Materials Included In Kit

Additional Materials Required

Prelab Preparation

Prepare a viable culture of living brine shrimp. Do this several days prior to the laboratory testing. Hatch the brine shrimp in numerous small containers instead of one large container if an aerator is not available. A small, clean fish bowl with an aerator works very well. Brine shrimp must be hatched in a clean container (e.g., rinsed completely with deionized water of any chemicals, soap, residues). Hatch the cysts only in the hatching salt solution provided in the kit. If all of the cysts are used at one time, thousands of brine shrimp will hatch all at once! When the hatch occurs, conduct the lab within several days.

To mix the hatching salt solution, add 36 grams of the salts to one liter of deionized or distilled water. Add the brine shrimp cysts to the hatching salt solution in the aerated container using approximately 1–2 grams of brine shrimp cysts per liter of salt solution. Make enough of the hatching salt solution to accommodate the number of student groups completing the lab. (Each group will use 50–100 mL of solution.)

Safety Precautions

Please review current Safety Data Sheets for additional safety, handling and disposal information. Read the hazards on the labels of the test chemicals and follow all safety precautions. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Be sure to read all of the precautions on the labels of the tested products as well as the suggested disposal procedures. Most will be disposed of following Flinn Suggested Disposal Method #26b.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Adequate time should be allowed for student experimentation. This will likely involve several successive class periods at a minimum.

• The brine shrimp should be tested soon after hatching. Dispense brine shrimp into smaller containers for each lab group prior to the laboratory. Do not have students work from your main brine shrimp culture. It might become contaminated with solutions and ruin the entire population.
• Be sure that the pipets used to transfer the brine shrimp are not used for other parts of the laboratory. Use the large pipets provided in the kit for transferring the brine shrimp.
• Cut the ends off the transfer pipets if the openings are too small to retrieve brine shrimp.
• The LD₅₀ assay does involve killing brine shrimp. There is a reasonable controversy over the use of animals for such purposes and some people have ethical and religious objections to using even “lower” organisms for testing. A pre- and post-lab discussion of these issues might be appropriate.
• Though the results will vary a great deal in this laboratory, students will be amazed at the sensitivity of brine shrimp to common chemicals and appreciate what a good biological indicator brine shrimp can be. Some products will be clearly more toxic to brine shrimp than others and some products will not be lethal even at very high doses.
• Determining the ppm of the chemicals surrounding the brine shrimp in the Petri dishes can be a challenge for students and they will need your assistance. For example, if 1 mL of a 50 ppm chemical solution is added to 50 mL of brine shrimp environmental solution, the net result would be a solution around the brine shrimp of approximately 1 ppm. The addition of another mL of chemical solution would raise the level to 2 ppm, etc.
  The labels on household chemicals will vary in how the concentrations of chemicals are listed. Usually they will not be listed in ppm but rather as a percentage concentration. A 1% solution, for example, represents 10,000 ppm. (10,000/1,000,000 = 1%). If very low ppm are desired, the chemicals will have to be diluted with large volumes of distilled water or by using a serial dilution scheme.
• This laboratory project can be a stimulus for many student-designed experiments. Be a facilitator and encourage student exploration.
• If you have any difficulty growing brine shrimp, your local aquarium shop is a good place to get help with your culture techniques. Aquarium shops typically grow brine shrimp by the thousands or millions to feed to their tropical fish.

Introduction

What is LD₅₀? How is it determined and what does it mean?

Concepts

• Toxicity

• LD₅₀

Background

Any substance can be harmful to living organisms. However, it is the dose that frequently determines the extent of the damage. Toxicologists have developed several tools to determine the toxicity of chemicals. Acute toxicity is the immediate killing effect of a substance from a single dose and is relatively easy to study. Chronic toxicity results from low doses repeated over long periods of time and is much more difficult to test.

Toxicologists use LD₅₀ values to estimate the acute toxicity of chemicals on humans. LD means the lethal dose and is expressed in milligrams of chemical per kilogram of body weight. The 50 in LD₅₀ represents a mortality rate of 50% of the test animals in a toxicity study. LD₅₀ values are calculated from dose–mortality curves. A typical statement reads as follows:

Aniline LD₅₀ oral–rat: 250 mg/kg

The above statement means that an oral dose of 250 mg of aniline per kg of body weight will kill 50% of the test sample of rats.

It should be noted that no LD₅₀ data exists for humans (for obvious reasons). Data from test animals is used to estimate the acute toxicity of chemicals on humans. Toxicity data should be used to understand the relative toxicity of chemicals and serve as a guide to asses relative danger in handling certain chemicals. In this experiment brine shrimp will be used. Brine shrimp are aquatic and their mass difficult to determine. They are extremely sensitive to very small amounts of chemicals. Thus, the concentration (in parts per million) of chemicals will be used in an attempt to rank chemicals in order of their relative toxicity to illustrate the LD₅₀ concept. Which chemical is potentially more dangerous to humans?

Benzoin: LD₅₀ oral–rat: 10,000 mg/kg
Acetaldehyde: LD₅₀ oral–rat: 661 mg/kg

Materials

Safety Precautions

Read the hazards on the labels of the test chemicals and follow all safety precautions. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Your teacher will help organize your research team and assign test chemicals to your team.
2. Study the chemical content labels on a variety of household chemicals. With your teacher’s help, determine how to approximate ppm of the active ingredient(s). (This usually involves adding a prescribed amount of the chemical to a measured amount of distilled water.)
3. Using a large transfer pipet, place 10 hatched brine shrimp into a Petri dish filled with ocean salt solution.
4. Test the effect of adding enough of the household chemical to achieve a 1 ppm concentration in the Petri dish. Stir the contents of the Petri dish and observe the brine shrimp for about two minutes.
5. If no major changes are observed, add more test chemical until 10 ppm is achieved in the Petri dish. Again, observe for two minutes.
6. Continue adding test chemical, stirring and observing, until an effect is noted in the activity or death of the brine shrimp. Carefully continue to add chemical to the Petri dish until an LD₅₀ value for the chemical can be determined (when half the brine shrimp die).
7. Record the LD₅₀ amounts for the various test chemicals. Pool the class results and discuss the findings. Discuss the sensitivity of brine shrimp to the various chemicals and what the LD₅₀ numbers represent. What, if any, inferences are there for human sensitivities?
8. Consult your instructor for appropriate disposal procedures.