Photolysis and photo-induced toxicity of pyraclostrobin to Vibrio fischeri: Pathway and toxic mechanism

Aquatic Toxicology 220 (2020) 105417

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Photolysis and photo-induced toxicity of pyraclostrobin to Vibrio fischeri: Pathway and toxic mechanism

T

Lingyun Fana, Ying Huanga, Tao Huanga, Kun Zhaob, Ya-Nan Zhanga, Chao Lia, Yuan Hui Zhaoa,* a

State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of Environment, Northeast Normal University, Changchun 130117, China b Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pyraclostrobin Photo-induced toxicity Vibrio fischeri Molecular docking Molecular dynamics simulation

Pyraclostrobin is a fungicide used widely across the world. However, its photolysis pathway and toxic mechanism is unclear. In this study, photolysis and photo-induced toxicity of pyraclostrobin to Vibrio fischeri were determined. The results showed that direct photolysis dominated the degradation of pyraclostrobin. Gas Chromatography-Mass spectrometry and quantum chemical calculation revealed that the pyraclostrobin was firstly photo-degraded into Methyl N-phenyl-carbamate and 1-(4-chlorophenyl)-3-hydroxy-1H-pyrzole, synthetic intermediates of pyraclostrobin, then into aniline, benzoquinone and acids. Toxicity assay showed that bioluminescent inhibition rate to V. fischeri fluctuated with radiation/illumination time and the toxicity curve can be classified into three phases (Phase I: 0–10 min, incline; Phase II: 10–60 min, decline; Phase III: 60–120 min, incline). The up-and-down curve indicates the change of parent compound during the photolysis. Simulation of molecular docking showed that the CDOCKER interaction energy of pyraclostrobin (-44.71) lower than other intermediate products (> -30.00), indicating that the parent compound is more toxic than its intermediates. An increased toxicity observed in the toxicity curve was attributed to the generation of benzoquinone with log1/ EC50 of 6.73, which can greatly change structure of target luciferase in Vibrio fischeri. In addition, the addition of radical scavengers can inhibit the bioluminescence of the tested solutions, indicating the involvement of radicals in the transformation of intermediates. This paper reveals that one of photochemical transformation products of pyraclostrobin can cause more toxic than its parent compound to bacteria. Environmental risk assessment should consider not only the parent compound, but also its metabolites.

1. Introduction Pyraclostrobin is a broad-spectrum fungicide and widely used in the fields of planting (Fernández-Ortuño et al., 2012; Chen et al., 2018). Due to the inhibition of mitochondrial respiration, pyraclostrobin is capable of effectively eliminating several plant fungi such as powdery mildew and macrophoma musae from cucumber and grey leaf, respectively. It is regarded as an environmental-friendly agricultural chemical because of low persistence in natural environment (Laugale et al., 2013; Mercader et al., 2013). It was listed as one of the fifty most extensively used active substances on grassland or landfill in the UK and the amount of its usage was increasing during the past several years (Garthwaite et al., 2016). A maximal concentration of 0.239 μg/l was observed in the surface water of Maine, Idaho and Wisconsin (Reilly et al., 2012) and 1.61 μg/l in Nebraska, USA (Mimbs et al., 2016). A much higher concentration of 17.24 μg/l was detected in the paddy ⁎

water of China (Guo et al., 2017). Although pyraclostrobin is a well-used fungicide for the targeted organisms, it does not indicate that it is safe for non-target organisms because the toxicity to an organism is related not only to the parent compound, but also to its degradation intermediates. During translocation and transformation, some chemicals might decompose into a variety of intermediates or final products that may pose a greater risk to the environment than the corresponding parent chemicals via multiple ways (Antonopoulou and Konstantinou, 2014). For some agricultural chemicals, photochemical transformation is a major way of migration and transform in natural environment because these chemicals can absorb sunlit radiation (McConkey et al., 1997; El-Alawi et al., 2001; Challis et al., 2013). Assessment of the ecological risk for 60 agricultural chemicals (e.g. insecticides, herbicides and fungicides) and 485 corresponding transformation products showed that 30 % of transformation products exhibited greater toxicity than the corresponding

Corresponding author. E-mail address: [email protected] (Y.H. Zhao).

https://doi.org/10.1016/j.aquatox.2020.105417 Received 28 August 2019; Received in revised form 16 December 2019; Accepted 12 January 2020 Available online 15 January 2020 0166-445X/ © 2020 Elsevier B.V. All rights reserved.

