Introduction

The family Noctuidae is the most diverse group within Lepidoptera, comprising a vast number of species that have a significant impact on the agricultural ecosystem (Caccia et al. 2014). Examples include the cotton leafworm, Spodoptera littoralis (Boisd.), and the black cutworm, Agrotis ipsilon (Hufnagel). Both insects are considered significant pests as they attack various crops during their seedling or vegetative stages (Ladhari et al. 2013).

The primary method for controlling S. littoralis or A. ipsilon has been the application of chemical insecticides (Awad et al. 2022; Moustafa et al. 2022a). However, the frequent and intensive use of these insecticides has resulted in the development of resistance to nearly all insecticide groups employed to control these pests (Abo-Elghar et al. 2005; Xu et al. 2016; Fouad et al. 2022; Moustafa et al. 2023a). This challenge led researchers to devote significant effort to develop alternative strategies for controlling these insects. These include (1) the use of botanical insecticides including plant extracts and essential oils (Khan et al. 2017; Moustafa et al. 2021a, 2023b; El-Shourbagy et al. 2023), which do not harm ecosystems (Rajendran and Sriranjini 2008), (2) bioinsecticides, such as microbial and fermentation products (Moustafa et al. 2022a), and (3) novel chemical insecticides (Moustafa et al. 2021b, 2023c).

Thiocyclam is based on the natural toxin of the marine worm Lumbriconereis heteropoda (Marenzeller). It has shown efficacy against coleoptera and lepidoptera pests (Ware and Whitacre 2004). The insecticide resistance action committee has classified thiocyclam as class 14 (IRAC 2022), which blocks the Nicotinic acetylcholine receptor (nAChR).

Chlorantraniliprole, on the other hand, is a promising new insecticide belonging to the diamide group that has demonstrated effectiveness against several lepidopteran insect pests (Lahm et al. 2005; Lanka et al. 2013). It has been classified as class 28 by IRAC (2022) and is known to modulate the function of the ryanodine receptor (Guo et al. 2013).

Environmental factors (Moustafa et al. 2018) or chemical reactions (Sanz-Asensio et al. 1997) may contribute to the degradation of insecticides. Therefore, dissipation studies are crucial in determining the pre-harvest interval (PHI) and ensuring that residue levels remain below the maximum residue limits (MRL) in food and environmental samples from each growing area under open field conditions (Malhat et al. 2012; El-Sheikh and Ashour 2022).

Nanotechnology offers a promising approach to countering the potential environmental impact of chemical insecticides (Bhattacharyya et al. 2010; Bharani and Namasivayam 2017) by improving their toxicity against insect pests (Kah 2015). Additionally, nanotechnology is being conceived as a rapidly evolving field that has the potential to reform agriculture and food systems (Namasivayam et al. 2018). Nano-insecticides are defined as pesticide formulations consisting of nano-sized engineered structures with insecticidal properties (Kah and Hofmann 2014; Yan et al. 2021). They are considered a potential alternative solution to reduce the environmental footprint of chemical insecticides (Yan et al. 2021). However, the increasing interest in nano-insecticides has raised questions about their toxicity, fate, and biodegradation (Chaturvedi et al. 2014).

Based on a previous study by Awad et al. (2022), both chlorantraniliprole and thiocyclam, as well as their nano-forms, have been identified as important candidates for the development of efficient nano-insecticides for controlling A. ipsilon.

In the current study, field experiments were conducted to compare the effectiveness of thiocyclam, chlorantraniliprole, and their nano-forms against S. littoralis and A. ipsilon. Additionally, the dissipation of both insecticides and their nano-forms was assessed in tomato fruits using the QuEChERs method and LC-ESI MS/MS.

Materials and methods

Insecticides, chemicals and reagents

The insecticides used in the study are detailed in Table 1. The preparation of the nano-forms of both tested insecticides, as performed by Awad et al. (2022), is shown in Figure (S1). Both of Nano-chlorine (chlorantraniliprole) and nano-sulfur (thiocyclam) were prepared by using a Hydrochloric acid (HCl) and orthorhombic bravais as a chlorine source, while for sulfur source sodium thiosulphate and sulfuric acid were used as described by Xu et al. (2006), Cota-Arriola et al. (2013) and De Oliveira et al. (2015).

