It was ensured that there was a large sample of Daphnia, from the same environment. This was controlled by buying the Daphnia from a pet shop, as the Daphnia were bred in the same conditions. From then onwards the Daphnia were kept in the water that they were bought in and stored in a fridge to maintain a constant temperature of 5°C.
The flex-cam and video recorder were set up and a single Daphnia was used to ensure that the heart could be seen through an appropriate magnification. As the heartbeat of Daphnia was very fast it was better to make a recording of each experiment carried out on the Daphnia. This meant that the video could be watched afterwards to count the number of heartbeats more accurately.
Once the apparatus had been set up, it was possible to carry out the experiments. Firstly, a single Daphnia was transferred from the large sample in the beaker, onto a cavity slide, using a pipette. It was ensured that there was enough water surrounding the Daphnia so that it was not harmed, but would not move around in the water too much. This slide was put under the microscope, so that the heart could be seen beating on the television clearly. Simultaneously the ‘record’ button was pressed on the video remote and ‘start’ on the stop clock.
After 1 minute, the video recording and timer were stopped and the slide was removed from under the microscope. Then 1ML of the 1x10g-1cm-3 ethanol solution was added into the cavity of the slide carrying the Daphnia. Immediately, the slide was put back under the microscope and the stop clock video recording were started. After 3 minutes, the video and stop clock were stopped. Using a small piece of a tissue paper as much of the ethanol solution was absorbed from the slide. Using a pipette, 1ml of water from the beaker containing the large sample of Daphnia was transferred onto the cavity. The slide was returned under the microscope so that the heart of the Daphnia could be clearly observed and once again the video recording and timer were started. After 3 minutes the recording was stopped and the Daphnia being experimented on was carefully returned into the water it removed from.
Using the 1x10g-1cm-3 ethanol solution the same process was repeated four more times. Then for the four remaining ethanol solutions the procedure was repeated five times for each concentration of ethanol. Altogether the experiment was carried out on 25 Daphnia.
The number of heartbeats of each Daphnia had to be counted- before, during and after submersion in alcohol. It was not accurate enough to count the number of heartbeats whilst viewing the video, so a grid was drawn up, of which there is a sample of on the following page. Each time the heart of the Daphnia was seen to beat a dot was drawn in the appropriate box, this was continued for each 10 second interval within the total time of each recording of 7 minutes in duration. This time interval was timed accurately by using a tape that a ‘click’ noise every 10 seconds. Then this procedure had to be carried out for each of the 24 remaining Daphnia. The number of dots in each of the boxes had to be counted to give the number of heartbeats for 10 seconds. The heartbeat in the 10 second intervals had to be counted for each of the 25 Daphnia. Then the final step was to transfer the number of heartbeats that were counted into a raw results table accurately and into the appropriate box.
CONCLUSIONS & DISCUSSIONS
From the graphs that I have drawn I have noticed a clear trend, which is as the concentration of alcohol that the Daphnia is submerged in increases the greater the decrease of their heart rate.
The general trend of the graphs A-E is that after the Daphnia have been submerged in alcohol their heart rate falls, but after returning to water the heart beat gradually rises. This trend is for the graphs drawn of the average number of heartbeats every 10 seconds, of the 5 Daphnia tested for each of the 5 concentrations. Alcohol has this effect on the heartbeat of Daphnia because alcohol is a depressant and therefore it affects the nervous system of the Daphnia. It is the nerve impulses that regulate the heartbeat in crustaceans, like Daphnia, as is the case with most other animals. The nerve impulses are generated by neurones that are found in nerve cells. These impulses are transported by motor neurones to the cardiac muscle causing the heart to beat.
Ethanol interferes with the nerves by binding with the receptor proteins that are located in the cell membranes of the postsynaptic node. This causes the communication that would normally take place between nerves to be halted. Once the neurotransmitters bind to the receptor sites the change is caused to take place. Receptors can bind with molecules that they should not, such as ethanol. Consequently, a decrease in nerve activity may take place. In the case of Daphnia the nerve fibres that take part in the transmission of the nerve impulse to the heart may contain receptors that bind to ethanol. This could therefore be the cause of a decrease in the activity of the nerves.
Between 0 seconds and 60 seconds the resting heartbeat on average remains at an almost constant rate. It is after the Daphnia are submerged in alcohol at 60 seconds that their heart rate falls. At 180 seconds, the heart rate of the Daphnia begins to rise gradually and at 240 seconds, when returned to water from their environment the heart rate increases further. However, this increase is not above the resting heart rate- it is just to below the Daphnia’s resting heart rate.
The general trend of the graph of the mean percentage decrease of heart rate when in alcohol from the resting heart rate against the log10concentration of alcohol is that at high concentrations, 1x10-1 gcm-3 to 1x10-4 gcm-3 the percentage decrease of heart rate is greatest. However, at a low concentration of 1x10-5gcm-3 the percentage decrease of heart rate is slightly lower, which does not fit with the trend of the rest of the graph.
