Ecotoxicity - Freshwater plants, Lemna spp.: Growth Inhibition test (ISO 20079: 2005 standard or OECD 221: 2006 guideline).

This test can be performed in accordance with the specifications of ISO 7346-1 standard or OECD 221 guideline. Both guidelines assess the toxicity of substances to freshwater aquatic plants using plants of the genus Lemna (duckweed). To carry out the test, exponentially growing cultures of plants of the Lemna genus (Lemna gibba and Lemna minor, generally), are allowed to grow in monocultures in the presence of the test product, for a period of seven days.

To perform the definitive test, at least 5 concentrations and three replicates per test concentration should be used. The separation factor between the concentrations should preferably not exceed 3.2, but a higher one can be used if the concentration-response curve is flat. The aim of the test is to quantify the toxic effects of the test product on the vegetative growth of aquatic plants during this period, through the evaluation of measurement variables (number of fronds and dry weight). To quantify test substance-related effects, growth in test solutions is compared to controls. From the average specific growth rate recorded in the series of solutions of the test product, the concentration that gives about a percentage rate of growth inhibition, expressed as ErCx (for example, ExC50), is determined. In addition, if the client indicates it, it is possible to determine the lowest concentration that causes a statistically significant effect on growth (LOEC: lowest observed effect concentration) and the highest concentration that does not cause a significant difference in growth (NOEC: no observed effect concentration). It must be taken into account that, if the EC50 is to be determined, it must fall within the range of test concentrations, with at least one concentration above and one below this value. If it is desired to estimate the LOEC or NOEC, the growth at the lower test concentration should be not significantly different from the control and at the higher concentration, the growth should be significantly lower. If the client does not know the test concentrations to request for the definitive test, he can request a preliminary test to determine the range of concentrations to be used in the final test.

The standard also admits the performance of a limit test, when a substance is expected to have no toxic effects at concentrations up to 100 mg/L (or up to its solubility limit). The test is performed with this concentration as the only treatment group, with at least 6 replicates.

To carry out the tests, the following information must be provided to the laboratory: the standard or guideline to be followed, the test to be evaluated (preliminary test, definitive test, limit test), the range of concentrations to be evaluated (a minimum of 5 concentrations for the test definitive or preliminary and one for the limit test, expressed in mg of product/L), if the determination of a specific parameter is desired, such as EC50 or NOEC/LOEC, the nature of the product and its solubility in water. The highest concentration of the test substance tested should not normally exceed the water solubility of the substance. The use of solvents other than water should be avoided. If for substances with low solubility in water, it may be necessary to prepare a concentrated stock solution or a dispersion of the substance using a solvent or an organic dispersant, in this case an additional control must be carried out to verify that the solvent does not cause toxicity.