Beauvericin and enniatin B mycotoxins alter aquatic ecosystems : Effects on green algae

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-NC-ND 4.0 h ps://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ Beau e icin and ennia in B myco oxins al e aqua ic ecosys ems : E ec s on g een algae © 2024 he Au ho s Published e sion Juan-Ga cía, Ana; Juan, C is ina; Taipale, Sami; Vehniäinen, Ee a-Riikka Juan-Ga cía, A., Juan, C., Taipale, S., & Vehniäinen, E.-R. (2024). Beau e icin and ennia in B myco oxins al e aqua ic ecosys ems : E ec s on g een algae. En i onmen al oxicology and pha macology, 107, A icle 104415. h ps://doi.o g/10.1016/j.e ap.2024.104415 2024 En i onmen al Toxicology and Pha macology 107 (2024) 104415 A ailable online 19 Ma ch 2024 1382-6689/© 2024 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by- nc-nd/4.0/). Beau e icin and ennia in B myco oxins al e aqua ic ecosys ems: E ec s on g een algae Ana Juan-Ga cía a , b , * , C is ina Juan a , Sami Taipale b , Ee a-Riikka Vehni¨ ainen b a Labo a o y o Food Chemis y and Toxicology, Facul y o Pha macy and Food Science, Uni e si y o Valencia, A . Vicen And ´ es Es ell´ es s/n, Val` encia, Bu jasso 46100, Spain b Depa men o Biological and En i onmen al Science, Uni e si y o Jy ¨ askyl¨ a, Su on ie 9C, Jy ¨ askyl¨ a FI-40014, Finland ARTICLE INFO Keywo ds: G een algae Beau e icin Ennia in B Phy o oxici y Mix u es ABSTRACT Myxo oxins can con amina e algal-based p oduc s and a i e o he ood chain o consume s p oducing ch onic oxici y e ec s. He e, we s udied phy o oxici y o myco oxins, beau e icin (BEA) and ennai in B (ENN B) in ou phy oplank on s ains: Acu odesmus sp., Chlamydomonas einha d ii, Haema ococcus plu ialis, and Mono aphidium g i i hii, which a e all g een algae. I was es ed he capaci y o clea ing he media o BEA and ENN B a di e en concen a ions by compa ing nominal and measu ed quan i ica ions. Resul s e ealed ha Acu odesmus sp. and C. einha d ii ended o low up and down g ow h a e wi hou eaching alues below 50% o 60%, espec i ely. On he o he hand, o H. plu ialis and M. g i i h, IC 50 alues we e eached. Rega ding he clea ance o media, in indi idual ea men a dec ease o he quan i ied myco oxin be ween nominal and measu ed alues was obse ed; while in bina y ea men , di e ences among bo h alues we e highe and mo e no ed o BEA han o ENN B. 1. In oduc ion De ec ing ans o ma ions in plan ecosys ems a low exposu e le els is challenging due o hei high sensi i i y o g ow h condi ions. How- e e , unicellula plan s possess cha ac e is ics ha make hem a mo e accessible sys em o conduc ing a ious s udies and moni o ing ecosys em impac s. This is pa icula ly ue o algae species ha ha e a well-de ined gene ic sys em and can be easily ans o med (Alexande e al., 1999). Aquacul u e has eme ged as a apidly g owing sec o in global ood p oduc ion o e he pas ew decades. Howe e , li le is known abou ood sa e y conce ns ela ed o po en ial oxigenic ungi in algal cul u es (Viegas e al., 2019), and e en less is unde s ood abou he en i on- men al associa ion be ween myco oxins and aqua ic o ganisms. Ce eals and o he plan s a e o en u ilized as componen s o supplemen o enhance ish meal in aquacul u e die s. This common p ac ice is employed in in ensi e o semi-in ensi e aquacul u e se ings due o i s ad an ages, including consis en quali y, op imal nu ien balance o g ow h, easy adminis a ion, and lowe o e all cos (Viegas e al., 2019). Howe e , his p ac ice also ca ies he isk o in oducing pollu an s gene a ed by ungi on ce eals in o he ood chain. Mo eo e , ce eal-based eeds ha e he po en ial o in oduce oxigenic ungi in o he aquacul u e en i onmen (Embaby e al., 2015; Viegas e al., 2019). While se e al esea che s ha e documen ed he nega i e impac o myco oxins on ish heal h (Pie sch e al., 2013; Tola e al., 2015), he e is a lack o knowledge ega ding he phy o oxic e ec s on unicellula plan s such as algae, which a e also he ounda ion o he aqua ic ophic chain. The p esence o myco oxins in eed, ood, and he en i onmen is a global p oblem o high signi icance ha equi es comp ehensi e s udy om a ious pe spec i es. I has been epo ed ha exposu e o hese myco oxins can lead o dea h and diseases in bo h animals and humans (Pie sch e al., 2013; Tola e al., 2015). Ch onic exposu e in humans can occu h ough he consump ion o plan -based, ish-based, and mea -based oods. Food au ho i ies ha e aken s eps o egula e myco- oxins wi h well-known oxici ies, as some ha e been classi ied by he In e na ional Associa ion o Resea ch in Cance (IARC) (EFSA, 2014; EFSA, 2019; EC, 2006). The impac o myco oxins on algae is o pa icula in e es and impo ance due o hei ole in he nu i ional basis o bo h aqua ic animals and he aqua ic en i onmen , especially in he con ex o ce eal- based eed. Any changes o al e a ions in algal g ow h, nu i ional * Co esponding au ho a : Labo a o y o Food Chemis y and