Overview
EN ISO 7346-3:1997 (ISO 7346-3:1996) specifies a flow-through method to determine the acute lethal toxicity of stable, non‑volatile, single substances soluble in water to the freshwater fish Brachydanio rerio (zebra fish). The test yields 24 h, 48 h, 72 h and 96 h LC50 values under defined water‑quality and exposure conditions. This part is the flow‑through edition of ISO 7346 and replaces the first edition.
Key topics and technical requirements
- Test organism: Brachydanio rerio (zebra fish), approx. 30 mm ± 5 mm (≈0.3 g ± 0.1 g); acclimatized at least 7 days in dilution water.
- Exposure design: Preliminary test to define concentration range, followed by a final test with at least five concentrations in a geometric series. Limit tests (10 fish) at solubility or 100 mg/L are permitted.
- Flow‑through system: Continuous or frequent renewal of test solution to maintain stable concentrations; recommended replacement ≥25 L/day (can be reduced to 12 L/day if dissolved oxygen remains >60% air saturation).
- Water quality: Standard dilution water composition and properties specified - pH ~7.8 ±0.2 and calcium hardness ≈250 mg/L as CaCO3. Aerate dilution water to ≥90% air saturation before use.
- Temperature and loading: Test temperature 23 °C ±1 °C; fish loading not to exceed 1.5 g fish/L.
- Analytical monitoring & validity: Where possible measure test substance concentrations. Nominal concentrations may be used only if stable (within about ±20%); otherwise use measured values. If concentrations decline significantly, LC50 cannot be reliably defined.
- Stock solutions & solvents: Prepare defined stock solutions; if organic solvent is needed, limit solvent to ≤0.1 mL/L and include solvent controls in addition to plain controls.
- Endpoints & observations: Record mortality at least twice daily; remove dead fish promptly. Estimate LC50 by probit or appropriate statistical method.
Applications and users
- Environmental and ecotoxicology laboratories performing regulatory acute toxicity testing.
- Chemical manufacturers and CROs conducting fish acute toxicity studies for registration (REACH, national regulations).
- Water quality and environmental monitoring agencies assessing hazard of single substances to freshwater fish.
- Researchers validating ecotoxicological test methods and comparing species sensitivity.
Related standards
- EN ISO 7346‑1: Static method (Part 1)
- EN ISO 7346‑2: Semi‑static method (Part 2)
- Guidance documents on LC50 estimation and laboratory QA/QC for ecotoxicity testing
Keywords: EN ISO 7346-3:1997, ISO 7346-3, flow-through method, acute lethal toxicity, Brachydanio rerio, zebra fish, LC50, water quality, toxicity testing.