Table 1 Tested insecticides and their rate of application (Awad et al. 2022)
Full size table

The reference standards for thiocyclam and chlorantraniliprole were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) with > 99% purity. HPLC grade Merck Acetonitrile and Methanol, Riedel–de Haen Ammonia solution (33%), and Formic acid (98–100%) were used for all experiments. LC-MS quality de-ionized water was produced using a Millipore instrument. Agilent technologies QuEChERS extraction reagents, which include Magnesium sulfate, sodium chloride, sodium citrate, and citric acid disodium salt were used. convenience. Finally, a 0.45 μm syringe filter made by Millipore was used.

Field trials

A field experiment was carried out during two consecutive spring seasons (2021 and 2022) at the agricultural experimental station of the Faculty of Agriculture, Cairo University, located in Giza, Egypt. The temperature during the experiment ranged from 18 to 32 °C, with a relative humidity of 55%. Tomato variety 023 was transplanted in an open field in double rows spaced 1.5 m apart and 0.75 m apart within the row, covering an area of 200 m2 (Kandil et al. 2023).

To evaluate the efficacy of the tested insecticides on S. littoralis and A. ipsilon, the experimental area was divided into five plots using a randomized complete block design with four replicates (Moustafa et al. 2022b). The leaves of the tomato plants were sprayed once with the recommended rate of the tested insecticides using a Knapsack sprayer cp-3 with one nozzle boom. In addition, five other plots were designated and sprayed when the tomato fruits were close to ripeness to determine the residues of the insecticides on the fruits. Samples were collected at zero, 1, 3, 5, 7, 10, 15 and 21 days after application.

Efficacy against S. littoralis and A. ipsilon

To evaluate the efficacy of thiocyclam, chlorantraniliprole, and their nano-forms against the 2nd instar larvae of S. littoralis and A. ipsilon, the protocol of the Egyptian Ministry of Agriculture was followed. Spraying of tomato plants was carried out using a manual Knapsack sprayer with a 20-liter capacity during the early morning hours to ensure uniform coverage. Leaves of the treated plants with the tested insecticides or water (control) were collected after two hours (zero time) and after 1, 3, 5, and 7 days from the application. The collected leaves were transferred into a glass container (0.25 L). Twenty-five larvae with four replications were added to each container and left to feed for 24 h. Afterward, the surviving larvae were fed untreated leaves and the mortality percentage was calculated ten days post-treatment using the Abbott formula (1925):

Mortality % = 1 - (Number of alive larvae in the treatment / Number of alive larvae in the control) x 100.

Tomatoes sample extraction

10 g of the tomato sample was added into a 50 ml polyethylene (PFTE) tube (Anastassiades et al. 2003). Then, 10 ml of acetonitrile was added. Homogenization was done using a Geno shaker at 700 rpm for 5 min. Afterward, a buffer-salt mixture was added to the tube, and homogenization was repeated. The resulting mixture was centrifuged at 4000 rpm for 5 min, and an aliquot from the upper layer was filtered using a 0.45 μm syringe filter before injecting 2 µl into the LC system. The horizontal shaker (Sample Prep 2010 − 230 Geno/Grinder) coupled with a 15- and 50-ml tube holder was used.

LC-MS/MS analysis

The Vanquish HPLC series connected to the TSQ Altis triple mass was used for separation, with an Agilent C18 Column Poroshell 120 EC (2.7 μm particle size and 3 × 50 mm). LC-MS/MS was established using an ESI (electrospray ionization) interface. Liquid-solid separation was performed at a flow rate of 0.3 ml/min, starting with 70% bottle A for 1 min, gradually changing to 10% over 5 min, and further decreasing to 2% in 3 min, with an additional minute at 2% before returning to 70% at 7.1 min for 2 min, resulting in a total run time of 9 min. Bottle A was prepared with 10 mM ammonium formate solution pH 4 in water-methanol (9:1), while bottle B contained only methanol. The SRM (Selected Reaction Monitoring) separation and detection mechanism was utilized to support quantitation by confirmation ion using positive ionization. Nitrogen gas was used for nebulizing, while argon gas was used for fragmentation.

