P ocedu es o he disin ec ion o
s ock cul u e o duckweed
Se ena Ca loni, Lau a Passa o e, Fab izio Pie ini, Massimo Zacchini
Na ional Resea ch Council o I aly, CNR
Resea ch Ins i u e on Te es ial Ecosys ems, IRET
No embe 2025
P ocedu es o he disin ec ion o s ock cul u e o duckweed
In oduc ion
The ulne abili y o aqua ic en i onmen s o he inc easingly widesp ead p esence o xenobio ic
compounds is cu en ly ep esen ing a majo global conce n. Va ious biological o ganisms a e
sensi i e o xenobio ics and, hus, make hem sui able as model species o oxici y s udies.
Eco oxicological s udies based on plan o ganisms in eshwa e ecosys ems a e an e icien ool o
e alua ing and moni o ing hei heal h, as well as ha o he en i e ood web up o he human die .
This is because plan o ganisms play a undamen al s uc u al and unc ional ole as p ima y
p oduce s (Gubbins e al., 2011).
Plan s belonging o he Lemnoideae sub amily a e ecognised as bioindica o s o wa e quali y
(Böcük e al., 2013; Mkandawi e e al., 2014; Pie ini e al., 2022). Lemnoideae, commonly known
as duckweeds, a e classi ied by The Angiospe m Phylogeny G oup (The Angiospe m Phylogeny
G oup, 2016) as a sub amily o A aceae. Howe e , mo e ecen s udies, based on molecula
analyses, sugges ha duckweeds a e phylogene ically and mo phologically dis inc om A aceae,
and should be es o ed o amily s a us as Lemnaceae (Tippe y e al., 2021).
Duckweeds a e aqua ic plan s ha play an impo an ecological ole wi hin eshwa e , es ua ine
and we land habi a s, o ming pa o he communi y o p ima y p oduce s (G eenbe g e al., 1992).
Duckweed plan s, pa icula ly hose belonging o he Lemna genus, a e among he smalles
ep esen a i e o he ascula plan s and a e cha ac e ized by he as es g ow h a es among
lowe ing plan s (S ee e al., 2015; Ziegle e al., 2015). They a e easy o cul i a e in labo a o y
condi ions, due hei as g ow h a e, which is d i en by mass p opaga ion ia asexual p oli e a ion
in ol ing he budding o daugh e onds (Pie ini e al., 2019).
Due o hei biological cha ac e is ics, duckweed plan s a e widely used o e alua e phy o oxici y o
pollu an s and assess hei en i onmen al isks. They ha e been selec ed as he model plan o
eco oxici y s udies on he eshwa e ecosys em (Baudo e al., 2015; Pie ini e al., 2015, 2016; Fo ni
and Tommasi, 2016; Iannilli e al., 2025) and a e o icially used in eco oxicological assays
(OECD/OCDE 221, 2006). Mo eo e , duckweed plan s ha e been indica ed o be e ec i e biological
ools o emo e oxic compounds om was ewa e (Hegazy e al., 2009; Ma amo os e al., 2012;
Ceschin e al., 2019).
O e 90% o he plan -based eco oxicological s udies a e conduc ed in a s ic ly labo a o y se ing
(Ceschin e al., 2020). Ob aining s ock o plan cul u es ee o pa asi es o pa hogens is a majo
challenge in he ield o eco oxicological assays. Fu he mo e, axenic condi ions a e desi able o
plan s employed in lab es s in o de o e alua e he espec i e oles o he plan s and hei
mic obio a in de oxi ying chemical compounds (Khella and Ze daoui, 2010; Liu e al., 2019).
To add ess hese challenges, p o ocols ha e been de eloped o ob ain plan cul u es a di e en
disin ec ion le els, anging om “clean” o s e ile, wi hou comp omising he su i al o
de elopmen po en ial o he plan s (Fi min e al., 2025).
Plan ma e ials and cul u e p o ocol
Th ee duckweed species a e cul i a ed in he g ow h chambe o he Resea ch Ins i u e on
Te es ial Ecosys ems, seconda y uni o Mon elib e i: Spi odela poly hiza (L.) Schleid. (Fig. 1),
Lemna mino L. (Fig. 2) and Lemna minu a Kun h (Fig. 3).
The onds o L. mino plan s we e pu chased om a company specialising in aqua ic cul i a ion
sys ems and main ained in s ock-cul u e condi ions. S. poly hiza and L. minu a, on he o he hand,
we e collec ed in he wild, cleaned, and acclima ised o s ock-cul u e condi ions. The g ow h
chambe is main ained a a cons an empe a u e o 25 ± 3 °C, wi h a pho osyn he ic pho on lux
densi y (PPFD) o 60–80 μmol m−2s−1 and a pho ope iod o 16h ligh /8h da k (Fig. 4). The ecipien s
con aining duckweeds, placed in he g ow h chambe , a e pa ially co e ed wi h a hin plas ic shee
o minimise con amina ion and a ligh ai low om a pump is gi en o oxygena ion.
