💧 Determination of the methylene blue active substances (MBAS) index by continuous flow analysis (CFA) — the sample in the stream is mixed with an alkaline methylene blue solution, the ion pairs formed (mainly with anionic surfactants) are extracted with trichloromethane, the organic phase is treated with an acidic methylene blue solution and measured photometrically at (650 ± 10) nm; result as sodium dodecyl sulfate, ranges (0,05–0,5) mg/l and (0,5–5,0) mg/l, PN-EN ISO 16265

Physicochemistry PN-EN ISO 16265

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 16265:2009 (English): from the title page through the contents, foreword, introduction and Clauses 1–4 up to and including 5.21 (methanol solution) on page 3. Outside the sample remain apparatus (6), sampling and sample pretreatment (7), procedure (8), calculation (9), expression of results (10), precision (11), test report (12) and Annexes A (example flow diagram) and B (precision data).

ACCORDING TO THE TEXT OF ISO 16265:2009 (foreword, introduction, Clauses 1–5). The standard was prepared by ISO/TC 147 “Water quality”, subcommittee SC 2 (first edition, 2009-03-01). Introduction: flow methods automate wet chemistry and suit large series (up to 100 samples per hour); flow injection analysis (FIA) and continuous flow analysis (CFA) are distinguished; the MBAS index is an analytical convention (a method-defined parameter) — it measures surfactants and other substances reacting with methylene blue under specified conditions. Warning: trichloromethane and methanol waste must be disposed of properly. Scope (1): the MBAS index in the ranges 0,05 mg/l to 0,5 mg/l and 0,5 mg/l to 5,0 mg/l in various waters (e.g. ground, drinking, surface water, waste water and leachates); anionic surfactants are the most important methylene blue active substances, so the method serves to estimate their content (including surfactants with carboxylate groups, e.g. soaps); other substances may also react and contribute to the result; case by case the range may be changed if it covers exactly one decade of concentration. References (2): ISO 648, ISO 1042, ISO 3696. Interferences (3): cationic compounds forming strong ion pairs with the active substances; humic acids above 20 mg/l; substances with high surface activity (e.g. non-methylene-blue-active surfactants above 50 mg/l); strongly reducing substances (e.g. S2−, SO32−, S2O32−, OCl−) — they must be removed before analysis (e.g. with H2O2); high concentrations of inorganic anions (e.g. nitrate, chloride, bromide) may cause positive bias; substances forming with the reagents coloured compounds soluble in trichloromethane. Filtration is advisable for particles larger than 100 µm (they may clog the tubes), but may cause significant losses of anionic surfactants by adsorption. The colour of the sample is compensated by a blank analysis without methylene blue in the reagents, and the difference in response is used in Equations (3) and (4). Samples with an MBAS index above 5 mg/l must be diluted before analysis. Principle (4): the sample in a continuous stream is mixed with an alkaline methylene blue solution — coloured ion pairs form with certain organic substances (e.g. anionic surfactants); the ion pairs are extracted with trichloromethane, the organic phase is treated with an acidic methylene blue solution and measured photometrically at (650 ± 10) nm; the result is expressed as sodium dodecyl sulfate concentration. Reagents (5): degassing with helium at 20 l/h for 15 min when bubbles appear, detergent added after degassing; grade 1 water according to ISO 3696; among others sodium tetraborate, sodium and potassium dihydrogen phosphates, sulfuric acid 1,84 g/ml, hydrochloric acid 1,18 g/ml, methylene blue, methanol, ethanol approximately 96 %, trichloromethane (stable 3 months in the dark, degassed 30 min before use), sodium dodecyl sulfate, petroleum ether, poly(ethylene glycol) dodecyl ether 30 %; alkaline borate solution (15,83 g tetraborate and 3,3 g NaOH per 1000 ml, stable one week); stock methylene blue solution (0,35 g in 500 ml ethanol, to 1000 ml, stable 6 months); alkaline methylene blue solution (100 ml stock solution + 50 ml borate solution, washed three times with 20 ml trichloromethane, extracted with 25 ml petroleum ether, filtered through a 0,45 µm filter, to 500 ml — prepared fresh before use); acidified methylene blue solution (6,8 ml sulfuric acid, 42,5 ml stock solution, 50 g sodium dihydrogen phosphate, to 1000 ml, stable one week); trichloromethane extraction solution (1 ml poly(ethylene glycol) dodecyl ether per 1000 ml, stable one week); sampler rinsing solution (1,1 g potassium dihydrogen phosphate per 1000 ml, stable one week); methanol solution (800 ml methanol + 100 ml hydrochloric acid, to 1000 ml, stable 6 months).

