Enantioselective Toxicity of Chiral Herbicide Metolachlor to Microcystis

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Agricultural and Environmental Chemistry

Enantioselective Toxicity of Chiral Herbicide Metolachlor to Microcystis aeruginosa Siyu Chen, Lijuan Zhang, Hui Chen, Zunwei Chen, and Yuezhong Wen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04813 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Journal of Agricultural and Food Chemistry

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Enantioselective Toxicity of Chiral Herbicide Metolachlor to

2

Microcystis aeruginosa

3 4

Siyu Chen,† Lijuan Zhang,† Hui Chen,‡ Zunwei Chen,§ Yuezhong Wen†,*

5 6

† Institute

7

Zhejiang University, Hangzhou 310058, China

8

‡

College of Science and Technology, Ningbo University, Ningbo 315211, China

9

§

Department of Veterinary Integrative Biosciences, Texas A&M University, College

10

of Environmental Health, College of Environmental and Resource Sciences,

Station, TX 77843, United States

1

ACS Paragon Plus Environment


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ABSTRACT:

12

The enantioselective effects of chiral herbicides on aquatic organisms have

13

received increasing attention. As one kind of freshwater algae responsible for most algal

14

blooms, Microcystis aeruginosa (M. aeruginosa) can produce hepatotoxic microcystin

15

and cause serious health concerns for drinking water. Thus, the effects of chiral

16

herbicides on M. aeruginosa are of vital significance but poorly understood, especially

17

as the structures of chiral herbicides become more complex. In this study, the

18

enantioselective effects of four metolachlor enantiomers based on carbon center and

19

axis chirality on M. aeruginosa were investigated for the first time at an enantiomeric

20

level. The results of investigation into algal growth inhibition, chlorophyll a (chl a)

21

content and cell integrity indicated that (S)-metolachlor ((S)-Met) was significantly

22

more toxic than any other isomer. The toxicity ranking of different enantiomers at the

23

highest concentration (15 mg/L) against M. aeruginosa was (S)-Met > (αR,1’S)-Met >

24

(αS,1’S)-Met > (αS,1’R)-Met > (αR,1’R)-Met, with (αS,1’S)-Met and (αR,1’S)-Met

25

displaying a synergistic effect. Additionally, the Fe distribution in M. aeruginosa

26

presented distinct enantioselectivity, which may contribute to the enantioselective

27

toxicity of metolachlor. Furthermore, metolachlor upregulated the expression of genes

28

mcyD and mcyH in an enantioselective manner, indicating that this herbicide can

29

potentially promote the synthesis and efflux of microcystin, thus aggravating

30

agricultural water contamination to different extents. Overall, this study will help to

31

understand the ecotoxicity of metolachlor at a deeper level and provide theoretical

32

insights into the enantioselective behaviors of metolachlor. 2


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Keywords: Metolachlor; enantioselectivity; Microcystis aeruginosa; microcystin; Fe

34

distribution.

35 36

INTRODUCTION

37

As an important group of agrochemicals, herbicides are applied to promote crop

38

yields by preventing the growth of weeds. The use of herbicides ranks first within

39

overall pesticide usage, accounting for up to 50% of agrochemicals.(1-3) Consequently,

40

herbicides unavoidably enter the aquatic ecosystem due to environmental factors such

41

as leaching to underground water and surface runoff, which may cause pleiotropic

42

effects on aquatic organism growth as well as physiological and biochemical processes

43

and even secondary metabolic processes.(4) Additionally, chirality is an inherent

44

attribute of nature, and over a quarter of herbicides possess chiral characteristics.(5)

45

Once they enter the ecosystem, most chiral pesticides with different enantiomers exhibit

46

different biological toxicity and activity toward organisms.(1)

47

The chiral herbicide metolachlor has increased its market share year after year due

48

to its strong herbicidal properties and low toxicity to animals. Researchers generally

49

believe that metolachlor interferes with the function of plant cell division, affects

50

photosynthesis, and disturbs the synthesis of lipids, flavonoids and proteins in plants.(6-

51

8) Due to the asymmetric carbon atom in the alkyl moiety and the chiral nitrogen axis,(9)

