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Nanoemulsion of cashew nut shell liquid bio-waste: Mosquito larvicidal activity and insights on possible mode of action
South African Journal of Botany 127 (2019) 293300
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Nanoemulsion of cashew nut shell liquid bio-waste: Mosquito larvicidal activity and insights on possible mode of action S. Kalaa,b, N. Soganc, Prveen Vermad, S.N. Naikb,*, A. Agarwala, P.K. Patanjalia, J. Kumara a
Formulation Division, Institute of Pesticide Formulation Technology (IPFT), Gurugram 122016, Haryana, India Center for Rural Development Technology (CRDT), Indian Institute of Technology (IIT), Hauz Khas, Delhi 110016, India c National Institute of Malaria Research (NIMR), Delhi 110077, India d Department of Zoology, Agra College, Agra 282004, India b
A R T I C L E
I N F O
Article History: Received 25 April 2019 Revised 4 September 2019 Accepted 5 October 2019 Available online xxx Edited by J Van Staden Keywords: Cashew nut shell liquid Waste Nanoemulsion Bio-pesticide Mosquitoes
A B S T R A C T
Cashew nut (Anacardium occidentale) shell liquid (CNSL) is a massive waste generated from the cashew nut processing industry. The Disposal of large volume of CNSL may become a concern in terms of ecological issues. CNSL is having insecticidal properties despite of this fact it has not been utilized to its full extent. CNSL is poorly soluble in water; in this context, we have utilized CNSL to develop a nanoemulsion for larvicidal applications; using spontaneous emulsification method. The monodispersed micelles of CNSL with mean diameter of 52 nm were produced. The comparative bioefficacy of bulk and nano CNSL was evaluated against the 3rd instar larva of Anopheles culicifacies, which was recorded with LC50 of 18.1 mg/L and 1.4 mg/ L, respectively. Morphological damages to the larva post exposure to CNSL nanoemulsion were examined using scanning electron microscope (SEM). Histopathological examination revealed detailed mode of toxicity of nano CNSL on larva. The result obtained showed that the CNSL nanoemulsion has enhanced larvicidal activity compared to bulk CNSL which allows effective utilization of waste. The utilization of CNSL nanoemulsion may also resolve disposal related issues of massive waste generated by cashew nut industry. Such approach may be suitable for enhanced efficacy against mosquito vector, at lower doses and also contribute towards reduced use of synthetic pesticides. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Vector borne diseases represent a key to threat for millions of people worldwide, which may lead to epidemics in population of humans and animals (Govindarajan et al., 2016). Among those vectors; mosquito is responsible for various life-threatening diseases with enormous economic impact, along with higher global disease burden (Doucoure et al., 2015). Mosquito belonging to genus Anopheles feed on vertebrate blood and transmit the protozoan parasites, causing malaria. The mosquitos have serious impact on human as well as animal health (Stuchin et al., 2016) and significantly influences the production from livestock (cattle) resulting in economic loses (Narladkar, 2018). The mosquitoes are carriers of deadly parasites, which are transmitted to humans through female Anopheles mosquito. Therefore, the control of these vectors is absolutely ‘essential’. Furthermore, the mosquito control will prevent the spread of mosquito-borne disease and improve quality of public health (Ghosh et al., 2012).
*Correspondence author. E-mail address: [email protected] (S.N. Naik). https://doi.org/10.1016/j.sajb.2019.10.006 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Although various vector control strategies involving synthetic pesticides are available, but the control on the mosquito population is not satisfactory, because the mosquitoes have developed resistance. Consequently, the increased use of pesticides has been employed which in turn has increased the load on environmental and come up with adverse impact on nature and mankind. Pesticides from plant origin, especially plant-based waste are currently receiving considerable attention because they are non-persistent, biodegradable and cost effective (Ghosh et al., 2012). Conventionally available formulations; emulsifiable concentrate (EC) is petroleum solvents based, whereas wettable powder (WP) produces lots of dust during usage (Knowels, 2008). With the advancement in nanotechnology, nanoemulsions provides the basis for formulating aqueous-based nanopesticides of poorly water-soluble active with improved and enhanced efficacy (Fernandes et al., 2014). The conversion of pesticide moiety to nano level through nanotechnology can reduce quantity of active ingredients, thereby reducing the dose, and cost and enhances the efficacy of the product (Ali et al., 2017). The cashew (Anacardium occidentale L.) is a tropical plant, native to Brazil and is widely distributed in many other countries around the world including India, Nigeria, Viet Nam, United Republic of Tanzania, Philippines, Indonesia, and Guinea-Bissau (Adeigbe et al.,
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2005; FAOSTAT, 2017). CNSL is a by-product from cashew industrial processing, it occurs in pericarp of the cashew fruit (Paiva et al., 2017), it has no added value and is considered as a waste. India is among top five countries producing cashew nuts (Karthic et al., 2014). CNSL comprises of 25% of total nuts production. In the year 2013, 1000 tons of CNSL was obtained as by-product from the industry. It is composed of phenolic components the disposal of such a large volume of CNSL may become a cause of concern in terms of ecological issues (Calo` et al., 2007; Lomonaco et al., 2009). CNSL is non-toxic, biodegradable; cheap and easily available. It contains cardanol, a monohydric phenol that is responsible for larvicidal activity (Mukhopadhyaya et al., 2010). Several reports have proved that CNSL is a potential larvicide against mosquitoes (Paiva et al., 2017; Raraswati et al., 2014). Although, the insecticidal potential of CNSL has not been utilized, fully, thus there is need for a suitable formulation technology, which may deliver enhanced bioefficacy at the lower doses. Moreover, CNSL was also found safe towards non-target organisms. In a study of impact on non-target organisms, CNSL exhibited no mortality on any of the organisms including those of fishes (Gambusia affinis and Poecilia reticulata), tadpoles (Bufo spp.), and water bugs (Heteroptera spp.) (Mukhopadhyaya et al., 2010). The increased interest in efficient, efficacious, ecofriendly and low cost pesticides practices has encouraged us to devolve nanoemulsion, utilizing CNSL. The bio-efficacy of the attained stable nano formulation of CNSL was comparatively investigated with its bulk emulsion form, against the malaria vector, Anopheles culicifacies. The judicious utilization of this agro-industrial by-product as a nano bio-pesticide will not only prevent the disposal-related problems, but also deal with the issues related to synthetic pesticides. 2. Materials and methods CNSL was procured from Plaza Chemical Industry, India. Tween80 (Polysorbate 80) and Span 20 (Sorbitan monolaurate) were purchased from Sigma-Aldrich and Propylene Glycol from HiMedia, India. All the chemicals used were of analytical grade. Larvae of Anopheles culicifacies were obtained from, National Institute of Malaria Research (NIMR), New Delhi, India. 2.1. Chemical composition of CNSL: (GCMS) The major components of CNSL were identified through GCMS (Shimadzu QP 2010 74,707 Plus) fitted with an FID and capillary column (0.32 mm i.d., length: 30 m, film thickness 0.25 mm). The sample was analyzed as per previously described method (Jesus et al., 2011). A 10-mg sample of CNSL was dissolved in 1 ml of hexane. A 1 ml aliquot of the hexane solution was injected GCMS temperature was programmed with initial oven temperature of 70 °C (hold time 5 min), which was increased at the rate of 10 °C/min to 300 °C (hold time 5 min). Injection temperature was 260 °C, with a split ratio of 10. The identification of chemical constituents of CNSL was carried out using GCMS. The peaks were identified by comparing the individual mass spectra with database of (NIST12 or NIST62- National Institute of Standards and Technology) and Wiley 229 mass spectrometry libraries. 2.2. Preparation of CNSL nanoemulsion Nanoemulsions were prepared using Low-energy emulsification method. CNSL, surfactant mixture (Tween80, Span20) and propylene glycol, were first mixed together in the different ratio and slowly added into aqueous phase drop wise followed by stirring (2000 rpm) for 2 h at room temperature. Different proportions, surfactant mixture and were standardized. The concentration of propylene glycol as co-surfactant was kept constant.
