Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372

https://doi.org/10.1007/s00128-022-03549-9

Duckweed Potential for the Phytoremediation of Linear Alkylbenzene

Sulfonate (LAS): Identification of Some Intermediate Biodegradation
Products and Evaluation of Antioxidant System
Zahra Masoudian1 · Seyed Yahya Salehi‑Lisar1 · Akbar Norastehnia2 · Sarieh Tarigholizadeh1

Received: 10 November 2021 / Accepted: 9 May 2022 / Published online: 7 June 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Duckweed (Lemna minor L.) has a high potential for wastewater treatment. Here, its capability for bioremoval of linear
alkylbenzene sulfonate (LAS) as one of the primary contaminants of water resources was evaluated. The effect of some
operational parameters on surfactant removal efficiency was determined. Also, the impact of LAS on several physiological
responses of Lemna was investigated. LAS remediation efficiency of L. minor was elevated with increasing LAS concentra-
tion, duckweed weight, and temperature. Furthermore, the optimal pH for removal was 7–8.5. The benzenesulfonate ring
and five homologs of sulfophenyl carboxylate were identified as intermediates in the LAS degradation pathway. A decrease
in relative growth rate and pigment contents was observed by increasing LAS concentration. In contrast, an increase in
hydrogen peroxide content and electrolyte leakage indicated oxidative stress by LAS. Induction of enzymatic/non-enzymatic
antioxidants was observed during the surfactant remediation process, indicating their role in overcoming free radicals gener-
ated under surfactant stress.

Keywords Bioremediation · Lemna minor · Oxidative stress · Surfactant

Industrialization of countries and the increase of world
population have led to the production and release of large
amounts of contaminants into the environment. Surfactants
or “surface-active substances” are widely used in many
fields of technology such as in laundry detergents (Bajpai
2007), pharmaceuticals (Mondal et al. 2015), agricultural
formulations (Castro et al. 2014), biotechnology (Singh et al.
2007), and textile industries (Rebello et al. 2014). The linear
alkylbenzene sulfonates (LAS) are the fundamental group of
anionic surfactants (Jardak et al. 2016). LAS is a mixture of
isomers and homologs consisting of a sulfonated aromatic
ring and a linear alkyl chain of various lengths (Rebello et al.
2014). Large amounts of surfactants are discharged into the
sewage. For example, anionic surfactant concentration in
* Zahra Masoudian
zahramasoodian@yahoo.com
1 2001; Hamada and Abe 2009; Costa et al. 2020) and plants
Department of Plant Sciences, Faculty of Natural Sciences,
University of Tabriz, Tabriz 5166616471, East Azerbaijan,
Iran
2
Department of Biology, Faculty of Sciences, University
of Guilan, Rasht, Iran
sewage is up to 10 mg ­L−1, and the mean concentrations
in wastewaters from surfactant production factories can be
over 300 mg ­L−1 (Zhang et al. 1999). Harmful effects of sur-
factants on the environment begin from the synthesis phase
and continue until the disposal into the environment (Rebello
et al. 2014). The formation of a foam layer on the water’s
surface by detergents reduces the penetration of oxygen into
the water and threatens aquatic life (Rajan 2015). Various
physical and chemical techniques have been shown to elimi-
nate surfactants (Beltrán et al. 2000; Bhatkhande et al. 2002;
Dehghani et al. 2010; Lopez-Vizcaino et al. 2012). How-
ever, the use of bioremediation methods is cost-effective and
environmentally friendly to other methods. In phytoremedia-
tion technology, specific plant species are used to treat con-
taminated sites, including air, land, and water (Pilon-Smits
2005). Previous research has shown that different organisms
such as algae (Chawla et al. 1987; Serejo et al. 2020), bac-
teria (Khleifat 2006; Asok et al. 2015), fungi (Yadav et al.
(Forni et al. 2008; Masoudian et al. 2020) can eliminate
surfactants. Despite the widespread use of this group of pol-
lutants, research on the mechanism of their biodegradation
is scarce. Former studies have illustrated that surfactants

