Preparation and characterization of enzyme-responsive emamectin benzoate microcapsules based on a copolymer matrix of silica–epichlorohydrin–carboxymethylcellulose

Abstract

Controlled release formulations of pesticides is highly desirable for maximising the utilization of the pesticide, as well as remarkably reducing environmental pollution. A stimuli-responsive controlled release formulation can intelligently respond to the stimuli produced by pests and trigger the release of the active ingredients to control pests effectively. In this work, a novel enzyme-responsive emamectin benzoate microcapsule was prepared using silica cross-linked with carboxymethylcellulose using epichlorohydrin. The results showed that the obtained microcapsules had a remarkable loading ability for emamectin benzoate (about 35% w/w) and could protect emamectin benzoate against photo- and thermal degradation effectively. The silica–epichlorohydrin–carboxymethylcellulose microcapsules displayed excellent cellulase stimuli-responsive properties and a sustained insecticidal efficacy against Myzus persicae. Allium cepa chromosome aberration assays demonstrated that the microcapsules had less genotoxicity than the technical grade emamectin benzoate (referred to throughout the manuscript as the technical). Given these advantages, the enzyme-responsive emamectin benzoate microcapsules are worth extending as a novel safe strategy for sustainable crop protection.

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1. Introduction

Controlled release technology is defined as a system in which the concentration of an active ingredient is preset, has sustained release properties and thus achieves the desired results.^1–5 It has increasingly gained scientific and commercial interest worldwide over the last few decades and has been identified as an emerging technology which is widely applied in the fields of medicine, coatings, pesticides, cosmetics, environmental engineering and food.^6–11 The controlled release formulation of pesticides is highly desirable for maximising the utilization of the pesticide, as well as remarkably reducing environmental pollution.^12–15 Due to the conservation and selective permeation properties of semi-permeable membranes, the use of pesticide microcapsules has been widely applied.^16–20 However, the active ingredient release of the existing pesticide microcapsules occurs typically through passive diffusion release, erosion of the capsule wall, or active diffusion via osmotic pressure, which results in poor control of the release of the core materials. Therefore, the development of new and advanced stimuli-responsive pesticide microcapsules with intelligently controlled release by internal and external stimuli has broad prospects.^21–26

The wall materials of microcapsules often include organic polymers and inorganic molecules. Mesoporous silica materials are always ideal candidates as drug carriers because of their large surface area, high drug loading capability, easy preparation and surface modification, good stability and biocompatibility.^27–31 Carboxymethylcellulose is an anionic linear polysaccharide derived from natural cellulose that has been widely used as a shell material for microcapsules due to its high biocompatibility, biofunctionality, and versatile chemical and physical properties.^32–34 Currently, many new stimuli-responsive systems for drug release combining the advantages of silica nanoparticles and polymers have been built. Different kinds of stimuli-responsive polymers have been functionalized on the surfaces of mesoporous silica materials via direct covalent bonds^35–38 or the method of assembly.^39–42

Stimuli-responsive systems for “on-demand” release respond to a range of stimuli, including enzymes, pH, temperature, photoirradiation, redox, competitive binding, magnetic fields and electric fields.^43–45 Among these stimuli, reduced pH and an increased level of cellulase are achieved when plants suffer from an insect pest which is already present or about to invade.^23,46 An alkaline condition is obtained in insects with an alkaline gut.²⁴ Despite many burgeoning achievements in human medicine, the application of stimuli-responsive systems in agriculture is still in its infancy. Therefore, the search for effective stimuli-responsive systems for pesticides with an “on-demand” release that, in particular, respond to internal biological stimuli still remains a big challenge in this field.