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the guideline of Determination of the Acute Toxicity-Luminescent Bacteria Test (GB/T15441-1995, http://kjs.mep.gov.cn/hjbhbz/bzwb/ jcffbz/199508/t19950801_67352.shtml). The V. fischeri and Microtox Toxicity Analyzer were obtained from the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China. The tested solutions were firstly adjusted to 3 % NaCl solutions by adding 2 ml of NaCl stock solution. Then, 1 ml of cultivated bacterial suspension was added into each tested solution. Bioluminescence was measured via Microtox Toxicity Analyzer after 15 min exposure and inhibition of bioluminescence was calculated by using 3 % NaCl solution as control. The more detailed information of measurement of bioluminescence can be found in the Supplementary materials.

parent compounds (Sinclair and Boxall, 2003). Acifluorfen, a diphenylether herbicide, exhibits low toxicity to Daphnia magna at a concentration of 0.1 mmol/L, but the toxicity remarkably increases if the sample is irradiated under UV radiation (Scrano et al., 2002). Triclosan, a widely used antimicrobial agent, exhibits significantly enhanced toxicity to Vibrio qingaiensis sp. nov. (Q67) after Xenon lamp radiation with 290 nm cut-off filter (Chen et al., 2016). The photochemical transformation pathway plays an important role in the evaluation of risk assessment for a chemical (Challis et al., 2013; Lagunas-Alluéa et al., 2012; Qu et al., 2017, 2019). Although there have been other studies that have documented the degradation pathways of pyraclostrobin (Zeng et al., 2019), no research has been carried out on the relationship between photolysis and photo-induced toxicity of pyraclostrobin in natural environment. Therefore, it is of great significance to figure out the transformation process and toxic mechanism of pyraclostrobin in aquatic environment. Theoretical chemistry, such as quantum chemistry and molecular mechanics, play an important role in the understanding of the photodegradation process and toxic mechanism of a compound because of simulation of the whole process. Quantum chemistry methods are usually engaged to calculate transition state and energy change (Hykrdová et al., 2018; Miao et al., 2018; Qu et al., 2018). Molecular mechanics methods are usually employed to figure out the interaction between receptors and ligands (Ciancetta et al., 2017; Marino et al., 2018). The computational simulation methods are helpful to fulfill the gap between the initial state and final state of a reaction, and to simulate the receptor variance of an intoxication process. These theoretical calculation methods can help us not only to reveal the process of photochemical transformation, but also to understand the toxic mechanism and explain the toxicokinetic process. The V. fischeri, characterized by its high toxicity sensitivity and convenient operation (5–30 min), is widely used as a model bacterium to investigate the acute toxicity of a chemical (Parvez et al., 2006). This advantage makes V. fischeri a good model/targeted organism to test the toxicity of illuminated agricultural chemical solution. In this study, photolysis kinetics and photo-induced toxicity assays to V. fischeri were conducted under simulated sunlit radiation. The concentrations of parent compound and bioluminescent inhibition rates of tested solutions were determined during photolysis period. The objective of this paper was to investigate the relationship of photolysis pathway and toxic mechanism of pyraclostrobin through the quantum chemistry calculations and in-silico simulations of molecular docking and dynamics. These analyses advance understanding toxic mechanism of pyraclostrobin and benefit the risk assessment of agricultural pollutants in aquatic environment.

2.3. Photolysis kinetics and photo-induced toxicity The photolysis kinetic and toxicity test experiments were performed in an XPA-7 merry-go-round photochemical reactor (Xujiang Electromechanical Plant, Nanjing, China) with a water-refrigerated Xenon lamp of 350 and 800 W equipped with 290 nm cutoff filter to simulate sunlight (EPA, 1998, 2008; OECD, 2008). The system was cooled by circulating cooling water to maintain temperature at 25.0 ± 2.0 °C. The light intensity detected by the TriOS-RAMSES spectroradiometer (TriOS GmbH, Germany, Fig. S4) was 1.871 × 10−10 and 7.535 × 10-10 Einstein/cm2/sec, respectively (Zhang et al., 2018a, 2018b). Gently stream 50 ml of pyraclostrobin solution (1 mg/l, pH = 6.02) was added into the photochemical reactor. The acetone used to prepare the stock solution was removed by nitrogen gas before adding ultrapure water. During the experiments, 0.5 and 2 ml solutions were sampled out from the irradiated solutions in the reactor at different time intervals, respectively. At the same time, 0.5 and 2 ml were also sampled from 50 ml non-irradiated control solution. Each 0.5 ml sample was simply filtered with polyethersulfone (0.45 um) and concentration of pyraclostrobin was determined by High Performance Liquid Chromatography (HPLC) as described below. Each 2 ml test sample was adjusted to 3 % NaCl and 1 ml of fresh bacterial culture was added into the solution. The inhibition rate of bioluminescence to V. fischeri was determined by Microtox Toxicity Analyzer after 15 min exposure. All the experiments were performed in three replicates. Photolysis rate of pyraclostrobin under simulated sunlit radiation were analyzed via pseudo-first order kinetics and details can be found in Supplementary materials. To investigate the effect of radicals and DOM on photolysis and toxicity, certain amount radical scavengers (e.g. t-BuOH and NaN3) and DOM was added into the 50 ml test solution of pyraclostrobin, respectively. The photolysis kinetic and photo-induced toxicity experiments were then carried out according to the procedure described above. At the same time, steady-state concentrations of reactive intermediates (e.g., singlet oxygen 1O2, hydroxyl radical %OH, and excited triplet dissolved organic matters 3DOM*) were determined and result were shown in Supplementary materials (Zhou et al., 2018).

