Glyphosate-Based Herbicide Toxicophenomics in Marine Diatoms: Impacts on Primary Production and Physiological Fitness

applied sciences A icle Glyphosa e-Based He bicide Toxicophenomics in Ma ine Dia oms: Impac s on P ima y P oduc ion and Physiological Fi ness Rica do C uz de Ca alho 1,2,* , Edua do Feijão1, Ana Ri a Ma os 3,4 , Ma ia Te esa Cab i a 5, Sa a C. No ais 6, Ma co F. L. Lemos 6, Isabel Caçado 1,4, João Ca los Ma ques 7, Pa ick Reis-San os 1,8 , Vanessa F. Fonseca 1,9 and Be na do Dua e 1,4 1MARE—Ma ine and En i onmen al Sciences Cen e, Faculdade de Ciências da Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisbon, Po ugal; [email p o ec ed] (E.F.); [email p o ec ed] (I.C.); [email p o ec ed] (P.R.-S.); [email p o ec ed] (V.F.F.); [email p o ec ed] (B.D.) 2cE3c, Cen e o Ecology, E olu ion and En i onmen al Changes, Facul y o Sciences, Uni e si y o Lisbon, Campo G ande, Edi ício C2, Piso 5, 1749-016 Lisbon, Po ugal 3 BioISI—Biosys ems and In eg a i e Sciences Ins i u e, Plan Func ional Genomics G oup, Depa amen o de Biologia Vege al, Faculdade de Ciências da Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal; [email p o ec ed] 4Depa amen o de Biologia Vege al da Faculdade de Ciências da Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal 5Cen o de Es udos Geog á icos (CEG), Ins i u o de Geog a ia e O denamen o do Te i ó io (IGOT) da Uni e sidade de Lisboa, Rua B anca Edmée Ma ques, 1600-276 Lisboa, Po ugal; [email p o ec ed] 6 MARE—Ma ine and En i onmen al Sciences Cen e, ESTM, Poly echnic o Lei ia, 2411-901 Lei ia, Po ugal; [email p o ec ed] (S.C.N.); [email p o ec ed] (M.F.L.L.) 7Uni e si y o Coimb a, MARE—Ma ine and En i onmen al Sciences Cen e, Depa men o Li e Sciences, 3000 Coimb a, Po ugal; [email p o ec ed] 8Sou he n Seas Ecology Labo a o ies, School o Biological Sciences, The Uni e si y o Adelaide, Adelaide, SA 5005, Aus alia 9Depa amen o de Biologia Animal da Faculdade de Ciências da Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal *Co espondence: c [email p o ec ed] Recei ed: 9 Sep embe 2020; Accep ed: 19 Oc obe 2020; Published: 22 Oc obe 2020   Fea u ed Applica ion: Applica ion o non-in asi e bio-op ical echniques o e alua e he eco oxici y o glyphosa e-based pes icide in ma ine dia oms wi h con i ma ion by classical biochemical ools. Abs ac : Glyphosa e is he main ac i e componen o he comme cial o mula ion Roundup ® , he mos widely used chemical he bicide wo ldwide. Howe e , i s po en ial high oxici y o he en i onmen and h oughou ophic webs has come unde inc easing sc u iny. The p esen s udy aims o in es iga e he applica ion o bio-op ical echniques and hei co ela ion o physiological and biochemical p ocesses, including p ima y p oduc i i y, oxida i e s ess, ene gy balance, and al e a ions in pigmen and lipid composi ion in Phaeodac ylum ico nu um, a ep esen a i e species o ma ine dia oms, using he case s udy o i s esponse o he he bicide glyphosa e-based Roundup ® o mula ion, a en i onmen ally ele an concen a ions. Cul u es we e exposed o he he bicide o mula ion ep esen ing e ec i e glyphosa e concen a ions o 0, 10, 50, 100, 250, and 500 µ g L −1 . Resul s showed ha high concen a ions dec eased cell densi y; u he mo e, he inhibi ion o pho osyn he ic ac i i y was no only caused by he impai men o elec on anspo in he hylakoids, bu also by a dec ease o an ioxidan capaci y and inc eased lipid pe oxida ion. Ne e heless, concen a ions o one o he plas idial ma ke a y acids had a posi i e co ela ion wi h he highes concen a ion as well as an inc ease in o al p o ein. Cell ene gy alloca ion also inc eased wi h concen a ion, ela i e o con ol and he lowes concen a ion, al hough cul u e g ow h was Appl. Sci. 2020,10, 7391; doi:10.3390/app10217391 www.mdpi.com/jou nal/applsci Appl. Sci. 2020,10, 7391 2 o 21 inhibi ed. Pigmen composi ion and a y acid p o iles p o ed o be e icien bioma ke s o he highes glyphosa e-based he bicide concen a ions, while bio-op ical da a sepa a ed con ols om in e media e concen a ions and high concen a ions. Keywo ds: pho obiology; ene ge ic me abolism; pes icide; oxida i e s ess; glyphosa e 1. In oduc ion In ecen yea s, he conce n o e eme ging pollu an s and hei e ec s on he ma ine bio a me abolism has g own exponen ially. Man-made con aminan s in a iably p esen new challenges in moni o ing e o s and isk p e en ion [ 1 ]. These chemicals, including pes icides, pha maceu icals, and pe sonal and household ca e p oduc s, a e used daily wo ldwide and hei p esence in he en i onmen s ems om an exponen ial inc ease in human ac i i ies ela ed o hei usage [ 2 ]. In he i s decade o he 21s cen u y, mo e han 50% o he o al p oduc ion o chemicals included en i onmen ally ha m ul compounds [ 3 ]. Fu he mo e, he speed o echnological ad ances made in syn he ic chemical p oduc ion con inues o inc ease he lis o hese no el subs ances [ 4 ], aising impo an ques ions and conce ns abou hei eco oxici y and e icien moni o ing me hodologies. Glyphosa e (N-(phosphonome hyl) glycine) is a phosphona e he bicide and he main ac i e ing edien in he comme cial mix u e Roundup ® , he mos used chemical he bicide wo ldwide [ 5 , 6 ]. Glyphosa e is a b oad-spec um he