Determination of thiamethoxam residues and dissipation kinetic in tomato plants and its efficacy against Bemisia tabaci under open field eco system

Abstract Whitefly, Bemisia tabaci cause important losses for many crops especially tomato, therefore farmers often use many chemical insecticides which had dangerous side effects on human beings and environment, thus there are need to determine the residues of these chemicals in plant . The dissipation of thiamethoxam in tomato leaves and fruits followed the first order kinetics; Half-lives were 2.91 and 3.15 days for fruits and leaves, respectively. The residues of thiamethoxam were determined using a QuEChERS method with HPLC-DAD and it was 0.14 mg/ kg in fruits after 7 days of treatment and less than the maximum residue limit (MRL 0.2 mg/kg. The residue in leaves was more than in fruits. Seedlings produced from treated seeds were more resistant to different stages of the whitefly population and protect tomato plants from whitefly infestation for 6-10 weeks after transplanting. The three sprays of thiamethoxam increased the reduction percentage of B. tabaci stages and the highest increase was recorded after the third spray in egg, nymph, and adult stages. It could be recommend that Thiamethoxam is considered an unconventional insecticide appropriate for B. tabaci control and safe for humans and the environment.


Introduction
Tomato, Solanum lycopersicum L. is considered one of the most widespread and usually used vegetables in the world (Dorais et al., 2008).Many pests and diseases infested tomato plants such as aphids and white fly.Large numbers of new pesticide compounds such as neonicotinoids developed as an alternative to conventional insecticides due to the extensive use of conventional insecticides which induced the development of resistance by the target insects Pawar et al. (2016).
Moreover, 3 months later, 35 g ammonia nitrate + 24 g potassium sulfate/plot were supplied.Finally, after the first fruit appearance, 35 g ammonia nitrate/plot was added as fertilizer regime.

Seed treatment with thiamethoxam
Ten grams of tomato seeds Castle rock was mixed in 20 ml solution of water + 0.2 g of thiamethoxam 25% WG and vigorously shaken for 10 minutes and left in shadow until dried.Three plots (20 m 2 ) were planted with seedlings produced from treated tomato seeds as mentioned above, while 3 plots were planted with seedlings produced from untreated tomato seeds as control.
Fifteen days after planting, weekly samples of twenty five tomato leaves were randomly collected early in the morning from each replicate, and transported to the laboratory for examination.The numbers of whitefly adults were counted in the field in the early morning before flight activity, while whitefly egg and nymph stages were counted in the laboratory using a dissecting microscope.Reduction percentages were calculated using Abbott Formula 1 (Abbott, 1925):

Foliar spraying with thiamethoxam
To determine the efficacy of thiamethoxam 25% WG on B. tabaci stages, six plots (each 20 m 2 ) were cultivated with tomato seedlings, and sprayed three times with thiamethoxam (20 g/100 L water) at 21 days interval starting from 30 days after planting, using a calibrated hand-held compression sprayer (Kwazar), while other three plots were sprayed with water and left as control.Samples of 25 leaves per replicate were randomly collected before spraying 0 time and 1, 3,5,7,9,11,13,15 & 21 days after pesticide spraying.Numbers of whitefly eggs, nymphs,, and adults were counted as previously mentioned.The reduction % of white fly stages was determined according to the Equation 2 of Henderson and Tilton (1955).(2)

Co =control, T=treatment
The experiments were arranged in a complete randomized block design (RCBD) with three replicates.

