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Di e ences in sul a e sensi i i y o ea ly de elopmen be ween b ackish and eshwa e
co egonines
© 2024 he Au ho s
Published e sion
Mäkinen, Mikko; Hu, Xiaoxuan; Ka jalainen, Juha
Mäkinen, M., Hu, X., & Ka jalainen, J. (2024). Di e ences in sul a e sensi i i y o ea ly
de elopmen be ween b ackish and eshwa e co egonines. In e na ional Jou nal o Limnology,
60, A icle 24. h ps://doi.o g/10.1051/limn/2024023
2024
RESEARCH ARTICLE
Di e ences in sul a e sensi i i y o ea ly de elopmen be ween
b ackish and eshwa e co egonines
Mikko Mäkinen
*
, Xiaoxuan Hu and Juha Ka jalainen
Depa men o Biological and En i onmen al Science, Uni e si y o Jy äskylä, Su on ie 9C, PO Box 35, FI 40014, Finland
Recei ed: 19 Decembe 2023; Accep ed: 30 Sep embe 2024
Abs ac –Sul a e is ound na u ally in he aqua ic en i onmen s, bu due o a ious an h opogenic
ac i i ies he sul a e concen a ions in su ace wa e s ha e inc eased globally. High le els o sul a e can
cause ad e se e ec s on aqua ic o ganisms. In his s udy we explo ed he e ec s o sul a e on he
ep oduc ion o wo co egonine species, whi efish (Co egonus la a e us (L.)) and endace (Co egonus
albula (L.)), in Bal ic Sea b ackish wa e and so bo eal eshwa e . The ch onic oxici y es s las ed om
e iliza ion o ha ching o he la ae, endpoin s being emb yonic and la al su i al, and size o newly
ha ched la ae. The ch onic 196–214-day es s we e conduc ed in di e en sodium sul a e (Na
2
SO
4
)
solu ions a wa e empe a u es simula ing na u al condi ions du ing he egg incuba ion om au umn o
sp ing. The sepa a e e iliza ion es s we e ca ied ou o measu e e iliza ion success (%). The e iliza ion
and ea ly emb yonic phase we e ound o be he mos sensi i e pe iods o sul a e oxici y. The su i al in
la e emb yonic phase, ha ching and 5-day la al phase was high (>80%). In he acu e e iliza ion es s wi h
b ackish wa e popula ions, he LC50- alues we e be ween 2554 and 2575 mg/L and wi h eshwa e
popula ions be ween 1107 and 1230 mg/L o sul a e. In he ch onic expe imen s om e iliza ion o
ha ching he LC10- alues o b ackish wa e popula ions we e be ween 1800 and 1820 mg/L and o
eshwa e popula ions be ween 335 and 624 mg/L o sul a e. The ole ance o sul a e in eshwa e
co egonines was significan ly lowe han b ackish wa e co egonines, bu i was in simila ange as o wha
has been obse ed in o he eshwa e species.
Keywo ds: Fish / emb yo / e iliza ion / le hal concen a ion / saliniza ion
1 In oduc ion
Sul a e (SO
4
2–
) is ound na u ally in aqua ic en i onmen s.
Due o an h opogenic ac i i ies, concen a ion o sul a e in
wa e ways can inc ease o le els which can be ha m ul o
aqua ic o ganisms. Poin sou ces o sul a e a e p ima ily
indus ial p ocesses ha u ilize sul u ic acid as pa o hei
p oduc ion cycle, d ainage om mining a eas and municipal
was ewa e s (Elphick e al., 2011;No ds om e al., 2015;
Ekholm e al., 2020). Majo di use sou ces a e un o s om
o es y a eas, e iliza ion uno s and illing o soil in
ag icul u al a eas, especially om fields loca ed on acid sul a e
soil (Elphick e al., 2011;Ekholm e al., 2020).
In bo eal eshwa e s, backg ound sul a e le els ange
om 3 o 30 mg/L, bu in a eas hea ily a ec ed by
an h opogenic ac i i ies sul a e le els can each alues as
high as o e 2000 mg/L (Ekholm e al., 2020). In he coas al
egions o he no he n Bal ic Sea he sul a e le els ange om
100 o 500 mg/L (Leh o an a e al., 2008;Zak e al., 2021).
Inc eased sul a e load in o su ace wa e s can lead o
saliniza ion and s a ifica ion, modi y he cycle o nu ien s
and cause eu ophica ion and anoxic condi ions in hypolimni-
on (Cañedo-A güelles e al., 2019;Wil se e al., 2020;Kaushal
e al., 2021;Zak e al., 2021). In addi ion, sul a e a ec s
aqua ic o ganisms by inducing osmo ic s ess which may lead
o inc eased consump ion o ene gy o main ain homeos asis
(G i fi h, 2017;Cañedo-A güelles e al., 2019) o by inducing
specific ion oxici y (Da ies and Hall, 2007). The oxici y
mechanism o sul a e is also linked o wa e ha dness, as low
wa e ha dness inc eases he oxic e ec s o sul a e (Soucek
and Kennedy, 2005;Da ies and Hall, 2007;Elphick e al.,
2011;Soucek e al., 2011).
