EN ISO 23753-1:2019 PDF

Overview

EN ISO 23753-1:2019 (CEN adoption of ISO 23753-1:2019) specifies a standardized laboratory method for measuring dehydrogenase activity in soils using 2,3,5‑triphenyltetrazolium chloride (TTC). Dehydrogenases are intracellular microbial enzymes linked to respiratory activity; their activity is used as an indicator of viable, physiologically active soil microbial communities and overall soil quality.

Key topics and technical requirements

• Principle: TTC is added to a soil sample; microbially mediated reduction produces triphenylformazan (TPF), which is extracted with ethanol and quantified by spectrophotometry at 485 nm.
• Core conditions:
  • Incubation at 25 °C ± 1 °C for 6 h.
  • Substrate (TTC) prepared in Tris buffer (100 mmol/l, pH 7.6); recommended TTC concentration in the procedure is noted (e.g., 300 mmol/l preparation, 10%).
  • Extraction solvent: analytical grade ethanol (96 %) is specified; other solvents (e.g., acetone) are noted as alternatives.
• Reagents and calibration:
  • Preparation of Tris buffer (100 mmol/l, pH 7.6), TTC substrate solution, and TPF stock/working solutions for standard curves.
  • Establishment of a TPF standard curve for quantification and validity checks.
• Sampling and handling:
  • Follow ISO 18400‑206 for soil collection and handling.
  • If sieved fresh samples cannot be analysed immediately, storage up to 7 days at 4 °C ± 2 °C is acceptable.
  • Dry matter determination per ISO 11465.
• Limitations and validity:
  • Abiotic reducers (e.g., Fe(II), sulfides), coal or carbonaceous materials, or very organic-rich substrates can bias results; mitigation checks (spiking with TPF and recovery) are recommended.
  • Storage time affects enzyme activity - samples with different storage times should not be compared.

Applications and users

This TTC-based soil enzyme assay is used for:

• Environmental and agricultural soil quality monitoring
• Research on soil microbial activity, biogeochemical cycles and soil management impacts
• Assessing effects of contamination, remediation and land-use changes Primary users include environmental laboratories, academic researchers, agronomists, soil ecologists, and regulatory agencies involved in soil health and ecosystem assessments.

Related standards

• ISO 11465 - Determination of dry matter and water content (gravimetric)
• ISO 18400‑206 - Sampling: collection, handling and storage for microbiological assessment
• EN ISO 23753‑1:2019 supersedes the previous edition (EN ISO 23753‑1:2011)

Keywords: soil quality, dehydrogenase activity, TTC method, triphenyltetrazolium chloride, soil enzyme assay, ISO 23753-1, microbial activity assay.