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
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