng 2008; Debelius e al. 2009; Ma kina and Aizdaiche 2006; Pawlik-Skow on
´ska
and Skow on
´ski 2001; S aube and Flo ence 1987; T ipa hi and Gau 2006). In Chlo ella
py enoidosa, Cu a ec s g ow h a es, pho osyn hesis, and con en o chlo ophyll-a s a ing
a concen a ions o 4 mmol L
1
(0.25 mg L
1
), 1.6 mmol L
1
(0.1 mg L
1
), and
1.6 mmol L
1
(0.1 mg L
1
), espec i ely (Wong and Chang 1991). Yan and Pan (2002)
epo ed he g ow h o Scenedesmus obliquus,C. py enoidosa, and Clos e ium lunula being
inhibi ed a concen a ions o 0.8, 1.0, and 3.0 mmol L
1
(50, 70, and 200 mgL
1
),
espec i ely. In his con ex , i should be men ioned ha unde labo a o y cul u e
condi ions, he onse o Cu oxici y depends also on ini ial cell densi y, composi ion o he
medium, and physical condi ions (Debelius e al. 2009).
The algal species Pa achlo ella kessle i used in his s udy is a common ood sou ce o
he bi o e consume s in eshwa e ecosys ems. They a e easy o cul u e in he labo a o y,
o en used in oxici y bioassays o p edic ing en i onmen al impac s o pollu an s, and
known o ha e a ema kable abili y o accumula e me als (Debelius e al. 2009; Kaduko a
´
and Vi c
ˇı´ko a
´2005; Mallick 2003). In o de o aise Cu-loaded algae o eeding
expe imen s wi h mussels, i is impo an o assess he oxicological and pa hophysiolog-
ical h eshold o Cu which does no a ec signi ican ly he nu i ional alue o Cu-loaded
algae compa ed o con ol algae, especially in espec o p o ein and ca bohyd a e
con en s. NOECs o Cu, i s e ec s on g ow h a e and biomass, on chlo ophyll-a and -b, in
he o ma ion o pheophy in-a, and on he p o ein and polysaccha ide con en s a e
de e mined.
Ma e ials and me hods
Glasswa e and eagen s
All glasswa e is insed wice wi h hal -concen a ed HNO
3
(65%; Sigma–Ald ich, Munich,
Ge many), deionized and bidis illed wa e , and s e ilized in an au ocla e (Ce ocla
CV-EL 18 O, Ce ocla S e ilize GmbH, T aun, Aus ia) a 120C o 15 min.
Lyophilized glycogen s anda d (Type VII, My ilus edulis), Coomassie blue solu ion,
bo ine se um albumin (BSA), and all o he chemicals (Sigma–Ald ich) a e o analy ical
g ade. Fo exposu e expe imen s, a Cu solu ion is p epa ed by dissol ing
538 A.P. Nug oho and H. F ank
21
0.1 g CuCl
2
2H
2
O in bidis illed wa e in a 100 mL olume ic lask yielding a concen-
a ion o 5.9 mmol L
1
.
Tes o ganism, cul u e condi ions, and oxici y es ing
Pa achlo ella kessle i is ob ained om he Cul u e Collec ion o Algae (SAG) o he
Uni e si y o Goe ingen, Ge many. An algal s ock cul u e is g own axenically in a
s e ilized K-medium (Kuhl and Lo enzen 1964), modi ied by con aining he mac onu i-
en s KNO
3
, NaH
2
PO
4
H
2
O, Na
2
HPO
4
2H
2
O, MgSO
4
7H
2
O a 1.5 imes, and CaCl
2
a
2.5 imes inc eased concen a ions, unde omission o Cu
2þ
and e hylenediamine e aace-
ic acid (EDTA), and wi h he pH alue lowe ed o 6.5. The algae a e kep suspended by
gen le shaking, main ained a 22 2C, and illumina ed con inuously wi h luo escen
ubes a a pho on in ensi y o 48–51 mmol m
2
s
1
in 2 L E lenmeye lasks connec ed o a
D echsel gas wash bo le o dis ibu e ai and CO
2
; he la e is illed wi h po assium
ca bona e bu e (2 mol L
1
KHCO
3
/K
2
CO
3
, 35/65 / ).
Fo oxici y es ing, aliquo s o he s ock cul u e a e added o 1.8 L modi ied K-medium
in 2 L E lenmeye lasks o es ablish an ini ial cell densi y o 1–2 10
5
cells mL
1
. The
cul u es a e main ained as desc ibed abo e. A day 3 a e inocula ion, Cu is added using
he s ock solu ion o es ablish he ollowing exposu e concen a ions: 0 (con ol), 5.9, 11.7,
23.5, 47, and 94 mmol L
1
. The algae a e g own a hese le els o 96 h, i.e., om day 3 o
day 7. G ow h is moni o ed daily and, in he end o he expe imen (day 7), biomass,
chlo ophyll-a and -b, pheophy in-a, p o ein, and polysaccha ide con en s a e de e mined,
as well as Cu accumula ion. Fo all analyses, h ee aliquo s a e aken.
De e mina ion o g ow h and biomass
G ow h is ollowed by measu ing op ical densi y. Th ee 3 mL aliquo s a e aken om each
cul u e using Pas eu pipe es connec ed wi h silicon ubing o 1000 mL mic opipe o s
(Ca l Ro h, Ka ls uhe, Ge many) and ans e ed o polys y ene cu e es wi h 1 cm ligh
pa h. The op ical densi y is ead a a wa eleng h o 686 nm (OD
686
) (UVIKON 930
Spec opho ome e , Kon on Ins umen s, Munich, Ge many). The speci ic g ow h a e is
calcula ed acco ding o Mei e al. (2006), aking he a e a he highes Cu concen a ion as
100% inhibi ion.
A he end o he expe imen , he whole algal cul u e is cen i uged in six 300 mL
cen i uga ion bo les a 10,000 pm a 4C o 10 min (Beckman A an i J25, o o
JA-16.50). The supe na an s a e disca ded and he algal pelle s a e washed by
esuspension/cen i uga ion, once wi h esh cul u e medium and once wi h phospha e-
bu e ed saline (PBS). The pelle s a e combined and he algae a e esuspended in 47 mL
bidis illed wa e , ans e ed o a 50 mL polyp opylene cen i uga ion ube o known
weigh , and cen i uged again (Beckman A an i J25, o o JA-16.50); he supe na an is
disca ded, and he esh weigh o he biomass is calcula ed by sub ac ing he weigh o
he emp y ube om he weigh o he ube con aining he algae. The pelle is ozen a
80C, eeze-d ied a 40C o 72 h, and weighed again o yield he d y weigh .
Coppe de e mina ion
Fo Cu de e mina ion, he lyophilized algae a e homogenized by acid diges ion as ollows:
h ee algal samples o 10 mg each a e placed in 55 mL bo osilica e glass ubes, and o each
Toxicological & En i onmen al Chemis y 539
22
ube, 5 mL o a mix u e (4 : 1) o sup apu e concen a ed HNO
3
and sup apu e
concen a ed HCl is added. The ubes a e kep in an o en a 40C o 1 h, ollowed by
95C o 3 h. The diges ed samples a e dilu ed wi h bidis illed wa e o 10 mL and il e ed
h ough a 0.45-mm cellulose sy inge il e (Ca l Ro h). Coppe is de e mined by induc i ely
coupled plasma mass spec ome y (Agilen 7500ce, Ce ac ASX-510, Agilen
Technologies, Waldb onn, Ge many) and exp essed pe kilog am d y weigh . Coppe
concen a ion pe kilog am we weigh is calcula ed by mul iplying he de e mined
concen a ion pe d y weigh wi h he a io o algal we weigh e sus d y weigh .
De e mina ion o chlo ophyll-a, chlo ophyll-b, and pheophy in-a con en s
Chlo ophyll-a and -b and pheophy in-a con en s a e de e mined acco ding o he APHA
me hod (APHA 1992). Lyophilized algae, 3 mg each, a e suspended in 12 mL ice-cold
ace one in 50 mL polyp opylene cen i uga ion ubes and sonica ed in an ice ba h a
20 kHz, acous ic powe 50 W (Labsonic U ip Sonica o , B. B aun Bio ech In e na ional,
Melsungen, Ge many) o 160 s in eigh 20 s pe iods, allowing equal ime o cooling on
ice. The homogena es a e kep o 2 h a 4C in he da k and cen i uged a 2500 pm a
4C o 15 min. The supe na an s, 3 mL each, a e ans e ed o 1 cm polys y ene cu e es,
and he op ical densi ies a 750 and 664 nm (OD
750b
and OD
664b
) a e ead (UVIKON 930
Spec opho ome e , Kon on Ins umen s); OD
664b
alue should lie be ween 0.1 and 1.0.
Subsequen ly, 0.1 mL o 0.1 mol L
1
HCl is added unde gen le agi a ion, and 90 s la e ,
he op ical densi ies a e ead again a 750 nm and, his ime, a 665 nm (OD
750a
and
OD
665a
). The OD
664b
/OD
665a
a io is calcula ed, and hen chlo ophyll-a and pheophy in-a
a e de e mined (APHA 1992):
Chlo ophyll-aðmg L1Þ¼26:7ðOD664b OD750bÞðOD665a ðOD750aÞ½
Pheophy in-aðmg L1Þ¼26:71:7ðOD665a ðOD750aÞðOD664b OD750bÞ½
Fo de e mina ion o chlo ophyll-b, 3 mL o he supe na an is ans e ed o a 1 cm
polys y ene cu e e and he op ical densi ies a 750, 664, 647, and 630 nm a e ead.
Chlo ophyll-b is calcula ed acco ding by he ich oma ic me hod (APHA 1992):
Chlo ophyll-bðmg L1Þ
¼21:03ðOD647 OD750Þ5:43ðOD664 OD750Þ2:66ðOD630 OD750Þ:
P o ein and polysaccha ide con en s
Lyophilized algae, 5 mg each, a e placed in 2 mL Eppendo ubes, 1 mL o PBS is added o
each ube, and he samples a e sonica ed o 160 s in eigh 20 s pe iods in an ice ba h a
20 kHz, acous ic powe 50 W, allowing equal ime o cooling on ice o a oid p o ein
dena u a ion. The homogena es a e cen i uged a 4C o 20 min a 15,000 pm (Beckman
A an i J25, o o JA-16.50).
The supe na an s a e used o de e mina ion o p o ein con en by he dye-binding
assay (K uge 1994). Aliquo s o 10 mL a e illed in o 1 mL disposable polys y ene cu e es
and 90 mL o bidis illed wa e and 1 mL o Coomassie blue solu ion a e added. A e gen le
bu ho ough mixing, he samples a e kep a oom empe a u e o 15 min be o e
540 A.P. Nug oho and H. F ank
23
abso bances a e ead a 595 nm. P o ein concen a ions a e de e mined om a calib a ion
cu e ob ained wi h BSA.
Ca bohyd a e is de e mined by he phenol–sul u ic acid assay (Masuko e al. 2005).
Aliquo s o he supe na an s, 50 mL each, a e placed in 2 mL Eppendo ubes, and 200 mL
o bidis illed wa e and 750 mL o concen a ed sul u ic acid a e apidly added o achie e
comple e mixing. Immedia ely a e wa d, 150 mL o a solu ion o 5% phenol in wa e is
added. A e incuba ion o 5 min a 90C in a s a ic wa e ba h, he ubes a e cooled o
oom empe a u e o 5 min in ano he wa e ba h and wiped d y o spec opho ome ic
measu emen a 490 nm. The concen a ions o polysaccha ides a e de e mined using a
calib a ion cu e ob ained wi h glycogen s anda d ype VII (Sigma–Ald ich).
