Effect of Salinity on the Degradation of Insect Repellent

Research Question

What is the effect of different concentrations of NaCl_(aq) (0.200 M, 0.400 M, 0.600 M, 0.800 M, 1.000 M) on the chemical degradation of Aerogard Tropical Strength Insect Repellent, measured in absorbance through UV-Vis spectrometry?

Hypothesis

In the presence of increased sweating, modelled by the increase in NaCl concentration, the efficacy of Aerogard’s insect repellent, represented in absorbance values, will decrease due to the degradation of the repellent caused by regulatory activities, such as thermoregulation (Strid, Hanson, Cross, Bond, & Jenkins, 2018). However, when exceeding a certain salinity concentration, the absorbance values would yield similar values and demonstrate no variance due to the maximum extent of degradation caused to Aerogard’s insect repellent.

Introduction

Expected to provide up to 6 hours of protection against insects, Aerogard Tropical Strength Insect Repellent is a chemical deterrent that can be applied to the skin of users (CSIRO, 2011). Used primarily to repel mosquitos, the main chemical constituent in Aerogard’s Tropical Strength repellent is N,N-diethyl-meta-toluamide, more commonly referred to as diethyltoluamide or DEET. First developed by the United States army in 1946, DEET is a man-made monocarboxylic acid amide formed from the condensation of the carboxy group of m-toluic acid with the nitrogen of diethylamine (Chemical Entities of Biological Interest, 2018). Acting as one of the most common active constituents in repellent formulations, Aerogard Tropical Strength Insect Repellent has a DEET concentration of approximately 19.1% (The Royal Children's Hospital Melbourne, 2018).

DEET is one of the most popular constituents in insect repellent formulations due to its efficient deterrent abilities against a range of insects such as mosquitos, flies and ticks (Interlandi, 2019). Additionally, DEET, along with other main active constituents in Aerogard Tropical Strength Insect Repellent like, N-octyl bicycloheptene dicarboximide, is biodegradable and should not persist in the environment, meaning that the main active chemicals in Aerogard’s insect repellent is able to break down into smaller molecules that demonstrate little to no repellent properties after a short period of time (Keith, et al., 2015).

The aim of this investigation is to explore the effect of the increased occurrence of sweating, represented by the increasing salinity concentrations, on the chemical degradation of Aerogard insect repellent. This topic is adequate to investigate as Aerogard’s Tropical Strength Insect Repellent states that the repellent is able to last for up to 6 hours but does not specify what conditions may limit the longevity of protection (Aerogard, 2021). Consequently, this investigation explores how a common characteristic of active insect repellent users (sweating) may influence the persistence of Aerogard’s repellent. Moreover, previous investigations explored the effect of DEET on thermoregulatory sweating of individuals but did not consider the effect of sweating on the efficacy and persistence of DEET (Kenefick, Cheuvront, Ely, Palombo, & Sawka, 2011). By investigating the proposed research question, a more thorough understanding of the impact of sweat on the chemical degradation of a DEET-based insect repellent, such as Aerogard’s Tropical Strength Repellent, can be established.

Background Information

Aerogard Tropical Strength Insect Repellent has two active constituents, N,N-diethyl-meta-toluamide and N-octyl bicycloheptene dicarboximide. N,N-diethyl-meta-toluamide, known as DEET, is the main active constituent responsible for the deterrent properties in Aerogard’s insect repellent, along with many other commercial insect repellents. DEET has a molecular formula of C₁₂H₁₇NO and a molecular weight of 191.27 g mol^-1 (National Center for Biotechnology Information, 2019). Formed from the condensation of m-toluic acid and diethylamine, DEET is a monocarboxylic acid amide with an octanol-water partition coefficient (log K_OW) of 2.02 and a solubility value of approximately >1.0 g dm^-3 at 25°C (Jackson, Luukinen, Buhl, & Stone, 2008). The octanol-water partition coefficient represents the hydrophobicity of a compound by identifying the ratio of solute concentration between water and octanol (European Centre for Ecotoxicology and Toxicology of Chemicals, 2014). DEET has a low log K_OW value of 2.02, suggesting that the compound is very hydrophilic and soluble in polar substances like water.

