Evaluation of Nanoemulsion of Eucalyptus globulus Oil as Potent Botanical Larvicide Against Malaria Vector, Anopheles stephensi and West Nile Vector, Culex pipiens Under Laboratory and Semi-field Conditions http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... Original Article Evaluation of Nanoemulsion of Eucalyptus globulus Oil as Potent Botanical Larvicide against Malaria Vector, Anopheles stephensi and West Nile Vector, Culex pipiens Under Laboratory and Semi-Field Conditions Sara Sabzalizade1, Amir Amani2,3, Hassan Vatandoost1,4, Fatemeh Hosseini1, Roya Najafi-Taher3, *Hamid Reza Basseri1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 3Medical Biomaterials Research Center (MBRC), Tehran University of Medical Sciences, Tehran, Iran 4Department of Chemical Pollutants and Pesticides, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Prof. Hamid Reza Basseri, email: basserih@tums.ac.ir (Received 31 Aug 2021; accepted 08 Dec 2021) Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences.Copyright © 2021 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/ by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited.by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. Abstract Background: Due to undesired environmental impact of insecticides as well as resistant of vectors to them, the development of organic and natural insecticides has been more considered. In the current study, we developed nanoemulsion of eucalyptus and investigated lavicidal activity of it against malaria vector, Anopheles stephensi and Culex pipiens under laboratory as well as semi-field conditions. Methods: An optimized nanoemulsion was prepared by mixing Eucalyptus oil, Tween 80 and ethanol at ratio of 1:2:1.5 in distilled water, then, stirred for 20 minutes at room temperature. The product was then used for bioassay tests against 3-4th instar larvae of Anopheles stephensi as well as Culex pipiens. Furthermore, a semi-field trial was carried out to evaluate larvicidal activity of nanoemulsion of eucalyptus. Results: Nanoemulsion of eucalyptus showed significantly high lavicidal activity comparing with bulk eucalyptus essential oil. The LC50 and LC90 value of nanoemulsion against An. stephensi were 111.0 and 180.8 ppm respectively and 29.5 and 73.7 ppm for Cx. pipiens, respectively. In the semi field condition, the Nanoemulsion of eucalyptus decreased 1-2nd instar larval density of Culicines and Anophelines to 90.1% and 85.2%, respectively. Conclusion: The nano formulation of eucalyptus oil showed high larvicidal activity. Therefore, nanoemulsion of eucalyptus oil can be used as an eco-friendly larvicide against mosquitoes. Keywords: Eucalyptus; Essential oil; Nanoemulsion; Larvicide; Anopheles stephensi; Culex pipiens Introduction Mosquitoes are a serious threat to public health as they act as vectors that help in transmission of diseases that can be lethal (1,2). Owing to the lack of proper medication and vaccines for treating mosquito-borne diseases, an alternative and effective approach used to control the vector population at the larvicidal stage is necessary because, during this stage, 381 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... the mosquitoes are in a stationary phase (3- 5). The resistance to synthetic pesticides and harmful effects of their accumulation in the environment has created the need for natural and non-persistent insecticides. The essential oils extracted from plants are suitable as they are economically reasonable and have high activity in certain cases and are also bio- degradable (4,6,7). Eucalyptus is a diverse genus of flowering trees and shrubs in the Myrtle family, Myrtaceae. The oil extracted from eucalyptus leaves possesses allelopathic property and prevents insects from attacking it, thereby, acting as a natural pesticide (8,9). The fumigation activity (10) and repellency (11) and insecticidal activity of eucalyptus oil has been demonstrated (12). Nanoemulsion of natural oils could enhance pesticide activity of the component (13). Nanoemulsions are emulsions whose droplet size is uniform and extremely small with the size ranging from 20 to 100 nm. Nanoemulsions are metastable and their stability is determined by the method of preparation (14). Nanoemulsions can be formulated by two kinds of methods such as high-energy and low-energy emulsification methods. The high- energy emulsification method comprises high- pressure homogenization and ultrasonication (15). Ultrasonication is the most