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Toxicity

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Toxicity tests of different plant crude extract on Mosquito Larvae

by
Martinez, Jose Djairus Mari C.
Oclarit, Jaymee
Navora, Jane Andrea
Baquiran, Sushmica
Cruz, Krystian Wilson Lee

A special problem submitted to Ma’am Teodora Ballancod
University of the Philippines Baguio in partial fulfillment of the requirements for Botany 109: Plant Taxonomy
Department of Biology
October 2011

Contents

Abstract………………………………………………………………………… 3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Background of the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Statement of Problem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Statement of Hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Significance of the Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Scopes and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Review of Related Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Research Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Recommendation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Works Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Abstract

This study aims to produce low cost, effective and environmental friendly mosquito wrigglers eradicator from 5 local plant of Baguio City. Extracts were tested for their effectiveness to kill mosquito wrigglers. Treatments were made with different concentrations such as 100%, 75% and 50% based on LD50 test. It was found out that the 100% concentration was the most effective. When added to water with 30 wrigglers however, the effectiveness of the pure extract decreases, such that it took more time and less number of killed wrigglers compared to the pure concentration. Though when compared to traditional larvicides, salt solution, result showed that the plant extracts were more effective than this. It is also comparable with commercial larvicides. The larvicidal effects of five plant extracts were tested against third instar (L3), of an Aedes aegypti. Probit analysis was used to analyze the data. The larval mortality was recorded at 24 hours with 1 hour interval basis. Neem (Azadirchta indica) proved to have the greatest toxicity against 3rd instar LD50 at 100% concentration. Aedes aegypti L. is the major vector of dengue fever, an endemic disease in the country. In an effort to find effective and affordable ways to control this mosquito, we have decided to conduct this research to help the community around Baguio City especially around those places belonging to dengue endemic places. The essential oils were extracted by larvicidal bioassay. These five local plants can be used as an effective substance and an alternative to kill mosquito wrigglers for it is environmental friendly, much cheaper to prepare and locally available.

Introduction
Dengue fever is endemic over large areas of tropics and subtropics. Outbreaks of dengue have repeatedly occurred in the Philippines over the last 10 years. The etiological agent is an arbovirus and the major vector is the Aedes aegypti mosquito.
Larviciding is one approach to vector control carried out at breeding centers of the vectors (Mohan and Ramaswamy, 2007). This is an effective way of reducing the population densities of mosquitoes before it emerge into an adult. Larval stages breed in water and are more easily dealt with in this habitat; therefore, they are attractive targets for pesticides (Chowdhury et al, 2008).
During the last decade, there have been attempts at development of alternative, environmentally friendly and sustainable way for mosquito control using natural products. These natural products utilized as mosquito insecticides limit the environmental impact of pesticides due to shorter latency, which may be beneficial for preventing the evolution of resistance (Hardin and Jackson, 2009). The control of mosquito larvae using indigenous medicinal plants is beneficial in developing countries, such as the Philippines. With all these factual things happening, this research is expected to give an alternative or a better ways of dealing with the problem. The purpose of this study was to investigate the potential for five plant crude extract, prepared of being used against the immature stages of mosquitoes. This research will help the community to used plant extract which are a way cheaper than commercial chemicals sold in markets.

Background of the study The alarming threat of malaria and dengue has grown for a few decades and about 2/5 of the world’s population is now at risk of this infirmity. As for the latest report of the Department of Health in their official website, a total of 38,876 dengue cases were reported from different disease reporting units nationwide from January 1 to July 23, 2011. This is 25.85% lower compared to the same time period last year (52,428). Weeks 28 and 29 contains partial data. Most of the cases were from the following regions: National Capital Region (23.74%), Region III (16.37%) and Region IV-A (14.53%). Ages of cases ranged from less than 1 month to 92 years old (median = 12 years). Majority (54%) of the cases reported were male. Most (36.76%) of the cases belonged to the 1 to 10 years age group (Fig. 2). Case fatality ratios greater than 1 were noted in the 1 to 10 years age group. There were 226 deaths (CFR 0.58%) reported. Reported cases with CFR greater than 1 came from Regions V, VI and ARMM. (http://www.doh.gov.ph)
In lined with these statistics and facts, the researchers have come up with a research that involves larvicidal application which is an effective way of reducing mosquito densities in their habitats before they develop into their adult stage. In this study, 5 plants, namely Gmelina (Gmelina arborea). Neem (Azadirachta indica), Oregano (Origanum vulgare), Lemon Grass (Andropogon citratus) and Basil (Ocimum basilicum), will be used with high toxic effect to these said organisms.
Larval stages occur primarily in water so it is easy for us to deal with it. Therefore this stage is an attractive target for pest-killers. Larvae, unlike adult mosquitoes, cannot change their behavior to avoid mosquito control measures (Killeen et al, 2002). During the last decade, there have been attempts at development of alternative, environmentally friendly and sustainable approaches for mosquito control using natural products with greater target specificity, lower bioaccumulation properties and reduction of the number of pests especially mosquitoes which is a problem of the world especially to the tropical countries (Benner, 1993)