bicide wi h a unique mode o ac ion: I ac s as a glycine analogue, inhibi ing he enzyme 5-enolpy u yl-shikima e-3-phospha e syn hase (EPSPS) o he shikima e pa hway, hus a ec ing he syn hesis o a oma ic amino acids [ 7 ]. The d ama ic inc ease in i s use globally is also associa ed wi h he de elopmen o glyphosa e- ole an c ops ha p esen a ole an EPSPS syn hase and/o a glyphosa e me abolism gene [ 8 ]. Being ela i ely unsuscep ible o chemical- and pho odecomposi ion, glyphosa e can easily each coas al and ma ine a eas ia a mul i ude o di ec and/o di use pa hways [ 9 ]. While pes icides a e mainly used in bo h ag icul u e and weed con ol, hei pe sis ence in any gi en aqua ic en i onmen can allow hem o be ca ied in o emo e ma ine en i onmen s, al hough he exac means by which his occu s is s ill a subjec unde deba e [10,11]. Ma ine phy oplank on is a key biomoni o in many ma ine ophic webs which, unde na u al condi ions, esponds o a wide ange o en i onmen al dis u bances and con amina ion e en s [ 12 – 16 ] and any impac s a his le el a e highly likely o lead o bo om-up impac s. Glyphosa e was shown o impai cyanobac e ia g ow h a concen a ions as low as 50 µ g L −1 [ 17 ], and in eshwa e , glyphosa e in luences mic obial communi y s uc u e, changing he communi y om g een algae and dia oms (glyphosa e sensi i e) o cyanobac e ia (glyphosa e ole an ) [ 18 , 19 ]. Mo eo e , s uc u al changes o ma ine communi ies ha e been associa ed wi h glyphosa e applica ion [ 20 ]. Ul ima ely, eco oxicological s udies on ma ine o ganisms a e o he u mos impo ance o es ablish guidelines o sa egua d local biodi e si y and he unc ioning o es ua ine, coas al, and ma ine en i onmen s [21,22]. Dia oms, as pa o phy oplank on, a e among he i s o ganisms o be a ec ed by con aminan s, quickly esponding o suspended oxican s due o hei small size (0.2–200 µ m) and high up ake a es [ 13 , 23 – 26 ]. Dia oms a e also cons an ly e-adjus ing he equilib ium be ween ene gy p oduc ion om pho osyn hesis and ene gy consump ion unde en i onmen al s ess condi ions, an abili y ha unde mines hei success in highly dynamic coas al and es ua ine en i onmen s [27]. While hey a e esponsible o hal o all he pho osyn hesis on Ea h and hus ha e an impo an ole in he global biological ca bon pump and he silica cycle [ 28 ], ou knowledge o ca bon alloca ion egula ion in dia oms is e y limi ed, pa icula ly because o hese p esen dis inc me abolic compa men a ions and addi ional me abolic pa hways in compa ison o he mo e widely s udied g een algae [29–32]. Dia oms a e also majo ma ine p oduce s o speci ic a y acids [ 32 ], including essen ial a y acids (EFA), linoleic acid (omega-6 [ ω -6] class), and α -linolenic acid (ALA). Dia oms also p oduce long-chain polyunsa u a ed a y acids (LC-PUFA) such as eicosapen aenoic acid (EPA) and docosahexaenoic acid Appl. Sci. 2020,10, 7391 3 o 21 (DHA) (omega-3 [ ω -3] class) [ 33 ]. Ve eb a es canno syn hesize essen ial a y acids and he abili y o p oduce LC-PUFA is also limi ed; hey do no ob ain hese h ough hei die [ 34 , 35 ]. Fu he mo e, LC-PUFA plays key oles in animal and human heal h, being majo componen s o neu ological issues [36]. Pho osyn hesis has been shown o be impai ed by glyphosa e-based he bicides wi hin wide anges o concen a ions, including in co dg ass Spa ina densi lo a om sp ay applica ion a concen a ions o 720–7200 g ha −1 [ 37 ], o di ec con ac o 10–80 mg L −1 in Anabaena e ilissima [ 38 ]. In bo h cases, he e was inhibi ion o CO 2 assimila ion and deple ion o in e media es om he pho osyn he ic ca bon educ ion cycle due o de egula ion o he shikima e pa hway [ 39 , 40 ]. In such scena ios, non-in asi e high- h oughpu bio-op ical sc eening ools, such as Pulse Ampli ude Modula ed (PAM) luo ome y, eme ge as in aluable echniques o e alua e eco oxici y in pho osyn he ic o ganisms [ 12 , 41 , 42 ]. Th ough PAM i is possible o ansla e he luo escence signals as p oxies o he bioene ge ics in ol ed in pho osyn hesis in a non-des uc i e o m [ 13 , 41 – 44 ] o e icien ly assess p ima y p oduc i i y [ 45 , 46 ] and he physiological e ec s in plan ma e ial a di e en concen a ions o con aminan s [ 12 , 41 , 42 , 44 ]. The hal -li e o glyphosa e in wa e a ies be ween 45 and 60 days [ 47 ] and phy oplank on ha e a as esponse in he p esence o con aminan s [ 13 , 25 ]. Thus, i is o he u mos impo ance o e alua e he po en ial e ec s o glyphosa e in he en i onmen , namely on p ima y p oduce s. The p esen s udy aimed o co ela e bio-op ical da a wi h he eco oxicological e ec s o he exposu e o he glyphosa e-based he bicide Roundup ® (i.e., cu en ly ound in es ua ine and ma ine sys ems) [ 48 – 51 ], wi h he wo king hypo hesis ha , simila ly o o he pho osyn he ic o ganisms, his he bicide will ha e a nega i e impac on p ima y p oduc i i y, an ioxidan enzyme ac i i y, ene gy balance, pigmen and a y acid composi ion o he model dia om Phaeodac ylum ico nu um, and i s po en ial implica ions on es ua ine o coas al ma ine ecosys ems. 2. Ma e ials and Me hods 2.1. Expe imen al Se up The model dia om P. ico nu um Bohlin (Bacilla iophyceae) (IO 108–01, IPMA, ALISU—Algae Collec ion o he Uni e si y o Lisbon, Lisbon, Po ugal) was