Journal Pre-proof Differential responses of two cyanobacterial species to R-metalaxyl toxicity: Growth, photosynthesis and antioxidant analyses Seham M. Hamed, Sherif H. Hassan, Samy Selim, Mohammed A.M. Wadaan, Mohamed Mohany, Wael N. Hozzein, Hamada AbdElgawad PII:
To appear in:
Received Date: 24 June 2019 Revised Date:
24 November 2019
Accepted Date: 26 November 2019
Please cite this article as: Hamed, S.M., Hassan, S.H., Selim, S., Wadaan, M.A.M., Mohany, M., Hozzein, W.N., AbdElgawad, H., Differential responses of two cyanobacterial species to R-metalaxyl toxicity: Growth, photosynthesis and antioxidant analyses, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.113681. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
R-metalaxylaccumulation Photosynthesis parameters Chl a RuBisCo PEPC Oxidative damage markers MDA H2O2 Protein peroxidation NADH oxidase
++ + + + + +
+ ----++ ++ ++ ++ ++
Antioxidant defense system FRAP GSH Tocopherols Flavenoids Polyphenols GR POX CAT GPX GSH ratio ASC ASC ratio Carotenoids APX DHAR MDHAR GST SOD
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + ++ +
+ + ++ + + + + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++
Differential responses of two cyanobacterial species to R-metalaxyl toxicity: growth,
photosynthesis and antioxidant analyses
Seham M. Hamed1*, Sherif H. Hassan2,6, Samy Selim2,3, Mohammed A.M. Wadaan4,
Mohamed Mohany5, Wael N. Hozzein4,6, Hamada AbdElgawad6,7 1
Soil Microbiology Department, Soils, Water and Environment Research Institute, Agricultural
Research Center, Giza, P.O. 175 El‒Orman, Egypt. 2
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Al jouf
University, Sakaka, P.O. 2014, Saudi Arabia 6
Box 41522, Egypt.
Riyadh, Saudi Arabia
Microbiology and Botany Department, Faculty of Science, Suez Canal University, Ismailia, P.O.
Bioproducts Research Chair, Department of Zoology, College of Science, King Saud University,
Riyadh, Saudi Arabia
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University,
Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef,
Integrated Molecular Plant Physiology Research, Department of Biology, University of
Antwerp, Antwerp, Belgium. 14
Email : [email protected]
Corresponding authors: Seham M. Hamed
17 18 1
Metalaxyl is a broad-spectrum chiral fungicide that used for the protection of plants,
however extensive use of metalaxyl resulted in serious environmental problems. Thus, a study on
the detoxification mechanism in algae/cyanobacteria and their ability for phycoremediation is
highly recommended. Here, we investigated the physiological and biochemical responses of two
cyanobacterial species; Anabaena laxa and Nostoc muscorum to R-metalaxyl toxicity as well as
their ability as phycoremediators. Two different levels of R-metalaxyl, at mild (10 mg/L) and
high dose (25 mg/L), were applied for one-week. We found that A. laxa absorbed and
accumulated more intracellular R-metalaxyl compared to N. muscorum. R-metalaxyl, which
triggered a dose-based reduction in cell growth, photosynthetic pigment content, and
photosynthetic key enzymes’ activities i.e., phosphoenolpyruvate carboxylase (PEPC) and
significantly less pronounced in A. laxa. On the other hand, R-metalaxyl significantly induced
oxidative damage markers, e.g., H2O2 levels, lipid peroxidation (MDA), protein oxidation and
NADPH oxidase activity. However, these increases were also lower in A. laxa compared to N.
muscorum. To alleviate R-metalaxyl toxicity, A. laxa induced the polyphenols, flavonoids,
tocopherols and glutathione (GSH) levels as well as peroxidase (POX), glutathione peroxidase
(GPX), glutathione reductase (GR) and glutathione-s-transferase (GST) enzyme activities. On the
contrary, the significant induction of antioxidants in N. muscorum was restricted to ascorbate,
catalase (CAT) and ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) enzyme
activities. Although A. laxa accumulated more R-metalaxyl, it experienced less stress due to
subsequent induction of antioxidants. Therefore, A. laxa may be a promising R-metalaxyl
phycoremediator. Our results provided basic data for understanding the ecotoxicology of R-
metalaxyl contamination in aquatic habitats and the toxicity indices among cyanobacteria.
Keywords: R-metalaxyl; Cyanobacteria; Phycoremediation; Toxicity; Photosynthetic reactions;
Oxidative stress; Antioxidant defense system
R-metalaxyl-induced toxicity in cyanobacteria was a function of dose and species type. Although
Anabaena laxa accumulated more R-metalaxyl, it experienced less stress due to its antioxidant
defense system. A. laxa may be a promising R-metalaxyl phycoremediator.
The use of fungicides in agriculture is still an efficient and most popular approach to
control fungal pathogens and to produce high-quality crops with optimal yields. However, the
frequent use of fungicides possibly poses an environmental danger, especially if residues remain
in the soil or migrate off-site and flow into rivers (Wightwick and Allinson, 2007; Komárek et
al., 2010). Consequently, their accumulation adversely impacts the health of terrestrial and
aquatic ecosystems as well as the long-term fertility of the soil and the productivity of crops
(Abass et al., 2007; Komárek et al., 2010; Wightwick et al., 2012).
