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PDF Files for PrintingLesson 2-The Dose Makes the Poison

At a Glance


Students observe beakers of water that contain different amounts of a mystery chemical. They discuss how each amount of the chemical might affect them if the chemical was beneficial or harmful to their bodies. Then, students set up investigations to test the effects of different doses of chemicals on seed germination and collect data for two consecutive days. Their investigations model the kinds of investigations toxicologists do to determine dose-response relationships in living systems.

Major Concepts

The total amount of chemical administered to, or taken by, an organism is called a dose, and the effect a chemical has on a living organism is called the response. The effect a chemical has on a living organism is related to its dose and the resultant concentration of chemical in the organism. Toxicity tests enable toxicologists to learn about responses of living organisms to doses of chemicals.


After completing this lesson, students will

Background Information

Dose, Concentration, and Threshold

The beneficial and harmful effects that a chemical has on an organism depend, in part, on the amount of the chemical that gets into the organism. The total amount of a chemical that is administered to, or taken by, the organism is called the dose. The effect of a chemical depends not only on the amount of the chemical that gets into the organism but also on the resulting concentration of the chemical in the body (the amount of chemical compared with the body size), the length of exposure to the chemical, and the route of exposure.

The measure of dose in toxicology is important; a large dose of a beneficial chemical can have a harmful effect, and a small dose of a harmful chemical can have no adverse effect. In the words of the 16th-century physician Paracelsus, "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy."1

Approximate Lethal Doses of Common Chemicals
(Calculated for a 160-lb. human from data on rats)
Chemical Lethal Dose

Sugar (sucrose)

3 quarts

Alcohol (ethyl alcohol)

3 quarts

Salt (sodium chloride)

1 quart

Herbicide (2,4-D)

1/2 cup

Arsenic (arsenic acid)

1–2 teaspoons


1/2 teaspoon

Food poison (botulism)


Source: Marczewski, A.E., and Kamrin, M. Toxicology for the citizen. Retrieved August 17, 2000, from the
World Wide Web:

A chemical is considered toxic if it produces adverse effects in a living organism at levels of exposure that are likely to occur. These adverse effects can range from slight symptoms, such as headache, nausea, or rashes, to severe symptoms, such as coma, convulsions, and death. Toxicologists recognize that, for most types of toxic responses to a chemical, there exists a dose threshold below which no toxicity is evident. As the dose increases, more severe toxic responses occur.

Toxicity Testing

How does a toxicologist know when a chemical is toxic to humans? When available, toxicologists study data from human populations that have been exposed to specific chemicals. The data usually come from studies of workplace exposure or incidents such as the Union Carbide chemical plant accident in India. In this way, toxicologists further their understanding of the effects of chemicals on humans. In the absence of human data, toxicologists test the toxicity of different doses of chemicals on cell and tissue cultures, plants, and other animals, such as rats and mice. These studies guide toxicologists in their understanding of which chemicals might be harmful to humans and in what amounts.

The use of animals in toxicology research is not taken lightly. Following is the Society of Toxicology's Animals in Research Policy Statement:

Toxicologists know that the kinds of questions they want to answer cannot always be answered by observing and describing humans exposed to chemicals.

They need to devise experiments that involve a system that resembles the human system. By studying a model of the human system, rats or mice, for example, toxicologists hope to apply the knowledge they gain to understanding the harmful effects of chemicals on humans. Although the basic tenet of toxicological studies is that "experimental results in animals, when properly qualified, are applicable to humans,"3 toxicologists recognize that different species can respond to doses of toxic substances differently. For example, on the basis of dose per unit of body surface, toxic effects in humans are usually about the same as for experimental animals; but on a body weight basis, humans are about 10 times more vulnerable than small experimental animals, such as mice.3

Because of both practicality and ethics, scientists who use animals in research carefully select the species and design experiments to achieve scientifically valid results. They obey strict regulations about the use of animals in experiments.2 Typically in these experiments, toxicologists expose experimental animals to high doses of toxic agents so that they minimize the number of animals they use. This experimental design assumes that the results of tests at high doses on a small number of animals can be extrapolated to estimate the risk of low doses to a large population of humans.

Guiding Principles in the Use of Animals in Toxicology

1. The use, care, and transportation of animals for training and for toxicological research and testing for the purpose of protecting human and animal health and the environment must comply with all applicable animal welfare laws.

2. When scientifically appropriate, alternative procedures that reduce the number of animals used, refine the use of whole animals, or replace whole animals (e.g., in vitro models, invertebrate organisms) should be considered.

3. For research requiring the use of animals, the species should be carefully selected and the number of animals kept to the minimum required to achieve scientifically valid results.

4. All reasonable steps should be taken to avoid or minimize discomfort, distress, or pain of animals.

5. Appropriate aseptic technique, anesthesia, and postoperative analgesia should be provided if a surgical procedure is required. Muscle relaxants or paralytics are not to be used in place of anesthetics.

