BACKGROUND:

Uncontrolled insect populations have been a problem for many people, as they eat agricultural yields, breed in households, and spread diseases. The American Mosquito Control Association states that mosquitos alone are responsible for over 1 million deaths annually from mosquito-borne diseases (AMCA, 2022). Insects are estimated to destroy 30% of all crops grown annually (SARE, 2022). For 4,500 years, humans have utilized chemical extracts to repel and kill unwanted insect pests (Unsworth, 2010). The main problem with synthetic insecticides is their dangerous health effects on animals and humans, especially young children (Zhang et al., 2019). Finding potent alternative insect repellents that are safe for humans is essential. A possible solution could be certain chemicals found in essential oils that are toxic to insects but safe for humans even when inhaled or ingested. These alternative insecticides are particularly attractive as many widely used insecticides are waning in effectiveness against target insect species.

Unfortunately, current synthetic insecticides are losing their potency over time, and the resistance of many important pest species to insecticides has become a global issue in the past few decades (Kissoum, 2020). Insects such as the Colorado Potato Beetle and Diamondback Moth are notorious for developing resistance to all synthetic insecticides registered against them. The Colorado Potato Beetle has been especially devastating to potato plants on Long Island and other parts of the Northeastern United States, while the Diamondback Moth has been uncontrollably destroying cruciferous vegetable crops throughout Southeast Asia (Forgash, 1984). These insects have naturally evolved an ability to overcome the toxic defenses of their hosts, which has helped them adapt to a wide range of human-made poisons (Alyokhin, 2008). These cases highlight the importance of new alternative insecticides to prevent the large-scale destruction of farmlands. Besides losing their potency against target pests, synthetic insecticides also have unintended off-target toxicity, which decreases animal biodiversity. 

Animals that unintentionally ingest synthetic insecticides show adverse health effects, such as the impacts observed from the DDT silent spring incident. Pesticides like DDT — dichlorodiphenyltrichloroethane — were sprayed excessively and indiscriminately to control crop pests. Populations of bald eagles and other birds crashed when DDT thinned their eggs, killing their embryos (Bienkowski, 2014). If an animal does not die directly from ingesting synthetic insecticides, it stays in its system, potentially weakening them and leaving them vulnerable to predators (Gangemi, 2016). This well-known incident brought global attention to insecticide safety and environmental impacts. These insecticides washed into water sources and disrupted the food chain, threatening delicate ecosystems for birds, fish, and ultimately humans (Haberman, 2017). 

Andrade and Imfeld’s experiments tested the rate of synthetic insecticide transport through rainwater. They determined that surface runoff caused by precipitation was a significant driver of pesticide transport from agricultural land into water sources (2021). Andrade et al. also found that pesticides in nearby water streams increased by up to 310% immediately after a rainfall event (2021). This increased level of pesticides only dissipates by 87% after seven days, which is enough time to permanently affect the ecosystem by killing animals and disrupting the food chain (Andrade et al., 2021). These synthetic insecticides, now contained in natural water, may eventually contaminate our potable water; there is already evidence that synthetic insecticides have adverse effects on human health (USGS, 2018). Due to the negative health effects of commonly used chemical pesticides, insect resistance, and the mortality of animals, it is vital to find safer alternatives.

Research has risen in the past few decades due to the insecticide health issue. The current use of insecticides sprayed in the atmosphere is increasingly under investigation due to their potential impacts on human health and ecosystems (Lichiheb et al., 2014). Jean-François Viel and colleagues designed an experiment measuring the health and mortality rate of farmers routinely exposed to pesticides and discovered they have a significantly higher risk for cancers, such as a 59% increase in disease rates for Multiple Myeloma and a 33% increase for Leukemia (Viel et al., 1993). Sule Isin argues that insecticide users must be aware of the possible undesirable effects on human health and seek alternatives, as evident from Viel’s data. (Isin et al., 2007). Insecticides have also been shown to cause reproductive and developmental effects in humans exposed to the chemicals by inhalation, ingestion, and direct contact with treated areas (Liu et al., 2014). One possible alternative to arise from recent research are natural essential oils.

