Enantioselective bioaccumulation and toxicity of the neonicotinoid

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Food Safety and Toxicology

Enantioselective bioaccumulation and toxicity of the neonicotinoid insecticide dinotefuran in the earthworms (Eisenia fetida) Tong Liu, Dan Chen, Yiqiang Li, Xiuguo Wang, and Fenglong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00285 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Journal of Agricultural and Food Chemistry

Enantioselective bioaccumulation and toxicity of the neonicotinoid insecticide dinotefuran in the earthworms (Eisenia fetida) Tong Liua, Dan Chena, Yiqiang Lia, Xiuguo Wanga,*, Fenglong Wanga,* a

Tobacco Research Institute of Chinese Academy of Agricultural Sciences (CAAS),

Qingdao 266101, P.R. China *Corresponding author: Xiuguo Wang and Fenglong Wang Tel./fax: +86 53288702136 E-mail: [email protected]; [email protected]

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ACS Paragon Plus Environment


Abstract: The enantioselective bioaccumulation and toxicity of dinotefuran in earthworms were studied in this study. The results showed that S-dinotefuran accumulated faster than Rac-dinotefuran and R-dinotefuran in earthworms. The acute toxicity of S-dinotefuran was 1.49 and 2.67 times that of the Rac-dinotefuran and R-dinotefuran in artificial soil during 14 d of exposure. At 1.0 mg/kg, the three tested chemicals inhibited the growth and reproduction as well as induced oxidative stress effects in earthworms; however, the toxic effects induced by S-dinotefuran were the most serious. The transcriptome sequencing results showed that S-dinotefuran had stronger interactions to biomacromolecules and influences on the endoplasmic reticulum (ER) than R-dinotefuran, which may be the main reason of enantioselectivities between the two enantiomers. The present results indicated that the risk of S-dinotefuran was higher than that of Rac-dinotefuran and R-dinotefuran in the soil environment to earthworms. Risk assessment of dinotefuran should be evaluated at the enantiomer level. Keywords: Chirality; Biomarkers; Residue Analysis; Transcriptome Sequencing; Molecular Mechanism

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INTRODUCTION Chirality refers to the property that material cannot overlap with its mirror image, which is an essential attribute of nature.1 Studies have shown that almost 30% of commercial pesticides have a chiral centre and the percentage of chiral pesticides in used pesticides may be more than 40%.2,3 The enantiomers were viewed as one compound for a long time due to the lack of awareness on chiral pesticides. However, the enantiomers of chiral pesticides showed different behaviour in absorption, metabolism and toxicity because the biomacromolecules, such as protein, nucleic acid and sugars, also have chiral structures.3,4 The enantiomers of chiral pesticides have dramatically different interactions with these biomacromolecules due to the different spatial configurations.4 An increasing number of studies have shown that the risk assessment on enantiomers cannot use the data of its racemate.3-6 Therefore, studying the environmental safety of chiral pesticides at the enantiomer level has become a current research hot spot in recent years. Neonicotinoid insecticides have been become the fastest developed synthesized pesticide in recent years.7,8 Dinotefuran, [(RS)-1-methyl-2-nitro-3-(tetrahydro-3furylmethyl)guanidine], is the latest generation of neonicotinoid insecticides with chiral structures.7,8 Dinotefuran has two enantiomers, R(-)-dinotefuran and S(+)-dinotefuran. Dinotefuran has some excellent properties in controlling numerous sucking and biting insects compared with traditional neonicotinoid insecticides, such as a wide insecticidal spectrum, high insecticidal activity and being environmentally safe.9 Therefore, dinotefuran is likely to become a worldwide insecticide with good