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Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372
can alter some plant properties, including a change in the
growth of Chara vulgaris L. (Liu and Wu 2018a), L. minor
L. (Forni et al. 2008), Myriophyllum spicatum L. (Liu et al.
2019), Potamogeton perfoliatus L. (Zhou et al. 2018), and
Azolla filiculoides L. (Masoudian et al. 2020). Furthermore,
a change in photosynthetic pigment content and antioxidant
enzymes activities (Liu and Wu 2018a; Zhou et al. 2018; Liu
et al. 2019; Masoudian et al. 2020) and damage to the cyto-
plasm, chloroplasts, and vacuoles were reported (Liu et al.
2004). Also, induction of lipid peroxidation (Liu and Wu
2018a; Zhou et al. 2018), reduction in membrane stability
(Liu and Wu 2018b; Masoudian et al. 2020), and necrosis
and chlorosis of Sagittaria lancifolia leaves (Fitrihidajati
et al. 2020) have been reported.
Lemna is a genus of free-floating aquatic plants growing
in slow-flowing or stagnant water. They are small aquatic
vascular plants consisting of submerged roots and floating
leaflets (Ceschin et al. 2020). The genus Lemna is a model
plant in ecotoxicological research. Most Lemna species have
a considerable ability to accumulate xenobiotics because of
large leaf area, high biomass production potential, and rapid
growth rate (Zezulka et al. 2013). The ability of Lemna to
remove a variety of pollutants, including nutrients (Ceschin
et al. 2020; Neidoni et al. 2020), heavy metals (Sree et al.
2015; Balasubramanian et al. 2019; Sun et al. 2020), and
agrochemicals (Wang et al. 2017; Panfili et al. 2019; Dos-
non-Olette et al. 2010; Anand et al. 2019) have been previ-
ously reported.
The purpose of this investigation was to evaluate the phy-
toremediation ability of L. minor to remove LAS surfactant
from water. In addition, the effect of experimental param-
eters on removal efficiency was investigated. Some possi-
ble by-products were identified using the liquid chroma-
tography–mass spectrometry (LC–MS) technique. Finally,
changes in some physiological parameters and the activity
of antioxidant enzymes, including peroxidase (POD, EC
1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), ascor-
bate peroxidase (APX, EC 1.11.1.11), and catalase (CAT,
EC 1.11.1.6), which can affect plant resistance to surfactants,
were evaluated.
Materials and Methods
Lemna minor were obtained from the Kiashahr jetty
(Kiashahr, Iran, N, 37° 25ʹ 10ʺ and E: 49° 56ʹ 56ʺ). Plants
were disinfected by 0.5% (v/v) NaClO, rinsed by distilled
water to remove unwanted organisms, and cultivated in a
large aquarium containing growth medium (in mg ­L −1:
­MgSO4⋅7H2O, 49.6; ­KH2PO4, 50.3; ­CaCl2, 11.1; ­KNO3
202; ­FeSO4⋅7H2O, 6; K­ 2HPO4, 27.8; K
­ 2SO4, 17.4; H
­ 3BO3,
5.72; ­Na 2-EDTA, 10; ­M nCl 2⋅4H 2O, 2.82; ­Z nSO 4, 0.6;
­CuCl2⋅H2O, 0.008; (­ NH4)6Mo7O24 ­4 H2O, 0.043; C ­ oCl2
365
­6H2O, 0.054) (Megateli et al. 2013). Plants were main-
tained under the following conditions (growth medium pH
7; temperature 25°C; and photoperiod 16/8 h (light/dark)
for a week). Before starting the experiments, sterilization
of the medium was performed by autoclave (for 1.5 h at
121°C) to remove biotic factors which might affect the
surfactant degradation. Physiological experiments were
performed with five concentrations of LAS surfactant (0,
10, 20, 30, and 40 mg ­L −1), fresh weights of L. minor
(4 g), and two treatment periods (3 and 7 days).
Sodium dodecyl benzene sulfonate surfactant
­(C18H29NaO3S; CAS number: 25155-30-0) was purchased
from Sigma (United States). Sodium dodecyl benzene sul-
fonate is a type of LAS, and its alkyl chain contains 12
carbons. Removal assays were performed with four con-
centrations of LAS surfactant (10, 20, 30 and 40 mg L ­ −1),
duration of treatment (1, 3, and 7 days), temperatures (5,
15 and 25°C), pH values (4, 5.5, 7 and 8.5), and initial
fresh weights of L. minor (0.5, 1, 2 and 4 g). The concen-
tration of surfactant in the culture medium was measured
using the Methylene Blue Active Substances (MBAS)
assay (Closceri et al. 1999). Percentage removal of the
LAS was represented by:
C0 − C1
LASremoval = × 100 (1)
C0
where ­C0 is the initial LAS concentration (mg ­L−1); ­C1 is the
final LAS concentration (mg ­L−1).
To identify metabolites produced by the biological puri-
fication process of surfactant, 4 g of the plant were treated
with the culture medium containing surfactant (20 mg L ­ −1)
for 7 days. After filtering the culture medium, solid-phase
extraction was performed. Chromatographic separations
were performed using an eclipse XDB-C18 column (5µ
particle size, 4.6 × 150 mm); with temperature of 35°C
at a flow rate of 0.5 mL ­min−1. The mass conditions were
mode: ­ESI−; cone volt: 35 V; capillary volt: 4.5 kV; gas
nebulizer: ­N2 (grade 5); flow gas rate: 250 L ­h−1; source
temperature: 120°C; and desolvation temperature: 300°C.
Intermediates from biological degradation of LAS by
Lemna were identified based on the comparison of their
molecular mass to charge ratio (m/z) with standard com-
pounds from Yadav et al. (2001).
Relative growth rates (RGR) of plants were measured
after 7 days of treatment and determined through Eq. (2)
(Radić et al. 2010):
ln(final fresh weight) − ln(initial fresh weight)
RGR(1∕day) =
day
(2)