Emamectin benzoate is a broad-spectrum, high-efficiency, and low-toxicity macrocyclic lactone insecticide derived from avermectins, produced by Streptomyces avermitilis, that has been applied for the control of pests on a variety of crops worldwide.⁴⁷ It is 18–80 400-fold more potent against Plutella xylostella, Trichoplusia ni and Spodoptera exigua, than other traditional insecticides such as fipronil, chlorfenapyr, and tebufenozide.⁴⁸ However, it is sensitive to light and strongly alkaline or strongly acidic conditions, so its biological activity is limited greatly in application.⁴⁹

In the present work, novel functionalized emamectin benzoate microcapsules were prepared using silica cross-linked with carboxymethylcellulose using epichlorohydrin. Initially, amino-functionalized silica microcapsules were synthesized by an emulsion polymerization method using alkoxysilane as a precursor, while epichlorohydrin modified carboxymethylcellulose was synthesized. Finally, the epichlorohydrin modified carboxymethylcellulose was conjugated on the surface of the silica microcapsules, resulting in emamectin benzoate microcapsules based on a copolymer matrix of silica–epichlorohydrin–carboxymethylcellulose. The preparation conditions of the emamectin benzoate microcapsules, the effects of pH, temperature and cellulase on the sustained-release performance, stability of emamectin benzoate in the microcapsules, and the bioactivity and genotoxicity of the emamectin benzoate microcapsules were investigated.

2. Experimental

2.1 Materials

The model pesticide, emamectin benzoate (95%), was supplied by Hebei Veyong Bio-Chemical Co., Ltd. (Hebei, China). Emamectin benzoate 1% emulsifiable concentrate (EC) was purchased from Syngenta crop protection Co., Ltd. (Suzhou, China). Sodium carboxymethylcellulose (CMC), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), ammonium hydroxide, hydrochloric acid, acetic acid, ethanol, ethyl acetate, epichlorohydrin, and hexadecyl trimethyl ammonium chloride (CTAC) were analytical chemicals purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). Carbol fuchsin and cellulase from Trichoderma viride with an activity of 3–10 units per mg of solid were obtained from Sigma Aldrich (St. Louis, USA). Acetonitrile and methanol were of HPLC grade and purchased from J. T. Baker (USA). Deionized water was used for all reactions and treatment processes. Myzus persicae were supplied by the insect laboratory at China Agricultural University.

2.2 Preparation of emamectin benzoate microcapsules

2.2.1 Synthesis of amino-functionalized silica microcapsules. The water phase was prepared by dissolving 1.5 g of CTAC in 100 mL deionized water (1.5% m/v of the water phase). The oil phase was obtained by dissolving 0.5 g of emamectin benzoate in a mixture of 5.0 mL of ethyl acetate and 4.0 mL of TEOS. The oil phase was dispersed in 84.5 mL of the water phase and the mixture was homogenized at 6000 rpm for 5 min to form an oil/water emulsion using a high-speed homogenizer (T18 digital ULTRA-TURRAX, IKA, Germany). The emulsion was poured into a three-necked flask equipped with a temperature controlled magnetic stirrer and continuously stirred at 600 rpm. Subsequently, 0.1 mL of the concentrated ammonia solution was added dropwise to the flask and constantly stirred for a further 2 h. After allowing the mixture to age overnight at room temperature, silica microcapsules were obtained. Finally, 0.8 mL of APTES was added to the flask and the reaction mixture was stirred at 50 °C for another 2 h. The precipitate was then filtered off, washed thrice with distilled water, centrifuged (6000 rpm) and dried at 60 °C.

2.2.2 Synthesis of epichlorohydrin modified carboxymethylcellulose (EMC). The aqueous solution of CMC was prepared by dissolving 2.5 g of CMC in 200 mL of distilled water. Thereafter, 2.5 mL of epichlorohydrin and 0.5 mL 6 M HCl were added and maintained under mechanical agitation for 24 h at 50 °C. Afterwards, ethanol was added to precipitate the modified CMC. After filtration, the modified CMC was lyophilized. The material obtained was triturated and stored in desiccators for later characterization and reaction.