Residues and dissipation of thiamethoxam in tomato leaves and fruits
Three plots each 20 m 2 were cultivated with 30 days old tomato seedlings prepared as previously mentioned and sprayed with thiamethoxam (25% WG) with field recommended rate (20 g/100 L water) at fruiting period (one spray only) using the calibrated Hand-Held compression sprayer.All agricultural practices were conducted as usual during the experimental period.
Thiamethoxam was used in soil, foliar and, seed treatments in most crops around the world and was active as contact, systemic activity (Maienfisch et al., 1999).Wettstein et al. (2016), Zhang et al. (2016) found that thiamethoxam was effective in sucking pests control such as aphids, mirids, thrips and, whiteflies.The sucking insect, Bemisia tabaci was controlled effectively on vegetables using neonicotinoids (Li et al., 2021).Also, Pandey (2018) found that thiamethoxam 25 WG was effective against aphids, leaf hopper and, whitefly.Maurya et al. (2015) found that Thiamethoxam 70% WS as tomato seed treatment protected tomato from aphid and thrips with significant increase in the fruit yield.Abdelatef et al. (2022) found that both spray ultra-low volume (ULV) and low volume (LV) techniques with thiamethoxam reduced Whitefly B. tabaci infestation and significantly increased cucumber fruit quality yield.Hafez and Singh (2016) reported that the initial residue of thiamethoxam was equal to the MRL after the 7th day of application at the recommended dose on tomato.Reddy et al. (2022) found that thiamethoxam at the recommended rate was safe for crops and the environment.Also, Yang et al. (2022) reported that thiamethoxam was safe and suitable for spinach under field conditions.
From the previous review, this study was conducted to throw light on the best method of applying thiamethoxam to achieve effective whitefly control, minimize the spraying numbers, reduce environmental pollution & human toxicity, as well as to compare seed treatment and foliar spraying, furthermore, to determine pesticide residues in leaves and fruits.
The standard of thiamethoxam (98% purity) was provided by Central Agricultural Pesticides Laboratory (CAPL) Giza, Egypt.Solvents, anhydrous magnesium sulfate and sodium chloride of analytical grade were supplied by Merck Ltd.Primary Secondary Amine (PSA) and Graphitized Carbon Black (GCB) was obtained from M/s Supelco, Bellefonte, PA, USA.

Field experiments and sampling procedure
Experiments were conducted at a private farm of Elbehira Governorate, Egypt from April 2022 to October 2022.
Thirty three plots of cultivation land were prepared, each plot (20 m 2 ) was divided into four rows of 10 m length and 0.5 m width.Twenty gram of tomato seeds, Solanum lycopersicum, and Variety Castle rock were sown in the nursery.Thirty days later, the growing seedlings were planted at requested plots, where each row was cultivated on both sides at 20 cm distance.Thirty days from planting, 35 g sodium sulfate + 12 g potassium sulfate + 47 g superphosphate/plot were added.One month later, 47 g sodium sulfate + 24 g potassium sulfate/plot were added.

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Residues of thiamethoxam in tomato and its efficacy against Bemisia tabaci One kg of tomato fruits and 100 leaves were randomly collected from each treatment at one hour (0 time), 1, 3, 5, 7, 9, 15 and 21 days after spraying the insecticide, and transferred to the laboratory where it was subjected to determine thiamethoxam residues.

Processes of pesticide analysis
QuEChERS methodology was used to extract and clean up tomato samples (Anastassiades et al., 2003).Homogenized tomato samples of leaves and fruits (15 g) were shaken with 15 ml of acetonitrile 1% acetic acid in a polyethylene tube 50 ml for 1 min., then the mixture was shacked vigorously for 5 min and a centrifuged for 10 min at 3000 rpm with 6 g of anhydrous magnesium sulfate and 1.5 g of sodium chloride.One ml of supernatant was transferred to centrifuge tube (15 ml) which contain 50 mg PSA, 25 mg graphitized carbon black and 150 mg Magnesium sulfate, then samples were mixed vigorously by vortexing for one minute and centrifuged for10 minutes at 6000 rpm.The final volume (2 ml) concentrated to dryness.

HPLC conditions
Agilent 1100 series HPLC system with a quaternary pump, thermostat compartment for the column and photodiode array detector was used.An ODS C18 HPLC column (250 × 4.6 mm, 5 μm) was used as the separation column.The mobile phase consisted of acetonitrile and water (60:40) with a flow rate of 0.8 ml/min.The retention time was 4.05 min.

