Differential responses of two cyanobacterial species to R-metalaxyl toxicity: Growth, photosynthesis and antioxidant analyses

Differential responses of two cyanobacterial species to R-metalaxyl toxicity: Growth, photosynthesis and antioxidant analyses

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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:

S0269-7491(19)33318-4

DOI:

https://doi.org/10.1016/j.envpol.2019.113681

Reference:

ENPO 113681

To appear in:

Environmental Pollution

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-metalaxyl

Anabaena laxa

Nostoc muscorum

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

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + ++ +

+ + ++ + + + + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++

1

Differential responses of two cyanobacterial species to R-metalaxyl toxicity: growth,

2

photosynthesis and antioxidant analyses

3 4

Seham M. Hamed1*, Sherif H. Hassan2,6, Samy Selim2,3, Mohammed A.M. Wadaan4,

5

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

3

7

Box 41522, Egypt.

8

4

9

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,

10

5

11

Riyadh, Saudi Arabia

12

6

13

Egypt 7

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

*

15

Email : [email protected]

16

[email protected]

Corresponding authors: Seham M. Hamed

17 18 1

19

Abstract

20

Metalaxyl is a broad-spectrum chiral fungicide that used for the protection of plants,

21

however extensive use of metalaxyl resulted in serious environmental problems. Thus, a study on

22

the detoxification mechanism in algae/cyanobacteria and their ability for phycoremediation is

23

highly recommended. Here, we investigated the physiological and biochemical responses of two

24

cyanobacterial species; Anabaena laxa and Nostoc muscorum to R-metalaxyl toxicity as well as

25

their ability as phycoremediators. Two different levels of R-metalaxyl, at mild (10 mg/L) and

26

high dose (25 mg/L), were applied for one-week. We found that A. laxa absorbed and

27

accumulated more intracellular R-metalaxyl compared to N. muscorum. R-metalaxyl, which

28

triggered a dose-based reduction in cell growth, photosynthetic pigment content, and

29

photosynthetic key enzymes’ activities i.e., phosphoenolpyruvate carboxylase (PEPC) and

30

ribulose‒1,5‒bisphosphate

31

significantly less pronounced in A. laxa. On the other hand, R-metalaxyl significantly induced

32

oxidative damage markers, e.g., H2O2 levels, lipid peroxidation (MDA), protein oxidation and

33

NADPH oxidase activity. However, these increases were also lower in A. laxa compared to N.

34

muscorum. To alleviate R-metalaxyl toxicity, A. laxa induced the polyphenols, flavonoids,

35

tocopherols and glutathione (GSH) levels as well as peroxidase (POX), glutathione peroxidase

36

(GPX), glutathione reductase (GR) and glutathione-s-transferase (GST) enzyme activities. On the

37

contrary, the significant induction of antioxidants in N. muscorum was restricted to ascorbate,

38

catalase (CAT) and ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) enzyme

39

activities. Although A. laxa accumulated more R-metalaxyl, it experienced less stress due to

40

subsequent induction of antioxidants. Therefore, A. laxa may be a promising R-metalaxyl

41

phycoremediator. Our results provided basic data for understanding the ecotoxicology of R-

42

metalaxyl contamination in aquatic habitats and the toxicity indices among cyanobacteria.

carboxylase/oxygenase

2

(RuBisCo).

These

decreases

were

43

Keywords: R-metalaxyl; Cyanobacteria; Phycoremediation; Toxicity; Photosynthetic reactions;

44

Oxidative stress; Antioxidant defense system

45 46

Main finding:

47

R-metalaxyl-induced toxicity in cyanobacteria was a function of dose and species type. Although

48

Anabaena laxa accumulated more R-metalaxyl, it experienced less stress due to its antioxidant

49

defense system. A. laxa may be a promising R-metalaxyl phycoremediator.

50

Introduction:

51

The use of fungicides in agriculture is still an efficient and most popular approach to

52

control fungal pathogens and to produce high-quality crops with optimal yields. However, the

53

frequent use of fungicides possibly poses an environmental danger, especially if residues remain

54

in the soil or migrate off-site and flow into rivers (Wightwick and Allinson, 2007; Komárek et

55

al., 2010). Consequently, their accumulation adversely impacts the health of terrestrial and

56

aquatic ecosystems as well as the long-term fertility of the soil and the productivity of crops

57

(Abass et al., 2007; Komárek et al., 2010; Wightwick et al., 2012).

58

Metalaxyl is a systemic acylalanine fungicide that commonly used to suppress a broad

59

spectrum of fungal pathogens in a number of vegetables and cereal crops (Sukul and Spiteller,

60

2000). Meanwhile, deposits of metalaxyl were detected on some vegetables and fruits (Malhat,

61

2017; Ibrahim et al., 2018). Although metalaxyl is poorly absorbed by the soil particles and other

62

organic materials (Komárek et al., 2010), it is highly soluble in water. Consequently, it can easily

63

leach to groundwater and flow into waterways by agricultural run-offs (Yao et al., 2009). In the

64

study of Ahmed et al. (2010) on samples from the Egyptian soils, metalaxyl was found to be

65

more stable in the microorganism poor soil. In this regard, the half-life of metalaxyl in water is

3

66

106 days, and several studies indicated its stability under neutral pH and in water exposed to

67

normal sunlight conditions (Yao et al., 2009). Thus, it could cause environmental risks due to its

68

high residual levels in agricultural crops under natural environmental conditions. Moreover, the

69

frequent application of metalaxyl was well documented to alter the soil microbial community

70

structure (Wang et al., 2019). Given the high toxicity of R-metalaxyl to some microorganisms

71

including microalgal species (Yao et al., 2009), it is necessary to develop environmentally

72

friendly approaches for phycoremediation of metalaxyl before it can reach the food webs.