Da a analysis
The da a o all pa ame e s a e s a is ically analyzed by one-way analysis o a iance
(ANOVA), ollowed by he Duncan mul iple compa ison es s i signi ican di e ences a e
ound. Da a a e ans o med o log uni s be o e s a is ical analysis o homogenei y o
a iance and no mali y. Linea eg ession analysis is pe o med o e alua ing he
ela ionships be ween Cu concen a ion and physiological and biochemical pa ame e s,
ollowed by Pea son co ela ion analysis o es ing he s eng h o linea ela ionships.
Toxici y is exp essed as NOEC, es ima ed using he Dunne ’s mul iple compa ison es
a e analysis by one-way ANOVA, while IC
10
and IC
50
alues a e de e mined using he
inhibi ion concen a ion (ICp) app oach (Ve sion 2.0, No be g-King 1993). Visual
MINTEQ so wa e is used o calcula e Cu specia ion in ela ion o pH o algal medium
(Ve sion 3.0, be a e sion; Gus a sson 2010).
Resul s
G ow h o P. kessle i is mode a e and s a is ically, non-signi ican ly inhibi ed (5% ela i e
o con ol, p40.05) by Cu gi en be ween day 3 and day 7 a a concen a ion o
5.9 mmol L
1
. Coppe exposu es a 11.7, 23.5, 47, and 94 mmol L
1
cause signi ican
dec eases by 27, 34, 87, and 100% ( p50.05). Inhibi ion plo ed on he p obi scale
(Figu e 1) shows a linea ela ionship o Cu concen a ion wi h a s ong, posi i e
co ela ion ( ¼0.96; p50.05). I should be kep in mind ha a he pH o he incuba ion
medium o 6.5, he ela i e pe cen age o ee Cu
2þ
is abou 89–91% o he nominal
concen a ion; dec ease o pH o 6.3 a he end o exposu e may inc ease he ee Cu
2þ
o
abou 95% (Gus a sson 2010).
Coppe exposu e a 5.9 mmol L
1
esul s in dec ease in chlo ophyll-a and -b con en s
and in OD
664b
/OD
665a
a io a day 7 (Table 1) hough s a is ically insigni ican (p40.05).
Reduc ions in chlo ophyll-a and -b (42% and 32%) a e ound o be signi ican a
11.7 mmol L
1
Cu, o he OD
664b
/OD
665a
a io a 23.5 mmol L
1
Cu. A he highes Cu
concen a ion (94 mmol L
1
), chlo ophyll-a and -b con en s a e s ongly lowe ed (97% and
95% ela i e o con ol), he OD
664b
/OD
665a
a io mode a ely. Reg ession and co ela ion
analysis show s ong, highly nega i e co ela ions be ween Cu exposu e concen a ion and
chlo ophyll-a ( ¼0.908; p50.01), chlo ophyll-b ( ¼0.906; p50.01), and OD
664b
/
OD
665a
a io ( ¼0.925; p50.01). Pheophy in-a is inc eased ( ¼0.912; p50.01) e en a
he lowes Cu concen a ion being signi ican ly di e en om con ol (44%, p50.05). A
he highes Cu concen a ion, pheophy in-a is inc eased by 800%. Biomass is educed by
Toxicological & En i onmen al Chemis y 541
24
15% ela i e o con ol (p40.05) a 5.9 mmol L
1
Cu; a 11.7 mmol L
1
and abo e,
educ ions by 20% and mo e (p50.05) a e ound.
Exposu e o algae o Cu esul s in s ong accumula ion o he me al (Figu e 2) a
abo e he na u al le el, he la e being abou 0.01 mmol kg
1
we weigh . On day 7 o he
expe imen , i.e., a e 4 days o Cu exposu e a 5.9 mmol L
1
, in acellula Cu eaches
2.5 mmol kg
1
we weigh , he 410- old o i s concen a ion in he wa e . A he highe Cu
exposu e concen a ions, simila accumula ion ac o s a e ound, i.e., 280- o 510- old.
The polysaccha ide con en is aised by 32% a 5.9 mmol L
1
Cu, he le el o p o ein is
sligh ly and insigni ican ly ele a ed (p40.05) (Figu e 3). Beyond a Cu exposu e le el o
Figu e 1. P obi plo o g ow h inhibi ion (be ween days 3 and 6) o P. kessle i a di e en CuCl
2
concen a ions.
No e: Iden ical le e s indica e ha di e ences a e no signi ican ( p40.05).
Table 1. E ec s o Cu exposu e on he con en s o chlo ophyll-a, pheophy in-a, chlo ophyll-b,
on OD
664b
/OD
665a
a io, and on biomass in P. kessle i on day 7, i.e., a e 4 days o exposu e.
Exposu e
CuCl
2
(mmol L
1
)
E ec s
Chlo ophyll-a
(mg g
1
dw)
Pheophy in-a
(mg g
1
dw)
Chlo ophyll-b
(mg g
1
dw)
OD
664b
/OD
665a
a io
Biomass
(g L
1
)
0 7.6
a
1.31 0.09
a
0.01 1.92
a
0.38 1.67
a
0.01 0.52
a
0.10
5.9 6.7
a
0.73 0.13
b
0.01 1.71
a
0.23 1.63
a
0.02 0.44
ab
0.04
11.7 4.4
b
0.37 0.15
b
0.02 1.30
b
0.05 1.59
a
0.01 0.41
b
0.05
23.5 4.0
b
0.46 0.25
c
0.03 1.00
c
0.04 1.48
b
0.08 0.38
b
0.02
47 0.3
c
0.04 0.71
d
0.05 0.14
d
0.02 1.18
c
0.01 0.30
c
0.01
94 0.2
c
0.04 0.73
d
0.03 0.10
d
0.02 1.13
c
0.06 0.25
c
0.01
No e: Means s anda d de ia ions (n¼3). Iden ical le e s indica e ha he alues a e s a is ically
no di e en (p40.05); dw ¼d y weigh .
542 A.P. Nug oho and H. F ank
25
5.9 11.7 23.5 47.0 94.0
CuCl2 concen a ion (mmol L–1)
0
10
20
30
40
50
60
Cu accumula ion (mmol kg–1 ww)
410 280
290
340
510
Figu e 2. Coppe accumula ion by P. kessle i a e 4 days o CuCl
2
exposu e.
No e: En ichmen ac o s a e gi en abo e each column.
Figu e 3. Concen a ion dependences o p o ein and polysaccha ide con en s in P. kessle i a e 4
days o CuCl
2
exposu e.
No e: Iden ical le e s indica e ha di e ences in hese pa ame e s a e no signi ican ( p40.05).
Toxicological & En i onmen al Chemis y 543
26
hemocyanin (Bi ge and Black 1979) and as co ac o o he p os he ic g oups o enzymes
such as o cy och ome-c oxidase, y osinase, dopamine -hyd oxylase, alcohol dehyd o-
genase, p olyl and lysyl oxidase, o o he s in ol ed in g ow h egula ion and de elopmen
(Amia d-T ique e al. 2006; Company e al. 2008). A highe concen a ions, Cu is oxic o
mussels, esul ing in al e ed calcium (Ca) homeos asis o blood cells (Via engo e al. 1994);
he 96-hou LC
50
o mollusks anges be ween 6 and 30 mmol L
1
(0.4–2 mg L
1
)
(C omp on 1998).
Coppe can be aken up by eshwa e mussels wi h he wa e o he ood. The ou e o
up ake in luences he dis ibu ion o he me al in he a ious o gans, de e mines he
dynamics o Cu-bioaccumula ion and elimina ion, and has consequences on he
pa hophysiology o coppe in he mussels (C o eau and Luoma 2005). In his wo k,
duck mussels (Anodon a ana ina) a e used as model species o s udy he oxicological
ele ance o coppe up ake ia bo h pa hways. The s able iso ope
63
Cu is used as ace o
ollow i s dis ibu ion wi hin he mussel and i s elimina ion upon depu a ion.
Ma e ials and me hods
Algal ood p epa a ion
Algae (Pa achlo ella kessle i) a e used as ood o he mussels and, when g own a a
63
Cu-concen a ion o 5.9 mmol L
1
(Nug oho and F ank 2010), o one expe imen al
g oup as Cu-exposu e sou ce. Algae a e g own in modi ied K-medium (Kuhl and
Lo enzen 1964) o 7 days o p oduce no mal o coppe -loaded algae. F eeze-d ied no mal
and coppe -loaded algae con ain 0.01 mmol kg
1
Cu (0.6 mg kg
1
Cu) and 40 mmol kg
1
Cu (2.4 mg kg
1
Cu) d y weigh (dw).
Iso opic Cu s ock solu ion p epa a ion and labwa e
A
63
Cu s ock solu ion (3.1 mmol L
1
, equi alen o 200 mg L
1
) is p epa ed by dissol ing
25 mg iso opically en iched (99%)
63
Cu oxide (Eu iso- op, Saa b u
¨cken, Ge many) in
1 mL sup apu HNO
3
(69%, Ca l Ro h, Ka ls uhe, Ge many) in a 100 mL glass beake ;
85 mL bidis illed wa e a e added, and he pH o he solu ion is adjus ed o 7.0 wi h
aqueous ammonia (25%, VWR, Da ms ad , Ge many). The solu ion is ans e ed o a
100 mL polyp opylene (PP) olume ic lask which is illed o he ma k wi h bidis illed
wa e . Glasswa e and plas ic equipmen s used o analy ical pu poses a e insed wice wi h
hal -concen a ed HNO
3
(65%; Sigma-Ald ich, Munich, Ge many), and deionized and
bidis illed wa e .
O ganisms
Abou 70 duck mussels (A. ana ina) (ZOO-E lebnis Online Shop, G osse ehn, Ge many)
wi h shell leng hs o 10–12 cm and weigh s o 100–200 g a e b ough o he labo a o y in
pond wa e . The mussels a e b ushed wi h dilu e KMnO
4
solu ion (0.1 mg L
1
), insed wi h
ap wa e , and placed in 38 L ae a ed ap wa e in 45 -L glass aqua ia a dim ligh o 7 days.
Du ing his pe iod hey a e no ed; e e y day, hal o he wa e is exchanged. Then he
mussels a e ma ked, weighed, and he shell leng hs a e measu ed. They a e ed wi h eeze-
d ied Cu- ee algae, 1.0 mg L
1
pe day, and acclima ized o u he 7 days o labo a o y
condi ions a a empe a u e o 17 1C wi h a pho ope iod o 12 h ligh pe day, a pho on
lux o 13–19 mmol m
2
s
1
, in 38 L a i icial pond wa e (APW) a pH 7.0 0.3
Toxicological & En i onmen al Chemis y 1839
33
(Ngo, Ge s mann, and F ank 2011) in 45 -L glass aqua ia co e ed wi h anspa en
polyp opylene lids. The aqua ia a e equipped wi h inne bio- il e s and s ainless s eel
ae a ion ubes. Eigh kilog ams glass beads a e used as subs a e. Two- hi d o he wa e is
exchanged e e y wo days; a comple e change is conduc ed on e e y six h day.
Expe imen al design
O hese mussels, 63 a e selec ed o ma ch in size and di ided in o h ee g oups consis ing
o 21 mussels each. They a e placed in h ee 45 L aqua ia con aining 38 L a i icial pond
wa e (APW). Two- hi d o he wa e is exchanged e e y second day; a comple e change is
conduc ed on e e y six h days. A con ol g oup (1) is kep in APW. Ano he g oup (2) is
exposed o 0.3 mmol L
1
(20 mgL
1
)
63
Cu in he wa e using he
63
Cu s ock solu ion; a e
each wa e change, he concen a ion is e-adjus ed by adding app op ia e olumes o he
s ock solu ion. A hi d g oup (3) ecei es daily 1.5 mg L
1
eeze-d ied
63
Cu-loaded algae
o 24 days, equi alen o a nominal coppe concen a ion o 0.06 mmol L
1
(3.6 mgL
1
).
