DESC:TECHNICAL FIELD
The present disclosure relates to a composition for controlling mosquitoes. The present disclosure also relates to a process for preparing a composition for controlling mosquitoes.

BACKGROUND
Mosquitoes are one of the deadliest animals in the world. Their ability to carry and spread disease to humans causes millions of deaths every year. As per a study by the World Health Organization (WHO), in 2015, malaria alone caused 438,000 deaths. The worldwide incidence of dengue has risen thirty-fold in the past thirty years and more countries are reporting their first outbreaks of the disease. Zika, dengue, chikungunya, and yellow fever are all transmitted to humans by the Aedes aegypti mosquito. More than half of the world’s population lives in areas where this mosquito species is present. Sustained mosquito control efforts are important to prevent outbreaks from these diseases.
Malaria is endemic in 91 countries, with about 40% of the world's population at risk. By undermining the health and working capacity of hundreds of millions, it is closely linked to poverty and stunts social and economic development. Up to 500 million cases occur every year, 90% of them in Africa, and there are up to 2.7 million deaths annually. Dengue is the world's most important mosquito-borne viral disease, with 2,500 million people worldwide at risk of infection and 20 million cases a year in more than hundred countries. In 1995, the worst dengue epidemic in Latin America and the Caribbean for 15 years struck at least 14 countries, causing more than 200,000 cases of dengue fever and almost 6,000 cases of the more serious dengue hemorrhagic fever. More than 524,000 dengue cases were officially registered in India in last 4 years.
Many major cities of the world in Asia, Africa, and the Latin Americas, are at risk of potentially devastating epidemics of yellow fever because they are infested with Aedes aegypti mosquitos which can transmit the disease.
A mosquito is a common insect in the family Culicidae. There are around 3,500 species of mosquitoes which may carry numerous diseases. Below are some of the common species of mosquitoes and the diseases they carry and spread in the Indian subcontinent.
• Aedes - Chikungunya, Dengue fever, Lymphatic filariasis, Rift Valley fever, Yellow fever, Zika
• Anopheles - Malaria, Lymphatic filariasis
• Culex - Japanese encephalitis, Lymphatic filariasis, West Nile fever
There are four stages in a mosquito’s lifecycle, namely: egg, larva, pupa, and adult or imago. A gravid (pregnant) female mosquito lays her eggs either individually or in attached groups called egg rafts on water surface or along its edges. Generally, an adult female mosquito lays a hundred to two hundred eggs at a time. However, this number may vary depending on species, climatic conditions, etc. These eggs can survive up to eight months before hatching. Most of the eggs hatch into larvae within the first 48 hours. As the larvae feed, they grow and undergo transformation by shedding their skin (molt) four times. The stages between these molts are called instars. Based on size and time of molt, the larval stage has four instars. Larvae feed on aquatic microorganisms and organic matter in the still water. Most of the larvae have an air tube called siphon for breathing and hang upside down from the water surface to breathe. The Anopheles mosquito larvae do not have a siphon tube; hence they lay parallel to water surface to get oxygen supply through a spiracular opening.
After the fourth instar, the larva turns in to a comma shaped being known as a pupa. Pupae dwell under the water surface and are pretty much mobile. In this stage, pupae do not feed, but come to the surface from time to time to breathe from two breathing tubes known as “trumpets”. Once it is ready to emerge, the pupal skin splits along the back allowing the adult mosquito to slowly come out and rest on the water surface for a couple of hours to harden its exoskeleton and dry its wings. Once dried, it spread its wings and flies. After couple of days, male and female mosquito begin to mate. After successful mating, the female adult mosquito feeds on human or animal blood for the egg development.
Mosquitoes serve as vectors for disease-causing viruses and parasites from person to person without being infected with the disease themselves. Various species of mosquitoes feed on humans.
The key components of mosquito control include surveillance (determining species and density of mosquitoes in a given area), physical control or source reduction (e.g. removing/ emptying stagnant water from breeding sites), larvicide (in cases where source reduction is not feasible)) and adulticide (since it is impossible to eliminate breeding habitat or control mosquito before they become adult, insecticides should be used). Mosquito control in early stages is a very effective way of reducing the incidence of mosquito borne diseases.
Use of chemical insecticides has been prevalent from long time now to control mosquitos. Use of organophosphorus and synthetic pyrethroid chemical-based insecticides has proved to be inefficient against mosquitos in aquatic stages. Use of such chemical compound for prolonged period has created resistance towards mosquitos. Additionally, excessive use of such compounds pollutes the overall aquatic habitat which is a serious environmental issue.
WO 2013/140274 A2 (priority from Indian Patent application - 335/KOL/2012) discloses a formulation comprising base oil/s and surface-active agents. The Mosquito Larvicidal Oil (MLO) product forms a thin oil layer on the water surface and kills mosquito by depriving them of oxygen. The MLO remains on the water surface without cracking and uniform for minimum 2 hours. This patent recommends MLO dosage rate of 9.6 ml/ sq. mt. As per Government of India guidelines MLO product needs application on weekly basis. The said patent indicates mortality rate of 90% after 24 hours, it also mentions MLO will remain on the water surface without cracking and uniform for minimum 2 hours.
US3499969A discloses a formulation with combination of ethoxylated 2,4-dinonyl phenol, diesel oil and paraffinic oil. The formulation has toxicity towards mosquito larvae and pupae. This patent uses ethoxylated nonylphenol in the formulation, which is environmentally hazardous. It causes feminization of aquatic organisms; decreases male fertility also decreases survival chance of young infant fish. It has estrogenic effect on human body hence not suitable for prolong human handling. For women it can create unbalanced cytokine which can result in implantation failure, pregnancy loss and other complication like breast cancer. The composition disclosed has no such effects and is environment friendly.
US4166112A discloses the use of biological spore forming bacteria of Bacillus Thuringinsis species. This product, once consumed, demonstrates larvicidal effect on target organisms i.e. mosquitoes. It uses biological agent which is not effective and is extremely costly. Most of this product is in powder form which at first needs to be dissolved in water to activate the bacteria and then sprayed into water. Hence there is additional cost of carrying out the procedure, using extra resources and wastage of water is involved. This invention would not work if the bacteria are not activated. The composition disclosed is a one-step solution and does not require additional resources for activation.
US4155995A discloses a formulation with larvicidal petroleum oil dispersed with 0.3 – 2.0 percent of ethoxylated phenol having 2-5 ethoxy group, 0.2-0.8 percent of dialkyl polysiloxane. Additionally, toxicants such as Dichlorodiphenyltrichloroethane (DDT), Chlordane, lindane, malathion and other additives may be present. The cited patent uses ethoxylated phenol and other toxicants like DDT, Chlordane, lindane, malathion and other additives. DDT, Chlordane, lindane, malathion are hazardous insecticides that have been banned due to harmful effects on human and environment. Ethoxylated phenol is extremely harmful as it can cause serious eye damage, and skin irritation.
Hence, there is a need in the art for a composition for controlling mosquitoes that has a high mortality rate, which is also non-toxic to humans, plants, aquatic animals such as fishes, free of harmful chemicals, safe to be handled by humans and environment friendly.

