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Year : 2019  |  Volume : 10  |  Issue : 1  |  Page : 69-72  

Larvicidal activity of chemically synthesized silver nanoparticles against Anopheles stephensi

1 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences; Radiation Biology Research Center, Iran University of Medical Sciences, Tehran, Iran
3 Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
4 Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences; Medical Biomaterials Research Center, Tehran University of Medical Sciences, Tehran, Iran

Date of Web Publication22-Aug-2019

Correspondence Address:
Amir Amani
Department of Medical Nanotechnology, Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpnr.JPNR_18_17

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Background: Silver nanoparticles (AgNPs) have wide applications in different fields such as medicine, food industry, and pest management. Larvicidal activity of many herbally biosynthesized AgNPs have been evaluated against main malaria vector, that is, Anopheles stephensi. However, results of the studies are very different. No report has been found on larvicidal activity of chemically synthesized AgNPs against A. stephensi. Materials and Methods: AgNPs were synthesized using chemical reduction and characterized by dynamic light scattering and transmission electron microscopy. Concentration of silver ion in the final solution was determined by ICP-AES. Turbidity of solutions of AgNPs at different concentrations (i.e., 0.2–100 ppm) was studied. Subsequently, larvicidal activity of nanoparticles was evaluated, in line with the WHO guideline for laboratory tests. Results: AgNPs were synthesized successfully and confirmed by ultraviolet analysis. Nanoparticles were spherical with a diameter of ~30 nm. AgNPs had no larvicidal activity up to 80 ppm and showed a small larvicidal effect (~20%) at 100 ppm. Conclusion: Chemically synthesized AgNPs are not proper candidates for control of larvae due to their low efficacy and effects on nontarget specious lived in stagnant water.

Keywords: Chemical reduction, larvicidal activity and Anopheles stephensi, silver nanoparticles, silver nitrate, sodium borohydride

How to cite this article:
Osanloo M, Amini SM, Sedaghat MM, Amani A. Larvicidal activity of chemically synthesized silver nanoparticles against Anopheles stephensi. J Pharm Negative Results 2019;10:69-72

How to cite this URL:
Osanloo M, Amini SM, Sedaghat MM, Amani A. Larvicidal activity of chemically synthesized silver nanoparticles against Anopheles stephensi. J Pharm Negative Results [serial online] 2019 [cited 2020 Jul 2];10:69-72. Available from:

   Introduction Top

Silver nanoparticles (AgNPs) are being increasingly used in various fields including medical sciences, health care,[1] food industry,[2] and also heavy metal absorbents.[3] Furthermore, biological activity of herbally biosynthesized AgNPs such as antioxidant [4] and hypoglycemic effect have been investigated.[5] Recently, larvicidal activity of herbally biosynthesized AgNPs has been evaluated against many mosquito populations such as Aedes,[6]Culex,[7] and Anopheles.[8] Among the mentioned mosquitoes, Anopheles stephensi is one of the main malaria vectors, thus, very important in public health, causing annually 500,000 death around the world.[9]

In the literature, various values have been reported for lethal concentration (LC50) of AgNPs which have been biosynthesized, against A. stephensi, ranging from 0.5[10] to 37.7 ppm,[7] showing a substantial difference (~75 times) in efficacy against larvae. However, there is no report about evaluation of larvicidal activity of pure AgNPs, which is synthesized with chemical reduction agents, against A. stephensi.

In this study, AgNPs were synthesized using silver nitrate and sodium borohydride (chemical reducer). First, larvicidal activity of sodium borohydride at applied concentration (40 mM) evaluated against A. stephensi. Then, larvicidal activity of AgNPs in range of 0.23–100 ppm was investigated.

