Determining the LD50 of carbamate propoxur on American nymph

cockroaches
A. Nguyen (99141754)

Introduction:
American cockroaches (Periplaneta Americana) are the largest of its type and
being the second most popular cockroach in the world [1] makes it one of the
most problematic household pests. The most common types being the German,
Australian and American cockroach, with the latter the focus of this experiment.
These cockroaches are reddish brown with a light-yellow border around their
heads and can grow up to 45mm in body length [2] and favour damp and humid
environments such as rubbish dumps, sewers, grease traps to food preparation
areas [2]. With a lifespan of up to one year, upon reaching adulthood they grow
wings and move on from the nymph stage [1]. Since their habitats are so
versatile similarly so are their diets, this includes excrement, fingernails/toenails
of sleeping persons or babies to their own skin casts [3]. Due to the nature of
their habitats and diets they are efficient carriers of pathogens such as
salmonella, E. coli and poliomyelitis [4] and hence can easily transmit disease
affecting public health. Furthermore, it has been shown from studies that
asthma, hay fever and dermatitis may be caused by cockroaches [4] so even
though they are not the direct cause, they are vectors for these disease [5] and
thus their population must be controlled to ensure the safety of society.

To combat this, insecticides play an important role in the domestic household

and industry as they exterminate common household pests with convenience
and affordability. A commonly used ingredient in insecticide is carbamate, which
is an organic compound derived from carbamic acid. Carbamates mechanism of
action is through the inhibition of acetylcholinesterase (AChE) activity [6]. When
the enzyme AChE is released from synaptic clefts, it is accountable for the
degradation of the neurotransmitter acetylcholine (ACh) after it has been
released from the presynaptic neuron. ACh is connected to receptors that
instigate contractions of the muscle thereby making it vital for the transmission
of nerve impulses. When the neuromuscular junction experiences excess ACh as
a result of AChE inhibition, the respiratory system is depressed and the body
undergoes parasympathetic activity in the gastrointestinal system as well as
smooth muscles of the heart and lungs [6]. This hyperpolarization of muscle cells
[7] would eventually result in myopathy and/or death [8].

An effective carbamate widely used is Propoxur (IUPAC name 2-propan-2-

yloxyphenyl N-methylcarbamate) or commercially known as Baygon, it is a
derivative of catechol which consists of the functional group isopropyl and
carbamate [7]. This insecticide has the capability of killing insects ranging from
spiders [9], snails, mosquitoes [10] to cockroaches [11]. Its high potency [12]
and ability to efficiently penetrate the body of the insect [8] as well as permeate
the cell membrane [13] make it a popular choice of insecticide.

However, like all marketed products propoxur must be tested for toxicity to
humans. This is determined through the LD50 which is a standard of
measurement of the lethal dose required to kill 50% of the sample population.
Hence to effectively maintain a safe cockroach population in society without
harming the environment and its inhabitants the ideal concentration needed to
do so is vital. Thus, in this study on the American Nymph Cockroach the LD50 of
propoxur is applied dermally, tested and compared between a class and an
individual group result.
Methods:
The method outlined in experiment 3: determination of the LD50 of an
insecticide in cockroaches of the pharmacology 1 manual was followed for this
experiment. Changes were made to the purity of propoxur to 97.6% hence the
mass of propoxur used was 0.2561g.

Results:

Table 1: cockroach mortality of sample 1 & 2 in response to different

propoxur dose concentrations (w/v %) and the average cockroach mass
in individual groups and the class as a whole
Propoxur dose concentrations (w/v %)

Group Contr 0.01 0.02 0.05 0.1 1 Average cockroach

ol mass (g)

S S S S S S S S S S S S
1 2 1 2 1 2 1 2 1 2 1 2

The 0 1 1 1 0 1 2 3 4 4 - - 0.522
Beatles

N.O 0 0 0 0 1 3 4 4 4 4 - - 0.596

The 1 2 0 0 2 3 4 3 4 4 - - 0.329
Husks

Adam 1 0 1 0 3 4 4 3 4 5 - - 0.384

FAM 0 0 - - 1 2 5 3 5 5 5 5 0.473

Tony 0 1 0 1 1 2 2 3 1 3 - - 0.545

Total 6 4 23 40 47 10 Average class

cockroach mass (g)

0.475
Table 1 shows the class results of number of cockroach deaths per different dose
concentrations of propoxur. Each dose had 2 samples (S1 and S2) and each
sample contained 5 cockroaches hence each group used a total of 50
cockroaches. All groups used the same propoxur dose concentrations; control
(0%), 0.01%, 0.05% and 0.1%, except for group FAM who did not use 0.01% but
instead used 1%. Unexpectedly all groups except FAM and N.O received at least
1 death in the control another unusual result is from group Tony who had the
least amount of deaths at the 0.1% dose. All groups had deaths at 0.01% except
for groups N.O and The Husks. Overall results for all groups indicate the highest
mortality at 0.1% compared to lower doses.