Validation procedure
The performance of the analytical methods validation parameters on tomato was evaluated using the following parameters: linearity, the limit of quantification (LOQs), accuracy, and precision.Recovery experiments were carried out by spiking untreated tomato leave and fruit samples with thiamethoxam at 0.25, 0.5 and, 1 mg/kg, processed as previously described, and analyzed by HPLC three times for each fortified concentration to establish the reliability and validity of the analytical method adopted.The calibration curve was drawn by plotting the mean peak area against the corresponding concentration ranging from 1 to 100 mg/kg of the target analysts in the mobile phase.Tomato -fortified samples were prepared using standard solutions.The precision of the method was determined based on the relative standard deviations (RSD).The limit of detection (LOD mg/kg) and limit of quantification (LOQ mg/kg) were evaluated based on 3:1 (LOD) while LQD was determined based on signal to noise ratio of 10:1 (Sahoo et al., 2012;Gao et al., 2014).The method which gave recovery of thiamethoxam in the range of 70-120% with a relative standard deviation < 20 was considered to be the ideal method and the lowest spiking level was considered as LOQ.

Statistical analysis
The obtained data were statistically analyzed using an analysis of variance (ANOVA) at 5% probability.The measurements were divided using Duncan's Multiple Range Test through the Costat software program (Version 6.400) 1989-2008.

Effect of tomato seed treatment with thiamethoxam on the population density of whitefly, Bemisia tabaci stages in the field
Regarding to the effect of seed treatment with thiamethoxam 25% WG, at the rate of 5g a.i /kg seeds , on the population density of whitefly, Bemisia tabaci stages in the field , the obtained results in Table 1 and Figure 1 revealed that there were significant decrease in white fly stages along more than 8 weeks compared with control.Means followed by different letters for each stage means significant difference at 5%.Reduction % according to Henderson and Tilton (1955).LSD =Least Significant Difference.
Statistical analysis of the obtained data revealed that there were significant differences in the numbers of eggs, nymphs and, adults between control and treated seeds along sampling periods.
The highest grand mean reduction percentage of B. tabaci was achieved with nymph stage (61.6%)followed by the adult stage (56.6%) and the egg stage (51.4%).

Effect of three sprays of thiamethoxam 20%WG (20 g/100 L water) against Bemisia tabaci infesting tomato seedlings under open field conditions
The obtained data in Tables 2, 3 and 4 and Figures 2, 3, and 4 indicated that there were significant differences in the mean number of whitefly egg, nymph, and adult stages between the treatment of three sprays with thiamethoxam25% WG and control.The highest grand mean reduction percentages after the first spray Table 2 and Figure 2 were achieved with the eggs stage (89.73%),followed by nymphs (80.33%), and adult stages (63.60%).The highest grand mean reduction percentages after the second spray Table 3 and Figure 3 were achieved with the eggs stage (97.10%),followed by nymphs (93.23%), and adult stages (74.30%).The highest grand mean reduction percentages after the second spray Table 4 and Figure 4 were achieved with the eggs stage (98.68%),followed by nymphs (97.07%), and adult stages (78.13%).
From the obtained results it could be reported that, the three sprays of thiamethoxam highly decreased B. tabaci stages, and the highest decrease in egg, nymph, and adult stages were achieved after the 3 rd spray with thiamethoxam at field recommended rate.F value P > 0.05% for eggs = 359.0for nymphs = 267.9for adults = 147.4 Means followed by different letters for each stage means significant difference at 5%.LSD =Least Significant Difference.Reduction % according to Henderson and Tilton (1955).Residues of thiamethoxam in tomato and its efficacy against Bemisia tabaci 3.3.Residues of thiamethoxam in tomato leaves and fruits sprayed with field recommended rate:

Linearity of the thiamethoxam insecticide
The calibration curve of thiamethoxam (Figure 5) shows good linearity and strong correlation between concentrations and peak area in the studied range (0-100 mg/kg) (r2 = 0.9886).The LOD and LOQ were 0.02 and 0.06 mg/kg at a signal to noise ratios of 3:1 and 10:1, respectively.These limits are below the maximum residue limits (MRLs) of 0.2 mg/kg for tomato fruits (Europe Union, 2005).

