Evaluation of toxicity of Bordeaux Mixture in Aedes aegypti larvae (L. 1672) (Diptera: Culicidae) and Gram-negative and Gram-positive bacteria  

Amandha Kaiser da Silva1 , Eduardo Jose de Arruda1 , Gustavo Graciano Fonseca2 , Cláudio Teodoro de Carvalho1 , Cicera Maria da Silva1 , Paulo César Cavalcante Vila Nova3 , Cleusa Rocha Garcia Gaban3 , Isaías Cabrini1
1. Faculty of Exact Sciences and Technology - Federal University of Grande Dourados, Dourados, Brazil
2. Faculty of Food Engineering - Federal University of Grande Dourados, Dourados, Brazil
3. Department of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, Brazil
Author    Correspondence author
Journal of Mosquito Research, 2015, Vol. 5, No. 6   doi: 10.5376/jmr.2015.05.0006
Received: 02 Mar., 2015    Accepted: 18 Apr., 2015    Published: 22 May, 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Silva et al., 2015, Evaluation of toxicity of Bordeaux Mixture in Aedes aegypti larvae (L. 1672) (Diptera: Culicidae) and Gram-negative and Gram-positive bacteria, Journal of Mosquito Research, Vol.5, No.6 1-8 (doi: 10.5376/jmr.2015.05.0006)

Abstract

Dengue is an infectious disease transmitted by the bite of female Aedes aegypti during blood feeding, for continuity of the reproduction process. Population mosquito control has been carried out for decades, through the intensive use of conventional insecticides. The Bordeaux Mixture (BM) is a colloidal substance with insecticidal activity, bactericidal, fungicidal and molluscicide and can act comprehensively to control immature forms of A. aegypti, in addition to altering the physical and chemical conditions in the breeding and food chain insect. The values for the lethal concentrations of BM were CL10 1.05 mg L-1 (confidence interval 0.70 - 1.34), LC50 3.06 mg L-1 (2.73 - 3.35) and LC90 8.94 mg L-1 (7.42 - 11.92). The results of this study allow us to propose the BM as insecticidal potential for population control insect vectors.