Toxicology, Facul y o Pha macy and Food Science, Uni e si y o Valencia, A . Vicen And ´ es Es ell´ es s/n, Val` encia, Bu jasso 46100, Spain. E-mail add ess: [email p o ec ed] (A. Juan-Ga cía). Con en s lis s a ailable a ScienceDi ec En i onmen al Toxicology and Pha macology jou nal homepage: www.else ie .com/loca e/e ap h ps://doi.o g/10.1016/j.e ap.2024.104415 Recei ed 14 July 2023; Accep ed 14 Ma ch 2024 En i onmen al Toxicology and Pha macology 107 (2024) 104415 2 p ope ies, o oxygen accumula ion can ha e ipple e ec s h oughou he ophic chain, ul ima ely impac ing a wide ange o species, om small o la ge. Global wa ming has al eady b ough abou changes in phy oplank on, zooplank on, and ish communi ies, mainly h ough cyanobac e ia blooms (Taipale e al., 2021). This, in u n, has a ec ed he p esence o ce ain myco oxins, which we e p e iously p esen a low le els bu ha e now s a ed o show a di e en p o ile in ood (Juan e al., 2020; El Jai e al., 2021). The li e a u e highligh s he i al ole o mic oalgae in he global ca bon cycle, p oduc ion o bio-based compounds, wa e pu i ica ion, and ood p oduc ion (Tho n on, 2012; De adas e al., 2021; Kusmayadi e al., 2021; Yada e al., 2021). Accu a e es ima ion o mic oalgae g ow h is c ucial o en i onmen al moni o ing and mic oalgae bio echnology. Howe e , he p esence o con aminan s can signi ican ly impac hei g ow h. The p esence o myco oxins in wa e is o en de i ed om leakage in plan issue, which is ca ied in o neighbo ing ponds h ough uno wa e . This, as men ioned ea lie , can a ec aqua ic species such as algae, ish, amphibians, and zooplank on (Goessens e al., 2021). Ag i- cul u al p ac ices ha e been iden i ied as he main sou ce o myco oxin con amina ion, as demons a ed in a s udy conduc ed by Schenzel e al. (2012), which de ec ed a ious myco oxins including 3-ace yl-deoxyni- alenol, deoxyni alenol, usa enone-X, ni alenol, HT-2 oxin, T-2 oxin, beau e icin, and zea alenone in wa e d ainage. F om he g oup o eme gen myco oxins, beau e icin (BEA) and ennia in B (ENN B) ha e inc eased hei p esence in ood and eed in he las decade (K íˇ zo ´ a e al., 2021). Bo h ha e been mainly s udied in di e en cell lines a qui e low mic omola concen a ions al hough e ealing se ious p oblems o consume s and animals. When assayed in mix u es syne gis ic and addi i e e ec s ha e been epo ed in i o (K íˇ zo ´ a e al., 2021; Agahi e al., 2020; Juan-Ga cía e al., 2019b; Juan-Ga cía e al., 2019a). BEA and ENN B wo k as ionopho es inco - po a ing in o he cell memb ane and c ea ing po es ha a e speci ic o ca ions (Kamya e al., 2004). Ennia ins (ENs) inhibi enzymes like acyl-CoA, choles e ol acyl ans e ase (ACAT), and cause a dec ease in iglyce ide p oduc ion and a smalle pool o ee a y acids in he cells a ising a hypolipidemic impac (Kamya e al., 2004). In addi ion, ROS p oduc ion by bo h BEA and ENN B has been demons a ed in di e en s udies (Juan-Ga cia e al., 2020b; P ospe ini e al., 2013). This s udy aims o in es iga e he biological e ec s o myco oxins in he aquacul u e en i onmen , speci ically ocusing on he po en ial exposu e o a ious aqua ic species o hese oxins (Viegas e al., 2019). Unde s anding hese e ec s is c ucial as i allows us o assess he p ox- imi y o myco oxins in edible issues and ma ine ood o consume s. Mo eo e , he impac o eme gen myco oxins and hei combina ions on algal species emains la gely unknown, unde sco ing he necessi y o comp ehensi ely con olling he e ec s o hese compounds. The co-occu ence o myco oxins wi h o he pollu an s signi ican ly in- c eases he eco oxicological isk. The e o e, in es iga ing he e ec s o myco oxins, bo h indi idually and in combina ion wi h o he con ami- nan s, is o u mos impo ance o holis ic isk assessmen and man- agemen in aqua ic en i onmen s. Fo he i s ime, i has been a emp ed o unde s and he ela ion- ship be ween myco oxins in aqua ic se ings and hei e ec s on algae. He e i is examined he e ec s o myco oxins in g ow h algae, hei accumula ion in algae, and how algae emo e such con aminan s om he media by clea ing. This has been ca ied ou by exposu e o beau- e icin (BEA) and ennai in B in ou phy oplank on g een algal s ains, including Acu odesmus sp., Chlamydomonas einha d ii, Haema ococcus plu ialis, and Mono aphidium g i i hii (ENN B). Assays we e pe o med by indi idual and mix u e exposu e. This las epo s a eal scena io as exposu e o con aminan s does no occu usually one by one compound. The s udy b ough us o highligh he nega i e implica ion ha myco- oxins can ha e in he en i onmen and in in e eb a es ha cons i u e he nu i ional basis o mos aqua ic animals. 2. Ma e ial and me hods 2.1. Reagen s The myco oxins used we e pu chased om Sigma Ald ich: beau- e icin (BEA, MW: 783.95 g/mol) and ennia in B (ENN B, MW: 639.82 g/mol). Me hanol (MeOH) was ob ained om Fishe Scien i ic (Helsinki, Finland), LC–MS sol en g ade. Deionized wa e (<18 MV cm esis i i y) was ob ained om milli-Q wa e pu i ica ion sys em (Milli- po e, Bed o d, MA, USA). S ock solu ions o myco oxins we e p epa ed in MeOH and main ained a −20ºC in he da k. The inal concen a ion o MeOH in he medium was ≤0.5% ( / ) as p e iously epo ed (Dom e al., 2012). 