To investigate the effect of caffeine on the heart rate of Daphnia (water fleas).

Background Knowledge:

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Daphnia are tiny water fleas with an average size of less than 3mm. Daphnia are translucent invertebrates (have no backbone) and their outer body is made up of a hard shell which protects the whole body except for the head. Daphnia’s are found in ponds, lakes, and calm streams where the temperature is between 21-24�C (68-71�F). Daphnia reproduce rapidly. Up to 13 billion related offspring can occur within 60 days for one Daphnia. In winter the eggs are thick-shelled and thin shelled in the summer. In warmer temperatures the eggs will hatch female, and in colder temperatures the eggs will be male.

Daphnia’s will be used to experiment the on as although they are tiny in size their heart beat can be examined through a microscope because of their transparency.

Caffeine is produced by plants as an insecticide. Cocoa in South America, coffee in Africa and tea in Asia has all been used for hundreds of years to produce “pick me up” drinks containing caffeine. These days, caffeine is used as a flavour enhancer in a wide range of cola and other soft drinks. In addition, it has medicinal uses in aspirin preparation, and is found in weight-loss drugs and as a stimulant in students’ exam-time favourites like Pro-plus and Red Bull.

In humans, caffeine acts as a stimulant drug, causing increased amounts of stimulatory neurotransmitters to be released. At high levels of consumption caffeine has been linked to restlessness, insomnia and anxiety, causing raised stress and blood pressure. This can lead to heart and circulation problems.


I hypothesise that the heart rate of the water fleas will increase if the concentration of caffeine is increased. I also predict that the aforementioned increase in heart rate will result in the flea dying.

I believe that this will occur because the heart muscle will not be able to cope with the increase in beats. The resulting stress on the heart causes it to stop and the flea will die.


* Culture of Daphnia: to conduct the experiment on.

* Cavity Slides: to place the flea and a few drops of water in.

* Dropping pipettes: 2 needed for dropping the solution and fleas

* Distilled water: needed to note down the original heart rate of the Daphnia.

* Caffeine tablets: needed to dissolve into water to make caffeine solution of varying concentrations.

* Cotton wool: to restrict the movements of the water flea.

* Measuring syringes: 2 needed to measure out solution.

* Test tubes: 5 needed to place solution in.

* Stopwatch: to ensure that inspection times are uniform.

* Paper towels or filter paper: to remove excess solution from around the flea.

* Microscope: to examine the flea accurately.

* Test tube rack: to place tubes containing the different solutions in.

Risk Assessment

In this experiment there are many risk factors posed to the student. The use of microscopes with a lighted base and glassware sometimes can cause injury to the student if they are not careful while handling such fragile equipment.

With the microscope, such heavy equipment must be carefully handled especially in a lab, where puddles may be found on the floor. Slips and trips can occur while holding heavy equipment. Also, microscopes that do not use a mirror but electrical lighting, need to be checked that the voltage of the light is at a comfortable level, and cannot blind someone. Low-level lighting will also help by not evaporating the solution on the cavity slides.

The glassware, such as beakers, test tubes, can easily shatter, so you have to be careful whilst handling such fragile equipment. Keep the glassware in a firm grip, and never carry more than you can handle. Once at the workstation, place gently on the table, and place the test tubes in the rack, so they cannot fall and roll around.

Also wear lab coats so that any spills onto your own clothes are prevented.

Ethical issues

The ethical issue that may relate to the conducting of this experiment is that of the animal rights issue. Investigating the affect of caffeine on the heart rate of Daphnia is in reality experimentation on animals. This has lead to a raging argument on whether or not experimenting on animals is morally and ethically acceptable.

Below are the ethical and moral rights, raised regarding animal experimentation.

