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OECD 208: Terrestrial Plant Testing - Seedling Emergence and Seedling Growth Test

OECD 208 is designed to assess effects on seedling emergence and early growth of higher plants following exposure to the test substance applied to the soil surface or into the soil. This guideline is applicable to the testing of general chemicals, biocides and crop protection products.

OECD 208 is also widely used as part of ASTM D6400 to evaluate the effects of composting products of plastic materials.

OECD 208 Key Summary

Seeds are placed in contact with soil treated with the test substance and evaluated for effects following usually 14 to 21 days after 50% emergence of the seedlings in the control group. Endpoints measured are visual assessment of seedling emergence, biomass measurements, shoot height, and the visible detrimental effects on different parts of the plant. The test can be conducted in order to determine the dose-response curve, or at a single concentration/rate as a limit test, according to the aim of the study. An appropriate statistical analysis is used to obtain effective concentration or effective application rate for the most sensitive parameter(s) of interest. Also, the no observed effect concentration and lowest observed effect concentration can be calculated in this test.

▶ Materials that can be tested:
     ➢ General chemicals, biocides and crop protection products, and so on;
▶ Test substance incorporation:
     ➢ The test substance should be incorporated into soil using an appropriate carrier to ensure even incorporation. For example, water soluble materials should be dissolved in water and then mix the solution with soil. Substances with low water solubility should be dissolved in a suitable volatile solvent (e.g. acetone, ethanol) and mixed with sand, followed by evaporation of solvent and addition of sand to soil. For solid, insoluble test substances, dry soil and the chemical are mixed in a suitable mixing device. Hereafter, the soil is added to the pots and seeds are sown immediately;
▶ Plant species: reasonably broad, such as carrot, sunflower, lettuce, cucumber, soybean, onion, rice, wheat, corn, and so on;
▶ Test substance dosage: up to 1,000 mg for 1,000 g dry soil;
▶ Natural soil: sandy loam, loamy sand, sandy clay loam, or commercial potting soil that contains up to 1.5 percent organic carbon. Sieve the soil to 2 mm and measure % organic carbon, pH and salt content as electronic conductivity;
▶ Pot size: 15 cm diameter;
▶ Number of pots: 3 for blank control and 3 for test substance;
▶ Testing conditions:
     ➢ Temperature: 22 °C ± 10 °C;
     ➢ Humidity: 70 % ± 25%;
     ➢ Photoperiod: minimum 16 hours of light;
     ➢ Light intensity: 350 ± 50 µE/m2/s;
     ➢ Light wavelength: 400 - 700 nm.
▶ Duration: 14-21 days;
▶ Measurement: visual assessment of seedling emergence, biomass measurements, shoot height, and the visible detrimental effects on different parts of the plant;
▶ Test validity criteria:
     ➢ Seedling emergence of blank is at least 70%;
     ➢ The seedlings of blank control do not exhibit visible phytotoxic effects;
     ➢ The mean survival of emerged control seedlings is at least 90% for the duration of the study.

▶ When OECD 208 is used as part of ASTM D6400, the sample compost and blank compost are mixed with a reference soil (substrate) at ratios of 25% and 50% (w/w or v/v) of compost. The mixtures are used for planting the seeds, and two different plant species are selected. Fill each tray with a minimum of 200 g of the mixture soil and add as a minimum 100 seeds on the top. Cover the seeds with a thin layer of inert material, such as siliceous sand or perlite. Perform the tests in three parallels for each mixture. Add water until 70% to 100% of the water holding capacity is reached. Supply water periodically during the whole testing duration as needed. The germination numbers (number of grown plants) and the plant biomass of the sample compost and the blank compost are compared in all mixing rates. Both germination rate and biomass are calculated as per cent of the corresponding values obtained with the blank compost.

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