2.2. Algae cul u e main enance The algae spp. Acu odesmus spp. (Uni e si y o Basel), Chlamydomo- nas ehniad i (UWCC), Haema occocus plu ialis, and Mono aphidium g i hii (NIVA-CHL 8) we e g own in MWC-medium (Modi ied W igh Chu-medium) (Guilla d e al., 1972) in 600 mL plas ic issue cul u e lasks. Each s ain was kep a +18ºC and unde a ligh :da k cycle o 16:8 h. The in ensi y o he illumina ion was selec ed based on known p e e ences which oscilla ed be ween 10 and 80 µmol quan a s −1 m −2 (lums) in o de o ensu e homogeneous g ow h condi ions. Pla es we e p epa ed by aking aliquo s om exponen ial g owing s ocks. 2.3. Myco oxin exposu e The concen a ion o he myco oxins and exposu e ime a e wo ac o s ha we e conside ed in his s udy. The algae spp. we e exposed o BEA and ENN B myco oxins indi idually o 24, 48, 72 and 96 h a a concen a ion in he anges o 0.1–64 µM o BEA and 0.05–25 µM o ENN B, all wi h 1:2 dilu ion. Concen a ions we e chosen based on s udies o aqua ic spp p e iously epo ed (Juan-Ga cía e al., 2020a, 2021). Algae we e also exposed o myco oxins in a bina y mix u e [BEA +ENN B] a a concen a ion ange om [8 +1.6] μ M o [0.06 +0.01] μ M, a ou exposu e imes (24, 48, 72 and 96 h) and including eigh dilu ions o each myco oxin concen a ion. The dilu ion a ios o he concen a ions o he bina y combina ions we e 5:1. Sol en con ol used was MeOH and main ained a ≤0.5% o all assays. 2.4. Phy o oxici y o BEA and ENN B in Acu odesmus spp., Chlamidomonas einha d ii, Haema occocus plu ialis, and Mono aphidium g i i hii Cul u e o algae s ains was main ained in MWC media as indica ed in Sec ion 2.2. Sub-cul u e o algae was p epa ed p e ious o expe i- men s wi h dilu ions main ained on an o bi al shake a 120 pm in E lenmeye s wi h cons an illumina ion (80 lums) and empe a u e (24 ºC). Only when g ow h cul u e was exponen ial (obse ed by up-g ow h in cell densi y) 96-well/pla es we e p epa ed a he ollowing densi y acco ding o he algae spp.: Acu odesmus spp and Chlamidomonas ein- a d ii a 5 ×10 4 cells/mL, Haema occocus plu ialis a 3 ×10 5 cells/mL and Mono aphidium g i hii a 1 ×10 5 cells/mL. A o al o 6 eplica es pe es solu ion we e used. The phy o oxici y assay was pe o med by measu ing he chlo ophyll luo escence pa ame e using a Fluo oskan Ascen (Labsys ems). B ie ly, 96 well/pla es we e algae seeded a densi ies epo ed abo e (100 µL/ well) join ly o myco oxin´s dilu ions. Pla es we e placed a 18 ºC and 80 lums and luo escen measu emen a 670 nm ook place a e 24, 48, 72, and 96 h. 2.5. Quan i ica ion p ocedu e o myco oxins by LC-MS/MS in aqua ic media om algae exposu e Fo quan i ying myco oxins in he aqua ic media o algae species a A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 3 long o he exposu e ime (24–96 h), a o al o 0.6 mL o media collec ed om six epea s om he 96-well pla e was allowed o p oceed. Myco- oxins we e con ained a di e en concen a ions (Sec ion 2.3). Ch oma og aphic sepa a ion was done by using an Agilen 1290 In ini y UHPLC sys em coupled o a 6460 T iple Quad upole mass spec ome e (Agilen Technologies, Waldb onn, Ge many). Sepa a ion was pe o med by using a column Agilen In ini yLab Po oshell 120 EC- C18 100 mm ×2.1 mm (inside diame e ), 2.7 μ m, he empe a u e was main ained a 30 ◦C. Mobile phases we e wa e con aining 0.1% o mic acid and 5 mM ammonium o ma e (phase A), and me hanol (phase B) wi h 0.1% o mic acid and 5 mM ammonium o ma e. An elu ion g adien o 15 min was made keeping he low a e o he mobile phase a 0.3 mL/min, The ollowing g adien was used: equilib a ion o 1 min a 70% A, 70–10% A in 10 min, 10% A o 2 min, 10–70% A in 0.5 min, 70% A o 4.5 min. and he injec ion olume was se a 3 μ L. The analysis was pe o med wi h a iple quad upole mass spec- ome e Agilen 6460 equipped wi h an elec osp ay ioniza ion sou ce (ESI) using Agilen Je S eam Technology. The ion sou ce pa ame e s we e se as ollows: shea h gas empe a u e, 340 ◦C; shea h gas low, 10 L/min, gas empe a u e, 200 ◦C; gas low, 8 L/min; nebulize , 40 psi; capilla y ol age, 3500 V; and nozzle ol age 500 V. The agmen ol age (FV), cell accele a o ol age (CAV), collision ene gy (CE) and mass ansi ions we e op imized o each me aboli e using MassHun e Op imize so wa e (Table 1). In all cases, he CAV pa ame e was se a 7. Analysis was done in dynamic mul iple eac ion moni o ing (dMRM) mode and wo mass ansi ions we e moni o ed o each oxin, one o quan i ica ion and ano he o con i ma ion. The quan i ica ion was done wi h MassHun e Quan i a e So wa e. 2.6. S a is ical analysis Mean inhibi ion concen a ion (IC 50 ) alues we e es ima ed by lineal in e pola ion. S a is ical analysis o da a we e ca ied ou using SPSS e sion 13 (SPSS, Chicago, IL, USA) s a is ical so wa e package. Da a we e exp essed as mean ±SD o h ee independen expe imen s. The s a is ical analysis o he esul s was pe - o med by s uden ’s T- es o pai ed samples. Di e ences be ween di e en ypes o cells we e analyzed s a is ically wi h ANOVA ollowed by he Tukey HDS pos hoc es o mul iple compa isons. The le el o p≤0.05 was conside ed s a- is ically signi ican . 