Overview
ISO 23753-2:2019 - Soil quality - Determination of dehydrogenases activity in soils - Part 2: Method using iodotetrazolium chloride (INT) specifies a standardized laboratory method to quantify microbial dehydrogenase activity in soils using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT). The method measures reduction of INT to iodonitrotetrazolium formazan (INTF), extracted with acetone and quantified by spectrophotometry (λ = 485 nm). Because INT reduction is less sensitive to oxygen than the TTC method, this approach is more robust for assessing soil microbial oxidative activity and viable microbial biomass.
Key topics and technical requirements
- Principle: INT added to sieved soil, incubated at 25 °C ± 1 °C for 4–6 h; INTF extracted with acetone and measured spectrophotometrically at 485 nm.
- Reagents: Tris buffer 100 mmol/L, pH 7.6; INT substrate solution (15 mmol/L); INTF stock (1 µmol/mL) for standard curve.
- Sample prep: Sieved soil, typically 2 g (reduced) or 5 g (large sample) per tube; substrate and control tubes in triplicate.
- Volumes & timings: Substrate or buffer volumes (2 or 5 mL) plus acetone extraction (8 or 20 mL); 1 h orbital agitation during extraction; centrifuge 5 min at 2 000 g, 20 °C.
- Standard curve & calculation: Calibration using INTF standards (0–80 nmol/mL) to derive INTF concentration and calculate activity in mU/g (nmol/min/g dry soil).
- Validity criteria: Standard curve R² ≥ 0.990; maximum absorbance for 80 nmol/mL between 2.3–2.6; sample triplicate CV ≤ 15%; absorbance difference sample vs control ≥ 0.02–0.05; solution color between pink and red.
- Limitations: Storage affects enzyme activity - compare only samples with equivalent storage. Method may give low/variable results for organic-rich forest humus layers.
Applications and users
- Practical applications: Routine laboratory assessment of soil biological activity, monitoring effects of land management, contamination, remediation, and agricultural practices; research on microbial respiration and soil health indicators.
- Who uses it: Soil testing laboratories, environmental consultancies, agronomy and forestry researchers, regulatory bodies and practitioners assessing soil quality and microbial activity.
- Why use this method: Provides a reproducible, oxygen-tolerant measure of viable microbial oxidative activity that complements respiration and other biological soil quality indicators.
Related standards
- ISO 23753-1 (TTC method) - alternative tetrazolium-based dehydrogenase test.
- ISO 18400-206 - soil sampling and handling for microbiological assessment.
- ISO 11465 - dry matter and water content determination.
Keywords: ISO 23753-2:2019, soil dehydrogenase activity, iodotetrazolium chloride (INT), INTF, soil quality testing, spectrophotometry 485 nm, Tris buffer pH 7.6, microbial activity indicator.