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To study the effects of caffeine on the heart rate To measure the heart rate of Daphnia at different concentration of caffeine.

INTRODUCTION The heart is a muscular organ responsible for pumping oxygenated blood through the blood vessels with repeat, rhythmic contractions.1 However, in arthropods and some other animals the heart does not pump blood. Instead, the heart pumps haemolymph which acts in a similar way as blood found in vertebrates. The fluid contains the copper-based protein called haemocyanin that acts as oxygen transporter. All living organism have their own heart. The heartrate is controlled by the hormones such as acetylcholine, noradrenaline and adrenaline. Acetylcholine slows down the heart rate while noradrenaline and adrenaline make the heart beat faster. Adrenaline increases the heart rate through the action of second messenger or transduction component, a chemical made in the cell known as cyclic adenosine monophosphate(cAMP). Transduction is the process that follows the action of a drug, hormone or neurotransmitter at a receptor, so when adrenaline activates the beta-1 adrenoceptor in the sinoatrial node, this leads to an increase in cAMP in the sinoatrial node and the result is an increase in heart rate.2 Some chemicals are also found to have a similar effect to these hormones such as caffeine and ethanol. Ethical issues might be raised if studies on the effect of these chemicals on the heart rate were done on humans. Therefore, experiments were carried out on simple organisms such as Daphnia as they may not suffer in the same way as higher animals would.
1 http://www.heart.com/the-heart.html 2 http://www.practicalbiology.org/areas/advanced/control-and-communication/control-ofheart-rate/investigating-factors-affecting-the-heart-rate-of-daphnia,92,EXP.html

Daphnia (or Daphnids) are members of a collection of animals that are broadly termed as "water fleas".3 They are predominantly small crustaceans and belong to a group known as Daphniidae. They got their common name because of their jerky movement through water and the resemblance to real fleas. The same species of Daphnia can sometimes look completely different in terms of both size and shape depending on some factors such as its place of origin and environmental factors at that location. There are approximately 150 known species in North America, and a similar number in Europe (many of these species are found on both continents, either through accidental introduction by man, or nature).4 Examples of

different species of Daphnia are Daphnia pulex and Daphnia

magna. Most Daphnia feed on

Figure 1: Anatomy of a Daphnia

particles floating in the water such as phytoplankton and some feed on zooplankton, but free-living algae, bacteria and fungi are the

http://www.practicalbiology.org/areas/advanced/cont rol-and-communication/control-of-heartrate/investigating-factors-affecting-the-heart-rate-ofdaphnia,92,EXP.html

predominant foods. Daphnia replicate by parthenogenesis, the ability to self-replicate without fertilisation of any form and the offspring are of the exact genetic content as of the parent. If any differences occur to the physical state of the offspring it is because of environmental factors.
3 http://www.caudata.org/daphnia/ 4 http://www.caudata.org/daphnia/

Parthenogenesis allows Daphnia to evolved and take good advantage of good conditions. During late spring, summer and early autumn, they reproduce on average ten live young individuals per Daphnia. Some eggs develop into males and the females produced eggs that must be fertilised when the food is scarce. The eggs can survive harsh conditions and can withstand if the pond dries up for a while and they can also even withstand freezing. The egg producing generations will start producing live young Daphnia again which are all female and the male will die out completely until the conditions worsen again. Daphnia has been used as tests subject intensely for over a century. The development of genomic infrastructure coupled with a wide range of phenotypic diversity make Daphnia a versatile model system to investigate fundamental mechanisms of inheritance and development, cellular function, physiological systems, immunity response, disease, macromolecular structure/function relationships, and the genetic basis of complex phenotypic traits.5 Daphnia is widely used as a model for ecotoxicological studies as they are a sensitive sentinel species in freshwater. Besides that, Daphnia is also used to understand genomic response to environmental stressors that are vital factors in human health and being. Daphnia have a wide, nearly cosmopolitan, distribution as they occur in a highly diverse set of habitats ranging from freshwater lakes to saline ponds and as a result of that they manifest extensive phenotypic diversity providing ample raw material to study gene function and genome by environment interactions.6 In this experiment to find out the effect of certain chemicals on the heart rate, caffeine was used on the Daphnia. Caffeine is a naturally occurring chemical stimulant called