While researching new insecticide alternatives, chemicals extracted from essential oils stood out with promising results. Essential oils are all-natural plant-based oils with a strong aroma extracted from raw plant materials traditionally used in food and folk medicines to ease stress and relieve pain (Johns Hopkins, 2022); (Tamburlin et al., 2021). Essential oils play a biological role in plant protection by reducing herbivores’ appetite. They also attract beneficial insects in favor of pollination and seed disposition and repel undesirable predatory insects. (Bakkali et al., 2008). Although the oils are entirely harmless to humans, they could ward off insects, according to the recent findings of Mansour and colleagues (2015). 

Essential oils showed potential insecticidal effects in some experiments comparable to chemical insecticides (Mansour et al., 2015; Silva et al., 2008). When Mansour et al. tested the toxicity of essential plant oils compared to conventional synthetic insecticides against the desert locust, they found Allium cepa essential oil had a low LD50 of 1.11 ppm. LD50 (lethal doses) is the ppm (parts per million) concentration of a material required for a 50% mortality rate. Thus, highly potent insecticides have very low LD50 values indicating strong insecticidal properties. Mansour’s experiment also tested synthetic insecticides from three classes: Organophosphates, Carbamates, and Pyrethroids. The most potent insecticide was Fenitrothion, an Organophosphate, at a similar but lower LD50 of 0.33 ppm, followed by Methomyl, a carbamate, with LD50 of 1.18 ppm, Carbosulfan, another carbamate, with LD50 of 1.19 ppm, then Fenvalerate, a Pyrethroid with LD50 of 1.48 ppm. Although natural oils do not have a higher potency than organophosphates, they compete directly with various chemical insecticides. To compensate for the lower potency, a higher concentration of essential oils is possible as there are few adverse effects, while synthetic insecticides must be regulated for safety. This research demonstrates that essential oils effectively control insect populations displaying similar toxicity levels to Carbamate and Pyrethroid insecticides. These results demonstrate that essential oils have similar toxicity levels as most tested insecticides; however, more research is needed to examine the specific active chemicals proving efficacious against other common pests. One crucial essential oil that warrants further investigation is L. angustifolia, a promising safe alternative with a published LD50 of 2.36 ppm. However, effective investigation of this essential oil requires appropriate laboratory models and tools such as the model research organism Drosophila melanogaster. It is crucial to confirm the effects of essential oils on a model research insect such as Drosophila melanogaster. 

Drosophila melanogaster is a common household pest that is a widely researched and tested model organism used to study the effects of insecticides on development, reproduction, and overall survival (Kissoum et al., 2020; Tang et al., 2022; Yi et al., 2021). Drosophila is an ideal model organism due to its ability to reproduce and mature quickly, allowing for extensive pool sample testing and leading to more accurate results. The short mean lifespan of 2-3 months and 10-day reproduction allows for the inexpensive but accurate data collection on the toxicity of essential oils. A vial of 60 Adult Drosophila is low-priced, and the flies have low maintenance requirements and multiply quickly, allowing larger culture sizes and little waiting time between experiments. This is important as the flies exposed to the oils and chemicals are expected to die and cannot be reused. Even if some flies survive, they cannot be reused due to the possibility of selecting resistant or weakened flies, which would skew the survivability rate. Thus, Drosophila provides an inexpensive but conclusive way to collect data on the toxicity of essential oils.