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application prospects.8,9 Neonicotinoid insecticides have also attracted great attention because of their high risks to bees.8 Additionally, studies have shown that neonicotinoid insecticides pose high risks to aquatic and terrestrial organisms, such as zebrafish and earthworm.10,11 As a promising insecticide, dinotefuran will inevitably enter the environment during its use. Due to dinotefuran is the latest generation of neonicotinoid insecticide, the database of risk assessment is limited. One study has shown that dinotefuran is relatively stable in the soil environment with a half-life of 50-100 d.8 The initial residual concentrations of dinotefuran in the environment ranged from 0.274 mg/kg to 0.907 mg/kg.12,13 Dinotefuran has good water solubility and mobility, and it will eventually enter the soil environment through direct application or underground runoff.8,9 Although dinotefuran is safe for birds, mammals and aquatic species, it may be harmful to soil organisms.9 Therefore, dinotefuran may be a potential soil pollutant, and its influences on the soil environment must be considered. It is worth noting that there are few studies on the influences of dinotefuran on the soil environment. Moreover, there is no report on the difference in environmental behaviour of dinotefuran enantiomers. Investigating the influences of dinotefuran on the soil environment at the enantiomer level is better to evaluate its environmental safety. In the present study, the influences of dinotefuran on the typical soil invertebrate--earthworm were studied at the enantiomer level. The differences in the bioaccumulation and toxicity of Rac-dinotefuran, R-dinotefuran and S-dinotefuran were investigated. The major objective of the present work was to evaluate the

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environmental safety of dinotefuran at the enantiomer level. Moreover, it may provide a basis for the better risk assessment of neonicotinoid insecticides.

MATERIALS AND METHODS Materials. Rac-dinotefuran with 99.0% purity was obtained from Dr. Ehrenstorfer GmbH (Germany). R-dinotefuran and S-dinotefuran with 99.0% purity was obtained from Shanghai Chiralway Biotech Co., Ltd (Shanghai, China). Earthworms (Eisenia fetida) were purchased from a breeding company located in Qingdao (China). The earthworms were cultured at 20 ± 2°C with a 12/12-h photo-period and fed with cow manure. Healthy adults with clitellum (350 ± 20 mg) were used for toxicological testing after 2 weeks. The selected earthworms were kept on moist filter paper for 12 h to void their guts before experiment. The tested soil was artificial soil and consisted of 70% sand, 20% kaolin clay and 10% sphagnum peat moss.14

Acute toxicity tests. The acute toxicity test, including the filter paper contact test and artificial soil contact test were performed according to the standard method of the Organization for Economic Cooperation and Development (OECD).15 For the filter paper contact test, a glass tube without a cover (8×3 cm) was used. The filter paper was put into the glass tube of a suitable size without overlapping. The Rac-dinotefuran, R-dinotefuran and S-dinotefuran were dissolved in distilled water at 1.0 mg/mL. Then, 1 mL of different concentrations of three tested chemical solutions were added into the glass tube at final concentrations of 13.28, 6.64, 3.32, 1.66, 0.83,

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0.42, 0.21, 0.11, 0.06 and 0.03 mg/cm2. For the control group, 1 mL of distilled water was added into the glass tube. Thereafter, one gut-cleaned earthworm was put into every tube, which was then sealed with a plastic film with small breathing holes. The earthworms were cultured in the dark at 20 ± 2°C for 48 h, and then the mortality of the earthworms was quantified. For each treatment, 20 replicates were prepared. For the acute toxicity test in artificial soil, a 1000-mL glass beaker and 500 g of artificial soil were used. The appropriate amounts of the three tested chemical solutions (1.0 mg/mL) were added into the artificial soil. After blending sufficiently, ten gut-cleaned earthworms were put into the soil and cultured at 20 ± 2°C in continuous light. On the 14th day, the lethal concentration of 50% (LC50) values and the no observed effect concentration (NOEC) values were calculated according to the mortality of the earthworms. For each treatment, 5 replicates were prepared.

Subchronic toxic test. Based on the NOEC values, the concentrations were set at 1.0 mg/kg to study the subchronic toxicity of the three tested chemicals on earthworms. Twenty-five earthworms were prepared in each beaker to determine the growth inhibitory effect and residue of the three tested chemicals. Another twenty-five earthworms were prepared in each beaker to determine the biochemical and genetic toxicity. Earthworms were cultured according to the method as described above. On days 2, 7, 14, 28 and 42, the soil and earthworms were randomly sampled for various analyses. For each treatment, 5 beakers were prepared.

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Growth and reproduction inhibitory effect. The initial average weight of five randomly selected earthworms was recorded as W0. On days 2, 7, 14, 28 and 42, the average weight of five randomly selected earthworms was recorded as Wt. The changes in the weight were calculated according to the formula as follows: Weight change rate (%) =

୛౪ష ୛బ

× 100%

୛బ

After 42 d exposure, the soil was transferred to a 1-mm aperture sieve. The soil was washed slowly using the running water, and the earthworm juveniles and earthworm cocoons were collected.