2. Materials and methods 2.1. Chemicals and reagents Pyraclostrobin (> 95 %, purity) was purchased from J&K scientific Ltd. Other chemicals used in the photo-degradation experiments, cultivation of V. fischeri and toxicity tests are detailed in the Supplementary materials. Suwannee River Natural Organic Matter (SRNOM) was purchased from International Humic Substances Society, USA. The SRNOM (1000 mg) was added into 1000 ml of ultrapure water and filtered through 0.45 μm membrane. The dissolved organic matter (DOM) of the solution determined via TOC-VCPH (SHIMADZU, Kyoto, Japan) was diluted into 300 mg/l and stocked in a 4 °C refrigerator as a stock solution. Ultrapure water (18 MΩ) was obtained from an ultrapure water system (UPT-II-10 T Ultrawater, Sichuan, China). Unless otherwise stated, all solutions were prepared by the ultrapure water.

2.4. Analysis of pyraclostrobin concentration and intermediates Each 0.5 ml solution sampled above was filtered with polyethersulfone (0.45 um) and concentration was quantified by using HPLC equipped with Waters 2489 UV/Visible detector and symmetry C18 (4.6 × 250 mm, particle size 5 μm ). HPLC detecting conditions of pyraclostrobin and some of intermediates can be found in Table S2. Identification of photolytic products was performed by a Thermo GC–MS (Trace 1310/TSQ8000) in electron ionization (EI) mode. A DB5MS capillary column (30 m × 0.25 mm × 0.25 μ m) was used with 1 ml/min helium as the carrier gas. The inlet, detector and source temperatures were set at 280, 250 and 300 °C, respectively. The oven temperature was set at 50 °C (4 min hold time), and then increased to 250 °C at 6 °C/min (20 min hold time). The sample pretreatment of

2.2. Acute toxicity to V. fischeri The acute toxicity tests to V. fischeri were conducted according to 2

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intermediate products for Supplementary materials.

GC–MS

analysis

was

detailed

in

2.5. In silico approaches Quantum chemical calculations were carried out by Gaussian 09 program (Frisch et al., 2009) (http://gaussian.com/) utilizing the B3LYP function (Hertwig and Koch, 1997) in combination with the 631+g(d, p) basis set based on density functional theory (DFT) (Koch and Holthausen, 2015). Geometrical relaxation in the excited states have been investigated for n = 5 employing fully optimized Time Dependent DFT (TDDFT) – B3LYP/6-31G* (Gross et al., 1996). Transition states (TSs) were characterized with only one imaginary vibrational frequency. Intrinsic reaction coordinate (IRC) analysis was executed to verify that each TS uniquely connects the designated reactant with the product (Fukui, 1981). The geometry optimization and TSs of all the parent compound, intermediates and products were obtained via the Berny optimization algorithm (Schlegel, 1982). All calculations were performed at temperature of 298.15 K and standard pressure of 101.325 kPa. The energy changes were defined as delta E for all the reactions. Molecular docking simulation was carried out by CDOCKER in DS 2.5 (http://www.3dsbiovia.com/products/collaborative-science/ biovia-discovery-studio/). The CHARMM force field was used for the energy minimizations in the docking process (Brooks et al., 1983; Chen et al., 2014). The 3D critical structure of luciferase (1BRL) was downloaded from RCSB PDB (http://www.rcsb.org/pdb). The structural file of 1BRL.pdb was optimized via deleting water and adding hydrogen at pH of 7. The selection of optimal docked conformations was based on the lowest CDOCKER energy and CDOCKER interaction energy. To analyze the structure variance of 1BRL, molecular dynamics simulation was performed by using sander program in AMBER 14 (http://ambermd.org/). The AMBER ff14SB protein force filed was used to model all peptide interactions and the general AMBER force filed was used for the ligands (Maier et al., 2015). To neutralize the 1BRL-ligand system, counter ions of Na+ was placed in the grids that had the largest negative Coulombic potential around the protein. The whole system was immersed in a rectangular box of 9 Å solute atom in all three dimensions. The particle mesh Ewald was employed for the long-range electrostatic interactions in the molecular mechanics minimization and molecular dynamics simulations. After minimization, the system was gradually heated in the NVT ensemble from 10 to 300 K over 50 ps. Initial velocities were assigned from a Maxwellian distribution at the starting temperature. Then 500 ps NPT equilibration with a target temperature of 300 K and a target pressure of 1 atm was performed before 5 ns molecular dynamics simulation. The SHAKE procedure was employed to constrain all hydrogen atoms with the time step of 2 fs. The coordinates of the complexes were saved every 2 ps. The trajectory was analyzed by VMD program (Humphrey et al., 1996). The displacement of aligned molecule was analyzed by TK console module in VMD via script “colordisplacement.tcl” downloaded from VMD Script Library (https://www.ks.uiuc.edu/Research/vmd/plugins/). The color scale range of displacement between the original conformation and extracted trajectory was set from 0 to 50 Å, and the color scale method was set in blue-white-red sequence.

