Cu-Zn and Ag-Cu Bimetallic Nanoparticles as Larvicide to Control Malaria Parasite Vector A comparative analysis
Savy P. Minal and Soam Prakash Department of Zoology, Dayalbagh Educational Institute, DEI Agra, India
[email protected]
Abstract— A single mosquitoes can threaten public health and wellbeing. With emerging mosquito borne and zoonotic diseases, there is a demand for new insecticidal formulations to control mosquito vector population. Monometallic nanometals like nanosilver, nanocopper and nanozinc nanoparticles are known to kill mosquito larvae efficiently. Our hypothesis is that the bimetallic combination of nanoparticles could be able to kill mosquito larvae much more efficiently than their respective monometallic form. To accomplish this we have done extracellular synthesis of monometallic Copper-, Zinc-, and Silver-nanoparticles, and bimetallic Copper-Zinc and Silver-Copper nanoparticles with the help of aqueous leaf extract of Ocimum sanctum (Linn.). The characterization was done through U.V.-Vis spectrophotometry and XRD which showed the formation of nanoparticles in the solution. Efficacy on 3 rd instar larvae of Anopheles mosquito was performed by following WHO guidelines. Lethal concentration values (LC) for each nanoparticles were obtained. Results of efficacy reflected that the bimetallic Silver-Copper nanoparticles followed by Copper-Zinc nanoparticles, both bimetallic nanoparticles have potential to kill mosquito larvae. Keywords—Larvicidal efficacy; Bimetallic nanoparticles; monometallic nanoparticles; Anopheles stephensi; Extracellular synthesis
I.
INTRODUCTION
In 2015 WHO reported nearly 214 million cases and 438000 deaths worldwide due to the transmission of malaria parasite through Anopheline species, however from India 0.85 million cases and 316 deaths were reported (NVBDCP annual report, 2014-15). Malaria fact sheet of WHO updated in April 2016, has reposted the ongoing transmission of malaria from 95 countries and territories. New data on malaria burden has highlighted the region with massive occurrence of 80% malaria cases and 90% malaria deaths in Sub-Saharan African regions, where mosquitoes are resistant to all the four classes of insecticides [1, 2]. WHO’s Integrated Vector Management (IVM) aims at the formulation of new vector control tools that provide improved efficacy that sustainably control disease vector, it should be cost-effective, safe for ecosystem and encourages the synergistic approach of such tools for multi-
disease control approach. IVM is focused on the use of appropriate measures to vector after the critical analysis of pattern of disease transmission, its severity, and need. These measures also aid to reduce further risk to increases insecticide resistance species in mosquito population. Firstly IVM favors environmental management strategies to reduce the breeding habitats of mosquito, secondly it employs the use of biological control method such as the use of bacterial larvicide, larvivorous fish. Lastly it employs the use of costly synthetic and toxic chemical insecticides through indoor residual spray (IRS), Long-lasting insecticidal nets (LLINs) chemical larvicides and adulticides [3]. In recent years a lot of work has been carried out on green synthesis and application of metal nanoparticles such as Nanosilver, nanogold, nanocopper, and nanozinc as mosquito larvicide [4, 5, 6, and 7]. In comparison with synthetic insecticides nanometal particles also gives promising results in terms of vector control [8]. These nanometals and nanometal oxides are synthesized from noble and transition metals most preferably silver, gold, platinum, titanium, palladium, copper, zinc and iron. Nanosilver has been used as algaecide and registered for its use in swimming pools with the Environmental Protection Agency (EPA). Nanosilver is also known to cause antimicrobial activity in a broad spectrum [9]. Nanometals can be synthesized with the help of precursor agent i.e. metal salt, reducing agent, and stabilizing agent. These reducing and stabilizing agents can be synthetic for example chemical agents, physical agents, synthetic surfactants and polymers, as well as natural polymers such as polysaccharides, polynucleotides, proteins and polypeptides. Leaf extract of plants and other natural active chemicals are also used for the synthesis of nanometals [10]. Bimetallic nanoparticles used for killing mosquito larvae may differ in terms of its effectiveness and toxicity from monometallic nanoparticles and varies among different species of mosquitoes. The green synthesis of bimetallic nanoparticles for mosquito control is not much explored and the property of the bimetallic nanoparticle significantly differ from their individual monometallic forms, therefore each new bimetallic nanoparticle of different non-identical metal salt differ from one another. The effectiveness of new combination of bimetallic nanoparticles can increase or decrease, depending upon the binding properties of the nanoparticles used to synthesize it [11, 12].
