EN ISO 7346-1:1997 PDF

Overview

EN ISO 7346-1:1997 (ISO 7346-1:1996) specifies a 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 test design and reporting for calculating 24 h, 48 h, 72 h and 96 h LC50 values (median lethal concentrations). It replaces the first edition and is part of a three‑part series covering static, semi‑static and flow‑through procedures for freshwater fish toxicity testing.

Key topics and technical requirements

• Test principle: Two-stage approach - preliminary range‑finding test followed by a definitive final test to allow LC50 estimation (probit or equivalent statistical methods).
• Test organism: Brachydanio rerio (zebra fish); typical specimen size ~30 mm ± 5 mm (≈0.3 g ± 0.1 g). Several other freshwater species are indicated as usable without modification.
• Static conditions: No renewal of test solution during exposure; applicable only when test concentrations remain relatively constant.
• Exposure times: 24, 48, 72 and 96 hours for determination of LC50 endpoints.
• Dilution water: Freshly prepared standard dilution water with pH 7.8 ± 0.2 and calcium hardness ≈ 250 mg/L expressed as CaCO3. Example salt composition provided (e.g. 294.0 mg/L CaCl2·2H2O; 123.3 mg/L MgSO4·7H2O; 63.0 mg/L NaHCO3; 5.5 mg/L KCl).
• Temperature and aeration: Temperature maintained at 23 °C ± 1 °C; dilution water aerated until dissolved oxygen ≥ 90% air saturation before use; during test DO must remain ≥ 60% ASV.
• Stock and test solutions: Guidance on preparing stock solutions, use of low‑toxicity organic solvents when required (maximum solvent concentration ≤ 0.1 mL/L or 0.1 g/L, whichever is greater) and limits on stock solution addition volumes.
• Quality criteria & validity: Control mortality ≤ 10% (or one per tank), control abnormal behaviour ≤ 10% (or one per tank); concentrations must be known or shown stable (within ~20% for nominal use) or analytical values used.
• Limit test: Procedure to show LC50 > limit of solubility or 100 mg/L using 10 fish.

Applications and who uses it

• Regulatory and compliance testing for chemical registration, environmental risk assessment and safety data sheets.
• Contract laboratories and academic researchers performing freshwater fish toxicity testing, water quality assessment and ecotoxicology studies.
• Chemical manufacturers, environmental consultants and agencies needing standardized LC50 data for zebra fish or comparable freshwater species.

Related standards

• EN/ISO 7346-2: Semi‑static method
• EN/ISO 7346-3: Flow‑through method

This standard is essential when conducting reproducible, internationally recognized tests for acute toxicity in freshwater fish under static exposure conditions and for generating LC50 endpoints used in environmental hazard assessment.