Development of imidacloprid and indoxacarb formulations to nanoformulations and their efficacy against Spodoptera littoralis (Boisd)

The conventional pesticide formulations have many side effects on environmental. These effects were due to the huge quantity of pesticides used. Using of nanopesticide formulations can be reduced the quantity of pesticides used and subsequently decreased the cost of pest control and environment contamination. Indoxacarb and imidacloprid nanoparticles were developed. The size of nanoparticles ranged between 200 and 400 nm. The obtained results showed that the percentage of mortalities in second instar larvae of Egyptian cotton leafworm, Spodoptera littoralis were 95 and 75% in indoxacarb and imidacloprid nanoparticles, respectively. On the other hand, these percentages were 76.7 and 91.7%, respectively, with conventional formulations. In spite of one fifth of pesticide concentrations were used in nanoformulations, the LC50, s in nanoformulations were 2.9 and 15.9 ppm for indoxacarb and imidacloprid, respectively, compared to 34.3 and 66.5 in conventional formulation. The loading capacity in both indoxacarb nanoparticle and imidacloprid nanoparticle were 60.7 and 52.0%, respectively. These results found that indoxacarb and imidacloprid nanoparticles were 12 and 4 times, respectively, more effective than indoxacarb and imidacloprid conventional formulations against the second instar larvae of S. littoralis. These results also showed that indoxacarb and imidacloprid nanoparticles can be used as effective formulations against the cotton leafworm larvae instead of the conventional formulation to reduce the environment contamination, control cost and pest population.


Background
The Egyptian cotton leafworm, Spodoptera littoralis was considered the key pest for most main crops in Egypt (Ragaei et al. 2019). This pest has been acquired resistance to many common insecticides. So, it's badly needed to develop a new formulation to suppress the highly population of this pest and reduce the environmental contamination. Recently, the global pesticides consumption is approximately 2 million tonnes of pesticides (De et al. 2014). The intensive use of conventional pesticide formulations involves the enormous amounts of different chemicals types to control weeds, insects, and pathogens of crops (Arnaud et al. 2005). However, many types of conventional pesticides formulations used were accumulated in biological system, contaminate soil and water environments, harm living organisms, and caused a disturbance in the balance of natural ecosystems (Carvalho 2017). Now there is a new trend to get new formulations for reducing the risk of side effects of conventional pesticide formulations. Nanotechnology is currently an important tool for increasing agricultural productivity. Nanotechnology makes a balance between minimal concentration and maximum pest control, safe concentration Page 2 of 7 Sabry et al. Bull Natl Res Cent (2021) 45:16 and reduces the cost of pest control (Oliveira et al. 2019). So, using of nanopesticides is very important in modern agriculture (He et al. 2019). There are many types of nanopesticide formulation such as nanoparticle pesticide formulations, nanosuspension pesticide formulations, nanoemulsion pesticides formulations and nanocapsules pesticide formulations (Sabry 2020). Imidacloprid is the promising insecticide in pest control. The mechanism of action of this insecticide concerned on the central nervous system of insects, with low side effect on mammals. The action of this insecticide is caused by interfering with the transmission of stimuli in the insect nervous system. This mechanism leads to blockage of the nicotinergic neuronal pathway. By blocking nicotinic acetylcholine receptors, imidacloprid prevents acetylcholine from transmitting impulses between nerves, resulting in the insect's paralysis and eventual death. El-Sheikh et al. (2018) used that imidacloprid against the second and forth instar larvae of S. littoralis. On the other hand, Sabry et al. (2013) used imidacloprid against the forth instar larvae S. littoralis. The LC 50 was 0.22 g/l.
Indoxacarb has also a new mode of action against insect pests. It works as a sodium channel blocker resulting in paralysis and death of targeted pests. This insecticide has been reported to have a good field activity against umber of Lepidoptera as well as certain Homoptera and Coleoptera insects. Also, these insecticide formulations are reducing of the pesticide risk on mammalian (Wing et al. 2000;McKinley et al. 2002).
This work aims to develop nanoparticle pesticide formulations of both imidacloprid and indoxacarb as new pest control trend and using it against the second instar larvae of cotton leafworm control.