ha es ed om monoclonal cul u es in 250 mL o /2 medium [ 52 ] in cul u e lasks (D echsel- ype gas washing bo les) unde con olled condi ions o 4 days (18 ± 1 ◦ C, unde cons an ae a ion and a 14 h ligh /10 h da k pho ope iod). The g ow h chambe was p og ammed o simula e sun ise and sunse using a sinusoidal unc ion wi h a ligh in ensi y a noon simula ing a na u al ligh en i onmen (RGB 1:1:1, Maximum PAR 80 µ mol pho ons m −2 s −1 , 14/10 h day/nigh hy hm). Acco ding o he O ganiza ion o Economic Coope a ion and De elopmen (OECD) guidelines o algae bioassays [ 53 ] and he ecommended ini ial cell densi y o mic oalgae cells wi h simila size o P. ico nu um, ini ial cell concen a ion was app oxima ely 2.7 ×105cells mL−1 . Fo y-eigh hou s a e inocula ion, cul u es we e exposed o 0, 10, 50, 100, 250, o 500 µ g L −1 glyphosa e o 48 h [ 24 , 41 , 46 ], ob ained om he glyphosa e-based he bicide “Roundup ® P on o” con aining 7.2 g L −1 o glyphosa e. Since no s udies ha e measu ed glyphosa e in ma ine wa e , he he bicide concen a ions we e chosen based on he ange o en i onmen al concen a ions ound in ag icul u al wa e s eams in ele an li e a u e [ 49 – 52 ], as i was ound ha his chemical p esen s he same hal -li e in sal wa e as i does in eshwa e [ 10 , 11 ]. While o he subs ances compose Roundup ® , glyphosa e is he main ac i e componen . Exposu e ook place 48 h a e inocula ion o ensu e ha he expe imen was pe o med du ing he cell exponen ial g ow h phase [ 24 , 41 , 46 ]. The e we e h ee eplica es o each he bicide concen a ion om a o al o 18 expe imen al uni s. To a oid con amina ion, he labwa e was washed wi h HNO 3 (20%) o 48 h, insed ho oughly wi h ul a-pu e wa e and au ocla ed. All cul u e manipula ions we e pe o med in a lamina ai low chambe using asep ic echniques. Appl. Sci. 2020,10, 7391 4 o 21 2.2. G ow h Ra es and Cell Ha es ing Du ing he exposu e ials, daily cell coun ing o P. ico nu um subjec ed o di e en glyphosa e-based he bicide concen a ions was pe o med using a Neubaue imp o ed coun ing chambe , wi h an Olympus BX50 (Tokyo, Japan) in e ed mic oscope, a 400- imes magni ica ion. Cul u e g ow h was de e mined om he di e ence be ween ini ial and inal loga i hmic cell densi ies di ided by he exposu e pe iod [ 54 ], exp essed as he mean speci ic g ow h a e pe day. Samples o pho ochemical and biochemical analysis we e collec ed a e 48 h o exposu e o glyphosa e-based he bicide (4 days a e inocula ion). Based on he glyphosa e-based he bicide concen a ions, we de e mined he No Obse ed E ec Concen a ion (NOEC) and he Lowes Obse ed E ec Concen a ion (LOEC) [ 55 ]. Fu he mo e, by applica ion o a sigmoidal dose- esponse cu e o he endpoin measu emen o each exposu e concen a ion, we also de e mined he E ec i e Concen a ion (EC) which inhibi ed g ow h by 10% (EC 10 ), 25% (EC 25 ), and 50% (EC 50 ) [ 55 ]. A he end o he exposu e ime and a e he chlo ophyll luo escence measu emen s (see nex sec ion), samples o 30 mL o cul u e we e cen i uged a 4000 × g o 15 min a 4 ◦ C (Sigma 2-16K, Sigma Labo zen i ugen GmbH, Ge many). The supe na an was emo ed, and pelle s we e immedia ely ozen in liquid ni ogen and s o ed a −80 ◦C un il analysis. 2.3. Bio-Op ical Assessmen h ough Chlo ophyll a Pulse Ampli ude Modula ed (PAM) Fluo ome y Pulse ampli ude modula ed (PAM) chlo ophyll luo escence measu emen s we e pe o med using a Fluo Pen FP100 (Pho o Sys em Ins umen s, D aso , Czech Republic) on 15 min da k-adap ed samples using a 1 mL cu e e. Cul u e cell densi y was assessed daily, using a non-ac inic ligh o measu e minimum chlo ophyll luo escence (F 0 ). Analysis o chlo ophyll ansien ligh cu es (Kau sky plo ) was ca ied ou using he OJIP es acco ding o [ 41 ]. Fluo ome ic analysis pa ame e s and hei desc ip ion can be accessed in Table 1. Table 1. Fluo ome ic analysis pa ame e s and hei desc ip ion. OJIP Tes A ea Co esponds o he oxidized quinone pool size a ailable o educ ion and is a unc ion o he a ea abo e he Kau sky plo N Reac ion cen e u no e a e SMCo esponds o he ene gy needed o close all eac ion cen e s M0Ne a e o PS II RC closu e γRC P obabili y ha a PS II chlo ophyll molecule will unc ion as an RC PGG ouping p obabili y be ween he wo PS II uni s ABS/CS Abso bed ene gy lux pe c oss-sec ion TR/CS T apped ene gy lux pe c oss-sec ion ET/CS Elec on anspo ene gy lux pe c oss-sec ion DI/CS Dissipa ed ene gy lux pe c oss-sec ion RC/CS Numbe o a ailable eac ion cen e s pe c oss-sec ion TR0/DI0 Con ibu ion o pa ial pe o mance due o he ligh eac ions o p ima y pho ochemis y δR0/(1 −δR0) Con ibu ion o PS I, educing i s end accep o s ψ0/(1 −ψ0) Con ibu ion o he da k eac ions om QA− o PC ψE0/(1 −ψE0) Equilib ium cons an o he edox eac ions be ween PS II and PS I RE0/RC Elec on anspo om PQH2 o he educ ion o PS I end elec on accep o s RC/ABS Reac ion cen e II densi y wi hin he an enna chlo ophyll bed o PS II Appl. Sci. 2020,10, 7391 5 o 21 2.4. Pigmen Analysis Pigmen ex ac ion was pe o med acco ding o me hodologies om p e ious wo ks [ 12 , 13 , 46 ]. Pu e ace one was added o sample pelle s and main ained in an ul a-sound cold ba h o 2 min, ensu ing o al disagg ega ion o cell ma e ial, and kep in he da k a − 20 ◦ C o 24 h o p e en deg ada ion. Samples we e cen i uged o 15 min a 4000 × gand 4 ◦ C and he supe na an s we e scanned by a dual-beam spec opho ome e om 350 nm o 750 nm a 0.5 nm s eps (Shimadzu UV-1603, Shimadzu Co., Kyo o, Japan). Using he SigmaPlo So wa e, he abso bance spec um was in oduced in he Gauss-Peak Spec a (GPS) i ing lib a y. Pigmen analysis was pe o med acco ding o [ 56 ], allowing he de ec ion o chlo ophyll aand c, pheophy in a, β -ca o ene, ucoxan hin, diadinoxan hin (DD), and dia oxan hin (DT). 