Keywords
Biological activity; Aedes aegypti; Dengue; Cooper; Chemical control; Brazil
Dengue has been a major challenge to global public health due to control difficulties of Aedes aegypti vector, different viral serotypes, and socio-environmental problems such as lack of health infrastructure, rapid urbanization, population mobility, cultural habits as the elimination of mosquito breeding, deforestation and climate change (Barreto et al., 2011). Bhatt et al. (2013), by modeling showed the global distribution of dengue risk whose spatial variations are strongly influenced by periods of rain, temperature elevation and the degree of urbanization. We estimated the occurrence of approximately 390 million cases/year, and of these only about 100 million are reported. It is a fact that underreporting values ??are high, and the WHO estimates cases of the disease is three times higher. In Brazil, dengue occurs periodically, with different serotypes and differently among regions, alternating periods of epidemics and low occurrence. The introduction of different serotypes of the disease in areas previously not reported, low population immunity and inefficient actions of population control of insects by zoonoses control units have high tax costs to the government and the population (Taliberti and Zucchi, 2010). The geographical area in dengue tends to increase due to mosquito dispersion and climate change. Dengue case notifications in children under 15 years are worrying. These children are subject to infections secondary heterologous dengue and are at higher risk for severe cases of the disease (Verhagen and Groot, 2014). Methods to minimize the incidence of the disease permeate the population mosquito control, mainly through insecticides and education. Currently a polyvalent vaccine is in advanced study phase. The study demonstrated clinical efficacy in children above 50% (Capeding et al., 2014). The pathophysiology, and the prospects of the vaccine and continuity of actions and strategies for vector control are essential to containing and reducing disease (Simmons et al., 2012; Villar et al., 2015). However, despite advances, the population reduction vector by applying biological or synthetic insecticide remains the main control strategy for surveillance in public health (WHO, 1981).
The integrated management strategies using tools such as the alternation of insecticides, reduction of breeding and health education are crucial points for disease reduction and socio-economic and environmental impacts (Lefevre et al. 2003; Lefevre et al., 2014; Macoris et al., 2014). Currently, the mosquito has also been proven as vector Chickungunya virus whose prevalence has increased rapidly by the lack of immunity of the human host (Brasil, 2014). Studies are important to guide the disease control strategies, encourage research into vaccines, new insecticides, improve vector control methods and allow to estimate the economic impact of the disease.
The Bordeaux Mixture (BM), first used in 1882 in Bordeaux, France, is a gelatinous colloidal suspension with fungicide property, bactericide and insecticide (Motta, 2008). It is produced by solubilization of pentahydrate copper sulphate (CuSO4·5H2O) followed by mixing the dispersion of quicklime (CaO) in defined proportions. The addition of calcium oxide (CaO) in water forms calcium hydroxide, Ca(OH)2, making it alkaline. This mixing process is the formation of precipitation membranes and/or colloidal vesicles and formation of complex compounds of copper and calcium. The mixture is held in non-metal container with a pH close to neutral setting (Pscheid and Ocamb, 1999). Several constituents of the BM are known: 10CuO·SO3·4CaO·SO3, CuO·SO3·CaO; 5CuO·SO3·2CaO, 5CuO·SO3 (Narayan, 1949). As a fungicide, the BM is marketed in the form of wettable powder with molecular formula Ca3Cu4H6O22S4.H2O tetracobre or tricalcium sulfate, Cu4Ca3(SO4)2, CAS: 8011-63-0, containing 74% to 77% solid and 14,80% to 20% (p/p) copper as copper sulfate and calcium. The molecular structure is presented 3Cu(OH)2·CuSO4 with 257,69 g mol-1. The action of the BM fungal spores in the enzymatic inactivation occurs by Cu (II) occurring osmotic absorption and accumulation of Cu (II) on spores or hyphae (Hislop and Mapother, 1969; PAN, 2015). The action of cupric compounds is comprehensive, but mainly on enzyme activity and alterations of vital metabolic pathways of the microorganism. The metal acts in cellular respiration, membrane permeability and activity of enzymes that have sulfhydryl groups, hydroxyl, amino or carboxyl. The inactivation of the enzymes of the fungus causing metabolic disorder increases the permeability of cell membranes and the disruption of cellular integrity with cell death and leakage of the microorganism. The metal activity is multi-site type, interfering simultaneously in the cells, caused by the comprehensive action of the compounds on different enzyme groups and acting in various metabolic pathways. The advantages of copper products such as copper oxychloride, copper hydroxide, cuprous oxide and the cupric compounds in the formulations of cupric compounds are fungal action by the increased resistance of plants and insect pest repellent, in addition to having the characteristic of protection and nutritional to vegetables (Hislop and Mapother, 1969; Rodrigues, 2006). BM has also been used as an herbicide for the control of aquatic plants invasive occurring near pipes (Rodrigues, 2006). The copper sulfate can also be used in low concentrations for the treatment of parasitic infections in fish, snails removal tanks, inhibition of most species of algae, as well as having bactericidal activity against microorganisms, for example, Escherichia coli (Broome and Donaldson, 2010). In this work the BM was evaluated as the insecticide and bactericide properties in A. aegypti larvae and Gram (+) and Gram (-).

 

1 Materials and Methods
1.1 Preparation of Bordeaux Mixture

 

BM was prepared using the methodology proposed by Motta (2008) and Penteado (2000) by mixing two solutions A and B: A) 100 g of copper sulfate pentahydrate (CuSO4. 5H2O) in water at 6.03% (w/v) and B) 100 g of calcium oxide (CaO) in water at 0.54% (w/v) for the formation of calcium hydroxide Ca(OH)2 and 180 g of calcium hydroxide/hydrated lime to 10 L of water. Solution A was slowly added to solution B, under stirring, with final adjustment of the pH between 7 and 7.5. the freshly prepared BM was frozen in a freezer at -6 °C for 24 h and dried in a lyophilizer for 72 h at -5 °C and 186 μHg.

 

1.2 Physical and Chemical Analysis
The overall reflectance spectra attenuated in the infrared (ATR) of BM and precursors CuSO4·5H2O e CaO were obtained from FTIR spectrophotometer Nicolet Is10 Thermo Scientific® using the ATR accessory with Germanium window. The determination of the measures of the average hydrodynamic radius of the particles of the BM in suspension were obtained by measures of Dynamic Light Scattering (DLS) in Zetasizer Nano ZS from Malvern® of FEQ/UNICAMP. BM samples were separated for analysis and aging stability in aqueous solution by SEM/EDS for a period of 24 h, 1, 2 and 3 months. The aged samples were frozen, freeze-dried and coated with Au-Pd Sputter Coater in Polaron, SC7620, VG Microtech (Uckfield, England) the LRAC/FEQ-Unicamp. The estimated thickness of the deposited layer of Au was measured by: Thickness = KiVt, where K = 0.17 Aº / mA.Volt.s; i = 3 mA; V = 1 V and t = 180 s. The thickness of coverage was estimated at 92Å. The micrographs and/or elemental microanalysis SEM/EDS was obtained in Scanning Electron Microscope with Energy Dispersive X-ray detector, Model SEM: Leo Model 440i and EDS: 6070 LEO Electron Microscopy/Oxford (Cambridge, England) with a voltage acceleration equal to 5 kV and beam current of 100 pA for the micrographs.
1.3 Bioassays of toxicity in Aedes aegypti larvae