Method validation

Method validation is an essential step in ensuring the accuracy and reliability of analytical data. To validate the method used in this study, the parameters and acceptance criteria were selected based on the Eurachem guideline on method validation, the Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed by Magnusson and Örnemark (2014), and the DG-SANTE Commission Regulation (2015).

Pesticide standards preparation

Stock solutions of thiocyclam and chlorantraniliprole were prepared in Toluene at a concentration of 1000 µg/ml. An intermediate solution was prepared by diluting the stock solution to a concentration of 10 µg/ml in acetonitrile. Calibration mixtures were prepared by serial dilutions of the intermediate solution to obtain concentrations of 0.005, 0.01, 0.05, 0.1, and 0.5 µg/ml in acetonitrile.

LC Mobile phase

1.73 mL of formic acid was added to 900 mL of deionized water. The mixture was then adjusted to pH 4 using an ammonia solution. Finally, the volume was made up to 1 L using methanol.

Data analysis

The efficacy of thiocyclam and chlorantraniliprole against S. littoralis and A. ipsilon was subjected to analysis of variance for randomized complete block design (RCBD) according to Steel et al. (1997). Least significance difference (LSD) was applied to detect statistical differences among treatments when the F-test for these treatments was significant at 5% probability level. All statistical analyses were carried out using MSTAT-C software package (Freed et al. 1989).

The degradation of thiocyclam and chlorantraniliprole was modeled using a first-order kinetic model: Ct = C0e_kt. where Ct represents the concentration at time t, and C0 represents the initial concentration. The dissipation rate constant k was used to evaluate the dissipation of the compounds over time.

Results

Efficacy of thiocyclam and chlorantraniliprole and their nano-forms against 2nd instar larvae of S. littoralis

The Efficacy of thiocyclam and chlorantraniliprole and their nano-forms applied to tomato plants in two consecutive seasons (2021 and 2022) was tested against the 2nd instar larvae of S. littoralis in laboratory. Percentage mortality was calculated at zero, 1, 3, 5 and 7 days after application. The results are tabulated in Tables (2 and 3).

In 2021, at zero time and 1 day after application, all insecticides caused 100% mortality. Concerning the residual effect, all insecticides remained highly toxic at 3, 5 and 7 days after application, causing mortality rates of 86.74%, 74.37%, and 55.58% for thiocyclam, 99.00%, 98.00% and 92.92% for nano-thiocyclam and 100%, 100%, and 76% for chlorantraniliprole, respectively. However, nano- chlorantraniliprole, resulted in 100% mortality at all time intervals (Table 2). As shown in Table 3, the same pattern was observed during the 2022 season.

Table 2 Mortality percentage of the 2nd instar larvae of S. littoralis fed tomato leaves treated with thiocyclam and chlorantraniliprole and their nano-forms during the 2021 season
Full size table
Table 3 Mortality percentage of the 2nd instar larvae of S. littoralis fed tomato leaves treated with thiocyclam and chlorantraniliprole and their nano-forms during the 2022 season
Full size table

Efficacy of thiocyclam and chlorantraniliprole and their nano-forms against 2nd instar larvae of A. ipsilon

The efficacy of thiocyclam and chlorantraniliprole and their nano-forms applied to tomato plants in two consecutive seasons (2021 and 2022) was also tested against the 2nd instar larvae of A. ipsilon in laboratory. Percentage mortality was calculated at zero, 1, 3, 5 and 7 days after application. The results are tabulated in Tables (4 and 5).