Enantioselective degradation in soil and bioaccumulation in earthworms. The concentrations of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in the soil and earthworms were determined using high-performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS). For soil, the three tested chemicals were extracted from 2 g of soil using 2 mL of distilled water and 4 mL of acetonitrile. For earthworms, five earthworms were homogenized using distilled water (w/v, 1:2), and then the three tested chemicals were extracted using acetonitrile (w/v, 1:2). Subsequently, 0.5 g of NaCl and 2 g of MgSO4 were added into the soil and earthworm samples. The samples were vigorously shaken for 2 min and centrifuged at 4000 g for 5 min. Thereafter, 1.5 mL of the sample was purged using 50 mg of C18 and was filtered using a 0.22-µm syringe filter. The separation was performed on a Hypersil GOLD C18 column (Thermo; 2.1×100 mm, 3.0 µm) using acetonitrile (A) and 0.1% formic acid water (B) as the mobile

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phase. The gradient elution program started at 90% A (0-2.0 min), decreased to 10% A (2.0-3.0 min), held at 10% A (3.0-8.0 min), increased to 90% A (8.0-8.1 min) and held at 90% A (8.1-10.0 min). The flow rate was 0.25 mL/min, and the injection volume was 10 µL. The Rac-dinotefuran, R-dinotefuran and S-dinotefuran contents were measured using a triple-quadrupole mass spectrometer (Thermo TSQ Quantum Ultra; Thermo Fisher Scientific Inc., San José, CA, USA). The detection was performed in the multiple reaction monitoring mode with the positive electrospray ionization (ESI+). The capillary voltage and capillary temperature were 3.0 kV and 350°C, respectively. The qualitative ion pair and quantitative ion pair were 203.1/113.1 (m/z) and 203.1/129.1 (m/z) with collision energies of 6 eV and 11 eV, respectively. The Rac-dinotefuran, R-dinotefuran and S-dinotefuran were added to the soil matrix solution and earthworm matrix solution at final concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 mg/kg to validate the linearity of the method. Meanwhile, the three tested chemicals were added to the soil matrix and earthworm matrix at three spiked levels (0.01, 0.1 and 2.0 mg/kg) to validate the recovery of the method.

Determination of the oxidative damage effect. One earthworm from each beaker was randomly selected and homogenized in 100 mM phosphate buffer (pH 7.8). The sample was centrifuged at 3000 g for 5 min, and the supernatant was stored at 4°C. The reactive oxygen species (ROS) levels in earthworms were assayed using 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as the fluorescence probe.16

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The supernatant was re-centrifuged at 20000 g for 25 min and then was reacted with DCFH-DA (5 µM) for 20 min at 37°C. Subsequently, the fluorescence intensity of the reaction sample was assayed using a fluorescence spectrophotometer (F-4600; Hitachi, Japan). The malonaldehyde (MDA) contents were assayed according to the chromogenic reaction between MDA and thiobarbituric acid (TBA).16 The reaction mixture contained supernatants (200 µL), 20% acetate buffer solution (1.5 mL), 1% TBA solution (1.5 mL), distilled water (1 mL) and sodium dodecyl sulphate solution (200 µL). The absorbance of samples was measured at 532 nm. The protein carbonyl (PCO) contents were assayed according to the reaction between 2,4-dinitrophenylhydrazine (DNPH) and carbonyl groups.17 The protein in the supernatants was reacted with DNPH and denatured using 20% trichloroacetic acid (TCA). The protein precipitate was dissolved in guanidine hydrochloride solution and the absorbance was measured at 370 nm. The DNA damage degree was evaluated according to the olive tail moment (OTM) values of the comet assay.18 The coelomocytes were collected from one earthworm and were mixed with an equal volume of low-melting agarose (LMA, 0.5%). The mixture of coelomocytes and LMA was coated onto a layer of normal-melting agarose (NMA, 0.8%) on a microslide. Subsequently, a layer of LMA was coated onto the microslide again. The cell membrane was cracked in cell lysis buffer for 60 min, and then the DNA was uncoiled in electrophoretic buffer for 15 min. The microslides were stained with EB for 15 min after electrophoresing at 25 V (300 mA) for 15 min and

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were neutralized in Tris-HCl buffer (0.4 M, pH 7.5) for 15 min. Finally, the OTM values were determined using a fluorescence microscope (Olympus, BX71, Japan).

