Abstract
This research explores the physiological effects of two popular energy drinks, Rockstar and Celsius, on the heart rate and survival of Daphnia magna, a transparent freshwater crustacean often used in toxicology testing. After exposing Daphnia magna to flat (non-carbonated) samples of Rockstar and Celsius, the research found a significant reduction in heart rate compared to a control group treated with culture water. Interestingly, no statistically significant difference was found between the two drinks, suggesting that both formulations, despite different branding and ingredients, may have a similar effect on cardiac function. These findings challenge the perception of certain energy drinks as healthier alternatives and highlight the importance of testing full formulations, not just isolated ingredients like caffeine. This research adds to the body of knowledge (BOK) by demonstrating how Daphnia magna can be a sensitive and informative model for showing the cardiovascular responses to common consumer products.
Introduction
With marketing campaigns pushing images of vitality, productivity, and athleticism, drinks like Rockstar and Celsius have become staples in convenience stores and vending machines across the globe. They’re marketed as performance boosters, but people rarely stop considering their physiological impact, particularly at the cellular or cardiovascular level. But how do these powerful mixtures of caffeine and other stimulants affect biological systems beyond our own? This question sparked my interest in exploring their physiological effects, not on humans, but on a small, transparent aquatic organism known as Daphnia magna.
Literature Review
Daphnia Magna Physiology
According to Dieter Ebert, a scientist from the National Center for Biotechnology Information, Daphnia magna (D. magna) are small crustaceans that belong to the Phyllopoda (Branchiopoda), a group comprised of entomostracans united by the morphology of flattened or leaflike legs used for swimming. Most Phyllopoda live in continental fresh water where they feed on plankton or browse over the bottom sediments. Characterized by a relatively large head with a compound eye, there are over 100 described Daphnia species (Ebert, 2022). Within Phyllopoda, D. magna belongs to the superorder of Cladocera, which is characterized by bodies enclosed by an uncalcified shell. This shell is known as the carapace. The carapace has a clear double wall largely made of chitin, a polysaccharide. The jointed limbs of Cladocera, are a form of apparatus used for feeding and respiration. Cladocerans swim with discontinuous and sudden movements using their antennae and limbs (Ebert, 2005). Many Daphnia including D. pulex and D. magna, have a darkly colored compound eye (Stoeckel & Siriwardena, 2008). This compound eye allows them to respond to shifts in light and motion, as well as exhibit different behaviors in response to various stimuli. As stated in Physiology of the Cladocera, they are one of the rare subgroups of animals that reproduce through parthenogenesis, a form of asexual reproduction where an embryo develops from an unfertilized egg (Smirnov, 2018). Parthenogenesis’s translation in Greek is “virgin birth”, with “partheno” equating to virgin and “genesis” to creation. In other words, they are born without the need for fertilization by a male partner (Figure 1).
What makes D. magna specifically valuable to research, is the ability to see through their carapace to their hearts. The heart of a D. magna is located dorsally and anterior to its brood chamber. Ebert defines the brood chamber as, “Space between the thorax and the dorsal carapace of Cladocera in which the oviduct ends and the eggs develop. It is in direct contact with the exterior medium.” (Ebert, 2005). The heart will beat an average of 200 times per minute at 68 degrees Fahrenheit. Because of their transparent bodies, the blood cells are visible as they move rapidly throughout the body cavity.
Natural Environment and Behavior
The name Daphnia, water flea, comes from the New Latin genus name, which was first used in 1847 (Miller, n.d.). The popular name for Daphnia is “water flea”, due to the organism’s jumping-like behavior throughout water. This behavior originates from the movement of the large antennae. The antennae are used to direct themselves through water, which produces a prompt upward movement, whereas the high density of their body creates a sinking effect (Ebert 2005). Daphnia in the absence of the large antennae, will sink rapidly to the ground.