73

Cyanobacteria, which are also known as blue green algae, are one of the most prevalent

74

photosynthetic prokaryotes and the major source of nitrogen fixation in paddy fields and

75

freshwater over decades (Singh et al., 2018). Recently, widespread and uncontrolled applications

76

of fungicides and pesticides in agro-ecosystems intensively altered the structure and functions of

77

soil and water microbes such as nitrogen-fixing cyanobacteria (Staley et al., 2015; Singh et al.,

78

2018). In this context, different cyanobacterial and algal species have significantly different

79

tolerance abilities to metalaxyl. Published reports showed that cyanobacteria were less sensitive

80

to metalaxyl compared to green algae (Kim and Lee, 2006; Jianyi et al., 2011). For example, the

81

96-h of the half maximal effective concentration (EC50) of metalaxyl to the green microalgae

82

Chlorella pyrenoidosa and Scenedesmus obliqnus were 21.16 and 7.48 mg/L, respectively (Ma et

83

al., 2002). In contrast, the cyanobacterial species Anabaena flosaquae and Mirocystis aeruginosa

84

showed higher EC50 values of 941.51 and 919.86 mg/L, respectively (Jianyi et al., 2011).

85

Metalaxyl toxicity inhibited mitotic cell division by impairing the biosynthesis of RNA

86

(Fisher and Hayes, 1982). It also inhibited the nitrogen fixation (Monkiedje et al., 2002) and the

87

photosynthetic reactions (de Sousa et al., 2017) and induced oxidative stress in M. aeruginosa

88

(Wang et al., 2018). On the other hand, higher plants could mitigate metalaxyl induced-oxidative

89

damage by increasing their cellular antioxidant defenses (Teixeira et al., 2011; de Sousa et al., 4

90

2013, 2017). In vivo and in vitro studies in S. nigrum suggested that the induced antioxidant

91

system was the major mechanism underlying the metalaxyl tolerance (Teixeira et al., 2011, de

92

Sousa et al., 2013, 2017). Among the microalgal species, studies showed differential sensitivities

93

to different fungicides including metalaxyl (Ma et al., 2002). However, data on metalaxyl toxicity

94

and the detoxification mechanism in algae/cyanobacteria is still unknown.

95

Studies to explore a potential metalaxyl phytoremediator have focused on several

96

organisms such as higher plants (Wang et al., 2015), microorganisms (Martins et al., 2017) and

97

freshwater worms (Di et al., 2013). These organisms were successfully able to degrade a large

98

variety of metalaxyl in soil and water. In this regard, high growth rate and large biomass

99

production of cyanobacteria make them potential outstanding phycoremediators (Kulasooriya and

100

Magana-Arachchi, 2016; Singh et al., 2018). Although they are extremely likely to abate the

101

adverse impacts of agrochemicals such as pesticides (Singh et al., 2018), up to date only a few

102

reports have been investigated their role in metalaxyl phycoremediation (Yao et al., 2009;

103

Baglieri et al., 2016). Moreover, there were no studies unrelieved the phycological and

104

biochemical mechanisms underlying the R-metalaxyl phycoremediation potential of these

105

microorganisms. Therefore, the present study was initiated to investigate the toxicity and

106

bioaccumulation of R-metalaxyl by two cyanobacterial species; Anabaena laxa and Nostoc

107

muscorum, under mild and high R-metalaxyl doses. Several parameters such as cell growth and

108

physiological and biochemical responses were assayed under R-metalaxyl exposure to highlight

109

its environmental risks to the aquatic organisms and the adaptive response of cyanobacteria to R-

110

metalaxyl.

111

2. Materials and methods

112

2.1. The cyanobacterial species and growth conditions

5

113

Anabaena laxa and Nostoc muscorum are hetercystous filamentous cyanobacterial

114

species, which were given by Jouf University, Kingdom of Saudi Arabia. The cyanobacterial

115

species were initially grown in BG110 medium (Stanier et al., 1971). All cultures were incubated

116

under controlled conditions of light intensity (35 µmol photons/m2/s with 16:8 h (light/dark)

117

photoperiods), and temperature (30±2 °C) for one week.

118

2.2. Growth inhibition test of the cyanobacterial species

119

The effective R-metalaxyl fungicide concentrations,

120

to mild and high levels, were determined by a growth inhibition test. The cyanobacterial cells at

121

exponential phase were collected by centrifugation at 3000 rpm at room temperature 25°C for ten

122

minutes. The obtained cell pellets were re-cultivated in a 250 mL conical flask capacity with 100

123

ml BG110 medium. The culture media were separately injected with seven distinct levels (mg/L)

124

of R-metalaxyl, 0 (negative control), 5, 10, 15, 20, 25 and 30 mg/L by adding the active

125

ingredient of R-metalaxyl directly to the medium. Cell growth of the two cyanobacterial species

126

was normalized at time zero to ~ 29 µg/L total Chl content in every treatment. The development

127

in cell growth of two cyanobacterial species was measured in basis of total Chl content (µg/L) at

128

each R-metalaxyl level. Each reading is an average of five replicates.

129

2.3. Experimental conditions

which

decrease

cyanobacterial growth

130

In the first untreated growth phase, A. laxa and N. muscorum were grown under

131

autotrophic growth conditions until exponential phase in a conical flask containing 1L of BG110

132

medium. The cyanobacterial cell pellets were harvested by centrifugation of 200 mL from each

133

culture for 20 min and under aseptic conditions. The obtained cell pellets were re-injected into

134

500 mL culture flask filled with 300 mL BG110 medium treated with mild (10 mg/L) and high

135

dose (25 mg/L), separately, of R-metalaxyl. These doses were selected based on the preliminary

6

136

toxicity test where, 10 and 25 mg/L of R-metalaxyl decreased the cell growth in terms of total

137

Chl by ~25% and 50%, respectively. The cell growth in each individual treatment was adjusted to

138

almost to 29 µg/L total Chl content for both cyanobacterial species. The cultures were incubated

139

for one-week under the same conditions of growth as detailed earlier. By the end of the

140

experiment, appropriate quantity from each individual treated culture was centrifuged, and the

141

obtained pellets were washed twice by 1 mM EDTA, then by miliQ water to remove any

142

adsorbed R-metalaxyl on the surface of the cell. Prior the metabolic analysis, the cyanobacterial

143

samples were freeze-dried in (Operon freeze-dryer, Operon Co. Ltd., Korea) at -80 °C for 48 h.

144

2.4. Intracellular bioaccumulation of R-metalaxyl

145

The intracellular R-metalaxyl contents were measured according to Viñas et al., (2008).