Table 4 Mortality percentage of 2nd instar larvae of A. ipsilon fed tomato leaves treated with thiocyclam and chlorantraniliprole and their nano-forms during the 2021 season
Full size table
Table 5 Mortality percentage of 2nd instar larvae of A. ipsilon fed tomato leaves treated with thiocyclam and chlorantraniliprole and their nano-forms during the 2022 season
Full size table

At zero-time, 1 day and 3 days after application, all insecticides caused nearly 100% mortality. Moreover, at 5 days after application, all insecticides sustained high to total mortality of the treated larvae. A similar trend was observed at 7 days after application for all insecticides except for thiocyclam in 2021 and thiocyclam and nano- thiocyclam in 2022.

Optimization and validation of the residue analysis

LC-ESI-MS/MS

LC-ESI-MS/MS was used with an ESI source adjusted to positive mode and connected to direct continuous infusion to optimize the SRM parameters for thiocyclam and chlorantraniliprole, in order to achieve the highest sensitivity. Two MRM transitions were chosen for quantitation and confirmation. The potential to create an acquisition method was optimized, and HPLC separation was conditioned prior to studying pesticide recovery. The injection volume was set at 2 µl, which reduced the matrix effect.

Matrix effect

To avoid any deviation in the analytic signal caused by the presence of sample components in the final extract, all results were calculated using standard in matrix.

Method linearity

For quantitative analysis, calculations were based on a six-point calibration curve (0.001, 0.002, 0.005, 0.01, 0.05, and 0.1 µg/ml). The correlation coefficient for thiocyclam and chlorantraniliprole were found to be 0.9982 and 0.9997, respectively.

Limit of quantitation

LOQ refers to the minimum concentration of the analyte in the test sample that can be determined with acceptable precision (repeatability) and recovery under the specified test conditions. To determine the lowest practical LOQ, repeated spiked samples were analyzed at the expected lowest quantitation level (0.01 mg/kg). The accepted recovery and relative standard deviation (RSD %) at LOQ are presented in Table 6.

Table 6 Evaluation of recovery and determination method for thiocyclam and chlorantraniliprole
Full size table

Recovery test and uncertainty evaluation

To evaluate the method’s performance, six replicates of spiked blank tomato samples were tested at three different concentrations (0.01, 0.05, and 0.1 mg/kg) using LC-MS/MS. The results presented in Table 6 showed an overall good recovery between 70% and 104% with ≤ 14.9% RSD.

The relative standard uncertainty due to precision investigation (Uprec), expressed as RSD of repeated spiked samples at different levels, was found to be 0.9%, 0.5%, and 1%. t calc was 2.18 while t_tab was 2.45 for 5 degrees of freedom. The bias of the analytical procedure was investigated for the lowest mean recovery of 80 ± 12.81%.

The uncertainties due to reference standard preparation, including analyte standard purity, balance, pipettes, micropipettes, solvents, and volumetric flasks, were found to be 0.7%.

To determine the combined uncertainty (UC), the positive square root of the sum of the squares of various uncertainty components was calculated. The final expanded uncertainty, obtained by multiplying the combined uncertainty using a coverage factor k = 2 for a confidence level of 95%, was found to be ± 23%.

Dissipation study

Residue concentrations of thiocyclam and chlorantraniliprole in tomato fruits are presented in Figs. 1 and 2. The results showed a classic dissipation pattern in chlorantraniliprole and a fast dissipation in thiocyclam using a power trend line, which can be attributed to the physico-chemical properties of the two insecticides.

Fig. 1
figure 1

Dissipation rates for thiocyclam and its nano-form in tomato fruits

Full size image
Fig. 2
figure 2

Dissipation rates for chlorantraniliprole and its nano-form in tomato fruits

Full size image

The half-lives of thiocyclam and its nano-form in tomatoes were 0.38 and 0.57 days, respectively, vs. 0.7 and 0.38 days for chlorantraniliprole and its nano-form, respectively. The dissipation rates of the insecticides were used to calculate the pre-harvest intervals (PHI). The calculated PHIs for thiocyclam and its nano-form using the EU maximum residue limit (MRL) of 0.01 mg/kg were 7 and 3 days, respectively. On the other hand, the calculated PHI of chlorantraniliprole in both forms was 1-day was using the Codex and EU MRL of 0.6 mg/kg, as reported by the European Commission (2008) and the Codex Alimentarius Commission (2018).