2.2.3 Synthesis of silica–epichlorohydrin–carboxymethylcellulose microcapsules. A solution of EMC was obtained by dissolving 1.0 g EMC in 100 mL distilled water. Then 1.0 g of the resulting amino-functionalized silica microcapsules and a certain amount of sodium bicarbonate were added and continuously stirred for 24 h at 50 °C. Finally, the solution was centrifuged and washed thrice with deionized water. The final emamectin benzoate microcapsules based on a copolymer matrix of silica–epichlorohydrin–carboxymethylcellulose were obtained after centrifugation, and dried at 60 °C in an oven.

2.3 Characterization

2.3.1 Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra, recorded on a Jasco FT-IR 5300 spectrophotometer, were employed to identify the different functional groups present in the samples. Samples were prepared as KBr pellets and scanned against a blank KBr pellet background at wavenumbers ranging from 4000 to 450 cm⁻¹ with a resolution of 4.0 cm⁻¹.

2.3.2 SEM observation. To observe the morphology and structure of emamectin benzoate microcapsules, scanning electron microscopy (SEM, Hitachi S4800, Japan) was used.

2.3.3 Thermogravimetric analysis. Thermogravimetric analysis (TGA) of emamectin benzoate microcapsules was performed with a SDT Q600 (TA Instruments-Waters LLC, USA) analyzer from 25 to 700 °C with a heat rate of 10 °C min⁻¹ to determine the loading efficiency of emamectin benzoate.

2.3.4 Particle size analysis. To determine the size and distribution of the emamectin benzoate microcapsules, a Mastersizer (Mastersizer 3000, Malvern Instruments Co., UK) was employed.

2.4 High performance liquid chromatography (HPLC) analysis

The concentration of emamectin benzoate was determined by HPLC with an ultraviolet detector (Shimadzu, Japan). The HPLC separation of emamectin benzoate was carried out on a Kromasil ODS C₁₈ column (250 mm × 4.6 mm, 5 μm; DIKMA, USA), which was equipped with a guard column (4 mm × 3 mm; Kromasil EasyGuard II C₁₈) and pre-equilibrated with a mobile phase composition of acetonitrile and water with 0.1% acetic acid (80 : 20, v/v) for 30 min. The flow rate was constant at 1 mL min⁻¹ and the column temperature was at room temperature. The injection volume was 20 μL and the detection wavelength was 245 nm. All the solvents were filtered with a 0.45 μm membrane filter.

2.5 Stability test

20 g of the prepared emamectin benzoate microcapsule solution (2%, w/v) was packed in glass tubes and stored at 40, 50, and 60 °C for a period of 60 days, and then the changes of the emamectin benzoate were analyzed. The stability of the emamectin benzoate microcapsules against UV radiation was tested by exposing the samples to a 36 W germicidal lamp (254 nm) at a distance of 20 cm and keeping the temperature at room temperature, and then analyzing the changes of the emamectin benzoate. The methanol solution of the technical grade emamectin benzoate was used as the control sample at the same time.

2.6 Controlled release kinetics

The resulting emamectin benzoate microcapsules were weighed and suspended in 500 mL of the methanol–water mixture (30 : 70, v/v), which was used as a release medium in order to dissolve the emamectin benzoate, and incubated at a stirring speed of 100 rpm for a given time at room temperature. The mixture, collected at different intervals, was then centrifuged, and the supernatant was determined by using HPLC and the cumulative release rate of the emamectin benzoate from the microcapsules was calculated to evaluate the sustained release properties. The release behaviors of the emamectin benzoate microcapsules at different pH values, temperatures and cellulase conditions were investigated. In an enzyme experiment, 10 mg of emamectin benzoate microcapsules were suspended in 18.75 mL of the methanol–water mixture (5 : 95, v/v) at pH 7.0 and then 6.25 mL of the enzyme solution (0.4 g of enzyme in 100 mL of water at pH 7.0) was added. For the release studies of the microcapsules without enzyme, 10 mg of the microcapsules were placed in 25 mL of the methanol–water mixture (5 : 95, v/v) at pH 7.0 and after a certain time an aliquot was separated, filtered and examined.

