1 Effects of Nicotine on Zebrafish Embryo Development Melissa Dunlap Greendale High School
2 Abstract It is recommended by doctors that women who are pregnant do not smoke because of the negative effects that, one of the main ingredients in cigarettes, can have on a developing embryo. An experiment was conducted to determine the effect that different concentrations of have on a developing zebrafish embryo. Fertilized zebrafish eggs were put into three different concentrations of solutions and a control, solution. Each day, the number of eggs dead, alive, and hatched were recorded along with any physical deformities.in conclusion, we found that those exposed to higher concentrations of were more likely to die sooner and more likely to have a curved spine once hatched. Introduction Nicotine, a chemical found in tobacco products, is one of the most heavily used addictive drugs in the U.S. (Nicotine, 2010). The Center for Disease Control estimates that nearly one in five adults smoke habitually (Fast Facts, 2011). Nicotine, which is highly addictive, leads to a release of chemicals from the brain which can cause heart, blood pressure, and sleep problems (McCall, 2008). The chemical has been found to be especially harmful to developing human embryos. The effects of are seen in every trimester of pregnancy, from increased spontaneous abortions in the first trimester, to increased premature delivery rates and decreased birth weights in the final trimester (Clark and Lambers, 1996). Additionally, up to 5% of infant deaths could be prevented if pregnant women did not smoke (Smoking can affect your baby's health, 2012). It is recommended by doctors that women who are pregnant not use tobacco products because of s harmful effects. The problem we tried to figure out in our study is how three different concentrations of will affect the development of zebrafish. In our experiment, zebrafish embryos are used to test the effects of embryonic exposure. These species were chosen because they are vertebrates, therefore their development can possibly be representative of how human embryos would develop if exposed to the same environmental factors. Three different concentrations of solutions are used, starting with the same number of eggs in each solution on the first day. Every day, the number dead, alive, and hatched are recorded. Any other observations are noted. We predicted that if we increase the concentration of, then the developmental effects of the zebrafish will be more severe, because is harmful to developing creatures. This hypothesis was correct. For the most part, our findings show us that compared to those in the no- salt water, the eggs exposed to generally die at a quicker rate and they are more likely to have curved spines. The severeness of the curved spines seems to be heightened by the higher concentrations. Generally, some type of effect or abnormality was noted in most embryos exposed to. The results observed in our experiment help us to get a better idea of what type of effects has in developing embryos of zebrafish, and since zebrafish have multiple similar characteristics as humans, we can vaguely see how may affect us as well. Through this, we can conclude that increased exposure to can lead to an increased chance death and possible birth defects in human embryos like we see in the
3 zebrafish. These results are significant because knowing the effects of on a developing baby can help educate women and convince them not to smoke while pregnant. Roughly one in ten women admitted to smoking during pregnancy (Tobacco Use and Pregnancy, 2010), so experiments like this can help to increase awareness and prevent embryonic exposure by promoting healthy lifestyle choices. Materials and Methods Materials used in the investigation include: 1 3x4 well tray 120 zebrafish eggs 100 ml of.05 mg/ml solution 100 ml of.1 mg/ml solution 100 ml of.2 mg/ml solution 500 ml dechlorinated water 500 ml of Instant Ocean Roughly 30 droppers notebook/ pen microscope Paper Towel Cell phone for taking pictures Incubator 10 ml graduated cylinder 100 ml graduated Cylinder ml waste beaker 3 closeable containers for storing solutions Safety: As a safety precaution, goggles were worn to prevent any harmful solutions from having contact with the eyes. If any was spilled on the skin, it was washed off right away. Any unwanted solution or eggs were disposed of through the use of a waste beakers as to assure that nothing toxic was poured down the sink and into the water supply. The independent variable was the concentration of the solution. This was either the control group exposed to a solution with 0.0 mg/ml of solution, or the experimental groups exposed to 0.05mg/mL, 0.1mg/mL and 0.2mg/mL of the solution. The dependent variable was the development of the zebrafish. Things that were kept constant throughout the experiment were the type of egg used, the methods of handling/ replacing solutions, solution temperatures, and the time at which observations were recorded. Procedure: Day 1 To begin the experiment, all materials were obtained (for more information on how the zebrafish were bred, see the zebrafish module). Each of the 12 wells in the 3x4 well plate were filled half full with instant ocean using a dropper. Using a different dropper, ten eggs were placed in each well, so that there were 120 eggs all together at the end of the first day. This number was used so that even if many died overnight (before the solutions were added), we would still have enough to work with. The 120 eggs were then examined under a microscope and dead embryos