Metalaxyl is a systemic acylalanine fungicide that commonly used to suppress a broad
spectrum of fungal pathogens in a number of vegetables and cereal crops (Sukul and Spiteller,
2000). Meanwhile, deposits of metalaxyl were detected on some vegetables and fruits (Malhat,
2017; Ibrahim et al., 2018). Although metalaxyl is poorly absorbed by the soil particles and other
organic materials (Komárek et al., 2010), it is highly soluble in water. Consequently, it can easily
leach to groundwater and flow into waterways by agricultural run-offs (Yao et al., 2009). In the
study of Ahmed et al. (2010) on samples from the Egyptian soils, metalaxyl was found to be
more stable in the microorganism poor soil. In this regard, the half-life of metalaxyl in water is
106 days, and several studies indicated its stability under neutral pH and in water exposed to
normal sunlight conditions (Yao et al., 2009). Thus, it could cause environmental risks due to its
high residual levels in agricultural crops under natural environmental conditions. Moreover, the
frequent application of metalaxyl was well documented to alter the soil microbial community
structure (Wang et al., 2019). Given the high toxicity of R-metalaxyl to some microorganisms
including microalgal species (Yao et al., 2009), it is necessary to develop environmentally
friendly approaches for phycoremediation of metalaxyl before it can reach the food webs.
Cyanobacteria, which are also known as blue green algae, are one of the most prevalent
photosynthetic prokaryotes and the major source of nitrogen fixation in paddy fields and
freshwater over decades (Singh et al., 2018). Recently, widespread and uncontrolled applications
of fungicides and pesticides in agro-ecosystems intensively altered the structure and functions of
soil and water microbes such as nitrogen-fixing cyanobacteria (Staley et al., 2015; Singh et al.,
2018). In this context, different cyanobacterial and algal species have significantly different
tolerance abilities to metalaxyl. Published reports showed that cyanobacteria were less sensitive
to metalaxyl compared to green algae (Kim and Lee, 2006; Jianyi et al., 2011). For example, the
96-h of the half maximal effective concentration (EC50) of metalaxyl to the green microalgae
Chlorella pyrenoidosa and Scenedesmus obliqnus were 21.16 and 7.48 mg/L, respectively (Ma et
al., 2002). In contrast, the cyanobacterial species Anabaena flosaquae and Mirocystis aeruginosa
showed higher EC50 values of 941.51 and 919.86 mg/L, respectively (Jianyi et al., 2011).
Metalaxyl toxicity inhibited mitotic cell division by impairing the biosynthesis of RNA
(Fisher and Hayes, 1982). It also inhibited the nitrogen fixation (Monkiedje et al., 2002) and the
photosynthetic reactions (de Sousa et al., 2017) and induced oxidative stress in M. aeruginosa
(Wang et al., 2018). On the other hand, higher plants could mitigate metalaxyl induced-oxidative
damage by increasing their cellular antioxidant defenses (Teixeira et al., 2011; de Sousa et al., 4
2013, 2017). In vivo and in vitro studies in S. nigrum suggested that the induced antioxidant
system was the major mechanism underlying the metalaxyl tolerance (Teixeira et al., 2011, de
Sousa et al., 2013, 2017). Among the microalgal species, studies showed differential sensitivities
to different fungicides including metalaxyl (Ma et al., 2002). However, data on metalaxyl toxicity
and the detoxification mechanism in algae/cyanobacteria is still unknown.
Studies to explore a potential metalaxyl phytoremediator have focused on several
organisms such as higher plants (Wang et al., 2015), microorganisms (Martins et al., 2017) and
freshwater worms (Di et al., 2013). These organisms were successfully able to degrade a large
variety of metalaxyl in soil and water. In this regard, high growth rate and large biomass
production of cyanobacteria make them potential outstanding phycoremediators (Kulasooriya and
Magana-Arachchi, 2016; Singh et al., 2018). Although they are extremely likely to abate the
adverse impacts of agrochemicals such as pesticides (Singh et al., 2018), up to date only a few
reports have been investigated their role in metalaxyl phycoremediation (Yao et al., 2009;
Baglieri et al., 2016). Moreover, there were no studies unrelieved the phycological and
biochemical mechanisms underlying the R-metalaxyl phycoremediation potential of these
microorganisms. Therefore, the present study was initiated to investigate the toxicity and
bioaccumulation of R-metalaxyl by two cyanobacterial species; Anabaena laxa and Nostoc
muscorum, under mild and high R-metalaxyl doses. Several parameters such as cell growth and
physiological and biochemical responses were assayed under R-metalaxyl exposure to highlight
its environmental risks to the aquatic organisms and the adaptive response of cyanobacteria to R-
2. Materials and methods
2.1. The cyanobacterial species and growth conditions
Anabaena laxa and Nostoc muscorum are hetercystous filamentous cyanobacterial
species, which were given by Jouf University, Kingdom of Saudi Arabia. The cyanobacterial
species were initially grown in BG110 medium (Stanier et al., 1971). All cultures were incubated
under controlled conditions of light intensity (35 µmol photons/m2/s with 16:8 h (light/dark)
photoperiods), and temperature (30±2 °C) for one week.
2.2. Growth inhibition test of the cyanobacterial species
The effective R-metalaxyl fungicide concentrations,
to mild and high levels, were determined by a growth inhibition test. The cyanobacterial cells at
exponential phase were collected by centrifugation at 3000 rpm at room temperature 25°C for ten
minutes. The obtained cell pellets were re-cultivated in a 250 mL conical flask capacity with 100
ml BG110 medium. The culture media were separately injected with seven distinct levels (mg/L)
of R-metalaxyl, 0 (negative control), 5, 10, 15, 20, 25 and 30 mg/L by adding the active
ingredient of R-metalaxyl directly to the medium. Cell growth of the two cyanobacterial species
was normalized at time zero to ~ 29 µg/L total Chl content in every treatment. The development
in cell growth of two cyanobacterial species was measured in basis of total Chl content (µg/L) at
each R-metalaxyl level. Each reading is an average of five replicates.