The mussels in he con ol and he exposu e g oups a e ed wi h algae in amoun s
adjus ed o hei ac ual numbe . Fo 18 mussels, 1.5 mg L
1
o eeze-d ied Cu- ee
(g oups 1 and 2) o
63
Cu-loaded algae (g oup 3) a e gi en pe day. When he numbe o
mussels is less han 18, 1.0 mg L
1
o eeze-d ied Cu- ee o
63
Cu-loaded algae a e gi en
daily (Ngo, Ge s mann, and F ank 2011) co esponding o a nominal concen a ion o
0.04 mmol L
1
(g oup 3). On day 24, he six mussels emaining in each g oup a e
ans e ed o APW- illed aqua ia o 12 days o depu a ion, ed wi h 1.0 mg L
1
o eeze-
d ied Cu- ee algae pe day.
Ac ual Cu concen a ions in he wa e including he suspended algae in each g oup a e
de e mined e e y second day. On he con ol g oup, Cu concen a ions in he APW du ing
expe imen a e below de ec ion limi . Fo he expe imen in ol ing Cu exposu e ia he
wa e , a e exchange o wa e he concen a ion is adjus ed o 0.32 0.006 mmol L
1
,
which alls o 0.03 0.01 mmol L
1
wi hin he nex 2 days. By he ood pa hway (g oup 3),
he nominal Cu concen a ion in he beginning and a e each wa e exchange is
0.07 0.01 mmol L
1
, alling o below de ec ion limi wi hin he nex 2 days.
Fo sampling, h ee mussels o each g oup a e aken o analysis a days 0, 6, 12, 18,
and 24 (exposu e), and a days 30 and 36 (depu a ion). The mussels a e anaes he ized wi h
an aqueous 2-phenoxye hanol solu ion (4 mL L
1
) o 30 min. Hemolymph (HML) and
ex apallial luid (EPF) a e wi hd awn using 5 mL sy inges wi h 0.55 25 mm needles
(B. B aun, Melsungen, Ge many), ans e ed in o 2-mL mic o ubes, and kep a 80C.
The mussels a e dissec ed on ice in o gills, man le, kidney, diges i e gland, oo , adduc o s,
and in es ines; he emainde is collec ed in a combined sample (GHL), i.e., gonads, hea ,
and labial palps. The issues a e washed wice wi h bidis illed wa e , d ied using il e
pape , placed in 15 mL polyp opylene (PP) ubes o known weigh s, weighed o ob ain he
we weigh s (ww), and lyophilized. A e lyophiliza ion, he ubes a e weighed again
o d y weigh s (dw). Tissue ac ions and body luids o he nine mussels aken a
day 0 a e used o calcula e he espec i e pe cen ages ela i e o he o al weigh o
so body ( wsb).
Me al analyses
Each lyophilized issue ac ion o abou 10–100 mg is placed in a 55 mL bo osilica e
glass ubes. 5 mL o a mix u e (4 þ1) o sup apu e concen a ed HNO
3
1840 A.P. Nug oho and H. F ank
34
(65%, Me ck, Da ms ad , Ge many) and sup apu e concen a ed HCl (30%, Me ck,
Da ms ad , Ge many) a e added o each ube. The ubes a e kep in an o en a 40C o
1 h and a 95C o 3 h. The diges ed samples a e dilu ed wi h bidis illed wa e o 10 mL
and il e ed h ough 0.45 mm cellulose sy inge il e s (Ca l Ro h, Ka ls uhe, Ge many).
Fo he de e mina ion o Cu in HML and EPF, 0.4–1 mL o each a e acidi ied wi h 0.5 mL
sup apu e concen a ed HNO
3
in PP ubes, dilu ed o 10 mL wi h bidis illed wa e , and
il e ed h ough 0.45 mm cellulose sy inge il e s. To al Cu and i s iso opes
63
Cu and
65
Cu
a e de e mined by induc i ely-coupled plasma mass spec ome y (ICP-MS, Agilen
7500ce, Ce ac ASX-510, Agilen Technologies, Waldb onn, Ge many). The de ec ion
limi s o o al Cu is 0.02 mmol L
1
o iso opic Cu 0.01 mmol L
1
.
To al coppe in each issue ac ion is calcula ed in mmol kg
1
ww by mul iplying he
analy ical da a wi h he a io o ww e sus dw. The concen a ion o exogenous coppe
C
63Cu
is calcula ed as C
63Cu
– 2.34 C
65Cu
, he concen a ion o endogenous coppe as
3.33 C
65Cu
, conside ing he na u al ela i e abundances o 69%
63
Cu and 31%
65
Cu. Fo
body luids, he Cu concen a ions a e gi en in mmol L
1
. To al and exogenous Cu-pools
in he issue ac ions and body luids a e calcula ed in mmol kg
1
wsb by mul iplying he
concen a ion da a wi h he weigh ac ion o he espec i e o gan o body luid.
S a is ical da a analyses
Da a a e ans o med o log uni s be o e s a is ical analysis o homogenei y o a iance
and no mali y. The da a o o al Cu a e s a is ically e alua ed by wo-way analysis o
a iance (ANOVA) conside ing exposu e ime and Cu exposu e pa hways as independen
a iables; i signi ican di e ences a e ound, hose be ween exposu e imes a e es ed by
he Dunne mul iple compa ison es s, be ween exposu e pa hways and con ols using he
Duncan mul iple compa ison es s. To assess he di e ences in exogenous Cu be ween
exposu e pa hways, he independen - es is pe o med.
Resul s
Exposu e o A. ana ina o Cu ia he wa e esul s in apid inc eases (Figu e 1) o he
concen a ions o o al Cu (solid lines) in he hemolymph (HML) and he ex apallial luid
(EPF) wi hin he i s 12 days, ollowed by slowe inc eases un il concen a ions o
0.38 mmol L
1
a e eached a day 24, abou he 2.5- old o con ol le el. F om he ood,
inc eases a e mo e mode a e, eaching abou 0.25 mmol L
1
, he 1.7- old o con ols. In
espec o exogenous Cu, he concen a ions in bo h body luids (do ed lines) inc ease
simila ly upon exposu e ia he wa e and he ood wi hin he i s 6 days al hough he
nominal concen a ion pe li e wa e olume in ood is conside ably lowe . La e on,
exposu e ia he wa e en ails as e up ake, especially in he HML be ween days 6–12 o
each 0.14 mmol L
1
, con inuing un il 0.17 mmol L
1
a day 24. O e all, inc eases du ing
he i s days a e as e o he EPF han o he HML.
Wi hin he 12 days o depu a ion, o al Cu concen a ions decline apidly in EPF and
in HML, in HML o animals ha ing ecei ed he me al by he wa e pa hway o abou
50% o e con ol; when ha ing been exposed ia he ood, he Cu concen a ions decline
almos ully back o con ol alues. Fo exogenous Cu, he concen a ions in he HML
and EPF o wa e - and ood-exposed animals decline in simila ela i e a es. A he end o
he depu a ion, he ac ion o exogenous Cu, i.e., he excess o
63
Cu o e he na u al
Toxicological & En i onmen al Chemis y 1841
35
abundance o his iso ope, ep esen s be ween 10% ( ood pa hway) and up o 30% (wa e
pa hway) o o al coppe .
In he o gan and issue ac ions (Figu es 2 and 3), concen a ions o endogenous Cu a
day 0 a e highes in he kidney and he diges i e gland (63 and 58 mmol kg
1
ww). In he
o he o gans, ini ial Cu le els a e much lowe , i.e., in man le, in es ines (bo h 15 mmol kg
1
ww), gills, and oo (bo h 12 mmol kg
1
ww). The mixed ac ion o he gonads, hea , and
labial palps (GHL) (20 mmol kg
1
ww) shows a ai ly high ini ial coppe le el al hough
no hing can be said abou he dis ibu ion be ween he issues con ained in i . The
adduc o s ha e he lowes concen a ion (7 mmol kg
1
ww), bu his is s ill much highe
han in HML and EPF (Figu e 1, 0.17 mmol L
1
).
The de elopmen o he o al coppe concen a ions is qui e di e se o he a ious
issues/o gans o e ime (Figu es 2 and 3; solid lines) in ela i e and absolu e e ms. Upon
up ake ia he wa e , s onges ela i e inc eases a e seen o he gills, he man le, and he
diges i e gland, especially wi hin he i s 6 days. When
63
Cu is adminis e ed ia he ood,
an almos equal inc ease o
63
Cu as ia wa e is ound o he diges i e gland, al hough i s
nominal ini ial concen a ion is only a i h o he concen a ion in he wa e in dissol ed
o m. Fo o he o gans, up ake om ood leads o mode a e ise in he man le, kidney,
in es ines, and GHL, almos none in he gills, adduc o s, and oo . In he diges i e gland,
highes concen a ions, i.e., 120–140 mmol kg
1
, a e eached wi hin 24 days i espec i e o
exposu e pa hway. Fo o he o gans, exposu e ia wa e esul s in peak concen a ions in
he gills o 75 mmol kg
1
(6.5 old ela i e o con ol), 70 mmol kg
1
in he man le
(4.2- old), and 70 mmol kg
1
in he mixed ac ion GHL (3.5– old); mode a e o low
ela i e inc eases a e seen in he oo , in es ines, adduc o s, and kidney (2.8-, 1.7-, 1.6-, and
1.4- old). Upon depu a ion, Cu concen a ions all immedia ely and s ongly in mos
Figu e 1. Concen a ions o o al (solid lines; ˙¼ ia wa e , #¼ ia ood; N¼con ol) and
exogenous (do ed lines; ¼ ia wa e , h¼ ia ood) Cu in hemolymph (HML) and ex apallial luid
(EPF) o A. ana ina du ing exposu e (days (d) 0–24) and depu a ion (days 24–36). Signi ican
di e ences in compa ison o con ol wi hin each g oup a e indica ed by
o
. The same le e s indica e
ha di e ences o Cu concen a ions a e no signi ican among g oups a each ime sampling (day)
while he di e en le e indica e p50.05. Signi ican di e ences be ween concen a ions o
exogenous Cu ia ood o wa e a e indica ed by
þ
.
1842 A.P. Nug oho and H. F ank
36
o gans, excep o he man le and he in es ines; o hese e en u he inc eases a e
obse ed wi hin he i s 6 days o depu a ion.
In espec o exogenous
63
Cu (Figu es 2 and 3; do ed lines), exposu e o
63
Cu ia wa e
leads o apid inc eases in he gills, man le, diges i e gland, and GHL wi hin he i s 6
days. In some o gans, i.e., diges i e gland, gills, and man le, he inc eases con inue un il
day 24 o each a maxima o abou 50 mmol kg
1
ww. Exogenous coppe in he kidney,
Figu e 2. Concen a ions o o al (solid lines; ˙¼ ia wa e , #¼ ia ood; N¼con ol) and
exogenous (do ed lines; ¼ ia wa e , h¼ ia ood) Cu in he gills, man le, diges i e gland, and
kidney o A. ana ina du ing Cu exposu e ia wa e and ood and du ing depu a ion. Signi ican
di e ences in compa ison o con ol wi hin each g oup a e indica ed by
o
. The same le e s indica e
ha di e ences o Cu concen a ions a e no signi ican among g oups a each ime sampling
(day (d)) while he di e en le e indica e p50.05. Signi ican di e ences be ween concen a ions o
exogenous Cu ia ood o wa e a e indica ed by
þ
. To al and exogenous Cu a e calcula ed by
mul iplica ion o he analy ical da a wi h he a io o d y weigh e sus we weigh .