2.3. Characterization of CNSL nanoemulsion 2.3.1. Droplet size (dynamic light scattering) Particle size and polydispesity index (PDI) of nanoemulsion was measure by Malvern Zetasizer Nano-ZS, UK. 100 mg/L sample solution was prepared in double distilled water was taken into transparent disposable polystyrene cuvettes and size was measured. Particle size of bulk emulsion was measured using PSA (Particle size analyzer) Malvern 2000S, UK. For stability studies nanoemulsion was centrifuged at 3500 rpm for 30 min. The stability was also checked at higher temperature (45 °C) and room temperature (25 °C). 2.3.2. Transmission electron microscopy (TEM) The morphology of CNSL nanoemulsuion was determined by TEM. One drop of an aqueous nanoemulsion was placed on the copper grid. The grid was air dried in a vacuum desiccator and then examined under Jeol TEM 1011 (Japan) at 80 eV and direct magnification of 20,000£. 2.4. Bioefficacy evaluation of CNSL nanoemulsion comparison with bulk emulsion 2.4.1. Collection and maintenance of Anopheles culicifacies The Laboratory reared larvae of Anopheles culicifacies were obtained from National Institute of Malaria Research, Insectary. The larvae were maintained in the standard condition of 25 § 2 °C and were kept in dechlorinated tap water in an enamel bowl. Larvae were fed on dog biscuit and yeast powder in the (3:1) ratio. 2.4.2. Dose response bioassays on Aopheles culicifacies The comparative larvicidal bioefficacy of Bulk CNSL and Nano CNSL was evaluated against 3rd instar larvae of Anopheles culicifacies according to the WHO standard larval susceptibility test method (WHO, 2005). The nanoemulsion was prepared using 5% CNSL, which was used to prepare the stock solution of 250 mg/L. Thereafter, serial dilution was done to obtain desired concentrations; 1.2 mg/L, 2.5 mg/ L, 5 mg/L, 10 mg/L and 15 mg/L (Nano) and similarly for bulk emulsion (5 mg/L, 8 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 30 mg/L, 40 mg/L). Larvae (20 individuals) were introduced into each test container containing test solution of 250 ml. A set up containing water, Tween 80: Span 20 (4:1) served as control. Each experiment was carried out in three replications. The number of deceased larvae was counted in the exposed population during the exposure period of 24 h. Corrected mortality was calculated using formulae (WHO, 2009). Mortality ð%Þ ¼
ðX Y Þ 100 ð100Y Þ
Where, X= Percentage mortality in treated sample, Y = Percentage mortality in control sample 2.5. Mode of action of CNSL nanoemulsion 2.5.1. External. morphology of treated larva: scanning electron microscopy The LC50 value of CNSL nanoemulsion estimated as described above was used to treat the Anopheles larvae and to understand the morphological impact of the CNSL nanoemulsion and thus its possible mode of action against larvae. Prior to SEM examination the larva was exposed to LC50 of CNSL nanoemulsion for 24 h. After exposure, the typical fixation process used in SEM to study biological system, which involves series of washing with ethanol, was omitted, because it might wash away the nanodroplets. The modified method was established so that nanodroplets could be retained and observed on larva body. The larva has been dried in the air thereafter placed upon adhesive stubs, and coated with Pt/Pd using sputter coater (Quorum SC7620, UK). The adhesion of CNSL nanoemulsion droplets on the
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cuticle of the larva was observed by scanning electron microscopy (SEM, Tescan Vega 3, Czech Republic). 2.5.2. Histopathology alterations in larva tissues induced by nanoformulation To study the histopathological impact of CNSL nanoemulsion on larva, the 3rd instar larvae of Anopheles were treated with Lethal Concentration 50 values (LC50) of Bulk and Nano CNSL. A setup containing tween, span and water served as control was also observed. After 24 h the larvae was removed from the treated solution and stored in buffered formalin reagent (pH 7.2). The tissues were then dehydrated by passing through graded ethanol series and embedded in paraffin wax (Mekhlafi, 2018). A thin longitudinal section (LS) of the tissue stained with Hematoxylin and Eosin (Hi-Media labs) was cut using microtome (Leica, Germany) and mounted on glass slide. The LS of the midgut region were examined under an Upright Olympus microscope. 2.6. Statistical analysis The results calculated here are means of three replicates. The standard deviation was calculated and reported as S.E. (standard error) in the tables. Analysis of variance (ANOVA) was performed on experimental data and means were compared using Duncan’s multirange test with SPSS 10.0 software. The significance level was p < 0.05. Lethal concentration (LC50) was determined at the 95% confidence level using probit analysis with SPSS software. 3. Results 3.1. Gas chromatographicmass spectrometry (GCMS) GCMS analysis of CNSL showed the presence of 32 components that formed 100% of the total liquid, with presence of monohydric phenol, cardanol (RT 28, % area 74.8) as major component. Other constituents identified as fatty acids (Table 1).
