Determination of Knock-down Time and Level of Toxicity of Panyawan (Tinospora rumphii L.) Leaf Extract against American Cockroach (Periplaneta americana)

This paper aims to determine the insecticidal activity of Panyawan (Tinospora rumphii) leaf extract against American cockroach (Periplaneta americana). A quantitative experimental research design was employed in the study. Cockroach strains were collected through trapping and hand catch methods. Panyawan leaf was collected and extracted to achieve seven different concentrations. The concentrations and controls were tested against the strains through the standard contact glass jar bioassay. Results revealed that the shortest knock-down time was achieved by the positive control at 1.08 minutes while the Extracts of Panyawan leaf reported a 1.42 to 20.25 minutes knock-down time. Probit Regression reported that the lethal concentration 50 of Panyawan leaf extract was 15.836%. One-Way ANOVA revealed that there is a significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls (p<0.01). Homogeneous subsets derived from Post Hoc Test using Tukey’s Honest Significant Difference reported that there is no significant difference between positive control and Extract 100% (p>0.01).


Background of the Study
According to the World Health Organization (2018), cockroaches have been present for about 360 million years. These insects are harmful pests because they spread filth and destroy fabrics, food, and book-bindings. They expel portions of their partially consumed food at intervals and drop excretions. They also release a nauseous secretion both from their mouths and from the gland openings on the body which gives an enduring, aggressive smell to areas or food visited by them.
Cockroaches are not typically the most important cause of a disease, but they may play an additional role in some allergic diseases as well as the spread of some infections. For instance, previous researches found out that cockroach sensitization is an important risk factor for the development of asthma, diarrhea, leprosy, and poliomyelitis (Do, Zhao, & Gao, 2016;Ifeanyi & Omowumi Odunayo, 2015;Paredes & Morales, 2016;Shahraki, Parhizkar, & Nejad, 2013).
Several species of Enterobacteriaceae including Escherichia spp., Klebsiella spp. and Salmonella spp. can be harbored in the cuticle of the cockroaches while 63% of the parasites taken from the pest were comprised of hookworm, a human intestinal parasite (Jalil et al., 2012;Oyeyemi, Agbaje, & Okelue, 2016). According to the findings of Tatang, Tsila, and Pone (2017), of the 844 adult cockroaches collected, the medical parasites carried by the pests were Ascaris (33.76%), Trichuris (11.97%), Capillaria (6.16%), Toxocara (4.86%), and Eimeria (2.73%). The Global Burden Disease (2017) found out that these parasites caused more than one million deaths in the year 2017.
The American cockroach, Periplaneta americana, is the largest of the common domestic cockroaches in the Philippines. Control of this species has been very difficult to achieve. To keep it under control, different synthetic insecticides have been used. However, the huge extent and continuous application of commercial insecticides resulted in many problems. Among them are toxic residues in food and humans, pollution of the environment, toxicity to non-target organisms, high cost, development of insect resistance strain and workers safety.
At present, resistance has become a significant problem that sometimes causes failure of control operations in many countries (Khan et al., 2015). Hence, there is a need to find alternative biodegradable insecticides derived from plant products that are easy to utilize, effective, and safe to environment as well as human health.
The abundance of plants in the Philippines and the huge potential to produce extracts using simple and affordable means is a very significant basis for conducting researches of the effects of natural products from plants against cockroaches. Panyawan, Tinospora rumphii, is a climbing vine plant commonly grown and matured in the wild places in the Philippines. Various researches revealed that the Panyawan plant possessed pesticidal and antimicrobial activities towards several pests. For instance, Gutierrez (2015) found out that T. rumphii is effective as grain protectant against adult maize weevil, Sitophilus zeamais M.
pest. Moreover, Aminul, Islam, and Mohammad (2011) stated that the methanolic and ethanolic extracts of the plant have antimicrobial, cytotoxicity and antioxidant activity. Further phytochemical analysis revealed that the plant leaf contains alkaloids, saponins, flavonoids and tannins that are known to possess medicinal and pesticidal properties (Ahmad, Jantan, & Bukhari, 2016). It provides an alternative to costly drugs and potentially toxic chemical pesticides.
Much of the work regarding insecticide has been done on German cockroach; however, only little data is available concerning American cockroach. Therefore, the present work was designed to evaluate the insecticidal efficacy of Panyawan (Tinospora rumphii) leaf extract on Periplaneta americana, and the susceptibility of the cockroaches to this formulated insecticide through laboratory bioassay using contact glass jar bioassay. The findings of the study will be valuable for the end users to choose better choices for the management of these pests.