Toxicological & En i onmen al Chemis y 1843
37
oo , in es ines, and GHL shows maximum concen a ions a day 12, ollowed by declines
un il he end o exposu e. Via he ood, exogenous
63
Cu ini ially inc eases in he diges i e
gland as as as ia he wa e , ollowed by sligh u he inc ease o each a maximum o
20 mmol kg
1
ww on day 24. In he gills, man le, adduc o s, and oo , a e sligh inc eases
du ing he i s 12 days o exposu e, exogenous Cu emains ela i ely unchanged un il he
end o he expe imen .
Du ing depu a ion, in he gills, man le, and diges i e gland, he le els o exogenous
coppe d ops wi hin he i s 6 days by 85, 70, and 60%. Fo animals ha ing ecei ed he
me al ia ood, simila pa e ns o dec ease a e obse ed o he kidney and GHL, only he
le els being lowe , i.e. abou a hi d.
The iso ope a ios o
63
Cu/
65
Cu and i s de ia ion om he na u al a io (2.33) a e also
moni o ed (Figu e 4). Complemen a y o Figu es 2 and 3, his allows o ollow he
mo emen o exogenous Cu wi hin he body. When
63
Cu is adminis e ed ia wa e , he
ela i e abundance o
63
Cu in he body luids HML and EPF ise up o 4.0 a day 12, hen
emain cons an . Upon depu a ion, he a io declines o abou 3.0. In he kidney, GHL,
oo , and in es ines, peaks o
63
Cu a e eached a day 12, while in he adduc o s, diges i e
gland, man le, and gills, maximum iso ope a ios a e ound a he end o exposu e a day
24. Du ing depu a ion, he ela i e abundance o
63
Cu in all o gans declines, bu no
Figu e 3. Concen a ions o o al (solid lines; ˙¼ ia wa e , #¼ ia ood; N¼con ol) and
exogenous (do ed lines; ¼ ia wa e , h¼ ia ood) Cu in he GHL (gonads, hea , and labial
palps), in es ines, oo , and adduc o s o A. ana ina du ing Cu exposu e ia wa e and ood and
du ing depu a ion. Signi ican di e ences in compa ison o con ol wi hin each g oup a e indica ed
by
o
. The same le e s indica e ha di e ences o Cu concen a ions a e no signi ican among
g oups a each ime sampling (day (d)) while he di e en le e indica e P50.05. Signi ican
di e ences be ween concen a ions o exogenous Cu ia ood o wa e a e indica ed by
þ
. To al and
exogenous Cu a e calcula ed by mul iplica ion o he analy ical da a wi h he a io o d y weigh
e sus we weigh .
1844 A.P. Nug oho and H. F ank
38
o ally back o he na u al a io emaining abou 25–45% highe han be o e exposu e.
When
63
Cu is adminis e ed ia ood, he inc ease in he iso ope a io is p onounced o he
diges i e gland, while all he o gans show only small inc eases.
Calcula ing he Cu-pools in he body compa men s gi es in e es ing insigh s (Figu e 5).
Al hough HML and EPF oge he cons i u e abou 70% (33 5% and 37 4%) o he o al
so body olume (Figu e 5, A), bo h a e insigni ican as Cu-pools. The man le, he gills, and
he in es ines a e he la ges solid o gans; oge he hey ep esen abou 18% (6.4 0.5,
5.9 0.6, and 5.4 0.8%) wsb. Smalle body ac ions a e he adduc o s (2.9 0.2% wsb),
he diges i e gland (2.8 0.3% wsb), he oo (2.2 0.4% wsb), he mixed ac ion GHL
(3.9 0.6% wsb), and he kidney (0.5 0.06% wsb). In he beginning (Figu e 5, B), he
o al pool o Cu (endogenous Cu) is abou 6 mmol kg
1
wsb, he la ges being in he
diges i e gland, ollowed by he man le, gills, in es ines, and GHL (Figu e 5, B). Upon
exposu e ia wa e (W), he o al Cu-pools inc eases, con inuing un il day 24 o each a
maximum o 25 mmol kg
1
wsb, i.e., he ou - old o he ini ial pool size; up ake ia he ood
(F) en ails a o al Cu-pool o only 9 mmol kg
1
wsb a day 24, i.e., sligh ly less han double
he con ol, he la ges pool being in he diges i e gland. The exogenous
63
Cu-pool inc eases
in pa allel o o al Cu-pool upon exposu e ia he wa e , eaching a maximum o abou
12 mmol kg
1
wsb a day 24. Fo he ood pa hway, i inc eases only sligh ly, he maximum
le el being a abou 1.5 mmol kg
1
wsb (Figu e 5, C). Du ing he 12 days o depu a ion, all
he pools a e apidly emp ied, pa icula ly he gills. The man le and he diges i e gland
e ain he Cu-pools ela i ely long (as also e lec ed in Figu es 2 and 3), in he la e mos
enaciously. Simila pa e ns a e ound o exogenous Cu.
Discussion
The expe imen s show ha
63
Cu is highly a ailable o A. ana ina (Figu es 2 and 3, a and
b), bo h in wa e -dissol ed o m o om
63
Cu-loaded algae. Calcula ion o Cu specia ion
Figu e 4. Iso ope a io o
63
Cu/
65
Cu in o gans and body luids o A. ana ina du ing exposu e o
63
Cu ia wa e (˙) o ood (#), and ollowing depu a ion (GHL ¼gonads, hea , and labial palps).
The ela i e inc eases o
63
Cu in pe cen o e he na u al a io upon exposu e ia wa e a e gi en
o day (d) 36.
Toxicological & En i onmen al Chemis y 1845
39
in a i icial pond wa e (APW) a a pH o 7.0 and a 17C shows ha he me al is
comple ely in he ee Cu
2þ
ionic o m, eady o up ake (Gus a sson 2010). By he ood
pa hway, he low nominal Cu concen a ion in he APW may be he main ac o
esponsible o he low Cu accumula ion in he mussel in absolu e e ms, bu in ela i e
e ms i is ob iously e en mo e e icien .
Du ing he 24 days o wa e -bo ne Cu exposu e, exogenous
63
Cu le els in he o gans
inc ease di e en ly, s onges in he gills o ep esen abou 70% o o al Cu (Figu es 2
and 3, a and b). The e is e idence o mobiliza ion and e-dis ibu ion o endogenous Cu
among he o gans de i ed om he ime pa e n o he
63
Cu/
65
Cu iso ope a io (Figu e 4).
The a ios a e highes in he gills, man le, and diges i e gland (bo h pa hways) a he six h
day o exposu e, showing ha he exogenous
63
Cu is ini ially aken up in o hese o gans.
The peaks o highes a ios a day 12 in he kidney, GHL, oo , and in es ines indica e ha
hese o gans i s ecei e ai ly high amoun s o exogenous Cu bu – as he exposu e
con inues – become ecipien s o endogenous coppe mobilized om he o he o gans,
p esumably mobilized by exogenous
63
Cu. La e , exogenous and mobilized endogenous
Cu is mainly s o ed in he diges i e gland, gills, adduc o s, and man le, he la e se ing as
ansien ecipien e en beyond he exposu e phase. The obse a ion o coppe being
pa icula ly s ongly e ained in he man le is no ewo hy as i is one o he mos impo an
o gans o egula ing he calcium household and o building he p o ec i e shell o he
bi al e (Lopes-Lima e al. 2008).
In he body luids, he iso ope a io emains ela i ely cons an a abou 4.0 du ing
days 12–24, e lec ing he oles o HML and EPF as ansi o y exchange and anspo
compa men s, being small as pools (Figu e 5). Upon depu a ion he iso ope a ios end o
all s ongly, indica ing ha a la ge ac ion o exogenous
63
Cu emains in a ela i ely
easily exchangeable o m while he endogenous Cu is mo e enaciously e ained.
Figu e 5. A: Pe cen ages o o al weigh o so body ( wsb) (EPF ¼ex apallial luid;
HML ¼hemolymph; GHL ¼gonads, hea , and labial palps), and B: o al and C: exogenous
Cu-pools ( igh o dina e) in A. ana ina du ing Cu exposu e ia ood (F) o wa e (W) and du ing
depu a ion. Signi ican di e ences in compa ison o con ol (day (d) 0) wi hin each g oup (wa e ¼
o
;
ood ¼*), and be ween Cu exposu e ia ood and wa e a e indica ed by
þ
. The o al Cu-pools in he
body ac ions a e calcula ed by mul iplica ion o he concen a ion da a (Figu e 2) wi h he
espec i e pe cen ages.
1846 A.P. Nug oho and H. F ank
40
Ne e heless, a he end o he depu a ion, he ela i e abundance o
63
Cu aken up ia he
wa e pa hway is be ween 25% and 45% highe han in he beginning in a ious body
compa men s, indica ing ha abou a hi d o he unc ional Cu pool has been exchanged
o exogenous
63
Cu.
Dis ibu ion o Cu in he mussel’s body allows o assess he ela i e impo ance o he
a ious coppe pools (Figu e 6). F om he wa e i is mainly compa men alized in o he
man le (30%), he gills (24%), and he diges i e gland (22%), al oge he h ee qua e s o
he o al Cu-pool. The o me wo o gans ha e la ge su ace a eas and in e ac di ec ly
wi h he wa e coming in o man le ca i y du ing il a ion (Ma igo
´mez e al. 2002); he
diges i e gland is he majo ecei ing o gan o he hemolymph pa hway. The man le has a
high sec e o epi helium lined wi h acid mucopolysaccha ides o diges ion o apped
small pa icles (Machado 2011). By he ood pa hway, he diges i e gland and he
in es ines a e he majo Cu- ecipien s. In addi ion, he o me o gan sec e es high amoun
o diges i e mucus o acili a e Cu s o age (Machado 2011). High Cu le els in GHL
sugges a ole o he hea as ion ecipien and i s close ana omical ela ion o he in es ines
and he kidney (Gosling 2003; Machado 2011).
Figu e 6. Scheme illus a ing he dis ibu ion o he o al Cu bu den (Cu-pools) among he body
ac ions (EPF ¼ex apallial luid; HML ¼hemolymph; GHL ¼gonads, hea , and labial palps;
LP ¼labial palps; F ¼ eces; U ¼u ine) o A. ana ina a e exposu e o Cu ia wa e (a. black a ow
( ou es o he hea ) and g ey a ow ( ou es om he hea o he o he o gans) o ood (b. whi e
a ow ( ou es o he hea ) and g ey/hea y-lined ( ou es om he hea o he o he o gans)) (pe kg
o o al weigh o so body). Whi e/hea y-lined a ows show he ou es o bo h Cu exposu e
pa hways. The igu e is adop ed om Ma igo
´mez e al. (2002).
Toxicological & En i onmen al Chemis y 1847
41
In ela ion o he o gan pools o A. ana ina, he size o he espec i e olumes is no
di ec ly ela ed o Cu bu den (Figu e 5, A, B, and C). Binding o speci ic compounds and
compa men aliza ion wi hin he o gans, and physiological and me abolic unc ions o he
o gans may play some oles (O che e 2003). In any case, he o gans which se e as
he p ima y si es o up ake, i.e., gills, man le, and diges i e gland, end o concen a e he
coppe .