Statement of the Problem This study aims to determine the toxicity differences among the five plant extracts as larvicides to mosquito wrigglers. The research specifically answers the following questions: 1. Which among the five plant extracts have the highest toxicity rate? 2. What active chemical components are present in these five plant extracts that effectively decrease mosquito wrigglers? 3. Is there a significant difference in the toxicity level among the five plants and commercially produced larvicides sold in the market?

Statement of the Hypothesis
Ho= There is no significant difference in the toxicity level of the five plant extracts subjected to different treatments.
Ha= There is a significant difference in the toxicity level of the five plant extracts subjected to different treatments.

Significance of the Study The relevance of this research lies on the possible result of wrigglers decreasing by the exploration of the different toxicity levels of the five plants in the production of the larvicides that is inexpensive and readily available to the environment. The use of these plants as larvicides may bring good health within the community specially, problems brought about by mosquitoes and its larvae that are uncontrollable and may bring much more damaging effects than it does now, if not treated. This study hopes to contribute to the upliftment of our country’s present economic and health status. This may bring about advantages in the use of natural resources that are locally available in the community, instead of relying on the chemical pest eradicators that are more often harmful to the environment and detrimental to human health. This project will also serve as a catalyst for further research in the utilization of other Philippine native plants.

Scopes and Limitations This research is confined only to the different toxicity of selected plants that are available in the Philippines, as an organic larvicide compared to commercial chemicals in the market today. The plants will be gathered within the vicinity of Baguio City same as the mosquito wrigglers that will be gathered from cultured laboratory of the Department of Science and Technology (DOST). Only the numbers of the wrigglers killed will be considered. To measure the effectiveness, time will be considered as the standard parameter. This study was conducted from the 4th of July 25th of September at the University of the Philippines Baguio, Baguio City. This study is limited to the following criteria: 1. Mosquito wrigglers were obtained from the cultured laboratory of the Department of Science and Technology Baguio City and some are from stagnant water. 2. This is limited in using 3 different concentrations: 100%, 75% and 50% based from the LD-50 test conducted.
Review of Related Literature

Mosquito species that share similar characteristics are separated by scientists into larger groups called genera, or genus as a singular name. Examples include Aedes(Ae.), Anopheles, Culex(Cx.), Culiseta, and Ochlerotatus (Och.). Aedes mosquitoes are daytime bites, which breed in floodwaters. Culex mosquitoes are evening/dusk feeders. They breed in both floodwaters and permanent bodies of water. Mosquitoes pass through four distinct stages-egg,larva,pupa,and adult. Mosquito larvae, also called “wrigglers”, survive best in still water, where they can come to the surface and breathe through a breathing tube. Most mosquito larvae feed on suspended plant material and organic particles in the water. In warm conditions, the larval stages develop in about five to seven days before transforming into the pupak, or “tumbler”, stages. True to their name, tumblers are highly active as they sink and rise to the surface. They do not feed.(Ogg, 2010)
Mosquito larvae, commonly called “wrigglers”, must live in water from 7 to 14 days depending on water temperature. Larvae must come to the surface at frequent intervals to obtain oxygen through a breathing tube called a siphon. The larve eats algae and small organisms which llive in the water.
During growth, the larva molts (shed its skin) four times. The stages between molts are called instar. At the 4th instar, the larva reaches a length of almost half inch. When the 4th instar larva molts it becomes a pupa. (http://www.mosquitoes.org/LifeCycle.html)
Larvae will emerge from egg within 2-3 days when the environment conditions are ideal. All mosquito larvae go through four developmental stages called instar. First instar is barely noticeable to the human eye. Last larval instar of some species can be approximately half inch (12.7mm) long. Larvae move through the water in a serpentine motion. When they sense a shadow or movement in their habitat, larvae will quickly dive to the bottom to avoid the source of disturbance.
Larvae have a well-formed head and lack legs. Upon careful inspection one can distinguish the wider thorax from the long and slender abdomen. A tube-like structure called a siphon, is located at the tip of the abdomen. Larvae use the siphon to breathe air from the water surface. Larvae possessing a siphon (Culicine) hold their body roughly at a 45

