# Daphnia Experiment

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### Introduction

In this experiment I aim to find out if caffeine has an effect on the heart rate of daphnia. Daphnia are water fleas that have a sort of heart that we can see in magnification. We can count the number of heart beats in a minute of a regular daphnia and try and get a new one with the same specifications as the old one that may have died. It is also a good idea to get a new one as we want to see how much it affects it from ordinary instead of adding the caffeine one after another. We have to be careful not to feed too much caffeine to the daphnia only a maximum of 1% as we may not be able to get a reading due to it dying. Planning

### Hypothesis

There is a correlation between concentration of caffeine solution and the heart rate of Daphnia. In this experiment, when the concentration of caffeine solution increases, the heart rate of Daphnia increases. Null Hypothesis

There is no correlation between concentration of caffeine solution and the heart rate of Daphnia. Scientific basis for the hypothesis 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. Statistical Test

Student’s T-test: it is used for continuous data because it shows a difference between the independent and dependent variable.

After working out my student-t test, my calculated value for t is greater than the critical value of t. If it is greater it states that I will need to reject my null hypothesis. This means that I can accept my hypothesis.

### Variables

The independent variable for the experiment is the presence of 5% caffeine that’s given to the daphnia which can be changed by adding a drop each time. The dependent variable is the heart rates of the daphnia which can be measured by finding the heart beat for 10s then multiply it by 6 to get it for 1 minute. This can be done by dotting the paper to count the heart beat. The controlled variable throughout the experiment are:

Temperature: if the temperature is high than it might change the heart rate. This can be controlled by using water bath. The daphnia should be on the slide for the same amount of time: this is because the heart increases the heart rate. Use similar size daphnia: the smaller the daphnia have a higher rate than bigger daphnia. The amount of water: dilute the caffeine solution.

In some experiments there are some limitations. These are the following: the heat produced when the lamp is on cannot be handled and might be the reason for the heart rate to increase the heart rate might increase because of the effort of the daphnia that is trying to escape from the cotton wool.

Equipment (other than standard glassware etc…)
Microscope
Cotton Wool
Pipette
O.5% caffeine solution
Dechlorinated water
Safety

In this experiment there are the following risks both for the lab partner that carries out the experiment and the daphnia: 1. We both should be cautious not to touch the lamp with wet hands since it uses electricity. 2. Also, be careful not to spill any of the pond water on the floor. 3. As for the daphnia, there is a chance for them to die as when they are exposed to light, they are also exposed to heat in which they aren’t used to.

### Ethical Issues

It should be understood that any experiment involving living organism always rises up bioethical issues. While performing experiment involving living organism can bring greater advances in science, it should not be forgotten
that the well-being of the living organisms involved in the experiment should be taken care of, so that they do not suffer for the experiment or at least their sufferings are minimized. Therefore, the Daphnia being used in the experiment ought to be treated with great care to avoid hurting or even killing them. If possible, the Daphnia should be returned back to their natural habitat after the experiment so that they can return to their normal lives. Method

1. These are the steps that we followed when doing the experiment:
2. Use fresh Daphnia for each different caffeine solution.
3. Use a pipette to transfer the daphnia to a cavity slide containing pond water of known volume.
4. Add some cotton wool to the cavity slide and place it in a way that the daphnia can’t move.
5. Turn the microscope lamp on and locate the heart of the daphnia through the microscope.
6. In a pair one will use a stopwatch to count 10 seconds which is the time during which the other person will measure the heart beat by dotting the beats onto a piece of paper.
7. Measure the beat rate.
8. Repeat steps 1-6 about 2 or 3 times and get an average without caffeine then after 6 readings repeat the steps with caffeine.
9. Clean the cavity slide using a tissue and make sure there is no pond water left.

#### Conclusion

In conclusion the heart rate of the daphnia increased when caffeine was added. From the graph it suggests that there is a significant difference between the heart rate without caffeine and with caffeine. The highest the heart rate per minute without caffeine was 306 beats which increased by 84 beats per minute when caffeine was added. When standard error (SE) bars do not overlap, you cannot be sure that the difference between two means is statistically significant. Even though the error bars do not overlap in the experiment, the difference is not statistically significant. Also as the concentration of caffeine increases, heart rate of Daphnia increases for caffeine concentration up to 0.5% because caffeine is a stimulant which raises the heart rate of animals. Assumption that a similar effect would result in human can be made.

However, some the results from the graph varied because we didn’t use the same size daphnia. Some of them were either pregnant or were having babies which could have affected the results because the bigger the daphnia the bigger the surface area the higher the heart rate. To improve the experiment I would have repeated the test three times in order to obtain higher accuracy. Random errors could be minimised in this way. Tapping a pencil on a piece of paper and count up the pencil marks at the end of the time period was a method which is more primitive and errors are more likely to occur because some pencil marks may be too close together and cause confusion in counting the number pencil mark.

The limited caffeine concentration for Daphnia is about 1%. That means high concentration of caffeine solution should not used for Daphnia may die of it. Hence, the highest concentration of caffeine solution used in the experiment is only 0.5%. There are a few unavoidable limitations in this experiment which may affect the accuracy of the results. The following are the limitations and their respective improvements: Temperature of the environment of the Daphnia is one of the factors that affect the heart rate of it. Prolong exposure of the Daphnia to the microscope light will overheat the Daphnia. To prevent the Daphnia from overheating, the microscope light should be turned off between observations and a heat sink should be used.

Heart beat of Daphnia can be very fast when caffeine solution was added. Missing a few beats when counting the heart beat or beating of legs is almost inevitable. An improvement to this is to set a video camera above the eyepiece so that the heart beat of Daphnia is recorded. Later, the video can be played in slow motion with the timeline of the video displaying on the screen. This helps to obtain a more accurate heart beat rate.

Since the Daphnia is not observed under its natural habitat, its stressed level in the experiment may be higher than usual. This might cause a change in its heart rate or an undesired change in the heart rate. The only thing to be done is to count the heart beat of the Daphnia as soon as it is ready to be observed.

Daphnia are selected as the model in this experiment as they are fairly easy to keep and the transparent body allows easy observation of changes in heart rate under a microscope without having to dissect it. On the minus side, experimenters have to be extremely cautious when dealing with them because they are delicate and vulnerable. Generally, when the concentration of caffeine solution increases, the heart rate of Daphnia increases. Prolong exposure of Daphnia to caffeine can result in the building up of tolerance of Daphnia towards caffeine.

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