BACKGROUND:

Key Concepts and Background for Planaria Pesticide Research

Synthetic pesticides are chemicals used to kill unwanted life and have been commonly used in agriculture since the late 19th century (Tudi et al., 2021). They are employed primarily against insects and plants that damage crops. The most common method of pesticide application is volatilization, the conversion of liquids into a vapor, and the application of the vapor across a broad area via unmanned vehicles or direct human application (Tudi et al., 2021). The vapor then condenses onto the desired crops, the surrounding farmland, and nearby bodies of water; it can also remain as a vapor in the atmosphere and be carried extremely long distances before condensing (Tudi et al., 2021). Insecticides, pesticides employed against insects, are selectively neurotoxic. Most act upon acetylcholinesterase and the acetylcholine receptor, critical components for signaling throughout the nervous system (Casida 2009, Tripathi et al., 2008). This neurotransmitter and receptor are the same ones targeted in humans with nerve gas. Similar to the effects of nerve gas on humans, insecticides bond with the acetylcholine receptor, causing the nervous systems of targeted insects to fail, leading to loss of coordinated mobility and death (and subsequent inability to damage crops) (Suryanarayanan, 2014). Herbicides, employed against plant life, are small molecules designed to act on sites specific to certain plant species, inhibiting photosynthesis, and amino acid synthesis (Traxler et al., 2023). They are designed to target specific sites in unwanted plants, ideally making them benign to the crops around which they are applied (Casida, 2009). Despite the targeted nature of synthetic pesticides, they have potential toxic effects against non-target species, including neurological damage, genotoxicity, birth defects, and carcinogenic effects (Parlakidis et al., 2023, Kumar et al., 2023).

Pesticides can pose a significant threat to animal, plant, and human life, potentially jeopardizing ecosystems with heavy contamination (Parlakidis et al., 2023). Aquatic ecosystems are particularly vulnerable to pesticide contamination because they have the most potential contaminant delivery routes. These routes include agriculture runoff, in which contaminated water enters ecosystems through irrigation systems (Jing et al., 2021), leaching, in which pesticides diffuse through soil (Pandey et al., 2020), spray-drift, in which airborne pesticides travel with the wind (Chen et al., 2019), and soil erosion (Vagı and Petsas, 2017). The presence of pesticides and their metabolites in aquatic bodies can cause toxic effects (cancer and birth defects in aquatic life, reduced ability for plants to photosynthesize, and death of plants and animals from decreased nutrient uptake and genetic damage) even at low concentrations (ng/L to mg/L) Rajapaksha et al., 2018; Brillas, 2021). This toxicity causes ecosystem fragility that is furthered by the inability of aquatic ecosystems to effectively break down and “cleanse themselves” of pesticides (Singh et al., 2023).

These chemicals persist within the environment and living tissue, and the levels of pesticide deposition in water, air, and soil are consistently increasing (Singh et al., 2023, Tudi et al., 2021). This is in part due to urbanization and industrialization driving up demand for increased crop yields, which are commonly improved with the use of synthetic pesticides and fertilizers (Matamoros and Rodriguez, 2016; Lam et al., 2021). These chemicals are not only effective in increasing yields but necessary in order to satisfy demand on the global agriculture industry (Jusoh et al., 2011). It is estimated that the immediate cessation of pesticide use would cause a 78% loss of fruit production, a 54% loss of vegetable production, and a 32% loss of cereal production (Tudi et al., 2021). This poses a massive challenge to a sustainable agriculture transition: as the population grows, pesticide use becomes more difficult to phase out, pesticide contamination becomes more pervasive, and affected ecosystems become more fragile (Shattuck 2021, Singh et al., 2023). Although concerns over pesticide use have existed in the scientific community since the interwar period (Bosso, 1987, Bohme, 2015, Pellow, 2007), their use is still supported by public policy (Stegmaier et al., 2014), as we have yet to develop an agricultural system independent of synthetic chemicals that would keep yields consistent with demand (Wuepper et al., 2023). In order to develop such a system, alternatives must be designed that would alleviate the harms of pesticides whilst serving the same functions (Goulet et al., 2023). 

