Scientific Research in School by Barker College

Science Science

Scientific Research in School Volume 5 Number 1 October 2023

Extension Journal

Senior Editor Dr Matthew Hill Creative Direction Mrs Susan Layton Dr Matthew Hill Research Supervisors Dr Katie Terrett Dr Alison Gates Dr Matthew Hill

The Barker Institute

About the Scientific Research in School Journal When the New South Wales Education Standards Authority announced a new course “Science Extension” to commence in 2019 we were thrilled that there was an opportunity for a formally-assessed capstone experience in Science for our students. From the perspective of the Barker Institute it was an exciting chance to support students doing academic research, alongside other subjects such as History Extension, Music Extension and English Extension 2. Where many capstone project courses fail is the at final step of the research process – dissemination. Research is not merely the process of conducing an investigation and writing a report, but sharing it with the wider community so that people can learn, critique, have other student researchers at multiple schools build on the projects published. I am so glad to be able to publish this journal each year now celebrating 84 articles each representing genuine contributions to science.

Dr Matthew Hill Director of The Barker Institute

Introduction

Over the last five years it has become clear that Scientific research is no longer restricted to universities and industry.

Science Extension is the capstone experience allowing our students to showcase all they have learned with real-world applications.

With the calibre of students and opportunities we have at Barker College there is no need to wait until post-school for our young people to be changing their world and, by doing so, enriching the lives of those around them. This is clearly evident in these student research articles (84 articles published in the first five editions of the journal) and I see it every day in the classrooms and corridors of our learning spaces.

The keynote speaker at Barker College’s celebration of Science Week 2023 was Laureate Professor Veena Sahajwalla, internationally recognised materials scientist, engineer and inventor revolutionising recycling science. She demonstrated how creativity and critical thinking through the Sciences can provide industry with effective and meaningful solutions to real-world problems. Our Year 12 Science Extension students are learning by doing just that.

Just as teachers and students alike continue to develop themselves and each other to be all they can be, teachers and students have meaningful contributions to make to those around them. I am proud of each of our students living out the school values as they make their mark.

These students are researchers who understand the impact that Science can have on the world. They have applied their scientific knowledge and skills, gained over the last 13 years of their schooling, in this capstone experience culminating in these published research reports.

Congratulations to our newest published authors. I am proud of all you have achieved in such a short space of time, am grateful that you share your knowledge and insights with the community, and look forward to your ongoing discoveries in your future fields of study.

I am grateful for the work of all Science staff at Barker for their investment in these students, especially the three research supervisors who along with lab staff supported them through the Science Extension research program.

Mr Phillip Heath AM Head of Barker College

Mrs Virginia Ellis Head of Science

Research Supervisors

Dr Matthew Hill Director, Barker Institute Physics Teacher

Dr Katie Terrett Science Extension Coordinator Chemistry Teacher

Dr Alison Gates Agriculture & Science Teacher Assistant Coordinator STEAM Term 4 2022 - Term 1 2023

In these 19 academic articles, students wrestle with the extant literature to accurately describe a gap in scientific understanding, before implementing valid methods to produce novel, first-hand results and findings to address this gap. It has been a pleasure to journey with them and wrestle with complex ideas and communication. Together we have endured through complications, celebrated breakthroughs, and explored implications. We are incredibly proud of them personally, and also the work that they have produced. We look forward to seeing the impact of this work on future research in Science.

Contents Part 1: Physics Salt, wood and a shocking result – Investigating Lichtenberg figures in wood Michael Ashworth The extended boundary of chaos: Testing the relationship between total energy and chaotic tendencies of a double pendulum system Jeslyn Tan

Post-processing of FFF non-polar polymer composites using microwave annealing Max Hanley

The impact of adding a duct to a toroidal vs traditional bladed propeller Bardia Lamyi-Arani

Part 2: Biology and Environmental Science What’s all the buzz about? An investigation into the diversity and distribution of pollen within the hive Julia Disney

Glyphosate and the effect that it has on rumen microbes and agricultural production Samuel Speed

Oh, it’s o-fish-al: An investigation into the sterilisation techniques of tilapia fish skin Alana Tully

Antibacterial properties and applications of raw Aloe vera to inhibit bacterial growth Joe Entwistle

To what extent can ocean sediment microflora withstand ocean acidification? Georgia Mantis

Bee venom extraction methods and their effect on potency Gus Opie

Ultraviolet light in the war against bacteria and antibiotic resistance Will Savage

Global recycling efforts: Zinc catalysts for efficient glycolysis of Polyethylene Terephthalate Henry Shortis

The effect of increased dietary energy and protein intake on mammalian skeletal muscle cells Sam Zibaee

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Contents Part 3: Chemistry Synthesis, isolation and purification of 4-iodoprimethamine as an anti-parasitic agent for P. Falciparum and Toxoplasma gondii Josh Botha

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Synthesis of a trifluoromethyl analogue of Pyrimethamine Max Graham

131

Synthesis of a 2-aminothiazole analogue Christian Kemp

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Synthesis of a new trifluoromethyl analogue of MMV006357 for the treatment of Mycetoma Jack Jeffress

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Synthesis of structures with the purpose of creating new analogues of Lopinavir Jonah Mills

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An exploration of the Belousov-Zhabotinsky chemical oscillator within a high school laboratory Isaac Denney

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Physics 2023 Physics research includes three projects building off research in previous volumes of this journal, and one completely novel exploration.

Physics underpins all we observe and the four students who explored this domain explored very different aspects of the physical universe. Michael’s project was the outworking of years of fascination with Lichtenberg figures (the lightning strike, fractal patterns when the surface of wood is electrocuted). It was exciting to find a way to explore this area of interest from a scientific perspective with visually beautiful results stemming from hours of trials enduring through various obstacles. The first of our three Physics projects building of previous work in this journal was from Jeslyn following recommendations from Harry’s 2021 project on chaotic motion in a double pendulum which was also published in the Australian Physics Magazine. Her research has also been submitted to Australian Physics. Max found effective ways to build on Jack’s 2022 project to more deeply explore post-processing methods of annealing 3D printed materials. There is more research to be done in this area and together these students have made valuable contributions. Finally, Bardia was inspired by research from Alex (2019) and Tom (2020) extending the methodology to explore the efficiency of adding a duct to a toroidal propellor. The design of the toroidal propellor already incorporates measures to improve efficiencies over a traditional propellor so it was fascinating to consider the impact of the added duct of varying lengths.

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Salt, wood, and a shocking result – Investigating Lichtenberg figures in wood Michael Ashworth Barker College Purpose: This paper aims to explore the production of Lichtenberg figures in wood due to electrocution from an applied high voltage (12 000 V). Specifically, the experiment investigates the relationship between the salt concentration of the electrolyte applied prior to electrocution on different qualitative and quantitative aspects of the produced burn patterns on the surface of the wood. Design/methodology/approach: 5 solutions of non-iodised table salt (NaCl) at varying concentrations (0, 70, 140, 210 and 280g/L) were applied to the surface of 10 290mmx135mm wood samples (Tasmanian oak). A 12,000 Volt step-up transformer was used to generate voltages high enough to generate Lichtenberg figures on the surface of the wood. Photos of the patterns were taken after the initial burning along with after the samples were processed which involve brushing and sanding away charring and any charcoal. This allowed for qualitative and quantitative observations. Findings: A linear relationship was found between the salt concentration and the width of the main channel length (r=0.98). Additionally, several qualitative observations were observed such as: at a salt concentration of 70g/L the most complex burn pattern was achieved, at salt concentrations of 140 and higher excessive charring occurs, and at extreme concentrations (0, 210, 280gL), patterns sometimes didn't connect. Research limitations/implications: Despite re-occurring pilot testing and trials on various occasions, producing reliable results (even any patterns at all) was found to be extremely difficult. Environmental factors such as temperature and humidity, along with the volume of water and how long it was left to soak impacted the successful trials in unexpected ways. Eventually, successful samples were produced (see Figure 7 and 8) however the intended 4 samples at each concentration were reduced to 2 due to these difficulties. Practical implications: Similar electrical patterns occur in lightning strikes and high-voltage equipment but the formation of these patterns in wood is ill-defined. This paper contributes to the currently limited field of comparing features of Lichtenberg figures under various conditions. Social implications: The production of these figures has recently gained interest in the wider community because of their unique shape and beauty. This paper is aware of the social context of the health risk and highlights precautions to reduce the risk of injury. Originality/value: While there is some literature on Lichtenberg figures, there is no reliable information on Lichtenberg figures in wood. This type of Lichtenberg creation has been more extensively described in blogs, videos, and other non-scientific websites. Keywords: Lichtenberg figures, Dielectric breakdown, Diffusion limited breakdown (DLA), Fractals, concentration Paper type: Research paper

Literature Review Diffusion limited aggregation, Lichtenberg figures and dielectric breakdown Diffusion Limited Aggregations (DLAs) are a growth model. The models follow basic rules involving a sequence of random walkers that become part of the model (Sander, 1986). These patterns can be found throughout nature such as crystal and coral growth, lightning, plant growth (Bourke, 2006), neuron growth (Amitrano et al., 1991) and even galaxy formations. The conceptual understanding of how these patterns are formed is important to the

understanding of this paper. The DLA was first written about in 1981 by T. A. Witten, Jr. and L. M. Sander in "Diffusion-limited aggregation: A kinetic critical phenomenon", where they provide a basic method of the modelling of these objects. Consider a point at the origin or a plane. Imagine a particle that moves out in a random direction of a unit. Now another particle is released, and it is allowed to move in any direction until it is a unit away from the previous points. When this process is repeated it produces a DLA pattern (Sander, 2000), as seen in Figure 1.

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Scientific Research in School Volume 5 Issue 1 2023 Dielectric breakdown is an electrical discharge that produces similar patterns to what is modelled through DLA. If a DLA was to be modelled electrically, there would be a voltage drop with a concentric potential difference around the diode. Dielectric breakdown, however, is a positive and negative point terminal separated by a distance and a burning or charring pattern attempts to complete the circuit by creating a path between them. The term "Lichtenberg figure" was named after the German physicist named, Georg Christoph Lichtenberg first written in his work "De nova methodo naturam ac motum fluidi electrici investigandi" or “On a new method of investigating the nature and motion of an electric fluid” (Lichtenberg, 1778). He was the first person to observe the phenomenon. In 1777, he used a large electrophorus, a machine to produce high voltage electricity, to discharge them on insulating materials to create the figures (Cherington et al., 2003). This was a precursor to the field of plasma physics. Lichtenberg figures can occur under both DLA and Dielectric breakdown configurations, depending on the circumstances of their creation or the origin of the voltage. For example, lightning strikes are a type of Lichtenberg figure but follow a DLA pattern, as it discharges into a grounded source. This present experiment involves voltage supplied by a transformer so it will follow a dielectric breakdown model where the positive and negative terminals are discrete points separated by a distance.

Figure 1: example of a modelled DLA using the abovementioned method with 100,000 particle iterations Source: (Sander, 2000 pg. 204)

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Examples of DLAs in nature and other similarities DLA’s are very common and are found throughout nature and are called Lichtenberg figures (Lichtenberg, 1777). Lichtenberg figures follow a diffusion limited aggregation, and form on insulating material such as wood, glass, resin, ebonite, the ground, and even human skin: Seen in Figure 2 B. These patterns can occur in all three states of matter: solids, liquids and gases. However, for this experiment, we will be looking at solids as they are the easiest to observe and create and will be permanent on the material.

Figure 2: Examples of Lichtenberg figures on different insulating material A - On a golf course from a lightning strike After: (Cherington et al., 2003) B - Discharge on human skin from lightning After: (Cherington et al., 2003) C - Discharge in wood from fractal wood burning After: (Russo et al., 2020) D - Discharge on a thin sheet After: (Wood, 2015)

We also see similar fractal patterns in nature, as seen in Figure 3, such as river delta systems (3A), neurons(3B), blood vessels (3C), tree branches (3D) and ant nests. While these examples are much broader than electrical discharge patterns, though electricity does play a part in neural patterns (Figure 3B), the visual similarities are obvious. These patterns have not been connected to DLA's or their formation, however, they do follow a similar formation method. That is a potential gradient in which a "particle" has a random chance to move in a certain direction. In the case of the river delta, the gradient is a change in elevation and gravity, with the particles being water, as it can move in any random direction. The mathematical relationship between these patterns and DLA's is an area that is worth further investigation.

Scientific Research in School Volume 5 Issue 1 2023 Fractals and fractal dimension DLA's and Dielectric breakdown both exhibit fractallike patterns (Niemeyer et al., 1984) following a Brownian tree shape. Fractals are a type of shape that is infinitely complex. These patterns follow simple patterns which is repeated infinitely many times to create a complex shape which is the same, no matter how far you zoom in or out (Benoit Mandelbrot, 1982). Fractal comes from the Latin word “fractus” which means an irregular surface like that of a broken stone, coined by Benoit Mandelbrot in 1975. Figure 5 shows some famous fractals including the Mandelbrot set, Sierpinski’s triangle, Koch snowflake and Dragon curve. Figure 3: Example of similar branching patterns in nature. A - River delta After: (“Learning Geology: Factors Controlling the Shape of a River Delta”) B - Neuron close up After: (Feja) C - Telangiectasia or spider vein on a knee After: (“What Are Spider Veins? — the Leg Vein Doctor | Brisbane | Varicose and Spider Vein Phlebology Clinic”) D - Tree canopy After: (“Fractals Explained: (Fractal Patterns in Nature) - the Conscious Vibe”)

Conduction and concentration: In this experiment, a salt solution (non-iodised table salt – NaCl) will be used as an electrolyte to aid the conductivity of electricity through the wood samples. A varying salt concentration will be the independent variable of the investigation. Figure 4 shows that the relationship between the concentration of salt in an aqueous solution is directly proportional to the conductivity of the solution. Understanding the relationship of the independent variable (salt concentration), to conductivity informs our understanding of the observations.

A fractal dimension is a way of quantifying the complexity of a shape as a ratio of the change in detail to the change in scale. There are several types of fractal dimensions determined theoretically and empirically. These fractional dimensional do not need to be integer values.

Figure 5: Examples of Fractals A - Mandelbrot set After: (Wikipedia Contributors, “Mandelbrot set”) B - Sierpinski’s triangle After: (Wikipedia Contributors, “Sierpiński Triangle”) C - Koch curve After: (Wikipedia Contributors, “Koch Snowflake”) D - Dragon curve After: (Wikipedia Contributors, “Dragon Curve”)

Scientific Research Question

Figure 4: The plot of the conductivity of NaCl aqueous at various percentages of NaCl mass. Source: (Widodo et al., 2018)

To what extent do different concentrations of a salt solution used to create Lichtenberg figures in wood affect qualitative aspects of these figures, along with quantitative aspects such as the width of the main channel?

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Scientific Hypothesis There will be relationships found between varying salt solutions and the qualitative aspects of these figures. Additionally, there will be a linear relationship between the salt concentration and the width of the main channel.

Methodology Wood sample Production Three 135mm x19mm x2.4m pieces of Tasmanian oak were sourced from a local timber supplier. They were run through a drum sander, removing approximately 1mm from the face of the wood to make one surface level and smooth. All three lengths were run through the sander to ensure consistency across all the pieces. The unsanded side was indicated with a piece of chalk. These lengths were then crosscut on a table saw to 290mm, creating a total of 24 pieces. Solution Five solutions were made from a mixture of distilled water and non-iodised table salt (NaCl). The solutions ranged from 0-280g/L of salt going up in 70g/L increments (0g/L, 70g/L, 140g/L, 210g/L, and 280g/L [saturated]). Salt was measured out on a scale to the approximate amount needed for half a litre (500ml) and then poured into a 500ml reagent bottle. This was repeated for all 5 concentrations, in their reagent bottle. Distilled water was then added filling it up to the 500ml marker on the bottle. Notes Before our experiment, I did some preliminary testing to determine the best method for creating the patterns. Initially there were issues generating the patterns, but after some experimentation I found that pre-soaking the wood with the salt solution, then allowing it to dry, and then rehydrating it with the same solution created the desired patterns. I ended up completing 10 tests, two for each salt concentration. Pre-soaking: For the pre-soaking, a sample was held vertically so that the sanded face was perpendicular to the ground. 10 squirts of the designated salt concentration were put on the surface, such that the maximum amount of water was put on the piece of wood. The wood was then marked to indicate which solution was applied to it. Once wetted, the water was spread using a finger to ensure an even coating of the solution. The wood was then left in a shaded area to dry out. Steps 1-5

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were repeated for all 10 samples, ensuring two wood samples per salt concentration were created. Burning For the burning, all 10 wood samples were collected, and a clear level surface was set up such to ensure that the sample was stable and level with the transformer next to it. Each wood sample was rehydrated with the appropriate salt concentration at the same time in pairs using the same procedure described in the soaking section (steps 1-4). The water was allowed to soak in for 2 minutes with the excess water then wiped off with paper towel. The wood was then placed next to the transformer and the diodes were placed on the surface. The two diodes were placed on opposite corners approximately 30mm diagonally from either edge. The transformer was then turned on and allowed to run until the pattern connected or nothing happened for 15 seconds. To ensure the safety of the tester a meter distance was maintained from the transformer and diodes. After the transformer was switched off and unplugged at the wall, one hand was used to move the diodes, and any residual charge was grounded on the transformer. This ensured there was no potential for electrocution. Photos of each sample were taken in even lighting conditions, with a Sony A7IV mirrorless camera. Cleaning To clean the samples, a hard brush with plastic bristles was used to scrub the surface and loosen any charcoal or carbon on the surface of the pattern. All 10 samples were scrubbed, and the loose dust was brushed off. After, brushing the pieces were then placed in a bucket of soapy water and again brushed. After that steel wool was used to remove the charring marks. This process was to highlight the channels and didn’t intend to remove all the charring, just make it lighter so it is easier to see the channels. The samples were then left to dry in the sun. The samples were then wet again and photographed in the same manner. The samples when wet gave the image higher contrast, making it easier to see the colour differences. The samples were also measured to find the largest main channel. A ruler was used to find the width closest to the nearest half millimetre.

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Results Concentration (g/l)

Test 1:

Test 2:

140

210

280

Figure 6: Photography of samples immediately after test, from salt concentrations ranging 0-280g/L

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Concentration (g/l)

Test 1:

Test 2:

140

210

280

Figure 7: Photography of samples after sanding, from salt concentrations ranging 0-280g/L

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Scientific Research in School Volume 5 Issue 1 2023 Table 1: Main channel width by sample salt concentration

Concentration (g/l) 0 70 140 210 280

Main channel width (mm) Test 1: Test 2: Average: 1.50 2.00 1.75 3.00 3.00 3.00 4.00 2.50 3.25 Outlier 4.00 4.00 4.50 Outlier 4.50

Max width of main channel (mm)

Main Channel Width vs Concentration 5 4 3 2 1 0

140

210

280

Salt concerntration (g/L)

Figure 8: Graph of concentration vs Max Width of Main channel. Test 1: Blue Column, Test 2: Grey Column, Average: Line

Discussion

current could join. For the high salt concentrations (Figure 6 G and J, 210 and 280g/L respectively), excessive salt crystallisation build-up was prominent on the wood samples. A possible reason for this is that the amount of salt caused the circuit to remain open and therefore the pattern was not produced. Another possible reason that the patterns did not form fully is the moisture content of the wood was too low for the process to occur. Water plays an important role as it allows the current to flow through the wood. However, during the burning process, the water is boiled off from the heat of the experiment. Observation 3: When excessive charring occurs, the main channel tends to be in a straight line, producing little to no branching. This is most evident in figure 7 H where the charring is visible along with the main channel. Additionally, this is also seen in samples E, F and I. Following on from observation 1, as the arch goes over the surface it burns a channel following the path of least resistance rather than a random walk expected from DLA, this would be along the grain. The grain of the wood can be seen as microscopic straws which run up and down forming the grain. When these are soaked, they absorb the liquid. For the current the easiest path would be through these channels as it has the least resistance, then goes across the grain to connect, which is what we see.

Qualitative Observations Observation 1: At salt concentrations of 140 and higher excessive charring occurs, seen in Figures 6 E, F, H, and I. After pre-soaking them it was observed that salt began to crystalise on the surface of the samples. Additionally, after reapplying the salt solution the salt crystals did not dissolve. During the testing, the arch was observed to go over the surface of the wood for these areas rather than through the wood which was seen in the non-charring sections. To combat this in the future, having appropriate measures in place to remove or reduce the crystalised salt should be implemented, such as scrapping the surface with a hard edge or using sandpaper to remove excess salt.

Observation 4: At 70g/L the most intricate branching occurred (Figure 7 D). Several factors may contribute to this intricate branching, however, it is probably a mix of multiple factors. The salt-to-water ratio was probably the most effective, having enough salt that the patterns would branch and enough water that it was able to complete the circuit and produce the pattern. However, due to the random walk nature of DLA’s, this is not a definitive result and is just an observation of this group of samples. Additionally, this idea of intricate is somewhat subjective in this context and further quantitative analysis, in the calculation of the fractal dimension, would need to occur for actual conclusions to be created.

Observation 2: At concentration extremes (0, 210, 280g/L) patterns sometimes didn't connect, seen in Figure 6 A, B, G and J. For the low salt concentrations (Figure 6 A and B, 0g/L) a possible reason for the pattern to not finish was that there was not enough salt. As previously mentioned, the salt-to-water ratio is important in the production of these patterns. During the experiment, the water boiled off and the circuit broke before the

Quantitative observations As seen in Figure 8, there is a linear relationship between salt concentration and the width of the main channel. The most likely reason for this is probably to do with the time it took for samples to complete. At lower salt concentrations the time taken for the paths to connect was low, meaning the amount of energy in the form of electrons going through the channels would be less than those with long burn

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Scientific Research in School Volume 5 Issue 1 2023 times. This would lead to a smaller channel. As the salt concentration increased for branching sections, the channel would constantly change so no large channels were produced. For excessive charring sections, it took a long time and consisted of one channel, explaining the thick channels we see for those samples. Two outliers were discounted as their patterns did not connect, so the channel was not fully formed. A Pearson's Correlation Coefficient test was performed resulting in a r-value of 0.98. This suggests a very strong positive correlation between these two variables, however, does not suggest causation as several spurious variables may cause it. Challenges and process During the initial testing stages of this experiment, the desired patterns were not produced in the manner that was anticipated. The patterns would only result in excessive charring, and even more strangely changing the location seemed to affect it. Through testing and trial, several variables were found that changed the creation of these figures and explained why the difference in location changed the pattern. Humidity seemed to influence the effective production of the patterns on the wood samples. When doing the initial testing, the experiment was run on overcast days and it resulted in excessive charring. This may be due to the difference in moisture between the wood and air being small enough that it was easier to go over the surface rather than through it, which is what is desired. Another factor that seemed to affect the production was the amount the salt was able to soak into the wood. Through testing, it was found that saturating the surface and letting it dry and then reapplying the solution was the most effective way of applying the solution. By pre-soaking it allowed an initial amount of salt to be absorbed into the wood. When the electrolyte was reapplied the salt in the wood was redissolved and seeped deeper into the wood, saturating the sample with more salt evenly throughout. Improvements and areas of research This study could have been improved by obtaining additional quantitative data documenting the time it took for the pattern to form, a comparison between the area of charring versus the channels through image analysis and potential calculation of the fractal dimension of the samples, to calculate the most intricate of the samples. Having greater control over the humidity would also remove any effect that this has on the creation of the figures. This could be achieved running the experiment in a humidity-

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controlled room. Additionally, more testing on different methods for applying the salt solutions to make it more consistent across samples would have been beneficial. This could be done by potentially soaking the samples in the solution for a set period. There are several different areas of research associated with this experiment which could be explored. For example, testing the affect different density woods and humidity have on the creation of these patterns and the conditions required for excessive charring to occur.

Conclusion In my research, I investigated Lichtenberg figures. Lichtenberg figures are a type of electrical discharge pattern that occurs in insulating material when exposed to high voltages, which follow a fractal pattern. This paper examined these patterns in wood samples using varying salt solution concentrations as an electrolyte and what effect it has on aspects of these patterns. These aspects were mainly qualitative in nature highlighted through visual images of these samples to create relationships, however quantitative data documenting the width of the main channel was collected. These patterns were created on small 290mm long wood slides with the aid of a 12,000 V step-up transformer. A total of 10 samples where produced, two for each concentration (0-280g/L in 70g/L increments). Photos of these samples were taken after the initial burning then after cleaning the samples, removing the carbon with a brush, and washing it. A linear relationship was found between the width of the main channel and salt concentration (r=0.98). Additionally, relationships between salt concentration and other aspects such as excessive charring, the shape of the pattern, intricacy and completion were found. This supported my hypothesis that there would be a relationship in visual aspects of the patterns, and that the salt concentration was proportional to the width of the main channel.

Acknowledgements I want to extend my thanks to Mr Cameron Dearn whom I consulted with before and during the project and who was graciously provided his insights and knowledge before he went on long service leave. I would also like to thank the Science Extension teachers, in particular Dr Matthew Hill who was my project supervisor and expertly guided me through the project. I extend my thanks to the technology department who allowed me to use their equipment

Scientific Research in School Volume 5 Issue 1 2023 to process my wood sample, in particular Mr Phil Barden who supervised me using the equipment.

impedance spectroscopy methods. AIP Conference Proceedings. doi:https://doi.org/10.1063/1.5062753.

References

Wikipedia Contributors (2019a). Mandelbrot set. [online] Wikipedia. Available at: https://en.wikipedia.org/wiki/Mandelbrot_set.

Sander, L.M. (1986). Fractal growth processes. Nature, 322(6082), pp.789–793. doi:https://doi.org/10.1038/322789a0.

Wikipedia Contributors (2019b). Sierpiński triangle. [online] Wikipedia. Available at: https://en.wikipedia.org/wiki/Sierpi%C5%84ski_triangle.

Bourke, P. (2006). Constrained diffusion-limited aggregation in 3 dimensions. Computers & Graphics, 30(4), pp.646–649. doi:https://doi.org/10.1016/j.cag.2006.03.011.

Wikipedia. (2020). Koch snowflake. [online] Available at: https://en.wikipedia.org/wiki/Koch_snowflake.

Amitrano, C., Coniglio, A., Meakin, P. and Zannetti, M. (1991). Multiscaling in diffusion-limited aggregation. Physical Review B, 44(10), pp.4974–4977. doi:https://doi.org/10.1103/physrevb.44.4974.

Wikipedia. (2021). Dragon curve. [online] Available at: https://en.wikipedia.org/wiki/Dragon_curve.

Sander, L.M. (2000). Diffusion-limited aggregation: A kinetic critical phenomenon? Contemporary Physics, 41(4), pp.203–218. doi:https://doi.org/10.1080/001075100409698. Georg Christoph Lichtenberg (2022). First treatise containing general experiments on a new method for researching the nature and movement of electrical matter presented at the public meeting of the Royal Society of Sciences on 21 February 1778. 13(1), pp.17–34. doi:https://doi.org/10.1386/pop_00037_1. Cherington, M., Olson, S. and Yarnell, P.R. (2003). Lightning and Lichtenberg figures. Injury, 34(5), pp.367– 371. doi:https://doi.org/10.1016/s0020-1383(02)00313-3. Russo, R.M., Pumiglia, L., Bettencourt, A.P., Roman, J. and Vercruysse, G. (2021). Shocked Though the Heart and YouTube Is to Blame—The Rising Incidence of Accidental Trans-cardiac Electrocution From Do-It-Yourself Fractal Wood Art, and a Call to Action. 42(2), pp.236–240. doi:https://doi.org/10.1093/jbcr/iraa172. Wood, M. (2015). Charging and Discharging of Lichtenberg Electrets. [online] Available at: https://www.proquest.com/openview/f99f8f2116a9eec093 b29131afad1822/1?pq-origsite=gscholar&cbl=18750. Learning Geology. (2019). Learning Geology: Factors Controlling the Shape of a River Delta. [online] Available https://geologylearn.blogspot.com/2019/04/factorsat: controlling-shape-of-river-delta.html. Feja, Koto (2020). Neuron cell close-up view stock photo. [Online image] istockphoto. Available at: https://www.istockphoto.com/photo/neuron-cell-close-upview-gm1222606221-358815424. The Leg Vein Doctor. (n.d.). What are Spider Veins? — The Leg Vein Doctor | Brisbane | Varicose and Spider Vein Phlebology Clinic. [online] Available at: https://www.thelegveindoctor.com/spider-veins. The conscious vibe. (2021). Fractals Explained: (Fractal Patterns in Nature) - the Conscious Vibe. [online] Available at: https://theconsciousvibe.com/this-is-whyfractals-are-so-important-most-people-in-the-world-dontknow-this/. Widodo, C.S., Sela, H. and Santosa, D.R. (2018). The effect of NaCl concentration on the ionic NaCl solutions electrical impedance value using electrochemical

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The extended boundary of chaos: Testing the relationship between total energy and chaotic tendencies of a double pendulum system Jeslyn Tan Barker College Purpose: This paper aims to investigate the impact of increasing the weights of the two masses on a double pendulum, in order to determine the relationship between the system’s total energy and its chaotic tendency (as indicated by the Lyapunov exponent). Design/methodology/approach: Due to the chaotic nature of a double pendulum system, it was necessary to use a computational simulation to ensure reliable results. A simulation by Shin (2021) was selected and amended to test a total of 3249 different double pendulum configurations, to find and compare total energies to Lyapunov exponents. The effect of increasing both masses (the top and bottom mass) on increasing energy and Lyapunov exponent was determined and analysed. Findings: It was found that in the case where both masses are of equal value, there is no relationship between the total energy of the system and its chaotic tendency (regardless of the increasing both masses, the Lyapunov exponent never changed). Increasing the upper and lower masses from 0.5kg to 29.0kg, continually increased total energy (though by different amounts) but chaotic motion was never achieved (as indicated by the Lyapunov exponent remaining negative). However, increasing the lower mass led to motion closer to the boundary of periodic and chaotic motion. This suggests that total energy is not related to a double pendulum’s chaotic tendency in all cases, and thus, cannot be the sole causation of chaotic motion. Research limitations/implications: While ultimately none of the situations tested in this project resulted in chaotic motion, this challenges current literature that suggests that increasing energy results in chaotic motion. This experiment found that the particular situation where a pendulum of length ratio, 1:2, with varying masses, chaotic motion is not dependent on total energy of the system. Practical implications: The study of the double pendulum allows for a practical understanding of non-linear dynamics, chaos theory, and thus, chaotic systems. Originality/value: Extant research in this journal suggests that energy (as opposed to pendulum length or mass ratio) was the determinant of whether a motion was chaotic or periodic (Breden, 2021). Whilst other papers have found a relationship between total energy and chaotic tendency when the time derivative of generalized momenta and angular velocity is altered (Bilargi & Jami, 2016), extant research suggests that there has not been any tests regarding this relationship whereby the time derivative of generalized momenta and angular velocity are substituted with the masses of the point masses of the double pendulum, to exhibit further situations where total energy is related to chaotic motion. Paper type: Research paper

Literature Review The Double Pendulum (DP) “A double pendulum (DP) consists of two-point masses attached on a rigid, weightless rod, with the top mass connected to the second point mass with a second rigid, weightless rod” (Breden, 2021, p. 51). The DP is a Hamiltonian system, inferring that total energy is conserved through the exchange of kinetic and potential energy (Schaft, 2006), and the law of conservation of energy states that under ideal conditions without external forces, the system will continue its trajectory forever, and thus, by using a simulation the system can be analysed over a duration of any desired time. Interestingly, this seemingly

simple system displays complex behaviours with respect to its chaotic tendencies in certain conditions.

Figure 1: Diagram of the simple double pendulum After: (‘Double Pendulum’, 2020)

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Scientific Research in School Volume 5 Issue 1 2023 Chaos Theory and the Lyapunov Exponent (Eigenvalue) Chaotic motion refers to unpredictable behaviour of a system due to sensitivity to initial conditions, thus, by introducing minor changes to its initial conditions, the system yields drastically different outcomes (Britannica, 2023). However, it is important to note that the motion of a DP is predictable in a controlled environment, contingent upon their initial conditions, entailing the creation of simulations based on differential equations to accurately reproduce its motion. The quantitative statement of chaotic motion lies in the Lyapunov exponents (λ), which characterises the rate of separation of infinitesimally close trajectories in phase space (Breden, 2021). Thus, if the rate of divergence is positive (λ > 0), then the trajectories will diverge exponentially, producing chaotic tendencies. Alternatively, if the rate of divergence is negative (λ < 0), the trajectories will converge to periodic motion (Calvão and Penna, 2015). Moreover, if the exponent is very close to zero or approaching periodicity, then the system is in a quasiperiodic or “almost periodic” state (Bilargi & Jami, 2016). The Lyapunov exponent equation is as shown in Equation 1. Equation 1: Lyapunov exponent as a function of time Source: (Danforth, 2017)

Hamiltonian Mechanics and Calculating Total Energy Given that a DP is a Hamiltonian system (a system where the total energy is constantly being exchanged and conserved), a method of calculating total energy in the system is through the Hamiltonian function (Indiati, Saefan and Marwoto, 2016). Similarly to Newtonian and Lagrangian, all three methods describe the motion of systems, and the same physical laws/problems can be described/solved through these methods, however unlike Newtonian or Lagrangian mechanics, Hamiltonian mechanics describes the motion of systems in terms of generalized coordinates and momenta, as opposed to position, acceleration, and velocity (Newtonian), or positions and generalized speeds (Lagrangian). The Hamiltonian function is essentially H = K + V where K denotes kinetic energy, and V denotes potential energy (Zain, 2019). The Hamiltonian of a double pendulum is given by the equation in Equation 2, where 𝑚𝑚𝑚𝑚 denotes mass, 𝑙𝑙𝑙𝑙 is length, 𝜃𝜃𝜃𝜃̇ is angular velocity.

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Equation 2: Hamiltonian equation for a DP Source: (Gupta et al., 2014)

Lagrangian Mechanics and the Euler-Lagrange Differential Equation Lagrangian mechanics was used to calculate the motion of the DP in the simulation. As mentioned previously, Lagrangian mechanics describes the motion of systems in terms of positions and generalized speeds. This infers that by using Lagrangian mechanics, constraint forces such as the tension of the strings do not need to be considered (as Newtonian mechanics does), and thus, is preferred in this situation. When simulating/solving the DP, or non-linear dynamic systems, there are several methods of doing so, such as the Range-Kutta method, the Adam Bashforth’s method, Euler Lagrange, and the Adams Moulton method (Calvão and Penna, 2015). Using these differential equations, the motion of the DP can be worked out using a simulation/computer program. The simulation provided uses the Euler-Lagrange differential equation given its simplicity to compute numerically. The Euler-Lagrange formula is expressed in Equation 3, and it is derived from the Lagrangian function of the DP which is L = K – V, where K denotes kinetic energy, and V denotes potential energy (Breden, 2021) Equation 3: Euler-Lagrange equation for a DP Source: (Deyst, 2003)

Extant Research Testing the Relationship Between Total Energy and Chaotic Tendencies of a Double Pendulum Breden (2021) builds on Gupta et al.’s (2014) results to increase the precision of the boundary of chaotic tendency in the specific situation of changing the length ratio on a DP system. Through numerical methods he increased the precision of the boundary of quasiperiodic state leading into a chaotic state, by a factor of 64 (1:2.34375 and 1:2.375). This was accomplished through the application of the bisection method on the previously predicted boundaries of chaos, as a result of the length ratio boundaries of 1:2 and 1:3. However, he does not explicitly mention the effects of energy on the chaotic tendency of the DP

Scientific Research in School Volume 5 Issue 1 2023 in his literature review other than in his discussion where he proposes that Energy may be the only or one of the reasons behind the change of chaotic tendency. Thus, the purpose of this paper was to discern whether energy was the main reason behind the change of chaotic tendency.

of state shown in the table to the left. Thus, this paper aims to find whether it is solely the change of energy altering the chaotic/periodic state or if the length and mass do effect the change of chaotic tendency as well.

Bilargi and Jami (2016) explored the reasoning behind the shift of chaotic tendencies due to energy levels and tested four energies to support their theory. They used a simulation, specifically MATLAB’s ode45 to analyse the DP system numerically, solving the system using the four first-order Hamilton’s Equations of Motion (non-linear differential equation to solve for a DP). In order to test different energies, length of the rigid weightless rods, and the masses remained constant, however, the initial angular velocity (𝑞𝑞𝑞𝑞̇1 , 𝑞𝑞𝑞𝑞̇2 ) and the time derivative of generalized momenta (𝑝𝑝𝑝𝑝̇1 , 𝑝𝑝𝑝𝑝̇2 ) for both pendula were altered. The first test was done at a low energy of 0.7809J, where the initial conditions were y = [𝑞𝑞𝑞𝑞̇1 , 𝑞𝑞𝑞𝑞̇2 , 𝑝𝑝𝑝𝑝̇1 , 𝑝𝑝𝑝𝑝̇2 ] = [0.2,0.2828,0,0], and the Lyapunov exponent is -3.426, deeming the system periodic. At the second energy level tested at 1.2807J, and initial conditions y = [0.7,0.3825,0,0], the system is quasiperiodic where the average Lyapunov exponent is -1.203. At an increased energy level of 29.4J, and initial conditions of [ℼ,ℼ, 0.5,0], the exponent is 1.906, inferring that the system is chaotic. Lastly, at a high energy of 104.25J at initial conditions [ℼ,0,0.5,0.5], the system returns to a quasi-periodic state denoted by its exponent of 0.4320. Based on their findings of the relationship between chaotic tendencies and total energy of a system when altering initial angular velocity and the time derivative of generalized momenta, this paper was aimed at testing whether there was a relationship between total energy and the change of chaotic tendencies when mass was the independent variable.

As the total energy of a double pendulum increases (by increasing either or both masses of the DP), are there any trends in relation to its chaotic tendency (calculated quantitatively by the Lyapunov exponent)?

Gupta et. al (2014), aimed to study the chaotic motion of the double pendulum, specifically, the relationship between the mass and length ratio of the double pendulum (DP), and the chaotic tendency of the system. To measure the changes in chaotic tendency, they used the Lyapunov exponent, a time series (for angular velocity), as well as Poincare maps to convey the motion of the DP. They concluded that chaotic tendency is directly proportional to the increase of mass and length ratio. This article explores the change of multiple variables on each or both pendula, however, it does not numerically specify the significance of the increase in mass and length. Furthermore, it tested very few points, and thus, it may not be accurate in the boundaries of the change

Scientific Research Question

Scientific Hypothesis As the total energy of a 1:2 length ratio double pendulum increases by changing either or both masses, the Lyapunov exponent would gradually move from negative (indicating periodic motion) to positive (indicating chaotic motion).

Methodology Part 1: Altering the Simulation A simulation was sourced from Shin (2021) and amended to conduct the experiment. The simulation was written in Python and included the EulerLagrange differential equation to simulate a DP and the Lyapunov exponent calculations and followed a logical iterative numerical process (Appendix 1). Shin (2021) offers further details on the derivation, implementation, and interpretation of the Lyapunov exponent and the Euler Lagrange Differential equation in the simulation used for this project. A line of code calculating the total energy using the Hamiltonian Equation was inserted. This calculation was based on the initial conditions and assumed constant for the duration of the motion as there were no external forces such as air resistance or friction to remove energy from the system. To test the reliability of the simulation with the additional calculations, the initial conditions: 𝑚𝑚𝑚𝑚1 = 1, 𝑚𝑚𝑚𝑚2 = 5, 𝑙𝑙𝑙𝑙1 = 1, 𝑙𝑙𝑙𝑙2 = 1, were inputted which produced a Lyapunov exponent of 0.0399, and a total energy of -104.68J, which conformed to the findings of Gupta et al., (2014). Following the test of the simulation’s reliability, the initial conditions for the experiment conducted were input as follows in Table 1.

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Scientific Research in School Volume 5 Issue 1 2023 Table 1: Initial conditions as input in the simulation

Parameter Length of the top pendulum rod Length of bottom pendulum rod Local acceleration due to gravity Initial angle of first rod Initial angle of second rod Initial angular velocity of first rod Initial angular velocity of second rod Simulation running time Upper Mass

Symbol 𝐿𝐿𝐿𝐿1

Value 1𝑚𝑚𝑚𝑚

𝑔𝑔𝑔𝑔

9.81𝑚𝑚𝑚𝑚𝑠𝑠𝑠𝑠

Lower Mass

𝑚𝑚𝑚𝑚2

𝐿𝐿𝐿𝐿2 𝜃𝜃𝜃𝜃1

2𝑚𝑚𝑚𝑚

Results −2

0.2 radians

𝜃𝜃𝜃𝜃2

0.2828 radians

𝜃𝜃𝜃𝜃1̈

0 radians

𝜃𝜃𝜃𝜃2̈

0 radians

t_stop

150 seconds

𝑚𝑚𝑚𝑚1

Array from 0.5kg29.0 kg Array from 0.5kg29.0 kg

Moreover, Shin (2021) altered both masses as well as the initial angle of both rods. For this experiment only the mass of each bob was modified. Part 2: Generating the Lyapunov Exponent and Hamiltonian Equation for Differing Upper and Lower Masses 3249 tests with each mass independently varying from 0.5-29kg (at graduations of 0.5kg) was conducted whereby each test produced a value for its Hamiltonian equation (total energy) and Lyapunov exponent (represents potential chaotic nature). In a manner similar to Figure 7 of Shin (2021), to effectively represent so many results, two heatmaps were produced. The first heatmap (Figure 2) was generated to find three extreme situations (Lyapunov exponent varies greatly as seen in the difference of colours in the three situations). The second heatmap (Figure 3) was generated to understand the change in total energy throughout the entire experiment. These heatmaps were combined to effectively test the hypothesis, by selecting three special cases to show the relationship between total energy and chaos (which are depicted in three lines superimposed in Figures 5 and 6). The first situation selected from the first heat map involved increasing lower mass from 0.5kg - 29.0kg and keeping upper mass constant at 0.5kg (as it has a high Lyapunov exponent comparatively as seen in Figure 2). The second situation selected was, increasing upper and lower

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mass at a constant rate (as it has a low Lyapunov exponent). The third situation involved increasing upper mass from 0.5kg -29.0kg, whilst lower mass was kept constant at 0.5kg.

The Lyapunov Exponent and total energy heat map (based on the changing masses) was generated.

Figure 2: Heatmap of Lyapunov Exponent values dependent for the 3249 tests of varying masses. The three lines on the figure indicate the three special cases that will be plotted in Figure 4.

Figure 3: Heatmap of total energy values dependent for the 3249 tests of varying masses. The three lines on the figure indicate the three special cases that will be plotted in Figure 4.

Using the data from these heat maps, a plot was created displaying the relationship between the Lyapunov exponent and total energy of the system (Figure 4) at the three extreme situations: when upper mass is 0.5 kg, and lower mass is varied from 0.5kg – 29.0kg, when lower mass is 0.5kg and upper mass

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Figure 4: A plot of Lyapunov Exponent (from Figure 2) as a function of total energy (from Figure 3) when increasing lower mass, when increasing upper mass, and when upper mass is equivalent to lower mass.

is varied from 0.5kg – 29.0kg, and when upper mass is equal to lower mass (exhibited in red in Figure 2). Increasing only lower mass: there is a visible increasing and curved relationship between total energy and its respective Lyapunov exponent. Initially, it seems as though the plot will potentially exceed periodic motion until 400J, where the trend of the plot appears to approach chaos but converges (like an asymptote) prior to reaching (λ > 0). Increasing only upper mass: initially, it follows a similar pattern to the increasing only lower mass plot, however, as energy increases, there is a prevalent decline, inferring that in this condition there is a different relationship to the plot of upper mass = 0.5kg. Increasing upper and lower masses at the same rate: There seems to be no relationship between the increase of total energy and the Lyapunov exponent. A notable feature of these results is that although the situation where lower mass increases towards 29.0kg and upper mass is kept constant at 0.5kg approaches chaotic motion, none of the plots reach chaotic motion (λ > 0). Moreover, the plots reach different maximum total energies as defined by the mass array (0.5kg – 29.0kg) where the plot of upper mass = lower mass reaches the greatest total energy (1138.705J), followed by the situation where upper mass is kept constant at 0.5kg, and lower mass is

ranging from 0.5kg – 29.0kg, reaching 855.0784J, and lastly, the situation where the upper mass is changing from 0.5kg – 29.0kg, and the lower mass is kept constant at 0.5kg, reaching 302.648J.

Discussion As seen in in Figure 4 the results did not support the hypothesis that as the total energy of a 1:2 double pendulum increases by changing either or both masses, the Lyapunov exponent would move from negative (indicating periodic motion) to positive (indicating chaotic motion). This is particularly evident in the case where upper mass = lower mass, as when the total energy increases, the Lyapunov exponent remains constant (at λ = -0.000933). This suggests that the total energy of the system may not be the causation of chaotic tendencies in every case, acting as evidence against the theory that total energy of a system is the determinant of chaotic tendencies. Notably, as evident in Figure 4, chaotic motion was never achieved in any of the 3249 cases tested. This raises the question whether varying masses of a DP can achieve chaos. This was an assumption derived from Gupta et al., (2014), who explores the relationship between changing mass and length ratios to chaotic motion. To which chaotic motion was achieved. Moreover, it was assumed that the distribution of mass along each pendulum would affect its rotational inertia, resulting in chaotic

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Scientific Research in School Volume 5 Issue 1 2023 motion. However, as both variables were altered by Gupta et al., (2014), varying masses alone may not be adequate to achieve chaotic motion.

project. I would also like to thank Harry Breden for providing advice and assistance when performing the experiment.

Although this does not discredit the disassociation of total energy and chaotic tendencies found in this paper, further research into this relationship could entail testing this relationship by varying length ratios which can induce chaotic tendencies in a DP, as verified by Breden (2021). Alternatively, testing whether the identical total energies produced by the change of length ratios in a DP and total energies produced by the time derivative of generalised momenta and angular velocity (as conducted by Bilargi & Jami (2016)), produce consistent Lyapunov exponents.

References

Through this proposed research, it may be concluded that the total energy of a DP may be related to its chaotic motion only in certain conditions, another variable other than energy may be found to be the primary justification of its changes in chaotic motion, or it may be found that the likely reason for the DP’s chaotic motion is a combination of a multitude of factors (Chen, 2008).

Conclusion This research project explored the relationship between total energy and chaotic tendencies of the double pendulum system. Through the use of an amended simulation of a double pendulum system by Shin (2021), the chaotic tendencies of the system were analysed numerically through the Lyapunov exponent. Moreover, given the DP’s Hamiltonian nature, the total energy was calculated through the Hamiltonian equations of motion. Previous research suggested that there was a relationship between total energy of a double pendulum the chaotic behaviour of the DP. However, upon analysing the 3249 sets of data on energy and Lyapunov exponent, it is evident that this is not always the case, apparent in (Figure 4) which exhibits that when the upper mass of the pendulum equals the lower mass of the pendulum, as total energy increases, the Lyapunov exponent remains constant. This indicates that the hypothesis was not supported, as while increasing the masses of a 1:2 double pendulum did increase the total energy, there were no tested circumstances when chaotic motion was observed.

Acknowledgements I would like to thank Dr Matthew Hill for providing assistance and advice throughout the duration of my

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Barnett, A. (2009). Numerical Analysis of the Dynamics of a Double Pendulum. Dartmouth College, [online] pp.1– 14. Available at: https://citeseerx.ist.psu.edu/document?repid=rep1&type=p df&doi=8e89690196235601fdc3db3d4e4e87fd33822dbd. Bilgari, H. and Jami, A.R. (2016). The Double Pendulum Numerical Analysis with Lagrangian ad the Hamiltonian Equations of Motion. S. International Conference on Mechanical and Aerospace Engineering, London, United Kingdom, pp.1–12. Breden, H. (2021). The Boundary of Chaos: An Investigation into the Length Ratio Dependent Chaotic Dynamics of a Planar Double Pendulum. 2021 Scientific Research in School, 3(1), pp.41–51. Calvão, A.M. and Penna, T. (2015). The double pendulum: a numerical study. European Journal of Physics. [online] Available at: https://www.semanticscholar.org/paper/Thedouble-pendulum%3A-a-numerical-studyCalv%C3%A3oPenna/13e719708abb510fa60fa6e107fcc626b525c666 [Accessed 31 Jan. 2023]. Chen, J. (2008). Chaos from simplicity : an introduction to the double pendulum. ir.canterbury.ac.nz. [online] Available at: https://ir.canterbury.ac.nz/handle/10092/12659 [Accessed 18 Jun. 2023]. D’Alessio, S. (2022). An analytical, numerical and experimental study of the double pendulum. European Journal of Physics, 44(1), p.015002. doi:https://doi.org/10.1088/1361-6404/ac986b. Danforth, C. (2017). Lecture 15: Double pendulum & Sharkovskii’s Theorem. [online] www.youtube.com. Available at: https://www.youtube.com/watch?v=r74SvaQomrw [Accessed 18 Jun. 2023]. Gupta, M.K., Bansal, K. and Singh, A.K. (2014). Mass and Length Dependent Chaotic Behavior of a Double Pendulum. IFAC Proceedings Volumes, 47(1), pp.297– 301. doi:https://doi.org/10.3182/20140313-3-in3024.00071. Hans Jürgen Korsch, Hans-Jörg Jodl and Hartmann, T. (2007). Chaos. Springer Science & Business Media. Indiati, I., Saefan, J. and Marwoto, P. (2016). Numerical Approach of Hamilton Equations on Double Pendulum Motion with Axial Forcing Constraint. [online] Available at: https://www.researchgate.net/publication/308532784_Nu merical_Approach_of_Hamilton_Equations_on_Double_ Pendulum_Motion_with_Axial_Forcing_Constraint [Accessed 18 Jun. 2023]. Jiwan Shin (2023). Double-Pendulum-Lyapunov/Effect of pendulum mass on the chaos motion of a double pendulum - Jihwan Shin.pdf at 053e2cf24e3ef20fbb500136340aa87c97f12045 ·

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shinben0327/Double-Pendulum-Lyapunov. [online] GitHub. Available at: https://github.com/shinben0327/Double-PendulumLyapunov/blob/053e2cf24e3ef20fbb500136340aa87c97f1 2045/Effect%20of%20pendulum%20mass%20on%20the %20chaos%20motion%20of%20a%20double%20pendulu m%20-%20Jihwan%20Shin.pdf [Accessed 18 Jun. 2023]. Rafat, M.Z., Wheatland, M.S. and Bedding, T.R. (2009). Dynamics of a double pendulum with distributed mass. American Journal of Physics, [online] 77(3), pp.216–223. doi:https://doi.org/10.1119/1.3052072. Schaft, A. van der (2006). Port-Hamiltonian systems: an introductory survey. [online] research.utwente.nl. Available at: https://research.utwente.nl/en/publications/porthamiltonian-systems-an-introductory-survey [Accessed 18 Jun. 2023]. The Editors of Encyclopaedia Britannica (2019). chaos theory | Definition & Facts | Britannica. In: Encyclopædia Britannica. [online] Available at: https://www.britannica.com/science/chaos-theory. Zain, S. (2019). ShieldSquare Captcha. [online] iopscience.iop.org. Available at: https://iopscience.iop.org/book/mono/978-0-7503-2076-4 [Accessed 18 Jun. 2023].

Appendix Appendix 1: Logic of Shin (2021) simulation

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Post-processing of FFF non-polar polymer composites using microwave annealing Max Hanley Barker College

Purpose: This paper aims to validate microwave annealing as a developing alternative to conventional 3D printing post-processing methods. The paper will be focusing on samples produced using acrylonitrile-butadiene-styrene (ABS) carbon composite. Design/methodology: Standardised ASTM-D638 dog bone specimens were 3D printed using an Anycubic Vyper™ FFF 3D printer and 3DX™ ABS carbon composite feedstock. Three treatment groups, each consisting of 10 specimens, a control, conventional process, and microwave annealing, were tested for tensile strength using a load-testing machine. Findings: It was found that for both 0.15mm layer height (t(8) = 2.60, p = 0.03 < 0.05) and 0.3mm layer height (t(8) = 2.39, p = 0.04 < 0.05), the interlayer tensile strength of the specimens was significantly improved via microwave annealing with consistent results for conventional processes. Limitations/implications: It was identified that limitations in the material chosen and/or the methodology led to results lower than expected based on manufacturer specifications. Improvements to the procedure to remove random errors were also identified. Practical applications (implications): This post-processing method has the potential to improve the strength significantly of FFF 3D printed parts and make them more isotropic in a timelier manner that does not negate the already existing time benefits of FFF printing. Which would greatly add to the technology and broaden its possible applications. Originality/value: This is, to the author’s knowledge, the first time that a commercially available ABS-CF composite has been tested with microwave annealing. Keywords: 3D printing; annealing; carbon composite filament; inter-layer tensile strength; microwave annealing. Paper type: Research paper

Literature Review 3d Printing Introduction As a technology, 3D printing is an umbrella term that describes several forms of additive manufacturing. Depending on the process, it can be used to create complex geometric parts using materials ranging from titanium to glass fibre (Blok et al. 2018). The most common form of 3D printing, and the focus of this paper, is known as fused filament fabrication (FFF. Also known by its genericised trademark, Fused Deposition Modelling, FDM) (Blanco 2020). FFF printing consists of a heated nozzle that melts a polymer-based filament feedstock to extrude a line of the given material (see Figure 1). These lines then make up layers which are stacked to form the 3D geometry. This contrasts with the second and third most common 3D printing methods, which use a photosensitive resin to create layers (stereolithography, SLA) or a powder material that is melted by a laser (selective laser sintering, SLS).

Figure 1: FFF Process Diagram Source: (Wichramasinghe Et Al. 2020)

Limitations of FFF printing The FFF 3D printing process creates two physical limitations on parts. The layered nature of the printing process leads to distinct anisotropic strength; specifically, the z-axis strength is limited by interlayer adhesion. This limitation makes the part up

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Scientific Research in School Volume 5 Issue 1 2023 to an order of magnitude weaker when loaded across the layers as opposed to along them (Kuznetsov et al., 2018). The second major limitation facing FFF printing is the reduced crystallinity in semicrystalline polymers resulting from the rapid cooling of plastic when it leaves the nozzle (Dong et al., 2020). Composites In 3d Printing Owing to the filament-style feedstock, FFF printing can use a diverse range of materials, most notably fibrous composites (Blok et al. 2018). The regular polymer feedstocks of FFF printing can be used as a matrix for short, chopped aramid fibres – making a composite with significantly altered physical properties. To fit through the nozzle of the printer and prevent clogging, the fibres are chopped to less than 400 μm (the typical nozzle diameter) (Blok et al. 2018). The following effects have been observed because of rigid fibre introduction: (a) decreased viscosity when molten (Wilson 2022), which leads to better dimensional accuracy but worse interlayer bonding, thus poorer z-axis strength. And, (b) improved strength parallel to the layer plane. Post-Processing (Annealing) It is this strength limitation in the FFF process, exacerbated by composite feedstock, that is the target of post-processing for 3D printed parts. A process by which interlayer strength can be increased is annealing. This is when the part is heated between its glass transition point (Tg) and its melting point (Tm),

enabling the layers to coalesce, thus increasing the bond strength. Additionally, by heating the plastic, the polymer chains become mobile and, through dispersion forces, will form larger crystalline structures (Wickramasinghe et al., 2020) Conventionally, annealing is done in an oven; however, the oven can only heat the material externally, meaning annealing times are long, thus negating one of the main advantages of FFF printing.

