🦠 Viable eggs of intestinal parasites (Ascaris, Trichuris, Toxocara) in sewage sludge — isolation by shaking, washing with centrifugation and flotation, microscopic examination
In short
The reference method for sludge intended for use in agriculture and for reclamation. The test is performed according to PN-Z-19005:2018-10; Indicator determined: the total number of viable eggs of Ascaris sp., Trichuris sp., Toxocara sp. in 1 kg of dry matter of the sludge. The procedure comprises 7 steps; it is used for: Sewage sludge before it is handed over for agricultural use — the test deciding on the acceptance of the batch, Sludge for the reclamation of land for agricultural purposes — the same threshold of zero viable eggs per kilogram of dry matter, Control of the effectiveness of the hygienisation of sludge (liming, composting, thermophilic digestion).
At a glance
- Standard: PN-Z-19005:2018-10
- Category: Microbiology
- Procedure steps: 7
- Indicator determined: the total number of viable eggs of Ascaris sp., Trichuris sp., Toxocara sp. in 1 kg of dry matter of the sludge
- Permissible value: 0 eggs/kg d.m. for sludge used in agriculture and for the reclamation of land for agricultural purposes
- Stages of the isolation: shaking or stirring → washing with centrifugation → flotation → microscopic examination; the wording of the annex since 14.01.2026 adds a density of the flotation solution not less than 1.2 g/cm³, an incubation of the eggs for a minimum of 14 days at 26–28 °C and an assessment of the viability of the eggs
Overview
The determination decides whether a sewage sludge may be used agriculturally, so it is one of the most important tests in sludge management. The regulation on municipal sewage sludge indicates as the reference method the isolation of viable eggs of intestinal parasites from a representative sample of the sludge by shaking or stirring, washing with the use of centrifugation and flotation, and then carrying out a microscopic examination. The indicator tested is the total number of viable eggs of Ascaris sp., Trichuris sp. and Toxocara sp. in a kilogram of dry matter of the sludge.
The limit values are strict: for sludge used in agriculture and for the reclamation of land for agricultural purposes the permissible number of viable eggs is zero per kilogram of dry matter. This means that a single viable egg in the sample tested excludes the agricultural use of that batch of sludge. The frequency of testing by the reference methods depends on the loading of the treatment plant expressed in population equivalent.
Why precisely these three genera: the human and the pig roundworm (Ascaris lumbricoides, Ascaris suum) and the whipworm (Trichuris trichiura) have a diagnostic value in the sanitary assessment of sludge; infection occurs by the alimentary route, and ascariasis in the case of a massive infection may lead to obstruction of the bowel. The eggs of these nematodes are exceptionally durable: the time of survival in soil is given as from 240–294 days to several years with the retention of the capacity to infect, and physical and chemical methods destroy them with little effectiveness. The survival of viable eggs in compost heaps at a temperature of 50–60 °C has been established, and even the capacity of Ascaris eggs to develop after thirty days of composting at a temperature of 60–70 °C in some layers of the heap. This explains why the hygienisation of sludge does not exempt one from the test.
The restriction to three genera of nematodes does not mean that other parasites do not occur — a human being may be the host of about one hundred and fifty species of parasitic worms; the indicator is a conventional but proven measure of the sanitary condition of the sludge. In laboratory practice the methodology of the Institute of Rural Health in Lublin was also used, on the assumption that sludges containing invasive eggs in a number of ten and above per kilogram are regarded as epidemiologically dangerous.
The critical points of the determination: the representativeness of the sample (the sludge is not homogeneous and the result is a yes-or-no one), the effectiveness of the flotation where the content of organic matter is high, and the distinction between viable and dead eggs under the microscope — the last requires practice and is the most frequent place of divergence between laboratories. A note on the wording of the regulation: the description above corresponds to the reference method in its wording of 2015. The annex in the wording in force since 14.01.2026 is richer by three operating parameters: flotation in a flotation solution of a density NOT LESS THAN 1.2 g/cm³, INCUBATION of the isolated eggs for a minimum of 14 days at a temperature of 26–28 °C and an ASSESSMENT OF THE VIABILITY OF THE EGGS after the microscopic examination. A laboratory working today applies the new wording.