Plant-derived pesticides are a promising alternative to synthetic pesticides, offering several mechanisms of pest deterrence along with biodegradability and a minimized ecological threat (Souto et al., 2021). Many natural plant-derived molecules (such as terpenes, flavonoids, alkaloids, polyphenols, cyanogenic glucosides, and quinones) serve important ecological functions (acting as repellants, antifeedants, insecticides, and growth regulators) that align with the uses of synthetic pesticides (Souto et al., 2021). Essential oils are concentrated plant compounds in a volatile liquid form and contain many bioactive molecules attributed to pesticidal function (Maldaner et al., 2023). Certain essential oils (such as eucalyptus and rosemary) have been shown to act as effective insect repellents, and have prospective use as alternatives to synthetics in grain preservation and antifungal applications (Souto et al., 2021, Azeem et al., 2022, Maldaner et al., 2023). Despite being a promising alternative to synthetic pesticides, the exact mechanisms of action of essential oils' pesticidal functions are still unclear, in part due to the lack of data on the complex chemical makeup of these oils (Lanzerstorfer et al., 2021). This means that more research needs to be done to ascertain the ecological impact of potential essential oil contamination before wide-scale implementation can be considered (Lanzerstorfer et al., 2021).

Performing a study on the potential impact of a contaminant on an ecosystem presents many challenges. Modeling an entire ecosystem in a lab would require more monetary and spatial resources than available to me in a high school lab environment, so a substitute is required to monitor the potential impact of a substance on an ecosystem. Biological indicators, or bioindicators, are living organisms that are used as a potential tool to monitor environmental health changes (either positive or negative) (Mothersill et al., 2016). The selection of an organism as a bioindicator depends on its physiological and behavioral changes: they must be common processes that are easy to study and apply directly to the sustainability of an ecosystem (Mothersill et al., 2016). Many research studies have used the entire lifecycle of an organism as a bioindicator, with the speed and manner of development acting as ecosystem health indicators (Huerta et al., 2022). By studying how the natural processes or life cycle of a bioindicator is altered, we can make assessments on how the ecosystem as a whole may be altered. 

Planarians are small invertebrate freshwater flatworms, and varieties of them are found worldwide (Pagán, 2017). Their characteristics (such as size and color) vary slightly from species to species, but their basic physiology remains constant. They live in freshwater, marine, and terrestrial environments (Pagán, 2017). They have a low tolerance against most chemical substances, making them potentially effective bioindicators for ecosystem damage due to chemical contamination (Manenti et al., 2014). Planarians also have extraordinary asexual regenerative capabilities, and small fragments of planaria are able to regenerate the entirety of the missing anatomy given 1 to 2 weeks (Alvarado et al., 2012). This means that if bisected, the front portion of the worm will regenerate the tail portion, and vice versa, leaving two genetically identical worms. This process is facilitated by the rapid production of stem cells, which differentiate to produce the missing tissues, and the “remodeling” of existing physiology in order to accommodate the newly generated tissue (Alvarado et al., 2012). These regenerative capabilities make planarian reproduction very straightforward to initiate and monitor in a lab environment, providing a bioindicator for possible damage to the reproduction capabilities of life within an ecosystem.

The study presented herein attempts to assess the possible negative effects of implementing essential oils as synthetic pesticide replacements. Two groups of D. Tigrina, an aquatic Planarian species, will be continuously exposed to low PPM (parts per million) concentrations of both traditional pesticides and essential oils over a period of two weeks. One of these groups will be in the process of asexual reproduction, and one will not. Survival rates of the non-reproducing group, along with reproduction success rates, will be recorded daily over the 2-week period. Subjects in the experimental groups will be immersed in 10 PPM solutions of Citrus sinensis (orange essential oil), Mentha piperta (peppermint essential oil), and a combination of Spinosad A & Spinosad D (consumer grade “organic farming” pesticides). The positive control group will be immersed in a 10 PPM solution of Cyflurthin, a synthetic pesticide characterized as highly toxic to invertebrates, and the negative control group will be immersed in pure spring water. I hypothesize that the alternatives to traditional pesticides will result in mortality and reproductive failure rates above those of the pure spring water group, but lower than those of the group exposed to the synthetic pesticide, because although some of the compounds in essential oils are potentially toxic to planaria, Cyflurthin was specifically designed to kill invertebrates. By recording how mortality and successful reproduction rates of D. Tigrina are affected by new pesticide alternatives, we will have a preliminary window into whether or not essential oils will have a significantly less destructive impact than synthetic pesticides. 