146

Mainly, 0.03‒0.05 g of the freeze-dried cyanobacterial samples was extracted with 5 mL of ethyl

147

acetate. The obtained extracts were filtered and evaporated under a nitrogen stream and the

148

residue reconstituted in water. The R-metalaxyl residues were subsequently micro-extracted

149

through direct-immersion solid-phase micro extraction (SPME, PDMS/DVB fiber, Supelco,

150

Bellefont, CA) at room temperature (25 oC) for 15 min. Eventually, R-metalaxyl residues were

151

adsorbed at a GC‒MS port for analysis. GC‒MS consisted of an ion-trap, detector (Saturn

152

2100T), injector (1177 split/splitless) and varian VF-5 ms capillary column (20 m length, 0.15

153

mm internal diameter, 0.15 ml film thickness) was used. The eluent flow was 0.7 mL/min and the

154

temperature program was 80 °C for 2 min, followed by a gradual increase in the temperature (15

155

°C/ min) up to 300 oC. Detection was performed at the µ SIS ion preparation mode with an

156

isolation window of 1 m/z units (selected m/z for R-metalaxyl: 206). Triphenylmethane in ethyl

157

acetate (1 mg/mL) was used as internal standard and as a QA/QC strategy. An aliquot of 10 µL of

158

triphenylmethane solution in ethyl acetate was added to the spiked cucumber sample. The

7

159

average recovery was 94%, the limit of detection (LOD) was 0.002 mg/kg, the limit of

160

quantification (LOQ) was 0.025 mg/kg and R2 was 0.997. The quantification relied on external

161

calibration with matrix-matching standards prepared from control cyanobacterial specimen

162

containing undetectable amounts of R-metalaxyl.

163

2.5. Physiological and biochemical assays

164

The metabolic measurements of cyanobacterial cells after one-week of the R-metalaxyl

165

stress were measured according to Hamed et al., (2017a,b) and Hamed et al., (2019). These

166

measurements included photosynthesis (Hällbom and Bergman, 1983) and photosynthetic-related

167

enzymes (e.g., ribulose‒1,5‒bisphosphate carboxylase/oxygenase (RuBisCo) (Sulpice et al.,

168

2007) and phosphoenolpyruvate carboxylase (PEPC) (Aoyagi and Bassham 1983) as well as

169

chlorophyll (Chl a) and carotenoids contents (Mackinney, 1941). Oxidative stress markers

170

(malondialdehyde (MDA) (Hodges et al., 1999), hydrogen peroxide (H2O2) (Jiang et al., 1990),

171

protein oxidation (Levine et al., 1994) and NADPH oxidase activity (Sarath et al., 2007) and

172

antioxidant metabolites (e.g., total antioxidant capacity (FRAP) (Benzie and Strain, 1999),

173

polyphenols (Chang et al., 2002), flavonoids (Zhang et al., 2006), tocopherols (AbdElgawad et

174

al., 2015), ascorbate (ASC) and glutathione (GSH) (Potters et al., 2004) and enzymes (e.g.,

175

peroxidase (POX) (Kumar and Khan, 1982), ascorbate peroxidase (APX), glutathione reductase

176

(GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR)

177

(Murshed et al., 2008), glutathione-s-transferase (GST) (Habig et al., 1974),

178

peroxidase (GPX), superoxide dismutase (SOD) (Dhindsa et al., 1981), catalase (CAT)

179

(Aebi,1984) were also analyzed.

180 181

2.6. Statistical analyses

8

glutathione

182

In this study, measurements were statistically compared using two-way ANOVA at

183

significance value of (P ≤ 0.05) by SPSS 20 software (IBM Corporation, New York, USA).

184

Before performing ANOVE test, variances were checked for normality and homogeneity using

185

Shapiro–Wilk and Levene’s testes, respectively. Cluster analysis of metabolic measurements was

186

conducted by Pearson distance metric of the Multi Experiment Viewer (MeV)™ 4 software

187

package (version 4.5, Boston, MA, USA).

188

3. Results

189

3.1. Cyanobacterial growth and doses selection

190

The impact of different R-metalaxyl concentrations (0, 5, 10, 15, 20, 25 and 30 mg/L) on

191

A. laxa and N. muscorum growth was investigated by analyzing the content of total Chl over one-

192

week of exposure. The result revealed that the cell growth was negatively correlated with the R-

193

metalaxyl concentration in a dose-dependent pattern, where a high concentration of R-metalaxyl

194

caused a substantial decrease in the chlorophyll content in both cyanobacterial species

195

(Supplementary Fig. 1). The highest R-metalaxyl concentration (30 mg/L) significantly decreased

196

total Chl level in A. laxa and N. muscorum by 74% and 76%, respectively. Upon exposure to 25

197

mg/L, the total Chl content of A. laxa and N. muscorum were declined by 53% and 59%,

198

respectively. In the cases of treatment with 15 and 20 mg/L of R-metalaxyl, total Chl content of

199

A. laxa was reduced by 28% and 36%, respectively, while N. muscorum showed a greater

200

decrease by 29% (15 mg/L) and 45% (20 mg/L) over one-week of exposure. Moderate growth

201

reduction was observed under 10 mg/L by 19% and 22% in A. laxa and N. muscorum,

202

respectively. Meanwhile, a concentration of 5 mg/L slightly decreased the total Chl content by

203

13% in A. laxa and 15% in N. muscorum. Overall, A. laxa was more tolerant to R-metalaxyl

9

204

compared to N. muscorum (P ≤ 0.05), and 10 and 25 mg/L were selected as mild and high doses,

205

respectively.

206

3.2. Bioaccumulation of R-metalaxyl in the cyanobacterial cells

207

Increasing the concentration of R-metalaxyl in the culture medium resulted in a

208

significant increase in intracellular R-metalaxyl content in both cyanobacterial strains but with

209

variable levels. Notably, A. laxa accumulated higher R-metalaxyl content by 26 and 66 µg/g fresh

210

weight (FW) as compared to 17 and 49 µg/g FW of N. muscorum under both mild and high R-

211

metalaxyl doses, respectively (Fig. 1a). While, the concentration of free R-metalaxyl in the media

212

was significantly high in N. muscorum medium by 25% and 41% at mild and high doses,

213

respectively compared to those of A. laxa (Fig. 1b). Indeed, data of intracellular and free R-

214

metalaxyl contents were consistent and reflecting the higher R-metalaxyl bioaccumulation ability

215

of A. laxa as compared to N. muscorum under R-metalaxyl exposure.

