Lethal Dose (LD50)

Introduction (Part 1):

We handle many materials daily that are toxic.  We are often unaware of the degree to which they are toxic.  For a variety of reasons, different animals respond differently to the same toxin.  Some animals may be very sensitive to a toxin, whereas others are relatively resistant to its effects.  Because species of animals vary, it is important to understand that what is toxic to one organism may not necessarily be toxic to other kinds of organisms to the same extent.

Many household items that we deal with on a regular basis are toxic materials, but we don’t usually think of them as being toxic.  It can be instructive to examine several such materials to determine their toxicity.

The commonly used term to describe acute ingestion toxicity is LD50.  LD means Lethal Dose (deadly amount) and the subscript 50 means that the dose was acutely lethal to 50% of the animals the chemical was administered to under controlled laboratory conditions.  The test animals (usually mice or rats) are given specific amounts of the chemical in either one oral dose or by a single injection and are then observed for 14 days.

Since LD50 values are measured from zero up, the lower the LD50, the more acutely 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)  For example, if the LD50 of the insecticide parathion is 4, a dose of 4 parts of parathion for every million parts of body weight would be lethal to at least half of the test animals.

An MSDS (Material Safety Data Sheet) is a document (for each chemical) with information on all the physical and chemical properties for that chemical, as well as information on reactions and safe disposal of the chemical waste.  The following information can usually be found in an MSDS:

• Identity of the organization responsible for creating the sheet and the date of issue
• The material’s identity, including its chemical and common names
• Hazardous ingredients
• Exposure limits
• Physical and chemical hazards and characteristics
• Health hazards
• Emergency and first aid procedures
• Spill and disposal procedures
• Spill and disposal procedures
• Precautions and safety equipment

Procedure (Part 1 – Researching and Getting Familiar with LD50)

1. Using YOUR OWN MASS in kg, figure out how many total grams would be required to kill 50% of perfect duplicates of yourself.  Be careful about units!  For your reference, a penny weighs around 3000mg or 3g.  You don’t need to show work for all of these problems, but write out ONE complete example of your calculations and conversion to LD50/person below the table so that I know how you did it.  Include your weight in pounds and kilograms.

Note:  1lb = 0.45359kg = 453.59g

2. Find a Material Safety Data Sheet (MSDS) for an ingredient in some household substance you have (e.g. toothpaste, shampoo, mouthwash, junk food additives, etc.) and give its LD50 for the oral route for a person in g/person.  Assume the LD50 of a rat or mouse will be the same as a human.  Don’t use any of the ones already listed below.  Search the MSDS’s at one of the following websites, and include the printed first page of the MSDS for the substance you have chosen.

http://www.msds.com  or  http://www.setonresourcecenter.com/MSDSs/comply1.htm  or

http://www.epa.gov/chemfact/

*Note:  These are Natural Substances

Show calculations below and/or attach another sheet showing your sample calculation(s):

Introduction (Part 2):

Over 15% of the Earth’s agricultural land is irrigated to maximize crop yields.  Irrigation water contains a variety of dissolved salts including NaCl, MgCl₂, CaCl₂, Na₂SO₄, CaSO₄, MgSO₄, Na₂CO₃, CaCO₃, and MgCO₃ among others.  These salts are leached from soil and rocks during percolation or runoff of surface water.  Groundwater can have significant amounts of salts, leached from sedimentary rocks comprising the aquifer.  When a field is irrigated, much of the water can evaporate, leaving these salts behind as a thin layer on top of the soil.  If you take a glass of tap water and leave it in the Sun until all the water evaporates, a film will be left on the glass.  These are salts.  Soil salinity can affect crop germination levels and yields.  Over time, salt builds up on fields until the soil is so salty (salinized) that seeds will no longer germinate in the soil.  Excessive salinity costs the United States billions of dollars each year.  As new land comes into use, it is often in arid areas, which are highly susceptible to the problems associated with soil salinization.

The use of a biological organism to test the toxicity of a chemical compound is termed bioassay.  In this method, it is assumed that a tested organism will react in a predictable way to increasing amounts of a particular chemical compound.  Bioassays have been used by drug companies to test new products on laboratory animals before humans.  Bioassays are also used in environmental testing.  They can determine the degree of harm to be expected from toxic soil, industrial effluents, agricultural runoffs, dredge spoils, and drilling and mining wastes, as well as testing for the effectiveness of the clean-up of a contaminated site.