4 were properly disposed of and replaced so that they wouldn t contaminate the others. Using distilled water, pure solution, and the graduated cylinders, the three different solution concentrations were prepared and then poured into the labeled, re closeable containers. The.05 mg/ml solution was made by mixing 5 ml of with 95 ml of Instant Ocean. The.1 mg/ml solution was made by mixing 10 ml of with 90 ml of Instant Ocean. The.2 mg/ml solution was made by mixing 20 ml of with 80 ml of Instant Ocean.The well tray was then put in an incubator overnight to keep the zebrafish at their necessary temperature and increase their chances of survival. Set up of well plate after first day: Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Instant Ocean w/ 10 embryos Day 2 The original 120 eggs were looked at under a microscope and dead ones were removed so the living ones wouldn t be contaminated. The number dead, alive, and hatched were recorded. The well tray was viewed so that there were three rows and four columns. The top row of 3 wells was labeled control and the instant ocean was sucked out of this row with a clean dropper and replaced with clean instant ocean. In the second row of three wells, the instant ocean was sucked out using a clean dropper. These three wells were then filled half full with the.05 mg/ml solution and that row was labeled.05. In the third row, the instant ocean was sucked out using a clean dropper and was replaced with.1 mg/ml solution and labeled accordingly. The same was done with the fourth row, except the instant ocean was replaced with.2 mg/ml solution and labeled appropriately. The well tray was then placed in an incubator overnight. Set up of well plate after second day and following days: Control Control Control.05 mg/ml solution.05 mg/ml solution.05 mg/ml solution.1 mg/ml solution.1 mg/ml solution.1 mg/ml solution.2 mg/ml solution.2 mg/ml solution.2 mg/ml solution Day 3 The wells were inspected under a microscope and the number dead, alive, and hatched were recorded. Any abnormalities and observations were also noted. The dead embryos were removed
5 and disposed of in a waste beaker as to not pollute water supply. The liquid in each well was sucked out using a clean dropper as to not contaminate and replaced with fresh liquid of the same kind and amount to say consistent. The well tray was again stored at an incubator overnight to allow zebrafish their ideal conditions. Day 4 Repeat of day three Day 5 Repeat of day three and four. All remaining eggs were disposed of in a waste beaker. The live fish were also cleaned up appropriately. The wells were rinsed cleaned using instant ocean, and all materials were cleaned and put away properly. The procedure was followed in accordance with the methods suggested by the SEPA module, which can be found online. Results Figure 1a. Eggs Living, Dead, Hatched After 24 hrs of sitting in Instant Ocean Solution Well 1 Well 2 Well 3 Average Alive Per Well Instant Ocean 6 alive, 4 dead, 7 alive, 3 dead, 5 alive, 5 dead, 6 Instant Ocean 8 alive, 2 dead, 4 alive, 6 dead, 8 alive, 2 dead, 6.67 Instant Ocean 7 alive, 3 dead, 5 alive, 5 dead, 8 alive, 2 dead, 6.67 Instant Ocean 4 alive, 6 dead, 5 alive, 5 dead, 7 alive, 3 dead, 5.33
6 Figure 1b. Eggs Living, Dead, Hatched After 24 Hours of being exposed to Nicotine Solution Well 1 Well 2 Well 3 Average Alive Per Well Control (no ) 5 alive, 1 dead,, visible movement in egg 7 alive, 0 dead, 1 hatched 5 alive, 0 dead mg/ml 8 alive, 0 dead,, visible blood pumping 3 alive, 0 dead, 8 alive, 0 dead, mg/ml 7 alive, 0 dead, spine curve visible in egg 5 alive, 0 dead, 5 alive, 2 dead, 0 hatched mg/ml 4 alive, 0 dead 5 alive, 0 dead, 4 dead, 3 alive, 0 hatched 4.33 Figure 1c. Eggs Living, Dead, Hatched After 48 Hours of Being Exposed to Nicotine Solution Well 1 Well 2 Well 3 Average Alive Per Well Control 4 alive, 1 dead 6 alive, 0 dead 2 hatched 4 alive, 1 dead 1 hatched mg/ml 6 alive, 0 dead 1 hatched (slight spine curve) 3 alive, 0 dead 8 alive, 0 dead 1 hatched (spine curved roughly 80 degrees) mg/ml 7 alive, 0 dead 1 hatched (extreme spinal curvature) 5 alive, 0 dead 5 alive, 0 dead mg/ml 4 alive, 0 dead 3 alive, 2 dead 1 hatched (curved spine) 3 alive, 0 dead 3.33
7 Figure 1d. Eggs Living, Dead, Hatched After 72 Hours of Being Exposed to Nicotine Solution Well 1 Well 2 Well 3 Average Alive Per Well Control 1 alive, 2 dead 2 hatched 6 alive, 1 dead 4 hatched 4 alive, 0 dead 1 hatched mg/ml 2 alive, 4 dead 2 hatched (slight spine curve) 1 alive, 1 dead both hatched 4 alive, 2 dead 2 hatched (spine curved roughly 80 degrees) mg/ml 0 alive, 7 dead 3 hatched (extreme spinal curvature) 0 alive, 5 dead 4 alive (2 seem to have a curved spine in the egg), 0 dead 1 hatched mg/ml 0 alive, 2 dead 2 hatched (very curved spine) 0 alive, 2 dead 1 hatched (curved spine) 0 alive, 2 dead 1 hatched 0 Figure 2a. ( Per Well)
8 Figure 2b. Average Eggs Hatched Per Well Over Time* *The.2 mg/ml line is underneath the.1 mg/ml line, as the data is the same for both Figure 3a: Control Group Fish Figure 3b:.05 mg/ml solution Fish