GMELINA Linnaeus, Sp. Pl. 2: 626. 1753.
Trees, tall shrubs, or rarely subshrubs, often climbing when young. Branchlets tomentose, often spiny. Leaves opposite, simple, sometimes lobed, usually with large gland patches near base, often abaxially gray mealy. Inflorescences usually terminal cymes or panicles, sometimes 1-flowered in leaf axils; cymules axillary, decussate, few flowered, short; bracts leaflike. Calyx persistent, campanulate, enlarged in fruit, truncate, 4- or 5-dentate or lobed, often somewhat unequal or oblique, usually with large glands. Corolla zygomorphic, ventricose funnelform, tube narrow at base, throat wide; limb oblique, ca. 2-lipped, upper lip 2-lobed or entire, lower lip 3-lobed with middle lobe larger. Stamens 4, distinctly didynamous, inserted on basal part of corolla tube, usually included, sometimes slightly exserted; anthers divaricate, opening by longitudinal slits. Ovary (2–)4-locular; ovules pendulous or laterally attached, often with a central cavity. Style slender, usually unequally 2-lobed or stigma awl-shaped. Fruit a succulent drupe, endocarp bony, mesocarp fleshy. Seeds 4 or by abortion 2 or 3.
Gmelina arborea Roxburgh, Pl. Coromandel 3: 41. 1815.
Trees ca. 15 m tall; bark grayish brown; branchlets, petioles, and inflorescences densely yellow-brown tomentose. Branchlets slightly 4-angled when young, becoming terete, lenticellate, leaf scars prominent. Petiole terete, 3.5–10 cm; leaf blade broadly ovate, 8–19 × 4.5–15 cm, thickly papery, base broadly cuneate to subcordate, apex acuminate; veins 3–5 pairs, abaxially prominent. Inflorescences terminal, narrow thyrses; peduncle 15–30 cm. Calyx 3–5 mm, with several black discoid gland patches; teeth 5, sharply triangular. Corolla yellow, 3–4 cm, 2-lipped, sparsely glandular; lower lip 3-lobed, outside yellowish brown puberulent, inside glabrous; upper lip entire or slightly 2-cleft. Ovary glabrous, glandular. Stigma unequally 2-cleft. Drupes yellow when ripe and black when dry, ellipsoid to obovoid-ellipsoid, 1.5–2 cm. Fl. Apr-May, fr. May-Jul.
Open forests along roadsides and near farm houses; below 1500 m. S Yunnan [Bangladesh, Bhutan, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Philippines, Sri Lanka, Thailand, Vietnam].
Allied to Gmelina asiatica but differs in having erect inflorescences and larger leaves.
Neem
Neem contains at least 35 biologically active principles,41 of which azadirachtin (AZA), a triterpenoid is the predominant insecticidal active ingredient in the seeds, leaves, and other parts of the tree. Neem products containing azadirachtin and other ingredients, have antifeedant, ovipositional deterrence, repellency, growth disruption, sterility and larvicidal action against insects42.
Neem based pesticides are now extensively used in agricultural practices all over the world. Neem oil and other commercial preparations of neem have been found as potential mosquito larvicide15. Dhar et al18 demonstrated the effect of neem oil volatiles on gonotropic cycle and inhibition of oviposition in An.stephensi and An. culicifacies. Control of mosquito breeding has also been demonstrated in the field in some confined habitats using indigenous methods of application of neem oil in water and neem oil coated on wooden scraps16,17. Wood scrap balls soaked in 5 to 20% neem oil in acetone were tested in overhead tanks of 0.50 cubic meter against An.stephensi breeding. Though it did not prohibit egg laying, it arrested pupal formation and eventually the adult emergence for about 45 days17. Neem oil emulsion in water was also found to control breeding of Cu.quinquefasciatus, An.stephensi and Ae. aegypti in pools, basement tanks and desert coolers, and the effective control lasted for 2 to 3 weeks16. Neem cake powder and urea coated with neem cake powder were evaluated for the control of mosquito breeding in rice fields19.
Application of neem cake powder alone or coated on urea resulted in drastic reduction in the late instar larvae and pupae of culicine mosquito for several weeks. Aqueous extract from deoiled neem seed kernels exhibited toxic and growth regulating activities against Cx. quinquefaciatus larvae with a 100% larval mortality especially during the first and second instars at all the tested concentrations43. Though neem products show high larvicidal activity, they do not show adulticidal action.
Zebitz44 suggested that azadirachtin acts as an antiecdysteroid and thus kills the larvae by growth inhibition effect. This, along with other delayed effects of neem products18 provides an alternative approach to chemical larvicides in mosquito control.