52

metolachlor has four enantiomers: (αS,1’S)-Met, (αR,1’S)-Met ， (αR,1’R)-Met, and

53

(αS,1’R)-Met (Figure 1). However, all metolachlor toxicity studies have focused on the

54

comparison between (Rac)-Met (Racemic metolachlor) and (S)-Met, which includes 3



55

the (αS,1’S)-Met and (αR,1’S)-Met enantiomers, and they indicate that (S)-Met

56

contributes to most of the toxicity of metolachlor. Information about the difference

57

between the four enantiomers is scarce. There had been no reports of baseline

58

separation of the four stereoisomers until recent years. The enantiomeric separation of

59

metolachlor was achieved by HPLC using a series of chiral columns,(9) which provides

60

a great opportunity to analyze the potential enantioselectivity of the four metolachlor

61

enantiomers and more accurately assess the ecological effects of metolachlor.

62

Algae have been frequently used in ecotoxicity studies, and, due to their bottom

63

position in the food chain of the aquatic environment, they also play an indispensable

64

role in the balance of the aquatic ecosystem. Microcystis aeruginosa (M. aeruginosa)

65

is a kind of freshwater algae responsible for algal bloom. Further risk lies in decay or

66

the disruption of cell integrity, upon which the cyclic heptapeptide compound

67

microcystin,(10) the most widely distributed hepatotoxin,(11) is released into the

68

aquatic environment and poses a threat to human health. Regarding this toxin, a

69

consensus has been reached that the mechanism of its synthesis is controlled by mcy

70

gene clusters.(12) The expression of these genes can be affected by external

71

environmental factors (13), and a previous study has shown that light has a positive

72

effect on mcyB and mcyD transcription and that the microcystin synthetase gene cluster

73

is regulated by light quality.(14) In addition, it was suggested that some toxic strains of

74

algae may enhance microcystin synthesis in a pesticide-polluted system. (15) However,

75

little is known about the effects of chiral herbicides on microcystin synthesis and release

76

in M. aeruginosa. 4


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In addition to the effects on toxin generation, other basic growth effects caused by

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metolachlor should also be taken into consideration. Trace element imbalance can cause

79

certain biological toxic effects.(16) The content and distribution of micronutrients in

80

plants are associated with the enantioselectivity of chiral herbicides.(17) It was reported

81

that the anomalous distribution of iron (Fe) in Arabidopsis thaliana is related to the

82

enantioselective phytotoxicity of the chiral herbicide dichlorprop.(18) In addition,

83

micronutrient Fe is essential for the growth of planktonic algae because it forms

84

metalloprotein complexes with heme or nonheme proteins that constitute important

85

components of the respiratory chain and photosynthetic system. Additionally, studies

86

have shown that Fe availability affects mcyD expression and microcystin synthesis.(19,

87

20) Therefore, we were interested in whether the effect of the enantioselectivity of

88

chiral herbicides on algae growth and microcystin behavior is related to Fe.

89

In the present study, we studied the enantioselective toxicities of the four isomers

90

of metolachlor on M. aeruginosa. The algal growth inhibition rate, chl a content and

91

cell integrity were determined to measure and compare the ecotoxicity of different

92

enantiomers. Additionally, the different effects of metolachlor enantiomers on the

93

distribution of trace element Fe was analyzed using scanning transmission X-ray

94

microscopy (STXM). Furthermore, the expression of microcystin synthesis-related

95

genes was examined. All of these experiments were performed at an enantiomeric level.

96

The results are conducive to further understanding the active mechanisms of

97

metolachlor and provide a new perspective to illuminate the enantioselective toxicity

98

of chiral pesticides with more complicated structures. 5



99 100

MATERIALS AND METHODS

101

Materials and reagents. M. aeruginosa (FACHB-912) isolated from Taihu Lake

102

in 1997 was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences

103

(Wuhan, China). Racemic metolachlor ((Rac)-Met) with 97.4% purity was purchased

104

from Qiaochang Chemical Co. LTD (Shandong, China). (S)-Met with 96% purity was

105

obtained from Syngenta (Switzerland). Different metolachlor enantiomers ((αR,1’S)-

106

Met, (αS,1’S)-Met, (αR,1’R)-Met and (αS,1’R)-Met) with 100% optical purity were

107

acquired from Phenomenex (Guangdong, China). Other reagents were analysis purity.