Figure 3: Tensile strength Vs time for oven and microwave annealing Source: (Dong Et Al. 2020)

A novel process known as microwave annealing uses a regular heating microwave to warm the part from the inside out. Many carbonaceous materials have high dielectric loss; suggesting the ability to convert microwave radiation into thermal energy. By using a carbon composite filament, 3D printed parts could be

Figure 2: “(a) Fracture surface of the untreated PLA specimen. (b) Bond between PLA filaments untreated specimen. (c) Surface of the untreated ABS specimen. (d) Fracture surface of the annealed PLA specimen. (e) Bond between PLA of the untreated specimen. (f) Surface annealed ABS specimen.” Source: (Wickramasinghe et al., 2020)

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Scientific Research in School Volume 5 Issue 1 2023 annealed using a microwave (Dong et al., 2020). Because the part is heated uniformly from the inside, the process is drastically faster than oven annealing. This process is a promising alternative that may overcome many of the limitations facing 3D printing that are yet to be effectively mitigated with current post-processing methods; however, the process is yet to be rigorously tested and has some disadvantages. Limitations/areas of investigation for microwave annealing As a novel process, few papers have been published that explore its potential. To the author’s knowledge, only two papers have attempted to test the process (Dong et al. 2020, and Wilson 2022). The first experiment for this technology used specialised, commercially unavailable filament – a limitation identified and overcome in the second experiment. However, as identified by the author, a limitation to the microwave annealing process is the secondary heating of polar polymers such as polyamide (nylon), theorised to be a result of rotation and energy absorption of amide groups in the polymer when it surpasses Tg (Wilson, 2022). While the degree of secondary heating will vary depending on the polymer composition and microwave frequency, it is nonetheless undesirable to perform microwave annealing on nylon. Acrylonitrile Butadiene Styrene (ABS) or Polyetheretherketone (PEEK) are ideal polymers to perform further testing on the efficacy of microwave annealing as they are non-polar and semipolar respectively; however, only ABS will be pursued for this paper due to its availability and the difficulties of printing in PEEK. More limitations of FFF printing Another major factor in the strength of FFF parts is the geometric parameters it is printed with. Specifically, layer height and nozzle diameter can drastically affect the strength of parts. (Kuznetsov et al., 2019) concludes that as nozzle diameter increases so does ultimate flexural stress (UFS), and as layer height increases, UFS decreases (Nomani et al. 2019). Unfortunately, a lower layer height significantly increases print time; as such, it is desirable to increase layer height; however, as Figure 4 demonstrates, such an increase leads to more voids in the part structure, which decreases strength. A combination of microwave annealing and increased layer height could lead to an even faster printing process without compromising functionality.

Figure 4: SEM cross sectional view of 0.6mm diameter nozzle prints with varying layer heights Source: (Kuznetsov et al., 2018)

Scientific Research Question Is microwave annealing an effective process for improving the interlayer strength of FFF 3D printed ABS-CF composite parts, regardless of layer height?

Scientific Hypothesis The microwave and conventional oven annealing processes will significantly improve the ultimate tensile strength in the perpendicular axis of FFF ABS-CF parts regardless of print layer height.

𝑯𝑯𝑯𝑯𝒂𝒂𝒂𝒂 : 𝜇𝜇𝜇𝜇𝑚𝑚𝑚𝑚 (0.15) 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝜇𝜇𝜇𝜇𝑜𝑜𝑜𝑜 (0.15) > 𝜇𝜇𝜇𝜇𝑐𝑐𝑐𝑐 (0.15)

and

𝜇𝜇𝜇𝜇𝑚𝑚𝑚𝑚 (0.3) 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝜇𝜇𝜇𝜇𝑜𝑜𝑜𝑜 (0.3) > 𝜇𝜇𝜇𝜇𝑐𝑐𝑐𝑐 (0.3) .

(where ‘m’ means microwave annealed, ‘o’ means oven annealed, ‘c’ means control, and (#) indicates the layer height in mm). 𝑯𝑯𝑯𝑯𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏𝒏 : any of the average tensile strengths for the post-processing methods are ≦ their corresponding layer height control.

Methodology To test the efficacy of microwave annealing in improving interlayer tensile strength, 30 standardised specimens were manufactured. The specimens, designed in the Solidworks™ Computer-Aided Design (CAD) platform, are in compliance with the ATSM D638 standard test method for tensile properties of plastics.

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Scientific Research in School Volume 5 Issue 1 2023 Treatment 2 – Conventional oven annealing Using a domestic convection oven, the samples were heated to their Tg (105°c) over 3 hours. The samples were placed on a tray with a layer of nonstick/insulating paper (see Figure 7).

Figure 5: Solidworks design environment with dogbone model

Using what is known as slicing software, the 3D models, arranged such that their layers are in the axis of loading (i.e. vertically), were prepared for printing using the following settings: Table 1: Printing parameters and slicing values

Parameter Nozzle temperature Bed temperature Printing speed Shells/infill Nozzle diameter Layer height

Value 245°c 110°c 35mm/s All shells 0.4mm 0.15mm & 0.3mm

The specimens were then printed on an Anycubic Vyper™ 3D printer using a carbon fibre & ABS composite filament from 3DX-Tech, a niche but commercially available material.

Figure 6: 3DX tech carbon fibre abs composite filament spool.

The specimens were printed in two batches of 15, with 5 going to each of the three treatment methods to mitigate the effect of printing inconsistencies. Treatment 1 – Control These specimens had no post-processing done on them and were tested as printed.

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Figure 7: Oven with four specimens loaded for annealing

Once the 3 hours had elapsed, the tray was removed from the oven, and the specimens were allowed to cool before being touched (this was done to prevent any unnecessary warping or damage). Treatment 3 – Microwave annealing Reflecting the methods outlined by Dong et al. 2020 & Wilson 2022, a domestic microwave (Model M8261 – 2220, 900w 2450MHz output) was used to irradiate the specimens. They were evenly placed on the microwave plater in a circle with a non-stick paper layer, avoiding the static centre (see Figure 8). The specimens were then microwaved in 1-minute pulses with a 40% duty cycle for a total irradiation time of 10 minutes (total time = 25min). This timing method was used to reduce the possibility of damaging the microwave’s magnetron whilst ensuring a total time similar to that of Dong et al. 2020 (force vs time graph Figure 3). The discrepancy between the timings of Wilson 2022 and Dong et al. 2020 is due to the runaway heating of Nylon, making heating for longer than 10 seconds impossible while maintaining any part geometry (the plastic erupts in smoke and rapidly deforms).

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to calculate the ultimate tensile strength (UTS), given the formula: 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑎𝑎𝑎𝑎𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑇𝑇𝑇𝑇𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠ℎ (𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑎𝑎𝑎𝑎) =

𝐹𝐹𝐹𝐹𝑜𝑜𝑜𝑜𝐹𝐹𝐹𝐹𝑐𝑐𝑐𝑐𝐹𝐹𝐹𝐹 (𝑁𝑁𝑁𝑁)

𝐴𝐴𝐴𝐴𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐴𝐴𝐴𝐴 (𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚2 )

An average was then calculated for each treatment & layer height, and 4 t-tests were evaluated (one for each process, for each layer height)

Results Figure 8: Microwave with 10 specimens loaded for annealing

To test the specimens, a load-testing machine from high school Design and Technology faculty was used. The machine used a DC linear actuator to apply a slow, steady load on the specimens, which were secured using G-clamps to the gantry and the base. (see Figure 9)

The specimens responded successfully to both the microwave and conventional annealing processes. The microwave process resulted in some undesirable changes to the specimen geometry/finish (see Figure 10). The 10 specimens did not consistently experience this warping/melting, with some experiencing none.

Figure 10: 4 warped microwave specimens on the bottom Figure 9: Load testing machine with a specimen loaded for testing

The steady application of load ensured that the specimens could undergo elastic deformation before ultimate tensile failure. A load cell (S-Type rated 2450N; model MT501) in the load-testing machine measured the force applied, and an electronic scale set to read peak value recorded the maximum force exerted on the specimen. Specimens were loaded individually, and the scale was tared between each test. The specimens were tested at a load rate of 5mm/s. The known cross-sectional area (16.8mm2) of each specimen and the maximum force required was used

The results found for specimens at 150μm layer height were consistent for 300μm, albeit less distinct. The comparison of each process, • Control vs Microwave(150μm) (t(8) = 2.60, p = 0.03 < 0.05) • Control vs Oven(150μm) (t(8) = 3.80, p = 0.005 < 0.05), for both layer heights, • Control vs Microwave(300μm) (t(8) = 2.39, p = 0.04 < 0.05) • Control vs Oven(300μm) (t(8) = 2.69, p = 0.03 < 0.05). demonstrated a significant increase in tensile strength for both processes regardless of the print layer height.

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Table 2: Force results

Figure 11: Box and wisker plot comparison of processes for 0.15mm layer height Table 3: Calculated tensile strength results

Figure 12: Box and wisker plot comparison of processes for 0.30mm layer height

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Discussion The results, in confirmation wth the hypothesis, demonstrate the positive effect of annealing, both microwave and conventional oven, on the interlayer tensile strength of parts. The control group (μ = 11.7 & 8.0), oven annealed (μ = 14.6 & 9.0), and microwave annealed (μ = 15.2, 9.8) all experienced an ultimate tensile strength (UTS) significantly lower than the advertised 46MPa (as identified by the supplier, 3DX-Tech). This is likely due to (a) the ‘explicit anisotropic strength’ of FFF parts (Kuznetsov et al. 2018) and interlayer testing (z-axis) in the weakest direction; and (b) the limitations of the printer and print environment used. ABS is somewhat notorious in FFF printing as a difficult material to work with; this is because of its higher working/printing temperature compared to more common alternatives like polylactic acid (PLA) – about 25% higher. The higher printing temperature results in worse interlayer bonding and printing artefacts/defects such as warping, cracking, and seam gaps where new lines start. A printing enclosure, a system that maintains a higher ambient temperature during the print, can help overcome this but was not implemented in this experiment. The annealing processes used were able to improve the tensile strength, as previously identified; however, they were largely ineffective in removing pre-annealed defects – as expected. Future experiments should attempt to address this experimental limitation by either improving the printing process or by utilising a lower printing temperature material such as PLA. An important factor of this experiment is the categorical nature of the post-processing. While the results demonstrate that oven annealing is more consistent and similarly effective, it is not the primary focus of this paper. Oven annealing has already been established as an effective method (Singh et al. 2018); as such, it serves as a comparison for microwave annealing in this experiment. A result of this is the difference in standard deviation (2x larger for 150μm and 1.7x larger for 300μm), a noteworthy difference likely due to the heterogeneous application of energy in a microwave (evident in the need for a rotating plate) or printing flaws exaggerated when the part is rapidly heated. The overlookable downside to the categorical comparison of the treatment methods is the time required; as identified in the method, the oven process was 3 hours compared to 25 minutes for microwaving. This has significant implications in the implementation of annealing on a commercial scale –

not to mention the substantially higher energy requirement of running an oven for an extended period (Wickramasinghe et al. 2020). One of the key advantages of 3D printing is its speed, something that may be negated by a lengthy annealing process. An interesting observation can be drawn from the warped/overheated microwave annealed specimens; the undesirable heating only occurred at the flared ends of the specimens, and a qualitative test of these demonstrated that they were several times stronger than the rest of the part. This suggests that a part of the specimen was exposed to a different amount of heating which resulted in a greater strength improvement. If the same process could be applied to the rest of the specimen its strength could improve significantly, therefore a more detailed analysis into this phenomenon could result in improvements to the microwave annealing process. Further research into this field should attempt to explore improvements to the experimental procedure to reduce the influence of printing defects on the final part and subsequently improve the reliability of results. In addition to this an exploration & subsequent explanation of the heating effect seen on the specimen flared ends could lead to a better overall process with significantly improved strength improvements.

Conclusion This investigation confirmed the efficacy of microwave annealing as a FFF post-processing method to improve interlayer tensile strength. It showed that the ultimate tensile strength of ABS carbon fibre composite could be increased by 30%, and that parts can be improved regardless of their layer height (tested for 0.15mm and 0.3mm). These results are consistent with those of Dong et al. in 2020 and Wilson J. in 2022 – with the only notable discrepancy being the reduced improvement shown between annealed and non-annealed; however, because oven annealing was also tested, and because those results were not significantly different, it can be concluded that this discrepancy is a result of ABS’s material properties, the experimental procedure (specifically, the printing set-up), or a combination of both. Data analysis involved multiple t-tests to determine the accuracy of the multifaceted hypothesis, the results of which confirmed the hypothesis.

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Acknowledgements This research has been made possible by the Barker College science extension team, robotics department, and design and technology department. I would like to thank Dr Matthew Hill for his insights and guidance, and his invaluable help with statistical analysis and terminology. Access to the load testing machine was also generously provided by Mr Darren Woodrow. And the project was made possible by the equipment and supplies donated by the Robotics department, thanks to Mr Jeser Becker.

References Blanco I., (2020) The Use of Composite Materials in 3D Printing. https://doi.org/10.3390/jcs4020042 Blok L., Longana M., Yu H., Woods B. (2018) An investigation into 3D printing of fibre reinforced thermoplastic composites. https://doi.org/10.1016/j.addma.2018.04.039 Butt, J., & Bhaskar, R. (2020). Investigating the Effects of Annealing on the Mechanical Properties of FFF-Printed Thermoplastics. Journal of Manufacturing and Materials Processing, 4(2), 38. https://doi.org/10.3390/jmmp4020038 Dong J., Huang X., Muley P., Wu T., Barekati-Goudarzi M., Tang Z., Li M., Lee S., Boldor D., Wu Q. (2019) Carbonised cellulose nanofibers as dielectric heat sources for microwave annealing 3D printed PLA composite. https://doi.org/10.1016/j.compositesb.2019.107640 Ferreira, R., Igor Cardoso Amatte, Thiago Assis Dutra, & Bürger, D. (2017). Experimental characterisation and micrography of 3D printed PLA and PLA reinforced with short carbon fibres. https://doi.org/10.1016/j.compositesb.2017.05.013 Kuznetsov V., Solonin A., Urzhumtsev O., Schilling R., Tavitov A., (2018) Strength of PLA Components Fabricated with Fused Deposition Technology Using a Desktop 3D Printer as a Function of Geometric Parameters of the Process. https://doi.org/10.3390/polym10030313 Nomani J., Wilson D., Paulino M., Mohammed M. (2019) Effect of layer thickness and cross-section geometry on the tensile and compressive properties of 3D printed ABS. https://doi.org/10.1016/j.mtcomm.2019.100626 Singh S., Singh M., Parkash C., Gupta M., Mia M., Singh R. (2018) Optimisation and reliability analysis to improve surface quality and mechanical characteristics of heattreated fused filament fabricated parts. https://doi.org/10.1007/s00170-018-03276-8 Wickramasinghe S., Truong D., Tran P. (2020) FDMBased 3D Printing of Polymer and Associated Composite: A review on Mechanical Properties, Defects and Treatments. https://doi.org/10.3390/polym12071529 Wilson J. (2022) Postprocessing of Commercially Available Thermoplastics by Micro- wave Annealing.

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Wong, K. V., & Hernandez, A. (2012). A Review of Additive Manufacturing. https://doi.org/10.5402/2012/208760 Material Sourced: https://www.3dxtech.com/product/carbonx-abs-cf/

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The impact of adding a duct to a toroidal vs traditional bladed propeller Bardia Lamyi-Arani Barker College Purpose: This paper aims to compare ducted toroidal propellers (a torus shaped propeller) to the more extensively researched ducted propellers Design/methodology/approach: In a direct comparison to previous research that explored the optimal duct length for a traditional propeller (Harper, 2020) this paper will follow an identical process however for the less-researched toroidal propeller which un-ducted already has efficiency benefits over the traditional propeller. A toroidal propeller and duct of five varying lengths was modelled using Computer Aided Design program SOLIDWORKS. Visual analysis of airflow heatmaps and numerical analysis of airspeed was used to determine efficiency. Findings: The introduction of a duct produces a far greater increase in efficiency for a traditional bladed propeller than a toroidal propeller, however it is argued that this may be due to the reasons why the non-ducted toroidal propeller is more efficient than the non-ducted traditional propeller. Interestingly, both propellers have the same optimal duct length of around 0.75, however they do not have the same maximal thrust at this ratio, such that the traditional propeller is more efficient. It was concluded that if applications of propellers don’t have enough room for the optimal ratio duct it is greatly advised to go toroidal propeller, however for applications where optimal ratio propeller to duct has enough room traditional bladed propellers are better. Research limitations/implications: Real-world modelling using 3D printed components based off these designs is the next step to further verify the conclusions as this research is strictly computational. Implications/Reasons: Despite their simplicity, propellers play a crucial role in the functionality of aircrafts. For both commercial and environmental reasons, small increases in efficiency (e.g. by slightly varying duct length ratios) can have large impacts in the world. Originality/value: Ducted traditional propellers and toroidal propellers are not new, however toroidal propellers are under-researched and there is no published literature about the implications of adding a duct to a toroidal propeller. Paper type: Research paper

Literature Review What is a Propeller? Propellers are rotating devices that force (or propel) fluids such as water or air in one direction, generating a thrust force on the propeller and the object attached to the propeller (Sheih & Morris, 2001; Sawale et al., 2018). With extensive applications in industries from planes to drones, to marine, it is unsurprising that efficiency of propellers has been a topic of extensive scientific research (Lee & Lin, 2002; Müller et al., 2014; Saimee, 2018; Red, 2015; Nagpurwala, 2015). Thrust is generated by a propeller by creating a pressure difference between the top and bottom of the blades (Bhuyan, 2021). This difference creates a twisting motion that makes the fluid, such as air go forward and create a lift force. An example of research into improving efficiency is by implementing a duct over the propeller in Figure 1 as a duct around the propeller helps to improve the flow of fluid by reducing turbulent flow and creating a

more laminar flow (Marlin, 2019). The increased flow of fluid leads to improved efficiency. However, this is on a traditionally bladed propeller Figure 2 an innovation has recently come out known as a toroidal propeller that theoretically address some of the inefficiencies (Blain, 2023). For example, wing tip vortices are less likely to be formed as the torus shape of the blades don’t cut the air as much. However, it is not a complete elimination of wing tip vortices and other inefficiencies will still be apparent. As well toroidal propeller will come with a much greater noise reduction (Sebastian et al., 2020) suggesting less turbulent air. Limitations can also occur as it is commonly said toroidal propellers may not be as effective and generating maximum thrust and top end speeds seem to fall off (Blain, L 2023; Carral et al., 2018).

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Figure 1: A traditional bladed ducted propeller

Figure 3: A velocity heat map of wing tip vortices on a c130 as the fluid is all at different pressures. Source: Lian, J. Z. (2003)

The fluid rotation of the wing tip vortices are caused by the swirling of air around the outside of the propeller decreasing its performance as it creates much denser air where the propeller spins (Lian, 2003). By the presence of a duct it is known that the wing tip vortices will decrease for a traditional propeller (Nagpurwala, 2015) this study will investigate wing tip vortices in ducted and unducted toroidal propellers.

Figure 2: A toroidal propeller

Propeller inefficiencies The three main inefficiencies include wing tip vortices, slow intake speed and turbulent air (Harper, 2020). Ducts are known to increase inefficiencies of propellers by decreasing wing tip vortices, increasing intake speed, and producing less turbulent air, producing a more laminar flow (Bishop, 1983; Marlin, 2019). Wing tip vortices Wingtip vortices is the swirling or circulation of air around the tip of the propeller which results to a decrease in efficiency (Miranda, & Brennan, 1986). They are a type of turbulent air as when the propeller moves through fluid there is a lower pressure of air on the upper surface and a high pressure on the lower surface this pressure difference creates the blades to have two counter rotating vortices at the wingtips (Newman, 1964) (Bishop, 1983).

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Turbulent air Turbulent air is the spread of air that produces erratic flow patterns, these patterns create turbulent air which raises inefficiencies (Sánchez-Caja, 2001). This swirling of air has a loss of thrust as it vibrates the propeller due to the multi-directional air incoming onto the propeller when spinning, this loses the energy produced by the propeller in the form of eddies, noise, and heat (Schetz & Jakubowski, 1975). The opposite of turbulent air is laminar air and research has concluded that the appearance of a duct produces more laminar flow (Miller, 2017) reducing the impact of performance on a propeller. Figure 4 demonstrates the laminar flow difference of the propeller as it is observed with the increase of a duct that the air becomes more streamlined however, unknowingly if this is the case for toroidal propellers

Scientific Research in School Volume 5 Issue 1 2023 enhance this effect to a greater extent (Muller et al., 2014; Kweder et al., 2014).

Figure 5: The Venturi Effect Source: (Vadaje, 2018)

Figure 6: the intake speed illustrated before a propeller and how the duct can curve air into the propeller better. Source: (Harper, 2020)

Figure 4: Velocity heat map of ducted-bladed propellers. Source: (Harper, 2020)

Slow intake speed When the air directed into the propeller is increased, propellers typically produce more thrust (Kweder et al., 2014). Therefore, when slow intake speed (the speed of air before the rotating blade) is close to the propeller it reduces the efficiency of traditional propellers. It is desired to increase the intake speed by creating a funnel like affect to the propeller. This increases the Venturi affect which is how the fluid flows laminar into a pipe (Vadaje, 2018), shown in Figure 5 which dictates the process of a duct reducing slow intake speed, resulting in an increase in air velocity towards the propeller Figure 6. By pre-accelerating the incoming air, the duct allows the propeller to further accelerate the air and produce higher thrust, making the propeller more efficient than a non-ducted propeller. Moreover, a longer duct can potentially

As these inefficiencies stand it was tested that a duct will reduce these inefficiencies (Saimee, 2018) and a previous paper found the ideal ratio of ducted propellers which is the ratio between the dimeter of the blades and duct is R=0.75 e.g a 0.75m propeller blade should have a 1m diameter duct (Harper, 2020). It remains to be seen how these inefficiencies may be present or addressed for a ducted toroidal propeller.

Scientific Research Question How does an introduction of a duct of varying length affect the efficiency of toroidal vs Traditional bladed propeller?

Scientific Hypothesis That while toroidal propellers are commonly more efficient than normal propellers, a duct of optimal length can result in traditional propeller being more efficient.

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Methodology Design of the propeller and duct In a recent study by Harper (2020) the efficiency of a duct of varying length added to a traditional propeller was measured. The exact same processes were used in this present study, however applied to the underresearched toroidal propeller, to allow for valid comparison between the effect of ducts on each type of propeller. An identical duct was used, named the ‘NACA 1415’ style duct (see Figure 7) as it has been proven to be most efficient (Bontempo & Marcello , 2016). Ducts of 5.0, 6.5, 8.0, 9.5 and 11.0 cm were generated and tested, this time around a toroidal propeller.

Figure 7: The ideal duct shape Source: Bontempo & Marcello, (2016)

Using identical software (computer aided design (CAD) program SOLIDWORKS – see Figure 8) six toroidal propellers, one with each of the previously mentioned duct lengths and one with no duct, were produced. They had identical propeller diameter (P=12.7cm).

Figure 8: The original design on a traditional bladed propeller source, modelled using CAD Source: Harper, (2020)

Determining efficiencies Using the flow simulation of SOLIDWORKS airflow around all the toroidal propellers could be computationally modelled. Specific parameters were as per Harper (2020) including 10m.s-1laminar airflow with a propeller angular velocity of 500 radians/s (~4800rpm). Repetition would be meaningless due to the computational model producing identical results for identical tests.

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As the program was able to determine velocities at all points in and around the propeller two types of results were able to be generated. Firstly, a velocity heat map for each propeller duct length was produced showing the cross-sectional air speed with colour representing the speed. This will be used to do a comparison and determine where the inefficacies have decreased or not between both the designs. The inefficiency which will be the focus? will be wing tip vortices, which show up as blue spots near the wing tips on a velocity map. Slow Intake speed which shows up behind the propeller as a colour of blue. Turbulent air can be determined by the consistency of air; the more different velocities shown on the velocity heat map will show as more turbulent air. Secondly numerical values at three specific locations could be recorded, averaged and plotted to determine relative and optimal efficiencies for varying duct lengths. The three locations were trailing the end of the duct by 10mm as pictured in Figure 9. Easy comparison between the results for the toroidal and traditional propeller will be facilitated by plotting the results on the same graph.

Figure 9: The velocity trap points for numerical analysis of efficiency. Source: Harper (2020)

These results were recorded and compared visually and graphically to Harper’s results for the ducted traditional propeller (2020) allowing valid comparison between the impact of a duct on a traditional and a toroidal propeller.

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Results

Figure 10: Velocity heatmaps allowing identification of slow-moving air (inefficiencies) of propellers with no duct and varying duct length including a traditional propeller (After: Harper, (2020), for comparison) and a toroidal propeller (results produced in this present research).

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Scientific Research in School Volume 5 Issue 1 2023 In Figure 10 the two-heat map velocity are shown. The left is the traditional propeller and right is a toroidal propeller. The dark blue colour is depicted undesirable and the richer in colour toward the red end is good as it generates more velocity. The ducted propeller shows a great amount of wing tip vortices in the non-ducted version but increases significant in performance with the implementation of a duct. The toroidal propeller shows a much-decreased wing tip vortices in the non-ducted propeller however gains are not as significant as a traditional propeller. Quantitative analysis The velocity trap points where averaged and produced Figure 11. Both propellers are most efficient at a duct length greater than 8cm and less than 11cm. Ideal ratio calculations R=Duct Length Propeller Diameter The idea ratio is calculated by this formula and both propeller diameter was a constant of 12.7cm. Traditional bladed propeller R=0.75

Toroidal propeller quantitates max performance could be considered from Figure 11 as 9.6cm resulting to: R=0.096 0.127 =0.76 (2 sig. fig.).

Discussion Traditional propellers In Figure 10 A1 (1 indicates traditional propeller results from previous research, 2 indicates results from this present study), the central area is prominently depicted in dark blue and is relatively largest compared to all the ducted, however still a big improvement over non-ducted. As we progress from A1 through D1, there is a noticeable reduction in the size of this central dark blue area, indicating an increase in overall air velocity. However, in Figure 10 E1, we observe a reversal of this trend as the central area increases again, suggesting that the total thrust has peaked between C1 and E1 and is now following a decreasing trend. The presence of the wing-tip vortices is most evident in Figure 10 A1, where a dark blue patch is clearly visible at the top. The size of this low-velocity patch

Figure 11: Average velocity of ducted toroidal vs ducted traditional propeller for various duct lengths

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Scientific Research in School Volume 5 Issue 1 2023 at the wingtip consistently varies with the central low-velocity patch, initially increasing from 5.09.5cm and then decreasing by 11cm.

effective, indicative that toroidal propellers may not have as much top end performance as expressed in literature review.

Figure 10 A1 reveals highly turbulent characteristics, as evidenced by the wavy boundary between the red/yellow and green/blue areas. However, as we progress towards D1, this boundary becomes much more linear and defined, indicating a trend towards faster and more laminar flow.

Implications The analysis of ducted and toroidal propellers provides valuable insights into their performance, efficiency, and how to mitigate their inefficacies.

Intake speeds seem to also improve throughout with the increase of a duct as behind the propeller blades the colour gets closer to red as the duct size increases. Toroidal propeller These figures provide great insight into the performance of toroidal propellers compared to the traditional propellers and non-ducted propeller. Comparing Figure 10 F1 and F2 (the non-ducted propellers) the toroidal propeller is far more efficient than the traditional propeller. This is evident as Figure 10 F2 (toroidal) there is less blue (slow, turbulent air), and wing tip vortices are almost not visible at all. This observation is consistent with previous findings in the literature (Sebastian et al, 2020). Comparing Figures A2 and A1, i.e. now with a duct added, it is apparent that the toroidal propeller (A2) performs better than the traditional propeller (A1) in terms of a lack of wing-tip vortices, faster intake speeds and less turbulent air. The central dark blue area in A2 is smaller than F2 (toroidal, non-ducted), indicating a higher overall air velocity and improved performance. However, as we progress from A2 through D2, the effectiveness of reducing the inefficiencies is not as substantial shown as in the ducted propellers. Visual analysis of the heatmaps (Figure 10) regarding impact of duct length on efficiency is greatly supported by the numerical results graphed in Figure 11. Firstly, a duct does increase efficiency for both propellers, increasing as the duct length increases up to a point (9.5cm) and secondly that the impact of the duct is far more substantial for a traditional propeller than a toroidal propeller. Therefore, these results provide supporting evidence for the hypothesis that a toroidal propeller is more effective than a traditional propeller however the implantation of a duct being closer to the optimal ratio will result to the traditional propeller being more

The results adhere to the background information as the toroidal propeller starts of more efficient however for optimal duct lengths the traditional propeller becomes more efficient. The amount that the traditional propeller with optimal duct length is more efficient than the toroidal propeller with optimal duct length is surprising, as it is much larger than expected. However, it is important to note that the specific performance characteristics and efficiency of toroidal propellers may vary depending on various factors such as design parameters (as these parameters was originally designed for traditional propellers), operating conditions, and application requirements. The study confirms the well-established understanding that wingtip vortices, turbulent air and slow intake speed have a detrimental effect on propeller efficiency. The presence of these vortices creates opposing forces to thrust (Miranda & Brennan, 1986; Bhuyan, 2021), resulting in decreased overall efficiency. Both ducted and toroidal propellers offer potential solutions to mitigate the inefficiencies and increase performance. Ducted propellers, as shown in the analysis, effectively reduce wingtip vortices by containing and directing the airflow. The duct helps in creating a more laminar flow, reducing turbulent flow and increasing overall efficiency. On the other hand, toroidal propellers provide an alternative design approach to minimize wingtip vortices generation. The unique torus shape of the blades allows for a different interaction with the fluid medium, resulting in reduced vortices, it can be said that the duct for toroidal propellers mostly just improved the turbulent air to laminar flow and intake speed as there was not much wing tip vortices to begin with. The analysis as well demonstrated that toroidal propellers exhibit improved performance compared to traditional nonduct propeller, due to the already efficient nature. However, it is perhaps unsurprising that adding a duct will have minimal effect when issues like wing-tip vortices have already been addressed.

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Scientific Research in School Volume 5 Issue 1 2023 The study also reveals that the efficiency of ducted toroidal propellers may not surpass that of ducted propellers when optimal ducts are included in the design. The findings suggest that the choice between ducted and toroidal propellers depends on various factors, including the specific application requirements, operating conditions, performance trade-offs, and in particular, if a duct is available and suitable for required applications. Toroidal propeller may be more suitable for applications where noise reduction and weight savings are crucial, such as marine and drones as ducts are not found on both boat propellers and drones. A ducted, traditional, propeller, on the other hand are more suitable for aeronautical applications and places which maximal thrust generation is necessary. In summary the findings of my experiment have concluded such observation that in applications where a duct cannot be used, it is recommended to go with toroidal propellers. However, if enough room for an optimal ratio duct can be fitted it is advised to go with traditional bladed propellers. Further Research It is worth noting that the analysis presented in this study is based on computational simulations. While computational methods provide very valuable insights, further experimental studies are necessary to validate the findings and provide a more comprehensive understanding of propeller performance. As well setting up a testing for both propellers in different environmental parameters can provide greater knowledge such as the propeller working in fluid such as water not air. Moreover, environmental considerations should not be overlooked in propeller design. The impact of propellers on noise pollution and marine life disturbance is of growing concern. Future research should explore the potential of both ducted and toroidal propellers to reduce these environmental impacts and contribute to sustainable propulsion solutions.

Conclusion My research project explored the difference in performance of toroidal and traditional bladed propellers when a duct of varying length was added. Computer simulations were used to produce velocity heat maps for visual analysis, and quantitative air flow velocities for a graphical comparison with previous research. It was found that the propeller

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increases in efficiency with the presence of a duct however, the increase in efficiency was less than as for a traditional bladed propeller. It was found that non-ducted variants showed that the toroidal propeller is far more efficient than the non-ducted propeller. However, for maximal thrust application a ducted propeller is better than a ducted toroidal propeller. Similar results happened with the process of a duct in a toroidal propeller such as decreased turbulent air, and slower intake speed improving its efficiency, leading me to accept the hypothesis that toroidal propellers are more efficient for non-duct applications and performance can increase with the presence of a duct, but the benefits are not as substantial as a ducted traditional propeller.

Acknowledgements I would like to thank Dr Matthew Hill for being an immense support throughout my project and providing reviews throughout this duration. I would also like to thank Mr Alastair Pilley for assisting through the testing in the computational simulation and lending me a license for SolidWorks.

References Bishop, R. E. D. (1983). The mechanics of vortex ring formation at the wing tips of hovering honey-bees. Journal of experimental biology, 107(1), 385-389. Newman, J. N. (1964). Marine hydrodynamics. The MIT Press. https://mitpress.mit.edu/9780262140263/marinehydrodynamics/ Bhuyan, S. (2021, January 5). Centrifugal Force and others in circular motion: Definition, Examples, and Equation. Retrieved January 18, 2023, from Science Facts website: https://www.sciencefacts.net/centrifugal-force.html Marlin, A 2019 ‘The Efficiency Of A Ducted Propeller’, Barker Science Extension Journal 2019. (2019). Retrieved January 7, 2023, from issuu website: https://issuu.com/barkercollege/docs/science_extension_jo urnal_final_18-10-2019_glenn/1?ff Harper, T 2020 ‘The Optimal Length Of A Propeller’ Science Extension Journal: Volume 2 |Number 1|September 2020. (2020). Retrieved January 7, 2023, from issuu website: https://issuu.com/barkercollege/docs/2020_science_ext_jo urnal_in_pdf_/1?ff Bontempo & Marcello , 2016. Wall pressure coefficient distribution along the duct NACA 1415. ResearchGate. https://www.researchgate.net/figure/Wall-pressurecoefficient-distribution-along-the-duct-NACA1415_fig11_293781540 Sawale, A. Archana, D. and Seshank, C. (2018). Development of underwater glider propulsion system based on toroidal propeller. Journal of Physics: Conference Series. https://doi.org/10.1088/1757-899X/455/1/012018

Scientific Research in School Volume 5 Issue 1 2023 NACA 1415 AIRFOIL 2020, Diagram, Airfoil Tools. http://airfoiltools.com/airfoil/naca4digit?MNaca4DigitFor m% (5Bcamber%5D=1&MNaca4DigitForm%5Bposition%5D =40&MNaca4DigitForm%5Bthick%5D=15&MNaca4Dig itForm%5BnumPoints%5D=81&MNaca4DigitForm%5Bc osSpace%5D=0&MNaca4DigitForm%5BcosSpace%5D= 1&MNaca4DigitForm%5BcloseTe%5D=0&yt0=Plot.

from New Atlas website: https://newatlas.com/aircraft/toroidal-quiet-propellers/ Sebastian et al, 2020. ‘United States patent Toroidal Propellers’ https://patentimages.storage.googleapis.com/d6/fe/53/bea 4417ed89176/US10836466.pdf

Lian, J. Z. (2003). MEMS: History and overview. Washington University in St Louis; Department of Mechanical Engineering and Materials Science Lee, Y. and Lin, C. 2002. Optimized Design of Composite Propeller'. Mechanics of Advanced Materials and Structures, Vol. 11, No. 1. Miller, C 2017, Bernoulli's Equation, Diagram, Owlication. https://owlcation.com/stem/Aerodynamics-The-Theoryof-Lift. Müller, L. Heinze, W. Kožulović, D. Hepperle, M. Radespiel, R. 2014. 'Aerodynamic Installation Effects of an Over-the- Wing Propeller on a High-Lift Configuration'. American Institute of Aeronautics and Astronautics Journal, Vol. 51, No. 1. Nagpurwala, Q, 2015. Ducted Fans and Propellers, Ramaiah School of Advanced Studies, Bangalore, pdf, http://164.100.133.129:81/econtent/Uploads/09%20Ducted%20Fans%20and%20Propellers%20%5BCom patibility%20Mode%5D.pdf. Red, U. 2015, Why Ducting A Propeller Makes It More Efficient, Flite Test, https://www.flitetest.com/articles/why- ducting-apropeller-makes-it-more-efficient. Reddit Aviation 2008, Simulation Graphic, Reddit. https://www.reddit.com/r/aviation/comments/cmq90i/sinc e_were_talking_c130_propeller_tip_vortices/. Saimee, A. 2018. 'Improvement of Airfoils Aerodynamic Efficiency by Thermal Camber Phenomenon at Low Reynolds Number'. Journal of Aerospace Technology and Management, Vol. 10, No. 1. Sánchez-Caja, A. 2001. 'Simulation of Incompressible Viscous Flow Around a Ducted Propeller Using a RANS Equation Solver'. Symposium on Naval Hydrodynamics (2001), Vol. 23, No. 1, pp. 527-538. Schetz, J.A. Jakubowski, A.K. 1975. 'Experimental studies of the turbulent wake behind self-propelled slender bodies'. American Institute of Aeronautics and Astronautics Journal, Vol. 13, No. 12. Shieh, C. Morris, P. 2001. Comparison of two- and threedimensional turbulent cavity flows'. American Institute of Aeronautics and Astronautics Journal, 39th Aerospace Sciences Meeting and Exhibit. Vadaje, Y. 2018, Venturi Effect Explained, Engineering Fact. https://www.engineeringfact.com/venturi-effectexplained/. Blain, L 2023 https://newatlas.com/author/loz-blain. (2023, January 27). Toroidal propellers: A noise-killing game changer in air and water. Retrieved June 8, 2023,

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Biology & Environmental Science Every year the scientific community gains a greater understanding of the impact of our actions on organisms and ecosystems. These nine projects provide further insight in this domain with benefits in food production, medicine, recycling, and sustainability.

In a growing tradition at Barker College, two students focused their study on bees. Julia developed a creative way to record and compare pollen diversity in bee hives, and Gus explored the difference that venom extraction methods makes on bee venom potency. Another project of interest to the Agricultural Science community was Samuel’s research into the impact of glyphosate (the active ingredient in Roundup®) on rumen microbes involving experimentation with an artificial rumen in the laboratory. Two projects explored antibacterial properties of preparing natural products for skin application. Tilapia fish skin and Aloe vera are known for topical applications and Alana and Joe used similar established techniques to provide scientific answers to open questions about these products that are often debated online. Ultraviolet light can also be used for antibacterial sterlisation and this was the topic of Will’s research in 2023. A particularly novel project, worthy of subsequent investigations, was undertaken by Sam where he sought to understand the impact of diet on mammalian cells through comparing muscle composition of grain and grass-fed beef. This opens the door for further investigation of the relationship between diet and muscle composition in beef cattle and considering the application across various mammalian species including humans. Finally Georgia cleverly modelled ocean acidification in a fish tank with interesting results, and Henry applied his Chemistry background to target novel methods of recycling PET plastics using various Zinc catalysts for maximum environmental impact.

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What’s all the buzz about? An investigation into the diversity and distribution of pollen within the hive Julia Disney Barker College Purpose: This paper aimed to examine how bees in urban beehives, with access to diverse floral sources, deposit, and store foraged pollen from the corbicula in frames within the brood box of managed hives. Design/methodology/approach: Two beehives from two locations in an urban setting were selected. Frames were removed and five cells were selected from each hive. The colour was recorded and counts of the 20 most common pollen species present were recorded using a specified protocol. Comparisons were able to be made within and between the hives. Findings: Two key findings of the paper included; that there was diversity between the pollen at the two hive locations, but also high diversity within each of the hives. Research and practical implications: Pollination is an unintended consequence of bee foraging behavior. A protocol for effectively measuring pollen in hives allows for better understanding of where and how bees forage. Considerable research related to biodiversity and agriculture can utilise the methods of this project. Social implications: Pollination from bees is relied on for one-third of global food supply. With increased monoculture farming, food supplies may be at risk especially in developing countries. Originality/value: As there was limited research and literature available, the protocol for determining species of pollen present in the cells allowed for a meaningful description and comparison had to be designed specifically for this project. It is hoped that further research will be able to utilise this technique to build on this novel field. Keywords: Pollen, diversity, bees, distribution, hive, flora, pollination Paper type: Research paper

Literature Review The European Honeybee (Apis mellifera) is crucial for the pollination of flora across the globe, with its importance required for both agricultural purposes (providing pollination) and economic purposes (producing honey, wax and royal jelly) (Wueppenhorst et al., 2022). As scientists turn their attention to restoring bee health (Goblirsch et al., 2018) understanding foraging and the deposition behaviour is a critical first step. The impact on bees from the lack of floral diversity Bees forage for pollen and nectar as a source of protein and carbohydrates and store it in the hive (Somerville, 2000). As shown in Figure 1, the pollen is collected in the corbicula which are located on the hind legs of bees (Jürgen Kottmann). Incidentally, when collecting pollen for foraging, they transfer pollen to the female part of the flower, pollinating the flower. Pollination is therefore an unintended consequence of their foraging behaviour, and it is relied on for one-third of global food supply (Food and Agriculture Organisation, 2021).

Figure 1: Corbicula or ‘pollen sacs’ on hind leg of bee (Jürgen Kottmann – Stock.adobe.com)

Sadly, we have realised too late the significance of bees due to their world-wide decline in relation to the crop production crisis (Environment UN, 2017). A major contributing factor in the decline of bees is colony collapse disorder. Through poor bee diet, brought about by limited diversity in the flora as a consequence of broadscale agricultural monocultures, that is a single plant/flower species dominating the foraging area (Watson & Stallins, 2016) Bees are limited in their range from the hive to a radius between 5km to 6km (Beekman & Ratnieks,

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Scientific Research in School Volume 5 Issue 1 2023 2000), so it is observed that in large scale monoculture plantations such as a canola field or pine plantation, the bees are restricted in their foraging with little diversity of pollen, leading to an inadequate nutritional supply and, in many cases, advances to colony collapse disorder in surrounding hives. Urban beehives are flourishing in comparison to rural beehives as urban colonies are able to forage on diverse flower species in gardens and reserves. It is unsurprising that rural beehives are worse affected by their urban counterparts, as in urban environments, the use of pesticides is limited, and floral diversity is stronger (through the availability of parks and gardens) compared to the monocultures and highly intensive use of pesticides of the agricultural environment (Mahé et al., 2021). Results from a study conducted by Samuelson et al. (2020), revealed that “honeybees exhibit lower colony performance in strength in rural areas, adding to the growing evidence that modern agricultural landscapes can constitute poor habitat for insect pollinators” (p.1). This underlines the importance of floral diversity available for bees and other pollinators.

Scientific Hypothesis Urban beehives will contain diverse groups of pollen, but the individual cells may be separated so that each cell has relatively uniform stores of pollen.

Methodology This project collected data from two managed flow hives (see honeyflow.com.au) in Sydney, Australia, one in Hornsby, New South Wales (located 34km from the Sydney Central Business District) and the other in Thornleigh, New South Wales (28km from the Sydney Central Business District). A single frame was extracted from Hive A (Hornsby) and Hive B (Thornleigh) as they were similar in urban setting with bushland in proximity and were both hives of Apis mellifera (European Honeybee). Once the frames were extracted from the beehives a transect was taken from each hive (shown in figure 2). As extracting the frames and removing honey is a typical process for commercial and hobby hives and no bees (invertebrates) were harmed or otherwise negatively impacted by the extraction of the brood frame containing the pollen, there were minimal ethical concerns.

Deposition patterns of pollen within the hive The pollen store of a beehive is vital for the survival of the colony, as the lipids (fats and oils) in the pollen are “important in honeybee development, nutrition and reproduction” (Manning, 2001 p.60). The pollen is stored as a protein-rich food source for developing larvae and for the rest of the colony and is generally located “on the frame where most of the unsealed brood is” (Dreller & Tarpy, 2000), for easy access. Discussions surrounding the foraging behaviours of bees and the deposition patterns and organisation of pollen within beehives globally, isn’t well understood in the scientific community, but is necessary in developing an understanding of colony development, nutrition potential impact on pollination. However, in 2007, Michigan State University released a website with information regarding pollination, where it was found that “Individual bees tend to focus on one kind (one species) of flower at a time”. This foraging behaviour could possibly connect with literature surrounding uniformity of pollen cells, and if a single bee deposits one load of pollen in one cell.

Scientific Research Question What is the distribution and diversity of pollen within cells in the pollen store of a brood frame in an urban beehive?

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Figure 2: Extracting the transect of pollen store from Hive A

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Figure 3: Pollen cells from ‘Hive 1’ in frame from brood box

When in the laboratory, five individual cells across the two transects were selected ensuring colour variation. This method of selection was chosen to potentially obtain variation of pollen within each cell (Figures 3 and 4). The Munsell Soil colour book (University of Georgia, 2016) was used to inspect the pollen cells to obtain the colour and hue of the pollen sample (Appendix 1) to precisely define the colours to allow for comparisons in future studies. Individual pollen cells were then sampled using a different sterile needle for each cell, ensuring that the sample was sterile and had not been contaminated. The pollen from each cell

was then mounted in glycerine on separate microscope slides (Appendix 2) following a method described by Wodehouse, 1933. Each slide was examined under a light microscope at a magnification of 100x, where an image was taken using a device that connected from the microscope to a laptop. A grid was added to the 10 images externally through a website where every second row and second column was blanked out, creating a checkerboard pattern (Appendix 3) establishing a replicable protocol for counting pollen. Pollen grains were then only counted if the grain was two-thirds inside of the

Figure 4: Pollen cells from ‘Hive 2’ in frame from brood box

Figure 4: Pollen cells from ‘Hive 2’ in frame from brood box Science Extension Journal • 43

Scientific Research in School Volume 5 Issue 1 2023 grid square. It was ignored if it was not at least twothirds inside. In this method, the counting of pollen continued until 100 grains were counted across the grid of each individual slide to analyse pollen diversity within the hive and obtain results, to receive a sufficient sample to be representative of the proportions of the pollen through the whole cell. In order to present the results to allow for meaningful analysis, the 20 most dominant (by number present in the 10 observed cells) species of pollen were counted. These 20 varieties of pollen grains were classified into distinct groups #1 - #20 by the naked eye rather than the correct identification which may allow for future identification of the specific names of the pollen (however this was beyond the scope of this investigation).

Results Table 1 Pollen Diversity Data - Grain # refers to individual pollen types, slide xx is the microscope slide and cell number, the colours are swatches from the pollen and matched with a hue and colour from the Munsell Soil Colour Book and the tone of green refers to the number of grains.

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Scientific Research in School Volume 5 Issue 1 2023 Table 1: Pollen Diversity Data - Grain # refers to individual pollen types, slide xx is the microscope slide and cell number, the colours are swatches from the pollen and matched with a hue and colour from the Munsell Soil Colour Book and the tone of green refers to the number of grains

Photo

Grain #

Location A Slides 1A

10A

#1 #2 #3

Location B Slides

15A

20A

10B

15B

20B

24 3

10 87

#8 #9

#10

#11

#12

#13

#14

#15

46 2

#16

#17

#18 #19 #20

2 75

1 3 78

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Discussion The data from Table 1 aligns with the literature and prior research suggesting that “urban areas may provide a diverse range of pollen sources” (Samuelson et al. 2020 p.747.) The data from Table 1 shows the diverse range of flora available in an urban setting shared between the two urban settings. As 20 distinct groups of pollen were identified (derived from just 10 cells), this indicated the sheer abundance of pollen within the urban environment that was tested, verifying the hypothesis of this paper. Figure 5 was created to visualise the microscopic images of the pollen cells from Hive A in Hornsby, this clearly displays the variety pollen types located within each cell. An interesting observation gathered from the data in Table 1, is how the abundance of one particular grain, Pollen Grain #14, in Cell 5A, is the majority amongst the other species of pollen in the cell. The exact type of pollen is unknown, but this could suggest that an individual bee returned to one cell to deposit the pollen from the same species of flower. This is consistent with literature which describes that “individual bees tend to focus on one kind of flower at a time” (Michigan State University 2007 paragraph. 2). Cells 5A and 15B are the only cells that show a high percentage of uniformity (over 85% of

the same pollen grain in one cell) within the table of results, being 94% and 89%, which is consistent with this literature. However, as eight out of ten pollen cells sampled did not have a high percentage of uniformity, indicating that in this set of data, the deposition of pollen by the bees, did not follow the behaviour suggested by Michigan State University, 2007, paragraph. 2. To further explore this varying consistency, a larger sample size would be beneficial to these results. With more hives tested from various locations around the country, more data could be tested to compare the types of pollen and if the deposition patterns are similar or different. By increasing the sample size and broadening the locations (e.g. urban, rural-urban, rural settings), it would be interesting to discover whether results differed and if there was uniformity of pollen within hives in rural settings, due to monocultures in certain rural areas. It is helpful to note that as Cell 5A has the highest uniformity (94% of the pollen grains in the cell were grain #14), the colour of the pollen cell was the brightest, more prominent colour of yellow amongst the other nine cells. This is to be expected, as most of the pollen cell is one species, making the colour more vivid. The Munsell Soil Colour Book was used in the determination of the correct colour of the individual

Figure 5: Image showing the variety of pollen from the same transect.

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Scientific Research in School Volume 5 Issue 1 2023 pollen cells. By adding the colour of each of the cells into the table it clearly shows immediately the difference in each of the pollen cells. There were similarities between three of the pollen cells, as 1A, 1B and 10B shared the same colour hue and number when matched against the Munsell Soil Colour Chart. Although none of the three cells all shared the same species of pollen, it could be suggested that with a variety of different pollen types, a similar colour was produced. Further research could involve swatching pollen cells and matching against a colour chart with more variety, improving the accuracy of the correct colour. It appeared that the colour of the pollen cells from Hive B in Thornleigh consisted of darker tones, whereas in Hive A, lighter tones were noted. Grains #1, #5 and #7 were present in most cells in Hive B, indicating that perhaps those grains, in particular, could have influenced the darker tones that were established across the cells of Hive B. Cell 20A was the most diverse among the other cells, with six different groups of pollen grains counted, compared to 10A and 15B only having a variation of three groups. Grain #5 appeared in seven out of the ten cells observed. It would be interesting to identify the exact type of pollen grain and examine it against the most common flora found in location A and location B to see whether the bees gravitate towards a certain species for a definite reason, for example, nutritional value. Or, if the flower of grain #5 is most common in the environment surrounding both of the hives and the bees forage for this particular type of pollen for convenience. The variation in the colour of the pollen cells between the two locations (indicated by the colour swatches at the top of Table 1) and the data presented in the table, indicates the variety of flora that is available in urban areas and, generally, that the pollen is deposited in a mix of species, rather than one specific type of pollen grain in each cell (aside from 5A). For future projects stemming from this research paper, future researchers may delve into identifying the pollen grains to create graphs and obtain statistical data that could be used to create a more indepth scientific report. Having the option to perform statistical tests on future projects would also benefit the project and future results with a chi-squared test comparing the categorical distributions of pollen being most likely to be helpful. For future papers incorporating similar ideas, the study of the nutritional value of pollen in urban and rural settings would be of value to scientists globally, to signify the importance of bee colony health and the impact that

monoculture environments may have on some of the most important pollinators on the planet.

Conclusion This research paper looked at the diversity and distribution of pollen in the pollen store of two urban beehives. High levels of diversity were found both within and between the two hives, which correlates with the current literature. A protocol for effectively measuring pollen in hives allows for better understanding of where and how bees forage, which is needed as pollination is necessary for the world's food supply. Since there is little information available on the distribution and storage of pollen in urban beehives, the methodology created in this study can be utilised for future research for measuring pollen in hives. The findings of this project showcase the diversity in flora in urban areas and discusses how limited diversity in flora, particularly in monoculture farming, affects bee colony health.

Acknowledgements I would like to thank Dr Alison Gates for helping to produce the idea for this paper and for all of the helpful insights, guidance, and encouragements throughout the process of this project. To Dr Matthew Hill I am very grateful for the assistance provided when structuring this report as well as the creative development of the results table. And finally, to Dr Katie Terrett, thank you for being a great mentor over the length of this process.

References Abou-Shaara, H 2014, ‘The foraging behaviour of honey bees, Apis mellifera: a review’, Veterinární Medicína, vol. 59, no. No. 1, pp. 1–10. Add Grid To An Image File - Online Tool 2021, Yo Motherboard. An Introduction To The Brood Nest 2020, PerfectBee, viewed 18 June 2023, <https://www.perfectbee.com/ahealthy-beehive/inspecting-your-hive/inspecting-andunderstanding-the-broodpattern#:~:text=Pollen%20is%20often%20placed%20imm ediately,as%20the%20source%20of%20protein.>. Anderson, KE, Carroll, MJ, Sheehan, T, Mott, BM, Maes, P & Corby‐Harris, V 2014, ‘Hive‐stored pollen of honey bees: many lines of evidence are consistent with pollen preservation, not nutrient conversion’, Molecular Ecology, vol. 23, no. 23, pp. 5904–5917. Beekman, M & Ratnieks, FLW 2000, ‘Long-range foraging by the honey-bee, Apis mellifera L.’, Functional Ecology, vol. 14, no. 4, pp. 490–496. Donkersley, P, Rhodes, G, Pickup, RW, Jones, KC & Wilson, K 2014, ‘Honeybee nutrition is linked to landscape

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Scientific Research in School Volume 5 Issue 1 2023 composition’, Ecology and Evolution, vol. 4, no. 21, pp. 4195–4206. Dreller, C & Tarpy, DR 2000, ‘Perception of the pollen need by foragers in a honeybee colony’, Animal Behaviour, vol. 59, no. 1, pp. 91–96. Environment, UN 2017, UNEP emerging issues: global honey bee colony disorder and other threats to insect pollinators, UNEP - UN Environment Programme. Ghosh, S, Jeon, H & Jung, C 2020, ‘Foraging behaviour and preference of pollen sources by honey bee (Apis mellifera) relative to protein contents’, Journal of Ecology and Environment, vol. 44, no. 1. Goblirsch, M, Lee, K, Jamestown, S, North, D, St, Paul, S, Paul, C, Otto, M, Smart, J, Wu-Smart, Hayes, G, Ramesh, S, Dennis & Louis, S 2018, Why Does Bee Health Matter? The Science Surrounding Honey Bee Health Concerns and What We Can Do About It, December, viewed 18 June 2023, <https://www.cast-science.org/wpcontent/uploads/2018/12/QTA20171_Bee_Health_565CB 839D149E.pdf>. Kottmann, J n.d., Portrait einer Honigbiene mit Pollenhöschen im Detail. Mahé, C, Jumarie, C & Boily, M 2021, ‘The countryside or the city: Which environment is better for the honeybee?’, Environmental Research, vol. 195, p. 110784. Manning, R 2001, ‘Fatty acids in pollen: a review of their importance for honey bees’, Bee World, vol. 82, no. 2, pp. 60–75. Michigan State University 2007, Pollination, Native Plants and Ecosystem Services, Michigan State University. Mihail Garbuzov & Francis 2013, ‘Quantifying variation among garden plants in attractiveness to bees and other flower-visiting insects’, vol. 28, no. 2, pp. 364–374, viewed 17 June 2023, <https://besjournals.onlinelibrary.wiley.com/doi/10.1111/ 1365-2435.12178>. Ruedenauer, FA, Wöhrle, C, Spaethe, J & Leonhardt, SD 2018, ‘Do honeybees (Apis mellifera) differentiate between different pollen types?’, in O Rueppell (ed.), PLOS ONE, vol. 13, no. 11, p. e0205821. Samuelson, AE, Gill, RD & Leadbeater, E 2020, ‘Urbanisation is associated with reduced Nosema sp. infection, higher colony strength and higher richness of foraged pollen in honeybees’, vol. 51, no. 5, pp. 746–762, viewed 17 June 2023, <https://link.springer.com/article/10.1007/s13592-02000758-1>. Somerville, D 2000, supplementary feeding.