widely used method owing to its ease of use and it is an economical method. The low-energy emulsification technique comprises methods that exploit the chemical properties of a system to convert a microemulsion into a nanoemulsion (16,17). The use of nanopesticides would be a contemporary measure for the control of pests and reducing the toxic effect of synthetic bulk pesticides on the environment (18). Recently, controlling for vector borne diseases using neem oil nanoemulsion will be of good alternative against Culex quinquefasciatus compared to the synthetic pesticides (13). The present study was carried out to develop a nano formulation of eucalyptus oil as an ecofriendly larvicide and evaluate its larvicidal activity against the larvae of malaria vector, Anopheles stephensi and vectors of West Nile virus (WNV), Culex pipiens in laboratory and semi-field conditions. Material and Methods Preparation of nanoemulsion Eucalyptus oil (Eucalyptus globulus) was purchased from Barich Co., Iran and stored at room temperature under laboratory conditions; Tween 80 (Polyoxyethylene 20 monooleate) was supplied from Sigma. All other chemicals used were of analytical reagent grade. The oil-in-water nanoemulsion was formulated using eucalyptus oil, non- ionic surfactant (tween 80) and water. The concentration of eucalyptus oil (6%, v/v) was fixed for all the formulations. Initially, coarse emulsion was prepared by adding water to organic phase containing oil, surfactant and co- surfactant in ratios 1:1: 1.5 (v/v) using a magnetic stirrer, which was then subjected to ultrasonic emulsification using a 20 kHz Sonicator (Ultrasonics, USA) with a power output of 750 W. Energy input was given through sonotrode containing a piezoelectric crystal with a probe diameter of 13 mm. Sonicator probe generates disruptive forces that reduce the droplet diameter converting coarse emulsion to nanoemulsion. Then the characterization of nanoemulsions was carried out and the emulsion stability was investigated. Droplet size distribution and polydisper- sity index The droplet size distribution (analysis by volume) and poly dispersity index (PDI) of eucalyptus oil nanoemulsion formulation (1:2) was determined using a 90-plus particle size analyzer. The PDI is a measure of the homogeneity and stability of the droplet size in the nanoemulsion system. PDI values below 0.2 indicate a narrow size distribution and thus provide long-term stability to the formulated nanoemulsion. Prior to experiment, formulated emulsion was diluted with milli-Q (Millipore corporation) double-distilled water to trim down multiple scattering effects. Morphology of emulsion droplets To visualize the shape and morphology of 382 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... the formulated nanoemulsions, Atomic force microscopy (AFM) was carried out. One drop of emulsion was negatively stained with phosphotungstic acid and positioned on a copper grid. Larvicidal bioassay Mosquito rearing: Mosquitoes consisting of An. stephensi and Cx. pipiens were reared under a uniform condition including larvae and adult nutrition, temperature (28±2 °C), humidity (70±10 %) and on a 12-h light- dark cycle. Larvae were grown in bowls at a density of 200 larvae / 500 ml of distilled water with 0.01% table salt, and fed on fish food. The pupae were transferred to cages made of muslin cloth before eclosion to the adult stage. The female mosquitoes were fed on 10% fructose and the females were fed on guinea pig blood. Larvicidal activity: The larvicidal activity of eucalyptus nanoemulsion against third instar larvae of An. stephensi and Culex pipiens were treated with different concentrations of nano and bulk eucalyptus oil emulsions. The bioassays were carried according to the guide line of World Health Organization for laboratory and field testing of mosquito larvicides (19). Initially, serial dilutions of nanoemulsion (5, 50, 100, 160, and 240 ppm) were prepared in sterile glass beakers (250 mL) containing 200 ml of water. Then 20 larvae (stage 3-4) of the mosquito species were placed into each beaker. The same procedure was conducted for the bulk of eucalyptus oil. The mortality rate of larvae was recorded after 24 and 48 hours. Each test was performed in four replicates. The percentages of larval mortality and standard deviation were calculated for each concentration of nanoemulsion and bulk emulsion. Semi-field larval bioassays Twelve artificial breeding places each 1×1 meters were provided in a