In his s udy, we aim o u he con ibu e o he knowledge
o he e ec s o sul a e on aqua ic o ganisms in so bo eal
wa e s. We con inued he wo k om ou p e ious esea ch
(Ka jalainen e al., 2021,2023) by examining he e iliza ion
and ea ly de elopmen o wo di e en co egonine (Co ego-
ninae) species, whi efish (Co egonus la a e us (L.)) and
endace (Co egonus albula (L.)), om bo h b ackish and
eshwa e en i onmen s. In his s udy in addi ion o
anad omous whi efish which we ha e s udied ea lie
Special issue - Biology and Managemen o Co egonid Fishes - 2023
Gues edi o s: O lane Anne ille, Chloé Goulon, Juha Ka jalainen, Jean Guilla d, Ja ed T. Mye s and Jason S ockwell
*Co esponding au ho : mikko. .j.makinen@jyu.fi
In . J. Lim. 2024, 60, 24
©M. Mäkinen e al., Published by EDP Sciences, 2024
h ps://doi.o g/10.1051/limn/2024023
A ailable online a :
www.limnology-jou nal.o g
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h ps://c ea i ecommons.o g/licenses/by/4.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
(Ka jalainen e al., 2021), new oxici y es s o sea-spawning
whi efish and endace in b ackish wa e and lake-spawning
whi efish and endace in eshwa e we e ca ied ou . Vendace
is mainly an inland species, bu i is ound in Gul o Bo hnia
and Gul o Finland egions o he Bal ic Sea whe e i spawns
in i e s, es ua ies o less saline coas al habi a s (HELCOM,
2013;Leh onen e al., 2023). In Bal ic Sea he endace is
comme cially caugh o i s oe which is a comme cially
impo an p oduc (Leh onen e al., 2023). In Finland, endace
gene a ed he la ges po ion o he e enue o he comme cial
inland fishe ies and oge he wi h pikepe ch (Sande lucio-
pe ca L.), i is one o he mos impo an a ge species o
comme cial fishe s in ecen yea s (Luke, 2023).
Whi efish popula ions a e ound om bo h inland lakes as
well as om he Bal ic Sea. In he Finnish coas al egions wo
eco ypes o whi efish a e p esen , a i e -spawning whi efish
and a sea-spawning whi efish (Leh onen, 1981). The aim o
his s udy was o compa e he new and p e iously published
sul a e oxici y es esul s o se e al whi efish and endace
popula ions o examine he di e ences in sul a e sensi i i y o
ea ly de elopmen be ween b ackish and eshwa e co ego-
nines. The endpoin a iables in he oxici y es s in ou s udy
we e: 1) e iliza ion success, 2) su i al in ea ly emb yonic
phase, 3) su i al in la e emb yonic phase, 4) su i al in
ha ching and as yolk sac la ae and 5) he size o he ha ched
la ae.
2 Ma e ials and me hods
2.1 Tes species
The oxici y es s we e ca ied ou using whi efish and
endace om bo h Bal ic b ackish wa e and inland eshwa e
(Tab. 1). Vendace and whi efish bo h spawn in la e au umn and
eggs incuba e un il sp ing. The incuba ion ime can las up o 6
mon hs. We ollowed he incuba ion pe iod om e iliza ion
o ha ching o b ackish wa e whi efish o 196 days and
anad omous whi efish o 165 days and eshwa e whi efish
and endace o 214 days and 203 days, espec i ely. The
pa en al fish o whi efish and endace we e caugh by
p o essional fishe s on 26
h
o Oc obe 2022 om Bal ic Sea
in La smo a chipelago (N63°72' 5418”,E22°62' 8513”).
Eggs and spe m o whi efish we e s ipped in ha bou and
s o ed in sepa a e chilled con aine s. Eggs om endace we e
s ipped in si e, bu males we e anspo ed in ice o he
uni e si y whe e he spe m was s ipped. In he whi efish es ,
we used game es o 7 emales and 7 males and in endace
expe imen , we used 6 emales and 9 males (Supplemen a y
ma e ial Tab. 1). Anad omous sea-mig a o y whi efish
game es om 5 emales and 5 males we e ob ained om
Ri e Kokemäenjoki (N61°33' 8384”,E22°11' 7176”)
(Ka jalainen e al., 2023)(Supplemen a y ma e ial Tab. 1).