2.5. An ioxidan Enzyme Assays The soluble p o ein ac ion was ex ac ed a 4 ◦ C om cell pelle s in 1 mL o 50 mM sodium phospha e bu e (pH 7.6) wi h 0.1 mM Na-EDTA and placed in an ul asound ba h o 1 min om he p e iously collec ed pelle s. The homogena e was cen i uged a 10,000 × g o 10 min a 4 ◦ C o emo e deb is and he supe na an collec ed o a new ube. P o ein concen a ion was de e mined acco ding o [ 57 ]. Ca alase (CAT) ac i i y was measu ed acco ding o [ 58 ], moni o ing H 2 O 2 consump ion and he consequen dec ease in abso bance a 240 nm ( ε =39.4 mM −1 cm −1 ). The eac ion mix u e con ained 50 mM o sodium phospha e bu e (pH 7.6), 0.1 mM o Na-EDTA, and 100 mM o H 2 O 2 wi h he eac ion being s a ed by he addi ion o 100 µ L o ex ac . Asco ba e pe oxidase (APX) was assayed acco ding o [ 59 ]. The eac ion mix u e con ained 50 mM o sodium phospha e bu e (pH 7.0), 5 µ M o H 2 O 2 , and 0.25 µ M L-asco ba e, and he eac ion was also ini ia ed wi h he addi ion o 100 µ L o he ex ac . The ac i i y was eco ded as he dec ease in abso bance a 290 nm and he amoun o asco ba e oxidized calcula ed om he mola ex inc ion coe icien ( ε =2.8 mM −1 cm −1 ). Supe oxide dismu ase (SOD) ac i i y was assayed acco ding o [ 60 ] by moni o ing he educ ion o py ogallol a 325 nm. The eac ion mix u e con ained 50 mM o sodium phospha e bu e (pH 7.0) and 0.24 mM o py ogallol and ul a-pu e wa e , wi h he eac ion being s a ed by he addi ion o 10 µ L o ex ac . Con ol assays we e done in he absence o subs a e o e alua e he au oxida ion o he subs a es. All he assays we e pe o med a 25 ◦C in a UV500 UV-Visible Spec ome e (Unicam, Wal ham, MA, USA). 2.6. Lipid Pe oxida ion Analysis Lipid pe oxida ion p oduc s we e de e mined acco ding o [ 61 ]. Sample pelle s we e homogenized b ie ly in 1.5 mL o 10% ( / ) T ichlo oace ic acid (TCA), con aining 0.4% (w/ ) hioba bi u ic acid (TBA) and placed in an ul asound ba h o 1 min. The eac ion was conduc ed a 100 ◦ C o 30 min; immedia ely a e i was hal ed h ough placemen in ice, and a e cen i uga ion a 15,000 × g o 10 min a 4 ◦ C, 1 mL o he supe na an was collec ed and mixed wi h 1 mL o 0.4% TBA and incuba ed again unde he same condi ions. A e again cooling down in ice and cen i uging, he abso bance a 532 nm and 600 nm o he supe na an was eco ded by spec opho ome y. The concen a ion o malondialdehyde (MDA) was de e mined using he mola ex inc ion coe icien ( ε =155 mM −1 cm −1 ). 2.7. Fa y Acid P o iles The a y acid analysis was pe o med acco ding o [ 46 ] by di ec ans-es e i ica ion o sample pelle s, in eshly p epa ed me hanol sul u ic acid (97.5:2.5, / ), a 70 ◦ C o 60 min, using he in e nal s anda d pen adecanoic acid (C15:0). Fa y acid me hyl es e s (FAMEs) we e eco e ed using pe oleum e he , d ied wi h an N 2 low, and e-suspended in an adequa e amoun o hexane. Th ough gas ch oma og aphy (Va ian 430-GC gas ch oma og aph equipped wi h a hyd ogen lame ioniza ion de ec o se a 300 ◦ C, Middelbu g, The Ne he lands), 1 µ L o he FAME solu ion was analyzed, se ing he injec o empe a u e o 270 ◦ C, wi h a spli a io o 50. The used-silica capilla y column (50 m × 0.25 mm; WCOT Fused Silica, CP-Sil 88 o FAME; Va ian, Middelbu g, The Ne he lands) Appl. Sci. 2020,10, 7391 6 o 21 was main ained a a cons an ni ogen low o 2.0 mL min −1 and he o en se o 190 ◦ C. Fa y acids iden i ica ion was pe o med by compa ison o e en ion imes wi h s anda ds (Sigma-Ald ich) and ch oma og ams analyzed by he peak su ace me hod, using he Galaxy so wa e. To de e mine he memb ane sa u a ion le els, he double bond index (DBI) was calcula ed acco ding o [46]: DBI =2×(% monoenes +2×% dienes +3×% ienes +4×% e aenes +5×% pen aenes) 100 (1) 2.8. Ene gy Balance Cell pelle s we e homogenized by ul asonica ion on ice (3x 10s a A =20%) using 1 mL o Milli-Q wa e . Aliquo s we e aken om each sample o he analysis o lipid, ca bohyd a e and p o ein con en s, and elec on anspo sys em (ETS) ac i i y. In all assays, ul apu e wa e was used as eac ion blank. The spec opho ome ic measu emen s we e pe o med in iplica es, a 25 ◦ C, using a syne gy H1 Hyb id Mul i-Mode mic opla e eade (Bio ek®Ins umen , Winooski, VT, USA). 