3. Resul s 3.1. Phy o oxici y o BEA, ENN B and hei mix u es in Acu odesmus spp., The e ec o BEA, ENN B, and hei mix u es on Acu odesmus spp., is epo ed in Fig. 1. Resul s e ealed a s ong dec ease o g ow h a e a all imes assayed o BEA and a concen a ions >8 µM anging om 50% o 11% o 24 h and 96 h, espec i ely espec o con ol(Fig. 1a). Simila ly, his happened o ENN B a concen a ions >6.25 µM wi h g ow h a e anging om 92% o 56% o 72 h and 96 h, espec i ely (Fig. 1b). IC 50 alues we e eached o he ollowing condi ions: i) o BEA: 18.0± 0.3 µM, 9.5 ±0.3 µM, 14.0 ±0.7 µM and, 13.1 ±0.5 µM o 24 h, 48 h, 72 h, and 96 h, espec i ely (Table 2); and ii) o ENN B: 12.0±0.5 µM, 10.6 ±0.6 µM, 5 ±0.4 µM o 48 h, 72 h, and 96 h, espec i ely (Table 2). Howe e , when mix u es we e assayed a se e al concen a- ion (<8 µM o BEA and <1.6 µM o ENN B) he e was a dec ease in g ow h a e when concen a ions inc eased bu he e was no a clea endency obse ed. Such dec eases we e abo e 60% and oscilla ions o all imes, concen a ions desc ibed he p o ile o his assay (Fig. 1c), and no IC 50 alues we e ob ained. In summa y, while in indi idual exposu e he IC 50 alues we e eached o almos all imes assayed, in bina y mix u e numbe o algae cells we e ne e abo e 50% o con ol (Fig. 1). 3.2. Phy o oxici y o BEA, ENN B and hei mix u es in Chlamydomonas einha d ii Fig. 2 epo s he e ec on Chlamydomonas einha d ii caused by myco oxins s udied he e. E ec o BEA esul ed in a dec ease o g ow h a e a e 8 µM o 24 h and 48 h in 14% and 22%, espec i ely; while a 72 h and 96 h an inc ease o g ow h a e was obse ed eaching maximum alues o 163% (32 µM) and 134% (64 µM), espec i ely (Fig. 2a). Fo ENN B inc eases o g ow h a e we e less no iceable al hough i was kep o all imes abo e 80% and almos a s aigh line de ines he p o ile o all imes assayed (Fig. 2b). A he highes con- cen a ion assayed (25 µM) he ollowing pe cen ages o g ow h a e we e epo ed 105%, 96%, 126% and, 149%, o 24 h, 48 h, 72 h, and 96 h espec i ely. Las ly, o BEA +ENN B di e en endency was obse ed wi h he exposu e ime and he g ow h a e was abo e 60% (Fig. 2b). A 24 h a dec ease in g ow h a e was obse ed (40%) a he highes concen a ion es ed [8 +1.6] µM; a 48 h and 96 h, i was obse ed sligh eco e y o g ow h a e wi h oscilla ion anges o 95–78% and 102–123%, espec i ely; while a 72 h g ow h a e anged om 89% o 134% a [0.12 +0.02] µM and [8 +1.6] µM, espec i ely (Fig. 2c). In summa y and in gene al e ms o Chlamydomonas ein- ha d ii ei he o indi idual o bina y ea men , he e e ec s on g ow h a e we e mild, wi hou ge ing alues below 60% (Fig. 2), so ha no IC 50 alues we e eached. 3.3. Phy o oxici y o BEA, ENN B and hei mix u es in Haema occocus plu ialis The p o ile e ec o myco oxins in algae Haema occocus plu ialis e- po ed in Fig. 3 is simila o Accu odesmus spp. (Fig. 1) o bo h scena ios s udied, indi idually and combined. Fo BEA, a he wo highes con- cen a ions s udied (64 µM and 32 µM), g ow h a e was below 37% o all imes s udied; howe e , a lowe concen a ions g ow h a e was abo e 41% excep o 24 h which a 8 µM g ow h a e dec eased un il 39% and o he nex concen a ion, 4 µM, g ow h a e inc eased un il 60% (Fig. 3a). IC 50 alues we e eached o all imes as ollows: 15.0 ±0.5 µM o 24 h and 22.5±0.8 µM o 48 h and 72 h, and 15.0 ±0.2 o 96 h (Table 2). Fo ENN B, dec eases in g ow h occu ed in a concen a ion-dependen manne wi h dec eases a he highes con- cen a ion (25 µM) o 60%, 73%, 85%, and 89% o 24 h, 48 h, 72 h, and 96 h, espec i ely (Fig. 3b). IC 50 alues we e eached o all imes as ollows: 21.5 ±0.5 µM, 18.0 ±0.3 µM, 16 ±0.3 µM and, 8 ±0.5 µM o 24 h, 48 h, 72 h, and 96 h, espec i ely (Table 2). Fo mix u es o BEA + ENN B dec eases espec o he con ol wen om 2% o 52%, om 1% o 34%, om 1% o 12%, and om 21% o 35% o 24 h, 48 h, 72 h, and 96 h, espec i ely (Fig. 3c). I is impo an o ema k ha g ow h a e in [BEA +ENN B] o 24 h and 72 h was abo e 100% in all concen a ions s udied excep o [4 +0.8] and [8 +1.6] µM (Fig. 3c). IC 50 alues we e eached only o 24 h a [8 +1.6] µM (Table 2). Table 1 IC 50 alues eached o algae spp a di e en exposu e imes. IC 50 (µM) - Exposu e ime Algae spp. 24 h 48 h 72 h 96 h Accu odesmus spp. BEA 18 ±0.3 9.5 ±0.3 14.0 ±0.7 13.1 ±0.5 ENN B n. . 