F eshwa e whi efish and endace we e ob ained om Lake
Konne esi (N62°61' 7519”,E26°35' 1125”)on2
nd
o
No embe 2022, whi efish by gill ne s and endace by seine
ne . The fish we e anspo ed in chilled con aine s o he
uni e si y whe e he eggs and spe m we e s ipped. In
eshwa e whi efish es , we used game es om 3 emales
and 6 males and in endace es we used 9 emales and 12
males (Supplemen a y ma e ial Tab. 1). Numbe o pa en al
fish a ied due o he ipeness o spawn o he fish caugh . The
oxici y es esul s o Ka jalainen e al. (2023) o oach
(Ru ilus u ilus L.) and b own ou (Salmo u a L.) we e
compa ed o whi efish and endace oxici y es esul s.
2.2 Tes se up and chemicals
Bo h he acu e and ch onic oxici y es s we e ca ied ou :
he acu e sho - e m e iliza ion es and he ch onic oxici y
es om e iliza ion o ha ching including he sho 5-day
incuba ion o ha ched la ae. In he acu e e iliza ion es ,
50–100 eggs om each sul a e ea men we e sampled o
de e mine e iliza ion success (%). In he ch onic incuba ion
es each ea men had 36 eggs which we e ollowed
h oughou he ea ly de elopmen . The es s we e applied in
same manne o each es species and popula ion and we e
analysed sepa a ely.
Table 1. Sul a e oxici y es s ca ied ou in so wa e s (RW = Ri e Kokemäenjoki wa e , LW = Lake Konne esi wa e ) and in b ackish wa e
(BW = Kokkola b ackish wa e ). Ch onic oxici y es s las ed om e iliza ion o he momen when he ha ched la ae eached age o 5 days
excep om e iliza ion o Salmo u a om e iliza ion o es ima ed eme gence momen (see Ka jalainen e al., 2023). Resul s o fi e oxici y
es s we e published fi s ime in his s udy and es o hem we e published p e iously in Ka jalainen e al. (2023).
Tes species and spawning habi a Tes ype and wa e Endpoin Sou ce
Co egonus la a e us, b ackish wa e Acu e, BW Fe iliza ion a e This s udy
Co egonus albula, b ackish wa e Acu e, BW Fe iliza ion a e This s udy
Co egonus la a e us, anad omous Acu e, RW Fe iliza ion a e Ka jalainen e al., 2023
Co egonus la a e us, esh wa e Acu e, LW Fe iliza ion a e Ka jalainen e al., 2023
Co egonus albula, esh wa e Acu e, LW Fe iliza ion a e Ka jalainen e al., 2023
Salmo u a, esh wa e Acu e, LW Fe iliza ion a e Ka jalainen e al., 2023
Ru ilus u ilus, esh wa e Acu e, LW Fe iliza ion a e Ka jalainen e al., 2023
Co egonus la a e us, b ackish wa e Acu e, BW Su i al This s udy
Co egonus la a e us, anad omous Acu e, RW Su i al Ka jalainen e al., 2023
Co egonus la a e us, eshwa e Acu e, LW Su i al This s udy
Co egonus albula, eshwa e Acu e, LW Su i al This s udy
Salmo u a, eshwa e Acu e, LW Su i al Ka jalainen e al., 2023
Ru ilus u ilus, eshwa e Acu e, LW Su i al Ka jalainen e al., 2023
Page 2 o 11
M. Mäkinen e al.: In . J. Lim. 2024, 60, 24
Expe imen al ea men solu ions we e made by dissol -
ing sodium sul a e (Na
2
SO
4
, Me ck, pu i y ≥99%) in o
fil e ed na u al wa e (Millipo e Pellicon & Du apo e GVPP,
0.22 mL). In b ackish wa e (BW) expe imen s we used
na u al wa e aken om he Bal ic Sea coas al a ea in
Kokkola (N63°92' 5371”,E23°03' 8227”, backg ound sul a e
240 mg/L) (Tab. 2). Fo eshwa e co egonine oxici y es s
na u al wa e was aken om Lake Konne esi (LW) (N62°61'
7519”,E26°35' 1125”, backg ound sul a e 5.0 mg/L) (Tab. 2)
and o anad omous whi efish es om Ri e Kokemäenjoki
(RW) (N61°33' 8384”,E22°11' 7176”, backg ound sul a e
12.2 mg/L) (Tab. 2)(Ka jalainen e al., 2023). The wa e used
as a base o ea men solu ions o e iliza ion and
h oughou he incuba ion was simila wa e om which
he pa en al fish o igina ed om. These na u al wa e s aken
om he di e en loca ions we e also he con ol ea men s
in he oxici y es s. S ock solu ions we e p epa ed om he
wa e s by dissol ing he Na
2
SO
4
in o fil e ed na u al wa e o
achie e he highes concen a ion. O he ea men solu ions
we e p epa ed by dilu ing he s ock solu ions wi h na u al
wa e o achie e he a ge concen a ions in each expe imen .
