Ecotoxicology and Environmental Safety 107 (2014) 77–83

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Preparation, characterization and efficiency of nanoencapsulated imidacloprid under laboratory conditions Nargess Memarizadeh a, Mohammad Ghadamyari a,n, Mohsen Adeli b,nn, Khalil Talebi c a

Department of Plant Protection, Faculty of Agricultural sciences, University of Guilan, Rasht, Iran Department of Chemistry, Faculty of Science, University of Lorestan, Khoramabad, Iran c Department of Plant Protection, Faculty of Agricultural and Natural Resources, University of Tehran, Karaj, Iran b

art ic l e i nf o

a b s t r a c t

Article history: Received 1 October 2013 Received in revised form 8 May 2014 Accepted 12 May 2014

In this work, nano-imidacloprid was prepared by direct encapsulation with ABA triblock linear dendritic copolymers composed of poly(citric acid) (PCA) as A block and poly(ethylene glycol) (PEG) as B block. Nanocapsules of imidacloprid were characterized using spectroscopy, microscopy and thermal analysis. The encapsulation process was performed by self-assembly of PCA–PEG–PCA in the presence of imidacloprid in different solvents. Comparison of the TEM images of nano-imidacloprid prepared in ethanol and water showed that, during the first day, self-assemblies appeared as small particles with an average size of 10–20 nm. Depending upon the type of solvent, the time and concentration, morphology and size of the nano-imidacloprid varied from fiber-like to globular to tubular from 10 nm to several mm in size. Higher loading capacity and slower release rate of imidacloprid from nano-imidacloprid at optimum pH of Glyphodes pyloalis's gut (pH ¼10) compared to neutral pH confirmed the selective and controllable action of nano-imidacloprid. Results of bioassays on the model insect showed that by using the nanoform of imidacloprid, essential dosage of pesticide and environmental risk decreased significantly and indicated good performance for this formulation. & 2014 Elsevier Inc. All rights reserved.

Keywords: Nanopesticide Biocompatible Molecular self-assembly Linear-dendritic Nanocapsule

1. Introduction The application of chemical pesticides has grown significantly worldwide as increased yields are sought by controlling pests. These compounds assure good agricultural production, but can raise serious concerns stemming from poor application procedures and lack of prudence in usage (Abhilash and Singh, 2009; Cooper and Dobson, 2007; Jones and Huang, 2003). The overuse of bulk pesticides can cause serious contamination to the ambient environment, mammalian toxicity, and threaten non-target organisms that are beneficial to the environment (Talebi et al., 2011). Since effective concentrations of the nanoparticular forms of pesticides are expected to be much lower than those of bulk materials, the application of pesticides in this form is a valuable solution to these problems. Controlled-release formulations of pesticides using nanoencapsulation can reduce the harmful effect of pesticides on non-target organisms. By maintaining an effective concentration

n

Corresponding author. Fax: þ 98 131 6690281. Corresponding author. Fax: þ 98 661 2202782. E-mail addresses: [email protected] (N. Memarizadeh), [email protected], [email protected] (M. Ghadamyari), [email protected] (M. Adeli), [email protected] (K. Talebi). nn

http://dx.doi.org/10.1016/j.ecoenv.2014.05.009 0147-6513/& 2014 Elsevier Inc. All rights reserved.

on the target for longer periods of time, controlled release of pesticides can significantly reduce the amount of active agent required for treatment and delay development of pesticide resistance (Anjali et al., 2010; Bhattacharyya et al., 2010; Ghormade et al., 2011; Khot et al., 2012; Reis et al., 2006; Wang et al., 2007). Nanoencapsulation of drugs or pesticides involves forming particles loaded with the desired material with diameters of less than 1000 nm. Nanoparticles are defined as solid, submicron-sized carriers that may or may not be biodegradable. Nanocapsules contain a cavity housing an inner liquid core surrounded by a polymeric membrane. The chemical agent is usually dissolved in the inner core, but may also be adsorbed onto the capsule surface (Reis et al., 2006). Studies have sought to develop the delivery of therapeutic drugs using nanoparticles and emphasize the advantages of nanoparticles over microparticles (Alle´mann et al., 1993; Couvreur, 1988; Couvreur et al., 1995; Reis et al., 2006; Singh et al., 2010). Few recent studies have reported on the development of nano-pesticide formulations (Anjali et al., 2010; Bhattacharyya et al., 2010; Guan et al., 2008, 2011; Jianhui et al. 2005; Kuzma et al., 2008; Popat et al., 2012). Although preparation of nanoparticles in pharmaceutics is common, research on nanopesticide formation and applications is scarce. Liu et al. (2008) prepared polymeric nanoparticles of the poorly soluble pyrethroid insecticide bifenthrin using flash nanoprecipitation to achieve a particle size in

