Dissipation and risk assessment of Solomon (300 OD), a combination product of beta-cyfluthrin and imidacloprid in lemon and onion samples

ABSTRACT Supervised field trials were conducted to study dissipation of combination product of beta-cyfluthrin and imidacloprid (Solomon) in various samples of lemon and onion. The pesticides were extracted and analysed using QuEChERS (with some modifications) based methods after validation of the method. Linearity, accuracy and precision data showed that the results were in the acceptable range as per SANTE 2019. The results of dissipation revealed that the initial accumulation of beta-cyfluthrin was 0.6 and 1.1 mg kg−1 in lemon and 0.24 and 0.97 mg kg−1 in onion samples at standard and double dose respectively. The residues of imidacloprid were 2.61 and 4 mg kg−1 in lemon and 0.36 and 0.53 in onion at two doses. The residues of beta-cyfluthrin followed first order dissipation in both the crops at standard dose and showed biphasic dissipation in double dose in both lemon and onion samples. Imidacloprid followed first-order dissipation in lemon in both the doses. Half-life of beta-cyfluthrin ranged from 3.6 to 23.2 days in lemon samples and that of imidacloprid was 15.1 and 21.5 days in two different doses. In onion the half-life ranged from 2.1 to 4.0 for beta-cyfluthrin and 2.8 to 5.2 days for imidacloprid in different doses. Theoretical maximum residue concentration (TMRC) was always less than maximum permissible intake (MPI) in all the samples suggesting that the use of combination product is safe.


Introduction
Lemon and onion are two very important crops with various medicinal values.The lemon (Citrus limon) and onion (Allium cepa)belongs to plant family Rutaceae and Amaryllidaceae respectively.Lemon is a small evergreen tree and is native to North eastern India.Its yellow fruit (lemons) is primarily used for juice and is as pickle where as onion is consumed along with leaves as spring onion as well as a bulb (onion).Both lemon and onion are excellent source of antioxidants (vitamin C in lemon) and flavonoids which help in preventing cell damage, boost immunity, reduces the risk of heart disease (ischaemic stroke) (https://www.medicalnewstoday.com/articles/283476#benefits, accessed 30 September 2020; https://www.healthline.com/nutrition/onion-benefits#:~:text=2.,and%20protect%20against%20blood%20clots, accessed 30 September 2020).In the present covid-19 pandemic situation inclusion medicinally important plants in our daily diet help in reducing the risk of infection.
However, these crops are also prone to infestation by pests.Insect pests affecting onion and lemon include bugs (mealybugs and pirate bugs) aphids, mites, thrips, and flies etc causing severe losses to crops.Beta-cyfluthrin and imidacloprid together offers a wide control on insect pests.Both are designed to be effective with contact or ingestion.Beta-cyfluthrin is non-systemic (pesticides which can be flushed or drained from the plant's root zone prior to harvest, leaving little to no residues behind) while imidacloprid is systemic (pesticides are absorbed by a plant on application and then circulate through the plant's tissues, killing the insects that feed on them) in nature.These belong to synthetic pyrethroids and neonicotinoids class of insecticides respectively.Both pesticides (beta-cyfluthrin and imidacloprid) act on the nervous system by interfering with transmission of stimuli in the nervous system [1], npic.orst.edu/factsheets/archive/imidacloprid.html].Beta-cyfluthrin act on sodium channels whereas imidacloprid acts on post-synaptic nicotinic acetylcholine receptors.Beta-cyfluthrin is a combination of two diastereoisomers and combines a rapid knock-down effect with long-lasting efficacy.Imidacloprid is one of the most widely applied insecticides in the world and can be applied by soil injection, tree injection, application to the skin of the plant, broadcast foliar, ground application as a granular or liquid formulation, or as a pesticide-coated seed treatment.Being systemic it translocates rapidly through plant tissues following application.Although many reports are available on molecular effects of pesticides on pests, there are very limited reports on effect of pesticides on plants at molecular level.Imidacloprid has been reported to lower key green volatiles (GLVs) in tea plant by suppressing critical GLVs synthesis-related genes [2].Pesticide application also lead to oxidative stress in plants by production of reactive oxygen species in turn leading to retarded growth and lowered photosynthetic efficiency of plants [3].Therefore, it becomes more important to study dissipation/degradation of pesticide residues in plants.
Studies have been conducted on efficacy of combined product of imidacloprid and beta-cyfluthrin on various pests.Athanassiou et al., 2013 [4] have shown that the efficacy of combination product of imidacloprid and beta-cyfluthrin (Temprid) showed improvement in insecticidal activity on concrete for control of seven storedproduct insect species as compared to when applied alone.In another study on wheat aphids it was found that Solomon 300 OD, a mixture of beta-cyfluthrin 9% and imidacloprid 21%, is more effective than individual compounds, is better to use as a combination formulation in the wheat ecosystem to manage aphids without any hazard of phytotoxicity [5].Solomon 300OD has been found to be effective against cashew tea mosquito bug and aphids [6] of cashew, insect-pest management in Chilli [7] and was effective against jassids and whitefly in/on cotton [8].Both betacyfluthrin and imidacloprid are effective in controlling large variety of pests infecting these crops.Therefore, present study was undertaken to study the behaviour of the combination product of beta-cyfluthrin and imidacloprid in two medicinally important crops of lemon and onion.Their impact on human health was also assessed as risk assessment so as to ensure safety of health of the consumers.

