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Introduction (Science):

Heart beat is a measurement of the one complete


pulsation of the heart (which is a measure of how often
the heart contracts in one cardiac cycle). A daphnia is a
water-flea and is a crustacean that can be found in pond
water. Due to the semi-transparent nature of the
Daphnia, the small heart of the Daphnia can be seen
under a low power microscope. We can then use this to
measure the effect of caffeine on the heart rate of the
Daphnia, by measuring how fast the heart beats. A
change in the heart beat of the Daphnia may not
necessarily mirror the effect that Caffeine has on the human heart but we can observe a rough
idea of how caffeine affects the metabolic processes.
Their heart rate increases due to the fact that chemicals that enter their body can affect their
heart rate by interfering with the chemicals that are used to transmit nerve signals around their
body. Chemicals that speed up the heart rate are known as stimulants and chemicals that
lower the heart rate are known as depressants.
Under normal conditions, the average heart rate of a Daphnia
is at 180 beats per minute.
Caffeine, which usually occurs as a white powder, has a molecular
formula of C8H10N4O2. It is unique in the sense that it is soluble in
water and in oil. Caffeine is usually extracted from the leaves of
plants and from beans or fruits of some plants. Interestingly, in the
perspective of science, caffeine is considered as a type of
recreational drug, although its effects are much milder of other
drugs such as marijuana, cocaine and heroin. The drug is extremely
popular, with statistics that show an estimation of global
consumption of caffeine at 120,000 tonnes per year. The recommended consumption for an
average adult is less than 300mg of caffeine, per day. Caffeine can be absorbed into our system
as we drink caffeinated drinks and the caffeine would be absorbed into our body through our
stomach and our small intestine. It is metabolised in our liver where it is then transported into
our tissues. Caffeine binds to our receptors on the surface of our heart muscle cells which leads
to an increase in the level of a certain kind of receptor in the cells, which then increases the
heart rate.
Different people have a different tolerance towards caffeine. Caffeine can have a positive but
also a negative effect on the body. Caffeine can be considered as a negative effect as it
increases anxiety, and restlessness and can cause insomnia, which can lead to heart and
circulation problems. It can, however, be considered positively as it can act as a diuretic, which

increases urine production (which reduces high blood pressure).


Hypothesis:
The higher the amount of caffeine in the blood, the faster the rate of the heartbeat of the
Daphnia as the Daphnia becomes much more active due to the caffeine.
Ethical Issues:
These animals, although small, deserve respect and must not be played with or treated roughly.
After observing the heart rate of these creatures, they must be promptly returned to the tank.
This is to ensure that the Daphnias return to their natural habitat after observations with them
have been made. Also, to prevent suffocation, the daphnias must always be immersed in water
or must be held down with a cotton wool that is wet with water. Also, chemicals must be diluted
well before experimenting on them with the Daphnia. Preliminary experiments should be made
to ensure that the conditions are suitable for the Daphnias. Also, prevent light from shining on
the Daphnia at all times to prevent the creature from being overheated. We have to be careful
not to harm them and to make sure that we do not stress them either. Ensure that tap water isnt
used to store the Daphnia as it is proven that the chlorine in the tap water can cause the death
of the Daphnia. The concentration of caffeine above 1% to be avoided as this kills daphnia.
Equipment:
Culture of Daphnia,
Pond water,
distilled water,
Different concentrations of caffeine solution (0.1%, 0.2 %, 0.3 %, 0.4 %, 0.5 %)
Cotton wool,
Microscope,
Glass slides used for the microscope,
Pipettes
Measuring cylinder (to ensure that accurate concentration of caffeine solutions are made)
Plan
1. Obtain one daphnia organism from the Pond water and place it on some wet
cotton wool. (Ensure the cotton wool is wet to ensure that the Daphnia has enough water
to survive) Make sure that when the Daphnia is placed on the cotton wool, the heart of
the Daphnia isnt covered by the cotton wool to ensure that the heart rate can be
counted.
2. Place the cotton wool on the glass slide.
3. Adjust the microscope and the slide to make sure that the heartbeat of the
Daphnia is visible in the microscope.