II.
MATERIAL AND METHODS
A. Material Metal salts namely Silver Nitrate (analytical grade), Zinc Sulphate and Copper (II) Sulphate pentahydrate Pure (cupric sulphate) were purchased from Merk. 3rd instar larvae of Anopheles stephensi were collected from the semi-urban region of Dayalbagh. Ocimum sanctum was collected from DEI campus. B. Preparation of Aqueous Extract Fresh and disease free leaves of Ocimum sanctum were washed using distilled water, air dried and finely chopped. 15 g of it was transferred in 250-mL flask containing 100 mL of triple deionized water to make 15% solution. Extract was done on hot plate at 65C for 1 h, and the resulting turbid crude extract was filtered using whatman-1 filter paper and resulting non-turbid filtrate was then stored at 4C for further use.
1971) [15]. The relationship between probit and log concentrations is plotted on graph from which probit regression line and probit equations were obtained from which the LC values were derived. III.
RESULTS
A. Visible Color Change of the Solution The molar solution immediately changed color after addition of plant leaf extract. In the so formed nanoparticle solution the plant extract got denatured turning the solution turbid. The nanoparticle solution of Cu Nps. and Cu-Zn Nps. solution turned greenish in appearance, while Zn Nps. solution turned brown and Ag Nps. solution turned turbid brown in appearance and Ag-Cu Nps. solution turned turbid greenish in appearance.
C. Synthesis of Nanoparticles Monometallic nanoparticles were synthesized weighing 50mM Copper Sulphate, 50mM Zinc Sulphate, and 10mM Silver Nitrate transferred in a 250-mL flask containing 10 mL of triple deionized water, to this molar solution 90 mL of prepared aqueous extract of Ocimum sanctum was added, to make the volume up to 100 mL in 1:9 ratio. Bimetallic Cu-Zn nanoparticles were synthesized by taking 25mM Copper Sulphate and 25mM of Zinc nitrate, while AgCu by taking 25mM Copper Sulphate and 5mM of Silver Nitrate separately in a 250-mL flask containing 10 mL of triple deionized water, to this molar solution 90 mL of prepared aqueous extract of Ocimum sanctum was added, to make the volume up to 100 mL in 1:9 ratio. These preparations were left at room temperature in dark condition for overnight.
Fig. 1: Molar solutions for making Ag-Cu Nps., Zn Nps., Cu-Zn Nps., Cu Nps., and Ag Nps.
D. Characterization of Nanoparticles Characterization study was done through U.V.-Vis spectroscopy, and X-ray diffraction (XRD). E. Mosquito Larvicidal Bioassay Larvicidal efficacy of prepared bimetallic and monometallic nanoparticles in laboratory was assessed as per W.H.O.’s guidelines [13] for laboratory testing of mosquito larvicides, and the mortality was recorded for 24 h and 48 h. Each test concentration contains ten 3rd instar larvae in 50 ml of distilled water. A positive control was set up by adding 8ml of plant extract to the distilled water containing ten 3 rd instar larvae and the test concentrations in ppm were prepared by converting molar concentrations into grams per liter which was then converted to milligrams per liter (ppm). F. Data Management and Statistical Analysis Obtained data on efficacy testing was subjected to corrected % mortality, using Abbott’s formula only when the mortality in positive control is more than 5%. (Abbott, 1925) [14]. Then the data was subjected to probit analysis (Finney,
Fig. 2: Reduced solution containing nanoparticles of Cu, Zn, Ag, Cu-Zn, and Ag-Cu Nps.