2.3. Experimental conditions
In the first untreated growth phase, A. laxa and N. muscorum were grown under
autotrophic growth conditions until exponential phase in a conical flask containing 1L of BG110
medium. The cyanobacterial cell pellets were harvested by centrifugation of 200 mL from each
culture for 20 min and under aseptic conditions. The obtained cell pellets were re-injected into
500 mL culture flask filled with 300 mL BG110 medium treated with mild (10 mg/L) and high
dose (25 mg/L), separately, of R-metalaxyl. These doses were selected based on the preliminary
toxicity test where, 10 and 25 mg/L of R-metalaxyl decreased the cell growth in terms of total
Chl by ~25% and 50%, respectively. The cell growth in each individual treatment was adjusted to
almost to 29 µg/L total Chl content for both cyanobacterial species. The cultures were incubated
for one-week under the same conditions of growth as detailed earlier. By the end of the
experiment, appropriate quantity from each individual treated culture was centrifuged, and the
obtained pellets were washed twice by 1 mM EDTA, then by miliQ water to remove any
adsorbed R-metalaxyl on the surface of the cell. Prior the metabolic analysis, the cyanobacterial
samples were freeze-dried in (Operon freeze-dryer, Operon Co. Ltd., Korea) at -80 °C for 48 h.
2.4. Intracellular bioaccumulation of R-metalaxyl
The intracellular R-metalaxyl contents were measured according to Viñas et al., (2008).
Mainly, 0.03‒0.05 g of the freeze-dried cyanobacterial samples was extracted with 5 mL of ethyl
acetate. The obtained extracts were filtered and evaporated under a nitrogen stream and the
residue reconstituted in water. The R-metalaxyl residues were subsequently micro-extracted
through direct-immersion solid-phase micro extraction (SPME, PDMS/DVB fiber, Supelco,
Bellefont, CA) at room temperature (25 oC) for 15 min. Eventually, R-metalaxyl residues were
adsorbed at a GC‒MS port for analysis. GC‒MS consisted of an ion-trap, detector (Saturn
2100T), injector (1177 split/splitless) and varian VF-5 ms capillary column (20 m length, 0.15
mm internal diameter, 0.15 ml film thickness) was used. The eluent flow was 0.7 mL/min and the
temperature program was 80 °C for 2 min, followed by a gradual increase in the temperature (15
°C/ min) up to 300 oC. Detection was performed at the µ SIS ion preparation mode with an
isolation window of 1 m/z units (selected m/z for R-metalaxyl: 206). Triphenylmethane in ethyl
acetate (1 mg/mL) was used as internal standard and as a QA/QC strategy. An aliquot of 10 µL of
triphenylmethane solution in ethyl acetate was added to the spiked cucumber sample. The
average recovery was 94%, the limit of detection (LOD) was 0.002 mg/kg, the limit of
quantification (LOQ) was 0.025 mg/kg and R2 was 0.997. The quantification relied on external
calibration with matrix-matching standards prepared from control cyanobacterial specimen
containing undetectable amounts of R-metalaxyl.
2.5. Physiological and biochemical assays
The metabolic measurements of cyanobacterial cells after one-week of the R-metalaxyl
stress were measured according to Hamed et al., (2017a,b) and Hamed et al., (2019). These
measurements included photosynthesis (Hällbom and Bergman, 1983) and photosynthetic-related
enzymes (e.g., ribulose‒1,5‒bisphosphate carboxylase/oxygenase (RuBisCo) (Sulpice et al.,
2007) and phosphoenolpyruvate carboxylase (PEPC) (Aoyagi and Bassham 1983) as well as
chlorophyll (Chl a) and carotenoids contents (Mackinney, 1941). Oxidative stress markers
(malondialdehyde (MDA) (Hodges et al., 1999), hydrogen peroxide (H2O2) (Jiang et al., 1990),
protein oxidation (Levine et al., 1994) and NADPH oxidase activity (Sarath et al., 2007) and
antioxidant metabolites (e.g., total antioxidant capacity (FRAP) (Benzie and Strain, 1999),
polyphenols (Chang et al., 2002), flavonoids (Zhang et al., 2006), tocopherols (AbdElgawad et
al., 2015), ascorbate (ASC) and glutathione (GSH) (Potters et al., 2004) and enzymes (e.g.,
peroxidase (POX) (Kumar and Khan, 1982), ascorbate peroxidase (APX), glutathione reductase
(GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR)
(Murshed et al., 2008), glutathione-s-transferase (GST) (Habig et al., 1974),
peroxidase (GPX), superoxide dismutase (SOD) (Dhindsa et al., 1981), catalase (CAT)
(Aebi,1984) were also analyzed.
2.6. Statistical analyses
In this study, measurements were statistically compared using two-way ANOVA at
significance value of (P ≤ 0.05) by SPSS 20 software (IBM Corporation, New York, USA).
Before performing ANOVE test, variances were checked for normality and homogeneity using
Shapiro–Wilk and Levene’s testes, respectively. Cluster analysis of metabolic measurements was
conducted by Pearson distance metric of the Multi Experiment Viewer (MeV)™ 4 software
package (version 4.5, Boston, MA, USA).