12.0 ±0.5 10.6 ±0.6 5.0 ±0.4 BEA +ENN B n. . n. . n. . n. . Haema ococcu plu ialis BEA 15.0 ±0.5 22.5 ±0.8 22.5 ±0.8 15 ±0.2 ENN B 21.5 ±0.5 18 ±0.3 16 ±0.3 8 ±0.5 BEA +ENN B [8 +1.6] n. . n. . n. . Mono aphidium g i hii BEA 14.0 ±0.7 n. . n. . n. . ENN B 5.0 ±0.5 9.0 ±0.3 8 ±0.5 8 ±0.5 BEA +ENN B [4 +0.8] n. . n. . n. . n. .: no eached A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 4 3.4. Phy o oxici y o BEA, ENN B and hei mix u es in Mono aphidium g i hii Resul s o Mono pahidium g i hii a e epo ed in Fig. 4. A 24 h bo h in indi idual and combined assays, he dec ease in g ow h a e was close o 50% o all concen a ions; while o 48 h, 72 h, and 96 h he p o ile desc ibed is e y simila among concen a ions assayed (Fig. 4). Fo BEA he highes dec ease o g ow h a e was de ec ed a 64 µM: 24%, 34%, and 39% o 48 h, 72 h, and 96 h, espec i ely (Fig. 4a) IC 50 alue was calcula ed only o 24 h and i was 14.0±0.7 µM (Table 2). Fo ENN B, he e was a alley e ec a 12.5 µM o exposu e imes o 48 h, 72 h, and 96 h, o inc ease a e wa d a 25 µM and eaching alues o 62% and 71% o g ow h a e o 72 h and 96 h, and 48 h, espec i ely (Fig. 3b). IC 50 alues we e eached o all imes as ollows: 5.0±0.5 µM, 9.0 ±0.3 µM, 8±0.5 µM o 24 h, 48 h, and bo h 72 h and 96 h, espec i ely (Table 2). Fo BEA +ENN B, he p o ile desc ibed o 48 h, 72 h, and 96 has an up and down p o ile shape al hough no below 80% and wi h dec eases o 2–39% (Fig. 4c). IC 50 alue could be calcula ed only o 24 h a [BEA +ENN B] [4 +0.8] µM (Table 2). 3.5. Quan i ica ion o myco oxins in algae spp Myco oxin concen a ions we e es ed a he beginning and he end o s udies wi h mean concen a ions o he exposu e pe iod calcula ed, whe e s udies o 24 h in leng h concen a ions we e based upon s a concen a ions only due o he small olume o solu ion used ac oss he eplica es. The quan i ied concen a ion o BEA and ENN B ound in he media a e he phy o oxici y assay is epo ed in Fig. 5. I is ep esen ed he nominal concen a ion and he measu ed concen a ions ha we e able o quan i y a e he exposu e. G aphics collec mean alues o ei he indi idually and mix u e scena ios a di e en condi ions es ed. I is also e ealing he capaci y ha he algae spp. had in clea ing he aqua ic media o bo BEA and ENN B myco oxin. In indi idual ea men exposu es (Figs. 5a and 5b), BEA showed dec ease o myco oxins quan i ied in 0.5 µM (dec ease o 36% espec o he con ol) o 16 µM (dec ease o 25% espec o he con ol), o he lowes and he highes concen a ions assayed, espec i ely (Fig. 5a); while o ENN B, hese dec ease we e om 0.2 µM (48%) o 2.5 µM (20%) co esponding o lowes and highes concen a ions assayed as well (Fig. 5b). On he o he hand, when bina y ea men was assayed he di e ences o nominal and measu ed concen a ions quan i ied educ ion o BEA 0.04 µM (40%) o 4 µM (50%) co esponding o only BEA in he mix u e BEA +ENN B a [0.12 +0.02] µM and [8 +1.6] µM, espec i ely (Fig. 5c). Fo ENN B le els quan i ied we e 0.006 µM (32%) o 0.4 µM (50%) co esponding o only ENN B in he mix u e BEA +ENN B a [0.12 +0.02] µM and [4 +0.8] µM, espec i ely (Fig. 5d). In summa y, in mix u e assays, he dec ease o myco oxins we e highe compa ed o indi idual ea men and mo e no ed o BEA han o ENN B (Fig. 5). 0 25 50 75 100 125 150 0 10 20 30 40 50 60 70 24h 48h 72h 96h 0 25 50 75 100 125 150 0 2 4 6 8 10 24h 48h 72h 96h 0 25 50 75 100 125 150 0 5 10 15 20 25 30 24h 48h 72h 96h a) b) c) BEA (µM) ENN B (µM) Concen a�on (µM) G ow h a e (%) G ow h a e (%) G ow h a e (%) 0 0.06 0.12 0.25 0.5 1 2 4 8 BEA 0 0.01 0.02 0.05 0.1 0.2 0.4 0.8 1.6 ENN B Concen a�on (µM) *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * *** *** *** **** *** * ** * *** Fig. 1. -E ec o BEA (a), ENN B (b) and hei mix u es (c) in he g ow h o Accu odesmus spp. a 24 h, 48 h, 72 h and 96 h. Da a co esponds o n=3 and eigh independen eplica es. *p≤0.05, **p≤0.01 and***p≤0.001 espec o he con ol. Table 2 UHPLC-MS/MS condi ions used o he de e mina ion o BEA and ENN B emaining in algae spp media. Myco oxin P ec Ion MS1 P od Ion MS2 F ag (V) CE (V) Re Time (min) BEA 801.5 262 180 32 10 244 36 ENNB 657.3 214 160 32 9.6 196 CE: collision ene gy; F ag: agmen a ion; P ec: p ecu so ; P od: p oduc ; V: ol age A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 5 0 20 40 60 80 100 120 140 160 180 0123456789 24h 48h 72h 96h 0 20 40 60 80 100 120 140 160 180 0 5 10 15 20 25 30 24h 48h 72h 96h 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 70 24h 48h 72h 96h a) b) c) BEA (µM) ENN B (µM) G ow h a e (%) G ow h a e (%) G ow h a e (%) 0 0.06 0.12 0.25 0.5 1 2 4 8 BEA 0 0.01 0.02 0.05 0.1 0.2 0.4 0.8 1.6 ENN B Concen a�on (µM) *** *** *** *** *** * *** *** *** *** *** *** *** *** *** *** ** ** *** *** *** *** Fig. 2. -E ec o BEA (a), ENN B (b) and hei mix u es (c) in he g ow h o Chlamydomonas eina d hiia 24 h, 48 h, 72 h and 96 h. Da a co esponds o n=3 and eigh independen eplica es. *p≤0.05, **p≤0.01 and***p≤0.001 espec o he con ol. 0 20 40 60 80 100 120 140 160 180 0123456789 24h 48h 72h 96h a) b) G ow h a e (%) G ow h a e (%) G ow h a e (%) 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 70 24h 48h 72h 96h 0 20 40 60 80 100 