Numbe o ea men s in BW expe imen including con ol
we e 10, in LW expe imen 11 and in RW expe imen 8
(Tab. 2). Wa e ha dness o na u al wa e s we e calcula ed
om he measu ed Ca
2þ
and Mg
2þ
concen a ions. The
ha dness alues o na u al wa e s we e 5.188 ± 0.147 mmol/L
(mean ± SD) (518.8 ± 14.7 mg/L as CaCO
3
)inBW,
0.139 ± 0.004 mmol/L (13.9 ± 0.4 mg/L) in LW and
0.284 ± 0.026 mmol/L (28.4 ± 2.6 mg/L) in RW. Thus, BW
wa e was ha d (>300 mg/L), while LW and RW wa e s we e
so (<30 mg/L).
Sul a e concen a ions we e moni o ed by aking samples
h oughou he expe imen s (Tab. 2). The unce ain y o he
sul a e analysis was 10%. The analysis was p o ided by an
independen acc edi ed labo a o y and done acco ding o
SFS-EN ISO 10304–1 (2009) s anda d. Wa e empe a u e
simula ed he na u al empe a u e hy hm and was moni o ed
h oughou he expe imen s wi h au oma ic empe a u e
logge s. The pH (Se enEasy, Me le -Toledo) (Table 2) and
oxygen (Mic ox 4 ace, P eSens P ecision Sensing GmbH)
le els we e moni o ed h oughou he expe imen s. The mean
oxygen concen a ion was 11.5 ± 0.1 mg/L (± SD, n= 18).
Table 2. The nominal and measu ed sul a e (mg/L SO
4
) concen a ions (mean ± SD), amoun o added Na
2
SO
4
in o expe imen al ea men s and
he pH (mean ± SD) in b ackish wa e con ol and expe imen al ea men s (numbe o samples aken, n= 5), in Lake Konne esi con ol and
expe imen al ea men s (n= 4) and in Ri e Kokemäenjoki con ol and expe imen al ea men s (n=5) (Ka jalainen e al., 2023).
Added Na
2
SO
4
Nominal SO
4
Measu ed SO
4
pH
B ackish wa e
Kokkola con ol 240.0 ± 7.1 7.6 ± 0.3
384 mg/L 500 mg/L 496.0 ± 13.6 7.5 ± 0.1
828 mg/L 800 mg/L 810.0 ± 16.7 7.5 ± 0.1
1567 mg/L 1300 mg/L 1280.0 ± 40.0 7.5 ± 0.1
2307 mg/L 1800 mg/L 1780.0 ± 40.0 7.5 ± 0.1
2898 mg/L 2200 mg/L 2180.0 ± 40.0 7.4 ± 0.0
3490 mg/L 2600 mg/L 2600.0 ± 70.7 7.5 ± 0.1
4081 mg/L 3000 mg/L 3050.0 ± 50.0 7.4 ± 0.0
4820 mg/L 3500 mg/L 3475.0 ± 43.3 7.4 ± 0.1
6299 mg/L 4500 mg/L 4500.0 ± 0.0 7.4 ± 0.0
Lake wa e
L. Konne esi con ol 5.0 ± 0.5 7.3 ± 0.2
436 mg/L 300 mg/L 307.5 ± 8.3 7.1 ± 0.1
880 mg/L 600 mg/L 615.0 ± 11.2 7.0 ± 0.1
1323 mg/L 900 mg/L 920.0 ± 18.7 7.0 ± 0.0
1767 mg/L 1200 mg/L 1175.0 ± 43.3 7.0 ± 0.0
2358 mg/L 1600 mg/L 1600.0 ± 0.0 6.9 ± 0.0
2654 mg/L 1800 mg/L 1800.0 ± 0.0 6.9 ± 0.0
2950 mg/L 2000 mg/L 2000.0 ± 0.0 6.9 ± 0.0
3541 mg/L 2400 mg/L 2500.0 ± 0.0 6.7 ± 0.0
4281 mg/L 2900 mg/L 2900.0 ± 81.6 6.8 ± 0.0
5168 mg/L 3500 mg/L 3600.0 ± 0.0 6.8 ± 0.0
Ri e wa e
R. Kokemäenjoki con ol 12.2 ± 2.8 7.3 ± 0.1
426 mg/L 300 mg/L 304.0 ± 8.9 7.2 ± 0.2
869 mg/L 600 mg/L 598.0 ± 22.8 7.1 ± 0.2
1313 mg/L 900 mg/L 892.0 ± 29.5 7.2 ± 0.2
1757 mg/L 1200 mg/L 1200.0 ± 0.0 7.1 ± 0.2
2200 mg/L 1500 mg/L 1500.0 ± 0.0 7.1 ± 0.1
2644 mg/L 1800 mg/L 1800.0 ± 0.0 7.1 ± 0.3
3531 mg/L 2400 mg/L 2380.0 ± 44.7 7.1 ± 0.2
Page 3 o 11
M. Mäkinen e al.: In . J. Lim. 2024, 60, 24
2.3 Fe iliza ion
App oxima ely 100 eggs pe each emale we e pu in o a
500 mL glass ja and 3–5mL o spe m om each male was
dis ibu ed e enly among all eggs om he emales. 25 mL o
wa e was added in o he ja o ac i a e he game es. The ja
was gen ly s i ed and le o si o 40 seconds a e which he
eggs we e insed wi h esh wa e o emo e he excess spe m.
