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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:
2016-02-12 to 2016-03-31
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)
Specific details on test material used for the study:
Batch: M15BB1451
Purity/composition: 98.8% (based on base titration assay)
Test item storage conditions At room temperature
Stable under storage conditions until 30 January 2021 (expiry date)
Analytical monitoring:
Details on sampling:
- Sampling method: samples for possible analysis were taken form all test concentrations and the control at t=0 and t=48h. 1.9 mL of volume was taken from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: the samples were stored in a freezer. Addionally, reserve samples of 1.9 mL were taken for possible analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying a 10-30 minute treatment period with ultrasonic waves followed by a 1.5-2.25 hour period of magnetic stirring to accelerate the dissolution of the test item in test medium. This resulted in a clear and colourless solution. The pH was adjusted from 9.2 (combined limit/range-finding test) and 9.1 (final test) to 8.4 with 1M HCl (Merck, Darmstadt, Germany). The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water-flea
- Source: in-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): <24h (from parental daphnids of more than 2 weeks old)
- Method of breeding: 250 newborn daphnids (i.e. less than 3 days old) are placed into 5 litres of medium in an all-glass culture vessel. Medium is M7. After 7 days of cultivation half of the medium is renewed twice a week.
- Feeding during test: no

- Acclimation period: max. 4 weeks
- Acclimation conditions (same as test or not): yes
- Type and amount of food: fresh water algae.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
180 mg/L as CaCO3
Test temperature:
At t= 0h: 8.1 - 8.2
At t= 48h: 7.9- - 8.0
Dissolved oxygen:
At t= 0h: 9.5 - 9.8
At t= 48h: 9.0 - 9.1
not relevant
Nominal and measured concentrations:
Final test
Nominal concentrations (mg/L): 2.2, 4.6, 10, 22, 46 mg/L
Measured concentrations (mg/L) at t=0h: 2.22, 4.72, 10.4, 23.5, 48.5
Measured concentrations (mg/L) at t=48h: 2.18, 4.67, 10.4, 23.3, 48.1
Details on test conditions:
- Test vessel: beaker
- Material, size, headspace, fill volume: 100 mL, glass beaker filled with 80mL
- Aeration: not during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility was defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test container and was monitored after 24 and 48 hours of continuous treatment with the test substance.

- Spacing factor for test concentrations: x2.1
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
8.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 3.2 - 21 mg/L
Details on results:
- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none
Results with reference substance (positive control):
- Results with reference substance valid?
- Mortality:
- EC50/LC50: 48h-EC50 = 0.29 mg/L (CI 95% 0.26-0.31 mg/L). The actual responses in this reference test with K2Cr2O7 were below the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was just below the expected range of 0.3 to 1.0 mg/L. Hence, the sensitivity of this batch of Daphnia magna was slightly above the sensitivity of batches previously tested at WIL Research Europe.
Reported statistics and error estimates:
The 24h and the 48h-EC50 value were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (nominal) using weighted linear regression.
Validity criteria fulfilled:
The acute toxicity of T000750 to Daphnia magna was determined in a 48-hour static test according to the OECD guideline 202.
Based on the results obtained during the present Daphnia study, the 48h-EC50 was 8.1 mg/L with a 95% confidence interval ranging from 3.2 to 21 mg/L. The results of the test can be considered reliable without restriction.

Description of key information

The study of Tabor-Kaplon (2016), investigating the acute toxicity of T000750 in Daphnia magna according to OECD guideline 202, was considered as the key study for endpoint coverage.  A LC50 of 8.1 mg/L (based on nominal test concentrations) was found after an exposure of 48 hours in a static test in freshwater. Based on the results it can be considered that T000750 is toxic to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8.1 mg/L

Additional information