Determination of the gene expression levels. The relative expression level of functional genes was measured using real-time polymerase chain reaction (RT-PCR). The primer sequences of target genes are listed in Table 1.19 The total RNA was extracted from one earthworm using the RNApure plant kit (Aidlab, China). The purity and concentration of the extracted RNA were determined using the NanoDrop 2000 (Thermo Fisher Scientific, USA). Subsequently, an appropriate amount of RNA was used reverse transcribed into cDNA using the 1st strand cDNA synthesis kit (Aidlab, China). RT-PCR was performed on the ABI 7500 PCR System in a 25-µL reaction system according to a 2 × SYBR Green qPCR Mix kit (Aidlab, China).

Transcriptomic analysis. After exposure, the total RNA of the earthworms in each treatment was extracted using the RNApure plant kit (Aidlab, China). The purity and fragment length of the extracted RNA were determined using the NanoDrop 2000 (Thermo Fisher Scientific, USA) and Agilent 2100 (Agilent, USA). The mRNA was enriched from the total RNA using Oligo (dT) magnetic beads. Subsequently, the mRNA was fragmented using fragmentation buffer. The RNA fragments were used to synthesize the cDNAs using random hexamers, and the cDNAs were purified using AMPure XP beads. The cDNAs were modified through end repair, addition of “A” bases and addition of “T” bases. Finally, the fragment size of the cDNAs was selected

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and amplified, followed by sequencing on the Illumina HiSeq 4000TM platform (Illumina, Inc., SanDiego, CA, USA). The total read number of the test samples ranged from 35702602 to 44847044. The reference annotation used in the present study was the transcriptome which was reconstructed using the Trinity method. The reads were remapped according to the reference annotation using the RSEM software, and the ratio of total mapped reads ranged from 55.53% to 59.08%. The fragments per kilobase of transcript sequence per million fragment (FPKM) method was used to determine the gene expression levels, and the DESeq package (ver.2.1.0) was used to determine the differentially expressed genes. Significant differences in gene expression were determined using the threshold of p-value < 0.05 and |log2FoldChange| > 1. The functions of the differential expressed genes were annotated using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Statistical analysis. SPSS software (Version 18.0, SPSS Inc.) was used to conduct all statistical analyses, and all data were expressed as the means ± SD. One-way analysis of variance along with the LSD test was performed for multiple comparisons at the p < 0.05 level.

RESULTS AND DISCUSSION Put Figure 1 here Enantioselective degradation and bioaccumulation in soil and earthworms. As

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shown in Table S1 and Table S2, the detection method have good linearity and high recovery in both soil and earthworm matrixes. The R2 of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in soil matrix and earthworm matrix ranged from 0.9996 to 0.9999. The limit of quantitation (LOQ) of the three tested chemicals in soil matrix and earthworm matrix was 0.01 mg/kg. The recoveries of the three tested chemicals in soil matrix and earthworm matrix ranged from 89.1% to 99.5%. During the entire exposure period, the concentrations of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in the tested soil decreased with increasing exposure time (Fig. 1). The degradation of Rac-dinotefuran, R-dinotefuran and S-dinotefuran showed no obvious difference at the beginning of exposure. From the 7th day, R-dinotefuran degraded faster than Rac-dinotefuran and S-dinotefuran. S-dinotefuran degraded the slowest among the three tested chemicals in soil. Therefore, we believe that the stability of the three tested chemicals in the soil is S-dinotefuran > Rac-dinotefuran > R-dinotefuran. This result indicated that there is selective behaviour in the degradation between S-dinotefuran and R-dinotefuran. The decrease in the contents of the three tested chemicals may be induced by the photolysis, microbial degradation or the absorption of earthworms. In the present study, the accumulation of the three tested chemicals in earthworms was detected during the exposure period (Fig. 1B). The present results showed that the concentrations of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in earthworms increased with increasing exposure time. However, S-dinotefuran accumulated faster than Rac-dinotefuran and R-dinotefuran from the 2nd day. R-dinotefuran accumulated the

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slowest among the three tested chemicals in earthworms. A similar trend was also observed in the biota-soil-accumulation factors (BSAFs) result (Fig. S1). These results indicated that there is selective behaviour in bioaccumulation between S-dinotefuran and R-dinotefuran. Similar results were also reported by Ye et al.20 who studied the toxicity and bioaccumulation of fenvalerate and esfenvalerate in earthworms. In their study, there was selective behaviour in the degradation and bioaccumulation between fenvalerate and esfenvalerate. Previous results and the present result showed that the environmental behaviour of chiral pesticide isomers may be different.