* Animals should be treated in the way that is best for the animals concerned – which may not be the way that suits human beings

* It is wrong for human beings to use animals for food, clothing, experiment or anything else

* It is wrong for humans to disregard the consequences to animals of their actions

* This applies to all animals, regardless of the way human beings feel about them

* It may sometimes be morally correct to violate an animal’s rights when these conflict with the rights of another animal or a human being – each case must be decided on its merits

During this experiment, all these will be taken into account. Firstly, Daphnia is used due to it being one of the most basic creatures in existence. They live in the water and thrive on unicellular algae and various sorts of organic detritus including protists and bacteria. They are mainly cultured as fish bait, but the translucent carapace is ideal to monitor the heart rate under the influence of caffeine, nicotine or adrenaline. They are tolerant of being observed under a cover slip and appear to suffer no harm when returned to open water.

Secondly, it is wrong for human beings to use animals for food or clothing. The Daphnia will not be used of either of these. However, if used for the advancement of knowledge and medical purposes, the use of such basic creatures is surely better than using much larger and complex animals.

Thirdly, this experiment on the changes in heart rate of Daphnia does not disregard the consequences to animals of human actions. It has been noted that the Daphnia are tolerant of being observed under a cover slip and appear to suffer no harm when returned to open water.

Finally, another option of finding out the affect of caffeine on the heart rate would be to use a human being. It therefore morally correct to violate an animal’s right when it is in conflict with the rights of a human being. The human being’s rights far outweigh that of a water flea’s.

Consequently the Daphnia should be respected and the least harm should be done to them as possible. Therefore, high concentrations of caffeine will not be used as this could be fatal for the Daphnia. Also when obtaining the Daphnia through a pipette care must be taken to ensure that no harm is caused to them. As the natural habitat of Daphnia is ponds, lakes, and calm streams, distilled water will be used to minimise any reaction to the Daphnia. Also, the Daphnia should not be left without water longer than a few seconds as lack of water could kill them. After the experiment is finished particular care must be taken in determining whether the Daphnia is dead or alive.

Therefore, using the Daphnia for this experiment is morally and ethically acceptable.

Independent variables

* The concentration of caffeine in the solution

Dependant variable

* The size of the water flea

* Amount of solution covering the water flea on the cavity slide

The independent variable will be controlled by measuring out the amount of distilled water and caffeine needed. In a solution of 10ml, it is possible to measure out exact concentrations of both distilled water and caffeine.

For 100% distilled water, 10ml of distilled water is poured into a test tube.

For a solution with a 10% concentration of caffeine, pour 9ml of distilled water and 1ml of caffeine into the test tube.

For a solution with a 30% concentration of caffeine, pour 7ml of distilled water and 3ml of caffeine into the test tube.

For a solution with a 50% concentration of caffeine, pour 5ml of distilled water and 5ml of caffeine into the test tube.

For a solution with a 70% concentration of caffeine, pour 3ml of distilled water and 7ml of caffeine into the test tube.

The size of the water fleas cannot realistically be measured, but because they are between 0.2 and 0.5 mm in length they do not pose a serious change in results.

The amount of solution covering the water flea on the cavity slide will be monitored by making sure that enough solution is dropped so that the flea and the cavity in the slide are immersed in the solution; this is about 4 drops of solution from the dropping pipettes.

Reliable and valid results will be ensured by doing the experiment in pairs. While one person is monitoring the time on the stopwatch, the other person should be counting the heart beats through the microscope. This will ensure correct and valid results because if it were to be one person monitoring the heart rate as well as the time, they would be distracted and not be able to devote enough concentration to either. That is why one person should monitor the time, while the other person should monitor the heart beats of the Daphnia.


Preparation of solutions

There are five different solutions. Distilled water: Take 10ml of distilled water with the syringe and empty into a test tube. 10% caffeine solution: take 9ml of distilled water and 1ml of caffeine and empty into a test tube. Use different syringes to ensure accurate and reliable results. 30% caffeine solution: take 7ml of distilled water and 3ml of caffeine and empty into a test tube. 50% caffeine solution: take 5ml of distilled water and 5ml of caffeine and empty into a test tube. 70% caffeine solution: take 3ml of distilled water and 7ml of caffeine and empty into a test tube.