Essential oils could potentially replace most synthetic pesticides if they prove to be less damaging to ecosystem health. Synthetic pesticides are one of many anthropogenic time bombs that we are completely reliant upon as a species, and it is imperative that we reduce that dependency before catastrophic damage is done to our biosphere. But before substituting apparent alternatives for synthetic pesticides, it is crucial to ensure that we are not replacing them with something that has the capacity to do equivalent or greater environmental damage. My study will give basic indications of whether or not some of these alternatives have the potential to do just as much damage to life, or whether they need further investigation. Simple toxicity assays with bioindicators such as D. Tigrina are critical first steps in investigating the next steps in the transition to sustainable agriculture. 

METHODS & PROCEDURE:

The current novel study builds on techniques and methods refined during last year’s replicate study. The replicate study attempted to replicate the findings of Byrne et al.'s 2018 study on the effects of ethanol on the negative phototaxis of D. Tigrina. The current study departs from the replicate in that it does not seek to investigate the neurological effects of a substance on the movement of D. Tigrina. Rather, the current study looks to investigate the possible effects of replacing traditional synthetic pesticides with essential oils, using the reproduction cycle and motility of D. Tigrina as a combined bioindicator. Plant-derived pesticides (such as essential oils) offer several mechanisms of pest deterrence along with biodegradability and a minimized ecological threat (Souto et al., 2021), so it is expected that they will not be as detrimental to reproduction or survival as synthetic pesticides, but may interfere with D. Tigrina physiology in novel ways.

The current novel study utilizes 2 control arms, one positive and one negative, alongside 3 experimental arms. Each of these arms involves two groups of D. Tigrina, one “regen” in which 9 subjects are bisected to stimulate asexual reproduction, and one “non-regen,” in which 9 subjects are kept unaltered. Each arm involves submerging each of these two groups in a solution dependent on the arm. In this study, the ability of D. Tigrina to regenerate asexually is a key indicator of solution toxicity. A lower successful regeneration rate in a given solution indicates a hindered ability to reproduce, and thus a high negative environmental impact of the solution. After a two-week period in each of these solutions, the subjects are removed, and data is collected with respect to their survival rates, and the success of the “regen” group in completing asexual reproduction. A basic negative phototaxis motility test is then run on the individuals from each arm and group as a measure of physical “health” and motility.

Each arm differs from the next only in the solution used. All solutions containing chemicals contain only parts per million of said chemical, with the rest of the solution consisting of spring water. This is done to simulate pesticide “runoff,” or the trace amounts of each substance that would appear in the environment should they replace synthetic pesticides on a large scale (Thinh et al., 2019).

The negative control arm submerges each group of D. Tigrina in pure spring water for the two-week period. D. Tigrina live in spring water, so it is expected that the two-week period in the “solution” will have no effect on either group. Two weeks is ample time for a bisected subject to fully complete the regeneration cycle (Alvarado et al., 2012), so a close to 100% successful regeneration rate and survival rate is predicted. Any major deviations from these values would indicate flaws in the experimental setup or the interference of random variables.

The positive control arm submerges each group of D. Tigrina in a 10 PPM Cyfluthrin solution, with the rest of the solution being composed of spring water. Cyfluthrin is a synthetic pesticide highly toxic to invertebrates (Soderlund, 1989), and even a trace amount is expected to have a significant negative impact on the subjects' ability to survive and reproduce. Significantly lower survival and successful reproduction rates are expected (<50%), and any unexpectedly high success/survival values would indicate flaws in the experimental setup or the interference of random variables. 