Once he wa e in he ja was clea i was filled up o 200 mL
and p o ided wi h ae ia ion. This was epea ed o each
di e en ea men concen a ion (Tab. 2). The e iliza ion
success a e (%) was de e mined a e 48–96 hou s pos
e iliza ion. A ound 50 o 100 andomly selec ed eggs om
each ea men we e obse ed unde a mic oscope o see i he
cell di isions had s a ed o de elop. The e iliza ion
empe a u e was 5 °C o b ackish wa e whi efish and endace
and 6 °C o eshwa e whi efish and endace. The e iliza ion
and i s success we e ega ded as an acu e exposu e expe imen .
2.4 Emb yonic pe iod and ha ching
A e 24-h pe iod eggs we e mo ed o 6-well pla es wi h
mo able inse s (VWR 6-well pla e ype 734–2717 6) wi h cu
ou Pas eu pipe e. The wells we e p efilled wi h 10 mL o
ea men solu ion co esponding wi h he ja om which he
eggs we e mo ed. Randomly selec ed eggs we e indi idually
placed in o he inse s wi hin he wells. The pla es we e
co e ed wi h lids o p e en e apo a ion du ing he expe i-
men s. Each ea men (Tab. 2) had o al o 36 eggs dis ibu ed
in o 6 pla es and di ided on wo sepa a e aluminium ays.
T ays we e placed on a shel in he cold oom. Placemen o he
ays was andomized. Tempe a u e o he cold oom was
adjus ed h oughou he expe imen o ollow he simula ed
na u al empe a u e cycle. The empe a u e a he e iliza ion
was 5 °C o b ackish wa e whi efish and endace and 6 °C o
eshwa e whi efish and endace. The a e age empe a u e
du ing he incuba ion pe iod was 2 °C o b ackish wa e
whi efish and o eshwa e whi efish and endace. The
a e age empe a u e du ing he ha ching and la al pe iod w
was 4 °C o b ackish wa e whi efish and 5 °C o eshwa e
whi efish and endace. The empe a u e was moni o ed wi h
wo sepa a e empe a u e logge s placed in o 6-well pla es
filled wi h ap wa e .
The wa e in he oxici y es s was changed weekly. Inse s
con aining he eggs we e gen ly mo ed om he old pla es o
new ones which we e p efilled wi h he co esponding sul a e
ea men . The ays housing he pla es we e also o a ed
du ing each wa e change o e en ou he possible empe a u e
fluc ua ions in he oom. The obse a ion o emb yonic pe iod
was di ided in o ea ly and la e pe iods. Once he eggs eached
he eyed s age dead eggs we e eco ded and emo ed om he
se up, hus ending he ea ly emb yonic pe iod. La e emb yonic
pe iod ended in o ha ching.
A e he fi s ha ching was obse ed pla es we e inspec ed
on daily basis o new ha chings. The da e o each ha ched
la ae was eco ded, and la ae was ollowed o 5 days
coun ing he day o ha ching as he fi s day. A e he 5-day
pe iod la ae we e emo ed om he well and p ese ed in
95% e hanol o u he analysis. The la ae we e collec ed
in o concen a ion specific ubes, espec i ely. The incuba ion
pe iod om e iliza ion o ha ching and o 5-day-old la ae
was ega ded as ch onic oxici y es .
2.5 To al leng h and mass measu emen s
To al leng h, esh and d y mass, and d y ma e (%, d y
mass di ided by esh mass) we e measu ed om he p ese ed
la ae. Be o e measu ing, la a was aken om e hanol and pu
in o a pe i dish filled wi h wa e o soak o 15 min o es o e
he size o issues (Ka jalainen, 1992). When aken ou he
la a was wiped gen ly wi h a mois pape owel o ge id o
he excess mois u e and leng h was measu ed immedia ely.
A e leng h measu emen , a la a was placed in a p e-weighed
aluminium cup and he esh mass was weigh ed wi h a
mic oscale. The la ae we e d ied a 60 °C o 24 h a e which
he cups we e eweighed o de e mine he d y mass o he
calcula ion o d y ma e .