120 140 160 180 0 5 10 15 20 25 30 24h 48h 72h 96h 0 0.06 0.12 0.25 0.5 1 2 4 8 BEA 0 0.01 0.02 0.05 0.1 0.2 0.4 0.8 1.6 ENN B c) BEA (µM) ENN B (µM) Concen a�on (µM) *** *** *** *** ** * *** *** ** *** *** * *** ** *** * * *** Fig. 3. -E ec o BEA (a), ENN B (b) and hei mix u es (c) in he g ow h o Haema occocus plu ialisa 24 h, 48 h, 72 h and 96 h. Da a co esponds o n=3 and eigh independen eplica es. *p≤0.05, **p≤0.01 and***p≤0.001 espec o he con ol. A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 6 4. Discussion The impo ance in moni o ing con aminan s in he en i onmen and he implica ions o myco oxins in aqua ic ecosys ems has become a end o egula o y au ho i ies. F om ou esul s, i can be obse ed how g ow h e ec s change acco ding o myco oxin, and he algae s ain; 0 20 40 60 80 100 120 0123456789 24h 48h 72h 96h 0 20 40 60 80 100 120 0 5 10 15 20 25 30 24h 48h 72h 96h a) b) c) ENN B (µM) G ow h a e (%) G ow h a e (%) G ow h a e (%) 0 20 40 60 80 100 120 0 10 20 30 40 50 60 70 24h 48h 72h 96h 0 0.06 0.12 0.25 0.5 1 2 4 8 BEA 0 0.01 0.02 0.05 0.1 0.2 0.4 0.8 1.6 ENN B BEA (µM) *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** * * *** *** ** *** *** ** * * ** ** * Fig. 4. -E ec o BEA (a), ENN B (b) and hei mix u es (c) in he g ow h o Mono aphidium g i hiia 24 h, 48 h, 72 h and 96 h. Da a co esponds o n=3 and eigh independen eplica es. *p≤0.05, **p≤0.01 and***p≤0.001 espec o he con ol. 0 10 20 30 40 50 60 70 0123456789 Theo ical Es�ma ed a) Well Numbe Concen a�on (µM) Nominal Es�ma ed 0 5 10 15 20 25 30 0 1 2 3 4 5 6 7 8 9 Theo ical Es�ma ed b) Well numbe Concen a�on (µM) Nominal Es�ma ed c) [BEA + ENN B] Concen a�on (µM) Well Numbe 0 1 2 3 4 5 6 7 8 9 0123456789 Theo ical Es�ma ed Nominal Es�ma ed 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0123456789 Theo ical Es�ma ed d) Well numbe [BEA + ENN B] Concen a�on (µM) Nominal Es�ma ed Fig. 5. -Theo e ical and es ima ed concen a ions o BEA (a), ENN B (b) and hei mix u es (BEA in c and ENN B in d) quan i ied by LC-MS. A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 7 which deno es a di e en sensibili y wi h he cha ac e is ics o each spp. es ed (Figs. 1 o 4). In ac , e ec s we e obse ed a e y low concen- a ions, which deno es he in e ac ion o myco oxins in na u e and speci ically in g een algae. This e eals he necessi y o explo ing hese compounds and sp ead he oxici y s udies. Acco ding o li e a u e, some s udies ha e pe o med he exposu e o algae o myco oxins (Alexande e al., 1999; Guo e al., Suzuki e al., 2014 and 2015) al hough i is sca ce and none can be ound o h ee o he spp. s udied in he e. In ac , he e a e only esul s in li e a u e o C. eina dh ii o s udying myco oxins. C. einha d ii is cha ac e ized by ha ing lagella mo emen and was shown o be a p omising plan icho hecene bioassay sys em when a o al o 14 myco oxins o icho hecene amily we e es ed a 80 µM (including DAS (4,15-diac- e oxysci penol), T-2 oxin, DON (deoxyni alenol), 3-ADON (3-ace- yldeoxyni alenol), 15-ADON (15-ace yldeoxyni alenol), FusX ( usa enon-X), NIV (ni alenol) and, TAS (3,4,15- iace oxysci penol)) (Alexande e al., 1999; Suzuki e al., 2015). I was demons a ed ha he chemical s uc u e o myco oxins de e mines he po en ial o phy o oxici y agains C. einha d ii and loss o mo emen happened. This coincided wi h wha i was epo ed ea lie ela ed o he p esence o a hyd oxyl g oup in C-3 o icho hecenes chemical s uc u e which p e- sen ed highe oxici y han hose wi h an ace yla ed g oup (Alexande e al., 1999); and no icing ha he glycosyla ion o myco oxins con- ibu es o a enua e he oxici y po en ial, as demons a ed wi h DON-3 G (Deoxyni alenol-3-glucoside) (Suzuki e al., 2015). In ano he s udy ca ied ou on C. einha d ii wi h di e en concen- a ions o myco oxins om 0.1 o 25 ppm including DON, 3-ADON, 15- ADON, NIV and FusX (Suzuki e al., 2014), i was shown ha he g ow h p o ile was e y simila o all es ed concen a ions. Howe e , di e - ences we e obse ed o 10 and 25 ppm on which g ow h dec eased, conside ably and mos ma kedly o FusX, NIV and 15-ADON (Suzuki e al., 2014). This ac coincides wi h he di e ences o oxici y epo ed be ween FusX and NIV in cellula es condi ions esul ing highe o FusX han o NIV bu wi hou ha ing clea i s mechanism (Sund ol e al., 2004; Suzuki e al., 2012). In he s udy on C. einha d ii by Suzuki e al. (2014), he e ec o DON and NIV was ma ked bu when compa ed be ween myco oxins, NIV had highe oxici y. Simila esul s o NIV happened when compa ed wi h he s udy o glucoside o m o DON (DON3G) (Suzuki e al., 2015). Wi h hese s udies, i was se he bes concen a ion o es ing Type B icho hecenes in C. einha d ii a 25 ppm o u u e esea ch (Suzuki e al., 2014; Alexande e al., 1999). On he o he hand, Va gas e al., (2015) s udied he e ec on he a e age o he cul u e on C. einha d ii a e 4 days exposed a 100 µM indi idually o 3-ADON, DON, NX-2 and NX-3 wi h a dec ease on i s g ow