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Enhanced mosquitocidal efficacy of pyrethroid insecticides by nanometric emulsion preparation towards Culex pipiens larvae with biochemical and molecular docking studies

Enhanced mosquitocidal efficacy of pyrethroid insecticides by nanometric emulsion preparation... Background: The growing threat of vector-borne diseases and environmental pollution with conventional pesticides has led to the search for nanotechnology applications to prepare alternative products. Methods: In the current study, four pyrethroid insecticides include alpha-cypermethrin, deltamethrin, lambda- cyhalothrin, and permethrin were incorporated into stable nanoemulsions. The optimization of nanoemulsions is designed based on the active ingredient, solvent, surfactant, sonication time, sonication cycle, and sonication energy by factorial analysis. The nanoscale emulsions’ droplet size and morphology were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The toxicity of nanoemulsions against Culex pipiens larvae was evaluated and compared with the technical and commercial formulations. The in vitro assay of adenosine triphosphatase (ATPase), carboxylesterase (CaE), and glutathione-S-transferase (GST) were also investigated. Furthermore, molecular docking was examined to assess the binding interactions between the tested pyrethroids and the target enzymes. Also, an ecotoxicological assessment of potential effects of the tested products on the freshwater alga Raphidocelis subcapitata was determined according to OECD and EPA methods. The emulsifible concentration (EC ) and NOEC (no observed effect concentration) values were estimated for each insecticide and graded according to the GHS to determine the risk profile in aquatic life. Results: The mean droplet diameter and zeta potential of the prepared pyrethroid nanoemulsions were found to be in the range of 72.00–172.00 nm and − 0.539 to − 15.40 mV, respectively. All insecticides’ nanoemulsions showed significantly high toxicity (1.5–2-fold) against C. pipiens larvae compared to the technical and EC. The biochemical activity data proved that all products significantly inhibited ATPase. However, GST and CaE were significantly activated. Docking results proved that the pyrethroids exhibited a higher binding affinity with CaE and GST than ATPase. The docking scores ranged from − 4.33 to − 10.01 kcal/mol. Further, the biosafety studies of the nanopesticides in comparison with the active ingredient and commercial EC were carried out against the freshwater alga R. subcapitata and the mosquitocidal concentration of nanopesticides was found to be non-toxic. * Correspondence: m_eltaher@yahoo.com Department of Pesticide Chemistry and Technology, Laboratory of Pesticide Residues Analysis, Faculty of Agriculture, Alexandria University, 21545-El-Shatby, Alexandria, Egypt Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 2 of 19 Conclusion: The mosquitocidal efficacy of nano-pyrethroids formulated in a greener approach could become an alternative to using conventional pesticide application in an environmentally friendly manner. Keywords: Culex pipiens, Pyrethroids, Nanoemulsion, Insecticidal activity, Biochemical studies, Molecular Docking, Ecotoxicity 1 Introduction relatively large droplet size. Emulsions are dynamically Culex pipiens is one of the many members of the unstable systems that tend to collapse through gravita- disease-carrying mosquito family. Specifically, C. pipiens tional separation, droplet aggregation, and Ostwald rip- is a well-known carrier of the West Nile virus, Saint ening. All these factors can negatively affect the end Louis encephalitis viruses, canine Dirofilaria worms, product’s efficiency and shelf life, reducing its pest con- avian malaria, and filarial worms. Since the early twenti- trol ability. Many of these problems can be overcome eth century, campaigns have been organized in many with the use of nanoemulsions, which are an effective countries to control this pest species [1]. For C. pipiens way to use pesticides efficiently, economically, and safely mosquitoes’ chemical control, pyrethroid insecticides [11]. Nanoemulsions consist of emulsifier-coated fine oil have been extensively used worldwide [2]. Their use in- droplets dispersed in water, having droplets covering the creased to represent from 18% in 2002 to 30% in 2017 of size range of 20–500 nm [9]. They are also referred to as the total global pesticide market [3]. These pesticides are mini-emulsions, ultrafine emulsions, submicron emul- synthetically modified analogs of the essential natural sions, and others. Due to their particular size, nanoemul- pyrethrins found in flowers of the Chrysanthemums sions are transparent or translucent to the naked eye genus. In general, many new pyrethroids are synthesized and are stable against sedimentation or creaming. The and added to the market to meet the enhanced global smaller size of the droplets increases their stability to the demand for food, vector-borne diseases, and pest species gravitational separation and accumulation of droplets. It resistant to other pesticides [4]. Pyrethroids are an es- increases their deposition, diffusion, and permeability to sential way to combat malaria and other mosquito-borne plant leaves and insect body surfaces [12]. The compos- diseases despite the risk of pyrethroids resistance in ition, characteristics, mechanism of formation, and sta- vector populations [5]. Pyrethroids are also common in- bility of pesticide nanoemulsions have essential gredients of household insecticides. The home environ- theoretical and practical significances on the promotion ment’s unregulated use increases the risk of exposure and application of pesticides compared to conventionally and adverse effects in the general population [6]. applied pesticides [13, 14]. Synthetic alpha-cyano pyrethroids such as alpha- The main objectives of this study were to prepare and cypermethrin, deltamethrin, and lambda-cyhalothrin are characterize O/W nanoemulsions of four pyrethroid in- potent environmentally compatible insecticides and have secticides (alpha-cypermethrin, deltamethrin, lambda- a wide margin of safety for mammals for preferential ap- cyhalothrin, and permethrin). Various factors were de- plication in agricultural, veterinary, and public health signed and investigated to prepare the nanoemulsions programs [7]. using a factorial design by Minitab software. Factors in- Pyrethroid products have been traded in some formu- clude the concentration of the active ingredient, solvent, lations, the most popular of which are emulsifiable con- surfactant, sonication time, sonication pulses, and centrate (EC), aerosol dispenser, wettable powder, dust sonication power. The prepared nanoemulsions’ charac- powder, and water dispersion granules [8]. The EC of terizations, including the droplet size distribution, poly- pyrethroids is usually two to nine times more toxic than dispersity index (PDI), viscosity, pH, stability, and the technical grade, likely due to synergistic reactions. It surface morphology by transmission electron microscopy is one of the most widely used delivery systems for (TEM) were investigated. The toxicity of the nanoemul- hydrophobic pesticides, accounting for 40–50% of total sions was investigated against Culex pipiens larvae com- formulations. However, about 300,000 tons per year of paring to the active ingredient and commercial EC. organic solvents are used to prepare the EC formulations Biochemical studies were also investigated in vitro on [9]. Besides, other common solvents and co-solvents can adenosine triphosphatase (ATPase), carboxylesterase also be used. These solvents have flammable, explosive, (CaE), and glutathione-S-transferase (GST). We further and toxic properties that make them harmful to humans applied the molecular docking of these insecticides in and crops and produce poisonous residues in the envir- conjunction with existing experimental data and onment [10]. In practice, some of the problems associ- enzyme-associated tested insecticides to hypothesize ated with using conventional emulsifiers as delivery how these compounds would interact with the target systems for hydrophobic pesticides relate to their proteins. Further, this study demonstrated the non-toxic Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 3 of 19 property of nanopesticides towards non-target species of in adult cages. Using a live pigeon in our study was ac- the freshwater alga Raphidocelis subcapitata. cording to the Ethics Committee of High Institute of Public Health’s acceptance and proved from Alexandria 2 Methods University under reference number 481. The egg rafts 2.1 Tested pyrethroids were transferred from adult cages to white trays contain- The technical grade of alpha-cypermethrin (97%), delta- ing de-chlorinated water for egg hatch [16]. methrin (98%), and lambda-cyhalothrin (95%) were ob- tained from Syngenta Agro. Co (6th of October, Giza, 2.4 Experimental design for nanoemulsions preparation Egypt), while permethrin technical grade (96%) was ob- The experimental design allows studying the effect of tained from Chema Industries (26 First Industrial Area, many variables with a limited number of experiments. EL-Nubariya, El-Beheira Governorate, Egypt). The The design relied on deltamethrin as an insecticide chemical structures and physicochemical properties of chosen from among the four types of pyrethroids tested. the tested pyrethroids are shown in Table S1. Formu- Windows version of MINITAB 19.1 software (2019 lated forms of alpha-cypermethrin (25% EC, Spar-kill®) Minitab Inc.) was used to design the experiments [17]. and lambda-cyhalothrin (10% EC, Lambda®) were pur- Statistical analysis of the results will reveal which vari- chased from El-Helb Pesticides and Chemicals Co. ables have a significant influence and correlate the de- (Dumyat Al Jadidah, Dumyat, Egypt. Deltamethrin for- sired response with the variables by the polynomial mulation (5% EC, Nu-tox®) was obtained from equation: Alexandria Co. for Pharmaceuticals & Chemical Y =A +A X +A X +A X +A X 1 0 1 1 2 2 3 3 n n industries, Co. Permethrin formulation (25% EC, Per- where Y is the dependent variable, A is a constant, gon®) was obtained from MEDMAC for Manufacturing and A –A are the coefficients of the independent 1 n Agricultural Chemicals & Veterinary Products Ltd (Um- values. X -X represent independent factors. 1 n Mutwa’ Al-Aslameya Street, Al-Jandaweel, Amman, A factorial experiment consists of two or more factors Jordan). from these designs, each with discrete possible values or “levels.” In this study, the factorial design methodology 2.2 Chemicals and reagents was used to study the effects of six independent vari- Adenosine triphosphate (ATP), bovine serum albumin ables: the concentration of deltamethrin (as an example (BSA), 1-chloro-2,4-dinitrobenzene (CDNB), dimethyl- of pyrethroids active ingredient), DMSO as a solvent, sulfoxide (DMSO), Folin-Ciocalteu phenol, L- and tween 80 as a surfactant, in addition to applied son- glutathione (GSH), α-naphthyl acetate, β-nicotinamide ication power, sonication time, and pulses of sonication adenine dinucleotide (β-NAD), tetrazotized O- on the droplet size, PDI, pH, and viscosity of prepared dianisidine (fast blue B salt), trichloroacetic acid (TCA), pyrethroid nanoemulsions. Droplet size, PDI, viscosity, Tris (hydroxymethyl)aminomethane), triton X-100 and and pH were determined as the dependent variables. tween 80 were purchased from Sigma-Aldrich Chemical Nine experimental trials involving six independent vari- Co. (St. Louis, MO, USA). Other commercially available ables were obtained from the software. Each variable solvents and chemicals such as ammonium molybdate, was tested at two levels, low (−) and high (+), in addition copper sulfate, EDTA (ethylenediaminetetraacetic acid), to the mean level of each variable was tested in only one ferrous sulfate, and sodium-potassium tartrate were of experiment. analytical grade and purchased from El-Gomhouria For Trading Chemicals And Medical Appliances Co., (Adeb 2.5 Preparation of nanoemulsions Ishak St, Manshia, Alexandria, Egypt) and used without To achieve the final optimized conditions, nine formula- further purification. tions of deltamethrin were prepared in different experi- mental setups, including the organic phase (active 2.3 Culture of Culex pipiens larvae ingredient and DMSO), an aqueous phase containing A susceptible strain of C. pipiens culture was obtained Tween 80, sonication time, sonication power, and sonic- from Research Institute of Medical Entomology, Minis- ation pulses. Deltamethrin was used as an example of try of Health, Dokki, Giza, Egypt, and reared in High In- pyrethroids in optimization experiments. However, four stitute of Public Health insectary, Alexandria University, insecticides, including alpha-cypermethrin, deltamethrin, Alexandria, Egypt [15]. The Larvae were fed on biscuits lambda-cyhalothrin, and permethrin, were prepared by [containing wheat flour and yeast powder mixed with the optimum method. Briefly, 0.5% a.i (w/v) of each in- milk powder (10: 1: 1, w/w, respectively)] until pupation secticide were dissolved in DMSO to form the organic in shallow trays containing 2–3 L of de-chlorinated phase. Tween 80 was dissolved in distilled water to form water. Male adults were fed on 30% sucrose solution, the polar phase. The organic phase was dropped into a and females were fed on pigeon blood four times a week polar phase to form the coarse emulsion by stirring at Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 4 of 19 room temperature for 30 min at 4000 rpm. The coarse 2.6.2 Droplet size, polydispersity index, and zeta potential emulsion was later converted into a nanoemulsion The droplet size, PDI, and zeta potential of pyrethroids through ultra-sonication using a high-energy ultrasonic nanoemulsion formulations were investigated using process by the ultrasonic probe (Ultrasonic Homoge- Zetasizer Nano ZS (Malvern Instruments, UK) at room nizers HD 2070 with HF generator (GM 2070), ultra- temperature. The mean particle size and PDI of nanoe- sonic converter UW2070, booster horn (SH 213 G), and mulsions were measured by the dynamic light scattering probe microtip MS 73, Ø 3 mm) (Fig. 1). The tip of the (DLS) technique. Emulsion droplet size was estimated by horn was symmetrically placed in the coarse emulsion, the average of three measurements and presented as and the ultra-sonication process was carried out at mean diameter in nm, while zeta potential was deter- pulses 9 cycles/sec, power 75 % for 15 min [18]. mined by the light scattering method [20]. The formula- tions were diluted with distilled water by 200-fold and 2.6 Characterization of the nanoemulsions sonicated for 5 min at pulses 9 cycles/s and 75 % power 2.6.1 Stability studies before the measurement to avoid multiple scattering The prepared nanoemulsions were subjected to stability effects. screening tests to select the most stable formulation. These stability tests, including centrifugation assay sta- 2.6.3 Transmission electron microscopy bility at a temperature of 25 and 40 °C and heating- Surface morphology, topology, and droplet size of four cooling test. Centrifugation assay in which three samples pyrethroids nanoemulsions were characterized by TEM from each prepared formulation were centrifuged for 30 (JEOL JEM-1400 Plus TEM, USA, Inc.) equipped with a min at 5000 rpm and noticed phase separation, cream- 20-mm aperture at 20 kV. Bright-field imaging increas- ing, and cracking. The nanoemulsions should have ing the magnification and diffraction modes was selected enough stability without phase separation. Stable formu- to reveal the nanoemulsions’ form and size. The nanoe- lations were exposed to other thermodynamic stability mulsion of each pyrethroid formulation (10 mL) was di- tests [19]. About 25 mL of freshly prepared nanoemul- luted with distilled water (1/100) and added to 200- sions were transferred to a transparent tube. The trans- mesh form war-coated copper TEM sample holders (EM formation from a steady state to creaming and Sciences, Hatfield, PA, Japan). coalescence was examined during the storage period of 3 months at 25 °C. After that, the heating-cooling test was 2.6.4 Viscosity and pH measurements investigated to show the effect of heating and cooling on The dynamic (absolute) viscosity of the nanoemulsion the prepared nanoemulsions’ stability. The prepared was determined using a digital viscometer (a Rotary Myr nanoemulsions were maintained at a temperature of 4 VR 3000) with an L3 spindle at 200 rpm at 25 °C. The °C and 40 °C with storage for 48 h for each temperature viscosity of the formulations was measured without fur- test. The formulations that remained stable at this ther dilution. Each reading was recorded after the equi- temperature were subjected to further investigation. librium of the sample for 2 min. The viscosity recording Fig. 1 Schematic illustration of the preparation and characterization of pyrethroid nanoemulsions Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 5 of 19 of samples was repeated three times, and the data 500 μg/L were tested in three replicates. Twenty C. expressed in mPa.s. In the present study, the digital pH pipiens larvae were put into plastic cups containing 100 meter (Crison pH Meter Basic 20, EU) was used to mL of de-chlorinated water. The larvae were treated sep- determine the prepared nanoemulsions’ pH values. arately with Tween 80 or DMSO, and larvae without any treatment were maintained as control. The larvae’s mor- 2.7 In vitro release of pyrethroids from nanoemulsions bidity and mortality were verified and recorded based on In vitro release experiments were carried out using the the larvae’s uncoordinated movement after investigating dialysis technique [21]. Two milliliters from each the cervical region with a needle. Larval mortality per- formulation (0.5%, v/v) was placed inside a dialysis bag centages were recorded after 24 and 48 h, and the me- (cellulose membrane, molecular weight cut-off 14,000, dian lethal concentration (LC ) values were calculated Sigma-Aldrich Co., St. Louis, MO), sealed, and from probit analysis with 95% confidence limits and immersed in a vessel containing 50 mL of 10 mM phos- other statistical parameters [22]. phate buffer solution (pH 7.4). The releasing system was maintained at 37 ± 1 °C under magnetic stirring (100 2.9 Biochemical studies rpm). One milliliter from the solution was taken out of 2.9.1 Preparation of enzyme homogenates and total protein the dissolution medium at predetermined time intervals, assay replaced with fresh buffer solution. Pyrethroids released Surviving larvae were homogenized in 10 mM NaCl (1%, were determined by ultra-high-performance liquid chro- w/v) Triton X-100, and 40 mM sodium phosphate buffer matography (UHPLC, UltiMate 3000 system, Thermo (pH 7.4) at 4°C to determine Adenosine triphosphatase Scientific, USA). The system was equipped with a DIO- (ATPase), carboxylesterase (CaE), and glutathione-S- NEX UltiMate 3000 variable wavelength ultraviolet de- transferase (GST) activities after 24 h of exposing to tector (VWD). The separation was performed on LC values of the tested pyrethroids. The homogenate analytical column ODS Hypersil C18 (250 × 4.6 mm was centrifuged