Research Project: AP Biology Name(s) Title N. Schaefer and J. Baerwald Effect of UV Exposure to Zebrafish Development Abstract 2 Introduction 3 Materials and Methods 5 Safety 5 Materials (list) 5 Procedure 5 Results 8 Discussion 15 References 17
Abstract The purpose of this experiment was to identify the effect of UV radiation on the zebrafish with applied sunscreen. In humans, increased UV light exposure has caused many people to develop skin cancer because the UV causes mutations in the DNA (Allen J., 2001). Knowing sunscreen is a method to prevent mutations, the experiment was designed to see which SPF sunscreen would work best in preventing mutations to the developing zebrafish. For the experimental group, the zebrafish were covered with plastic wrap sprayed with either 15, 30, or 50 SPF sunscreen. The positive control group had no protection from the UV light. Then these 4 groups were exposed to the same amount of UV light by placing them into the Crosslinker fb uvxl 00 UV box. To see the effect of the experiment, the zebrafish were monitored, specifically paying attention to abnormal growth patterns and deaths, similar to the experiment done by Gonzalez (2017). The predicted results were that if there is an increase in UV exposure, then there will be more deformities and/or deaths in the zebrafish because light exposure can cause damage at a molecular level and damage DNA.The result from the experiment was that with too little UV protection there were more deaths and deformities, and with too much UV protection there was an increase in fungal related deaths. The results tell us that, while avoiding UV radiation can lower the chances of developing cancer, it also may be that not encountering a certain amount of UV radiation may have adverse effects in itself. While most people have access to showers and other personal hygiene tools, those who do not may benefit from being exposed to some UV radiation, which can be received from the sun, in order to prevent surface fungal growth.
Introduction Background Information According to researchers at UW Milwaukee, zebrafish are good models because they develop outside of the mother, are produced in large numbers, develop in sync in a clutch, are optically clear, develop rapidly, and the fish are able to change their UV light absorption (Petering, D. H., 2003). It is important to note that just because they can change their UV light absorption does not mean that they will survive UV light exposure as explained by A. Gonzalez s lab which found that there is a an amount of UV exposure on the fish that will kill/hurt them (2017). The fish develop in clutches that provide a large number of eggs with little to no genetic differentiation, meaning genetic differences will not be a variable that contributes to the results. Also, they develop in sync, making it is easier to see the effects of the environmental factors among the experimental groups (Petering, D. H., 2003). Development of the Embryos:
Identifying development stages: (Petering, D. H.) UV Light According to Allen (2001), as stratospheric ozone decreases, UV radiation is allowed to pass through, and exposure at the Earth s surface increases. This idea that ozone is decreasing leads to The United Nations Environment Program [estimating] that a sustained 1% depletion of ozone will ultimately lead to a 2-3% increase in the incidence of skin cancer (Allen, 2001). The cause of the increase in skin cancer would be due to less UVB radiation being absorbed by the atmosphere, this causes skin cancer because the UVB s wavelength can cause breaks in the DNAs bonding, leading to irregularities in cell division (Allen, 2001). A counter to the reduced ozone and increase in UV radiation is the amount of dust and smoke in the air because of large fires, like the one in California in 2017. Also, the increased water depth, due to glaciers melting, could affect how much sunlight gets to the fish, since the increased amount of hydrogen atoms will increase the absorbance of the UV light before it gets to the fish. With the potential change in UV exposure, one may ask how they can protect themselves and/or their growing fish from this UV exposure and how much different levels of sunscreen really changes the amount of
