Parasitol Res DOI 10.1007/s00436-011-2277-8
ORIGINAL PAPER
Acaricidal, pediculocidal and larvicidal activity of synthesized ZnO nanoparticles using wet chemical 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 of the anti-parasitic activities to determine the efficacies of synthesized zinc oxide nanoparticles (ZnO NPs) prepared by wet chemical method using zinc nitrate and sodium hydroxide as precursors and soluble starch as stabilizing agent against the larvae of cattle tick Rhipicephalus (Boophilus) microplus, Canestrini (Acari: Ixodidae); head louse 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 to filter paper envelopes impregnated with different ZnO NP concentrations. Direct contact method was conducted to determine the potential of pediculocidal activity. Parasite larvae were exposed to varying concentrations of synthesized ZnO NPs for 24 h. The results suggested that the mortality 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 results showed that the optimal times for measuring mortality effects 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 was observed at 10 mg/L treated for 6 h. The mortality was confirmed after 24 h of observation period. The larval mortality effects of synthesized ZnO NPs were 37%, 72%, 100% and 43%, 78% and 100% at 6, 12, and 24 h against A. V. Kirthi : A. A. Rahuman (*) : G. Rajakumar : S. Marimuthu : T. Santhoshkumar : C. Jayaseelan : K. Velayutham Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. Abdul Hakeem College, Melvisharam-632 509, Vellore District, Tamil Nadu, India e-mail:
[email protected]
A. subpictus and C. quinquefasciatus, respectively. It is apparent that the small size and corresponding large specific surface area of small nanometer-scale ZnO particles impose several effects that govern its parasitic action, which are size dependent. ZnO NPs were synthesized by wet chemical process, and it was characterized with the UV showing peak at 361 nm. X-ray diffraction (XRD) spectra clearly shows that the diffraction peaks in the pattern indexed as the zinc oxide with lattice constants a=3.249 and c=5.206 Å. The FTIR spectrum showed the range of 400–4,000 cm−1. The band at 899.56 cm−1; 1,151.87 cm−1; 1,396 cm−1; and these bands showed the complete composition of ZnO NPs. SEM micrograph showed 60–120-nm size and aggregates of spherical shape nanoparticles. EDX showed the complete chemical composition of the synthesized nanoparticles of zinc oxide. The maximum efficacy was observed in zinc oxide against the R. microplus, P. humanus capitis, and the larvae of A. subpictus, C. quinquefasciatus with LC50 values of 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 NPs showed 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 the concurrent assay. This is the first report on anti-parasitic activity of the synthesized ZnO NPs.
Introduction Rhipicephalus (Boophilus) microplus, known as the cattle tick, generally only parasitizes bovines; however, when the tick population reaches high levels of pasture infestations
Parasitol Res
its parasitism may extend to other mammal species. Economic losses caused by ectoparasites in Brazil are estimated to be approximately 2.65 billion dollars a year, in which R. microplus is responsible for more than 75% of the losses (Grisi et al. 2002). R. microplus is responsible for losses in milk, meat, and leather production and for the death of a number of animals, which results in economic losses associated with cattle production. A recent survey on acaricide resistance conducted through questionnaire reported a large-scale acaricide resistance in India (FAO 2004). Continuous and indiscriminate use of acaricides leads to the selection of chemical-resistant ticks along with contamination of the environment and animal products (Graf et al. 2004). The cattle tick is a vector of several diseasecausing pathogens such as Babesia bovis, B. bigemina, and Anaplasma marginale (Rosario-Cruz et al. 2005). Chemical acaricides have played a major role in controlling R. microplus in Mexico; however, their intensive use has led to the development of resistant tick populations within Mexico, and recently this resistance has spread beyond the northern 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 obligate ectoparasite which is found exclusively on humans. These lice have evolved with mankind and, thus, were distributed all over the world (Aspöck and Walochnik 2007; Burgess 2004; Mehlhorn and Mehlhorn 2009). Head lice are ectoparasites and its infestation due to unhygienic conditions has negatively affected the society for decades. Infestations are prevalent worldwide and especially common among school children in both developed and developing countries (Gratz 1997). The control of human head lice worldwide depends primarily on the continued applications of organochlorine (dichlorodiphenyltrichloroethane (DDT) and lindane), organophosphorus (malathion), carbamate (carbaryl), pyrethrin, pyrethroid (permethrin and δ-phenothrin), and avermectin (ivermectin originated from Streptomyces avermitilis) insecticides (Gratz 1997; Dolianitis and Sinclair 2002). Commercially available topical cream known as Licatack® smoothens the hair, is skin-safe, and smells good; it offers a very efficient and positive alternative to toxic, gluing, flammable, or skin-irritating products found on the market of anti-louse products (AbdelGhaffar et al. 2010a). Infestations are prevalent worldwide and especially common among school children in both developed and developing countries (Gratz 1997). AbdelGhaffar et al. (2010b) reported that the product Licatack® proved its efficacy on larvae and adult head lice after its efficacy was shown in intense in vitro screening tests. Head louse infestation may result in social embarrassment when infested children and their families become mobbed as “dirty” or “antisocial” (Mehlhorn et al. 1995; Mehlhorn and Mehlhorn 2009; Toloza et al. 2010b).