Replicate Study:

The most relevant component of methodology from last year’s replicate study as it relates to this year’s novel study is the motility test. Most other aspects of this year's study were developed independently of the replicate study’s procedure, as it differs so vastly from this novel study in aspects such as exposure time, solution concentration, and chemical type.

The replicate study looked to quantify the effects of ethanol exposure on D. Tigrina with a basic motility test (Byrne et al., 2018). The motility test is designed to elicit a photophobic motor response from D. Tigrina by placing them in a brightly lit area and providing each subject access to a shaded area ~5 cm away. The speed and efficiency of each subject's movement to the shaded area is recorded and is a useful quantitative indicator of neurological and physiological capability (Byrne et al., 2018).

A 10 cm EZ BioResearch petri dish served as the basic platform for the test. A ruler was used to find a diameter of the dish's lid (note: “the diameter” in this case refers to a line across the lid that bisects it evenly), the diameter line was then marked with a Sharpie. AmazonCommercial black electrical tape was then used to tape over half of the dish’s lid, leaving the lid on one side of the diameter occluded from outside light, and the other half clear. The ruler was then used to find a second diameter line across this lid, perpendicular to the first diameter (the line across the dish lid represented by the ruler's position should cross the center). The point on the edge of the non-occluded side of the dish lid that the ruler lies on was then marked with a dot using a Sharpie, and designated as the “starting point.” The petri dish is now fully “bisected,” and should resemble Figure a. when viewed from above.

A table or flat surface measuring at least 2’ by 4’ was then designated as the “working surface,” and was free of all materials unless otherwise specified. An ENOCH 14 Watt Swing Arm Desk Lamp With Clamp was then clamped to the working surface, and plugged into a 120W wall outlet. The head of the lamp was then angled to be flush with the working surface, and the lamp's arm was adjusted such that it was 64 cm above the working surface. A bisected petri dish was then placed on the working surface, directly beneath the head of the lamp. A 25 mL graduated cylinder was then used to measure out 25 mL of spring water, which was then poured into the bisected petri dish. An ALPTOY 64-inch Extendable Tripod was then placed on the working surface, and a recording device was affixed to the tripod. The tripod’s position and height were then adjusted such that the recording device had a clear top-down view of the bisected dish (it was ensured that the tripod/recording device didn’t come between the lamp and the dish). The lamp was then turned on, and the recording device was activated. A 1 mL transfer pipette was used to place a single test subject in the bisected dish, directly beneath the “starting point.” The subject was then recorded until it had completely relocated beneath the occluded side of the dish. Time from placement in the dish to relocation was then recorded by analyzing the recording, and designated “relocation time.”

Model Organisms:

Gloves and a lab coat were used when handling D. Tigrina. D. Tigrina were kept in 10 cm EZ BioResearch petri dishes, with 25 mL of water in each dish. The number of D. Tigrina in a single dish did not exceed 30. A 1 mL pipette was used to transfer D. Tigrina across petri dishes, and the time D. Tigrina spent outside of water/solution was minimized. When transferring D. Tigrina into a dish with a different solution, they were briefly deposited onto a paper towel, to ensure no cross-contamination of solutions during the transfer process. 

D. Tigrina were selected as the subjects of this study for their high chemical sensitivity and resulting potential to be effective bioindicators, as well as their low maintenance when cultured in a lab (Martinez et al., 2021). Regular maintenance of D. Tigrina consisted of both dish cleaning and routine feeding. Cleaning was performed by using 1 mL pipettes to transfer the D.Tigrina in the selected petri dish into a fresh “interim” petri dish with 25 mL of spring water. The selected dish was then washed with dish soap and a sponge, or simply disposed of depending on time and resource availability. If the original dish was disposed of, the interim dish was redesignated as the habitation dish and placed into storage, along with the D. Tigrina inside. If the original dish was washed, it was rinsed and refilled with spring water, and the D. Tigrina in the interim dish were placed inside the freshly cleaned dish with a 1 mL pipette. All dishes containing living D. Tigrina were cleaned at least once a week. 