semi-field condition in the Kazeroun area, southern Iran and allow the wild mosquitoes to lay their eggs on the surface of breeding places. The density of larvae 10 per dipper were measured in each breeding place before any intervention. The spray of three times of obtained lethal concentration (LC90) of bulk-eucalyptus oil (≈114 ppm) and nano- eucalyptus oil (≈90 ppm) were sprayed randomly on the surface of each breeding place. Each treatment was replicated in four breeding places and four breeding places of free oils as the control. Statistical analysis The mortality quantities of 50% and 90 % of imagicides (LC50 and LC90) and the level of confidence of 95%, the equation of the regression line were estimated using a regression probit analysis as described by Finney (20). Mosquito identifications All the adults females were identified using morphological identification key identification (21). Results Characterization of the selected nano- emulsion Based on the thermodynamic stability study, 1:2 ratio nanoemulsions was selected, and their average diameter is 18 nm and their size distribution as presented in Fig. 1. The polydispersity index (PI) of the nanoemulsions is 0.060 which shows that it is uniform. The particles are presented in Atomic force microscopy (AFM) from 22 to 40 nm, and the droplets are spherical in nature (Fig. 2). Larvicidal activity of eucalyptus oil nano- emulsion The larvicidal activity of both nanoemulsion eucalyptus oil and bulk eucalyptus oil against larvae of An. stephensi and Cx. pipiens was varied. High mortality rate was observed among larvae of An. stephensi exposed to 160 ppm of nanoemulsion of eucalyptus oil within 24 hours, while the mortality rate for eucalyptus oil was 74% at the concentration of 160 ppm after 24 hours (Table 1). Moreover, 383 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... 100% mortality occurred for the larvae exposed to both nanoemulsion eucalyptus oil and eucalyptus oil at a concentration of 240 ppm. Overall, the larvicidal activity of nanoemulsion oil against larvae of An. stephensi within 24 hours was significantly higher than eucalyptus at 160 ppm, but absolute mortality was observed among the larvae of An. stephensi after 48 hours in both nanoemulsion formulation and bulk oil of eucalyptus (Table 1). Similarity, the larvicidal activity of nanoemulsion oil against larvae of Cx. pipiens was higher than eucalyptus oil at 160 ppm within 24 hours while 100% mortality occurred by 240 ppm for both formulations of eucalyptus oils (Table 2). Lethal concentration (LC50and LC90) for nanoemulsion of eucalyptus oil and eucalyptus oil were significantly different within 24 and 48 hours after treatment (Table 3). Moreover, LC50 and LC90 of nanoemulsion of eucalyptus oil against larvae of An. stephensi significantly lower than eucalyptus oil within both 24 and 48 hours treatment (Table 3). Similarly, lower LC50 and LC90 was observed for larvae of Cx. pipense exposed to nanoemulsion of eucalyptus oil which was significantly different with LC50 and LC90 for the larvae exposed to eucalyptus oil (Table4). Semi field larval bioassays Both nano-eucalyptus and bulk eucalyptus oils decreased the density of mosquito larvae in the treated breeding places one day after Fig. 1. Size distribution of eucalyptus nanoemulsion oil measured by Dynamic Light Scattering Fig. 1. Size distribution of eucalyptus nanoemulsion oil measured by Dynamic Light Scattering Fig. 2. Atomic force microscopy image and size of eucalyptus nanoemulsion oil Fig. 2. Atomic force microscopy image and size of eucalyptus nanoemulsion oil 384 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... spraying. The high larvae population at stage 1 and 2 in the control breeding sites indicated that the wild mosquitoes laid eggs frequently during the bioassay period (Fig. 3). Therefore, the density of mosquito larvae was relatively high in the control breeding site while rapidly declining in the treated sites. However, nano- eucalyptus emulsion kept the larvae density low for 6 days post spraying while the number of larvae at stage 1 and 2 increased two days after eucalyptus oil spraying (Fig. 3). The results indicating that nano-eucalyptus oil Fig. 3. Residual larvicidal activity of Nanoemulsion of eucalyptus oil and bulk eucalyptus oil against different stages of mosquito larvae at semi-field condition. Twelve artificial breeding places (1×1 meters) were prepared and then allowed the wild mosquitoes laid eggs. L1, 2 = Larvae stage 1 & 2, L 3,4= Larvae stage 3 & 4 Fig. 3. Residual larvicidal activity of Nanoemulsion of eucalyptus oil and bulk eucalyptus oil against different stages of mosquito larvae at semi-field condition. Twelve artificial breeding places (1×1 meters) were prepared and then allowed the wild mosquitoes laid eggs. L1, 2 = Larvae stage 1 & 2, L 3,4= Larvae stage 3 & 4 Table 1. Probit analysis of larvicidal activity of essential and nanoemlusion of Eucalyptus globulus oil on 3rd and 4th instar larvae of Anopheles stephensi after 24 hours Concentration (ppm) Essential oil Nanoemlusion oil Mortality (%) Observed probit mortality Expected probit mortality Mortality (%) Observed probit mortality Expected probit mortality 60 9 3.659 3.246 8 3.595 3.384 160 74 5.643 5.658 91 5.025 5.204 240 100 7.526 6.654 100 7.576 7.024 360 100 7.526 7.651 100 7.576 8.089 Control 0 - - 0 - - Table 1. Probit analysis of larvicidal activity of essential and nanoemlusion of Eucalyptus globulus oil on 3rd and 4th instar larvae of Anopheles stephensi after 24 hours Table 2. Probit analysis of larvicidal activity of essential and nanoemlusion of Eucalyptus globulus oil on 3rd and 4th instar larvae of Culex pipiens after 24 hours Concentration (ppm) Essential oil Nanoemlusion oil Mortality (%) Observed probit mortality Expected probit mortality Mortality (%) Observed probit mortality Expected probit mortality 60 8 3.595 3.384 11 3.773 3.520 160 51 5.025 5.204 86 4.900 5.161 240 100 7.576 7.024 100 7.576 6.802 360 100 7.576 8.089 100 7.576 8.443 Control 0 - - 0 - - Table 2. Probit analysis of larvicidal activity of essential and nanoemlusion of Eucalyptus globulus oil on 3rd and 4th instar larvae of Culex pipiens after 24 hours 385 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... has slightly longer residual larvicidal activity rather than bulk eucalyptus. Discussion Due to numerous limitations to control of adult mosquitoes, the ideal method to control them is targeting the larval stage. Control of mosquito larvae is based largely on the use of synthetic chemicals. Generally, synthetic pesticides have some disadvantages such as health problems, harmful to the environment, pests may develop tolerance to certain chemicals over time and contamination of soil and water resources (22-24). However, the indiscriminate and injudicious use of pesticides has led to the widespread development of resistance among pests as well as insect vectors (25). Thus, alternative components to be needed for controlling mosquito vectors. Botanical pesticides are considered as safe, easily Table 3. Lethal concentration (LC50 and LC90) and in the 24 and 48 hours bioassay tests of Eucalyptus oil and Nano- Eucalyptus against 3rd and 4th instar larvae of Anopheles stephensi b ± SE LC90 (ppm) ± 95%C.L. LC50 (ppm) .± 95%C.L χ2 (Heterogeneity) χ2 table (df) p-Value Exposure time (hours) 5.6608 ± 0.764 169.1937 206.2336 295.0632 103.7845 122.8343 145.0833 15.611* 15.086 (5) 0.01 24 E uc al yp tu s oi l 2.399 ± 1.023 142.402 155.973 175.732 93.761 101.663 145.0833 14.623* 15.035 (5) 0.00 48 6.0457 ± 0.539 102.8420 111.0171 119.7206 63.7924 80.8730 105.7719 5.1802* 9.210 (2) 0.01 24 N an o- E uc al yp tu s oi l 7.0457 ± 0.652 46.235 58.595 65.548 68.235 76.869 95.582 4.258* 9.145 (2) 0.00 48 Table 3. Lethal concentration (LC50 and LC90) and in the 24 and 48 hours bioassay tests of Eucalyptus oil and Nano-Eucalyptus against 3rd and 4th instar larvae of Anopheles stephensi Table 4. Lethal concentration (LC50 and LC90) and in the 24 and 48 hours bioassay tests of Eucalyptus oil and Nano- Eucalyptus against 3rd and 4th instar larvae of Culex pipiens b ± SE LC90 (ppm) ± 95%C.L. LC50 (ppm) ± 95%C.L χ2 (Heterogeneity) χ2 table (df) p-Value Exposure time (hours) 6.5324 ± 1.034 20.3120 39.4112 78.6321 47.3526 68.6321 76.1356 10.546* 8.235 (2) 0.01 24 E uc al yp tu s oi l .4508 ± 1.127 14.7673 37.3711 86.4146 42.4037 64.2180 74.0175 10.678* 9.210 (2) 0.01 48 3.2200 ± 0.313 25.7675 29.4644 33.0781 43.3775 53.6733 70.0869 15.611* 9.210 (2) 0.01 24 N an o- E uc al yp tu s oi l 5.1234 ± 0.4123 19.9645 23.2523 26.2879 31.6885 34.5681 39.0263 15.646* 9.451 (2) 0.01 48 Table 4. Lethal concentration (LC50 and LC90) and in the 24 and 48 hours bioassay tests of Eucalyptus oil and Nano-Eucalyptus against 3rd and 4th instar larvae of Culex pipiens 386 http://jad.tums.ac.ir Published Online: Dec 31, 2021 J Arthropod-Borne Dis, Dec 2021, 15(4): 380–388 S Sabzalizade et al.: Evaluation of