Chemicals, reagents and solvents
All chemicals (reagents and solvents) were of high purity and were pesticide reagent (PR) or LC-MS/MS grade.Wherever needed, the solvents were distilled before use.All chemicals were checked for any impurity before putting into use.

Certified reference materials (CRMs) and preparation of standards
All the CRMs were purchased from Sigma Aldrich, USA.Primary and intermediate standard solutions of beta-cyfluthrin and imidacloprid were prepared in toluene and acetonitrile respectively.Earlier study has demonstrated that dilution of samples significantly reduces matrix effect on gas chromatography and liquid chromatography in most of the cases in various commodities [9].Therefore, working standards for method validation and dissipation studies were prepared in diluted corresponding matrix by standard addition method.

Supervised field trial
Details of field trials, climatic conditions and samples collection for lemon and onion are provided in Table 1 and Table 2 respectively.Samples were collected following validated statistical methods as described in [10].
Randomised field trial was performed for trials.Three plots each, for control, standard and double dose applications were taken.Schematic layout is given as Figure 1.Plants were plotted in nine plots arranged in three rows (R1, R2 and R3), every row with three.In first row (R1) standard dose application was performed in first plot.Control plot was in the centre.Plot with double dose application was in the last.In second row (R2), plot arrangement was control followed by standard dose and double dose application plots respectively.In third row (R3), plot arrangement was double dose followed by standard dose and control plots respectively.Control plots were not treated with any kind of chemical/solvent/pesticides and were thus untreated plots.No residues were detected in the control plots.

Sample preparation, extraction and clean-up
For lemon samples, approximately 500 g samples of immature and mature fruits were collected and homogenised with peel.For experiment with mature fruits without peel, 750 g samples were collected, peel removed and homogenised.For fruit juice, 1 kg sample was collected, juice extracted.Representative samples were taken and analysed.

Control
Standard dose For onion samples, approximately 2 kg of immature onion bulb and leaves (whole plant) and mature onion bulb were collected, homogenised and a representative sample was drawn.

Control
For Soil, representative sample of 1 kg soil was collected, dried under shade and ground to pass through 2 mm sieve and sub-samples was drawn for residue analysis.
Modified QuEChERS [11] was used for extraction of pesticide residues from the onion and lemon samples.

