The test requires three test concentrations and controls.
Two vessels per treatment for zebrafish (each vessel containing 5 males and 5 females).
Four vessels per treatment for fathead minnow (each vessel containing 2 males and 4 females due to the territorial behaviour of male fathead minnow).
Four vessels or replicates per treatment are used for Japanese medaka (each vessel containing 3 males and 3 females).
The following parameters are assessed:
Analytical chemistry – Weekly water sample collections from tanks to analyse test chemical levels.
Vitellogenin (Vg) – ELISA methods are used to determine the amount of this egg yolk pre-cursor protein. Vg is normally expressed only in female fish and dormant in males. However, when male fish are exposed to endocrine disruptors the Vg gene is expressed in a dose dependent manner. Therefore, the Vg gene expression in male fish has been used as a marker of exposure to oestrogenic endocrine disruptors.
Morphological observations – Appearance and observation of secondary sex characteristics including; male colouration and fat pad (presence and weight at termination), nuptial tubercle mapping, daily assessment of survival and behaviour compared to the controls.
Fertilisation success is assessed, embryos are visually inspected, infertile and fertile eggs counted.
Fecundity – Daily egg production is assessed.
Survival, behaviour and appearance – Fish are observed daily for mortality and abnormal behaviour relative to the controls
Observation of secondary sex characteristics – secondary specialised sex characteristics are also observed such as nuptial tubercles in male fathead minnow and papillary processes in Japanese medaka.