The water flea Daphnia pulex (see figure 1) is a small (1.1-3.5mm long ) crustacean commonly found in ponds in the United Kingdom. The full taxonomy of Daphnia pulux is as follows:
Kingdom – Branchiopoda
Phylum – Diplostraca
Class – Cladocera
Order – Anomopoda
Family – Daphniidae
Genus – Daphnia
Subgenus – pulex
Figure 1. Daphnia pulex, showing the clear exoskeleton and inside the body (Cladoceran website 2005)
Whilst being crustaceans and having an exoskeleton Daphnia are easy to study as their exoskeleton is actually clear so it is possible to see through it and study the working of the inside of their body. In particular it is possible to see the flow of the circulatory system and the heart beating. Therefore they are useful to test the effects of specific drugs on heartbeat, as it is easy to see the changes via an obvious change in heartbeat.
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Daphnia have been used in the present experiment as they are readily available and it is simple to study the effects of drugs on their systems. They are also robust to a variety of chemicals being present in their environment so can be used to assess the effects of these chemicals without fear that the chemicals will kill them instantly.
Aspirin (acetylsalicylic acid), alcohol (ethanol), and caffeine are drugs that all commonly used by people. All 3 drugs have an effect on the human heart, mostly through indirect effects on the circulation and factors that affect circulation – eg platelet clotting. Alcohol causes a lowering of heart beat due to expanding the veins through vasodilatation. Aspirin also lowers the heart rate via its effects on platelets and the way that they cause veins to constrict. Caffeine, however, acts to increase heart beat.
It is unfortunately not practical to test the effect of any of the drugs on the heartbeat of people as the drugs have a lot of other effects within the body as well. Instead Daphnia are used as they have a small body so the drug effects are less widespread and the effect on the heart is easy to assess. The effects are also evident very quickly so it is possible to carry out a large number of investigations in a relatively short space of time.
Hypothesis
The experimental hypotheses for each of the three drugs are as follows:
Aspirin will cause the heart rate of Daphnia to lower in a concentration dependent manner.
Alcohol will cause the heart rate of Daphnia to lower in a concentration dependent manner.
Caffeine will cause heart rate of Daphnia to increase in a concentration dependent manner.
The null hypothesis in all cases is that each of the drugs will have no effect on the concentration rate of Daphnia.
Variables
In order to see whether it is actually the drugs that are causing the change in the heart rate of Daphnia the only variable that can change is the concentration of the drug. As well as this each drug will have to be tested separately to make sure that it is only the effect of that drug which is causing the change, if any. A fourth set of results also needs to be taken, which is the heart rate of daphnia without any of the drugs added at all.
For this experiment the independent variable will be the drug concentration. An independent variable is one that is chosen and set specifically by the experimentor.
For this experiment the dependent variable – the one that changes as a result of the independent variable changing – will be the heart rate of the Daphnia.
Constant variables will be:
The number of Daphnia (approx 100)
The volume of water (100ml)
The temperature (same room temperature of approx 21 °C in all cases)
The species of Daphnia (Daphnia pulux)
The amount of water transferred to microscope slide for each test (1ml)
The same length of time the Daphnia would be left in the chemical (1 hour)
Method
Daphnia will be collected from a pond, using a fine mesh sive and kept in a contained with pond water until they can be counted and the correct number can be transferred to a petri dish containing 100ml of distilled water for each experiment. Distilled water will be used so that there are no other chemicals present which might have an effect on heart rate.
Approximately 100 Daphnia will be used for each experiment, although it would not be possible to count exactly that number so an approximate number will be used in each case.
Aspirin will come in a readily available over the counter soluble form (Boots 300mg soluble aspirin)
Caffeine will also come in a readily available over the counter form of ProPlus (50 mg Caffeine)
Alcohol will come from vodka (Smirnoff Blue label 45% vol).
The drug will be added to the distilled water in the petri dish before adding the daphnia and stirred with a clean glass rod to make sure that it is fully dissolved and spread throughout the liquid. Then the daphnia will be transferred to the petri dish using a pipette.