2.7 Bioactivity

The bioactivity of the emamectin benzoate microcapsules against M. persicae was assayed using a modification of the pot test described by Lowery et al.⁵⁰ An insecticide-susceptible strain of the green peach aphid (M. persicae) was maintained in the laboratory on Chinese cabbage (Brassica pekinensis (Lour) Rupr) at 25 °C, 60% relative humidity and an L16:D8 photoperiod. Groups of 100 adult aphids were inoculated on the leaves of each Chinese cabbage at the four-leaf stage in a cage (60 × 60 × 60 cm) covered with gauze on all sides. 10 mL of different concentrations of the microcapsules and emamectin benzoate 1% EC (3, 6, and 12 mg L⁻¹ of emamectin benzoate) were sprayed on each Chinese cabbage with a microaerosol sprayer, and with water as the control. Each concentration was performed in triplicate. The decline rate of the insect density and control efficiency were calculated at 1, 7, 14, and 21 days after spraying.

2.8 Allium cepa chromosome aberration assays

Allium cepa assays were carried out based on the protocol to evaluate the genotoxicity of chemicals proposed by Rank and Nielsen.⁵¹ A. cepa seeds were germinated in distilled water until the roots had reached a length of about 2 cm, then they were subjected to different concentrations of emamectin benzoate (10, 50, and 100 mg L⁻¹), either free or encapsulated in the microcapsules, for periods of 24 h. Distilled water was used as the control. The roots were fixed using the ethanol–acetic acid mixture (3 : 1, v/v). After fixing, the samples were washed in distilled water and then immersed in a solution of 1 M HCl at 60 °C for 9 min. The meristematic region of the root tip was cut, placed on a slide together with a drop of carbol fuchsin, and covered with a cover slip which was used to gently squash and spread the cells. About 500 cells were observed on each slide under an optical microscope to determine the mitotic index, which considers the proportion of cells in division, and the chromosomal aberration index, which considers the proportion of divisions with chromosomal aberrations. Each treatment was performed in triplicate.

3. Results and discussion

3.1 Preparation of emamectin benzoate microcapsules

In this work, novel functionalized emamectin benzoate microcapsules were prepared using silica cross-linked with carboxymethylcellulose using epichlorohydrin. Emamectin benzoate was inserted into the silica shell formed by the hydrolysis of TEOS using the emulsion polymerization method. Silica shells were modified with APTES to obtain amino-functionalized silica microcapsules. Meanwhile, EMC was synthesized. Finally, EMC was mixed and cross-linked with the resulting amino-functionalized silica microcapsules. The formation mechanism of the emamectin benzoate microcapsules is presented in Scheme 1.

Scheme 1 The possible mechanism for the preparation of the silica–epichlorohydrin–carboxymethylcellulose microcapsules.

3.1.1 Effects of CTAC. The preliminary experiment showed that CTAC was the optimal surfactant to stabilize the oil/water emulsion. It was proved that the formation and diameter of the microcapsules was greatly influenced by the concentration of CTAC. When the concentration of CTAC was lower than 1%, a stable emulsion was not obtained, which resulted in the separation of the oil phase and water phase, and ruptured the microcapsule walls without the emamectin benzoate coating (Fig. 1A1). With the concentration of CTAC increasing, the dispersion and stability of the emulsion droplets improved. When the concentration of CTAC was higher than 1.0%, spherical and intact microcapsules were achieved (Fig. 1A2 and A3). The mean particle sizes of the emulsion were 1.0 μm and 3.0 μm when the concentrations of CTAC were 2.0% and 1.5%, respectively. When the concentration of CTAC was higher than 2.0%, the particle size distribution and morphology of the emulsion was no different. The particle size distribution of the microcapsules is shown in Fig. 2.

Fig. 1 SEM images of the silica microcapsules prepared with different amounts of CTAC, TEOS and EMC. (A1) CTAC 0.5%, (A2) CTAC 1.5%, (A3) CTAC 2.0%, (B1) TEOS 4 mL, (B2) TEOS 4.5 mL, (B3) TEOS 5 mL, (C1) EMC 0.5 g, (C2) EMC 1.0 g, (C3) EMC 1.5 g.