2.6 S a is ical analysis
The e ec s o sul a e on he e iliza ion a e (%) and he
emb yonic su i al we e analysed by using K uskall–Wallis H
es and pai wise compa isons by Bon e oni es be ween he
con ol and added sul a e ea men s. The e ec s on he la al
leng h and esh mass we e analysed wi h one-way ANOVA
and he e ec s on d y ma e a io we e analysed wi h
K uskall–Wallis H es , pai wise compa isons we e done o
bo h by Bon e oni es . These analyses we e done in IBM
SPSS ( 28.0.1.1) s a is ics so wa e. The concen a ion-
esponse analysis was done by using ‘d c’package (Ri z
e al., 2015)inR(R Co e eam, 2022) o analyse he oxici y
da a. The le hal concen a ion LC10 ( he concen a ion, which
was le hal o 10% o es ed indi iduals) wi h he 95% confidence
in e als was calcula ed o ch onic exposu e ( oxici y es
om e iliza ion o 5-day-old la ae) and he le hal concen a-
ion LC50 ( he concen a ion, which was le hal o 50% o es ed
indi iduals) wi h he 95% confidence in e als was es ima ed
o acu e exposu e ( e iliza ion es ) (Supplemen a y ma e ial
Tab. 3). The s a is ical analyses we e made o each oxici y
es sepa a ely. The LC alues o he sepa a e oxici y es s
we e compa ed o e alua e he di e ences in sul a e sensi i i y
o di e en whi efish and endace popula ions o igina ed in
b ackish and eshwa e en i onmen s.
3 Resul s
3.1 Sul a e concen a ion a ec s e iliza ion and
ea ly emb yo de elopmen
Sul a e concen a ion significan ly a ec ed e iliza ion
a e o whi efish and endace in all b ackish and eshwa e
expe imen s (Fig. 1,Supplemen a y ma e ial Tab. 2). Sul a e
dis u bed ea ly cell di ision and emb yo de elopmen , and
majo i y o he mo ali y in all expe imen s occu ed in he
ea ly emb yonic phase om e iliza ion o he eyed s age
(Fig. 2,Supplemen a y ma e ial Tab. 2). In he la e emb yonic
phase om he eyed s age o 5-day-old newly ha ched la ae,
sul a e did no significan ly a ec he su i al o he
de eloping emb yos o li ing ha ched la ae (Fig. 3,
Supplemen a y ma e ial Tab. 2). The su i al o endace in
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M. Mäkinen e al.: In . J. Lim. 2024, 60, 24

Fig. 1. P opo ion o success ully e ilized eggs in b ackish wa e endace (A) (%, n = 51–104 eggs pe ea men ), b ackish wa e whi efish (B)
(%, n = 51–104 eggs pe ea men ), anad omous sea-mig a o y whi efish (C) (%, n = 50–51 eggs pe ea men ), eshwa e endace (D) (%,
n=55–80 eggs) and eshwa e whi efish (E) (%, n = 55–80 eggs). Expe imen s done in di e en sul a e (SO
4
2–
) ea men s and in con ol
ea men s (BW = b ackish wa e , RW = R. Kokemäenjoki wa e , LW = L. Konne esi wa e ) wi h backg ound sul a e concen a ions. The
significance le els a e abo e he ba s (K uskall-Wallis): =P >0.05, * = P <0.05, ** = P <0.01. Ve ical lines on ba s ep esen he 95%
confidence limi s.
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M. Mäkinen e al.: In . J. Lim. 2024, 60, 24
he LW expe imen a sul a e concen a ion o 920 mg/L was
low (0%, Fig. 3C) bu also he indi iduals a e he sul a e
exposu e by he eyed s age was low, wi h only one ali e
emb yo.
Fo he en i e du a ion o he expe imen om he
e iliza ion o he end o la al pe iod, a s a is ically significan
di e ence in su i al was ound be ween ea men s in BW
(H=108.35, DF = 9, P<0.001), RW (H=50.11, DF = 7, P<
0.001) and LW whi efish (H=255.94, DF = 10, P<0.001) and
LW endace (H=146.86, DF = 10, P<0.001) (Supplemen a y
ma e ial Tab. 2,Supplemen a y ma e ial Fig. 1).
3.2 Sensi i i y o eshwa e fish and b ackish wa e
fish
The sul a e a ec ed he e iliza ion o eshwa e whi efish
and endace in lowe concen a ions han he e iliza ion o
anad omous whi efish o b ackish wa e whi efish and endace
(Fig. 1,Supplemen a y ma e ial Tab. 3). The acu e LC50
( e iliza ion a e) o eshwa e fishes a ied om 1107 o
1353 mg/L while he LC50 o anad omous whi efish and sea-
spawning whi efish and endace we e o e 2500 mg/L.
Simila ly, he ch onic LC10 o eshwa e fishes o he en i e
incuba ion expe imen ( om e iliza ion o 5-day-old la ae)
was clea ly lowe (335–1139 mg/L) han he LC10 o Bal ic
Sea whi efish and endace (Fig. 4,Supplemen a y ma e ial
Tab. 3).