Put Figure 2 here Enantioselective effects on the growth and reproduction of earthworms. Highly toxic substances can affect the growth of organisms, resulting in the death of organisms.20 In the present study, the mortalities using the filter paper contact test and soil contact test were increased with increasing concentrations of Rac-dinotefuran, R-dinotefuran and S-dinotefuran (Fig. 2A and 2B). Among the three tested chemicals, the mortalities of earthworms following S-dinotefuran treatments were higher than those

following

Rac-dinotefuran

treatments

and

R-dinotefuran

treatments.

Additionally, the mortalities of earthworms in S-dinotefuran treatments first reached 100%. For the filter paper contact test, the LC50 values for Rac-dinotefuran, R-dinotefuran and S-dinotefuran were 0.188, 0.698 and 0.050 µg/cm2, respectively. For the soil contact test, the acute toxicity of S-dinotefuran was 1.49 and 2.67 times

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that of the Rac-dinotefuran and R-dinotefuran. The LC50 values for the three tested chemicals were 4.885, 8.786 and 3.286 mg/kg, respectively. These results indicated that the toxicity sequence of the three tested chemicals was S-dinotefuran > Rac-dinotefuran > R-dinotefuran. In the present study, the growth and reproduction were seriously inhibited by Rac-dinotefuran, R-dinotefuran and S-dinotefuran after 42 d of exposure (Fig. 2C and 2D). At the beginning of exposure, Rac-dinotefuran and S-dinotefuran showed little influence on the weight change rate of earthworms. However, significant differences were observed from the 14th day compared with those of the control group (p < 0.05). For R-dinotefuran, significant differences in the weight change rate were observed from the 28th day compared with those of the control group (p < 0.05). For the reproduction of earthworms, the cocoon crop number and juvenile number following Rac-dinotefuran, R-dinotefuran and S-dinotefuran treatments were significantly decreased after 42 d of exposure compared with those in the control group (p < 0.05). Studies have shown that the weight and reproductive rate of earthworms would be seriously inhibited by highly toxic chemicals.20,21 Similar results were also reported by Wang et al.22 who studied the toxicity of a neonicotinoid insecticide, guadipyr, in earthworms. In their study, the growth and reproduction were also inhibited by guadipyr from 10 mg/kg.

Put Figure 3 here Enantioselective effects on the oxidative damage to earthworms. ROS are

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continuously generated in living organisms and play an essential role in intracellular signalling systems.23 However, environmental stress, such as drought, low temperature and toxic chemicals, could induce the excessive accumulation of ROS in cells, resulting in oxidative damage to cell components.24 In the present work, the ROS levels following S-dinotefuran exposure were markedly enhanced from the 7th day (Fig. 3A). Regarding Rac-dinotefuran and R-dinotefuran exposure, a significant increase in ROS levels were first observed on the 14th day and 28th days, respectively. Similar change trends were also observed in the MDA content, PCO content and DNA damage degree. Regarding the MDA content, significant increases were observed following S-dinotefuran and Rac-dinotefuran exposure from the 14th day (Fig. 3B). On the 28th day and 42nd day, the MDA content following R-dinotefuran exposure was also significantly higher than that of the control group. Regarding the PCO content, no significant differences were observed following Rac-dinotefuran, R-dinotefuran and S-dinotefuran exposure until the 14th day compared with that in the control group (p > 0.05, Fig. 3C). The PCO contents following Rac-dinotefuran, R-dinotefuran and S-dinotefuran exposure were markedly higher than those of the control group from days 28, 42 and 28, respectively (p < 0.05). For the DNA damage degree, significant differences were first observed on the 7th day following Rac-dinotefuran and S-dinotefuran exposure compared with that in the control group (Fig. 3D). For R-dinotefuran, a significant increase in the DNA damage degree was observed since from the 14th day. These significant changes in the MDA content, PCO content and DNA damage degree indicated that Rac-dinotefuran, R-dinotefuran and S-dinotefuran

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had caused oxidative damage in earthworms. Moreover, the differences in the ROS level, MDA content, PCO content and DNA damage degree induced by the three tested chemicals indicated that Rac-dinotefuran, R-dinotefuran and S-dinotefuran had shown selective behaviour in earthworms.