4. Using the stopwatch, count the heart beats of the Daphnia by putting paper to
pen every time you see the heart of the Daphnia beat. This is useful as it ensures
minimal human error due to the fact that you arent clicking anything which could
malfunction. Make sure that you are dotting a different part of the paper at every time.
This will be a control to be compared with the other results as it they are all affected by
caffeine. (Make sure the same person does it everytime to reduce Random errors)
5. Place a couple of Daphnias in another small container with pond water and place
0.1% of Caffeine (or the other concentrations of caffeine) in that measuring cylinder.
Keep the Daphnia in it for 3 minutes.
6. Then repeat steps 1-5 for every concentration of Daphnia.
7. Repeat the experiments 3 times with different organisms of Daphnia for the
different concentrations.
8. Obtain the results and record the average.
Safety Precautions:
The Daphnias must be handled carefully and not roughly. Handle all the experiments carefully
and if any chemical touches skin, ensure that the area exposed is immediately washed. Wear
safety goggles to prevent chemicals from splashing into the eye. Make sure hair is tied back at
all times and it does not touch any of the chemicals. When handling liquid with temperatures,
take precaution needed (such as using tongs) to prevent scalding of hands. Also, when handling
liquid, ensure that the liquid doesnt spill anywhere that might cause someone to trip and fall.
Hygienic precautions are also very necessary as a culture is used. Always make sure that the
microscope is carried by 2 hands, to ensure that it doesnt fall and break.

C - The amount of caffeine experimented on each daphnia


O - Daphnia
R - Repeat the experiment 3 times to increase reliability.
M - Measure the heart rate of the Daphnia creatures by tapping pencil on to paper every time
we notice a heartbeat
S - Keep the temperature of the water the same as an increase in temperature could cause an
increase in the heart rate of the Daphnia. The volume of water provided to the Daphnia under
the microscope (or how the wet cotton wool is placed on the Daphnia) - a deficiency in water
can cause the heart rate of the Daphnias to increase as it may be suffocating. Also make sure
that the time in which each Daphnia is kept in the pond water that contains the caffeine is the
same (3 minutes) as if the Daphnia was left in the caffeine for a longer time, it could have a
stronger effect on the Daphnia, which could prove your results unreliable. The type of organism
should also be kept the same to ensure that the results are comparable as different organisms
could have a different resistance to caffeine. The oxygen concentration of the water surrounding
the Daphnia should also be kept as similar as possible.
Results:

Graph:

Analysis/Discussion:
The results from our experiment shows us that the caffeine concentration has various effects on
the Daphnias. At the start, there is a 3.5 % decrease of the mean beats/minute of the Daphnias
from the Caffeine concentrations of 0.1 % to 0.2 % which suggests that caffeine is a depressant
rather than a stimulant. The trend then increases from 0.2% to 0.4% with there being an 11.8%
increase of the Daphnias heart rate from 0.2 % to 0.4 %. However, after that, there is a
decrease in the heart rate, from 186 to 170, which is an 8.6 % decrease in the mean
beats/minute of the Daphnias. The results of the control experiments, however, were much
more varied. There is a 38.6% difference between the fastest heart rate of the Daphnias and the
slowest heart rate. However, the results of the Daphnia with the mean of 194 beats/minute can
be considered an anomaly as it is extremely high and does not match the other results and has
a 14 beat/minute difference with the average heart beat of a Daphnia, which can be considered
to be quite high. However, the slowest heart rate of the Daphnias that were under the control
condition, with only 140 beats/minute can also be considered an anomaly as it is too small and
is too low compared with the other results, with there being a 22 beats/minute difference with
the second lowest result and is also 20 beats/minute off the average heartbeat of a Daphnia,
which is at 180 beats/minute.
The result of this experiment also does not match our hypothesis in the sense that the average
heart beat of the Daphnia is at 180 beats/minute but almost every result, except 1, is lower than
the average heart rate, despite the concentration of the caffeine increasing. The only result that
matches our hypothesis could then be regarded as an anomaly as it is the only one that has
surpassed the average heart rate. However, based on the control in this experiment (as these
Daphnias could have been obtained in a different pond or a different region to the Daphnias that
were tested to prove the average heart rate and those Daphnias could have a higher tolerance
to caffeine), which then suggests that the average heart rate is too high for the Daphnias that
were used in these experiments.
The science behind this experiment should be that the caffeine acts as a stimulant to the
Daphnias and increase their heart rate, thus making them more active. As their heart rate
increases, their heart should pump faster, which should be observable under the light
microscope. The higher the concentration of caffeine, the faster the heart rate as there is more
of the caffeine running in the bloods of the Daphnia.

