Article pubs.acs.org/JAFC
Phoxim Microcapsules Prepared with Polyurea and Urea− Formaldehyde Resins Differ in Photostability and Insecticidal Activity Da-xia Zhang,†,‡ Bei-xing Li,†,‡ Xian-peng Zhang,†,# Zheng-qun Zhang,⊥ Wei-chang Wang,†,# and Feng Liu*,†,# †
Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection; ‡Research Center of Pesticide Environmental Toxicology; #Key Laboratory of Pesticide Toxicology & Application Technique; and ⊥College of Horticultural Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, People’s Republic of China S Supporting Information *
ABSTRACT: The application of pesticide microcapsules (MCs) in agriculture is becoming more and more popular. In this study, the effects of different wall materials on the stomach toxicity, contact toxicity, length of efficacy, and photolysis characteristics of pesticide microcapsules were investigated. The results showed that microencapsulation reduced the stomach and contact toxicities of phoxim and prolonged the efficacy of this light-sensitive chemical in the greenhouse test. Neither of the degradation curves for microencapsulated phoxim under ultraviolet light fit a first-order model, although the emulsifiable concentrate (EC) degradation curve fit it well. The phoxim-loaded polyurea microcapsules (PUA-MCs) showed significantly increased UV-resistance ability, stomach toxicity, and contact toxicity compared with the phoxim-loaded urea−formaldehyde microcapsules (UF-MCs). These experiments indicated that it is crucial to select the appropriate wall materials for pesticide microcapsules on the basis of application sites and physicochemical properties of pesticide active ingredients. KEYWORDS: phoxim, microcapsule, urea−formaldehyde resin, polyurea, contact toxicity, stomach toxicity
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INTRODUCTION
polyurea, polyurethane, and polyamide are usually used for interfacial polymerization. The capsule wall material is one of the most important components because it is a key factor that influences the physical and chemical properties of the microcapsules, including their release characteristics and mechanical strength.14,15 Yin et al.16 prepared nanocapsules using methyl methacrylate and styrene as wall material and improved their nematicidal activity. Asrar et al.17 prepared tebuconzaole microcapsules with poly(methyl methacrylate) or poly(styrene-co-maleic anhydride) as wall material and demonstrated that controlled-release formulations exhibited better biological activity than traditional foliar applications. Yang et al.18 and Yuan et al.19 successfully encapsulated a mixture of fipronil and chlorpyrifos with a melamine−formaldehyde resin and proved that these microcapsules had better activity against white grubs than conventional formulations. However, López et al.20 reported that microencapsulation of Schinus molle (Anacardiaceae) essential oil using a mixture of maltodextrin and gum arabic as wall material reduced control of flies. Therefore, microencapsulation may increase or reduce pesticide biological activity due to differences in wall materials. Thus, it is crucial to select appropriate wall materials to develop successful pesticide microcapsules. How insecticides kill pests primarily depends on the mode of action of the stomach and contact toxicities. In addition, when pesticides are sprayed onto the plant surface, light is a key
Chemical control has been the most common strategy for controlling pests in past decades. Some pesticides are so easily degraded by microorganisms and sunlight that it has been necessary to increase the initial amounts and application frequencies of pesticides to promote the effective duration for protecting crops from being damaged. However, this not only increases the economic investment and labor of farmers but also adds more chemicals into the environment. Microencapsulation technology has received extensive attention in the domain of agrochemicals.1,2 It provides isolation from the external environment by using various wall materials, which consequently improve the chemical stability of the active ingredients. Rapid advancements in microencapsulation are closely related to the following advantages: (1) microencapsulation can protect the active ingredients from being affected by environmental factors, such as air, sunlight, rain, and microorganisms;3 and (2) it can control the release rate of pesticides from microcapsules to prolong the duration of the pesticides and reduce their application frequency.4 Microcapsules (MCs) can be prepared through numerous methods, including phase separation,5−7 interfacial polymerization,8−10 in situ polymerization,11,12 and layer-by-layer polyelectrolyte deposition.13 To date, more than 150 pesticide microcapsule preparations have been officially registered by the Ministry of Agriculture of China, including insecticides, fungicides, and herbicides. In the future, there will be more pesticide microcapsule products used in agriculture. Among them, in situ and interfacial polymerization are the most commonly used microencapsulation technologies. Urea−formaldehyde and melamine resins are common wall materials for in situ polymerization, whereas © XXXX American Chemical Society
Received: January 15, 2016 Revised: March 21, 2016 Accepted: March 24, 2016
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DOI: 10.1021/acs.jafc.6b00231 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Characterization of the microcapsules containing phoxim: (A) particle size distribution; (B) SEM micrograph of UF-MC; (C) SEM micrograph of PUA-MC (magnified 4500×; acceleration voltage of 20 kV). 25 ± 1 °C and 70 ± 5%, respectively. Larva mortality was assessed after 8 and 24 h. The criterion for death was that a larva did not move when prodded with a camel hair brush. To enhance the experimental precision, each experiment was repeated in triplicate. Stomach Toxicity Evaluation. The feeding bioassays for the three samples on A. ipsilon fourth-instar larvae were performed using the leaf sandwich method.26 Phoxim EC, UF-MCs, and PUA-MCs were diluted to concentrations of 400, 500, 600, and 700 mg/L. Then, 1 μL of diluted solution was distributed on a cabbage leaf with a diameter of 0.5 cm. Subsequently, another leaf was used to fabricate the insecticide-loaded sandwiched leaves with the help of gelatinized corn starch. The insecticide-loaded leaves were used to feed larvae (within a weight range of 0.1252−0.1505 g) that had been starved for 24 h. For each treatment, 48 larvae were utilized, including a control group that was fed leaf sandwiches without phoxim. The assay was repeated three times. After treatment for 24 and 48 h, the relevant larva mortality was investigated. The eventually obtained frass from each treatment was collected and separately stored in the dark at 0 °C. The frass was ground and transferred to a brown volumetric flask with methanol to a final volume of 25 mL. The solution was sonicated for 1 h, and then phoxim was detected by HPLC. Conditions used for HPLC detection are described in the Supporting Information. UV Irradiation. Phoxim EC, UF-MCs, and PUA-MCs were diluted to a concentration of 3600 mg/L (active ingredient). Then, 1 mL of diluted solution was evenly smeared onto Petri dishes (6 cm diameter). After exposure to ultraviolet light with an average irradiation intensity of 18 μW/cm2 (25 ± 2 °C), the Petri dishes were removed every 30 min for further detection. The dishes were washed with methanol and filled to a constant volume of 25 mL. Then, the amount of phoxim present was detected using an HPLC system. How the duration of ultraviolet radiation influences contact toxicity was also investigated. Samples were diluted with distilled water to yield a 3600 mg/L solution. Then, 50 μL of diluted solution was transferred to 24-cell culture plates placed on a table concentrator (100 rpm, dark condition, 25 °C) to dry the solution. Subsequently, the plates were placed 30 cm under an ultraviolet lamp (18 μW/cm2). Irradiated plates were removed at different time points of 0, 1, 2, 3, 4, and 5 h to determine the toxicity of remaining phoxim on A. ipsilon fourth-instar larvae. Bioassay was performed in the same way as described above. Verification Test. Verification tests were performed in a greenhouse. The common phoxim concentration for foliar treatment was determined to be 200−600 mg/L. To determine the differences among UF-MCs, PUA-MCs, and EC and MCs, higher concentrations were also adopted. The samples were diluted to concentrations of 175, 350, 700, and 1400 mg/L for foliar treatment. The dilutions were then evenly sprayed on cabbage leaves. To promote foliage spreadability, 0.05% silicone additives (Silwet 408, Momentive Performance Materials, USA) were added to the dilution. Cabbage leaves from the untreated group were sprayed with an aqueous solution containing 0.05% Silwet 408. The cabbage was planted in a greenhouse with average daytime and night temperatures of 25 and 15 °C, respectively. Each treatment was randomly arranged with three replications. Treated cabbage leaves were picked periodically and cut with a 1 cm
factor that could result in the rapid degradation of the active ingredients. The above factors play vital roles in insecticide efficacy. In this study, phoxim was studied as an insecticide model. Phoxim, an organophosphate insecticide, has high stomach and contact toxicities to Lepidoptera larvae.21 However, it is unstable when exposed to light.22,23 In the current study, phoxim MCs were prepared with two different wall materials (PUA and UF), and their stomach and contact toxicities to the black cutworm, Agrotis ipsilon, were investigated. Ultraviolet irradiation experiments were carried out to examine the effect of UV irradiation on the remaining amounts of phoxim and the contact toxicity of PUA-MCs, UFMCs, and emulsifiable concentrate (EC). Finally, a greenhouse experiment was performed as a proof to study the effective duration of PUA-MCs and UF-MCs compared with EC.
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MATERIALS AND METHODS
Chemicals. The technical material (purity = 95%) of phoxim was kindly provided by Hubei Xianlong Chemical Industry Co. (Hubei, China). Other chemicals used to fabricate two different microcapsules are listed in the Supporting Information. Insects. A. ipsilon Rottemberg were continuously reared in a constant-environment room under standard conditions of 25 ± 1 °C, 70 ± 5% relative humidity, and a 16:8 light/dark cycle. Larvae were fed an artificial diet containing corn starch and vitamins. Fourth-instar A. ipsilon larvae were used for the bioassay experiments. Microencapsulation Procedure. UF-MCs were prepared using an in situ polymerization method according to previously reported procedures.11 PUA-MCs fabricated with the polyurea wall material were obtained using an interfacial polymerization method.24 The standard procedures used are described in the Supporting Information. Microcapsule Characterization. The particle size and size distribution of the MCs were measured with a laser particle size analyzer (LS-POP 6; Omec Instruments Co., Ltd., Guangdong, China) with D90, D50, and D10 representing the particle sizes at 90, 50, and 10%, respectively, the cumulative distribution in the sample. The appearance of MCs was observed using a scanning electron microscope (SEM) (JSM-6610LV, JEOL, Tokyo, Japan). Contact Toxicity Evaluation. A modified dry film bioassay method (derived from the typical glass-vial bioassay25) was adopted to assess the contact toxicity of the microencapsulated and unencapsulated phoxim in A. ipsilon fourth-instar larvae. The three samples, Phoxim-EC, UF-MC, and PUA-MC, were diluted with distilled water to yield concentrations of 2000, 1500, 1000, and 500 mg/L. After the transfer of 50 μL of the diluted solutions to each cell in 24-cell culture plates, the plates were placed on a table concentrator (100 rpm, 25 °C, dark treatment). Once the diluted solutions were thoroughly dried, 24 A. ipsilon fourth-instar larvae were placed in the plate cells for treatment. The tested insects were transferred to plates without phoxim an hour later. Fresh cabbage leaves were used to feed the larvae while the temperature and relative humidity were maintained at B
DOI: 10.1021/acs.jafc.6b00231 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry puncher. The A. ipsilon fourth-instar larvae were selected and placed into 24-cell plates containing treated leaves. Experiments were carried out at constant temperature (25 ± 1 °C) and repeated three times. Larva mortality was determined at 24 h. Statistical Analysis. All of the data were statistically analyzed using a SPSS software (version 16.0) and were displayed as means and standard deviations.