   Materials and Methods Top


Silver nitrate (AgNO3, 99%) was obtained from Dr. Mojallali Chemicals Co. (Iran). Sodium borohydride (NaBH4, 99%), nitric acid (reagent grade, 65%), and hydrochloric acid (ACS reagent, 37%) were purchased from Merck Chemicals (Germany). Glassware were washed with Aqua regia and rinsed with deionized (DI) water. All aqueous solutions were prepared with DI water (Barnsted E-Pure TM 18.3 MΩ water). A. stephensi (Beach strain) third and fourth instars larvae were used in this study, obtained from the Department of Medical Entomology, Tehran University Medical Sciences. Colonies were maintained at 28°C ± 2°C with 12:12 light and dark photoperiods and 65% ± 5% relative humidity.

Synthesis of silver nanoparticles

AgNPs were synthesized based on the chemical reduction route. Briefly, 0.5 mL of ice-cooled sodium borohydride solution (40 mM) was injected in 12 mL of silver nitrate solution (0.5 mM) under vigorous stirring for 15 min. Prepared AgNPs were concentrated through sequences of centrifugation at 3000 rpm for 4 min with 100 kDa centrifugation filter tube.

Characterization of silver nanoparticles

Particle size and particle size distribution of AgNPs were determined by dynamic light scattering (DLS, Scatteroscope, K-ONE. LTD, Korea). D50 (median) was taken as particle size and particle size distribution was calculated using the equation (1).

AgNPs were also studied by transmission electron microscopy (TEM, Zeiss EM 900 Germany). TEM pictures were analyzed by Digimizer software (MedCalc Software bvba, Belgium).

The exact concentration of silver ions was investigated by inductively coupled plasma atomic emission spectroscopy (ICP-AES, VISTA-PRO, Varian, USA). For confirming the successful synthesis of AgNPs, an extinction spectrum of the colloidal solution of nanoparticles was recorded by using a Lambda 35 (Perkin-Elmer USA) spectrophotometer.

Turbidity of solution of AgNPs was measured using Turbidimeter (2100AN Laboratory Turbidimeter, EPA, 115 Vac, USA).

Evaluation larvicidal activity of silver nanoparticles

Larvicidal activity of AgNPs was evaluated with the WHO guideline for laboratory test with some modifications.[11] Solution of AgNPs with concentration of 100 ppm was employed as stock solution. By dilution of stock solution with DI water, different concentrations of AgNPs were prepared (i.e., 0.23–100 ppm). Batches of 25 larvae were added to containers containing 200 mL AgNPs at mentioned concentrations. The tests were repeated 16 times at 4 different replications. Two control groups were also considered: sodium borohydride (40 mM) and DI water. After 24 h of exposure, all larvae (live or dead) were transferred to distilled water using a textile filter and then dead larvae were counted.

   Results Top

Characterization of silver nanoparticles

As shown in [Figure 1], synthesized AgNPs exhibit a sharp extinction peak at 390 nm, concluded successfully of synthesis process. Using DLS analysis, particle size and particle size distribution of AgNPs were 35 nm and 2 nm, respectively [Figure 2]. AgNPs had spherical shapes with size of 29 ± 4.5 nm, using TEM image [Figure 2].
Figure 1: Dynamic light scattering (left) and transmission electron microscopy (right) images of silver nanoparticles. Determined particle sizes were 35.0 nm and 29.0 nm ± 4.5 nm, respectively

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Figure 2: Ultraviolet spectrum of synthesized silver nanoparticles

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Evaluation of larvicidal activity of silver nanoparticles

Turbidity of solutions of AgNPs at different concentrations and their corresponded larvicidal activity against A. stephensi are shown in [Table 1]. Turbidity decreased from 1040 nephelometric turbidity units (NTU) at concentration of 100 ppm, to 2.23 NTU, for sample with concentration of 0.23 ppm. No larvicidal activity was shown for different concentrations of AgNPs up to 80 ppm, and also, for sodium borohydride (40 mM) (data not shown). At concentration of 100 ppm, 21% mortality was observed. Higher concentrations of AgNPs were not tested due to production of high viscosity and osmotic pressure as well as the need to using chemical stabilizers.
Table 1: Larvicidal activity of silver nanoparticles against Anopheles stephensi and related turbidity at different concentrations

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   Discussion Top

Results of characterization of synthesized AgNPs are illustrated in [Figure 1] and [Figure 2]. Synthesized AgNPs had a sharp extinction peak at 390 nm, this value is comparable with recent reports: reported ranges of extinction peak for AgNPs were 390–420 nm.[12],[13],[14] Results of TEM analysis show that they have spherical shape, with an average size of 29.3 ± 4.5 nm, showing appropriate particle size distribution.