STATUS (as of 03.10.2026). The PKN search (term “PN-EN ISO 16265”, 03.10.2026) shows PN-EN ISO 16265:2012 (English version, “Introduces: EN ISO 16265:2012 [IDT], ISO 16265:2009 [IDT]”) without a withdrawal note; the second item is PN-EN 16265:2016-01 on pyrotechnic articles — a different standard with the same number. In the iTeh catalogue (read on 03.10.2026) ISO 16265:2009 has the status “Published” and stage 90.93 “International Standard confirmed” (start date 07.04.2026). The manual method for anionic surfactants with methylene blue is described in a separate entry on ISO 7875-1, and non-ionic surfactants in the entry on PN-ISO 7875-2.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read on 03.10.2026). The designation “ISO 16265” (excluding the standards on pyrotechnic articles with the same number) appears in 5 records at 4 laboratories: AB 005, AB 313, AB 1095 (2 records), AB 1448. In the current PCA documents (read on 03.10.2026, issues from 18.12.2025 to 31.07.2026) it appears in 5 rows of the same laboratories (AB 1095 — 2, the others 1 each). The form “PN-EN ISO 16265:2012” — at all of them; at AB 1095 for the sum of surfactants together with procedure PB-12/LF issue 4 of 7.01.2026. None of these items is suspended (AB 1448 has a suspension in another part of its scope). The “Laboratories” tab (“PN-EN ISO 16265”) shows the same 4 laboratories — checked with the tab query on the production server labcoda.pl on 03.10.2026.

WHAT THE LABORATORIES TEST (items in PCA). Water and waste water (AB 005, AB 313, AB 1448), water, water intended for human consumption and waste water (AB 1095) — concentration of anionic surface-active substances (detergents, anionic surfactants), at AB 1448 “anionic surfactants (MBAS index)”; ranges: (0,010–100) mg/l (AB 005), (0,05–50) mg/l (AB 313, AB 1448), (0,050–50) mg/l (AB 1095); technique — continuous flow analysis (CFA) with spectrophotometric detection. At AB 1095 also waste water — sum of anionic and non-ionic surfactants (calculated). The upper limits of 50 and 100 mg/l exceed the 5,0 mg/l of Clause 1 — the standard requires such samples to be diluted (3); the lower limit of 0,010 mg/l at AB 005 lies below the 0,05 mg/l of Clause 1 (Clause 1 allows other ranges covering one decade).

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. With interpretation: the MBAS index is a conventional parameter — it also includes other substances reacting with methylene blue, and cationic compounds and humic acids above 20 mg/l interfere (introduction, 1, 3). With the sample: strongly reducing substances must be removed before analysis; filtration for particles > 100 µm may cause surfactant losses by adsorption; above 5 mg/l — dilution (3). With sample colour: blank analysis without methylene blue (3). With reagents: the alkaline methylene blue solution prepared fresh, the other solutions have specified shelf lives (one week, 6 months), trichloromethane degassed before use (5).

WHAT WE DO NOT GIVE. We did not read the apparatus and flow diagram, sample preparation, course of the determination, Equations (3) and (4), precision data or the content of the test report. Nor do we give limit values for surfactants from water and waste water legislation.

Method principle

The sample in a continuous stream is mixed with an alkaline methylene blue solution; methylene blue active substances (mainly anionic surfactants) form coloured ion pairs, which are extracted with trichloromethane. The organic phase is treated with an acidic methylene blue solution and measured photometrically at (650 ± 10) nm; the result is expressed as sodium dodecyl sulfate.

Applications

• Ground, drinking and surface water, waste water and leachates — MBAS index (0,05–0,5) and (0,5–5,0) mg/l (1)
• Estimation of anionic surfactant content, including soaps (1)
• Water, water intended for human consumption and waste water — anionic surfactants by CFA in the accreditation scopes of 4 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 16265:2012

Title (PL)

Jakość wody — Oznaczanie indeksu substancji aktywnych względem błękitu metylenowego (MBAS) — Metoda z zastosowaniem ciągłej analizy przepływowej (CFA)

Title (EN)

Water quality — Determination of the methylene blue active substances (MBAS) index — Method using continuous flow analysis (CFA)

Step-by-step procedure

1. Sample

   Remove strongly reducing substances; above 5 mg/l dilute; filter if necessary. PN-EN ISO 16265:2012 without a withdrawal note (03.10.2026).

2. Reagents

   Prepare the methylene blue, borate and extraction solutions; degas if bubbles appear.

3. Reaction and extraction

   In the stream: sample + alkaline methylene blue, extraction of ion pairs with trichloromethane.

4. Measurement

   Organic phase with acidified methylene blue; photometry at (650 ± 10) nm.

5. Result

   As sodium dodecyl sulfate; we do not give the equations and precision data (Clauses 9–11).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Trichloromethane and methanol — dispose of waste properly (warning of the standard); work in a fume hood
• Concentrated sulfuric and hydrochloric acid — eye and skin protection

Frequently asked questions