1.3.9 Describe how the effect of caffeine on heart rate in Daphnia can be investigated practically, and discuss whether there are ethical issues in the use of invertebrates.

the invertebrate daphnia is suitable for experiment because:

• it is abundant in nature and easily obtained (isn't endangered).
• is transparent so its heart can be seen without the need for dissection (procedure is non-invasive).
• has a very simple nervous system and will not suffer ‘stress’ such as mammals might, which makes it ethically more appropriate.

precautions:

• immobilise daphnia (physical activity affects heart rate): use strands of cotton wool in a small dish of the experimental solution.
• make sure other variables are controlled, such as water temperature and daphnia size: temperature should be monitored with a thermometer in the water, use daphnia of similar size for all measurements.
• accurate measurement of heart rate: a dot is put on a piece of paper, or clicking a button on a calculator to keep count of heart beats.
• repeatability: ensure that variables other than caffeine concentration are controlled.  

procedure: 

• place daphnia on cavity slide that has caffeine, without the cover slip to ensure sufficient oxygen supply. 
• place cavity slide filled with ice water under the slide with daphnia to act as a heat sink (daphnia are ectotherms so change in external temperature affects their metabolism and enzyme activity, which in turn affects their heat rate).
• repeat experiment at each concentration to get the mean heart rate (to reduce effect of anomalies, increase reliability, and average out errors).
• repeat procedure using several different concentration of caffeine.
• record and represent results in a graphical format. 
• have a control experiment (to find heart rate under normal conditions without caffeine and to compare treated animal with untreated).