[LD50 AND PHYTOREMEDIATION LAB WRITE UP] Kezia Mitchell Per.

1
Introduction: LD50 is the median lethal dose, where half of the population dies due to a toxin. All toxins have different LD50s that vary due to the substances that make them up. Determining the lethal dose of toxicity is different in every sample. First, LD50 is measured in milligrams of the substance per kilogram of body weight so the first step is to convert the measurements of body weight from pounds to kilograms (1 kg= 2.2 lb), secondly by using the cross divide and multiply method, the LD50 can be discovered. Finding out the LD50 of a toxin could be important to humans, animals and the environment because it can prove deadly if you go past the LD50 limit, bypassing this information can cause death among all three subjects. By knowing the LD50 humans, animals, and the environment can steer into a positive direction by knowing how much is okay for them to handle. Otherwise, this information could be deadly if ignored. Materials: Stock 0.0025 M copper sulfate solution Distilled water Graduated cylinders 5 petri dishes (capable of holding at least 25 mL) each group Grease pencil 50 Black worms each group Timer each group

Procedures: 1. Lay out each petri dish on your bench top and pre-label them with the following concentrations using the grease pencil: a. 0% CuSO4 b. 0.01 % CuSO4 c. 0.1% CuSO4 d. 1% CuSO4 e. 10% CuSO4 2. Place 22.5 mL of distilled water into the 0, 0.01, 0.1,1 and 10% CuSO4 petri dishes. You will be doing a serial dilution for the CuSO4 concentrations in this lab. To start, add 2.5 mL of the CuSO4 standard (found on the lab bench for the class to share) to the 10% CuSO4 petri dish. Next, take 2.5 mL of the 10% CuSO4 petri dish and place it in the 1% dish. Then take 2.5 mL of the 1% dish and place it in the 0.1% CuSO4 petri dish. Then take 2.5 mL of the 0.1% dish and place it in the 0.01% CuSO4 petri dish. Take 2.5 mL from the 0.01% dish and discard that into the waste bin. All the beakers should now have 22.5 mL of solution in them. 3. Place 10 blackworms into the 0, 0.01, 0.1, 1 and 10% CuSO4 solutions and record your initial observations into the table below in the column labeled Initial Observations. Gently prod and poke the blackworms and note how they move. Next, observe the blackworms after 5,10,15,20 and 30 minutes. Record your observations for each petri dish in the columns labeled with the appropriate time. Note how many blackworms in each treatment are moving, and how they

[LD50 AND PHYTOREMEDIATION LAB WRITE UP] Kezia Mitchell Per.1

look. Be specific and quantitative. At the end of the class period, count how many blackworms have died and record that in the column labeled Final.

When you do serial dilutions, you multiply together all of the dilution factors. Make sure you are clear on what constitutes a dilution factor. When I add a small amount of the concentrated stuff to an empty container, "top it off" with saline/water/whatever, then remove some small amount, this constitutes a dilution. Data: Time (min) 5 10 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead

Treatment Initial Observations Few 0% clumped Slow 0.01% movement Few 0.1% clumped Medium 1% movement Sudden 10% dispersal

15 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead

20 0 Dead 0 Dead 0 Dead 0 Dead 0 Dead

30 0 Dead 0 Dead 0 Dead 1 Dead 3 Dead

Final 0 Dead 1 Dead 1 Dead 3 Dead 9 Dead

[LD50 AND PHYTOREMEDIATION LAB WRITE UP] Kezia Mitchell Per.1

Data Analysis:

# Of Dead Worms vs Dosage of Copper

10 # Of dead worms (not moving) 9 8 7 6 5 4 3 2 1 0 0% 0.01% 0.10% Dosage of Copper 1% 10% # of dead worms

The LD50 was found by finding the point where half of the population died and matching it down to the dosage of copper. Discussion/Conclusion: My data showed me that our LD50 was in the middle of 1% and 10%, however it is closer to the 10% side, so we concluded that 10% of Copper was our LD50 for the blackworms. I learned from this lab that even an amount thats not too significant such as 1% can prove harmful to species. I also learned that some species take a while to show affects, our worms didnt start dying until well after 15 minutes in the experiment. I dont think me and my partner had any experimental errors, next time maybe there should be less diluted concentrations to show the effects better. I know some groups who had no blackworms die through their experimental time. You could expand this lab to making it longer and studying further into the blackworms, also studying what in the copper is affecting the blackworms. That probably has something to do with how the worms started to turn red and slowly release blue particles prior to their death.

