🌿 Acute toxicity test — Daphnia magna

Environmental PN-EN ISO 6341

Overview

STATUS (as of 02.11.2026). PN-EN ISO 6341:2013-04 — English version (Polish title in the PKN catalogue: “Jakość wody — Oznaczanie hamowania mobilności Daphnia magna Straus (Cladocera, Crustacea) — Test toksyczności ostrej”), published on 17.04.2013, implements EN ISO 6341:2012 and ISO 6341:2012 [IDT]; it replaced PN-EN ISO 6341:2002 (Polish version). The card has no withdrawal note — the standard is current; in the ISO catalogue the standard is confirmed (stage 90.93). ISO 6341:2012 is the fourth edition of 15.10.2012, which replaced ISO 6341:1996 and incorporated its Technical Corrigendum Cor 1:1998.

SCOPE (clause 1). Determination of acute toxicity to Daphnia magna Straus (Cladocera, Crustacea) — of chemical substances soluble under the test conditions or maintained as a stable suspension or dispersion, of industrial and sewage effluents, treated or untreated waste water, aqueous extracts and leachates, fresh surface and ground waters, and eluates and pore water of freshwater sediments. Crustaceans are of ecotoxicological interest as primary consumers and a major component of the zooplankton (introduction).

PRINCIPLE AND DEFINITIONS (clauses 3–4). An animal is immobilized when it is not able to swim during the 15 s following gentle agitation of the solution, even if it can still move its antennae; a neonate is a first-instar animal less than 24 h old. The initial concentration that immobilizes 50 % of the daphnids in 24 h or 48 h is determined — the 24 h EC50 or 48 h EC50; the lowest concentration immobilizing all and the highest immobilizing none are also desirable. The test has one or two stages: a preliminary test to find the range of concentrations and a definitive test; for substances a limit test at 100 mg/l or at a lower concentration of solubility can be performed.

CONDITIONS AND ORGANISMS (clauses 5–6). Exposure in the dark or under a photoperiod of 16 h light and 8 h dark, at (20 ± 2) °C, in an atmosphere free from toxic vapours and dusts. Neonates less than 24 h old, preferably not first-brood progeny, from a healthy culture — without mortality above 20 % in 2 days, males, ephippia or discoloured animals; they may also hatch from dormant eggs (ephippia) or come from a specialized supplier. Pure water — conductivity below 10 µS/cm. Natural water, reconstituted water or dechlorinated tap water is acceptable for culturing and dilution if the daphnids survive in it without signs of stress; pH 6–9 and hardness 140–275 mg/l CaCO₃ are recommended. The example reconstituted water has pH 7.8 ± 0.5, hardness (225 ± 50) mg/l CaCO₃, a molar Ca : Mg ratio close to 4 : 1 and dissolved oxygen above 7 mg/l: 25 ml each of solutions of calcium chloride dihydrate (11.76 g/l), magnesium sulfate heptahydrate (4.93 g/l), sodium bicarbonate (2.59 g/l) and potassium chloride (0.23 g/l) are made up to 1 l, aerated until saturation and stable pH, and then not aerated further. M4 medium (Annex A) is for culturing — it is not used as dilution water for samples with bivalent metal ions or of unknown composition, because EDTA reduces metal bioavailability.

SAMPLES AND DILUTIONS (clause 8). Testing as soon as possible, preferably within 12 h of collection; otherwise the sample is cooled to 0–5 °C and tested within 24 h, and if testing within 72 h is impossible it is frozen below −18 °C and tested within 2 months (thawing at not more than 30 °C, not in a microwave). Suspended matter may be allowed to settle for at most 2 h, centrifuged (e.g. 10 min at 5 000 g) or filtered; aeration only if dissolved oxygen is below 40 % saturation, for at most 20 min. The test is carried out without pH adjustment; pH is measured at the beginning and the end, and if toxic effects occur at pH outside 6.0–9.0 the test may be repeated with pH adjustment. At least five concentrations in a geometric series: for waste waters, waters, pore water and extracts usually with a factor of 2, for substances in the definitive test not more than 3.2 (2.2 for steep curves); each concentration and the control preferably in four replicates; solvent at most 0.1 ml/l, with controls without and with solvent.