3.1. Cyanobacterial growth and doses selection
The impact of different R-metalaxyl concentrations (0, 5, 10, 15, 20, 25 and 30 mg/L) on
A. laxa and N. muscorum growth was investigated by analyzing the content of total Chl over one-
week of exposure. The result revealed that the cell growth was negatively correlated with the R-
metalaxyl concentration in a dose-dependent pattern, where a high concentration of R-metalaxyl
caused a substantial decrease in the chlorophyll content in both cyanobacterial species
(Supplementary Fig. 1). The highest R-metalaxyl concentration (30 mg/L) significantly decreased
total Chl level in A. laxa and N. muscorum by 74% and 76%, respectively. Upon exposure to 25
mg/L, the total Chl content of A. laxa and N. muscorum were declined by 53% and 59%,
respectively. In the cases of treatment with 15 and 20 mg/L of R-metalaxyl, total Chl content of
A. laxa was reduced by 28% and 36%, respectively, while N. muscorum showed a greater
decrease by 29% (15 mg/L) and 45% (20 mg/L) over one-week of exposure. Moderate growth
reduction was observed under 10 mg/L by 19% and 22% in A. laxa and N. muscorum,
respectively. Meanwhile, a concentration of 5 mg/L slightly decreased the total Chl content by
13% in A. laxa and 15% in N. muscorum. Overall, A. laxa was more tolerant to R-metalaxyl
compared to N. muscorum (P ≤ 0.05), and 10 and 25 mg/L were selected as mild and high doses,
3.2. Bioaccumulation of R-metalaxyl in the cyanobacterial cells
Increasing the concentration of R-metalaxyl in the culture medium resulted in a
significant increase in intracellular R-metalaxyl content in both cyanobacterial strains but with
variable levels. Notably, A. laxa accumulated higher R-metalaxyl content by 26 and 66 µg/g fresh
weight (FW) as compared to 17 and 49 µg/g FW of N. muscorum under both mild and high R-
metalaxyl doses, respectively (Fig. 1a). While, the concentration of free R-metalaxyl in the media
was significantly high in N. muscorum medium by 25% and 41% at mild and high doses,
respectively compared to those of A. laxa (Fig. 1b). Indeed, data of intracellular and free R-
metalaxyl contents were consistent and reflecting the higher R-metalaxyl bioaccumulation ability
of A. laxa as compared to N. muscorum under R-metalaxyl exposure.
3.3. Effect of R-metalaxyl on photosynthetic pigments
After one-week of 10 and 25 mg/L R-metalaxyl exposure, Chl a was declined by 14% and
43% in A. laxa as compared to 30% and 65% in N. muscorum, respectively (Fig. 2a). In contrast,
carotenoids content was gradually increased with increasing R-metalaxyl concentrations by 73%
and 149% in A. laxa and by 20% and 93% in N. muscorum at 10 and 25 mg/L R-metalaxyl,
respectively. Meanwhile, N. muscorum had higher absolute carotenoids content under control
conditions compared to A. laxa (Fig. 2b).
3.4. Effect of R-metalaxyl on photosynthetic-related enzymes
In accordance with the growth inhibition results, photosynthesis process and the activity
of its key enzymes phosphoenolpyruvate carboxylase (PEPC) and ribulose‒1,5‒bisphosphate 10
carboxylase/oxygenase (RuBisCo) were considerably reduced in cyanobacterial species treated
with R-metalaxyl. These decreases were strengthened at high concentration of R-metalaxyl,
compared to the respective control. Treatment with the mild R-metalaxyl dose considerably
decreased the photosynthetic activity by 53% and 43% (p≤ 0.05) in A. laxa and N. muscorum,
respectively, compared to 70% and 82% (p≤ 0.05) decrease at the high dose, respectively (Fig.
2c). Consistently, the RuBisCo activity was also declined in A. laxa and N. muscorum by 13%
and 50% (p≤ 0.05), respectively, at 10 mg/L and markedly decreased by 27% and 68% (p≤ 0.05),
respectively, at 25 mg/L (Fig. 2d). A slight decrease was noticed for the PEPC activity at 10
mg/L by 8% and 16% in A. laxa and N. muscorum, respectively, a decrease percent which was
magnified at 25 mg/L to 29% and 73%, respectively (Fig. 2e). Our result revealed that A. laxa
was more tolerant to R-metalaxyl stress than N. muscorum, where the activities of RuBisCo and
PEPC were less decreased in A. laxa. For example, activities of RuBisCo and PEPC of A. laxa at
10 mg/L R-metalaxyl were 77% and 130% higher than of those of N. muscorum. Furthermore, at
25 mg/L R-metalaxyl the activities of RuBisCo and PEPC of A. laxa were 130% and 194%
higher than those of N. muscorum.
3.5. Oxidative stress
Our result indicated an increase in the oxidative damage markers induced by the fungicide
R-metalaxyl in the two
cyanobacterial species. Where, a considerable increase in H2O2
formation, lipid peroxidation (malondialdehyde (MDA)), NADPH oxidase and protein oxidation
was recorded, and this increase was dose-related. After one-week exposure, the H2O2 level was
increased (8% and 17%) by mild dose of R-metalaxyl dose in A. laxa and N. muscorum,
respectively. At the high R-metalaxyl dose, H2O2 level was notably increased in A. laxa and N.
muscorum by 46% and 71% (p ≤ 0.05), respectively (Fig. 3a).
Similarly, at the mild dose MDA content was elevated by 4% and 31% (p ≤ 0.05) in A.
laxa and N. muscorum, respectively. On the other hand, the maximum MDA content was
observed at the high dose of R-metalaxyl, i.e., 28% and 77% (p ≤ 0.05) in A. laxa and N.
muscorum, respectively (Fig. 3b). NADPH oxidase was also slightly increased under the mild
dose, while the high dose induced a notable increase by 30% and 47% (p ≤ 0.05) in A. laxa and
N. muscorum, respectively (Fig. 3c). Similarly, the mild dose lightly increased the protein
oxidation by 3% and 7% in A. laxa and N. muscorum and the high R-metalaxyl dose continued to
increase it by 14% and 29% (p ≤ 0.05), respectively (Fig. 3d). Obviously, the two cyanobacterial
species experienced a cellular oxidative damage in response to R-metalaxyl treatment, but R-
metalaxyl-induced oxidative damage was noticeably less in A. laxa compared to N. muscorum
under mild and high R-metalaxyl doses.
