Hypothesis/ Null hypothesis :
As the caffeine concentration rises, so does the mean heart rate of daphnia.
Caffeine does not affect the heart rate of daphnia.

CAFFEINE:
Caffeine mimics some of the effects of adrenaline and noradrenaline in the heart. It also increases the amount of cAMP in the senatorial node. Caffeine has additional effects on the heart. Like adrenaline and noradrenaline, it can affect the main pumping chambers (ventricles), leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart's individual beats are associated with an increased volume of blood ejected into the circulation per unit time. This is called increasing cardiac output.

Risk Assessment:
In this experiment there are the following risks both for the human that carries out the experiment and the daphnia:
The human that carries out the experiment should be cautious not to touch the lamp with wet hands since it uses electricity.
Also, be careful not to spill any of the pond water on the floor.
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.
Moreover, when they are given caffeine of a high concentration their heart may beat so fast that will cause their death.
Furthermore, there is the risk of the daphnia to dry up, which may kill it in the middle of the experiment.

Method:
These are the steps that we followed when doing the experiment:
1. Use a pipette to transfer the daphnia to a dish slide containing pond water of known volume.
2. Add some cotton wool to the dish slide and move them with a fine paint brush in such a way that the daphnia can’t move.
3. Use a calculator to calculate the heart rate. Set its reading to zero and add 1 to the answer.
4. One student will use a stopwatch to count 1 minute which is the time during which the measurement of the heart rate will take place.
5. Turn the microscope lamp on and locate the heart of the daphnia through the microscope.
6. Count the heartbeats for 1 minute. Do this 3 times, get an average and record the bpms in the Data Table.
7. Clean the dish slide using a tissue and make sure there is no pond water left.
8. Use a pipette to transfer a known volume of caffeine solution of concentration 0.1% to the dish slide.
9. Transfer the daphnia to the dish slide using the pipette.
10. Measure the heart beats of daphnia, following the same procedure as before.
11. Repeat steps 9-11 using caffeine solutions of concentrations 0.2% and 0.3%

ETHICAL ISSUES (Notes for the experiment):
Pond water is used as the daphnia are used to living there and we want to keep as many aspects from their normal life as we can, so that the results are more accurate. This is also the reason for which they should be kept in pond water not taking from the wild
.You can purchase live cultures from suppliers, including pet shops and local aquarists. Some scientific suppliers sell viable dried Daphnia eggs and culture kits. Alternatively, you can collect adult Daphnia by pond dipping; in this case you must observe strict hygiene procedures, since pollutants and the bacteria causing Weil’s disease may contaminate pond water. Stock purchased from aquarists is usually free from this hazard.
The microscope lamp should only be on while counting the beat rate as it might alter the temperature and when the temperature is increased the heart rate may go up because of this and not because of the aspect we are investigating.
A dish slide and NOT an ordinary flat microscope slide shall be used, as the dish slide will help keep the daphnia in a small volume of either caffeine solution or pond water and not letting it escape.

Cotton is used to keep the daphnia still so that the heart rate can be easily counted.
This experiment involves a living organism so we need to be careful with it. So, we should leave it in conditions similar to those that it