Mosquitoes are the most important single group of insects in terms of public health importance, which transmit a number of diseases, such as malaria, filariasis, dengue, Japanese encephalitis, etc., causing millions of deaths every year (Das et al. 2007). Anopheles is an important vector for the transmission of malaria (Gutiérrez et al. 2008) and Culex is known for transmission of filariasis in human and lumbar paralysis in cattle (Kwong-Chung et al. 2004). A. subpictus is the most abundant anopheline in most parts of the Indian subcontinent (Rao 1984) and recognized as a primary or secondary vector of malaria, a disease of great socioeconomic importance in different parts of the world (Panicker et al. 1981; Kulkarni 1983; Chatterjee and Chandra 2000). C. quinquefasciatus has been recorded round the year in different parts of the country (Chand et al. 1988). A careful and prolonged control of the vector can eliminate filariasis, but it is not an easy task due to its natural tolerance and early development of resistance to available insecticides (Brown and Pal 1971). India contri butes about 40% of the total global burden and accounts for about 50% of the people at risk of infection. Of the people exposed to the risk of infection, individuals with microfilaraemia, suffering from lymphoedema and hydrocele cases 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, Brugia malayi, and Brugia timori, and it spreads by the bite of an infected Culex mosquito. Thenmozhi et al. (2006) reported this species as a vector of Japanese encephalitis virus in Cuddalore, an area of Tamil Nadu, India, endemic for the disease. A. subpictus breeds in a variety of habitats like flowing or stagnant waters, clear or turbid waters, water with or without vegetation, unshaded or slightly shaded water bodies, wells, burrow pits, channels, ponds, tanks ground pools, fallow and freshly flooded rice fields, cement cisterns, tree holes, lake margins, fresh or brackish waters, etc. and the adult has a flight range of 1.5–6 km (Nagpal and Sharma 1995). Alternative control agents with novel modes of action and low mammalian toxicity and environmental impact are badly needed. The harmful effects of chemicals on non-target populations, ever growing resistance to chemical insecticides along with the recent resurgence of different mosquito-borne diseases have induced scientists to explore alternative, simple, sustainable methods of mosquito control. The eradication of adult mosquitoes using chemical insecticides is not a prudent strategy, as for the occurrence of adult stage alongside human habitation, and the adults can easily escape remedial mea sures. So the exploration of more effective and eco-friendly techniques like application of cost-effective, natural that can adapt 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 the environment would be more promising. This lack of efficacy is due to the emergence of resistance by the head louse to synthetic compounds, and researchers aimed on the search of new substitutes to synthetic ingredients, such as synthesized zinc oxide nanoparticles (ZnO NPs). ZnO nanostructured material has gained much interest owing to its wide applications for various devices such as solar cells, varistors, transducers, transparent conducting electrodes, sensors, and catalysts, since it is an abundant and inexpensive material (Van de Pol 1990; Mayo 1996). Highly ionic nanoparticulate metal oxides such as ZnO NPs are unique in that they can be produced with high surface areas and with unusual crystal structures (Klabunde et al. 1996). Compared to organic materials, inorganic materials such as ZnO possess superior durability, greater selectivity, and heat resistance (Padmavathy and Vijayaraghavan 2008). Moreover, zinc is a mineral element essential to human health and ZnO is a form in the daily supplement for zinc. ZnO NPs also have good biocompatibility to human cells (Padmavathy and Vijayaraghavan 2008). The bulk ZnO powders has demonstrated antibacterial and antifungal activity (Yamamoto 2001; Sawai and Yoshikawa 2004). In agriculture, zinc compounds are mainly used as fungicides (Waxman 1998). Recent studies have shown that NP of some materials, including metal oxides, can induce cell death in eukaryotic cells (Everts et al. 2006; Nel et al. 2006; Gupta and Gupta 2005; Magrez et al. 2006) and growth inhibition in prokaryotic cells (Brayner et al. 2006; Thill et al. 2006) due to its cytotoxicity. ZnO was among the most widely used NPs since they have applications in a large variety of sectors ranging from personal care products to coatings and catalysts in environmental remediation (Choopun et al. 2009; Kamat and Meisel 2003; Wang 2004). Manzo et al. (2010) have reported that ZnO nanoparticles exert toxic and genotoxic effects upon terrestrial organisms like plants (Lepidium sativum, Vicia faba), crustaceans (Heterocyipris