 

Toxicity bioassays were performed according to the methodology described by WHO (1981). Groups of 20 larvae of third instar A. aegypti (Rockefeller) were placed in 40 ml aqueous solution of 0.5% DMSO with different concentrations of BM. As positive and negative control we used 0.5% DMSO and water, respectively. After 24 h of exposure was registered mortality being considered dead larvae that had no movement or did not respond to stimuli with a Pasteur pipette (Hartberg and Craig-Jr, 1970; Gadelha and Toda, 1985). For each toxicity test were used seven concentrations of from 0 to 100 mg L-1 in quadruplicate. Data were analyzed by Probit method (Finney, 1974) to obtain the LCs 10%, 50% and 90% with 95% confidence interval. The experiments were repeated three times every other day.

 

1.4 Bioassays of toxicity in gram-negative and gram-positive

 

Antibiograms were performed by Kirby-Bauer method (Bauer, 1966) to assess the BM in comparison with the antibiotics penicillin and streptomycin for gram-positive Gram (+) and Gram negative (-). The microorganisms were grown in Mueller-Hinton broth for 24 h at 37 °C. For inoculation, it used rates of 100 uL of the inoculum with an optical density of 1.0 and a wavelength of 600 nm. Disks of 6 mm diameter filter paper were impregnated with concentrations in quadruplicate, 1 mg L-1 to 1000 mg L-1 BM. Gram (-) bacteria were: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Salmonella typhimurium (ATCC 14028) and Gram (+): Listeria monocytogenes (ATCC 7644) and Staphylococcus aureus (ATCC 25923). The concentration and availability of Cu (II) BM, CuSO4(OH)2Cu3 is larger and potentially more toxic to the larvae of A. aegypti.

 

2 Results
2.1 Physical and Chemical Analysis

 

ATR copper sulfate pentahydrate,[Cu(H2O)4SO4].5H2O showed bands at 866 cm-1 to H2O in coordination, band 1089 cm-1 to ion SO4-2 free and band 3169 cm-1 to H2O uncoordinated (Figure 1). CaO in the spectrum can be observed in a band 3641 cm-1 concerning the oxygen-metal bond. In the BM spectrum showed a band at 1112 cm-1 concerning the ion SO4-2 free, band 1620 cm-1 HOH on the folding bands, and 3535 cm-1 and 3404 cm-1 referring to stretch OH. These results are consistent with the literature, and suggest the formation of copper sulphate dibasic CuSO4(OH)2Cu3·H2O (Pscheid and Ocamb, 1999).In DLS, the average Z value can be used for product quality control purposes. The results showed a narrow, unimodal distribution with a polydispersity index of 0.152, Z-average mean diameter of 2,563 nm and of 5,126 microns particle, which is higher than the value reported for colloidal particles. It can be concluded that the sample is a heterogeneous suspension of particle aggregates and/or agglomerates colloid. BM when prepared in the form of a colloidal dispersion has activity for up to 3 months depending on the form of preparation, storage and pH. The freshly prepared and dried product has activity for up to two years. The aging of the colloidal dispersion and/or loss of activity of BM was investigated by SEM/EDS from the modifications and structural composition of the metastable compounds and/or morphological changes. In Figure 2 are shown the structures of the sols from 0 to 3 months after freezing and freeze drying.The micrographs (Figure 2) show the aging in aqueous medium of BM 24 h samples (A) up to 3 months after preparation (D). It can be seen that specimen 1 month (B) still has a number of crystals needle type. After this period, the samples at 2 months (C) and 3 months (D) have a lower number of needle-like crystals and a larger number of agglomerates/ aggregates. The needle-like crystals are crystals of copper sulphate (CuSO4).


