Potential Risk and Mechanism of Microcystin Induction by Chiral

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The Potential Risk and the Mechanism of Microcystin Induction by Chiral Metalaxyl Cui Wang, Zhen Yang, Lihua Tang, Ximing Wang, and Quan Zhang Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00507 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 2, 2018

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Environmental Science & Technology Letters

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The Potential Risk and Mechanism of Microcystin Induction by Chiral

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Metalaxyl

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Cui Wang1, Zhen Yang1, Lihua Tang1, Ximing Wang2, 3, Quan Zhang2, 3*

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Zhejiang, People’s Republic of China

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Zhejiang, People’s Republic of China

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Zhejiang Province, Hangzhou 310032, Zhejiang, People’s Republic of China

College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053,

College of Environment, Zhejiang University of Technology, Hangzhou 310032,

Key Laboratory of Microbial Technology for Industrial Pollution Control of

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*To whom correspondence should be addressed. Phone: +86 571 8887 1579;

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Fax: +86-571-88320265; E-mail: [email protected] (Q Zhang).

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1

ACS Paragon Plus Environment


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Abstract Pesticide-induced oxidative stress has been widely observed in aquatic algae. The

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microcystin production caused by Microcystis aeruginosa would intensify under the

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oxidative stress induced by solar radiation during algae bloom. However, whether

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oxidative stress caused by the pollutants is also contributed to the toxic microcystin

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generation and the potential mechanism remain unknown, especially for chiral

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environmental contaminants. Metalaxyl, a high-volume chiral fungicide, was selected

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as the model to compare the risks to M. aeruginosa. Our results revealed a significant

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induction of algae growth or photosynthetic pigments at 30-50 mg/L R-metalaxyl

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(fungicidally active enantiomer) compared with 300-400 mg/L rac-metalaxyl at 48 h.

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At the non-toxic time point, the lowest observed effect concentration (LOEC) of

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reactive oxygen species (ROS) induction was 10 mg/L in R-metalaxyl, which was 30

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times lower than the corresponding value for the rac. The LOEC of extra and intra

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-microcystin induction of R-metalaxyl was 3.3 and 30.0 times higher than that of

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rac-metalaxyl, respectively. R-metalaxyl enantioselectively induced M. aeruginosa

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genes encoding components of microcystin synthesis and secretion. The potency

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estimation revealed that only the level of ROS and genes encoding microcystin

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synthesis were positively correlated with the upregulation of intra-microcystin.

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Furthermore, we confirmed that attenuation of ROS by N-acetylcysteine mostly

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alleviated the intra-microcystin induction. This demonstrates that enantiomer which

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can cause oxidative stress may be prone to pose risks to the aquatic system, especially

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during algal bloom.

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1. Introduction

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The well-known harmful algal bloom (HAB) events involve toxic or otherwise

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harmful phytoplankton. The cyanobacteria Microcystis aeruginosa (M. aeruginosa) is

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the primary producer of the toxicant microcystin during bloom outbreaks 1. In a

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natural context, algal blooms contain both toxic and nontoxic species. Algae strains

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capable of microcystin production are predominant under environmental stress 2.

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Researchers are appealed to the protective role of microcystin under the oxidative

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stress response of M. aeruginosa during natural blooming 3. Additionally,

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environmental pesticide-induced oxidative stress to aquatic algae has also been widely

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observed 4, 5.

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Chiral agrochemicals have shown a strong appeal in recent decades owing to their

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high efficacy and low usage of the pure enantiomer. Aquatic phytoplankton is

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sensitive to these chemicals which are intensively released into the groundwater and

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surface runoff. Most research on the toxic effects of chiral herbicides on

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phytoplankton has focused on algae species such as Chlorella pyrenoidosa (C.

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pyrenoidosa), C. vulgaris, Scenedesmus obliquus (S. obliquus) and M. aeruginosa 6-9.

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Several studies have found that chiral herbicides are enantioselectively toxic to M.