Du ing depu a ion, Cu is elimina ed ai ly as om he body (Figu es 2 and 3, a and b),
due o he la ge di e ences in g adien Cu concen a ion be ween he mussel and APW. Han
e al. (1993) epo ed ha he ini ial apid elimina ion can be caused by deso p ion o loosely
bound, unassimila ed coppe , whe eas slowe elimina ion e lec s he loss om pools
(endogenous Cu) whe e coppe is mo e igh ly bound o issue componen s. Rapid
elimina ion is also obse ed in he gills and diges i e gland o he ma ine clam R. decussa us
wi hin he i s 10 days o depu a ion (Se a im and Bebianno 2009). In espec o Cu
elimina ion om he diges i e gland, he le el in A. ana ina d ops o abou 60% o e con ol
wi hin 6 days (Figu es 2 and 3, a). A simila pa e n is obse ed in he ma ine mussels
My ilus gallop o incialis exposed o Cu a 0.63 mmol L
1
(40 mgL
1
) ia wa e o 3 days
(Via engo e al. 1981). This con i ms ha he diges i e gland is he main o gan o me al
elimina ion in bi al es (Ma igo
´mez e al. 2002). Acco ding o Ma igo
´mez e al. (2002), he
elease o me als om mussel body can occu ia he diges i e ac as a componen o eces
o ia he kidney oge he wi h exc e o y conc e ions as a componen o u ine (Figu e 6).
Coppe accumula ion in A. ana ina du ing exposu e ia wa e o ood ep esen s wo
di e en p ocesses, i.e., bioconcen a ion (wa e ) and biomagni ica ion ( ood). Calcula ion
o he bioconcen a ion and biomagni ica ion le els allows o assess he ela i e impo ance
o exposu e ia wa e o ood. Bioconcen a ion can be exp essed as en ichmen ac o (EF),
i.e., he a io o he concen a ion o exogenous Cu kg
1
wsb (Figu e 5, C) o he
concen a ion in he wa e . Biomagni ica ion is no mally assessed as ans e ac o (TF).
The en ichmen ac o in he mussel a he end o he exposu e (day 24) is abou 43, by he
ood pa hway a TF o 25 is eached (Cu concen a ion in he APW-added algal ood is
equi alen o 0.06 mmol L
1
, exogenous Cu-pools in he mussel ¼1.5 mmol kg
1
wsb;
Figu e 5, C). Thus, exposu e ia wa e is mo e e ec i e om his poin o iew. In espec o
biomagni ica ion, he TF is lowe han he EF o algae which is abou 400- old (Nug oho
and F ank 2010), indica ing only weak biomagni ica ion o coppe along he ood chain
om he algae o he mussel. O e all, dis ibu ion and accumula ion o coppe in A. ana ina
a e he esul s o exposu e ime, exposu e pa hways, and physiological unc ions o he
espec i e o gans. Food up ake is mo e e icien aking he i e- old lowe nominal
concen a ion o coppe in hese expe imen s in o conside a ion.
These expe imen s will help unde s and he isks associa ed wi h coppe exposu e o
eshwa e mussels. Coppe accumula ion may p omo e he si ua ion o me abolic acidosis
leading o he dissolu ion o CaCO
3
deposi s, inducing he inc ease o Ca concen a ion in
he EPF (An unes e al. 2002; Faubel e al. 2008; Lopes-Lima e al. 2008). In e e ence
wi h Ca homeos asis by he inhibi ion o Ca-ATPase by Cu (San ini e al. 2011) may lead
o physiological s ess. These ac o s oge he wi h he in ol emen o coppe in he
o ma ion o eac i e oxygen species (Company e al. 2008) may be a con ibu o y ac o
in he o e all Eu ope-wide obse ed decline o eshwa e bi al es.
Conclusions
Exposu e o A. ana ina o Cu ia he wa e o ia he ood leads o en ichmen o he
ansi ion me al in he mussel. Coppe is mainly s o ed in he diges i e gland, gills,
1848 A.P. Nug oho and H. F ank
42
inc eases we e mo e mode a e due o he lowe Cu-in ake, i.e., abou he 1.2- old o
con ol a day 24. Ca le els in HML and EPF we e co ela ed o o al Cu concen a ion ( -
ood (HML) ¼0.85, -wa e (HML) ¼0.90, - ood (EPF) ¼0.61, -wa e (EPF) ¼0.73;
p50.05). Upon depu a ion, Ca concen a ions in he body luids declined as , e u ning
o con ol alues du ing he i s six days al hough Cu was s ill ele a ed.
In he o he o gans and issues (Figu e 2), highes Ca concen a ions a day 0 we e
ound in he man le (90 mmol kg
1
ww), he gills, he diges i e gland, he mixed ac ion
con aining gonads, hea , and labial palps (GHL) (all abou 80 mmol kg
1
ww), and in he
in es ines (70 mmol kg
1
ww); much lowe we e he Ca-le els in he kidney, he adduc o s
(bo h 20 mmol kg
1
ww), and he oo (10 mmol kg
1
ww). Upon Cu exposu e ia wa e o
ood, Ca le els inc eased in all o gans excep o he adduc o s. Highes Ca le els we e
ound in he gills, man le, and diges i e gland upon exposu e ia he wa e , eaching abou
140–160 mmol kg
1
ww (2- old con ol) a day 24, highes ela i e inc ease being ound in
he kidney (4- old, 80 mmol kg
1
ww). Fo GHL and he in es ines, maximum Ca le els
we e a 110 (1.3- old o con ol) and 100 (1.4- old) mmol kg
1
ww, espec i ely. In he
oo , Ca eached abou he 4- old (40 mmol kg
1
ww) o con ol a day 24 upon Cu-
exposu e ia he wa e . When Cu-exposu e ook place ia he ood wi h i s nominally
lowe Cu-le els pe li e APW, Ca concen a ions in he diges i e gland, he in es ines, he
Figu e 2. Concen a ions o Ca ( igh o dina e, do ed lines; S¼upon exposu e ia wa e ,
h¼upon exposu e ia ood, D¼con ol) and Cu (le o dina e, solid lines; ^¼ ia wa e , #¼ ia
ood; N¼con ol) in he a ious o gans o A. ana ina du ing Cu exposu e (E) and depu a ion (D)
(GHL ¼gonads/hea /labial palps). Signi ican di e ences in compa ison o con ol wi hin each
g oup a e indica ed by
o
. Simila le e s indica e ha di e ences o Ca concen a ions a e no
signi ican among g oups a each ime sampling (day, d) while di e en le e s indica e p50.05.
Concen a ion o Ca is calcula ed by mul iplica ion o he analy ical da a wi h he a io o d y weigh
e sus we weigh . Concen a ions o coppe a e he same as in Nug oho and F ank (2011b).
Toxicological & En i onmen al Chemis y 103
49
kidney, and he GHL inc eased almos equally as in he animals ha ing ecei ed Cu ia he
wa e . In he adduc o s, Ca le els emained almos unchanged, as also ound o Cu. In he
gills, man le, diges i e gland, and kidney upon exposu e ia he wa e o he ood, Ca
concen a ions we e s ongly and posi i ely co ela ed o he Cu le els in he espec i e
o gans ( 40.7; p50.05).
Upon depu a ion, Ca le els declined slowly, excep o he kidney wi h as elimina ion
o excessi e Ca. Upon he 12 days o depu a ion, Ca in he kidney, adduc o s, in es ines,
and GHL e u ned almos ully back o con ol alues while in he gills, man le, diges i e
gland, and oo he Ca le els declined o abou 20–70% abo e con ol alues.
Soluble ca bohyd a es and p o eins in all o gans we e lowe ed upon Cu exposu e and
in pa allel o i s concen a ions (Figu e 3), s onges e ec s being obse ed when Cu was
supplied ia he wa e . Ca bohyd a e le els dec eased d as ically un il he end o exposu e
wi h wa e -dissol ed Cu, i.e. by 80% (gills) and 70% (kidney). Exposu e o ood-con ained
Cu had mode a e e ec s excep o he diges i e gland and he kidney; hese wo o gans
showed only li le di e ences be ween he wo exposu e pa hways. Fo all o he solid
o gans (no shown in Figu e 3), soluble ca bohyd a es we e dec eased by 5–10%.
Ca bohyd a es in HML and EPF a day 24 we e lowe ed by only 5–10% upon Cu
exposu e ia he ood, he e ec s again being sligh ly s onge when Cu was aken up om
he wa e (8–12%). Co ela ion analyses con i med s ong nega i e ela ionships be ween
Cu and ca bohyd a e ( 40.6; p50.05) in he gills, man le (wa e pa hway), diges i e
gland, and kidney (bo h pa hways).
Figu e 3. Con en s o soluble ca bohyd a es and p o eins (N¼con ol; #¼ ia ood; ^¼ ia wa e )
in he gills, man le, diges i e gland, and kidney o A. ana ina du ing Cu exposu e (E) and
depu a ion (D). Signi ican di e ences in compa ison o con ol wi hin each g oup a e indica ed
by
o
. Simila le e s indica e ha di e ences o Ca concen a ions a e no signi ican among g oups
a each ime sampling (day, d) while di e en le e s indica e p50.05.
104 A.P. Nug oho and H. F ank
50
Soluble p o eins in he gills, man le, diges i e gland, and kidney declined o be ween
20% and 45% o con ol le els a day 24. Fo he diges i e gland and he kidney, he
e ec s we e almos equal o bo h exposu e pa hways while o he gills and he man le
g ea di e ences we e ound. Soluble p o ein le els in all o he issue compa men s we e
dec eased by no mo e han 5–10% (no shown in Figu e 3). In he HML and EPF, le els
o soluble p o eins a day 24 we e lowe ed by only 5–10% (HML: 550 40 mg L
1
; EPF:
390 40 mg L
1
;n¼3) o bo h pa hways. Signi ican ela ionships o Cu and Ca wi h
soluble p o ein le els ( 40.6; p50.05) exis ed in he gills, man le (wa e pa hway),
diges i e gland, and kidney (bo h pa hways). Upon depu a ion, soluble ca bohyd a e and
p o ein le els in he s udied o gans s a ed o inc ease, al hough no ully back o con ol
wi hin he 12 days.
Discussion
Exposu e o A. ana ina o coppe ia wa e o ood causes inc eases o Ca le els in all body
compa men s, by he wa e pa hway being s onge han ia he ood pa hway.
Ne e heless, when conside ing he nominally i e- old lowe Cu concen a ion con ib-
u ed by he algal suspension in he APW, he e ec s o he la e exposu e pa hway on he
diges i e gland and he kidney a e su p isingly s ong. Inc ease o Ca in all o gans
(Figu e 2) sugges s ha Cu no only a ec s he enzymes egula ing he in e nal Ca balance
bu also he o e all Ca bu den is s ongly inc eased; such a looding o he o ganism wi h
excess Ca, mainly o he gills, man le, and diges i e gland, can only esul om
mobiliza ion o CaCO
3
om he shell, mos likely due o Cu-induced me abolic acidosis
(An unes e al. 2002; Faubel e al. 2008; Lopes-Lima e al. 2008). This is con i med by he
inc ease o Ca in he EPF be ween days 6 and 24. In e e ence o Cu wi h Ca homeos asis
by a ec ing he mechanisms o Ca ex usion ac oss cellula memb anes may be ano he
complica ion (Via engo e al. 1994; Via engo, Bu lando, and Bolognesi 2002; Pa naik,
Chainy, and Jena 2007). Inc ease o Ca in he HML has also been epo ed by Via engo
(1994) o exposu e o My ilus edulis o Cu a 0.5–2 mmol L
1
.
