General toxicity tests evaluate how chemicals affect organisms and the environment through short-term (acute) and longer-term (chronic) exposures. Acute studies estimate concentrations that cause 50% mortality, while chronic assays (often using zebrafish) monitor endpoints such as hatching, growth and survival across several exposure levels to derive NOEC and LOEC.
Fish are exposed to the test chemical for 96 hours. Mortality is recorded at 24, 48, 72 and 96 hours and used to estimate the LC50 – the concentration that kills half of the exposed fish.
At least five concentrations in a geometric series and a minimum of seven fish per level are typically tested to obtain a clear concentration–response curve.
Test Guideline 204 described a 14-day prolonged toxicity study in fish. It has been withdrawn as a stand-alone method but is still referenced when interpreting older datasets and legacy studies.
Early life stages from fertilised egg to freely feeding larvae are exposed to several concentrations of the test substance under flow-through or semi-static conditions.
Endpoints include hatching success, survival, growth, behaviour and external appearance, and the data are used to derive NOEC, LOEC or ECx values.
Embryos and sac-fry are exposed to several concentrations of the substance in flow-through or semi-static systems until just before yolk-sac absorption or before starvation occurs in controls.
Lethal and sub-lethal effects, behaviour, hatching and survival are compared with controls to derive NOEC/LOEC or ECx.
Juvenile fish in exponential growth are exposed for about 28 days to at least five sub-lethal concentrations plus controls, usually under flow-through conditions.
Growth rates, body weight and condition at the end of the test are analysed to determine ECx, NOEC and LOEC for growth.
Sexually mature males and spawning females are held together and exposed to graded concentrations of the test substance for 21 days.
Daily egg production and reproductive biomarkers such as vitellogenin and secondary sexual characteristics are measured to detect endocrine-related effects.
This 21-day assay identifies chemicals with oestrogenic, androgenic or aromatase-inhibiting activity in fish using species such as zebrafish, Japanese medaka and fathead minnow.
Biomarkers including vitellogenin and secondary sexual characteristics are evaluated separately in males and females to characterise the endocrine mode of action.
Fish are exposed from fertilisation through sexual differentiation (around 60 days after hatching) to at least three concentrations of suspected endocrine-active substances.
Sex ratio, intersex incidence and vitellogenin levels are combined to reveal how the substance affects sexual development.
Freshly fertilised zebrafish embryos are exposed individually in multi-well plates for 96 hours to several concentrations plus a control.
Four defined lethal endpoints are checked every 24 hours and used to calculate an acute LC50, alongside measurements of water quality and exposure.
Fish are exposed to a constant concentration of the substance in a flow-through system and then transferred to clean water for a depuration phase.
Chemical levels in water and fish tissues are measured over time to calculate uptake and depuration rate constants and the bioconcentration factor (BCF).