ISO 7346-1:1996

Overview

ISO 7346-1:1996 - Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio) - Part 1: Static method - specifies a standardized laboratory procedure for measuring acute lethal toxicity (24 h, 48 h, 72 h and 96 h LC50) of stable, non‑volatile, single substances that are soluble in water. The test uses the zebra fish (Brachydanio rerio) as the reference organism (but may be adapted to other freshwater, marine or brackish species). This second edition updates and replaces the 1984 edition and is one part of a three‑part series (static, semi‑static and flow‑through methods).

Key topics and requirements

• Test principle: Two‑stage approach - a preliminary range‑finding test followed by a final definitive test to determine LC50 values (graphical, probit or other statistical analysis).
• Organism: Zebra fish (B. rerio), typical size ~30 mm ±5 mm (~0.3 g ±0.1 g). Other acceptable species listed in the standard include Lepomis macrochirus, Oryzias latipes, Pimephales promelas and Poecilia reticulata.
• Test conditions:
  • Static exposure: no renewal of test solution; suitable only where test concentrations remain relatively constant.
  • Temperature: 23 °C ± 1 °C.
  • Dissolved oxygen: initially aerate dilution water to ≥90% air saturation; during test DO must be ≥60% ASV.
  • Loading rate: not to exceed 1 g fish per litre.
  • Dilution water composition: pH 7.8 ± 0.2, calcium hardness ≈ 250 mg/L as CaCO3; typical salts specified (e.g., CaCl2·2H2O 294.0 mg/L, MgSO4·7H2O 123.3 mg/L, NaHCO3 63.0 mg/L, KCl 5.5 mg/L).
• Stock solutions & solvents: stock solutions prepared in deionized/distilled water; where low solubility requires a solvent, organic solvent concentration in tests must not exceed 0.1 mL/L (or 0.1 g/L), and solvent controls are required.
• Vessels & loading: vessels sized to provide large air–water interface (example 800 cm² per 10 L) and close‑fitting covers; cleanliness and inert materials required.
• Validity criteria: control mortality ≤10%, control abnormal behaviour ≤10%; concentration stability criteria (nominal vs measured) and reference chemical checks (e.g., potassium dichromate) for laboratory performance.
• Endpoints & reporting: report 24/48/72/96 h LC50, confidence limits or slope if required; use appropriate graphical/probit analysis and document environmental and analytical conditions.

Applications and users

• Environmental toxicology laboratories performing aquatic toxicity testing and water quality assessments
• Regulatory agencies and risk assessors establishing acute toxicity endpoints for chemical registration, environmental monitoring and compliance
• Chemical manufacturers, formulators and consultants generating toxicity data to support safety dossiers, product stewardship and environmental impact studies

Related standards

• ISO 7346-2: Semi‑static method (renewal every 24–48 h)
• ISO 7346-3: Flow‑through method (continuous replenishment)
• Other ISO/OECD aquatic toxicity methods used for comparative regulatory testing

Keywords: ISO 7346-1:1996, zebra fish, Brachydanio rerio, static method, LC50, acute lethal toxicity, water quality testing, aquatic toxicity testing, environmental monitoring.