Biology Lab Report

NAME NRIC GROUP STUDENT ID TITTLE OF PRACTICAL DATE LECTURER

: : : : : : :

NURAINA SAKINA BINTI ZAKRI 951010-11-5076 11SC2 1311171218 THE EFFECT OF CAFFEIN ON HEART RATE 1ST OCTOBER 2013 MR WILLIAM NGU

THE EFFECT OF CAFFEIN ON HEART RATE OBJECTIVE To investigate the effect of caffeine on the heart rate of Daphnia sp. (water fleas) To highlight experimental and investigative skills

INTRODUCTION

The structural formula of a caffeine molecule

The space-filling model of a caffeine molecule

In 1819, caffeine (IUPAC nomenclature: 1,3,7-trimethyl- 1H-purine- 2,6(3H,7H)dione) was discovered by a German chemist, Friedrich Ferdinand Runge. It is a bitter white crystalline xanthine that acts as a psychoactive stimulant drug and a mild diuretic in humans and other animals. Caffeine, an odourless and slightly bitter alkaloid, is found in coffee, tea, kola nuts, ilex plants, and, in small amounts, in cocoa, where it acts as a natural pesticide that paralyzes and kills certain insects feeding on the plants. It is also known as guaranine when found in guarana, mateine when found in mate, and theine when found in tea; all of these names are synonyms for the same chemical compound. It is most commonly consumed by humans in infusions extracted from the cherries of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks containing products derived from the kola nut. Other sources include yerba mate, guarana berries, and the Yaupon Holly.

Caffeine is absorbed rapidly into the bloodstream from the gastro-intestinal tract in human whenever consumed. It reaches maximum concentration in circulatory system within about an hour. The blood distributes it throughout the body. It even manages to pass through the blood-brain barrier. Early experiments showed that low concentrations of caffeine may produce small decreases in heart rate in human, whereas higher concentrations may make the heart beat abnormally fast. In the brain it constricts the cerebral blood vessels. If one is used to drinking several cups of coffee a day but quit drinking later, those blood vessels will dilate, maybe enough to give that person a powerful headache. It is one of the best known withdrawal symptoms. In humans, caffeine is a central nervous system (CNS) stimulant, having the effect of temporarily warding off drowsiness and restoring alertness. It is the most commonly used mind-altering drug in the world. When used in moderation, caffeine acts as a mild stimulant to the nervous system, blocking the neurotransmitter adenosine and resulting in a feeling of well-being and alertness. According to one study, caffeine in the form of coffee significantly reduces the risk of heart disease in epidemiological studies. However, the protective effect was found only in participants who were not severely hypertensive (i.e. patients that are not suffering from a very high blood pressure). Furthermore, no significant protective effect was found in participants aged less than 65 years or in cerebrovascular disease mortality for those aged equal or more than 65 years. Excessive intake of caffeine can result in restlessness, insomnia, and heart irregularities. The effects of caffeine vary from person to person, as people excrete it at different rates. Physical dependence and unpleasant symptoms upon withdrawal (headache, fatigue and depression) are common in regular caffeine users. A study of effect of caffeine in human heart rate will be very valuable. However, there are many difficulties in conducting an experiment that involves human as guinea pig. Thus, an alternative is to use a model organism to represent human. The results of the experiment can then be used as a reference to the effect of caffeine on human. Daphnia are selected as the model as they can be easily handled in many ways in the investigation. Daphnia sp. are small, planktonic crustaceans. They, between 0.2 to 5mm in length, are common freshwater cladocerans, often classified with other tiny crustaceans as
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microcrustaceans. Cladocerans are commonly known as water fleas not only due to their salutatory swimming style, but also because of their resemblance to real fleas, though real fleas are insects and share only an extremely distant common ancestry with Daphnia, since both crustaceans and insects are arthropods. Most species in the Order Cladocera are freshwater species, although there are some marine species. The classification of Cladocera is as an order within the Subclass Diplostraca within the Class Branchiopoda within the Subphylum Crustacea. Daphnia sp. live in various aquatic environments ranging from acidic swamps to freshwater lakes, ponds, streams and rivers.