Recovery test
The mean recoveries of thiamethoxam in tomato fruit and leaf samples have been presented in Table 5.The achieved recoveries for thiamethoxam in spiked tomato fruits and leaves were ranged from 98.4 to 103.6% for fruits and from 96.4 to 102.7% for leaves.The (Relative Standard Deviation % RSD ) value was ranged from 2.232 to 4.32 and 1.27 to 8.53 for tomato fruits and leaves, respectively.

Kinetic study
The thiamethoxam disappearance kinetics in tomato fruit and leaves were evaluated by plotting thiamethoxam residues against time.The dissipation kinetics of thiamethoxam followed the first-order rate Equation 3: Where Ct is the total amount of pesticide concentration at time t, C 0 is the initial residues (the total amount of pesticides present at time t = 0) and k is the rate constant on day-1.The half-life time was determined from the k value for each experiment,t 1/2 = ln 2/k.Half-life time (t 1/2 ) in days was calculated according to Equation 4 (Moye et al., 1987).
( ) Where t = time in days, m = residue at x time, and a = initial residue.

Thiamethoxam residues and disappearance % in tomato leaves
The obtained results in Table 6 and Figure 6a & b show the residues and disappearance % of thiamethoxam in tomato leaves.The initial residue was 0.91 mg/kg one hour after the application of the field recommended rate and the residues dissipated to 48.89% after 3 days of treatment.After that, the residual dissipated by 66.67 and 97.77% after 5 and 21 days, respectively.The Half-life value (t½) for degradation of thiamethoxam was 3.15 days and the dynamics described by the equation C = 0.90 e -0.220 t, with R2 = 0.86385.The RSD% value ranged from 2 to 9.1%.

