🦠 Microcystins (cyanobacterial toxins) in water — solid-phase extraction and HPLC with UV detection at 238 nm
In short
Water with cyanobacterial biomass is filtered, the biomass is extracted with a methanol/water 75/25 mixture, and the filtrate and the treated water are concentrated on a C-18 SPE cartridge; the microcystins are eluted with methanol/water 90/10 containing 0. The test is performed according to ISO 20179:2005; Range of the standard solutions: 0.2; 0.4; 1.0; 2.0 and 3.0 µg/ml of each microcystin (Table 3 of the standard). The procedure comprises 10 steps (about 55 min in total); it is used for: Control of water intended for consumption during a cyanobacterial bloom in the water source (Journal of Laws 2026 item 748: parametric value 1.0 µg/l for microcystin-LR), Raw water from surface intakes — lakes and impoundment reservoirs liable to blooms, Assessment of the efficiency of water treatment with regard to the removal of cyanobacterial toxins.
At a glance
- Standard: ISO 20179:2005
- Category: Microbiology
- Procedure steps: 10
- Total time of stages: 55 min
- Validated analytes: MCYST-RR, MCYST-YR, MCYST-LR; other structural variants — determinable, but without unambiguous identification (no commercial standards, co-elution)
- Matrix: Raw water containing biomass and treated water (e.g. tap water)
- Detection: RP-HPLC with UV / diode-array detection at 238 nm (alternatively mass spectrometry — Annex A of the standard)
Overview
Microcystins are toxins produced by cyanobacteria; the standard opens with a warning that they are strongly hepatotoxic to humans. The names of the variants come from two variable amino acids: MCYST-LR contains leucine (L) and arginine (R), MCYST-RR two arginine units, and MCYST-YR tyrosine (Y) and arginine. For microcystin-LR the World Health Organization has proposed a threshold value of 1 µg/l in water; the standard states that after concentration of the sample by SPE this value can be controlled.
ISO 20179:2005 describes the determination and quantification of microcystins in raw water containing biomass and in treated water such as tap water. The method is validated for three variants: MCYST-RR, MCYST-YR and MCYST-LR. It can also be applied to several other structural variants of these microcystins, but their unambiguous identification is not possible — commercial standards are lacking and the peaks co-elute. In Annex A the standard permits mass spectrometry as an alternative detection.
In Poland microcystin-LR is a chemical parameter of water intended for consumption: the Regulation of the Minister of Health of 22 May 2026 (Journal of Laws 2026 item 748) sets a parametric value of 1.0 µg/l with the note that „parametr ten mierzy się jedynie w przypadku potencjalnych zakwitów w źródle wody (zwiększenie gęstości komórek sinicowych lub potencjał tworzenia zakwitu)" [this parameter is measured only in the case of potential blooms in the water source (an increase in the density of cyanobacterial cells or a bloom-forming potential)], and sets the permissible measurement uncertainty at 30 % of the parametric value. The regulation does not indicate a reference method for this parameter — reference methods are named only for the microbiological parameters. ISO 20179:2005 itself has not been adopted into the collection of Polish Standards; the first edition remains current and was confirmed by ISO in a systematic review completed on 7 April 2026 (stage 90.93 "International Standard confirmed"). In the life-cycle card of the standard neither a superseding standard nor a corrigendum (Cor/Amd) has been recorded.
THE POLISH TITLE IN THIS ENTRY IS OUR WORKING TRANSLATION, not an official title: the standard has not been adopted into the collection of Polish Standards.
Method principle
Water samples containing cyanobacterial material (biomass) are first filtered. The biomass retained is extracted separately with a methanol/water mixture, the extract is filtered, diluted and cleaned up by solid-phase extraction (SPE); the filtrate after the separation of the biomass is treated like a sample of clean water. Samples of clean water, e.g. tap water, are concentrated directly on an SPE cartridge. The microcystins are eluted from the cartridge with a methanol/water 90/10 (v/v) mixture containing 0.1 % by volume of trifluoroacetic acid (TFA), and determined quantitatively by reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet / diode-array detection at a wavelength of 238 nm.