The figure below shows the anatomy of a typical Daphnia sp.

In terms of nutritional information, Daphnia have a protein content of around 50% dry weight and a fat content of 20-27% for adults (4-6% for juveniles), nevertheless some species have been reported to have a higher protein content. Daphnia tend to be almost kidney shaped, possessing only a single compound eye (though they have an ocellus, a simple eye), two doubly-branched antennae (frequently half the length of the body or more), and leaf-like limbs inside the carapace that produce a current of water which carries food and oxygen to
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the mouth and gills. The carapace covers the body, including the 4 to 6 pairs of thoracic appendages, and is used as a brood chamber. The abdomen and post-abdomen (distal to the anus) is generally bent forward under the thorax. The post-abdomen bears two large claws used primarily for cleaning debris out of the carapace. Swimming is accomplished mainly by downward strokes of the large second set of antennae. The action of this second set of antennae is responsible for the jumping motion too. Their bodies are almost transparent and with a microscope the heart beating can be observed easily, and sometimes even their last meal (the gut may appear green if the individual has been feeding on algae). As a result they make excellent subjects for the microscope as one can observe the beating heart. The heart is at the top of the back, just behind the head, and the average heart rate is approximately 180 bpm under normal conditions. A few Daphnia prey on tiny crustaceans and rotifers, but most are filter feeders, ingesting mainly unicellular algae (e.g. Chlamydomonas sp.) and various sorts of organic detritus including protists and bacteria. In the summer months, they can often be seen "blooming" in ponds and lakes as the concentration of algae builds up. They also eat forms of yeast, but mostly in laboratories or controlled environments. The life span of Daphnia, from the release of the egg into the brood chamber until the death of the adult, is highly variable depending on the species and environmental conditions. Typically, the lifespan of a Daphnia does not exceed one year and is largely temperature dependent. It is discovered that the life span increases as temperature decreases, due to lowered metabolic activity. Daphnia reproduce by parthenogenicity (the ability to self-replicate without fertilisation of any form), during the late spring, summer and early autumn (depending on temperature, food availability and presence of waste products of their metabolism). The entire race is made up of females during this period. Developing embryos are often visible in the mother's body without the aid of a microscope. However, when food is scarce, some eggs develop into males and the females produce eggs that must be fertilised. These eggs develop into small embryos which then go into suspended animation, and are shed with the carapace as dark brown/black saddle-shaped cases known as ephippia. These can survive harsh conditions. When conditions improve again, the egg producing generations begin producing live young once again (all females), and the male sex dies out completely until it is needed when conditions worsen once again.
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Similar to many animals, Daphnia sp. are prone to alcohol intoxication, and make excellent subjects for studying the effects of the depressant on the nervous system due to the translucent exoskeleton, and the visibly altered heart rate. They are tolerant of being observed live under a cover slip and appear to suffer no harm when returned to open water. In this experiment, it is intended to study the effect of different concentration of caffeine solutions on the heart rate of Daphnia.

PROBLEM STATEMENT What is the effect of caffeine on the heart rate of water fleas (Daphnia sp.)? HYPOTHESIS There is a relationship between the concentrations of caffeine on the heart rate of water fleas (Daphnia sp.). In this experiment, the greater the concentration of caffeine, the higher the heart rate of water fleas (Daphnia sp.). VARIABLE Independent variable Dependent variable Constant variable : : : The concentration of caffeine solution The heart rate of Daphnia sp. Temperature and pH of the environment of Daphnia sp., time to record the heartbeat of Daphnia sp., the type of Daphnia sp. used APPARATUS Pippeters, cavity slides, scientific calculator, spatula, weighing dish, electronic mass balance, 250 ml beakers, 100 ml measuring cylinders, glass rod, stopwatch and light microscope. MATERIALS A culture of Daphnia sp., filter paper or tissue paper, distilled water or pond water, caffeine powder (coffee powder)

