Determination of the LD50 of Propoxur on American Nymph Cockroaches

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08/02/20 Sciences Reference this

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  1. Introduction

Periplaneta Americana or American cockroaches are the largest, most recurrent and almost the most abundant domestic pest in households. The cockroaches can adapt to any living conditions, regardless of temperature, cleanliness, exposure to light etc. In terms of temperature, they function optimally in temperatures around 21°C. They are usually located in sewers, drainage systems, rubbish and areas where food is present. Their diet is their defying factor, in the sense that they are an opportunistic eater. They are able to consume anything, as well as an organic matter which has decayed. Their eerie habitual nature allows them to transmit pathogens and germs E.coli and transmit them to humans and the general public.

This study examines the strength of insecticides in controlling the spread of such pathogens to the general public, and ultimately reducing the population of the American cockroaches. The organic compound, Carbamate, is commonly used to terminate cockroaches. The organic compound is derived from carbamic acid and can inhibit the activity of the enzyme acetylcholinesterase (AChE) (Silberman & Taylor 2018). The carbamic acid group targets the Acetyl-Cholinesterase enzyme in cockroaches and thereby, inhibiting this enzyme ability to activate in the nervous system. The inhibition of the enzyme will result in a decline in cholinesterase enzymatic activity, and upon exposure to the carbamate, the cockroach will experience toxicity to neurons and thus paralysing their nervous system (Corbel et al. 2006).

This study specifically examines Propoxur, which contains carbamate as its active constituent. The presence of the organic compound allows for the inhibition of the enzyme which is associated with neurotransmission, acetylcholinesterase and thus produces neurotoxic effects when it hits target organisms (Vale & Lotti 2015). Propoxur is commercially used due to its strenuous penetrative ability and thus, in order to determine the insecticide is not toxic, but yet safe for humans to handle, it must be tested for toxicity. Lethal Dose at 50%, known as LD50, is the evaluation of toxicity undergone through the calculation of the lethal dose when 50% of the sample population has been terminated. The evaluative process of toxicity ensures that safety to the human population, as well as ensuring the penetrative ability to the insecticides remains. In addition to this, theoretically, an increase in LD50 will result in an increase in mortality rates. Conversely, research has suggested that a decrease in LD50 will result in an increase in toxicity of the insecticide and thus it will reduce the time of exposure required to eliminate 50% of the population (University of Minnesota Extension 2015. Thus, analysis of the toxicity of a substance and understanding the LD50 is of significance.

LD50 is a measurement used to test chemicals that could potentially be toxic, as it examines how much of a substance is required to kill 50% of the sample population. This experiment explores this concept, as a range of 0.01% w/v and 0.1% w/v of carbamate propoxur concentrations were used as a means to determine the LD50  (mg/kg). Theoretically, an increase in cockroach mortality rates will be due to a higher concentration of propoxur dose.

The central objective of this study is to examine the LD50 of propoxur on American cockroaches in their Nymph stage, at various concentrations. In addition, the mortality results will be compared amongst the class of 2017 results. This study hypothesised that a positive relationship between propoxur concentration and mortality rates.

  1. Materials and Methodology

A detailed methodology used in this experiment is located in the ‘91707 Pharmacology 1 Subject Manual’ on pages LD1 to LD8. In summary, this methodology examined a suitable range of carbamate propoxur (% w/v) in order to determine a precise and accurate LD50 on the American Cockroaches. The purity of the sample listed in the book was 100%, however it was altered to 97.6%.

The range was set up with serial dilutions in 4 vials of EMK and Carbamate Propoxur stock at 0.1, 0.05, 0.02 and 0.01% w/v. The fifth vial was the control, which did not contain the insecticide, rather just EMK. The methodology required each ten of the plastic cups, which were lathered in Fluon to contain a sample population of 5 cockroaches within the size range of 2cm. There was a total of 50 cockroaches and the average weight of each cockroach obtained was 0.431grams. The American Cockroaches were administered with the insecticide of the corresponding dilution dose, and the fatality rate of each dose was averaged and recorded.

The results calculated were then used to create a Log-linear and log dose-probit graph which assisted with the determination of LD50 through the use of coefficient of determination (R2) and the trendline equation.

