LD50 Lab
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There are many ways to evaluate health threats from toxic substances in the environment. Toxins may affect cells,
tissues, organs, organ systems, whole organisms, populations, and ecological communities. Toxins may be either
synthetic or naturally occurring.
In the science of toxicology, the study of toxic substances, dose-response analysis are often used to evaluate the
direct lethal effects of substances on organisms. A dose-response analysis is a test that uses living organisms as
an indicator. A common measure of acute (lethal) toxicity is called the LD50 (lethal dose 50). This is the
experimentally determined concentration of the substance that will kill fifty percent of the individuals exposed to
it.
The reason that some, but not all of the exposed individuals are killed is twofold. First, there is genetic variation
among individuals in a population. Some individuals are more susceptible and some are more resistant to the
lethal effects of any substance. Second, exposure to the toxin is uneven. The toxin is likely not to be evenly
distributed in the food or environment of the individuals. One of the challenges of a good experiment is making
sure that all the experimental individuals are evenly exposed to the toxin.
Also, it is extremely important to remember that different species of animals will vary in their response to a
particular toxin. What may be extremely poisonous to one animal may have no harmful effect on another. This is
why test studies using laboratory animals cannot be extrapolated to humans with 100% certainty. Generally
speaking, the more closely related two animals are, the more the results can be extrapolated from one to another
with confidence. Universities and government agencies use vertebrate animals such as rats, rabbits, and
chimpanzees for drug and toxicology testing.
Before analyzing the results of a dose response analysis, we need to become familiar with the units commonly used
in toxicology such as parts per million and parts per trillion. In studying toxicology, AP Environmental Science
students sometimes become confused about the concept of percent, parts per million (ppm), parts per billion (ppb), and
parts per trillion (ppt). These quantifications are traditionally based upon mass (weight) and not volume or numbers.
For example, to make a one part per million salt mixture in sugar you could take 999,999 grams of sugar and add 1 gram
of salt. The individual size and mass of the salt and sugar grains doesn’t matter; only the accumulated mass matters. Also
notice that you are not starting with 1,000,000 grams of sugar. The salt has to be incorporated into the total mass of (in
this case) 1,000,000 grams.
PPM is equivalent to milligrams per liter (mg/l). This is because there are a 1000 milligrams in a gram. One milliliter
of water weighs one gram. There are 1000 milliliters in a liter. Therefore 1000 x 1000 = 1,000,000. There are a 1,000
millions in 1 billion and 1,000 billions in 1 trillion, therefore, there are 1,000,000 millions in a trillion. Furthermore, 1
ppm = 1,000 ppb = 1,000,000 ppt. So if you have a backyard pond with water that contains 6,000 ppb of dissolved
oxygen that would be the same as 6 ppm of dissolved oxygen or 6,000,000 ppt of dissolved oxygen.
The state standard for mercury in drinking water is 5 ppb. My tap water tested at .002 ppm. Is it safe? .002 x 1,000 = 2
ppb Yes, according to the state, the level of mercury in your water is below the threshold. If ocean water has a 3.5 %
concentration of salt, how many ppm is that? 3.5 % is 3.5 parts per 100. What do we have to multiply 100 by to get
1,000,000? We have to multiply by 10,000. So that means that we have to multiply 3.5 by 10,000 as well. A 3.5 % salt
solution is equivalent to 35,000 ppm.
Complete the dilution activity below to get familiar with these units before analyzing the results of the dose-response
analysis
Dilution Activity
In this activity you are going to perform a serial dilution using a solution of Congo Red (a natural stain). The Congo Red
solution was created by adding 10 grams of Congo Red to 90 ml of water thus creating a one-part-per-ten solution of
Congo Red. This is equivalent to 100,000 parts per million (ppm) because ten times 100,000 is equal to one million. It is
also equal to 100,000,000 parts per billion (ppb) or 100,000,000,000 parts per trillion (ppt).
1. Obtain a dropper and a beaker of tap water
2. Obtain a well tray and put three or four drops of the one-part-per-ten solution of Congo Red solution into well one
(top left corner). Carefully carry the well tray back to your bench without spilling or sloshing the congo red
solution into any of the other wells.
3. Put nine drops of water into each of the other wells.
4. Now take the dropper and carefully transfer one drop of the Congo Red solution from well one into well two.
Squirt ant remaining Congo red back into well one. Use a toothpick (not the tip of the dropper!) to stir up well
two. Observe what you see.
5. Now you need to write some information down in the table on the next page. What is the new ratio of Congo Red
to water? (Remember, the ratio in the first well was 1:10), How many parts per million, billion, or trillion does
this represent? Finally, what is the color density? In other words, what color of red or pink do you see? Or do you
see any color at all?
6. After you are done with well two, rinse the dropper in the water that remains in the beaker. Now transfer a drop
from well two into well three and squirt the remaining liquid in the dropper back into well two. Answer the same
questions for well three as you did for well two.
7. Continue the serial dilution all the way through to the last well making sure to carefully wash stirring toothpick
before making a transfer to the next well.
8. When you are finished, thoroughly wash the well tray.
Answer the following questions in your own words:
1. How many parts per billion were in your last well?
2. How many parts per trillion were in your last well?
3. What was the last well in which you could see any trace of the Congo Red dye?
4. Were there any Congo Red molecules in the last well? Explain.
5. From what you have learned in this activity, do think it is possible that clean, clear drinking water could be contaminated
with chemical toxins such as pesticides that wash off of agricultural fields?
Herbicide Toxicity- A Dose-Response Analysis
An herbicide is suspected of causing toxic effects in the human population and may even be responsible for several deaths
in the community. As a result toxicologists have been requested to analyze the herbicide and present a formal risk
assessment of the chemicals in this herbicide. As part of the assessment they have performed a dose response assessment
using mice. Seven groups of 100 mice were each exposed to various concentrations of the herbicide and the number
surviving in each group was recorded. The data is shown below, but unfortunately the dosage is reported in percent
instead of ppm, making it hard to compare the results of this study to other similar studies. Start by converting the percent
concentrations to parts per billion and recording the answer in the data table. Additionally, the number surviving will
need to be converted to % mortality in order to create a dose response curve. Record the % mortality in the data table.
Dose of Herbicide
(% Concentration)
0.00000001
0.00000005
0.0000001
0.0000005
0.0000009
0.000005
0.00001
0.00005
0.00009
0.0001
0.0005
0.0009
0.001
0.005
Dose-Response Data For Mice Exposed to Pesticide
Dose of Herbicide
Dose of Herbicide
# Surviving 24
(% Concentration)
(PPB)
Hours After
expressed in
Exposure
Scientific Notation
96
95
93
93
92
92
71
60
38
4
1
0
0
0
% mortality
Analysis
Plot the results of the LD50 test on the six cycle, semi-log graph paper at the back of this lab. Label the graph axes. (%
Mortality v. Dose in ppb)
Conclusion Questions
Answer the following questions in your own words.
1. Is there a threshold of toxicity for this herbicide on mice? How can you tell? If so, label this point on the graph.
2. What is the LD50 concentration of this herbicide in mice? Label this point on the graph.
3. In order to develop a complete risk assessment of the effect of this herbicide on a human population what else
would you need to know about the herbicide as a pollutant?
4. Cadmium Nitrate is a white crystal chemical that is a known carcinogen that may affect kidneys and lungs. LD
50 testing has been done on rats. The LD 50 for rats was found to be 300 ppm. If cadmium nitrate is metabolized
in the same way in humans as it is in rats, what would be the lethal amount of cadmium nitrate for the average 70
kg human being? Show all work, circle your answer.