Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) (ISO 14669:1999).
ISO 14669 describes a method for determining the acute toxicity to one of three specific species of marine copepods (Copepoda, Crustacea) of soluble chemicals or substances that can be maintained as a stable suspension or dispersion, in industrial effluents or wastewater (treated or untreated, after settling, filtration, or centrifugation if necessary), or in marine or estuarine waters. The test copepods can be Acartia tonsa, Tisbe battagliai, or Nitocra spinipes. The copepod used is exposed to various concentrations of the test sample in synthetic seawater at 20°C ± 2°C with a photoperiod of 16 hours of light and 8 hours of darkness. Copepod mortality is recorded at 24 and 48 hours. The median lethal concentration (LC50), which kills 50% of the exposed copepods at 24 or 48 hours, is determined. Additionally, for certain purposes, it may be convenient to extend the exposure period to 96 hours and determine the LC50 at 96 hours.
The test is performed in one or two stages: a preliminary test that determines the range of concentrations to be tested in the final test and provides an approximate LC50 value at 48 hours, and a final test, performed when the approximate value from the preliminary test is insufficient, which allows for the calculation of the LC50 at 24 and 48 hours and determines the concentrations corresponding to 0% and 100% mortality. Additionally, if the test is used for chemical substances, a limit test can be performed at 100 mg/L or at a lower concentration that is the maximum at which the substance is soluble or in stable dispersion under the test conditions.
For the preliminary test, a wide range of sample concentrations should be selected in geometric progression (a factor of 10 or 3.2 between concentrations is recommended), with a minimum of five copepods per concentration. For the final assay, a range of concentrations should be selected based on the results of the preliminary test, but using a smaller factor (typically 1.8 or 2) between concentrations. It is desirable that the selected concentrations result in two or three mortality percentages between 10% and 90%. For each concentration and each control, a minimum of 20 copepods are used (four replicates with five copepods each). Finally, for the limit assay, 20 copepods are exposed to a single concentration of 100 mg/L or lower (the maximum concentration at which the substance is soluble or in stable dispersion under the test conditions).