At a high concentration of ethanol solution the heartbeat of the Daphnia does not return to the resting heart rate by the end of the experiment. Therefore, at an alcohol concentration of 1x10-1gcm-3 the recovery time for the heartbeat of the Daphnia to return to the resting heart rate is long.
EVALUATION
The experiment that I carried out was repeated five times for each of the five different concentrations of alcohol that were used. In total there were 25 individual experiments that were carried out. These experiments were sufficient enough to enable me to construct line graphs and therefore find out the relationship between alcohol and its effect on the heart rate of Daphnia. I measured the effect of alcohol on the heart rate of Daphnia by calculating the percentage decrease of heart rate from the resting heart rate.
VARIABILITY ANAYSIS
The largest error bar that was drawn on the graphs that were plotted was +/-6 heartbeats. But this was only on a single occurrence, at 250 seconds. This was when the Daphnia had just been removed from the 1x10-2gcm-3 concentration of ethanol solution and returned to water from the environment it came from. The error caused means that the heartbeat of the Daphnia could have been 6 heartbeats higher or lower than the calculated mean heartbeat of 34. However, I previously noticed that there was an anomalous result which occurred when Daphnia number 7 was being tested at 250 seconds. The heartbeat of the Daphnia was considerably higher than the other heart heartbeats counted for that particular Daphnia. Therefore, the large error bar was probably due to the anomalous result that I have described. The remainder of the error bars that were drawn were between 0 and +/-5 heartbeats. Due to these relatively small error bars I think that it is fair to say that the results I obtained from carrying out the experiment were generally accurate. This means that the conclusions made are also valid as there were little inaccuracies that took place whilst carrying out the experiments.
LIMITATIONS AND ERRORS
As I was using living organisms, not all of the Daphnia were the same size. This is because the size of Daphnia is not fixed, therefore contributing to a significant error in the experiments that were carried out. Different sized Daphnia may respond to alcohol in different ways, so this was a limitation to the experiment that I carried out.
The Daphnia were kept in a separate beaker filled with water prior to being placed onto the cavity slide. This meant that when I removed the Daphnia using a pipette water was being removed from the beaker at the same time. Therefore the Daphnia remaining in the being that had not yet been tested were not in a constant environment. The water levels were continually being changed as a Daphnia was removed to take part in an experiment. This means that the water conditions were not controlled as well as they could have been. Also, with regard to the water conditions other pupils were also carrying out experiments involving the use of Daphnia. This means that cross contamination was likely to have taken place. Once again this contributed to altering the environment that the Daphnia were kept in. temperature changes also took place in the duration of testing the Daphnia. The room temperature was considerably higher than the temperature of the fridge and therefore the Daphnia needs a certain amount of time to adjust to the temperature into which the beaker was placed. However, whilst carrying out the experiment this was not measured accurately.
Errors may have taken place during the preparation of the alcohol solutions. This is because small errors probably occurred when using the 100cm3 measuring cylinder because it only measures to the nearest 1ml. This would have created a percentage error of +/-1%. This percentage error may not have been significant if the measuring cylinder was used on one occasion. However, the 100cm3 measuring cylinder was used several times and therefore may have had a large contribution to the percentage error.
The amount of water that the Daphnia were surrounded in prior to submersion in alcohol was not measured accurately. The amount of water was 1ml, but the Daphnia had to be kept in a small amount of water before the 1ml was added to ensure that the Daphnia was not harmed. So this may have contributed to some inaccuracies as the Daphnia probably were immersed in slightly different amounts of water.
I would also ensure that the Daphnia were tested for a longer period after being returned to water from their environment. This is because after 3 minutes of recording the heartbeat some of the Daphnia had not returned to their resting heart rate. It would be interesting to find out how long it takes for the Daphnia’s heartbeat to return to the resting heart rate after submersion in different concentrations of alcohol solution.
I would use more than five concentrations of ethanol solution to test the Daphnia with, in a future experiment. Possible concentrations of ethanol that could be used are 5x10-1gcm-3, 5x10-2gcm-3, 5x10-3gcm-3, 5x10-4gcm-3 and 5x10-5gcm-3.
Another limitation to the experiment that I carried out was that not all of the Daphnia were tested on one day. Therefore, by the following day the age of the Daphnia was different to the Daphnia involved in the experiment on the previous day. As the Daphnia age it is possible that changes take place in the way that the heart functions.
If I were to carry out a further investigation into the effect of alcohol on the heartbeat of Daphnia I would want to determine where exactly in the body of the Daphnia the alcohol affects. To find out if the alcohol has a direct effect on the heart or if it affects the nervous system I would dissect the heart from several Daphnia and submerge only the heart in alcohol.
In future experiments it would also be interesting to find out the effect of caffeine on the heart rate of the Daphnia and whether it produces similar results to the experiment using alcohol or not. It would also be interesting
REFERNCES
BOOKS
INTERET- WEBSITE ADDRESS
CONTENTS PAGE
Content Page
Abstract
Introduction
Method
Results
Conclusion & Discussions
Evaluation- Variability Analysis