216

3.3. Effect of R-metalaxyl on photosynthetic pigments

217

After one-week of 10 and 25 mg/L R-metalaxyl exposure, Chl a was declined by 14% and

218

43% in A. laxa as compared to 30% and 65% in N. muscorum, respectively (Fig. 2a). In contrast,

219

carotenoids content was gradually increased with increasing R-metalaxyl concentrations by 73%

220

and 149% in A. laxa and by 20% and 93% in N. muscorum at 10 and 25 mg/L R-metalaxyl,

221

respectively. Meanwhile, N. muscorum had higher absolute carotenoids content under control

222

conditions compared to A. laxa (Fig. 2b).

223

3.4. Effect of R-metalaxyl on photosynthetic-related enzymes

224

In accordance with the growth inhibition results, photosynthesis process and the activity

225

of its key enzymes phosphoenolpyruvate carboxylase (PEPC) and ribulose‒1,5‒bisphosphate 10

226

carboxylase/oxygenase (RuBisCo) were considerably reduced in cyanobacterial species treated

227

with R-metalaxyl. These decreases were strengthened at high concentration of R-metalaxyl,

228

compared to the respective control. Treatment with the mild R-metalaxyl dose considerably

229

decreased the photosynthetic activity by 53% and 43% (p≤ 0.05) in A. laxa and N. muscorum,

230

respectively, compared to 70% and 82% (p≤ 0.05) decrease at the high dose, respectively (Fig.

231

2c). Consistently, the RuBisCo activity was also declined in A. laxa and N. muscorum by 13%

232

and 50% (p≤ 0.05), respectively, at 10 mg/L and markedly decreased by 27% and 68% (p≤ 0.05),

233

respectively, at 25 mg/L (Fig. 2d). A slight decrease was noticed for the PEPC activity at 10

234

mg/L by 8% and 16% in A. laxa and N. muscorum, respectively, a decrease percent which was

235

magnified at 25 mg/L to 29% and 73%, respectively (Fig. 2e). Our result revealed that A. laxa

236

was more tolerant to R-metalaxyl stress than N. muscorum, where the activities of RuBisCo and

237

PEPC were less decreased in A. laxa. For example, activities of RuBisCo and PEPC of A. laxa at

238

10 mg/L R-metalaxyl were 77% and 130% higher than of those of N. muscorum. Furthermore, at

239

25 mg/L R-metalaxyl the activities of RuBisCo and PEPC of A. laxa were 130% and 194%

240

higher than those of N. muscorum.

241

3.5. Oxidative stress

242

Our result indicated an increase in the oxidative damage markers induced by the fungicide

243

R-metalaxyl in the two

cyanobacterial species. Where, a considerable increase in H2O2

244

formation, lipid peroxidation (malondialdehyde (MDA)), NADPH oxidase and protein oxidation

245

was recorded, and this increase was dose-related. After one-week exposure, the H2O2 level was

246

increased (8% and 17%) by mild dose of R-metalaxyl dose in A. laxa and N. muscorum,

247

respectively. At the high R-metalaxyl dose, H2O2 level was notably increased in A. laxa and N.

248

muscorum by 46% and 71% (p ≤ 0.05), respectively (Fig. 3a).

11

249

Similarly, at the mild dose MDA content was elevated by 4% and 31% (p ≤ 0.05) in A.

250

laxa and N. muscorum, respectively. On the other hand, the maximum MDA content was

251

observed at the high dose of R-metalaxyl, i.e., 28% and 77% (p ≤ 0.05) in A. laxa and N.

252

muscorum, respectively (Fig. 3b). NADPH oxidase was also slightly increased under the mild

253

dose, while the high dose induced a notable increase by 30% and 47% (p ≤ 0.05) in A. laxa and

254

N. muscorum, respectively (Fig. 3c). Similarly, the mild dose lightly increased the protein

255

oxidation by 3% and 7% in A. laxa and N. muscorum and the high R-metalaxyl dose continued to

256

increase it by 14% and 29% (p ≤ 0.05), respectively (Fig. 3d). Obviously, the two cyanobacterial

257

species experienced a cellular oxidative damage in response to R-metalaxyl treatment, but R-

258

metalaxyl-induced oxidative damage was noticeably less in A. laxa compared to N. muscorum

259

under mild and high R-metalaxyl doses.

260

3.6. Antioxidant defense system

261

3.6.1. Molecular antioxidants

262

We found that R-metalaxyl-induced cellular oxidative damage was associated with

263

induction of antioxidant molecules in both cyanobacterial species. However, exposure to the high

264

R-metalaxyl concentration resulted in a higher antioxidant capacity in A. laxa than in N.

265

muscorum. The total antioxidant capacity measured by ferric reducing/antioxidant power assay

266

(FRAP), was higher in A. laxa than N. muscorum at the mild dose of R-metalaxyl (42% and 6%,

267

respectively). While, the FRAP was measured under the high R-metalaxyl dose and it was

268

increased by 89% and 41% (p≤ 0.05) in A. laxa and N. muscorum, respectively, compared to the

269

control (Fig. 4J). Furthermore, the antioxidant molecules were differentially increased in the two

270

cyanobacterial species under different doses of R-metalaxyl. For instance, the mild R-metalaxyl

271

dose induced glutathione (GSH), polyphenols, flavonoids and tocopherols in A. laxa by 67%, 12

272

62%, 8% and 78% and these antioxidant molecules were further increased by 149%, 134%, 45%

273

and 174% (p≤ 0.05), respectively at the high R-metalaxyl dose (Fig. 4l, o, p & q). As anticipated,

274

the high R-metalaxyl concentration induced high levels of FRAP, GSH, polyphenols, and

275

tocopherols levels in A. laxa by 25%, 17%, 52% and 36%, respectively, compared to the induced

276

levels of these parameters in N. muscorum. Conversely, at both mild and high doses of R-

277

metalaxyl, N. muscorum showed more ASC content by 36% and 118%, respectively (Fig. 4K),

278

which were 25% and 56% greater than those of A. laxa.

279

3.6.2. ROS scavenging enzymes

280

R-metalaxyl significantly induced the activity of antioxidant enzymes in a dose-based

281

response (Fig. 4). On the other hand, there were variable responses between the two

282

cyanobacterial species. For example, the mild dose increased peroxidase (POX), glutathione

283

peroxidase (GPX), glutathione reductase (GR), and glutathione-s-transferase (GST) enzymes

284

activities in A. laxa by 44%, 53%, 63% and 78% (p ≤ 0.05), respectively. These enzymes

285

activities were further elevated by 109%, 93%, 95% and 182% (p ≤0.05), respectively, at the

286

high R-metalaxyl dose, compared to the control (Fig. 4a, c, d, e).