PROCEDURE 1. Five 250 ml beakers containing 100 ml distilled water is prepared by measuring 100 ml distilled water using a 100 ml measuring cylinder and placing it in the beaker. 2. Using an electronic balance, caffeine powder of mass 0.1g, 0.2g, 0.3g, 0.4g and 0.5g is measured. Firstly, an empty weighing dish is placed on the electronic balance and the mass of the weighing dish is noted. Then, with the weighing dish still on the electronic balance, the measured mass is set to 0g. After that, by using a spatula, a small amount of caffeine powder is placed inside the weighing dish and the desired mass of caffeine powder is obtained. 3. To prepare 0.1% caffeine solution, 0.1g of caffeine powder is dissolved in 100ml distilled water. A glass rod is used to ensure that all the caffeine powder has dissolved. This step is repeated by dissolving caffeine powder of mass 0.2g, 0.3g, 0.4g and 0.5g into 100ml distilled water respectively to obtain caffeine solutions of 0.2%, 0.3%, 0.4% and 0.5%. 4. A light microscope is set up. A low power objective lens of 40 magnification is used. 5. One single Daphnia sp. is selected from the Daphnia sp. culture. By using a pippeter, the selected Daphnia sp. is carefully sucked out of the culture and then transferred to a cavity slide along with an appropriate amount of pond water. 6. By using a filter paper, the excess water on the cavity slide is absorbed so that the Daphnia sp. lies on its side and has limited movement so that it can be easily viewed under the microscope. However a small amount of water is left so that the Daphnia sp. can survive. 7. The cavity slide is then place on the microscope stage and held in position using stage clips. 8. The microscope is adjusted by first adjusting the coarse focusing knob and the fine focusing knob until a fine image of the Daphnia sp. is observed. The position of the cavity slide is adjusted until the heart of the Daphnia sp. can be seen clearly. 9. .One student from the group is assigned to observe the Daphnia sp. using a microscope and count the number of heart beat or leg beat made by the Daphnia sp. within 15 seconds. A scientific calculator is programmed so that every successive tapping of the = button records one heart beat or beating of legs. Another student is

assigned to record the time by using a stopwatch. This step is repeated again to obtain a second reading. The average of value of the readings is worked out. 10. The Daphnia sp. is then returned to a beaker containing the same pond water as the Daphnia sp. culture. 11. Then another Daphnia sp. from the Daphnia sp. culture is obtained using the same methods in step 5 and 6. 12. A few drops of caffeine solution of concentration 0.1% is added to the Daphnia sp.. The Daphnia sp. is left in the caffeine solution for 1 minute. After that, its heartbeat is counted by the same student and using same methods as in step 9. 13. Steps 10 to 12 are repeated by using caffeine solutions 0.2%, 0.3%, 0.4% and 0.5% respectively. 12. Each average value of heart beat of Daphnia sp. is multiplied by four to obtain the value of heart beat of Daphnia sp. in unit of beats per minute. 13. The results of the experiment are tabulated in Table 1. 14. A bar graph showing the comparisons of the heartbeat of Daphnia sp. with treatments (addition of caffeine solution) and without treatments is plotted 15. A line graph of the heartbeat of Daphnia sp. per minute against the concentration of caffeine solution is drawn

RESULT Concentration of caffeine solution (%) 0.0 (control) 0.1 0.2 0.3 0.4 0.5 The heart rate of Daphnia sp. (beats per minute) Reading 1 328 376 392 396 376 372 Reading 2 336 384 400 412 384 368 Average 332 380 396 404 380 370

Table 1 : Shows the heart rate of Daphnia when treated with caffeine solutions of different concentrations

Bar Graph of Heart Rate of Daphnia sp. (beats per minute) against The Concentration of Caffeine Solution (%)
Heart rate of Daphnia sp. (beats per minute) 450

400

350

300

250

200

150

100

50

0 0.1 0.2 0.3 0.4 0.5 Concentration of caffeine solution (%)