To determine the effect index (EI), first, the tolerance
index (TI) was measured based on the relative growth
13
366
rate and calculated through Eq. (3). Effect index (EI) was
determined using Eq. (4) (Masoudian et al. 2020).
RGR at higher LAS concentration
TI = (3)
RGR under control conditions
EI = 1 − TI (4)
Concentrations of chlorophyll a, b, and total carotenoids
were evaluated using the method reported by Lichtenthaler
(1987). The plant was homogenized with 80% acetone.
Samples were stored in the refrigerator for 24 h. Absorb-
ance of the solvent was determined at 663 nm, 646 nm,
and 470 nm with CamSpec M501 model UV/Visible
spectrophotometer.
To determine enzyme activity, the plant was homoge-
nized using 0.01 M of potassium phosphate buffer (pH 7)
containing 0.2% of polyvinyl pyrrolidone. The extract was
centrifuged at 10,000×g at 4°C, and the supernatant was
used to measure enzymatic activity and total protein con-
tent. POD activity was measured using phosphate buffer
(10 mM, pH 7.0), 4 mM H ­ 2O2, 20 mM guaiacol, and 100
µL of enzyme extract. Guaiacol oxidation and tetraguaiacol
production were determined as POD activity at 470 nm
(Chance and Maehly 1955). PPO activity was measured
using 10 mM potassium phosphate buffer, 20 mM pyro-
gallol, 0.74 mM hydrogen peroxide, and enzyme extract.
Purpurogallin production rate was measured as PPO activity
at 420 nm (Chance and Maehly 1955). APX activity was
measured using 0.1 mM EDTA, 100 µL of enzyme extracts,
0.1 mM ­H2O2, 0.5 mM ascorbic acid, and phosphate buffer
(50 Mm). Oxidation of ascorbic acid was recorded as APX
activity at 290 nm (Nakano and Asada 1981). CAT activ-
ity was measured using enzyme extract, 10 mM ­H2O2, and
phosphate buffer (50 mM). H ­ 2O2 reduction was determined
as catalase activity at 240 nm (Chance and Maehly 1955).
Enzyme activity was expressed as U ­mg−1 protein. Protein
concentration was estimated using the method reported by
Bradford (1976).
Anthocyanin content was measured using the method
reported by Wagner (1979). Plants were homogenized
with a solution of hydrochloric acid (1%) in methanol.
Homogenates were centrifuged at 10,000×g. Absorb-
ance of the samples was read at 550 nm. Anthocyanin
content was measured by the extinction coefficient of
33,000 ­mol−1 ­cm−1.
To assay hydrogen peroxide ­(H2O2) content, plants were
homogenized in trichloroacetic acid (0.1% W/V) and centri-
fuged at 12,000×g for 15 min. Then, 500 µL of the extract
was added to 2.5 mL of reaction mixture containing 1 M
potassium iodide and 100 mM potassium phosphate buffer.
Mixtures were incubated for 60 min in the dark. Next, the
absorbance of the mixtures was determined at 390 nm. ­H2O2
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Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372
concentration was calculated using the standard ­H2O2 curve
(Alexieva et al. 2001).
To measure DPPH scavenging ability (antioxidant activ-
ity), plant fresh samples were homogenized in methanol
(80% v/v) and centrifuged at 10,000×g for 15 min. Then,
the antioxidant activity was measured based on free radi-
cal scavenging 2, 2-diphenyl-1-picrylhydrazyl (DPPH).
The mixture containing the ethanolic solution of 0.1 μM
DPPH and the methanolic extract was shaken vigorously
and kept at room temperature. Sample absorbance was
recorded at 517 nm. DPPH scavenging activity ­(DPPHSC
%) was computed by Eq. (5). The lower absorbance of the
sample demonstrates higher free radical scavenging activ-
ity (Selvaraj et al. 2013).
DPPHsc%
(absorbance of the control − absorbance of the sample)
= × 100
absorbance of the control
(5)
To assay electrolyte leakage, 200 mg of the fresh plant
were cut into small fragments and placed in test tubes con-
taining deionized water (10 mL). Tubes were closed and kept
in a water bath at 33°C. After 120 min, the primary electri-
cal conductivity of the solution ­(EC1) was determined using
an electrical conductivity meter (EC-400L, iSTEK, Korea).
Tubes were autoclaved at 121°C for 20 min to release all
electrolytes. Afterward, tubes reached room temperature,
and the terminal electrical conductivity ­(EC2) was read. EL
was computed by Eq. (6) (Dionisio-Sese and Tobita 1998).
Primary electrical conductivity
EL(%) = × 100 (6)
Terminal electrical conductivity
Experiments were done under controlled conditions
using a completely randomized design (CRD) with three
replications. Data were analyzed by SPSS 22.0, and results
were reported as the means ± standard error (SE). One Way
ANOVA was used to compare the means by Duncan’s test,
and differences were considered significant at p ≤ 0.01.
Results
L. minor diminished LAS concentrations in the medium. On
day one bioremoval rate was increased in the 30 and 40 mg
­L−1 treatments compared to the 10 mg ­L−1 treatment. On
day three, the percentage of bioremediation in the 40 mg ­L−1
treatment was higher than the 10 mg ­L−1 treatment. How-
ever, on day seven, the percentage of bioremoval showed a
significant increase only in the 20 mg L­ −1 treatment com-
−1
pared to the 40 mg ­L treatment. In all treatments, the
bioremoval efficiency was not significantly different between
days 1 and 3. (Fig. 1a). Bioremoval efficiency was enhanced
by increasing L. minor biomass, as bioremoval efficiency in
Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372 367