3.6. Antioxidant defense system
3.6.1. Molecular antioxidants
We found that R-metalaxyl-induced cellular oxidative damage was associated with
induction of antioxidant molecules in both cyanobacterial species. However, exposure to the high
R-metalaxyl concentration resulted in a higher antioxidant capacity in A. laxa than in N.
muscorum. The total antioxidant capacity measured by ferric reducing/antioxidant power assay
(FRAP), was higher in A. laxa than N. muscorum at the mild dose of R-metalaxyl (42% and 6%,
respectively). While, the FRAP was measured under the high R-metalaxyl dose and it was
increased by 89% and 41% (p≤ 0.05) in A. laxa and N. muscorum, respectively, compared to the
control (Fig. 4J). Furthermore, the antioxidant molecules were differentially increased in the two
cyanobacterial species under different doses of R-metalaxyl. For instance, the mild R-metalaxyl
dose induced glutathione (GSH), polyphenols, flavonoids and tocopherols in A. laxa by 67%, 12
62%, 8% and 78% and these antioxidant molecules were further increased by 149%, 134%, 45%
and 174% (p≤ 0.05), respectively at the high R-metalaxyl dose (Fig. 4l, o, p & q). As anticipated,
the high R-metalaxyl concentration induced high levels of FRAP, GSH, polyphenols, and
tocopherols levels in A. laxa by 25%, 17%, 52% and 36%, respectively, compared to the induced
levels of these parameters in N. muscorum. Conversely, at both mild and high doses of R-
metalaxyl, N. muscorum showed more ASC content by 36% and 118%, respectively (Fig. 4K),
which were 25% and 56% greater than those of A. laxa.
3.6.2. ROS scavenging enzymes
R-metalaxyl significantly induced the activity of antioxidant enzymes in a dose-based
response (Fig. 4). On the other hand, there were variable responses between the two
cyanobacterial species. For example, the mild dose increased peroxidase (POX), glutathione
peroxidase (GPX), glutathione reductase (GR), and glutathione-s-transferase (GST) enzymes
activities in A. laxa by 44%, 53%, 63% and 78% (p ≤ 0.05), respectively. These enzymes
activities were further elevated by 109%, 93%, 95% and 182% (p ≤0.05), respectively, at the
high R-metalaxyl dose, compared to the control (Fig. 4a, c, d, e).
Our results also revealed that the two cyanobacterial species exhibited a differential
pattern of the enzymes activity responses under R-metalaxyl treatments. For instance, the high
dose of R-metalaxyl considerably elevated POX, GR and GPX activities in A. laxa by 49%, 59%
and 41%, respectively, compared to N. muscorum (p ≤0.05). On the other hand, more increases in
catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and
monodehydroascorbate reductase (MDHAR) activities were observed in N. muscorum by 154%,
128%, 146% and 109% (p ≤ 0.05), respectively, at the high dose (Fig. 4b, g, h, i).
In this study, certain ROS scavenging enzymes were found to be significantly increased in
N. muscorum under only the high R-metalaxyl dose. For instance, POX, SOD activities were
increased by 45% and 58% (p ≤ 0.05), respectively (Fig. 4f, a). Our results also showed that
CAT, APX and DHAR activities were more pronounced in N. muscorum by 132%, 76% and
95%, respectively, as compared to A. laxa at the high concentration of R-metalaxyl.
3.7. Cluster analysis
The clustered heat map represented in (Fig. 5) revealed that R-metalaxyl-induced toxicity
in cyanobacteria was a function of dose and species type. The measured metabolites were divided
into four primary clusters based on their response to R-metalaxyl treatments. The first cluster
primarily comprises of photosynthetic activity and the involved photosynthetic enzymes, which
were progressively decreased in the two cyanobacterial species in a dose-dependent response,
however this reduction was markedly higher in N. muscorum at the high R-metalaxyl
concentration (25 mg/L). The second cluster consists of ascorbate and glutathione cycle related
metabolites and enzymes (GSH, ASC, DHAR and MDHAR enzymes) and the oxidative damage
markers (H2O2, protein oxidation and NADH oxidase) as well as CAT and APX enzymes, which
were considerably increased in N. muscorum especially by the higher R-metalaxyl dose. In
contrast, the third cluster shows a pronounced increase in total antioxidant capacity (FRAP),
polyphenols content, and POX, GR and GPX enzymes activities at the high dose R-metalaxyl in
A. laxa compared to N. muscorum. This result assumes their possible role in mitigating R-
metalaxyl-induced oxidative stress in A. laxa. The fourth cluster consisted of tocopherols,
flavonoids, malondialdehyde (MDA) and the two enzymes SOD and GST. These cellular
metabolites showed a significant increase under the high dose of R-metalaxyl in comparison to
the mild dose, with a comparable value in A. laxa and N. muscorum. These results assume the
possible role of these metabolites in alleviating the R-metalaxyl stress in both cyanobacterial
Fungicides such as metalaxyl-contaminated terrestrial and aquatic ecosystems
concentrations have gradually increased worldwide (Komárek et al., 2010; Wang et al., 2019).
Our theoretical understanding postulates that metalaxyl accumulates in aquatic and soil
organisms and consequently induces growth reduction and cellular damage. Although,
cyanobacteria were successfully able to degrade a large variety of metalaxyl in soil and water, a
deep understanding of their detoxification mechanisms not well studied. Unrevealing these
mechanismsis an important step to select and develop an environmentally friendly
microorganism for phycoremediation of metalaxyl before it can reach the food webs. Therefore,
in this study we investigated the physiological and biochemical mechanisms underlying R-
metalaxyl phycoremediation in two species of cyanobacteria.
4.1. R-metalaxyl decreased cell growth by reducing the photosynthesis efficiency
In the present study, R-metalaxyl inhibited cell growth and proceeded cell death at the
used high concentration. Consistently, R-metalaxyl and rac-metalaxyl induced dose-dependent
effects (0.05‒900 mg/L) on Scenedesmus quadricauda (Yao et al., 2009). Growth inhibition by
metalaxyl was explained by its interfering with RNA synthesis by reducing the activity of RNA
polymerase I (Buchenauer, 1990). Moreover, fungicides were found to induce other abnormal
growth and morphological changes such as plasmolysis, thylakoids destruction, nuclei and
chloroplasts disappearance and lipid droplets accumulation (Prasad et al., 2010; Liu et al., 2016;
Du et al., 2019).