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Table 2 Composition of CNSL identified by GCMS. Peak
R. time
Area%
Name of compound
1 2 3 4 5 6 7 8 9
6.498 8.443 10.333 12.128 14.268 16.733 17.726 18.181 18.338
0.04 0.10 0.10 0.05 0.03 0.05 0.17 0.52 0.42
10 11 12
18.601 20.318 20.651
1.43 0.08 0.05
13 14 15
21.467 22.748 23.222
0.03 0.22 0.65
16 17 18 19 20 21
23.861 24.705 24.980 25.435 25.551 26.151
0.10 0.06 0.04 0.06 2.18 0.03
22
26.457
0.16
23 24 25 26 27
26.634 27.690 28.351 29.530 30.418
0.03 2.17 74.8 0 0.05 0.10
28 29 30 31 32 Total
30.614 30.680 31.202 31.680 32.000
0.98 0.99 1.34 11.38 1.21 100.00
Decane Undecane DODECANE Tridecane (R)-(-)-14-Methyl-8-hexadecyn-1-ol Caryophyllenyl alcohol 1,2,3-Butanetriol, 3TMS derivative D-2-Deoxyribose, 3TMS derivative 3,8-DIOXA-2,9-DISILADECANE, 2,2,9,9-TETRAMETHYL-5,6-BIS[(TRIMETHYLSILY Ribitol, 5TMS derivative Myristic acid, TMS derivative D-(+)-Xylose, tetrakis(trimethylsilyl) ether, methyloxime (anti) E-11-Tetradecenol, trimethylsilyl ether Palmitic Acid, TMS derivative TRIMETHYL ({2,3,4,5,6-PENTAKIS[(TRIMETHYLSILYL)OXY]CYCLOHEXYL}OXY)S cis-Vaccenic acid Oleic Acid, (Z)-, TMS derivative Stearic acid, TMS derivative (Z)-3-(pentadec-8-en-1-yl) phenol 3-Tridecylphenol XYLITOL, 1,2,3,4,5-PENTAKIS-O-(TRIMETHYLSILYL)D-GLUCITOL, 1,2,3,4,5,6-HEXAKIS-O(TRIMETHYLSILYL)D-(-)-Tagatose, 5TMS derivative Ginkgol (TMS) (Z)-3-(pentadec-8-en-1-yl)phenol (Cardanol) (Z)-3-(pentadec-8-en-1-yl)phenol 7,10,13-HEXADECATRIENOIC ACID, METHYL ESTER 1,8,11,14-Heptadecatetraene, (Z,Z,Z)(Z)-3-(Heptadec-10-en-1-yl)phenol Bilobol C15:1 (1TMS) 2,6,8-Trimethylbicyclo[4.2.0]oct-2-ene-1,8-diol 1,4-Benzenediol, 2,5-dimethyl-
3.2. Preparation and characterization of nanoemulsion The nanoemulsion was prepared by low energy spontaneous emulsification method. In order to predict the best surfactant ratio, several emulsions were prepared by varying the relative amounts of Tween-80 and Span-20 (F1-F5 Table 2). The nanoemulsions F1, F2, F3 and F4 showed phase separation at higher temperature (45 °C) therefore did not characterize further. The nanoemulsion (F5) was found to be stable with Z-average diameter of 52 nm and PDI of 0.27. In stability test after storage at an ambient temperature for two months there was slight increase in the mean particle size, which was recorded as 56 nm. At higher temperature (45 °C) particle size was recorded as 60 nm (Table 3). Typical size distribution of nanoemulsion recorded initially and after storage is shown in Fig. 1. For comparison, bulk emulsion yielded much larger particles, with an average size of 20 mm. There was no phase separation observed in the formulation during storage period. The morphology of the nanoemulsion was visualized by TEM (Fig. 2). The TEM images revealed that the droplets had spherical morphology. Table 1 Composition of nanoemulsions. Formulation
CNSL
Propylene glycol
(Tween: Span) (10)
Water
F1 F2 F3 F4 F5
5 5 5 5 5
3 3 3 3 3
0:1 1:0 1:1 3:2 4:1
82 82 82 82 82
Table 3 Characterization of nanoemulsion values are given as mean standard error (n = 3). Code
Size (nm/microns)
PDI
Initial After 2 months period at room temp 25 °C. After storage at higher temperature 45 °C
52 § 3.9 56 § 2.1 60 § 2.9
0.27 § 0.06 0.22 § 0.03 0.46 § 0.05
3.3. Larvicidal bioassay The larval mortality was dose and time dependent (Table 4). No mortality was observed in the control. The LC50 (24 h) for Bulk and Nano CNSL was found to be 18.1 mg/L and 1.4 mg/L, respectively. The lethal indices for 24 h were found to be statistically significant (p < 0.05) (Table 4). 3.4. Mode of action: morphological observations of larva (scanning electron microscopy) The present study provided a comprehensive evaluation of the effect of CNSL nanoemulsion on the external morphology of larva. SEM examination revealed the larva was more intact in the control treatment (Fig. 3(A)); whereas, the completely deformed larva was observed from treatment with nano CNSL (Fig. 3(B)). The uptake and impregnation with nanoemulsion droplets through the cuticle of the larva (Fig. 3(C)) can be clearly observed in treated larva (inside circles). It is worth noting that nano droplets were present on the body of larva treated with nanoemulsion, whereas the control larva
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Fig. 1. DLS measurement of particle size distribution of NE A (after perpetration), B (after storage at ambient temperature), C (after storage at higher temperature).
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was devoid of droplets (Fig. 3(D)). Image analysis showed that nanoemulsion adhered to the body of larva. 3.5. Histopathological alterations LS examination of the control larval tissues displayed intact and undamaged assembly of the epithelial cells (EC) lying upon the completely unbroken peritrophic membrane (PM) (Fig 4(A)). However the larval exposed to Bulk CNSL displayed partial damage in EC and PM (Fig. 4(B)). The Nano CNSL treated larval tissues displayed significant damaged and broken EC and PM with extensive midgut content (MC) leakage (Fig. 4(C)). 4. Discussion The overuse of pesticides in bulk form has led to the contamination of the environment (Anjali et al., 2010). The application of nanotechnology in insecticide delivery can enhance the efficacy of the product, thereby reducing the load of pesticides in environment
Fig. 2. TEM of nanoemulsion showing spherical droplets.