In this investigation, you will perform what is called a dose-response study.  This method requires you to increase the dose of a chemical incrementally and record how the organism responds to the exposures.  For a test organism, you will use radish or lettuce seeds.  Radishes and lettuce seeds are commonly used in bioassays because their root growth, rather than just germination rate, is especially sensitive to many chemicals.  For a variety of reasons, salt solutions will be used as toxins.  Salt is inexpensive and safe to use and is a widespread environmental problem for plants.

Materials

• Radish/Lettuce seeds
• Salt water solutions (3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, 0.1%, 0.05%, 0.01%) Note: 1%=1gNaCl : 100mL H₂O
• Distilled water
• Paper towel
• Baggies
• Salt Water

Procedure (Day 1)

1. Prepare your salt solutions, then label 10 baggies with your group number and pre-selected salt concentrations.  Nine will be test solutions and one will be a control with distilled water.
2. Soak a paper towel with your first salt solution (just enough to moisten the paper towel-not dripping wet) and place in the appropriately labeled baggie.
3. Place 10 radish or lettuce seeds into the paper towel and loosely fold the paper towel over them.  Place the folded paper towel in the baggie and seal it shut.  You want to minimize the amount of air in the baggie.
4. Repeat Steps 2 and 3 for the remainder of the test solutions.  (Don’t forget your control group.)
5. Place your baggies at the back of your lab station for at least 4 days.
6. Create a data table in the space provided.  Be sure to include:

• • The type of seed you used
  • The number of seeds you started with
  • The number of seeds that did not germinate
  • The percent non-germinating seeds
  • The average length of the roots called the radicle.
  • Percent mortality (this is shown in the analysis)

Procedure (Day 5)

1. After at least 4 days, collect your data.  Count how many seeds in each bag germinated and measure the radicle in millimeters.  Be sure that you measure only the root, from the seed remnant to the tip of the root; not the shoot and beginnings of leaves.
2. Calculate the percent non-germinating. (Show one calculation.)
3. Calculate the average length of the radicle and record on data table.  (Show one calculation.)
4. Calculate the percent mortality.  (Show one calculation.)

% Mortality = [(Initial – Germinated) x 100] / Initial

Calculations:

Percent Non-Germinating:

Average Length of Roots:

Percent Mortality:

Graphing:

1. Graph the average length of the radicle versus salt solution strength.
2. Graph the percent non-germinated versus salt solution strength.
3. Graph the percent mortality versus salt solutions strength.
   1. Estimate the LD50 by drawing a horizontal line from the 50 percent mortality point on the graph to the plotted line and then a vertical line from that point to the concentration of the pollutant scale on the x-axis.  The intersecting point on your plotted line is the LD50 estimate.  Label this as using a different color.
   2. Use the same procedure from A to determine the LD25 and LD75.  Label these points using the same color you did for LD50.

Analysis/Conclusion:

1. What is meant by the term threshold of toxicity?  In what other contexts have you seen this term?
2. On your graph of percent non-germination vs. solution strength, label the Threshold of Toxicity.
3. What is meant by LD-50?  Describe some situations in which it is used.  What do the LD25 and LD50 represent?
4. Label LD50 on your graph of percent non-germination vs. solution strength.
5. Discuss three environmental effects of using sodium chloride (NaCl) on roads and highways during ice and snow storms.
6. Define salinization and describe how it affects agricultural land and developing crops.
7. What are the sources of salts on the salinized lands?
8. Discuss three specific ways that farmers can remediate the salinization of their cropland.
9. Looking at your answers to number 6, give one negative aspect of each of the remediation techniques listed above.
10. Why is it that only a certain percentage of organisms die in a given population when exposed to a certain concentration of a toxic chemical?
11. What are the variables in this experiment? (Independent, Dependent & Control)
12. What is the purpose of the baggie where only distilled water was used?  Be specific!
13. Based on your data from this lab, what is the safe concentration of salt for radish/lettuce seed germination?  What is the Lowest Observable Effect Concentration (LOEC)?  Explain your answer.
14. Often, indicator species are used to study the overall health of an ecosystem.  If you were to study an ecosystem containing radish or lettuce plants, would you use it as an indicator species?  Why or why not?  Explain your reasoning.
15. What possible sources of error were present?  How might they have specifically influenced your data and how would you resolve them in future experiments.
16. What changes would you make to this lab to advance your studies on LD50 and bioassays?
17. What changes would you make to this lab to advance your studies on salinization?