B. U.V.-Vis. Spectrophotomery Results U.V. Spectrophotometry of turbid and colloidal nanoparticle solution was performed, after assigning the plant extract as blank. The peak for Cu Nps. can be located on 406nm at 3.472 a.u. absorbance (Graph 1), for Zn Nps. (Graph 2) peak was observed at 411.5nm with the absorbance of 3.718 a.u., peaks for Ag Nps. (Graph 3) was located at 400.5nm with the absorbance of 0.721 a.u., dual peaks in CuZn Nps. (Graph 4) solution was observed at 401nm (copper Nps.) with absorbance of 4 a.u., and at 408.5nm (zinc Nps.) with absorbance of 4 a.u., dual peaks were also seen for Ag-
Cu Nps. (Graph 5) at 400.05nm (silver Nps.) with an absorbance of 3.274 a.u., and at 406nm (copper Nps.) with 4 a.u. absorbance.
Graph 5.
Graph 3.
C. XRD Results XRD diffraction patterns of nanoparticles was compared with JCPDS cards for respective standard metal nanoparticles and the diffraction data showed monoclinic and cubic configuration and the obtained concentric rings corresponding to the 2θ values are in good coordination with JCPDS card of corresponding standard metal nanoparticles. High noise diffraction peaks suggests the presence of leaf extract in the test sample for XRD. Diffraction peaks for copper nanoparticles (Graph 6.) were observed at 32.55 and 77.45 of 2θ which corresponds to (110) and (222) crystal planes and in good coordination with JCPDS card no. 89-5899 for copper nanoparticles. Diffraction peaks for zinc nanoparticles (Graph 7.) were observed at 28.1 and 47.6 and 56.9 of 2θ which corresponds to (111), (220) and (311) crystal planes and in good coordination with JCPDS card no. 5-0566 for zinc nanoparticles. Diffraction peaks for silver nanoparticles (Graph 8.) were observed at 37.45 and 44.15 of 2θ which corresponds to (111) and (200) crystal planes and in good coordination with JCPDS card no. 4-0783 for silver nanoparticles. Diffraction peaks for Copper-Zinc nanoparticles (Graph 9.) were observed at 27.87 (Zn), 33.9 (Zn) and 39.4 (Cu) of 2θ which corresponds to (111) and (200) crystal planes and in good coordination with JCPDS cards for Copper and Zinc Nps. Diffraction peaks for silver-copper nanoparticles (Graph 10.) were observed at 32.9 (Cu), 35.8 (Cu) and 64.4 (Ag) of 2θ which corresponds to (110), (111) and (220) crystal planes and in good coordination with JCPDS card for Silver and Copper Nps.
Graph 4.
Graph 6.
Graph 1.
Graph 2.
Graph 7.
D. Efficacy Results Efficacy of monometallic and bimetallic nanoparticle test concentration (TABLE I.) was carried out on 3rd instar larvae of Anopheles mosquito. LC50 is called the lethal concentration value at which half of test population dies. Anti-log of log LC values gives ppm concentration per liter. For Cu Nps LC50 value was observed at 177.378 ppm after 24h and at 118.032 ppm after 48h and LC90 value was observed at 240.159 ppm after 48h. For Zn Nps. LC90 value was observed at 408.695 ppm after 48h. For Silver Nps, LC50 value was observed at 15.591 ppm after 24h, LC90 value was observed at 11.235 ppm after 48h, LC99 value was observed at 16.676 ppm after 48h. For Cu-Zn Nps. LC50 value was observed at 444.733 ppm after 24h. For Ag-Cu Nps. LC90 value was observed at 192.930 ppm after 24h, LC99 value was observed at 191.513 ppm after 48h. From these results we can say that silver-copper bimetallic Nps. are superior over copper-zinc Nps. In our previous study (Savy, 2015) we have observed that copperzinc Nps. and copper Nps. test conc. yielded nearly equal % mortality result for Culex quinquefasciatus 3rd instar larvae, while a very low yield in zinc Nps. test concentrations. DISCUSSION
Graph 8.
Graph 9.