Graph 1 shows a bar chart which compares the heart rate of Daphnia sp. when treated with caffeine solutions and when the Daphnia sp. are not treated

Graph of heart rate of Daphnia sp. (beats per minute) against the concentration of caffeine solution (%)
Heart Rate Of Daphnia sp. (beats per minute) 450

400

350

300

250

200

150

100

50

0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of Caffeine solution (%)

Graph 2 shows a line graph that shows the heart rates of Daphnia sp. at different concentrations of caffeine solutions

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Increment of Heart Rate of Daphnia against the Concentration Of Caffeine Solution (%)

Increment of Heart Rate of Daphnia 80

70

60

50

40

30

20

10

0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration Of Caffeine Solution (%)

Graph 3 shows the increment of the heart rate of Daphnia sp. when treated with caffeine solutions of different concentrations

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DISCUSSION The aim of this experiment is to investigate the effects of concentration of caffeine on the heart rate of living organisms. In this case, to investigate the effect of concentration of caffeine on the heart rate of Daphnia sp. or more commonly known as water fleas. The hypothesis is that the greater the concentration of caffeine solution the higher the heart rate of Daphnia sp.. While conducting this experiment, we can also equip ourselves with laboratory skills and investigative skills. The independent variable of this experiment is the concentration of caffeine solution. This variable is controlled by measuring different amounts of caffeine powder (coffee powder) that is 0.1g, 0.2g, 0.3g, 0.4g and 0.5g using an electronic balance and dissolving the caffeine powder in exactly 100ml of distilled water. By using this method, we obtain caffeine solutions of different known concentrations that is 0.1%, 0.2%, 0.3%, 0.4% and 0.5%. Distilled water is used instead of tap water because distilled water is pure and does not contain any other organisms that may disturb our view of the Daphnia sp.. The concentration of caffeine solution is set from 0.1% to 0.5% only because in this concentration the Daphnia sp. can still stay alive and carry out its metabolic functions as usual thus making the experiment possible. If caffeine solution of concentration higher than 1.0% is used, the Daphnia sp. will automatically die when exposed to this solution, making it impossible to carry out the experiment. The dependent variable in this experiment is the heart rate of Daphnia sp.. The technique used to measure this is by observing the heart beats of the Daphnia sp. which can be seen clearly when viewed under a microscope. The heart rate of Daphnia sp. when in its natural habitat that is in the pond water without any treatments is set as a control so that comparisons between the heart rate in different caffeine concentration can be made. The heart beats of the Daphnia sp. can sometimes be hard to observe. Another method that can be used to count the heart beat is by observing the leg beat of Daphnia sp. which is proportional to its heart beat. To make observations and counting of the heart beat easier, a scientific calculator is used. Another method involves tapping a pencil on a piece of paper and counting the dots made afterwards. The best way to count the heartbeat is actually by capturing a video of the Daphnia sp. in the 15 seconds time frame and replays it in slow motion to count the heart beats. However because of limited resources, the calculator method is chosen.