Oregano
Oregano (Scientific name: Origanum vulgare) is also known as Wild Marjoram, Mountain Mint, Origanum, Wintersweet and Winter Marjoram. This erectly spreading plant has strong aromatic characteristics, with leaves and stems that are fleshy. The leaves of oregano are heart-shaped, with toothed edges, and which, grow for up to 9 meters in length. In other countries, the plant is primarily used as a culinary ingredient. However, in countries like the Philippines, Oregano is a known herbal medicine for its strong anti-oxidant properties. Oregano contains a rosmarinic acid compound, thymol, and carvacrol that are responsible for its anti-inflammatory, anti-bacterial, anti-oxidant, anti-fungal and anti-viral properties. Oregano also contains flavinoids, triterpenoids, sterols, vitamin C, and vitamin A. Its anti-bacterial properties have been proven by recent studies to treat infections of the reproductive tracts, and which make it ideal to be given to women who have just given birth.
The volatile oils in oregano and its properties are believed to be responsible for slowing the process of spoilage of food and thus minimizing the risk of ingesting harmful bacteria, parasites and fungi.
Based on a study conducted on the various types of oregano, it was determined that these herbs were able to kill bacteria due to the heavy content of phenolic substances found in their cellular structure. Oregano, by connecting the free radicals released from the cells within its contents, is very healthy and improves the body's cell protection system. (http://www.healthtweak.com/oregano-origanum-vulgare)
Basil
Ocimum basilicum, otherwise known as basil or sweet basil, are of the family Leguminosae. It is commonly seen in garden and is anative of India. It is an annual herb that can grown up to 1m. I t has an erect stem, round below and squared above. It has an ovate or lanceolate leaves, opposites until 5cm long. Its petiole is long, darker above, very odorous flowers grouped in spikes, loose vetricils with 6 flowers each. It has a white or pink corolla and white stamens. Its active components are essence with linanol, estragole and eugenol.They may be used as mosquito repellent due to the odor given off by some of its components like eugenol.
Sweet basil contains a volatile oil (about 1%), which consists principally of linalool and methyl chavicol, along with small quantities of methyl cinnamate, cineole, and other terpenes.(http://www.botanical-online.com/medicinalsocimumangles.htm)
Lemon Grass
Cymbopogon citratus, generally known as lemongrass, is a resourceful herb, a natural source of aroma, mosquito repellent as well as a plant that is widely used to decorate gardens. Lemongrass belongs to the grass or Poaceae family (formerly known as Gramineae) and has several functions - an effective herb, aromatic or container garden, or as a medication for various conditions. One may find a number of variety of lemongrass and each of them possessing dissimilar chemical compositions. However, citral is the major chemical ingredient found in all varieties of essential oils of lemongrass.
The chemical composition of the essential oil extracted from lemongrass comprises huge quantities of citral (geranial, neral) as well as several other monoterpenoids. The main elements of citronella oil are geraniol, citronellal and citronellol. Lemongrass is considered to be of low toxicity at low doses (http://sharonfalsetto.suite101.com/lemongrass-essential-oil-a132780.)
Research Methodology
Plant materials were collected. After that plant were cleaned, segregated, chopped, and air-dried in a shady place. All plants used were then kept safe as zip lock bag to avoid in contact to parasites and other air-borne particles. Dried materials were then ground in a blender or a simple mortar and pestle. The ground plant materials were dipped in solvents (methanol and ethanol) in tightly capped jars separately for 48 hours. The solvents along with extracts were drained out and filtered. The filtrated solutions were then placed in a glass bottle. Larvicidal bioassay was carried out as per standard WHO techniques in 500 ml glass beakers containing 250 ml of water and 30 numbers of late III cultured mosquito larvae for various concentrations. (Appendix A). Three different concentrations of each extract were tried out at a time with three replicates. One control was kept with each set of experiment and mortality was recorded after 24 hours. Three sets of experiments were conducted for each extract. Tests were carried out under controlled laboratory conditions.