108

All glassware was sterilized in an autoclave.

109

Algal culture and growth inhibition assay. M. aeruginosa was cultured in BG-

110

11 medium.(21) M. aeruginosa was incubated at 25 ± 1°C with 12 h light (2000 lux)

111

and 12 h dark. The cultures were shaken twice per day to ensure optimal growth. The

112

absorbance of the algae suspension was measured at 680 nm with a Shimadzu UV-

113

2401PC spectrophotometer (Tokyo, Japan). The cell density was determined using an

114

Olympus BX-53 microscope and Sedgwick-Rafter Counting Chamber. The regression

115

equation between cell density (Y, ×104 cells/mL) and OD680 (X) was calculated as

116

Y=2000 X + 55.0142 (R2=0.9972). Algae suspensions in the exponential growth phase

117

were transferred into the medium mixed with metolachlor to reach a density of 1.1×106

118

cells/mL in the final 50.0 mL solution, and the final pH was adjusted to 7.2 ± 0.1. The

119

exposure concentrations of metolachlor were set as 1, 5, 7, 10, and 15 mg/L, and all the

120

treatments were performed in triplicate. Each flask was incubated as described above. 6


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After 72 h of incubation, when the toxic effects could be observed obviously and clearly,

122

cell density was determined based on OD680 according to the equation mentioned above.

123

Subsequently, the growth inhibition rates were calculated according to the following

124

formula:

125

Inhibition rate =

126 127

(𝐶0 ― 𝐶𝑇) 𝐶0

× 100%,

where C0 represents the cell densities of the control groups, while CT stands for cell densities of the treated groups.(22)

128

Chlorophyll a content. A 10 mL volume of algae suspension of each enantiomer

129

treatment group was centrifuged for 5 min at 8000 rpm to collect M. aeruginosa cells.

130

Then, an equal volume of 95% ethanol was added to obtain chlorophylls. After

131

extraction for 8 h at 4 °C, the supernatant was obtained and quantiﬁed photometrically

132

by a UV-2401 PC spectrophotometer (Shimadzu Corp., Japan) at 665 and 649 nm. The

133

chl a concentration was calculated according to the widely accepted formula as follows

134

(23):

135

Chl a content = 13.7 × OD665 ― 5.76 × OD649

136

Determination of cell integrity. M. aeruginosa cell integrity was determined by

137

flow cytometry (Becton Dickinson ， USA). After 72 h of incubation, 1.0 mL of

138

microalgae suspension of each treatment group was injected into the sample tube. Then,

139

0.2 μM of SYTOX Green nucleic acid dyes was added to the sample tube under dark

140

conditions. The incubation time for each sample was 10 min to ensure sufficient

141

staining. The excitation wavelength was set at 488 nm. Two fluorescence channels (FL1

142

and FL3) were used to collect and record data. FL1 (530 nm) detected green 7



143

fluorescence for SYTOX Green fluorescence intensity detection, and FL3 (670 nm)

144

detected red fluorescence. Forward scatter (FSC) reflected the cell volume, and side

145

scatter (SSC) mirrored the granularity of the cells.(24) FL1 and FSC were used to

146

identify cyanobacterial cells. The FL1 and FL3 channels must detect at least 10,000

147

algal cells. The data were analyzed using WinMDI2.9 software.

148

Analysis of gene expression. The three step PCR procedure was conducted as

149

described previously.(18) Total RNA was extracted using Trizol reagent (Invitrogen)

150

according to manufacturer instructions. RNA was reverse-transcribed to cDNA using a

151

reverse transcriptase kit (Toyobo, Tokyo, Japan); the analysis was conducted using a

152

SybrGreen Real-time PCR Master Mix (Toyobo, Tokyo, Japan). The relative gene

153

expression among the treatment groups was quantified using the 2–ΔΔCt method.(25)

154

Three replicates were used in each treatment. Genes involved in microcystin synthesis

155

were selected as the target genes, and 16S rRNA was selected as the housekeeping gene.

156

Genes and primer sequences are displayed in Table 1.