Drugs will be in the quantities shown in tables 1-3
Table 1. A table showing the number of caffeine tablets used in each experiment and the resulting caffeine concentration
Number of tablets
Amount of caffeine (mg)
Amount of water (l)
Caffeine concentration (mg / l)
0
0
0
0.1
1
50
0.1
5
2
100
0.1
10
3
150
0.1
15
4
200
0.1
20
Table 2. A table showing the number of aspirin tablets used in each experiment and the resulting aspirin concentration
Number of tablets
Amount of aspirin (mg)
Amount of water (l)
Caffeine concentration (mg / l)
0
0
0.1
0
1
300
0.1
30
2
600
0.1
60
3
900
0.1
90
4
1200
0.1
120
Table 3. A table showing the amount of vodka used in each experiment and the resulting alcohol concentration
Amount of vodka (ml)
Amount of alcohol (mg)
Amount of water (l)
Alcohol concentration (mg / l)
0
0
0.1
0
50
22.5
0.1
2.25
100
45
0.1
4.5
150
67.5
0.1
6.75
200
90
0.1
9
There would actually be only 1 control experiment, to check for differences caused by the drug, so the top row of each table actually corresponds to the same experiment, where there is just water in the petri dish and no added chemicals.
As the daphnia are small organisms with a relatively permeable exoskeleton they would be able to absorb the caffeine present in the water and it could have its effect upon the body.
In each case the Daphnia would be left in the water and drug for a time of 1 hour to ensure that the drug has had any effect. As the Daphnia are only going to be used for a short period of time they would receive no food as it would not be necessary.
Whilst whole Daphnia are visible to the naked eye, a microscope is necessary to see the actually heart beats. Therefore a clean pipette will be used to transfer a droplet of the treated water and daphnia onto a microscope slide. This would then be covered using a cover slip to prevent the liquid from moving. This will then be looked at under the microscope and the number of times the Daphnia’s heart beats per minute recorded. Each time that the heart beat is counted it will be repeated 3 times to obtain an average.
Different microscope slides would be used in each case to avoid cross contamination by different drugs or drug concentrations.
Equipment list
Fine mesh sieve
Plastic container to store daphnia in
Distilled water (13 x 100 ml)
13 clean plastic pipettes
13 clean petri dishes
13 microscope slides and cover slips
Light microscope
Caffeine tablets (ProPlus)
Aspirin tablets (Boots soluble aspirin)
Alcohol (Smirnoff blue label vodka)
Measuring cylinder
Risk assessment
There are a number of specific areas which involve risk within this experiment. These are the initial collection of daphnia, the handling of daphnia and the handling of chemicals. Specific risk assessment are as follows:
If alcohol / drug is ingested then rinse out mouth immediately and seek medical advice.
If alcohol / drug solution is spilt on clothes rinse affected area and remove clothes if necessary.
If alcohol / drug solution gets into eyes then rinse out immediately and seek medical advice.
An adult (over 18) must purchase the vodka and be present during its use.
Care should be taken when obtaining the daphnia from a pond and an adult should ideally do the collecting.
If pond water is ingested then rinse out mouth immediately and seek medical advice.
Results
It was found that, as Daphnia heart beat was very fast it was easier and more reliable to count in 15 second sections and then convert this to beats per minute. So this was done in all cases.
The heart rate of daphnia under control conditions was found to be an average of 350 beats / minute. The results for each of the 3 drugs are considered separately below.
Caffeine
The results from the experiment to investigate the effect of caffeine upon the heart rate of Daphnia are shown in figure 2 below.
Figure 2. The effect of caffeine on the heart rate of Daphnia Pulex (average of 3 counts)
It can be seen that there is a positive correlation between caffeine concentration and Daphnia heart rate. The percentage increase in heart rate, when compared to control, is shown in table 4 below.
Table 4. A table showing the effect of increasing caffeine concentration on daphnia heart rate
Caffeine concentration (mg/l)
Heart rate increase (beats/minute)
Percentage increase
50
13 bpm
3.5%
100
15 bpm
4.3%
150
17 bpm
4.9%
200
20 bpm
5.8%
Aspirin
Unfortunately the concentrations of aspirin used actually killed the daphnia whilst they were in the solution for the required hour. Therefore it was necessary to alter the concentrations and repeat the experiment. As the daphnia died at all concentrations of aspirin even 30 mg / litre must have been too concentrated for them. Therefore the concentration was reduced to 10% of original values, and the experiment repeated. The new concentrations were obtained by diluting the dissolved tablets in more distilled water, eg 10 times the amount to obtain concentrations of 1/10. unfortunately, due to time constraints, the counts could not be repeated 3 times so the figure are for a single count at each concentration.
