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Compa ison o h ee unionid mussel species in emo ing g een mic oalgae g own in
eci cula ing aquacul u e sys em e luen
© The Au ho (s) 2024
Published e sion
Julkunen, Ville; S e čić, Čedomi ; Pi honen, Juhani; Pulkkinen, Ka ja
Julkunen, V., S e čić, Č., Pi honen, J., & Pulkkinen, K. (2024). Compa ison o h ee unionid
mussel species in emo ing g een mic oalgae g own in eci cula ing aquacul u e sys em
e luen . Hyd obiologia, Ea ly online. h ps://doi.o g/10.1007/s10750-024-05547-w
2024
Vol.: (0123456789)
1 3
Hyd obiologia
h ps://doi.o g/10.1007/s10750-024-05547-w
PRIMARY RESEARCH PAPER
Compa ison o  h ee unionid mussel species in emo ing
g een mic oalgae g own in eci cula ing aquacul u e sys em
e luen
VilleJulkunen · Čedomi S e čić·
JuhaniPi honen· Ka jaPulkkinen
Recei ed: 2 Oc obe 2023 / Re ised: 4 Ap il 2024 / Accep ed: 4 Ap il 2024
© The Au ho (s) 2024
dec eased o did no change in anks wi h M. g i -
i hii, bu inc eased in anks con aining Selenas um
sp. These esul s sugges ha o he es ed species
Anodon a mussels and M. g i i hii show bes po en-
ial o RAS e luen bio emedia ion applica ion. We
conclude ha a co-cul u e o mic oalgae and unio-
nid mussels could be used o ecycling nu ien s in
aquacul u e.
Keywo ds Biological ha es ing· Mussel
bio il a ion· Nu ien ecycling· Reci cula ing
aquacul u e sys em (RAS)· E luen bio emedia ion
In oduc ion
Aquacul u e p oduc ion inc eased h ee old du ing
he i s wo decades o wen y- i s cen u y and he
inc ease in p oduc ion has c ea ed p oblems such as
nu ien pollu ion (Naylo e  al., 2021). T adi ional
cage o low- h ough aquacul u e sys ems o e ew
oppo uni ies o nu ien eco e y, because nu ien s
eleased om was e and ish exc e a a e dilu ed in
a la ge olume o wa e . Reci cula ing aquacul u e
sys ems (RAS) a e land-based sys ems o a ming
aqua ic o ganisms whe e wa e use is signi ican ly
educed compa ed o adi ional low- h ough sys ems
due o cons an e luen ea men and ecycling
(B egnballe, 2015). In RAS, ypically only 5–10%
o he o al wa e olume is enewed daily wi h
esh wa e o p e en he accumula ion o dissol ed
Abs ac Global inc ease in aquacul u e p oduc-
ion has c ea ed a need o educe i s en i onmen al
impac s. Nu ien s could be ecycled especially a
land-based eci cula ing aquacul u e sys ems (RAS)
by cul i a ing g een mic oalgae in aquacul u e e lu-
en . Howe e , mic oalgae a e di icul o ha es . As
a mul i- ophic solu ion, mussels could be used in
ha es ing mic oalgae. We es ed h ee Eu opean
eshwa e mussels (duck mussel Anodon a ana ina,
swan mussel A. cygnea, and swollen i e mussel
Unio umidus) o il e ing wo common g een mic o-
algae (Mono aphidium g i i hii and Selenas umsp.)
g own in RAS e luen . Mussels dec eased mic oalgal
concen a ions in he anks 42–83% o e h ee con-
secu i e ials. Algal concen a ions a he end o each
ial we e lowes o bo h mic oalgae in anks con ain-
ing Anodon a mussels. Clea ance a es we e highe
o Anodon a mussels han o U. umidus. Mussels
biodeposi ed mo e mic oalgae o ank bo oms when
M. g i i hii was il e ed. Ammonium concen a ion
Handling edi o : Manuel Lopes-Lima
Supplemen a y In o ma ion The online e sion
con ains supplemen a y ma e ial a ailable a h ps:// doi.
o g/ 10. 1007/ s10750- 024- 05547-w.
