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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:
2015-08-03 to 2015-08-26
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:
- Source and lot/batch No.of test material: A14JB3442
- Expiration date of the lot/batch: 2016-09-30 (retest date)
- Purity test date: 2014-09-30 (certificate of analysis release date)

- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: 0.21 g/L, stability in water not indicated

Analytical monitoring:
Details on sampling:
- Sampling method: singular samples for possible analysis were taken from all test concentrations and the control at t=0 and t=48h. 3.0 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. Additionally, reserve samples of 3.0 mL were taken for possible analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an expected low water solubility of the test item saturated solutions (SS) were prepared. The preparation of the test solutions started with the highest concentration of 100 mg/L applying two days of magnetic stirring at room temperature to ensure maximal dissolution of the test item in the test medium. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate. The dispersion was consequently filtered through a 0.45µm membrane filter to remove any non-dissolved material. The resulting undiluted filtrate was used as highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS 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: 7.7 - 7.8
At t= 48h: 8.1
Dissolved oxygen:
At t= 0h: 7.9-8.4
At t= 48h: 9.0-9.2
not relevant
Nominal and measured concentrations:
Final test
Nominal concentrations (% of the SS): 10, 18, 32, 56, 100 % of the SS prepared at 100 mg/L
Measured concentrations (mg/L) at t=0h: 9.88, 16.9, 31.0, 52.9, 97.4
Measured concentrations (mg/L) at t=48h: 9.61, 30.2, 16.6, 52.3, 95.6
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, defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test container. This was monitored after 24 and 48 hours of continuous treatment with the test substance.

- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 1.0 and 10% of a SS prepared at a loading rate of 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.:
42 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 38-46mg/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? yes (should lie between 0.3 and 1.0 mg/L)
- EC50/LC50: 48h-EC50 = 0.32 mg/L. the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe.
Reported statistics and error estimates:
The 24 and 48h-EC50 values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding nominal test item concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.0.0. (ToxRat Solutions gmbh, Germany) was used to perform the analyses.
Validity criteria fulfilled:
The acute toxicity of T001036 to Daphnia magna was determined in a 48-hour static test according to the OECD guideline 202.
The 48-h EC50 was determined to be 42 mg/L based on analytically confirmed nominal concentrations (95% CL 38-46 mg/L). The results of the test can be considered reliable without restriction.

Description of key information

The study of Migchielsen (2016), investigating the acute toxicity of T001036 to Daphnia magna according to OECD guideline 202, was considered as the key study for endpoint coverage. The measured 48-hour EC50 was determined to be 42 mg/L. Based on these findings, it can be considered that T001036 is harmful to aquatic invertebrates.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
42 mg/L

Additional information

The acute toxicity of T001036 with the water flea Daphnia magna STRAUS was investigated in a 48-hour static test according to OECD 202 and EU Method C.2 (Migchielsen, 2016). The study was rated a Klimisch score of 1. Daphnia magna were exposed to different concentrations of test substance during 48 hours.

Daphnids were exposed to an untreated control and to test groups representing 10, 18, 32, 56 and 100% of a saturated solution prepared at nominally 100 mg/L. Analysis of the samples taken at the start of the final test showed that measured test item concentrations were in agreement with the nominal concentrations (94-99%). All concentrations remained stable during the test period and therefore results were expressed in terms of analytically confirmed nominal concentrations.

The 48-hour EC50 was determined to be 42 mg/L (95% confidence interval between 38 and 46 mg/L). Based on the results it can be considered that T001036 is harmful to aquatic invertebrates.