Dose/Response Experiments on Lettuce Seeds

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1 Dose/Response Experiments on Lettuce Seeds Name: Date: Lab# Bioassays: Background Information on Dose/Response Experiments A bioassay involves use of a biological organism to test for chemical toxicity. Perhaps the oldest and most commonly known example is the canary in the coal mine. Traditionally, coal miners have taken caged canaries down into the mines to help ensure a safe air supply. Canaries are more sensitive than humans to methane, an odorless gas released during the mining process, so they were used to provide an advanced warning of when methane was building up to dangerous levels in the mines. If the canary died, it meant the miners should leave the mine as quickly as possible. Another sort of bioassay is used to test the effects of compounds being considered for use in drugs or skin care products. Before a chemical compound receives FDA approval as an ingredient in products for human use, it must be thoroughly tested on laboratory animals. For environmental testing, bioassays provide an integrated picture of overall toxicity of an effluent or a sample of water, sediment, or soil from a contaminated site. Fathead minnows, various aquatic invertebrates, earthworms, protozoans, and seeds all are used for bioassays of aquatic samples (see Keddy et al., 1995, for an extensive review). The idea behind these bioassays is that the test organism will react in a predictable way to various types of environmental contaminants. Several studies have compared the sensitivies of various types of seeds to common pollutants (for example, Wang and Williams, 1988; Wang, 1987; Wang, 1986). The following are a few examples of ways in which lettuce seed bioassays have been used by scientists for environmental testing purposes: to map areas for clean-up of Superfund sites (Thomas et al., 1986) to screen industrial effluents (Wang and Williams, 1988) to test the effectiveness of clean-up of lead-contaminated soil (Chang et al., 1997) to design clean-up strategies at a site contaminated by treatment of lumber with creosote and other compounds (Athey et al., 1989) Why Lettuce Seeds? Of all the possible water quality bioassay organisms, lettuce seeds might be one of the last you would think of using. Lettuce doesn't live in water, so why would it even be considered? The answer is that lettuce seed bioassays have proven to be an easy and inexpensive means of testing the toxicity of some types of contaminants of concern in water and sediments, including heavy metals and some pesticides and other organic toxicants. Lettuce seeds provide distinct advantages over most other test organisms: they are inexpensive, easy to culture, and require no upkeep between experiments. Although any variety of lettuce might work, Lactuca sativa L. var. Buttercrunch is the standard species recommended for bioassays by the U.S. Environmental Protection Agency, the Food and Drug Administration, and the Organization for Economic Cooperation and Development. Within any one species, individuals respond differently when exposed to any particular chemical. Between species, there are even greater differences in chemical sensitivities. You can test this idea by comparing your lettuce seed results with bioassays using another organism such as water fleas (Daphnia sp.). 1

