Overview
EN ISO 7346-2:1997 (ISO 7346-2:1996) specifies a semi-static laboratory 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 (24 h, 48 h, 72 h and 96 h LC50) by renewing test solutions at regular intervals (every 24 h or 48 h) - a compromise between static and flow‑through methods.
Key topics and technical requirements
- Test organism: Brachydanio rerio (zebra fish); recommended specimen length ~30 mm ± 5 mm (≈0.3 g ± 0.1 g). Other freshwater species may be used with appropriate modifications.
- Scope of substances: Stable, non‑volatile, single substances soluble in water. Special arrangements required for highly volatile substances.
- Test design:
- Two-stage procedure: preliminary range‑finding test, then a final definitive test.
- Limit test option at the solubility limit or 100 mg/L (whichever is lower) using 10 fish per concentration and corresponding controls.
- Typical dosing: select an approximately geometric series of at least five concentrations to produce 0–100% mortality across concentrations.
- Semi‑static renewal: Renew test solutions every 24 or 48 hours; transfer survivors to fresh solution without delay.
- Controls: Include a control with dilution water and, if a solvent is used, a solvent control at the maximum solvent concentration. Solvent concentration shall not exceed 0.1 mL/L (or 0.1 g/L as applicable).
- Environmental conditions & monitoring:
- Test temperature: 23 °C ± 1 °C.
- Standard dilution water: pH ~7.8 ± 0.2, calcium hardness ~250 mg/L as CaCO3; specified salt composition is provided.
- Measure dissolved oxygen, pH and temperature at start and before/after renewals; chemical analysis of test substance concentrations is required when stability is in doubt.
- Validity criteria: Concentrations should remain reasonably constant (about ±20% of nominal) or be analytically quantified; otherwise LC50 cannot be reliably defined with this method.
- Reporting: Report 24, 48, 72 and 96 h LC50 values (and analytical concentrations if measured).
Applications and users
- Who uses it: Environmental laboratories, regulators, chemical manufacturers, ecotoxicologists and quality control teams performing aquatic toxicity assessment.
- Practical uses:
- Screening and hazard classification of chemicals for acute fish toxicity.
- Regulatory submissions, chemical safety assessments and comparisons with other test methods.
- Supplementary method when flow‑through testing is impractical, but static testing is insufficient due to concentration instability.
Related standards
Keywords: EN ISO 7346-2, ISO 7346-2, zebra fish toxicity test, semi-static method, acute lethal toxicity, LC50, Brachydanio rerio, water quality testing, aquatic ecotoxicology.