Nanoemulsion... biodegradable, environmentally friendly and with low toxicity (26,27). However, usage of them is often limited due to instability and rapid degradation, application frequency as well as requiring higher application rates. Hence, the use of botanical insecticides associated with nanotechnology offers considerable potential for increasing efficacy of plant based insecticide (27-29). Eucalyptus oil is the oil distilled from the leaves of eucalyptus, a genus of the plant family Myrtaceae. The repellent activity of eucalyptus oil was demonstrated against Cx. quinquefasciatus (30,31). The seed and leaf extract of eucalyptus oil contain compounds that are toxic to mosquito larvae (32,33). Since they do not cause any adverse health effects; they are used as insect repellents and a safe and eco-friendly alternative to synthetic pesticides (34). Therefore, in this study, we used eucalyptus oil as a safe and non-toxic larvicide. Generally, botanic pesticides have low effective durability; therefore, we utilized nanotechnology to overcome this problem. We synthesized nano-eucalyptus oil to increase larvicidal durability and effectiveness. The method of preparation determines the stability of the formulated nanoemulsion. Based on atomic force microscopy analysis, the size of our product was from 22 to 40 nm (Fig. 2), and the larvae mortality we achieved in laboratory and semi field bioassays (Tables 1 , 2 and Fig. 3) was as the results of these nano-size. Nevertheless, the larvicidal effects of nano-eucalyptus oil are likely to change as the nano-particle size changes. The stabilization of the nanoemulsions is also dependent on the steric effect of the non-ionic surfactant (35). Therefore, the effectiveness of nano-eucalyptus oil may be influenced by chemical component of water in the breeding sites. In semi-field assay, we did not observe 100% mortality of mosquito larvae a day after treatment of nano-eucalyptus oil and eucalyptus. This result may be due to components of water in the breeding sites. In addition, only a single concentration of nano-eucalyptus oil (≈90 ppm) was tested against wild mosquito larvae. According to WHO guide line, the larvicide dosage for the field trial should be three times of LD90 concentration in a laboratory scale. However, we did not observe convenient results for larvicidal effect of nano-eucalyptus oil in the field condition, Thus, more serial concentrations of the nano emulsion should be assays to overcome the highest larvae mortality in the field condition. In this study, eucalyptus oil (6%, v/v) is mixed with a non-ionic surfactant, tween 80 along with water, which is used as an aqueous phase. This emulsion is then subjected to ultrasonication that breaks down the bulk emulsion into an emulsion comprising droplets having the size in the nanometer range. This increases the surface area of the droplets, thereby, increasing the reactivity; thus, making nanoemulsions more effective than its bulk counterpart. However, the insecticidal and antimicrobial activity of eucalyptus oil is linked to its chemical composition such as 8-cineole (eucalyptol), which its concentration ratio varies in eucalyptus trees from different locations (36). Conclusion The nano formulation of eucalyptus oil showed high larvicidal activity against mosquito larvae when compared to its bulk counterpart. Therefore, nanoemulsion of eucalyptus oil can be used as an eco-friendly larvicide against mosquitoes. From this study, it can be concluded that eucalyptus oil nanoemulsion is a safe and effective alternative larvicide to control mosquito- borne diseases. Acknowledgements The authors would like to thank the School of Public Health, Tehran University of Medical Sciences, for their support in carrying out this research. This study was supported by the Deputy of Research, Tehran University of Medical Sciences, Grant No. 29565. 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Evaluation of Nanoemulsion of Eucalyptus globulus Oil as Potent Botanical Larvicide Against Malaria Abstract Keywords Introduction Material and Methods Preparation of nanoemulsion Droplet size distribution and polydisper- sity index Morphology of emulsion droplets Larvicidal bioassay Semi-field larval bioassays Statistical analysis Mosquito identifications Results Characterization of the selected nano- emulsion Larvicidal activity of eucalyptus oil nano- emulsion Semi field larval bioassays Discussion Conclusion Acknowledgements Competing interests References