287

Our results also revealed that the two cyanobacterial species exhibited a differential

288

pattern of the enzymes activity responses under R-metalaxyl treatments. For instance, the high

289

dose of R-metalaxyl considerably elevated POX, GR and GPX activities in A. laxa by 49%, 59%

290

and 41%, respectively, compared to N. muscorum (p ≤0.05). On the other hand, more increases in

291

catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and

292

monodehydroascorbate reductase (MDHAR) activities were observed in N. muscorum by 154%,

293

128%, 146% and 109% (p ≤ 0.05), respectively, at the high dose (Fig. 4b, g, h, i).

13

294

In this study, certain ROS scavenging enzymes were found to be significantly increased in

295

N. muscorum under only the high R-metalaxyl dose. For instance, POX, SOD activities were

296

increased by 45% and 58% (p ≤ 0.05), respectively (Fig. 4f, a). Our results also showed that

297

CAT, APX and DHAR activities were more pronounced in N. muscorum by 132%, 76% and

298

95%, respectively, as compared to A. laxa at the high concentration of R-metalaxyl.

299

3.7. Cluster analysis

300

The clustered heat map represented in (Fig. 5) revealed that R-metalaxyl-induced toxicity

301

in cyanobacteria was a function of dose and species type. The measured metabolites were divided

302

into four primary clusters based on their response to R-metalaxyl treatments. The first cluster

303

primarily comprises of photosynthetic activity and the involved photosynthetic enzymes, which

304

were progressively decreased in the two cyanobacterial species in a dose-dependent response,

305

however this reduction was markedly higher in N. muscorum at the high R-metalaxyl

306

concentration (25 mg/L). The second cluster consists of ascorbate and glutathione cycle related

307

metabolites and enzymes (GSH, ASC, DHAR and MDHAR enzymes) and the oxidative damage

308

markers (H2O2, protein oxidation and NADH oxidase) as well as CAT and APX enzymes, which

309

were considerably increased in N. muscorum especially by the higher R-metalaxyl dose. In

310

contrast, the third cluster shows a pronounced increase in total antioxidant capacity (FRAP),

311

polyphenols content, and POX, GR and GPX enzymes activities at the high dose R-metalaxyl in

312

A. laxa compared to N. muscorum. This result assumes their possible role in mitigating R-

313

metalaxyl-induced oxidative stress in A. laxa. The fourth cluster consisted of tocopherols,

314

flavonoids, malondialdehyde (MDA) and the two enzymes SOD and GST. These cellular

315

metabolites showed a significant increase under the high dose of R-metalaxyl in comparison to

316

the mild dose, with a comparable value in A. laxa and N. muscorum. These results assume the

14

317

possible role of these metabolites in alleviating the R-metalaxyl stress in both cyanobacterial

318

species.

319

4. Discussion

320

Fungicides such as metalaxyl-contaminated terrestrial and aquatic ecosystems

321

concentrations have gradually increased worldwide (Komárek et al., 2010; Wang et al., 2019).

322

Our theoretical understanding postulates that metalaxyl accumulates in aquatic and soil

323

organisms and consequently induces growth reduction and cellular damage. Although,

324

cyanobacteria were successfully able to degrade a large variety of metalaxyl in soil and water, a

325

deep understanding of their detoxification mechanisms not well studied. Unrevealing these

326

mechanismsis an important step to select and develop an environmentally friendly

327

microorganism for phycoremediation of metalaxyl before it can reach the food webs. Therefore,

328

in this study we investigated the physiological and biochemical mechanisms underlying R-

329

metalaxyl phycoremediation in two species of cyanobacteria.

330

4.1. R-metalaxyl decreased cell growth by reducing the photosynthesis efficiency

331

In the present study, R-metalaxyl inhibited cell growth and proceeded cell death at the

332

used high concentration. Consistently, R-metalaxyl and rac-metalaxyl induced dose-dependent

333

effects (0.05‒900 mg/L) on Scenedesmus quadricauda (Yao et al., 2009). Growth inhibition by

334

metalaxyl was explained by its interfering with RNA synthesis by reducing the activity of RNA

335

polymerase I (Buchenauer, 1990). Moreover, fungicides were found to induce other abnormal

336

growth and morphological changes such as plasmolysis, thylakoids destruction, nuclei and

337

chloroplasts disappearance and lipid droplets accumulation (Prasad et al., 2010; Liu et al., 2016;

338

Du et al., 2019).

15

339

Numerous studies reported that pesticides and fungicides induced inhibition in

340

photosynthesis at the biochemical and molecular levels. In this context, Prasad et al., (2010)

341

correlated the fungicide endosulfan-induced reduction in the photosynthesis activity in N.

342

muscorum and N. calcicola with the reduced supply of the assimilatory power ATP and NADPH.

343

It was also reported that different pesticides induced electron flow blockage at the QB binding

344

site of the freshwater green algae Chlorella vulgaris, Scenedesmus obliquus and

345

Pseudokirchneriella subcapitata (Chalifour et al., 2009). Indeed, different genes involved in the

346

respiratory electron transport chain were significantly inhibited in Phaeodactylum tricornutum

347

after treatment with the fungicide azoxystrobin (Du et al., 2019).

348

Microalgal photosynthetic pigments content is very sensitive to the toxicity of fungicides.

349

Exposure of algal cells to organic xenobiotics, i.e., fungicides and heavy metals could damage the

350

photosynthetic pigments (Huang et al., 2012a,b; Du et al., 2019). In the current study, R-

351

metalaxyl reduced the photosynthesis of algal cells by decreasing the synthesis of Chl a in a

352

dose-dependent manner. To reduce the oxidative damage in pigments, algae accumulate

353

carotenoids upon exposure to elevated R-metalaxyl content. Carotenoids possess detoxification

354

potential against excited molecules e.g., triplet carbonyls (Woodall et al., 1997). Carotenoids also

355

protect cell components through scavenging free radicals (Czerpak et al., 2006).

356

In the present study, A. laxa experienced less R-metalaxyl stress compared to N.

