Effects of mixed application of avermectin, imidacloprid and carbendazim on soil degradation and toxicity toward earthworms

The application of pesticides in mixtures often exerts multiple pressures on agricultural soils in the short term. Therefore, it is necessary to assess the effects of mixed application on the environmental behavior and ecotoxicity of pesticides in soil. In this study, we assessed the effects of three common pesticides through mixed application on soil degradation and toxicity toward the earthworm Eisenia fetida. Compared with the degradation half-lives (DT50) the single pesticide, the DT50 values of avermectin, imidacloprid and carbendazim in the binary mixtures were similar. However, their DT50 values in the ternary mixtures were approximately 1.5 times longer than those in the individual applications, enhancing their stable in soil after two or three applications. The ternary mixtures of the pesticides showed significantly synergistic toxicity toward E. fetida, while their binary mixtures exhibited a changing interaction throughout the entire effect level range. The ternary mixtures activated higher SOD and CAT activities in E. fetida than the individual treatments, confirming their synergistic effects. By conducting avoidance tests with E. fetida, ternary toxic interactions were effectively assessed within a relatively short testing period. In summary, the three pesticides in ternary mixtures exhibited longer degradation half-lives and synergistic toxicity toward earthworms compared to individual or binary mixtures.

Field trial.The residue study of these three pesticides and their mixtures was carried out in the field at the experimental base at Hainan University (Haikou, China).The trial areas had no history of the use of the three pesticides.A 30-m 2 zone (10 m × 3 m) was used as a treatment plot, and another 30-m 2 zone was defined as a control according to the guidelines of the pesticide analytical method of China 19 .A 5-m 2 protective barrier was established between the plots.Each treatment and control (sprayed with water) were designed with three replicates.The three pesticides were applied as a spray in the single and combined treatments.The application concentration of each pesticide product was two times the dose recommended by the company (60 ml/ha for avermectin treatment, 40 ml/ha for imidacloprid treatment, and 200 g a.i./ha for carbendazim treatment).Each dose was sprayed three times at an interval of 10 d for the single and combined treatments.Representative soil samples were collected at 2 h and 1, 2, 3, 7, 10, 14, 21, and 28 days after the first application.Additional soil samples were collected 14 days after the second and third application.All soil samples were stored at − 20 °C until use.

Sample preparation and extraction.
Ten grams of soil were put into a 100-mL centrifuge tube, to which 30 mL acetonitrile was added and vortexed for 2 h.Then, 4 g of anhydrous MgSO 4 and 2 g of NaCl were put into the tube, followed by shaking for 2 min.The tube was centrifuged at 4000 r/min for 5 min.After that, an aliquot of the upper organic layer was collected and dried by a rotary evaporator.The extract residues were redissolved in 2 mL of methanol and filtered with a 0.22-μm nylon syringe filter for high-performance liquid chromatography (HPLC) analysis.

HPLC analysis.
Chromatographic separation was performed on a Waters e2695 HPLC (Waters Associates, USA) with a TechMate C18-ST (5 μM, 4.6 × 250 mm) column (TechMate Technology Co., Ltd.).To detect avermectin, imidacloprid and carbendazim, methanol, water and acetonitrile were used as mobile phase A, mobile phase B and mobile phase C, respectively.A ternary solvent system in gradient mode was operated with a flow rate of 1 mL/min.The linear mobile phase gradient started at 35% A, 45% B and 20% C (0-8 min) and was then maintained at 95% A and 5% B (8-18 min).The column was kept at 30 °C with the photodiode array detection at 245 nm.
Validation and calculation method.The method linearity was evaluated with a series of calibration standards.Ten microliters of the working standard solutions were injected into the HPLC system, and elution was carried out as described above.The calibration curves were prepared by plotting the peak area versus the concentration of the standard compound.The reproducibility of the method was determined by the intra-and interday precisions.The intra-and interday relative standard deviations (RSDs) were calculated with the pesticides spiked at three different concentrations (0.2, 0.5 and 1 mg/kg) in soil.The limit of quantification (LOQ) Enzyme activity assays.The enzyme activity test was performed according to the method described by Chen et al. 23 .Briefly, the exposure concentration of each pesticide was 10% of the LC 50 value of the acute toxicity of the artificial soil test.The joint toxicity concentration of three pesticides was conducted at an equal-toxic proportion (1:1:1), which was set as 3.3% of the LC 50 value of each pesticide in the mixture.The exposure method of the earthworm E. fetida was the same as the artificial soil test.After 14 days of exposure, three earthworms were collected, rinsed with distilled water and then weighed.The earthworms were homogenized manually in a vitreous tissue homogenizer with 5 ml phosphate buffer (pH 7.4) and were centrifuged at 3000 r/min for 10 min at 4 °C.The supernatants were collected to determine protein contents and enzyme activities.The protein concentration was measured using a Bradford protein assay kit (Sangon Biotech Co., Shanghai, China).Superoxide dismutase (SOD) activity was determined by inhibiting the photochemical reduction of nitroblue tetrazolium (NBT) as described by Beauchamp and Fridovich 24 .One unit was considered the amount of enzyme that inhibited NBT reduction by 50%, and the results were expressed as U per mg protein.The catalase (CAT) activity was determined by the rate of decomposition of H 2 O 2 , which was evaluated by the decrease in absorbance at 240 nm and expressed as U per mg protein 25 .
Avoidance test.The avoidance test was designed according to ISO guidelines 26 .Five tested concentrations were prepared based on the LC 50 values from the acute tests (Tables S2, S3).All test treatments were conducted with three replicates.A two-choice chamber was used for the test.One half of the chamber was filled with 300 g of treated soil, and the other half was filled with 300 g of control soil.Ten earthworms were then placed in the middle of the chamber.The test was performed in the dark for 48 h at 20 °C.Then, the divider was reinserted, and the number of individuals on each side of the container was recorded.Individuals found in the middle of the sections were counted as 0.5 for each side.The avoidance rate A (%) was calculated according to the equation (A = [(C − T)/10] × 100, where C = earthworms observed in the control soil and T = earthworms observed in the treated soil).

