🌿 Chlorophyll a in Water

Physicochemistry PN-ISO 10260

Overview

STATUS (as of 03.11.2026). PN-ISO 10260:2002 — Polish version, published on 20.12.2002, implements ISO 10260:1992 [IDT]; the PKN catalogue card has no note of withdrawal. ISO 10260:1992 (first edition) has the stage “confirmed after review” (90.93) in the ISO catalogue. Since 08.01.2025 there is also PN-EN 17899:2025-01 (English version; implements EN 17899:2024) — spectrophotometric determination of chlorophyll-a by ethanol extraction for routine monitoring of water quality, corrected for phaeopigments, for all types of surface water including marine water; its card does not state that it replaces PN-ISO 10260. This description concerns PN-ISO 10260.

SCOPE (clause 1). Chlorophyll-a in phytoplankton of natural surface waters and in algal growth tests; with appropriate sampling — also in phytobenthic communities (Annex A). Chlorophyll-b, chlorophyll-c and some metabolites do not contribute to the result; phaeopigments are determined semiquantitatively — to correct the chlorophyll-a determination and as an indicator of the portion of inactive algal biomass. Chlorophyll is sensitive to light and oxygen, especially after extraction, so samples are not exposed to bright light or to air. The pigment of certain rarely occurring phototrophic bacteria (e.g. Chlorobium) interferes with the determination.

PRINCIPLE (clause 3). Algae and other suspended matter are collected on a filter, the pigments are extracted with hot ethanol, and the concentrations of chlorophyll-a and phaeopigments are calculated from the difference in absorbance at 665 nm before and after acidification of the extract. Heating with ethanol to 75 °C for 5 min inactivates chlorophyllase and accelerates the lysis of the pigments. The absorbance at 665 nm is corrected by the absorbance at 750 nm, because slight turbidity may remain in the extract, also after acidification.

PROCEDURE (clauses 6 and 7). Samples are taken according to ISO 5667-1 and ISO 5667-2; refrigerated storage in the dark for less than 8 h is acceptable but should be avoided — filtration and extraction are best done immediately. A measured volume of sample (usually 0.1–2 l) is filtered, the filter is dried by vacuum and placed in the extraction vessel. Variant A: 30–40 ml of ethanol heated to 75 °C, grinding of the filter with a rod-shaped homogenizer, extraction for at least 3 min (usually several hours or overnight at room temperature), filtration into a 50 ml or 100 ml flask and making up with ethanol — the extract volume Ve. Variant B: an exactly measured volume of ethanol Ve (usually 20 ml or 25 ml) in a tightly closed vessel, 5 min in a 75 °C bath, 15 min cooling, filtration without rinsing or centrifugation. Extracts may be kept in a refrigerator overnight (no more than 3 days), and raw extracts in air-tight brown glass vessels below −25 °C for up to 30 days; frozen water samples and filters with solids are not stored.

MEASUREMENT AND CALCULATION (7.4, clause 8). The absorbance at 665 nm and 750 nm is measured against ethanol, then part of the extract is acidified (0.01 ml of hydrochloric acid 3 mol/l per 10 ml) and measured again after 5–30 min. The absorbance at 665 nm should be 0.01–0.8 — to start with, the standard suggests 0.5 l of sample, a 50 mm filter, 20 ml of ethanol and a 5 cm cuvette. The chlorophyll-a concentration ρc = (A − Aa) × 29.6 × Ve/(Vs × d), and that of phaeopigments ρp = Aa × 20.8 × Ve/(Vs × d) − ρc, where A and Aa are the absorbances (665 nm minus 750 nm) before and after acidification, Ve — the extract volume in ml, Vs — the volume of filtered sample in l, d — the optical path length in cm. The factors result from the operational absorption coefficient of chlorophyll-a in 90 % ethanol (82) and the ratio A/Aa = 1.7 for pure chlorophyll-a. The result is reported in µg/l (mg/m³) with at most two significant figures or one decimal place. In interlaboratory trials the variation between laboratories was 5–11 % for chlorophyll-a and 6–46 % for phaeopigments.

WHY ETHANOL (Annex A.2). Acetone, recommended since 1966 by UNESCO-SCOR, extracts poorly in some cases; hot ethanol and methanol are equally efficient and considerably more efficient than cold acetone, but methanol causes problems with acidification and is toxic — therefore the standard recommends ethanol as the reference extractant for freshwater and marine samples. The absorbance of chlorophyll-a in acetone at 665 nm is 2–3 % higher than in ethanol.