Conclusion:

The results from this experiment do not support our data by much due to the Daphnias and that
they can be affected easily by temperature (which could easily change due to the surroundings
and the fact that this experiment was not done in a professional laboratory. The temperature
could also change due to the light from the microscope, which increases the temperature of the
water). The anomalies, illustrated by the control (194 beats/min), could also be due to the high
stress levels as the Daphnias are not in their natural environment. However, if an average of all
the control is taken, it can be concluded that the hear rate of the Daphnia does increase, as a
general trend, as the concentration of caffeine increases.
Evaluation:
Due to time constraints, we couldnt select the Daphnia that was the biggest, which would have
helped us as we measured the heart rate as the heart would have been bigger or it would have
been more obvious, which would have been more obvious. Besides that, due to the fact that all
the Daphnias were of different size, some heart rates were easier to measure than others.
However, larger daphnia will have a slower response to change in caffeine than a smaller
daphnia as the surface area to volume ratio is smaller due to the longer diffusion distance. It
also would have been better if we purchased or kept the age of the Daphnia the same (as far as
possible), by keeping them from when they have been hatched. This is because the various
caffeine concentrations could have a different effect as the age of some Daphnias increase as
(with humans), the arteries harden and lose its elasticity, and therefore it is harder for the
arteries to accommodate to the higher blood pressure surging through. This then reduces the
validity of our results as the Daphnias are of different age and react to the caffeine
concentrations differently and we do not know the age of the Daphnia sample that we
experimented on.
Besides that, the validity of the experiment could have been improved on as the temperature of
the solutions are kept the same. This is because a high temperature could affect the Daphnia by
making it more active (the enzymes would be closer to its optimum temperature, thus working
faster and making the reactions in the Daphnia to happen at a quicker rate), thus increasing its
heart rate. If the temperature gets too high, then the Daphnias could potentially die due to the
high temperature and the inability for the body of the Daphnia to accommodate to it.
We controlled the reliability of our results by ensuring that the confounding variables are kept
the same as far as possible. This could include the temperature of the water, the pond that the
Daphnias were obtained from and this could also include the type of Organism.
Besides that, to improve the accuracy of our results, we could have also made sure that the
Daphnias were kept at 10 degree celcius before experimenting on them as this is when the
heart rate of the Daphnias are at their slowest rate (this also ensures that the heart rate of the
Daphnias before they are mixed with the caffeine is as similar as possible). To improve the
accuracy of our results, we also need to make sure that the used Daphnias are not returned
back into the container with the original Daphnias as this could cause a mix up and we could
accidentally re use the same Daphnia for another experiment of a different caffeine
concentration. To also improve on the reliability of our results, the volume of caffeine placed with
the Daphnia needs to be kept as similar as possible to ensure that there is no overexposure of

the caffeine to certain Daphnia molecules, which could affect the heart rate of the Daphnia by
increasing it, thus decreasing the reliability of our results as the result wouldnt be as accurate
as possible. We also have to make sure that we do not keep the Daphnias under the
microscope for too long as if left too long under light microscope, temperature increases due to
heat of lamp and causes increase in heart rate.
To improve the precision of our results, we had used a stopwatch instead of a clock to ensure
that the time is read to the last second to make sure that it is as similar as possible for every
repeat. Also make sure to turn off the light on the microscope when not observing daphnia
during period of acclimatization to prevent overheating of daphnia. Start stop clocks at the same
time to ensure that the result is as precise and as accurate as possible. We could also improve
the precision of our results by using a clicker instead of just putting pen to paper and this could
minimize human error.
In addition to that, since the Daphnia is not observed under its natural habitat, its stress level in
the experiment may be higher than usual. This might cause fluctuations in its heart rate or an
undesired change in the heart rate. The only thing to be done is to count the heart beat or
beating of legs of the Daphnia as soon as it is ready to be observed. Weak daphnia or pregnant
daphnia may be more easily stressed than normal healthy daphnia.
Besides this, standard deviation shows how close the values are to the mean (how spread the
data is). Our data lacks accuracy as the values are spread and far from the mean.

Further Experiments:
To further analyse the effect of a drug, like caffeine, on the heart rate of a living organism, like
the daphnia, these are some changes in the experiment that could have been done:
Use another species of daphnia, to see if the results are the same. This could be
done on brine shrimps or other organisms with a similar circulatory system.
Use different concentrations of caffeine, such as 0.3% and 0.6%. This would
validate our results even more as it proves our results to be more reliable.
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 heart beat 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.

Bibliography:
http://www.sciencebuddies.org/Files/3462/5/Pharm_img004.gif

http://www.ukessays.com/essays/biology/the-effect-of-caffeine-on-heart-rate-biologyessay.php#ixzz3Jsddsbmv
http://www.chem.purdue.edu/gchelp/molecules/caff.gif
http://www.scribd.com/doc/29218203/Heart-Rate-of-Daphnia#scribd
http://people.uwec.edu/janikds/biol312/lab%20discussion%20docs/312%20daphnia%20lab.pdf
http://brainstormthoughts147.blogspot.com/2011/05/study-notes-for-biology-3-practical-a1.html
http://www.writework.com/essay/caffeine-affects-heart-rate-daphnia

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