Fig. 1. Photolysis kinetics of pyraclostrobin under the photo-radiation of 350 and 800 W Xenon lamps.

sunlight radiation. The photolysis rate of pyraclostrobin in aqueous solution can well be described by first-order kinetics (Zeng et al., 2019). The photolysis rate increases with increasing of the radiation energy with the measured first-order kinetics rate constants of 0.011 and 0.124 and half-life periods of 63.6 and 5.6 min for 350 and 800 W Xenon lamp radiations, respectively (Fig. 1). Inspection of the UV–vis absorption spectrum of pyraclostrobin (Fig. S4) shows that the pyraclostrobin has characteristic absorption band from 275 to 320 nm, indicating that pyraclostrobin is capable of absorbing short wave radiation in UV–vis light, leading to the photolysis of pyraclostrobin. 3.2. Identification of intermediate products To identify the intermediate products during the photolysis, the tested solution was analyzed by GC–MS sampling at 5, 15, 60, 180 and 240 min during photolysis experiment under the radiation of 800 W Xenon lamp. Six intermediates were detected from the tested solution of pyraclostrobin. The names of parent and intermediate compounds, as well as CAS number, name abbreviation (Abb.), molecular weight (MW), octanol/water partition (KOW), are listed in Table 1. More detailed information on the Chromatographic retention time and structures of the products detected at 5, 15, 60, 180 and 240 min can be found in Supplementary materials (Fig. S5). The baseline toxicity is calculated from the baseline model log 1/ EC50 = 0.938 log KOW + 0.833 (Wang et al., 2017). The toxicity value to pyraclostrobin was determined in this paper. The measured toxicity values to benzoquinone and aniline was obtained from published references, respectively (Kaiser and Palabrica, 1991; Wang et al., 2017). The log KOW values of pyraclostrobin and intermediate products were obtained from the database of EPI Suite (version 4.0, http://www.epa. gov/oppt/exposure/pubs/episuitedl.htm). 3.3. Toxicity of pyraclostrobin to V. fischeri The dose-response curve of pyraclostrobin to V. fischeri is shown in Fig. S1. The toxicity value calculated from the dose-response curve (Fig. S2) and expressed in the negative of the logarithm of the molar concentration (log 1/EC50) to V. fischeri is 5.50 in. mol/L (Table 1). Table 1 also lists the baseline toxicity (i.e. 4.57) predicted from baseline model for pyraclostrobin. The baseline model developed from the baseline compounds was widely used to compare the mode of action of compounds from baseline level (Wang et al., 2017). Compared with the baseline toxicity, the measured toxicity (5.50) is greatly higher than baseline level, indicating that pyraclostrobin can be identified as a specifically-acting compound to V. fischeri. This compound exhibits excess toxicity because of the specific interaction with certain receptors, such as luciferase. Although pyraclostrobin has a considerably great

3. Results 3.1. Photolysis kinetics Fig. 1 is the plots of concentration varying with time for pyraclostrobin under Xenon lamp radiation of 350 and 800 W, respectively. In dark control, the concentration of pyraclostrobin was stable with less than 4.5 % variation, indicating that pyraclostrobin cannot be hydrolyzed and is stable under dark laboratory condition. In contrast, pyraclostrobin is not stable and can be photo-degraded under simulated 3

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Table 1 The MW, log KOW, estimated baseline and measured toxicity (log 1/EC50) for pyraclostrobin and its intermediate products. CAS

Compound

Abb.

MW

logKOW

log1/EC50 (exp)

log1/EC50 (pred)

175013-18-0 2603-10-3 76205-19-1 62-53-3 123-31-9 106-51-4

Pyraclostrobin Methyl N-phenyl-carbamate 1-(4-chlorophenyl)-3-hydroxy-1H-pyrzole Aniline Hydroquinone Benzoquinone

PYR MPC CHP AN HQ BQ

388 151 194 93 110 108

3.99 1.75 2.74 0.90 0.59 0.20

5.50 2.51 4.49 2.49 NA 6.73

4.57 2.47 3.40 1.68 1.38 1.02

toxicity value to V. fischeri, the exposure concentration of pyraclostrobin may decrease more rapidly in the aquatic environment due to phototransformation. The toxicity value determined from pyraclostrobin cannot well reflect to true toxic effect because its intermediates and final products may have different toxic effect to V. fischeri.

