ORIGINAL PAPER

Acaricidal, pediculocidal and larvicidal activityof synthesized ZnO nanoparticles using wetchemical route against blood feeding parasites

Arivarasan Vishnu Kirthi & Abdul Abdul Rahuman & Govindasamy Rajakumar &

Sampath Marimuthu & Thirunavukkarasu Santhoshkumar &

Chidambaram Jayaseelan & Kanayairam Velayutham

Received: 3 January 2011 /Accepted: 28 January 2011# Springer-Verlag 2011

Abstract The present study was based on assessments ofthe anti-parasitic activities to determine the efficacies ofsynthesized zinc oxide nanoparticles (ZnO NPs) preparedby wet chemical method using zinc nitrate and sodiumhydroxide as precursors and soluble starch as stabilizingagent against the larvae of cattle tick Rhipicephalus(Boophilus) microplus, Canestrini (Acari: Ixodidae); headlouse Pediculus humanus capitis, De Geer (Phthiraptera:Pediculidae); larvae of malaria vector, Anopheles subpictus,Grassi; and filariasis vector, Culex quinquefasciatus, Say(Diptera: Culicidae). R. microplus larvae were exposed tofilter paper envelopes impregnated with different ZnO NPconcentrations. Direct contact method was conducted todetermine the potential of pediculocidal activity. Parasitelarvae were exposed to varying concentrations of synthe-sized ZnO NPs for 24 h. The results suggested that themortality effects of synthesized ZnO NPs were 43% at 1 h,64% at 3 h, 78% at 6 h, and 100% after 12 h against R.microplus activity. In pediculocidal activity, the resultsshowed that the optimal times for measuring mortalityeffects of synthesized ZnO NPs were 38% at 10 min, 71%at 30 min, 83% at 1 h, and 100% after 6 h against P.humanus capitis. One hundred percent lice mortality wasobserved at 10 mg/L treated for 6 h. The mortality wasconfirmed after 24 h of observation period. The larvalmortality effects of synthesized ZnO NPs were 37%, 72%,100% and 43%, 78% and 100% at 6, 12, and 24 h against

A. subpictus and C. quinquefasciatus, respectively. It isapparent that the small size and corresponding large specificsurface area of small nanometer-scale ZnO particles imposeseveral effects that govern its parasitic action, which are sizedependent. ZnO NPs were synthesized by wet chemicalprocess, and it was characterized with the UV showing peak at361 nm. X-ray diffraction (XRD) spectra clearly shows thatthe diffraction peaks in the pattern indexed as the zinc oxidewith lattice constants a=3.249 and c=5.206 Å. The FTIRspectrum showed the range of 400–4,000 cm−1. The band at899.56 cm−1; 1,151.87 cm−1; 1,396 cm−1; and these bandsshowed the complete composition of ZnO NPs. SEMmicrograph showed 60–120-nm size and aggregates ofspherical shape nanoparticles. EDX showed the completechemical composition of the synthesized nanoparticles ofzinc oxide. The maximum efficacy was observed in zincoxide against the R. microplus, P. humanus capitis, and thelarvae of A. subpictus, C. quinquefasciatus with LC50 valuesof 29.14, 11.80, 11.14, and 12.39 mg/L; r2=0.805, 0.876,0.894, and 0.904, respectively. The synthesized ZnO NPsshowed the LC50 and r2 values against the R. microplus(13.41 mg/L; 0.982), P. humanus capitis (11.80 mg/L;0.966), and the larvae of A. subpictus (3.19; 0.945 mg/L),against C. quinquefasciatus (4.87 mg/L; 0.970), respectively.The control (distilled water) showed nil mortality in theconcurrent assay. This is the first report on anti-parasiticactivity of the synthesized ZnO NPs.

Introduction

Rhipicephalus (Boophilus) microplus, known as the cattletick, generally only parasitizes bovines; however, when thetick population reaches high levels of pasture infestations

A. V. Kirthi :A. A. Rahuman (*) :G. Rajakumar :S. Marimuthu : T. Santhoshkumar : C. Jayaseelan :K. VelayuthamUnit of Nanotechnology and Bioactive Natural Products,Post Graduate and Research Department of Zoology,C. Abdul Hakeem College,Melvisharam-632 509,Vellore District, Tamil Nadu, Indiae-mail: abdulrahuman6@hotmail.com

Parasitol ResDOI 10.1007/s00436-011-2277-8

its parasitism may extend to other mammal species.Economic losses caused by ectoparasites in Brazil areestimated to be approximately 2.65 billion dollars a year, inwhich R. microplus is responsible for more than 75% of thelosses (Grisi et al. 2002). R. microplus is responsible forlosses in milk, meat, and leather production and for the deathof a number of animals, which results in economic lossesassociated with cattle production. A recent survey onacaricide resistance conducted through questionnairereported a large-scale acaricide resistance in India (FAO2004). Continuous and indiscriminate use of acaricides leadsto the selection of chemical-resistant ticks along withcontamination of the environment and animal products (Grafet al. 2004). The cattle tick is a vector of several disease-causing pathogens such as Babesia bovis, B. bigemina, andAnaplasma marginale (Rosario-Cruz et al. 2005). Chemicalacaricides have played a major role in controlling R.microplus in Mexico; however, their intensive use has ledto the development of resistant tick populations withinMexico, and recently this resistance has spread beyond thenorthern border of Mexico (Rosario-Cruz et al. 2005;Rodriguez-Vivas et al. 2006a, b; http://www.tahc.state.tx.us).