Fig. 2 Particle size distribution of silica microcapsules.

3.1.2 Effects of TEOS. TEOS was used as a wall material and the amount used had a significant influence on the wall thickness and morphology of the microcapsules. It was observed that the wall thickness of the microcapsules rose with an increased amount of TEOS (Fig. 1B1 and B2). However, the results did not conclusively demonstrate that more TEOS can result in better microcapsules. When the amount of TEOS reached 5 mL, grapelike clusters of microcapsules were fabricated (Fig. 1B3).

3.1.3 Effects of EMC. EMC, used as the outer wall material which intelligently responded to internal and external stimuli, was a vital component of the stimuli-responsive microcapsules. Different amounts of EMC (0.5, 1.0, 1.5 g) were dissolved in 100 mL of distilled water to obtain solutions of EMC. Afterwards, the solutions of EMC were conjugated with the resulting amino-functionalized silica microcapsules using sodium bicarbonate as the acid binding agent. When the amount of EMC was less than 1 g, the silica microcapsules were partially conjugated with EMC, and the unstable outer wall can be easily disaffiliated from the surface of silica microcapsules (Fig. 1C1). When the silica microcapsules were conjugated with a larger amount (1.5 g) of EMC, a large number of microcapsules were merged into a chunk (Fig. 1C3). The desired microcapsules with a stable outer wall were obtained by using 1.0 g EMC (Fig. 1C2). It was found that the particle size of the microcapsules increased with an increase in the amount of EMC. For this process, a certain amount of sodium bicarbonate was used to maintain pH 7.5–8.0 to achieve completion of the conjugation reaction. The excess sodium bicarbonate and sodium chloride would not affect the particle size and morphology of the microcapsules.

3.2 Characterization of emamectin benzoate microcapsules

3.2.1 Results of Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra of silica, amino-functionalized silica, EMC, silica–epichlorohydrin–carboxymethylcellulose and sodium carboxymethylcellulose are shown in Fig. 3. Compared with silica (Fig. 3a), the spectrum of the amino-functionalized silica showed that the characteristic stretching vibration bands of the amine (–NH₂) at 3401 cm⁻¹ and the absorption band of the methylene group (–CH₂–) at 2985 cm⁻¹ appeared (Fig. 3b), which indicated that the APTES was successfully attached on the surface of the silica. The sodium carboxymethylcellulose showed a broad band at 3416 cm⁻¹, corresponding to the stretching vibration modes of the –OH groups. The peak at 2877 cm⁻¹ was attributed to the C–H stretching vibration. Two absorption bands at 1587 and 1427 cm⁻¹ confirmed the presence of the COO⁻ groups (Fig. 3e). The spectrum of EMC exhibited a new absorption band of the carbonyl group (C=O) of the ester at about 1748 cm⁻¹ compared with sodium carboxymethylcellulose, demonstrating that the oxirane group of epichlorohydrin successfully reacted with the carboxyl of the carboxymethylcellulose (Fig. 3c). The silica–epichlorohydrin–carboxymethylcellulose exhibited the absorption band of the methylene group (–CH₂–) at 2985 cm⁻¹ and the carbonyl group (C=O) of the ester at about 1748 cm⁻¹. The absorption band of the amine (–NH₂) at 3401 cm⁻¹ was also replaced by a broad band of the carboxymethylcellulose at 3416 cm⁻¹, and the intensity decrease of the absorption band at 1550 cm⁻¹ (N–H bending of the primary amine salt) demonstrated that EMC was linked with the amino-functionalized silica (Fig. 3d).

Fig. 3 FT-IR spectra of silica (a), amino-functionalized silica (b), EMC (c), silica–epichlorohydrin–carboxymethylcellulose (d) and sodium carboxymethylcellulose (e).

