Differences in sulfate sensitivity of early development between brackish and freshwater coregonines

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ 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 Page 4 o 11 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. Page 5 o 11 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. Page 6 o 11 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. Page 7 o 11 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. Page 8 o 11 M. Mäkinen e al.: In . J. Lim. 2024, 60, 24