357

muscorum. Increased stress tolerance of A. laxa was possibly due to high phycoremediation

358

ability. Our finding is in accordance with that of Baglieri et al., (2016) where, Chlorella vulgaris

359

and Scenedesmus quadricauda showed great and quick potential for removal of six

360

agrochemicals from water and the removal of metalaxyl was strongly correlated with the rate of

361

its degradation. In this context, the green microalga Scenedesmus obliquus showed

16

362

enantioselective biodegradation to benalaxyl enantiomers (rac-, S-(+)-, R-(‒)-), a similar

363

chemical group to metalaxyl, where the half-life of R-benalaxyl was 5.04 days (Huang et al.,

364

2012 a).

365

4.2. R-metalaxyl induced cellular oxidative damage and modulated the antioxidant status

366

Our results revealed that R-metalaxyl treatment induced significant oxidative damage in

367

both A. laxa and N. muscorum. In agreement, Wang et al. (2018) reported that R-metalaxyl

368

enantioselectively induced oxidative stress in the cyanobacterium M. aeruginosa. Metalaxyl

369

stress induced a cell damage e.g., lipid peroxidation and DNA damage that was mainly mediated

370

through ROS generation (de Sousa et al., 2013, 2017). Many researchers found that fungicides

371

with different chemical groups remarkably increased MDA content and lipid peroxidation in

372

microalgae (Huang et al., 2012a; Lu et al., 2018; Du et al., 2019). For instance, Huang et al.

373

(2012 a,b) reported that benalaxyl and hexaconazole induced peroxidative damages in pigments

374

and membrane lipids by reactive oxygen species (ROS) in Scenedesmus obliquus. The overall

375

pattern of oxidative damage was largely consistent with our findings in the current study.

376

Similar to previous studies, marked increases in the antioxidant molecules and enzymes

377

activities in response abiotic stresses such as metalaxyl stress and heavy metals (de Sousa et al.,

378

2013, 2017, Hamed et al., 2017a,b). High antioxidant capacity has been linked to improved

379

environmental stress tolerance (Hamed et al., 2017a,b). As a strategy to mitigate oxidative stress

380

impact, microalgal species and higher plants treated with fungicides increased antioxidant

381

metabolites such as proline, polyphenols, tocopherols, glutathione and/or ascorbate levels

382

(Teixeira et al., 2011; de Sousa et al., 2013, 2017; Lu et al., 2018).

383

Fungicides and pesticides-induced antioxidant enzymes activities such as SOD, CAT,

384

APX, GPX and GST were also previously recorded in several microalgal species, e.g.,

17

385

Scenedesmus obliquus, Chlorella pyrenoidosa and Phaeodactylum tricornutum (Liu et al., 2016;

386

Lu et al., 2018; Du et al., 2019). It has been well documented that genes encoding for several

387

antioxidant enzymes are activated by isoproturon pesticide exposure in the green microalga

388

Chlamydomonas reinhardtii (Bi et al., 2012). Overall, high lipid peroxidation level and damage

389

to the membrane structure suggest that the induced antioxidant enzymes may not have been able

390

to maintain ROS below the toxic level. In the current study, we observed that R-metalaxyl

391

induced oxidative damages, however, A. laxa showed less oxidative damage and less growth

392

inhibition than N. muscorum. This stress tolerance was attributed to the high antioxidant defense

393

system of A. laxa, and its biodegradation ability.

394

4.3. Cellular adaptation to R-metalaxyl toxicity is a species-specific response and a function

395

of R-metalaxyl concentration

396

We performed cluster analysis to highlight the species-specific responses of A. laxa and

397

N. muscorum to R-metalaxyl stress at the physiological and biochemical levels (Fig. 5). The

398

differential species stress-tolerance was observed at the photosynthetic related parameters, where

399

it was less affected in A. laxa. R-metalaxyl increased the oxidative damage was more pronounced

400

in N. muscorum. Reduced oxidative damage in A. laxa was attributed to improved antioxidant

401

defense system such as increased FRAP, GSH, tocopherols and polyphenols levels and POX, GR

402

and GPX enzymes levels. In agreement to our results, other abiotic stresses induced by Zn and

403

Cu resulted in differential growth inhibition, oxidative damage and antioxidant responses in

404

Chlorella sorokiniana and Scenedesmus acuminatus (Hamed et al., 2017a,b).

405

Regarding N. muscorum, R-metalaxyl stress induced the activity of APX, CAT, DHAR

406

and MDHAR activities than those in A. laxa. The toxicity of R-metalaxyl towards A. laxa and N.

407

muscorum was a function of the applied R-metalaxyl concentration and species type. Many

18

408

studies investigated the responses of microalgae and higher plants to fungicides and heavy metal

409

toxicities found that increased stress level induced high damage and high antioxidants (Hamed et

410

al., 2017a,b; de Sousa et al., 2013, 2017). Different agrochemicals markedly decreased the

411

photosynthetic processes in the microalgae Chlamydomonas reinhardtii and Phaeodactylum

412

tricornutum (Bi et al., 2012; Du et al., 2019).

413

4.4. R-metalaxyl bioremoval capacity using cyanobacteria

414

Indiscriminate application of metalaxyl poses serious environmental problems against the

415

aquatic and soil organisms as well as for human health (Monkiedje et al., 2002; Sukul, 2006; de

416

Sousa et al., 2017; Wang et al., 2019). Microbial detoxification of metalaxyl in contaminated

417

sites has been elucidated in several reports (Massoud et al., 2008; Martins et al., 2017).

418

Phycoremediation using green microalgae is well-known to be an effective tool for the removal

419

of many agrochemicals (Liu et al., 2016; Bi et al., 2012; Huang et al., 2012 a,b). Recently,

420

Baglieri et al., (2016) found that the green microalgae, Chlorella vulgaris and Scenedesmus

421

quadricauda showed high potential for degradation of pesticides, e.g. fenhexamid, metalaxyl,

422

triclopyr and iprodione. In this regard, simultaneous biocides biodegradation is considered as an

423

important detoxification mechanism in microalgae (Jin et al., 2012; Liu et al., 2016).

424

Cyanobacteria have ubiquitous distribution since they can adapt to several climates, thus

425

they have a great potential to decrease the negative effects of pesticides (Singh et al., 2018).

