Toxicity and LD50and LC50 Lab Design Bodas APES Background: from OSHAhttp://www.ccohs.ca/oshanswers/chemicals/ld50.html What does LD50 mean? LD stands for "Lethal Dose". LD50 is the amount of a material, given all at once, which causes the death of 50% (one half) of a group of test animals. The LD50 is one way to measure the short-term poisoning potential (acute toxicity) of a material. Toxicologists can use many kinds of animals but most often testing is done with rats and mice. It is usually expressed as the amount of chemical administered (e.g., milligrams) per 100 grams (for smaller animals) or per kilogram (for bigger test subjects) of the body weight of the test animal. The LD 50 can be found for any route of entry or administration but dermal (applied to the skin) and oral (given by mouth) administration methods are the most common. What does LC50 mean? LC stands for "Lethal Concentration". LC values usually refer to the concentration of a chemical in air but in environmental studies it can also mean the concentration of a chemical in water. For inhalation experiments, the concentration of the chemical in air that kills 50% of the test animals in a given time (usually four hours) is the LC50 value. Why study LD50's? Chemicals can have a wide range of effects on our health. Depending on how the chemical will be used, many kinds of toxicity tests may be required. Since different chemicals cause different toxic effects, comparing the toxicity of one with another is hard. We could measure the amount of a chemical that causes kidney damage, for example, but not all chemicals will damage the kidney. We could say that nerve damage is observed when 10 grams of chemical A is administered, and kidney damage is observed when 10 grams of chemical B is administered. However, this information does not tell us if A or B is more toxic because we do not know which damage is more critical or harmful. Therefore, to compare the toxic potency or intensity of different chemicals, researchers must measure the same effect. One way is to carry out lethality testing (the LD50 tests) by measuring how much of a chemical is required to cause death. This type of test is also referred to as a "quantal" test because it is measures an effect that "occurs" or "does not occur". Why are LD50 and LC50 values a measure of acute toxicity? Acute toxicity is the ability of a chemical to cause ill effects relatively soon after one oral administration or a 4-hour exposure to a chemical in air. "Relatively soon" is usually defined as a period of minutes, hours (up to 24) or days (up to about 2 weeks) but rarely longer. How are LD/LC50 tests done? In nearly all cases, LD50 tests are performed using a pure form of the chemical. Mixtures are rarely studied. The chemical may be given to the animals by mouth (oral); by applying on the skin (dermal); by injection at sites such as the blood veins (i.v.- intravenous), muscles (i.m. - intramuscular) or into the abdominal cavity (i.p. - intraperitoneal). The LD50 value obtained at the end of the experiment is identified as the LD50 (oral), LD50 (skin), LD50 (i.v.), etc., as appropriate. Researchers can do the test with any animal species but they use rats or mice most often. Other species include dogs, hamsters, cats, guinea-pigs, rabbits, and monkeys. In each case, the LD50 value is expressed as the weight of chemical administered per kilogram body weight of the animal and it states the test animal used and route of exposure or administration; e.g., LD50 (oral, rat) - 5 mg/kg, LD50 (skin, rabbit) - 5 g/kg. So, the example "LD50 (oral, rat) 5 mg/kg" means that 5 milligrams of that chemical for every 1 kilogram body weight of the rat, when administered in one dose by mouth, causes the death of 50% of the test group. If the lethal effects from breathing a compound are to be tested, the chemical (usually a gas or vapour) is first mixed in a known concentration in a special air chamber where the test animals will be placed. This concentration is usually quoted as parts per million (ppm) or milligrams per cubic metre (mg/m3). In these experiments, the concentration that kills 50% of the animals is called an LC50 (Lethal Concentration 50) rather than an LD50. When an LC50 value is reported, it should also state the kind of test animal studied and the duration of the exposure, e.g., LC50 (rat) - 1000 ppm/ 4 hr or LC50 (mouse) - 5mg/m3/ 2hr. Answer the following questions below after reading the introductory information. Common Name(s): DL-Ephedrine Approved Chemical Name 1. Define LD50 and differentiate it from LC50. 