the concentration of Phase I products reaches the peak, the toxicity of illuminated solution declines; Afterwards the Phase II products transform to tertiary products with higher toxicity, the toxicity of illuminated solution inclines as the concentration of Phase III products incline. 4. Discussion

3.4. Photo-induced toxicity of pyraclostrobin

4.1. Direct photolysis and photo-sensitization

To investigate the toxicity variation during the photolysis, the inhibition rate to V. fischeri was determined at different time during the photolysis. The initial concentration was chosen based on the EC50 obtained from the acute toxicity test. The results in Fig. 2 show that inhibition rate of the tested samples increases rapidly with increasing of photolysis time (Phase I) and reaches peak at 120 min for 350 W photoradiation, and then decreases with increasing of photolysis time (Phase II). To further investigate the trend of toxicity, high energy lamp (800 W) was used in the toxicity test experiment. It shows that, after decreasing in Phase II, the inhibition rate to V. fischeri increases again and reach steady after 240 min (Phase III). The variation of the inhibition rate to V. fischeri can be classified into three phases (Fig. 2): Phase I, the toxicity increases with the radiation time (0–120 min for 350 W or 0∼15 min for 800 W Xenon lamp); Phase II, the toxicity decreases with the radiation time (120–360 min for 350 W or 15–60 min for 800 W Xenon lamp); Phase III, the toxicity gradually increases with the radiation time (60–240 min for 800 W Xenon lamp) and the remains steady (240–360 min for 800 W Xenon lamp). The up and down fluctuation curve of toxicity indicates that the pyraclostrobin is not simply photo-degraded into one or two photolysis products. Some intermediates and final products are even more toxic to V. fischeri than the parent compound, pyraclostrobin. During the photolysis of pyraclostrobin, it firstly degrades to intermediate products, the joint effect of pyraclostrobin and intermediate products is more toxic than the pure parent compound solution (Phase I). The toxicity of illuminated solution inclines with the inclining concentration of products of Phase I; Then the Phase I products transform to secondary products with lower toxicity to V. fischeri (Phase II), After

There are two mechanisms during the photo-chemistry transformation process, direct-photolysis and photo-sensitization. Ascertaining the effect of photochemically produced reactive intermediates (PPRIs) during simulated sunlit radiation is conducive to clarifying the photolysis process of a chemical. Photosensitization generally leads to the production of PPRIs, such as singlet oxygen (1O2) and hydroxyl radical (·OH), and play an important role in photolysis. The ·OH and 1O2, as two of the common PPRIs occurred in the photolysis, may be involved in the photolysis of pyraclostrobin (Lindsey and Tarr, 2000a, 2000b; Hammes et al., 2006; He et al., 2013). To investigate the photosensitization and effect of PPRIs on the photolysis reactions, tertiary butanol (t-BuOH) and sodium azide (NaN3) were added into the pyraclostrobin solution in the photolysis reactor, respectively. It is well known that ·OH and 1O2 are two common PPRIs and t-BuOH and NaN3 can well scavenge ·OH and 1O2, respectively, and inhibit their oxidation to reactants or intermediates. 4.1.1. Effect of %OH on photolysis and toxicity Fig. 3 shows the photolysis kinetics and photo-induced toxicity of pyraclostrobin with and without the t-BuOH, respectively. No difference is observed in photolysis curves of pyraclostrobin between the tested solutions in presence and absence of %OH scavenger (t-BuOH) (Fig. 3a), suggesting that the reaction of ·OH with pyraclostrobin is not significant as compared to other reactions with PPRIs. However, toxicity tests show that ·OH can affect the photolysis reactions of intermediates (Fig. S6). Different toxicity variance curves were observed between the tested solutions in presence and absence of %OH scavenger (t-BuOH) (Fig. 3b). The toxicity increases much slower in the presence of t-BuOH as compared with the ·OH scavenger free solution. In comparison with the experiments without radical scavenger, the decreased inhibition rate is attributed to the relatively slow photochemical transformation of intermediates because ·OH radicals are scavenged by t-BuOH. This is supported by the observation of determined concentrations of three main intermediates varying with the time (Fig. S6). 4.1.2. Effect of 1O2 on photolysis and toxicity Fig. 3 also shows the photolysis kinetics and photo-induced toxicity of pyraclostrobin with and without the NaN3. The Fig. 3a reveals that the addition of NaN3 can significantly reduce the photolysis rate of pyraclostrobin with the half-life of pyraclostrobin photolysis increased from 5.6 to 13.6 min under 800 W Xenon lamp radiation, indicating that 1O2 can affect photolysis of parent compound, pyraclostrobin. The decreased photolysis rate in the presence of 1O2 scavenger indicates that PPRIs is involved in photolysis of pyraclostrobin, although the direct photolysis plays dominant role in the pyraclostrobin photolysis.