Toxicity of chemically synthesized AgNPs for humans and animals has already been evaluated. For instance, toxicity of AgNPs with size of 35 nm appeared at 500 ppm in hepatocytes and at 1000 ppm in Caco-2 cells.[15],[16] In another study, after oral administration of AgNPs (60 nm, 1000 mg/kg/day) to Sprague Dawley Rats, liver toxicity was observed.[17] Thus, in this study, larvicidal activity of AgNPs was investigated at below of toxic concentration, that is, in range of 0.2–100 ppm.

Reviewing the literature, many reports may be found about evaluation of larvicidal activity of herbally biosynthesized AgNPs against A. stephensi. In the followings, larvicidal activity of herbally biosynthesized AgNPs is classified by their size: LC50 of AgNPs with size <35 nm were 4.60, 19.13, and 28.22 ppm for nanoparticles biosynthesized by Ulva lactuca,[8]Murraya koenigii,[18] and Nerium oleander,[19] respectively. Calculated LC50 for AgNPs in range of 35–60 nm were as 8.21, 4.98, and 21.51 ppm, for biosynthesized ones from Aristolochia indica,[20]Aloe vera,[21] and Euphorbia hirta,[22] respectively. Furthermore, reported LC50 for AgNPs with sizes >60 nm were 15.28, 26.35, and 1.39 ppm, for the ones biosynthesized by materials extracted from Annona muricata,[7]Cassia roxburghii,[6] and Musa paradisiaca,[23] respectively.

Considering the abovementioned reported, size does not appear to be important in determining the larvicidal activity of AgNPs. In two different studies on larvicidal activity of AgNPs with similar sizes (i.e., ~105 nm) reported LC50 values were substantially different, while LC50 of AgNPs biosynthesized by Plumeria rubra against A. stephensi was 1.74 ppm,[24] corresponding value for AgNPs biosynthesized by A. muricata was documented as 37.7 ppm.[7] A second factor which may contribute to the larvicidal activity of biosynthesized AgNPs is the activity of plant extract itself. However, LC50 of two different AgNPs which were biosynthesized with E. hirta and Plumeria rubra showed similar LC50 values (i.e., 169 and 171 ppm, respectively) while the LC50 of the extracts were hugely different (i.e., 21.51 and 1.74 ppm, respectively).[22],[24] It is now well known that AgNPs easily interact with live organs, functional groups, or even other particles.[25] Considering the mentioned reports and results of our work, it is arguable that larvicidal activity of AgNPs is related to functional groups on the surface of the nanoparticles which are formed during the biosynthesis process. Thus, the chemically synthesized nanoparticles which do not have surface groups are not expected to show larvicidal activity, a finding which has not been reported so far.

Besides that, increase in AgNPs concentration makes the water turbid [Table 1]. High turbidity could affect other nontarget organisms such as larvae of diving beetle or bacteria in stagnant water.[26],[27] This item greatly reduces usability of chemically synthesized AgNPs as larvicide.

Evaluation of larvicidal activity of herbally biosynthesized AgNPs is not limited to A. stephensi. Its larvicidal activity has been evaluated against other species of mosquitos such as Aedes aegypti,[24]Culex quinquefasciatus,[28] and Anopheles subpictus.[29] Such activities may also be investigated for chemically synthesized AgNPs, in the future researches.

   Conclusion Top

Although herbally biosynthesized AgNPs have shown various degrees of larvicidal activities, chemically synthesized AgNPs do not show toxicities against larvae of A. Stephensi. This could be due to lack of functional groups on the surface of prepared particles.


This research was supported by Tehran University of Medical Sciences and Health Services grant No. 95-01-87-31860.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2]

  [Table 1]


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