Numerous studies reported that pesticides and fungicides induced inhibition in
photosynthesis at the biochemical and molecular levels. In this context, Prasad et al., (2010)
correlated the fungicide endosulfan-induced reduction in the photosynthesis activity in N.
muscorum and N. calcicola with the reduced supply of the assimilatory power ATP and NADPH.
It was also reported that different pesticides induced electron flow blockage at the QB binding
site of the freshwater green algae Chlorella vulgaris, Scenedesmus obliquus and
Pseudokirchneriella subcapitata (Chalifour et al., 2009). Indeed, different genes involved in the
respiratory electron transport chain were significantly inhibited in Phaeodactylum tricornutum
after treatment with the fungicide azoxystrobin (Du et al., 2019).
Microalgal photosynthetic pigments content is very sensitive to the toxicity of fungicides.
Exposure of algal cells to organic xenobiotics, i.e., fungicides and heavy metals could damage the
photosynthetic pigments (Huang et al., 2012a,b; Du et al., 2019). In the current study, R-
metalaxyl reduced the photosynthesis of algal cells by decreasing the synthesis of Chl a in a
dose-dependent manner. To reduce the oxidative damage in pigments, algae accumulate
carotenoids upon exposure to elevated R-metalaxyl content. Carotenoids possess detoxification
potential against excited molecules e.g., triplet carbonyls (Woodall et al., 1997). Carotenoids also
protect cell components through scavenging free radicals (Czerpak et al., 2006).
In the present study, A. laxa experienced less R-metalaxyl stress compared to N.
muscorum. Increased stress tolerance of A. laxa was possibly due to high phycoremediation
ability. Our finding is in accordance with that of Baglieri et al., (2016) where, Chlorella vulgaris
and Scenedesmus quadricauda showed great and quick potential for removal of six
agrochemicals from water and the removal of metalaxyl was strongly correlated with the rate of
its degradation. In this context, the green microalga Scenedesmus obliquus showed
enantioselective biodegradation to benalaxyl enantiomers (rac-, S-(+)-, R-(‒)-), a similar
chemical group to metalaxyl, where the half-life of R-benalaxyl was 5.04 days (Huang et al.,
4.2. R-metalaxyl induced cellular oxidative damage and modulated the antioxidant status
Our results revealed that R-metalaxyl treatment induced significant oxidative damage in
both A. laxa and N. muscorum. In agreement, Wang et al. (2018) reported that R-metalaxyl
enantioselectively induced oxidative stress in the cyanobacterium M. aeruginosa. Metalaxyl
stress induced a cell damage e.g., lipid peroxidation and DNA damage that was mainly mediated
through ROS generation (de Sousa et al., 2013, 2017). Many researchers found that fungicides
with different chemical groups remarkably increased MDA content and lipid peroxidation in
microalgae (Huang et al., 2012a; Lu et al., 2018; Du et al., 2019). For instance, Huang et al.
(2012 a,b) reported that benalaxyl and hexaconazole induced peroxidative damages in pigments
and membrane lipids by reactive oxygen species (ROS) in Scenedesmus obliquus. The overall
pattern of oxidative damage was largely consistent with our findings in the current study.
Similar to previous studies, marked increases in the antioxidant molecules and enzymes
activities in response abiotic stresses such as metalaxyl stress and heavy metals (de Sousa et al.,
2013, 2017, Hamed et al., 2017a,b). High antioxidant capacity has been linked to improved
environmental stress tolerance (Hamed et al., 2017a,b). As a strategy to mitigate oxidative stress
impact, microalgal species and higher plants treated with fungicides increased antioxidant
metabolites such as proline, polyphenols, tocopherols, glutathione and/or ascorbate levels
(Teixeira et al., 2011; de Sousa et al., 2013, 2017; Lu et al., 2018).
Fungicides and pesticides-induced antioxidant enzymes activities such as SOD, CAT,
APX, GPX and GST were also previously recorded in several microalgal species, e.g.,
Scenedesmus obliquus, Chlorella pyrenoidosa and Phaeodactylum tricornutum (Liu et al., 2016;
Lu et al., 2018; Du et al., 2019). It has been well documented that genes encoding for several
antioxidant enzymes are activated by isoproturon pesticide exposure in the green microalga
Chlamydomonas reinhardtii (Bi et al., 2012). Overall, high lipid peroxidation level and damage
to the membrane structure suggest that the induced antioxidant enzymes may not have been able
to maintain ROS below the toxic level. In the current study, we observed that R-metalaxyl
induced oxidative damages, however, A. laxa showed less oxidative damage and less growth
inhibition than N. muscorum. This stress tolerance was attributed to the high antioxidant defense
system of A. laxa, and its biodegradation ability.
4.3. Cellular adaptation to R-metalaxyl toxicity is a species-specific response and a function
of R-metalaxyl concentration
We performed cluster analysis to highlight the species-specific responses of A. laxa and
N. muscorum to R-metalaxyl stress at the physiological and biochemical levels (Fig. 5). The
differential species stress-tolerance was observed at the photosynthetic related parameters, where
it was less affected in A. laxa. R-metalaxyl increased the oxidative damage was more pronounced
in N. muscorum. Reduced oxidative damage in A. laxa was attributed to improved antioxidant
defense system such as increased FRAP, GSH, tocopherols and polyphenols levels and POX, GR
and GPX enzymes levels. In agreement to our results, other abiotic stresses induced by Zn and
Cu resulted in differential growth inhibition, oxidative damage and antioxidant responses in
Chlorella sorokiniana and Scenedesmus acuminatus (Hamed et al., 2017a,b).