D. Tigrina were fed with the yolk of boiled chicken eggs, but are also commonly fed raw chicken, ox liver, and beef in a lab setting (Byrne et al., 2019). Small portions of egg yolk were placed in the petri dishes containing D. Tigrina, and left until all, or the vast majority, of the subjects appeared sated. In D. Tigrina, this “sated appearance” is indicated by the subjects taking on the color of their food, and widening slightly (Apyari et al., 2021). The cleaning procedure outlined above was then performed, ensuring no biological contamination from decomposing food matter. 

` Optimally, D. Tigrina should be stored in a mostly dark, temperature-regulated area (Martinez et al., 2021). In this study, D. Tigrina were stored in a closet in a temperature-controlled facility. The closet was accessed somewhat regularly by researchers during the day, so all habitation dishes were placed in a small cardboard box within the closet. This allowed for a continuously dark environment for the subjects when in storage. The cardboard box was perforated with small holes to allow ample airflow, ensuring subjects did not suffocate in their dishes. To the same effect, habitation dishes were never stacked on top of each other, and no objects were ever placed atop habitation dishes while they were in storage. This was done to ensure no airtight seal was accidentally made in a petri dish, which would result in the suffocation and death of all subjects within the dish. 

D. Tigrina can reproduce asexually by fission, and said fission can be easily induced in a lab (Alvarado et al., 2012). To induce this asexual reproduction, D.Tigrina were placed on a paper towel with a 1 mL pipette, and then bisected width-wise with a LabAider #11 Surgical Grade Blade, producing two viable fragments. When using blades to bisect subjects, eye protection was always worn. No blade was carried from the working surface across the lab, and extreme caution was exercised when handling the blade. Blades were disposed of in the puncture-resistant packaging they were shipped in. Each fragment was then transferred into a dish containing the trial’s current solution. 

Solution Creation:

The potential impact of 3 different chemical solutions was assessed in this study, along with a traditional synthetic pesticide as a positive control. The three potential alternatives to traditional pesticides that were tested are GreenHealth 100% Pure Citrus sinensis (orange essential oil), GreenHealth 100% Pure Mentha piperta (peppermint essential oil), and Captain Jack’s Deadbug Brew (a .001% solution of Spinosad A & Spinosad D (MSDS), consumer-grade “organic farming” pesticides). The positive control was performed with BioAdvanced Cyfluthrin Insect Killer, which contained .75% Cyfluthrin (MSDS), a synthetic pesticide characterized as highly toxic to invertebrates, and the negative control group was immersed in pure spring water. All dilutions of Spinosad (MSDS) and Cyfluthrin (MSDS) were done under the LabAire Systems Fume Hood, wearing nitrile gloves, goggles, and a lab coat. These chemicals present little risk in their diluted form, but can be acutely toxic, so extreme caution was used in their handling.

The dilution of the 2 essential oils into 10 PPM solutions was accomplished by micropipetting 5 μL of each oil (using a generic .5 to 10 μL Pipette Controller and .5 to 10 μL universal pipette tips) into 499.995 mL of spring water. 400 mL of spring water was measured into a 1 L beaker using a 100 mL graduated cylinder, 90 mL was measured into the same beaker with a 25 mL graduated cylinder, 990 μL was measured into the beaker with a generic 10 to 100 μL Pipette Controller and 200 μL universal pipette tips. The final 5 μL was measured into the beaker with a generic .5 to 10 μL Pipette Controller and .5 to 10 μL universal pipette tips. In retrospect, this procedure could have created a more accurate solution using volumetric flasks and only a single micropipette measurement, as the many discrete measurements and no final volume check leave a lot of room for error. 