Statistical analysis.
Statistical analysis was conducted using the statistical software package SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA).The differences between the pesticide-treated and control groups were determined with Student's t test and Fisher's least significant difference (LSD) test.P values below 0.05 were considered statistically significant.

Results
Assay validation.The residual analysis method for avermectin, imidacloprid and carbendazim in soil was established and validated.The baseline separation of the three pesticides was determined with an HPLC system containing a C18 column (Fig. S1).Good linearity was achieved within the concentration range of 0.02-20 mg/L with linear equations y = 22804x + 50.46 (R 2 = 0.9997), y = 51339x + 706.75 (R 2 = 0.9993), and y = 26858x + 108.92 (R 2 = 0.9995) for avermectin, imidacloprid, and carbendazim, respectively.The LOQ values for these pesticides in soil were all below 0.01 mg/kg.The average recoveries of the three pesticides from soils were between 80-104% with relative standard deviations of 2.0-10.1% (Table S4), indicating repeatability of the method.

Degradation of the binary and ternary mixtures of the three pesticides in soil. As shown in
Fig. 1 and Table 1, the dissipation of avermectin, imidacloprid, carbendazim and their mixtures in soils followed first-order kinetics with high correlation coefficients (R 2 ≥ 0.9132).The degradation half-life values of the pesticides ranged from 4.10 to 10.19 days under the individual and combined applications.Compared with the DT50 the single pesticide, the DT50 values of avermectin, the DT50 values of these pesticides in the binary mixtures were similar.However, their DT50 values in the ternary mixtures were approximately 1.5 times higher than those in the single-use group.We further applied solutions of avermectin, imidacloprid, carbendazim and their ternary mixture either two or three times in soil, and the pesticide residue amounts at 14 days after application are shown in Fig. 2. The amount of residues of avermectin, imidacloprid and carbendazim in soil with a single application were 0.086-0.091mg/kg, 0.113-0.122mg/kg and 2.350-2.386mg/kg, respectively.The amount of residues of avermectin, imidacloprid and carbendazim in soil with mixed application were 0.117-0.125mg/kg, 0.124-0.142mg/kg and 2.646-2.982mg/kg, respectively.The amount of residues of these pesticides after mixed application were significantly higher than those after application alone.
Joint acute toxicity of the three pesticides to the earthworm E. fetida.The CI-isobologram method was used to determine the nature of the toxicological interactions among avermectin, imidacloprid and carbendazim toward the earthworm E. fetida by the acute toxicity test.Table 2 shows the dose-effect curve parameters (Dm, m, and r) of the individual pesticides and their mixtures, as well as the mean CI values of the mixtures.Dm is the median lethal concentration and equals the LC 50 value 26 .The Dm values of avermectin, imidacloprid and carbendazim were 26.83, 3.18 and 8.46 mg/kg, respectively.All dose-effect curves were sigmoidal (m > 1) and conformed to the median effect principle (r > 0.93).CI values plotted as a function of the mortality rate (f a ) shows the types of interaction (synergism, antagonism and additive effect) as a function of the level of www.nature.com/scientificreports/ the effect of pesticide mixtures toward E. fetida (Fig. 3).The binary mixtures of the pesticides showed changing interactions throughout the entire effect level range.In contrast, stable synergistic responses were noted for their ternary mixtures with CI values less than 1.
Joint toxicity of the three pesticides on the SOD and CAT activities of E. fetida.To further confirm the joint acute toxicity of avermectin, imidacloprid and carbendazim to E. fetida, the effects of the three pesticides and their mixtures on two antioxidative enzyme activities of E. fetida were investigated, and the results   Table 2. Dose-effect parameters and CI values of the three pesticides and their binary and ternary mixtures on acute toxicity toward Eisenia fetida.a The parameters Dm, m, and r represent the potency (LC 50 ), the linear correlation coefficient of the median-effect plot, and the accordance of data to the mass-action law, respectively.b CI < 1, CI = 1, and CI > 1 mean synergism (Syn), additive effect (Add), and antagonism (Ant), respectively.LC 10 , LC 50 and LC 90 represent the doses causing 10, 50, and 90% mortality rate of Eisenia fetida, respectively.4. SOD activity was activated with the single pesticide, while CAT activity showed no change.