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The constant variables in this experiment include temperature and pH of the environment of Daphnia sp., time to record the heartbeat of Daphnia sp. and the type of Daphnia sp. used. The temperature and pH of the environment of Daphnia sp. is controlled by carrying out the experiment in an air conditioned laboratory where the temperature is set to 25oplacing the Daphnia sp. in a culture containing pond water from its natural habitat. Other than that, distilled water is used to make the caffeine solutions to ensure a stable pH where the Daphnia sp. can survive in. the time to record the heart beats of the Daphnia sp. is set to 15 seconds by using a stopwatch. The counted heart beats in 15 seconds is then multiplied by 4 to obtain the number of heart beats per minute. The same type of Daphnia sp. from the same species and breed is used for every repetitions of the experiment. As we can from Table 1, the heart rate of Daphnia sp. increases as the concentration of caffeine solution increases. When the concentration of caffeine solution is 0.1% the heart rate of Daphnia sp. is 380 beats per minute followed by 396 beats per minute in caffeine solution of 0.2% and 404 beats per minute in caffeine solution of 0.3%. However as the concentration of caffeine solution increases to 0.4 %, the heart rate of Daphnia decreases to 380 bpm. The same trend is also seen in the heart rate of Daphnia sp. treated with caffeine concentration of 0.5% that is at 370 bpm. Graph 1 shows a bar graph which compares the heart rate of Daphnia sp. when treated with caffeine solutions of different concentrations and with the heart rate of Daphnis.so that is not treated with caffeine. As we can observe, there is a significant increase in the heart rates of the Daphnia sp. when treated with caffeine for every different concentration. Table 2 shows a line graph which records the heart rates of Daphnia sp. at different concentration of caffeine solutions. It can be observed that the trend on the line graph is proportional to the results that is obtained in the Table 1. Graph 3 shows a line graph which represents the increment of heart rate of Daphnia sp. at every concentration of caffeine solutions. It can be seen that from 0.1% until 0.3% the increment of heart rate increases steadily. As the concentration of caffeine increases, heart rate of Daphnia increases for caffeine concentration up to 0.3% because caffeine is a stimulant which raises the heart rate of animals. Assumption that a similar effect would result in human can be made. However, there is a drop in the heart rate for 0.4% and 0.5% of caffeine solution. This is an anomaly shown in the experiment. Reference to other research papers and the
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results from other students doing the same experiment as well as consultation to experienced lecturers reveals that the heart rate of the Daphnia should increase from 0.0% up to 0.5% of caffeine solution. This is because 0.0 0.5% of caffeine solution is still within the concentration range which a typical Daphnia can tolerate. The probable explanation to this occurrence is that the Daphnia selected for this experiment is a weak one and thus has a narrower concentration range which it can tolerate, most probably from 0.0-0.3%. Another explanation to this anomaly is that throughout the experiment, the same Daphnia is used instead of using fresh Daphnia for each different caffeine solution of different concentration. Due to the fact that caffeine is primarily an antagonist of the central nervous system's receptors for the neurotransmitter adenosine, the body of Daphnia which regularly consume caffeine adapt to the continual presence of the drug by substantially increasing the number of adenosine receptors in the central nervous system. This increase in the number of the adenosine receptors makes the body much more sensitive to adenosine. Consequently, the stimulatory effects of caffeine are substantially reduced, a phenomenon known as a tolerance adaptation, thus the heart rate reduces even though the concentration of caffeine solution increases.

FURTHER STUDIES Besides concentration of caffeine solution, factors like surrounding temperature, amount of alcohol and amount of some drugs such as aspirin can affect the heart rate of Daphnia. Daphnia are very sensitive to poor water conditions too. Further studies can be done by testing how the different pollutants in the water affect the heart rate of Daphnia. These tests can be done by using the same methods as we used in this experiment. The difference that can be made is that instead of using caffeine as the variable, alcohol solutions are used instead. By using ethanol or other organic alcohol, the heart rate of Daphnia sp. can be observed just the same as in this experiment. The higher the concentration of alcohol, the higher the heart rate of Daphnia sp.

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SAFETY PRECAUTION Throughout the experiment, some safety precautions are exercised to ensure the accuracy and reliability of the results. 1. Lab coats and closed toe shoes must be worn at all times inside the laboratory when carrying out the experiment to protect ourselves from any injuries caused by broken apparatus. 2. Eye protections must be worn to protect ourselves from any toxic chemicals or any microorganisms that is found inside the pond water of the Daphnia sp. culture. 3. The experiment must be done in an air conditioned room which temperature is set to below 30oC. This is because Daphnia sp. are cold climate creatures and may die from high temperatures. 4. When carrying out the experiment, students must treat the Daphnia sp. very carefully as the number of Daphnia sp. available in the laboratory is very limited. Therefore students need to be extra careful when handling the Daphnia sp. to avoid them from dying. 5. When observing the Daphnia sp. under microscope, ensure that the light used is not so bright. This is to prevent the Daphnia sp. from dying from overheating due to the exposure to bright light. 6. When placing the Daphnia sp. on the cavity slide, make sure that there is only a small amount of water on it. This is to prevent the Daphnia sp. from moving around on the cavity slide which makes it hard to observe under the microscope. 7. In contrast, ensure that there is enough water on the cavity slide to ensure that the Daphnia sp. can survive. Too little water causes the Daphnia sp. to suffocate and die. 8. Filter paper should be used in this experiment instead of muslin cloth as suggested by the manual. This is because the muslin cloth is too absorbent and may remove the Daphnia sp. from the glass side. In addition, muslin cloth has fibres that may stain the cavity slide and make observations of Daphnia sp. harder. 9. The experiment for each concentration must be repeated at least twice to obtain the average count of heart beat. This is to increase the validity and reliability of the experiments