OBJECTIVE
A primary objective of the present disclosure is to provide a composition for controlling mosquitoes by effectively killing mosquitoes.

Another objective of the present disclosure is to provide a composition for killing mosquitoes in aquatic habitat.

A preferred objective of the present disclosure is to provide a composition comprising of at least one mineral oil, silicon oil, and surfactant.

Yet another objective of the present disclosure is to provide a process for preparing a composition for killing mosquitoes.

A further objective of the present disclosure is to provide a composition which is free from harmful chemicals (insecticides, pesticides) and is biodegradable.

SUMMARY
In an aspect of present disclosure, there is provided a composition for killing mosquitoes in an aquatic habitant, wherein said formulation comprises of a low viscosity mineral oil in the range of 65 to 95 percent by volume (vol%), a medium viscosity mineral oil in the range of 17 to 23 vol%, silicon oil in the range of 1.5 to 5 vol% and surfactants in the range of 0.5 to 3 vol% and wherein the vol% is based on total volume of the composition.

In a preferred aspect of the present disclosure, there is provided a composition having an optimum kinematic viscosity range as well as an optimum surface tension value for forming an uninterrupted and uniform layer that does not crack on the water surface, resulting in effective suffocation and enhanced mortality of the mosquito larvae.

In another aspect of the present disclosure, there is provided a composition which forms a thin, unbreakable, and long-lasting layer on the water surface.

In an aspect of present disclosure, there is provided a process of preparing a composition for killing mosquitoes, described herein, wherein the process comprises
(a) Blending silicon oil and low viscosity mineral oil at 100 – 120 rpm for 20 to 30 minutes to obtain a mixture;
(b) Pouring the medium viscosity mineral oil in the mixture obtained in step (a);
(c) Adding the surfactant to the mixture obtained in step (b);
(d) Admixing the mixture of step (c) at 60 - 100 rpm for 30 to 45 minutes to obtain the composition.

These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

DETAILED DESCRIPTION
While the disclosed composition and method is susceptible to various modifications and alternative forms, specific embodiments thereof are described in detail in examples section below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.

The present disclosure provides a composition for controlling mosquitoes. The present disclosure provides a composition for controlling mosquitoes by effectively killing mosquitoes.
In one embodiment, the present disclosure provides a composition with a very high mortality rate. In another embodiment, the composition yields a mortality of 100 %. In another embodiment, the composition yields a mortality of at least 90 %. In another embodiment, the composition yields a mortality of 95 to 100%.