2 How to start the bioassay experimental design Ask a question When you want to do an experiment, a good way to start is by asking a question. For bioassays, the questions might be "Does salt inhibit germination of lettuce seeds?" or "Is salt toxic to Daphnia?" Form your hypothesis After you decide on a research question, then you are ready to state your hypothesis or prediction of what you think will happen. An example hypothesis: "The number of seeds that germinate will decrease with increasing concentration of NaCl." Choose the variables Your independent variable is the factor that you will change in your experiment. For example, in a lettuce seed bioassay the usual independent variable is the concentration of the solution to which the seeds are exposed. The dependent variable is the factor that you predict will change as a result of variation in your independent variable. The number of seeds that germinate and the lengths of their roots are examples of two different dependent variables in lettuce seed bioassay experiments. If you are confused about the independent and dependent variables, it may help to think back to your research question and then think about how you might want to present the results of your experiment. For example, for a bioassay using Daphnia, you might decide to set up a bar graph to display your results. On the x-axis (the horizontal axis), you would put your independent variable. These are the numbers that you know in advance, such as the concentrations of your test solutions. On the y-axis (the vertical axis), you would put your dependent variable. This is the factor you will be measuring in your experiment, such as the length of the lettuce roots or the number of Daphnia that die at each concentration. Plan the treatments A treatment is a factor that affects the outcome of a scientific experiment. In a bioassay, the experimental treatment usually is the concentration of the solution to which the seeds or organisms are exposed. (If you want to test the effect of temperature on seed germination or Daphnia growth, the treatment would be temperature rather than solution concentration.) Choose a control In a scientific experiment, the control is the group that serves as a standard of comparison. It is exposed to the same conditions as the treatment groups, except for the variable being tested. In bioassays, the control group is the set of seeds or Daphnia grown in distilled or culture water rather than in a test solution. Decide the number of replicates Replicates are individuals or groups that are exposed to exactly the same conditions in an experiment. In a lettuce seed bioassay, you might test 6 different solution concentrations, plus a distilled water control. For each of these concentrations, you might use 3 Petri dishes, each containing 5 lettuce seeds, or 3 beakers, each containing 10 Daphnia. In this experiment, you would have 3 replicates at each concentration (the 3 Petri dishes or beakers). Specify the constants The constants in an experiment are the factors that do not change. What your constants will be will depend on what question you are asking. In a bioassay experiment, the temperature usually is kept constant. However, if your question were, "How does temperature affect the life span of Daphnia?" then in this case the temperature would be a variable rather than a constant. 2

3 Conducting a Reference Toxicity Test with Lettuce Seeds The idea behind a reference toxicity test is that the test organism, in this case lettuce seeds, will respond in a predictable manner to varying concentrations of a particular chemical compound. At some threshold concentration, all of the test organisms will be killed (or in this case, none of the lettuce seeds will sprout). In solutions that are more dilute, some level of inhibition will occur in seed germination and/or radicle length. If the concentration is low enough, no response will be detectable. This is called a dose/response experiment. You vary the dose of a selected compound, then measure the response of the bioassay organism. Using NaCl as the Reference Toxicity Test In order to determine whether lettuce seeds provide a good bioassay for salt toxicity, you can conduct a reference test using known concentrations of NaCl (table salt). First, make a 0.2M NaCl solution by mixing 12 g NaCl with enough deionized or distilled water to make 1 liter. This will be your 100% concentration reference solution. Second, label a series of beakers with the following concentrations: 100%, 10%, 1%, 0.1%, 0.01%, 0.001% and Control Preparing Serial Dilutions: You will need ml beakers for each environmental sample being tested. Label each from the table below before starting. From the beaker containing the 100% conc. Sample, remove 10 ml s and add to a beaker containing 90 ml of distilled water. This is your 10% test solution. From this 10% test solution beaker remove 10 ml s and add to a beaker containing 90 ml s of distilled water. This is your 1% test solution. Continue serial dilutions as shown in the table below. Concentration example Percent Concentration 12 g/l= 12,000 mg/l= 12 parts per thousand 1.2 g/l= 1,200 mg/l = 1.2 parts per thousand 100% 0.12 g/l= 120 mg/l = 120 parts per million 1% g/l = 12 mg/l = 12 parts per million 0.1% g/l = 1.2 mg/l = 1.2 parts per million g/l =.12 mg/l = 120 parts per billion 10% 0.01% 0.001% Control 0% Reference Bioassay Procedure: 1. Treat the lettuce seeds in a 10% bleach solution for 20 minutes, then rinse five times with deionized or distilled water. This kills fungal spores that can interfere with seed germination. Note: Tap water can be used if you do not have access to deionized or distilled water, but it will introduce more variability into your experiment because of the varied minerals and other compounds it contains. 3

