A study area should be selected with milkweed stems which satisfy the four following criteria.

1. The stems should be at least 100 feet from any road to avoid confounding effects of vehicle exhaust, road salts and, most importantly, road side cuttings. Abandoned lots, fields, levees streamways or prairies are ideal sites.

2. Each study area should contain at least 30-50 milkweed stems. The stems should be within 100 to 150 feet of each other to minimize the influence of changing soil or site conditions.

3. For statistical purposes a minimum of ten stems must be randomly chosen. Each stem should have 10 or more leaves. Most plants have mature leaves at least 4 inches in length. Some milkweed plants, however, have leaves that are only 2-3 inches long even when full grown! Look at a mature leaf on the mid to lower stem to confirm maximum leaf size. Stems with small leaves are acceptable for this study. Leaves missing more than 10% of their surface area due to disease or insect chewing should not be evaluated.

4. Each milkweed should only have one main stem. Multiple stemmed/branched plants should be avoided.

Your study area should be afforded some kind of protection (e.g., from mowing, etc.) to minimize losses. Staking your plots and running string to the corner stakes may be required to prevent unintentional destruction or disturbance. Study areas need not be made permanent, but it is desirable to have them in secure areas so they can be studied in the future if needed.

Only one formal evaluation for ozone injury is required. You should locate the study area(s) in late June or July to ensure the milkweed will be of sufficient size and abundance to meet the sampling requirements. You may want to examine the stems for visual evidence of ozone injury in July. You need not record your results. The formal evaluation should occur between September 1 - September 15. This will allow us to make a meaningful comparison of injury data from our research community.

If you intend to investigate the stems and leaves in detail over the summer, mark each study plant with paper tags, colored yarn/string or twist ties. Please mark as inconspicuously as possible. If you want to track specific leaves on a stem, use permanent marker to number each leaf on its lower surface or you may want to place a twist tie on the bottom and/or top pairs of leaves on the stems you are studying. Marking the plant and leaves will enable you to relocate the same stems and leaves during later visits. Marking also provides a valuable reference point for assessing stem growth, leaf loss or other phenomena.

Very important--contact the property owner or manager to make sure the study area is acceptable to them. A cooperative land owner/manager can protect your study area from disturbance during the growing season.

In the early fall, please have the following information:

1. Data sheet showing latitude and longitude of the study area location.

2. All leaves from your ten plants, injured and uninjured leaves for your study area. These leaves should be collected and pressed between September 1st and September 15th. Remember--many of you will probably not see ozone injury on your plants, but your sample leaves will help to verify the occurrence and severity of ozone injury.

Evaluating Ozone Injury

Ozone injury on milkweed leaves typically results in sharply defined, small dot like lesions (stipples) on the upper surface of the leaves. These lesions are observed only on the upper leaf surface and are black-dark' purple. Veins are usually not affected. The small purple dots may be observed on a leaf showing ozone damage.

Injury on the leaves may vary considerably! In general, the location of ozone injury on a leaf is determined by the maturity of the leaf. Acute ozone injury tends to develop towards the tip of young leaves, in the center of fully grown leaves, and at the base of the oldest leaves. Foliage frequently exposed to ozone may exhibit injury symptoms all over the upper leaf surface. Ozone damage appears as sharply defined, small dot-like lesions (stipples) on the upper surface of the leaves. These lesions are observed only on the upper leaf surface and are black-dark purple. Veins are usually not affected. If the injury is severe, it may produce an overall dark discoloration of the upper leaf surface as the lesions coalesce.

NIH Image/ Scion Image Protocol for Measuring Milkweed Damage

Using ArcView Image Analyst for Measuring Milkweed Damage

Estimating damage - Alternate Low Technology Procedure - If you do not have access to NIH Image/Scion Image or ArcView IA, you may use the procedure below to estimate the percentage of leaf area injured, enter the code numbers below on to the data sheet for all of the leaves for each of the ten sample plants. This estimating procedure is not as accurate but will help with a relative index of damage.