Honey

bee

nutrition

and

University of Georgia 2016, Munsell.pdf. Vaughan, DM & Calderone, NW 2002, ‘Assessment of pollen stores by foragers in colonies of the honey bee, Apis mellifera L.’, Insectes Sociaux, vol. 49, no. 1, pp. 23–27. Watson, K & Stallins, JA 2016, ‘Honey Bees and Colony Collapse Disorder: A Pluralistic Reframing’, Geography Compass, vol. 10, no. 5, pp. 222–236.

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Wodehouse, RP 1933, ‘Preparation of Pollen for Microscopic Examination’, Bulletin of the Torrey Botanical Club, vol. 60, no. 6, p. 417. Wueppenhorst, K, Eckert, JH, Steinert, M & Erler, S 2022, ‘What about honey bee jelly? Pesticide residues in larval food jelly of the Western honey bee Apis mellifera’, Science of The Total Environment, vol. 850, p. 158095.

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Appendices

Appendix 2:Pollen from Hive A mounted in Glycerine on microscope slides.

Appendix 1: Munsell Soil Colour + Hue Data sampled from pollen cells. Pollen slide #

Colour Hue + Number:

2.5Y 7/8

5Y 8/8

10A

2.5Y 8/8

15A

10YR 7/8

20A

10YR 6/8

2.5Y 7/8

10YR 5/8

10B

2.5Y 7/8

15B

7.5YR 5/8

20B

10YR 8/8

Colour:

Appendix 1: Gridded Pollen Image of cell 20A

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Glyphosate and the effect that it has on rumen microbes and agricultural production Samuel Speed Barker College Purpose: This paper aims to test the effect of Glyphosate the active ingredient in Roundup® on the diversity of rumen microbes. This is significant as if Roundup®, a widely used herbicide kills Rumen microbes or inhabits their function, it would have a large impact on the cow’s ability to digest carbohydrates and proteins, and hence reduce their productivity. Methodology: To test this two experimental groups were set up, each with three samples, one where glyphosate in the form of Roundup® was added, and one where it was not. Samples from the six rumens were used to inoculate agar plates, which were then observed and counts of diversity were taken. Findings: Upon comparing the results from the roundup and non-roundup groups, it was found that on average the roundup group had a significantly lower diversity of microorganisms than the nonroundup group, 2.3333 microorganisms per agar plate compared to the 4 microorganisms per agar plate in the non-roundup group. Research Limitations: In this experiment there was a lack of time, a larger sample size produced by further repetition would improve the reliability of this research. Practical and Social implications: The fact that glyphosate impacts microorganism variety in the rumen, means that it should not be used in agriculture, hence the use of it as a herbicide should be banned. Value: This paper investigates the impact of glyphosate on the diversity of rumen microbes and the implications of the reduced diversity on cow productivity and efficiency. Keywords: Artificial rumen, Glyphosate, Microorganisms Paper type: Research paper

Literature Review Herbicides are chemicals used to manipulate or control undesirable vegetation. Herbicide application occurs most frequently in row-crop farming, where they are applied before, during or after planting to maximise crop productivity by minimizing other vegetation, and later by improved harvesting (US EPA, 2015). Glyphosate is the active agent in Roundup®, a nonselective herbicide that kills most plants (NPIC, 2018). The introduction of herbicide tolerant genetically engineered (GE) crops in 1996, made it possible to utilise glyphosate as a post-emergence herbicide, hence dramatically extending the time period during which glyphosate-based herbicides could be applied. Alongside the increases in crop productivity, this led to a dramatic increase in the use of glyphosate in agriculture. In the United States of America, the use of herbicides with glyphosate as the active ingredient increased from 18,144 tonnes in 1995, to 125,384 tonnes in 2014 (Benbrook, 2016).

Glyphosate is absorbed into the foliage and transported with sugars to metabolically active regions in the plant where it interferes with the shikimic acid pathway, which is responsible for the metabolism of carbohydrates and Amino acids (Manthey et al., 2022; Leistner, 1999). Since glyphosate disrupts protein and carbohydrate digestion in plants there is potential for it to do the same in the cow’s rumen when ingested via grazing, by interrupting microbes that are also responsible for the digestion and absorption of Amino acids and carbs in the Rumen. Since carbohydrates are a vital source of energy (Dairy NZ, 2023), Amino acids act as building blocks to form proteins needed for body functions, growth, reproduction and milk production (Pescatore, 2017). If Glyphosate does impact microbe functioning or variety, it could reduce the efficiency and productivity of farms, the very thing that it is meant to increase. Which alongside the International Agency for Research on Cancer (IARC), classifying glyphosate as “probably carcinogenic to humans” in March 2015, where regular users of Roundup® had a 41% greater chance of developing lymphoma, than those who never used

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Figure 1: Standard RUSITEC system setup. Source: (Riede, 2016)

it, could lead to the phasing out of glyphosate in agriculture (Stokstad, 2021). One way of testing the effects of glyphosate on microbes in the rumen is via an artificial rumen (AR) setup knows as Rumen Simulation Technique (RUSITEC). The RUSITEC has been present for many years and is now a well-established and tested semi-continuous ‘in vitro model’ (models that provide insight into cells and microorganism’s behavior). They have been used for investigating ruminal fermentation, the digestion of feed, gases produced, the microbiota of the rumen, metabolism changes and the effect of pharmaceuticals (Admin, 2022). Figure 2 is a standard RUSITEC system setup, with a buffer solution, an electromotor, effluent flask, gas bag, solid phase and a liquid phase. Food in the rumen is chewed and mixed with saliva in the mouth, it then moves down through the esophagus into the Rumen. The digestive system is broken down into four compartments, the rumen, reticulum, omasum and abomasum. (Moran, 2005) This can be seen in Figure 3 which shows a diagram of a cow’s digestive system.

Figure 2: A Cow’s digestive system. Source: (Linn & Otterby, 2021)

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The rumen which is the first compartment, is full of microbes and enzymes secreted by them. There are three main microbes, bacteria, protozoa and fungi, which are responsible for carrying out fermentation in the rumen. These microbes release enzymes that split peptide bonds, which are covalent bonds, that hold together amino acid chains which form proteins, hence they break down protein into amino acids and then ammonia. The amino acids are then absorbed through the rumen wall into the bloodstream, and microbial protein is synthesised from nitrogen sources like ammonia and urea in the liver. The microbes are then digested as microbial protein in the Abomasum (Moran, 2005). In the Abomasum, fats are digested by hydrochloric acid, pepsin and lipase. They start off as complex carbohydrates but are broken down into simple sugars by enzymes secreted by microbes. These simple sugars are then taken up by microbes and turned into Volatile fatty acids (VFA) which provide the cow with 70% of its energy (Moran, 2005). Microflora in the rumen are influenced by the combination of substrate, physical structure, and pH environment. Hence the pH of rumen is highly influential on types and number of each rumen microbe and therefore needs to be controlled. With more acidic pH greatly reducing the number of microbes in the liquid and solid state of rumen fluid. (Guo et al., 2021). The pH of the rumen usually sits at around 6 to 7. However VFA produced from microbes breaking down carbohydrates can bring down the pH impacting the functioning of microbes and therefore the cow’s ability to break down feed. Saliva has several roles in digestion, it makes chewing and swallowing easier, but significantly it acts as a buffering agent against acidity, with a pH of

Scientific Research in School Volume 5 Issue 1 2023 around 8.5 it counteracts acidity produced by VFA, keeping the rumen’s pH in the ideal range of 6 to 7 (Moran, 2005).

food in grams a cow gets daily) you get 32 grams of feed, hence it is proportional to the amount of feed a cow would get.

The artificial saliva in this experiment was created following the suggested composition for synthetic saliva outlined by McDougall in 1948: Table 1: The components and their concentration in a synthetic saliva

Components of saliva Sodium bicarbonate Disodium phosphate Sodium chloride Potassium chloride Anhydrous calcium chloride Anhydrous magnesium

Concentration (g/L) 9.8 9.3 0.47 0.57 0.04 0.06

With a high pH of around 8.2 (due to the large amount of sodium bicarbonate), it solves the problem of acidity in the artificial rumen.

Figure 3: Frozen feed capsules

After the 2 weeks, a microscope slide was prepared with the contents of the AR and a methylene blue stain; it was then viewed and photos were taken. Figures 4, 5, and 6 show examples of microorganisms seen. The presence of the microorganisms allowed for the next steps of the method to proceed.

Scientific Research Question Does Glyphosate in Roundup® inhibit the growth and function of microbes in a ruminant digestive system?

Scientific Hypothesis The artificial rumen samples that contain Roundup® will have a lower variety of microbes than those where Roundup® is not present.

Methodology Pilot Study A single reagent bottle was autoclaved, and inoculated with 10 grams of solid inoculum, collected from a cow’s rumen at an abattoir in northern New South Wales, eating a ration with a ratio of 80:20 grain to grass ratio. The conical flask was then placed into the AR setup for 2 weeks, being ‘fed’ with a capsule twice, one at the start and one a week in. This food consisted of 100 grams of grass, 200 grams of grain and 500ml’s of artificial saliva pulverised in a food processor, then separated into six equal amounts, and frozen. This was done so that they were the same volume, around 32 grams per capsule. This amount was chosen as cows eat between 22.5kg and 25kg’s of food daily (Fischer & Hutjens, 2019), and the average Rumen capacity of a mature cow is 120 litres (Moran, 2005). 500ml divided by 120 litres is 0.00416667, multiplied by the 7500 (the amount of

Figure 4: Bacteria stained with methylene blue stain, under a microscope on 100x magnification.

Figure 5: Fungi stained with methylene blue under a microscope on 100x magnification

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Figure 6: Protozoa stained with methylene blue stain, under a microscope on 100x magnification

Preparation of samples Six 500ml reagent bottles were filled with 300ml of artificial saliva each, they were then autoclaved with the lid on loosely, so the saliva and bottle were both autoclaved in one go. The AR’s content from the pilot study was then stirred thoroughly and the contents were measured and separated into six equal amounts of 50ml, which were then poured into each of the six reagent bottles, hence inoculating each of the reagent bottles with microflora originally present in the rumen inoculum, turning them into AR’s. A stopper and swan neck was fitted onto each of the AR’s to prevent microorganisms other than those from the rumen inoculum skewing the results. Rumination and feeding The reagent bottles were then placed onto an orbital shaker, inside of an incubator as seen in Figure 7. The orbital shaker was set to 181 rotations per minute and the incubator was set to 38 degrees Celsius. The artificial rumens were then 'fed' one frozen capsule every three days, for the remainder of the study, the contents of which were previously outlined in the pilot study.

The six reagent bottles were then split evenly into two separate experimental groups, one where Roundup® was added and one where it was not added. The Roundup® in this experiment had a glyphosate concentration of 360 grams per litre. Roundup® with this concentration of glyphosate is generally applied at a rate of 10ml per litre of water (Sinochem Australia 2015). For the experimental group with Roundup®, this application rate was applied, 5ml of Roundup® was added to a reagent bottle containing 500mls of distilled water. This bottle was then shaken to mix the two liquids, a 1 ml pipette was then used to transfer one millilitre into the three artificial rumens, in the Roundup® experimental group. They were then left in the incubator for a week, being fed once over that time period. Preparation of Agar plates Sampling from the AR’s occurred twice, once after the first week of feeding and again a week after Roundup® was added. The process of sampling was as follows; A sterile swab was dunked in rumen fluid (RF) from the first artificial rumen, it was then used to inoculate the tryptic soy agar plate, spreading the RF in even strips along the base plate. The lid was then tapped to the base plate and the base plate was labelled either Rumen test 1, 2 or 3 or Rumen control 4, 5, or 6 control, depending on which experimental group they were in ‘test’ being the roundup group and ‘control’ being the non-roundup group. An agar plate without any rumen fluid was also set up, to ensure the purity of the agar. The agar plates were then placed in an incubator set to 38 degrees Celsius for 5 days, to allow for the microorganisms to grow. Analysis methodology: After the 5 days the agar plates were observed, and photographs were taken. Two counts of the diversity of microorganism colonies were taken and recorded in a table, One before (count 1) and one after (count 2). Different colonies were determined by observing their characteristics of shape, size, colour, surface appearance and texture, and finding differences between them (CDC, 2023). Figure 9 provides an example of some of the different colonies. Counts of abundance were not recorded as there was a large level of abundance on all six of the agar plates, and no observable difference between them. A t-test was then conducted on the diversity counts collected.

Figure 7: Incubator and Orbital shakeup for the artificial rumens

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Scientific Research in School Volume 5 Issue 1 2023 Table 2: Diversity counts of microbes on the agar plates from the Roundup® artificial rumens

Test 1 Test 2 Test 3 Test Average

Diversity count Week 1 Week 2 5 2 5 3 4 2 4.6666 2.3333

Table 3: Diversity counts of microbes on the agar plate from the non-Roundup artificial rumens

Figure 8: A photo of an Agar plate with microbes on it grown from AR Test 2

Results The results of this experiment appear below as Tables 2-4. Table 2 shows 2 diversity counts of microbes grown on agar plates from the three test AR’s which were treated with Roundup®. The first diversity count was taken before the group was treated with Roundup® 1 week into the experiment, the second count was taken a week after the AR’s had been treated with Roundup®. Table 3 shows diversity counts of the microbes grown on the three agar plates, sampled from the AR’s not treated with Roundup®, taken at the same time as the AR’s with Roundup®. Table 4 shows a t-test used to determine whether there is a significant difference between the means of the second diversity count of the AR’s treated with Roundup® and the ones not. The results of this experiment appear below as Tables 2-4. Table 2 shows 2 diversity counts of microbes grown on agar plates from the three test AR’s which were treated with Roundup®. The first diversity count was taken before the group was treated with Roundup® 1 week into the experiment, the second count was taken a week after the AR’s had been treated with Roundup®. Table 3 shows diversity counts of the microbes grown on the three agar plates, sampled from the AR’s not treated with Roundup®, taken at the same time as the AR’s with Roundup®. Table 4 shows a t-test used to determine whether there is a significant difference between the means of the second diversity count of the AR’s treated with Roundup® and the ones not.

Test 1 Test 2 Test 3 Test Average

Diversity count Week 1 Week 2 3 5 5 3 5 4 4.3333 4

Table 4: A T-test comparing the second diversity counts of both the Roundup and non-roundup AR

Sample size Mean Standard deviation Alpha value T-value P value

AR Roundup 3 2.3333

AR no Roundups 3 4

0.67

2 0.05 -2.5 0.033383

Null hypothesis: the means are the same. Alternative hypothesis: the means are different. The P-value = 0.033383, and since 0.033383 is less than the alpha value of 0.05, the null hypothesis is rejected and the alternative hypothesis is accepted, hence the result is significant.

Discussion The hypothesis is that the artificial rumen samples that contain Roundup® will have a lower variety of microbes than those where Roundup® is not present. The results showed that when grown on an agar plate, there was a significant decrease in the level of diversity where Roundup® was and was not present. Using a student’s t-test the p-value was found to be 0.03383, which is less than the alpha value of 0.05, hence the null hypothesis that the means are the same could be rejected, and the alternative hypothesis that the means are different could be accepted. The mean in the experimental group where Roundup® was added, decreased from 4.66 to 2.33 microorganisms per agar plate when Roundup® was added. Compared to the decrease of 4.33 to 4 microorganisms per agar plate in the experimental group where Roundup® was not added, there is a

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Scientific Research in School Volume 5 Issue 1 2023 significant difference. This difference and the rejection of the null hypothesis, means that it can be reliably concluded that on average when Roundup® is added to an artificial rumen, it will significantly decrease the variety of microorganisms present, hence the results support the hypothesis of the experiment. The decrease in the variety of microorganisms present in the artificial rumen is significant, due to the implications that it could have on a cow’s digestion of feed and therefore its ability to extract nutrients from the food. Since each of the main micro biotic groups of fungi, protozoa and bacteria, are important in the digestive process. Fungi degrades fibre in the form of plant cell walls totally, by producing large amounts of the enzymes cellulases and hemicellulases, which breakdown cellulose and hemicellulose, the main components of plant fibre (Akin & Borneman, 1990; Matthews et al., 2018). This releases the energy stored in the complex plant carbohydrates, otherwise inaccessible to the animal, converting them to short-chain fatty acids that are absorbed by the animal for its energy needs through the rumen wall (Moraïs & Mizrahi, 2019). Protozoa are important for carbohydrate breakdown in the rumen, due to the carbohydrate-active enzymes they produce like glycosyl hydrolases 5. They also produce enzymes that digest rumen fungi, releasing chitin from the fungi’s cell membrane (Williams et al., 2020). This is important as chitin, can easily be converted into chitosan via enzymatic or chemical deacetylation, which improves feed efficiency in cows (Ocampo et al., 2019; Elieh-Ali-Komi & Hamblin, 2016). Bacteria carry out most of the digestion of sugars, starch, fibre and protein for the cow. (Russell, 2007) Microbial proteins, are derived from several species of microorganisms but are most commonly derived from fungi, bacteria, yeast or microalgae (Bojana Bajić et al., 2022). Hence a decrease in the variety of one or all of the micro biotic groups would have a large impact on the cow’s ability to convert feed into energy and protein, which would then have implications for the efficiency and productivity of production for farmers. Therefore, the fact that glyphosate likely leads to a decrease in the variety of microorganisms present in the artificial rumen is significant. Alongside the International Agency for Research on Cancer (IARC), classifying glyphosate as “probably carcinogenic to humans” in March 2015, where regular users of Roundup® had a 41% greater chance of developing lymphoma than those who never used it, (Stokstad, 2021) should lead to the phasing out of glyphosate as a widely used herbicide.

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In this experiment there were two unexpected experimental results; the increase in microorganism diversity on the agar plate swapped with RF from AR control four from 3 in diversity count one to 5 in the second diversity count, and the overall decrease of the control group’s average from 4.333 to 4. These results could have occurred due to a number of reasons. The samples taken did not effectively represent the diversity of microorganisms present in the rumen contents of the AR’s leading to a lower diversity being shown on the agar plates, or in the second agar plate swapped from AR control 4 contamination could have occurred, hence increasing the number of microorganisms present. To determine whether these events were anomalies or representative of the results, it would have been preferable to do an increased level of replication for each of the experimental groups, increasing the sample size and the reliability of the results. However, due to a limited amount of time to complete the experiment, this increased level of repetition was not possible. There are three key ways, to continue my research, enriching the scientific literature of Glyphosate’s impact on rumen microbes diversity and cows in general. The first is increasing the repetition which would improve the reliability of the results and provide a more definitive idea of glyphosate’s impact. The second is identifying the colonies lost when glyphosate is added, growing a pure culture of that organism, lawn a plate with that pure culture, then using a glyphosate-soaked filter paper disk to do zone of inhibition tests on that organism, to determine whether it was glyphosate that killed the organism and how much of the organism it killed, and hence the impact that glyphosate has had on the cow. Then finally do this method with inoculum from cows that are on a fully grass-fed ration to determine whether glyphosate effects the microbe population in grassfed cows differently or the same as those on a mixture of grain and grass.

Conclusion In conclusion, this study has demonstrated that the diversity of rumen microorganisms in artificial rumens is negatively impacted by the addition of Roundup® and its active ingredient glyphosate. These results indicate that the hypothesis can be cautiously accepted. However, unexpected results, highlighted the need for an increase in the repetition within experimental groups, where increasing the sample size would make the results more reliable.

Scientific Research in School Volume 5 Issue 1 2023 The method employed in this research task effectively answers the research question of does glyphosate in Roundup® inhibit the growth and function of microbes in a ruminant digestive system? It found via the use of a student’s t-test that there was a significantly lower diversity of rumen microbes sampled from an artificial rumen-containing Roundup® when compared to samples from an artificial rumen that did not contain Roundup®. The implication of glyphosate decreasing the variety of rumen microorganisms in artificial rumens could potentially have a large impact on the cow’s ability to produce energy and protein, and hence its productivity and efficiency. This is due to the importance of microorganisms, particularly bacteria, protozoa and fungi in a cow’s digestive system. Where decreases in the variety of the microorganisms impacts their ability to function and hence the cow’s ability to digest. Hence further repetition of this experiment should be done to provide a full picture of glyphosate’s effects on microbe diversity, if this yields the same results as this experiment. Glyphosate should no longer be used as a herbicide due to the negative implications that it has on a cow’s productivity and efficiency, and it being a probable carcinogen in humans.

Acknowledgements I would like to thank Dr Alison Gates for helping me with formulating and carrying out my method, and Dr Matthew Hill for proofreading and provide feedback on my project write up.

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Stokstad, E 2021, Why Europe may ban the most popular weed killer in the world, Science.org, viewed 6 February 2023, <https://www.science.org/content/article/whyeurope-may-ban-most-popular-weed-killer-world>. Williams, CL, Thomas, BJ, McEwan, NR, Pauline Rees Stevens, Creevey, CJ & Huws, S 2020, ‘Rumen Protozoa Play a Significant Role in Fungal Predation and Plant Carbohydrate Breakdown’, vol. 11, viewed 18 June 2023, <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC720098 9/>.

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Oh, it’s o-fish-al: An investigation into the sterilisation techniques of tilapia fish skin Alana Tully Barker College Purpose: This paper aims to investigate the most effective sterilisation technique for fish skin, specifically for its application in burns, to determine the method that results in the least amount of microbial growth after 48 hours. Nine different sterilisation techniques were identified from relevant literature and thoroughly examined for their efficacy in this context. Methodology: For each technique, a sterilised scalpel was used to cut one 2.0cm² square off the Tilapia fish’s skin. The fish-skin samples then underwent one of 9 sterilisation treatments, and these included Betadine (10% Povidone Iodine), Chlorhexidine Gluconate (2%), Ethanol (100%), Boiling Water, Hydrogen Peroxide (30%), Methylated Spirits (70% ethanol), UV Light, Autoclave, Ultrasound. Each treated sample was then placed on an agar plate to culture for 48 hours at 37.5°c. The microbial growth was then observed and recorded. Findings: Sterilisation treatments such as Hydrogen Peroxide and Autoclaving produced almost no microbial growth, while techniques such as Boiling Water and Ultrasound produced significant microbial growth. Implications/Limitations: Although sterilisation techniques hold promise, their potential effects on collagen, crucial for burn healing, remain not entirely comprehended. This necessitates additional research to validate their appropriateness for treating burn wounds. Keywords: Tilapia, sterilisation, skin, burns treatment Paper Type: Research Paper

Literature Review Burns A burn is an injury to the skin or other organic tissue primarily caused by heat or due to radiation, radioactivity, electricity, friction or contact with chemicals (World Health Organisation, 2018). In the United States, an estimated 1.25 million individuals sustain a burn injury each year (Summer et al., 2007). Human skin is a complex arrangement of layers containing distinct anatomical features, as shown in Figure 1. Severity varies from first-degree burns affecting only the skin's outer layer to third-degree burns damaging all skin layers and underlying tissues, potentially causing life-threatening complications (Mayo Clinic, 2021). Prompt treatment is crucial to mitigate risks and promote healing. The seriousness of burns is determined by their depth, extent, location, the patient's age and health, and the cause of the burn (MedlinePlus, 2023). Burn depth is shown in Figure 2.

Figure 2: Layers of Human Skin Source: (Byju.com, 2020)

Figure 1: SEQ Figure \* ARABIC 2 - Different degrees of burns Source: (Wikimedia.com, 2022)

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Scientific Research in School Volume 5 Issue 1 2023 Current treatment The field of burn treatment has seen substantial advancements in recent years, as various studies offer a wide range of techniques. Implementing rigorous isolation protocols and employing inventive antibiotic therapies is paramount in managing severe burns, particularly in cases where patients are afflicted with multidrug-resistant gram-negative microorganisms (Branski et al., 2009). Foreseeing the intensity of thermal injuries and directing therapeutic protocols is a crucial facet that leverages a particular illness severity scoring system. (Fouchard et al., 2000). In addition, the study of percutaneous microwave ablation vs radiofrequency introduces thermal injury treatments like radiofrequency (RFA) and microwave ablation (MWA) as viable options for burn care (Poulou et al., 2015). The latter aims to destroy diseased tissue while preserving the surrounding healthy tissues. In addition, the discourse extends to ground-breaking approaches for burn injuries that are not conducive to primary closure. This investigation underscores the application of bioengineered skin substitutes to expedite wound resolution, with the primary aim being tissue regeneration that reinstates the impacted region's architectural and functional properties. This comprehensive methodology to burn management symbolises burn care's multidisciplinary and progressive character. (Macri & Clark, 2009). Issues with treatment Current treatments for burns, while innovative, are not without their issues and challenges. A study of hydrogels presents the use of hydrogels in wound dressings. While hydrogels offer unique properties such as high-water content, softness, flexibility, and biocompatibility, sterilisation under optimal conditions may not always be achievable with these materials (Caló & Khutoryanskiy 2015). Photodynamic therapy (PDT) exhibits its own limitations. Although PDT can eliminate pathogenic microorganisms, its antimicrobial effect subsides once the light source is deactivated. Furthermore, PDT may not sufficiently differentiate between microbial cells and host tissue (Hamblin & Hasan, 2004; Dai, Huang & Hamblin, 2009). Quinacrine is a molecule proposed to sterilise burn sites, as shown in Figure 3. Using quinacrine for sterilisation faces another set of issues (Lippes, 2002). Despite quinacrine's safe and effective application, the interruption of clinical trials due to concerns from the World Health Organization underscores potential complications with this method. Lastly, Silver compounds have been

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commonly used as antimicrobial agents, but the emergence of bacterial resistance to these compounds could undermine the effectiveness of silver-based sterilisation methods. These challenges highlight the complexities of burn treatment, emphasising the need for continuous innovation and improvement in the field. (Ibrahim et al., 2020).

Figure 3: Quinacrine Molecule

Benefits of fish skin Research in recent years has highlighted the potential benefits of using fish skin in treating burns and other wounds. In their comprehensive review, Subhan et al., (2021) stress the high collagen content in fish skin, ranging from 70-80%, and its instrumental role in various phases of wound healing, including haemostasis, inflammation, proliferation, and remodelling. Collaborating with different research facilities in Pakistan, the authors suggest that fish collagen holds substantial promise as a biomaterial for developing medical products and therapeutics due to its unique characteristics. Further highlighting the advantages of fish skin in wound management revealed that burns addressed with fish skin recuperate significantly more rapidly than those treated with porcine small-intestine submucosa following a 28-day period. This insinuates that fish skin might potentially serve in treating other wound categories, such as diabetic foot ulcers and other persistent full-thickness wounds, considering the observed expedited healing durations (Baldursson et al., 2015). Complementing this investigation, a comprehensive evaluation of collagen hydrogels extracted from Nile tilapia skin was performed for wound dressing applications. While the study was oriented towards hydrogel fabrication rather than immediate skin application, it was discerned that the collagen hydrogel dressing significantly hastened the recuperative process of deep second-degree burn wounds compared to commercially available

Scientific Research in School Volume 5 Issue 1 2023 products. This research, led by diverse Chinese institutions, further bolsters the prospective use of fish skin, particularly its collagen constituent, as a plausible alternative for enhancing wound healing procedures and results (Ge et al., 2020). Fish skin applications and collagen structure Researchers have been exploring the potential of fish skin, particularly its high collagen content, for use in burn treatments. Fish skin waste contains a notable collagen content of 5%-30% and may be applied as wound dressings for burn injuries. There is a substantial amount of fish waste produced in Indonesia (23.52 million tons in 2016), and there is a possibility of repurposing this waste into burn dressings. Researchers emphasise the economic benefits of using collagen as a wound dressing, given its low cost and the reduced time required for application, making it a feasible option for use in developing countries and educational settings (Afifah et al., 2019). A study by Hamada, Nagashima, and Shiomi (2001) from the Department of Food Science and Technology in Japan provides a detailed method for extracting collagen from fish skin. This replicable procedure, which utilises accessible chemicals such as sodium chloride, is critical for the broader application of fish skin in wound treatments. In a more specific case study, Tilapia fish skin was used as a xenograft to treat burns on a 3-year-old boy. It was found that Tilapia skin adhered well to the burned area, exhibited no antigenicity or toxicity, and promoted complete re-epithelialization of the wound, further highlighting the potential of fish skin, and particularly its collagen content, in burn treatment applications (Almeida Costa et al., 2019). No mention was made of the sterilisation techniques used. Despite this considerable body of research on the applications of Tilapia fish skin on burns patients, it is evident that a significant gap remains in the current scientific literature, specifically pertaining to the understanding and application of sterilisation techniques for fish, highlighting a critical area that requires further exploration. Pilot study and collagen structure Initially, this report aimed to investigate which sterilisation technique would produce the highest amount of collagen retained in the sample. Various methodologies were investigated to see whether collagen could be accurately measured in the high school laboratory. We investigated various protocols involving water and fat-soluble dyes and centrifuging samples, hoping to visualise the collagen component

of the sample. Unfortunately, the laboratory techniques that were suggested in the literature required laboratory equipment that was not available in the school context. Accordingly, whilst the literature review on collagen was retained, the scientific investigation focussed on the relative efficacy of the sterilisation techniques, intending to provide a foundation for later work to determine the impact of each technique on collagen degradation. The most likely technique for this future work would involve preparing paraffin mounted thin sections and the structural investigation of collagen-containing cells using light microscopy.

Scientific Research Question What is the most effective sterilisation technique for fish skin?

Scientific Hypothesis Common sterilisation techniques that are beneficial in other circumstances can be applied to the novel case of fish skin sterilisation with similar or improved results to conventional iodine sterilisation.

Methodology In order to compare the efficacy of fish skin sterilisation techniques, a standard preparation methodology needed to be established, as well as clear metrics to define and measure efficacy. Preparation of samples For each technique, a sterilised scalpel was used to cut 1x 2.0 cm2 square off the Tilapia fish’s skin. This particular sample had been obtained from Hornsby Seafood Shop, stored in the Barker College Science Laboratory Freezer, and undergone no further cleaning (other than prior to purchase). The fish-skin samples then underwent one of 9 sterilisation treatments (and one control) described below. They were placed approximately in the centre of a clean agar plate using sterilised forceps. The plates were sealed and incubated at 37.5°c for 48 hours before being removed for photography and observation. Sterilisation methods The nine sterilisation techniques are described below, and the alphabet notation (A, B, C etc.) was used to denote each group: 1

(A) Betadine (10% Povidone Iodine), (B) Chlorhexidine Gluconate (2%), (C) Ethanol (100%), (D) Boiling Water, (E) Hydrogen

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Scientific Research in School Volume 5 Issue 1 2023 Peroxide (30%), (F) Methylated Spirits (70% ethanol): The sample was placed in a sterile petri-dish and fully submerged with the liquid for 10 minutes. 2

(G) UV Light: The sample was placed in a sterile petri-dish and placed into a UV chamber at 254 nanometres for 10 minutes.

(H) Autoclave: The sample was placed in a sterile petri-dish and placed into the autoclave at 121ºC and 40 Psi for 15 minutes

(I) Ultrasound: The sample was placed in a sterile glass tube and fully submerged in an Ultrasound bath for 10 minutes.

(J) Control: The sample was placed in a sterile petri-dish

Determining efficacy In order to evaluate the efficacy of the study results, a critical assessment of the white microbial growth was executed. Microbial growth was visually examined for its size and structure, indicating the sterilisation treatment's effectiveness. The collected data was then juxtaposed with that in Figure A, which served as a baseline for the experiment due to iodine being the most common technique currently. The comparative analysis facilitated a better understanding of the efficacy of the fish skin sterilisation techniques.

Figure 4: Group A - Iodine Fish Sample - Baseline for other results

Results The results are presented in Figure 5. Each sample was ranked in order from best (most effective sterilisation) to worst (least effective sterilisation) as follows: 1. 2. 3. 4. 5. 6. 7. 8. 9.

E C H B F G J I D

Figure 4: Fish skin sample results for each Group B-J

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Discussion The study's primary focus was to ascertain the efficacy of different sterilisation techniques, as indicated by the zones of microbial growth, and assess their suitability for the application of fish skin to heal burns. The collected data shows that figures 5-C and 5-E demonstrated the most effective sterilisation, while figures 5-D and 5-I had the worst outcomes. Figure 5-G was an outlier with an unusual observation of the presence of bugs yet a lack of microbial growth. Figures 5-C and 5-E demonstrated the most effective sterilisation, the almost non-existent zones of microbial growth in these figures illustrate that the applied sterilisation techniques were successful. This suggests that the sterilisation techniques used in these instances successfully eliminated or significantly reduced the presence of microorganisms. Hydrogen peroxide (Figure 5- E) is a good sterilisation technique because it can kill a wide range of microorganisms, including bacteria, viruses, fungi, and spores. When hydrogen peroxide comes into contact with biological material, it breaks down into water and oxygen molecules, releasing free radicals that can damage the cellular components of microorganisms and disrupt their metabolic processes. This oxidative effect makes hydrogen peroxide an effective sterilising agent. Depending on the application, it can be used in various forms, such as vaporised hydrogen peroxide (VHP) or hydrogen peroxide solution. Moreover, hydrogen peroxide is suitable for sterilising delicate and heat-sensitive materials that cannot withstand the high temperatures of autoclaving. Autoclaving (Figure 5-C) is a good sterilisation technique because it uses high-pressure steam to kill microorganisms. The combination of high temperature and pressure inside the autoclave creates an environment that effectively denatures proteins and disrupts cell membranes, ensuring the destruction of most microorganisms, including heat-resistant spores. Autoclaving is highly effective in sterilising a wide variety of materials, including those that are heat-resistant and nonporous. It provides a quick and efficient sterilisation process. However, autoclaving is unsuitable for heat-sensitive materials, such as plastics, specific electronic components, and some chemicals, as they may be damaged or altered by high temperature and pressure.

Figure 5-G, interestingly, was an outlier. The bugs suggest that UV light is not a good sterilisation technique; however, the lacking presence of microbial growth suggests that it was successful. The bugs could be non-pathogenic; hence, their presence does not equate to microbial contamination. Alternatively, these bugs might be pathogens, but the growth medium or conditions might not support their proliferation. Another possibility is that the sample contained antimicrobial substances, inhibiting microbial growth but ineffective against the bugs. A detailed identification of the bugs and testing the sample for antimicrobial substances could provide more insights into this anomaly. Figures 5-D and 5-I, on the other hand, showed the worst sterilisation results; they showed large zones of microbial growth, signifying ineffective sterilisation. Potential reasons could include improper handling or storage conditions after sterilisation, leading to recontamination. Alternatively, the initial microbial load on the fish skin might have been too high for the sterilisation process to manage effectively. The sterilisation techniques used were not as effective, perhaps due to insufficient duration or intensity or because they were inappropriate for the types of microbes on the fish skin. It is also possible that the fish skin in these samples had properties that made it more susceptible to microbial colonisation, such as a lower collagen content or a different collagen structure. Boiling water (Figure 5-D) effectively kills many microorganisms but may not provide complete sterilisation for all types of materials and microorganisms. Although it can kill most vegetative bacteria, viruses, and fungi, it is ineffective against heat-resistant bacterial spores and certain viruses. Furthermore, boiling water is primarily suitable for heat-resistant materials and may cause damage or alterations to heat-sensitive materials like plastics, certain chemicals, and delicate equipment. As a result, boiling water may not achieve the required level of sterility, particularly in critical applications such as medical and laboratory settings. Ultrasound (Figure 5-I) is not a direct sterilisation technique but is commonly used for cleaning and disinfection. While ultrasound can help dislodge and remove debris, it is ineffective in killing all microorganisms. Its primary function involves generating high-frequency sound waves that create microscopic bubbles in a liquid called cavitation. When these bubbles collapse, localised pressure and agitation occur, aiding in cleaning and removing

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Scientific Research in School Volume 5 Issue 1 2023 contaminants. However, the effectiveness of ultrasound in killing microorganisms is limited. It may not eliminate all bacteria, viruses, and spores, particularly in complex or hard-to-reach areas. While the findings of this study present promising implications for sterilisation techniques, they come with inherent limitations that must be acknowledged. A significant concern arises from the fact that these sterilisation methods may adversely affect collagen, a vital component in the wound healing process, particularly in burns. Despite our understanding of the impact these techniques have on the pathogenic load, the effect they might have on the structural integrity and biological functionality of collagen is not thoroughly studied in the context of burn wounds. This creates an inherent uncertainty, as potential negative impacts on collagen could impede wound healing or lead to sub-optimal healing outcomes. The results of this experiment open several avenues for further research. More extended incubation periods could be investigated as some microorganisms have a longer doubling time and may not form visible colonies within the initial incubation period. Extending this period could reveal a more comprehensive spectrum of microorganisms present on the fish skin. More fish skin samples and different sterilisation techniques could also make for interesting further studies; techniques such as silver nanoparticles and ethylene oxide may be used, and utilising a broader array of samples and sterilisation methods could identify a more effective technique or combination of methods. Finally, fish skin is rich in collagen, an essential component for wound healing. Assessing how sterilisation techniques influence the collagen content or structure could offer valuable information. For instance, some methods might damage the collagen structure, reducing its efficacy for burn treatment. Similarly, high collagen content might act as a protective barrier against microbial contamination. Expanding on these research directions can significantly enhance our understanding of fish skin sterilisation for its application in burn healing. Such insights can provide the basis for developing standardised processing methods that maintain the therapeutic properties of fish skin while ensuring its microbiological safety.

Conclusion My research project explored the most effective sterilisation technique for fish skin that exhibited the least amount of microbial growth after 48 hours. Each sample was sterilised using a different technique and

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then incubated for 48 hours. Each sample was then observed for its microbial growth and subsequently photographed. Data was collected by observing the level of growth of the samples and comparing them to the current Iodine treatment. The data showed that Hydrogen Peroxide and Autoclave techniques produced better sterilisation than Iodine.

References Admin 2020a, Skin Diagram with Detailed Illustrations and Clear Labels, BYJUS, BYJU’S, viewed 17 June 2023, <https://byjus.com/biology/skin-diagram/>. 2020b, Skin Diagram with Detailed Illustrations and Clear Labels, BYJUS, BYJU’S, viewed 18 June 2023, <https://byjus.com/biology/skin-diagram/>. Afifah, A, Suparno, O, Haditjaroko, L & Tarman, K 2019, ‘Utilisation of fish skin waste as a collagen wound dressing on burn injuries: a mini review’, IOP Conference Series: Earth and Environmental Science, vol. 335, no. 1, p. 012031. Almeida Costa, B, Maciel, E, Júnior, L, Odorico De, M, Filho, M, Fechine, F, Amaral De Moraes, M, Raimundo, F, Júnior, S, Araújo, M, Soares, N, Becker, M & Rocha, S 2019, Use of Tilapia Skin as a Xenograft for Pediatric Burn Treatment: A Case Report. Baldursson, BT, Kjartansson, H, Konrádsdóttir, F, Gudnason, P, Sigurjonsson, GF & Lund, SH 2015, ‘Healing Rate and Autoimmune Safety of Full-Thickness Wounds Treated With Fish Skin Acellular Dermal Matrix Versus Porcine Small-Intestine Submucosa’, The International Journal of Lower Extremity Wounds, vol. 14, no. 1, pp. 37–43. Branski, LK, Al-Mousawi, A, Rivero, H, Jeschke, MG, Sanford, AP & Herndon, DN 2009, ‘Emerging Infections in Burns’, Surgical Infections, vol. 10, no. 5, pp. 389–397. Burns: MedlinePlus Medical Encyclopedia 2023, Medlineplus.gov, viewed 18 June 2023, <https://medlineplus.gov/ency/article/000030.htm#:~:text =Calm%20and%20reassure%20the%20person,help%20re lieve%20pain%20and%20swelling.>. Caló, E & Khutoryanskiy, VV 2015, ‘Biomedical applications of hydrogels: A review of patents and commercial products’, European Polymer Journal, vol. 65, pp. 252–267. Chen, J, Gao, K, Liu, S, Wang, S, Elango, J, Bao, B, Dong, J, Liu, N & Wu, W 2019, ‘Fish Collagen Surgical Compress Repairing Characteristics on Wound Healing Process In Vivo’, Marine Drugs, vol. 17, no. 1, p. 33, viewed 16 October 2021, <https://www.mdpi.com/16603397/17/1/33>. Dai, T, Huang, Y-Y & Hamblin, MR 2009, ‘Photodynamic therapy for localized infections—State of the art’, Photodiagnosis and Photodynamic Therapy, vol. 6, no. 34, pp. 170–188. Demling, RH 2008, ‘Burns: what are the pharmacological treatment options?’, Expert Opinion on Pharmacotherapy, vol. 9, no. 11, pp. 1895–1908.

Scientific Research in School Volume 5 Issue 1 2023 File:Burn Degree Diagram.svg - Wikimedia Commons 2022a, Wikimedia.org, viewed 17 June 2023, <https://commons.wikimedia.org/wiki/File:Burn_Degree_ Diagram.svg>. File:Burn Degree Diagram.svg - Wikimedia Commons 2022b, Wikimedia.org, viewed 18 June 2023, <https://commons.wikimedia.org/wiki/File:Burn_Degree_ Diagram.svg>. Fouchard, N, Bertocchi, M, Roujeau, J-C, Revuz, J, Wolkenstein, P & Bastuji-Garin, S 2000, ‘SCORTEN: A Severity-of-Illness Score for Toxic Epidermal Necrolysis’, Journal of Investigative Dermatology, vol. 115, no. 2, pp. 149–153. Ge, B, Wang, H, Li, J, Liu, H, Yin, Y, Zhang, N & Qin, S 2020, ‘Comprehensive Assessment of Nile Tilapia Skin (Oreochromis niloticus) Collagen Hydrogels for Wound Dressings’, Marine Drugs, vol. 18, no. 4, p. 178. HAMADA, Y, NAGASHIMA, Y & SHIOMI, K 2001, ‘Identification of Collagen as a New Fish Allergen’, Bioscience, Biotechnology, and Biochemistry, vol. 65, no. 2, pp. 285–291. Hamblin, MR & Hasan, T 2004, ‘Photodynamic therapy: a new antimicrobial approach to infectious disease?’, Photochemical & Photobiological Sciences, vol. 3, no. 5, p. 436. Ibrahim, A, Hassan, D, Kelany, N, Kotb, S & Soliman, M 2020, ‘Validation of Three Different Sterilization Methods of Tilapia Skin Dressing: Impact on Microbiological Enumeration and Collagen Content’, Frontiers in Veterinary Science, vol. 7.

Moiemen, N, Lee, K & Joory, K 2014, ‘History of burns: The past, present and the future’, Burns & Trauma, vol. 2, no. 4, p. 169. Muthumari, K, Anand, M & Maruthupandy, M 2016, ‘Collagen Extract from Marine Finfish Scales as a Potential Mosquito Larvicide’, The Protein Journal, vol. 35, no. 6, pp. 391–400. Poulou, LS, Botsa, E, Thanou, I, Ziakas, PD & Thanos, LT 2015, ‘Percutaneous microwave ablationvsradiofrequency ablation in the treatment of hepatocellular carcinoma’, World Journal of Hepatology, vol. 7, no. 8, p. 1054. Sikorski, ZE, Scott, DN, Buisson, DH & Love, RM 1984, ‘The role of collagen in the quality and processing of fish’, C R C Critical Reviews in Food Science and Nutrition, vol. 20, no. 4, pp. 301–343. Subhan, F, Hussain, Z, Tauseef, I, Shehzad, A & Wahid, F 2020, ‘A review on recent advances and applications of fish collagen’, Critical Reviews in Food Science and Nutrition, vol. 61, no. 6, pp. 1027–1037. Summer, GJ, Puntillo, KA, Miaskowski, C, Green, PG & Levine, JD 2007, ‘Burn Injury Pain: The Continuing Challenge’, The Journal of Pain, vol. 8, no. 7, pp. 533–548. World Health Organization 2018, Burns, Who.int, World Health Organization: WHO, viewed 18 June 2023, <https://www.who.int/news-room/factsheets/detail/burns>.

Jafari, H, Lista, A, Siekapen, MM, Ghaffari-Bohlouli, P, Nie, L, Alimoradi, H & Shavandi, A 2020, ‘Fish Collagen: Extraction, Characterization, and Applications for Biomaterials Engineering’, Polymers, vol. 12, no. 10, p. 2230. Kirsner, RS, Margolis, DJ, Baldursson, BT, Petursdottir, K, Davidsson, OB, Weir, D & Lantis, JC 2019, ‘Fish skin grafts compared to human amnion/chorion membrane allografts: A double‐blind, prospective, randomized clinical trial of acute wound healing’, Wound Repair and Regeneration, vol. 28, no. 1, pp. 75–80. Lima, E, Moraes, M, Costa, B, Uchôa, A, Martins, C, Moraes, M, Rocha, M & Fechine, F 2001, ‘Treatment of deep second-degree burns on the abdomen, thighs, and genitalia: use of tilapia skin as a xenograft’, Revista Brasileira de Cirurgia Plástica, vol. 35, no. 2, pp. 243–248, viewed 12 March 2022, <http://rbcp.org.br/details/2755/en-US/treatment-of-deepsecond-degree-burns-on-the-abdomen--thighs--andgenitalia--use-of-tilapia-skin-as-a-xenograft>. Lippes, J 2002, ‘Quinacrine sterilization: the imperative need for American clinical trials’, Fertility and Sterility, vol. 77, no. 6, pp. 1106–1109. Macri, L & Clark, RAF 2009, ‘Tissue Engineering for Cutaneous Wounds: Selecting the Proper Time and Space for Growth Factors, Cells and the Extracellular Matrix’, Skin Pharmacology and Physiology, vol. 22, no. 2, pp. 83– 93. Mayo Clinic 2018, Burns - Symptoms and causes, Mayo Clinic.

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Antibacterial properties and applications of raw Aloe vera to inhibit bacterial growth Joe Entwistle Barker College Purpose: This paper aims to examine antibacterial properties of raw Aloe vera when applied as the skin of the plant or the inner pulp. There is a presence of the anthraquinone aloe-emodin in both the skin and inner pulp of the plant and both products are used for their antibacterial properties. Shelflife issues limit the feasibility of extracting of anthraquinones, therefore it is important to determine the efficacy of natural application of different components of the raw plant to inhibit bacterial growth. Design/methodology/approach: Fourteen nutrient agar plates were prepared with a Staphylococcus epidermidis solution spread on the surface. Five plates had three pieces of Aloe vera skin (fifteen in total) placed on each plate while five others instead had Aloe vera pulp placed in the same manner. The five from each treatment along with four controls were incubated for 48 hours and then observed for inhibition zones where bacteria did not grow. Findings: The results concluded that the pulp had an observable zone of inhibition against Staphylococcus epidermidis after 48 hours with an average of 1.82mm radius, however the skin had no zone of inhibition with some plates having bacteria build up around the skin. There was a significant difference in the mean zones of inhibition (t=13.8869, p<0.0001). Additionally, it was found that the zone of inhibition created by the pulp decreased the longer it was left after extraction and before application. Research limitations/implications: Additional research is needed increasing the scale of the experiment with more samples and also relating the potential impacts of water content on antibacterial potency, slight alterations that could increase anthraquinones present in the skin, and how efficacy may decrease over time. Practical implications: The results demonstrate a potential of the aloe pulp to be used as a raw and natural alternative to other anti-bacterial substances while still lending to further research on utilising compounds present in the skin via raw application. Social implications: In developing countries, Aloe vera may be able to be used as a community science option to reduce the use of antibiotics or other antibacterial substances that are expensive or becoming less effective due to adaptation. Originality/value: To the author's knowledge, previous works had not focused on Staphylococcus epidermidis and the effects of applying the Aloe vera pulp vs the skin. This research was valuable at adding to the information on natural Aloe vera application as lots of focus had been previously put on extracting and isolating the compound without attempting to maximise the effectiveness of the raw, fresh plant with the presence of the compound. Keywords: Aloe vera, aloe-emodin, anthraquinone, antibiotic resistance, antibacterial. Paper type: Research paper

Literature Review Aloe vera background Community science and education is a cheap resource for developing practical use and understanding of scientific concepts through empowering individuals to improve health practices (Wandersman, 2003). Aloe vera (barbadensis miller) (Figures 1 & 2) is a perennial succulent medicinal plant part of the Liliaceae family with over 400 species in the world (Boudreau and Beland, 2006). It is proven to have antimicrobial properties which can be effective in the treatment of common epidermal bacterial infections (Coopoosamy and Magwa, 2006;

Park and Jo, 2006; Olaleye and Bello-Michael 2005; Malik et al., 2016; Choi and Chung 2003; Bashir et al., 2011; Dong et al., 2019). Aloe vera is used primarily as a topical medicinal treatment with uses including, healing and soothing of sunburn, skin wounds, and even anti-fungal treatments (Maan et al., 2017). The plant has been linked to historical uses in ancient cultures in Africa, Asia and South America to heal external wounds (Park and Jo, 2006). This relates to the project as it describes the varying applications and uses for the plant and its potential to be used as a more wide-scale anti-bacterial substance that is readily available and doesn’t need manufacturing or altering. The presence of

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Scientific Research in School Volume 5 Issue 1 2023 anthraquinones aloe-emodin in the plant’s leaves has been studied and recognised as an active ingredient responsible for antibacterial properties, validifying further research in this area (Borges-Argáez et al., 2017).

Figure 1: Aloe vera plants, Source: (Melchor, 2021)

Figure 3: Decrease in approved antibacterial drugs, Source: (Hill, 2016)

Figure 2: Cross section of Aloe vera Source: (Maan et al,. 2019)

Antibacterial resistance There has been a significant increase in antibiotic resistance amongst common bacteria and hence the need to test other options for antimicrobial properties (Frieri et al., 2017). There has been discourse around this problem of antibiotic resistance and the need for further research into novel treatment options such as naturally derived treatments. This relates to Aloe vera-related research on the antibacterial efficacy of Aloe vera and its practical use as a substitute for commonly used antibiotics. Similarly on this Hutchings et al.’s (2019) article on the history of antibiotics looks at the initial implementation of antibiotics as an anti-bacterial medicine in 1928 due to the discovery of penicillin. They write about the gradual decline in antibiotic resistance and how the decreased discovery of new antibiotics has led to an “antimicrobial resistance crisis”. All of this means it's a highly worthwhile pursuit to seek to find effective alternatives such as the antibacterial properties of Aloe vera.

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Past research Much research has been conducted on Aloe vera with Coopoosamy and Magwa (2006) investigating aloeemodin in a variety of Aloe vera called Aloe excelsa which has had limited scientific studies on its properties, unlike other varieties. The report tested nine bacteria and determined there is antibacterial activity and inhibitory effects from aloe-emodin against all tested bacteria including Staphylococcus epidermidis which will be used in the investigation. Despite the brief amount of information, it is a relevant reference as it demonstrates the antimicrobial activity of Aloe vera which allows for further investigation on how to best apply this medicinal plant to achieve maximum inhibitory results. Olaleye and Bello-Michael (2005) investigate the varying microbial activity between the aloe skin and aloe pulp by measuring the appearance of zones of inhibition. The data shows how the effect varies depending on the bacteria tested. This is similar to the research project as it is testing the difference in microbial properties between different parts of the plant. However, they did not test Staphylococcus epidermidis which allows for this research project to investigate the inhibitory effect on this specific bacterial pathogen, one that is a common skin bacterium causing infections to open wounds. Anthraquinones have the potential to be used as pharmacological tools due to their antimicrobial activity. Aloe-emodin which is a type of

Scientific Research in School Volume 5 Issue 1 2023 anthraquinone naturally occurs in the Aloe vera plant and therefore reflects the inhibitory potential of aloe against bacterial pathogens. This relates to this research project due to the discussion on the key component of Aloe vera which gives it its antibacterial potency. Aloe vera has been researched regarding the varying components of the and the role these have in the acclaimed antimicrobial properties with discussion being on the potential of the plant for further medical use but noting the need for further investigation and evaluation on the effect of each component in the plant (Choi et al., 2003). Bashir et al. (2011) study the comparison between Aloe vera and traditional antibiotics in inhibiting skin infections. The study showed the ability of Aloe vera extracts to inhibit the growth of both gram-positive and negative bacteria. This study relates to this research project as it demonstrates the ability of bacterial inhibition in Aloe vera and its need for further research. Additional research has examined the impacts of extracting the anthraquinones from the plant material, with Sadiq et al. (2022) discovering a decrease in anthraquinone aloin concentration by 50% at a temperature of 50 degrees Celsius. This article also notes a ‘substantial reduction’ in aloin concentration when the pH was raised to 6.7. Due to this, the efficacy of isolating the active ingredient is reduced and hence an approach to a more natural application is validified.

Scientific Research Question What is the most effective method of application of Aloe vera to inhibit Staphylococcus epidermidis bacterial growth?

Scientific Hypothesis Direct application of pulp of Aloe vera will produce a larger zone of inhibition on a Staphylococcus epidermidis lawned plate than peeled skin applied over the same area.