REGULATIONS AND GUIDELINES. Monitoring of water bodies: the reference method for the indicator chlorophyll “a” (1.1.5) is the spectrophotometric method according to PN-ISO 10260 and the HELCOM guide to chlorophyll-a monitoring (Dz.U. 2021 item 1576, Annex 8). In the classification of ecological status, chlorophyll-a is an indicator of the “phytoplankton” element for transitional and coastal waters, with class boundary values in µg/l (Dz.U. 2021 item 1475, Annexes 9 and 10). The HELCOM guidelines (2017; second source) are based on ISO 10260 but use ethanol of at least 96 % (10 ml, without drying the filter), GF/F filters, centrifugation of the extract and measurement without correction for phaeopigments, with an absorption coefficient of 83 for 96 % ethanol and an absorbance of 0.1–0.8 at 663–665 nm.

WHAT WE DO NOT GIVE HERE. Trophic classes of waters or “typical” chlorophyll concentrations — the standard does not give them; the class boundary values for transitional and coastal waters are contained in the regulation Dz.U. 2021 item 1475.

Method principle

Algae from a measured volume of water are collected on a glass-fibre filter, and the pigments are extracted with hot ethanol (90 %, 75 °C). The absorbance of the extract is measured at 665 nm and at 750 nm (turbidity correction) before and after acidification with hydrochloric acid. The chlorophyll-a concentration is calculated from the difference in absorbance, and that of phaeopigments from the absorbance after acidification: ρc = (A − Aa) × 29.6 × Ve/(Vs × d).

Applications

• Phytoplankton of natural surface waters (clause 1)
• Algal growth tests (clause 1)
• Phytobenthos — with appropriate sampling (clause 1, Annex A)
• Monitoring of water bodies — reference method for the indicator chlorophyll “a” (Dz.U. 2021 item 1576, Annex 8 point 1.1.5)
• Classification of the ecological status of transitional and coastal waters — indicator of the “phytoplankton” element (Dz.U. 2021 item 1475, Annexes 9 and 10)

Key parameters

Standard

Standard number

PN-ISO 10260:2002

Title (PL)

Jakość wody — Pomiar parametrów biochemicznych — Spektrometryczne oznaczanie stężenia chlorofilu a

Title (EN)

Water quality — Measurement of biochemical parameters — Spectrometric determination of the chlorophyll-a concentration

Step-by-step procedure

1. Sampling

   Take the sample according to ISO 5667-1 and ISO 5667-2; protect it from bright light and air; refrigerated storage in the dark for less than 8 h is acceptable, but filtration and extraction are best done immediately (1.3, clause 6).

2. Filtration

   Mix the sample and filter a measured volume Vs (usually 0.1–2 l) through a glass-fibre filter; dry the filter by vacuum and place it in the extraction vessel without touching it with fingers (7.1).

3. Extraction — variant A

   Pour 30–40 ml of ethanol heated to 75 °C onto the filter, after a few minutes grind it with the homogenizer, extract for at least 3 min, filter into a 50 ml or 100 ml flask, rinse the vessel with ethanol and make up to the mark — this is the volume Ve (7.2).

4. Extraction — variant B

   Add an exactly measured volume of ethanol Ve (usually 20 ml or 25 ml), close tightly, heat for 5 min in the 75 °C bath, cool for 15 min, filter without rinsing or centrifuge to a clear extract (7.3).

5. Measurement before acidification

   Measure the absorbance of the clear extract at 665 nm and 750 nm against ethanol; the absorbance at 665 nm should be 0.01–0.8 — choose the sample volume, extractant volume and cuvette accordingly (7.4.1).

6. Acidification and second measurement

   Acidify part of the extract (usually 5–10 ml) with hydrochloric acid 3 mol/l — 0.01 ml per 10 ml — and after 5–30 min measure the absorbance at 665 nm and 750 nm again (7.4.2).

7. Calculation

   ρc = (A − Aa) × 29.6 × Ve/(Vs × d), ρp = Aa × 20.8 × Ve/(Vs × d) − ρc, where A = A665 − A750 before acidification and Aa — after acidification; result in µg/l with at most two significant figures (clause 8).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Ethanol — harmonised CLP classification: H225 (highly flammable liquid and vapour); in variant B the extraction in the 75 °C bath is carried out in tightly closed vessels
• Hydrochloric acid for acidification is prepared according to its safety data sheet

Frequently asked questions