Understanding The Problem
Most of your students have heard something about ozone depletion and know that ozone (O3) protects us from dangerous ultraviolet radiation. At the start of this unit, you should make clear that the same ozone that protects us many miles above the planet can be extremely harmful when it appears on the ground as part of human-made smog. In Tropospheric Ozone you will find background material on the structure, formation, and effects of stratospheric ("good") and tropospheric ("bad") ozone. Make sure that your students understand that tropospheric ozone is a result of photochemical reactions between sunlight and the products of combustion (from car exhaust, fires, etc.).
Many large cities with high ozone levels have established expensive monitoring stations for physico-chemical measuring of ozone levels. But ozone can easily travel away from these areas into surrounding rural areas that are not adequately monitored. In addition, many countries have little capacity for ozone monitoring at all. The use of bioindicators - specific living organisms that exhibit an easily measureable response when exposed to low levels of ozone - is a solution to this world-wide need.1
1 Another inexpensive device for monitoring ozone levels is the TERC ozonometer. These methods can be used together for greater accuracy.
Why Use Tobacco As An Ozone Bioindicator?
Tobacco (Nicotiana tabacum L.) is a particularly good bioindicator for the presence of ozone because:
· its response to ozone is highly visible and easily measurable by students.
· one variety, Bel-W3, visibly responds to very low concentrations of ozone.
· another variety, Bel-B, visibly responds only to higher levels.
Together, these varities can be used as a simple scale.
How Ozone Harms Plants
Ozone enters leaves through the stomata during the normal exchange of gases between the plant and the atmosphere. Once inside, the reactive ozone binds to the plasma membranes of cells, altering their metabolism, reducing the levels of chlorophyll, and eventually inhibiting photosynthesis. A large enough exposure will result in colored marks and dried out lesions on the leaves (see sidebar). Smaller exposures may not cause such obvious marks yet still will weaken plants.
A plant that exhibits a visible injury when exposed to dangerous concentrations of ozone can be used as an ozone-indicator plant, signaling dangerous levels of pollution.
Why We Care --- Boris and Luda Berenfeld
At an International Conference on Ecological Indicators in October, 1990, in Fort Lauderdale, Florida, we met two young scientists from Mexico City who used tobacco plants as air pollution monitors (especially atmospheric ozone monitors). After having read about such work, we finally had a chance to speak with people for whom measuring ozone levels was not a laboratory exercise, but a real-life problem. Their job was to estimate how polluted their native city is in order to warn people about the high level of atmospheric ozone - one of the most dangerous components of smog.
We saw pictures of their ozone monitoring stations. You might think that these "stations" contained thousands of dollars of sophisticated equipment, such as computers, meters, etc. But no! Their stations were wooden crates with one side replaced with a wire screen (to protect against insects and provide some shade) with a dozen simple green tobacco plants inside. I held some leaves from these plants in my handsthey were covered with tiny white spots; some were covered over 80% of their surface, others 20%. These leaves came from plants that were exposed for several days in the most polluted places in Mexico City. The number of their spots correlated with the levels of ozone.
Imagine this huge city of 20 million people. There is traffic 24 hours a day, little control of exhaust fumes, and a hot sun that creates smog from the mixture of gases in the air. This city exists in a valley surrounded by mountains which trap the smog. This smog can cause problems such as disease.
When we returned from the conference, we called Professor Manning of the University of Massachusetts, one of the leading experts in using plants as indicators of air pollution. Dr. Manning visited TERC and we discussed the possibility of our Global Lab schools using tobacco plants as monitors for ozone levels in their areas. He gave us some seeds of two varieties of tobacco plants, as well as a protocol he has used in many places, including Mexico City.
Flecking - A common symptom of O3 injury, flecking usually indicates acute damage. Small spots or flecks occur due to the death of palisade cells. These flecks are metallic or brown and often bleach to tan or white with age.