A total of 30 late III cultured mosquito larvae were introduced in 500 ml glass beaker containing various concentrations of different plant extracts. The treatments were replicated three times, and each replicate set contained one control. Mortalities were reported after 24 hours of the exposure period under laboratory condition. The declining and dead larvae in three replicates were combined and expressed as percentage mortality for each concentration. Dead larvae were acknowledged when they failed to move after probing with a needle or a stick. Declining larvae were those unable of rising to the surface within reasonable period of time, usually 1 minute after touching. The percentage mortality was calculated and analysis of data was carried out by employing probit analysis (Finney, 1971) and corrections for mortality if needed were done by using Abbott formula (Abbotts, 1925).
Percentage of mortality= Number of dead larvaeNumber of larvae introduced X 100 (1)
Corrected percentage of mortality= 1- n in T after treatmentn in C after treatment X 100 (2)
Where n= number of larvae, T= treatment and C= control.
Experimental Design
T1- 100% Plant Extract
T2- 75% Plant Extract + 25% Water
T3- 50% Plant Extract + 50% Water
T4- Control Setup (Commercial Larvicides)
T5-Salt Solution
*Each treatment was done with three replicates

Results

Table 1 Physical Characteristic of Five Plant Extracts. Plant Extract | Odor | Color | Gmelina arborea(Gmelina) | Foul | Yellow Brown | Azadirachta indica(Neem) | Foul | Dark Green | Origanum vulgare(Oregano) | Pleasant | Light Green | Andropogon citrates(Lemon Grass) | Pleasant | Light Brown | Ocimum basilicum(Basil) | Pleasant | Dark Green |

Table 2 Mortality Rate of Larvae in Three Replicates Using Different Solutions/ Mixtures in Different Concentrations | Replicates | Treatment | Mean | Average% | | | T1 | T2 | T3 | | | Plant AGmelina | 1 | 28 | 26 | 25 | 26.33 | 87.78 | | 2 | 28 | 23 | 23 | 24.67 | 82.23 | | 3 | 30 | 25 | 23 | 26 | 86.67 | Plant BNeem | 1 | 30 | 28 | 26 | 28 | 93.33 | | 2 | 30 | 27 | 25 | 27.33 | 91.11 | | 3 | 30 | 28 | 27 | 28.33 | 94.45 | Plant COregano | 1 | 26 | 24 | 26 | 25.33 | 84.44 | | 2 | 26 | 23 | 25 | 24.67 | 82.22 | | 3 | 25 | 24 | 22 | 23.67 | 78.89 | Plant DLemon Grass | 1 | 26 | 23 | 24 | 24.33 | 81.11 | | 2 | 25 | 23 | 25 | 24.33 | 81.11 | | 3 | 25 | 22 | 23 | 23.33 | 77.78 | Plant EBasil | 1 | 26 | 25 | 24 | 25 | 83.33 | | 2 | 25 | 24 | 25 | 24.67 | 82.23 | | 3 | 25 | 25 | 24 | 24.67 | 82.23 | Commercial Larvicide | 1 | 29 | 96.67 | | 2 | 30 | 100 | | 3 | 30 | 100 | SaltSolution | 1 | 10 | 33.33 | | 2 | 14 | 46.67 | | 3 | 17 | 56.67 |

