OECD 239: Water-sediment Myriophyllum spicatum toxicity test

The purpose of this test is to determine the inhibitory effect of the test item on the vegetative growth of the freshwater plant Myriophyllum spicatum. Data for rooted macrophytes may be required in cases where algae and Lemna tests are not sensitive to the mode of action or if partioning to sediment is a concern, leading to exposure via root uptake.  M. spicatum will be exposed in a a static or semi-static test to various concentrations of the test item under defined conditions. The inhibition of growth compared to control cultures will be determined over a period of 14 days.

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

For the 7 days pre-rooting phase five healthy shoot tips without flowering shoots at a determined length are planted in pots with artificial sediment and reconstituted water. Without any exposure via water or sediment root development is induced during this phase.

At test start two of the plants are removed from the pots to obtain three largely homogenous individuals for testing. For the control six replicates and for each treatment group three replicates were prepared. One replicate contains generally one pot with three plants.

After application of the test item concentrations in a geomtric series the exposure phase lasts for two weeks. The exposure takes place at a light intensity of 8000-10000 lux with a light:dark ratio of 16:8 h and a temperature of 20 ± 2°C.

At test start and test end the length and biomass of every plant will be determined. For the analytical dose verification the test water and the sediment can be analysed at test initiation and termination.

Generally, the method can be used for other higher plant species as well, e.g. M. aquaticum or Glyceria maxima.

Taylor-made study designs

The exposure of the test item can occur via water or sediment. In exceptional cases the application of the test compound via sediment is possible.

In case of fast degradation of the test item during the exposure phase, periodical water renewals are possible (semi-static test design).

An additional recovery periode after the exposure can be affiliated, to show that the effects are reversible.


The endpoints of the study are growth of total shoot length as well as fresh and dry weight. Based on these parameters NOEC, LOEC and EC50 (EC20, EC10) can be calculated for yield and growth rate. Moreover sublethal effects, such as chlorosis are determined.

Guidelines and literature

  • OECD Guideline 239: Water-Sediment Myriophyllum spicatum Toxicity Test, September 26, 2014