Put Figure 4 here Enantioselective effects on the function genes. Studies have shown that studying the influence of chemicals on the relative expression of functional genes could effectively reflect the toxicity of hazardous chemicals.25,26 SOD and CAT are important antioxidant enzymes, that could remove the excess superoxide anion-free radical (O2·-) and hydrogen peroxide (H2O2) in the cells of organisms.27 In the present work, the relative expression quantities of the SOD gene following Rac-dinotefuran and S-dinotefuran exposure were markedly higher than those following R-dinotefuran exposure and the control group at the beginning of exposure (Fig. 4A). However, the up-regulation trend following Rac-dinotefuran and S-dinotefuran exposure disappeared from the 28th day and 7th day, respectively. From the 28th day, the relative expression quantity of the SOD gene following S-dinotefuran exposure was significantly lower than that in the control group. Regarding R-dinotefuran, significant up-regulation of the SOD gene was observed on the 28th and 42nd days. For the CAT gene, there were no significant changes in the Rac-dinotefuran, R-dinotefuran and S-dinotefuran treatments on the 2nd day and 14th day compared with that in the control group (Fig. 4B). Significant up-regulation in the

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CAT gene following Rac-dinotefuran and S-dinotefuran treatments were observed on days 7, 28 and 42. For R-dinotefuran, significant up-regulation in the CAT gene was observed from the 28th day. The up-regulated SOD gene and CAT gene indicated that earthworms have begun to remove the excess ROS in cells. The down-regulated SOD gene and CAT gene may be due to excess ROS beyond the scavenging capacity of the antioxidant enzymes, resulting in cell dysfunction. TCTP is related to cell growth and division, which play an important role in organism growth.28 ANN is related to the reproduction of earthworms, which could induce egg-legging behaviour in earthworms.19,29 In the present work, the relative expression quantities of the TCTP gene and ANN gene following Rac-dinotefuran, R-dinotefuran and S-dinotefuran exposure showed no significant changes on the 2nd and 7th days compared with those in the control group (Fig. 4C and 4D). Significant up-regulation in TCTP gene and significant down-regulation in ANN gene were first observed on days 14, 28 and 14 after exposure to Rac-dinotefuran, R-dinotefuran and S-dinotefuran compared with those in the control group. The significant up-regulated expression quantity of the TCTP gene indicated that the growth and division of cells were inhibited. The significant down-regulated expression quantity of the ANN gene indicated that the reproduction of earthworm was inhibited. Accordingly, the present results also showed that the growth and reproduction of earthworms were significantly inhibited by the three tested chemicals (Fig. 2C and 2D). These results indicated that the three tested chemicals had growth and reproduction inhibitory effect on earthworms once again, especially the S-dinotefuran.

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Put Figure 5 here Enantioselective effects on the transcriptomic profiles. The sequencing data have been deposited into sequence read archive (SRA) of NCBI under BioProject accession PRJNA399235 (alias: SRP115867). As shown in Fig. 5, the number of differential expressed genes was 5983 between the Rac-dinotefuran-exposed treatments and control groups. Among them, the number of up regulated genes was 2414,

and

the

number

of

down-regulated

genes

was

3569.

Between

R-dinotefuran-exposed treatments and the control group, the number of differentially expressed genes was only 1555. Among them, the number of up-regulated genes was 807, and the number of down-regulated genes was 748. However, the number of differentially expressed genes between the S-dinotefuran-exposed treatments and control group was significantly higher than that of the other two groups. The number of up-regulated genes was 5795, and the number of down-regulated genes was 4891 among 10686 differentially expressed genes. The number of differentially expressed genes induced by S-dinotefuran was higher than the total number of the differentially expressed genes induced by Rac-dinotefuran and R-dinotefuran, indicating that S-dinotefuran had stronger influences on earthworms than Rac-dinotefuran and R-dinotefuran. Moreover, this result indicated that there is enantioselective toxicity in earthworms between R-dinotefuran and S-dinotefuran.