157

Distribution of Fe in algal cells. After 72 h of exposure, algae suspensions from

158

different treatments were centrifuged at 1,000 rpm for 2 min to collect algae cells, after

159

which the supernatant was discarded. Collected algal cells were fixed overnight with

160

2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0). Samples with a thickness of

161

2 μm were acquired by ultramicrotome (Leica UC7) in the Bioultrastructure Analysis

162

Laboratory (Zhejiang University). Subsequently, algae cell slices were placed on a

163

carbon film molybdenum grid and observed at beamline 08U (BL08U) at Shanghai

164

Synchrotron Radiation Facility (SSRF) of the Chinese Academy of Sciences to examine 8


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the distribution of Fe in algae cells by scanning transmission soft X-ray microscopy

166

(STXM). The step size was 50 nm, and the scanning range was 7 μm × 7 μm. The

167

experiment was performed with a 0.1 eV scan to record the L-side NEXAFS spectrum

168

of Fe. The two-dimensional distribution image of the Fe element was plotted using its

169

absorption difference at 707.2 eV and 703 eV.

170

Statistical analysis. The data were analyzed using Origin 8.0 software (OriginLab,

171

Northampton, MA, USA) according to the methods provided by the manufacturer in

172

the test kit. The comparison was made using one-way analyses of variance (ANOVA)

173

followed by multiple comparison tests of means (Tukey’s test). The differences were

174

considered statistically significant when the P value was less than 0.05.

175 176

RESULTS AND DISCUSSION

177

Enantioselective effects on M. aeruginosa growth inhibition. Enantioselective

178

effects of chiral herbicides on algae have long been recognized (26-28), and toxicity of

179

metolachlor to algae has been discussed in-depth only in terms of carbon center chirality.

180

Much higher toxicity for (S)-Met is indicated compared with the toxicity of (Rac)-Met

181

and (R)-Met.(29, 30) In this study, the toxicities of different enantiomers of metolachlor,

182

based on both carbon center and axis chirality, were compared. As shown in Figure 2,

183

(S)-Met produced the most severe inhibitory effects on M. aeruginosa compared to the

184

effects of other enantiomers, which was consistent with the confirmed conclusion that

185

the herbicidal activity of metolachlor comes from (S)-Met.(31) When the exposure

186

concentration was 1 mg/L, inhibition rates were all below 10%. (αS,1’S)-Met, (αS,1’R)9



187

Met and (αR,1’R)-Met even promoted the growth of M. aeruginosa, which could be

188

contributed to stimulatory effects caused by low-level toxic agents as described in a

189

previous study.(32) The inhibition rate showed a tendency to increase with an increase

190

in the exposure concentration of different enantiomers of metolachlor. The inhibition

191

rate was lower at lower herbicide concentrations but increased rapidly beginning at a

192

concentration 7 mg/L. When the exposure concentration was 15 mg/L, the toxicity

193

ranking of different enantiomers against M. aeruginosa was obviously as follows: (S)-

194

Met > (αR,1’S)-Met > (αS,1’S)-Met > (Rac)-Met > (αS,1’R)-Met > (αR,1’R)-Met.

195

Interestingly, with an inhibition rate of 63.7%, the toxicity of (S)-Met was significantly

196

greater than the toxicity of (αR,1’S)-Met and (αS,1’S)-Met at the same concentration,

197

indicating possible synergism between these two enantiomers separated from (S)-Met

198

based on axis chirality. This is the first study to compare the enantioselective toxicities

199

of all metolachlor enantiomers. To better understand the potential mechanism involved

200

in the different toxicities, more aspects other than growth inhibition have been

201

investigated.

202

Enantioselective effects on chlorophyll a content. Chlorophyll is an

203

indispensable component of plant photosynthesis. In this study, we considered the chl

204

a content to be one of the basic indicators used to compare the effect of metolachlor

205

enantiomers on algal photosynthesis. The results presented in Figure 3 show that after

206

72-h-exposure to 15 mg/L of different metolachlor enantiomers, the chl a content of

207

algal cells in all treated groups was significantly lower than that in the control group.