Fig. 1  Bioremoval efficiency of a b

linear alkylbenzene sulfonate 100
1 Day 3 Day 7 Day 100
(LAS) by L. minor (a) at various 80 80

Bioremoval (%)
LAS concentrations (T = 25°C,

Bioremoval (%)
a a
ab
Treatment period = 1, 3, 7 days, 60 a-d
a-d a-d
a-c
a-d b-d 60 ab
pH 7, L. minor biomass = 4 g), c-e b-d
40 bc
b at different plant biomass e
de 40 c
[Treatment period = 7 days, 20 20
(LAS)0 = 20 mg ­L−1, T = 25°C,
pH 7], c at various temperatures 0 0
(pH 7, L. minor biomass = 4 g, 10 20 30 40 0.5 1 2 4
­[LAS]0 = 20 mg ­L−1, Treat- LAS (mg L-1) wieght(g)
ment period = 7 days) and d
at different pH (Treatment c d
period = 7 days, L. minor 100 100
biomass = 4 g, ­[LAS]0 = 20 mg 80
Bioremoval (%)

80
­L−1). Values are mean ± SE of

Bioremoval (%)
a a
ab
three replicates. Different letters 60 ab 60 b
indicate a statistical difference 40
b b b
40
between treatments at p ≤ 0.01
20 20
0 0
5 15 25 4 5.5 7 8.5 10
Temperature
T pH
202

the presence of 4 g of L. minor was 1.2 times higher than to the results, LAS is converted to sulfophenyl carboxylate
medium containing 0.5 g of a plant (Fig. 1b). Bioremedia- (SPC) by alkyl chains of different lengths (­ C2- to C
­ 6-SPC).
−
tion increased with increasing temperature (Fig. 1c). The Benzenesulfonate ring ­(C6H5SO3 ) and five homologous
percentage of removal was significantly higher in pH 7 and sulfophenyl carboxylate were detected (­ C 6H 5SO 3: 157,
8.5 in comparison with other pH values (Fig. 1d). ­C2-SPC: 215, C ­ 3-SPC: 229, C
­ 4-SPC: 243, C ­ 5-SPC: 257,
Using LC–MS analysis, the intermediate compounds ­C6-SPC: 271 m/z) (Fig. 2).
during bioremediation of LAS by L. minor L. were recog- L. minor growth was reduced in the presence of sur-
nized according to their mass to charge ratio. According factant. In all treatments, except for the 10 mg L ­ −1