Regarding N. muscorum, R-metalaxyl stress induced the activity of APX, CAT, DHAR
and MDHAR activities than those in A. laxa. The toxicity of R-metalaxyl towards A. laxa and N.
muscorum was a function of the applied R-metalaxyl concentration and species type. Many
studies investigated the responses of microalgae and higher plants to fungicides and heavy metal
toxicities found that increased stress level induced high damage and high antioxidants (Hamed et
al., 2017a,b; de Sousa et al., 2013, 2017). Different agrochemicals markedly decreased the
photosynthetic processes in the microalgae Chlamydomonas reinhardtii and Phaeodactylum
tricornutum (Bi et al., 2012; Du et al., 2019).
4.4. R-metalaxyl bioremoval capacity using cyanobacteria
Indiscriminate application of metalaxyl poses serious environmental problems against the
aquatic and soil organisms as well as for human health (Monkiedje et al., 2002; Sukul, 2006; de
Sousa et al., 2017; Wang et al., 2019). Microbial detoxification of metalaxyl in contaminated
sites has been elucidated in several reports (Massoud et al., 2008; Martins et al., 2017).
Phycoremediation using green microalgae is well-known to be an effective tool for the removal
of many agrochemicals (Liu et al., 2016; Bi et al., 2012; Huang et al., 2012 a,b). Recently,
Baglieri et al., (2016) found that the green microalgae, Chlorella vulgaris and Scenedesmus
quadricauda showed high potential for degradation of pesticides, e.g. fenhexamid, metalaxyl,
triclopyr and iprodione. In this regard, simultaneous biocides biodegradation is considered as an
important detoxification mechanism in microalgae (Jin et al., 2012; Liu et al., 2016).
Cyanobacteria have ubiquitous distribution since they can adapt to several climates, thus
they have a great potential to decrease the negative effects of pesticides (Singh et al., 2018).
Previous studies assumed that cyanobacteria are less sensitive to metalaxyl than green algae in
terms of the toxicity indices EC50 and LOEC (Jianyi et al., 2011). In this study, we applied
metalaxyl in higher concentrations than that expected in runoff ranges (5 to 47 µg/L, Wilson et
al., 2001). Since target cyanobacteria were able to grow under high R-metalaxyl concentrations
up to 25 mg/L, they can be used as excellent bioinoculants for purification of agricultural 19
wastewater. The selection of a tolerant cyanobacterial species for the purification of wastewater
is an efficient and inexpensive approach for fungicides removal from polluted water bodies. Data
obtained in our study suggest that the cyanobacterium A. laxa could be used as an excellent
phycoremediator for R-metalaxyl in different contaminated sites.
The present study revealed that R-metalaxyl had a differential toxic effect on the
cyanobacterial species A. laxa and N. muscorum. R-metalaxyl significantly inhibited cell growth,
photosynthetic pigments content and photosynthesis-related enzymes at high concentrations. A.
laxa exposed to high R-metalaxyl over a week showed higher R-metalaxyl bioaccumulation and
experienced less R-metalaxyl oxidative injury than N. muscorum. In this study, we provided a
holistic integrated framework of the physiological and molecular responses in the studied species
to R-metalaxyl. Higher intracellular accumulation of antioxidant enzymes (peroxidase,
glutathione reductase, glutathione peroxidase, glutathione-s-transferase and superoxide
dismutase) and antioxidant molecules (glutathione, polyphenols, flavonoids and tocopherols)
were the main metabolites used by A. laxa to alleviate the R-metalaxyl toxicity. Overall, the
capacity of A. laxa antioxidant defense system is most probably contributing to use it as a
The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for funding this work through research group No. (205).
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Supplementary Fig. 1. Growth response in terms of total Chl content of Anabaena laxa and
Nostoc muscorum cultured in BG110 medium supplemented with different initial R-metalaxyl
concentrations (0‒30 mg/L) for one-week of exposure. Different letters on columns indicate
significant differences (P ≤ 0.05); lower-case letters to compare between different R-metalaxyl
concentrations within the same species, and capital letters allow comparisons between different
species within the same R-metalaxyl concentration. Data presented are the average of five
replicates ± standard error (SE).
Fig. 1. (a) intracellular and (b) free R-metalaxyl contents in the culture media of A. laxa and N.
muscorum grown in BG110 medium supplemented with different R-metalaxyl concentrations;
control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for one week. *Asterisks indicate
significances between treatments and control conditions within the same species (p ≤ 0.05).
**Asterisks indicate significances between the two species exposed to different treatments (p ≤
0.05). Data presented are the average of five replicates ± standard error (SE).
Fig. 2. Photosynthetic pigments, (a) chlorophyll a content (b) carotenoids content (c)
photosynthesis activity (b) RuBisCo activity (d) and phosphoenolpyruvate carboxylase (PEPC)
activity (e) of A. laxa and N. muscorum grown in BG110 medium supplemented with different R-
metalaxyl concentrations; control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for one-week of
exposure.*Asterisks indicate significances between treatments and control conditions within the
same species (p ≤ 0.05). Data presented are the average of five replicates ± standard error (SE).