Understanding how D. magna respond to their natural environment is important for comparing their responsiveness to energy drinks. Although D. magna primarily live in freshwater, their adaptability to survive in saltwater highlights their resilience and versatility as a crustacean. Sunil Siriwardena, an aquaculture master’s and PhD graduate from the Institute of Aquaculture and the University of Stirling, adds to this by identifying how D. magna adapt their behavior in response to environmental stressors (Stoeckel & Siriwardena, 2008). Their bodies can adapt depending on predator presence to increase survival rate. For example, a D. magna can develop a crown of thorns, an elongated tail/spine, or a fortified exoskeleton. These phenotype adaptations can help to increase their survival rate. D. magna have found the balance between energy saving and optimized fitness. According to Ebert, “…Daphnia species found in lakes with planktivorous fish are smaller and more transparent than species found in fishless water bodies.” (Ebert, 2005). Moreover, smaller species such as D. galeata, D. cucullate, and D. hylalina are usually not found in fishless waters due to their smaller size, whereas large species such as D. magna and D. pulex, are commonly found in fishless waters. Ebert adds to this by observing, “It has been proposed that in water bodies without predation, the composition of Daphnia species is influenced by size-dependent competition, with larger species out-competing smaller species.” (Ebert, 2005). This phenomenon emphasizes D. magna versatility as a species.
Rockstar and Celsius Energy Drink
Advertised as boosting physical and mental stimulation, energy drinks are a type of functional beverage containing stimulant compounds. As stated on the Rockstar website, Rockstar specifically contains guarana, taurine, and caffeine (Rockstar Energy, n. d.). Guarana, (Paullinia Cupana), is a climbing plant native to the Amazon and has historically served as a stimulant and traditional medicine among Brazil’s Indigenous peoples. Guarna seeds may have several benefits, but the potential is lost in processing (Cleveland Clinic, 2023). “The guarana seed itself is a natural food that could have some health benefits. But when it’s processed, packaged and mixed with sugar and other additives, it loses its integrity,” dietitian Beth Czerny, observed (Cleveland Clinic, 2023). This Amazon plant is packed double the amount of caffeine in coffee. The caffeine inside guarana is more potent because it reacts differently inside the body. Andrea Costantino and colleagues from the Department of Surgical, Medical, and Molecular Pathology at the University of Pisa, adds to this by saying, “Guarana seeds notably surpass coffee beans in caffeine content, containing additional xanthine alkaloids such as theobromine and theophylline.” (Costantino et al., 2023). This herbal additive increases and enhances the caffeine content as well as the stimulatory attributes of energy drinks. While Rockstar is promoted as being able to enhance focus, the guarana it contains can cause headaches, anxiety, and shakiness. Due to guarana’s status as an herbal supplement, its caffeine content goes unlisted on product labels.
In addition to the generous serving of herbal caffeine, Rockstar is formulated with extremely high amounts of sugar. The American Heart Association (AHA) recommends adults consume no more than 25-36 grams of added sugar in one day (American Heart Association, n.d.). The AHA recommendation suggests that higher levels of added sugars may increase the risk of heart disease. However, a single 16.0 fluid ounce of Rockstar Energy Drink contains 63 grams of added sugars, practically double the recommended amount by the AHA. Harvard Health Publishing, a peer reviewed publisher, expands on this by saying, “Consuming too much added sugar can raise blood pressure and increase chronic inflammation… Consumption of sugar, especially in sugary beverages, also contributes to weight gain by tricking your body into turning off its appetite-control system because liquid calories are not as satisfying as calories from solid foods.” (Harvard Health Publishing, 2022). This explains why it is easy for people to add more calories and sugar to their regular diet when consuming energy drinks. High blood pressure and chronic inflammation both are pathological pathways to heart disease.
Current research suggests Celsius may be a heathier alternative to energy drinks. Celsius is a popular energy drink marketed with numerous health benefits. It is designed to boost metabolism, burn calories, and provide sustained energy. The company, Celsius, advertises its product as a pre-workout supplement and metabolism booster, often promoting it alongside images of athletic individuals, gym settings, and active lifestyles (Celsius, n.d.). This health-oriented image has helped Celsius gain popularity among individuals, such as teens and young adults, seeking energy-boosting beverages without the perceived risks of traditional energy drinks. According to Jamie N, a registered dietitian, “Among the ingredients are green tea, extract, guarana seed extract, caffeine, taurine, glucuronolactone, ginger extract, and green tea leaf extract.” (Jamie. N., 2023). While Celsius is formulated with guarana, similar to Rockstar, it is advertised as a healthy alternative due to its perceived ability to increase metabolism and improve exercise response. According to Celsius’ website, “Six publishes university studies have been conducted on Celsius by U.S. accredited scientific research facilities rendering strict scientific standards.” (Celsius, n.d.). The six studies were presented at scientific conferences and published in peer reviewed publications. All six of the studies observed and claimed that Celsius has thermogenic properties. In other words, Celsius causes your body to burn more calories and fat than without.
