1. Effect of Analgesic Medications on the Heart Rate of Daphnia magna Bin Huang Aberdeen Central High School Purpose Hypothesis Methods Discussion H ₁ : The Daphnia magna will show an increase in heart rate for both types of analgesic medications, however, there will be a much higher increase for the prescription painkillers. H ₂ : The Daphnia magna will also show an increase in heart rate for the higher concentrated solutions of medications for each individual painkiller. The purpose of the experiment is to explore the effect of analgesic medications, both prescription and non-prescription painkillers, on the heart rate of Daphnia magna. Does the heart rate increase or decrease when the subjects are exposed to dilute amounts of the medications? What correlation can be seen between different concentrations of the same medications? Materials Conclusion Photos Aspirin Acetaminophen Hydrocodone Ibuprofen Morphine Weigh Boats Pipettes Latex Gloves Goggles Sharpie Markers Daphnia magna Analytic Scale Light Microscope Spring Water Graduated Cylinder Powder Scoop Magnetic Stir Bar Erlenmeyer Flasks Petri Dishes Containers 1)Goggles and gloves were obtained and worn throughout the experiment. 2)Dilutions of the analgesic medications were made using a graduated cylinder, a powder scoop, weigh boats, Erlenmeyer flasks, and an analytic scale. 3)To make the first concentration, one milligram of the medication was measured out on a weigh boat using an analytic scale, while another scale is zeroed out with an Erlenmeyer flask on top. 4)The one milligram of medication is transferred into the Erlenmeyer flask by first washing the contents of the weigh boat with a pipette using spring water, then pouring that directly into the flask. 5)Spring water is then added minutely until the scale reaches one hundred grams or one hundred milliliters. 6)A magnetic stir bar is then dropped into the solution and the flask is placed on a stir plate and left until the solution becomes homogeneous. 7)The solution in the flask is then carefully transferred into a container labeled with a Sharpie marker. 8)To make the second concentration, an Erlenmeyer flask is placed on a scale and zeroed out, one milligram of the first concentration is pipetted into the flask. 9)Steps five through seven are then repeated, but the container is labeled differently. 10)To make the third concentration, an Erlenmeyer flask is again placed on a scale and zeroed out; one milligram of the second concentration is then pipetted into the flask. 11)Steps five through seven are again repeated with a different label. 12)Steps three through eleven are repeated for each of the five analgesic medications rendering three concentrations for each. 13)Thirty milliliters of each concentration are then pipetted into separate Petri dishes. 14)The heart rate of three Daphnia magna specimens from regular spring water were then measured using a light microscope. 15)To record their heart rate, a timer was set and every time their heart beat, a dot was made on a blank sheet of paper to represent one beat. 16)Once the initial heart rates were recorded, they were placed into the Petri dish solution. 17)A timer was then set for one hour. 18)After the hour, the specimen is transferred out and their heart rates are measured again and recorded. 19)These steps are then repeated for three other specimens in each concentration. 20)Collect data on an Excel spreadsheet to be transferred into a statistical analysis program. 21)Then use Minitab to run an ANOVA test (one way unstacked). 22)The ANOVA test will then determine whether the null hypothesis can be accepted or rejected. One-way ANOVA: Ace, Asp, Ibu, Hyd, Mor (1/100) (Key: Ace = Acetaminophen, Asp = Aspirin, Ibu = Ibuprofen, Hyd = Hydrocodone, Mor = Morphine) An ANOVA one-way (unstacked) test was run on data from the table that contained the change in heart beats for the D. magna that were exposed to the 1/100 concentrated analgesic solution. The test shows that there is a significant difference as the P-value is way below 0.05. Source DF SS MS F P Factor 4 583012 145753 848.72 0.000 Error 10 1717 172 Total 14 584730 S = 13.10 R-Sq = 99.71% R-Sq(adj) = 99.59% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---------+---------+---------+---------+ Ace 3 69.33 16.65 (-*) Asp 3 76.00 6.93 (*-) Ibu 3 -309.33 6.11 (*-) Hyd 3 92.00 6.93 (-*) Mor 3 -336.00 21.17 (*) ---------+---------+---------+---------+ -240 -120 0 120 Pooled StDev = 13.10 One-way ANOVA: Ace, Asp, Ibu, Hyd, Mor (1/10,000) (Key: Ace = Acetaminophen, Asp = Aspirin, Ibu = Ibuprofen, Hyd = Hydrocodone, Mor = Morphine) An ANOVA one-way (unstacked) test was run on data from the table that contained the change in heart beats for the D. magna that were exposed to the 1/10,000 concentrated analgesic solution. The test shows that there is a significant difference as the P-value is way below 0.05. Source DF SS MS F P Factor 4 287132 71783 205.80 0.000 Error 10 3488 349 Total 14 290620 S = 18.68 R-Sq = 98.80% R-Sq(adj) = 98.32% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev --------+---------+---------+---------+- Ace 3 20.00 12.00 (-*-) Asp 3 64.00 4.00 (-*-) Ibu 3 -317.33 16.65 (-*-) Hyd 3 25.33 4.62 (-*-) Mor 3 -81.33 35.85 (-*-) --------+---------+---------+---------+- -240 -120 0 120 Pooled StDev = 18.68 One-way ANOVA: Ace, Asp, Ibu, Hyd, Mor (1/1,000,000) (Key: Ace = Acetaminophen, Asp = Aspirin, Ibu = Ibuprofen, Hyd = Hydrocodone, Mor = Morphine) An ANOVA one-way (unstacked) test was run on data from the table that contained the change in heart beats for the D. magna that were exposed to the 1/1,000,000 concentrated analgesic solution. The test shows that there is a significant difference as the P-value is way below 0.05. Source DF SS MS F P Factor 4 263588.3 65897.1 726.81 0.000 Error 10 906.7 90.7 Total 14 264494.9 S = 9.522 R-Sq = 99.66% R-Sq(adj) = 99.52% Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---+---------+---------+---------+------ Ace 3 16.00 8.00 (-*) Asp 3 13.33 8.33 (*-) Ibu 3 -316.00 16.00 (*-) Hyd 3 12.00 4.00 (*) Mor 3 20.00 6.93 (*) ---+---------+---------+---------+------ -300 -200 -100 0 Pooled StDev = 9.52 Photos taken by Bin Huang. All tables, charts, graphs and images are created by Bin Huang. Beats Per Minute (BPM) Acetaminophen (Ace) Aspirin (Asp) Ibuprofen (Ibu) Hydrocodone (Hyd) Morphine (Mor) Initial C1308324316 320316308304316312304312320360328 Final C1372412372388392400000396400408000 Initial C2312332312320308 312304336328336340320 324 Final C2332364340380376372000348364368216220284 Initial C3300304324 332300332316300328 340320332344 Final C3308328340 336320000344340348344356 KeyC1C2C3 Description One milligram diluted until one hundred milliliters (1/100) One milligram of C1 diluted until one hundred milliliters (1/10,000) One milligram of C2 diluted until one hundred milliliters (1/1,000,000) Change in Heart Rate (BPM) C1AceAspIbuHydMor Trial 16472-30884-320 Trial 28872-30496-360 Trial 35684-31696-328 C2AceAspIbuHydMor Trial 12060-31220-104 Trial 23268-30428-100 Trial 3864-33628-40 C3AceAspIbuHydMor Trial 1816-3321624 Trial 2244-3161224 Trial 31620-300812 Raw Data After data collection, statistical tests were run on the numbers to conclude the experiment. One of my hypotheses were that the analgesic medications, both prescription and non-prescription, would increase the heart rate in Daphnia magna and that the prescription medications would increase the heart rate more than the non-prescription ones because they are the stronger painkillers. The other hypothesis is that the D. magna will show an increase in heart rate for the higher concentrated solutions. Both H ₁ and H ₂ were supported by parts of the data but looking at the entirety of the experiment, though, the H ₁ cannot be supported but H ₂ was proven correct. On top of that, I am also able reject my null hypothesis because the results calculated by the ANOVA one- way (unstacked) test provides the experiment with P-values that are less than 0.05. This shows that the experiment’s results were not of random chance and the figures collected are significant. The results show that many of the medications increased the heart rate of the test subjects. They also show that aspirin out of the five painkillers consistently had the highest increase in heart beat per minute over all the concentrations. Since both the different concentrations of ibuprofen and morphine had signs of killing the D. magna, it actually warrants further inquiry and experimentation for maybe different time intervals to see the actual change in heart rate for the two. This leads me to believe that there might be other effects the painkillers are having on the specimen with regards to their chemical compositions because the data collected do not seem consistent across the spectrum of different medications. The experiment in the project was designed to investigate the effects of analgesic medications on the heart rate of Daphnia magna water fleas. D. magna were chosen because their heart rate could be measured under a light telescope without much difficulty. The body of the specimen are enclosed by a carapace that are clear which makes one able to count the amount of times its heart beats just by examination. A change in Daphnia heart rate may not be an indicator of a similar change in human or vertebrate heart rate under the same conditions, but the experiment proposes an interesting technique for studying the effects of different substances on a simple metabolic process. And, increasingly enough the Daphnia species are now being used to understand genomic responses to ecological stressors that are imperative factors in the health and well-being of humans. At the end, a conclusion was drawn that most of the medications did either increase the heart rate of the specimen or kill them off depending on which painkiller was used. Therefore, with certainty the medications indeed have a significant effect on the heart rate of the species. There are not very many weaknesses that could undermine the validity of this project; however, data collection could be done smoother with a camera that could videotape microscopic images. This way, the video could be slowed down to count the beats more accurately. Also if further inquiry could be a possibility then different time intervals would be something interesting to experiment with. One could also look at even more concentrations of the different medications, but the time limitations imposed on the project made such experimentation challenging. Data Analysis