5 http://www.nih.gov/science/models/daphnia/ 6 http://www.nih.gov/science/models/daphnia/

trimethylxanthine and its chemical formula is C8H10N4O27. Caffeine is a drug and has similar traits with other drugs such as amphetamines, cocaine, and heroin. Caffeine stimulates the brain function in the same biochemical mechanism as these drugs do. Caffeine causes the increase number of stimulatory neurotransmitters to be released by the body. Caffeine tastes very bitter and is a white crystalline powder in its pure form. Caffeines energizing properties was

discovered around 800 a.d. according to African folklore and coffee was the local staple food in Europe and Asia as early as the 1400s along with tea. Plants get rid of insects by making a chemical we now know as caffeine. Cocoa, coffee and tea which we know to have contained caffeine has been used
Figure 2: Chemical structure of caffeine

for a long time to give us the energy boost that we need to keep going. The amount of caffeine can vary


from food to food and depends on the type, serving size and how the food or beverage is prepared. Caffeine content can range from as much as 160 milligrams in some energy drinks to as little as 4 milligrams in a 1-ounce serving of chocolate-flavored syrup. Even decaffeinated coffee isn't completely free of caffeine.8 Some over-the-counter pain relievers, cold medications, and diet pills may also contain caffeine ranging from 16 milligrams to over as much as 200 milligrams. This is because caffeine is a mild painkiller and it also increases the effectiveness of other pain relievers.

7 http://science.howstuffworks.com/caffeine1.htm 8 http://www.webmd.com/balance/caffeine-myths-and-facts

What is the effect of caffeine on the heart rate of Daphnia?

The higher the concentration of caffeine, the higher the heart rate of Daphnia.

Manipulated variable : The concentration of caffeine used. Responding variable : The heart rate of Daphnia. Constant variable : The type of caffeine. : Type of Daphnia.

APPARATUS Stop watch, beakers, cavity slide, electronic balance, dropping pipettes, microscope, pencil, filter paper, cotton wools.

MATERIALS Distilled water, caffeine solutions, Daphnia,

PROCEDURE 1. A few of cotton wool was placed on a slide to restrict the movement of Daphnia. 2. One large Daphnia was transferred to the slide by using a pipette. The water was removed using filter paper then 1-2 drops of distilled water was added into it. 3. The numbers of heart beats per minute was recorded by using a stopwatch.
4. Work in pair, the beat was counted by a person while the heart beat was counted by the

other person by using pencil mark.

5. The heart rate intervals of 15 seconds for 1 minute were recorded. Do a blind study. 6. The procedure was repeated by using other Daphnia with similar size and the slide was

cleaned. The water was replaced with caffeine solution (1%). The Daphnia and caffeine solution were equilibrated for 1 minute before putting under the microscope. 7. The results were recorded in a suitable format and appropriate graph. 8. The treatments were compared. RESULTS Concentration of Caffeine (%) Heart beats of Daphnia sp in 15 seconds time intervals Avera ge Control 0.1 0.2 0.3 0.4 28.0 62.0 73.0 76.0 66.0 24.0 60.0 72.0 78.0 72.0 34.0 60.0 75.0 88.0 65.0 32.0 62.0 76.0 79.0 62.0 29.5 61.0 74.0 80.3 66.3 118.0 244.0 296.0 321.0 265.0 BPM








Table 1 shows the readings of heart beats of Daphnia sp correspond to the different concentrations of Caffeine solution

Graph 1 shows the graph of heart rate at time intervals against the correspond different concentrations

Graph 2 shows the graph of average heart rate against the different correspond concentrations