Table 4 Statistical evaluation of the percentage mortality data of CNSL nanoemulsion in comparison to bulk emulsion (24 h). § S.E.: standard error. Means (§S.E.) followed by the same letters (ae) within columns indicate no significant difference (p < 0.05) (Duncan’s multi-range test) and comparative bioefficacy LC50 of bulk and nanoemulsion of CNSL against Anopheles culicifacies after 24 h. Concentration bulk (mg/L)
Number of dead larva (n = 20)
Mortality (%)
(LC50 mg/L) LCL-UCL
LC90 mg/L LCL-UCL
Slope
Intercept
1.2 2.5 5 10 15 Concentration bulk (mg/L) 5 8 10 15 20 30 40
9.7 § 0.6d 11.7 § 1.5c 15.3 § 0.6b 16.7 § 0.6ab 18.3 § 1.5a
48.3 § 2.9d 58.3 § 7.6c 76.7 § 2.9b 83.3 § 2.9ab 91.7 § 7.6a
1.4 (0.66- 2.9)
14.89 (7.330.2)
1.2 § 0.02
4.8 § 0.1
1.3 § 0.6e 2.0 § 1.0e 6.0 § 1.0d 8.7 § 0.6cd 9.7 § 1.5c 13.7 § 1.5b 17.7 § 1.5a
6.7 § 2.9e 10.0 § 5.0e 30.0 § 5.0d 43.3 § 2.9cd 48.3 § 7.6c 70.0 § 5.0b 86.7 § 7.6a
18.1 (13.823.7)
56.5 (40.379.1)
2.9 § 0.6
1.2 § 0.6
LC Lethal Concentration; LCL-95 Lower Confidential Limit; ULC-95 Upper confidential Limit § S.E.: standard error. Mean (§S.E.) followed by the same letters (ae) within columns indicate no significant difference (p < 0.05) (Duncan’s multi-range test).
Fig. 3. Scanning electron microscopy (SEM) images of air-dried Anopheles culicifacies larva exposed to the CNSL nanoemulsion. (A1) Untreated intact larva, (A2) external surface of larva devoid of nanodroplets. (B1) Treated deformed/damaged larva and (B2) external surface of larva with targeted nanodroplets (with in circles).
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Fig. 4. L.S of larvae treated with showing midgut region EC (epithelial cells), (PM) peritrophic membrane and MC midgut content of (A) control (B) LC50 (24 h) of Bulk CNSL and LC50 (24 h) of Nano CNSL observed at 100£ magnification.
(Ali et al., 2017). In the present work, CNSL (waste from cashew nut shell industry) nanoformulation have been proposed, in order to minimize disposal related issues of waste and to enhance its efficacy as a bio-pesticide. The phenolic compound, cardanol in CNSL is known to exhibit strong larvicidal activity against mosquitoes (Paiva et al., 2017; Raraswati et al., 2014). The concentration of cardanol may vary and reported up to 67.8% (Lomonaco et al., 2017). The higher percentage
of cardanol 74.8%, identified in our CNSL may be attributed to extraction process, which involves the excess of heat (180200 °C) during the roasting process, due which anacardic acid present in CNSL, undergoes decarboxylation and is converted to cardanol, the statement is supported by previous report (Jesus et al., 2011). The spontaneous emulsification occurs when an organic phase and an aqueous phase are mixed (Bouchemal et al., 2004). A combination of non-ionic surfactants, sorbitan monolaurate (HLB 8.6) and
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Polysorbate 80 (HLB 15.6) were selected, since non-ionic surfactants do not affect pH and ionic strength (Ali et al., 2017). The increase in the concentration of T-80 improved the stability of the nanoemulsions. The stabilization of nanodroplets in the emulsion with surfactant ratio of 4:1 would be due to reduction of interfacial free energy, which provides a mechanical barrier to coalescence. The propylene glycol as co-surfactant further reduces the interfacial tension between two distinct phases. The lower PDI value (0.27) is indicative of higher uniformity of droplet size; it is in accordance with previous literature (Anjali et al., 2010). In the present study, the combination of non-ionic low and high HLB and propylene glycol as co-surfactant might have also favored the stability of the CNSL nanoemulsions. Despite the particle grown up, in the storage stability test, which was still in nano range. The reason for maintaining stability was attributed to the large head group size of Tween 80 that produced great repulsive forces between droplets (Akbas et al., 2018). The dose response bioassay result signifies the improved larvicidal efficacy of nano CNSL as compared to the bulk CNSL; at low exposure concentrations. This may be because nanoparticles exhibit much higher ability to penetrate the membranes of the cells compared to bulk particles (Anjali et al., 2010, 2012). Due smaller size the nano CNSL it provided better adsorption and facilitates easier penetration into the cuticle of larvae, which is further confirmed by morphological study of larva. These dynamic features of the nano CNSL have enhanced the larval mortality as compared to bulk CNSL. The mode of action on pesticide is essential in order to improve the quality, sustainability and applicability of a product. The major CNSL constituent; cardanol is capable of exerting deep effects on insects. The excellent effect exhibited by cardanol because of the unsaturation on the alkyl side chain that increases its liposolubility facilitating passage through the cell membrane, this correlation between liposolubility and penetration was described in earlier report (Lomonaco et al., 2009). The similar effect of adherence of nanoformulation was also observed on the stored pest through SEM imaging (Hashem et al., 2018). The histopathological observations also confirmed the improved penetration of Nano CNSL as compared to Bulk CNSL into the larval tissues. 5. Conclusion The CNSL nanoemulsion can be effectively utilized as pesticide and use of waste material as pesticide may reduce the disposal issues. The improved bioefficacy possessed by the nano CNSL even at lower concentrations may significantly reduce the pesticide load and toxicity to the environment. This study also showed that CNSL nanoemulsion might have potential to control immature stages of mosquitoes, which are easy to target as compared to adult. The nano CNSL exerted toxicity through external penetration and caused damage to the larva. The usage of CNSL as larvicide would be cost effective, in particular for controlling mosquito larvae in breeding places like stagnant water sources. Funding by external sources This article does not use any fund from any of the external sources/Agencies/Organizations. Involvement of human/animal in the study N/A. Submission declaration The manuscript is original has not been published before and it is not under consideration for publication anywhere else. The
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Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex Quinquefasciatus and Culex Tritaeniorhynchus (Diptera: Culicidae). Research in Veterinary Science 104, 77–82. Hashem, A.S., Awadalla, S.S., Zayed, G.M., Maggi, F., Benelli, G., 2018. Pimpinella anisum essential oil nanoemulsions against Tribolium castaneum—insecticidal activity and mode of action. Environmental Science and Pollution Research 25 (19), 18802– 18812. https://doi.org/10.1007/s11356-018-2068-1. Karthic, P.K., Sinija, V.R., Alagusundaram, K., 2014. Indian cashew processing industry an overview. Journal of Food Research and Technology 2 (2), 60–66. Knowells, A., 2008. Recent developments of safer formulations of agrochemicals. Environmentalist 28, 35. https://doi.org/10.1007/s10669-007-9045-4. Mukhopadhyaya, A.K., Hatib, T.W., Satya, B.P., 2010. Larvicidal properties of cashew nut shell liquid (Anacardium occidentale L) on immature stages of two mosquito species. Journal of Vector Borne Diseases 47, 257–260. Narladkar, W., 2018. Projected economic losses due to vector and vector-borne parasitic diseases in livestock of India and its significance in implementing the concept of integrated practices for vector management. Veterinary World 11 (2), 151–160. Raraswati, G.R.S., Mulyaningsih, B., 2014. Larvicidal activity of a mixture of cashew nut shell liquid and water-soluble extract of soap nut fruit (Sapindus rarak DC.). Against
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(Eds.) WHO/Department of control of neglected tropical diseases, 2009. Guideline for Efficacy Testing of Insecticide for Indoor and Outdoor Ground Applied Space Spray Application. In: Zaim, D.R. (Ed.), Guideline for Efficacy Testing of Insecticide for Indoor and Outdoor Ground Applied Space Spray Application. WHOPES, p. 83. (Eds.) WHO reference number: WHO/HTM/NTD/WHOPES/ GCDPP/2009.6. World Health Organization 2005. laboratory and field-testing of mosquito larvicides (CDS/WHOPES/GCDPP/05.13). Geneva. https://www.who.int/whopes/resources/ who_cds_whopes_gcdpp_2005.13/en/. (Accessed on February 15, 2018).
