To expose Brassica rapa seeds to varying concentrations of a toxicant.
This lesson is part of a three-part series on toxicology, the scientific study of poisons and their affect on biological systems. These lessons are based on Toxicology Enrichment Materials developed by Suzanne Conklin and found on the Society of Toxicology’s website.
In the first lesson, Toxicology 1: Toxicology and Living Systems, students are introduced to the basic concepts and terminology of the science. Toxicology 2: Finding the Toxic Dose allows students the opportunity to conduct a toxicology experiment on a plant. Specifically, students determine the toxic dose of a chemical that will inhibit seed germination in Brassica rapa, a relative to cabbages and mustards. In the third lesson, Toxicology 3: Toxicology and Human Health, students investigate the effect of environmental tobacco smoke on human lung development. These lessons can be done in a series or they can stand alone.
In the United States, thousands of chemicals are consumed and utilized in everyday items, such as food, personal care products, prescription drugs, household cleaners, and garden/lawn care products. The effects of many of these chemicals on humans are unknown. Moreover, our use and disposal of these products ultimately contaminates our planet’s soil, water, and air. Safeguarding public health and the quality of the environment depends on identifying the effects of these chemicals and the levels of exposure at which they may become hazardous.
As high-school students study the flow of matter and energy in natural systems, they develop an understanding that important molecules and elements are continually recycled. By studying toxicology, students add to this understanding by examining that hazardous chemicals in natural systems also are passed along the food chain. Thus, an herbicide used to kill weeds or a pesticide used to kill insects are not isolated to their target organisms. We are, in fact, all connected on this planet to each other and the risk to our own health must always be weighed against the benefit attained by using hazardous chemicals.
This lesson is an experimental hands-on activity in which students determine the toxic dose of a chemical that will inhibit seed germination in Brassica rapa, a relative of cabbages and mustards. Neither the teacher nor the students are provided with a set procedure to conduct the investigation. Rather, the lesson provides a set of guidelines forcing students to brainstorm ideas, contribute their thoughts, and determine through dialogue with their peers how a hypothesis can be tested. Thus, through this activity, students will gain an understanding of toxicology as well as the basic principles of “doing”
This simple activity allows students to conduct their own investigation in small groups of two to three. Therefore, before beginning this activity, students should be familiar with group work and the principles of scientific investigation, including hypothesis generation, describing procedures, conducting an experiment, collecting data, recording results, and generating conclusions.
Students are allowed to choose their own chemical to deduce its toxicity to a germinating seed. Students are responsible for obtaining most of the readily available materials. The only materials that you should provide are the seeds and measuring instruments such as rulers, graduated cylinders, and beakers for the serial dilutions. Standard Brassica rapa seeds can be purchased from Carolina Biological Supply Company.
The experiment takes a small amount of space and it takes a limited time over the course of one week. Simple algebraic principles are emphasized when students make serial dilutions to establish a dose range. Students also should have a basic understanding of plant germination and seed anatomy (e.g., radicle, hypocotyls, cotyledon, root hairs). Review this terminology before beginning this lesson.
If available, use a digital or Polaroid camera to take pictures of students’ results. Ensure that students have all their samples labeled and then line them up to take a picture. Students can include these pictures in their lab reports.
Information about Brassica rapa and other rapid cycling plants can be found at the official Fast Plants website. The site offers ordering information, growing instructions, instructional materials, online resources, and a picture gallery.
Provide students with the Plant Bioassays student sheet. Have the class read the student sheet and examine the pictures. Based on this reading, ask students these questions:
- Why is sample soil taken from both areas suspected of having herbicides and areas free of herbicide?
- (The soil from areas free of herbicide serves as a control in the experiment. By having a control, it can be deduced that any lack of growth observed by the experimental plants is due to the herbicide in the soil and not anything else.)
- Why do you think soil samples are taken from the upper surface?
- (Most residual herbicides are bound in the upper 5 cm or 2 inches of the soil.)
- Why are several samples taken from the same area and combined rather than just one?
- (This reduces variability in the experimental soil sample.)
- Why should different samples be kept apart when drying?
- (To avoid cross contamination.)
- A plant toxicologist does a bioassay using oat seeds on sample soil suspected of having residues of an unknown herbicide. It is found that in the experimental samples, oat growth is stunted as compared to the controls. What conclusions can be drawn from these results?
- (The experimental soil samples have an herbicide that is affecting the growth of oat plants.)
- What group of herbicides do you suspect is in the sample soil?
- (The sample soil probably has either residues of Group 3 or Group 5 herbicides. To determine the exact group, the experiment should be repeated with oat, green foxtail, and cucumber. If both the oat and green foxtail is affected, but not the cucumber, we can deduce that the residues belong to Group 3. If oat and cucumber are affected according to the description in Table 1 but not green foxtail, we can deduce that the residues belong to Group 5 herbicides. If all three plants are affected, the residues might contain both groups of herbicides.)
- Why are three or four pots made for one sample?
- (This reduces variability in plant growth.)
- Why should assay pots contain no drainage holes at the bottom?
- (To prevent the leaching of herbicide chemicals.)
- Why are 10 to 15 seeds planted in each sample pot?
- (To reduce variability.)
- Why are pots also made using sterilized, herbicide-free potting soil?
- (This is another laboratory control and is used as a further check for the experimental plants.)
- Describe the results of the faba bean bioassay in Figure 2.
- (As the concentration of picloram in the soil increases, faba bean growth is stunted. The plant becomes less green, indicating that there is less chlorophyll for photosynthesis, the leaves become thinner and begin to curl over on themselves, and at 62.5 ppb, the stem begins to bend and twist.)
