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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
Study period:
20 February 2017 - 27 February 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
according to
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
GLP compliance:
Specific details on test material used for the study:
Completely soluble in test medium up to 100 mg/L.
Analytical monitoring:
Details on sampling:
- Concentrations: all test concentrations and the control
- Sampling method: 1.8 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: in a freezer (≤-15°C) at the analytical laboratory of the Test Facility
- At the end of the exposure period, the replicates were pooled at each concentration before sampling
Details on test solutions:
- Direct addition method: A 100 mg/L stock solution of test item in test medium was prepared by applying a 10- minute period of ultrasonic waves to accelerate dissolution of the test item. This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: > two weeks old
- Age of test organisms at test start: < 24 hours
- Feeding during test: no
- Other: Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

- Acclimation period: none
Daphnids were introduced to the test solutions within 32 minutes after preparation of the test solutions.

- Method of breeding: start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7, as prescribed by Dr. Elendt-Schneider) in an all-glass culture vessel. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week.
Test type:
Water media type:
Total exposure duration:
48 h
180 mg/L expressed as CaCO3
Test temperature:
start: 8.2
end: 8.1
Dissolved oxygen:
start: 9.2 mg/L
end: 8.9 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L (combined limit/range-finding test)
- Measured concentrations: only samples taken from the control and the highest concentration, at t=0 and t=48h, were analysed. At t=0 and t=48, the measured concentration of the 100 mg/L test solution was at the level of nominal (97% on both occasions). Therefore, the effect parameters were expressed based on the analytically confirmed nominal concentration.
Details on test conditions:
- Test vessel: 100 mL, all-glass, fill volume: 80 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the limit concentration, 2 for the remaining concentrations
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1 daphnid/16 mL test solution

- Medium: Adjusted ISO medium. The following chemicals (analytical grade) were dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands):
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L
- Culture medium different from test medium: Culture medium had extra trace elements, macro nutrients and vitamins added as specified in Medium M7.
- Intervals of water quality measurement:
* temperature continuously in a temperature control vessel
* pH and dissolved oxygen at test start (t=0) and test end (t=48 h), for the highest test concentration and the control

- Adjustment of pH: no
- Photoperiod: 16 hours per day

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility at 0, 24 and 48 hours.

Combined limit/range-finding test: 0.10, 1.0, 10 and 100 mg/L.
Based on the results a definitive study was not required.
Reference substance (positive control):
potassium dichromate (performed February 2017)
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: Nominal test concentration confirmed by analysis.
Details on results:
- Immobilization of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Other: One daphnid was found to be immobile at the end of the test in one of the replicates at the 1.0 mg/L concentration level. Immobilization of ≤10% is allowed for the control treatment and therefore, considered biologically not relevant. Hence the effect observed in the 1.0 mg/L replicate was also considered biologically not relevant. Moreover, as no immobilization was observed at higher concentrations, this was not regarded as a test item related effect.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 48h-EC50 = 0.48 mg/L, i.e. within the expected range based on historical data collected at the testing laboratory (48h-EC50 between 0.3 and 1.0 mg/L)
- Limit test: no
- Dose-response test: yes (test concentrations: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L)
Reported statistics and error estimates:
No statistics/error estimates calculated because no relevant effects were observed at the highest test concentration.

Acceptability of the test:

- In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.

- The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

- The concentration of the substance was satisfactorily maintained throughout the study period.

- Constant test conditions (pH and temperature) were maintained throughout the study period.

Validity criteria fulfilled:
The 48h-EC50 of the substance to Dapnhia magna was determined to be >100 mg/L, based on analytically confirmed nominal test concentrations.
Executive summary:

In a study performed in accordance with OECD 202 (2004) and according to GLP principles, the acute toxicity of the substance to Daphnia magna was investigated. In a combined limit/range-finding test, performed under static conditions, daphnids were exposed for 48 hours to an untreated control and nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L (control and 100 mg/L - 4 replicates of 5 daphnids; 0.10, 1.0 and 10 mg/L - 2 replicates of 5 daphnids).

As the substance was completely soluble in test medium up to 100 mg/L, a stock solution of the substance at 100 mg/L was prepared (applying a 10 -minute period of ultrasonication to accelerate dissolution of the test item). This stock solution was used as highest test concentration and lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Samples for analytical confirmation of actual exposure concentrations were taken at t = 0 and 48 hours from all test levels, but only the control and the highest concentration samples were analysed. The measured concentration in the highest test concentration was found to be at the level of the nominal concentration at test start and test end (i.e. 97% of nominal). Since the test concentration remained stable during the test (99% of initial) the effect parameter was reported in terms of the analytically confirmed nominal concentration.

No biologically relevant immobilization was observed in any of the test groups. Therefore, the 48h-EC50 of the substance to Daphnia magna is concluded to be > 100 mg/L.

The study met the OECD validity criteria.

Description of key information

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information