This Test Guideline describes an acute immobilisation assay on chronomids and is designed to complement existing Test Guidelines for chironomid chronic toxicity assays (TG 218, 219 and 233). The test method is based on TG 202: Daphnia sp. Acute Immobilisation Test. First instar Chironomus sp.. larvae are exposed to a range of concentrations of the test substance in water-only vessels for a period of 48 hours. C. riparius is the preferred species but C. dilutus or C. yoshimatsui may also be used for the test. At least 20 larvae, preferably divided into four groups of five larvae each, should be used for each test concentration and for controls. In the definitive test, at least five test concentrations should be used, with a dilution water control and solvent control (if appropriate). Immobilisation is recorded at 24 and 48 hours, and if data allow, the EC50 is calculated at 24 and 48 hours. A limit test with a single concentration may also be performed at 100 mg/L of test substance or up to the practical limit of solubility (whichever is lowest) in order to demonstrate that the EC50 is greater than this concentration.