DISCUSSION Based on the results, the heart rate of Daphnia every 1 minute increases as the concentration of the caffeine increases except when the concentration of caffeine is 0.4%. This may be due to some errors in the experiment which will be discussed later. First set of experiment was done with o% concentration of caffeine. The heart rate of the Daphnia used in this set acts as a control and used as a baseline for experiments using o.1%, 0.2%, 0.3%, 0.4% and 0.5% caffeine concentration. The heart rate obtained later in this experiment was compared with the control and a graph was drawn (Graph 1). The drop in the heart rate at 0.4% caffeine solution may be due to the size of the Daphnia. As different sizes of Daphnia have their heart beat at different rates, the bigger Daphnia have slower heart rate while the smaller ones have faster heart rate it can be said that maybe the Daphnia used during the concentration of caffeine

is 0.4% is larger than the others used as they are small organisms and it is hard to see them before putting them under the microscope. The heart beats per minute (BPM) was obtained (Table 1) by multiplying the results by four. By doing this we assumed that the same result will be obtained if the experiments were to be repeated. The BPM for 0.1%, 0.2%, 0.3% 0.4% and 0.5% caffeine solution are 118, 244, 296, 321, 265 and 343 respectively. The heart of a Daphnia will be affected in the same way as a human heart would in the presence of caffeine. Caffeine belongs to a class of compound called methylxanthines9. The amount of cAMP in the sinoatrial (SA) node is increased by caffeine this will lead to an increase in electrical activity of SA node. This causes the SA node to depolarize and beat faster. Caffeine is similar to adrenaline and noradrenaline as caffeine can lead to the increase in the rate of contraction and relaxation of each heart beat by affecting the main pumping chambers which are the ventricles. Caffeine also binds to the A1 adenosine receptors in the brain and the heart blocking the receptors from binding to adenosine as caffeine looks similar to adenosine to a nerve cell. Caffeine also inhibit a class of enzyme known as cyclic nucleotide phosphodiestererases which is responsible for degrading a stimulatory signal produced when excitatory neurotransmitters activate different neurons in the central nervous system (CNS).10 The stimulatory signal remains active longer when they are inhibited by caffeine. This causes a greater sense of alertness along with higher heart rate, blood pressure and respiratory rate. The pituitary gland in the brain senses the effect of caffeine on the brain which causes increased neuron firing. The pituitary gland will release hormones to tell the adrenal glands to produce adrenaline, the fight or flight hormone.
9 http://www.madsci.org/posts/archives/jul99/931925101.Zo.r.html 10 http://www.madsci.org/posts/archives/jul99/931925101.Zo.r.html

The concentration of the caffeine solution used was manipulated and other factors such as time taken to measure the heart rate, pond water, size of Daphnia and temperature are kept constant. To get the concentration of 0.1%, 0.2 %, 0.3%, 0.4% and 0.5% caffeine solution, the mass of caffeine that was mix with 100ml distilled water are 0.1g, 0.2g, 0.3g, 0.4g and 0.5g respectively.

EVALUATION Limitations and Errors There are a few sources of errors in this experiment. Firstly, the temperature of the surrounding is not constant for all slides and it may not be constant throughout the experiment for each slide. The slides were put under the light microscope. As we know, not all the electricity supplied to the bulb is converted to light energy. Some are converted to heat energy and this heat energy will transfer to the slide causing the temperature to increase. Increase in temperature will causes the heart rate of the Daphnia to increase and this will lead to an inaccurate result. We can use a heatsink, a cavity tile filled with iced water placed on the microscope under the slide to maintain a constant temperature. Next, the counting of the heart rate will have errors. Human reaction will certainly cause some errors in this experiment especially during the transition from the first 15 seconds to the next. The person counting the beat and the person counting the heart beat are not synchronized