The results from these new concentrations are shown in figure 3 below.
Figure 3. The effect of aspirin on the heart rate of Daphnia
It can be seen from figure 3 that there is no particular pattern to the results, other than the fact that aspirin is linked to a reduction in heart rate. However this reduction does not appear to be concentration dependent, or at least not at the concentrations used in this experiment.
Alcohol
The effect of increasing alcohol concentration is shown in figure 4.
Figure 4. The effect of alcohol on the heart rate of Daphnia (average of 3 counts)
It can be seen that alcohol had quite a large effect on the heart rate of daphnia and there was an inverse correlation between alcohol concentration and heart rate. The percentage decrease in heart rate, when compared to control, is shown in table 5 below.
Table 5. A table showing the effect of increasing alcohol concentration on daphnia heart rate
Alcohol concentration (mg/l)
Heart rate decrease (beats/minute)
Percentage decrease
2.25
104 bpm
29.8 %
4.5
120 bpm
34.3 %
6.75
134 bpm
38.3 %
9
148 bpm
42.3 %
Statistical analysis of results
It was not possible to undertake accurate statistical analysis of the aspirin results as only 1 count was taken at each concentration level on the repeated experiment. However it was possible to undertake a t test analysis on the alcohol and caffeine results, using the original data as opposed to the mean results shown in the graphs. Statistical analysis was carried out using the software available from Graphpad quickcalcs. These statistically analysed results are shown in table 6 and 7 below.
Table 6. A table to show the relative statistical significance of varying concentrations of caffeine upon the heart rate of daphnia
Concentration compared with control (mg/l)
2 tailed p value
Statistical significance
5 mg/l
0.2567
None
10 mg/l
0.0301
Yes
15 mg/l
0.0575
None
20 mg/l
0.0579
None
Table 7. A table to show the relative statistical significance of varying concentrations of alcohol upon the heart rate of daphnia
Concentration compared with control (mg/l)
2 tailed p value
Statistical significance
2.5 mg/l
0.0001
extremely
4.5 mg/l
0.0012
very
6.75 mg/l
extremely
9 mg/l
0.0019
very
Discussion
The results from this experiment indicate that both caffeine and alcohol affect heart rate of daphnia. Aspirin may affect heart rate but the results obtained here do not provide enough evidence to support this view fully.
Why does caffeine affect heart rate?
The results from this experiment show that caffeine causes heart rate to increase by around 0.05% per mg of caffeine. Whilst it cannot be known exactly why this happens there are various possible reasons.
Caffeine if a methylxanthine which acts by inhibiting the enzyme (phosphodiesterase) that metabolises cAMP. The resulting rise in cAMP levels has the same effect as increasing levels of adenylate cyclase, which is one of the principal effects of adrenaline. Adrenaline is known to increase heart rate via its effects on the ¢1 adrenoceptors so it is likely that caffeine has a similar effect. Whilst these effects are known about in humans, and daphnia are not the same organisms, it is likely that the effects are mediated for similar reasons – eg the involvement of enzymes and inhibition / stimulation of these.
It is possible that the effects of caffeine were not as pronounced as might have occurred due to a reflex reaction. In humans an increase in heart rate is often rapidly followed by a reflexive compensatory mechanism to slow the heart rate again. This compensation can involve the veins dilating and a stimulation of enzymes such as phosphodiesterase in order to reduce cAMP levels. In the case of the daphnia it might have been the case that a more significant rise in heart rate occurred as soon as the caffeine was absorbed by the daphnia but then there was a compensatory mechanism to reverse this effect. However as the solution of caffeine was present around the daphnia for the whole duration of the experiment then this is unlikely as each time new caffeine was absorbed it would cause heart rate to increase, and the daphnia’s system would not be able to constantly undergo the reflexive compensations.
How does aspirin affect heart rate
Aspirin has its principal effects on an enzyme called cyclo-oxygenase. Aspirin acts by binding to the active site of the enzyme, thus preventing it from binding to the eiocosanoid and having its required effect. The inhibition is irreversible as aspirin binds permanently to the active site of the enzyme. Cyclo-oxygenase (COX) is the main enzyme involved in the conversion of eiocosanoids found in membrane fatty acids, into the inflammatory mediators prostaglandins and leuoktrienes. COX acts to convert arachadonic acid into the prostaglandin precursor prostaglandin G2 and also to convert this precursor into prostaglandin H2. It is from PGH2 that the main inflammatory mediators are generated. Therefore the prevention of COX prevents even the first part of the inflammatory cycle from taking place.