V.Julkunen(*)· Č.S e čić· J.Pi honen· K.Pulkkinen
Depa men o Biological andEn i onmen al
Science, Uni e si y o Jy äskylä, P.O. Box35,
40014Jy äskylänyliopis o, Finland
e-mail: [email p o ec ed]
Hyd obiologia
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nu ien s and suspended solids o igina ing om eces,
me abolic was e p oduc s, and unea en eed o le els
ha m ul o ish (Pied ahi a, 2003; B egnballe, 2015).
As a esul , he concen a ion o dissol ed nu ien s
is much highe in RAS e luen han in low- h ough
aquacul u e, enabling nu ien eco e y om he
e luen o ecycling (Pied ahi a, 2003; Ma ins e al.,
2010).
One o he ongoing de elopmen s in RAS is he
up ake o nu ien s om e luen ia he in eg a ed
mul i- ophic aquacul u e (IMTA) app oach (Ma ins
e  al., 2010). IMTA e e s o he simul aneous
cul i a ion o wo o mo e compa ible o ganisms om
di e en ophic le els in a single sys em, whe e he
was es om he ed o ganism, usually ish, a e used as
sou ces o nu i ion o o he cul u ed o ganisms such
as mic oalgae o il e - eede s (T oell e  al., 2009;
Neo i e al., 2017). Mic oalgae a e among he mos
sui able o ganisms o IMTA as hey o e sus ainable
and cos -e icien bio emedia ion o aquacul u e
e luen , and impo an biomolecules om he
p oduced biomass (p o eins, lipids, ca bohyd a es,
pigmen s) can be la e used in human oods, animal
eeds, e ilize s, bio uels, and o he p oduc s (Ma a
e al., 2010, 2012; A bib e al., 2012).
Mic oalgal ha es ing is he main limi a ion o
he u iliza ion o mic oalgae in IMTA as chemical
and mechanical ha es ing o mic oalgae can be up
o 90% o he o al in es men cos s (Molina G ima
e al., 2003; La ino ičs & Juhna, 2017). The di ec
use o mic oalgae as ish eed in IMTA is pa ly
limi ed by he small cell size o mic oalgae and i s
poo diges ibili y, especially o some ca ni o ous ish
species (Shah e  al., 2018; Tibbe s, 2018; Dou ou
e  al., 2020). Consequen ly, hese cons ain s could
be mi iga ed wi h biological ha es ing o mic oalgae
by il e - eeding o ganisms such as mussels and
u ilizing mussels as a ishmeal eplacemen in eeds
o ish (Sicu o e al., 2010) o chicken (McLaughlan
e  al., 2014). Concen a ed nu ien - ich eces and
pseudo eces p oduced by mussels il e ing mic oalgae
g own in he e luen ( an de Mee e al., 2023) could
be a p omising esou ce o be used as e ilize and in
bio uel and biogas p oduc ion (Vaughn e al., 2004).
Bi al es, including he eshwa e mussels o
he o de Unionida, a e highly e icien in il e ing
all pa icula e ma e om hei su oundings
(Vaughn e al., 2004; Vaughn, 2018). While il e ing
he su ounding wa e , mussels emo e ino ganic
and o ganic pa icles, such as g een mic oalgae
and dia oms (K yge & Riisgå d, 1988; Vaughn
& Hakenkamp, 2001; Bon es e  al., 2007), bu
also ha m ul mic oo ganisms and pa icles, such
as la obac e ia (Hajisa a ali e  al., 2022), oxic
cyanobac e ia (Bon es e  al., 2007), ish pa asi es
(Gopko e  al., 2017), and mic oplas ics (Be glund
e  al., 2019). F eshwa e mussels can selec which
o he il e ed pa icles hey inges (Lopes-Lima
e al., 2014). This enables he mussels o selec ood
pa icles hey equi e a any gi en ime and disca d
po en ially ha m ul o non-nu i ional pa icles
encapsula ed in mucus as pseudo eces (Bon es
e al., 2007). Pseudo eces p oduc ion is a me hod by
which some bi al es, including all unionid mussels,
deposi pa icles om he su ounding wa e o he
sedimen wi hou he pa icles going h ough he
mussel gu . F eshwa e mussels can be oppo unis ic
in hei g azing and can use he o ganic ma e ial
in he sedimen , including p e iously deposi ed
pseudo eces, as ood (Nichols e al., 2005).