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Nanoemulsion of cashew nut shell liquid bio-waste: Mosquito larvicidal activity and insights on possible mode of action S. Kalaa,b, N. Soganc, Prveen Vermad, S.N. Naikb,*, A. Agarwala, P.K. Patanjalia, J. Kumara a
Formulation Division, Institute of Pesticide Formulation Technology (IPFT), Gurugram 122016, Haryana, India Center for Rural Development Technology (CRDT), Indian Institute of Technology (IIT), Hauz Khas, Delhi 110016, India c National Institute of Malaria Research (NIMR), Delhi 110077, India d Department of Zoology, Agra College, Agra 282004, India b
A R T I C L E
I N F O
Article History: Received 25 April 2019 Revised 4 September 2019 Accepted 5 October 2019 Available online xxx Edited by J Van Staden Keywords: Cashew nut shell liquid Waste Nanoemulsion Bio-pesticide Mosquitoes
A B S T R A C T
Cashew nut (Anacardium occidentale) shell liquid (CNSL) is a massive waste generated from the cashew nut processing industry. The Disposal of large volume of CNSL may become a concern in terms of ecological issues. CNSL is having insecticidal properties despite of this fact it has not been utilized to its full extent. CNSL is poorly soluble in water; in this context, we have utilized CNSL to develop a nanoemulsion for larvicidal applications; using spontaneous emulsification method. The monodispersed micelles of CNSL with mean diameter of 52 nm were produced. The comparative bioefficacy of bulk and nano CNSL was evaluated against the 3rd instar larva of Anopheles culicifacies, which was recorded with LC50 of 18.1 mg/L and 1.4 mg/ L, respectively. Morphological damages to the larva post exposure to CNSL nanoemulsion were examined using scanning electron microscope (SEM). Histopathological examination revealed detailed mode of toxicity of nano CNSL on larva. The result obtained showed that the CNSL nanoemulsion has enhanced larvicidal activity compared to bulk CNSL which allows effective utilization of waste. The utilization of CNSL nanoemulsion may also resolve disposal related issues of massive waste generated by cashew nut industry. Such approach may be suitable for enhanced efficacy against mosquito vector, at lower doses and also contribute towards reduced use of synthetic pesticides. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Vector borne diseases represent a key to threat for millions of people worldwide, which may lead to epidemics in population of humans and animals (Govindarajan et al., 2016). Among those vectors; mosquito is responsible for various life-threatening diseases with enormous economic impact, along with higher global disease burden (Doucoure et al., 2015). Mosquito belonging to genus Anopheles feed on vertebrate blood and transmit the protozoan parasites, causing malaria. The mosquitos have serious impact on human as well as animal health (Stuchin et al., 2016) and significantly influences the production from livestock (cattle) resulting in economic loses (Narladkar, 2018). The mosquitoes are carriers of deadly parasites, which are transmitted to humans through female Anopheles mosquito. Therefore, the control of these vectors is absolutely ‘essential’. Furthermore, the mosquito control will prevent the spread of mosquito-borne disease and improve quality of public health (Ghosh et al., 2012).
*Correspondence author. E-mail address: [email protected] (S.N. Naik). https://doi.org/10.1016/j.sajb.2019.10.006 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Although various vector control strategies involving synthetic pesticides are available, but the control on the mosquito population is not satisfactory, because the mosquitoes have developed resistance. Consequently, the increased use of pesticides has been employed which in turn has increased the load on environmental and come up with adverse impact on nature and mankind. Pesticides from plant origin, especially plant-based waste are currently receiving considerable attention because they are non-persistent, biodegradable and cost effective (Ghosh et al., 2012). Conventionally available formulations; emulsifiable concentrate (EC) is petroleum solvents based, whereas wettable powder (WP) produces lots of dust during usage (Knowels, 2008). With the advancement in nanotechnology, nanoemulsions provides the basis for formulating aqueous-based nanopesticides of poorly water-soluble active with improved and enhanced efficacy (Fernandes et al., 2014). The conversion of pesticide moiety to nano level through nanotechnology can reduce quantity of active ingredients, thereby reducing the dose, and cost and enhances the efficacy of the product (Ali et al., 2017). The cashew (Anacardium occidentale L.) is a tropical plant, native to Brazil and is widely distributed in many other countries around the world including India, Nigeria, Viet Nam, United Republic of Tanzania, Philippines, Indonesia, and Guinea-Bissau (Adeigbe et al.,
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2005; FAOSTAT, 2017). CNSL is a by-product from cashew industrial processing, it occurs in pericarp of the cashew fruit (Paiva et al., 2017), it has no added value and is considered as a waste. India is among top five countries producing cashew nuts (Karthic et al., 2014). CNSL comprises of 25% of total nuts production. In the year 2013, 1000 tons of CNSL was obtained as by-product from the industry. It is composed of phenolic components the disposal of such a large volume of CNSL may become a cause of concern in terms of ecological issues (Calo` et al., 2007; Lomonaco et al., 2009). CNSL is non-toxic, biodegradable; cheap and easily available. It contains cardanol, a monohydric phenol that is responsible for larvicidal activity (Mukhopadhyaya et al., 2010). Several reports have proved that CNSL is a potential larvicide against mosquitoes (Paiva et al., 2017; Raraswati et al., 2014). Although, the insecticidal potential of CNSL has not been utilized, fully, thus there is need for a suitable formulation technology, which may deliver enhanced bioefficacy at the lower doses. Moreover, CNSL was also found safe towards non-target organisms. In a study of impact on non-target organisms, CNSL exhibited no mortality on any of the organisms including those of fishes (Gambusia affinis and Poecilia reticulata), tadpoles (Bufo spp.), and water bugs (Heteroptera spp.) (Mukhopadhyaya et al., 2010). The increased interest in efficient, efficacious, ecofriendly and low cost pesticides practices has encouraged us to devolve nanoemulsion, utilizing CNSL. The bio-efficacy of the attained stable nano formulation of CNSL was comparatively investigated with its bulk emulsion form, against the malaria vector, Anopheles culicifacies. The judicious utilization of this agro-industrial by-product as a nano bio-pesticide will not only prevent the disposal-related problems, but also deal with the issues related to synthetic pesticides. 