29 and 80%, which is significantly higher than that of both MC groups. Twenty-four hours after treatment (Figure 2B), the mortality increased dramatically. With regard to mortality, the different treatments ranked as UF-MCs < PUA-MCs < EC, which demonstrated that microencapsulation could significantly reduce phoxim contact toxicity. PUA-MCs also showed higher contact toxicity than UF-MCs. This phenomenon may be greatly owed to the microcapsule release mechanism. When the microcapsule particles were exposed to air, the core released the phoxim gradually by osmosis. The UF main structure is made of hydrophilic carbonyls and imino groups, whereas the PUA main structure consists of long hydrophobic carbon chains (Schemes S1 and S2 in the Supporting Information). As a result, phoxim is released more easily through PUA than UF. Stomach Toxicity. Stomach toxicities were determined using the leaf sandwich method.26 The results are shown in Figure 3. At 24 h after treatment, when the treatment dose is
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RESULTS AND DISCUSSION Microcapsule Characterization. The size distributions of the PUA-MCs and UF-MCs were found to be unimodal (Figure 1A). The PUA-MCs D50 was 5.14 μm, whereas the D10 and D90 were 1.47 and 11.40 μm, respectively. The UF-MCs size distribution was similar to that of the PUA-MCs, with D10, D50, and D90 being 1.33, 4.52, and 9.83 μm, respectively. Both wall materials are nonporous membranes, which is apparent in the SEM images (Figure 1B,C). With regard to the PUA-MCs and UF-MCs containing phoxim, which is the active ingredient encapsulated in the polymeric shell, they do not have significant bioactivity unless the phoxim is released from the MCs. The most critical factors influencing the release properties of the core materials include the MC particle size, the wall structure, and the diffusivity of the active ingredients passing through the wall.27 In this study, the particle sizes of the two MC types were similar, so the efficacy of the MCs depended on the last two factors. Contact Toxicity. The mortality of the test insects was investigated after being treated for 8 or 24 h (Figure 2). At 8 h after treatment (Figure 2A), the PUA-MC group mortality ranged from 18 to 61%, whereas that of the UF-MC group ranged from 8 to 55%. The EC group mortality ranged between
Figure 3. Stomach toxicity of PUA-MCs, UF-MCs, and EC of phoxim to the A. ipsilon larvae using the leaf sandwich method (A) 24 and (B) 48 h after treatment. Data with different lower case letters are significantly different at the p < 0.05 level by Duncan’s multiple-range test.
the same, the mortality ranked as UF-MCs < PUA-MCs < EC. Forty-eight hours after treatment, the mortality of all of the treatments increased compared with that at 24 h. The mortalities of the PUA-MC and UF-MC groups were similar to and lower than that of the EC group. As the MCs did not show increased rapid activity, we wondered whether they had better residual activity. However, all of the living A. ipsilon larvae stopped dying 72 h after treatment. This indicates that microencapsulation can reduce pesticide stomach toxicity. We supposed that MCs ingested by insects through the mouthparts would not rupture immediately, thus exhibiting insecticide efficacy. In terms of the higher stomach activity of the PUAMCs compared with the UF-MCs, the ability of the phoxim to better penetrate PUA than UF may have been a key factor.
Figure 2. Contact toxicity of PUA-MCs, UF-MCs, and EC of phoxim to the A. ipsilon larvae at (A) 8 and (B) 24 h after treatment. Data with different lower case letters are significantly different at the p < 0.05 level by Duncan’s multiple-range test. C
DOI: 10.1021/acs.jafc.6b00231 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry To identify why the stomach toxicity of the microcapsules was lower than that of EC, the phoxim content in frass was monitored. Phoxim was recovered from frass fortified with EC, PUA-MCs, and UF-MCs at concentrations of 100, 200, 400, and 800 mg/kg ranging from 86.06 to 96.56%, with a standard deviation (SD)