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10. A new Daphnia sp. should be used for every different concentration of caffeine solution. This is to avoid the Daphnia sp. from developing tolerance towards caffeine which can affect the results greatly.

LIMITATIONS There is unavoidable existence of limitations that could affect the outcomes of the experiments. They are 1. When the Daphnia sp. is placed on the microscope stage and exposed to the microscope light, it can cause the Daphnia sp. to be overheated. This can increase the heart rate of Daphnia sp. which affects the validity of the results. This can also cause the Daphnia sp. to die from overheating. 2. The heartbeat of Daphnia sp. when immersed in caffeine solution of 0.3% to 0.5% is very fast. Missing a few heartbeats when counting is inevitable. Therefore the results obtained may or may not be accurate to the actual number of heartbeats of the Daphnia sp.. 3. The use of scientific calculators may also affect the number of heartbeats counted. Because the tapping of the calculator is very fast because of the fast Daphnia sp. heartbeat, the number of tapping and the number that is shown on the calculator screen may not be the same thus affects the results of the experiment. 4. The same Daphnia sp. is used by two or more groups therefore it may be injured or hurt while being transferred from one container to the other. Therefore the heartbeat rate may change and affects the results. 5. Long exposure of the Daphnia sp. to caffeine solution causes the Daphnia sp. to develop tolerance against the caffeine solution where the heart rate does not increase even when the concentration of caffeine solution is increased. Therefore the results is not accurate or obey the theory/hypothesis anymore. 6. Since the Daphnia sp. is not observed under its natural habitat, its stress level may increase which causes a fluctuation in its heart rate. Therefore the counting of the heartbeat must be done as soon as the Daphnia sp. is ready to be observed.

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CONCLUSION Generally, when the concentration of caffeine solution increases, the heart rate of Daphnia increases also increases. The hypothesis is therefore accepted. However prolong exposure of Daphnia to the caffeine solution can result in the building up of tolerance of Daphnia towards caffeine. Thus the results do not obey the hypothesis.

REFERENCE 1. Edexcel AS Biology, Students book, Pearson company, 2008 2. Edexcel Biology for AS, CJ Clegg, Hodder Education, 2008 3. Text PRE U Biology Volume 1, Lee Ching, PEARSON Longman, 2008 4. International Edition Biology Seventh Edition, Campbell, Reece, Pearson Benjamin Cummings, 2005 5. Advanced Biology Principles & Applications Study Guide, CJ Clegg with DG Mackean, PH Openshaw and RC Reynolds, John Murray (Publishers) Ltd, 1996 6. Carpenter, SR, & Kitchell, JF (1993) The Trophic Cascade In Lakes. Cambridge University Press, London, England. 7. Davidson College Department of Biology (1999)

http://www.bio.davidson.edu/index.html. 8. Rith, J (1988) Plant succession on abandoned railways in rural New York State. Proceedings of the 73rd Annual Meeting of the Ecological Society of America, Davis, CA. 9. www.sciencebuddies.org/mentoring/project_ideas/Pharm_p009.shtml 10. http://en.wikipedia.org/wiki/Daphnia sp. 11. http://en.wikipedia.org/wiki/Caffeine 12. http://www.caudata.org/Daphnia sp./

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