2. Materials and methods CNSL was procured from Plaza Chemical Industry, India. Tween80 (Polysorbate 80) and Span 20 (Sorbitan monolaurate) were purchased from Sigma-Aldrich and Propylene Glycol from HiMedia, India. All the chemicals used were of analytical grade. Larvae of Anopheles culicifacies were obtained from, National Institute of Malaria Research (NIMR), New Delhi, India. 2.1. Chemical composition of CNSL: (GCMS) The major components of CNSL were identified through GCMS (Shimadzu QP 2010 74,707 Plus) fitted with an FID and capillary column (0.32 mm i.d., length: 30 m, film thickness 0.25 mm). The sample was analyzed as per previously described method (Jesus et al., 2011). A 10-mg sample of CNSL was dissolved in 1 ml of hexane. A 1 ml aliquot of the hexane solution was injected GCMS temperature was programmed with initial oven temperature of 70 °C (hold time 5 min), which was increased at the rate of 10 °C/min to 300 °C (hold time 5 min). Injection temperature was 260 °C, with a split ratio of 10. The identification of chemical constituents of CNSL was carried out using GCMS. The peaks were identified by comparing the individual mass spectra with database of (NIST12 or NIST62- National Institute of Standards and Technology) and Wiley 229 mass spectrometry libraries. 2.2. Preparation of CNSL nanoemulsion Nanoemulsions were prepared using Low-energy emulsification method. CNSL, surfactant mixture (Tween80, Span20) and propylene glycol, were first mixed together in the different ratio and slowly added into aqueous phase drop wise followed by stirring (2000 rpm) for 2 h at room temperature. Different proportions, surfactant mixture and were standardized. The concentration of propylene glycol as co-surfactant was kept constant.
2.3. Characterization of CNSL nanoemulsion 2.3.1. Droplet size (dynamic light scattering) Particle size and polydispesity index (PDI) of nanoemulsion was measure by Malvern Zetasizer Nano-ZS, UK. 100 mg/L sample solution was prepared in double distilled water was taken into transparent disposable polystyrene cuvettes and size was measured. Particle size of bulk emulsion was measured using PSA (Particle size analyzer) Malvern 2000S, UK. For stability studies nanoemulsion was centrifuged at 3500 rpm for 30 min. The stability was also checked at higher temperature (45 °C) and room temperature (25 °C). 2.3.2. Transmission electron microscopy (TEM) The morphology of CNSL nanoemulsuion was determined by TEM. One drop of an aqueous nanoemulsion was placed on the copper grid. The grid was air dried in a vacuum desiccator and then examined under Jeol TEM 1011 (Japan) at 80 eV and direct magnification of 20,000£. 2.4. Bioefficacy evaluation of CNSL nanoemulsion comparison with bulk emulsion 2.4.1. Collection and maintenance of Anopheles culicifacies The Laboratory reared larvae of Anopheles culicifacies were obtained from National Institute of Malaria Research, Insectary. The larvae were maintained in the standard condition of 25 § 2 °C and were kept in dechlorinated tap water in an enamel bowl. Larvae were fed on dog biscuit and yeast powder in the (3:1) ratio. 2.4.2. Dose response bioassays on Aopheles culicifacies The comparative larvicidal bioefficacy of Bulk CNSL and Nano CNSL was evaluated against 3rd instar larvae of Anopheles culicifacies according to the WHO standard larval susceptibility test method (WHO, 2005). The nanoemulsion was prepared using 5% CNSL, which was used to prepare the stock solution of 250 mg/L. Thereafter, serial dilution was done to obtain desired concentrations; 1.2 mg/L, 2.5 mg/ L, 5 mg/L, 10 mg/L and 15 mg/L (Nano) and similarly for bulk emulsion (5 mg/L, 8 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 30 mg/L, 40 mg/L). Larvae (20 individuals) were introduced into each test container containing test solution of 250 ml. A set up containing water, Tween 80: Span 20 (4:1) served as control. Each experiment was carried out in three replications. The number of deceased larvae was counted in the exposed population during the exposure period of 24 h. Corrected mortality was calculated using formulae (WHO, 2009). Mortality ð%Þ ¼
ðX Y Þ 100 ð100Y Þ
Where, X= Percentage mortality in treated sample, Y = Percentage mortality in control sample 2.5. Mode of action of CNSL nanoemulsion 2.5.1. External. morphology of treated larva: scanning electron microscopy The LC50 value of CNSL nanoemulsion estimated as described above was used to treat the Anopheles larvae and to understand the morphological impact of the CNSL nanoemulsion and thus its possible mode of action against larvae. Prior to SEM examination the larva was exposed to LC50 of CNSL nanoemulsion for 24 h. After exposure, the typical fixation process used in SEM to study biological system, which involves series of washing with ethanol, was omitted, because it might wash away the nanodroplets. The modified method was established so that nanodroplets could be retained and observed on larva body. The larva has been dried in the air thereafter placed upon adhesive stubs, and coated with Pt/Pd using sputter coater (Quorum SC7620, UK). The adhesion of CNSL nanoemulsion droplets on the
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cuticle of the larva was observed by scanning electron microscopy (SEM, Tescan Vega 3, Czech Republic). 2.5.2. Histopathology alterations in larva tissues induced by nanoformulation To study the histopathological impact of CNSL nanoemulsion on larva, the 3rd instar larvae of Anopheles were treated with Lethal Concentration 50 values (LC50) of Bulk and Nano CNSL. A setup containing tween, span and water served as control was also observed. After 24 h the larvae was removed from the treated solution and stored in buffered formalin reagent (pH 7.2). The tissues were then dehydrated by passing through graded ethanol series and embedded in paraffin wax (Mekhlafi, 2018). A thin longitudinal section (LS) of the tissue stained with Hematoxylin and Eosin (Hi-Media labs) was cut using microtome (Leica, Germany) and mounted on glass slide. The LS of the midgut region were examined under an Upright Olympus microscope. 2.6. Statistical analysis The results calculated here are means of three replicates. The standard deviation was calculated and reported as S.E. (standard error) in the tables. Analysis of variance (ANOVA) was performed on experimental data and means were compared using Duncan’s multirange test with SPSS 10.0 software. The significance level was p < 0.05. Lethal concentration (LC50) was determined at the 95% confidence level using probit analysis with SPSS software. 3. Results 3.1. Gas chromatographicmass spectrometry (GCMS) GCMS analysis of CNSL showed the presence of 32 components that formed 100% of the total liquid, with presence of monohydric phenol, cardanol (RT 28, % area 74.8) as major component. Other constituents identified as fatty acids (Table 1).
