The higher the concentration of caffeine the higher the heart rate will become. After a few minutes the daphnia will become accustomed to the environment and heart beat will likely regulate – this is remembering that it is not excessively high concentration for the daphnia to die. This would be due to the fact that they are cold blooded animals and react accordingly to their surroundings. Safety This is always a concern when working with any kind of toxin so points such as the following must be maintained to keep working safely:-
When heating water ensure all usual precautions are applied to Bunsen burner usage
Do not contaminate daphnia ‘home’ water with any substance.
Fair Test/Failures To create a higher level of accuracy when obtaining results, the clicker counter was used for 20 seconds and multiplied by 3 to get the results for one minute.
This was done three times and an average was taken to create more acurate results
Preliminary results were also taken just to ensure all anomalies will be seen and accounted for.
An attempt is made to use the same daphnia for all experiments as daphnia’s vary in size and metabolic rates therefore this must have every attempt made to be avoided. Although occasionally when using high concentrations, death is unavoidable.
Also the age of the daphnia can have some effect, although some only live a matter of minutes, like any other animals, their metabolic rates vary depending on age, size and characteristics .
With the equipment in use, it is more or less impossible to know whether the daphnia is ill or in a poor state, so if unlucky an ill daphnia could alter the entire bioassay. Further equipment such as extremely acurate thermal probes, or other physical assessment tools would be necessary if conducting an extremely acurate bioassay.
Although the female daphnia population is in excess of the male, if a male daphnia was taken for the experiment it would make results differ due to the fact that It is difficult to distinguish male and female Daphnia. They are basically identical except that males are generally smaller in size, have larger antenules, and the first legs have a stout hook used in clasping the female during mating. Rather than trying to identify males to detect stressful environments, it is much simpler to inspect the brood chambers of females). Ie. If a male daphnia was used, the results would differ greatly.
Daphnia stress levels can also alter results, in as much as if the daphnia had a high blood pressure (hyper tension) caffeine would heighten this and alcohol lower it, although starting from a plane incorrect to the necessary average.
Also factors such as the heat from the lamp raising the temperature must be considered as it is known that reactions with enzymes occur faster when the temperature is risen.
If a petri dish, or pipette was contaminated with another substance or a high concentration of alcohol or caffeine this would also affect this bioassay in a negative fashion.
All Daphnia are extremely sensitive to metal ions and many dissolved
toxins in the water. Moina shows the highest resistance levels. these are likely to be apparent dissolved in the oxygen to be used. Analysis Of Results It can be seen from both graph and table, that as the levels of caffeine in the NaCl solution increase, the daphnia’s heart beats per minute increase at a high rate, which seems to peak at about 340 bpm and then levels off until the daphnia die at around 0.8%. With relation to the alcohol, this showed that the BP dropped from 186 BPM and leveled off at around 100 BPM before they die at a concentrarionof close to 0.9%. It seems that the daphnia have a higher tolerance levels to the drug alcohol than they seem to caffeine, this is obtained due to the fact it takes a higher concentration of solution for it to stop the daphnia’s metabolism completely. To find out more and why these affect the heart so much, more detailed research must go into the heart of this crustacean. The crustacean hearts are known to be neurogenic. The cardiac rhythm is originated in cardiac ganglion.
The cardiac ganglion is composed of the pacemaker (small) and follower (large) neurons. The pacemaker neurons exhibit spontaneous bursting activity, and induce firing of follower neurons through excitatory synapses. The follower neurons make excitatory neuromuscular junctions onto the cardiac muscle and cause muscle contraction, the heartbeat.
The cardiac muscles are simply driven by the motor neurons (followers) in the cardiac ganglion just like other skeltal muscles that are driven by their own motor neurons in the CNS.
In crayfish, the cardiac ganglion is often buried under the cardiac muscle fibers. From the knowledge of this it shows how the pacemaker and follower and motor neurons adapt to this new environment and this is why alcohol and caffeine can effect the crustacean heart so quickly and effectively.
So how does alcohol and caffeine interfere with nerves? All cells have a type of protein embedded in their cell membranes called receptor proteins. These receptors allow communication between cells to occur. Nerves can receive communication from cells far away or from nerve cells near by. Cells far away secrete hormones, which find their way to the target nerve’s hormone-receptors. Once these hormones attach to the receptor, changes in the nerve take place. The hormone-receptors, when activated, can change how quickly a nerve generates an impulse, or alter how fast the nerve passes impulses along. Some hormones stimulate an increase in heart rate (such as during exercise) and other hormones decrease heart rate (such as during sleep) which shows how an increase in stress applied to the cardiac muscle of the heart, ie. From caffeine can make it beat faster, and a decrease such as forced by a depressant such as alcohol can make it beat more slowly .