Method principle
From a representative sample of the sludge the viable eggs of intestinal parasites are isolated in three successive stages: shaking or stirring, which releases the eggs from the solid phase of the sludge, washing with the use of centrifugation, which separates them from fine particles and organic matter, and flotation in a solution of a suitable density, in which the eggs pass into the surface phase and the heavier particles fall to the bottom. The material obtained is examined under a microscope, counting and differentiating the eggs of Ascaris sp., Trichuris sp. and Toxocara sp. and assessing their viability. The result is given as the total number of viable eggs in a kilogram of dry matter of the sludge, and for that reason the dry matter is determined in parallel (PN-EN 15934 or PN-EN 12880) and the result is converted to it. For sludge intended for use in agriculture and for the reclamation of land for agricultural purposes the permissible value is zero viable eggs per kilogram of dry matter. A note on the wording of the regulation: the description above corresponds to the reference method in its wording of 2015. The annex in the wording in force since 14.01.2026 is richer by three operating parameters: flotation in a flotation solution of a density NOT LESS THAN 1.2 g/cm³, INCUBATION of the isolated eggs for a minimum of 14 days at a temperature of 26–28 °C and an ASSESSMENT OF THE VIABILITY OF THE EGGS after the microscopic examination. A laboratory working today applies the new wording.
Applications
- Sewage sludge before it is handed over for agricultural use — the test deciding on the acceptance of the batch
- Sludge for the reclamation of land for agricultural purposes — the same threshold of zero viable eggs per kilogram of dry matter
- Control of the effectiveness of the hygienisation of sludge (liming, composting, thermophilic digestion)
- Sanitary assessment of the land on which the sludge is to be used
- Sanitary monitoring of a treatment plant — the frequency depending on the population equivalent
Key parameters
| Parameter | Value |
|---|---|
| Indicator determined | the total number of viable eggs of Ascaris sp., Trichuris sp., Toxocara sp. in 1 kg of dry matter of the sludge |
| Permissible value | 0 eggs/kg d.m. for sludge used in agriculture and for the reclamation of land for agricultural purposes |
| Stages of the isolation | shaking or stirring → washing with centrifugation → flotation → microscopic examination; the wording of the annex since 14.01.2026 adds a density of the flotation solution not less than 1.2 g/cm³, an incubation of the eggs for a minimum of 14 days at 26–28 °C and an assessment of the viability of the eggs |
| Accompanying determination | dry matter (PN-EN 15934 / PN-EN 12880) — the result is converted to a kilogram of dry matter |
| Durability of the eggs | survival in soil from 240–294 days to several years; they survive in compost heaps at 50–60 °C |
| Critical point | the distinction between viable and dead eggs under the microscope — the main source of divergence between laboratories |
Standard
- Standard number
- PN-Z-19005:2018-10
- Title (PL)
- Jakość gleby — Oznaczanie liczby żywych jaj pasożytów jelitowych Ascaris sp., Trichuris sp., Toxocara sp. w komunalnych osadach ściekowych
- Title (EN)
- Soil quality — Determination of the number of viable eggs of intestinal parasites Ascaris sp., Trichuris sp., Toxocara sp. in municipal sewage sludge
Step-by-step procedure
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Taking a representative sample
Take a sample representative of the whole batch of the sludge — where the result is a yes-or-no one, an unrepresentative sample is the greatest risk. In parallel take material for the determination of the dry matter.
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Release of the eggs
Subject the sample to shaking or stirring, so as to release the eggs bound to the solid phase of the sludge.
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Washing with centrifugation
Wash the material with the use of centrifugation, separating the eggs from fine mineral particles and organic matter. Repeat in accordance with the procedure until a clean, readable sediment is obtained.
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Flotation
Carry out the flotation in a solution of a suitable density — the eggs pass into the surface phase, the heavier impurities fall to the bottom. Collect the surface phase for the microscopic examination. According to the wording of the annex since 14.01.2026 the flotation solution is to have a density not less than 1.2 g/cm³, and the isolated eggs are incubated for a minimum of 14 days at a temperature of 26–28 °C before the microscopic examination.
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Microscopic examination
Examine the preparation under a microscope, distinguishing the eggs of Ascaris sp., Trichuris sp. and Toxocara sp. and assessing their viability. Count the viable eggs.
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Conversion to dry matter
Convert the result to the total number of viable eggs in a kilogram of dry matter of the sludge, using the dry matter determined in parallel.
