Overview
EN ISO 23753-2:2019 - "Soil quality - Determination of dehydrogenases activity in soils - Part 2: Method using iodotetrazolium chloride (INT)" - is a standardized laboratory method adopted by CEN/ISO for measuring soil dehydrogenase activity, an indicator of viable microbial respiratory activity. The method adds an INT substrate to soil, incubates the mixture (25 °C ± 1 °C for 4–6 h), extracts the iodonitrotetrazolium formazan (INTF) with acetone and quantifies the product by spectrophotometry (485 nm). The INT method is more robust to oxygen interference than the TTC method (ISO 23753-1).
Key topics and technical requirements
- Scope: Quantitative determination of dehydrogenase activity in soils using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT).
- Principle: Microbial enzymes reduce INT to INTF; INTF is extracted with acetone and measured spectrophotometrically.
- Critical reagents: INT substrate (15 mmol/L prepared in Tris buffer 100 mmol/L, pH 7.6), Tris buffer (100 mmol/L, pH 7.6), INTF stock (1 µmol/mL in acetone), analytical acetone, HCl (1 mol/L).
- Apparatus: Visible-light spectrophotometer (λ = 485 nm), incubator (25 °C ± 1 °C), centrifuge (≈2 000 g, 20 °C ± 1 °C), ultrasonic probe, orbital shaker, glass tubes/cuvettes.
- Procedure highlights: Follow ISO 18400‑206 for sampling and ISO 11465 for dry matter determination; establish INTF calibration curve (duplicates/triplicates); incubate soil + INT; extract and measure.
- Limitations & validity:
- Storage affects enzyme activity - compare only samples with similar storage conditions.
- Upper forest humus layers or soils with very high organic matter may give low/variable results.
- Method includes validity criteria and standard-curve requirements to ensure data quality.
Applications and users
- Practical uses:
- Assess microbial activity and soil biological health in agriculture and forestry.
- Monitor soil response to management practices, organic amendments, or contamination/remediation.
- Baseline and trend monitoring in environmental impact assessments.
- Typical users:
- Environmental and soil testing laboratories
- Soil microbiologists and ecologists
- Agronomists and land managers
- Environmental consultants and regulatory bodies
Related standards
- ISO 23753-1 - TTC method for dehydrogenase activity (useful for method comparison; INT is less O2-sensitive).
- ISO 11465 - Determination of dry matter and water content (gravimetric).
- ISO 18400-206 - Soil sampling, handling and storage for microbiological assessment.
Keywords: EN ISO 23753-2:2019, soil quality, dehydrogenase activity, iodotetrazolium chloride, INT method, soil microbiology, enzyme activity assay, spectrophotometry.