Figure 1: Chemical Structure of N,N-diethyl-meta-toluamide with labelled functional groups (Sigma-Aldrich, 2018)

diethylamine (secondary amine)

m-toluic acid (monocarboxylic acid)

Peptide bond between carboxy group of m-toluic acid and nitrogen of diethylamine

Serving as the most popular active constituent in commercial insect repellents, DEET temporarily reduces the volatility of 1-octen-3-ol, an alcohol present in human breath and sweat that attracts mosquitos, thereby reducing mosquito attraction towards humans (Afify, Betz, Riabinina, Lahondère, & Potter, 2019). Theoretically, with the increased occurrence of sweat present on the human skin, the efficacy of DEET in reducing the volatility of 1-octen-3-ol will diminish as a result of the dilution and breakdown of the chemical, therefore allowing for mosquito attraction to humans to return to a higher state. As DEET is synthetically formed from the condensation of m-toluic acid, a carboxylic acid, and diethylamine, an amine, the addition of water may result in the compound undergoing hydrolysis reactions to form two separate compounds (Chemical Entities of Biological Interest, 2018). Additionally, the dilution of NaCl in water results in the formation of Na⁺ ions and Cl^- ions. These ions may cause peptide bond fission due to their ionic charge, leading to the degradation of DEET and the formation of two compounds.

Method

While this experiment was designed, consideration was placed on the handling of the Aerogard’s insect repellent which is composed of active constituents like DEET and N-octyl bicycloheptene dicarboximide. Upon further research, it was discovered that if exposed to the skin for extended periods of time, DEET may cause skin irritation, redness, rash and/or swelling. Additionally, if ingested DEET may cause stomach discomfort, vomiting and nausea (National Pesticide Information Center, 2008). Consequently, as the experiment was performed, skin contact with Aerogard’s insect repellent was minimised by wearing Personal Protective Equipment (PPE) in the form of latex gloves, a laboratory coat, safety glasses and closed leather shoes. Furthermore, though N-octyl bicycloheptene dicarboximide does not cause skin irritation and is not a skin sensitiser, appropriate safety protocols in the form of PPE were worn to limit the exposure of N-octyl bicycloheptene dicarboximide on the skin, should this chemical cause any unexpected safety hazards (National Pesticide Information Center, 2016). Throughout the duration of the experiment, Aerogard’s insect repellent was not inhaled or placed near exposed organs like the eyes to mitigate the risk of potential DEET and N-octyl bicycloheptene dicarboximide contamination. A risk assessment was conducted on www.riskassess.com.au and was approved by school laboratory technicians and a chemistry teacher.

Following the experimental process, the remaining Aerogard solution along with all NaCl solutions was significantly diluted prior to disposal into the laboratory sink. This was done to mitigate the risk of harming aquatic animals through the disposal of concentrated solutions of Aerogard and NaCl. Additionally, as the main active ingredients in Aerogard Tropical Strength Repellent is biodegradable, the disposal of the repellent into the laboratory sink did not pose a substantial threat to marine wildlife.

To ensure that there was variation in the results obtained in the proposed investigation, a preliminary experiment was conducted where only two concentrations of NaCl (1.000 M and 2.000 M) was tested at 300nm. The preliminary experiment served to determine whether variation across different NaCl concentrations was present and allowed for the identification of potential flaws within the methodology that may have potentially impacted the experimental results obtained. The results of the preliminary experiment indicated a decrease in absorbance from 1.000 M to 2.000 M, with 2.000 M producing an absorbance reading of 0 at 300nm, thereby suggesting that varying concentrations of NaCl does result in the degradation of Aerogard’s insect repellent at 300nm.

Based on the results obtained from the preliminary experiment, independent variables were chosen. The preliminary experiment allowed for the identification that an NaCl concentration of 2.000 M was too high as that concentration obtained an absorbance reading of 0. From this, the independent variable of NaCl concentration was chosen at 0.000 M, 0.200 M, 0.400 M, 0.600 M, 0.800 M and 1.000 M with 0.000 M acting as the control experiment.

The dependent variable of this investigation is the concentration of Aerogard which was calculated through the use of a calibration curve and compared across each independent variable to identify the extent of degradation caused by increasing NaCl concentrations. The equation generated in the calibration curve was rearranged to calculate the concentration of Aerogard repellent at a given absorbance level. Based on the absorbance values obtained, the concentration of Aerogard repellent at each NaCl concentration could be predicted using the rearranged formula. This was then compared across each independent variable to identify the correlation between increasing NaCl concentrations and the concentration of Aerogard repellent. Additionally, it should be noted that the samples were processed at 300nm as the absorbance of the sample failed to produce an absorbance reading below approximately 250nm.