Bio il e ing applica ions o en use species ha a e
easily a ailable and ha e high il e ing e iciency.
Fo example, he d eissenid zeb a mussel [D eissena
polymo pha (Pallas, 1771)] has been used in ea ing
indus ial and municipal was ewa e s (Ellio e  al.,
2008; Mezzano e e al., 2016), and in bio emedia ion
o eu ophic na u al wa e s (Pi es e al., 2009). The
d eissenid quagga mussel [D. bugensis (And uso ,
1897)] has been used in ha es ing g een mic oalgae
cul u ed in was ewa e plan e luen o emo al
o nu ien s om he e luen ( an de Mee e  al.,
2023). Howe e , D. polymo pha and D. bugensis
a e in asi e species ha can eplace local auna and
cause de imen al changes in hei in aded eshwa e
ecosys ems (S aye e al., 1998; Rowe e al., 2015;
Ożgo e al., 2020). As he e a e s ill a eas in Eu ope,
whe e he in asi e eshwa e mussel is no p esen ,
na i e mussel species should be used in bi al e-based
bio emedia ion applica ions. Duck mussel [Anodon a
ana ina (Linnaeus, 1758)], swan mussel [A. cygnea
(Linnaeus, 1758)], and swollen i e mussel (Unio
umidus Philipsson, 1788), all belonging o he amily
Unionidae, a e common and eadily a ailable om
lakes, ponds, and slow- lowing i e s in No he n
Eu ope (Vaughn & Hakenkamp, 2001; Lopes-Lima
e  al., 2017). They ha e been shown o ha e high
il e ing capaci ies (K yge & Riisgå d, 1988; Pusch
e al., 2001; Bon es e al., 2007; Moëzzi e al., 2013),
Hyd obiologia
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which makes hem sui able candida es o es ing
bio emedia ion applica ions in he No dic coun ies.
This expe imen was ca ied ou as a easibili y
s udy o compa e h ee na i e and common
eshwa e mussel species, A. ana ina, A. cygnea, and
U. umidus, and wo species o g een mic oalgae in
he emo al o nu ien s om RAS e luen . The wo
g een mic oalgae es ed, Mono aphidium g i i hii
(Be k.) Komá k.-Legn. and Selenas um sp., a e
e icien a abso bing dissol ed nu ien s om RAS
e luen and a e common in Eu opean eshwa e
sys ems (S e čić e al., 2019; Calde ini e al., 2021).
Howe e , as hese mic oalgae di e in shape and
size and in hei pala abili y o il e - eede s such as
Daphnia magna S aus, 1820 (S e čić e  al., 2020),
hei sui abili y o bio emedia ion applica ion wi h
mussels was in es iga ed. As he g een mic oalgae
a e di icul and expensi e o ha es , his s udy also
aimed o de e mine i and o wha ex en he mussels
would biodeposi he il e ed mic oalgae in o a highly
condensed mucus-co e ed mic oalgae sludge ha
could be easily emo ed om he ank bo oms.
Addi ionally, since he applica ion aims o educe he
concen a ion o nu ien s in he was ewa e , le els o
phospha e and ammonium we e moni o ed as hey a e
eleased by mussels and mic obial me abolic ac i i y.