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aeruginosa 8. R-Diclofop acid was reported to inhibit the growth and induce oxidative

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stress and greater toxin release of M. aeruginosa owing to its destruction of the cell

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membrane 7. However, microcystin generation undergoes sophisticated modulation by

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M. aeruginosa, not merely as a result of the acute cellular damage. Whether mild

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stress acts as a sensitive stimulant of the M. aeruginosa response to chiral pollutants 3



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remains unknown. Compared with herbicide and insecticide industries, the fungicide industry has

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shown strong innovation ability and the trend of rapid development. Metalaxyl

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(methyl N-(methoxyacetyl)-N-(2, 6-xylyl)-DL-alaninate) is an acylalanine fungicide

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with systemic function and is frequently used for tropical and subtropical crops to

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eliminate soil-borne downy mildew pathogens. In vitro biological testing against

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Phythophtora infestants and Pythium ultimum showed that the R-enantiomer was

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approximately 1,000 times more active than the S-enantiomer 10. Metalaxyl is

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relatively photolytically stable in water and soil when exposed to natural sunlight 11.

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The reported toxicity tests have proved that the R-enantiomer is 20 times and 4 times

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more toxic than the racemate to algae and Daphnia magna, respectively 12. However,

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the enantioselective effect of chiral fungicides on M. aeruginosa has not been

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investigated well.

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In this study, we selected the commercially widely used fungicide metalaxyl and

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its active enantiomer as the model chemicals. The non-toxic concentration (no

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significant cell death was caused at that concentration) at a certain time point was the

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main focus of our investigations. The enantioselective effects of R-metalaxyl and

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rac-metalaxyl on the growth, ROS production, and microcystin-LR (MC-LR)

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production by M. aeruginosa were evaluated. We also detected the genes encoding

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components of the synthesis and secretion of microcystin. Finally, the potency was

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evaluated to determine the major contributors to microcystin generation induced by

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metalaxyl. The results provided in this work demonstrate the potential ecological risk 4


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of the pure enantiomers of fungicides during algal blooms and will also be helpful in

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promoting the regulation of the use of chiral pesticides in the future.

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2. Materials and Methods

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2.1. Chemicals and reagents

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The M. aeruginosa strain FACHB-905 (obtained from the Institute of

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Hydrobiology, Chinese Academy of Sciences, Wuhan, China) was routinely cultured

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in Blue-Green Medium 11 (BG11) prior to the experiments. All the chemicals and

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experimental kits were listed in supporting information (S1.1).

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2.2. Cell growth measurements

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The cell density, chlorophyll a (CLA) and carotenoid (CAR) were measured

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according to our published method and the details modified at the present study can

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be found in SI 1.2.

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2.3 Measured concentration of the treatment groups

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The acute concentration of both rac-and R-metalaxyl was detected during the

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exposure time. The details were listed in SI 1.3.

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2.4 ELISA procedure

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After exposure to metalaxyl with or without 1 mM NAC for 48 h, the culture

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medium was collected for determination the extra-MC-LR. Twenty millilitres of cells

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was collected, and intracellular (intra-) MC-LR was extracted by 70% methanol after

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three freeze-thaw cycles. The MC-LR levels were determined according to the

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manufacturer’s instructions. For each treated group, five replicates were used. 5



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2.5 ROS detection The level of intracellular ROS was detected by a fluorescence probe as described

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previously 13. Briefly, after treatment, algal cells were centrifuged, and the pellets

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were re-suspended in a phosphate-buffered saline (PBS) solution. The probe

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DCFH-DA (Beyotime, Shanghai, China) was added to each sample with three

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replicates at a final concentration of 10 µM. After incubation at 25 °C for 60 min in

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the dark, the samples were completely washed with fresh PBS to remove the unbound

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probe. The fluorescence intensity was recorded using a fluorescence microplate reader

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(SpectraMax®M5/M5e, America) with an excitation wavelength of 485 nm and an

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emission wavelength of 525 nm.

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2.6 RNA extraction and real-time RT-PCR analysis

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To explore the possible mechanism of the enantioselectivity of metalaxyl in

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microcystin production, mcyA, mcyD, and mcyH were quantified in this study. The

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primer sequences and RNA extraction procedure were the same as in our previous

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reports 14 and were designed by Sangon Biotech (Shanghai, China). The 16S rRNA

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gene was used as the housekeeping gene. The relative mRNA was expressed by 2-∆∆ct.

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All the treatment groups have at least three replicates.

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2.7 Potency estimation

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The method of potency estimation was the same as in our previous report 13.

