Puff at your own risk! The dangerous effects of vaping chemicals on zebrafish embryos

I wanted to do this project to find out whether vaping a flavoured e-cigarette poses significant health risks. Canada has one of the highest rates of teen vaping in the world. More than one in four Grade 12 students and over one in five Grade 11 students vape regularly. Youth mistakenly believe that flavoured e-cigarettes are safer than traditional cigarettes.

Vaping liquids contain: a vape base, flavors which contain chemicals and possibly nicotine. Youth find flavours enticing and nicotine keeps them hooked. I decided to  investigate how both vaping liquids and their aerosols affect the survival rates and development of zebrafish embryos.

My research questions were:

How do nicotine, blueberry-flavoured, and vape base liquids and their aerosols affect zebrafish embryo survival rates?

Are vape aerosols more harmful to zebrafish embryos than their starting liquids in terms of survival rates?

My project benefits the public as it provides information on the dangerous effects of vaping. Many teens use vapes to manage stress, anxiety and boredom. Youth who vape are three times as likely to start smoking. Vaping is often a gateway to future cigarette smoking. They are  more likely to use alcohol and cannabis and have poor mental health. In addition, youth vaping is associated with increased risk of pulmonary and cardiovascular disease, gastrointestinal symptoms, headaches and dizziness. My project makes the world a better place as it adds to the growing body of research that finds vaping harmful. It justifies strict regulations on vaping, similar to those for tobacco products.

I learned about vaping in Canada from newspaper articles, the Canadian Pediatric Society research papers, the Health Canada Website and from academic articles. I use zebrafish embryos as a developmental model to research e-cigarette toxicology.

When the vaping liquid in an e-cigarette is heated, the chemicals present in it undergo reactions that produce new chemicals that are also harmful to health. Some research has shown that vaping liquid flavours such as blueberry have harmful health impacts. The chemical composition of vape flavours is proprietary.

I did my research at the  Berman Zebrafish Lab, at the University of Ottawa/CHEO Research Institute. They provided laboratory facilities, equipment and materials to conduct this experiment.

My research hypotheses were:

Zebrafish embryos exposed to the nicotine liquid and aerosol will exhibit the lowest survival rates, followed by those exposed to blueberry-flavoured liquid and aerosol, and then vape base liquid and aerosol.

Exposure to vaping aerosols will result in lower zebrafish embryo survival rates than exposure to their starting liquids.

The project materials were:

Zebrafish embryos

Erlenmeyer flasks

One airlock

60mL syringe

One permanent marker

One waste-beaker for disposal

96 well plates

Pipettes

Graduated cylinders

Embryo medium solution

One dissecting and compounding microscope

One incubator (set to 28.5°C)

One e-cigarette

Vaping liquids

Fume-hood

My procedure had the following steps:

Build a vaping machine that converts vaping liquid to aerosol and place inside fume-hood to avoid inhalation.

Collect aerosol from vaping liquid to produce a stock buffer solution.

Dilute vaping liquid and aerosol as needed.

Treat zebrafish embryos two days post-fertilization with different dilutions and make observations (3 independent experiments, 5 concentrations).

Check for survival and count live embryos on day 5.

Observe embryos under a microscope and take images.

Conduct ethical euthanasia of zebrafish embryos.

My main research findings were:

Vaping liquids

Zebrafish embryo survival rates decreased as concentrations of nicotine and blueberry-flavoured liquids increased.

Nicotine liquid was the most toxic with an LD50 of ~0.02mg/mL. Small error bars (SD) show highly consistent results and the decrease in survival rates was statistically significant.

Blueberry-flavoured liquid was less toxic with an LD50 of ~0.07mg/mL. Medium error bars show greater variability in survival rates, although results at higher concentrations were still statistically significant.

Vape base liquid showed no significant toxicity, as zebrafish embryos did not reach a 50% mortality rate. Small error bars indicate consistent and reliable results.

Vaping aerosols

Zebrafish embryo survival rates decreased as concentrations of all vaping aerosols increased, with statistically significant results.

The nicotine aerosol was the most toxic, with an LD50 of ~0.005mg/mL. Very small error bars indicate highly consistent results.

Blueberry-flavoured aerosol was less toxic, with an LD50 of ~0.01mg/mL. Error bars were smaller at higher doses, showing increased consistency.

Vape base aerosol was the least toxic, with an LD50 ~0.04mg/mL. Moderate error bars show some variation, but the overall trend is clear.