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the range 60–200 nm. Jianhui et al. (2005) reported the sodium dodecyl sulfate (SDS) modified photocatalytic TiO2/Ag nanomaterial conjugated with dimethomorph as a nanopesticide. Guan et al. (2008) directly encapsulated imidacloprid microcrystals using the natural polysaccharides chitosan and sodium alginate through layer-by-layer self-assembly. In another study, Guan et al. (2011) produced microcrystals of avermectin by recrystallization in the presence of a stabilizer. Popat et al. (2012) successfully used the mesoporous silica nanoparticles with different pore sizes, morphologies and mesoporous structures to load imidacloprid and termite control. They reported the dependence of adsorption amount and release profile of imidacloprid on type of a mesoporous structure and surface area of silica particles and proved the efficacy of silica nanoparticles in delivery of biopesticides. The formulation of water dispersible nanopermethrin was investigated for its larvicidal properties by Anjali et al. (2010). It was prepared by solvent evaporation of oil in a water microemulsion obtained by mixing an organic and aqueous phase. Supramolecular organizational characteristics of block copolymers as a result of nanophase segregation and their interfacial and adhesive properties indicate that these copolymers are useful materials for different applications. The formation of nanometer-sized patterns, encapsulation and controlled release of other compounds are among the most important (Fréchet, 1994; Wurm and Frey, 2011). Noncovalent interaction between the building blocks of these supramolecules enables them to degrade back into individual molecules and makes them promising multidisciplinary materials (Naeini et al., 2010). Citric acid molecules form a dendritic structure of PCA–PEG– PCA copolymers with a high degree of molecular uniformity and monodispersity as well as a highly functional surface. It is one of the most versatile and important carboxylic acid intermediates of metabolism in most plants and animals (Dhillon et al., 2011). Poly (ethylene glycol) (PEG) as linear polyether hydrophilic blocks is used to modify dendrimers in the design of solubilizing and drug delivery systems. This compound is typically conjugated to the surface of a dendrimer to provide high water solubility, biocompatibility and the ability to modify the biodistribution of carriers (D’Emanuele and Attwood, 2005). Imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a neonicotinoid insecticide and is a systemic contact insecticide and a nicotinic acetylcholine receptor stimulator. Imidacloprid is less toxic to humans and highly active against pests; it is known worldwide for these qualities (Guan et al., 2008; Zhou et al., 2006). The present work encapsulated imidacloprid in nanomaterials and produced a novel pesticide. Imidacloprid was directly encapsulated with PCA–PEG–PCA ABA type linear-dendritic copolymers as a biocompatible compound. As previously proven, aqueous solutions of PCA–PEG–PCA linear-dendritic copolymers lead to molecular self-assemblies (Naeini et al., 2010). The characteristics of the prepared molecular self-assemblies in ethanol (as a basic solvent) and in water (as a secondary solvent) were investigated for encapsulation of imidacloprid in this study. The lesser mulberry pyralid (Glyphodes pyloalis Walker) is a major pest on the mulberry. For this study, it was reared under laboratory conditions and bioassayed using topical and leaf dip bioassay techniques to determine the efficacy of the novel nanoimidacloprid in each solvent. Ethanol and water were used as solvents for topical and leaf dip bioassay methods, respectively. 2. Materials and methods 2.1. Insects The first population of G. pyloalis was collected from infested mulberry orchards near the city of Rasht in Iran. Mass rearing of the insects was done in