Test insect
Laboratory colony of Egyptian cotton leafworm, Spodoptera littoralis was reared under laboratory conditions (26 ± 1 °C and 70 ± 5 RH) for many generations. The second instar larvae of S. littoralis were used as a target test against both indoxacarb and imidacloprid conventional and nanoformulations.

Test chemicals
Two common insecticides were used.

Indoxacarb (Avaunt 15% EC), produced by Du Pont
De Nemours. Indoxacarb belong to a new class of insecticides called oxadiazine and it works as a sodium channel blocker. The recommended field rate is 200 ml/feddan (4200 m 2 ) 2. Imidacloprid (Trade name is Commando 35% SC) produced by Vapco Company Jordan. This insecticide belongs to neonicotinoids group. The recommended field rate is 250 ml/feddan (4200 m 2 )

Preparing of imidacloprid and indoxacarb nanoparticles
Chitosan with a high molecular was used as a carrier for active ingredient of imidacloprid and indoxacarb. Both imidacloprid and indoxacarb nanoparticles were prepared according to Vaezifar et al. (2013). The chitosan was taken and dissolved in the acetic acid (2% v/v) followed by continuous stirring with the help of magnetic stirrer for 15-20 min. The 0.8% (w/v) tripolyphosphate solution containing insecticides (imidacloprid or indoxacarb) were added to the chitosan solution (chitosan + acetic acid) followed by 5-10 min of stirring. The suspension was centrifuged at10,000 RPM for 30 min. The pellet was collected and lyophilized to obtain imidacloprid and indoxacarb nanoparticles. Photography of nanoparticles was achieved by scan electronic microscope (Fig. 1a, b). After the imidacloprid and indoxacarb nanoparticles were prepared the loading capacity of both imidacloprid and indoxacarb were calculated according to He et al. (2017): Loading capacity is defined as the mass percentage of the loaded LC to the total solids in the imidacloprid or indoxacarb/chitosan nanoparticles. This loading capacity was determined by about 30 mg of the samples (indoxacarb or imidacloprid nanoparticles) were weighed and dissolved in 50 ml of acetonitrile, and the mixture remained in a shaking tank overnight at a constant temperature to completely dissolve the carrier material. After the solution was filtered, the mass concentration of indoxacarb or imidacloprid in acetonitrile was examined by HPLC (The HPLC system was equipped with an XTerra RP18 column, 5 µm particle size, 4.6 mm internal diameter × 250 mm length (Waters ® , USA) under a detection wavelength of 278 nm (He et al. 2017) (Fig. 2). The loading capacity of imidacloprid was calculated by division of 2.600/5.000 × 100 = 52%. The loading capacity of indoxacarb was 3.083/5.000 × 100 = 60.8%.

Bioassay
Three concentrations of both indoxacarb and imidacloprid were used; the field rate and other two lower concentrations (Table 1).

=
Mass of loaded insecticide Mass of insecticide nanoparticles × 100 Page 3 of 7 Sabry et al. Bull Natl Res Cent (2021) 45:16 Each concentration has three replicates and ten healthy larvae per replicate were used. Other three replicates sprayed by water as a control. Lettuce leaves were used as natural diet to larvae. Deeping technique was used with larvae treatment. The percentages of mortalities were recorded after one, two four and seven days. After seven days the lethal concentrations for 50% of population LC 50 s were recorded and the dead larvae were photographed.

Statistical analysis
Data were subjected to the analysis of variance test (ANOVA) via Randomized Complete Block Design (RCBD) (F test) and analysis of variance (one ways classification ANOVA) followed by a least significant difference (LSD) at 5% (Costat Statistical Software 1990).

