Determination of LD50 of an Insecticide in Cockroaches PDF

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Determination of the LD50 of an Insecticide in American Nymph Cockroaches Abstract Cockroaches can be pest to society in which their population need to be monitored and controlled. Insecticides can be used to help control and monitor their population, however in order to do so, the LD50 value must be determined. The aim of this study was to determine the LD50 of propoxur on the American Nymph Cockroaches. Different concentration of insecticide was constructed and tested on the cockroaches. The data obtained were graphed and LD50 was determined. The LD50 of propoxur on American cockroaches were 0.02399%w/v obtained from the sigmoidal graph and 0.03823%w/v obtained from the linear graph. The investigations is only limited to the American cockroaches; further research is suggested to determine if different type of cockroaches would affect the LD50 value. Introduction Pests have been detrimental to humans and the environment. Pest can include terminates, cockroaches, slugs, snails, mice, rat, pigeons, ticks, flea, mosquito and bed bugs to name a few (Department of Health, 2010). The most common pest are the cockroaches in particular the American cockroaches Periplaneta americana (Linnaeus) (The Pennsylvania State University, 2013). These cockroaches can be found almost everywhere in particular in dark, warm and moist places such as rubbish dumps, sewers, basement or in commercial buildings in which food are stored or prepared (Michigan State University, n.d.). Cockroaches are classified as pest as they can trigger many reactions to an individual such as allergic and asthmatic reactions (WHO & Rozendaal, 1997). The American cockroaches are a common cockroach species in which it is native to Africa and the Middle East and were introduce to other countries like America (Bell & Adiyodi, 1981). The American cockroaches are a reddish brown in colour and 3-4cm in length when they are fully grow adults thus one of the largest species of cockroaches (The Pennsylvannia State University, 2013, Michigan State University, n.d. & University of Florida, 2017). There are three stages in the life cycle of an American cockroach, the egg stage, the nymph stage and the adult stage (The Pennsylvannia State University, 2013 & University of Florida, 2017). The nymph stage of an American cockroach starts when the egg hatches in which the cockroaches are white in colour (University of Florida, 2017). The cockroaches will continue to molt until they are a reddish brown colour which usually takes about 6-12 months (University of Florida, 2017). The adult American cockroaches are reddish brown in colour in which it would have developed its wings (The Pennsylvannia State University, 2013 & University of Florida, 2017). American cockroaches mainly feed on decaying organic matter however will eat about almost everything. Their fecal matter can cause and trigger asthma and allergic reaction in humans (WHO & Rozendaal, 1997). They can also carry eggs of parasitic worms in which they can cause allergic reactions such as dermatitis, swelling of eye lids and respiratory conditions (WHO & Rozendaal, 1997). Also, since cockroaches are found almost everywhere, it can pick up bacteria such Salmonella on their legs in which these bacteria can cause serve reactions such as food poisoning, infections, diarrhea, dysentery, leprosy and typhoid fever (WHO & Rozendaal, 1997). Thus it is important to monitor the American cockroaches and have control measures prepared. Insecticides can be used as a control measure. Insecticides can be used to harm, repel or kill insects (Department of Agriculture and Food, n.d.). Propoxur is an active ingredient in insecticides (Baygon). Propoxur (2-isopropoxyphenyl methylcarbamate) is a carbamate and