the laboratory under controlled conditions at 25 7 1 1C, 707 5 percent RH, and 16:8 L:D. Newly-ecdysed fifth instar larvae of G. pyloalis were used for the bioassay experiments. 2.2. Materials PEG (molecular weight [Mn]¼ 1000), citric acid, tetrahydrofuran and diethyl ether were purchased from Merck. Dialysis bags (Mn cutoff 2000) were purchased from Sigma-Aldrich (St Louis, Missouri). Imidacloprid (95 percent) was obtained from Kavosh Kimia (Kerman, Iran). 2.3. Synthesis of PCA–PEG–PCA copolymers PEG (Mn¼ 1000) and citric acid (molar ratio of CA/PEG¼ 1/10) were used for synthesis of PCA–PEG–PCA copolymers using the method described by Naeini et al. (2010). Three steps were involved in the polymerization process. In the first step, the temperature of polymerization was increased to 110 1C for 20 min so that a transparent viscose compound was formed. In the second step, the temperature was increased to 130 1C to the melting state for 15 min. In the third step, the temperature was maintained at 150 1C for 30 min and mixture was stirred vigorously. During these processes, water was removed from the reaction medium by a vacuum pump. After these steps, the ampule contents were dissolved in tetrahydrofuran and then precipitated in diethyl ether several times. The PCA–PEG– PCA copolymers formed a viscous yellow compound with a yield of eight percent. 2.4. Encapsulation of imidacloprid by PCA–PEG–PCA copolymers molecular selfassembly Imidacloprid dissolved in acetone (1 g/100 ml) and PCA–PEG–PCA copolymers dissolved in ethanol as a basic solvent (1 g/20 ml) were mixed at room temperature and stirred for 8 h. Applying dialysis bag (Mn cutoff 2000), free imidacloprid was separated and then resultant solution containing nano-imidacloprid was maintained at 4 1C. Specific volumes of this solution were diluted in the two solvents (ethanol and deionized water) for bioassay and other experiments. 2.5. Characterization Nuclear magnetic resonance (NMR) spectra were recorded in acetone-d6 for imidacloprid, D2O for the copolymers, and a mixture of these solvents for encapsulated imidacloprid on a Bruker DRX 400 MHz apparatus (Vernon Hills, Illinois) with the solvent proton signal as a reference. Infrared (IR) spectra were recorded using a Nicolet 320 FTIR (Nicolet Instrument, Madison, Wisconsin). Transmission electron microscopic (TEM) analyses were performed using a Philips (Model CM120) electron microscope (Netherlands). Thermo gravimetric analyses (TGA) were carried out in a thermal analyzer (Model DSC 60; Shimadzu, Tokyo, Japan) under a dynamic atmosphere of N2 as an inert gas at 10 ml/min at room temperature. Optical microscopy images were recorded using an Olympus BH2 (Tokyo, Japan). 2.6. Determination of loading capacity of PCA–PEG–PCA copolymers A specific volume of nano-imidacloprid coated with PCA–PEG–PCA copolymers was prepared and sealed in a dialysis bag (Mn cutoff 2000). Then dialysis bag was immersed in the phosphate-buffered saline solution (PBS) which was stirred with a magnetic stirrer at a fixed speed at room temperature for about 30 min. So that the release of free imidacloprid from dialysis bag to PBS buffer becomes possible. Then the concentration of encapsulated imidacloprid (inside of the dialysis bag) was determined using UV absorbance for imidacloprid at λmax ¼268 nm. This experiment was done at two pH values (pH of 7 and 10) for the PBS. The percentage of encapsulation was calculated as % encapsulation ¼ {(total imidacloprid concentration free imidacloprid concentration)/total imidacloprid concentration}n100 2.7. Determination of in vitro release rate of imidacloprid from nanocapsules The fluid released from the dialysis bag contained a specific volume of encapsulated imidacloprid which was separated from free imidacloprid (as described above) into the PBS buffer. The release media were stirred using a magnetic stirrer at a fixed speed at room temperature; after 1, 2, 3, 4, 6, 8, 24, 48 and 72 h, certain volumes of the buffer in the outside of the dialysis bag were sampled and the concentration of released imidacloprid was determined using UV absorbance at 268 nm. This was done at a neutral pH (7) for the PBS buffer and the common pH of Lepidopteran gut, (10). The percentage of imidacloprid released was calculated using following equation: % release¼ (free imidacloprid concentration at each sample/total imidacloprid concentration)n100

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2.8. Bioassay 2.8.1. Leaf dip bioassay The efficiency of the encapsulated imidacloprid diluted in water for the newlyecdysed fifth instar larvae of G. pyloalis was assayed using leaf dip bioassay (Memarizadeh et al., 2011). The technical imidacloprid was dissolved in acetone and diluted to generate six serial dilutions with distilled water. Mulberry leaf disks (diameter: 3.5 cm) were formed so that each petiole was placed in a vial containing water that provided moisture to the leaf disks for several days. These disks were then immersed in the dilutions for 45 s. After drying, synchronized fifth instar larvae of G. pyloalis were placed on each treated leaf disk. Up to five larvae were placed on each disk. Mortality was assessed after the treated larvae were maintained at 257 1 1C, 707 10 R.H. for 24, 48, 72, 96 and 120 h. Each experiment was replicated ten times. The criterion for death was that a larva did not move when prodded with a camel hair brush.