at 5000 rpm for 20 min at 4°C. The diameter, 5-micron particle size, Thermo scientific, supernatant was used immediately for enzymatic assay USA). Data were managed using a Chromeleon™ Chro- or stored at – 20 °C. Total protein was determined ac- matography Data System Software. The system consists cording to Lowry et al.’s[23] method, and the concen- of a binary gradient solvent pump to control the mobile trations were calculated by comparing with the standard phase’s flow rate and an autosampler for automatic in- curve of BSA. jection, a vacuum degasser, and a column oven (5–80 °C). The detection of tested pyrethroids was with a flow 2.9.2 ATPase assay rate of 1 mL/min, injection volume of 10 μL, and gradi- ATPase activity was performed according to Koch’s[24] ent solvent system, as shown in Table S2. The tested py- method. The reaction mixture, which contained 400 rethroids’ release profile was expressed as a cumulative + + +, mM Na ,20mMK , 5 mM Mg and 5 mM ATP, was concentration (mg/L ± SE) and plotted versus time. The prepared, and 200 μL of the crude enzyme was added to experiments were carried out in triplicate for each tested this mixture. Then, the volume was completed to 950 μL compound. The analytical grade of tested pyrethroids with Tris-HCl buffer (pH 7.4). After 10 min incubation was used for standard preparation. The calibration curve at 37 °C, the reaction was stopped with 200 μL of TCA. obtained from each insecticide’s analytical standard was A fresh color reagent (5 g ferrous sulfate in 10 mL am- used to determine the final concentrations released from monium molybdate solution prepared in 10 N sulfuric the nanoemulsions. acid) was added to the reaction mixture. The absorbance of the developed blue color was measured at 740 nm, 2.8 Toxicity assay against C. pipiens larvae −1 and the enzyme activity was calculated as OD min According to the World Health Organization, the larval −1 mg protein . bioassay was performed to compare the effect of nanoe- mulsions of selected pyrethroids with their active ingre- dient and commercial EC formulations on the C. pipiens 2.9.3 CaE assay larva recommendations [16]. Third instar larvae were CaE activity was determined according to Van Asperen’s used in the evaluation by a direct contact method. The [25] method, which used α-naphthyl acetate as a sub- three forms of tested insecticides (technical, commercial strate. The assay mixture contained 50 μL of homogen- EC formulation, and nanoemulsion) were tested to ob- ate enzyme, 2.1 mL of 50 mM sodium phosphate buffer tain the LC values. Technical pyrethroids were dis- (pH 7.4), and 25 μLof5mM α-naphthyl acetate solu- solved in DMSO and mixed with Tween-80 (0.05%), tion. The mixture incubation was done at 37 °C for 15 while the EC and nanoemulsions were dissolved in dis- min. Finally, 25 μL of 0.3% Fast blue B salt dissolved in tilled water. Different concentrations ranging from 0.5 to 3.5% SDS was added and incubated for 15 min at 37°C. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 6 of 19 The absorption was measured at 555 nm. The enzyme An acute algal growth inhibition test was conducted −1 −1 activity was expressed as OD min mg protein . using different concentrations of each insecticide in ster- ile AAM in a final volume of 50 mL. Tested concentra- tions of a pesticide were prepared from stock solutions 2.9.4 GST assay on an arithmetic progression covering an expected range GST assay was performed using reduced glutathione (2.5 of toxicity from 0 to 90%. Stock solutions of technical mM) by Saint-Deniset et al. [26]. The assay mixture con- insecticides were prepared in 1% DMSO and a corre- tained 100 μL of 1.5 mM CDNB, 200 μL of reduced sponding control was included. Stock solutions of the glutathione, and 1.5 ml of pH 7.4 phosphate buffer. A EC and NE were prepared. Algal suspensions were ex- total of 200 μL of the enzyme was added to the above posed to different concentrations (0.0005–500 mg/L) mixture, shaken gently, and incubated for 15 min at 37 prepared in the same medium of algae culture. All assays °C. The absorbance was recorded at 340 nm using a UV/ were conducted in triplicate. An inoculum of the Visible spectrophotometer (Alpha-1502. Laxco Inc, exponentially-growing culture of R. subcapitata (har- USA). One unit of enzyme activity attributed to the vested from 4–7 days stock culture) was prepared no quantity of conjugated enzyme with 1 mmol of GSH per −1 more than 2–3 h before the beginning of the test. Initial min. The enzyme activity was expressed as OD min −1 cell density for the growth inhibition test was 10,000 mg protein . cells/ml in both test and control flasks. Zero-time begins at inoculation of all flasks with the algal cells followed 2.10 Molecular docking by incubation for 96 h in a temperature-controlled (25 The modeled protein structure, ATPase (PDB ID: 4BYG) °C) orbital shaker set at 100 rpm under continuous illu- and detoxifying enzymes CaE (PDB ID: 5W1U) and GST mination via white fluorescent lamps. After 96 h, algal (PDB ID: 5FT3) in their PDB formats were downloaded growth in terms of viable cell concentration was exam- from the protein data bank (PDB) (http://www.rcsb.org) ined in a Neubauer hemocytometer using a phase- and imported on to the Molecular Operating Environ- contrast microscope. Growth rate inhibition of the alga ment (MOE) 2014.13 software (Chemical Computing was used as the endpoint in this assessment. The per- Group Inc, Montreal, Quebec, Canada). The structure of cent inhibition values were calculated after 96 h, and the each enzyme was visualized by the MOE [27]. The pro- median effective concentration (EC ) values were calcu- tein chemistry of the missing hydrogen was corrected, lated from the probit analysis with 95% confidence limits after which the heteroatoms and the crystallographic [22]. The no observed effect concentration (NOEC) after water molecules were removed from the protein. Chem- algal exposure to each tested insecticide was calculated ical structures of the tested pyrethroids were drawn by by the formula: NOEC = EC/10 [31]. Furthermore, the ChemDraw Professional Ultra Version 15 (PerkinElmer, hazard statement of each tested insecticide was esti- Informatics, Inc., USA). The structures were converted mated according to UNECE GHS (2019) [32]. to 3D, and the energy was minimized by the MMFF94 function [28]. The triangle-matching algorithm was se- 2.12 Statistical analysis lected from MOE for docking the compounds into the Statistical analysis was performed using the IBM SPSS selected enzymes’ active sites. Free energy of binding software version 25.0 (SPSS, Chicago, IL, USA) [33]. was calculated from the contributions of hydrophobic, Mortality percentages were calculated for each treatment ionic, hydrogenated, and van der Waals interactions. A and corrected using Abbott’s equation [34]. Means and ligand was considered adequate for a minimum docking standard error (SE) were obtained from three independ- score value (or interaction energy calculation) of an ent replications performed for each treatment. The log enzyme-ligand complex. dose-response (LdP) lines were used in the determin- ation of the LC values for the mosquito’s bioassay and 2.11 Bio-efficacy experiment on the freshwater green alga EC values for the algal bioassay according to the probit The freshwater green alga Raphidocelis subcapitata was analysis [22]. The least-square regression analysis was obtained from the Faculty of Science; Mansoura Univer- used to determine the 95% confidence limits. Analysis of sity, Egypt. The stock culture was maintained in 250-mL variance (ANOVA) of the biochemical data was con- borosilicate Erlenmeyer flasks containing culture ducted and means property values were separated (p ≤ medium at 24 ± 2 °C, under a continuous white fluores- 0.05) with Student-Newman-Keuls (SNK). cent light of 3000–4000 lux, and manually shaken twice a day [29]. The axenic culture was maintained for the 3 Results provision of a continuous supply of “healthy” cells for 3.1 Physiochemical properties of the tested pyrethroids the tests in a standard algal assay medium (AAM) as de- The chemical structure and physicochemical proper- scribed in Miller et al. [30]. ties of the tested pyrethroids are shown in Table S1. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 7 of 19 Permethrin from type I pyrethroids lacks a cyano formulations 4, 8, and 9 showed significant differences group and three insecticides from type II pyrethroids in their viscosity values (81.00, 160.23, and 70.67 (alpha-cypermethrin, deltamethrin, and lambda- mPa.s, respectively). The pH of the prepared formula- cyhalothrin) in which an alpha-cyano group is present tions was in the range of 7.78-8.18. at thephenylbenzylalcohol position.The tested Based on these quantitative data, the first-order poly- compounds’ molecular weight was 416.3, 505.21, nomial equations (1-4) and their corresponding coeffi- 449.85, and 505 g/mol for alpha-cypermethrin, delta- cients were generated for each of the response variables methrin, lambda-cyhalothrin, and permethrin, respect- in the factorial design test. The models indicated the in- ively. The polar surface area (PSA) of all tested dividual parameters’ behavior in others’ presence on the pyrethroids was 59.32, except permethrin was 35.53, viscosity, particle size, PDI, and pH for deltamethrin while the hydrophobicity factor (ALogP) of all tested nanoemulsions. compounds was around 6. There are no hydrogen bond donors (HBD) in the tested pyrethroids, while Viscosity = − 110 + 21.09 a.i − 0.67 solvent + 5.11 surfactant ……….(1) + 9.86 sonication pulses + 1.91 sonication time + 0.255 the number of hydrogen bond acceptors (HBA) sonication power ranged from 3 to 7. 2 s = 23.0714, r = 89.49% Droplet size = 20951 + 1524 a.i − 180 solvent − 514 ……….(2) 3.2 Optimization of the nanoemulsions preparation surfactant − 417 sonication pulses − 216 sonication time + 25.5 sonication power The different experimental setup using Minitab soft- s = 1959.47, r = 87.19% ware was used to determine the influence of six inde- PDI = − 0.76–0.0336 a.i + 0.0256 solvent − 0.0010 surfactant ……….(3) pendent variables on the pyrethroid nanoemulsions’ + 0.0541 sonication pulses + 0.0244 sonication time − characterization (dependent variable) (Table S3). Del- 0.00548 sonication power tamethrin was selected as a model of the tested pyre- s = 0.2112, r = 82.45% throids for the optimization experiments. During the pH = 6.52 − 0.0187 a.i + 0.0304 solvent + 0.0155 surfactant ……….(4) nanoforming process, emulsification was achieved in − 0.0006 sonication pulses − 0.0048 sonication time − 0.00235 sonication power the context of the droplet shearing phenomenon. The s = 0.1863, r = 62.06% sound waves (frequency 25–75 Hz) generated by the sonotrode (a tool that creates ultrasonic vibrations) Also, the influence of each factor on the response were applied to induce a mechanical vibration. variableswas shownasParetocharts inFig. 2.Itwas Followed by acoustic cavitation, which could lead to a noted that the active ingredient, sonication pulses, further collision and cause strong shock waves to and surfactant were more significant factors than the shear the largest droplets to a nanometer size. The others on the nanoemulsion viscosity (Fig. 2A). In visual appearance of the nine deltamethrin nanoemul- comparison, the active ingredient, surfactant, and sionsisshown in Figure S1. The quantitative results sonication time showed the highest effect on the including the droplet size (nm), PDI, and viscosity prepared nanoemulsions’ particle size (Fig. 2B). In the (mPa.s) are presented in Table S4.There are signifi- case of the PDI value, the sonication power, cant differences in the droplet size of the nine pre- sonication time, and sonication pulses, respectively, pared deltamethrin formulations. Formulations 1, 5, had a significant effect (Fig. 2C). On the contrary, the and 7 presented 172.46, 364, and 417 nm, respect- solvent was the most significant factor in the pH ively, while the other six formulations showed droplet value (Fig. 2D). sizes larger than 500 nm. In the PDI case, there are Among the 9 different experimental setup (Table S3), no significant differences between the formulations formulation 1 with 0.5 % a.i, 44% DMSO, 15% tween 80, (0.516–0.964) except formulation 2 (PDI = 0.158). 40.5% water, 9 cycle/s sonication pulses, and 75% Nanoemulsions were exposed to extreme storage con- sonication power for 15 min was the best. The resulted ditions to predict the samples’ ability to be physically nanoemulsion 1 was in clear visual appearance with a stable for up to three months. All prepared delta- smaller droplet size of 234 nm ± 4.13. Therefore, these methrin formulations did not pass the centrifugation parameters were selected to prepare the other test at 5000 rpm except formulations 1 and 5, while pyrethroids nanoemulsions (alpha-cypermethrin, all prepared formulations did not pass the heating- lambda-cyhalothrin, and permethrin). cooling test. The viscosity and pH measurements of prepared deltamethrin formulations. There is no sig- nificant difference between the viscosity values of for- 3.3 Characterizations of the pyrethroid nanoemulsions mulations 1, 2, 5, and 6 (74.67, 80.16, 90.23, and 3.3.1 Droplet size and polydispersity index 90.23 mPa.s, respectively). Formulations 3 and 7 have The droplet size of alpha-cypermethrin, deltamethrin, 40.32 and 37.67 mPa.s, respectively. However, lambda-cyhalothrin, and permethrin nanoemulsions Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 8 of 19 Fig. 2 Pareto charts representing the effect of factors and process variables on viscosity (A), droplet size (B), PDI (C), and pH (D) for deltamethrin nanoemulsions at α = 0.05 were 90.26, 172.00, 168, and 72, respectively (Table 3.3.3 Viscosity and pH 1). However, the PDI values were 0.337, 0.827, 0.448, The viscosity values of alpha-cypermethrin, deltameth- and 0.295 for alpha-cypermethrin, deltamethrin, rin, lambda-cyhalothrin, and permethrin nanoemulsions lambda-cyhalothrin, and permethrin, respectively. were 60.15, 74.67, 53.76, and 50.68 mPa.s, respectively (Table 1). The pH measurements were 8.51, 7.84, 8.20, 3.3.2 Zeta potential and 8.17 for alpha-cypermethrin, deltamethrin, lambda- The prepared nanoemulsions revealed negative values of cyhalothrin, and permethrin, respectively. zeta potential (− 0.603, − 0.669, − 0.539, and − 15.4 mV for alpha-cypermethrin, deltamethrin, lambda- 3.3.4 Thermodynamic stability studies cyhalothrin, and permethrin, respectively) (Table 1 and The stability results after the centrifugation and heating- Figure S2). cooling cycle are presented in Table 1. The results Table 1 The observed visual stability, droplet size, polydispersity index (PDI), zeta potential, dynamic (absolute) viscosity, and pH of prepared pyrethroid nanoemulsions Insecticide Visual Droplet size Polydispersity Zeta Viscosity pH Stability after appearance (nm) ± SE index (PDI) ± potential (mPa.s) ± Centrifugation at 5000 Heating-cooling SE (mV) SE rpm cycle b c b Alpha- Clear 90.26 ± 3.78 0.337 ± 0.01 − 0.603 60.15 ± 8.51 √ × cypermethrin 0.12 a a a Deltamethrin Clear 172.00 ± 0.827 ± 0.10 − 0.669 74.67 ± 7.84 √ × 34.07 7.86 a b c Lambda- Clear 168.00 ± 0.448 ± 0.05 − 0.539 53.76 ± 8.20 √ × cyhalothrin 4.08 0.20 c d c Permethrin Clear 72.00 ± 8.30 0.295 ± 0.02 − 15.40 50.68 ± 8.17 √ × 1.200 Different letters in the same column indicate significant differences according to the Student-Newman-Keuls (SNK) test (P ≤ 0.05). (√) refer to the stable state, (×) refer to the non-stable state. Preparation condition: 0.5 % a.i, 44% solvent (DMSO), 15% surfactant (tween 80) and 40.5% water with sonication pulses 9 cycle/s at 75% power for 15 min Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 9 of 19 showed that all nanoemulsions were transparent and and other statistical parameters in Table 2. The results stable at 5000 rpm of centrifugation and 25 °C for up to showed that alpha-cypermethrin nanoemulsion gave the 3 months (Figure S3), while these products were sepa- LC value of 20 μg/L that was more significant than the rated under a heating-cooling cycle test. EC formulation (40 μg/L) and the technical form (43 μg/ L) after 24 h of the experiment. The technical form of 3.3.5 Transmission electron microscopy deltamethrin proved an LC value of 28 μg/L, while the The morphological study of the structure of pyrethroid EC and nanoemulsion gave the LC of 26 and 28 μg/L, nanoemulsions was carried out by TEM. Figure 3 shows respectively, after 24 h. Lambda-cyhalothrin technical, the TEM micrograph of pyrethroid nanoemulsions, EC, and nanoemulsion gave LC values 27, 23, and 15 demonstrating the spherical shape. The droplets had a μg/L after 24 h and 18, 13, and 10 μg/L after 48 h, re- uniform shape and size. TEM analyses also confirmed spectively. However, according to the 95% confidence the nanometric droplet diameter of formulated limit based on the probit analysis, there is no significant pyrethroids at magnification 20,000×. difference between lambda-cyhalothrin and deltameth- rin. The LC values at 24 h of both insecticides interfere 3.4 Pyrethroids released from nanoemulsions with the lower and upper 95% confidence limits. How- The release profile assay was carried out using in vitro ever, permethrin nanoemulsion proved the lowest tox- dialysis experiment. Cumulative amounts (mg/L) of the icity with LC values 233 and 127 μg/L after 24 and 48 tested pyrethroids released from their nanoemulsions h, respectively. In permethrin, the LC values of EC and into phosphate buffer solution per time are shown in technical forms were 280 and 322 μg/L after 24 h, re- Fig. 4. Initial burst release was measured after 30 min, spectively, while the nanoemulsion was the most active and the concentrations 60.60, 25.29, 103.58, and 303.60 form (LC = 233 and 127 μg/L after 24 and 48, mg/L were quantified for alpha-cypermethrin, delta- respectively). methrin, lambda-cyhalothrin, and permethrin, respect- ively. It was noted that the rate of permethrin released 3.6 Enzymatic activity −1 from the nanoemulsion (60%) was greater than lambda- The data are shown in Table 3, as OD mg protein cyhalothrin (20%), alpha-cypermethrin (12%), and delta- min. The untreated larvae have 1.40, 3.31, and 2.70 for methrin (5%) after 30 min of the dialysis. After 180 min ATPase, CaE, and GST, respectively. By estimating the of the experiment, each compound’s release concentra- level of ATPase, CaE, and GST, it was found that the tion slightly increased to 82, 112, and 314 mg/L for insecticides in nanometric formulas had a significant alpha-cypermethrin, lambda-cyhalothrin, and permeth- effect as compared to control, technical form, and EC rin, respectively, whereas the concentration released treatment. The data proved that the activity of all tested from deltamethrin nanoemulsion reached only 76.58 enzymes was significantly increased except ATPase. The mg/L (15%) after 180 min of the experiment. insecticides caused a significant ATPase inhibition up to −1 0.50 OD mg protein min compared to 1.40 in control. 