Figure 1 Spectra ATR Bordeaux Mixture, pentahydrate copper sulphate and calcium oxide


Figure 2 Micrographs of Bordeaux mixture (BM) in different periods of storage: A = 24 h, B = 1 month, C = 2 months and D = 3 months. Bar: 10μm and Magnification = 1.50 kx

2.2 Bioassays of toxicity in Aedes aegypti larvae
The values found for the BM of Lethal Concentrations (LC) were: LC10 1.05 mg L-1 (confidence interval 0.70 -1.34), LC50 3.06 mg L-1 (2.73 - 3.35) and LC90 8.94 mg L-1 (7.42 - 11.92). These results LC50 can be compared to other cupric compounds of the literature, showing its most toxic to mosquitoes. The LC50 for larvae of 3rd and 4th stages of A. aegypti available in the literature are: Rayms-Keller et al. (1998) gave 33 mg L-1 for Cu (II), Arruda et al. (2011) to Na2[Cu(II)(EDTA)] obtained 32.65 mg L-1, Nardeli et al. (2014) acetate to Cu (II) 26.91 mg L-1 and Raes et al. (2000) 120 to 160 mg L-1. Gopinathan and Arumugham (2015) to Anopheles subpictus and Culex quinquefasciatus obtained LC50 in the range from 10.0 to 0.625 mg L-1.
2.3 Bactericidal activity
Results of the agar diffusion test for inhibition zones for the BM and antibiotics patterns are shown in Figures 3 and 4. Possibly, the heterogeneity of colloidal particles and the size distribution influence the spread of the gel and thus interfere with the results bactericidal activity and justify the reduction in activity with increasing concentration. At the concentration of 1 mg L-1 BM exhibited the same inhibition zone diameter in relation to the standard antibiotics.


Figure 3 Diameter of inhibition halos of the Bordeaux mixture for gram (-) bacteria and Gram (+)