2.8.2. Topical bioassay To determination the efficacy of the encapsulated nano-imidacloprid prepared using ethanol, a topical bioassay was performed as described by Scott et al. (1990). In the preliminary tests, five concentrations of bulk and nano-imidacloprid were tested. The lowest and highest doses were 14.5 and 60 ppm for bulk-imidacloprid, respectively, and 10 and 48 ppm, respectively, for nano-imidacloprid. Fifty larvae per concentration were used for all experiments and each experiment was replicated four times; 1 μl of the desired concentration was topically applied to the metathoracic tergum. The controls received 1 μl of ethanol in the same way. The treated larvae were allowed to eat fresh leaves. After 24, 48, 72 and 96 h, the numbers of dead larvae were recorded. The criterion for death was that a larva did not move when prodded with a camel hair brush.

3. Results and discussion 3.1. Characterization of nano-imidacloprid The 1H NMR spectrum of PCA–PEG–PCA copolymers (Fig. 1a of Supporting information) shows chemical shifts at 4.7 ppm for end methylene groups of PEG, 3.6 ppm for PEG methylene groups, and 2.6–2.8 ppm for methylene groups of PCA (Naeini et al., 2010). The 1 H NMR spectrum of free imidacloprid (Fig. 1b of Supporting information) shows signals at 1.4, 2.8 and 3.81 ppm for the methylene groups. It also shows chemical shifts at 2 ppm for the amine group and at 7.84, 8.47 and 8.65 for protons of the pyridine ring. The 1H NMR spectrum for nano-imidacloprid shows chemical shifts correlating with the pyridine ring of imidacloprid at 7.74, 8.17 ppm and chemical shifts at 1.5–4.2 correlating to the methylene groups of imidacloprid and all signals for the PCA–PEG–PCA copolymers. The chemical shifts at 2 ppm for the amine group of imidacloprid are also shown (Fig. 2(A) of Supporting information). The appearance of signals of the copolymers and imidacloprid with small shifts in the 1H NMR spectrum of the nano-imidacloprid confirms encapsulation of the imidacloprid inside the molecular selfassemblies of copolymers and the successful preparation of nanoimidacloprid (Guzsvány et al., 2006; Namazi and Adeli, 2003, 2005). In the 13C NMR spectra, chemical shifts at 175, 170, 64 and 42 ppm assigned the carbonyl and carbons of the PCA blocks. Chemical shifts at 67–70 ppm correspond to the PEG carbons and end methylene groups of the PEG (Naeini et al., 2010). The 13C NMR spectrum of imidacloprid shows signals at 123.5, 138.5, 145.9, 150.3, and 163 ppm that correspond to the carbons of the aromatic ring. The 13C NMR spectrum of nano-imidacloprid shows chemical shifts at 35, 45, 47, 51, 63 and 65 ppm that correspond to the aliphatic methylene groups of the PCA–PEG–PCA copolymers and imidacloprid (Fig. 2(B) of Supporting information). The chemical shifts at 131, 149, 152 and 172 ppm correspond to the pyridine of the imidacloprid and at 176 ppm to the carbonyl groups of the PCA blocks of copolymers (Guzsvány et al., 2006; Namazi and Adeli, 2003, 2005). The FTIR spectrum of the copolymers shows the peaks at 2400– 3600 cm  1 and 1730 cm  1 corresponding to the hydroxyl and carbonyl groups of the PCA blocks. The peak at 3354 cm  1 in the