4 2. In each of 7, petri dishes, place a piece of filter paper. Label the dishes according to the first column in the following table. Note: Absorbent paper towels or coffee filters can be substituted for the filter paper, as long as they are first shown to be nontoxic. (Bleached paper may contain dyes or chlorine.) 3. To each petri dish, add 2 ml of the appropriate test solution. In the control dishes, use deionized water as your test solution. 4. To each dish, add 5 lettuce seeds, spaced evenly on the filter paper so that they do not touch each other or the sides of the dish. 5. Place the dishes in a plastic bag and seal it to retain moisture. Incubate the seeds in the dark at constant temperature (preferably 24.5 degrees C) for 4-5 days. 6. At the end of this time, count how many seeds in each dish have germinated, and measure the root length of each to the nearest mm. Look carefully at the plants to make sure you are measuring just the root, not the shoot as well. (See Diagram, Page 6) 7. Record this data on the data sheets provided. Discuss the dose/response outcome. Using Other Toxic Compounds To be useful, a bioassay must be sensitive to the types of compound you are interested in evaluating. For example, if you are worried about herbicide contamination of ponds or streams, a bioassay based on seed germination might prove to be more sensitive than one based on death of fish or invertebrates. On the other hand, fish are likely to be much more sensitive than seeds to a compound that is a nerve toxin, for example. To determine the sensitivity of an organism to a chemical compound, scientists carry out reference toxicity tests. To do this, you measure the response of the organism to a wide range of concentrations of the selected chemical. What concentrations should you use? That of course depends on both the bioassay organism and the chemical being tested. Before scientists begin an experiment, usually they search through published scientific literature for papers that relate to the procedure they have in mind. With too high a concentration, the test organisms will all die, or in the case of seeds, none will sprout. With too low a concentration, you will not be able to detect any difference between your samples and your control. Ideally, you want to test concentrations that cover both of these endpoints plus a range of concentrations in between. Then you will be able to conclude whether your test organism responds in a predictable way to the compound you are testing. Serial dilutions are one way to set up a broad range of concentrations. For example, suppose you suspect that in a 100 mg/l solution of a selected compound, no lettuce seeds will sprout, and you are interested in narrowing this down to find out the range of concentrations in which germination will occur. You might decide to start with a 10-fold dilution series, testing solutions of 100, 10, 1, 0.1 and 0.01 mg/l. Another possibility would be a dilution series in which each solution is half the strength of the previous solution in the series: 100, 50, 25, 12.5, and 6.25 mg/l. Bioassays: Dose Response Testing with Lettuce Seeds Group 1 NaCl 12 g/l 100% solution Group 2 KCl 12 g/l 100% solution Group 3 CuSO g/l 100% solution 4

5 1. Soak lettuce seeds for 20 minutes in a 10% bleach solution (add 1 part household bleach to 9 part deionized or distilled water). Then rinse five times. This kills fungal spores that can interfere with seed germination. Note: Tap water can be used if you do not have access to deionized or distilled water, but it will introduce more variability into your experiment because of the varied minerals and other compounds it contains. 2. Place a piece of filter in each of 7 petri dishes. Add 2 ml of each serial dilution sample to each dish 3. For sediment or soil samples, place 3 grams of sample in the bottom of each petri dish and cover with filter paper. If the sample does not contain enough moisture to saturate the filter paper, add up to 2 ml deionized water as needed. 4. Prepare a control by setting up dishes using 2 ml deionized or distilled water as your test solution. 5. To each dish, add 5 lettuce seeds, spaced evenly on the filter paper so that they do not touch each other or the sides of the dish. 6. Place the dishes in a plastic bag, and seal it to retain moisture. Incubate in the dark at constant temperature (preferably 24.5 degrees C) for 5 days (120 hours). 7. At the end of this time, count how many seeds in each dish have germinated, and measure the root length of each to the nearest mm. Look carefully at the plants to make sure you are measuring just the root, not the shoot as well. Taking Measurements At the end of the growth period, count and record how many seeds in each dish have germinated. For each sprout, measure the radicle length to the nearest mm. (The radicle is the embryonic root). Look carefully at the plants to make sure you are measuring just the radicle, not the shoot as well. For example, in the picture below, you would measure just the part between the two arrows, not the shoot and cotelydons to the left. How Good are Your Data? Once you have counted how many seeds germinated, and measured their radicle lengths, then what? How can you interpret these results? Comparison to the Control The first thing to check is your control (the dishes that contain deionized or distilled water rather than a sample). The purpose of the control is to identify how well the seeds will grow without any added contaminants. Would you expect all of the seeds in your control dishes to germinate? Probably not, just like a gardener does not expect all the seeds in a garden to sprout. If fewer than 80% of the seeds in your control dishes sprouted, something may have gone wrong in your experiment. Perhaps the seeds were too old or stored improperly, so they were no longer viable. Or maybe something went wrong with the conditions for growth. Did the dishes get too hot, too dry, 5

