Caffeine can be absorbed rapidly into the bloodstream from the gastro-intestinal tract. It reaches maximum concentration within an hour. The blood distributes it throughout the body. One effect of caffeine is to interfere with adenosine at multiple sites in the brain. Caffeine also acts at other sites in the body to increase heart rate, constrict blood vessels, relax air passages to improve breathing and allow some muscles to contract more easily. Caffeine is also an addictive substance and is associated with many withdrawal symptoms. Typical withdrawal symptoms include headache, fatigue and muscle pain. These symptoms can occur within 24 hours after the last dose of caffeine. This is because without caffeine, the blood vessels of the head to dilate, leading to an excess of blood in the head and causing a and .
On top of that, caffeine binds to receptors on the surface of heart muscle cells. This has the overall effect of increasing the rate of and increases the amount of available for muscle contraction and relaxation. In large amounts, and especially over extended periods of time, caffeine can lead to a condition known as ‘caffeinism’. Symptoms of caffeinism include , , , and . An acute overdose of caffeine, usually in excess of about 300 milligrams, can cause the central nervous system to be over-stimulated. This condition is known as caffeine intoxication. It may include restlessness, , insomnia, , disturbance, , irritability and a .
Other than using Daphnia, other organisms can also be used to study the effects of different caffeine concentration on their behaviour. Examples include bacteria such as Escherichia coli and Pseudomonas fluorescens. E.coli are found in the human intestines, while Ps. Fluorescens are found in a wide range of terrestrial and aquatic habitats. According to a study using these bacteria, at a high caffeine concentration (1.0%), the growth of both bacteria is inhibited. This shows that caffeine also exhibits antibacterial properties.
Anatomy of Daphnia
- Problem statement:
How does the concentration of caffeine solution affect the heart rate of Daphnia?
- Aim:
To determine the effect of concentration of caffeine solution on the heart rate of Daphnia.
2. Hypothesis:
The higher the concentration of caffeine solution, the higher the heart rate of Daphnia.
3.1 Apparatus:
- Cavity slides
- Dropping pipettes
- Muslin cloth
- Petri dish
- Spatula
- Stopwatch
- Paper towels
- Light microscope
3.2 Materials:
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Culture of Daphnia (water fleas)
- Pond water
- Caffeine solution
4. Techniques:
4.1 Mount the Daphnia into the cavity of the slide
- The cavity slide has a concave depression at the centre that can hold a large drop of water. A Daphnia is placed in the cavity and viewed using a light microscope.
4.2 Calculating the heart rate of Daphnia using a calculator
- The heart beats are counted by setting up a basic calculator to add one digit repetitively (key in 1+1=, then key in +1=. Subsequently, press the = key in time with heart beat)
- This is done for 15-second periods, which are then multiplied by 4 to obtain the number of heart beats in one minute.
5. Variables:
- Manipulated variable: Concentration of caffeine
- In this experiment, the concentration of caffeine is varied by adding different amounts of caffeine to the same amount of distilled water. To prepare 0.1% caffeine solution, 0.1g of caffeine is dissolved in 100ml of distilled water. The mixture is shaken to form a solution. Subsequently, 0.2 g of caffeine powder is dissolved in the same amount of distilled water to make 0.2% caffeine solution. Then, the amount of caffeine is altered accordingly to obtain caffeine solutions of concentrations of 0.3%, 0.4% and 0.5%.
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Dependent variable: heart rate of Daphnia
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This is recorded by counting the number of heart beats of Daphnia for 15 seconds. The measurement is then multiplied by 4 to obtain the number of heart beats per minute. This is the heart rate of Daphnia.
5.3 Constant variable: Volume of caffeine solution, Daphnia used
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The volume of caffeine solution is kept constant and the same Daphnia is used throughout the experiment to obtain accurate results.
6. Procedure:
- A Daphnia is placed on a cavity slide lined with muslin cloth.