Fig. 2  (-)-LC–ESI–MS spectra

during bioremediation of linear
alkylbenzene sulfonate (LAS)
surfactant after 7 days

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368 Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372

Table 1  RGR and EI in the L. minor was exposed to Linear Alkylb-
enzene Sulfonate (LAS) solution for 7 days
LAS (mg ­L−1) RGR​
0 0.09 ± 0.003a
10 0.07 ± 0.004ab
20 0.05 ± 0.007bc
30 0.04 ± 0.01c
40 0.02 ± 0.004c
Values are mean ± SE of three replicates. Different letters indicate a
statistical difference between treatments at p ≤ 0.01
treatment, RGR was reduced, while the effect index (EI)
increased (Table 1).
On day three, the amount of chlorophyll a and total
chlorophyll decreased in treatments of 30 and 40 mg L ­ −1
compared to the control. On day seven, it was reduced in
all treatments except 10 mg ­L−1 treatment in comparison
with control. However, chlorophyll b concentration was
unchanged in all applied concentrations of LAS. On both
days three and seven, total carotenoids content decreased in
all treatments except 10 mg ­L−1 treatment (Table 2).
On day three, PPO activity improved in the 30 and 40 mg
−1
­L treatments compared to the control. On day seven, PPO
activity increased in all treatments except 40 mg L­ −1 treat-
ment (Table 3). On day three, no change in POD activity
was detected in all treatments, but on day seven, the activ-
ity of this enzyme increased in all treatments except for
10 mg ­L−1 treatment (Table 3). On day three, APX activity
increased in the 40 mg L ­ −1 treatment. On day seven, APX
EI activity increased in all treatments except 10 mg L ­ −1 treat-
ment (Table 3) . On day three, CAT activity did not change
0c
in any of the treatments, but on day seven, it was increased
0.15 ± 0.04bc
in all treatments compared to the control (Table 3).
0.36 ± 0.09ab
On day three, anthocyanin concentration in L. minor was
0.56 ± 0.12a
increased in the 20, 30, and 40 mg ­L−1 treatments. However,
0.69 ± 0.05a
on day seven, it did not change in any treatments compared
to the control (Table 4). On day three, DPPH scavenging
ability increased in all treatments except 10 mg L ­ −1 treat-
ment. On day seven, it was raised in all treatments in com-
parison with control (Table 4). On day three, ­H2O2 content
did not change in all treatments, but on day seven, it signifi-
cantly increased in the 30 and 40 mg ­L−1 treatments in com-
parison with control (Table 4). On day three, the electrolyte
leakage was elevated in the presence of 40 mg L ­ −1 of LAS
compared to control, and on day seven in all treatments, it
was higher than the control (Table 4).
Discussion
Removal of surfactant by L. minor was considerably affected
by the initial concentration of surfactant. In previous find-
ings, the bioremoval efficiency increased by increasing
of surfactant concentration (Masoudian et al. 2020; Forni
et al. 2008) and by increasing of dye concentration (Vafaei
et al. 2012; Torbati 2016). This may be due to the fact that

Table 2  Photosynthetic pigments concentration (µg ­g−1 FW) in the L. minor exposed to linear alkylbenzene sulfonate (LAS) solution for 3 and
7 days
­ −1)
LAS (mg L Chlorophyll a Chlorophyll b Total chlorophyll Total carotenoids

Day 3 Day 7 Day 3 Day 7 Day 3 Day 7 Day 3 Day 7

Control 225.07 ± 12.8ab 238.4 ± 4.9a 72.39 ± 5.99a 70.91 ± 14.90a 297.47 ± 18. ­1ab 309.31 ± 10.1a 102.7 ± 3.93a 99.85 ± 11.22a
abc a a a abc a ab
10 207.43 ± 2.2 261.37 ± 8.6 65.5 ± 2.88 66.85 ± 3.48 272.93 ± 5.0 328.22 ± 5.2 91.68 ± 0.64 79.16 ± 4.35abc
20 181.65 ± 25.9bcd 138.89 ± 14.3de 49.79 ± 13.8a 60.82 ± 6.09a 231.4 ± 35.6bcd 199.71 ± 20.3 cd 72.07 ± 7.9bc 72.61 ± 7.88bc
30 162.75 ± 16.8cde 114.01 ± 5.5e 40.72 ± 9.69a 50.16 ± 5.44a 203.46 ± 26.5 cd 164.17 ± 8.9d 72.6 ± 5.49bc 59.74 ± 2.74c
40 148.93 ± 10.0de 119.09 ± 6.2e 51.89 ± 4.67a 46.69 ± 3.29a 200.82 ± 12.5 cd 165.78 ± 9.4d 65.41 ± 5.15bc 62.87 ± 3.41c

