Ecosystems have a way of maintaining themselves under ordinary conditions but when a new factor is added that will most likely threaten the environment action needs to be put forth. Our environment is affected by many pollutants on a daily bases forming a threat to natural stability. For example some of the most polluted parts of the environment are bodies of water like that explained in our lab. Once pollution occurs animals can be affected in many ways even to the brink of death.
In the lab the stream is getting pumped with salty potassium filled water that can kill daphnia, habitants of the stream, if the concentration gets too high. To prevent this a real-time assay can be performed. This is a short timed test that we use to measure the effect of pollutant on organism by finding the smallest concentration possible that could still be a bother. This is a valuable tool because we can get direct results from a living organism by detecting stress signals all in a short period of time allowing us o fix the problem quicker.
Using an organism for the assay is the best choice because there are many variables that can be used to identify stress on the subject because of a specific input.
Materials and Methods
Everything is kept the same in every experiment except when concentration differences need to be changed. Meaning all lighting, heating, vibration daphnia numbers will not change to make sure there are minimal changes and differences in the experiments. The sizes of the daphnia are also better to be kept around the same. Daphnia are really quick so picking something to measure in a two-hour interval made it easier to narrow it down to heart rate. Since we can control their space, meaning less area of movement we can see their heart better. This also meant it wouldn’t allow us to measure other things that require a larger area of movement which were the majority of the variables. Chasing down the critters is very hard.
Week one was the period of observation where all materials were used while looking at the daphnia so that the variables to be measured are chosen and the methods in how they will be measured are ruled out. Before the lab a handout with information on daphnia should be read to familiarize oneself with the little organisms. For observation we are given a regular microscope, to use with it regular shallow white well plates and a dissecting scope, to use with it a see through 24-well plate. A small amount of water should be added to the both the white wells and about three or four of the clear wells. Now that everything is ready for the daphnia they are to be transferred to the wells be using a pipet that has had its tip cut of to a diameter of about 2-3 mm so the critters aren’t damaged. Write down any observations you see such as variables and movements and any ideas for a possible assay.
Week two an assay system will be created using the variable that is chosen versus the concentration of KCl. The concentration we started with, was given, a 1:1 ratio of KCl to pond water meaning that half of the mixture is KCl and half is pond water. To perform this experiment we also need a control that would be 100% pond water. Two regular microscopes are used with the three well white trays. In one tray pond water is added to the three wells, half of the group will look at these and in the other the mixture of 1:1 KCl : pond water is added, the other half of the group will look at these. Daphnia are then transferred using a transfer pipet that has the tip cut to a diameter of 2-3 mm. The experiment needs to start after a 15-minute exposure period maybe shorter because they may start reacting to the different concentrations.
Once the slides are in focus view the daphnia to see if there are any changes in the variable if so one partner should start counting while the other is keeping time and writing down results. Every once in a while to keep thing consistent switch who counts and switch around the wells. After the observation period make sure the lights are off so the daphnia aren’t heated and they have enough water, and make sure they don’t get moved around too much. Once the experiment is compare results of KCl concentration versus the control. If something did occur to the daphnia do the KCl concentration experiment but half it, If nothing happened double the concentration. Once you are done take our results and plot them using a T-Test in excel.
Week three the daphnia are tested under different concentrations of an herb to see if the herb causes any types of stress on the variable. All of the same methods are done as in week one and two except in preparing the herbal solutions. To prepare the herbal solution the herb needs to be grinded mixed with methanol and left to sit for a few minutes so it can react and separate the important contents of the herb out. In large clear wells the mixture is measured in microliters of 500, 50 and 5, we also measure out 500 of methanol and all of these are left to dry out. Then 5ml of water is added to 5 wells the four including the substances and one without. The methanol only and water only wells are our controls once this is finished the variables are measured like in the previous experiments.
(on graphs and tables)
Daphnia are very easily affected by even the smallest amount of KCl slowing down their heart rate compared to our controls. Our 1:4 ratio of KCl still slightly affected the heart rate of our daphnia. The daphnia give a clear and quick report on the environmental condition this is why they are good to experiment on.