Put Figure 6 here

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Differential mechanism analysis between R-dinotefuran and S-dinotefuran using GO database. As shown in Fig. 6, 30 altered GO biological process terms are listed. For biological process, there are 5 altered GO biological process terms. Among them, only the number of differentially expressed genes in the protein metabolic process terms was more than 500, significantly higher than that in other terms. This result indicated that S-dinotefuran had a more serious influence on the protein metabolic process in earthworms than R-dinotefuran. For the cellular component, there were only 3 altered GO biological process terms, and none of them had more than 500 differentially expressed genes. For molecular function, there are 22 altered GO biological process terms, and 7 of these terms had more than 500 differentially expressed genes. Among them, the catalytic activity term had the largest number of differentially expressed genes, followed by the hydrolase activity term. These two terms are related to enzymes in earthworms, which belongs to the protein. Additionally, the numbers of differentially expressed genes in nucleotide binding, purine nucleotide binding, ribonucleotide binding, purine ribonucleotide binding and purine ribonucleotide triphosp terms were also more than 500. These 5 terms are related to nucleic acids in earthworms. Previous studies have shown that enzymes, proteins and nucleic acids, had chiral structures, which might induce the selective behaviour of enantiomers based on their different spatial configurations.4,20,30,31 Therefore, we believe that biomacromolecules in earthworms, such as enzymes, proteins and nucleic acids, induced the selective behavior between S-dinotefuran and R-dinotefuran, resulting in S-dinotefuran had stronger interactions to these

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biomacromolecules.4 Finally, this strong interaction damaged the normal functions of these biomacromolecules, leading to selective toxic effects on earthworms.

Put Figure 7 here Differential mechanism analysis between R-dinotefuran and S-dinotefuran using KEGG database. As shown in Fig. 7, 20 of the most significantly enriched pathways are listed. Among these 20 pathways, the enrichment degree and the number of differentially expressed genes in protein processing in the endoplasmic reticulum pathway was the most. The endoplasmic reticulum (ER) is a subcellular organelle that could be divided into the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER).32 The RER could synthesize proteins and transfer them to every position of the cell.33 In the present study, a total of 8 protein metabolism-related pathways were seriously influenced among the total 20 pathways, likely because the function of RER was damaged. The SER could participate in the synthesis of carbohydrates, alcohols and lipids, which has more complex functions than the RER.32 In the present study, 2 alcohol metabolism-related pathways, 3 carbohydrate metabolism-related pathways and 3 lipid metabolism-related pathways were seriously influenced among the 20 pathways, which may be due to the function of SER being damaged. Additionally, SER could inactivate the toxic substance and detoxify them through the liver.34,35 In the present study, 2 cytochrome P450 metabolism-related pathways were seriously influenced. Cytochrome P450 is a type of terminal oxygenase that is mainly distributed in the ER and mitochondria.36 Studies

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have shown that cytochrome P450 is the key enzyme in the metabolic process of toxic substances.37 That the cytochrome P450 metabolism pathways were influenced indicated that the detoxification capability of earthworms was inhibited, possibly because the function of SER was damaged. Additionally, the damage in the function of ER indicated that the metabolism of energy in earthworms may be influenced. Studies have shown that the ER membrane is closely connected with the outer mitochondrial membrane, whose connected region is called the mitochondriaassociated membrane (MAM).38,39 The mitochondria are the main energy metabolism sites in the cells, called “power houses”.39 Moreover, the joint action between the ER and mitochondria plays an important role in regulating calcium signals, lipid synthesis, ER stress and cell apoptosis.40 The abnormal function of the ER could induce damage in the MAM region, leading to an influence on the function of mitochondria, metabolism of energy and function of cells.41 Additionally, the mitochondria are the main organelles to generate ROS.42 In the present study, the ROS levels in earthworms changed significantly after exposure to the three tested chemicals, especially after exposure to S-dinotefuran, which may be due to the function of ER being damaged. In the present study, S-dinotefuran has stronger interactions to biomacromolecules, such as carbohydrate, lipid and protein, resulting in the functions of ER were destroyed. Meanwhile, the damage in ER function caused the abnormal in synthesis and metabolism of these biomacromolecules, finally leading to the growth and reproduction of earthworms were influenced. However, the reason that S-dinotefuran

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had stronger interactions to biomacromolecules should be studied deeply in the future. In

conclusion,

the

present

results

showed

that

there

were

significant

enantioselectivities between R-dinotefuran and S-dinotefuran in degradation, accumulation and toxicity. S-dinotefuran posed a higher risk in the soil environment to earthworms than Rac-dinotefuran and R-dinotefuran. Moreover, the present results indicated that the risk assessment of dinotefuran should be evaluated at the enantiomer level.