protection one may have. So, in this experiment the differences in protection from exposure of UV light provided by sunscreen(15, 30, 50) will be compared with the effects the development of the zebrafish embryos. The predicted results were that a lower SPF will provide less protection from UV radiation and result in more deformities and death in the fish, because less protection from sunscreen will result in more damage to DNA, which may lead to death and deformity. Development of Methods Original data from a previous lab was used: Irradiation of the Zebrafish Embryo by Gonzalez (2017) which explained, there was a very statistically significant drop in the amount of living embryo in said dish in comparison to the control dish. Furthermore on the infosheet for the UV box, it was found that the box itself doesn t give off the same amount of energy over time, so to get an exact amount, a test set the amount of energy expended very high, then let it run approximately -15 seconds. The fish were exposed to 516X0 microjoules/cm 2 every time. To compare this to the intensity of the sun which gives off about 00 joules/sec/m 2 (Murphy. 2012), the energy the fish were exposed to was 5.16 joules/m 2 meaning if the fish were exposed for seconds of the sun at full energy the fish would have our data at an extreme as the fish only experience.0516% of the UV light it would if on land. Materials and Methods Safety Do not drink or eat by the lab station, this will prevent accidently drinking or eating the fish. Do not try to blind yourself by looking at the UV light. Properly expose the water by dumping it down the drain. Do not accidentally drink the methyl blue, this should be prevented by not having food or drinks in the labstation. Material Zebrafish embryos 2 Well Plates Crosslinker fb UVXL 00 UV Box Spray Sunscreens (15, 30, 50) Aluminum foil Plastic wrap/ Seran wrap Scissors Paper towel Incubator box Sharpie
Procedure 1. Spawning tank setup 2. Obtain rinsed embryos from your teacher. 3. Label your plate with your name and class hour. Use the Sharpie provided. 4. Fill each well of the plate with Instant Ocean/Embryo Media solution and methyl blue solution (to help reduce fungal death) using the transfer pipette. Divide the embryos so there is an equal amount in each plate. 5. Label the columns of the well plate as a positive control, SPF 15, SPF 30, and SPF 50. Use another well plate to use as the negative control (well plate will not receive UV exposure). Use aluminum foil to cover the bottom of the well. *UV light should only come into the well plate through the top. Use the Sharpie to label the Aluminum foil to proper orientation to prevent confusion and help with labeling. See Figure 1. (Figure 1) 6. Record exact numbers of live embryos, on student data sheet. Observe your embryos under the dissecting microscope. Record observations on student data sheet. 7. Place each plate in the incubator overnight. 8. The next day, remove the plate from incubator. Remove dead embryos from the plate using the transfer pipette. Squirt dead embryos into waste beaker. Count remaining embryos, hatched fish, and record in data table. Remove Instant Ocean/Embryo Media solution from the Falcon dish. NOTE: Tilt the plate so the embryos settle and remove the liquid from the top. Add fresh Embryo Media liquid 9. Place plate under dissecting microscope and record observations on student data sheet. Note/describe any developmental markers and abnormalities.. Remove 1-2 embryos and place on the depression slide with cover slip. Observe the embryos using the compound microscope. Record observations on student data sheet. Repeat for all light types. 11. Return the embryos to their well in the plate.
12. Cover the well plate with clear plastic wrap (to protect sunscreen from getting into the wells). Cut 4 even strips of additional cling wrap and spray each strip with either 15,30, or 50 SPF sunscreen. Place the strips over the corresponding labelled column. 13. Set the Crosslinker fb UVXL 00 UV Box to expose the fish to 516 X0 microjoules/cm 2 14. Start the UV Box 15. Take the well plate out when the fish have been exposed. Remove all of the cling wrap and place the cover back onto the well plate. Return the plates to the appropriate incubator 16. Repeat steps 6-15 Record all data. 17. Use a T-test to determine statistical significance, in the instance Graphpad was used. 18. Place all embryos and fish in waste container. Your teacher will properly dispose of the organisms
Results The experimental groups consisted of the wells that were exposed to UV light but protected by SPF 15, 30, and 50. The positive control was the fish that were fully exposed to UV light. The negative control was the fish that were not exposed to any UV light. The fish protected by SPF 30 showed no abnormalities and little death occurred. It was found that the fish with full exposure and SPF 15 had the most observable physical deformities usually related to scoliosis of the spine or spacing of the eyes shown by the pictures below. SPF 15 had a difference in nutrient sack growth than all the others shown by pictures below. Fully exposed fish and fish protected by SPF 50 experience embryo death at a higher rate than the others shown by the graphs. It was also seen that fully exposed fish and fish protected by SPF 50 had a lower hatch rate than the other cells. Day 1 Level of sunscreen Full exposure (B) 15 SPF(C) 30 SPF(D) 50 SPF (E) Observed Amount -All unhatched -one half-hatched Observations: Starting pictures no noticable differences between all the fish.