  1. Results

Dose Concentration (% w/v)

Mortality rate for Group (%)

Mortality rate for Class 2017 (%)

0.1

55

73

0.05

50

58

0.02

35

30.7

0.01

25

0

Table 1: Tabulation of percentage Mortality rates of the American Cockroaches under the exposure of different dose concentrations (w/v) across 2 groups.

Table 1 illustrates the difference in mortality rates in percentages between Class of 2017 and the group. The table depicts what exposed dose concentration (0.01, 0.02, 0.05 and 0.1) is able to exterminate the American Cockroaches, thus exploring that the lower the dose concentration, the lower the mortality rate is. By confirming the total amount of how many American cockroaches had deceased after the infestation of Propoxur, the mortality rate percentage was calculated by converting the raw amount into a percentage.

It is shown that the dose concentration 0.1 (%w/v) resulted in a mortality rate of 55% in the group, whereas the mortality rate for the Class of 2017, at the same dose concentration, was at 73%. Furthermore, the dose concentration at 0.05 (%w/v) has a mortality rate of 50% compared to the mortality rate of the class of 2017 at 58%. In addition to this, the dose concentration of 0.02 (%w/v) resulted in 35% of deaths for the group, parallel to the mortality rate of the class of 2017 being 30.7%. Lastly, the group mortality rate at 0.01(%w/v) resulted in 25% of deaths in comparison to an overwhelmingly distinctive 0% for the class of 2017.

 

Figure 1: Dose-response curve of Log10 dose of carbonate propoxur versus the mortality rate percentage of American cockroaches. Lines on each curve signify the LD50. It was found to be 0.03% (w/v) for the class of 2017 and 0.05% (w/v) respectively for the group.

The data presented on the dose-response curve in figure 1 represents the mortality rate percentage (%) versus the Log10 dose of carbonate propoxur. This figure enabled the finding of the LD50 for both the class of 2017 and the group, finding 0.04% (w/v) for the class of 2017 and 0.05% (w/v) for the group, respectively. Additionally, the class of 2017 results indicated that at log10 -1.42, 50% of the population were deceased and at -1.3, 50% of the group’s population died, correspondingly. Overall, the figure illustrated a close relationship, as an increase in  Log10 dosages  resulted in an increase in the mortality percentage.

Figure 2:  Probit percentage of deaths (%) in American cockroaches against the Log10 dose of carbonate propoxur for Class of 2017 and group.

Figure 2 is a probit graph which assisted in converting the mortality percentage as stated in table 1 into a probability unit and further into a linear figure from a sigmoid graph. The R2 value for the group is 0.99 and the 50% mortality percentage is found at 5% in figure 2. The 5% was used with the trend line equation depicted on the figure 2 to calculate the Log10 probit which was found to be -1.218 for the group and -1.46 for the Class of 2017, respectively. In addition to this, the median effective dose (EC50) was 0.06 % w/v for the group and 0.04% w/v for the class of 2017.

Graph/Group

Calculated LD50 (mg/kg)

Group/Fig. 1

0.91mg/kg

Class/Fig. 1

0.72 mg/kg

Group/Fig. 2

1.1mg/kg

Class/Fig. 2

0.72 mg/kg

Average:

0.86 mg/kg

Table 2: LD50 results based on calculations in table 2. The average LD50 for both the class and group was calculated to 0.86mg/kg (2 significant figures) and the LD50 values (mg/kg) were calculated respectively.

Figure 1. Group

Figure 1 C. 2017

Figure 2. Group

Figure 2 C.2017

 

0.05

0.04

0.06 grams

0.04 grams

0.05g/100mL

0.04g/100mL

0.06g/100mL

0.04g/100mL

0.0005g/mL

0.0004g/mL

0.0006g/mL

0.0004g/mL

0.0005mg/uL

0.0004mg/uL

0.0006mg/uL

0.0004mg/uL

0.0005mg/0.5503g

0.0004mg/0.716g

0.0006mg/0.5503g

0.0004mg/0.716g

0.00908595

0.00071556

0.00109031

0.00071556

0.908×10^-3mg/g

0.716×10^-4mg/g

1.0903× 10^-3mg/g

0.716×10^-4mg/g

0.908

0.716

1.090

0.716

Average

0.908mg/kg

0.716mg/kg

1.090mg/kg

0.716mg/kg

0.857575mg/kg

Table 3: The table depicts the LD50 calculation of the Class and the group based on Figures 1 and 2. The final value was rounded to two significant figures where applicable and units were set in mg/kg. The final values were inputted into table 2 and used for analysis in section 4 of the report.