Pediculus humanus capitis, head louse, is an obligateectoparasite which is found exclusively on humans. Theselice have evolved with mankind and, thus, were distributedall over the world (Aspöck and Walochnik 2007; Burgess2004; Mehlhorn and Mehlhorn 2009). Head lice areectoparasites and its infestation due to unhygienic con-ditions has negatively affected the society for decades.Infestations are prevalent worldwide and especially com-mon among school children in both developed anddeveloping countries (Gratz 1997). The control of humanhead lice worldwide depends primarily on the continuedapplications of organochlorine (dichlorodiphenyltrichloro-ethane (DDT) and lindane), organophosphorus (malathion),carbamate (carbaryl), pyrethrin, pyrethroid (permethrinand δ-phenothrin), and avermectin (ivermectin originatedfrom Streptomyces avermitilis) insecticides (Gratz 1997;Dolianitis and Sinclair 2002). Commercially available topicalcream known as Licatack® smoothens the hair, is skin-safe,and smells good; it offers a very efficient and positivealternative to toxic, gluing, flammable, or skin-irritatingproducts found on the market of anti-louse products (Abdel-Ghaffar et al. 2010a). Infestations are prevalent worldwideand especially common among school children in bothdeveloped and developing countries (Gratz 1997). Abdel-Ghaffar et al. (2010b) reported that the product Licatack®proved its efficacy on larvae and adult head lice after itsefficacy was shown in intense in vitro screening tests. Headlouse infestation may result in social embarrassment wheninfested children and their families become mobbed as“dirty” or “antisocial” (Mehlhorn et al. 1995; Mehlhornand Mehlhorn 2009; Toloza et al. 2010b).

Mosquitoes are the most important single group ofinsects in terms of public health importance, which transmita number of diseases, such as malaria, filariasis, dengue,Japanese encephalitis, etc., causing millions of deaths everyyear (Das et al. 2007). Anopheles is an important vector forthe transmission of malaria (Gutiérrez et al. 2008) andCulex is known for transmission of filariasis in human andlumbar paralysis in cattle (Kwong-Chung et al. 2004). A.subpictus is the most abundant anopheline in most parts ofthe Indian subcontinent (Rao 1984) and recognized as aprimary or secondary vector of malaria, a disease of greatsocioeconomic importance in different parts of the world(Panicker et al. 1981; Kulkarni 1983; Chatterjee andChandra 2000). C. quinquefasciatus has been recordedround the year in different parts of the country (Chand et al.1988). A careful and prolonged control of the vector caneliminate filariasis, but it is not an easy task due to itsnatural tolerance and early development of resistance toavailable insecticides (Brown and Pal 1971). India contributes about 40% of the total global burden and accounts forabout 50% of the people at risk of infection. Of the peopleexposed to the risk of infection, individuals with micro-filaraemia, suffering from lymphoedema and hydrocelecases in the globe, India alone accounts for 39.0%,37.9%, 46.4%, and 48.1%, respectively (Michael et al.1996). Filariasis is caused by Wuchereria bancrofti, Brugiamalayi, and Brugia timori, and it spreads by the bite of aninfected Culex mosquito. Thenmozhi et al. (2006) reportedthis species as a vector of Japanese encephalitis virus inCuddalore, an area of Tamil Nadu, India, endemic for thedisease. A. subpictus breeds in a variety of habitats likeflowing or stagnant waters, clear or turbid waters, waterwith or without vegetation, unshaded or slightly shadedwater bodies, wells, burrow pits, channels, ponds, tanksground pools, fallow and freshly flooded rice fields, cementcisterns, tree holes, lake margins, fresh or brackish waters,etc. and the adult has a flight range of 1.5–6 km (Nagpaland Sharma 1995).

Alternative control agents with novel modes of action andlow mammalian toxicity and environmental impact are badlyneeded. The harmful effects of chemicals on non-targetpopulations, ever growing resistance to chemical insecticidesalong with the recent resurgence of different mosquito-bornediseases have induced scientists to explore alternative, simple,sustainable methods of mosquito control. The eradication ofadult mosquitoes using chemical insecticides is not a prudentstrategy, as for the occurrence of adult stage alongside humanhabitation, and the adults can easily escape remedial measures. So the exploration of more effective and eco-friendlytechniques like application of cost-effective, natural that canadapt to describe habitats of common pests like R. microplus,P. humanus capitis, and the larvae of A. subpictus, C.quinquefasciatus without having no adverse effect to human

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population as well as the non-target population of theenvironment would be more promising.