6 or contaminated in some way? Did you use tap water for your control, rather than deionized or distilled water? In many cases this works fine, but since tap water is highly variable from source to source, it gives less predictable results. Sources of Variability Within each treatment, how much variability did you find in your results? Did the replicate dishes show similar numbers of seeds sprouting, and similar average radicle lengths? If you think the data are much more variable than you would expect, you might want to explore the potential sources of variability for this type of experiment. There are many sources of variability inherent in lettuce seed bioassays. The following table outlines a few of these sources and how they might affect your experiments. Potential Sources of Variability Viability (health) of the seeds Definition of "germination" Precision of measurement Bias Questions to Ask What percentage of the seeds will sprout under ideal conditions? Does everyone agree on how germination is defined? If a seed sprouts but has no distinct radicle that can be measured, do you count it as having germinated or not? If several people measure the same radicle, do they come up with exactly the same measurement? Is there greater variability in the data if several people take the measurements, compared with having them all done by one person? Are you tempted to choose the best looking seeds for your control, and smaller seeds for treatments that you don't expect to grow much anyway? If you knew you would get a higher grade if your data indicated clear differences between the control and the treatments, would this affect how you selected which seeds to put in each dish? Points to Consider If fewer than 80% sprout in your control, you may have a problem with your seeds or growing conditions. This is the kind of decision that each experimenter can make, but in order to compare the results of your experiments with those of other scientists, you want to clearly state what decisions you have made. Judgement is often important in scientific measurements. At what exact point do you start measuring the radicle, and how tightly do you stretch it while measuring its length? Would everyone make the same decisions? Would you do it the same way every time? Scientists continually have to be on the lookout for sources of bias in their experiments. They are much more likely to get their results published and to continue receiving funding if they get good results from their experiments. Why do you think scientists might not want to be biased, in spite of these pressures? 6

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12 Name Some questions to consider: (Please answer on separate paper using full sentences.) 1. Did at least 80% of the seeds in the control dishes germinate? If not, what would you recommend doing differently next time to try to get a better germination rate? 2. Did lettuce seed germination respond in a predictable way to concentration? Describe any trends you observed. 3. Does any of your data not fit the trends you observed? If so, can you think of any reasons why these data might lie outside the range you would expect? Note: In bioassays there are two ways to reports results: TC50, the toxic concentration that causes organisms to grow 50% as well as the control LC50, the lethal concentration that kills 50% of the test organisms 4. What is your estimate of the TC50 based on your lettuce seed germination data? TC50 = What is your estimate of the TC50 based on your radicle length data? TC50 = Which shows a greater response to the chemical you tested: germination rate or radicle length? Describe any similarities or differences that you noticed in trends between these two indicators of toxicity. 5. What can you conclude about the toxicity of the substance you tested? Is this what you expected? Was your hypothesis supported by the data? 6. If other students carried out a dose/response experiment using a different chemical, did their data follow the same trends as yours? How would you rate the relative toxicities of the different chemicals tested? 7. Based on this experiment, would you say that lettuce seed germination or root length would provide a useful bioassay for water samples from the environment? Why or why not? 8. If you were going to repeat this experiment, what would you do differently? How might you improve the experimental design to reduce the variability of your data or lead to more reliable results? excerpted from: