Persistence, dissipation and dietary risk assessment of sulfoxaflor in wheat agro-ecosystem under tropical climatic conditions

ABSTRACT Sulfoxaflor is a systemic sulfoximine insecticide, used to control sucking pests in different agricultural crops including wheat. However, the information regarding its residual fate in wheat agroecosystem under tropical environment and the associated dietary risk are still obscure. Therefore, a supervised field experiment was conducted in wheat comprising the application of sulfoxaflor 12% (w/v) SC at 30 and 60 g a.i. ha−1 in two consecutive seasons. An optimised method for estimation of sulfoxaflor residues using modified QuEChERS technique was developed and validated. The extracting solvent ethyl acetate + cyclohexane (9 + 1 v/v) along with the clean-up combinations viz. 75 mg PSA+25 mg GCB+150 mg magnesium sulphate for wheat plant and straw and 75 mg PSA+75 mg C18 + 150 mg magnesium sulphate for grain produced satisfactory recovery, minimum matrix interference and optimum peak shape. The average recovery, repeatability and within-laboratory reproducibility of sulfoxaflor in wheat substrates ranged between 90.66% and 103.55%, 2.67–5.17% and 3.05–8.15%, respectively, and the limit of quantification was estimated 0.01 mg kg−1. Sulfoxaflor was stable up to 60 days in wheat matrices under storage (−20°C). The dissipation of sulfoxaflor in wheat plants followed first-order kinetics and the overall shorter persistence in Season I and II ranged between 1.05–1.14 and 2.44–2.47 days (T1 – T2), respectively. The deviation in the persistence of the compound between two seasons due to variation in climatic parameters was observed to be statistically significant. The estimated pre-harvest interval of sulfoxaflor ranged between 4.95–5.34 (Season I) and 11.37–11.55 days (Season II). Besides, the terminal residues of sulfoxaflor were below quantification limit in the harvested wheat products, and the estimated long-term dietary risk of sulfoxaflor was observed to be negligible. Therefore, it was expected that the application of this insecticide in wheat cultivation should pose no residual toxicity and the harvested wheat products can be considered safe for human consumption.


Introduction
Wheat (Triticum aestivum L.), popularly known as the 'king of cereals', is an integral part of Indian food habits.Consumption of wheat serves an excellent source of energy and numerous health promoting substances, like: flavonoids, alkylresorcinols, phenolic acid, vitamins, carotenoid, phytosteroid and dietary fibre etc.These bioactive components exert protective effects against various chronic diseases, such as: cardio-vascular diseases, colorectal cancer, type-2 diabetes and obesity [1].India is the second-largest wheat producing country, securing an annual production of 103.596 million tones [2].But, wheat production in India is severely affected in terms of quality and quantity due to pest infestation, especially aphids [3].The aphid infestation is more prominent during cold and cloudy winters, reducing the yield of wheat to the extent of 10-50% [4].
With the advantages of greater efficacy, faster action and less time and labourintensive attributes, insecticides offer an economic approach to the Indian farmers for management of insect pest population.Sulfoxaflor ([methyl -oxo -[1 -[6 -(trifluoromethyl) pyridin -3 -yl] ethyl] -λ 6 -sulfanylidene] cyanamide), a novel insecticide, is used to effectively control various aphid complexes in wheat under Indian agro-climatic conditions with no phytotoxicity symptoms and minimal adverse effects to the natural enemies [4].Besides, sulfoxaflor is also reported to offer significant efficacy against broad spectrum of sap feeders viz.aphids, whiteflies and hoppers in wheat, barley, leafy and fruiting vegetables, soybean, tubers, oilseeds and fruit crops [5].The molecule belongs to the sulfoximine chemical class and acts as nicotinic acetylcholine receptor (nAchR) competitive modulator by producing excitatory nerve responses in the target insect, followed by paralysis and ultimately death [5].
The growing concern regarding consumer health protection necessitates the critical evaluation of pesticide residues in agricultural commodities.Pesticides, though significantly contributing to global food security via minimising pest interference, may pose threat to the food safety because of their intrinsic toxic properties [6].Insecticides, which are introduced directly to the crop canopy, may be absorbed within plant matrix [7] and may remain stable depending on the matrix composition, physico-chemical properties of the active ingredient (a.i.) and environmental condition [8].The molecules may contaminate agricultural products by exceeding the maximum residue limit (MRL), thereby inviting trade barriers as a consequence of failing to satisfy stringent food safety regulations of national and international markets and numerous human health problems.For example, sulfoxaflor is reported to produce potent toxicological effects, causing developmental, neuromuscular and reproductive abnormalities; decreased neonatal survival and hepatotoxicity [5].Therefore, to perceive the environmental fate of pesticides, characterisation of its persistence and dissipation behaviour in the natural agroecosystem is crucial.So far, only a few studies have investigated the fate and dissipation kinetics of sulfoxaflor in various matrices viz.cucumber [9], greenhouse vegetables [10], Asian pear and oriental melon [11], spinach and Korean cabbage [12] and puer tea [13].However, no data exists regarding the dissipation of sulfoxaflor in wheat and its food safety assessment under Indian tropical agro-climatic conditions.Besides, the degradation of a pesticidal molecule is also reported to vary depending upon the seasonal variability [11].
Therefore, a two season field dissipation study was conducted in wheat to assess the persistence and degradation behaviour of sulfoxaflor in the wheat agroecosystem.The terminal residues of sulfoxaflor in wheat co-products viz.wheat grain and straw were determined at harvest and the dietary risk was also estimated to predict the impact of the toxicant on consumer health.

