ISO 23753-1:2019

Overview

ISO 23753-1:2019 - "Soil quality - Determination of dehydrogenases activity in soils - Part 1: Method using triphenyltetrazolium chloride (TTC)" specifies a standardized enzymatic assay to quantify soil microbial dehydrogenase activity. The method adds TTC to soil, incubates to form triphenylformazan (TPF), extracts the TPF and measures it by spectrophotometry at 485 nm. Dehydrogenase activity (DHA) is a widely used indicator of viable microbial oxidative activity and overall soil biological functioning.

Key topics and requirements

• Scope and principle: Measures intracellular dehydrogenases via TTC reduction to TPF; TPF quantified spectrophotometrically (λ = 485 nm).
• Sampling and sample prep: Follow ISO 18400-206 for collection/handling and ISO 11465 for dry matter determination. Sieved fresh soils may be stored up to 7 days at 4 °C ± 2 °C.
• Reagents: Tris buffer (100 mmol/l, pH 7.6), TTC substrate solution (300 mmol/l stock; final assay concentration 60 mmol/l), analytical ethanol (96 %), and TPF standard solutions for calibration.
• Apparatus: Visible-light spectrophotometer, incubator (25 °C ± 1 °C), orbital shaker, centrifuge (2 000 g, 20 °C ± 2 °C), and appropriate U‑bottom tubes.
• Procedure highlights:
  • Test portion: typically 5.00 g moist soil with 4 ml Tris buffer and 1 ml TTC (final 60 mmol/l).
  • Incubation: 6 h at 25 °C ± 1 °C.
  • Extraction: 25 ml ethanol, 1 h orbital agitation (250 rpm) in the dark; add TTC to control after extraction, centrifuge 5 min at 2 000 g, read supernatant at 485 nm.
  • Standard curve: TPF working concentrations (0, 11, 22, 55, 110 nmol/ml).
• Calculations & validity: DHA reported in nmol/min/g dry soil using the standard curve. Validity criteria include R² ≥ 0.990 for the calibration, maximum absorbance for 110 nmol/ml between 2.3–2.6, triplicate CV ≤ 15 %, and sample–control absorbance difference ≥ 0.02–0.05 (instrument-dependent).
• Limitations: Storage time affects activity; abiotic reducers (e.g., Fe(II), sulfides) and coal-like materials can give false positives; method may be unsuitable for peat, organic litter or carbon-rich sediments without preliminary testing.

Applications and users

• Who uses it: Environmental and soil testing laboratories, agronomy and land‑management researchers, contaminated land assessors, remediation consultants, and regulatory agencies.
• Why it’s used: To assess soil microbial health, monitor impacts of land management or contamination, evaluate biological recovery in remediation, and support research into soil biogeochemical processes.

Related standards

• ISO 11465 - Determination of dry matter and water content (gravimetric).
• ISO 18400-206 - Soil sampling for microbiological assessments.
• Other parts of the ISO 23753 series (see ISO website) for complementary DHA methods and contexts.

Keywords: ISO 23753-1:2019, dehydrogenase activity, TTC, triphenyltetrazolium chloride, TPF, soil quality, soil microbiology, enzyme assay, spectrophotometry.