This lack of efficacy is due to the emergence of resistanceby the head louse to synthetic compounds, and researchersaimed on the search of new substitutes to synthetic ingre-dients, such as synthesized zinc oxide nanoparticles (ZnONPs). ZnO nanostructured material has gained much interestowing to its wide applications for various devices such assolar cells, varistors, transducers, transparent conductingelectrodes, sensors, and catalysts, since it is an abundant andinexpensive material (Van de Pol 1990; Mayo 1996). Highlyionic nanoparticulate metal oxides such as ZnO NPs areunique in that they can be produced with high surface areasand with unusual crystal structures (Klabunde et al. 1996).Compared to organic materials, inorganic materials such asZnO possess superior durability, greater selectivity, and heatresistance (Padmavathy and Vijayaraghavan 2008). More-over, zinc is a mineral element essential to human health andZnO is a form in the daily supplement for zinc. ZnO NPsalso have good biocompatibility to human cells (Padmavathyand Vijayaraghavan 2008). The bulk ZnO powders hasdemonstrated antibacterial and antifungal activity(Yamamoto 2001; Sawai and Yoshikawa 2004). In agricul-ture, zinc compounds are mainly used as fungicides (Waxman1998). Recent studies have shown that NP of some materials,including metal oxides, can induce cell death in eukaryoticcells (Everts et al. 2006; Nel et al. 2006; Gupta and Gupta2005; Magrez et al. 2006) and growth inhibition inprokaryotic cells (Brayner et al. 2006; Thill et al. 2006)due to its cytotoxicity. ZnO was among the most widely usedNPs since they have applications in a large variety of sectorsranging from personal care products to coatings and catalystsin environmental remediation (Choopun et al. 2009; Kamatand Meisel 2003; Wang 2004). Manzo et al. (2010) havereported that ZnO nanoparticles exert toxic and genotoxiceffects upon terrestrial organisms like plants (Lepidiumsativum, Vicia faba), crustaceans (Heterocyipris incon-gruens), and insects (Folsomia candida).

Certain novel properties of NPs could lead to adversebiological effects, with the potential to create toxicity(Oberdörster et al. 2005). The forecasted huge increase inthe manufacture and use of NPs makes it likely thatincreasing human and environmental exposure to NPs willoccur (Nowack and Bucheli 2007). A lot of toxicity data forsoil invertebrates exposed to zinc salts became available(Lock and Janssen 2001). Beyer and Anderson (1985)assessed the toxicity of zinc oxide to the woodlice Porcellioscaber and they found that soil litter spiked with 1,600 mgof Zn per kilogram dry weight of soil litter causedsignificant negative effects. The toxicity study for zincoxide to the earthworm Eisenia fetida in soil showed DNAdamage to earthworm, activity of cellulase, and damage tomitochondria of gut cells were investigated after acute

toxicity test (Hu et al. 2010). Rekha et al. (2010) reportedthat the toxicity of nano-sized ZnO and Mn-doped ZnOwere investigated using both Gram-positive and Gram-negative bacteria. Heng et al. 2010 demonstrated that initialexposure of BEAS-2B cells to oxidative stress sensitizedtheir subsequent response to cytotoxic challenge with ZnOnanoparticles.

However, detailed studies to investigate the toxicitythreshold of identical nanoparticles to different biologicalsystems and to obtain a fundamental understanding of thefactors controlling the interaction of nanostructures withbiological systems and their mode of toxicity are necessaryto aid in the design of better and safer materials and moreefficient biomedical applications. Unlike insoluble nano-particles such as nano-TiO2 and nano-SiO2, the solubility ofnano ZnO may play a more important role in its toxicity.However, inconsistent results on the solubility of nano ZnOin water were presented in the literatures (Franklin et al.2007; Lin and Xing 2007; Wang et al. 2008). Two differentmechanisms compete to control the zinc NPs toxic action:(1) a chemical effect based on the chemical composition, e.g., release of (toxic) ions; and (2) stress or stimuli causedby the surface, size, and/or shape of the nanoparticle itself(Brunner et al. 2006). These stimuli can be either due to amechanical hindrance to biological functions or to adifferent interaction of the chemical compound in thenanostructured form with the biological environment.

In the present study, we reported the zinc oxide NPswould be useful in promoting research aiming at thedevelopment of new agent for acaricidal, pediculocidal,and mosquito larvicidal activity.

Materials and methods

Materials

The chemicals zinc nitrate, sodium hydroxide, and starchwere purchased from Sigma Company and the solutionswere prepared by using double distilled water.