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imidacloprid IR spectrum corresponds the symmetric stretching vibrations of N–H and the peaks at 1437–1596 are the C ¼C and NO2 stretching frequencies. The presence of nano-imidacloprid particles led to a shift in copolymers, some peaks or changes in their relative intensities. In particular, the peak at 1730 cm  1 shifted to 1723 cm  1, confirming hydrogen bonds between the PCA blocks of the copolymers and imidacloprid. The new peak appeared at 3389 cm  1, which again indicates hydrogen bonds between the N–H groups of imidacloprid and the carboxyl functional groups of the copolymers. These obvious changes suggest that imidacloprid and the copolymers interacted strongly and that imidacloprid became part of the nano-imidacloprid molecular self-assembly (Fig. 2(C) of Supporting information). Thermal analysis was carried out at 0–600 1C at a heating rate of 10 1C/min under nitrogen flow to compare the thermal stability of the synthesized nano-imidacloprid with its components, the copolymers and free imidacloprid. TGA diagram of PCA–PEG–PCA copolymers shows three main weight-loss temperatures: the first (110–180 1C) is for the evaporation of water and decarboxylation of the carboxyl functional groups of the PCA blocks. The second weight-loss (190–260 1C) is for decomposition of the PCA blocks, and the third stage (380–430 1C) is for the decomposition of the PEG block (Naeini et al., 2010). TGA diagram of imidacloprid shows two stages for weight-loss that correspond to the start of decomposition at 250 1C and where decomposition reached 66.4 percent at 550 1C. The TGA diagram of nano-imidacloprid shows that decomposition started at 150 1C and the weight of the compound slowly decreased to eleven percent at 440 1C. The water molecules were replaced by imidacloprid when the starting point for decomposition increased to 150 1C. Since weight loss for the PCA–PEG–PCA copolymers at 260 1C decreased from 72 percent to 58 percent for nano-imidacloprid, it can be deduced that amount of encapsulated imidacloprid was about fourteen percent (Fig. 2(D) of Supporting information). The molecular self-assembly of the copolymers in ethanol and water in the presence of imidacloprid was investigated using TEM. Fig. 1(A) shows the TEM images of spherical nanoparticles of imidacloprid encapsulated in copolymers prepared in ethanol on the first day of the synthesis. As seen in the figure, nanoparticles joined as time passed, leading to the aggregation of nanoparticles that produced microspherical capsules (Fig. 1(C)). Fig. 1(B) shows the TEM image of nano-imidacloprid prepared by self-assembly of copolymers in water in the presence of imidacloprid on the first day. The results show that molecular self- assembly of synthesized nano-imidacloprid in water led to formation of semispherical nanoparticles. Fig. 1(D) is optical microscopy image which shows that the nanoparticles joined to form large strands and, finally, fibers by 2.1 m in width and several mm in length. The inset in Fig. 1(A) shows the nano-imidacloprid prepared in ethanol with the imidacloprid particle in the center and the copolymers assembled around it. It is evident that the imidacloprid is encompassed in the core by PCA–PEG–PCA copolymers when water was used as solvent (Fig. 1(B)). Previous studies have shown that the size and shape of supramolecular assemblies of PCA–PEG–PCA copolymers depend on the structure of the copolymers. Copolymers with larger PCA blocks produce fibers with greater diameters (Naeini et al., 2010). The results of the present study also show that the type of solvent, the concentration of copolymers and the presence of imidacloprid during encapsulation as a small guest molecule affected the size and morphology of the supramolecular assemblies. When an ethanol solution with high concentrations of imidacloprid (2.5 gr/L) and copolymers (12.5 gr/L) was left at 4 1C, globular supramolecular assemblies gradually appeared. Fig. 1(C) shows the globular microcapsules resulting from molecular self-assembly of the copolymers in the presence of imidacloprid prepared in ethanol after approximately twenty days. As seen, imidacloprid was encapsulated in the core of

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Fig. 1. Molecular self-assembly of PCA–PEG–PCA. (A) TEM image of encapsulated imidacloprid by products of PCA–PEG–PCA copolymers molecular self-assembly in ethanol. (B) TEM image of encapsulated imidacloprid by products of PCA–PEG–PCA copolymers molecular self-assembly in water. (C) Optical microscopy images of globular segments resulting from ethanol solution of PCA–PEG–PCA copolymers (12.5 g/l) and imidacloprid (2500 mg/l), after twenty days. (D) Optical microscopy images of fibers resulting from water solution of PCA–PEG–PCA copolymers (250 mg/l) and imidacloprid (50 mg/l), after ten days. (E) Optical microscopy images of tube particles resulting from water solution of PCA–PEG–PCA copolymers (12.5 g/l) and imidacloprid (2500 mg/l), after three days.

the globular capsules. In the presence of water as an initial solvent or for dilution, the size and shape of the supramolecular assemblies of the copolymers in the presence of imidacloprid differed strongly from that for ethanol. For high concentrations of the copolymers and imidacloprid, large tubular particles formed after two or three days (Fig. 1(E)). At low concentrations of the copolymers in the presence of imidacloprid after about ten days, fiber-shaped supramolecular assemblies appeared (Fig. 1(D)) and the imidacloprid was encapsulated in segments of the fibers. It is likely that the fiber-shaped, globular and tubular segments contained assembled PCA blocks, because these blocks as dendritic architecture contain several functional groups that interact with imidacloprid. The open nature of dendritic architecture has led to the encapsulation drug molecules within the branches of a dendrimer (Naeini et al., 2010; Wurm and Frey, 2011). The hydrophobicity of imidacloprid caused its attachment to PCA–PEG–PCA copolymers by electroestatic or other interactions in water and forces hydrophilic segments of copolymers (citric acid branches) to cover imidacloprid more completely than in the presence of an organic solvent such as

