OECD 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment

As chemicals may not only have toxic effects but due to prolonged exposure also effects on population level and / or sexual development (e.g. as potential endocrine disruptors) life-cycle tests of suitable organisms in water and sediment become more and more important. The spiked water or spiked sediment test design gives the possibility to address to the different exposure scenarios of chemicals in water. Among the invertebrates the endocrine system of insects is best described therefore chironomids are appropriate test organisms to investigate potential endocrine effects in sexually reproducing organism.

Study Design

Test organisms

Chironomus species occur in aquatic habitats in high abundance and diversity. Larvae of these midges are typical sediment dwellers and an important prey for fish, the adults also for birds. Chironomus riparius is bred in a laboratory culture.

Course of the test

Taking into account the specific behaviour of the test item (e.g. physical chemical properties, adsorption effects, degradation) the test can be performed in a spiked water or spiked sediment design. A schematic illustration of a chironomid life-cycle test is shown in fig. 1. The test starts by placing first instar larvae (1st generation) into the test vessels. As soon the adults emerge they are  transferred carefully to special breeding cages to facilitate swarming, mating and oviposition into crystallising dishes containing the same test item concentration as the corresponding test vessels. Egg ropes produced by the adult midges are transferred to micro plates together with a small amount of culture medium from the crystallising dish to start the observation of the 2nd generation. The hatched larvae are transferred carefully to test vessels which are freshly prepared with test substance and are examined under similar conditions as the parent generation.

ibacon and ring tests

The OECD Guideline 233 is an extension of the existing OECD guidelines 218 and 219 which have been implemented at ibacon in numerous GLP studies since many years. Due to our in-depth experience in the performance of spiked water and spiked sediment tests ibacon has been chosen to participate in the chironomid two generation life cycle ring test, in 2009. In July 2010 the ring test resulted in the implementation of the OECD 233. In the guideline both the spiked sediment and the spiked water exposure scenarios are described. The mentioned test designs can be adapted to consider special physical chemical properties of the test item (e.g. adsorption effects, degradation) or if other exposure scenarios e.g. via contaminated food are of interest.

study design of a chironomid  full life-cycle test




  • Total numbers of adults emerged (1st and 2nd generation)
  • Development rate (1st and 2nd generation)
  • Sex ratio of fully emerged and alive adults (1st and 2nd generation)
  • Number of egg ropes per female (1st generation)
  • Fertility of the egg ropes (1st generation)

Guidelines and literature

  • OECD Guidelines for Testing of Chemicals, No. 233: “Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment”