Synthesis of Nanopesticides by Encapsulating ... - ACS Publications

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Synthesis of Nanopesticides by Encapsulating Pesticide Nanoparticles Using Functionalized Carbon Nanotubes and Application of New Nanocomposite for Plant Disease Treatment Nahid Sarlak,*,† Asghar Taherifar,† and Fatemeh Salehi§ †

Department of Chemistry, Faculty of Sciences, Lorestan University, Khorram Abad, Iran Department of Molecular Biology, Faculty of Biology, Tehran University, Tehran, Iran

§

ABSTRACT: Polymerization of citric acid onto the surface of oxidized multiwall carbon nanotubes led to MWCNT-graftpoly(citric acid) (MWCNT-g-PCA) hybrid materials. Because of the presence of conjugated citric acid branches, synthesized MWCNT-g-PCA hybrid materials were not only soluble in water but also able to trap water-soluble chemical species and metal ions. Trapping of pesticides such as zineb and mancozeb in aqueous solution by MWCNT-g-PCA hybrid materials led to encapsulated pesticide (EP) in the polycitric acid shell. Optimum conditions for encapsulation of zineb and mancozeb in hyperbranched polycitric acid such as pH, time of stirring, and temperature were investigated by the UV−vis spectroscopy method. Encapsulation of pesticides on CNT-g-PCA hybrid material was confirmed via TEM analysis. Experiments indicated that new the CNT-g-PCA-EP hybrid material in comparison with bulk pesticide had a superior toxic influence on Alternaria alternata fungi. KEYWORDS: zineb, mancozeb, pesticide, multiwalled carbon nanotubes, nanocomposite

1. INTRODUCTION

conditions that would otherwise break it down, yielding capsules ranging from less than one micrometer to several hundred micrometers in size.7 Encapsulation can be achieved by several techniques with different purposes in mind. Encapsulation of materials may occur with the intention that the core material be confined within capsule walls for a specific period of time. Alternatively, core substances can be encapsulated so that the core material will be released either gradually through the capsule walls, known as controlled release or diffusion, or when external conditions activate the capsule walls to break, melt, or dissolve.8 There are many applications for encapsulated materials in various fields such as agriculture, pharmaceuticals, foods, cosmetics and fragrances, textiles, paper, paints, coatings and adhesives, printing applications, and many other industries. Pesticides may be encapsulated to be released over time, allowing farmers to apply the pesticides less often rather than requiring very highly concentrated and perhaps toxic initial applications followed by repeated applications to battle the loss of efficacy due to leaching, evaporation, and degradation.9 Protection of pesticides from full contact with the elements lessens the risk to the environment and those that might be exposed to the chemicals and provides a more efficient strategy for pest control.10 In the past decade, there has been increasing interest in the studies of polymer/CNT nanocomposites due to the unique combination of promising properties and construction of multifunctional structures of each component.11 Previously

Organic pesticides including insecticides and herbicides are widely used in agriculture because of their powerful biological activity.1 Mancozeb and zineb were first introduced during the 1940s and widely used. Mancozeb as a pesticide protects many fruit, vegetable, nut, and agricultural crops against a wide range of fungal diseases. It is a grayish-yellow powder, and its chemical name is manganese ethylene bis(dithiocarbamate) (polymeric). Zineb is a light-colored powder or crystal the ch em i c al n a m e o f w h i ch i s z in c et h y le n e bi s(dithiocarbamate).2−6 Figures 1a and 1b show the chemical structures of mancozeb and zineb molecules, respectively. Encapsulation is a process of surrounding or enveloping one substance within another material on a very small scale. Another definition of encapsulation is the process by which a material is surrounded and protected from extraneous

Received: May 6, 2013 Revised: April 12, 2014 Accepted: May 2, 2014

Figure 1. Structure of (a) mancozeb molecule and (b) zineb molecule. © XXXX American Chemical Society

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dx.doi.org/10.1021/jf404720d | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 2. Synthetic scheme of CNT-g-PCA-EP.

Figure 3. FTIR data for (a) MWCNT−COOH and (b) MWCNT-g-PCA hybrid material.

Trimnell et al.12 investigated the encapsulation of pesticide within a starch−borate complex. In this work the starch, pesticide, and water were mixed and alkali was added to gelatinize the starch. Then the mixture was treated with boric acid. In our study zineb and mancozeb were encapsulated into MWCNT-g-PCA hybrid material, which process leads to conversion of bulk pesticide to pesticide nanoparticles. Effective parameters in the encapsulation process, such as pH, temperature, and time of stirring, were optimized via the UV−vis spectroscopic method. The influence of encapsulated pesticide was studied on the malady Alternaria alternate, so, for this purpose, potato dextrose agar (PDA) was used as a growth medium. Results show that pesticide nanoparticles in contrast with bulk pesticide have extraordinary function. To the best of our knowledge, this paper is the first report about using watersoluble polymerized carbon nanotubes for encapsulation of pesticides and application the new nanopesticides in malady agent treatment.

In comparison with previous reports there are some novel and specific advantages as follows: (a) Pesticide encapsulated into CNT-g-PCA hybrid material is more stable and effective than bulk pesticide. (b) Pesticide usage will be decreased in agriculture. (c) Water solubility of encapsulated pesticides will be increased. (d) Multiwall carbon nanotubes will be dissolved in the water. This product can be used extensively in several greenhouse and field studies and agriculture.

2. EXPERIMENTAL SECTION 2.1. Reagents. The MWCNT was purchased from Nutrino. The outer diameter of MWCNT was between 20−40 nm. Monohydrate citric acid, sulfuric and nitric acid, tetrahydrofuran, and cyclohexane were purchased from Merck. Pesticide standard was purchased from Delta Green South Corporation of Iran. 2.2. Apparatus. Fourier transform infrared (FT-IR) spectroscopic measurements were performed using a FT-IR spectrometer (Thermo B



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Figure 4. Effect of pH on the encapsulation of mancozeb (a) and zineb (b) with CNT-g-PCA. Nicolet Magna-IR 560 spectroscope, USA). The FTIR spectra of acid modified CNTs and CNT-g-PCA were obtained in transmittance mode by placing a small amount of the materials in KBr pellets. The UV−vis measurements were obtained using a double beam spectrophotometer (Shimadzu 1650 UV−vis spectrophotometer, Japan) with 1 cm quartz cells. In order to well disperse pesticide in the polymeric shell of hybrid material, an ultrasonic bath (22 kHz) was used. The TEM study was carried out using a microscope operating at 100 kV. The samples for the TEM analysis were prepared by dilution of nanocomposite with distilled water. A drop of the suspension was applied onto a carbon-coated copper grid and was dried in air. A centrifuge (Eppendorf 5810) was used for separation of CNT-g-PCAEP from pesticide molecules. 2.3. Preparation of MWCNT-g-PCA Hybrid Materials. MWCNTs were opened according to reported procedures in the literature.13 Briefly, MWCNTs (2 g) were added to 40 mL of sulfuric and nitric acid mixture (3/1) in a reaction flask and refluxed for 24 h at 120 °C. The mixture was cooled and diluted with distilled water, and then it was filtrated and washed with distilled water until the pH reached 5; finally it was dried by vacuum oven. MWCNT-g-PCA hybrid materials were prepared according to the reported procedure.14 2.4. Preparation of MWCNT-g-PCA-EP. A water solution of MWCNT-g-PCA (39 mg/mL) with a mancozeb water solution (5.12 × 10−4 M) and also a water solution of MWCNT-g-PCA (28 mg/mL) with a zineb water solution (1.45 × 10−4 M) were mixed separately and placed in an ultrasonic bath for 10 min in order to well disperse pesticide nanoparticles in the polycitric acid shell. Then the mixtures were stirred for complete encapsulation of mancozeb and zineb into CNT-g-PCA for 35 and 80 min, respectively. Encapsulated pesticide nanoparticles were purified by centrifugation. Pesticide molecules which were not reacted with CNT-g-PCA were separated from encapsulated pesticide nanoparticles using a

centrifuge with rotating speed of 2000 rpm within 7 min. During the process of centrifugation the sample was separated into two phases, a pellet consisting of MWCNT-g-PCA-EP and a supernatant that was unreacted pesticide molecules. Then the supernatant was separated and discarded. Figure 2 shows the synthetic scheme of the CNT-gPCA-EP. 2.5. Preparation of Potato Dextrose Agar (PDA) Medium. Usually for PDA medium preparation, 250 g of potatoes was boiled for 20 min and the sap of potato was taken. Afterward 20 g of dextrose agar was added to the sap under continuous stirring and slowly warmed until it was well dissolved. Then the medium was diluted to 1 L by warmed distilled water and autoclaved for 15 min at 121 °C and pressure 1.5 kg/cm2. Finally PDA medium was chilled to 45 °C and was poured into a Petri dish.15−17

3. RESULTS AND DISCUSSION 3.1. Nanohybrid Characterization. The FTIR spectra shown in Figure 3 confirm the presence of functional groups in MWCNT−COOH and MWCNT-g-PCA hybrid materials. The carbonyl stretching frequency in MWCNT−COOH was seen at 1701 cm−1 (COOH); The 3200−3500 cm−1 band present in the MWCNT−COOH spectrum was attributed to the hydroxyl vibration of the carboxylic acid group attached through the opening. The peak around 1400−1500 cm−1 was assigned to the CC stretching of the carbon skeleton. As shown in Figure 3b a broad absorbance band at 2600−3573 cm−1 was attributed to hydroxyl functional groups of grafted PCA. In this spectrum, a band of carbonyl groups of citric acid was seen at 1718 cm−1. Also the CC band of MWCNT appeared at 1400−1500 cm−1. C



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3.2. Optimization of Effective Parameters on Synthesis of CNT-g-PCA-EP. The effect of various parameters on encapsulation of pesticides into CNT-g-PCA was studied. Figure 4 shows the role of pH in the encapsulation of pesticides by MWCNT-g-PCA hybrid material. The maximum encapsulation of pesticide molecules into polymeric shell corresponds to maximum absorption in UV−vis spectra. Maximum encapsulation for mancozeb and zineb according to Figure 4 takes place at pH 3 and 8 respectively. Furthermore, temperature had no significant effect on the encapsulation reaction. The required time for complete encapsulation of pesticides in CNT-g-PCA is very important. Figure 5 shows the effect of

Figure 6. Color change during time progression of the encapsulation between pesticide zineb and MWCNT-g-PCA: (a) initial; (b) after 2 h; (c) after 8 h.

Figure 5. Effect of stirring time on encapsulation of pesticides into CNT-g-PCA hybrid material. Mancozeb (a) and zineb (b).

stirring time on the reaction between pesticides and CNT-gPCA. As shown in Figure 5a,b with the addition of pesticide aqueous solutions to CNT-g-PCA the respective absorption peak increases. In other words, maximum encapsulation for mancozeb and zineb in hyperbranched polycitric acid shell takes place after 35 and 80 min, respectively. Figure 6 shows the color change during the progress of the complexation reaction between zineb and CNT-g-PCA hybrid material under optimum conditions. As shown in Figure 6, the light yellow color of solution turned gradually to pale yellow after 2 h. The darkening of the solution continued and fixed after 8 h, which indicated that the encapsulation process was completed. 3.3. Encapsulation of the Pesticides by CNT-g-PCA: UV−Vis Study. The encapsulation of the zineb and mancozeb by polymeric shell of CNT-g-PCA was investigated spectrophtometrically. Figure 7a(iii),b(iii) shows the UV−vis spectra for encapsulated zineb and mancozeb by CNT-g-PCA respectively.

Figure 7. UV spectra for encapsulation of (a) zineb and (b) mancozeb by CNT-g-PCA: (i) aqueous solution of CNT-g-PCA; (ii) aqueous solution of pesticide; (iii) after addition of pesticide to CNT-g-PCA.

A simple route which clearly approves the encapsulation of pesticides in hyperbranched polycitric acid shell was attained with addition of various amounts of CNT-g-PCA to each pesticide and monitoring of the UV spectra. As shown in Figure 8, addition of CNT-g-PCA led to increasing the peak intensity while the fine structures of pesticide spectra follow the same pattern. These behaviors confirm the encapsulation of pesticides into polycitric acid hyperbranched shell. D



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3.4. TEM Images of Synthesized CNT-g-PCA-Pesticide. Finally encapsulation of pesticides by the CNT-g-PCA was confirmed by TEM analysis. Figure 9 shows TEM images of zineb and mancozeb encapsulation by the CNT-g-PCA hybrid material which clearly indicates polycitric acid (PCA) shell around CNTs and encapsulated pesticide nanoparticles. 3.5. Application of Encapsulated Zineb Nanoparticles on Alternaria alternate Fungi. Encapsulation of pesticide nanoparticles in hyperbranched polycitric acid results in the appearance of extraordinary characteristics. In this study, the influence of CNT-g-PCA-EP on the malady Alternaria alternata fungi was studied. For this purpose, potato dextrose agar (PDA) medium was used to grow the malady Alternaria alternate. Bulk and encapsulated zineb were added to the control medium, and their effects on growth of colonies were studied after 3 and 10 days (Figure 10). The results show that encapsulated zineb nanoparticles have more effect on growth restriction in comparison to the bulk zineb. This effect after 10 days reaches the maximum intensity. Probably this phenomenon is attributable to increases in the contact area which was formed between encapsulated pesticide and fungi. 3.6. Conclusion. In summary, mancozeb and zineb were successfully encapsulated into CNT-g-PCA hybrid material. Results showed that encapsulation of pesticides into hybrid material are dependent on pH and time of stirring but temperature has no significant effect on the encapsulation process. Experiments show that the optimum pH for encapsulation of mancozeb and zineb is 3 and 8 respectively.

Figure 8. Addition of various amounts of a water solution of CNT-gPCA to pesticides (a) mancozeb and (b) zineb.