3.3 Size o newly ha ched la ae
Sul a e significan ly a ec ed leng h and esh mass o he
5-day-old la ae in anad omous whi efish and eshwa e
endace and whi efish (Fig. 5,Supplemen a y ma e ial Tab. 4).
Leng h o he la ae in he concen a ion o 1175 mg/L di e ed
s a is ically om he con ol ea men in bo h eshwa e
species, bu in e ms o esh mass none o he sul a e
Fig. 2. Su i al (%) o b ackish wa e whi efish (A) (34 days om he e iliza ion, n = 36), anad omous sea-mig a o y whi efish (B) (25 days
om he e iliza ion, n = 36), eshwa e endace (C) (34 days om he e iliza ion, n = 36) and eshwa e whi efish (D) (34 days om he
e iliza ion, n = 36) in he ea ly emb yonic phase om e iliza ion o eyed s age in di e en sul a e (SO
4
2–
) concen a ions wi h con ol
ea men s (BW = b ackish wa e , RW = R. Kokemäenjoki wa e , LW = L. Konne esi wa e ) and hei backg ound SO
4
2–
concen a ions. The
significance le els a e abo e he ba s (K uskall-Wallis): =P >0.05, * = P <0.05, ** = P <0.01. Ve ical lines on ba s ep esen he 95%
confidence limi s.
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M. Mäkinen e al.: In . J. Lim. 2024, 60, 24
concen a ions di e ed s a is ically om con ol ea men
(Fig. 5). In anad omous whi efish expe imen a significan
di e ence was ound in leng h and esh mass measu emen s
o he 5-day-old la ae, bu no in d y ma e (Fig. 5,
Supplemen a y ma e ial Tab. 4).
4 Discussion
In his s udy he e iliza ion and he ea ly emb yonic phase
o co egonines we e he mos sensi i e pe iods o sul a e
exposu e in he e ms o su i al. In p e ious s udies
Ka jalainen e al. (2021) epo ed ha he e iliza ion and
he ea ly pe iod o emb yonic de elopmen o a mig a o y
anad omous whi efish we e ound o be sensi i e o he sul a e
exposu e. Meays and No din (2013) no ed in hei s udy ha
he sensi i e phase o sul a e in de elopmen o ainbow ou
(Onco hynchus mykiss (Walbaum)) emb yo was om eyed
s age o ale in, bu hey also men ioned ha p e-eyed s age
could be e en mo e sensi i e endpoin . Ou esul s showed ha
he p e-eyed s age and e iliza ion we e indeed he mos
sensi i e pe iods in ea ly de elopmen o fish. The mechanism
o sul a e oxici y is ei he h ough osmo ic s ess o specific
ion oxici y (Da ies and Hall, 2007;G i fi h, 2017;Cañedo-
A güelles e al., 2019). Sul a e can dis u b he osmo ic
homeos asis o he cell and c ea e imbalance in wa e
egula ion wi hin, hus causing oxic e ec s o he o ganism
(Da ies and Hall, 2007). Compa ed wi h o ganisms de eloped
in saline en i onmen s, o ganisms de eloped in hypo-osmo ic
condi ions o eshwa e en i onmen s a e mo e h ea ened by
inc eases o salini y and a e mo e suscep ible o he ad e se
e ec s o osmo ic s ess and specific ion oxici y (Cañedo-
A güelles e al., 2019). The osmo egula o y adap a ion o
saline en i onmen s (Ha e al., 1991;Koel and Pe e ka, 1995;
Fyhn e al., 1999) can explain he di e ence ound in ou s udy
Fig. 3. Su i al (%) o b ackish wa e whi efish (A) (169 days om he end o ea ly emb yonic pe iod, n = 36), anad omous sea-mig a o y
whi efish (B) (140 days om he end o ea ly emb yonic pe iod, n = 36), eshwa e endace (C) (175 days om he end o ea ly emb yonic
pe iod, n = 36) and eshwa e whi efish (D) (186 days om he end o ea ly emb yonic pe iod, n = 36) du ing he la e emb yonic phase, ha ching
and la al pe iod in di e en sul a e (SO
4
2–
) concen a ions wi h con ol ea men s (BW = b ackish wa e , RW = R. Kokemäenjoki wa e ,
LW = L. Konne esi wa e ) and hei backg ound SO
4
2–
concen a ions. The significance le els a e abo e he ba s (K uskall-Wallis): =P >
0.05, * = P <0.05, ** = P <0.01. Ve ical lines on ba s ep esen he 95% confidence limi s.
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M. Mäkinen e al.: In . J. Lim. 2024, 60, 24
be ween he b ackish wa e and eshwa e o ms. The
anad omous sea-mig a o y whi efish and bo h b ackish wa e
endace and whi efish exhibi ed high ole ance (i.e., ha ing high
LC alues) agains sul a e exposu e in he acu e e iliza ion es s
aswellasin hech onicincuba ion expe imen s. The ole anceo
he eshwa e o ms was lowe , and he LCs we e a he le el o
common oach and b own ou epo ed in Ka jalainen e al.