For the Spinosad and Cyfluthrin solutions, the process was slightly more complex, as the chemicals arrived in pre-diluted solutions for agricultural use. The Cyfluthrin solution came prediluted to .75% Cyfluthrin by volume, so .133 mL of this solution was measured with a  generic 10 to 100 μL Pipette Controller and 200 μL universal pipette tips, along with a generic .5 to 10 μL Pipette Controller and .5 to 10 μL universal pipette tips. The measured Cyfluthrin was then mixed with 99.866 mL of spring water (measured with a combination of 25 mL and 10 mL graduated cylinders, along with a generic 10 to 100 μL Pipette Controller and 200 μL universal pipette tips, and a generic .5 to 10 μL Pipette Controller and .5 to 10 μL universal pipette tips), yielding a 10 PPM Cyfluthrin solution. The prediluted “organic farming” pesticide had a .001% Spinosad concentration. Thus to achieve the desired 10 PPM concentration, 1 mL of the .001% solution was micropipetted (using a generic 1000 μL pipette controller and 1000 μL pipette tips) into 99 mL of spring water (measured with 25 and 10 mL graduated cylinders). All solutions were stored in EISCO Reagent 1000 mL chemical storage bottles after dilution, and stored alongside other lab materials (the especially low concentrations means that these solutions, despite containing potentially dangerous substances, pose little threat to human health). After being used, waste solution containing Spinosad or Cyfluthrin was poured into an EISCO Reagent 1000 mL chemical storage bottle marked “waste,” and replaced alongside the other solutions. 

Experimental Procedure:

The potential environmental impact assay used in this study relied on the asexual reproduction and survival rates of D. Tigrina when continuously exposed to “runoff-level” amounts of each substance. 10 PPM represents the LD50 of modern synthetic pesticides for many freshwater fish species and would be an unusually high level of runoff contamination (Abas et al., 2022, Lefranq et al., 2017). Pesticide use is expected to rise proportionally to the human population (and thus, exponentially), so in the interest of accounting for the ever-growing agriculture industry, this concentration was selected (Ren et al., 2024).

 Two groups were exposed to each substance, no less than 2 days after feeding, with the two exposure groups constituting one arm of the experiment. The first group was denoted by “[arm abbreviation]-REGEN” on their petri dish, and the second simply by “[arm abbreviation].” Examples of arm abbreviations include “NC” for negative control, “PC” for positive control, “EXP-1” for the first experimental group, and so on. 

The REGEN group was bisected in order to induce asexual reproduction, and the non-REGEN group was simply moved from their habitation dish to their experimental dish without any alterations. The non-REGEN group consisted of nine D. Tigrina, and the bisected REGEN group consisted of 18 “fragments” undergoing asexual regeneration.  Each experimental dish was first labeled with a sharpie following the scheme outlined above, and then filled with 25 mL of their respective solution. For the negative control, this solution was pure spring water, and for the positive control it was the 10 PPM Cyfluthrin solution. The experimental groups used 10 PPM Citrus sinensis, Mentha piperta, and Spinosad.  

After two weeks in solution each group was examined, and each of the members had their conditions categorized. The fragments in the REGEN group were examined and placed into one of three categories: “Successful,” meaning the fragment had achieved a full regeneration including a developed head with photoreceptors and a tapered tail that displayed a photophobic response, “Viable,” meaning the tail/head were not fully formed, but the fragment was alive and able to locomote, and “Unsuccessful,” meaning the fragment was dead. The members of the non-REGEN group were grouped into two categories: “Alive,” characterized by the individual's ability to locomote and display a photophobic response, or “Dead,” categorized by a complete lack of response to stimuli and deliquescing body. If there were 9 or more living members of a given group after the 2-week exposure period, the motility and photophobia test outlined in last year's study was performed. Relocation times were then analyzed with a 2-tailed equal variance T-Test.

RESULTS AND DISCUSSION:

The Planaria Vitality Index (PVI) system was developed late in the experimental process to quantify Planaria health based on collected data. The difficulty of testing data constructed from binary outcomes for significance wasn’t fully considered until large amounts of data had been collected, and so the main purpose of the PVI was to produce analog data that could be tested for significance. 

