Caffeine is a drug considered to be a stimulant as well as a mild diuretic. The substance itself can be sourced in beans, leaves and even the fruit of some plants where it exists as a natural pesticide.

Upon consumption by humans, caffeine acts as a metabolic stimulant upon the central nervous system (CNS), resulting in the temporary relief from drowsiness along with an increase in general mental alertness.

Caffeine, even though it’s addictive nature is widely known, is still regularly consumed by most adults daily and resides in a wide range of readily available store-bought products. Currently, in the UK there exist no laws restricting the purchase of caffeine to certain groups of individuals despite many conducted studies concluding that the substance may result in negative implications upon particular persons such as young children or pregnant women.

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The following experiment will investigate the effects of caffeine upon the heart rate of daphnia (water flea) via the use of specific, controlled methods and the restriction of both variables and fixed conditions.


I predict that as the concentration of caffeine to which the daphnia is exposed increases, the heart rate of the daphnia will increase likewise. The two will be both correlational and proportional to one another.


* Culture of Daphnia

* Cavity Slide

* Heat Lamp

* Dropping Pipette

* Distilled Water

* Caffeine

* Cotton Wool

* Paper

* Pencil

* Standard Glassware

* Filter Paper

* Microscope

* Stopwatch

Cavity Slide

Cotton Wool Ring

Distilled Water



* The petri dish to be used will first be cooled in order to prevent dangerous overheating of the daphnia by the heat lamp during the experiment.

* The heat lamp will then be angled to an appropriate position to ensure that full lighting can be applied throughout the experiment.

* A pipette will be used to transfer the daphnia onto the cavity slide along with a very small amount of water which is then carefully absorbed using filter paper.

* Two drops of distilled water will then be pipetted onto the daphnia.

* Cotton wool will be carefully positioned in a ring surrounding the daphnia in order to prevent the organism from swimming directly off the slide.

* The cavity slide containing the daphnia should then be placed under the microscope where its beating heart can be located.

* Using a pen and a piece of paper, each heartbeat of the daphnia will be recorded via a series of dots for a period of one minute which can be determined using the stopwatch.

* Once the said period of time is up, the daphnia will be removed from under the microscope and away from the heat source, into a hospitable environment alternate to that of the rest of the remaining culture so as not to confuse the unused daphnia to the tested ones.

* The above method should then be repeated exactly except from the replacement of distilled water for alternate caffeine solutions.

o Each of the solutions including the control should be repeated three times in order to obtain reliable results, reduce the chances of anomalous results and also to acquire an average.

o For every new test, a new daphnia should be used.


By pursuing the following measures, I can assume greater validity of my to-be-obtained results. First, the general size of each f the daphnia used will be regulated by subjective concurring selection via the intervention of an outside party opinion. This will ensure that the daphnia used for my experiments are generally of a similar size and are not deemed so by the investigator alone. Also, the light intensity and therefore the heat distribution of the lamp will be kept identical for each experiment as the same heat lamp will continuously be used for each experimental daphnia. In addition to this, the distance of the heat lamp from the microscope will also remain continuous as neither apparatus will be moved or altered in any way for each new test. The measurement of the heart beats per minute will also be judged by the same person throughout the investigation to disallow for any variations in subjective results.

Furthermore, a blind study will be conducted where the individual who determines the heart beats of the daphnia will remain unaware of the concentration in which the organism is currently submerged. This means that experimenter effects by which the expectance of a result influences the actual result gained, thus the use of a blind study will eliminate such occurrences. It is also to be noted that the period of time for which the daphnia is left in the solution can be controlled by the implementation of a stop watch to time all procedural occurrences throughout the investigation. Finally, the amount of solution in which the daphnia is submerged can be standardised by allowing only 2 drops of solution drip from the pipette onto the cavity slide each time.

Independent Variable: Concentration of Solution

Dependant Variable: Heart rate of Daphnia

Potential Extraneous Variables

* Caffeine tolerance of organism

* Exposed surface area of daphnia

* Stress tolerance of daphnia

Health and Safety Measures

* Take care when handling glassware.

* Take care of handling heat lamp, allow to cool before handling and do not touch when turned on.

* Quickly clean up any liquid spillages to prevent malfunction of any equipment.

* Refrain from placing electrical equipment in close proximity to any liquids.

* Ensure work space is clear before commencing experiment.


Daphnia are living organisms, therefore it s imperative that they are treated as such and are not exposed to any unnecessary prolonged stressful situations that may cause harm to their physical well-being. As such, measures and precautions must be undertaken in order to prevent the arisal of any such unethical proceedings. Firstly, the daphnia must be kept in conditions similar to that of their natural habitat. These include the correct temperatures and water type to which they will be exposed. This will hopefully minimise the stress that the daphnia will be placed under and help to achieve greater accuracy in the results.

Secondly, the heat used on the daphnia during the investigation must be kept to a minimal intensity in order to prevent the overheating of the organisms by which they may dry up and die. Therefore, as previously mentioned the cavity slides upon which the daphnia will be placed are to be cooled to prevent the above occurrence, also the heat lamp will only be turned on for minute intervals during which the heart rate of the daphnia will be recorded. Cotton wool has also been implemented into the method to guarantee that the water flea does not simply swim off the edge of the cavity slide and die.


Percentage of Caffeine Present in Solution

Heart beats per minute

Mean Heartbeats per Minute


































Analysis of Results

It can be seen from the table of results and more clearly from the graph that a definite pattern or trend emerges between the comparative data. There exists a fairly strong positive correlation between the heart rate of the daphnia and the caffeine concentrations to which they were exposed. A regression line has been included to demonstrate this correlation. As the percentage of caffeine in the solution increases, the heart rate of the daphnia does likewise. There do however exist limitations inflicting upon these results such as the sensitivity of the organisms to stressful situations or the fact that one of the water fleas used may possess an existing physical disorder that may ultimately affect their heart rate thus altering the results. (Other factors have been discussed in greater detail in Validity).


Although the experiment was conducted with the upmost care and attention, there are several factors which, should the opportunity arise, could be improved upon and altered.

First, the conditions in which the daphnia were kept could have represented greater accuracy to the organism’s natural habitat to alleviate the stress of environmental change and generate greater result accuracy. Also, continuing with this point, the daphnia could have been left in an undisturbed state for 48 hours previous to the start of the investigation to allow them time to settle and once again remove the stress factor of new surroundings affecting their heart rate.

Also, the heat produced from the heat lamp was much too fierce and as a result it would be of benefit to the experiment as a whole to alter the light source used in order to reduce the risk o overheating to the water fleas.

Finally, it may also prove advantageous to use water from the daphnia’s natural habitat rather than distilled water in the cavity slides so that the organisms have greater access to Oxygen supplies when necessary. Yet again, this would help to reduce the risk of death or physical impairment to the daphnia and improve the ethical status of the investigation.

Going further

Should time allowances be made it may be interesting to take matters further and pursue other aspects of the experiment such as;

* Using a greater variation of caffeine solution concentrations

* Investigating the role of other substances upon heart rate

* Using alternate daphnia species to conclude whether the effects prove different or dissimilar.


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