Lethal Toxicity and Sublethal Metabolic Interference Effects of

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Agricultural and Environmental Chemistry

Lethal toxicity and sub-lethal metabolic interference effects of sulfoxaflor on the earthworm (Eisenia fetida) Song Fang, Yizhi Zhang, Xiangwei You, Peng Sun, Jun Qiu, and Fanyu Kong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04633 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Journal of Agricultural and Food Chemistry

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Lethal toxicity and sub-lethal metabolic interference effects of sulfoxaflor on the

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earthworm (Eisenia fetida)

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Song Fang*, Yizhi Zhang, Xiangwei You, Peng Sun, Jun Qiu, Fanyu Kong*

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Laboratory of Tobacco Quality and Safety Risk Assessment, Ministry of Agriculture and

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Rural Affairs, Tobacco Research Institute of Chinese Academy of Agricultural Sciences,

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Qingdao, 266101, China

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*Corresponding author: Song Fang and Fanyu Kong

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Tel./fax: +86 53288701916

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Email: [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Testing for effects of pesticides on non-target organisms is an integral part

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of ecological risk assessment. In the present study, the acute toxicity of sulfoxaflor to

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earthworms was evaluated using an artificial soil toxicity test, and sub-lethal effects were

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assessed through oxidative stress and metabolomics. Sulfoxaflor is a super toxic pollutant

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to earthworms that easily bioaccumulates in earthworms, and contains LC2, LC10, and

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LC50 value of 0.08 (0.04 - 0.13), 0.19 (0.11 - 0.25) and 0.54 (0.45 - 0.65) mg/kg

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respectively. Sub-lethal doses of sulfoxaflor resulted in oxidative damage to earthworms

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in which antioxidant enzymatic activities including SOD, CAT, GST were significantly

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inhibited, and MDA content accumulated. Metabolomics analysis suggested the energy

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metabolism and the urea cycle in earthworms were significantly activated, while

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nucleotide metabolism was depressed which could cause DNA damage. Results suggest

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earthworms have the potential to be a new entry point for sulfoxaflor into the wildlife

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food chain. Since earthworms significantly contribute to soil function and ecosystems,

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the high safety risks of sulfoxaflor to the earthworm could extend to the environment. In

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view of these findings, more attention should be given to the risks sulfoxaflor poses on

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the environment through its effects on earthworms.

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KEYWORDS: environmental toxicology; acute toxicity; residue analysis; oxidative

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stress; metabolomics

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INTRODUCTION

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Neonicotinoid insecticides have played a very important role in agricultural pest

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management worldwide.1 As a fourth-generation neonicotinoid, sulfoxaflor performs

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equally well or better against a wide range of sap-feeding insects than other neonicotinoid

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insecticides, including acetamiprid, imidacloprid, and thiamethoxam.2 Additionally,

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sulfoxaflor acts on the insect nicotinic acetylcholine receptor (nAChR) in a distinct

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manner compared to other neonicotinoids, and it is considered an important new tool in

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insecticide resistance management programs as it lacks insecticidal cross-resistance.3,4

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Since its commercialization by Dow Agro Sciences in 2013, sulfoxaflor has been

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registered and approved for use in more than 40 countries worldwide.

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The impact of pesticides on the environment is a global issue of growing concern, and

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there is increasing evidence that neonicotinoids have profound effects on non-target

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organisms, such as honeybees and earthworms.5,6 Although studies have shown that

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sulfoxaflor is safe for humans, fish and other aquatic species, sulfoxaflor residues can

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bioaccumulate in such animals’ bodies and have a high long-term risk for the small

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herbivorous mammals that usually found in field use to grow vegetables or cotton.7

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Additionally, with the worldwide application of sulfoxaflor, it is bound to affect

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non-target organisms and the ecological environment.8 Some researchers have shown that

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sulfoxaflor poses moderate risk to honeybees,9 and sub-lethal concentrations of

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sulfoxaflor are likely to have a negative impact on ants.10 Up to now, the study of

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sulfoxaflor mostly focused on biological characterization,11,12 its mechanism of 3



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action,13,14 metabolism and residual detection.15,16 The environmental safety risk of

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sulfoxaflor on earthworms remains unknown based on the review of past reports.