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Assessment and test report
Compare the result with the permissible value: for sludge used in agriculture and for the reclamation of land for agricultural purposes it is zero viable eggs per kilogram of dry matter. In the test report give the number and the genus of the eggs detected and the dry matter to which the result was converted.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Microscope — a microscopic examination concludes the determination according to the reference method | differentiation of the eggs of Ascaris, Trichuris and Toxocara and assessment of the viability | — |
| Laboratory centrifuge | washing with the use of centrifugation — a stage of the reference method | — |
| Shaker or stirrer | shaking or stirring — the first stage of the reference method | — |
| Vessels for flotation | flotation — the third stage of the reference method | — |
| Drying oven at 105 °C and an analytical balance | the parallel determination of the dry matter (PN-EN 15934) for the conversion of the result | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Flotation solution | — | the reference method names flotation as a stage of the isolation; neither the regulation nor the methodology referred to specifies the kind of the solution — it is chosen by the procedure of the laboratory; the density is given only by the annex in the wording since 14.01.2026: not less than 1.2 g/cm³ (the wording of 2015, which the rest of this entry describes, gave no density) |
| Water for washing | — | washing with the use of centrifugation — a stage of the reference method |
| Disinfectants for the working place and the glassware | — | infectious material — disinfection after every series |
Health and safety (OHS)
- Sewage sludge and the eggs of parasites are infectious material — work in gloves and a laboratory coat, without contact of the hands with the face, with disinfection of the working place after every series.
- The eggs of Ascaris are exceptionally resistant — ordinary disinfectants and the temperature of composting do not guarantee their destruction; sterilise the glassware and the equipment in accordance with the procedure.
- The test must not be carried out at a working place where culture media are prepared or where other microbiological cleanliness tests are carried out.
- Treat the waste after the flotation as infectious material — do not pour it into the drains without disinfection.
- When pipetting the surface phase avoid the formation of an aerosol.
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-Z-19005:2018-10 — “Soil quality — Determination of the number of viable eggs of intestinal parasites Ascaris sp., Trichuris sp., Toxocara sp. in municipal sewage sludge”.
How does this method work?
From a representative sample of the sludge the viable eggs of intestinal parasites are isolated in three successive stages: shaking or stirring, which releases the eggs from the solid phase of the sludge, washing with the use of centrifugation, which separates them from fine particles and organic matter, and flotation in a solution of a suitable density, in which the eggs pass into the surface phase and the heavier particles fall to the bottom. The material obtained is examined under a microscope, counting and differentiating the eggs of Ascaris sp.
What is the measuring range and accuracy?
Indicator determined: the total number of viable eggs of Ascaris sp., Trichuris sp., Toxocara sp. in 1 kg of dry matter of the sludge; Permissible value: 0 eggs/kg d.m. for sludge used in agriculture and for the reclamation of land for agricultural purposes; Stages of the isolation: shaking or stirring → washing with centrifugation → flotation → microscopic examination; the wording of the annex since 14.01.2026 adds a density of the flotation solution not less than 1.2 g/cm³, an incubation of the eggs for a minimum of 14 days at 26–28 °C and an assessment of the viability of the eggs; Accompanying determination: dry matter (PN-EN 15934 / PN-EN 12880) — the result is converted to a kilogram of dry matter.
How long does the test take?
The procedure comprises 7 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Microscope — a microscopic examination concludes the determination according to the reference method, Laboratory centrifuge, Shaker or stirrer, Vessels for flotation, Drying oven at 105 °C and an analytical balance.
Where is this test used?
Sewage sludge before it is handed over for agricultural use — the test deciding on the acceptance of the batch; Sludge for the reclamation of land for agricultural purposes — the same threshold of zero viable eggs per kilogram of dry matter; Control of the effectiveness of the hygienisation of sludge (liming, composting, thermophilic digestion); Sanitary assessment of the land on which the sludge is to be used; Sanitary monitoring of a treatment plant — the frequency depending on the population equivalent.
What safety precautions apply?
Sewage sludge and the eggs of parasites are infectious material — work in gloves and a laboratory coat, without contact of the hands with the face, with disinfection of the working place after every series.; The eggs of Ascaris are exceptionally resistant — ordinary disinfectants and the temperature of composting do not guarantee their destruction; sterilise the glassware and the equipment in accordance with the procedure.; The test must not be carried out at a working place where culture media are prepared or where other microbiological cleanliness tests are carried out.; Treat the waste after the flotation as infectious material — do not pour it into the drains without disinfection.; When pipetting the surface phase avoid the formation of an aerosol.
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 PN-Z-19005.