The Harmful Effects of Nicotine on Zebrafish Embryos Waukesha North High School Anna Merten Abstract This experiment was done in order to draw attention to the dangerous effects of nicotine on developing human fetuses and human health in general. This experiment also brings attention to the severity of the medical issues that nicotine can cause, an understanding of these dangers can decrease the amount of people that have them. For this experiment zebrafish embryos were put in five different nicotine concentration solutions for 72 hours, so that the negative effects of the different amounts could be recorded over time. This was done to understand the developmental delays and deaths caused by nicotine. The data collected shows that the higher the amount of nicotine the more deaths and developmental delays there will be. The eggs that did hatch in the nicotine, were smaller and had less movement. These results relate to human health because they show how quickly nicotine can affect someone and how severe this effect can be. These effects could possibly be used to have a better understanding of how to medically treat health issues from nicotine as well. Introduction Nicotine is an extremely powerful, dangerous, and addictive substance, the effects of nicotine on the body can be devastating and even life threatening. From scientific observation and experiments scientists have been able to figure out that nicotine enters the brain and disrupts normal function. This could mean a number of different things including stunted size and growth, weakened bones, and a lack of tissue building and repairing (Danish 2013). Nicotine mainly enters the body through smoking. It travels through the bloodstream and goes to the brain in 7-15 seconds (ucanquit2.org). For this experiment the effects of nicotine on zebrafish was tested, to get an understanding of what it does and how extreme it actually is. Zebrafish were chosen because they are genetically similar to humans, and the impact of drugs and other things is very easy to see in them. They also hatch fairly quickly so the abnormalities after being hatched were easier to see. The effects were able to be seen in a couple of days instead of weeks. It was hypothesized that if more nicotine was in one well than another, then the zebrafish in the well with more nicotine would have more developmental issues and a larger death toll because, from previous knowledge, nicotine causes multiple severe deformities and death.
Material and Methods 1 Multi-well Tray 4 Stock Solutions of Nicotine (0.01, 0.05, 0.1, 0.2 mg/ml Nicotine) 1 0.00 mg/ml Nicotine Solution 1 Beaker 1 Sharpie 1 Roll of Masking Tape 3 Small and Large Bore Pipettes 50 Zebrafish Embryos 1 Dissecting Scope First, a tray with five wells was selected, next, 50 zebrafish eggs were retrieved, and a large pipet was used to place ten eggs in each well. Wearing gloves during this process is a necessary safety precaution to protect from any potential dangers. This placing of the eggs was done under a dissecting scope in order to have accuracy in numbers and make sure the eggs were all alive. The healthy embryos appeared to have a black dot under the dissecting scope and were light in color. Next, the bad eggs were removed and replaced with good eggs using the large bore pipette. After there were ten healthy eggs in each well the small bore pipette was used to carefully suck the control solution out of each well, doing so under the dissecting microscope to avoid sucking up any embryos. Once each well had no excess liquid they were labeled 1-5, as a way to keep track of the solution in each one. Then, carefully, 1 ml of assigned stock solution nicotine was squirted into each well. The first well was the control and got 0.00 mg/ml nicotine concentration, the second well got 0.01 mg/ml, the third well got 0.05 mg/ml, the fourth well got 0.1 mg/ml of nicotine, and lastly the fifth well got 0.2 mg/ml. After this was completed, the microscope was used to take pictures and record baseline data and observations of the zebrafish eggs in each well. These baseline observations recorded the sizing, coloring, movement, deformities, and amount hatched or deadly the embryos. This is because the development and survival rates of the zebrafish eggs are what is being tested overall. Things like size, coloring, movement, and deformities all help determine these overall measures. If an embryo was small or not moving, it meant that it was delayed and possibly close to death. The coloring also helps determine health, if the embryo was clear or grayish it was most likely dead or close to death. Next, the top was placed on the tray and it was placed in a safe place for 24 hours. After the 24 hours the zebrafish were observed, again taking notes and pictures under the microscope. The dead eggs were removed out of each well with a pipette and the number of hatched and dead embryos were counted and recorded. Next, using the small pipette, the solution was removed from each well, doing so under the dissecting