208

In detail, the chl a content of the (S)-Met-treated group (340.7 μg/L) was less than half 10


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that of the control group (897.4 μg/L) and the (Rac)-Met group (700.9 μg/L). In addition,

210

compared to (αS,1’S)-Met and (αR,1’S)-Met, (S)-Met displayed a stronger inhibitory

211

effect on chl a content, which is consistent with the inhibitory effects on growth and

212

further confirms the possibility of synergy between the chiral axis enantiomers

213

(αS,1’S)-Met and (αR,1’S)-Met.

214

Chlorophylls are essential pigment species for light-harvesting and energy

215

transduction in higher plants and algae.(33) Thus, the content of chlorophyll is closely

216

related to the activity of photosynthesis.(34) In addition, the chlorophyll content is

217

easily affected by external pollutants. Therefore, this growth indicator also reflects

218

external stress as well as the toxicity of xenobiotics. For example, it was demonstrated

219

that the chlorophyll content in Chlorella vulgaris decreased because of the presence of

220

heavy metal copper and cadmium.(35) In addition, another chiral herbicide,

221

imazethapyr, was also reported to enantioselectively affect plant photosynthesis

222

through chlorophyll synthesis.(36) Thus, the sharp decrease in chlorophyll content in

223

this study indicated the highest toxicity of (S)-Met and reflected the different toxicities

224

of the four enantiomers, in which both enantiomers from (S)-Met ((αS,1’S)-Met and

225

(αR,1’S)-Met) were more toxic than those from (R)-Met ((αS,1’R)-Met and (αR,1’R)-

226

Met) in terms of their effects on chl a content.

227

Enantioselective effects on cell integrity. Algal cell integrity is another

228

indication of the level of environmental contamination. A previous study demonstrated

229

that xenobiotic contaminants such as green solvents and chiral ionic liquids can damage

230

aquatic algae cell membrane integrity.(37) Similarly, herbicides have also been 11



231

reported to impact the integrity of algal cells.(27, 38) Therefore, the effects of different

232

metolachlor enantiomers on M. aeruginosa cell integrity were investigated and

233

compared for the first time to evaluate growth conditions and toxic effects. According

234

to the results, different metolachlor enantiomers affected the integrity of M. aeruginosa

235

cells to varying degrees. The proportion of intact cells in the (S)-Met group was only

236

27.4% compared with that of the control group. Compared to the two (S)-isomer

237

((αS,1’S)-Met and (αR,1’S)-Met) groups, (R)-isomer ((αS,1’R)-Met and (αR,1’R)-Met)

238

groups had less of an impact on cell integrity. Notably, the cell integrity percentage in

239

the (S)-Met treatment group was 56.7% and 52% lower than those in the (αS,1’S)-Met

240

and (αR,1’S)-Met groups, respectively, indicating the high toxicity of (S)-Met and the

241

synergy between (αS,1’S)-Met and (αR,1’S)-Met.

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Cyanobacteria can produce various toxic compounds, and most of the cyanotoxins

243

are intracellular in healthy algal cells.(39) However, once the algal cell integrity is

244

destroyed by xenobiotic substances, the intracellular toxins can be released into the

245

surrounding aquatic environment and become extracellular toxins that are difficult to

246

remove,(40) posing a great challenge to the safety of drinking water. In this study, (S)-

247

Met produced the greatest effects on the integrity of M. aeruginosa compared to the

248

effects of the other enantiomers (Figure 4). Additionally, a synergetic effect was

249

observed between both enantiomers from (S)-Met. All these results indicate that, on the

250

one hand, the disruption of cell integrity can be a major contributor to the cytotoxicity

251

of metolachlor enantiomers, which showed significant enantioselective behaviors and

252

synergetic effects, and on the other hand, given the production of extracellular toxins, 12


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further considerations should be taken during ecotoxicity evaluation and risk

254

assessments of metolachlor and other environmental contaminants.