Research Gap
A peer reviewed study published in the National Library of Medicine, aimed to investigate the effects of selected central nervous system agents on the heart rate of D. magna. The researchers tested the effect of dopamine on the heart rate of D. magna and compared its effect with caffeine and ethanol alone in combination. Findings from this article report both caffeine and dopamine were found to instantly increase of heart rate of D. magna in a dose-dependent manner. The caffeine synergized with dopamine to increase the Daphnia’s heart rate (Kundu & Singh, 2018). Therefore, I assumed that both drinks would increase the heart rate because of their caffeine content. However, I predicted that Rockstar, with its higher sugar and additive content, would have a more extreme effect, and potentially a higher mortality rate. While Kundu’s and Singh’s study provides valuable insight into the effects of isolated stimulants like caffeine and dopamine on D. magna, it does not specifically address the more complex and realistic scenario presented by commercial energy drinks. These beverages are not single agent solutions, rather, they contain a wide range of additional ingredients, including taurine, artificial sweeteners, herbal extracts, electrolytes, and vitamins. These ingredients can interact in unknown ways to influence biological systems. As a result, prior research leaves a critical gap in understanding how the full formulation of these energy drinks affects cardiac function and survival in model organisms. Therefore, this study aims to answer the question: What are the physiological effects of Rockstar energy drink compared to Celsius energy drink on the heart rate and survival of Daphnia Magna? Unlike pure caffeine, energy drinks are comprised of a complex mixture of ingredients. This study’s goal is to fill that gap by analyzing how the complete formulation of Rockstar and Celsius influence heart function beyond the known effects of caffeine alone. While many studies isolate the effects of caffeine or other compounds, few, if any, have compared the real-world physiological effects of full brand formulations. By doing so, this research contributes to the body of knowledge and to public health awareness by offering consumers evidence into the potential risks that come with consuming energy drinks.
Method
The method of my research addresses the question: What are the physiological effects of Rockstar energy compared to Celsius Energy drink on the heart rate and behavior of Daphnia Magna? The research will utilize a comparative experimental method, which is defined as a scientific investigation that compares the results of different conditions on a variable. In the context of this research, the ‘different conditions’ will be the two types of energy drinks, and the variable will be D. magna. Additionally, the data produced will be graphed, and a performance og multiple statistical tests to determine whether there is a significant difference within the data. This research questions exists on the assumption that physiological effects from energy drinks will be observed in D. magna and that they will be comparable. This implies the research believes one drink may have a more negative effect on D. magna than the other.
This method helps establish systematic similarities and differences between the effects of the energy drinks, making the comparison more effective than a standard experimental approach, which may not allow for clear distinctions between them.
Ethics
Because this research involves the testing and sacrifice of D. magna; ethics must be considered. The protocol of submerging the D. magna in energy drinks was IRB approved. Furthermore, they are invertebrates who lack a complex central nervous system; therefore, they cannot feel pain, minimizing their suffering. While D. magna may exhibit avoidance behaviors when exposed to stimuli, these responses are most likely instinctive reactions rather than indications of pain. To kill the D. magna humanely, a small amount of bleach will be added to the culture water. This method has been practiced in multiple alike experiments and will be replicated as so.
Daphnia magna Culture
- magna were cultured in a plastic cups containing stock solution, or distilled water. The D. magna cultures were supplemented with active yeast added as needed. Yeast or bacteria are the preferred food for D. magna. The cultures were routinely monitored to ensure production of healthy D. magna.
Counting of Daphnia magna’s Heart Rate
To investigate the effect of certain variables on the heart rate and behavior of D. magna, real-time monitoring of changes is required. Each D. magna was placed on a glass depression slide with 1 drop of a variable solution. The slide was observed in real time under a standard microscope at 4x magnification, and the heart rate was counted using a slow-motion camera (Figure 2). A slow-motion camera is necessary due to the very fast pace of D. magna’s hearts. In the control trial, measurements were taken quickly to ensure accurate data collection and to prevent stress on the D. magna, as prolonged exposure to the limited water volume and heat from the microscope could potentially skew the results. In the variable trials, a 30-second waiting period was observed after adding the variable solution. This allowed for sufficient time for the energy drinks to begin affecting the D. magna and induce any potential physiological changes, such as change in heart rate. This period ensures that the drinks have enough time to interact with the organism before measurements are taken, allowing for more accurate assessment of the effects. The heart rates were recorded in a data table inside of Excel, a spreadsheet program that helps users organize and analyze data.