An uncontrollable variable evident in this investigation is the presence of UV light on the Aerogard insect repellent. As the experiment was conducted over numerous days, the Aerogard insect repellent was subjected to UV light which may have caused photodegradation in the repellent, thereby reducing its duration of protection and impacting the absorbance levels obtained. However, as the Aerogard repellent was contained in a single glass beaker, the photodegradation caused by UV light most likely would have been consistent across the entire Aerogard solution, thereby avoiding differing UV light exposures across each trial and independent variable. An additional uncontrollable variable would be the opacity of the NaCl and Aerogard solution in the microplate prior to processing. Due to the chemical composition of Aerogard, the repellent increased in opacity when it was deposited from the micropipette. As the Aerogard repellent was deposited into the microplate wells at varying time intervals, this provided differing amounts of time for the repellent to return back to a clear state. Consequently, solutions located at the far right (1.000 M NaCl) may have had a higher opacity than samples located at the far left (0.000 M NaCl) of the microplate. This would have provided inaccurate absorbance values as a varied level of opacity would have influenced the absorbance level of the sample.

Steps

Preparing 1.000 M NaCl_(aq) Stock Solution:

1. Ensure that the experimenter is wearing protective eyewear, rubber gloves and a laboratory coat.
2. Place a weighing boat onto the electronic balance and tare the scale.
3. Using a metal spatula, measure 2.922 ± 0.001 g of NaCl_(s) into a disposable weighing boat, taring the balance between each interval.
4. Using a permanent marker, label a 50.00 ± 0.05 cm³ volumetric flask with 1.000 M.
5. Using a glass funnel, transfer the NaCl granules from the weighing boat into the volumetric flask.
6. Add distilled water into the volumetric flask, ensuring to rinse the glass funnel to collect NaCl_(s) residue.
7. Add additional distilled water into the volumetric flask to create a 50.00 ± 0.05 cm³ solution.
8. Place a volumetric flask stopper on the volumetric flask and agitate the solution until homogenous, ensuring that the NaCl granules are adequately dissolved.

Preparing NaCl_(aq) Solutions (0.000 M, 0.200 M, 0.400 M, 0.600 M, 0.800 M):

1. Using a measuring cylinder, obtain 40 ± 1 cm³ of 1.000 M solution and deposit it into a separate 50.00 ± 0.05 cm³ volumetric flask.
2. Add distilled water into the volumetric flask to create a 50 cm³ solution.
3. Using a permanent marker, label the volumetric flask with 0.800 M.
4. Using a measuring cylinder, obtain 37.5 ± 1.0 cm³ of 0.800 M solution and deposit it into a separate 50.00 ± 0.05 cm³ volumetric flask.
5. Add distilled water into the volumetric flask to create a 50 cm³ solution.
6. Using a permanent marker, label the volumetric flask with 0.600 M.
7. Using a measuring cylinder and a micropipette, obtain 33.3 ± 1.0 cm³ of 0.600 M solution and deposit it into a separate 50 ±0.05 cm³ volumetric flask.
8. Add distilled water into the volumetric flask to create a 50 cm³ solution.
9. Using a permanent marker, label the volumetric flask with 0.400 M.
10. Using a measuring cylinder, obtain 25 ± 1 cm³ of 0.400 M solution and deposit it into a separate 50.00 ± 0.05 cm³ volumetric flask.
11. Add distilled water into the volumetric flask to create a 50 cm³ solution.
12. Using a permanent marker, label the volumetric flask with 0.200 M.
13. Deposit 50.00 ± 0.05 cm³ of distilled water into a separate 50.00 ± 0.05 cm³ volumetric flask.
14. Using a permanent marker, label the volumetric flask with 0.000 M.

Preparing Aerogard Solution:

1. Sufficiently spray the Aerogard repellent into a 100 cm³ glass beaker until approximately 20 cm³ of the repellent has been obtained.
2. Ensure that appropriate eyewear is worn and that the Aerogard repellent is sprayed away from open fires and exposed organs, like the eyes.
   1. It should be noted that some chemicals in the Aerogard repellent, such as diethyltoluamide, may stain clothing and, thus, should be avoided on fabrics.

Measuring Absorbance Samples:

1. Turn on the BMG Labtech SPECTROstar Nano microplate reader.
2. Using a multichannel micropipette, deposit 100 ± 1 µl (0.100 cm³) of 0.000 M NaCl solution from the glass beaker into the first column of the Greiner 96-well microplate.
3. Utilising the same micropipette tips, draw 150 ± 1 µl (0.150 cm³) of distilled water and deposit in a waste beaker. Repeat this step thrice.
4. Repeat Step 26 and Step 27 for each independent variable (0.200 M, 0.400 M, 0.600 M, 0.800 M, 1.000 M).
5. After rinsing the micropipette tip, obtain 200 ± 1 µl (0.200 cm³) of distilled water and deposit the solution in the last column of the Greiner microplate. This serves as the blank for the microplate reader to calibrate readings against.
6. Draw 100 ± 1 µl (0.100 cm³) of Aerogard solution and deposit the solution in the column of wells containing 0.000 M NaCl solution.
7. Repeat Step 30 for each independent variable (0.200 M, 0.400 M, 0.600 M, 0.800 M, 1.000 M).
8. After 5 minutes, place the Greiner microplate into the microplate reader and process the sample.
9. Record the absorbance levels of the sample at 300nm.
10. Rinse the Greiner microplate with distilled water after processing the sample.