Ma e ials andme hods
Mic oalgae cul i a ion
The g een mic oalgae we e cul u ed in was ewa e
o igina ing om a labo a o y-scale RAS loca ed a
he Depa men o Biological and En i onmen al
Science, Uni e si y o Jy äskylä, Finland. Fish kep
in he RAS ( o al olume c. 4.5 m3) we e whi e ish
[Co egonus la a e us (Linnaeus, 1758)] and ainbow
ou [Onco hynchus mykiss (Walbaum, 1792)],
and hey we e ed wi h d y pelle s (Ci cui ed,
Raisioaqua, Raisio, Finland) using bel eede s. The
mic oalgae we e g own in 60-L ubula bubble-
column pho obio eac o s (diame e 20 cm, heigh
200cm) illed wi h RAS was ewa e and inocula ed
wi h mic oalgae om s ock cul u es. The eshwa e
mic oalgal s ains M. g i i hii (NIVA-CHL 8) and
Selenas um sp. (K–1877 (NIVA)) we e acqui ed
om he No wegian Cul u e Collec ion o Algae
(NORCCA). S ock cul u es we e main ained in
mic oalgae medium MWC (Modi ied W igh ’s
C yp ophy e based on Guilla d & Lo enzen (1972)
as desc ibed p e iously in S e čić e al. (2019)). Two
pho obio eac o s, each con aining a monocul u e o
mic oalgae, we e cons an ly illumina ed wi h wo
e ically moun ed LED g ow ligh s (AP67 spec um,
18 W, T8 ubes; Valoya Oy, Helsinki, Finland) wi h
an in ensi y o 100µmol pho ons m–2 s–1 measu ed a
he side o he pho obio eac o by a high- esolu ion
spec o adiome e (HP-350; HiPoin Inc, Taichung
Ci y, Taiwan). The pho obio eac o s we e co e ed
o minimize e apo a ion and wa e sp aying due
o ae a ion. Sys ems we e cons an ly supplied wi h
comp essed ai om he bo om h ough pe o a ed
coiled ubes o keep he mic oalgae in suspension and
o p o ide CO2. The ai supply was no measu ed bu
se o be simila in bo h pho obio eac o s based on
isual wa e bubbling. The mic oalgae cul u es used
in he expe imen we e g own in he pho obio eac o s
o ou days un il hey had exhaus ed he phospha e
in he was ewa e (S e čić e al., 2019) be o e each
ial wi h he mussels.
Mussel collec ion and main enance
Mussels we e cap u ed by sno keling om he li o-
al zone o Lake Jy äsjä i (62°14′31″N, 25°46′12″E)
om he app oxima e dep h o one me e on June
20 h, 2019. Mussels we e iden i ied isually as A.
ana ina, A. cygnea, and U. umidus. As mussels
we e cap u ed haphaza dly, hey we e assumed o
ep esen ypical adul specimens o each species
ega ding indi idual mussel size in he local popu-
la ion. The mussels we e kep in wo low- h ough
(400 mL min–1) holding anks in 40 L o bo ehole
wa e ae a ed wi h ai pumps (Eheim ai 400; Eheim
GmbH & Co. KG, Deizisau, Ge many) a app ox.
33mL min−1, in a empe a u e-con olled labo a o y
(17.0 ± 0.3 °C). Mussels used o M. g i i hii, and
Selenas um sp. il e ing ials we e kep in sepa a e
anks. No ood was o e ed o mussels be ween he
ials. Each mussel was ma ked by d illing a unique
code on he shell wi h a o a y cu e (D emelS ylo + ;
Robe Bosch GmbH, Ge lingen, Ge many). Mussel
leng hs we e measu ed be o e and a e he se ies o
h ee ials wi h Ve nie calipe s o he nea es mm.
D y weigh es ima es we e calcula ed o each mus-
sel om he a e age o he wo shell leng h meas-
u emen s (Table1). The d y weigh es ima es o A.
Hyd obiologia
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ana ina we e calcula ed acco ding o Zapi is e  al.
(2021)
The d y weigh es ima es o A. cygnea and U.
umidus we e calcula ed as
and
Respec i ely (Ra e a & Sp oca i, 1997). In he
o mulae, l is mussel leng h in cm.
Expe imen al se up
The e iciency o he h ee mussel species in emo -
ing he wo mic oalgal species cul i a ed in RAS
was ewa e was e alua ed in a se ies o h ee ials.