Rhipicephalus microplus collection and bioassay test

The newly attached larvae of R. microplus (Canestrini)(Acari: Ixodidae) were collected from the softer skin insidethe thigh, flanks, abdomen, brisket, and forelegs ofnaturally infested cattle. R. microplus larvae have a short,straight capitulum and a brown to cream body. The parasiteswere identified in the Department of Veterinary Parasitology,Madras Veterinary College, Tamil Nadu Veterinary andAnimal Sciences University, Chennai, Tamil Nadu. Theapplied method in the present study to verify the acaricidalactivity of ZnO NPs against the larvae of R. microplus was

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developed as per the method of FAO (2004), incorporatingslight modifications to improve practicality and efficiency oftested materials (Fernandes 2001; Fernandes et al. 2005).From the stock solution, 20 mg/L was prepared and a series offilter paper envelopes (Whatman filter paper no.1; 125 mm indiameter) with micropores were treated with each concentra-tion of ZnO. The synthesized ZnO NPs were impregnatedwith 10 mg/L of which 3 ml solution of the stock wasuniformly distributed with a pipette on internal surfaces. Fiveenvelopes were impregnated with each tested solution. Thecontrol papers were impregnated with distilled water only.The opening of the envelopes (treated and inoculated withlarval ticks) was folded (10 mm) and re-sealed with a metallicclip, with its identification mark (tested solution and concen-tration) on the outside. The packets are placed in the BODincubator at a temperature of 28–30°C and 80–90% RH for24 h. The envelopes were opened 24 h after exposure and thenumber of live and mortality larvae were recorded (Fernandesand Freitas 2007). The experimental media, in which 100%mortality of larvae occurs alone, were selected for a dose–response bioassay.

Collection of head lice

Adults of P. humanus capitis were collected from apopulation of children between the ages of 3 and 12, withthe approval of their guardians, by raking a metal lousecomb through sections of the scalp. Adults were obtainedand pooled by carefully removing them from the metalteeth of the comb into clean plastic boxes. Once collected,the head lice were transported to our laboratory (Picolloet al. 1998, 2000). The children had not been treated withany pediculicide solution for at least the preceding month,using only the louse comb. The head lice were identified byDr. A. Sangaran, Department of Veterinary Parasitology,Madras Veterinary College, Tamil Nadu Veterinary andAnimal Sciences University, Chennai, Tamil Nadu.

Pediculocidal activity

The synthesized ZnO NPs solutions were diluted usingdouble distilled water as a solvent according to the desiredconcentrations of 25, 20, 15, 10, and 5 mg/L and the zincoxide of 50, 25, 12.5, and 6.25 mg/L. Each test included aset of control group (distilled water) with five replicates foreach individual concentration. Each louse was carefullytransferred into a glass dish and 0.02 ml of the synthesizedZnO NPs was applied directly on the dorsal part of thelouse using a 1-ml micropipette. For ZnO, 0.02 ml wasapplied directly on the dorsal part of the louse. After 15 s ofcontact with the agent, the louse was transferred into a petridish lined with filter paper and observed using a hand lensuntil dead or otherwise. All the petri dishes were set aside

in a dark chamber at 26±0.5°C and 70±1% humidity. Theelapsed time was recorded for each test agent as the“knockdown” time. The death of the louse was confirmedwhen there was cessation of motility or waggling of theappendages on touching with a needle. Ten lice were usedfor each determination (Oladimeji et al. 2000).

Mosquito culture

A. subpictus and C. quinquefasciatus larvae were collectedfrom a rice field to a stagnant water area of Melvisharam(12°56′23″ N, 79°14′23″ E) and identified in the ZonalEntomological Research Centre, Vellore (12°55′48″ N, 79°7′48″ E), Tamil Nadu, to start the colony, and the larvaewere kept in plastic and enamel trays containing tap water.They were maintained and reared in the laboratory as perthe method of Kamaraj et al. (2009).

Larvicidal bioassay

During preliminary screening with the laboratory trial, thelarvae of A. subpictus and C. quinquefasciatus werecollected from the insect-rearing cage and identified in theZonal Entomological Research Centre, Vellore. For thebioassay test, mosquito larvae were taken in five batches of20 in 249 mL of water and 1.0 mL of ZnO. The control wasset up with dechlorinated tap water and ZnO. The numberof dead larvae was counted after 24 h of exposure, and thepercentage of mortality was reported from the average offive replicates. The experimental media in which 100%mortality of larvae occurs alone were selected for dose–response bioassay.

A synthesized ZnO NPs toxicity test was performedby placing 20 mosquito larvae into 200 mL of sterilizeddouble distilled water with nanoparticles into a 250-mLbeaker (Borosil). The nanoparticle solutions were dilutedusing double distilled water as a solvent according to thedesired concentrations (4.0, 2.0, 1.0, and 0.5 mg/L).Each test included a set of control group (zinc oxide anddistilled water) with five replicates for each individualconcentration. Mortality was assessed after 24 h todetermine the acute toxicities on fourth instar larvae ofA. subpictus and C. quinquefasciatus. In order to comparethe mortality of ZnO NPs to that of dissolved H2O2

released and the mosquito larvae were exposed to a rangeof dissolved H2O2 concentrations so as to cover the rangereleased from all doses of ZnO NPs. To avoid settling ofparticles especially at higher doses, all treatment solutionswere sonicated for an additional 5 min prior to addition ofthe mosquito larvae. Since this additional sonicationappeared to significantly decrease the settling of particles,we tested the effects of ZnO NPs without sonication(stirred only) or with sonication.