3.5 Larvicidal efficacy of pyrethroid nanoemulsions The most effective compound on the ATPase was The larvicidal activity of the technical, EC, and permethrin with specific activities of 0.50, 0.64, and 0.83 nanoemulsion of each insecticide was evaluated against for nanoemulsion, technical, and EC, respectively. C. pipiens larvae to record the mortality after 24 and 48 However, the lowest effective compound was alpha- h of the exposure. The data are presented as LC values cypermethrin, with activities of 0.86, 1.38, and 1.18 for Fig. 3 Transmission electron micrograph of prepared pyrethroid nanoemulsions alpha-cypermethrin (A), deltamethrin (B), lambda-cyhalothrin (C) permethrin (D): The TEM was performed on a JEOL JEM-1400 Plus, transmission electron microscope operating at an acceleration voltage of 80.0 kV with a 20-mm aperture. Print magnification 20,000× Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 10 of 19 Fig. 4 Release concentration of alpha-cypermethrin (A), deltamethrin (B), lambda-cyhalothrin (C), and permethrin (D) after time intervals for 3 h and standard error (n = 3) technical, commercial EC, and nanoemulsion, respect- with ATPase were ranged from − 4.33 to − 5.46 kcal/ ively. Deltamethrin inhibited the ATPase to 0.69, 0.77, mol (Table 4). The results revealed that all insecticides and 0.75 for the technical, EC, and nanoemulsion, re- exhibited H-bonding with amino acids in the active spectively. Lambda-cyhalothrin gave a specific activity of pockets of ATPase. α-Cypermethrin, deltamethrin, and 0.62 for the technical and 1.03 for the nanoemulsion. permethrin exhibited H-bonding with amino acid Asn All tested pyrethroids caused activation of the CaE, A112 by distances 3.54, 3.26, and 3.21 Å, respectively. which ranged from 3.32 to 7.02 compared to 3.31 in the Simultaneously, lambda-cyhalothrin exhibited H- untreated larvae. Alpha-cypermethrin nanoemulsion was bonding with Asn A112-N18 and Trp A116-N18 with the most active with a specific activity of 7.02. It was 3.38 and 3.56 Å, respectively. The binding confirmation followed by deltamethrin with specific activities of 4.53, of the tested pyrethroids with ATPase is shown in Figure 3.44, and 3.63 for the technical, EC nanoemulsion, S4. α-Cypermethrin (Figure S4A) and deltamethrin (Fig- respectively. ure S4C) interacted with ATPase by van der Waals (Glu For the GST activity, permethrin was the most 181, Gly 182, Leu 168, Pro 170, Trp 116, Val 167, and effective insecticide in activating this enzyme with the Val 183) and (Gly 113, Gly 171, Gly 182, Leu 168, Pro specific activities of 6.32, 7.85, and 7.86 for the technical, 170, Trp 116, Trp 169, and Val 167), respectively. Both commercial EC, and nanoemulsion, respectively, compounds interacted by H-arene bond with amino acid compared to 2.70 in control. It was followed by alpha- Asn A112 with 3.59 and 4.17 Å, respectively. In contrast, cypermethrin that caused the specific activity of 5.31, lambda-cyhalothrin and permethrin interacted with 7.54, and 11.94 for the technical, EC, and nanoemulsion, ATPase by van der Waals (Glu 181, Gly 182, Leu 168, respectively. The specific activity of GST treated with Pro 170, and Val 167) and (Gly 113, Phe 108, Leu 168, deltamethrin was higher than 5 for the three products. and Trp 116), respectively (Figure S4B and D). Lambda-cyhalothrin gave activity of 4.57 for the tech- Tested pyrethroids exhibited binding affinity ranged nical, 6.55 for the EC, and 6.15 for the nanoemulsion. from − 7.44 to − 10.01 kcal/mol on the active sites of CaE (Table 5). Lambda-cyhalothrin was the highest (ΔG 3.7 Molecular docking = − 10.01 kcal/mol) followed by α-cypermethrin, The docking scores and binding mechanism include H- deltamethrin, and then permethrin ΔG values of − 9.35, bonds, Van der Waals, and hydrophobic interactions of − 8.72, and − 7.44 kcal/mol, respectively. Alpha- the tested pyrethroids with ATPase (4BYG), CaE cypermethrin interacted with the CaE enzyme through (5W1U), and GST (5FT3) are shown in Tables 4, 5, and two hydrogen bonds (Asp 279-CL11 and Leu 328-N15) 6, respectively. Analysis of the docking results showed with distances of 3.32 and 0.6 Å, respectively. Besides, that the pyrethroids showed a higher binding affinity some van der Waals bonds ( Arg 73, Arg 392, Asp 279, with CaE and GST than ATPase. The docking scores Glu 118, Gly 109, Gly 110, Gln 330 His 442, Leu 327, Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 11 of 19 Table 2 Larvicidal activity of the technical, EC, and nanoemulsion of alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin against C. pipiens larvae a b c 2 d Insecticide Time of Type of LC 95% confidence limits (μg/L) Slope ± Intercept (χ ) exposure formulation (μg/ SE ±SE Lower Upper (h) L) Alpha-cypermethrin 24 T 43 26 46 2.47 ± 0.15 3.66 ± 0.26 20.69 EC 40 35 64 3.22 ± 0.18 4.31 ± 0.27 35.35 NE 20 15 26 2.30 ± 0.14 3.94 ± 0.26 22.45 48 T 40 26 46 2.47 ± 0.15 3.66 ± 0.26 20.69 EC 38 32 63 3.20 ± 0.18 4.21 ± 0.27 35.32 NE 18 15 24 2.51 ± 0.15 4.37 ± 0.28 20.59 Deltamethrin 24 T 28 23 36 2.23 ± 0.13 3.45 ± 0.22 21.02 EC 26 20 31 1.31 ± 0.08 1.52 ± 0.08 73.20 NE 16 12 21 2.52 ± 0.16 4.50 ± 0.27 43.97 48 T 25 19 34 2.12 ± 0.13 3.39 ± 0.21 29.62 EC 24 20 28 1.30 ± 0.08 1.57 ± 0.09 57.09 NE 13 9 16 2.51 ± 0.17 4.76 ± 0.30 35.17 Lambda-cyhalothrin 24 T 27 17 45 1.47 ± 0.09 2.31 ± 0.17 30.16 EC 23 12 41 1.25 ± 0.08 2.06 ± 0.14 37.22 NE 15 11 20 2.05 ± 0.12 3.75 ± 0.23 18.42 48 T 18 12 27 1.30 ± 0.09 2.27 ± 0.16 18.84 EC 13 8 18 1.43 ± 0.07 2.72 ± 0.17 17.77 NE 10 7 14 1.42 ± 0.09 2.85 ± 0.18 19.7 Permethrin 24 T 322 221 506 1.36 ± 0.089 0.67 ± 0.081 22.655 EC 280 206 392 1.64 ± 0.103 0.91 ± 0.09 20.66 NE 233 201 270 1.68 ± 0.10 1.06 ± 0.09 07.20 48 T 225 134 423 1.26 ± 0.08 0.81 ± 0.08 40.21 EC 196 134 288 1.97 ± 0.12 1.39 ± 0.10 37.04 NE 127 76 215 1.43 ± 0.08 1.29 ± 0.09 44.54 T technical, EC emulsifiable concentrate, NE nanoemulsion. The Median lethal concentration. The LC value of each compound between the other compound’s confidence limits is not significantly different. However, if the fit confidence intervals (95%) are non-overlapping, there is a significant difference between the b c d compounds. Slope of the concentration mortality regression line ± error (SE). Intercept of the regression line ± SE. Chi-squared value Leu 328, Lys 331, Phe 281 Ser 191, Trp 224, Tyr 428, Glu 113, Glu 268 Gly 109, Leu 328, Leu 328, Lys 331, and Val 393) are included (Figure S5A). Lambda- Phe 281, and Thr 112) and Pi-cation interaction (Arg cyhalothrin bonded through HBD, HBA (Arg 73-F11 73-6-ring, 3.90 Å). and Glu 113-C16, respectively) with 2.9 Å for both and The docking results with GST (Table 6) indicated that van der Waals interactions (Arg 73, Arg 74, Asn 452, lambda-cyhalothrin was the highest affinity binding with Ala 443, Gln 330, Glu 113, Gly 109, Gly 110, His 427, the lowest energy value − 9.95 kcal/mol. It was followed His 442, Leu 120, Leu 327, Leu 446, Met 432, Phe 281, by alpha-cypermethrin, permethrin, deltamethrin with Ser 191, Ser 447, Thr 112, Tyr 121, Tyr 428, and Phe energy values − 8.55, − 8.53, and − 8.24 kcal/mol. alpha- 281) (Figure S5B). Three hydrogen bonds (Ser 191-Br8, cypermethrin interacted with GST through van der His 442-Br8, and Arg 73-O1) with distances of 3.46, Waals with 14 amino acids (Arg 112, Glu 116, His 41, 3.62, and 2.95 Å, respectively, and thirteen van der Leu 42, Leu 111, Leu 119, Lys 39, Phe 108, Phe 120, Pro Waals (Arg 73, Glu 113, Glu 268, Gly 109, Gly 110, Gln 13, Thr 54, Val 11, Val 55, and Val 207) with docking 330, His 442, Leu 327, Leu 334, Lys 331, Phe 281, Ser score of − 8.55 kcal/mol (Figure S6A). Figure S6B shows 191, and Tyr 428) were formed between deltamethrin the interactions between lambda-cyhalothrin and GST and CaE (Figure S5C). Figure S5D shows the interactions through van der Waals with 15 amino acids (Arg A112, between permethrin and CaE, which was through one Cys A115, Glu A116, His A41, His A53, Leu A36, Leu HBA (Lys 331–O1), ten van der Waals (Arg 73, Gln 330, A111, Leu A119, Lys A39, Lys B136, Phe A108, Pro A13, Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 12 of 19 Table 3 Biochemical effects of tested pyrethroids on some enzymes activity in C. pipiens larvae after 24 h of the treatment with LC of each compound −1 Treatment Type of Specific activity (OD mg protein min) ± SE formulation ATPase CaE GST Untreated sample – 1.40 ± 0.04 3.31 ± 0.01 2.70 ± 0.27 Alpha-cypermethrin T 0.86 ± 0.02 5.06 ± 0.15 5.31 ± 0.70 EC 1.38 ± 0.01 7.02 ± 0.50 7.54 ± 0.88 NE 1.18 ± 0.00 4.53 ± 0.25 11.94 ± 0.84 Deltamethrin T 0.69 ± 0.02 3.44 ± 0.19 5.49 ± 0.60 EC 0.77 ± 0.04 3.63 ± 0.71 5.63 ± 0.80 NE 0.75 ± 0.03 4.12 ± 0.09 5.90 ± 0.30 Lambda-cyhalothrin T 0.62 ± 0.05 1.51 ± 0.09 4.57 ± 0.48 EC 1.19 ± 0.02 3.32 ± 0.10 6.55 ± 0.85 NE 1.03 ± 0.01 3.35 ± 0.01 6.15 ± 0.45 Permethrin T 0.64 ± 0.00 2.31 ± 0.09 6.32 ± 0.59 EC 0.83 ± 0.13 2.66 ± 0.08 7.85 ± 0.44 NE 0.50 ± 0.01 3.37 ± 0.12 7.86 ± 0.37 T technical, EC emulsifiable concentrate, NE nanoemulsion, OD optical density, SE standard error, ATPase adenosine triphosphatase, CaE carboxylesterase, GST glutathione-S-transferase Ser A12, Val A11, and Val A207) and arene H-bond with 3.8 Ecotoxicity study against the freshwater green alga Phe A120. Deltamethrin reacted with GST through 10 The toxicity endpoint values after acute exposure of R. van der Waals (Glu A116, Leu A36, Leu A42, Leu A111, subcapitata to different forms of pyrethroids used as Lys A39, Phe A120, Phe A108, Pro A13, Thr A54, and mosquito larvicides are illustrated in Table 7. The Val A55), 3 H-bonds with amino acids ( His A41, His sensitivity of R. subcapitata to insecticides; expressed as A53, and Arg A112 ) and H-arene bond with amino acid EC , ranged from 0.76 to > 100 mg/L. Based on these ASN A112 (Figure S6C). Permethrin interacted with the values, the decreasing order of the sensitivity was pocket of GST through H-bond with amino acid Ser commercial EC > NE > technical form. The current data A12 and 14 amino acids through van der Waals bonds disclosed that the commercial EC of the tested (Arg 112, Cys 115, Glu 116, His 53, Leu 36, Leu 111, insecticides were more toxic to R. subcapitata and Leu 119, Phe 108, Phe 120, Pro 13, Thr 54, Val 11, Val recorded 0.76, 4.92, 5.03, and 16.98 mg/L for 55, and Val 207) (Figure S6D). deltamethrin, permethrin, lambda-cyhalothrin, and Table 4 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of ATPase (PDB ID: 4BYG) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid-ligand (Å) acid-ligand (Å) mol) atom atom Alpha- − 4.85 Glu 181, Gly 182, Leu 168, Pro Asn A112- HBA 3.54 Asn A112-6- Arene-H 3.59 1.66 cypermethrin 170, Trp 116, Val 167, Val 183 N15 ring Lambda- − 5.46 Glu 181, Gly 182, Leu 168, Pro Asn A112- HBA 3.38 –– – 1.88 cyhalothrin 170, Val 167 N18 HBA 3.56 TRP A116- N18 Deltamethrin − 4.33 Gly 113, Gly 171, Gly 182, Leu Asn A112- HBA 3.26 Asn A112-6- Arene-H 4.17 1.96 168, Pro 170, Trp 116, Trp 169, Val N15 ring Permethrin − 4.61 Gly 113, Phe 108, Leu 168, Trp Asn A112- HBA 3.21 - - - 3.36 116 O1 RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 13 of 19 Table 5 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of CaE (PDB ID: 5W1U) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid- (Å) acid- (Å) mol) ligand ligand atom atom Alpha- − 9.35 Arg 73, Arg 392, Asp 279, Glu 118, Gly 109, Asp HBD 3.32 –– – 1.14 cypermethrin Gly 110, Gln 330 His 442, Leu 327, Leu 328, 279- HBA 0.6 Lys 331, Phe 281 Ser 191, Trp 224, Tyr 428, Cl11 Val 393 Leu 328- N15 Lambda- − 10.01 Arg 73, Arg 74, Asn 452, Ala 443, Gln 330, Glu HBD 2.90 –– – 2.03 cyhalothrin Glu 113, Gly 109, Gly 110, His 427, His 442, 113- HBA 2.90 Leu 120, Leu 327, Leu 446, Met 432, Phe C16 281, Ser 191, Ser 447, Thr 112, Tyr 121, Tyr Arg 73- 428, Phe 281 F11 Deltamethrin − 8.72 Arg 73, Glu 113, Glu 268, Gly 109, Gly 110, Ser HBD 3.46 –– – 2.21 Gln 330, His 442, Leu 327, Leu 334, Lys 331, 191-Br8 HBD 3.62 Phe 281, Ser 191, Tyr 428 His 442- HBA 2.95 Br8 Arg 73- O1 Permethrin − 7.44 Arg 73, Gln 330, Glu 113, Glu 268 Gly 109, Lys HBA 3.04 Arg 73- Pi-cation 3.90 1.93 Leu 328, Leu 328, Lys 331, Phe 281, Thr 112 331–O1 6-ring RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules are participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Table 6 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of GST (PDB ID: 5FT3) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid- (Å) acid- (Å) mol) ligand ligand atom atom Alpha- − 8.55 Arg 112, Glu 116, His 41, Leu 42, Leu 111, Leu HBD 3.56 –– – 1.66 cypermethrin Leu 119, Lys 39, Phe 108, Phe 120,Pro 13,Thr A36- HBA 2.58 54, Val 11, Val 55, Val 207 Cl11 His 53- N15 Lambda- − 9.95 Arg A112, Cys A115, Glu A116, His A41, His Thr A54- HBA 3.19 Phe Arene-H 4.06 1.50 cyhalothrin A53, Leu A36, Leu A111, Leu A119, Lys A39, N18 HBA 3.53 A120-6- Lys B136, Phe A108, Pro A13, Ser A12, Val Val A55- ring A11, Val A207 N18 Deltamethrin − 8.24 Glu A116, Leu A36, Leu A42, Leu A111, Lys His A41- HBA 3.09 ASN Arene-H 4.17 0.58 A39, Phe A120, Phe A108, Pro A13, Thr A54, O1 HBA 3.68 A112-6- Val A55 His A53- HBA 2.76 ring N15 Arg A112- N15 Permethrin − 8.53 Arg 112, Cys 115, Glu 116, His 53, Leu 36, Ser A12- HBA 3.24 –– – 1.76 Leu 111, Leu 119, Phe 108, Phe 120, Pro 13, O1 Thr 54, Val 11, Val 55, Val 207 RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules are participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 14 of 19 Table 7 EC and NOEC (mg/L) of nanoformulations compared with technical and commercial formulated pyrethroids to freshwater microalga R. subcapitata. The 95% confidence limits of the EC values are indicated in parentheses Insecticide Formulation Toxicity endpoint 96 h EC (mg/L) NOEC (mg/L) GHS hazard statement Alpha-cypermethrin T 69.33 (38.15–145.54) 6.932 H402 EC 16.98 (10.99–29.01) 1.698 H402 NE 101.11 (38.08–409.14 1.011 S Lambda-cyhalothrin T 33.89 (15.93–94.27) 3.389 H402 EC 5.03 (2.96–9.62) 0.503 H401 NE 11.29 (1.83–121.71) 1.129 H402 Deltamethrin T > 100 > 10 S EC 0.76 (0.56–1.05) 0.076 H400 NE 13.14 (6.49–30.74) 1.314 H402 Permethrin T 14.94 (9.72–23.72) 1.494 H402 EC 4.92 (3.31–7.75) 0.492 H401 NE 20.55 (12.66–36.25) 2.055 H402 T technical, EC emulsifiable concentrate, NE nanoemulsion, NOEC no observed effect concentration on algal growth rate, H hazard statement. H400 very toxic to aquatic life (hazardous to the aquatic environment, acute hazard, category 1; ≤ 1mg/L); H401 toxic to aquatic life (hazardous to the aquatic environment, acute hazard, category 2; > 1–≤ 10 mg/L); H402 harmful to aquatic life (hazardous to the aquatic environment, acute hazard, category 2; > 10–≤ 100 mg/L). S: Safe use (no hazard statement is suggested) since acute toxicity > 100 mg/L alpha-cypermethrin, respectively. The potency of com- 4 Discussion mercial EC may be attributed to the additives in the for- 4.1 Physicochemical properties of the tested pyrethroids mulation rather than the active ingredient. According to Lipinski’s “rule of five” [35], good intestinal Toxicity of nanoformulations showed a different absorption and oral bioavailability of compounds reflect pattern where alpha-cypermethrin exhibited a safe effect RB and MR’s acceptable values. The stereo-specificity of on R. subcapitata (EC > 100 mg/L) while the EC for the drug molecule is a property of nRB, which was found 50 50 the other insecticides recorded 11.29, 13.14, and 20.55 to be < 10. There are no hydrogen bond donors (HBD) mg/L for lambda-cyhalothrin, deltamethrin, and per- in the tested pyrethroids, while the number of hydrogen methrin, respectively. The sensitivity of R. subcapitatata bond acceptors (HBA) ranged from 3 to 7. The literature towards nanoformulations was lambda-cyhalothrin > has also documented that excellent absorption in the in- deltamethrin > permethrin. The differential toxicity of testine is induced by PSA < 140 [36]. The Log S value nanoformulations depends on their nanostructure and for all insecticides is between − 6.84 and − 7.22, indicat- high surface to mass ratio as well as the nature of their ing low water solubility. constitutive element. On the other hand, the EC values for the technical form of the tested insecticides were 4.2 Characterizations of the pyrethroid nanoemulsions 33.89, 69.33, >100, and 14.94 mg/L for lambda- Several studies prepared and characterized pyrethroid cyhalothrin, cypermethrin, deltamethrin, and permeth- nanoemulsions, such as alpha-cypermethrin, deltameth- rin, respectively. The sensitivity of R. subcapitata was rin, lambda-cyhalothrin, and permethrin [13, 14]. The permethrin > lambda-cyhalothrin > cypermethrin > droplet size of the prepared nanoemulsions is in agree- deltamethrin. ment with other studies. Mishra and others reported For subsequent characterization of the potentially that nano-sized permethrin’s mean particle size was hazardous effects of the tested insecticides and 175.3 nm [13], whereas the TEM analysis investigated by addressing safety issues of the developed nanopesticides, Patel et al. [37] revealed that cypermethrin particle size’s both NOEC and hazard statements were evaluated encapsulation was ranged between 115 and 119 nm. (Table 7). The data showed that all nanoformulations However, the droplet size of beta cypermethrin nanosus- represent category acute III with harmful effects to pension prepared by Zeng et al. [38] was 168 nm. It was aquatic life (H402) compared with the commercial EC observed no phase separation, creaming, and sedimenta- forms which represent category acute I and II with very tion under room temperature (25 °C) and accelerated toxic and/or toxic hazardous effects to the aquatic life stability evaluation [8]. The long-term physical stability (H400 and H410). of a nanoemulsion related to its small droplets makes Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 15 of 19 this type of formulation being referred to as “ap- h, and the accumulated releases were over 90%. How- proaching thermodynamic stability.” The average droplet ever, the release rate of lambda-cyhalothrin nanoemul- size of the nanoemulsions typically falls within the range sion was rapid within the first 30 h, and then, it slowed of 20–500 nm [19]. The small size of the droplets in down and maintained a stable release until equilibrium nanoemulsions gives them some advantages over con- after 80 h. ventional emulsions. These advantages include higher optical clarity, higher stability to droplet aggregation and 4.4 Toxicity against C. pipiens larvae gravitational separation, and higher bioactivity of encap- The effect of the three forms of the tested insecticides sulated components. The nanoemulsions have emerged was significantly different against C. pipiens larvae. It as alternative drug carriers because they increase the dis- can be noted that the technical form exhibited the solution rates and bioavailability of many poorly soluble lowest larvicidal activity. However, the EC of all tested drugs in water [9]. insecticides slightly improved the toxic action against PDI reflects the distribution of the particle size in a the larvae. However, all insecticides’ nanoemulsions formulation. The PDI is a dimensionless measure of the showed significantly high toxicity (1.5–2-fold) compared width of size distribution calculated from the cumulated to the technical and EC. This finding led to a significant analysis and ranges from zero to one [39]. A lower PDI decrease in the field application rate by half-value, value (near zero) indicates the existence of a uniform resulting in low environmental pollution and hazards. distribution of droplet size and homogenous The nanoscale form of pesticides