ISO 21115:2019

Overview

ISO 21115:2019 - Water quality: Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1) describes a standardized in vitro assay to assess fish acute toxicity using the permanent rainbow trout gill cell line RTgill-W1. The method exposes confluent monolayers in 24‑well plates to water samples (surface waters, effluents) or chemicals for 24 hours, then quantifies cell viability by fluorescence. Results are reported as percent viability relative to unexposed controls and summarized with dose/response metrics (e.g., ECx).

Key technical topics and requirements

• Cell model and format: RTgill‑W1 permanent cell line seeded in 24‑well tissue culture plates; requires routine sterile cell culture techniques.
• Exposure media: Use of protein‑free L‑15/ex for chemical testing and adjusted whole‑water sample/ex (w‑ws/ex) for direct water samples to control osmolality.
• Exposure time: 24 hours (standardized acute exposure).
• Viability assessment: Trio of fluorescent indicator dyes measuring complementary endpoints:
  • alamarBlue (resazurin) - metabolic activity
  • CFDA‑AM - cell membrane integrity
  • Neutral red - lysosomal membrane integrity
• Controls and validity:
  • Negative (L‑15/ex) and solvent controls (e.g., DMSO when used)
  • Positive control (e.g., 3,4‑Dichloroaniline used in validation) to demonstrate assay performance
  • No‑cells controls to determine background fluorescence
• Data handling: Fluorescence readings expressed as percent viability vs control; toxicity quantified by response curves and reported ECx values (e.g., EC50).
• Interferences: Guidance on matrix effects from effluents and potential chemical interference with fluorescent dyes; includes sampling and chemical analysis steps.
• Quality criteria: Performance criteria and acceptance limits for controls and assay reproducibility.

Practical applications and users

ISO 21115:2019 is designed for:

• Environmental testing laboratories assessing effluent or surface water toxicity
• Regulatory and compliance monitoring for water quality and discharge permits
• Chemical safety testing and ecotoxicology screening by manufacturers and CROs
• Research groups developing alternative methods to reduce animal testing

Benefits include reduced animal use, faster turnaround, smaller sample volumes and less waste compared to whole‑animal assays. Users must have cell culture facilities and trained personnel.

Related standards

• ISO 15088 and OECD 236 - zebrafish embryo assays for fish acute toxicity (comparable alternative tests)
• ISO 21115 complements these methods in regulatory or screening contexts where cell‑based assays are preferred.

Keywords: ISO 21115:2019, RTgill‑W1, fish gill cell line, water quality, acute toxicity, cell viability assay, alamarBlue, CFDA‑AM, neutral red, L‑15/ex, effluent testing, EC50.