In one embodiment, the composition yields high mortality rates in a short duration of time. In one embodiment, the composition yields at least 90% mortality in less than or equal to 3 hours of application. In a preferred embodiment, the composition yields mortality of 90 to 100% of Aedes, Anopheles and Culex species mosquito larvae within 6 to 8 hours and pupae within 4 to 6 hours of application.

In yet another embodiment, the composition is effective in controlling mosquitoes in low doses. In another embodiment, the dosage of the composition is 2.5 ml or less per square meter of area. In a preferred embodiment, the dosage of the composition is 2.5 to 5 ml per square meter of area.

In another embodiment, the composition requires less frequency of application for effectively controlling the mosquitoes. In an embodiment, the recommended application frequency is once in weeks. In another embodiment, the recommended application frequency is once in 3 weeks or less. In a preferred embodiment, the recommended application frequency is once in 2 to 3 weeks.

The composition disclosed is non-toxic to plants and humans, in particular fish, that are present in the area of its application. The composition does not affect the environment and has no adverse effect on non-target organisms. Additionally, the composition is safe for handling without any specialized safety equipment.

The composition disclosed is free from harmful chemicals and is biodegradable.

In one embodiment, the present disclosure provides a composition for killing mosquitoes in aquatic habitat. In another preferred embodiment, the present disclosure provides a composition for killing Aedes, Anopheles, Culex and like.

In one preferred embodiment, the composition gives mortality rate of greater than 95% against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus mosquito.

In one embodiment, the composition comprises of at least one mineral oil, silicon oil, and surfactant. In one embodiment, the composition comprises of at least one mineral oil, silicon oil, surfactant, and emulsifier.

In a preferred embodiment, the composition for killing mosquitoes in an aquatic habitat comprises of at least one mineral oil, silicon oil, and surfactant. In another preferred embodiment, the composition comprises of two types of mineral oil, silicon oil, and at least one surfactant.

In one embodiment, the mineral oils are of varied viscosity. In another embodiment, the mineral oil is selected from mineral oil of high viscosity, mineral oil of medium viscosity, mineral oil of low viscosity, or a combination thereof.

In one embodiment, the mineral oils are of varied flash points.
Raw Material Viscosity Flash Point
Oil 1 2.5 cSt to 7.5 cSt >70oC
Oil 2 9.5 cSt to 15 cSt >200oC
Surfactant - >250oC
Silicon oil 350 Cps >250?

In an embodiment, the present disclosure relates to a composition for killing mosquito larvae or pupae, or both, in an aquatic habitat, the composition comprising a low viscosity mineral oil, a medium viscosity mineral oil, silicon oil, and one or more surfactants
In a preferred embodiment the composition comprises:
(a) a low viscosity mineral oil in the range of 60 to 98 percent by volume,
(b) a medium viscosity mineral oil in the range of 10 to 30 percent by volume,
(c) silicon oil in the range of 0.5 to 10 percent by volume, and
(d) one or more surfactants in the range of 0.1 to 10 percent by volume,
wherein, the percent by volume is with reference to the volume of the composition.

In another preferred embodiment the composition comprises:
(a) a low viscosity mineral oil in the range of 65 to 95 percent by volume,
(b) a medium viscosity mineral oil in the range of 17 to 23 percent by volume,
(c) silicon oil in the range of 1.5 to 5 percent by volume and
(d) one or more surfactants in the range of 0.5 to 3 percent by volume
wherein, the percent by volume is with reference to the volume of the composition.

The term “composition” used herein refers to the composition for killing mosquitoes, preferably mosquitoes in larvae or pupae stage.

A mere mixture of mineral oils and surfactant/ emulsifiers is not sufficient for making a good quality composition with desired spreading and film formation characteristics. Therefore, an optimized combination of mineral oil/s and surfactants/emulsifiers was selected based on the chemistry and nature of the emulsifiers to get a composition suitable for spreading and film formation characteristics. Optimum concentration of surfactant/emulsifier along with required kinematic viscosity of base oil/s were observed to achieve good spreading and film characteristics of the composition.

The present composition regulates the mosquito larvae and pupae population. The mosquito control composition disclosed demonstrates high efficacy and consistent results after application. It eliminates mosquito larvae and pupae present in water within 5-6 hours and 4-5 hours of application respectively by cutting off the oxygen supply to them and suffocating them, thereby leading to mortality rate of 100%. No prior art publication claims any composition which has an optimum kinematic viscosity range as well as an optimum surface tension value for forming an uninterrupted and uniform layer that does not crack on the water surface, resulting in effective suffocation and enhanced mortality of the mosquito larvae.