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Microcystin generation (extra-MC-LR and intra-MC-LR), cell volume, genes

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encoding microcystin (mcyA, mcyD and mcyH), and ROS were normalized to the

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average of the respective controls to calculate the fold-change value at each 6


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concentration of R- and rac-metalaxyl. The potency estimates (b) were calculated

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according to the equation fold-change = (dose+1)β, where β represented the slope of

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the dose-effect relationship on a logarithmic scale plotted using Origin 13.0

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(OriginLab, USA).

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2.8 Statistical analysis

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Statistical analysis of the data was carried out by the statistical program package

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GraphPad Prism 7.0 (GraphPad Software, USA). All data are expressed as the mean ±

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standard deviation (SD) from three independent replications. Significant differences

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between the control and the treated group were determined using One-way ANOVA.

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Statistical significance was determined by p< 0.05 and 0.01.

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3. Results and Discussion

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3.1 Enantioselective effect of metalaxyl on the growth of M. aeruginosa

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The measured concentrations of each test solution during the exposure time was

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shown in Table S1.There was no significant difference between the nominal

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concentration and the tested concentration during the test. The growth patterns

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including algae viability and the contents of photo-pigments including chlorophyll a

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and carotenoid of M. aeruginosa after treatment with R- and rac-metalxyl at both 48

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and 96 h are provided in S2. Result. Overall, at 48h there was stimulation in algae

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growth and inhibition trend at 96h. The effective threshold for R and rac-metalaxyl

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displayed their enantioselectivity.

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3.2 Metalaxyl enantioselectively induced the production of ROS, intra- , and 7



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extra-MC-LR

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Subsequent UV-irradiation-induced oxidative stress may intensify microcystin

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secretion by M. aeruginosa 3. Thus, we detected both ROS and microcystin which

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may induce by the two chemicals. The intensity of green fluorescence detected in a

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microplate manifesting ROS accumulation in algae induced by metalaxyl is plotted in

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Figure 1. We observed a mild but significant increase in ROS at or above 300 mg/L in

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rac-metalaxyl. The obvious induction of ROS was detected at or above10 mg/L in

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R-metalaxyl, with a maximum induction of 1.4-fold compared to the control. The

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lowest observed effect concentration (LOEC) of the R-enantiomer was 30-fold lower

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than that of the racemate for ROS induction.

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The intra- and extra-MC-LR were detected at 48 h according to algae growth.

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Both the R and rac forms caused a dose-dependent induction of MC-LR. As depicted

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in Figure 2A, a significant increase in extra-MC-LR was observed at 100 mg/L for

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the rac form and at 20 mg/L for the R form (Figure 2C). The maximum induction of

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extra-MC-LR was 1.4-fold compared to the control for the rac treatment and 1.2-fold

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compared to the control for the R groups (Figure 2 B, D). The intra-MC-LR

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maximum induction was much more obvious than that of the extra-MC-LR. A

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2.6-fold increase was observed for rac at 400 mg/L, while a 3.3-fold increase was

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observed for R at 40 mg/L. The LOEC was 300 mg/L in rac treatment which was

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higher than that of R-metalaxyl (10 mg/L), indicating the enantioselective effect on

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the MC-LR secretion and synthesis.

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3.3 Metalaxyl enantioselectively induced the transcription of genes encoded for 8


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microcystin synthesis and secretion Genes involved in MC-LR synthesis and secretion was selected to investigate the

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potential reason for the enantioselective production of MC-LR. Figure 2 E, F shows

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that mcyA, mcyD, and mcyH were enantioselectively upregulated when M. aeruginosa

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was exposed to metalaxyl at the tested ranges. The expression of mcyD was

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significantly upregulated only after treatment with 400 mg/L rac. The level of mcyA

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was dose-dependently upregulated by rac, with the maximum induction at 400 mg/L,

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which was nearly 5.3-fold greater than that of the control. For the mcyH, the overt

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induction was observed at or above 100 mg/L, an approximately 2.0-fold increase

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compared to the control group. Significant mcy gene modulation occurred at 10 mg/L

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or above for R-metalaxyl. The maximum induction of mcyD, mcyA and mcyH was

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4.6-fold, 2.2-fold and 2.6-fold, respectively for R-metalaxyl at 50 mg/L. R-metalaxyl

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was more potent than the racemate form because the LOEC was 10-fold lower for

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R-metalaxyl than for the rac form. The content of microcystin was suggested to be correlated with the microcystin

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synthase genes 15, 16. Of the two transcribed operons, namely, mcyA-C and mcyD-J,

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mcyA encoded microcystin synthetase, while mcyD encoded polyketide synthetase