Statement of the Problem
This study aims to determine the insecticidal activity of Panyawan (Tinospora rumphii) leaf extract against American cockroach (Periplaneta americana). Specifically, this study attempts to the answer the following questions: 1. What is the knock-down time of the cockroaches exposed to Panyawan leaf extract in terms of the following concentrations: 3. Is there a significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls?

Hypothesis
The null hypothesis was formulated and tested at 0.01 level of significance stating: H1: There is no significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls.

Independent Variable
Dependent Variable

Figure 1. Research paradigm of the study showing the type of treatment will directly affect the knock-down time and level of toxicity
Presented in Figure 1 is the research paradigm of the study showing the type of treatment will directly affect the knock-down time and level of toxicity. As shown in the table, the independent variable is the type of treatment (Panyawan leaf extract), positive control and negative control while the dependent variables are the mortality rate and level of toxicity.

Significance of the Study
This study is important to the following: Household. The findings of the study will find an alternative insecticide that is cheaper and environment-friendly against the cockroaches. The households in the community can use the product to control the proliferation of the pests. Students. The study will add to the body of scientific knowledge and hence, will allow the students to explore scientific developments about Panyawan leaf.
Future Researchers. The proposed study will benefit and help the future researchers. The ideas presented may be used as reference data in conducting new researches or in testing the validity of other related findings. This can also open in the development of the study.

Definition of Terms
The following terms were defined operationally for ease and clarity: Toxicity. This refers to the degree to which a chemical substance can harm humans or animals.

American Cockcroach (Periplaneta americana)
As stated in the Columbia Encyclopedia 6 th ed. Cockroaches are considered to be the most irritating household pests since it is one of the unhygienic creatures that causes great destruction and contamination of food supplies.
According to the study of Solilan (2015), to support the claims above, they prefer to live in warm, dark, wet areas, like sewers and basements. They often enter structures through drains and pipes.
According to Healthline (2017), in connection with the study of Solilan, moderate to high numbers of cockroaches' can affect rural businesses like restaurants since they can spoil food with feces and urine and can transmit diseases and parasites to humans and livestock (eg salmonella). Although American cockroaches can be found in homes, they are also common in large commercial buildings, grocery stores and hospitals.
As cited by Mella (2010) According to Strattford (2010), in connection with Somerton, cockroaches are well recognized by people because of its reputation of being dirty and for invading households. Cockroaches can also cause damage in crop paddocks immediately after sowing by digging into loose soil to find larger seeds such as maize, sunflower, wheat, oats, barley, pulses, pumpkin and marrow. They also eat the newly sprouted seedlings before and after they emerge from the soil. The impact of cockroaches' is not as great on plants beyond the seedling stage, at least not until seeds or grains begin to mature. Plants such as wheat are then damaged by cockroaches gnawing at the nodes on the stems causing developing seed heads to fall. In maturing crops of wheat, oats, barley, pulses, sorghum and maize, losses of up to 30% have been reported. Heavy losses can also occur in vineyards and vegetable crops from eating and fouling of produce.
On the word of Matte (2016), cockroaches can ruin an organization's reputation. If clients and customers spot evidence of insect infestation in the premises you manage, they are likely want not to do business with you.

