OECD 238: Sediment-free Myriophyllum spicatum toxicity test

The sediment-free higher tier study with Myriophyllum spicatum  is designed to assess the toxicity of chemicals to higher plants. Test with higher aquatic plants species can be necessary for the risk assessment of plant protection products and biocides. The toxicity of different test compounds can be easily determined with the sediment-free system independent of the distribution behaviour  of the compound between water and sediment. This test system has a low analytical comlexity and the obtained results can be compared with those of Lemna test results.

Study Design

Test Organisms

Myriophyllum spicatum (Eurasian water milfoil) is a submerged, rooted species which tolerates a wide range of conditions and is found in both static and flowing water bodies. M. spicatum is bred in our own sterile laboratory culture.

Course of the test

The sediment-free Myriophyllum test is a sterile test system where static or semi-static studies can be easily conducted. It is recommended for test items that e.g. degrade rapidly or which are volatile.

The objective of this 14 day-exposure test is the quantification of chemical related effects on the vegetative growth over this period. Therefore, a series of geometric test concentrations (à five replicates) and a control group (ten replicates) are assessed.   Each replicate consists of one lateral branch shortened to 2.5 cm that is inserted into a test tube under aseptic conditions. At test start and test end the length and biomass of every plant will be determined.

The exposure takes place at a light intensity of 6000-9000 lux with a light:dark ratio of 16:8 hours. The temperature should be 23 ±  2 °C and the pH value  should remain between 6 – 9.


Growth of shoot length, lateral branches and roots  as well as fresh and dry weight can be easily determined with the help of the sediment-free test system. Moreover, sublethal effects such as chlorosis or necrosis are recorded. The test concentrations of at least the highest and one lower concentrations are verified at test initiation and termination.

Guidelines and literature

  • OECD Guideline 238: Sediment-free Myriophyllum spicatum Toxicity Test, September 26, 2014