426

Previous studies assumed that cyanobacteria are less sensitive to metalaxyl than green algae in

427

terms of the toxicity indices EC50 and LOEC (Jianyi et al., 2011). In this study, we applied

428

metalaxyl in higher concentrations than that expected in runoff ranges (5 to 47 µg/L, Wilson et

429

al., 2001). Since target cyanobacteria were able to grow under high R-metalaxyl concentrations

430

up to 25 mg/L, they can be used as excellent bioinoculants for purification of agricultural 19

431

wastewater. The selection of a tolerant cyanobacterial species for the purification of wastewater

432

is an efficient and inexpensive approach for fungicides removal from polluted water bodies. Data

433

obtained in our study suggest that the cyanobacterium A. laxa could be used as an excellent

434

phycoremediator for R-metalaxyl in different contaminated sites.

435

5. Conclusion

436

The present study revealed that R-metalaxyl had a differential toxic effect on the

437

cyanobacterial species A. laxa and N. muscorum. R-metalaxyl significantly inhibited cell growth,

438

photosynthetic pigments content and photosynthesis-related enzymes at high concentrations. A.

439

laxa exposed to high R-metalaxyl over a week showed higher R-metalaxyl bioaccumulation and

440

experienced less R-metalaxyl oxidative injury than N. muscorum. In this study, we provided a

441

holistic integrated framework of the physiological and molecular responses in the studied species

442

to R-metalaxyl. Higher intracellular accumulation of antioxidant enzymes (peroxidase,

443

glutathione reductase, glutathione peroxidase, glutathione-s-transferase and superoxide

444

dismutase) and antioxidant molecules (glutathione, polyphenols, flavonoids and tocopherols)

445

were the main metabolites used by A. laxa to alleviate the R-metalaxyl toxicity. Overall, the

446

capacity of A. laxa antioxidant defense system is most probably contributing to use it as a

447

phycoremediator.

448

Acknowledgment

449

The authors extend their appreciation to the Deanship of Scientific Research at King Saud

450

University for funding this work through research group No. (205).

451

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Microcystin Induction by Chiral Metalaxyl. Environmental Science & Technology

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Letters, doi: 10.1021/acs.estlett.8b00507.

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Wang F., Zhou T., Zhu L., Wang X., Wang J., Wang J., Du Z., Li B. (2019) Effects of successive

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metalaxyl application on soil microorganisms and the residue dynamics. Ecological

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Indicators 103, 194–201.

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Wang M., Hua X., Zhang Q., Yang Y., Shi H., Wang M. (2015) Enantioselective degradation of metalaxyl in Grape, Tomato, and Rice Plants. Chirality 27, 109–114 . 27

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Surface Waters of a Horticultural-Production Catchment in South easten Australia.

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Wilson P.C., Whitwell T., Klaine S.J. (2001) Metalaxyl toxicity, uptake, and distribution in

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several ornamental plant species. Journal of Environmental Quality 30, 411–417.

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Woodall A.A., Britton G., Jackson M.J. (1997) Carotenoids and protection of phospholipids in

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ability. Biochimica and Biophysica Acta 7, 617–635.

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metalaxyl in acute, chronic, and sublethal effect on aquatic organisms: Daphnia magna,

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Scenedesmus quadricanda, and Danio rerio. Environmental Toxicology 24, 148–156.

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Zhang Q., Zhang J., Shen J., Silva A., Dennis D.A., Barron C.J. (2006) A simple 96‒well

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632

Figure captions

633

Supplementary Fig. 1. Growth response in terms of total Chl content of Anabaena laxa and

634

Nostoc muscorum cultured in BG110 medium supplemented with different initial R-metalaxyl

635

concentrations (0‒30 mg/L) for one-week of exposure. Different letters on columns indicate

636

significant differences (P ≤ 0.05); lower-case letters to compare between different R-metalaxyl

637

concentrations within the same species, and capital letters allow comparisons between different

28

638

species within the same R-metalaxyl concentration. Data presented are the average of five

639

replicates ± standard error (SE).

640 641

Fig. 1. (a) intracellular and (b) free R-metalaxyl contents in the culture media of A. laxa and N.

642

muscorum grown in BG110 medium supplemented with different R-metalaxyl concentrations;

643

control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for one week. *Asterisks indicate

644

significances between treatments and control conditions within the same species (p ≤ 0.05).

645

**Asterisks indicate significances between the two species exposed to different treatments (p ≤

646

0.05). Data presented are the average of five replicates ± standard error (SE).

647 648

Fig. 2. Photosynthetic pigments, (a) chlorophyll a content (b) carotenoids content (c)

649

photosynthesis activity (b) RuBisCo activity (d) and phosphoenolpyruvate carboxylase (PEPC)

650

activity (e) of A. laxa and N. muscorum grown in BG110 medium supplemented with different R-

651

metalaxyl concentrations; control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for one-week of

652

exposure.*Asterisks indicate significances between treatments and control conditions within the

653

same species (p ≤ 0.05). Data presented are the average of five replicates ± standard error (SE).

654 655

Fig. 3. Oxidative damage markers (a) H2O2, (b) MDA, (c) NADPH oxidase and (d) protein

656

oxidation of Anabaena laxa and Nostoc muscorum cultured in BG110 medium supplemented with

657

different R-metalaxyl concentrations; control (0 mg/L), mild (10 mg/L) and high (25 mg/L) for

658

one-week exposure. *Asterisks indicate significances between treatments and control conditions

659

within the same species (p ≤ 0.05). Data presented are the average of five replicates ± standard

660

error (SE).

661 29

662

Fig. 4. Response of the antioxidant enzymes activities and antioxidant molecules of A. laxa and

663

N. muscorum grown in BG110medium under different R-metalaxyl concentrations; control (0

664

mg/L), mild (10 mg/L) and high (25 mg/L) for one week exposure; (a) peroxidase (POX), (b)

665

catalase (CAT), (c) glutathione reductase (GR), (d) glutathione peroxidase (GPX), (e)

666

glutathione-s-transferase (GST), (f) superoxide dismutase (SOD), (g) ascorbate peroxidase

667

(APX), (h) dehydroascorbate reductase (DHAR) and (i) monodehydroascorbate reductase

668

(MDHAR); (j) total antioxidant capacity (ferric reducing/antioxidant power assay, FRAP), (k)

669

ascorbate (ASC), (l) glutathione (GSH), (m) ASC/tASC, (n) GSH/tGSH, (o) polyphenols, (p)

670

flavenoids and (q) tocopherols. *Asterisks indicate significances between treatments and control

671

conditions within the same species (p ≤ 0.05).**Asterisks indicate significances between the two

672

species exposed to different treatments (p ≤ 0.05). Data presented are the average of five

673

replicates ± standard error (SE).