Table Summary 1: Mortality Rate of the Different Treatments MIXTURES AND SOLUTIONS | TREATMENT | | | T1 | T2 | T3 | TREATMENT MEAN % | | Gmelina | 86 | 74 | 71 | 85.56 | | Neem | 90 | 83 | 78 | 92.96 | | Oregano | 77 | 71 | 73 | 81.85 | | Lemon Grass | 76 | 68 | 72 | 80 | | Basil | 76 | 74 | 73 | 82.59 | | Commercial | 89 | 98.89 | Salt Solution | 41 | 45.56 | TOTAL | 1272/1,530 | MEAN | 83.14 |

Discussions As we can see in the table, commercial larvicide (the artificial) is still the best larvicide when it comes to eradicating the vectors of dengue and malaria. The traditional solution is not that effective based on the experiment. About the five plants tested, Neem is the best source of extract in killing the larvae; the others are not that significantly different but still have the potential to kill larvae. Neem vs. Commercial- The data says that the commercial larvicide kills primarily 98.89% of larvae significantly higher than the 92.96% of Neem extract. In numbers we can say that Commercial kills more but as we stated earlier this artificial larvicide has harmful effects thus making the Neem extract the must choice.

SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
Extracts from the five plants, namely Gmelina (Gmelina arborea), Neem (Azadirachta indica), Oregano (Origanum vulgare), Lemon Grass (Andropogon citratus) and Basil (Ocimum basilicum), were tested for their physical properties, specifically the odor and the color and found out that the one with pleasant smell and light colored extract were less effective than those that are foul and dark colored ones.
The extracts were also tested for their effectiveness to kill mosquito wrigglers. Treatments were made with different concentration such as 100%, 75% and 50% based on a LD50 test conducted. It was found out that the 100% plant extract yield the most effective larvicide and among the five plant extract, neem is the most effective.
When added to water with 30 wrigglers however, the effectiveness of the extract decreased, such that it only kill a number of mosquito less than the number compared to the pure extract. When compared to commercial and traditional method, results showed that the extracts were more effective than salt solution. It is also comparable to commercial larvicides. This shows that natural products from plants would serve as one of the cheapest larvicides in the city to help eliminate mosquito wrigglers that causes diseases to human race.

Conclusion This project sought to prove the effectiveness of the five plant extracts to eradicate mosquito wrigglers. After the experiments the researchers had proven the effectivity of these plants. The plant extracts are more effective than the traditional one, which is salt solution. They also found out that Neem (Azadirachta indica) was the most efficient larvicides among the five plants that were tested. Though commercial larvicide gave the most successful yield, the researchers conclude that these plant extract can be used as an alternative because it is environmental friendly, much cheaper and locally available.
Recommendation
Based on the results of the study the following are recommended: 1. A comparative study should be done to compare extract of the five plants based on different plant parts. 2. Other species of mosquito should also be tested in the study. 3. The extract should also be conducted on the natural habitat of this test animal. 4. The extracts should be tried to mix to test whether there is a significant difference on the number and time of killing. 5. The extract should be tried to other farm pests and insects.

Works Cited

Chaudhuri, S.R., M. Mishra, P. Nandy and A.R. Thakur, 2008. Waste management: A case study of ongoing traditional practices at East Calcutta Wetland. Am. J. Agric. Biol. Sci., 3: 315-320.

Dubey, N.K. 2011 Natural Products in Plant Pest Management, London, U.K., pp 48-50.

Jesse A. Hardin, J. A., Jackson, F., 2009, Applications of natural products in the control of mosquito-transmitted diseases:African Journal of Biotechnology Vol. 8 (25), pp. 7373-7378, 29 December 2009

Ong, Barbara P. Ogg. Residential Mosquito Control. 2010

Raj M, Ramaswamy., 2007 Evaluation of larvicidal activity of plant extracts: African Journal of Biotechnology Vol. 6 (5), pp. 631-638.

http://www.doh.gov.ph (accessed September 24, 2011)

http://www.mosquitoes.org/LifeCycle.html (accessed September 24, 2011)

http://www.healthtweak.com/oregano-origanum-vulgare (accessed September 24, 2011)

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