incongruens), and insects (Folsomia candida). Certain novel properties of NPs could lead to adverse biological effects, with the potential to create toxicity (Oberdörster et al. 2005). The forecasted huge increase in the manufacture and use of NPs makes it likely that increasing human and environmental exposure to NPs will occur (Nowack and Bucheli 2007). A lot of toxicity data for soil invertebrates exposed to zinc salts became available (Lock and Janssen 2001). Beyer and Anderson (1985) assessed the toxicity of zinc oxide to the woodlice Porcellio scaber and they found that soil litter spiked with 1,600 mg of Zn per kilogram dry weight of soil litter caused significant negative effects. The toxicity study for zinc oxide to the earthworm Eisenia fetida in soil showed DNA damage to earthworm, activity of cellulase, and damage to mitochondria of gut cells were investigated after acute
toxicity test (Hu et al. 2010). Rekha et al. (2010) reported that the toxicity of nano-sized ZnO and Mn-doped ZnO were investigated using both Gram-positive and Gramnegative bacteria. Heng et al. 2010 demonstrated that initial exposure of BEAS-2B cells to oxidative stress sensitized their subsequent response to cytotoxic challenge with ZnO nanoparticles. However, detailed studies to investigate the toxicity threshold of identical nanoparticles to different biological systems and to obtain a fundamental understanding of the factors controlling the interaction of nanostructures with biological systems and their mode of toxicity are necessary to aid in the design of better and safer materials and more efficient biomedical applications. Unlike insoluble nanoparticles such as nano-TiO2 and nano-SiO2, the solubility of nano ZnO may play a more important role in its toxicity. However, inconsistent results on the solubility of nano ZnO in water were presented in the literatures (Franklin et al. 2007; Lin and Xing 2007; Wang et al. 2008). Two different mechanisms 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 caused by the surface, size, and/or shape of the nanoparticle itself (Brunner et al. 2006). These stimuli can be either due to a mechanical hindrance to biological functions or to a different interaction of the chemical compound in the nanostructured form with the biological environment. In the present study, we reported the zinc oxide NPs would be useful in promoting research aiming at the development of new agent for acaricidal, pediculocidal, and mosquito larvicidal activity.
Materials and methods Materials The chemicals zinc nitrate, sodium hydroxide, and starch were purchased from Sigma Company and the solutions were 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 inside the thigh, flanks, abdomen, brisket, and forelegs of naturally infested cattle. R. microplus larvae have a short, straight capitulum and a brown to cream body. The parasites were identified in the Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu. The applied method in the present study to verify the acaricidal activity of ZnO NPs against the larvae of R. microplus was
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developed as per the method of FAO (2004), incorporating slight modifications to improve practicality and efficiency of tested materials (Fernandes 2001; Fernandes et al. 2005). From the stock solution, 20 mg/L was prepared and a series of filter paper envelopes (Whatman filter paper no.1; 125 mm in diameter) with micropores were treated with each concentration of ZnO. The synthesized ZnO NPs were impregnated with 10 mg/L of which 3 ml solution of the stock was uniformly distributed with a pipette on internal surfaces. Five envelopes were impregnated with each tested solution. The control papers were impregnated with distilled water only. The opening of the envelopes (treated and inoculated with larval ticks) was folded (10 mm) and re-sealed with a metallic clip, with its identification mark (tested solution and concentration) on the outside. The packets are placed in the BOD incubator at a temperature of 28–30°C and 80–90% RH for 24 h. The envelopes were opened 24 h after exposure and the number of live and mortality larvae were recorded (Fernandes and 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 a population of children between the ages of 3 and 12, with the approval of their guardians, by raking a metal louse comb through sections of the scalp. Adults were obtained and pooled by carefully removing them from the metal teeth of the comb into clean plastic boxes. Once collected, the head lice were transported to our laboratory (Picollo et al. 1998, 2000). The children had not been treated with any pediculicide solution for at least the preceding month, using only the louse comb. The head lice were identified by Dr. A. Sangaran, Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu. Pediculocidal activity The synthesized ZnO NPs