Water (Control)
Using a pipette, 1 D. magna was removed from the culture jar and placed onto a depression microscope slide containing 1 drop of water. The microscope was focused to 4x on the beating heart. Using a slow-motion camera, the heart was recorded for 10 seconds. This measurement was taken quickly, so the D. magna did not become stressed and overheated in the small volume of water. Using the slow-motion video, the number of beats were counted and multiplied by 6 to get the number of beats per minute. This was repeated 15 times using 15 different individual Daphnia. To avoid using the same D. magna, the used D. magna were placed in a separate container.
Energy Drinks
First, carbonation was removed from both Rockstar and Celsius before testing. Originally, the drinks were tested fully carbonated, but it was quickly noticed that the D. magna died almost instantly upon exposure. This immediate mortality made it impossible to collect any heart rate data, so the decision was made to let the drinks go flat to isolate the effects of the non–carbonated ingredients.
Using a pipette, 1 D. magna was removed from the culture jar and placed onto a depression microscope slide. Using a paper towel, the culture water was quickly absorbed and replaced it with flat (non-carbonated) Original Rockstar to ensure it does not get diluted. The microscope was focused to 4x on the beating heart. A 20-second waiting period was observed after adding the variable solution. This allowed for sufficient time for Rockstar to begin affecting the D. magna and induce any potential physiological changes, such as change in heart rate. Using a slow-motion camera, the heart was recorded for 10 seconds. Using the slow-motion video, the number of beats were counted and multiplied by 6 to get the number of beats per minute. The data was recorded in the table below. This was repeated 15 times using 15 different individual Daphnia. To avoid using the same D. magna, the organisms used were placed in a separate container. The following steps were repeated a second time using flat (non-carbonated) Celsius. The Celsius used was a regular, standard version of the drink, similar to how Original Rockstar represents the standard version of Rockstar energy drinks.
Results
After obtaining the heart rate number of all 42 D. magna, the results were plotted on a box and whisker graph and analyzed in detail (Figure 3). The D. magna who were tested using their culture water as a control had a mean heart rate of 406.6 beats per minute, those tested with flat Rockstar had a mean heart rate of 227.6, and those tested with Flat Celsius had a mean heart rate of 245.1.
A one-way ANOVA test was conducted to indicate whether there were significant differences between the three exposure groups. The test showed a statistically significant difference between the three exposure groups, F(2, 39) = 50.34, p > 0.001. This means at least one group had a significantly different mean heart rate than the others. The p-value suggests that the differences between groups are highly significant and unlikely due to chance. To determine exactly which groups are significantly different from each other, a Post-hoc Tukey’s HSD test was performed. This test revealed that Flat Rockstar (M = 227.57, SD = 57.56) and Flat Celsius (M = 406.64, SD = 33.69) lowered heart rate significantly compared to Water (M = 406.64, SD = 33. 69). However, no significant difference between Flat Rockstar and Flat Celsius was reported. Independent t-tests were used to further confirm these differences. The t-test between Water and Rockstar showed a significant difference, p < 0.001, as did the test between Water and Celsius, p < 0.001. However, the t-test confirmed that there is no significant difference between Rockstar and Celsius, p = 0.443, suggesting that both energy drinks had a similar effect on the D. magna.
Analysis
The quantitative findings from this experiment suggest that both Flat Rockstar and Flat Celsius caused a significant reduction in the heart rate of D. magna when compared to the control group (culture water). Although no significant difference was observed between the two drinks, the similar depressive effect on heart rate suggests that shared components such as caffeine, taurine, or other stimulants may be responsible for the observed physiological response. An analytical point is the lack of significant difference between Rockstar and Celsius (p = 0.443), which implies that their effects on cardiac activity are statistically comparable. This is particularly notable given that Celsius is often marketed and perceived as a “healthier” energy drink. Despite Celsius being advertised as an “healthier” alternative, the data indicates that both beverages have similar impacts on D. magna heart rate in the absence of carbonation. Therefore, the assumption that Celsius is a less physiologically disruptive alternative is not supported by these findings, furthermore, highlighting that health claims based on branding may not accurately reflect actual biological effects.
