BIOLOGY LAB REPORT

TITLE PREPARED BY I/C NUMBER STUDENT ID GROUP LECTURERS NAME PRACTICAL DATE SUBMISSION DATE

: THE EFFECT OF CAFFEINE ON HEART RATE : : : : : : :

OBJECTIVE To investigate the effect of caffeine on the heart rate of Daphnia (water fleas). To highlight experimental and investigative skills.

Introduction DAPHNIA(1)

Figure 1 : Daphnia(2) Daphnia is a large genus comprising about 150 species, belonging to the cladoceran family Daphniidae . Daphnia are small, planktonic crustaceans, between 0.2 and 5 mm in length. They are commonly called water fleas because of their saltatory swimming .They live in various aquatic environments ranging from acidic swamps to freshwater lakes, ponds, streams and rivers. The most important features of Daphnia are the compound eyes, the second antennae, and a pair of abdominal setae. In many species, the carapace is translucent or nearly so and as a result they make excellent subjects for the microscope as one can observe the beating heart. Even under relatively low power microscope, the eye being moved by the ciliary muscles can be seen, as well as its heart beat. The heart is at the top of the back, behind the head, and the average heart rate is approximately 180 bpm under normal conditions. Daphnia, like many animals, are prone to alcohol intoxication, and make excellent subjects for studying the effects of the depressant on the nervous system and 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.

Figure 2 : Reproduction of Daphnia(2) Daphnia reproduce parthenogenetically usually in the spring until the end of the summer. In addition to the production of eggs capable of overwintering, this switch to sexual reproduction has also been proposed to allow greater offspring variation that is useful in varied or unpredictable conditions. The lifespan of a Daphnia does not exceed one year and is largely temperature dependent. For example, individual organisms can live up to 108 days at 3 C (37 F) while some organisms live for only 29 days at 28 C (82 F). A clear exception to this trend is during the winter time in which harsh conditions limit the population in which females have been recorded to live for over six months. These females generally grow at a slower rate but in the end are larger than ones under normal conditions. Sometimes Daphnia may be used in certain environments to test the effects of toxins on an ecosystem. This makes Daphnia an indicator species, particularly useful in that area because of its short lifespan and reproductive capabilities. As they are nearly transparent, their internal organs are easy to study in live specimens . They are often fed to tadpoles or small species of amphibians such as the African Dwarf Frog. Daphnia are also a popular live food in tropical and marine fish keeping.

CAFFEINE(3)

Figure 3 : Caffeine formula(2) Caffeine is a bitter, white crystalline xanthine alkaloid that acts as a stimulant drug. Caffeine is found in seeds, leaves, and fruit of some plants.Caffeine acts as a natural pesticide that paralyzes and kills certain insects feeding on the plants. It is most commonly consumed by humans in infusions extracted from the bean of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks containing products derived from the kolanut.

Figure 4 : Side effects of Caffeine in human(2) Figure 5 : Effect of moderate caffeine consumption(2)

Caffeine can have both positive and negative effects on anxiety disorders depending on the dose. At high doses ( greater than 300 mg) , it can both cause and make anxiety worse. At low doses it may reduce symptoms of anxiety. Caffeine withdrawal, on the other hand, can cause an increase in anxiety level. In moderate doses caffeine typically does not affect learning or memory. It does however improve cognitive function in people who are fatigued, due to its effect on alertness. Some studies have however found a modest protective against Alzheimer disease, but the evidence is inconclusive. Caffeine is also used as a flavour enhancer in cola and other soft drinks also it has medicinal uses in aspirin preparations and is found in weight-loss drug and as a stimulant in normal people in everyday to keep them alert in their work.

Problem Statement : How does the concentration of caffeine affect the heart rate of Daphnia? Hypothesis : The higher the caffeine concentration, the higher the number of heart beat of Daphnia. Variables :

Types of Variables Manipulated Variable: Concentration of caffeine

Ways to control the variables

Use different amount of caffeine powder, measured with electronic balance and mix with fixed amount of water needed.

Responding Variables: Number of heart beat of Daphnia/ 15 seconds Number of heart beat of Daphnia was calculated by making marks on paper while counting the heart beat and later calculate and record.

Fixed Variables: Temperature of surrounding of Daphnia Hold experiment at room temperature

throughout experiment Daphnia used Use same Daphnia throughout the experiment.