Chemicals and reagents
Both the analytical standard of sulfoxaflor with 99.7% purity and the formulation (sulfoxaflor 12% (w/v) SC) were supplied by Dow AgroSciences India Pvt. Ltd.The organic solvents used in the analysis viz.acetonitrile, ethyl acetate and cyclohexane were purchased from J.T. Baker, Avantor, USA.The salts of ACS grade viz.magnesium sulphate, sodium sulphate and sodium chloride were purchased from Merck Life Sciences Pvt. Ltd., Germany.The dispersive clean-up reagents, that is, primary secondary amine or PSA and C18 were obtained from Agilent Technologies Inc., USA, and graphitised carbon black (GCB) was procured from United Chemical Technology, Bellefonte, PA, USA.

Field experimental design
A supervised field trial of sulfoxaflor 12% (w/v) SC on wheat (variety: Sonalika) was carried out in two consecutive Rabi seasons viz.2016-17 and 2017-18 at the Central Research Farm of Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal, India (altitude 12 m, latitude 22ᵒ59' N, longitude 88ᵒ27' E).The insecticide formulation (sulfoxaflor 12% (w/v) SC) was applied on 4 January 2017 in Season I and 4 January 2018 in Season II following two treatment doses viz.30 g a.i.ha −1 (T1) and 60 g a.i.ha −1 (T2).Each treatment (T1 and T2) along with the untreated control (T3) were replicated thrice, and the samples were collected at 0 (2 hr after application), 1, 2, 3, 5, 7, 10 and 15 days after application.Besides, the wheat products (viz.wheat grain and straw) were also collected at harvest for the estimation of end-point residues of sulfoxaflor.The meteorological data of both the seasons including maximum and minimum temperature (ᵒC), relative humidity (%), total rainfall (mm), cloud octa and bright sunshine hour were acquired from the regional meteorological station of Bidhan Chandra Krishi Viswavidyalaya, Nadia, West Bengal, India and UV index was obtained from the Tropospheric Emission Monitoring Internet Service (TEMIS) satellite system (Figure 1).