In one embodiment, the composition is sprayed or directly poured into the aquatic habitat.

In a preferred embodiment, the aquatic habitat is mosquito breeding sites, stagnant waters and larvae and pupae infested sites. The stagnant waters include but are not limited to, gutters, swamps, rain pools, stream shoreline, ponds, puddles, marshes, as well as artificial water containers like abandoned tires, flowerpot, buckets, or discarded toys.

In one embodiment, “Mineral oil” relates to the commonly known product of the same name, which is a by-product of the distillation of petroleum (crude oil) to make gasoline, cosmetics, pharmaceuticals, and many other products. Synonymous names for mineral oil include “paraffin oil” or “white mineral oil” or “petroleum oil” among other common names.

In one embodiment, the low viscosity mineral oil has a viscosity in the range of 2.5 cSt to 7.5 cSt.

In one embodiment, the medium viscosity mineral oil has a viscosity in the range of 9.5 cSt to 15 cSt.

In one embodiment, the silicones include polymers that include any synthetic compound made up of repeating units of siloxane, or a combination thereof. Some common forms of silicone used in the composition disclosed include, but not limited to, silicon oil, silicone grease, and silicone resin. The silicon oil is a non-toxic silicon-based oil having high quality, thermal stability, oxidation stability, chemical inertness, low flammability, low surface tension, and shear stability. Additionally, the silicon oil is non-corrosive, highly compressible, and exhibits small degree of change over a wide temperature range as compared to petroleum oil. Viscosity of the silicon oil is in a range of 200 cSt to 400 cSt.

In a preferred embodiment, the silicon oil is selected from the group consisting of methyl silicone, methylphenyl silicone, methyl hydrogen silicone, fluorosilicone, methylalkyl silicone, amino silicone, and vinyl silicone oil.

In one embodiment, the surfactants may include, but are not limited to, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, or a combination thereof. The surfactant in the present specification particularly is non-ionic, has C10 – C16 alkyl group, soluble in water, ethanol, carbon tetra chloride, toluene etc. It has having excellent stability to hard water, resistance to meter salts, acids and alkali, flash point greater than 250? and an HLB value range between 8 – 16.

In a preferred embodiment, the surfactants are selected from the group consisting alkyl and alkylphenyl polyethylene glycol ether, fatty acid alkanolamides, sucrose fatty acid esters, alkyl polyglucosides, trialkylamine oxides, alkylaryl polyglycol ethers and alkyl ether sulfates.

The composition disclosed has an optimum kinematic viscosity range as well as an optimum surface tension value for forming a stable uniform layer that does not crack on the water surface.

In one embodiment, the kinematic viscosity of the composition is in the range of 3 to 15 cSt at 28°C.

In another preferred embodiment, the composition has a surface tension less than 25 N/m at 28°C.

The invention disclosed cuts off atmospheric oxygen supply (air) to larvae and pupae who dwell under water. They come to the water surface at frequent intervals to get fresh oxygen supply. The composition forms an impregnable oil layer/ film which cuts off supply of oxygen to larvae and pupae. Unlike chemical and biological insecticides, larvae and pupae do not develop resistance to the composition, as resistance developments would need morphological changes or physical mutation of overall mosquito species which would enable them to penetrate the oil layer/ film.

Usage of only low viscous oil
Low viscosity mineral oil has volatile properties, high biodegradability, and low density which will form a very thin non-uniform layer of oil/ film that can be easily penetrated and broken by larvae and pupae during their respiration process from their siphon tube (in case of Aedes and Culex Larvae), piracular opening (in case of anopheles larvae), trumpets (in case of Pupae) or due to their wriggling, tumbling movement on the water surface.

Usage of Silicon Oil, Medium Viscous Oil and Surfactant
Silicon oil is viscoelastic and acts like an elastic solid at low temperature. Additionally, polydimethylsiloxane (PDMS) has excellent thermal and oxidation stability. PDMS fluid has uncommonly low surface tension which allows efficient spreading, high surface activity and low internal cohesive energies between each molecule. Lower viscosity PDMS are expensive and have low flashpoint and volatile weight loss of 6% at certain temperature. The oil layer/ film formed by this product is pregnable due to very low surface tension. Maintaining optimum surface tension for a prolonged time is key for any oil layer/ film to work efficiently. Direct usage of PDMS/ silicon Oil having viscosity greater than 200 cSt will cause the composition to settle under water and not produce the desired result.
Medium viscosity mineral oil has better volatile, thermal and oxidation stability than low viscosity mineral oil. Medium viscosity mineral oil does not have efficient spreading properties. Therefore, a composition with only medium viscosity mineral oil is not self-sufficient to form oil layer/ film.
The inventors have surprisingly found that combining low and medium viscosity oil provides better environmental and oxidation stability of the mixture formed.
Surfactants allow low viscosity mineral oil, medium viscosity mineral oil and silicon oil to emulsify and reduce internal cohesive force between various molecule thus providing synergistic effect between all the components which further enable overall product to maintain optimum surface tension, spreadability and stability of oil layer/ film for at least two weeks.
The composition disclosed is used in the preservation of vector bone diseases. The vector born disease include, but are not limited to, chikungunya, dengue fever, lymphatic filariasis, rift valley fever, yellow fever, zika, malaria, lymphatic filariasis, Japanese encephalitis, lymphatic filariasis, west Nile fever and onchocerciasis

The present disclosure also relates to a process for preparation of a composition for killing mosquito larvae or pupae, or both, in an aquatic habitat.