3.2.2 Thermogravimetric analysis. The TGA curves of blank silica microcapsules, silica microcapsules with emamectin benzoate, silica–epichlorohydrin–carboxymethylcellulose microcapsules, and silica–epichlorohydrin–carboxymethylcellulose microcapsules with emamectin benzoate are depicted in Fig. 4. The results showed that the weight loss before 180 °C was probably attributed to the evaporation of water in the microcapsules, and the weight loss between 180 and 270 °C might be attributed to the evaporation and decomposition of emamectin benzoate, whereas the weight loss over 270 °C could be due to the decomposition of the carboxymethylcellulose shells. The total weight losses of the microcapsules in the range of 180–700 °C were about 60.0% and 25.0%, so the loading efficiency of emamectin benzoate was about 35%.

Fig. 4 TGA curves of silica microcapsules (a), silica microcapsules with emamectin benzoate (b), silica–epichlorohydrin–carboxymethylcellulose microcapsules (c), and silica–epichlorohydrin–carboxymethylcellulose microcapsules with emamectin benzoate (d).

3.3 Thermal and light stability

The prepared emamectin benzoate microcapsule solution was stored at 40, 50 and 60 °C for a period of 60 days to determine the effects of temperature variation on the stability of microcapsules. Fig. 5a shows that emamectin benzoate microcapsules are more stable than the technical under high temperatures. The decomposition rate of the technical at 40, 50 and 60 °C over 60 days was higher than that of the microcapsules which exhibited less than 3% decomposition. Fig. 5b shows the effects of UV radiation on the stability of the emamectin benzoate microcapsules at pH 7 and 25 °C. Emamectin benzoate was sensitive to light, and the irradiated technical samples were degraded completely within 48 h. Nevertheless, the decomposition rate of the emamectin benzoate wrapped in microcapsules was found to be less than 25% after 72 h of UV radiation. These results evidently showed that emamectin benzoate could be protected by the microcapsule wall, demonstrating the excellent UV-shielding properties of the resulting microcapsules for emamectin benzoate.

Fig. 5 Stability of the resulting emamectin benzoate microcapsules and the technical affected by (a) temperature and (b) UV radiation .

3.4 Controlled release kinetics

In order to develop the intelligent, self-regulated controlled release formulation of pesticides, the novel stimuli-responsive systems that respond to internal biological stimuli produced by pests were prepared using mesoporous silica materials cross-linked with natural polymers via direct covalent bonds. The stimuli-responsive microcapsules respond to the stimuli produced by pests and trigger the release of the active ingredients to control the pests effectively. The possible mechanism of formation of the enzyme-responsive microcapsules and triggered release by cellulase is schematically presented in Scheme 2.

Scheme 2 The possible mechanism of the formation of the enzyme-responsive microcapsules and triggered release by cellulase.

3.4.1 Effects of pH. Fig. 6a shows the release behaviors of emamectin benzoate from the microcapsules (3.0 μm) under different pH values (5.0, 7.0 and 9.0) at 25 °C. The cumulative release rates were 33.17%, 9.79% and 52.77% at pH 5.0, 7.0, 9.0 after 36 h and eventually reached 57.52%, 15.17% and 80.13% after 120 h, respectively. The cumulative release rate of the emamectin benzoate was the lowest at pH 7.0, demonstrating that the hydrolysis rate of the ester bond was low under medium conditions. The release of the emamectin benzoate from the microcapsules at pH 9.0 was faster than that at pH 5.0. That was probably because the microcapsule wall was hydrolyzed more easily at a high pH, which made the emamectin benzoate release more rapidly from the microcapsules.

Fig. 6 Effect of the pH value (a), temperature (b) and cellulase (c) on the release behaviors of the emamectin benzoate from the emamectin benzoate microcapsules.

3.4.2 Effects of temperature. Fig. 6b shows the release behaviors of the emamectin benzoate from the microcapsules (3.0 μm) at different temperatures at pH 7.0. The cumulative release rates increased from 55.21% to 66.87% in the temperature range of 25 to 45 °C after 60 days, which showed that the presence of high temperatures could increase the speed of the decomposition of the microcapsule wall.