Discussion

The fate and degradation rate of pesticides in various matrices in open fields are influenced by a combination of factors, including the physical and chemical properties of the pesticide, the environmental variables, the frequency and rate of pesticide application, and the growth dilution factor (Moustafa et al. 2018; Kandil et al. 2023). All of these factors significantly affect the dissipation of pesticides and alter their half-lives (Li et al. 2019; Malhat and Abdallah 2019). Therefore, Nanotechnology could plays a vital role in the expansion of different fields (Rabel et al. 2019; Namasivayam et al. 2021) including; engineering, biomedicine and pesticides (Namasivayam et al. 2020a; Awad et al. 2022) due to their distinctive size-dependent chemical and physical properties (Namasivayam et al. 2020b).

Over the past decade, next-generation agrichemical technologies, including new pesticide active ingredients or formulation technologies, have been developed to explore alternatives to traditional pesticides (Zhang et al. 2023). Nano-pesticide formulations, for instance, have the potential to alter the toxicity and residual activity of pesticides, thereby reducing the need for frequent applications (Gopal et al. 2012).

The aim of the present study was to investigate the efficacy of thiocyclam, chlorantraniliprole, and their nano-forms on S. littoralis and A. ipsilon, as well as to determine their residues in tomato fruits, with the goal of enhancing tomato productivity while ensuring public health.

In general, our results showed that thiocyclam, chlorantraniliprole and their nano-forms were significantly efficient against the 2nd instar larvae of S. littoralis and A. ipsilon, compared to the control. Chlorantraniliprole was more effective than thiocyclam, and the nano-forms of both insecticides caused a higher mortality rate than the traditional ones. The residual efficacy of chlorantraniliprole was higher than any other insecticides that had been reported to date (Adams et al. 2016; Hosseinzaden et al. 2019; Batola et al. 2020; Mian et al. 2022). On the other hand, the larval population of maize steam borer, Chilo partellus (C. Swinhoe), ranged from 52 to 91 after thiocyclam application (Gunewardena and Madugalla 2011).

The higher insecticidal activity caused by chlorantraniliprole and its nano-form against both insects may be due to its novel mode of action through ryanodine receptor modulation (IRAC 2022). This could be attributed to the variability in insecticide characteristics influencing the structure and preparation.

Concerning residue analysis, the method’s compatibility for analyzing various sample types led to good recoveries. The physico-chemical properties of the analytes, such as their log octanol water values (solubility in acetonitrile: 1.2 and 0.71 g/L, log Pow − 0.07 and 2.76 at 22 °C for thiocyclam and chlorantraniliprole, respectively) indicate that these are moderately polar analytes with a high partition coefficient in acetonitrile compared to water. Additionally, the dissociation constant (pKa = 3.95 and 10.88 at 20 °C for thiocyclam and chlorantraniliprole, respectively), plays a vital role in ionization, and supports the use of a mobile phase with a lower pH than the analyte’s pKa for greater sensitivity (Krishnan 2009).

Moreover, the low melting point and vapor pressure of thiocyclam (125–128 °C and 0.545 mPa at 20 °C, (E-Pesticide Manual 2002) compared to those of chlorantraniliprole (208–210 °C and 1.2 × 10− 14 mm Hg at 25 °C as reported by the National Center for Biotechnology Information (2023) supports the fast degradation of thiocyclam vs. chlorantraniliprole under field conditions. This finding was in agreement with the reported half- lives of both pesticides in soil (1 day at pH 6.8 and 22 °C for thiocyclam vs. 223–886 days at 25ºC 44–50% MWHC soil moisture for chlorantraniliprole (E-Pesticide Manual 2002; Lewis et al. 2016).

Considering the concentration of the active ingredient in the formulation, the application rate of thiocyclam and chlorantraniliprole on tomatoes was 595 and 28.56 g of active ingredient per hectare, respectively. However, the initial residue concentration (at zero time) of thiocyclam and chlorantraniliprole were 0.22 and 0.95 mg/Kg, respectively.