Generating a Calibration Curve:

1. Using a set of unused micropipette tips, use a multichannel micropipette to draw 250 ±1 µl (0.250 cm³) of distilled water and deposit the solution into the first column of the Greiner microplate.
2. Repeat Step 35 with decreasing increments of distilled water (200 ± 1 µl, 150 ± 1 µl, 100 ± 1 µl, 50 ± 1 µl), depositing the distilled water in columns from left to right.
3. Using a set of clean micropipette tips, use a multichannel micropipette to draw 50 ± 1 µl (0.050 cm³) of Aerogard repellent into the first column of the Greiner microplate.
4. Repeat Step 37 with increasing increments of Aerogard repellent (100 ± 1 µl, 150 ± 1 µl, 200 ± 1 µl, 250 ± 1 µl), depositing the solution in columns from left to right.
   1. The total volume in each well of the Greiner microplate should be 300 ± 2 µl
5. At the far-right column of the Greiner microplate, deposit 300 ± 1 µl (0.300 cm³) of distilled water. This serves as the blank for the microplate reader to calibrate readings against.
6. Place the Greiner microplate into the microplate reader and process the sample.
7. Record the absorbance levels of the sample at 300nm.
8. Rinse and dry the Greiner microplate.

Raw Data

Qualitative Data

When depositing the Aerogard repellent into the microplate, the solution increased in opacity from clear to colourless. This was mitigated by processing the microplate after five minutes to allow for the Aerogard solution to return to a clear state. However, the opacity of trials in the far-right column of the microplate most likely would have been greater than the opacity of trials in the far-left column due to different resting periods, thereby potentially providing inaccurate results.

Quantitative Data

Table 1: Absorbance levels of Aerogard repellent subjected to various salinity concentrations (0.000 mol dm^-3, 0.200 mol dm^-3, 0.400 mol dm^-3, 0.600 mol dm^-3, 0.800 mol dm^-3, 1.000 mol dm^-3), measured by a UV-Vis spectrometer at 300nm.

Salinity (± 4.3% mol dm-3)	Absorbance Level (± 0.001)
	Trial 1	Trial 2	Trial 3	Trial 4	Trial 5	Trial 6	Trial 7	Trial 8
0.000	0.894	0.981	0.888	0.997	0.864	0.806	0.954	0.952
0.200	0.798	0.967	0.916	0.852	0.837	0.753	0.814	0.864
0.400	0.609	0.716	0.654	0.751	0.707	0.641	0.732	0.649
0.600	0.573	0.489	0.468	0.443	0.505	0.493	0.441	0.461
0.800	0.315	0.310	0.254	0.332	0.411	0.379	0.375	0.286
1.000	0.355	0.219	0.309	0.327	0.317	0.293	0.330	0.344

Processing Data

Propagation of Uncertainty

The propagated uncertainty calculated below illustrates the possibility of random error present in the experiment, displayed as a percentage. The percentage uncertainty calculations below correspond to the uncertainty of the NaCl solutions used to obtain absorbance readings.

$\%\mathit{uncertainty\; of}1.000\mathit{M\; NaCl\; solution}=\left(\frac{0.001}{2.939}*100\right)+\left(\frac{0.050}{50}*100\right)$

$\mathit{}=0\%+0.1\%$

$\mathit{}=0.1\%$

$\%\mathit{uncertainty\; of}0.800\mathit{M\; NaCl\; solution}=0.1\%+\left(\frac{0.050}{50}*100\right)+\left(\frac{1.0}{50}*100\right)$

$\mathit{}=0.1\%+0.1\%+2\%$

$\mathit{}=2.2\%$

$\%\mathit{uncertainty\; of}0.600\mathit{M\; NaCl\; solution}=2.2\%+\left(\frac{0.050}{50}*100\right)+\left(\frac{1.0}{50}*100\right)$