h and anging be ween 6.25% and 96% espec o he con ol (Va gas e al., 2015). Concen a ions in ou s udy did no ollow he limi epo ed in p e ious publica ion because he e a e no s udies o BEA and ENN B in his meaning o compa e di ec ly ou esul s wi h he algae spp. s udied. Howe e , g ow h a es o C. eina d hii in indi idual and combined ea men we e highe o BEA han o ENN B. The sligh endency and oscilla ions obse ed can be associa ed wi h he iono- pho ic p ope ies o hese compounds. Thus, ion pump mechanisms as lagella mobili y o enzyme ac i a ion o clea ing he media o oxican s a e being a ec ed by he p ope ies o hese compounds, and a di e en concen a ion and ime poin , al e a ions in g ow h a e a e obse ed; ne e heless, no IC 50 alues we e eached (Fig. 2). Acco ding o li e a- u e, only speci ic dose poin s ha e been s udied in C. eina dh ii algae (Suzuki e al., 2014 and 2015). Compa ison wi h ou IC 50 alues is no possible o be associa ed; howe e , o NIV, 15-ADON, 3-ADON and DON 3 G inhibi o y g ow h a e below 50% has been epo ed a 80 µM o C. eina dh ii (Suzuki e al., 2015). Rega ding H. plu ialis, i is cha ac e ized by ha ing a s ong na u al ca o enoid an ioxidan , as axan hin (Mula czyk e al., 2020); in ac , mos o he applica ions o H. plu ialis a e associa ed o nu i ion and heal h as well as low cos o i s p oduc ion. Applica ions in he aqua- cul u e indus y o H. plu ialis ( ich in as axan hin) ela ed o he pigmen a ion o ish ha e been also epo ed; as i imp o es he colo - a ion o many aqua ic animal spp (lobs e s, sh imps, salmon, e c) and he p ope ies associa ed o an ioxidan s as ole ance o s ess and g ow h pe o mance (Shah e al., 2016; Mula czyk e al., 2020; Lim e al., 2018). The e ec o o he ca o enoids (lu ein and zeaxan hin) ha e been s udied in i o in Caco-2 cells and in SH-SY5Y cells in alle ia ing my- co oxins e ec s o BEA (Juan-Ga cía e al., 2019b; Mon esano e al., 2020). As axan hin om H. plu ialis has demons a ed o p o ec mesenchymal s em cells o m oxida i e s ess and i has been pos ula ed as a good candida e in con olling he ha m ul impac s o oxida i e s ess (Mohammadi e al., 2021), educing TNF- α o exe ing neu o- p o ec i e e ec s (Pe ei a e al., 2021). The e ec s epo ed in he e o H. plu ialis a he lowes concen a- ion o myco oxins es ed could be ela ed o he p o ec i e e ec ha his sou ce o an ioxidan exe s which ends up eaching IC 50 alues. A ac ha has been demons a ed as bo h BEA and ENN B p omo e he p oduc ion o ROS in di e en in i o cell lines (Juan-Ga cía e al., 2020a; P ospe ini e al., 2013). The esul s o phy o oxici y we e s onge o BEA han o ENN B in indi idual ea men eaching IC 50 alues; while in combina ion and o he concen a ions s udied, he ac i a ion o mechanism o esis ance o myco oxins seems o be a oiding he inhibi o y concen a ion e ec . Besides he esul s o phy o oxic e ec o myco oxins in he ou algae s ains, i ook us o go a s ep u he and explo e he unc ion o p o ec ion associa ed o some o hem. In ac a dis inguish unc ion o some mic oalgae is o emo e con aminan s o he en i onmen . The capaci y o mic oalgae o emo e such con aminan s may depend on bo h hei indi idual quali ies and how hey a e o ganized in he en i- onmen , as epo ed o M. g i i hii (de Bashan and Bashan, 2010). Fo ins ance, because unicellula o ms ha e la ge su ace o he adso p- ion p ocess, hey should po en ially ha e be e emo al abili ies. Howe e , hey equen ly o ganize in o cell agg ega es ha p oduce bio ilms, which may co e po ions o he su ace egions (De Philippis e al., 2011; Lakmali e al., 2022). The abili y in emo ing me als o ole ance o me als in wa e wi h algae has been he ocus o se e al yea s o s udy. Fo some algae spp i is known ha me al ions emo ed se e as nu ien s in hei me abolic p ocesses, as i is he case o M. g i i hii (de Bashan and Bashan, 2010). To men ion ha M. g i i hii emo es zinc om he en i onmen by binding signi ican amoun s (Bacsi e al., 2015; B inza e al., 2007; Chong e al., 2000) simila ly as i happens o o he mic oalgae o ch omium (VI) (Takami e al., 2012), o ho ium (de Quei oz e al., 2012) o o nickel (Chong e al., 2000). In mos cases, low le els o cleanse s suppose a widesp ead wo ldwide p oblem as i can accumula e in li ing o ganisms as do no biodeg ade (Long e al., 2014). Rela ed o his unc ion o clea ance, he use o M. g i i hii o e alua ing ecosys em heal h in was ewa e wi h pes i- cides in lakes om Rumania ( egion o Cluj-Napoca) was ca ied ou due o i s cha ac e iza ion as dominan phy oplank on in his egion and gi ing accep able esul s (Lakmali e al., 2022). The e a e no s udies o BEA and ENN B ca ied ou in his di ec ion al hough i has been obse ed some esis ance o his algae spp. agains some compounds, which will a ec he ecosys ems and he nu i ional basis o he ood chain and hose loca ed abo e (Houssou e al., 2020). S udies o he emo al o con aminan s conclude ha algae spp. p esen in su ace wa e s can help