Vaping aerosols compared to liquids

All aerosols had lower LD50 values than their corresponding liquids, showing that vaping increases the toxicity of their chemicals.

All aerosols caused a significant decrease in zebrafish embryo survival rates, even at low concentrations.

These results are statistically significant, showing that aerosols were more toxic than liquids.

Many embryos develop more edema, deformed skeletons, and misshapen jaws when exposed to aerosols and higher concentrations of liquids.

Based on my research I found that:

Hypothesis 1 is supported: Zebrafish embryos exposed to the nicotine vaping liquid and aerosol had the lowest survival rates, followed by those exposed to the blueberry-flavoured liquid and aerosol, and then the vape base liquid and aerosol.

Hypothesis 2 is supported: Vaping aerosols significantly reduce zebrafish embryo survival than their starting liquids.

These conclusions were significant for many reasons. It shows how the nicotine, flavoring liquids and aerosols were very toxic, and the vape base is less toxic. This matters as people only vape with a flavoring or possibly a nicotine add-on, never just the vape base. My experiment also demonstrates that aerosols are far more toxic than their starting liquids. What surprised me the most, was how the Vape Base liquid (which is propylene-gycol, a food addictive) was non-toxic as a starting liquid. When it was converted into an aerosol, it suddenly became drastically more toxic than before. This makes it apparent how aerosolizing makes non-toxic chemicals toxic, and increases the toxicity of already harmful substances.

Sources of error:

Unhealthy Fish: Fish can have undetected health issues despite being screened for health, and

Pipetting error: Pipettes sometimes don’t collect the exact amount of liquid that they are supposed to, due to air bubbles.

Possible changes could be:

More replicates and exposure testing: I would conduct additional replicates and test whether adding the liquid or aerosol daily to fish leads to a more significant effect on survival rates.

Increase the frequency of embryo checks: I would check my embryo development over each of the five days. This would show me the embryo developmental change over time.

Sincere thanks to the Berman Zebrafish Lab, University of Ottawa/CHEO Research Institute for providing the laboratory facilities, equipment and materials to conduct this experiment. Heartfelt gratitude to Dr. Jason Berman and Nadine Azzam for their invaluable mentorship and supervision. Nadine, thank you for helping me with statistical analysis. Thank you to all the lab members for their advice.

Thank you to the University of Wisconsin-Milwaukee. My research method is based on one developed by the Science Education Partnership Award (SEPA) Program. I am grateful to Renee Hesselbach for sending me extensive study materials.

Thank you to Dr. Roma Rambaran, Associate Director of Incyte Corporation, USA, who helped me to brainstorm my research direction.

References

An updated overview of e‑cigarette impact on human health. (n.d.).

Canadian Paediatric Society, Adolescent Health Committee. (2021). Protecting children and adolescents against the risks of vaping. https://cps.ca/en/documents/position/protecting-children-and-adolescents-against-the-risks-of-vaping

Carvan, M. J., III, Hansen, T., Hesselbach, R., Zientek, A., Berg, C., & Petering, D. H. (2024). Bringing real inquiry‑based science to diverse secondary educational environments: A virtual zebrafish laboratory to investigate environmental health. Zebrafish, 21(2), 73–79. https://pmc.ncbi.nlm.nih.gov/articles/PMC11035841/#B11

CBC News. (n.d.). Vaping in Canada: What we know — Canada has some of the highest teen vaping rates in the world, new data shows. https://www.cbc.ca

Health Canada. (2018). Infographic: Vaping – the mechanics. Government of Canada. https://www.canada.ca/en/services/health/publications/healthy-living/vaping-mechanics-infographic.html

Heart and Stroke Foundation of Canada. (n.d.). Protecting youth from the vaping crisis.https://www.heartandstroke.ca/-/media/pdf-files/position-statements/vaping-crisis-position-statement.pdf?rev=325ec1c4d23942058acf0d55d2b97989

Springer Nature. (2020). Pictures of normal zebrafish development [PDF]. https://link.springer.com/content/pdf/10.1186/s12302-020-00398-3.pdf

Tomasiewicz, H., & Petering, D. (2022). Electronic cigarette toxicology research using zebrafish embryos as a developmental model. WInSTEP‑SEPA Program, University of Wisconsin–Milwaukee; National Institute of General Medical Sciences, Science Education Partnership Award Program.