What issue may affect the validity of an experiment investigating heart rate in Daphnia?

Fatigue of the subject can significantly affect the validity of an experiment investigating heart rate in Daphnia. When subjects experience fatigue, their physiological responses may alter, leading to variations in heart rate that are not related to the experimental manipulation. For example, if Daphnia are overstimulated or if they experience prolonged exposure to stressors, their heart rates might increase or decrease unpredictably due to the fatigue rather than the conditions being tested. This can introduce variability that complicates the interpretation of the results.

While excessive light exposure, limited water volume, and incorrect temperature measurement can also impact the overall health and behavior of Daphnia, the direct consequence of fatigue on heart rate specifically makes it a more pertinent issue in the context of ensuring valid and reliable data in such experiments. Fatigue directly impacts the physiological state of the organism being studied, making it a critical factor to control for to draw accurate conclusions from the experimental data.