Introduction: Phytoremediation describes the treatment of environmental problems through the use of plants that mitigate the environmental problem without the need to excavate the contaminant material and dispose of it elsewhere. It is beneficial to the environment because it is a cheaper way to clean out the hazardous toxins that our in our water. It works by inserting these hungry plants into the water, and they get inserted into contaminated water and suck in all the toxins, removing them from the water

[LD50 AND PHYTOREMEDIATION LAB WRITE UP] Kezia Mitchell Per.1

source. This is connected to the LD50 because the copper concentrations in that lab will help determine the ability the water lettuce plant can withhold. Materials: 1 water lettuce place per group 1 beaker per group (holding at least 50 mL) 0.0025 M Copper sulfate (CuSO4) solution 10, 1, 0.1, 0.01% CuSO4 stocks Distilled water Plant nutrient solution Graduated cylinder Grease pencil per group Plastic Spoon per group 10 black worms per group Cu test kit Time per group

Procedures: 1. Look at your LD-50 lab with blackworms and determine the concentration of CuSO4 that killed 50% of the blackworm population (the LD-50). Your value should be close to one of the values that were tested during that lab (0, 0.01, 0.1 1, and 10% CuSO4). Once you determined your LD50, write that concentration on your beaker. Please round your LD-50 to one of the values listed in the table below if it was not exact. 2. Once you determined the CuSO4 concentration you will use in this lab, look at the table to see how to make your CuSO4 solution. You should have a total of 50mL solution. 3. Make your copper solution and place it in the beaker. Take an initial copper measurement using the copper kit provided in the class. Follow the directions on the copper measuring kit. Record your first copper reading in the data table below in the Day 1 section. 4. Now add one drop of nutrient solution to the beaker, and 1 water lettuce plant. The water lettuce plants are fragile, so carefully scoop them up out of the class tank with a spoon and place them in your beaker. Record any initial observations you might have. 5. Place your beaker with the water lettuce in a sunny spot in the classroom. 6. Record copper measurements every day for the next 2-3 days in the table. When removing solution for the copper measurement, make sure not to disturb the water lettuce. 7. Graph the changed in the observed copper concentrations over time on the graph provided below. Use the day as the x-axis and the copper concentration as the y-axis.

[LD50 AND PHYTOREMEDIATION LAB WRITE UP] Kezia Mitchell Per.1

Data:

Day
1 2 3

Cu [ppm]
3.0 0.6 0.3

Observations
Big, one dead leaf, very green with long roots They look like theyre dying! It looks weird, like its slowly loosing its color

Data Analysis:

Cu [ppm] asborption in Water lettuce

3.5 3 2.5 Cu [ppm] 2 1.5 1 0.5 0 1 2 Days 3 Cu [ppm]

Discussion/ Conclusion: My data shows that the amount of copper in the water actually went down. This proves that the water lettuce sucked it up because there is no other place for the copper to go besides in the plant. I learned that the phytoremediation idea is wonderful except there are some kinks to it; I noticed that my water lettuce plant especially started to look limp throughout the days and seem as if it was dying. I am not sure if this was due to lack of sunlight because it was in a classroom and not outside, but it definitely shows that maybe too high of concentrations are just as harmful to the plants being used for phytoremediation. Next time, I think I would have my plant sit outside in a safe place so it would be more comparable to the actual environment outside. This lab could be expanded to each copper solution being tested for each group so they can see the difference of how the plant reacts towards the toxin. In my opinion, hyper accumulators are helpful in remediating a toxic site but I also fear that after they accumulate all the toxins, there is no place to put them. I feel as if, the only way to get rid of them would to be putting them back in the environment, which might cause another leak of the toxin. I think the idea has a good start but definitely needs to find an efficient method to handling the plants after they have finished their jobs.