solutions were diluted using double distilled water as a solvent according to the desired concentrations of 25, 20, 15, 10, and 5 mg/L and the zinc oxide of 50, 25, 12.5, and 6.25 mg/L. Each test included a set of control group (distilled water) with five replicates for each individual concentration. Each louse was carefully transferred into a glass dish and 0.02 ml of the synthesized ZnO NPs was applied directly on the dorsal part of the louse using a 1-ml micropipette. For ZnO, 0.02 ml was applied directly on the dorsal part of the louse. After 15 s of contact with the agent, the louse was transferred into a petri dish lined with filter paper and observed using a hand lens until dead or otherwise. All the petri dishes were set aside
in a dark chamber at 26±0.5°C and 70±1% humidity. The elapsed time was recorded for each test agent as the “knockdown” time. The death of the louse was confirmed when there was cessation of motility or waggling of the appendages on touching with a needle. Ten lice were used for each determination (Oladimeji et al. 2000). Mosquito culture A. subpictus and C. quinquefasciatus larvae were collected from a rice field to a stagnant water area of Melvisharam (12°56′23″ N, 79°14′23″ E) and identified in the Zonal Entomological Research Centre, Vellore (12°55′48″ N, 79° 7′48″ E), Tamil Nadu, to start the colony, and the larvae were kept in plastic and enamel trays containing tap water. They were maintained and reared in the laboratory as per the method of Kamaraj et al. (2009). Larvicidal bioassay During preliminary screening with the laboratory trial, the larvae of A. subpictus and C. quinquefasciatus were collected from the insect-rearing cage and identified in the Zonal Entomological Research Centre, Vellore. For the bioassay test, mosquito larvae were taken in five batches of 20 in 249 mL of water and 1.0 mL of ZnO. The control was set up with dechlorinated tap water and ZnO. The number of dead larvae was counted after 24 h of exposure, and the percentage of mortality was reported from the average of five 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 performed by placing 20 mosquito larvae into 200 mL of sterilized double distilled water with nanoparticles into a 250-mL beaker (Borosil). The nanoparticle solutions were diluted using double distilled water as a solvent according to the desired concentrations (4.0, 2.0, 1.0, and 0.5 mg/L). Each test included a set of control group (zinc oxide and distilled water) with five replicates for each individual concentration. Mortality was assessed after 24 h to determine the acute toxicities on fourth instar larvae of A. subpictus and C. quinquefasciatus. In order to compare the mortality of ZnO NPs to that of dissolved H2O2 released and the mosquito larvae were exposed to a range of dissolved H2O2 concentrations so as to cover the range released from all doses of ZnO NPs. To avoid settling of particles especially at higher doses, all treatment solutions were sonicated for an additional 5 min prior to addition of the mosquito larvae. Since this additional sonication appeared 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 and synthesized ZnO NPs were subjected to dose–response bioassay for larvicidal activity against the larvae of A. subpictus and C. quinquefasciatus. Different concentrations ranging from 25 to 3.12 mg/L (pure zinc oxide) and 4.0 to 0. 5 mg/L (for synthesized ZnO NPs) were prepared for larvicidal activity of parasites. The number of dead larvae was counted after 24 h of exposure, and the percentage of mortality was reported from the average of five replicates. However, at the end of 24 h, the selected test samples turned out to be equal in their toxic potential. Synthesis of ZnO nanoparticles The ZnO NPs were prepared by wet chemical method using zinc nitrate and sodium hydroxide as precursors and soluble starch as stabilizing agent. Soluble starch (0.1%) was dissolved in 500 mL of distilled water by using a microwave oven. Zinc nitrate, 14.874 g (0.1 mol), was mixed with starch solution. The solution was kept under constant stirring using a magnetic stirrer to completely dissolve the zinc nitrate. After complete dissolution of the zinc nitrate, 0.2 mol of sodium hydroxide solution was added under constant stirring, drop by drop touching the walls of the vessel. The reaction was allowed to proceed for 2 h after complete addition of sodium hydroxide. After the completion of reaction, the solution was allowed to settle overnight, and the supernatant solution was then discarded care fully. The remaining solution was centrifuged at 10,000 g for 10 min and the supernatant was discarded. Thus, obtained nanoparticles were washed three times using distilled water. Washing was carried out to remove the by-products and the excessive starch that were bound with the nanoparticles. After washing, the nanoparticles were dried at 80°C overnight. During drying, complete conversion 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 by periodic sampling of aliquots (1 mL) of the aqueous component after 20 times dilution and measuring the UV– Vis spectra of the solution. UV–visible spectroscopy of these aliquots were monitored as a function of time of reaction on a Schimadzu 1601 spectrophotometer in 200–700-nm range operated at a resolution of 1 nm. Further, the reaction mixture was subjected to centrifugation at 60,000 rpm for 40 min; the resulting pellet was dissolved in de-ionized water and filtered through Millipore filter (0.45 μm). The synthesized nano-
