Drugging Daphnia
by Lizzy Irvin; assisted by: Jojo Gager, Maggie Becker, Denali Mokris, and Christiana delBene
Mr. Jaeger's classroom, West Ottawa North High School
November 20, 2014
Objective
This experiment was conducted to investigate the effects of red food coloring of Daphnia. My
hypothesis was that as the concentration of food coloring increased, so would the heart rate of
the Daphnia.
Introduction
The Daphnia magna, the water flea, will be observed during this lab. The bodies of Daphnias
semi-transparent, allowing direct observation of the beating of the heart with a microscope.
The heart of the Daphnia is located just behind the head, in the crook of their digestive track.
Since the food coloring is lipid soluble, it will be able to pass through the plasma membrane,
directly affecting the Daphnia. Daphnia are used in investigation because they are an indicator
species, meaning the effects of different substances on them are very similar to the effects on
humans. Before coming up with our own experiments, the students in our class all investigated
the effects of alcohol and caffeine on Daphnia. We concluded that as the concentration of
caffeine increased, so did the heart rate, because caffeine is a stimulant, and that as the
concentration of alcohol increased, the heart rate decreased, because alcohol is a depressant.
These results coordinate very well with the effects of alcohol on humans. So the intent is that
we will be able to learn how red food coloring affects humans by experimenting with different
concentrations of it on Daphnia. Red 40 food coloring is a petroleum byproduct. Red 40 has
been linked to attention disorders. In addition, red food coloring can lead to asthma,
respiratory problems, heart problems, and cancer. The purpose of this lab is to investigate if
Red 40 can quickly change the heart rate of the organism.
Pre-Lab Procedure
1. Using the instructions in the “Measurements” section, make the 4 different solutions of
0%, 10%, 20% & 30% food coloring solutions and place in dropper bottles.
2. Place a petri dish half full of cold water on the stage of the dissecting microscope to
serve as a heat sink.
3. Place a slide on top of the petri dish and spread a thin network of cotton fibers in the
middle of the slid to “trap” the Daphnia.
Control Procedure
1. Transfer a fresh Daphnia and several drops of spring water into the network of cotton
fibers on a clean slide.
2. Count the heartbeats for 15 seconds. Record your results in Table 12.1
3. Remove the springwater using a Kimwipe. Test the response of Daphnia to 3 or 4 drops
of spring water using exactly the same procedure and time intervals you used in testing
the food coloring. Perform 6 trials, dabbing up the water between trials.
Experimental Procedure
1. Use the edge of a Kimwipe to remove the water from the slide.
2. Place 3 or 4 drops of 10% food coloring on the Daphnia. Daphnia should be completely
submerged. Wait exactly 1 minute. Then, count the heartbeats for 15 seconds and
record your data in Table 12.2 and 12.3
3. Use a Kimwipe to mop up the 10% alcohol. Then, test 20% and 30% alcohol using
exactly the same time intervals and procedure. Be sure to remove each solution before
adding a new solution.
4. Place the Daphnia in the recovery bowl.
5. Repeat procedure with another Daphnia
6. Wash and dry the slide and clean up your area.
Measurements
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Place 10 mL spring water in a 10 mL graduated cylinder.
o Pour the mixture into a dropper bottle and label it as the 0% solution
Place 1 mL red food coloring and 9 mL spring water in a 10 mL graduated cylinder, shake
until the dye is evenly distributed.
o Pour the mixture into a dropper bottle and label it as the 10% solution
Place 2 mL red food coloring and 8 mL spring water in a 10 mL graduated cylinder, shake
until the dye is evenly distributed.
o Pour the mixture into a dropper bottle and label it as the 20% solution
Place 3 mL red food coloring and 7 mL spring water in a 10 mL graduated cylinder, shake
until the dye is evenly distributed.
o Pour the mixture into a dropper bottle and label it as the 30% solution
Materials
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24 mL spring water
7 mL Stonemill Essentials Food Coloring
10 mL graduated cylinder
4 dropper bottles
Cotton Fibers
Daphnia
Counter
Microscope & Clean Slides
Petri Dish
Timer
Remember
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If your Daphnia dies part way through the experiment, start over with a new live
Daphnia to ensure consistent results
Alway start with the lowest concentration of food coloring and increase, to avoid higher
percentage contamination when switching solutions
Control Data
Table 12.1
Drop No. % Food Coloring
Beats/15 sec Beat/min
1
0% (spring water) 60
240
2
0% (spring water) 58
232
3
0% (spring water) 61
244
4
0% (spring water) 62
248
5
0% (spring water) 64
256
6
0% (spring water) 63
252
Average Beats/Min
~245
Experimental Data
Trial #1- Table 12.2
Drop No. % Food Coloring
Beats/15 sec Beats/min
0
0% (spring water)
67
268
1
10% food coloring 73
292
2
20% food coloring 63
252
3
30% food coloring 54
216
Trial #2- Table 12.3
Drop No. % Food Coloring
Beats/15 sec Beats/min
0
0% (spring water) 76
304
1
10%
70
280
2
20%
70
280
3
30%
65
260
% Food Coloring Average Beats/min
0%
286
10%
286
20%
266
30%
238
Calculations
All measurements were recorded out of 15 seconds. Since we want the number of beats per
minute, one should simply multiply the number of beats in 15 seconds by 4. The reason you
multiply by 4 is because there is 60 seconds in a minute and 15X4=60.