Extraction and clean up of sulfoxaflor
Representative samples of wheat plant (2 g), grain (2 g) and straw (2 g) were taken in 50 mL polypropylene centrifuge tubes separately.Each sample was added with 10 mL milli-Q water and vortexed for 1 min.A solvent mixture (10 mL) of ethyl acetate + cyclohexane (9 + 1 v/v) was added and subjected to vortex for 1 min.The salt mixture of sodium sulphate (5 g) + sodium chloride (1.5 g) was added afterwards and mixed thoroughly for 1 min., followed by rotospin at 50 rpm for 10 min.Then the samples were centrifuged at 5000 rpm for 10 min.and the supernatant liquid (4 mL) was collected.The extracts of wheat plant and straw were cleaned up by transferring 2 mL aliquot into a micro-centrifuge tube pre-filled with 75 mg PSA, 25 mg GCB and 150 mg magnesium sulphate.Whereas, for wheat grain samples, a combination of 75 mg PSA, 75 mg C18 and 150 mg magnesium sulphate clean-up reagents was used.The tubes were vortexed for 1 min., followed by centrifugation at 10,000 rpm for 10 min.The cleaned extracts (1.5 mL) were evaporated to dryness and reconstituted with 1.5 mL acetonitrile.Finally, the samples were filtered through a 0.2 μm membrane filter and analysed in LC-MS/MS for sulfoxaflor residues.

Recovery experiment
The recovery experiment for sulfoxaflor was conducted in the wheat substrates viz.wheat plant, grain and straw by fortifying separately at 0.01, 0.05 and 0.10 mg kg −1 concentration levels in order to validate the acceptance of the optimised analytical method.A calibration curve comprising 0.0015, 0.003, 0.01, 0.02, 0.05, 0.1, 0.25, 0.50 and 1.0 mg kg −1 concentration levels was used for quantification of sulfoxaflor residues in wheat matrices.

Storage stability experiment
Since pesticides are prone to degradation upon exposure to environmental factors, such as temperature, humidity and light; a stability test of the target analyte is necessary to confirm its degradation in stored samples is within acceptable range.The storage stability of sulfoxaflor was assessed by fortifying blank wheat plant, straw and grain samples separately at 10× LOQ level, that is, 0.1 mg kg −1 [14].The samples were stored in 50 mL centrifuge tubes at −20°C under dark condition and the recovery of sulfoxaflor was determined at a periodic interval of 0, 30 and 60 days.The maximum acceptable loss of sulfoxaflor residues during storage was considered as 30% [14], below which the analyte is stable for storage before analysis.

Data analysis
The dissipation of sulfoxaflor was evaluated following single first-order (SFO) kinetics: Where, 'C t ' indicates the concentration (mg kg −1 ) of sulfoxaflor at time 't' (days), 'C 0 ' represents the initial concentration (mg kg −1 ) of sulfoxaflor at time t = 0 days and 'k' is the dissipation rate constant (day −1 ).The persistence of sulfoxaflor was evaluated by estimating the half-life (t 1/2 ) of the molecule: The time required for the initial residues of sulfoxaflor to dissipate below the recommended maximum residue limit (MRL), that is, the pre-harvest interval (PHI) was estimated following the equation [15]: Besides, the impact of seasonal variation and treatment doses on the dissipation of sulfoxaflor was estimated by Two-way Analysis of Variance (Two-way ANOVA).

Food safety assessment
The food safety to the consumers was evaluated in terms of the end-point residue estimation and dietary risk assessment.The end-point residues of sulfoxaflor were estimated in the wheat substrates (wheat grain and straw) to investigate the risk of food contamination associated with the application of the aforesaid insecticide.Dietary risk can be assessed following two different established procedures viz.deterministic and probabilistic methods.The probabilistic method involves statistical data and probability theory [16], whereas the deterministic method of dietary risk estimation is widely used in research studies and regulatory purposes [17,18].In our experiment, the chronic dietary risk associated with the application of the insecticide sulfoxaflor was assessed for three critical population subgroups viz.infants (6-12 months), children (4-6 years) and adults by using the deterministic model as follows: The national estimated daily intake (NEDI) was calculated as follows: The acceptable daily intake (ADI) of sulfoxaflor was reported to be 0.04 mg kg −1 body weight day −1 [19] and F i represents the recommended wheat consumption per day per person.The resultant value of RQ d >1 signifies the chance of high dietary risk and vice versa.