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Table 2 Composition of CNSL identified by GCMS. Peak
R. time
Area%
Name of compound
1 2 3 4 5 6 7 8 9
6.498 8.443 10.333 12.128 14.268 16.733 17.726 18.181 18.338
0.04 0.10 0.10 0.05 0.03 0.05 0.17 0.52 0.42
10 11 12
18.601 20.318 20.651
1.43 0.08 0.05
13 14 15
21.467 22.748 23.222
0.03 0.22 0.65
16 17 18 19 20 21
23.861 24.705 24.980 25.435 25.551 26.151
0.10 0.06 0.04 0.06 2.18 0.03
22
26.457
0.16
23 24 25 26 27
26.634 27.690 28.351 29.530 30.418
0.03 2.17 74.8 0 0.05 0.10
28 29 30 31 32 Total
30.614 30.680 31.202 31.680 32.000
0.98 0.99 1.34 11.38 1.21 100.00
Decane Undecane DODECANE Tridecane (R)-(-)-14-Methyl-8-hexadecyn-1-ol Caryophyllenyl alcohol 1,2,3-Butanetriol, 3TMS derivative D-2-Deoxyribose, 3TMS derivative 3,8-DIOXA-2,9-DISILADECANE, 2,2,9,9-TETRAMETHYL-5,6-BIS[(TRIMETHYLSILY Ribitol, 5TMS derivative Myristic acid, TMS derivative D-(+)-Xylose, tetrakis(trimethylsilyl) ether, methyloxime (anti) E-11-Tetradecenol, trimethylsilyl ether Palmitic Acid, TMS derivative TRIMETHYL ({2,3,4,5,6-PENTAKIS[(TRIMETHYLSILYL)OXY]CYCLOHEXYL}OXY)S cis-Vaccenic acid Oleic Acid, (Z)-, TMS derivative Stearic acid, TMS derivative (Z)-3-(pentadec-8-en-1-yl) phenol 3-Tridecylphenol XYLITOL, 1,2,3,4,5-PENTAKIS-O-(TRIMETHYLSILYL)D-GLUCITOL, 1,2,3,4,5,6-HEXAKIS-O(TRIMETHYLSILYL)D-(-)-Tagatose, 5TMS derivative Ginkgol (TMS) (Z)-3-(pentadec-8-en-1-yl)phenol (Cardanol) (Z)-3-(pentadec-8-en-1-yl)phenol 7,10,13-HEXADECATRIENOIC ACID, METHYL ESTER 1,8,11,14-Heptadecatetraene, (Z,Z,Z)(Z)-3-(Heptadec-10-en-1-yl)phenol Bilobol C15:1 (1TMS) 2,6,8-Trimethylbicyclo[4.2.0]oct-2-ene-1,8-diol 1,4-Benzenediol, 2,5-dimethyl-
3.2. Preparation and characterization of nanoemulsion The nanoemulsion was prepared by low energy spontaneous emulsification method. In order to predict the best surfactant ratio, several emulsions were prepared by varying the relative amounts of Tween-80 and Span-20 (F1-F5 Table 2). The nanoemulsions F1, F2, F3 and F4 showed phase separation at higher temperature (45 °C) therefore did not characterize further. The nanoemulsion (F5) was found to be stable with Z-average diameter of 52 nm and PDI of 0.27. In stability test after storage at an ambient temperature for two months there was slight increase in the mean particle size, which was recorded as 56 nm. At higher temperature (45 °C) particle size was recorded as 60 nm (Table 3). Typical size distribution of nanoemulsion recorded initially and after storage is shown in Fig. 1. For comparison, bulk emulsion yielded much larger particles, with an average size of 20 mm. There was no phase separation observed in the formulation during storage period. The morphology of the nanoemulsion was visualized by TEM (Fig. 2). The TEM images revealed that the droplets had spherical morphology. Table 1 Composition of nanoemulsions. Formulation
CNSL
Propylene glycol
(Tween: Span) (10)
Water
F1 F2 F3 F4 F5
5 5 5 5 5
3 3 3 3 3
0:1 1:0 1:1 3:2 4:1
82 82 82 82 82
Table 3 Characterization of nanoemulsion values are given as mean standard error (n = 3). Code
Size (nm/microns)
PDI
Initial After 2 months period at room temp 25 °C. After storage at higher temperature 45 °C
52 § 3.9 56 § 2.1 60 § 2.9
0.27 § 0.06 0.22 § 0.03 0.46 § 0.05
3.3. Larvicidal bioassay The larval mortality was dose and time dependent (Table 4). No mortality was observed in the control. The LC50 (24 h) for Bulk and Nano CNSL was found to be 18.1 mg/L and 1.4 mg/L, respectively. The lethal indices for 24 h were found to be statistically significant (p < 0.05) (Table 4). 3.4. Mode of action: morphological observations of larva (scanning electron microscopy) The present study provided a comprehensive evaluation of the effect of CNSL nanoemulsion on the external morphology of larva. SEM examination revealed the larva was more intact in the control treatment (Fig. 3(A)); whereas, the completely deformed larva was observed from treatment with nano CNSL (Fig. 3(B)). The uptake and impregnation with nanoemulsion droplets through the cuticle of the larva (Fig. 3(C)) can be clearly observed in treated larva (inside circles). It is worth noting that nano droplets were present on the body of larva treated with nanoemulsion, whereas the control larva
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Fig. 1. DLS measurement of particle size distribution of NE A (after perpetration), B (after storage at ambient temperature), C (after storage at higher temperature).
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was devoid of droplets (Fig. 3(D)). Image analysis showed that nanoemulsion adhered to the body of larva. 3.5. Histopathological alterations LS examination of the control larval tissues displayed intact and undamaged assembly of the epithelial cells (EC) lying upon the completely unbroken peritrophic membrane (PM) (Fig 4(A)). However the larval exposed to Bulk CNSL displayed partial damage in EC and PM (Fig. 4(B)). The Nano CNSL treated larval tissues displayed significant damaged and broken EC and PM with extensive midgut content (MC) leakage (Fig. 4(C)). 4. Discussion The overuse of pesticides in bulk form has led to the contamination of the environment (Anjali et al., 2010). The application of nanotechnology in insecticide delivery can enhance the efficacy of the product, thereby reducing the load of pesticides in environment
Fig. 2. TEM of nanoemulsion showing spherical droplets.
