For brine shrimp exposed to increasing incinerations of three chemicals, ammonia will have the highest LLC-50 (toxicity), bleach will have the intermediate LLC-50 (toxicity) and vinegar will have the lowest LLC-50 (toxicity). MATERIALS AND METHODS: For this experiment we use the following materials: Petri dishes (4 per group) Stop watch or clock Safety goggles (1 for each person) Brine shrimp Brine solution (3. % w/v NCAA and water) need 600 ml per class Vinegar- need about 50 ml in small dropper bottle (1 per group) Ammonia- need about 50 ml in small dropper bottle (1 per group) Bleach- need about 50 ml in small roper bottle (1 per group) 3 pipettes (1 ml) – each labeled for the respective contaminants Pipette pumps Grease pencils Beakers (5 or 10 ml) Graduated cylinders (25 ml) Magnifiers or hand lens (minimum 2 per group) Beakers (Extra large) for waste- one each labeled for Vinegar, Bleach and Ammonia Beaker (Extra large) for tested Brine Shrimp Our procedures consisted of eight steps.
Step 1- We all put on our safety goggles before gathering our materials. Step 2- We gathered our 4 Petri dishes and labeled them using a grease pencil. One Petri dish labeled “C” for Control, ‘V” for Vinegar, “A” for Ammonia and “B” for Bleach. Step 3- Once the Petri dishes were labeled, we took a 25 ml cylinder and used a pipette to gather 12 brine shrimp to put inside of the cylinder. Using the measurement lines on the side of the cylinder, we had to make sure the measurement of water and brine shrimp reached a level of 20 ml.
Once we gathered exactly 12 brine shrimp, we then dumped the contents of the cylinder, including the brine shrimp, into the Petri dish labeled “Cm for control. We continued to use the pipette to gather 12 brine shrimp and measured the level to 20 ml for each of the other Petri dishes until each Petri dish contained 12 urine shrimp each. Step 4- Before adding any of the concentrations to the Petri dishes, we put the lids on all four of the Petri dishes and set our stop watch for 5 minutes to observe the brine shrimp in their controlled atmosphere.
Once 5 minutes ended, we then counted the number of live brine shrimp and recorded the information on a chart for each individual Petri dish. This was very important because brine shrimp are known for cannibalistic behaviors and losing a brine shrimp to cannibalism would throw off the count of live shrimp after the concentrations have been added. Step 5- We discussed how affective we thought each concentration would be towards the brine shrimp. Our hypothesis was that ammonia will have the highest LLC-50 (toxicity), bleach will have the intermediate LLC-50 (toxicity) and vinegar will have the lowest LLC-50 (toxicity).
Step 6- Once the hypothesis was made, we gathered our dropper bottles of ammonia, vinegar and bleach. Leaving the “C” (control) Petri dish aside, we began adding the proper chemicals to the assigned Petri dish using a clean pipette pump assigned to each chemical. Each person was responsible for one Petri dish because the chemicals ad to be added at the same time to get accurate results. The Petri dish labeled ‘V” was given 0. 5 ml of vinegar, the Petri dish labeled “B” was given 0. 5 ml of bleach, the dish labeled “A” was given 0. Ml of ammonia at the same time and then we gently swirled the liquid in the Petri dishes and quickly replaced the lids and began the stop watch for 5 minutes. Step 7- Once 5 minutes was up we counted the number of live brine shrimp in each Petri dish and calculated the concentration percent and mortality rate percentage. Using the number of live Hiram, we were able to calculate the number of dead brine shrimp, which then allowed us to calculate the percent mortality rate at each concentration level.
Because the brine shrimp were so small it was optional to use the magnifying glass or hand lens to be able to count the number of live brine shrimp in each dish. Formula for calculating concentration %: NCAA (ml) + brine ml + amount of chemical added- Total Volume NEXT (amount of chemical) / (total volume) x Formula for calculating mortality rate %: (total dead) / (total starting number) x % Step 8- We added another 0. Ml of each chemical to their assigned Petri dishes a second time and set the stop watch for 5 minutes again. After the five minutes, we counted the number of live brine shrimp and calculated the mortality rate percentage.
Step 9- Next we began adding 1 ml of each chemical to their assigned Petri dishes, set the stop watch for 5 minutes and when the 5 minutes was over, we recorded the number of live shrimp and calculated the mortality rate percentage. We continued repeating this step, until a total of (five) I-ml aliquots had been added and the data was recorded. Step 10- Since the mortality rate for the brine shrimp in the Petri dishes labeled “V” (vinegar) and “A” (ammonia) had reached at least 80%, we could then end the test with that particular group of brine shrimp.
The remaining Petri dish, “B” (bleach), had not reached at least 80% mortality rate so we continued to test it. Only this time, we had to begin adding 2 ml of bleach to the Petri dish and set the stop watch for 5 minutes and record data. This step was done 2 times. Step 11- The addition of 2 doses of 2 mil’s was not changing the mortality rate, so we had to begin adding 5 mil’s and recording the number of live shrimp after minutes and calculated the mortality rate percentage. This was done twice.