1. Anti-Oxidant Remediation of UV-Stressed Earthworms By: Pat Ebbert Pittsburgh Central Catholic PJAS 2010

2. Introduction Vitamin C acts as an anti-oxidant to slow the cell-crippling effects of free radicals in organisms. UV exposure can promote free radical production.

3. Vitamin C Exists as ascorbic acid in its reduced form and dehydroascorbic acid in its oxidized form. Acts as a reducing agent (anti-oxidant) by neutralizing oxidative species such as hydrogen peroxide. Reduced Form Oxidized Form

4. Ultra-Violet Radiation Electromagnetic radiation ranging in wavelength from about 10nm-400nm. Photolysis of molecules can cause the production of free radicals in cells. Direct UV effects, as well as free radicals can disrupt DNA synthesis and production of proteins needed for tissue growth and regeneration.

5. Anti-Oxidant Role in Tissue Regeneration Because anti-oxidants remove free radicals that can have damaging cell effects, including disruption of protein production, they may play a role in promoting the re-growth of lost tissue. Practical Application: Administration of anti-oxidants could improve recovery rates in humans suffering from tissue damage.

6. Lumbricus terrestris Commonly known as the “Canadian Night- crawler”, abundant in Europe and North America. Have the ability of regenerating segments after severe tissue damage has occurred. UV exposure may disrupt tissue regeneration ability, and introduction of Vitamin C may help to lessen these effects.

7. Purpose To determine the difference in regeneration ability between earthworm heads and tails. To determine the effect of UV radiation exposure on the regeneration ability of earthworms. To determine the effect of UV radiation and ascorbic acid on the regeneration ability of earthworms exposed to UV radiation.

8. Hypothesis #1 Null: There will be NO significant variation between the regeneration abilities of the heads of the earthworms and the tails of the earthworms. Alternative: There WILL be significant variation between the regeneration abilities of the heads of the earthworms and the tails of the earthworms.

9. Hypothesis #2 Null: UV radiation exposure will have NO significant effect on the regeneration abilities of the earthworms, and introduction of ascorbic acid will have NO significant effect on either the UV or no-UV groups. Alternative: UV radiation exposure WILL have a significant effect on the regeneration abilities of the earthworms, and introduction of ascorbic acid WILL have a significant effect on either the UV or no-UV groups.

10. Materials 32 live Canadian night-crawler earthworms 48 paper cups Large screen to fit over cups (prevent worms from escaping) Large supply of topsoil (enough to fill 48 cups half- way) Plastic spoon 50ml conical Distilled water 1000mg Vitamin C powder tablets 5ml and 10ml macro-pipettes Pair of scissors Pair of rubber gloves (for handling) Gram scale Plastic weigh-boat UV radiation machine Notebook and pencil (for data recording) Sharpie marker (for labeling)

11. General Procedure 1. 48 paper cups were filled roughly halfway with topsoil. 2. Concentrations of 20g/L of Vitamin C were prepared from 1000mg Vitamin C capsules. 3. 10mL of new Vitamin C solution were pipetted into each of 24 paper cups. 4. 16 live worms were placed in Vitamin C cups, 16 in soil-only cups; a screen was placed over all the cups to ensure the worms wouldn’t escape (Worms were stored in a garage, with temperatures ranging from 4.5 to 6.6 degrees Celsius). 5. After 3 days, half of the worms (8-VC, 8-NVC) were cut medially, dividing the worms into “head” and “tail” segments, and then tail segments were placed in the leftover cups in their respective spots. 6. Cut worms were all weighed using a gram scale, and data was recorded. 7. On this same day, half of all worms (including non-cut) were transferred from soil cups to empty plastic cups and given a 30-second UV exposure, then re- transferred back to their respective soil cups and covered with the screen. 8. Survivorship was checked after one and two week intervals for ALL worms, and cut worms were weighed at one and two week intervals, with all data recorded.

12. General Procedure (cont.) Earthworms CutHeadsUVVCNo VCNo UVVCNo VCTailsUVVCNo VCNo UVVCNo VCNot CutUVVCNo VCNo UVVCNo VC *12 total groups, x4 replicates of each = 48 total data points

14. 2-Factor ANOVA Statistical Analysis ANOVA Source of VariationSSdfMSF P-value F crit Sample0.14177730.0472590.939540.4369933.008787 Columns (H, T)0.7627661 15.16424 0.00068 4.259677 Interaction0.05649930.0188330.3744110.7722163.008787 Within1.207207240.0503 Total2.16824931 Was there significant variation between the Head and Tail groups, controlling for other variables?

15. Further Analysis ANOVA Source of VariationSSdfMSF P-value F crit Sample0.0134670410.0134670.768385 0.39792 4.747225 Columns0.0539672410.0539673.079193 0.10477 4.747225 Interaction0.005660710.0056610.322981 0.58030 4.747225 Within0.21031709120.017526 Total0.2834120715 Was there significant variation among the “Heads” group, comparing the Vitamin C factor with the UV factor?

16. Further Analysis (cont.) ANOVA Source of VariationSSdfMSF P-value F crit Sample0.0197681 0.237962 0.63447 4.747225 Columns6.71E-051 0.000808 0.97779 4.747225 Interaction0.1053461 1.268095 0.28214 4.747225 Within0.99689120.083074 Total1.12207115 Was there significant variation among the “Tails” group, comparing the Vitamin C factor with the UV factor?

17. Results and Conclusions Hypothesis #1: Because the p-value received from the data was <.05, the null hypothesis was rejected, and so the alternative hypothesis was accepted. There appeared to be significant variation between the head and tail groups’ changes in body mass. Hypothesis #2: Because all p-values received from the head and tail data sets respectively were >.05, the null hypothesis was accepted, and so the alternative hypothesis was rejected. The presence of neither UV radiation nor Vitamin C appeared to have a significant effect on the change in body mass of either the head or tail groups.

18. Limitations and Extensions Limitations Only one Vitamin C concentration and one UV exposure time were used. Worms may have lost large volumes of blood or have been exposed to infection when cut in half. Other methods of Vitamin C action besides its anti-oxidant properties may have been at work Other methods of UV damage besides free radical production may have been present. Extensions Allow for additional exposure times and VC concentrations. Keep worms in more controlled, nutritious environment. More accurate gram scale. Additional replicates.

19. References “Anti-Oxidants and Free Radicals”. Rice.edu.. “Lumbricus Terrestris”. Lander.edu.. “Oxidative Stress”. University of Valencia.. “Ultra-Violet Radiation”. Hps.org.. “Vitamin C”. Wikipedia.org..