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Dose–response bioassay

Based on the preliminary screening results, zinc oxide andsynthesized ZnO NPs were subjected to dose–responsebioassay for larvicidal activity against the larvae of A.subpictus and C. quinquefasciatus. Different concentrationsranging from 25 to 3.12 mg/L (pure zinc oxide) and 4.0 to0. 5 mg/L (for synthesized ZnO NPs) were prepared forlarvicidal activity of parasites. The number of dead larvaewas counted after 24 h of exposure, and the percentage ofmortality was reported from the average of five replicates.However, at the end of 24 h, the selected test samplesturned out to be equal in their toxic potential.

Synthesis of ZnO nanoparticles

The ZnO NPs were prepared by wet chemical methodusing zinc nitrate and sodium hydroxide as precursorsand soluble starch as stabilizing agent. Soluble starch(0.1%) was dissolved in 500 mL of distilled water byusing a microwave oven. Zinc nitrate, 14.874 g(0.1 mol), was mixed with starch solution. The solutionwas kept under constant stirring using a magnetic stirrerto completely dissolve the zinc nitrate. After completedissolution of the zinc nitrate, 0.2 mol of sodiumhydroxide solution was added under constant stirring,drop by drop touching the walls of the vessel. Thereaction was allowed to proceed for 2 h after completeaddition of sodium hydroxide. After the completion ofreaction, the solution was allowed to settle overnight,and the supernatant solution was then discarded carefully. The remaining solution was centrifuged at 10,000 gfor 10 min and the supernatant was discarded. Thus,obtained nanoparticles were washed three times usingdistilled water. Washing was carried out to remove theby-products and the excessive starch that were boundwith the nanoparticles. After washing, the nanoparticleswere dried at 80°C overnight. During drying, completeconversion of Zn(OH)2 into ZnO takes place (Yadav et al.2006).

Characterization of ZnO nanoparticles

The bioreduction of the ZnO NPs solutions was monitored byperiodic sampling of aliquots (1 mL) of the aqueouscomponent after 20 times dilution and measuring the UV–Vis spectra of the solution. UV–visible spectroscopy of thesealiquots were monitored as a function of time of reaction on aSchimadzu 1601 spectrophotometer in 200–700-nm rangeoperated at a resolution of 1 nm. Further, the reaction mixturewas subjected to centrifugation at 60,000 rpm for 40 min; theresulting pellet was dissolved in de-ionized water and filteredthrough Millipore filter (0.45 μm). The synthesized nano-

particles were identified by XRD spectroscopy (Perkin-ElmerSpectrum One instrument, PW 1830 instrument operating at avoltage of 40 kV and a current of 30 mA with Cu Kαradiation). Fourier transform infrared (FTIR) spectra of thesamples were measured using Perkin Elmer Spectrum Oneinstrument in the diffuse reflectance mode at a resolution of4 cm−1 in KBr pellets. Powder samples for the FTIR wereprepared similar to powder diffraction measurements. TheFTIR spectra of synthesized ZnO NPs taken were analyzed,which discussed for the possible functional groups for theformation of nanoparticles. For the scanning electronmicroscopic studies, 25 μL of sample was sputter-coatedon copper stub, and the images of nanoparticles were studiedusing scanning electron microscopy (SEM; JEOL, ModelJFC-1600).

Data analysis

Mean percent larval mortality data were subjected toanalysis of variance and compared with Duncan’s multiplerange tests to determine any differences between ZnO NPsand within species and concentration (SPSS 2007). Prior toanalysis, mortality in treatments was corrected for controlsusing the formula of Abbott (1925). LC50 and theirassociated confidence intervals were estimated from24-h concentration mortality data using probit analysis(Finney 1971). Lethal concentrations at 50% and slopelevels were considered significantly different if theirassociated confidence intervals did not overlap. All differences were considered significant if P≤0.05.

Results

In the present study, parasite larvae were exposed tovarying concentrations of synthesized ZnO NPs for 24 h.The results suggested that the mortality effects ofsynthesized ZnO NPs were 43% at 1 h, 64% at 3 hand 78% at 6 h, and 100% after 12 h against R.microplus activity. In pediculocidal activity, the resultsshowed that the optimal times for measuring mortalityeffects of synthesized ZnO NPs were 38% at 10 min, 71%at 30 min, 83% at 1 h, and 100% after 6 h against P.humanus capitis. One hundred percent lice mortality wasobserved at 10 mg/L treated for 6 h. The mortality wasconfirmed after 24 h of observation period. The larvalmortality effects of synthesized ZnO NPs were 37%, 72%,100% and 43, 78, and 100 at 6, 12, and 24 h against A.subpictus and C. quinquefasciatus, respectively. Anti-liceactivity of zinc oxide and ZnO NPs showed the 29.14,13.41 mg/L; r2=0.805 and 0.982, respectively. Thepediculocidal activity results showed that the highestmortality in ZnO NPs than zinc oxide. Among these, the