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which is directly responsible for the toxin production 17. However, these toxic strains

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always have the capability to turn on/off the genes depending on their circumstances

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the aquatic nitrate levels 16. Environmental chemicals such as arsenic, glufosinate,

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copper sulfate, and N-phenyl-2-naphthylamine can turn on the microcystin genes,

. Several studies have revealed that the production of microcystin is independent of

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inducing microcystin generation 5, 14, 19. The data presented herein demonstrated that

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the R form enantioselectively upregulated mcyD genes even at low doses, indicating

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its higher aquatic risk during algal blooms compared to the racemate. Additionally, researchers have found that exposure to paraquat and glufosinate

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resulted in a 50% to 90% increase in MC-LR owing to oxidative stress on M.

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aeruginosa 14, 20, indicating the potency role of ROS in MC induction. Generation of

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oxidative species is a common feature in chemical-induced toxicity under laboratory

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pure culture conditions. Chiral herbicides and algaecides can induce ROS in algae 4, 7,

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14, 20

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fungicides. As confirmed by our study and others, the fungicidal active R-metalaxyl

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was proven to be more toxic to algae than the racemate 12. The hormesis and the mild

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ROS induction indicated an enantioselective stress to M. aeruginosa at 48 h 21.

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However, whether or not all of the factors including ROS, microcystin genes, and

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algae growth have the contribution to MC is largely unknown. Thus, we did the

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further estimation and verification.

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3.4 Potency estimation of metalaxyl-induced MC-LR and verification

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. As an ancient bacterium, cyanobacteria may also be a potential target for

Factors such as ROS, cell contents, genes encoding components of extra-MC-LR

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release, and genes encoding components of intra-MC-LR synthesis were used to

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estimate the potency. The results show that the potency of ROS and mcy genes

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encoding factors involved in synthesis were positively correlated with intra-MC-LR,

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with R2=0.9441 and 0.8388, respectively (Figure 3A, C). However, mcyH induction

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and ROS did not show significant correlation with extra-MC-LR. Both intra-MC-LR 10


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and extra-MC-LR are not dependent on cell numbers (Figure 3B). Furthermore, we

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confirmed the role of ROS in intra-MC-LR induction. Figure 4 revealed that NAC

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partially and significantly attenuated intra-MC-LR induction at 40 mg/L of R and 400

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mg/L of rac. Several studies supported the relationship between the algal growth rate

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and the toxin content 16, 22. Based on the potency estimation and the ROS inhibition

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assay, our study speculated that the production of intra-microcystin in M. aeruginosa

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induced by metalaxyl primary originated from ROS and genetic transcription.

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Previous study indicated that mcy gene knockout algae were more vulnerable to H2O2

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than the normal toxic strain, which indicated a potential relationship between ROS

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and mcy genes 23.

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To date, there have been few reports regarding the enantioselective impact on the

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MC-LR induction of M. aeruginosa by chiral compounds. The few reports in the

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literature have been primarily focused on the enantioselective growth inhibition,

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physiological disruption, and photosynthetic pigment synthesis of M. aeruginosa

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between the enantiomers 7, 8. Regarding its specific properties in algae species, M.

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aeruginosa has been widely investigated for the ecological risks under eutrophication

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conditions 24, 25. Recently, several studies found that exposure to herbicides, trace

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metals, and plant growth regulators resulted in a high induction of microcystin 14, 15, 25.

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The present study revealed that at the non-cytotoxic exposure duration, R-metalaxyl

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gave rise to greater oxidative stress than rac-metalaxyl. For microcystin induction, the

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LOEC of R-metalaxyl was several times lower than that of rac-metalaxyl, displaying

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a remarkably enantioselective effect between the two forms. Both the induction of 11



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genes encoding factors involved in MC-LR synthesis and ROS could account for the

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upregulation of MC-LR. Our study has for the first time observed the enantioselective

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impact on M. aeruginosa of chiral metalaxyl, suggesting the high risk of toxic

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fungicidal enantiomers in aquatic systems.

