Ecotoxicity - Terrestrial plants: Germination and seedling growth tests (OECD 208: 2006).
This test is designed to evaluate the effects on seedling germination and early plant growth after exposure to the soil-incorporated test substance (if exposure to the chemical is through the soil and there is concern for uptake by roots) or applied to the soil surface (in the case of testing plant protection products).
The seeds are placed in contact with soil treated with the test substance by one of the two indicated methods (incorporation into the soil or application on the surface) and are evaluated for the effects between 14 and 21 days after germination occurred in 50% of the seedlings in the control group, not exposed to the product under evaluation. Evaluation criteria are visual observation of seedling germination, measurements of biomass (dry or wet) of plants, height of shoots, and visible detrimental effects on different parts of the plant.
This Standard does not indicate the number of species to be included in the tests, since it depends on the relevant regulatory requirements, which you should consult with the competent authorities. The Standard does indicate that the plant species selected for the test must correspond to different plant families to consider the taxonomic diversity in the plant kingdom and to obtain reliable and reproducible results. In the choice of species, their taxonomic diversity in the plant world, their distribution, the abundance of specific characteristics of the life cycle of the species and the regional natural existence must be taken into account. In general, crop species can be used, as well as other species not usually cultivated. Among the crop species, the following can be considered as usual: tomato, cucumber, lettuce, soybean, cabbage, carrot, oats, corn, onion or fodder. For species not normally cultivated, we recommend requesting information from the regulatory authorities to include those that are really suitable for the area in which the product is used.
Depending on the objective of the study, a test with multiple concentrations can be carried out to determine the dose-response curve, or with a single concentration as a limit test (with a concentration of 1,000 mg/Kg of dry soil). For each concentration, 4 replicates are analyzed, analyzing at least a total of 20 seeds per concentration.
The test with multiple concentrations must be carried out with a minimum of 5 concentrations in a geometric series, with a factor not greater than 3. This test allows to determine, through an appropriate statistical analysis, the concentration or proportion that causes a toxic effect (ECx: Effect concentration; ERx: Effect rate) with an undesirable alteration or damage for each species (germination, biomass weight, final number of plants present, visible alteration). Test concentrations should be suitable, eg. if EC50 is desired, test concentrations should include those that cause 20 to 80% effect. If the client knows the range of concentrations that causes said effect, they can request the test indicating the test concentrations. If you do not know them, you will need to do the range search test first. Also, if requested, the Non Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) can be calculated. To determine these concentrations, a larger number of replicates are required to obtain the desired statistical power.
If the client does not know the concentrations to be tested in the multi-concentration test, they can perform a preliminary range-finding test to determine the concentrations to be tested in the definitive dose-response study. For the range-finding test, the test concentrations should be widely spaced (eg, 0.1, 1.0, 10, 100, and 1,000 mg/kg dry soil). For plant protection products, concentrations could be based on the recommended or maximum application concentration or rate, eg. 1:100, 1:10, 1:1 of the recommended/ maximum application concentration.
In order to perform the tests, the client must provide the laboratory with the following information: test to be evaluated according to the expected route of exposure (incorporation into soil; application on surface); type of test to be carried out (preliminary, multiple concentrations or limit test) range of concentrations to be evaluated (expressed in mg of product/kg of dry soil); nature of the product and its solubility (if it is insoluble in water, the recommended organic solvent should be indicated); type of plants against which it is desired to evaluate (of habitual crops or others), if possible consulting with the pertinent registration authorities in case they require any special ones; type of parameter to determine (EC50, NOEC…). It must be taken into account that, if the solvent used is different from water, an additional control of the solvent must be added.