The danger of commonly used pesticides, the potential of essential oils, and the advantages of testing on Drosophila melanogaster motivate my current work investigating the fumigation toxicity of linalool and linalyl acetate commonly found in promising oils such as L. angustifolia on Drosophila melanogaster. Preliminary data testing the toxicity of essential oils mixed the compounds into the fly media, resulting in ingestion, contact, and fumigation toxicity, so it was impossible to identify how each exposure method impacted the mortality rate individually. The testing of only fumigation toxicity is to limit the possible variables of contact and ingestion toxicity. It is essential to research which chemical, linalool or its derivative linalyl acetate, elicited most of the repulsive and toxic effects displayed in natural lavender oil against Drosophila Melanogaster. Linalool and linalyl acetate was chosen for my experiment as the chemicals are safe for humans and have also been shown to be a potential alternative to synthetic insecticides against pests (Kheloul et al., 2020; Silva, 2021; Api et al., 2022). The chemicals do not present a concern for genotoxicity, repeated dose toxicity, reproductive toxicity, skin sensitization, phototoxicity/photo allergenicity, local respiratory toxicity, or environmental toxicity (RIFM, 2022). The chemicals are also abundant in L. angustifolia, making up 71% of the oil, with linalool at 34% and linalyl acetate at 37% (Boelens, 1995). 

For this experiment, three different chemical fumigation conditions will be tested in 300 mL airtight jar arenas, each containing ten male and ten female flies. The negative control consists of pure acetone, which will be present in all cohorts and hypothesized to have little to no effect on the mortality rate; the positive control contains lavender oil, a commonly hypothesized and used alternative insecticide/insect repellent containing high concentrations of linalool and linalyl acetate; and two experimental groups, testing the fumigation effectiveness of linalool at 5.5 μL and linalyl acetate at 6.0 μL. The two chemical compounds will then be compared on their effectiveness based on insect life expectancy, concentration, and cost-effectiveness. 

The positive control of pure lavender oil is expected to elicit the most potent effects as it contains both linalool and linalyl acetate. Either the linalool or linalyl acetate experiments should yield higher toxicity, or a mixture of the two may elicit the toxic effects found in lavender oil. Due to a knowledge gap in research, it is unknown which chemical between linalool and linalyl acetate will display higher fumigation toxicity. These chemicals contribute to the floral aroma of lavender oil with similar concentrations. The results of this experiment can provide further information on easy-access and economically affordable, safe insecticide alternatives. Suppose the fumes of linalool or linalyl acetate are confirmed to be toxic to fruit flies. In that case, future studies may be conducted to determine the contact and ingestion toxicity and the repellent capabilities. This future study would offer more insecticide methods to limit insect resistance and decrease reliance on synthetic insecticides.

METHODS & PROCEDURE:

Preliminary Study:

Preliminary research replicated S. Buentello Wong et al.’s 2016 investigation on the toxicity of essential oil formulations against the Mexican fruit fly, Anastrepha ludens. Although the toxicity of three essential oils (clove, basil, and thyme) was initially tested, only one experimental formulation was replicated due to time constraints. The experiment aimed to compare clove oil’s toxicity, an oil proven to display increased mortality, to basil oil to determine if basil could be sufficiently used as a safer alternative as the chemicals it contains are less harmful and irritating if inhaled or ingested (Mansour et al., 2015). However, effective investigation of this essential oil requires appropriate laboratory models and tools such as the model research organism Drosophila melanogaster. The species of fly used in S. Buentello Wong et al.’s experiment, Anastrepha ludens, was changed to Drosophila melanogaster to confirm the effects of essential oils on a model organism readily available and commonly researched. Changing the organism experimented on would change the data for the mortality rate found in Mansour’s experiment, so it was important to replicate the effects on a widely researched model organism. The experiment consisted of a positive control using clove oil to determine a baseline LD50, a negative control with no oil to eliminate any undesired variables in the arena setup that could cause a change in mortality rate, and an experimental arm testing the basil oil at the same concentration as the positive control clove oil. The testing arena was a culture vial where the oil formulation was mixed into the media using 1% Tween 20 as an oil-water emulsifier. 1.125 mL of the oil was mixed into 120 mL of water using Tween 20 and stirred for 30 seconds with a glass stir rod. After thoroughly mixing the oil and water, 30 g of instant fly media was added to contain the oil in the fly food, exposing the flies to the oil over the experiment’s duration. The negative control substituted the oil for water to ensure there was not another variable impacting the mortality rate of the flies. 14 g of media containing oil was placed in each experiment vial along with a mesh net to allow flies to feed through capillary action without drowning. Each experimental arm contained nine vials with ten male and ten female flies exposed to the testing conditions for 96 hours. Every 24 hours after transferring flies into the testing vials, qualitative and quantitative data on the behavior and mortality rate of the 20 flies per each vial were recorded in the data tables provided under the procedure section.