Fig. 2. Photo-induced toxicity of pyraclostrobin under radiations of 350 and 800 W Xenon lamps, respectively. 4

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plays a more fundamental role than photo-sensitization of pyraclostrobin. 4.1.3. Influence of DOM on photolysis and toxicity The Fig. 3a reveals that DOM can significantly affect the photolysis of parent compound (i.e. pyraclostrobin). The photolysis rate in the presence of DOM is much slower than that in pure pyraclostrobin solution. At the same time, DOM can significantly decrease the toxicity to V. fischeri although DOM itself has a certain toxic effect (Fig. 3b). The photolysis of pyraclostrobin in the presence of DOM shows a decrease in the degradation kinetics, which is owing to chromophoric DOM decreases the transparency of solution and influence the light absorption of pyraclostrobin (Gemmell et al., 2013). This screening effect reduces the number of pyraclostrobin molecules that become photo-chemically exited and hence reduces the direct photolysis of pyraclostrobin. In addition, DOM plays a role of %OH scavenger, which inhibits the subsequent reactions. Thus, the toxicity variance trend is very similar to that in the presence of NaN3. It is important to note that the dissolved organic carbon/matter can also act as a photosensitizers (i.e. triplet state of dissolved organic matter, 3DOM*). The determined steady concentration of 3DOM*, 1O2 and ∙OH are 7.72 × 10–15, 1.68 × 10–13 and 2.31 × 10−15 M, respectively (Fig. S7). Under the sunlit irradiation in the natural aquatic environment, these radicals can increase the photolysis rate of pyraclostrobin and its intermediate products. However, slightly reduced photolysis rate in the presence of DOM (Fig. 3) indicates the 3DOM* does not play an important role in the photolysis of pyraclostrobin as compared with the direct photolysis and screening effect. 4.2. Photolysis pathway To investigate the photolysis pathway of pyraclostrobin, the reaction free energy was calculated by G09 program and intermediates were identified by GC–MS (Fig. S5) (Doll and Frimmel, 2003; Gautam et al., 2005). The results show that pyraclostrobin is firstly photo-degraded into some synthetic intermediates, such as MPC and CHP shown in Table 1 (Mercader et al., 2008; Nakamura et al., 2017). Then, the PPRIs generated during photolysis oxidizes the intermediates into aniline, benzoquinone and low molecular weight acids (Palominos et al., 2008; Pimentel et al., 2008), leading to the decline of pH in the tested solution (Fig. S8). Based on GC–MS results and quantum calculation, the photolysis pathway of pyraclostrobin was deduced and shown in Fig. 4(a). During the radiation process, pyraclostrobin firstly absorbs light photon and transforms into the excited state (2.58 eV, Fig. 4(b)). After demethoxylation and PPRIs oxidation, the parent compound degrades to MPC, CHP and other intermediate products. Then under the Xenon lamp radiation, these intermediate products photo-chemically transform into AN. Afterwards, the AN and other intermediates are transformed to BQ and low molecular weight acid by PPRIs oxidation. The pathway shown in Fig. 4(a) suggests that the increased toxicity in Phase I is mainly attributed to the formation of the intermediates (e.g. MPC and CHP). The total concentration in the solution (the pyraclostrobin and its intermediates) at the initial stage of photolysis is greatly higher than the initial concentration (pyraclostrobin only) (Fig. S6). There are many intermediates that were not detected but contributed to the overall toxicity and their joint toxicity (pyraclostrobin and its intermediates) is supposed to be greater than the parent compound (i.e. pyraclostrobin). The decreased toxicity in Phase II is mainly attributed to the formation of aniline and its toxicity is lower than the joint toxicity of intermediates. The increased toxicity in Phase III is mainly attributed the formation of benzoquinone and acids (Andrade et al., 2006; Sirés et al., 2007; McFerrin et al., 2008). Experiments show that benzoquinone is more toxic than aniline (see toxicity values in Table 1). The variation of the inhibition rate to V. fischeri during photolysis reflects not only the change of intermediates, but also change of

Fig. 3. Influence of PPRIs (·OH and 1O2) and DOM on the photolysis (a) and photo-induced toxicity to V. fischeri (b) of pyraclostrobin under Xenon lamp (800 W) radiation.