Thiamethoxam residues and disappearance in tomato fruits
The obtained results in Table 6 and Figure 7a & b show the residue and disappearance % of thiamethoxam in tomato fruits, where the initial residue in fruits was 0.645 mg/kg one hour after the application of the field recommended rate.The residues were dissipated to 52.74% after 3 days showing residues of 0.23 mg/kg.Furthermore, the residual amount of thiamethoxam dissipated calculated in Table 2 and Figure 4 a was 66.18 and 99.85% after 5 and 21 days, respectively.The relative standard deviations RSD% value ranged from 1.43 to 8.82%.The residue of thiamethoxam in tomato fruits was below MRL 0.2 mg/ kg after 7 days of its application at the recommended rate.Half-life time value (t½) for degradation of thiamethoxam in tomato fruits was 2.91 days, after the application of recommended dose and the dynamics could be described by the equation C = 0.680 e -0.238 t, with R2 = 0.86385.
From the previous results, it could be concluded that the average residues in tomato fruits were less than the leaves, and thiamethoxam dissipated in fruits more than in leaves.The residues of thiamethoxam in tomato fruits were below its established MRL 0.2 mg kg-1 after 7 days of its application at the recommended rate.Residues of thiamethoxam in tomato and its efficacy against Bemisia tabaci whitefly population and achieved the highest fruit yield (126.14 q/ha).Also, Liang et al. (2015) found that thiamethoxam significantly reduced the average number of aphids on plants.Recently, Karthik et al (2020) found that thiamethoxam 25% WG at 25 g a.i./ha was highly effective against whitefly after the first and second spray and minimized the spraying numbers, so that, it reduces environmental pollution and human hazards.
In addition, Abdelatef et al. (2022) successfully control Bemisia tabaci infested cucumber plants by spraying thiamethoxam resulting a significant increase in cucumber fruit quality yield.
According to DG SANCO (2013), the obtained recovery of thiamethoxam was in the range of 70-120% with a relative standard deviation (RSD %) value within the acceptable range of ≤ 20% so, the values show that the method was a good performance, sensitive and suitable for the determination of thiamethoxam residues in tomatoes.As for thiamethoxam residues, Hafez and Singh (2016) found that the residue of thiamethoxam in tomato fruits was declined progressively with time, where more than 36% of initial residues of thiamethoxam were dissipated one day after pesticide application with the recommended rate, raised to 90.91% on the 7 th day of application.Also, Wang et al. (2013) reported that the residues of thiamethoxam declined progressively in tomato fruits with time, the initial deposits were found to be equal to the MRL on the 7 th day at the recommended rate, furthermore, the half-life time values of thiamethoxam ranged from 3.9 to 4.4 days in tobacco leaves, and added that the use of thiamethoxam at low effective rate does not cause any hazard to the consumers up to 7 days of treatment.Karmakar and Kulshrestha (2009) found that the thiamethoxam residues were dissipated to 59 and 73% in 5 and 7 days and were not detected at 15 days, the half-life value was 3.5 days when thiamethoxam applied at 140 g a.i./ha and reported waiting 8 days safe consumption of treated tomato.Hafez and Singh (2016) found that the initial deposits of thiamethoxam on tomatoes were found to be equal to the MRL on the 7 th day at the recommended rate.Ramadan et al. (2016) revealed that the initial deposits of thiamethoxam in tomato plants were 0.647 mg kg-1, and residue levels decreased to 0.03 mg kg-1 after 15 days from application indicating that 95.4% of thiamethoxam dissipated.Abd El-Zaher et al. (2011) reported that kidney bean horns could be safely consumed after 7 days of thiamethoxam application according to the recommended maximum residue limit for thiamethoxam in kidney bean (0.2 ppm).Also, Rabea et al. (2018) found that the half-life period of thiamethoxam on pepper fruits was 3.11 days.In addition, Abd-Alrahman (2014) reported that the recovery was 99.4% for potato tubers and that the terminal residues of thiamethoxam were below the maximum residue limit (MRL 0.2 mg/ kg) after 6 days, and safe for human consumption, in addition, the half-life time value (t½) of thiamethoxam was 2.92 days, after spraying recommended rate.Yang et al. (2022) considered that thiamethoxam is the safe and proper application for spinach grown in field conditions.

Discussion
The obtained results are in agreement with Naveed et al. (2010) who reported that cotton seed treated with thiamethoxam kept the population of B. tabaci below the economic threshold level (6 leaf -1 ) up to 50 days after sowing seed compared with the control.Also, Maurya et al. (2015) found that tomato seed treated with Thiamethoxam 70% WS at 4.2 g a.i /kg of seed reduced the early season insect pests (aphids and thrips) as well as significantly increased the fruit yield.Recently, Ding et al. (2018) treated successfully corn seeds with thiamethoxam against early-season thrips and reduced yield losses under field conditions.In addition, (Wettstein et al., 2016, Zhang et al., 2016) reported that thiamethoxam is highly effective in the control of piercing-sucking pests (aphids, mirids, trips and whiteflies).The present findings are in harmony with those of El-Naggar (2006) who reported that thiamethoxam was effective against thrips for 7 weeks after planting.Furthermore, El-Zahi and Aref (2011) reported that thiamethoxam was very effective against cotton aphids under field conditions and Patil et al. (2014) reported that, foliar spray of thiamethoxam 25 WG @ 0.006% was effective against whitefly population and achieved the highest fruit yield (126.14 q/ha).Also, Liang et al. (2015) found that thiamethoxam significantly reduced the average number of aphids on plants.On okra, Pawar et al. (2016) reported that thiamethoxam was effective in aphids, jassids, and whiteflies control.Pandey (2018) found that thiamethoxam 25 WG @ 0.006% was effective against aphids, leaf hoppers, and white fly on okra plants.Also, Karthik et al. (2020) found that thiamethoxam 25% WG at 25 g a.i./ha was highly effective against whitefly after the first and second spray and minimized the spraying numbers, so that, it reduces environmental pollution and human hazards, as well as to compare seed treatment and foliar spraying.Recently, Abdelatef et al. (2022) sprayed thiamethoxam to control Bemisia tabaci on cucumber plants and found that it reduced B. tabaci infestation and caused a significant increase in cucumber fruit quality yield.Naveed et al. (2010) who reported that cotton seed treated with thiamethoxam kept the population of B. tabaci below the economic threshold level (6 leaf -1 ) up to 50 days after sowing seed compared with the control.Also, Maurya et al. (2015) found that tomato seed treated with Thiamethoxam 70% WS at 4.2 g a.i /kg of seed reduced the early season insect pests (aphids and thrips) as well as significantly increased the fruit yield.Recently, Ding et al. (2018) treated successfully corn seeds with thiamethoxam against early-season thrips and reduced yield losses under field conditions.In addition, (Wettstein et al., 2016, Zhang et al., 2016) reported that thiamethoxam is highly effective in the control of piercing-sucking insects.El-Naggar (2006) who reported that thiamethoxam was effective against thrips for 7 weeks after planting.Furthermore, El-Zahi and Aref (2011) reported that thiamethoxam was very effective against cotton aphids under field conditions and Patil et al. (2014) reported that, foliar spray of thiamethoxam 25 WG @ 0.006% was effective against