Setting up experiment

1. Place a few strands of cotton wool on a cavity slide; this will help restrict the movement of the water flea. Using a pipette, transfer one large water flea to a cavity slide. Remove the water from around the water flea using filter paper, then add three or four drops of distilled water. Use as much water as you can and do not use a cover slip. Together these precautions will help maintain a sufficient oxygen supply to the flea. A cavity slide filled with iced water will act as a heat sink. View the water flea under low power. Focus on its heart which can be seen through its translucent body. The location of the heart is shown below:

2. Use a stopwatch to record the number of heartbeats per 10 seconds. This is made easier by working in a pair, with one person counting beats while the other person tells them the time-period. Tap a pencil on a piece of paper and count up the pencil marks at the end of the time period. Record the heart rate at intervals of 2 minutes over a 10 minute period. It is a good idea to do a “blind” study to avoid bias in the results. The person counting the heartbeats should be unawares as to whether the Daphnia is in water or a caffeine solution.

3. Repeat the procedure using the other water fleas from the culture solution and fresh, clean slides. Repeat the water with caffeine solution. Repeat the procedure using different concentrations of caffeine.

Observation of results

What I have observed in this experiment has been noted down below.

Concentration of caffeine

Heart rate (per 10 seconds)

Mean heart rate


23, 22, 24, 24, 24



21, 23, 23, 25, 25



26, 28, 30, 32, 33



26, 29, 33, 29, 25



28, 26, 29, 26, 30


I have drawn a table to show my results.

Analysis of results

Below are graphs that map the change in the heart rate of the water fleas in different solutions:

Mean heart rate: 23.83 � 24.0

Mean heart rate: 24.0

Mean heart rate: 30.0

Mean heart rate: 26.5

Mean heart rate: 27.83 � 28.0


Although the graphs do not show a directly proportional link, the results from the graphs agree with the hypothesis. The link is positively correlated and overall there is a clear pattern. This indicates that as the caffeine concentration increases the heart rate increases. This is because the affect of caffeine on Daphnia is the same as humans, and stimulated the heart and nervous system in the Daphnia. Higher concentrations meant more adenosine receptors in the brain were blocked, which slowed down the nerve cells. Therefore more adrenalin neurotransmitters were released which increased the activity of the receptors which increased the heart rate.

The fault in a complete directly proportional link can be the fact that the heart beats were not the exact true value as they were only timed for ten seconds. Nevertheless the results are good enough to support a valid conclusion. The figures go up steadily which shows a systematic relationship between the concentration and the heart rate. Also because there are no extreme anomalies the results are more reliable.


The investigation has given the desired results, however many improvements or changes could be made to the procedure to make the results more accurate.

The culture of Daphnia used could affect the rate. This is because different cultures may be stored in different conditions with a different Ph, organic matter, humidity, oxygen concentration and temperature and therefore have a different lifestyle. To make the experiment reliable, subjectivity should be removed and a random sample of Daphnia could have been obtained from all places of the river e.g. upstream-downstream. However even with different cultures there is bound to be a personal difference between the Daphnia.

When applying drops of the solution on the cotton wool the same pipette was used for each repeated concentration and for the different concentrations. This is not very accurate as some solution may have remained in the pipette and could have transferred some solution from one beaker to another. Also with dropping pipettes it was difficult to control the drops and sometimes several drops will come out. Also many times the end of the pipette was taken out of the solution before the bulb was released so a little air had got drawn into the pipette.

Personal interpretation of the Daphnia’s heartbeats could not be 100% accurate. To overcome this problem of interpretation a video microscope could be used so that the beats could have been slowed down and counted more accurate, or a devise such as an ECG sensor would provide most accurate results.

Also through personal bias a systematic error could have been created. Because the effect of caffeine on the heart rate was already known this could cause an expectation of higher results. The best way to avoid personal bias is to conduct a blind test.

Furthermore, whilst starting or stopping the clock the reaction time is bound to have been slightly slower than intended.

Although equipment was checked for cleanliness the microscope was not taken into account and could have also caused systematic errors values differing from the true value by the same amount as the lens, eyepiece and stage were not checked for cleanliness.

To improve the reliability, the experiment should be repeated a number of times. Results that vary considerably should be discounted or repeated. This would allow us to see the degree of error, because the greater the variation of results the greater the possibility of error.


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