Fig. 1 – Spi odela poly hiza Fig. 2 – Lemna mino Fig. 3 – Lemna minu a
Fig. 4 – G ow h chambe
The cul u es a e ed wi h Hoagland solu ion a hal s eng h o L. mino and one- en h s eng h o
S. poly hiza and L. minu a ( he concen a ions o he medium we e de e mined h ough
expe imen al ials). Sub-samples o each duckweed species a e ans e ed e e y 10 days o esh,
clean medium, o minimise con amina ion by o he o ganisms, as indica ed in he OECD 221
guidelines (OECD, 2006). Only uncon amina ed onds a e used o u he cul i a ion a each
ans e . The Hoagland nu ien medium comp ises o mac onu ien s (MgSO4.7H2O, KNO3,
CaCl2.2H2O), mic onu ien s (Fe(EDTA)Na, H3BO3, ZnSO4.7H2O, MnCl2.4H2O, (NH4)6Mo7O24.4H2O,
CoCl2.6H2O, CuSO4.5H2O) and phospha e (KH2PO4) mixed in deionised wa e . These nu ien s a e
p epa ed and added as six solu ions (Table 1). The pH o he solu ion is adjus ed o 6.5 (McLay, 1976;
Ceschin e al., 2017).
Full
s eng h
Full
s eng h
Hal
s eng h
One- i h
s eng h
Solu ion
Nu ien s
Concen a ion
ml in 1 L
ml in 10 L
ml in 1 L
ml in 1 L
Sol. 1
KH2PO4
13,6 g/L
5,00
50,00
2,5
1,00
Sol. 2
MgSO4.7H2O
49,3 g/L
10,00
100,00
5
2,00
Sol. 3
KNO3
60,66 g/L
30,00
300,00
15
6,00
Sol. 4
CaCl2.2H2O
73,5 g/L
16,00
160,00
8
3,20
Sol. 5
Fe(EDTA)Na
7,5 g/250 ml
0,75
7,5
0,375
0,15
Sol. 6
H3BO3
715 mg/250 ml
1,00
10,00
0,5
0,20
ZnSO4.7H2O
55 mg/250 ml
MnCl2.4H2O
452 mg/250 ml
(NH4)6Mo7O24.4H2O
5 mg/250 ml
CoCl2.6H2O
25 mg/250 ml
CuSO4.5H2O
20 mg/250 ml
Table 1 - Recipe o p epa ing Hoagland solu ion.
Disin ec ion p ocedu e
Plan s selec ed o expe imen al es ing mus be cleaned and disin ec ed. This condi ion equi es a
wo-s ep p o ocol in ol ing a b ie inse in a 0,5% bleach solu ion (sodium hypochlo i e, 7% o ac i e
Cl), ollowed by a ho ough washing in dis illed, s e ilised wa e (Pie ini e al., 2015).
Fi s s ep - Disin ec ion s ep o cul u e s ock main enance
This s ep is applied o he en i e plan colony and is only necessa y i algae con amina ion is e iden
All he passages desc ibed below a e in ended o an inoculum o 6 g o S. poly hiza e L. mino , 4 g
o L. minu a. Repea each passage as many imes as necessa y.
Add 200 ml o deionised wa e o 2 Magen a boxes. Add 199 ml o deionised wa e o 1 Magen a
boxes.
S e ilise in an au ocla e:
- Tweeze s
- Spa ulas
- Fo ks
- Tips 1 ml
- Magen a boxes p e iously p epa ed
In a lamina low hood:
1. add 1 ml o unscen ed bleach o he Magen a box con aining 199 ml o wa e ;
2. using a o k, subme ge he plan inoculum in he 0,5% bleach solu ion o 1 minu e, s i ing
gen ly wi h a spa ula o he o k;
3. using a o k, ans e he plan s o a Magen a box con aining deionised, s e ilised wa e and
lea e o soak o 2 minu es, s i ing gen ly wi h a spa ula o he o k;
4. using a o k, ans e he plan s o a new Magen a box con aining deionised, s e ilised wa e
and lea e o soak o 2 minu es, s i ing gen ly wi h a spa ula o he o k.
Be o e ans e ing he cleaned colony, clean he ecipien s con aining duckweed plan colonies
wi h bleach and inse ho oughly.
Keep he plan s in Hoagland solu ion a he co ec concen a ion o one week: one- i h o S.
poly hiza ( a he han one- en h o he no mal amoun used o cul u e main enance), one-hal o
L. mino and one- en h o L. minu a.
Fig. 5 – T ans e ing plan s Fig. 6 – S i ing gen ly
Second s ep - Disin ec ion s ep o expe imen al ial
This s ep is only applied o a pa o he plan colony, esul ing om he selec ion o he heal hies
and cleanes indi iduals. All he passages desc ibed below a e in ended o an inoculum o 6 g o S.
poly hiza e L. mino , 4 g o L. minu a. Repea each passage as many imes as necessa y.