scope prevented any harm to the zebrafish. Each well was replaced with 1 ml of the correct nicotine stock solution that it had For the first 24 hours, making sure the correct one went in each well. The wells were stored in a safe place and the process was repeated every 24 hours for 72 hours. After the 72 hours, final assessments and conclusions were made. A chi-square analysis was run at the end of the experiment to ensure that the data collected was statistically significant and not based on possible chance. Results The effects of nicotine on zebrafish embryos was researched in order to better understand what nicotine does to a developing human being. It was hypothesized that the more nicotine present, the more zebrafish embryos there would be that died or did not hatch. We set up our experiment with observation every 24 hours because, based on background research, it was felt that this would give enough time for change in the embryos and not too much time where something would be missed. Ten embryos were chosen to be put in each solution to ensure accurate data was collected for every concentration. There were two dependent variables in the experiment, the number of zebrafish dead and the number of zebrafish hatched. The independent variable in the experiment was the nicotine solutions, this would be the solutions of 0.01 mg/ml, 0.05 mg/l, 0.1 mg/ml, 0.2 mg/ml. The control variable in the experiment was the 0.00 mg/ml nicotine solution. These variables are related because the strength of the nicotine solution (independent variable) causes there too either be a smaller or larger amount of either embryo deaths or embryo hatchings (dependent variable). There were a few controls present in the experiment including the temperature in which the zebrafish were kept. It was ensured that they weren't exposed to any different amounts of light either. Each well was also exposed to the nicotine for the same amount of time to make sure that the results weren't influenced by exposure time. To make sure that the data collected was accurate and not based on chance a chi square analysis was run. The results of the two chi-squares showed that both the number of zebrafish embryos hatched and the number of zebrafish that died in each concentration of nicotine was statistically significant and not based on pure chance. The first chi-square that was calculated was for the data on the amounts of dead and alive zebrafish. The chi-square value for mortality was 25.21. The data was interpreted by looking at the degree of freedom (4) and following it on the Chi-Square Table to the 0.05 category. The number in that area was 9.49 and since 25.21 is greater than 9.49 the Null Hypothesis was rejected, meaning the data collected on the number of zebrafish live and dead was statistically significant and not based on chance. The same process was repeated in order to find the validity of the number of zebrafish hatched and unhatched. The expected number for all concentrations of nicotine hatched came out to be 4. The chi-square value for hatched and unhatched embryos was 14.18. When the number
was checked on the chart with a degree of freedom of 4 the conclusion was made that the Null Hypothesis was rejected and the data was confirmed to be valid. As shown in Figure 1 after 48 hours there was a large increase in zebrafish deaths in all concentrations except the control. Table 2 and 3 demonstrate that a fair amount of zebrafish died after they had already hatched. It is observed that the zebrafish in the nicotine solutions were smaller in size and had less physical development then the control. The severity of the development and size delays is larger in the higher concentrations of nicotine as well. All of this can be seen in Table 1 and in the visuals in Figure 3. It is also shown that the higher the nicotine concentration is, the smaller amount of embryos there will be that hatch (Table 5). Also seen, is that almost all of the zebrafish in all of the nicotine solutions were dead by the end of the 72 hour period. Figure 1 Figure 1 demonstrates that as the nicotine concentration increased the more zebrafish died at the end of the 72 hour time period. It also shows that there was a large increase in death after the 48 hour period.
Figure 2 Figure 2 shows that the more nicotine present, the less number of embryos there were that hatched. Also shown is that the majority of eggs hatched after 48 hours. Table 1: Observations of Embryos Over 72 Hours Hours Post Fertilization Observations 0 hours A few of the embryos were seen moving around inside the eggs. All eggs appeared to have little tails. 24 hours All embryos were a little bit darker. The tails were much more distinct. A few had black spots in the eggs. 48 hours A zebrafish in well #2 was observed to be hatching. All embryos has large black eyes. The hatched zebrafish moved slower in wells 2-4 (none yet hatched in 5). The more nicotine in a well, the smaller the eggs. 72 hours All zebrafish in well #1 were observed to be moving well. All hatched were smaller than the control. The higher the concentration of nicotine, the more limp and shapeless the hatched zebrafish became. Protists were seen to be attacking in wells #2-5. At the start all embryos were not hatched and showed no movement, this gradually increased over the time period, although most died and did not hatch in the higher concentrations.