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Soil is the most important environmental mediator for the transport and transformation

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of pesticides into the environment. Earthworms are a vital species in soil ecosystems,

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playing important roles in pollutant decomposition, soil nutrient mineralization and

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formation.17 The response of earthworms to soil contaminated with pesticides is an

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important indicator for assessing soil ecological toxicity.18 Studies on the ecotoxicology

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of pesticides to earthworms frequently focus on apparent indices, such as mortality,

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growth and reproduction rates.19,20 These studies investigated the overall toxicity of

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pesticides, however, they did not reveal the toxicity mechanism in earthworms.

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Metabolomics, a popular technique for studying metabolic mechanisms, has been used

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in recent years to study metabolites and their dynamic effects on organisms before and

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after interference.21,22 The major analytical tools used for metabolomic studies are

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high-field proton nuclear magnetic resonance (NMR) spectroscopy and chromatography

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tandem mass spectrometry (MS) based techniques. Compared to NMR techniques, MS

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methods have higher separation capacity and sensitivity, and can identify small molecule

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metabolites from complex mixtures in a high-throughput mode.23,24 Although

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metabolomics have been used in the identification of metabolic pathway perturbations

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related to environmental heavy metals or organic contaminants exposure,25,26 there is

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little research investigating the metabolic response of earthworms to sulfoxaflor toxicity.

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The present study aims to evaluate the lethal toxicity and sub-lethal metabolic

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interference mechanism of sulfoxaflor in the earthworms. Acute toxicity tests were

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performed and the lethal concentration (LC) values of sulfoxaflor on earthworms were

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calculated. The effects of oxidative stress and metabolic disturbance of sulfoxaflor on

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earthworms were then evaluated under sub-lethal dose exposure. This research

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contributes to a greater understanding of the toxicity and metabolic interference

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mechanisms in earthworms, and it provides effective information for risk management of

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sulfoxaflor.

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MATERIALS AND METHODS

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Materials. Sulfoxaflor with 99% purity was obtained from J&K Scientific (China).

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Mass spectroscopy grade methanol, acetonitrile, ammonium acetate and ammonium

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hydroxide were obtained from CNW Technologies (Germany). Ultrapure water was

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prepared using a Milli-Q system of Merck Millipore (Germany). Commercial assay kits

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for antioxidant enzymatic activities including superoxide dismutase (SOD), catalase

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(CAT), glutathione peroxidase (GPX) and malonaldehyde (MDA) were obtained from

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Suzhou Comin Biotech (China). All other chemicals were analytical grade and obtained

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from Qingdao Quanchang Biotech (China). Earthworms (Eisenia fetida) with visible

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clitellum and wet weight between 0.3 - 0.5 g were obtained from Shandong Hongda

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Biotech (China), and acclimated to the experimental conditions for two weeks before the

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experiment. The artificial soil was consisted of 70% sand, 20% kaolin clay and 10%

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sphagnum peat moss. 5



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Instruments. The concentration of sulfoxaflor in the soil and earthworms was

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determined using a UPLC system (Waters, USA) equipped with Orbitrap MS (AB

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SCIEX, USA). Antioxidant enzymatic activities were detected by ELx 800 TM

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Microplate Spectrophotometer (Biotek, USA). Metabolomics analysis was performed on

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a 1290 UHPLC system (Agilent Technology, USA) equipped with a Q Exactive Orbitrap

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MS (Thermo Fisher Scientific, USA). The System utilized an ACQUITY UPLC HSS T3

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column (2.1 * 100 mm * 1.8 μm, Waters, USA).

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Earthworm culture and exposure experiments. Earthworm culture and exposure

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experiments were performed according to the Organization for Economic Cooperation

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and Development’s guidelines (OECD).27 Based on previous experimental results, a

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range of concentrations of test substances was prepared using water as the solvent. For

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each exposure experiment, the desired amount of sulfoxaflor was mixed into 20 g sand.

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The mixed sand was set aside for 1 h to evaporate water and then mixed thoroughly with

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980 g artificial soil (dry weight) in a mixer. The soil was put into a glass beaker, and the

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moisture of the soil was adjusted to 35%. Earthworms were transferred into the soil and

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cultured at 20 ± 1 °C for 12 h in light and 12 h in the dark.

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Acute toxicity tests. The acute toxicity of sulfoxaflor to earthworms was tested by an

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artificial soil contact toxicity assay. Based on previous experimental results, the

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determined concentrations of sulfoxaflor in soil sample were 0.05, 0.1, 0.2, 0.4, 0.6, 0.8,

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and 1 mg/kg. Each soil sample had four replicates at each experimental concentration,

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and each treatment contained 18 earthworms. On days 7 and 14, both the mortality rate of

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earthworms was determined and LC values were calculated.