Figure 9. TEM images of encapsulation of zineb (a) and mancozeb (b) with MWCNT-g-PCA. E



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Figure 10. Influence of zineb encapsulation on Alternaria alternate fungi. Medium control (a and d), medium control with encapsulated zineb (b and e), and medium control with bulk zineb (c and f). Top, after 3 days; bottom, after 10 days. (7) Del Carmen Giménez-López, M.; Moro, F.; Torre, A. L.; GómezGarcía, C. J.; Brown, P. D.; Slageren, J. V.; Khlobystov, A. N. Encapsulation of single-molecule magnets in carbon nanotubes. Nat. Commun. 2011, 2, 407. (8) Doane, W. M. Encapsulation of pesticides in starch by extrusion. Ind. Crops Prod. 1992, 1, 83−87. (9) Lewis, D. H.; Cowsar, D. R. Principles of Controlled Release Pesticides. In Controlled Release Pesticides; Scher, H. B., Ed.; American Chemical Society: Washington, DC, 1977; pp 1−16 (10) Ulbricht, H.; Hertel, T. Dynamics of C60 Encapsulation into Single-Wall Carbon Nanotubes. J. Phys. Chem. B 2003, 107, 14185− 14190. (11) Ajayan, P. M. Nanotubes from Carbon. Chem. Rev. 1999, 99, 1787−1800. (12) Trimnell, D.; Shasha, B. S.; Wing, R. E.; Otey, F. H. Pesticide encapsulation using a starch−borate complex as wall material. J. Appl. Polym. Sci. 1982, 27, 3919−3928. (13) Stobinski, L.; Lesiak, B.; Kövér, L.; Tóth, J.; Biniak, S.; Trykowski, G.; Judek, J. Multiwall carbon nanotubes purification and oxidation by nitric acid studied by the FTIR and electron spectroscopy methods. J. Alloys Compd. 2010, 501, 77−84. (14) Adeli, M.; Bahari, A.; Hekmatara, H. Carbon Nanotube-graftpoly(citric acid) nanocomposites. Nano 2008, 03, 37−44. (15) Kuo, C.-F.; Chyau, C.-C.; Wang, T.-S.; Li, C.-R.; Hu, T.-J. Enhanced Antioxidant and Anti-inflammatory Activities of Monascus pilosus Fermented Products by Addition of Turmeric to the Medium. J. Agric. Food Chem. 2009, 57, 11397−11405. (16) Xu, M.-J.; Yang, Z.-L.; Liang, Z.-Z.; Zhou, S.-N. Construction of a Monascus purpureus Mutant Showing Lower Citrinin and Higher Pigment Production by Replacement of ctnA with pks1 without Using Vector and Resistance Gene. J. Agric. Food Chem. 2009, 57, 9764− 9768. (17) Zheng, Z.; Shetty, K. Solid-State Production of Beneficial Fungi on Apple Processing Wastes Using Glucosamine as the Indicator of Growth. J. Agric. Food Chem. 1998, 46, 783−787.

Furthermore, the time of stirring for complete encapsulation of mancozeb and zineb was 35 and 80 min, respectively. As a result, in this work we could produce pesticide nanoparticles. So encapsulated zineb as pesticide nanoparticles in comparison with bulk zineb showed an extraordinary effect on the malady Alternaria alternata fungi, and its effect reached maximum after 10 days.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Al-Degs, Y. S.; Al-Ghouti, M. A.; El-Sheikh, A. H. Simultaneous determination of pesticides at trace levels in water using multiwalled carbon nanotubes as solid-phase extractant and multivariate calibration. J. Hazard. Mater. 2009, 169, 128−135. (2) Scher, H. B.Advances in Pesticide Formulation Technology. In Advances in Pesticide Formulation Technology; Scher, H. B., Ed.; American Chemical Society: Washington, DC, 1984; pp 1−7 (3) Matthews, G. A. Pests, Pesticides and Pest Management. In Highlights In Environmental Research; Mason, J., Ed.; Imperial College Press: London, 2000; pp 165−189. (4) Rijtema, P. E.; Groenendijk, P.; Kroes, J. G. Environmental Influences on Processes. In Environmental Impact Of Land Use In Rural Regions; Imperial College Press: London, 1999; pp 167−203. (5) Tordoir, W. F. The Development of Safe Chemical Pesticides. In Occupational Health In National Development; World Scientific: 1994; pp 27−42. (6) Honeycutt Richard, C.; Zweig, G.; Ragsdale, N.; Durham William, F. Preface, Introduction. In Dermal Exposure Related to Pesticide Use; American Chemical Society: Washington, DC, 1985; pp xi−xiv. F


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