OECD 212: Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages

The Short-term Toxicity Test on Fish Embryo and Sac-Fry Stages is a test in which the life stages from the newly fertilized egg to the end of the sac-fry stage are exposed. During the test no feeding is provided in the embryo and sac-fry test, and the test should thus be terminated while the sac-fry are still nourished from the yolk-sac.

This test can provide useful information in order to combine the information of lethal and sublethal tests and can be used as a screening for a Fish Early Life Stage Test (OECD 210). The short exposure of fish to chemicals can reduce the sensitivity and thus underestimate the chronic toxicity. In general this test is expected to be less sensitive than the OECD 210 and does not replace the OECD 210.

Study Design

Test Organisms

  • Danio rerio (zebrafish)
  • Pimephales promelas (fathead minnow)
  • Oryzias latipes (Japanese medaka)

Course of the test

The embryo and sac-fry stages of fish are exposed to five concentrations of the test substance dissolved in water. The test starts with placing at least 30 fertilised eggs divided equally between at least three replicate test chambers per concentration, and is terminated just before the yolk-sac of any larvae in any of the test chambers has been completely absorbed or before mortalities by starvation start in controls. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect concentration and hence the no observed effect concentration. Hatching, mortality, abnormal appearance and behavior are recorded daily. Concentrations of the substance higher than 96 hour LC50 or 100 mg/L, whichever is the lower, need not to be tested. The test concentrations are verified analytically at regular time intervals. During the course of the test the dissolved oxygen concentrations, pH and water temperature are measured


Reported Endpoints include survival, hatching, abnormal appearance, behaviour and days until hatching. Additionally, at the end of the test the individual length of all surviving fish is determined and all surviving fish are individually weighed (wet weight blotted dry or dry weights). The lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) are derived by assessing and comparing the lethal and sub-lethal effects with the control values.

Guidelines and literature

  • OECD Guideline for Testing of Chemicals, No. 212: “Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages “, adopted September 21, 1998