isopropyl insecticide (Kovacic & Somanathan 2012). Propoxur inhibits acetylcholinesterase activities in an insect (Cornell University, 1993) which is an important enzyme in the nervous system. Propoxur paralyses the nervous system which harms or kills the insect. At low doses of propoxur, motor activity can be lowered (Kovacic & Somanathan 2012). Thus different concentrations of insecticides can have different effects on the cockroaches. The aim of this investigation was to determine the LD50 of a carbamate insecticide (propoxur) in cockroaches (American Nymph Cockroaches). LD50 is the estimate amount of poison that under control conditions will be a lethal dose to kill 50% of the population of the organism. In this investigation, the ideal concentration of insecticide is identified by using assay with wide range of concentrations (Rang & Hill, 2013). Material and Method Insecticides of different concentrations were prepared and tested on the American Nymph cockroaches. There was an alteration in the original experimental methodology in which the purity of the propoxur used was 97.6%. Materials and methods used can be obtained from the Pharmacology 1 Subject Manual 91707 (Autumn Semester 2018) pages LD1-LD8. Results Class results were obtained for the percentage of death of cockroaches at different concentrations of propoxur used in which mean, standard deviation, log10 dose response and probit analyses were performed. Results from log10 dose response and probit analyses were expressed in graph and table format as seen in table 1, figure 1 and figure 2. Table 1. Table 1 shows the percentage of cockroaches’ death at different concentrations of propoxur of two different sample, our results and last year class results as well as the standard deviation. It can be seen that the higher the concentration of propoxur used, the larger the death percentage is. 0.1% w/v had the highest death percentage while 0.01% w/v had the lowest death percentage. Control 0.01% w/v 0.02% 0.05% 0.1% w/v w/v w/v Our Result Log10Dos -2 -1.69897 -1.30103 -1 e Average Percentage 10 30 40 100 100 0 0 14.142135 42.426406 0 Standard 9 6 Deviation Log10Dos -2 -1.69897 -1.30103 -1 Last Year e Class Percentage 0 18 30.7 58 73 Average Standard 0 3.5 7 15 20 Deviation Table 2. Table 2 shows the average weight of cockroaches used in this investigation from two sample, our results and last year class results. Average Weight of Cockroaches Our Result Average 0.53082g Last Year Class Average 0.431g

Table 3. Table 3 shows the LD50 from the two sample, our results and last year class results in %w/v and in mg/kg. Table 3 shows that last year class results had a higher LD 50 compared to our results. LD50 Determined by LD50 (mg/kg) Graph (% w/v) Our Results Log10 Dose Response 0.023988329 0.4519108 mg/kg Probit Graph 0.03823030995 0.72021231mg/kg 0.90265694mg/kg Last Year Class Log10 Dose Response 0.038001894 Results Probit Graph 0.03952184267 0.9169801mg/kg

Figure 1. Log-Dose Response Curve for our results (Blue) and last year class results (Orange). Figure 1 shows that at the death percentage of 50%, the log10Dose of our results were -1.62 and the log10Dose of last year class results were -1.41.

Figure 2. Probit Curve for our results (Blue) and last year class results (Orange). Figure 2 shows that our result had a linear regression of 1 and last year class result has a linear regression of 0.99707. The linear regression of both line were greater than 0.95, hence results were valid. Table 4. Table 4 shows the conversion of LD50 from %w/v to mg/kg in both sample from figure 1, our results and last year class results. Determining LD50 From Figure 1 Figure 1: Our Values Figure 1: Last Year Class Result At Death Percentage = 50, Log10Dose = -1.62 At Death Percentage = 50, Log10Dose = -1.41 LD50 = 10log10 dose = 10-1.62 LD50 = 10log10 dose = 10-1.41 LD50 = 0.02398832919 ≈ 0.02399% w/v LD50 = 0.0389045145 ≈ 0.03890% w/v LD50 = 0.02398832919% w/v LD50 = 0.0389045145% w/v = 0.02398832919g/100mL = 0.0389045145g/100mL = 0.0002398832919g/mL = 0.000389045145g/mL = 0.0002398832919mg/L = 0.000389045145mg/L = 0.0002398832919mg /0.53082g (from table = 0.000389045145mg /0.431g (from table 2) = 9.0265694 x 10-4 mg/g 2) -4 = 4.519108 x 10 mg/g = 0.90265694mg/kg = 0.4519108 mg/kg Table 5. Table 5 shows the conversion of LD50 from %w/v to mg/kg in both sample from figure 2, our results and last year class results. Determining LD50 From Figure 2 Figure 2: Our Values Figure 2: Last Year Class Result y=0.9004x +1.2764 y=1.5619x + 2.1916 LD50 = y=0 LD50 = y=0 -1.2764 = 0.9004x -2.1916 = 1.5619x −1.2764 −2.1916 = -1.417592181 = -1.403162815 x= x= 0.9004 1.5619 LD50 = 10log10 dose = 10-1.417592181 LD50 = 10log10 dose = 10-1.403162815 LD50 = 0.03823030995 ≈ 0.03823% w/v LD50 = 0.03952184267 ≈ 0.03952% w/v