Filipe (1972) isolated West Nile Virus (WNV) in 1969, from a pool of Anopheles maculipennis s.l. mosquitoes that were collected from southern region of Alentejo, Portugal [16]. Fernandes et al. (1998) isolated West Nile Virus (WNV) in 1996 from a pool of unfed Anopheles atroparvus collected from the wetlands of the Tejo river estuary near Lisbon, Portugal [17]. Thenmozhi et al. (2006) analyzed wild male and female anopheles mosquito samples for presence of Japanese Encephalitis Virus (JEV). These samples were collected from Cuddalore an endemic area for Japanese Encephalitis Virus, situated in Tamil Nadu state of India. Both male and female pools of Anopheles subpictus Grassi were found positive for JEV. Researchers proposed natural transovarial transmission of virus must be responsible for infecting male Anopheles subpictus mosquitoes. Although their data was not significant since out of 19 positive of female pools JEV was confirmed from only four isolates by insect bioassay [18]. Lehrer (2010), proposed his hypothesis in medical hypothesis journal that occurrence of malaria outbreak of United States in 2004 was co-related with occurrence of malignant brain tumor from 19 US states. He proposed transmission of virus responsible for malignant glioma either SV40 or cytomegalovirus (CMV) through anopheles mosquitoes. These data shows that Anopheles mosquito can also transmit other diseases apart from the only transmission of sporozoite of malaria parasite [19]. Marimuthu et al. (2010) synthesized silver nanoparticles using aqueous leaf extract of Mimosa pudica, and checked their efficacies at varying concentration, on larvae of mosquitoes and ticks, and concluded that low doses of silver nanoparticles were more efficient in compare to high doses of aqueous leaf extract alone, hence mortality in their nanoparticle test concentration was also due to some the larvicidal properties of plant extract [20].
Graph 10.
Rizwan et al. (2014) in his review articles has discussed the bioremediation process through nanomaterials like
TABLE I. EFFICACY RESULTS
Nanoparticle test concentration (ppm)
% Mortality after
Probit equation (ŷ)
Copper Nps.
24h
y = 1.9837x + 0.5387
48h Zinc Nps.
Silver NPs.
Copper-Zinc Nps.
Silver-Copper Nps.
R2
Log LC50
Log LC90
Log LC99
(ppm)
(ppm)
(ppm)
0.5637
2.2489
2.8942
3.4235
y = 4.1484x - 3.5956
0.2453
2.0720
2.3805
2.6336
24h
y = 3.895x - 5.6727
0.4585
2.7401
3.0687
3.0687
48h
y = 2.0604x + 0.8994
0.6661
1.9901
2.6114
3.1210
24h
y = 6.5595x - 2.825
0.9437
1.1929
1.3880
1.5481
48h
y = 6.1199x - 0.1497
0.6594
0.8414
1.0506
1.2221
24h
y = 3.3289x - 3.8153
0.9548
2.6481
3.0326
3.3480
48h
y = 0.7031x + 4.1336
0.0868
1.2322
3.0527
4.5461
24h
y = -1.9292x + 10.689
0.6097
2.9488
2.2854
1.7411
48h
y = 4.1895x - 2.2314
0.5901
1.7260
2.0316
2.2822
nanopalladium, and nanoiron for heavy metals, solid waste, hydrocarbons, waste water and uranium remediation. Nanoiron can remove silver from contaminate water and other pollutants like mercury, arsenic, cadmium etc. Bimetallic iron-nickel nanoparticles are known for their dehalogenation properties and used to degrade trichloroethylene (TCE) [9]. According to He and Zhao (2005), nanometals like iron-palladium nanoparticles can dechlorinate as they degrade chlorinated organic compounds such as dichloroethylene (DCE), trichloroethylene (TCE), and Polychlorinatedbiphenyls (PCB) found in polluted water. Hence from the above statements we can say that nanoparticles not only prevent nanometal and metal pollution in the water bodies but also helps in breaking down of the complex molecules that are harmful for nature [22]. In our study we have reported the use of bimetallic silver-copper nanoparticles in mosquito control nanoformulations, and also that silver-copper pair is superior over the copper-zinc bimetallic nanoparticles.
CONCLUSION In the present investigation we have reported that the combination of Silver-Copper bimetallic nanoparticles efficiently kills the 3rd instars of Anopheles mosquito larvae at lower concentration as compare to Copper-Zinc bimetallic nanoparticles. In spite of having equal molar concentration of precursor agent, efficacy results of bimetallic nanoparticles were advanced over the monometallic nanoparticles. ACKNOWLEDGMENT We sincerely express our gratitude to Prof. P.S. Satsangi Sahab, Chairman of Advisory Committee on Education, Dayalbagh Educational Institute, Agra, India. We are thankful to Prof. P.K. Kalra, Director, Dayalbagh Educational Institute (DEI), Agra, India. We sincerely thank Prof. Sahab Das (Head of Department, Chemistry, DEI) for
their support in instrument Spectrophotometry and XRD.