Values are mean ± SE of three replicates. Different letters indicate a statistical difference between treatments at p ≤ 0.01

Table 3  Effect of increasing LAS on the activity of PPO, POD, APX, and CAT (U ­mg−1 protein) in the L. minor after 3 and 7 days
PPO POD APX CAT​
­ −1) Day 3
LAS (mg L Day 7 Day 3 Day 7 Day 3 Day 7 Day 3 Day 7

Control 290.71 ± ­38d 308.07 ± ­25d 147.31 ± ­41c 150.4 ± ­13c 339.06 ± ­29d 346.94 ± ­24d 0.68 ± 0.06bc 0.66 ± 0.02bc
10 423.25 ± ­26 cd 604.18 ± ­61ab 220.19 ± ­9abc 261.02 ± ­37abc 378.99 ± ­34 cd 539.1 ± ­38bcd 0.38 ± 0.07 cd 1.13 ± 0.03a
20 491.24 ± ­58bcd 758.43 ± ­95a 167.11 ± ­27bc 298.45 ± ­49ab 431.65 ± ­77bcd 609.83 ± ­32b 0.25 ± 0.04 cd 1.2 ± 0.09a
30 614.6 ± ­31abc 629.71 ± ­80abc 184.11 ± ­3bc 339.21 ± ­44a 490.37 ± ­35bcd 589.48 ± ­60bc 0.5 ± 0.06bcd 1.38 ± 0.12a
40 736.37 ± ­49ab 525.65 ± ­41abcd 189.95 ± ­17bc 323.84 ± ­20a 571.01 ± ­85bc 821.58 ± ­21a 0.74 ± 0.1b 1.36 ± 0.08a

Values are mean ± SE of three replicates. Different letters indicate a statistical difference between treatments at p ≤ 0.01