(2023) (Supplemen a y ma e ial Tab. 3).
High sul a e concen a ions a ec ed he ha ching leng h o
eshwa e endace and whi efish. The leng h o 5-day old
la ae was sho e in high sul a e concen a ions. The dec ease
in size and mass in ela ion o he inc ease o sul a e
concen a ion migh be he esul o inc eased osmo ic s ess
which may hinde g ow h and de elopmen (Cañedo-
A güelles e al., 2019). On he o he hand, he leng h o he
5-day-old la ae inc eased in high sul a e concen a ions in he
anad omous whi efish expe imen , eflec ing again highe
sul a e ole ance o sea-mig a o y whi efish. In all, he
di e ences in ha ching size o la ae be ween he sul a e
concen a ions we e small, which indica ed ha he emb yonic
g ow h o endace and whi efish a e eyed s age was no e y
sensi i e o sul a e. Ka jalainen e al. (2021) epo ed ha size
di e ences in la ae ha ched om a 31-day egg incuba ion
sul a e-exposed expe imen e en ually e ened ou du ing he
ollowing sul a e- ee depu a ion p ocess. Fu he esea ch is
equi ed o find ou how ex ended sul a e exposu e a ec s he
g ow h o whi efish la ae.
The e iliza ion success and su i al in emb yonic
de elopmen in ou con ol g oups o na u al eshwa e o
b ackish wa e we e a he same le el as epo ed in ea lie
s udies (Ka jalainen e al., 2015;Ka jalainen e al., 2021;
S ewa e al., 2021). In p esen s udy, whi efish had >94%
e iliza ion success, and 53–92% su i al du ing he
emb yonic and la al pe iod. Fo compa ison, in Ka jalainen
e al. (2015) whi efish had >85% emb yonic su i al in
eshwa e expe imen and in S ewa e al. (2021) he su i al
was 51%. In ou p e ious s udy (Ka jalainen e al., 2021), he
e iliza ion a e o whi efish was >88% and he emb yonic
su i al 58–85%. In he p esen sul a e expe imen s, endace
e iliza ion success was >69% in con ol ea men s and
su i al in emb yonic pe iod was 53%. In Ka jalainen e al.
(2015) he emb yonic su i al o endace was much lowe
(35–50%) while in S ewa e al. (2021) he emb yonic
su i al o whi efish in Konne esi con ol wa e was >80%.
Al oge he , he e iliza ion success and he su i al o
emb yonic de elopmen in co egonines ha e been e y
a iable in he di e en expe imen al s udies and i is well-
known ha he ep oduc ion success o whi efish can be
a ec ed by male- emale pai ings and hei gene ic compa i-
bili y (Wedekind e al., 2001,2008;Wedekind and Mülle ,
2004). Thus, andom selec ion o emales and males in
c ossing may lead o a iable e iliza ion success and
emb yonic su i al, e en in he cases whe e he eggs and
mil o se e al indi iduals ha e been mixed. Acco ding o
Wedekind e al. (2001), ma e selec ion and pai spawning
beha iou s o whi efish play an impo an ole in selec ing
compa ible genes o u u e o sp ing. Fu he mo e, male-
emale in e ac ions influence ea ly emb yonic su i al
(Wedekind and Mülle , 2004;Wedekind e al.,2008)bu in
he la e emb yonic s age, su i al seemed o be a ec ed by male
ai s such as size o he b eeding ube cles which co ela ed wi h
he su i al(Wedekind e al., 2008). The ep oduc i e beha iou
o endace has been s udied much less. Vendace has
pai spawning bu no b eeding ube cles (Ka jalainen and
Ma jomäki, 2017). I is possible ha hey ha e also ma e
selec ion, hough mechanisms and selec ion cues a e unknown.
Ou expe imen s wi h b ackish and eshwa e co egonines
om Kokkola egion, Lake Konne esi and Ri e Kokemäen-
joki, las ing om e iliza ion o ha ching and la al s age,
showed ha he success o e iliza ion and he su i al du ing
Fig. 4. The LC50 alues o e iliza ion (A) and LC10 alues o en i e incuba ion pe iod (B) in di e en sul a e (SO
4
2–
) concen a ions wi h
esul s om ea lie expe imen s (Ka jalainen e al., 2021;Ka jalainen e al., 2023). The species in he cha a e endace (C.alb), whi efish
(C.la ), oach (R. u ) and b own ou (S. u ) wi h di e en backg ounds (LW = lake wa e , RW = i e wa e , BW = b ackish wa e ). Ve ical
lines on ba s ep esen he 95% confidence limi s.
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