Download Pre-Lab Questions

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Lab Activity
Introduction:
If you have ever taken any form of medicine, one important study that must be conducted before that
medicine is put on the market is that of the LD50 test. LD50 (lethal dose 50%) is a toxicological value that
measures the dose that kills 50% of a test population over the course of 14 days. In other words, if you
are exposed to this dosage, you have a 50% chance of dying within two weeks. Every substance is toxic
if the dose is sufficient. Botulinum neurotoxins (botox is part of this classification) are known to have
LD50 values as low as 0.000005 mg/kg (milligrams of substance per kilogram of body mass). Even water
can be toxic - its LD50 is about 90,000 mg/kg, meaning the average person would have to drink roughly 6
Liters of water in one sitting for a 50% chance of death. Most commonly, LD50 test are performed on
mice because they have many physiological similarities to humans. Dose-response curves typically take
the form of:
Materials List:

Computer with NetLogo and Excel (or Numbers) installed
Procedure:
Background:
If you are not familiar with NetLogo and Excel, your instructor will walk you through the features
you will use prior to the lab.
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Preparation:


Open the NetLogo program titled LD50 provided by your instructor.
Open the Excel file titled LD50 provided by your instructor.
Pre-Lab Questions:
Before you begin, answer the following questions about toxicology and LD50’s. You may need to refer to
the dose-response curve in the introduction to answer some of the questions.
1. Consider the LD50 graph of Drug X above. Draw a dashed horizontal line starting at 50% on the
y-axis, ending on the graphed line. From the point where this dashed line intersects with the
graphed line, draw a dashed vertical line down, ending on the x-axis. This x-value is the LD50 of
Drug X.
2. What is the LD50 of Drug X?
3. A different drug (Drug Y) is tested and found to have an LD50 of 40 mg/kg. Would Drug Y be
considered more dangerous or less dangerous than Drug X? Explain your answer.
4. Using the table in the Further Research and Challenge Questions sections rank the following
compounds from 1-4, most to least toxic, based on LD50 levels.
Nicotine ______
Alcohol ______
(ethanol)
Sodium Chloride ______
(table salt)
Sucrose _______
(table sugar)
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Lab Activity:
NetLogo Setup
1. Once the LD50 program is open in NetLogo, take a moment to familiarize yourself with the
layout.
Figure 1 – Sample Screen in the Toxicity Program
Things to notice:
 Two buttons – 1) New Trial resets the program to start a new trial with the chosen
parameters and 2) Next Day advances the tick counter by one
 A chooser titled Chemical (with options SodiumCyanide and Arsenic)
 A slider titled Conc (changes the concentration of the substance being tested)
 A monitor titled Dead Mice (telling the total number of dead mice for that trial).
 You will also notice at the top of the black window with the mice a “tick counter.” The
ticks represent the number of days that have passed since a dose of your chosen
chemical and concentration was administered. (How many days are we interested in?)
White mice are living, red mice have, sadly, perished in the experiment.
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Data Collection
1. Choose chemical you will test in the chooser.
2. Slide the concentration slider to the concentration you wish to test (start with 0). Record this
value in your Excel spreadsheet.
3. Click the New Trial button to start a new trial with the chosen chemical and concentration.
4. You will notice you have all white mice. Click the Next Day button to advance the tick counter.
Be sure to make observations about the numbers of dead mice with each click of the Next Day
button. Continue clicking the Next Day button until the tick counter reaches 14.
5. Read the number of dead mice from the Dead Mice monitor. Record this total in your Excel
spreadsheet.
6. Increase the value of the concentration slider by 1mg/kg.
7. Repeat steps 1-6 for all concentrations (1-20 mg/kg) and for both Sodium Cyanide and Arsenic.
Data Analysis Procedure:
1. You should now have collected data for dead mice for concentrations 1 mg/kg to 20 mg/kg for
both sodium cyanide and arsenic. In Excel, you should create a line graph that plots the number
of dead mice (y-axis) vs. dose concentration (x-axis).
2. Be sure to title your graph and label (with units) your axes.
a. To add a title, double click on the title until it is highlighted, then type the name.
b. To label axes, click on Chart Tools on the tool bar, and then Design.
c. Once under the design menu, click add chart element, axis titles, then choose either
horizontal or vertical.
d. Format the x- and y-axis gridlines by right clicking on the axis and choose add minor
gridlines.
3. Save your Excel file and graphs to turn in to your instructor.
Results
1. What is the LD50 of arsenic in mg/kg?
2. What is the LD50 of sodium cyanide in mg/kg?
3. Based on the data you collected, which chemical is more dangerous, arsenic or sodium cyanide?
Explain your answer referencing your data and results.
Conclusions
1.
Why is LD50 data so useful in dealing with human health and environmental toxins?
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2. Keeping in mind how LD50 data is collected, what are the limitations of using LD50 data in dealing
with human health and environmental toxins?
3. In the course of collecting LD50 data, many thousands of mice and rats have been killed. Write a
paragraph describing whether you consider this to be ethical research? Include in your
argument:
 Why is data collected on lab mice instead of humans?
 What other options do we have besides using mice?
 How closely do the results from testing on mice correlate with human LD50 values?
 Would it be more or less ethical to use a species that is more closely related to us (like
chimpanzees).
 How does time and cost factor into choosing a test subject for LD50 testing?
Further Research and Challenge Questions
The table below shows the LD50 of several substances, toxic and non-toxic. For each substance, calculate
the amount of each substance that statistically would have a 50% chance of killing you.
1. Record your weight in pounds here (you can lie if you don’t want to use your real weight, but
then you’ll never know how much of each substance may be fatal…) : _____________________
2. Convert your weight to kilograms by dividing by 2.2 : ___________________
3. Calculate your lethal dose in the chart below. Since LD50 is in mg/kg, you must multiply your
mass in kg by the LD50 to determine the lethal dose in mg. Report your answer in grams.
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Substance
Sucrose
29,700
Ethanol
7,060
Sodium Chloride (table
salt)
3,000
THC (found in cannabis)
1,270 (males)
730 (females)
Aspirin
200
Caffeine (from coffee,
chocolate, and most
sodas)
192
Nicotine (from tobacco
products)
50
Arsenic (often leached
into water supplies
from mining)
14
Sodium Cyanide (often
used in gold mining)
6.4
Venom of the
Australian Inland
Taipan snake
4. The
LD50 (mg/kg)
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Lethal Human Dose (grams)
= mass x LD50 /1000
0.025
VX Nerve Gas
0.0023
Batrachotoxin (from
Poison Dart frogs)
0.0020
accepted
human
values for
arsenic and sodium cyanide LD50 are listed in the table above. How do these values compare to
your graphically determined LD50 values for rats. Calculate the percent error for both sodium
cyanide and arsenic.
Polonium-210 (nuclear
fission waste product)
0.00001
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5.
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Challenge Question! The EPA has set the maximum acceptable arsenic level in drinking water at
0.010 mg/L. If your drinking water has the maximum allowable arsenic amount, how much
water would you have to drink to have a statistical 50% chance to die from arsenic poisoning?
What about a statistical 100% chance of death (use your collected data to help answer the
second part of this question).
6. Challenge Question! Apple seeds contain a small amount of amygdalin, which can produce
cyanide when metabolized in your body. The typical apple seed produces about 0.49 mg of
cyanide in the body. Based on your body weight, how many apple seeds would you need to eat
to have a statistical 50% chance of death? (DO NOT attempt to eat a bunch of apple seeds to
prove this concept! PLEASE!)