has been applied to populations, whereas a PDI value closer to 1 (one) control the developed resistance in insect species, displays a wide range of droplet sizes (heterogeneity of attributed to conventional pesticides’ excessive use. the system). The PDI value around 0.2 indicates the Compared to the traditional pesticides, the higher droplet population’s homogeneity in prepared efficacy of nano pesticides was observed. In agreement formulations. Besides other important criteria, zeta with our results, other studies proved that pyrethroids’ potential is another essential characteristic of the preparation in nanoemulsion form made them more nanoemulsions and an indicator of the nanoemulsion active than the conventional forms [9, 14]. Mishra et al. stability associated with the droplets’ surface potential. [13] prepared nano-sized permethrin in its colloidal state The negative values are necessary for droplet-droplet re- and studied its effect on C. tritaeniorhynchus larvae. pulsion and thus enhanced nanoemulsion stability [40]. They found that the LC of the bulk permethrin was The high stability of formulations with zeta potential 442 μg/L. In contrast, the LC of the nano-permethrin values is associated with repulsive forces that exceed was 57 μg/L. The present study also supports nano pes- attracting van der Waals forces, resulting in dispersed ticides’ ability to control mosquito vectors. Reducing particles and a deflocculated system. The range of pH nanoemulsions and elevating their surface area could fa- value of nanoemulsion has a strong effect on its stability. cilitate their passive penetration into the target pest, thus The different pH value levels lead to a change in the enhancing their toxicity [13]. As the results presented, globules’ surface charge and their stability during stor- alpha-cypermethrin, deltamethrin, and lambda- age. Keeping different nanoemulsions under environ- cyhalothrin were the most toxic insecticides (LC mental storage conditions may be an essential criterion ranged from 10 to 43 μg/L) compared to the permethrin for judging effectiveness, potency, and stability [19]. (LC ranged from 127 to 322 μg/L) against C. pipiens larvae. This finding refers to the pyrethroid type’s chem- 4.3 Release studies of pyrethroid nanoemulsions ical structure that the alpha-cypermethrin, deltamethrin, The efficiency of nanoformulation to extend residence and lambda-cyhalothrin are cyano-derivatives. However, time, reduce insecticide losses, and reduce overuse. It permethrin is a non-cyano-derivative. As well-known also makes the pesticide’s continuous and stable release from the literature, cyano-derivatives of the pyrethroids possible [8]. Our results agree with the results obtained were more active against different pests than the non- by Nguyen et al. [41], who proved that the release rate cyano derivatives [42]. of deltamethrin nanoemulsion was lower than 20% in the first 3 h of the experiment. In addition, it confirmed 4.5 Biochemical studies that lambda-cyhalothrin /polyurethane nanoemulsion To elucidate some biochemical actions of the tested had a slower release rate than the traditional formula- pyrethroids on C. pipiens larvae, the effect of the LC tions. In addition, the release profile of the lambda- values on the ATPase, CaE, and GST isolated from the cyhalothrin-loaded nanoemulsion was compared to its survived treated larvae after 24 h was examined. In EC and WP formulations at 25 °C [8]. The results re- agreement with our findings, Kakko et al. [43] proved ported that the lambda-cyhalothrin released from EC that cypermethrin was the most toxic against ATPase, and WP was very fast and reached equilibrium after 48 followed by permethrin and natural pyrethrin. The cell Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 16 of 19 toxicity was dependent on the chemical structure of structures. Kumar et al. illustrated the molecular interac- pyrethroids. The pyrethroids without the α-cyano group tions of some pyrethroids including cypermethrin to- show the weakest physiological effect. Clark and Matsu- wards adaptive immune cell receptors of T (CD4 and + ++ mura [44] measured Na -Ca ATP hydrolysis and Ca- CD8) and B (CD28 and CD45) [51]. They found that Mg ATP hydrolysis in cockroach brain tissue under fenvalerate (− 5.534 kcal/mol: CD8), fluvalinate (− 4.644 in vitro conditions. They found that the non-cyano- and − 4.431 kcal/mol: CD4 and CD45), and cyperme- + ++ containing pyrethroids inhibited Na -Ca -ATP hydroly- thrin (− 3.535 kcal/mol: CD28). Data exhibited less sis mostly than their cyano-containing counterparts. The docking energy or more affinity for B cell and T cell im- ++ ++ reverse is true for pyrethroid action on Ca -Mg -ATP mune receptors, which may later result in immunosup- hydrolysis. pressive and hypersensitivity reactions. Markus et al also As well known, CaEs hydrolyze numerous endogenous elucidated the inhibitory activity of deltamethrin against and exogenous ester-containing compounds. Therefore, human GST [49]. They found that deltamethrin appears they play a vital role in the detoxification of pyrethroids, to fit well in an eccentric cavity located at the GST ho- strongly related to the resistance phenomenon. Identifi- modimer, likely causing conformational changes at the cation of CaE genes associated with pyrethroid resist- enzyme’s substrate binding sites such that the enzyme is ance was investigated in the malaria vector Anopheles no longer able to effectively convert GSH and CDNB. sinensis [45] and the mosquito Aedes aegypti [46]. In disagreement with our results, Kostaropoulos et al. 4.7 Biosafety evaluation against the freshwater green [47] proved that the pyrethroids bind with the active site alga of GST, resulting in a significant decrease of its activity Treating the aquatic environment with nanomaterials to towards CDNB in a competitive manner, but was not control mosquito larvae or other pests may lead to conjugated with GSH. Grant and Matsumura [48] found important risks for non-target aquatic organisms [52]. a variation in the action and level of GST due to its Both physicochemical and toxicological properties of interaction with pyrethroids, studied GST as an nanomaterials would permit and control environmental antioxidant defense agent confer pyrethroid resistance in risk assessment and safety of these materials [53]. Micro- Nilaparvata lugens and demonstrated that lambda- algae are widely used in bioassay toxicity testing of cyhalothrin and permethrin induced oxidative stress and aquatic pollutants since they are sensitive organisms lipid peroxidation in insects. For these reasons, they hy- with a high capacity of bioaccumulation due to their pothesized that the prominent role of elevated GSTs in high surface of contact [54]. conferring resistance in N. lugens is through protecting A concentration-response ratio established for R. sub- tissues from oxidative damage. Markus et al. also eluci- capitata and 96 h EC values are shown in Table 7. dated the inhibitory activity of deltamethrin against hu- Considering the values obtained for EC , it was ob- man GST [49]. They proved that deltamethrin was a served that this organism was more sensitive and highly potent inhibitor of GST-P1-1, and it inhibited the homo- affected by the commercial form (EC) of all tested insec- dimeric enzyme in a non-competitive manner. Thus, the ticides after acute exposure, followed by technical form purpose of determining ATPase, CaE, and GST levels as and/or nanoformulations. It is worthy to mention that essential parameters to study the toxic effect of nano- the synthesized nanoformulation are readily soluble in pesticides on insect vector species. water with no agglomeration and proved to be safe to algae and aquatic organisms when tested as alpha- 4.6 Docking studies cypermethrin nanoemulsion and less toxic (2–17-fold) It is well known that molecular docking is a method to than the commercial EC in case of lambda-cyhalothrin, predict and understand molecular recognition, find the deltamethrin and permethrin nanoemulsions. predominant binding mode and binding affinity between Similar results were obtained by Grillo et al. [55] who the protein and ligand, and give a three-dimensional stated that paraquat-loaded chitosan nanoparticles structural explanation of the protein-ligand interaction. showed less toxicity than paraquat (96 h EC s were 1.15 The bond interactions were useful for elucidation of sev- and 0.48 mg/L; respectively). Also, other ecotoxicity eral biological activities of tested compounds as larvi- studies demonstrated that thiamethoxam nanoparticles cides [50]. Zeng et al. studied the interactions of pepsin were less toxic than commercial formulations for R. sub- with deltamethrin and cyhalothrin by multi- capitata and non-toxic for A. salina under the condi- spectroscopic approaches and molecular docking [50]. tions of the study. Based on the existing knowledge, the They approved that the tested pyrethroids bounded dir- method of green synthesis of nanoparticles and several ectly into the enzyme cavity site. The binding was influ- green-fabricated metal nanoparticles failed to show tox- enced by the active site’s microenvironment resulting in icity against different aquatic organisms. Plumeria the extension of peptide strands with loss of α-helix rubra-and Pergularia daemia-synthesized Ag Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 17 of 19 nanoparticles did not exhibit any evident toxicity against different toxicity parameters establishes the non-toxic fishes after 48 h of exposure to concentrations corre- behavior of insecticide concentrations applied against sponding to the LC and LC values on IV instar larvae non-target species. This confirms environmental safety 50 90 of Ae. aegypti and An. stephensi [56]. Subarani et al. [57] with strong efficacy as a mosquitocidal agent against lar- also did not report the toxicity effects of Catharanthus vae. Also, the data proved the greatest effect of the roseus-synthesized Ag nanoparticles against fish and nanoemulsions as alternatives to the conventional mosquito predators; G. affinis after 72 h of exposure. pesticide formulations. As related to NOEC values among tested insecticides Abbreviations (Table 7), it represents the highest test concentration at ANOVA: Analysis of variance; a.i: Active ingredient; ATP: Adenosine which no toxic effects are observed, and went parallel to triphosphate; ATPase: Adenosine triphosphatase; BSA: Bovine serum albumin; C. pipiens: Culex pipiens; CaE: Carboxylesterase; CDNB: 1-Chloro-2,4- the EC pattern recorded in the current study. dinitrobenzene; DLS: Dynamic light scattering; DMSO: Dimethylsulfoxide; However, NOEC can be regarded as a chronic endpoint, EC: Emulsifiable concentrate; EDTA: Ethylenediaminetetraacetic acid; GSH: L- and values indicated in this study reflect the glutathione; GST: Glutathione-S-transferase; LC : Median lethal concentration; MOE: Molecular Operating Environment; O/W: Oil/water; concentrations that can offer minimum protection to PDB: Protein data bank; PDI: Polydispersity index; SE: Standard error; the test organism; R. subcapitata against tested SPSS: Statistical Package for the Social Sciences; TCA: Trichloroacetic acid; insecticides particularly on a long-term basis. Further- TEM: Transmission electron microscopy; WHO: World Health Organization; β- NAD: β-Nicotinamide adenine dinucleotide more, classification of tested insecticides according to their potential hazard statements to the aquatic ecosys- 6 Supplementary Information tem, an only commercial form of deltamethrin can be The online version contains supplementary material available at https://doi. considered highly hazardous to R. subcapitata (category org/10.1186/s42506-021-00082-1. acute I; H400) and is not recommended for application in waterways, whereas its nanoformulation exhibited a Additional file 1: SUPPLEMENTARY MATERIALS (DATA IN BRIEF). Table S1. Chemical structure and physicochemical properties of the tested less hazardous effect on the test alga. Additionally, all pyrethroids. Description of data: This table shows the chemical structure the tested nanoformulations showed only harmful effects and physicochemical properties of the tested compounds. The tested to aquatic life (category acute III; H402) compared with compounds' molecular weight was 416.3, 505.21, 449.85, and 505 g/mol for alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, and permeth- very toxic or toxic hazardous effects (category I or II; H rin, respectively. The polar surface area (PSA) of all tested pyrethroids was 400 or 401) of commercial forms of the same insecti- 59.32, except permethrin was 35.53,. wWhile the hydrophobicity factor cides to aquatic life. (ALogP) of all tested compounds was around 6. There are no hydrogen bond donors (HBD) in the tested pyrethroids, while the number of hydro- It can be concluded that, for safety purposes, gen bond acceptors (HBA) ranged from 3 to 7. Table S2. HPLC gradient nanopesticides can be recommended for use in vector solvent system for separation of alpha-cypermethrin, deltamethrin, control programs in waterways and can be considered lambda-cyhalothrin and permethrin. Description of data: This table shows the HPLC conditions used for the separation of pyrethroids understudy. highly promising for the development of safe insecticides These conditions include the gradient solvent system and the optimum against mosquitoes. The nanopesticides are less harmful wavelength used in the separation process. Table S3. Experimental fac- to the environment and more efficient (in terms of cost torial design for preparation and optimization of deltamethrin nanoemul- sions. Description of data: This table shows the different experimental and performance) than the existing formulations. setup using Minitab software was used to determine the influence of six Nevertheless, only further research will show whether independent variables on the pyrethroid nanoemulsions' characterization the research results can find their way to application in (dependent variable). In these optimization experiments, deltamethrin was selected as a model of the tested pyrethroids. Table S4. The ob- practice. served visual stability, droplet size, polydispersity index (PDI), zeta poten- tial, dynamic (absolute) viscosity, and pH of prepared deltamethrin 5 Conclusion nanoemulsions. Description of data: This table presents the quantitative results of nanoemulsion pyrethroids include the droplet size (nm), PDI, Permethrin from type I (non-cyano) and three pH, and viscosity (mPa.s). The data proved that there are significant differ- pyrethroids from type II (alpha-cypermethrin, ences in the droplet size of the nine prepared deltamethrin formulations. deltamethrin, and lambda-cyhalothrin) were prepared in In the PDI case, there are no significant differences between the formula- tions (0.516-0.964) except formulation 2 (PDI = 0.158). There is no signifi- nanoemulsions. The modification of these compounds cant difference between the viscosity values of formulations 1, 2, 5, and to nanoform increased the insecticidal properties. 0.5% 6. However, formulations 4, 8, and 9 showed significant differences in a.i, 44% DMSO, 15% tween 80, 40.5% water, 9 cycle/s of their viscosity values. The pH of the prepared formulations was in the range of 7.78-8.18. Figure S1. The visual appearance of prepared delta- sonication pulses, 75% power for 15 min were selected methrin nanoemulsions. The code number represents the experimental as the optimal conditions for preparation of the insecti- factorial design shown in Table 2. Description of data: This figure presents cide nanoemulsions. The remarkable stable behavior of the visual appearance of the nine produced deltamethrin nanoemulsions formulations. Figure S2. Zeta potential distribution graph of pyrethroid prepared nanopesticides with adequate larvicidal activity nanoemulsions of alpha-cypermethrin (A), deltamethrin (B), lambda- at the lowest exposure concentration makes it a suitable cyhalothrin (C), and permethrin (D). Description of data: This figure pre- and effective mosquito control agent. In addition, the sents a zeta potential distribution graph of prepared pyrethroid nanoe- mulsions. Figure S3. The visual appearance of pyrethroid nanoemulsions evaluation of the biosafety of nanoscale pesticides of alpha-cypermethrin (1), deltamethrin (2), lambda-cyhalothrin (3), and against freshwater alga R. subcapitata by calculating Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 18 of 19 Received: 7 February 2021 Accepted: 2 June 2021 permethrin (4). Description of data: This figure presents the visual appear- ance of alpha-cypermethrin (1), deltamethrin (2), lambda-cyhalothrin (3), and permethrin (4) nanoemulsions. Figure S4. Docking view of the tested pyrethroids on the binding sites of ATPase (PDB ID: 4byg). Alpha- References cypermethrin (A), lambda-cyhalothrin (B), deltamethrin, (C), and permeth- 1. Vinogradova EB. Culex pipiens pipiens mosquitoes: taxonomy, distribution, rin (D). Description of data: This figure presents the docking view of the ecology, physiology, genetics, applied importance and control: Pensoft tested pyrethroids on the binding sites of ATPase (PDB ID: 4byg). Left is Publishers; 2000. the 2D interaction diagram structure and right is the complex structure 2. Scott JG, Yoshimizu MH, Kasai S. Pyrethroid resistance in Culex pipiens in stereo view (3D). Figure S5. Docking view of the tested pyrethroids mosquitoes. Pestic Biochem Physiol. 2015;120:68–76. on the binding sites of CaE (PDB ID: 5w1u). Alpha-cypermethrin (A), 3. Saxena PN, Bhushan B. Estimation of median lethal dose of commercial lambda-cyhalothrin (B), deltamethrin, (C), and permethrin (D). Description formulations of some type II pyrethroids. Jordan J Biol Sci. 2017;10(3):193–7. of data: This figure presents the docking view of the tested pyrethroids 4. Li Y, Zhou G, Zhong D, Wang X, Hemming-Schroeder E, David RE, et al. on the binding sites of CaE (PDB ID: 5w1u). Left is the 2D interaction dia- Widespread multiple insecticide resistance in the major dengue vector gram structure, and right is the complex structure in stereo view (3D). Aedes albopictus in Hainan Province. China Pest Manage Sci. 2021;77(4): Figure S6. Docking view of the tested pyrethroids on the binding sites of GST (PDB ID: 5ft3). Alpha-cypermethrin (A), lambda-cyhalothrin (B), del- 1945–53. 5. Jones RT, Ant TH, Cameron MM, Logan JG. Novel control strategies for tamethrin, (C), and permethrin (D). Description of data: This figure pre- mosquito-borne diseases: The Royal Society; 2021. sents the docking view of the tested pyrethroids on the binding sites of 6. Dzib-Florez S, Ponce-García G, Medina-Barreiro A, González-Olvera G, GST (PDB ID: 5ft3). Left is the 2D interaction diagram structure, and right is the complex structure in stereo view (3D). 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Enhanced mosquitocidal efficacy of pyrethroid insecticides by nanometric emulsion preparation towards Culex pipiens larvae with biochemical and molecular docking studies

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Copyright © The Author(s) 2021
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2090-262X
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10.1186/s42506-021-00082-1