Looking at both samples as a whole there was 1 death in the control and 0.01%,
7 deaths in 0.02% and 0.05% and 9 deaths in 0.1%. This is consistent as dose
increased so did toxicity. The average mass of cockroaches for each group is also
noted which was then used to calculate the average class mass of cockroach.
Table 2: Average mortality rate (%) of group Adam and class against
different propoxur dose concentrations (w/v %)
Propoxur dose Average mortality rate (%) Average mortality rate
concentrations (w/v %) of group Adam (%) of class

Control 10 10

0.01 10 8

0.02 70 38.33

0.05 70 66.67

0.1 90 78.33

1 - 100

Table 2 Calculated from the results for group Adam and class in table 1, table 2
shows the cockroach deaths as a percentage of the group and class as a whole
as different propoxur doses were used. For class average mortality rate the total
death for each concentration was divided by the total amount of cockroach used
which was 60 except for 0.01% it was 50 because group FAM did not use this
dose concentration. At 1% only group FAM used this and experienced 10 deaths
out of 10 cockroaches used. hence the mortality rate was 100%.
Figure 1. Group Adam and class average of American cockroach
mortality against different propoxur dose concentrations on probit-log
graph

Figure 2. Group adam and class average of american cockroach

mortality against different dose concentrations on linear-log graph

From both graphs the LD50 of group Adam and the class can be seen as a weight
per volume percentage of propoxur. The LD50 determined as a w/v% for group
Adam and the class from the probit log graph is 0.033 and 0.025 and from the
linear-log graph is 0.032 and 0.028 respectively. These w/v% values have been
changed to mg/kg and noted in table 3.

Figure 3. Sample w/v to mg/kg conversion

Class probit LD50 = 0.025% w/v
= 0.025g/100ml divide by 1000
= 0.025mg/100l divide by 100
= 0.00025mg/l propoxur per cockroach

Use group average mass of cockroach to convert from mg to mg/kg (see table 1)

0.00025mg/0.475g of cockroach
= 0.00053 mg/g multiply by 1000

LD50 = 0.53 mg/kg

Table 3. LD50 of group Adam and class in mg/kg, determined from
probit-log and linear-log graphs

Probit-log graph Linear-log graph

LD50 Group Adam (mg/kg) 0.86 0.83

LD50 Class (mg/kg) 0.53 0.60

Table 3 shows all the LD50 calculated from the probit and linear log curves. The
class LD50 results from both curves are lower than the group results.

Discussion:

The purpose of this experiment was to determine the lethal dose of American
nymph cockroaches via dermal application of the carbamate propoxur. From the
probit curve the LD50 of the class calculated was 0.53 mg/kg which is lower than
the group which was 0.86 mg/kg hence making it more toxic.

Results from our group Adam indicate that the insecticide propoxur which has a
low LD50 is an effective chemical to kill cockroaches. Nevertheless, it is still
important to consider the possibility of insecticide resistance as a result of the
cockroaches metabolism boost [14]. Not many studies have been carried out on
the American cockroach but there has been extensive research on the German
cockroach. From these studies, it can be seen that the nymph stage is the most
resistant to insecticides such as propoxur, more so than during their adulthood
[15]. Therefore, the particular use of nymph cockroaches for this experiment
allows the maximum LD50 to be determined. Although it has not been confirmed
that American cockroaches are resistant to propoxur, alternative studies have
suggested that they are resistant to other insecticides because of multiple
exposure to that insecticide over time [16].

In addition to propoxur many other insecticides have been studied such as

organophosphates and fipronil. Organophosphates have become increasingly
redundant as pest control as their frequent reuse in the domestic and agricultural
environment has seen their efficacy drop [16]. Fipronil unlike organophosphates
are highly effective in German and American cockroaches at lose dose
concentrations with high mortality rates due to little resistance [17]. The
effectiveness of an insecticide depends on which cockroach population and
environment it is tested on and this can be inferred from two contradicting
studies of cockroach resistance to pyrthroids by Shafiqur-Rahman and Akter
(2006) and Syed et al (2013). Shafiqur-Rahman and Akter (2006) found that the
American cockroach is resistant to pyrthroids whilst Syed et al (2013) did not.
Further studies on propoxur is ideal since they are relatively new as a pest
control thus this limited exposure may mean the American cockroach has not yet
developed resistance. Hence more studies are needed to confirm whether this
experiment's results are consistent with propoxur having a low LD50 and thus
low resistance in the American nymph cockroach.

The class LD50 extrapolated from both the probit and lose-dose curves are 0.53
mg/kg and 0.60 mg/kg respectively. This is lower than that of the groups which
was 0.86 mg/kg hence making it more toxic. To improve accuracy of the LD50,
the probit graph was used to convert the sigmoidal log-dose curve into a linear
line. Again, to improve accuracy for the class result, 12 samples for each
different propoxur dose concentrations were used however since there were 6
individual groups picking cockroaches, making up dilutions and dermally
applying propoxur, the reliability is counteracted. This can be seen in group FAM
having no death in 0.01% and 100% death in 1% as they failed to make up the
correct dilutions. As a result of this the class average calculated for 0.01% was
only out of a total of 50 cockroaches instead of the 60 like every other dose
concentration, making the class average mortality rate slightly lower. The same
mistake is also suspected of group Tony as they have the least mortalities in
0.1% propoxur concentration compared to all the other groups. Groups N.O and
The Husks experienced the same results for 0.01% with no deaths recorded
when all other groups recorded at least 1 death, this would have again lowered
the class average results. Another discrepancy for the class average is that at
0.01% the mortality rate is lower than that of the controls this can be accounted
for by The Husks as they had significantly high deaths recorded for their control
sample. Thus, it can be said that groups The Husks and Tony create
discrepancies in the class average.

Referring to our group Adam results, the mortality induced by the propoxur dose
is relatively low. Our cockroach deaths did not make a gradual increase but
instead stayed the same for the control and 0.01% then made a jump at 0.02%
and stayed the same for 0.05% only then did it make a gradual increase at 0.1%.
Dilutions were unlikely to be the cause as sample 4 and 5 provided accurate
results. This has raised awareness for other possible errors we could have made,
including overexposure when dermally applying the propoxur to the bellies of the
cockroaches as well as obtaining cockroaches that were not in the nymph stage
thereby lowering the resistance to propoxur.

Literature LD50 values obtained from Butts & Davidson (1955) [18] showcases
the variable LD50 values in cockroaches (specifically the female nymph Blattella
germanica of 70-100 g), based on species, weights and gender. They obtained an
LD50 value of 1.01 mg/kg whilst the LD50 from this study was 0.53 mg/kg, this
highlights the many variables that affect the toxicity of insecticides. As females
have additional fat it makes sense that they would metabolise chemicals
differently to males and the different insecticide used in the study would further
cause the variation in LD50 result.

The test subject of this experiment was American nymph cockroaches with no
specificity for gender hence it is not known which gender was tested for each
sample which like the age of the cockroaches could have impacted the results.
Difficulties of handpicking the right size and right age against the race of time
significantly lowered the consistency and accuracy of the results for both the
class and individual group Adam. This makes for a promising future study where
the gender, size and age of the cockroach can be taken into account alongside
the use of one set of diluted propoxur concentrations to avoid dilution errors.
Another improvement that could have been made was to use more than 2
samples in order to observe more closely the effect of each different dose
concentration as well as increasing reliability.

Conclusion:

It has been demonstrated that a relatively low dose of carbamate insecticide,

propoxur is efficient in managing the American nymph cockroach population, as
indicated by the LD50 obtained. This LD50 obtained is lower than literature
values hence from this study it has been found that a lower LD50 is efficient in
killing the American nymph cockroach. At this point in time American nymph
cockroaches are still likely to have resistance to propoxur hence making it an
effective insecticide in comparison to other marketed ones. Future improvements
have been suggested in discussion to manage this disease vector causing
organism.

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