As insecticides, thiocyclam and chlorantraniliprole work through different mechanisms of action. On one hand, thiocyclam has a limited systemic activity, which means that it is only able to move within the plant in an upward direction, towards the growing tips or apical parts of the plant. It is not effective in controlling insects that feed on the lower parts of the plant. In addition, thiocyclam undergoes a fast degradation that is accelerated by light (E-Pesticide Manual 2002).

On the other hand, chlorantraniliprole has a full systemic action and can move both upwards and downwards within the plant. It can also move laterally from the point of application, providing good coverage throughout the plant. The low aqueous solubility and low volatility of chlorantraniliprole support its trans-locationality into the plant, allowing it to reach all parts of the plant including the roots. The mechanism of infusion of non-ionic particles through the plant cuticle is influenced by the polarity characteristics of the particles, which affects their ability to dissolve in the cuticular waxes and oils (Schreiber 2005).

In addition, chlorantraniliprole translocation into xylem is more active than into phloem tissues, which supports the limited translocation from fruits to other parts of plants. This suggests that when foliar spraying is applied, the fruit tends to store the insecticide in its tissue with less mobility to other parts. Moreover, chlorantraniliprole does not translocate from the sprayed leaves to the new leaves, regardless of the stage of application (Pes et al. 2020).

As shown in Figs. 1 and 2, diverse behaviors were unexpectedly found when comparing traditional and nano-forms of the two pesticides, especially in the first 3 days. The dissipation rate of thiocyclam reached 94% in its traditional form and 82% in its nano-form, whereas for chlorantraniliprole, it reached 63% in its traditional form and 93% in its nano-form.

It is evident that thiocyclam became more stable in its nano-form, while chlorantraniliprole lost 30% of its stability in its nano-form and these results suggest that the stability of pesticides is significantly affected by their formulations.

A fast degradable pesticide that is affected by light, like thiocyclam, became more stable in the environment when prepared as nano particles. Nano-pesticide engineering enhances pesticide stability and enables the development of a controlled-release system based on humidity sensitivity. It improves stability even in unfavorable environmental conditions (Chaud et al. 2021).

On the other hand, a compound with poor water solubility and a high melting point, such as chlorantraniliprole, shows a slow-release mechanism with a high persistence effect. However, when prepared in nanoparticle formulation, it exhibits an opposite behavior. It is clear that the nanoparticle form maintains water solubility and enhances bioactivity. This explanation supports the previous findings that the nanoparticle form reduces the retardation effect and increases the release and activity of the compound. The solid nano dispersion is a promising candidate for improving pesticide solubility and efficacy, and its application in crop protection will reduce the pesticide residue in food and the environmental pollution (Cui et al. 2016).

A rapid dissipation of thiocyclam was observed, particularly in its nano form. After 21 days of treatment, the residue amount decreased to 0.005 and 0.002 mg/kg, representing a 98% and 93% reduction in thiocyclam and its nano-form, respectively.

Furthermore, a rapid dissipation of chlorantraniliprole was observed, particularly in its traditional form. After 21 days of treatment, the residue levels declined to 0.105 and 0.002 mg/kg, with a corresponding loss of 89% and 98% in chlorantraniliprole and its nano-form, respectively. These findings are consistent with a study by Malhat et al. (2012), which reported that only 4% of the initial residue remained 15 days after application.

Regarding pre-harvest interval (PHI), there was a difference in behavior between thiocyclam and chlorantraniliprole in their traditional forms. Thiocyclam required a PHI of 7 days, while chlorantraniliprole required only 1 day, despite thiocyclam having a lower initial concentration than chlorantraniliprole. In fact, thiocyclam is a restricted pesticide in the EU with a lower maximum residue limit (MRL) of 0.01 mg/kg compared to chlorantraniliprole’s MRL of 0.6 mg/kg. Consistent with our results, a high PHI (up to 14 days) was reported for thiocyclam in tomatoes with an application rate of 800 g/hectare (Weerakoon 2019).

In conclusion, the use of nano-formulations of thiocyclam in pest control may carry some risks due to its extended half-life time. However, chlorantraniliprole can be a safer and more effective alternative in its nano-form.