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Enantioselective expression patterns of genes involved in microcystin

256

synthesis. In addition to exogenous factors, e.g., cell integrity, as discussed above,

257

which can result in algal toxin pollution, the effect of endogenous factors, for example,

258

gene expression involved in microcystin synthesis, was also investigated here. The

259

genes for microcystin synthesis are arranged in a certain order and are composed of

260

domains with different roles in microcystin synthesis. The synthesis of microcystin is

261

completed through step-by-step reactions carried out mainly by the mcy gene

262

cluster.(12) The essential chemical group involved in the expression of microcystin, 3-

263

amino-9-methoxy-10-phenyl-2,6,8-trimethyl-decca-4,6-dienoic acid, which is known

264

as Adda, is synthesized through the joint effects of mcyG, mcyD, mcyE and mcyJ.(41,

265

42) Among these genes, the mcyD gene functions as the polyketide synthase (PKS) in

266

Adda synthesis. Additionally, mcyH in the microcystin gene cluster functions as a

267

transporter gene.(43) Therefore, the gene expression patterns of mcyD and mcyH were

268

selectively detected in this study.

269

After treatment with different enantiomers of metolachlor for 72 h, both mcyD and

270

mcyH were significantly upregulated compared to mcyD and mcyH expression in the

271

control, with significant enantiomeric differences (Figure 5). For example, in terms of

272

mcyD expression, the relative transcriptional abundance of mcyD in the (S)-Met group

273

was 12.36 times that in the control group, indicating that (S)-Met promoted Adda

274

synthesis as well as microcystin production. Meanwhile, with regard to the expression 13



275

of mcyH, which encodes the microcystin transmembrane protein, the transcriptional

276

level was 22.83 times higher than that of the control, which implied that (S)-Met

277

increased the release of microcystin. Previous studies have shown that environmental

278

stress has an impact on the behavior of microcystin,(13-15) and the results in this study

279

suggested that metolachlor enantiomers have some potential to promote both the

280

formation and efflux of microcystin, which is likely to increase the concentration of

281

microcystin in a similar pattern. The effects on the expression of genes related to

282

microcystin synthesis and efflux seem to be opposite to the effects on the inhibition

283

rate. In fact, M. aeruginosa is responsible for algae bloom, which is a severe problem

284

leading to water contamination. However, further risk lies in microcystin, which is

285

released into the aquatic ecosystem with decay or cell integrity disruption in the algae.

286

The toxicity of metolachlor destroyed algal cell integrity and even killed the algae cells,

287

reducing the cell density and resulting in the inhibition of algal growth. The microcystin

288

in decaying algae cells was released into the aquatic environment. Additionally, the

289

expression of genes mcyD and mcyH in relatively healthy algae cells was upregulated.

290

In other words, mcy gene expression was positively correlated with the toxicity of

291

metolachlor enantiomers, indicating that mcy genes can act as biomarkers when

292

investigating the effects of contaminant toxicity on M. aeruginosa.

293

Enantioselective effects on Fe distribution in algal cells. Iron (Fe) is an essential

294

trace metal nutrient for the growth of phytoplankton, and its deficiency, excess or

295

changes in its distribution can influence biological growth and even lead to toxicity.(44,

296

45) In this study, the effects of metolachlor enantiomers on Fe distribution in M. 14


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297

aeruginosa were investigated. As shown in Figure 6, after treatment with 15 mg/L of

298

different enantiomers of metolachlor for 72 h, the Fe distribution in algal cells displayed

299

a significant enantioselective difference. In the control group, iron was evenly

300

distributed (Figure 6 a), while in the (S)-Met treatment group, Fe was aggregated around

301

the edge of the cell (Figure 6 c). Similarly, Fe also showed a distinct aggregation pattern

302

in the (αS,1’S)-Met and (αR,1’S)-Met groups (Figure 6 d & e). Additionally, according

303

to the change of intensity, two (R)-stereoisomers caused a certain degree of Fe

304

accumulation (Figure 6 f & g).

305

Over the past few decades, studies have shown that nutrient elements, such as

306

nitrogen, phosphorus and iron, have an impact on microcystin(10) synthesis by M.