The index was designed to produce a figure between zero and one for each solution tested. Zero indicates an almost complete loss of Planarian reproduction/survival ability and was the expected outcome for the positive control arm, which consisted of a 10 PPM Cyfluthrin solution. Cyfluthrin has been proven to be extremely toxic to invertebrates even in trace amounts, causing irreversible neuroendocrine system damage by interfering with sodium channels (Soderlund et al., 1989). With such a low concentration, the lethal dose of even highly toxic chemicals such as Cyfluthrin would not be expected to be present within the subjects in 2 weeks. The exception to this is when high rates of stem cell differentiation are taking place, as stem cells are more vulnerable to cell death and genotoxicity when exposed to even small amounts of toxins (Kang et al., 2010). High rates of stem cell differentiation take place in Planaria regeneration, so it was expected that (if methods were sound and no random variables were present) 10 PPM Cyfluthrin exposure would completely hinder the Planaria’s asexual reproduction, whilst leaving the non-regenerating group alive, yielding a PVI score of 0 (Tu et al., 2015). A PVI score of one would indicate successful survival as well as a fully successful asexual reproduction cycle. This was the expected value for the negative control, in which whole Planaria survived in pure spring water and the fragments successfully regenerated. 

It is worth noting, before the results of the PVI testing are discussed, that none of these results could be analyzed for statistical significance. Using the PVI system, each group of 18 Planaria was treated as a cohort and produced one data point, meaning that these results are supported by only one biological replicate per solution. Rather than acting as the results of a definitive scientific investigation, the data produced by the regeneration tests can act as a preliminary view into the possible effects of these chemicals. In publishing this data, we hope to open the door to further (and more definitive) studies on the interactions between Planaria regeneration and chemical runoff. 

The incomplete PVI data for both control arms supports the effectiveness of the experimental setup. The PVI score for the positive control (10 PPM Cyfluthrin) was the expected 0, where no successful reproductions were recorded. Qualitatively, the surviving planaria in the non-regen group also displayed signs of physical stress including reduced size and wounds across the body that appear to be the result of either corrosion or decay (example here). Physical stress was later quantitatively analyzed in the Motility Assay. The PVI score for the negative control arm was 1.055, slightly above the expected value. This is due to the random fission of one worm in the non-regen group, and subsequent regeneration of two worms. Thus the recorded n value was 10, allowing the PVI value to exceed 1. This indicated that the Planaria in this group had a slightly above-expected ability to survive and reproduce, most likely due to their size when shipped from the biological supplier. The departure from the expected value of 1 is largely insignificant, but in the future the size and vitality of the worms selected for experimentation must be taken into account. These preliminary results being so close to the expected values indicate that the 10 PPM concentration used in this study, along with all other aspects of the experimental setup and the PVI, are well suited for studying the effects of pesticide runoff on D. Tigrina.

The methodology departed somewhat from the system outlined in Figure 1 in the cases of the two experimental groups (submerged in 10 PPM Orange Oil and 10 PPM Peppermint Oil solutions respectively). Initially, the procedure outlined in Figure One was followed, but spontaneous fission and regeneration occurred several times, leading to outlying results and a PVI score far greater than even the negative control (~1.44 for the 10 PPM Orange oil, and ~1.55 for the 10 PPM Peppermint oil). Upon a retroactive analysis of these groups, it was determined that their original size and health before exposure was abnormally large, and that their size and health were a direct result of variation between batches as they were raised in a lab with variable conditions prior to their shipment and arrival to Berkeley Carroll. The average length range of D. Tigrina in the wild is 9 to 15 mm, and many of the worms used in these trials exceeded 25 mm in length (Pickavance, 1971). In order to collect data from these groups, the smaller offspring from the first trials were re-bisected and once again placed in solution for two weeks. The worms are now the second generation to spend the entirety of their life in solution, with a total exposure period of 4 weeks. The results of these second round trials are displayed in Figure 2, and fall into the expected range. 

The second-round group submerged in orange oil had a PVI Score of .945, and the second-round group submerged in peppermint oil had a PVI score of .778. This would seem to indicate that long-term, multigenerational exposure to peppermint oil is somewhat harmful to Planaria reproduction, but more data is needed to confirm this. In the future, these same tests need to be run across 9 groups for each solution to attain a measurement that can be analyzed for significance. Alternatively, a test with continuous variables would allow for less testing time and more biological replicates, and would ideally quantify subject length, motility, and physical abnormalities