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Abstract

Background: The growing threat of vector-borne diseases and environmental pollution with conventional pesticides has led to the search for nanotechnology applications to prepare alternative products. Methods: In the current study, four pyrethroid insecticides include alpha-cypermethrin, deltamethrin, lambda- cyhalothrin, and permethrin were incorporated into stable nanoemulsions. The optimization of nanoemulsions is designed based on the active ingredient, solvent, surfactant, sonication time, sonication cycle, and sonication energy by factorial analysis. The nanoscale emulsions’ droplet size and morphology were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The toxicity of nanoemulsions against Culex pipiens larvae was evaluated and compared with the technical and commercial formulations. The in vitro assay of adenosine triphosphatase (ATPase), carboxylesterase (CaE), and glutathione-S-transferase (GST) were also investigated. Furthermore, molecular docking was examined to assess the binding interactions between the tested pyrethroids and the target enzymes. Also, an ecotoxicological assessment of potential effects of the tested products on the freshwater alga Raphidocelis subcapitata was determined according to OECD and EPA methods. The emulsifible concentration (EC ) and NOEC (no observed effect concentration) values were estimated for each insecticide and graded according to the GHS to determine the risk profile in aquatic life. Results: The mean droplet diameter and zeta potential of the prepared pyrethroid nanoemulsions were found to be in the range of 72.00–172.00 nm and − 0.539 to − 15.40 mV, respectively. All insecticides’ nanoemulsions showed significantly high toxicity (1.5–2-fold) against C. pipiens larvae compared to the technical and EC. The biochemical activity data proved that all products significantly inhibited ATPase. However, GST and CaE were significantly activated. Docking results proved that the pyrethroids exhibited a higher binding affinity with CaE and GST than ATPase. The docking scores ranged from − 4.33 to − 10.01 kcal/mol. Further, the biosafety studies of the nanopesticides in comparison with the active ingredient and commercial EC were carried out against the freshwater alga R. subcapitata and the mosquitocidal concentration of nanopesticides was found to be non-toxic. * Correspondence: m_eltaher@yahoo.com Department of Pesticide Chemistry and Technology, Laboratory of Pesticide Residues Analysis, Faculty of Agriculture, Alexandria University, 21545-El-Shatby, Alexandria, Egypt Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 2 of 19 Conclusion: The mosquitocidal efficacy of nano-pyrethroids formulated in a greener approach could become an alternative to using conventional pesticide application in an environmentally friendly manner. Keywords: Culex pipiens, Pyrethroids, Nanoemulsion, Insecticidal activity, Biochemical studies, Molecular Docking, Ecotoxicity 1 Introduction relatively large droplet size. Emulsions are dynamically Culex pipiens is one of the many members of the unstable systems that tend to collapse through gravita- disease-carrying mosquito family. Specifically, C. pipiens tional separation, droplet aggregation, and Ostwald rip- is a well-known carrier of the West Nile virus, Saint ening. All these factors can negatively affect the end Louis encephalitis viruses, canine Dirofilaria worms, product’s efficiency and shelf life, reducing its pest con- avian malaria, and filarial worms. Since the early twenti- trol ability. Many of these problems can be overcome eth century, campaigns have been organized in many with the use of nanoemulsions, which are an effective countries to control this pest species [1]. For C. pipiens way to use pesticides efficiently, economically, and safely mosquitoes’ chemical control, pyrethroid insecticides [11]. Nanoemulsions consist of emulsifier-coated fine oil have been extensively used worldwide [2]. Their use in- droplets dispersed in water, having droplets covering the creased to represent from 18% in 2002 to 30% in 2017 of size range of 20–500 nm [9]. They are also referred to as the total global pesticide market [3]. These pesticides are mini-emulsions, ultrafine emulsions, submicron emul- synthetically modified analogs of the essential natural sions, and others. Due to their particular size, nanoemul- pyrethrins found in flowers of the Chrysanthemums sions are transparent or translucent to the naked eye genus. In general, many new pyrethroids are synthesized and are stable against sedimentation or creaming. The and added to the market to meet the enhanced global smaller size of the droplets increases their stability to the demand for food, vector-borne diseases, and pest species gravitational separation and accumulation of droplets. It resistant to other pesticides [4]. Pyrethroids are an es- increases their deposition, diffusion, and permeability to sential way to combat malaria and other mosquito-borne plant leaves and insect body surfaces [12]. The compos- diseases despite the risk of pyrethroids resistance in ition, characteristics, mechanism of formation, and sta- vector populations [5]. Pyrethroids are also common in- bility of pesticide nanoemulsions have essential gredients of household insecticides. The home environ- theoretical and practical significances on the promotion ment’s unregulated use increases the risk of exposure and application of pesticides compared to conventionally and adverse effects in the general population [6]. applied pesticides [13, 14]. Synthetic alpha-cyano pyrethroids such as alpha- The main objectives of this study were to prepare and cypermethrin, deltamethrin, and lambda-cyhalothrin are characterize O/W nanoemulsions of four pyrethroid in- potent environmentally compatible insecticides and have secticides (alpha-cypermethrin, deltamethrin, lambda- a wide margin of safety for mammals for preferential ap- cyhalothrin, and permethrin). Various factors were de- plication in agricultural, veterinary, and public health signed and investigated to prepare the nanoemulsions programs [7]. using a factorial design by Minitab software. Factors in- Pyrethroid products have been traded in some formu- clude the concentration of the active ingredient, solvent, lations, the most popular of which are emulsifiable con- surfactant, sonication time, sonication pulses, and centrate (EC), aerosol dispenser, wettable powder, dust sonication power. The prepared nanoemulsions’ charac- powder, and water dispersion granules [8]. The EC of terizations, including the droplet size distribution, poly- pyrethroids is usually two to nine times more toxic than dispersity index (PDI), viscosity, pH, stability, and the technical grade, likely due to synergistic reactions. It surface morphology by transmission electron microscopy is one of the most widely used delivery systems for (TEM) were investigated. The toxicity of the nanoemul- hydrophobic pesticides, accounting for 40–50% of total sions was investigated against Culex pipiens larvae com- formulations. However, about 300,000 tons per year of paring to the active ingredient and commercial EC. organic solvents are used to prepare the EC formulations Biochemical studies were also investigated in vitro on [9]. Besides, other common solvents and co-solvents can adenosine triphosphatase (ATPase), carboxylesterase also be used. These solvents have flammable, explosive, (CaE), and glutathione-S-transferase (GST). We further and toxic properties that make them harmful to humans applied the molecular docking of these insecticides in and crops and produce poisonous residues in the envir- conjunction with existing experimental data and onment [10]. In practice, some of the problems associ- enzyme-associated tested insecticides to hypothesize ated with using conventional emulsifiers as delivery how these compounds would interact with the target systems for hydrophobic pesticides relate to their proteins. Further, this study demonstrated the non-toxic Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 3 of 19 property of nanopesticides towards non-target species of in adult cages. Using a live pigeon in our study was ac- the freshwater alga Raphidocelis subcapitata. cording to the Ethics Committee of High Institute of Public Health’s acceptance and proved from Alexandria 2 Methods University under reference number 481. The egg rafts 2.1 Tested pyrethroids were transferred from adult cages to white trays contain- The technical grade of alpha-cypermethrin (97%), delta- ing de-chlorinated water for egg hatch [16]. methrin (98%), and lambda-cyhalothrin (95%) were ob- tained from Syngenta Agro. Co (6th of October, Giza, 2.4 Experimental design for nanoemulsions preparation Egypt), while permethrin technical grade (96%) was ob- The experimental design allows studying the effect of tained from Chema Industries (26 First Industrial Area, many variables with a limited number of experiments. EL-Nubariya, El-Beheira Governorate, Egypt). The The design relied on deltamethrin as an insecticide chemical structures and physicochemical properties of chosen from among the four types of pyrethroids tested. the tested pyrethroids are shown in Table S1. Formu- Windows version of MINITAB 19.1 software (2019 lated forms of alpha-cypermethrin (25% EC, Spar-kill®) Minitab Inc.) was used to design the experiments [17]. and lambda-cyhalothrin (10% EC, Lambda®) were pur- Statistical analysis of the results will reveal which vari- chased from El-Helb Pesticides and Chemicals Co. ables have a significant influence and correlate the de- (Dumyat Al Jadidah, Dumyat, Egypt. Deltamethrin for- sired response with the variables by the polynomial mulation (5% EC, Nu-tox®) was obtained from equation: Alexandria Co. for Pharmaceuticals & Chemical Y =A +A X +A X +A X +A X 1 0 1 1 2 2 3 3 n n industries, Co. Permethrin formulation (25% EC, Per- where Y is the dependent variable, A is a constant, gon®) was obtained from MEDMAC for Manufacturing and A –A are the coefficients of the independent 1 n Agricultural Chemicals & Veterinary Products Ltd (Um- values. X -X represent independent factors. 1 n Mutwa’ Al-Aslameya Street, Al-Jandaweel, Amman, A factorial experiment consists of two or more factors Jordan). from these designs, each with discrete possible values or “levels.” In this study, the factorial design methodology 2.2 Chemicals and reagents was used to study the effects of six independent vari- Adenosine triphosphate (ATP), bovine serum albumin ables: the concentration of deltamethrin (as an example (BSA), 1-chloro-2,4-dinitrobenzene (CDNB), dimethyl- of pyrethroids active ingredient), DMSO as a solvent, sulfoxide (DMSO), Folin-Ciocalteu phenol, L- and tween 80 as a surfactant, in addition to applied son- glutathione (GSH), α-naphthyl acetate, β-nicotinamide ication power, sonication time, and pulses of sonication adenine dinucleotide (β-NAD), tetrazotized O- on the droplet size, PDI, pH, and viscosity of prepared dianisidine (fast blue B salt), trichloroacetic acid (TCA), pyrethroid nanoemulsions. Droplet size, PDI, viscosity, Tris (hydroxymethyl)aminomethane), triton X-100 and and pH were determined as the dependent variables. tween 80 were purchased from Sigma-Aldrich Chemical Nine experimental trials involving six independent vari- Co. (St. Louis, MO, USA). Other commercially available ables were obtained from the software. Each variable solvents and chemicals such as ammonium molybdate, was tested at two levels, low (−) and high (+), in addition copper sulfate, EDTA (ethylenediaminetetraacetic acid), to the mean level of each variable was tested in only one ferrous sulfate, and sodium-potassium tartrate were of experiment. analytical grade and purchased from El-Gomhouria For Trading Chemicals And Medical Appliances Co., (Adeb 2.5 Preparation of nanoemulsions Ishak St, Manshia, Alexandria, Egypt) and used without To achieve the final optimized conditions, nine formula- further purification. tions of deltamethrin were prepared in different experi- mental setups, including the organic phase (active 2.3 Culture of Culex pipiens larvae ingredient and DMSO), an aqueous phase containing A susceptible strain of C. pipiens culture was obtained Tween 80, sonication time, sonication power, and sonic- from Research Institute of Medical Entomology, Minis- ation pulses. Deltamethrin was used as an example of try of Health, Dokki, Giza, Egypt, and reared in High In- pyrethroids in optimization experiments. However, four stitute of Public Health insectary, Alexandria University, insecticides, including alpha-cypermethrin, deltamethrin, Alexandria, Egypt [15]. The Larvae were fed on biscuits lambda-cyhalothrin, and permethrin, were prepared by [containing wheat flour and yeast powder mixed with the optimum method. Briefly, 0.5% a.i (w/v) of each in- milk powder (10: 1: 1, w/w, respectively)] until pupation secticide were dissolved in DMSO to form the organic in shallow trays containing 2–3 L of de-chlorinated phase. Tween 80 was dissolved in distilled water to form water. Male adults were fed on 30% sucrose solution, the polar phase. The organic phase was dropped into a and females were fed on pigeon blood four times a week polar phase to form the coarse emulsion by stirring at Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 4 of 19 room temperature for 30 min at 4000 rpm. The coarse 2.6.2 Droplet size, polydispersity index, and zeta potential emulsion was later converted into a nanoemulsion The droplet size, PDI, and zeta potential of pyrethroids through ultra-sonication using a high-energy ultrasonic nanoemulsion formulations were investigated using process by the ultrasonic probe (Ultrasonic Homoge- Zetasizer Nano ZS (Malvern Instruments, UK) at room nizers HD 2070 with HF generator (GM 2070), ultra- temperature. The mean particle size and PDI of nanoe- sonic converter UW2070, booster horn (SH 213 G), and mulsions were measured by the dynamic light scattering probe microtip MS 73, Ø 3 mm) (Fig. 1). The tip of the (DLS) technique. Emulsion droplet size was estimated by horn was symmetrically placed in the coarse emulsion, the average of three measurements and presented as and the ultra-sonication process was carried out at mean diameter in nm, while zeta potential was deter- pulses 9 cycles/sec, power 75 % for 15 min [18]. mined by the light scattering method [20]. The formula- tions were diluted with distilled water by 200-fold and 2.6 Characterization of the nanoemulsions sonicated for 5 min at pulses 9 cycles/s and 75 % power 2.6.1 Stability studies before the measurement to avoid multiple scattering The prepared nanoemulsions were subjected to stability effects. screening tests to select the most stable formulation. These stability tests, including centrifugation assay sta- 2.6.3 Transmission electron microscopy bility at a temperature of 25 and 40 °C and heating- Surface morphology, topology, and droplet size of four cooling test. Centrifugation assay in which three samples pyrethroids nanoemulsions were characterized by TEM from each prepared formulation were centrifuged for 30 (JEOL JEM-1400 Plus TEM, USA, Inc.) equipped with a min at 5000 rpm and noticed phase separation, cream- 20-mm aperture at 20 kV. Bright-field imaging increas- ing, and cracking. The nanoemulsions should have ing the magnification and diffraction modes was selected enough stability without phase separation. Stable formu- to reveal the nanoemulsions’ form and size. The nanoe- lations were exposed to other thermodynamic stability mulsion of each pyrethroid formulation (10 mL) was di- tests [19]. About 25 mL of freshly prepared nanoemul- luted with distilled water (1/100) and added to 200- sions were transferred to a transparent tube. The trans- mesh form war-coated copper TEM sample holders (EM formation from a steady state to creaming and Sciences, Hatfield, PA, Japan). coalescence was examined during the storage period of 3 months at 25 °C. After that, the heating-cooling test was 2.6.4 Viscosity and pH measurements investigated to show the effect of heating and cooling on The dynamic (absolute) viscosity of the nanoemulsion the prepared nanoemulsions’ stability. The prepared was determined using a digital viscometer (a Rotary Myr nanoemulsions were maintained at a temperature of 4 VR 3000) with an L3 spindle at 200 rpm at 25 °C. The °C and 40 °C with storage for 48 h for each temperature viscosity of the formulations was measured without fur- test. The formulations that remained stable at this ther dilution. Each reading was recorded after the equi- temperature were subjected to further investigation. librium of the sample for 2 min. The viscosity recording Fig. 1 Schematic illustration of the preparation and characterization of pyrethroid nanoemulsions Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 5 of 19 of samples was repeated three times, and the data 500 μg/L were tested in three replicates. Twenty C. expressed in mPa.s. In the present study, the digital pH pipiens larvae were put into plastic cups containing 100 meter (Crison pH Meter Basic 20, EU) was used to mL of de-chlorinated water. The larvae were treated sep- determine the prepared nanoemulsions’ pH values. arately with Tween 80 or DMSO, and larvae without any treatment were maintained as control. The larvae’s mor- 2.7 In vitro release of pyrethroids from nanoemulsions bidity and mortality were verified and recorded based on In vitro release experiments were carried out using the the larvae’s uncoordinated movement after investigating dialysis technique [21]. Two milliliters from each the cervical region with a needle. Larval mortality per- formulation (0.5%, v/v) was placed inside a dialysis bag centages were recorded after 24 and 48 h, and the me- (cellulose membrane, molecular weight cut-off 14,000, dian lethal concentration (LC ) values were calculated Sigma-Aldrich Co., St. Louis, MO), sealed, and from probit analysis with 95% confidence limits and immersed in a vessel containing 50 mL of 10 mM phos- other statistical parameters [22]. phate buffer solution (pH 7.4). The releasing system was maintained at 37 ± 1 °C under magnetic stirring (100 2.9 Biochemical studies rpm). One milliliter from the solution was taken out of 2.9.1 Preparation of enzyme homogenates and total protein the dissolution medium at predetermined time intervals, assay replaced with fresh buffer solution. Pyrethroids released Surviving larvae were homogenized in 10 mM NaCl (1%, were determined by ultra-high-performance liquid chro- w/v) Triton X-100, and 40 mM sodium phosphate buffer matography (UHPLC, UltiMate 3000 system, Thermo (pH 7.4) at 4°C to determine Adenosine triphosphatase Scientific, USA). The system was equipped with a DIO- (ATPase), carboxylesterase (CaE), and glutathione-S- NEX UltiMate 3000 variable wavelength ultraviolet de- transferase (GST) activities after 24 h of exposing to tector (VWD). The separation was performed on LC values of the tested pyrethroids. The homogenate analytical column ODS Hypersil C18 (250 × 4.6 mm was centrifuged at 5000 rpm for 20 min at 4°C. The diameter, 5-micron particle size, Thermo scientific, supernatant was used immediately for enzymatic assay USA). Data were managed using a Chromeleon™ Chro- or stored at – 20 °C. Total protein was determined ac- matography Data System Software. The system consists cording to Lowry et al.’s[23] method, and the concen- of a binary gradient solvent pump to control the mobile trations were calculated by comparing with the standard phase’s flow rate and an autosampler for automatic in- curve of BSA. jection, a vacuum degasser, and a column oven (5–80 °C). The detection of tested pyrethroids was with a flow 2.9.2 ATPase assay rate of 1 mL/min, injection volume of 10 μL, and gradi- ATPase activity was performed according to Koch’s[24] ent solvent system, as shown in Table S2. The tested py- method. The reaction mixture, which contained 400 rethroids’ release profile was expressed as a cumulative + + +, mM Na ,20mMK , 5 mM Mg and 5 mM ATP, was concentration (mg/L ± SE) and plotted versus time. The prepared, and 200 μL of the crude enzyme was added to experiments were carried out in triplicate for each tested this mixture. Then, the volume was completed to 950 μL compound. The analytical grade of tested pyrethroids with Tris-HCl buffer (pH 7.4). After 10 min incubation was used for standard preparation. The calibration curve at 37 °C, the reaction was stopped with 200 μL of TCA. obtained from each insecticide’s analytical standard was A fresh color reagent (5 g ferrous sulfate in 10 mL am- used to determine the final concentrations released from monium molybdate solution prepared in 10 N sulfuric the nanoemulsions. acid) was added to the reaction mixture. The absorbance of the developed blue color was measured at 740 nm, 2.8 Toxicity assay against C. pipiens larvae −1 and the enzyme activity was calculated as OD min According to the World Health Organization, the larval −1 mg protein . bioassay was performed to compare the effect of nanoe- mulsions of selected pyrethroids with their active ingre- dient and commercial EC formulations on the C. pipiens 2.9.3 CaE assay larva recommendations [16]. Third instar larvae were CaE activity was determined according to Van Asperen’s used in the evaluation by a direct contact method. The [25] method, which used α-naphthyl acetate as a sub- three forms of tested insecticides (technical, commercial strate. The assay mixture contained 50 μL of homogen- EC formulation, and nanoemulsion) were tested to ob- ate enzyme, 2.1 mL of 50 mM sodium phosphate buffer tain the LC values. Technical pyrethroids were dis- (pH 7.4), and 25 μLof5mM α-naphthyl acetate solu- solved in DMSO and mixed with Tween-80 (0.05%), tion. The mixture incubation was done at 37 °C for 15 while the EC and nanoemulsions were dissolved in dis- min. Finally, 25 μL of 0.3% Fast blue B salt dissolved in tilled water. Different concentrations ranging from 0.5 to 3.5% SDS was added and incubated for 15 min at 37°C. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 6 of 19 The absorption was measured at 555 nm. The enzyme An acute algal growth inhibition test was conducted −1 −1 activity was expressed as OD min mg protein . using different concentrations of each insecticide in ster- ile AAM in a final volume of 50 mL. Tested concentra- tions of a pesticide were prepared from stock solutions 2.9.4 GST assay on an arithmetic progression covering an expected range GST assay was performed using reduced glutathione (2.5 of toxicity from 0 to 90%. Stock solutions of technical mM) by Saint-Deniset et al. [26]. The assay mixture con- insecticides were prepared in 1% DMSO and a corre- tained 100 μL of 1.5 mM CDNB, 200 μL of reduced sponding control was included. Stock solutions of the glutathione, and 1.5 ml of pH 7.4 phosphate buffer. A EC and NE were prepared. Algal suspensions were ex- total of 200 μL of the enzyme was added to the above posed to different concentrations (0.0005–500 mg/L) mixture, shaken gently, and incubated for 15 min at 37 prepared in the same medium of algae culture. All assays °C. The absorbance was recorded at 340 nm using a UV/ were conducted in triplicate. An inoculum of the Visible spectrophotometer (Alpha-1502. Laxco Inc, exponentially-growing culture of R. subcapitata (har- USA). One unit of enzyme activity attributed to the vested from 4–7 days stock culture) was prepared no quantity of conjugated enzyme with 1 mmol of GSH per −1 more than 2–3 h before the beginning of the test. Initial min. The enzyme activity was expressed as OD min −1 cell density for the growth inhibition test was 10,000 mg protein . cells/ml in both test and control flasks. Zero-time begins at inoculation of all flasks with the algal cells followed 2.10 Molecular docking by incubation for 96 h in a temperature-controlled (25 The modeled protein structure, ATPase (PDB ID: 4BYG) °C) orbital shaker set at 100 rpm under continuous illu- and detoxifying enzymes CaE (PDB ID: 5W1U) and GST mination via white fluorescent lamps. After 96 h, algal (PDB ID: 5FT3) in their PDB formats were downloaded growth in terms of viable cell concentration was exam- from the protein data bank (PDB) (http://www.rcsb.org) ined in a Neubauer hemocytometer using a phase- and imported on to the Molecular Operating Environ- contrast microscope. Growth rate inhibition of the alga ment (MOE) 2014.13 software (Chemical Computing was used as the endpoint in this assessment. The per- Group Inc, Montreal, Quebec, Canada). The structure of cent inhibition values were calculated after 96 h, and the each enzyme was visualized by the MOE [27]. The pro- median effective concentration (EC ) values were calcu- tein chemistry of the missing hydrogen was corrected, lated from the probit analysis with 95% confidence limits after which the heteroatoms and the crystallographic [22]. The no observed effect concentration (NOEC) after water molecules were removed from the protein. Chem- algal exposure to each tested insecticide was calculated ical structures of the tested pyrethroids were drawn by by the formula: NOEC = EC/10 [31]. Furthermore, the ChemDraw Professional Ultra Version 15 (PerkinElmer, hazard statement of each tested insecticide was esti- Informatics, Inc., USA). The structures were converted mated according to UNECE GHS (2019) [32]. to 3D, and the energy was minimized by the MMFF94 function [28]. The triangle-matching algorithm was se- 2.12 Statistical analysis lected from MOE for docking the compounds into the Statistical analysis was performed using the IBM SPSS selected enzymes’ active sites. Free energy of binding software version 25.0 (SPSS, Chicago, IL, USA) [33]. was calculated from the contributions of hydrophobic, Mortality percentages were calculated for each treatment ionic, hydrogenated, and van der Waals interactions. A and corrected using Abbott’s equation [34]. Means and ligand was considered adequate for a minimum docking standard error (SE) were obtained from three independ- score value (or interaction energy calculation) of an ent replications performed for each treatment. The log enzyme-ligand complex. dose-response (LdP) lines were used in the determin- ation of the LC values for the mosquito’s bioassay and 2.11 Bio-efficacy experiment on the freshwater green alga EC values for the algal bioassay according to the probit The freshwater green alga Raphidocelis subcapitata was analysis [22]. The least-square regression analysis was obtained from the Faculty of Science; Mansoura Univer- used to determine the 95% confidence limits. Analysis of sity, Egypt. The stock culture was maintained in 250-mL variance (ANOVA) of the biochemical data was con- borosilicate Erlenmeyer flasks containing culture ducted and means property values were separated (p ≤ medium at 24 ± 2 °C, under a continuous white fluores- 0.05) with Student-Newman-Keuls (SNK). cent light of 3000–4000 lux, and manually shaken twice a day [29]. The axenic culture was maintained for the 3 Results provision of a continuous supply of “healthy” cells for 3.1 Physiochemical properties of the tested pyrethroids the tests in a standard algal assay medium (AAM) as de- The chemical structure and physicochemical proper- scribed in Miller et al. [30]. ties of the tested pyrethroids are shown in Table S1. Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 7 of 19 Permethrin from type I pyrethroids lacks a cyano formulations 4, 8, and 9 showed significant differences group and three insecticides from type II pyrethroids in their viscosity values (81.00, 160.23, and 70.67 (alpha-cypermethrin, deltamethrin, and lambda- mPa.s, respectively). The pH of the prepared formula- cyhalothrin) in which an alpha-cyano group is present tions was in the range of 7.78-8.18. at thephenylbenzylalcohol position.The tested Based on these quantitative data, the first-order poly- compounds’ molecular weight was 416.3, 505.21, nomial equations (1-4) and their corresponding coeffi- 449.85, and 505 g/mol for alpha-cypermethrin, delta- cients were generated for each of the response variables methrin, lambda-cyhalothrin, and permethrin, respect- in the factorial design test. The models indicated the in- ively. The polar surface area (PSA) of all tested dividual parameters’ behavior in others’ presence on the pyrethroids was 59.32, except permethrin was 35.53, viscosity, particle size, PDI, and pH for deltamethrin while the hydrophobicity factor (ALogP) of all tested nanoemulsions. compounds was around 6. There are no hydrogen bond donors (HBD) in the tested pyrethroids, while Viscosity = − 110 + 21.09 a.i − 0.67 solvent + 5.11 surfactant ……….(1) + 9.86 sonication pulses + 1.91 sonication time + 0.255 the number of hydrogen bond acceptors (HBA) sonication power ranged from 3 to 7. 2 s = 23.0714, r = 89.49% Droplet size = 20951 + 1524 a.i − 180 solvent − 514 ……….(2) 3.2 Optimization of the nanoemulsions preparation surfactant − 417 sonication pulses − 216 sonication time + 25.5 sonication power The different experimental setup using Minitab soft- s = 1959.47, r = 87.19% ware was used to determine the influence of six inde- PDI = − 0.76–0.0336 a.i + 0.0256 solvent − 0.0010 surfactant ……….(3) pendent variables on the pyrethroid nanoemulsions’ + 0.0541 sonication pulses + 0.0244 sonication time − characterization (dependent variable) (Table S3). Del- 0.00548 sonication power tamethrin was selected as a model of the tested pyre- s = 0.2112, r = 82.45% throids for the optimization experiments. During the pH = 6.52 − 0.0187 a.i + 0.0304 solvent + 0.0155 surfactant ……….(4) nanoforming process, emulsification was achieved in − 0.0006 sonication pulses − 0.0048 sonication time − 0.00235 sonication power the context of the droplet shearing phenomenon. The s = 0.1863, r = 62.06% sound waves (frequency 25–75 Hz) generated by the sonotrode (a tool that creates ultrasonic vibrations) Also, the influence of each factor on the response were applied to induce a mechanical vibration. variableswas shownasParetocharts inFig. 2.Itwas Followed by acoustic cavitation, which could lead to a noted that the active ingredient, sonication pulses, further collision and cause strong shock waves to and surfactant were more significant factors than the shear the largest droplets to a nanometer size. The others on the nanoemulsion viscosity (Fig. 2A). In visual appearance of the nine deltamethrin nanoemul- comparison, the active ingredient, surfactant, and sionsisshown in Figure S1. The quantitative results sonication time showed the highest effect on the including the droplet size (nm), PDI, and viscosity prepared nanoemulsions’ particle size (Fig. 2B). In the (mPa.s) are presented in Table S4.There are signifi- case of the PDI value, the sonication power, cant differences in the droplet size of the nine pre- sonication time, and sonication pulses, respectively, pared deltamethrin formulations. Formulations 1, 5, had a significant effect (Fig. 2C). On the contrary, the and 7 presented 172.46, 364, and 417 nm, respect- solvent was the most significant factor in the pH ively, while the other six formulations showed droplet value (Fig. 2D). sizes larger than 500 nm. In the PDI case, there are Among the 9 different experimental setup (Table S3), no significant differences between the formulations formulation 1 with 0.5 % a.i, 44% DMSO, 15% tween 80, (0.516–0.964) except formulation 2 (PDI = 0.158). 40.5% water, 9 cycle/s sonication pulses, and 75% Nanoemulsions were exposed to extreme storage con- sonication power for 15 min was the best. The resulted ditions to predict the samples’ ability to be physically nanoemulsion 1 was in clear visual appearance with a stable for up to three months. All prepared delta- smaller droplet size of 234 nm ± 4.13. Therefore, these methrin formulations did not pass the centrifugation parameters were selected to prepare the other test at 5000 rpm except formulations 1 and 5, while pyrethroids nanoemulsions (alpha-cypermethrin, all prepared formulations did not pass the heating- lambda-cyhalothrin, and permethrin). cooling test. The viscosity and pH measurements of prepared deltamethrin formulations. There is no sig- nificant difference between the viscosity values of for- 3.3 Characterizations of the pyrethroid nanoemulsions mulations 1, 2, 5, and 6 (74.67, 80.16, 90.23, and 3.3.1 Droplet size and polydispersity index 90.23 mPa.s, respectively). Formulations 3 and 7 have The droplet size of alpha-cypermethrin, deltamethrin, 40.32 and 37.67 mPa.s, respectively. However, lambda-cyhalothrin, and permethrin nanoemulsions Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 8 of 19 Fig. 2 Pareto charts representing the effect of factors and process variables on viscosity (A), droplet size (B), PDI (C), and pH (D) for deltamethrin nanoemulsions at α = 0.05 were 90.26, 172.00, 168, and 72, respectively (Table 3.3.3 Viscosity and pH 1). However, the PDI values were 0.337, 0.827, 0.448, The viscosity values of alpha-cypermethrin, deltameth- and 0.295 for alpha-cypermethrin, deltamethrin, rin, lambda-cyhalothrin, and permethrin nanoemulsions lambda-cyhalothrin, and permethrin, respectively. were 60.15, 74.67, 53.76, and 50.68 mPa.s, respectively (Table 1). The pH measurements were 8.51, 7.84, 8.20, 3.3.2 Zeta potential and 8.17 for alpha-cypermethrin, deltamethrin, lambda- The prepared nanoemulsions revealed negative values of cyhalothrin, and permethrin, respectively. zeta potential (− 0.603, − 0.669, − 0.539, and − 15.4 mV for alpha-cypermethrin, deltamethrin, lambda- 3.3.4 Thermodynamic stability studies cyhalothrin, and permethrin, respectively) (Table 1 and The stability results after the centrifugation and heating- Figure S2). cooling cycle are presented in Table 1. The results Table 1 The observed visual stability, droplet size, polydispersity index (PDI), zeta potential, dynamic (absolute) viscosity, and pH of prepared pyrethroid nanoemulsions Insecticide Visual Droplet size Polydispersity Zeta Viscosity pH Stability after appearance (nm) ± SE index (PDI) ± potential (mPa.s) ± Centrifugation at 5000 Heating-cooling SE (mV) SE rpm cycle b c b Alpha- Clear 90.26 ± 3.78 0.337 ± 0.01 − 0.603 60.15 ± 8.51 √ × cypermethrin 0.12 a a a Deltamethrin Clear 172.00 ± 0.827 ± 0.10 − 0.669 74.67 ± 7.84 √ × 34.07 7.86 a b c Lambda- Clear 168.00 ± 0.448 ± 0.05 − 0.539 53.76 ± 8.20 √ × cyhalothrin 4.08 0.20 c d c Permethrin Clear 72.00 ± 8.30 0.295 ± 0.02 − 15.40 50.68 ± 8.17 √ × 1.200 Different letters in the same column indicate significant differences according to the Student-Newman-Keuls (SNK) test (P ≤ 0.05). (√) refer to the stable state, (×) refer to the non-stable state. Preparation condition: 0.5 % a.i, 44% solvent (DMSO), 15% surfactant (tween 80) and 40.5% water with sonication pulses 9 cycle/s at 75% power for 15 min Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 9 of 19 showed that all nanoemulsions were transparent and and other statistical parameters in Table 2. The results stable at 5000 rpm of centrifugation and 25 °C for up to showed that alpha-cypermethrin nanoemulsion gave the 3 months (Figure S3), while these products were sepa- LC value of 20 μg/L that was more significant than the rated under a heating-cooling cycle test. EC formulation (40 μg/L) and the technical form (43 μg/ L) after 24 h of the experiment. The technical form of 3.3.5 Transmission electron microscopy deltamethrin proved an LC value of 28 μg/L, while the The morphological study of the structure of pyrethroid EC and nanoemulsion gave the LC of 26 and 28 μg/L, nanoemulsions was carried out by TEM. Figure 3 shows respectively, after 24 h. Lambda-cyhalothrin technical, the TEM micrograph of pyrethroid nanoemulsions, EC, and nanoemulsion gave LC values 27, 23, and 15 demonstrating the spherical shape. The droplets had a μg/L after 24 h and 18, 13, and 10 μg/L after 48 h, re- uniform shape and size. TEM analyses also confirmed spectively. However, according to the 95% confidence the nanometric droplet diameter of formulated limit based on the probit analysis, there is no significant pyrethroids at magnification 20,000×. difference between lambda-cyhalothrin and deltameth- rin. The LC values at 24 h of both insecticides interfere 3.4 Pyrethroids released from nanoemulsions with the lower and upper 95% confidence limits. How- The release profile assay was carried out using in vitro ever, permethrin nanoemulsion proved the lowest tox- dialysis experiment. Cumulative amounts (mg/L) of the icity with LC values 233 and 127 μg/L after 24 and 48 tested pyrethroids released from their nanoemulsions h, respectively. In permethrin, the LC values of EC and into phosphate buffer solution per time are shown in technical forms were 280 and 322 μg/L after 24 h, re- Fig. 4. Initial burst release was measured after 30 min, spectively, while the nanoemulsion was the most active and the concentrations 60.60, 25.29, 103.58, and 303.60 form (LC = 233 and 127 μg/L after 24 and 48, mg/L were quantified for alpha-cypermethrin, delta- respectively). methrin, lambda-cyhalothrin, and permethrin, respect- ively. It was noted that the rate of permethrin released 3.6 Enzymatic activity −1 from the nanoemulsion (60%) was greater than lambda- The data are shown in Table 3, as OD mg protein cyhalothrin (20%), alpha-cypermethrin (12%), and delta- min. The untreated larvae have 1.40, 3.31, and 2.70 for methrin (5%) after 30 min of the dialysis. After 180 min ATPase, CaE, and GST, respectively. By estimating the of the experiment, each compound’s release concentra- level of ATPase, CaE, and GST, it was found that the tion slightly increased to 82, 112, and 314 mg/L for insecticides in nanometric formulas had a significant alpha-cypermethrin, lambda-cyhalothrin, and permeth- effect as compared to control, technical form, and EC rin, respectively, whereas the concentration released treatment. The data proved that the activity of all tested from deltamethrin nanoemulsion reached only 76.58 enzymes was significantly increased except ATPase. The mg/L (15%) after 180 min of the experiment. insecticides caused a significant ATPase inhibition up to −1 0.50 OD mg protein min compared to 1.40 in control. 