Apparatus Stop watch, dropper, cavity slide, microscope, measuring cylinder, electronic balance, 100ml beaker

Materials Daphnia, distilled water, coffee powder, cotton wool, tissue paper

Technique used The Daphnia was observed under light microscope. The heart beats off Daphnia was counted directly by observing the heart of the transparent Daphnia by one person. The experiment was done by each group with different concentration to prevent contamination of different concentration of caffeine on Daphnia, thus result in alteration of result, causing less validity of result.

Procedure : 1. A cotton wool was placed on cavity of microscope slide to restrict the movement of Daphnia. 2. A daphnia was transferred from a beaker containing pond water to the cavity of microscope slide using a dropper. 3. Excess liquid was removed using tissue paper. Two drops of distilled water was placed on Daphnia specimen using dropper. 4. Another slide was placed under the specimen slide to act as heat sink, works by preventing loss of water. 5. The slide was then placed on microscope stage. 6. Daphnia was examined in low power objective lens and the location of eye, heart and other part was determined. The heart of Daphnia was observed . 7. A stop clock was used to record the number of heartbeats for every 15 seconds for 10 minutes. Heartbeats were counted under normal condition (distilled water) at intervals of 2 minutes over a 10 minute period. Daphnias heart rate was counted within 15 seconds for every 2 minutes. A pencil was tapped by observant on a piece of paper and the pencil marks was counted at the end of the time period. 8. It is a good idea to do a blind study to avoid bias in the results. 9. Different caffeine concentration was prepared using coffee powder. 0.1g of coffee powder was diluted in 100ml for preparation of 1% of caffeine concentration. 10. Step 8 was repeated using of 0.4g, 0.6g and 0.8g coffee powder and diluted with 100ml for preparation of 0.4% , 0.6% and 0.8%. 11. Steps 1 until 7 was repeated using same Daphnia under different concentration of caffeine as stated in table below. 12. At the end of the experiment, the Daphnia was returned to the stock culture. 13. The results were tabulated. 14. The increase in heart rate of Daphnia was obtained by calculating the difference in the heart rate of Daphnia before and after put in targeted solution. 15. A graph of time against heart rate of Daphnia was plotted. 16. A bar graph plotted for caffeine concentration against number of heart beat.

RESULTS DATA COLLECTION Number of heart beat/ 15 seconds Length of time in solution / minutes (min) 2 4 6 8 10 Mean

Treatment

Caffeine (0.4%) Caffeine (0.6%) Caffeine (0.8%) Caffeine (1.0%) Mean Control Control Control Control Mean

53

54

56

56

58

55.4

59

60

61

63

63

61.2

62

65

65

69

68

65.8

73

73

74

76

75

74.2

61.75 56 67 63 66 63

63 55 67 65 61 62

64 55 66 65 62 62

66 54 64 62 64 61

66 54 65 64 65 62 54.8 65.8 63.8 63.6

Table 1 : Treatment used for 10 minutes against number of heart beat

Time against Number of Hearbeat/15 seconds

67 66

Number of Heartbeat / 15 seonds

65
64

63
62

Control

61
60 59 58 2 4 6 Time (min) 8 10 With Caffeine Concentration

Graph 1 : Time(min) against number of heartbeat / 15 seconds

Graph 2 : Caffeine concentration (%) against number of heart beat/15 seconds

DISCUSSION Analysis of data An experiment was held to determine the effect of caffeine concentration on number of heart beat of Daphnia. A Daphnia was obtained from pond water using dropper and placed inside cavity of slide. Cotton wool is used to restrict the movement of Daphnia. The heartbeat of Daphnia in distilled water was observed and recorded for ten minutes and for every 2 minutes, it was recorded for 15 seconds. This step was carried out with different concentration of caffeine solution that is diluted according to the masses in procedure above. The heart beat was recorded and at last, both results were compared. Used Daphnia was returned to the pond water, reducing ethical issues. Since usage of higher concentration of caffeine will kill the Daphnia, thus solution used is diluted first and the highest concentration used is 1%. In graph 2, there is steady increase of heart beat number with caffeine concentration from 0.4% to 0.8%. However, there is tremendous increase from 0.8% to 1.0% caffeine concentration. This may due to the Daphnia being immersed too long in caffeine solution before the heart beat is calculated. Another possible cause is may be the caffeine concentration higher than stated value as the solution is being added without wiped first for preventing the Daphnia from die. At the beginning of the experiment, the heartbeats of Daphnia is obtained by calculating the heartbeats of Daphnia in 15 seconds timed by a stopwatch, while observing under light microscope. Without the presence of caffeine (in distilled water), the initial heartbeat of Daphnia obtained by making marks on paper and counting it. Since every group is doing control study, thus all readings were collected and the mean is obtained. Restricting the Daphnia using cotton wool from swimming around increase the validity of data as the observer can calculate the heart beat with full concentration. It also can be observed that the number of heart beat of Daphnia in distilled water decreasing, remaining the same and then decrease again and at last increase. It shows that the heart beat of Daphnia in distilled water is also altered and since the Daphnia used are not same, thus the data is taken for mean.

