Overview
ISO 7346-2:1996 specifies a semi-static method for determining the acute lethal toxicity of stable, non-volatile, single substances soluble in water to the freshwater fish Brachydanio rerio (zebra fish). The standard defines procedures to estimate median lethal concentrations (LC50) at 24 h, 48 h, 72 h and 96 h under controlled laboratory conditions. It replaces the first edition and is part of the ISO 7346 series (Part 1: static method; Part 3: flow-through method).
Key keywords: ISO 7346-2:1996, water quality, semi-static method, acute lethal toxicity, Brachydanio rerio, LC50, zebra fish, toxicity testing.
Key topics and technical requirements
- Test principle: Use a two-stage test (preliminary range-finding, then final test) to determine concentrations that cause 50% mortality (LC50) after specified exposure times.
- Test organism: Zebra fish (Brachydanio rerio), ~30 mm ±5 mm total length (≈0.3 g ±0.1 g). Fish must be from one stock, acclimatized at least 7 days and disease-free.
- Environmental conditions: Temperature controlled at 23 °C ± 1 °C; laboratory illumination with 12–16 h photoperiod; avoid airborne contaminants.
- Semi-static regime: Test solutions renewed every 24 h or 48 h (compromise between static and flow-through); transfer live fish to freshly prepared vessels on renewal.
- Dilution water: Standard dilution water with pH 7.8 ± 0.2 and calcium hardness ≈ 250 mg/L as CaCO3 (specific salt recipe given in the standard).
- Stock solutions & solvents: Stock solutions prepared in deionized/glass-distilled water; if organic solvents are used, limit solvent concentration to ≤ 0.1 mL/L (or 0.1 g/L). Include both a solvent control and a water-only control.
- Data quality / validity criteria: Dissolved oxygen ≥ 60% air saturation value during the test; control mortality ≤ 10%; control abnormal behaviour ≤ 10%. Reference chemical (e.g., potassium dichromate) used to check lab performance.
- Concentration measurement: If test concentrations change by > ~20% from nominal, measured analytical values must be used; if concentrations decline substantially or unpredictably, this method may not define an LC50 reliably.
- Limit test: A limit-test at aqueous solubility or 100 mg/L (whichever is lower) using 10 fish can demonstrate LC50 is greater than that concentration if no mortality occurs.
Applications and users
- Environmental and ecotoxicology laboratories conducting acute fish toxicity tests.
- Chemical manufacturers and registrants assessing aquatic toxicity for regulatory submissions.
- Water quality specialists and environmental regulators performing risk assessments and prioritizing chemicals.
- Research groups studying mode-of-action or comparative species sensitivity using standardized methods.
Related standards
- ISO 7346-1: Static method (same test species)
- ISO 7346-3: Flow-through method (for unstable or continuously replenished exposures)
ISO 7346-2:1996 provides a standardized, reproducible semi-static protocol suitable for regulatory and research contexts where maintaining test concentration by periodic renewal is required.