Insecticide Study
According to Ware, G.W et al (2004) Insecticides are agents of chemicals or biological origin that control insects. That may result from killing the insect or else preventing it from having a large population. Insecticides may be natural or manmade and are applied to target pests in a myriad of formulations and delivery systems. The science of biotechnology has, in recent years, even incorporated bacterial genes coding for insecticidal proteins into various crop plants that deal death to unsuspecting pests that feed on them.
According to Whitacre, D. et al (2004) Some 10,000 species of the more than 1 million species of insects are crop-eating, and of these, approximately 700 species worldwide cause most of the insect damage to man's crops, in the field and in storage. and Humans have been on earth for more than 3 million years, while insects have existed for at least 250 million years. We can assume that among the first approaches used by our primitive ancestors to reduce insect annoyance was hugging smoky fires or spreading mud and dust over their skin to repel biting and tickling insects. Today, such approaches would be classified as repellents, a category of insecticides.
As stated on the Encyclopaedia Britannica (2018)  increased frequencies, and led to the appearance of new units which were not present in untreated control insects. They also caused transitory phases of conduction blocks, the length of which increased as intoxication proceeded. The DDT analog tested could be characterized by pronounced repetitive dischanges, whereas the neuroactivity of pyrethroids was typically associated with a very regular spike pattern in continuous phases of extremely high frequencies. Also chlordimeform, a formamidine insecticide, was found to increase the spike frequency, but to a much lesser extent than that observed for the other insecticides tested.
Deng, L., et al (2007). The effects of an organophosphorous insecticide, methamidophos, on the pest control potential of the spider Hylyphantes graminicola (Sundevall) (Araneae: Linyphiidae) were investigated in the laboratory with the fruit flies (Drosophila melanogaster Meigen). The influence of methamidophos on predation by H. graminicola was very obvious in female spiders, which preyed on fewer prey in the 8 h after exposure to the insecticide but subsequently recovered. On the other hand, the predation rates in male spiders were not affected by the insecticide within 24 h of treatment. However, a 10% lethal dose (LD10) of methamidophos resulted in an enhanced predation rate per day for male spiders, whereas a 50% lethal dose reduced the predation rate. In addition, it was shown that the functional response of H. graminicola to the fruit fly was a type II response, and the type of functional response of insecticide-treated females changed from type II to type I, with no change in the response of male spiders. The attack rate of males treated with the LD10 dosage of insecticide was significantly higher than the controls, which suggests that the insecticide stimulates the performance of spiders. Prey utilization of males treated with low doses of insecticide was lower than the control, which indicates that the insecticide did not result in these spiders eating more prey, but killing more. in situ, on abundance, drift, community structure, or dynamics. Azinphos-methyl, chlorpyrifos, and endosulfan were frequently detected at levels above those reported to reveal effects in the field; however, knowledge about effects of insecticides in the field is still sparse.

Panyawan (Tinospora rumphii L.)
(Panyawan is a native plant from tropical and subtropical rain forest in India, Chapter 2

Research Design
In this study, a quantitative experimental research design was utilized.
According to Creswell (2013), this design is a systematic and scientific approach

Collection of Plant Materials
The Panyawan (Tinospora rumphii) plants were taken from an authorized plant shop at Bankerohan Public Market, Building Number 2, Marfori Street, Barangay 5-A, Davao City, Philippines. These plants were specified and verified by Maria Theresa C. Baslot, a Botanist. Leaves were separated from the stem and roots of the collected Tinospora rumphii plant. The leaves were then washed with distilled water to remove any debris. These were air dried at room temperature for several days until the leaves became brittle and moisture-free. The plant materials were inspected daily until optimum dryness was achieved.

Preparation of Extract
Collected dried leaves were ground using a house blender and the extracted material was then subjected to the extraction process. The extraction process followed the procedures written in the book of Guevara (2005)  was stored with a tight stopper in the cold, at temperatures between 0°-5°C.

Contact Glass Jar Bioassay
Tests were conducted on adult male strains of cockroach using the World Health Organization standard method (WHO, 1970). Each of the nine treatments was prepared in acetone and 2.5 mL was pipetted into a 0.4-L glass jar (inner surface area = 302.6 cm 2 ). Nine jars containing different treatments were rolled horizontally over a flat surface until all of the acetone had evaporated, so that the treatment was deposited evenly over the inner surface of the jar. Negative control group received acetone only. Insecticidal activity of each treatment was evaluated by adding one adult cockroach per glass jar. Knock-down time was determined by subtracting the time of mortality and time the treatment was given. Mortality criterion was as follows: if a cockroach was unable to return back to its normal position within 2-3 minutes after being touched with forceps, it was considered dead and counted in mortality data. To calculate the lethal toxicity concentration, another set of bioassays was carried out but this time, ten cockroaches were added per glass jar. The duration of contact was 1 hour and cockroaches were then transferred to holding cups after the contact. The readings of mortality were taken during this time and expressed as percentage mortality. The transferred cockroaches were still provided with rodent diet, a cotton plugged water vial and a cardboard as a harborage. Three replicates were set up for the treated and controls.