Figure 1 .
Figure 1.Effect of tomato seed treatment with field recommended rate of thiamethoxam on B. tabaci stages ± SE infecting tomato plants under open field conditions.

Figure 2 .
Figure 2. Effect of the first spray on tomato plants with field recommended rate of thiamethoxam on B. tabaci stages ± SE under open field conditions.

Figure 3 .
Figure 3.Effect of the second spray on tomato plants with field recommended rate of thiamethoxam on B. tabaci stages ± SE under open field conditions.

Figure 4 .
Figure 4. Effect of the third spray on tomato plants with field recommended rate of thiamethoxam on B. tabaci stages ± SE under open field conditions.

Figure 6 .
Figure 6.(a) Residues and (b) disappearance % of thiamethoxam in leaves of tomato plants after different periods of treatment.

Figure 7 .
Figure 7. (a) Residues and (b) disappearance% of thiamethoxam in fruits of tomato plants after different periods of treatment.

Table 1 .
Effect of tomato seed treatment with thiamethoxam on white fly,Bemisia tabaci stages under open field conditions.

Table 2 .
Effect of the 1 st spray on tomato plants with field recommended rate of thiamethoxam 25%WG on B. tabaci stages under open field conditions.

Period after spray (days) 1 st spray (20 g/100 L water) Control Reduction % mean numbers of white fly stages / 25 leaves egg nymph adult egg nymph adult egg nymph adult
Henderson and Tilton (1955) letters for each stage means significant difference at 5%.LSD =Least Significant Difference.Reduction % according toHenderson and Tilton (1955).Brazilian Journal of Biology, 2023, vol.83, e2731055/11Residues of thiamethoxam in tomato and its efficacy against Bemisia tabaci

Table 3 .
Effect of the 2nd spray on tomato plants with field recommended rate of thiamethoxam 25%WG on B. tabaci stages under open field conditions.

Table 4 .
Effect of the 3 rd spray on tomato plants with field recommended rate of thiamethoxam 25%WG on B. tabaci stages under open field conditions.
Means followed by different letters for each stage means significant difference at 5%.LSD =Least Significant Difference.Brazilian Journal of Biology, 2023, vol.83, e273105 6/11 Dhafar et al.

Table 5 .
Recovery percentages and %RSD of thiamethoxam from spiked leaves and tomato fruits samples.

Table 6 .
Residues of thiamethoxam and loss % in the leaves and fruits of tomato plants cultivated under field conditions.