facility
for
U.V.-Vis
REFERENCES [1] [2]
[3] [4] [5]
[6]
[7]
[8] [9]
[10] [11]
[12]
[13] [14] [15] [16] [17]
[18]
[19] [20]
[21]
WHO | Malaria http://www.who.int/mediacentre/factsheets/fs094/en/ NATIONAL VECTOR BORNE DISEASE CONTROL PROGRAMME http://nvbdcp.gov.in/doc/annual-report-nvbdcp-2014-15.pdf PAHO WHO | Integrated Vector Management (IVM) http://www1.paho.org/english/GOV/CD/cd48-13-e.pdf?ua=1 Soni N, Prakash S. Possible mosquito control by silver nanoparticles synthesized by soil fungus (Aspergillus niger 2587). 2013. Soni N, Prakash S. Efficacy of fungus mediated silver and gold nanoparticles against Aedes aegypti larvae. Parasitology research. 2012 Jan 1;110(1):175-84. Ramyadevi J, Jeyasubramanian K, Marikani A, Rajakumar G, Rahuman AA, Santhoshkumar T, Kirthi AV, Jayaseelan C, Marimuthu S. Copper nanoparticles synthesized by polyol process used to control hematophagous parasites. Parasitology research. 2011 Nov 1;109(5):1403-15. Vijayakumar S, Vinoj G, Malaikozhundan B, Shanthi S, Vaseeharan B. Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2015 Feb 25;137:886-91. Benelli G. Research in mosquito control: current challenges for a brighter future. Parasitology research. 2015 Aug 1;114(8):2801-5. Rizwan M, Singh M, Mitra CK, Morve RK. Ecofriendly application of nanomaterials: Nanobioremediation. Journal of Nanoparticles. 2014 Jun 19;2014. Brașoveanu M, Nemțanu MR. Natural polymers used as matrices for metallic nanoparticle synthesis by ionizing irradiation. S.P. Minal, “Efficacy of Bimetallic Copper-Zinc Nanoparticles against Larvae of Microfilariae Vector Culex quinquefasciatus (Say) in Laboratory”, M.Sc. Dissertation, Dayalabgh Educational Institute (DEI), Agra, India - 2015 S.P. Minal “Comparative Study of Two Bimetallic Nanoparticles for Controlling Mosquito Vectors of Public Health Concerns” M.Phil. Dissertation, Dayalabgh Educational Institute (DEI), Agra, India 2016 LARVICIDES, M., “Guidelines for laboratory and field testing of mosquito larvicides.” 2005 Abbott WS. A method of computing the effectiveness of an insecticide. J. econ. Entomol. 1925 Apr 1;18(2):265-7. Finney DJ. Probit Analysis: 3d Ed. Cambridge University Press; 1971. Filipe AR. Isolation in Portugal of West Nile virus from Anopheles maculipennis mosquitoes. Acta virologica. 1972 Jul;16(4):361. Fernandes T, Clode MH, Simões MJ, Ribeiro H, Anselmo ML. Isolation of virus West Nile from a pool of unfed Anopheles atroparvus females in the Tejo River estuary. Acta Parasitol Port. 1998;5(1):7. Thenmozhi V, Rajendran R, Ayanar K, Manavalan R, Tyagi BK. Long‐term study of Japanese encephalitis virus infection in Anopheles subpictus in Cuddalore district, Tamil Nadu, South India. Tropical Medicine & International Health. 2006 Mar 1;11(3):288-93. Lehrer S. Anopheles mosquito transmission of brain tumor. Medical hypotheses. 2010 Jan 31;74(1):167-8. Marimuthu S, Rahuman AA, Rajakumar G, Santhoshkumar T, Kirthi AV, Jayaseelan C, Bagavan A, Zahir AA, Elango G, Kamaraj C. Evaluation of green synthesized silver nanoparticles against parasites. Parasitology research. 2011 Jun 1;108(6):1541-9. He F, Zhao D. Preparation and characterization of a new class of starch-stabilized bimetallic nanoparticles for degradation of
chlorinated hydrocarbons in water. Environmental science & technology. 2005 May 1;39(9):3314-20.