LD50 = 0.03823030995% w/v = 0.03823030995g/100mL = 0.0003823030995g/mL = 0.0003823030995mg/L = 0.0003823030995mg/0.53082g (from table 2) = 7.2021231 x 10-4 mg/g = 0.72021231mg/kg

LD50 = 0.03952184267 % w/v = 0.03952184267 g/100mL = 0.0003952184267 g/mL = 0.0003952184267mg /L = 0.0003952184267mg/0.431g (from table 2) = 9.169801 x 10-4 mg/g = 0.9169801mg/kg

Discussion Insecticide plays an important role in monitoring and controlling the levels of insects in particular the American Nymph Cockroaches. Monitoring and controlling the levels of these cockroaches is very important, as the American cockroaches can trigger many reactions to the human body such as allergic reactions and asthma. The American cockroaches are also carriers of many diseases, parasitic worms and bacteria which can have detrimental effects on human with symptoms such as food poisoning or diarrhea. The LD50 value from our result were 0.4519mg/kg obtained from the sigmoidal graph and 0.7202mg/kg obtained from the linear graph as seen in table 3. There is quite a difference in these LD50 values, however, it can be said that the linear probit graph produced a more accurate LD50 value. The linear probit graph allowed for better extrapolation compared to the sigmoidal graph which was difficult to extrapolate due to the curve nature of the graph. This can also be said for the results obtained by last year’s class. The LD50 value from last year class were 0.9026mg/kg obtained from the sigmoidal graph and 0.9170mg/kg obtained from the linear probit graph as seen in table 3. These LD50 values obtained from last year’s class were quite similar indicating that both graph produced fairly accurate values. The LD50 values could have been affected by many external factors such as temperature, length, the stage of life cycle the cockroaches were in and how healthy the cockroaches may be. The LD50 values could also have been affected by the graph in which the LD50 values were extrapolated from. External factors can play a huge part in determining the LD50 value. Each cockroach was measured and chose accordingly, however, it was difficult to keep the length consistent as some cockroaches were slightly curved thus could have affected the LD50 value. In future investigations and studies, the exact length of the cockroaches would be recorded and cockroaches of 2cm with a 1mm leeway would be chosen. The stage of the life cycle the cockroaches were in may have also affected the LD50 value as some cockroaches could have been more susceptible or resistant to the insecticide, i.e. cockroaches which may have just recently been in the egg stage of the life cycle or cockroaches approaching the adult stage in the life cycle which could have been more resistant to the insecticide. It is very difficult to keep this consistent as well as determine the stages the cockroaches may be in. In future investigations and studies, monitoring the exact growth of the cockroaches once they hatch from the egg would be helpful in determine the stage in which the cockroaches may be in thus would not affect the value of LD50. Similar to the stage of life cycle the cockroaches may be in, how healthy a cockroach may be could affect the LD50 value as weaker cockroaches may be more susceptible to the insecticide while healthier cockroaches may be more resistant to the insecticides. It would be difficult to pick out healthier cockroaches in future investigation, thus averaging a larger quantity of cockroaches would help obtain a more accurate LD50.