Senior Study:

Overview:

The senior study was similar to the preliminary study; however, instead of exposing the flies directly to media containing the chemical formulations leading to ingestion, contact, and inhalation toxicity, the experiment was modified using Benelli et al.’s method only to expose flies to fumigation toxicity. The isolation of fumigation toxicity eliminates other toxicity variables and provides data on one form of toxicity. The chemicals tested were also altered to a positive control of lavender oil, and two experimental arms testing prominent chemicals found in lavender: linalool and linalyl acetate (Shellie et al., 2002). To determine which chemical elicits the insecticidal effects of lavender oil, the chemicals were tested at concentrations corresponding to their percent abundance in Bulgarian lavender oil. Linalool was tested at 34% concentration by volume, and linalyl acetate was tested at 37% concentration of the amount of Bulgarian lavender oil tested (Boelens et al., 2012).

General Safety and Fly Care:

Before entering a research session, hands were washed with soap, and safety equipment, including a lab coat, nitrile gloves, and safety goggles, were put on. Any research requiring chemicals that produced harmful or distracting fumes was conducted inside a LabAire Systems DynamicFLO Fume Hood. Before using any chemicals, the MSDS was reviewed. Flammable chemicals, including ethanol (Item #: 861261, MSDS), acetone (Item #: 270725, MSDS), and ether (Flinn Scientific Item #: E0003, MSDS), were stored in a locked flammable cabinet when not in use. Drosophila melanogaster was bought from Carolina Biological in pre-made vials consisting of fly media, netting, and a foam cap (Item#: 172100). The culture vials were placed in a storage cabinet at room temperature to allow the flies to reproduce, allowing for the number of flies required for the experiment. If a culture vial was overcrowded or ran out of food, the experimental procedure’s general fly care and relocation portion was executed. A new culture vial was created by replicating Carolina Biological’s method with an empty culture vial, 12 mL of water added to 3 grams of instant fly media (4:1 ratio) measured on a Flinn Scientific Electronic Balance scale (Item #OB2142), fly netting, and a foam cap (Sadeghi, 2022). The flies in the original shipping vial were knocked out using FlyNap and transferred into the new vial. The flies were exposed to FlyNap using the included fly wand in the Drosophila Culture kit for approximately 3 minutes in a fume hood (FlyNap MSDS). The knockout was conducted on a fly stand upside down to prevent knocked-out flies from falling and drowning in the media. If the original vial was out of food, any remaining flies were knocked out using FlyNap and killed using the fly morgue provided in the Drosophila Culture Kit (Item #: 173050) filled with 50% ethanol and 50% water. The remaining larvae were killed using a 50% bleach-water solution (Bleach MSDS), and the contents were discarded in the trash. If plenty of food was left, the original vial was capped and placed back into the storage cabinet to allow the eggs and larvae to mature into adult flies and continue reproducing.

