Toxicology LD 50 Lab

You will analyze data collected from a lab to explain the concept of toxicity and demonstrate how to
calculate the LD50 for a particular organism. Be sure to read the lab completely before beginning your
investigation. Submit your completed assignment according to your teacher's instructions.

Background: LD50
It is important for environmental scientists to examine the influence of toxic substances on
organisms. This does not mean that they superfluously expose organisms to hazardous
materials; this means that they learn to recognize what levels of substances become threats in
order to take preventative measures.

LC is shorthand for Lethal Concentration. Typically, LC refers to toxic chemicals found in the
air, but in environmental science the LC is also used when studying toxic chemicals
found in water. LC50 is the concentration of toxin that kills 50% of the observed organisms.
When testing for LC50, scientists must consider, those organisms that die of natural causes
versus those organisms that die due to the concentration of toxins present. To account for this,
many trials are conducted at several concentrations to account for errors.

The commonly used term to describe ingestion toxicity is LD50. LD means Lethal Dose
and the subscript 50 means that the dose was lethal to 50% of the animals to which the
chemical was administered under controlled laboratory conditions. LD50 values are measured
from zero up, therefore, the lower the LD50, the more toxic the chemical. Therefore, a chemical
with an oral LD50 of 500 would be much less toxic than a chemical with an LD50 of 5. LD50
values are expressed as milligrams per kilogram (mg/kg), which means mg of chemical per kg
of body weight of the animal. Mg/kg is the same as ppm.

The amount of a substance that is harmful is referred to as the dose. Dosage can depend
on the amount per exposure, the number of exposures, the age, sex, body size, etc. of the
individuals that are exposed, how well an individual's body can cope with the substance,
and the sensitivity to the substance. Risk management would look at making levels depending
on the most sensitive individuals or the average individuals.

Dosage depends on the following characteristics of the substances:

1. Solubility - the substance may be either water-soluble or oil-soluble
2. Persistence - how long the substance and its effects last
3. Bioaccumulation - substances that are absorbed and stored at higher levels than
normal
4. Biomagnification - the increase in the levels of the substance as it goes up the food
chain
5. Chemical interactions - the mixing with other chemicals to increase or decrease the
effects of the substance

Overview
You will prepare a lab report using provided data to test the LD50 of brine shrimp. Herbal tea is
not toxic to humans; however, compounds commonly found in herbal tea are toxic to brine
shrimp.

The investigation described below will determine the lethal concentration of herbal tea
compounds for this organism.
Procedure
This is a play-along lab. If you cannot obtain/afford these supplies, you can complete the lab
without purchasing. You MUST read through the procedure below to understand where the
data (below) originated, because all students are expected to submit the Analysis and
Discussion.

1. Place four herbal teabags onto the bottom of a cup. Place 80 ml of hot artificial seawater
(brine) into the cup and let it steep for 15 minutes, shaking gently every 5 minutes.
Squeeze each teabag between two spoons. A cup of tea contains 200mL of water per
teabag; so, four teabags in 80mL of water will make a 10x solution.
2. As the tea is seeping, label two of the container lids 10x, two 7.5x, two 5x, two, 2.5x, two
1x, two 0.5x, two 0.1x, and two 0x or control. Using a spoon or pipette, transfer 10
shrimp to each of your 16 containers.
3. After your tea has steeped for 15 minutes (be careful not to over steep), you will make
each of the following solutions, or concentrations:
10x: This solution is your original tea solution.
7.5x = 7.5ml of 10x solution + 2.5ml of seawater
5x = 5ml of 10x solution + 5ml of seawater
2.5x = 2.5ml of 10x solution + 7.5ml of seawater
1x = 10 ml of 10x stock solution + 90 ml of seawater
0.5x = 5ml of 1x stock solution + 5ml of seawater
0.1x = 1ml of 1x stock solution + 9ml of seawater
0x: This is the brine seawater
Let your solutions cool to room temperature before continuing.
4. You must minimize the dilution error that occurs due to the shrimp being in a salt
solution, so just before you add the tea extract, use the pipette to remove as much of the
brine as possible. This will prevent the tea from becoming dilute from the excess brine.
5. Add 5ml of the appropriate solutions to each of your labeled containers with the brine
shrimp. Cover and place containers where they will not be disturbed.
6. After 24 hours, count the surviving brine shrimp. Calculate the mortality.

Data
Use the data below to complete your lab report. The number of deaths (# dead) at each
concentration was entered for you. You will calculate mortality based on the equation shown in
the table. The first row is calculated for you.

Concentration Initial number of Number dead after 24 Mortality

brine shrimp hours (number dead/initial
number)
10 10 10 10/10 = 1
 10 10 10
7.5 10 10
7.5 10 10
5.0 10 8
5.0 10 10
2.5 10 6
2.5 10 7
1.0 10 3
1.0 10 1
0.5 10 1
0.5 10 0
0.1 10 1
0.1 10 0
0 10 0
0 10 0

Analysis
Plot a scatter graph of concentration (x-axis) vs. mortality (y-axis). Hint: Using a logarithmic
scale for the x-axis does a good job of spreading out the lower concentrations. If you use a
logarithmic scale, do not plot the 0-concentration data.

Try to find the general trend curve of the data. Trace this curve on your graph.

Locate (interpolate) and mark the LC50 on your graph.

Discussion Questions
1. Based on your data in this lab what is the safe concentration of herbal tea for brine
shrimp (the LOEC, or Lowest Observable Effect Concentration)? How can you be sure
this is based on the toxicity of herbal tea and not natural causes?
2. Brine Shrimp have a higher tolerance for many pollutants than does another crustacean,
the Daphnia, also called a water flea. Indicator species are used to study the overall
health of an ecosystem. If you were to study an ecosystem, would you use the Brine
Shrimp or the Daphnia as the indicator species? Explain your reasoning.
3. In this lab, you are (technically) determining the LC50 (Lethal concentration) rather than
the LD50 (Lethal dose). What is the difference?
4. If you repeated this experiment to publish your results in a scientific journal, what would
you do to improve upon this lab?
5. Select a substance from the table below. Using your own mass (in kg), figure out how
many total grams of that substance would be required to kill 50% of clones of yourself.
Be careful about units.

Substance LD50 (mouse or rat)

(source or product) mg/kg or g/kg
benzaldehyde (Cherry Flavor) * 4.8 mg/kg
Tetrahydrocannabinol (THC from marijuana) * 110 mg/kg
Ethyl acetate (Cherry Flavor) * 6100 mg/kg
propylene glycol (Cherry Flavor) 20 g/kg
 Caffeine (Mountain Dew) * 0.13 g/kg
malic acid (sour candy) * 1.6 g/kg
Methanol (wood alcohol) * 5628 mg/kg
Nicotine (through mouth) * 190 mg/kg
Botulinum toxin (bacteria)* 3 x 10N8 mg/kg
potassium nitrate (fertilizer) 190 mg/kg
sodium fluoride (toothpaste) 52 mg/kg
parathion (pesticide) 6.0 mg/kg
Vx (nerve gas) 2 x 10N2 mg/kg
tetrodotoxin (poison from puffer fish) 334 x 10N6 g/kg
diazinon (ant killer dust) 0.076 g/kg
gamma hydroxybutyrate (date rape drug) 2.0 g/kg

Submit your Data Table, Graph and Discussion questions for this lab report.