ethanol. As expected, when nano-imidacloprid was prepared in ethanol, only a globular self-assembly was produced. In spite of the interesting supramolecular self-assembly systems in the presence of water that verify good interaction between nano-imidacloprid particles in water, the results also showed that, even after several months, the new nano-imidacloprid formulation produced in ethanol show no deposits, which confirmed its stability. 3.2. Determination of loading capacity of PCA–PEG–PCA copolymers for imidacloprid The prevailing pHs of the model insect gut are 9–11; thus, in addition to neutral pH (7) the effect of pH 10, as the optimal gut pH of G. pyloalis, on loading capacity and release rate was examined. The loading capacity of PCA–PEG–PCA copolymers was investigated using UV spectroscopy and a calibration curve was obtained by plotting the concentration of imidacloprid versus λmax. This calibration curve was used to determine the highest concentration of imidacloprid needed for a certain concentration

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of PCA–PEG–PCA copolymers to produce nano-imidacloprid. This concentration was considered to be the maximum loading capacity of the copolymers. The results showed that the loading capacity of the copolymers at pH 7 was 53 percent and at pH 10 was 80 percent. During the preparation time of imidacloprid nanocapsules using 1000 mg/L imidacloprid and 5000 mg/L PCA– PEG–PCA copolymers, 53 percent of the free imidacloprid was loaded on the PCA–PEG–PCA copolymers and converted to nanoimidacloprid at a PBS pH of 7 and 80 percent was loaded at a PBS pH of 10. Thermal analysis of the nanocapsule using 10 g/L imidacloprid and 5 g/L copolymers prepared by ethanol showed that the amount of the encapsulated imidacloprid inside selfassemblies was around fourteen percent. In general, since the number of guest molecules incorporated into a dendrimer is somewhat dependent on the architecture of the dendrimer, loading capacity may be enhanced by the formation of a complex containing a large number of groups on the dendrimer surface. The external surfaces of the citric acid blocks of PCA–PEG–PCA have been investigated as dendrimers for potential sites of interaction with imidacloprid. The number of surface groups available for drug interactions doubles with each increasing generation of dendrimer (D’Emanuele and Attwood, 2005); three generations of PCA–PEG–PCA copolymers were effective for increasing potential sites of interaction with imidacloprid.

Fig. 2. Release rate percentage of imidacloprid from nanocapsule, at neutral pH and optimum pH of G. pyloalis larva's gut.

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3.3. Determination of release rate of imidacloprid from nanoimidacloprid The concentration of imidacloprid released from the imidacloprid nanocapsules into the PBS buffer (pH ¼ 7) is shown in Fig. 2. The percentage of free imidacloprid in the PBS buffer (pH ¼7) showed that imidacloprid release at this pH slowly increased for 6 h and then remained constant. The release rate of imidacloprid into high pH PBS buffer (pH ¼10) increased up to 24 h. The results showed that, at the higher pH (pH ¼ 10), the percentage of imidacloprid released was higher than at pH ¼7. The selective action of nano-imidacloprid at the pH of G. pyloalis gut (pH¼10) (higher loading capacity and releasing time) confirms that it can be used as a pesticide formulation compatible with the objectives of precision agriculture. The results of the leaf dip bioassay further verify this claim. 3.4. Determination of insecticide efficiency of synthesized nanoimidacloprid To study the insecticidal efficiency of nano-imidacloprid over the bulk imidacloprid diluted in water, leaf dip bioassay tests on the G. pyloalis were performed (Table 1). For the 500 ppm concentration of free imidacloprid and an exposure time of four days, 100 percent mortality was observed. At the 300 ppm concentration of nano-imidacloprid with an exposure time of five days, 100 percent mortality was also observed (Table 1). The results of ANOVA and mean comparison showed that the effect of concentration, time and interaction of time and concentration for nano-imidacloprid was significant. Mortality significantly increased only when nano-imidacloprid was used in the bioassay; the effect of concentration correlated with the time of exposure and an increase in concentration over time (five days). Despite the reduction of imidacloprid concentration, the time for control increased when nano-imidacloprid was used. These results verify that the effective materials were slowly released from the polymeral nanocapsules and remained on the target site for a greater length of time. When time for control was increased, the consumption of pesticide decreased. A comparison of LC50 (Lethal Concentration 50 percent or median concentration lethal) for bulk and nanoimidacloprid showed that as exposure time increased, LC50 for the nano-imidacloprid interestingly decreased over free imidacloprid. So, after four and five days of exposure, LC50 of nanoimidacloprid decreased to 4.82 and 9.05-fold less than the bulk form of imidacloprid, respectively (Fig. 3(A)). This result indicates that the nanoform of imidacloprid shows better performance over bulk imidacloprid.