6. Care and handling of all animals used for research purposes must be directed by veterinarians or other individuals trained and experienced in the proper care, handling, and use of the species being maintained or studied. Veterinary care is to be provided in a timely manner when needed.

7. Investigators and other personnel shall be qualified and trained appropriately for conducting procedures on living animals, including training in the proper and humane care and use of laboratory animals.

8. Protocols involving the use of animals are to be reviewed and approved by an institutional animal care and use committee before being initiated. The composition and function of the committee shall be in compliance with applicable animal welfare laws, regulations, guidelines, and policies.

9. Euthanasia shall be conducted according to the most current guidelines of the American Veterinary Medical Association (AVMA) Panel on Euthanasia or similar bodies in different countries.

Source: Society of Toxicology. March 1999. Retrieved August 14, 2000, from the World Wide Web:

Toxicity testing is not designed to demonstrate that a chemical is safe for humans, but is used to identify the types of toxic effects a chemical can produce. One early test performed on a chemical is the Ames test, named after Bruce Ames of the University of California–Berkeley. In this test, specially engineered bacteria are exposed to a chemical. If the bacteria mutate, the chemical reacted with DNA and is a potential mutagen or carcinogen. Scientists use the Ames test to economically weed out mutagenic chemicals because it avoids testing on higher animals.

Often, scientists use cell cultures in toxicology testing. Scientists expose isolated cells to a chemical and observe the response. If the cells die during the experiment, the chemical may be too toxic for use by humans. As with the Ames test, tests on cell cultures help scientists narrow the list of chemicals they need to test further on animals by eliminating those that are clearly too toxic.

If these preliminary tests suggest a chemical might be used safely with humans, scientists consider testing with animals. One of the first animal tests that scientists perform on a new chemical determines its acute toxicity. Toxicologists determine what dose of the chemical, under the intended route of exposure, causes 50 percent of the animals (mice or rats) to die (lethal dose, or LD50). Toxicologists also determine the effective concentration at which 50 percent of the animals exhibit a measurable response (EC50).

Scientists perform subacute toxicity tests to learn about the toxicity of a chemical after repeated doses. To test a chemical that is likely to enter the body through ingestion, scientists add doses of a chemical (high, low, and intermediate) to the feed for the experimental animals, usually rats or mice. Each animal receives a specified dose over the course of 90 days. Scientists observe the animals once or twice daily for signs of toxicity, including changes in body weight, diet consumption, changes in fur color or texture, respiratory or cardiovascular distress, motor and behavioral abnormalities, or palpable masses. They record premature death and collect blood and tissue samples from all animals for further study. If the chemical is likely to pose a risk to humans through skin contact or inhalation, scientists perform tests that incorporate those routes of exposure. They conduct long-term or chronic exposure studies in a similar manner, but the exposure time is increased to a time period that can range from six months to two years.

Efforts are under way to reduce the use of animals in some kinds of toxicity testing.4 For example, researchers have developed a collagen matrix barrier that serves as a kind of artificial skin. If a chemical or chemical mixture penetrates the artificial skin, it is likely to irritate, corrode, or burn human skin.

For example, in the illustration below, the drawing shows how a chemical is tested using the collagen matrix barrier. A sample of the test chemical is dropped onto the matrix. If no chemical penetrates the matrix, the solution in the bottle below the matrix remains clear. If the chemical penetrates the matrix, it will cause a color change in the solution in the bottle below. The photo on the right shows the indicator solution changing color after the test chemical has penetrated the matrix. This method, using artificial skin, can replace the current practice of using three animals to test every new chemical. Because more than 2,000 chemicals are introduced each year and many are tested before they are introduced on the market, this replacement means a significant reduction in the number of experimental animals used in toxicity testing.4

syringe expelling a drop of test chemical into a bottle of indicator solution with a collagen matrix stopper detail of indicator solution bottle
Photo: Courtesy  InVitro International

Researchers are also developing techniques that are more accurate than the traditional methods. In the past, when researchers wanted to know if a blood pressure medicine was working in an animal, they inserted a catheter into an artery in the animal's leg. The animal then needed to be restrained so that scientists could take readings over a four- to five-hour period. Today, a sensor implanted in the animal's abdominal cavity allows researchers to continually measure results while the animal can move freely and remain with its family. Its heart rate is more relaxed and normal, so the results do not mirror the compounding effect of stress.

The rate at which new technology is being used to help researchers reduce their reliance on laboratory animals is accelerating. People who are concerned about animal welfare are working with researchers to encourage better experimental design and more humane techniques. Together, they are working to replace laboratory animals with scientifically valid alternatives, reduce their numbers, and refine techniques to minimize pain and suffering.