Table 2: Number of Zebrafish in Different Concentrations of Nicotine Dead Over a Period of 72 Hours Treatment # of Starting Embryos # Dead After 24 Hours # Dead After 48 Hours # Dead After 72 Hours 0.00 mg/ml 10 1 1 1 0.01 mg/ml 10 1 3 8 0.05 mg/ml 10 0 0 8 0.1 mg/ml 10 0 0 9 0.2 mg/ml 10 0 0 10 Overall almost all eggs were dead by 72 hours in every well except for the control. Most of the zebrafish did not die until after the 48 hour mark. Table 3: Number of Zebrafish in Different Concentrations of Nicotine Hatched Over a Period of 72 Hours Treatment # of Starting Embryos # Hatched After 24 Hours # Hatched After 48 Hours # Hatched After 72 Hours 0.00 mg/ml 10 1 2 9 0.01 mg/ml 10 1 2 3 0.05 mg/ml 10 0 2 2 0.1 mg/ml 10 1 3 4 0.2 mg/ml 10 0 0 2 Table 3 shows that the majority of embryos did not hatch at all, except for in the control. there was a steady gradual increase of hatched eggs over the 72 hours, again not including the control.
Figure 3: Visual Records of Zebrafish Embryos Over 72 Hours 0.00 mg/ml 0.01 mg/ml 0.05 mg/ml 0.1 mg/ml 0.2 mg/ml 24 Hours 48 Hours 72 Hours All of the eggs start out unhatched then gradually hatch or die between the 24 hour and 48 hour marks. The size and development of the embryos is noticeably small and delayed in the higher nicotine concentrations. Discussion The results of this experiment supported the original hypothesis that the higher the nicotine concentrations are in a well then the higher the amounts of unhatched or dead embryos there will be. This related to the research of the effect nicotine has, because it was known how quickly nicotine travels to the brain and cuts off the oxygen flow. The death of the embryos in such a short period of time, where the majority of the embryos died at or a little after 48 hours (Figure 1), showed how much this is actually true and how quick the poison of nicotine can become dangerous. The delays in size and hatching in the zebrafish showed that the nicotine slows down the brain and body growth systems and causes mutations or errors in development. The more a person smokes or ingests nicotine the worse that person's health and development will be was proven by the fact that zebrafish in higher concentrations of nicotine had worse effects. This is also a trend in the data seeing that the fish continually got worse as the
concentration of nicotine steadily increased. From prior research it was learned that nicotine can slow down growth significantly. This was proven in the research because the more nicotine present in a well, the smaller the hatched zebrafish in it (Table 1). A suggestion for improving this experiment would be adding more concentrations, to all observation of more subtle changes. Having more zebrafish in each concentration would also bring more accurate results, seeing that there is larger amount of test subjects, and a smaller amount of chance. An error that could have possibly been made in this experiment is extracting zebrafish that were still alive from the wells. This could have happened when a zebrafish had the characteristics of being dead, but was actually just very small or did not have any movement or much change. In this case, a dissecting scope or microscope that allows closer magnification or another method of determining life could improve the experiment. A question that could be answered with further in depth research would be what exactly happens in the brain that causes these developmental delays. Overall, nicotine causes devastating developmental delays in growth and ultimately results in deaths, these deaths could easily be avoided by not consuming nicotine and having an understanding of how deadly it can be. References and Literature Cited Danish, E. (n.d.). Does Smoking Stunt Your Growth? Retrieved from http://www.healthguidance.org/entry/15558/1/does-smoking-stunt-your-growth.html Effects of Nicotine. (n.d.). Retrieved from https://www.ucanquit2.org/nicotineeffects?p=1 Mandel, A. (2010, March 11). Nicotine Effects. Retrieved from http://www.newsmedical.net/health/nicotine-effects.aspx