Knockout Sorting Dish and Jar Arena Setup:

To prepare for the experiment, it was necessary to create a controlled knockout area where flies could be sorted by sex. This was achieved by hot gluing a 5 cm by 5 cm piece of a washcloth to the inside of a 15 cm petri dish lid. Based on preliminary research, flies were knocked out using ether instead of FlyNap to minimize death during knockout and transfer. Flies in the selected culture vial were exposed to a fly wand from the Drosophila Culture Kit dipped in ether for approximately one minute or until the flies were visibly knocked out. Flies were sorted using a brush into piles according to sex using the bottom piece of the 15 cm petri dish. Flies are easily sexed by observing the color of the fly’s abdomen. Males have darker abdomens, while females are typically larger and have peach-colored abdomen. Using a petri dish ensured that flies waking up from the ether knockout before completing sorting could be re-anesthetized by adding 1 mL of ether to the glued washcloth using a 1 mL pipette and placing it on the petri-dish until the flies cease movement.

To prepare the testing jars using Benelli et al.’s method, a 1 cm by 1 cm filter paper square was cut out for each 300 mL screw top jar. A hot glue gun was used to glue the filter paper on the middle of the inside surface of the jar lid. Fly media was also prepared using the 4:1 water ratio to instant fly media and placed in one scintillation cap per jar to prevent starvation and dehydration throughout the 96-hour experiment. Vial labels were used to organize the jars for future data collection using the format “Cohort # - Experiment arm.” After preparing the experiment jar arenas, a culture vial was knocked out by exposing the flies to a fly wand from the Drosophila Culture kit dipped in the ether until the flies were visually knocked out. A fly stands to prevent knocked-out flies from drowning in the media, as directed by Chen et al.’s experiment (2013). The knocked-out flies were placed on the prepared knockout petri dish and were carefully sorted by sex, as previously described. Once all the flies were sorted, ten male and ten female flies were isolated and moved into a 300 mL jar using a small weigh boat (as recommended by Buentello et al.’s experiment, 2016). The open jar top was covered with sterile gauze to allow easy chemical application on the filter paper once all the flies were awake. The lid was placed on the gauze, sealing the jars until ready. This fly transfer process was then repeated for all remaining jars in the experiment. The flies were left to recover for 24 hours, as preliminary research showed that the fly’s resistance to fumigation toxicity was weakened directly after ether application and transfer to the jar experiment arenas.

Chemical Application on Filter Paper:

After determining the optimal concentration for lavender of 54 µL/L through a concentration test to match the ideal LD50 as directed in Benelli et al.’s experiment, a 20 µL to 200 µL micropipette was used to measure 162 µL of Plant Guru Bulgarian lavender oil and 38 µL of acetone into a 1.5 mL Eppendorf tube and mixed by shaking for 15 seconds (2012). LD50 (lethal doses) is the ppm (parts per million) concentration of a material required for a 50% mortality rate. Thus, highly potent insecticides have low LD50 values indicating solid insecticidal properties. Rubber bands were placed around the loose gauze of the jar to act as a temporary lid when the lid with the filter paper was removed. 20 µL of the 81% oil-acetone solution was placed on each positive control experiment jar lid for 30 seconds to allow the acetone to evaporate. The lids were placed back onto the jars with the gauze between the filter paper and the flies to prevent contact toxicity throughout the experiment. The excess gauze outside the jar was cut off, and parafilm was wrapped around the area between the lid and the jar to prevent fumes from escaping. This procedure was used for the negative control. However, 20 µL of pure acetone was used for each jar lid. This eliminated the possibility of acetone as an unwanted variable and verified that the flies would survive in the arena, as conducted in Benelli et al.’s experiment (2012). The linalool group used 145 µL of acetone and 55 µL of linalool to replicate Bulgarian Lavender oil’s 34% linalool composition (Boelens et al., 2012). The linalyl acetate group used 140 µL of acetone and 60 µL of linalyl acetate to match the 37% of linalyl acetate composition of Bulgarian Lavender oil (Boelens et al., 2012). The results of the experimental groups were then compared to the positive control that displayed an LD50 at 54 µL/L to determine which chemical (linalool or linalyl acetate), together or alone, elicits the insecticidal properties of lavender oil.