The heart beat number before the Daphnia is soaked in concentrated but still diluted caffeine solution has an obvious effect. This can be seen in Graph 1. The number of heart beat of Daphnia with caffeine water is higher that without caffeine (control which is distilled water). This perfectly shows that caffeine will increase number of heart beat of Daphnia (likewise in human). It is also can be observed as time increases the number of heart beat also increase. But at 8 th minute till 10th minute it can be seen that the number of heart beat stays same. This may be due to maximum tolerance of Daphnia towards the caffeine concentration. The Daphnia also may suffer from stress due to the culture solution. It is also possible that not same species used by all the group. Thus it can be concluded that different Daphnia species have different tolerance toward caffeine concentration, results in effecting the data. Thus as a conclusion, it can be said that increase in caffeine concentration will increase number of heart beat off Daphnia over time. There is clear difference between number of heart beat when the Daphnia was cultured in caffeine solution and distilled water. This further stresses that the presence of caffeine will affect the heart beat of Daphnia. This can be said that the caffeine is working as a stimulant thus increasing the number of heart beat.

Evaluation Limitation and improvement Several limitation are found in this experiment. Sources of error can be avoided totally at times or reduced to minimum most of the time. But, in this experiment , the limitations are bit harder to overcome. The daphnia used in experiment may suffer from a genetic defect or have an abnormally high or low tolerance to caffeine, leading to inaccurate results. Thus, using one daphnia for control and the same daphnia for specific concentration of caffeine and finding the mean for both constant values and different caffeine concentration minimizing inaccuracy of data. Another limitation is there may be some error in calculating the heart beats of Daphnia. Since the experiment were done by a group of student consisting three, it is possible for human error to happen. Stopwatch may be start late or earlier than stated. The observer also may start counting the heart beat before the stop clock switch on and stop later after the stop clock is stopped. Counting of the pencil marks also can be limited since in the rush of plotting the marks, the points may overlap causing the reader to not to able to count the overlapping point, resulting in inaccuracy of result. Taking mean reading (for 5 readings) may increase the data improvement. Furthermore, using heat sink to keep the Daphnia in a cool environment is not accurate because as the water eventually evaporate over time because of the microscopes light, the Daphnias environment is warmer resulting in a higher initial heart rate as usual. Heart rate of Daphnia may increased not only due to deprivation of water but also putting it into new environment (caffeine) might increase the stress level of the Daphnia. This is overcome by putting drops of water in between some time, thus decreasing the effect of water deprivation, improving the data.

Validity and reability For ensure the validity of the results, only one Daphnia was used per group and other groups used same size of Daphnia as possible as can. Before the experiment was carried out with caffeine solution, the Daphnias heart beat was determined with distilled water as constant and the average reading was obtained. Heart beat of Daphnia when cultured with caffeine solution was obtained and average reading was recorded. Since only one Daphnia is used , thus it increase the validity and reability of the data. Furthermore, the caffeine used may be an overdose for the Daphnia despite using measured coffee powder, causing adverse effect on it rather than the desired effect. Using different amount of caffeine on only one specific Daphnia and finding mean increase the datas validity and reability. Extra care was taken when activity of reading data was taken place. Since overlapping of points marked by observer may affect the validity of data, thus when the observer start to plot the marks on the paper, another person or the reader of data start counting the sound of the pencil hitting the paper. This will, for sure, give arise to two readings afterwards and most likely to happen result is chosen .This ensures the heart beat of Daphnia in both distilled water and caffeine solution to be continuous and logical ,guarantee the validity and reability of data.