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Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372
Table 4  Anthocyanin content (µmol g­ −1 FW), DPPH scavenging ability (%), hydrogen peroxide concentration (µmol g­ −1 FW), and EL (%) in the
L. minor was grown in medium containing LAS surfactant for 3 and 7 days
Anthocyanine DPPH scavenging ability
−1
LAS (mg ­L ) Day 3 Day 7 Day 3
Control 10.50 ± 1.6b 10.3 ± ­2b 40.43 ± ­4d
10 14.24 ± ­3ab 17.3 ± 4.5ab 48.74 ± ­3 cd
20 23.03 ± 2.3a 18.08 ± ­3ab 62.05 ± ­2ab
30 22.62 ± ­2a 16.57 ± 1.3ab 60.61 ± 2.4ab
40 24.34 ± 2.6a 15.5 ± ­3ab 70.58 ± ­3a
Values are mean of three replicates ± SE. Different letters indicate a statistical difference between treatments at p < 0.01
with increasing LAS concentration, the possibility of con-
tact between LAS molecules and the surface of L. minor
increases. Moreover, the rate of absorption is dependent
on the number of molecules in solution, because penetra-
tion of organic substances into plant cell apoplasts happens
via diffusion (Movefeghi et al. 2016). In the present study,
increasing the plant biomass had a positive effect on reme-
diation. This result was consistent with the results of previ-
ous reports for removal of sodium dodecyl benzene sulfonate
(SDBS) by A. filiculoides (Masoudian et al. 2020), LAS by
Sagittaria lancifolia (Fitrihidajati et al. 2020), an azo dye by
green macroalga Enteromorpha (Khataee et al. 2013), and
Spirodela polyrhiza (Movafeghi et al. 2016; Torbati 2016).
There are two reasons for this finding: (1) increasing in plant
weight providing a wider area for absorption of surfactant;
and (2) increasing plant biomass can increase intracellular
and extracellular enzymes effective in pollutant purification
(Kabra et al. 2011). In this study, increased temperature had
a positive effect on remediation. The highest bioremoval
efficiency of surfactant by Azolla (Masoudian et al. 2020)
and malachite green by Spirodella (Torbati 2016) at 25°C
has been reported. Rising temperatures could lead to the
increase of cell membrane permeability and consequently
enhance diffusion of water and water-soluble compounds
from the medium into plant tissues. Temperature influences
the reaction kinetics and evolvability of enzymes (Bunzel
et al. 2019). According to literature, the 22.5–27.5°C tem-
perature range is optimum for the growth of L. minor (Xiao
et al. 2013). Consequently, a high plant growth rate is also
involved in more effective remediation of contaminants from
water. Removal of surfactant in the pH range of 7.0–8.5 was
more than in acidic and basic (> 8.5) mediums. Results of
our previous study showed that pH 7.0 was optimum for
the bioremoval of SDBS by A. filiculoides (Masoudian et al.
2020). The pH is an influential parameter in the solubil-
ity and absorption capacity of molecules and it can help
regulate the availability and mobility rate of ions and their
different forms (Devlin and Witham 1986).
According to this study, sulfophenyl carboxylates
are probably the essential intermediate compounds of
369
H2O2 EL
Day 7 Day 3 Day 7 Day 3 Day 7
41.65 ± 5.0d 2.90 ± 0.42c 3.00 ± 0.28c 10.43 ± 0.9e 9.57 ± ­1e
53.77 ± 6.0abc 3.41 ± 0.26c 4.83 ± 0.35bc 17.82 ± 0.9de 29.45 ± ­5bcd
60.5 ± 4.2ab 3.76 ± 0.26c 5.58 ± 0.62bc 19.25 ± 3.1de 36.9 ± ­2bc
59.46 ± 2.6ab 5.54 ± 0.35bc 7.99 ± 1.21ab 23.49 ± 3.0cde 43.16 ± ­6b
59.98 ± 4.1ab 4.96 ± 1.11bc 9.27 ± 1.32a 27.13 ± 3.2 cd 63.82 ± ­6a
surfactant degradation by L. minor. Detected intermedi-
ate compounds (­ C2 to C ­ 6-SPC) are similar to intermedi-
ate compounds from SDBS degradation by A. filiculoides
(Masoudian et al. 2020) and Phanerochaete chrysosporium
(Yada et al. 2001). The proposed mechanism for break-
down of the alkyl chain of LAS by L. minor is in accord-
ance with Scott and Jones (2000). The breakdown of the
alkyl chain begins with the oxidation of the terminal
methyl group (ω-oxidation) to the carboxylic acid. Sulfo-
phenyl carboxylic acids (SPCs) can then be subjected to
β-oxidation, and the two-carbon components enter the tri-
carboxylic acid cycle as acetyl Co-A. The next stage is the
loss of the sulfonate group (Scott and Jones 2000). León
et al. (2004) reported biodegradation rates of C­ 11-SPC and
­C5-SPC of over 99%. In previous studies, at 10 mg ­L−1
concentrations of C ­ 11-SPC, unlike ­C11 -LAS, no lethality
was observed (Hampel and Blasco 2002). LAS biodeg-
radation intermediates have also been shown to be 10 to
10,000 times less toxic than the parent molecule (Henau
et al. 1986).
In this study, the potential of L. minor in bioremoval of
LAS was studied. One of the most striking characteristics
of L. minor is its ability to co-existing with various micro-
organisms (Kristanti et al. 2012). Therefore, it can be con-
cluded that bioremediation of LAS was done by the plant as
well as co-exist microorganisms. Some microorganisms can
improve the process of removal of contaminants by plants
through stimulating plant growth and enhancing the bio-
availability of contaminants (Yousaf et al. 2010; Oliveira
et al. 2014). Also, they can produce a variety of enzymes
that can reduce the toxicity of organic pollutants (Wang and
Dai 2011; Rehman et al. 2018) . In previous studies, several
cases of the role of rhizobacteria in phytoremediation have