Chemicals and mixtures (ratio)
In contrast, a considerable increase in SOD and CAT activities was found under the ternary treatment.

Joint toxicity of the three pesticides on the avoidance behavior of E. fetida.
To explore a method for assessing joint toxicity within a shorter test period, we evaluated the avoidance behavior of E. fetida in response to the three pesticides and their ternary mixture.Our results showed a significant difference between individual and combined applications at LC 50 values of 0.5% and 1%.There was a positive correlation between the toxicity of these pesticides and the avoidance behavior of E. fetida.The number of individuals observed in the control soil was greater than 80% in the treatment, which means that this soil has a limited habitat function and is unsuitable for this species 27 .The concentrations of avermectin, imidacloprid, carbendazim, and their ternary mixture were 10%, 5%, 5%, and 1% of the LC 50 values, respectively, with approximately 80% of the individuals avoiding the treatment (Fig. 5).The avoidance effects increased considerably under combined treatments of the three pesticides compared to the individual treatments.

Discussion
Mixtures of pesticides in agricultural fields are common.Insecticides and fungicides are widely applied together as a combined pesticide to control insect pests and plant diseases simultaneously 28 .The current risk assessment is based on a single chemical, which is not effective in evaluating its environmental behavior and ecotoxicology in the natural system because of the compound interaction in a mixture system 29 .In this study, we assessed the effects of three common pesticides through mixed application on soil degradation and toxicity towards earthworm.
Degradation is an important environmental behavior of pesticides in soil, which is directly related to the residence time and the degree of impact on the soil environment 30 .In this study, we investigated the dissipation dynamics and residues of avermectin, imidacloprid and carbendazim and compared the differences between their individual and combined applications.All dissipation of the three pesticides and their binary and ternary mixtures in soils followed first-order kinetics.The degradation half-life values of the single and mixed pesticides were not higher than 10.19 days.According to the Chinese National Standard GB/T 31270.1-2014,all these pesticides are easily degraded pesticides (half-life values < 30 d) under both individual and combined applications.However, the DT50 values of the three pesticides in the ternary mixtures increased considerably compared with those in the individual application, which led to higher residues in soil after several applications.This is in accordance to the scientific literature, which states that mixed application prolongs the degradation time of certain pesticides in soil.For example, the fungicide chlorothalonil significantly inhibits the degradation of the herbicide metolachlor in soil, and the inhibition rate is up to two times 31 .The fungicide mancozeb and its mixture with the insecticide thiamethoxam significantly suppress the degradation of the herbicide pendimethalin in soil 2 .A possible reason may be that microbial degradation of a compound in a mixture can be strongly impacted by other compounds in the mixture 32,33 .Thus, the combined application of pesticides might suppress the degradative activity of soil microorganisms.Our hypothesis is that the ternary mixture of avermectin, imidacloprid and carbendazim significantly hampers the survival of essential microorganisms responsible for degrading these pesticides, leading to a substantial decline in pesticide degradation efficiency.However, the underlying mechanism still needs further exploration.
Earthworms are often regarded as a key indicator species for ecotoxicological assessments of soil pollution because of their high biomass and ecological importance in soil 34 .To obtain information about the ecotoxicological effects of avermectin, imidacloprid and carbendazim in soil, the earthworm E. fetida was exposed to artificial soil containing each pesticide or their mixture, and the acute toxicity was measured.The LC 50 values of the three pesticides are consistent with previous reports, which showed that avermectin was classified as low toxic, while imidacloprid and carbendazim were classified as moderately toxic 10,12 .However, some reports indicate that imidacloprid is highly toxic to earthworms and is prohibited for both seed coating and soil treatment within the European Union 35,36 .The LC 50 values of their binary and ternary mixtures fall between the maximum and minimum LC 50 values of the three pesticides, indicating that the combined use balances their respective toxicities.The CI equation method has been widely used to calculate the predictions of mixture toxicity 15,20,37 .In this study, the joint toxicity of the three pesticides was determined with the CI model and visualized by isobolograms.The ternary mixture of the pesticides showed stable synergistic behavior throughout the entire effect level range.Thus, these multicomponent mixtures might pose a larger threat to soil organisms.Two antioxidative enzymes, SOD and CAT, have been used as important biochemical biomarkers of exposure to pollutants that trigger oxidative stress 38 .We evaluated the toxicity of the ternary mixture towards E. fetida by measuring the enzyme activities.The ternary mixture elicited a significant increase in SOD and CAT activities compared to individual treatments at equitoxic concentrations.The enzyme test indicated that treatment with the ternary mixtures www.nature.com/scientificreports/had more adverse effects on the survival of E. fetida than the individual treatments.These results also indicated that the co-occurrence of these pesticides caused more negative effects on the soil organisms than expected.In recent years, a growing body of research has shown that the toxicity effects of pesticide mixtures on nontarget organisms are synergistic [39][40][41] .The predicted synergism in most pesticide mixtures at low-effect levels indicates a potential ecotoxicological risk associated with the co-occurrence of these chemicals even at low concentrations 15 .
The synergistic effects of these pesticides should be of great concern to regulatory authorities and the public.It is necessary to reconsider the current procedures for determining the quality of soils and effects of chemicals on behavior, which likely underestimate the negative effects of chemical mixtures.The earthworm mortality test is frequently used in current standardized laboratory test systems.The test provides information on the toxic effects of contaminants on exposed organisms but does not offer firsthand insight into the reactions and behaviors of these organisms when exposed to contaminants in the soil 42 .Earthworms are also a suitable early warning system due to their rapid response to both natural and anthropogenic stressors 43 .Various chemicals have been studied for toxicity by earthworms using avoidance test [44][45][46] .In addition, the mortality test requires a longer period and is much more labor-intensive than the avoidance test.Therefore, the avoidance test is a promising candidate for a short-time screening test to determine the joint toxicity of chemicals toward earthworms.In this study, there was a positive correlation between the toxicity of pesticides and the avoidance behavior of E. fetida under both individual and combined applications.Some studies have shown that the avoidance behaviors toward pesticides at concentrations in a range are similar to the acute test results 17,47 .Therefore, the avoidance test is useful as a screening tool for the preliminary assessment of pesticides and their mixtures in soils.