2.8.1. Ene gy A ailable The ene gy a ailable (Ea) was measu ed by de e mining he o al p o ein, ca bohyd a e, and lipid con en s and ans o ming he esul s in o ene ge ic equi alen s (combus ion ene gies: 17,500 mJ mg ca bohyd a es −1 , 24,000 mJ mg p o ein −1 , and 39,500 mJ mg lipid −1 ) [ 61 ]. Ex ac ion and quan i ica ion o o al lipids, p o eins, and ca bohyd a es we e pe o med acco ding o [ 62 , 63 ] wi h mino modi ica ions [ 64 ]. Cell pelle s we e esuspended in 50 mM sodium phospha e bu e (pH 7) con aining 1 mM phenylme hylsul onyl luo ide (PMSF). Cell dis up ion was pe o med wi h 0.42–0.6 mm glass beads (Sigma-Ald ich) o 15 min a 6.5 ms –1 (Fas P ep-24, MP Biomedicals). Cell ex ac was cen i uged a 10,000 × g o 20 min a 4 ◦ C. The supe na an was s o ed a − 80 ◦ C un il u he analysis. To al p o ein con en in he samples was de e mined using B ad o d’s me hod [ 56 ]. To al lipids we e ex ac ed by adding 250 µ L o chlo o o m (spec opho ome ic g ade, Sigma-Ald ich), 250 µ L o me hanol (spec opho ome ic g ade, Sigma-Ald ich) and 125 µ L Milli-Q wa e o 150 µ L o he sample. A e cen i uga ion a 1000 × g o 5 min, he o ganic phase and in e phase we e emo ed and 500 µ L o H 2 SO 4 was added o 100 µ L o lipid ex ac and cha ed o 15 min a 200 ◦ C. The mix u e was cooled down o 20 ◦ C, 1.5 mL o deionized wa e was added, and o al lipid con en was de e mined by measu ing he abso bance a 375 nm and compa ed o a calib a ion cu e using ipalmi in alues as s anda d. To al ca bohyd a e con en was de e mined by adding 50 µ L o 15% TCA o he 150 µ L o sample and subjec ed o − 20 ◦ C o 10 min. A e cen i uga ion a 1000 × g o 10 min, he o al ca bohyd a e con en o he supe na an ac ion was quan i ied by adding 50 µ L o 5% ( / ) phenol and 200 µ Lo 18MH 2 SO 4 o 50 µ L ex ac [ 65 ]. Following 30 min o incuba ion a 20 ◦ C, abso bance was measu ed a 492 nm and compa ed o a calib a ion cu e using glucose as s anda d. 2.8.2. Ene gy Consump ion Cellula oxygen consump ion and me abolism a e di ec ly linked o mi ochond ial ETS ac i i y. As such, ETS was de e mined acco ding o [ 66 ] wi h majo modi ica ions [ 65 ]. In 30 µ L o sample o blank, 20 µ L o homogenizing bu e [0.3 M T is, 15% (w/ ) poly inyl py olidone (PVP), 459 µ M MgSO4, 1.5 mL T i on X-100, pH 8.5], 100 µ L o bu e ed subs a e solu ion ( educed nico inamide adenine dinucleo ide (NADH) (1.79 mM) and educed nico inamide adenine dinucleo ide phospha e (NADPH) (280 µ M) in 0.13 M T is, 0.3% (w/ ) T i on X-100, pH 8.5) we e added. The eac ion was ini ia ed by adding 50 µ L o 8 mM p-iodoni o e azolium (INT), ollowing he change in abso bance a 490 nm o e a 3 min pe iod a 20 ◦ C. The o mazan o med was calcula ed by using he ex inc ion coe icien , ε=15,900 mM–1 cm–1. The cellula ene gy consump ion (Ec) was de e mined by using he ETS da a ( o each 2 µ mol o INT- o mazan o med, 1 µ mol o O 2 was consumed in he ETS), ans o ming he calcula ed quan i y Appl. Sci. 2020,10, 7391 7 o 21 o oxygen consumed in o ene ge ic equi alen s by using he speci ic oxyen halpic equi alen s o an a e age lipid, p o ein, and ca bohyd a e mix u e o 480 kJ mol O2−1[62]. 2.8.3. Cellula Ene gy Alloca ion The cellula ene gy alloca ion (CEA), a me hodological app oach ha in eg a es he ene gy a ailable and ene gy consump ion o an o ganism, was s anda dized o 10 6 cells and calcula ed based on measu emen s o lipid, ca bohyd a e, and p o ein con en and ETS ac i i y o each sample as ollows [67]: CEA =Ea Ec (2) whe e: Ea (a ailable ene gy)=ca bohyd a e +lipid +p o ein mJ 10−6cells(3) Ec (ene gy consump ion)=ETS ac i i y mJ h−110−6cells(4) 2.9. S a is ical Analysis Each a iable was e alua ed h ough one-way ANOVA wi h Tukey’s mul iple compa isons es (G aphPad P ism 6.03 o Windows, G aphPad So wa e, San Diego, CA, USA), ega ding di e ences among glyphosa e-based he bicide concen a ions. The da a ob ained om he Kau sky plo s, pigmen , and a y acid p o iles, we e used as he basis o he cons uc ion o he espec i e esemblance ma ixes based on he Euclidean dis ances be ween samples. To classi y and sepa a e he di e en ea men g oups, s a is ical mul i a ia e models based on he Kau sky plo , pigmen composi ion, and a y acid p o ile a iable we e gene a ed using Canonical Analysis o P incipal Coo dina es (CAP), h ough he non-pa ame ic mul i a ia e analysis packages in P ime 6 so wa e as desc ibed p e iously in o he wo ks [12,45,68,69]. 3. Resul s 3.1. Cell G ow h Ra es Rega ding cell densi y, a e 48 h exposu e P. ico nu um cul u es we e nega i ely a ec ed, in pa icula by he wo highes concen a ions o glyphosa e-based he bicide (250 and 500 µ g L −1 ), wi h sligh dec eases in cell densi y obse ed only a lowe concen a ions (50 and 100 µ g L −1 ) (Figu e 1A). Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 o 23 2.8.3. Cellula Ene gy Alloca ion The cellula ene gy alloca ion (CEA), a me hodological app oach ha in eg a es he ene gy a ailable and ene gy consump ion o an o ganism, was s anda dized o 106 cells and calcula ed based on measu emen s o lipid, ca bohyd a e, and p o ein con en and ETS ac i i y o each sample as ollows [67]: 𝐶𝐸𝐴 = 𝐸𝑎 𝐸𝑐 (2) whe e: 𝐸𝑎 (𝑎𝑣𝑎𝑖𝑙𝑎𝑏𝑙𝑒 𝑒𝑛𝑒𝑟𝑔𝑦)= 𝑐𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒 + 𝑙𝑖𝑝𝑖𝑑 + 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 (𝑚𝐽 10−6 𝑐𝑒𝑙𝑙𝑠) (3) 𝐸𝑐 (𝑒𝑛𝑒𝑟𝑔𝑦 𝑐𝑜𝑛𝑠𝑢𝑚𝑝𝑡𝑖𝑜𝑛)=𝐸𝑇𝑆 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑚𝐽 ℎ−1 10−6 𝑐𝑒𝑙𝑙𝑠) (4) 2.9. S a is ical Analysis Each a iable was e alua ed h ough one-way ANOVA wi h Tukey’s mul iple compa isons es (G aphPad P ism 6.03 o Windows, G aphPad So wa e, San Diego, CA, USA), ega ding di e ences among glyphosa e-based he bicide concen a ions. The da a ob ained om he Kau sky plo s, pigmen , and a y acid p o iles, we e used as he basis o he cons uc ion o he espec i e esemblance ma ixes based on he Euclidean dis ances be ween samples. To classi y and sepa a e he di e en ea men g oups, s a is ical mul i a ia e models based on he Kau sky plo , pigmen composi ion, and a y acid p o ile a iable we e gene a ed using Canonical Analysis o P incipal Coo dina es (CAP), h ough he non-pa ame ic mul i a ia e analysis packages in P ime 6 so wa e as desc ibed p e iously in o he wo ks [12,45,68,69]. 