PROCEDURE — SECOND SOURCE. Method C.2 of Regulation (EC) No 440/2008 (Daphnia sp. acute immobilisation test, consistent with OECD Test Guideline 202 of 2004): at least 20 animals per concentration and control in four groups of five, at least 2 ml of solution per animal; 18–22 °C without variation of more than ± 1 °C in a test; no aeration, no pH adjustment and no feeding; readings after 24 h and 48 h; oxygen and pH at the beginning and the end in the control and the highest concentration. The test is valid when not more than 10 % of the daphnids are immobilized in the controls and dissolved oxygen at the end exceeds 3 mg/l; the reference substance is tested monthly, at least twice a year.

WHAT WE DO NOT GIVE HERE. Of ISO 6341:2012 the text up to 9.1 is available; the preliminary and definitive tests, the sensitivity check with potassium dichromate (9.4) with the required 24 h EC50 range, the limit test, the estimation of the EC50 and the validity criteria (clause 10), the test report and precision data (Annex E) are outside it — therefore we give neither the dichromate EC50 range nor the validity criteria of the standard (the second source gives the criteria of method C.2).

Method principle

Young Daphnia magna less than 24 hours old are exposed at (20 ± 2) °C, in the dark or under a 16 h + 8 h photoperiod, to a series of concentrations of the sample or substance prepared in dilution water; after 24 h or 48 h the animals unable to swim for 15 s after gentle agitation are counted.

From the percentage of immobilized animals the 24 h EC50 or 48 h EC50 is determined — the initial concentration that immobilizes half of the animals (clauses 3–5).

Applications

• Chemical substances soluble or maintained as a stable suspension or dispersion under the test conditions (clause 1)
• Industrial and sewage effluents, treated and untreated (clause 1)
• Aqueous extracts and leachates (clause 1)
• Fresh surface and ground waters; eluates and pore water of freshwater sediments (clause 1)
• Acute toxicity of substances to daphnids — method C.2 of Regulation (EC) No 440/2008, consistent with OECD Test Guideline 202

Key parameters

Standard

Standard number

PN-EN ISO 6341:2013-04

Title (PL)

Jakość wody — Oznaczanie hamowania mobilności Daphnia magna Straus (Cladocera, Crustacea) — Test toksyczności ostrej

Title (EN)

Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test

Step-by-step procedure

1. Culture and neonates

   Healthy culture — without mortality above 20 % in 2 days, males, ephippia or discolouration; gravid females are isolated and the young collected within 24 h; with different culture conditions — acclimation of one generation for about a week (6.1).

2. Sample

   Testing preferably within 12 h of collection (at 0–5 °C — within 24 h; frozen below −18 °C — within 2 months); homogenization by hand; suspended matter — settling for at most 2 h, centrifugation or filtration; measurement of pH and oxygen; no pH adjustment (8.1).

3. Concentration series

   At least five concentrations in a geometric series in dilution water — for waste waters and waters usually with a factor of 2; each concentration and the control preferably in four replicates (8.2.2, 9.1).

4. Exposure

   At least 20 animals per concentration and control, in groups of five, at least 2 ml of solution per animal; (20 ± 2) °C, darkness or 16 h + 8 h; no feeding and no aeration (clause 5; method C.2, 1.8.1).

5. Reading

   After 24 h and 48 h the number of immobilized animals — unable to swim for 15 s after gentle agitation, even if they move their antennae; dissolved oxygen and pH at the beginning and the end of the test in the control and the highest concentration (3.3, 8.1; method C.2, 1.8.2–1.8.3).

6. Result and validity

   EC50 from the percentage immobilized as a function of concentration (e.g. probit analysis) with a 95 % confidence interval; the test is valid when not more than 10 % of the animals are immobilized in the controls and oxygen at the end exceeds 3 mg/l (method C.2, 1.6 and 2).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard states that the test requires familiarity with normal laboratory practice and suitably qualified staff, and that safety practices are established by the user (warning of the standard)
• Potassium dichromate is a carcinogenic substance, toxic via inhalation — a ready-made solution of defined concentration reduces the risk of inhaling the dust (6.4)

Frequently asked questions