Table 1 Mortality percentage of newly ecdysed fifth instar larvae of G. pyloalis by leaf dip bioassay method over the fifth day. Insecticides

Concentrations (ppm)

Percent mortality at times (h) after treatment 24

Nano-imidacloprid

Imidacloprid

300 183 111 68 42 25 500 316 200 126 80 50

48 kl

45.75 7 2.52 427 1.22l 22.55 7 2.63m 20.417 1.71m 16.83 7 1.45mn 7.62 7 1.43n 57.5 7 2.04defg 39.167 4.13hijk 34.167 2.09jkil 21.85 7 3.07mnl 8.82 7 1.38no 2.5 7 2o

72 ghij

657 2.04 61.25 7 2.39hij 46.5 7 2.36kl 44.75 7 2.28kl 40.75 7 3.33l 197 1mn 77.58 7 4b 607 3.16cde 557 3.19efgh 43.75 7 2.39fghi 23.8 7 2.52mno 15.617 1.72mno

96 abcd

88.75 7 1.25 76.25 7 2.39efg 70.417 1.71fghi 72.337 2.33fgh 557 1.45jk 38.75 7 3.33l 86.08 7 1.63ab 757 3.2bc 71.667 2.04bcd 58.65 7 3.41defg 39.58 7 3.33hijk 277 3.33jklm

Means followed by similar letters showed no significantly difference from each other by Tukey's test (p o0.05).

120 ab

95.5 7 2.02 93 7 1.91abc 85 7 2.04bcde 81.25 7 2.52cdef 75.75 7 2.46efg 59.25 7 1.65ij 100 7 0a 84.25 7 3.37ab 78.75 7 2.39b 76.25 7 3.17b 57.75 7 3.17defg 42.5 7 2.5ghij

100 7 0a 96 7 1.58ab 92.25 7 1.31abc 90.75 7 0.75abc 85.5 7 2.02bcde 78.25 7 1.18def 100 7 0a 86.25 7 2.39ab 80 7 2.04b 78.75 7 3.75b 58.75 7 3.75def 46.25 7 2.39efghi

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Table 2 Mortality percentage of newly ecdysed fifth instar larvae of G. pyloalis by topical bioassay method over the fourth day. Insecticides

Nano-imidacloprid

Imidacloprid

Concentrations (ppm)

10 14.5 22 32 48 14.5 22 32 47.5 60

Percent mortality at times (h) after treatment 24

48

72

96

13.1777 0.0968r 21.3557 0.1937n 31.322 7 0.1927j 34.1667 0.0881i 67.1477 0.0866d 16.1887 0.240q 18.1337 0.088p 33.0447 0.155l 49.9667 0.088h 75.9667 0.1452d

15.2337 0.1452q 23.1447 0.0987m 35.1337 0.0881h 38.3 7 0.1732g 70.0337 0.0881c 18.0617 0.1428p 20.026 7 0.0371o 35.0337 0.0881k 52.067 0.124g 78.0167 0.095c

16.488 7 0.2474p 26.622 7 0.1746l 38.144 7 0.0987g 42.2 7 0.115f 72.819 7 0.117b 20.023 7 0.033o 22.016 7 0.0440n 36.542 7 0.0632j 54 7 0.0577f 80.043 7 0.058b

18.277 70.0909o 30.23370.1452k 41.655 70.0867f 48.23370.1452e 78.790 70.123a 20.016 70.0218o 24.046 70.0785m 38.244 70.072i 56.026 70.0648e 82 70.0346a

Means followed by similar letters showed no significantly difference from each other by Tukey's test (po 0.05).

Fig. 3. Comparison of LC50 values of bulk- and nano-imidacloprid. (A) Comparison of LC50 values of bulk and nanoform of imidacloprid using leaf dip bioassay over five days on G. pyloalis larva. (B) Comparison of LC50 values of bulk and nanoform of imidacloprid using topical bioassay over four days on G. pyloalis larva.

