LD50 of the Carbamate Insecticide Propoxur on America Nymph Cockroaches

IntroductionRoughly 3,500 type of cockroaches have been recognised, including Periplaneta Americana, commonly referred to as the American cockroach (Cockroaches 1849). Cockroaches adaptability to their environments have enabled them to live in harsh conditions and spread to many places. American cockroaches are the largest of the species measuring 35-40 mm long with a reddish-brown colour, and have wings that cover their entire abdomen, the nymphs are smaller and do not have wings (Cockroaches 1849) (Imamura, S. 2003). Compared to the German cockroach that is 10-15 mm in length, it’s body is a shade of brown ranging from dark to light, and has two very distinct black striped running down it’s back and it has wings (Valles, S. 2017). They are found in dark, warm and moist areas of buildings where there is food and water available, their main food source being decaying food matter (Cockroaches 1849). Cockroaches have three main stages in their life cycle: egg 1-3 months, nymph lasts several months to a year, then adult (Imamura, S. 2003). During the nymph cycle the cockroaches grow by continuously shedding the cuticle or skin (Imamura, S. 2003).

Cockroaches are vectors for transmitting diseases including: Escherichia coli, diarrhoea, Pseudomonas aeruginosa, plague, fungi, parasites, dysentery, leprosy, cholera, typhoid fever, and viral diseases (Imamura, S. 2003) (Stankus R, 1990). As cockroaches spread disease and they carry the eggs of parasitic worms, and furthermore they could be the cause of allergic reactions including; itching and swelling of the eyelids, dermatitis, and respiratory conditions (Stankus R, 1990). Cockroaches lay a sort of trail with their pheromones or chemicals, that help other cockroaches identify where food or mates may be found this will increase the number of cockroaches in an area and can lead to an infestation (Jeanson, R, 2006). Cockroaches are common insects that plague society with disease and an unsettling feeling of seeing the cockroaches so the control of cockroaches in an area needs to be controlled to stop the spread of disease. The number of cockroaches in an area are managed through several ways including: glue strips, caulk gaps, gel bait, roach hotels, boric acid powder, sprays, and pet management professionals. One such class of insecticide used is carbamate and is derived from carbamic acid (Weiden, M 1965).

Carbamate is a successful insecticide by having a brief effect of inhibiting cholinesterase enzymes on the target pest and because they are used widely due to its benefits of having a low mammalian, oral and dermal toxicity and it influences a broad range of insects (Weiden, M 1965). Their structures complement the active site of acetylcholinesterase, therefore once carbamate binds to the AChE receptor it behaves as a neurohormone and induces a toxic reaction and ACH accumulates in the synaptic junction (Kurtz, A.1987). One pesticide in the carbamate family is propoxur, it blocks the chemical action and production of cholinesterase enzyme and causes the nervous system of insects to be paralysed (Propoxur 2017). To measure the effects of pesticides on American nymph cockroaches we used LD50.

LD50 measures an amount of the substance being tested that kills 50 per cent of the test sample in mg/kg meaning milligrams of pesticide per kilogram of the cockroach body weight. The aim of this experiment is to measure the LD50 of Baygon, which has the active ingredient propoxur from the carbamate class of insecticides, on American Nymph cockroaches.

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MethodsThe methods of this experiment can be found in the Pharmacology Subject Manual pages 28-35. The only change is the Propoxur used is 97.6% pure concentration compared to 100%.

Results

% Response vs Propoxur Dose Concentration on a Probit curve graph

Figure 1: The relationship between % Response (death of cockroaches) and Propoxur Dose Concentration (% w/v) using a probit curve and a line of best fit. The group data collected by group Batman uses only 2 points at, 0.01% and 0.05% due to the 0.02% data point being an outlier. The group LD50 is measured at 0.05% Propoxur dose concentration (% w/v). The class LD50 is measured at 0.03% Propoxur dose concentration (5 m/v).

LD50 Calculations Group resultsLD50 = 0.05% w/v

= 0.05g/100mL= 0.05mg/100L= 0.0005mg/L (per cockroach)= 0.0005mg/0.35g= 0.0014mg/g= 0.14mg/kg

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LD50 Calculations Class average resultsLD50 = 0.03% w/v

= 0.03g/100mL= 0.03mg/100L= 0.0003mg/L (per cockroach)= 0.0003mg/0.48g= 0.000625mg/g= 0.0625mg/kg

% Response vs Propoxur Dose Concentration on a log-dose response curve

Figure 2: The relationship between % Response (death of cockroaches) and Propoxur Dose Concentration (% w/v) using a log-dose response curve. The group data collected by group Batman uses only 2 points at, 0.01% and 0.05% due to the 0.02% data point being an outlier. The group LD50 is measured at 0.05% Propoxur dose concentration (% w/v). The class LD50 is measured at 0.03% Propoxur dose concentration (5 m/v).

LD50 Calculations Group resultsLD50 = 0.05% w/v

= 0.05g/100mL= 0.05mg/100L= 0.0005mg/L (per cockroach)= 0.0005mg/0.35g= 0.0014mg/g

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= 0.14mg/kg

LD50 Calculations Class average resultsLD50 = 0.03% w/v

= 0.03g/100mL= 0.03mg/100L= 0.0003mg/L (per cockroach)= 0.0003mg/0.48g= 0.000625mg/g= 0.625mg/kg

Probit-log Linear- logLD50 Class 0.625mg/kg 0.625mg/kgLD50 Group 0.14mg/kg 0.14mg/kg

Figure 3: Propoxur LD50 of American Nymph Cockroaches categorized by class and group according to the log and probit graphs.