Onion
Whole plant (immature onion bulb and leaves)/dry onion bulb (2.0 kg) were homogenised on high volume homogeniser at 3000 rpm for 2.0 minutes.From this 15 g sample was taken and extracted using 15 mL of acidified acetonitrile (1% acetic acid in acetonitrile).Magnesium sulphate(MgSO 4 ) (6 g) and Sodium acetate(NaCl) (1.5 g) were added, followed by centrifugation at 3500 rpm for 2 minutes.Supernatant was taken (6 mL) and dispersive cleanup was performed using 0.3 g Primary Secondary Amine (PSA) and 0.9 g MgSO 4 .The samples were then again centrifuged at 2500 rpm for 2 minutes.The supernatant was divided into two parts.For first part 100 µL was taken and diluted 10 times with acetonitrile, filtered through 0.22 µm syringe filter and analysed for imidacloprid as described below under instrumentation.Another aliquot of 2.0 mL aliquot was taken into 15 mL capacity test tube and concentrated under gentle stream of N 2 at 45°C in a turbovap and reconstituted to 2.0 mL with petroleumspirit: acetone (3:1, v/v) and analysed for beta-cyfluthrin as described under instrumentation.

Lemon fruit
Approximately 500 g samples were cut into small pieces and homogenised using high volume homogeniser at 3000 rpm for 2.0 minutes.A representative 15 g homogenised sample (citrus fruits with peel, without peel & juice) was extracted using 15 mL acetonitrile.pH of the solution was maintained at 6.0 using minimum amount of Sodium hydroxide (NaOH).Anhydrous magnesium sulphate (4 g), sodium chloride (NaCl, 1.0 g), trisodium citrate (1.0 g) and disodium hydrogen citrate sesquihydrate (0.5 g) were added and shaken vigorously for 1.0 minute.Sample was centrifuged at 3000 rpm for 5.0 minutes.The co-extracted wax of citrus fruits was removed by low temperature (4°C) overnight treatment in the refrigerator followed by centrifugation at 3000 rpm for 5.0 minutes.From this an aliquot of 6.0 mL was drawn and transferred to 15 mL capacity centrifuge tube containing 0.9 g MgSO 4 and 0.15 g PSA for cleanup, vortexed for 2.0 minutes and centrifuged at 3000 rpm for 5.0 minutes.Supernatant (4 mL) was acidified with 40 µL 5% formic acid and divided into two parts and analysed for imidacloprid and betacyfluthrin as mentioned above.

Soil
The field soil samples were extracted as earlier [12].A representative sample of 10 g soil was taken in 50 mL capacity centrifuged tube and extracted using 20 mL acetonitrile.Anhydrous magnesium sulphate (MgSO 4 ) (4 g) and sodium chloride (1.0 g) were added and centrifuged at 3500 rpm for 2.0 minutes.Supernatant (10.0 mL) was taken and cleaned-up by adding 1.5 g MgSO 4 and 0.25 g PSA for cleanup followed by centrifugation at 2500 rpm for 2.0 minutes.Supernatant was divided into two parts and analysed for imidacloprid and beta-cyfluthrin as mentioned above.

Calculations of analyte concentration
Concentration of the analyte was calculated against linearity graphs.Matrix matched standards (standard addition method) were used .

Method performance check
Linearity (as R 2 ), accuracy (as % recovery) and precision (as relative standard deviation) were studied as per the requirements and acceptance criteria of SANTE, 2019 [13].Linearity curves were drawn using 5 point curve from standards prepared in diluted (10×) lemon matrix for both beta-cyfluthrin and imidacloprid.The concentrations were 0.05, 0.1, 0.25, 0.5 and 1.0 mg kg −1 .For onion samples 6 point curves were prepared with 0.025, 0.05, 0.1, 0.25, 0.5 and 1.0 mg kg −1 for beta-cyfluthrin and 0.001, 0.0025, 0.005, 0.01, 0.025 and 0.05 mg kg −1 for imidacloprid.Accuracy and precision for both beta-cyfluthrin and imidacloprid in onion and lemon samples was studied at 0.05, 0.25 and 0.50 mg kg −1 spiking levels with five replicates each.Limit of Quantitation (LOQ) was established at the lowest concentration where all the method validation parameters were within the acceptable limits in accordance with SANTE 2019 [13].