Topical bioassay was used to evaluate the performance of nano-imidacloprid prepared in ethanol (Table 2). In this method, ethanol was used as a volatile solvent. The ANOVA and mean comparison in a split-plot in time design showed that as exposure time increased for both bulk and nano-imidacloprid, the mortality rate of G. pyloalis increased significantly. These results indicate that the effect of concentration, time and the interaction of time and concentration for nano- and bulkimidacloprid were significant. Comparison of LC50 at 24, 48, 72 and 96 h showed that at the all periods of exposure, LC50 was significantly lower for nano-imidacloprid than free imidacloprid. This means that less nano-imidacloprid was necessary as an essential dose for pest control, which decreases probable environmental risk significantly (Fig. 3(B)). The topical bioassays confirmed that using the nanoform allowed successful delivery to the target sites and decreased loss of pesticide. In particular, the increased penetration of the effective material by means of citric acid molecules to the metathoracic tergum membrane cells of G. pyloalis larvae enhanced the effectiveness of nanoimidacloprid prepared with ethanol.

Molecular self-assemblies of PCA–-PEG–PCA copolymers enabled it to encapsulate imidacloprid. As the bioassay results show, this property of PCA–PEG–PCA copolymers improved the insecticidal efficiency of imidacloprid, decreased the dosage and frequency of pesticide use and increased the length of effectiveness. Furthermore, the potential of dendrimers to interact with labile or poorly soluble drugs may enhance drug stability and bioavailability. Encapsulation of a drug within a dendrimer may also provide a means of controlling its release (Wurm and Frey, 2011). These biodegradable and biocompatible copolymers do not add pollutants to the environment and can be recommended for use. It is necessary to investigate the effects of residues of imidacloprid and nano-imidacloprid in a model plant field ecosystem to ensure its safety. Research in this area is currently in progress.

4. Conclusion Technologies such as nanoencapsulation and controlled release methods enable bulk pesticides to dissolve in water more effectively

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and increase their efficacy. Nanotechnology research in the field of agriculture also aims to reduce pollution from pesticides and make agriculture more environment friendly (Bhattacharyya et al., 2010). Because it is necessary to use water to dilute pesticides in agricultural applications, the investigation of nano-imidacloprid prepared in ethanol and diluted in water was the focus of this study. in vitro and in vivo experiments verified the preparation of controlled release formulation of imidacloprid applying PCA–PEG–PCA copolymers as biocompatible and biodegradable linear-dendritic copolymers. This formulation is more advisable and is introduced here as a promising and eco-friendly pesticide formulation in accordance with the objectives of precision farming to maximize output (crop yields) while minimizing input (pesticides). Acknowledgment Authors would like to thank Iran National Science Foundation for their financial support of this project. We also extend our appreciation to Professor Nosratallah Mahmoodi. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.05.009. References Abhilash, P.C., Singh, N., 2009. Pesticide use and application: an Indian scenario. J. Hazard. Mater. 165, 1–12. Alle´mann, E., Gurny, R., Doekler, E., 1993. Drug-loaded nanoparticles preparation methods and drug targeting issues. Eur. J. Pharm. Biopharm. 39, 173–191. Anjali, C.H., SudheerKhan, S., Margulis-Goshen, K., Magdassi, S., Mukherjee, A., Chandrasekaran, N., 2010. Formulation of water-dispersible nanopermethrin for larvicidal applications. Ecotoxicol. Environ. Saf. 73, 1932–1936. Bhattacharyya, A., Bhaumik, A., Rani, P.U., Mandal, S., Epidi, T., 2010. Nano-particles —a recent approach to insect pest control. Afr. J. Biotechnol. 9, 3489–3493. Cooper, J., Dobson, H., 2007. The benefits of pesticides to mankind and the environment. Crop Prot. 26, 1337–1348. Couvreur, P., 1988. Polyalkylcyanoacrylates as colloidal drug carriers. C.R.C. Crit. Rev. Ther. Drug Carrier Syst. 5, 1–20. Couvreur, P., Dubernet, C., Puisieux, F., 1995. Controlled drug delivery with nanoparticles: current possibilities and future trends. Eur. J. Pharm. Biopharm. 41, 2–13. D’Emanuele, A., Attwood, D., 2005. Dendrimer–drug interactions. Adv. Drug Deliv. Rev. 57, 2147–2162. Dhillon, G.S., Brar, S.K., Verma, M., Tyagi, R.D., 2011. Utilization of different agroindustrial wastes for sustainable bioproduction of citric acid by Aspergillus niger. Biochem. Eng. J. 54, 83–92. Fréchet, J.M.J., 1994. Functional polymers and dendrimers: reactivity, molecular architecture, and interfacial energy. Science 263, 1710–1715.

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