🌿 Chlorophyll a in Water

Physicochemistry PN-ISO 10260

Overview

Chlorophyll a is the main photosynthetic pigment present in all photoautotrophic organisms — algae, cyanobacteria, and aquatic plants. Its concentration in water is a widely used indicator of phytoplankton biomass and degree of eutrophication of water bodies. Trophic classification of waters is largely based on chlorophyll a concentration: oligotrophic waters (<2.5 µg/L), mesotrophic (2.5–8 µg/L), eutrophic (8–25 µg/L), and hypereutrophic (>25 µg/L).

The spectrophotometric method according to PN-ISO 10260 involves concentrating phytoplankton on glass fiber filter (GF/C or GF/F), extracting pigments with acetone (90%), and measuring absorbance at 665 nm (chlorophyll a absorption maximum) with turbidity correction at 750 nm. Acidification of extract with HCl allows distinguishing active chlorophyll a from its degradation products (pheophytin a).

Chlorophyll a monitoring is required by Water Framework Directive (2000/60/EC) as element of biological assessment of surface water status. In Poland, this parameter is mandatory in ecological status assessment of lakes and dam reservoirs (Journal of Laws 2019 item 2149).

Alternatively, chlorophyll a can be determined fluorometrically (more sensitive, LOD ~0.1 µg/L) or chromatographically (HPLC — separation of individual pigments). However, the spectrophotometric method remains reference method due to simplicity and low cost.

Method principle

Phytoplankton from defined water volume (0.5–2 L) is concentrated on glass fiber filter (GF/C, porosity 1.2 µm or GF/F, 0.7 µm) by vacuum filtration. Filter with residue is homogenized in 90% acetone and extracted in darkness at 4°C for 12–24 hours. Extract is clarified by centrifugation (500 g, 20 min). Absorbance is measured at 665 nm (chlorophyll a) and 750 nm (turbidity correction). Then extract is acidified with 0.1 M HCl (0.1 mL per 10 mL extract) and after 90 seconds absorbance is measured again at 665 and 750 nm. Difference in absorbance before and after acidification allows calculation of active chlorophyll a and pheophytin a concentrations. Calculations: Chl a [µg/L] = 26.7 × (A665₀ - A665a) × V_extract / (V_sample × l), where l — optical path length.

Applications

• Eutrophication monitoring of lakes and dam reservoirs (WFD 2000/60/EC)
• Trophic classification of surface waters (oligotrophic → hypereutrophic)
• Ecological status assessment of waters — biological element (Journal of Laws 2019 item 2149)
• Algal and cyanobacterial bloom monitoring (toxin risk)
• Water quality control at surface water intakes
• Limnological and oceanographic research

Key parameters

Standard

Standard number

PN-ISO 10260:2002

Title (PL)

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

Title (EN)

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

Step-by-step procedure

1. Water sample collection

   Collect representative water sample (1–5 L) from established depth. Store in darkness at 4°C. Filter within 4 hours of collection.

   🌡 Temperature: 4°C

2. Filtration

   Place GF/C filter (47 mm) on vacuum filtration apparatus. Add pinch of MgCO₃ to filter. Filter measured sample volume (0.5–2 L) under vacuum (<0.3 bar).

   ⏱ Time: 15–60 min

3. Acetone extraction

   Transfer filter with residue to centrifuge tube. Add 10 mL 90% acetone. Homogenize filter with Ultra-Turrax (30 s). Close, wrap with aluminum foil.

   ⏱ Time: 5 min

4. Incubation in darkness

   Place tubes in refrigerator (4°C) for 12–24 hours in darkness. Acetone penetrates cells and extracts pigments.

   ⏱ Time: 12–24 h • 🌡 Temperature: 4°C

5. Extract clarification

   Centrifuge extract (500 g, 20 min) or filter through GF/F filter. Transfer clear extract to clean vial.

   ⏱ Time: 25 min

6. Absorbance measurement (before acidification)

   Measure absorbance at 665 nm and 750 nm against 90% acetone (blank). Record values A665₀ and A750₀.

   ⏱ Time: 3 min

7. HCl acidification

   Add 0.1 mL 0.1 M HCl to cuvette with 10 mL extract. Gently mix by inversion. Wait exactly 90 seconds.

   ⏱ Time: 90 s

8. Absorbance measurement (after acidification)

   Measure absorbance at 665 nm and 750 nm. Record values A665a and A750a. Acidification converts chlorophyll → pheophytin.

   ⏱ Time: 3 min

9. Calculations

   Chl a [µg/L] = 26.7 × [(A665₀ - A750₀) - (A665a - A750a)] × V_extract [mL] / (V_sample [L] × l [cm]). Pheophytin a = 26.7 × [1.7 × (A665a - A750a) - (A665₀ - A750₀)] × V_e / (V_p × l).

   ⏱ Time: 5 min

10. Quality control

    Chlorophyll a standard in acetone (known concentration) — recovery 90–110%. Acetone blank A665 < 0.005. Duplicate: CV < 15%.

    ⏱ Time: 10 min

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Acetone — flammable (flash point -20°C), vapors form explosive mixtures, work under fume hood!
• Hydrochloric acid — corrosive, irritant vapors, use in ventilated area
• Water samples — potentially contain cyanobacterial toxins (microcystins), gloves
• Safety glasses, lab coat, and nitrile gloves mandatory
• Collect acetone waste in flammable organic waste container

Frequently asked questions