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Degradation in soil and bioaccumulation in earthworms. For degradation and

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bioaccumulation tests, the concentration of sulfoxaflor in soil was 0.2 mg/kg in four

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replicates experimental samples. Five earthworms and about 10 g soil form each

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treatment were randomly sampled on days 0, 1, 3, 5, 7, 10, 14, 21 and 28. For soil, 3 mL

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of distilled water and 5 mL of acetonitrile were added to 5 g soil. For earthworms, five

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earthworms were weighed and homogenized using 10 mL distilled water. Then, the

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pretreated sample was vortex treated for 10 min and centrifuged at 4000 g for 5 min.

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Thereafter, 1.5 mL of supernatant was filtered using a 0.22 μm syringe filter for further

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UPLC-MS analysis, and the injection volume was 5 μL. The parameters for UPLC and

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MS are shown in Table S1 and S2 respectively.

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Oxidative damage test. For oxidative stress testing, the concentrations of sulfoxaflor

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in soil were 0.01, 0.05 (non-lethal dose), 0.1 (approximate value of LC2) and 0.2

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(approximate value of LC10) mg/kg. Each of these experimental concentrations were

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tested in four replicates of soil sample. Five earthworms from each treatment were

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randomly sampled on days 1, 7, 14 and 28, and the antioxidant enzymatic activities were

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determined using assay kits according to the kit instructions.

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Metabolic interference tests. For metabolomics experiments, the concentration of

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sulfoxaflor in soil was 0.2 mg/kg. Eight earthworms from each treatment were randomly

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sampled on day 14. A method based on previous reports was used to extract and analyze 7



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metabolites in earthworms.28,29 Individual earthworms were placed in a centrifuge tube

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containing 2 mL of extraction liquid (V methanol: V acetonitrile: V water = 2: 2: 1) and

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20 μL of internal standard. The mixture was homogenized for 4 min and then underwent

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ultrasound treatment for 5 min, for a total of three times. After incubating at -20 °C for 1

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h, the sample was centrifuged at 12000 rpm for 15 min, and the fresh supernatant was

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dried under a vacuum. Then 200 μL of extraction liquid (V acetonitrile: V water= 1: 1)

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was added and the extracts were reconstituted. After vortex treatment for 30 s and being

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sonicated for 10 min, the extracts were centrifuged at 12000 rpm for 15 min. The

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supernatant (60 μL) was transferred into a glass vial for further UHPLC-MS analysis,

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with an injection volume of 2 μL. Quality control (QC) samples were obtained by pulling

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10 μL of supernatant from each sample. The parameters of UHPLC and MS are shown in

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Table S3 and S4 respectively.

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Statistical analysis. Probit analysis was used to calculate the LC values of sulfoxaflor

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for earthworms. ANOVA was performed along with LSD testing for multiple

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comparisons among treatments (p < 0.05), and results were expressed as mean ± SD. All

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analyses were performed using SPSS software (V 22.0).

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For metabolomics analysis, MS raw data files were converted into the mzXML format

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using ProteoWizard (V 3.0.6428), and XCMS (V 1.46) was employed for peak detection,

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noise filtering, and peak alignment. The resulting three-dimensional data including the

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peak number, sample name, and normalized peak area were fed to SIMCA (V 14.1) for

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principal

component

analysis

(PCA)

and 8

orthogonal


projections

to

latent

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structures-discriminate analysis (OPLS-DA). Differential compounds were screened

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using variable importance in the projection (VIP > 1) in the first principal component of

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the OPLS-DA model, and then were analyzed by ANOVA (P < 0.05). The matching

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information of different metabolites was confirmed by retrieving KEGG and PubChem

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databases. In addition, commercial databases including KEGG of the corresponding

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species

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MetaboAnalyst http://www.metaboanalyst.ca/ were utilized to search for metabolite

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pathways.

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RESULTS AND DISCUSSION

Caenorhabditis

elegans

(nematode)

http://www.genome.jp/kegg/

and

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Acute toxicity in earthworms. In the present study, the mortality of earthworms in the

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sulfoxaflor exposure treatments showed a significant dose-effect relationship, and linear

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regression correlation coefficients (R2) were > 0.92 (Figure S1). Table 1 summarizes the

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results from the specific effect levels (LC2, LC10 and LC50) used in the artificial soil tests.

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According to the toxicity classes,30 extreme toxicity of sulfoxaflor in earthworms was

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observed.