Other receptors allow communication between adjacent nerve cells (such as between the pacemaker neurons and the follower neurons). Instead of hormones, this type of receptor binds to neurotransmitters.
Receptors of any type, however, are not perfect. Sometimes they accidentally bind molecules that they shouldn’t. This can cause an inappropriate increase in nerve activity, or it may cause an inappropriate decrease in nerve activity. (The change depends on the type of receptor involved, and the type of binding the molecule uses.) The nerve fibers conducting pulses to the hearts of Daphnia may contain receptors that inappropriately bind to ethanol (or a product of ethanol after it is broken down) causing an inappropriate decrease in nerve activity. Which could be another form of anomylais result.
In relation to my prediction, my estimated graph was proved to be more or less correct and what was stated about caffeine being related to hypertension was also proved to be seemingly also correct in as much as the fact that the blood pressure greatly rose as the caffeine percentage increased. The rate at which they both increased was similar which shows that caffeine and alcoholism can have and equally adverse effect on ones health although us as humans bodies are more accepting of caffeine than we are alcohol – the fact that daphnia is a cold blooded crustacean shows that crustaceans such as daphnia can intake more capacity in comparison to their body mass than we can as humans and the fact that they are cold blooded makes them more at risk to the drug caffeine.
It was also clear that the beat did become irregular as the dosage increased due to the fact they were close to death or very ill.
Also although not presented in a table or graph form it was shown that the heartbeat did regulate after three minutes of it being in the solution (at concentrations of 0%-0.6%)
My relation predictions such as ‘as the dosage increased so will the heart rate’ however basic, remain the important key to the enzymatic reactions taking place inside the water-flea’s body.
The fact that caffeine is a stimulant, and will kill daphnia at high dosage has been undoubtedly proved, and that alcohol is a depressant and lowers BP, and when In high dosage will kill, is likewise proved
The questions posed in the plan have all been provided an answer to, except, due to the lack of time it was impossible to vary the temperature with the accuracy necessary for this bioassay. Evaluation My plan worked, it proved to be one of the optimum ways to test the effects of such drugs on a small crustacean with the limited time, equipment and background research, and for this account, to improve this bioassay further variables such as temperature combined with one of the drugs, and/or adding nicotine, another depressant that is said to be addictive to humans who smoke cigarettes, over a prolonged period of time, it could be seen if the small cold blooded crustacean would also become mentally dependant on such a drug as nicotine, this could be done, in combination with a percentage of tar to see how this effects the daphnia’s respiratory system and enzyme reactions.
The apparent faults that could occur when doing an experiment are included under the heading ‘fair test/failures’.
There was one or two anomylais results and these are circled and highlighted on both graph and table, this could be due to any number of faults (most of which stated under ‘fair test/failures’) these include, ill daphnia, poorly measured concentration, another substance apparent in petri dish or pipette and so on.
On one or two occasions it was impossible (with this equipment) to count the daphnia’s heart. Neuronic sensors would be ideal for counting the BPM’s of the daphnia’s heart
The plan was also accurate and my knowledge of the daphnia’s background and ideal conditions were crucial in deducting, time, measures and temperatures for the experiment to be carried out at.
The results proved to be clear, the double graph, helping in seeing how the two sets of figures overlap at room temperature. They were sufficiently good for acurate conclusions to be drawn and an efficient bioassay to occur.
There is Definitive room for further investigation into caffeine and alcohol acting as stimulants and depressants respectively, but to fully investigate a caffeine molecule (C8H10O2N4H2O) fig 5
must be taken apart and seen how varying combinations of carbon, oxygen, hydrogen, effect the daphnia’s heart rate. To do this, more advanced equipment and higher levels of skill and time must be incorporated to make the bioassay a success. Daphnia BioassayPreliminary alcohol exp.
Percentage of alcohol in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)
0%186198184
20%176178181
40%157148150
60%142144137
80%12011097
100%deaddead71
Actual Alcohol exp. Percentage of alcohol in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)
0%187184189
20%179184182
40%154157149
50%143144139
60%137131142
70%127125124
80%121116101
90%1178489
100%deaddeaddead
Preliminary caffeine exp. Percentage of caffeine in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)
0%207207207
20%244247241
40%300309307
60%333339345
80%toomany tocount347349
100%deaddeaddead
Actual caffeine exp. Percentage of caffeine in water solutionTest 1(amount of beats in one minute)Test 2(amount of beats in one minute)Test 3(amount of beats in one minute)
0%204211209
20%247249254
40%299304311
50%310316319
60%334339347
70%344340339
80%341344347
90%301deaddead
100%deaddeaddead