Methodology Primary investigation: Comparing application of skin vs pulp The workspace was sterilized with ethanol before interaction with the agar plates. Fourteen nutrient agar plates were then labelled according to the treatment. A sterile pipette was then used to drop 0.5mL of Staphylococcus epidermidis (debilitated strain) from Southern Biological onto fourteen nutrient agar plates. The bacteria were then spread using a sterile plate spreader to form a thin layer of

the bacteria solution. Four of the fourteen agar plates were left as the controls (i.e., only bacterial lawn). Aloe vera was prepared by slicing the skin into fifteen 1cm 2 pieces on a dissection board using a scalpel. The pulp was then also collected by slicing it using a scalpel into fifteen 1 cm2 pieces. To set up each treatment, five bacteria agar plates had three pieces of skin applied using sterile tweezers and evenly placed on each plate. Then, the same was repeated with the pulp to achieve five plates with a pulp treatment also. The plates were then all sealed using tape and left inverted. They were placed in the incubator and left for 48 hours at 37 degrees Celsius. After this time, observations were recorded, and zone of inhibition radii (mm) were recorded using vernier callipers. Analysis methodology A t-test (two-tailed, independent samples) was used to determine the significance between the zone of inhibitions. Data was entered into an online calculator to receive the p-value, t statistic and alpha value to determine the significance between the means of the two groups. Further testing: Shelf-life of Aloe vera pulp In response to the results of the Phase 1 testing, it was deemed important to see if these results were consistent even after the pulp was extracted and stored for 24, 48 and 72 hours. The workspace was sterilized with ethanol. Aloe vera pulp was cut into nine 1cm2 pieces using a sterile scalpel. They were then placed in a sterilised petri dish which was sealed using tape. After 24 hours, a sterile pipette was then used to drop 0.5mL of Staphylococcus epidermidis onto 1 nutrient agar plate. The bacteria were then spread using a sterile plate spreader to form a thin layer of the bacteria solution. Three pieces of the Aloe vera pulp were applied using sterile tweezers onto the prepared agar plate and distributed evenly before sealing. The agar plate was then incubated at 37 degrees Celsius for 48 hours. The zone of inhibition radii (mm) was recorded after this time using vernier callipers. The process was then repeated, with the pulp being left for 48 hours and then 72 hours before being placed on the prepared agar plates.

Results Results from testing Primary testing demonstrated an observable zone of inhibition against the Staphylococcus epidermidis bacterium from the Aloe vera pulp application ( average= 1.82mm) (Figure 3). Results from the Aloe vera skin application showed no observable zone of

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Scientific Research in School Volume 5 Issue 1 2023 inhibition against the bacterium however an increased buildup was observed around the edges of the skin (Figure 4). Also, the control with no Aloe vera present displayed an even bacterial lawn, validating control of external variables and the ability of pulp to inhibit bacterial growth.

Further testing: Shelf life Further testing was conducted to determine the effect of storing the pulp of Aloe vera once extracted from the plant and its impacts on the inhibitory ability against Staphylococcus epidermidis. Pulp stored for 24 hours recorded an inhibition zone of 2.8mm on average across the three pieces. Both pulp stored for 48 (see Figure 5) and 72 hours (see Figure 6) displayed an average zone of inhibition of 0.4mm. All clearly had a zone of inhibition; however, it decreased after being stored for further than 24 hours. Table 2: Averaged results from further testing

Figure 4: Zone of inhibition of one pulp plate

Time left before application (hours) 0 (n= 15) 24 (n=3) 48 (n=3) 72 (n=3)

Average zone of inhibition (mm) 1.82 2.8 0.4 0.4

Figure 5: Zone of inhibition of one skin plate Table 1: Averaged results from the trial

Application of Aloe vera Pulp (n= 15) Skin (n=15) Control

Average zone of inhibition (mm) 1.82 0 0

Figure 6: Zone of inhibition of pulp stored for 48 hours.

Data analysis A student’s t-test (two-tailed, independent samples) was carried out to determine the significance between the zone of inhibition of the pulp compared to the skin. It was found that t=14.0649 with a p-value of <0.0001 which is less than the alpha value of 0.05. Therefore, it can be said that the mean zone of inhibition of Aloe vera pulp is significantly greater than that of Aloe vera skin.

Figure 7: Zone of inhibition of pulp stored for 24 hours.

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Discussion Primary testing A valid methodology was undertaken to receive the given results, following one similar to that of Olaleye and Bello-Michael (2005) whereby plates were inoculated with a bacterial culture prior to application and zones of inhibition were observed and calculated after incubation. As seen in Table 2 and confirmed by the t-test, the zone of inhibition created by the Aloe vera (1.82mm) pulp against Staphylococcus epidermidis was significantly different to that of the skin which did not produce an observable zone of inhibition (t=14.06949, p<0.0001). Figures 3 and 4 demonstrate one of five plates tested for each group and reflect an overall similarity across each plate and corresponding application. It is evident that the active ingredient of aloe-emodin was able to inhibit bacterial growth through pulp application while not in the skin, despite being present in both parts of the raw plant (Dong et al., 2019). Through replication, the pulp is shown to result in zones of inhibition across each pulp application plate, however, varying measurements across each plate (see Appendix 1) suggest that possible errors during the set-up or data collection may result in the slight variation. For example, the movement of the pulp on the plate before measurement may have slightly skewed the reality of the inhibition zones. Despite this, there is still evidence to apply to the research question which supports the hypothesis that the pulp will result in a higher zone of inhibition against a Staphylococcus lawned plate than that of the skin. A proposed reason for the difference despite the presence of aloe-emodin in the whole Aloe vera leaf is that the water concentration between the two components whereby the pulp had a higher content allowed the anthraquinone of aloe-emodin to work more effectively through the aqueous component of the plant (Malik and Muller, 2016). Hence, it is logical in further testing to potentially examine how to still utilise the skin but through other methods such as grinding it into a paste and adding distilled water in order to simulate the water concentration that may have assisted the ability of the pulp to inhibit bacterial growth. Further testing The decrease in efficacy, as measured by the smaller zones of inhibition after 24, 48 and 72 hours, could be attributed to the desiccation of the pulp once extracted and hence the drop off in the inhibition zone after 24 hours. Also, there was visual evidence of desiccation between 24 hours and 48 hours (see

figures 6 & 7) which supports the claim that water concentration influences the ability of aloe-emodin to demonstrate its antibacterial potency as proposed earlier in this discussion. This increases the importance of future research on the influence of water content on the antibacterial potency of Aloe vera. Additionally, in terms of raw application, storage after extraction is shown to impact the inhibitory effects negatively suggesting that emphasis should be placed on extracting the components from the plant for immediate use. However, the results in further testing are limited due to the lack of repetition that was carried out, unlike the primary method. This could help further determine the impact of storage and allow further time intervals to be recorded in terms of their effect on antibacterial activity. Limitations As discussed previously, further replication could have been carried out on the second set of testing to increase validity and also receive a better understanding of the impact of storage on the pulp and the relation this had to the water content. Also, the slight variation in the results may have been a cause of inconsistent measurements due to the movement of the pulp on the agar plates, creating slight changes in the appearance and size of the zone of inhibitions. Because the zones were measured based off the pulp on the plate, the movement may alter the actual zone of inhibition measurement as the zone observed may have been caused by sliding of the pulp. Another limitation of this project was the limited time frame. This limited the ability to test multiple strains of bacteria and restricted the scope of the experiment. Despite receiving results on Staphylococcus epidermidis, it would’ve been beneficial to investigate the varying effects between strains as conducted in other experiments surrounding this topic (Olaleye and Bello-Michael, 2005). Despite the use of a common skin infection-causing bacteria, including bacteria that may cause internal infections could allow further consideration towards application (Surjushe et al., 2008). Implications and further research This research and other pieces of research in this field highlight the validity of medicinal plant application as an alternative antibacterial to traditional antibiotics, particularly with the rise in bacterial resistance to antibiotics. An examination of the impact of Aloe vera on other pathogens such as

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Scientific Research in School Volume 5 Issue 1 2023 viruses and protists could provide further ability to treat diseases caused by these pathogens and others. Further research into maximising the antibacterial potency of the natural plant would benefit the community science sphere and allow for simple and accessible treatments that are able to treat common infections and potentially more. Hence, further testing could include determining how to potentially use the Aloe vera skin to achieve inhibitory results, potentially through increasing water content and grinding the component to release the active ingredients. Similarly, as discussed above, more research could be carried out on the storage life of Aloe vera pulp once extracted as temperature has an effect on the extracted compounds rendering them difficult to keep in this isolated form. This could draw a relation to the effects of oxidisation or water content on the ability of the anthraquinones to effectively inhibit bacterial growth. The implications of this experiment could lead to a better response to the ongoing and growing antibiotics resistance crisis and provide relief to scientists attempting to manufacture further variations of antibiotics that do not have bacteria resistant to them in populations.

Conclusion This research project explored the effect of the application of raw Aloe vera on its antibacterial potency. The zone of inhibition created by the pulp against five agar plates with a Staphylococcus epidermidis was tested, with the same being repeated with the Aloe vera skin. Data was collected using vernier callipers to measure the zone of inhibition and collated in a table. A t-test was run and determined the average zone of inhibition caused by the pulp (1.82mm) was significantly higher than the average zone of inhibition (0mm) caused by the skin (t=13.8869, p<0.0001). This led me to accept the hypothesis that the direct application of pulp of Aloe vera will produce a larger zone of inhibition on a Staphylococcus epidermidis lawned plate than peeled skin applied over the same area. Also, research was conducted on the impact of storing the pulp after extracting it and prior to applying whereby the zone decreased from 2.8mm after 24 hours to 0.4 after both 48- and 72-hour intervals.

Acknowledgements I would like to thank Dr Alison Gates for her valuable assistance in the carrying out and testing component

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of this research and her guidance towards possible entry points for this topic. Also, I would like to thank Dr Matthew Hill for his support in collating data, data analysis and the scientific writing process.

References Bashir, A., Saeed, B., Mujahid, T.Y. and Jehan, N. (2011). Comparative study of antimicrobial activities of Aloe vera extracts and antibiotics against isolates from skin infections. African Journal of Biotechnology, [online] 10(19), pp.3835–3840. doi:https://doi.org/10.4314/ajb.v10i19. Borges-Argáez, R., Chan-Balan, R., Cetina-Montejo, L., Ayora-Talavera, G., Sansores-Peraza, P., Gómez-Carballo, J. and Cáceres-Farfán, M. (2019). In vitro evaluation of anthraquinones from Aloe vera (Aloe barbadensis Miller) roots and several derivatives against strains of influenza virus. Industrial Crops and Products, 132, pp.468–475. doi:https://doi.org/10.1016/j.indcrop.2019.02.056. Boudreau, M.D. and Beland, F.A. (2006). An Evaluation of the Biological and Toxicological Properties ofAloe Barbadensis(Miller), Aloe Vera. Journal of Environmental Science and Health, Part C, 24(1), pp.103–154. doi:https://doi.org/10.1080/10590500600614303. Choi, S. and Chung, M.-H. (2003). A review on the relationship between aloe vera components and their biologic effects. Seminars in Integrative Medicine, 1(1), pp.53–62. doi:https://doi.org/10.1016/s15431150(03)00005-x. Coopoosamy, R.M. and Magwa, M.L. (2006). Antibacterial activity of aloe emodin and aloin A isolated from Aloe excelsa. African Journal of Biotechnology, [online] 5(11). doi:https://doi.org/10.4314/ajb.v5i11.42978. Dong, X., Zeng, Y., Liu, Y., You, L., Yin, X., Fu, J. and Ni, J. (2019). Aloe‐emodin: A review of its pharmacology, toxicity, and pharmacokinetics. Phytotherapy Research, 34(2), pp.270–281. doi:https://doi.org/10.1002/ptr.6532. Frieri, M., Kumar, K. and Boutin, A. (2017). Antibiotic resistance. Journal of Infection and Public Health, [online] 10(4), pp.369–378. doi:https://doi.org/10.1016/j.jiph.2016.08.007. Futureearth.org.au. (2020). Community Science for Sustainability | Future Earth Australia. [online] Available at: https://www.futureearth.org.au/initiatives/communityscience-sustainability [Accessed 4 Feb. 2023]. Hill, S. (2016). 5.2 Natural selection. [online] The nature of science. Available at: https://natureofscienceib.wordpress.com/2016/12/11/5-2natural-selection/ [Accessed 14 Jun. 2023]. Hutchings, M.I., Truman, A.W. and Wilkinson, B. (2019). Antibiotics: past, Present and Future. Current Opinion in Microbiology, [online] 51(1), pp.72–80. doi:https://doi.org/10.1016/j.mib.2019.10.008. Kammoun, M., Miladi, S., Ali, Y., Damak, M., Gargouri, Y. and Bezzine, S. (2011). In vitro study of the PLA2 inhibition and antioxidant activities of Aloe vera leaf skin extracts. Lipids in Health and Disease, 10(1), p.30. doi:https://doi.org/10.1186/1476-511x-10-30.

Scientific Research in School Volume 5 Issue 1 2023 Maan, A.A., Nazir, A., Khan, M.K.I., Ahmad, T., Zia, R., Murid, M. and Abrar, M. (2018). The therapeutic properties and applications of Aloe vera : A review. Journal of Herbal Medicine, 12, pp.1–10. doi:https://doi.org/10.1016/j.hermed.2018.01.002. Malik, E.M. and Müller, C.E. (2016). Anthraquinones As Pharmacological Tools and Drugs. Medicinal Research Reviews, 36(4), pp.705–748. doi:https://doi.org/10.1002/med.21391. Melchor, L.O. (2021). Tips for Growing Aloe Vera Outdoors | Gardener’s Path. [online] Gardener’s Path. Available at: https://gardenerspath.com/plants/succulents/grow-aloeoutdoors/. Munita, J.M. and Arias, C.A. (2016). Mechanisms of antibiotic resistance. Virulence Mechanisms of Bacterial Pathogens, Fifth Edition, [online] 4(2), pp.481–511. doi:https://doi.org/10.1128/microbiolspec.vmbf-00162015. Olaleye, M.T., Olaleye, M.T. and Bello-Michael, C.O. (2005). Comparative antimicrobial activities of aloe vera gel and leaf. African Journal of Biotechnology, [online] 4(12). doi:https://doi.org/10.4314/ajb.v4i12.71436. Park, Y. and Tae Geun Jo (2006). Perspective of industrial application of Aloe vera. pp.191–200. doi:https://doi.org/10.1007/978-0-387-34636-6_6. Sadiq, U., Gill, H. and Chandrapala, J. (2022). Temperature and pH Stability of Anthraquinones from Native Aloe vera Gel, Spray-Dried and Freeze-Dried Aloe vera Powders during Storage. Foods, 11(11), p.1613. doi:https://doi.org/10.3390/foods11111613. Surjushe, A., Vasani, R. and Saple, D. (2008). Aloe vera: A short review. Indian Journal of Dermatology, [online] 53(4), p.163. doi:https://doi.org/10.4103/00195154.44785.

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To what extent can ocean sediment microflora withstand ocean acidification? Georgia Mantis Barker College Purpose: To observe and recount the effects of ocean acidification on the growth of bacteria in the ocean by mimicking ocean environments using samples from a domestic fish tank. Design/methodology/approach: The number and variety of bacteria present in ocean-like samples at various pH levels were observed and documented. Findings: Rather than having a negative effect on the number of bacteria that was able to grow, by altering the pH, it was observed that ocean acidification has rather had a detrimental effect on the biodiversity of the bacteria that was able to grow. Research limitations/implications: Real-world modelling using 3D printed components based off these designs is the next step to further verify the conclusions as this research is strictly computational. Implications/reasons: This project will be beneficial for researchers observing the effects of ocean acidification on the food chain as well as the biodiversity of species. Being at the bottom of the food chain, by decreasing the biodiversity of bacteria, you are removing its ability to survive future environmental changes which could lead to a continual 'domino effect' up the food chain. It would be most beneficial for the next step in this area of research to be taken in the direction of testing how to either increase bacteria's defence against a changing pH or prevent ocean acidification and the changing pH. Originality/value: Being a growing threat to our oceans, by observing the effect of ocean acidification and global warming on the microfauna and bacteria living in the sediment, this experiment is able to draw attention to the rising threat that a drastic change in pH could have on the biodiversity of bacteria and therefore the knock-on effect it could have on the entire food chain. Paper type: Research paper

Literature Review

Figure 1: graph showing the increase in C02 emissions and the effect it has had on the pH of the ocean.

Due to rising concentrations of carbon dioxide in the air, contributed to by human actions and fossil fuel emissions, the pH of the ocean has fallen by 0.1 pH units since 1990, equaling around a 30% increase in acidity. (As shown in figure 1). The Ocean absorbs around 30% of the CO2 that is released in the atmosphere and therefore, as levels of C02 increase due to human activity, the amount absorbed by the ocean will simultaneously increase as well. Ocean

acidification occurs when a series of reactions occur, catalyzed by a higher concentration of CO2, resulting in an increase in the number of hydrogen ions in the ocean. This has extensive implications and impacts on marine life, such as chemical erosion of corals, crabs and other ocean crustations and their shells. As the acidity of the ocean rises, marine life is forced to use more energy to maintain healthy body fluid chemistry (US EPA 2016). Due to this increased expulsion of energy, crustations like crabs and sea urchins start to dissolve their protective shells to counteract the increased acidity, decreasing its protective capacity therefore impairing the organism’s overall health. Due to the damaging effects of ocean acidification, the aim of this experiment was to observe and test to what extent microorganisms in the sediment can survive ocean acidification and from this, whether the effects caused by the decreased pH are reversable. However, due to the restriction of being in a school lab, the time and resources needed test the change in pH overtime or revert the acidification back to its original pH to see if the effects are reversible weren’t available. Therefore, it would be beneficial if someone with the time and resources were to build off this experiment

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Scientific Research in School Volume 5 Issue 1 2023 and complete it with these alterations. As well as this, when observing the results from this experiment, it is important to take to account that only a sample of the sediment is being extracted and tested, meaning that it is not considering an ecosystems resilience and natural ability to regenerate and build off itself. As well as this, the experiment more tests and observes the effects of a pH ‘shock’ as it is not monitoring the slow change in pH overtime but rather a sudden change to the acidity. The main effect caused by the decrease in microbial colonies is the effect it has on the food chain. Microfauna is considered a ‘producer’ in the food chain, as they are at the bottom and sustain all the predators that fall above them. This highlights that if their numbers decrease it will cause a knock-on effect as all the organisms that fall above them will lose the abundance of their prey and therefore have more competition for a limited food supply. It is also important to consider when observing the growth occurring that there are several different kinds of microbes living in the sediment, all of which having different resistances to the changing pH, meaning that even if some species survive, due to sensitive strands becoming wiped out, the natural biodiversity will be impacted, decreasing the microbe’s ability to combat sudden or major environment changes in the future. When testing changes in pH you must also consider the natural change in pH that occurs across the 4 seasons. There is a natural variance of around 0.11 in the ocean’s pH from the highest being in spring of an average pH of 8.26 and the lowest being in autumn, having an average of 8.15 (Krause et al., 2012). As well as this, when monitoring ocean acidification and the change in the ocean’s pH across the planet, it is key to observe hotspots and understand why they are occurring. As shown by the map in (figure 3), it can be observed that on average, there is a considerably lower pH of the ocean around the equator than at the higher latitudes. This is because warmer waters retain carbon dioxide better than colder waters meaning these warmer waters are more susceptible to ocean acidification (NOAA, 2016). Which is problematic as many of these warmer areas are home to some of the largest coral reefs in the world, including the Great Barrier Reef off the coast of Australia. Therefore, it is so important to test the effects of this acidification and in doing so how long we have left until our oceans are beyond salvation.

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Figure 2: diagram showing the pH of the oceans due to human-caused ocean acidification.

The rising concentration of CO2 in the ocean is affecting not only coral growth but is also confusing fish and other animals travel and breeding patterns which is affecting their ability to reproduce and grow their populations. This is having a detrimental effect negative on biodiversity and is unbalancing the food chain as the numbers of these fish decreasing effects the animals that feed on them and causes a knock-on effect along the food chain. This is the same effect that is being caused by the decrease in biodiversity of microorganisms as they hold a critical role in the food chain being producers for an ecosystem and feeding all the organisms that fall above them on the chain. However, despite the negative effects ocean acidification is having on marine wildlife including the slowing of coral growth, it is strangely enough having some positive effects including aiding in the growth of seagrass populations (Ocean acidi cation, n.d.). This highlights how although ocean acidification is having detrimental effects on many organisms across the globe, it is aiding in then growth and abundance of other organisms.

Scientific Research Question To what extent can ocean sediment microflora withstand ocean acidification?

Scientific Hypothesis It is hypothesised that acidification of marine samples will result in a reduction of both the relative variety (biodiversity) and abundance of marine sediment microorganisms.

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Methodology

Results

Sample extraction 18 samples of sediment were taken from the fish-tank in the science building, which mimics a small-scale ocean ecosystem. These sediments then had their pH tested confirming they were all the same, ensuring validity. Two of these samples were be kept as controls to make sure there was reliability and that nothing had contaminated the water or the sediments in the fish tank altering the results. Alteration of pH The remaining 15 samples were each altered in groups of 3 ensuring consistency of results and quickly isolating outliers, and unexpected results. 15 mL’s (10mL water, 5mL of sediment) of each of the samples were placed in test tubes and the pH was changed by running carbonic acid through them until the desired pH is reached (demonstrated by the pH probe). This method of acidification was chosen over hydrochloric acid as it mimics the effects of ocean acidification more realistically than running acid through the samples, potentially killing or damaging the bacteria and microfauna. The samples were changed to pH of 8, 7.5, 7, 6.5 and 6 to model the pH change that is predicted to occur before 2100. Growth of microbes Once the samples were changed, a few drops from each were removed and grown on agar plates highlighting both the abundance and variation of bacteria grown on the control plates versus the changed samples, allowing a visual representation of the effects that ocean acidification is having on the ocean's microorganisms. Samples were stored in the incubator for 7 days at a stable temperature of 27 degrees Celsius, mimicking the temperature of coastal waters which is usually around 25-30 degrees Celsius, therefore promoting as much bacterial growth as possible.

Figure 4 : Key Microbes

Each pH was tested (sampled) three times to ensure validity and consistency of results. (Each sample represents an agar plate on which bacteria was grown)

Figure 3: example of the agar plate post incubation. Demonstrates the growth that has occurred.

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Scientific Research in School Volume 5 Issue 1 2023 Microbe 1 results

Microbe 3 results

Table 1: the results of the growth of microbe 1 at different pH levels

Table 3: the results of the growth of microbe 3 at different pH levels

8.0 59 85 189 111

7.5 210 150 70 143.3

7 44 108 90 80.6

6.5 69 59 35 54.3

6 141 100 95 112

Count of Microbe s

pH Sample 1 Sample 2 Sample 3 Average

8.0 3 6 3 4

7.5 9 8 5 7.3

7 6 3 1 3.3

6.5 5 1 2 2.6

6 8 3 6 5.6

Figure 5: graph demonstrating the results on the abundance of microbe 1 across different pH levels

Figure 7: graph demonstrating the results on the abundance of microbe 3 across different pH levels

Microbe 2 results

Microbe 4 results

Table 2: the results of the growth of microbe 1 at different pH levels

Table 4: the results of the growth of microbe 5 at different pH levels

8.0 59 85 189 111

7.5 210 150 70 143.3

7 44 108 90 80.6

6.5 69 59 35 54.3

Figure 6: graph demonstrating the results on the abundance of microbe 2 across different pH levels

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6 141 100 95 112

Count of Microbe s

pH Sample 1 Sample 2 Sample 3 Average

8.0 4 2 3 3

7.5 4 1 2 2.3

7 0 2 2 1.3

6.5 0 0 0 0

Figure 8: graph demonstrating the results on the abundance of microbe 4 across different pH levels

6 0 0 0 0

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Discussion

Table 5: the results of the growth of microbe 5 at different pH levels

The results from the experiment were moderately similar to what was hypothesised, with two of the microbes; microbes 2 and 3 (figures 6 and 7 and tables 2 and 3) not being heavily affected by the changing acidity. In fact, both displayed signs of increased average abundance with microbe 2 having an average abundance of 111 microbes per plate at pH 8 and 112 at pH 6 and microbe 3 having an average of 4 microbes per plate at pH 8 and 5.6 at pH 6.

Count of Microbe s

Microbe 5 results

pH Sample 1 Sample 2 Sample 3 Average

8.0 44 0 0 14.6

7.5 0 0 0 0

7 0 0 0 0

6.5 0 0 0 0

6 0 0 0 0

Figure 9: graph demonstrating the results on the abundance of microbe 5 across different pH levels

Average Result Table 6: demonstrates the average abundance of each microbe across different pH levels

# of Microbes

pH Microbe 1 Microbe 2 Microbe 3 Microbe 4 Microbe 5 Total

8.0 47.6 111 4 3 14.6 180.3

7.5 17 143.3 7.3 2.3 0 170

7 7 80 3.3 1.3 0 116

6.5 6.3 54.3 2.6 0 0 63

6 1.3 112 5.6 0 0 119

Figure 10: graph demonstrating the results on the average abundance of each microbe across different pH levels

This supports the theory that when other species decrease in number and abundance, those species remaining can thrive and grow in numbers. However, the changing pH level had a larger effect on microbes 1 and 4 (figures 5 and 8 and tables 1 and 4) with microbe 4 having no growth on any of the pH6 or 6.5 plates and microbe 1 decreasing significantly, having an average abundance of 47.6 at pH of 8 to an average abundance of 1.3 per plate at pH of 6, supporting the hypothesis that altering the pH will lead to a decrease in the biodiversity of the microfauna, however doesn’t complete support the hypothesis that it will have a large impact on the abundance of overall organisms as only the susceptible types decrease in numbers and abundance, however the other types are able to survive and thrive. Backed up by (Krause et al, 2013), it can be concluded that bacterial abundance was not overly influenced by pH, but rather the biodiversity was. Their findings suggested that by changing the oceans pH, rather than destroying all the bacteria, instead "those with the ability to survive in more acidic waters will survive and thrive changing the composition and type of bacterial colonies". This decrease in biodiversity is highly detrimental to the survival of the bacteria as it decreases its chance of surviving future environmental changes as having a higher range of alleles in a population increases its chance of some organisms in the population possessing a favourable trait allowing them to survive. This decrease in biodiversity also has the potential to send knock on affects up the food chain as bacteria and microorganisms are the base of the chain and supply nutrients all the way up. Therefore, indicating that a decrease in biodiversity of microfauna could potentially impact many other organisms including those that feed off the bacteria and so on.

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Scientific Research in School Volume 5 Issue 1 2023 ocean reef to ensure the same microfauna and the same defence system. As well as this, by changing the samples pH gradually over a long period of time rather than drastically altering them as this experiment has due to time restrictions, it would more accurately mimic the gradual change in pH caused by ocean acidification and may give the bacteria a better chance of survival as by altering the samples drastically and immediately, this experiment mimicked an ecosystem 'shock' which is a sudden change in the environment rather than the long and gradually changing process of ocean acidification.

Conclusion

Figure 11: sample 1 of pH 8 with the unexpected result of microbe 5

Highlighted in (figure 9 and table 5), there was an unexpected result on one of the agar plates for pH 8 in the experiment. It displayed different symptoms to the rest of the plates and contained microbe 5 (figure 11). This result was classified as an outlier as none of the other plates displayed these symptoms or contained this microbe leading to the belief that the sample may have been contaminated at some point throughout the experiment. Being an outlier, two results tables were created, one including this result (found in results section (table 5)) and one not including it (found in appendix) in order to observe whether it altered the trends demonstrated in the tables and graphs. However, since three samples were taken at each pH level it was immediately identified that this was an outlier and therefore the results didn’t disrupt the findings. Following this investigation, it would be most beneficial for someone to do further research in this area by, following the growth of the microorganisms, reverting the samples to the oceans natural pH of 8 to observe whether the damage done to the organisms and the biodiversity are beyond salvageable and at which point the manmade destruction has overpowered the oceans natural defence and revival system and pushed it beyond recovery. As well as thus, when observing the results, it is important to consider the limitations of the experiment including the restrictions of working in a school laboratory as well as the fact that the samples were all taken from a school fish tank, which may not include the same microfauna as an ocean reef or have the same natural defence system. Therefore, for future experiments, it may be beneficial to extract the samples from an

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It is predicted that by the year 2100, the oceans will have dropped to a pH of 6, which is a very large decrease from the average of 8 pH of our oceans currently. This drastic change in acidity is having a major effect on the oceans and marine life, harming corals, wildlife and ocean plants. These events inspired this experiment which aimed to test the resistance of microfauna to a changing pH, and in doing so, observe their natural defence system and see how large of an effect it is having on the microbes. From this, conclusions can be drawn which demonstrate the effect this change will have on biodiversity and abundance and therefore the possible impacts it could have on the food chain and the microfauna’s future survival. This experiment found that rather than having an overwhelming effect on the abundance of bacteria, it rather had a major effect on the biodiversity of the microfauna with 2 of the microorganisms being heavily affected by the change in pH, but another two hardly being affected. This emphasises how due to the rising acidity; the more sensitive strands will die out and the strands with a natural defence to the higher pH will thrive and remain unaffected. This partially supported the null hypothesis that the change in pH would influence both the abundance and variation of the bacteria, as it rather had a much larger effect on the biodiversity and variation over the abundance.

Acknowledgements I would like to firstly thank Dr Alison Gates for all her support throughout my research and experiment and for her advice, I wouldn’t have been able to do any of it without her. I would also like to thank Dr Katie Terrett and Dr Matthew Hill for their constant support and for being my point of call especially in the second half of the year. I have been so lucky to be surrounded by such amazing scientists, they have assisted me so much with my scientific understanding

Scientific Research in School Volume 5 Issue 1 2023 and nurtured my love of the subject. I would also like to thank Amanda Pardini for her encouragement and support of me in the beginning of my project.

References McMinn, A. (2017). Reviews and syntheses: Ice acidification, the effects of ocean acidification on sea ice microbial communities. Biogeosciences, 14(17), 39273935. Krause, E., Wichels, A., Giménez, L., Lunau, M., Schilhabel, M.B. and Gerdts, G. (2012). Small Changes in pH Have Direct Effects on Marine Bacterial Community Composition: A Microcosm Approach. PLoS ONE, [online] 7(10), p.e47035. doi: 10.1371/journal.pone.0047035.

Figure 13: sample of an agar plate kept at a pH of 6.5

Oceans2Earth.(2020). Marine Conservation Oceans2Earth NOAA Fisheries (2020). Understanding Ocean Acidification. [online] NOAA. Available at: Woods (2018). Scientists Pinpoint How Ocean Acidification Weakens Coral Reefs. [online] Woods Hole Oceanographic Institution. Ocean acidification. (n.d.). Available at: https://www.aims.gov.au/sites/default/files/Acidification.p df

Figure 14: sample of an agar plate kept at a pH of 7

Appendices Table 7: demonstrates the average abundance of each microbe across different pH levels excluding microbe 5

Count

pH Microbe 1 Microbe 2 Microbe 3 Microbe 4 Total

8.0 47.6 111 4 3 165.7

7.5 17 143.3 7.3 2.3 170

7 7 80 3.3 1.3 116

6.5 6.3 54.3 2.6 0 63

6 1.3 112 5.6 0 119

Figure 15: sample of an agar plate kept at a pH of 7.5

Figure 16: sample of an agar plate kept at a pH of 8 Figure 12: sample of an agar plate kept at a pH of 6

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Bee venom extraction methods and their effect on potency Gus Opie Barker College Purpose: Existing research has shown anti-microbial properties of Apis Mellifera’s venom, with pharmaceutical products using venom in their ingredients. This project aims to determine whether the extraction of the bee’s venom (which has so been under-studied) affects the potency of the venom's anti-microbial effects on selected bacteria. Design/methodology/approach: The experiment was conducted using two different extraction methods on local Apis Mellifera’s hives. A non-destructive method of electro-stimulation and a destructive method of manual venom removal was then applied onto lawned agar plates of bacteria (Escherichia coli K12 strain and Staphylococcus epidermis). The relationship between antimicrobial effects was recorded. This method was ethically conducted, using humane euthanisation methods of freezing. Findings: The investigation results suggest a relationship between the destructive method inhibiting the growth of Escherichia coli K12 strain and Staphylococcus epidermis more effectively than the non-destructive method. Research limitations: Insufficient venom vole and contamination of the non-destructive venom allow for unreliable results. However, the results suggest a purer sample of the destructive venom samples due to the isolation of the venom sac from manual extraction. Thus, further research is needed to further the reliability pilot experiment results. Practical implications: Due to the increase in anti-bacterial medication, this report aims to allow further research on natural anti-microbial medicines. The report suggests that the bee’s venom is effective in inhibiting the growth of Escherichia coli K12 strain and Staphylococcus epidermis, allowing for further research in bee venom in modern medicine. Key Words: Bee’s venom, Antimicrobial, Non-destructive extraction, Destructive extraction, Antibacterial Paper type: Research paper

Literature Review Venom research Venom research of animals have shown that it should not be feared but admired (Shaw, 2022). For thousands of years venom has been used in the formulation of medical practices around the globe and is still being used and researched to this day (Thomas, 2022). As antibiotic resistance has increased, so has the push for adapted medical ingredients, with a spike in natural anti-microbial sources such as venom (Thomas, 2001). Bee’s venom Bees are venom producing apoidea of the Insecta class. There are approximately 20,000 species of Bees, with Australia being the habitat of over 2000 species of domestic and wild Bees (Wheen Bee Foundation, 2020). Bee diversity has allowed the species to be distributed on every continent except Antarctica and are found in a wide range of habitats from deserts to rainforests and even alpine climates (Fründ et al., 2013). Bees from around the world have

different venoms to suit the environment and defensive needs, with many Bee species adapting their defensive stingers for individual threats in their environment. Bee venom. Bee venom, or apitoxin, is a defensive secretion produced by a gland in the abdominal cavity of bees. Bee venom is a largely studied substance due to the complexity of its biological properties. The main component of Bee venom (See figure 1) is melittin, a pain producing substance, however this substance is an antimicrobial peptide (Dotimas et al., 1987). This is due to the melittin causing membrane disruption, activation of the immune system and inhibition of enzymes within the cells. Hegazi et al. (2014) states that “The antimicrobial activity of honeybee venom may be due to the presence of several peptides like melittin, apamin, adolapin, mast cell degranulating peptide, enzymes, biologically active amines and non-peptide component” (p268). Bee venom has been long been studied for its medicinal properties including venombased creams and lozenges and is widely used in alternative medicine (Lee et al., 2005).

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Scientific Research in School Volume 5 Issue 1 2023 Bees are eminently suitable for secondary school research as bees are invertebrates, thus not falling under the restrictions of Animals in School and ethical scientific restrictions. Secondly, Apis Mellifera’s venom is a mildly toxic, causing minimal danger to humans if stung (Bogdanov, 2012). Thirdly, Apis Mellifera’s bees are an abundant species that are inexpensive to purchase and house. To capture the bees, safe bee keeping practices must be followed. Complete personal protection equipment following the agriculture of Victoria’s guidelines is worn during experimentation (Agriculture Victoria, 2021). To uphold ethical practice, captured bees are placed into a freezer to be euthanise.

Figure 1: Composition of bee’s venom

The antimicrobial activity of Bee venom has been documented using the most common, domesticated Bee species, Apis Mellifera, which is a Western Honeybee that is often used as a model organism of scientific research on Bee Venom and behaviour (Abou-Shaara, 2014). Boutrin et al. (2008) investigates bees' venom, antimicrobial effect on specific gram-negative bacteria, Trypanosoma brucei and an enterobacteriaceae experimental parasitology. The experiment concluded that there were significant antimicrobial effects on both bacteria subjects, highlighting the antigenic possibilities that bee venom has in medical treatments. Further the investigation discusses the difference in gramnegative and gram-positive bacterial, highlighting the importance of testing both in an experiment. Hegazi et al. (2014) investigates the effect of Bee venom on gram-positive bacteria including Staphylococcus epidermis, concluding the venoms anti-bacteria activity on the tested bacteria. Hegazi et al. (2014) conclude that Bees venom has antimicrobial activity that has medical uses (e.g acupuncture, rash creams), yet discusses how the extraction method of the venom can differ the antimicrobial potency, thus leading to thus pilot investigation (Nelson Honey and Marketing, 1997). Gram-positive and gram-negative bacteria are two categories that describe the structure of bacterial cell walls, therefore, both bacterium must be tested to have a generalisable conclusion of the medical use of Bees venom (Park et al., 2013).

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Escherichia coli K12 strain Escherichia coli K12 strain (E-Coli) is a gramnegative bacterium that is commonly found in the human intestines and gut. It is a lab safe infectious bacterium that commonly used as a model organism for scientific research (Kuhnert et al., 1995) Staphylococcus epidermis Staphylococcus epidermis (S. albus) is a grampositive bacterium that is commonly found in the human skin and nostril. The bacteria are lab safe and is used as a model organism for gram-positive bacteria. (Namvar et al., 2014).

Scientific Research Question Does the extraction method of Apis Mellifera’s venom alter its potency as an antibacterial agent against both gram-positive (Staphylococcus epidermis) and gram-negative (Escherichia coli K12 strain) bacterium?

Scientific Hypothesis The manual, destructive removal of the Bee venom sac will produce venom that is more potent (ie a greater zone of inhibition) against pathogenic bacteria tested.

Method Keeping bees A pre-established hive was purchased, and venom was extracted with an electro-stimulant apparatus in Spring to all for least disruption of the hive (Orkin, 2023). The hive must have a sustainable population to allow for the euthanisation of a minimum of one hundred bees without causing a disruption in the hive.

Scientific Research in School Volume 5 Issue 1 2023 Destructive extraction method Following the method described by Hegazi et al. (2014), frozen bees were placed into a sterile petri dish. The lab bench was sterilised using 70% ethanol. A beaker was filled with 70% ethanol, and one pair of forceps were submerged in the ethanol solution. Before extraction the forceps were flamed using a Bunsen burner, allowing for complete sterilisation of extraction equipment. Gloves were worn while handling bees to ensure no contamination of subjects. The stinger of with the venom sac were grasped by the forceps and were extracted using a parallel pull of the abdomen, ensuring only the venom sac and stinger is removed from the bee as seen in figure 2. The extracted venom sac and stinger were placed into a second sterile petri dish, with a lid placed on top after the venom sac is placed inside to ensure no airborne contamination. The forceps were submerged into the beaker with the ethanol solution and flamed before repeating the extraction steps. The method was repeated until a minimum of fifty venom sacs were extracted, as seen in figure 3. The remaining bee bodies were responsibly disposed of or composted. The venom sac was then crushed using the flat side of a flamed scalpel. The crushed venom sacs were then transported into a sterile test tube with 10mL of distilled water. The distilled water and venom solution was then electrically mixed using a magnetic mixer for 24 hours.

Figure 2: Venom Sac and Sting

Figure 3: Venom sacs removed

Non-destructive extraction method The non-destructive method of venom extraction is commercially used. This experiment was influenced by the literature of how to extract bees venom using a marketed electro-stimulation device. A Whale Labs Bee Venom Collector (MK2 4 collection frame) was purchased. The devices set up instructions and safety precautions were followed to ensure proper use of the device. A portable power source (e.g a car battery) was placed in an undercover area near the hive where the venom collector was installed. Before instillation of the venom extractor, complete personal protection equipment must be worn, following the agriculture of Victoria’s guidelines of bee keeping safety (Agriculture Victoria, 2021). As seen in figure 4, the electro-stimulation extraction frame was secured at the entrance of the hive. Once installed, the provided agitation spray was sprayed onto the hive to produce a defence pheromone that disrupted the communication of the hive and promote the bees to sting the frame (Bortolotti et al., 2014). The frame was installed for a minimum of two hours, or until the glass frame is coated with venom. The frame was supervised and was sprayed with the agitation spray when needed. Once the frame has been coated with venom, the glass top plate was removed. In a sterilised lab using 70% ethanol to wipe the lab benches, a razor blade was submerged in a beaker of a 70% ethanol solution. The razor will then be flamed using a Bunsen burner to

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Scientific Research in School Volume 5 Issue 1 2023 ensure sterilisation. Gloves were worn when handling the glass plate. Using the sterilised blaze, the layer of venom on the glass plate was scraped into a sterile test tube containing 10 mL of distilled water. Once all of the venom is transported into the test tube, the distilled water and venom solution was then electrically mixed using a magnetic mixer for 24 hours.

Figure 5: Lab setup

Figure 4: Extraction of bees

Zone of inhibition testing After the 24 hours of mixing both solutions of the non-destructive and destructive extracted venom were moved to a 70% ethanol wiped sterile bench, placed into a test tube holder. A lab coat, gloves and safety glasses were used while handling the bacteria and venom solutions. Ten agar plate dishes were be acquired. Each agar plate was labeled with the bacteria that was cultured in it and which type of extraction solution was measured. The plate was also be divided into three section which was labeled S1, S2 and S3. This was repeated for 8 of the agar plates. The remaining two agar plate was be labelled control, with one being for the E. coli bacterium, and the second for S.albus. A Petri dish with a 70% ethanol solution was made, with a glass cell spreader (L shaped) and sterile forceps being submerged. A contained broth of the E-coli and S.albus was acquired. A heat proof mat and a Bunsen burner was alight in safety mode for flaming the glass cell spreader and forceps. The lab set up is seen in figure 5.

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Plate culturing The glass cell spreader (L shaped) was used to culture the agar plates. Using a sterilised pipette, 10 mL of the E.Coli broth was dripped onto a labeled agar plate. The glass cell spreader was removed from the ethanol solution and then flamed. Using the short L side of the glass cell spreader, the 10 mL of E. coli broth was evenly spread to culture the agar plate. Once evenly spread, the glass cell spreader was submerged in the ethanol solution and the agar plate lid was placed on to prevent airborne contamination. Using a sterilised pipette, the 10 mL of the E.Coli broth was repeated and dripped onto the next labeled agar plate. Then, the glass cell spreader was removed from the ethanol solution and then flamed, then was used to repeat the spreading technique to culture the agar plate. Once completed, the glass cell spreader was submerged in the ethanol solution and the agar plate lid was placed on. These steps were repeated for all six E. Coli labeled agar plates. This method was repeated for the S.albus labeled agar plates, repeating the same amount of S.albus broth and the method of culturing the agar plate. The sterilisation method will also be completed for all six agar plates, with all the S.albus labelled plates being cultured. Filter paper impregnation A labeled sterilised Petri dish will contain twelve sterile filter paper discs being spread in the Petri dish. A sterilised pipette was used to extract the nondestructive venom solution. The solution was dripped onto the paper discs, impregnating the discs. All paper discs were completely absorbed with the nondestructive venom solution. This method was repeated for the destructive venom solution in a secondary sterilised Petri dish.

Scientific Research in School Volume 5 Issue 1 2023 Placement of filter paper The forceps were removed from the ethanol solution and flamed before use as sterilisation. The impregnated filter paper of the non-destructive solution is picked up, with the lid of the Petri dish containing the filter papers being placed back on after filter paper is removed to prevent contamination. The filter paper that is grasped was transported to the labeled non-destructive cultured agar plate, avoiding being out of the Petri dish for an extended period of time to reduce contamination. The filter paper is placed in the centre of section 1 of the labeled nondestructive agar plate. The forceps will then be submerged in the ethanol solution and flamed again. These steps were repeated for the non-destructive filter papers for sections 2 and 3, with the forceps being serialised and flamed between each paper placement. Once all three sections of the agar plates contain an impregnated filter paper containing the non-destructive venom solution, the lid of the agar plate was secured with tape. These steps were repeated for the five labelled non-destructive agar plates, with each of the three-section containing a centred filter paper. All five agar plates were sealed using tape, with the sixth agar plate (the control plate) being sealed with tape containing no filter paper. The same method of placement of the impregnated filter paper was used with the destructive venom solution. The forceps was sterilised and flamed between each placement of the filter paper, with the five agar plates containing three centred filter papers in section 1, 2 and 3. The sixth plate (the control plate) will contain no filter paper. All plates were sealed using tape. Incubation The twelve cultured agar plate with ten having three filter paper discs and two being experimental controls was placed into an incubator. Described by Krell (1996), the incubator was set at 37 degrees Celsius to mimic the temperature of the human body. The agar plates will remain the in incubator for 48 hours to allow for a clear zone of inhibition. Zone of inhibition method Using a vernier callipers, the zone of inhibition of each filter paper disc was measure, taking three measurements around each disc and averaging the inhibition measurement. The measurement method was repeated for all three sections of each disc and was displayed in a results table. A mean done diameter was calculated for each group and compared to answer the experiments aim.

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Results Table 1: Zone of inhibition based on extraction method and bacteria.

Figure 6a,b 6c,d 6e,f 6g,h 6i,j 6k,l

Extraction Method Destructive Destructive NonDestructive NonDestructive Control Control

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Bacteria

Zone of inhibition (mm) Dish 2 3 1 2 1 3.1 1.4 0.5 1.3 0.2

3 1.3 0

E-coli S.albus

1 2.33 0

Dish 1 2 1.6 0.1

E-coli

S.albus

E-coli S.albus

0 0

Mean 1.8 0.4

Scientific Research in School Volume 5 Issue 1 2023 Intended data analysis Due to complication in the results with the nondestructive solution promoting bacterial growth, complete data analysis could not be conducted, however a clear result was able to be concluded. It had been intended for an ANOVA test to compare the means of the two group, and to conclude if there was a significant difference in the means of the mean zone of inhibition. The ANOVA would have allowed for a scientific conclusion if a difference had been found between the different extraction method against the two bacteria. Though the means were unable to be used due to the results being skewed by contamination, a significant zone of inhibition was observable in the destructive venom extraction, which indicates that there are anti-microbial relationships between the venom and the bacterium that were tested.

Discussion This study investigated the effect of bee venoms potency using two different extraction methods, with the purpose to investigate the research gap in this field. Using the method of Hegazi et al. (2014) where a full-scale study of Apis Mellifera’s venom against a variety of gram-positive and gram-negative bacteria was carried out the methods for full bee venom extract (a mixture of blended bees and ethanol) and destructive venom sac extraction were employed for this investigation, as detailed in the methodology section. This experiment used the secondary school safe bacteria E. coli and S.albus to test the venom on a variety of bacteria. The results were inconclusive due to the lack of data and aroused many flaws in the method of the experiment. The minimal zone of inhibition for the four destructive extraction E. Coli and S.albus agar plates align with the results of Foster et al., with the results suggesting there are antimicrobial properties in Apis Mellifera’s venom. Moreover, the results in figures 6.a and 6.b indicate more of a resistance to the E. coli bacteria strain, which could be evidence that bee venom could be more effective as an anti-bacterial medicine to gramnegative bacteria. Unfortunately, as no zone of inhibition was recorded for the non-destructive method the results are inconclusive and a definitive conclusion about the difference in anti-microbial potency of the two extraction methods of bee's venom was not able to be determined. However, this study has served as a pilot experiment allowing insight into the limitations of a non-destructive extraction method. The raw data supports the hypothesis that the manual, destructive removal of the Bee venom sac will produce venom that is more potent against

pathogenic bacteria, however the investigation cannot be used as conclusive evidence of this statement. Limitations The most significant hurdle of this research was contamination of the non-destructive venom. Due to the electro-stimulus apparatus being outside in an uncontrolled environment, the prevention of venom contamination was extremely difficult, leading to inaccurate results. Sanad et al. (2013) discuss the limitations of the Whale Lab electrical stimulating device stating that the “contamination of honey leads to a food source for bacteria”. Figures 6.e, 6.f, 6.g and 6.h are visual representations of this with promoted yellow bacterial growth around the non-destructive venom solution. The hypothesis for this yellow growth is that both the E. coli and S.albus bacteria were resistant due to a low concentration of venom. Also due to the Electrical stimulation device being installed at the door of the hive, honey from the hive contaminated the venom solution allowing for the bacteria to feed on the sugar. Thus, the nondestructive venom solution was extremely ineffective causing more bacterial growth with no zone of inhibition. Moreover, another limitation of this experiment was the effectiveness of the manual destructive extraction of the venom sac. The experiment exposed the limitations of this extraction method with the manual extraction taking extended periods of time. Further, the destructive extraction method was extremely hard to get a non-contaminant sample without the bee’s intestines, creating a significant amount of failed extraction attempts. These limitations mean that the destructive method isn’t an economically sound, thus meaning if the destructive extraction method is more potent, it wouldn’t be effective as a commercially used method of venom extraction. Implications This research, along with other studies has shown the importance of studying venom and its properties as a medical tool. As antibiotic resistance is increasing globally, a push for alternate methods of treatment against bacterial diseases is needed (McShan et al., 2015). Experimental research into bee venom as an alternate antibiotic has increased in popularity and funding, however, has many experimental gaps, including the extraction methods, thus leading to the current pilot experiment. The use Apis Mellifera’s venom against bacteria has been researched, however the extraction method is an understudied aspect of this topic. Although the current experimental data

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Scientific Research in School Volume 5 Issue 1 2023 does not indicate conclusive results that can answer the investigations hypothesis, the current experiment has demonstrated the effects of hive contamination on venom extraction and may suggest that the destructive venom may have a greater potency on gram-negative bacteria. Further research should be conducted to determine if this difference in antimicrobial potency for the gram-negative bacteria is significant as an addition to the current results. Moreover, further research should be conducted to compose a more effective methodology of electroextraction that minimises the contamination of honey into the venom solution. This research could be implemented into mainstream venom farming for medical use as the non-destructive extraction method appears to be more of a sustainable method compared to the destructive extraction method, increasing its research value to the scientific community.

Conclusion This research project aimed to determine if there was a significant difference in venom potency against bacteria compèred with the method of extraction. The Apis Mellifera’s venom was extracted using the destructive manual method and was concluded to have an antibacterial effect on both E. coli and S.albus bacteria. The non-destructive results were inconclusive, with the venom solution promoting willow bacterial growth due to contaminates including honey being a food source for the bacterium. Though a limited amount of data was able to be recorded, the findings of the experiment have allowed for an opportunity for further research in this field and can be used to determine methodology flaws in the non-destructive electro-extraction device, leading to contamination and promotion of bacterial growth. The results suggest more potency towards gram-negative bacteria (S.albus), which can be further studied in future investigations. By improving the method using the non-destructive electro-extraction device and using a wider range of both gram-positive and gram-negative bacteria, enough data may be collected to answer the hypothesis in the future. The implications of this experiment may lead to further research which can expand our understanding of adapted antibiotic medicines, answering the ongoing antibiotic resistance crisis.

References Abou-Shaara, H. (2014). The foraging behaviour of honey bees, Apis mellifera: a review. Veterinární Medicína, 59(No. 1), pp.1–10. doi:https://doi.org/10.17221/7240vetmed.

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Bogdanov, S. (2014). Bee Venom: Composition, Health, Medicine: A Review. [online] Available at: http://www.pacificdomes.com/site/wpcontent/uploads/2012/08/www.beehexagon.net_files_file_fileE_Health_VenomBookReview. pdf [Accessed 18 Jun. 2023]. Bortolotti, L. and Costa, C. (2014). Chemical Communication in the Honey Bee Society. [online] Nih.gov. Available at: https://www.ncbi.nlm.nih.gov/books/NBK200983/. Boutrin, M.-C. . F., Foster, H.A. and Pentreath, V.W. (2008). The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria. Experimental Parasitology, [online] 119(2), pp.246–251. doi:https://doi.org/10.1016/j.exppara.2008.02.002. Clark, R. (2009). Bid to sell bee venom as functional ingredient. [online] New Hope Network. Available at: https://www.newhope.com/supplements/bid-sell-beevenom-functional-ingredient. Department of Jobs, P. and R. (2021). Safe beekeeping practices - Agriculture. [online] Agriculture Victoria. Available at: https://agriculture.vic.gov.au/livestock-andanimals/honey-bees/handling-and-management/safebeekeepingpractices#:~:text=Beekeeping%20coveralls%20and%20be e%20suits [Accessed 18 Jun. 2023]. Fründ, J., Dormann, C.F., Holzschuh, A. and Tscharntke, T. (2013). Bee diversity effects on pollination depend on functional complementarity and niche shifts. Ecology, 94(9), pp.2042–2054. doi:https://doi.org/10.1890/121620.1. Habermann, E. (1972). Bee and Wasp Venoms. Science, [online] 177(4046), pp.314–322. doi:https://doi.org/10.1126/science.177.4046.314. Kuhnert, P., Nicolet, J. and Frey, J. (1995). Rapid and accurate identification of Escherichia coli K-12 strains. Applied and environmental microbiology, [online] 61(11), pp.4135–4139. doi:https://doi.org/10.1128/aem.61.11.4135-4139.1995. Kumerage, N. (2022). 2022 Scientific Research in School. [online] issuu. Available at: https://issuu.com/barkercollege/docs/2022_science_journa l/144?ff [Accessed 4 Feb. 2023]. Lee, J.-D., Park, H.-J., Chae, Y. and Lim, S. (2005). An Overview of Bee Venom Acupuncture in the Treatment of Arthritis. Evidence-based Complementary and Alternative Medicine, [online] 2(1), pp.79–84. doi:https://doi.org/10.1093/ecam/neh070. Maitip, J., Mookhploy, W., Khorndork, S. and Chantawannakul, P. (2021). Comparative Study of Antimicrobial Properties of Bee Venom Extracts and Melittins of Honey Bees. Antibiotics, 10(12), p.1503. doi:https://doi.org/10.3390/antibiotics10121503. McShan, D., Zhang, Y., Deng, H., Paresh Chandra Ray and Yu, H. (2015). Synergistic Antibacterial Effect of Silver Nanoparticles Combined with Ineffective Antibiotics on Drug ResistantSalmonella typhimuriumDT104. Synergistic Antibacterial Effect of Silver Nanoparticles Combined with Ineffective Antibiotics on Drug Resistant Salmonella

Scientific Research in School Volume 5 Issue 1 2023 typhimurium DT104, 33(3), pp.369–384. doi:https://doi.org/10.1080/10590501.2015.1055165. Namvar, A.E., Bastarahang, S., Abbasi, N., Ghehi, G.S., Farhadbakhtiarian, S., Arezi, P., Hosseini, M., Baravati, S.Z., Jokar, Z. and Chermahin, S.G. (2014). Clinical characteristics of Staphylococcus epidermidis: a systematic review. GMS hygiene and infection control, [online] 9(3), p.Doc23. doi:https://doi.org/10.3205/dgkh000243. NHMRC (2021). Australian code for the care and use of animals for scientific purposes | NHMRC. [online] www.nhmrc.gov.au. Available at: https://www.nhmrc.gov.au/aboutus/publications/australian-code-care-and-use-animalsscientificpurposes#:~:text=The%20use%20of%20animals%20for. Orkin (2023). Honeybee Colony: Facts About Roles Within Honey Bee Hives | Orkin. [online] www.orkin.com. Available at: https://www.orkin.com/pests/stingingpests/bees/honey-bees/honey-bee-colony# [Accessed 18 Jun. 2023]. Sanad, Read.E, Mohanny and Karem (2013). The Efficacy of a New Modified Apparatus for Collecting Bee Venom in Relation to Some Biological Aspects of Honeybee Colonies. [online] Available at: https://www.researchgate.net/profile/Karem-MMohanny/publication/315628567_The_Efficacy_of_a_Ne w_Modified_Apparatus_for_Collecting_Bee_Venom_in_ Relation_to_Some_Biological_Aspects_of_Honeybee_Co lonies/links/58d667a092851c44d4748d3d/The-Efficacyof-a-New-Modified-Apparatus-for-Collecting-BeeVenom-in-Relation-to-Some-Biological-Aspects-ofHoneybee-Colonies.pdf. says, M.O. (2022). The Medical Uses of Venom. [online] News-Medical.net. Available at: https://www.newsmedical.net/health/The-Medical-Uses-ofVenom.aspx#:~:text=Cobra%20venom%20is%20among %20the. The Wheen Bee Foundation. (2020). Australian Native Bees. [online] Available at: https://www.wheenbeefoundation.org.au/about-beespollination/australian-nativebees/#:~:text=Australia%20has%20over%202%2C000%2 0species.