A s ong dec ease o ca bohyd a e le els in A. ana ina upon Cu exposu e indica es ha
his is ano he sensi i e oxicological endpoin associa ed wi h he dis u bance o Ca
homeos asis. A simila pa e n is ound in he gills and man le o he eshwa e mussel
Lamellindens ma ginalis unde coppe s ess a 2 mmol L
1
(133 mgL
1
) o 3 days. This has
been a ibu ed o cell hypoxia (Sa yapa ameshwa , Reddy, and Kuma 2006) leading o
inc eased ac i i ies o glycoly ic enzymes in ol ed in anae obic ATP p oduc ion (Ma ı´nez
e al. 2006). Fu he dec ease obse ed be ween days 12 and 24 (Figu e 3) sugges s ha a
lowe ca bohyd a e le els gluconeogenesis is s imula ed.
S ong co ela ion be ween inc eased Ca and low p o ein le els illus a e he key ole o
he elec oly e as in acellula signaling ac o . Acco ding o Via engo e al. (1994),
ele a ed cy osolic Ca le els ac i a ed p o ein deg ada ion. Dec ease in p o ein le els was
also ound in he eshwa e mussel Anodon a woodiana a e exposu e o Cu a
0.9 mmol L
1
(0.06 mg L
1
) o 4 weeks (Ku nia, Pu wan o, and Mahajoeno 2010). Simila
o ou indings, only mino e ec s on he soluble p o ein le els in HML and EPF we e
obse ed wi h he eshwa e mussel Anodon a cygnea upon exposu e o CuSO
4
a
10
6
mol L
1
o 1 mon h (Mou a, Vila inho, and Machado 2000).
O e all, Cu exposu e a en i onmen ally ele an le els leads o inc eased Ca le els in
all body compa men s o A. ana ina, indica ing ha Cu in e e es wi h Ca homeos asis.
Dissolu ion o Ca om he shell upon Cu-induced me abolic acidosis (An unes e al. 2002;
Toxicological & En i onmen al Chemis y 105
51
Lopes-Lima e al. 2008) and dis ibu ion o he elec oly e h oughou he o he body
compa men s a e likely o con ibu e o i s ele a ed le els. This can lead o he ac i a ion
o Ca-dependen ca abolic p ocesses such as lipid hyd olysis, DNA agmen a ion, and
p o ein deg ada ion, ul ima ely leading o cell dea h (Via engo e al. 1994). Dec eased
ca bohyd a e and p o ein le els, being s ongly in e sely co ela ed o Ca le els, sugges
ha he mussels may no ha e su icien ene gy and essen ial nu ien s o no mal
ep oduc ion, g ow h, and de elopmen . A he same ime, ca bohyd a es and p o eins a e
impo an componen s o he o ganic ma ix which con ols CaCO
3
polymo phism, size,
and shape o he c ys alli es (Ma in and Luque 2004). These s ong pa hophysiological
esponses o en i onmen -like le els o Cu may be one o he many ac o s in ol ed in he
p esen ly obse ed decline o many Eu opean eshwa e bi al es, including he eshwa e
pea l mussel Ma ga i i e a ma ga i i e a (Baue 1986).
Conclusions
Coppe exposu e esul s in inc eases o Ca le els in all body compa men s, accompanied
by dec eases in he le els o soluble p o eins and ca bohyd a es. These e ec s may esul in
dis u bance o mussel’s ep oduc ion, g ow h and de elopmen , and shell o ma ion,
leading o popula ion decline.
Acknowledgmen s
We would like o hank P o . D . Jo ge P. Machado (Labo a o y o Applied Physiology, Uni e si y
o Po o, Po ugal) o help ul discussion. Financial suppo by he Di ec o a e Gene al o Highe
Educa ion, Minis y o Na ional Educa ion o he Republic o Indonesia, is highly app ecia ed.
Re e ences
Albe , B., D. B ay, J. Lewis, M. Ra , K. Robe , and J.D. Wa son. 1994. Molecula biology o he
cell. 3 d ed. New Yo k: Ga land Publishing.
An unes, C., T. Magalha
˜es-Ca doso, G. Mou a, D. Gonc¸ al es, and J. Machado. 2002. E ec s o Al,
Ni, Co, Zn, Cd, and Cu me als on he ou e man le epi helium o Anodon a cygnea (Unionidae).
Halio is 31: 71–84.
Baue , G. 1986. The s a us o he eshwa e pea l mussel Ma ga i i e a ma ga i i e a in he sou h o
i s Eu opean ange. Biological Conse a ion 38: 1–9.
Besse , J.M., C.G. Inge soll, and J.P. Giesy. 1996. E ec s o spa ial and empo al a ia ion o acid-
ola ile sul ide on he bioa ailabili y o coppe and zinc in eshwa e sedimen s. En i onmen al
Toxicology and Chemis y 15: 286–93.
Bi ge, W.J., and J.A. Black. 1979. E ec s o coppe on emb yonic and ju enile s ages o aqua ic
animals. In Coppe in he en i onmen . Pa II. Heal h e ec s, ed. J.O. N iagu, 373–99. New Yo k:
John Wiley & Sons.
Demayo, A., and M.C. Taylo . 1981. Guidelines o su ace wa e quali y. Vol. 1: Ino ganic chemical
subs ances – Coppe . O awa: Wa e Quali y B anch, Inland Wa e s Di ec o a e, En i onmen
Canada.
Faubel, D., M. Lopes-Lima, S. F ei as, L. Pe ei a, J. And ade, A. Checa, H. F ank, T. Ma suda,
and J. Machado. 2008. E ec s o Cd
2þ
on he calcium me abolism and shell mine aliza ion o
bi al e Anodon a cygnea.Ma ine and F eshwa e Beha iou and Physiology 41: 93–108.
Honkoop, P.J.C., J.V. de Mee , J.J. Beukema, and D. Kwas . 1999. Rep oduc i e in es men in he
in e idal bi al e Macoma bal hica.Jou nal o Sea Resea ch 41: 203–12.
106 A.P. Nug oho and H. F ank
52
Julshamn, K., E.K. To pe, C. Bø nes, L.J. Sæ h e, and A. Maage. 2001. Cadmium, lead, coppe and
zinc in blue mussels (My ilus edulis) sampled in he Ha dange jo d, No way. Jou nal o
En i onmen al Moni o ing 3: 539–42.
K uge , N.J. 1994. The B ad o d me hod o p o ein quan i a ion. In Me hods in molecula biology:
Basic p o ein and pep ide p o ocols, Vol. 32, ed. J.M. Walke , 9–15. New Je sey: Humana P ess,
Inc.
Ku nia, A.I., E. Pu wan o, and E. Mahajoeno. 2010. Exposu e coppe hea y me al (Cu) on
eshwa e mussel (Anodon a woodiana) and i s ela ion o Cu and p o ein con en in he body
shell. Bioscience 2: 48–53.
Lopes-Lima, M., R. Blehe , T. Fo g, M. Ha ne , and J. Machado. 2008. S udies on a PCMA-like
p o ein in he ou e man le epi helium o Anodon a cygnea: Insigh s on calcium anscellula
dynamics. Jou nal o Compa a i e Physiology B 178: 17–25.
Ma ie, B., G. Luque , J.P.D. Ba os, N. Guicha d, S. Mo el, G. Alca az, L. Bollache, and F. Ma in.
2007. The shell ma ix o he eshwa e mussel Unio pic o um (Paleohe e odon a, Unionoida):
In ol emen o acidic polysaccha ides om glycop o eins in nac e mine aliza ion. FEBS Jou nal
274: 2933–45.
Ma in, F., and G. Luque . 2004. Molluscan shell p o eins. Comp es Rendus Pale ol 3:
469–92.
Ma ı´nez, M.L., C. Land y, R. Boehm, S. Manning, A.O. Cheek, and B.B. Rees. 2006. E ec s o
long- e m hypoxia on enzymes o ca bohyd a e me abolism in he Gul killi ish, Fundulus g andis.
The Jou nal o Expe imen al Biology 209: 3851–61.
Masuko, T., A. Minami, N. Iwasaki, T. Majima, S. Nishimu a, and Y.C. Lee. 2005. Ca bohyd a e
analysis by a phenol–sul u ic acid me hod in mic opla e o ma . Analy ical Biochemis y 339:
69–72.
Mou a, G., L. Vila inho, and J. Machado. 2000. The ac ion o Cd, Cu, C , Zn, and Pb on luid
composi ion o Anodon a cygnea (L.): O ganic componen s. Compa a i e Biochemis y and
Physiology B 127: 105–12.
Nug oho, A.P., and H. F ank. 2011a. P oducing Cu-loaded algae o eeding
expe imen s: E ec s o coppe on Pa achlo ella kessle i.Toxicological and En i onmen al
Chemis y 93: 537–48.
Nug oho, A.P., and H. F ank. 2011b. Up ake, dis ibu ion, and bioaccumula ion o
coppe in he eshwa e mussel Anodon a ana ina. Toxicological and En i onmen al Chemis y
93: 1838–50.
Pa naik, S., G.B.N. Chainy, and J.K. Jena. 2007. Cha ac e iza ion o Ca
2þ
-ATPase ac i i y in gill
mic osomes o eshea e mussel, Lamellidens ma ginalis (Lama ck) and hea y me al modula ions.
Aquacul u e 270: 443–50.
San ini, O., N. Chahbane, P. Vasseu , and H. F ank. 2011. E ec s o low-le el coppe exposu e on
Ca
2þ
-ATPase and ca bonic anhyd ase in he eshwa e bi al e Anodon a ana ina. Toxicological
and En i onmen al Chemis y 93: 1826–37.
Sa yapa ameshwa , K., T.R. Reddy, and N.V. Kuma . 2006. S udy o ca bohyd a e me abolism in
selec ed issues o eshwa e mussel, Lamellidens ma ginalis unde coppe sulpha e oxici y.
Jou nal o En i onmen al Biology 27: 39–41.
S ei , B., and S. Win e . 1993. Cadmium up ake and compa men al ime cha ac e is ics in he
eshwa e mussel Anodon a ana ina.Chemosphe e 26: 1479–90.
Vian , M.R., J.H. Wal on, P.L. TenB ook, and R.S. Tjee dema. 2002. Suble hal ac ions o coppe
in abalone (Halio is u escens) as cha ac e ized by in i o
31
P NMR. Aqua ic Toxicology 57:
139–51.
Via engo, A. 1994. Hea y me al cy o oxici y in ma ine o ganisms: E ec s on Ca
2þ
homeos asis and
possible al e a ion o signal ansduc ion pa hways. Ad ances in Compa a i e En i onmen al
Physiology 20: 85–110.
Via engo, A., B. Bu lando, and C. Bolognesi. 2002. Cellula esponses o coppe in aqua ic
o ganisms. In Handbook o coppe pha macology and oxicology, ed. E.J. Massa o, 417–27.
New Je sey: Humana P ess, Inc.
Toxicological & En i onmen al Chemis y 107
53
Via engo, A., L. Canesi, M.N. Moo e, and M. O unesu. 1994. E ec o Hg
2þ
and Cu
2þ
on he
cy osolic Ca
2þ
le el in molluscan blood cells e alua ed by con ocal mic oscopy and spec o-
luo ime y. Ma ine Biology 119: 557–64.
Vino , I., and J.C. Pihan. 2005. Ci cula ion o coppe in he bio ic compa men s o a eshwa e
dammed ese oi . En i onmen al Pollu ion 133: 169–82.
W igh , D.A., and P. Welbou n. 2002. En i onmen al oxicology. Camb idge: Camb idge Uni e si y
P ess.