  1. Discussion

The significance of this study was to determine the effectiveness of the lethal dose (LD50) of the carbamate insecticide, propoxur, on the death of lymph American Cockroaches. Propoxur insecticide is composed of an organic compound, carbamate which is used to a great extent into organisms, due to its low toxicity to humans (Beyond Pesticides 2010) and due to its penetrative ability (Campbell, A. & Chapman, M., 2008). This study explored the effectiveness of various concentrations (0.1%w/v to 0.01% w/v) of propoxur upon exposure on two sets of five cockroaches for each concentration of the insecticide. The group data presented in section 3 of this report is not the data in which the author of this paper obtained. The reasoning behind this is because the data obtained was invalid and inaccurate as all the sample population had died. The data presented in section 3 of this report illustrated a strong relationship between the dose concentration (w/v) and the mortality rate (% w/v) whereby an increase in dose concentration, resulted in a concurrent increase in the mortality rate (% w/v) as depicted in figures 1 and 2. Correspondingly, the R2 value illustrated in figure 2 for the class of 2017 was 0.99 and 0.98 for the group, respectively, further proving the validity and accuracy of this case.

Figure 1 and 2 enabled the calculation for the LD50 values (mg/kg) as 0.72 for the class of 2017 and 0.91 (mg/kg) for the group of figure 1. Further to this, the average LD50 value as depicted in table 2 is 0.86 (mg/kg). Respective of the tightly bonded values formulated, the class of 2017 results represented a more precise LD50 of propoxur. The class of 2017 R2 value of 0.99 as well as the gradual positive linear relationship in contrast to the group, proves the accuracy and validity of between the mortality percentage and the log10 dose. 

There are a number of elements which could have contributed to the slight variance of the LD50 across the data obtained from the group. These include the difference of the average weights of the cockroaches calculated, as well as the difference in the size of the subjects not being kept constant at 0.5

 ±

2cm. The inconsistency in ensuring the accuracy of sizing and weighing the cockroaches was kept constant are possibly due to the weight being taken after the insecticide was exposed and the sizing using the human eye. This human error could have been evaded through the use of a measuring the length via a ruler and the weight of each cockroach being taken individually prior to the exposure of the propoxur. By ensuring this control measure was followed, the accuracy and precision of the LD50 result for the group would have increased substantially.

However, both sets of data (figure 1 and 2) illustrate a slight difference in the coefficient of determination (R2). In saying this, indicating that the results obtained were significant and valid and sources of human error were controlled and eradicated as the values were close to be equal to 1.   

In addition, table 1 depicts an increase in variance between the effectiveness of the dose concentration to the mortality rate in the group compared to the class of 2017. Although the purity of the propoxur samples were not 100%, the decrease in effectiveness in the dose could be due to the cockroach’s distinctive metabolic response. Furthermore, immunity and resistance to a range of toxicity levels fluctuate and increase when cockroaches are in their lymph stage of their life cycle (Roush 1991).

Research has found that the larger a cockroach is, the more surface area in the abdomen is present (where the insecticide was exposed) and the more tissue volume present, therefore altering the effectiveness of the propoxur (Martinez 2011). Thus, as their weight and size increases, the LD50 varies as they become more prone to the sense of propoxur, further illuminating to the gist that the smaller subjects found were more prone to die from the doses propoxur (White & Kearner 2014).

The varied result of the LD50 could be also a component of human error such as the absence of repetition conducted throughout the study from the group leading to insufficient amounts of data. The data obtained for the group was not sparse enough to generate a valid standard deviation and hence, altering the integrity of the data of the group. The repetition of an experiment will create a direct relationship between an increase in the precision and validity of results and a decline in human error (Hu et al. 2011).