In one embodiment, the process comprises of blending silicon oil and mineral oils of varied viscosity to obtain a mixture; adding surfactant(s) and/or emulsifier to the obtained mixture of oils, followed by mixing to obtain the composition.
In a preferred embodiment, the process comprises of:
(a) Blending silicon oil and low viscosity mineral oil at 100 – 120 rpm for 20 to 30 minutes to obtain a mixture;
(b) Pouring the medium viscosity mineral oil in the mixture obtained in step (a);
(c) Adding the surfactant to the mixture obtained in step (b);
(d) Admixing the mixture of step (c) at 60 - 100 rpm for 30 to 45 minutes to obtain the composition.

Application:
The composition disclosed creates a single, uniform, and homogenous film over the water surface. The composition works in two ways as given below.
1. The oil film prevents the larvae and pupae from penetrating the oil film, thus cutting off the atmospheric oxygen supply, resulting in suffocation and death of the larvae and pupae.
2. Once the larvae and pupae come in contact with the sticky oil film composition, it saturates the larvae’s siphon tube and further the tracheal system. In case of anopheles larvae, the oil film composition saturates the spiracular opening. In case of pupae, the composition chokes the two breathing tubes or “trumpets”. Larvae may unsuccessfully try to remove the blockage at the siphon through mouth. The larvae and pupae eventually die due to exertion and lack of oxygen.

The experimentation indicated that pupae die earlier than larvae. Therefore, it is the most effective and practical solution for mosquito control.

EXAMPLES
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1:
The composition and process involved are illustrated below:
Table 1: Composition for killing mosquitoes
Ingredients Quantity
Low viscosity mineral oil 65 vol%
Medium viscosity mineral oil 27 vol%
Silicon oil 5 vol%
Surfactant 3 vol%

Table 2: Composition for killing mosquitoes
Ingredients Quantity
Low viscosity mineral oil 75 vol%
Medium viscosity mineral oil 20 vol%
Silicon oil 4 vol%
Surfactant 1 vol%

Table 3: Composition for killing mosquitoes
Ingredients Quantity
Low viscosity mineral oil 85 vol%
Medium viscosity mineral oil 10 vol%
Silicon oil 3 vol%
Surfactant 2 vol%

Characteristics of the composition:
• Relative density lower than 0.95
• Flash point greater than 70?
• Kinematic viscosity in a range of 3 cSt to 15 cSt
• Spreading pressure of 1.8 X 10 -2 N/m
• Pour point till 7?

Example 2:
Preparation of the composition
The manufacturing process was carried out at normal environmental temperature (around 28°C) / condition in an enclosed facility. Low viscosity mineral oil was pumped from a storage tank to a mixing tank. Silicon oil was transferred inside the mixing tank. Agitation in the mixing tank was carried out at 110 rpm for 25 minutes using specialized impeller. This allows high viscosity silicon oil to disperse into low viscosity mineral oil to form a uniform mixture. Once consistency is achieved, medium viscosity mineral oil and surfactants were transferred in the mixing tank. Agitation in the mixing tank was carried out at 80 rpm for 40 minutes. Slow speed continuous mixing for elongated periods of time allows each component to efficiently distribute the particles and create a more homogeneous solution. While the manufacturing process is going on, the resultant product is tested in the laboratory to make sure that it meets all the desired specifications. Once desired results are achieved, product is passed through series of fine filters to remove any solid / foreign particle and is stored further in packaging tank.