108 A.P. Nug oho and H. F ank
54
Toxicological & En i onmen al Chemis y
Vol. 94, No. 5, May 2012, 918–929
E ec s o coppe on lipid pe oxida ion, glu a hione, me allo hionein,
and an ioxida i e enzymes in he eshwa e mussel Anodon a ana ina
Andhika Puspi o Nug oho
ab
and Ha mu F ank
a
*
a
En i onmen al Chemis y and Eco oxicology, Uni e si y o Bay eu h, D-95440 Bay eu h,
Ge many;
b
Labo a o y o Ecology, Facul y o Biology, Gadjah Mada Uni e si y,
Yogyaka a 55281, Indonesia
(Recei ed 20 Augus 2011; inal e sion ecei ed 8 Ma ch 2012)
Coppe is an essen ial elemen o all animals. A ele a ed concen a ions, i is
oxic and can pa icipa e in he o ma ion o eac i e oxygen species, leading o
cellula damage. In his s udy, he eco oxicological ele ance o coppe was
in es iga ed wi h eshwa e mussels, Anodon a ana ina. When he mussels we e
exposed o coppe a en i onmen ally ealis ic concen a ions, ei he ia he wa e
(0.3 mmol L
1
Cu) o ed wi h Cu-loaded algae (equi alen o 0.06 mmol L
1
Cu),
he le el o hioba bi u ic acid- eac i e subs ances ose and glu a hione
dec eased. This was associa ed wi h he induc ion o me allo hionein and,
ela i e o o al p o ein, o glu a hione educ ase and he an ioxida i e enzymes
supe oxide dismu ase, ca alase, and glu a hione pe oxidase. Bu , since he o e all
p o ein-syn he ic capaci y was hampe ed by he coppe insul , he ac i i ies o he
enzymes ela i e o issue weigh and coppe concen a ions we e dep essed.
Du ing depu a ion, mos pa ame e s s a ed o no malize al hough no e u ning
o con ol alues wi hin 12 days.
Keywo ds: coppe ; Anodon a ana ina; hioba bi u ic acid- eac i e subs ances;
glu a hione; me allo hionein; an ioxida i e enzymes
In oduc ion
Me als a e b ough o he ea h’s su ace by mining o a mul i ude o ag icul u al,
indus ial, and echnological applica ions. One o he echnologically impo an me als is
coppe (Cu), used o elec ical powe ins alla ions and in he building sec o , as animal
eed addi i e o ungicide, as pa o machine ies, ehicles, elec ic appliances, and in many
o he consume p oduc s. Du ing i s use, i is eleased by co osion and/o ab asion,
mobilized as pa icula e ma e and d y o we deposi ed, o some ex en ending up in he
sedimen s o eshwa e ecosys ems (Smolde s e al. 2003). In non-con amina ed
eshwa e ecosys ems, i s concen a ions ange om 0.02 o 0.3 mmol L
1
(1–20 mgL
1
)
(Momc
ˇilo ic
´2004). Close o mining ac i i ies, aqua ic coppe pollu ion can each le els o
up o 30 mmol L
1
(1.7 mg L
1
) (Smolde s e al. 2003).
Mussels li e a he in e ace o ee- lowing wa e s and sedimen s and may be
ch onically exposed o coppe o long ime pe iods o in e mi en ly a luc ua ing le els,
depending upon empo a y hyd ological condi ions and ex en o sedimen oxygena ion
*Co esponding au ho . Email: [email p o ec ed]
ISSN 0277–2248 p in /ISSN 1029–0486 online
ß2012 Taylo & F ancis
h p://dx.doi.o g/10.1080/02772248.2012.675156
h p://www. and online.com
55
(Bhadu i e al. 2000; Poo , Gillissen, and Koelmans 2007). Coppe is an essen ial elemen
o hei ci cula o y oxygen ca ie hemocyanin (Momc
ˇilo ic
´2004) and plays a ole as
co ac o o a numbe o enzymes such as cy och ome oxidase, supe oxide dismu ase
(SOD), alcohol dehyd ogenase, dopamine hyd oxylase, y osinase, and lysyl oxidase
(Se a im and Bebianno 2009). Howe e , a excessi e concen a ions coppe can pa icipa e
in he o ma ion o eac i e oxygen species (ROS) h ough a Habe –Weiss cycle,
p oducing hyd oxyl adicals (
OH) om hyd ogen pe oxide (H
2
O
2
) and supe oxide (O
2)
(Bigo e al. 2011; Company e al. 2008). ROS may cause cellula damage by lipid
pe oxida ion when he an ioxida i e de ense sys ems o aqua ic animals a e o e whelmed,
leading o inac i a ion o memb ane enzymes, des uc ion o p o eins (Remme e al.
1989), and changes in he DNA s uc u e (Company e al. 2008; Lackne 1998; Se a im
and Bebianno 2009).
Mussels can cope wi h mode a ely ele a ed coppe in a ious ways (Se a im and
Bebianno 2009). In he cy osol, glu a hione (GSH) and me allo hionein (MT), he la e a
amily o cys eine- ich p o eins (I anko ic
´e al. 2010), p o ide p o ec ion agains
inc eased concen a ions h ough binding he coppe ions o he hiol g oups o hei
cys eine esidues (Company e al 2008; F eedman, Ci iolo, and Peisach 1989). O he
s a egies agains coppe -induced oxida i e oxici y is he induc ion o enzymes such as
SOD, ca alase (CAT), glu a hione pe oxidase (GPX), and glu a hione educ ase (GR)
(Isani e al. 2003).
Coppe has been obse ed in high concen a ions in he issue o eshwa e pea l
mussels Ma ga i i e a ma ga i i e a (F ank and Ge s mann 2007) and o he Eu opean
eshwa e mussel species (Tallandini e al. 1986). Thei popula ions a e s ongly a ec ed
Eu ope-wide and some a e h ea ened wi h ex inc ion (Cu elod, Seddon, and Neube
2011). Unde s anding he po en ial in ol emen o Cu in his phenomenon is he majo
mo i a ion o his s udy.
In his wo k, Anodon a ana ina is used as model species. In p e ious publica ions,
i has been shown ha A. ana ina can accumula e coppe om he wa e o by eeding
on coppe -con aining algae (Nug oho and F ank 2011b). This a icle ocuses on he
e ec s o coppe on MT and GSH and on an ioxida i e enzymes as esponse o
oxida i e s ess, signaled by inc eased le els o hioba bi u ic acid- eac i e subs ances
(TBARS).
Ma e ials and me hods
Chemicals
Iso opically en iched (99%)
63
Cu oxide (Eu iso- op, Saa b u
¨cken, Ge many) was used.
Concen a ed HNO
3
(69%) and concen a ed HCl (30%) we e o sup apu e g ade (Me ck,
Da ms ad , Ge many); o he chemicals (Ca l Ro h, Ka ls uhe, Ge many; Sigma-Ald ich,
Munich, Ge many) we e o analy ical g ade. Cleaning o labwa e and p epa a ion o
he Cu
2þ
s ock solu ion a e desc ibed in a p e ious publica ion (Nug oho and
F ank 2011b).
Animals and expe imen al design
Se en y duck mussels (A. ana ina) (ZOO-E lebnis Online Shop, G osse ehn, Ge many)
wi h shell leng hs o 10–12 cm and weigh s be ween 100 and 200 g we e b ough o he
labo a o y in pond wa e . Mussel handling, acclima iza ion, and expe imen al design
Toxicological & En i onmen al Chemis y 919
56
(Nug oho and F ank 2011b) as well as he p epa a ion o no mal and Cu-loaded algae
ha e been desc ibed ea lie (Nug oho and F ank 2011a). The mussels we e di ided in o
h ee g oups consis ing o 21 mussels each. The i s g oup was kep in a i icial pond
wa e (APW); he second one was exposed o 0.3 mmol L
1
(20 mgL
1
)
63
Cu
2þ
in he wa e ;
he hi d g oup ecei ed daily 1.5 mg L
1
eeze-d ied
63
Cu-loaded algae (40 mmol
63
Cu
pe kg d y weigh ) o 24 days, equi alen o a nominal concen a ion o 0.06 mmol
(3.6 mgL
1
)
63
Cu pe li e APW.
Fo sampling, h ee mussels o each g oup we e aken o analysis a days 0, 6, 12, 18,
and 24 (exposu e), and a days 30 and 36 (depu a ion). The mussels’ so bodies we e
dissec ed on ice in o gills, man le, kidney, and diges i e gland. Two aliquo s o e e y issue
ac ion, abou 5–10 mg each, we e placed in sepa a e 2-mL mic o ubes o known weigh .
The i s aliquo was used o he de e mina ion o MT and he second one o he
de e mina ion o TBARS, GSH, enzyme ac i i ies, and p o eins. All mic o ubes we e kep
in a eeze a 80C un il u he analysis. The emainde s o he issues we e placed in
15-mL polyp opylene (PP) ubes o known weigh s and we e lyophilized o coppe
de e mina ion.
Analy ical me hods
Sample p epa a ion
F ozen issue samples in mic o ubes we e hawed and immedia ely mixed wi h 500 mL
suc ose (0.5 mol L
1
)/T is-HCl (20 mmol L
1
; pH 8.6) bu e , o which leupep ine
(6 mmol L
1
) and phenylme hanesul onyl luo ide (PMSF) (0.5 mmol L
1
) we e added as
an i p o eoly ic agen s and -me cap oe hanol (0.01%) as educing agen . The mix u es
we e sonica ed in an ice ba h wi h 12 s okes o a sonica o (Labsonic U ip sonica o , B.
B aun Bio ech In e na ional, Melsungen, Ge many) a 20 kHz, acous ic powe 50 W. The
homogena es we e cen i uged a 4C o 30 min a 10,000 g(He aeus Mul i uge 1L-R,
The mo Scien i ic, Os e ode, Ge many). Supe na an s we e used o MT de e mina ion.
Fo he de e mina ion o TBARS, GSH, enzyme ac i i ies, and p o eins, ozen issue
samples o 5–10 mg we e hawed and immedia ely mixed wi h 500 mL phospha e bu e
(50 mmol L
1
; pH 7.4) con aining 150 mmol L
1
KCl, 1 mmol L
1
e hylenediamine e a-
ace ic acid (EDTA), 1 mmol L
1
di hio h ei ol (DTT), and 0.01% (w/ ) PMSF. The
samples we e homogenized in an ice ba h wi h 12 s okes o a sonica o a 20 kHz, acous ic
powe 50 W, and cen i uged a 4C o 30 min a 10,000 g. The supe na an s we e used
o analysis.
To al coppe
To al coppe in lyophilized issues and eeze-d ied algal ood, and – e e y second day –
he ac ual coppe concen a ions in APW we e de e mined by induc i ely-coupled plasma
mass spec ome y. De ails ha e been desc ibed p e iously (Nug oho and F ank 2011b).
Lipid pe oxida ion
Lipid pe oxida ion was de e mined ollowing he me hod o Buege and Aus (1978) by
measu ing TBARS, exp essed as malondialdehyde (MDA) equi alen s. Abso bances o
samples we e ead a 535 nm wi h a mic opla e eade (Bio ek Syne gy HT, Bad
F ied ichshall, Ge many). TBARS le els we e es ima ed using a s anda d cu e ob ained
920 A.P. Nug oho and H. F ank
57
wi h 1,1,3,3- e ame hoxyp opane (99%; VWR, Da ms ad , Ge many) as s able p ecu so
o MDA and exp essed as mmol kg
1
issue we weigh ( ww).
Glu a hione
GSH was de e mined acco ding o Ande son (1985). Abso bances o samples we e
measu ed a 412 nm wi h a mic opla e eade . The GSH con en was es ima ed using a
s anda d cu e ob ained wi h educed GSH and exp essed as mmol kg
1
ww.
Me allo hioneins
MT concen a ions we e de e mined by he spec opho ome ic me hod o Via engo e al.
