OECD 234 Fish Sexual Development Test

This test assesses the effects on early-life stages and sexual development of fish starting with newly fertilised eggs until the fish are sexually differentiated. The main endpoints of the study are vitellogenin (VTG) concentration and sex ratio. The combination of these endpoints allows the detection of the mode of action of an endocrine disruptor.

Test Organisms

  • Oryzias latipes (Japanese medaka), recommended species because of genetic sex markers,
  • Danio rerio (zebrafish)
  • Gasterosteus aculeatus (three spined stickleback)

The Japanese medaka is the recommended species because genetic sex markers can be used to identify the genetic sex, it is fully validated and has analysable secondary sex characteristics.


Course of the test

This study is a long-term study of approximately 60 days post-hatch for zebrafish, three-spined sticklebacks and medaka. Fertilised eggs are exposed to five different concentrations of a chemical in a flow-through system. Four replicates with 30 eggs in each replicate are prepared and observed until gonad development of the juveniles.


The endpopints of the study enables the detection of chemicals with androgenic or oestrogenic properties as well as anti-androgenic, anti-oestrogenic and steroidogenesis inhibiting properties.

  • Vitellogenin
  • sex ratio (the sex ratio is based on genetic sex (for Japanese medaka) or phenotypic sex by gonadal histology examination)
  • survival
  • hatching success
  • lenght
  • body weight
  • abnormal behaviour
  • morphological abnormalities
  • Secondary sex characteristics are observed for Japanese medaka.

Guidelines and literature

  •  OECD Guideline for Testing of Chemicals, Test No. 234: Fish Sexual Development Study.