There is a lack of research currently undergone on American cockroaches, in specific, the effect of different insecticides on the pests. In contrast, there has been major study done on the effect of insecticides on the German cockroaches. A study of particular interest investigated the efficacy of multiple insecticides on the German cockroaches (Valles, Koehler & Brenner 1999). The insecticides tested were propoxur and chlorpyrifos. The study discovered that the LD50 of propoxur was 0.65mg/kg, and the LD50 of chlorpyrifos at 0.68 mg/kg, correspondingly.  Another research also studied the efficacy of  chlorpyrifos and found a LD50 of 0.713mg/kg (Rust & Reierson 1991). The difference in these values were found to be due to females and males both inheriting diverse physiological features, as well as females having a higher fat percentage – and thus metabolising slower.

Although no research has been undertaken on the resistance of American cockroaches to the insecticide tested in this experiment, the trends and results obtained can suggest that the cockroaches are not resistant to Propoxur but can be resistant to other insecticides. Factors including lifestyle, weight, body fat, localisation of insecticides and sex should be examined and considered in future research to attain a precise and valid LD50.

  1. References
  • Beyond Pesticides 2010, Propoxur, United States of America, viewed 28 May 2018, <https://www.beyondpesticides.org/assets/media/documents/pesticides/factsheets/Propoxur.pdf>
  • Campbell, A. & Chapman., M. 2008, Handbook of poisoning in Dogs and Cats, 1st edn, Wiley Backwell, Oxford, United Kingdom.
  • Corbel, V., Stankiewicz, M., Bonnet, J., Grolleau, F., Hougard, J. & Lapied, B. 2006, Synergism between insecticides permethrin and Propoxur occurs through activation of presynaptic muscarinic negative feedback of acetylcholine release in the insect central nervous system’, Neurotoxicology, vol. 218, pp. 141-150.

         Hu, LP., Bao, XL. & Wang, Q. 2011, ‘The repetition principle in scientific research’, Journal of Chinese Integrative Medicine, no. 9, pp. 937-940

  • Martinez, M.N., 2011. ‘Factors influencing the use and interpretation of animal models in the development of parenteral drug delivery systems’, The AAPS journal, vol. 13, no. 4, pp.632-649.
  • Roush, R.T. 1991, Management of pesticide resistance, CRC Press, Florida.

         Silberman, J., & Taylor, A. 2018, ‘ Carbamate Toxicity’, Stat Pearls, pp. n/a.

  • University of Minnesota Extension 2015, ‘Appendix A: Pesticides Toxicities’, Minneapolis, viewed 27 May 2019.
  • Vale, A. & Lotti, M. 2015, ‘Chapter 10 – Organophosphorus and carbamate insecticide poisoning’, in M. Lotti & M.L. Bleecker (eds), Handbook of Clinical Neurology, vol. 131, Elsevier, pp. 149-68.
  • Valles, S.M., Koehler, P.G. & Brenner, R.J. 1999, ‘Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea’, Comp Biochem Physiol C Phamacol Toxicol Endocrinol, vol. 124, no. 3, pp. 227-232.
  • White, R. & Kearner, R. 2014,’ Metabolic scaling in animals: methods, empirical results, and theoretical explanations’, Comprehensive Physiology, vol. 4, no. 8, pp. 231 – 256.
  1. Appendix

All data was obtained and inputted in the software, Microsoft Excel for data analysis

  1. Table 4: Class of 2017 Data. Average mortality percentage (%) and the standard deviation upon corresponding concentration of propoxur.

2017 DATA

Concentration of Propoxur 

0.1

0.05

0.02

0.01

control

% death

73

58

30.7

18

0

STDEV

20

15

7

3.5

0

  1. Table 5: Group Data. Average mortality percentage (%) and the standard deviation upon corresponding concentration of propoxur.

Concentration of Propoxur 

0.1

0.05

0.02

0.01

AV % (DEATH)

55

50

35

25

STDEV

44.3471157

41.63332

30

30

  1. Table 6: EC50 Calculation for probit graph

LogEC50

-1.218

EC50

0.06053409

y = 0.8246x + 0.0044

x = (5-6.0044)/0.8246

x = -1.218

ANTI-LOG = 0.065 % w/v

  1. Table 7: LD50 calculation for Figure 1

Group:

Log 10 (LD50)

-1.3

LD50 (%w/v)

0.05

2017

Log 10(LD50)

-1.42

LD50 (%w/v)

0.03801894

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