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It has been generally accepted that neonicotinoids pose great safety risks to

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environmental organisms, such as honeybees and earthworms.5,6 Although sulfoxaflor

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has little effect on honeybees, it is a super toxic pollutant to earthworms. Additionally,

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sulfoxaflor was highly toxic to the larvae of a natural predator of sap-feeding pests

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(Adalia bipunctata),31 and it has not only lethal, but also sub-lethal effects on red

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imported fire ant (Solenopsis invicta) populations.32 Therefore, more attention should also

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be given to the ecological risks of sulfoxaflor. 9



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Degradation in soil and bioaccumulation in earthworms. The residual amounts of

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sulfoxaflor in soil and earthworms during the whole exposure period are shown in Figure

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1. In the present study, the sulfoxaflor concentration in soil decreased slowly as exposure

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time increased. On the 21st day, the degradation rate exceeded 90%. The concentration of

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sulfoxaflor in earthworms first increased and then decreased as exposure time increased.

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On the 7th day, the concentration of sulfoxaflor in earthworms reached maximum height.

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Fourteen days later, the concentration of sulfoxaflor in the earthworms was relatively

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stable, but still at a high level.

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The degradation of pollutants in soil is influenced by microorganisms, light and soil

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properties. Studies have shown that microbial degradation is the main mechanism of

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sulfoxaflor degradation in soil.15 The artificial soil is rich in organic matter, and vigorous

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microbial activity accelerated the degradation of sulfoxaflor. However, earthworms

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undergo strong bioaccumulation of sulfoxaflor. The concentration of sulfoxaflor in

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earthworm remains over 10 times higher than that in soil even after 14 days. Similar

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results revealed that selective bioaccumulation in adult and juvenile earthworms of other

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neonicotinoids such as imidacloprid, acetamiorid, and thiachloprid.33 The accumulation

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of organochlorine pesticides in earthworms was also reported.34 Our study reveals a new

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potential point of entry of sulfoxaflor into the wildlife food chain.

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Effects on the oxidative damage to earthworms. The activities of SOD, CAT, GST,

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and MDA concentration in earthworms are shown in Figure 2. For the non-lethal dose

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(0.01, 0.05 mg/kg) treatments, sulfoxaflor had few effects on SOD activity during the

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exposure period. For sub-lethal (0.1, 0.2 mg/kg) treatments, the SOD activity did not

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change significantly on the 1st day. With prolonged exposure time, the SOD activity

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significantly increased and then considerably decreased. A similar biphasic change in

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CAT and GST activities was also observed. The MDA content did not significantly

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change in non-lethal dose treatments, however, it increased significantly with the

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prolongation of exposure time in the sub-lethal treatments.

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Antioxidant system played an important role in maintaining the function of system,

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and the induction of the antioxidant enzymatic activities has been considered a biomarker

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of environmental pollution in earthworm toxicology.35 SOD and CAT are the first line of

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antioxidative defense against reactive oxygen species (ROS) in earthworms, which may

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cause oxidative damage to biological macromolecules, like DNA, protein and lipid.

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Normally, ROS levels and the antioxidant enzyme contents coexist in a dynamic balance.

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However, excessive ROS production exceeding the antioxidant capacity causes oxidative

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stress in the organism.36 In the present study, the antioxidant enzymatic activities in

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earthworms increased, and then decreased after longer sub-lethal exposure. The activities

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of antioxidant enzymes were activated by sulfoxaflor, which could resist antioxidant

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damage. However, longer sub-lethal dose exposure caused irreversible antioxidant

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damage, and the activities of antioxidant enzymes were subsequently inhibited.

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Antioxidant damage further leads to lipid peroxidation, which damages the cell

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membrane and leads to destruction of membrane lipids. In addition, as the product of

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lipid peroxidation, MDA can react with deoxyadenosine and deoxyguanosine in DNA, 11



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forming DNA adducts which can cause mutagenesis and carcinogenesis.37 Our results

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indicated that exposure sulfoxaflor resulted in serious oxidative damage to earthworms.

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Effect on metabolic interference to earthworms. Good data quality and repeatability

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of analytical methods are the basis for metabolomics. Base peak chromatograms (BPC)

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showed that the retention time and peak area of QC samples overlapped well, and no

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significant peaks were detected in the blank samples (Figure S2). The results of PCA

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analysis showed the distribution of QC samples was intensive and the results of PCA-X

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are distributed within 2 STD (Figure S3). The OPLS-DA model showed clear separation

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and discrimination, revealing a visible perturbation of the earthworm metabolic profiles

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between the two groups (Figure 3). All samples fell within the 95% confidence interval

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and the results of a permutation test (n = 200) confirmed the good quality of the

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OPLS-DA model, which has good robustness and no over-fitting. All results indicated

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that the stability and repeatability of the analytical methods were acceptable.