Even as progress is made in the name of animal welfare, however, conflicting pressures arise from the public's interest in knowing more about the health and safety data on major industrial chemicals. For example, in October 1998, Vice President Al Gore announced his plan to collect data on 2,800 high-production-volume chemicals. Animal rights groups recognized that such testing would require the destruction of more than 1 million animals. For a year, to minimize the number of animals and avoid needless testing, animal welfare activists lobbied to halt or modify the plan. One year later, the U.S. Environmental Protection Agency (EPA) made new recommendations for high-production-volume chemical testing that should reduce animal use. They now will consider previous results from chemical safety databases to ensure that testing is not redundant and will postpone the testing of some chemicals in the hope that nonanimal tests will become available. To that end, the National Institute of Environmental Health Sciences (NIEHS) will invest at least $4.5 million over two years to develop and validate nonanimal protocols, and the EPA will contribute $250,000 each year for two years.5

Notes about Lesson 2

In this lesson, students perform toxicity tests on seeds, paying careful attention to the dose and concentration of chemicals. Students might not be aware that plants differ from animals in many ways: They have no nervous system or efficient circulatory system, and they have a photosynthetic mechanism and cell walls that animals do not. Therefore, the students' results from toxicity tests on seeds cannot be extrapolated to suggest a chemical's risk or safety to humans without further testing on animal systems, which is inappropriate for the classroom. However, students can understand the importance of using model systems in science when human subjects cannot be used because of the potential risk. Students can understand that many questions in science suggest a variety of investigation methods and that their use of models in scientific inquiry can help them establish relationships based on evidence from their own observations.

In Advance

Web-Based Activities
Activity Number Web Version

Activity 1


Activity 2


Activity 3


Activity 4


Extension Activity


Activity Number Master Number Number of Copies

Activity 1

Master 2.1, Opening Questions

1 transparency

Activity 2

Master 2.2, Making Solutions for Toxicity Testing

1 for each studenta

Activity 3

Master 2.3, Toxicity Testing on Seeds

1 for each student

Activity 4



Extension Activity



a If you want students to calculate percent concentration on their own, mask the numbers in the concentration column before copying.

Activity 1

For the class:

  • overhead projector
  • transparency of Master 2.1, Opening Questions
  • shoe box from Lesson 1
  • 1 small jar containing the mystery chemical from Lesson 1a
  • 1 eyedropper
  • 1 pair of safety glasses
  • 1 pair of latex gloves
  • 3 1,000-mL beakers or 3 large jars of the same size, each containing 500 mL of water
  • 1 piece of white poster board to use as a backdrop for the demonstration
  • 1 resealable plastic sandwich bag containing radish seeds
  • 1 beaker containing 250 mL of water (optional)

For each student:

  • science notebook
Activity 2

For the class:

  • Web site address
  • computer with Internet access
  • chemicals from Lesson 1
  • mystery chemical from Lesson 1
  • 1 resealable plastic sandwich bag containing radish seeds

For each student:

  • 1 copy of Master 2.2, Making Solutions for Toxicity Testing
  • science notebook
Activity 3

For each team of 3 students:

  • 3 copies of Master 2.2, Making Solutions for Toxicity Testing
  • 3 copies of Master 2.3, Toxicity Testing on Seeds
  • 3 pairs of safety glasses
  • 3 pairs of latex glovesb
  • 1 100-mL beaker filled with 50 mL of a chemical; see Preparation for Activity 3
  • 1 permanent marker
  • length of masking tape
  • 6 50-mL beakersc
  • 1 50-mL graduated cylinder
  • 1 10-mL graduated cylinder
  • 100 mL of purified water in a beaker
  • 1 eyedropper
  • 6 resealable plastic sandwich bags
  • 12 paper napkinsd
  • 60 radish seeds in a resealable plastic sandwich bag
  • 1 tray

For each student:

  • science notebook
Activity 4

For each team of 3 students:

  • bags of seeds treated with chemicals from Activity 3
  • 1 copy of Master 2.3, Toxicity Testing on Seeds, from Activity 3
  • Web version of data for Day 2 (optional; see Preparation for Activity 4)

For each student:

  • science notebook
Extension Activity

For the class:

  • computers with Internet access
  • materials for designing a bulletin board display

a The mystery chemical is the solution of blue food coloring and water used in Lesson 1.
b Check that no students are allergic to latex. If any are, assign their team a chemical that you know will not irritate the skin, such as sugar water or cola. The team members with the latex allergy then can work safely without gloves. Alternatively, they can use vinyl gloves, if available.
c Alternatively, you could use 6 clean baby-food jars or 6 test tubes set up in a rack made out of a shoe box.
d Use regular, white, one-ply napkins (12 x 11 5/8 inches, unfolded) that you can buy in bulk at the grocery store. If you use something different, test your setup to make sure that the napkins or paper towels you use can absorb 20 mL of liquid in a plastic bag.

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