Example 3:
Evaluation of efficacy of the composition
Environmental Conditions
Larvae (4th instar), pupa and eggs of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus maintained in insect breeding laboratory were used for the experiment. The experimental room temperature and humidity were recorded. The mean maximum temperature (ºC) during study period was 27.6 ± 1ºC. The mean maximum relative humidity (%) during study was 67.8 ± 5% respectively. In the experimental room, 12 hours of artificial lighting and 12 hours of darkness were maintained.
The experimental room was cleaned and mopped daily with Liquid disinfectant.
Diet and Water
For larvicidal and pupicidal effect, the bioassay duration was six hour no need of larval food. In case of oviposition assay only cotton soaked in glucose water was kept in cage overnight.
Grouping
Larvicidal study: A batch of 25 , 4th stage larvae/formulation /trial/ replicate was used.
Pupicidal study : A batch of 25 pupa/formulation /trial/ replicate was used.
Oviposition study: A batch of 10-15 female mosquito /trial/replicate was used.
Persistency study: A batch of 25, 4th stage larvae/replicate was used every week.
Efficacy testing of sample involves -1. Larvicidal Effect 2. Pupicidal Effect 3. Effect on Oviposition 4. Persistency study for Single dose of the sample used for the experiment.
The test sample comprised of polydimethylsiloxane 2%, low viscosity and medium viscosity mineral oil and a surfactant.

Larvicidal Effect
The larvicidal effect of test sample was tested against fourth instars (4–8 days old) separately. Four glass beakers (500ml) were filled with 250ml of water and 25 larvae were added to each beaker. Using the recommended dose of 2.5mL/m2, sample was applied to all beaker. One beaker remained untreated and served as the control. The beakers were allowed to stand undisturbed condition for observations. All larvae (4th instar) were observed for mortality and conversion (if any) to next stage for 6 hours. Mortality was checked after every hour. Two experiments with three replicates were performed. All three species were tested as above.

Table 4: Evaluation of the efficacy of sample against 4th stage larvae of Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus
Mosquito species Experiment R Percentage of mortality of larvae at time intervals (In hrs.)
4th instar larvae
Aedes aegypti Experiment I Hrs. 30 min 1 2 3 4 %M
R1 4 48 80 92 100 100%
R2 8 56 80 100 100 100%
R3 0 48 76 92 100 100%
Mean 4 50.66 78.66 94.66 100 100%
Aedes aegypti Experiment II R1 4 36 68 92 100 100%
R2 4 44 88 100 100 100%
R3 0 52 84 100 100 100%
Mean 2.66 44 80 97.33 100 100%
Anopheles stephensi Experiment I R1 4 36 76 92 100 100%
R2 0 32 80 100 100 100%
R3 12 48 92 100 100 100%
Mean 5.33 38.66 82.66 97.33 100 100%
Anopheles stephensi Experiment II R1 0 48 88 100 100 100%
R2 4 56 92 100 100 100%
R3 8 64 88 100 100 100%
Mean 4 56 89.33 100 100 100%
Culex quinquefasciatus Experiment I R1 8 56 92 100 100 100%
R2 12 48 96 100 100 100%
R3 0 48 80 92 100 100%
Mean 6.66 50.66 89.33 97.33 100 100%
Culex quinquefasciatus Experiment II R1 4 56 92 100 100 100%
R2 8 64 88 100 100 100%
R3 8 56 88 100 100 100%
Mean 6.66 58.66 89.33 100 100 100%
Control 0 0 0 0 0 0 0

Table 5: Evaluation of the efficacy of control against 4th stage larva of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus
Mosquito species Expt. Percentage of mortality of larva at time intervals Mean values (In hours)

4th instar larvae
Aedes aegypti Hrs. 1 2 3 4 %M
Experiment I 4 50.66 78.66 94.66 100 100%
Experiment II 2.66 44.00 80.00 97.33 100 100%
Anopheles stephensi Experiment I 5.33 38.66 82.66 97.33 100 100%
Experiment II 4.00 56.00 89.33 100 100 100%
Culex quinquefasciatus Experiment I 6.66 50.66 89.33 97.33 100 100%
Experiment II 6.66 58.66 89.33 100 100 100%

Results: In Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus hundred percent (100%) mortality was observed in 3 to 4 hours, 3 hours, and 3 hours, respectively. In case of Larvicidal effect, mortality of Anopheles and Culex was faster than Aedes.
Pupicidal Effect – Three glass beakers (500ml) were filled with 250 mL of water and 25 pupae were added to each beaker. Using the recommended dose, 2.5mL/m2 sample was applied to all beaker. One beaker remained untreated and served as the control. Beakers were allowed to stand in undisturbed conditions for observations. All pupae were observed for mortality and conversion (if any) of pupa to mosquito. Mortality was checked after every hour. Two experiments with three replicates were performed. All three species were tested as above. A pupa was considered dead if it did not show the characteristic stretching reaction on slight dipping. All the treated and control beakers were covered with mosquito netting to prevent escaping of emerged adult mosquitoes. The entire experiment was repeated by using the same test sample.