Data Collection:

Immediately after sealing the jars with parafilm, qualitative and quantitative data were collected on day one. The starting date and the percent fly mortality were recorded in the quantitative data table provided underneath the procedure section. Flies not moving were considered dead and counted towards the mortality rate. To calculate the mortality percentage of the flies, the number of dead flies was divided by 20 and multiplied by 100, giving the percentage of dead flies. Qualitative observations included notes of fly behavior, such as movement, flight, and speed. Data were recorded every 24 hours finishing at the 96-hour mark. If the chemical tested displayed a similar or greater fly mortality rate than the lavender oil using a two-tailed, independent statistics t-test, it would be determined to reflect the same insecticidal properties.

Fly Extermination and Cleaning:

When the experiment was finished after 96 hours, the parafilm sealing each jar was removed, and the lid was taken off carefully; the gauze remained on the jar opening, acting as a temporary lid with a rubber band holding it down to the jar to prevent surviving flies from escaping. Using a plastic transfer pipette, ether was directly applied to the gauze, and the lid was placed back on the jar. Once all the remaining flies were knocked out after approximately one minute, they were transferred into the fly morgue, and the food containing the eggs and larvae was washed with bleach, as previously mentioned in the General Safety and Fly Care section. The jars, lids, and scintillation caps were thoroughly washed with soap and water and dried on a drying rack. The filter paper on the lids was disposed of in a waste basket.

RESULTS AND DISCUSSION:

Figure 1 shows a direct correlation between the concentration of Bulgarian lavender oil and the percent mortality of Drosophila. The concentration test was conducted to discover the LD50 (Lethal Dose 50), the optimal concentration (ppm) of Bulgarian lavender oil, to elicit a 50% mortality rate within 72 hours. 2.7 μL of Bulgarian lavender oil was fumigated into a 300 mL jar to replicate the nine μL/L concentration in Benelli et al.'s method, which displayed a 50% mortality rate (2012). However, preliminary research showed that the 2.7 μL concentration did not display fly mortality. This may result from the highly variable composition of the lavender oil tested, so it was essential to conduct a concentration test for Bulgarian lavender oil to determine the LD50 concentration. A concentration test was conducted testing multiplicative values from the original 2.7 μL concentration: 1x at 2.7 μL, 2x at 5.4 μL, 3x at 8.1 μL, 6x at 16.2 μL, and 12x at 32.4 μL. Based on the results, the 16.2 μL concentration test displayed the most promising results as it was closest to the LD50 value for Bulgarian lavender oil on Drosophila: 2.7 μL displayed 0% mortality after 72 hours, 5.4 μL displayed 15% mortality, 8.1 μL displayed 25% mortality, 16.2 μL displayed 75% mortality, and 32.4 μL displayed 100% mortality by 24 hours. 32.4 μL would kill the flies too fast, resulting in a lack of data for prolonged toxicity. Although both the 8.1 μL and 16.2 μL doses displayed mortality levels equally distant from the desired 50% mortality rate, a higher mortality rate is desired for the positive control in this experiment, so 16.2 μL was chosen. This concentration will provide a fly mortality baseline for future experimental groups to compare. Groups that display a mortality rate similar to Bulgarian lavender oil will be the chemical that elicits most of the toxic effects found in the oil.

As represented in Figure 2, The negative control group was tested first to confirm that the experimental setup would not cause an increase in mortality from any unknown external variables. Preliminary data showed that external variables included weakened flies from fly etherization, lack of food causing the flies to starve, and contact toxicity when the filter paper with the chemical applied is directly exposed to the flies. The negative control results show an average mortality rate of 0% after 72 hours, confirming that the current arena design was fit for testing the fumigation toxicity of chemicals on Drosophila. If any other groups tested displayed a mortality rate, it was solely caused by the introduced chemicals. The negative control group also eliminated the possibility that acetone would impact the fly mortality rate as the filter paper in each jar had 20 μL of acetone added. This confirmation allows future groups to use acetone to supplement the missing microliters in each jar, standardizing 20 μL. 

