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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)
Version / remarks:
April 2004
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Single Samples for analysis were taken from all test concentrations and the control.
Frequency: at t=0 h and t=48 h
Volume: 0.2 mL from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Storage: Not applicable, samples were analysed on the day of sampling
Details on test solutions:
The test item was not completely soluble at the loading rate of 100 mg/L. Thus, preparation of the test solution started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to reach the maximum dissolution of the test item in medium. Thereafter, the Water Accommodated Fraction (WAF) was collected by means of filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation) and used as test concentration. The test solution was clear and colorless at the end of the preparation procedure. No correction was made for the purity/composition of the test item.
The method of preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: Test medium without test item or other additives
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flee
- Strain/clone: Straus, 1820
- Age at study initiation (mean and range, SD): less than 24 hours
- Method of breeding: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of Elendt M7 medium in an all-glass culture vessel. The maximum age of the cultures was 4 weeks. After 7 days of cultivation, half of the medium was renewed twice a week. The temperature of medium was 18-22°C.
- Source: In-house laboratory culture with a known history
- Age of parental stock (mean and range, SD): 2nd to 5th brood
- Feeding during test: none

- Acclimation period: none; breeding and test conditions were comparable.
- Health during acclimation/breeding (any mortality observed): no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals; no delay in the production of the first brood.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
1.8 mM (180 mg CaCO3/L)
Test temperature:
20 - 21 °C
7.7 - 8.2
Dissolved oxygen:
8.4 - 9.5 mg O2/L
Nominal and measured concentrations:
nominal loading rate: 100 mg/L (WAF)
measured test concentrations: test start and end: < LOQ (0.001 mg/L)
Details on test conditions:
- Test vessel: 60 mL, all-glass vessels, containing 50 mL of test 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: 10 mL/animal

- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands); Composition: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: yes (daphnids were cultured in Elendt M7 medium)
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning and at the end of the test, for all concentrations and the control. Temperature was measured continuously in a temperature control vessel, beginning at the start of the test.

- Adjustment of pH: not necessary
- Photoperiod: daily photoperiod of 16 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Immobility (including mortality) and signs of disease or stress (e.g. discolouration, unusual behaviour such as surface trapping, etc.) after 24 and 48 hours of exposure.

- Test concentrations: combined limit/range-finding study testing WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no biologically significant immobility at any of the WAFs throughout the exposure period.
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Key result
48 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Details on results:
Behavioural abnormalities:
- Other biological observations: At the end of the test, a mortality of 5% was observed in the 100 mg/L-WAF. Since ≤ 10% immobility is allowed in the control, this effect was considered biologically not relevant.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
All test solutions were clear and colorless at the end of the preparation procedure.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 48-hour EC50: 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L (historical data of 48h EC50 are between 0.3 and 1.0 mg/L)
Reported statistics and error estimates:
EL50-values and NOELR were determined directly from the raw data.
Validity criteria fulfilled:
In the control, no daphnids have been immobilised or showed other sub-lethal effects; the dissolved oxygen concentration at the end of the test was > 3 mg/L in control and test vessels.

Description of key information

effect value for Daphnia magna (OECD 202): 48-h EL50: > 100 mg/L WAF

Key value for chemical safety assessment

Additional information

LC50 for freshwater invertebrates: > 100 mg/L

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