Optimisation of analytical methods
Initially, the analytical method of sulfoxaflor was optimised based on ethyl acetate + cyclohexane (M1), acetonitrile (M2) and ethyl acetate (M3) based QuEChERS and modified QuEChERS methods.Clean-up of M1, M2 and M3 was optimised using different combinations of PSA, GCB, C18 and magnesium sulphate.Higher interference of matrix coextractives associated with lower recovery and improper peak shape of sulfoxaflor was observed in M3 compared to M2 (Figure 2-3).However, addition of cyclohexane with ethyl acetate, that is, ethyl acetate + cyclohexane (9 + 1) (M1) improved the nature of the chromatogram with satisfactory recovery of the molecule (Figure 2-3) and was therefore used as the extractant of sulfoxaflor in our experiment.It was observed that the clean-up of wheat plant, straw and grain with 25 mg PSA (C1, C6) and 50 mg PSA (C2, C7 and C8), though produced comparatively higher response of sulfoxaflor, could not significantly remove matrix impurities compared to 75 mg PSA (C3, C4, C5 and C9) which resulted in slightly lower response but sharp peak without matrix interference (Fig. S1-S2).In the case of wheat plant and straw, clean-up without GCB (C5) resulted in the elution of matrix impurities (Fig. S1).Using GCB up to 25 mg (C1, C2 and C3) to remove plant pigments was observed to be satisfactory in this case (Fig. S1), since the higher amount, that is, 35 mg of GCB (C4) had no significant impact on clean-up (Fig. S1) but reduced both the response and recovery of the molecule (Figure 2).Hence, the combination C3 (75 mg PSA +25 mg GCB + 150 mg magnesium sulphate) was selected for clean-up of wheat plant and straw samples (Fig. S1).However, for wheat grain, a combination of 75 mg C18 along with 75 mg PSA and 150 mg magnesium sulphate (C9) produced optimum results in terms of both peak shape and percent recovery of the molecule (Fig. S2).

Performance of the optimised analytical method
The performance of the optimised analytical methods was evaluated based on the validation parameters: linearity, recovery (%), precision (repeatability RSD r and reproducibility RSD wR ), matrix effect (%), specificity and limit of quantification (LOQ) [20].The calibration curve of sulfoxaflor offered satisfactory linearity with the coefficient of determination (R 2 ) 0.99.The limit of quantification (LOQ) was assessed as the lowest spiked level of sulfoxaflor in the matrix, which satisfied the method performance criteria [20,21], that is, 0.01 mg kg −1 in our experiment.The LOQ was also in compliance with the necessary regulatory criteria as it was lower than the maximum residue limit (MRL) proposed for sulfoxaflor by the European Union (EU), that is, 0.2 mg kg −1 .The average percent recovery of sulfoxaflor in wheat substrates viz.plant, grain and straw ranged between 90.66% and 103.55% with repeatability (RSD r ) and within-laboratory reproducibility (RSD wR ) in the range of 2.67-5.17%and 3.05-8.15%,respectively (Table 1).The matrix effect (ME %) was calculated by using the formula: [ME (%) = {(peak area of the analyte in matrix -peak area of the analyte in solvent)/peak area of the analyte in solvent}] and the ME (%) of sulfoxaflor in wheat plant, grain and straw ranged between 1.37% and 3.95% (Table 1).The specificity was also found to be acceptable as the response of sulfoxaflor was <30% of the matrix-matched standard at the desired retention time in control samples.Thus, the optimised analytical method was adopted for residue estimation of sulfoxaflor in wheat samples.