Whilst it is not immediately obvious how the prevention of inflammatory mediators should reduce heart rate, it becomes obvious when the effect of the inflammation on heart rate is known. Whilst each individual prostanoid has slightly different effects, the overall one if to cause vasoconstriction and also an increase in myocardial contractility. This latter particularly results in heart rate being increased as the heart muscle in contracting more frequently.
This means that decreases heart rate by preventing this increased heart muscle contraction, as well as preventing the vasculature from constricting so much.
Again, it is the effects of aspirin in humans that is known about, but it could be expected that the effect is similar in daphnia.
Why might alcohol affect heart rate
It is not known exactly how ethanol acts to decrease heart rate, but it is believed to be a knock on effect from the changes in peripheral vasculature. Ethanol is known to cause vadodilatation – eg dilation of blood vessels. If blood vessels are dilated then there is more space thus a greater volume for blood so the heart has to beat fewer times to deliver the same amount of blood.
It is also possible that ethanol has its effects on heart rate via a central nervous system effect. Ethanol initially increase catecholamine release, including dopamine. This acts to stimulate heart rate. However following this initial effect ethanol actually reduces the concentration of catecholamines, principally noradrenaline, by increasing the rate at which it is removed from cells. As the daphnia had been left in the ethanol containing water for an hour it would be expected that they had reached this second stage by the time heart rate was being measured. However it is possible that the conflict between the initial and subsequent effects of alcohol could be the reason why the results did not follow a particular pattern.
Sources of error
Whilst statistical significance appears to be present in a number of results it is very difficult to be certain with such a small population number. In order to be more certain of results, a much larger sample size should be used in each case. When a result has been shown to be significant and is actually due to chance this is known as a type I statistical error.
Temperature is another variable that affects the heart rate of daphnia, evident by the fact that daphnia can be preserved alive at low temperatures due to a slowing of heart rate. Therefore it is possible that the heat from the light microscope lamp caused an increase in the heart rate. However, as the light was present on each counting session this variable, whilst possibly affecting heart rate, would affect all counts by a similar amount. Therefore all counts would be raised from what they should be. This would serve to make the results from caffeine more significant and alcohol less significant. Given that the results from alcohol showed an inverse correlation then this source of error could have only made the results less significant. However in the case of the caffeine it is possible that they were responsible for the change in heart rate observed, rather than the caffeine, as the changes were rather small anyway. If the experiment were to be repeated this could be controlled by careful monitoring of temperature and ensuring that this remained constant throughout the experiment, a variable that was no measured in this experiment.
The fact that alcohol had to be used from an initially diluted form, and in a non pure ethanol formulation may have caused the results to be affected. Whilst calculations were made to find the overall concentration of ethanol into which the daphnia were placed, it is not certain whether this was correct. The alcohol could have contained other chemicals in addition to ethanol which could have affected the daphnia and their heart rate.
The formulation of caffeine used was in the form of proplus tablets. These are also not caffeine in a pure format and would contain other padding chemicals such as sugar and talcum powder to make up the tablet. Sugar may well affect the heart rate of daphnia as it is a food stuff for them, and their heart rate may change whilst feeding, or following a substantial amount of food.
Whilst daphnia reproduce asexually they are present in male and female forms. It was not possible to ascertain which gender was being counted on each occasion. It is the females which actually give birth to young. However pregnant females have a lower heartbeat than non pregnant females and males. Therefore if some of the daphnia that were being counted were actually pregnant females then this could have artificially lowered the heart rate result. Without studying each daphnia and ensuring that all were of the same gender and pregnant / not it is not possible to tell whether this factor adversity affected the results.
Whilst the daphnia were held in place loosely on the microscope slide they were not fixed in place. Therefore it was not always possible to check that it was the same daphnia that was being used for the repetitions of counts for each of the drug treatments. It is therefore possible that the variation within each of the 3 counts was due to different daphnia being used, rather than a different heart rate at different times.
It was not possible to ascertain how old each daphnia was which may have had an effect on heart rate as juveniles usually have a higher heart rate than older organisms.
It was assumed that all daphnia obtained from the pond were actually of the common daphnia pulex species. However it is possible that there were a mix of different species within the sample collected. If an organism from a different species was used for each of the counts then this could also give rise to altered results.
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