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Ultraviolet light in the war against bacteria and antibiotic resistance Will Savage Barker College Purpose: This paper aims to determine the difference between the ultraviolet resistance between Staphylococcus epidermidis and Escherichia coli bacteria. This will allow for the safe sterilisation by UV light of these two bacteria and can potentially be used as a possible indicator of the difference in UV susceptibility between gram positive and negative bacteria. Design/methodology/approach: A total of 24 lawned agar plates of bacteria cultures (12 S. epidermidis, 12 E. coli) were exposed to 254nm UV. Three from each group were exposed to the light for 0, 4, 7, and 10 minutes. Findings: It was found all E. coli samples were much less susceptible than the S. epidermidis at all time periods bar the group at 4 minutes. Research Limitations/Implications: This suggests that gram negative bacteria will have higher resistance to ultraviolet light than gram positive. However, S. epidermidis and E. coli were the only safe bacteria that could be grown in the laboratory which prevents a conclusive answer to that question. Social Implications: As medically important bacteria recognised by the World Health Organisation, it is possible to develop safe methods of UV sterilisation of E. coli and S. epidermidis in relation to wavelength and time of exposure. This could be applied in medical, industrial, agricultural, or household areas. Originality/value: Many other papers have studied how UV light can be used to kill bacteria, but few have recognised the relation of gram-positive and negative cell walls. Paper type: Research paper

Literature Review Bacterial infection is and always has been a leading cause of death in the world, currently second, with an estimated 7.7 million deaths every year (7.7 Million People Die from Bacterial Infections Every Year – 2022 – ReAct, 2022). Antibiotics have always been the main method of treating such infections since their invention. But as medicine has progressed, so have bacteria and their ability to survive such treatments with antibiotic resistance through natural selection resulting in many of the deaths occurring in people prescribed with antibiotics as seen in Figure 1

Figure 1: Deaths due to antimicrobial resistance every year by 2050 Source: (GDARB, 2019)

This combined with low supply and high costs of many antibiotics have raised the need for alternate methods of treating infections that also don’t drive the development of resistance. Until those technologies arise, preventing an infection in the first place is the best method of controlling bacteria. But again, common ways of disinfecting areas of bacteria use chemicals like alcohols and bleach which are limited in some areas of the world especially developing nations where they are in low supply and are expensive. An alternative to chemicals for disinfection is the use of UV light. The use of UV light as a disinfectant is a method mostly used for water treatment, but by utilizing UV light as a surface disinfectant, chemicals will not be needed to prevent bacterial infection (Katara et al., 2008). Katara et al., attempt to create a standardised procedure for hospital disinfection with ultraviolet light. There is no global standard set procedure in terms of time, distance, and wavelength. By using a germicidal UV tube at 260nm wavelength, it was determined that at a maximum distance of 8 feet for 30 mins was sufficient to properly sterilise an operating theatre of all E. coli in positions that could cause infection. The investigation is very specific in

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Scientific Research in School Volume 5 Issue 1 2023 the use of UV sterilisation that does not apply to all the cases in which it could be used and is only tested against one bacterium which weakens the applicability of this source. Nevertheless, the investigation allowed for the understanding of how there is a significant difference in the sterilisation powers of UV between an agar plate and a whole room. Yet, bacteria are all different and require different treatments to inactivate and kill. By studying S. epidermidis and E. coli (both recognised as medically important by WHO), the method of treatment with UV light can be determined for safe disinfection as they both have vastly different surface properties (Sonohara et al., 1995). Sonohara et al. (1995), investigate the surface properties of both E. coli and S. aureus in comparison. The investigation examined the hydrophobicity, electrical properties, and pH of each cell wall. The data was then applied to various properties of each bacteria including survival in acidic environments, surface adhesion and charge. The results found that despite the differing grampositive and negative surface structures, each bacteria provided mixed results with no clear interpretation. The investigation was extensive as each test was repeated 64 times and a mean taken, proving the method reliable. The experiment relates to this project as it explored E. coli and S. aureus which is extremely similar to S. epidermidis. This provided insight that the differing cell walls may not affect the bacteria’s UV susceptibility but as this wasn’t tested it is unknown. This experiment will examine different aspects of the UV light and its ability to inactivate bacteria. UV light can inactivate bacteria by forming pyrimidine dimers (Oguma et al., 2001), which are lesions between thymine and cytosine bases in the DNA (Refer to Fig. 1). However, if DNA damage is minimal, it is possible the presence of the RecA DNA repair protein in E. coli could mend the any pyrimidine dimers that form (Taylor et al., 2020). This will allow for the safe and broad application of UV surface disinfection to kill all dangerous bacteria by applying the right wavelength of light for the correct time period. There is extensive research into how UV light effects DNA and a whole organism (Davies, 1995) but little on its potential application to surface disinfection and specifically targeting bacteria (Rass et al., 2008).

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Scientific Research Question Do Staphylococcus epidermidis (albus) and Escherichia coli differ in their susceptibility to ultraviolet light?

Scientific Hypothesis Null Hypothesis: There will be no recorded difference in the ultraviolet susceptibility between S. epidermidis and E. coli. Alternative Hypothesis: The dense outer lipid membrane (present in gram negative bacteria) of E. coli bacteria and the presence of DNA repair protein RecA, will offer greater resistance to ultraviolet light than the thick but less dense peptidoglycan cell wall (present in gram positive bacteria) of S. epidermidis, thus making E. coli bacteria less susceptible to ultraviolet light than S. epidermidis bacteria.

Methodology 1.

The base of 12 agar plates were labelled with a marker indicating date and number of seconds of exposure or control. 2. A sterile pipette was used to sample and put 2 drops of an E. coli culture solution on an agar plate. 3. A plate spreader was used to fully cover an agar plate with the bacteria culture solution on each agar plate. 4. A lid was placed on each plate and tape was wrapped around the sides of each plate and lid to secure it, while leaving a small gap for air flow. 5. Steps 1-4 were repeated 11 more times. 6. The base of the agar plate was labelled with a marker indicating date, bacterial sample, being S. epidermidis, and number of seconds of exposure or control. 7. A sterile pipette was used to sample and put 2 drops of a S. epidermidis culture solution on an agar plate. 8. A plate spreader was used to fully cover the agar with the bacteria culture solution. 9. The lid was placed on the plate and tape was wrapped around the sides of the plate and lid to secure it, while leaving a small gap for air flow. 10. Steps 6-8 were repeated 11 more times. 11. 3 of the E. coli and 3 of the S. epidermidis plates were then exposed to 4 minutes of UV light, one at a time in the CAMAG UV-Cabinet II at 254nm wavelength.

Scientific Research in School Volume 5 Issue 1 2023 12. 3 of the E. coli and 3 of the S. epidermidis plates were then exposed to 7 minutes of UV light, one at a time CAMAG UV-Cabinet II at 254nm wavelength. 13. 3 of the E. coli and 3 of the S. epidermidis plates were then exposed to 10 minutes of UV light, one at a time CAMAG UV-Cabinet II at 254nm wavelength (middle of recommended 200300nm in industrial applications). 14. All plates were placed in an incubator at 37°C for 3 days, including a third control group of three plates with no bacterial samples. 15. The plates were removed, and observations recorded by counting the number of bacterial colonies remaining with a marker and ROCKER Galaxy 330 Colony Counter.

Results The results of my experimental method appear below in Figures 2-4. Tables 1-4 show the results of the ANOVA test results from each exposure time. Figures 1-3 and Tables 1 and 2 illustrate the relationship between number of colonies and time of UV light exposure for each bacterium. Tables 1-4 reveal that there is a significant difference in the number of colonies of each bacterium at each exposure time bar 4 minutes.

Figure 4: Comparison of Mean Number of Colonies for each Bacterium

The graphing and ANOVA tests performed for each group revealed that S. epidermidis is significantly more susceptible to UV light than E. coli. The ANOVA tests resulted with the f-statistic varying greatly between each group and the p-value being less than the alpha-value in every case except in the 4 minutes exposure group. This group had a p-value of 0.1152 which is greater than the alpha-value of 0.01. E. coli had a greater standard deviation than S. epidermidis in every group as well as E. coli having 4 degrees of freedom and S. epidermidis having 1 degree of freedom in every group all as seen in Tables 1-4. Table 1: ANOVA Results for 0 minutes of Exposure

Figure 2: Experimental Data for S. epidermidis

Groups

Mean

S. epidermidis

1567.67 8166.67

Standard Standard Deviation Error 249.03 143.78 763.76 440.96

Source Between Groups Within Groups

D of F

F-Stat

P-Value

202.43

0.0001

E. coli

Figure 3: Experimental Data for E. coli

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Scientific Research in School Volume 5 Issue 1 2023 Table 2: ANOVA Results for 4 minutes of Exposure

Groups

Mean

S. epidermidis

2.33 475.67

Standard Standard Deviation Error 2.52 1.45 408.43 235.81

Source Between Groups Within Groups

D of F

F-Stat

P-Value

4.03

0.1152

E. coli

et al., 1990) as seen in Figure 6. This also includes pyrimidine dimers, refer to Figure 7, which are the primary type of DNA damage due to UV light (Kemp et al., 2012).

Table 3: ANOVA Results for 4 minutes of Exposure

Standard Standard Deviation Error 1 0.58 7 4.04

Groups

Mean

S. epidermidis

2 26

Source Between Groups Within Groups

D of F

F-Stat

P-Value

34.56

0.0042

E. coli

Figure 5: Difference in Gram-positive and Gram-negative cell walls Source: (Differences between Gram-Negative and GramPositive Bacteria., 2022)

Table 4: ANOVA Results for 7 minutes of Exposure

Standard Standard Deviation Error 1 0.58 1.73 1

Groups

Mean

S. epidermidis

1 9

Source Between Groups Within Groups

D of F

F-Stat

P-Value

0.0023

E. coli

Figure 6: Process by which RecA repairs mutations Source: (Roca et al., 1990)

Discussion The E. coli and S. epidermidis both yielded very different results at each exposure time with E. coli always forming more colonies. With the p-value being less than the alpha-value in every instance bar the 4 minutes exposure group, which can be interpreted as an outlier, the null hypothesis that there will be no difference in ultraviolet susceptibility can be rejected, and the alternate hypothesis that E. coli will be less susceptible than S. epidermidis can be accepted. However, the high standard deviation in E. coli indicates that the process used to culture the bacteria could be flawed. This could be found in the method of counting colonies which relied heavily on human perception. E. coli forms many small colonies that can be difficult to distinguish by eye when close together. The main differences between E. coli and S. epidermidis are the cell walls (E. coli having a gramnegative and S. epidermidis having a gram-positive) as seen in Figure 5, as well as E. coli possessing the RecA protein. RecA is a DNA repair protein that can identify point mutations and replace base pairs (Roca

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Figure 7: Pyrimidine Dimers in DNA Source: (Kemp et al., 2012)

As stated in the hypothesis, despite being thicker with much more peptidoglycan, the cell wall of S. epidermidis does not provide much protection from harmful ultraviolet waves as the peptidoglycan does not absorb much of their energy (Coohill et al., 2009). E. coli’s thinner but much denser cell wall with a double membrane provides much more absorbance of the UV waves, decreasing the number of pyrimidine dimers that can potentially form in the bacterium’s DNA. This paired with the 3-day period in which the cultures are left to grow provides plenty of time in which RecA can mend most of the pyrimidine dimers that form gives E. coli much greater protection from UV light than S. epidermidis.

Scientific Research in School Volume 5 Issue 1 2023 The method for this experiment is flawed and favours the growth of more E. coli colonies compared to S. epidermidis. By determining the number of bacteria that has grown in an agar dish by counting the number of colonies relies on human perception (as referenced before) but also favours E. coli as it naturally grows smaller but more colonies than S. epidermidis. This means that a plate with E. coli can form many more colonies than a plate with an equal population of S. epidermidis. Another variable that favours E. coli are the incubator temperatures which were set to 37°C. E. coli being a digestive tract infection has evolved to grow optimally at the human core temperature. S. epidermidis being a skin infection has evolved to grow optimally at human skin temperature which fluctuates at 30-35°C. While not preventing the growth of S. epidermidis, the temperature prevents it from growing as fast as possible which the E. coli can do as it is at optimal temperature.

Acknowledgements

To improve the method, a process other than colony counting should be devised to determine the number of bacteria grown on any given plate. The improve the validity, the lid of the agar plate should be removed when exposing to UV as it adds a layer of protection that bacteria would not receive in a realworld scenario. The thicker outer rim of the lid clearly mitigated the effect of the UV light as a ring of bacteria can be seen around the edges of the plate as seen in Figure 7. With more options of bacteria the experiment could shift to determine the difference in UV susceptibility between gram-negative and grampositive bacteria which was not possible with only 2 types of bacteria available. If more time and resources were available, more than 3 repeats of each group and more groups could be implemented to determine if the method is reliable and to improve the accuracy of the means to provide more detail in terms of specification for sterilisation.

Taylor, W., Camilleri, E., Craft, D. L., Korza, G., Granados, M. R., Peterson, J., ... & Setlow, P. (2020). DNA damage kills bacterial spores and cells exposed to 222nanometer UV radiation. Applied and Environmental Microbiology, 86(8), e03039-19.

Conclusion In conclusion my research project explored how susceptible both E. coli and S. epidermidis are to UV light. I grew 12 colonies of each bacterium and exposed them to UV light for 0, 4, 7 and 10 mins. I collected data by counting the colonies surviving on each agar plate after 3 days. My data analysis involved a one-way ANOVA test for each exposure time to compare the means of each. The results of my data showed that significantly more colonies of E. coli survived than S. epidermidis, leading me to accept my alternative hypothesis that E. coli is less susceptible to UV light than S. epidermidis.

I would like to thank Dr Alison Gates for helping me establish and execute my project idea.

References Bank, H. L., John, J., Schmehl, M. K., & Dratch, R. J. (1990). Bactericidal effectiveness of modulated UV light. Applied and Environmental Microbiology, 56(12), 38883889 Sonohara, R., Muramatsu, N., Ohshima, H., & Kondo, T. (1995). Difference in surface properties between Escherichia coli and Staphylococcus aureus as revealed by electrophoretic mobility measurements. Biophysical chemistry, 55(3), 273-277. Oguma, K., Katayama, H., Mitani, H., Morita, S., Hirata, T., & Ohgaki, S. (2001). Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Applied and Environmental microbiology, 67(10), 4630-4637.

Katara, G., Hemvani, N., Chitnis, S., Chitnis, V., & Chitnis, D. S. (2008). Surface disinfection by exposure to germicidal UV light. Indian journal of medical microbiology, 26(3), 241-242. Maclean, M., MacGregor, S. J., Anderson, J. G., & Woolsey, G. (2009). Inactivation of bacterial pathogens following exposure to light from a 405-nanometer lightemitting diode array. Applied and environmental microbiology, 75(7), 1932-1937. Coohill, T. P., & Sagripanti, J. L. (2009). Bacterial inactivation by solar ultraviolet radiation compared with sensitivity to 254 nm radiation. Photochemistry and Photobiology, 85(5), 1043-1052. Santos, A. L., Oliveira, V., Baptista, I., Henriques, I., Gomes, N., Almeida, A., ... & Cunha, Â. (2013). Wavelength dependence of biological damage induced by UV radiation on bacteria. Archives of microbiology, 195(1), 63-74. Roca, A. I., Cox, M. M., & Brenner, S. L. (1990). The RecA Protein: Structure and Functio. Critical reviews in biochemistry and molecular biology, 25(6), 415-456. Livneh, Z., & Lehman, I. R. (1982). Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli. Proceedings of the National Academy of Sciences, 79(10), 3171-3175. Davies, R. J. (1995). Ultraviolet radiation damage in DNA. Biochemical society transactions, 23. Rass, K., & Reichrath, J. (2008). UV damage and DNA repair in malignant melanoma and nonmelanoma skin cancer. Sunlight, Vitamin D and Skin Cancer, 162-178. Bannerman, D. D., Paape, M. J., Lee, J. W., Zhao, X., Hope, J. C., & Rainard, P. (2004). Escherichia coli and

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Scientific Research in School Volume 5 Issue 1 2023 Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clinical and Vaccine Immunology, 11(3), 463-472. Kemp, M. G., & Sancar, A. (2012). DNA excision repair: where do all the dimers go?. Cell Cycle, 11(16), 29973002. Differences between Gram-negative and Gram-positive bacteria. (2022). ResearchGate; ResearchGate. https://www.researchgate.net/figure/Differences-betweenGram-negative-and-Gram-positivebacteria_fig2_357901900 Global Disruption of Antibiotic-Resistant Bacteria. (2019, July 6). Public Health Post. https://www.publichealthpost.org/databyte/antibioticresistant-bacteria/ 7.7 million people die from bacterial infections every year – 2022 – ReAct. (2022, December 16). ReAct. https://www.reactgroup.org/news-and-views/news-andopinions/year-2022/7-7-million-people-die-frombacterial-infections-every-year/

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Scientific Research in School Volume 5 Issue 1 2023

Global recycling efforts: Zinc catalysts for efficient glycolysis of polyethylene terephthalate Henry Shortis Barker College Purpose: This research paper will outline the use of different zinc catalysts to break down the Polyethylene terephthalate (PET) polymer, their effect on the rate of glycolysis reaction and the yield of the Bis(2-Hydroxyethyl) terephthalate (BHET) degradation product. Design/methodology/approach: Glycolysis was done using ethylene glycol with an active zinc catalyst to break down the PET into other monomers and oligomers as well as the desired BHET. This was done at ~170 °C for 2 hours to achieve optimal results. Findings: The zinc sulfate catalyst was the most effective in terms of yield. The zinc acetate catalyst, which is said but current literature suggests is the most effective, showed relatively low yields compared to the other catalysts. Zinc chloride provided the highest consistent yield out of all the catalysts. Zinc nitrate gave the lowest yields while also creating a terracotta colour in the solution and product. Research limitations/implications: Testing the reliability of the results was a limitation of this research due to time constraints, which meant that the consistency of the data obtained was not assessed. Additionally, limited available lab equipment meant that the accuracy of the results was not optimised in this study and should be further optimised in the future. Practical implications: The practical implication of this paper is to help further the research into the chemical recycling of PET plastics specifically the catalysts involved in the glycolysis reaction. This research can help to investigate ways in which discarded plastics can be recycled in an effort to reduce landfill dumping and environmental issues associated with burning waste. Social implications: The social implication of this paper is to assist in the global effort towards improving recycling of plastics by providing an effective method for doing so, as well as to determine the possibility of a closed-loop recycling system turning PET into BHET then back into PET for different moulds. Originality/value: This paper is building on other research papers by further testing catalysts as well as testing unique catalysts not previously investigated. Keywords: Bis(2-Hydroxyethyl) terephthalate, Polyethylene terephthalate, glycolysis, catalyst Paper Type: Research paper

Literature Review Introduction Poly(ethylene terephthalate) (PET) is a versatile thermoplastic resin and is extensively used for various products in the forms of fibres, films, etc. PET is either produced through the esterification of Terephthalic acid with ethylene glycol followed by polycondensation, transesterification using dimethyl terephthalate with ethylene glycol, or bis(hydroxyethyl) terephthalate (BHET) and a catalyst. Global plastic recycling The recycling of plastics has been a long-standing endeavour being undertaken by many scientists. Worldwide polymer production was estimated to be 260 million metric tonnes per annum in the year 2007 (Hopewell, Dvorak & Kosior, 2009) and all the plastic must go somewhere. A study conducted (Gourmelon, 2015) indicated that in the United States

alone, only 9% (2.8 million tons) of total plastics consumed were recycled in 2012. The rest of the plastics were either dealt with in the nation or shipped to other nations such as China for them to deal with, effectively or otherwise. Within the respective nations, the majority was either put into landfill which accounted for 13% of the nation’s municipal solid waste stream (Thiounn & Smith, 2020). In terms of the waste shipped to China, which receives 56% of world plastic waste imports, it isn’t well known what occurs from there. The International Solid Waste Association reports that indirect evidence suggests that most plastic is still being reprocessed using family-run, low-tech businesses with no environmental protection controls. The Chinese government, however, has started to work to reduce the amount of these unregulated facilities through its Green Fence Operation in 2010 (Gourmelon, 2015).

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Scientific Research in School Volume 5 Issue 1 2023 This puts forward concerns about the transparency of countries in terms of where the waste goes and its impact on the local environments, as well as nations shipping their waste to China to consider whether the waste can be effectively dealt with in their own country. The exportation of plastic waste to China also creates another major issue with the world relying on China to deal with all its plastic exports, this creates a situation with one country controlling the fate of the world's recycling situation with no room for an alternative. Scientists across the globe have been trying to develop more effective procedures for recycling to allow individual nations to deal with their waste within their own borders. One such method is closed-loop recycling. The overall effort of closed-loop recycling is to allow for the continual production of plastics while reducing the overall waste they create (Figure 1). Within Australia and New Zealand, the company Closed Loop is making strides in a circular economy of products, materials, and resources. Their service provides consultation with businesses to determine the most effective method of creating a circular economy that fits the practical nature of the company. Closed Loop has direct support from the Australian Government to champion a new age of upcycling products to reduce overall waste.

Figure 2: Closed-loop system for PET plastics Source: (Damayanti & Wu, 2021)

Chemical recycling of plastics The chemical recycling of polymers, specifically PET, is a process where the polymer is depolymerised into its original components or monomers in which can then be repolymerised into new oligomers or even be turned back into PET. There are a multitude of pathways to get back to the desired PET polymer (Figure 2).

Figure 1: Different methods of PET recycling through chemical processes and their products

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Scientific Research in School Volume 5 Issue 1 2023 Research into closed-loop systems with PET The method this paper used was glycolysis is the reaction of depolymerization of PET with ethylene glycol leading to Bis(2-Hydroxyethyl) terephthalate (BHET) and PET oligomers. The glycolysis reaction creates a crystallized product that can then be used in repolymerisation to return to PET. Repolymerisation can be done through a mixture of BHET and a catalyst and can be isolated after being washed with methanol and tetrahydrofuran (THF). The insoluble fraction can be isolated by centrifugation and dried under a vacuum (Shah et al., 1984). According to Jeswani et al., (2021) another method known as Pyrolysis, which involves superheating biomass without the presence of oxygen, emits 50% less CO2 than other energy recovery methods. Environmental impacts Even with the global effort towards the cycling of plastic waste, there is still a great disparity between the amount of waste and how much is recycled. Research has shown that the plastic waste generated in the United States during 2018 was 35.7 million tons, however, only 4.5% of this was recycled (Benyathiar et al. 2022). The overall waste buried in landfills still contribute to air, water, and soil pollution. Even with chemical recycling, it still isn’t completely safe for the environment yet. According to Lee and Liew, (2020), methods of chemical recycling including hydrolysis and sometimes glycolysis, produce unwanted waste in terms of catalysts and by-products, namely acids, which need to be safely handled or else it could have drastic impacts on the environment. Although chemical recycling reduces the amount of CO2 waste, the use of chemicals and catalysts can create unwanted issues that can be toxic to the environment. However, in terms of overall ethics involved in using chemical recycling over primary and secondary methods, the benefits of chemical recycling outweigh the negative impacts presented by primary and secondary methods. As outlined by Lee et al., (2021), the prospect of chemical recycling will play a crucial role in sustainable waste management. Although the economic uncertainty and constraints make people sceptical about large-scale recycling, the overall potential is there to allow for eco-friendly plastic recycling with future possibilities for closed-loop systems and net zero emissions. Building on current literature This research paper will help to expand the current literature on this topic by going further in-depth into different zinc-based catalysts, which is shown by the literature to be the most effective catalyst, and show

which specific ones are the most effective and efficient. Overall, finding out the most effective catalysts for this reaction can figure out how efficient the chemical recycling process is as well as consider other factors such as cost, availability, and yield. My project revolves around the chemical recycling of plastics, specifically the polymer PET, which is most common for plastic bottles of commercial drinks. This paper will be investigating the interactions between the chemical process of breaking down the PET polymer into a BHET polymer and using different catalysts to help speed up the reaction to break down the PET polymers into BHET. Previous research suggests that zinc acetate is likely to prove a useful catalyst for this reaction. This paper will be using the zinc acetate catalyst as a control, building on the current research, and using other catalysts such as zinc chloride, zinc nitrate, and zinc sulfate to compare to the zinc acetate.

Scientific Research Question How does the use of different zinc-based catalysts affect the yield of the BHET (Bis (2-Hydroxyethyl) terephthalate) polymer from the chemical decomposition of the PET (Polyethylene terephthalate) polymer through a glycolysis reaction (Figure 3)?

Scientific Hypothesis The catalyst zinc acetate will be the most efficient in the glycolysis of PET and provide the highest yield.

Figure 3: Chemical pathway of PET to BHET through glycolysis

Methodology Chemicals The list of chemicals and their respective weights per test are as follows: Polyethylene terephthalate (10g), ethylene glycol (27.825 ml), zinc acetate (0.25g), zinc chloride (0.18g), zinc sulfate (0.22g), zinc nitrate (0.26g). All catalysts were done at a 1:38 molar ratio

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Scientific Research in School Volume 5 Issue 1 2023 of PET to Catalyst and the Ethylene Glycol was done at a 1:7.6 molar ratio of PET to Ethylene Glycol. These ratios were outlined in previous research from Khoonkari et al., (2015) and López-Fonseca et al., (2011). Process The PET granules first had to be cleaned of surface impurities to ensure that foreign matter would interfere with the experiment. This was done by washing the granules using dilute (1%) sodium hydroxide for roughly an hour (Figure 4). The granules were then filtered out and left to dry in an oven at 80 °C. Each reaction included the chemicals and weights as per listed above and were put into a 250ml beaker. The beakers were then wrapped in cotton balls and aluminium foil to help with insulation (Figure 5). Each beaker was then placed on a heating mantle with a stir bar and set to ~170 °C. A watchglass was then placed on the beakers to further assist with insulation as well as to protect from potentially volatile reactions as part of a safety precaution. The reaction was then left for 2 hours to allow for both the heating mantle to reach the desired temperature and for the reaction to take place.

After the 24 hours, the crystallised BHET was then put through the sintered-glass filter again to remove the excess distilled water, with the residue being the final BHET product.

Figure 5: Insulation of the beakers

An initial weight was recorded, and then the residue was put into a vacuum chamber with drying agent to get rid of any potential distilled water still in the residue. Repeated weighing was then conducted until a consistent weight was reached. This weight was then put into the formula to determine yield of BHET.

Figure 6: Reaction filtrate containing BHET and distilled water initially inside the refrigerator

Figure 4: Process of cleaning the PET granules

After this period, the reactions were let to cool, then were transferred over to 1000ml beakers with the use of hot distilled water (400ml). While the reactions were still hot, they were then filtered through the use of a sintered-glass filter under vacuum. The collected filtrate was then transferred into a conical flask where it would then be placed in a refrigerator at 4 °C for 24 hours to allow for the crystallisation of the BHET (Figure 6,7).

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Figure 7: Crystallised BHET submersed in distilled water

Scientific Research in School Volume 5 Issue 1 2023 Calculations The formula being used can be shown in this equation (López-Fonseca et al., 2011): 𝑤𝑤𝑤𝑤ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 / 𝑀𝑀𝑀𝑀𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝐹𝐹𝐹𝐹𝑤𝑤𝑤𝑤ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 Yield % = 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐼𝐼𝐼𝐼𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝐹𝐹𝐹𝐹 × 100 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝐹𝐹𝐹𝐹𝑤𝑤𝑤𝑤ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 / 𝑀𝑀𝑀𝑀𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑀𝑀𝑀𝑀 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝐹𝐹𝐹𝐹𝑤𝑤𝑤𝑤ℎ𝑡𝑡𝑡𝑡 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑃𝑃𝑃𝑃𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵

In terms of this formula the assumption is made that the total product from the reaction is 100% pure BHET which, within the limitations of the research paper, is out of our scope. Further research will be done to investigate possible alternatives for extraction to help ensure the purity of the BHET within our limitations. This was limited through the use of the hot distilled water which researched shows can help dissolve any unreacted catalyst as well as potentially some higher oligomers such as dimers and trimers (López-Fonseca et al., 2011).

Results Table 1: Yield percentages of each catalyst across the trials

Catalyst Zinc acetate Zinc nitrate Zinc chloride Zinc sulfate

Trial 1 15.82 17.35 70.76 N/A*

Yield (%) Trial 2 Average 41.38 28.60 25.04 21.20 72.57 71.67 94.01 94.01**

*Trial 1 of zinc sulfate encountered an issue where the PET didn’t properly undergo glycolysis. However, since this issue can be prevented in future experiments, it is being deemed as N/A. **Moreover, since trial 1 is counted as N/A and not 0, then the average of the zinc sulfate is 94.01 even though it’s only a single test. A more detailed explanation will be provided in the discussion.

Discussion Overall, the differing anions of each catalyst created different conditions for the reactions which caused the difference in each respective yield. Zinc nitrate’s terracotta colour is also another reason as to its lack of use. If the recycling process turns the BHET a off shade of beige, it would make processes more time consuming returning its colouration to the typical white that polymers are. Zinc acetate provided a relatively low yield in the second experiment of 41% which goes against what other research papers had found. Current literature showed that zinc acetate would provide yields ranging from 74% (Liu et al., 2018) up to 80% (Guo, Lindqvist & de la Motte,

2018) which goes against the results found in this paper as well as the proposed hypothesis. An explanation as to this discrepancy is that the ratios used in this paper are different to those used in the aforementioned papers. This difference could be the reason behind the differing yields and that the yields of BHET can alter based on the molar ratios across the experiment. In contrast to this, as shown in Table A, Zinc Chloride provided consistent yields of 70% to ~73% and zinc sulfate providing a single significant yield of 94% which gives reason for its effectiveness. Zinc chloride’s relatively high and consistent yield shows that it has some potential for the glycolysis of PET on a larger scale. Alongside this, in figure 7, the zinc sulfate produced the finest crystalised BHET which gives a visual confirmation of the quality of the yield produced. Yields between 70-79% are classified as very good for PET glycolysis with papers generally getting just shy of 70% (Khoonkari et al, 2015). Zinc sulfate’s yield is the highest out of all catalysts at 94.01%. The first trial of the zinc sulfate encountered an issue during the heating process in which the reaction didn’t properly undergo the glycolysis and the PET granules remained solid, albeit slightly softened. This issue can easily be overcome which is what occurred in the second trial achieving the highest yield. Zinc sulfate was a lesser used catalyst in majority of the papers so further research into the catalyst and its potential to be more effective than both zinc acetate and zinc chloride. Overall, a reason as to the differences in yields compared to the current literature is due the limitations within the experimentation. A major limitation encountered during the experiments was availability of lab equipment such as a glass thermometer inside the reaction vessel. This meant that the temperature had to be checked using an external temperature probe which would provide inaccurate readings as to the temperature the reactants are at. Another limitation is that of time, meaning that only 2 rounds of testing could be conducted. This meant that although trends can be seen in some of the catalysts, an overall conclusion is hard to draw as there isn’t enough data to use to establish a definite conclusion. However, with the limited data in this paper, the experiment was still valid, and observations can be taken from the results gathered.

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Scientific Research in School Volume 5 Issue 1 2023 In terms of furthering this experiment, continuations should aim to control and overcome the limitations of this experiment. The understanding of the procedure before running the experiment and gathering results is vital. Being able to gather more results will be able to definitely determine which of the four catalysts is the most effective, as well as the potential to test other non-zinc-based catalysts to see if there is another cation which is more effective for the glycolysis of PET.

Conclusion I investigated the efficacy of four zinc compounds, namely, zinc chloride, etc. for their potential as glycolysis catalysts for the decomposition of PET into BHET. Zinc chloride proved to be the most effective catalyst, affording the highest consistent yield of BHET from PET, however, zinc sulfate had the highest yield in a single test. Although it was hypothesised that Zinc acetate would produce the highest yield of BHET, this was not the case in this experiment. With further experimentation, there is potential for Zinc sulfate to be the optimal catalyst to use for the glycolysis of PET into BHET in industrial production of chemical recycling.

Acknowledgements I would like to thank Dr Katie Terrett for helping develop and conduct my project idea, and Dr Matthew Hill for assisting in structuring the report.

References Al-Sabagh, AM, Yehia, FZ, Eshaq, Gh, Rabie, AM & ElMetwally, AE 2016, ‘Greener routes for recycling of polyethylene terephthalate’, Egyptian Journal of Petroleum, vol. 25, no. 1, pp. 53–64. Benyathiar, P, Kumar, P, Carpenter, G, Brace, J & Mishra, DK 2022, ‘Polyethylene Terephthalate (PET) Bottle-toBottle Recycling for the Beverage Industry: A Review’, Polymers, vol. 14, no. 12, p. 2366. Damayanti & Wu, H-S 2021, ‘Strategic Possibility Routes of Recycled PET’, Polymers, vol. 13, no. 9, p. 1475. Davidson, MG, Furlong, RA & McManus, MC 2021, ‘Developments in the life cycle assessment of chemical recycling of plastic waste – A review’, Journal of Cleaner Production, vol. 293, no. 2, p. 126163.

Hopewell, J, Dvorak, R & Kosior, E 2009, ‘Plastics recycling: Challenges and Opportunities’, Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 364, no. 1526, pp. 2115–2126. Jeswani, H, Krüger, C, Russ, M, Horlacher, M, Antony, F, Hann, S & Azapagic, A 2021, ‘Life cycle environmental impacts of chemical recycling via pyrolysis of mixed plastic waste in comparison with mechanical recycling and energy recovery’, Science of The Total Environment, vol. 769, no. 3, p. 144483. Khoonkari, M, Haghighi, AH, Sefidbakht, Y, Shekoohi, K & Ghaderian, A 2015, ‘Chemical Recycling of PET Wastes with Different Catalysts’, International Journal of Polymer Science, vol. 2015, no. 3, pp. 1–11. Lee, A & Liew, MS 2020, ‘Tertiary recycling of plastics waste: an analysis of feedstock, chemical and biological degradation methods’, Journal of Material Cycles and Waste Management, vol. 23, no. 1. Lee, J, Kwon, EE, Lam, SS, Chen, W-H, Rinklebe, J & Park, Y-K 2021, ‘Chemical recycling of plastic waste via thermocatalytic routes’, Journal of Cleaner Production, vol. 321, no. 2, p. 128989. Liu, B, Lu, X, Ju, Z, Sun, P, Xin, J, Yao, X, Zhou, Q & Zhang, S 2018, ‘Ultrafast Homogeneous Glycolysis of Waste Polyethylene Terephthalate via a DissolutionDegradation Strategy’, Industrial & Engineering Chemistry Research, vol. 57, no. 48, pp. 16239–16245. López-Fonseca, R, Duque-Ingunza, I, de Rivas, B, FloresGiraldo, L & Gutiérrez-Ortiz, JI 2011, ‘Kinetics of catalytic glycolysis of PET wastes with sodium carbonate’, Chemical Engineering Journal, vol. 168, no. 1, pp. 312– 320. Ragaert, K, Delva, L & Van Geem, K 2017, ‘Mechanical and chemical recycling of solid plastic waste’, Waste Management, vol. 69, no. 2, pp. 24–58. Schwarz, AE, Ligthart, TN, Godoi Bizarro, D, De Wild, P, Vreugdenhil, B & van Harmelen, T 2021, ‘Plastic recycling in a circular economy; determining environmental performance through an LCA matrix model approach’, Waste Management, vol. 121, no. 3, pp. 331–342. Shah, TH, Bhatty, JI, Gamlen, GA & Dollimore, D 1984, ‘Aspects of the chemistry of poly(ethylene terephthalate): 5. Polymerization of bis(hydroxyethyl)terephthalate by metallic catalysts’, Polymer, vol. 25, no. 9, pp. 1333–1336. Soong, Y-HV, Sobkowicz, MJ & Xie, D 2022, ‘Recent Advances in Biological Recycling of Polyethylene Terephthalate (PET) Plastic Wastes’, Bioengineering, vol. 9, no. 3, p. 98. Thiounn, T & Smith, RC 2020, ‘Advances and approaches for chemical recycling of plastic waste’, Journal of Polymer Science, vol. 58, no. 10, pp. 1347–1364.

Gourmelon, G 2015, Global Plastic Production Rises, Recycling Lags, 27 January, Vital Signs, Washington, DC, pp. 91–95.

Xin, J, Zhang, Q, Huang, J, Huang, R, Jaffery, QZ, Yan, D, Zhou, Q, Xu, J & Lu, X 2021, ‘Progress in the catalytic glycolysis of polyethylene terephthalate’, Journal of Environmental Management, vol. 296, no. 2, p. 113267.

Guo, Z, Lindqvist, K & de la Motte, H 2018, ‘An efficient recycling process of glycolysis of PET in the presence of a sustainable nanocatalyst’, Journal of Applied Polymer Science, vol. 135, no. 21, p. 46285.

Xochitl, Q-P, María del Consuelo, H-B, María del Consuelo, M-S, Rosa María, E-V & Alethia, V-M 2021, ‘Degradation of Plastics in Simulated Landfill Conditions’, Polymers, vol. 13, no. 7, p. 1014.

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Scientific Research in School Volume 5 Issue 1 2023

The effect of increased dietary energy and protein intake on mammalian skeletal muscle cells Sam Zibaee Barker College Purpose: This paper aims to investigate the changes that mammalian skeletal muscle cells undergo on a higher intake of dietary energy and apply the findings to sports medicine specifically looking to slow muscle atrophy in athletes during periods of injury. Design/methodology/approach: In order to test this, samples that were taken from grass-fed and grain-fed beef and were stained on slides. Photographs were taken of the samples under x100 magnification on a Light Microscope (LM) cell area, cell abundance, endomysium and perimysium width, and nuclei were recorded across each sample. These results were compared to determine the differences in tissues and cell structure as influenced by higher dietary protein and energy consumption (grain-fed) to the control group (grass-fed). Findings: It was found that the muscle cells nourished with a high intake of dietary energy and protein (grain-fed) had a significantly increased cross-sectional area when compared to the samples that were on a traditional pasture diet (grass-fed) (t = 3.10497, p = 0.005578). Research limitations/implications: The paper was limited to using tissue samples from cows, a small sample size of 6 was used from each treatment – 12 total. The results have the implication that future research should a test wider range of samples from cows, and also consider other mammals and humans in ethical, controlled, and appropriate settings. Practical implications: These findings may be applied in sports medicine to manage injured athletes with reduced exercise and redirect focus to nutrition. This paper could be improved if it were conducted again with greater control on any factors with potential to impact the net protein balance. Originality/value: At the time of research previous papers have investigated exercise induced hypertrophy, and sarcopenia (muscle atrophy related to aging) in men aged 68-71 years. The findings from this paper aim to demonstrate the significance of strictly nutrition in hypertrophy. Keywords: hypertrophy, muscle protein synthesis, muscle protein breakdown, nutrition, net protein balance Paper type: Research paper

Literature Review Muscle function and injury prevalence A long-term injury can be devastating to an athlete’s performance and career. During elongated periods of inactivity, muscle atrophy occurs, this is the thinning or loss of muscle tissue (MedlinePlus, 2021) and strength (Cao et al., 2018). This makes the recovery process for them even longer as they need to rebuild that muscle and coordination to the level it once was prior to their injury. A mean of 62.49 injuries per 100 players per season was recorded across the Major League Baseball, National Basketball Association, National Football League, and National Hockey League from 2007 to December 2019 (Bullock et al., 2019). With more than half of the world’s best athletes sustaining injuries in an increasingly competitive pool of players, methods of rehabilitation should be investigated.

The base function of skeletal muscle is to contract to produce movement, stabilise, maintain body posture/position, and support joints (McCuller et al., 2023). A muscle is made up from bundles of muscle fibres, skeletal muscle myoblasts differentiate and fuse into multinucleated cells which behave as one unit to contract or lengthen (Braithwaite & Al Khalili, 2022). The skeletal muscle aids in maintaining the body's posture and offers structural support. Additionally, the skeletal muscle serves as a repository for amino acids that are used by the body's many organs to create proteins that are specific to those organs, skeletal muscle is also essential for preserving thermostasis and serves as an energy source during starvation (Dave, Varacallo & Shook, 2018). Muscular atrophy vs hypertrophy Atrophy is a result of when the rate of muscle protein synthesis (MPS) is inferior to muscle protein breakdown (MPB) (Ding et al., 2018), this means that

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Scientific Research in School Volume 5 Issue 1 2023 lack of exercise is not the sole cause of atrophy, inadequate levels of nutrition are another major component in the muscle protein degradation process. Thus, for recovering athletes as well as the greater population who are limited in their movement, atrophy has the potential to be minimised with proper nutrition. Skeletal muscle growth referred to as skeletal muscle hypertrophy, manifests as increases in muscle mass, muscle thickness, muscle area, muscle volume, and (importantly for this study) increases in muscle fibre cross-sectional area (Haun et al., 2019). An individual’s rate of muscle protein synthesis must exceed the rate of their muscle protein breakdown to result in a positive net protein balance, hypertrophy (Krzysztofik et al., 2019). In a rested and fasted state, rates of muscle protein breakdown (MPB) exceed those of muscle protein synthesis (MPS) and thus skeletal muscle is in a state of negative net protein balance (Biolo et al., 1995). The balance of MPS & MPB and consequence for muscles is summarised in Table 1. However, in response to protein feeding, it is shown there is a significant but short-term increase in rates of MPS and no significant change in MPB rendering skeletal muscle in a state of positive net protein balance (NBAL) (Biolo et al., 1997; Phillips, 2004). Table 1: understanding components NBAL

MPS < MPB MPS = MPB MPS > MPB

=> negative net protein balance (atrophy) => zero net protein balance => positive net protein balance (hypertrophy)

Things that affect MPB Muscle atrophy occurs when MPB rates exceed MPS. This can be induced from a variety of conditions, one notably being inadequate levels of nutrition (Schiafinno et al., 2013). It must be understood that the lack of nutrition isn’t ‘stimulating’ MPB, but rather MPB achieves a more substantial impact on NBAL as MPS is inhibited resulting in a NBAL < 0. Tipton, Hamilton & Gallagher (2018) describe exercise as a ‘powerful mediator of MPB’ and that ‘training status’ (exercise frequency/experience) is also a determinant, finding that resistance-trained individuals saw a ‘little, if any’ increase in MPB postexercise, the opposite was found in untrained individuals who demonstrated measurable increases of MPB with exercise at the same relative intensity. Insulin availability and carbohydrate intake has demonstrated to show a clear role in reducing MPB

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post exercise (Abdulla et al., 2016; Glynn et al., 2010). Things that effect MPS According to research by Tipton, Hamilton & Gallagher (2018), despite exercise is a signal for MPB, it has a much more potent increase on MPS, this increase can last 24-48 hours post-exercise (Phillips, 2013) depicted by the dashed line in Figure 1, whereas increased levels of MPB will end after a 24-hour period. Essential amino acid (EAA) ingestion has been found to show increases in muscle protein anabolic response, a net gain in protein balance (Walker et al., 2013), depicted by the solid peaks in Figure 1.

Figure 1: elevated rate of MPS 48hrs post-exercise Source: (Phillips, 2013).

When EAA levels are at or above their postabsorptive concentrations, it is detected that insulin stimulates MPS and reduces protein degradation (Everman et al., 2016) resulting in an overall positive NBAL. Nutrition From Kumar et al. (2009), when exercising in a fasted state, NBAL will not achieve a positive value despite an increase in MPS, because the rate of MPB which exceeded that of MPS has also risen. Protein ingestion facilitates post-exercise recovery and supports the growth and maintenance of skeletal muscle (Mazzulla et al., 2018), it is crucial for MPS, Nutritional content in typical cattle feeds This study will use muscle from store bought steaks to meet ethics protocols and investigate within the bounds of high school equipment limitations. Hence it is important to understand the differences in cattle feed and the nutritive values of a standard grain ration vs a grass ration. Table 2 below shows the energy requirements for beef steers at different production states (200kg = young, fast growing & 450kg = finishing/slaughter weight) for maintenance, this will

Scientific Research in School Volume 5 Issue 1 2023 supply the animal with enough energy to maintain functions and structure, but falls short for muscle growth, NBAL = 0. Table 2: average daily energy requirements for steers to have a NBAL = 0, Source: (Brown, Hindmarsh and Mcgregor, 2015, p.86)

Animal and its production state Beef steer (200 kg) Maintenance Beef steer (450 kg) Maintenance

(MJ) 27.0 49.0

From Meat and Livestock Australia, often the high protein requirements of fast-growing young steers cannot be met without additional protein supplementation (MLA, 2006). A grain ration can cater to this as additional protein dense sources can be supplied; however, this is where a strict grass/pasture ration will be stretched to meet such requirements, see Table 3.

of Dry Matter (DM), which tends to be 2%-3% of the cattle’s liveweight per day. Table 4: metabolisable energy (MJ/kg DM) of grain and grass feeds, available from equal gross energy intake Source: (MLA, 2006, p.8).

Gross energy (MJ/kg DM) Digestibility (%) Digestible energy (MJ/kg DM) Metablised (%) Metabolisable energy (MJ/kg DM)

Type of feed Grain Straw 18

90%

40%

16.25

7.2

80%

5.8

Table 3: nutritive value of a typical pasture vs. soybean meal, Source: (Brown, Hindmarsh and Mcgregor, 2015, p.84)

Ryegrass/clover pasture

Soybean meal

4.2

15.6 40%

Phosphorus (P) Ca:P ratio

40% 12%, supplies 9 of 10 essential amino acids 4.3g/kg dry matter 1.0-4.0/kg dry matter 2:1

Uses

Roughage

Food Energy MJ/kg dry matter Carbohydrate Protein Calcium (Ca)

46%, has all essential amino acids 30g/kg dry matter 7.2g/kg dry matter 1:2.4 Protein source but unpalatable in quantity

Ryegrass and clover (most commonly white clover) are strongly recommended for use in cattle grazing pastures (Griffiths, 2011) and soybean has been a common staple in grain rations for its nutritive value (Lehmkuhler and VanValin, 2021), Table 3 compares these two industry-standard sources of nutrition. The soybean meal is superior to pasture in energy (3.7 x pasture MJ/kg), protein (3.8 x protein and supplies all EAA), and other micronutrients (Ca & P) explaining its popularity in the beef industry. Additionally, grain-feeds are processed and engineered to produce high yield in nutrients, Table 4 provides insight to the significantly higher nutritive figures showing that grain is more digestible thus greater energy and protein is yielded from the cattle from the same mass

Figure 2: Dietary energy flow hierarchy for cattle. Source: (MLA, 2005, p.8)

Scientific Research Question How nutrition (specifically energy/caloric and protein per volume) affects mammalian muscular hypertrophy and atrophy.

Scientific Hypothesis That grain-fed cows (high caloric and protein concentration) will have more hypertrophied muscles (as measured by the cell cross-sectional area) than grass-fed cows.

Methodology 3 grass-fed rump steaks and 3 grain-fed rump steaks were bought from separate supermarkets, 1 of each from Woolworths, Coles, and Aldi to maximise variety. The unopened steak packages were sent to Douglass Hanly Moir Pathology for further processing and slide preparation. Each steak had

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Scientific Research in School Volume 5 Issue 1 2023 three biopsies taken from it: 2 cross-sectional to the muscle fibres and 1 transversal to the muscle fibres. These blocks of tissue were embedded in paraffin wax and labelled (see Figure 3).

Quantitative analysis involved the 6 cross-sectional cell areas being averaged for the grass-fed and the grain-fed slides, a student’s t-test was performed to compare the two means. An alpha value of 0.05 was used therefore if the t-test returned a p value less than 0.05 the difference in means would be deemed significant.

Figure 3: tissue block embedded in paraffin wax

2μm thin layers were shaved off the tissue blocks with a microtome; the thin shavings were stained use Haematoxylin and Eosin (H&E) – the most widely used stain in histology for tissue biopsies and were then aligned on a microscope slide as shown in Figure 4. The same process was conducted for all other cross-section blocks, producing 6 samples for each treatment, 12 total.

Figure 4: Slide 1A fully prepared

A light microscope (LM) was used to collect data and other observations. Using a microscope camera mounted to the eyepiece, images were taken of the slides under x100 magnification, at which they would be observed at to count cell number. A μm scale bar slide at x100 magnification was referred upon to calculate the area of the field of view (FOV) of the images to be 24.19μm2. By dividing 24.19μm2 by however many cells were counted, the approximate cross-sectional area for a cell on that slide was determined.

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Figure 5: image used for cell count, Slide 6B (x100).

Results Table 5: grain-fed cells had a larger cross-sectional area than grass-fed cells

Number of cells in 24.19𝝁𝝁𝝁𝝁𝝁𝝁𝝁𝝁2 area

Cross-sectional area of cells (μm2)

Sample

Grain

Grass

Grain

Grass

0.81

0.71

0.97

0.83

0.58

0.50

1.21

0.49

0.97

0.58

0.97

0.39

27.8

44.0

0.92

0.59

Scientific Research in School Volume 5 Issue 1 2023 2015), the 3.7 x MJ/kg DM more in grain than in pastures enables the maximisation of MPS found in the results.

Figure 6: column graph with standard deviation error bars

A students t-test was conducted to analyse these values: The t-value is 3.10497. The p-value is .005578. The result is significant at p < .05. Grain-fed cell Variance, s2: 0.0438567 Standard Deviation, s: 0.2094198 Grass-fed cell Variance, s2: 0.021655555 Standard Deviation, s: 0.14715826

Discussion The results showed that the grain-fed muscle cells had a larger cross-sectional area (= 0.92 μm2) than the grass-fed muscle cells (= 0.56 μm2), with a p-value = 0.005578 significant at p < 0.05. Thus, the primary hypothesis that the grain-fed cattle would have more hypertrophied cells has been supported. This result can be attributed to the higher dietary energy and protein intake of grain-fed cattle compared to the grass-fed cattle. It is known that when carbohydrates are ingested post-exercise it will inhibit MPB rates (Abdulla et al., 2016; Glynn et al., 2010) and EAA availability will stimulate increases MPS (Walker et al., 2013). When EAA levels are at or above their post-absorptive concentration, reduces protein degradation (Everman et al., 2016), reduced MPB and increased MPS will result in an overall positive NBAL. As the that are cattle fed with grain have high protein, energy, and EAA intake (Table 3), despite minimal exercise, the abundance of the required nutrients for MPS will mean that even in a rested state they will be in a state of positive NBAL. An increase in dietary energy is required to maximise MPS to supply sufficient glucose, stores of glycogen, and to fuel the ‘costly protein synthetic process’ (Slater et al., 2019; Morton, McGlory and Phillips,

However, this is only true if the assumption that both grain-fed and grass-fed cattle are consuming similar masses is met. If it is not, then other factors must be considered like exercise and regularity of feeding. Figure 1 is strong evidence that these factors could skew the data, it displays intense spikes of MPS increase following feeding/protein ingestion. Protein anabolism (synthesis) is favoured when amino acids are supplied evenly and steadily throughout the day (Mosoni and Mirand, 2003), this paper is limited as it is unaware of the specific rations fed, times fed, regularity of feeding, exercise, age, and etc. It is unknown how many kg of DM each of the cattle was consuming per day, there is also a possibility that some cattle were provided with a more palatable diet incentivising greater DM consumption. Further research should look at these factors in more detail, it can aim to control as many variables as possible or investigate the effect they individually have to compose a greater understanding of interactions between these factors behind the process of hypertrophy. A deeper analysis of nutrition has the potential to differentiate carbohydrates and protein by how pronounced their contributions are to a positive NBAL. This knowledge would have meaningful implications in sports medicine for injury recovery as well as in agriculture, possibly elucidating more efficient methods feeding in the beef industry.

Conclusion This paper aimed to investigate the efficacy and importance of nutrition to slow skeletal muscle atrophy and support hypertrophy with minimal nonresistance exercise in order to apply the findings to assist injured athletes and those living a sedentary lifestyle. Data was collected from 6 grass-fed rump samples (representing a regular diet) and 6 grain-fed rump samples (representing a high energy and high protein diet) using a microtome, they were then stained with Hematoxylin and Eosin on slides, then examined under a Light Microscope at x100 magnification. The data analysis involved a t-test to compare the cross-sectional area of the cells in each treatment, these results showed that there is a significant difference between the two (t = 3.10497, p = .005578) supporting the primary hypothesis that the proliferation of muscular hypertrophy will

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Scientific Research in School Volume 5 Issue 1 2023 increase when exposed to a higher dietary energy intake.

Acknowledgements I would like to thank Dr Katie Terrett and especially Dr Matthew Hill for guiding me through the process of constructing a research paper and taking me on as their mentee. I extend my gratitude to Dr Alison Gates who supported me through the early stages of making a project I would be passionate about and helping me throughout the data collection. A special thanks to Mrs Virginia Ellis (Head of Science) for outsourcing the help from Douglass Hanly Moir Pathology who kindly made the data collection possible, and to Agriculture teacher Mrs Lucy Pitkin for providing her knowledge in this field.