2. Why would a scientist or a person from the general public want Structure: to know about the LD50? 3. What is acute toxicity? 4. How is a LD50 or LC50 designed in general? 5. Assume you get hired out of college at an environmental science company named ToxoBodii that specializes in toxicological testing and chemical analysis of new products. A company comes to ToxoBodii with a new diet supplement called “Infinity” that contains the compound ephedra (ephedrine). Write a brief lab protocol for testing the toxicity of this new diet supplement, and develop a working hypothesis. 6. List some of the materials you would need to conduct your experiment. 7. Do you think that humans would be precluded as experimental subjects? Why or why not? Scenario A colleague of yours at ToxoBodii is testing a new pH based insecticide product in terms of its efficacy and toxicity. Your colleague at work asks you to work with her and conduct some of the data analysis. One of the major ingredients of this “new” insecticide with the marketing name of “H-Bomb” is Ammonium Hydroxide. Here is a copy of your colleague’s data and lab work so far using crickets as the experimental model to test the new “H-Bomb” product. Purpose : To determine the LD-50 of “H-Bomb” and analyze its effectiveness as marketable insecticide. Procedure: 1. Make one of the assigned ammonia dilutions: 2. Obtain a 1000 mL beaker and add 10 healthy crickets to the beaker. 3. Place 10 drops of the dilution on a cotton ball. 4. Place a cotton ball in the beaker. CAUTION – do not allow any liquid to drip directly onto the crickets – we’re testing the fumes, not direct touch. 5. Cover the beaker snugly with aluminum foil. 6. After 30 minutes, record the # of dead crickets. 7. Make, and record, observations of the living crickets. Dilution (Concentration) 100% 10% 1% 0.1% 0.01% 0% Amount of ammonium hydroxide (ml) 100 10 1 0.10 0.010 0 Amount of distilled water (mL) 0 90 99 99.9 99.99 100 Data Dilution # of dead crickets % dead crickets Observations of living crickets 100 9/10 All but one cricket appears dead.. Lone cricket struggling 10 7/10 Crickets showing signs of slowing/stress. Many mortalities 1 3/10 3 apparent cricket mortalities, 7 still alive, unusual abdomen movements 0.1 1/10 0.01 1/10 Normal activity overall, one cricket appears deceased due to labored respiration? Normal activity overall, one cricket appears to be moving much slower 0 0/10 Normal activity, crickets moving about, making cricket sounds Your job is to do as follows!!! 8. Calculate the % of dead crickets for the “H-Bomb” experiment. 9. What variable was being manipulated in this lab? What variables were being controlled? 10. What was the control set up for this experiment? Explain 11. Graph the % dead vs the dilution concentration. Watch your scale you range includes .01-100! Use semi-log paper. 12. Determine the LC50 from your graph. 13. Graph the % dead vs the amount in (ml) of Ammonium Hydroxide. 14. Determine the LD50 from your graph. Watch your scale you range includes 0, 0.010, 0.10-100! Use semi-log paper. 15. Analyze your data/graphs and relate what they say by explaining how this data supports or refutes “HBomb”’’s effectiveness and toxicity? 16. Would this lab work for all chemicals? Justify your answer. 17. Should this data be extrapolated directly to snails? Humans? Why or why not? 18. List a few non-toxic alternatives or strategies that might work well in lieu of using “H-Bomb”. 19. Are you in favor of animal model based testing? Why or why not? 20. If the compound in “H-Bomb” was also found to have anti-cancer properties (like a chemotherapy agent), and it could perhaps be used a treatment for someone in your family in the future, would that alter your response to question #19? Explain. 21. Write a one paragraph/abstract of the “H-Bomb” experiment to pass on to your colleague. Be sure to label both the X and Y axis. Be sure to give your graphs appropriate titles. Remember that your are plotting on Semi-Log graph paper (see the back for directions) Be sure to include any appropriate units!!!! Remember each dark vertical line is ten times the previous dark line!!! Graph the concentrations and amounts on the X axis!