been reported, including effects of Arthrobacter sp. in SDS
decomposition (Margesin and Schinner 1999), Exiguobac-
terium in Cr removal by L.minor (Tang et al. 2015), and
Pseudomonas and Rhodococcus strains in the degradation of
the nitrophenols (Kristanti et al. 2012). However, the effect
of symbiotic bacteria with duckweed on surfactant remedia-
tion requires further research.
13
370
The L. minor growth rate was reduced in the presence
of surfactant. The inhibitory impact of surfactants on the
growth of A. filiculoides (Masoudian et al. 2020), Myrio-
phyllum spicatum L. (Liu et al. 2019), Chara vulgaris L.
(Liu and Wu 2018b), and L. minor (Forni et al. 2008) was
reported. This phenomenon can be due to reduced photosyn-
thetic pigments or increased ­H2O2 formation (Tables 2, 3).
Metabolism disruption and cellular substructures degrada-
tion may reduce growth and tolerance index (TI) (Markina
2010). In this study, total carotenoids and chlorophyll a were
more effected (decreased) than chlorophyll b, which are sim-
ilar to results reported for A. filiculoides (Azizullah et al.
2012; Masoudian et al. 2020). Ruban et al. (1999) suggested
the more stability of chlorophyll b than other pigments are
related to its tighter connection to proteins existing in the
thylakoids. Decreased content of photosynthetic pigments
may be due to their oxidative degradation and damage to
the protein-pigment complex. Contrary to our findings, Liu
and Wu (2018b) have shown that surfactant reduces chloro-
phyll b in Ceratophyllum demersum L. Our findings showed
an increase in the concentration of H ­ 2O2 by increasing the
surfactant dosage and treatment time, which can indicate
the occurrence of oxidative stress. This finding is consist-
ent with Masoudian et al. (2020). Oxygen-free radicals can
damage the plant's respiratory and photosynthetic systems
and increase the activity of plant antioxidant enzymes as a
compatible reaction. (Bhattacharjee 2012). These phenom-
ena were confirmed by the results of photosynthetic pigment
content and activity of APX, PPO, POD, and CAT (Tables 2,
3). Surfactant concentration and treatment time altered anti-
oxidant enzyme activity. Changes in the activity of antioxi-
dant enzymes support their role in responding to the adverse
effects of SDBS. Modification of the activity of PPO, POD,
CAT, and APX in A. filiculoides (Masoudian et al. 2020) and
an increase in POD and SOD activity in Chara vulgaris in
the presence of surfactant have been reported (Liu and Wu
2018a). Oxidoreductase enzymes play a role in reducing the
reactive oxygen species; they are also involved in reactions
related to converting contaminants to other compounds or
their degradation (Schröder et al. 2008; Husain 2010). In
the present study, increasing the amount of anthocyanin in
the presence of surfactant emphasized the role of these sub-
stances in scavenging free radicals. The antioxidant function
of flavonoid compounds is performed in two ways: (1) par-
ticipation in the redox reactions, or (2) chelation of iron ions
and inactivation of superoxide derivatives produced during
the Fenton reaction (Oturan et al. 2011). Similar findings
were observed in the anthocyanin content of A. filiculoides
treated with SDBS (Masoudian et al. 2020). In this study,
an increase in the DPPH scavenging ability (antioxidant
activity) of L. minor was observed, which may be due to
an increase in the content of non-enzymatic antioxidants
such as anthocyanins and free proline (data not shown) or
13
Bulletin of Environmental Contamination and Toxicology (2022) 109:364–372
increased activity of enzymatic antioxidants (Table 3). In
this study, electrolyte leakage increased with increasing
stress. This phenomenon may be due to increased free radi-
cal formation, such as the increased ­H2O2 content (Table 3)
and subsequently increased lipid peroxidation (data not
shown).
Findings of this investigation indicate that L. minor can
eliminate LAS surfactant from the medium. Bioremoval effi-
ciency is related to parameters such as surfactant concentra-
tion, pH, and temperature. Some intermediate compounds of
degradation surfactant were detected, although the impact
of symbiotic microorganisms involved in this process needs
more investigation. This study showed that the antioxidant
defense system of L. minor was increased, but the harmful
effects of oxidative stress were not completely eliminated.
Decreasing plant growth, total chlorophyll, total carotenoids,
and increasing damage to cell membranes confirm this fact.
Acknowledgements The authors thank the University of Tabriz for
financial support and the University of Guilan for providing labora-
tory facilities.
Author Contributions Conceptualization, SYSL and AN; method-
ology and data curation, SYSL, AN and ZM; writing-original draft
preparation, ZM and ST; project administration SYSL and AN; funding
acquisition, SYSL and ZM. All authors have read and agreed to the
published version of the manuscript.
Data Availability The datasets used and/or analyzed under the cur-
rent study are available from the corresponding author on reasonable
request.
Declarations
Conflict of interest The authors declare no competing interests.
Consent to Publish Not applicable.
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