Fig. 3. Oxidative damage markers (a) H2O2, (b) MDA, (c) NADPH oxidase and (d) protein
oxidation of Anabaena laxa and Nostoc muscorum cultured in BG110 medium supplemented with
different R-metalaxyl concentrations; control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for
one-week exposure. *Asterisks indicate significances between treatments and control conditions
within the same species (p ≤ 0.05). Data presented are the average of five replicates ± standard
Fig. 4. Response of the antioxidant enzymes activities and antioxidant molecules of A. laxa and
N. muscorum grown in BG110medium under different R-metalaxyl concentrations; control (0
mg/L), mild (10 mg/L) and high (25 mg/L) for one week exposure; (a) peroxidase (POX), (b)
catalase (CAT), (c) glutathione reductase (GR), (d) glutathione peroxidase (GPX), (e)
glutathione-s-transferase (GST), (f) superoxide dismutase (SOD), (g) ascorbate peroxidase
(APX), (h) dehydroascorbate reductase (DHAR) and (i) monodehydroascorbate reductase
(MDHAR); (j) total antioxidant capacity (ferric reducing/antioxidant power assay, FRAP), (k)
ascorbate (ASC), (l) glutathione (GSH), (m) ASC/tASC, (n) GSH/tGSH, (o) polyphenols, (p)
flavenoids and (q) tocopherols. *Asterisks indicate significances between treatments and control
conditions within the same species (p ≤ 0.05).**Asterisks indicate significances between the two
species exposed to different treatments (p ≤ 0.05). Data presented are the average of five
replicates ± standard error (SE).
Fig. 5. A cluster analysis (heat map) of several parameters related to photosynthesis, oxidative
damage, antioxidant analyzed in A. laxa and N. muscorum after one-week of exposure to; control
(0 mg/L), mild (10 mg/L) and high (25 mg/L) R-metalaxyl levels. The patterns shown in the heat
map were the average of at least five replicates for each metabolite. Blue and yellow colors
indicate lower and higher concentrations, respectively.
680 681 682
** Control 10 mg/L 25 mg/L
R-metalaxyl content in media (mg/mL)
Internal R-metalaxyl content (µg/g fresh wt)
** Control 10 mg/L 25 mg/L
0 A. laxa
a) Chl a (µg/L)
Control 10 mg/L 25 mg/L
(nmol /mg protein.min)
0 N. muscorum
Control 10 mg/L 25 mg/L
0.2 0.0 A. laxa
d) RuBisCo activity
Control 10 mg/L 25 mg/L
A. laxa 150 (nmol /mg protein.min)
(CO 2 fixation (nmol /mg DW. min)
0 A. laxa
Control 10 mg/L 25 mg/L
Control 10 mg/L 25 mg/L
0 A. laxa
Fig.3. Control 10 mg/L 25 mg/L
(nmol /g fresh wt)
60 (nmol /g fresh wt)
0 A. laxa
(nmol /mg protein)
6 4 2
Control 10 mg/L 25 mg/L
(nmol reduced NBT/mg protein.min)
Control 10 mg/L 25 mg/L
Control 10 mg/L 25 mg/L
3 2 1 0
0 A. laxa
0 A. laxa
4 2 0
Control 10 mg/L 25 mg/L
Control 10 mg/L 25 mg/L
Control 10 m g/L 25 m g/L
** GPX activity
0.15 0.10 0.05 0.00 A. laxa
(µmol GSH-CDNB conj./mg protein.min)
(a) (µmol NADPH/ mg (protein).min)
Control 10 mg/L 25 mg/L
(µmol oxi. pyrogallol/mg (protein)/min)
Control 10 mg/L 25 mg/L
(µmol NADPH/mg protein/min)
(µmol H2O 2/ mg (protein).min)
Control 10 m g/L 25 m g/L
2 1 0
C ontrol 10 m g/L 25 m g/L
Control 10 mg/L 25 mg/L
(n) Control 10 m g/L 25 m g/L
30 20 10
0 A. laxa
Control 10 m g/L 25 m g/L
0.00 A. laxa
** Control 10 m g/L 25 m g/L
(µmol/g fresh wt)
(l) A. laxa
Control 10 mg/L 25 mg/L
(c) (µmol NADPH/mg (protein)/min)
Control 10 mg/L 25 mg/L
(µg/g fresh wt)
** C o n tro l 1 0 m g/ L 2 5 m g/ L
Quercitin equivalent (mg/g fresh wt)
(p) Control 10 mg/L 25 mg/L
(µmol trolox/ g fresh wt)
GSH (µmol/g fresh wt)
Control 25 mg/L 10 mg/L
(µmol AsA/mg protein.min)
C ontrol 10 m g/L 25 m g/L
(µmol NADH/ mg (protein).min)
Gallic acid equivalent (mg/gFW)
0 A. laxa
1- R-metalaxyl-induced toxicity in cyanobacteria was a function of dose and species type. 2- Although Anabaena laxa accumulated R-metalaxyl, it experienced less stress than Nostoc muscorum. 3- R-metalaxyl induced photosynthesis inhibition and oxidative damage which were less pronounced in A. laxa 4- Our analyses implicated the role of antioxidant defence system in R-metalaxyl stress tolerance in A. laxa. 5- A. laxa is a promising species for R-metalaxyl phycoremediation.
Author Contribution Statement SMH and HAG designed the experimental approach, contributed in the metabolic measurements, analyzed the data, generated all the figures, statistical analyses and wrote the original draft of the manuscript. SHH contributed in the metabolic analyses, the experimental setup and revised the manuscript. WNH contributed in data analysis, revised and validated the manuscript. SS , MAMW, MM contributed in funding acquisition, data analysis and revising the manuscript.
Conflict of Interest and Authorship Conformation Form Please check the following as appropriate:
● All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. ● This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. ● The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript ● The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:
Seham M. Hamed1, Soil Microbiology Department, Soils, Water and Environment Research Institute, Agricultural Research Center, Giza, P.O. 175 El‒Orman, Egypt. Sherif H. Hassan2, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Al jouf University, Sakaka, P.O. 2014, Saudi Arabia Samy Selim3, Microbiology and Botany Department, Faculty of Science, Suez Canal University, Ismailia, P.O. Box 41522, Egypt. Mohammed A.M. Wadaan4, Bioproducts Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia Mohamed Mohany5, Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Wael N. Hozzein Botany and Microbiology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt Hamada AbdElgawad6, Integrated Molecular Plant Physiology Research, Department of Biology, University of Antwerp, Antwerp, Belgium.