307

aeruginosa(20, 46, 47). Furthermore, crosstalk between the behaviors of micronutrients

308

(uptake and distribution) in plants and the enantioselectivity of chiral herbicides has

309

been discussed in previous studies.(17, 18) In this study, the patterns of Fe aggregation

310

in differently treated algal cells were consistent with the toxicity caused by each

311

enantiomeric group. The toxicity of metolachlor enantiomers to M. aeruginosa was

312

positively correlated with abnormal Fe distribution in algal cells, which indicated that

313

enantioselective Fe distribution might contribute to enantioselective toxicity and further

314

confirmed the relationship between microelement behavior and enantioselective effects

315

of chiral herbicides. In addition, considering Fe availability can regulate microcystin

316

synthesis-related gene expression and thus the synthesis of microcystin,(19) these

317

results provide another angle to help explain the possibility that M. aeruginosa releases

318

microcystin into the environment under xenobiotic stress. 15



319

In summary, the enantioselective effects of the chiral herbicide metolachlor on the

320

algae M. aeruginosa have been investigated based on both axis and carbon center

321

chirality for the first time. The effects of different metolachlor enantiomers on the

322

inhibition of basic growth rate, photosynthesis and factors that may affect harmful

323

microcystin production and release, including cell integrity, microcystin synthesis-

324

related gene expression, and Fe distribution, were determined and compared. In detail,

325

(S)-Met exhibited the highest inhibitory effects on M. aeruginosa growth and chl a

326

content, followed by (αS,1’S)-Met and (αR,1’S)-Met. Regarding changes to cell

327

integrity, (S)-Met caused the greatest damage to algal cells, which might induce

328

microcystin release. The same concern has been further confirmed by changes in

329

microcystin synthesis-related gene expression, in which the (S)-Met stimulated the

330

expression of both mcyD and mcyH genes. Interestingly, there is also a significant

331

enantioselective effect of metolachlor enantiomers on the regulation of Fe distribution,

332

in which toxicity was positively correlated with Fe aggregation in algal cells.

333

Furthermore, synergistic effects were observed between both enantiomers ((αS,1’S)-

334

Met and (αR,1’S)-Met) from (S)-Met in all aspects examined in this study, which helps

335

explain the toxicity of (S)-Met. Overall, the results discussed above provide new insight

336

into the enantioselective effects of chiral pesticides and can help increase the efficiency

337

of chiral pesticides during real application.

338

Implications. These results have important implications for agricultural chemistry,

339

public health and environmental protection. The enantioselective differences between

340

four enantiomers of metolachlor raise new questions with respect to the application of 16


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(S)-Met, which includes (αS,1’S)-Met and (αR,1’S)-Met) enantiomers. While these two

342

enantiomers have enantioselective toxic effects, they also display a synergistic effect.

343

(S)-Met ((αS,1’S)-Met and (αR,1’S)-Met) is known to be an active herbicide. These

344

results show that the application of (S)-Met could pose a risk to public health and the

345

environment. Although this study considered only one small molecule (metolachlor),

346

the concept is relevant for other complex chiral compounds. Overall, our study

347

indicated that more complex structures require more accurate toxic assessment.

348 349

AUTHOR INFORMATION

350

*Corresponding Author: Phone: (86)-88982421. E-mail: [email protected]

351

Institute of Environmental Health, College of Environmental and Resource Sciences,

352

Zhejiang University, Hangzhou 310058, China

353

Notes

354

The authors declare no competing financial interest.

355

ACKNOWLEDGMENT

356

This work was supported by the National Natural Science Foundations of China

357

(NSFC, No. 21876150 and 21677124), the Zhejiang Provincial Education Department

358

Foundation of China (No. LQ19B070002) and the Ningbo Municipal Natural Science

359

Foundation of China (No. 2018A610209).

17



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Table 1. Sequences of the primer pairs used in real-time PCR Genes

Primer sequence Forward: TGACACTCAGGGACGAAAGC

16SrRNA Reverse: CCACATACTCCACCGCTTGT Forward: TCGAGGGGCAGAAGGAGTTA mcyD Reverse: GCAATGTGAAAAACGCCTCG Forward: TCCCAAGGAACTTCCGCATC mcyH Reverse: GAGTAAAGGGGAGCCACCAC 498

25



499

Figure Captions

500

Figure 1. Molecular structure of metolachlor enantiomers.

501

Figure 2. Inhibition rate of M. aeruginosa after 72 h treatment with different

502

metolachlor enantiomers.

503

Figure 3. Chlorophyll content of M. aeruginosa cells treated with 15 mg/L metolachlor

504

enantiomers. (Different letters represent significant differences, P

Enantioselective Toxicity of Chiral Herbicide Metolachlor to Microcystis

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