OECD 202: Daphnia sp., Acute Immobilization Test

Summary

This test is a standard ecotoxicity guideline used to assess the short-term toxic effects of chemical substances on freshwater invertebrates. The test is a critical component of environmental risk assessments for chemical substances that may enter into aquatic environments.

The OECD 202 Testing Method

This is a 48 hour static (or semi-static/flow-through) test that measures the percentage of juvenile Daphnia that are immobilized after exposure to at least 5 graded concentrations of a chemical substance. In this test, immobilization can be defined as the inability of the daphnid to swim within 15 seconds after gentle agitation of the test vessel. A control group (no test substance) is used for comparison. The most common test organism used is Daphnia magna Straus. These organisms are ecologically important and sensitive to a wide range of toxicants, making them an ideal test organism. It results in a calculation of the 48 hour EC50 (and optionally the 24 hour EC50), NOEC/LOEC and dose-response curves.

Here are some additional details about the OECD 202 test method:

• Substances that can be tested:
  • Water soluble or dispersible chemicals
  • Water solubility and vapor pressure should be known
• Test organisms:
  • Daphnia magna Straus are preferred (other Daphnia sp. may be acceptable if culture performance is demonstrated).
  • Juveniles <24 hours old, preferably not first brood
• Exposure period: 48 hours (immobilization measured at 24 hours and 48 hours)
• Test design:
  • At least 5 geometrically spaced concentrations with control vessels
  • 4 replicates with 5 daphnids at each concentration level
  • Optionally, a limit test at 100 mg L⁻¹ or solubility limit is allowed
• Medium: Reconstituted or natural water with pH of 6-9 and hardness of 140–250 mg CaCO₃ L⁻¹
• Volume: 20 mL animal⁻¹ minimum (e.g., 50-100 mL beakers loosely covered)
• Environmental conditions:
  • 20 ± 2 °C
  • Photoperiod is commonly 16 hours light/8 hours dark
• Feeding: None during 48 hour test
• Observations:
  • Daphnids are counted as immobilized if they are unable to swim within 15 seconds of gentle agitation
  • Sub-lethal effects are also recorded (e.g. surfacing, spasms)
• Endpoints:
  • 48 hour EC50 (optionally, 24 hour EC50)
  • NOEC/LOEC via ANOVA
  • Any other abnormal behavior is reported
• Validity criteria:
  • Less than 10% immobilization in controls
  • DO at least 3 mg L⁻¹ in all vessels
• Reference toxicant:
  • Potassium dichromate (24 hour EC50  of 0.6-2.1mg L⁻¹ in all vessels)
  • Tested every 1-2 months to confirm test integrity

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