Conclusion
In this study, we determined the effects of mixture interactions of avermectin, imidacloprid and carbendazim on their degradation in soil and toxicity toward E. fetida.Their ternary combinations significantly increased the DT50 of each pesticide.The ternary mixtures of these pesticides showed stable synergistic toxicity toward the earthworms.Our findings contribute to understanding the complexity of effects from pesticide mixtures on non-target organisms and provide useful information about the interactions of pesticides in soil.

Figure 1 .
Figure 1.Dissipation of avermectin (AVE), imidacloprid (IMI) and carbendazim (CAR) individually and combined in soil.Solid lines show the first-order kinetics results.Values are expressed as the means ± standard errors (SEs) of three replicates.

Table 1 .
Degradation parameters of the three pesticides used individually or in combination in soil.a The three pesticides were applied as a foliar spray in the single and combined treatments.b Fold change = Half-lives combined used in soil/ half-lives individual used in soil.

Figure 2 .
Figure 2. Residues of avermectin, imidacloprid and carbendazim used individually and in combination in soil.Soil samples were collected 14 days after two or three applications of each treatment.Values are expressed as the means and SEs of three replicates.Statistical analyses were performed using Student's t test: *P < 0.05.

Figure 3 .
Figure 3. Combination index plot for binary and ternary mixtures of the three pesticides in the artificial soil acute toxicity test.CI values are plotted as a (f a ) of the earthworms by computer simulation.CI < 1, = 1 and > 1 indicates synergism, an additive effect and antagonism, respectively.

Figure 4 .
Figure 4. Effects of avermectin, imidacloprid, carbendazim and their ternary mixture on SOD activity and CAT activity in E. fetida.Values are the means and SEs of three replicates.Means with different letters are significantly different according to the LSD test (P < 0.05).

Figure 5 .
Figure 5. Avoidance responses of E. fetida to avermectin, imidacloprid, carbendazim and their ternary mixture.Values are expressed as the means and SEs of three replicates.

-effect parameters a CI values b Dm (mg/kg) m R LC 10 LC 50 LC 90
are shown in Fig.