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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: nominal 4.27, 9.39, 20.7, 45.5, and 100 mg/L and control
- Sampling intervals: start and end of the renewal periods
- Sampling method: Samples from expired media were taken from the pooled media from replicate vessels.
- Sample storage conditions before analysis: no storage prior to analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
At each concentration, the test substance was dispersed in dilution medium in an aspirator. The bulk preparations (4 L) were stirred for approximately 48 hours in the dark, after which a syphon was added to the mid-vessel portion of each aspirator and the preparations were then left to stand for a further approximate 48 hours in the dark. An aliquot (2 L) of the mid-vessel portion was siphoned from each preparation vessel and discarded, after which a further 1 L was removed mid-vessel to provide the Water Accommodated Fractions (WAFs).
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: in-house culture originating from a strain supplied by the National Institute for Applied Chemical Research (IRCHA), France
- Age at study initiation: < 24 h
- Method of breeding: Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.5 to 0.8 litres of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. The culture medium was renewed three times each week.
Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial two days when a slightly lower ration was given. Culture conditions ensured that the stock animals reproduced by parthenogenesis.
The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels, held in a separate holding vessel and used in the test.
- Feeding during test: none

- Acclimation period: not relevant, conditions were the same for maintenance and testing
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
268 , 247and 254 mg/L as CaCO3 for the three batches of Elendt M4 medium used
Test temperature:
19.6-20.6 °C
Dissolved oxygen:
97-104% air saturation value (>3 mg/L)
Nominal and measured concentrations:
nominal loading rates: 4.27, 9.39, 20.7, 45.5 and 100 mg test material/L
At all concentrations, the levels of carbon ranged between 0.00 and 0.2 mg C/L after background correction.
Details on test conditions:
- Test vessel: covered glass jars containing 100 mL of medium
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 20 mL medium per organism

- Source/preparation of dilution water: deionised, reverse osmosis water
- Total organic carbon: 0.9-1.2 mg C/L
- Alkalinity: 59, 43 and 46 mg /L as CaCO3 for the three batches of Elendt M4 medium used
- Ca/Mg ratio: 3.7:1
- Culture medium different from test medium: no

- Adjustment of pH: no
- Photoperiod: 16 hours light : 8 hours dark with periods of subdued lighting at the beginning and end of each light phase

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : numbers of mobile, immobile and floating Daphnia counted approx. 24 and 48 hours after the start of the study

- Test concentrations: nominal loading rates of 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 10% immobility at 10 and 100 mg/L after 48 hours. Not considered significant.
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: results are expressed in terms of loading rates
48 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: results are expressed in terms of loading rates
Details on results:
- Behavioural abnormalities: none
- Other biological observations: After 48 hours, at a nominal loading rates of 20.7 and 45.5 mg/L two Daphnia were swimming at the surface, which was not considered significant; no immobility occurred at any other test concentration.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

At all concentrations, the test media were colourless. In expired medium, particulate matter was observed on the surface of test vessels at nominal loading rates of 20.7 to 100 mg/L.
Reported statistics and error estimates:
The “no observed effect loading rate” (NOELR) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.
Validity criteria fulfilled:
The number of floating and/or immobile Daphnia in the control group did not exceed 10% and the concentration of dissolved oxygen was > 3 mg/L (≥60% of the air saturation value at the temperature used) at the end of the test.

Description of key information

48-h EC50 (based on loading rates) >100 mg/L for Daphnia magna (OECD TG 202, EU C.2)

Key value for chemical safety assessment

Additional information

The results indicate that the test material is not toxic to daphnia up to the limits of its water solubility (<1 mg/L) and a NOEC and thus a PNEC cannot be determined.