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Differential metabolites were screened through comprehensive analysis by VIP of the

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OPLS-DA model and P values of ANOVA. This screening was confirmed by retrieving

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data from the KEGG and PubChem databases. A total of 26 metabolites were identified,

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and their information, including VIP values, P values and fold changes, are given in

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Table 2. As is considered common knowledge, complex metabolic reactions are affected

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by complex metabolic pathways and networks. Through a comprehensive pathway

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analysis, some key metabolic interference pathways were found and summarized, as

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shown in the Figure 4. The sub-lethal dose exposure of sulfoxaflor mainly affected 12


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energy metabolism, urea cycle, homeostasis of amino acid metabolism and nucleotide

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metabolism of the earthworms.

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Specifically, metabolite alterations in the carbohydrate metabolism, TCA cycle, and

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changes in some amino acids indicated that sulfoxaflor affects earthworm energy

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metabolism. A carbohydrate metabolism related metabolite, glucose-6-phosphate, was

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up-regulated. Glucose-6-phosphate is a common intermediate product of carbohydrate

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metabolism, and it is the crossing point of various metabolic pathways38. Up-regulation

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of malic acid and citric acid showed inhibition of the TCA cycle. The TCA cycle is the

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hinge of sugar, fat and protein metabolism, and it is also the main metabolic process for

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supplying energy39. The metabolism of some amino acids and derivatives have obviously

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changed. Glutamate, glutamine, and histidine were also up-regulated. The α-ketoglutaric

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acid generated by the decomposition of amino acids can be converted into sugar or lipids,

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and it can resynthesize some nonessential amino acids. Additionally, α-ketoglutaric acid

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and can also participate in the energy metabolism of the TCA cycle.40 Leucine and valine

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were up-regulated as well. The branched chain amino acid can be an important source of

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calories, and it is superior as a fuel to the ubiquitous intravenous glucose.41 All these

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results showed that energy metabolism of earthworms was affected after sulfoxaflor

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exposure. Similar to our results, interference in energy metabolism were also observed in

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earthworms42, land snail43 and zebrafish larvae44 after exposure to other pesticides.

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Some metabolites related to the urea cycle had obvious disturbances, which is the main

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way to remove ammonia toxicity in animals. In the earthworms, aspartic acid and 13



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asparagine were down-regulated, while arginine and ornithine were up-regulated.

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Aspartic acid is the donor of nitrogen during the urea synthesis process, and the urea

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cycle is linked to the TCA cycle by aspartic acid and fumaric acid. Arginine and ornithine

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are important intermediate products in the urea cycle and play an important role in urea

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synthesis.45 The disturbances in all these metabolites reflect the up-regulation of the urea

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cycle, which possibly causes the abnormality of the excretory function in earthworms.

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The purine and pyrimidine metabolisms were affected based on relevant metabolites

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indicating the perturbation of nucleotide metabolism. Deoxyguanosine, deoxyinosine,

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deoxyuridine and guanine, which are all nucleoside components of DNA, were

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down-regulated. Deoxyguanosine can be converted into 8-hydroxydeoxyguanosine,

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which is regarded as a critical biomarker of oxidative stress and oxidative DNA

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damage.46 The formation of 8-hydroxydeoxyguanosine induces the reduction of

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deoxyguanosine, indicating that oxidative stress and DNA damage occurred after

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sulfoxaflor exposure.47 Guanine is transformed to guanosine monophosphate by

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hypoxanthine

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down-regulation of HGPRT activity imply that the DNA base repair might be blocked.48

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The changes in these metabolites indicate that sulfoxaflor interfered with nucleotide

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metabolism and could cause DNA damage in earthworms. DNA damage is also

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significantly increased through exposure of earthworms to thiacloprid.49 Therefore, one

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of the reasons for long-term toxicity of neonicotinoids to earthworms may be the DNA

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damage.

guanine

phosphoribosyl

transferase

14


(HGPRT)

catalysis.

The

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In summary, sulfoxaflor is a super toxic pollutant to earthworms. Earthworms

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bioaccumulate sulfoxaflor in high concentrations and may be a new potential point of

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entry of sulfoxaflor into the wildlife food chain. Sub-lethal doses of sulfoxaflor resulted

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in oxidative damage, which mainly affected energy metabolism, the urea cycle,

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homeostasis of amino acid metabolism and nucleotide metabolism within earthworms.