Be o e he i s ial, he mussels we e acclima ed o
he expe imen al se up o se en days. In each ial,
mussels we e placed indi idually in glass es anks
(wid h 17, leng h 17, heigh 24cm), ha we e illed
wi h 4L o algae-con aining wa e aken om he
pho obio eac o s (see Sec . "Calcula ions and da a
analyses".). Tanks we e moni o ed o se en days
o a change in algal densi y. The se up emained he
same h oughou he consecu i e ials, and he same
indi idual mussels we e used in all ials and we e
p o ided wi h he same mic oalgae. The a ionale o
using he same indi idual in h ee sepa a e ials was
o moni o he consis ency in he il e ing beha io o
he mussels. In each ial, he e we e h ee anks o
A. ana ina and A. cygnea indi iduals, ou anks o
U. umidus indi iduals, and wo anks wi hou mus-
sels as con ols o bo h species o mic oalgae, o al-
ing 24 anks. The labo a o y was illumina ed dimly
by ou luo escen ubes (TL-D 36W/16 Yellow, T8;
dw =
9.66
∗
10
−4∗l2−
0.09
l+2.31
dw =0.12e0.31l
dw =0.04e0.51l
Koninklijke Philips N.V., Eindho en, The Ne he -
lands) wi h 12:12 ligh :da k pe iod, 0.09 ± 0.01µmol
pho on m–2 s–1 ligh in ensi y a wa eleng h ange o
570–590 nm. The dim ligh ing and he wa eleng h
ange we e chosen o minimize mic oalgal g ow h
in he es anks and o mimic na u al ligh ing con-
di ions o he mussels. The anks we e co e ed wi h
anspa en plas ic shee s o p e en wa e e apo a ion
and mic oalgal c oss-con amina ion be ween anks.
Tes anks had cons an ae a ion om an in eg a ed
ai comp esso di ided in o each ank ia ubing and
ai s ones. The ae a ion a e was no measu ed bu
se isually o be simila in each ank and kep low
o educe s ess on he mussels and sp aying o wa e
om he anks. Tes anks we e in he same labo a-
o y as main enance anks, a 17.0 ± 0.3°C.
Concen a ions o mic oalgae we e measu ed
be o e, du ing, and a e each ial om samples aken
wi h a pipe e om a dep h o app oxima ely 5cm
om he su ace o he wa e so as no o dis u b he
sedimen ed mic oalgae on he bo om o he anks.
Sedimen ed mic oalgae deposi ed by mussels we e
quan i ied a he end o he hi d ial, and dissol ed
phospha e and ammonium in he wa e we e analyzed
a he beginning and he end o he second ial
(Table2).
Calcula ions and da a analyses
Mic oalgal cell densi y and o al cell olume (mL
mic oalgae in mL H2O) in he es anks we e
measu ed wi h a CASY Elec onic Cell Coun e and
Analyze (OLS-OMNI Li e Science GmbH & Co.
B emen, Ge many, la e e e ed o as CASY), wi h a
capilla y po e size o 60µm. The selec ed measu ing
ange was 3.5–7.5µm o M. g i i hii and 2.4–3.8µm
o Selenas um sp.
Table 1 D y weigh (g) es ima es (mean ± SE) o Anodon a ana ina, Anodon a cygnea, and Unio umidus used in Mono aphidium
g i i hii and Selenas um sp. ea men s based on hei shell leng hs (cm)
Mussel species Mono aphidium g i i hii Selenas um sp.
Leng h (cm) D y weigh (g) Leng h (cm) D y weigh (g)
Anodon a ana ina (n = 3) 9.07 ± 0.71 2.19 ± 0.58 9.10 ± 0.29 2.14 ± 0.25
Anodon a cygnea (n = 3) 11.10 ± 0.78 3.98 ± 1.04 10.87 ± 0.86 3.72 ± 0.87
Unio umidus (n = 4) 7.44 ± 0.33 1.85 ± 0.31 7.70 ± 0.70 2.5 ± 0.99

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Clea ance a es (CR) o mussels we e calcula ed
o each combina ion o mussel and mic oalgal spe-
cies om changes in o al mic oalgal cell olume in
he es anks. The clea ance a es we e calcula ed
using he o mula (F os , 1972):
in which V s ands o es ank olume (4000 mL).