Ex.) For Drop No. 1 of the control data, the Daphnia measure 60 beats per 15 seconds.
60x4=240, so 240 would be the number of beats per minute.
Next, we did several trials for the control as well as for the experimental experiment.
Averages are more reliable than single trials because they remove outliers and show the
general trend. To calculate averages, one should simply add up all of the values of the same
measurement, and then divide the sum by the number of values used in the addition.
Ex.) For the control experiment, the averages were 240, 232, 244, 248, 256, 242. To find
the average you add all these numbers together. The sum is 1472. Since 6 values were added
you divide the sum by 6. 1472/6=245.3. So the average is about 245 beats per minute.
Finally, for our solutions, we chose a mL graduated cylinder to measure in because 10 is
easily divisible by 100, so it is easy to calculate percentages. 1 mL food coloring to 9 mL spring
water equals a 10% concentration of red food coloring. We realize that not all 10% of the food
coloring is red 40, but that is also made of water, propylene, and glycol. We just wanted to see
a darker color each time and increase the percentage of red 40 in the solution by increasing
concentrations. Therefore, when we put 2 mL food coloring to 8 mL spring water it was a 20%
concentration, and when we put 3 mL food coloring to 7 mL spring water it was a 30%
concentration.
Results
As the concentration of red food coloring increased, the heart rate of the Daphnia decreased.
The results of the experiment falsifies our hypothesis. This would suggest that red 40 is a
depressant, and causes the heart rate of the Daphnia to decrease. However, to get more
accurate, significant results, further testing and more trials should be conducted
Analysis
The results of the experiment would suggest that red food coloring acts as a depressant in
daphnia as well as caused an irregular heartbeat. A possible problem with the lab is that the
counter is not precise because it is controlled by a human, based on their hand-eye
coordination. Another possible problem is that the other additives in the food coloring caused
the change, not only the Red 40. In addition, another concern may be that the color of the
water that was changed with concentrations, it got darker, affected the Daphnia not necessarily
just the concentration of the dye. Another area of concern is the differences between Daphnia.
Some may just respond differently to the food coloring than others. For example, for the first
specimen we tested, we noted an irregular heart beat at 30% concentration, but we did not
observe the same phenomenon in the second specimen. This would lead us to believe that
more testing should be conducted to get better results.
Conclusions
We feel like we followed the procedure well, which is supported by our data. Since are control
data and experimental data were significantly different, it would suggest that the change in
heart rate was caused by the chemicals, not the procedure. In order to get better results, we
should repeat the experiment again to get more data to average. In addition, we should find a
better way to record the number of beats, such as videotaping the daphnia and rewatching the
footage slower to get a more precise measurement. For further testing, we should try to find
pure Red 40 with only added water, or red 40 that does not cause a change of color in spring
water. This would ensure that Red 40 caused the change, not the additives not the color of the
water. It also would be interesting to test different red dyes, or even different colored dyes to
see if they also acted as a depressant on Daphnia as the Red 40 food coloring. It may also be a
good idea to do more longer-term testing. Since we only had class hours to investigate, testing
longer term may produce different results, such as adaptation, or even death. Our results did
not tell us much about the results it could have on humans, except possibly that Red 40 can
cause heart problems. If we tested long term we may be able to see a link to cancer or
respiratory problems. More testing is needed.
References
Stevens, Laura. "Are Fruit Loops Evil? ADHD Hyperactivity and Food Dyes." ADDitude Magazine.
Web. 20 Nov. 2014. .
Snoek, Ben, and Robert Ganzi. "Drugging Daphnia." Google Documents. 17 Nov. 2014. Web. 20
Nov. 2014.
Acknowledgements
Mr. Jaeger, AP Biology Teacher
West Ottawa High School
Author Information
Lizzy Irvin is an AP Biology student at West Ottawa High School