Discussion
The data from this research suggests that Flat Rockstar and Flat Celsius both significantly reduce the heart rate compared to water. This indicates that the drinks ingredients have similar physiological effects despite Celsius being marketed as healthier. However, neither drink acted as initially expected. D. magna have been shown to respond to doses of caffeine with an increased heart rate, yet both drinks resulted in cardiac suppression.
Research has shown that certain artificial sweeteners can have adverse effects on the heart rate of D. magna. According to the journal Neurotoxicology and Teratology, sucralose (which is found in Celsius), induced dose dependent increases in acetylcholinesterase (AChE) activity. AChE is an important enzyme in the nervous system, it helps transmit signals in the body, especially in muscle movement and heart function. If an artificial sweetener like sucralose increases AChE activity too much, it can interfere with normal nerve signals, potentially leading to an altered heart rate (Wiklund, et al. 2023). This suggests that the drinks may be slightly toxic, causing a stress response that slows heart rate as a protective mechanism followed by a compensatory reduction in heart rate.
These findings demonstrate the complex interactions between caffeine, additional energy drink ingredients, and small aquatic organisms such as D. magna. Furthermore, this complexity may contribute to the unexpected cardiac suppression observed in D. magna. These results contribute to the body of knowledge by demonstrating how sensitive and valuable Daphnia magna‘s physiological systems are for studying the effects of chemical substances.
Limitations and Future Research
While this research provides valuable insight into how energy drinks affect D. magna heart rate, it is important to address some present limitations. One key limitation is the number of D. magna tested may not be large enough to fully represent population-wide effects or account for natural variability. A larger sample could improve statistical validity and reduce the impacts of outliers on the overall trend. Another limitation is the results translation to human physiology. D. magna are commonly used in toxicological studies, but their cardiovascular system differs significantly from humans. Therefore, the findings may not fully translate to the human heart. Second, carbonation was removed from both Rockstar and Celsius by letting them go flat before testing. This was done to control the physical effect of carbonation on D. magna, but it also means that I didn’t test the full experience of consuming an energy drink as people normally would. Carbonation might interact with other ingredients and potentially affect heart rate differently. Lastly, a major limitation is measurement variability. Heart rate in D. magna can be affected by many factors, including temperature, age, and stress from handling, all of which could potentially skew the results.
Future research could explore the individual effects of specific energy components. Such as artificial sweeteners, taurine, guarana, or electrolytes. Furthermore, testing different concentrations of energy drinks may help determine if a lower dose would produce the anticipated stimulatory effect. Including more sample groups can improve statistical reliability and reduce the impact of outliers. Larger sample sizes could also reveal more subtle trends that smaller groups might miss. Additionally, future experiments could use mammalian models, such as mice or rats, which have more complex systems that better represent human physiology. This would allow for stronger connections between the findings and potential effects on humans. Lastly, since D. magna are a model organism, future research could test how similar energy drinks affect human cardiovascular function. This would help implicate these results into real world human application.
Conclusion
To conclude, this research found that both Rockstar and Celsius significantly lowered the heart rate of Daphnia magna compared to the control group. Interestingly, there was no significant difference between the effects of the two drinks, despite their branding and ingredient differences. This suggests that both beverages may have similar physiological impacts on heart function, even though Celsius is often marketed as the “healthier” option. These findings highlight the importance of looking beyond marketing claims and considering how complex ingredient combinations in energy drinks can influence biological systems. While this study focused on Daphnia magna, it raises important questions about how these effects might translate to other organisms and potentially, to humans. This study does not just contribute to the BOK, it challenges the assumptions we make about so-called “healthier” alternatives and emphasizes the importance of testing complex real-world mixtures, not just isolated ingredients. It leaves consumers with an important question: If these energy drinks can suppress heart function in a model organism within minutes, what could the long-term effects be in humans after months or years of use? In the end, this research revealed more than just numbers; it uncovered how profoundly a living system like D. magna can be to substances many of us consume without a second thought. While Daphnia magna may be small, their reactions offer big insights, reminding us that even the smallest life forms can expose the biggest truths.
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