LD50 lab

Ant and Roach Killer-safe for cats? (Erica Brauer)

Author

Erica Brauer, Granada Hills Charter High School, California State University Northridge

(Lab adapted from Dan Hyke)

Principles

lethal dose limits, applications to wildlife and humans

Standards

Include the science standards which are addressed by this equipment. If no specific content stards are addressed, use the investigation and experimentation standards.

Materials needed

List any other materials that are necessary or helpful when using this demonstration device

Procedure

PROCEDURE:

First, obtain 7 plastic soufflé containers and a water soluble marker.

1. Label the 7 containers as follows: Control, 100ppm, 10ppm, 1ppm, 100ppb, 10ppb, 1ppb.

2. Add 45ml of pond water to the Control and 45ml of pond water to the other six containers.

3. Using a 10ml graduated cylinder and an eyedropper pipet (both of which have been rinsed in distilled water), carefully transfer 5ml of the copper sulfate (1,000 ppm) stock solution to the soufflé container labeled 100ppm. Stir gently.

4. Carefully rinse the glassware and then transfer 5ml of the 100ppm container to the container marked 10ppm. Stir gently.

5. Complete the serial dilution for the other four containers. (The solutions should be prepared before the Daphnia arrive. Cover the solutions if they are to be left out overnight.)

6. Using an eyedropper pipet, transfer 10 viable Daphnia magna into each of the plastic containers. Try to pick Daphnia of a consistent size.

7. Expose the Daphnia to the experimental conditions for six to twenty-four hours depending on what is most convenient.

8. After the exposure, carefully count the Daphnia to determine the percent mortality.

9. Graph the mortality rate on six-cycle semi-log graphing paper. Use the log axis for plotting the concentrations of copper sulfate and the “normal” axis for plotting the percent mortality.

10. The LC₅₀ can be determined by following the 50% mortality line over to where it intersects the mortality curve. The intersection will be your LC₅₀ concentration.

Explanation

PURPOSE: To determine that concentration of a ant and roach spray that will kill fifty percent of a given population of Daphnia magna. In short: to find the ant and roach - LC₅₀ for Daphnia magna.

BACKGROUND: 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, bioassay techniques are often used to evaluate the direct lethal effects of substances on organisms. A bioassayis a test that uses living organisms as an indicator. A common measure of acute (lethal) toxicity is called the LC₅₀ (lethal concentration 50). LD₅₀ is also used but refers to when a toxin is ingested by an organisms as opposed to being absorbed through its skin or outer membrane. 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. Since it is illegal for high schools to use vertebrate animals for toxicology testing, we will be using a small invertebrate species called Daphnia magna commonly known as the water flea. Daphnia magna is the largest member of the genus Daphnia and is good for this experiment because it is easy to see and count. Daphnia are crustaceans in the suborder Cladocera.

Teacher Notes Concerning Daphnia magna: This is one of the best organisms to use for this experiment. Another organism that can be used is the California blackworm which is far less expensive than Daphnia magna and can be purchased at your local aquarium store. Some toxicology labs suggest using brine shrimp, but I have found that they have a high mortality rate and that it is a hassle making sure that the correct concentration of salt water is used in all test conditions.

Daphnia magna can be ordered through your favorite supply house. Make sure they arrive the day before you plan on starting the lab. Under good conditions they will last for several days. Their mortality rate tends to go up if the water they are in gets too warm, so keep them in a cool area (20 to 5 degrees Celsius). I store them in my refrigerator until I am ready to use them. Do not place them in direct sunlight. Make sure to open their shipping containers to the air as soon as they arrive, but do not aerate their water with an air pump (they get caught in the air bubbles and die).

Daphnia are excellent organisms to use for this lab since they are animals that live in the water. They readily absorb most chemicals that are in the water with them. Therefore, it is important not to use tap water (because of the chlorine) or distilled water (because it will upset their osmotic balance) during this experiment.

Ideally, you will use the exact same water that they were cultured in for your experimental conditions. If you ask, sometimes suppliers will send you an extra bag of the same water that the Daphnia were cultured in. If not, you will need to get some pond water or spring water and then filter it using a fine mesh screen or cloth.

Always make sure that before you transfer Daphnia from one water media to another water media, that both have been brought to the same temperature. Otherwise, your Daphnia may undergo thermal shock and die. The easiest way to do this, of course, is to just leave the Daphnia along with all the sources of water that will be used during the lab in the same room together for a few hours.

If you plan on using your Daphnia within a day or two, you will not need to feed them, but if you plan on trying to keep them alive for several weeks or if you plan on culturing them, then they will need to be fed. Daphnia eat bacteria, protists, and unicellular algae.

You can feed them by taking a tiny piece of yolk from a hard- boiled egg, mashing it up, and then dropping it into their water. This will start the vigorous bacterial growth on which Daphnia feed. Caution: Do not add too much egg yolk. Excessive bacterial growth will kill the Daphnia. You may want to start several cultures to ensure better survival of your Daphnia. They may also be fed them finely powdered algae. Do not clean out the debris that accumulates on the bottom of the containers.

Questions

1. What is your lab groups LD50?
2. What is the class LD50?
3. Convert from ppm or ppb to mg/ml.
4. Is it likely that your ant and roach spray killed the cat?
5. How much roach spray would the cat need to ingest for it to have a 50 % chance at dying?

Everyday examples of the principles illustrated

Your neighbor accuses you of killing her cat. She saw you using raid ant and roach spray in and around your house, and then she found her cat dead. She claims that you poisoned her cat. How can we demonstrate that this is unlikely?