o clea , elimina e and be easible in he ea men o was ewa e s (Bacsi e al., 2015). Checking ou esul s, i is deno ed ha M. g i i hii was he algae spp. wi h he highes capabili y in clea ing he media o myco oxins as a e he s ong educ ion o g ow h a e a 24 h, in he ollowing exposu e imes i inc eases and was kep o sligh ly dec ease a he highes concen a ions es ed (Fig. 4). Fo all ou algae, he educ ion o myco oxins in he media was obse ed a e y low doses; his could be associa ed o he ac ha a he highes concen- a ions he algae cells die o losses he lagella mobili y (as o C. eina d hii) so he clea ance unc ion does no ake place (Fig. 5). Las ly, Accu odesmus spp. ha e ga ne ed a en ion as a po en ial s ain o bio uel p oduc ion due o hei apid g ow h and high A. Juan-Ga cía e al. En i onmen al Toxicology and Pha macology 107 (2024) 104415 8 pho osyn he ic capaci y compa ed o plan s (Chis i, 2007). These algae a e known o hei high con en o sa u a ed a y acids, which is essen ial o his applica ion (Piligae e al., 2015). Howe e , hey also possess he unique abili y o emo e zinc om he en i onmen (Güçlü and E an, 2012) and phenolic compounds (Lindne e al., 2022). Simila o o he unicellula algae, Accu odesmus spp. se e as excellen subjec s o s udying he di ec e ec s o ex e nal g ow h agen s (Tala ek-Ka wel e al., 2020). The in luence o a ious cul i a ion ac o s, such as nu ien s, em- pe a u e, ligh spec a, and ni ogen, has been in es iga ed o enhance a y acid con en and imp o e indus ial and ood- ela ed p ope ies (Helamieh e al., 2021; B eue e al., 2012; Mando a e al., 2016). I has been obse ed ha cul i a ing mic oalgae unde low empe a u es and g een ligh spec a can yield a subs an ial biomass and a y acid ou pu sui able o he ood indus y (Helamieh e al., 2021). Phy oho mones play a signi ican ole in biological p ocesses, a ec ing g ow h, s ess esponse, and physiological unc ions. In his con ex , myco oxin exposu e, pa icula ly he impac o BEA and ENN B as epo ed in p e ious s udies, may c ea e s ess condi ions ha lead o lowe le els o endogenous phy oho mones. Insu icien amoun s o hese ho mones may con ibu e o he inabili y o mi iga e he nega i e e ec s o myco oxin-induced s ess (Fig. 1), as e idenced by he IC 50 alues being eached only a e su passing a ce ain concen a ion h eshold. The obse ed s ong e ec s o BEA and ENN B on Accu o- desmus could po en ially be a ibu ed o he decline and educed e ec i eness o endogenous phy oho mones c ucial o s ess adap a ion (Tala ek-Ka wel e al., 2020). 5. Conclusions The e idence o phy o oxici y o BEA and ENN B highligh s he nega i e implica ions ha myco oxins can ha e on he g ow h o algae, which a e c ucial nu i ional componen s o many aqua ic animals. The in oduc ion o hese compounds in o he ood chain esul s in an in- c ease in ch onic exposu e o consume s, e en a e y low le els. This emphasizes he impo ance o ood au ho i ies ensu ing ood sa e y and paying a en ion o ood con aminan s ha a e no ye egula ed. Indi idual exposu e o algae o BEA and ENN B demons a ed sig- ni ican phy o oxici y in Accu odesmus spp and Haema ococcus plu ialis, as indica ed by he IC 50 alues. Howe e , when hese myco oxins we e combined, phy o oxici y was obse ed only a e y speci ic concen a- ions o BEA and ENN B. The p ope ies associa ed wi h he di e en algae species, such as hei cleaning abili ies o an ioxidan ichness, we e no su icien o alle ia e he e ec s o myco oxin exposu e. Consequen ly, he p esence o myco oxins in he en i onmen , e en a low le els, can dis up ecosys ems. Special a en ion should be gi en o p ese ing and add essing he changes p esen ed by he changing clima e and he in oduc ion o myco oxins in o aqua ic en i onmen s. Decla a ion o Compe ing In e es The au ho s decla e ha he e a e no con lic s o in e es . Da a A ailabili y The da a ha has been used is con iden ial. Acknowledgemen s This wo k has been suppo ed by he Spanish Minis y o Science and Inno a ion PID2020-115871RB-100. AJG would like o exp ess he g a i ude o Uni e si y o Jy ¨ askyl¨ a o he JYU Visi ing Fellow P og am G an (444/2021) ecei ed and o FORTHEM Alliance P ojec (REF: 612489-EPP-1–2019-1-DEEPPKA2-EUR-UNIV) o he suppo p o ided in his g an . Re e ences Agahi, F., Fon , G., Juan, C., Juan-Ga cía, A., 2020. Indi idual and combined e ec o zea alenone de i a es and beau e icin myco oxins on SH-SY5Y cells. Toxins 12, 212. h ps://doi.o g/10.3390/ oxins12040212. Alexande , N.J., McCo mick, S.P., Ziegenho n, S.L., 1999. Phy o oxici y o selec ed icho hecenes using Chlamydomonas einha d ii as a model sys em . Na . Toxins 7, 265–269. B´ acsi, I., No ak, Z., Janoszky, M., B-Be es, M., G igo szky, I., Nagy, S.A., 2015. The sensi i i y o wo Mono aphidium species o zinc: hei possible u u e ole in bio emedia ion. In J. En i on. Sci. Technol. 12 (8), 2455–2466. de Bashan, L.E., Bashan, Y., 2010. 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