Half-life and risk assessment
Half-life, t 1/2 = ln(2)/k, indicates the time required to reduce the concentration by 50% from any concentration point in time.For beta-cyfluthrin and imidacloprid in onion and lemon samples, it was calculated as described earlier [14,15].Risk assessment was performed by comparing Theoretical maximum residue concentration (TMRC) and Maximum Permissible Intake (MPI).TMRC was calculated as product of maximum concentrations of residues obtained from trial and that of per capita consumption per day.MPI was calculated by multiplying acceptable daily intake (ADI) https://www.fao.org/fao-who-codexalimentarius/codex-texts/dbs/pestres/pesticide-detail/en/?p_id=206; https://www.fao.org/fao-who-codexalimentarius/codextexts/dbs/pestres/pesticide-detail/en/?p_id=157) with average body weight of 60 kg (http://icmr.nic.in/final/rda-2010.pdf)and expressed as mg residue/per person/day.TMRC<MPI was considered to be safe.

Method validation
The linearity data from 5-point curve from lemon matrix showed that the regression coefficient R 2 was 0.998 and 0.990 for beta-cyfluthrin and imidacloprid respectively.In onion matrix the R 2 values were 0.999 and 0.993 for beta-cyfluthrin and imidacloprid respectively.These values are in acceptable range of R 2 ≥ 0.99 and showed less than 20% residual variation as per SANTE 2019 [13].The accuracy data (Table 3) reveals that the recovery of beta-cyfluthrin ranges from 97 to 112% in various lemon samples and 85 to 104% in onion samples.Similarly the recovery of imidacloprid ranged from 94 to 111% in lemon and 83 to 100% in onion samples (Table 3).The precision data shows that the relative standard deviation (RSD) was less than 20% in all the cases.Both the accuracy and precision data are in the acceptable range of 70-120% and RSD ≤20% in accordance with SANTE, 2019 [13] confirming the suitability of the method for intended use.The diluted matrix has been found to eliminate matrix effect in most of the cases yielding linear response in calibration graphs [3].

Dissipation of beta-cyfluthrin and imidacloprid in lemon and onion samples
Initial accumulation of beta-cyfluthrin was 0.6 and 1.1 mg kg −1 in immature lemon fruits with peel (Table 4).The residues decreased slightly by 5% and 22% after 1 day of application in standard and double dose respectively.Thereafter, the residues showed a large decrease of approximately 40% in both the doses on 3 rd day after application.The residues remained almost steady (with marginal change in residue level) in standard dose till 10 th day.However, in double dose the residues levels showed marginal increase on 5 th and 7 th day after application.Thereafter the residues decreased by 46% on 10 th day in double dose also.
The positioning of lemon fruits on the tree is such that these are variedly covered by leaves.Therefore, on foliar application some lemon fruits may get more application of spray than others.This may contribute to abnormal degradation pattern observed on few days.Other reason could be environmental factors like wind which may have lead to drift in the spray droplets causing more accumulation at some places.The residues thereafter decreased by 86.7 and 72.6% in standard and double dose on 30 th day after application.When the mature samples were analysed without peel the residues were less than limit of quantitation (LOQ) of 0.05 mg kg −1 .The residues were less than LOQ in juice also.As beta-cyfluthrin is surface pesticide (http://pmep.cce.cornell.edu/profiles/extoxnet/carbaryl-dicrotophos/cyfluthrinext.html#:~:text=It%20is%20a%20non%2Dsystemic,potato%20beetle%2C%20and%20many%20others., accessed on 11 September 2020) and does not show translocation in plants [16] removal of peel resulted in significant reduction in residue levels in both the samples.As the application was foliar the accumulation of beta-cyfluthrin was less than LOQ in soil in both the doses.The residues of imidacloprid showed initial accumulation of 2.61 and 4 mg kg −1 in standard and double dose.The residues decreased thereafter by 40% on 5 th day and 20 th day in standard and double dose respectively (Table 4).The residues persisted till 30 th day showing 77 and 66% decrease in both the doses respectively in lemon fruits with peel.When lemon fruits were analysed without peel and in lemon juice the residues of imidacloprid were observed (Table 3).This can be attributed to systemic nature of imidacloprid (http://npic.orst.edu/factsheets/archive/imidacloprid.html,accessed on 11 September 2020) and translocation of imidacloprid within the plant tissues [17].
In onion samples beta-cyfluthrin residues showed initial accumulation of 0.24 and 0.97 mg kg −1 in standard and double dose respectively (Table 5).The residues showed a linear degradation trend with 58.33% decrease on 5 th day after application in standard dose.The residues thereafter, were below limit of quantitation of 0.05 kg kg −1 in all the onion samples and in soil in standard dose.In double dose the residues linearly decreased reaching to 0.10 mg kg −1 by 10 th day after application showing 89% decrease in the residue.Thereafter the residues were less LOQ in all the samples.Imidacloprid residues were 0.36 and 0.53 mg kg −1 in standard and double dose respectively on 0 day after application.In standard dose the residues decreased linearly by 79% on 5 th day after application.Thereafter, the residues were less than LOQ of 0.05 mg kg −1 in all the onion    samples and in soil.In double dose the residues decreased by 90% on 15 th day after application and were below LOQ thereafter in all the onion samples (Table 5).In double dose the residues of imidacloprid persisted longer than beta-cyfluthrin.This can be attributed to systemic nature of imidacloprid.However, in standard dose imidacloprid dissipation was faster as compared to beta-cyfluthrin.The difference in the dissipation of two pesticides in these crops can be due to variation in the positioning of the crop part under investigation.The lemon fruits are aerial where as the onion bulbs grow below the soil.As a result, the foliar application received will be more in the lemon fruit as compared to onion bulb leading to higher initial accumulations.Earlier studies on combined product of imidacloprid and betacyfluthrin in other crops have also been investigated.In chickpea the residues of betacyfluthrin for corresponding doses persisted till 7th and 10 th day after application in standard and double dose and till 10 th day in imidacloprid in both the doses.The residues reached below limit of quantitation of 0.01 μg g −1 on the 10th and 15th day in betacyfluthrin and 15 th day in imidacloprid in chick pea green pods [18].However in okra the residues reached below limit of quantitation of 0.05 μg g −1 by 3 rd day in standard dose and 5 th and 7 th day in double dose [19].A lower persistence has been reported in brinjal also [20,21] demonstrating that the dissipation of the residues depend on crop (part)pesticide combinations.