Ethical Considerations
The researchers secured the Plant Ethics Checklist before conducting the study involving plant subjects. The form was filled-up to guide the researchers on the ethical way of utilizing plant subjects that were used in the experiment of this research.

Data Analysis
The following statistical tools were employed in the study using the SPSS Software 23.0 version: Mean. This was used to determine the mortality rate of Periplaneta americana after application of nine treatments.

One-Way Analysis of Variance (ANOVA).
This was used to know if there is significant difference in the mortality rate of cockroaches when exposed to

RESULTS AND DISCUSSION
Knock-down times of the cockroaches exposed to different Panyawan leaf extract concentrations, negative control and positive control  An LC50 value is the concentration of a material in air that will kill 50% of the test subjects when administered as a single exposure. This value gives the relative acute toxicity of an inhalable material and is related to the lowest concentration reported to have killed the test subjects. Using Probit Regression to analyze the data, it was found out in Table 2 that the leaf extract has a lethal concentration 50 of 15.836%. This means that if a cockroach would receive a Panyawan Leaf

Level of toxicity of the Panyawan leaf extract on the cockroaches in terms of LC50 using Probit Regression
Extract with a concentration of 15.836%, there is a 50% chance the pest will die and if this survives, it will not be in good shape.

Summary
The present study aimed to determine the insecticidal activity of Panyawan (Tinospora rumphii) leaf extract against American cockroach (Periplaneta americana). Specifically, the study attempts to know the knock-down times of the cockroaches exposed to different Panyawan leaf extract concentrations, negative control and positive control, to determine the level of toxicity of the Panyawan leaf extract on the cockroaches in terms of LC50 using Probit Regression, and to determine if there is a significant difference in the knock-down times of cockroaches when exposed to various Panyawan leaf extract concentrations. The null hypothesis was then formulated and tested at 0.01 alpha level of significance.
A quantitative experimental research design was used in the study. The experiment was conducted at the University of Immaculate Conception -Bonifacio Campus, De Jesus Street, Poblacion District, Davao City. The cockroach strains were collected from different parts of the household of the researchers. Panyawan leaves were collected and extracted to obtain seven different concentrations.
Efficacy of the concentrations were measured through contact glass jar bioassay.
After a series of experimentation, the data were tabulated and analyzed using the SPSS 23.0 version.
The findings of the study were summarized and arranged based on the formulated research questions: 3. One-Way Analysis of Variance (ANOVA) was used to determine if there is a significant difference in the knock-down times of cockroaches when exposed to various Panyawan leaf extract concentrations. Because the pvalue of 0.00 was lesser than 0.01 alpha level of significance, the null hypothesis is rejected; therefore, there is a significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls. Homogeneous subsets derived from Post Hoc Test using Tukey's Honest Significant Difference reported that there is no significant difference between positive control and Extract 100% as well as between Extract 75% and Extract 50%; however, both these two groups are significantly different from the Extract 25%, Extract 10%, Extract 5%, negative control and Extract 0.1%

Conclusion
Based on the findings of the study, the following conclusions were drawn: 1. The shortest knock-down time was achieved by positive control while the Extracts of Panyawan leaf reported a 1.42 to 20.25 minutes knock-down time.
2. The concentration of Panyawan leaf extract required to kill 50% of the cockroaches was 15.386%.
3. There is a significant difference in the knock-down times of cockroaches when exposed to different Panyawan leaf extract concentrations, negative and positive controls. There is no significant difference in the knock-down times of cockroaches when exposed to positive control and 100% Panyawan leaf extract.

Recommendations
Based on the findings and conclusion of the study, the following recommendations were formulated: 1. Panyawan leaf extract concentrations of 5% to 100% were found to kill the cockroaches faster than the negative control. Therefore, the households can utilize the extract as an alternative to much expensive commercial pesticide.
2. The lethal toxicity of Panyawan leaf extract was 15.386%. Therefore, less concentrations are required to control the proliferation of the pests.
3. Future researches should be done to determine other factors that could affect the mortality of the cockroaches.