Table 6: Evaluation of the efficacy of sample against Pupa of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus
Mosquito species Experiment R Percentage of mortality of Pupa at time intervals (In hours)
Pupa
Aedes aegypti Experiment I Hours ½ 1 2 3 4 %M
R1 8 12 72 100 100 100%
R2 12 16 76 100 100 100%
R3 28 44 100 100 100 100%
Mean 16 24 82.66 100 100 100%
Aedes aegypti Experiment II R1 24 40 100 100 100 100%
R2 16 32 68 100 100 100%
R3 16 24 72 100 100 100%
Mean 18.66 32 80 100 100 100%
Anopheles stephensi Experiment I R1 0 4 52 88 100 100%
R2 4 8 44 100 100 100%
R3 8 16 56 100 100 100%
Mean 4 9.33 50.66 96 100 100%
Anopheles stephensi Experiment II R1 0 4 72 100 100 100%
R2 8 12 80 100 100 100%
R3 4 12 68 92 100 100%
Mean 4 9.33 73.33 97.33 100 100%
Culex quinquefasciatus Experiment I R1 24 44 80 100 100 100%
R2 28 48 84 100 100 100%
R3 28 56 100 100 100 100%
Mean 26.66 49.33 88 100 100 100%
Culex quinquefasciatus Experiment II R1 24 36 72 100 100 100%
R2 32 52 100 100 100 100%
R3 28 48 100 100 100 100%
Mean 28 45.33 90.66 100 100 100%
Control 0 0 0 0 0%

Table 7: Evaluation of the efficacy of control against pupa of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus
Mosquito species Experiment Percentage of mortality of Pupa at time intervals Mean values (In hours)

Pupa Stage
Aedes aegypti Hours 1 2 3 4 %M
Experiment I 16 24 82.66 100 100 100%
Experiment II 18.66 32 80 100 100 100%
Anopheles stephensi Experiment I 4 9.33 50.66 96 100 100%
Experiment II 4 9.33 73.33 97.33 100 100%
Culex quinquefasciatus Experiment I 26.66 49.33 88 100 100 100%
Experiment II 28 45.33 90.66 100 100 100%

Results: In Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus hundred percent (100%) mortality was observed in 3 hours, 3 to 4 hours, and 2 to 3 hours respectively and no emergence of adult mosquito was observed in all three mosquito species. In case of Pupicidal effect, mortality of Culex was faster than Aedes and Anopheles. The results of the visual assessment showed that test sample possesses Larvicidal property and Pupicidal property.

Effect on Oviposition - Female mosquitoes, 4–6 days old, were blood fed. After 2 days, 10-15 gravid females were transferred to a cage that contained a 6% glucose-soaked cotton ball and oviposition plastic bowl (500 ml). For double choice experiment, the bowl was treated with test sample (2.5mL/m 2) and other served as the control. The bowls were placed at two opposite corners of the cage (24 ×24 × 24 cm). In test bowl, egg laying capacity of mosquito was observed, and number of eggs were counted. After 24 hours, the eggs laid in oviposition bowl were counted. Two experiments with three replicates were performed for oviposition assay. In the second experiment, the females were placed in a no choice situation. In this case, each cage contained only one oviposition bowl, a glucose-soaked cotton and 10-15 gravid females. Bowl was treated with test sample (2.5mL/m2). Two experiments with three replicates were performed. After 24 hours, the eggs laid in each oviposition bowl were counted.

Table 8: Oviposition assay
Mosquito species Experiment R* Single Choice Test Double Choice Test
Test Control Percentage of deterrence Test Control Percentage of deterrence
Aedes aegypti

Experiment I R1 36 322 88.81 20 282 92.90
R2 40 308 87.01 18 301 94.01
R3 39 318 87.73 24 290 91.72
Mean 38.33 316
87.85 20.66 291 92.87

Aedes aegypti

Experiment II

R1 44 326 86.50 22 300 92.66
R2 50 310 83.87 16 292 94.52
R3 36 316 88.60 14 284 95.07
Mean
43.33
317.33
86.32
17.33
292
94.08

Anopheles stephensi
Experiment I R1 10 240 95.83 04 220 98.18
R2 12 236 94.91 06 212 97.16
R3 07 225 96.88 03 202 98.51
Mean 9.66 233.66
95.87 4.33 211.33 97.95
Anopheles stephensi Experiment II R1 11 260 95.76 03 210 98.57
R2 06 230 97.39 03 218 98.62
R3 08 242 96.69 05 204 97.54
Mean 8.33 244
96.61 3.66
210.66 98.24

Culex quinquefasciatus
Experiment I R1 02 26 92.30 01 11 90.90
R2 03 20 85 02 15 86.66
R3 02 22 90.90 01 12 91.66
Mean 2.33 22.66 89.4 1.33 12.66 89.74
Culex quinquefasciatus
Experiment II R1 01 18 94.44 01 10 90
R2 02 24 91.66 01 14 92.85
R3 01 16 93.75 01 13 92.30
Mean 1.33 19.33 93.28 01 12.33 91.71

Results: Good results were obtained in single choice experiment as compared to double choice experiment. In double choice, it was observed that maximum eggs were laid on control as compared to test. In the single choice experiment, very less eggs were laid. In all three species, very less eggs were laid in test bowl as compared to control.