Storage stability experiment
The stability of sulfoxaflor was evaluated as per cent recovery representing the relative ratio between the determined concentration and the spiked concentration that is, 0.1 mg kg −1 .The recovery of sulfoxaflor ranged within 88.30-98.72%,86.19-96.51%and 85.43-97.21% in wheat plant, straw and grain samples, respectively (Table S1).The per cent degradation of sulfoxaflor in wheat plant, straw and grain was estimated as 2.60, 2.77 and 2.80%, respectively, up to 30 days (Table S1).A maximum of 8.47, 7.94 and 9.88% loss of sulfoxaflor residues was recorded in wheat plant, straw and grain, respectively, up to 60 days (Table S1).Since, the rate of degradation of sulfoxaflor was below 30% of the initial residues, the results indicated no significant loss of sulfoxaflor in stored samples up to 60 days.Similarly, Thompson et al. [22] reported stability of sulfoxaflor up to 64 days in stored groundwater samples.

Dissipation of sulfoxaflor in wheat
The average initial concentration of sulfoxaflor in wheat plants ranged between 1.78-2.78and 1.87-3.10mg kg −1 (T 1 -T 2 ) in Season I and Season II, respectively (Figure 4).The initial residues then dissipated up to 65.31-74.25%within 2 days after application in Season I.However, in Season II, the initial residues of sulfoxaflor dissipated up to 69.98-75.07%within 5 days after application.The dissipation of sulfoxaflor followed single first-order kinetics in our experiment (Figure 4) and the half-life values were estimated as 1.05-1.14days (T 1 -T 2 ) in Season I and 2.44-2.47days (T 1 -T 2 ) in Season II (Table 2).The lower stability of sulfoxaflor under open field conditions has been documented by several researchers.For example, Kim et al. [23] reported first-order dissipation kinetics of sulfoxaflor, resulting in 1.5 days half-life in lettuce.Similarly, Chen et al. [10] reported 2.65 ± 0.08 to 3.21 ± 0.16 days half-life of sulfoxaflor in cucumber fruit.Besides, we observed a significant deviation in the half-life of sulfoxaflor between two seasons due to fluctuation in environmental condition (Table 3), resulting in shorter persistence in Season I than Season II.The fate of pesticides in the environment is reported to vary with climatic conditions as well as with the nature of the pesticidal molecule [11].Earlier, Gauthier and Mabury [24] observed rapid photolysis of sulfoxaflor (t 1/2 : 39.61 ± 1.3 hours) and Bello et al. [25] reported oxidation of cyano-carbon bond and loss of sulphur side chain are the two photo degradative pathways of sulfoxaflor.Similarly, cyano hydrolysis of sulfoxaflor in presence of cytochrome P450 enzyme is also reported as the primary metabolic pathway of the molecule in plant [26,27] and sulfoxaflor urea (1 -{1 -[(6 -Trifluoromethyl -3 - pyridyl) ethyl] (methyl) oxido -λ4 -sulfanylidene} -urea) is therefore identified as the major metabolite in wheat [28].Now, increased enzymatic activity at high temperature causing faster metabolism of pesticides is already documented [29] and accordingly, Kabir et al. [11] also observed faster degradation of sulfoxaflor at high temperature.Hence, higher UV index, bright sunshine hour (BSSH) and atmospheric temperature in Season I (UV index: 6.4,BSSH: 7.5 h, maximum temperature: 24.1 ᵒC, minimum temperature: 8.3 ᵒC) may have accelerated the rate of photo as well as enzymatic degradation of sulfoxaflor, resulting in lower persistence in Season I than Season II (UV index: 5.3, BSSH: 5.8 h, maximum temperature: 20.7 ᵒC, minimum temperature: 6.8 ᵒC) (Figure 1).This was also the reason why a shorter PHI of sulfoxaflor of 4.95-5.34days was observed in Season I than Season II, that is, 11.37-11.55days.Therefore, as the cases of uncontrolled application of pesticides is often reported in India, the results indicate a minimum 15 days interval should be maintained between the date of application and harvest for safe and proper use of sulfoxaflor.Accordingly, a pre-harvest interval of 14-21 days for sulfoxaflor application in winter wheat is recommended by the European Food Safety Authority [30].

