The Effect of the Acid in Mountain Dew On Zebrafish Embryo’s Development and Hatching Rate Austin Westberg Union Grove Union High School Mrs. Aleid Allen Abstract This experiment was designed to test the effects of Mountain Dew on the development and hatch rate of zebrafish embryos. The fertilized eggs were put into one of twelve wells, with four different concentrations of Mountain Dew (made from the same can), the concentrations were 0% Mountain Dew (the control), 0.5%, 1%, and 10%. There was 3 wells for each concentration. This study was done over a time period of five days. Every day, the solutions were taken out and replaced with fresh solution. The dead embryos were also taken out each day. High concentrations of the soda were observed to eliminate a lot more of the embryos. There was not many birth defects with the fish, but the ones that had defects were very detrimental. This experiment could help understand how drinking a sugary, caffeinated, and acidic drink could affect the health and development of a human embryo. Introduction Danio rerio, commonly known as zebrafish, are native to the Ganges River (“Zebrafish FAQ’s”). These are used to model human development because the embryos only take about 120 hours from fertilization to develop. They also are easy to observe because the eggs are clear and they develop outside of the mother. There is large amounts of offspring so a lot of fish can be tested at once. Vertebrate development is divided into roughly three stages: an early phase of rapid cell divisions, the establishment of three body axes, and the development of organ systems (“Background Information - Zebrafish”). All of these stages happen in a short period of time making them easily observed. This experiment was designed to test the effects of PepsiCo’s Mountain Dew on the development of zebrafish embryos. Mountain Dew has many different ingredients. Some of these include sugar, caffeine, and concentrated orange juice. Inside of a 12 fl oz. can, there is a total of 54 mg of caffeine and 46g of sugar ("Official Site for PepsiCo Beverage Information | Product"). Due to the citric acid found in the drink, it has a low pH-about 3. Although small amounts of these kinds of beverages is safe, too much may be harmful. In children and adults without proper tooth care, the acidity can easily erode tooth enamel. The caffeine in the drink may also have an effect on the development of an embryo. The main reason that Mountain Dew was chosen was because a test showed that when a whole mouse was submerged in Mountain Dew and sealed, it only took a mere 30 days to dissolve everything except the tail, leaving a red mush (Andrew, 2015). If it can dissolve a whole mouse, large quantities may have the ability to affect an embryo’s health or development. The purpose of this experiment was to determine the effect of Mountain Dew on zebrafish offspring’s embryonic development. It was hypothesized that if an embryo is put in an area with a high Mountain Dew concentration, the embryos will be more likely to be deformed or die because of the high acidity of the soda. Materials & Methods 120 fertilized Instant Ocean Mountain Dew Dissecting zebrafish eggs (one can) microscope Pipettes 4 500 mL beakers to one small beaker for graduated cylinder make dilutions waste 1L flask stirring rod pH meter sharpie goggles Deionized water tape for labels compound microscope and slides Incubator (optimal temperature-28 ℃) Procedure: The first step in this experiment was to obtain rinsed embryos and label the well plate in sharpie with the concentration for each well. The wells were labelled A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, and C4. One liter of instant ocean solutions was made. Dilutions were then made by taking first, 10mL of Mt. Dew and adding it to 90mL of instant ocean. The other dilutions were made: 1mL Mt. Dew in 99mL instant ocean and 0.5mL Mt. Dew in 99.5mL instant ocean. The pH of these solutions was recorded with the (Figure 1) pH meter. Each well was filled halfway with its solution. Three wells were designated for the three dilutions and a solution of just instant ocean-for a total of twelve wells. Some embryos are observed under the microscope. Next, ten fertilized eggs were put into each well, making 30 per dilution. The fish were left overnight inside the wells and the well plate was placed in the incubator. The next day, to gather data, the number of hatched fish, unhatched fish, and dead fish were counted. Then