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3.5 Environmental Implication

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Development of the pure enantiomers of chiral compounds, especially for the

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wide used pesticides, meets the requirements of green chemistry 26, 27. Chemicals that

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show large toxic gaps between/among enantiomers are manufactured as “highly

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active” and thus “low environmentally abundant” green compounds. Though

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R-metalaxyl is 20 to 100 times more active than the S and rac forms, the R enantiomer

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shows higher toxicity to S. obliquus, Daphnia Magana and zebrafish 12. Many

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effective enantiomers of fungicides, such as tradimefon 28, tebuorazle 29 and

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hexaconazole 30, also show higher toxicity to aquatic organisms. Excluding the

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common aquatic toxic endpoints, our study observed that the toxic enantiomer

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especially the one that can induce the oxidative stress may be prone to promote the

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microcystin production of cyanobacteria. Thus, aquatic risk assessment for the

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enantiomeric fungicides should be emphasized and re-considered, especially for M.

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aeruginosa.

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Supporting Information: The table shows 1) the measured concentrations of rac-metalaxyl and

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R-metalaxyl during experiment; 2) the effects of rac-Metalaxyl and R-Metalaxyl on the growth of

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M. aeruginosa. The figures show the effects of rac-metalaxyl and R-metalaxyl on the growth of M.

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aeruginosa. This information is available free of charge on the ACS Publications website. 12


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Acknowledgement Funding supports National Natural Science Foundation of China (21677130,

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21777147) and Zhejiang Province Nature Science Foundation of China

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(LY18B070007). All authors declare no competing financial interest and have no

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conflicts of interest to disclose.

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25. Alexova, R.; Fujii, M.; Birch, D.; Cheng, J.; Waite, D.; Ferrari, B. C.; Neilan, B.

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A. Iron uptake and toxin synthesis in the bloom-forming Microcystis aeruginosa

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under iron limitation. Environ. Microbiol. 2011, 13, 1064-1077.

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26. Garrison, A. W. On the issue of enantioselectivity of chiral pesticides: A green chemistry opportunity. Green Chem. 2004, 6, G77-G78. 27. Lu, C.S.; Chang, C.H.; Palmer, C.; Zhao, M.R.; Zhang, Q. Neonicotinoids

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residues in fruits and vegetables: an integrated dietary exposure assessment

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approach. Environ. Sci. & Technol. 2018, 52, 3175-3184.

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28. Li, Y. B.; Dong, F. S.; Liu, X. G.; Xu, J.; Han, J.; Han, Y. T.; Zheng, Y. Q. Chiral

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fungicide triadimefon and triadimenol: Stereoselective transformation in

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greenhouse crops and soil, and toxicity to Daphnia magna. J. Hazard. Mater.

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2014, 265, 115-123.

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29. Qi, S. Z.; Chen, X. F.; Liu, Y.; Jiang, J. Z.; Wang, C. J. Comparative toxicity of rac- and S-tebuconazole to Daphnia magna. J Environ. Sci. Health., Part B. 2015, 16


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50, 456-462. 30. Han, J. J.; Jiang, J. Z.; Su, H.; Sun, M. J.; Wang, P.; Liu, D. H.; Zhou, Z. Q.

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Bioactivity, toxicity and dissipation of hexaconazole enantiomers. Chemosphere.

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2013, 93, 2523-2527.

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Figure 1 Metalaxyl causes reactive oxygen species in Microcystis aeruginosa. M.

368

aeruginosa cells were exposed to varying concentrations of rac- (A) and R-metalaxyl

369

(B). The probe DCFH-DA was added, and the fluorescence was detected in a

370

microplate. * Statistical significance with p < 0.05 compared to the control. **

371

Statistical significance with p < 0.01 compared to the control.

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18


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Figure 2 The effects of different rac-metalaxyl and R-metalaxyl concentrations on

376

intra-MC-LR, extra-MC-LR(A, B, C, D) and mcyA, mcyD, mcyH genes at 48 hours of

377

incubation (E, F). The heat map values were used in HemI (Heatmap Illustrator,

378

version 1.0). The X- and Y-axes represent the concentrations of metalaxyl and the mcy

379

genes, respectively. * Statistical significance with p < 0.05 compared to the control. 19



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** Statistical significance with p < 0.01 compared to the control.

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Figure 3 Potential of metalaxyl as a function of genes (A), cell density (B), ROS (C)

384

and microcystin production potency in Microcystis aeruginosa. The blue circle

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represents rac-metalaxyl, while the yellow circle represents R-metalaxyl. The

386

significance level and the correlation coefficient are given in the upper left corner.

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p

Potential Risk and Mechanism of Microcystin Induction by Chiral

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