Using pond water / Daphnia culture solution, in this case , using pond water for the Daphnia to be cultured before the experiment starts also increases the reability of data. Since the pond water is the suitable surrounding for Daphnia, thus its heart beat will be in normal rate and culturing the Daphnia in either distilled water and caffeine solution will show the perfect differences leading to increasing in validity and reability of data.

Risk Assessment Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any chemical substance. Furthermore, the glassware such as beakers and boiling tubes should be handled with full care because they are fragile. The microscopes used are very fragile and the light bulbs can get hot, precautionary steps should taken. For the Daphnia, make sure to be careful, not to feed too much caffeine to the Daphnia only a maximum of 1% as we may not be able to get reading due to it dying. The concentration of the solution is controlled so that the Daphnia wont be affected. Ethical issue To determine the effects of a human life, we have to take a substitute of a human being and then infer that any result that we obtain from this experiment will be the same as what will happen in a human. Obviously we have to take into count that the amount of caffeine consumption of the Daphnia and that of human will be different as the scales as to how big we are different. We hve to be careful not to feed too much caffeine to the Daphnia only a maximum of 1% as we may not be able to get a reading due to it dying.Daphnia are a great model for this experiment because they can be easily handled and manipulated. Daphnia are also visible under the microscope and it is easier to see their heart beat. Daphnia are advantageous when it comes to experiments because they are easy to keep and maintain, they are also easy to study plus they are inexpensive. But, h umans cant be compared to daphnia due to their difference in the central nervous system. It should also be assumed that daphnia are small and the caffeine concentration that increased the daphnia heart rate to max will not increase the heart rate of the human. Another important fact is that, daphnia have a neurogenic heart meaning it doesnt contract but instead conducts impulses versus hum an hearts; they are myogenic meaning they contract. Although the Daphnia are simple organisms that may not suffer in the same way as higher animal, they still deserve respect. These Daphnia should be returned promptly to the holding tank after being examined.

Further Investigation A similar experiment can be carried out to investigate the relationship between number of Daphnias heart beat and alcohol concentration. This investigation is run to see the effect of varying amount of alcohol concentration on Daphnias heart beat. Since the alcohol is known as depressant , two valid hypothesis can be made. First of all, in the presence of alcohol, the number of heart beat of Daphnia may decrease. And for the second hypothesis, as the amount of alcohol increases, the number of heart beat will decrease. The procedure for this experiment is the same with above and all we need to do is substituting the caffeine concentration with alcohol amount or concentration of 10%. 20%, 30%, 40% and 50%. Control should be taken with distilled water or better in pond water solution to get the real number of heart beat. There should be a decrease in number of heart beta for 15 seconds for 10 minutes as alcohol introduced because of the depressant property of alcohol, slowing ddown the number of heart beat.

Conclusion In conclusion, as the caffeine increased, the daphnias heart rates also increased and the hypothesis is accepted. Through the labs results it is believed that there is a relationship between the daphnias and humans heart rate therefore if an increase in the daphnias heart rate is due to high caffeine concentration, then humans also have a high heart rate after consumption of caffeine. This is dangerous as mentioned before because this makes human prone to numerous diseases in the body that correlate with caffeine. First of all, increase in heart rate means that blood is being pumped in the human body at a higher rate which increases the chance of damaged or erupted veins and arteries which further will increases the humans chance of getting atherosclerosis.

Reference 1. http://en.wikipedia.org/wiki/Daphnia 2. http://www.google.com.my/search?q=daphnia+culture&hl=en&safe=off&biw=1366& bih=681&prmd=imvnsfd&source=lnms&tbm=isch&ei=j8ijTqWxKoXorQeWkrH9Dw&sa =X&oi=mode_link&ct=mode&cd=2&sqi=2&ved=0CA8Q_AUoAQ#hl=en&safe=off&tbm =isch&sa=X&ei=msijTqjLFcjlrAf0zNzhAQ&ved=0CDoQvwUoAQ&q=daphnia+and+caffei ne&spell=1&bav=on.2,or.r_gc.r_pw.,cf.osb&fp=efa7cf78339474ac&biw=1366&bih=68 1 3. http://en.wikipedia.org/wiki/Caffeine