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ACKNOWLEDGMENTS The present study was supported by grants from the National Natural Science Foundation of China [No. 41771352] and the Agricultural Science and Technology Innovation Program [No. ASTIP-TRIC04].

Notes The authors declare no competing ﬁnancial interest.

Supporting Information The results of method validation and the BSAFs for Rac-dinotefuran, R-dinotefuran and S-dinotefuran. This material is available free of charge via the Internet at http://pubs.acs.org.

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Table legends Table 1 Primer sequences used for quantitative real-time PCR analysis.

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Figure captions Fig. 1 Residues of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in the artificial soil (A) and earthworms (B). Bars are the means ± SD of five replicates. Fig. 2 Dose-effect curves for the filter paper contact test (A) and soil contact test (B), and inhibitory effects on the weight (C) and reproduction (D) of earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments. Fig. 3 Effects of Rac-dinotefuran, R-dinotefuran and S-dinotefuran on the ROS level (A), MDA content (B), PCO content (C) and DNA damage degree (D) in earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments. Fig. 4 Effects of Rac-dinotefuran, R-dinotefuran and S-dinotefuran on the relative expression levels of the SOD (A), CAT (B), TCTP (C) and ANN (D) genes in earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments. Fig. 5 The volcanic figure of differentially expressed genes after exposure to Rac-dinotefuran, R-dinotefuran and S-dinotefuran compared with the control group. Fig. 6 The function annotation of differentially expressed genes using GO database between S-dinotefuran and R-dinotefuran. Fig. 7 The function annotation of differentially expressed genes using KEGG database between S-dinotefuran and R-dinotefuran.

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List of Table Table 1 Primer sequences used for quantitative real-time PCR analysis. Gene

Primers sequences (5′-3′) F

TCCATCGTCCACAGAAAG

R

AAATGTCCTCCGCAAGCT

F

TGCTCACTTCAACCCATTT

R

TTGGCAACACCACTTTCA

F

CATTGCGGATGGAAACTA

R

TTCGGATTACGATTGAGA

F

TTTCTTCCGCCTGCTTTG

R

ACCGACCTACCACCGACA

F

TCGAATATGCCCTCAGCA

R

TGGACTCGCCACAGAAGA

β-actin

Accession no. GU177854

SOD

GU177856

CAT

GU177857

ANN

GU177859

TCTP

GU177860

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List of Figure

Fig. 1 Residues of Rac-dinotefuran, R-dinotefuran and S-dinotefuran in the artificial soil (A) and earthworms (B). Bars are the means ± SD of five replicates.

33



Fig. 2 Dose-effect curves for the filter paper contact test (A) and soil contact test (B), and inhibitory effects on the weight (C) and reproduction (D) of earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments.

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Fig. 3 Effects of Rac-dinotefuran, R-dinotefuran and S-dinotefuran on the ROS level (A), MDA content (B), PCO content (C) and DNA damage degree (D) in earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments.

35



Fig. 4 Effects of Rac-dinotefuran, R-dinotefuran and S-dinotefuran on the relative expression levels of the SOD (A), CAT (B), TCTP (C) and ANN (D) genes in earthworms. Bars are the means ± SD of five replicates. Different letters indicate significant differences (p < 0.05) between treatments.

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Fig. 5 The volcanic figure of differentially expressed genes after exposure to Rac-dinotefuran, R-dinotefuran and S-dinotefuran compared with the control group.

37



Fig. 6 The function annotation of differentially expressed genes using GO database between S-dinotefuran and R-dinotefuran.

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Fig. 7 The function annotation of differentially expressed genes using KEGG database between S-dinotefuran and R-dinotefuran.

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TOC Graphic

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Enantioselective bioaccumulation and toxicity of the neonicotinoid

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