$\mathit{}=2.2\%+0.1\%+2\%$

$\mathit{}=4.3\%$

$\%\mathit{uncertainty\; of}0.400\mathit{M\; NaCl\; solution}=4.3\%+\left(\frac{0.050}{50}*100\right)+\left(\frac{1.0}{50}*100\right)$

$\mathit{}=4.3\%+0.1\%+2\%$

$\mathit{}=6.4\%$

$\%\mathit{uncertainty\; of}0.200\mathit{M\; NaCl\; solution}=6.4\%+\left(\frac{0.050}{50}*100\right)+\left(\frac{1.0}{50}*100\right)$

$\mathit{}=6.4\%+0.1\%+2\%$

$\mathit{}=8.5\%$

The uncertainty below corresponds to the uncertainty of the volume in each well of the microplate prior to being sampled (multichannel pipette used twice). As the BMG UV-Vis spectrometer processes samples from a top-down view, differing volumes in each well can influence the absorbance level of trials within the sample.

$\%\mathit{uncertainty\; of\; microplate\; volume}=\left(\frac{1}{100}*100\right)*2$

$\mathit{}=1\%*2$

$\mathit{}=2\%$

Processed Data

Table 2: Calibration curve used to determine the absorbance level of increasing concentrations of Aerogard repellent (0.149 mol dm^-3, 0.298 mol dm^-3, 0.447 mol dm^-3, 0.596 mol dm^-3, 0.745 mol dm^-3), measured using a UV-Vis spectrometer at 300nm.

Concentration of Aerogard (mol dm-3)	Absorbance Level (± 0.001)
0.149 (50ul) ± 2.1%	0.417
0.298 (100ul) ± 4.2%	0.914
0.447 (150ul) ± 6.3%	1.310
0.596 (200ul) ± 8.4%	1.895
0.745 (250ul) ± 10.5%	2.044

Graph 1: Calibration curve used to determine the absorbance level of increasing concentrations of Aerogard repellent (0.149 mol dm^-3, 0.298 mol dm^-3, 0.447 mol dm^-3, 0.596 mol dm^-3, 0.745 mol dm^-3), measured using a UV-Vis spectrometer at 300nm.

A calibration curve was produced by measuring the absorbance level of increasing concentrations of Aerogard repellent, ranging from 0.149 mol dm^-3 to 0.745 mol dm^-3. Consequently, a trendline and its formula was determined which was rearranged to predict the concentration of Aerogard from absorbance values obtained in Table 1.

A sample calculation involving the linear trendline equation in Graph 1 is rearranged to isolate the concentration of Aerogard as demonstrated below:

Linear trendline equation rearranged: $x=\frac{y-0.0459}{2.8414}$ Where: $x=$ Aerogard concentration $y=$Absorbance of given sample

Sample calculation of Trial 1 with 0.000 mol dm-3 NaCl: $x=\frac{0.894-0.0459}{2.8414}$ $x$ = 0.298 mol dm-3

Table 3: Concentration of Aerogard repellent after subjected to increasing salinity concentrations (0.000 mol dm^-3, 0.200 mol dm^-3, 0.400 mol dm^-3, 0.600 mol dm^-3, 0.800 mol dm^-3, 1.000 mol dm^-3), calculated from the rearranged equation generated from the calibration curve.

Salinity (± 4.3% mol dm-3)	Concentration of Aerogard (± 6.3% mol dm-3)
	Trial 1	Trial 2	Trial 3	Trial 4	Trial 5	Trial 6	Trial 7	Trial 8
0.000	0.298	0.329	0.296	0.335	0.288	0.268	0.320	0.319
0.200	0.265	0.324	0.306	0.284	0.278	0.249	0.270	0.288
0.400	0.198	0.236	0.214	0.248	0.233	0.209	0.241	0.212
0.600	0.186	0.156	0.149	0.140	0.162	0.157	0.139	0.146
0.800	0.095	0.093	0.073	0.101	0.128	0.117	0.116	0.085
1.000	0.109	0.061	0.093	0.099	0.095	0.087	0.100	0.105

The concentration of Aerogard was predicted based on the regression equation produced by the calibration curve in Graph 1. The regression equation was rearranged to determine the concentration of Aerogard repellent with consideration of the absorbance value of a sample. Additionally, as there are insufficient trials to conduct further statistical analysis like standard deviation, random error for each independent variable was calculated by halving the range of samples. Sample calculations determining the mean and random error of 0.000 mol dm^-3 NaCl is displayed in Table 4.

Table 4: Statical analysis calculations to determine the mean concentration of Aerogard repellent and the random error associated with Aerogard concentration as salinity concentration increases.