Fig. 3b shows that the 1O2 can also affect the photolysis of intermediates because of slowly increased toxicity rate as compared with the 1 O2 scavenger free solution. This is supported by the observation of determined concentrations of some intermediates varying with the time (Fig. S6). Different transformation rates have been observed for the some intermediate products between the tested solutions in presence and absence of 1O2 scavenger (NaN3) (Fig. S6). Compared to the toxicity variance curve in presence of %OH scavenger, the increased toxicity is much slower in the presence of 1O2 scavenger. The reason is partly due to the slower photolysis of parent compound caused by the 1O2 scavenger. In comparison with t-BuOH which has no effect to V. fischeri (Fig. 3b), NaN3 has a certain toxic effect to V. fischeri with bioluminescent inhibition rate varying from 5 to 8 % during the photolysis. If radicals 1O2 and %OH have same reaction rate to the intermediates, in theory, the inhibition rate in presence of NaN3 will be greater than that in presence of t-BuOH because of the higher toxicity for NaN3 than the tBuOH. The much more slowly increased toxicity for the tested solution in presence of NaN3 as compared to t-BuOH suggests that 1O2 radical play a more important role than ·OH for the photolysis of intermediate products in this case (Fig. S6). This can also been seen from the photolysis of the parent compound in the presence of t-BuOH and NaN3 (Fig. 3a). Above results indicate that photo-sensitization (indirect) photolysis can affect the degradation of pyraclostrobin because of the decreased photolysis rate to parent compound and the increased toxicity inhibition rate to V. fischeri in the presence of radical scavengers (t-BuOH and NaN3). On the other hand, still very fast photolysis rate in the presence of radical scavengers (t-BuOH and NaN3) suggests that direct photolysis 5

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Fig. 4. Photolysis pathway (a) and energy barriers of major photochemical process (b) of pyraclostrobin under Xenon lamp (800 W) radiation (The value in the figure represents the free energy change (kJ·mol−1) between compounds, positive value indicates absorbing energy and negative indicates releasing energy. The structures labeled with a crosshair indicate that these products were detected by GC–MS, and the structures labeled with a square bracket indicate that these products were inferred from Gaussian 09 program).

pyraclostrobin and intermediates at the luciferase binding site can be found in the Figs. S9 and S10. The interaction energy (Eb) reflects the interaction stability of a receptor with a ligand. The lower Eb value indicates the more strength of the interaction between a compound and luciferase and greater toxic effect to V. fischeri from the compound (Wang et al., 2014). Inspection of the calculated Eb values in Table 2 suggests that the pyraclostrobin has the lowest Eb value involving many H-bond and Pi-bond interactions. It is supposed to have the greatest toxicity as comparing with the intermediates. Table 1 lists the single toxicity values experimentally determined for three compounds. To fill in the data gaps for other compounds and compare the chemical toxicity, the baseline toxicity values were calculated from baseline model. In principle, the baseline toxicity is minimum toxicity that compounds exhibit and should be less than the measured values. Comparison of the data in Tables 1 and 2 shows that, except benzoquinone, toxicity is well correlated with the Eb, indicating that luciferase is the target receptor in V. fischeri of original and intermediate compounds. If we neglect the joint toxic effect, the toxicity will decrease with increasing of photo-radiation time. However, opposite situation observed in the toxicity variance in Phase II (Fig. 2) suggests that the increased toxicity with increasing photo-radiation time is attributed to

exposure concentrations. It is well known that the acids with low pH are very toxic to a range of bacteria and widely used in food preservative. Although the toxicity in each phase is mainly attributed to one to two intermediates, all the reactions shown in Fig. 3 can occur at the same time and all the intermediates can be detected from the tested solution sampling at the different time (Fig. S5 – 6). The concentrations of three main intermediates varied with time can be found in Supplementary materials (Fig. S6). 4.3. Toxic mechanisms 4.3.1. Molecular docking Previous investigations suggested that luciferase, a vital enzyme catalyzing the fluorescent reaction in V. fischeri, was a putative receptor target of many toxicants in V. fischeri (Hastings et al., 1973). To investigate toxic mechanisms and compare the toxicity of parent compounds with intermediates, the interaction energy of receptor in V. fischeri (i.e. luciferase) with a ligand (i.e. parent or transformation products) was calculated by CDOCKER program and the values are listed in Table 2. The H-bonds and Pi-bonds involved in the molecular docking are also listed in the Table 2. The detailed docking views of 6

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Table 2 CDOCKER interaction energy of pyraclostrobin and its major intermediates. Name

Sitea

CDOCKER energy

CDOCKER interaction energy (Eb)

H-bond

Pi-bond

PYR

1

−6.1434

−44.7082

HIS82:HN (LUX A) HIS81:HD1 (LUX B)

MPC

3

−26.3724

−29.2018

LYS2:HZ3 (LUX B)

AN

3

−15.6999

−17.5287

LYS225:NZ (LUX B)

ARG119:NH1 (LUX B) ARG119:NH2 (LUX B) ARG85:NH2 (LUXB) ARG119:NH1 (LUX B) ARG119:NH2 (LUX B) ARG119:NH1 (LUX B) LYS225:NZ (LUX B) LYS2:NZ (LUX B) LYS70:NZ (LUX B) ASP294:OD2 (LUX B) ASP37:OD1 (LUX B)

HQ BQ

2 3

−19.1983 −23.5737

−20.8278 −23.9053

THR80:O (LUX A) LYS2:HZ2 (LUX B) LYS70:HZ2 (LUX B)

a

LYS225:NZ (LUX B)

Docking sites are shown in Fig. S9.