and therefore there will be a slight delay when doing the experiment. The counting of the dots which represent the heart beat of the Daphnia contributes to the error. Some of the dots may overlap each other and it may cause misinterpretation during counting. Therefore, the results might be lower or even higher than expected. We can project the image of the slide onto a large screen by using an ICam. We can also use a stroboscope to freeze the motion even though this is not recommended as it is very tricky and can cause eye strain and dizziness. Some of the limitations in this experiment are we cannot see the heart of the Daphnia clearly as they are very small. Some might say the heart is at one place while the other might say it is somewhere else. The Daphnia is also constantly moving which makes it even harder to observe its heart. This could cause the beat that they counted is wrong. To be on the safe side, we can count the rate at which the legs beat as they are proportional to the heart beat. We can also place a cotton wool to restrict the movement of the Daphnia, but this is not very effective. Therefore, we can use starch acetate or petroleum jelly which can reduce the movement of Daphnia effectively so that the counting process can be done much more accurately. This experiment was only done once for each concentration and the result was multiplied by 4. The results do not represent the true value for the experiment as they are only assumptions. For each concentration it should be repeated at least three times and calculate the average to ensure the results are accurate and precise. Other than that, the caffeine concentration used might not be exactly as the concentration stated. The coffee powder might not dissolve completely thereby reducing the concentration of the caffeine solution and affecting the data. We just assume that the concentration used is exactly the same as the one stated.

In this experiment, the Daphnia used was not the same for all concentration. They may differ in size and species. The metabolic rate might differ and also does its heart rate. Some will have a naturally slow or fast heart rate. For each concentration of caffeine solution and the control slide we used distilled water. This may caused the Daphnia to become stressful and their heart rate increases. This is because distilled water lacks oxygen. We should use pond water or Daphnia culture solution so that the Daphnia are in their best condition.

SAFETY PRECAUTION Throughout this experiment students should always lab coats. Caffeine stain is hard to remove. Therefore, we must wear lab coats so that our clothes will not get stained. The apparatus used in this experiment are fragile and expensive. They should be handled with care as not to break or damage the apparatus.

FURTHER INVESTIGATIONS There are other factors that can affect the heart rate that can be investigated. Caffeine can be replaced with alcohol and we can also investigate what effect temperature can do to our heart rate.

Ethanol can slows down the heart rate and at similar concentrations used for caffeine solutions; ethanol can depresses the nervous system by acting as nonselective neurodepressant. The concentrations used are sufficient to depress the respiratory centres of the brain resulting in death in humans. We can investigate whether this is true or false. Other than that, we can investigate the effect of temperature on heart rate. The temperatures that can be used to investigate this are 0oC, 25oC, 35oC, 45oC, 55oC and 65oC. When the temperature increases, the metabolic rate also increases and therefore the heart rate also increases. This experiment can be carried out by putting the Daphnia in a culture solution which has been immersed at different temperatures. This experiment using caffeine solution was only up to 0.5% caffeine solution. We can investigate further by increasing the concentration of caffeine up to 1% at observe what happen to the heart rate of the Daphnia.

CONCLUSION The hypothesis is accepted as the rate of concentration of caffeine increase the rate of heartbeat of daphnia also increase. This is shown clearly based on the result and graph above.

Caffeine myths ad facts. Retrieved October 21, 2011, from http://www.webmd.com/balance/caffeine-myths-and-facts Daphnia. Retrieved October 20, 2011, from National Institute of Health: http://www.nih.gov/science/models/daphnia/

John Clare, B. P. Daphnia: An Aquarist's Guide. Retrieved October 20, 2011, from Daphnia: http://www.caudata.org/daphnia/ Marshall Brain, C. W. How Caffeine Works. Retrieved October 21, 2011, from Howstuffworks: http://science.howstuffworks.com/caffeine1.htm Terry Hebert, F. U. MadSci Network:Zoology. Retrieved October 22, 2011, from MadSci: http://www.madsci.org/posts/archives/jul99/931925101.Zo.r.html The Human Heart. Retrieved October 20, 2011, from Articles of the Heart: http://www.heart.com/the-heart.html