particles were identified by XRD spectroscopy (Perkin-Elmer Spectrum One instrument, PW 1830 instrument operating at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation). Fourier transform infrared (FTIR) spectra of the samples were measured using Perkin Elmer Spectrum One instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets. Powder samples for the FTIR were prepared similar to powder diffraction measurements. The FTIR spectra of synthesized ZnO NPs taken were analyzed, which discussed for the possible functional groups for the formation of nanoparticles. For the scanning electron microscopic studies, 25 μL of sample was sputter-coated on copper stub, and the images of nanoparticles were studied using scanning electron microscopy (SEM; JEOL, Model JFC-1600). Data analysis Mean percent larval mortality data were subjected to analysis of variance and compared with Duncan’s multiple range tests to determine any differences between ZnO NPs and within species and concentration (SPSS 2007). Prior to analysis, mortality in treatments was corrected for controls using the formula of Abbott (1925). LC50 and their associated confidence intervals were estimated from 24-h concentration mortality data using probit analysis (Finney 1971). Lethal concentrations at 50% and slope levels were considered significantly different if their associated confidence intervals did not overlap. All diffe rences were considered significant if P≤0.05.
Results In the present study, parasite larvae were exposed to varying concentrations of synthesized ZnO NPs for 24 h. The results suggested that the mortality effects of synthesized ZnO NPs were 43% at 1 h, 64% at 3 h and 78% at 6 h, and 100% after 12 h against R. microplus activity. In pediculocidal activity, the results showed that the optimal times for measuring mortality effects 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 was observed at 10 mg/L treated for 6 h. The mortality was confirmed after 24 h of observation period. The larval mortality 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-lice activity of zinc oxide and ZnO NPs showed the 29.14, 13.41 mg/L; r2 = 0.805 and 0.982, respectively. The pediculocidal activity results showed that the highest mortality 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 oxide and ZnO NPs are noted; however, the highest mortality was found in synthesized ZnO NPs and zinc oxide against the larvae of A. subpictus (LC50 =11.14, 3.19 mg/L; r2 = 0.894and 0.945) and against the larvae of C. quinquefasciatus (LC50 =12.39, 4.87 mg/L; r2 =0.904 and 0.970), respectively. The control (distilled water) showed nil mortality in the concurrent assay. The chi-square value was significant at p≤0.05 level. The complete mortality
was observed for synthesized ZnO NPs for R. microplus and 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 synthesized shows peak absorption at 361 nm (Fig. 1). Figure 2 shows the X-ray diffraction pattern of the grown ZnO NPs prepared using the above conditions. The spectra clearly shows the diffraction peaks in the pattern indexed as the zinc oxide with lattice constants a=3.249 and c=5.206 Å and 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 of Pediculus humanus capitis, and larvae of Anopheles subpictus, Culex quinquefasciatus Test sample
Species
Zinc oxide
R. microplus
P. humanus capitis
A. subpictus
C. quinquefasciatus
Synthesized ZnO NPs
R. microplus
P. humanus capitis
A. subpictus
C. quinquefasciatus
Concentrations (mg/L)
Percent mortalitya (mg/L)±SD
50.0 25.0 12.5 6.25 50.0 25.0 12.5 6.25 25.0 12.5 6.25 3.12 25.0
80±0.987 74±1.022 39±1.880 28±1.002 78±0.810 66±0.868 36±0.594 24±1.262 91±0.825 78±1.242 49±0.922 39±1.025 96±0.825
12.5 6.25 3.12 10.0 5.0 12.5 6.25 10.0 5.0 12.5 6.25 4.0 2.0 1.0 0.5 4.0 2.0 1.0
63±1.242 56±0.922 26±1.025 100±0.00 86±1.29 56±1.59 31±0.84 100±0.00 91±1.509 63±0.914 41±1.238 100±0.00 84±1.796 72±1.231 38±1.185 100±0.00 85±1.796 49±1.231
0.5
25±1.185
LC50 (mg/L)
UCL–LCL (mg/L)
Slope
r2
29.14
24.21–35.08
39
0.805
33.88
27.89–41.17
36
0.876
11.14
8.18–15.17
49
0.894
12.39
10.18–15.07
63
0.904
13.41
12.22–14.71
56
0.982
11.80
10.37–13.44
63
0.966
3.19
3.69–2.77
84
0.945
4.87
4.04–5.17
85
0.970
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
