🌿 Acute toxicity test — Daphnia magna
In short
Biological acute toxicity test using Daphnia magna (water flea) — determination of EC50 (concentration causing 50% immobilization) after 24 and 48 h exposure. The test is performed according to PN-EN ISO 6341:2013; Test organism: Daphnia magna Straus, neonates <24 h. The procedure comprises 6 steps (about 2 h in total); it is used for: Chemical classification for aquatic toxicity (REACH, CLP), Assessment of industrial and municipal wastewater toxicity, Surface water quality monitoring.
At a glance
- Standard: PN-EN ISO 6341:2013
- Category: Environmental
- Procedure steps: 6
- Total time of stages: 2 h
- Test organism: Daphnia magna Straus, neonates <24 h
- Exposure time: 24 and 48 h
- Endpoint: EC50 (immobilization)
Overview
The Daphnia magna test is one of the most widely used ecotoxicological bioassays worldwide. Daphnia magna (large water flea) is a small (2–5 mm) freshwater crustacean which, due to its sensitivity to pollutants, short life cycle, and ease of laboratory culture, is a model organism in aquatic toxicology.
The test involves exposing young Daphnia magna individuals (<24 h) to a series of dilutions of the tested substance/sample and observing immobilization after 24 and 48 hours. The result is EC50 — concentration causing 50% immobilization. The test is required in chemical classification (REACH), wastewater and water quality assessment, and waste classification.
ISO 6341 is harmonized with OECD 202 guidelines and is part of the ecotoxicological test battery used in aquatic environmental impact assessment.
Method principle
Young Daphnia magna individuals (neonates <24 h, 5 pcs. per replicate, 4 replicates) are exposed to 5–7 concentrations of the tested substance (geometric series) and control (pure culture medium). After 24 and 48 h, the number of immobilized individuals (unable to swim within 15 s after gentle shaking) is observed. EC50 is calculated by probit or logistic method with 95% confidence interval.
Applications
- Chemical classification for aquatic toxicity (REACH, CLP)
- Assessment of industrial and municipal wastewater toxicity
- Surface water quality monitoring
- Waste classification (aquatic environment toxicity)
- Environmental impact assessment (EIA)
- Wastewater treatment plant efficiency studies
Key parameters
| Parameter | Value |
|---|---|
| Test organism | Daphnia magna Straus, neonates <24 h |
| Exposure time | 24 and 48 h |
| Endpoint | EC50 (immobilization) |
| Temperature | 20 ± 2°C |
| Photoperiod | 16 h light / 8 h dark |
| Validation criterion | Immobilization in control ≤10% |
Standard
- Standard number
- PN-EN ISO 6341:2013
- Title (PL)
- Jakość wody — Oznaczanie hamowania ruchliwości Daphnia magna Straus (Cladocera, Crustacea) — Test ostrej toksyczności
- Title (EN)
- Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test
Step-by-step procedure
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Daphnia magna culture
Maintain culture in ISO medium, 20°C, 16/8 h photoperiod. Feed daily with algae. Collect neonates <24 h for test.
-
Dilution preparation
Prepare 5–7 sample concentrations in geometric series (e.g., 100, 50, 25, 12.5, 6.25%). Control = pure medium.
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Exposure
To each vessel (50 mL solution) transfer 5 neonates with Pasteur pipette. 4 replicates per concentration = 20 individuals. Do not feed during test.
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Reading after 24 h
Observe under stereomicroscope. Count immobilized individuals (unable to swim within 15 s after gentle shaking).
-
Reading after 48 h
Repeat observation. Record number of immobilized per concentration. Measure pH and O₂ in control samples.
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EC50 calculation
Calculate EC50 by probit or logistic method with 95% confidence interval. Check validation: control ≤10% immobilization.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Climate chamber/incubator | Binder KBF 240, Memmert ICH260L (20°C, photoperiod) | 15,000–40,000 PLN |
| Stereomicroscope | Olympus SZX7, Leica S9i, Zeiss Stemi 305 | 5,000–25,000 PLN |
| Oxygen meter | WTW Oxi 3310, Hach HQ40d | 3,000–12,000 PLN |
| pH meter | Mettler Toledo SevenCompact | 3,000–8,000 PLN |
| Test vessels (beakers 50–100 mL) | Borosilicate glass beakers, glass lids | 10–30 PLN/pc. |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Culture medium (ISO water) | — | CaCl₂ (294 mg/L) + MgSO₄ (123 mg/L) + NaHCO₃ (65 mg/L) + KCl (5.75 mg/L) in deionized water |
| Potassium dichromate K₂Cr₂O₇ | 7778-50-9 | Reference substance for validation test. EC50 24h: 0.6–2.1 mg/L |
| Daphnia food | — | Chlorella vulgaris culture (green algae), 0.1–0.2 mg C/daphnia/day |
Health and safety (OHS)
- Potassium dichromate — carcinogen cat. 1B, mutagenic. Use fume hood and gloves.
- Samples may contain toxic substances — nitrile gloves mandatory
- Test waste — collect in chemical waste containers
- Biological culture — maintain sterility, avoid contamination
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 6341:2013 — “Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test”.
How does this method work?
Young Daphnia magna individuals (neonates <24 h, 5 pcs. per replicate, 4 replicates) are exposed to 5–7 concentrations of the tested substance (geometric series) and control (pure culture medium).
What is the measuring range and accuracy?
Test organism: Daphnia magna Straus, neonates <24 h; Exposure time: 24 and 48 h; Endpoint: EC50 (immobilization); Temperature: 20 ± 2°C.
How long does the test take?
The times given for the individual stages add up to approximately 2 h. The procedure comprises 6 steps.
What equipment is required?
Climate chamber/incubator, Stereomicroscope, Oxygen meter, pH meter, Test vessels (beakers 50–100 mL). Indicative cost of the core instrument (Climate chamber/incubator): 15,000–40,000 PLN.
Where is this test used?
Chemical classification for aquatic toxicity (REACH, CLP); Assessment of industrial and municipal wastewater toxicity; Surface water quality monitoring; Waste classification (aquatic environment toxicity); Environmental impact assessment (EIA); Wastewater treatment plant efficiency studies.
What safety precautions apply?
Potassium dichromate — carcinogen cat. 1B, mutagenic. Use fume hood and gloves.; Samples may contain toxic substances — nitrile gloves mandatory; Test waste — collect in chemical waste containers; Biological culture — maintain sterility, avoid contamination.
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-EN ISO 6341.