Results
To make sure that the size of insecticide particles in nano size, scanning electron microscope (SEM) (JSM-7401 F, JEOL Ltd., Akishima-shi, Japan) was used to determine the size of pesticide particles (Fig. 1). Also, to make sure that the pesticide particles are deposited on polymer particles (chitosan), loading capacities of both tested insecticides are determined (Fig. 2). The obtained results showed that the loading capacities of both imidacloprid and indoxacarb were 52 and 60.8%, respectively.

Using of imidacloprid and indoxacarb conventional formulations
Imidacloprid and indoxacarb are promising insecticides belong to different insecticide groups. Imidacloprid and indoxacarb conventional formulations are used against the second instar larvae of Egyptian cotton leafworm under laboratory conditions ( Table 2). As clear in Table 2 the first concentration (recommended field rate) for imidacloprid is more effective than indoxacarb (the percent of larvae mortalities are 91.7 and 76.7%, respectively.
With the second and the third concentration imidacloprid also is more effective than indoxacarb. The statistical analysis shows that there is a significant difference between the first concentrations in both imidacloprid and indoxacarb but there is no difference in the second and third concentrations. The LC 50 , s of imidacloprid is less than and indoxacarb. The LC 50 , s are 66.5 and 34.3 ppm, respectively.

Using of imidacloprid and indoxacarb nanoparticles
The efficacy of imidacloprid and indoxacarb nanoparticles against the second instar larvae of S. littoralis are examined (Table 3). One fifth of the concentrations which used with conventional formulations are used (Fig. 1).
The efficacy indoxacarb nanoparticles are more than imidacloprid nanoparticles with all concentrations used. The percent of mortalities with the first, second and third concentrations are 95, 80, 58.3 and 75, 50, 35 in indoxacarb and imidacloprid, respectively. The LC 50 , s are 2.9 and 15.9 ppm, respectively (Table 3) (Fig. 3). The statistical analysis shows that there are significant differences between indoxacarb and imidacloprid nanoparticles.

Discussion
The intensive use of conventional formulations of pesticides caused many problems to environment. Using of nanoparticles formulations may be reducing these problems. Using of imidacloprid and indoxacarb nanoparticles against the second instar larvae of Spodoptera littoralis reduces the amount of insecticides uses and increases the efficacy of pest control. The quality of insect control by nanoparticles formulation was better than conventional formulations. The LC 50 , s of both indoxacarb and imidacloprid nanoparticles were 2.9 and 15.9 ppm, respectively. While the LC 50 , s of indoxacarb and imidacloprid conventional formulations were 34.3 and 66.5 ppm, respectively. This means that the Page 5 of 7 Sabry et al. Bull Natl Res Cent (2021) 45:16 indoxacarb nanoparticles was most effective than the conventional formulations by 12 times. And also, imidacloprid nanoparticles were more effective than the conventional one by 4 times. The insecticide concentrations in nanoparticles were one fifth of the concentrations in conventional formulations. This result also showed that the nanoparticles formulations were less toxic to nontarget organisms such as human and natural enemies of tested pest. The same result was found by Assemi et al. (2014). The authors found that the nanoimidacloprid was 8 times more effective than imidacloprid conventional formulations against tobacco aphids, Myzus persicae. Rouhani et al. (2012) found that the imidacloprid nanoparticles were more effective than Ag and Ag-Zn nanoparticles against Aphis nerii. Ahmed et al. (2019) used lambda-cyhalothrin nanoparticles against the second instar larvae of S. littoralis. The authors found that the tested concentrations of lambda-cyhalothrin nanoparticles decreased the insect population to 37 times compare with the

Conclusion
The conventional formulations of pesticides have many side effects on environment due to their residues in soil and plants. Nanopesticide formulations are the best solution for reducing the pesticide restudies in soil, human and plants and also reduces the cost of pest control. In this work this strategy was used with indoxacarb and imidacloprid pesticides. The one fifth concentration of nanopesticide was more effective than the conventional formulation. So, this work recommended that the using of nanotechnology in pesticide formulations development to reduce the side effects of pesticides. This strategy also can be used as a part of integrated pest management (IPM) program.

Abbreviations
LC 50 : Lethal Concentrations for 50% of pest population; LSD: Less Significant Difference.