Variables:

a) Independent: caffeine concentrations

b) Dependent: heart rate of daphnia
c) Fixed: temperature of test solutions, volume of solutions, stress degree of daphnia, size of
daphnia, time for acclimatization, pH, absence of other foreign stimulants, light intensity,
duration of exposure to caffeine
Method:
A single daphnia was placed in a beaker containing the test solution for 5 minutes
(either pond water or pond water with0.5 % caffeine)

Place a few cotton strands on cavity slide. This is to immobilize the daphnia and enable
more consistent readings to be taken (prevents misjudgments, reduces random errors).

Remove water from daphnia with filter paper and then add pond water to it to prevent
dehydration (reduce stress, do not use distilled water as it lacks oxygen). Withdraw as
much water as possible from the slide, leaving just enough to keep the organism alive.
Do not use a cover slip. This is to maintain sufficient supply of oxygen to daphnia.

View daphnia under low power lens of light microscope and focus on its heart. Use
stopwatch and pencil-tapping method to record number of heartbeats per 20-second
interval.

Repeat procedure using other similar daphnia from the culture solution using fresh
slides. Then, repeat using different test concentrations of caffeine.

Compare with the control (plain pond water) and record data in table, calculate mean
and present in line graph.

Control all fixed variables and only change one variable at a time. Use similar daphnia Validity:
for each experiment and accurate measurement methods to record heart rate. (How would you
ensure a successful experiment?) Place cavity slide with iced water under it to act as heat sink.
A heat sink maintains a relatively lower temperature, which prevents unnecessary stress on
daphnia, which may result in unreliable results.
control the caffeine concentrations (manipulated variable); replicate the Reliability:
measurements, dealing with anomalies by ignoring anomalous results, large amount of data by
having more samples, immobilize daphnia
pencil-tapping, video recording Accuracy:
repeats and average calculations, use same daphnia, off light microscope when not Precision:
observing daphnia during period of acclimatization to prevent overheating of daphnia, remove
test solution during observation, start stop clocks at the same time
Carry out simultaneous experiments using the same variables (testing the same Replicates:
concentration).
repeat using a few Daphnia of similar size and species for each concentration and Repeats:
then repeat for different concentrations of caffeine.

Issues:
For:
1. Abundant in nature and breed rapidly. No threat of extinction.
2. Clones, hence no loss in genetic variation.
3. Simple nervous system, heart consists of nerves called cardiac ganglion.
4. Reduced awareness of pain due to lack of developed nervous system.
5. Transparent. No need for dissection.
6. Bred as fish food, and will hence die anyway.
7. Inexpensive.
Against:
1. Use of any animal is wrong. Right to live.
2. Daphnia may feel a degree of pain.
3. Not closely related to humans. Risky to extrapolate heart rate findings from daphnia to
humans without first validating the model. It only has a simple heart.
4. Easily stressed.

Discussion:
1. Daphnia may be killed by chlorine in tap water. Any tap water used should be left to
stand for 24 hours first to let the chlorine dissipate.

2. To prepare approximately 5 cm3 caffeine solution : Dissolve 0.5 g caffeine in 100 cm3
water. Instant coffee can be used(0.5%). Make up the solution with culture water or
distilled water.

3. Stroboscope can be used to increase accuracy of heart rate readings. Do not confuse
beating of legs or moving embryos in the egg sac with the heart rate. However, if the
heart cannot be spotted, then one may use leg-beatings, as it is proportional to the
metabolic rate / cardiac frequency of daphnia.

4. Concentration of caffeine above 1% to be avoided as this kills daphnia

5. If left too long under light microscope, temperature increases due to heat of lamp and
causes increase in heart rate

6. Daphnia were cooled on ice before the experiment. Beakers containing Daphnia in pond
water were put on ice for about half an hour. This had the effect of slowing the heart
rate and thus facilitating counting.

7. To immobilize the daphnia, you may also use petroleum jelly.

8. The results of the experiment would be more accurate and valid if a blind study is
done. This is because the experimenter who is counting the heartbeat of the Daphnia is
unaware of the concentration of caffeine in the solution it is in. This can prevent bias
due to the observers expectations. Studies have shown that observers expectations
can significantly influence the results.

9. Usage of same daphnia for each round affects accuracy, as it would have developed
acclimatization to the different caffeine concentrations.

10. Larger daphnia will have a slower response to change in caffeine than a smaller daphnia
as the surface area to volume ratio is smaller. Longer diffusion distance.