3. Resul s 3.1. Cell G ow h Ra es Rega ding cell densi y, a e 48 h exposu e P. ico nu um cul u es we e nega i ely a ec ed, in pa icula by he wo highes concen a ions o glyphosa e-based he bicide (250 and 500 µg L−1), wi h sligh dec eases in cell densi y obse ed only a lowe concen a ions (50 and 100 µg L−1) (Figu e 1A). Figu e 1. G ow h indica o s ((A) cell densi y and (B) de i ed g ow h pa ame e s) o Phaeodac ylum ico nu um ollowing exposu e o a he bicide o mula ion ep esen ing di e en glyphosa e-based he bicide concen a ions o (A) 24 h and 48 h, and (B) 48 h (mean ± s.d., n = 3, di e en le e s indica e signi ican di e ences a p < 0.05). Howe e , a e 48 h exposu e only dec eases in speci ic g ow h a es, wi h lowe di isions pe day and highe doubling ime, we e obse ed a he highes glyphosa e-based he bicide concen a ions (>250 µg L−1) (Figu e 1B). The e o e, a nega i e e ec was obse ed in he wo highes he bicide o mula ion concen a ions ega ding all he de e mined g ow h pa ame e s. Based on he Figu e 1. G ow h indica o s (( A ) cell densi y and ( B ) de i ed g ow h pa ame e s) o Phaeodac ylum ico nu um ollowing exposu e o a he bicide o mula ion ep esen ing di e en glyphosa e-based he bicide concen a ions o ( A ) 24 h and 48 h, and ( B ) 48 h (mean ± s.d., n =3, di e en le e s indica e signi ican di e ences a p<0.05). Appl. Sci. 2020,10, 7391 8 o 21 Howe e , a e 48 h exposu e only dec eases in speci ic g ow h a es, wi h lowe di isions pe day and highe doubling ime, we e obse ed a he highes glyphosa e-based he bicide concen a ions ( >250 µg L−1) (Figu e 1B). The e o e, a nega i e e ec was obse ed in he wo highes he bicide o mula ion concen a ions ega ding all he de e mined g ow h pa ame e s. Based on he glyphosa e-based he bicide concen a ions p esen in he en i onmen su ounding he dia om popula ion used in he expe imen , we de e mined he espec i e NOEC (10 µ g L −1 ), he LOEC (50 µg L−1), he EC10 (15.4 µg L−1), he EC25 (94.4 µg L−1), and he EC50 (225.9 µg L−1). 3.2. Bio-Op ical Assessmen o Dia om Pho ochemis y Apa om he lowes concen a ion (10 µ g L −1 ), he Kau sky plo s showed dec easing luo escence alues wi h inc easing he bicide quan i ies (Figu e 2), pa icula ly a he highes glyphosa e-based he bicide concen a ions (250 and 500 µg L−1). Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 o 23 glyphosa e-based he bicide concen a ions p esen in he en i onmen su ounding he dia om popula ion used in he expe imen , we de e mined he espec i e NOEC (10 µg L−1), he LOEC (50 µg L−1), he EC10 (15.4 µg L−1), he EC25 (94.4 µg L−1), and he EC50 (225.9 µg L−1). 3.2. Bio-Op ical Assessmen o Dia om Pho ochemis y Apa om he lowes concen a ion (10 µg L−1), he Kau sky plo s showed dec easing luo escence alues wi h inc easing he bicide quan i ies (Figu e 2), pa icula ly a he highes glyphosa e-based he bicide concen a ions (250 and 500 µg L−1). Figu e 2. Chlo ophyll ansien kine ics (OJIP cu es) in Phaeodac ylum ico nu um ollowing a 48 h exposu e o a glyphosa e-based he bicide o mula ion in di e en concen a ions (mean ± s.d., n = 3). The analysis o he pho ochemical p ocess om ligh -ha es ing elec onic anspo , ep esen ed by he ou main ene gy luxes, demons a es he e ec s o exposu e o he di e en he bicide concen a ions in P. ico nu um cul u es (Figu e 3). Figu e 2. Chlo ophyll ansien kine ics (OJIP cu es) in Phaeodac ylum ico nu um ollowing a 48 h exposu e o a glyphosa e-based he bicide o mula ion in di e en concen a ions (mean ± s.d., n =3). The analysis o he pho ochemical p ocess om ligh -ha es ing elec onic anspo , ep esen ed by he ou main ene gy luxes, demons a es he e ec s o exposu e o he di e en he bicide concen a ions in P. ico nu um cul u es (Figu e 3). The amoun o ene gy abso bed by he pho osys em II (PS II) an ennae (ABS/CS), he ene gy lux ha was e ec i ely apped inside he PS II (TR/CS) and anspo ed wi hin he elec on anspo chain (ETC) (ET/CS), as well as in he ene gy dissipa ion lux (DI/CS) and he educ ion o he numbe o oxidized PS II eac ion cen e s (RC/CS), all showed he same pa e n wi h inc easing concen a ions. O e all, no e ec s ela i e o he con ol we e obse ed a he lowes glyphosa e-based he bicide concen a ion (10 µ g L −1 ), bu a small dec easing e ec o in e media e concen a ions (50 and 100 µ g L −1 ) and a high dec ease in he highes glyphosa e-based he bicide concen a ions (250 and 500 µ g L −1 ) was e iden . These changes can be u he analyzed h ough inspec ion o he unc ioning o di e en componen s o he pho osys ems and ETC in esponse o glyphosa e-based he bicide concen a ion (Supplemen a y Figu e S1). Rega ding he oxidized quinone pool size, he e was a dec ease only in he highes glyphosa e-based he bicide concen a ions (250 and 500 µ g L −1 ). Howe e , he e we e no signi ican changes in he numbe o Q A edox u no e s un il maximum luo escence was eached (N), excep o he highes concen a ion in which enhancemen was obse ed. The same pa e n occu ed in he ene gy needed o close ( educe) all RCs (S M ). A he highes glyphosa e-based Appl. Sci. 2020,10, 7391 9 o 21 he bicide exposu e concen a ions (250 and 500 µ g L −1 ), a dec ease in he p obabili y o a PS II chlo ophyll molecule unc ioning as an RC ( γRC ) was de ec ed. Howe e , no di e ences we e obse ed in he QA educ ion a e (M0). Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 o 23 glyphosa e-based he bicide concen a ions p esen in he en i onmen su ounding he dia om popula ion used in he expe imen , we de e mined he espec i e NOEC (10 µg L−1), he LOEC (50 µg L−1), he EC10 (15.4 µg L−1), he EC25 (94.4 µg L−1), and he EC50 (225.9 µg L−1). 3.2. Bio-Op ical Assessmen o Dia om Pho ochemis y Apa om he lowes concen a ion (10 µg L−1), he Kau sky plo s showed dec easing luo escence alues wi h inc easing he bicide quan i ies (Figu e 2), pa icula ly a he highes glyphosa e-based he bicide concen a ions (250 and 500 µg L−1). Figu e 2. Chlo ophyll ansien kine ics (OJIP cu es) in Phaeodac ylum ico nu um ollowing a 48 h exposu e o a glyphosa e-based he bicide o mula ion in di e en concen a ions (mean ± s.d., n = 3). The analysis o he pho ochemical p ocess om ligh -ha es ing elec onic anspo , ep esen ed by he ou main ene gy luxes, demons a es he e ec s o exposu e o he di e en he bicide concen a ions in P. ico nu um cul u es (Figu e 3). Figu e 3. The ene gy luxes (abso bed (ABS/CS), apped (TR/CS), anspo ed (ET/CS) and dissipa ed (DI/CS)) and he numbe o a ailable eac ion cen e s pe c oss-sec ion (RC/CS) in Phaeodac ylum ico nu um ollowing a 48 h exposu e o a glyphosa e-based he bicide o mula ion in di e en concen a ions (mean ±s.d., n =3, di e en le e s indica e signi ican di e ences a p<0.05). While he ac i e OECs showed a dec ease only unde he highes glyphosa e-based he bicide concen a ion (500 µ g L −1 ), he P G , he g ouping p obabili y ha co ela es wi h he disconnec ion be ween he wo PS II uni s, inc eased wi h he highes glyphosa e-based he bicide concen a ion (250 and 500 µg L−1) (Figu e 4). Be ween PS II and PS I, pho ochemical p ocesses showed a signi ican dec ease in he con ibu ion o ligh (TR 0 /DI 0 ) and da k ( ψ0 /1 −ψ0 ) eac ions o he pho ochemical cycle in he highes glyphosa e-based he bicide concen a ion (250 and 500 µ g L −1 ). A simila pa e n was obse ed in he eac ion cen e densi y wi hin he PS II an enna chlo ophyll bed (RC/ABS). On he o he hand, a he PS I le el he e was a signi ican enhancemen in he ac i i y o his pho osys em ( δR0 /1 −δR0 ) in esponse o he highes glyphosa e-based he bicide concen a ions, also leading o an inc ease in he equilib ium cons an o he edox eac ion be ween bo h pho osys ems owa ds he PS II ( ψE0 /(1 −ψE0 )). Fu he mo e, ega ding PS I, in insic changes led, in u n, o an inc ease o he elec on anspo om PQH 2 o he educ ion o he PS I end accep o s (RE0/RC). In he apid ligh cu e (RLC)-de i ed pa ame e s, he pho osyn he ic e iciency ( α ) and he maximum elec on anspo a e (ETR max ) only dec eased wi h he highes glyphosa e-based he bicide concen a ion (500 µ g L −1 ), wi h no e ec on pho oinhibi ion ( β ) and ligh sa u a ion (E k ), wi h only a sligh educ ion a 250 µ g L −1 glyphosa e-based he bicide exposu e in he la e pa ame e (Supplemen a y Figu e S2). Appl. Sci. 2020,10, 7391 16 o 21 Ye , a se e e shi in he edox equilib ium be ween pho osys ems owa ds he PS II ( ψE0 /(1 −ψE0 )) was s ill obse ed, dec easing he edox po en ial eaching he PS I, and hus impai ing he ligh and da k eac ions o pho osyn hesis and he enewal o subs a es in he PS I [45]. Ne e heless, he a ailable ene gy (Ea) inc eased in he highes glyphosa e-based he bicide concen a ions, pa icula ly a 500 µ g L −1 , p obably due o an inc ease in lipid and p o ein concen a ions. The inc ease in Ea has been obse ed in g een algae, which accumula ed ene gy ese es as a esponse o en i onmen al s ess [ 64 , 80 , 81 ]. A possible explana ion could be ela ed o he need o algae o coun e ac he inc ease in ene gy expendi u e, as obse ed in he p esen s udy. The e o e, ollowing a simila pa e n as Ea, he ene gy consump ion demons a ed by he mi ochond ial ETS o P. ico nu um inc eased signi ican ly a he highes concen a ions, eaching a 300% inc ease in he highes concen a ion ela i e o con ol. This inc ease in ene gy consump ion has been obse ed in o he mic oalgae [ 65 ]. None heless, he Ea was highe han he ETS, which led o a sligh inc ease o he CEA in he highes concen a ions bu only allowing a di e en ia ion o he highes concen a ions om he con ol and he lowes concen a ions. This inc ease in CEA may e lec a highe impai men in mi ochond ia unc ioning ela i e o chlo oplas unc ioning ha can ul ima ely impac he la e due o biophysical dis u bances [ 82 ]. While hese ene gy balances appea o indica e an inc ease in he ene gy a ailable o g ow h o cell di ision, he highes glyphosa e-based he bicide concen a ions inhibi ed dia om cul u e g ow h. Based on his da a, s ess o ces he ene gy o shi om pho osyn