5. Conclusions

the joint toxicity. If the parent compound and intermediates have similar inhibition effect to V. fischeri, the joint inhibition rate of MPC and CHP (synthetic intermediates of pyraclostrobin) estimated from the added toxicity values in Table 1 is apparently greater than the toxicity of pyraclostrobin to V. fischeri. Furthermore, the joint toxicity is caused not only by the two compounds. A number of transformation products can be formed during the short photo-radiation time (Fig. 4). That is why there is a sharply increased toxicity in Phase I. The decreased toxicity in Phase II reflects the decreasing number of the intermediates. The increased toxicity in Phase III is attributed to the formation of benzoquinone and acids. They are more toxic than aniline. Although molecular docking can well explain why pyraclostrobin has a great toxicity to V. fischeri. It cannot explain why benzoquinone is more toxic than pyraclostrobin (Table 2). Furthermore, the docking sites in luciferase determined by CDOCKER energy are not same for all the studied compounds (Fig. S9). Besides that, the Eb value is the characterized by Van der waals force and electrostatic force, which is highly related to molecular weight and volume. This indicates that Eb values cannot well reflect the interaction ability of ligands with target enzyme if there is great difference of molecular weight and volume or different optimal docking sites between molecules.

Degradation of pyraclostrobin is dominated by photo-modification or direct photodegration and the can well be described by first-order kinetics. Photo-sensitization also plays an important role in the degradation of pyraclostrobin. Addition of radical 1O2 scavenger (NaN3) into the tested solution decreases the photolysis rate of parent compound, indicating that photo-sensitization takes part in the degradation of pyraclostrobin. The GC–MS results reveal that the pyraclostrobin is firstly photo-degraded into some intermediates (e.g. synthetic intermediates of pyraclostrobin), then into aniline, benzoquinone and acids with low molecular weight. The toxicity variance can be classified into three phases: The joint toxicity of pyraclostrobin and some intermediates (increased toxicity in Phase I); the formation of aniline with a lower toxicity than other intermediates (decreased toxicity in Phase II); the formation and accumulation of benzoquinone and acids (increased toxicity in Phase III). The addition of radical scavengers (t-BuOH and NaN3) significantly affects the bioluminescent inhibition rate, which indicates that radicals involve in the photodegradation of intermediates. The simulation of molecular docking reveals that pyraclostrobin has stronger interaction ability with target receptors in luciferase and can well explain why pyraclostrobin is more toxic than most intermediates to V. fischeri. The simulation of molecular dynamics reveals that benzoquinone can greatly change the structure of luciferase and explain why it is more toxic than pyraclostrobin. Above results are very valuable for the risk assessment of pyraclostrobin in the aquatic environment.

4.3.2. Molecular dynamics Molecular dynamics is another way to investigate the toxic mechanism of a compound. Unlike molecular docking which evaluate the toxic effect through the receptor-ligand interaction stability, it evaluates the toxic effect by simulating structural change of receptor (e.g. luciferase) before and after docking by a ligand through molecular dynamics. Greater change in the structure caused by a ligand indicates the greater effect to receptor, leading to a greater bioluminescent inhibition rate to V. fischeri. Fig. 5 shows the changed luciferase structures docked by pyraclostrobin (a) and benzoquinone (b), respectively. The original structure of 1BRL and the conformation of 1BRL docked by pyraclostrobin and benzoquinone (Figs. S9 – 10), respectively. This luciferase enzyme is composed of two parts, LUX A and LUX B with a pretty compact structure (Fig. S11) (Englander et al., 2003; Fisher et al., 1995; Rachman et al., 2014; Tanner et al., 1997). After simulation with docked PYR and BQ, however, the luciferase structure becomes unconsolidated and is twisted greatly by benzoquinone as compared with pyraclostrobin (Fig. 5). This indicates that benzoquinone should have greater toxic impact to V. fischeri than pyraclostrobin. It can also explain why there is an increased toxicity observed in the toxicity variance (Phase III in Fig. 2).

CRediT authorship contribution statement Lingyun Fan: Conceptualization, Investigation, Formal analysis, Writing - original draft. Ying Huang: Methodology, Data curation. Tao Huang: Visualization. Kun Zhao: Software, Validation. Ya-Nan Zhang: Writing - review & editing. Chao Li: Software. Yuan Hui Zhao: Funding acquisition, Supervision, Writing - review & editing. Declaration of Competing Interest None to declare. Acknowledgements This work was supported by National Natural Science Foundation of China (21976026 and 21777022) and Jilin Province Science and Technology Development Project (20180520078JH). We are very grateful to Prof. Jingwen Chen for the molecular docking simulation by using Discovery Studio 2.5. 7

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Fig. 5. The changed luciferase structures docked by pyraclostrobin (a) and benzoquinone (b), respectively (Blue: unchanged and slightly changed part of luciferase structure; White: Moderately changed part of luciferase structure; Red: greatly changed part of luciferase structure).

Appendix A. Supplementary data

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