Half-life, pre harvest interval (PHI) and risk assessment
The equations for dissipation kinetics of beta-cyfluthrin and imidacloprid in lemon and onion are shown in Figure 2 and 3.In lemon, the residues of beta-cyfluthrin showed linear degradation following first order kinetics in standard dose with R 2 = 0.947 and half-life of 10.4 days.In double dose, the residues of beta-cyfluthrin followed first order biphasic dissipation with half-life of 3.6 and 23.2 days in first and second phase (Table 4).Imidacloprid, however, showed linear first-order dissipation with R 2 0.820 and 0.951 in standard and double dose with half-life of 15.1 and 21.5 days in standard and double dose respectively (Table 4).
In onion beta-cyfluthrin followed similar trend with linear first-order dissipation in single dose and biphasic dissipation in double dose with half-life of 4.0, 2.1 and 3.5 days in standard and double dose phase-I and phase-II respectively (figure 1 and 2).The R 2 was more than 0.9 in all the cases.Imidacloprid also followed linear dissipation with R 2 = 0.903 and 0.934 respectively in standard and double dose (figure 1 and 2).The half-life ranged from 2.8 and 5.2 in single and double dose (Table 5).
Earlier study on chickpea has also reported longer half-life in the range of 6-9 days for both these compounds [18].However, in okra and brinjal the half-lives ranged from 0.49 to 3.8 days [18][19][20][21] suggesting that the degradation of these pesticides varies among plant systems.
The consumption of lemon in rural and urban India is 1.211 and 2.117 numbers per month per person respectively.With average weight of lemon as 75 g per lemon, per day consumption of lemon comes out to be 0.003 and 0.005 kg respectively (Table 6).The consumption of onion is 0.842 and 0.951 kg per person per month in rural and urban regions respectively.Raw lemon fruit is slightly bitter in taste and is not consumed.Mature lemon fruits are consumed as pickle (with peel) or as juice.Pickle preparation processing involving addition of many ingredients, the theoretical maximum residue concentration (TMRC) was calculated from  the residues in mature fruit with peel and in juice at 30 day after application.The TMRC was much lower than the maximum permissible intake (MPI) for both imidacloprid and betacyfluthrin (Table 6) for both rural and urban population suggesting it is safe to use a combination product of beta-cyfluthrin and imidacloprid on lemon provided a Pre harvest interval (PHI) of 30 days is followed.The maximum residue limits (MRL) for beta-cyfluthrin and imidacloprid are 0.3 and 1 mg kg −1 in citrus fruits as per Codex https://www.fao.org/fao-whocodexalimentarius/codex-texts/dbs/pestres/pesticide-detail/en/?p_id=206; https://www.fao.org/fao-who-codexalimentarius/codex-texts/dbs/pestres/pesticide-detail/en/?p_id=157).The residues reached below these levels in mature lemon fruit after 30 days of application.Therefore, a PHI of 30 days is proposed.
Onion is consumed as spring onion as well as mature whole onion.Therefore, the TMRC was calculated at 0 day after application.Again the TMRC was much lower than MPI, suggesting that it is safe to use combination product of beta-cyfluthrin and imidacloprid on onion.MRL for imidacloprid in onion is 0.1 mg kg −1 as per codex and there is no MRL for beta-cyfluthrin https://www.fao.org/fao-who-codexalimentarius/codex-texts/dbs/pestres/pesticide-detail/en/?p_id=206; https://www.fao.org/fao-who-codexalimentarius/codextexts/dbs/pestres/pesticide-detail/en/?p_id=157).Therefore, considering the LOQ of 0.05 mg kg −1 , which is attained by 7 th and 15 th day in standard and double dose for betacyfluthrin and on 10 th and 20 th day for imidacloprid in standard and double dose a PHI of 20 days is proposed for onion.Risk assessment studies based on TMRC [22] and hazard Quotient [18] on chickpea have also found the use of the combination product to be safe provided good agricultural practices (GAP) are followed.