Applications
- Control of water intended for consumption during a cyanobacterial bloom in the water source (Journal of Laws 2026 item 748: parametric value 1.0 µg/l for microcystin-LR)
- Raw water from surface intakes — lakes and impoundment reservoirs liable to blooms
- Assessment of the efficiency of water treatment with regard to the removal of cyanobacterial toxins
- Tap water distributed in the network (samples of clean water concentrated directly on SPE)
- Determination of the variants MCYST-RR and MCYST-YR alongside MCYST-LR — the method is validated for all three
Key parameters
| Parameter | Value |
|---|---|
| Validated analytes | MCYST-RR, MCYST-YR, MCYST-LR; other structural variants — determinable, but without unambiguous identification (no commercial standards, co-elution) |
| Matrix | Raw water containing biomass and treated water (e.g. tap water) |
| Detection | RP-HPLC with UV / diode-array detection at 238 nm (alternatively mass spectrometry — Annex A of the standard) |
| Reference value | 1 µg/l MCYST-LR according to the WHO proposal, attainable after SPE concentration; in Poland the parametric value is 1.0 µg/l with a permissible measurement uncertainty of 30 % |
| Requirements for the SPE cartridge | Capacity of at least 100 µg of each microcystin; recovery not less than 80 % for MCYST-LR and 70 % for MCYST-RR and -YR at a loading of 0.05 µg of each |
| Range of the standard solutions | 0.2; 0.4; 1.0; 2.0 and 3.0 µg/ml of each microcystin (Table 3 of the standard) |
Standard
- Standard number
- ISO 20179:2005
- Title (PL)
- Jakość wody — Oznaczanie mikrocystyn — Metoda z zastosowaniem ekstrakcji do fazy stałej (SPE) i wysokosprawnej chromatografii cieczowej (HPLC) z detekcją w nadfiolecie (UV)
- Title (EN)
- Water quality — Determination of microcystins — Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection
Step-by-step procedure
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Taking the sample
Take samples in accordance with the standards cited in ISO 20179: ISO 5667-4 (natural and artificial lakes) and ISO 5667-5 (drinking water from treatment works and from the network). For preparing the reagents use grade 3 water according to ISO 3696.
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Limiting losses on plastics
Throughout the analysis avoid, wherever possible, components made of plastics (pipettes, tubing, plastic cartridges) — the microcystins adsorb on their walls and the result is lowered by the losses.
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Separating the biomass
Filter the water sample containing cyanobacterial material through a glass microfibre filter with a retention capacity of 1–2 µm (diameter up to 47 mm). This step concerns only samples with phytoplankton; the filtrate is treated further as a sample of clean water.
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Extracting the biomass
Extract the biomass from the filter separately with a methanol/water 75/25 (v/v) mixture. For disrupting the cells and dispersing the suspension the standard provides an ultrasonic probe (about 60 W, about 20 kHz), an ultrasonic bath, a horizontal shaker and a centrifuge of at least 4000 min⁻¹ and an RCF of at least 10 000 g. Filter and dilute the extract.
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Concentration on the SPE cartridge
Pass the filtrate of the sample and the samples of clean water (e.g. tap water) through a C-18 SPE cartridge from a 500 ml reservoir with the assistance of a vacuum pump. The cartridge must have a capacity of at least 100 µg of each microcystin and give a recovery of at least 80 % for MCYST-LR and 70 % for MCYST-RR and -YR.
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Washing and elution
Wash the cartridge with a methanol/water 20/80 (v/v) mixture and elute the microcystins with a methanol/water 90/10 (v/v) mixture with the addition of 0.1 % by volume of TFA. After concentration redissolve the eluate in a methanol/water 20/80 (v/v) mixture.
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Preparing the standards
Establish the exact concentration of each stock standard spectrophotometrically: dissolve it in pure methanol and record the absorption curve 220–250 nm in a 1 cm quartz cell against methanol, then calculate the concentration from equation (1) with ε = 39 800 l/(mol·cm) and a molar mass of 994 (LR), 1044 (YR) or 1037 g/mol (RR). From the mixed stock solution of 2.5 µg/ml (stored below −16 °C) prepare five standards: 0.2; 0.4; 1.0; 2.0 and 3.0 µg/ml.
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Chromatographic separation
Carry out the separation in the reversed-phase mode. The standard gives an EXAMPLE gradient (Table 1, described in the standard as "an example"; this is a starting point for optimisation on one’s own column, not a requirement): 0 min 30 % A / 70 % B, 10 min 35/65, 40 min 70/30, 42–44 min 100/0, 46–55 min return to 30/70; flow 0.3–1.0 ml/min depending on the column (A — acetonitrile with TFA, B — water with TFA; degas both phases before use).
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Detection and calculation of the result
Determine the microcystins quantitatively from UV / DAD detection at 238 nm, relating the peak areas to the calibration curve. Relate the result to the value of 1 µg/l of microcystin-LR (WHO; in Poland the parametric value is 1.0 µg/l).