P epa e 500 ml o Hoagland solu ion, wi h he equi ed concen a ion o he chosen duckweed
plan o he ial (one- i h o S. poly hiza, one-hal o L. mino , one- en h o L. minu a), wi hou
adding i on (Fe(EDTA)Na), o p e en i om p ecipi a ing du ing he s e ilisa ion p ocess in he
au ocla e.
Add 200 ml o deionised wa e o 2 Magen a boxes. Add 199 ml o deionised wa e o 1 Magen a
box.
S e ilise in an au ocla e:
- Tweeze s
- Spa ulas
- Fo ks
- Tips 1 ml
- Tips 200 µl
- Magen a boxes p e iously p epa ed
- 2 emp y Magen a boxes
- Hoagland solu ion wi hou i on p e iously p epa ed
Place unde a UV ligh in a lamina low hood o 15-20 minu es:
- Magne ic s i e
- Pipe e (in he ange o 200 µl ips)
- 1 Beake 500 ml illed wi h deionised wa e
- Po able pH me e
- Deionised wa e o cleaning pH me e p obe
- NaOH 1M solu ion
- Pas eu disposable pipe e
- Some shee s o lab pape
T ans e he plan inoculum de i ed om he colony selec ion o he beake con aining deionised
wa e ha has been placed unde UV ligh (Fig. 5).
In a lamina low hood:
1) P epa e Hoagland solu ion:
a) add he co ec p opo ion o i on (Fe(EDTA)Na), p e iously il e ed wi h a s e ile,
disposable sy inge il e , po e size o 0.22 μm, o he s e ilised Hoagland solu ion,
aking in o accoun he concen a ion and he inal olume;
b) adjus he pH o 6.5 by adding 1M NaOH using s e ile ips;
c) add 200 ml o he p epa ed Hoagland solu ion o he emp y, s e ilised Magen a
boxes;
2) add 1 ml o unscen ed bleach o he Magen a box con aining 199 ml o wa e ;
3) using a o k, subme ge he plan inoculum in he 0,5% bleach solu ion o 1 minu e, s i ing
gen ly wi h a spa ula o he o k;
4) using a o k, ans e he plan s o a Magen a box con aining deionised, s e ilised wa e and
lea e o soak o 2 minu es, s i ing gen ly wi h a spa ula o he o k;
5) using a o k, ans e he plan s o a new Magen a box con aining deionised, s e ilised wa e
and lea e o soak o 2 minu es, s i ing gen ly wi h a spa ula o he o k;
6) ans e plan s o he Magen a boxes illed wi h he p epa ed, s e ilised Hoagland solu ion;
7) close he Magen a boxes wi h pa a ilm.
Fig. 7 – Ma e ials eady o disin ec ion Fig. 8 – Magen a boxes se ( om le o igh : inal
eco e y, wo deionised wa e s, one bleach solu ion)
Keep he duckweed plan s in Magen a boxes and ensu e he e is a sligh , cons an mo emen o 50
pm unde con olled condi ions (cons an empe a u e o 25 ± 3 °C, pho ope iod o 16h ligh /8h
da k), o 3 days be o e s a ing he expe imen al ial.
Final ema ks/ ecommenda ions
Du ing he p ocedu e he plan s expe ience bo h chemical and physical s ess. The chemical s ess
is caused by insing he plan s in bleach solu ion, while he physical s ess depends on how
igo ously he plan s a e shaken inside he Magen a boxes du ing his p ocess. I is ele an ha he
plan s a e in good physiological condi ion, as well as clean, a he s a o he expe imen al ial.
Some ips o educing s ess:
- pay a en ion when insing in he bleach solu ion: he o al ime should be no mo e han 1
minu e;
- handle he plan s wi h g ea ca e: his is especially impo an du ing insing;
- espec he lapse ime be ween he wo s eps (1 week) and be ween he second s ep and
he s a o he ial (3 days): his ime is impo an o allowing he plan s o eco e om
s ess.
Following his wo-s eps p o ocol is possible o ob ain duckweed plan cul u es nea ly ee o
pa asi es o pa hogens in lab condi ions, in o de o conduc eco oxicological assays o
phy o emedia ion s udies.
Au ho s
Se ena Ca loni, Lau a Passa o e, Fab izio Pie ini, Massimo Zacchini.
Na ional Resea ch Council o I aly – Resea ch Ins i u e on Te es ial Ecosys ems, CNR-IRET.
Regional Resea ch Campus o Rome 1, S ada P o inciale 35d, 9 – 00010 Mon elib e i (RM).
Pho o C edi s: Se ena Ca loni, CC BY-SA 4.0
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