References Abdulla, H., Smith, K., Atherton, P.J. and Idris, I. (2015). Role of insulin in the regulation of human skeletal muscle protein synthesis and breakdown: a systematic review and meta-analysis. Diabetologia, 59(1), pp.44–55. doi:https://doi.org/10.1007/s00125-015-3751-0. Biolo, G., Maggi, S.P., Williams, B.D., Tipton, K.D. and Wolfe, R.R. (1995). Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. American Journal of PhysiologyEndocrinology and Metabolism, 268(3), pp.E514–E520. doi:https://doi.org/10.1152/ajpendo.1995.268.3.e514. Biolo, G., Tipton, K.D., Klein, S. and Wolfe, R.R. (1997). An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. The American Journal of Physiology, [online] 273(1 Pt 1), pp.E122-129. doi:https://doi.org/10.1152/ajpendo.1997.273.1.E122. Brown, L., Hindmarsh, R. and Mcgregor, R. (2015). Dynamic agriculture : years 11-12. 3rd ed. South Melbourne, Vic. Cengage Learning Australia, p.86. Bullock, G.S., Murray, E., Vaughan, J. and Kluzek, S. (2021). Temporal trends in incidence of time-loss injuries in four male professional North American sports over 13 seasons. Scientific Reports, 11(1). doi:https://doi.org/10.1038/s41598-021-87920-6. Cao, R.Y., Li, J., Dai, Q., Li, Q. and Yang, J. (2018). Muscle Atrophy: Present and Future. Advances in Experimental Medicine and Biology, pp.605–624. doi:https://doi.org/10.1007/978-981-13-1435-3_29. Dave, H.D., Varacallo, M. and Shook, M. (2018). Anatomy, Skeletal Muscle. [online] Nih.gov. Available at: https://www.ncbi.nlm.nih.gov/books/NBK537236/. Ding, S., Dai, Q., Huang, H., Xu, Y. and Zhong, C. (2018). An Overview of Muscle Atrophy. Advances in Experimental Medicine and Biology, [online] pp.3–19. doi:https://doi.org/10.1007/978-981-13-1435-3_1. Everman, S., Meyer, C., Tran, L., Hoffman, N., Carroll, C.C., Dedmon, W.L. and Katsanos, C.S. (2016). Insulin does not stimulate muscle protein synthesis during

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increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans. American Journal of Physiology-Endocrinology and Metabolism, 311(4), pp.E671–E677. doi:https://doi.org/10.1152/ajpendo.00120.2016. Glynn, E.L., Fry, C.S., Drummond, M.J., Dreyer, H.C., Dhanani, S., Volpi, E. and Rasmussen, B.B. (2010). Muscle protein breakdown has a minor role in the protein anabolic response to essential amino acid and carbohydrate intake following resistance exercise. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 299(2), pp.R533–R540. doi:https://doi.org/10.1152/ajpregu.00077.2010. Griffiths, N. (2011). Pasture and winter forage crop sowing guide -Hawkesbury-Nepean, Hunter and Manning Valleys. [online] Available at: https://www.dpi.nsw.gov.au/__data/assets/pdf_file/0008/7 6319/Pasture-and-winter-forage-crop-sowing-guide-forhawkesbury-nepean-hunter-and-manning-valleys.pdf. Haun, C.T., Vann, C.G., Roberts, B.M., Vigotsky, A.D., Schoenfeld, B.J. and Roberts, M.D. (2019). A Critical Evaluation of the Biological Construct Skeletal Muscle Hypertrophy: Size Matters but So Does the Measurement. Frontiers in Physiology, [online] 10. doi:https://doi.org/10.3389/fphys.2019.00247. Krzysztofik, M., Wilk, M., Wojdała, G. and Gołaś, A. (2019). Maximizing Muscle Hypertrophy: A Systematic Review of Advanced Resistance Training Techniques and Methods. International journal of environmental research and public health, 16(24), p.E4897. doi:https://doi.org/10.3390/ijerph16244897. Kumar, V., Atherton, P., Smith, K. and Rennie, M.J. (2009). Human muscle protein synthesis and breakdown during and after exercise. Journal of Applied Physiology, [online] 106(6), pp.2026–2039. doi:https://doi.org/10.1152/japplphysiol.91481.2008. Lehmkuhler, J. and VanValin, K. (2021). Feeding soybeans to beef cattle. [online] ASC-245: Feeding Soybeans to Beef Cattle. Available at: http://www2.ca.uky.edu/agcomm/pubs/ASC/ASC245/AS C245.pdf. Mazzulla, M., Volterman, K.A., Packer, J.E., Wooding, D.J., Brooks, J.C., Kato, H. and Moore, D.R. (2018). Whole-body net protein balance plateaus in response to increasing protein intakes during post-exercise recovery in adults and adolescents. Nutrition & Metabolism, 15(1). doi:https://doi.org/10.1186/s12986-018-0301-z. McCuller, C., Jessu, R. and Callahan, A.L. (2022). Physiology, Skeletal Muscle. [online] PubMed. Available at: https://www.ncbi.nlm.nih.gov/books/NBK537139/#:~:text =The%20main%20functions%20of%20skeletal. Medline Plus (2019). Muscle atrophy: MedlinePlus Medical Encyclopedia. [online] Medlineplus.gov. Available at: https://medlineplus.gov/ency/article/003188.htm. MLA (2015). Beef cattle nutrition An introduction to the essentials Grazing Land Management Nutrition Managing the Breeder Herd Selling Marketing. [online] Available at: https://www.mla.com.au/globalassets/mla-corporate/beefcattle-nutrition2.pdf.

Scientific Research in School Volume 5 Issue 1 2023 Morton, R.W., McGlory, C. and Phillips, S.M. (2015). Nutritional interventions to augment resistance traininginduced skeletal muscle hypertrophy. Frontiers in Physiology, 6(245). doi:https://doi.org/10.3389/fphys.2015.00245. Mosoni, L. and Mirand, P.P. (2003). Type and timing of protein feeding to optimize anabolism. Current Opinion in Clinical Nutrition & Metabolic Care, 6(3), pp.301–306. doi:https://doi.org/10.1097/01.mco.0000068961.34812.77. Phillips, S.M. (2004). Protein requirements and supplementation in strength sports. Nutrition, 20(7-8), pp.689–695. doi:https://doi.org/10.1016/j.nut.2004.04.009. Phillips, S.M. (2013). Protein Consumption and Resistance Exercise: Maximizing Anabolic Potential. [online] Gatorade Sports Science Institute. Available at: https://www.gssiweb.org/sports-scienceexchange/article/sse-107-protein-consumption-andresistance-exercise-maximizing-anabolic-potential. Schiaffino, S., Dyar, K.A., Ciciliot, S., Blaauw, B. and Sandri, M. (2013). Mechanisms regulating skeletal muscle growth and atrophy. FEBS Journal, [online] 280(17), pp.4294–4314. doi:https://doi.org/10.1111/febs.12253. Slater, G.J., Dieter, B.P., Marsh, D.J., Helms, E.R., Shaw, G. and Iraki, J. (2019). Is an Energy Surplus Required to Maximize Skeletal Muscle Hypertrophy Associated With Resistance Training. Frontiers in nutrition, [online] 6, p.131. doi:https://doi.org/10.3389/fnut.2019.00131. Tipton, K.D., Hamilton, D.L. and Gallagher, I.J. (2018). Assessing the Role of Muscle Protein Breakdown in Response to Nutrition and Exercise in Humans. Sports Medicine, [online] 48(1), pp.53–64. doi:https://doi.org/10.1007/s40279-017-0845-5. WALKER, D.K., DICKINSON, J.M., TIMMERMAN, K.L., DRUMMOND, M.J., REIDY, P.T., FRY, C.S., GUNDERMANN, D.M. and RASMUSSEN, B.B. (2011). Exercise, Amino Acids, and Aging in the Control of Human Muscle Protein Synthesis. Medicine & Science in Sports & Exercise, [online] 43(12), pp.2249–2258. doi:https://doi.org/10.1249/mss.0b013e318223b037.

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Appendices Table 6: raw data collected for area and number of cells

Grain

Sample 1 2 3 Total Grass Sample 4 5 6 Total

No. of cells in FOV (counted) Slide A B Mean 30 25 27.5 20 25 22.5 42 25 33.5 27.8

Approximate area of single cell ( 𝜇𝜇𝜇𝜇𝑚𝑚𝑚𝑚2 ) Slide A B Mean 0.81 0.97 0.89 1.21 0.97 1.09 0.58 0.97 0.77 0.92

No. of cells in FOV (counted) Slide A B Average 34 49 41.5 29 42 35.5 48 62 55 44

Approximate area of single cell ( 𝜇𝜇𝜇𝜇𝑚𝑚𝑚𝑚2 ) Slide A B Average 0.71 0.49 0.60 0.83 0.58 0.71 0.50 0.39 0.45 0.59

Figure 7: area graph showing difference in cross-sectional area

Figure 8: pie chart ratio representation of total cells counted from both groups

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Scientific Research in School Volume 5 Issue 1 2023 Table 7: cell nuclei counted in the photographs

Grain Grass

1A 60 4A 78

1B 53 4B 112

2A 62 5A 68

Nuclei counted in area 2B 3A 78 70 5B 6A 79 73

3B 57 6B 79

Average 63.3 Average 81.5

Figure 9: the steaks used for samples

Figure 10: shows Slide 5 Transverse (x400), blue dots are nuclei

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Chemistry The constant threat of new and evolving drug resistant pathogens continues to inspire the Chemistry projects we choose. Five projects in the field of medicinal chemistry have explored the development of new drug analogues as contributors to the Breaking Good project. One completely new project explored a fascinating and complex oscillating chemical reaction.

Continuing our commitment to the open-source drug discovery initiative, Josh and Max focused on synthesising new analogues of the previously potent antimalarial drug, pyrimethamine. Josh explored the synthesis optimisation and purification of an iodine analogue that was the subject of Maxine (2021) and Kai’s (2020) projects. Max focused on a whole new analogue, introducing the trifluoromethyl group into the molecule with the hope of exploring how this effects the activity of the original antimalarial drug. As part of a whole new drug discovery effort, Christian and Jack synthesized related analogues of a lead compound in the search for new treatments for the skin infection, mycetoma. Both compounds were successfully synthesized and are awaiting testing against the disease-causing pathogen. Jonah also paved new territory with his investigation of the synthesis of new fragments en route to new analogues of Lopinavir, a promising drug for the treatment of COVID-19. Lastly, Isaac was fascinated by oscillating chemical reactions and dedicated his time to understanding the Belousov-Zhabotinsky reaction. The predicted oscillations between blue and red coloured compounds proved elusive initially but were a sight to behold once the ideal conditions were found.

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Synthesis, isolation and purification of 4iodopyrimethamine as an anti-parasitic Agent for P. Falciparum and Toxoplasma gondii Josh Botha Barker College Purpose: This paper illustrates the attempted increase in yield, quantity and purity of 4iodopyramethamine by investigating the isolation of the product, ultimately aiming to provide sufficient material to test against plasmodium falciparum and toxoplasma gondii. Design/methodology/approach: Synthesising 4-iodopyramethamine was conducted through the pyrimethamine synthetic pathway proposed by Sydney Grammar School in 2016. Findings: It was found that the isolation process could be improved to afford an increased quantity of 4-iodopyramethamine product, however, in an impure crude reaction product with no yield was achieved. Thus, biological testing against plasmodium falciparum and toxoplasma gondii was not conducted. Research limitations/implications: This project aspires to produce sufficient product to test the efficacy of the pyrimethamine analogue against plasmodium falciparum and toxoplasma gondii to determine if the product is a more efficient enzyme inhibitor, thus increasing efficacy in clinical applications. The project also briefly investigates the future possibilities of using the iodine atoms on the R-4 position for perfuming cross coupling reactions to synthesis alternative compounds. The research was limited by time constraints. Practical implications: This project aims to remedy the declined efficacy of Daraprim (pyrimethamine), which consequently resulted from mutations in the dihydrofolate reductase (DHFR) in parasites, leading to the in emergence of drug resistant parasites such as P. falciparum and T. gondii. Social implications: Decreased efficacy of pyrimethamine has the resulted in the global malaria morality rate decrease to slow and even rise, resulting in more malaria related deaths. In 2018, there were 405 000 global deaths (WHO, 2022) as a consequence of malaria. The African Region is carrying the highest malaria burden with with 95% of cases and 96% of deaths occurring within the region. Originality/value: This report is valuable as it contributes to the open-source malaria project established on GitHub, however, specifically extends on the research conducted by Barker College students in 2020 and 2021. Their research explored the synthesis of the 4-iodopyrimethamine analogue (Wong, 2020) and further optimising the synthesis (Wu, 2021). However, neither trail was able to product a sufficient quantity of product to be used for further research. This report outlines the process taken to achieve a sufficient quantity of the 4-iodopyrimethamine analogue. Keywords: Synthesis, isolation, purification, pyrimethamine, 4-iodopyrimethamine, malaria, toxoplasmosis Paper Type: Research Report

Literature Review Malaria Malaria is an acute febrile protozoan vector-borne disease caused by Plasmodium parasites and is transmitted through an infected female Anopheles mosquito; however, it may also be transmitted through blood transfusion and contaminated needles. There are five Plasmodium parasites causing malaria with the most virulent being Plasmodium falciparum, the most severe and widespread. If left untreated, P. falciparum can progress to critical health

complications, including fatality (World Health Organization, 2023). P. falciparum causes disease once an infected female Anopheles mosquito’s ‘bite’ punctures the skin, releasing the parasites into the newly infected organism’s bloodstream where it will travel to the liver. Within the infected liver cell, P. falciparum will undergo the exoerythrocytic cycle where it will mature to reach the schizont stage (producing an average of 20 new daughter cells) causing the cell to rupture and thus releasing the parasites. P. falciparum

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Figure 1: Global trends in malaria mortality rate. Source: (WHO, 2022

then invades the hosts red blood cells and continues to develop initially through the immature trophozoite stage (ring stage). The intraerythrocytic P. falciparum parasite consumes up to 70% of the host cell hemoglobin in an acidic digestive vacuole, generating a crystalline by-product called hemozoin. During this stage, adhesion of P. falciparum-infected RBCs to blood vessel walls helps avoid splenic clearance and contributes to the clinical symptoms of the disease, including fatal complications. (Tilley et al., 2011) P. falciparum will then reach maturity within the trophozoite stage leading to the schizont stage, leading to a ruptured schizont, killing the red blood cell. (Centers for Disease Control and Prevention, 2020) Essentially, P. falciparum attacks the body’s red blood cells, impacting its ability to supply blood to its organs, leading to extremely threatening health complications such as anemia, respiratory distress, organ failure (kidney and liver), coma, seizures, brain damage and even fatality.

According to the World Health Organization, in 2021, there were an estimate of 247 million cases and 619 000 deaths occurring worldwide. Comparatively, in 2018, there were 405 000 global malaria deaths, which is an approximately 50% increase. Additionally, the African region carried a high share of the global malaria prevalence with 95% of cases and 96% of deaths occurring within the region, and within the region, children under 5 years of age accounted for 80% of the deaths (World Health Organization, 2023). Since 2000, the incidence and mortality rate for malaria has decreased due to developments of antimalarial medication and prevention methods. Despite the progress in malaria treatment, the rate of reduction has dramatically slowed since 2015, even increasing again in 2020. The decrease in rate of reduction (Figure 2 & Figure 3) is consequential of

Figure 2: WHO African Region malaria incidence and mortality rate. Source: (WHO, 2022)

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Scientific Research in School Volume 5 Issue 1 2023 drug resistant P. falciparum. Due to rapid mutations in the parasites, efficacy of all anti-malarial drugs has drastically decreased (Nattee et al., 2017). Toxoplasmosis Toxoplasmosis is a disease caused by the protozoan Toxoplasma gondii, a unicellular eukaryotic parasite. T. gondii is in the Apicomplexa family and contains unique organelles at one end of the cell, the apical end, that are used for host cell invasion. The plasmodium species are in the Apicomplexa family and are similar to T. gondii as both parasites have an apicoplast, a unique algae-derived organelle that is dedicated to specific metabolic functions. T. gondii is transmitted by eating contaminated food, such as undercooked meat, or most commonly through contact with an infected cats' faeces. Whilst T. gondii isn’t as virulent as the plasmodium species, it still induces health complications for millions of people worldwide. (CDC, 2019) T. gondii can produce both sexually and asexually. Sexual reproduction only occurs within cats, which are the parasites definitive hosts. Within the cat’s gut epithelium, the parasites differentiate into the male and female gametocytes, allowing for sexual reproduction. The infected cat will shed millions of oocytes, containing four haploid sporozoites, in the faeces (S. Al-Malki, 2021). Thus, organisms can contract infection through accidental ingestion of oocyst. In addition to definitive hosts, T. gondii can asexually reproduce after infecting a range of warm blooded ‘intermediate hosts’, where the parasite replicates its haploid genomes through endodyogeny, dividing it into two daughter cells. (Kochanowsky & Koshy, 2012) Once infected, T. gondii invades the host cell’s plasma membrane but does not require a specific host receptor or protein enabling the parasite capable of invading any nucleated host cell. During acute infection, T. gondii disseminates throughout the host as a tachyzoite, the fast-replicating form of the parasite, which is targeted by the host immune response. T. gondii then converts to slowly replicating bradyzoite, which encysts, and lead to the chronic stage of infection withing the organism. This transition is thought to enable T. gondii to evade immune responses, leading to persistent infection. (S. Al-Malki, 2021) (Mendez & Koshy, 2017)

otherwise may present a mild flu-like illness (CDC, 2019). However, T. gondii can asymptomatically persist in the CNS of immunocompetent individual. This tropism for the CNS leads to health complications for those with deficient immune responses, such as developing fetuses or AIDS patients, causing damage to the brain, eyes, or other organs. (Mendez & Koshy, 2017) Pyrimethamine Pyrimethamine is a specific derivative of 2,4 – diaminopyrimidine and is commercially sold under the trade name ‘Daraprim.’ Since its introduction in 1952, the drug was predominately widespread as an anti-malarial, used to treat malaria (Stanford, 2015). However, since then pyrimethamine has been further used as an anti-parasitic and anti-protozoal agent, treating other diseases such as toxoplasmosis, caused by T. gondii parasites and imposes major health concerns for those with weakened immune systems (CDC, 2019). Additionally, the price of Daraprim has increased from $13.50 to $750 a pill (Stanford, 2015).

Figure 2: Pyrimethamine (1)

Pyrimethamine has been an effective prevention and treatment drug for these diseases due to its ability to be a competitive enzyme inhibitor of dihydrofolate reductase (DHFR; EC 1.5.1.3). Specifically in P. falciparum, DHFR is one domain of a bifunctional protein that also contains thymidylate synthase. “DHFR is a key enzyme in the redox cycle for production of tetrahydrofolate, a cofactor that is required for the transfer of C1 units used in the biosynthesis of DNA and protein (Sibley et al., 2001).” Essentially, in P. falciparum, pyrimethamine would inhibit the DHFR of the parasite, preventing the parasites' ability for biosynthesis of DNA and protein, eliminating its ability to reproduce (Bloland, 2001).

Despite this, in humans, T. gondii doesn’t pose much threat to a healthy human as the immune response is likely to prevent the parasite from causing illness or

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Table 1: Principal polymorphisms in codons of P. falciparum DHFR and DHPS with the resulting amino acid changes Source: (Sibley et al., 2001)

Figure 3: A) Structure of pyrimethamine B) 3D modelling of enzyme-inhibitor complex at the active site between pyrimethamine and wild-type DHFR ligand Source: (Tropak et al., 2015)

More recently, researchers have observed a declined efficacy of clinical treatments. “Compared with the wild-type sequence, a point mutation changing Ser108 to Asn108 (S108N) in the dhfr gene increases resistance to pyrimethamine about 100-fold (Sibley et al., 2001).” Thus, due to the declined efficacy of pyrimethamine, the rate of reduction in malaria deaths has dramatically slowed since 2015, even increasing again in 2020 (Figure 1). Therefore, outlining a need for the development of new affordable drugs to remedy the decreasing efficacy of pyrimethamine. Analogues Mutations in the parasites that are causing drug resistance are likely to be a continuous burden on the precedence of malaria and toxoplasmosis. Thus, an approach to overcoming the mutating parasites is the development of new analogues. Analogues are small structural changes in a compound to improve its efficacy. Previous 4-iodopyrimethamine research This report is an addition to the open-source malaria project, public on GitHub, specifically extending on

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the research conducted by Barker College students in 2020 (Wong, 2020) and 2021 (Wu, 2021). This research aims to increase the yield, quantity, and purity of the novel 4-iodopyrimethamine analogue that was initially synthesised by a Barker College student in 2020 (Wong, 2020), using the methodology improved by a Barker College student in 2021 (Wu, 2021). The iodine analogue is of interest due to the larger atomic radius of the atom and the lower electronegativity of compared to the chlorine atom. Replacing the chlorine atom with the larger iodine atom at the R-4 position has the potential to improve biological activity through enhanced binding interactions in the enzyme active site. Additionally, it is an appropriate ‘leaving group’ (Schnürch et al., 2006) to be used as a “synthetic handle” for metal catalysed cross-coupling reactions such as the Suzuki or Nigishi coupling (Biajoli, 2004), thus enabling the compound to be further used in the synthesis of more analogues. The iodo-analogue was successfully produced (Wong, 2020) using the synthetic pathway developed by Sydney Grammar School (SGS, 2016) for the synthesis of pyrimethamine (Figure 6). The students at Sydney Grammar School collaborated with the University of Sydney through the ‘Breaking Good’ project to successfully synthesis pyrimethamine in their school laboratory.

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Figure 4: Synthetic pathway for 4-iodopyrimethamine analogue (2)

Having access to the same recourses, the Barker College student in 2020 was able to successfully synthesis the iodine analogue (2) from 4iodophenylacetonitrile (3) in our school laboratory, producing <10% and insufficient material to conduct any further testing. As a result, another Barker College student aimed to improve the methodology for the synthesis of the iodo analogue, achieving an increased yield for step 1 and for step 2 but not affording any yield in step 3 of the synthesis. It is found that the isolation of the pyrimethamine analogue (2) in previous attempts has proved to be challenging with the attempt in 2020 producing <10% yield (Wong, 2020) and the 2021 attempt not producing any yield (Wu, 2021). The previous attempts at isolation of the pyrimethamine analogue (2) were unsuccessful due to the large quantity of DMSO, which is highly polar solvent that is used to pull out the impurities in solution. In solution with water and the compound (2), the DMSO would begin to dissolve in the water but also in the desired compound (2) which therefore led to difficulties regarding the crystallisation of the pyrimethamine analogue (2) as the organic solvent was not able polar enough to separate the desired compound (2) out of the crude reaction product. This report aims to

remedy the inability to isolate the product by increasing the relative proportion of water to DSMO.

Scientific Research Question Can the yield, quantity, and purity of the 4iodopyrimethamine analogue be increase and its effectiveness as an anti-parasitic agent against plasmodium falciparum and toxoplasma gondii be tested?

Scientific Hypothesis It is hypothesized that the yield, quantity and purify of 4-iodopyrimethamine analogue can be increased and thus be tested as an effective anti-parasitic agent against plasmodium falciparum and toxoplasma gondii.

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Scientific Research in School Volume 5 Issue 1 2023 quartet, m = multiplet) and assignment. Atom labels on structures are to illustrate 1H NMR spectral assignments and do not necessarily correspond to the IUPAC names given. The solvent peak for acetone (δ 29.84) was used as an internal reference for 13C NMR spectra. Mass spectra were recorded by the Mass Spectrometry Unit of the School of Chemistry, The University of Sydney, Sydney. Analytical thin layer chromatography was performed with Merck Kieselgel 60 F254 (0.2 mm) pre-coated aluminium sheets, and visualisation was achieved by inspection under UV light. Throughout the reaction process Thin Layer Chromatography (TLC) was conducted to gauge the progress of the reaction and determine the point of completion. TLC analysis was conducted with pure dichloromethane or 20:80 ethyl acetate/hexanes. Step 1: Synthesis of 2-(4-iodophenyl)-3oxopentanenitrile

Figure 5: 2-(4-iodophenyl)-3-oxopentanenitrile (4)

4-iodophenylacetonitrile (15.045g, 0.0619mol, 1 equiv.), ethyl propionate (7.5 g, 0.0735 mol, 1.05 equiv.) and potassium tert-butoxide (15.705g, 0.13995 mol, 2 equiv.) were combined in THF (150 mL) at room temperature, with stirring in a round bottom flask. The reaction mixture turned to a dark red and heated up rapidly. The reaction was sealed and stirred for 2 hours. The reaction mixture was worked up by the addition of 1.0 M HCl (150 mL) to the reaction vessel. The acidified reaction mixture was transferred to a separating funnel and the aqueous layer was extracted with DCM (3 x 97.5 mL). The combined organic layer was washed with brine (150mL), dried with anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 2-(4-iodophenyl)-3oxopentanenitrile (21.412g, 0.0716 mol, ≈ > 90%) as a reddish oil. TLC was conducted with 100% DCM as the eluent. The crude 2-(4-iodophenyl)-3oxopentanenitrile (4) was used without purification in the second step of the synthesis.

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Step 2: Synthesis of 2-(4-iodophenyl)-3-(2methylpropoxy)-pent-2-enenitrile

Figure 6: 2-(4-iodophenyl)-3-(2-methylpropoxy)-pent-2enenitrile (5)

2-(4-iodophenyl)-3-oxopentanenitrile (21.412g, 0.0716 mol, 1 equiv.) was dissolved in a mixture of toluene (97.5mL) and 2-methylpropan-1-ol (9.75mL, 0.1315 mol). 18M H2SO4 (1.5 mL) was added, and the mixture was refluxed for 10 hours in a Dean Stark apparatus. The reaction mixture was poured onto a saturated sodium hydrogen carbonate in a separating funnel and the aqueous phase was extracted with DCM (2 x 75mL). The combined organic extracts were dried over anhydrous sodium sulfate. Addition of triethylamine (7.5mL, 0.0741 mol) to the reaction mixture converted the unreacted starting material to its very polar triethylammonium enolate salt. Chromatography silica (75g) was added to the organic phase, which was made up to 300mL with dichloromethane and stirred for 1.5 hours. The organic phase was filtered using vacuum filtration and rinsed with 1M HCL (2 x 75mL) and deionised water (75 mL) in a separating funnel to remove all traces of triethylamine. The combined organic extracts were dried over anhydrous sodium sulfate and filtered. The solvent was removed in vacuo to yield 2-(4-iodophenyl)-3-(2-methylpropoxy)-pent-2enenitrile (5) (16.828g, 0.047 mol, 66%) as a red oil. This product was used in the next step of synthesis. Step 3: Synthesis of 4-iodopyrimethamine analogue

Figure 7: 4-iodopyrimethamine analogue (2)

2-(iodophenyl)-3-(2-methylpropoxy)-pent-2enenitrile (16.828g, 0.047 mol, 1 equiv.) was

Scientific Research in School Volume 5 Issue 1 2023 dissolved in DMSO (135.0 mL). Guanidine hydrochloride (9.6g, 0.1004 mol, 2 equiv.) was stirred into the solution followed by sodium methoxide powder (6.00 g, 0.111 mol, 2.2 equiv.). The solution became dark red in colour on addition of the sodium methoxide, which dissolved into the solution within an hour. No precipitation of sodium chloride was observed. The solution was allowed to stand at room temperature for 48 hours. No crystals appeared in the reaction mixture. To optimise the isolation of the product, a portion (a third of the volume) of the reaction mixture was poured onto water (approx.150 mL) and DCM (50 mL) was added. An emulsion formed in the separating funnel, so brine (50 mL) added to disperse the emulsion. The resulting aqueous layer was extracted with another portion of DCM (50 mL) to afford the crude reaction product.

Figure 10: TLC after synthesis of 2-(4-iodophenyl)-3-(2methylpropoxy)-pent-2-enenitrile

Step 3: Synthesis of 4-iodopyrimethamine analogue

Figure 11: TLC after synthesis of 4-iodopyrimethamine analogue

Figure 8: Synthetic pathway for 4-iodopyrimethamine

Results Step 1: Synthesis of 2-(4-iodophenyl)-3oxopentanenitrile

Figure 12: 1H NMR Spectra after synthesis of 4iodopyrimethamine analogue

Figure 9: TLC after synthesis of 2-(4-iodophenyl)-3oxopentanenitrile

Step 2: Synthesis of 2-(4-iodophenyl)-3-(2methylpropoxy)-pent-2-enenitrile

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Figure 13: Mass Spectra after synthesis of 4-iodopyrimethamine analogue

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Discussion Step 1: Synthesis of 2-(4-iodophenyl)-3oxopentanenitrile

previous 2021 report (Figure 18) and 2020 report (Figure 17), the TLC displayed no starting material left in the product, whereas both the previous trails had starting material remaining in the end product. (Wu, 2021; Wong, 2020)

Figure 14: Reaction of 4-iodophenylacetonitrile (3) to form 2-(4-iodophenyl)-3-oxopentanenitrile (4)

A condensation reaction between 4-iodoacetonitrile (3) and ethyl propionate to afford compound 4. More specifically, addition of the strong base potassium tert-butoxide caused deprotonation at the CH2 group of Compound (3), and reaction with ethyl propionate led to the elimination of ethanol (Figure 16).

Figure 16: 2020 report’s TLC after step 1 Source: (Wong, 2020)

Figure 15: Mechanism of the reaction between 4iodophenylacetonitirile and ethyl propionate

This reaction was performed with a scale up of reaction material, by starting with 15.045g of 4iodophenylacetonitrile, a 1.5x increase from the 11.33g previously used in the 2021 report (Wu, 2021). The yield obtained for this step cannot be accurately determined as not all of the solvent was removed from the product in solution, however, the excess solvent is not of concern as the product is transferred into the next step of synthesis immediately. However, the estimated yield for this reaction would be >90%, which obtained similar results to the previous 2021 report which achieved a 91% yield (Wu, 2021) and further still a significant increase to the yield obtained in 2020 (Wong, 2020). The scaledup reaction further resulted in minimal loss of the target compound (4) during the extraction and separation from the aqueous impurities. Furthermore, filtration of compound (4) from sodium sulphate was conducted using a sintered funnel as proven to be optimal by the previous report (Wu, 2021), however as above, there was still solvent remaining in the target compound (4) after filtration. Additionally, the appearance of polar baseline material on the TLC (Figure 10) corresponded to the formation of the more polar compound (4). In comparison to the

Figure 17: 2021 report’s TLC after step 1 Source: (Wu, 2021)

H NMR and mass spectra was not conducted for the reaction of 4-iodophenylacetonitrile (3) to form 2-(4iodophenyl)-3-oxopentanenitrile (4), however, the previous trails for the reaction had conducted 1H NMR (Figure 27) and mass spectra (Figure 28) on the product, and thus had sufficient evidence to conclude the confirmed formation of 2-(4-iodophenyl)-3oxopentanenitrile. Therefore, it was not necessary to conduct 1H NMR and mass spectra as the compound formed is likely to be the target compound (4). 1

Step 2: Synthesis of 2-(4-iodophenyl)-3-(2methylpropoxy)-pent-2-enenitrile

Figure 18: Reaction of 2-(4-iodophenyl)-3oxopentanenitrile (4) to form 2-(4-iodophenyl)-3-(2methylpropoxy)-pent-2-enenitrile (5)

Step 2 of the synthesis was performed under reflux whereby a substitution reaction led to the formation of Compound (5) from Compound (4). A Dean Stark

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Scientific Research in School Volume 5 Issue 1 2023 apparatus was used to remove water and drive the equilibrium reaction in the forward direction. The addition of triethylamine to the reaction mixture converted the unreacted starting material to its very polar triethylammonium enolate salt, removing the remaining starting material. Addition of silica gel at this stage facilitated the removal of this salt from the reaction mixture. Re-protonation of the triethylammonium enolate salt stuck to the silica gel would have occurred after addition of HCl, reforming the enol tautomer of Compound 4 (Figure 20).

Figure 19: Mechanism of the reaction between 4iodophenylacetonitirile and ethyl propionate

Following the successful optimisation of the reaction achieved in the 2021 trail (Wu, 2021), the sintered funnel was incorporated as an alternative to separate the silica gel from the crude product mixture. The TLC (Figure 11) revealed that the level purity achieved in the previous 2021 attempt (Figure 22) was not obtained in this reaction as the starting material reaction spot was more apparent than the 2021 attempt. However, the starting material spot was less apparent than the 2020 attempt (Figure 21), thus the optimization of the reaction outlined in the 2021 report (Wu, 2021), still resulted in an increase in purity.

Figure 20: 2020 report’s TLC after work up in step 2 Source: (Wong, 2020)

Comparatively, this achieved a yield of 66% which is a 9% decrease from the 2021 trail (Wu, 2021), which achieved a 75% yield. Despite this, the reaction still obtained a 9% increase in comparison to the 2020 trail which achieved a yield of 57%. H NMR and mass spectra was not conducted for the reaction of 2-(4-iodophenyl)-3-oxopentanenitrile (4) to form 2-(4-iodophenyl)-3-(2-methylpropoxy)-pent2-enenitrile (5), however, the previous trails for the reaction had conducted 1C NMR (Figure 29) and mass spectra (Figure 30) on the product, and thus had sufficient evidence to conclude the confirmed formation of 2-(4-iodophenyl)-3-(2-methylpropoxy)pent-2-enenitrile. Therefore, it was not necessary to conduct 1C NMR, 1H NMR and mass spectra as the compound formed is likely to be the target compound (5), in this case the target compound still contained starting material as it was not pure. 1

Step 3: Synthesis of 4-iodopyrimethamine analogue

Figure 22: Reaction of 4-iodophenylacetonitrile (5) to form 2-(4-iodophenyl)-3-oxopentanenitrile (2)

Step 3 of synthesis was initiated by the deprotonation of the guanidine ion by sodium methoxide. Following this, guanidine reacted at the nitrile carbon of Compound (5), and subsequent electron rearrangement and the elimination of 2methylpropanol afforded the pyrimidine ring seen in Compound (2). No crystals appeared in the reaction mixture. To improve the isolation of the desired compound, the proportion of water to DMSO was increased, as a portion (a third of the volume) of the reaction mixture was poured onto an increased volume of water (approx.150 mL) and DCM (50 mL) was added. An emulsion formed in the separating funnel, so brine (50 mL) added to disperse the emulsion. The resulting aqueous layer was extracted with another portion of DCM (50 mL) to afford the crude reaction product. Essentially, when the DMSO concentration was high in solution relative to the water, the organic solvent couldn’t extract the product as there the high

Figure 21: 2021 report’s TLC after work up in step 2 Source: (Wu, 2021)

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Figure 23: Previous report’s 1H NMR spectra after synthesis of 4-iodopyrimethamine analogue Source: (Wong, 2020)

concentration of DMSO resulted in the DMSO holding onto the water and the desired product. Thus, by increasing the proportion of water to DMSO, the DMSO would be of lower relative concentration to the water, allowing the organic solvent to extract the compound (2). The appearance of polar baseline material in the TLC, the product candidate, indicated the formation of the pyrimidine rings which contains polar NH2 groups. The TLC displayed starting material still present in the final product, likely due to the isolation difficulties resulting from the polarity of the compound (2). H NMR (400MHz, (CDCl3) δ 7.78 (2H, d , H1), 6.99 (2H, d, H2), 4.79 (2H, br s, NH2), 4.47 (2H, br s, NH2), 2.27 (2H, q ,CH2), 1.05 (3H, t, CH3); MS (+ESI) [M + H+] m/z = 341.03. (Wong, 2020) 1

Figure 24: 1H NMR Spectra after synthesis of 4iodopyrimethamine analogue (5.0 – 8.0 ppm)

Figure 25: 1H NMR Spectra after synthesis of 4iodopyrimethamine analogue (0.0 – 4.0 ppm)

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Scientific Research in School Volume 5 Issue 1 2023 Due to the inability to isolate a pure product, the crude product produced a complex 1H NMR spectrum that was difficult to analyse. However, in the 2020 product 1H NMR spectrum (Figure 24), doublet signals at 7.79 and 6.99 ppm were assigned to aromatic H1 and H2 hydrogen environments. This is consistent in the crude product 1H NMR spectrum (Figure 25), confirming the aromatic H1 and H2 hydrogen environments. Additionally, the 2020 1H NMR spectrum (Figure 24), displayed two characteristic broad singlets at 4.79 and 4.47 ppm were indicative of the NH2 groups. The broad singlet at 4.79 ppm is consistent with the crude product 1H NMR spectrum (Figure 26), however, the singlet at 4.47 ppm is extremely insignificant in the crude product 1H NMR spectrum, but still present and thus confirming the NH2 groups in the crude reaction product. The splitting pattern of the quartet at 2.27 ppm was characteristic of the CH2 from the ethyl group, and the splitting pattern of the triplet at 1.05 ppm was indicative of the CH3 on the ethyl group which are both consistent with the 2020 trail (Figure 24) product and the crude reaction product (Figure 25, Figure 26). 4-iodopyrimethamine has a molar mass of 340g/mol, mass spectroscopy of the crude reaction product signals a base peak at 341.02 m/z (Figure 14), and the 2020 product signals at 341.03 m/z (Figure 31). Therefore, confirming the presence and successful synthesis of the 4-iodopyrimethamine analogue within the crude reaction product. No yield was achieved but the isolation afforded a large quantity of crude product. Comparatively, the isolation attempt in 2021 did not produce any yield (Wu, 2021). However, the previous attempt in 2020 produced <10% yield (Wong, 2020), but the yield and quantity were insufficient to use for further research. The inability to obtain a yield is a result of the continued difficulties regarding the isolation of the 4-iodopyrimethamine analogue. As the desired compound (2) is dissolved in DMSO, the solvent used (DCM) was not able to extract the product and separate it due to the DMSO holding onto the desired compound. Despite increasing the relative proportion of water to DMSO in attempt to remedy the isolation difficulties, time restraints limited the possibility of conducting further purification to the crude reaction product. Thus, purity was insufficient and therefore the product could not be used to conduct biological testing against P. falciparum and T. gondii. The quantity of crude reaction product should be used for future considerations such as column

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chromatography to purify the compound to achieve a yield of the 4-iodopyrimethamine analogue. Future Research Extending on the research obtained in this report, the large quantity of crude reaction product can be used for future research regarding the purification of the final crude reaction compound using column chromatography. Column chromatography can be performed used to separate/purify liquid and solid sample compounds based on polarity or hydrophobicity. It involves passing a mixture through a column packed with a stationary phase material, typically a solid adsorbent. The different components in the mixture interact with the stationary phase differently, polar molecules ‘stick’ allowing nonpolar compounds to pass more easily (Srivastava et al., 2021). To purify the 4-iodopyrimethamine analogue, a more polar solvent than DCM would need to be used to sperate the crude reaction product and obtain a pure yield that then is able to be used for biological testing to conclude whether the 4iodopyrimethamine analogue is of increased efficacy to the original pyrimethamine. Additionally, future possibilities of the novel 4iodopyrimethamine could extend past its potential as a new and more effective inhibitor of DHFR. The 4iodopyrimethamine analogue applications could include the preparation of new analogues via substitution at the R4 position. (Richardson and Stevens, 2002). A Suzuki cross coupling involves the ‘substitution’ of a boronic acid or a boronic ester with an organic halide or triflate in the presence of a palladium catalyst to form a new carbon-carbon bond (Suzuki, 2011). Iodine is an acceptable ‘leaving group’ in which enables the boronic acid or boronic ester to react, thus indicating the use of the novel 4iodopyrimethamine in cross coupling reactions. Additionally, chlorine is also found to eb an acceptable and versatile ‘leaving group’ which could lead to further research regarding the original molecule, pyrimethamine (Schnürch et al., 2006). Thus, if the 4-iodopyrimethamine analogue proves to provide no improvement to the efficacy against P. falciparum and T. gondii, its simplicity regarding cross coupling reactions are applications regarding new synthetic pathways for alternative pyrimethamine analogues. Ultimately, this could lead to the development of more competitive DHFR enzyme inhibitors.

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Conclusion The research conducted resulted in the successful increase in quantity of the 4-iodopyrimethamine (2) analogue after increasing the starting material to 15.045g and increasing the relative proportion of water to DMSO in the isolation stage of step 3. However, no yield was obtained, and the final product was impure as the 4-iodopyrimethamine analogue was in a crude reaction product. Structural conformation was conducted using mass spectrometry (Figure 14) and 1H NMR (Figure 13) after the synthesis of the 4-iodopyrimethamine analogue (2), however, it was complex due to the impurities resulting from isolation difficulties. The results were compared to the pure 4iodopyrimethamine (2) mass spectrometry (Figure 27) and 1H NMR (Figure 24) spectra results obtained in 2020 (Wong, 2020), which showed results consistent with the structure and mass of the desired compound (2). Thus, it was concluded that the 4iodopyrimethamine analogue was successfully synthesised and present within the crude reaction product. Due to time restrains, column chromatography was not perfumed to further purify the crude reaction product. Therefore, due to impurity and lack of yield, biological testing against P. falciparum and T. gondii was not able to be conducted. Overall, quantity of the desired compound (2) was increased, allowing future research to be conducted with the crude reaction product material, performing column chromatography to further purify the crude reaction product, or investigate cross-coupling reactions to synthesis alternative analogues to increase drug efficacy with enzyme inhibition.

Acknowledgments I would like to extend my gratitude to the Barker College Science Extension staff, specifically Dr Katie Terrett for her invaluable contribution through her extensive knowledge, expertise, supervision of the synthesis and ultimately the completion of this report. I would also like to acknowledge and extend my appreciation to the collaborators of Breaking Good at Sydney University who provided the 1H NMR experimental data.

References Bloland, P. (2001). WHO/CDS/CSR/DRS/2001.4 Drug resistance in malaria. [online] Available at: https://www.who.int/csr/resources/publications/drugresist/ malaria.pdf.

CDC (2019). CDC - Toxoplasmosis - General Information - Frequently Asked Questions (FAQs). [online] CDC. Available at: https://www.cdc.gov/parasites/toxoplasmosis/gen_info/faq s.html. Centers for Disease Control and Prevention (2020). CDC Malaria - About Malaria - Biology. [online] CDC. Available at: https://www.cdc.gov/malaria/about/biology/index.html. Kochanowsky, J. and Koshy, A. (2012). Current Biology. Current Biology, 22(3), pp.R79–R80. doi:https://doi.org/10.1016/j.cub.2011.12.035. Mendez, O.A. and Koshy, A.A. (2017). Toxoplasma gondii: Entry, association, and physiological influence on the central nervous system. PLOS Pathogens, [online] 13(7), p.e1006351. doi:https://doi.org/10.1371/journal.ppat.1006351. Nations, U. (2023). Climate Change and Malaria - A Complex Relationship. [online] United Nations. Available at: https://www.un.org/en/chronicle/article/climatechange-and-malaria-complexrelationship#:~:text=Climate%20change%20greatly%20in fluences%20the. Nattee, C., Khamsemanan, N., Lawtrakul, L., Toochinda, P. and Hannongbua, S. (2017). A novel prediction approach for antimalarial activities of Trimethoprim, Pyrimethamine, and Cycloguanil analogues using extremely randomized trees. Journal of Molecular Graphics and Modelling, 71, pp.13–27. doi:https://doi.org/10.1016/j.jmgm.2016.09.010. reference.medscape.com. (2023). Daraprim (pyrimethamine) dosing, indications, interactions, adverse effects, and more. [online] Available at: https://reference.medscape.com/drug/daraprimpyrimethamine342668#:~:text=USES%3A%20This%20medication%20i s%20used. Richardson, M.L. and Stevens, M.F.G. (2002). Structural Studies on Bioactive Compounds. Part 37.1 Suzuki Coupling of Diaminopyrimidines: A New Synthesis of the Antimalarial Drug Pyrimethamine. Journal of Chemical Research, 2002(10), pp.482–484. doi:https://doi.org/10.3184/030823402103170664. S. Al-Malki, E. (2021). Toxoplasmosis: stages of the protozoan life cycle and risk assessment in humans and animals for an enhanced awareness and an improved socio-economic status. Saudi Journal of Biological Sciences, 28(1), pp.962–969. doi:https://doi.org/10.1016/j.sjbs.2020.11.007. Schnürch, M., Radoslav Flasik, Ather Farooq Khan, Spina, M., Mihovilovic, M.D. and Stanetty, P. (2006). Cross‐Coupling Reactions on Azoles with Two and More Heteroatoms. European Journal of Organic Chemistry, 2006(15), pp.3283–3307. doi:https://doi.org/10.1002/ejoc.200600089. School, S.L. (2015). Daraprim and Predatory Pricing: Martin Shkreli’s 5000% Hike. [online] Stanford Law School. Available at: https://law.stanford.edu/2015/10/05/daraprim-and-drugpricing/#:~:text=Daraprim%20was%20developed%20in% 20the [Accessed 18 Jun. 2023].

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Synthesis of a trifluoromethyl analogue of Pyrimethamine Max Graham Barker College Purpose: This research paper aims to illustrate the procedure taken to create a new analogue of pyrimethamine in a school laboratory. Design/ methodology/approach: The procedure to create standard pyrimethamine is followed, yet using a different starting material, as well as other smaller alterations like changes in solvents. Findings: It cannot be confirmed whether the final product was the 3-trifluoromethyl analogue of pyrimethamine. Biological analysis suggests that the sample may have contained the desired compound, however it was highly impure with a significantly low yield. Research limitations/implications: The main limitation of this experiment was that this compound has never been made before, which is why only small quantities were used: the aim was to see if it was possible for this compound to be created. Yet, this is also why this research is so valuable, as it involves real experimental data surrounding the creation of the compound. Practical implications: With the growing risk of antimicrobial resistance, new forms of treatments are needed to combat evolving diseases such as malaria and toxoplasmosis. These new treatments have large applications in endemic countries where high drug pressure leads to resistance developing. Social implications: Reducing the disease burden in these countries will increase economic output and reduce mortality rates. Originality/value: This research paper follows the synthesis of a new analogue of pyrimethamine, where there is currently no literature surrounding this synthesis. It may be assumed that this compound has never been created before. Keywords: antimicrobial resistance, 3-triflouromethyl analogue, pyrimethamine, Dihydrofolate reductase (DHFR). Paper type: Research paper

Literature Review Malaria is an infectious protozoan disease which in humans, is caused by five plasmodium species of parasite. The transmission of malaria is restricted to the pregnant female anopheles mosquito, which acts as a vector for the disease (CDC, 2022). Initially, the disease develops within the mosquito and is subsequently transmitted to humans through the insect's saliva during the process of the mosquito feeding on blood. Once the disease enters the blood

stream, it attacks the liver and red blood cells, which can cause serious health issues for individuals, in which some may lead to death. Malaria can be extremely dangerous if pregnant woman or children are infected, and higher mortality rates are seen in these demographics (Centers for Disease Control and Prevention, 2020). Malaria remains a significant global public health issue, and in endemic countries, it is of major concern, prompting governments to take

Figure 1: Trends in: a) malaria case incidence (cases per 1000 population at risk) and b) mortality rate (deaths per 100,000 population at risk). Source: (WHO, 2022)

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Figure 2: Incidence of malaria, global distribution, 2019 Source: (WHO, 2020)

comprehensive action (World Health Organisation, 2020). Countries with high malaria infection rates typically have poor healthcare infrastructure, as well as humid climates ideal for mosquito development. In 2019, there were 229 million cases of malaria globally, which led to 409,000 deaths in the same year (WHO, 2020). The most prominent and violent strain of malaria is caused by Plasmodium Falciparum, which is the dominant species of malaria in most endemic continents, making it the ideal focus of this research paper. Improving access to antimalarial drugs in developing countries can greatly diminish the prevalence of the disease, therefore reducing the burden of malaria and promoting better health outcomes in developing countries, potentially leading to reduced poverty rates, lower infant mortality rates, and higher life expectancy. Toxoplasmosis is an infectious disease which is caused by the parasite Toxoplasma gondii. It is present in undercooked meats; such as pork and lamb, as well as contaminated water (Mayo Clinic, 2020). Toxoplasmosis is also found in cat feces and most warm blooded animals. The disease can be spread through the consumption of contaminated products, handling of contaminated objects such as needles or cat feces, and in rare cases through blood transfusions with infected blood (Montazeri et al., 2018). Oocysts develop inside cats or on raw meats, which once ingested, will undergo binary fission and produce

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tachyzoites which will infect the host and can be spread through the blood or to a fetus if one is present (CDC, 2019). All cats and other warm blood animals, and most humans that are infected are asymptomatic, and this will remain the case for their entire life. A small percentage of infected people will experience a multitude of symptoms, including head and muscle aches, swollen nymph nodes and a fever. People with compromised immune systems may be vulnerable to permanent retina, brain and organ damage. It is predicted that over 50% of the world’s population are infected with toxoplasmosis yet show no symptoms (Flegr et al., 2014). Unborn children are also at high risk of toxoplasmosis as the disease is easily spread from the child’s mother, and 200000 congenial cases occur globally each year (Torgerson and Mastroiacovo, 2013). Antimicrobial drugs are the most common forms of malaria and toxoplasmosis treatment, as well as being the most effective. However, one of the main issues associated with the use of these drugs is antimicrobial resistance. This is when microorganisms are given the chance to develop resistance to common treatments through mutation (Tse, Korsik and Todd, 2019). The microbes that develop such resistance are able to reproduce, increasing the prevalence of this new strain. This is an issue because this results in the drug being ineffective in certain areas of the world or amongst smaller human populations where the resistance strain is prominent, causing an increase in

Scientific Research in School Volume 5 Issue 1 2023 healthcare costs and severity of a disease on a country (WHO, 2021). Pyrimethamine is an antiparasitic drug sold under the name Daraprim, as well as others. It is used as a treatment for serious toxoplasmosis cases, as well as a preventative measure against toxoplasmosis for people with HIV/AIDS (Florey, 1983). It is used in conjunction with Sulfadoxine to treat severe malaria cases, although use for the treatment of malaria has declined due to resistance developing in p. falciparum strains, as well as adverse side effects, which include; blood dyscrasias, rash and, very rarely, seizures or shock (Kuhlmann and Fleckenstein, 2017). Due to the potential severity of these conditions, the use of other alternatives is examined before pyrimethamine is advised. Pyrimethamine is a competitive inhibitor and works by preventing the reproduction of parasitic cells (Hastings and Sibley, 2002). All cells, including human and parasitic cells require a type of vitamin B called folate, which is crucial for the replication and maintenance of cells. Folate is derived from folic acid, through a series of reactions, one of which is catalysed by the enzyme Dihydrofolate reductase (DHFR). In humans, folic acid needs are primarily meet through their diet, however in parasitic cells this is not the case, so they must synthesise their own in order to survive. Pyrimethamine shares a similar structure to dihydrofolate, the final substrate used in the creation of folate. This results in pyrimethamine competing for the active site on the DHFR enzyme, rendering the enzyme dysfunctional if pyrimethamine successfully attaches. This greatly

decreases the production of folate in a cell, and therefore prevents the cell from growth and reproduction. The active site for human DHFR varies slightly to the active site for the malaria DHFR, this along with precise doses, this significantly reduces the damage to folate production pyrimethamine causes in humans. Both Malaria and toxoplasmosis strains are known for developing resistance to antifolate treatments. Gene mutations in the DHFR enzyme cause smaller changes in the chemical structure of the active site. These point mutations lead to a reduction of the binding efficiency of pyrimethamine. In both cases, pyrimethamine becomes quite ineffective, so the parasite continues to reproduce, and these now resistant strains of the parasite spread through a population (Peterson, Walliker and Wellems, 1988). The creation of new analogues of synthetic drugs is crucial in order to minimise the effects of resistance. By only slightly altering the chemical structure of common treatments, the effect of point mutations in microbes can be reduced. Changes in structure, as well as new atoms entirely can have a significant impact. Alterations of atoms contained on the molecule will modify its effectiveness, as the molecular weight and electronegativity (ability to attract electrons) will vary between atoms, and this will cause unique interactions with parasitic cells. In the case of this experiment, a chlorine atom has been

Figure 3: Reduction reaction of Dihydrofolate to Tetrahydrofolate catalysed by DHFR.

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Figure 4: Chemical Structure of Pyrimethamine

replaced by a carbon atom, with 3 fluorine atoms attached, and this 3-triflouromethyl group has also been moved to an adjacent carbon. Due to the increased electronegativity of fluorine atoms, as well as the increased mass of the 3-triflouoromethyl group, compared to a single chlorine atom, this new analogue of pyrimethamine has the potential to interact with the DHFR enzyme of resistant malaria and toxoplasmosis parasites (Ray, Das and Suar, 2017). However, there is very limited information on this molecule because it hasn’t been synthesised before.

Figure 5: Proposed Chemical Structure of 3triflouromethyl analogue of pyrimethamine.

This report aims to illustrate the procedure taken to create a new analogue of pyrimethamine in a school laboratory. This form of scientific research is highly valuable as it is develops the understanding of new forms of antimalarial and toxoplasmosis drugs. With the increasing concern of antimicrobial resistance, scientific research into alternative drug treatments is necessary to combat disease as resistance continues to develop. The synthetic pathway produced by Sydney Grammar in 2016 will be altered to suit the 3-triflouromethyl group (figure 6). The ‘breaking good’ team at Sydney university, who help the public conduct scientific research, will assist by using Mass spectroscopy, Hydrogen Nuclear Magnetic Resonance (NMR), and Carbon NMR to conduct biological testing. This will provide information on the structure and purity of the products from each of the three steps.

Figure 6: 1: Structural Chemical Equations for the Proposed Synthesis

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Scientific Research Question Can a 3-triflouromethyl analogue of pyrimethamine be successfully synthesised, with a sufficient yield for chemical analysis of effectiveness against Plasmodium falciparum.

Scientific Hypothesis That a 3-triflouromethyl analogue of pyrimethamine will be synthesised in a school laboratory, yielding a sufficient amount of product to undergo chemical analysis.