Table 4 Statistical evaluation of the percentage mortality data of CNSL nanoemulsion in comparison to bulk emulsion (24 h). § S.E.: standard error. Means (§S.E.) followed by the same letters (ae) within columns indicate no significant difference (p < 0.05) (Duncan’s multi-range test) and comparative bioefficacy LC50 of bulk and nanoemulsion of CNSL against Anopheles culicifacies after 24 h. Concentration bulk (mg/L)
Number of dead larva (n = 20)
Mortality (%)
(LC50 mg/L) LCL-UCL
LC90 mg/L LCL-UCL
Slope
Intercept
1.2 2.5 5 10 15 Concentration bulk (mg/L) 5 8 10 15 20 30 40
9.7 § 0.6d 11.7 § 1.5c 15.3 § 0.6b 16.7 § 0.6ab 18.3 § 1.5a
48.3 § 2.9d 58.3 § 7.6c 76.7 § 2.9b 83.3 § 2.9ab 91.7 § 7.6a
1.4 (0.66- 2.9)
14.89 (7.330.2)
1.2 § 0.02
4.8 § 0.1
1.3 § 0.6e 2.0 § 1.0e 6.0 § 1.0d 8.7 § 0.6cd 9.7 § 1.5c 13.7 § 1.5b 17.7 § 1.5a
6.7 § 2.9e 10.0 § 5.0e 30.0 § 5.0d 43.3 § 2.9cd 48.3 § 7.6c 70.0 § 5.0b 86.7 § 7.6a
18.1 (13.823.7)
56.5 (40.379.1)
2.9 § 0.6
1.2 § 0.6
LC Lethal Concentration; LCL-95 Lower Confidential Limit; ULC-95 Upper confidential Limit § S.E.: standard error. Mean (§S.E.) followed by the same letters (ae) within columns indicate no significant difference (p < 0.05) (Duncan’s multi-range test).
Fig. 3. Scanning electron microscopy (SEM) images of air-dried Anopheles culicifacies larva exposed to the CNSL nanoemulsion. (A1) Untreated intact larva, (A2) external surface of larva devoid of nanodroplets. (B1) Treated deformed/damaged larva and (B2) external surface of larva with targeted nanodroplets (with in circles).
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Fig. 4. L.S of larvae treated with showing midgut region EC (epithelial cells), (PM) peritrophic membrane and MC midgut content of (A) control (B) LC50 (24 h) of Bulk CNSL and LC50 (24 h) of Nano CNSL observed at 100£ magnification.
(Ali et al., 2017). In the present work, CNSL (waste from cashew nut shell industry) nanoformulation have been proposed, in order to minimize disposal related issues of waste and to enhance its efficacy as a bio-pesticide. The phenolic compound, cardanol in CNSL is known to exhibit strong larvicidal activity against mosquitoes (Paiva et al., 2017; Raraswati et al., 2014). The concentration of cardanol may vary and reported up to 67.8% (Lomonaco et al., 2017). The higher percentage
of cardanol 74.8%, identified in our CNSL may be attributed to extraction process, which involves the excess of heat (180200 °C) during the roasting process, due which anacardic acid present in CNSL, undergoes decarboxylation and is converted to cardanol, the statement is supported by previous report (Jesus et al., 2011). The spontaneous emulsification occurs when an organic phase and an aqueous phase are mixed (Bouchemal et al., 2004). A combination of non-ionic surfactants, sorbitan monolaurate (HLB 8.6) and
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Polysorbate 80 (HLB 15.6) were selected, since non-ionic surfactants do not affect pH and ionic strength (Ali et al., 2017). The increase in the concentration of T-80 improved the stability of the nanoemulsions. The stabilization of nanodroplets in the emulsion with surfactant ratio of 4:1 would be due to reduction of interfacial free energy, which provides a mechanical barrier to coalescence. The propylene glycol as co-surfactant further reduces the interfacial tension between two distinct phases. The lower PDI value (0.27) is indicative of higher uniformity of droplet size; it is in accordance with previous literature (Anjali et al., 2010). In the present study, the combination of non-ionic low and high HLB and propylene glycol as co-surfactant might have also favored the stability of the CNSL nanoemulsions. Despite the particle grown up, in the storage stability test, which was still in nano range. The reason for maintaining stability was attributed to the large head group size of Tween 80 that produced great repulsive forces between droplets (Akbas et al., 2018). The dose response bioassay result signifies the improved larvicidal efficacy of nano CNSL as compared to the bulk CNSL; at low exposure concentrations. This may be because nanoparticles exhibit much higher ability to penetrate the membranes of the cells compared to bulk particles (Anjali et al., 2010, 2012). Due smaller size the nano CNSL it provided better adsorption and facilitates easier penetration into the cuticle of larvae, which is further confirmed by morphological study of larva. These dynamic features of the nano CNSL have enhanced the larval mortality as compared to bulk CNSL. The mode of action on pesticide is essential in order to improve the quality, sustainability and applicability of a product. The major CNSL constituent; cardanol is capable of exerting deep effects on insects. The excellent effect exhibited by cardanol because of the unsaturation on the alkyl side chain that increases its liposolubility facilitating passage through the cell membrane, this correlation between liposolubility and penetration was described in earlier report (Lomonaco et al., 2009). The similar effect of adherence of nanoformulation was also observed on the stored pest through SEM imaging (Hashem et al., 2018). The histopathological observations also confirmed the improved penetration of Nano CNSL as compared to Bulk CNSL into the larval tissues. 5. Conclusion The CNSL nanoemulsion can be effectively utilized as pesticide and use of waste material as pesticide may reduce the disposal issues. The improved bioefficacy possessed by the nano CNSL even at lower concentrations may significantly reduce the pesticide load and toxicity to the environment. This study also showed that CNSL nanoemulsion might have potential to control immature stages of mosquitoes, which are easy to target as compared to adult. The nano CNSL exerted toxicity through external penetration and caused damage to the larva. The usage of CNSL as larvicide would be cost effective, in particular for controlling mosquito larvae in breeding places like stagnant water sources. Funding by external sources This article does not use any fund from any of the external sources/Agencies/Organizations. Involvement of human/animal in the study N/A. Submission declaration The manuscript is original has not been published before and it is not under consideration for publication anywhere else. The
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