(1997) modi ied by Ve leca , Jena, and Chainy (2008). Abso bances o samples we e ead
a 412 nm wi h a mic opla e eade . The MT con en was de e mined using GSH (Ca l
Ro h, Ka ls uhe, Ge many) as s anda d, assuming ha 1 mmol GSH is equi alen o
0.055 mmol MT. Concen a ions o MT we e exp essed as mmol kg
1
ww.
Enzyme ac i i ies
SOD ac i i ies we e de e mined by he p ocedu e o Beauchamp and F ido ich (1971),
based on he inhibi ion o ni o e azolium blue educ ion and measu ing sample
abso bances a 560 nm. CAT ac i i ies we e assayed spec opho ome ically acco ding o
Rao, Paliya h, and O m od (1996) by moni o ing he dec ease in he abso bance o H
2
O
2
a 240 nm. GPX ac i i ies we e de e mined acco ding o Paglia and Valen ine (1967) and
GR ac i i ies acco ding o Massey and William (1965) in he p esence o GSSG, in bo h
cases ollowing he a e o NADPH oxida ion a 340 nm. Abso bances we e measu ed wi h
a mic opla e eade ; enzyme ac i i ies we e calcula ed in uni s pe millig am p o ein and
pe g am ww.
P o eins
P o eins we e de e mined by he dye-binding assay (K uge 1994). Abso bances o he
samples we e ead a 595 nm wi h a mic opla e eade . The concen a ions we e
de e mined using BSA (96%; Sigma-Ald ich, Munich, Ge many) o calib a ion.
S a is ical da a analyses
The a iabili y o he obse ed pa ame e s and o o al Cu concen a ion in he di e en
o gans we e es ed by wo-way analysis o a iance (ANOVA) conside ing exposu e ime
and coppe exposu e pa hways as independen a iables, ollowed by he Duncan mul iple
compa ison es s ( p<0.05) i signi ican di e ences we e ound. Da a we e ans o med o
log(Xþ1) uni s be o e s a is ical analysis o he homogenei y o a iance and no mali y.
Linea eg ession analysis was pe o med o e alua ing he ela ionship be ween Cu
concen a ion and he obse ed pa ame e s, ollowed by Pea son co ela ion analysis o
es ing he s eng h o linea ela ionship.
Toxicological & En i onmen al Chemis y 921
58
Bonne is, E., O. Pe ce al, S. Masson, L. Ha e, and P.G.C. Campbell. 2005. Sub-cellula pa i ioning
o Cd, Cu and Zn in issues o indigenous unionid bi al es li ing along a me al exposu e g adien
and links o me al-induced e ec s. En i onmen al Pollu ion 135: 195–208.
Bouskill, N.J., R.D. Handy, T.E. Fo d, and T.S. Galloway. 2006. Di e en ia ing coppe and a senic
oxici y using biochemical bioma ke s in Asellus aqua icus and D eissena polymo pha.
Eco oxicology and En i onmen al Sa e y 65: 342–9.
Buege, J.A., and S.D. Aus . 1978. Mic osomal lipid pe oxida ion. Me hods in Enzymology 52:
302–10.
Canesi, L., A. Via engo, C. Leonzio, M. Filippelli, and G. Gallo. 1999. Hea y me als and
glu a hione me abolism in mussel issues. Aqua ic Toxicology 46: 67–76.
Company, R., A. Se a im, R.P. Cosson, A. Fiala-Me
´dioni, L. Camus, A. Colac¸ o, R. Se a
˜o-San os,
and M.J. Bebianno. 2008. An ioxidan biochemical esponses o long- e m coppe exposu e in
Ba hymodiolus azo icus om Menez-Gwen hyd o he mal en . Science o he To al En i onmen
389: 407–17.
Cu elod, A., M. Seddon, and E. Neube . 2011. Eu opean ed lis o non-ma ine molluscs.
Luxembou g: Publica ions O ice o he Eu opean Union.
de La on aine, Y., F. Gagne
´, C. Blaise, G. Cos an, P. Gagnon, and H.M. Chan. 2000. Bioma ke s in
zeb a mussels (D eissena polymo pha) o he assessmen and moni o ing o wa e quali y o he S
Law ence Ri e (Canada). Aqua ic Toxicology 50: 51–71.
Doyo e, A., C. Cossu, M. Jacquin, M. Babu , and P. Vasseu . 1997. An ioxidan enzymes,
glu a hione and lipid pe oxida ion as ele an bioma ke s o expe imen al o ield exposu e in he
gills and he diges i e gland o he eshwa e bi al e Unio umidus.Aqua ic Toxicology 39:
93–110.
Eisle , R. 1993. Zinc haza ds o ish, wildli e, and in e eb a es: A synop ic e iew. Lau el, MD: U.S.
Depa men o he In e io , Fish, and Wildli e Se ice, Pa uxen Wildli e Resea ch Cen e .
F ank, H., and S. Ge s mann. 2007. Declining popula ions o eshwa e pea l mussels
(Ma ga i i e a ma ga i i e a) a e bu dened wi h hea y me als and DDT/DDE. Ambio 36: 571–4.
F eedman, J.H., M.R. Ci iolo, and J. Peisach. 1989. The ole o glu a hione in coppe me abolism
and oxici y. The Jou nal o Biological Chemis y 264: 5598–605.
Isani, G., M. Mona i, G. And eani, M. Fabb i, and E. Ca pene
`. 2003. E ec o coppe exposu e on
he an ioxidan enzymes in bi al e mollusk Scapha ca inaequi al is.Ve e ina y Resea ch
Communica ions 27: 691–3.
I anko ic
´, D., J. Pa ic
ˇic
´, V. Bea o ic
´, R.S. Klobuc
ˇa , and G.I.V. Klobuc
ˇa . 2010. Inducibili y o
me allo hionein biosyn hesis in he whole so issue o zeb a mussels D eissena polymo pha
exposed o cadmium, coppe , and pen achlo ophenol. En i onmen al Toxicology 25: 198–211.
K uge , N.J. 1994. The B ad o d me hod o p o ein quan i a ion. In Me hods in molecula biology:
Basic p o ein and pep ide p o ocols, Vol. 32, ed. J.M. Walke , 9–15. New Je sey: Humana P ess.
Lackne , R. 1998. ‘‘Oxida i e s ess’’ in ish by en i onmen al pollu an s. In Fish eco oxicology, eds.
T. B aunbeck, D.E. Hin on and B. S ei , 203–24. Swi ze land: Bi kha
¨use Ve lag.
Massey, V., and C.H. Williams. 1965. On he eac ion mechanism o yeas glu a hione educ ase.
The Jou nal o Biological Chemis y 240: 4470–81.
Momc
ˇilo ic
´, B. 2004. Coppe . In Elemen s and hei compounds in he en i onmen : Me als and hei
compounds, Vol. 2, eds. E. Me ian, M. Anke, M. Ihna and M. S oepple , 731–50. Weinheim:
Wiley-VCH Ve lag.
Nug oho, A.P., and H. F ank. 2011a. P oducing Cu-loaded algae o eeding expe imen s: E ec s o
coppe on Pa achlo ella kessle i.Toxicological and En i onmen al Chemis y 93: 537–48.
Nug oho, A.P., and H. F ank. 2011b. Up ake, dis ibu ion, and bioaccumula ion o coppe in he
eshwa e mussel Anodon a ana ina.Toxicological and En i onmen al Chemis y 93: 1838–50.
Nug oho, A.P., and H. F ank. 2012. E ec s o coppe exposu e on calcium, ca bohyd a e, and
p o ein le els in he eshwa e mussel Anodon a ana ina.Toxicological and En i onmen al
Chemis y 94: 99–108.
Paglia, D.E., and W.N. Valen ine. 1967. S udies on quan i a i e and quali a i e cha ac e iza ion o
e y h ocy e glu a hione pe oxidase. Jou nal o Labo a o y and Clinical Medicine 70: 158–69.
928 A.P. Nug oho and H. F ank
65
Pe ce al, O., Y. Couilla d, B. Pinel-Alloul, E. Bonne is, and P.G.C. Campbell. 2006. Long- e m
ends in accumula ed me als (Cd, Cu and Zn) and me allo hionein in bi al es om lakes wi hin a
smel e -impac ed egion. Science o he To al En i onmen al 369: 403–18.
Poo , A., F. Gillissen, and A.A. Koelmans. 2007. E ec s o low egime and looding on hea y
me al a ailabili y in sedimen and soil o a dynamic i e sys em. En i onmen al Pollu ion 148:
779–87.
Rao, M.V., G. Paliya h, and D.P. O m od. 1996. Ul a iole -B and ozone-induced biochemical
changes in an ioxidan enzymes o A abidopsis haliana.Plan Physiology 110: 125–36.
Remme , H., W. Kessle , H. Einsele, Th. Hin ze, G. Diaz de To anzo, A.M. Gha aibeh, and
H. F ank. 1989. E hanol p omo es oxygen- adical a ack on p o eins bu no on lipids. D ug
Me abolism Re iews 20: 219–32.
Saba ini, S.E., I. Rocche a, D.E. Nahabedian, C.M. Luque , M.R. Eppis, L. Bianchi, M. del,
and C. Rı´os de Molina. 2011. Oxida i e s ess and his ological al e a ions p oduced by
die a y coppe in he eshwa e bi al e Diplodon chilensis.Compa a i e Biochemis y and
Physiology C 154: 391–8.
Se a im, A., and M.J. Bebianno. 2009. Me allo hionein ole in he kine ic model o coppe
accumula ion and elimina ion in he clam Rudi apes decussa us.En i onmen al Resea ch 109:
390–9.
Smolde s, A.J.P., R.A.C. Lock, G. Van de Velde, R.I.M. Hoyos, and J.G.M. Roelo s. 2003. E ec s
o mining ac i i ies on hea y me al concen a ions in wa e , sedimen , and mac oin e eb a es in
di e en eaches o he Pilcomayo Ri e , Sou h Ame ica. A chi es o En i onmen al
Con amina ion and Toxicology 44: 314–23.
Tallandini, L., A. Cassini, N. Fa e o, and V. Albe goni. 1986. Regula ion and subcellula
dis ibu ion o coppe in he eshwa e mussel Anodon a cygnea (L.) and Unio elonga ulus (P .).
Compa a i e Biochemis y and Physiology C 84: 43–9.
Ve leca , X.N., K.B. Jena, and G.B.N. Chainy. 2008. Modula ion o an ioxidan de ences in
diges i e gland o Pe na i idis (L.), on me cu y exposu es. Chemosphe e 71: 1977–85.
Via engo, A., E. Ponzano, F. Donde o, and R. Fabb i. 1997. A simple spec opho ome ic me hod
o me allo hionein e alua ion in ma ine o ganisms: An applica ion o Medi e anean and
An a c ic mollusks. Ma ine En i onmen al Resea ch 44: 69–84.
Toxicological & En i onmen al Chemis y 929
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DECLARATION
I he eby decla e ha his submission is my own accoun o my own esea ch and ha ,
o he bes o my knowledge and belie , i con ains nei he ma e ial p e iously published
o w i en by ano he pe son no ma e ial which o a subs an ial ex en has been
accep ed o he awa d o any o he deg ee o diploma o a uni e si y o any o he
ins i u e o highe lea ning, excep whe e due acknowledgmen has been made in he
ex .
ERKLÄRUNG
Hie mi e klä e ich, dass ich die A bei selbs s ändig e ass und keine ande en als die
angegebenen Hil smi el e wende habe.
Wei e hin e klä e ich, dass ich nich ande wei ig mi ode ohne E olg e such habe,
eine Disse a ion einzu eichen ode mich eine Dok o p ü ung zu un e ziehen.
Bay eu h, den 9 No embe 2011 ____________________________
Andhika Puspi o Nug oho