3.5 Larvicidal efficacy of pyrethroid nanoemulsions The most effective compound on the ATPase was The larvicidal activity of the technical, EC, and permethrin with specific activities of 0.50, 0.64, and 0.83 nanoemulsion of each insecticide was evaluated against for nanoemulsion, technical, and EC, respectively. C. pipiens larvae to record the mortality after 24 and 48 However, the lowest effective compound was alpha- h of the exposure. The data are presented as LC values cypermethrin, with activities of 0.86, 1.38, and 1.18 for Fig. 3 Transmission electron micrograph of prepared pyrethroid nanoemulsions alpha-cypermethrin (A), deltamethrin (B), lambda-cyhalothrin (C) permethrin (D): The TEM was performed on a JEOL JEM-1400 Plus, transmission electron microscope operating at an acceleration voltage of 80.0 kV with a 20-mm aperture. Print magnification 20,000× Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 10 of 19 Fig. 4 Release concentration of alpha-cypermethrin (A), deltamethrin (B), lambda-cyhalothrin (C), and permethrin (D) after time intervals for 3 h and standard error (n = 3) technical, commercial EC, and nanoemulsion, respect- with ATPase were ranged from − 4.33 to − 5.46 kcal/ ively. Deltamethrin inhibited the ATPase to 0.69, 0.77, mol (Table 4). The results revealed that all insecticides and 0.75 for the technical, EC, and nanoemulsion, re- exhibited H-bonding with amino acids in the active spectively. Lambda-cyhalothrin gave a specific activity of pockets of ATPase. α-Cypermethrin, deltamethrin, and 0.62 for the technical and 1.03 for the nanoemulsion. permethrin exhibited H-bonding with amino acid Asn All tested pyrethroids caused activation of the CaE, A112 by distances 3.54, 3.26, and 3.21 Å, respectively. which ranged from 3.32 to 7.02 compared to 3.31 in the Simultaneously, lambda-cyhalothrin exhibited H- untreated larvae. Alpha-cypermethrin nanoemulsion was bonding with Asn A112-N18 and Trp A116-N18 with the most active with a specific activity of 7.02. It was 3.38 and 3.56 Å, respectively. The binding confirmation followed by deltamethrin with specific activities of 4.53, of the tested pyrethroids with ATPase is shown in Figure 3.44, and 3.63 for the technical, EC nanoemulsion, S4. α-Cypermethrin (Figure S4A) and deltamethrin (Fig- respectively. ure S4C) interacted with ATPase by van der Waals (Glu For the GST activity, permethrin was the most 181, Gly 182, Leu 168, Pro 170, Trp 116, Val 167, and effective insecticide in activating this enzyme with the Val 183) and (Gly 113, Gly 171, Gly 182, Leu 168, Pro specific activities of 6.32, 7.85, and 7.86 for the technical, 170, Trp 116, Trp 169, and Val 167), respectively. Both commercial EC, and nanoemulsion, respectively, compounds interacted by H-arene bond with amino acid compared to 2.70 in control. It was followed by alpha- Asn A112 with 3.59 and 4.17 Å, respectively. In contrast, cypermethrin that caused the specific activity of 5.31, lambda-cyhalothrin and permethrin interacted with 7.54, and 11.94 for the technical, EC, and nanoemulsion, ATPase by van der Waals (Glu 181, Gly 182, Leu 168, respectively. The specific activity of GST treated with Pro 170, and Val 167) and (Gly 113, Phe 108, Leu 168, deltamethrin was higher than 5 for the three products. and Trp 116), respectively (Figure S4B and D). Lambda-cyhalothrin gave activity of 4.57 for the tech- Tested pyrethroids exhibited binding affinity ranged nical, 6.55 for the EC, and 6.15 for the nanoemulsion. from − 7.44 to − 10.01 kcal/mol on the active sites of CaE (Table 5). Lambda-cyhalothrin was the highest (ΔG 3.7 Molecular docking = − 10.01 kcal/mol) followed by α-cypermethrin, The docking scores and binding mechanism include H- deltamethrin, and then permethrin ΔG values of − 9.35, bonds, Van der Waals, and hydrophobic interactions of − 8.72, and − 7.44 kcal/mol, respectively. Alpha- the tested pyrethroids with ATPase (4BYG), CaE cypermethrin interacted with the CaE enzyme through (5W1U), and GST (5FT3) are shown in Tables 4, 5, and two hydrogen bonds (Asp 279-CL11 and Leu 328-N15) 6, respectively. Analysis of the docking results showed with distances of 3.32 and 0.6 Å, respectively. Besides, that the pyrethroids showed a higher binding affinity some van der Waals bonds ( Arg 73, Arg 392, Asp 279, with CaE and GST than ATPase. The docking scores Glu 118, Gly 109, Gly 110, Gln 330 His 442, Leu 327, Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 11 of 19 Table 2 Larvicidal activity of the technical, EC, and nanoemulsion of alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, and permethrin against C. pipiens larvae a b c 2 d Insecticide Time of Type of LC 95% confidence limits (μg/L) Slope ± Intercept (χ ) exposure formulation (μg/ SE ±SE Lower Upper (h) L) Alpha-cypermethrin 24 T 43 26 46 2.47 ± 0.15 3.66 ± 0.26 20.69 EC 40 35 64 3.22 ± 0.18 4.31 ± 0.27 35.35 NE 20 15 26 2.30 ± 0.14 3.94 ± 0.26 22.45 48 T 40 26 46 2.47 ± 0.15 3.66 ± 0.26 20.69 EC 38 32 63 3.20 ± 0.18 4.21 ± 0.27 35.32 NE 18 15 24 2.51 ± 0.15 4.37 ± 0.28 20.59 Deltamethrin 24 T 28 23 36 2.23 ± 0.13 3.45 ± 0.22 21.02 EC 26 20 31 1.31 ± 0.08 1.52 ± 0.08 73.20 NE 16 12 21 2.52 ± 0.16 4.50 ± 0.27 43.97 48 T 25 19 34 2.12 ± 0.13 3.39 ± 0.21 29.62 EC 24 20 28 1.30 ± 0.08 1.57 ± 0.09 57.09 NE 13 9 16 2.51 ± 0.17 4.76 ± 0.30 35.17 Lambda-cyhalothrin 24 T 27 17 45 1.47 ± 0.09 2.31 ± 0.17 30.16 EC 23 12 41 1.25 ± 0.08 2.06 ± 0.14 37.22 NE 15 11 20 2.05 ± 0.12 3.75 ± 0.23 18.42 48 T 18 12 27 1.30 ± 0.09 2.27 ± 0.16 18.84 EC 13 8 18 1.43 ± 0.07 2.72 ± 0.17 17.77 NE 10 7 14 1.42 ± 0.09 2.85 ± 0.18 19.7 Permethrin 24 T 322 221 506 1.36 ± 0.089 0.67 ± 0.081 22.655 EC 280 206 392 1.64 ± 0.103 0.91 ± 0.09 20.66 NE 233 201 270 1.68 ± 0.10 1.06 ± 0.09 07.20 48 T 225 134 423 1.26 ± 0.08 0.81 ± 0.08 40.21 EC 196 134 288 1.97 ± 0.12 1.39 ± 0.10 37.04 NE 127 76 215 1.43 ± 0.08 1.29 ± 0.09 44.54 T technical, EC emulsifiable concentrate, NE nanoemulsion. The Median lethal concentration. The LC value of each compound between the other compound’s confidence limits is not significantly different. However, if the fit confidence intervals (95%) are non-overlapping, there is a significant difference between the b c d compounds. Slope of the concentration mortality regression line ± error (SE). Intercept of the regression line ± SE. Chi-squared value Leu 328, Lys 331, Phe 281 Ser 191, Trp 224, Tyr 428, Glu 113, Glu 268 Gly 109, Leu 328, Leu 328, Lys 331, and Val 393) are included (Figure S5A). Lambda- Phe 281, and Thr 112) and Pi-cation interaction (Arg cyhalothrin bonded through HBD, HBA (Arg 73-F11 73-6-ring, 3.90 Å). and Glu 113-C16, respectively) with 2.9 Å for both and The docking results with GST (Table 6) indicated that van der Waals interactions (Arg 73, Arg 74, Asn 452, lambda-cyhalothrin was the highest affinity binding with Ala 443, Gln 330, Glu 113, Gly 109, Gly 110, His 427, the lowest energy value − 9.95 kcal/mol. It was followed His 442, Leu 120, Leu 327, Leu 446, Met 432, Phe 281, by alpha-cypermethrin, permethrin, deltamethrin with Ser 191, Ser 447, Thr 112, Tyr 121, Tyr 428, and Phe energy values − 8.55, − 8.53, and − 8.24 kcal/mol. alpha- 281) (Figure S5B). Three hydrogen bonds (Ser 191-Br8, cypermethrin interacted with GST through van der His 442-Br8, and Arg 73-O1) with distances of 3.46, Waals with 14 amino acids (Arg 112, Glu 116, His 41, 3.62, and 2.95 Å, respectively, and thirteen van der Leu 42, Leu 111, Leu 119, Lys 39, Phe 108, Phe 120, Pro Waals (Arg 73, Glu 113, Glu 268, Gly 109, Gly 110, Gln 13, Thr 54, Val 11, Val 55, and Val 207) with docking 330, His 442, Leu 327, Leu 334, Lys 331, Phe 281, Ser score of − 8.55 kcal/mol (Figure S6A). Figure S6B shows 191, and Tyr 428) were formed between deltamethrin the interactions between lambda-cyhalothrin and GST and CaE (Figure S5C). Figure S5D shows the interactions through van der Waals with 15 amino acids (Arg A112, between permethrin and CaE, which was through one Cys A115, Glu A116, His A41, His A53, Leu A36, Leu HBA (Lys 331–O1), ten van der Waals (Arg 73, Gln 330, A111, Leu A119, Lys A39, Lys B136, Phe A108, Pro A13, Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 12 of 19 Table 3 Biochemical effects of tested pyrethroids on some enzymes activity in C. pipiens larvae after 24 h of the treatment with LC of each compound −1 Treatment Type of Specific activity (OD mg protein min) ± SE formulation ATPase CaE GST Untreated sample – 1.40 ± 0.04 3.31 ± 0.01 2.70 ± 0.27 Alpha-cypermethrin T 0.86 ± 0.02 5.06 ± 0.15 5.31 ± 0.70 EC 1.38 ± 0.01 7.02 ± 0.50 7.54 ± 0.88 NE 1.18 ± 0.00 4.53 ± 0.25 11.94 ± 0.84 Deltamethrin T 0.69 ± 0.02 3.44 ± 0.19 5.49 ± 0.60 EC 0.77 ± 0.04 3.63 ± 0.71 5.63 ± 0.80 NE 0.75 ± 0.03 4.12 ± 0.09 5.90 ± 0.30 Lambda-cyhalothrin T 0.62 ± 0.05 1.51 ± 0.09 4.57 ± 0.48 EC 1.19 ± 0.02 3.32 ± 0.10 6.55 ± 0.85 NE 1.03 ± 0.01 3.35 ± 0.01 6.15 ± 0.45 Permethrin T 0.64 ± 0.00 2.31 ± 0.09 6.32 ± 0.59 EC 0.83 ± 0.13 2.66 ± 0.08 7.85 ± 0.44 NE 0.50 ± 0.01 3.37 ± 0.12 7.86 ± 0.37 T technical, EC emulsifiable concentrate, NE nanoemulsion, OD optical density, SE standard error, ATPase adenosine triphosphatase, CaE carboxylesterase, GST glutathione-S-transferase Ser A12, Val A11, and Val A207) and arene H-bond with 3.8 Ecotoxicity study against the freshwater green alga Phe A120. Deltamethrin reacted with GST through 10 The toxicity endpoint values after acute exposure of R. van der Waals (Glu A116, Leu A36, Leu A42, Leu A111, subcapitata to different forms of pyrethroids used as Lys A39, Phe A120, Phe A108, Pro A13, Thr A54, and mosquito larvicides are illustrated in Table 7. The Val A55), 3 H-bonds with amino acids ( His A41, His sensitivity of R. subcapitata to insecticides; expressed as A53, and Arg A112 ) and H-arene bond with amino acid EC , ranged from 0.76 to > 100 mg/L. Based on these ASN A112 (Figure S6C). Permethrin interacted with the values, the decreasing order of the sensitivity was pocket of GST through H-bond with amino acid Ser commercial EC > NE > technical form. The current data A12 and 14 amino acids through van der Waals bonds disclosed that the commercial EC of the tested (Arg 112, Cys 115, Glu 116, His 53, Leu 36, Leu 111, insecticides were more toxic to R. subcapitata and Leu 119, Phe 108, Phe 120, Pro 13, Thr 54, Val 11, Val recorded 0.76, 4.92, 5.03, and 16.98 mg/L for 55, and Val 207) (Figure S6D). deltamethrin, permethrin, lambda-cyhalothrin, and Table 4 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of ATPase (PDB ID: 4BYG) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid-ligand (Å) acid-ligand (Å) mol) atom atom Alpha- − 4.85 Glu 181, Gly 182, Leu 168, Pro Asn A112- HBA 3.54 Asn A112-6- Arene-H 3.59 1.66 cypermethrin 170, Trp 116, Val 167, Val 183 N15 ring Lambda- − 5.46 Glu 181, Gly 182, Leu 168, Pro Asn A112- HBA 3.38 –– – 1.88 cyhalothrin 170, Val 167 N18 HBA 3.56 TRP A116- N18 Deltamethrin − 4.33 Gly 113, Gly 171, Gly 182, Leu Asn A112- HBA 3.26 Asn A112-6- Arene-H 4.17 1.96 168, Pro 170, Trp 116, Trp 169, Val N15 ring Permethrin − 4.61 Gly 113, Phe 108, Leu 168, Trp Asn A112- HBA 3.21 - - - 3.36 116 O1 RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 13 of 19 Table 5 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of CaE (PDB ID: 5W1U) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid- (Å) acid- (Å) mol) ligand ligand atom atom Alpha- − 9.35 Arg 73, Arg 392, Asp 279, Glu 118, Gly 109, Asp HBD 3.32 –– – 1.14 cypermethrin Gly 110, Gln 330 His 442, Leu 327, Leu 328, 279- HBA 0.6 Lys 331, Phe 281 Ser 191, Trp 224, Tyr 428, Cl11 Val 393 Leu 328- N15 Lambda- − 10.01 Arg 73, Arg 74, Asn 452, Ala 443, Gln 330, Glu HBD 2.90 –– – 2.03 cyhalothrin Glu 113, Gly 109, Gly 110, His 427, His 442, 113- HBA 2.90 Leu 120, Leu 327, Leu 446, Met 432, Phe C16 281, Ser 191, Ser 447, Thr 112, Tyr 121, Tyr Arg 73- 428, Phe 281 F11 Deltamethrin − 8.72 Arg 73, Glu 113, Glu 268, Gly 109, Gly 110, Ser HBD 3.46 –– – 2.21 Gln 330, His 442, Leu 327, Leu 334, Lys 331, 191-Br8 HBD 3.62 Phe 281, Ser 191, Tyr 428 His 442- HBA 2.95 Br8 Arg 73- O1 Permethrin − 7.44 Arg 73, Gln 330, Glu 113, Glu 268 Gly 109, Lys HBA 3.04 Arg 73- Pi-cation 3.90 1.93 Leu 328, Leu 328, Lys 331, Phe 281, Thr 112 331–O1 6-ring RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules are participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Table 6 Molecular docking, binding scores and binding interactions of tested pyrethroids within the active sites of GST (PDB ID: 5FT3) Insecticide Docking van der Waals H-bond Hydrophobic interactions (π- RMSD score (S) interactions) ΔG Amino Interaction Distance Amino Interaction Distance (kcal/ acid- (Å) acid- (Å) mol) ligand ligand atom atom Alpha- − 8.55 Arg 112, Glu 116, His 41, Leu 42, Leu 111, Leu HBD 3.56 –– – 1.66 cypermethrin Leu 119, Lys 39, Phe 108, Phe 120,Pro 13,Thr A36- HBA 2.58 54, Val 11, Val 55, Val 207 Cl11 His 53- N15 Lambda- − 9.95 Arg A112, Cys A115, Glu A116, His A41, His Thr A54- HBA 3.19 Phe Arene-H 4.06 1.50 cyhalothrin A53, Leu A36, Leu A111, Leu A119, Lys A39, N18 HBA 3.53 A120-6- Lys B136, Phe A108, Pro A13, Ser A12, Val Val A55- ring A11, Val A207 N18 Deltamethrin − 8.24 Glu A116, Leu A36, Leu A42, Leu A111, Lys His A41- HBA 3.09 ASN Arene-H 4.17 0.58 A39, Phe A120, Phe A108, Pro A13, Thr A54, O1 HBA 3.68 A112-6- Val A55 His A53- HBA 2.76 ring N15 Arg A112- N15 Permethrin − 8.53 Arg 112, Cys 115, Glu 116, His 53, Leu 36, Ser A12- HBA 3.24 –– – 1.76 Leu 111, Leu 119, Phe 108, Phe 120, Pro 13, O1 Thr 54, Val 11, Val 55, Val 207 RMSD the root mean square deviation of the pose in Å, from the original ligand. This field is present if the site definition was identical to the ligand definition. Residues/water molecules are participating in hydrogen bonds and close van der Waals contacts (< 4 Å) with the inhibitors Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 14 of 19 Table 7 EC and NOEC (mg/L) of nanoformulations compared with technical and commercial formulated pyrethroids to freshwater microalga R. subcapitata. The 95% confidence limits of the EC values are indicated in parentheses Insecticide Formulation Toxicity endpoint 96 h EC (mg/L) NOEC (mg/L) GHS hazard statement Alpha-cypermethrin T 69.33 (38.15–145.54) 6.932 H402 EC 16.98 (10.99–29.01) 1.698 H402 NE 101.11 (38.08–409.14 1.011 S Lambda-cyhalothrin T 33.89 (15.93–94.27) 3.389 H402 EC 5.03 (2.96–9.62) 0.503 H401 NE 11.29 (1.83–121.71) 1.129 H402 Deltamethrin T > 100 > 10 S EC 0.76 (0.56–1.05) 0.076 H400 NE 13.14 (6.49–30.74) 1.314 H402 Permethrin T 14.94 (9.72–23.72) 1.494 H402 EC 4.92 (3.31–7.75) 0.492 H401 NE 20.55 (12.66–36.25) 2.055 H402 T technical, EC emulsifiable concentrate, NE nanoemulsion, NOEC no observed effect concentration on algal growth rate, H hazard statement. H400 very toxic to aquatic life (hazardous to the aquatic environment, acute hazard, category 1; ≤ 1mg/L); H401 toxic to aquatic life (hazardous to the aquatic environment, acute hazard, category 2; > 1–≤ 10 mg/L); H402 harmful to aquatic life (hazardous to the aquatic environment, acute hazard, category 2; > 10–≤ 100 mg/L). S: Safe use (no hazard statement is suggested) since acute toxicity > 100 mg/L alpha-cypermethrin, respectively. The potency of com- 4 Discussion mercial EC may be attributed to the additives in the for- 4.1 Physicochemical properties of the tested pyrethroids mulation rather than the active ingredient. According to Lipinski’s “rule of five” [35], good intestinal Toxicity of nanoformulations showed a different absorption and oral bioavailability of compounds reflect pattern where alpha-cypermethrin exhibited a safe effect RB and MR’s acceptable values. The stereo-specificity of on R. subcapitata (EC > 100 mg/L) while the EC for the drug molecule is a property of nRB, which was found 50 50 the other insecticides recorded 11.29, 13.14, and 20.55 to be < 10. There are no hydrogen bond donors (HBD) mg/L for lambda-cyhalothrin, deltamethrin, and per- in the tested pyrethroids, while the number of hydrogen methrin, respectively. The sensitivity of R. subcapitatata bond acceptors (HBA) ranged from 3 to 7. The literature towards nanoformulations was lambda-cyhalothrin > has also documented that excellent absorption in the in- deltamethrin > permethrin. The differential toxicity of testine is induced by PSA < 140 [36]. The Log S value nanoformulations depends on their nanostructure and for all insecticides is between − 6.84 and − 7.22, indicat- high surface to mass ratio as well as the nature of their ing low water solubility. constitutive element. On the other hand, the EC values for the technical form of the tested insecticides were 4.2 Characterizations of the pyrethroid nanoemulsions 33.89, 69.33, >100, and 14.94 mg/L for lambda- Several studies prepared and characterized pyrethroid cyhalothrin, cypermethrin, deltamethrin, and permeth- nanoemulsions, such as alpha-cypermethrin, deltameth- rin, respectively. The sensitivity of R. subcapitata was rin, lambda-cyhalothrin, and permethrin [13, 14]. The permethrin > lambda-cyhalothrin > cypermethrin > droplet size of the prepared nanoemulsions is in agree- deltamethrin. ment with other studies. Mishra and others reported For subsequent characterization of the potentially that nano-sized permethrin’s mean particle size was hazardous effects of the tested insecticides and 175.3 nm [13], whereas the TEM analysis investigated by addressing safety issues of the developed nanopesticides, Patel et al. [37] revealed that cypermethrin particle size’s both NOEC and hazard statements were evaluated encapsulation was ranged between 115 and 119 nm. (Table 7). The data showed that all nanoformulations However, the droplet size of beta cypermethrin nanosus- represent category acute III with harmful effects to pension prepared by Zeng et al. [38] was 168 nm. It was aquatic life (H402) compared with the commercial EC observed no phase separation, creaming, and sedimenta- forms which represent category acute I and II with very tion under room temperature (25 °C) and accelerated toxic and/or toxic hazardous effects to the aquatic life stability evaluation [8]. The long-term physical stability (H400 and H410). of a nanoemulsion related to its small droplets makes Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 15 of 19 this type of formulation being referred to as “ap- h, and the accumulated releases were over 90%. How- proaching thermodynamic stability.” The average droplet ever, the release rate of lambda-cyhalothrin nanoemul- size of the nanoemulsions typically falls within the range sion was rapid within the first 30 h, and then, it slowed of 20–500 nm [19]. The small size of the droplets in down and maintained a stable release until equilibrium nanoemulsions gives them some advantages over con- after 80 h. ventional emulsions. These advantages include higher optical clarity, higher stability to droplet aggregation and 4.4 Toxicity against C. pipiens larvae gravitational separation, and higher bioactivity of encap- The effect of the three forms of the tested insecticides sulated components. The nanoemulsions have emerged was significantly different against C. pipiens larvae. It as alternative drug carriers because they increase the dis- can be noted that the technical form exhibited the solution rates and bioavailability of many poorly soluble lowest larvicidal activity. However, the EC of all tested drugs in water [9]. insecticides slightly improved the toxic action against PDI reflects the distribution of the particle size in a the larvae. However, all insecticides’ nanoemulsions formulation. The PDI is a dimensionless measure of the showed significantly high toxicity (1.5–2-fold) compared width of size distribution calculated from the cumulated to the technical and EC. This finding led to a significant analysis and ranges from zero to one [39]. A lower PDI decrease in the field application rate by half-value, value (near zero) indicates the existence of a uniform