Discussion

Upon evaluation of the probit-log graph and linear-log graph the data shows an exponential rise in the deaths of the American nymph cockroaches as the amount of propoxur increases. The significance of the results shows that both the log-dose response curve and the probit curve are equally reliable for determining the LD50 of Propoxur on American nymph cockroaches. The results obtained we’re the same for both graphs, therefore either graph would be suitable to use when determining the LD50. The log-dose response curve and probit curve determined the LD50 for the class was 0.625mg/kg and for the group it was 0.14mg/kg according to Figure 3. Although the graphs both determined that either the probit-log or the linear-log graph to be equally reputable sources to find the LD50, the probit graph should be the chosen graph used to analyse the dose-response relationships. This is because probit analysis is used to test the relative toxicity of the tested chemicals on a living sample. The probit analysis uses regression to analyse binomial response variables by taking the sigmoid curve and transforming it to a linear relationship for analysis when they run a regression (Gaddum, J. 1948).

One data point for 0.02% has been omitted from the group data points that were graphed because it was an outlier being 0. The suspected causes were mishandling of the cockroaches while applying the propoxur concentrations, the propoxur wasn’t applied correctly or the cockroaches did not undergo treatment with the pesticide. This collected data has been determined to be an outlier due to comparing results with the rest of the classes collected data and them having had deaths at 0.02%. Another reason this collected data is a mistake is because in the data cockroaches were killed at 0.01% dilution, and due to preliminary studies done by the UTS staff we know that the range for LD50 is between 0.01% w/v and up to 0.1% w/v so there had to have been deaths at 0.02% propoxur concentration.

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Another source of error that could affect the results obtained is the weight of the cockroaches. The weight of a nymph is less than that of an adult American cockroach and may influence the time it takes for death of the cockroach. The nymph is constantly shedding its outer cuticle as it grows, leaving more delicate skin underneath whereas an adult cockroach would have a hard cuticle that can withstand more external pressures (Imamura, S. 2003). The varying weights can be seen with the class average weight being 0.48 g and the group average cockroach weight being 0.35. Our group cockroaches may adhere more to the guidelines of using the nymph size for this experiment, whereas the class may have used larger adult sized cockroaches. This theory is backed up by a study showing that male and female German cockroaches had a different LD50 due to different weights between the sexes, but when the measurements we’re changed to microgram per gram per insect both male and females had a similar LD50 (Scott, J.,1990).

The weight error may be solved by being more stringent on which cockroaches are chosen, or better separating the cockroaches by size and sex and adjusting the dosage given according to weight. In accordance to the outlier that our group managed, I would suggest better planning for the group members with more obvious labelling of dose concentrations, and being more aware while preparing the dilutions.

According to the collected data, propoxur is reliable insecticide for killing half of the population using amounts between 0.14 mg/kg and 0.625mg/kg for the American nymph cockroach population. Although propoxur is a reliable insecticide for that of the American cockroach it is not a reliable insecticide for that of the German cockroach population. According to studies, the LD50 of propoxur in German cockroaches ranges between 14.50 mg/kg and 17.25 mg/kg much higher than that of the American cockroach LD50 (Lee, C. 1996). The toxicity of the insecticide can be influenced my many factors including physiological resistance, age and sex of the cockroaches (Lee, C. 1996). The pesticide resistant cockroaches have an enzyme that allows their body to break down the pesticide and excrete it with no harm to the insect, this advantage is passed on to its offspring therefore leading to pesticide resistant cockroaches. A different class of pesticide is necessary to kill these resistant cockroaches because their body has not adapted to the new form of the toxin. This resistance is to the carbamate class of pesticides because other class of pesticides such as pyrethroids and organophosphates had an LD50 much lower than that of the carbamate class, ranging between 0.17 mg/kg to 8.58 mg/kg (Lee, C. 1996).

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References

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Lee, C. 1996, Insecticide toxicity on the adult German cockroach, BZuttella germanica (L.) (Dictyoptera: Blattellidae), Malaysian Journal of Science, vol 17A 1-9, pp.1-9, viewed 26 May 2017, <http://www.chowyang.com/uploads/2/4/3/5/24359966/012.pdf>.

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Scott, J., Cochran, D. and Siegfried, B. 1990, Insecticide Toxicity, Synergism, and Resistance in the German Cockroach (Dictyoptera: Blattellidae), Journal of Economic Entomology, vol 83, no 5, pp.1698-1703,.

STANKUS, R., HORNER, W. and LEHRER, S. 1990, Identification and characterization of important cockroach allergens2, Journal of Allergy and Clinical Immunology, vol 86, no 5, pp.781-787, viewed 25 May 2017, <http://www.entomoljournal.com/archives/2015/vol3issue1/PartF/3-1-46-828.pdf>.

Valles, S. 2017, German cockroach - Blattella germanica (Linnaeus), Entnemdept.ufl.edu. viewed 25 May 2017, <http://entnemdept.ufl.edu/creatures/urban/roaches/german.htm>.

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