An alternative method of quantifying Planarian reproductive health had to be developed due to the statistical insignificance of the PVI measurements. The Motility Assay was developed to satisfy this need. Planaria have a strong photophobic response and will attempt to locomote away from any light source (Paskin et al., 2014). In the Motility Assay, the exposure procedure from the PVI testing was done on naive worms for the negative control solution, the positive control solution, and the Peppermint oil solution. After exposure, 9 worms from each non-regen and regen group were moved into motility testing. The worms from each group were placed into an array of 9 Petri dishes under a lamp with a grid of 1 cm graph paper below the dishes. Their motion was recorded for 5 minutes. Since planarians, when healthy, exhibit a powerful photophobic response, the lamp should elicit rapid planarian motion from healthy subjects. The worm's motility score was quantified as the number of lines on the graph paper the worms crossed in the 5 minute period. Healthy worms were expected to have higher motor function, and thus more motion beneath the light of the lamp, more grid lines crossed, and a higher motility score.

The average motility score for each group tested is shown in Figure 3. The motility scores of each group were compared against the rest using an independent, 2-tailed T-test. The results of the T-test showed that the negative control non-regenerating group (avg. Motility score 81) performed significantly better than all other groups (max p-value .00035, vs. ExpPep-NR group). The negative control regenerating group (avg. Motility score 46) significantly outperformed both positive control groups and the regenerating peppermint oil group (max p-value ~.0000008 vs. PC-NR) and didn’t perform significantly differently when compared to the non-regenerating peppermint oil group (with a p-value of ~.15). The non-regenerating positive control group (avg. Motility score 7) performed significantly better than the regenerating positive control group (avg. Motility score 0), with a p-value of ~.048. Most interestingly, there was no significant difference between the performance of the regenerating positive control group and the regenerating peppermint oil group (avg. Motility score 2, p-value .08). 

This data shows that the freshly regenerated worms were consistently less motile than their non-regenerated counterparts, indicating that locomotive function undergoes some kind of improvement during the period after physical regeneration is complete (completion was quantified as the presence of a tail tapering to a point, and the characteristic arrow-shaped frontal structure). This invites future research into the non-visible aspects of Planarian reproduction and development- a study ought to be done in the future that looks to quantify the average period in which Planaria regain full motor function after physical regeneration appears complete. 

In answering the initial experimental question as to what the possible negative effects of implementing essential oils as synthetic pesticide replacements are, the data from the Motility assay seems definitive in one regard. Peppermint oil, present in the same amounts as industrial synthetic pesticides, has the capability to alter Planarian health on a similar scale. This is clearly indicated by the lack of significant difference between the motility performance of the group reproducing in 10 PPM Cyfluthrin and the group reproducing in 10 PPM Peppermint oil. These results suggest that wide-scale deployment of Peppermint oil in pesticidal applications could be just as catastrophic for affected ecosystems as the current synthetic pesticides. In the process of transitioning towards a more sustainable agricultural system, conclusions such as this are crucial as the list of candidates for pesticide replacement is narrowed. To find what works, we must first identify what doesn’t work. 

Future research into potential synthetic pesticide replacements should first and foremost focus on testing as vast a range of compounds and mixtures as possible. Pesticide contamination is one of the most pressing issues facing humanity at large, and improving upon our current agricultural systems with new, less destructive pesticidal compounds is a crucial step toward a sustainable future. To locate these new compounds, a massive research effort must be undertaken to thin the pool of candidates as much as possible before evaluating implementation processes. To enable this wide-scale research effort, effective and easily obtainable bioindicators such as Planaria must be used to evaluate the potential environmental impacts of different compounds. In order to use Planaria as a widely applicable research tool, a more effective system of reproductive data quantification must be developed. The main limitation of the PVI system was that it only provided binary outcomes for reproductive success when in reality planarian reproduction can manifest itself in some irregular ways due to the ability of any body portion to regenerate the entire anatomy of the worms. Future studies surrounding Planaria as a bioindicator should have a system of quantifying reproductive success with continuous variables, accounting for initial vs final size, physical defects, irregular reproduction patterns, etc. Such a system would have swaths of applications in ecology and toxicology research, and may be crucial for preserving the future of our environment.

Link to Lab Notebook

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