Fo calcula ing indi idual CR, n is 1, as he e was
1 mussel pe es ank. Fo calcula ing he size-
dependen CR, n deno es he calcula ed d y weigh
o a mussel in he es ank. Δ is he ime pe iod
be ween measu emen s in hou s. A0 is he mic oalgal
cell olume (mL mic oalgae in mL H2O) in he es
ank and A’0 is he co esponding cell olume a e age
in he con ol anks a he beginning o he ial. Fo
es ima ion o sus ained CR, he s a ing poin o he
CR
=V∕n∗Δ ∗
{
lnA0∕A −lnA
�
0
∕A
�
}
measu emen o calcula ing Δ was day 0, and he
cell olumes o A0 and A’0 we e om samples o day
0. A and A’ we e he olumes a he ime o sampling
om he es ank and he a e age cell olume in he
con ol anks. In his s udy, A and A’ we e measu ed
on days 4 and 7, o calcula e he mussels’ abili y o
main ain il a ion o a pe iod o 4 and 7 days o
es ing (sus ained CRs). When calcula ing maximum
CR (la e e e ed o as CRmax), A0 and A we e he
s a ing and he end olumes o he highes obse ed
dec ease in mic oalgal cell olume in each es ank
in he daily sampling be ween days 1–4 o he second
and hi d ials, wi h A’0 and A’ being he a e age
alues in he con ol anks a co esponding ime. The
i s ial was no included in he calcula ion o CRmax
because o some missing da a poin s on days 2–3.
Mic oalgal deposi s we e measu ed a he end o
he hi d ial (Table 2) by pho og aphing he es
anks a e d aining hem o wa e . ImageJ ( 1.50e,
Na ional Ins i u e o Heal h, Be hesda, Ma yland,
USA) was used o de e mine he a ea o mic oalgal
deposi s in each ank, including he con ols. Pho os
we e c opped o include only he a ea o es ank bo -
oms (Fig.1). Fo s a is ical analysis, he deposi ed
mic oalgal a ea in he es anks was calcula ed by
mul iplying he obse ed ela i e mic oalgal deposi
a ea by he ela i e a ea ee o mic oalgae in he con-
ol anks o accoun o algal deposi ion no ela ed o
mussel p esence in he es anks.
Phospha e-phospho us (PO4-P), ni a e-ni ogen
(NO3-N), and ammonium-ni ogen (NH4-N) we e
measu ed a he beginning and a he end o he
second ial om he ank wa e wi h a mobile
Table 2 S a a ion ime o mussels be o e he s a o he
h ee ials and he measu emen s aken (deno ed wi h x) du -
ing each ial
CR clea ance a e
T ial 1 T ial 2 T ial 3
S a a ion ime (days) 3 7 7
Mic oalgal cell olume change
o sus ained CR calcula ion
×××
Mic oalgal cell olume change
o CRmax calcula ion
– × ×
Mic oalgal deposi a ea [cm2] – – ×
PO4-P – × –
NH4-N – × –
Fig. 1 Example o mic o-
algal deposi s on he bo om
o a es ank a he end o
he hi d 7-day ial wi h
a ank con aining Mono-
aphidium g i i hii mic o-
algae and an Anodon a
cygnea mussel. (a) O iginal
pho o (da k g een a eas)
and (b) pho o a e image
analysis wi h ImageJ p o-
g am (black a eas c opped
om (a)). Bo om a eas
wi h deposi ed eces and
pseudo eces we e included
in he analyses
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labo a o y spec opho ome e (LASA 100, D .
Lange, Ge many) using es ing ki s (LCK 349
o PO4-P, LCK 339 o NO3-N, and LCK304 o
NH4-N; Hach Lange GmbH, Düsseldo , Ge many).
The samples we e p e- il e ed h ough 0.22 µm
sy inge il e s o sepa a e mic oalgal biomass
om he solu ion be o e he nu ien analyses
o he il e ed wa e . All nu ien analyses we e
made acco ding o he manu ac u e ’s ins uc ions.
Nu ien concen a ion a he beginning was
measu ed only once be o e he wa e con aining he
mic oalgae was di ided be ween he es anks.
S a is ical analyses we e pe o med wi h IBM
SPSS S a is ics 26. As he same mussel indi iduals
we e used in epea ed ials, he da a om he
h ee ials we e conside ed dependen eplica es.