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Control of the method
Examine in parallel a spiked sample: a control solution with a concentration of 1 µg/l of each of the three microcystins, prepared by diluting 200 µl of the mixed stock solution to 500 ml with tap water or with toxin-free lake water.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with UV or DAD detector | A reversed-phase column, detection at 238 nm, mobile phase flow of 0.3–1.0 ml/min chosen to suit the column | — |
| Set for solid-phase extraction | A 500 ml SPE reservoir with a connector for cartridges and a vacuum pump for SPE | — |
| Laboratory centrifuge | At least 4000 min⁻¹ and an RCF of at least 10 000 g; because of the flammable extraction solvents an explosion-proof centrifuge is recommended | — |
| Ultrasonic probe and ultrasonic bath | A probe of about 60 W power and about 20 kHz frequency — for disrupting the cyanobacterial cells | — |
| Glass microfibre filters | Retention capacity 1–2 µm, diameter not greater than 47 mm; needed only for samples with phytoplankton | — |
| Horizontal shaker with adjustment | Needed only when analysing samples containing phytoplankton | — |
| Spectrophotometer with 1 cm quartz cells | For establishing the exact concentration of the standards from the absorption curve 220–250 nm | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Methanol | 67-56-1 | HPLC purity; a component of the extraction solution, of the solvent for the standards and of the SPE elution solution |
| Acetonitrile | 75-05-8 | HPLC purity; the basis of mobile phase A (800 ml of acetonitrile + 500 µl of TFA, made up with acetonitrile to 1000 ml; stability about 3 weeks at room temperature) |
| Trifluoroacetic acid (TFA) | 76-05-1 | An addition to the mobile phases (500 µl per 1000 ml) and to the SPE elution solution (0.1 % by volume) |
| Extraction solution methanol/water 75/25 (v/v) | — | For extracting the microcystins from the cyanobacterial biomass retained on the filter |
| SPE elution solution — methanol/water 90/10 (v/v) with 0.1 % by volume of TFA | — | For eluting the microcystins from the SPE cartridge |
| Methanol/water solution 20/80 (v/v) | — | The solvent for diluting the standards, for washing the SPE cartridge and for redissolving the eluate |
| Microcystin standards MCYST-LR, -YR and -RR | — | Commercial, in ampoules; the exact concentration of the stock solution is established spectrophotometrically from the absorption curve 220–250 nm in methanol, at molar masses of 994, 1044 and 1037 g/mol and a molar absorption coefficient of 39 800 l/(mol·cm) at 238 nm |
Health and safety (OHS)
- Microcystins are strongly hepatotoxic to humans — the standard places the warning already in its heading; work with the standard solutions in gloves, in a fume cupboard
- Collect laboratory waste containing microcystins separately and dispose of it as highly toxic chemical waste; prolonged decontamination with a concentrated sodium hypochlorite (NaClO) solution is admissible
- The examination may be carried out only by suitably trained persons — the standard sets this as an absolute condition
- Methanol and acetonitrile are flammable and toxic, trifluoroacetic acid is corrosive — work in a fume cupboard, in gloves and protective goggles
- Because of the flammable extraction solvents the standard strongly recommends an explosion-proof centrifuge
Frequently asked questions
Which standard describes this test?
The test is performed according to ISO 20179:2005 — “Water quality — Determination of microcystins — Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection”.
How does this method work?
Water samples containing cyanobacterial material (biomass) are first filtered. The biomass retained is extracted separately with a methanol/water mixture, the extract is filtered, diluted and cleaned up by solid-phase extraction (SPE); the filtrate after the separation of the biomass is treated like a sample of clean water.
What is the measuring range and accuracy?
Validated analytes: MCYST-RR, MCYST-YR, MCYST-LR; other structural variants — determinable, but without unambiguous identification (no commercial standards, co-elution); Matrix: Raw water containing biomass and treated water (e.g. tap water); Detection: RP-HPLC with UV / diode-array detection at 238 nm (alternatively mass spectrometry — Annex A of the standard); Reference value: 1 µg/l MCYST-LR according to the WHO proposal, attainable after SPE concentration; in Poland the parametric value is 1.0 µg/l with a permissible measurement uncertainty of 30 %.
How long does the test take?
The times given for the individual stages add up to approximately 55 min. The procedure comprises 10 steps.
What equipment is required?
HPLC system with UV or DAD detector, Set for solid-phase extraction, Laboratory centrifuge, Ultrasonic probe and ultrasonic bath, Glass microfibre filters, Horizontal shaker with adjustment, Spectrophotometer with 1 cm quartz cells.
Where is this test used?
Control of water intended for consumption during a cyanobacterial bloom in the water source (Journal of Laws 2026 item 748: parametric value 1.0 µg/l for microcystin-LR); Raw water from surface intakes — lakes and impoundment reservoirs liable to blooms; Assessment of the efficiency of water treatment with regard to the removal of cyanobacterial toxins; Tap water distributed in the network (samples of clean water concentrated directly on SPE); Determination of the variants MCYST-RR and MCYST-YR alongside MCYST-LR — the method is validated for all three.
What safety precautions apply?
Microcystins are strongly hepatotoxic to humans — the standard places the warning already in its heading; work with the standard solutions in gloves, in a fume cupboard; Collect laboratory waste containing microcystins separately and dispose of it as highly toxic chemical waste; prolonged decontamination with a concentrated sodium hypochlorite (NaClO) solution is admissible; The examination may be carried out only by suitably trained persons — the standard sets this as an absolute condition; Methanol and acetonitrile are flammable and toxic, trifluoroacetic acid is corrosive — work in a fume cupboard, in gloves and protective goggles; Because of the flammable extraction solvents the standard strongly recommends an explosion-proof centrifuge.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number ISO 20179.