Statistical Analysis	Formulae	Sample Calculation of Aerogard Concentration (0.000 mol dm-3 NaCl)
Mean	$\overline{X}=$ $\frac{\sum \mathit{sample\; values}\mathrm{}}{\mathit{sample\; size}}$	$\overline{X}=$ $\frac{0.298+0.329+0.296+0.335+0.288+0.268+0.320+0.319\mathrm{}}{8}$ = 0.307 mol dm-3
Random Error	$\mathit{random\; error}=\frac{\mathit{max}.\mathit{value}-\mathit{min}.\mathit{value}}{2}$	$\mathit{random\; error}=\frac{0.335-0.268}{2}$ = 0.034

Table 5: Mean concentration of Aerogard repellent with random error values associated with increasing salinity concentrations (0.000 mol dm^-3, 0.200 mol dm^-3, 0.400 mol dm^-3, 0.600 mol dm^-3, 0.800 mol dm^-3, 1.000 mol dm^-3).

Salinity (± 4.3% mol dm-3)	Mean Aerogard Concentration (± 6.3% mol dm-3)	Random Error
0.000	0.307	0.034
0.200	0.283	0.038
0.400	0.224	0.025
0.600	0.154	0.008
0.800	0.101	0.028
1.000	0.094	0.024

Graph 2: Mean concentration of Aerogard repellent after exposed to increasing salinity concentrations (0.149 mol dm^-3, 0.298 mol dm^-3, 0.447 mol dm^-3, 0.596 mol dm^-3, 0.745 mol dm^-3).

Based on the mean concentration of Aerogard calculated in Table 5, Graph 2 illustrates the mean concentration of Aerogard repellent as the concentration of NaCl increases from 0.000 mol dm^-3 to 1.000 mol dm^-3. The trendline observed in Graph 2 indicates that a negative correlation is present between increasing salinity concentrations and the concentration of Aerogard, thereby suggesting that Aerogard insect repellent undergoes degradation as the concentration of NaCl increases. A coefficient of determination (R²) value of 0.96 signifies that a strong negative correlation is present between the independent variable and the dependent variable. The vertical error bars represent the calculated random error that was present throughout the experiment. It should be noted that error bars across some independent variables overlapped which consequently impacts the trends identified from this graph, therefore potentially invalidating the conclusions developed from this investigation.

Analysis and Discussion

With reference to Graph 2, the declining trendline suggests that there is a positive correlation between the increase in salinity and the chemical degradation of Aerogard’s Tropical Strength Insect Repellent. A coefficient of determination (R²) value of 0.96 indicates a strong, negative correlation between salinity concentrations and the concentration of Aerogard repellent. This demonstrates that the relationship between the independent variable (increasing salinity concentrations) and the dependent variable (concentration of Aerogard repellent) is significant and that a change in the independent variable demonstrates an impact on the dependent variable. Seen in the concentration of Aerogard in 0.000 mol dm^-3 NaCl solution of 0.358 mol dm^-3 compared to the concentration of Aerogard in 1.000 mol dm^-3 NaCl solution of 0.085 mol dm^-3, the increase in NaCl concentration as a representation of the increased occurrence of sweat consequently results in the degradation of the chemical constituents, most notably DEET, in Aerogard’s insect repellent.

As a result of the increase in salinity concentration from 0.000 mol dm^-3 to 1.000 mol dm^-3, the concentration of Aerogard insect repellent has decreased by approximately 69%, thereby indicating that approximately 69% of the repellent has undergone chemical degradation as a result of the increased salinity concentrations. A potential reason as to why the increased occurrence of sweat by a user may impact the concentration of Aerogard’s repellent could be that the presence of sweat dilutes the repellent, resulting in a lower concentration and efficacy of the repellent. As stated in a scientific research journal investigating the efficacy of insect repellents on various species of mosquitoes, findings concluded that the loss of repellent effectiveness can be caused by numerous activities such as friction from clothing, washing of the skin and the presence of sweat (Lupi, Hatz, & Schlagenhauf, 2013). Additionally, the increased presence of sweat along with water may separate the peptide bond present between the carboxylic acid of m-toluic acid and the amine of diethylamine, thereby resulting in the chemical degradation of DEET (Chemical Entities of Biological Interest, 2018).

Evaluation

Based on R² value displayed in Graph 2, the reliability of this investigation is supported as an R² value of 0.96 suggests that approximately 96% of the variance within the independent variable demonstrates an effect on the dependent variable. However, demonstrated in Table 5, the random error across each independent variable is somewhat significant which may oppose to the reliability of this experiment. Represented through the use of vertical error bars in Graph 2, the overlap of error bars across some independent variables suggests that the difference between Aerogard concentration at some salinity concentrations may not be significantly correlated to the increase in NaCl concentration, thereby impacting the validity of the conclusions drawn from this investigation.