- At what chemical concentration do you think the plant starts to show the toxic symptoms?
- (1.9 ppb.)
- What would a dose-response curve for this plant look like?
- (Students should draw a sigmoid curve with dose increasing on the x-axis and extent of response on the y-axis.)
- Extrapolate the dose-response curve past 250 ppb of picloram.
- (The most negative response will be death of plant after which any further concentration increase in the soil will produce no worse effects. This is reflected in a dose-response curve by a leveling off.)
- Toxicologists would say that a bioassay looks at the sublethal effect of chemicals on plant growth and development. What do you think is meant by sublethal effects?
- (The impact of a toxic chemical on plant growth and development at a concentration that does not kill the plant.)
- Why do you think toxicologists study and examine the sublethal effects of toxic chemicals on living organisms?
- (It is very easy to understand that a large dose of a toxic chemical will cause the death of the organism. However, lower concentrations of a toxic chemical may not be safe either. Sublethal concentrations of a toxic chemical may impair growth, development, reproduction, and other normal functioning of an organism. Without the ability to function properly, survival is often temporary. Therefore, sublethal effects of pollutants and other toxic chemicals may be as important in the long run as lethal doses in modifying populations and balances within an ecosystem.)
Tell students that they will work in groups of two to three to determine the toxic dose of a chemical on seed germination. The plant they will study is Brassica rapa. Provide students with the Find the Toxic Dose student sheet. Go over all the directions with the students and set a deadline for the experiment.
Divide students into their laboratory groups and provide each student with Designing Your Toxicity Experiment student sheet. Allow students time to brainstorm with their group partners, decide upon a chemical they would like to test, and outline a procedure for the investigation. After students are done brainstorming, have one student from each group present their ideas to the rest of the class as well as the chemical they would like to test. Encourage students to ask each other questions, clarifying procedures and testing methods. Use guiding questions, such as:
- What is your control?
- Why are you using a control in this experiment?
- How many seeds will you use?
- Why are we using serial dilutions of the chemical?
- Are we examining lethal or sublethal effects of the chemical?
- Do you expect all seeds to react the same way to the same dose of chemical? Why or why not?
- What data are you going to collect?
- How will you collect this data?
After all groups have presented, allow students to regroup with their lab partners and make changes to their procedures as necessary.
Tell students that they must bring their own supplies and materials, with the exception of the seeds, graduated cylinders, beakers, rulers, and any other supplies that you can provide. Allow students at least two days to gather all the necessary materials.
As a class, go over the procedure for making serial dilutions. Then allow students to begin their investigations. It should take students one to two class periods to set up the experiment. Ensure that students develop a schedule for daily observations and data collection. The seeds will germinate within three to five days. If available, use a camera to take pictures of the students’ results. They can include these pictures in their lab reports.
As a class, discuss the results of each group’s experiment. Ask students:
- How do you think the plant was absorbing the toxicant? (Toxicants are absorbed through the root system of the plant.)
- Can the results of your experiment be applied to all plants? Why or why not? (Not directly. The results can only be extrapolated to be similar if the two plants are similar to one another. Some plants may not absorb the toxicant at all, others might require a smaller concentration for a toxic dose, while others may be more resilient to the chemical and show toxic effects at a much higher concentration.)
- Our test was conducted on seeds and examined germination. Do you think your results would have been different if you tested the chemical on growing plants? Adult plants? Why or why not? (Answers will vary.)
- Was your experiment examining acute or chronic toxicity? (Acute toxicity – one dose delivered at a single time.)
- How could you redesign your experiment to examine chronic toxicity? (Small doses would have to be delivered over a long period of time.)
- From your results, can you determine at what chemical concentration your seeds began to show the effects of a toxic dose? (This will depend on students’ results.)
- How can you graph your data? (Students should practice making dose-response curves with increasing chemical concentration on the x-axis and labeled responses on the y-axis. Students should also include a descriptive title.)
Have students write up their investigation in the form of a scientific paper. Provide students with the Finding the Toxic Dose: Presenting Results student sheet as a set of guidelines in writing their paper. Their papers should include these elements:
- Introduction (purpose, choice of chemical, dose range, information hoped to gain from the investigation)
- Materials and Methods (source of chemical, treatment, solutions, seeds, containers, wicks, etc.)
- Results (organized presentation of all observations, graphs, tables, drawings)
- Discussion (conclusions, significance of results, proposal of further experiments)
Toxicity Testing in Brine Shrimp offers a middle- and high-school level lesson on toxicity testing in brine shrimp. The short activity requires a maximum of two, 60-minute class periods. Students work in teams and examine the toxic effects of household chemicals on adult brine shrimp. The lab includes background information on brine shrimp and toxicology, a list of materials, procedures, a student worksheet, and discussion questions.
Charles Drewes, in the Department of Zoology and Genetics at Iowa State University, has published numerous articles and activities to promote the use of California Blackworms in the pre-college science classroom. These publications can be viewed online:
- Lumbriculus variegatus: A Biology Profile provides background information about the taxonomy, lifestyle, reproduction, muscle, circulation, and behavior of California Blackworms.
- Biological Smoke Detectors is a useful and informative online, toxicology mini-manual, or primer, for students and teachers. This manual gives background information and ideas for using invertebrates (e.g., Lumbriculus and earthworms) for ecotoxicity testing and is designed to assist student research or science fair projects.
- Through a Looking Glass is an inquiry-based lab exercise that allows viewing of the internal and external features and functions in whole worms, Lumbriculus, or worm fragments. The lab requires the use of commercially available, flat-tipped culture tubes.