Di e ences in ela i e mic oalgal concen a ions
a e day 7 and di e ences be ween obse ed
maximum clea ance a e and sus ained clea ance
a es on days 4 and 7 we e he e o e analyzed
wi h epea ed measu es ANOVA wi h mic oalgal
ea men s and mussel species as independen
ac o s. Pos hoc compa isons we e made wi h
Fishe ’s LSD es . Di e ences in mic oalgal
deposi s in he anks we e analyzed wi h one-way
ANOVA. Di e ences in nu ien concen a ions
be ween day 0 and day 7 in each ea men we e
de e mined wi h one sample es s. The h eshold
o s a is ical signi icance was 0.05.
Resul s
Rela i e concen a ions o mic oalgae a e 1, 4, and
7days
Mic oalgal concen a ion in he es anks ela i e o
he s a ing le el di e ed be ween mussel species on
days 1, 4, and 7, and wi hin mussel species on days
4 and 7 o bo h species o mic oalgae (Table 3).
Mic oalgal concen a ion by day 7 was educed o
17–58% ela i e o day 0, depending on he ea -
men (Fig.2) i.e., mussels emo ed 42–83% o algae.
Anodon a cygnea was he as es o s a il e ing wi h
some indi iduals clea ing he es anks o below 10%
algal concen a ion du ing i s 24h, and bo h Ano-
don a species dec eased algal concen a ions mo e
han Unio umidus on all measu emen days, excep
on day 1 in he M. g i i hii ial, when he A. ana ina
ea men did no di e om he U. umidus ea men
(Fig.2, Suppl. 1 and 2). By he end o he ials, ela-
i e concen a ions we e lowes in anks wi h Ano-
don a mussels when compa ed o ei he U. umidus o
con ol anks o bo h mic oalgae (Fig.2).
Mic oalgal concen a ions did no di e wi hin
each mussel ea men be ween he wo mic oalgae
(Fishe ´s LSD, P > 0.05) o all h ee mussel species
on ela i e concen a ions measu ed a days 1, 4, and
7. Wi hin con ol anks, he mic oalgal concen a ions
we e, howe e , highe o M. g i i hii han o
Selenas um sp. on days 4 and 7 (P = 0.038 and 0.015,
espec i ely) (Suppl. 1 and 2.).
Table 3 Resul s o a epea ed measu es ANOVA be ween
subjec e ec s (BSE) es s o de e mine di e ences, and wi hin
subjec e ec s (WSE) es s o de e mine consis ency in mic o-
algal concen a ions on days 1, 4, and 7 ela i e o s a ing con-
cen a ion be ween used mic oalgal species, mussel species,
and hei in e ac ions
S a is ically signi ican di e ences (P < 0.05) a e bolded
S a is ical es Day Algae Mussel species Algae × Mussel species
d , E o F alue Pd , E o F alue Pd , E o F alue P
BSE 1 1, 16 2.522 0.132 3, 16 24.327 < 0.001 3, 16 0.893 0.466
4 1, 16 8.710 0.009 3, 16 55.409 < 0.001 3, 16 0.699 0.566
7 1, 16 1.772 0.202 3, 16 64.101 < 0.001 3, 16 2.618 0.087
WSE 1 2, 32 19.787 < 0.001 6, 32 2.034 0.090 6, 32 1.927 0.107
4 2, 32 1.894 0.167 6, 32 4.318 0.003 6, 32 1.538 0.198
7 2, 32 0.901 0.413 6, 32 5.404 < 0.001 6, 32 1.915 0.109
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Maximum and sus ained clea ance a es (CR) pe
indi idual mussel
Bo h he CRmax pe indi idual mussel calcula ed
om ials 2–3 and he sus ained CRs pe indi idual
mussel o e 4 o 7days di e ed be ween he mus-
sel species (Table 4). Bo h CRmax and sus ained
CRs we e highe o A. cygnea and A. ana ina when
compa ed o U. umidus o bo h mic oalgal species
(Fig.3). The clea ance a es did no di e be ween
Fig. 2 Rela i e mic oal-
gal concen a ion on days
1, 4, and 7 (mean % o
day 0 ± SE o 3 ials) o
con ol (n = 2), Anodon a
ana ina (n = 3), A. cygnea
(n = 3), and Unio umidus
(n = 4) ea men s o (a)
Mono aphidium g i i hii
and (b) Selenas um sp.