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present synthesized ZnO NPs against P. humanus capitis(LC50=11.80 mg/L, r2=0.966).The larvicidal zinc oxideand ZnO NPs are noted; however, the highest mortalitywas found in synthesized ZnO NPs and zinc oxide againstthe larvae of A. subpictus (LC50=11.14, 3.19 mg/L; r2=0.894and 0.945) and against the larvae of C. quinquefas-ciatus (LC50=12.39, 4.87 mg/L; r2=0.904 and 0.970),respectively. The control (distilled water) showed nilmortality in the concurrent assay. The chi-square valuewas significant at p≤0.05 level. The complete mortality

was observed for synthesized ZnO NPs for R. microplusand P. humanus capitis at 10 mg/L, the larvae of A.subpictus and C. quinquefasciatus at 4 mg/L (Table 1).

UV–visible spectrum taken for ZnO NPs synthesizedshows peak absorption at 361 nm (Fig. 1). Figure 2shows the X-ray diffraction pattern of the grown ZnO NPsprepared using the above conditions. The spectra clearlyshows the diffraction peaks in the pattern indexed as thezinc oxide with lattice constants a=3.249 and c=5.206 Åand well matched with the available joint committee on

Table 1 Parasitic activity of zinc oxide and synthesized zinc oxide nanoparticles against the larvae of Rhipicephalus microplus, adult ofPediculus humanus capitis, and larvae of Anopheles subpictus, Culex quinquefasciatus

Test sample Species Concentrations(mg/L)

Percent mortalitya

(mg/L)±SDLC50 (mg/L) UCL–LCL

(mg/L)Slope r2

Zinc oxide R. microplus 50.0 80±0.987

25.0 74±1.022

12.5 39±1.880 29.14 24.21–35.08 39 0.805

6.25 28±1.002

P. humanus capitis 50.0 78±0.810

25.0 66±0.868

12.5 36±0.594 33.88 27.89–41.17 36 0.876

6.25 24±1.262

A. subpictus 25.0 91±0.825

12.5 78±1.242

6.25 49±0.922 11.14 8.18–15.17 49 0.894

3.12 39±1.025

C. quinquefasciatus 25.0 96±0.825

12.5 63±1.242

6.25 56±0.922 12.39 10.18–15.07 63 0.904

3.12 26±1.025

Synthesized ZnO NPs R. microplus 10.0 100±0.00

5.0 86±1.29

12.5 56±1.59 13.41 12.22–14.71 56 0.982

6.25 31±0.84

P. humanus capitis 10.0 100±0.00

5.0 91±1.509

12.5 63±0.914 11.80 10.37–13.44 63 0.966

6.25 41±1.238

A. subpictus 4.0 100±0.00

2.0 84±1.796

1.0 72±1.231 3.19 3.69–2.77 84 0.945

0.5 38±1.185

C. quinquefasciatus 4.0 100±0.00

2.0 85±1.796

1.0 49±1.231 4.87 4.04–5.17 85 0.970

0.5 25±1.185

Control (distilled water), nil mortality

LC50 lethal concentration that kills 50% of the exposed larvae, UCL upper confidence limit, LCL lower confidence limit, r2 regression coefficient

*P<0.05, significant levelaMean value of five replicates

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powder diffraction standards (JCPDS 36–1451). The broadreflection at 15° is due to the low crystallinity of the solublestarch. The functional or composition quality of the synthe-sized product was analyzed by the FTIR spectroscopy. TheFTIR spectrum showed the range of 400–4,000 cm−1 (Fig. 3).The band at 899.56 cm−1; 1,151.87 cm−1; 1,396 cm−1; andthese bands showed that complete composition of ZnO NPs.The surface appearance of the ZnO NPs with spherical andaggregates were formed depicting a scanning electronmicrograph (Figs. 4a, b). Further observations revealed that

the ZnO NPs were in the nanoscale range of 60–120 nm.The energy dispersive X-ray (EDX) spectroscopy of the ZnONPs depicting the chemical components present in thesample was identified to be zinc oxide (Fig. 4c).

Discussion

Martinez-Velazquez et al. (2010) have reported that therepellency activity of the Ocimum basilicum essential oilagainst R. microplus would be important, since the majorcomponents were linalool (30.61%), estragole (20.04%),α-farnesene (6.96%), eugenol (6.61%), and 1,8-cineole(6.2%). Fifty percent hydroethanolic extracts of Bonning-hausenia albiflora whole plant, Calotropis procera root,Citrus maxima flower, Acorus calamus rhizome, andWeidelia chinensis whole plant showed acaricidal efficacyranging from 4% to 35% within 24 h of application on R.(B.) microplus, rhizome extract of A. calamus revealed that79.31% of correlation with log concentration in probitmortality could be assigned to the concentration of theextract, and the regression line of the extract showed theLC85 as 11.26% (Ghosh et al. 2010). In anti-lice activity ofaqueous crude leaf extracts, silver, and synthesized silvernanoparticles of Mimosa pudica, the highest mortality wasfound in synthesized silver nanoparticles against R. micro-plus (LC50=8.89, 11.82, and 0.69 ppm), respectively(Marimuthu et al. 2010).