Stipples - These are punctate spots consisting of a few palisade cells injured or killed by O3. Stipples may be white, black, red or reddish-purple (e.g. white stipple of ash leaves, cucumber, grapes.)
Change in color - Leaves exposed to low concentration of O3 take on an extensive reddish-brown or bronze color (e.g. bronzing of beans, ash leaves)
Chlorosis (loss of chlorophyll) - This can occur over a long time period and may be the only symptom of chronic O3 injury. This can result in premature leaf fall (e.g. beans, morning glory).
Adapted from Manning & Feder,
1980.
General Procedure
The procedure has four stages: starting the seeds; first transplantation; second transplantation; and placement at the outdoor site. Since plants at the outdoor sites will be replaced every two weeks, new seeds should be started every two weeks and moved through the sequence.
The details of this laboratory procedure have been adapted from the work of Manning & Feder who have extensive experience in using tobacco and other indicator plants to monitor air quality.
Materials
Tobacco seeds
Growing chamber (Aquarium, environmental chamber) with the following characteristics:
· Plant light such as Gro-lite®, approximately 2000 foot-candles
· a timer
· activated charcoal filter (for ozone free conditions)
· a fan to provide strong air-flow to prevent overheating
· temperature maintained at approximately 25-30°C.
Plastic pots 2", 4", 8". (The 2" pots could be peat pots or paper egg cartons)
Large shallow tray
Individual saucers for 4" pots
Screened box
Starter soil mixes
Redi-earth®
Terra-lite®
Fertilizers (commercially available)
Hoagland's solution (Contains a variety of macro- and micro-elements that together supply all the nutrients a plant needs.)
Soluble fertilizer (15-15-15 N-P-K) (15% nitrogen, 15% phosphorous, and 15% potassium, by weight, each in soluble form.)
Labels
Wooden or plastic stakes
Notebook
Starting the Seeds
1. Label two plastic 4" pots Bel-W3 and Bel-B.
2. Fill the pots not less than 1/2" from the rim with thoroughly moistened Redi-Earth® starter soil.
3. Place the labelled pots with soil in saucers.
4. Sprinkle 50-60 Bel-W3 seeds and 50-60 Bel-B seeds onto the soil surface in the appropriate pots. Try to spread them out evenly. Gently press the seeds onto the surface of the soil, but do not cover them with soil.
5. Cover the labelled trays with their lids and place the entire arrangement in a growing chamber. Leave the Gro-lite® on at all times.
6. Fill the saucers not more than 1/4 inch (1/2 cm) with Hoagland's solution diluted with water in a 1:1 ratio. Reapply this fertilizer once a week.
7. The seeds should germinate in about two weeks.
First Transplantation
Three to four weeks after planting, the seedlings should be about 2.5 cm. or taller and should have four small 1/4" leaves (about 7 mm. long). The seedlings must be transfered into 2" pots; they are still very delicate and easily damaged, so handle them with care. Allow some soil to remain on the roots.
1. Separate the seedlings and transfer each one to an individual plastic, paper, or peat 2" pot filled with Redi-Earth®. Make sure that each pot is labelled with the appropriate strain, Bel-W3 or Bel-B, the date of sowing, and an identification number. In a notebook, record each identification number and write the strain, the date of sowing, and the date of first transplantation next to it.
2. Soak each pot thoroughly with full-strength Hoagland's solution immediately after transplantation.
3. Place the pots onto saucers and place them inside the growing chamber.
4. Use the timer to alternate between 8 hours of light and 16 hours of dark in the chamber.
5. Once a week, wash the tray out with water to prevent the growth of algae and refill it with full-strength Hoagland's solution.
Second Transplantation
In two to three weeks, the seedlings will be about 7.5 cm. tall, and must be transferred from the 2" pots to 4" pots. For each seedling:
1. Fill a 4" plastic pot 1/3 full of Tera-lite®.
2. If the seedling is growing in a peat or paper pot, place the whole thing into the 4" pot. If the seedling is in a plastic pot, you must remove it and then put it in the 4" pot.