- Excess liquid is removed using paper towels to allow the Daphnia to lie on its side.
- The Daphnia is observed using low power magnification (40x).
- The heart is identified and the heart beat is counted for 15 seconds. The values are multiplied by 4 to obtain the number of heart beats per minute.
- Step 3 is repeated to obtain another reading.
- The Daphnia is then removed and placed into a Petri dish filled with pond water.
It is left to swim in the pond water for about one minute.
- One drop of 0.1% caffeine solution is added into the cavity of the slide. The
Daphnia is then placed onto the slide lined with muslin cloth.
- The heart is identified and the heart beat is counted for 15 seconds. The values are
multiplied by 4 to obtain the number of heart beats per minute.
- The measurement is repeated to obtain another reading.
- The experiment is repeated using 0.2%, 0.3%, 0.4% and 0.5% caffeine solution.
- The results obtained are recorded. A graph of heart rate against concentration of caffeine solution is drawn to illustrate the relationship between the concentration of caffeine solution and heart rate of Daphnia.
7. Precautions:
- The same volume of caffeine solution is used throughout the experiment.
- Other factors affecting the heart rate such as the temperature of the solution is kept constant throughout the experiment.
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The same Daphnia is used throughout the experiment to minimise errors.
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The Daphnia is a very delicate creature. It should be handled with extra care.
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Muslin cloth is used to ensure that the Daphnia remains in position in the cavity of the slide.
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Excess water on the cavity slide is absorbed using paper towels. Too much solution on the slide will cause the Daphnia to move about vigorously and this affects the heart rate.
- The experiment for each concentration of caffeine is repeated once to improve accuracy.
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As temperature affects the heart rate of Daphnia, the microscope is turned off when not in use as the microscope light radiates heat.
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The Daphnia is placed into a petri dish containing pond water after each experiment. Thus, a better heart rate count can be obtained.
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Distilled water and not tap water is used to dissolve caffeine. This is because tap water usually contains chlorine which will kill the Daphnia.
- As a light microscope with separate illumination is used, direct sunlight should not be reflected through the optics.
8. Risk factor:
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A web search on "caffeine lethal dose Daphnia" gives the median lethal concentration of caffeine for Daphnia as 182 mg/L of water. This is the concentration at which 50% of the organisms die when exposed to that concentration.
- Thus, the concentration of the caffeine solution used in this experiment must not exceed the 182mg/L of water.
Use a transfer pipette to pick up an individual Daphnia, and transfer it to the Petri dish. Put the pipette under the surface of the solution in the Petri dish and pipette carefully to avoid introducing air bubbles beneath the carapace of the Daphnia. (Air bubbles make Daphnia float at the surface, unable to swim, which will eventually kill them.)
9. Results:
Table 1
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A graph of heart rate of Daphnia against concentration of caffeine solution is plotted according to the readings in table 1.
10. Discussion:
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Explanation for graph of heart rate of Daphnia against the concentration of caffeine solution
Based on the graph of heart rate of Daphnia against the concentration of caffeine solution, it can be seen that the heart rate increases when the concentration of caffeine solution increases. There is an overall increase in heart rate and the graph peaks at 204 beats per minute, which corresponds to the caffeine concentration of 0.4%. As mentioned, caffeine is a stimulant which will stimulate the nervous systems of animals, thus increasing the heart rate.
When the concentration of caffeine solution is 0.5%, there is a slight drop in the heart rate of Daphnia. This may be due to the high concentration of caffeine solution used, which causes the heart rate to slow down instead of speeding up. Also, as the same Daphnia is used, the animal may be exhausted as it is placed out of its natural habitat for a long time. Due to exhaustion, the heart rate decreases.
Moreover, there is a greater increase in heart rate from 0.0% to 0.1% caffeine solution and from 0.2% to 0.3% caffeine solution. This is because the recovery rate of Daphnia when placed into pond water at these concentrations is higher. Replacing the Daphnia in its natural habitat will enable the Daphnia to ‘recover’ to improve the accuracy of the measurement of its heart rate.
On the other hand, there is a smaller increase in heart rate of daphnia from 0.1% to 0.2% caffeine solution and also from 0.3% to 0.4% caffeine solution. This is due to the lower recovery rate of Daphnia. It may also be due to errors such as taking the Daphnia out of the pond water to quickly, which shortens the recovery period of the Daphnia. This causes the subsequent reading to be almost similar to the reading before, which contributes to the small increase in heart rate.
- Suggestions to improve the method of carrying out the experiment
- The experiment can be repeated at least twice to obtain 3 readings. From these readings, the average is calculated to improve the accuracy of the results.
Better overall results can be obtained by repeating the experiment because any errors in one experiment can be compensated for by the other two experiments.
10.3 Short comings and other sources of error in the procedure:
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As the Daphnia is placed in a different environment and deprived of water, its stress level may rise. This contributes to the inaccuracy of the results as high stress levels can affect its heart rate.
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Effects of caffeine on Daphnia may be different than that on humans.
Daphnia are evolutionarily far-removed from humans, so the results when Daphnia are exposed to caffeine may not be the same as that for humans.
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Using too many chemicals on one Daphnia sample will stress them out and lead to erroneous results.
- Due to fairly slow human reactions, it is only possible to measure the time of the reaction to the nearest 0.1 second even though the stopwatch shows measurements to the nearest 0.01 second.
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Miscalculations may occur during the calculation of the heart beat. This is because the heart of the Daphnia beats fairly fast.
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The microscope light radiates heat. As temperature affects the heart rate of Daphnia, the rise in temperature due to heat from the microscope will cause the results to be inaccurate.
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Some caffeine solution may adhere to the Daphnia. When it is placed onto the slide with caffeine solution of another concentration, some of the caffeine adhering to the Daphnia will mix with the new caffeine solution. This will affect the actual concentration of the caffeine solution being tested.
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The solution on the slide will cause the Daphnia to move about vigorously and this affects the heart rate.
10.4 Ethical issues to be considered
This experiment deals with live organisms, which are ‘water fleas’ or Daphnia. Thus, there are many ethical issues pertaining to the use of animals for the sake of science. However, the use of Daphnia does not raise as many ethical issues as compared to using human specimens in this experiment.
Nevertheless, experiments on invertebrates are largely unregulated and not included in statistics. Most animals are euthanized after being used in an experiment. There are some organisations that oppose the use of animals in such experiments. This is based on a range of arguments; that it is cruel, poor scientific practice, cannot reliably predict effects in humans, poorly regulated, that the costs outweigh the benefits, or that animals have an intrinsic right not to be used for experimentation.
Also, if every biology student uses the Daphnia for this experiment, the population of Daphnia in that particular area would decrease drastically and may cause a major disruption in the food chain of the aquatic habitat. This is only if all the Daphnia are euthanized after the experiment. Thus, the solution to this problem would be to avoid killing these animals and to return all the Daphnia to its original habitat.
10.5 Further investigations
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This experiment can also be done using or to replace caffeine and observing the effects on the heart rate of Daphnia.
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Apart from stimulants, a depressant such as alcohol can also be used to replace caffeine in this experiment. Daphnia are prone to intoxication and make excellent subjects for studying the effects of the depressant on the .
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The effect of temperature on heart rate in Daphnia can also be investigated. To carry out the experiment, identical culture solutions at temperatures of 5, 10, 15, 20, 25 and 30 degrees Celsius are used. The Daphnia is then placed into these cultures in turn, allowing 5 minutes to elapse before taking a new heart rate determination. Similarly, a graph of heart rate of Daphnia against temperature can be plotted.
- Conclusion:
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The higher the caffeine concentration, the higher the heart rate of Daphnia.
Sources:
Books
1. Title: Edexcel Biology for AS
Author: C.J.Clegg
Publisher: Hodder Education, 2008
Website
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