Extreme temperature can affect the cockroaches’ behavior as they are cold blooded organisms (Ogg, Ogg & Ferraro, 2006). Cockroaches like warm and moist places (Michigan State University, n.d.), hence extremely hot or extremely cold places can affect the cockroaches’ system causing them to be slowed down or killed as seen by the control from our results in table 1. Even though there were no insecticide used, there was still a death of a cockroach as the cockroach’s body cannot adapt to the change of the sudden change in temperature. Also since the cockroaches were in placed in cold temperature, this could have made them weak and hence more susceptible to the effects of the insecticide. In future investigations and studies, a gradually change in temperature to allow the cockroaches’ body to adapt to the cold conditions could be performed in which these cold condition would still slow them down so the insecticide can be placed onto the cockroaches thus allowing us to obtain a more accurate LD50 value. Using two set of graphs to extrapolate the LD50 value can help obtain a more accurate LD50 value as seen in table 3, figure 1 and figure 2, however the shape of the curve and the amount of data used for the graph can affect the LD50 value. As seen in figure 1, last year class result did not produce a sigmoidal graph; it was more like a linear graph. This could have affected the LD50 value as it would be similar to the linear probit graph as illustrated by the result of the LD50 value of last year class result as seen in table 3. It is very difficult to determine the percentage of death from insecticide to product a sigmoidal graph thus more replicates must be conducted to obtain a more accurate LD50 value. The amount of data used for the graph can affect the LD50 value as seen in table 3 as the LD50 value obtained had a huge difference. The linear probit graph only contained two data which was used to construct the linear graph. This is not very accurate as the graph is not a line of best fit but just connecting the dots to form a line. If there are more values to graph, the equation of the line would be different in which a more accurate LD50 could be obtained. Similarly, to the percentage of death from insecticide to product a sigmoidal graph, it is very difficult to determine the percentage of death and the amount of data collected, thus more replicates must be conducted to obtain a more accurate LD50 value.

The LD50 values can vary due to the different type of insecticide used and the type of cockroaches tested on. Studies done by Ruhma et al, (Ruhma et al, 2014) indicated that the LD50 value can vary depending on the different type of cockroaches. In the study, Imidacloprid was used as the insecticide and the American cockroaches the specimen tested upon however the cockroaches were obtained from three different locations. In this study, the LD50 of Imidacloprid on cockroaches obtain from sewage were 2.712l/mL while the LD50 value of Imidacloprid on cockroaches obtained from kitchens and basement were 1.960l/mL demonstrating that external factors can affect the LD50 value. The LD50 values can also vary between females and males. Studies done by Lee, Yap & Chong, (Lee, Yap & Chong, 1996) indicated the LD50 of propoxur of male German cockroaches was 15.60g/g and LD50 of propoxur of female German cockroaches was 15.85g/g demonstrating that gender of cockroaches can affect the LD50 values. The LD50 values from both studies differ from the LD50 value obtained in this investigation. The difference in the LD50 indicates that many factors and variable can affect the effects of insecticides. Limitation that were encountered during this investigation included not being able to replicate the investigation under stricter conditions such as keeping the temperature of laboratory the same as temperature can affect the cockroaches. Although the investigation was replicated,

there were large difference in result thus results were not consistent. Even though class data was used in the investigation, people may approach the method differently which may alter the results of the investigation, i.e. the handling of cockroaches. Further directions for this investigation includes repeating the investigation with smaller increment of concentration to obtain a more accurate LD50 value. Also using adult cockroaches and different type of cockroaches to see how the LD50 value would change would be beneficial in understanding LD50. Conclusion The purpose of the investigation was to determine the LD50 value of propoxur on the American cockroaches. The investigation determined that LD50 of propoxur on American cockroaches were about 0.02399%w/v and 0.03823%w/v. More research should be conducted as LD50 result varied even though the same method was followed. The results only demonstrated the LD50 on one type of cockroaches, replicating the investigation with other breed of cockroaches would be beneficial to see how the LD50 values would change.

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Rang H.P. & Hill R.G., 2013, Drug Discovery and Devleopment, 2nd edition, Churchill Livingstone Elsevier, London Ruhma, S., Manzoor, F., Adalat, R., Abdul-Sattar, A. & Syed, A., 2014, Laboratory Evaluation of Toxicity of Insecticide Formulations from Different Classes Against American Cockroach (Dictyoptera: Blattellidae), Iranian Journal of Arthropod-Borne Diseases, vol. 8, no. 1, pp. 21-34. The Pennsylvania State University, 2013, American Cockroaches, Viewed 26/05/2018 https://ento.psu.edu/extension/factsheets/american-cockroaches University of Florida, 2017, American Cockroaches, Viewed 26/05/2018 http://entnemdept.ufl.edu/creatures/urban/roaches/american_cockroach.htm World Health Organisation (WHO) & Rozendaal J.A., 1997, Vector Control: Methods For Use By Individuals and Communities, World Health Organisation, Geneva...