Fig. 1 UV–visible spectroscopy of synthesized zinc oxide nano-particles at different time intervals. Wavelength peak was observed at361 nm

Fig. 2 XRD pattern of synthesized zinc oxide nanoparticles

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The LD50 values of DDT obtained for each permethrin-resistant head louse population was 0.0354 μg per insectagainst P. humanus. The values obtained demonstrated thatthere is a DDT resistance phenomenon in the pyrethroid-resistant populations of head lice (Barrios et al. 2010). InPongamia pinnata leaf extracts, a filter paper diffusionmethod was conducted for determining the potentialpediculocidal activity of petroleum ether extracts possessexcellent anti-lice activity with values ranging between50.3% and 100%, whereas chloroform and methanolextracts showed moderate pediculocidal effects (Sunilsonet al. 2009). Significant differences in fumigant activityagainst head lice were found among the essential oils fromthe native and exotic plant species and the most effectiveessential oils were Cinnamomum porphyrium, followed byAloysia citriodora (chemotype 2), and Myrcianthes pseu-domato, with KT50 values of 1.12, 3.02 and 4.09,respectively (Toloza et al. 2010a). From extract and oilfrom the fruits of Melia azedarach, treatments on adult licewith solutions of extract, oil, and combinations of the twodisplayed significantly higher mortality values, (rangingfrom 62.9% to 96.5%, than the respective control, whichshowed 21.9% mortality (F=15.31; df=12, 74; P≤0001)(Carpinella et al. 2007).

The larvicidal effect of aqueous crude leaf extracts, silver,and synthesized silver nanoparticles ofM. pudica showed thatthe highest mortality was found in synthesized AgNPsagainst the larvae of A. subpictus (LC50=8.89, 11.82, and

0.69 ppm) and against the larvae of C. quinquefasciatus(LC50=9.51, 13.65, and 1.10 ppm) (Marimuthu et al. 2010).The highest mortality was found in methanol, aqueous, andsynthesized AgNPs, which used Nelumbo nucifera plantextract against the larvae of A. subpictus (LC50=8.89, 11.82,and 0.69 ppm; LC90=28.65, 36.06, and 2.15 ppm) andagainst the larvae of C. quinquefasciatus (LC50=9.51, 13.65,and 1.10 ppm; LC90=28.13, 35.83, and 3.59 ppm), respec-tively (Santhoshkumar et al. 2010).

ZnO NPs have some excellent properties like exceptional mechanical strength, antistatic, antibacterial, and UVabsorption properties (Thuenemann and Ruland 2000). TheUV sharp peak may be ascribed to the monodispersed, ZnONPs while the slope line corresponds to a UV absorbancecaused by larger NP aggregates that persist in the solution(Santilli et al. 2007). XRD confirms the presence of ZnO inthe synthesized material. The presence of starch in thecompletely washed nano ZnO indicates their strong bindingnature (Yadav et al. 2006). No other peak related toimpurities were detected in the spectra within the detectionlimit of the X-ray diffraction, which further confirms thatthe synthesized powder is pure ZnO. In FTIR, the band at899.56 cm−1 was correlated to zinc oxide (Wahab et al.2007a), 1,151.87 was towards CO2, 1,396 cm−1 was relatedto C–O and C=O, respectively (Wahab et al. 2007a, b,2008). This shows the complete composition of the nano-particle which was made in the experiment. The peaksassigned to diffractions from various planes correspond to

899.

56

1025

.6111

51.8

7

1384

.15

1638

.14

3409

.65

-10

0

10

20

30

40

50

60

70

80

90

100

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig. 3 FTIR spectrum of silver nanoparticles-synthesized zinc oxide nanoparticles

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the hexagonal closely packed structure of zinc oxide. TheSEM analyses on ZnO also revealed the presence ofagglomerates of the primary nanoparticles. In order toreduce the size of the large primary particles and to breakthe nanoparticle agglomerates and the use of dispersantsdoes not enhance the size reduction (Zhang et al. 2007).Embryo toxicity test revealed that nano ZnO killed zebra-fish embryos (50 and 100 mg/L), retarded the embryo