resulting in low environmental pollution and hazards. distribution of droplet size and homogenous The nanoscale form of pesticides has been applied to populations, whereas a PDI value closer to 1 (one) control the developed resistance in insect species, displays a wide range of droplet sizes (heterogeneity of attributed to conventional pesticides’ excessive use. the system). The PDI value around 0.2 indicates the Compared to the traditional pesticides, the higher droplet population’s homogeneity in prepared efficacy of nano pesticides was observed. In agreement formulations. Besides other important criteria, zeta with our results, other studies proved that pyrethroids’ potential is another essential characteristic of the preparation in nanoemulsion form made them more nanoemulsions and an indicator of the nanoemulsion active than the conventional forms [9, 14]. Mishra et al. stability associated with the droplets’ surface potential. [13] prepared nano-sized permethrin in its colloidal state The negative values are necessary for droplet-droplet re- and studied its effect on C. tritaeniorhynchus larvae. pulsion and thus enhanced nanoemulsion stability [40]. They found that the LC of the bulk permethrin was The high stability of formulations with zeta potential 442 μg/L. In contrast, the LC of the nano-permethrin values is associated with repulsive forces that exceed was 57 μg/L. The present study also supports nano pes- attracting van der Waals forces, resulting in dispersed ticides’ ability to control mosquito vectors. Reducing particles and a deflocculated system. The range of pH nanoemulsions and elevating their surface area could fa- value of nanoemulsion has a strong effect on its stability. cilitate their passive penetration into the target pest, thus The different pH value levels lead to a change in the enhancing their toxicity [13]. As the results presented, globules’ surface charge and their stability during stor- alpha-cypermethrin, deltamethrin, and lambda- age. Keeping different nanoemulsions under environ- cyhalothrin were the most toxic insecticides (LC mental storage conditions may be an essential criterion ranged from 10 to 43 μg/L) compared to the permethrin for judging effectiveness, potency, and stability [19]. (LC ranged from 127 to 322 μg/L) against C. pipiens larvae. This finding refers to the pyrethroid type’s chem- 4.3 Release studies of pyrethroid nanoemulsions ical structure that the alpha-cypermethrin, deltamethrin, The efficiency of nanoformulation to extend residence and lambda-cyhalothrin are cyano-derivatives. However, time, reduce insecticide losses, and reduce overuse. It permethrin is a non-cyano-derivative. As well-known also makes the pesticide’s continuous and stable release from the literature, cyano-derivatives of the pyrethroids possible [8]. Our results agree with the results obtained were more active against different pests than the non- by Nguyen et al. [41], who proved that the release rate cyano derivatives [42]. of deltamethrin nanoemulsion was lower than 20% in the first 3 h of the experiment. In addition, it confirmed 4.5 Biochemical studies that lambda-cyhalothrin /polyurethane nanoemulsion To elucidate some biochemical actions of the tested had a slower release rate than the traditional formula- pyrethroids on C. pipiens larvae, the effect of the LC tions. In addition, the release profile of the lambda- values on the ATPase, CaE, and GST isolated from the cyhalothrin-loaded nanoemulsion was compared to its survived treated larvae after 24 h was examined. In EC and WP formulations at 25 °C [8]. The results re- agreement with our findings, Kakko et al. [43] proved ported that the lambda-cyhalothrin released from EC that cypermethrin was the most toxic against ATPase, and WP was very fast and reached equilibrium after 48 followed by permethrin and natural pyrethrin. The cell Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 16 of 19 toxicity was dependent on the chemical structure of structures. Kumar et al. illustrated the molecular interac- pyrethroids. The pyrethroids without the α-cyano group tions of some pyrethroids including cypermethrin to- show the weakest physiological effect. Clark and Matsu- wards adaptive immune cell receptors of T (CD4 and + ++ mura [44] measured Na -Ca ATP hydrolysis and Ca- CD8) and B (CD28 and CD45) [51]. They found that Mg ATP hydrolysis in cockroach brain tissue under fenvalerate (− 5.534 kcal/mol: CD8), fluvalinate (− 4.644 in vitro conditions. They found that the non-cyano- and − 4.431 kcal/mol: CD4 and CD45), and cyperme- + ++ containing pyrethroids inhibited Na -Ca -ATP hydroly- thrin (− 3.535 kcal/mol: CD28). Data exhibited less sis mostly than their cyano-containing counterparts. The docking energy or more affinity for B cell and T cell im- ++ ++ reverse is true for pyrethroid action on Ca -Mg -ATP mune receptors, which may later result in immunosup- hydrolysis. pressive and hypersensitivity reactions. Markus et al also As well known, CaEs hydrolyze numerous endogenous elucidated the inhibitory activity of deltamethrin against and exogenous ester-containing compounds. Therefore, human GST [49]. They found that deltamethrin appears they play a vital role in the detoxification of pyrethroids, to fit well in an eccentric cavity located at the GST ho- strongly related to the resistance phenomenon. Identifi- modimer, likely causing conformational changes at the cation of CaE genes associated with pyrethroid resist- enzyme’s substrate binding sites such that the enzyme is ance was investigated in the malaria vector Anopheles no longer able to effectively convert GSH and CDNB. sinensis [45] and the mosquito Aedes aegypti [46]. In disagreement with our results, Kostaropoulos et al. 4.7 Biosafety evaluation against the freshwater green [47] proved that the pyrethroids bind with the active site alga of GST, resulting in a significant decrease of its activity Treating the aquatic environment with nanomaterials to towards CDNB in a competitive manner, but was not control mosquito larvae or other pests may lead to conjugated with GSH. Grant and Matsumura [48] found important risks for non-target aquatic organisms [52]. a variation in the action and level of GST due to its Both physicochemical and toxicological properties of interaction with pyrethroids, studied GST as an nanomaterials would permit and control environmental antioxidant defense agent confer pyrethroid resistance in risk assessment and safety of these materials [53]. Micro- Nilaparvata lugens and demonstrated that lambda- algae are widely used in bioassay toxicity testing of cyhalothrin and permethrin induced oxidative stress and aquatic pollutants since they are sensitive organisms lipid peroxidation in insects. For these reasons, they hy- with a high capacity of bioaccumulation due to their pothesized that the prominent role of elevated GSTs in high surface of contact [54]. conferring resistance in N. lugens is through protecting A concentration-response ratio established for R. sub- tissues from oxidative damage. Markus et al. also eluci- capitata and 96 h EC values are shown in Table 7. dated the inhibitory activity of deltamethrin against hu- Considering the values obtained for EC , it was ob- man GST [49]. They proved that deltamethrin was a served that this organism was more sensitive and highly potent inhibitor of GST-P1-1, and it inhibited the homo- affected by the commercial form (EC) of all tested insec- dimeric enzyme in a non-competitive manner. Thus, the ticides after acute exposure, followed by technical form purpose of determining ATPase, CaE, and GST levels as and/or nanoformulations. It is worthy to mention that essential parameters to study the toxic effect of nano- the synthesized nanoformulation are readily soluble in pesticides on insect vector species. water with no agglomeration and proved to be safe to algae and aquatic organisms when tested as alpha- 4.6 Docking studies cypermethrin nanoemulsion and less toxic (2–17-fold) It is well known that molecular docking is a method to than the commercial EC in case of lambda-cyhalothrin, predict and understand molecular recognition, find the deltamethrin and permethrin nanoemulsions. predominant binding mode and binding affinity between Similar results were obtained by Grillo et al. [55] who the protein and ligand, and give a three-dimensional stated that paraquat-loaded chitosan nanoparticles structural explanation of the protein-ligand interaction. showed less toxicity than paraquat (96 h EC s were 1.15 The bond interactions were useful for elucidation of sev- and 0.48 mg/L; respectively). Also, other ecotoxicity eral biological activities of tested compounds as larvi- studies demonstrated that thiamethoxam nanoparticles cides [50]. Zeng et al. studied the interactions of pepsin were less toxic than commercial formulations for R. sub- with deltamethrin and cyhalothrin by multi- capitata and non-toxic for A. salina under the condi- spectroscopic approaches and molecular docking [50]. tions of the study. Based on the existing knowledge, the They approved that the tested pyrethroids bounded dir- method of green synthesis of nanoparticles and several ectly into the enzyme cavity site. The binding was influ- green-fabricated metal nanoparticles failed to show tox- enced by the active site’s microenvironment resulting in icity against different aquatic organisms. Plumeria the extension of peptide strands with loss of α-helix rubra-and Pergularia daemia-synthesized Ag Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 17 of 19 nanoparticles did not exhibit any evident toxicity against different toxicity parameters establishes the non-toxic fishes after 48 h of exposure to concentrations corre- behavior of insecticide concentrations applied against sponding to the LC and LC values on IV instar larvae non-target species. This confirms environmental safety 50 90 of Ae. aegypti and An. stephensi [56]. Subarani et al. [57] with strong efficacy as a mosquitocidal agent against lar- also did not report the toxicity effects of Catharanthus vae. Also, the data proved the greatest effect of the roseus-synthesized Ag nanoparticles against fish and nanoemulsions as alternatives to the conventional mosquito predators; G. affinis after 72 h of exposure. pesticide formulations. As related to NOEC values among tested insecticides Abbreviations (Table 7), it represents the highest test concentration at ANOVA: Analysis of variance; a.i: Active ingredient; ATP: Adenosine which no toxic effects are observed, and went parallel to triphosphate; ATPase: Adenosine triphosphatase; BSA: Bovine serum albumin; C. pipiens: Culex pipiens; CaE: Carboxylesterase; CDNB: 1-Chloro-2,4- the EC pattern recorded in the current study. dinitrobenzene; DLS: Dynamic light scattering; DMSO: Dimethylsulfoxide; However, NOEC can be regarded as a chronic endpoint, EC: Emulsifiable concentrate; EDTA: Ethylenediaminetetraacetic acid; GSH: L- and values indicated in this study reflect the glutathione; GST: Glutathione-S-transferase; LC : Median lethal concentration; MOE: Molecular Operating Environment; O/W: Oil/water; concentrations that can offer minimum protection to PDB: Protein data bank; PDI: Polydispersity index; SE: Standard error; the test organism; R. subcapitata against tested SPSS: Statistical Package for the Social Sciences; TCA: Trichloroacetic acid; insecticides particularly on a long-term basis. Further- TEM: Transmission electron microscopy; WHO: World Health Organization; β- NAD: β-Nicotinamide adenine dinucleotide more, classification of tested insecticides according to their potential hazard statements to the aquatic ecosys- 6 Supplementary Information tem, an only commercial form of deltamethrin can be The online version contains supplementary material available at https://doi. considered highly hazardous to R. subcapitata (category org/10.1186/s42506-021-00082-1. acute I; H400) and is not recommended for application in waterways, whereas its nanoformulation exhibited a Additional file 1: SUPPLEMENTARY MATERIALS (DATA IN BRIEF). Table S1. Chemical structure and physicochemical properties of the tested less hazardous effect on the test alga. Additionally, all pyrethroids. Description of data: This table shows the chemical structure the tested nanoformulations showed only harmful effects and physicochemical properties of the tested compounds. The tested to aquatic life (category acute III; H402) compared with compounds' molecular weight was 416.3, 505.21, 449.85, and 505 g/mol for alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, and permeth- very toxic or toxic hazardous effects (category I or II; H rin, respectively. The polar surface area (PSA) of all tested pyrethroids was 400 or 401) of commercial forms of the same insecti- 59.32, except permethrin was 35.53,. wWhile the hydrophobicity factor cides to aquatic life. (ALogP) of all tested compounds was around 6. There are no hydrogen bond donors (HBD) in the tested pyrethroids, while the number of hydro- It can be concluded that, for safety purposes, gen bond acceptors (HBA) ranged from 3 to 7. Table S2. HPLC gradient nanopesticides can be recommended for use in vector solvent system for separation of alpha-cypermethrin, deltamethrin, control programs in waterways and can be considered lambda-cyhalothrin and permethrin. Description of data: This table shows the HPLC conditions used for the separation of pyrethroids understudy. highly promising for the development of safe insecticides These conditions include the gradient solvent system and the optimum against mosquitoes. The nanopesticides are less harmful wavelength used in the separation process. Table S3. Experimental fac- to the environment and more efficient (in terms of cost torial design for preparation and optimization of deltamethrin nanoemul- sions. Description of data: This table shows the different experimental and performance) than the existing formulations. setup using Minitab software was used to determine the influence of six Nevertheless, only further research will show whether independent variables on the pyrethroid nanoemulsions' characterization the research results can find their way to application in (dependent variable). In these optimization experiments, deltamethrin was selected as a model of the tested pyrethroids. Table S4. The ob- practice. served visual stability, droplet size, polydispersity index (PDI), zeta poten- tial, dynamic (absolute) viscosity, and pH of prepared deltamethrin 5 Conclusion nanoemulsions. Description of data: This table presents the quantitative results of nanoemulsion pyrethroids include the droplet size (nm), PDI, Permethrin from type I (non-cyano) and three pH, and viscosity (mPa.s). The data proved that there are significant differ- pyrethroids from type II (alpha-cypermethrin, ences in the droplet size of the nine prepared deltamethrin formulations. deltamethrin, and lambda-cyhalothrin) were prepared in In the PDI case, there are no significant differences between the formula- tions (0.516-0.964) except formulation 2 (PDI = 0.158). There is no signifi- nanoemulsions. The modification of these compounds cant difference between the viscosity values of formulations 1, 2, 5, and to nanoform increased the insecticidal properties. 0.5% 6. However, formulations 4, 8, and 9 showed significant differences in a.i, 44% DMSO, 15% tween 80, 40.5% water, 9 cycle/s of their viscosity values. The pH of the prepared formulations was in the range of 7.78-8.18. Figure S1. The visual appearance of prepared delta- sonication pulses, 75% power for 15 min were selected methrin nanoemulsions. The code number represents the experimental as the optimal conditions for preparation of the insecti- factorial design shown in Table 2. Description of data: This figure presents cide nanoemulsions. The remarkable stable behavior of the visual appearance of the nine produced deltamethrin nanoemulsions formulations. Figure S2. Zeta potential distribution graph of pyrethroid prepared nanopesticides with adequate larvicidal activity nanoemulsions of alpha-cypermethrin (A), deltamethrin (B), lambda- at the lowest exposure concentration makes it a suitable cyhalothrin (C), and permethrin (D). Description of data: This figure pre- and effective mosquito control agent. In addition, the sents a zeta potential distribution graph of prepared pyrethroid nanoe- mulsions. Figure S3. The visual appearance of pyrethroid nanoemulsions evaluation of the biosafety of nanoscale pesticides of alpha-cypermethrin (1), deltamethrin (2), lambda-cyhalothrin (3), and against freshwater alga R. subcapitata by calculating Taktak et al. Journal of the Egyptian Public Health Association (2021) 96:21 Page 18 of 19 Received: 7 February 2021 Accepted: 2 June 2021 permethrin (4). Description of data: This figure presents the visual appear- ance of alpha-cypermethrin (1), deltamethrin (2), lambda-cyhalothrin (3), and permethrin (4) nanoemulsions. Figure S4. Docking view of the tested pyrethroids on the binding sites of ATPase (PDB ID: 4byg). Alpha- References cypermethrin (A), lambda-cyhalothrin (B), deltamethrin, (C), and permeth- 1. Vinogradova EB. Culex pipiens pipiens mosquitoes: taxonomy, distribution, rin (D). Description of data: This figure presents the docking view of the ecology, physiology, genetics, applied importance and control: Pensoft tested pyrethroids on the binding sites of ATPase (PDB ID: 4byg). Left is Publishers; 2000. the 2D interaction diagram structure and right is the complex structure 2. Scott JG, Yoshimizu MH, Kasai S. Pyrethroid resistance in Culex pipiens in stereo view (3D). Figure S5. Docking view of the tested pyrethroids mosquitoes. Pestic Biochem Physiol. 2015;120:68–76. on the binding sites of CaE (PDB ID: 5w1u). Alpha-cypermethrin (A), 3. Saxena PN, Bhushan B. Estimation of median lethal dose of commercial lambda-cyhalothrin (B), deltamethrin, (C), and permethrin (D). Description formulations of some type II pyrethroids. Jordan J Biol Sci. 2017;10(3):193–7. of data: This figure presents the docking view of the tested pyrethroids 4. Li Y, Zhou G, Zhong D, Wang X, Hemming-Schroeder E, David RE, et al. on the binding sites of CaE (PDB ID: 5w1u). Left is the 2D interaction dia- Widespread multiple insecticide resistance in the major dengue vector gram structure, and right is the complex structure in stereo view (3D). Aedes albopictus in Hainan Province. China Pest Manage Sci. 2021;77(4): Figure S6. Docking view of the tested pyrethroids on the binding sites of GST (PDB ID: 5ft3). Alpha-cypermethrin (A), lambda-cyhalothrin (B), del- 1945–53. 5. Jones RT, Ant TH, Cameron MM, Logan JG. Novel control strategies for tamethrin, (C), and permethrin (D). Description of data: This figure pre- mosquito-borne diseases: The Royal Society; 2021. sents the docking view of the tested pyrethroids on the binding sites of 6. Dzib-Florez S, Ponce-García G, Medina-Barreiro A, González-Olvera G, GST (PDB ID: 5ft3). Left is the 2D interaction diagram structure, and right is the complex structure in stereo view (3D). 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Journal

Journal of the Egyptian Public Health AssociationSpringer Journals

Published: Jul 15, 2021

Keywords: Culex pipiens; Pyrethroids; Nanoemulsion; Insecticidal activity; Biochemical studies; Molecular Docking; Ecotoxicity

References