mic oalgae. Di e en le e s
(a–c) deno e signi ican
di e ences (Fishe ’s LSD,
P < 0.05) be ween mussel
and con ol ea men s
wi hin used mic oalgae
Table 4 Resul s o a epea ed measu es ANOVA be ween
subjec e ec s (BSE) es s o de e mine di e ences, and wi hin
subjec e ec s (WSE) es s o de e mine consis ency in indi-
idual maximum clea ance a e, and sus ained clea ance a es
om day 0 o day 4 and om day 0 o day 7, be ween mic oal-
gal species, mussel species and hei in e ac ions
S a is ically signi ican di e ences (P < 0.05) a e bolded
S a is ical es Clea ance a e Algae Mussel species Algae × Mussel species
d , E o F alue Pd , E o F alue Pd , E o F alue P
BSE Maximum 1, 14 0.005 0.946 2, 14 14.199 < 0.001 2, 14 0.462 0.640
4days sus ained 1, 14 0.029 0.868 2, 14 31.889 < 0.001 2, 14 0.659 0.516
7days sus ained 1, 14 2.615 0.128 2, 14 20.187 < 0.001 2, 14 0.118 0.889
WSE Maximum 1, 14 9.535 0.008 2, 14 3.782 0.049 2, 14 2.239 0.143
4days sus ained 2, 28 8.520 0.001 4, 28 2.828 0.053 4, 28 2.226 0.092
7days sus ained 2, 28 15.279 < 0.001 4, 28 4.693 0.005 4, 28 3.144 0.030
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he Anodon a species o ei he o he mic oalgal
species (Fig.3).
Size-dependen maximum and sus ained clea ance
a es
Size-dependen CRmax calcula ed om ials 2–3
did no di e be ween he ea men s, bu he size-
dependen CR sus ained ei he o 4- o 7-day pe i-
ods di e ed be ween he mussel species o bo h spe-
cies o mic oalgae (Table5). Unio umidus had lowe
sus ained CR when compa ed o Anodon a ana ina
o e bo h 4- and 7-day pe iods when il e ing M.
g i i hii, and o e 7days when il e ing Selenas um
sp. (Fig.4). Sus ained CRs did no di e be ween U.
umidus and A. cygnea, no be ween he wo Ano-
don a species (Fig.4).
Mic oalgal deposi ion in he es anks a e 7days
The a ea o deposi ed mic oalgae on he bo om
o he es anks a he end o he hi d ial di e ed
be ween mic oalgal species (ANOVA F1,18 = 13.165,
P = 0.002). The mic oalgal deposi a ea was signi i-
can ly la ge o M. g i i hii (ca. 15–30%) han o
Selenas um sp. (ca. 5–15% o ank bo om a ea) in
Anodon a mussel anks, bu no in anks con aining
U. umidus (Fig.5).
Nu ien s
All measu ed PO4-P concen a ions on days 0 and
7 we e below he lowes de ec ion limi o he used
es ki (0.05mg L–1), hence no s a is ical es s we e
pe o med. The e was no s a is ically signi ican
change du ing he 7-day expe imen in NO3-N in
any o he anks, wi h ni a e-ni ogen le els being
unde ec able in anks con aining M. g i i hii and
Fig. 3 Boxplo s o
maximum and 4- and 7-day
sus ained indi idual a e age
clea ance a es o e 3 ials
o (a) Mono aphidium g i -
i hii and (b) Selenas um
sp. mic oalgae o Anodon a
ana ina (n = 3), A. cygnea
(n = 3) and Unio umidus
(n = 4). Di e en le e s
(a and b) deno e s a is i-
cally signi ican di e ences
(Fishe ’s LSD, P < 0.05)
be ween he mussel species
o da a se s wi h s a is i-
cal di e ences be ween
subjec s. Median, in e qua -
ile ange, min/max, and
ou lie s a e displayed, wi h
a e age indica ed wi h ×