some of the fish were observed under the microscope and observations were recorded. The fish were also observed under the compound microscope. Any abnormalities were recorded in the lab notebook. With a clean pipette for each well, the solution drained from each and replaced with clean solution. The dead embryos were also removed and placed in the “waste” beaker. To determine which were dead, the embryos were observed under the microscope. After every day, the fish were counted and the solution was replaced. For safety, no Mountain Dew was consumed, goggles were worn, and all glass was handled with care. The number of living, hatched, and percent living was observed and graphed over time. (Figure 1) shows how the well plate was set up and what concentrations of Mountain Dew went where. Results The purpose of this experiment was to test how a zebrafish embryo’s environment affects its health and development. In this setup, Mountain Dew was mixed into the environment in different concentrations to test whether that the acidity would help or halt development in the fish. The fish were then evaluated over the course of five days. The independent variable in this experiment were the different concentrations of Mountain Dew, while the dependent variable was the number of unhatched, hatched, and dead embryos, along with any deformities found in the fish. The experimental groups were the three wells with 10% Mt. Dew, the three with 1% Mt. Dew, and the three with 0.5% Mt. Dew. The control of this experiment was the 0% Mountain Dew, instant ocean solution. The control was tested as a “standard” so the other groups could be compared to it after the results were collected. Any data inside of tables or graphs came directly from data directly observed in the experiment. This experiment can show how large amounts of acidity can affect the development and health of embryos. Data Presentation Day 1 Results & Observations: On day 1, everything in the experiment was set up including making dilutions, taking the pH of each solution, finding enough fertilized eggs, observing the eggs under a microscope, and placing them into their respective wells. All of the fish were alive and unhatched. The pH of just instant ocean was 6.00, the 10% Mt. Dew was 3.36, the 1% Mt. Dew was 4.06, and the 0.5% Mt. Dew was 4.10. Day 2 Results & Observations (Table 1: Zebrafish on day 2) (Day 2) Living Living Hatched Dead Unhatched A1 (control) 9/10 0/10 1/10 B1 (control) 10/10 0/10 0/10 C1 (control) 9/10 0/10 1/10 A2 (10%) 6/10 0/10 4/10 B2 (10%) 9/10 0/10 1/10 C2 (10%) 5/10 0/10 5/10 A3 (1%) 9/10 0/10 1/10 B3 (1%) 6/6 0/6 0/6 C3 (1%) 10/10 0/10 0/10 A4 (0.5%) 10/10 0/10 0/10 B4 (0.5%) 9/10 0/10 1/10 C4 (0.5%) 10/10 0/10 0/10 In well C1, the dead embryo lysed open and emptied into the solution. In some of the live eggs, some movement was observed. In C3, one of the eggs had developed a head and tail. In wells A2, B2, and C2, some of the eggs had absorbed the Mountain Dew and turned a yellow/green color. It was also observed that a mistake was made in the well B3. Only 6 fertilized eggs were placed into that well instead of 10. Day 3 Results & Observations Table 2: Zebrafish on day 3 (Day 3) Living Unhatched Living Hatched Dead A1 (control) 9/10 0/10 1/10 B1 (control) 10/10 0/10 0/10 C1 (control) 9/10 0/10 1/10 A2 (10%) 0/10 0/10 10/10 B2 (10%) 0/10 0/10 10/10 C2 (10%) 0/10 0/10 10/10 A3 (1%) 9/10 0/10 1/10 B3 (1%) 5/5* 0/5 0/5 C3 (1%) 10/10 0/10 0/10 A4 (0.5%) 9/10 1/10 0/10 B4 (0.5%) 8/10 1/10 1/10 C4 (0.5%) 9/10 1/10 0/10 *One embryo from B3 was lost and it was not known whether or not it died, so the total in that well was dropped from 6 to 5. Every embryo from the 10% Mountain Dew wells turned yellow/green and died. The hatched fish from B4 was accidentally put in the waste beaker for about a minute while switching out the solutions. Upon retrieval from the waste beaker, it was rinsed in instant ocean and returned to its well. Most of the unhatched fish have developed heads & tails and also black pigment. Day 4 Results & Observations Table 3: Zebrafish on day 4 (Day 4) Living Unhatched Living Hatched Dead A1 (control) 7/10 2/10 1/10 B1 (control) 9/10 1/10 0/10 C1 (control) 8/10 0/10 2/10 A2 (10%) 0/10 0/10 10/10 B2 (10%) 0/10 0/10 10/10 C2 (10%) 0/10 0/10 10/10 A3 (1%) 0/10 9/10 1/10 B3 (1%) 0/5 5/5 0/5 C3 (1%) 0/10 9/10 1/10 A4 (0.5%) 2/10 8/10 0/10 B4 (0.5%) 0/10 9/10 1/10 C4 (0.5%) 3/10 6/10 1/10 One fish from C4 had massive swelling with an irregular heart shape.