Methodology General experiment details 1H spectra were recorded at 300 K using a Bruker Avance DRX500 NMR spectrometer in deuterated solvents. Residual acetone (δ 2.05) was used as internal reference for 1H NMR spectra. The data is reported as chemical shift (δH ppm), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and assignment. Atom labels on structures are to illustrate 1H NMR spectral assignments and do not necessarily correspond to the IUPAC names given. The solvent peak for acetone (δ 29.84) was used as an internal reference for 13C NMR spectra. Mass spectra were recorded by the Mass Spectrometry Unit of the School of Chemistry, The University of Sydney, Sydney. The molecular ion [M + H+], [M + Na+] or [M - H+] is listed. Analytical thin layer chromatography was performed with Merck Kieselgel 60 F254 (0.2 mm) pre-coated aluminium sheets, and visualisation was achieved by inspection under UV light. Throughout the reaction process Thin Layer Chromatography (TLC) was conducted to gauge the progress of the reaction and determine the point of completion. TLC analysis was conducted with pure dichloromethane or 20:80 ethyl acetate/hexanes. Step 1: Synthesis oxopentanenitrile

2-(3-trifluoromethyl)-3-

Figure 2: Chemical Structure of 2-(3-trifluoromethyl)-3oxopentanenitrile

3-triflouromethylphonylacetonitrile (5.00 g, 0.027 mol, 1 equiv.), ethyl propionate (2.9 g, 0.02835 mol, 1.05 equiv.) and potassium tert-butoxide (6.06 g, 0.054 mol, 2 equiv.) were combined in THF (50 mL) at room temperature, with stirring in a round bottom flask. The mixture changed to a blood red colour, and its temperature increased. After 30 minutes, stirring was turned off as the reaction mixture appeared homologous. In a fume hood, the reaction was sealed and left for 2 hours. The reaction mixture was worked up by the addition of 1.0 M HCl (100 mL) to the reaction vessel. The acidified reaction mixture was moved into a separating funnel and the aqueous layer was extracted with Dichloromethane (3 x 65 mL). The combined organic layer was washed with brine (100mL) and dried with anhydrous sodium sulfate. This solution was then filtered, and concentrated in vacuo to produce a red oil substance which was 2-(3trifluoromethyl)-3-oxopentanenitrile ( 8.363 g, 0.0346 mol, 60%). TLC was conducted with 50:50 DCM : hexane as the eluent. The product: 2-(3trifluoromethyl)-3-oxopentanenitrile was not purified and used in the second step of the synthesis. Step 2: Synthesis of (2-(3-trifluoromethylphenyl)-3(2-methylpropoxy)-pent-2-enenitrile)

Figure 3: Chemical Structure of (2-(3trifluoromethylphenyl)-3-(2-methylpropoxy)-pent-2enenitrile)

The product from step 1: 2-(3-trifluoromethyl)-3oxopentanenitrile (8.363 g, 0.0346 mol) was dissolved in a mixture of 2-methylpropan-1-ol (3.125 mL) and toluene (75.0 mL). After the addition of 18M Sulfuric Acid (1.00 mL)., the mixture was refluxed for 10 hours in a Dean Stark apparatus. This reaction mixture was poured onto a saturated sodium hydrogen carbonate in a separating funnel. The aqueous phase was then extracted with DCM (3 x 50 mL), and the combined organic extracts were dried over anhydrous sodium sulfate. 3.0 mL of triethylamine was added to this reaction mixture to convert the unreacted starting material to

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Scientific Research in School Volume 5 Issue 1 2023 a triethylammonium enolate salt, which is highly polar. Chromatography silica (25g) was added to the organic mixture, DCM was added to bring the mixture to 200 mL and this was stirred for two hours. This organic phase was decanted and rinsed with 1M HCL (2 x 50 mL) and deionised water (50 mL), to remove all traces of triethylamine. The solvent was removed from the organic solution via evaporation in a fume hood to yield (2-(3-trifluoromethylphenyl)-3(2-methylpropoxy)-pent-2-enenitrile) (3.767g, 0.0127 mol, 35%) as a dark red oil.

The solid was then dissolved in 100% ethanol and after 48 hours, no crystallisation had occurred.

Step 3: Synthesis of 3-trifluoromethyl pyrimethamine

Figure 5: Dean Stark apparatus from step 2. Figure 4: Chemical Structure of 3-triflouromethyl pyrimethamine.

(2-(3-trifluoromethylphenyl)-3-(2-methylpropoxy)pent-2-enenitrile) (3.767g, 0.0127 mol) was dissolved in DMSO (45.0 mL). Guanidine hydrochloride (2.5 g, 0.0262 mol) was stirred into this solution. Sodium methoxide powder (1.55 g, 0.0279 mmol) is added and the solution turned a dark red colour. After 1 hour, all sodium methoxide had dissolved, and no sodium chloride precipitate was present. The solution remained in a fume hood to stand for 48 hours. The reaction mixture was poured onto water and extracted with DCM. The solution was concentrated in vacuo to afford a red oily solid.

Figure 6: Extraction of organic layer via Filter funnel: step 3.

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Results Step 1: Synthesis of 2-(3-trifluoromethyl)-3-oxopentanenitrile

Figure 7: H-NMR Spectra of 2-(3-trifluoromethyl)-3-oxopentanenitrile.

H NMR (400MHz, CDCl3) δ 8.37 (1H, s, CH), δ 8.29 (H, d, CH), δ 7.86 (1H, d, CH), δ 7.60 (2H, q, CH2), δ 4.76 (1H, s, CH), δ 3.82 (1H, s, CH), δ 1.07 (3H, t, CH3). 1

Figure 8: C-NMR Spectra of 2-(3-trifluoromethyl)-3-oxopentanenitrile.

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Figure 9: Mass Spectrum of 2-(3-trifluoromethyl)-3-oxopentanenitrile.

(Left) Figure 10: TLC of 2(3-trifluoromethyl)-3oxopentanenitrile.

(Right) Figure 11: Labelled expanded Chemical structure of 2-(3-trifluoromethyl)-3oxopentanenitrile for reference.

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Step 2: Synthesis of (2-(3-trifluoromethylphenyl)-3-(2-methylpropoxy)-pent-2-enenitrile

Figure 12: H-NMR Spectra of (2-(3-trifluoromethylphenyl)-3-(2-methylpropoxy)-pent-2-enenitrile).

(Left) Figure 13: Labelled expanded Chemical Structure of (2-(3trifluoromethylphenyl)-3-(2methylpropoxy)-pent-2enenitrile) for reference. (Right) Figure 14: TLC of (2(3-trifluoromethylphenyl)-3(2-methylpropoxy)-pent-2enenitrile).

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Figure 15: Mass Spectrum of (2-(3-trifluoromethylphenyl)-3-(2-methylpropoxy)-pent-2-enenitrile).

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Step 3: Synthesis of 3-trifluoromethyl pyrimethamine

Figure 16: H-NMR Spectra of 3-trifluoromethyl pyrimethamine.

Figure 17: TLC of 3-trifluoromethyl pyrimethamine.

Figure 18: TLC of 3-trifluoromethyl pyrimethamine.

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Figure 19: Mass Spectrum of 3-trifluoromethyl pyrimethamine.

Figure 20: Labelled expanded chemical structure of 3-trifluoromethyl pyrimethamine for reference.

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Discussion Step 1: Synthesis of 2-(3-trifluoromethyl)-3oxopentanenitrile

Figure 21: Reaction of 3(trifluoromethyl)phenylacetonitrile to form 2-(3trifluoromethyl)-3-oxopentanenitrile.

Step 1 of the synthesis was completed to afford 2-(3trifluoromethyl)-3-oxopentanenitrile in a condensation reaction between 3(triflouromethyl)phenylacetonitrile and ethyl propionate. Potassium tert-butoxide, a strong base, was also added. This allowed for the deprotonation of the CH2 group in the starting material. This subsequently led to ethanol being removed from this compound. The yield for this first step was 60%, which is slightly lower than most previous pyrimethamine synthesis experiments done in school laboratories. However the product from this step was not purified, and analysis of H and C NMR data suggests that some impurities were present. The multiplet at δ 2.70 did not align with any hydrogen environments present on the product of this step. All other peaks on the HNMR spectra represent hydrogens present on 2-(3-trifluoromethyl)-3oxopentanenitrile. Two separate runs of thin layer chromatography suggest that this reaction occurred and a new product formed. Reaction spots at the very top of both TLC sheets, that are only present in the samples containing the product, suggest the intended chemical reaction occurred. Similar to the ones to follow, the C-NMR spectrum of this experiment is quite complex, containing many peaks which leave interpretation quite difficult. The molecular weight of the intended product of step 1 is 240.1. The Mass spectrum has a intense peak at 240, representing the fragment that is the entire compound. All biological analysis of step 1 suggests that 2-(3-trifluoromethyl)-

3-oxopentanenitrile was successfully however it contained many impurities.

created,

Step 2: Synthesis of (2-(3-trifluoromethylphenyl)3-(2-methylpropoxy)-pent-2-enenitrile)

Figure 22: Reaction of 2-(3-trifluoromethyl)-3oxopentanenitrile to form (2-(3-trifluoromethylphenyl)-3(2-methylpropoxy)-pent-2-enenitrile)

Step 2 of this synthesis was conducted under reflux conditions, which allowed a substitution reaction to take place between 2-(3-trifluoromethyl)-3oxopentanenitrile and 2-methylpropanol to create (2(3-trifluoromethylphenyl)-3-(2-methylpropoxy)pent-2-enenitrile). This second step was done in a dean stark apparatus, specifically to push the equilibrium to the right by removing water; a byproduct of this reaction. This was done to maximise the yield of step 2. To remove unreacted starting material, triethylamine was added after the first stage of the reaction, and silica gel assisted with the further removal of this newly formed salt. However, TLC analysis suggests that this reaction did not progress to a sufficient standard, and the percentage yield of 35% encourages the same conclusion. The hydrogen NMR and the mass spectrum of step two were both highly contaminated, leading to the interruption that this product contained many impurities. However it can be inferred that some starting material reacted to form the desired compound, so this impure mixture was then used for step 3. Step 3: Synthesis pyrimethamine

3-trifluoromethyl

Figure 23: Reaction of (2-(3-trifluoromethylphenyl)-3-(2methylpropoxy)-pent-2-enenitrile)to form a 3triflouromethyl analogue of pyrimethamine.

The final step of the synthesis intended to make the desired drug. The deprotonation of guanidine hydrochloride is assisted by sodium methoxide. This allows for the electron rearrangement and subsequent removal of 2-methylpropanol group, and for the

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Scientific Research in School Volume 5 Issue 1 2023 guanidine molecule to attach, creating the final compound. May TLC analyses were conducted with different solvents in order to confirm whether this reaction had taken place. A reaction spot can be seen on most TLC pieces, leading to the conclusion that a highly impure product has been made. The mass spectrum of step 3 contained a variety of different peaks, with one signal at the desired 282.11 m/z. This could potentially be the fragment of the 3triflouromethyl analogue, however it cannot be stated with certainty. The HNMR spectrum was again highly complex, and this result again confirms the following notion that this procedure should include the purification of products after each step for a more successful yield. Future research Although it is unclear whether a 3-triflouromethyl analogue of pyrimethamine was created, this experiment has given new insight into the creation of this unique compound. There are many areas of improvement in order to optimise the synthesis of this molecule. Firstly, one of the main reasons why the yield of products continued to decline as the synthesis progressed was the build up of impurities. Due to time constraints, the products of each step were unable to be properly purified. A strong conclusion can be made that these impurities continued to accumulate, which resulted in the NMR and mass spectrums to have limited clarity. More extensive research could also be made into the behaviour of this compound and its reactants, and how a 3triflouromethyl group causes changes in reactivity. Future experimentation and the optimisation of the creation of new analogue of pyrimethamine has the ability to reduce the burden of resistant malaria and toxoplasmosis parasites.

Conclusion This research project explored the synthesis of a 3triflouromethyl analogue of pyrimethamine; an antimalarial drug, in a school laboratory. A three-step experiment was conducted, a similar procedure to create standard pyrimethamine, but with a different starting material, as well as other smaller changes like different solvents suited to the three fluorine atoms on the molecule. TLC analysis was used extensively throughout the investigation, as a simple way to confirm if chemical reactions had occurred and new compounds had been formed. Samples were taken from the products of all three steps, as well as from the starting material. These samples were used for biological analysis: H-NMR, C-NMR and mass spectroscopy was performed at Sydney university.

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This provided structural information on what was created, as well as presenting insight into the purity of the products. Analysis of this data is inconclusive whether a 3-triflouromethyl pyrimethamine was created, with potentially an extremely low yield that was highly impure and therefore the hypothesis can be neither accepted nor rejected. Future experimentation and research could improve yields and purities of this unique antimalarial compound by purifying the product of each step.

Acknowledgments I would like to thank Dr Katie Terrett for her supervision of this experiment. Her extensive knowledge and clear scientific methodology was invaluable. A special thanks is also necessary for the team at Sydney University, managing breaking good projects. There help in biological analysis and providing the data for this experiment was much appreciated.

References Agarwal, A., Srivastava, K., Puri, S.K. and Chauhan, P.M., 2005. Antimalarial activity and synthesis of new trisubstituted pyrimidines. Bioorganic & medicinal chemistry letters, 15(12), pp.3130-3132 Ashley, E.A., Phyo, A.P. 2018. 'Drugs in Development for Malaria'. Springer Open Choice, vol. 78, no. 12, pp. 1-19. Bloland, P.B. and World Health Organization, 2001. Drug resistance in malaria (No. WHO/CDS/CSR/DRS/2001.4). World Health Organization. CDC (2019). CDC - Toxoplasmosis - Biology. [online] Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/parasites/toxoplasmosis/biology.htm l. CDC (2022). CDC - Malaria - About Malaria - FAQs. [online] Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/malaria/about/faqs.html. Centers for Disease Control and Prevention (2020). CDC Malaria - About Malaria - Biology. [online] CDC. Available at: https://www.cdc.gov/malaria/about/biology/index.html. Chan, D.C., Laughton, C.A., Queener, S.F. and Stevens, M.F., 2002. Structural studies on bioactive compounds. Part 36: design, synthesis and biological evaluation of pyrimethamine-based antifolates against Pneumocystis carinii. Bioorganic & medicinal chemistry, 10(9), pp.30013010. Florey, K. (1983). (Analytical Profiles of Drug Substances 12) Klaus Florey (Eds.) - Academic Press (1983) PDF | PDF | Amine | Proton Nuclear Magnetic Resonance. [online] Scribd. Available at: https://www.scribd.com/document/423001705/12Analytical-Profiles-of-Drug-Substances-12-Klaus-FloreyEds-Academic-Press-1983-pdf [Accessed 18 Jun. 2023].

Scientific Research in School Volume 5 Issue 1 2023 Hastings, M.D. and Sibley, C.H. (2002). Pyrimethamine and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax. Proceedings of the National Academy of Sciences, 99(20), pp.13137–13141. doi:https://doi.org/10.1073/pnas.182295999. Loutfy, M.A. and Aboul-Enein, H.Y., 1983. Pyrimethamine. In Analytical Profiles of Drug Substances (Vol. 12, pp. 463-482). Academic Press. Mayo Clinic (2020). Toxoplasmosis - Symptoms and Causes. [online] Mayo Clinic. Available at: https://www.mayoclinic.org/diseasesconditions/toxoplasmosis/symptoms-causes/syc20356249#:~:text=Toxoplasmosis%20(tok%2Dso%2Dpla z. Montazeri, M., Mehrzadi, S., Sharif, M., Sarvi, S., Tanzifi, A., Aghayan, S.A. and Daryani, A. (2018). Drug Resistance in Toxoplasma gondii. Frontiers in microbiology, [online] 9, p.2587. doi:https://doi.org/10.3389/fmicb.2018.02587. Murphy, S.C. 2006, 'Malaria and Global Infectious Diseases: Why Should We Care?", AMA Journal of Ethics, vol. 8, no. 4, pp. 245-50. Peterson, D.L., Walliker, D. and Wellems, T.E. (1988). Evidence that a point mutation in dihydrofolate reductasethymidylate synthase confers resistance to pyrimethamine in falciparum malaria. 85(23), pp.9114–9118. doi:https://doi.org/10.1073/pnas.85.23.9114.

WHO 2020, World Malaria Report 2020, viewed 20 February 2021, <https://www.who.int/publications/i/item/9789240015791 >. WHO (2022). World malaria report 2022. [online] www.who.int. Available at: https://www.who.int/teams/global-malariaprogramme/reports/world-malaria-report-2022. Yuthavong, Y., Tarnchompoo, B., Vilaivan, T., Chitnumsub, P., Kamchonwongpaisan, S., Charman, S.A., McLennan, D.N., White, K.L., Vivas, L., Bongard, E., Thongphanchang, C., Taweechai, S., Vanichtanankul, J., Rattanajak, R., Arwon, U., Fantauzzi, P., Yuvaniyama, J., Charman, W.N. & Matthews, D. 2012, 'Malarial dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target', Proceedings of the National Academy of Sciences, vol. 109, no. 42, pp. 16823-8. Yuthavong, Y., Yuvaniyama, J., Chitnumsub, P., Vanichtanankul, J., Chusacultanachai, S., Tarnchompoo, B., Vilaivan, T. & Kamchonwongpaisan, S. 2005, Malarial (Plasmodium falciparum) dihydrofolate reductasethymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors', Parasitology, vol. 130, no. Pt 3, pp. 249-59.

Ray, S., Das, S. and Suar, M. (2017). Molecular Mechanism of Drug Resistance. Drug Resistance in Bacteria, Fungi, Malaria, and Cancer, pp.47–110. doi:https://doi.org/10.1007/978-3-319-48683-3_3. Roper, C., Pearce, R., Nair, S., Sharp, B., Nosten, F. and Anderson, T., 2004. Intercontinental spread of pyrimethamine-resistant malaria. Science, 305(5687), pp.1124-1124. Sydney Grammar School Synthesis 2016, 'Daraprim Synthesis', Pyrimethamine Synthesis: Status at the end of 2016, viewed February 20 <https://malaria.ourexperiment.org/daraprim_synthesis>.2 021, Weiss, D.J., Lucas, T.C.D., Nguyen, M., Nandi, A.K., Bisanzio, D., Battle, K.E., Cameron, E., Twohig, K.A., Pfeffer, D.A., Rozier, J.A., Gibson, H.S., Rao, P.C., Casey, D., Bertozzi-Villa, A., Collins, E.L., Dalrymple, U., Gray, N., Harris, J.R., Howes, R.E., Kang, S.Y., Keddie, S.H., May, D., Rumisha, S., Thorn, M.P., Barber, R., Fullman, N., Huynh, C.K., Kulikoff, X., Kutz, M.J., Lopez, A.D., Mokdad, A.H., Naghavi, M., Nguyen, G., Shackelford, K.A., Vos, T., Wang, H., Smith, D.L., Lim, S.S., Murray, C.J.L., Bhatt, S., Hay, S.I. & Gething, P.W. 2019, Mapping the global prevalence, incidence, and mortality of Plasmodium falciparum, 2000-17: a spatial and temporal modelling study', The Lancet, vol. 394, no. 10195, pp. 32231. White, N.J., 1998. Drug resistance in malaria. British medical bulletin, 54(3), pp.703-715. White, N.J. 2004, 'Antimalarial drug resistance', Journal of Clinical Investigation, vol. 113, no. 8, pp. 1084-92. WHO (2021). Antimicrobial resistance. [online] Who.int. Available at: https://www.who.int/news-room/factsheets/detail/antimicrobial-resistance.

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Synthesis of a 2-aminothiazole analogue Christian Kemp Barker College Purpose: This research project is focused on the synthesis of a 2-aminothiazole compound to test its inhibitory capacity against Madurella Mycetomatis (M. Mycetomatis) fungi to assess the viability of this new compound as an alternative treatment for the subcutaneous skin disease, mycetoma. In addition, this project aimed to test the effectiveness of Sydney University’s synthetic procedure for 2-aminothiazoles in synthesizing one specific analogue which had previously not been synthesised. Design/methodology/approach: The experimental design for this project was an adaptation of the synthesis pathway for 2-aminothiazole compounds devised by Sydney University in 2012. The targeted analogue was obtained by adjusting the original synthetic pathway to replace the methyl group of the pyridine compound in step three with a trifluoromethyl group, which would theoretically change the electron distribution and thereby the inhibitory capacity of my final compound. Findings: Although due to time restraints the 2-aminothiazole analogue could not be sent to Sydney University to be tested in-vitro against M. Mycetomatis, relatively high yields of 86.67%, 73.23% and 93.77% were achieved for steps one, two and three respectively of the experimental procedure, thereby supporting the effectiveness of Sydney University’s synthetic procedure in obtaining my desired compound. This was backed up using Nuclear Magnetic Resonance (NMR) spectroscopy as well as mass spectroscopy, which both provided data to indicate a successful synthesis of the desired analogue. Practical implications: The research carried out in this project has major practical implications as it is based around the development of an alternative treatment for eumycetoma, which was identified as a Neglected Tropical Disease (NTD) by the World Health Assembly (WHA) in 2016 which currently most often requires surgical excision as the primary form of treatment. Research limitations/implication – Due to time constraints the 2-aminothiazole analogue that was successfully synthesized could not be sent to Sydney University for testing against M. Mycetomatis, meaning the inhibitory capacity of the analogue for eumycetoma remains the subject of future work. Originality/value: This paper builds on past research, mainly relating to findings from the MycetOS project launched in conjunction with the Open Access Boxes initiative launched by the Medicines for Malaria Venture (MMV) in 2015 to synthesize an analogue that has never been synthesized or tested in the past and test it as an alternative treatment for eumycetoma. Paper type: Research paper

Literature Review Mycetoma refers to a chronic, slow growing, progressively destructive subcutaneous infection that can spread to deep tissues and bone (DNDI, 2022). As it is a neglected disease, many epidemiological features of mycetoma remain uncharacterized, although it is mainly found in areas around the equator in what is known as the ‘mycetoma belt’ (Watson et. al, 2022). Africa is the continent most affected by Mycetoma, with 10608 of the 19494 listed cases of the disease coming from Sudan (Develoux, 2022) (Figure 1). Although the disease’s specific etiological factors remain largely ambiguous, it is known that causative agents of mycetoma are present in the environment and move into the body due to injuries or trauma (Develoux, 2022). The causal agents and modes of

Figure 1: Prevalence of Mycetoma. Source: (van de Sande, 2013)

treatment for mycetoma vary across the disease’s two distinct etymologies. Actinomycetoma, which is caused by bacteria, can be treated by antimicrobial combination therapy using compounds such as

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Scientific Research in School Volume 5 Issue 1 2023 amikacin sulfate and co-trimoxazole (Figures 2 and 3) with a 90% success rate (Scolding et. Al, 2018). Eumycetoma, on the other hand, is caused by the fungus M. Mycetomatis (Figure 4) and is treated using long-term antifungal medicine in combination with surgical excision/amputation, which is often the only available treatment for advanced infections (WHO, 2022).

Figure 2: Chemical structure of amikacin sulfate. Source: (Pubchem, n.d.)

Figure 3 : Chemical structure of co-trimoxazole. Source: (Toronto Research Chemicals, n.d.)

Figure 4: Microscopic image of M. Mycetomatis fungus. Source: (University of Adelaide, 2022)

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Drug therapy for eumycetoma has a mean duration of 12.9 months and a duration range of between 3-36 months (Turiansky et. Al, 2022). A study conducted by the Mycetoma Research Centre in Sudan reveals the inadequacy of the current treatments for eumycetoma, finding that from a sample of 1,242 patients, 54% of these patients did not attend outpatient follow up due to treatment side effects and dissatisfaction with therapy outcome (RSTMH, 2019). As well as this, eumycetoma is often endemic to impoverished areas so the high cost of long-term drug therapy can either cause significant financial strain or simply isn’t an option for those affected due to economic reasons as well as limited access to healthcare and medication (CDC, 2020). In 2016, Mycetoma was recognized by the World Health Assembly (WHA) as a neglected tropical disease (NTD) (Fahal et. Al, 2022). This proved an important step in raising awareness as well as initiating global partnerships and research programs geared towards a better understanding of the disease and potential treatments. Open-Source Drug Screening Programs have proved the main mode of discovery for alternative treatments of mycetoma, involving the use of radically transparent working practices encouraging public interaction and prepublication sharing of data and ideas in determining potential new ways to treat the disease (Balasegaram et. Al, 2017). An example of this is the Mycetoma Open-Source project (MycetOS). Launched in 2018 by the University of Sydney in conjunction with Erasmus University Medical Centre (Erasmus MC) and the Drugs for Neglected Diseases Initiative (DNDI), uses an ‘open-source pharma’ approach to assess potential new treatments for eumycetoma (DNDI, 2023). Another example of this is the Open Access Boxes Initiative opened by the Medicines for Malaria Venture in 2015 by providing researchers with free access to 400 diverse compounds to catalyze the discovery of new treatments for neglected diseases such as mycetoma (MMV, 2019). These initiatives work in conjunction, as the MycetOS project screens drugs made available by the Open Access Boxes initiative to identify starting points for mycetoma drug discovery (DNDI, 2023). A website providing an overview of compounds in the Pandemic Response box from the Open Access Boxes Initiative with activity against fungal NTDs noted that the 2-aminothiazole class of organic material displays good inhibitory activity against M. Mycetomatis, with a Minimum Inhibitory Capacity (MIC) of 0.25 ug/ml (Lim et. Al, 2022). As well as

Scientific Research in School Volume 5 Issue 1 2023 this, compounds which contain an aminothiazole scaffold have shown antitumor, antiviral, antibacterial, anti-prion, anti-inflammatory and antifungal properties (Elsadek et. Al, 2021). Most notably, the aminothiazole scaffold has provided the basis for clinically applied anticancer drugs such as dasatinib and alpelisib thanks to its potent nanomolar activity against a wide range of human cancerous cell lines (Alizedeh and Hashemi, 2021). According to the literature, the 2-aminothiazole mode of action against M. Mycetomatis is as a potassiumcalcium (KCa) channel inhibitor, stimulating hyperpolarization in these channels to affect the cells’ ability to function (Gentles et. Al, 2008). Other classes of compounds from the Pandemic Response Box showing particularly good inhibitory activity against M. Mycetomatis included fenarimols, strobilurins and benzimidazole carbamates, (Lim et. Al, 2022). Having been tested against M. Mycetomatis in vitro, the fenarimol analogue, MMV689244 (Figure 5) demonstrated a Minimum Inhibitory Capacity (MIC) of one ug/ml (Microgram per millilitre), meaning the compound was able to completely inhibit the growth of M. Mycetomatis with a minimum concentration of one ug/ml. As well as this the strobilurin compound, azoxystrobin (Figure 6) is a broad-spectrum antifungal showed a MIC value of 0.06um against M. Mycetomatis and the benzamide compounds fenbendazole (Figure 7) and carbendazim (Figure 8) both had a promising MIC of 0.5um when tested in vitro against M. Mycetomatis (Lim et. Al, 2022).

Figure 6: General structure of the strobilurin compound, azoxystrobin. Source: (Wikimedia commons, 2013)

Figure 7: General structure of benzamide compound, fenbendazole. Source: (Pubchem, n.d.)

Figure 8: General structure of benzamide compound, carbendazim. Source: (Pubchem, n.d.) Figure 5: General structure of the MMV689244 fenarimol analogue. Source: (MycetOS, 2019)

To this point, research on 2-aminothiazole compounds has been concentrated mainly on the MMV006357 analogue (1) (Figure 9) from the stasis box of the MMV’s Open Source Mycetoma initiative (Github, 2023). This 2-aminothiazole analogue has undergone Malaria screening by pharmaceutical companies GlaxoSmithKline (GSK) and Novartis and Leishmania screening by Australian school, Saint Jude, and has been successfully synthesized by Sydney University using the synthetic pathway that my methodology is based on (Github, 2023).

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Figure 9: The chemical structure for the lead compound, 2-aminothiazole analogue MMV006357 (1) Source: (Github, n.d.)

Figure 12 : The chemical structure for the 2aminothiazole analogue to be synthesized, 4-(pyridine2-yl)-N-(5-(trifluoromethyl)pyridine-2-yl)thiazol-2amine (2)

This project focuses on the synthesis of a 2aminothiazole analogue (2) because the lead compound (1) shows promising MIC values of 0.25 ug/ml against M. Mycetomatis (Lim et. Al, 2022). Synthetic procedures for it are readily available and able to be carried out in a high school lab. As well as this, although many compounds have been analyzed

Figure 10: Sydney University’s synthetic procedure for 2-aminothiazole analogues

Figure 11: My adaptation of Sydney University’s synthetic pathway for the 2-aminothiazole compound

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Scientific Research in School Volume 5 Issue 1 2023 against M. Mycetomatis, I have not yet come across my specific analogue in mycetoma literature. The synthetic procedure I am using for my 2aminothiazole analogue (2) is based on the reaction scheme formulated by Sydney University (Figure 11). I chose this procedure because it has already been used to successfully synthesize several aminothiazole compounds. My compound replaces a methyl group on one of the compound’s benzene rings with a CF3 group, which I will achieve by substituting the pyridine compound used in step one of Sydney Uni’s procedure (3) with one that contains a CF3 group (4) in place of one of the original pyridine compound’s methyl groups (Figures 13, 14). The resulting difference between my analogue and the lead compound can be seen in figures 9 and 10. From step two onwards, my synthetic procedure will replicate that devised by Sydney University. My adaptation of Sydney University’s synthetic pathway can be seen in figure 12.

Figure 13: The pyridine compound used in my adaptation of Sydney University’s synthetic procedure, 5-(trifluoromethyl)pyridine-2-amine) (4)

In this investigation I aim to determine the effect this has on the compound’s inhibitory activity against M. Mycetomatis.

Scientific Research Question Can the 2-aminothiazole analogue (2) be synthesised in a school laboratory and be tested as an anti-fungal agent against M. Mycetomatis?

Scientific Hypothesis That the 2-aminothiazole analogue (2) can be synthesised in a school laboratory and tested as an anti-fungal agent against M. Mycetomatis.

Methodology H and 13C NMR spectra were recorded at 300 K using a Bruker Avance DRX400 NMR spectrometer. Residual acetone (δ 2.05) and chloroform (δ 7.26) were used as internal reference for 1H NMR spectra. The data is reported as chemical shift (δH ppm), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and assignment. Atom labels on structures are to illustrate 1H NMR spectral assignments and do not necessarily correspond to the IUPAC names given. The solvent peak for chloroform (δ 77.0) was used as an internal reference for 13C NMR spectra. Mass spectra were recorded by the Mass Spectrometry Unit of the School of Chemistry, The University of Sydney, Sydney. The molecular ion [M + H+] or [M - H+] is listed. 1

Analytical thin layer chromatography was performed with Merck Kieselgel 60 F254 (0.2 mm) pre-coated aluminium sheets, and visualisation was achieved by inspection under UV light. Throughout the reaction process Thin Layer Chromatography (TLC) was conducted to gauge the progress of the reaction and determine the point of completion. TLC analysis was conducted using dichloromethane. Step 1: Synthesis of N-((5-(trifluoromethyl)pyridine2-yl)carnamothioyl)benzamide (5)

Figure 14: The pyridine compound used in Sydney University’s original synthetic procedure, 2-amino-4methylpyridine (3) Source: (Merck, n.d.)

The addition of a CF3 group to the original 2aminothiazole structure (1) in my compound will change the electron distribution drastically due to the difference in electronegativity between fluorine and hydrogen, increasing the reactivity of the molecule.

Figure 15 : N-((5-(trifluoromethyl)pyridine-2yl)carnamothioyl)benzamide (5)

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Scientific Research in School Volume 5 Issue 1 2023 In a round bottom flask fitted with a reflux condenser, benzoyl chloride (1.912g, 13.60mmol, 1 equiv.) was added to a suspension of potassium thiocyanate (1.436g, 14.78mmol, 1 equiv.) in acetone (35mL). The reaction was then heated to reflux (60 ºC) and stirred for 20 minutes. The reaction was then cooled slightly and 5-(trifluoromethyl)pyridine-2-amine (4) (2.000g, 12.33mmol, 1 equiv.) was added. The reaction was returned to reflux and stirred for another 30 minutes. After cooling the reaction was poured over ice water (100mL) and stirred for 10 minutes. The precipitate was then collected via vacuum filtration and washed with cold water (10mL). Step 2: Synthesis of 1-(5-(trifluoromethyl)pyridine-2yl)thiourea

Figure 16: 1-(5-(trifluoromethyl)pyridine-2-yl)thiourea (6)

In a round bottom flask fitted with a reflux condenser, the benzamide compound (5) (3.438g, 10.37mmol, 1 equiv.) was added to a 2.5M aq. NaOH solution (49.35mL, 105.7mmol, 10 equiv.) and the reaction was heated to 80°C. The pH was adjusted to 4-5 using 1M HCl to quench the remaining NaOH followed by adjusted to pH 8 using a sat. Na2CO3 solution to precipitate the product. The precipitate was collected via vacuum filtration and washed with cold water before being dried in a desiccator overnight. Step 3: Synthesis of 4-(pyridine-2-yl)-N-(5(trifluoromethyl)pyridine-2-yl)thiazol-2-amine

Figure 17: 4-(pyridine-2-yl)-N-(5(trifluoromethyl)pyridine-2-yl)thiazol-2-amine (7)

The thiourea compound (6) (1.696g, 7.667mmol, 1 equiv.) was added to 2-bromo-1-(pyridine-2yl)ethan-1-one (2.154g, 7.667mmol, 1 equiv.) in ethanol (40mL). The reaction was heated to reflux (80C) and stirred until completion as determined via TLC (20% EtOAc/hexane). After cooling the

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reaction mixture was poured over ice water (25mL). The pH was adjusted to pH 8 using a sat aq. Na2CO3 solution to precipitate the product. The precipitate (7) was collected via vacuum filtration and washed with cold water (2 x 10mL) and cold hexane (2 x 10mL).

Scientific Research in School Volume 5 Issue 1 2023 Data analysis

Figure 18: mass spectra after step 3

Figure 19: HNMR spectra after step 3

1H NMR (500 MHz, acetone-d6): δ 10.98 (1H, br s, NH), 8.60-8.56 (1H, m, Ar-H), 8.00 (1H, d, J = 7.6 Hz, ArH), 7.81 (1H, t, J = 6.9 Hz, Ar-H), 7.74 (1H, s, Ar-H), 7.60 (1H, s, Ar-H), 7.27-7.24 (1H, m, Ar-H), 7.22 (1H, d, J = 4.66 Hz, Ar-H). 13C NMR (100 MHz, acetone-d6): δ 182.3 (C), 155.9 (C), 144.1 (CH), 135.9 (CH), 125.1 (C), 112.6 (CH). MS (+ESI): m/z 220.02 (M – H+), 222.03 (M + H+), (M + Na+).

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Figure 20: TLC after step 2

Figure 21: TLC after step 1 (Brown product)

translucent needles, so Thin Layer Chromatography (TLC) had to be used to identify which was the desired benzamide compound. Both TLCs were run in 50:50 dichloromethane and revealed that the appearance of the polar baseline material for the brown product’s TLC corresponded with the polar nature of the benzamide product (5), due to two amine groups and a double bonded oxygen. Therefore, it was concluded that the brown product was the desired benzamide compound (5) and translucent needles were residual starting material. The yield obtained for this step was 86.67%, which is very good, especially given the time constraints posed on the experimental procedure due to the project deadline, therefore proving the effectiveness of Sydney University’s synthetic pathway. However, the high percentage yield could potentially also be attributed to remaining impurities that were present when weighing the product, which could be further evidenced by the presence of bands on the benzamide product’s TLC that could not be attributed to the starting material or the product itself.

Figure 23: the mechanism for the reaction that occurs between benzoyl chloride and potassium thiocyanate

Step 2: Synthesis of 1-(5-(trifluoromethyl)pyridine-2yl)thiourea (6) Figure 22: TLC after step 1 (Needles)

Discussion Step 1: Synthesis of N-((5-(trifluoromethyl)pyridine2-yl)carnamothioyl)benzamide (5) The reaction for step one was a substitution reaction between benzoyl chloride and potassium thiocyanate to produce benzoyl isothiocyanate, which was then reacted with the pyridine compound (4) to produce a benzamide compound (5). More specifically, the CCl bond of the benzoyl chloride was cleaved by the nitrogen atom of the potassium thiocyanate to facilitate the substitution reaction (Figure 23). The isothiocyanate compound was then able to react with the starting material (4), in an addition reaction that produced the step 1 product, (5) Interestingly, this step resulted in the formation of two distinctly different products, a brown substance and thin

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Step two was a hydrolysis reaction that occured between the benzamide compound (5) and NaOH resulting in the formation of a thiourea compound (6). The product from this step again appeared as a brown, homologous substance. TLC for this step was conducted using 50:50 dichloromethane and the appearance of polar baseline material indicated that the thiourea compound had been synthesized, as the removal of the non-polar benzyl group from the benzamide compound (5) would have resulted in an increased polarity. In this step, I was instructed in the synthetic procedure to rinse the precipitate with cold water (2 x 25mL) following vacuum filtration, however in a momentary lapse of reason the bottle containing acetone was mistook for the bottle containing distilled water. As a result of this, the synthetic procedure had to be started from step one. This did not have any significant impact on the project as my experimental values and yields were approximately the same across my first and second

Scientific Research in School Volume 5 Issue 1 2023 run. The yield obtained for this step of the experimental procedure was 73.23%, which is significantly less than that of the previous step, which could be due to some of the product (6) not precipitating with Na2CO3 and being collected as filtrate in vacuum filtration. As well as this the TLC did indicate some residual starting material affecting the purity of the thiourea product (6), therefore indicating that the reaction did not go to completion. As the experimental procedure instructed the addition of NaOH in a high 10:1 ratio to the benzamide compound (5) this can likely be accounted for by the relatively short reaction time of 15 minutes that may not have allowed the reaction to go to completion. future attempts to repeat this step could allow for a longer reaction time (ie. 30 minutes) to ensure that the reaction does go to completion.

Figure 24: the general mechanism for the reaction that occurred between the benzamide compound (5) and NaOH to form the thiourea compound (6).

Step 3: Synthesis of 4-(pyridine-2-yl)-N-(5(trifluoromethyl)pyridine-2-yl)thiazol-2-amine (7) Step three of synthesis was a ring formation that occurred between the thiourea compound (6) and alpha bromoketone to form the desired 2aminothiazole analogue (7). Due to time constraints this was the only step of the synthetic procedure that mass spectroscopy and NMR spectroscopy data was able to be obtained, which required sending our product for analysis by Sydney University for analysis due to a lack of available equipment. In the 1 H NMR spectrum, the broad singlet at 10.98ppm was allocated to the NH group on the final product as its position downfield can be attributed to the electron shielding of the nitrogen atom. Singlets at 7.60 ppm, 7.74 ppm, 8.00 ppm, 7.81 ppm and 7.22 ppm were all assigned to aromatic H1 environments. The splitting pattern of doublet signals at 8.60-8.56 and 7.27-7.24 correlated with two of the aromatic hydrogens on the second benzene ring of the desired 2-aminothiazole analogue (7). The 13C NMR results indicated peaks at 182.3 ppm, 144.1 ppm, 135.9 ppm, 125.1 ppm and 112.6 ppm which corresponds with the number of carbon environments for my desired compound. As well as this mass spectroscopy signals at 220.2m/z and 222.03 m/z corresponded to (M – H+) and (M +

H+), therefore further confirming successful synthesis of the desired 2-aminothiazole analogue (7). Future projects As well as its potential as a cheaper and more effective alternative to currently available treatments for eumycetoma, the successful synthesis of the 2aminothiazole analogue (7) could be applied to develop other analogues through cross-coupling reactions. Although literature shows that the CF3 functional group is generally stable and not susceptible to chemical manipulation, alkyl fluorides often take part in nucleophilic substation with metal or carbon nucleophiles, in which the C-F bonds can be cleaved through single electron transfer and therefore can serve as a leaving group in crosscoupling reactions under specific conditions (Iwasaki, 2023). Therefore, if the 2-aminothiazole analogue itself is not potent against M. Mycetomatis, it shows analogues whose activity could also be tested against M. Mycetomatis and in other applications. Another aim for future projects could be to biologically test this analogue or other analogues in vivo against M. Mycetomatis to determine its inhibitory capacity, which wasn’t achieved in this project due to time constraints posed by the project deadline.

Conclusion The research conducted resulted in successful synthesis of the desired 2-aminothiazole analogue (7), via the synthetic pathway developed by Sydney University. Structural confirmation was carried out using mass spectroscopy, 1H NMR and 13C NMR to confirm that the desired product was synthesised after each step. Despite this, due to time constraints posed by the project deadline, the compound could not undergo in vitro testing against M. Mycetomatis. Biological testing of the analogue could be completed in future to provide biological data on its efficacy as a cheaper and more effective alternative to current treatments for eumycetoma.

Acknowledgements I would like to thank Dr Katie Terrett for her support in conducting research and developing the idea and method, as well as ordering and gathering chemicals required for my project. Thanks to Dr Matthew Hill and Dr Alison Gates for assistance in research and idea generation.

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References Alizadeh, S.R. and Hashemi, S.M. (2021). Development and therapeutic potential of 2-aminothiazole derivatives in anticancer drug discovery. Medicinal Chemistry Research, 30(4), pp.771–806. doi:https://doi.org/10.1007/s00044020-02686-2. Anon, (n.d.). Cotrimoxazole-13C-d3 | C1313CH15D3N4O3 • C10H11N3O3S C 13 | TRC. [online] Available at: https://www.trc-canada.com/product-detail/?C725304 [Accessed 18 Jun. 2023]. Balasegaram, M., Kolb, P., McKew, J., Menon, J., Olliaro, P., Sablinski, T., Thomas, Z., Todd, M.H., Torreele, E. and Wilbanks, J. (2017). An open source pharma roadmap. PLOS Medicine, 14(4), p.e1002276. doi:https://doi.org/10.1371/journal.pmed.1002276. Develoux, M. (2022). Epidemiologic Aspects of Mycetoma in Africa. Journal of Fungi, [online] 8(12), p.1258. doi:https://doi.org/10.3390/jof8121258. dndi.org. (2017). MycetOS | DNDi. [online] Available at: https://dndi.org/research-development/portfolio/mycetos/ [Accessed 18 Jun. 2023]. Eumycetoma (Fungal Mycetoma) Treatment & Management: Medical Care, Surgical Care, Long-Term Monitoring. (2019). eMedicine. [online] Available at: https://emedicine.medscape.com/article/1090738treatment. Fahal, A.H., Ahmed, K.O., Saeed, A.A., Elkhawad, A.O. and Bakhiet, S.M. (2022). Why the mycetoma patients are still neglected. PLOS Neglected Tropical Diseases, 16(12), p.e0010945. doi:https://doi.org/10.1371/journal.pntd.0010945. Farouk Elsadek, M., Mohamed Ahmed, B. and Fawzi Farahat, M. (2021). An Overview on Synthetic 2Aminothiazole-Based Compounds Associated with Four Biological Activities. Molecules, [online] 26(5), p.1449. doi:https://doi.org/10.3390/molecules26051449. Gentles, R.G., Grant-Young, K., Hu, S., Huang, Y., Poss, M.A., Andres, C., Fiedler, T., Knox, R., Lodge, N., Weaver, C.D. and Harden, D.G. (2008). Initial SAR studies on apamin-displacing 2-aminothiazole blockers of calcium-activated small conductance potassium channels. Bioorganic & Medicinal Chemistry Letters, [online] 18(19), pp.5316–5319. doi:https://doi.org/10.1016/j.bmcl.2008.08.023. GitHub. (n.d.). Mycetoma Monthly Meeting 22nd Nov 2022 · Issue #83 · OpenSourceMycetoma/Series-1Fenarimols. [online] Available at: https://github.com/OpenSourceMycetoma/Series-1Fenarimols/issues/83 [Accessed 18 Jun. 2023]. GitHub. (2021). Series-2-Aminothiazoles. [online] Available at: https://github.com/OpenSourceMycetoma/Series-2Aminothiazoles [Accessed 18 Jun. 2023]. Lim, W., Melse, Y., Konings, M., Duong, H., Eadie, K., Fahal, A., Laleu, B., Perry, B., Todd, M., Ioset, J.-R. and Van De Sande, W. (2019). Using the MycetOS approach to pinpoint chemical properties of fenarimols for in vivo efficacy in Madurella mycetomatis mycetoma. [online] Available at: https://dndi.org/wpcontent/uploads/2019/01/WLim_MycetOSApproach_Sixt

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hInternationalConferenceMycetoma_2018.pdf [Accessed 18 Jun. 2023]. Lim, W., Verbon, A. and van de Sande, W. (2022). Identifying novel drugs with new modes of action for neglected tropical fungal skin diseases (fungal skinNTDs) using an Open Source Drug discovery approach. Expert Opinion on Drug Discovery. doi:https://doi.org/10.1080/17460441.2022.2080195. Medicines for Malaria Venture. (2019). MMV and DNDi make 400 compounds available to stimulate research into new drugs for pandemic diseases. [online] Available at: https://www.mmv.org/newsroom/news-resourcessearch/mmv-and-dndi-make-400-compounds-availablestimulate-research-new [Accessed 18 Jun. 2023]. n.a., n.a. (2023). 2-amino-4-metylpyridine. [online] 2Amino-4-methylpyridine. Available at: https://www.sigmaaldrich.com/AU/en/product/aldrich/123 080 [Accessed 3 Mar. 2023]. PubChem (n.d.). Amikacin sulfate. [online] pubchem.ncbi.nlm.nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Amikacinsulfate. PubChem (n.d.). Azoxystrobin. [online] pubchem.ncbi.nlm.nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Azoxystrobi n. PubChem (n.d.). Carbendazim. [online] pubchem.ncbi.nlm.nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Carbendazi m#section=Animal-Concentrations [Accessed 18 Jun. 2023]. PubChem (n.d.). Fenbendazole. [online] pubchem.ncbi.nlm.nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Fenbendazol e. van de Sande, W.W.J. (2013). Global Burden of Human Mycetoma: A Systematic Review and Meta-analysis. PLoS Neglected Tropical Diseases, 7(11), p.e2550. doi:https://doi.org/10.1371/journal.pntd.0002550. Watson, A.K., Kepplinger, B., Bakhiet, S.M., Mhmoud, N.A., Chapman, J., Allenby, N.E., Mickiewicz, K., Goodfellow, M., Fahal, A.H. and Errington, J. (2022). Systematic whole-genome sequencing reveals an unexpected diversity among actinomycetoma pathogens and provides insights into their antibacterial susceptibilities. PLOS Neglected Tropical Diseases, 16(7), p.e0010128. doi:https://doi.org/10.1371/journal.pntd.0010128. www.cdc.gov. (2020). Mycetoma | Fungal Diseases | CDC. [online] Available at: https://www.cdc.gov/fungal/diseases/mycetoma/index.htm l#:~:text=The%20treatment%20for%20mycetoma%20incl udes. www.rstmh.org. (n.d.). A new treatment for mycetoma? | RSTMH. [online] Available at: https://www.rstmh.org/news-blog/blog/a-new-treatmentfor-mycetoma [Accessed 18 Jun. 2023]. www.who.int. (n.d.). Mycetoma. [online] Available at: https://www.who.int/news-room/factsheets/detail/mycetoma.

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Synthesis of a new trifluoromethyl analogue of MMV006357 for the treatment of Mycetoma Jack Jeffress Barker College Purpose: This research paper aims to describe the synthesis of a new 2-aminothiazole analogue as a new potential treatment for mycetoma skin infections. The new analogue was successfully synthesised following the synthesis pathway developed by the open source mycetoma consortium, comprising research scientists from all over the world. Design/methodology/approach: The proposed methodology involves the synthesis of a specific analogue following three steps. In step 1, from benzoyl chloride an addition and substitution reaction was used to form a benzamide intermediate, which after hydrolysis and the formation of a thiazole ring, afforded the desired analogue. Findings: The product was analysed using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectroscopy and showed that the product was pure and reflected the desired product with an overall yield of 59%. Research limitations/implications: Time constraints posed limitations to this project as we were unable to test the analogues effectiveness against the fungus Madurella mycetomatis. Practical implications: The practical implications of this project arise from the new structure activity information that biological testing will provide. With this new insight, the potential of this new analogue can be explored as a new drug candidate. Originality/value: To date, there has not yet been any trifluoromethyl analogues synthesised prior to this report, and the synthesis of these new analogues will expand our understanding of this structure as a potential drug candidate. Keywords: Mycetoma, 2-aminothiazole. Paper type: Research Paper

Literature Review Mycetoma is an inflammatory and chronic infectious disease which causes large subcutaneous masses that discharges pus containing grains, usually affecting the lower limbs. This disease can either be caused by bacteria (actinomycetoma) or Fungi (eumycetoma). Mycetoma is most commonly caused by the fungus Madurella mycetomatis (M. mycetomatis) (Fahal et al., 2018). M. mycetomatis is the most common pathogen which causes the disease Eumycetoma. M. mycetomatis can be found in the soil and water and these pathogens then enter the body through wounds or minor skin injuries (Hashizume et al., 2022). Mycetoma does not spread between people. Treatment of Actinomycetoma is possible, with success rates of up to 90%. While Eumycetoma can sometimes be treated with a combination of prolonged medication and surgery with it being less than 27% effective. (Beer et al., 2018). Mycetoma is not a reportable disease, and as such there is no official incidence and prevalence data. In 2013-2017 a rough estimate of the global burden of disease was obtained through a literature search. A total of 17,076 cases were recorded in this study,

however the actual number of cases is likely much higher (van de Sande et al., 2018). Mycetoma has a worldwide distribution, but its prevalence is unevenly distributed. It is most commonly found in tropical and subtropical regions, with Africa having the highest prevalence (Figure 1). Studies have shown that in some parts of central Africa, M. Mycetomatis accounts for more than 70% of all mycetoma infections. (Ahmed et al., 2004). Treatment options for mycetoma are generally limited. There has been a need for new drugs to be used as treatment as, the most effective and commonly used drug against mycetoma is ketoconazole (Fahal, 2010). However, this drug has recently been restricted by the United States Food and Drug Administration (FDA), European Medicines Agency (EMA) and the government of Sudan. (Lim et al., 2018). This is due to it causing potentially fatal side effects such as liver injury and adrenal gland problems. This has caused a dilemma as there are other drugs able to treat the disease to

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Figure 1: Geographical distribution of some eumycetoma agents Source: (Ahmed et al., 2004)

some extent, but these are too expensive to be used as a standard treatment. Therefore, there is urgent need to find new drugs which are effective, safe and costefficient that can combat Mycetoma. (Lim et al., 2018). In 2016 the World Health Organisation (WHO) identified mycetoma as a neglected tropical disease (NTD) and like most neglected diseases, it lacked the many necessary provisions and support required to implement successful treatments. However, the organisation Medicines for Malaria Venture (MMV) released the Open Access Boxes initiative in order to reduce the burden of neglected diseases. In this initiative over 1600 compounds were tested for activity against M. mycetomatis (Lim et al., 2022). As a result, the effectiveness of several drugs from the Open Access Boxes were tested to find new drugs that can be used as a treatment against this disease by members of the global scientific community. From the open access boxes initiatives the Stasis and Pathogen boxes 800 compounds were screened for their inhibitability in vitro growth of M. mycetomatis. To begin, 800 compounds from the Pathogen or Stasis Boxes were screened in vitro at a concentration of 100 micromoles per litre (μM) (Lim et al., 2018).

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This is a moderate concentration which gives an indication of the potential of the compounds as inhibitors of fungal growth. For the 215 compounds which were inhibited at 100 μM, their IC50s were determined. IC50 is the concentration of compound in μM where 50% reduction in fungal growth was obtained. In total, 13 compounds had an IC50 of 5 μM or lower. (Lim et al., 2022). In vitro screening revealed MMV006357 (a 2- aminothiazole compound) was one of the most potent exploratory compounds against mycetoma.

Figure 2: MMV006357 compound's chemical structure Source: (Lim et al., 2018).

2-aminothiazole is a five membered heterocyclic unit with sulphur and nitrogen and a exocyclic amine (Jakopin et al., 2020). The 2- aminothiazole class of compounds inhibits ion channels and electron transfers (Kesicki et al., 2016). To be more specific, 2-aminothiazole compound are generally potent

Scientific Research in School Volume 5 Issue 1 2023 calcium-activated potassium (KCa) channel inhibitor. In the KCa channel, this protein regulates calcium ions that bind to calmodulin, a protein in the domain of the KCa channel. A higher occupancy leads to an opening of the channel. This opening of the channel leads to hyperpolarization of the plasma membrane. Thereby affecting the rate and pattern of neuron firing, which will kill the organism and therefore the disease can’t progress. (Gentles et al., 2008). In the literature there have been various reports on the usefulness of 2-aminothiazole containing compounds to treat various diseases. These compounds have been shown to act as anticancer, antifungal, antioxidant, antimicrobial and anti-inflammatory agents (Farouk et al., 2001). Examples of drugs which contain this structural feature are Famotidine, Cefdinir and Meloxcam, used to treat stomach and intestine ulcers, infections caused by bacteria and relieves pain and inflammation caused by osteoarthritis, respectively. (Das, 2016).

Figure 4: Chemical structure of Cefdinir Source: (Guay, 2002)

Figure 5: Chemical structure of Famotidine Source: (Basavaiah et al., 2011)

Figure 3: Chemical structure of Meloxicam Source: (PubChem, 2023).

Therefore, in the hopes of adding to the existing body of research about the structure-activity relationships of the 2-aminothiazole compounds, we plan to synthesise an analogue of MMV006357 as part of the Open Source Mycetoma research consortium. Eventually, if this synthesis is successful, the compound can be tested against the M. Mycetomatis fungus. The synthetic pathway for the 2aminothiazole analogue developed by the opensource consortium will be the focus of this methodology (Figure 6).

Figure 6: Original reaction pathway

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Figure 7: Bromoketones previously used to developed analogues of 2-aminothiazole.

Figure 7 shows a number of different bromoketones that have already been used to develop different analogues by the open source consortium on GitHub (Scroggie, 2022) Previously, the analogues made only varied at the bromoketone and therefore only changed the structure the left aromatic ring. However, in my lead compound (figure 9) the left aromatic ring is unchanged and instead the structure of the aromatic ring on the right is altered by changing a methyl group to a trifluoromethyl substituent.

Figure 9: Lead compound (1)

The synthesis of the target analogue in this procedure will follow the same synthesis pathway (figure 6), however, in step 1 the pyridine used is 5(trifluromethyl)pyridin-2-amine, then in step 3 the bromoketone will be replace with 2-bromo-1(pyridine-1-yl)-ethan-1-one to reflect the desired trifluoromethyl analogue, 4-(pyridin-2-ly)-N-(4(trifluoromethyl)pyridin-2-ly)thiazole-2-amine (Figure 10).

Figure 8: Original compound

Figure 10: Reaction pathway used to synthesis the trifluoromethyl analogue of MMV00635

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Scientific Research Question Can the trifluoromethyl analogue (1) of MMV006357 be synthesised in a school laboratory, and its effectiveness as an anti-fungal agent against M. Mycetomatis be tested?

Scientific Hypothesis That the trifluoromethyl analogue of MMV00635 can be synthesized and its effectiveness as an anti-fungal agent against can be tested.