The general trend of Aerogard concentration displayed in Table 5 and the trendline in Graph 2 highlights a negative correlation between increasing salinity concentrations and Aerogard concentration. The strong decreasing trend in Aerogard concentration as salinity concentration increases indicates that the hypothesis of how the increased presence of sweat, represented by the increased salinity concentrations, evidently promotes the chemical degradation of Aerogard insect repellent. This demonstrates how the increased presence of sweat affects the repellent’s efficacy and persistence, causing users to more frequently apply Aerogard’s repellent in order to reap continuous deterrent properties.

The random error and propagated uncertainty identified represent the overall reliability of this investigation. Propagated uncertainty depicts the potentiality for random error to occur within the experiment and is calculated by considering the uncertainty of the equipment used. Random error communicates the experimental error that occurred during this investigation and is calculated by halving the range between the highest and lowest Aerogard concentration value across an independent variable. By calculating the random error percentage of the Aerogard concentration (dividing random error by mean concentration, multiplied by 100), it was identified that nearly all independent variables had a higher random error percentage than propagated uncertainty, thereby suggesting that random error is present within this investigation that cannot be attributed to the equipment utilised. It should be noted that at 0.600 mol dm^-3 NaCl concentration, the propagated uncertainty was ± 6.3% while the percentage error was ± 5.2%. However, across other independent variables, the random error percentage was significantly greater than the propagated uncertainty, suggesting that there is a degree of random error that cannot be attributed to the equipment used. This may have been caused by the inconsistent use of the equipment when conducting the serial dilution to obtain the various NaCl concentrations.

The propagated uncertainty of the concentration of Aerogard was calculated in various steps involved in the investigation, such as the percentage uncertainty of the volume in the microplate, which directly influences the absorbance values obtained. As a serial dilution was performed to obtain the various salinity concentrations, the propagated uncertainty accumulated as the concentration of NaCl decreased, resulting in 10.5% propagated uncertainty in 0.200 mol dm^-3 NaCl solution. It should be noted that nearly all independent variables had a random error higher than the propagated uncertainty which suggests that there is more random error present that cannot be attributed to the equipment utilised throughout the experiment.

A factor that may have contributed to the random error of this experiment could be how the micropipette tips was not replaced during the serial dilution or when depositing solutions into the microplate due to the limited supply of micropipette tips. To mitigate this source of error, the micropipette tips was rinsed thrice with distilled water by drawing up a greater volume of distilled water (150 µl) and disposing the water into a waste beaker. However, rinsing the micropipette tips may have impacted the concentration of the NaCl solution drawn as there may have been excess distilled water within the micropipette tips, thereby diluting the concentration of the NaCl solutions and thus impacting the reliability of the experiment.

Additionally, another factor that may be attributed to the random error of this experiment could be the inconsistent opacity of the solutions in the wells of the microplate. Due to the chemical composition of Aerogard insect repellent, the solution increased in opacity from clear to colourless as it was deposited from the micropipette into the microplate wells. As the BMG SPECTROstar Nano microplate reader processes samples from a top-down view, inconsistencies concerning the volume or opacity of the solutions in each well would have provided imprecise absorbance values. This may have accounted for the inconsistent absorbance values of some trials across the independent variables, thereby contributing to the random error present in this experiment. Ideally, the Aerogard repellent should have been deposited from the micropipette at a much slower rate to avoid the increased opacity and the microplate should have been provided with an extended period of time to allow for the solutions to establish a consistent opacity. It should be considered, however, that if the solutions in the microplate were to be left for an extended period of time, this may have resulted in the additional degradation of Aerogard’s repellent caused by other factors such as UV light which may have impacted the conclusions drawn from this investigation.

It should be considered that significant statistical analysis could not be conducted due to the insufficient number of trials. Statistical analysis such as standard deviation, coefficient of variation or t-test could not be conducted which would have provided a more accurate and statistically significant representation of random error/variation evident in this investigation. Ideally, by increasing the number of trials conducted, this would have allowed for more statistically significant analysis to be conducted as opposed to calculating random error by halving the range of the highest and lowest Aerogard concentration recorded at each independent variable. Furthermore, as there was no knowledge of a fixed concentration of Aerogard at a specific NaCl concentration, further statistical analysis such as percentage error could not be determined as this investigation lacked a comparison value.