Persistency Study
Persistency study was carried out for four weeks. For the persistency study, two glass beakers (500ml) were filled with 250ml of water and 25 larvae were added to each beaker. Using the recommended dose, 2.5mL/m 2 samples was introduced only on Day 1 of Week 1 in all the beakers. One beaker remained untreated and served as the control. All the beakers were allowed to stand in undisturbed condition for observation. Fourth instar Aedes aegypti larvae were used for this study. All Aedes aegypti larvae (4th instar) were observed for mortality and conversion (if any) to next stage for 4 weeks. Every week, 25 new 4th stage larvae were added in the treated beaker. Mortality for the larvae were recorded for each week. Two Experiments were carried out for the shared sample.

Table 9: Persistency study
Experiment Test/Control Percentage mortality of 4th instar larvae in Hours
1st week 2nd week 3rd week 4th week
Experiment. I Test
(2.5ml) 100% within 4 hours 100% within 24 hours 100% after 48 hours 80% after 72 hours
Control 00% 00% 00% 00%
Experiment. II Test
(2.5ml) 100% within 3.5 hours 100% after 22 hours 100% after 44 hours 80% after 70 hours
Control 00% 00% 00% 00%

Results: The sample was found effective against Aedes Aegypti 4th stage larvae. The Larval mortality resulted 100% during first three weeks and about 80% at week four of the study.

Based on the results of the study:
It may be concluded that the composition (Dose – 2.5ml/m2) was effective against larvae and pupae of mosquito species, namely, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The sample has good deterrence effect against gravid female mosquitoes of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus species. Additionally, the sample was found effective against Aedes aegypti larvae for 2-3 weeks.
,CLAIMS:1. A composition for killing mosquito larvae or pupae, or both, in an aquatic habitat, the composition comprising a low viscosity mineral oil, a medium viscosity mineral oil, silicon oil, and one or more surfactants.

2. The composition as claimed in claim 1, wherein the composition comprises:
(a) a low viscosity mineral oil in the range of 60 to 98 percent by volume,
(b) a medium viscosity mineral oil in the range of 10 to 30 percent by volume,
(c) silicon oil in the range of 0.5 to 10 percent by volume, and
(d) one or more surfactants in the range of 0.1 to 10 percent by volume,
wherein, the percent by volume is with reference to the volume of the composition.

3. The composition as claimed in claim 1, wherein the composition comprises:
(a) a low viscosity mineral oil in the range of 65 to 95 percent by volume;
(b) a medium viscosity mineral oil in the range of 17 to 23 percent by volume;
(c) silicon oil in the range of 1.5 to 5 percent by volume; and
(d) one or more surfactants in the range of 0.5 to 3 percent by volume
wherein, the percent by volume is with reference to the volume of the composition.

4. The composition as claimed in claims 1 to 3, wherein the low viscosity mineral oil has a viscosity in the range of 2.5 cSt to 7.5 cSt.

5. The composition as claimed in claims 1 to 3, wherein the medium viscosity mineral oil has a viscosity in the range of 9.5 cSt to 15 cSt.

6. The composition as claimed in claims 1 to 3, wherein the silicon oil is selected from the group consisting of methyl silicone, methylphenyl silicone, methyl hydrogen silicone, fluorosilicone, methylalkyl silicone, amino silicone, and vinyl silicone oil.

7. The composition as claimed in claims 1 to 3, wherein one or more surfactants are selected from the group consisting of alkyl and alkylphenyl polyethylene glycol ether, fatty acid alkanolamides, sucrose fatty acid esters, alkyl polyglucosides, trialkylamine oxides, alkylaryl polyglycol ethers and alkyl ether sulfates.

8. The composition as claimed in claims 1 to 7, wherein the composition has a kinematic viscosity in the range of 3 to 15 cSt at 28°C.

9. The composition as claimed in claims 1 to 8, wherein the composition has a surface tension less than 25 N/m at 28°C.

10. A method for preparing a composition for killing mosquito larvae or pupae, or both, in an aquatic habitat, the method comprising:
(a) mixing silicon oil and a low viscosity mineral oil at 100 – 120 rpm for 20 to 30 minutes for obtaining a mixture;
(b) mixing the medium viscosity mineral oil and the mixture obtained in step (a); and
(c) mixing the one or more surfactants and the mixture obtained in step (b) at 60 - 100 rpm for 30 to 45 minutes for obtaining the composition.

11. The process as claimed in claim 10, wherein the silicon oil, the low viscosity mineral oil, the one or more surfactant, the medium viscosity mineral oil are as claimed in claims 1 – 9.