Figure 4 Diameter of the inhibition zones for antibiotics standards

3 Discussion
The storage BM in aqueous media allows the acid-base type reactions continue to occur and leading to formation of microcrystalline needles and aggregates in the form of plates. The adsorption and reaction of the Cu (II) is accompanied by change of habit of the crystals. These reactions are similar to the formation of tabular crystals, equivalent to that obtained by the addition of NaOH and Cu (II). Reactions for the formation of sulfates hydroxides are slow, but cause decomposition of the blue crystals, becoming greenish. This fact can be explained by reaction with copper hydroxides until the formation of carbonates. The images A, B, C and D (Figure 2) show that the aging of the BM in the aqueous medium, the consumption of copper sulfate crystals in the formation of more complex compounds. The formation of agglomerates/aggregates, possibly by coalescence occurred with changes in size and shape. The process of formation of more complex compounds, justifies the loss of biological activity of BM within 1 to 2 months, possibly by unwillingness of the Cu (II).
A study with clones of Aedes albopictus cells C6/36 as a model system for analysis of metals in insects shows the toxic effects of Cu (II) on C6/36 cells. The effects of Cu (II) were similar to Cd (II) and Hg (II). The Cu (II) induced microtubule hyperpolymerization, cell aggregation and apoptosis. The reported process of apoptosis is dependent on the cell density and reaches a maximum in the concentration range 0.75 to 1 mM Cu (II) (120 to 160 mg L-1. The Cu (II) in concentrations induced cell death by necrosis in concentration of 0.75 mM, apoptosis was started after 18 h of exposure and the number of apoptotic cells increased almost linearly up to 42 h exposure level achieved in 70-80% of apoptotic cells (Raes, 2000). Study for analysis of metal tolerance was performed with eggs that hatch during the rainy season, which did not experience the quiescence or prolonged effect of latency. The results showed that this generation of larvae can regain competence to metal tolerance. This behavior can be extended to the mosquito's response to other forms of stress: pollution, oxidative stress and metabolism of insecticide tolerance and larval competition. The study showed that prolonged standing of the eggs can contribute to the increase of the viral vector and may have implications load control strategies. The mosquito larvae from newly hatched eggs at the end of the rainy season and early dry are physiologically compromised, and therefore more susceptible to vector control strategies than the larvae that hatch during rainy periods. The larva has reduced tolerance to metals, which affects larval development. These facts may be disclosed as a result of lack of sufficient nutritional reserves.
Alternative scenarios may account for the reduced mortality due to increased development and availability of metals causing direct effect on the availability of metals or storage of nutrients and/or through additional metal induced physiological demands. The metal ions can be complex with amino acids and proteins and affect metal bioavailability in the insect, interfere with enzymatic processes, cause peroxidation of polyunsaturated fatty acids, reductions in reserve lipids and damage to the digestive system of insects (Perez and Noriega, 2014). Cu (II) were evaluated for larvicidal activity against Anopheles subpictus and Culex quinquefasciatus. Bioassays were performed with mosquito larvae with the complex [Cu (phen) (L-Thr) (H2O)] (ClO4) with semicarbazide, thiosemicarbazide, urea and thiourea. Dose-response experiments were performed at concentrations ranging from 10.0 to 0.625 mg L-1 metal complexes. The complex with urea showed larvicidal activity against C. quinquefasciatus and A. subpictus at all concentrations. The complexes of Cu (II) showed larvicidal activity and concentration-dependent activity (Perez and Noriega, 2012). Copper is a micronutrient and constituent of enzymes involved in the breathing process, protection against oxidative stress, pigmentation and iron metabolism, however, is toxic in high concentrations (Egli et al., 2006; Gopinathan and Arumugham, 2015).
The studies with BM showed that the toxicity of the compounds Cu (II) is related to the destruction of the peritrophic matrix, digestive system and/or severe and irreversible cell damage in the insect's metabolism (Rayms-Keller et al., 1998; Raes et al., 2000). The Cu (II) was active for the larvae of A. aegypti by destruction of peritrophic matrix and induction "in vivo" oxidative stress by free radicals and oxidizing species with cellular damage and insect death. These damages are similar to those reported for plant extracts, tannins and essential oils (Gusmão et al., 2002; Trexler et al., 2003; Levy et al., 2004; Abed et al., 2007; Andreini et al., 2008; Kato et al., 2008; Magalhães, 2014). One can propose that BM has similar toxicity mechanism that caused by plant extracts, tannins and essential oils.
The high toxicity of BM can be explained by the amount and availability of 20% of Cu (II) to the larvae of A. aegypti (Sapec, 2012; Bayer, 2007). Other studies of metal complexes of Cu (II) showed that the APCs toxicity of Cu (II) compounds is related to the destruction of peritrophic matrix (PM) and damage to the digestive system and/or metabolic activity at the cellular level. The metal complexes (metal-insecticides) cause severe and irreversible damage to the insect's metabolism, mainly to the midgut (Arruda et al., 2011). The Cu (II) shows activity to the larvae of A. aegypti by destruction of peritrophic matrix and induction "in vivo" oxidative stress producing free radicals and oxidizing species induced by metal ion, resulting in irreversible cell damage and insect death in the larval stage (Rayms-Keller et al., 1998; Nardeli et al., 2014; Magalhães, 2014). Thus, one can propose that the BM has the same toxicity similar mechanism in relation to the metal ion Cu (II) whereas the BM has high concentration and metal availability for induction of oxidative stress and cell damage to the insect's metabolism.
BM showed insecticide and biological activity against gram (-) bacteria and gram (+). The BM can be proposed as a low cost and easy to use insecticide for the control of immature mosquito’s present toxicity, composition with micro and macronutrients for plants and microorganisms, reducing the environmental impact. The results showed that the compound has insecticidal and bactericidal activity, and can be used with advantage for the control and imposition of unfavorable conditions for the insect toxicity, changes in the breeding water parameters, control of microorganisms and the food chain of insects.
The presence of microorganisms in the container, as well as products of metabolism and high concentration of organic material tend to decrease the concentration of oxygen and favors larvae hatching. In the control of A. aegypti is considered that the availability of breeding and larval food are important factors, and for oviposition and continuity lifecycle availability of organic matter and microorganisms directly influence the growth and the emergence of the adult insect (vector) (Levy et al., 2004; Abed et al., 2007). Oviposition response of pregnant females of Ae. albopictus for attractive chemical factors, drawn by organic infusions with bacteria was evaluated. Water containing Psychrobacter immobilis (from a nursery), Sphingobacterium multivorum (from ground) and Bacillus spp. (from infusion of oak leaves) resulted in greater oviposition rate than treatment with water and bacteria (Trexler et al., 2003).
4 Conclusions
BM showed insecticide and biological activity against gram (-) bacteria and gram (+). The BM can be proposed as a low cost and easy to use insecticide for the control of immature mosquito’s present toxicity, composition with micro and macronutrients for plants and microorganisms, reducing the environmental impact. The results showed that the compound has insecticidal and bactericidal activity, and can be used with advantage for the control and imposition of unfavorable conditions for the insect toxicity, changes in the breeding water parameters, control of microorganisms and the food chain of insects.
Acknowledgment
FUNDECT/MS, CNPq, CAPES/PROCAD 2007 e 2008, LRAC/FEQ-UNICAMP, Dengue Group MS, BASF and UFGD.
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WHO World Heath Organization, 1981, WHO-VBC., 81, 807

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