hesis in o ene gy consump ion. This may change pho osyn he ic elec on pa hways, leading o Ea down egula ion and an inc ease in ETS o a oid ca bon was e, as a s a egy o handle an excess o me abolic ene gy. The e a e di e en me abolic pa hways unde which ene gy spillo e can be managed, bu cu en ele an knowledge is s ill e y sca ce [32]. The CAP analyses we e e y e icien in he assessmen o he e ec s o he glyphosa e-based he bicide on he pho ochemis y, pigmen composi ion, and lipid me abolism o P. ico nu um, as well as on he po en ial o hese me abolic ea u es as oxici y bioma ke s. Howe e , only he use o bio-op ical echniques (e.g., Kau sky plo ) allowed an e icien iden i ica ion o all he di e en glyphosa e-based he bicide exposu e ea men s (con ol, low, in e media e, and high concen a ions). As in p e ious s udies, mul i a ia e analysis e icien ly classi ied g oups subjec ed o di e en exposu e le els, in compa ison o he o en-used uni a ia e analysis [ 12 , 45 , 69 ]. While glyphosa e is he main componen o Roundup ® , o he subs ances a e also in i s composi ion and i is o g ea in e es in u u e wo ks o assess syne gies and/o an agonisms be ween he he bicide o mula ion e sus glyphosa e in i s pu e o m, compa ing he alues o hose measu ed in he habi a s. 5. Conclusions Glyphosa e-based pes icides, pa icula ly a high concen a ions, ha e a clea e ec on se e al me abolic pa hways in ma ine dia oms (changes in pigmen p o ile, pho osyn he ic impai men , and dec eased an ioxidan capaci y). E en hough pho op o ec i e mechanisms we e induced, memb ane damage s ill occu ed. These ac s highligh he applica ion o pulse ampli ude modula ed chlo ophyll luo escence bio-op ical me hods as a p omising ool o inclusion in eco oxicological s udies wi h se e al ad an ages ela i e o he classical biochemical app oaches, namely as da a acquisi ion, lowe mone a y cos s, and a high numbe o a iables ob ained while ensu ing a high deg ee o accu acy. Thus, bio-op ical ools appea as a as , inexpensi e, and eliable me hod o oxicophenomic assessmen o he impac s o his widesp ead pes icide in ma ine dia oms, wi h a clea dose- esponse pa e n. Mo eo e , he high olume o da a p oduced by hese bio-op ical echniques can be e icien ly applied in mul i a ia e analysis, p o iding a classi ica ion wi h a high deg ee o accu acy. Wi h his in mind, PAM luo ome y appea s o be a p omising ool o inclusion in eco oxicological s udies aiming owa ds a possible use in long- ange de ec ion h ough sa elli e image y in a global chemical pollu ion-moni o ing ne wo k. Appl. Sci. 2020,10, 7391 17 o 21 Supplemen a y Ma e ials: The ollowing a e a ailable online a h p://www.mdpi.com/2076-3417/10/21/7391/s1, Figu e S1: Pho osys em II and ETC ela ed pho ochemical ai s, Figu e S2: Rapid ligh cu es and de i ed pa ame e s, Figu e S3: Majo a y acids classes, Figu e S4: Fa y acid a ios, Figu e S5: Omega 6 o omega 3 a y acid a io, Table S1: Fluo ome ic analysis pa ame e s and hei desc ip ion. Au ho Con ibu ions: Concep ualiza ion, B.D., V.F.F. and P.R.-S.; me hodology, B.D.; o mal analysis, R.C.d.C. and B.D.; in es iga ion, R.C.d.C., E.F. and S.C.N.; da a cu a ion, R.C.d.C.; w i ing—o iginal d a p epa a ion, R.C.d.C.; w i ing— e iew and edi ing, E.F., A.R.M., M.T.C., S.C.N., M.F.L.L., I.C., J.C.M., P.R.-S., V.F.F., B.D.; supe ision, B.D.; p ojec adminis a ion, B.D.; unding acquisi ion, B.D. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was unded by Fundaç ã o pa a a Ci ê ncia e a Tecnologia (FCT) ia p ojec g an s PTDC/CTA-AMB/30056/2017 (OPTOX), UID/MAR/04292/2019, UID/MULTI/04046/2019. Wo k was also unded by he In eg a ed P og amme o SR&TD Sma BioR ( e e ence Cen o-01-0145-FEDER-000018), co- unded by Cen o 2020 p og am, Po ugal 2020, Eu opean Union, h ough he Eu opean Regional De elopmen Fund. B. Dua e and V. Fonseca we e suppo ed by in es iga ion con ac s (CEECIND/00511/2017 and DL57/2016/CP1479/CT0024). P. Reis-San os was suppo ed by FCT h ough a pos doc o al g an (SFRH/BPD/95784/2013). Acknowledgmen s: The au ho s would like o hank Ca oline Casey and Joana Roma o e ising he English language, g amma , punc ua ion, and spelling o he manusc ip . Con lic s o In e es : The au ho s decla e no con lic o in e es . Abb e ia ions ALA α-linolenic acid; APX asco ba e pe oxidase; CAP Canonical Analysis o P incipal Coo dina es; CAT ca alase; CEA cellula ene gy alloca ion; DBI double bond index; DD diadinoxan hin; DHA docosahexaenoic acid; DT dia oxan hin; Ea ene gy a ailable; Ec ene gy consump ion; EC E ec i e Concen a ion; EFA essen ial a y acids; EPA eicosapen aenoic acid; EPSPS 5-enolpy u yl-shikima e-3-phospha e syn hase; ETC elec on anspo chain; ETS elec on anspo sys em; FAME a y acids me hyl es e s; FCP ucoxan hin-chlo ophyll p o ein; LC-PUFA polyunsa u a ed a y acids; LOEC Lowes Obse ed E ec Concen a ion; MDA malondialdehyde; NOEC No Obse ed E ec Concen a ion; OEC oxygen-e ol ing complexes; PAM Pulse Ampli ude Modula ed; PS I pho osys em I; PS II pho osys em II; RC eac ion cen e s; RLC apid ligh cu es; SOD supe oxide dismu ase Re e ences 1. Ga ilescu, M.; Demne o á , K.; Aamand, J.; Aga hos, S.; Fa a, F. 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