Conclusion
The method used in the present study for estimation of pesticide residues from lemon can be extended to other acid rich plant commodities.Results from the present study show that in the combination product of beta-cyfluthrin and imidacloprid on lemon and onion the residues of imidacloprid and beta-cyfluthrin followed first order dissipation in both the crops at standard dose.However in double dose where imidacloprid followed first-order dissipation, beta-cyfluthrin showed biphasic dissipation in double dose in both lemon and onion samples.The risk assessment studies show that the combination product can be safely used in lemon and onion following proper PHI provided GAP are followed.The data from the present study can be used to establish maximum residue limits (MRL) for these plants (parts) by Food Safety and Standards Authority of India (FSSAI) and CODEX Alimentarius.

Figure 1 .
Figure 1.Dissipation kinetics of beta-cyfluthrin in [A] lemon and [B] onion at standard and doubledose.

Figure 2 .
Figure 2. Dissipation kinetics of beta-cyfluthrin in [A] lemon and [B] onion at standard and doubledose.

Figure 3 .
Figure 3. Dissipation kinetics Imiacloprid in [A] lemon and [B] onion at standard and double-dose.

Table 1 .
Details of supervised field trials in lemon and onion.

Table 2 .
Details of formulation application, climatic conditions and sample collection.
ESI) mode.Column used for separation of analytes was Reprosil gold XBD, 50 × 2.1 mm length, i.d.1.8 µm pore size.Mobile phase has 5 mM Ammonium formate in water as solvent A and Acetonitrile-Methanol mixture (1:1, v/v) as solvent B. Column and sample temperatures were 40°C and 5°C respectively.Injection volume was 5.0 µL.Total run time for analyte separation in column was 5.0 minutes.Details of column flow are as follows:

Table 3 .
Accuracy (as recovery) and precision (as relative standard deviation-RSD) (n = 5) in lemon and onion samples.

Table 4 .
Dissipation and harvest time residues in lemon samples (n = 5).

Table 5 .
Dissipation and harvest time residues in onion samples (n = 3).

Table 6 .
Risk assessment of residues of beta-cyfluthrin and imidacloprid as theoretical maximum residue concentration (TMRC) in lemon and onion.Body wt (kg) MPI (mg/per person/day)