3. Add Tera-lite® until the soil level is 1/2" from the rim of the 4" pot.
4. Wash any soil from leaves with water.
5. Transfer the label from the 2" pot to the 4" pot and record the date of the second transplantation next to the identification number in the notebook.
6. Soak each pot throughly with one teaspoon of a solution of 15-15-15 fertilizer in a gallon of water.
7. Continue to add this fertilizer once a week and provide tap water as needed.
8. Let the plants grow in the chamber under the previous conditions for about 3-4 weeks until they are about 20 cm tall and have at least four well-extended leaves 5-7.5 cm in length.
Pairing (optional)
We think growing a sensitive and tolerant plant under the same conditions (in the same pot) for a long time is a good method for comparing their reactions. Thus we modified the Second Transplantation step of Manning & Feder's procedure. At TERC, we transferred a pair of 7.5 cm. seedlings (one Bel-W3 and one Bel-B) into one 8" pot. Using this pairing method, we put the plants in an ozone-containing chamber and got a strong response from the sensitive Bel-B strain and no response from the tolerant Bel-W3 strain.
Outdoor Exposure
The 20 cm. plants with four or more large leaves are ready to be placed in the monitoring sites of your choice. All plants must be placed in a screened box for shading.
1. Put eight plants (four of each strain) at each location.
2. Water each plant thoroughly as often as needed.
3. Once a week add 15-15-15 ferilizer solution.
Important: Keep the plants well watered and well fertilized. Plants that are nutrient- or moisture-deprived will lose their sensitivity to ozone.
4. After the first four pairs of plants have been outdoors for two weeks, replace two pairs with new ones.
5. Thereafter, replace the two oldest pairs with new pairs every two weeks.
Data Collection
1. All plants should be regularly read at least twice a week.
2. Each student should have their own leaves to read for damage. This is a subjective measurement.
3. At each reading, the student should estimate what percent of the leaf surface is injured.
Identifying Ozone Damage: A sun-, wind-, or dryness-damaged leaf will look wilted and weak. Parts of it will be discolored, usually along the edges. In contrast, an ozone-damaged leaf looks morphologically healthy. It will not look wilted or weak. The discoloration of the ozone-damaged leaf is not limited to one area. Rather, the leaf will be speckled all over with little dots.
Dr. Manning evaluates injury this by visual inspection, grading leaf injury from 1 to 5.
numerical rating % leaf injury
1 0-20
2 20-40
3 40-60
4 60-80
5 80-100
4. Tobacco leaves that are not well-watered, dried out, or weak by wind and sun may exhibit damage that is not ozone related. Students should note this kind of damage but not count it as ozone related.
5. The students should keep a notebook in which they record at each reading the percentage of injury and any notes about the leaf condition, such as wilting or insect injury, etc.
6. After each plant is removed from the site, each leaf should be labeled, photocopied, then dried and stored in a folder (one folder per pair of plants). Each label should point to the pages of the notebook that contains all the information about this leaf and plant.
Analysis
1. The new injury for each leaf at each reading period is the difference between the percentage of injury of the end of the prior period and the beginning of the next. Calculate the new injury for each leaf at each reading period.
2. For each reading period at every location, average the new injury of all the leaves on all four plants of the same strain.
3. Make a graph for each strain at each site of the average new injury over the reading period.
Brainstorming questions:
1. How can you tell which levels of ozone produce what degree of damage to your tobacco plants? Why is it necessary to know this?
(Teacher's Note: You need to calibrate your biological "instrument" against a standard. This could be an established ozone-monitoring station. Building the TERC Ozonometer in the Global Laboratory Notebook describes a cheap, simple method for building a physical ozone measuring device.)
2. Other substances lower air quality. Name as many as you can. You can use bioindicators to investigate some of these pollutants in your area.