Day 2 Level of Exposure No Exposure (A) Full Exposure (B) SPF 15 (C) SPF 30 (D) SPF 50 (E) 2 hatched 9 total 1 hatched 0 2 hatched 1 hatched Observations Fish 4 hatched 8 total No hatch 1 hatched 0 0 hatched and 1 unhatched 11 total No hatch 3 hatched 2 hatched 0 Observations: Noticed a changed yolk sack and eyes seem to be more spaced out. Unexposed group added as positive control.
Day 3 Level of Exposure No Exposure (A) Full Exposure (B) SPF 15 (c) SPF 30 (D) SPF 50 (E) 2 unhatched and 9 total 9/ Dead- Fungus 1 still aliveunhatched 1 dead - 2 unhatched - 7 alive and well 6 unhatched alive - 1 unhatched dead - 3 hatched and alive 3 unhatched dead - 7 alive hatched Observations total, 2 unhatched 9 alive- 5 unhatched 4 hatched Curved spines Spaced eyeballs 1 dead-unhatched 6 unhatched 4 hatched 6 unhatched - 1 unhatched and dead - 3 alive 7 unhatched- 1 dead unhatched 1 hatched -1 hatched dead 11 hatched alive- 6 unhatched 4 hatched 4 unhatched - all have messed up eyes others alive 3 unhatched and dead - 7 unhatched alive 4 unhatched- dead 3 unhatched alive - 3 hatched alive Many showing curved spines Some curved spines shown Notes: To combat the fungal deaths we are adding methyl blue. This will hopefully kill the fish without harming the fish. Noticeable eyes more more spaced out
Day 4 Level of Exposure No Exposure (A) Full Exposure (B) SPF 15 (C) SPF 30 (D) SPF 50 (E) Observation 11 hatched alive Well emptied because of fungus yesterday 8 hatched alive - 1 unhatched alive - 1 unhatched dead 1 unhatched dead - 2 unhatched alive - 7 alive hatched 1 unhatched alive - 9 hatched alive 6 hatched alive - 2 unhatched alive 5 alive unhatched - 1 alive hatched - 4 dead hatched alive hatched 1 dead unhatched - 9 alive hatched 9 unhatched dead - 1 hatchd dead 11 hatched alive 2 dead from fungus hatched - 5 unhatched alive - 3 unhatched alive 9 hatched alive - 1 unhatched unhatched alive 4 dead unhatched - 1 unhatched alive - 5 hatched alive 4 alive and hatched - 6 unhatched dead (fungus) Observations: Full exposure and SPF 15 show curved spines
Graph 1. Graph 2.
Data The data used is the average results for the cells and the data used to make the graphs. Since each well is technically independent of the other, the average of the data was used for graphing. The equation used is (A r +B r +C r )/3=Average
Discussion There was statistical significance in two sets of data: the well protected by SPF 50 and the well with full exposure compared to well with no UV exposure. Using Graph 1, the average number of hatched fish between the wells protected by SPF 50 and the well with no UV exposure, there statistical significance(p=.0194). Continuing there was statistical significance between the wells with no UV exposure and the well with full UV exposure(p=0.0029). Both sets of data could be used to imply that there are amounts that are too much UV light and too little UV light for these fish to grow normally. A conclusion can be made, as the statistical significance shows the likeliness that our data appeared randomly was extremely low. Although the amount of unhatched deaths in comparison to alive unhatched did not have statistical significance(graph 2), on their own it is suggested to repeat the experiment in the future to increase the sample size. If the results were similar but larger causing statistical significance between the positive and negative control, it would mean that a conclusion could be drawn that the deaths of the embryos were caused by too much UV light. It is important to note all p values were calculated using graphpad. The intensity of UV radiation getting through to organisms on Earth is changing. Whether it is decreasing due to changes in oceanic levels (heating of the ocean), or increasing due to harm to the ozone layer, it can be related to parts of our experiment where 50 SPF change enough UV exposure in the well that it caused an unexpected result. The well with no protection exposed to.0516% extra UV in comparison to the fish with no exposure also had a significant result. To expand this research one could test the same UV light exposure on other fish embryos to see how they are affected. If this were to come out with the same results, it could lead into better understanding on how to raise fish sustainably in fisheries, and lead to research on how to protect fish in their natural habitat from some of the effects, like change in UV intensity, of climate change. The previous statement is important to humans because fishing is a big part of our economy and a fish provides environmental benefits like: [contributing] nutrients to marine ecosystems and perhaps medicinal purposes (Gavrilles, 2012). Originally the lab was to test the effects of different levels of sunscreen on the fish, but with further research it was found that the extra protection of using SPF 50 instead of SPF 30 is very minimal. Furthermore, SPF is the measurement of UVB blocked radiation (Wang, 20). Since SPF 30 to 50 should have little extra protection they only really block UVB radiation, why was there only significant data for the 50 SPF and not the 30 SPF? The discrepancy between our project and research conducted could be due to our sources of error: some deaths resembled fungal deaths but it was unclear if they were natural infections or caused by the UV light. Or it could be due to the hatched fish that hatched faster than expected; they hatched in our first day. Another issue could be from the spray sunscreen. Since it was a spray, it is possible that the particles spread out or dropped into the wells. To minimize the
error caused, different levels (15, 30, 50 SPF) were kept away from each other when spraying. Tape was also added onto the wells before applying the plastic wrap in order to prevent sunscreen from dripping in the wells. A limitation to our lab is the amount of time that was given to go through the process during class because of our limited amount of time in class. There was not ample time to complete the procedure with care and appropriate attention to detail, potentially leading to other unseen errors.
References Allen, J. (2001, September 6). Ultraviolet Radiation: How It Affects Life on Earth : Feature Articles. Retrieved December 16, 2017, from https://earthobservatory.nasa.gov/features/uvb/uvb_radiation3.php Gavrilles, B., (2012) Fish have enormous nutrient impacts on marine ecosystems, study finds. Retrieved december 16 2017, from http://news.uga.edu/releases/article/fish-enormous-nutrient-impacts-121112/ Gonzalez, A. (2017). Irradiation of the Zebrafish Embryo. Retrieved December 7, 2018, from https://www.google.com/url?q=https://people.uwm.edu/winstep/files/2017/05/go0nzalez_zf_ Greendale-1gtb030.pdf&sa=D&ust=1512864253977000&usg=AFQjCNHea8s1iJW7iMNV0 j7g7bi2_ajsnw GraphPad. (2017). Retrieved Nov. & dec., 2017, from https://www.graphpad.com/quickcalcs/ttest1/ Murphy, T., (January, 17, 2012). Basking in the Sun. Retrieved December 11, 2018, from https://dothemath.ucsd.edu/2012/01/basking-in-the-sun/ Petering, D. H., Berg, C., Tomasiewicz, H., Caravan, M., Hesselbach, R., & Petering, L. (2016). ZEBRAFISH AS MODELS: STUDYING THE EFFECTS OF ENVIRONMENTAL AGENTS ON HUMAN HEALTH. Retrieved Sept. & oct., 2017, from http://guides.library.uwm.edu/ld.php?content_id=20971 Ultra-Violet Productions (n.d.). Ultraviolet Crosslinkers: Operating Instructions and Service Manual. Retrieved November 20, 2017, from https://www.uvp.com/manuals/811201.pdf Wang, S., (20). ASK THE EXPERT: Does a higher- SPF (sun protection factor) sunscreen always protect your skin better? Retrieved December 16, 2017, from http://www.skincancer.org/skin-cancer-information/ask-the-experts/does-a-higher-spf-sunsc reen-always-protect-your-skin-better