674 675

Fig. 5. A cluster analysis (heat map) of several parameters related to photosynthesis, oxidative

676

damage, antioxidant analyzed in A. laxa and N. muscorum after one-week of exposure to; control

677

(0 mg/L), mild (10 mg/L) and high (25 mg/L) R-metalaxyl levels. The patterns shown in the heat

678

map were the average of at least five replicates for each metabolite. Blue and yellow colors

679

indicate lower and higher concentrations, respectively.

680 681 682

30

Fig.1

80 60

** Control 10 mg/L 25 mg/L

b) 20

* *

40

* 20

*

R-metalaxyl content in media (mg/mL)

Internal R-metalaxyl content (µg/g fresh wt)

a)

15

** Control 10 mg/L 25 mg/L

* *

10 5

*

*

0

0 A. laxa

N. muscorum

A. laxa

N. muscorum

a) Chl a (µg/L)

80 70

Control 10 mg/L 25 mg/L

*

60 50

*

40

*

30

*

20 10

b)

30

Carotenoids (µg/L)

Fig. 2.

25

10

*

* *

50

PEPC activity

(nmol /mg protein.min)

*

0 N. muscorum

Control 10 mg/L 25 mg/L

0.8

*

0.6

*

0.4

*

0.2 0.0 A. laxa

N. muscorum

d) RuBisCo activity

Control 10 mg/L 25 mg/L

150

1.0

*

A. laxa 150 (nmol /mg protein.min)

Photosynthesis

(CO 2 fixation (nmol /mg DW. min)

200

1.2

*

5

N. muscorum

A. laxa

e)

*

15

0 A. laxa

100

*

20

0

c)

Control 10 mg/L 25 mg/L

N. muscorum

Control 10 mg/L 25 mg/L

100

* *

50

*

0 A. laxa

N. muscorum

Fig.3. Control 10 mg/L 25 mg/L

* *

* MDA

40

b) 10

20

(nmol /g fresh wt)

60 (nmol /g fresh wt)

H2O2

a)

0 A. laxa

* *

4 2

(nmol /mg protein)

6

6 4 2

d) 4

Control 10 mg/L 25 mg/L

*

A. laxa

Protein peroxidation

NADPH oxidase

(nmol reduced NBT/mg protein.min)

8

8

*

*

0

N. muscorum

c)

Control 10 mg/L 25 mg/L

N. muscorum

Control 10 mg/L 25 mg/L

3 2 1 0

0 A. laxa

N. muscorum

A. laxa

N. muscorum

Fig. 4.

0.0

0 A. laxa

A. laxa

N. muscorum

SOD

6

*

4 2 0

N. muscorum

H2O2

*

Control 10 mg/L 25 mg/L

A. laxa

Control 10 mg/L 25 mg/L

1.0

* *

* 0.5

0.0

N. muscorum

APX

1.5

A. laxa

CAT

(e)

*

0.25

Control 10 m g/L 25 m g/L

*

0.20

GST activity

0.2

8

** GPX activity

0.4

**

10

(d)

**

0.15 0.10 0.05 0.00 A. laxa

N. muscorum

POX

N. muscorum

(µmol GSH-CDNB conj./mg protein.min)

50

*

(a) (µmol NADPH/ mg (protein).min)

100

*

POX activity

150

Control 10 mg/L 25 mg/L

0.6

(µmol oxi. pyrogallol/mg (protein)/min)

0.8

*

*

(b)

**

CAT activity

Control 10 mg/L 25 mg/L

APX activity

200

(g)

(µmol NADPH/mg protein/min)

SOD activity

O2.-

(U/mg protein/min)

250

(f)

(µmol H2O 2/ mg (protein).min)

R-metalaxyl

Control 10 m g/L 25 m g/L

4

*

3

* *

*

2 1 0

A. laxa

N. muscorum

H2O

GPX

(o)

(j)

(i)

2

0.00

MDHAR

GR activity

0.5

*

C ontrol 10 m g/L 25 m g/L

*

Control 10 mg/L 25 mg/L

0.15

*

0.5 0.0

ASC

*

*

80

0.6 0.4

* *

*

0.2

A. laxa

N. muscorum

0.05

A. laxa

(n) Control 10 m g/L 25 m g/L

50 40

60 40

30 20 10

0 A. laxa

* *

0.10

N. muscorum

Control 10 m g/L 25 m g/L

20

0.0

N. muscorum

100

*

0.00 A. laxa

(m)

** Control 10 m g/L 25 m g/L

*

0.1

GSH/tGSH

0.8

1.0

A. laxa

0.20

GR

**

GSSG

*

N. muscorum

N. muscorum

(k)

**

*

A. laxa

0.2

N. muscorum

(µmol/g fresh wt)

1.5

20

(l) A. laxa

0.3

0.0

Control 10 mg/L 25 mg/L

*

0.1

0.0

2.0

*

*

40

0

(c) (µmol NADPH/mg (protein)/min)

1.0

Control 10 mg/L 25 mg/L

*

0.2

0.4

1.5

*

*

0.3

DHAR

(q)

(µg/g fresh wt)

** C o n tro l 1 0 m g/ L 2 5 m g/ L

0.0

*

A. laxa

Tocopherols

DHA

N. muscorum

N. muscorum

*

FRAP

(h) 0.4

ASC/tASC

Flavonoids

Quercitin equivalent (mg/g fresh wt)

2.0

MDHA

0.02

(p) Control 10 mg/L 25 mg/L

GSH

0.04

A. laxa

A. laxa

*

(µmol trolox/ g fresh wt)

*

0.06

0

2.5

*

GSH (µmol/g fresh wt)

4

Control 25 mg/L 10 mg/L

0.08

DHAR

ASC

*

0.10

(µmol AsA/mg protein.min)

*

6

MDHAR

C ontrol 10 m g/L 25 m g/L

*

8

(µmol NADH/ mg (protein).min)

Polyphenols

Gallic acid equivalent (mg/gFW)

10

60

N. muscorum

0 A. laxa

N. muscorum

N. muscorum

Fig.5.

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:

Author’s name

Affiliation

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.