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The lethal toxicity results and sub-lethal metabolic interference effects on earthworms

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may have implications for requiring a better assessment of the soil environmental risks of

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sulfoxaflor. Due to the earthworm’s significant contribution to soil function and the

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ecosystem, more attention should be given to the high safety risks sulfoxaflor may pose

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to the environment at large.

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ACKNOWLEDGMENTS

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This work was financially supported by the Agricultural Science and Technology

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Innovation Program (ASTIP-TRIC06).

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Supporting Information

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The parameters of UPLC and MS for detecting the sulfoxaflor concentration in soil

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and earthworms (Table S1, S2), the parameters of UHPLC and MS for detecting

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metabolites in earthworms (Table S3, S4), linear regression curve of sulfoxaflor toxicity

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to earthworm (Figure S1), Base peak chromatograms (BPC) of QC and blank samples

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(Figure S2), PCA and PCA-X distribution diagram of QC samples (Figure S3).

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Notes

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The authors declare no competing financial interest. 15



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459

Table legends

460

Table 1. LC values of sulfoxaflor for earthworms.

461

Table 2. Information of the characteristic metabolites in earthworms.

24


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462

List of Tables

463

Table 1. LC values of sulfoxaflor for earthworms Pesticide days sulfoxaflor

LC Values(mg/kg dry soil)

Toxicity grade

LC2

LC10

LC50

7

0.12 (0.04 - 0.19)

0.24 (0.13 - 0.33)

0.61 (0.49 - 0.76)

Extremely toxic

14

0.08 (0.04 - 0.13)

0.19 (0.11 - 0.25)

0.54 (0.45 - 0.65)

Extremely toxic

25



465

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Table 2. Information of the characteristic metabolites in earthworms. Compound

KEGG ID

VIP Values

P-Values

Fold change

L-Arginine

C00062

1.79

0.018

2.07

L-Histidine

C00135

1.55

0.042

1.72

L-Leucine

C00123

1.99

0.028

1.30

L-Methionine

C00073

1.51

0.049

1.21

D-Glutamine

C00819

1.45

0.048

1.19

L-Glutamate

C00025

1.95

0.020

1.17

L-Asparagine

C00152

1.80

0.019

0.87

L-Valine

C01799

1.67

0.025

1.32

L-Phenylalanine

C00079

1.27

0.050

0.74

L-Aspartic acid

C00049

1.54

0.037

0.81

L-Ornithine

C00077

1.80

0.032

1.37

Spermidine

C00315

1.65

0.042

1.39

N-Acetylglutamic acid

C00624

2.09

0.004

1.25

D-Glucose-6-phosphate

C00092

1.83

0.017

1.68

Malic acid

C00711

1.60

0.033

1.24

Maleic acid

C01384

1.66

0.036

1.16

Hydroxypropionic acid

C01013

1.26

0.050

0.83

Citric acid

C00158

1.90

0.019

1.38

Propionic acid

C00163

1.57

0.040

0.73

4-Pyridoxic acid

C00847

1.56

0.040

1.27

Deoxyguanosine

C00330

1.28

0.045

0.71

Deoxyinosine

C05512

1.91

0.010

0.69

Deoxyuridine

C00526

1.47

0.033

0.84

Guanine

C00242

1.46

0.049

0.82

Serotonin

C00780

1.25

0.032

0.74

Xanthurenic acid

C02470

2.08

0.026

1.42

26


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467

Figure captions

468

Figure 1. Sulfoxaflor residues in artificial soil and earthworms.

469

Figure 2. Effects of sulfoxaflor on the oxidative damage to earthworms. Data with

470

different letters means significant difference at P < 0.05.

471

Figure 3. Score scatter plot and permutation test of OPLS-DA model for earthworms.

472

Figure 4. Perturbed metabolic pathways and metabolites in earthworms.

27



473

List of Figures

474 475

Figure 1. Sulfoxaflor residues in artificial soil and earthworms.

28


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476 477

Figure 2. Effects of sulfoxaflor on the oxidative damage to earthworms. Data with

478

different letters means significant difference at P < 0.05.

29



479 480

Figure 3. Score scatter plot and permutation test of OPLS-DA model for earthworms.

30


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481 482

Figure 4. Perturbed metabolic pathways and metabolites in earthworms.

31



483

TOC Graphic

484

32


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Lethal Toxicity and Sublethal Metabolic Interference Effects of

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