When testing the negative control, preliminary data determined that the following arena conditions must be met to ensure a 0% mortality rate when flies are not exposed to chemicals. This was provided by filling a scintillation jar cap with food. Fly media must be in the jar to prevent the flies from starving. A sterilized gauze must be placed between the jar lid and the flies in the jar to prevent contact toxicity with the filter paper. When applying the chemicals to the filter paper, the acetone must be allowed to evaporate before sealing the lid on the jar to prevent the acetone's increasing mortality rate. Acetone supplemented the remaining volume to match the standardized 20 μLapplied to every filter paper. The jars were then sealed with parafilm to prevent fumes from escaping the testing arena. This method optimized the negative control arena design to limit fly mortality and any external variables that would impact the results.

The positive control was tested next using 16.2 μL of Bulgarian lavender oil and 3.8 μL of acetone to gather a fly mortality baseline, as shown in Figure 3. The mortality rates of the linalool, linalyl acetate, and the mixture groups using concentrations corresponding to the percent by volume found in Bulgarian Lavender oil can be directly compared to the lavender oil's toxicity to determine individual chemical effectiveness. The positive control displayed an average of 84.17% mortality after 72 hours of exposure. The results of the linalool and linalyl acetate groups can be directly compared to the positive control mortality to determine which, if either, elicits the majority of the toxic effects found in Bulgarian lavender oil. 

Next, the linalool group was tested at 5.5 μL of linalool and 14.5 μL of acetone to match the 34% concentration by volume found in 16.2 μL of Bulgarian lavender oil, as displayed in Figure 3 (Boelens, 1995). This concentration was tested to determine if linalool was the primary toxic component found in Bulgarian lavender oil when isolated. The experiment's results displayed an average mortality rate of 12.5% after 72 hours, significantly less than the Bulgarian lavender oil. A two-tailed, independent T-Test was conducted to determine if the mortality rate of the linalool group and the positive control were statistically different. A p-value greater than 0.05 is insignificant and would mean that the two groups are statistically similar, and a p-value less than 0.05 would mean that the two groups are statistically different. The two groups were significantly different when testing the difference at the 72-hour mark (p = 0.0000003, p < .001). Based on this data, linalool does not elicit most of the fumigation toxicity found in Bulgarian lavender oil when isolated.

Next, the linalyl acetate group was tested at 6.0 μL of linalyl acetate and 14.0 μL of acetone to match the 47% concentration by volume found in 16.2 μL of Bulgarian lavender oil, as displayed in Figure 3 (Boelens, 1995). This concentration was tested to determine if linalyl acetate was the primary toxic component found in Bulgarian lavender oil when isolated. The experiment's results displayed an average mortality rate of 9.17% after 72 hours, significantly less than the Bulgarian lavender oil. A two-tailed, independent T-Test was then conducted to determine if the mortality rate of the linalyl acetate group and the positive control was statistically different. The two groups were significantly different when comparing the average percent mortality at the 72-hour mark (p = 0.0000001, p < .001). Another two-tailed independent T-Test was conducted to determine if the mortality rate of the linalool group and the linalyl acetate groups were statistically similar. When testing the average percent mortality at the 72-hour mark, the two groups were not significantly different (p = 0.32, p > .05). The linalool group and the linalyl acetate group started with a seemingly higher similarity in mortality rate with a higher p-value after 24 hours (p = 0.73), which increased further by 48 hours (R = 1). Thus, as the experiment continued, the similarity between the two groups' mortality rates decreased as the linalool group's mortality increased at later time points. Based on these results, linalyl acetate does not elicit the fumigation toxicity found in Bulgarian lavender oil when isolated. Since neither the linalool group nor the linalyl acetate group produced a mortality rate similar to the Bulgarian lavender oil, there is either a different component in Bulgarian lavender oil producing the fumigation toxicity or the linalool and linalyl acetate have synergistic toxicity.