End-point residue estimation at harvest
The terminal residues of sulfoxaflor in harvested wheat grain and straw were below the limit of quantification (BLOQ, i.e., <0.01 mg kg −1 ) irrespective of seasons and treatment dosage (Figure 5).The interval between the application of sulfoxaflor and sampling of the harvested wheat products in season I and II was 60 days.Moreover, the terminal residues of sulfoxaflor in harvested wheat grain were significantly lower than the reported maximum residue limit (MRL), that is, 0.2 mg kg −1 as proposed by the EU.Therefore, the application of sulfoxaflor 12% (w/v) SC at the recommended (30 g a.i.ha −1 ) as well as double the recommended (60 g a.i.ha −1 ) doses in wheat crops was expected to promote no regulatory issues for national as well as international trade.

Dietary risk assessment
The estimation of NEDI was done by considering the LOQ value (0.01 mg kg −1 ) of sulfoxaflor in our experiment as the terminal residues of sulfoxaflor were found to be BLOQ at harvest, which was earlier proposed by Yang et al. [31] and Yu et al. [32].The recommended cereal consumption per day per person is 15 g for Indian infants (6-12 months) and 120 g for children (4-6 years), as proposed by the National Institute of Nutrition (NIN) [33].Besides, an intake of 310 g wheat per day per person is also reported for adult men [33].The acceptable daily intake (ADI) of sulfoxaflor is 0.04 mg kg −1 bw day −1 [19] and the average body weight of an infant, child and adult man are reported to be 8.4, 18 and 60 kg, respectively [33].The estimated chronic dietary risk quotient (RQ d ) of sulfoxaflor was 0.0004, 0.0017 and 0.0012 for infants, children and adult men, respectively.Since, the RQ d values were below one for the critical Indian population sub-groups, it was expected that the application of the insecticide at recommended doses will not pose any long-term dietary risk to the consumers.

Conclusion
The study portrays the residual fate and dissipation dynamics of sulfoxaflor 12% (w/v) SC in wheat agroecosystem under tropical climatic conditions.An effective analytical method was developed and optimised for extracting solvents and clean-up combinations and finally validated to assess sulfoxaflor residues in wheat samples.The method showed satisfactory linearity, recovery and precision along with appropriate peak shape of sulfoxaflor with lower matrix interference.The residues of sulfoxaflor were stable up to 60 days in stored wheat substrates, which assured the validity of the final residue data.The residues of sulfoxaflor dissipated following single first-order kinetics irrespective of the seasons and treatment doses.However, a significant influence of climatic deviations on the persistence of the molecule was observed resulting in shorter half-life in season I than season II.However, the variation in sulfoxaflor persistence was non-significant for treatment doses.Besides, absence of end-point residues at harvest as a consequence of relatively shorter persistence of sulfoxaflor in wheat plants as well as lower chronic dietary toxicity ensures the harvested wheat products are safe for human consumption.The research will help to understand the dissipation dynamics of sulfoxaflor in tropical agro-climatic environments and provide safe and proper use of the aforesaid insecticide in wheat agroecosystem.

Figure 2 .
Figure 2. Mean recovery (%) comparison of sulfoxaflor in wheat plant, straw and grain in different extracting solvents (a) and clean-up combinations (b).

Figure 5 .
Figure 5. Chromatogram of sulfoxaflor in wheat grain (a) and straw (b) at harvest.

Table 1 .
Results of method validation of sulfoxaflor in wheat plant, grain, and straw.

Table 2 .
Coefficient of determination (R 2 ), regression equation and half-life (t 1/2 ) of sulfoxaflor in wheat plant at two treatment doses (T1 and T2) in Season I and Season II.

Table 3 .
Two-way analysis of variance (Two-way ANOVA) of sulfoxaflor persistence in wheat plant at two treatment doses (T1 and T2) in Season I and Season II.
a SEM: Standard Error of Mean; b CD: Critical Difference at 95% confidence level