hatching (1–25 mg/L), reduced the body length of larvae,and caused tail malformation after 96 h of exposure (Bai etal. 2009). The LC50 of nano ZnO and ZnO/bulk aqueoussuspensions on the zebrafish survival were 1.793 and1.550 mg/L, respectively after 96 h; the EC50 values on thezebrafish embryo hatching rate were 2.065 and 2.066 mg/L,respectively after exposure of 84 h (Zhu et al. 2008).Franklin et al. (2007) who reported that the EC50 values forthe same algal species were 0.063 mg Zn/L for bulk ZnOand 0.068 mg Zn/L for nano ZnO after 72 h. Moos et al.(2010) reported that nano-sized ZnO was more cytotoxicthan the micrometer-sized ZnO with LC50 values of 15±1and 29±4 μg/cm2, respectively. Berardis et al. (2010)demonstrated that the cytotoxicity, oxidative stress, apopto-sis, and proinflammatory mediator release induced by ZnOnanoparticles on human colon carcinoma LoVo cells.Prolonged exposure to ZnO NPs at 10 μg mL(−1) results indecreased mitochondrial activity, loss of normal cell mor-phology, and disturbances in cell cycle distribution (Kocbeket al. 2010). Manusadžianas et al. (2009) reported that thelethality response of aquatic organisms (macrophytic algaecells of Nitellopsis obtusa, shrimps Thamnocephalus platyu-rus, and rotifer Brachionus calyciflorus) induced by sonicat-ed and non-sonicated nano ZnO suspensions with variousparticle sizes (10 and 20–30 nm) and nano ZnO particlesshowed LC50 values of 438, 0.21, and 0.6 mg/L for 20–30 nm, respectively. The soluble zinc ions do exert toxiceffects against the feeding organisms, freshwater algaPseudokirchneriella subcapitata, and that this toxic actionmay, in turn, affect the overall toxicity against H. incon-gruens resulting in acute and chronic responses up to 21%and 34%, respectively. Heinlaan et al. (2008) reported thatthe toxicity of ZnO nanoparticles in freshwater has beenthoroughly investigated and reported for Vibrio fischeri(EC50=1.9 mg L−1), Daphnia magna (LC50 3.2 mg L−1),and Tamnocephalus platyurus (LC50=0.18 mg L−1). In mostcases, soluble zinc ions (Zn2+) from ZnO seem to be themain source for the toxicity (Aruoja et al. 2009; Heinlaan etal. 2008). For T. platyurus, the toxicity is attributed to thesoluble, dissociated Zn ions, and the LC50 values for D.magna were up to threefold lower for ZnO NPs than for bulkZnO, suggesting a clear toxic effect of the nano ZnO due tothe peculiarity of the nanometric size.

However, in order to explain the 100%mortality observed,we should hypothesize some other toxic mechanism. It hasbeen reported, indeed, that NPs may adsorb on phytoplankton(Rhee and Thomson 1992) and onto the algal cell surface(Navarro et al. 2008) or even that adhesion of NPsaggregates to the exoskeleton of crustaceans (Baun et al.2008) may cause physical effects and/or loss of mobility. It isinteresting to note that chronic zinc toxicity for F. candida(EC50) in standard artificial soil has been found at concen-trations from 487 mg kg−1 dry weight (Smit and Gestel

Fig. 4 SEMmicrographic image of synthesized zinc oxide nanoparticles.a Image magnification, ×5,000; b Image magnification, ×10,000; cEDX of the zinc oxide nanoparticles showing chemical composition

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1998) to 900 mg kg−1 dry weight (Sandifer and Hopkin1997). This study is, to the best of our knowledge, the first toreport the effect of ZnO NPs upon parasites.

No aggregation of ZnO particles was observed during theassay possibly because most of the ZnOwas dissolved at theselow concentrations. As a result of their small size, nano-particles may offer other advantages to the biomedical fieldthrough improved biocompatibility (Kim et al. 2007). This isthought to occur because metals may act on a broad range ofpesticidal targets, and many mutations would have to occurfor insects to resist their antipesticidal activity. The possiblemechanisms for the cytotoxicity of the cells were reactiveoxygen species production, dissolution, and release of toxiccations, lysosomal damage, and inflammation against ZnONPs (Nel et al. 2009). The soluble fraction of the ZnO NPs(i.e., the Zn2+ ion) the toxic actions, ZnO NPs exert a highertoxic effect in its insoluble form compared to that of thesame amount of ionic zinc. The NPs toxic action can belinked to a chemical effect and/or stress or stimuli caused bythe peculiar physical characteristics of the nano state (Manzoet al. 2010). Lice are extremely intolerant of zinc because itweakens their shell. It has been reported that adhesion of NPaggregates to the exoskeleton of parasites may causephysical effects and/or loss of mobility (Baun et al. 2008).This study is, to the best of our knowledge, the first to reportthe effect of ZnO NPs upon human parasites. Therefore,further research is needed to be carried out in this work.

Conclusion

Many efforts have been made to overcome the emergingproblem of pesticidal resistance among a variety of disease-causing pests, and advances in the field of entomology mayoffer a great opportunity of research in this field. In thispresent work, ZnO NPs were prepared by wet chemicalmethod. From the XRD analysis, it showed that Zn2+ ionswere formed without affecting the crystal structure of zinc.The EDX spectra showed the purity of the material. Fromthe above-mentioned studies, it can be concluded that ZnONPs prepared by wet chemical method may be used as apromising material in parasitical applications.

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