After determining that linalool and linalyl acetate, when tested alone, do not elicit the toxic effects found in Bulgarian lavender oil; it was hypothesized that a mixture of the two chemicals might interact with each other to elicit a combined fumigant toxicity. To create this compound, 5.5 μL of linalool and 6.0 μL of linalyl acetate were mixed to represent the 34% and 37% found in Bulgarian lavender oil, respectively, and the remaining 8.5 μL to reach 20 μL for consistency was filled by acetone as displayed in Figure 3 (Boelens, 1995). If all other chemicals were removed, this new mixture would contain 11.5 μL of linalool and linalyl acetate representing the Bulgarian lavender oil. If the mixture has a mortality rate statistically similar to the Bulgarian lavender oil, it could be inferred that the combined effects of linalool and linalyl acetate are responsible for the majority, if not all, of the fumigation toxicity found in Bulgarian lavender oil. The experiment's results displayed an average mortality rate of 68.33% after 72 hours. Three two-tailed, independent T-Tests were conducted to determine if the mortality rate of the mixed compound group was similar or different from the linalool, linalyl acetate, and positive control groups. At 72 hours, the average percent mortality of the linalool and the linalyl acetate groups were each significantly different than the mixed compound group (p = 0.00000002, p < .001; p = 0.0000000006, p < .001). The final two-tailed, independent T-Test was conducted to determine if the average percent mortality of the mixed compound group and the positive control were statistically similar as predicted by the hypothesis. However, the two groups were statistically different at the 72-hour mark (p = 0.019, p < .05). While the mixed compound group easily outperformed the linalool and linalyl acetate groups, it did not elicit the same level of toxicity as the positive control after 72 hours. However, at the 24-hour mark, the mixed compound group and the positive control were statistically similar (p = 0.113560505, p > .05). They only became statistically different by the 48-hour mark (p = 0.025, p < .05). Based on this data, one can conclude that, while the mixed compound group and the Bulgarian lavender oil elicit similar levels of toxicity in the short term (ie.24 hours), the Bulgarian lavender oil has prolonged toxicity and eventually outperforms the mixed compound group. A likely reason is that the Bulgarian Lavender oil contains other chemicals in addition to linalool and linalyl acetate that may increase toxicity, although further research must be conducted to determine the exact reason. 

While this experiment has provided a baseline for knowledge on a topic with crucial knowledge gaps, further research must be conducted to confirm and expand upon the findings of this study. The experimental design may be altered to omit other variables, such as variable vapor pressure or different evaporation speeds resulting in some chemicals evaporating more readily or being less prone to filling the air within the jar must be regulated as it may impact mortality rates. A method to regulate and result in uniform vapor pressure would result in more accurate results. Due to the limitations of this study's workplace, the chemical composition of the Bulgarian lavender oil could not be confirmed to contain 34% linalool and 37% linalyl acetate. Those percentages were determined using Boelens's 1995 chemical composition study on Bulgarian lavender oil; however, based on research, lavender oil's composition tends to vary greatly. A form of verification, such as gas chromatography, to determine what concentrations of linalool and linalyl acetate to use based on the selected lavender oil would benefit the accuracy of this study. Future steps include expanding the experimental design to isolate other toxicity exposure methods, such as contact toxicity and ingestion toxicity. While linalool and linalyl acetate did not elicit the toxic effects of Bulgarian lavender oil when isolated, the chemicals may be more proficient in contact and ingestion toxicity. Other essential oils such as Basil, Clove, and Thyme also warrant further investigation into their Drosophila melanogaster toxicity. The cost-effectiveness of each oil and exposure method may be compared to determine which is most effective to use in an agricultural setting.

Link to Lab Notebook

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