9 Figure 3c:.1 mg/ml solution Fish Figure 3d:.2 mg/ml solution Fish Description of Results: In the control group our results show us that after 72 hours of being exposed to the instant ocean, this group had the highest average number of eggs hatched per well as seen in figure 2b. Not only did our control group have the highest amount of living eggs as time went on, but of the living eggs, all of them seemed to have normal physical growth, like the fish depicted in figure 3a. Their spines were all straight, and they could be found swimming around their wells like they normally would be. The control group had the lowest average fish dead per well per day with.78 fish dying per well each day on average. The group exposed to the.05 mg/ml solution had the second highest average fish hatched per well by the end of 72 hours, this again can be seen in figure 2b. This group also had the second highest average amount of fish alive per well by the end, with the second lowest rate of fish death. We can again see a connection between amount of living fish and amount of hatched fish. Of the three groups, the fish in this group were less likely to die before the 72 hours. On average, 1.44 fish died per well each day in this group. We also found that of the fish that hatched in this.05mg/ml solution, about 20% of them had some type of spine curvature. Most of the curves in this group were not very extreme, as illustrated by figure 3b. The majority of the hatched eggs in this group, however, appeared to have a normal physical appearance. Those that had a curved spine from this group still were actively moving within their well. The group exposed to the.1 mg/ml solution were tied with the.2 mg/ml
10 solution with average eggs hatched by the end of 72 hours. The.1 mg/ml solution group had more living eggs at the end of the 72 hours than the.2 mg/ml solution. While these two groups started with and ended with different amounts of eggs, their rate of average fish deaths per day were the same with roughly 1.78 fish dying per well each day on average. The.2 mg/ml group was the only group to have no living fish by the end of the 72 hours. Both the.1 mg/ml group and the.2 mg/ml group had fish develop with spinal curvatures. About 25% of the fish in the.1 mg/ml solution had a curved spine, and about 30% of the fish in the.2 mg/ml solution had a curved spine. We found, however, that for the most part, the curved spines of the fish in the higher concentration were much more severe than those in lower concentrations, as seen in figures 3c and 3d. The fish were equally as likely to die in the.1 mg/ml solution as the.2 mg/ml solution. Discussion In our experiment we sought to find the effect that different concentrations of would have on developing zebrafish embryos. Eggs were placed in three different concentrations of and one solution containing no. Overall we found that different concentrations had different effects on the embryos, especially when it came to embryo death, hatching rate, spine formation. Our data supported the hypothesis, because as concentration increased, the negative impact on the fish also increased, in this case it was death rate. Based off of our results and data, we can conclude that concentration can be attributed to the death of the zebrafish, with more leading to a higher death rate.those in the lower concentration were less likely to die than those in the higher concentration. The control group may have had more eggs hatched simply because they had more eggs that stayed alive, as the embryos exposed to more were more likely to die. Death among the control group eggs could be attributed to contaminated instant ocean or fungus, among other things.we can also conclude that increased exposure lead to the physical deformities in the fish, most notably a curved spine. The higher the concentration of, the more severe the spinal curvature is. Some errors that may have affected the accuracy of our data is that when we were taking the liquid out of the wells each day, we may have not gotten all of it so the replaced liquid may not have been completely fresh. Another error could be that if we thought an egg was alive that was actually dead, it may have lead to the death of the other fish in its well. A third could be that we may have forgotten to use a clean dropper every time we switched concentrations, so there may have been some cross contamination there. The conclusions found in this study are very relevant to our real lives because they convey the possible effects of exposure to developing human embryos, as findings here can be applied to other vertebrates. It is true that is equally as harmful to developing human babies as it was to the zebrafish. Other studies have even found that prenatal exposure to
11 can cause mitotic abnormalities and that abstinence from all forms of should be advised to pregnant women for the entirety of gestation (Wickström, 2007). Continuous findings about the negative effects of are important because as stated earlier, many women smoke while pregnant, possibly because they don t truly understand the damage that they are doing. It is important we use information like this to educate others and to be a wake up call for those who find it harmless to expose developing embryos to cigarettes or other tobacco products.
12 References Fast Facts. (2011, June 5). Centers for Disease Control and Prevention. Retrieved November 30, 2013, from Lambers, D., & Clark, K. (2013, April 5). The maternal and fetal physiologic effects of. Science Direct. Retrieved October 9, 2013, from McCall, B. (2008, November 20). Nicotine: why it s bad for you. COSMOS magazine Nicotine why its bad for you Comments. Retrieved December 2, 2013, from Nicotine. (2010, May 13). Psychology Today. Retrieved November 30, 2013, from Smoking can affect your baby's health. (2012, November 8). American Cancer Society. Retrieved November 29, 2013, from Tobacco Use and Pregnancy. (2010, September 23). Centers for Disease Control and Prevention. Retrieved December 1, 2013, from Wickstrom, R. (2007, September 3). Effects of Nicotine During Pregnancy: Human and Experimental Evidence. National Center for Biotechnology Information. Retrieved October 10, 2013, from
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