3.4.3 Effects of cellulase. Fig. 6c shows the release behaviors of the emamectin benzoate from the microcapsules (3.0 μm) in the presence of cellulase at 25 °C and pH 7.0. A relatively high release rate of the emamectin benzoate was found in the presence of cellulase. The active ingredient was released, with 28.66% being released after 1 h, and reached a maximum at 20 h. When the microcapsules were treated with enzyme, EMC, which was used as the outer wall material was degraded by cellulase, which randomly cleaved the cellulose into smaller fragments.⁵² However, there was no active ingredient observed in the presence of non-related enzymes (pronase, amidase). Therefore, the release of the active ingredient from the emamectin benzoate microcapsules was triggered by cellulase.

3.5 Bioactivity

Fig. 7 shows the bioactivity of the emamectin benzoate microcapsules against M. persicae in concentrations ranging from 3–12 mg L⁻¹. The results indicated that the control efficiency of emamectin benzoate 1% EC 1 day after spraying against M. persicae was better than that of the microcapsules at the same concentrations, while the control efficiency of the microcapsules against M. persicae was better than that of the emamectin benzoate 1% EC at the same concentrations since 7 days after spraying. That was probably because the stimuli, including pH and the cellulase produced by M. persicae, were weak at 1 day after spraying, and then the stimuli-responsive microcapsules, responding to the intensifying stimuli, released the active ingredient more and faster over time. The stimuli-responsive emamectin benzoate microcapsules exhibited a better and more sustained insecticidal efficacy against M. persicae than emamectin benzoate 1% EC.

Fig. 7 Insecticidal activity of the emamectin benzoate microcapsules against Myzus persicae.

3.6 Allium cepa chromosome aberration assays

Allium cepa assays are widely used in the analysis of general genotoxicity and are sufficiently sensitive for monitoring purposes.⁵¹ Genotoxic compounds reduce the mitotic index of the A. cepa cells, as indicated by chromosome abnormalities that increase as a function of the concentration of the chemical. Fig. 8 shows the genotoxicity of the emamectin benzoate microcapsules against A. cepa at the concentrations of 10, 50, and 100 mg L⁻¹.

Fig. 8 Genotoxicity of the emamectin benzoate microcapsules against A. cepa. (A) mitotic index; (B) chromosome aberration index. The statistical analysis was performed by comparison with (a) the control, and (b) the technical. * Indicates a statistically significant difference at 0.05 level.

There was a decreased mitotic index for the microcapsules and the technical compared with the control. The mitotic index diminished as the concentration of the microcapsules and the technical increased, and the difference between the technical at a concentration of 100 mg L⁻¹ and the control was statistically significant. The mitotic index of the microcapsules at a concentration of 100 mg L⁻¹ was significantly higher than that of the technical at the same concentration, demonstrating that the microcapsules reduced the cytotoxicity of the technical (Fig. 8A). The chromosomal aberration index of the microcapsules and the technical was higher than that of the control. Higher concentrations of the microcapsules and the technical increased the chromosomal aberration index. Significantly greater index values were obtained for the treatments using the technical at concentrations of 50 and 100 mg L⁻¹, compared to the control and the microcapsules at the same concentration, while the index values between the microcapsules and the control were similar, suggesting that the microcapsule wall provided a degree of protection against genotoxic effects (Fig. 8B).

4. Conclusions

In the present work, we prepared a novel enzyme-responsive emamectin benzoate microcapsule using silica cross-linked with carboxymethylcellulose using epichlorohydrin. The results showed that the resulting microcapsules had a remarkable loading ability for emamectin benzoate (about 35% w/w) and could protect emamectin benzoate against photo- and thermal degradation effectively. The silica–epichlorohydrin–carboxymethylcellulose microcapsules displayed excellent cellulase stimuli-responsive properties and a sustained insecticidal efficacy against M. persicae. A. cepa chromosome aberration assays demonstrated that the microcapsules caused less chromosome damage compared to the technical. It was concluded that the enzyme-responsive controlled release formulation provided a useful means of controlling agricultural pests, simultaneously reducing the risk of harm to the environment and human health.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31471799, 31171888) and the National Department Public Benefit Research Foundation of China (201303031).

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