Methodology 1H spectra were recorded at 300 K using a Bruker Avance DRX500 NMR spectrometer in deuterated solvents. Residual acetone (δ 2.05) was used as internal reference for 1H NMR spectra. The data is reported as chemical shift (δH ppm), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and assignment. Atom labels on structures are to illustrate 1H NMR spectral assignments and do not necessarily correspond to the IUPAC names given. Analytical thin layer chromatography was performed with Merck Kieselgel 60 F254 (0.2 mm) pre-coated aluminium sheets, and visualisation was achieved by inspection under UV light. Throughout the reaction process Thin Layer Chromatography (TLC) was conducted to gauge the progress of the reaction and determine the point of completion. TLC analysis was conducted with pure DCM. The solvent peak for acetone (δ 29.84) was used as an internal reference for 13C NMR spectra. Mass spectra were recorded by the Mass Spectrometry Unit of the School of Chemistry, The University of Sydney, Sydney. The molecular ion [M + H+], [M + Na+] or [M - H+] is listed. Step 1: Synthesis of N-((4-trifluromethyl)pyridin2yl)carbamothioyl)Benzamide

In a round bottom flask with a reflux condenser, Benzoyl chloride (1.9g, 13.602 mmol, 1 equiv.) was added to Potassium thiocyanate (1.4g, 14.777 mmol, 1 equiv.) in 35 mL of acetone. The reaction was heated to a reflux (60 °C) and stirred for 20 minutes. The reaction was cooled slightly and 5(trifluromethyl)pyridin-2-amine (2.0g, 12.337 mmol. 1 equiv) was added. The reaction was heated to reflux (60 °c) and stirred for 30mins. The reaction was poured over ice water (100mL) to cool the reaction down and stirred for a further 10mins. The precipitate was collected by using vacuum filtration and washed with 10 ml of ice water to afford N-((4trifluromethyl)pyridin2yl)carbamothioyl)Benzamide as a yellow solid (2.389 g, 60%).TLC was conducted with 100% DCM as the eluent. The crude N-((4trifluromethyl)pyridin2yl)carbamothioyl)Benzamide (3) was used without purification in the second step of the synthesis. Step 2: Synthesis of 1-(4(trifluoromethyl)pryridin-2-yl)thiourea

Figure 12: 1-(4-(trifluoromethyl)pryridin-2-yl)thiourea (4)

In a round bottom flask with a reflux condenser, N((4-trifluoromethyl)pyridin2yl)carbamothioyl)Benzamide (2.389 g, 7.344 mmol, 1 equiv.) was added to a 2.5M solution of NaOH (30mL) and was heated to reflux for 15mins (80 °𝑐𝑐𝑐𝑐). The reaction was then cooled. The pH was adjusted to 4 using 1M of HCl. After that the pH was adjusted to 8 using a saturated solution of Na2OH3 (sodium carbonate) to precipitate the product. The precipitate was collected using vacuum filtration and rinsed with 25mL of cold water. The precipitate was left to dry overnight to afford 1-(4-(trifluoromethyl)pryridin-2yl)thiourea as a brown solid (0.702g, 43%). TLC was conducted with 100% DCM as the eluent. The crude 1-(4-(trifluoromethyl)pryridin-2-yl)thiourea (4) was used without purification in the third step of the synthesis.

Figure 11: N-((4-trifluoromethyl)pyridin2yl)carbamothioyl)Benzamide (3)

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Scientific Research in School Volume 5 Issue 1 2023 Step 3: Synthesis of 4-(pyridin-2-ly)-N-(4(trifluoromethyl)pyridin-2-ly)thiazole-2-amine

Figure 13: 4-(pyridin-2-ly)-N-(4(trifluoromethyl)pyridin-2-ly)thiazole-2-amine (1)

In a round bottom flask with a reflux condenser, 1(4-(trifluoromethyl)pryridin-2-yl)thiourea (0.702 g, 3.17mmol, 1 equiv.) was added to 2-bromo-1(pyridin-2-yl)ethan-1-one (0.721g, 1 equiv.) in ethanol (40mL). The reaction was heated to reflux (80°𝑐𝑐𝑐𝑐) and stirred to completion using TLC. The reaction was then cooled down by pouring ice water (25mL) over the mixture. The pH was adjusted to 8 using a saturated solution of CO3. The precipitate was then collected using vacuum filtration and rinsed with cold water (2 x 10mL) to afford 4-(pyridin-2-ly)-N(4-(trifluoromethyl)pyridin-2-ly)thiazole-2-amine (0.746 g, 73% yield). The crude 4-(pyridin-2-ly)-N(4-(trifluoromethyl)pyridin-2-ly)thiazole-2-amine (1) was not purified.

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Results Step 1: Synthesis of N-((4-trifluromethyl)pyridin-2yl)carbamothioyl)Benzamide

Figure 14: 1H NMR spectra after step 1

1H NMR (500 MHz, acetone-d6): δ 13.60 (1H, br s, NH), 10.60 (1H, br s, NH), 8.80 (1H, m, Ar-H), 8.27 (1H, dd, J = 2.77, 8.91 Hz, Ar-H), 8.11 (2H, dd, J = 0.96, 8.50 Hz), Ar-H), 7.44-7.71(1H, m, AR-H), 7.61(3H,t, J= 7.93 Hz).

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Figure 15: 13C NMR spectra after step 1

C NMR (100 MHz, acetone-d6): δ 179.4 (C), 155.6 (C), 146.6 (C), 136.4 (CH), 134.4 (CH), 129.7 (CH), 129.3 (CH), 129.3 (CH), 125.6 (C), 123.6 (C), 115.6 (CH). MS (+ESI): m/z 324.04 (M – H+), 348.04 (M + Na).

Figure 16: Mass spectra after step 1

Figure 17: TLC after step 1

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Step 2: Synthesis of 1-(4-(trifluoromethyl)pryridin-2-yl)thiourea

Figure 18: 1H NMR spectra after step 2

1H NMR (500 MHz, acetone-d6): δ 10.64 (1H, br s, NH), 9.88 (1H, br s, NH), 8.66 (1H, m, Ar-H), 8.26 (1H, br s, NH), 8.13 (1H, dd, J = 2.43, 9.02 Hz, Ar-H), 7.43 (1H, d, J = 7.97 Hz), Ar-H).

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Figure 19: 13C NMR spectra after step 2

C NMR (100 MHz, acetone-d6): δ 182.3 (C), 155.9 (C), 144.1 (CH), 135.9 (CH), 125.1 (C), 112.6 (CH). MS (+ESI): m/z 220.02 (M – H+), 222.03 (M + H+). 13

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Figure 20: Mass spectra after step 2

Figure 21: TLC after step 2

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Scientific Research in School Volume 5 Issue 1 2023 Step 3: Synthesis of 4-(pyridin-2-ly)-N-(4-(trifluoromethyl)pyridin-2-ly)thiazole-2-amine

Figure 22: 1H NMR spectra after step 3

1H NMR (500 MHz, acetone-d6): δ 10.85 (1H, br s, NH), 8.70 (1H, s, Ar-H), 8.59 (1H, d, Ar-H), 8.03 (2H, m, Ar-H), 7.81 (1H, dt, J = 1.6, 7.8 Hz, Ar-H), 7.77 (1H, s, Ar-H), 7.43 (1H, d, J = 8.8 Hz, Ar-H), 7.28 (1H, dd, J = 5.0, 7.6 Hz, Ar-H).

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Figure 23: 13C NMR spectra after step 2

C NMR (100 MHz, acetone-d6): δ 159.6 (C), 155.5 (C), 153.8 (CH), 150.8 (CH), 150.3 (CH), 145.4 (C), 137.6 (CH), 135.7 (CH), 126.5 (C), 124.3 (C), 123.3 (CH), 121.1 (CH), 111.6 (CH), 111.4 (CH). MS (+ESI): m/z 321.04 (M – H+), 323.06 (M + H+), 345.04 (M + Na).

Figure 24: Mass spectra after step 3

Figure 25: TLC after step 3

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Figure 26: Reaction of Benzoyl chloride (2) to form N-((4-trifluoromethyl)pyridin-2yl)carbamothioyl)Benzamide (4)

Discussion Step 1: Synthesis of N-((4-trifluromethyl)pyridin2yl)carbamothioyl)Benzamide Step 1 was used to afford compound 4 from a substitution reaction between compound 2 and Potassium thiocyanate to form compound 3 followed by an addition reaction between compound 3 and 5(trifluromethyl)pyridin-2-amine. More specifically the C-Cl bond of benzoyl chloride was cleaved by potassium thiocyanate’s nitrogen atom to facilitate this substitution reaction (figure 27).

Figure 27: Mechanism behind the formation of compound 3

In the next part of this step the nitrogen bond breaks and attacks the carbon of the isothiocyanate group, the pair of electrons then form a bond with a hydrogen to form compound 4 (figure 28).

Figure 28: Mechanism behind formation of compound 4

The yield obtained for this step was 60% which is a significantly low yield which is likely due to a lack of reaction optimisation due to time constraints and the limited equipment available. Another factor that may have attributed to such a yield may simple be due to structural differences between compound 4 and the starting material (2), as the reaction may have acted differently with the replacement of the CH3 group with a CF3 group. Therefore, in order to improve the yield for later experiments it is important to optimise reaction conditions such as reaction times and temperature as well as an improved isolation step that minimises loss of products.

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The TLC (figure 17) indicated that there was no or very little residual starting material and a more polar product formed, which corresponds to compound 4. In the 1H NMR spectrum (figure 14) the singlet at 13.60ppm and the singlet at 10.60ppm were assigned to the 1H NH protons. This was expected as the chemical shift should be much more down field than the other signals. The signals between 8.10ppm and 7.44ppm were assigned to the aromatic protons on the two rings as these were expected in this region. The two signals at 8.80ppm and 8.27ppm were assigned to the three protons on the pyridine ring as they are relatively more downfield as the CF3 group is very electron withdrawing which causes more deshielding of protons in those environments. These three protons were expected as doublet of doublet signals which corresponds to figure 14 at 8.80ppm as there is a doublet of doublet and at 8.27ppm there is an overlapping doublet of doublet. The mass spectroscopy (figure 16) indicated the molecular mass to be 324.04 (M – H+) and 348.04 (M + Na) which corresponds to its molecular mass of 325.31. The 13C NMR (figure 15) displays 11 peaks, which matches the number of carbon environments in compound 4. The peak which is significantly more downfield than the other signals at 179.4ppm was assigned to the Carbon double bond Sulfur (C=S) as it is adjacent to a double bond Oxygen (C=O) and a nitrogen which will cause deshielding of that carbon environment. Overall, in combination of all results, it can be determined that the synthesis of step 1 was successful and the desired compound 4 was synthesised. Step 2: Synthesis of 1-(4(trifluoromethyl)pryridin-2-yl)thiourea

Figure 29: Reaction of N-((4-trifluoromethyl)pyridin2yl)carbamothioyl)Benzamide (4) to form of 1-(4(trifluoromethyl)pryridin-2-yl)thiourea (5)

Scientific Research in School Volume 5 Issue 1 2023 In Step 2 of the synthesis, a hydrolysis reaction performed under reflux was used to form compound 5 from compound 4. In this process the Hydroxide ion forms a water molecule with the oxygen which causes the aromatic ring to detach and allow a hydrogen atom to attach to the nitrogen (figure 30).

Figure 30: Figure 30: Mechanism behind the formation of compound 5.

The yield of this step was extremely low with 43%. This low yield is most likely due to the reaction not going to completion. Another difficulty faced during this step was a faulty pH meter. While using this equipment the pH meter was continuously needing to be recalibrated, therefore making the measurement very unreliable when adjusting the pH of the reaction mixture, therefore causing less precipitate to form and residual Sodium hydroxide. Future improvements to increase the yield of this step include optimising reaction time and temperature and a more precise pH meter to control and regulate the pH more accurately. The TLC indicates the product is pure as there is very little or no residual starting material found in the product. The TLC corresponds to compound 5 as it is less polar than compound 4 (figure 21). In the 1H NMR spectrum (figure 18), the two most downfield signals at 10.64ppm 9.88ppm and 8.26ppm were assigned to the 1H NH protons as their chemical shift was expected to be very downfield in comparison to other signals. The other signal in this region of the

spectrum, at 8.66ppm was assigned to the aromatic proton as it is adjacent the CF3 group is which is electronegative and causes more deshielding of the protons. The remaining peaks were assigned to aromatic protons as they are more upfield relative to the other signals. The 13C NMR (figure 19) indicates that there are 6 carbon environments as there are 6 peaks which corresponds to compound 5. Similar to step 1, the peak most downfield at 182.3ppm was assigned to the Carbon double bond Sulfur (C=S) as it is adjacent to a double bond Oxygen (C=O) and a nitrogen which causes deshielding of the carbon environment. The mass spectroscopy (figure 20) displays the parent peak to be 220.02 (M – H+) and 222.03 (M + H+), corresponding to its molecular mass of 221.20. Therefore, despite this steps low yield it is clear that the desired product compound 5 was synthesised. Step 3: Synthesis of 4-(pyridin-2-ly)-N-(4(trifluoromethyl)pyridin-2-ly)thiazole-2-amine

Figure 31: Reaction of 1-(4-(trifluoromethyl)pryridin-2yl)thiourea (5) to form 4-(pyridin-2-ly)-N-(4(trifluoromethyl)pyridin-2-ly)thiazole-2-amine (1)

In step 3 of the synthesis a 2-aminothiazole ring formation is involved to form compound 1. In this process the sulfur reacts with the carbon as the bromine and carbon bond (C-Br) is weak, causing the loss of a bromide ion. A hydrogen is then picked up which allows water to form due to it being an energetically favourable reaction. Thus, leading to the formation of compound 1 (figure 32).

Figure 32: Mechanism behind the formation of compound 1

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Scientific Research in School Volume 5 Issue 1 2023 Despite the yield of step 3 being a significant improvement from step 1 and 2 with 73% yield of compound 1, the loss of product is most likely attributed to the reaction not going to completion or the product not precipitating with Na2CO3 and instead collected as filtrate in the vacuum filtration. The appearance of a less polar product than the starting material in the TLC (figure 25) indicated corresponds to compound 1 due to the formation of a 2-aminothiazole ring. In the 1H NMR (figure 22), similar to step 1 and 2 the most downfield signal at 10.85ppm was assigned to the to the 1H NH proton. The signals between 8.70ppm and 7.28ppm were assigned as aromatic protons on the 3 rings as these are expected to be in this region. The 13C NMR (figure 23) indicated 14 carbon environment which is representative of compound 1. The mass spectroscopy (figure 24) indicated the molecular mass to be 321.04 (M – H+), 323.06 (M + H+), 345.04 (M + Na) which corresponds to it molecular mass of 322.31. By observing the results, it is clear that during step the desired product, compound 1 was synthesised. Future research The successful synthesis of the trifluoromethyl analogue of MMV006357 provides a greater understanding of the synthesis process developed on GitHub (Scroggie, 2022). To improve this scientific report there are particular areas that need to be focused on. One such improvement to this report is to further optimise the synthesis in order to obtain a higher yield. Also, due to time restraints, this report was unable to determine the effectiveness of the 2aminothiazole analogue produced. Therefore the changes that may have occurred to the compounds potency against M. Mycetomatis due to the change in the CH3 group to CF3 is still undetermined. Further research could investigate the effect of replacing the CH3 with a CF3 group and even if compound 1 is not particularly potent against M. Mycetomatis, it will help to develop the future research of other analogues which may exhibit higher and more effective inhibition of M. Mycetomatis.

Conclusion The research conducted in this scientific report resulted in the successful synthesis of the trifluoromethyl analogue (1) of MMV006357 using the synthesis pathway developed by the scientific community on GitHub (Scroggie, 2022). After each step, to confirm that the correct product was synthesised an 1H NMR, mass spectroscopy, 13C NMR and TLC was obtained and analysed. However,

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due to time restraints the analogue was unable to be sent to Sydney University for biological testing on M. Mycetomatis to obtain its enzyme inhibition data. The reaction pathway should also be further developed to produce a greater yield for future research. In doing so more data can be collected on the inhibition and interaction of the 2-aminothiazole groups on M. Mycetomatis, with the purpose of developing a new treatment for Eumycetoma.

Acknowledgements I would like to thank Dr Katie Terrett for guiding me throughout this project and for explaining the concepts and intricacies behind this research. I would also like to thank the collaborators at Sydney University for performing the NMR’s and mass spectroscopy after each step of the synthesis.

References Ahmed, A.O., van Leeuwen, W., Fahal, A., van de Sande, W., Verbrugh, H. and van Belkum, A., 2004. Mycetoma caused by Madurella mycetomatis: a neglected infectious burden. The Lancet infectious diseases, 4(9), pp.566-574. Basavaiah, K. and Zenita, O., 2011. Spectrophotometric determination of famotidine using sulphonphthalein dyes. Química Nova, 34, pp.735-742. Beer, K.D., Blaney, D.D., Kadzik, M., Asiedu, K.B., Shieh, W.J., Bower, W., Jackson, B.R., Walke, H. and Chiller, T., 2018. A call to action for mycetoma. Current Fungal Infection Reports, 12, pp.99-104. Das, D., Sikdar, P. and Bairagi, M., 2016. Recent developments of 2-aminothiazoles in medicinal chemistry. European Journal of Medicinal Chemistry, 109, pp.89-98. Fahal, A.H., 2010. Management of mycetoma. Expert Review of Dermatology, 5(1), pp.87-93. Fahal, A.H., Suliman, S.H. and Hay, R., 2018. Mycetoma: the spectrum of clinical presentation. Tropical medicine and infectious disease, 3(3), p.97. Farouk Elsadek, M., Mohamed Ahmed, B. and Fawzi Farahat, M., 2021. An overview on synthetic 2aminothiazole-based compounds associated with four biological activities. Molecules, 26(5), p.1449. Gentles, R.G., Grant-Young, K., Hu, S., Huang, Y., Poss, M.A., Andres, C., Fiedler, T., Knox, R., Lodge, N., Weaver, C.D. and Harden, D.G., 2008. Initial SAR studies on apamin-displacing 2-aminothiazole blockers of calcium-activated small conductance potassium channels. Bioorganic & medicinal chemistry letters, 18(19), pp.53165319. Guay, D.R., 2002. Cefdinir: an advanced-generation, broad-spectrum oral cephalosporin. Clinical therapeutics, 24(4), pp.473-489. Hashizume, H., Taga, S., Sakata, M.K., Taha, M.H.M., Siddig, E.E., Minamoto, T., Fahal, A.H. and Kaneko, S., 2022. Detection of multiple mycetoma pathogens using

Scientific Research in School Volume 5 Issue 1 2023 fungal metabarcoding analysis of soil DNA in an endemic area of Sudan. PLOS Neglected Tropical Diseases, 16(3), p.e0010274. Jakopin, Ž., 2020. 2-aminothiazoles in drug discovery: Privileged structures or toxicophores?. ChemicoBiological Interactions, 330, p.109244. Kesicki, E.A., Bailey, M.A., Ovechkina, Y., Early, J.V., Alling, T., Bowman, J., Zuniga, E.S., Dalai, S., Kumar, N., Masquelin, T. and Hipskind, P.A., 2016. Synthesis and evaluation of the 2-aminothiazoles as anti-tubercular agents. PLoS One, 11(5), p.e0155209. Lim, W., Melse, Y., Konings, M., Phat Duong, H., Eadie, K., Laleu, B., Perry, B., Todd, M.H., Ioset, J.R. and van de Sande, W.W., 2018. Addressing the most neglected diseases through an open research model: The discovery of fenarimols as novel drug candidates for eumycetoma. PLoS neglected tropical diseases, 12(4), p.e0006437. Lim, W., Verbon, A. and van de Sande, W., 2022. Identifying novel drugs with new modes of action for neglected tropical fungal skin diseases (fungal skinNTDs) using an Open Source Drug discovery approach. Expert Opinion on Drug Discovery, 17(6), pp.641-659. PubChem 2023, Meloxicam, National Library of Medicine, PubChem, viewed 18 June 2023, <https://pubchem.ncbi.nlm.nih.gov/compound/meloxicam >. Scroggie , K. (2022). General Synthesis Route for the Synthesis of 2-aminothiazoles. [online] GitHub. Available at: https://github.com/OpenSourceMycetoma/Series-2Aminothiazoles/wiki/General-Synthesis-Route [Accessed 17 Jun. 2023]. van de Sande, W., Fahal, A., Ahmed, S.A., Serrano, J.A., Bonifaz, A., Zijlstra, E. and Eumycetoma Working Group, 2018. Closing the mycetoma knowledge gap. Medical mycology, 56(suppl_1), pp.S153-S164.

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Synthesis of structures with the purpose of creating new analogues of Lopinavir Jonah Mills Barker College Purpose: This research paper aims to synthesise trifluoromethyl phenoxy acetic acid and phenoxy acetic acid in a school laboratory in order to create new analogues for Lopinavir. Findings: This paper describes the successful synthesis of 2-phenoxy acetic acid and the failed synthesis of 2-(2-(trifluoromethyl)phenoxy) acetic acid both being new analogues of Lopinavir. Research implications: A higher yield and successful synthesis of 2-(2-(trifluoromethyl)phenoxy) acetic acid may be achieved with a change in methodology and better purifying techniques. Originality/value: The present studies highlight the importance of synthesis and the potential for different analogues of Lopinavir to be a viable antiviral option against SARS-CoV-2. Paper type: Research paper

Literature Review The global coronavirus disease 2019 (COVID-19) epidemic abruptly presented itself as a significant threat to public health worldwide. A large number of cases of pneumonia were identified in Wuhan, a city in the Hubei Providence, China. These cases were connected to COVID-19, a highly contagious respiratory illness caused by Severe Acute Respiratory Syndrome Coronavirus – 2 (SARS CoV2) (Health, 2023). COVID-19 is highly contagious, as it can be spread up to 48 hours before symptoms begin. Being unaware of having the virus, yet still being able to spread it, paired with being 10 times more contagious than that of the typical seasonal flu, has resulted in over 200 countries around the globe being affected by the virus. Individuals have been affected as many with COVID-19 have been left with ongoing immune and respiratory compromises, long COVID, and it has even resulted in death. Those individuals who are most affected have been regarded as at-risk individuals. These include people over 50 with additional risk factors, including obesity, neurological disease, liver or kidney diseases, diabetes, respiratory-related issues, and those with active cancer. It has also affected economic growth as Australia experienced two significant falls in GDP during the height of restrictions in Australia (Economic Gains and Losses over the COVID-19 Pandemic, 2022). WHO declared the end of the global health emergency in May 2023 (Ghebreyesus, 2019), yet it is an ongoing pandemic in Australia and many other countries today.

Figure 1: Image of SARS CoV-2 Cell. Source: (News-Medical, 2022)

The coronavirus entry into host cells is directed by the spike glycoproteins or (S protein), which plays an essential role in the attachment of the virus, fusion and entry into the host cells. SARS CoV-2 depends on these spike glycoproteins (Figure 1) for the virus entry and thus has been the main antiviral target. The S protein comprises 2 sections of the S1 and S2 subunit (Figure 2). S1 is responsible for binding to the receptor of the host cell. S2, on the other hand, is to fuse the virus membranes to host cells. The site at the border of these 2 subunits is called the S1/S2 protease binding site (Wang et al., 2020). For all coronaviruses, protease binds to the site and is critical for fusing the host cells to the virus membrane, which results in an irreversible conformational change.

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Scientific Research in School Volume 5 Issue 1 2023 replication, both only are effective against the specific side effects of SARS CoV-2, compared to Lopinavir which targets SARS CoV-2 in its viral life cycle and thus preventing further infection.

Figure 2: S Protein subunits S1 and S2. Source: (Mansbach et al., 2021)

As this is only a recent discovery, work is underway to investigate the structure-activity relationship between Lopinavir and the protease Mpro enzyme. Due to the relevancy of coronavirus, many other drugs have been studied, synthesised, and tested; however, they have yet to emerge as viable options for controlling the pandemic. The 4 main drugs which went through stage 3-4 clinical trials included Hydroxychloroquine (HCQ), Baricitinib, Dexamethasone and Remdesivir (US National Library of Medicine, 2020). HCQ is an antimalarial drug that has a potential blocker of viral maturation. After these clinical trials, the short- and long-term side effects were illuminated, the major being polymorphic ventricular tachycardia (Drożdżal et al., 2021). Baricitinib is an anti-inflammatory, and after 113 patients were tested with this drug, it showed a significantly lower fatality window. Those who wrote the study concluded that doctors should give this drug to those diagnosed with moderate to severe coronavirus cases. Dexamethasone, a steroid antiinflammatory, and Remdesivir, which blocks viral

Figure 3: Structure of Lopinavir

Lopinavir, an antiretroviral peptide protease inhibitor, has predominately been paired with ritonavir, another protease inhibitor that is used against the human immunodeficiency virus (HIV) as it prevents HIV from continuing to reproduce within the body (Shuter & Chandwani, 2022a). To complete its role in the viral life cycle, HIV-1 protease must cleave structural and functional proteins into viral polypeptide strands (Apostolopoulos et al., 2021). Lopinavir is a potent inhibitor of HIV-1 protease as it prevents the completion of the infectious virions' life cycle and prevents subsequent cellular infection. More recent studies have shown that the SARS-CoV2 virus also relies on a protease enzyme, specifically Mpro, for reproduction. Many existing drugs have been screened against SARS-CoV-2 for their ability to inhibit protease Mpro. Lopinavir had the highest result with a 66.67% binding site similarity (Dayer et al., 2017), thus resulting in significant focus from researchers and scientists on its ability to combat SARS-CoV-2.

Figure 3: Binding Site Similarities to Protease Mpro

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Scientific Research in School Volume 5 Issue 1 2023 Lopinavir has been established as a potential drug against SARS-CoV-2 due to its potent ability to inhibit the enzyme protease. A lot of research has dug into why it can inhibit the enzyme and the possible parallels that can be drawn from Lopinavir against HIV and COVID-19. Studies into analogues seem to be limited, with many sources only analysing certain analogues of Lopinavir using various software, as opposed to the physical synthesis, which was suggested by (Rafi et al., 2020) as “we propose more studies, including synthesis and biological activities of these compounds (pg.18)”. When looking at the binding site capabilities of the analogues studied through software, many showed similar, if not worse, similarities to that of Lopinavir. Thus, there is a significant gap in research on the synthesis of analogues of Lopinavir. In an attempt to add to this investigation, I will be aiming to synthesise two structurally altered fragments of a small section of Lopinavir, highlighted in Figure 3, to work together with scientists at the University of Sydney to use these new fragments to synthesise new analogues of Lopinavir on the path to discovering new drugs for SARS CoV-2 with new modes of action. As the research on the synthesis of new Lopinavir analogues is limited, there is no precise method to follow. This has made it very difficult when looking into the synthesis of Compound 1: 2-phenoxy acetic acid and Compound 2: 2-(2(trifluoromethyl)phenoxy)acetic acid as there were no other studies on which I can base my method. However, to obtain the best results possible, a method of a structurally similar compound founded the basis of the methodology.

internal reference for 1H NMR spectra. The data is reported as chemical shift (δH ppm), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and assignment. Atom labels on structures are to illustrate 1H NMR spectral assignments and do not necessarily correspond to the IUPAC names given. The solvent peak for acetone (δ 29.84) was used as an internal reference for 13C NMR spectra. Mass spectra were recorded by the Mass Spectrometry Unit of the School of Chemistry, The University of Sydney, Sydney. The molecular ion [M - H+] is listed. Analytical thin layer chromatography was performed with Merck Kieselgel 60 F254 (0.2 mm) pre-coated aluminium sheets, and visualisation was achieved by inspection under UV light. Throughout the reaction process Thin Layer Chromatography (TLC) was conducted to gauge the progress of the reaction and determine the point of completion. TLC analysis was conducted with pure dichloromethane. Step 1: Synthesis of Compound 1

Figure 5: 2-phenoxyacetic acid

Can 2-phenoxyacetic acid and 2-(2-(trifluoromethyl) phenoxy) acetic acid be synthesised in a school laboratory and used for the synthesis of two new analogues of lopinavir?

Chloroacetic acid (5.20 g, 55mmol, 1 equiv.) was added to a round bottomed flask and 10 M sodium hydroxide solution in water was added (10 mL, 250mmol, 4.55 equiv.). Phenol (5.19 g, 55 mmol, 1 equiv) was added to the flask and the resulting reaction was heated under reflux for 24 hours. After this time, concentrated hydrochloric acid (approx. 15 mL) was added to neutralise the sodium hydroxide and cause the acid product to precipitate. After 3 days, crystals were observed in the flask, which were collected under vacuum filtration to afford the desired product as a white solid (4.50 g, 53.6 %).

Scientific Hypothesis

Step 2: Synthesis of Compound 2

Scientific Research Question

That 2-phenoxyacetic acid and 2-(2-(trifluoromethyl) phenoxy) acetic acid be synthesised in a school laboratory and used for the synthesis of two new analogues of lopinavir.

O O

CF3

Methodology

Figure 6: 2-(2-(trifluoromethyl)phenoxy)acetic acid

1H spectra were recorded at 300 K using a Bruker Avance DRX500 NMR spectrometer in deuterated solvents. Residual acetone (δ 2.05) was used as

Chloroacetic acid (5g, 53mmol, 0.98 equiv.) was added to a round bottomed flask and 10 M sodium hydroxide solution in water was added (10 mL,

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250mmol, 4.72equiv.). Trifluoromethyl phenol (8.5g, 52mmol, 1 equiv.) was added to the flask and the resulting reaction was heated under reflux for 24 hours. After this time, concentrated hydrochloric acid (approx. 15 mL) was added to neutralise the sodium hydroxide and cause the acid product to precipitate. After 3 days, no crystals were observed and the product was unable to be isolated.

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Results Compound (1) Table 1: Step 1

Name Molecular Weight (g/mol) Mass (g) No. Moles (mol) Density

Starting Material

Reagent 1

Reagent 2

Reagent 3

Product

Phenol

Chloroacetic Acid

Sodium Hydroxide

Hydrochloric Acid

2-phenoxyacetic acid

94.11

94.5

39.997

36.458

152.15

5.19

5.20

4.50

0.055

0.25

0.41

0.030

1.07g/cm3

1.58g/cm3

2.13g/cm3

1.2g/mL

1.21g/cm3

1H NMR (500 MHz, acetone-d6): δ 7.32-7.29 (2H, m, Ar-H), 6.99-6.95 (3H, m, Ar-H), 4.72 (3H, s, CH2); 13C NMR (100 MHz, acetone-d6): δ 170.3 (C=O), 159.2 (C), 130.2 (CH), 122.0 (CH), 115.3 (CH), 65.4 (CH2). MS (+ESI): m/z 151.06 (M – H+).

Figure 4: 13C NMR spectra of Compound 1

Figure 5: 1H NMR spectra of Compound 1

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Figure 7: TLC of Compound 1

Figure 6: Mass Spectra of Compound 1

Figure 8: TLC of Compound 2 (Starting material – left, Compound 2 – right)

Compound (2)

Table 2: Compound 2

Name Molecular Weight (g/mol) Mass (g) No. Moles (mol) Density

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Starting Material Reagent 1 2-(trifluoromethyl) Chloroacetic phenol Acid

Reagent 2 Sodium Hydroxide

Reagent 3 Hydrochloric Acid

Product 2-(2-(trifluoromethyl) phenoxy) acetic acid

162.11

94.5

39.997

36.458

220.15

8.5

N/A

0.052

0.053

0.25

0.41

N/A

1.333g/mL

1.58g/cm3

2.13g/cm3

1.2g/mL

N/A

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Discussion Step 1: Synthesis of Compound 1

Figure 9: Synthesis of Compound 1

Step 2: Synthesis of Compound 2

Figure 10: Synthesis of Compound 2

The synthesis in Step 2 was performed but could not afford Compound 2 from a condensation reaction between 2-(trifluoromethyl) phenol and chloroacetic acid. More specifically, in the presence of aqueous sodium hydroxide and then a neutralisation of the strong base with hydrochloric acid, phenol and chloroacetic acid was unsuccessful in producing Compound 2. This result was likely due to the need for a structured methodology to base the experiment on. Due to this lack of methodology, some guesswork had to be done regarding the mass of reactants rather than a structured and proven method. Due to time constraints, a second experiment was unable to be conducted. Thus, future trials should seek to optimise the methodology to allow for the successful synthesis of Compound 2. The appearance of a less polar starting material on the TLC (Figure 11) corresponds with the very polar Compound 2. As no precipitate was formed, we can assume that this polar end-product is not Compound 2. No other tests were conducted as no crystals were observed, and a product could not be produced. Further Research In addition to its potential as a new and more effective inhibitor of protease Mpro, the successful synthesis of 2-phenoxyacetic acid may have important applications in the guidelines for the development of other analogues. Due to the synthesis of 2phenoxyacetic acid (1) being easier than that of 2-(2(trifluoromethyl) phenoxy) acetic acid (2), a way of obtaining a higher yield of Compound 1 could be done through more purification. Using this research paper, it has created opportunities for further research to develop other analogues of Lopinavir which may exhibit more effective inhibition of protease Mpro through an increase in binding site affinity. Further trials may develop the methodology through finding

a set quantity of each substance to then result in a more reliable and accurate experiment producing a better yield of Compound 1 while also allowing for the successful synthesis of Compound 2.

Conclusion In addition to its potential as a new and more effective inhibitor of protease Mpro, the successful synthesis of 2-phenoxyacetic acid may have critical applications in the guidelines for developing other analogues. Due to the synthesis of 2-phenoxyacetic acid (1) being more accessible than that of 2-(2(trifluoromethyl) phenoxy) acetic acid (2), a way of obtaining a higher yield of Compound 1 could be done through more purification. This research paper has created opportunities for further research to develop other analogues of Lopinavir, which may exhibit more effective inhibition of protease Mpro through increased binding site affinity. Further trials may develop the methodology through further research to produce a better yield of Compound 1. As Compound 2 was not successfully synthesised, further trials can hopefully, with a more developed methodology, successfully synthesise Compound 2.

Acknowledgements I would like to acknowledge the Barker College Science Extension staff who helped develop my project idea and supporting me through my project. I would like to thank Dr Katie Terrett for her help in the structure of my report as well as her expertise in the synthesis of my 2 compounds.

References Apostolopoulos, V, Bojarska, J, Chai, T-T, Elnagdy, S, Kaczmarek, K, Matsoukas, J, New, R, Parang, K, Lopez, OP, Parhiz, H, Perera, CO, Pickholz, M, Remko, M, Saviano, M, Skwarczynski, M, Tang, Y, Wolf, WM, Yoshiya, T, Zabrocki, J & Zielenkiewicz, P 2021, ‘A Global Review on Short Peptides: Frontiers and Perspectives’, Molecules, vol. 26, no. 2, p. 430, viewed 23 January 2022, <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC783066 8/>. Dayer, MR, Taleb-Gassabi, S & Dayer, MS 2017, ‘Lopinavir; A Potent Drug against Coronavirus Infection: Insight from Molecular Docking Study’, Archives of Clinical Infectious Diseases, vol. 12, no. 4. Drożdżal, S, Rosik, J, Lechowicz, K, Machaj, F, Szostak, B, Przybyciński, J, Lorzadeh, S, Kotfis, K, Ghavami, S & Łos, MJ 2021, ‘An update on drugs with therapeutic potential for SARS-CoV-2 (COVID-19) treatment’, Drug Resistance Updates, vol. 59, no. 1, p. 100794, viewed 7 February 2023,

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<https://www.sciencedirect.com/science/article/pii/S1368 764621000546>.

Lopinavir’, Scientia Pharmaceutica, vol. 83, no. 1, pp. 49– 63.

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FENNER, F, BACHMANN, PA, GIBBS, EPJ, MURPHY, FA, STUDDERT, MJ & WHITE, DO 1987, ‘Structure and Composition of Viruses’, Veterinary Virology, vol. 1, no. 1, pp. 3–19, viewed 28 January 2023, <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC717359 9/>. Health 2023, About Coronavirus (COVID-19), Australian Government Department of Health and Aged Care, viewed 18 June 2023, <https://www.health.gov.au/topics/covid19/about#:~:text=COVID%2D19%20is%20a%20disease,r ange%20from%20mild%20to%20severe.>. Hoaston, G & Vold, R 2023, Sci-Hub | 2H-NMR Spectroscopy of Solids and Liquid Crystals. Solid-State NMR III Organic Matter, 1–67 | 10.1007/978-3-64261223-7_1, Sci-hub.ru, viewed 7 February 2023, <https://sci-hub.ru/10.1007/978-3-642-61223-7_1>. Jin, Z, Du, X, Xu, Y, Deng, Y, Liu, M, Zhao, Y, Zhang, B, Li, X, Zhang, L, Peng, C, Duan, Y, Yu, J, Wang, L, Yang, K, Liu, F, Jiang, R, Yang, X, You, T, Liu, X & Yang, X 2020, ‘Structure of Mpro from COVID-19 virus and discovery of its inhibitors’, Nature, vol. 582, no. 582, pp. 289–293. Lopinavir and Ritonavir: MedlinePlus Drug Information 2019, Medlineplus.gov, viewed 8 June 2023, <https://medlineplus.gov/druginfo/meds/a602015.html>. Malik, D, Baboota, S, Ahuja, A, Hasan, S & Ali, J 2023, Sci-Hub | Recent Advances in Protein and Peptide Drug Delivery Systems. Current Drug Delivery, 4(2), 141– 151 | 10.2174/156720107780362339, Sci-hub.ru, viewed 3 February 2023, <https://scihub.ru/https://doi.org/10.2174/156720107780362339>. Mansbach et al., 2021, Sci-Hub | The SARS-CoV-2 Spike variant D614G favors an open conformational state. Science Advances, 7(16) | 10.1126/sciadv.abf3671, Scihub.se, viewed 18 June 2023, <https://scihub.se/10.1126/sciadv.abf3671>. News-Medical 2022, SARS-CoV-2 can evade Paxlovid by multiple mechanisms, News-Medical.net, viewed 18 June 2023, <https://www.newsmedical.net/news/20220630/SARS-CoV-2-can-evadePaxlovid-by-multiple-mechanisms.aspx>. Rafi, MdO, Bhattacharje, G, Al-Khafaji, K, Taskin-Tok, T, Alfasane, MdA, Das, AK, Parvez, MdAK & Rahman, MdS 2020, ‘Combination of QSAR, molecular docking, molecular dynamic simulation and MM-PBSA: analogues of lopinavir and favipiravir as potential drug candidates against COVID-19’, Journal of Biomolecular Structure and Dynamics, pp. 1–20. Raghava Reddy, AV 2015, ‘Synthesis and Characterization of Impurities in the Production Process of

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Health 2023,

Scientific Research in School Volume 5 Issue 1 2023

An exploration of the Belousov-Zhabotinsky chemical oscillator within a high school laboratory Isaac Denney Barker College Purpose: Existing research has shown that the rate of oscillations of the Belousov-Zhabotinsky Reaction (BZ reaction) increases with an increase of ethanol in the reaction system. This research paper aimed to explore the effects of ethanol on the BZ reaction within a school laboratory, which could provide potential for future application in the gasohol or alcohol industries. Design/methodology/approach: The experiment originally intended to optimise the reaction within the lab, before introducing the independent variable of ethanol. After initial testing, the decision was made to instead focus on the optimisation of the reaction as a visual demonstration through the test of multiple methods found in literature. Findings: This paper was able to demonstrate that the BZ chemical oscillator can be successfully replicated within a high school laboratory. Research limitations/implications: Limits in the levels of controls and time constraints have meant that the correlation between ethanol and mean oscillation time was not able to be demonstrated. Originality/value: This is the first paper to attempt to quantify ethanol levels using the BZ reaction in a high school laboratory, which could provide potential industry applications in the gasohol and alcohol industries. Keywords: Belousov-Zhabotinsky Reaction; Oscillator; Ethanol; Oscillation Period Paper type: Scientific research paper

Glossary Belousoz-Zhabotinsky Reaction: a chemical oscillator which oscillates between red and blue, as a result of the oxidation and reduction of a metal catalyst. Chemical Oscillator: a periodically repeating chemical reaction, involving reactants, intermediates, and products. Ethanol Quantification: the detection of ethanol concentration in a solution. Oscillation Period: the time taken (in seconds) between oscillations.

Literature Review What is a chemical oscillator? A chemical oscillator involves a periodically repeating chemical reaction, during which the concentration of reactants decreases, and the concentration of products increase as in a regular chemical reaction, however, the concentration of intermediates or catalysts oscillates as the conversion of reactants to products occurs. These oscillations can occur as there is a constant decrease in free energy as the reaction occurs, and were thought impossible as the non-equilibrium conditions appeared to break the second law of thermodynamics, as the reactions do not progress directly to equilibrium, but instead remain far

from it for a large amount of time. Examples of these include the Briggs-Rauscher Reaction, which oscillates between blue, colourless, and a pale yellow several times a minute, and the well-known example of the Belousov-Zhabotinsky (BZ) Reaction, which this paper focuses on, which either oscillates from red to a blue (in a test tube or beaker) or oscillates with blue rings in an orange solution (in a petri dish) (Field & Schneider, 1989). The best understood category of chemical oscillators are the ones driven by the oxidation of organic material by a bromate ion (BrO3-) in a highly acidic, aqueous state. The reaction is generally catalysed by a metal ion with two oxidation states separated by a single electron, eg. Ce(IV)/Ce(III) or Fe(phen)3+3/Fe(phen)2+3 (Field & Schneider, 1989). As the charge and therefore ion of the catalyst changes, the colour can also change. Early history of the BZ Reaction The BZ reaction was first discovered by Boris Belousov in the 1950s at Moscow’s All-Union Institute of Sanitation and Chemistry. While Belousov was not the first to discover a genuine oscillatory reactor in a homogenous phase (generally accredited to William Bray’s discovery of the ‘Bray-Liebhafsky’ reaction in 1921), the BZ reaction would become the catalyst for a widespread acceptance of chemical.

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Scientific Research in School Volume 5 Issue 1 2023 oscillators as a theoretically possible phenomena throughout the scientific community (Kiprijanov, 2016). This did not occur until after his research was furthered by Anatoly Zhabotinsky and his team throughout the 1960s. The group was not only able to provide explanations for the complex chemical processes and reaction rates but was also able to prove that the reaction occurs in a closed homogenous system. The research began to gain traction within the USSR in the mid-1960s, but only found widespread attention in the Western world in the late 60s and early 70s (Kiprijanov, 2016). Chemistry of the BZ Reaction The overarching reaction for the BZ reaction is given by: 3CH2(COOH)2 + 4BrO3-  4Br- + 9CO2 + 6H2O Malonic Acid + Bromate  Bromide + Carbon Dioxide + Water However, the reaction is far more complex, involving over 40 reactions within the system. The 10 main reactions are as follows:

Figure 1: Main reactions of the BZ system Source: (Barzykina, 2020)

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Figure 2: Ferroin Indicator Structure Source: (Sigma Aldrich, n.d.)

The colour change occurs as the ferroin ion (an iron compound intermediary with a ferrous 2+ ion) of the ferroin indicator (Figure 2) which is an orange colour is oxidised to the ferric ion (3+), which has a blue colour. In a beaker or test tube, the colour change occurs as the concentration of ferric passes that of the ferroin but is quickly changed back to orange as the ferric ions are reduced by either malonic acid or 2bromomalonic acid (Figure 1). Models of the BZ reaction The most widespread and referenced model of the BZ reaction is the Oregonator model, which provides a simulation of the most significant kinetic features of the chemical mechanism. It was the first model to demonstrate how a region of oxidation could propagate through the system as a wave (Field & Noyes, 1974). However, this model was very basic, and has been replaced by many other, more advanced models. Generally, these focus on the nonlinear dynamics of the system, and how the many systems demonstrate deterministic chaos (Györgyi & Field, 1992). Factors affecting oscillation rate Internal changes within the reaction system will have significant impacts on the structure and dynamics of the reaction (Masia et al., 2001). This means that many factors, such as volume and concentration, as well as surface contaminations, such as surfactants (Paul, 2005) and stir rate when the system is run in a beaker with a stir bar (Kalishyn et al., 2010) will all have significant effects on the system’s dynamics. Further, external changes in the environment, such as temperature and humidity (Masia et al., 2001), air flow, and oxygen levels in the environment (Wang et al., 1996) have been found to have significant effects on the system.

Scientific Research in School Volume 5 Issue 1 2023 Practical applications of the BZ Reaction Currently, the BZ reaction has applications in physics, as a result of the deterministic chaos dynamics and formation of spatial patterns (Field, 2015), and as a model for biological systems as a result of these movements being a simplification of biological systems (Shanks, 2001). However, it currently has only one possible practical application, in the form of ethanol detection in gasohols and alcohols. The BZ reaction as a method of quantifying levels of alcohols works because primary alcohols can change the BZ reaction by reacting with the acidic bromate ions to form the autocatalytic intermediary of bromous acid. By increasing the rate at which the bromous acid is produced, the rate of overall reaction and therefore oscillations is able to be increased (Pelle et al., 2003), while the visibility of the oscillations decreases, as the system is diluted, creating an important consideration when choosing a catalyst for the ethanol detection using this reaction (Sansuk et al., 2019). Thus, given a set humidity and temperature level (which may affect reaction rate), the trend between reaction rate and ethanol concentration can be graphed and a correlation can be found, resulting in a potential to work backwards and find ethanol concentration based on reaction rates.

Scientific Research Question Research question 1 Can the highly sensitive BZ reaction be successfully replicated within a school laboratory? Research question 2 Is there a significant correlation between the concentration of ethanol in a BZ reaction system and the oscillation rate of the reaction?

Scientific Hypothesis Hypothesis 1 That the BZ reaction can successfully be replicated within a school laboratory. Hypothesis 2 That there is a significant negative correlation between the concentration of ethanol and oscillation rate in the BZ reaction.

Methodology Initial method: (NileRed, 2017) The starting point to test the BZ reaction within the school laboratory was chosen to be the mixture used by NileRed, and was chosen as the mixture was

shown in video to result in clear blue rings and minimal carbon dioxide bubbles. To replicate, 3 initial solutions were created - solutions A, B and C. -

Solution A consisted of 33.5mL distilled water, 1mL conc. Sulfuric Acid and 2.5g Sodium Bromate. Solution B consisted of 0.5g Malonic Acid and 5mL distilled water. Solution C consisted of 0.5g Sodium Bromide and 5mL distilled water.

To replicate the reaction, the solutions were combined in a small petri dish using 6mL of solution A, 1mL of solution B and 0.5mL of solution C, and were stirred until the system decolourised. Then, 1mL ferroin indicator was added to the mixture and stirred just until evenly distributed. The system was then left and recorded on an iPhone camera. This was repeated under medium to high stirring in a test tube. Multiplying the volume of each substance by two, the reaction was repeated again both in a large petri dish and small beaker (under medium-high stirring). Second method: (Sansuk & Sombook, 2018) The second method tested was used by Sansuk and Sombook (2018), and involved a significantly lower concentration of Bromate, as well as just 40% the amount of indicator in a solution 83% of the volume, as compared to the initial method. To replicate the method, 6mL 1M Sodium Bromate, 1.2mL 1M Malonic Acid, 0.9mL 1M Sodium Bromide, 1.8mL 5M Sulfuric Acid, 5.1mL distilled water and 1.2mL indicator were combined in a large petri dish and the system was left covered and recorded. As in the initial method, the reaction was repeated in a small beaker (under high stirring). This was repeated, but with 3mL Bromate, 0.6mL Malonic Acid, 0.45mL Bromide, 0.9mL Sulfuric Acid, 2.55mL distilled water and 0.6mL indicator in a test tube. Final method: Original – combination of both previous methods The final method tested was composed of the same Solutions A, B and C from the initial method in a small beaker. 12mL of solution A, 2mL B and 1mL C were combined in a small beaker under constant, medium-high stirring. After the system decolourised, 1mL of indicator was added to the system, which is half the amount for the same volume of solution compared to the initial method. This was repeated in a large petri dish (with no stirring). Where possible, a lid was placed over the system to minimise the potential disturbances due to air flow from the fume

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Scientific Research in School Volume 5 Issue 1 2023 hood. The reaction was left and recorded on an iPhone. Simple demonstration of ethanol quantification potential Three ethanol solutions were prepared: 5%, 10% and 15% (v/v) ethanol in water. Four small beakers were set up on a single stir plate (in order to keep stir speed constant), such that they were evenly spaced from the centre of the plate, and a medium-sized stir bar was placed in each. The stirring was turned on to mediumhigh. The reaction was replicated in the first beaker as in the final method, using 12mL of Solution A, 2mL of Solution B and 1mL of Solution C, as well as 1mL of distilled water. This was left to decolourise before 1mL of ferroin indicator was added to the system. This was repeated for the other 3 beakers, replacing the water with 1mL of the 5%, 10% and 15% ethanol solutions in each beaker respectively. The experiment was recorded using an iPhone and the average time between oscillations was found for each system. Given time restraints, this experiment was only run once per ethanol solution.

Figure 5: Initial Method in Test Tube

Second method

Results Initial method

Figure 6: Second Method in Large Petri Dish at different times

Figure 3: Initial Method in Small Petri Dish

Figure 7: Second Method in Test Tube

Figure 4: Initial Method in Large Petri Dish

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Scientific Research in School Volume 5 Issue 1 2023 Final method

Table 1: Oscillation Rates vs Ethanol Concentration Ethanol Concentration (%) 0 5 10 15

Average oscillation time (s) 1-12 4-9 61 55 19 18 27 27 50 50

Figure 8: Final Method in Petri Dish at Different times

Figure 11: Graph of Ethanol Concentration vs Oscillation Times Figure 9: Final Method in Beaker

Simple testing with Ethanol

Data Analysis – Pearson’s correlation coefficient Table 2: Pearson’s Correlation Parameters Parameter Pearson’s correlation coefficient (r) P-value Covariance Sample size Statistic

Value -0.04346 0.9565 -5 4 -0.06152

Discussion

Figure 10: Testing with Ethanol in Beakers

Optimization of the BZ reaction in a high school laboratory The first hypothesis tests whether the BZ reaction can be successfully replicated and optimised within a school laboratory. For the first method, the initial results appeared promising, with blue rings appearing when the reaction was run within a petri dish (Figure 3 & 4). However, these rings were either too spaced apart (Figure 3) or the lines of the rings were unclear (Figure 4), and the system itself was often too dark. Within a test tube or beaker, the reaction would oscillate once to blue, but would then become too dark to distinguish any colour changes from that point onwards (Figure 5). Thus, a second method was found which used a lower volume of indicator, which is very dark coloured, and is likely the reason for the solution going dark under

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Scientific Research in School Volume 5 Issue 1 2023 the initial method. This second method for recreating the reaction tested was taken from the literature (Sansuk & Sombook, 2018). The only major differences between this and the initial method was in the concentration of bromide/bromate ions and the amount of indicator, which were both lower. The results from the second method were less promising, with hardly visible oscillations and movements occurring within the petri dish, while the system stayed mostly a pale blue colour (Figure 6). In the test tube and beaker replications, the bromine produced from the initial combination of solutions would not decolourise, resulting in a murky brown mixture after the indicator was added (Figure 7). The paler colour is likely due to the fact that the system is more dilute, and the lack of clear, visible oscillations is a result of not being able to reduce the ferric ion back into the original (red) ferrous ion, an example of how the structure and dynamics of the reaction change (Masia et al., 2001). Thus, to optimise the reaction, a method was developed which combined aspects of both methods. The method also involved ensuring the system was covered, to minimise the effects and thus errors of airflow on the surface of the reaction. The final method was found to be far more successful than either of the initial two methods tested, with clear and distinct rings formed when the reaction was run in a petri dish (Figure 8), and visible oscillations from red to blue and then from blue to dark red and back when run in a beaker (Figure 9). The system was still too dark, but the oscillations were visible and the time between oscillations were able to be calculated. Simple testing with ethanol The second hypothesis predicts that there exists an inverse relationship between mean oscillation time and ethanol concentration. The expected results from the testing of various ethanol concentrations is an inverse logarithmic relationship (Figure 13), and as ethanol concentration decreases, the period of oscillations is expected to decrease at a decreasing rate (Sansuk & Somboon, 2018). However, the results found from the testing of ethanol concentrations did not align with the results of the literature. As seen in Figure 11 and Table 1, the results did not appear to have any correlation, which was confirmed through the use of a Pearson’s correlation test. The average times of oscillations 4-9 were used as this was found to be the period where the oscillation time was the most consistent (Table 3). The test used to analyse the results was a Pearson’s correlation test (Table 2). The r-value found was 0.04346, which indicates a very weak negative

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correlation. The r2 value calculated was 0.00189, which means that only 0.189% of the variance of one of the variables is explained by the other. The P-value of 0.9565, which is greater than 0.05, indicating there is no significant correlation between ethanol concentration and mean oscillation period. This is likely explained by the sensitive nature of the BZ reaction, and the lack of proper controls when testing. While simple things, such as the level of stirring, volume of system and temperature, and the order that reactants were added were kept constant, the airflow and cleanliness of beakers, which both have significant effects on the dynamics of the reaction (Paul, 2005), were not able to be controlled given the limitations of working in a high school laboratory. Further, due to time constraints, each ethanol concentration was only tested once, meaning reliability cannot be assessed. Another key thing that should be controlled for future experiments is the time taken between the decolourisation of the system and the addition of indicator, and time between the additions of each of the reactants. This is the most likely explanation of the time disparities between the addition of indicator and the first colour change to blue (Table 3 – See Appendices). As a result of the positive qualitative results of the final method reaction system within the petri dish, as well as the successful, clear oscillations between blue and dark red of the system in the beaker, the first hypothesis can be accepted and the null hypothesis can be rejected, as the system was successfully optimised within a school laboratory. The expected trends between mean oscillation periods and ethanol concentration did not reflect an inverse relationship, and no significant correlation was found, and as a result the second hypothesis cannot be accepted. However, as a result of the level of errors and lack of repetition, the null hypothesis cannot be accepted either, and further testing is required to validate any potential correlations.

Scientific Research in School Volume 5 Issue 1 2023 addition times of each solution and the indicator, the airflow of the environment and the cleanliness of the surfaces of the beakers, which all have significant impacts on the dynamics of the system. As a result, while the second hypothesis cannot be accepted as the expected results were not found, the null hypothesis could not be rejected either.

Figure 12: Ethanol Concentration vs Log of Oscillation Period, Source: (Sansuk et al., 2018, p5)

Thus, there is significant room for future research, and this paper has shown that given proper controls, there is potential for future high school research in ethanol quantification utilising the BZ chemical oscillator.

Acknowledgements I would like to thank Dr Katie Terrett, Dr Matthew Hill and Dr Alison Gates for assistance developing the project idea, and Dr Katie Terrett for assistance in conducting the experiment and writing the project.

References Barzykina, I. (2020). Chemistry and Mathematics of the Belousov–Zhabotinsky Reaction in a School Laboratory. Journal of Chemical Education, 97(7), 1895–1902. https://doi.org/10.1021/acs.jchemed.9b00906

Figure 13: Ethanol Concentration vs Oscillation Period, Source: (Sansuk et al., 2018, p5)

Conclusion This research paper set out to test whether the BZ chemical oscillator could be optimised within a high school laboratory. Further, the paper set out to test the correlation between ethanol concentration within the system and the oscillation rate of the system, as described by Sansuk et al., 2019, who found an inverse logarithmic relationship. The results of the testing to attempt to optimise were qualitatively successful, and a method was found which was able to demonstrate clear, visible rings (Figure 8), which were observed to move across the system slowly over the timespan of over half an hour. However, the attempts to find the correlation between ethanol concentration and oscillation rate were unsuccessful, with the Pearson’s correlation coefficient calculated to be -0.04346. While a negative correlation was anticipated (Figure 13), this coefficient indicates a very weak correlation, and, with a P-value of 0.9565, an insignificant one. However, the most likely explanation of these findings is a lack of proper controls, such as the

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