Hypothesis

I hypothesize that beverages with higher acidity levels result in a higher staining of the teeth. Given the higher acidity of Iced Tea and Gatorade, they will stain teeth significantly more than Coca-Cola and apple juice.  

Because eggshells have a similar chemical composition to our tooth enamel, making them react similarly with other chemicals. This can help us understand what stains tooth enamel. Therefore, we did the experiment on eggshells given the unavailability of human teeth within the study duration.

The primary outcome: Level of staining after 3 hours of the eggs being in the beverages. Secondary outcome: Level of staining after 7 days of being in the beverages

Variables

Controlled variable(s): The controlled variable in my experiment is the white hard-boiled eggs at baseline before entering the beverages.

Responding variable(s): The responding variable in my experiment is the level of staining at each time point.

Manipulated variable(s): The manipulated variable in my experiment is the different levels of acidity (beverages) of drinks which are Gatorade, Iced Tea, Coca-Cola, and Apple juice.

Procedure

1. Preparation of the materials: We gathered 20 eggs and boiled them to ensure stability. We bought 5 different beverages (Gatorade, Iced tea, Coca Cola, and Apple juice). We also gathered 20 transparent plastic cups (5 cups per beverage) 
2. ImageJ app was used to analyze eggs staining (JImageJ is a Java-based image processing program developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, BSD-2, USA, December 2023)
3. We took photos of all 20 eggs before they went into the beverages to calculate the color before the experiment (Figure 1). All photos were taken using iPhone 14 camera (12MP Main: 26 mm, ƒ/1.5 aperture, sensor‑shift optical image stabilization, 100% Focus Pixels).
4. We labeled each cup with the abbreviation of each of the beverages (example for Coca Cola: C-1, C-2, C-3, etc).
5. We poured all the beverages into the cups and then put the eggs into each beverage.
6.  We let the eggs sit together in the same environment (closed cooler).
7. At 3 hours we took the eggs out, dried them then took photos from the same angle while the eggs were put upright. From that point, we took photos every day for 7 days using the same technique, at the same time, and in the same place to ensure all environmental factors were the same. 
8. All photos were processed using the ImageJ app. 
9. All results were recorded on an Excel sheet.

                                                                 Observations

After 3 hours:

• Coca-Cola: Slight staining observed, with some darker spots.
• Iced Tea: Significant staining evident, with visible stains on the eggs.
• Apple Juice: Minimal to no staining observed on all eggs.
• Gatorade: Pronounced staining observed, with darker spots evident.

After 7 days:

• Coca-Cola: Significant staining observed, particularly at the top of the eggs.
• Iced Tea: Most heavily stained beverage, with numerous dark spots evident.
• Apple Juice: Least stained beverage, although slight yellowing observed on the sides.
• Gatorade: Pronounced staining similar to Iced Tea, particularly concentrated at the top.

Analysis

Means, medians, and proportions were used to describe the egg staining level. Chi Sequard and test were used to compare pre-and post-beverage exposure for 3 hours. Repeat measure ANOVA was used to examine the different levels of staining between the beverages over time. All tests were 2-tailed with a 5% level of significance. Statistical analyses were done using STATA software.

Conclusion

In conclusion, beverages with high acidities such as Iced Tea and Coca-Cola Cola stains eggshells (and presumably teeth enamel) more than beverages with lower acidities such as Coca-Cola and apple juice.

Application

Since the Gatorade and Iced Tea stained the eggs significantly, they will stain the teeth quite a lot over time and the Coca-Cola also comes close to staining the teeth as much as the Gatorade and the iced tea so the safest option to drink is apple juice which doesn’t stain as much as the other beverages.

Some ways I would improve for my scientific method is that I would use a microscope instead of iPhone 14 Pro-max zoom for taking photos of the eggs.

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Acknowledgments

Acknowledgments to my dad, and my science fair teachers for helping me along the way in doing this science fair project.