OECD 226: Predatory mite reproduction test in soil

The reproduction of Hypoaspis aculeifer will be determined in a laboratory experiment. Therefore the mites are exposed to soil that is treated with the test substance and compared to a control and to a reference item. The Directive 1107/2009 requires assessment of effects on soil organisms for all test compounds. For substances applied directly to soil testing on Hypoaspis aculeifer is mandatory, as they are considered to be a relevant representative of soil fauna. The reproduction provides information about inhibition of the specific population growth rate.

Study Design

Test Organisms

Hypoaspis aculeifer is a widespread predatory mite in soil ecosystems, feeding on Enchytraeids, Collembola, other Mites and Nematodes. Its ecological importance in agriculture is the potential to regulate plant parasitic nematodes. It is worldwide distributed and can easily be collected and reared in the laboratory. The mites are bred in in-house laboratory cultures to assure availability of the test organisms all over the year.


Course of the Test (OECD 226)

The studies are performed in controlled environmental rooms with defined temperature. Illumination follows a night and day rhythm.

If there is no information about the toxicity of the test substance, a range finding pre-test helps to identify the suitable concentration range. Preferably the tested concentrations should affect reproduction and not mortality of the adult female mites. The number of test concentrations and replicates depends on the experimental design.

Usually 5 test item concentrations (8 concentrations for ECx-design) with four replicates and a negative control comprising eight replicates are tested. A toxic reference substance is tested in a dose response design at least once per year.

The test substance is mixed into an artificial soil consisting of quartz sand, sphagnum peat, kaolin clay and calcium carbonate. Moisture and pH are adjusted.

Ten adult female mites from synchronised breeding are introduced in each replicate. The female mites are exposed in the soil for 14 days. The mites are fed with living prey, e.g. cheese mites (Tyrophagus putrescentiae). The exposure takes place via dermal and alimentary uptake.

At the end of the study the surviving female mites and the juveniles are extracted via a heat gradient. After extraction numbers of juveniles and adults are counted separately under binoculars.



ibacon performs studies on H. aculeifer according to GLP for many years. ibacon’s study director participated in the ring tests with H. aculeifer and contributed to the working group establishing the OECD 226.



[soil mites 002] As the focus of this study lies on reproduction, the tested concentration range mostly results in slight or no mortality. The number of offspring of the mites exposed to the test item is compared to that of the control group.
Results on reproduction are used to determine a NOEC (no observed effect concentration) or to calculate an EC10 and EC20.


Guidelines and Literature

  • OECD Guidelines for the Testing of Chemicals Test No. 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil, adopted guideline October 3, 2008.