A factor that may have contributed to the propagated uncertainty across the independent variables could have been the process of conducting a serial dilution to obtain the various NaCl concentrations (0.000 M, 0.200 M, 0.400 M, 0.600 M, 0.800 M). A serial dilution was conducted from a stock NaCl solution (1.000 M) with 0.1% propagated uncertainty. This propagated uncertainty was carried forward to diluted NaCl concentrations, creating a cumulative effect involving the propagated uncertainty of NaCl solutions with the most diluted NaCl solution (excluding control) of 0.200 M having an 8.5% propagated uncertainty. The cumulative effect involving propagated uncertainty of the NaCl solutions could have been mitigated by creating the solutions independently. By measuring various masses of NaCl and creating the solutions separately, this would have minimised the propagated uncertainty of the solutions, resulting in only the uncertainty of the electronic balance and 50 cm³ volumetric flask contributing to the propagated uncertainty.

Conclusion

This investigation explored the effect of increasing concentrations of NaCl (0.000 M, 0.200 M, 0.400 M, 0.600 M, 0.800 M, 1.000 M) on the chemical degradation of Aerogard Tropical Strength insect repellent. This was done by subjecting a constant volume of Aerogard insect repellent in various salinity concentrations and recording the absorbance levels of the samples. Additionally, a calibration curve was constructed where the regression equation was rearranged in order to determine the concentration of Aerogard insect repellent from an absorbance value. From this, the mean Aerogard concentration at each salinity concentration was recorded and plotted to observe the effect of such concentrations on Aerogard insect repellent. In response to the research question, “What is the effect of different concentrations of NaCl_(aq) (0.000 M, 0.200 M, 0.400 M, 0.600 M, 0.800 M, 1.000 M) on the chemical degradation of Aerogard Tropical Strength Insect Repellent, measured in absorbance through UV-Vis spectrometry?” it was concluded that there is a negative correlation between the independent variable and dependent variable, suggesting that as the presence of perspiration increases, the efficacy and persistence of Aerogard Tropical Strength insect repellent deteriorates.

Supported in Graph 2, the declining trendline concludes that an increase in salinity concentrations results in the chemical degradation of Aerogard insect repellent. By exposing Aerogard repellent to increasing concentrations of NaCl solutions, the salinity most likely initiates the breakdown of Aerogard insect repellent by diluting and disrupting the chemical bonds between DEET molecules. This leads to the degradation of DEET as seen in the decrease in concentration of Aerogard insect repellent, with DEET being the main active constituent.

The significance of this investigation serves to advise Aerogard insect repellent users, and users of repellents containing DEET, how the efficacy and persistence of deterrent properties of a repellent containing DEET may be impacted as a result of the increased occurrence of thermoregulatory sweating. This may provide some general information as to how often the insect repellent should be reapplied, regardless of how long the repellent should last. However, due to ethical guidelines which limited the use of bodily fluids, the increased presence of sweat was represented with increasing concentrations NaCl solutions. This may decrease the validity of this investigation as human sweat consists of various constituents beyond salt, such as ammonia and urea, which was not considered in this investigation (Rekstis, 2020).

Further Investigation

In order to develop a more thorough understanding of what mechanisms of action DEET undergoes as a result of increasing salinity concentrations, further research should be conducted to observe the chemical properties of DEET in saline environments. By conducting additional relevant research into this area, a more thorough understanding as to why DEET undergoes chemical degradation in increasingly saline environments can be developed. Moreover, a study published in 2016 on the Journal of Dermatological Treatment concluded that insect repellents containing DEET causes a decrease in sunscreen SPF (Rodriguez & Maibach, 2016). Consequently, further research could be conducted to identify the extent to which increasing salinity concentrations impacts the efficacy and persistence of sunscreen. The conclusions drawn from identifying the extent of degradation caused in sunscreen could be compared to the data obtained in this investigation to suggest whether insect repellent or sunscreen should be applied first onto the skin, with the option that degrades the most in saline environments being applied on top. This would theoretically allow for the prolonged efficacy and persistence of both insect repellent and sunscreen as the presence of perspiration increases. However, the presence of UV light should be considered as UV light may cause the insect repellent or sunscreen to undergo photodegradation.

Additionally, experimentation involving the degradation of insect repellents with picaridin, a common active constituent in many commercial insect repellents, could be conducted and compared to the results obtained in this investigation to identify the greater degree of degradation that the active constituent (picaridin or DEET) undergoes. This allows individuals to identify which range of insect repellents, containing picaridin or DEET, has the greater efficacy and persistence in saline environments, thereby providing a more reliable and durable insect repellent that is able to withstand a more extensive level of thermoregulatory sweating.

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